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Andrea COSSARIZZA

Professore Ordinario
Dipartimento di Scienze Mediche e Chirurgiche materno infantili e dell'adulto - Sede ex Scienze Biomediche

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Pubblicazioni

2024 - Advances and Challenges in Sepsis Management: Modern Tools and Future Directions [Articolo su rivista]
Santacroce, Elena; D’Angerio, Miriam; Ciobanu, Alin Liviu; Masini, Linda; Lo Tartaro, Domenico; Coloretti, Irene; Busani, Stefano; Rubio, Ignacio; Meschiari, Marianna; Franceschini, Erica; Mussini, Cristina; Girardis, Massimo; Gibellini, Lara; Cossarizza, Andrea; De Biasi, Sara
abstract

: Sepsis, a critical condition marked by systemic inflammation, profoundly impacts both innate and adaptive immunity, often resulting in lymphopenia. This immune alteration can spare regulatory T cells (Tregs) but significantly affects other lymphocyte subsets, leading to diminished effector functions, altered cytokine profiles, and metabolic changes. The complexity of sepsis stems not only from its pathophysiology but also from the heterogeneity of patient responses, posing significant challenges in developing universally effective therapies. This review emphasizes the importance of phenotyping in sepsis to enhance patient-specific diagnostic and therapeutic strategies. Phenotyping immune cells, which categorizes patients based on clinical and immunological characteristics, is pivotal for tailoring treatment approaches. Flow cytometry emerges as a crucial tool in this endeavor, offering rapid, low cost and detailed analysis of immune cell populations and their functional states. Indeed, this technology facilitates the understanding of immune dysfunctions in sepsis and contributes to the identification of novel biomarkers. Our review underscores the potential of integrating flow cytometry with omics data, machine learning and clinical observations to refine sepsis management, highlighting the shift towards personalized medicine in critical care. This approach could lead to more precise interventions, improving outcomes in this heterogeneously affected patient population.

2023 - A Comprehensive Analysis of Cytokine Network in Centenarians [Articolo su rivista]
Pinti, M.; Gibellini, L.; Lo Tartaro, D.; De Biasi, S.; Nasi, M.; Borella, R.; Fidanza, L.; Neroni, A.; Troiano, L.; Franceschi, C.; Cossarizza, A.
abstract

Cytokines have been investigated extensively in elderly people, with conflicting results. We performed a comprehensive analysis of the plasma levels of 62 cytokines and growth factors involved in the regulation of the immune system, in healthy centenarians, and middle-aged controls. We confirmed the previously observed increase in the levels of several pro-inflammatory cytokines, such as TNF-α and IL-6, and found that several other cytokines, directly or indirectly involved in inflammation (such as IFN-α, IL-23, CCL-5), were present at higher levels in centenarians. We did not observe any increase in the levels of anti-inflammatory cytokines, with the notable exception of the Th2-shifting cytokine IL-19. No relevant difference was observed in cytokines regulating T cell immunity. Several growth factors having a role in regulating immunity, such as G-CSF, GM-CSF, EGF, and VEGF, were upregulated in centenarians, too. Principal component analysis of the cytokine dataset showed that pro and anti-inflammatory cytokines were the variables that contributed the most to the variability of the data we observed.

2023 - Analysis of Antigen-Specific T and B Cells for Monitoring Immune Protection Against SARS-CoV-2 [Articolo su rivista]
De Biasi, Sara; Paolini, Annamaria; Lo Tartaro, Domenico; Gibellini, Lara; Cossarizza, Andrea
abstract

: Immunological memory is the basis of protection against most pathogens. Long-living memory T and B cells able to respond to specific stimuli, as well as persistent antibodies in plasma and in other body fluids, are crucial for determining the efficacy of vaccination and for protecting from a second infection by a previously encountered pathogen. Antigen-specific cells are represented at a very low frequency in the blood, and indeed, they can be considered "rare events" present in the memory T-cell pool. Therefore, such events should be analyzed with careful attention. In the last 20 years, different methods, mostly based upon flow cytometry, have been developed to identify such rare antigen-specific cells, and the COVID-19 pandemic has given a dramatic impetus to characterize the immune response against the virus. In this regard, we know that the identification, enumeration, and characterization of SARS-CoV-2-specific T and B cells following infection and/or vaccination require i) the use of specific peptides and adequate co-stimuli, ii) the use of appropriate inhibitors to avoid nonspecific activation, iii) the setting of appropriate timing for stimulation, and iv) the choice of adequate markers and reagents to identify antigen-specific cells. Optimization of these procedures allows not only determination of the magnitude of SARS-CoV-2-specific responses but also a comparison of the effects of different combinations of vaccines or determination of the response provided by so-called "hybrid immunity," resulting from a combination of natural immunity and vaccine-generated immunity. Here, we present two methods that are largely used to monitor the response magnitude and phenotype of SARS-CoV-2-specific T and B cells by polychromatic flow cytometry, along with some tips that can be useful for the quantification of these rare events. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of antigen-specific T cells Basic Protocol 2: Identification of antigen-specific B cells.

2023 - Circulating and Tumor-Associated Neutrophils in the Era of Immune Checkpoint Inhibitors: Dynamics, Phenotypes, Metabolism, and Functions [Articolo su rivista]
Gibellini, L.; Borella, R.; Santacroce, E.; Serattini, E.; Boraldi, F.; Quaglino, D.; Aramini, B.; De Biasi, S.; Cossarizza, A.
abstract

Simple Summary Neutrophils are the most abundant leukocytes in the circulation, represent the first line of defense in the immune system and mediate inflammation. Increasing evidence suggests that neutrophils constitute a large population of cells with phenotypic and functional heterogeneity. In this review, we summarize and discuss new findings delineating that both circulating neutrophils and tumor-associated neutrophils have a role in tumor prognosis and resistance to immune checkpoint inhibitors. Neutrophils are the most abundant myeloid cells in the blood and are a considerable immunological component of the tumor microenvironment. However, their functional importance has often been ignored, as they have always been considered a mono-dimensional population of terminally differentiated, short-living cells. During the last decade, the use of cutting-edge, single-cell technologies has revolutionized the classical view of these cells, unmasking their phenotypic and functional heterogeneity. In this review, we summarize the emerging concepts in the field of neutrophils in cancer, by reviewing the recent literature on the heterogeneity of both circulating neutrophils and tumor-associated neutrophils, as well as their possible significance in tumor prognosis and resistance to immune checkpoint inhibitors.

2023 - Critical COVID-19 Patients Through First, Second And Third Wave: Retrospective Observational Study Comparing Outcomes In ICU. [Articolo su rivista]
Coloretti, Irene; Farinelli, Carlotta; Biagioni, Emanuela; Gatto, Ilenia; Munari, Elena; Dall'Ara, Lorenzo; Busani, Stefano; Meschiari, Marianna; Tonelli, Roberto; Mussini, Cristina; Guaraldi, Giovanni; Cossarizza, Andrea; Clini, Enrico; Girardis, Massimo
abstract

Introduction- The time-course of the COVID-19 pandemic was characterized by subsequent waves identified by peaks of Intensive Care Unit (ICU) admission rates. During these periods, progressive knowledge of the disease led to the development of specific therapeutic strategies. This retrospective study investigates whether this led to improvement in outcomes of COVID-19 patients admitted to ICU. Methods- Outcomes were evaluated in consecutive adult COVID19 patients admitted to our ICU, divided into three waves based on the admission period: the first wave from February 25th, 2020, to July 6th, 2020; the second wave from September 20th, 2020, to February 13th, 2021; the third wave from February 14th, 2021 to April 30th, 2021. Differences were assessed comparing outcomes and by using different multivariable Cox models adjusted for variables related to outcome. Further sensitivity analysis was performed in patients undergoing invasive mechanical ventilation. Results- Overall, 428 patients were included in the analysis: 102, 169 and 157 patients in the first, second and third wave. The ICU and in-hospital crude mortalities were lower by 7% and 10% in the third wave compared to the other 2 waves (p>0.05). A higher number of ICU and hospital free days at day 90 was found in the third wave when compared to the other 2 waves (p=0.001). Overall, 62.6% underwent invasive ventilation, with decreasing requirement during the waves (p=0.002). The adjusted Cox model showed no difference in the Hazard Ratio for mortality among the waves. In the propensity-matched analysis the hospital mortality rate was reduced by 11% in the third wave (p=0.044). Conclusions - With application of best practice as known by the time of the first three waves of the pandemic, our study failed to identify a significant improvement in mortality rate when comparing the different waves of the COVID-19 pandemic, notwithstanding, the sub-analyses showed a trend in mortality reduction in the third wave. Rather, our study identified a possible positive effect of dexamethasone on mortality rate reduction and the increased risk of death related to bacterial infections in the three waves.

2023 - Detailed characterization of SARS-CoV-2-specific T and B cells after infection or heterologous vaccination [Articolo su rivista]
Lo Tartaro, Domenico; Paolini, Annamaria; Mattioli, Marco; Swatler, Julian; Neroni, Anita; Borella, Rebecca; Santacroce, Elena; Di Nella, Alessia; Gozzi, Licia; Busani, Stefano; Cuccorese, Michela; Trenti, Tommaso; Meschiari, Marianna; Guaraldi, Giovanni; Girardis, Massimo; Mussini, Cristina; Piwocka, Katarzyna; Gibellini, Lara; Cossarizza, Andrea; De Biasi, Sara
abstract

: The formation of a robust long-term antigen (Ag)-specific memory, both humoral and cell-mediated, is created following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination. Here, by using polychromatic flow cytometry and complex data analyses, we deeply investigated the magnitude, phenotype, and functionality of SARS-CoV-2-specific immune memory in two groups of healthy subjects after heterologous vaccination compared to a group of subjects who recovered from SARS-CoV-2 infection. We find that coronavirus disease 2019 (COVID-19) recovered patients show different long-term immunological profiles compared to those of donors who had been vaccinated with three doses. Vaccinated individuals display a skewed T helper (Th)1 Ag-specific T cell polarization and a higher percentage of Ag-specific and activated memory B cells expressing immunoglobulin (Ig)G compared to those of patients who recovered from severe COVID-19. Different polyfunctional properties characterize the two groups: recovered individuals show higher percentages of CD4+ T cells producing one or two cytokines simultaneously, while the vaccinated are distinguished by highly polyfunctional populations able to release four molecules, namely, CD107a, interferon (IFN)-γ, tumor necrosis factor (TNF), and interleukin (IL)-2. These data suggest that functional and phenotypic properties of SARS-CoV-2 adaptive immunity differ in recovered COVID-19 individuals and vaccinated ones.

2023 - Do all critically ill patients with COVID-19 disease benefit from adding tocilizumab to glucocorticoids? A retrospective cohort study. [Articolo su rivista]
Mussini, Cristina; Cozzi-Lepri, Alessandro; Meschiari, Marianna; Franceschini, Erica; Jole Burastero, Giulia; Faltoni, Matteo; Franceschi, Giacomo; Iadisernia, Vittorio; Volpi, Sara; Dessilani, Andrea; Gozzi, Licia; Conti, Jacopo; DEL MONTE, Martina; Milic, Jovana; Borghi, Vanni; Tonelli, Roberto; Brugioni, Lucio; Romagnoli, Elisa; Pietrangelo, Antonello; Corradini, Elena; Girardis, Massimo; Busani, Stefano; Cossarizza, Andrea; Clini, Enrico; Guaraldi, Giovanni
abstract

2023 - Dysfunctional subsets of CD39+ T cells, distinct from PD-1+, driven by leukemic extracellular vesicles in myeloid leukemias [Articolo su rivista]
Swatler, Julian; Lo Tartaro, Domenico; Borella, Rebecca; Brewinska-Olchowik, Marta; Paolini, Annamaria; Neroni, Anita; Turos-Korgul, Laura; Wiech, Milena; Kozlowska, Ewa; Cysewski, Dominik; Grabowska-Pyrzewicz, Wioleta; Wojda, Urszula; Basak, Grzegorz; Argüello, Rafael J; Cossarizza, Andrea; De Biasi, Sara; Piwocka, Katarzyna
abstract

2023 - Evaluating immunological and inflammatory changes of treatment-experienced people living with HIV switching from first-line triple cART regimens to DTG/3TC vs. B/F/TAF: the DEBATE trial [Articolo su rivista]
Cossarizza, A.; Cozzi-Lepri, A.; Mattioli, M.; Paolini, A.; Neroni, A.; De Biasi, S.; Tartaro, D. L.; Borella, R.; Fidanza, L.; Gibellini, L.; Beghetto, B.; Roncaglia, E.; Nardini, G.; Milic, J.; Menozzi, M.; Cuomo, G.; Digaetano, M.; Orlando, G.; Borghi, V.; Guaraldi, G.; Mussini, C.
abstract

Background: The aim of this randomized clinical trial (RCT) was to compare immunological changes in virally suppressed people living with HIV (PLWH) switching from a three-drug regimen (3DR) to a two-drug regimen (2DR).Methods: An open-label, prospective RCT enrolling PLWH receiving a 3DR who switched to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) or dolutegravir/lamivudine (DTG/3TC) was performed. Blood was taken at baseline and months 6 and 12. The primary outcome was the change in CD4+ or CD8+ T-cell counts and CD4/CD8 ratio over time points. The secondary outcomes were the changes in immunological and inflammatory parameters. Parametric mixed-linear models with random intercepts and slopes were fitted separately for each marker after controlling for potential confounders.Results: Between the two arms (33 PLWH each), there was no difference in CD4+ or CD8+ T cells, CD4/CD8 ratio, and IL-6 trajectories. PLWH switching to DTG/3TC had increased levels of both transitional memory and terminally differentiated CD4+ T cells (arm-time interaction p-value = 0.02) and to a lesser extent for the corresponding CD8+ T-cell subsets (p = 0.09). Significantly lower levels of non-classical monocytes were detected in the B/F/TAF arm at T6 (diff = -6.7 cells/mm(3); 95% CI; -16, +2.6; p-value for interaction between arm and time = 0.03). All differences were attenuated at T12.Conclusion: No evidence for a difference in absolute CD4+ and CD8+ T-cell counts, CD4/CD8 ratio, and IL-6 trajectories by study arm over 12 months was found. PLWH on DTG/3TC showed higher levels of terminally differentiated and exhausted CD4+ and CD8+ T lymphocytes and non-classical monocytes at T6. Further studies are warranted to better understand the clinical impact of our results.

2023 - Healthy preterm newborns: Altered innate immunity and impaired monocyte function [Articolo su rivista]
De Biasi, Sara; Neroni, Anita; Nasi, Milena; Lo Tartaro, Domenico; Borella, Rebecca; Gibellini, Lara; Lucaccioni, Laura; Bertucci, Emma; Lugli, Licia; Miselli, Francesca; Bedetti, Luca; Neri, Isabella; Ferrari, Fabrizio; Facchinetti, Fabio; Berardi, Alberto; Cossarizza, Andrea
abstract

: Birth prior to 37 completed weeks of gestation is referred to as preterm (PT). Premature newborns are at increased risk of developing infections as neonatal immunity is a developing structure. Monocytes, which are key players after birth, activate inflammasomes. Investigations into the identification of innate immune profiles in premature compared to full-term infants are limited. Our research includes the investigation of monocytes and NK cells, gene expression, and plasma cytokine levels to investigate any potential differences among a cohort of 68 healthy PT and full-term infants. According to high-dimensional flow cytometry, PT infants have higher proportions of CD56+/- CD16+ NK cells and immature monocytes, and lower proportions of classical monocytes. Gene expression revealed lower proportions of inflammasome activation after in vitro monocyte stimulation and the quantification of plasma cytokine levels expressed higher concentrations of alarmin S100A8. Our findings suggest that PT newborns have altered innate immunity and monocyte functional impairment, and pro-inflammatory plasmatic profile. This may explain PT infants' increased susceptibility to infectious disease and should pave the way for novel therapeutic strategies and clinical interventions.

2023 - Immunological signature in human cases of monkeypox infection in 2022 outbreak: an observational study [Articolo su rivista]
Agrati, Chiara; Cossarizza, Andrea; Mazzotta, Valentina; Grassi, Germana; Casetti, Rita; DE BIASI, Sara; Pinnetti, Carmela; Gili, Simona; Mondi, Annalisa; Cristofanelli, Flavia; LO TARTARO, Domenico; Notari, Stefania; Maffongelli, Gaetano; Gagliardini, Roberta; Gibellini, Lara; Aguglia, Camilla; Lanini, Simone; D'Abramo, Alessandra; Matusali, Giulia; Fontana, Carla; Nicastri, Emanuele; Maggi, Fabrizio; Girardi, Enrico; Vaia, Francesco; Antinori, Andrea
abstract

2023 - Multiparametric analysis of tumor infiltrating lymphocytes in solid tumors [Capitolo/Saggio]
Borella, R.; Paolini, A.; Aramini, B.; Gibellini, L.; Masciale, V.; Lo Tartaro, D.; Dominici, M.; De Biasi, S.; Cossarizza, A.
abstract

The use of single-cell technologies in characterizing the interactions between immune and cancer cells is in continuous expansion. Indeed, the combination of different single-cell approaches enables the definition of novel phenotypic and functional aspects of the immune cells infiltrating the tumor microenvironment (TME). This approach is promoting the discovery of relevant and reliable predictive biomarkers, along with the development of new promising treatments. In this chapter, we describe the main subsets of tumor-infiltrating lymphocytes from a phenotypical and functional point of view and discuss the use of single-cell technologies used to characterize these cell populations within TME.

2023 - Prognostic immune markers identifying patients with severe COVID-19 who respond to tocilizumab [Articolo su rivista]
DE BIASI, Sara; Mattioli, Marco; Meschiari, Marianna; LO TARTARO, Domenico; Paolini, Annamaria; Borella, Rebecca; Neroni, Anita; Fidanza, LUCIA MICHELA PIA; Busani, Stefano; Girardis, Massimo; Coppi, Francesca; Mattioli, Anna Vittoria; Guaraldi, Giovanni; Mussini, Cristina; Cossarizza, Andrea; Gibellini, Lara
abstract

Introduction: A growing number of evidences suggest that the combination of hyperinflammation, dysregulated T and B cell response and cytokine storm play a major role in the immunopathogenesis of severe COVID-19. IL-6 is one of the main pro-inflammatory cytokines and its levels are increased during SARS-CoV-2 infection. Several observational and randomized studies demonstrated that tocilizumab, an IL-6R blocker, improves survival in critically ill patients both in infectious disease and intensive care units. However, despite transforming the treatment options for COVID-19, IL-6R inhibition is still ineffective in a fraction of patients. Methods: In the present study, we investigated the impact of two doses of tocilizumab in patients with severe COVID-19 who responded or not to the treatment by analyzing a panel of cytokines, chemokines and other soluble factors, along with the composition of peripheral immune cells, paying a particular attention to T and B lymphocytes. Results: We observed that, in comparison with non-responders, those who responded to tocilizumab had different levels of several cytokines and different T and B cells proportions before starting therapy. Moreover, in these patients, tocilizumab was further able to modify the landscape of the aforementioned soluble molecules and cellular markers. Conclusions: We found that tocilizumab has pleiotropic effects and that clinical response to this drug remain heterogenous. Our data suggest that it is possible to identify patients who will respond to treatment and that the administration of tocilizumab is able to restore the immune balance through the re-establishment of different cell populations affected by SARS-COV-2 infection, highlighting the importance of temporal examination of the pathological features from the diagnosis.

2023 - Randomized, double-blind, placebo-controlled trial of rapamycin in amyotrophic lateral sclerosis [Articolo su rivista]
Mandrioli, J.; D'Amico, R.; Zucchi, E.; De Biasi, S.; Banchelli, F.; Martinelli, I.; Simonini, C.; Lo Tartaro, D.; Vicini, R.; Fini, N.; Gianferrari, G.; Pinti, M.; Lunetta, C.; Gerardi, F.; Tarlarini, C.; Mazzini, L.; De Marchi, F.; Scognamiglio, A.; Soraru, G.; Fortuna, A.; Lauria, G.; Bella, E. D.; Caponnetto, C.; Meo, G.; Chio, A.; Calvo, A.; Cossarizza, A.
abstract

In preclinical studies rapamycin was found to target neuroinflammation, by expanding regulatory T cells, and affecting autophagy, two pillars of amyotrophic lateral sclerosis (ALS) pathogenesis. Herein we report a multicenter, randomized, double-blind trial, in 63 ALS patients who were randomly assigned in a 1:1:1 ratio to receive rapamycin 2 mg/m2/day,1 mg/m2/day or placebo (EUDRACT 2016-002399-28; NCT03359538). The primary outcome, the number of patients exhibiting an increase >30% in regulatory T cells from baseline to treatment end, was not attained. Secondary outcomes were changes from baseline of T, B, NK cell subpopulations, inflammasome mRNA expression and activation status, S6-ribosomal protein phosphorylation, neurofilaments; clinical outcome measures of disease progression; survival; safety and quality of life. Of the secondary outcomes, rapamycin decreased mRNA relative expression of the pro-inflammatory cytokine IL-18, reduced plasmatic IL-18 protein, and increased the percentage of classical monocytes and memory switched B cells, although no corrections were applied for multiple tests. In conclusion, we show that rapamycin treatment is well tolerated and provides reassuring safety findings in ALS patients, but further trials are necessary to understand the biological and clinical effects of this drug in ALS.

2023 - Reduced Graphene Oxide Electrolyte-Gated Transistor Immunosensor with Highly Selective Multiparametric Detection of Anti-Drug Antibodies [Articolo su rivista]
Sensi, M.; de Oliveira, R. F.; Berto, M.; Palmieri, M.; Ruini, E.; Livio, P. A.; Conti, A.; Pinti, M.; Salvarani, C.; Cossarizza, A.; Cabot, J. M.; Ricart, J.; Casalini, S.; Gonzalez-Garcia, M. B.; Fanjul-Bolado, P.; Bortolotti, C. A.; Samori, P.; Biscarini, F.
abstract

The advent of immunotherapies with biological drugs has revolutionized the treatment of cancers and auto-immune diseases. However, in some patients, the production of anti-drug antibodies (ADAs) hampers the drug efficacy. The concentration of ADAs is typically in the range of 1-10 pm; hence their immunodetection is challenging. ADAs toward Infliximab (IFX), a drug used to treat rheumatoid arthritis and other auto-immune diseases, are focussed. An ambipolar electrolyte-gated transistor (EGT) immunosensor is reported based on a reduced graphene oxide (rGO) channel and IFX bound to the gate electrode as the specific probe. The rGO-EGTs are easy to fabricate and exhibit low voltage operations (& LE; 0.3 V), a robust response within 15 min, and ultra-high sensitivity (10 am limit of detection). A multiparametric analysis of the whole rGO-EGT transfer curves based on the type-I generalized extreme value distribution is proposed. It is demonstrated that it allows to selectively quantify ADAs also in the co-presence of its antagonist tumor necrosis factor alpha (TNF-alpha), the natural circulating target of IFX.

2023 - Risk of SARS-CoV-2 reinfection by vaccination status, predominant variant and time from prior infection: a cohort study, Reggio Emilia province, Italy, February 2020 to February 2022 [Articolo su rivista]
Vicentini, Massimo; Venturelli, Francesco; Mancuso, Pamela; Bisaccia, Eufemia; Zerbini, Alessandro; Massari, Marco; Cossarizza, Andrea; De Biasi, Sara; Pezzotti, Patrizio; Bedeschi, Emanuela; Giorgi Rossi, Paolo
abstract

: BackgroundUnderstanding the epidemiology of reinfections is crucial for SARS-CoV-2 control over a long period.AimTo evaluate the risk of SARS-CoV-2 reinfection by vaccination status, predominant variant and time after first infection.MethodsWe conducted a cohort study including all residents in the Reggio Emilia province on 31 December 2019, followed up until 28 February 2022 for SARS-CoV-2 first infection and reinfection after 90 days. Cox models were used to compare risk of first infection vs reinfection, adjusting for age, sex, vaccine doses and comorbidities.ResultsThe cohort included 538,516 residents, 121,154 with first SARS-CoV-2 infections and 3,739 reinfections, most in the Omicron BA.1 period. In the pre-Omicron period, three doses of vaccine reduced risk of reinfection by 89% (95% CI: 87-90), prior infection reduced risk by 90% (95% CI: 88-91), while two doses and infection reduced risk by 98% (95% CI: 96-99). In the Omicron BA.1 period, protection estimates were 53% (95% CI: 52-55), 9% (95% CI: 4-14) and 76% (95% CI: 74-77). Before Omicron, protection from reinfection remained above 80% for up to 15 months; with Omicron BA.1, protection decreased from 71% (95% CI: 65-76) at 5 months to 21% (95% CI: 10-30) at 22 months from the first infection. Omicron BA.1 reinfections showed 48% (95% CI: 10-57) lower risk of severe disease than first infections.ConclusionsNatural immunity acquired with previous variants showed low protection against Omicron BA.1. Combined vaccination and natural immunity seems to be more protective against reinfection than either alone. Vaccination of people with prior infection reduced the risk of severe disease.

2023 - Role of selective digestive decontamination in the prevention of VAP in COVID-19 patients: a pre-post observational study. [Articolo su rivista]
Biagioni, Emanuela; Ferrari, Elena; Gatto, Ilenia; Serio, Lucia; Farinelli, Carlotta; Coloretti, Irene; Talamonti, Marta; Tosi, Martina; Meschiari, Marianna; Tonelli, Roberto; Venturelli, Claudia; Mussini, Cristina; Clini, Enrico; Sarti, Mario; Cossarizza, Andrea; Busani, Stefano; Girardis, Massimo.
abstract

The aim of our study was to evaluate whether the introduction of SDD in a structured protocol for VAP prevention was effective in reducing the occurrence of ventilator associated pneumonia (VAP) in COVID19 patients without changes in the microbiological pattern of antibiotic resistances. This observational pre-post study including adult patients requiring invasive mechanical ventilation (IMV) for severe respiratory failure related to SARS-CoV-2 admitted in three COVID19 intensive care units (ICUs) in an Italian hospital from February 22, 2020, to March 8, 2022. Selective digestive decontamination (SDD) was introduced from the end of April 2021 in the structured protocol for VAP prevention. The SDD consisted of a tobramycin sulphate, colistin sulphate and amphotericin B suspension applied in the patient oropharynx and the stomach via nasogastric tube. Three-hundred forty-eight patients were included in the study. In the 86 patients (32,9%) who received SDD the occurrence of VAP decreased by 7,7% (p = 0,192) compared to patients who did not receive SDD. Onset time of VAP, the occurrence of multidrug-resistant microorganisms AP, the length of invasive mechanical ventilation and hospital mortality were similar in patients who received and who did not receive SDD. The multivariate analysis adjusted for confounders showed that the use of SDD reduces the occurrence of VAP (HR 0,536, CI 0,338-0,851; p = 0,017) Our pre-post observational study indicates that the use of SDD in a structured protocol for VAP prevention seems to reduce the occurrence of VAP without changes in the incidence of multidrug-resistant bacteria in COVID19 patients.

2023 - The association of procalcitonin and C- reactive protein with bacterial infections acquired during ICU stay in COVID-19 critically ill patients. [Articolo su rivista]
Campani, Simone; Talamonti, Marta; Dall’Ara, Lorenzo; Coloretti, Irene; Gatto, Ilenia; Biagioni, Emanuela; Tosi, Martina; Meschiari, Marianna; Tonelli, Roberto; Clini, Enrico; Cossarizza, Andrea; Guaraldi, Giovanni; Mussini, Cristina; Sarti, Mario; Trenti, Tommaso; Girardis, Massimo
abstract

In COVID-19 patients, procalcitonin (PCT) and C-reactive protein (CRP) performance in identify-ing bacterial infections remains unclear. Our study aimed to evaluate the association of PCT and CRP with secondary infections acquired during ICU stay in critically ill COVID-19. This observa-tional study included adult patients admitted to three COVID-19 intensive care units (ICU) from February 2020 to May 2022 with respiratory failure caused by SARS-CoV-2 infection and ICU stay≥ 11 days. The values of PCT and CRP collected on the day of infection diagnosis were com-pared to those collected on day 11 after ICU admission, the median time for infection occurrence, in patients without secondary infection. The receiver operating characteristic curve (ROC) and multivariate logistic model were used to assess PCT and CRP association with secondary infec-tions. Two hundred and seventy-nine patients were included, of whom 169 (60,6%) developed secondary infection after ICU admission. The PCT and CRP values observed on the day of the in-fection diagnosis were larger (p< 0,001) than those observed on day 11 after ICU admission in pa-tients without secondary infections. The ROC analysis calculated an AUC of 0,744 (95%CI 0,685-0,803) and 0,754 (95%CI 0,695-0,812) for PCT and CRP, respectively. Multivariate logistic models showed that PCT ≥ 0,16 ng/ml and CRP≥ 1,35 mg/dl were associated (p<0,001) with infections acquired during ICU stay. Our results indicated that PCT and CRP values were associated with developing secondary infections in COVID-19 patients with an ICU stay > 11 days with an ac-ceptable level of diagnostic accuracy using cut-off values lower than those commonly used in no-COVID-19 patients.

2023 - Ultra-rare RTEL1 gene variants associate with acute severity of COVID-19 and evolution to pulmonary fibrosis as a specific long COVID disorder [Articolo su rivista]
Bergantini, L.; Baldassarri, M.; D'Alessandro, M.; Brunelli, G.; Fabbri, G.; Zguro, K.; Degl'Innocenti, A.; Mari, F.; Daga, S.; Meloni, I.; Bruttini, M.; Croci, S.; Lista, M.; Maffeo, D.; Pasquinelli, E.; Serio, V. B.; Antolini, E.; Basso, S. L.; Minetto, S.; Tita, R.; Mencarelli, M. A.; Rizzo, C. L.; Pinto, A. M.; Ariani, F.; Montagnani, F.; Tumbarello, M.; Rancan, I.; Fabbiani, M.; Cameli, P.; Bennett, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Guerrini, S.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Raffaelli, C. S.; Emiliozzi, A.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Romani, D.; Piacentini, P.; Bandini, M.; Desanctis, E.; Cappelli, S.; Canaccini, A.; Verzuri, A.; Anemoli, V.; Pisani, M.; Ognibene, A.; Lorubbio, M.; Pancrazzi, A.; Vaghi, M.; Monforte, A. D. A.; Miraglia, F. G.; Mondelli, M. U.; Mantovani, S.; Bruno, R.; Vecchia, M.; Maffezzoni, M.; Martinelli, E.; Girardis, M.; Busani, S.; Venturelli, S.; Cossarizza, A.; Antinori, A.; Vergori, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Riva, A.; Francisci, D.; Schiaroli, E.; Pallotto, C.; Parisi, S. G.; Basso, M.; Panese, S.; Baratti, S.; Scotton, P. G.; Andretta, F.; Giobbia, M.; Scaggiante, R.; Gatti, F.; Castelli, F.; Quiros-Roldan, E.; Antoni, M. D.; Zanella, I.; Monica, M.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Fiorentino, G.; Carella, M.; Castori, M.; Merla, G.; Squeo, G. M.; Aucella, F.; Raggi, P.; Perna, R.; Bassetti, M.; Di Biagio, A.; Sanguinetti, M.; Masucci, L.; Guarnaccia, A.; Valente, S.; Di Florio, A.; Mandala, M.; Giorli, A.; Salerni, L.; Zucchi, P.; Parravicini, P.; Menatti, E.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Lacerenza, G.; Mussini, C.; Tavecchia, L.; Crotti, L.; Parati, G.; Mene, R.; Sanarico, M.; Gori, M.; Raimondi, F.; Stella, A.; Biscarini, F.; Bachetti, T.; La Rovere, M. T.; Bussotti, M.; Ludovisi, S.; Capitani, K.; Dei, S.; Ravaglia, S.; Giliberti, A.; Gori, G.; Artuso, R.; Andreucci, E.; Pagliazzi, A.; Fiorentini, E.; Perrella, A.; Bianchi, F.; Bergomi, P.; Catena, E.; Colombo, R.; Luchi, S.; Morelli, G.; Petrocelli, P.; Iacopini, S.; Modica, S.; Baroni, S.; Micheli, G.; Falcone, M.; Urso, D.; Tiseo, G.; Matucci, T.; Grassi, D.; Ferri, C.; Marinangeli, F.; Brancati, F.; Vincenti, A.; Borgo, V.; Lombardi, S.; Lenzi, M.; Di Pietro, M. A.; Vichi, F.; Romanin, B.; Attala, L.; Costa, C.; Gabbuti, A.; Bellucci, A.; Colaneri, M.; Casprini, P.; Pomara, C.; Esposito, M.; Leoncini, R.; Cirianni, M.; Galasso, L.; Bellini, M. A.; Gabbi, C.; Picchiotti, N.; Furini, S.; Fallerini, C.; Bargagli, E.; Renieri, A.
abstract

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a novel coronavirus that caused an ongoing pandemic of a pathology termed Coronavirus Disease 19 (COVID-19). Several studies reported that both COVID-19 and RTEL1 variants are associated with shorter telomere length, but a direct association between the two is not generally acknowledged. Here we demonstrate that up to 8.6% of severe COVID-19 patients bear RTEL1 ultra-rare variants, and show how this subgroup can be recognized. Methods: A cohort of 2246 SARS-CoV-2-positive subjects, collected within the GEN-COVID Multicenter study, was used in this work. Whole exome sequencing analysis was performed using the NovaSeq6000 System, and machine learning methods were used for candidate gene selection of severity. A nested study, comparing severely affected patients bearing or not variants in the selected gene, was used for the characterisation of specific clinical features connected to variants in both acute and post-acute phases. Results: Our GEN-COVID cohort revealed a total of 151 patients carrying at least one RTEL1 ultra-rare variant, which was selected as a specific acute severity feature. From a clinical point of view, these patients showed higher liver function indices, as well as increased CRP and inflammatory markers, such as IL-6. Moreover, compared to control subjects, they present autoimmune disorders more frequently. Finally, their decreased diffusion lung capacity for carbon monoxide after six months of COVID-19 suggests that RTEL1 variants can contribute to the development of SARS-CoV-2-elicited lung fibrosis. Conclusion: RTEL1 ultra-rare variants can be considered as a predictive marker of COVID-19 severity, as well as a marker of pathological evolution in pulmonary fibrosis in the post-COVID phase. This notion can be used for a rapid screening in hospitalized infected people, for vaccine prioritization, and appropriate follow-up assessment for subjects at risk. Trial Registration NCT04549831 (www.clinicaltrial.org)

2022 - 4-1BBL-containing leukemic extracellular vesicles promote immunosuppressive effector regulatory T cells [Articolo su rivista]
Swatler, J.; Turos-Korgul, L.; Brewinska-Olchowik, M.; De Biasi, S.; Dudka, W.; Le, B. V.; Kominek, A.; Cyranowski, S.; Pilanc, P.; Mohammadi, E.; Cysewski, D.; Kozlowska, E.; Grabowska-Pyrzewicz, W.; Wojda, U.; Basak, G.; Mieczkowski, J.; Skorski, T.; Cossarizza, A.; Piwocka, K.
abstract

Chronic and acute myeloid leukemia evade immune system surveillance and induce immunosuppression by expanding proleukemic Foxp31 regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse, and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector proleukemic Tregs. Using mouse model of leukemia-like disease, we found that Rab27adependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, and IL21R and included 2 distinct effector Treg (eTreg) subsets: CD301CCR8hiTNFR2hi eTreg1 and CD391TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive Tregs and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms.

2022 - A first update on mapping the human genetic architecture of COVID-19 [Articolo su rivista]
Pathak, Ga; Polimanti, R; Karjalainen, J; Daly, M; Ganna, A; Daly, Mj; Stevens, C; Kanai, M; Liao, Rg; Trankiem, A; Balaconis, Mk; Nguyen, H; Solomonson, M; Veerapen, K; Ripatti, S; Nkambul, L; Bryant, S; Sankaran, Vg; Neale, Bm; Karczewski, Kj; Martin, Ar; Atkinson, Eg; Tsuo, K; Baya, N; Turley, P; Gupta, R; Walters, Rk; Palmer, Ds; Sarma, G; Cheng, N; Lu, W; Churchhouse, C; Goldstein, Ji; King, D; Zhou, W; Seed, C; Finucane, H; Satterstrom, Fk; Andrews, Sj; Sloofman, Lg; Sealfon, Sc; Hoggart, C; Underwood, Sj; Cordioli, M; Pirinen, M; Donner, K; Kivinen, K; Palotie, A; Kaunisto, M; Harerimana, N; Chwialkowska, K; Wolford, B; Roberts, G; Park, D; Ball, Ca; Coignet, M; Mccurdy, S; Knight, S; Partha, R; Rhead, B; Zhang, M; Berkowitz, N; Gaddis, M; Noto, K; Ruiz, L; Pavlovic, M; Hong, El; Rand, K; Girshick, A; Guturu, H; Baltzell, Ah; Niemi, Mek; Pigazzini, S; Rahmouni, S; Georges, M; Belhaj, Y; Guntz, J; Claassen, S; Beguin, Y; Gofflot, S; Nkambule, L; Nkambul, L; Cusick, C; Moutschen, M; Misset, B; Darcis, G; Guiot, J; Azarzar, S; Malaise, O; Huynen, P; Meuris, C; Thys, M; Jacques, J; Leonard, P; Frippiat, F; Giot, Jb; Sauvage, As; Von Frenckell, C; Lambermont, B; Nakanishi, T; Morrison, Dr; Richards, Jb; Butler-Laporte, G; Forgetta, V; Ghosh, B; Laurent, L; Henry, D; Abdullah, T; Adeleye, O; Mamlouk, N; Kimchi, N; Afrasiabi, Z; Rezk, N; Vulesevic, B; Bouab, M; Guzman, C; Petitjean, L; Tselios, C; Xue, Xq; Afilalo, J; Adra, D; Mooser, V; Li, R; Belisle, A; Lepage, P; Ragoussis, J; Auld, D; Lathrop, Gm; Afilalo, M; Oliveira, M; Brenner, B; Brassard, N; Durand, M; Chasse, M; Kaufmann, De; Schurr, E; Hayward, C; Richmond, A; Baillie, Jk; Glessner, Jt; Hakonarson, H; Chang, X; Shaw, Dm; Below, J; Polikowski, H; Lauren, Pe; Chen, Hh; Zhu, Wy; Davis, L; Kerchberger, Ve; Campbell, A; Porteous, Dj; Fawns-Ritchie, C; Morris, M; Mccormick, Jb; North, K; Glessner, Jr; Gignoux, Cr; Wicks, Sj; Crooks, K; Barnes, Kc; Daya, M; Shortt, J; Rafaels, N; Chavan, S; Timmers, Prhj; Wilson, Jf; Tenesa, A; Kerr, Sm; D'Mellow, K; Shahin, D; El-Sherbiny, Ym; El-Jawhari, Jj; von Hohenstaufen, Ka; Sobh, A; Eltoukhy, Mm; Mohamed, Aas; Elhadidy, Ta; Abd Elghafar, Ms; Elnagdy, Mh; Samir, A; Hegazy, Maf; Abdel-Aziz, M; Khafaga, Wt; El-Lawaty, Wm; Torky, Ms; Moahmed, Hs; El-shanshory, Mr; Yassen, Am; Okasha, K; Eid, Ma; Medina-Gomez, C; Uitterlinden, Ag; Ikram, Ma; Magi, R; Milani, L; Metspalu, A; Laisk, T; Lall, K; Lepamets, M; Esko, T; Reimann, E; Alavere, H; Metsalu, K; Puusepp, M; Naaber, P; Laane, E; Pesukova, J; Peterson, P; Kisand, K; Tabri, J; Allos, R; Hensen, K; Starkopf, J; Ringmets, I; Tamm, A; Kallaste, A; Batini, C; Tobin, Md; Venn, Ld; Lee, Ph; Shrine, N; Williams, At; Guyatt, Al; John, C; Packer, Rj; Ali, A; Wang, X; Wain, Lv; Bee, Ce; Adams, El; Free, Rc; Hollox, Ej; Ruotsalainen, S; Kristiansson, K; Koskelainen, S; Perola, M; Rivolta, C; Quinodoz, M; Kamdar, D; Bochud, Py; Boillat, N; Bibert, S; Nussle, Sg; Albrich, W; Suh, N; Neofytos, D; Erard, V; Voide, C; Friolet, R; Vollenweider, P; Pagani, Jl; Oddo, M; zu Bentrup, Fm; Conen, A; Clerc, O; Marchetti, O; Guillet, A; Guyat-Jacques, C; Foucras, S; Rime, M; Chassot, J; Jaquet, M; Viollet, Rm; Lannepoudenx, Y; Portopena, L; Bochud, Py; Desgranges, F; Filippidis, P; Guery, B; Haefliger, D; Kampouri, Ee; Manuel, O; Munting, A; Papadimitriou-Olivgeris, M; Regina, J; Rochat-Stettler, L; Suttels, V; Tadini, E; Tschopp, J; Van Singer, M; Viala, B; Boillat-Blanco, N; Brahier, T; Hugli, O; Meuwly, Jy; Pantet, O; Nussle, Sg; Bochud, M; D'Acremont, V; Younes, Se; Albrich, Wc; Suh, N; Cerny, A; O'Mahony, L; von Mering, C; Frischknecht, M; Kleger, Gr; Filipovic, M; Kahlert, Cr; Wozniak, H; Negro, Tr; Pugin, J; Bouras, K; Knapp, C; Egger, T; Perret, A; Montillier, P; di Bartolomeo, C; Barda, B; de Cid, R; Carreras, A; Galvan-Femenia, I; Blay, N; Farre, X; Sumoy, L; Cortes, B; Moreno, V; Kogevinas, M; Garcia-Aymerich, J; Castano-Vinyals, G; Dobano, C; Mercader, Jm; Mercader, J; Guindo-Martinez, M; Torrents
abstract

2022 - Acid base disorders in patients with COVID-19 [Articolo su rivista]
Alfano, Gaetano; Fontana, Francesco; Mori, Giacomo; Giaroni, Francesco; Ferrari, Annachiara; Giovanella, Silvia; Ligabue, Giulia; Ascione, Elisabetta; Cazzato, Silvia; Ballestri, Marco; Di Gaetano, Margherita; Meschiari, Marianna; Menozzi, Marianna; Milic, Jovana; Andrea, Bedini; Franceschini, Erica; Cuomo, Gianluca; Magistroni, Riccardo; Mussini, Cristina; Cappelli, Gianni; Guaraldi, Giovanni; De Biasi, Sara; Cossarizza, Andrea; Gibellini, Lara
abstract

Purpose Acid-base derangement has been poorly described in patients with coronavirus disease 2019 (COVID-19). Considering the high prevalence of pneumonia and kidneys injury in COVID-19, frequent acid-base alterations are expected in patients admitted with SARS-Cov-2 infection. The study aimed to assess the prevalence of acid-base disorders in symptomatic patients with a diagnosis of COVID-19. Methods The retrospective study enrolled COVID-19 patients hospitalized at the University Hospital of Modena from 4 March to 20 June 2020. Baseline arterial blood gas (ABG) analysis was collected in 211 patients. In subjects with multiple ABG analysis, we selected only the first measurement. A pH of less than 7.37 was categorized as acidemia and a pH of more than 7.43 was categorized as alkalemia. Results ABG analyses revealed a low arterial partial pressure of oxygen (PO2, 70.2 +/- 25.1 mmHg), oxygen saturation (SO2, 92%) and a mild reduction of PO2/FiO(2) ratio (231 +/- 129). Acid-base alterations were found in 79.7% of the patient. Metabolic alkalosis (33.6%) was the main alteration followed by respiratory alkalosis (30.3%), combined alkalosis (9.4%), respiratory acidosis (3.3%), metabolic acidosis (2.8%) and other compensated acid-base disturbances (3.6%). All six patients with metabolic acidosis died at the end of the follow-up. Conclusion Variations of pH occurred in the majority (79.7%) of patients admitted with COVID-19. The patients experienced all the type of acid-base disorders, notably metabolic and respiratory alkalosis were the most common alterations in this group of patients.

2022 - An explainable model of host genetic interactions linked to COVID-19 severity [Articolo su rivista]
Onoja, A.; Picchiotti, N.; Fallerini, C.; Baldassarri, M.; Fava, F.; Mari, F.; Daga, S.; Benetti, E.; Bruttini, M.; Palmieri, M.; Croci, S.; Amitrano, S.; Meloni, I.; Frullanti, E.; Doddato, G.; Lista, M.; Beligni, G.; Valentino, F.; Zguro, K.; Tita, R.; Giliberti, A.; Mencarelli, M. A.; Rizzo, C. L.; Pinto, A. M.; Ariani, F.; Di Sarno, L.; Montagnani, F.; Tumbarello, M.; Rancan, I.; Fabbiani, M.; Rossetti, B.; Bergantini, L.; D'Alessandro, M.; Cameli, P.; Bennett, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Guerrini, S.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Raffaelli, C. S.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Romani, D.; Piacentini, P.; Bandini, M.; Desanctis, E.; Cappelli, S.; Canaccini, A.; Verzuri, A.; Anemoli, V.; Pisani, M.; Ognibene, A.; Pancrazzi, A.; Lorubbio, M.; Vaghi, M.; D'Arminio Monforte, A.; Miraglia, F. G.; Bruno, R.; Vecchia, M.; Girardis, M.; Venturelli, S.; Busani, S.; Cossarizza, A.; Antinori, A.; Vergori, A.; Emiliozzi, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Riva, A.; Francisci, D.; Schiaroli, E.; Paciosi, F.; Tommasi, A.; Zuccon, U.; Vietri, L.; Scotton, P. G.; Andretta, F.; Panese, S.; Baratti, S.; Scaggiante, R.; Gatti, F.; Parisi, S. G.; Castelli, F.; Quiros-Roldan, E.; Antoni, M. D.; Zanella, I.; Della Monica, M.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Fiorentino, G.; Carella, M.; Castori, M.; Aucella, F.; Raggi, P.; Perna, R.; Bassetti, M.; Di Biagio, A.; Sanguinetti, M.; Masucci, L.; Guarnaccia, A.; Valente, S.; De Vivo, O.; Bargagli, E.; Mandala, M.; Giorli, A.; Salerni, L.; Zucchi, P.; Parravicini, P.; Menatti, E.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Lacerenza, G.; Coviello, D. A.; Mussini, C.; Martinelli, E.; Tavecchia, L.; Belli, M. A.; Crotti, L.; Parati, G.; Sanarico, M.; Biscarini, F.; Stella, A.; Rizzi, M.; Maggiolo, F.; Ripamonti, D.; Suardi, C.; Bachetti, T.; La Rovere, M. T.; Sarzi-Braga, S.; Bussotti, M.; Capitani, K.; Dei, S.; Ravaglia, S.; Artuso, R.; Andreucci, E.; Gori, G.; Pagliazzi, A.; Fiorentini, E.; Perrella, A.; Bianchi, F.; Bergomi, P.; Catena, E.; Colombo, R.; Luchi, S.; Morelli, G.; Petrocelli, P.; Iacopini, S.; Modica, S.; Baroni, S.; Segala, F. V.; Menichetti, F.; Falcone, M.; Tiseo, G.; Barbieri, C.; Matucci, T.; Grassi, D.; Ferri, C.; Marinangeli, F.; Brancati, F.; Vincenti, A.; Borgo, V.; Lombardi, S.; Lenzi, M.; Di Pietro, M. A.; Vichi, F.; Romanin, B.; Attala, L.; Costa, C.; Gabbuti, A.; Mene, R.; Colaneri, M.; Casprini, P.; Merla, G.; Squeo, G. M.; Maffezzoni, M.; Mantovani, S.; Mondelli, M. U.; Ludovisi, S.; Colombo, F.; Chiaromonte, F.; Renieri, A.; Furini, S.; Raimondi, F.
abstract

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as “Respiratory or thoracic disease”, supporting their link with COVID-19 severity outcome.

2022 - Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma [Articolo su rivista]
Golinelli, G.; Grisendi, G.; Dall'Ora, M.; Casari, G.; Spano, C.; Talami, R.; Banchelli, F.; Prapa, M.; Chiavelli, C.; Rossignoli, F.; Candini, O.; D'Amico, R.; Nasi, M.; Cossarizza, A.; Casarini, L.; Dominici, M.
abstract

Background: Ewing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES. Methods: The anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario. Findings: In vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver. Interpretation: We here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies.

2022 - Cardiovascular Effects of Whole-Body Cryotherapy in Non-professional Athletes [Articolo su rivista]
Coppi, Francesca; Pinti, Marcello; Selleri, Valentina; Zanini, Giada; D'Alisera, Roberta; Latessa, Pasqualino Maietta; Tripi, Ferdinando; Savino, Gustavo; Cossarizza, Andrea; Nasi, Milena; Mattioli, Anna Vittoria
abstract

2022 - Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity [Articolo su rivista]
Fallerini, C.; Picchiotti, N.; Baldassarri, M.; Zguro, K.; Daga, S.; Fava, F.; Benetti, E.; Amitrano, S.; Bruttini, M.; Palmieri, M.; Croci, S.; Lista, M.; Beligni, G.; Valentino, F.; Meloni, I.; Tanfoni, M.; Minnai, F.; Colombo, F.; Cabri, E.; Fratelli, M.; Gabbi, C.; Mantovani, S.; Frullanti, E.; Gori, M.; Crawley, F. P.; Butler-Laporte, G.; Richards, B.; Zeberg, H.; Lipcsey, M.; Hultstrom, M.; Ludwig, K. U.; Schulte, E. C.; Pairo-Castineira, E.; Baillie, J. K.; Schmidt, A.; Frithiof, R.; Mari, F.; Renieri, A.; Furini, S.; Montagnani, F.; Tumbarello, M.; Rancan, I.; Fabbiani, M.; Rossetti, B.; Bergantini, L.; D'Alessandro, M.; Cameli, P.; Bennett, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Guerrini, S.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Raffaelli, C. S.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Spagnesi, M.; Romani, D.; Piacentini, P.; Bandini, M.; Desanctis, E.; Cappelli, S.; Canaccini, A.; Verzuri, A.; Anemoli, V.; Pisani, M.; Ognibene, A.; Pancrazzi, A.; Lorubbio, M.; Vaghi, M.; Monforte, A. D.; Miraglia, F. G.; Mondelli, M. U.; Girardis, M.; Venturelli, S.; Busani, S.; Cossarizza, A.; Antinori, A.; Vergori, A.; Emiliozzi, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Riva, A.; Francisci, D.; Schiaroli, E.; Paciosi, F.; Tommasi, A.; Scotton, P. G.; Andretta, F.; Panese, S.; Baratti, S.; Scaggiante, R.; Gatti, F.; Parisi, S. G.; Castelli, F.; Quiros-Roldan, E.; Antoni, M. D.; Zanella, I.; Monica, M. D.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Fiorentino, G.; Carella, M.; Castori, M.; Aucella, F.; Raggi, P.; Perna, R.; Bassetti, M.; Biagio, A. D.; Sanguinetti, M.; Masucci, L.; Guarnaccia, A.; Valente, S.; Vivo, O. D.; Doddato, G.; Tita, R.; Giliberti, A.; Mencarelli, M. A.; Rizzo, C. L.; Pinto, A. M.; Perticaroli, V.; Ariani, F.; Carriero, M. L.; Sarno, L. D.; Alaverdian, D.; Bargagli, E.; Mandala, M.; Giorli, A.; Salerni, L.; Zucchi, P.; Parravicini, P.; Menatti, E.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Lacerenza, L. G.; Coviello, D. A.; Mussini, C.; Martinelli, E.; Mancarella, S.; Tavecchia, L.; Belli, M. A.; Crotti, L.; Parati, G.; Sanarico, M.; Raimondi, F.; Biscarini, F.; Stella, A.; Rizzi, M.; Maggiolo, F.; Ripamonti, D.; Suardi, C.; Bachetti, T.; Rovere, M. T. L.; Sarzi-Braga, S.; Bussotti, M.; Capitani, K.; Dei, S.; Ravaglia, S.; Artuso, R.; Andreucci, E.; Gori, G.; Pagliazzi, A.; Fiorentini, E.; Perrella, A.; Bianchi, F.; Bergomi, P.; Catena, E.; Colombo, R.; Luchi, S.; Morelli, G.; Petrocelli, P.; Iacopini, S.; Modica, S.; Baroni, S.; Segala, F. V.; Menichetti, F.; Falcone, M.; Tiseo, G.; Barbieri, C.; Matucci, T.; Grassi, D.; Ferri, C.; Marinangeli, F.; Brancati, F.; Vincenti, A.; Borgo, V.; Stefania, L.; Lenzi, M.; Pietro, M. A. D.; Vichi, F.; Romanin, B.; Attala, L.; Costa, C.; Gabbuti, A.; Roberto, M.; Zuccon, U.; Vietri, L.; Ceri, S.; Pinoli, P.; Casprini, P.; Merla, G.; Squeo, G. M.; Maffezzoni, M.; Bruno, R.; Vecchia, M.; Colaneri, M.; Ludovisi, S.; Marincevic-Zuniga, Y.; Nordlund, J.; Luther, T.; Larsson, A.; Hanslin, K.; Gradin, A.; Galien, S.; Anderberg, S. B.; Rosen, J.; Rubertsson, S.; Clohisey, S.; Horby, P.; Millar, J.; Knight, J.; Montgomery, H.; Maslove, D.; Ling, L.; Nichol, A.; Summers, C.; Walsh, T.; Hinds, C.; Semple, M. G.; Openshaw, P. J. M.; Shankar-Hari, M.; Ho, A.; Mcauley, D.; Ponting, C.; Rowan, K.; Griffiths, F.; Oosthuyzen, W.; Meikle, J.; Finernan, P.; Furniss, J.; Mcmaster, E.; Law, A.; Paterson, T.; Wackett, T.; Armstrong, R.; Murphy, L.; Fawkes, A.; Clark, R.; Coutts, A.; Donnelly, L.; Gilchrist, T.; Hafezi, K.; Macgillivray, L.; Maclean, A.; Mccafferty, S.; Morrice, K.; Weaver, J.; Boz, C.; Golightly, A.; Ward, M.; Mal, H.; Szoor-McElhinney, H.; Brown, A.; Hendry, R.; Stenhouse, A.; Cullum, L.; Law, D.; Law, S.; Law, R.; Swets, M.; Day, N.; Taneski, F.; Duncan, E.; Zechner, M.; Parkinson, N.; Klaric, L.; Bretherick, A.
abstract

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management.

2022 - Cytomegalovirus blood reactivation in COVID-19 critically ill patients: risk factors and impact on mortality. [Articolo su rivista]
Gatto, Ilenia; Biagioni, Emanuela; Coloretti, Irene; Farinelli, Carlotta; Avoni, Camilla; Caciagli, Valeria; Busani, Stefano; Sarti, Mario; Pecorari, Monica; Gennari, William; Guaraldi, Giovanni; Franceschini, Erica; Meschiari, Marianna; Mussini, Cristina; Tonelli, Roberto; Clini, Enrico; Cossarizza, Andrea; Girardis, Massimo; Gibellini, Lara
abstract

Purpose: Cytomegalovirus (CMV) reactivation in immunocompetent critically ill patients is common and relates to a worsening outcome. In this large observational study, we evaluated the incidence and the risk factors associated with CMV reactivation and its effects on mortality in a large cohort of COVID-19 patients admitted to the intensive care unit (ICU). Methods: Consecutive patients with confirmed SARS-CoV-2 infection and acute respiratory distress syndrome admitted to three ICUs from February 2020 to July 2021 were included. The patients were screened at ICU admission and once or twice per week for quantitative CMV-DNAemia in the blood. The risk factors associated with CMV blood reactivation and its association with mortality were estimated by adjusted Cox proportional hazards regression models. Results: CMV blood reactivation was observed in 88 patients (20,4%) of the 431 patients studied. SAPS II score (HR 1,031, 95% CI 1,010-1,053, p=0,006), platelet count (HR 0,0996, 95% CI 0,993-0,999, p=0,004), invasive mechanical ventilation (HR 2,611, 95% CI 1,223-5,571, p=0,013) and secondary bacterial infection (HR 5,041; 95% CI 2,852-8,911, p<0,0001) during ICU stay were related to CMV reactivation. Hospital mortality was higher in patients with (67,0%) than in patients without (24,5%) CMV reactivation but the adjusted analysis did not confirm this association (HR 1,141, 95% CI 0,757-1,721, p=0,528). Conclusion: The severity of illness and the occurrence of secondary bacterial infections were associated with an increased risk of CMV blood reactivation, which, however, does not seem to influence the outcome of COVID-19 ICU patients independently.

2022 - Decay pattern of anti–SARS–CoV–2 antibodies in PWH [Abstract in Atti di Convegno]
Milić, Jovana; Tili, Alessandro; Renzetti, Stefano; Motta, Federico; Meschiari, Marianna; Fogliani, Rossella; Ferrari, Filippo; Meccugni, Barbara; Mimmi, Stefano; Borsari, Silvana; Calza, Stefano; Cossarizza, Andrea; Mussini, Cristina; Guaraldi, Giovanni
abstract

2022 - Effective Treatment of Patients Experiencing Primary, Acute HIV Infection Decreases Exhausted/Activated CD4+ T Cells and CD8+ T Memory Stem Cells [Articolo su rivista]
Lo Tartaro, D.; Camiro-Zuniga, A.; Nasi, M.; De Biasi, S.; Najera-Avila, M. A.; Jaramillo-Jante, M. D. R.; Gibellini, L.; Pinti, M.; Neroni, A.; Mussini, C.; Soto-Ramirez, L. E.; Calva, J. J.; Belaunzaran-Zamudio, F.; Crabtree-Ramirez, B.; Hernandez-Leon, C.; Mosqueda-Gomez, J. L.; Navarro-Alvarez, S.; Perez-Patrigeon, S.; Cossarizza, A.
abstract

Several studies have identified main changes in T- and B-lymphocyte subsets during chronic HIV infection, but few data exist on how these subsets behave during the initial phase of HIV infection. We enrolled 22 HIV-infected patients during the acute stage of infection before the initiation of antiretroviral therapy (ART). Patients had blood samples drawn previous to ART initiation (T0), and at 2 (T1) and 12 (T2) months after ART initiation. We quantified cellular HIV-DNA content in sorted naïve and effector memory CD4 T cells and identified the main subsets of T- and B-lymphocytes using an 18-parameter flow cytometry panel. We identified correlations between the patients’ clinical and immunological data using PCA. Effective HIV treatment reduces integrated HIV DNA in effector memory T cells after 12 months (T2) of ART. The main changes in CD4+ T cells occurred at T2, with a reduction of activated memory, cytolytic and activated/exhausted stem cell memory T (TSCM) cells. Changes were present among CD8+ T cells since T1, with a reduction of several activated subsets, including activated/exhausted TSCM. At T2 a reduction of plasmablasts and exhausted B cells was also observed. A negative correlation was found between the total CD4+ T-cell count and IgM-negative plasmablasts. In patients initiating ART immediately following acute/early HIV infection, the fine analysis of T- and B-cell subsets has allowed us to identify and follow main modifications due to effective treatment, and to identify significant changes in CD4+ and CD8+ T memory stem cells.

2022 - Effects of Energy Drink Acute Assumption in Gastrointestinal Tract of Rats [Articolo su rivista]
Nasi, Milena; De Gaetano, Anna; Carnevale, Gianluca; Bertoni, Laura; Selleri, Valentina; Zanini, Giada; Pisciotta, Alessandra; Caramaschi, Stefania; Reggiani Bonetti, Luca; Farinetti, Alberto; Cossarizza, Andrea; Pinti, Marcello; Manenti, Antonio; Mattioli, Anna Vittoria
abstract

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2022 - Evidence for mitochondrial Lonp1 expression in the nucleus [Articolo su rivista]
Gibellini, Lara; Borella, Rebecca; De Gaetano, Anna; Zanini, Giada; Tartaro, Domenico Lo; Carnevale, Gianluca; Beretti, Francesca; Losi, Lorena; De Biasi, Sara; Nasi, Milena; Forcato, Mattia; Cossarizza, Andrea; Pinti, Marcello
abstract

The coordinated communication between the mitochondria and nucleus is essential for cellular activities. Nonetheless, the pathways involved in this crosstalk are scarcely understood. The protease Lonp1 was previously believed to be exclusively located in the mitochondria, with an important role in mitochondrial morphology, mtDNA maintenance, and cellular metabolism, in both normal and neoplastic cells. However, we recently detected Lonp1 in the nuclear, where as much as 22% of all cellular Lonp1 can be found. Nuclear localization is detectable under all conditions, but the amount is dependent on a response to heat shock (HS). Lonp1 in the nucleus interacts with heat shock factor 1 (HSF1) and modulates the HS response. These findings reveal a novel extramitochondrial function for Lonp1 in response to stress.

2022 - First and second wave among hospitalized COVID-19 patients with severe pneumonia: a comparison of 28-day mortality over 1-year pandemic in a tertiary university hospital in Italy. [Articolo su rivista]
Meschiari, M; Cozzi-Lepri, A; Tonelli, R; Bacca, E; Menozzi, M; Franceschini, E; Cuomo, G; Bedini, A; Volpi, S; Milic, J; Brugioni, L; Romagnoli, E; Pietrangelo, A; Corradini, E; Coloretti, I; Biagioni, E; Busani, S; Girardis, M; Cossarizza, A; Clini, E; Guaraldi, G; Mussini, C.
abstract

Objectives: The first COVID-19-19 epidemic wave was over the period February-May 2020. Since October 1st, 2020 Italy, as many other European countries, faced a second wave. The aim of this analysis was to compare the 28-day mortality between the two waves among COVID-19 hospitalised patients. Design: Observational cohort study. Standard survival analysis was performed to compare all-cause mortality within 28 days after hospital admission in the two waves. Kaplan-Meier curves as well as Cox regression model analysis were used. The effect of wave on risk of death was shown by means of hazard ratios (HRs) with 95% confidence intervals (CI). A sensitivity analysis around the impact of the circulating variant as a potential unmeasured confounder was performed. Setting: University Hospital of Modena, Italy. Patients admitted to hospital for severe COVID-19 pneumonia during the first (February 22nd – May 31st, 2020) and second wave (October 1st- December 31st, 2020) were included. Results: During the two study periods, a total of 1,472 patients with severe COVID-19 pneumonia were admitted to our hospital, 449 during the first wave and 1,023 during the second. Median age was 70 years (IQR:56-80), 37% females, 49% with PaO /FiO < 250 mmHg, 82% with ≥1 comorbidity, median duration of symptoms was 6 days. 28-day mortality rate was 20.0% (95% CI:16.3-23.7) during the first wave vs. 14.2% (95% CI:12.0-16.3) in the second (log-rank test p-value= 0.03). After including key predictors of death in the multivariable Cox regression model, the data still strongly suggested a lower 28-day mortality rate in the 2nd wave (aHR=0.64, 95% CI: 0.45, 0.90, p- value=0.01). Conclusions: In our hospitalized COVID-19 patients with severe pneumonia, the 28-day mortality appeared to be reduced by 36% during the second as compared to the first wave. Further studies are needed to identify factors that may have contributed to this improved survival.

2022 - Innate immunity changes in soccer players after whole-body cryotherapy [Articolo su rivista]
Selleri, Valentina; Mattioli, Marco; Lo Tartaro, Domenico; Paolini, Annamaria; Zanini, Giada; De Gaetano, Anna; D'Alisera, Roberta; Roli, Laura; Melegari, Alessandra; Maietta, Pasqualino; Tripi, Ferdinando; Guerra, Emanuele; Chester, Johanna; Savino, Gustavo; Trenti, Tommaso; Cossarizza, Andrea; Mattioli, Anna Vittoria; Pinti, Marcello; Nasi, Milena
abstract

Whole-body cryotherapy (WBC) consists of short exposure (up to 2-3 min) to dry air at cryogenic temperatures (up to -190 degrees C) and has recently been applied for muscle recovery after injury to reduce the inflammation process. We aimed to determine the impact of cryotherapy on immunological, hormonal, and metabolic responses in non-professional soccer players (NPSPs). Nine male NPSPs (age: 20 +/- 2 years) who trained regularly over 5 consecutive days, immediately before and after each training session, were subjected to WBC treatment (WBC-t). Blood samples were collected for the evaluation of fifty analytes including hematologic parameters, serum chemistry, and hormone profiles. Monocytes phenotyping (Mo) was performed and plasmatic markers, usually increased during inflammation [CCL2, IL-18, free mitochondrial (mt)DNA] or with anti-inflammatory effects (IL2RA, IL1RN), were quantified. After WBC-t, we observed reduced levels of ferritin, mean corpuscular hemoglobin, mean platelet volume, testosterone, and estradiol, which however remain within the normal ranges. The percentage of the total, intermediates and non-classical Mo increased, while classical Mo decreased. CXCR4 expression decreased in each Mo subset. Plasma IL18 and IL2RA levels decreased, while IL1RN only exhibited a tendency to decrease and CCL2 showed a tendency to increase. Circulating mtDNA levels were not altered following WBC-t. The differences observed in monocyte subsets after WBC-t may be attributable to their redistribution into the surrounding tissue. Moreover, the decrease of CXCR4 in Mo subpopulations could be coherent with their differentiation process. Thus, WBC through yet unknown mechanisms could promote their differentiation having a role in tissue repair.

2022 - Metabolic reprograming shapes neutrophil functions in severe COVID-19 [Articolo su rivista]
Borella, Rebecca; De Biasi, Sara; Paolini, Annamaria; Boraldi, Federica; Tartaro, Domenico Lo; Mattioli, Marco; Fidanza, Lucia; Neroni, Anita; Caro-Maldonado, Alfredo; Meschiari, Marianna; Franceschini, Erica; Quaglino, Daniela; Guaraldi, Giovanni; Bertoldi, Carlo; Sita, Marco; Busani, Stefano; Girardis, Massimo; Mussini, Cristina; Cossarizza, Andrea; Gibellini, Lara
abstract

: To better understand the mechanisms at the basis of neutrophil functions during SARS-CoV-2 we studied patients with severe COVID-19 pneumonia. They had high blood proportion of degranulated neutrophils and elevated plasma levels of myeloperoxidase (MPO), elastase and MPO-DNA complexes, which are typical markers of neutrophil extracellular traps (NET). Their neutrophils display dysfunctional mitochondria, defective oxidative burst, increased glycolysis, glycogen accumulation in the cytoplasm, and increase glycogenolysis. Hypoxia-inducible factor 1α (ΗΙF-1α) is stabilized in such cells, and it controls the level of glycogen phosphorylase L (PYGL), a key enzyme in glycogenolysis. Inhibiting PYGL abolishes the ability of neutrophils to produce NET. Patients displayed significant increases of plasma levels of molecules involved in the regulation of neutrophils' function, including CCL2, CXCL10, CCL20, IL-18, IL-3, IL-6, G-CSF, GM-CSF, IFN-γ. Our data suggest that metabolic remodelling is vital for the formation of NET and for boosting neutrophil inflammatory response, thus suggesting that modulating ΗΙF-1α or PYGL could represent a novel approach for innovative therapies. This article is protected by copyright. All rights reserved.

2022 - Molecular and cellular immune features of aged patients with severe COVID-19 pneumonia [Articolo su rivista]
Lo Tartaro, D.; Neroni, A.; Paolini, A.; Borella, R.; Mattioli, M.; Fidanza, L.; Quong, A.; Petes, C.; Awong, G.; Douglas, S.; Lin, D.; Nieto, J.; Gozzi, L.; Franceschini, E.; Busani, S.; Nasi, M.; Mattioli, A. V.; Trenti, T.; Meschiari, M.; Guaraldi, G.; Girardis, M.; Mussini, C.; Gibellini, L.; Cossarizza, A.; De Biasi, S.
abstract

Aging is a major risk factor for developing severe COVID-19, but few detailed data are available concerning immunological changes after infection in aged individuals. Here we describe main immune characteristics in 31 patients with severe SARS-CoV-2 infection who were >70 years old, compared to 33 subjects <60 years of age. Differences in plasma levels of 62 cytokines, landscape of peripheral blood mononuclear cells, T cell repertoire, transcriptome of central memory CD4+ T cells, specific antibodies are reported along with features of lung macrophages. Elderly subjects have higher levels of pro-inflammatory cytokines, more circulating plasmablasts, reduced plasmatic level of anti-S and anti-RBD IgG3 antibodies, lower proportions of central memory CD4+ T cells, more immature monocytes and CD56+ pro-inflammatory monocytes, lower percentages of circulating follicular helper T cells (cTfh), antigen-specific cTfh cells with a less activated transcriptomic profile, lung resident activated macrophages that promote collagen deposition and fibrosis. Our study underlines the importance of inflammation in the response to SARS-CoV-2 and suggests that inflammaging, coupled with the inability to mount a proper anti-viral response, could exacerbate disease severity and the worst clinical outcome in old patients.

2022 - Patients Recovering from Severe COVID-19 Develop a Polyfunctional Antigen-Specific CD4+ T Cell Response [Articolo su rivista]
Paolini, A.; Borella, R.; Neroni, A.; Lo Tartaro, D.; Mattioli, M.; Fidanza, L.; Di Nella, A.; Santacroce, E.; Gozzi, L.; Busani, S.; Trenti, T.; Meschiari, M.; Guaraldi, G.; Girardis, M.; Mussini, C.; Gibellini, L.; De Biasi, S.; Cossarizza, A.
abstract

Specific T cells are crucial to control SARS-CoV-2 infection, avoid reinfection and confer protection after vaccination. We have studied patients with severe or moderate COVID-19 pneumonia, compared to patients who recovered from a severe or moderate infection that had occurred about 4 months before the analyses. In all these subjects, we assessed the polyfunctionality of virus-specific CD4+ and CD8+ T cells by quantifying cytokine production after in vitro stimulation with different SARS-CoV-2 peptide pools covering different proteins (M, N and S). In particular, we quantified the percentage of CD4+ and CD8+ T cells simultaneously producing interferon-γ, tumor necrosis factor, interleukin (IL)-2, IL-17, granzyme B, and expressing CD107a. Recovered patients who experienced a severe disease display high proportions of antigen-specific CD4+ T cells producing Th1 and Th17 cytokines and are characterized by polyfunctional SARS-CoV-2-specific CD4+ T cells. A similar profile was found in patients experiencing a moderate form of COVID-19 pneumonia. No main differences in polyfunctionality were observed among the CD8+ T cell compartments, even if the proportion of responding cells was higher during the infection. The identification of those functional cell subsets that might influence protection can thus help in better understanding the complexity of immune response to SARS-CoV-2.

2022 - Phenotypic, functional, and metabolic heterogeneity of immune cells infiltrating non–small cell lung cancer [Articolo su rivista]
Aramini, B.; Masciale, V.; Samarelli, A. V.; Dubini, A.; Gaudio, M.; Stella, F.; Morandi, U.; Dominici, M.; De Biasi, S.; Gibellini, L.; Cossarizza, A.
abstract

Lung cancer is the leading cancer in the world, accounting for 1.2 million of new cases annually, being responsible for 17.8% of all cancer deaths. In particular, non-small cell lung cancer (NSCLC) is involved in approximately 85% of all lung cancers with a high lethality probably due to the asymptomatic evolution, leading patients to be diagnosed when the tumor has already spread to other organs. Despite the introduction of new therapies, which have improved the long-term survival of these patients, this disease is still not well cured and under controlled. Over the past two decades, single-cell technologies allowed to deeply profile both the phenotypic and metabolic aspects of the immune cells infiltrating the TME, thus fostering the identification of predictive biomarkers of prognosis and supporting the development of new therapeutic strategies. In this review, we discuss phenotypic and functional characteristics of the main subsets of tumor-infiltrating lymphocytes (TILs) and tumor-infiltrating myeloid cells (TIMs) that contribute to promote or suppress NSCLC development and progression. We also address two emerging aspects of TIL and TIM biology, i.e., their metabolism, which affects their effector functions, proliferation, and differentiation, and their capacity to interact with cancer stem cells.

2022 - Plasma Cytokine Atlas Reveals the Importance of TH2 Polarization and Interferons in Predicting COVID-19 Severity and Survival [Articolo su rivista]
Gibellini, L.; De Biasi, S.; Meschiari, M.; Gozzi, L.; Paolini, A.; Borella, R.; Mattioli, M.; Lo Tartaro, D.; Fidanza, L.; Neroni, A.; Busani, S.; Girardis, M.; Guaraldi, G.; Mussini, C.; Cozzi-Lepri, A.; Cossarizza, A.
abstract

Although it is now widely accepted that host inflammatory response contributes to COVID-19 immunopathogenesis, the pathways and mechanisms driving disease severity and clinical outcome remain poorly understood. In the effort to identify key soluble mediators that characterize life-threatening COVID-19, we quantified 62 cytokines, chemokines and other factors involved in inflammation and immunity in plasma samples, collected at hospital admission, from 80 hospitalized patients with severe COVID-19 disease who were stratified on the basis of clinical outcome (mechanical ventilation or death by day 28). Our data confirm that age, as well as neutrophilia, lymphocytopenia, procalcitonin, D-dimer and lactate dehydrogenase are strongly associated with the risk of fatal COVID-19. In addition, we found that cytokines related to TH2 regulations (IL-4, IL-13, IL-33), cell metabolism (lep, lep-R) and interferons (IFNα, IFNβ, IFNγ) were also predictive of life-threatening COVID-19.

2022 - Preliminary testing of a simplified methodology for indoor environments evaluation correlated to airborne transmission: The case of a university classroom with vertical low-velocity ventilation [Relazione in Atti di Convegno]
Puglia, M.; Ottani, F.; Muscio, A.; Cossarizza, A.; Tartarini, P.
abstract

2022 - Redistribution of CD8+ T cell subsets in metastatic renal cell carcinoma patients treated with anti-PD-1 therapy [Articolo su rivista]
De Biasi, S.; Guida, A.; Lo Tartaro, D.; Fanelli, M.; Depenni, R.; Dominici, M.; Finak, G.; Porta, C.; Paolini, A.; Borella, R.; Bertoldi, C.; Cossarizza, A.; Sabbatini, R.; Gibellini, L.
abstract

Renal-cell carcinoma (RCC) is responsible for the majority of tumors arising from the kidney parenchyma. Although a progressive improvement in median overall survival has been observed after the introduction of anti-PD-1 therapy, many patients do not benefit from this treatment. Therefore, we have investigated T cell dynamics to find immune modification induced by anti-PD-1 therapy. Here, we show that, after therapy, RCC patients (5 responders and 14 nonresponders) are characterized by a redistribution of different subsets across the memory T cell compartment.

2022 - Remodeling of T Cell Dynamics During Long COVID Is Dependent on Severity of SARS-CoV-2 Infection [Articolo su rivista]
Wiech, M.; Chroscicki, P.; Swatler, J.; Stepnik, D.; De Biasi, S.; Hampel, M.; Brewinska-Olchowik, M.; Maliszewska, A.; Sklinda, K.; Durlik, M.; Wierzba, W.; Cossarizza, A.; Piwocka, K.
abstract

Several COVID-19 convalescents suffer from the post-acute COVID-syndrome (PACS)/long COVID, with symptoms that include fatigue, dyspnea, pulmonary fibrosis, cognitive dysfunctions or even stroke. Given the scale of the worldwide infections, the long-term recovery and the integrative health-care in the nearest future, it is critical to understand the cellular and molecular mechanisms as well as possible predictors of the longitudinal post-COVID-19 responses in convalescent individuals. The immune system and T cell alterations are proposed as drivers of post-acute COVID syndrome. However, despite the number of studies on COVID-19, many of them addressed only the severe convalescents or the short-term responses. Here, we performed longitudinal studies of mild, moderate and severe COVID-19-convalescent patients, at two time points (3 and 6 months from the infection), to assess the dynamics of T cells immune landscape, integrated with patients-reported symptoms. We show that alterations among T cell subsets exhibit different, severity- and time-dependent dynamics, that in severe convalescents result in a polarization towards an exhausted/senescent state of CD4+ and CD8+ T cells and perturbances in CD4+ Tregs. In particular, CD8+ T cells exhibit a high proportion of CD57+ terminal effector cells, together with significant decrease of naïve cell population, augmented granzyme B and IFN-γ production and unresolved inflammation 6 months after infection. Mild convalescents showed increased naïve, and decreased central memory and effector memory CD4+ Treg subsets. Patients from all severity groups can be predisposed to the long COVID symptoms, and fatigue and cognitive dysfunctions are not necessarily related to exhausted/senescent state and T cell dysfunctions, as well as unresolved inflammation that was found only in severe convalescents. In conclusion, the post-COVID-19 functional remodeling of T cells could be seen as a two-step process, leading to distinct convalescent immune states at 6 months after infection. Our data imply that attenuation of the functional polarization together with blocking granzyme B and IFN-γ in CD8+ cells might influence post-COVID alterations in severe convalescents. However, either the search for long COVID predictors or any treatment to prevent PACS and further complications is mandatory in all patients with SARS-CoV-2 infection, and not only in those suffering from severe COVID-19.

2022 - The interplay of post-acute COVID-19 syndrome and aging: a biological, clinical and public health approach [Articolo su rivista]
Guaraldi, Giovanni; Milic, Jovana; Cesari, Matteo; Leibovici, Leonard; Mandreoli, Federica; Missier, Paolo; Rozzini, Renzo; Cattelan, Anna Maria; Motta, Federico; Mussini, Cristina; Cossarizza, Andrea
abstract

The post-acute COVID-19 syndrome (PACS) is characterized by the persistence of fluctuating symptoms over three months from the onset of the possible or confirmed COVID-19 acute phase. Current data suggests that at least 10% of people with previously documented infection may develop PACS, and up to 50-80% of prevalence is reported among survivors after hospital discharge. This viewpoint will discuss various aspects of PACS, particularly in older adults, with a specific hypothesis to describe PACS as the expression of a modified aging trajectory induced by SARS CoV-2. This hypothesis will be argued from biological, clinical and public health view, addressing three main questions: (i) does SARS-CoV-2-induced alterations in aging trajectories play a role in PACS?; (ii) do people with PACS face immuno-metabolic derangements that lead to increased susceptibility to age-related diseases?; (iii) is it possible to restore the healthy aging trajectory followed by the individual before pre-COVID?. A particular focus will be given to the well-being of people with PACS that could be assessed by the intrinsic capacity model and support the definition of the healthy aging trajectory.

2022 - The “Elastic Perspective” of SARS-CoV-2 Infection and the Role of Intrinsic and Extrinsic Factors [Articolo su rivista]
Boraldi, F.; Lofaro, F. D.; Cossarizza, A.; Quaglino, D.
abstract

Elastin represents the structural component of the extracellular matrix providing elastic recoil to tissues such as skin, blood vessels and lungs. Elastogenic cells secrete soluble tropoelastin monomers into the extracellular space where these monomers associate with other matrix proteins (e.g., microfibrils and glycoproteins) and are crosslinked by lysyl oxidase to form insoluble fibres. Once elastic fibres are formed, they are very stable, highly resistant to degradation and have an almost negligible turnover. However, there are circumstances, mainly related to inflammatory conditions, where increased proteolytic degradation of elastic fibres may lead to consequences of major clinical relevance. In severely affected COVID-19 patients, for instance, the massive recruitment and activation of neutrophils is responsible for the profuse release of elastases and other proteolytic enzymes which cause the irreversible degradation of elastic fibres. Within the lungs, destruction of the elastic network may lead to the permanent impairment of pulmonary function, thus suggesting that elastases can be a promising target to preserve the elastic component in COVID-19 patients. Moreover, intrinsic and extrinsic factors additionally contributing to damaging the elastic component and to increasing the spread and severity of SARS-CoV-2 infection are reviewed.

2022 - Validation of a Novel Three-Dimensional (3D Fusion) Gross Sampling Protocol for Clear Cell Renal Cell Carcinoma to Overcome Intratumoral Heterogeneity: The Meet-Uro 18 Study [Articolo su rivista]
Brunelli, M.; Martignoni, G.; Malpeli, G.; Volpe, A.; Cima, L.; Raspollini, M. R.; Barbareschi, M.; Tafuri, A.; Masi, G.; Barzon, L.; Ammendola, S.; Villanova, M.; Cerruto, M. A.; Milella, M.; Buti, S.; Bersanelli, M.; Fornarini, G.; Rebuzzi, S. E.; Vellone, V. G.; Gaggero, G.; Procopio, G.; Verzoni, E.; Bracarda, S.; Fanelli, M.; Sabbatini, R.; Passalacqua, R.; Perrucci, B.; Giganti, M. O.; Donini, M.; Panni, S.; Tucci, M.; Prati, V.; Ortega, C.; Calio, A.; Eccher, A.; Alongi, F.; Pappagallo, G.; Iacovelli, R.; Mosca, A.; Umari, P.; Montagnani, I.; Gobbo, S.; Atzori, F.; Munari, E.; Maruzzo, M.; Basso, U.; Pierconti, F.; Patriarca, C.; Colombo, P.; Lapini, A.; Conti, G.; Salvioni, R.; Bollito, E.; Cossarizza, A.; Massari, F.; Rizzo, M.; Franco, R.; Zito-Marino, F.; Plata, Y. A.; Galuppini, F.; Sbaraglia, M.; Fassan, M.; Dei Tos, A. P.; Colecchia, M.; Moch, H.; Scaltriti, M.; Porta, C.; Delahunt, B.; Giannarini, G.; Bortolus, R.; Rescigno, P.; Banna, G. L.; Signori, A.; Obispo, M. A. L.; Perris, R.; Antonelli, A.
abstract

We aimed to overcome intratumoral heterogeneity in clear cell renal cell carcinoma (clear-RCC). One hundred cases of clearRCC were sampled. First, usual standard sampling was applied (1 block/cm of tumor); second, the whole tumor was sampled, and 0.6 mm cores were taken from each block to construct a tissue microarray; third, the residual tissue, mapped by taking pieces 0.5 × 0.5 cm, reconstructed the entire tumor mass. Precisely, six randomly derived pieces of tissues were placed in each cassette, with the number of cassettes being based on the diameter of the tumor (called multisite 3D fusion). Angiogenic and immune markers were tested. Routine 5231 tissue blocks were obtained. Multisite 3D fusion sections showed pattern A, homogeneous high vascular density (10%), pattern B, homogeneous low vascular density (8%) and pattern C, heterogeneous angiogenic signatures (82%). PD-L1 expression was seen as diffuse (7%), low (33%) and absent (60%). Tumor-infiltrating CD8 scored high in 25% (pattern hot), low in 65% (pattern weak) and zero in 10% of cases (pattern desert). Grading was upgraded in 26% of cases (G3–G4), necrosis and sarcomatoid/rhabdoid characters were observed in, respectively, 11 and 7% of cases after 3D fusion (p = 0.03). CD8 and PD-L1 immune expressions were higher in the undifferentiated G4/rhabdoid/sarcomatoid clearRCC subtypes (p = 0.03). Again, 22% of cases were set to intermediate to high risk of clinical recurrence due to new morphological findings of all aggressive G4, sarcomatoid/rhabdoid features by using 3D fusion compared to standard methods (p = 0.04). In conclusion, we propose an easy-to-apply multisite 3D fusion sampling that negates bias due to tumor heterogeneity.

2022 - Whole-genome sequencing reveals host factors underlying critical COVID-19 [Articolo su rivista]
Kousathanas, A.; Pairo-Castineira, E.; Rawlik, K.; Stuckey, A.; Odhams, C. A.; Walker, S.; Russell, C. D.; Malinauskas, T.; Wu, Y.; Millar, J.; Shen, X.; Elliott, K. S.; Griffiths, F.; Oosthuyzen, W.; Morrice, K.; Keating, S.; Wang, B.; Rhodes, D.; Klaric, L.; Zechner, M.; Parkinson, N.; Siddiq, A.; Goddard, P.; Donovan, S.; Maslove, D.; Nichol, A.; Semple, M. G.; Zainy, T.; Maleady-Crowe, F.; Todd, L.; Salehi, S.; Knight, J.; Elgar, G.; Chan, G.; Arumugam, P.; Patch, C.; Rendon, A.; Bentley, D.; Kingsley, C.; Kosmicki, J. A.; Horowitz, J. E.; Baras, A.; Abecasis, G. R.; Ferreira, M. A. R.; Justice, A.; Mirshahi, T.; Oetjens, M.; Rader, D. J.; Ritchie, M. D.; Verma, A.; Fowler, T. A.; Shankar-Hari, M.; Summers, C.; Hinds, C.; Horby, P.; Mcauley, D.; Montgomery, H.; Openshaw, P. J. M.; Elliott, P.; Walsh, T.; Tenesa, A.; Fawkes, A.; Murphy, L.; Rowan, K.; Ponting, C. P.; Vitart, V.; Wilson, J. F.; Yang, J.; Bretherick, A. D.; Scott, R. H.; Hendry, S. C.; Moutsianas, L.; Law, A.; Caulfield, M. J.; Baillie, J. K.; Begg, C.; Ling, L.; Millar, J.; Pereira, A. C.; Aravindan, L.; Armstrong, R.; Biggs, H.; Boz, C.; Brown, A.; Clark, R.; Coutts, A.; Coyle, J.; Cullum, L.; Das, S.; Day, N.; Donnelly, L.; Duncan, E.; Finernan, P.; Fourman, M. H.; Furlong, A.; Furniss, J.; Gallagher, B.; Gilchrist, T.; Golightly, A.; Hafezi, K.; Hamilton, D.; Hendry, R.; Law, D.; Law, R.; Law, S.; Lidstone-Scott, R.; Macgillivray, L.; Maclean, A.; Mal, H.; Mccafferty, S.; Mcmaster, E.; Meikle, J.; Moore, S. C.; Murphy, S.; Hellen, M.; Zheng, C.; Chen, J.; Paterson, T.; Schon, K.; Stenhouse, A.; Das, M.; Swets, M.; Szoor-McElhinney, H.; Taneski, F.; Turtle, L.; Wackett, T.; Ward, M.; Weaver, J.; Wrobel, N.; Arbane, G.; Bociek, A.; Campos, S.; Grau, N.; Jones, T. O.; Lim, R.; Marotti, M.; Ostermann, M.; Whitton, C.; Alldis, Z.; Astin-Chamberlain, R.; Bibi, F.; Biddle, J.; Blow, S.; Bolton, M.; Borra, C.; Bowles, R.; Burton, M.; Choudhury, Y.; Collier, D.; Cox, A.; Easthope, A.; Ebano, P.; Fotiadis, S.; Gurasashvili, J.; Halls, R.; Hartridge, P.; Kallon, D.; Kassam, J.; Lancoma-Malcolm, I.; Matharu, M.; May, P.; Mitchelmore, O.; Newman, T.; Patel, M.; Pheby, J.; Pinzuti, I.; Prime, Z.; Prysyazhna, O.; Shiel, J.; Taylor, M.; Tierney, C.; Wood, S.; Zak, A.; Zongo, O.; Bonner, S.; Hugill, K.; Jones, J.; Liggett, S.; Headlam, E.; Bandla, N.; Gellamucho, M.; Davies, M.; Thompson, C.; Abdelrazik, M.; Bakthavatsalam, D.; Elhassan, M.; Ganesan, A.; Haldeos, A.; Moreno-Cuesta, J.; Purohit, D.; Vincent, R.; Xavier, K.; Kumar, R.; Frater, A.; Saleem, M.; Carter, D.; Jenkins, S.; Lamond, Z.; Wall, A.; Fernandez-Roman, J.; Hamilton, D. O.; Johnson, E.; Johnston, B.; Martinez, M. L.; Mulla, S.; Shaw, D.; Waite, A. A. C.; Waugh, V.; Welters, I. D.; Williams, K.; Cavazza, A.; Cockrell, M.; Corcoran, E.; Depante, M.; Finney, C.; Jerome, E.; Mcphail, M.; Nayak, M.; Noble, H.; O'Reilly, K.; Pappa, E.; Saha, R.; Saha, S.; Smith, J.; Knighton, A.; Antcliffe, D.; Banach, D.; Brett, S.; Coghlan, P.; Fernandez, Z.; Gordon, A.; Rojo, R.; Arias, S. S.; Templeton, M.; Meredith, M.; Morris, L.; Ryan, L.; Clark, A.; Sampson, J.; Peters, C.; Dent, M.; Langley, M.; Ashraf, S.; Wei, S.; Andrew, A.; Bashyal, A.; Davidson, N.; Hutton, P.; Mckechnie, S.; Wilson, J.; Baptista, D.; Crowe, R.; Fernandes, R.; Herdman-Grant, R.; Joseph, A.; O'Connor, D.; Allen, M.; Loveridge, A.; Mckenley, I.; Morino, E.; Naranjo, A.; Simms, R.; Sollesta, K.; Swain, A.; Venkatesh, H.; Khera, J.; Fox, J.; Andrew, G.; Barclay, L.; Callaghan, M.; Campbell, R.; Clark, S.; Hope, D.; Marshall, L.; Mcculloch, C.; Briton, K.; Singleton, J.; Birch, S.; Brimfield, L.; Daly, Z.; Pogson, D.; Rose, S.; Nown, A.; Battle, C.; Brinkworth, E.; Harford, R.; Murphy, C.; Newey, L.; Rees, T.; Williams, M.; Arnold, S.; Polgarova, P.; Stroud, K.; Meaney, E.; Jones, M.; Ng, A.; Agrawal, S.; Pathan, N.; White, D.; Daubney, E.; Elston, K.; Grauslyte, L.; Hussain, M.; Phull, M.; Pogreban, T.; Rosaroso, L.; Salciute, E.; Franke, G.; Wong, J.
abstract

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease.

2021 - Aging of immune system [Capitolo/Saggio]
Pinti, M.; De Biasi, S.; Gibellini, L.; Lo Tartaro, D.; De Gaetano, A.; Mattioli, M.; Fidanza, L.; Nasi, M.; Cossarizza, A.
abstract

In old people, many alterations of innate and adaptive immunity have been described and viewed as deleterious, hence the term immunosenescence. Immunosenescence is a complex process involving multiple reorganizational and developmentally regulated changes, rather than a simple unidirectional decline of the whole function. Whereas innate immunity is relatively well preserved in older people, adaptive immunity is more susceptible due both to the functional decline associated with the passage of time and to antigen burden to which an individual has been exposed during lifetime. Although it is conceivable that global immune changes may lead to various diseases, it is also obvious that these changes may be needed for extended survival/longevity. This chapter summarizes recent data on the dynamic reassessment of immune changes with aging.

2021 - Anti-drug antibody detection with label-free electrolyte-gated organic field-effect transistors [Articolo su rivista]
Sensi, Matteo; Berto, Marcello; Gentile, Sara; Pinti, Marcello; Conti, Andrea; Pellacani, Giovanni; Salvarani, Carlo; Cossarizza, Andrea; Bortolotti, Carlo Augusto; Biscarini, Fabio
abstract

The efficacy of immunotherapy can be undermined by the development of an immune response against a drug/antibody mediated by anti-drug antibodies (ADAs) in treated patients. We present the first label-free EGOFET immunosensor that integrates a biological drug, Nivolumab (Opdivo©), as a specific recognition moiety to quantitatively and selectively detect ADAs against the drug. The limit of detection is 100 fM. This demonstration is a prelude to the detection of ADAs in a clinical setting in the treatment of different pathologies, and it also enables rapid screening of biological drugs for immunogenicity.

2021 - Apoptosis of Hepatocytes: Relevance for HIV-Infected Patients under Treatment [Articolo su rivista]
Gruevska, A.; Moragrega, Ab.; Cossarizza, A.; Esplugues, J. V.; Blas-Garcia, A.; Apostolova, N.
abstract

Due to medical advances over the past few decades, human immunodeficiency virus (HIV) infection, once a devastatingly mortal pandemic, has become a manageable chronic condition. However, available antiretroviral treatments (cART) cannot fully restore immune health and, consequently, a number of inflammation-associated and/or immunodeficiency complications have manifested themselves in treated HIV-infected patients. Among these chronic, non-AIDS (acquired immune deficiency syndrome)-related conditions, liver disease is one of the deadliest, proving to be fatal for 15-17% of these individuals. Aside from the presence of liver-related comorbidities, including metabolic disturbances and co-infections, HIV itself and the adverse effects of cART are the main factors that contribute to hepatic cell injury, inflammation, and fibrosis. Among the molecular mechanisms that are activated in the liver during HIV infection, apoptotic cell death of hepatocytes stands out as a key pathogenic player. In this review, we will discuss the evidence and potential mechanisms involved in the apoptosis of hepatocytes induced by HIV, HIV-encoded proteins, or cART. Some antiretroviral drugs, especially the older generation, can induce apoptosis of hepatic cells, which occurs through a variety of mechanisms, such as mitochondrial dysfunction, increased production of reactive oxygen species (ROS), and induction of endoplasmic reticulum (ER) stress and unfolded protein response (UPR), all of which ultimately lead to caspase activation and cell death.

2021 - Association of toll-like receptor 7 variants with life-threatening COVID-19 disease in males: Findings from a nested case-control study [Articolo su rivista]
Fallerini, C.; Daga, S.; Mantovani, S.; Benetti, E.; Picchiotti, N.; Francisci, D.; Paciosi, F.; Schiaroli, E.; Baldassarri, M.; Fava, F.; Palmieri, M.; Ludovisi, S.; Castelli, F.; Quiros-Roldan, E.; Vaghi, M.; Rusconi, S.; Siano, M.; Bandini, M.; Spiga, O.; Capitani, K.; Furini, S.; Mari, F.; Renieri, A.; Mondelli, M. U.; Frullanti, E.; Valentino, F.; Doddato, G.; Giliberti, A.; Tita, R.; Amitrano, S.; Bruttini, M.; Croci, S.; Meloni, I.; Mencarelli, M. A.; Rizzo, C. L.; Pinto, A. M.; Sarno, L. D.; Beligni, G.; Tommasi, A.; Iuso, N.; Montagnani, F.; Fabbiani, M.; Rossetti, B.; Zanelli, G.; Bargagli, E.; Bergantini, L.; D'Alessandro, M.; Cameli, P.; Bennett, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Guerrini, S.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Spertilli, C.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Spagnesi, M.; Romani, D.; Piacentini, P.; Desanctis, E.; Cappelli, S.; Canaccini, A.; Verzuri, A.; Anemoli, V.; Ognibene, A.; D'Arminio Monforte, A.; Miraglia, F. G.; Girardis, M.; Venturelli, S.; Busani, S.; Cossarizza, A.; Antinori, A.; Vergori, A.; Emiliozzi, A.; Gabrieli, A.; Riva, A.; Scotton, P. G.; Andretta, F.; Panese, S.; Scaggiante, R.; Gatti, F.; Parisi, S. G.; Baratti, S.; Antoni, M. D.; Monica, M. D.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Fiorentino, G.; Carella, M.; Castori, M.; Merla, G.; Squeo, G. M.; Aucella, F.; Raggi, P.; Marciano, C.; Perna, R.; Bassetti, M.; Biagio, A. D.; Sanguinetti, M.; Masucci, L.; Valente, S.; Mandala, M.; Giorli, A.; Salerni, L.; Zucchi, P.; Parravicini, P.; Menatti, E.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Coviello, D. A.; Mussini, C.; Bosio, G.; Martinelli, E.; Mancarella, S.; Tavecchia, L.; Gori, M.; Crotti, L.; Parati, G.; Gabbi, C.; Zanella, I.; Rizzi, M.; Maggiolo, F.; Ripamonti, D.; Bachetti, T.; Rovere, M. T. L.; Sarzi-Braga, S.; Bussotti, M.; Chiariello, M.; Belli, M. A.; Dei, S.
abstract

Background: Recently, loss-of-function variants in TLR7 were identified in two families in which COVID-19 segregates like an X-linked recessive disorder environmentally conditioned by SARS-CoV-2. We investigated whether the two families represent the tip of the iceberg of a subset of COVID-19 male patients. Methods: This is a nested case-control study in which we compared male participants with extreme phenotype selected from the Italian GEN-COVID cohort of SARS-CoV-2-infected participants (<60 y, 79 severe cases versus 77 control cases). We applied the LASSO Logistic Regression analysis, considering only rare variants on young male subsets with extreme phenotype, picking up TLR7 as the most important susceptibility gene.

2021 - Author Correction: Endogenous control of inflammation characterizes pregnant women with asymptomatic or paucisymptomatic SARS-CoV-2 infection (Nature Communications, (2021), 12, 1, (4677), 10.1038/s41467-021-24940-w) [Articolo su rivista]
De Biasi, S.; Tartaro, D. L.; Gibellini, L.; Paolini, A.; Quong, A.; Petes, C.; Awong, G.; Douglas, S.; Lin, D.; Nieto, J.; Galassi, F. M.; Borella, R.; Fidanza, L.; Mattioli, M.; Leone, C.; Neri, I.; Meschiari, M.; Cicchetti, L.; Iannone, A.; Trenti, T.; Sarti, M.; Girardis, M.; Guaraldi, G.; Mussini, C.; Facchinetti, F.; Cossarizza, A.
abstract

The original version of this Article contained an error in Table 1. The correct version of the first row of the 2nd, 3rd, 5th and 7th columns states ‘CTR’, ‘PN’, ‘CTR vs PN’ and ‘PN vs PP’, instead of the original, incorrect ‘HD’, ‘NP’, ‘CTR vs NP’ and ‘CTR vs PP’. This has been corrected in both the PDF and HTML versions of the Article.

2021 - Better prognosis in females with severe COVID-19 pneumonia: possible role of inflammation as potential mediator. [Articolo su rivista]
Mussini, C; Cozzi-Lepri, A; Menozzi, M; Meschiari, M; Franceschini, E; Rogati, C; Cuomo, G; Bedini, A; Iadisernia, M; Volpi, S; Milic, J; Tonelli, R; Brugioni, L; Pietrangelo, A; Girardis, M; Cossarizza, A; Clini, E; Guaraldi, G.; De biasi, S; Gibellini, Lara
abstract

Objectives: Sex differences in COVID-19 severity and mortality have been described. Key aims of this analysis were to compare the risk of invasive mechanical ventilation (IMV) and mortality by sex and to explore whether variation in specific biomarkers could mediate this difference. Methods: This was a retrospective, observational cohort study among patients with severe COVID- 19 pneumonia. A survival analysis was conducted to compare time to the composite endpoint of IMV or death by sex. Interaction was formally tested to compare the risk difference by sex in subsets. Mediation analysis with a binary endpoint IMV or death (yes/no) by end of follow-up for a number of inflammation/coagulation biomarkers in the context of counterfactual prediction was also conducted. Results: Among 415 patients, 134 were females (32%) and 281 males (67%), median age 66 years (IQR 54-77). At admission, females showed a significantly less severe clinical and respiratory profiles with a higher PaO2/FiO2 (254 mmHg vs 191 mmHg; p=0.023). By 28 days from admission, 49.2% (95% CI: 39.6-58.9%) of males vs. 31.7% (17.9-45.4%) of females underwent IMV or death (log-rank pvalue<0.0001) and this amounted to a difference in HR of 0.40 (0.26-0.63, p=0.0001). The AUC in Creactive protein (CRP) over the study period appeared to explain 85% of this difference in risk by sex. Conclusions: Our analysis confirms a difference in the risk of COVID-19 clinical progression by sex and provides a hypothesis for potential mechanisms leading to this. CRP showed a predominant role to mediate the difference in risk by sex.

2021 - C9orf72 intermediate repeats confer genetic risk for severe covid-19 pneumonia independently of age [Articolo su rivista]
Zanella, I.; Zacchi, E.; Piva, S.; Filosto, M.; Beligni, G.; Alaverdian, D.; Amitrano, S.; Fava, F.; Baldassarri, M.; Frullanti, E.; Meloni, I.; Renieri, A.; Castelli, F.; Quiros-Roldan, E.; Mari, F.; Daga, S.; Benetti, E.; Furini, S.; Fallerini, C.; Valentino, F.; Doddato, G.; Giliberti, A.; Tita, R.; Bruttini, M.; Croci, S.; Pinto, A. M.; Mencarelli, M. A.; Rizzo, C. L.; Montagnani, F.; Tumbarello, M.; Rancan, I.; Sarno, L. D.; Palmieri, M.; Carriero, M. L.; Fabbiani, M.; Rossetti, B.; Bargagli, E.; Bergantini, L.; D'Alessandro, M.; Cameli, P.; Bennett, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Guerrini, S.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Raffaelli, C. S.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Spagnesi, M.; Piacentini, P.; Bandini, M.; Desanctis, E.; Cappelli, S.; Canac-Cini, A.; Verzuri, A.; Anemoli, V.; Ognibene, A.; Pancrazi, A.; Lorubbio, M.; Vaghi, M.; Monforte, A. D.; Miraglia, F. G.; Mondelli, M. U.; Bruno, R.; Marco, V.; Mantovani, S.; Ludovisi, S.; Girardis, M.; Venturelli, S.; Busani, S.; Cossarizza, A.; Antinori, A.; Vergori, A.; Emiliozzi, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Riva, A.; Francisci, D.; Schiaroli, E.; Tommasi, A.; Paciosi, F.; Scotton, P. G.; Andretta, F.; Panese, S.; Scaggiante, R.; Gatti, F.; Parisi, S. G.; Antoni, M. D.; Monica, M. D.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Fiorentino, G.; Carella, M.; Castori, M.; Merla, G.; Squeo, G. M.; Aucella, F.; Raggi, P.; Marciano, C.; Perna, R.; Bassetti, M.; Biagio, A. D.; Sanguinetti, M.; Masucci, L.; Valente, S.; Mandala, M.; Giorli, A.; Salerni, L.; Zucchi, P.; Parravicini, P.; Menatti, E.; Baratti, S.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Coviello, D. A.; Mussini, C.; Bosio, G.; Martinelli, E.; Mancarella, S.; Tavecchia, L.; Belli, M. A.; Crotti, L.; Parati, G.; Picchiotti, N.; Gori, M.; Gabbi, C.; Sanarico, M.; Ceri, S.; Pinoli, P.; Raimondi, F.; Bis-Carini, F.; Stella, A.; Rizzi, M.; Maggiolo, F.; Ripamonti, D.; Suardi, C.; Bachetti, T.; Rovere, M. T. L.; Sarzi-Braga, S.; Bussotti, M.; Capitani, K.; Zguro, K.; Dei, S.; Ravaglia, S.; Artuso, R.; Perrella, A.; Bianchi, F.; Bergomi, P.; Catena, E.; Colombo, R.; Perticaroli, V.; Gennarelli, M.; Magri, C.; Basiotto, G.; Zizioli, D.; Giliani, S.; Monti, E.; Foca, E.; Carriero, C.; Latronico, N.; Padovani, A.; Brugnoni, D.
abstract

A cytokine storm, autoimmune features and dysfunctions of myeloid cells significantly contribute to severe coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Genetic background of the host seems to be partly responsible for severe phenotype and genes related to innate immune response seem critical host determinants. The C9orf72 gene has a role in vesicular trafficking, autophagy regulation and lyso-some functions, is highly expressed in myeloid cells and is involved in immune functions, regulating the lysosomal degradation of mediators of innate immunity. A large non-coding hexanucleotide repeat expansion (HRE) in this gene is the main genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), both characterized by neuroinflammation and high systemic levels of proinflammatory cytokines, while HREs of intermediate length, although rare, are more frequent in autoimmune disorders. C9orf72 full mutation results in haploinsufficiency and intermediate HREs seem to modulate gene expression as well and impair autophagy. Herein, we sought to explore whether intermediate HREs in C9orf72 may be a risk factor for severe COVID-19. Although we found intermediate HREs in only a small portion of 240 patients with severe COVID-19 pneumonia, the magnitude of risk for requiring non-invasive or mechanical ventilation conferred by harboring intermediate repeats >10 units in at least one C9orf72 allele was more than twice respect to having shorter expansions, when adjusted for age (odds ratio (OR) 2.36; 95% confidence interval (CI) 1.04–5.37, p = 0.040). The association between intermediate repeats >10 units and more severe clinical outcome (p = 0.025) was also validated in an independent cohort of 201 SARS-CoV-2 infected patients. These data suggest that C9orf72 HREs >10 units may influence the pathogenic process driving more severe COVID-19 phenotypes.

2021 - COVID-19 in the tonsillectomised population [Articolo su rivista]
Capriotti, V.; Mattioli, F.; Guida, F.; Marcuzzo, A. V.; Manto, A. L.; Martone, A.; Molinari, G.; Fabbris, C.; Menegaldo, A.; Calvanese, L.; Latini, G.; Cingolani, C.; Gradoni, P.; Nata, F. B.; De Sisti, C.; Selle, V.; Leone, G.; Indelicato, P.; Pilolli, F.; Mevio, N.; Roncoroni, L.; Papi, S.; Meschiari, M.; Tominz, R.; D'Ascanio, L.; Dragonetti, A.; Torelli, L.; Trenti, L.; Spinato, G.; Boscolo-Rizzo, P.; Bussi, M.; Cossarizza, A.; Presutti, L.; Tirelli, G.
abstract

Objective. Interactions between SARS-CoV-2 and pharyngeal associated lymphoid tissue are thought to influence the manifestations of COVID-19. We aimed to determine whether a previous history of tonsillectomy, as a surrogate indicator of a dysfunctional pharyngeal associated lymphoid tissue, could predict the presentation and course of COVID-19. Methods. Multicentric cross-sectional observational study involving seven hospitals in Northern and Central Italy. Data on the clinical course and signs and symptoms of the infection were collected from 779 adults who tested positive for SARS-CoV-2, and analysed in relation to previous tonsillectomy, together with demographic and anamnestic data. Results. Patients with previous tonsillectomy showed a greater risk of fever, temperature higher than 39°C, chills and malaise. No significant differences in hospital admissions were found. Conclusions. A previous history of tonsillectomy, as a surrogate indicator of immunological dysfunction of the pharyngeal associated lymphoid tissue, could predict a more intense systemic manifestation of COVID-19. These results could provide a simple clinical marker to discriminate suspected carriers and to delineate more precise prognostic models.

2021 - Cell Death in Coronavirus Infections: Uncovering Its Role during COVID-19 [Articolo su rivista]
Paolini, A.; Borella, R.; De Biasi, S.; Neroni, A.; Mattioli, M.; Lo Tartaro, D.; Simonini, C.; Franceschini, L.; Cicco, G.; Piparo, A. M.; Cossarizza, A.; Gibellini, L.
abstract

Cell death mechanisms are crucial to maintain an appropriate environment for the functionality of healthy cells. However, during viral infections, dysregulation of these processes can be present and can participate in the pathogenetic mechanisms of the disease. In this review, we describe some features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and some immunopathogenic mechanisms characterizing the present coronavirus disease (COVID-19). Lymphopenia and monocytopenia are important contributors to COVID-19 immunopathogenesis. The fine mechanisms underlying these phenomena are still unknown, and several hypotheses have been raised, some of which assign a role to cell death as far as the reduction of specific types of immune cells is concerned. Thus, we discuss three major pathways such as apoptosis, necroptosis, and pyroptosis, and suggest that all of them likely occur simultaneously in COVID-19 patients. We describe that SARS-CoV-2 can have both a direct and an indirect role in inducing cell death. Indeed, on the one hand, cell death can be caused by the virus entry into cells, on the other, the excessive concentration of cytokines and chemokines, a process that is known as a COVID-19-related cytokine storm, exerts deleterious effects on circulating immune cells. However, the overall knowledge of these mechanisms is still scarce and further studies are needed to delineate new therapeutic strategies.

2021 - Circulating mucosal-associated invariant T cells identify patients responding to anti-PD-1 therapy [Articolo su rivista]
De Biasi, S.; Gibellini, L.; Lo Tartaro, D.; Puccio, S.; Rabacchi, C.; Mazza, E. M. C.; Brummelman, J.; Williams, B.; Kaihara, K.; Forcato, M.; Bicciato, S.; Pinti, M.; Depenni, R.; Sabbatini, R.; Longo, C.; Dominici, M.; Pellacani, G.; Lugli, E.; Cossarizza, A.
abstract

Immune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8+ T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8+ T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8+ population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy.

2021 - Corrigendum: To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis (Front. Immunol., (2021), 12, (653974), 10.3389/fimmu.2021.653974) [Articolo su rivista]
Di Rosa, F.; Cossarizza, A.; Hayday, A. C.
abstract

In the original article, there was a mistake in the legend for Figure 1 as published. On the Viable cells, no “dump” gate, “CD16” was written instead of “CD19”. The correct legend appears below. “HD PBMCs were stained with the viability dye eFluor 780 (eF780), the DNA dye Hoechst-33342, and fluorochrome conjugated mAbs against surface markers and Ki-67, as described (16). An example of flow cytometry analysis is shown. (A) Gating of viable single CD8 T cells in 6 steps: 1) DNA-A/-W singlets. Single cells having 2n≤ DNA content ≤4n were selected on the DNA-area (A) versus (vs) DNA-width (W) plot; 2) Time exclusion. Stable acquisition over time (seconds) was monitored on the time vs DNA-A plot and any events collected in case of pressure fluctuations were excluded; 3) Viable cells, no “dump”. Cells expressing CD4, CD14 and CD19, and dead cells were excluded; 4) FSC-A/SSC-A “relaxed” gate. A “relaxed” gate was used on the FSC-A vs SSC-A plot, to include highly activated and cycling lymphocytes (15); 5) CD8 T cells. CD8 T cells were gated on the CD3 versus CD8 plot; 6) Refined singlets. A few remaining doublets composed by one cell sitting on top of another (so called “shadow” doublets) were excluded as Ki-67int/- events having > 2n DNA content (16). This gating strategy was used as a base for the subsequent gates. (B) The following naïve/memory subsets of CD8 T cells were identified: CD45RA+ CCR7+ Naïve, CD45RA- CCR7+ central memory (CM), CD45RA- CCR7- effector memory (EM), and CD45RA+ CCR7- (EMRA). (C) Cell cycle phases of each naïve/memory CD8 T cell subset were defined on DNA-A vs Ki67-A plot as follows: cells in G0 were identified as DNA 2n/Ki67- (bottom left quadrant); cells in G1 as DNA 2n/Ki67+ (upper left quadrant); cells in S-G2/M (or TDS cells) as DNA>2n/Ki67+ (top right quadrant). Unpublished data in relation to (16).” In the original article, there was also amistake in the legend for SupplementaryTable 1 as published. The peptide- HLA-A*02 tetramer list was incorrectly formatted, there was missing information about numbers in the table (they represent average percentages); missing information about the number of mice (panel A) and number of human donors (panel B and C); and a missing citation of original references at the end. The corrected Supplementary Material File is linked below. In the original article, there was also a mistake in Figure 1 as published. There was an incorrect y-axis label in panel A, third graph from left. The corrected Figure 1 appears below. (Figure presented.) The authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated.

2021 - Darunavir/Cobicistat Is Associated with Negative Outcomes in HIV-Negative Patients with Severe COVID-19 Pneumonia [Articolo su rivista]
Milic, Jovana; Novella, Alessio; Meschiari, Marianna; Menozzi, Marianna; Santoro, Antonella; Bedini, Andrea; Cuomo, Gianluca; Franceschini, Erica; Digaetano, Margherita; Carli, Federica; Ciusa, Giacomo; Volpi, Sara; Bacca, Erica; Franceschi, Giacomo; Yaacoub, Dina; Rogati, Carlotta; Tutone, Marco; Burastero, Giulia; Faltoni, Matteo; Iadisernia, Vittorio; Dolci, Giovanni; Cossarizza, Andrea; Mussini, Cristina; Pasina, Luca; Guaraldi, Giovanni
abstract

The aim of this study was to evaluate both positive outcomes, including reduction of respiratory support aid and duration of hospital stay, and negative ones, including mortality and a composite of invasive mechanical ventilation or death, in patients with coronavirus disease 2019 (COVID-19) pneumonia treated with or without oral darunavir/cobicistat (DRV/c, 800/150mg/day) used in different treatment durations. The secondary objective was to evaluate the percentage of patients treated with DRV/c who were exposed to potentially severe drug-drug interactions (DDIs) and died during hospitalization. This observational retrospective study was conducted in consecutive patients with COVID-19 pneumonia admitted to a tertiary care hospital in Modena, Italy. Kaplan-Meier survival curves and Cox proportional hazards regression were used to compare patients receiving standard of care with or without DRV/c. Adjustment for key confounders was applied. Two hundred seventy-three patients (115 on DRV/c) were included, 75.8% males, mean age was 64.6 (±13.2) years. Clinical improvement was similar between the groups, depicted by respiratory aid switch (p>.05). The same was observed for duration of hospital stay [13.2 (±8.9) for DRV/c vs. 13.4 (±7.2) days for no-DRV/c, p=.9]. Patients on DRV/c had higher rates of mortality (25.2% vs. 10.1%, p<.0001. The rate of composite outcome of mechanical ventilation and death was higher in the DRV/c group (37.4% vs. 25.3%, p=.03). Multiple serious DDI associated with DRV/c were observed in the 19 patients who died. DRV/c should not be recommended as a treatment option for COVID-19 pneumonia outside clinical trials.

2021 - Development and validation of a prediction model for tocilizumab failure in hospitalized patients with SARS-CoV-2 infection [Articolo su rivista]
Mussini, C; Cozzi-Lepri, A; Menozzi, M; Meschiari, M; Franceschini, E; Milic, J; Brugioni, L; Pietrangelo, A; Girardis, M; Cossarizza, A; Tonelli, R; Clini, E; Massari, M; Bartoletti, M; Ferrari, A; Cattelan, Am; Zuccalà, P; Lichtner, M; Rossotti, R; Girardi, E; Nicastri, E; Puoti, M; Antinori, A; Viale, Pl; Guaraldi, G.
abstract

Background: The aim of this secondary analysis of the TESEO cohort is to identify, early in the course of treatment with tocilizumab, factors associated with the risk of progressing to mechanical ventilation and death and develop a risk score to estimate the risk of this outcome according to patients’ profile. Methods: Patients with COVID-19 severe pneumonia receiving standard of care + tocilizumab who were alive and free from mechanical ventilation at day6 after treatment initiation were included in this retrospective, multicenter cohort study. Multivariable logistic regression models were built to identify predictors of mechanical ventilation or death by day-28 from treatment initiation and β-coefficients were used to develop a risk score. Secondary outcome was mortality. Patients with the same inclusion criteria as the derivation cohort from 3 independent hospitals were used as validation cohort. Results: 266 patients treated with tocilizumab were included. By day 28 of hospital follow-up post treatment initiation, 40 (15%) underwent mechanical ventilation or died [26 (10%)]. At multivariable analysis, sex, day-4 PaO2/FiO2 ratio, platelets and CRP were independently associated with the risk of developing the study outcomes and were used to generate the proposed risk score. The accuracy of the score in AUC was 0.80 and 0.70 in internal validation and test for the composite endpoint and 0.92 and 0.69 for death, respectively. Conclusions: Our score could assist clinicians in identifying, early after tocilizumab, patients who are likely to progress to mechanical ventilation or death so that they could be selected for eventual rescue therapies.

2021 - Effects of cytokine blocking agents on hospital mortality in patients admitted to ICU with acute respiratory distress syndrome by SARS-CoV2 infection: retrospective cohort study. [Articolo su rivista]
Coloretti, I; Busani, S; Biagioni, E; Venturelli, S; Munari, E; Marco, S; Dall’Ara, L; Tosi, M; Clini, E; Tonelli, R; Fantini, R; Mussini, C; Meschiari, M; Guaraldi, G; Cossarizza, A; Alfano, G; Girardis, M; Gibellini, Lara
abstract

Background- The use of cytokine-blocking agents has been proposed to modulate the inflammatory response in patients with COVID19. Tocilizumab and Anakinra were included in the local protocol as an optional treatment in critically ill patients with acute respiratory distress syndrome (ARDS) by SARS-CoV2 infection. This cohort study evaluated the effects of therapy with cytokine blocking agents on in-hospital mortality in COVID19 patients requiring mechanical ventilation and admitted to intensive care unit. Methods- The association between therapy with Tocilizumab or Anakinra and in-hospital mortality was assessed in consecutive adult COVID19 patients admitted to our ICU with moderate to severe ARDS. The association was evaluated by comparing patients who receive to those who did not receive Tocilizumab or Anakinra and by using different multivariable Cox models adjusted for variables related to poor outcome, for the propensity to be treated with Tocilizumab or Anakinra and after patient matching. Results- Sixty-six patients who received immunotherapy (49 Tocilizumab, 17 Anakinra) and 28 patients who did not receive immunotherapy were included. The in-hospital crude mortality was 30,3% in treated patients and 50% in non-treated (OR 0,77, 95% CI 0,56-1,05, p=0,069). The adjusted Cox model showed an association between therapy with immunotherapy and in-hospital mortality (HR 0,40, 95% CI 0,19-0,83, p=0,015). This protective effect was further confirmed in the analysis adjusted for propensity score, in the propensity-matched cohort and in the cohort of patients with invasive mechanical ventilation within 2 hours after ICU admission. Conclusions- Although important limitations, our study showed that cytokine-blocking agents seem to be safe and to improve survival in COVID-19 patients admitted to ICU with ARDS and the need of mechanical ventilation.

2021 - Employing a systematic approach to biobanking and analyzing clinical and genetic data for advancing COVID-19 research [Articolo su rivista]
Daga, S.; Fallerini, C.; Baldassarri, M.; Fava, F.; Valentino, F.; Doddato, G.; Benetti, E.; Furini, S.; Giliberti, A.; Tita, R.; Amitrano, S.; Bruttini, M.; Meloni, I.; Pinto, A. M.; Raimondi, F.; Stella, A.; Biscarini, F.; Picchiotti, N.; Gori, M.; Pinoli, P.; Ceri, S.; Sanarico, M.; Crawley, F. P.; Birolo, G.; Montagnani, F.; Di Sarno, L.; Tommasi, A.; Palmieri, M.; Croci, S.; Emiliozzi, A.; Fabbiani, M.; Rossetti, B.; Zanelli, G.; Bergantini, L.; D'Alessandro, M.; Cameli, P.; Bennet, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Guerrini, S.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Spertilli, C.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Spagnesi, M.; Piacentini, P.; Bandini, M.; Desanctis, E.; Cappelli, S.; Canaccini, A.; Verzuri, A.; Anemoli, V.; Ognibene, A.; Vaghi, M.; D'Arminio Monforte, A.; Merlini, E.; Mondelli, M. U.; Mantovani, S.; Ludovisi, S.; Girardis, M.; Venturelli, S.; Sita, M.; Cossarizza, A.; Antinori, A.; Vergori, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Riva, A.; Francisci, D.; Schiaroli, E.; Scotton, P. G.; Andretta, F.; Panese, S.; Scaggiante, R.; Gatti, F.; Parisi, S. G.; Castelli, F.; Quiros-Roldan, M. E.; Magro, P.; Zanella, I.; Della Monica, M.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Fiorentino, G.; Carella, M.; Castori, M.; Merla, G.; Aucella, F.; Raggi, P.; Marciano, C.; Perna, R.; Bassetti, M.; Di Biagio, A.; Sanguinetti, M.; Masucci, L.; Gabbi, C.; Valente, S.; Meloni, I.; Mencarelli, M. A.; Rizzo, C. L.; Bargagli, E.; Mandala, M.; Giorli, A.; Salerni, L.; Zucchi, P.; Parravicini, P.; Menatti, E.; Baratti, S.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Coviello, D. A.; Mussini, C.; Bosio, G.; Mancarella, S.; Tavecchia, L.; Renieri, A.; Mari, F.; Frullanti, E.
abstract

Within the GEN-COVID Multicenter Study, biospecimens from more than 1000 SARS-CoV-2 positive individuals have thus far been collected in the GEN-COVID Biobank (GCB). Sample types include whole blood, plasma, serum, leukocytes, and DNA. The GCB links samples to detailed clinical data available in the GEN-COVID Patient Registry (GCPR). It includes hospitalized patients (74.25%), broken down into intubated, treated by CPAP-biPAP, treated with O2 supplementation, and without respiratory support (9.5%, 18.4%, 31.55% and 14.8, respectively); and non-hospitalized subjects (25.75%), either pauci- or asymptomatic. More than 150 clinical patient-level data fields have been collected and binarized for further statistics according to the organs/systems primarily affected by COVID-19: heart, liver, pancreas, kidney, chemosensors, innate or adaptive immunity, and clotting system. Hierarchical clustering analysis identified five main clinical categories: (1) severe multisystemic failure with either thromboembolic or pancreatic variant; (2) cytokine storm type, either severe with liver involvement or moderate; (3) moderate heart type, either with or without liver damage; (4) moderate multisystemic involvement, either with or without liver damage; (5) mild, either with or without hyposmia. GCB and GCPR are further linked to the GCGDR, which includes data from whole-exome sequencing and high-density SNP genotyping. The data are available for sharing through the Network for Italian Genomes, found within the COVID-19 dedicated section. The study objective is to systematize this comprehensive data collection and begin identifying multi-organ involvement in COVID-19, defining genetic parameters for infection susceptibility within the population, and mapping genetically COVID-19 severity and clinical complexity among patients.

2021 - Endogenous control of inflammation characterizes pregnant women with asymptomatic or paucisymptomatic SARS-CoV-2 infection [Articolo su rivista]
De Biasi, S.; Tartaro, D. L.; Gibellini, L.; Paolini, A.; Quong, A.; Petes, C.; Awong, G.; Douglas, S.; Lin, D.; Nieto, J.; Galassi, F. M.; Borella, R.; Fidanza, L.; Mattioli, M.; Leone, C.; Neri, I.; Meschiari, M.; Cicchetti, L.; Iannone, A.; Trenti, T.; Sarti, M.; Girardis, M.; Guaraldi, G.; Mussini, C.; Facchinetti, F.; Cossarizza, A.
abstract

SARS-CoV-2 infection can affect all human beings, including pregnant women. Thus, understanding the immunological changes induced by the virus during pregnancy is nowadays of pivotal importance. Here, using peripheral blood from 14 pregnant women with asymptomatic or mild SARS-CoV-2 infection, we investigate cell proliferation and cytokine production, measure plasma levels of 62 cytokines, and perform a 38-parameter mass cytometry analysis. Our results show an increase in low density neutrophils but no lymphopenia or gross alterations of white blood cells, which display normal levels of differentiation, activation or exhaustion markers and show well preserved functionality. Meanwhile, the plasma levels of anti-inflammatory cytokines such as interleukin (IL)-1RA, IL-10 and IL-19 are increased, those of IL-17, PD-L1 and D-dimer are decreased, but IL-6 and other inflammatory molecules remain unchanged. Our profiling of antiviral immune responses may thus help develop therapeutic strategies to avoid virus-induced damages during pregnancy.

2021 - Finding predictive factors for immunotherapy in metastatic renal-cell carcinoma: What are we looking for? [Articolo su rivista]
Guida, A.; Sabbatini, R.; Gibellini, L.; De Biasi, S.; Cossarizza, A.; Porta, C.
abstract

A major breakthrough in cancer immunotherapy was the development of monoclonal antibodies targeting inhibitory immune checkpoint proteins. This approach demonstrated significant antitumor activity and efficacy in different cancer types, including metastatic renal cell carcinoma (mRCC). In the majority of patients, this drug is able to restore the patient's tumour-specific T-cell-mediated response thus improving both overall survival and objective response rate. However, a lack of clinical response occurs in a number of patients, raising questions about how to predict and increase the number of patients who receive long-term clinical benefit from immune checkpoint therapy or not. The aim of this review is to summarize available data about immune biomarkers in patients with mRCC treated with immunotherapy.

2021 - Gene expression analysis of T-cells by single-cell RNA-seq [Capitolo/Saggio]
Lo Tartaro, D.; De Biasi, S.; Forcato, M.; Gibellini, L.; Cossarizza, A.
abstract

During the last decade, the rapid progress in the development of next-generation sequencing (NGS) technologies has provided relevant insights into complex biological systems, ranging from cancer genomics to microbiology. Among NGS technologies, single-cell RNA sequencing is currently used to decipher the complex heterogeneity of several biological samples, including T cells. Even if this technique requires specialized equipment and expertise, nowadays it is broadly applied in research. In this chapter, we will provide an optimized protocol for the isolation of T cells and the preparation of RNA sequencing libraries by using droplet digital technology (ddSEQ, Bio-Rad Laboratories). We will also illustrate a guide to the main steps of data processing and options for data interpretation. This protocol will support users in building a single-cell experimental framework, from sample preparation to data interpretation.

2021 - Genetic mechanisms of critical illness in COVID-19 [Articolo su rivista]
Pairo-Castineira, E.; Clohisey, S.; Klaric, L.; Bretherick, A. D.; Rawlik, K.; Pasko, D.; Walker, S.; Parkinson, N.; Fourman, M. H.; Russell, C. D.; Furniss, J.; Richmond, A.; Gountouna, E.; Wrobel, N.; Harrison, D.; Wang, B.; Wu, Y.; Meynert, A.; Griffiths, F.; Oosthuyzen, W.; Kousathanas, A.; Moutsianas, L.; Yang, Z.; Zhai, R.; Zheng, C.; Grimes, G.; Beale, R.; Millar, J.; Shih, B.; Keating, S.; Zechner, M.; Haley, C.; Porteous, D. J.; Hayward, C.; Yang, J.; Knight, J.; Summers, C.; Shankar-Hari, M.; Klenerman, P.; Turtle, L.; Ho, A.; Moore, S. C.; Hinds, C.; Horby, P.; Nichol, A.; Maslove, D.; Ling, L.; Mcauley, D.; Montgomery, H.; Walsh, T.; Pereira, A. C.; Renieri, A.; Millar, J.; Nichol, A.; Walsh, T.; Shankar-Hari, M.; Ponting, C.; Meikle, J.; Finernan, P.; Mcmaster, E.; Law, A.; Baillie, J. K.; Paterson, T.; Wackett, T.; Armstrong, R.; Clark, R.; Coutts, A.; Donnelly, L.; Gilchrist, T.; Hafezi, K.; Macgillivray, L.; Maclean, A.; Mccafferty, S.; Morrice, K.; Weaver, J.; Boz, C.; Golightly, A.; Ward, M.; Mal, H.; Szoor-McElhinney, H.; Brown, A.; Hendry, R.; Stenhouse, A.; Cullum, L.; Law, D.; Law, S.; Law, R.; Swets, M.; Day, N.; Taneski, F.; Duncan, E.; Parkinson, N.; Collier, D.; Wood, S.; Zak, A.; Borra, C.; Matharu, M.; May, P.; Alldis, Z.; Mitchelmore, O.; Bowles, R.; Easthope, A.; Bibi, F.; Lancoma-Malcolm, I.; Gurasashvili, J.; Pheby, J.; Shiel, J.; Bolton, M.; Patel, M.; Taylor, M.; Zongo, O.; Ebano, P.; Harding, P.; Astin-Chamberlain, R.; Choudhury, Y.; Cox, A.; Kallon, D.; Burton, M.; Hall, R.; Blowes, S.; Prime, Z.; Biddle, J.; Prysyazhna, O.; Newman, T.; Tierney, C.; Kassam, J.; Shankar-Hari, M.; Ostermann, M.; Campos, S.; Bociek, A.; Lim, R.; Grau, N.; Jones, T. O.; Whitton, C.; Marotti, M.; Arbane, G.; Bonner, S.; Hugill, K.; Reid, J.; Welters, I.; Waugh, V.; Williams, K.; Shaw, D.; Roman, J. F.; Martinez, M. L.; Johnson, E.; Waite, A.; Johnston, B.; Hamilton, D.; Mulla, S.; Mcphail, M.; Smith, J.; Baillie, J. K.; Barclay, L.; Hope, D.; Mcculloch, C.; Mcquillan, L.; Clark, S.; Singleton, J.; Priestley, K.; Rea, N.; Callaghan, M.; Campbell, R.; Andrew, G.; Marshall, L.; Mckechnie, S.; Hutton, P.; Bashyal, A.; Davidson, N.; Summers, C.; Polgarova, P.; Stroud, K.; Pathan, N.; Elston, K.; Agrawal, S.; Battle, C.; Newey, L.; Rees, T.; Harford, R.; Brinkworth, E.; Williams, M.; Murphy, C.; White, I.; Croft, M.; Bandla, N.; Gellamucho, M.; Tomlinson, J.; Turner, H.; Davies, M.; Quinn, A.; Hussain, I.; Thompson, C.; Parker, H.; Bradley, R.; Griffiths, R.; Scriven, J.; Nilsson, A.; Bates, M.; Dasgin, J.; Gill, J.; Puxty, A.; Cathcart, S.; Salutous, D.; Turner, L.; Duffy, K.; Puxty, K.; Joseph, A.; Herdman-Grant, R.; Simms, R.; Swain, A.; Naranjo, A.; Crowe, R.; Sollesta, K.; Loveridge, A.; Baptista, D.; Morino, E.; Davey, M.; Golden, D.; Jones, J.; Moreno Cuesta, J.; Haldeos, A.; Bakthavatsalam, D.; Vincent, R.; Elhassan, M.; Xavier, K.; Ganesan, A.; Purohit, D.; Abdelrazik, M.; Morgan, J.; Akeroyd, L.; Bano, S.; Lawton, T.; Warren, D.; Bromley, M.; Sellick, K.; Gurr, L.; Wilkinson, B.; Nagarajan, V.; Szedlak, P.; Cupitt, J.; Stoddard, E.; Benham, L.; Preston, S.; Laha, S.; Slawson, N.; Bradshaw, Z.; Brown, J.; Caswell, M.; Melling, S.; Bamford, P.; Faulkner, M.; Cawley, K.; Jeffrey, H.; London, E.; Sainsbury, H.; Nagra, I.; Nasir, F.; Dunmore, C.; Jones, R.; Abraheem, A.; Al-Moasseb, M.; Girach, R.; Padden, G.; Egan, J.; Brantwood, C.; Alexander, P.; Bradley-Potts, J.; Allen, S.; Felton, T.; Manna, S.; Farnell-Ward, S.; Leaver, S.; Queiroz, J.; Maccacari, E.; Dawson, D.; Delgado, C. C.; Saluzzio, R. P.; Ezeobu, O.; Ding, L.; Sicat, C.; Kanu, R.; Durrant, G.; Texeira, J.; Harrison, A.; Samakomva, T.; Scriven, J.; Willis, H.; Hopkins, B.; Thrasyvoulou, L.; Jackson, M.; Zaki, A.; Tibke, C.; Bennett, S.; Woodyatt, W.; Kent, A.; Goodwin, E.; Brandwood, C.; Clark, R.; Smith, L.; Rooney, K.; Thomson, N.; Rodden, N.; Hughes, E.; Mcglynn, D.; Clark, C.; Clark, P.; Abel, L.; Sundaram, R.; Gemmell, L.; Brett, M.; Hornsby, J.;
abstract

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10−8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10−8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 × 10−12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10−8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte–macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice.

2021 - Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition) [Articolo su rivista]
Cossarizza, A.; Chang, H. -D.; Radbruch, A.; Abrignani, S.; Addo, R.; Akdis, M.; Andra, I.; Andreata, F.; Annunziato, F.; Arranz, E.; Bacher, P.; Bari, S.; Barnaba, V.; Barros-Martins, J.; Baumjohann, D.; Beccaria, C. G.; Bernardo, D.; Boardman, D. A.; Borger, J.; Bottcher, C.; Brockmann, L.; Burns, M.; Busch, D. H.; Cameron, G.; Cammarata, I.; Cassotta, A.; Chang, Y.; Chirdo, F. G.; Christakou, E.; Cicin-Sain, L.; Cook, L.; Corbett, A. J.; Cornelis, R.; Cosmi, L.; Davey, M. S.; De Biasi, S.; De Simone, G.; del Zotto, G.; Delacher, M.; Di Rosa, F.; Santo, J. D.; Diefenbach, A.; Dong, J.; Dorner, T.; Dress, R. J.; Dutertre, C. -A.; Eckle, S. B. G.; Eede, P.; Evrard, M.; Falk, C. S.; Feuerer, M.; Fillatreau, S.; Fiz-Lopez, A.; Follo, M.; Foulds, G. A.; Frobel, J.; Gagliani, N.; Galletti, G.; Gangaev, A.; Garbi, N.; Garrote, J. A.; Geginat, J.; Gherardin, N. A.; Gibellini, L.; Ginhoux, F.; Godfrey, D. I.; Gruarin, P.; Haftmann, C.; Hansmann, L.; Harpur, C. M.; Hayday, A. C.; Heine, G.; Hernandez, D. C.; Herrmann, M.; Hoelsken, O.; Huang, Q.; Huber, S.; Huber, J. E.; Huehn, J.; Hundemer, M.; Hwang, W. Y. K.; Iannacone, M.; Ivison, S. M.; Jack, H. -M.; Jani, P. K.; Keller, B.; Kessler, N.; Ketelaars, S.; Knop, L.; Knopf, J.; Koay, H. -F.; Kobow, K.; Kriegsmann, K.; Kristyanto, H.; Krueger, A.; Kuehne, J. F.; Kunze-Schumacher, H.; Kvistborg, P.; Kwok, I.; Latorre, D.; Lenz, D.; Levings, M. K.; Lino, A. C.; Liotta, F.; Long, H. M.; Lugli, E.; Macdonald, K. N.; Maggi, L.; Maini, M. K.; Mair, F.; Manta, C.; Manz, R. A.; Mashreghi, M. -F.; Mazzoni, A.; Mccluskey, J.; Mei, H. E.; Melchers, F.; Melzer, S.; Mielenz, D.; Monin, L.; Moretta, L.; Multhoff, G.; Munoz, L. E.; Munoz-Ruiz, M.; Muscate, F.; Natalini, A.; Neumann, K.; Ng, L. G.; Niedobitek, A.; Niemz, J.; Almeida, L. N.; Notarbartolo, S.; Ostendorf, L.; Pallett, L. J.; Patel, A. A.; Percin, G. I.; Peruzzi, G.; Pinti, M.; Pockley, A. G.; Pracht, K.; Prinz, I.; Pujol-Autonell, I.; Pulvirenti, N.; Quatrini, L.; Quinn, K. M.; Radbruch, H.; Rhys, H.; Rodrigo, M. B.; Romagnani, C.; Saggau, C.; Sakaguchi, S.; Sallusto, F.; Sanderink, L.; Sandrock, I.; Schauer, C.; Scheffold, A.; Scherer, H. U.; Schiemann, M.; Schildberg, F. A.; Schober, K.; Schoen, J.; Schuh, W.; Schuler, T.; Schulz, A. R.; Schulz, S.; Schulze, J.; Simonetti, S.; Singh, J.; Sitnik, K. M.; Stark, R.; Starossom, S.; Stehle, C.; Szelinski, F.; Tan, L.; Tarnok, A.; Tornack, J.; Tree, T. I. M.; van Beek, J. J. P.; van de Veen, W.; van Gisbergen, K.; Vasco, C.; Verheyden, N. A.; von Borstel, A.; Ward-Hartstonge, K. A.; Warnatz, K.; Waskow, C.; Wiedemann, A.; Wilharm, A.; Wing, J.; Wirz, O.; Wittner, J.; Yang, J. H. M.; Yang, J.
abstract

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

2021 - Hypokalemia in Patients with COVID-19 [Articolo su rivista]
Alfano, G.; Ferrari, A.; Fontana, F.; Perrone, R.; Mori, G.; Ascione, E.; Magistroni, R.; Venturi, G.; Pederzoli, S.; Margiotta, G.; Romeo, M.; Piccinini, F.; Franceschi, G.; Volpi, S.; Faltoni, M.; Ciusa, G.; Bacca, E.; Tutone, M.; Raimondi, A.; Menozzi, M.; Franceschini, E.; Cuomo, G.; Orlando, G.; Santoro, A.; Di Gaetano, M.; Puzzolante, C.; Carli, F.; Bedini, A.; Milic, J.; Meschiari, M.; Mussini, C.; Cappelli, G.; Guaraldi, G.; Borghi, V.; Burastero, G.; Corradi, L.; Di Gaetano, M.; Dolci, G.; Fantini, R.; Iadisernia, V.; Larne, D.; Pellegrino, F.; Rogati, C.; Santoro, A.; Tonelli, R.; Yaacoub, D.; Alfan, S.; Marco, B.; Pulizzi, R.; Leonelli, M.; Facchini, F.; Damiano, F.; Girardis, M.; Andreotti, A.; Biagioni, E.; Bondi, F.; Busani, S.; Chierego, G.; Scotti, M.; Cossarizza, L. S. A.; Bellinazzi, C.; Borella, R.; De Biasi, S.; De Gaetano, A.; Fidanza, L.; Gibellini, L.; Iannone, A.; Tartaro, D. L.; Mattioli, M.; Nasi, M.; Paolini, A.; Pinti, M.
abstract

Background: Patients with COVID-19 experience multiple clinical conditions that may cause electrolyte imbalances. Hypokalemia is a concerning electrolyte disorder closely associated with severe complications. This study aimed to estimate prevalence, risk factors and outcome of hypokalemia in a cohort of patients with confirmed COVID-19. Methods: A retrospective analysis was conducted on 290 non-ICU admitted patients with COVID-19 at the tertiary teaching hospital of Modena, Italy, from February 16 to April 14, 2020. Results: Hypokalemia was detected in 119 out of 290 patients (41%) during hospitalization. Mean serum potassium was 3.1 ± 0.1 meq/L. The majority of patients (90.7%) patients experienced only a mild decrease in serum potassium level (3–3.4 mEq/L). Hypokalemia was associated with hypocalcemia, which was detected in 50% of subjects. Urine potassium-to-creatinine ratio, measured in a small number of patients (n = 45; 36.1%), revealed an increase of urinary potassium excretion in most cases (95.5%). Risk factors for hypokalemia were female sex (odds ratio (OR) 2.44; 95% CI 1.36–4.37; P 0.003) and diuretic therapy (OR 1.94, 95% CI 1.08–3.48; P 0.027). Hypokalemia, adjusted for sex, age and SOFA score, was not associated with ICU transfer (OR 0.52; 95% CI 0.228–1.212; P = 0.131), in-hospital mortality (OR, 0.47; 95% CI 0.170–1.324; P = 0.154) and composite outcome of ICU transfer or in-hospital mortality (OR 0.48; 95% CI 0.222–1.047; P = 0.065) in our cohort of patients. Conclusions: Hypokalemia was a frequent disorder in subjects with COVID-19. Female sex and diuretic therapy were identified as risk factors for low serum potassium levels. Hypokalemia was unrelated to ICU transfer and death in this cohort of patients.

2021 - Identification and characterization of a SARS-CoV-2 specific CD8+ T cell response with immunodominant features [Articolo su rivista]
Gangaev, Anastasia; Ketelaars, Steven L C; Isaeva, Olga I; Patiwael, Sanne; Dopler, Anna; Hoefakker, Kelly; De Biasi, Sara; Gibellini, Lara; Mussini, Cristina; Guaraldi, Giovanni; Girardis, Massimo; Ormeno, Cami M P Talavera; Hekking, Paul J M; Lardy, Neubury M; Toebes, Mireille; Balderas, Robert; Schumacher, Ton N; Ovaa, Huib; Cossarizza, Andrea; Kvistborg, Pia
abstract

The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8+ T cells. Here, we analyze samples from 31 patients with COVID-19 for CD8+ T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8+ T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8+ T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8+ T cell responses are detectable up to 5 months after recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8+ T cells during convalescence.

2021 - Mapping the human genetic architecture of COVID-19 [Articolo su rivista]
Niemi, M. E. K.; Karjalainen, J.; Liao, R. G.; Neale, B. M.; Daly, M.; Ganna, A.; Pathak, G. A.; Andrews, S. J.; Kanai, M.; Veerapen, K.; Fernandez-Cadenas, I.; Schulte, E. C.; Striano, P.; Marttila, M.; Minica, C.; Marouli, E.; Karim, M. A.; Wendt, F. R.; Savage, J.; Sloofman, L.; Butler-Laporte, G.; Kim, H. -N.; Kanoni, S.; Okada, Y.; Byun, J.; Han, Y.; Uddin, M. J.; Smith, G. D.; Willer, C. J.; Buxbaum, J. D.; Mehtonen, J.; Finucane, H.; Cordioli, M.; Martin, A. R.; Zhou, W.; Pasaniuc, B.; Julienne, H.; Aschard, H.; Shi, H.; Yengo, L.; Polimanti, R.; Ghoussaini, M.; Schwartzentruber, J.; Dunham, I.; Chwialkowska, K.; Francescatto, M.; Trankiem, A.; Balaconis, M. K.; Davis, L.; Lee, S.; Priest, J.; Renieri, A.; Sankaran, V. G.; van Heel, D.; Deelen, P.; Brent Richards, J.; Nakanishi, T.; Biesecker, L.; Eric Kerchberger, V.; Kenneth Baillie, J.; Mari, F.; Bernasconi, A.; Ceri, S.; Canakoglu, A.; Wolford, B.; Faucon, A.; Dutta, A. K.; Schurmann, C.; Harry, E.; Birney, E.; Nguyen, H.; Nasir, J.; Kaunisto, M.; Solomonson, M.; Dueker, N.; Vadgama, N.; Limou, S.; Rahmouni, S.; Mbarek, H.; Darwish, D.; Uddin, M. M.; Albertos, R.; Perez-Tur, J.; Li, R.; Folkersen, L.; Moltke, I.; Koelling, N.; Teumer, A.; Kousathanas, A.; Utrilla, A.; Verdugo, R. A.; Zarate, R.; Medina-Gomez, C.; Gomez-Cabrero, D.; Carnero-Montoro, E.; Cadilla, C. L.; Moreno-Estrada, A.; Garmendia, A.; Moya, L.; Sedaghati-Khayat, B.; Boua, P. R.; Fave, M. -J.; Francioli, L.; Lemacon, A.; Migeotte, I.; Patel, S.; Varnai, R.; Szentpeteri, J. L.; Sipeky, C.; Colombo, F.; von Hohenstaufen, K.; Lio, P.; Vallerga, C.; Wang, Q.; Tanigawa, Y.; Im, H.; Han, C.; Song, H.; Lim, J.; Lee, Y.; Kim, S.; Im, S.; Atanasovska, B.; Ahmad, H. F.; Boer, C.; Jansen, P.; Kaja, E.; Pasko, D.; Kennis-Szilagyi, I.; Kornilov, S. A.; Prijatelj, V.; Prokic, I.; Sivanadhan, I.; Perumal, S.; Esmaeeli, S.; Pearson, N. M.; Auton, A.; Shelton, J. F.; Shastri, A. J.; Filshtein-Sonmez, T.; Coker, D.; Symons, A.; Esparza-Gordillo, J.; Aslibekyan, S.; O'Connell, J.; Ye, C.; Weldon, C. H.; Perera, M.; O'Leary, K.; Tuck, M.; O'Brien, T.; Meltzer, D.; O'Donnell, P.; Nutescu, E.; Yang, G.; Alarcon, C.; Herrmann, S.; Mazurek, S.; Banagan, J.; Hamidi, Z.; Barbour, A.; Raffat, N.; Moreno, D.; Friedman, P.; Ferwerda, B.; van de Beek, D.; Brouwer, M. C.; Vlaar, A. P. J.; Joost Wiersinga, W.; Posthuma, D.; Tissink, E.; Koos Zwinderman, A. H.; Uffelmann, E.; van Agtmael, M.; Algera, A. G.; van Baarle, F.; Bax, D.; Beudel, M.; Bogaard, H. J.; Bomers, M.; Bonta, P. I.; Bos, L.; Botta, M.; de Brabander, J.; de Bree, G.; de Bruin, S.; Bugiani, M.; Bulle, E.; Chouchane, O.; Cloherty, A.; Dongelmans, D.; Elbers, P.; Fleuren, L.; Geerlings, S.; Geerts, B.; Geijtenbeek, T.; Girbes, A.; Goorhuis, B.; Grobusch, M. P.; Hafkamp, F.; Hagens, L.; Hamann, J.; Harris, V.; Hemke, R.; Hermans, S. M.; Heunks, L.; Hollmann, M.; Horn, J.; Hovius, J. W.; de Jong, M. D.; Koning, R.; van Mourik, N.; Nellen, J.; Nossent, E. J.; Paulus, F.; Peters, E.; van der Poll, T.; Preckel, B.; Prins, J. M.; Raasveld, J.; Reijnders, T.; Schinkel, M.; Schultz, M. J.; Schuurman, A.; Sigaloff, K.; Smit, M.; Stijnis, C. S.; Stilma, W.; Teunissen, C.; Thoral, P.; Tsonas, A.; van der Valk, M.; Veelo, D.; de Vries, H.; van Vugt, M.; Wouters, D.; Minnaar, R. P.; Kromhout, A.; van Uffelen, K. W. J.; Wolterman, R. A.; Roberts, G.; Park, D.; Ball, C. A.; Coignet, M.; Mccurdy, S.; Knight, S.; Partha, R.; Rhead, B.; Zhang, M.; Berkowitz, N.; Gaddis, M.; Noto, K.; Ruiz, L.; Pavlovic, M.; Hong, E. L.; Rand, K.; Girshick, A.; Guturu, H.; Baltzell, A. H.; Guntz, J.; Beguin, Y.; Pigazzini, S.; Nkambule, L.; Bouysran, Y.; Busson, A.; Peyrassol, X.; Wilkin, F.; Pichon, B.; Smits, G.; Vandernoot, I.; Goffard, J. -C.; Georges, M.; Moutschen, M.; Misset, B.; Darcis, G.; Guiot, J.; Jadot, L.; Azarzar, S.; Dellot, P.; Gofflot, S.; Claassen, S.; Bertrand, A.; Parzibut, G.; Clarinval, M.; Moermans, C.; Malaise, O.; El Kandoussi, K.; Thonon, R.; Huynen, P.; Mesdagh, A.; Melo, S.
abstract

The genetic make-up of an individual contributes to the susceptibility and response to viral infection. Although environmental, clinical and social factors have a role in the chance of exposure to SARS-CoV-2 and the severity of COVID-191,2, host genetics may also be important. Identifying host-specific genetic factors may reveal biological mechanisms of therapeutic relevance and clarify causal relationships of modifiable environmental risk factors for SARS-CoV-2 infection and outcomes. We formed a global network of researchers to investigate the role of human genetics in SARS-CoV-2 infection and COVID-19 severity. Here we describe the results of three genome-wide association meta-analyses that consist of up to 49,562 patients with COVID-19 from 46 studies across 19 countries. We report 13 genome-wide significant loci that are associated with SARS-CoV-2 infection or severe manifestations of COVID-19. Several of these loci correspond to previously documented associations to lung or autoimmune and inflammatory diseases3–7. They also represent potentially actionable mechanisms in response to infection. Mendelian randomization analyses support a causal role for smoking and body-mass index for severe COVID-19 although not for type II diabetes. The identification of novel host genetic factors associated with COVID-19 was made possible by the community of human genetics researchers coming together to prioritize the sharing of data, results, resources and analytical frameworks. This working model of international collaboration underscores what is possible for future genetic discoveries in emerging pandemics, or indeed for any complex human disease.

2021 - Measles-induced immune amnesia likely recorded in the 18th century [Articolo su rivista]
Galassi, F. M.; Varotto, E.; Mussini, C.; Cossarizza, A.
abstract

2021 - Methylprednisolone as rescue therapy after tocilizumab failure in patients with severe COVID-19 pneumonia [Articolo su rivista]
Guaraldi, Giovanni; Banchelli, Federico; Milic, Jovana; Dolci, Giovanni; Massari, Marco; Corsini, Romina; Meschiari, Marianna; Girardis, Massimo; Busani, Stefano; Cossarizza, Andrea; Salvarani, Carlo; Mussini, Cristina; D'Amico, Roberto
abstract

2021 - Mitochondrial DNA and exercise: Implications for health and injuries in sports [Articolo su rivista]
Zanini, G.; De Gaetano, A.; Selleri, V.; Savino, G.; Cossarizza, A.; Pinti, M.; Mattioli, A. V.; Nasi, M.
abstract

Recently, several studies have highlighted the tight connection between mitochondria and physical activity. Mitochondrial functions are important in high-demanding metabolic activities, such as endurance sports. Moreover, regular training positively affects metabolic health by increasing mitochondrial oxidative capacity and regulating glucose metabolism. Exercise could have multiple effects, also on the mitochondrial DNA (mtDNA) and vice versa; some studies have investigated how mtDNA polymorphisms can affect the performance of general athletes and mtDNA haplogroups seem to be related to the performance of elite endurance athletes. Along with several stimuli, including pathogens, stress, trauma, and reactive oxygen species, acute and intense exercise also seem to be responsible for mtDNA release into the cytoplasm and extracellular space, leading to the activation of the innate immune response. In addition, several sports are characterized by a higher frequency of injuries, including cranial trauma, associated with neurological consequences. However, with regular exercise, circulating cell-free mtDNA levels are kept low, perhaps promoting cf-mtDNA removal, acting as a protective factor against inflammation.

2021 - Mitochondrial toxicity induced by plant molecules [Capitolo/Saggio]
De Gaetano, Anna; Solodka, Kateryna; Cossarizza, Andrea; Pinti, Marcello
abstract

2021 - Mitophagy and oxidative stress: The role of aging [Articolo su rivista]
De Gaetano, A.; Gibellini, L.; Zanini, G.; Nasi, M.; Cossarizza, A.; Pinti, M.
abstract

Mitochondrial dysfunction is a hallmark of aging. Dysfunctional mitochondria are recognized and degraded by a selective type of macroautophagy, named mitophagy. One of the main factors contributing to aging is oxidative stress, and one of the early responses to excessive reactive oxygen species (ROS) production is the induction of mitophagy to remove damaged mitochondria. However, mitochondrial damage caused at least in part by chronic oxidative stress can accumulate, and autophagic and mitophagic pathways can become overwhelmed. The imbalance of the delicate equilibrium among mitophagy, ROS production and mitochondrial damage can start, drive, or accelerate the aging process, either in physiological aging, or in pathological age-related conditions, such as Alzheimer’s and Parkinson’s diseases. It remains to be determined which is the prime mover of this imbalance, i.e., whether it is the mitochondrial damage caused by ROS that initiates the dysregulation of mitophagy, thus activating a vicious circle that leads to the reduced ability to remove damaged mitochondria, or an alteration in the regulation of mitophagy leading to the excessive production of ROS by damaged mitochondria.

2021 - Modulation of tregs and inkt by fingolimod in multiple sclerosis patients [Articolo su rivista]
Ferraro, D.; De Biasi, S.; Simone, A. M.; Orlandi, R.; Nasi, M.; Vitetta, F.; Pinti, M.; Fogliani, M.; Meletti, S.; Cossarizza, A.; Sola, P.
abstract

The altered numbers and functions of cells belonging to immunoregulatory cell networks such as T regulatory (Tregs) and invariant Natural Killer T (iNKT) cells have been reported in Multiple Sclerosis (MS), an immune-mediated disease. We aimed to assess the frequencies of Tregs and iNKT cells in MS patients throughout a one-year treatment with fingolimod (FTY) and to correlate immunological data with efficacy and safety data. The percentage of Tregs (defined as Live Dead-CD3 + CD4 + FoxP3 + CD25++/CD127− cells) increased steadily throughout the year, while there was no significant difference in the absolute number or percentage of iNKT cells (defined as CD3 + CD14−CD19− Vα24-Jα18 TCR+ cells). However, out of all the iNKT cells, the CD8+ iNKT and CD4−CD8− double-negative (DN) cell percentages steadily increased, while the CD4+ iNKT cell percentages decreased significantly. The mean percentage of CD8+ T cells at all time-points was lower in patients with infections throughout the study. The numbers and percentages of DN iNKT cells were more elevated, considering all time-points, in patients who presented a clinical relapse. FTY may, therefore, exert its beneficial effect in MS patients through various mechanisms, including the increase in Tregs and in iNKT subsets with immunomodulatory potential such as CD8+ iNKT cells. The occurrence of infections was associated with lower mean CD8+ cell counts during treatment with FTY.

2021 - Monocyte Distribution Width (MDW) as novel inflammatory marker with prognostic significance in COVID-19 patients [Articolo su rivista]
Riva, G.; Castellano, S.; Nasillo, V.; Ottomano, A. M.; Bergonzini, G.; Paolini, A.; Lusenti, B.; Milic, J.; De Biasi, S.; Gibellini, L.; Cossarizza, A.; Busani, S.; Girardis, M.; Guaraldi, G.; Mussini, C.; Manfredini, R.; Luppi, M.; Tagliafico, E.; Trenti, T.
abstract

Monocyte Distribution Width (MDW), a new cytometric parameter correlating with cytomorphologic changes occurring upon massive monocyte activation, has recently emerged as promising early biomarker of sepsis. Similar to sepsis, monocyte/macrophage subsets are considered key mediators of the life-threatening hyper-inflammatory disorder characterizing severe COVID-19. In this study, we longitudinally analyzed MDW values in a cohort of 87 COVID-19 patients consecutively admitted to our hospital, showing significant correlations between MDW and common inflammatory markers, namely CRP (p < 0.001), fibrinogen (p < 0.001) and ferritin (p < 0.01). Moreover, high MDW values resulted to be prognostically associated with fatal outcome in COVID-19 patients (AUC = 0.76, 95% CI: 0.66–0.87, sensitivity 0.75, specificity 0.70, MDW threshold 26.4; RR = 4.91, 95% CI: 1.73–13.96; OR = 7.14, 95% CI: 2.06–24.71). This pilot study shows that MDW can be useful in the monitoring of COVID-19 patients, as this innovative hematologic biomarker is: (1) easy to obtain, (2) directly related to the activation state of a fundamental inflammatory cell subset (i.e. monocytes, pivotal in both cytokine storm and sepsis immunopathogenesis), (3) well correlated with clinical severity of COVID-19-associated inflammatory disorder, and, in turn, (4) endowed with relevant prognostic significance. Additional studies are needed to define further the clinical impact of MDW testing in the management of COVID-19 patients.

2021 - NLRP3 and IL-1β gene expression is elevated in monocytes from HIV treated patients with neurocognitive disorders [Articolo su rivista]
Mazaheri-Tehrani, Elham; Mohraz, Minoo; Nasi, Milena; Chester, Johanna; De Gaetano, Anna; Lo Tartaro, Domenico; Seyedalinaghi, Seyedahmad; Gholami, Mohammad; De Biasi, Sara; Gibellini, Lara; Mattioli, Anna Vittoria; Pinti, Marcello; Mussini, Cristina; Cossarizza, Andrea
abstract

Systemic immune activation and inflammation in chronic HIV infection are driving factors of non-AIDS related events, including neurocognitive impairment. The role of inflammasome in monocytes from patients with HIV infection has been extensively studied but its association with the extent of neurocognitive dysfunction has been poorly investigated.

2021 - One-year persistence of neutralizing anti-SARS-CoV-2 antibodies in dialysis patients recovered from COVID-19 [Articolo su rivista]
Alfano, Gaetano; Fontana, Francesco; Morisi, Niccolò; Giaroni, Francesco; Mori, Giacomo; Guaraldi, Giovanni; Magistroni, Riccardo; Cappelli, Gianni; Cossarizza, Andrea; Gibellini, Lara
abstract

The immunological mechanisms that modulate immune response to SARS-CoV-2 infection remain elusive. Little is known on the magnitude and the durability of antibody response against COVID-19. There is consensus that patients with immune dysfunction, such as dialysis patients, may be unable to mount a robust and durable humoral immunity after infections. Recent studies showed that dialysis patients seroconverted after COVID-19, but data on the durability of the immune response are missing. We reported the data of a durable anti-spike protein seroconversion after natural SARS-CoV-2 infection in three patients on hemodialysis with a mean age of 67.2 +/- 13.8 years. A mean antibody titer of 212.6 +/- 174.9 UA/ml (Liaison (R), DiaSorin) was found after one year (range, 366-374 days) from the diagnosis of COVID-19. In conclusion, this case series provided evidence that patients receiving hemodialysis who recovered from severe COVID-19 were able to mount a long-lasting immune response against SARS-CoV-2. Although the protective capacity of this long-term immunity remains to be determined, these patients did not report signs of reinfection after recovery from COVID-19.

2021 - Protective role of a tmprss2 variant on severe covid-19 outcome in young males and elderly women [Articolo su rivista]
Monticelli, M.; Mele, B. H.; Benetti, E.; Fallerini, C.; Baldassarri, M.; Furini, S.; Frullanti, E.; Mari, F.; Andreotti, G.; Cubellis, M. V.; Renieri, A.; Daga, S.; Fava, F.; Valentino, F.; Doddato, G.; Giliberti, A.; Tita, R.; Amitrano, S.; Bruttini, M.; Croci, S.; Meloni, I.; Pinto, A. M.; Mencarelli, M. A.; Rizzo, C. L.; Montagnani, F.; Sarno, L. D.; Tommasi, A.; Palmieri, M.; Carriero, M. L.; Alaverdian, D.; Beligni, G.; Iuso, N.; Inchingolo, G.; Fabbiani, M.; Rossetti, B.; Zanelli, G.; Bargagli, E.; Bergantini, L.; D'Alessandro, M.; Cameli, P.; Bennet, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Guerrini, S.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Spertilli, C.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Spagnesi, M.; Piacentini, P.; Bandini, M.; Desanctis, E.; Cappelli, S.; Canaccini, A.; Verzuri, A.; Anemoli, V.; Ognibene, A.; Pancrazi, A.; Lorusso, M.; Vaghi, M.; D'Arminio Monforte, A.; Merlini, E.; Miraglia, F. G.; Mondelli, M. U.; Bruno, R.; Marco, V.; Mantovani, S.; Ludovisi, S.; Girardi, M.; Venturelli, S.; Sita, M.; Cossarizza, A.; Antinori, A.; Vergori, A.; Emiliozzi, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Riva, A.; Francisci, D.; Schiarol, E.; Paciosi, F.; Scotton, P. G.; Andretta, F.; Panese, S.; Scaggiante, R.; Gatti, F.; Parisi, S. G.; Castelli, F.; Quiros-Roldan, E.; Antoni, M. D.; Zanella, I.; Monica, M. D.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Fiorentino, G.; Carella, M.; Castori, M.; Merla, G.; Squeo, G. M.; Aucella, F.; Raggi, P.; Marciano, C.; Perna, R.; Bassetti, M.; Biagio, A. D.; Sanguinetti, M.; Masucci, L.; Valente, S.; Mandala, M.; Giorli, A.; Salerni, L.; Zucchi, P.; Parravicini, P.; Menatti, E.; Baratti, S.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Coviello, D. A.; Mussini, C.; Bosio, G.; Martinelli, E.; Mancarella, S.; Tavecchia, L.; Crotti, L.; Parati, G. -F.; Picchiotti, N.; Gori, M.; Gabbi, C.; Sanarico, M.; Ceri, S.; Pinoli, P.; Raimondi, F.; Bis-Carini, F.; Stella, A.; Rizzi, M.; Maggiolo, F.; Ripamonti, D.; Suardi, C.; Bachetti, T.; Rovere, M. T. L.; Sarzi-Braga, S.; Bussotti, M.; Bergomi, M.; Capitani, K.; Zguro, K.; Dei, S.
abstract

The protease encoded by the TMPRSS2 gene facilitates viral infections and has been implicated in the pathogenesis of SARS-CoV-2. We analyzed the TMPRSS2 sequence and correlated the protein variants with the clinical features of a cohort of 1177 patients affected by COVID-19 in Italy. Nine relatively common variants (allele frequency > 0.01) and six missense variants which may affect the protease activity according to PolyPhen-2 in HumVar-trained mode were identified. Among them, p.V197M (p.Val197Met) (rs12329760) emerges as a common variant that has a deleterious effect on the protease and a protective effect on the patients. Its role appears particularly relevant in two subgroups of patients—young males and elderly women—and among those affected by co-morbidities, where the variant frequency is higher among individuals who were mildly affected by the disease and did not need hospitalization or oxygen therapy than among those more severely affected, who required oxygen therapy, ventilation or intubation. This study provides useful information for the identification of patients at risk of developing a severe form of COVID-19, and encourages the usage of drugs affecting the expression of TMPRSS2 or inhibiting protein activity.

2021 - Rare variants in Toll-like receptor 7 results in functional impairment and downregulation of cytokine-mediated signaling in COVID-19 patients [Articolo su rivista]
Mantovani, S.; Daga, S.; Fallerini, C.; Baldassarri, M.; Benetti, E.; Picchiotti, N.; Fava, F.; Galli, A.; Zibellini, S.; Bruttini, M.; Palmieri, M.; Croci, S.; Amitrano, S.; Alaverdian, D.; Capitani, K.; Furini, S.; Mari, F.; Meloni, I.; Montagnani, F.; Tumbarello, M.; Rancan, I.; Fabbiani, M.; Rossetti, B.; Bergantini, L.; D'Alessandro, M.; Cameli, P.; Bennett, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Guerrini, S.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Raffaelli, C. S.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Romani, D.; Piacentini, P.; Bandini, M.; Desanctis, E.; Cappelli, S.; Canaccini, A.; Verzuri, A.; Anemoli, V.; Pisani, M.; Ognibene, A.; Pancrazzi, A.; Lorubbio, M.; Vaghi, M.; D'Arminio Monforte, A.; Miraglia, F. G.; Bruno, R.; Vecchia, M.; Girardis, M.; Venturelli, S.; Busani, S.; Cossarizza, A.; Antinori, A.; Vergori, A.; Emiliozzi, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Riva, A.; Francisci, D.; Schiaroli, E.; Paciosi, F.; Tommasi, A.; Scotton, P. G.; Andretta, F.; Panese, S.; Baratti, S.; Scaggiante, R.; Gatti, F.; Parisi, S. G.; Castelli, F.; Quiros-Roldan, E.; Antoni, M. D.; Zanella, I.; Della Monica, M.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Fiorentino, G.; Carella, M.; Castori, M.; Aucella, F.; Raggi, P.; Perna, R.; Bassetti, M.; Di Biagio, A.; Sanguinetti, M.; Masucci, L.; Guarnaccia, A.; Valente, S.; De Vivo, O.; Doddato, G.; Lista, M.; Beligni, G.; Valentino, F.; Zguro, K.; Tita, R.; Giliberti, A.; Mencarelli, M. A.; Rizzo, C. L.; Pinto, A. M.; Ariani, F.; Di Sarno, L.; Bargagli, E.; Mandala, M.; Giorli, A.; Salerni, L.; Zucchi, P.; Parravicini, P.; Menatti, E.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Lacerenza, G.; Mussini, C.; Martinelli, E.; Tavecchia, L.; Belli, M. A.; Crotti, L.; Parati, G.; Sanarico, M.; Raimondi, F.; Biscarini, F.; Stella, A.; Bachetti, T.; La Rovere, M. T.; Ludovisi, S.; Bussotti, M.; Dei, S.; Ravaglia, S.; Artuso, R.; Andreucci, E.; Gori, G.; Pagliazzi, A.; Fiorentini, E.; Perrella, A.; Bianchi, F.; Bergomi, P.; Catena, E.; Colombo, R.; Luchi, S.; Morelli, G.; Petrocelli, P.; Iacopini, S.; Modica, S.; Baroni, S.; Segala, F. V.; Falcone, M.; Tiseo, G.; Barbieri, C.; Matucci, T.; Grassi, D.; Ferri, C.; Marinangeli, F.; Brancati, F.; Vincenti, A.; Borgo, V.; Lombardi, S.; Lenzi, M.; Di Pietro, M. A.; Vichi, F.; Romanin, B.; Attala, L.; Costa, C.; Gabbuti, A.; Mene, R.; Colaneri, M.; Casprini, P.; Merla, G.; Squeo, G. M.; Maffezzoni, M.; Frullanti, E.; Mondelli, M. U.; Renieri, A.
abstract

Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.

2021 - SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues [Articolo su rivista]
D'Antonio, M.; Nguyen, J. P.; Arthur, T. D.; Matsui, H.; D'Antonio-Chronowska, A.; Frazer, K. A.; Neale, B. M.; Daly, M.; Ganna, A.; Stevens, C.; Pathak, G. A.; Andrews, S. J.; Kanai, M.; Cordioli, M.; Ganna, A.; Karjalainen, J.; Pathak, G. A.; Polimanti, R.; Andrews, S. J.; Cordioli, M.; Pirinen, M.; Kanai, M.; Harerimana, N.; Veerapen, K.; Wolford, B.; Nguyen, H.; Solomonson, M.; Stevens, C.; Liao, R. G.; Chwialkowska, K.; Trankiem, A.; Balaconis, M. K.; Hayward, C.; Richmond, A.; Campbell, A.; Morris, M.; Fawns-Ritchie, C.; Glessner, J. T.; Shaw, D. M.; Chang, X.; Polikowski, H.; Lauren, P. E.; Chen, H. -H.; Wanying, Z.; Hakonarson, H.; Porteous, D. J.; Below, J.; North, K.; Mccormick, J. B.; Timmers, P. R. H. J.; Wilson, J. F.; Tenesa, A.; D'Mellow, K.; Kerr, S. M.; Niemi, M. E. K.; Cordioli, M.; Nkambul, L.; von Hohenstaufen, K. A.; Sobh, A.; Eltoukhy, M. M.; Yassen, A. M.; Hegazy, M. A. F.; Okasha, K.; Eid, M. A.; Moahmed, H. S.; Shahin, D.; El-Sherbiny, Y. M.; Elhadidy, T. A.; Abd Elghafar, M. S.; El-Jawhari, J. J.; Mohamed, A. A. S.; Elnagdy, M. H.; Samir, A.; Abdel-Aziz, M.; Khafaga, W. T.; El-Lawaty, W. M.; Torky, M. S.; El-Shanshory, M. R.; Batini, C.; Lee, P. H.; Shrine, N.; Williams, A. T.; Tobin, M. D.; Guyatt, A. L.; John, C.; Packer, R. J.; Ali, A.; Free, R. C.; Wang, X.; Wain, L. V.; Hollox, E. J.; Venn, L. D.; Bee, C. E.; Adams, E. L.; Niemi, M. E. K.; Niavarani, A.; Cordioli, M.; Nkambul, L.; Sharififard, B.; Aliannejad, R.; Amirsavadkouhi, A.; Naderpour, Z.; Tadi, H. A.; Aleagha, A. E.; Ahmadi, S.; Moghaddam, S. B. M.; Adamsara, A.; Saeedi, M.; Abdollahi, H.; Hosseini, A.; Chariyavilaskul, P.; Chamnanphon, M.; Suttichet, T. B.; Shotelersuk, V.; Pongpanich, M.; Phokaew, C.; Chetruengchai, W.; Jantarabenjakul, W.; Putchareon, O.; Torvorapanit, P.; Puthanakit, T.; Suchartlikitwong, P.; Hirankarn, N.; Nilaratanakul, V.; Sodsai, P.; Brumpton, B. M.; Hveem, K.; Willer, C.; Wolford, B.; Zhou, W.; Rogne, T.; Solligard, E.; Asvold, B. O.; Abedalthagafi, M.; Alaamery, M.; Alqahtani, S.; Baraka, D.; Al Harthi, F.; Alsolm, E.; Safieh, L. A.; Alowayn, A. M.; Alqubaishi, F.; Al Mutairi, A.; Mangul, S.; Alshareef, A.; Sawaji, M.; Almutairi, M.; Aljawini, N.; Albesher, N.; Arabi, Y. M.; Mahmoud, E. S.; Khattab, A. K.; Halawani, R. T.; Alahmadey, Z. Z.; Albakri, J. K.; Felemban, W. A.; Suliman, B. A.; Hasanato, R.; Al-Awdah, L.; Alghamdi, J.; Alzahrani, D.; Aljohani, S.; Al-Afghani, H.; Alrashed, M.; Aldhawi, N.; Albardis, H.; Alkwai, S.; Alswailm, M.; Almalki, F.; Albeladi, M.; Almohammed, I.; Barhoush, E.; Albader, A.; Massadeh, S.; Almalik, A.; Alotaibi, S.; Alghamdi, B.; Jung, J.; Fawzy, M. S.; Lee, Y.; Magnus, P.; Trogstad, L. -I. S.; Helgeland, O.; Harris, J. R.; Mangino, M.; Spector, T. D.; Emma, D.; Smieszek, S. P.; Przychodzen, B. P.; Polymeropoulos, C.; Polymeropoulos, V.; Polymeropoulos, M. H.; Fernandez-Cadenas, I.; Perez-Tur, J.; Llucia-Carol, L.; Cullell, N.; Muino, E.; Carcel-Marquez, J.; Dediego, M. L.; Iglesias, L. L.; Planas, A. M.; Soriano, A.; Rico, V.; Aguero, D.; Bedini, J. L.; Lozano, F.; Domingo, C.; Robles, V.; Ruiz-Jaen, F.; Marquez, L.; Gomez, J.; Coto, E.; Albaiceta, G. M.; Garcia-Clemente, M.; Dalmau, D.; Arranz, M. J.; Dietl, B.; Serra-Llovich, A.; Soler, P.; Colobran, R.; Martin-Nalda, A.; Martinez, A. P.; Bernardo, D.; Rojo, S.; Fiz-Lopez, A.; Arribas, E.; de la Cal-Sabater, P.; Segura, T.; Gonzalez-Villa, E.; Serrano-Heras, G.; Marti-Fabregas, J.; Jimenez-Xarrie, E.; de Felipe Mimbrera, A.; Masjuan, J.; Garcia-Madrona, S.; Dominguez-Mayoral, A.; Villalonga, J. M.; Menendez-Valladares, P.; Chasman, D. I.; Buring, J. E.; Ridker, P. M.; Franco, G.; Sesso, H. D.; Manson, J. E.; Chang, X.; Glessner, J. R.; Hakonarson, H.; Hayward, C.; Richmond, A.; Porteous, D. J.; Campbell, A.; Fawns-Ritchie, C.; Medina-Gomez, C.; Uitterlinden, A. G.; Arfan Ikram, M.; Kristiansson, K.; Koskelainen, S.; Perola, M.; Donner, K.; Kivinen, K.; Palotie, A.; Ripatti, S.; Ruotsalainen, S.; Kaunisto, M.; Nakanishi, T.; B
abstract

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types.

2021 - Sensing Inflammation Biomarkers with Electrolyte-Gated Organic Electronic Transistors [Articolo su rivista]
Burtscher, B.; Manco Urbina, P. A.; Diacci, C.; Borghi, S.; Pinti, M.; Cossarizza, A.; Salvarani, C.; Berggren, M.; Biscarini, F.; Simon, D. T.; Bortolotti, C. A.
abstract

An overview of cytokine biosensing is provided, with a focus on the opportunities provided by organic electronic platforms for monitoring these inflammation biomarkers which manifest at ultralow concentration levels in physiopathological conditions. Specifically, two of the field's state-of-the-art technologies—organic electrochemical transistors (OECTs) and electrolyte gated organic field effect transistors (EGOFETs)—and their use in sensing cytokines and other proteins associated with inflammation are a particular focus. The overview will include an introduction to current clinical and “gold standard” quantification techniques and their limitations in terms of cost, time, and required infrastructure. A critical review of recent progress with OECT- and EGOFET-based protein biosensors is presented, alongside a discussion onthe future of these technologies in the years and decades ahead. This is especially timely as the world grapples with limited healthcare diagnostics during the Coronavirus disease (COVID-19)pandemic where one of the worst-case scenarios for patients is the “cytokine storm.” Clearly, low-cost point-of-care technologies provided by OECTs and EGOFETs can ease the global burden on healthcare systems and support professionals by providing unprecedented wealth of data that can help to monitor disease progression in real time.

2021 - Sex Difference in Access to Sports: A 1-Year Retrospective Study [Articolo su rivista]
Nasi, M.; D'Alisera, R.; Cossarizza, A.; Guerra, E.; Savino, G.; Mattioli, A. V.
abstract

Purpose. Regular physical activity is a cornerstone in the prevention and treatment of cardiovascular disease thanks to its anti-inflammatory effects. Thus, favoring the access to sports is of importance for promoting well-being. The aim of the present study was to investigate how the practice of different sports is distributed among different age groups and between men and women, by taking a picture of the medical certificate request in 2017 for sports in the population of the province of Modena, Italy. Methods. We analyzed the difference in distribution of requested medical certificate from 18 874 males and 7625 females stratified for age (<18 years, 18-40 years, and >40 years) and for different sporting disciplines (athletics, football, bike, swimming, basketball, volleyball, tennis, other team sports, other individual sports, and disabled sports). Results. Men requested medical certificates more than women (more than 2.5 times). The distribution of requested certificates differs significantly (chi-square test P <.0001) at different ages and between males and females of same age. Certificate for men aged less than 18 years were 7550 and for women were 4131 and the difference increase with age. Conclusions. In order to decrease the imbalance between men and women access to sports, it is mandatory to promote a healthy lifestyle and reduce, as consequence, cardiovascular risks, mostly in women after 40 years.

2021 - Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males [Articolo su rivista]
Baldassarri, M.; Picchiotti, N.; Fava, F.; Fallerini, C.; Benetti, E.; Daga, S.; Valentino, F.; Doddato, G.; Furini, S.; Giliberti, A.; Tita, R.; Amitrano, S.; Bruttini, M.; Croci, S.; Meloni, I.; Pinto, A. M.; Iuso, N.; Gabbi, C.; Sciarra, F.; Venneri, M. A.; Gori, M.; Sanarico, M.; Crawley, F. P.; Pagotto, U.; Fanelli, F.; Mezzullo, M.; Dominguez-Garrido, E.; Planas-Serra, L.; Schluter, A.; Colobran, R.; Soler-Palacin, P.; Lapunzina, P.; Tenorio, J.; Pujol, A.; Castagna, M. G.; Marcelli, M.; Isidori, A. M.; Renieri, A.; Frullanti, E.; Mari, F.; Montagnani, F.; Di Sarno, L.; Tommasi, A.; Palmieri, M.; Fabbiani, M.; Rossetti, B.; Zanelli, G.; Sestini, F.; Bergantini, L.; D'Alessandro, M.; Cameli, P.; Bennett, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Guerrini, S.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Spertilli, C.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Spagnesi, M.; Piacentini, P.; Bandini, M.; Desanctis, E.; Cappelli, S.; Canaccini, A.; Verzuri, A.; Anemoli, V.; Ognibene, A.; Vaghi, M.; Monforte, A. D.; Merlini, E.; Miraglia, F. G.; Mondelli, M. U.; Mantovani, S.; Ludovisi, S.; Girardis, M.; Venturelli, S.; Sita, M.; Cossarizza, A.; Antinori, A.; Vergori, A.; Emiliozzi, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Riva, A.; Francisci, D.; Schiaroli, E.; Paciosi, F.; Scotton, P. G.; Andretta, F.; Panese, S.; Baratti, S.; Scaggiante, R.; Gatti, F.; Parisi, S. G.; Castelli, F.; Quiros-Roldan, E.; Antoni, M. D.; Zanella, I.; Monica, M. D.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Fiorentino, G.; Carella, M.; Castori, M.; Merla, G.; Aucella, F.; Raggi, P.; Marciano, C.; Perna, R.; Bassetti, M.; Di Biagio, A.; Sanguinetti, M.; Masucci, L.; Valente, S.; Mencarelli, M. A.; Lo Rizzo, C.; Bargagli, E.; Mandala, M.; Giorli, A.; Salerni, L.; Zucchi, P.; Parravicini, P.; Menatti, E.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Coviello, D. A.; Mussini, C.; Bosio, G.; Martinelli, E.; Mancarella, S.; Tavecchia, L.; Crotti, L.; Parati, G.; Aguilera-Albesa, S.; Albu, S.; Casasnovas, C.; Velez-Santamaria, V.; Horcajada, J. P.; Villar, J.; Rodriguez-Palmero, A.; Ruiz, M.; Seijo, L. M.; Troya, J.; Valencia-Ramos, J.; Gut, M.
abstract

Background: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. Methods: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. Findings: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing). Interpretation: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats. Funding: MIUR project “Dipartimenti di Eccellenza 2018-2020” to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and “Bando Ricerca COVID-19 Toscana” project to Azienda Ospedaliero-Universitaria Senese. Private donors for COVID-19 research and charity funds from Intesa San Paolo.

2021 - The COVID-19 puzzle: deciphering pathophysiology and phenotypes of a new disease entity [Articolo su rivista]
Osuchowski, M. F.; Winkler, M. S.; Skirecki, T.; Cajander, S.; Shankar-Hari, M.; Lachmann, G.; Monneret, G.; Venet, F.; Bauer, M.; Brunkhorst, F. M.; Weis, S.; Garcia-Salido, A.; Kox, M.; Cavaillon, J. -M.; Uhle, F.; Weigand, M. A.; Flohe, S. B.; Wiersinga, W. J.; Almansa, R.; de la Fuente, A.; Martin-Loeches, I.; Meisel, C.; Spinetti, T.; Schefold, J. C.; Cilloniz, C.; Torres, A.; Giamarellos-Bourboulis, E. J.; Ferrer, R.; Girardis, M.; Cossarizza, A.; Netea, M. G.; van der Poll, T.; Bermejo-Martin, J. F.; Rubio, I.
abstract

The zoonotic SARS-CoV-2 virus that causes COVID-19 continues to spread worldwide, with devastating consequences. While the medical community has gained insight into the epidemiology of COVID-19, important questions remain about the clinical complexities and underlying mechanisms of disease phenotypes. Severe COVID-19 most commonly involves respiratory manifestations, although other systems are also affected, and acute disease is often followed by protracted complications. Such complex manifestations suggest that SARS-CoV-2 dysregulates the host response, triggering wide-ranging immuno-inflammatory, thrombotic, and parenchymal derangements. We review the intricacies of COVID-19 pathophysiology, its various phenotypes, and the anti-SARS-CoV-2 host response at the humoral and cellular levels. Some similarities exist between COVID-19 and respiratory failure of other origins, but evidence for many distinctive mechanistic features indicates that COVID-19 constitutes a new disease entity, with emerging data suggesting involvement of an endotheliopathy-centred pathophysiology. Further research, combining basic and clinical studies, is needed to advance understanding of pathophysiological mechanisms and to characterise immuno-inflammatory derangements across the range of phenotypes to enable optimum care for patients with COVID-19.

2021 - The impact of tocilizumab on respiratory support states transition and clinical outcomes in COVID-19 patients. A Markov model multi-state study [Articolo su rivista]
Milic, J.; Banchelli, F.; Meschiari, M.; Franceschini, E.; Ciusa, G.; Gozzi, L.; Volpi, S.; Faltoni, M.; Franceschi, G.; Iadisernia, V.; Yaacoub, D.; Dolci, G.; Bacca, E.; Rogati, C.; Tutone, M.; Burastero, G.; Raimondi, A.; Menozzi, M.; Cuomo, G.; Corradi, L.; Orlando, G.; Santoro, A.; Digaetano, M.; Puzzolante, C.; Carli, F.; Bedini, A.; Busani, S.; Girardis, M.; Cossarizza, A.; Miglio, R.; Mussini, C.; Guaraldi, G.; D'Amico, R.
abstract

Background The benefit of tocilizumab on mortality and time to recovery in people with severe COVID pneumonia may depend on appropriate timing. The objective was to estimate the impact of tocilizumab administration on switching respiratory support states, mortality and time to recovery. Methods In an observational study, a continuous-time Markov multi-state model was used to describe the sequence of respiratory support states including: no respiratory support (NRS), oxygen therapy (OT), non-invasive ventilation (NIV) or invasive mechanical ventilation (IMV), OT in recovery, NRS in recovery. Results Two hundred seventy-one consecutive adult patients were included in the analyses contributing to 695 transitions across states. The prevalence of patients in each respiratory support state was estimated with stack probability plots, comparing people treated with and without tocilizumab since the beginning of the OT state. A positive effect of tocilizumab on the probability of moving from the invasive and non-invasive mechanical NIV/IMV state to the OT in recovery state (HR = 2.6, 95% CI = 1.2–5.2) was observed. Furthermore, a reduced risk of death was observed in patients in NIV/IMV (HR = 0.3, 95% CI = 0.1–0.7) or in OT (HR = 0.1, 95% CI = 0.0–0.8) treated with tocilizumab. Conclusion To conclude, we were able to show the positive impact of tocilizumab used in different disease stages depicted by respiratory support states. The use of the multi-state Markov model allowed to harmonize the heterogeneous mortality and recovery endpoints and summarize results with stack probability plots. This approach could inform randomized clinical trials regarding tocilizumab, support disease management and hospital decision making.

2021 - To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis [Articolo su rivista]
Di Rosa, F.; Cossarizza, A.; Hayday, A. C.
abstract

This study discusses substantive advances in T cell proliferation analysis, with the aim to provoke a re-evaluation of the generally-held view that Ki-67 is a reliable proliferation marker per se, and to offer a more sensitive and effective method for T cell cycle analysis, with informative examples in mouse and human settings. We summarize recent experimental work from our labs showing that, by Ki-67/DNA dual staining and refined flow cytometric methods, we were able to identify T cells in the S-G2/M phases of the cell-cycle in the peripheral blood (collectively termed “T Double S” for T cells in S-phase in Sanguine: in short “TDS” cells). Without our refinement, such cells may be excluded from conventional lymphocyte analyses. Specifically, we analyzed clonal expansion of antigen-specific CD8 T cells in vaccinated mice, and demonstrated the potential of TDS cells to reflect immune dynamics in human blood samples from healthy donors, and patients with type 1 diabetes, infectious mononucleosis, and COVID-19. The Ki-67/DNA dual staining, or TDS assay, provides a reliable approach by which human peripheral blood can be used to reflect the dynamics of human lymphocytes, rather than providing mere steady-state phenotypic snapshots. The method does not require highly sophisticated “-omics” capabilities, so it should be widely-applicable to health care in diverse settings. Furthermore, our results argue that the TDS assay can provide a window on immune dynamics in extra-lymphoid tissues, a long-sought potential of peripheral blood monitoring, for example in relation to organ-specific autoimmune diseases and infections, and cancer immunotherapy.

2020 - ACE2 gene variants may underlie interindividual variability and susceptibility to COVID-19 in the Italian population [Articolo su rivista]
Benetti, E.; Tita, R.; Spiga, O.; Ciolfi, A.; Birolo, G.; Bruselles, A.; Doddato, G.; Giliberti, A.; Marconi, C.; Musacchia, F.; Pippucci, T.; Torella, A.; Trezza, A.; Valentino, F.; Baldassarri, M.; Brusco, A.; Asselta, R.; Bruttini, M.; Furini, S.; Seri, M.; Nigro, V.; Matullo, G.; Tartaglia, M.; Mari, F.; Elisa, F.; Chiara, F.; Sergio, D.; Susanna, C.; Sara, A.; Francesca, F.; Montagnani, F.; Di Sarno, L.; Tommasi, A.; Palmieri, M.; Emiliozzi, A.; Fabbiani, M.; Rossetti, B.; Zanelli, G.; Bergantini, L.; D'Alessandro, M.; Cameli, P.; Bennet, D.; Anedda, F.; Marcantonio, S.; Scolletta, S.; Franchi, F.; Mazzei, M. A.; Conticini, E.; Cantarini, L.; Frediani, B.; Tacconi, D.; Feri, M.; Scala, R.; Spargi, G.; Corridi, M.; Nencioni, C.; Caldarelli, G. P.; Spagnesi, M.; Piacentini, P.; Bandini, M.; Desanctis, E.; Canaccini, A.; Spertilli, C.; Donati, A.; Guidelli, L.; Croci, L.; Verzuri, A.; Anemoli, V.; Ognibene, A.; Vaghi, M.; D'Arminio Monforte, A.; Merlini, E.; Mondelli, M. U.; Mantovani, S.; Ludovisi, S.; Girardis, M.; Venturelli, S.; Sita, M.; Cossarizza, A.; Antinori, A.; Vergori, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Riva, A.; Francisci, D.; Schiaroli, E.; Scotton, P. G.; Andretta, F.; Panese, S.; Scaggiante, R.; Parisi, S. G.; Castelli, F.; Quiros-Roldan, M. E.; Magro, P.; Minardi, C.; Castelli, D.; Polesini, I.; Della Monica, M.; Piscopo, C.; Capasso, M.; Russo, R.; Andolfo, I.; Iolascon, A.; Carella, M.; Castori, M.; Merla, G.; Aucella, F.; Raggi, P.; Marciano, C.; Perna, R.; Bassetti, M.; Di Biagio, A.; Sanguinetti, M.; Masucci, L.; Gabbi, C.; Valente, S.; Guerrini, S.; Meloni, I.; Mencarelli, M. A.; Rizzo, C. L.; Bargagli, E.; Mandala, M.; Giorli, A.; Salerni, L.; Fiorentino, G.; Zucchi, P.; Parravicini, P.; Menatti, E.; Baratti, S.; Trotta, T.; Giannattasio, F.; Coiro, G.; Lena, F.; Coviello, D. A.; Mussini, C.; Renieri, A.; Pinto, A. M.
abstract

In December 2019, an initial cluster of interstitial bilateral pneumonia emerged in Wuhan, China. A human-to-human transmission was assumed and a previously unrecognized entity, termed coronavirus disease-19 (COVID-19) due to a novel coronavirus (SARS-CoV-2) was described. The infection has rapidly spread out all over the world and Italy has been the first European country experiencing the endemic wave with unexpected clinical severity in comparison with Asian countries. It has been shown that SARS-CoV-2 utilizes angiotensin converting enzyme 2 (ACE2) as host receptor and host proteases for cell surface binding and internalization. Thus, a predisposing genetic background can give reason for interindividual disease susceptibility and/or severity. Taking advantage of the Network of Italian Genomes (NIG), here we mined whole-exome sequencing data of 6930 Italian control individuals from five different centers looking for ACE2 variants. A number of variants with a potential impact on protein stability were identified. Among these, three more common missense changes, p.(Asn720Asp), p.(Lys26Arg), and p.(Gly211Arg) were predicted to interfere with protein structure and stabilization. Rare variants likely interfering with the internalization process, namely p.(Leu351Val) and p.(Pro389His), predicted to interfere with SARS-CoV-2 spike protein binding, were also observed. Comparison of ACE2 WES data between a cohort of 131 patients and 258 controls allowed identifying a statistically significant (P value < 0.029) higher allelic variability in controls compared with patients. These findings suggest that a predisposing genetic background may contribute to the observed interindividual clinical variability associated with COVID-19, allowing an evidence-based risk assessment leading to personalized preventive measures and therapeutic options.

2020 - Acute myeloid leukemia in patients living with HIV infection: Several questions, fewer answers [Articolo su rivista]
Forghieri, F.; Nasillo, V.; Bettelli, F.; Pioli, V.; Giusti, D.; Gilioli, A.; Mussini, C.; Tagliafico, E.; Trenti, T.; Cossarizza, A.; Maffei, R.; Barozzi, P.; Potenza, L.; Marasca, R.; Narni, F.; Luppi, M.
abstract

Both human immunodeficiency virus (HIV) infection and acute myeloid leukemia (AML) may be considered relatively uncommon disorders in the general population, but the precise incidence of AML in people living with HIV infection (PLWH) is uncertain. However, life expectancy of newly infected HIV-positive patients receiving anti-retroviral therapy (ART) is gradually increasing, rivaling that of age-matched HIV-negative individuals, so that the occurrence of AML is also expected to progressively increase. Even if HIV is not reported to be directly mutagenic, several indirect leukemogenic mechanisms, mainly based on bone marrow microenvironment disruption, have been proposed. Despite a well-controlled HIV infection under ART should no longer be considered per se a contraindication to intensive chemotherapeutic approaches, including allogeneic hematopoietic stem cell transplantation, in selected fit patients with AML, survival outcomes are still generally unsatisfactory. We discussed several controversial issues about pathogenesis and clinical management of AML in PLWH, but few evidence-based answers may currently be provided, due to the limited number of cases reported in the literature, mainly as case reports or small retrospective case series. Prospective multicenter clinical trials are warranted to more precisely investigate epidemiology and cytogenetic/molecular features of AML in PLWH, but also to standardize and further improve its therapeutic management.

2020 - Altered bioenergetics and mitochondrial dysfunction of monocytes in patients with COVID-19 pneumonia [Articolo su rivista]
Gibellini, L.; De Biasi, S.; Paolini, A.; Borella, R.; Boraldi, F.; Mattioli, M.; Lo Tartaro, D.; Fidanza, L.; Caro-Maldonado, A.; Meschiari, M.; Iadisernia, V.; Bacca, E.; Riva, G.; Cicchetti, L.; Quaglino, D.; Guaraldi, G.; Busani, S.; Girardis, M.; Mussini, C.; Cossarizza, A.
abstract

In patients infected by SARS-CoV-2 who experience an exaggerated inflammation leading to pneumonia, monocytes likely play a major role but have received poor attention. Thus, we analyzed peripheral blood monocytes from patients with COVID-19 pneumonia and found that these cells show signs of altered bioenergetics and mitochondrial dysfunction, had a reduced basal and maximal respiration, reduced spare respiratory capacity, and decreased proton leak. Basal extracellular acidification rate was also diminished, suggesting reduced capability to perform aerobic glycolysis. Although COVID-19 monocytes had a reduced ability to perform oxidative burst, they were still capable of producing TNF and IFN-γ in vitro. A significantly high amount of monocytes had depolarized mitochondria and abnormal mitochondrial ultrastructure. A redistribution of monocyte subsets, with a significant expansion of intermediate/pro-inflammatory cells, and high amounts of immature monocytes were found, along with a concomitant compression of classical monocytes, and an increased expression of inhibitory checkpoints like PD-1/PD-L1. High plasma levels of several inflammatory cytokines and chemokines, including GM-CSF, IL-18, CCL2, CXCL10, and osteopontin, finally confirm the importance of monocytes in COVID-19 immunopathogenesis.

2020 - COVID-19 pandemic: usefulness of telemedicine in management of arrhythmias in elderly people [Articolo su rivista]
Mattioli, Anna Vittoria; Cossarizza, Andrea; Boriani, Giuseppe
abstract

In March 2020, the WHO defined the diffusion of novel coronavirus, Severe Acute Respiratory Syndrome-Coronavirus- 2 (SARS-CoV-2) as pandemic.[13] As a consequence, the Italian Government among others has enforced quarantine on the population to contain the diffusion of the infection.

2020 - COVID-19-associated vasculitis and thrombotic complications: from pathological findings to multidisciplinary discussion [Articolo su rivista]
Vacchi, Caterina; Meschiari, Marianna; Milic, Jovana; Marietta, Marco; Tonelli, Roberto; Alfano, Gaetano; Volpi, Sara; Faltoni, Matteo; Franceschi, Giacomo; Ciusa, Giacomo; Bacca, Erica; Tutone, Marco; Raimondi, Alessandro; Menozzi, Marianna; Franceschini, Erica; Cuomo, Gianluca; Orlando, Gabriella; Santoro, Antonella; Di Gaetano, Margherita; Puzzolante, Cinzia; Carli, Federica; Bedini, Andrea; Cossarizza, Andrea; Castaniere, Ivana; Ligabue, Guido; De Ruvo, Nicola; Manco, Gianrocco; Rolando, Giovanni; Gelmini, Roberta; Maiorana, Antonino; Girardis, Massimo; Mascia, Maria Teresa; Mussini, Cristina; Salvarani, Carlo; Guaraldi, Giovanni
abstract

Neutrophilic arterial vasculitis in COVID-19 represents a novel finding and could be responsible for thrombotic complications.

2020 - Circulating Mitochondrial DNA and Lipopolysaccharide-Binding Protein but Not Bacterial DNA Are Increased in Acute Human Immunodeficiency Virus Infection. [Articolo su rivista]
Nasi, Milena; Pecorini, Simone; DE BIASI, Sara; Digaetano, Margherita; Chester, JOHANNA MARY; Aramini, Beatrice; Lo Tartaro, Domenico; Pinti, Marcello; De Gaetano, Anna; Gibellini, Lara; Mattioli, Anna Vittoria; Mussini, Cristina; Cossarizza, Andrea
abstract

Microbial translocation has been suggested as a major driver of chronic immune activation HIV infection. Thus, we compared the extent of microbial translocation in patients with acute HIV infection and patients followed after CD4-guided structured treatment interruption (STI) by measuring different circulating markers: (1) lipopolysaccharide (LPS)-binding protein (LBP), (2) bacterial DNA, (3) soluble CD14 (sCD14), and (4) mitochondrial DNA (mtDNA). Bacterial DNA and sCD14 levels were similar in all groups. Patients in acute phase showed higher levels of LBP and mtDNA. In STI, we found a positive correlation between the percentage of CD8+ T cells and bacterial DNA levels. Considering all patients, LBP was positively correlated with the percentage and the absolute count of CD8+ T cells, and with mtDNA stressing the importance of mitochondrial products in sustaining chronic immune activation.

2020 - Clinical and molecular characterization of COVID-19 hospitalized patients [Articolo su rivista]
Benetti, E.; Giliberti, A.; Emiliozzi, A.; Valentino, F.; Bergantini, L.; Fallerini, C.; Anedda, F.; Amitrano, S.; Conticini, E.; Tita, R.; D'Alessandro, M.; Fava, F.; Marcantonio, S.; Baldassarri, M.; Bruttini, M.; Mazzei, M. A.; Montagnani, F.; Mandala, M.; Bargagli, E.; Furini, S.; Renieri, A.; Mari, F.; Doddato, G.; Croci, S.; Di Sarno, L.; Tommasi, A.; Daga, S.; Palmieri, M.; Fabbiani, M.; Rossetti, B.; Zanelli, G.; Cameli, P.; Bennett, D.; Scolletta, S.; Franchi, F.; Cantarini, L.; Frediani, B.; Tacconi, D.; Spertilli, C.; Feri, M.; Donati, A.; Scala, R.; Guidelli, L.; Ognibene, A.; Spargi, G.; Corridi, M.; Nencioni, C.; Croci, L.; Caldarelli, G. P.; Spagnesi, M.; Piacentini, P.; Canaccini, A.; Verzuri, A.; Anemoli, V.; Vaghi, M.; Monforte, A. D.; Merlini, E.; Mondelli, M. U.; Mantovani, S.; Ludovisi, S.; Girardis, M.; Venturelli, S.; Cossarizza, A.; Antinori, A.; Vergori, A.; Rusconi, S.; Siano, M.; Gabrieli, A.; Francisci, D.; Schiaroli, E.; Scotton, P. G.; Andretta, F.; Panese, S.; Scaggiante, R.; Parisi, S. G.; Castelli, F.; Roldan, M. E. Q.; Magro, P.; Minardi, C.; della Monica, M.; Piscopo, C.; Capasso, M.; Carella, M.; Castori, M.; Merla, G.; Aucella, F.; Raggi, P.; Bassetti, M.; Di Biagio, A.; Sanguinetti, M.; Masucci, L.; Gabbi, C.; Valente, S.; Guerrini, S.; Frullanti, E.; Meloni, I.; Mencarelli, M. A.; Rizzo, C. L.; Pinto, A. M.
abstract

Clinical and molecular characterization by Whole Exome Sequencing (WES) is reported in 35 COVID-19 patients attending the University Hospital in Siena, Italy, from April 7 to May 7, 2020. Eighty percent of patients required respiratory assistance, half of them being on mechanical ventilation. Fiftyone percent had hepatic involvement and hyposmia was ascertained in 3 patients. Searching for common genes by collapsing methods against 150 WES of controls of the Italian population failed to give straightforward statistically significant results with the exception of two genes. This result is not unexpected since we are facing the most challenging common disorder triggered by environmental factors with a strong underlying heritability (50%). The lesson learned from Autism-Spectrum-Disorders prompted us to re-analyse the cohort treating each patient as an independent case, following a Mendelian-like model. We identified for each patient an average of 2.5 pathogenic mutations involved in virus infection susceptibility and pinpointing to one or more rare disorder(s). To our knowledge, this is the first report on WES and COVID-19. Our results suggest a combined model for COVID-19 susceptibility with a number of common susceptibility genes which represent the favorite background in which additional host private mutations may determine disease progression.

2020 - Effects of whole-body cryotherapy on the innate and adaptive immune response in cyclists and runners [Articolo su rivista]
Nasi, Milena; Bianchini, Elena; Lo Tartaro, Domenico; De Biasi, Sara; Mattioli, Marco; Paolini, Annamaria; Gibellini, Lara; Pinti, Marcello; De Gaetano, Anna; D’Alisera, Roberta; Roli, Laura; Chester, Johanna; Mattioli, Anna Vittoria; Polverari, Tomassina; Maietta, Pasqualino; Tripi, Ferdinando; Stefani, Omar; Guerra, Emanuele; Savino, Gustavo; Trenti, Tommaso; Cossarizza, Andrea
abstract

2020 - Efficient T cell compartment in HIV+ patients receiving orthotopic liver transplant and immunosuppressive therapy [Articolo su rivista]
Franceschini, Erica; De Biasi, Sara; Digaetano, Margherita; Bianchini, Elena; Lo Tartaro, Domenico; Gibellini, Lara; Menozzi, Marianna; Zona, Stefano; Tarantino, Giuseppe; Nasi, Milena; Codeluppi, Mauro; Guaraldi, Giovanni; Magistri, Paolo; Di Benedetto, Fabrizio; Pinti, Marcello; Mussini, Cristina; Cossarizza, Andrea
abstract

In patients undergoing orthotopic liver transplant (OLT), immunosuppressive (IS) treatment is mandatory and infections are leading causes of morbidity/mortality. Thus, it is essential to understand the functionality of cell-mediated immunity after OLT. The aim of the study was to identify changes in T cell phenotype and polyfunctionality in HIV+ and HIV- patients treated with IS after OLT.

2020 - Expansion of plasmablasts and loss of memory B cells in peripheral blood from COVID-19 patients with pneumonia [Articolo su rivista]
De Biasi, S.; Lo Tartaro, D.; Meschiari, M.; Gibellini, L.; Bellinazzi, C.; Borella, R.; Fidanza, L.; Mattioli, M.; Paolini, A.; Gozzi, L.; Jaacoub, D.; Faltoni, M.; Volpi, S.; Milic, J.; Sita, M.; Sarti, M.; Pucillo, C.; Girardis, M.; Guaraldi, G.; Mussini, C.; Cossarizza, A.
abstract

Studies on the interactions between SARS-CoV-2 and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVID-19 pneumonia. COVID-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and naïve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM+ and IgM− plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, D-dimer, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov2, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies.

2020 - Handling and processing of blood specimens from patients with Covid-19 for safe studies on cell phenotype and cytokine storm [Articolo su rivista]
Cossarizza, Andrea; Gibellini, Lara; DE BIASI, Sara; LO TARTARO, Domenico; Mattioli, Marco; Paolini, Annamaria; Fidanza, Lucia; Bellinazzi, Caterina; Borella, Rebecca; Castaniere, Ivana; Meschiari, Marianna; Sita, Marco; Manco, Gianrocco; Clini, Enrico; Gelmini, Roberta; Girardis, Massimo; Guaraldi, Giovanni; Mussini, Cristina
abstract

The pandemic caused by SARS-CoV-2 heavily involves all those working in a laboratory. Samples from known infected patients or donors who are considered healthy can arrive, and a colleague might be asymptomatic but able to transmit the virus. Working in a clinical laboratory is posing several safety challenges. Few years ago, ISAC published guidelines to safely analyze and sort human samples that were revised in these days. We describe the procedures that we have been following since the first patient appeared in Italy, which have only slightly modified our standard one, being all human samples associated with risks.

2020 - Impaired Mitochondrial Morphology and Functionality in Lonp1wt/- Mice [Articolo su rivista]
De Gaetano, Anna; Gibellini, Lara; Bianchini, Elena; Borella, Rebecca; De Biasi, Sara; Nasi, Milena; Boraldi, Federica; Cossarizza, Andrea; Pinti, Marcello
abstract

LONP1 is a nuclear-encoded mitochondrial protease crucial for organelle homeostasis; mutations ofLONP1have been associated with Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome. To clarify the role of LONP1 in vivo, we generated a mouse model in whichLonp1was ablated. The homozygousLonp(-/-)mouse was not vital, while the heterozygousLonp1(wt/-)showed similar growth rate, weight, length, life-span and histologic features as wild type. Conversely, ultrastructural analysis of heterozygous enterocytes evidenced profound morphological alterations of mitochondria, which appeared increased in number, swollen and larger, with a lower complexity. Embryonic fibroblasts (MEFs) fromLonp1(wt/-)mice showed a reduced expression ofLonp1andTfam, whose expression is regulated by LONP1. Mitochondrial DNA was also reduced, and mitochondria were swollen and larger, albeit at a lesser extent than enterocytes, with a perinuclear distribution. From the functional point of view, mitochondria from heterozygous MEF showed a lower oxygen consumption rate in basal conditions, either in the presence of glucose or galactose, and a reduced expression of mitochondrial complexes than wild type. In conclusion, the presence of one functional copy of theLonp1gene leads to impairment of mitochondrial ultrastructure and functions in vivo.

2020 - Increased Plasma Levels of Mitochondrial DNA and Normal Inflammasome Gene Expression in Monocytes Characterize Patients With Septic Shock Due to Multidrug Resistant Bacteria [Articolo su rivista]
Busani, Stefano; De Biasi, Sara; Nasi, Milena; Paolini, Annamaria; Venturelli, Sophie; Tosi, Martina; Girardis, Massimo; Cossarizza, Andrea
abstract

Introduction: The activity and regulation of inflammasome is receiving increasing attention in septic shock. Moreover, there is a growing body of evidence suggesting that mitochondrial DNA (mtDNA) can play a role as biomarker of disease severity and even mortality both in adults and children in critically ill setting. However, no data are available on the amount of circulating mtDNA and inflammasome gene expression in multi-drug resistant (MDR) bacteria septic shock. For this reason, the aim of this study was to determine whether plasma mtDNA levels and inflammasome gene expression in monocytes could be related to severity in patients admitted to intensive care unit (ICU) with septic shock due to MDR pathogens. Materials and Methods: Peripheral blood mononuclear cells (PBMC) and plasma were isolated from up to 20 ml of venous blood by density gradient centrifugation in patients admitted to ICU with the diagnosis of septic shock due to MDR-bacteria. Then, CD14+ monocytes were sorted, and RNA and DNA were extracted. NLRP3, PYCARD, AIM2 and NAIP expression level was analyzed by RT-PCR. Plasma circulating mtDNA levels were quantified by digital droplet PCR. Basal and outcome characteristics of the patients were collected. Age-matched healthy subjects were chosen as controls. Results: Nineteen patients with septic shock and 20 healthy subjects were enrolled in the study. A small trend toward an increased expression of inflammasome genes was observed in septic shock patients, who also displayed a marked tendency to an increased expression of IL-18 and IL-1β genes. Circulating mtDNA levels were significantly higher in septic shock patients if compared to healthy subjects, and patients who died in ICU were characterized by higher level of mtDNA if compared to those who were dismissed after 7 days. No correlations were found between mtDNA and inflammasome level and other clinical variables. Conclusion: Despite many limitations, our data suggest that in patients with septic shock caused by MDR pathogens the expression of main inflammasome genes was comparable to that of healthy patients without infection. Furthermore, our data evidence a possible role of mtDNA as a prognostic marker of severity in septic shock from MDR.

2020 - Increased plasma levels of mitochondrial DNA and pro-inflammatory cytokines in patients with progressive multiple sclerosis [Articolo su rivista]
Nasi, M.; Bianchini, E.; De Biasi, S.; Gibellini, L.; Neroni, A.; Mattioli, Marco; Pinti, M.; Iannone, A.; Mattioli, A. V.; Simone, A. M.; Ferraro, D.; Vitetta, F.; Sola, P.; Cossarizza, A.
abstract

The role of damage-associated molecular patterns in multiple sclerosis (MS) is under investigation. Here, we studied the contribution of circulating high mobility group box protein 1 (HMGB1) and mitochondrial DNA (mtDNA) to neuroinflammation in progressive MS. We measured plasmatic mtDNA, HMGB1 and pro-inflammatory cytokines in 38 secondary progressive (SP) patients, 35 primary progressive (PP) patients and 42 controls. Free mtDNA was higher in SP than PP. Pro-inflammatory cytokines were increased in progressive patients. In PP, tumor necrosis factor-α correlated with MS Severity Score. Thus, in progressive patients, plasmatic mtDNA and pro-inflammatory cytokines likely contribute to the systemic inflammatory status.

2020 - Machine learning in predicting respiratory failure in patients with COVID-19 pneumonia - challenges, strengths, and opportunities in a global health emergency. [Articolo su rivista]
Ferrari, D; Milic, J; Tonelli, R; Ghinelli, F; Meschiari, M; Volpi, S; Faltoni, M; Franceschi, G; Iadisernia, V; Yaacoub, D; Ciusa, G; Bacca, E; Rogati, C; Tutone, M; Burastero, G; Raimondi, A; Menozzi, M; Franceschini, E; Cuomo, G; Corradi, L; Orlando, G; Santoro, A; Di Gaetano, M; Puzzolante, C; Carli, F; Borghi, V; Bedini, A; Fantini, R; Tabbì, L; Castaniere, I; Busani, S; Clini, E; Girardis, M; Sarti, M; Cossarizza, A; Mussini, C; Mandreoli, F; Missier, P; Guaraldi, G.
abstract

Aims- The aim of this study was to estimate a 48 hour prediction of moderate to severe respiratory failure, requiring mechanical ventilation, in hospitalized patients with COVID-19 pneumonia. Methods- This was an observational study that comprised consecutive patients with COVID-19 pneumonia admitted to hospital from 21 February to 6 April 2020. The patients’ medical history, demographic, epidemiologic and clinical data were collected in an electronic patient chart. The dataset was used to train predictive models using an established machine learning framework leveraging a hybrid approach where clinical expertise is applied alongside a data-driven analysis. The study outcome was the onset of moderate to severe respiratory failure defined as PaO 2 /FiO 2 ratio <150 mmHg in at least one of two consecutive arterial blood gas analyses in the following 48 hours. Shapley Additive exPlanations values were used to quantify the positive or negative impact of each variable included in each model on the predicted outcome. Results- A total of 198 patients contributed to generate 1068 usable observations which allowed to build 3 predictive models based respectively on 31-variables signs and symptoms, 39-variables laboratory biomarkers and 91-variables as a composition of the two. A fourth “boosted mixed model” included 20 variables was selected from the model 3, achieved the best predictive performance (AUC=0.84) without worsening the FN rate. Its clinical performance was applied in a narrative case report as an example. Conclusion- This study developed a machine model with 84% prediction accuracy, which is able to assist clinicians in decision making process and contribute to develop new analytics to improve care at high technology readiness levels.

2020 - Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with Covid-19 pneumonia. [Articolo su rivista]
De Biasi, S; Meschiari, M; Gibellini, L; Bellinazzi, C; Borella, R; Fidanza, L; Gozzi, L; Iannone, A; Lo Tartaro, D; Mattioli, M; Paolini, A; Menozzi, M; Milić, J; Franceschi, G; Fantini, R; Tonelli, R; Sita, M; Sarti, M; Trenti, T; Brugioni, L; Cicchetti, L; Facchinetti, F; Pietrangelo, A; Clini, E; Girardis, M; Guaraldi, G; Mussini, C; Cossarizza, A.
abstract

We provide an in-depth investigation of the T cell compartment and functionality, cytokine production and plasma levels in a total of 39 patients affected by Covid-19 pneumonia. At admission, patients were lymphopenic; for all, SARS-CoV-2 was detected in a nasopharyngeal swab specimen by real-time RT-PCR, and pneumonia was subsequently confirmed by X-rays. Detailed 18-parameter flow cytometry coupled with unsupervised data analysis revealed that patients showed similar percentages of CD4+ and CD8+ T cells, but a decreased absolute number in both populations. For CD4+ T lymphocytes, we found a significant decrease in the number of naïve, central and effector memory cells and an increased percentage of terminally differentiated cells, regulatory T cells, and of those that were activated or that were expressing PD1 and CD57 markers. Studies on chemokine receptors and lineage-specifying transcription factors revealed that, among CD4+ T cells, patients displayed a lower percentage of cells expressing CCR6 or CXCR3, and of those co-expressing CCR6 and CD161, but higher percentages of 62 CXCR4+ or CCR4+ cells. No differences were noted in the expression of T-bet or GATA-3. Analyses of patients' CD8+ T cells showed decreased numbers of naïve and central memory and increased amounts of activated cells, accompanied by increased percentages of activated cells and of lymphocytes expressing CD57, PD1, or both. CD8+ T cells expressed lower percentages of CCR6+, CXCR3+ or T-bet+ cells and of CXCR3+,T-bet+ or CCR6+,CD161+ lymphocytes. We also found higher percentages of cells expressing CCR4+, CXCR4 or GATA-3. Analyses of lymphocyte proliferation revealed that terminally differentiated CD4+ and CD8+ T cell from patients had a lower proliferative index than controls, whereas cellular bioenergetics, measured by the quantification of mitochondrial oxygen consumption and extracellular acidification rate, was similar in CD4+ T cells from both groups. We measured plasma level of 31 cytokines linked to inflammation, including T helper (TH)type-1 and TH2 cytokines, chemokines, galectins, pro- and anti-inflammatory mediators, finding that most were dramatically increased in Covid-19 patients, confirming the presence of a massive cytokine storm. Analysis of the production of different cytokines after stimulation by anti-CD3/CD28 monoclonal antibodies revealed that patients not only had a high capacity to produce tumour necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-2, but also showed a significant skewing of CD4+ T cells towards the TH17 phenotype. A therapeutic approach now exists based on the administration of drugs that block IL-6pathway, and seems to improve the disease. IL-17 is crucial in recruiting and activating neutrophils, cells that can migrate to the lung and are heavily involved in the pathogenesis of Covid-19. We show here that a skewing of activated T cells towards the TH17 functional phenotype exists in Covid-19 patients. We therefore suggest that blocking the IL-17 pathway by biological drugs that are already used to treat different pathologies could provide a novel, additional strategy to improve the health of patients infected by SARS-CoV-2.

2020 - Mitochondria, oxidative stress, cancer, and aging [Capitolo/Saggio]
Pecorini, S.; Gibellini, L.; Biasi, S. D.; Bianchini, E.; Nasi, M.; Cossarizza, A.; Pinti, M.
abstract

In human cells, the main source of reactive oxygen species (ROS) and oxidative stress are mitochondria, the organelles where oxidative phosphorylation take place. Although ROS are an inevitable by-products of respiration, they do not necessarily have detrimental effects; low doses of ROS can have beneficial effects on cells, and their production can be finely regulated in mitochondria. Increasing ROS levels and products of the oxidative stress, which occur in aging and age-related disorders, are related to progressive dysfunction of mitochondria, due to damage to mitochondrial DNA or to oxidation and damage of mitochondrial proteins, and are also present in cancer. This chapter focuses on the regulation of ROS production in mitochondria and on the mechanisms that lead to its dysregulation in aging and cancer.

2020 - Mitochondrial damage-associated molecular patterns stimulate reactive oxygen species production in human microglia [Articolo su rivista]
Nasi, Milena; De Gaetano, Anna; Bianchini, Elena; De Biasi, Sara; Gibellini, Lara; Neroni, Anita; Mattioli, Marco; Pinti, Marcello; Tartaro, Domenico Lo; Borella, Rebecca; Mattioli, Anna Vittoria; Chester, Johanna; Melegari, Alessandra; Simone, Anna Maria; Ferraro, Diana; Vitetta, Francesca; Sola, Patrizia; Cossarizza, Andrea
abstract

Microglia are the resident innate immune cells of the central nervous system and exert functions of host defence and maintenance of normal tissue homeostasis, along with support of neuronal processes in the healthy brain. Chronic and dysregulated microglial cell activation has increasingly been linked to the status of neuroinflammation underlying many neurodegenerative diseases, including multiple sclerosis (MS). However, the stimulus (or stimuli) and mechanisms by which microglial activation is initiated and maintained MS are still debated. The purpose of our research was to investigate whether the endogenous mitochondrial (mt)-derived damage-associated molecular patterns (MTDs) mtDNA, N-formyl peptides and cardiolipin (CL) contribute to these phenomena. We characterized the effects of the abovementioned MTDs on microglia activation in vitro (i.e. using HMC3 cells) by evaluating the expression of gene coding for proteins involved in their binding and coupled to downstream signaling pathways, the up-regulation of markers of activation on the cell surface and the production of pro-inflammatory cytokines and reactive oxygen species. At the transcriptional level, significant variations in the mRNA relative expression of five of eleven selected genes were observed in response to stimulation. No changes in activation of antigenic profile or functional properties of HMC3 cells were observed; there was no up-regulation of HLA-DR expression or increased secretion of tumor necrosis factor-α and interleukin-6. However, after stimulation with mtDNA and CL, an increase in cellular oxidative stress, but not in the mt ROS O2-, compared to control cells, were observed. There were no effects on cell viability. Overall, our data suggest that MTDs could cause a failure in microglial activation toward a pro-inflammatory phenotype, possibly triggering an endogenous regulatory mechanism for the resolution of neuroinflammation. This could open a door for the development of drugs selectively targeting microglia and modulating its functionality to treat MS and/or other neurodegenerative conditions in which MTDs have a pathogenic relevance.

2020 - Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma. [Articolo su rivista]
Marchetto, A; Ohmura, S; Orth, Mf; Knott, Mml; Colombo, Mv; Arrigoni, C; Bardinet, V; Saucier, D; Wehweck, Fs; Li, J; Stein, S; Gerke, Js; Baldauf, Mc; Musa, J; Dallmayer, M; Romero-Pérez, L; Hölting, Tlb; Amatruda, Jf; Cossarizza, A; Henssen, Ag; Kirchner, T; Moretti, M; Cidre-Aranaz, F; Sannino, G; Grünewald, Tgp.
abstract

2020 - Peritoneal dialysis in the time of coronavirus disease 2019 [Articolo su rivista]
Alfano, Gaetano; Fontana, Francesco; Ferrari, Annachiara; Guaraldi, Giovanni; Mussini, Cristina; Magistroni, Riccardo; Cappelli, Gianni; Bacca, Erica; Bedini, Andrea; Borghi, Vanni; Burastero, Giulia; Carli, Federica; Ciusa, Giacomo; Corradi, Luca; Cuomo, Gianluca; Digaetano, Margherita; Dolci, Giovanni; Faltoni, Matteo; Fantini, Riccardo; Franceschi, Giacomo; Franceschini, Ericad; Iadisernia, Vittorio; Larnõ, Damiano; Menozzi, Marianna; Meschiari, Marianna; Milic, Jovana; Orlando, Gabriella; Pellegrino, Francesco; Raimondi, Alessandro; Rogati, Carlotta; Santoro, Antonella; Tonelli, Roberto; Tutone, Marco; Volpi, Sara; Yaacoub, Dina; Aten, G.; Marco, Ballestri; Mori, Giacomo; Girardis, Massimo; Andreotti, Alberto; Biagioni, Emanuela; Bondi, Filippo; Busani, Stefano; Chierego, Giovanni; Scotti, Marzia; Serio, Lucia; Cossarizza, Andrea; Bellinazzi, Caterina; Borella, Rebecca; de Biasi, Sara; de Gaetano, Anna; Fidanza, Lucia; Gibellini, Lara; Iannone, Anna; Lo Tartaro, Domenico; Mattioli, Marco; Nasi, Milena; Paolini, Annamariag; Pinti, Marcello
abstract

In the current setting of global containment, peritoneal dialysis (PD) and home haemodialysis are the best modalities of renal replacement therapy (RRT) to reduce the rate of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Considering the shorter and easier training programme of PD compared to home haemodialysis, PD appears a practical solution for patients with end-stage renal disease to reduce the risk of hospital-acquired infection. PD offers the advantage of minimizing the risk of viral transmission through interpersonal contact that commonly occurs during the haemodialysis session and while travelling from home to the haemodialysis facility using public transport services. To overcome barriers to health care access due to the containment measures for this emerging disease, telemedicine is a useful and reliable tool for delivering health care without exposing patients to the risk of contact. However, novel issues including handling of potentially infected dialysate, caregivers' infectious risk and adequacy of PD in critically ill patients with acute respiratory distress syndrome remain to be clarified. In conclusion, PD should be preferred to the other modalities of RRT during the coronavirus disease 2019 (COVID-19) outbreak because it can be a solution to cope with the increased number of infected patients worldwide.

2020 - Prevalence of HIV-associated Neurocognitive Disorder (HAND) and its subgroups among HIV-positive persons on anti-retroviral therapy in Iran [Articolo su rivista]
Elham, M. -T.; Vahid, N.; Seyed Ahmad, S. A.; Omid, D.; Cossarizza, A; Mussini, C.; Sara, A. N.; Leila, S.; Mohammad, G.; Hanieh, G.; Minoo, M.
abstract

This study aimed to determine the prevalence and determinants of HIV-associated neurocognitive disorder (HAND) and its subgroups in HIV-positive patients in Tehran, Iran. Ninety-three HIV-positive individuals were assessed; the majority were male (60%) and the mean age of patients was 36.5 years (SD = 9), with 8 years as the median duration of HIV infection. The relationship between demographic and clinical variables was examined using logistic regression analysis. The overall prevalences of HAND and cognitive complaints were 50.5% and 73%, respectively. Lower nadir CD4 counts (≤ 200), lower educational levels (≤ 12 years), longer disease duration (≥ 5years), and higher depression rates were positively associated with the presence of HAND. This study shows that the prevalence of HANDs in Iran is high, but similar to the prevalence levels found in Western societies. Further studies are needed to longitudinally evaluate the presence of HAND, in particularly to recognize new biomarkers and specific neurocognitive domains in HIV.

2020 - Publisher Correction: Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma (Nature Communications, (2020), 11, 1, (2423), 10.1038/s41467-020-16244-2) [Articolo su rivista]
Marchetto, A.; Ohmura, S.; Orth, M. F.; Knott, M. M. L.; Colombo, M. V.; Arrigoni, C.; Bardinet, V.; Saucier, D.; Wehweck, F. S.; Li, J.; Stein, S.; Gerke, J. S.; Baldauf, M. C.; Musa, J.; Dallmayer, M.; Romero-Perez, L.; Holting, T. L. B.; Amatruda, J. F.; Cossarizza, A.; Henssen, A. G.; Kirchner, T.; Moretti, M.; Cidre-Aranaz, F.; Sannino, G.; Grunewald, T. G. P.
abstract

The original version of this Article contained an error in Fig. 3b. In Fig. 3b the “<” symbol in the labelling for light grey circles was replaced by “>”. This has been corrected in both the PDF and HTML versions of the Article.

2020 - SARS-CoV-2, the Virus that Causes COVID-19: Cytometry and the New Challenge for Global Health [Articolo su rivista]
Cossarizza, A.; De Biasi, S.; Guaraldi, G.; Girardis, M.; Mussini, C.
abstract

2020 - Safety and efficacy of the Russian COVID-19 vaccine: more information needed [Articolo su rivista]
Bucci, E.; Andreev, K.; Bjorkman, A.; Calogero, R. A.; Carafoli, E.; Carninci, P.; Castagnoli, P.; Cossarizza, A.; Mussini, C.; Guerin, P.; Lipworth, B.; Sbardella, G.; Stocki, T.; Tuosto, L.; van Tulleken, C.; Viola, A.
abstract

2020 - Single-Cell Approaches to Profile the Response to Immune Checkpoint Inhibitors [Articolo su rivista]
Gibellini, L.; De Biasi, S.; Porta, C.; Lo Tartaro, D.; Depenni, R.; Pellacani, G.; Sabbatini, R.; Cossarizza, A.
abstract

Novel treatments based upon the use of immune checkpoint inhibitors have an impressive efficacy in different types of cancer. Unfortunately, most patients do not derive benefit or lasting responses, and the reasons for the lack of therapeutic success are not known. Over the past two decades, a pressing need to deeply profile either the tumor microenvironment or cells responsible for the immune response has led investigators to integrate data obtained from traditional approaches with those obtained with new, more sophisticated, single-cell technologies, including high parameter flow cytometry, single-cell sequencing and high resolution imaging. The introduction and use of these technologies had, and still have a prominent impact in the field of cancer immunotherapy, allowing delving deeper into the molecular and cellular crosstalk between cancer and immune system, and fostering the identification of predictive biomarkers of response. In this review, besides the molecular and cellular cancer-immune system interactions, we are discussing how cutting-edge single-cell approaches are helping to point out the heterogeneity of immune cells in the tumor microenvironment and in blood.

2020 - Surfactant replacement might help recovery of low-compliance lung in severe COVID-19 pneumonia. [Articolo su rivista]
Busani, S; Dall’Ara, L; Tonelli, R; Clini, E; Munari, E; Venturelli, S; Meschiari, M; Guaraldi, G; Cossarizza, A; Ranieri, Mv; Girardis, M.
abstract

It has been hypothesized that there is a reduced AT2 cells number with low ability to synthesize and secrete endogenous surfactant in COVID-19 patients. To our knowledge, exogenous surfactant replacement has not been described so far in COVID-19 patients. We here report five cases of critically ill COVID-19 undergoing exogenous surfactant instillation through the airways.

2020 - The biology of Lonp1: More than a mitochondrial protease [Capitolo/Saggio]
Gibellinia, Lara; De Gaetano, Anna; Mandrioli, Mauro; Van Tongeren, Elia; Bortolotti, Carlo Augusto; Cossarizza, Andrea; Pinti, Marcello
abstract

Initially discovered as a protease responsible for degradation of misfolded or damaged proteins, the mitochondrial Lon protease (Lonp1) turned out to be a multifaceted enzyme, that displays at least three different functions (proteolysis, chaperone activity, binding of mtDNA) and that finely regulates several cellular processes, within and without mitochondria. Indeed, LONP1 in humans is ubiquitously expressed, and is involved in regulation of response to oxidative stress and, heat shock, in the maintenance of mtDNA, in the regulation of mitophagy. Furthermore, its proteolytic activity can regulate several biochemical pathways occurring totally or partially within mitochondria, such as TCA cycle, oxidative phosphorylation, steroid and heme biosynthesis and glutamine production. Because of these multiple activities, Lon protease is highly conserved throughout evolution, and mutations occurring in its gene determines severe diseases in humans, including a rare syndrome characterized by Cerebral, Ocular, Dental, Auricular and Skeletal anomalies (CODAS). Finally, alterations of LONP1 regulation in humans can favor tumor progression and aggressiveness, further highlighting the crucial role of this enzyme in mitochondrial and cellular homeostasis.

2020 - The importance of advanced cytometry in defining new immune cell types and functions relevant for the immunopathogenesis of HIV infection [Articolo su rivista]
Agrati, C.; de Biasi, S.; Fidanza, L.; Gibellini, L.; Nasi, M.; Pinti, M.; Cossarizza, A.
abstract

In the last years, novel, exciting immunological findings of interest for HIV research and treatment were identified thanks to different cytometric approaches. The analysis of the phenotypes and functionality of cells belonging to the immune system could clarify their role in the immunopathogenesis of HIV infection, and to elaborate key concepts, relevant in the treatment of this disease. Important discoveries have been made concerning cells that are important for protective immunity like lymphocytes that display polyfunctionality, resident memory T cells, innate lymphoid cells, to mention a few. The complex phenotype of myeloid-derived suppressor cells has been investigated, and relevant changes have been reported during chronic and primary HIV infection, in correlation with changes in CD4þ T-cell number, T-cell activation, and with advanced disease stage. The search for markers of HIV persistence present in latently infected cells, namely those molecules that are important for a functional or sterilizing cure, evidenced the role of follicular helper T cells, and opened a discussion on the meaning and use of different surface molecules not only in identifying such cells, but also in designing new strategies. Finally, advanced technologies based upon the simultaneous detection of HIV-RNA and proteins at the single cell level, as well as those based upon spectral cytometry or mass cytometry are now finding new actors and depicting a new scenario in the immunopathogenesis of the infection, that will allow to better design innovative therapies based upon novel drugs and vaccines.

2020 - Tocilizumab in patients with severe COVID-19: a retrospective cohort study [Articolo su rivista]
Guaraldi, G; Meschiari, M; Cozzi-Lepri, A; Milic, J; Tonelli, R; Menozzi, M; Franceschini, E; Cuomo, G; Orlando, G; Borghi, V; Santoro, A; Di Gaetano, M; Puzzolante, C; Carli, F; Bedini, A; Corradi, L; Fantini, R; Castaniere, I; Tabbì, L; Girardis, M; Tedeschi, S; Giannella, M; Bartoletti, M; Pascale, R; Dolci, G; Brugioni, L; Pietrangelo, A; Cossarizza, A; Pea, F; Clini, E; Salvarani, C; Massari, M; Viale, Pl; Mussini, C.
abstract

Background- There is no approved therapy for COVID-19 pneumonia. The aim of this multicentre cohort study was to assess the role of tocilizumab in reducing the risk of invasive mechanical ventilation and/or death in patients with severe COVID-19 pneumonia who received standard of care (SoC) treatment. Methods- The TESEO Cohort Study is a retrospective, multicentre observational cohort study of patients with COVID-19 severe pneumonia treated with SoC with or without tocilizumab using intravenous (IV) or subcutaneous (SC) formulations, identifying respectively treated and comparator groups. Survival analysis was performed with participants’ follow-up accruing from the date of entry into clinics until initiation of invasive mechanical ventilation or death, used as a composite outcome. Treatment groups were compared using Kaplan-Meier curves and Cox regression analysis after adjusting for gender, age and baseline Sequential Organ Failure Assessment (SOFA) score. Findings- Of 544 patients included, 179 patients were treated with tocilizumab: 88 with the IV (16.1%) and 91 with SC formulation (16.7%). Mortality was significantly higher in the comparator group (20%) as opposed to tocilizumab IV (6.8%) and tocilizumab SC (7.7%) (p<0.001). A reduced risk of invasive mechanical ventilation/death was shown for participants treated with tocilizumab from fitting a Cox regression analysis adjusted for gender, age and SOFA score (aHR=0.61, 95% CI:0.40-0.92; p=0.02). We found no evidence for a difference between IV and SC administration route of tocilizumab. With regards to the mortality endpoint alone, a reduced risk was observed comparing tocilizumab with the comparator group (aHR=0.38 95% CI:0.17-0.83, p=0.02) . Interpretation- Tocilizumab, regardless of IV or SC administration may be capable of reducing invasive mechanical ventilation or death in severe COVID-19 pneumonia. Our observations should be confirmed in randomised studies. Funding- This study was not funded.

2020 - Two fatal cases of acute liver failure due to HSV-1 infection in COVID-19 patients following immunomodulatory therapies. [Articolo su rivista]
Busani, S; Bedini, A; Biagioni, E; Serio, L; Tonelli, R; Meschiari, M; Franceschini, E; Guaraldi, G; Cossarizza, A; Clini, E; Maiorana, A; Gennari, W; De Maria, N; Luppi, M; Mussini, C; Girardis, M.; Gibellini, Lara
abstract

We reported two fatal cases of acute liver failure secondary to Herpes Simplex Virus 1 infection in COVID-19 patients, following tocilizumab and corticosteroid therapy. Screening for and prompt recognition of Herpes Simplex Virus 1 reactivation in these patients, undergoing immunomodulatory treatment, may have potentially relevant clinical consequences.

2019 - Altered Expression of PYCARD, Interleukin 1β, Interleukin 18, and NAIP in Successfully Treated HIV-Positive Patients With a Low Ratio of CD4+ to CD8+ T Cells [Articolo su rivista]
Nasi, Milena; Pecorini, Simone; De Biasi, Sara; Bianchini, Elena; Digaetano, Margherita; Neroni, Anita; Lo Tartaro, Domenico; Pullano, Rosalberta; Pinti, Marcello; Gibellini, Lara; Mussini, Cristina; Cossarizza, Andrea
abstract

The expression and activity of main inflammasome components in monocytes from successfully treated HIV+ patients are poorly studied. Thus, we enrolled 18 patients with low and 17 with normal CD4/CD8 ratio compared to 11 healthy donors. Our results show that patients with low ratio have a decreased CCR2 expression among classical and intermediate monocytes and an increased CCR5 expression among classical, compared to whose with normal ratio. They also showed higher NAIP and PYCARD mRNA levels after LPS-stimulation suggesting an altered ability to control immune activation that could affect their immune reconstitution.

2019 - Current gaps in sepsis immunology: new opportunities for translational research [Articolo su rivista]
Rubio, Ignacio; Osuchowski, Marcin F; Shankar-Hari, Manu; Skirecki, Tomasz; Winkler, Martin Sebastian; Lachmann, Gunnar; La Rosée, Paul; Monneret, Guillaume; Venet, Fabienne; Bauer, Michael; Brunkhorst, Frank M; Kox, Matthijs; Cavaillon, Jean-Marc; Uhle, Florian; Weigand, Markus A; Flohé, Stefanie B; Wiersinga, W Joost; Martin-Fernandez, Marta; Almansa, Raquel; Martin-Loeches, Ignacio; Torres, Antoni; Giamarellos-Bourboulis, Evangelos J; Girardis, Massimo; Cossarizza, Andrea; Netea, Mihai G; van der Poll, Tom; Scherag, André; Meisel, Christian; Schefold, Joerg C; Bermejo-Martín, Jesús F
abstract

Increasing evidence supports a central role of the immune system in sepsis, but the current view of how sepsis affects immunity, and vice versa, is still rudimentary. The European Group on Immunology of Sepsis has identified major gaps that should be addressed with high priority, such as understanding how immunological alterations predispose to sepsis, key aspects of the immunopathological events during sepsis, and the long-term consequences of sepsis on patient's immunity. We discuss major unmet topics in those three categories, including the role of key immune cells, the cause of lymphopenia, organ-specific immunology, the dynamics of sepsis-associated immunological alterations, the role of the microbiome, the standardisation of immunological tests, the development of better animal models, and the opportunities offered by immunotherapy. Addressing these gaps should help us to better understand sepsis physiopathology, offering translational opportunities to improve its prevention, diagnosis, and care.

2019 - Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition) [Articolo su rivista]
Cossarizza, A.; Chang, H. -D.; Radbruch, A.; Acs, A.; Adam, D.; Adam-Klages, S.; Agace, W. W.; Aghaeepour, N.; Akdis, M.; Allez, M.; Almeida, L. N.; Alvisi, G.; Anderson, G.; Andra, I.; Annunziato, F.; Anselmo, A.; Bacher, P.; Baldari, C. T.; Bari, S.; Barnaba, V.; Barros-Martins, J.; Battistini, L.; Bauer, W.; Baumgart, S.; Baumgarth, N.; Baumjohann, D.; Baying, B.; Bebawy, M.; Becher, B.; Beisker, W.; Benes, V.; Beyaert, R.; Blanco, A.; Boardman, D. A.; Bogdan, C.; Borger, J. G.; Borsellino, G.; Boulais, P. E.; Bradford, J. A.; Brenner, D.; Brinkman, R. R.; Brooks, A. E. S.; Busch, D. H.; Buscher, M.; Bushnell, T. P.; Calzetti, F.; Cameron, G.; Cammarata, I.; Cao, X.; Cardell, S. L.; Casola, S.; Cassatella, M. A.; Cavani, A.; Celada, A.; Chatenoud, L.; Chattopadhyay, P. K.; Chow, S.; Christakou, E.; Cicin-Sain, L.; Clerici, M.; Colombo, F. S.; Cook, L.; Cooke, A.; Cooper, A. M.; Corbett, A. J.; Cosma, A.; Cosmi, L.; Coulie, P. G.; Cumano, A.; Cvetkovic, L.; Dang, V. D.; Dang-Heine, C.; Davey, M. S.; Davies, D.; De Biasi, S.; Del Zotto, G.; Dela Cruz, G. V.; Delacher, M.; Della Bella, S.; Dellabona, P.; Deniz, G.; Dessing, M.; Di Santo, J. P.; Diefenbach, A.; Dieli, F.; Dolf, A.; Dorner, T.; Dress, R. J.; Dudziak, D.; Dustin, M.; Dutertre, C. -A.; Ebner, F.; Eckle, S. B. G.; Edinger, M.; Eede, P.; Ehrhardt, G. R. A.; Eich, M.; Engel, P.; Engelhardt, B.; Erdei, A.; Esser, C.; Everts, B.; Evrard, M.; Falk, C. S.; Fehniger, T. A.; Felipo-Benavent, M.; Ferry, H.; Feuerer, M.; Filby, A.; Filkor, K.; Fillatreau, S.; Follo, M.; Forster, I.; Foster, J.; Foulds, G. A.; Frehse, B.; Frenette, P. S.; Frischbutter, S.; Fritzsche, W.; Galbraith, D. W.; Gangaev, A.; Garbi, N.; Gaudilliere, B.; Gazzinelli, R. T.; Geginat, J.; Gerner, W.; Gherardin, N. A.; Ghoreschi, K.; Gibellini, L.; Ginhoux, F.; Goda, K.; Godfrey, D. I.; Goettlinger, C.; Gonzalez-Navajas, J. M.; Goodyear, C. S.; Gori, A.; Grogan, J. L.; Grummitt, D.; Grutzkau, A.; Haftmann, C.; Hahn, J.; Hammad, H.; Hammerling, G.; Hansmann, L.; Hansson, G.; Harpur, C. M.; Hartmann, S.; Hauser, A.; Hauser, A. E.; Haviland, D. L.; Hedley, D.; Hernandez, D. C.; Herrera, G.; Herrmann, M.; Hess, C.; Hofer, T.; Hoffmann, P.; Hogquist, K.; Holland, T.; Hollt, T.; Holmdahl, R.; Hombrink, P.; Houston, J. P.; Hoyer, B. F.; Huang, B.; Huang, F. -P.; Huber, J. E.; Huehn, J.; Hundemer, M.; Hunter, C. A.; Hwang, W. Y. K.; Iannone, A.; Ingelfinger, F.; Ivison, S. M.; Jack, H. -M.; Jani, P. K.; Javega, B.; Jonjic, S.; Kaiser, T.; Kalina, T.; Kamradt, T.; Kaufmann, S. H. E.; Keller, B.; Ketelaars, S. L. C.; Khalilnezhad, A.; Khan, S.; Kisielow, J.; Klenerman, P.; Knopf, J.; Koay, H. -F.; Kobow, K.; Kolls, J. K.; Kong, W. T.; Kopf, M.; Korn, T.; Kriegsmann, K.; Kristyanto, H.; Kroneis, T.; Krueger, A.; Kuhne, J.; Kukat, C.; Kunkel, D.; Kunze-Schumacher, H.; Kurosaki, T.; Kurts, C.; Kvistborg, P.; Kwok, I.; Landry, J.; Lantz, O.; Lanuti, P.; Larosa, F.; Lehuen, A.; LeibundGut-Landmann, S.; Leipold, M. D.; Leung, L. Y. T.; Levings, M. K.; Lino, A. C.; Liotta, F.; Litwin, V.; Liu, Y.; Ljunggren, H. -G.; Lohoff, M.; Lombardi, G.; Lopez, L.; Lopez-Botet, M.; Lovett-Racke, A. E.; Lubberts, E.; Luche, H.; Ludewig, B.; Lugli, E.; Lunemann, S.; Maecker, H. T.; Maggi, L.; Maguire, O.; Mair, F.; Mair, K. H.; Mantovani, A.; Manz, R. A.; Marshall, A. J.; Martinez-Romero, A.; Martrus, G.; Marventano, I.; Maslinski, W.; Matarese, G.; Mattioli, A. V.; Maueroder, C.; Mazzoni, A.; Mccluskey, J.; Mcgrath, M.; Mcguire, H. M.; Mcinnes, I. B.; Mei, H. E.; Melchers, F.; Melzer, S.; Mielenz, D.; Miller, S. D.; Mills, K. H. G.; Minderman, H.; Mjosberg, J.; Moore, J.; Moran, B.; Moretta, L.; Mosmann, T. R.; Muller, S.; Multhoff, G.; Munoz, L. E.; Munz, C.; Nakayama, T.; Nasi, M.; Neumann, K.; Ng, L. G.; Niedobitek, A.; Nourshargh, S.; Nunez, G.; O'Connor, J. -E.; Ochel, A.; Oja, A.; Ordonez, D.; Orfao, A.; Orlowski-Oliver, E.; Ouyang, W.; Oxenius, A.; Palankar, R.; Panse, I.; Pattanapanyasat, K.; Paulsen, M.; Pavlinic, D.; Penter,
abstract

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

2019 - ISAC 34th International Congress Vancouver, Canada June 22 – 26, 2019 [Articolo su rivista]
Chattopadhyay, P. K.; Cossarizza, A.
abstract

2019 - Inducing cancer indolence by targeting mitochondrial Complex I is potentiated by blocking macrophage-mediated adaptive responses [Articolo su rivista]
Kurelac, I.; Iommarini, L.; Vatrinet, R.; Amato, L. B.; De Luise, M.; Leone, G.; Girolimetti, G.; Umesh Ganesh, N.; Bridgeman, V. L.; Ombrato, L.; Columbaro, M.; Ragazzi, M.; Gibellini, L.; Sollazzo, M.; Feichtinger, R. G.; Vidali, S.; Baldassarre, M.; Foriel, S.; Vidone, M.; Cossarizza, A.; Grifoni, D.; Kofler, B.; Malanchi, I.; Porcelli, A. M.; Gasparre, G.
abstract

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.

2019 - Italian expert panel consensus statements on two-drug antiretroviral regimens to treat naïve and virologically suppressed HIV-1 infected patients [Articolo su rivista]
Antinori, Andrea;  , ; Santoro, Maria Mercedes; Gagliardini, Roberta;  , ; Marchetti, Giulia;  , ; Mondi, Annalisa;  , ; Cento, Valeria;  , ; Perno, Carlo Federico;  , ; Andreoni, Massimo;  , ; Ceccherini-Silberstein, Francesca;  , ; Cossarizza, Andrea;  , ; Clerici, Mario;  , ; Monforte, Antonella D'Arminio;  , ; Luca, Andrea;  , ; Di Biagio, Antonio;  , ; Di Perri, Giovanni;  , ; Galli, Massimo;  , ; Girardi, E.  ;  , ; Gori, Andrea;  , ; Lazzarin, Adriano;  , ; Lichtner, Miriam;  , ; Lo Caputo, Sergio;  , ; Madeddu, Giordano;  , ; Maggiolo, Franco;  , ; Mussini, Cristina;  , ; Re, Maria Carla;  , ; Ripamonti, Diego;  , ; Rizzardini, Giuliano;  , ; Zazzi, Maurizio
abstract

New strategies for HIV treatment able to reduce drug-exposure, medium-long term toxicities and costs are a recognized clinical need. The availability of newer drugs with improved potency and tolerability, as well as high genetic barrier to resistance, makes antiretroviral-sparing strategies with two-drug regimens (2DRs) particularly attractive. Substantial evidence has been generated over the last few years supporting 2DR in virologically suppressed HIV infected patients for whom a therapy switch is planned. More recently, very promising data on 2DR in naïve patients have also been reported. The main purpose of this consensus is to provide an overview of guideline indications and recommendations, and the most recent data from clinical studies of 2DR in both naïve and virologically suppressed patients. As an expert consensus, suggestions and indications on the use and management of 2DR are also provided.

2019 - Mitochondrial functionality and metabolism in T cells from progressive multiple sclerosis patients [Articolo su rivista]
De Biasi, Sara; Simone, Anna Maria; Bianchini, Elena; Lo Tartaro, Domenico; Pecorini, Simone; Nasi, Milena; Patergnani, Simone; Carnevale, Gianluca; Gibellini, Lara; Ferraro, Diana; Vitetta, Francesca; Pinton, Paolo; Sola, Patrizia; Cossarizza, Andrea; Pinti, Marcello
abstract

Patients with primary progressive (PP) and secondary progressive (SP) forms of multiple sclerosis (MS) exhibit a sustained increase in the number of Th1, T cytotoxic type-1 and Th17 cells in peripheral blood, suggesting that the progressive phase is characterized by a permanent peripheral immune activation. As T cell functionality and activation are strictly connected to their metabolic profile, we investigated the mitochondrial functionality and metabolic changes of T cell subpopulations in a cohort of progressive MS patients. T cells from progressive patients were characterized by low proliferation and increase of terminally differentiated/exhausted cells. T cells from PP patients showed lower Oxygen Consumption Rate and Extracellular Acidification Rate, lower mitochondrial mass, membrane potential and respiration than those of SP patients, a downregulation of transcription factors supporting respiration and higher tendency to shift towards glycolysis upon stimulation. Furthermore, PP effector memory T cells were characterized by higher Glucose transporter -1 levels and a higher expression of glycolytic-supporting genes if compared to SP patients. Overall, our data suggest that profound differences exist in the phenotypic and metabolic features of T cells from PP and SP patients, even though the two clinical phenotypes are considered part of the same disease spectrum.

2019 - Modulating the Faradic Operation of All-Printed Organic Electrochemical Transistors by Facile in Situ Modification of the Gate Electrode [Articolo su rivista]
Sensi, Matteo; Berto, Marcello; Candini, Andrea; Liscio, Andrea; Cossarizza, Andrea; Beni, Valerio; Biscarini, Fabio; Bortolotti, Carlo Augusto
abstract

Organic electrochemical transistors (OECTs) operated in the faradic regime were shown as outperforming transducers of bioelectric signals in vitro and in vivo. Fabrication by additive manufacturing techniques fosters OECTs as ideal candidates for point-of-care applications, as well as imposes limitations on the choice of materials and their processing conditions. Here, we address the question of how the response of fully printed OECTs depends on gate electrode material. Toward this end, we investigate the redox processes underlying the operation of OECTs under faradic regime, to show OECTs with carbon gate (C-gate) that exhibit no current modulation gate voltages <1.2 V. This is a hallmark that no interference with the faradic operation of the device enabled by redox processes occurs when operating C-gate OECTs in the low-voltage range as label-free biosensors for the detection of electroactive (bio)molecules. To tune the faradic response of the device, we electrodeposited Au on the carbon gate (Au-C-gate), obtaining a device that operates at lower gate voltage values than C-gate OECT. The presence of gold on the gate allowed further modification of the electrical performances by functionalization of the Au-C-gate with different self-assembled monolayers by fast potential-pulse-assisted method. Moreover, we show that the presence in the electrolyte solution of an external redox probe can be used to drive the faradic response of both C- and Au-C-gate OECTs, impacting on the gate potential window that yields effective drain current modulation. The results presented here suggest possible new strategies for controlling the faradic operation regime of OECTs sensors by chemical modification of the gate surface.

2019 - Mortality in Patients With Septic Shock by Multidrug Resistant Bacteria: Risk Factors and Impact of Sepsis Treatments [Articolo su rivista]
Busani, Stefano; Serafini, Giulia; Mantovani, Elena; Venturelli, Claudia; Giannella, Maddalena; Viale, Pierluigi; Mussini, Cristina; Cossarizza, Andrea; Girardis, Massimo
abstract

Background: Patients with septic shock by multidrug resistant (MDR) microorganism maybe considered a specific population of critical patients at very high risk of death in whom the effects of standard sepsis treatment has never been assessed. The objective of this retrospective analysis was to evaluate the risk factors for 30-day mortality and the impact of sepsis management in patients with septic shock caused by MDR bacteria. Methods: Patients with septic shock by MDR bacteria admitted to the mixed intensive care unit (ICU) of Modena University Hospital during a 6-year period were studied. The clinical and microbiological characteristics and sepsis treatments provided were analyzed and compared between survivors (S) and nonsurvivors (NS) at 30 days after septic shock appearance. Results: Ninety-four patients were studied. All therapeutic interventions applied to patients during their ICU stay did not show statistical significance between S and NS groups, except for administration of immunoglobulin M (IgM) preparation which were provided more frequently in S group (P <.05). At the multivariate adjusted analysis, preexisting cancer (odds ratio [OR] = 2.965) and Acinetobacter baumannii infections (OR = 3.197) were independently correlated with an increased risk of 30-day mortality, whereas treatment with IgM preparation was protective (OR = 0.283). Conclusions: This retrospective study showed that in patients with septic shock caused by MDR bacteria, history of cancer and infection sustained by A baumannii increase the risk of mortality and that standard sepsis treatments do not seem to provide any protective effect. Adjunctive therapy with IgM preparation seems to be beneficial, but further appropriate studies are needed to confirm the results observed.

2019 - Sporadic and hereditary hemangioblastoma: The role of endothelial cells [Articolo su rivista]
Feletti, A.; Bianchini, E.; De Gaetano, A.; Gibellini, L.; De Biasi, S.; Pavesi, G.; Mattioli, A. V.; Nasi, M.; Cossarizza, A.; Pinti, M.
abstract

2019 - Synthesis and anticancer activity of CDDO and CDDO-me, two derivatives of natural triterpenoids [Articolo su rivista]
Borella, R.; Forti, L.; Gibellini, L.; De Gaetano, A.; De Biasi, S.; Nasi, M.; Cossarizza, A.; Pinti, M.
abstract

Triterpenoids are natural compounds synthesized by plants through cyclization of squalene, known for their weak anti-inflammatory activity. 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), and its C28 modified derivative, methyl-ester (CDDO-Me, also known as bardoxolone methyl), are two synthetic derivatives of oleanolic acid, synthesized more than 20 years ago, in an attempt to enhance the anti-inflammatory behavior of the natural compound. These molecules have been extensively investigated for their strong ability to exert antiproliferative, antiangiogenic, and antimetastatic activities, and to induce apoptosis and differentiation in cancer cells. Here, we discuss the chemical properties of natural triterpenoids, the pathways of synthesis and the biological effects of CDDO and its derivative CDDO-Me. At nanomolar doses, CDDO and CDDO-Me have been shown to protect cells and tissues from oxidative stress by increasing the transcriptional activity of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). At doses higher than 100 nM, CDDO and CDDO-Me are able to modulate the differentiation of a variety of cell types, both tumor cell lines or primary culture cell, while at micromolar doses these compounds exert an anticancer effect in multiple manners; by inducing extrinsic or intrinsic apoptotic pathways, or autophagic cell death, by inhibiting telomerase activity, by disrupting mitochondrial functions through Lon protease inhibition, and by blocking the deubiquitylating enzyme USP7. CDDO-Me demonstrated its efficacy as anticancer drugs in different mouse models, and versus several types of cancer. Several clinical trials have been started in humans for evaluating CDDO-Me efficacy as anticancer and anti-inflammatory drug; despite promising results, significant increase in heart failure events represented an obstacle for the clinical use of CDDO-Me.

2019 - Thymus Imaging Detection and Size Is Inversely Associated With Metabolic Syndrome and Frailty in People With HIV [Articolo su rivista]
Guaraldi, Giovanni; Franconi, Iacopo; Milic, Jovana; Besutti, Giulia; Pintassilgo, Ines; Scaglioni, Riccardo; Ligabue, Guido; Riva, Nicoletta; Raimondi, Alessandro; Menozzi, Marianna; Carli, Federica; Zona, Stefano; Santoro, Antonella; Malagoli, Andrea; Borghi, Vanni; Torricelli, Pietro; Cossarizza, Andrea; Mussini, Cristina
abstract

People with HIV (PWH) may experience accentuating aging in relation to immuno-activation. Little is known regarding thymus (THY) involution in this process. We sought to investigate the relationship between THY imaging detection/size and clinically relevant aging outcomes such as metabolic syndrome (MetS), multimorbidity (MM), and frailty in PWH.

2018 - DNA Topoisomerase I differentially modulates R-loops across the human genome [Articolo su rivista]
Manzo, S. G.; Hartono, S. R.; Sanz, L. A.; Marinello, J.; De Biasi, S.; Cossarizza, A.; Capranico, G.; Chedin, F.
abstract

Background: Co-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown. Results: Here, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains are characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay. Conclusions: Our findings reveal new properties of Top1 in regulating R-loop homeostasis in a context-dependent manner and suggest a potential role for Top1 in modulating the replication process via R-loop formation.

2018 - EGOFET Peptide Aptasensor for Label-Free Detection of Inflammatory Cytokines in Complex Fluids [Articolo su rivista]
Berto, Marcello; Diacci, Chiara; D'Agata, Roberta; Pinti, Marcello; Bianchini, Elena; Lauro, Michele Di; Casalini, Stefano; Cossarizza, Andrea; Berggren, Magnus; Simon, Daniel; Spoto, Giuseppe; Biscarini, Fabio; Bortolotti, Carlo A.
abstract

Organic electronic transistors are rapidly emerging as ultrahigh sensitive label-free biosensors suited for point-of-care or in-field deployed applications. Most organic biosensors reported to date are based on immunorecognition between the relevant biomarkers and the immobilized antibodies, whose use is hindered by large dimensions, poor control of sequence, and relative instability. Here, an electrolyte-gated organic field effect transistor (EGOFET) biosensor where the recognition units are surface immobilized peptide aptamers (Affimer proteins) instead of antibodies is reported. Peptide aptasensor for the detection of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) with a 1 × 10−12 M limit of detection is demonstrated. Ultralow sensitivity is met even in complex solutions such as cell culture media containing 10% serum, demonstrating the remarkable ligand specificity of the device. The device performances, together with the simple one-step immobilization strategy of the recognition moieties and the low operational voltages, all prompt EGOFET peptide aptasensors as candidates for early diagnostics and monitoring at the point-of-care.

2018 - Exploring viral reservoir: The combining approach of cell sorting and droplet digital PCR [Articolo su rivista]
Gibellini, Lara; Pecorini, Simone; De Biasi, Sara; Pinti, Marcello; Bianchini, Elena; DE GAETANO, Anna; Digaetano, Margherita; Pullano, Rosalberta; Lo Tartaro, Domenico; Iannone, Anna; Mussini, Cristina; Cossarizza, Andrea; Nasi, Milena
abstract

Combined antiretroviral therapy (cART) blocks different steps of HIV replication and maintains plasma viral RNA at undetectable levels. The virus can remain in long-living cells and create a reservoir where HIV can restart replicating after cART discontinuation. A persistent viral production triggers and maintains a persistent immune activation, which is a well-known feature of chronic HIV infection, and contributes either to precocious aging, or to the increased incidence of morbidity and mortality of HIV positive patients. The new frontier of the treatment of HIV infection is nowadays eradication of the virus from all host cells and tissues. For this reason, it is crucial to have a clear and precise idea of where the virus hides, and which are the cells that keep it silent. Important efforts have been made to improve the detection of viral reservoirs, and new techniques are now giving the opportunity to characterize viral reservoirs. Among these techniques, a strategic approach based upon cell sorting and droplet digital PCR (ddPCR) is opening new horizons and opportunities of research. This review provides an overview of the methods that combine cell sorting and ddPCR for the quantification of HIV DNA in different cell types, and for the detection of its maintenance.

2018 - HIV prevalence and correlations in prisons in different regions of the world: A review article [Articolo su rivista]
Golrokhi, R.; Farhoudi, B.; Taj, L.; Pahlaviani, F. G.; Mazaheri-Tehrani, E.; Cossarizza, A.; Seyedalinaghi, S.; Mohraz, M.; Voltarelli, F. A.
abstract

The prevalence of HIV is substantially higher among prisoners than the general population, while the incidence varies considerably in different regions around the world. If we consider Sub-Saharan Africa as one region with the highest prevalence of HIV, data on African prisoners would be limited. Despite the low prevalence of HIV in the Middle East and North Africa, its incidence is rising in these regions with a few exceptions; there are insufficient data on HIV prevalence in prisons. A similar situation is present in both Pacific and Central Asia as well as in Eastern Europe. A high rate of infection is mainly observed among prisoners in Western and Central parts of Europe, since the data from these are more available than other parts. Nowadays, the sexual transmission mode and tattooing are important ways in HIV risks among prisoners after injecting drug use as the most common route of HIV transmission in all regions. However, it is difficult to compare and analyze the prevalence of HIV among prisoners in different regions regarding the limited data and different methods which they used in collecting data. Eventually, it can certainly be said that prisons are one of the high-risk places for HIV transmission; on the other hand, can be a suitable place for implementing HIV case-finding, linkage to treatment and harm reduction programs.

2018 - High speed flow cytometry allows the detection of circulating endothelial cells in hemangioblastoma patients [Articolo su rivista]
De Biasi, Sara; Gibellini, Lara; Feletti, Alberto; Pavesi, Giacomo; Bianchini, Elena; Lo Tartaro, Domenico; Pecorini, Simone; De Gaetano, Anna; Pullano, Rosalberta; Nasi, Milena; Pinti, Marcello; Cossarizza, Andrea; Boraldi, Federica
abstract

Circulating endothelial cells (CECs) detach from the intima monolayer after endothelial damages. Their circulating endothelial progenitors (CEPs) represent less than 0.01% of nucleated blood cells. Increased levels of CECs and CEPs have been detected in patients with several types of cancer, suggesting that they could be a useful blood-based marker for detecting a tumor, or for monitoring its clinical course. However, their routine monitoring is time consuming and technically challenging. Here, we present a flow cytometry method for quantifying such cells in a cohort of patients with hemangioblastoma (HB). HB is a rare benign tumor, responsible for 1-2.5% of primary intracranial tumors and up to 10% of spinal cord tumors, and for which no tools are available to predict the onset or recurrence in patients undergoing surgical removal of tumor mass. This method allowed us to accurately quantifying CEC and CEP before and after surgery. CEPs are present at high levels in HB patients than control before intervention, and decrease after tumor removal, suggesting that their percentage could represent a valid tool to monitor the disease onset and recurrence.

2018 - Human dental pulp stem cells expressing STRO-1, c-kit and CD34 markers in peripheral nerve regeneration [Articolo su rivista]
Carnevale, Gianluca; Pisciotta, Alessandra; Riccio, Massimo; Bertoni, Laura; DE BIASI, Sara; Gibellini, Lara; Zordani, Alessio; Cavallini, Gian Maria; LA SALA, Giovanni Battista; Bruzzesi, Giacomo; Ferrari, Adriano; Cossarizza, Andrea; DE POL, Anto
abstract

Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The application of stem cells able to differentiate in Schwann cell-like cells in vitro and in vivo, could represent an attractive therapeutic approach for the treatment of nerve injuries. Further, stem cells sources sharing the same embryological origin as Schwann cells might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal subpopulation of human STRO-1(+) /c-Kit(+) /CD34(+) DPSCs, expressing P75(NTR) , nestin and SOX-10, to differentiate into Schwann cell-like cells in vitro and to promote axonal regeneration in vivo, which led to functional recovery as measured by sustained gait improvement, in animal rat model of peripheral nerve injury. Transplanted human dental pulp stem cells (hDPSCs) engrafted into sciatic nerve defect, as revealed by the positive staining against human nuclei, showed the expression of typical Schwann cells markers, S100b and, noteworthy, a significant number of myelinated axons was detected. Moreover, hDPSCs promoted axonal regeneration from proximal to distal stumps 1 month after transplantation. This study demonstrates that STRO-1(+) /c-Kit(+) /CD34(+) hDPSCs, associated with neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues. Copyright © 2016 John Wiley & Sons, Ltd.

2018 - Indications for starting ART [Articolo su rivista]
Mussini, Cristina; Cossarizza, Andrea
abstract

Invited Editorial for explaining when. starting antiretroviral therapy.

2018 - LonP1 Differently Modulates Mitochondrial Function and Bioenergetics of Primary Versus Metastatic Colon Cancer Cells [Articolo su rivista]
Gibellini, L; Losi, L; De Biasi, S; Nasi, M; Lo Tartaro, D; Pecorini, S; Patergnani, S; Pinton, P; De Gaetano, A; Carnevale, G; Pisciotta, A; Mariani, F; Roncucci, L; Iannone, A; Cossarizza, A; Pinti, M.
abstract

Mitochondrial Lon protease (LonP1) is a multi-function enzyme that regulates mitochondrial functions in several human malignancies, including colorectal cancer (CRC). The mechanism(s) by which LonP1 contributes to colorectal carcinogenesis is not fully understood. We found that silencing LonP1 leads to severe mitochondrial impairment and apoptosis in colon cancer cells. Here, we investigate the role of LonP1 in mitochondrial functions, metabolism, and epithelial-mesenchymal transition (EMT) in colon tumor cells and in metastasis. LonP1 was almost absent in normal mucosa, gradually increased from aberrant crypt foci to adenoma, and was most abundant in CRC. Moreover, LonP1 was preferentially upregulated in colorectal samples with mutated p53 or nuclear β-catenin, and its overexpression led to increased levels of β-catenin and decreased levels of E-cadherin, key proteins in EMT, in vitro. LonP1 upregulation also induced opposite changes in oxidative phosphorylation, glycolysis, and pentose pathway in SW480 primary colon tumor cells when compared to SW620 metastatic colon cancer cells. In conclusion, basal LonP1 expression is essential for normal mitochondrial function, and increased LonP1 levels in SW480 and SW620 cells induce a metabolic shift toward glycolysis, leading to EMT.

2018 - Rapamycin treatment for amyotrophic lateral sclerosis protocol for a phase II randomized, double-blind, placebo-controlled, multicenter, clinical trial (RAP-ALS trial) [Articolo su rivista]
Mandrioli, J.; D'Amico, R.; Zucchi, E.; Gessani, A.; Fini, N.; Fasano, A.; Caponnetto, C.; Chio, A.; Bella, E. D.; Lunetta, C.; Mazzini, L.; Marinou, K.; Soraru, G.; De Biasi, S.; Lo Tartaro, D.; Pinti, M.; Nichelli, P.; Vicini, R.; Cabona, C.; Calvo, A.; Moglia, C.; Manera, U.; Fuda, G.; Canosa, A.; Ilardi, A.; Lauria, G.; Dalla Bella, E.; Gerardi, F.; Scognamiglio, A.; De Marchi, F.; Mora, G.; Gizzi, M.; Cossarizza, A.
abstract

Introduction: Misfolded aggregated proteins and neuroinflammation significantly contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, hence representing therapeutic targets to modify disease expression. Rapamycin inhibits mechanistic target of Rapamycin (mTOR) pathway and enhances autophagy with demonstrated beneficial effects in neurodegeneration in cell line and animal models, improving phenotype in SQSTM1 zebrafish, in Drosophila model of ALS-TDP, and in the TDP43 mouse model, in which it reduced neuronal loss and TDP43 inclusions. Rapamycin also expands regulatory T lymphocytes (Treg) and increased Treg levels are associated with slow progression in ALS patients. Therefore, we planned a randomized clinical trial testing Rapamycin treatment in ALS patients. Methods: RAP-ALS is a phase II randomized, double-blind, placebo-controlled, multicenter (8 ALS centers in Italy), clinical trial. The primary aim is to assess whether Rapamycin administration increases Tregs number in treated patients compared with control arm. Secondary aims include the assessment of safety and tolerability of Rapamycin in patients with ALS; the minimum dosage to have Rapamycin in cerebrospinal fluid; changes in immunological (activation and homing of T, B, NK cell subpopulations) and inflammatory markers, and on mTOR downstream pathway (S6RP phosphorylation); clinical activity (ALS Functional Rating Scale-Revised, survival, forced vital capacity); and quality of life (ALSAQ40 scale). Discussion: Rapamycin potentially targets mechanisms at play in ALS (i.e., autophagy and neuroinflammation), with promising preclinical studies. It is an already approved drug, with known pharmacokinetics, already available and therefore with significant possibility of rapid translation to daily clinics. Findings will provide reliable data for further potential trials. Ethics and dissemination: The study protocol was approved by the Ethics Committee of Azienda Ospedaliero Universitaria of Modena and by the Ethics Committees of participating centers (Eudract n. 2016-002399-28) based on the Helsinki declaration.

2018 - Switching to dual/monotherapy determines an increase in CD8+ in HIV-infected individuals: An observational cohort study [Articolo su rivista]
Mussini, C.; Lorenzini, P.; Cozzi-Lepri, A.; Marchetti, G.; Rusconi, S.; Gori, A.; Nozza, S.; Lichtner, M.; Antinori, A.; Cossarizza, Andrea; d'Arminio Monforte, A.; Castagna, A.; Castelli, F.; Cauda, R.; Di Perri, G.; Galli, M.; Iardino, R.; Ippolito, G.; Lazzarin, A.; Marchetti, G. C.; Perno, C. F.; Rezza, G.; von Schloesser, F.; Viale, P.; Ceccherini-Silberstein, F.; Girardi, E.; Lo Caputo, S.; Puoti, M.; Andreoni, M.; Ammassari, A.; Balotta, C.; Bandera, A.; Bonfanti, P.; Bonora, S.; Borderi, M.; Calcagno, A.; Calza, L.; Capobianchi, M. R.; Cingolani, A.; Cinque, P.; De Luca, A.; Di Biagio, A.; Gianotti, N.; Guaraldi, G.; Lapadula, G.; Madeddu, G.; Maggiolo, F.; Marcotullio, S.; Monno, L.; Quiros Roldan, E.; Rossotti, R.; Santoro, M. M.; Saracino, A.; Zaccarelli, M.; Fanti, I.; Galli, L.; Rodano, A.; Shanyinde, M.; Tavelli, A.; Carletti, F.; Carrara, S.; Di Caro, A.; Graziano, S.; Petrone, F.; Prota, G.; Quartu, S.; Truffa, S.; Giacometti, A.; Costantini, A.; Barocci, V.; Angarano, G.; Santoro, C.; Suardi, C.; Donati, V.; Verucchi, G.; Minardi, C.; Quirino, T.; Abeli, C.; Manconi, P. E.; Piano, P.; Cacopardo, B.; Celesia, B.; Vecchiet, J.; Falasca, K.; Sighinolfi, L.; Segala, D.; Blanc, P.; Vichi, F.; Cassola, G.; Viscoli, C.; Alessandrini, A.; Bobbio, N.; Mazzarello, G.; Mastroianni, C.; Pozzetto, I.; Caramma, I.; Chiodera, A.; Milini, P.; Rizzardini, G.; Moioli, M. C.; Piolini, R.; Ridolfo, A. L.; Salpietro, S.; Tincati, C.; Puzzolante, C.; Abrescia, N.; Chirianni, A.; Borgia, G.; Orlando, R.; Bonadies, G.; Di Martino, F.; Gentile, I.; Maddaloni, L.; Cattelan, A. M.; Marinello, S.; Cascio, A.; Colomba, C.; Baldelli, F.; Schiaroli, E.; Parruti, G.; Sozio, F.; Magnani, G.; Ursitti, M. A.; Cristaudo, A.; Vullo, V.; Acinapura, R.; Baldin, G.; Capozzi, M.; Cicalini, S.; Fontanelli Sulekova, L.; Iaiani, G.; Latini, A.; Mastrorosa, I.; Plazzi, M. M.; Savinelli, S.; Vergori, A.; Cecchetto, M.; Viviani, F.; Bagella, P.; Rossetti, B.; Franco, A.; Fontana Del Vecchio, R.; Francisci, D.; Di Giuli, C.; Caramello, P.; Orofino, G. C.; Sciandra, M.; Bassetti, M.; Londero, A.; Pellizzer, G.; Manfrin, V.; Starnini, G.; Ialungo, A.
abstract

Background: The CD4/CD8 ratio has been associated with the risk of AIDS and non-AIDS events. We describe trends in immunological parameters in people who underwent a switch to monotherapy or dual therapy, compared to a control group remaining on triple antiretroviral therapy (ART). Methods: We included patients in Icona who started a three-drug combination ART regimen from an ART-naïve status and achieved a viral load ≤ 50 copies/mL; they were subsequently switched to another triple or to a mono or double regimen. Standard linear regression at fixed points in time (12-24 months after the switch) and linear mixed model analysis with random intercepts and slopes were used to compare CD4 and CD8 counts and their ratio over time according to regimen types (triple vs. dual and vs. mono). Results: A total of 1241 patients were included; 1073 switched to triple regimens, 104 to dual (72 with 1 nucleoside reverse transcriptase inhibitor (NRTI), 32 NRTI-sparing), and 64 to monotherapy. At 12 months after the switch, for the multivariable linear regression the mean change in the log10 CD4/CD8 ratio for patients on dual therapy was -0.03 (95% confidence interval (CI) -0.05, -0.0002), and the mean change in CD8 count was +99 (95% CI +12.1, +186.3), taking those on triple therapy as reference. In contrast, there was no evidence for a difference in CD4 count change. When using all counts, there was evidence for a significant difference in the slope of the ratio and CD8 count between people who were switched to triple (points/year change ratio = +0.056, CD8 = -25.7) and those to dual regimen (ratio = -0.029, CD8 = +110.4). Conclusions: We found an increase in CD8 lymphocytes in people who were switched to dual regimens compared to those who were switched to triple. Patients on monotherapy did not show significant differences. The long-term implications of this difference should be ascertained.

2018 - The Contribution of Poliomyelitis to President Roosevelt’s Heart Failure. A Lesson on the Importance of Vaccinations for Cardiovascular Prevention [Articolo su rivista]
Varotto, E.; Cossarizza, A.; Galassi, F. M.
abstract

2018 - The degree of HIV-1 amino acid variability is strictly related to different disease progression rates [Articolo su rivista]
Scutari, Rossana; Faieta, Monica; D’Arrigo, Roberta; Fabeni, Lavinia; Mussini, Cristina; Cossarizza, Andrea; Casoli, Claudio; Perno, Carlo Federico; Svicher, Valentina; Alteri, Claudia; Aquaro, Stefano
abstract

The aim of this study is to evaluate the amino acid variability of HIV-1 Gp41, C2–V3, and Nef in a group of patients characterized by different disease progression rates. HIV-1 sequences were collected from 19 Long term non progressor patients (LTNPs), 9 slow progressors (SPs), and 11 rapid progressors (RPs). Phylogenetic trees were estimated by MEGA 6. Differences in amino acid variability among sequences belonging to the 3 groups have been evaluated by amino acid divergence, Shannon entropy analysis, and the number of amino acid mutations (defined as amino acid variations compared with HxB2). The involvement of amino acid mutations on epitope rich regions was also investigated. The population was mainly composed of males (74.3%) and HIV-1 subtype B strains (B: 92.32%, CRF_12BF, A1, C: 2.56% each). Viral load (log10 copies/mL) and CD4+T cell count (cells/mm3) were 3.9 (3.5–4.2) and 618 (504–857) in LTNPs, 3.3 (2.8–4.7) and 463 (333–627) in SPs, and 4.6 (4.3–5.3) and 201 (110–254) in RPs. Gp41 and C2–V3 amino acid divergence was lower in LTNP and SP strains compared to RPs (median value: 0.085 and 0.091 vs. 0.114, p = 0.005 and 0.042) and a trend of lower variability was observed for Nef (p = 0.198). A lower entropy value was observed at 10, 3, and 7 positions of Gp41, C2–V3, and Nef belonging to LTNPs and at 7, 3, and 1 positions of Gp41, C2–V3, and Nef belonging to SPs compared with RPs (p < 0.05). Focusing on epitope rich regions, again a higher degree of conservation was observed in Gp41 and C2–V3 sequences belonging to LTNPs and SPs compared to those belonging to RPs. This study shows that the extent of amino acid variability correlates with a different HIV-1 progression rate. This variability also involves CTL epitope rich regions, thus suggesting its involvement in the immune escape process modulation.

2018 - The role of latency reversal agents in the cure of HIV: A review of current data [Articolo su rivista]
Bashiri, Kiandokht; Rezaei, Nima; Nasi, Milena; Cossarizza, Andrea
abstract

The definitive cure for human immunodeficiency virus type-1 (HIV) infection is represented by the eradication of the virus from the patient's body. To reach this result, cells that are infected but do not produce the virus must become recognizable to be killed by the immune system. For this purpose, drugs defined "latency reverting agents" (LRA) that reactivate viral production are under investigation. A few clinical studies have been performed in HIV-infected patients treated with LRA and combined antiretroviral therapy (cART). The strategy is thus to combine cART and LRA to reactivate the virus and unmask latently infected cells that, because of cART, cannot produce a fully competent form of the virus. Unmasked cells can present viral antigens to the immune system, that ultimately recognizes and kills such latently infected cells. This review reports and discusses recent studies that have been published on this topic.

2018 - Welcome to Prague CYTO 2018 [Articolo su rivista]
Cossarizza, A.; Wallace, P. K.
abstract

2017 - Activation of Fas/FasL pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells [Articolo su rivista]
CARNEVALE, Gianluca; Carpino, Guido; Cardinale, Vincenzo; PISCIOTTA, ALESSANDRA; RICCIO, Massimo; Bertoni, Laura; GIBELLINI, Lara; DE BIASI, SARA; Nevi, Lorenzo; Costantini, Daniele; Overi, Diletta; COSSARIZZA, Andrea; DE POL, Anto; Gaudio, Eugenio; Alvaro, Domenico
abstract

Intrahepatic cholangiocarcinoma (iCCA) represents a heterogeneous group of malignancies emerging from the biliary tree, often in the context of chronic bile ducts inflammation. The immunological features of iCCA cells and their capability to control the lymphocytes response have not yet been investigated. The aims of the present study were to evaluate the interaction between iCCA cells and human peripheral blood mononuclear cells (PBMCs) and the role of Fas/FasL in modulating T-cells and NK-cells response after direct co-culture. iCCA cells express high levels of Fas and FasL that increase after co-culture with PBMCs inducing apoptosis in CD4(+), CD8(+) T-cells and in CD56(+) NK-cells. In vitro, c-FLIP is expressed in iCCA cells and the co-culture with PBMCs induces an increase of c-FLIP in both iCCA cells and biliary tree stem cells. This c-FLIP increase does not trigger the caspase cascade, thus hindering apoptotis of iCCA cells which, instead, underwent proliferation. The increased expression of Fas, FasL and c-FLIP is confirmed in situ, in human CCA and in primary sclerosing cholangitis. In conclusion our data indicated that iCCA cells have immune-modulatory properties by which they induce apoptosis of T and NK cells, via Fas/FasL pathway, and escape inflammatory response by up-regulating c-FLIP system.

2017 - Ageing and inflammation in patients with HIV infection [Articolo su rivista]
Nasi, Milena; DE BIASI, Sara; Gibellini, Lara; Bianchini, Elena; Pecorini, S.; Bacca, V.; Guaraldi, Giovanni; Mussini, Cristina; Pinti, Marcello; Cossarizza, Andrea
abstract

Nowadays, HIV+ patients have an expected lifespan that is only slightly shorter than healthy individuals. For this reason, along with the fact that infection can be acquired at a relatively advanced age, the effects of ageing on HIV+ people have begun to be evident. Successful anti-viral treatment is, on one hand, responsible for the development of side effects related to drug toxicity; on the other hand, it is not able to inhibit the onset of several complications caused by persistent immune activation and chronic inflammation. Therefore, patients with a relatively advanced age, i.e. aged more than 50 years, can experience pathologies that affect much older citizens. HIV+ individuals with non-AIDS-related complications can thus come to the attention of clinicians because of the presence of neurocognitive disorders, cardiovascular diseases, metabolic syndrome, bone abnormalities and non-HIV-associated cancers. Chronic inflammation and immune activation, observed typically in elderly people and defined as 'inflammaging', can be present in HIV+ patients who experience a type of premature ageing, which affects the quality of life significantly. This relatively new condition is extremely complex, and important factors have been identified as well as the traditional behavioural risk factors, e.g. the toxicity of anti-retroviral treatments and the above-mentioned chronic inflammation leading to a functional decline and a vulnerability to injury or pathologies. Here, we discuss the role of inflammation and immune activation on the most important non-AIDS-related complications of chronic HIV infection, and the contribution of aging per se to this scenario.

2017 - Basic science and pathogenesis of ageing with HIV: Potential mechanisms and biomarkers [Articolo su rivista]
Lagathu, Claire; Cossarizza, Andrea; Bã©rã©ziat, Vã©ronique; Nasi, Milena; Capeau, Jacqueline; Pinti, Marcello
abstract

The increased prevalence of age-related comorbidities and mortality is worrisome in ageing HIV-infected patients. Here, we aim to analyse the different ageing mechanisms with regard to HIV infection. Ageing results from the time-dependent accumulation of random cellular damage. Epigenetic modifications and mitochondrial DNA haplogroups modulate ageing. In antiretroviral treatment-controlled patients, epigenetic clock appears to be advanced, and some haplogroups are associated with HIV infection severity. Telomere shortening is enhanced in HIV-infected patients because of HIV and some nucleoside analogue reverse transcriptase inhibitors. Mitochondria-related oxidative stress and mitochondrial DNA mutations are increased during ageing and also by some nucleoside analogue reverse transcriptase inhibitors. Overall, increased inflammation or 'inflammageing' is a major driver of ageing and could result from cell senescence with secreted proinflammatory mediators, altered gut microbiota, and coinfections. In HIV-infected patients, the level of inflammation and innate immunity activation is enhanced and related to most comorbidities and to mortality. This status could result, in addition to age, from the virus itself or viral protein released from reservoirs, from HIV-enhanced gut permeability and dysbiosis, from antiretroviral treatment, from frequent cytomegalovirus and hepatitis C virus coinfections, and also from personal and environmental factors, as central fat accumulation or smoking. Adaptive immune activation and immunosenescence are associated with comorbidities and mortality in the general population but are less predictive in HIV-infected patients. Biomarkers to evaluate ageing in HIV-infected patients are required. Numerous systemic or cellular inflammatory, immune activation, oxidative stress, or senescence markers can be tested in serum or peripheral blood mononuclear cells. The novel European Study to Establish Biomarkers of Human Ageing MARK-AGE algorithm, evaluating the biological age, is currently assessed in HIV-infected patients and reveals an advanced biological age. Some enhanced inflammatory or innate immune activation markers are interesting but still not validated for the patient's follow-up. To be able to assess patients' biological age is an important objective to improve their healthspan.

2017 - Geriatric-HIV medicine: A science in its infancy [Articolo su rivista]
Guaraldi, Giovanni; Cossarizza, Andrea
abstract

not available

2017 - Guidelines for the use of flow cytometry and cell sorting in immunological studies [Articolo su rivista]
Cossarizza, Andrea; Chang, Hyun-dong; Radbruch, Andreas; Andrã¤, Immanuel; Annunziato, Francesco; Bacher, Petra; Barnaba, Vincenzo; Battistini, Luca; Bauer, Wolfgang M.; Baumgart, Sabine; Becher, Burkhard; Beisker, Wolfgang; Berek, Claudia; Blanco, Alfonso; Borsellino, Giovanna; Boulais, Philip E.; Brinkman, Ryan R.; Bã¼scher, Martin; Busch, Dirk H.; Bushnell, Timothy P.; Cao, Xuetao; Cavani, Andrea; Chattopadhyay, Pratip K.; Cheng, Qingyu; Chow, Sue; Clerici, Mario; Cooke, Anne; Cosma, Antonio; Cosmi, Lorenzo; Cumano, Ana; Dang, Van Duc; Davies, Derek; De Biasi, Sara; Del Zotto, Genny; Della Bella, Silvia; Dellabona, Paolo; Deniz, Gã¼nnur; Dessing, Mark; Diefenbach, Andreas; Di Santo, James; Dieli, Francesco; Dolf, Andreas; Donnenberg, Vera S.; Dã¶rner, Thomas; Ehrhardt, Gã¶tz R. A.; Endl, Elmar; Engel, Pablo; Engelhardt, Britta; Esser, Charlotte; Everts, Bart; Falk, Christine S.; Fehniger, Todd A.; Filby, Andrew; Fillatreau, Simon; Follo, Marie; Fã¶rster, Irmgard; Foster, John; Foulds, Gemma A.; Frenette, Paul S.; Galbraith, David; Garbi, Natalio; Garcã­a-godoy, Maria Dolores; Ghoreschi, Kamran; Gibellini, Lara; Goettlinger, Christoph; Goodyear, Carl S.; Gori, Andrea; Grogan, Jane; Gross, Mor; Grã¼tzkau, Andreas; Grummitt, Daryl; Hahn, Jonas; Hammer, Quirin; Hauser, Anja E.; Haviland, David L.; Hedley, David; Herrera, Guadalupe; Herrmann, Martin; Hiepe, Falk; Holland, Tristan; Hombrink, Pleun; Houston, Jessica P.; Hoyer, Bimba F.; Huang, Bo; Hunter, Christopher A.; Iannone, Anna; Jã¤ck, Hans-martin; Jã¡vega, Beatriz; Jonjic, Stipan; Juelke, Kerstin; Jung, Steffen; Kaiser, Toralf; Kalina, Tomas; Keller, Baerbel; Khan, Srijit; Kienhã¶fer, Deborah; Kroneis, Thomas; Kunkel, Dã©sirã©e; Kurts, Christian; Kvistborg, Pia; Lannigan, Joanne; Lantz, Olivier; Larbi, Anis; Leibundgut-landmann, Salome; Leipold, Michael D.; Levings, Megan K.; Litwin, Virginia; Liu, Yanling; Lohoff, Michael; Lombardi, Giovanna; Lopez, Lilly; Lovett-racke, Amy; Lubberts, Erik; Ludewig, Burkhard; Lugli, Enrico; Maecker, Holden T.; Martrus, Glã²ria; Matarese, Giuseppe; Mauerã¶der, Christian; Mcgrath, Mairi; Mcinnes, Iain; Mei, Henrik E.; Melchers, Fritz; Melzer, Susanne; Mielenz, Dirk; Mills, Kingston; Mjã¶sberg, Jenny; Moore, Jonni; Moran, Barry; Moretta, Alessandro; Moretta, Lorenzo; Mosmann, Tim R.; Mã¼ller, Susann; Mã¼ller, Werner; Mã¼nz, Christian; Multhoff, Gabriele; Munoz, Luis Enrique; Murphy, Kenneth M.; Nakayama, Toshinori; Nasi, Milena; Neudã¶rfl, Christine; Nolan, John; Nourshargh, Sussan; O'connor, Josã©-enrique; Ouyang, Wenjun; Oxenius, Annette; Palankar, Raghav; Panse, Isabel; Peterson, Pã¤rt; Peth, Christian; Petriz, Jordi; Philips, Daisy; Pickl, Winfried; Piconese, Silvia; Pinti, Marcello; Pockley, A. Graham; Podolska, Malgorzata Justyna; Pucillo, Carlo; Quataert, Sally A.; Radstake, Timothy R. D. J.; Rajwa, Bartek; Rebhahn, Jonathan A.; Recktenwald, Diether; Remmerswaal, Ester B. M.; Rezvani, Katy; Rico, Laura G.; Robinson, J. Paul; Romagnani, Chiara; Rubartelli, Anna; Ruland, Jã¼rgen; Sakaguchi, Shimon; Sala-de-oyanguren, Francisco; Samstag, Yvonne; Sanderson, Sharon; Sawitzki, Birgit; Scheffold, Alexander; Schiemann, Matthias; Schildberg, Frank; Schimisky, Esther; Schmid, Stephan A; Schmitt, Steffen; Schober, Kilian; Schã¼ler, Thomas; Schulz, Axel Ronald; Schumacher, Ton; Scotta, Cristiano; Shankey, T. Vincent; Shemer, Anat; Simon, Anna-katharina; Spidlen, Josef; Stall, Alan M.; Stark, Regina; Stehle, Christina; Stein, Merle; Steinmetz, Tobit; Stockinger, Hannes; Takahama, Yousuke; Tarnok, Attila; Tian, Zhigang; Toldi, Gergely; Tornack, Julia; Traggiai, Elisabetta; Trotter, Joe; Ulrich, Henning; Van Der Braber, Marlous; Van Lier, Renã© A. W.; Veldhoen, Marcello; Vento-asturias, Salvador; Vieira, Paulo; Voehringer, David; Volk, Hans-dieter; Von Volkmann, Konrad; Waisman, Ari; Walker, Rachael; Ward, Michael D.; Warnatz, Klaus; Warth, Sarah; Watson, James V.; Watzl, Carsten; Wegener, Leonie; Wi
abstract

Nessun abstract disponibile

2017 - HIV-DNA content in different CD4+ T-cell subsets correlates with CD4+ cell : CD8+ cell ratio or length of efficient treatment [Articolo su rivista]
Gibellini, Lara; Pecorini, Simone; DE BIASI, Sara; Bianchini, Elena; Digaetano, Margherita; Pinti, Marcello; Carnevale, Gianluca; Borghi, Vanni; Guaraldi, Giovanni; Mussini, Cristina; Cossarizza, Andrea; Nasi, Milena
abstract

Objectives: HIV establishes a latent infection at different degrees within naïve (TN) or central (TCM) and effector memory (TEM) CD4+ T cell. Studying patients in whom HIV production was suppressed by combined antiretroviral therapy, our main aim was to find which factors are related or can influence intracellular viral reservoir in different CD4+ T-cell subsets. Methods: We enrolled 32 HIV+ patients successfully treated for more than 2 years, with a CD4+ T-cell count more than 500 cells/μl and plasma viremia undetectable from at least 1 year. Proviral HIV-DNA, the amount of cells expressing signal-joint T-cell receptor rearrangement excision circles and telomere length were quantified by droplet digital PCR in highly purified, sorted CD4+ T-cell subsets; plasma IL-7 and IL-15 were measured by ELISA. Results: HIV-DNA was significantly lower in TN cells compared with TCM or to TEM. Conversely, TN cells contained more signal-joint T-cell receptor rearrangement excision circles compared with TCM or to TEM; no appreciable changes were observed in telomere length. HIV-DNA content was significantly higher in TN and TCM cells, but not in TEM, from patients with shorter time of treatment, or in those with lower CD4+ : CD8+ ratio. Conclusion: Length of treatment or recovery of CD4+ : CD8+ ratio significantly influences viral reservoir in both TN and TCM. Measuring HIV-DNA in purified lymphocyte populations allows a better monitoring of HIV reservoir and could be useful for designing future eradication strategies.

2017 - Injury-induced immunosuppression: we are finally on the right track? [Articolo su rivista]
Busani, Stefano; Cossarizza, Andrea; Girardis, Massimo
abstract

Non abstract

2017 - Invariant natural killer T cells and mucosal-associated invariant T cells in multiple sclerosis [Articolo su rivista]
Bianchini, Elena; DE BIASI, Sara; Simone, ANNA MARIA; Ferraro, Diana; Sola, Patrizia; Cossarizza, Andrea; Pinti, Marcello
abstract

Multiple sclerosis (MS) is a chronic progressive inflammatory demyelinating disorder of the central nervous system, and in several countries is a leading cause of permanent neurological disability in young adults, particularly women. MS is considered an autoimmune disease, caused by an aberrant immune response to environmental triggers in genetically susceptible subjects. However, the contribution of the innate or of the adaptive immune system to the development and progression of the disease has not yet been fully elucidated. Innate-like T lymphocytes are unconventional T cells that bridge the innate and adaptive arms of the immune system, because they use a T cell receptor to sense external ligands, but behave like innate cells when they rapidly respond to stimuli. These cells could play an important role in the pathogenesis of MS. Here, we focus on invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells, and we review the current knowledge on their biology and possible involvement in MS. Although several studies have evaluated the frequency and functions of iNKT and MAIT cells both in MS patients and in experimental mouse models, contradictory observations have been reported, and it is not clear whether they exert a protective or a pro-inflammatory and harmful role. A better understanding of how immune cells are involved in MS, and of their interactions could be of great interest for the development of new therapeutic strategies.

2017 - Novelties in evaluation and monitoring of human immunodeficiency virus-1 infection: Is standard virological suppression enough for measuring antiretroviral treatment success? [Articolo su rivista]
Svicher, V.; Marchetti, G.; Ammassari, A.; Ceccherini-Silberstein, F.; Sarmati, L.; Andreoni, M.; Angarano, G.; Antinori, A.; Antonelli, G.; Bandera, A.; Bonfanti, P.; Bonora, S.; Borderi, M.; Calcagno, A.; Capobianchi, M. R.; Castagna, A.; Castelli, F.; Cauda, R.; Celesia, B. M.; Chirianni, A.; Cicalini, S.; Cingolani, A.; Cinque, P.; Clementi, M.; Cossarizza, A.; Monforte, A. D.; D'Ettorre, G.; De Luca, A.; Di Biagio, A.; Di Perri, G.; Galli, M.; Gianotti, N.; Girardi, E.; Guaraldi, G.; Lazzarin, A.; Lichtner, M.; Lo Caputo, S.; Madeddu, G.; Maggi, P.; Maggiolo, F.; Marchetti, G. C.; Mastroianni, C.; Mussini, C.; Nozza, S.; Nunnari, G.; Parisi, S.; Parruti, G.; Perno, C. F.; Piconi, S.; Carla, M.; Ripamonti, D.; Rusconi, S.; Santoro, M.; Sterrantino, G.; Svicher, V.; Vullo, V.; Zaccarelli, M.; Zazzi, M.
abstract

The high potency and tolerability of the currently available antiretroviral drugs has modified HIV-1 infection from a life-threatening disease to a chronic illness. Nevertheless, some issues still remain open to optimize the management of HIV-1 infected patients in term of maintenance of virological suppression over time, identifying patients that could benefit from simplification therapy, and reducing co-mordibities driven by chronic inflammation. The availability of robust and affordable virological and immunological markers can help in solving these issues by providing information on the burden of HIV-1 reservoir in all the anatomical compartments in which the virus replicates as well as on persistent inflammation, immune activation and senescence despite successful virological suppression. In this light, this review is aimed at providing new insights (arising from a two-day Italian expert meeting hold in Rome in March 2016) in evaluation and monitoring of HIV-1 infection from a virological, immunological and clinical perspective. Particular attention has been focused on role of novel parameters (such as total HIV-1 DNA, residual viremia, and immunological markers) in optimizing treatment strategies, enhancing medical adherence, and individualizing monitoring.

2016 - Aging of the immune system: Focus on inflammation and vaccination [Articolo su rivista]
Pinti, Marcello; Appay, Victor; Campisi, Judith; Frasca, Daniela; Fülöp, Tamas; Sauce, Delphine; Larbi, Anis; Weinberger, Birgit; Cossarizza, Andrea
abstract

Major advances in preventing, delaying, or curing individual pathologies are responsible for an increasingly long life span in the developed parts of our planet, and indeed reaching eight to nine decades of life is nowadays extremely frequent. However, medical and sanitary advances have not prevented or delayed the underlying cause of the disparate pathologies occurring in the elderly: aging itself. The identification of the basis of the aging processes that drives the multiple pathologies and loss of function typical of older individuals is a major challenge in current aging research. Among the possible causes, an impairment of the immune system plays a major role, and indeed numerous studies have described immunological changes which occur with age. Far from the intention of being exhaustive, this review will focus on recent advances and views on the role that modifications of cell signalling and remodelling of the immune response play during human aging and longevity, paying particular attention to phenomena which are linked to the so called inflammaging process, such as dysregulation of innate immunity, altered T-cell or B-cell maturation and differentiation, as well as to the implications of immune aging for vaccination strategies in the elderly.

2016 - Anti-TNF-α drugs differently affect the TNFa-sTNFR system and monocyte subsets in patients with psoriasis [Articolo su rivista]
Gibellini, Lara; De Biasi, Sara; Bianchini, Elena; Bartolomeo, Regina; Fabiano, Antonella; Manfredini, Marco; Ferrar, Federica; Albertini, Giuseppe; Trenti, Tommaso; Nasi, Milena; Pinti, Marcello; Iannone, Anna; Salvarani, Carlo; Cossarizza, Andrea; Pellacani, Giovanni
abstract

TNF-a has a central role in the development and maintenance of psoriatic plaques, and its serum levels correlate with disease activity. Anti-TNF-a drugs are, however, ineffective in a relevant percentage of patients for reasons that are currently unknown. To understand whether the response to anti-TNF-a drugs is influenced by the production of anti-drug antibodies or by the modulation of the TNFa-TNFa receptor system, and to identify changes in monocyte phenotype and activity, we analysed 119 psoriatic patients who either responded or did not respond to different anti-TNF-a therapies (adalimumab, etanercept or infliximab), and measured plasma levels of TNF-a, TNF-a soluble receptors, drug and anti-drug antibodies. Moreover, we analyzed the production of TNF-a and TNF-α soluble receptors by peripheral blood mononuclear cells (PBMCs), and characterized different monocyte populations. We found that: i) the drug levels varied between responders and non-responders; ii) anti-infliximab antibodies were present in 15% of infliximab-treated patients, while anti-etanercept or anti-adalimumab antibodies were never detected; iii) plasma TNF-a levels were higher in patients treated with etanercept compared to patients treated with adalimumab or infliximab; iv) PBMCs from patients responding to adalimumab and etanercept produced more TNF-a and sTNFRII in vitro than patients responding to infliximab; v) PBMCs from patients not responding to infliximab produce higher levels of TNF-a and sTNFRII than patients responding to infliximab; vi) anti- TNF-a drugs significantly altered monocyte subsets. A complex remodelling of the TNFa-TNFa receptor system thus takes place in patients treated with anti-TNF-α drugs, that involves either the production of anti-drug antibodies or the modulation of monocyte phenotype or inflammatory activity.

2016 - Biorecognition in Organic Field Effect Transistors Biosensors: The Role of the Density of States of the Organic Semiconductor [Articolo su rivista]
Berto, Marcello; Casalini, Stefano; Di Lauro, Michele; Marasso, Simone L; Cocuzza, Matteo; Perrone, Denis; Pinti, Marcello; Cossarizza, Andrea; Pirri, Candido F; Simon, Daniel T; Berggren, Magnus; Zerbetto, Francesco; Bortolotti, Carlo Augusto; Biscarini, Fabio
abstract

Biorecognition is a central event in biological processes in the living systems that is also widely exploited in technological and health applications. We demonstrate that the Electrolyte Gated Organic Field Effect Transistor (EGOFET) is an ultrasensitive and specific device that allows us to quantitatively assess the thermodynamics of biomolecular recognition between a human antibody and its antigen, namely, the inflammatory cytokine TNFα at the solid/liquid interface. The EGOFET biosensor exhibits a superexponential response at TNFα concentration below 1 nM with a minimum detection level of 100 pM. The sensitivity of the device depends on the analyte concentration, reaching a maximum in the range of clinically relevant TNFα concentrations when the EGOFET is operated in the subthreshold regime. At concentrations greater than 1 nM the response scales linearly with the concentration. The sensitivity and the dynamic range are both modulated by the gate voltage. These results are explained by establishing the correlation between the sensitivity and the density of states (DOS) of the organic semiconductor. Then, the superexponential response arises from the energy-dependence of the tail of the DOS of the HOMO level. From the gate voltage-dependent response, we extract the binding constant, as well as the changes of the surface charge and the effective capacitance accompanying biorecognition at the electrode surface. Finally, we demonstrate the detection of TNFα in human-plasma derived samples as an example for point-of-care application.

2016 - Cellular Senescence, Immunosenescence and HIV [Capitolo/Saggio]
Fulop, T.; Herbein, G.; Cossarizza, A.; Witkowski, J. M.; Frost, E.; Dupuis, G.; Pawelec, G.; Larbi, A.
abstract

Aging is a complex biological process that leads to several physiological changes. Among these changes, the most striking are those involving the innate and adaptive parts of the immune system. Furthermore, these changes are associated with a low-grade inflammation called inflamm-aging, which is the result of several lifelong antigenic stimulations, including chronic viral infections such as cytomegalovirus. Immunosenescence, concomitantly with inflamm-aging, is considered as the leading cause of age-related diseases including cardiovascular, neurodegenerative and metabolic diseases, and cancer. HIV infection, once considered a unique deadly infectious disease, has now become a chronic disease with efficacious highly active antiretroviral therapy. This signifies that the treatment transforms HIV infection from a chronic infection to a chronic inflammatory disease. Most people with HIV infection become aged, and older adults have been contracting HIV infection. Thus, there is a great interest to study HIV infection in relation to immunosenescence and inflamm-aging to determine whether immunosenescence contributes to HIV infection, or if HIV is causing immunosenescence and, as such, represents a premature immunosenescence and accelerated aging. Although there are many similarities in the immune and inflammatory changes and the occurrence of age-related chronic diseases between normal aging and HIV infection, the interaction between these processes is not well understood, and consequently the concept that HIV infection is an accelerated aging model is questioned. Future studies are needed to effectively answer this question for the better care of HIV-infected elderly patients.

2016 - Decreased circulating mtDNA levels in professional male volleyball players [Articolo su rivista]
Nasi, Milena; Cristani, Alessandro; Pinti, Marcello; Lamberti, Igor; Gibellini, Lara; DE BIASI, Sara; Guazzaloca, Alessandro; Trenti, Tommaso; Cossarizza, Andrea
abstract

Purpose: Exercise exerts various effects on the immune system, and evidence is emerging on its anti-inflammatory effects; the mechanisms on the basis of these modifications are poorly understood. Mitochondrial DNA (mtDNA) released from damaged cells acts as a molecule containing the so-called damage-associated molecular patterns and can trigger sterile inflammation. Indeed, high plasma levels of mtDNA are associated to several inflammatory conditions and physiological aging and longevity. The authors evaluated plasma mtDNA in professional male volleyball players during seasonal training and the possible correlation between mtDNA levels and clinical parameters, body composition, and physical performance. Methods: Plasma mtDNA was quantified by real-time PCR every 2 mo in 12 professional volleyball players (PVPs) during 2 consecutive seasons. As comparison, 20 healthy nonathlete male volunteers (NAs) were analyzed. Results: The authors found lower levels of mtDNA in plasma of PVPs than in NAs. However, PVPs showed a decrease of circulating mtDNA only in the first season, while no appreciable variations were observed during the second season. No correlation was observed among mtDNA, hematochemical, and anthropometric parameters. Conclusions: Regular physical activity appeared associated with lower levels of circulating mtDNA, further confirming the protective, anti-inflammatory effect of exercise.

2016 - Emerging role of Lon protease as a master regulator of mitochondrial functions [Articolo su rivista]
Pinti, Marcello; Gibellini, Lara; Nasi, Milena; De Biasi, Sara; Bortolotti, Carlo Augusto; Iannone, Anna; Cossarizza, Andrea
abstract

Lon protease is a nuclear-encoded, mitochondrial ATP-dependent protease highly conserved throughout the evolution, crucial for the maintenance of mitochondrial homeostasis. Lon acts as a chaperone of misfolded proteins, and is necessary for maintaining mitochondrial DNA. The impairment of these functions has a deep impact on mitochondrial functionality and morphology. An altered expression of Lon leads to a profound reprogramming of cell metabolism, with a switch from respiration to glycolysis, which is often observed in cancer cells. Mutations of Lon, which likely impair its chaperone properties, are at the basis of a genetic inherited disease named of the cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

2016 - Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356 [Articolo su rivista]
Klionsky, D. J.; Abdelmohsen, K.; Abe, A.; Abedin, M. J.; Abeliovich, H.; Arozena, A. A.; Adachi, H.; Adams, C. M.; Adams, P. D.; Adeli, K.; Adhihetty, P. J.; Adler, S. G.; Agam, G.; Agarwal, R.; Aghi, M. K.; Agnello, M.; Agostinis, P.; Aguilar, P. V.; Aguirre-Ghiso, J.; Airoldi, E. M.; Ait-Si-Ali, S.; Akematsu, T.; Akporiaye, E. T.; Al-Rubeai, M.; Albaiceta, G. M.; Albanese, C.; Albani, D.; Albert, M. L.; Aldudo, J.; Algul, H.; Alirezaei, M.; Alloza, I.; Almasan, A.; Almonte-Beceril, M.; Alnemri, E. S.; Alonso, C.; Altan-Bonnet, N.; Altieri, D. C.; Alvarez, S.; Alvarez-Erviti, L.; Alves, S.; Amadoro, G.; Amano, A.; Amantini, C.; Ambrosio, S.; Amelio, I.; Amer, A. O.; Amessou, M.; Amon, A.; An, Z.; Anania, F. A.; Andersen, S. U.; Andley, U. P.; Andreadi, C. K.; Andrieu-Abadie, N.; Anel, A.; Ann, D. K.; Anoopkumar-Dukie, S.; Antonioli, M.; Aoki, H.; Apostolova, N.; Aquila, S.; Aquilano, K.; Araki, K.; Arama, E.; Aranda, A.; Araya, J.; Arcaro, A.; Arias, E.; Arimoto, H.; Ariosa, A. R.; Armstrong, J. L.; Arnould, T.; Arsov, I.; Asanuma, K.; Askanas, V.; Asselin, E.; Atarashi, R.; Atherton, S. S.; Atkin, J. D.; Attardi, L. D.; Auberger, P.; Auburger, G.; Aurelian, L.; Autelli, R.; Avagliano, L.; Avantaggiati, M. L.; Avrahami, L.; Azad, N.; Awale, S.; Bachetti, T.; Backer, J. M.; Bae, D. -H.; Bae, J. -S.; Bae, O. -N.; Bae, S. H.; Baehrecke, E. H.; Baek, S. -H.; Baghdiguian, S.; Bagniewska-Zadworna, A.; Bai, H.; Bai, J.; Bai, X. -Y.; Bailly, Y.; Balaji, K. N.; Balduini, W.; Ballabio, A.; Balzan, R.; Banerjee, R.; Banhegyi, G.; Bao, H.; Barbeau, B.; Barrachina, M. D.; Barreiro, E.; Bartel, B.; Bartolome, A.; Bassham, D. C.; Bassi, M. T.; Bast, R. C.; Basu, A.; Batista, M. T.; Batoko, H.; Battino, M.; Bauckman, K.; Baumgarner, B. L.; Bayer, K. U.; Beale, R.; Beaulieu, J. -F.; Beck, G. R.; Becker, C.; Beckham, J. D.; Bedard, P. -A.; Bednarski, P. J.; Begley, T. J.; Behl, C.; Behrends, C.; Behrens, G. M. N.; Behrns, K. E.; Bejarano, E.; Belaid, A.; Belleudi, F.; Benard, G.; Berchem, G.; Bergamaschi, D.; Bergami, M.; Berkhout, B.; Berliocchi, L.; Bernard, A.; Bernard, M.; Bernassola, F.; Bertolotti, A.; Bess, A. S.; Besteiro, S.; Bettuzzi, S.; Bhalla, S.; Bhattacharyya, S.; Bhutia, S. K.; Biagosch, C.; Bianchi, M. W.; Biard-Piechaczyk, M.; Billes, V.; Bincoletto, C.; Bingol, B.; Bird, S. W.; Bitoun, M.; Bjedov, I.; Blackstone, C.; Blanc, L.; Blanco, G. A.; Blomhoff, H. K.; Boada-Romero, E.; Bockler, S.; Boes, M.; Boesze-Battaglia, K.; Boise, L. H.; Bolino, A.; Boman, A.; Bonaldo, P.; Bordi, M.; Bosch, J.; Botana, L. M.; Botti, J.; Bou, G.; Bouche, M.; Bouchecareilh, M.; Boucher, M. -J.; Boulton, M. E.; Bouret, S. G.; Boya, P.; Boyer-Guittaut, M.; Bozhkov, P. V.; Brady, N.; Braga, V. M. M.; Brancolini, C.; Braus, G. H.; Bravo-San-Pedro, J. M.; Brennan, L. A.; Bresnick, E. H.; Brest, P.; Bridges, D.; Bringer, M. -A.; Brini, M.; Brito, G. C.; Brodin, B.; Brookes, P. S.; Brown, E. J.; Brown, K.; Broxmeyer, H. E.; Bruhat, A.; Brum, P. C.; Brumell, J. H.; Brunetti-Pierri, N.; Bryson-Richardson, R. J.; Buch, S.; Buchan, A. M.; Budak, H.; Bulavin, D. V.; Bultman, S. J.; Bultynck, G.; Bumbasirevic, V.; Burelle, Y.; Burke, R. E.; Burmeister, M.; Butikofer, P.; Caberlotto, L.; Cadwell, K.; Cahova, M.; Cai, D.; Cai, J.; Cai, Q.; Calatayud, S.; Camougrand, N.; Campanella, M.; Campbell, G. R.; Campbell, M.; Campello, S.; Candau, R.; Caniggia, I.; Cantoni, L.; Cao, L.; Caplan, A. B.; Caraglia, M.; Cardinali, C.; Cardoso, S. M.; Carew, J. S.; Carleton, L. A.; Carlin, C. R.; Carloni, S.; Carlsson, S. R.; Carmona-Gutierrez, D.; Carneiro, L. A. M.; Carnevali, O.; Carra, S.; Carrier, A.; Carroll, B.; Casas, C.; Casas, J.; Cassinelli, G.; Castets, P.; Castro-Obregon, S.; Cavallini, G.; Ceccherini, I.; Cecconi, F.; Cederbaum, A. I.; Cena, V.; Cenci, S.; Cerella, C.; Cervia, D.; Cetrullo, S.; Chaachouay, H.; Chae, H. -J.; Chagin, A. S.; Chai, C. -Y.; Chakrabarti, G.; Chamilos, G.; Chan, E. Y. W.; Chan, M. T. V.; Chandra, D.; Chandra, P.; Chang, C. -P.; Chang,
abstract

2016 - Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) [Articolo su rivista]
Klionsky, Daniel J; Abdelmohsen, Kotb; Abe, Akihisa; Abedin, Md Joynal; Abeliovich, Hagai; Acevedo Arozena, Abraham; Adachi, Hiroaki; Adams, Christopher M; Adams, Peter D; Adeli, Khosrow; Adhihetty, Peter J; Adler, Sharon G; Agam, Galila; Agarwal, Rajesh; Aghi, Manish K; Agnello, Maria; Agostinis, Patrizia; Aguilar, Patricia V; Aguirre Ghiso, Julio; Airoldi, Edoardo M; Ait Si Ali, Slimane; Akematsu, Takahiko; Akporiaye, Emmanuel T; Al Rubeai, Mohamed; Albaiceta, Guillermo M; Albanese, Chris; Albani, Diego; Albert, Matthew L; Aldudo, Jesus; Algül, Hana; Alirezaei, Mehrdad; Alloza, Iraide; Almasan, Alexandru; Almonte Beceril, Maylin; Alnemri, Emad S; Alonso, Covadonga; Altan Bonnet, Nihal; Altieri, Dario C; Alvarez, Silvia; Alvarez Erviti, Lydia; Alves, Sandro; Amadoro, Giuseppina; Amano, Atsuo; Amantini, Consuelo; Ambrosio, Santiago; Amelio, Ivano; Amer, Amal O; Amessou, Mohamed; Amon, Angelika; An, Zhenyi; Anania, Frank A; Andersen, Stig U; Andley, Usha P; Andreadi, Catherine K; Andrieu Abadie, Nathalie; Anel, Alberto; Ann, David K; Anoopkumar Dukie, Shailendra; Antonioli, Manuela; Aoki, Hiroshi; Apostolova, Nadezda; Aquila, Saveria; Aquilano, Katia; Araki, Koichi; Arama, Eli; Aranda, Agustin; Araya, Jun; Arcaro, Alexandre; Arias, Esperanza; Arimoto, Hirokazu; Ariosa, Aileen R; Armstrong, Jane L; Arnould, Thierry; Arsov, Ivica; Asanuma, Katsuhiko; Askanas, Valerie; Asselin, Eric; Atarashi, Ryuichiro; Atherton, Sally S; Atkin, Julie D; Attardi, Laura D; Auberger, Patrick; Auburger, Georg; Aurelian, Laure; Autelli, Riccardo; Avagliano, Laura; Avantaggiati, Maria Laura; Avrahami, Limor; Awale, Suresh; Azad, Neelam; Bachetti, Tiziana; Backer, Jonathan M; Bae, Dong Hun; Bae, Jae Sung; Bae, Ok Nam; Bae, Soo Han; Baehrecke, Eric H; Baek, Seung Hoon; Baghdiguian, Stephen; Bagniewska Zadworna, Agnieszka; Bai, Hua; Bai, Jie; Bai, Xue Yuan; Bailly, Yannick; Balaji, Kithiganahalli Narayanaswamy; Balduini, Walter; Ballabio, Andrea; Balzan, Rena; Banerjee, Rajkumar; Bánhegyi, Gábor; Bao, Haijun; Barbeau, Benoit; Barrachina, Maria D; Barreiro, Esther; Bartel, Bonnie; Bartolomé, Alberto; Bassham, Diane C; Bassi, Maria Teresa; Bast, Robert C; Basu, Alakananda; Batista, Maria Teresa; Batoko, Henri; Battino, Maurizio; Bauckman, Kyle; Baumgarner, Bradley L; Bayer, K. Ulrich; Beale, Rupert; Beaulieu, Jean François; Beck, George R; Becker, Christoph; Beckham, J. David; Bédard, Pierre André; Bednarski, Patrick J; Begley, Thomas J; Behl, Christian; Behrends, Christian; Behrens, Georg Mn; Behrns, Kevin E; Bejarano, Eloy; Belaid, Amine; Belleudi, Francesca; Bénard, Giovanni; Berchem, Guy; Bergamaschi, Daniele; Bergami, Matteo; Berkhout, Ben; Berliocchi, Laura; Bernard, Amélie; Bernard, Monique; Bernassola, Francesca; Bertolotti, Anne; Bess, Amanda S; Besteiro, Sébastien; Bettuzzi, Saverio; Bhalla, Savita; Bhattacharyya, Shalmoli; Bhutia, Sujit K; Biagosch, Caroline; Bianchi, Michele Wolfe; Biard Piechaczyk, Martine; Billes, Viktor; Bincoletto, Claudia; Bingol, Baris; Bird, Sara W; Bitoun, Marc; Bjedov, Ivana; Blackstone, Craig; Blanc, Lionel; Blanco, Guillermo A; Blomhoff, Heidi Kiil; Boada Romero, Emilio; Böckler, Stefan; Boes, Marianne; Boesze Battaglia, Kathleen; Boise, Lawrence H; Bolino, Alessandra; Boman, Andrea; Bonaldo, Paolo; Bordi, Matteo; Bosch, Jürgen; Botana, Luis M; Botti, Joelle; Bou, German; Bouché, Marina; Bouchecareilh, Marion; Boucher, Marie Josée; Boulton, Michael E; Bouret, Sebastien G; Boya, Patricia; Boyer Guittaut, Michaël; Bozhkov, Peter V; Brady, Nathan; Braga, Vania Mm; Brancolini, Claudio; Braus, Gerhard H; Bravo San Pedro, José M; Brennan, Lisa A; Bresnick, Emery H; Brest, Patrick; Bridges, Dave; Bringer, Marie Agnès; Brini, Marisa; Brito, Glauber C; Brodin, Bertha; Brookes, Paul S; Brown, Eric J; Brown, Karen; Broxmeyer, Hal E; Bruhat, Alain; Brum, Patricia Chakur; Brumell, John H; Brunetti Pierri, Nicola; Bryson Richardson, Robert J; Buch, Shilpa; Buchan, Alastair M; Budak
abstract

Guidelines for studying autophagy

2016 - Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction [Articolo su rivista]
Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela
abstract

Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascu- lar manifestations. A number of studies highlighted the role of these cells in age-related dis- eases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentia- tion markers in healthy subjects and in patients affected by peripheral vascular manifesta- tions associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accu- rately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vas- cular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context.

2016 - Mitochondrial proteases as emerging pharmacological targets [Articolo su rivista]
Gibellini, Lara; DE BIASI, Sara; Nasi, Milena; Iannone, Anna; Cossarizza, Andrea; Pinti, Marcello
abstract

The preservation of mitochondrial function and integrity is critical for cell viability. Under stress conditions, unfolded, misfolded or damaged proteins accumulate in a certain compartment of the organelle, interfering with oxidative phosphorylation and normal mitochondrial functions. In stress conditions, several mechanisms, including mitochondrial unfolded protease response (UPRmt), fusion and fission, and mitophagy are engaged to restore normal proteostasis of the organelle. Mitochondrial proteases are a family of more than 20 enzymes that not only are involved in the UPRmt, but actively participate at multiple levels in the stress-response system. Alterations in their expression levels, or mutations that determine loss or gain of function of these proteases deeply impair mitochondrial functionality and can be associated with the onset of inherited diseases, with the development of neurodegenerative disorders and with the process of carcinogenesis. In this review, we focus our attention on six of them, namely CLPP, HTRA2 and LONP1, by analysing the current knowledge about their functions, their involvement in the pathogenesis of human diseases, and the compounds currently available for inhibiting their functions.

2016 - Optimized Cryopreservation and Banking of Human Bone-Marrow Fragments and Stem Cells [Articolo su rivista]
Carnevale, Gianluca; Pisciotta, Alessandra; Riccio, Massimo; De Biasi, Sara; Gibellini, Lara; Ferrari, Adriano; La Sala, Giovanni Battista; Bruzzesi, Giacomo; Cossarizza, Andrea; De Pol, Anto
abstract

Adult mesenchymal stem cells are a promising source for cell therapies and tissue engineering applications. Current procedures for banking of human bone-marrow mesenchymal stem cells (hBM-MSCs) require cell isolation and expansion, and thus the use of large amounts of animal sera. However, animal-derived culture supplements have the potential to trigger infections and severe immune reactions. The aim of this study was to investigate an optimized method for cryopreservation of human bone-marrow fragments for application in cell banking procedures where stem-cell expansion and use are not immediately needed. Whole trabecular fragments enclosing the bone marrow were stored in liquid nitrogen for 1 year in a cryoprotective solution containing a low concentration of dimethyl sulfoxide and a high concentration of human serum (HuS). After thawing, the isolation, colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, cell senescence, apoptosis, and multi-lineage differentiation potential of hBM-MSCs were tested in media containing HuS compared with hBM-MSCs isolated from fresh fragments. Human BM-MSCs isolated from cryopreserved fragments expressed MSC markers until later passages, had a good proliferation rate, and exhibited the capacity to differentiate toward osteogenic, adipogenic, and myogenic lineages similar to hBM-MSCs isolated from fresh fragments. Moreover, the cryopreservation method did not induce cell senescence or cell death. These results imply that minimal processing may be adequate for the banking of tissue samples with no requirement for the immediate isolation and use of hBM-MSCs, thus limiting cost and the risk of contamination, and facilitating banking for clinical use. Furthermore, the use of HuS for cryopreservation and expansion/differentiation has the potential for clinical application in compliance with good manufacturing practice standards.

2016 - Quantification of mitochondrial reactive oxygen species in living cells by using multi-laser polychromatic flow cytometry [Articolo su rivista]
DE BIASI, Sara; Gibellini, Lara; Bianchini, Elena; Nasi, Milena; Pinti, Marcello; Salvioli, Stefano; Cossarizza, Andrea
abstract

Reactive oxygen species (ROS) are constantly produced in cells, mainly by mitochondria, as a consequence of aerobic respiration. Most ROS derive from superoxide, which is rapidly converted to hydrogen peroxide. ROS are involved in the regulation of several physiological and pathological processes, and the possibility to measure them simultaneously is needed, when the redox status of the cells is modified by experimental/biological conditions. Flow cytometry is the main technology that generates multiple information at the single cell level in a high-throughput manner, and gives rapid and quantitative measurements of different ROS with high sensitivity and reproducibility. Here, we describe a novel approach to detect simultaneously mitochondrial hydrogen peroxide and mitochondrial superoxide in living cells. The staining has been performed by using the fluorescent dyes MitoSOX Red Mitochondrial Superoxide Indicator, Mitochondria Peroxy Yellow 1, Annexin-V Pacific Blue conjugate, TO-PRO-3 iodide, anti-CD4-APC-Cy7 and -CD8-Pacific Orange mAbs. We used this approach to quantify mitochondrial ROS in CD4+ and CD8+ T cells form patients affected by Down syndrome and age- and sex-matched healthy donors.

2016 - TRANSAUTOPHAGY: European network for multidisciplinary research and translation of autophagy knowledge [Articolo su rivista]
Casas, Caty; Codogno, Patrice; Pinti, Marcello; Batoko, Henri; Morán, María; Proikas Cezanne, Tassula; Reggiori, Fulvio; Sirko, Agnieszka; Soengas, María S; Velasco, Guillermo; Lafont, Frank; Lane, Jon; Faure, Mathias; Cossarizza, Andrea
abstract

abstract: A collaborative consortium, named “TRANSAUTOPHAGY,” has been created among European research groups, comprising more than 150 scientists from 21 countries studying diverse branches of basic and translational autophagy. The consortium was approved in the framework of the Horizon 2020 Program in November 2015 as a COST Action of the European Union (COST means: CO-operation in Science and Technology), and will be sponsored for 4 years. TRANSAUTOPHAGY will form an interdisciplinary platform for basic and translational researchers, enterprises and stakeholders of diverse disciplines (including nanotechnology, bioinformatics, physics, chemistry, biology and various medical disciplines). TRANSAUTOPHAGY will establish 5 different thematic working groups, formulated to cooperate in research projects, share ideas, and results through workshops, meetings and short term exchanges of personnel (among other initiatives). TRANSAUTOPHAGY aims to generate breakthrough multidisciplinary knowledge about autophagy regulation, and to boost translation of this knowledge into biomedical and biotechnological applications.

2016 - Th1 and Th17 pro-inflammatory profile characterizes iNKT cells in virologically suppressed HIV+ patients with low CD4/CD8 ratio [Articolo su rivista]
DE BIASI, Sara; Bianchini, Elena; Nasi, Milena; Digaetano, Margherita; Gibellini, Lara; Carnevale, Gianluca; Borghi, Vanni; Guaraldi, Giovanni; Pinti, Marcello; Mussini, Cristina; Cossarizza, Andrea
abstract

INTRODUCTION:: Scanty data exist on the phenotype and functionality of invariant natural killer T (iNKT) cells in HIV+ patients (pts). METHODS:: By flow cytometry, we studied iNKT cells from 54 HIV+ pts who started combined antiretroviral therapy (cART) and had undetectable viral load for >1 year. Twenty-five maintained a CD4/CD8 ratio <0.4, while 29 reached a ratio >1.1; 32 age- and sex-matched subjects were healthy controls (CTR). RESULTS:: Pts with low ratio had lower percentage of CD4+ iNKT cells compared to pts with high ratio, and higher CD8+ iNKT cell percentage; double negative (DN) iNKT cells were lower in HIV+ pts compared to CTR. Pts with low ratio had higher percentage of CD4+ and DN iNKT cells expressing CD38 and HLA-DR compared to pts with high ratio. CD4+ iNKT cells expressing PD-1 were higher in pts with CD4/CD8 ratio <0.4, while DN iNKT cells expressing PD-1 were lower compared to pts with ratio >1.1. Pts with low ratio had higher CD4+ iNKT cells producing IL-17, CD8+ iNKT cells producing IFN-γ, TNF-α or IFN-γ plus TNF-α, and DN iNKT cells producing IL-17 or IL-17 plus IFN-γ compared to CTR. Activated CD4+ (or CD8+) T cells correlated with activated CD4+ (or CD8+) iNKT cells, as well as the percentages of CD4+ (or CD8+) T cells expressing PD-1 was correlated to that of CD4+ (or CD8+) iNKT cells expressing PD-1. CONCLUSIONS:: Low CD4/CD8 ratio despite effective cART is associated with altered iNKT cell subsets, enhanced activation and prominent Th1/Th17 pro-inflammatory profile.

2016 - iNKT cells in secondary progressive multiple sclerosis patients display pro-inflammatory profiles [Articolo su rivista]
DE BIASI, Sara; Simone, ANNA MARIA; Nasi, Milena; Bianchini, Elena; Ferraro, Diana; Vitetta, Francesca; Gibellini, Lara; Pinti, Marcello; DEL GIOVANE, Cinzia; Sola, Patrizia; Cossarizza, Andrea
abstract

Background. Multiple Sclerosis (MS), an autoimmune disease with neurodegeneration and inflammation, is characterized by several alterations of different T cell subsets. However, few data exist on the role of iNKT lymphocytes. Objective. To identify possible changes in the phenotype of iNKT cells in patients with different clinical forms of MS, and find alterations in their polyfunctionality (i.e., ability to produce simultaneously up to 4 cytokines such as IL‐17, TNF‐α, IFN‐γ, IL‐4). Methods. We studied a total of 165 patients, 91 with a Relapsing Remitting form [RR; 31 were treated with interferon (IFN)1‐β, 25 with natalizumab (Nat), 29 with glatiramer acetate (Gla); 17 were newly-diagnosed RR without treatment, 19 not active RR without treatment]. Forty-four patients had a Progressive MS: 20 Primary Progressive (PP), 24 Secondary Progressive (SP). A total of 55 age- and sex-matched subjects represented healthy controls (CTR). Among fresh peripheral blood mononuclear cells (PBMC) iNKT cells were identified by flow cytometry. Moreover, the capability of iNKT cells to produce different cytokines (IL‐17, TNF‐α, IFN‐γ, and IL‐4) after in vitro stimulation were evaluated in 18 RR (11 treated with Nat and 7 with IFN), 4 PP, 6 SP and 16 CTR. Results. No main differences were found in iNKT cell phenotype among MS patients with different MS forms, or during different treatments. However, the polyfunctional response of iNKT cells showed Th1 and Th17 profiles. This was well evident in patients with secondary progressive form, who are characterized by high levels of inflammation and neurodegeneration, and exhibited a sustained increase in the production of Th17 cytokines. Patients treated with natalizumab displayed lower levels of iNKT cells producing IL‐17, TNF‐α and IFN‐γ. Conclusion. Our data suggest that the progressive phase of the disease is characterized by permanent iNKT activation and a skewing towards an inflammatory phenotype. Compared to other treatments, natalizumab was able to modulate iNKT cell function.

2015 - Analysis of inflammasomes and antiviral sensing components reveals decreased expression of NLRX1 in HIV-positive patients assuming efficient antiretroviral therapy [Articolo su rivista]
Nasi, Milena; DE BIASI, Sara; Bianchini, Elena; Digaetano, Margherita; Pinti, Marcello; Gibellini, Lara; Pecorini, Simone; Carnevale, Gianluca; Guaraldi, Giovanni; Borghi, Vanni; Mussini, Cristina; Cossarizza, Andrea
abstract

Objective: Few studies have investigated the importance of different components of the inflammasome system and of innate mitochondrial sensing (IMS) pathways in HIV infection and its treatment. We analysed the expression of several components of the inflammasome and of the IMS in HIV-positive patients taking successful combination antiretroviral therapy (cART). Methods: We enrolled 20 HIV-positive patients under cART, who achieved viral suppression since at least 10 months and 20 age and sex-matched healthy donors. By RT-PCR, using peripheral blood mononuclear cells (PBMCs), we quantified the mRNA expression of 16 genes involved in inflammasome activation and regulation (AIM2, NAIP, PYCARD, CASP1, CASP5, NLRP6, NLRP1, NLRP3, TXNIP, BCL2, NLRC4, PANX1, P2RX7, IL-18, IL-1β, SUGT1) and eight genes involved in IMS (MFN2, MFN1, cGAS, RIG-I, MAVS, NLRX1, RAB32, STING). Results: Compared with controls, HIV-positive patients showed significantly lower mRNA levels of the mitochondrial protein NLRX1, which plays a key role in regulating apoptotic cell death; main PBMC subpopulations behave in a similar manner. No differences were observed in the expression of inflammasome components, which however showed complex correlations. Conclusion: The decreased level of NLRX1 in HIV infection could suggest that the virus is able to downregulate mechanisms linked to triggering of cell death in several immune cell types. The fact that HIV-positive patients did not show altered expression of inflammasome components, nor of most genes involved in IMS, suggests that the infection and/or the chronic immune activation does not influence the transcriptional machinery of innate mechanisms able to trigger inflammation at different levels.

2015 - Different origin of adipogenic stem cells influences the response to antiretroviral drugs [Articolo su rivista]
Gibellini, Lara; DE BIASI, Sara; Nasi, Milena; Carnevale, Gianluca; Pisciotta, Alessandra; Bianchini, Elena; Bartolomeo, Regina; Polo, Miriam; DE POL, Anto; Pinti, Marcello; Cossarizza, Andrea
abstract

Lipodystrophy (LD) is a main side effect of antiretroviral therapy for HIV infection, and can be provoked by nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). LD exists in different forms, characterized by fat loss, accumulation, or both, but its pathogenesis is still unclear. In particular, few data exist concerning the effects of antiretroviral drugs on adipocyte differentiation. Adipose tissue can arise either from mesenchymal stem cells (MSCs), that include bone marrow-derived MSCs (hBM-MSCs), or from ectodermal stem cells, that include dental pulp stem cells (hDPSCs). To analyze whether the embryonal origin of adipocytes might impact the occurrence of different phenotypes in LD, we quantified the effects of several antiretroviral drugs on the adipogenic differentiation of hBM-MSCs and hDPSCs. hBM-MSCs and hDPSCs were isolated from healthy donors. Cells were treated with 10 and 50μM stavudine (d4T), efavirenz (EFV), atazanavir (ATV), ritonavir (RTV), and ATV-boosted RTV. Viability and adipogenesis were evaluated by staining with propidium iodide, oil red, and adipoRed; mRNA levels of genes involved in adipocyte differentiation, i.e. CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ), and in adipocyte functions, i.e. fatty acid synthase (FASN), fatty acid binding protein-4 (FABP4), perilipin-1 (PLIN1) and 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2), were quantified by real time PCR. We found that ATV, RTV, EFV, and ATV-boosted RTV, but not d4T, caused massive cell death in both cell types. EFV and d4T affected the accumulation of lipid droplets and induced changes in mRNA levels of genes involved in adipocyte functions in hBM-MSCs, while RTV and ATV had little effects. All drugs stimulated the accumulation of lipid droplets in hDPSCs. Thus, the adipogenic differentiation of human stem cells can be influenced by antiretroviral drugs, and depends, at least in part, on their embryonal origin.

2015 - Dynamics of adaptive and innate immunity inpatients treated during primary human immunodeficiency virus infection: Results from Maraviroc in HIV Acute Infection (MAIN) randomized clinical trial [Articolo su rivista]
Ripa, M.; Pogliaghi, M.; Chiappetta, S.; Galli, L.; Pensieroso, S.; Cavarelli, M.; Scarlatti, G.; DE BIASI, Sara; Cossarizza, Andrea; De Battista, D.; Malnati, M.; Lazzarin, A.; Nozza, S.; Tambussi, G.
abstract

We evaluated the dynamics of innate and adaptive immunity in patients treated with combined antiretroviral therapy (cART) during primary human immunodeficiency virus infection (PHI), enrolled in a prospective randomized trial (MAIN, EUDRACT 2008-007004-29). After 48 weeks of cART, we documented a reduction in activated B cells and CD8+ T cells. Natural killer cell and dendritic cell frequencies were measured and a decrease in CD16+ CD56dim with a reciprocal rise in CD56high natural killer cells and an increase in myeloid and plasmacytoid dendritic cells were recorded. In conclusion, 48 weeks of cART during PHI showed significant benefits for both innate and adaptive immunity.

2015 - Inhibition of Lon protease by triterpenoids alters mitochondria and is associated to cell death in human cancer cells [Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Bartolomeo, Regina; De Biasi, Sara; Cormio, Antonella; Musicco, Clara; Carnevale, Gianluca; Pecorini, Simone; Nasi, Milena; De Pol, Anto; Cossarizza, Andrea
abstract

Mitochondrial Lon protease (Lon) regulates several mitochondrial functions, and is inhibited by the anticancer molecule triterpenoid 2-cyano-3, 12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), or by its C-28 methyl ester derivative (CDDO-Me). To analyze the mechanism of action of triterpenoids, we investigated intramitochondrial reactive oxygen species (ROS), mitochondrial membrane potential, mitochondrial mass, mitochondrial dynamics and morphology, and Lon proteolytic activity in RKO human colon cancer cells, in HepG2 hepatocarcinoma cells and in MCF7 breast carcinoma cells. We found that CDDO and CDDO-Me are potent stressors for mitochondria in cancer cells, rather than normal non-transformed cells. In particular, they: i) cause depolarization; ii) increase mitochondrial ROS, iii) alter mitochondrial morphology and proteins involved in mitochondrial dynamics; iv) affect the levels of Lon and those of aconitase and human transcription factor A, which are targets of Lon activity; v) increase level of protein carbonyls in mitochondria; vi) lead to intrinsic apoptosis. The overexpression of Lon can rescue cells from cell death, providing an additional evidence on the role of Lon in conditions of excessive stress load.

2015 - Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments [Articolo su rivista]
DE BIASI, Sara; Cerri, Stefania; Bianchini, Elena; Gibellini, Lara; Persiani, Elisa; Montanari, Gloria; Luppi, Fabrizio; Carbonelli, Cristiano Matteo; Zucchi, Luigi; Bocchino, Marialuisa; Zamparelli, Alessandro Sanduzzi; Vancheri, Carlo; Sgalla, Giacomo; Richeldi, Luca; Cossarizza, Andrea
abstract

Background: It has been suggested that circulating fibrocytes and endothelial cells actively participate in the intense remodelling of the pulmonary vasculature in patients with idiopathic pulmonary fibrosis (IPF). Indeed, fibrotic areas exist that have fewer blood vessels, whereas adjacent non-fibrotic tissue is highly vascularized. The number of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) might reflect the balance between vascular injury and repair. Thus, fibrocytes as well as endothelial cells could potentially be used as biomarkers of disease progression and treatment outcome. Methods: Peripheral blood samples were collected from 67 patients with a multidisciplinary diagnosis of IPF and from 45 age-matched and sex-matched healthy volunteers. Buffy coat was isolated according to standard procedures and at least 20 million cells were stained with different monoclonal antibodies for the detection of CEC, EPC and circulating fibrocytes. For the detection of CEC and EPC, cells were stained with anti-CD45, anti-CD34, anti-CD133, anti-CD14, anti-CD309 and with the viability probe Far-Red LIVE/DEAD. For the detection of circulating fibrocytes, cells were first stained with LIVE/DEAD and the following monoclonal antibodies: anti-CD3, anti-CD19, anti-CD45, anti-CD34 and anti-CD14, then cells were fixed, permeabilized and stained with fluorochrome-conjugated anti-collagen I monoclonal antibodies. Results: Patients with IPF displayed almost undetectable levels of circulating fibrocytes, low levels of CEC, and normal levels of EPC. Patients treated with nintedanib displayed higher levels of CEC, but lower levels of endothelial cells expressing CD309 (the type II receptor for vascular endothelial growth factor). Treatment with both nintedanib and pirfenidone reduced the percentage of CEC and circulating fibrocytes. Conclusions: Levels of CEC were reduced in patients with IPF as compared to healthy individuals. The anti-fibrotic treatments nintedanib and pirfenidone further reduced CEC levels. These findings might help explain the mechanism of action of these drugs and should be explored as predictive biomarkers in IPF.

2015 - Mitochondrial Lon protease at the crossroads of oxidative stress, ageing and cancer [Articolo su rivista]
Pinti, Marcello; Gibellini, Lara; Liu, Yongzhang; Xu, Shan; Lu, Bin; Cossarizza, Andrea
abstract

Lon protease is a nuclear DNA-encoded mitochondrial enzyme highly conserved throughout evolution, involved in the degradation of damaged and oxidized proteins of the mitochondrial matrix, in the correct folding of proteins imported in mitochondria, and in the maintenance of mitochondrial DNA. Lon expression is induced by various stimuli, including hypoxia and reactive oxygen species, and provides protection against cell stress. Lon down-regulation is associated with ageing and with cell senescence, while up-regulation is observed in tumour cells, and is correlated with a more aggressive phenotype of cancer. Lon up-regulation contributes to metabolic reprogramming observed in cancer, favours the switch from a respiratory to a glycolytic metabolism, helping cancer cell survival in the tumour microenvironment, and contributes to epithelial to mesenchymal transition. Silencing of Lon, or pharmacological inhibition of its activity, causes cell death in various cancer cells. Thus, Lon can be included in the growing class of proteins that are not responsible for oncogenic transformation, but that are essential for survival and proliferation of cancer cells, and that can be considered as a new target for development of anticancer drugs.

2015 - Natural Compounds Modulating Mitochondrial Functions [Articolo su rivista]
Gibellini, Lara; Bianchini, Elena; DE BIASI, Sara; Nasi, Milena; Cossarizza, Andrea; Pinti, Marcello
abstract

Mitochondria are organelles responsible for several crucial cell functions, including respiration, oxidative phosphorylation, and regulation of apoptosis; they are also the main intracellular source of reactive oxygen species (ROS). In the last years, a particular interest has been devoted to studying the effects on mitochondria of natural compounds of vegetal origin, quercetin (Qu), resveratrol (RSV), and curcumin (Cur) being the most studied molecules. All these natural compounds modulate mitochondrial functions by inhibiting organelle enzymes or metabolic pathways (such as oxidative phosphorylation), by altering the production of mitochondrial ROS and by modulating the activity of transcription factors which regulate the expression of mitochondrial proteins. While Qu displays both pro- and antioxidant activities, RSV and Cur are strong antioxidant, as they efficiently scavenge mitochondrial ROS and upregulate antioxidant transcriptional programmes in cells. All the three compounds display a proapoptotic activity, mediated by the capability to directly cause the release of cytochrome c from mitochondria or indirectly by upregulating the expression of proapoptotic proteins of Bcl-2 family and downregulating antiapoptotic proteins. Interestingly, these effects are particularly evident on proliferating cancer cells and can have important therapeutic implications.

2015 - Neural crest derived niche of human dental pulp stem cells promotes peripheral nerve regeneration and remyelination in animal model of critical sized sciatic nerve injury [Articolo su rivista]
Carnevale, Gianluca; Pisciotta, Alessandra; DE BIASI, Sara; Gibellini, Lara; Cossarizza, Andrea; Bruzzesi, Giacomo; Ferrari, Adriano; DE POL, Anto
abstract

ABSTRACT Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The use of stem cells, easily accessible, capable of rapid expansion in culture as well as fully integrate into the host tissue and capable to differentiate in myelinating cells of the peripheral nervous system, represent an attractive therapeutic approach for the treatment of nerve injuries. Farther, stem cells sources sharing the same embryological origin of Schwann cells, might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal sub-population of STRO-1+/c-Kit+/CD34+ hDPSCs (1, 2), most of which being positive for neural crest (P75NTR) and neural progenitor cells (nestin) markers, to differentiate into Schwann cells-like cells in vitro and to promote axonal regeneration in vivo. As a matter of fact, following culture in appropriate induction medium, STRO-1+/c-Kit+/CD34+ hDPSCs were able to commit towards Schwann cells express- ing P75NTR, GFAP and S100b. After transplantation in animal model of sciatic nerve defect, hDPSCs promoted axonal regeneration from proximal to distal stumps, providing guidance to newly formed myelinated nerve fibers, which led to functional recovery as measured by sustained gait improvement. Particularly, transplanted hDP- SCs engrafted into critical sized sciatic nerve defect, as revealed by the positive stain- ing against human nuclei, showed the expression of typical Schwann cells markers, S100b and GFAP. In conclusion this study demonstrates that STRO-1+/c-Kit+/CD34+ hDPSCs, associated to neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues.

2015 - Overcoming challenges in cellular analysis: Multiparameter analysis of rare cells [Articolo su rivista]
Cossarizza, A.; Cousins, D.
abstract

2015 - P-Cresol and Cardiovascular Risk in Kidney Transplant Recipients [Articolo su rivista]
Ligabue, Giulia; Damiano, Francesca; Cuoghi, Alberto; DE BIASI, Sara; Bellei, Enrica; Granito, Maria; Aldo, T.; Cossarizza, Andrea; Cappelli, Gianni
abstract

p-Cresol Sulphate (pCS) is a uremic toxin that originates exclusively from dietary sources and has a high plasma level related to chronic kidney disease (CKD) and cardiovascular disease (CVD). The aim of our study was to evaluate the plasma levels of pCS in kidney transplant recipients (KTRs) related to estimated glomerular filtration rate (eGFR), traditional risk factors, cardiovascular clinical events and endothelial progenitor cells (EPCs), bone marrow-derived cells for the vascular repair system. We considered 51 KTRs and 25 healthy blood donors (HBDs). pCs levels were analyzed using high-performance liquid chromatography (HPLC) coupled with mass spectrometry with an electrospray ionization (ESI) (LC/ESI-MS/MS) on a triple-quadrupole; EPCs were analyzed using flow cytometric analysis. eGFR was 52.61 ± 19.9 mL/min/1.73 m<sup>2</sup> in KTRs versus 94 ± 21 mL/min/1.73 m<sup>2</sup> in HBDs. We did not find differences in pCS levels between KTRs and HBDs. Levels of pCS were inversely related with eGFR in KTRs and pCS levels were significantly lower in KTRs with eGFR <30 mL/min/1.73 m<sup>2</sup> versus eGFR >30 mL/min/1.73 m<sup>2</sup>. Furthermore, there was a difference in pCS levels between eGFR <30 mL/min/1.73 m<sup>2</sup> of KTRs compared with HBDs. Levels of pCS were almost significantly influenced by the presence of a previous vascular event and were inversely related with mature EPCs. These findings suggest that KTRs should not have higher CVD risk than HBDs and their physiological vascular repair system appears to be intact. In KTRs the reduction of eGFR also increased pCS levels and reduced EPCs numbers and angiogenesis capacity. In summary, pCS acts as an emerging marker of a uremic state, helping assess the global vascular competence in KTRs.

2015 - Polymorphisms of large effect explain the majority of the host genetic contribution to variation of HIV-1 virus load [Articolo su rivista]
Mclaren, P. J.; Coulonges, C.; Bartha, I.; Lenz, T. L.; Deutsch, A. J.; Bashirova, A.; Buchbinder, S.; Carrington, M. N.; Cossarizza, A.; Dalmau, J.; De Luca, A.; Goedert, J. J.; Gurdasani, D.; Haas, D. W.; Herbeck, J. T.; Johnson, E. O.; Kirk, G. D.; Lambotte, O.; Luo, M.; Mallal, S.; Van Manen, D.; Martinez-Picado, J.; Meyer, L.; Miro, J. M.; Mullins, J. I.; Obel, N.; Poli, G.; Sandhu, M. S.; Schuitemaker, H.; Shea, P. R.; Theodorou, I.; Walker, B. D.; Weintrob, A. C.; Winkler, C. A.; Wolinsky, S. M.; Raychaudhuri, S.; Goldstein, D. B.; Telenti, A.; De Bakker, P. I. W.; Zagury, J. -F.; Fellay, J.
abstract

Previous genome-wide association studies (GWAS) of HIV-1Cinfected populations have been underpowered to detect common variants with moderate impact on disease outcome and have not assessed the phenotypic variance explained by genome-wide additive effects. By combining the majority of available genome-wide genotyping data in HIV-infected populations, we tested for association between ¡«8 million variants and viral load (HIV RNA copies per milliliter of plasma) in 6,315 individuals of European ancestry. The strongest signal of association was observed in the HLA class I region that was fully explained by independent effects mapping to five variable amino acid positions in the peptide binding grooves of the HLA-B and HLA-A proteins. We observed a second genome-wide significant association signal in the chemokine (C-C motif) receptor (CCR) gene cluster on chromosome 3. Conditional analysis showed that this signal could not be fully attributed to the known protective CCR5'32 allele and the risk P1 haplotype, suggesting further causal variants in this region. Heritability analysis demonstrated that common human genetic variation¡mostly in the HLA and CCR5 regions¡explains 25% of the variability in viral load. This study suggests that analyses in non-European populations and of variant classes not assessed by GWAS should be priorities for the field going forward.

2015 - Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and “CD4 Easy Count Kit-Dry”, as Revealed by the Comparison with the Gold Standard Dual Platform Technology [Articolo su rivista]
Nasi, Milena; De Biasi, Sara; Bianchini, Elena; Gibellini, Lara; Pinti, Marcello; Scacchetti, Tiziana; Trenti, Tommaso; Borghi, Vanni; Mussini, Cristina; Cossarizza, Andrea
abstract

An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the "gold standard" for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the "CD4% Count Kit-Dry".

2015 - Uncompensated polychromatic analysis of mitochondrial membrane potential using JC-1 and multilaser excitation [Capitolo/Saggio]
DE BIASI, Sara; Gibellini, Lara; Cossarizza, Andrea
abstract

The lipophilic cation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-etraethylbenzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (δψm). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual-laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC-1 can be utilized by polychromatic flow cytometry to simultaneously detect, without any compensation between fluorescences, δψm along with other biological parameters, such as apoptosis and the production of reactive oxygen species.

2014 - Aging and longevity: an immunological perspective [Articolo su rivista]
Cossarizza, Andrea; Frasca, Daniela
abstract

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2014 - Aging with HIV infection: a journey to the center of inflammAIDS, immunosenescence and neuroHIV [Articolo su rivista]
Nasi, Milena; Pinti, Marcello; DE BIASI, Sara; Gibellini, Lara; Ferraro, Diana; Mussini, Cristina; Cossarizza, Andrea
abstract

In the last years, a significant improvement in life expectancy of HIV+ patients has been observed in Western countries. The parallel increase in the mean age of these patients causes a parallel increase in the frequency of non-AIDS related complications (i.e., neurocognitive, cardiovascular, liver and kidney diseases, metabolic syndrome, osteoporosis, non-HIV associated cancers, among others), even when antiviral treatment is successful. Immune activation and persistent inflammation characterizes both HIV infection and physiological aging, and both conditions share common detrimental pathways that lead to early immunosenescence. Furthermore, HIV-associated neurocognitive disorders represent important consequences of the infection. The persistent systemic immune activation, the continuous migration of activated monocytes to the central nervous system and progressive patients' aging contribute to develop neuronal injuries, that are in turn linked to HIV-associated neurocognitive disorders, which can persist despite successful antiretroviral treatment.

2014 - Circulating mitochondrial DNA increases with age and is a familiar trait: implications for "inflamm-aging" [Articolo su rivista]
Pinti, Marcello; Cevenini, E; Nasi, Milena; De Biasi, Sara; Salvioli, S; Monti, Daniela; Benatti, Stefania; Gibellini, Lara; Cotichini, R; Stazi, Ma; Trenti, T; Franceschi, C; Cossarizza, Andrea
abstract

Mitochondrial components, including mitochondrial DNA (mtDNA), when released extracellularly, can act as "damage-associated molecular pattern" (DAMP) agents and cause inflammation. As many elderly people are characterized by a low-grade, chronic inflammatory status defined "inflamm-aging", we evaluated if circulating mtDNA can contribute to this phenomenon. Eight hundred and thirty-one Caucasian subjects were enrolled in the study, including 429 siblings aged 90-104 years (90+ siblings). MtDNA plasma levels increased gradually after the fifth decade of life. In 90+ subjects, mtDNA values of two members of the same sibling relationship were directly correlated, suggesting a role for familiar/genetic background in controlling the levels of circulating mtDNA. The subjects with the highest mtDNA plasma levels had the highest amounts of TNF-??, IL-6, RANTES and IL-1ra; the subjects with the lowest mtDNA levels had the lowest levels of the same cytokines. In vitro stimulation of monocytes with mtDNA concentrations similar to the highest levels observed in vivo resulted in an increased production of TNF-??, suggesting that mtDNA can modulate the production of proinflammatory cytokines. Our findings therefore show that circulating mtDNA increases with age, and can significantly contribute to the maintenance of the low grade, chronic inflammation observed in elderly people This article is protected by copyright. All rights reserved.

2014 - Cytometry for immunology: the marriage continues [Articolo su rivista]
Cossarizza, Andrea; Radbruch, Andreas
abstract

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2014 - HUMAN BILIARY TREE STEM/PROGENITOR CELLS (hbTSCS) FROM PERIBILIARY GLANDS (PBGS) OF ADULT LIVER DISPLAY IMMUNOMODULATORY PROPERTIES THROUGH Fas/Fas LIGAND INDUCED T-CELL LYMPHOCYTE APOPTOSIS [Abstract in Rivista]
Carnevale, G; Riccio, M; Cardinale, V; Gibelini, L; De Biasi, S; Pisciotta, A; Carpino, G; Gentile, R; Berloco, Pb; Brunelli, R; Bastianelli, C; Cossarizza, A; Gaudio, E; Alvaro, D; De Pol, A
abstract

Background and Aims: hBTSCs have the potential for regenerative medicine in liver and pancreas diseases. T-cell control was recently demonstrated for mesenchymal stem cells. The aims of this study were to evaluate Fas-L expression within the stem cell niches of adult biliary tree (PBGs), and to study the interaction between hBTSCs and human lymphocytes. Methods: HLA antigens, Fas and Fas-L expression were evaluated by immunofluorescence and western blotting (WB) in cells of human biliary tree in comparison with fibroblast cells, dental pulp stem cells and bone marrow mesenchymal stem cells. The influence of hBTSCs on lymphocytes’ activation and apoptosis were assessed by co-culturing experiments. Results: Adult hBTSCs expressed both class I and class II HLA antigens, whereas fetal hBTSCs only class I HLA antigens. 10 to 30% of the hBTSCs in PBGs were positive for Fas-L. Fas-L+ cells were mostly located at the bottom of PBGs and co-expressed EpCAM (Epithelial-Cell-AdhesionMolecule) and proliferation marker (PCNA:Proliferating-CellNuclear-Antigen). Mature cells at the bile duct surface epithelium (mature cholangiocytes) were almost all negative for Fas-L. In culture experiments confocal microscopy demonstrated that Fas-L expression was restricted to EpCAM+/LGR5+ (a marker associated with endodermal stem cells) hBTSCs. WB confirmed that hBTSCs constitutively expressed high level of Fas-L which increased after co-culture with T-cells. FACS analysis reveled that activated CD4+ and CD8+ T-cells co-cultured with hBTSCs underwent to a massiveinduction of apoptosis. Fas receptor appeared over-expressed in T-cells co-cultured with hBTSCs respect to resting T-cells. Conclusions: Our data demonstrated that hBTSCs can induce “premature” apoptosis in T-cells trough the activation of Fas/Fas-L pathway

2014 - Human biliary tree stem/progenitor cells (hBTSCs) from peribiliary glands (PBGs) of adult liver display immunomodulatory properties through Fas/Fas ligand induced T-cell lymphocyte apoptosis [Abstract in Rivista]
Carnevale, G.; Riccio, M.; Cardinale, V.; Gibelini, L.; De Biasi, S.; Pisciotta, A.; Carpino, G.; Gentile, R.; Berloco, P. B.; Brunelli, R.; Bastianelli, C.; Cossarizza, A.; Gaudio, E.; Alvaro, D.; De Pol, A.
abstract

Background and aim: hBTSCs have been retrieved in peribiliary glands (PBGs) of adult and fetal biliary tree, and have the potential for regenerative medicine in liver, biliary tree, and pancreas diseases. The ability of stem cells to control T-cells’ immune responses was recently demonstrated by human mesenchymal stem cells. The aims of the present study were to evaluate Fas-L expression within the stem cell niches of adult biliary tree, and to study the in vitro interaction between hBTSCs and human lymphocytes. Material and methods: HLA antigens, Fas and Fas-L expression were evaluated in situ and in vitro by immunofluorescence and Western blots in cells of the human biliary tree in comparison with fibroblast cells, dental pulp stem cells and bone marrow mesechymal stem cells. Co-cultures of hBTSCs with human leucocytes were used to analyze the influence of hBTSCs on lymphocytes’ activation and apoptosis. Results: Adult hBTSCs expressed both class I and class II HLA antigens, whereas fetal hBTSCs had class I HLA antigens only. In PBG niche 10-30% BTSCs were positive for Fas-L. Fas-L positive cells were mostly located at the bottom of PBGs and co-expressed EpCAM (epithelial cell adhesion molecule) and a marker of proliferation (PCNA: Proliferating Cell Nuclear Antigen). Conversely, mature cells at the surface epithelium and cholangiocytes of large intrahepatic ducts were almost all negative for Fas-L. In culture experiments confocal microscopy demonstrated that Fas-L expression was restricted to EpCAM+/LGR5+(a marker associated with endodermal stem cells) cells. Western blot data confirmed that hBTSCs constitutively expressed high level of Fas-L that increased after co-culture with T-cells. FACS analysis of T-cells co-cultured with hBTSCs indicated that hBTSCs were able to induce apoptosis in activated CD4+ and CD8+ T-cell populations. Moreover, Fas receptor appears to be more expressed in T-cells co-cultured with hBTSCs than in resting T-cells. Conclusions: In conclusion our data suggest that hBTSCs could modulate the T-cells response through the production of Fas-L, which influences the lymphocyte Fas/Fas-L pathway by inducing “premature” apoptosis in CD4+ and CD8+ T-cells.

2014 - Life Expectancy in the Immune Recovery Era [Articolo su rivista]
Guaraldi, Giovanni; Cossarizza, Andrea; C., Franceschi; A., Roverato; E., Vaccher; G., Tambussi; E., Garlassi; Menozzi, Marianna; Mussini, Cristina; A., DʼArminio Monforte
abstract

INTRODUCTION: National cohort and intercohort studies have been set to describe the differences of life expectancy (LE) of HIV-infected individuals. OBJECTIVE: The aim of this study was to assess the impact of immune recovery (IR) on LE of patients with HIV undergoing combination antiretroviral therapy. METHODS: In this retrospective observational study, outcome measure was LE of patients with HIV compared with LE of northern Italian population. Group categorizations were as follows: patients with no immune recovery (nIR), patients with IR, patients who are immune maintained, and pre-highly active antiretroviral therapy (HAART) and post-HAART. Abridged life tables were constructed from age-specific mortality rates (per 1000 person years) to estimate LE from the age of 20-55 years. RESULTS: A total of 9671 patients, 71% men, were included. After 2005, we assisted to a rapid increase in the overall rate of patients attaining IR in the community coupled with a progressive decrease of AIDS death, but not of non-AIDS deaths. In a 40-year-old patient, LE was 38.10 years [standard error (SE) = 2.60], 30.08 years (SE = 0.98), and 22.9 (SE = 0.69) in the IR, post-HAART group and nIR, respectively, compared with 41.38 years of the general Italian population. An approximately 5-year gap in LE was observed in IR patients. DISCUSSION: We describe IR at a "community" level, related to calendar year and apparent 10 years after HAART introduction. HAART community IR is significantly influencing LE and is associated with the changing clinical picture of HIV disease. An increasing gradient of LE exists between nIR, post-HAART, and IR groups, with the latter, above the age of 40 years only, reaching LE of general population.

2014 - Mitochondria hyperfusion and elevated autophagic activity are key mechanisms for cellular bioenergetic preservation in centenarians. [Articolo su rivista]
Sgarbi, G; Matarrese, P; Pinti, Marcello; Lanzarini, C; Ascione, B; Gibellini, Lara; Dika, E; Patrizi, A; Tommasino, C; Capri, M; Cossarizza, Andrea; Baracca, A; Lenaz, G; Solaini, G; Franceschi, C; Malorni, W; Salvioli, S.
abstract

Mitochondria have been considered for long time as important determinants of cell aging because of their role in the production of reactive oxygen species. In this study we investigated the impact of mitochondrial metabolism and biology as determinants of successful aging in primary cultures of fibroblasts isolated from the skin of long living individuals (LLI) (about 100 years old) compared with those from young (about 27 years old) and old (about 75 years old) subjects. We observed that fibroblasts from LLI displayed significantly lower complex I-driven ATP synthesis and higher production of H2O2 in comparison with old subjects. Despite these changes, bioenergetics of these cells appeared to operate normally. This lack of functional consequences was likely due to a compensatory phenomenon at the level of mitochondria, which displayed a maintained supercomplexes organization and an increased mass. This appears to be due to a decreased mitophagy, induced by hyperfused, elongated mitochondria. The overall data indicate that longevity is characterized by a preserved bioenergetic function likely attained by a successful mitochondria remodeling that can compensate for functional defects through an increase in mass, i.e. a sort of mitochondrial "hypertrophy".

2014 - Persistent inflammation in HIV infection: Established concepts, new perspectives. [Articolo su rivista]
Nasi, Milena; Pinti, Marcello; Mussini, Cristina; Cossarizza, Andrea
abstract

Immune activation is now considered a main driving force for the progressive immune failure in HIV infection. During the early phases of infection, a rapid depletion of gastrointestinal CD4+ T cells occurs that is followed by a deterioration of the gut epithelium and by the subsequent translocation of microbial products into the blood. Activation of innate immunity results in massive production of proinflammatory cytokines, which can trigger activation induced cell death phenomena among T lymphocytes. Moreover, persistent antigenic stimulation and inflammatory status causes immune exhaustion. The chronic immune activation also damages lymphoid tissue architecture, so contributing to the impairment of immune reconstitution. Recently, new mechanisms were identified, so opening new perspective on the innate immune sensing in HIV-1 infection. Cell death is followed by the release of molecules containing "damage-associated molecular patterns", that trigger a potent innate immune response through the engagement of Toll-like receptors. Then, also different types of HIV-related nucleic acids can act as potent stimulators of innate immunity. All these events contribute to the loss of T cell homeostatic regulation and to the failure of adaptive immunity.

2014 - Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells. [Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Boraldi, Federica; Giorgio, Valentina; Bernardi, Paolo; Bartolomeo, Regina; Nasi, Milena; DE BIASI, Sara; Missiroli, Sonia; Carnevale, Gianluca; Losi, Lorena; Tesei, Anna; Pinton, Paolo; Quaglino, Daniela; Cossarizza, Andrea
abstract

Lon is a nuclear-encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up-regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon-silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to β-oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5-fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2-deoxy-d-glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3- and 1.3-fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells.-Gibellini, L., Pinti, M., Boraldi, F., Giorgio, V., Bernardi, P., Bartolomeo, R., Nasi, M., De Biasi, S., Missiroli, S., Carnevale, G., Losi, L., Tesei, A., Pinton, P., Quaglino, D., Cossarizza, A. Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells.

2014 - Sirtuin 3 interacts with Lon protease and regulates its acetylation status. [Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Beretti, Francesca; Pierri, Cl; Onofrio, A; Riccio, Massimo; Carnevale, Gianluca; DE BIASI, Sara; Nasi, Milena; Torelli, F; Boraldi, Federica; DE POL, Anto; Cossarizza, Andrea
abstract

Lon is a mitochondrial protease that degrades oxidized damaged proteins, assists protein folding and participates in maintaining mitochondrial DNA levels. Changes in Lon mRNA levels, protein levels and activity are not always directly correlated, suggesting that Lon could be regulated at post translational level. We found that Lon and SIRT3, the most important mitochondrial sirtuin, colocalize and coimmunoprecipitate in breast cancer cells, and silencing or inhibition of Lon did not alter SIRT3 levels. Silencing of SIRT3 increased the levels of Lon protein and of its acetylation, suggesting that Lon is a target of SIRT3, likely at K917.

2014 - Small RNAs in prokaryotes and eukaryotes: A lesson for human immunologists: Comment on "Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems" by Edwin L. Cooper and Nicola Overstreet. [Articolo su rivista]
Pinti, Marcello; Cossarizza, Andrea
abstract

No abstract available

2014 - Successful treatment of HIV-1 infection increases the expression of a novel, short transcript for IL-18 receptor α chain [Articolo su rivista]
Nasi, Milena; Alboni, Silvia; Pinti, Marcello; Tascedda, Fabio; Benatti, Cristina; Benatti, Stefania; Gibellini, Lara; DE BIASI, Sara; Borghi, Vanni; Brunello, Nicoletta; Mussini, Cristina; Cossarizza, Andrea
abstract

The importance of interleukin (IL)-18 in mediating immune activation during HIV infection has recently emerged. IL-18 activity is regulated by its receptor (IL-18R), formed by an α and a β chain, the IL-18-binding protein, and the newly identified shorter isoforms of both IL-18R chains. We evaluated gene expression of the IL-18/IL-18R system in peripheral blood mononuclear cells from HIV+ patients. Compared with healthy donors, IL-18 expression decreased in patients with primary infection. The IL-18Rα short transcript expression was strongly upregulated by successful highly active antiretroviral therapy. HIV progression and its treatment can influence the expression of different components of the complex IL-18/IL-18R system.

2014 - Switching to darunavir/ritonavir monotherapy vs. triple-therapy on body fat redistribution and bone mass in HIV-infected adults: Monarch randomized controlled trial [Articolo su rivista]
Guaraldi, Giovanni; Zona, Stefano; Cossarizza, Andrea; L., Vernacotola; Carli, Federica; A., Lattanzi; Nardini, Giulia; Orlando, Gabriella; E., Garlassi; R., Termini; M., Garau
abstract

BackgroundChanges in body fat distribution and bone mass in HIV-infected patients may be associated with long-term use of nucleoside analogues.MethodsThe Monarch trial recruited 30 patients receiving non-nucleoside reverse transcriptase inhibitor or protease inhibitor-based highly active antiretroviral therapy, with HIV RNA <40 copies/mL. Patients were randomized to either darunavir/ritonavir 800/100 mg once daily monotherapy or darunavir/ritonavir 800/100 mg once daily + 2NRTIs. Bone mass, peripheral lipoatrophy and central fat accumulation were assessed using dual-energy X-ray absorptiometry scanning, supplemented by computed tomography scans.ResultsMedian age was 43 years, 77% were males. Visceral adipose tissue remained stable from baseline to Week 48 in the whole group (p = 0.261) with no significant difference between arms (p = 0.56). There was a significant reduction in insulin resistance (HOMA-IR, p = 0.013) over 48 weeks in the whole group, but not of body mass index (p = 0.24). In the darunavir/ritonavir monotherapy arm, there was a small but significant increase in both lumbar and femur bone mineral density at 48 weeks and was observed after correction for baseline values. The absolute change in lumbar bone mineral density at 48 weeks was more pronounced in the darunavir/ritonavir arm compared with the darunavir/ritonavir + 2NRTIs arm.ConclusionsIn this study, discontinuing nucleoside analogues and switching to darunavir/ritonavir monotherapy was associated with a small but statistically significant increase in bone mineral density, but stable levels of limb fat and visceral adipose tissue.

2014 - The Fas/Fas ligand apoptosis pathway underlies immunomodulatory properties of human biliary tree stem/progenitor cells [Articolo su rivista]
Riccio, Massimo; Carnevale, Gianluca; Cardinale, Vincenzo; Gibellini, Lara; DE BIASI, Sara; Pisciotta, Alessandra; Carpino, Guido; Gentile, Raffaele; Berloco, Pasquale B; Brunelli, Roberto; Bastianelli, Carlo; Napoletano, Chiara; Cantafora, Alfredo; Cossarizza, Andrea; Gaudio, Eugenio; Alvaro, Domenico; DE POL, Anto
abstract

Human biliary tree stem/progenitor cells (hBTSCs) are multipotent epithelial stem cells, easily obtained from the biliary tree, with the potential for regenerative medicine in liver, biliary tree, and pancreas diseases. Recent reports indicate that human mesenchymal stem cells are able to modulate the T cell immune response. However, no information exists on the capabilities of hBTSCs to control the allogeneic response. The aims of this study were to evaluate FasL expression in hBTSCs, to study the in vitro interaction between hBTSCs and human lymphocytes, and the role of Fas/FasL modulation in inducing T cell apoptosis in hBTSCs/T cell co-cultures.

2013 - A Janus role for MerTK in the outcome of septic shock [Articolo su rivista]
Girardis, Massimo; Cossarizza, Andrea
abstract

No

2013 - Absence of liver steatosis in HIV-HCV co-infected patients receiving regimens containing tenofovir or abacavir [Articolo su rivista]
Borghi, V; Bisi, Luca; Manzini, L; Cossarizza, Andrea; Mussini, Cristina
abstract

In human immunodeficiency virus-hepatitis C virus (HIV-HCV) co-infected patients, steatosis has been independently associated with a number of antiretroviral drugs, including stavudine, especially in patients with non-3 HCV genotypes. We retrospectively investigated the presence of steatosis among HIV-HCV co-infected and HCV mono-infected patients, and the role of tenofovir disoproxil fumarate (TDF) or abacavir (ABC) in determining hepatic steatosis. METHODS: Liver steatosis was retrospectively evaluated in all consecutive biopsies performed in the period 2000-2008 in HCV mono-infected and HIV-HCV co-infected patients. A steatosis rate of >5 % was considered to be significant, and a multivariate logistic analysis was performed to evaluate factors associated with steatosis. RESULTS: In total, 393 HCV-infected patients underwent liver biopsy during the study period, of whom 205 (52.2 %) were co-infected with HIV. A steatosis rate of >5 % was diagnosed in 33.0 % of HCV mono-infected and in 47.8 % of HIV-HCV co-infected patients (P = 0.003). The rate of steatosis was higher in patients resuming antiretroviral therapy (54.7 %) than in naïve patients (33.3 %; P = 0.006). When the overall population was considered, steatosis was associated to HCV genotype 3 [odds ratio (OR) 4.53, 95 % confidence interval (CI) 2.71-7.58; P < 0.001]. In terms of the use of nucleos(t)ide drugs in HIV co-infected patients, multivariate analysis showed that only in patients with HCV genotypes other than genotype 3 was steatosis related to the use of stavudine (OR 5.38, 95 % CI 1.18-24.53; P = 0.03). The use of TDF (OR 1.07, 95 % CI 0.39-2.88; P = 0.898) or ABC (OR 0.592, 95 % CI 0.09-4.07; P = 0.594) was not associated with steatosis. CONCLUSION: In HCV mono-infected and HIV-HCV co-infected patients, steatosis appears to be a virus-mediated effect of HCV genotype 3. In HIV patients infected with HCV genotypes other than genotype 3, the risk of developing steatosis was higher in those patients resuming antiretroviral regimens containing old drugs rather than the new antiretrovirals.

2013 - Association Study of Common Genetic Variants and HIV-1 Acquisition in 6,300 Infected Cases and 7,200 Controls [Articolo su rivista]
Mclaren, P. J.; Coulonges, C.; Ripke, S.; van den Berg, L.; Buchbinder, S.; Carrington, M.; Cossarizza, A.; Dalmau, J.; Deeks, S. G.; Delaneau, O.; De Luca, A.; Goedert, J. J.; Haas, D.; Herbeck, J. T.; Kathiresan, S.; Kirk, G. D.; Lambotte, O.; Luo, M.; Mallal, S.; van Manen, D.; Martinez-Picado, J.; Meyer, L.; Miro, J. M.; Mullins, J. I.; Obel, N.; O'Brien, S. J.; Pereyra, F.; Plummer, F. A.; Poli, G.; Qi, Y.; Rucart, P.; Sandhu, M. S.; Shea, P. R.; Schuitemaker, H.; Theodorou, I.; Vannberg, F.; Veldink, J.; Walker, B. D.; Weintrob, A.; Winkler, C. A.; Wolinsky, S.; Telenti, A.; Goldstein, D. B.; de Bakker, P. I. W.; Zagury, J. -F.; Fellay, J.
abstract

Multiple genome-wide association studies (GWAS) have been performed in HIV-1 infected individuals, identifying common genetic influences on viral control and disease course. Similarly, common genetic correlates of acquisition of HIV-1 after exposure have been interrogated using GWAS, although in generally small samples. Under the auspices of the International Collaboration for the Genomics of HIV, we have combined the genome-wide single nucleotide polymorphism (SNP) data collected by 25 cohorts, studies, or institutions on HIV-1 infected individuals and compared them to carefully matched population-level data sets (a list of all collaborators appears in Note S1 in Text S1). After imputation using the 1,000 Genomes Project reference panel, we tested approximately 8 million common DNA variants (SNPs and indels) for association with HIV-1 acquisition in 6,334 infected patients and 7,247 population samples of European ancestry. Initial association testing identified the SNP rs4418214, the C allele of which is known to tag the HLA-B*57:01 and B*27:05 alleles, as genome-wide significant (p = 3.6×10-11). However, restricting analysis to individuals with a known date of seroconversion suggested that this association was due to the frailty bias in studies of lethal diseases. Further analyses including testing recessive genetic models, testing for bulk effects of non-genome-wide significant variants, stratifying by sexual or parenteral transmission risk and testing previously reported associations showed no evidence for genetic influence on HIV-1 acquisition (with the exception of CCR5Δ32 homozygosity). Thus, these data suggest that genetic influences on HIV acquisition are either rare or have smaller effects than can be detected by this sample size.

2013 - Cytometry for immunology: A stable and happy marriage [Articolo su rivista]
Cossarizza, A.; Radbruch, A.
abstract

2013 - Cytometry, immunology, and HIV infection: Three decades of strong interactions. [Articolo su rivista]
Cossarizza, Andrea; DE BIASI, Sara; Gibellini, Lara; Bianchini, E; Bartolomeo, Regina; Nasi, Milena; Mussini, Cristina; Pinti, Marcello
abstract

Flow cytometry (FCM) has been extensively used to investigate immunological changes that occur from infection with the human immunodeficiency virus (HIV). This review describes some of the most relevant cellular and molecular changes in the immune system that can be detected by FCM during HIV infection. Finally, it will be discussed how this technology has facilitated the understanding not only of the biology of the virus but also of the mechanisms that the immune system activates to fight HIV and is allowing to monitor the efficacy of antiretroviral therapy.

2013 - Daunomycin, an antitumor DNA intercalator, influences histone-DNA interactions. [Articolo su rivista]
Wójcik, K; Zarębski, M; Cossarizza, Andrea; Dobrucki, J. w.
abstract

Although daunomycin and adriamycin are considered effective antitumor drugs and have been used in the clinic for over 40 years, their mechanism of action is still a matter of debate. We investigated the influence of daunomycin on interaction between linker or core histones and DNA in live HeLa cells in vitro, using image and flow cytometry. Exposure to daunomycin at clinically relevant concentrations (25-250 nM) caused dissociation of wild-type H1.1 as well as 4 H1 point mutants from DNA, followed by their accumulation in nucleoli and aggregation of chromatin. A detectable dissociation of H2B core histones occurred only at much higher concentrations of the drug (500 nM). Replication of DNA and synthesis of RNA were not halted by daunomycin (up to 2500 nM); however the characteristic subnuclear distribution of sites of transcription and replication was lost. Dissociation of the H1.1 linker histones and subsequent loss of higher order chromatin structures may constitute an important component of the mechanism of cytotoxicity of daunomycin.

2013 - Early alterations of B cells in patients with septic shock: another piece in the complex puzzle of the immune response in sepsis [Articolo su rivista]
Girardis, Massimo; Cossarizza, Andrea
abstract

Impairment of the inflammatory-immune response is currently accepted as a hallmark of severe sepsis even in the early stages of the disease. In this context, the alterations of the circulating B-lymphocytes have never been described in detail. The study by Monserrat and colleagues in the previous issue of Critical Care indicated that, in patients with septic shock, the B-cell compartment is early and deeply altered with different patterns in subset distribution and activation between survivors and non-survivors.

2013 - Frequent early multiple sclerosis relapses during treatment with fingolimod: a paradoxical effect? [Articolo su rivista]
Ferraro, Diana; Federzoni, Lucia; Vitetta, F; Simone, ANNA MARIA; Cossarizza, Andrea; Nichelli, Paolo Frigio; Sola, P.
abstract

This is the case report of a multiple sclerosi patient who presented frequent relapses early after beginning a treatment with fingolimod

2013 - Life expectancy in the immune-recovery era: the evolving scenario of HIV epidemic in Northern Italy [Abstract in Rivista]
Guaraldi, Giovanni; Cossarizza, Andrea; C., Franceschi; A., Roverato; E., Vaccher; G., Tambussi; Garlassi, Elisa; Menozzi, Marianna; Mussini, Cristina; A., D’Arminio Monforte
abstract

National cohort and inter-cohort studies have been set to describe differences of Life Expectancy (LE) of HIV-infected individuals. the aim of this study was to assess the impact of immune-recovery on LE of HIV-patients undergoing cART.

2013 - Monocytic population in chronic lymphocytic leukemia shows altered composition and deregulation of genes involved in phagocytosis and inflammation [Articolo su rivista]
Maffei, Rossana; Bulgarelli, Jenny; Fiorcari, Stefania; Bertoncelli, L; Martinelli, Silvia; Guarnotta, C; Castelli, Ilaria; Deaglio, S; Debbia, Giulia; DE BIASI, Sara; Bonacorsi, G; Zucchini, Patrizia; Narni, Franco; Tripodo, C; Luppi, Mario; Cossarizza, Andrea; Marasca, Roberto
abstract

Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B-cells and the extent of infiltration is associated with adverse prognostic factors. Blood monocyte population was studied by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate T cell proliferation in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes treated with leukemia were also evaluated. Chronic lymphocytic leukemia patients showed an increase in the absolute number of monocytes compared to normal controls (792+/-86 cells/mL vs. 485+/-46 cells/mL, p=0.003). Higher number of nonclassical CD14+CD16++ and Tie-2 expressing monocytes (TEMs) was also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment in phagocytosis. We also detected gene alterations such as the down-regulation of PTGR2, a reductase able to inactivate the prostaglandin E2, indicating an immunosuppressive activity. Accordingly, T cell proliferation was inhibited in contact with monocytes from patients compared to normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated 'education' of immune elements may also include the establishment of a skewed phenotype in monocyte/macrophage population.

2013 - N-acetyl-cysteine prevents toxic oxidative effects induced by IFN-α in human neurons. [Articolo su rivista]
Alboni, Silvia; Gibellini, Lara; Montanari, Claudia; Benatti, Cristina; Benatti, Stefania; Tascedda, Fabio; Brunello, Nicoletta; Cossarizza, Andrea; Pariante, Carmine M.
abstract

Currently IFN-α is widely used for effective treatment of viral infections and several malignancies. However, IFN-α can cause neuropsychiatric disturbances and mental impairments, including fatigue, insomnia, depression, irritability and cognitive deficits. Molecular and cellular mechanisms leading to such side-effects are still poorly understood. Neurons seem to be an important target in mediating cellular effects induced by exposure to this cytokine, but so far little is known about IFN-α-induced effects on these cells. We have investigated the ability of IFN-α (2-100 ng/ml) to induce damage and toxicity to the human neuroblastoma SH-SY5Y cell line, commonly used for studying such phenomena, and the mechanisms underlying these effects. After 24 h treatment, IFN-α increased mitochondrial activity, whereas cell density was reduced in a dose- and time-dependent manner. This effect did not depend on reduced cell proliferation, but rather the activation of apoptosis, as revealed by an increased Bax:Bcl-2 mRNA ratio after 72-h IFN-α exposure. At this time-point, IFN-α also reduced the expression of the brain-derived neurotrophic factor gene, and induced an increase in reactive oxygen species (ROS). A co-treatment with N-acetyl-cysteine (NAC; 5 mm), a potent antioxidant and mitochondrial modulator, was able to counteract all of these IFN-α-induced effects. These findings demonstrated that IFN-α induces neurotoxicity and apoptosis that is, in part, very likely due to mitochondrial damages and production of ROS. We suggest that NAC, already tested for the treatment of psychiatric disorders, may be useful to prevent IFN-α-induced central side-effects in a safe and effective way.

2013 - Novel genetic association of TNF-α-238 and PDCD1-7209 polymorphisms with long-term non-progressive HIV-1 infection. [Articolo su rivista]
Nasi, Milena; Riva, A; Borghi, V; D'Amico, Roberto; DEL GIOVANE, Cinzia; Casoli, C; Galli, M; Vicenzi, E; Gibellini, Lara; DE BIASI, Sara; Clerici, M; Mussini, Cristina; Cossarizza, Andrea; Pinti, Marcello
abstract

About 2-5% of HIV-1-infected subjects, defined as long-term non-progressors (LTNPs), remain immunologically stable for a long time without treatment. The factors governing this condition are known only in part, and include genetic factors. Thus, we studied 20 polymorphisms of 15 genes encoding proinflammatory and immunoregulatory cytokines, chemokines and their receptors, genes involved in apoptosis, and the gene HCP5. METHODS: We analyzed 47 Caucasian LTNPs infected for &gt;9 years, compared with 131 HIV-1-infected Caucasian patients defined as 'usual progressors'. The genotypes were determined by methods based upon PCR, and the statistical analysis was performed by univariate logistic regression. RESULTS: The well-known CCR5Δ32 del32 allele, the cell death-related TNF-α-238 A and PDCD1-7209 T alleles, and HCP5 rs2395029 G, a non-coding protein associated with the HLA-B*5701, were found positively associated with the LTNP condition. No association was observed for other single nucleotide polymorphisms (SDF-1-801, IL-10-592, MCP-1-2518, CX3CR1 V249I, CCR2V64I, RANTES-403, IL-2-330, IL-1β-511, IL-4-590, FASL IVS3nt-169, FAS-670, FAS-1377, FASL IVS2nt-124, PDCD1-7146, MMP-7-181, and MMP7-153). CONCLUSIONS: The novel genetic associations between allelic variants of genes TNF-α-238 and PDCD1-7209 with the LTNP condition underline the importance of host genetic factors in the progression of HIV-1 infection and in immunological preservation.

2013 - Randomized Trial to Evaluate Cardiometabolic and Endothelial Function in Patients with Plasma HIV-1 RNA Suppression Switching to Darunavir/Ritonavir with or without Nucleoside Analogues [Articolo su rivista]
Guaraldi, Giovanni; Zona, Stefano; Cossarizza, Andrea; L., Vernacotola; Carli, Federica; A., Lattanzi; Beghetto, Barbara; Orlando, Gabriella; DE BIASI, Sara; R., Termini; M., Garau
abstract

Background: We performed a study to evaluate change in cardiometabolic and endothelial function in HIV-infected patients switching to darunavir/ritonavir (DRV/r) monotherapy versus triple therapy. Methods: The MONARCH trial recruited 30 patients who were taking triple combination therapy and with HIV RNA<40 copies/ mL. Patients were randomized to either DRV/r 800/100 mg once daily (OD) monotherapy or DRV/r 800/100 mg OD plus 2 nucleoside reverse transcriptase inhibitors (NRTIs). The primary objective was to assess endothelial function change from baseline to 24 and 48 weeks in brachial artery flow-mediated dilation (FMD) test; changes in endothelial precursor cells (EPCs) and circulating endothelial cells (CECs) were secondary objectives. Results: At baseline, the median age of participants was 43 years, 77% were men, and median CD4 cell count was 585 cells/μL. The median FMD (%) decreased in both arms in the study period (P ≯ .05), with no statistically significant difference between arms (10.7% at baseline and 6.7% at week 48 in the DRV/r + 2 NRTIs arm; 11.1% at baseline and 8.8% at week 48 in the DRV/r arm). The changes at week 48 were similar in the 2 arms for EPCs and CECs. Total cholesterol and low-density lipoprotein (LDL) cholesterol showed larger rises to week 48 in the DRV/r arm monotherapy group than in the triple-therapy group (+26 vs +9 mg/dL for total cholesterol and +14 vs +5 mg/dL for LDL cholesterol). Conclusions: In the MONARCH trial, switching from triple combination treatment to DRV/r, with or without nucleoside analogues, did not translate into clinically meaningful reductions in endothelial function as measured by FMD.

2013 - Recurrent varicella following steroids and fingolimod in a Multiple Sclerosis patient [Articolo su rivista]
Ferraro, Diana; DE BIASI, Sara; Vitetta, F; Simone, A. M; Federzoni, Lucia; Borghi, Valentina; Cossarizza, Andrea; Nichelli, Paolo Frigio; Sola, P.
abstract

This is the case report of a multiple sclerosis patient who presented recurrent varicella following steroid and fingolimod therapy

2012 - Advancing cytometry for immunology. [Articolo su rivista]
Cossarizza, Andrea; Nolan, J; Radbruch, A; Tárnok, A.
abstract

Cytometry is a key technology for immunology. It allows researchers to scrutinize the cells of the immune system in molecular detail, and to assess phenotype and function at the level of individual cells, no matter how rare these cells may be. The International Society for the Advancement of Cytometry, ISAC, by way of its meetings, online resources and publications (e.g. Cytometry Part A and Current Protocols in Cytometry, which are all published by Wiley) track the ever advancing developments regarding cytometry instrumentation and reagents, and the analysis of complex data sets. In June this year in Leipzig, Germany, ISAC held its annual conference "CYTO 2012", a marketplace of innovation in cytometry.

2012 - Herpes Simplex I virus impairs regenerative outcomes of periodontal regenerative therapy in intrabony defects. A pilot study [Articolo su rivista]
Bertoldi, Carlo; Pellacani, Chiara; Lalla, Michele; Consolo, Ugo; Pinti, Marcello; Cortellini, P.; Cossarizza, Andrea
abstract

Aim: To evaluate the impact of herpesvirus type-1 and -2 on the clinical outcomes of periodontal regenerative procedures in isolated deep intrabony pockets, in an experimental population with no detectable periodontal pathogens. Materials and Methods: Seventeen periodontal intraosseous defects in 17 moderate- to-advanced periodontitis patients were treated with regenerative therapy and amelogenins. Microbiological evaluation was performed at baseline (after the completion of initial therapy) and at 1 year to exclude the presence of periodontal pathogens. Herpesviruses-1 and -2 DNA were quantified in the pocket tissues associated to the intrabony defect using molecular assays. Clinical attachment level (CAL), probing pocket depth (PPD) and gingival recession (REC) were recorded at baseline and at 1 year. Results: After 1 year, the 17 defects resulted in significant CAL gain, PPD reduction and REC increase. HSV-1 was detected in five patients. Herpesvirus-2 was never found. The two subpopulations positive or negative to herpesvirus-1 were homogeneous at baseline. At 1 year, the five herpesvirus-1 positive patients resulted in lower amounts of CAL-gain and PPD reduction and greater amount of REC with respect to the 12 herpesvirus-1 negative patients. Conclusions: The presence of herpesvirus-1 at baseline is associated with poor clinical outcomes following regenerative therapy. PMID: 22292785 IDS Number: 907NW

2012 - Immunological advantages of everolimus versus cyclosporin A in liver-transplanted recipients, as revealed by polychromatic flow cytometry. [Articolo su rivista]
Roat, Erika; DE BIASI, Sara; Bertoncelli, Linda; Rompianesi, Gianluca; Nasi, Milena; Gibellini, Lara; Pinti, Marcello; DEL GIOVANE, Cinzia; A., Zanella; DI BENEDETTO, Fabrizio; Gerunda, Giorgio Enrico; Cossarizza, Andrea
abstract

Several immunosuppressive drugs with different mechanisms of action are available to inhibit organ rejection after transplant. We analyzed different phenotypic and functional immunological parameters in liver-transplanted patients who received cyclosporin A (CsA) or Everolimus (Evr). In peripheral blood mononuclear cells (PBMC) from 29 subjects receiving a liver transplant and treated with two different immunosuppressive regimens, we analyzed T cell activation and differentiation, regulatory T cells (Tregs) and Tregs expressing homing receptors such as the chemokine receptor CXCR3. T cell polyfunctionality was studied by stimulating cells with the superantigen staphylococcal enterotoxin B (SEB), and measuring the simultaneous production of interleukin (IL)-2 and interferon (IFN)-γ, along with the expression of a marker of cytotoxicity such as CD107a. The analyses were performed by polychromatic flow cytometry before transplantation, and at different time points, up to 220 days after transplant. Patients taking Evr had a higher percentage of total CD4⁺ and naïve CD4⁺ T cells than those treated with CsA; the percentage of CD8⁺ T cells was lower, but the frequency of naïve CD8⁺ T cells higher. Patients taking Evr showed a significantly higher percentage of Tregs, and Tregs expressing CXCR3. After stimulation with SEB, CD8⁺ T cells from Evr-treated patients displayed a lower total response, and less IFN-γ producing cells. The effects on the immune system, such as the preservation of the naïve T cell pool and the expansion of Tregs (that are extremely useful in inhibiting organ rejection), along with the higher tolerability of Evr, suggest that this drug can be safely used after liver transplantation, and likely offers immunological advantages.

2012 - Mitochondrial DNA haplogroups and incidence of lipodystrophy in HIV-infected patients on long-term antiretroviral therapy. [Articolo su rivista]
De Luca, A; Nasi, Milena; Di Giambenedetto, S; Cozzi Lepri, A; Pinti, Marcello; Marzocchetti, A; Mussini, Cristina; Fabbiani, M; Bracciale, L; Cauda, R; Cossarizza, Andrea
abstract

We investigated the rates of lipodystrophy events, according to mitochondrial DNA haplogroup, in 187 patients starting combination antiretroviral therapy and following it. Incidence rates of lipoatrophy and fat accumulation were 8.2 and 4.8 per 100 person-years of follow-up, respectively. In multivariable models, patients with haplogroup K were at higher risk of any lipodystrophy [adjusted relative risk (aRR) 4.02, P = 0.0009], lipoatrophy (competing-risk aRR 2.42, P = 0.09; cause-specific aRR 2.99, P = 0.031), and fat accumulation (competing-risk aRR, 2.63, P = 0.11; cause-specific aRR 5.27, P = 0.019) than those with haplogroup H. Mitochondrial haplogroups may explain part of the genetic predisposition to lipodystrophy during combination antiretroviral therapy.

2012 - Mitochondrial DNA: a proinflammatory 'enemy from within' during HIV infection? [Articolo su rivista]
Pinti, Marcello; Mussini, Cristina; Cossarizza, Andrea
abstract

Mitochondria are crucial in cell life, as they are the main intracellular source of energy, and have a main role in cell death as they contain molecules able to trigger apoptosis. However, mitochondria can also release molecules called ‘damage-associated molecular patterns’ (DAMPs) that trigger a potent innate immune response and cause inflammation through the engagement of the Toll-like receptors (TLR). During human immunodeficiency virus (HIV) infection, a proinflammatory status is present that is not completely explained by the activity of the virus per se, or by the effective immune response against the virus. However, the presence of significant amounts of DAMPs of mitochondrial origin, such as extracellular plasmatic mtDNA, in the peripheral blood of subjects with HIV infection suggests that part of the inflammation typical of such infection could be because of the activity of these molecules, and thus opens new therapeutic perspectives.

2012 - SIICA-DGfI 2011 Joint Meeting: Front-line immunology with sensorial stimulations, neuromuscular activity and phago/pinocytosis [Articolo su rivista]
Cossarizza, A.; Radbruch, A.
abstract

2012 - STEAP1 Is Associated with the Invasive and Oxidative Stress Phenotype of Ewing Tumors. [Articolo su rivista]
Grunewald, Tg; Diebold, I; Esposito, I; Plehm, S; Hauer, K; Thiel, U; da Silva Buttkus, P; Neff, F; Unland, R; Müller Tidow, C; Zobywalski, C; Lohrig, K; Lewandrowski, U; Sickmann, A; da Costa, Op; Görlach, A; Cossarizza, Andrea; Butt, E; Richter, Gh; Burdach, S.
abstract

Ewing tumors comprise the second most common type of bone-associated cancer in children and are characterized by oncogenic EWS/FLI1 fusion proteins and early metastasis. Compelling evidence suggests that elevated levels of intracellular oxidative stress contribute to enhanced aggressiveness of numerous cancers, possibly including Ewing tumors. Using comprehensive microarray analyses and RNA interference, we identified the six-transmembrane epithelial antigen of the prostate 1 (STEAP1)-a membrane-bound mesenchymal stem cell marker of unknown function-as a highly expressed protein in Ewing tumors compared with benign tissues and show its regulation by EWS/FLI1. In addition, we show that STEAP1 knockdown reduces Ewing tumor proliferation, anchorage-independent colony formation as well as invasion in vitro and decreases growth and metastasis of Ewing tumor xenografts in vivo. Moreover, transcriptome and proteome analyses as well as functional studies revealed that STEAP1 expression correlates with oxidative stress responses and elevated levels of reactive oxygen species that in turn are able to regulate redox-sensitive and proinvasive genes. In synopsis, our data suggest that STEAP1 is associated with the invasive behavior and oxidative stress phenotype of Ewing tumors and point to a hitherto unanticipated oncogenic function of STEAP1. Mol Cancer Res; 10(1); 52-65. ©2011 AACR.

2012 - Single-Nucleotide Polymorphism-Defined Class I and Class III Major Histocompatibility Complex Genetic Subregions Contribute to Natural Long-term Nonprogression in HIV Infection. [Articolo su rivista]
Guergnon, J; Dalmasso, C; Broet, P; Meyer, L; Westrop, Sj; Imami, N; Vicenzi, E; Morsica, G; Tinelli, M; Zanone Poma, B; Goujard, C; Potard, V; Gotch, Fm; Casoli, C; Cossarizza, Andrea; Macciardi, F; Debré, P; Delfraissy, Jf; Galli, M; Autran, B; Costagliola, D; Poli, G; Theodorou, I; Riva, A.
abstract

We performed a genome-wide association study comparing a cohort of 144 human immunodeficiency virus (HIV type 1-infected, untreated white long-term nonprogressors (LTNPs) with a cohort of 605 HIV-1-infected white seroconverters. Forty-seven single-nucleotide polymorphisms (SNPs), located from class I to class III major histocompatibility complex (MHC) subregions, show statistical association (false discovery rate, <0.05) with the LTNP condition, among which 5 reached genome-wide significance after Bonferonni correction. The MHC LTNP-associated SNPs are ordered in ≥4 linkage disequilibrium blocks; interestingly, an MHC class III linkage disequilibrium block (defined by the rs9368699 SNP) seems specific to the LTNP phenotype.

2012 - Stable changes in CD4+ T lymphocyte miRNA expression after exposure to HIV-1. [Articolo su rivista]
F., Bignami; E., Pilotti; Bertoncelli, Linda; P., Ronzi; M., Gulli; N., Marmiroli; Magnani, Giacomo; Pinti, Marcello; L., Lopalco; Mussini, Cristina; R., Ruotolo; M., Galli; Cossarizza, Andrea; C., Casoli
abstract

MicroRNAs (miRNAs) inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4(+) T cells from HIV-1 élite long-term nonprogressors (éLTNPs), naive patients, and multiply exposed uninfected (MEU) patients, and we observed that the éLTNP patients clustered with naive patients, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated éLTNP from MEU patients and 23 miRNAs distinguished naive from MEU patients, whereas only 1 miRNA (miR-155) discriminated éLTNP from naive patients. We proposed that miRNA expression may discriminate between HIV-1-infected and -exposed but negative patients. Analysis of miRNAs expression after exposure of healthy CD4(+) T cells to gp120 in vitro confirmed our hypothesis that a miRNA profile could be the result not only of a productive infection but also of the exposure to HIV-1 products that leave a signature in immune cells. The comparison of normalized Dicer and Drosha expression in ex vivo and in vitro condition revealed that these enzymes did not affect the change of miRNA profiles, supporting the existence of a Dicer-independent biogenesis pathway.

2012 - T cell activation but not polyfunctionality after primary HIV infection predicts control of viral load and length of the time without therapy. [Articolo su rivista]
Cossarizza, Andrea; Bertoncelli, Linda; Nemes, Elisa; Lugli, Enrico; Pinti, Marcello; Nasi, Milena; DE BIASI, Sara; Gibellini, Lara; Montagna, Jp; Vecchia, M; Manzini, Lisa; Meschiari, Marianna; Borghi, Valentina; Guaraldi, Giovanni; Mussini, Cristina
abstract

Immune changes occurring after primary HIV infection (PHI) have a pivotal relevance. Our objective was to characterize the polyfunctionality of immune response triggered by PHI, and to characterize immune activation and regulatory T cells, correlating such features to disease progression. PATIENTS AND METHODS: We followed 11 patients experiencing PHI for 4 years. By polychromatic flow cytometry, we studied every month, for the first 6 months, T lymphocyte polyfunctionality after cell stimulation with peptides derived from HIV-1 gag and nef. Tregs were identified by flow cytometry, and T cell activation studied by CD38 and HLA-DR expression. RESULTS: An increase of anti-gag and anti-nef CD8+ specific T cells was observed 3 months after PHI; however, truly polyfunctional T cells, also able to produce IL-2, were never found. No gross changes in Tregs were present. T lymphocyte activation was maximal 1 and 2 months after PHI, and significantly decreased in the following period. The level of activation two months after PHI was strictly correlated to the plasma viral load 1 year after infection, and significantly influenced the length of period without therapy. Indeed, 80% of patients with less than the median value of activated CD8+ (15.5%) or CD4+ (0.9%) T cells remained free of therapy for &gt;46 months, while all patients over the median value had to start treatment within 26 months. CONCLUSIONS: T cell activation after PHI, more than T cell polyfunctionality or Tregs, is a predictive marker for the control of viral load and for the time required to start treatment.

2012 - The STEAP protein family: Versatile oxidoreductases and targets for cancer immunotherapy with overlapping and distinct cellular functions [Articolo su rivista]
Grunewald, T. G. P.; Bach, H.; Cossarizza, A.; Matsumoto, I.
abstract

The human six-transmembrane epithelial antigen of the prostate (STEAP) protein family contains at least five homologous members. The necessity of multiple homologous STEAP proteins is still unclear, but their peculiar and tissue-specific expression suggests that they are assigned to distinct functional tasks. This concept is supported by the fact that especially STEAP1, and to a lesser extent STEAP2 and -4, are highly over-expressed in many different cancer entities, while being only minimally expressed in a few normal tissues. Despite their very similar domain organisation, STEAP3 seems to act as a potent metalloreductase essential for physiological iron uptake and turnover, while in particular STEAP4 appears to be rather involved in responses to nutrients and inflammatory stress, fatty acid and glucose metabolism. Moreover, individual STEAP proteins possess overlapping functions important for growth and survival of cancer cells. Due to their membrane-bound localisation and their high expression in many different cancers such as prostate, breast and bladder carcinoma as well as Ewing's sarcoma, STEAP proteins have been recognised and utilised as promising targets for cell- and antibody-based immunotherapy. This review summarises our present knowledge of the individual members of the human STEAP family and highlights the functional differences between them. © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

2012 - The protease inhibitor atazanavir triggers autophagy and mitophagy in human preadipocytes [Articolo su rivista]
Gibellini, Lara; DE BIASI, Sara; Pinti, Marcello; Nasi, Milena; Riccio, Massimo; Carnevale, Gianluca; Cavallini, Gian Maria; F. J., Sala De Oyanguren; J. E., O’Connor; Mussini, Cristina; DE POL, Anto; Cossarizza, Andrea
abstract

Background: The association betweenHAARTand lipodystrophy iswell established, but lipodystrophy pathogenesis is still poorly understood. Drugs, and in particular protease inhibitors, accumulate in adipose tissue affecting adipocyte physiology and gene expression by several mechanisms. Recent studies have identified autophagy as another process affected by these classes of drugs, but no studies have been performed in adipose cells. Methods: SW872 preadipocytic human cell line was used to evaluate changes induced by amprenavir (APV), ritonavir (RTV), or atazanavir (ATV), all used at 10–200mmol/l. A subline was stably transfected with murine stem cell virus (pMSCV)-enhanced green fluorescent protein (EGFP)-LC3 plasmid (to obtain a fluorescent LC3 protein) and treated with ATV at different doses. The distribution of LC3 and the colocalization of mitochondria, lysosome, and autophagosome were assessed by confocal microscopy. Transmission electron microscopy of ATV-treated cells was also performed. The cellular content of lysosomes was assessed using Lysotracker Green; apoptosis was evaluated by annexin V/propidium iodide staining, and mitochondrial superoxide anion (mtO2-) was analyzed by mitoSOX red. Lysosomes, apoptosis, and mtO2 - were studied by flow cytometry and multispectral imaging flow cytometry. Results: In SW872 cells, RTV caused massive apoptosis, more than autophagy, whereas APV was almost ineffective. ATV induced both apoptosis (high doses) and autophagy (low doses). ATV-treated cells displayed LC3-specific punctae, suggesting the formation of autophagosomes that enclosed mitochondria, as revealed by electron microscopy. At low doses, ATV promoted mitochondrial superoxide generation, whereas at high doses, it induced mitochondrial membrane depolarization. Conclusion: Autophagy/mitophagy can be considered a mechanism triggered by ATV in SW872 preadipocytes.

2011 - A randomized, controlled trial to evaluate change in cardiometabolic and endothelial function in HIV-infected patients with optimal viral suppression on Darunavir/ritonavir monotherapy vs. triple-therapy. [Abstract in Rivista]
Guaraldi, Giovanni; Zona, Stefano; Cossarizza, Andrea; L., Vernacotola; Carli, Federica; A., Lattanzi; Beghetto, Barbara; Orlando, Gabriella; R., Termini; M., Garau
abstract

MONET and MONOI have shown a small rise in cholesterol after stopping tenofovir in darunavir/ritonavir arm, without providing any information regarding endothelial function. We performed a study to compare the change in brachial artery flow mediated vasodilatation in patients receiving darunavir/ritonavir monotherapy versus triple therapy.

2011 - CD4+ T-cell differentiation, regulatory T cells and gag-specific T lymphocytes are unaffected by CD4-guided treatment interruption and therapy resumption. [Articolo su rivista]
Nemes, Elisa; Lugli, Enrico; Bertoncelli, Linda; Nasi, Milena; Pinti, Marcello; Manzini, S; Prati, Francesca; Manzini, Lisa; DEL GIOVANE, Cinzia; D'Amico, Roberto; Cossarizza, Andrea; Mussini, Cristina
abstract

OBJECTIVES:Despite limiting exposure to antiretroviral drugs, structured treatment interruptions can influence multiple aspects of T-cell immunity, particularly those regarding CD4(+) T lymphocytes. We evaluated the impact of CD4-guided treatment interruption (CD4-GTI) and treatment resumption on regulatory T cells (Tregs), T-lymphocyte activation, differentiation and polyfunctional gag-specific response.METHODS:Patients were analyzed just prior to treatment interruption, at 2 and 6 months after treatment interruption, just prior to treatment resumption and at 2 and 6 months after treatment resumption. Thawed peripheral blood mononuclear cells were stained immediately for phenotype analysis or stimulated with HIV-gag peptides and analyzed by polychromatic flow cytometry.RESULTS:Treatment interruption resulted in a CD4(+) cell count decrease and plasma viral load (pVL) increase, but did not preclude a good immune reconstitution and a complete suppression of pVL after treatment resumption. Treatment interruption did not influence CD4(+) T-cell differentiation and Treg subsets. During treatment interruption, gag-specific CD4(+) T cells were not lost, although the frequency of HIV-specific CD8(+) cells increased. Most gag-specific CD4(+) T cells were potentially cytotoxic (CD107a(+)) and were not influenced by pVL or by HAART. Most helper (CD154(+)) gag-specific CD4(+) T lymphocytes did not produce interferon-γ or interleukin-2.CONCLUSION:CD4-GTI did not cause depletion of memory cells, Tregs or HIV-specific CD4(+) cells and, on the contrary, could induce HIV-specific responses. If guided by CD4(+) T-cell count, treatment interruption does not provoke irreversible immune damages.

2011 - CD4saurus Rex & HIVelociraptor vs. development of clinically useful immunological markers: a Jurassic tale of frozen evolution. [Articolo su rivista]
A., De Maria; Cossarizza, Andrea
abstract

One of the most neglected areas of everyday clinical practice for HIV physicians is unexpectedly represented by CD4 T cell counts when used as an aid to clinical decisions. All who care for HIV patients believe that CD4+ T cell counts are a reliable method to evaluate a patient immune status. There is however a fatalistic acceptance that besides its general usefulness, CD4+ T cell counts have relevant clincal and immunological limits. Shortcomings of CD4 counts appear in certain clinical scenarios including identification of immunological nonresponders, subsequent development of cancer on antiretroviral teatment, failure on tretment simplification. Historical and recently described parameters might be better suited to advise management of patients at certain times during their disease history. Immunogenotypic parameters and innate immune parameters that define progression as well as immune parameters associated with immune recovery are available and have not been introduced into validation processes in larger trials. The scientific and clinical community needs an effort in stimulating clinical evolution of immunological tests beyond "CD4saurus Rex" introducing new parameters in the clinical arena after appropriate validation.

2011 - Decline of CD4⁺ T-cell count before start of therapy and immunological response to treatment in antiretroviral-naive individuals. [Articolo su rivista]
Mussini, Cristina; Cossarizza, Andrea; Sabin, C; Babiker, A; De Luca, A; Bucher, Hc; Fisher, M; Rezza, G; Porter, K; Dorrucci, M.
abstract

OBJECTIVE:Treatment guidelines recommend initiation of therapy for individuals experiencing rapid CD4 cell decline. It is not known, however, whether the rate of CD4 cell decline before combination antiretroviral therapy (cART) is related to immunological response following cART.METHODS:We estimated precART and postcART CD4 cell slopes by mixed models and categorized patients into two groups according to whether estimated precART slopes were above or below the 75th percentile. We compared immunological responses of the two groups through both mixed models and survival techniques. Models were stratified by CD4 cell at baseline, adjusted for HIV RNA, age, sex, HIV transmission group, year of seroconversion, initiation during primary infection, hepatitis C virus and hepatitis B virus serostatus, and cART class.RESULTS:Of 2038 eligible patients, 1531 and 507 experienced median (interquartile range) precART CD4 cell slope of −105 (−471 to −61) and −42 (−62 to −80) cells/μl, respectively, over 2 years. After adjusting for potential confounders, individuals with shallower decline experienced a slower rate of CD4 cell recovery following cART initiation of +9.5 [95% confidence interval (CI) +6.6 to +12.2] compared to +13.9 (+13.0 to +14.8) cells/μl per month among those with steeper precART decline (P &lt; 0.001). After stratifying by the baseline CD4 cell count, the adjusted relative hazard of an increase from baseline of more than 50 cells/μl was 0.70 (95% CI 0.62−0.79) for those with a shallower vs. steeper precART decline.CONCLUSION:Findings highlight the existence of a subgroup of individuals with shallower precART CD4 cell decline who experience poorer CD4 cell increases after cART; new studies in this group may provide information to optimize responses to therapy.

2011 - Decreased mitochondrial DNA content in subcutaneous fat from HIV-infected women taking antiretroviral therapy as measured at delivery. [Articolo su rivista]
Nasi, Milena; Pinti, Marcello; Chiesa, E; Fiore, S; Manzini, S; DEL GIOVANE, Cinzia; D'Amico, Roberto; Palai, N; Campatelli, C; Sabbatini, F; Roccio, M; Tibaldi, C; Masuelli, G; Mussini, Cristina; Ferrazzi, E; d'Arminio Monforte, A; Cossarizza, Andrea
abstract

BACKGROUND: Increasing numbers of pregnant HIV-positive women are receiving combination antiretroviral regimens for preventing mother-to-child virus transmission or for treating the infection itself. Several studies have demonstrated that nucleoside reverse transcriptase inhibitors (NRTIs) induce mitochondrial toxicity by several mechanisms, including depletion of mitochondrial DNA (mtDNA). By the quantification of mtDNA levels, we studied mitochondrial toxicity in HIV-positive women at delivery and the possible correlations with antiretroviral regimens, viroimmunological and metabolic parameters.METHODS: We analysed 68 HIV-positive women enrolled in the Italian Prospective Cohort Study on Efficacy and Toxicity of Antiretroviral in Pregnancy (TARGET Study); all were taking ≥1 NRTI. We quantified mtDNA copies per cell in subcutaneous fat samples collected during delivery. At the 3rd, 6th and 9th month of pregnancy, we collected data concerning CD4(+) T-cell count, plasma HIV RNA, total and high-density lipoprotein (HDL) cholesterol, fasting plasma glucose and triglycerides. As a control, we analysed mtDNA levels in abdominal subcutaneous fat samples from 23 HIV-seronegative women at delivery.RESULTS: mtDNA content was significantly lower in HIV-infected women when compared with HIV-negative controls. mtDNA content varied independently from viroimmunological, lipid and glucose parameters at the different months, with the exceptions of triglycerides at the 9th month and of HDL at the 6th month of pregnancy.CONCLUSIONS: In subcutaneous tissue from women taking NRTI-based antiretroviral regimens, we observed a significant decrease of mtDNA content, compared with uninfected women not on antiviral treatment. Moreover, a significant correlation was noted between mtDNA content and HDL cholesterol and triglycerides.

2011 - EXTRACELLULAR MITOCHONDRIAL DNA PLASMA LEVELS INCREASE DURING AGING AND LONGEVITY: IMPLICATIONS FOR "INFLAMMAGING" [Abstract in Rivista]
Cossarizza, Andrea; Cevenini, E.; Stazi, M. A.; Cotichini, R.; Monti, Daniela; Nasi, Milena; Gibellini, Lara; DE BIASI, Sara; Benatti, Stefania; Pinti, Marcello; Franceschi, Claudio
abstract

Mitochondrial (mt) DNA or degraded mitochondrial peptides are involved in the pathogenesis of the systemic inflammatory response syndrome (SIRS), a condition that often affects patients who survive a trauma and that resembles sepsis. Indeed, SIRS is due, at least partially, to molecules called "damage-associated molecular patterns" (DAMPs), a family of conserved molecules, conceptually similar to PAMPs, that includes hyaluronan fragments, heat shock proteins, S100 proteins, beta-amyloid, uric acid, IL-1 alpha, IL-33, and the DNA-binding nuclear protein HMGB1. DAMPs can trigger an innate immune response, causing inflammation through the engagement of several TLRs. Mitochondrial DAMPs (MTDs) can be released by damaged tissues, and are highly present in plasma of patients with SIRS. MTDs are represented by formyl peptides, that bind the formyl peptide receptor-1 (FPR-1), and mtDNA, that binds TLR-9, whose activation causes a potent inflammatory reaction.In the last years, we have shown that the production of proinflammatory cytokines is highly increased during the aging process, which is characterized by the accumulation of cellular and molecular defects, and involutive phenomena occurring concomitantly to a hyperstimulation of both innate and adaptive immunity (accumulation of expanded clones of memory and effector T cells, shrinkage of the TCR repertoire, progressive activation of macrophages). These phenomena result in a low-grade, chronic state of inflammation defined as “inflammaging”.To investigate the possible role of extracellular mtDNA during inflammaging, by an original real time PCR assay we have measured plasma levels of mtDNA in individuals who have been followed for 5 years: 429 siblings >90 years old (including 20 ultracentenarians) and 231 controls (50-75 y.o.). Moreover, we have studied 50 young donors (<30 years). A highly significant age-related increase of mtDNA plasma levels was observed. In donors >90 years, mtDNA plasma levels were significantly similar within families, suggesting the existence of a possible genetic component that controls this parameter. mtDNA was not correlated to classical inflammatory markers (CRP, VES), nor to main hematochemical conventional risk factors (among which ALT, albumin, cholesterol and glycemia). Finally, individuals who died within one year from the plasma collection had a lower level of mtDNA, while those who survived >5 years had much higher mtDNA plasma content.We can hypothesize that the age-related increase in plasma mtDNA could be either a marker of an optimal elimination of potentially dangerous cells, or the result of a complex remodeling of the entire organism, that makes use of potentially proinflammatory molecules like MTDs.

2011 - Functional characterization of the promoter of the human Lon protease gene. [Articolo su rivista]
Pinti, Marcello; Gibellini, Lara; DE BIASI, Sara; Nasi, Milena; Roat, Erika; O'Connor, Je; Cossarizza, Andrea
abstract

Lon, a nuclear-encoded mitochondrial enzyme, degrades oxidized proteins of the mitochondrial (mt) matrix, and participates in the replication of mtDNA. Lon is upregulated in the presence of substances such as stavudine (d4T), D-deoxyribose (dRib), that increase the intracellular reactive oxygen species (ROS) levels, or in the presence of H(2)O(2.) Here we show the promoter region -623/+1 is essential for response to ROS, and that in SW872, HepG2 and WI-38 cell lines the region -1230/-623 represses transcription, while the region -2023/-1230 increases promoter activity. D4T upregulates Lon promoter activity in all cell lines while dRib upregulates Lon mainly in HepG2 cells, and in shorter incubation times. These data confirm that Lon can be considered a stress responsive protein.

2011 - HIV-1 Infection and the Aging of the Immune System: Facts, Similarities and Perspectives [Articolo su rivista]
DE BIASI, Sara; Pinti, Marcello; Nasi, Milena; Gibellini, Lara; Bertoncelli, Linda; Manzini, Serena; Mussini, Cristina; Cossarizza, Andrea
abstract

During infection with the human immunodeficiency type-1 virus (HIV), the immune system has to cope with the exposure to an unexpected number of different and new antigens that are generated by continuous mutations of the virus. This phenomenon causes a profound derangement of the immune response, which is similar to that defined immunosenescence, a complex remodeling, whereby clonotypical immunity deteriorates, and ancestral and innate immunity is largely preserved. Either in HIV+ patients or in elderly individuals, the lifelong chronic antigenic stress, along with the involution of the thymus, causes the accumulation of memory/effector T cells and the exhaustion of naïve T cells. Furthermore, in both these conditions a chronic inflammatory status exists in the aging process, which has been defined as "inflammaging" and is characterized by an enhanced production of proinflammatory cytokines. In this review, we will underline the similarities that exist between immunological changes present during the physiological aging process and HIV infection. © 2011.

2011 - INCREASED LEVELS OF PLASMATIC MITOCHONDRIAL DNA DURING HIV INFECTION REVEAL A NEW ROLE FOR MITOCHONDRIAL DAMAGE-ASSOCIATED MOLECULAR PATTERNS [Abstract in Rivista]
Cossarizza, Andrea; Pinti, Marcello; Nasi, Milena; Gibellini, Lara; DE BIASI, Sara; Montagna, J. P.; Bertoncelli, Linda; Bisi, Luca; Manzini, Lisa; Benatti, Stefania; Borghi, V.; Mussini, Cristina
abstract

Recently, it has been shown that mtDNA or degraded mitochondrial peptides can act as “damage-associated molecular patterns" (DAMPs), that are conceptually and functionally similar to PAMPs. Mitochondrial DAMPs, defined MTDs, can be involved in the pathogenesis of the systemic inflammatory response syndrome (SIRS), a condition that often affects patients who survive a trauma, characterized by the presence of shock and compromised function of several organs. During SIRS, mtDNA released from damaged or dead cells can bind TLR-9, whose activation causes a potent inflammatory reaction, with the production of proinflammatory cytokines. Since HIV infection is characterized by a proinflammatory status, and by a hyperproduction of proinflammatory cytokines, we asked whether soluble mtDNA circulating in the plasma could play a role in determining such condition.Thus, we have studied plasma levels of mtDNA in HIV+ patients showing a different course of the infection, and have correlated such levels to the activation of the immune system and to the plasma viremia. We analyzed individuals during acute, primary HIV infection (AHI), patients with an advanced infection (including those with full blown acquired immunodeficiency syndrome, AIDS) but still naive for antiretroviral therapy (ART), and those defined “long term non progressors” (LTNPs), who had been infected since at least 8 years, always out of treatment, but with a normal number of CD4+ T cells, a low grade of apoptosis and a good immunological control of the virus.In all HIV+ patients but LTNP plasma levels of mtDNA were significantly higher than in healthy controls. Furthermore, in naive patients, 6 months of efficient ART (able to increase CD4+ T cell count, decrease viral load and reduce T cell activation) did not modify mtDNA plasma levels. These levels were not correlated to CD4+ T cell count, nor to markers of immune activation, but had a significant correlation with plasma viral load, revealing a possible role for mtDNA not only as a molecule able to trigger inflammation, but also as a novel biomarker of virus-induced damage.The identification of the role of MTDs could relevant not only to identify possible new biomarkers of disease progression, but also in designing new therapeutic strategies that regard soluble mtDNA, as novel treatments could target either soluble MTDs, or the receptors they use. Thus, in HIV infection, as in other diseases characterized by excessive inflammation, interfering with MTDs could likely become a novel strategy to reduce the harmful immune activation.

2011 - Increased plasma levels of extracellular mitochondrial DNA during HIV infection: a new role for mitochondrial damage-associated molecular patterns during inflammation. [Articolo su rivista]
Cossarizza, Andrea; Pinti, Marcello; Nasi, Milena; Gibellini, Lara; Manzini, S; Roat, Erika; DE BIASI, Sara; Bertoncelli, Linda; Montagna, Jp; Bisi, Luca; Manzini, Lisa; Trenti, T; Borghi, V; Mussini, Cristina
abstract

HIV infection is characterized by a chronic inflammatory state. Recently, it has been shown that mitochondrial DNA (mtDNA) released from damaged or dead cells can bind Toll like receptor-9 (TLR9), an intracellular receptor that responds to bacterial or viral DNA molecules. The activation of TLR9 present within monocytes or neutrophils results in a potent inflammatory reaction, with the production of proinflammatory cytokines. We measured plasma levels of mtDNA in different groups of HIV(+) patients, i.e., those experiencing an acute HIV infection (AHI), long term non progressors (LTNP), late presenters (LP) taking antiretroviral therapy for the first time, and healthy controls. We found that in AHI and LP mtDNA plasma levels were significantly higher than in healthy individuals or in LTNP. Plasma mtDNA levels were not correlated to peripheral blood CD4(+) T cell count, nor to markers of immune activation, but had a significant correlation with plasma viral load, revealing a possible role for mtDNA in inflammation, or as a biomarker of virus-induced damage.

2011 - NK-cell phenotype at interruption underlies widely divergent duration of CD4+-guided antiretroviral treatment interruption. [Articolo su rivista]
Bozzano, F; Nasi, Milena; Bertoncelli, Linda; Nemes, Elisa; Prati, F; Marras, F; Mussini, Cristina; Moretta, L; Cossarizza, Andrea; De Maria, A.
abstract

Long-term side effects may represent a relevant burden of antiretroviral treatment (ART) in HIV-infected patients with good CD4 immune reconstitution over extended time spans. CD4-guided treatment interruption (TI) has been evaluated to address this point and may result in a wide spectrum of time off ART in different patient cohorts. We studied whether differences in innate immune responses, in particular NK cells, are associated to patterns of longer (LoTI) or a shorter (ShTI) TI. Clinical cohort parameters were analyzed on a group of patients widely diverging for TI duration (<9 versus >18 months) on samples before TI, including NK-cell analysis and function by natural cytotoxicity receptor (NCR)-triggered γ-IFN production. Although persistently reduced NCR expression (NKp30) and function were observed in both LoTI and ShTI patients on ART compared with healthy donors, relevant differences were observed at baseline TI in those patients who subsequently developed LoTI course. Lower expression of NKG2D and NKp46 on NK cells. This also translates in reduced γ-IFN production in redirected functional assays. Thus, differences in innate immune balance exist during ART, may be associated to differential control of HIV infection and their understanding could explain clinical differences in individual patients that are not reflected by CD4(+) cell counts alone.

2011 - Quality assessment of human mitochondrial DNA quantification: MITONAUTS, an international multicentre survey. [Articolo su rivista]
Côté, Hc; Gerschenson, M; Walker, Ua; Miro, O; Garrabou, G; Hammond, E; Villarroya, J; Giralt, M; Villarroya, F; Cinque, P; Garcia Arumi, E; Andreu, Al; Pinti, Marcello; Cossarizza, Andrea
abstract

Mitochondrial DNA quantification by qPCR is used in the context of many diseases and toxicity studies but comparison of results between laboratories is challenging. Through two multigroup distributions of DNA samples from human cell lines, the MITONAUTS group anonymously compared mtDNA/nDNA quantification across nine laboratories involved in HIV research worldwide. Eight of the nine sites showed significant correlation between them (mean raw data R(2)=0.664; log(10)-transformed data R(2)=0.844). Although mtDNA/nDNA values were well correlated between sites, the inter-site variability on the absolute measurements remained high with a mean (range) coefficient of variation of 71 (37-212) %. Some variability appeared cell line-specific, probably due to chromosomal alterations or pseudogenes affecting the quantification of certain genes, while within cell line variability was likely due to differences in calibration of the standard curves. The use of two mtDNA and two single copy nDNA genes with highly specific primers to quantify each genome would help address copy number variants. Our results indicate that sample shipment must be done frozen and that absolute mtDNA/nDNA ratio values cannot readily be compared between laboratories, especially if assessing cultured cell mtDNA content. However, within laboratory and relative mtDNA/nDNA comparisons between laboratories should be reliable.

2011 - Quercetin and cancer chemoprevention [Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Nasi, Milena; J. P., Montagna; DE BIASI, Sara; Roat, Erika; Bertoncelli, Linda; E. L., Cooper; Cossarizza, Andrea
abstract

Several molecules present in the diet, including flavonoids, can inhibit the growth of cancer cells with an ability to act as "chemopreventers". Their cancer-preventive effects have been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis as well as the antioxidant functions. The antioxidant activity of chemopreventers has recently received a great interest, essentially because oxidative stress participates in the initiation and progression of different pathological conditions, including cancer. Since antioxidants are capable of preventing oxidative damage, the wide use of natural food-derived antioxidants is receiving greater attention as potential anti-carcinogens. Among flavonoids, quercetin (Qu) is considered an excellent free-radical scavenging antioxidant, even if such an activity strongly depends on the intracellular availability of reduced glutathione. Apart from antioxidant activity, Qu also exerts a direct, pro-apoptotic effect in tumor cells, and can indeed block the growth of several human cancer cell lines at different phases of the cell cycle. Both these effects have been documented in a wide variety of cellular models as well as in animal models. The high toxicity exerted by Qu on cancer cells perfectly matches with the almost total absence of any damages for normal, non-transformed cells. In this review we discuss the molecular mechanisms that are based on the biological effects of Qu, and their relevance for human health.

2010 - Apoptosis in HIV/AIDS [Capitolo/Saggio]
Cossarizza, A.
abstract

The immune system can use several strategies to fight viral infections. Among these, the death of infected cells has ultimately the role of avoiding the spread of infection, even if in most cases either the cells containing the virus or ‘innocent’ cells are killed. Thus, in diff erent situations, as defence mechanisms, every living organism can trigger apoptosis, autophagy and eventually necrosis. In turn, viruses are able to evade the immune response by interfering with several mechanisms and components. The human immunodeficiency virus (HIV), that causes the acquired immunodeficiency syndrome (AIDS), infects a huge number of cells expressing the CD4 molecule, including monocytes and T helper lymphocytes, that can trigger apoptosis. HIV encodes proteins with pro-apoptotic activity, such as gp120, gp160, Tat, Nef, Vpr, Vpu, Vif and the viral protease. These proteins can provoke the death of infected and uninfected lymphocytes through several mechanisms, including the action of host-encoded molecules belonging to the tumor necrosis factor (TNF) family, or via the mitochondrial apoptotic pathway. The acute and chronic phases of HIV infection are characterized by a proinflammatory status that facilitates cell death, and is a further cause of immune damage. Nowadays the therapy for HIV/AIDS includes potent antiretroviral drugs, that are able to decrease apoptosis by diff erent mechanisms. Indeed, not only drugs diminish the production of the virus by infected cells, and thus reduce the inflammation and the production of apoptogenic molecules, but also have a direct action on the viral protease. Finally, the host’s genetic background plays a main role in the entire process of cell death in HIV infection.

2010 - Cytotoxic granule release dominates gag-specific CD4+ T-cell response in different phases of HIV infection. [Articolo su rivista]
Nemes, Elisa; Bertoncelli, Linda; Lugli, Enrico; Pinti, Marcello; Nasi, Milena; Manzini, Lisa; Manzini, Simona; Prati, Francesca; Borghi, V; Cossarizza, Andrea; Mussini, Cristina
abstract

BACKGROUND: The activity of virus-specific T lymphocytes, among which those capable of a polyfunctional response against the viral protein gag, is crucial to control HIV infection. OBJECTIVE: The objective of this study is to investigate the polyfunctionality of gag-specific T cells in different phases of HIV infection, analyzing markers related to T-helper cell 1 (Th1) and degranulation/cytotoxicity, and the production of Th1 cytokines in peripheral blood lymphocytes from patients experiencing an acute primary infection, long-term nonprogressors, patients naive for antiretroviral drugs, and patients taking HAART. MATERIALS AND METHODS: Cells were stimulated with a pool of gag-derived peptides or with a superantigen (staphylococcal enterotoxin B). Using eight-color polychromatic flow cytometry, we analyzed the expression of interleukin-2, interferon-gamma, CD154, and CD107a by CD4 and CD8 T cells. RESULTS: The main finding was that in all HIV-positive patients, about half gag-specific CD4 T cells were CD107a, that is, able to degranulate. CD4CD154 cells unable to produce Th1 cytokines were the second most represented population. Truly polyfunctional CD4 T cells were very rare and present only in a few long-term nonprogressors. Superantigen stimulation showed that CD4 T lymphocytes from all patients displayed a typical Th response, including interleukin-2 and interferon-gamma production, lacking CD107a expression. CONCLUSION: In all the aforementioned phases of HIV infection, the large majority of gag-specific CD4 T lymphocytes cannot be identified by the sole expression of interleukin-2 and interferon-gamma, which is early impaired. Degranulation and helper functions other than Th1 cytokine production are the predominant features of HIV-specific CD4 lymphocytes.

2010 - Data analysis in flow cytometry: the future just started. [Articolo su rivista]
E., Lugli; M., Roederer; Cossarizza, Andrea
abstract

In the last 10 years, a tremendous progress characterized flow cytometry in its different aspects. In particular, major advances have been conducted regarding the hardware/instrumentation and reagent development, thus allowing fine cell analysis up to 20 parameters. As a result, this technology generates very complex datasets that demand for the development of optimal tools of analysis. Recently, many independent research groups approached the problem by using both supervised and unsupervised methods. In this article, we will review the new developments concerning the use of bioinformatics for polychromatic flow cytometry and propose what should be done to unravel the enormous heterogeneity of the cells we interrogate each day.

2010 - Effect of antioxidants on mitochondrial function in HIV-1-related lipoatrophy: a pilot study. [Articolo su rivista]
Milazzo, L; Menzaghi, B; Caramma, I; Nasi, Milena; Sangaletti, O; Cesari, M; Poma, Bz; Cossarizza, Andrea; Antinori, S; Galli, M.
abstract

We investigated the effect of antioxidant supplementation on mitochondrial function, fat distribution, and lipid and glucose metabolism in HIV-1-infected patients with antiretroviral therapy (ART)-related lipoatrophy. 61 ART-treated HIV-1-infected patients with lipoatrophy were randomized to receive either n-acetyl-L-carnitine (n = 21), lipoic acid + n-acetylcisteine (LA/NAC) (n = 20), or no supplementation (n = 20) for 48 weeks. At baseline and at the end of treatment, mitochondrial function was studied by (13)C-methionine breath test and by mitochondrial (mt)-DNA quantification on circulating T-cells and subcutaneous adipose tissue. Body composition was assessed by dual-energy X-ray absorpiometry (DEXA). (13)CO(2)-exhalation increased between baseline and week 48 in both supplementation arms as evidenced by a higher delta over baseline excretion at 45 min (from mean ± SEM of 7.8 ± 1.08 to 9.9 ± 0.6, p = 0.04 in the n-acetyl-carnitine arm, and from 7.4 ± 0.8 to 11.5 ± 1.6, p = 0.01 in LA/NAC arm). Cumulative (13)CO2 excretion increased from median (interquartile range; IQR) of 3.25 (2.55-4.2) to 4.51 (4.12-5.2) in the carnitine arm; from 3.79 (2.67-4.37) to 4.83 (4.25-5.56) in the LA/NAC arm; p = 0.004, 0.02, respectively. mtDNA content increased in CD4+ T-cells from patients who received n-acetyl-carnitine (+30 copies/cell; p = 0.03), without significant difference by the overall comparison of the study groups. Fat body mass and lipid profile did not change significantly in any of the arms. Our study showed that antioxidant supplementation may have a protective role on mitochondrial function, with limited effects on the reversal of clinical lipodystrophic abnormalities in HIV-1-infected patients.

2010 - HIV infection in the era of cytomics [Articolo su rivista]
Cossarizza, A.
abstract

2010 - Interfering with ROS metabolism in cancer cells: the potential role of quercetin. [Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Nasi, Milena; DE BIASI, Sara; Roat, Erika; Bertoncelli, Linda; Cossarizza, Andrea
abstract

Abstract: A main feature of cancer cells, when compared to normal ones, is a persistent pro-oxidative state that leads to an intrinsic oxidative stress. Cancer cells have higher levels of reactive oxygen species (ROS) than normal cells, and ROS are, in turn, responsible for the maintenance of the cancer phenotype. Persistent ROS stress may induce adaptive stress responses, enabling cancer cells to survive with high levels of ROS and maintain cellular viability. However, excessive ROS levels render cancer cells highly susceptible to quercetin, one of the main dietary flavonoids. Quercetin depletes intracellular glutathione and increases intracellular ROS to a level that can cause cell death.

2010 - Membrane lipid defects are responsible for the generation of reactive oxygen species in peripheral blood mononuclear cells from vitiligo patients [Articolo su rivista]
Dell'Anna, M. L.; Ottaviani, M.; Bellei, B.; Albanesi, V.; Cossarizza, A.; Rossi, L.; Picardo, M.
abstract

The pathogenesis of vitiligo, an acquired depigmenting disease of the skin, involves oxidative stress. Based on that, the generation of reactive oxygen species (ROS) by the mitochondria may be relevant in the pathogenesis of vitiligo. Here, we evaluate the biochemical and functional alterations involved in the defective activity that has been previously described both in melanocytes and peripheral blood mononuclear cells (PBMC) from vitiligo patients. Moreover, we used a freeze-thaw test as a mild stress stimulus to disclose any latent defects in the assembly of membrane lipids that may compromise the functionality of the membrane itself. We show that the lipid constitution of the membrane is altered in vitiligo. Specifically, the cardiolipin (CL) level in the mitochondrial inner membrane is reduced and the level of cholesterol is increased. Furthermore, an increase in the expression level of 3-hydroxy-3methyl-glutaryl-CoenzymeA-reductase (HMG-CoA reductase), the rate-limiting enzyme for cholesterol biosynthesis, was also seen. Associated with that, the expression of electron transport chain (ETC) lipid-dependent subunits was also modified, and their expression was further affected by the freeze-thaw stress. The expression of CL-independent mitochondrial proteins, such as porin and Bcl2, were unaffected in vitiligo PBMC. These data confirm that ETC protein expression mainly correlates with lipid arrangement and that loss of their expression is not due to generalized or random oxidative-mediated damage.Wesuggest that the modification of membrane lipid components in vitiligo cells may be the biochemical basis for the mitochondrial impairment and the subsequent production of intracellular ROS following the exposure to a mild stress. © 2010 Wiley-Liss, Inc.

2010 - Mitochondrial changes during D-drug-containing once-daily therapy in HIV-positive treatment-naive patients. [Articolo su rivista]
Maggiolo, F.; Roat, Erika; Pinti, Marcello; Nasi, Milena; Gibellini, Lara; DE BIASI, Sara; Airoldi, M.; Ravasio, V.; Mussini, Cristina; Suter, F.; Cossarizza, Andrea
abstract

BACKGROUND: Antiviral drugs of the category of nucleoside reverse transcriptase inhibitors (NRTIs), largely used for the treatment of HIV infection, can have toxic effects on mitochondria. We performed a cross-sectional study on mitochondrial toxicity in a randomized group of patients belonging to a larger randomized study on different NRTI-based once-daily regimens by quantifying mitochondrial DNA (mtDNA), three different mitochondrial RNAs (mtRNAs) and functional parameters in highly purified peripheral CD4+ and CD8+ T-cells.METHODS: A total of 49 previously treatment-naive patients treated for a mean of 15 months with efavirenz plus didanosine plus lamivudine (group 1), or tenofovir disoproxil fumarate plus lamivudine (group 2), or didanosine plus abacavir (group 3) were considered. The groups were matched for sex, age, CDC classification, risk factor for HIV, nadir CD4+ T-cell count and baseline viral load. mtDNA and mtRNA were quantified by using real-time PCR assays.RESULTS: No patient showed any clinical symptom; however, the amount of mtDNA in CD4+ and CD8+ T-cells was significantly lower in groups 1 and 3; similarly, the expression of different mtRNAs in both CD4+ and CD8+ T-cells showed significant differences that were dependent upon the drug used. No differences were found in mitochondrial membrane potential and mitochondrial mass in peripheral lymphocytes. The amount of total HIV DNA in CD4+ T-cells did not differ among the groups, who displayed a similar immune reconstitution and control of the virus.CONCLUSIONS: An efficient didanosine-containing once-daily therapy can have more mitochondrial toxicity than regimens devoid of this drug.

2010 - Predictive Value of Intracellular HIV-1 DNA Levels During CD4-Guided Treatment Interruption in HIV(+) Patients [Articolo su rivista]
Nasi, Milena; Pinti, Marcello; Manzini, S; Gibellini, Lara; Manzini, Lisa; Bisi, Luca; DE BIASI, Sara; DEL GIOVANE, Cinzia; D'Amico, Roberto; Borghi, V; Mussini, Cristina; Cossarizza, Andrea
abstract

The amount of HIV-1 DNA within peripheral blood mononuclear cells is an important marker of viral activity. We studied intracellular HIV-1 DNA content in purified CD4(+) T cells from 28 chronically HIV-1-infected adults with sustained CD4(+) T cell counts (>500 cells/microl) and undetectable plasma viral load (<50 copies/ml), who underwent CD4-guided treatment interruption (TI). Patients were followed up for 18 months during TI, and for 6 months after treatment resumption (TR). Six naïve HIV(+) patients starting therapy were also enrolled and followed up for 6 months. All patients were studied every 2 months; HIV-1 DNA copy number was quantified with real-time PCR. Considering all patients remaining off-treatment, in the first 18 months of TI, intracellular HIV-1 DNA levels (expressed as Log(10) copies/million cells) remained stable (mean, 3.82 and 3.77 at time 0 and after 18 months, respectively). Similarly, HIV-1 DNA values, either in patients who restarted treatment after TI (time 0, 4.90) or in naïve patients who started treatment for the first time (time 0, 4.37), did not change significantly in the first 6 months of therapy (4.42 and 3.67, respectively). Evaluating HIV-1 DNA variations during the first 2 months of TI, we found that patients with a stable level had a lower risk to reach a CD4(+) T cell count <350 cells/microl, and thus to restart therapy, whereas this risk was significantly higher in those with a marked increase of HIV-1 DNA. In conclusion, intracellular HIV-1 DNA is a predictive marker for the length of CD4-guided TI.

2010 - T cell homeostasis in centenarians: from the thymus to the periphery. [Articolo su rivista]
Pinti, Marcello; Nasi, Milena; Lugli, Enrico; Gibellini, Lara; Bertoncelli, Linda; Roat, Erika; DE BIASI, Sara; Mussini, Cristina; Cossarizza, Andrea
abstract

The immune system undergoes a process of profound remodelling during aging, referred to as immunosenescence, and characterized by complex modifications of several components. In this review, we discuss recent developments and observations regarding the generation of T cells in the thymus during aging and longevity, and the regulation and maintenance of peripheral blood lymphocytes. The generation of new T cells is indeed crucial to maintain a functional immune system, and is a fundamental step to avoid unsuccessful aging, thus reaching longevity in good health. Mechanisms will be described that are related to the production and maintenance of those lymphocytes defined "recent thymic emigrants", and to the detection of the so called "T cell receptor rearrangement excision circles (TREC)", along with the presence in the periphery of naïve and memory T cells, that can be influenced and regulated by several different mechanisms. Several strategies aimed at improving thymic functionality are currently receiving a growing interest, and some of them are based on molecules that are produced by, and/or act on immune cells. Data on the possible use of these molecules, including cytokines like interleukin (IL)-7, IL-15 and keratinocyte growth factor, to restore thymic function are reviewed and discussed.

2010 - The Role of Mitochondria in HIV Infection and Its Treatment [Articolo su rivista]
Pinti, M.; Nasi, M.; Gibellini, L.; Roat, E.; De Biasi, S.; Bertoncelli, L.; Cossarizza, A.
abstract

Mitochondria play a dual role in the life of the cell, being capable of producing either energy (in the form of ATP) or potentially dangerous reactive oxygen species (ROS), and they also contain molecules that, when released into the cytoplasm, cause apoptosis. There is a growing interest in the importance of these organelles during the infection caused by the human immunodeficiency virus (HIV), as well as during its treatment. Indeed, several drugs that are capable of blocking HIV can also interact with the enzyme responsible for the replication of mitochondrial DNA and inhibit its activity. Cytokines produced by the immune system can alter ROS production. Furthermore, the virus as such can trigger different mechanisms that interfere with mitochondrial functionality and induce alterations, ultimately causing cell death. As a result, mitochondria can be severely altered by HIV infection and by its treatment. © 2010 Taiwan Medical University.

2010 - Upregulation of nuclear-encoded mitochondrial LON protease in HAART-treated HIV-positive patients with lipodystrophy: implications for the pathogenesis of the disease [Articolo su rivista]
Pinti, Marcello; Gibellini, Lara; Guaraldi, Giovanni; Orlando, Gabriella; Gant, Tw; Morselli, Eugenia; Nasi, Milena; Salomoni, Paolo; Mussini, Cristina; Cossarizza, Andrea
abstract

BACKGROUND: HAART can provoke metabolic changes and body fat redistribution, resulting in lipodystrophy, a side effect significantly involving mitochondrial function. Mitochondrial DNA (mtDNA) depletion caused by nucleosidic reverse transcription inhibitors is supposed to be a crucial mechanism in the pathogenesis of mitochondrial damages. METHODS: In adipose tissue from 22 HIV-positive patients with lipodystrophy and 20 healthy controls, we analyzed gene expression by microarray analysis and real-time PCR. The most upregulated gene was further studied in the human adipocytic cell line SW872 by real-time PCR, western blot, transient transfection assays and flow cytometry. RESULTS: We identified 18 genes differently expressed between lipodystrophy patients and controls, and focused our attention on the nuclear-encoded mitochondrial protease LON, essential in mtDNA maintenance. In SW872 cells, treatment with stavudine (d4T) doubled LON levels, in parallel with mtDNA depletion. As d4T increased reactive oxygen species (ROS) intracellular content, we measured LON in presence of deoxyribose, which causes oxidative stress but not mtDNA depletion, and observed LON upregulation. Ethidium bromide, which markedly depletes mtDNA, did not alter LON levels. The antioxidant glutathione inhibited the increase of intracellular ROS and the increase in LON caused by d4T or deoxyribose. CONCLUSION: LON upregulation was due to d4T-induced ROS production, rather than due to mtDNA depletion, and represents a response to an oxidative stress. Other mechanisms than mtDNA depletion thus exist that explain nucleosidic reverse transcription inhibitors toxicity. This observation provides a rationale for possible therapeutic interventions aimed at reducing intracellular ROS content in patients assuming HAART.

2010 - Γ ν over(ω, ̃) θ ι Σ ε α υ τóν (know thyself) and recognize dangers: An evolutionistic view [Articolo su rivista]
Cossarizza, A.
abstract

2009 - Analisi di rilevanza del riscontro intralesionale degli Herpes simplex virus nei risultati della terapia parodontale rigenerativa [Articolo su rivista]
Bertoldi, Carlo; Pellacani, C.; Pinti, Marcello; Lalla, Michele; Lucchi, A.; Zaffe, Davide; Consolo, Ugo; Cossarizza, Andrea
abstract

Lo scopo del presente studio è quello di indagare la presenza degli herpes simplex virus 1 & 2 (HSVs) nelle lesioni angolari parodontali ed analizzarne l’influenza nella finalizzazione in terapia chirurgica rigenerativa parodontale. Materiali e Metodi: Sono stati complessivamente valutati 7 pazienti di cui 5 di sesso femminile e 2 di sesso maschile, di età compresa tra i 22 e i 60 anni sistemicamente sani. Dopo decontaminazione causale e di eventuali altri siti patologici, venivano eseguite indagini microbiologiche in ogni paziente con tamponi per escludere uno specifico ruolo batterico o micologico nell’ambito dei casi trattati. Immediatamente prima dell’intervento, un test biomolecolare (Meridol® Perio Diagnostics), basato sulla tecnologia realt-time polymerase chain reaction (RT-PCR), è stato utilizzato in rapporto al difetto angolare su cui intervenire ed ai solchi circostanti per escludere la presenza di batteri sicuramente parodontopatogeni (la cui presenza avrebbe portato all’esclusione del paziente dallo studio; ad egli, comunque, sarebbe stata garantita l’assistenza necessaria al caso). Il sito veniva trattato mediante minimally invasive surgical technique (MIST) e con enamel matrix protein (EMP – Emdogain , Biora AB, Malmö, Sweden). Il connettivo infiammatorio patologico presente nel difetto angolare veniva asportato, veniva inserito in provetta sterile e refrigerato immediatamente a -20 C°. Tale prelievo, in seguito, veniva preparato per la fase biomolecolare di rilevazione virale mediante sonde per acidi nucleici e real-time polymerase chain reaction (RT-PCR). Durante tutto il periodo di follow-up quindi il paziente veniva mantenuto in terapia di mantenimento e tutte le diverse misurazioni (FMPS, FMBS, PPD, REC, CAL) erano compiute in singolo-cieco da uno stesso operatore. In particolare le misurazioni erano effettuate prima dell’intervento (fase T0), a 6 mesi dall’intervento (T1) ed a una anno (T2); il follow-up era quindi a 12 mesi. Pur essendo registrati i diversi indici parodontali necessari alla terapia, la PPD rappresentava la variabile finale principale misurabile. L’analisi biomolecolare era condotta mediante estrazione del DNA virale e tecnica RT-PCR, come precedentemente descritto (7). Risultati I valori di PPD raccolti a T1 indicavano un miglioramento (non statisticamente significativo) degli stessi difetti per 11 delle tasche parodontali prese in esame, con una riduzione della profondità media di 1,5 mm, mentre in 3 casi non si registrava alcun sensibile miglioramento. I dati raccolti a T2 (fine del periodo di follow-up) mostravano una riduzione statisticamente significativa della PPD di tutti i difetti parodontali analizzati in rapporto a T0 ed, in particolare, il miglioramento medio era di 5,5 mm in termini di PPD. In 3 difetti angolari, tuttavia, tale miglioramento era stato piuttosto scarso e la profondità di tali difetti, anche in seguito al trattamento chirurgico, risultava ancora non fisiologica, ovvero persisteva una tasca. Le indagini biomolecolari hanno evidenziato che 6 dei siti tra i difetti parodontali valutati, erano positivi all’infezione da HSVs (si può considerare l’infezione da HSVs significativa con valori ≥ di 10 copie di DNA appartenente a tale agente su 1000 cellule analizzate). Il confronto tra la presenza e la concentrazione di HSVs con la profondità del difetto parodontale misurato in T2 ha indicato che i casi con un più alta concentrazione di copie virali coincidevano con i difetti parodontali che non avevano presentato un miglioramento significativo dopo il trattamento chirurgico dello stesso. In fase T0 non si erano riscontrate differenze statisticamente significative in termini di PPD tra i siti infettati da HSVs ed i restanti. Si può osservare anche una associazione lineare positiva e significativa tra PPD in T1 e PPD in T0. Mediante l’analisi dei coefficienti di correlazione anche in scala logaritmica si è p

2009 - HIV-infected long-term nonprogressors display a unique correlative pattern between the interleukin-7/interleukin-7 receptor circuit and T-cell homeostasis [Articolo su rivista]
Marchetti, G; Riva, A; Cesari, M; Bellistrì, Gm; Gianelli, E; Casabianca, A; Orlandi, C; Magnani, M; Meroni, L; d'Arminio Monforte, A; Mussini, Cristina; Cossarizza, Andrea; Galli, M; Gori, A.
abstract

BACKGROUND:We hypothesized that there may be a correlation between the interleukin-7 (IL-7)/IL-7 receptor (IL-7R) regulatory system and parameters of T-cell homeostasis in HIV-infected long-term nonprogressors (LTNPs) as compared with patients with disease progression.METHODS:The possibility of a correlation between T-cell homeostatic parameters and IL-7/IL-7R was investigated in 22 LTNPs (CD4 count > or =500 cells/microL for >10 years) vs. HIV-positive patients at different disease stages [12 early: CD4 count > or =400 cells/microL ; 15 late (AIDS-presenters): CD4 count < or =150 cells/microL ].RESULTS:Compared with early-stage HIV-positive patients, LTNPs displayed a higher circulating IL-7 concentration (P=0.05), which was positively associated with higher IL-7Ralpha expression and a higher T-cell receptor excision circle (TREC) content specifically within CD4 cells (P<0.05). Compared with late-stage disease patients, early-stage disease patients displayed a lower IL-7 concentration (P<0.01) and higher percentages of IL-7Ralpha+ CD4 and CD8 cells (P=0.05). IL-7 was positively correlated with the percentage of TREC+ CD4 cells (P<0.01), which translated into a higher percentage of naïve CD4 cells in early-stage disease patients than in late-stage disease patients; however, the CD4 cells in early-stage disease patients were less enriched in recent thymic emigrants (RTEs) compared with LTNPs (P<0.05). In late-stage AIDS-developing patients, substantially increased IL-7 was correlated with a decreased percentage of IL-7Ralpha+ CD4 cells (P=0.01), which resulted in these patients having a significantly lower percentage of naïve T cells (P<0.01) and a significantly lower content of TREC (P<0.01) than the other patients.CONCLUSIONS:The maintenance of high CD4 cell counts in LTNPs was associated with a specific IL-7/IL-7R pattern characterized by increased IL-7 and highest IL-7Ralpha-expressing CD4 cells relative to other patients. Compared with patients with late-stage disease, LTNPs displayed a phenotypically naïve, less activated CD4 cell pool highly enriched in RTEs, suggesting the existence of a compensatory IL-7-mediated pathway specifically sustaining peripheral CD4 counts.

2009 - HOXD13 binds DNA replication origins to promote origin licensing and is inhibited by geminin. [Articolo su rivista]
Salsi, Valentina; Ferrari, Silvia; Ferraresi, Roberta; Cossarizza, Andrea; Grande, Alexis; Zappavigna, Vincenzo
abstract

HOX DNA-binding proteins control patterning during development by regulating processes such as cell aggregation and proliferation. Recently, a possible involvement of HOX proteins in replication origin activity was suggested by results showing that a number of HOX proteins interact with the DNA replication licensing regulator geminin and bind a characterized human origin of replication. The functional significance of these observations, however, remained unclear. We show that HOXD13, HOXD11, and HOXA13 bind in vivo all characterized human replication origins tested. We furthermore show that HOXD13 interacts with the CDC6 loading factor, promotes pre-replication complex (pre-RC) proteins assembly at origins, and stimulates DNA synthesis in an in vivo replication assay. HOXD13 expression in cultured cells accelerates DNA synthesis initiation in correlation with the earlier pre-RC recruitment onto origins during G(1) phase. Geminin, which interacts with HOXD13 as well, blocks HOXD13-mediated assembly of pre-RC proteins and inhibits HOXD13-induced DNA replication. Our results uncover a function for Hox proteins in the regulation of replication origin activity and reveal an unforeseen role for the inhibition of HOX protein activity by geminin in the context of replication origin licensing.

2009 - Investigating T cells by polychromatic flow cytometry [Capitolo/Saggio]
Lugli, E; Troiano, L; Cossarizza, Andrea
abstract

Since its development, flow cytometry gave a relevant contribution to the field of Immunology. Its unique potential to analyse multiple parameters at the single cell level allowed the identification of unknown cell subsets with specific roles in immunoregulation as well as in the pathogenesis of several diseases. More recently, with the advent of new equipments and fluorochromes, the possibility exists to analyse simultaneously a large number (up to 19) of parameters in a single cell. This strategy, defined polychromatic flow cytometry (PFC), has been widely utilised in the last years for the fine analysis of immune cell phenotypes, including antigen-specific T lymphocytes, B cell subsets, and the intracellular phosphoproteome, among others. A huge amount of data can be generated by such an approach, and their interpretation could become a very complex and time-consuming task. Protocols for performing PFC will be discussed in this chapter, together with some guidelines for data interpretation and analysis.

2009 - Lymphocytes sub-types and functions in centenarians as models for successful ageing [Capitolo/Saggio]
Lugli, E.; Troiano, L.; Pinti, M.; Nasi, M.; Roat, E.; Ferraresi, R.; Bertoncelli, L.; Gibellini, L.; Nemes, E.; Cossarizza, A.
abstract

Several cell subsets participate to the immune response, and their close interplay is fundamental for the successful elimination of harmful pathogens. In addition, a tight regulation of the immune response has to occur in order to avoid excessive inflammation and potential autoreactivity towards self components. In the last years, the discovery and the characterization of new lymphocytes subsets, including regulatory T (Treg)-cells and Natural Killer T (NKT)-cells allowed a better understanding of how an effector immune response is induced and therefore down-modulated. During the ageing of the immune system, a process termed immunosenescence, these subsets undergo a profound remodelling, both in phenotype and function. In this chapter, we will describe the essential features of lymphocyte populations in centenarians and the differences that occur with unsuccessfully aged people.

2009 - Magnitude and Determinants of CD4 Recovery After HAART Resumption After 1 Cycle of Treatment Interruption [Articolo su rivista]
Mussini, Cristina; Touloumi, G; Bakoyannis, G; Sabin, C; Castagna, A; Sighinolfi, L; Erikson, Le; Bratt, G; Borghi, V; Lazzarin, A; Cossarizza, Andrea; Esposito, R.
abstract

OBJECTIVE:The extent of immune reconstitution following HAART resumption after 1 cycle of treatment interruption (TI) is not well known.METHODS:Multicenter retrospective analysis of patients who discontinued HAART with a CD4 &gt; 500 cells/microL. Cox proportional hazards models were used to identify prognostic factors for immunologic response after treatment resumption. CD4 trends were investigated using linear mixed models.RESULTS:One hundred and eighty-three individuals were included. Median CD4 at TI and at treatment restart were 748 and 459 cells/microL, respectively. Median time from TI to treatment restart was 5.52 months. Ninety percent of the patients reached an undetectable viral load. One hundred and twenty-five subjects experienced immunologic response; 66 patients reached their pre-TI CD4 levels. At 3, 6, 12, and 24 months after treatment restart, the median CD4 increase was 149, 153, 161, and 178 cells/microL, respectively. Subjects with less steep CD4 declines during TI tended to have a lower initial CD4 increase, as did those reinitiating HAART with viral loads &lt; 5000 copies/mL, whereas subjects who had experienced a virologic response to their initial HAART regimen had slower CD4 increases.CONCLUSIONS:Patients willing to discontinue treatment should be advised that immune reconstitution to pre-TI values is possible in fewer than 50% of patients at 2 years after treatment restart.

2009 - Morphologic, histochemical, and functional analysis of platelet-rich plasma activity on skeletal cultured cells [Articolo su rivista]
Bertoldi, Carlo; Pinti, Marcello; Zaffe, Davide; Cossarizza, Andrea; Consolo, Ugo; G. B., Ceccherelli
abstract

BACKGROUND:Platelet-rich plasma (PRP) is a medium containing concentrated amounts of growth factors in a form that is easy to handle in regenerative sites. The aim of this study was to assess the effect of PRP on the differentiation of cultured skeletal cells and the capability of PRP to induce the production of some osteogenesis-related molecules and mineralization.STUDY DESIGN AND METHODS:Flow cytometry (cellular antigens), real-time quantitative polymerase chain reaction (RT-qPCR; bone morphogenetic protein [BMP] messengers), alkaline phosphatase (ALP; osteogenic expression), and calcification analyses were performed on 24- and 48-hour human bone cells (HBCs) and 143B and SaOS-2 (osteosarcoma) cell cultures to study the effect of PRP on proliferation and differentiation of skeletal cultured cells. PRP was added using different protocols since no studies are available on bone cultures treated in the long term with PRP.RESULTS:Flow cytometry showed PRP induction toward a nonhemopoietic lineage in HBCs; RT-qPCR showed enhanced mRNA encoding for BMP2 in HBCs, BMP6 and BMP7 in 143B cultures, and BMP2 and BMP7 in SaOS-2 cultures. Better ALP and calcification results were obtained in SaOS-2 cultures when PRP was added more frequently at shorter intervals while poor results were obtained after single PRP addition.CONCLUSIONS:The results highlight induction of bone cell proliferation and differentiation by PRP. Since repeated administration of PRP is needed to achieve the best results, an almost continuous delivery system of PRP, or better a controlled release of growth and differentiation factors, using biomaterials might provide increased performance at bone regeneration sites. IDS Number: 478LS PMID: 19413738

2009 - Quercetin inhibits lymphocyte activation and proliferation without inducing apoptosis in peripheral mononuclear cells. [Articolo su rivista]
Lugli, Enrico; Ferraresi, Roberta; Roat, Erika; Troiano, Leonarda; Pinti, Marcello; Nasi, Milena; Nemes, Elisa; Bertoncelli, L; Gibellini, Lara; Salomoni, Paolo; Cooper, El; Cossarizza, Andrea
abstract

Toxicity of chemotherapeutic drugs towards normal cells is a serious side effect of cancer treatment. Thus, finding of molecules with low toxicity for normal cells is crucial. Several natural compounds, such as flavonoid quercertin, are receiving a growing attention as “chemopreventers”. Quercetin kills tumour-derived cell lines, but little is known about its effects on normal cells. Here we show that although quercetin exerts a higher apoptotic potential on leukemic cell lines than on peripheral blood mononuclear cells (PBMCs) and does not sensitize PBMCs to CD95-induced apoptosis, it is able to inhibit normal immune functions such as T cell proliferation and activation. Quercetin sensitivity is independent on cell cycle progression since it was not abrogated in serum-starved U937 cells, nor proliferating PBMCs underwent apoptosis after quercetin treatment. However, quercetin prevented PHA-induced PBMC proliferation and SEB-induced upregulation of activation markers. Our data suggest that quercetin, while incapable of inducing apoptosis in normal cells under several conditions, could interfere with effector T cell function.

2009 - Relevance of CD38 expression on CD8 T cells to evaluate antiretroviral therapy response in HIV-1-infected youths [Articolo su rivista]
Rosso, R.; Fenoglio, D.; Terranova, M. P.; Lantieri, F.; Risso, D.; Pontali, E.; Setti, M.; Cossarizza, A.; Ravetti, J. L.; Viscoli, C.; Kunkl, A.
abstract

Surrogate markers for monitoring immuno-virological discordant responders, in addition to plasma viral load and CD4 cells, are still lacking. We assessed the diagnostic utility of CD38 expression on CD8 T cell assay, alone or in association with lymphocyte proliferation to mycotic antigens, in evaluating antiretroviral response. 28 vertically HIV-infected youths, 21 HAART- and seven 2 nucleotide reverse transcriptase inhibitors-treated, were enrolled in a retrospective study. Responders (57.1%) and non-responders (42.9%) to stable antiretroviral therapy for a minimum of 6 months, on the basis of viral load and CD4 T cells, comprehensively evaluated by CD38 expression on CD8 T lymphocytes [measured as CD38 antibody bound per CD8 T cell (CD38 ABC) and Í38+ of total CD8 T cells (Í38/CD8)] and lymphocyte proliferation to P. jiroveci, C. albicans, C. neoformans, A. fumigatus at a single time point after treatment, were selected. CD38 expression ≥2401 CD38 ABC and ≥85% CD38/CD8 cut-off points, accurately discriminates responders versus non-responders, both measures resulting in 75.0% (CI 42.8-94.5) sensitivity (identification of non-responder) and 93.8% (CI 69.8-99.8) specificity (identification of responder), when considered as single assays. The association '≥2401 CD38 ABC or ≥85% CD38/CD8' improved sensitivity to 83.3% (CI 51.6-97.9), while the association '&lt;2401 CD38ABC (or &lt;85% CD38/CD8) and lymphoproliferative response positive to ≥2 tested organisms' improved specificity to 100% (CI 79.4-100). In conclusions, CD38 expression and mycotic antigen-specific T-cell proliferation may be used as additional parameters to existing criteria to evaluate antiretroviral response in immuno-virological discordant patients. © 2010 Blackwell Publishing Ltd.

2009 - Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry [Articolo su rivista]
Cossarizza, Andrea; Ferraresi, Roberta; Troiano, Leonarda; Roat, Erika; Gibellini, Lara; Bertoncelli, Linda; Nasi, Milena; Pinti, Marcello
abstract

Reactive oxygen species (ROS) are continuously produced in the cell as a consequence of aerobic metabolism, and are controlled by several antioxidant mechanisms. An accurate measurement of ROS is essential to evaluate the redox status of the cell, or the effects of molecules with the pro-oxidant or antioxidant activity. Here we report a cytofluorimetric technique for measuring simultaneously, at the single-cell level, hydrogen peroxide and superoxide anion, reduced glutathione (a main intracellular antioxidant) and cell viability. The staining is performed with the fluorescent dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA), hydroethidine (HE), monobromobimane (MBB) and TO-PRO-3. This analysis is possible with new-generation flow cytometers equipped with several light sources (in our case, four lasers and an UV lamp), which excite different fluorochromes. This approach is extremely useful to study the balance between ROS content and antioxidants in cells receiving different stimuli, and to analyze the relationship between oxidative stress and cell death.

2008 - Altered clonogenic capability and stromal cell function characterize bone marrow of HIV-infected subjects with low CD4+ T cell counts despite viral suppression during HAART [Articolo su rivista]
Isgrò, A; Leti, W; DE SANTIS, W; Marziali, M; Esposito, A; Fimiani, C; Luzi, G; Pinti, Marcello; Cossarizza, Andrea; Aiuti, F; Mezzaroma, I.
abstract

Inflammatory cytokines in bone marrow may impair hematolymphopoiesis in human immunodeficiency virus (HIV)-infected subjects who do not experience reconstitution of CD4(+) T cells despite suppression of virus replication while receiving highly active antiretroviral therapy (HAART) (immunological nonresponders). METHODS: Bone marrow samples from 12 immunological nonresponders receiving HAART were studied and compared with samples from 11 immunological responders. The mean CD4(+) T cell count (+/- standard deviation) was 174 +/- 68 cells/mm(3) and plasma HIV RNA levels had been &lt;50 copies/mL for at least 1 year for individuals enrolled in the study. The clonogenic capability of bone marrow samples was evaluated using the colony forming cell assay and the long-term culture-initiating cell assay. CD34(+) cells from the colony forming cell assay were pooled for real-time polymerase chain reaction analysis of Fas and Fas ligand. Bone marrow cytokine production (interleukin-2 and tumor necrosis factor-alpha) and stromal interleukin-7 levels were analyzed by enzyme-linked immunosorbent assay in both groups. Flow cytometric analysis of CD4(+) and CD8(+) T cell subsets was performed. RESULTS: A reduced clonogenic capability and a decrease in the level of more primitive progenitor cells were observed in parallel with lower production of interleukin-2 and increased tumor necrosis factor-alpha levels. A significant upregulation of Fas and Fas ligand on CD34(+) cells and a higher stromal interleukin-7 production were observed. Impairment of the naive T cell compartment and persistent T cell activation were observed in peripheral blood. CONCLUSIONS: Samples from immunological nonresponders show reduced growth of in vitro colonies and an altered cytokine production in bone marrow. The cytokine pattern observed and the altered Fas and Fas ligand pathway may determine stem cell apoptosis and low CD4(+) cell recovery. These features, which are similar to those observed in HIV-infected subjects before starting therapy, persist despite treatment.

2008 - Apoptosis and HIV infection: about molecules and genes. [Articolo su rivista]
Cossarizza, Andrea
abstract

During the evolution, the immune system has developed several strategies to fight viral infections. Apoptosis, autophagy and necrosis are different types of cell death that play a main role in the interactions between infective agents and the host, since they are often important defence mechanisms that have to avoid the spreading of the infection. In turn, viruses have evolved numerous ways to evade the host immune system by influencing the behaviour and functionality of several components. HIV infects and kills CD4+ T helper lymphocytes, preferentially those that are antigen-specific, but also encodes proteins with apoptotic capacities, including gp120, gp160, Tat, Nef, Vpr, Vpu, Vif and, last but not least, the viral protease. This latter protein can kill infected and uninfected lymphocytes through the action of several host molecules, mainly members of the tumor necrosis factor family, or via the mitochondrial apoptotic pathway. The proinflammatory state that is characteristic of both the acute and chronic phase of HIV infection facilitates cell death, and is an additional cause of immune damage. Potent antiretroviral drugs that are largely use in therapy can reduce apoptosis by different mechanisms, that not only include the diminished production of the virus by infected cells and the subsequent reduction of inflammation, but also a direct action on the viral protease. The role of the host genetic background is finally crucial in understanding the process of cell death in HIV infection.

2008 - Effects of the change from Stavudine to tenofovir in human immunodeficiency virus-infected children treated with highly active antiretroviral therapy: studies on mitochondrial toxicity and thymic function. [Articolo su rivista]
Rosso, Sabina; Nasi, Milena; Di Biagio, A.; Repetto, E.; Dentone, C.; Pinti, Marcello; Nemes, Elisa; Ferraresi, Roberta; Mussini, Cristina; Esposito, Roberto; Viscoli, C.; Cossarizza, Andrea
abstract

BACKGROUND:Changing from drugs that have significant mitochondrial toxicity to less toxic compounds may be of benefit in human immunodeficiency virus (HIV)-positive patients who receive highly active antiretroviral therapy. Few data on mitochondrial toxicity of antiviral drugs are available in HIV-positive children. METHODS: Eighteen HIV-positive children (median age, 10.9 years) receiving a stavudine-containing regimen were randomized to maintain stavudine (arm A) or change to tenofovir (arm B), while preserving the remaining drugs. Glucose, lipidic, and viro-immunologic factors were assessed at months 0, 1, 3, 6, 12, and 18. Thymic output and mtDNA content were measured in peripheral blood mononuclear cells at 0 and 6 months, mtDNA in isolated CD4+ and CD8+ T cells after 18 months. RESULTS: From baseline to month 6, arms A and B showed similar thymic output and mtDNA. After 18 months, a significant decrease in plasma HDL was observed in arm B, along with a small increase in blood glucose; mtDNA showed no difference. In the 2 arms other factors did not show significant differences from the baseline and from the previous values at 18 months. CONCLUSIONS: Changing from stavudine to tenofovir was well-tolerated, and viro-immunologic success was maintained.

2008 - Gastrointestinal dysmotility in mitochondrial neurogastrointestinal encephalomyopathy is caused by mitochondrial DNA depletion [Articolo su rivista]
Giordano, C.; Sebastiani, M.; De Giorgio, R.; Travaglini, C.; Tancredi, A.; Valentino, M. L.; Bellan, M.; Cossarizza, Andrea; Hirano, M.; d’Amati, G.; Carelli, V.
abstract

Chronic intestinal pseudo-obstruction is a life-threatening condition of unknown pathogenic mechanisms. Chronic intestinal pseudo-obstruction can be a feature of mitochondrial disorders, such as mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a rare autosomal-recessive syndrome, resulting from mutations in the thymidine phosphorylase gene. MNGIE patients show elevated circulating levels of thymidine and deoxyuridine, and accumulate somatic mitochondrial DNA (mtDNA) defects. The present study aimed to clarify the molecular basis of chronic intestinal pseudo-obstruction in MNGIE. Using laser capture microdissection, we correlated the histopathological features with mtDNA defects in different tissues from the gastrointestinal wall of five MNGIE and ten control patients. We found mtDNA depletion, mitochondrial proliferation, and smooth cell atrophy in the external layer of the muscularis propria, in the stomach and in the small intestine of MNGIE patients. In controls, the lowest amounts of mtDNA were present at the same sites, as compared with other layers of the gastrointestinal wall. We also observed mitochondrial proliferation and mtDNA depletion in small vessel endothelial and smooth muscle cells. Thus, visceral mitochondrial myopathy likely causes gastrointestinal dysmotility in MNGIE patients. The low baseline abundance of mtDNA molecules may predispose smooth muscle cells of the muscularis propria external layer to the toxic effects of thymidine and deoxyuridine, and exposure to high circulating levels of nucleosides may account for the mtDNA depletion observed in the small vessel wall. --------------------------------------------------------------------------------Chronic intestinal pseudo-obstruction (CIPO) is a highly morbid and often life-threatening condition characterized by marked dysfunction of gut propulsive motility, which results in a clinical picture mimicking mechanical obstruction.1,2,3 Patients with CIPO usually complain of severe symptoms including abdominal pain and distension, early satiety, bloating, and vomiting, as well as constipation and/or diarrhea. CIPO is an important cause of chronic intestinal failure, because affected individuals become unable to maintain normal nutrition and body weight. Concerning etiologies, CIPO may be primary or secondary to a variety of systemic diseases.2,3 Primary CIPO may be due to abnormalities of smooth muscle cells of muscularis propria (ie, visceral myopathy) and/or enteric neuronal supplies of gastrointestinal (GI) wall (ie, visceral neuropathy).3 In addition, abnormalities of the GI pacemaker cells, the interstitial cells of Cajal have been reported.4 CIPO is an increasingly recognized clinical feature of mitochondrial encephalomyopathies.5 This heterogeneous group of genetic disorders is caused by dysfunction of the mitochondrial respiratory chain that usually affects highly energy dependent tissues such as brain and muscle.6 Among mitochondrial encephalomyopathies, one most frequently associated with GI dysmotility and CIPO is mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive syndrome due to mutations in the thymidine phosphorylase gene TYMP.7 MNGIE is defined clinically by severe GI dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy, white matter changes in brain magnetic resonance imaging, and mitochondrial abnormalities.7 GI dysmotility leads to progressive weight loss and cachexia of MNGIE patients, and diverticulosis of small intestine complicated by inflammation and perforation often causes their death in early adulthood. Biochemical abnormalities in MNGIE include drastically reduced thymidine phosphorylase activity leading to accumulation of thymidine (dThd) and deoxyuridine (dUrd) in blood and tissues.8,9 Toxic levels of dThd and dUrd induce nucleotide pool imbalances that in turn lead to mtDNA abnormalities (point mutations, multiple deletions, and depletion).8,10-12 The pathogenic

2008 - Genetic polymorphisms differently influencing the emergence of atrophy and fat accumulation in HIV-related lipodystrophy [Articolo su rivista]
Zanone Poma, B.; Riva, A.; Nasi, Milena; Cicconi, P.; Broggini, V.; Cozzi Lepri, A.; Mologni, D.; Mazzotta, F.; D’Arminio Monforte, A.; Mussini, Cristina; Cossarizza, Andrea; Galli, M.
abstract

Objective and design: The present study aims at evaluating the influence of geneticpolymorphisms on antiretroviral therapy (ART)-associated lipodystrophy. We includedin the study 255 ICoNA. patients and we assessed the distribution of Fas 670 AGpolymorphism, ApoC3 455 CT and 482 CT polymorphisms, C161T silent substitutionin the PPAR g gene, the Adrenergic b3 Receptor (ARb3) codon 64 TC variant, andtwo polymorphisms in the Adrenergic b2 Receptor (ARb2) codon 16 AG and codon 27CG. Crude rates of lipoatrophy and fat accumulation and adjusted relative rates werecalculated using Poisson regression.Results: In a multivariate model after adjusting for gender, HIV exposure, age, currentviral load, hepatitis C virus (HCV) serology, nucleoside reverse-transcriptase inhibitor(NRTI) pair/‘third drugs’ currently used, months of pre-highly active antiretroviraltherapy (HAART) exposures to NRTI, the following genotypes resulted protectiveagainst lipoatrophy: ApoC3 455 CC genotype [adjusted relative risks (ARR) 0.2,95% confidence interval (CI) 0.046–0.91 vs CT/TT, P¼0.037], ARb3 codon 64 TTgenotype (ARR 0.39, 95% CI 0.14–1.06 vs TC/CC, P¼0.066), and Fas 670 GGgenotype (ARR 0.51, 95% CI 0.26–1.01 vs AG/AA, P¼0.053). With regard to fataccumulation, in the multivariate model, the ARb2 codon 27 CC genotype resultedprotective (ARR 0.21, 95% CI 0.08–0.51 vs CG/GG, P¼0.0006), whereas the ARb2codon 16 AA genotype resulted associated with higher risk (ARR 3.72, 95% CI 1.58–8.76 vs AG/GG, P¼0.0026).Conclusion: Our study suggests that genetic polymorphisms of genes involved inapoptosis and adipocyte metabolism are significantly related to ART associated lipodystrophy.Particularly, we evidenced a role for ApoC3 455 in lipoatrophy and for thetwo variants of ARb2 in fat accumulation.

2008 - HIV coinfection and antiretroviral therapy enhances liver steatosis in patients with hepatitis C, but only in those infected by HCV genotype other than 3. [Articolo su rivista]
Borghi, V; Puoti, M; Mussini, Cristina; Bellelli, S; Angeletti, C; Sabbatini, Francesca; Prati, Francesca; Cossarizza, Andrea; Esposito, Roberto
abstract

Liver steatosis is a common finding in hepatitis C virus (HCV) infection and is associated with an increased progression of the disease. However, HCV genotype 3 steatosis presents a peculiar and virus-induced pathogenesis. We analysed the effect of HIV coinfection and antiretroviral therapy on hepatic steatosis and the effect of the steatosis on fibrosis in patients with or without HCV genotype 3 infection. METHODS: All consecutive HIV-infected and uninfected patients who had undergone a liver biopsy for evaluation of HCV infection at the Infectious Diseases Clinic (Modena, Italy) were included in this study. Primary outcomes were the presence or absence of steatosis or the presence of moderate or advanced fibrosis. RESULTS: A total of 284 patients were enrolled: 187 infected by HCV and 97 coinfected with HIV and HCV. In HCV genotype 3 patients, only HCV-related variables, such as plasma HCV RNA levels (odds ratio [OR] per log10 1.68, P < 0.001) and estimated duration of HCV infection (OR per year 1.17, P = 0.004) were associated with steatosis. In patients infected with other HCV genotypes, steatosis was associated with older age (OR per 5 years 1.47, P < 0.001), with exposure to d-drugs in HIV-HCV-coinfected patients (OR 2.60, P = 0.04) and specifically exposure to stavudine (OR 2.76 HIV-HCV-coinfected versus not HIV-infected patients, P = 0.04). Steatosis was independently associated with bridging fibrosis only in patients infected by HCV genotype other than 3 (OR 4.03, P = 0.01). CONCLUSIONS: Hepatic steatosis, in both HCV-monoinfected and in HIV-HCV-coinfected patients, is strongly correlated with HCV genotype 3, probably through interactions between HCV virus and liver cells. HIV-related increase of steatosis in patients with HCV is probably related to antiretroviral drugs, especially stavudine, in patients infected by HCV genotype other than 3.

2008 - Herpes simplex virus type 1 dysregulates anti-fungal defenses preventing monocyte activation and downregulating toll-like receptor-2 [Articolo su rivista]
Cermelli, Claudio; Orsi, Carlotta Francesca; Ardizzoni, Andrea; Lugli, Enrico; Cenacchi, Valeria; Cossarizza, Andrea; Blasi, Elisabetta
abstract

We investigated the interplay occurring between pathogens in the course of dual infections, using an in vitro model in which the THP-1 monocytic cell line is first infected with HSV-1 and then exposed to Ca or Cn. These three pathogens share some pathogenic features: they cause opportunistic infections, target macrophages and are neurotropic. Here, we show that HSV-1-infected THP-1 cells exhibited augmented phagocytosis against the two opportunistic fungi but reduced capability to counteract fungal infection: the better ingestion by monocytes was followed by facilitated fungal survival and replication. Reduced IL-12 production was also observed. Cytofluorimetric analysis showed that HSV-1-infected monocytes exhibit: (i) downregulated TLR-2 and TLR-4, critical structures in fungal recognition; (ii) reduced expression of CD38 and CD69, known to be important markers of monocyte activation; and (iii) enhanced expression of apoptosis and necrosis markers, in the absence of altered cell proliferation. Overall, these findings imply that HSV-1 infection prevents monocyte activation, thus leading to a significant dysfunction of the monocyte-mediated anti-Candida response; HSV-1 induced apoptosis and necrosis of monocytes further contribute to this impairment.

2008 - Homeostatic cytokines and expansion of regulatory T cells accompany thymic impairment in children with Down syndrome [Articolo su rivista]
Roat, E; Prada, N; Lugli, E; Nasi, Milena; Ferraresi, R; Troiano, L; Giovenzana, C; Pinti, Marcello; Biagioni, O; Mariotti, M; Di Iorio, A; Consolo, Ugo; Balli, F; Cossarizza, Andrea
abstract

Down syndrome (DS), the most common chromosomal abnormality in humans, is characterized by precocious immunologic aging that results, among other things, in alterations of B and T lymphocyte subsets and natural killer cells, defective phagocytosis, and chemotaxis of polymorphonuclear leukocytes. We studied 30 children affected by DS, compared them to 29 healthy controls, and evaluated the functionality of the thymus (by measuring the amount of lymphocytes that express the signal-joint T cell receptor rearrangement excision circles [sj-TREC+]), the plasma levels of interleukin (IL)-7 and IL-15, the proliferative T cell response to these cytokines, the expression of the alpha chain of the IL-7 receptor (CD127), the extrathymic differentiation of T lymphocytes, and the presence of natural regulatory T cells (Tregs) in peripheral blood. We found that DS children had a significantly lower number of sj-TREC+ lymphocytes, the levels of which were strongly correlated with age. We found higher plasma levels of IL-7 and IL-15 than in healthy controls, and a higher proliferative T cell response to IL-15. DS children also showed a lower percentage of CD4(+) cells and profound alterations of T cell differentiation, along with increased amount of Tregs and of cells expressing markers of apoptosis. We can thus hypothesize that the precocious thymic involution occurring in DS is mirrored by a high production of IL-7 and IL-15, which is crucial for cell survival and proliferation. The complex alterations present in the periphery are likely the result of a compensatory mechanism: the overproduction of homeostatic cytokines could be a reaction to the impaired intrathymic production of T lymphocytes and/or to the expansion of Treg in the periphery, and could be required to allow the survival of T cells.

2008 - Mitochondrial DNA Haplogroups and Highly Active Antiretroviral Therapy–Related Lipodystrophy [Articolo su rivista]
Nasi, Milena; Guaraldi, Giovanni; Gabriella, Orlando; Caterina, Durante; Pinti, Marcello; Elisa, Nemes; Nardini, Giulia; Giuseppe, Passarino; Cocchi, Marina; Esposito, Roberto; Mussini, Cristina; Cossarizza, Andrea
abstract

Background. The combination of different point mutations in mitochondrial DNA (mtDNA), which are defined as haplogroups, may cause modification in organelle function and may be involved in several pathologies. We analyzed the distribution of mtDNA polymorphisms in human immunodeficiency virus (HIV)–infected patients with lipodystrophy, a relevant adverse event caused by highly active antiretroviral therapy, and their correlation with metabolic and viroimmunologic parameters. Methods. The frequency of the 9 most common European haplogroups was investigated in 346 white, HIV- infected patients with lipodystrophy. Haplogroups were identified on the basis of classic methods. Statistical analysis was performed with use of 1-way analysis of variance, the x2 test, and principal-components analysis. Results. The distribution of mtDNA haplogroups among patients with lipodystrophy was similar to that among the general European population. We found no differences between patients with different haplogroups with regard to viroimmunologic results (plasma HIV load, CD4+ T cell count, and nadir CD4+ T cell count), glucose data (glucose, insulin, C-peptide, and glycosylated hemoglobin concentrations and insulin resistance), lipid data (levels of triglycerides, total cholesterol, high- and low-density lipoproteins, and apolipoprotein A1 and B), acid-base balance parameters (lactate level and anion gap), or anthropometric measures (weight, body mass index, and waist- to-hip ratio). No differences were observed in trunk fat levels, leg-fat ratio (which was determined by dual-energy X-ray absorptiometry), or exposure to different drug classes. Principal-components analysis confirmed that the spatial distribution of patients belonging to a given haplogroup was not influenced by different clinical parameters. Conclusions. Our study indicates that, in HIV-infected patients with lipodystrophy, mtDNA haplogroups are not related to major metabolic changes or to particular viroimmunologic features.

2008 - OPA1 mutations associated with dominant optic atrophy impair oxidative phosphorylation and mitochondrial fusion [Articolo su rivista]
Zanna, C.; Ghelli, A.; Porcelli, A. M.; Karbowski, M.; Youle, R. J.; Schimpf, S.; Wissinger, B.; Pinti, Marcello; Cossarizza, Andrea; Vidoni, S.; Valentino, M. L.; Rugolo, M.; Carelli, V.
abstract

Dominant optic atrophy (DOA) is characterized by retinal ganglion cell degeneration leading to optic neuropathy. A subset of DOA is caused by mutations in the OPA1 gene, encoding for a dynamin-related GTPase required for mitochondrial fusion. The functional consequences of OPA1 mutations in DOA patients are still poorly understood. This study investigated the effect of five different OPA1 pathogenic mutations on the energetic efficiency and mitochondrial network dynamics of skin fibroblasts from patients. Although DOA fibroblasts maintained their ATP levels and grew in galactose medium, i.e. under forced oxidative metabolism, a significant impairment in mitochondrial ATP synthesis driven by complex I substrates was found. Furthermore, balloon-like structures in the mitochondrial reticulum were observed in galactose medium and mitochondrial fusion was completely inhibited in about 50% of DOA fibroblasts, but not in control cells. Respiratory complex assembly and the expression level of complex I subunits were similar in control and DOA fibroblasts. Co-immunoprecipitation experiments revealed that OPA1 directly interacts with subunits of complexes I, II and III, but not IV and with apoptosis inducing factor. The results disclose a novel link between OPA1, apoptosis inducing factor and the respiratory complexes that may shed some light on the pathogenic mechanism of DOA.

2008 - OPA1 mutations induce mitochondrial DNA instability and optic atrophy ‘plus’ phenotypes [Articolo su rivista]
Amati Bonneau, P.; Valentino, M. L.; Reynier, P.; Gallardo, M. E.; Bornstein, B.; Boissière, A.; Campos, Y.; Rivera, H.; Gonzalez de la Aleja, J.; Carroccia, R.; Iommarini, L.; Labauge, P.; Figarella Branger, D.; Marcorelles, P.; Furby, A.; Beauvais, K.; Letournel, F.; Liguori, R.; La Morgia, C.; Montagna, P.; Liguori, M.; Zanna, C.; Rugolo, M.; Cossarizza, Andrea; Wissinger, B.; Verny, C.; Schwarzenbacher, R.; Martin, M. A.; Arenas, J.; Ayuso, C.; Garesse, R.; Lenaers, G.; Bonneau, D.; Carelli, V.
abstract

Mutations in OPA1, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA). We here report on eight patients from six independent families showing that mutations in the OPA1 gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPA1 protein in mtDNA stability. The five OPA1 mutations associated with these DOA ‘plus’ phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLG1, PEO1 (Twinkle) and SLC25A4 (ANT1) were ruled out. Our results show that certain OPA1 mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability.

2008 - Physiology and immunology of the thymus gland [Capitolo/Saggio]
Nasi, Milena; Pinti, Marcello; Troiano, Leonarda; Cossarizza, Andrea
abstract

The thymus is a giand located in the upper anteriorportion of the chest cavity just behind the sternurn,Under the evolutionary pressure exerted by the emergence of adaptive immunityAnd its inherent risk to from receptorsThat recognize self molecules, this gland appeared about 500 milT cells in order to prevent autoimmunity and orchestrate selftolerance.The thymus has thus become a crucial lymphoid organ in which cells arriving from the bone marrow undergo a finely tuned process of selection based on the specificity of T-cell receptors (TCRs) and differentiate into mature T-cells.The development of thymocitesn involves a stringent selection in which only 1-3% of these cells succeed in survival and can leave the gland to colonize the periphery and give origin to effective immune cells. Duting maturation in thymus, T cells are first positively selected for uselfulness and then negatively selected against autoreactivity. These intrathymis events are governed by sequential interactions of thymocites with different stromal cell types during the migration through the thymus essentially from the external to the internal part of the thymic lobuli. The mature T cells called naïve T cells leave the organ and contribute to the peripheral T cell poll. The complex process of intrathymic maturation of T-lymphocites involvesvarious thymic specific factors and several other molecules. Indeed T cell maturation requires either direct cell-to-cell or paracrine interactions that occur via cytokines or thymic hormones produced by the cells of the thymic microenvironment. For a long toime the functions of the thymus have remained obscure. The first demonstration of its crucial role in the ontogeny and development of the immune system was provided in 1961 when it was shown that mice thymectomized immediately after birth had poorly developed lymphoid tisses impaired immune responses and susceptibility to infections. Althought cells present in the thymus were believed to be immunoincompetent a few years later it was shown that they could proliferate after an antigenic challenge and produce cells unable to synthetize antibodies. Such cells were capable of enabling other lymphocyte to differentiate to antibody-forming cells. This was the first demonstration in mammalians of the existence of two major subsets of lymphocytes now known as T- and B-cells. It required a re-evaluation of many immunological phenomena such as tolerance memory and autoimmunity and it was followed by a huge number of studies elucidating many of the mysteries of the immune system.

2008 - Plasma HIV load and proviral DNA decreases after two standard antiretroviral regimens in HIV-positive patients naïve to antiretrovirals [Articolo su rivista]
Torti, C; Quiros Roldan, Me; Cologni, G; Nichelatti, M; Ceresoli, F; Pinti, Marcello; Nasi, Milena; Cossarizza, Andrea; Lapadula, G; Costarelli, S; Manca, N; Gargiulo, F; Magoni, M; Carosi, G.
abstract

To compare early decrease of HIV plasma viral load (pVL) after two standard combinations of highly active antiretroviral therapy (HAART). (ii) To evaluate variations of proviral HIV-DNA load on conditions of sustained pVL undetectability. Two different sub-studies of a multicentre prospective randomized controlled trial which compared two first-line HAART (i.e., zidovudine+lamivudine+lopinavir/ritonavir versus tenofovir+lamivudine+ efavirenz). Only patients enrolled at the coordinating centre (University of Brescia) were included in the two sub-studies. In the first sub-study, we calculated pVL decrease with respect to baseline at any of the following time-points: days 1, 3, 7, 14 and 28. Decreases of the pVL were compared between the two treatment groups. In the second sub-study, we analyzed variation of proviral HIV-DNA load in CD4+ T-cells from baseline to week 52 only in patients who maintained the same treatment regimen and had sustained undetectable pVL. In either studies, linear regression analysis was used to investigate what factors could influence variations of pVL and of proviral HIV-DNA load. (i) 64 patients were studied. A significant decrease of pVL was found from day 3 on, without statistically significant differences between the two study groups. However, after adjusting for possible confounders, tenofovir+lamivudine+efavirenz resulted to be associated with greater pVL decreases. (ii) 45 patients were studied. Mean proviral HIV-DNA load decreased from 1,610 (95%CI: 879-2,341) to 896 (95% CI 499-1,293) copies/10(6) cells (P=0.05). Linear regression analysis showed that the decrease of proviral DNA load during follow-up was independently and inversely correlated with age. Further studies are needed to compare pVL decay between antiretroviral regimens and assess whether proviral HIV-DNA load is a surrogate marker of treatment effectiveness.

2008 - Resistance of mtDNA-depleted cells to apoptosis [Articolo su rivista]
Ferraresi, Roberta; Troiano, Leonarda; Pinti, Marcello; Roat, Erika; Lugli, Enrico; Quaglino, Daniela; D., Taverna; D., Bellizzi; G., Passarino; Cossarizza, Andrea
abstract

Cells lacking mitochondrial genome (defined as rho(0)) are useful models in studies on cancer, aging, mitochondrial diseases and apoptosis, but several of their functional aspects have been poorly characterized. Using different clones of rho(0) cells derived from the human osteosarcoma line 143B, we have tested the effects of different apoptogenic molecules such as staurosporine (STS), doxorubicin, daunomycin and quercetin, and have analyzed apoptosis, mitochondrial membrane potential (MMP), levels of oxygen free radicals, reduced glutathione (GSH) content, and expression of P-glycoprotein (P-gp). When compared to parental cells, rho(0) cells resulted much less sensitive to apoptosis. MMP was well maintained in rho(0) cells, and remained unchanged after adding apoptogenic agents, and did not change after treatment with molecules able to depolarize mitochondria such as valinomycin. After adding STS, the production of reactive oxygen species was similar in both cell types, but rho(0) cells maintained higher levels of GSH. In rho(0) cells, P-gp was strongly over-expressed both at mRNA and protein level, and its functionality was higher. The resistance to apoptosis of rho(0) cells could be not only due to an increased scavenger capacity of GSH, but also due to a selection of multidrug resistant cells that hyperexpress P-gp.

2007 - A whole-genome association study of major determinants for host control of HIV-1 [Articolo su rivista]
Fellay, J; SHIANNA K., V; Ge, D; Colombo, S; Ledergerber, B; Weale, M; Zhang, K; Gumbs, C; Castagna, A; Cossarizza, Andrea; COZZI LEPRI, A; DE LUCA, A; Easterbrook, P; Francioli, P; Mallal, S; MARTINEZ PICADO, J; MIR J., M; Obel, N; SMITH J., P; Wyniger, J; Descombes, P; ANTONARAKIS S., E; LETVIN N., L; MCMICHAEL A., J; HAYNES B., F; Telenti, A; Goldstein, D. B.
abstract

Understanding why some people establish and maintain effective control of HIV-1 and others do not is a priority in the effort to develop new treatments for HIV/AIDS. Using a whole-genome association strategy, we identified polymorphisms that explain nearly 15% of the variation among individuals in viral load during the asymptomatic set-point period of infection. One of these is found within an endogenous retroviral element and is associated with major histocompatibility allele human leukocyte antigen (HLA)–B*5701, whereas a second is located near the HLA-C gene. An additional analysis of the time to HIV disease progression implicated two genes, one of which encodes an RNA polymerase I subunit. These findings emphasize the importance of studying human genetic variation as a guide to combating infectious agents.

2007 - Analisi dell’espressione delle Bone Morphogenetic Proteins in presenza di Platelet-Rich Plasma: studio in vitro su un modello di differenziamento osseo. [Articolo su rivista]
Bertoldi, Carlo; Pinti, Marcello; Cossarizza, Andrea; Lucchi, A.; Bencivenni, D.; Consolo, Ugo
abstract

Lo scopo di questo studio è di verificare se il PRP è in grado indurre modificazioni nella trascrizione genica di alcune BMPs strettamente coinvolte nel differenziamento osseo. Materiali e Metodi: Il PRP veniva allestito mediante doppia centrifugazione a 1200 ed a 4400 giri. Una parte del PRP ottenuto era utilizzato per allestire la trombina autologa. Quote di PRP e di trombina non utilizzate clinicamente, con il consenso informato dei pazienti, venivano impiegate per i test in vitro”. L’attivazione del PRP, con il rilascio degli -granuli, era ottenuta unendo il PRP con la trombina (3:1 vol/vol). Le cellule 143B (osteosarcoma umano) sono coltivate in mezzo completo (DMEM+FBS 5 %) 37°c in atmosfera umidificata con 5% di CO2. Le cellule (2,0 * 106 per pozzetto) sono state incubate in presenza o meno di PRP (5% vol/vol) per 24 o 48 ore e successivamente raccolte ed utilizzate per l’estrazione dell’RNA. Ogni esperimento è effettuato in triplicato. Il dosaggio delle BMP2 e BMP7 avveniva mediante sonde nucleotidiche e tecniche di RT-PCR. I risultati sono stati normalizzati rispetto ai livelli di espressione di un gene housekeeping ( HBP/L13) già utilizzato nel nostro laboratorio.(CIT Circulation ) (16). L’espressione relativa di BMP2 BMP6 e BMP7 è stata calcolata rispetto al campione di controllo non trattato con PRP il cui valore di espressione è stata arbitrariamente posto pari a 1, utilizzando il cosiddetto metodo del “delta Ciclo Risultati: Abbiamo quantificato i livelli di espressione dei tre messaggeri codificanti per BMP2, BMP6 e BMP7 in cellule 143B in seguito a trattamento o meno con PRP. In nessuno dei casi (sia dopo 24 che dopo 48 ore di induzione) è stato possibile evidenziare l’espressione di BMP2 nelle cellule 143B. Per quanto riguarda l’espressione di BMP6 e BMP7, il trattamento con PRP determina un incremento significativo di espressione per entrambi i trascritti, sia dopo 24 che 48 ore. In particolare, l’effetto è risultato massimo dopo 24 ore di trattamento nel caso di BMP7. Dopo un picco massimo di espressione a 24 ore, i livelli dei due trascritti diminuiscono a 48 ore, pur mantenendosi superiori ai livelli basali. Conclusioni: In conclusione, i risultati ci fanno ritenere che il PRP possa giocare un ruolo nella induzione di BMP7, mentre altri studi a tempi più prolungati sarebbero necessari per definirne la cinetica di BMP2 e BMP6.

2007 - Differential course of HIV-1 infection and apolipoprotein E polymorphism [Articolo su rivista]
Corder, E. H.; Galeazzi, L.; Franceschi, C.; Cossarizza, A.; Paganelli, R.; Pinti, M.; Mussini, C.; Borghi, V.; Pinter, E.; De Cristofaro, R.; Galeazzi, R.; Perini, M.; Aiuti, F.; Giunta, S.
abstract

We studied the course of infection with human immunodeficiency virus type 1 (HIV-1) in relation to apolipoprotein E (APOE) polymorphism found for 209 Italians treated at Infectious Disease Clinics in Rome and Modena. Clinically, patients were classified into four groups according to the yearly rate of decline in CD4+ cell count (LTNP: long-term non-progression; SLOW, 'NORMAL' or RAPID). Patients at both extremes of the clinical spectrum, i.e. those who rapidly progressed to AIDS and those with stable high CD4 cell counts, had few APOE ε4 and ε2 alleles (P = 0.04). Detailed clinical information was then used to construct four model-based clinical profiles using grade-of-membership analysis (GoM), predictive of APOE genotypic frequencies: 1. The clinical profile associated with good long-term prognosis lacked ε2 (P=0.01); 2. Disease progression to AIDS was associated with ε4 and ε2, most evident for zidovudine-lamivudine regimens without a protease inhibitor (P = 0.03); and, 3. AIDS patients had low ε4 and ε2 frequencies, consistent with a high mortality rate among ε4+ and ε2+ AIDS patients. These findings suggest allele-specific immunomodulatory effects involving inherited APOE isoform important enough to alter the clinical course of HIV infection and, possibly, drug efficacy. They imply a connection between lipid metabolism and immunity potentially relevant to common disorders. © 2007 Versita Warsaw and Springer-Verlag.

2007 - Identification and characterization of an aspartyl protease from Cryptococcus neoformans [Articolo su rivista]
Pinti, Marcello; Orsi, Carlotta Francesca; Gibellini, Lara; Esposito, Roberto; Cossarizza, Andrea; Blasi, Elisabetta; Peppoloni, Samuele; Mussini, Cristina
abstract

Abstract Cryptococcosis, caused by Cryptococcus neoformans, is an invasive infection often occurring in AIDS patients. Potent therapy against HIV, which includes protease inhibitors (PIs), has beneficial effects also on opportunistic infections by pathogens such as C. neoformans and C. albicans. PIs inhibit growth of C. albicans by affecting the activity of its aspartyl proteases. We identified, cloned and sequenced a cDNA from C. neoformans encoding for a putative aspartyl protease (CnAP1), and the corresponding genomic region. The gene cnap1 codifies for a protein of 505 aa, with a canonical aspartyl protease structure. We purified the recombinant protein and analyzed its activity in the presence of PIs (Indinavir, Lopinavir, Ritonavir), but did not evidence any inhibition of protease activity. The transcriptional level of cnap1 in C. neoformans is constant in different media. The absence of any inhibition activity by PIs suggests that other targets for PIs might exist in C. neoformans.

2007 - Induction of Mitochondrial Biogenesis Is a Maladaptive Mechanism in Mitochondrial Cardiomyopathies [Articolo su rivista]
M, Sebastiani; C, Giordano; C, Nediani; C, Travaglini; E, Borchi; M, Zani; M, Feccia; M, Mancini; V, Petrozza; Cossarizza, A.; P, Gallo; W, Taylor R.; G, Damati
abstract

Defects of the mitochondrial genome cause a heterogeneous group of clinical disorders, including mitochondrial cardiomyopathies (MIC). The molecular events linking mtDNA defects to cardiac remodeling are unknown. Energy derangements and increase of mitochondrial-derived reactive oxygen species (ROS) could both play a role in the development of cardiac dysfunction in MIC. In addition, mitochondrial proliferation could interfere with sarcomere alignment and contraction

2007 - Membrane lipid alterations as a possible basis for melanocyte degeneration in vitiligo [Articolo su rivista]
DELLANNA M., L; Ottaviani, M; Albanesi, V; PARO VIDOLIN, A; Leone, G; Ferraro, C; Cossarizza, Andrea; Rossi, L; Picardo, M.
abstract

The occurrence of oxidative stress has been proposed as a pathogenetic mechanism for melanocyte degeneration in vitiligo. In order to evaluate this possible correlation we focused on the lipid component of cell membranes. We observed in vitiligo melanocytes, through FACS methods, an increased median fluorescence intensity of rhodamine 123 and C11-BODIPY581/591 indicating a spontaneous higher production of reactive oxygen species (ROS) and membrane lipoperoxidation, associated with an altered pattern of cardiolipin (CL) distribution, defined on the basis of the fluorescence pattern after staining with 10-nonyl acridine orange. We confirmed membrane peroxidation by confocal and contrast-phase microscopes and demonstrated impaired activity of the mitochondrial electron transport chain (ETC) complex I. Finally, we observed increased apoptotic events following exposure to the pro-oxidant cumene hydroperoxide by Annexin V/propidium iodide fluorescence. We hypothesize that in vitiligo melanocytes lipid instability, with a defect in the synthesis or recycling of CL, induces ETC impairment and ROS production. In basal conditions melanocytes maintain the redox balance whereas following chemical or physical stress ROS-mediated membrane peroxidation is increased with a possible further CL oxidation, leading to cell death or detachment.

2007 - Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome. [Articolo su rivista]
Roat, Erika; Prada, Nicole; Ferraresi, Roberta; Giovenzana, Chiara; Nasi, Milena; Troiano, Leonarda; Nemes, Elisa; Pinti, Marcello; Lugli, Enrico; O., Biagioni; M., Mariotti; L., Ciacci; Consolo, Ugo; Balli, Fiorella; Cossarizza, Andrea
abstract

Different types of cells from subjects with Down syndrome (DS) have an increased susceptibility to cell death. We have studied apoptosis and mitochondrial (mt) membrane potential (Delta Psi(m)) in peripheral blood mononuclear cells (PBMC) from DS children and age-matched healthy donors after in vitro treatment with apoptogenic molecules, along with mtDNA content. We found that PBMC from DS and healthy controls had a similar tendency to undergo apoptosis and a similar amount of mtDNA. However, in cells from DS subjects, mitochondria showed a higher loss of Delta Psi(m), underlying the presence of an increasing susceptibility of these organelles to damaging agents. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.</div>

2007 - Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry. [Articolo su rivista]
Troiano, Leonarda; Ferraresi, Roberta; Lugli, Enrico; Nemes, Elisa; Roat, Erika; Nasi, Milena; Pinti, Marcello; Cossarizza, Andrea
abstract

The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters. Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. In the model of U937 cells incubated with the chemopreventive agent quercetin (3,3',4',5,7-pentahydroxyflavone), we describe the detection at the single cell level of changes in MMP (by JC-1), early apoptosis (exposition of phosphatidylserine on the plasma membrane detected by annexin-V), late apoptosis and secondary necrosis (decreased DNA content by Hoechst 33342 and permeability of the plasma membrane to propidium iodide). The procedure can be completed in less than 2 h.

2007 - Polychromatic analysis of mitochondrial membrane potential using JC-1 [Capitolo/Saggio]
Lugli, Enrico; Troiano, Leonarda; Cossarizza, Andrea
abstract

Dissipation of the mitochondrial membrane potential (m) has been accepted as a hallmark of some apoptotic processes. Depending on the model studied, it can occur during the early stages of cell death or later on, after loss of DNA integrity. Discordant data in the literature could be the consequence of using improper probes such as rhodamine 123 (R123) or DiOC6(3), which are not always appropriate for the study of m. The lipophilic cation JC-1, a specific probe for measuring m, is currently the gold standard. Thanks to recently developed instruments and additional probes for cell surface and intracellular markers, it can be used in polychromatic flow cytometric assays to simultaneously detect m along with other biological parameters.

2007 - Subject classification obtained by cluster analysis and principal component analysis applied to flow cytometric data [Articolo su rivista]
Lugli, Enrico; Pinti, Marcello; Nasi, Milena; Troiano, Leonarda; Ferraresi, Roberta; Mussi, Chiara; Salvioli, Gianfranco; Patsekin, V; Robinson, Jp; Durante, Caterina; Cocchi, Marina; Cossarizza, Andrea
abstract

BACKGROUND: Polychromatic flow cytometry (PFC) allows the simultaneous determination of multiple antigens in the same cell, resulting in the generation of a high number of subsets. As a consequence, data analysis is the main difficulty with this technology. Here we show the use of cluster analysis (CA) and principal component analyses (PCA) to simplify multicolor data visualization and to allow subjects' classification. METHODS: By eight-colour cytofluorimetric analysis, we investigated the T cell compartment in donors of different age (young, middle-aged, and centenarians). T cell subsets were identified by combining positive and negative expression of antigens. The resulting data set was organized into a matrix and subjected to CA and PCA. RESULTS: CA clustered people of different ages on the basis of cytofluorimetric profile. PCA of the cellular subsets identified centenarians within a different cluster from young donors, while middle-aged donors were scattered between these groups. These approaches identified T cell phenotypes that changed with increasing age. In young donors, memory T cell subsets tended to be CD127+ and CD95- whereas CD127-, CD95+ phenotypes were found at higher frequencies in people with advanced age. CONCLUSIONS: Our data suggest the use of bioinformatic approaches to analyze large data-sets generated by PFC and to obtain the rapid identification of key populations that best characterize a group of subjects. (c) 2007 International Society for Analytical Cytology.

2007 - The interaction between HIV and its host [Articolo su rivista]
Cossarizza, Andrea
abstract

Since the beginning of the AIDS epidemics, a relevantnumber of scientists of different disciplines have beeninvestigating the interactions between HIV and its host fromthe immunological and virological perspective. It was understoodthat the first moments of the infection are crucial indetermining the entire course of the disease. Indeed, thevirus is immediately able to provoke a variety of damagesand alterations in several compartments, including not onlythe peripheral blood and lymph nodes, but also the mucosalimmune system. Several studies performed in patients experiencingacute, primary infection had shown the importanceof a massive immune activation as a key mechanism that,through the induction of apoptosis, could lead to the depletionof CD4þ T cells that is typical of HIV infection.More recent data demonstrate that acute HIV infectionis accompanied by a marked and selective loss of memoryCD4þ T cells, predominantly from the mucosal surfaces.Indeed, in the simian model of acute infection, 30–60% ofthese cells throughout the body are infected by the virus,and most of these infected cells disappear within 4 days.Highly active antiretroviral therapy (HAART) has significantlychanged the course of HIV infection either by prolongingboth survival or and the time free from opportunisticinfections. However, everyday life of HIVþ patients hasbeen modified by the administration of complex therapeuticregimens. It is common opinion that when started, antiretroviraltherapy for HIV has to be continued life-long, butlong-term treatment with multiple drugs often leads to theonset of serious side effects and unacceptable toxicity, and,in most patients, to treatment fatigue. In the attempt toavoid either the onset of side effects or a poor adherenceto therapy, and to maintain an efficient HIV-specific immuneresponse, several groups have started clinical studies evaluatingdifferent strategies of therapy interruptions. Sincemany years, our group has been conducting several studiesto investigate the main features of the CD4 cell-guided interruptions.This strategy allows the patient to remain off-therapyfor periods that are extremely much longer than thoseof the structured therapy interruptions, and also representsan ideal model to study not only the immediate reaction ofthe immune system to the reactivation of the virus, but alsothe evolution of the immune response with time. We haverecently investigated the changes of several immunologicalmarkers during CD4 cell monitored therapy interruption ina group of 17 patients. Viral load and activation of CD8þlymphocytes increased significantly in the first two monthsafter treatment discontinuation, then decreased and remainedstable. CD4þ cell count decreased in the first4 months, then remained stable; naı¨ve, central memory,effector memory, and terminally differentiated CD4þ cellschanged proportionally in a similar manner. Patients whohad to restart therapy within 1 year always presented more CD4þ naı¨ve cells and less CD4þ effector memory lymphocytes,while the others had a higher expression of CD127on peripheral T cells. Data will be presented on the importanceof immune activation and of the interleukin-7 systemin these situations.

2006 - A cytoplasmic PML mutant inhibits p53 function [Articolo su rivista]
C., Bellodi; K., Kindle; F., Bernassola; Cossarizza, Andrea; D., Dinsdale; G., Melino; D., Heery; P., Salomoni
abstract

The promyelocytic leukaemia gene (Pml) is a tumor suppressor identified in acute promyelocytic leukaemia (APL), where it is fused to RAR alpha gene as a result of the chromosomal translocation t( 15; 17). Pml encodes both nuclear and cytoplasmic isoforms. While nuclear PML has been intensively investigated, cytoplasmic PML proteins are less characterized. PML nuclear isoforms (nPML) are the essential components of sub-nuclear structures referred to as PML nuclear bodies (PML-NB). In response to cellular insults such as DNA damage and oncogenic activation, nPML modulates p53 activity through CBP-mediated acetylation and activates its pro-apoptotic and growth suppressive functions. Two missense mutations resulting in truncated PML cytoplasmic proteins (Mut PML) have been identified in aggressive APL cases. Here we report that cytoplasmic PML is able to induce the relocation of nPML to the cytoplasm, thus reducing the number of PML-NBs. Remarkably, Mut PML inhibits p53 transcriptional, growth suppressive, and apoptotic functions, thus suggesting that cytoplasmic expression of PML has an impact on survival through inhibition of nuclear PML. Overall our findings shed new light on the role of PML cytoplasmic proteins in the regulation of p53.

2006 - Anti-HIV drugs and the mitochondria [Articolo su rivista]
Pinti, Marcello; Salomoni, P; Cossarizza, Andrea
abstract

Several drugs are currently used that can significantly prolong the course of the infection with the human immunodeficiency virus (HIV), the cause of the acquired immunodeficiency syndrome (AIDS). Among these drugs, the nucleosidic inhibitors of viral reverse transcriptase can alter mitochondrial (mt) function by inhibiting the mitochondrial DNA polymerase gamma (the enzyme responsible for the replication of mtDNA). Decreased mtDNA content provokes a diminished synthesis of respiratory chain enzymes, leading to alterations in rut function. These are in turn responsible for a variety of side effects frequently observed in HIV+ patients, that range from hyperlactatemia and lactic acidosis to lipodystrophy, a pathology characterized by accumulation of visceral fat, breast adiposity, cervical fat-pads, hyperlipidemia, insulin resistance and fat wasting in face and limbs. In this paper, data concerning the effects of different compounds on mitochondria, their role in the pathogenesis of lipodystrophy, and problems related to studies on the mt toxicity of antiviral drugs are reviewed and thoroughly discussed.

2006 - Cytoplasmic function of mutant PML and PML-RARalpha [Articolo su rivista]
Bellodi, C; Kindle, K; Bernassola, F; Dinsdale, D; Cossarizza, Andrea; Melino, G; Heery, D; Salomoni, P.
abstract

The promyelocytic leukemia (PML) tumor suppressor of acute promyelocytic leukemia (APL) regulates major apoptotic and growth-suppressive pathways. In APL, PML is involved in a chromosomal translocation generating the PML-retinoic acid receptor-{alpha} (RAR{alpha}) fusion protein. Two missense mutations in the remaining PML alleles have been identified, which give rise to a truncated cytoplasmic PML protein (Mut PML). APL patients carrying these mutations display resistance to retinoic acid (RA) and very poor prognosis. Here we show that Mut PML associates with the cytoplasmic regions we refer to as PML-cytoplasmic bodies (PML-CBs). Mut PML interacts with PML-RAR{alpha} in PML-CB and potentiates PML-RAR{alpha}-mediated inhibition of RA-dependent transcription. Remarkably, Mut PML stabilizes PML-RAR{alpha} and inhibits differentiation induced by pharmacological doses of RA. A mutant form of PML-RAR{alpha} that accumulates in the cytoplasm inhibits RA-dependent transcription and differentiation, thus suggesting that cytoplasmic localization of PML-RAR{alpha} may contribute to transformation. Finally, we show that the bcr3 PML-RAR{alpha} form is predominantly cytoplasmic and accumulates in PML-CBs. Taken together, these findings reveal novel insights into the molecular mechanisms contributing to APL.

2006 - Espressione differenziale di BMPs in presenza di PRP: studio pilota. [Abstract in Rivista]
Bertoldi, Carlo; Pinti, Marcello; Cossarizza, Andrea; Zaffe, Davide; Consolo, Ugo
abstract

Vedi allegato

2006 - Immunophenotype of HIV plus patients during CD4 cell-monitored treatment interruption: role of the IL-7/IL-7 receptor system [Articolo su rivista]
Nemes, E.; Lugli, E.; Nasi, M.; Ferraresi, R.; Pinti, M.; Bugarini, R.; Borghi, V.; Prati, F.; Esposito, R.; Cossarizza, A.; Mussini, C.
abstract

Objective: To investigate immunological changes during CD4-guided therapy interruption in HIV+ patients who suspended HAART. Patients: Seventeen patients aged &gt; 18 years, who had received HAART for at least 12 months, and had a pre-interruption CD4+ cell count &gt; 500 cells/mu d, interrupted treatment. Median nadir CD4+ cell count was 288 cells/mu l. HIV plasma viral load at discontinuation was &lt; 50 or &gt; 50 copies/ml. Criteria for restarting treatment were: a CD4+ T-lymphocyte count &lt; 350 cells/ d on two separate occasions, a clinical manifestation of AIDS, and the patient's desire to resume HAART. Eleven patients were still off therapy after 12 months (group A); according to the first criterion, six patients restarted therapy within 12 months (group B). Methods: Haernatological, viro-immunological, cytofluorimetic and molecular assays were performed at baseline and every 2 months following standard methods. Statistical analysis was performed under Stata 7.0. Results: In the first 2 months of treatment interruption, a significant increase in viral load and CD8+ lymphocyte activation occurred. Then such parameters decreased and remained stable. In all patients, a decrease in CD4+ lymphocytes took place as well, that affected in a similar manner naive, central memory, effector memory and terminally differentiated cells. Group B always presented lower amounts of CD4+ effector memory lymphocytes. The expression of CD`127 was always higher in group A. Conclusions: The loss of CD4+ lymphocytes upon viral rebound is equal among naive and memory subsets. Patients with higher expression of CD127, who are likely to exert a better capacity to utilize endogenous interleukin-7 by T cells, could remain off therapy for longer periods. (c) 2006 Lippincott Williams &amp; Wilkins

2006 - Mitochondrial neurogastrointestinal encephalomyopathy: Evidence of mitochondrial DNA depletion in the small intestine [Articolo su rivista]
Giordano, C.; Sebastiani, M.; Plazzi, G.; Travaglini, C.; Sale, P.; Pinti, Marcello; Tancredi, A.; Liguori, R.; Montagna, P.; Bellan, M.; Valentino, M. L.; Cossarizza, Andrea; Hirano, M.; D'Amati, G.; Carelli, V.
abstract

Background & Aims: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease clinically defined by gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy, white-matter changes in brain magnetic resonance imaging, and mitochondrial abnormalities. Loss-of-function mutations in thymidine phosphorylase gene induce pathologic accumulations of thymidine and deoxyuridine that in turn cause mitochondrial DNA (mtDNA) defects (depletion, multiple deletions, and point mutations). Our study is aimed to define the molecular basis of gastrointestinal dysmotility in a case of MNGIE. Methods: By using laser capture microdissection techniques, we correlated histologic features with mtDNA abnormalities in different tissue components of the gastrointestinal wall in a MNGIE patient and ten controls. Results: The patient's small intestine showed marked atrophy and mitochondrial proliferation of the external layer of muscularis propria. Genetic analysis revealed selective depletion of mtDNA in the small intestine compared with esophagus, stomach, and colon, and microdissection analysis revealed that mtDNA depletion was confined to the external layer of muscularis propria. Multiple deletions were detected in the upper esophagus and skeletal muscle. Site-specific somatic point mutations were detected only at low abundance both in the muscle and nervous tissue of the gastrointestinal tract. Analysis of the gastrointestinal tract from 10 controls revealed a non-homogeneous distribution of mtDNA content; the small intestine had the lowest levels of mtDNA. Conclusion: Atrophy, mitochondrial proliferation, and mtDNA depletion in the external layer of muscularis propria of small intestine indicate that visceral myopathy is responsible for gastrointestinal dysmotility in this MNGIE patient.

2006 - Modulation of CD38 expression in human longevity: A flow cytometric study [Abstract in Atti di Convegno]
Lugli, Enrico; Pinti, Marcello; Troiano, Leonarda; Nasi, Milena; Ferraresi, Roberta; Roat, Erika; Durante, Caterina; Cocchi, Marina; Cossarizza, Andrea
abstract

The dynamics of CD38 expression innewborns and young healthy donors hasbeen widely investigated for many years.However, little is known about the modulationof this marker during humanageing. We analyzed the changes inCD38 expression in peripheral bloodlymphocytes (PBL) from subjects whowere centenarians. For this purpose weused polychromatic flow cytometry, apowerful technology that allows the determinationof multiple antigens (in ourcase, up to 8) present in the same cell.Among the subsets within CD4+ andCD8+ T cell populations identified bythis approach, we investigated the expressionof CD38 together with markersrelated to extrathymic T cell differentiation(CD45RA and CCR7), T cell survival(CD127/IL-7rα) and activation/apoptosis(CD95). The groups analysed includedyoung donors (21±2 years old),middle-aged individuals (60±1.5 yearsold) and centenarians.By automatic boolean gating, we identifiedall the possible subsets obtained bythe combination of positive and negativeexpression for each marker indicatedabove. Moreover, we could distinguish betweendim or bright expression of CD38.CD38 expressed by CD4+ T cells doesnot show significant modifications in thethree samples either in of the virgin ormemory subsets.A slight increase in CD38 expressionwas found in PBL CD8+ T cells from centenarians.These CD8+/38dim T cells displayeda CD45RA-/CCR7+ central memoryor CD45RA-/CCR7- effector memoryphenotype. Further, CD38 expressionwas associated with the presence ofCD95 and the absence of CD127/IL-7rα.These results were also confirmed byCluster Analysis (CA) and PrincipalComponent Analysis (PCA) of the highnumber of T cell populations identifiedby flow cytometry. These bioinformatictechniques cluster the individuals accordingto the flow cytometric profile,which confirmed that the subsets with anincreased expression of CD38 (CD38bright) are more frequent in the sampleof centenarians.In conclusion, our data indicate amodulation of CD38 expression in CD8+T cells during human ageing. In particular,the preferential coexpression of thisantigen with CD95 but not CD127/IL-7r_suggests an age-dependent acquisition ofan effector phenotype of CD8+ T cellswhich could, at least in part, explain thechronic pro-inflammatory status presentin centenarians.

2006 - Polymorphisms of Fas gene: Relationship with Alzheimer's disease and cognitive decline [Articolo su rivista]
M., Chiappelli; Nasi, Milena; Cossarizza, Andrea; E., Porcellini; E., Tumini; Troiano, Leonarda; Pinti, Marcello; M., Franceschi; F., Licastro
abstract

The Fas antigen (CD95) is a cell surface receptor that mediates cell apoptosis signalling. Recent investigations have shown that Fas-regulated apoptosis was linked to neurodegenerative lesions in the brain of patients with Alzheimer's disease ( AD). Here data regarding the association of two polymorphisms of the Fas promoter region with AD patient's cognitive deterioration are reported. The polymorphism at position - 1377 was associated with the risk of developing AD and with a differential rate of cognitive decline during a 2-year follow-up. The polymorphism at position - 670 was not associated with the risk of AD and with the cognitive decline during the follow-up. Our data suggest that different genetic background in the Fas gene may influence the risk and clinical progression of the disease by affecting neurodegenerative processes leading to neuronal loss. Copyright (C) 2006 S. Karger AG, Basel.

2006 - Protective effect of acetyl-L-carnitine against oxidative stress induced by antiretroviral drugs [Articolo su rivista]
Ferraresi, Roberta; Troiano, Leonarda; Roat, Erika; Nemes, Elisa; Lugli, Enrico; Nasi, Milena; Pinti, Marcello; M., Calvani; M., Iannuccelli; Cossarizza, Andrea
abstract

Both HIV infection per se and antiretroviral drugs might contribute to oxidative stress and mitochondrial dysfunctions. In this study we assess zidovudine, stavudine and didanosine on U937 and CEM cell lines. All these drugs induced apoptosis and increased intracellular hydrogen peroxide but not superoxide anions. The addition of acetyl-L-carnitine (ALC) was able to prevent the pro-oxidant effect of the drugs tested. Supplementation with ALC, deficient in certain cohorts of HIV-infected individuals, especially on high active antiretroviral therapy regimen, has been associated with favourable effects. These data suggest that one of these effects could be a direct anti-oxidant action. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

2006 - Role of cytoplasmic forms of PML and PML-RAR alpha in the response to therapy [Articolo su rivista]
Bellodi, C; Kindle, K; Bernassola, F; Dinsdale, D; Cossarizza, Andrea; Melino, G; Heery, D; Salomoni, P.
abstract

The PML tumour suppressor of acute promyelocytic leukaemia (APL) regulates major apoptotic and growth suppressive pathways. In APL, one PML allele is involved in a chromosomal translocation generating the PML-RARα fusion protein. Two missense mutations in the remaining PML allele have been identified, which give rise to a truncated cytoplasmic PML protein (Mut PML). APL patients carrying these mutations display resistance to retinoic acid (RA) and very poor prognosis.The aim of this study was to analyze the subcellular distribution of Mut PML and determine its role in the regulation therapy-induced differentiation. Finally, we set to determine the relationship between Mut PML and PML-RARα.Both non-haematopoietic and haematopoietic cell lines were used in this study. Retroviral transduction was achieved using retroviral vectors encoding Mut PML, wild type and mutant PML-RARα. Both bcr1 and bcr3 PML-RARα variants were used in this study. PML-RARα nuclear localization signals were mutagenized in order to induce its exclusive cytoplasmic localization. Differentiation was determined both by surface markers expression and morphological analysis. Transcriptional assays were performed using multimerized retinoic-acid (RA) responsive elements cloned upstream the luciferase cDNA, and transcriptional activities were measured in transient transfection experiments.We found that Mut PML associates with cytoplasmic regions we refer to as PML-cytoplasmic bodies (PML-CBs). Mut PML interacted with PML-RARα in PML-CB and potentiated PML-RARα-mediated inhibition of RA-dependent transcription. Remarkably, Mut PML stabilized PML-RARα and inhibited differentiation induced by pharmacological doses of RA. A mutant form of PML-RARα that accumulates in the cytoplasm inhibits RA-dependent transcription and differentiation, thus suggesting that cytoplasmic localization of PML-RARα may contribute to transformation. Finally, we show that the bcr3 PML-RARα form is predominantly cytoplasmic and accumulates in PML-CBs.Taken together, these findings reveal novel insights into the molecular mechanisms contributing to APL and the response to therapy.

2006 - Thymic output and functionality of the IL-7/IL-7 receptor system in centenarians: implications for the neolymphogenesis at the limit of human life [Articolo su rivista]
Nasi, Milena; Troiano, Leonarda; E., Lugli; Pinti, Marcello; Ferraresi, Roberta; Monterastelli, Elena; Mussi, Chiara; Salvioli, Gianfranco; Franceschi, Claudio; Cossarizza, Andrea
abstract

During aging, the thymus undergoes a marked involution that is responsible for profound changes in the T-cell compartment. To investigate the capacity of the thymus to produce new cells at the limit of human lifespan, we analyzed some basic mechanisms responsible for the renewal and maintenance of peripheral T lymphocytes in 44 centenarians. Thymic functionality was analyzed by the quantification of cells presenting the T-cell receptor rearrangement excision circles (TREC). A new method based upon real-time PCR was used, and we found that most centenarians (84%) had undetectable levels of TREC+ cells. Six-color cytofluorimetric analysis revealed that centenarians had an extremely low number of naive T cells; central memory and effector memory T cells were greatly increased, while terminally differentiated cells were as numerous as in young (aged 20-45) or middle-aged (aged 58-62) donors. Interleukin (IL)-7 and IL-7 receptor alpha-chain (CD127) levels were the same at all ages, as shown by ELISA, flow cytometry and real-time PCR. However, IL-7 plasma levels were higher in centenarian females than males. The presence of TREC+ cells and of very few naive T lymphocytes suggests that in centenarians such cells could either derive from residues of thymic lymphopoietic islets, or even represent long-living lymphocytes that have not yet encountered their antigen. IL-7 could be one of the components responsible, among others, for the higher probability of reaching extreme ages typical of females.

2005 - Altered mithocondrial RNA production in adipocytes from HIV-infected individuals with lipodystrophy [Abstract in Rivista]
Galluzzi, L.; Pinti, Marcello; Guaraldi, Giovanni; Mussini, Cristina; Troiano, L.; Roat, E.; Giovenzana, C.; Nemes, E.; Nasi, M.; Orlando, G.; Salomoni, P.; Cossarizza, Andrea
abstract

Damage to mitochondria (mt) is a major side effect of highly active antiretroviral therapy (HAART) tha includes a nucleoside reverse transcriptase inhibitor (NRTI).Such damage is associated with the onset of lipodystrophy in HAART-treated HIV+ patients. To further investigate mt changes during this syndrome, we analysed the expression of mtRNA in adipocytes from lipodystrophic HIV+ patients taking NRTI-containing HAART and compared it with similar cells from healthy individuals.

2005 - Altered mitochondrial RNA production in adipocytes from HIV-infected individuals with lipodystrophy [Articolo su rivista]
Galluzzi, Lorenzo; Pinti, Marcello; Guaraldi, Giovanni; Mussini, Cristina; Troiano, Leonarda; Roat, Erika; Giovenzana, Chiara; Nemes, Elisa; Nasi, Milena; Orlando, Gabriella; Salomoni, P; Cossarizza, Andrea
abstract

Background: Damage to mitochondria (mt) is a major side effect of highly active antiretroviral therapy (HAART) that includes a nucleoside reverse transcriptase inhibitor (NRTI). Such damage is associated with the onset of lipodystrophy in HAART-treated HIV+ patients. To further investigate mt changes during this syndrome, we analysed the expression of mtRNA in adipocytes from lipodystrophic HIV+ patients taking NIRTI-containing HAART and compared it with similar cells from healthy individuals. Materials and methods: Total RNA was extracted from adipocytes collected from different anatomical locations of 11 HIV+ lipodystrophic patients and seven healthy control individuals. RNA was reverse transcribed and Taqman-based real-time PCR was used to quantify three different mt transcripts (ND1, CYTB and ND6 gene products). mtRNA content was normalized versus the housekeeping transcript L13. Results: ND1, CYTB and ND6 expression was significantly reduced in HIV+ lipodystrophic patients. HIV+ men and women did not differ in a statistically significant way regarding the levels of ND1 and ND6, whereas the opposite occurred for CYTB. Conclusions: Lipodystrophy following treatment with NRTI-containing HAART is associated with a decrease in adipose tissue mtRNAs.

2005 - Biological importance of the two Toll-like receptors, TLR2 and TLR4, in macrophage response to infection with Candida albicans [Articolo su rivista]
Blasi, Elisabetta; Mucci, Anna; Neglia, Rachele Giovanna; Pezzini, Francesco; Colombari, Bruna; D., Radzioch; Cossarizza, Andrea; E., Lugli; G., Volpini; G., Del Giudice; Peppoloni, Samuele
abstract

The aim of this study was to assess the role of TLR2, TLR4 and MyD88 accessory molecule in the effector and secretory response of macrophages to viable microbial agents. Using TLR-deleted macrophage cell lines generated from the bone marrow of genetically engineered mice (TLR4 gene-deficient, MyD88- and TLR2-knockout mice) and wild-type control mice, we found that TLR2-deleted macrophages exhibit increased ability to contain Candida albicans infection compared to TLR2+/+ counterpart. In contrast, both MyD88-/- and TLR4-/- macrophages retain levels of functional activity comparable to that of the respective wild-type MyD88+/+ and TLR4+/+ controls. The difference in anticandidal effector functions observed between TLR2-/- and TLR2+/+ macrophages is abrogated upon opsonization of the fungal target and interestingly is not observed when using other microbial targets, such as Streptococcus pneumoniae and Helicobacter pylori. When tested for secretory response to C albicans, TLR2-deleted macrophages show a pattern of cytokine production similar to that of TLR2+/+ controls. Finally, flow cytometry analysis reveals that TLR2-deleted macrophages express only TLR4, while, as expected, TLR2+/+ macrophages are both TLR2 and TLR4 positive; in no cases, modulation of such markers occurs in macrophages exposed to C albicans infection. In conclusion, these data indicate that TLR2 and TLR4 have different biological relevance, in which TLR2 but not TLR4, is involved in the accomplishment of macrophage-mediated anticandidal activity, while the secretory response to C albicans appears to be TLR4 but not TLR2-dependent. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

2005 - CD4 cell-monitored treatment interruption in patients with a CD4 cell count > 500 x 10(6) cells/I [Articolo su rivista]
Mussini, Cristina; Bedini, Andrea; V., Borghi; Guaraldi, Giovanni; Esposito, Roberto; E., Barchi; R., Emilia; A., Cozzi Lepri; A. N., Phillips; P., Ortolani; Cossarizza, Andrea; G., Bratt; L. E., Eriksson; L., Sighinolfi; A. D., Monforte; A., De Luca; S., Di Giambenedetto; A., Antinori
abstract

Background: Little is known about CD4 cell count changes in patients with high CD4 cell counts who interrupt antiretroviral therapy, especially in those with a nadir of 250350 x 10(6) cells/I. Methods: Data derived from 139 patients from seven prospective cohorts who had > 12 months highly active antiretroviral therapy (HAART), CD4 cell count nadir of > 250 x 10(6) cells/I and at pre-interruption of > 500 x 106 cells/I. Endpoint was time to CD4 cell count < 350 x 10(6) cells/I or reinitiation of treatment. Results: At interruption, the median CD4 cell count was 800 x 106 cells/I, median viral load was 1.7 log(10) copies/ml. At the time of analysis, 63 (45.3%) had resumed therapy or experienced < 350 x 10(6) cells/I CD4 cells over a median interruption of 75 weeks. Of these, 33 (52.4%) experienced a decline to < 350 x 10(6) cells/I and 30 (47.6%) restarted therapy before their CD4 cell count had fallen below this level. In 43 patients with CD4 cell nadir of 251-350 x 10(6) cells/I, median time to therapy resumption or CD4 cell count < 350 x 10(6) cells/I was 61 weeks. Higher CD4 cell count nadir, longer duration of viral load suppression on therapy, and higher viral load level at interruption were independently associated with longer time to restart therapy. The risk of clinical events was 5 per 1000 person-years of follow-up. Conclusions: Patients who started therapy with CD4 cell count of 250-350 x 10(6) cells/I and who later interrupted therapy appear able to remain off therapy with a CD4 cell count > 350 x 10(6) cells/I for a substantial period of time.

2005 - Changes in mitochondrial RNA production in cells treated with nucleoside analogues. [Articolo su rivista]
Galluzzi, Lorenzo; Pinti, Marcello; Troiano, Leonarda; Prada, Nicole; Nasi, Milena; Ferraresi, Roberta; Salomoni, P; Mussini, Cristina; Cossarizza, Andrea
abstract

BACKGROUND: To investigate mitochondrial (mt) toxicity of antiretroviral drugs further, we developed a novel real-time PCR-based assay for the quantification of mtRNA. We analysed the effects of stavudine (d4T), didanosine (ddl) and zidovudine (AZT) on the production of mtRNAs in different human cell lines and compared the production with the amount of mtDNA present in the same cells. MATERIALS AND METHODS: HUT78, CEM and U937 cells were exposed to different nucleoside reverse transcriptase inhibitors (NRTIs) for 7 days. Thereafter, nucleic acids were isolated and Taqman-based real-time PCR was used to quantify mtDNA and three different mtRNAs (ND1, CYTB and ND6 gene products). RESULTS: Different amounts of mtRNAs exist in different cell lines. When mtRNA was measured in cells exposed to an NRTI, a marked decrease was observed in cells treated with d4T, but not with ddl or AZT. Changes in mtRNA production did not always correspond to modifications in mtDNA content: 1 microM d4T significantly changed mtRNA but not mtDNA content. CONCLUSIONS: d4T, but not ddl or AZT, significantly alters mtRNA quantity and quality. The method we have developed can reveal changes that are not observed by measuring mtDNA content only, and can be used for ex vivo studies on drug toxicity.

2005 - Characterization of cells with different mitochondrial membrane potential during apoptosis [Articolo su rivista]
E., Lugli; Troiano, Leonarda; Ferraresi, Roberta; Roat, Erika; Prada, Nicole; Nasi, Milena; Pinti, Marcello; El, Cooper; Cossarizza, Andrea
abstract

Background: Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (Delta Psi), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different Delta Psi during apoptosis. Methods: U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect AT, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracker Deep Red for the determination of mitochondrial mass. Results: Treatment with Qu provoked the onset of three cell populations with different Delta Psi: (1) healthy cells, with normal Delta Psi, DNA content and physical parameters, high mitochondrial mass, PI- and Annexin V-negative; (2) cells with intermediate Delta Psi and normal DNA content, but with physical parameters typical of apoptotic cells and low mitochondrial mass; most of them were PI+ and Annexin V+; (3) cells with collapsed Delta Psi that had low mitochondrial mass and were Annexin-V+, PI+; half of them showed diminished DNA content. Similar results, i.e. the presence of cells with intermediate Delta Psi, were observed in other models of apoptosis. Conclusions: During Qu-induced apoptosis, loss of Delta Psi, PS exposure, and decrease of mitochondrial mass are early events that precede permeability to PI and loss of DNA. Populations of cells with different Delta Psi, as revealed by flow cytometry after JC-1 staining, differed also for other parameters associated to apoptosis. Thus, the simultaneous analysis of several parameters by polychromatic flow cytometry permits a better identification of many stages of cell death, and, more in general, allows to evaluate the eventual heterogenic sensibility of the population under study to a given compound.

2005 - Depletion of mtDNA limited to the external layer of muscularis propria induces gastrointestinal dysmotility in a MNGIE patient [Abstract in Atti di Convegno]
Bellan, M.; Giordano, C.; Sebastiani, M.; Plazzi, Giuseppe; Travaglini, C.; Pinti, M.; Sale, P.; Zani, M.; Liguori, Rocco; Montagna, Pasquale; Baruzzi, Agostino; Valentino, M. L.; Cossarizza, A.; Hirano, M.; D'Amati, G.; Carelli, Valerio
abstract

2005 - Direct analysis of thymic function in children with Down's syndrome. [Articolo su rivista]
Nicole, Prada; Nasi, Milena; Troiano, Leonarda; Erika, Roat; Pinti, Marcello; Elisa, Nemes; Enrico, Lugli; Roberta, Ferraresi; Ciacci, Luigi; Davide, Bertoni; Ornella, Biagioni; Milena, Gibertoni; Cristina, Cornia; Liviana, Meschiari; Elisabetta, Gramazio; Mauro, Mariotti; Consolo, Ugo; Balli, Fiorella; Cossarizza, Andrea
abstract

BACKGROUND: Down's syndrome (DS) is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes (the so called "recent thymic emigrants", RTE) in children with DS. METHODS: We studied 8 children affected by DS, aged 2-7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE, i.e. peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC). RESULTS: In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found. CONCLUSIONS: The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects.

2005 - Effect of treatment interruption monitored by CD4 cell count on mitochondrial DNA content in HIV-infected patients: a prospective study [Articolo su rivista]
Mussini, Cristina; Pinti, Marcello; R., Bugarini; V., Borghi; Nasi, Milena; Nemes, Elisa; Troiano, Leonarda; Guaraldi, Giovanni; Bedini, Andrea; C., Sabin; Esposito, Roberto; Cossarizza, Andrea
abstract

Background: HIV infection per se and HAART can alter mitochondrial functionality, leading to a decrease in mitochondrial DNA content. Objective: To evaluate whether treatment interruption monitored by CD4 cell count can restore mitochondrial DNA content in peripheral blood lymphocytes. Methods: Mitochondrial DNA content was measured in platelet-free CD4 and CD8 T cells by real-time polymerase chain reaction; flow cytometry was used to identify and quantify activated CD4 and CD8 T lymphocytes. Results: The 30 patients had been treated for a mean of 107 months (range, 27-197). Median CD4 cell count at discontinuation was 702 cells/mu l (range, 547-798). Median observational time from HAART discontinuation was 11.3 months (range, 4-26). Discontinuation of treatment provoked significant increases in mitochondrial DNA in CD8 T cells, which started only 6 months after therapy cliscontinuation [5.12 copies/ cell per month from 0 to 6 months (P = 0.3 7) and 2 6.96 copies/cel I per month from 6 to 12 months (P < 0.0001)]. Conclusions: This study is the first showing that mitochondrial DNA content can increase in peripheral blood lymphocytes during treatment interruption, but only after at least 6 months of interruption. Consequently, interruptions of shorter periods, whether by clinician or patient decision, are unlikely to allow restoration of mitochondrial DNA and so decrease HAART-related toxicity.

2005 - Effects on mitochondria and thymus of the switch from stavudine to tenofovir in HIV-infected children [Abstract in Atti di Convegno]
Rosso, R; Nasi, Milena; Pinti, Marcello; Nemes, Elisa; Roat, Erika; Di Biagio, A; Repetto, E; Bassetti, M; Cossarizza, Andrea; Bassetti, D.
abstract

Drug-associated dysfunction of mitochondria (mt) is believed to play a role in the aetiology of the various adverse symptoms that occur in HIV-infected patients treated with the nucleoside reverse transcriptase inhibitors (NRTIs). Switching to drugs with less mt toxicity may be of benefit in HIV-infected patients receiving antiretroviral therapy containing stavudine (d4T), but the efficacy and the safety of this strategy in paediatric population is unknown. We have analysed the effect of the switch from d4T to tenofovir (TDF), paying particular attention to the maintenance of the immunovirological status and lipid profile.

2005 - Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin [Articolo su rivista]
Ferraresi, Roberta; Troiano, Leonarda; Roat, Erika; E., Lugli; Nemes, Elisa; Nasi, Milena; Pinti, Marcello; M. I. G., Fernandez; E. L., Cooper; Cossarizza, Andrea
abstract

We have analyzed the anti- or pro-oxidant effects of the flavonoid quercetin (QU) by evaluating, in U937 cell line, hydrogen peroxide (H2O2), superoxide anion (O-2), reduced glutathione (GSH) content, mitochondrial membrane potential, DNA content, phosphatidylserine exposure on the outer face of the plasma membrane and cell viability. Polychromatic flow cytometry was used to evaluate in the same cells several functional parameters. For short periods of treatment QU exerted an anti-oxidant effect (decrease in H2O2 levels), whereas for long periods it showed a pro-oxidant activity (increase in O-2). In these conditions, GSH content was reduced, and this correlated with a lack of anti-oxidant activity of QU, which in turn could be correlated with proapoptotic activity of this molecule. Thus, QU can exert different effects (anti-/prooxidant) depending on exposure times and oxidative balance, and in particular on stores of GSH.

2005 - Flow Cytometry as a tool for analyzing invertebrate cells. [Articolo su rivista]
Cossarizza, Andrea; Pinti, Marcello; Troiano, Leonarda; Cooper, E. L.
abstract

Flow cytometry (FCM) is a powerful tool that allows analysis of thousand of cells in a few seconds,at the single cell level. In the last 15 years, researchers have used FCM to investigate the cellularmachinery of invertebrates. Analyses have focused on functions linked to innate immunity, such asphagocytosis and natural killer cell activity, as well as on the sensitivity of invertebrate cells to aparticular stress or to a toxic agent. Further, FCM has been employed to recognize antigens, or atleast immunodominant epitopes, shared in common with mammalian cells, including humanleukocytes. In this review, main studies that have utilized FCM to investigate either phenotype andfunctions of invertebrate cells are reported and discussed.

2005 - Genetic polymorphisms of Fas (CD95) and Fas ligand (CD178) influence the rise in CD4+ T cell count after antiretroviral therapy in drug-naive HIV-positive patients. [Articolo su rivista]
Nasi, Milena; Pinti, Marcello; Bugarini, R; Troiano, Leonarda; Lugli, E; Bellodi, C; Mussini, Cristina; Borghi, V; Trenti, T; Balli, Fiorella; Esposito, Roberto; Cossarizza, Andrea
abstract

Fas and Fas ligand (FasL) are the main genes that control cell death in the immune system. Indeed, they are crucial for the regulation of T lymphocyte homeostasis because they can influence cell proliferation. A strong debate exists on the importance of Fas/FasL system during HIV infection, which is characterized by the loss of CD4+ T cells directly, or indirectly, caused by the virus. To investigate whether the genetic background of the host plays a role in the immunoreconstitution, we studied the influence of different Fas and FasL polymorphisms on CD4+ T lymphocyte count and plasma viral load following initiation of highly active antiretroviral therapy (HAART) in drug-naive HIV+ patients. We studied 131 individuals, who were compared to 136 healthy donors. Statistical analysis was performed by using X-2 test, Fischer's Exact Test, and analysis for repeated measurements. The group of HIV+ patients had an unexpected lower frequency of FasLnt169 polymorphism (delT allele) than healthy controls (p=0.039). We then observed no significant differences in the immune reconstitution, in terms of CD4+ T cell increase, when the influence of single alleles of the gene Fas or FasL was considered. However, the combination of some polymorphisms of Fas or FasL significantly influenced CD4+ T cell production and viral load decrease, showing that these genes can play a role in the immunoreconstitution triggered by antiretroviral therapy.

2005 - HIV type 1 protease inhibitors enhance bone marrow progenitor cell activity in normal subjects and in HIV type 1-infected patients [Articolo su rivista]
A., Isgrò; A., Aiuti; I., Mezzaroma; L., Ruco; Pinti, Marcello; Cossarizza, Andrea; F., Aiuti
abstract

HIV- 1 protease inhibitors ( PIs) may improve hematopoietic functions owing to their direct effects on bone marrow ( BM) progenitor cells. In this study we investigated this hypothesis evaluating the effect of adding ritonavir ( RTV) and indinavir ( IND) on hematopoietic colony formation assays by colony- forming cell ( CFC) and long- term culture- initiating cell ( LTC- IC) assays, on apoptosis, on cytokine production and stromal cells, in subjects with HIV- 1 infection, and in seronegative controls. After PI addition, CFC and LTC- IC assays in HIV- 1- infected patients showed levels of colony growth significantly higher than those observed at baseline; the same PI activity on colony formation was observed in healthy subjects. No significant modifications on Fas, the membrane form of Fas ( mFas) and Fas- ligand ( FasL) expression, and on cytokine production were observed at BM level after the addition of PIs. At baseline, in HIV- 1- infected patients, the majority of the stromal cells appeared as large and rounded, whereas after the addition of RTV or IND the stromal cells exhibited a fibroblast- like morphology and produced higher stem cell factor ( SCF) and lower MIP- 1alpha levels when compared with the stromal production without the addition of IND. RTV and IND increased colony growth of BM obtained either from HIV- 1- infected patients or from normal individuals, in parallel with the normalization of functional and morphological characteristics of stromal cells.

2005 - Immunotherapy of HIV disease [Capitolo/Saggio]
Lori, F.; Kelly, L. M.; Cossarizza, A.; Lisziewicz, J.
abstract

2005 - Impairment of recent thymic emigrants in HCV infection. [Articolo su rivista]
Cianci, R; Nasi, Milena; Pinti, Marcello; Starnino, S; Florio, A; Cammarota, G; De Luca, A; Cauda, R; Grieco, A; Rapaccini, G; Gasbarrini, G; Cossarizza, Andrea; Pandolfi, F.
abstract

Hepatitis C Virus (HCV) often has a more favorable course in younger patients. Considering the involution of the thymic function with age, we investigated the output of recent thymic emigrants (RTE) in HCV patients. To evaluate RTE, we used a competitive quantitative PCR in order to determine the percentages of cells with cj-T cell receptor excision circles (TREC). This study was performed in 14 HCV patients at diagnosis and before any anti-HCV treatment. The results obtained in this group were compared to those obtained in a group of age-matched controls. We found that in the 14 HCV patients naive for anti-HCV treatment the mean percentage of cj-TREC was 3%. We could not detect a correlation between the percentages of cj-TREC and age or patients' viremia. In contrast, in the 26 age-matched controls mean percentage of cj-TREC was 5.6% (P=0.01). Our study describes a novel immune defect in HCV patients. Additional studies are needed to get further insight in the possible role of TREC defect in the pathogenesis and prognosis of the disease.

2005 - MMP-7 promoter polymorphisms do not influence CD4+ recovery and changes in plasma viral load during antiretroviral therapy for HIV-1 infection. [Articolo su rivista]
E., Lugli; Pinti, Marcello; Nasi, Milena; Troiano, Leonarda; Prada, Nicole; Mussini, Cristina; V., Borghi; Esposito, Roberto; Cossarizza, Andrea
abstract

Matrix metalloproteinase-7 (MMP-7) generates soluble Fas Ligand (FasL), which is involved in the apoptotic loss of CD4(+) T cells during HIV infection. We evaluated whether two polymorphisms in MMP-7 promoter could influence CD4(+) recover in response to antiretroviral therapy, and found that these polymorphisms are ineffective.

2005 - Mitochondria, HIV infection and its treatment: where do we go from here? [Articolo su rivista]
Cossarizza, Andrea; Reiss, P.
abstract

The idea of organizing a meeting on different aspectsof the interactions between mitochondria, HIV infectionand its treatment emerged as a result of thegrowing interest the scientific community has shown inthis topic during recent years. Many of the scientistswho have been conducting clinical as well as morebasic research in this field responded enthusiastically tothe invitation to present and discuss their ongoingresearch at the first international conference on“Mitochondrial toxicity and HIV infection: understandingthe pathogenesis for a therapeutic approach”,held in Modena (Italy) from May 19-21, 2005. Theinitiative for this conference was taken by ProfessorAndrea Cossarizza, Chair of Immunology at theUniversity of Modena and Reggio Emilia, and hiscolleagues from the Department of Infectious Diseasesat the same University, Professor Roberto Esposito andDr Cristina Mussini.The meeting was made possible by un unrestrictededucational grant from Gilead Sciences, Italy, andpublication of part of the proceedings of the meeting inthis supplement of Antiviral Therapy was madepossible by an unrestricted grant from Sigma Tau, Italy.We wish to underline that the conference organizershad full responsibility for the content of theprogramme as well as the choice of speakers andsession chairs, without any interference from the pharmaceuticalsponsors. All papers appearing in thissupplement underwent peer-review prior to beingconsidered for publication.The meeting started with a lectura magistralis given byProfessor Vladimir Skulachev (State University ofMoscow, Russia), one of the founding fathers of thefield of “bioenergetics”, who provided an overview onprogrammed death in relation to ageing, at the level ofindividual cells (apoptosis), whole organisms (phenoptosis)as well as subcellular organelles includingmitochondria (mitoptosis). This stimulated discussionon whether several of the adverse effects of treatmentfor HIV infection might be a reflection of “acceleratedageing” by mechanisms including mitochondrialtoxicity.The talks and related discussions that took place in thefollowing days revealed that several issues concerningthe detrimental effects which HIV, other concomitantinfections such as those with hepatitis C, and HIVtherapy may have on mitochondria warrant furtherinvestigation and clarification in the years to come.What is becoming increasingly clear is that antiretrovirals,and nucleoside analogue reverse transcriptaseinhibitors (nRTI) in particular, may affect mitochondriain more ways than one. Inhibition by nRTI ofDNA γ-polymerase resulting in mitochondrial DNA(mtDNA) depletion remains one of the cornerstones bywhich nRTI may induce mitochondrial toxicity. It isevident however that nRTI and even other classes ofantiretrovirals such as HIV protease inhibitors, mayhave additional effects on mitochondria, for instanceby way of exerting direct effects on mtRNA transcriptionand mitochondrial enzymes. Differences betweenindividual agents as well as differences in the extent towhich these effects may play a role in different celltypes remain to be further delineated.An important problem still remaining is the choice ofthe type of cell in which to best measure mitochondrialmarkers as a possible reflection of treatment toxicity.Blood obviously remains the easiest tissue to obtainfrom patients, but contamination with platelets whichcontain numerous mitochondria may yield poorlyinterpretable results when using whole blood orperipheral blood mononuclear cells (PBMCs). Betterpurified cell populations such as isolated CD4+ orCD8+ T lymphocytes, or platelet-depleted lymphocytes or monocytes may provide more reliable results andneed to be investigated further. Nevertheless, othertissues and cells coming directly from the organsystems affected by the treatment toxicity being studied(for instance adipose, hepatic or renal) may be moreinformative, and more appropriate when trying toassess the p

2005 - Protective effect of acetyl-L-carnitine on oxidative damage induced by antiretroviral drugs [Articolo su rivista]
Ferraresi, Roberta; Troiano, Leonarda; Roat, Erika; Nemes, Elisa; Iannuccelli, M; Calvani, M; Cossarizza, Andrea
abstract

Both HIV infection per se and antiretroviral drugs might contribute to oxidative stress and mitochondrial dysfunctions. In this study we assess zidovudine, stavudine and didanosine on U937 and CEM cell lines. All these drugs induced apoptosis and increased intracellular hydrogen peroxide but not superoxide anions. The addition of acetyl-l-carnitine (ALC) was able to prevent the pro-oxidant effect of the drugs tested. Supplementation with ALC, deficient in certain cohorts of HIV-infected individuals, especially on high active antiretroviral therapy regimen, has been associated with favourable effects. These data suggest that one of these effects could be a direct anti-oxidant action.

2005 - RUOLO BIOLOGICO DI DUE RECETTORI TOLL-SIMILI, TLR2 E TLR4, NELLA RISPOSTA MACROFAGICA A CANDIDA ALBICANS [Abstract in Atti di Convegno]
Pezzini, Francesco; A., Mucci; Neglia, Rachele Giovanna; Colombari, Bruna; Cossarizza, Andrea; Lugli, Enrico; G., Del Giudice; Peppoloni, Samuele; Blasi, Elisabetta
abstract

RUOLO BIOLOGICO DI DUE RECETTORI TOLL-SIMILI, TLR2 E TLR4, NELLA RISPOSTA MACROFAGICA A CANDIDA ALBICANSIntroduzione: I TLR sono una classe di recettori coinvolti nel riconoscimento di agenti microbici da parte di cellule dell’immunità innata e nella conseguente risposta effettrice. La loro funzione si è rivelata complessa e profondamente diversificata in relazione anche al microrganismo con cui entrano in contatto. L’obiettivo di questo studio è stato valutare il ruolo delle molecole TLR2, TLR4 e MyD88 nella risposta effettrice e secretoria del macrofago nei riguardi di C. albicans.Materiali e metodi: Linee macrofagiche generate dal midollo osseo di topi geneticamente modificati (TLR2-/-, TLR4-/-, MyD88-/-) e dai rispettivi controlli (TLR2+/+, TLR4+/+, MyD88+/+) sono state saggiate per attività fagocitica, attività antimicrobica e produzione di citochine dopo infezione con C. albicans. Risultati: I macrofagi TLR2-/- mostrano una fagocitosi significativamente aumentata e una maggiore capacità di contenere l’infezione da C. albicans rispetto alla controparte TLR2+/+. In contrasto, sia i macrofagi TLR4-/- che quelli MyD88-/- mantengono livelli di attività funzionale sostanzialmente invariati rispetto ai controlli. La differenza nelle funzioni effettrici anticandida osservata tra macrofagi TLR2-/- e TLR2+/+ viene annullata con l’opsonizzazione del micete e non viene osservata nei riguardi di altri microrganismi. In risposta all’ infezione con C. albicans, i macrofagi TLR2-/- producono livelli di citochine comparabili a quelli dei controlli TLR2+/+. Infine, l’analisi in citofluorimetria a flusso rivela che, come atteso, i macrofagi TLR2-/- esprimono solo il TLR4, mentre i macrofagi TLR2+/+ sono positivi sia al TLR2 che al TLR4; in nessun caso, vi è modulazione di questi markers nei macrofagi esposti all’infezione con C. albicans.Conclusioni: Questi dati indicano che il TLR2 e il TLR4 hanno differente ruolo biologico; in particolare, il TLR2, e non il TLR4, è coinvolto nell’espletamento dell’attività anticandida da parte del macrofago, mentre la risposta secretoria sembra essere dipendente dal TLR4 e non dal TLR2.

2004 - Antiretroviral nucleoside and nucleotide analogues and mitochondria [Articolo su rivista]
Cossarizza, Andrea; G., Moyle
abstract

This review is intended to provide understanding ofthe function of mitochondria, the advantages anddisadvantages of available techniques for assessing mitochondrialfunction and quantity and to discuss the mainclinical toxicities thought to be associated with mitochondrialdysfunction and how they may be managedor their prevalence reduced.Mitochondria are the key organelles in energy productionin all human cells except erythrocytes. Energy, inthe form of ATP, is produced through the highlyefficient oxidative phosphorylation pathway. Additionally,mitochondria perform a range of other biologicalfunctions and carry a number of factors involved in cellapoptosis. Both HIV infection and antiretroviral nucleosideanalogues (nucleoside reverse transcriptaseinhibitors; NRTI) are known to affect mitochondrialDNA content and other aspects of mitochondrial function.A number of important clinical events occurringin individuals with HIV infection and on antiretroviraltherapy have been linked to mitochondrial injury anddysfunction. In vitro studies have demonstrated thatNRTI may differ in their effects on mitochondria andmay affect mitochondria in different cell lines in differentways. This is likely to influence the clinicalsyndromes associated with toxicity to these agents.Dideoxy-NRTI have the greatest affinity for mitochondrialDNA polymerase-ª, the enzyme responsiblefor mitochondrial DNA replication, whereas othernucleoside analogues may influence mitochondrialfunction also through other mechanisms. These differencesmay be important in choosing techniquesto evaluate the impact of antiretroviral agents onmitochondria.

2004 - Balanced regulation of mRNA production for Fas and Fas ligand in lymphocytes from centenarians: how the immune system starts its second century. [Articolo su rivista]
Pinti, Marcello; Troiano, Leonarda; Nasi, Milena; Ferraresi, Roberta; Mussi, Chiara; Bellodi, C; Salvioli, Gianfranco; Cossarizza, Andrea
abstract

The functionality of the immune system during aging is crucial for protection against the most common age-related diseases. Apoptosis plays a central role in the senescence of the immune system, as evidenced by the increased plasma membrane expression of a key molecule like Fas protein. We analyzed the mRNA levels of different forms of Fas (total [tFas] and membrane [mFas]) and of its ligand (FasL) in peripheral blood lymphocytes from centenarians, the best example of successful aging, who were compared with young and middle-aged donors.

2004 - Complementary and alternative medicine during HIV infection. [Relazione in Atti di Convegno]
Nasi, Milena; Pinti, Marcello; Troiano, Leonarda; Cossarizza, Andrea
abstract

According to the Joint United Nations Program of HIV/AIDS (UNAIDS) and the World Health Organization (WHO) (Joint United Nations Program of HIV/AIDS, 2001), as of the end of 2001, there were about 40 million adults and children living with human immunod-eficiency virus (HIV) infection. This total does not include the 20 million people around the world who already died of AIDS. Of the 40 million currently alive, 37.2 are adults, 17.6 are women, and more than 2.7 are children. In 2001, there were 5 million new cases of HIV infection in the world, and 3 million AIDS related deaths. The large majority (almost three quarters) live in Sub-Saharan Africa where the prevalence rate of the infection among adults is 8.4%; more than 55% of infected individuals are women. The second major pocket of HIV infection is in South and Southeast Asia, with more than 6 million people infected. In North America where the epidemic was first described, there are 940,000 individuals who are HIV- , and in Western Europe, 540,000. Furthermore, South America, China, and East-ern Europe are characterized by a rapid increase in infection rates. These dramatic numbers clearly indicate that the fight against HIV/AIDS is an absolute health, social, economical and political priority in all parts of the world.

2004 - Decreased apoptosis of bone marrow progenitor cells in HIV-1-infected patients during highly active antiretroviral therapy [Articolo su rivista]
A., Isgro; I., Mezzaroma; A., Aiuti; A., Fantauzzi; Pinti, Marcello; Cossarizza, Andrea; F., Aiuti
abstract

Impaired haematopoiesis during HIV-1 infection may be caused by the overproduction of inflammatory cytokines by immune cells at the bone marrow level inducing Fas-mediated apoptosis of stem progenitors. In this study, we evaluated the effects of highly active antiretroviral therapy on apoptosis of CD34+ stem cells derived from the bone marrow of HIV-1-infected patients, and observed decreased Fas expression on progenitor cells, in parallel with the diminution of TNF-alpha levels and the amelioration of clonogenic parameters.

2004 - Discontinuation of maintenance therapy for cryptococcal meningitis in patients with AIDS treated with highly active antiretroviral therapy: An international observational study [Articolo su rivista]
Mussini, Cristina; P., Pezzotti; J., MIRO' MEDA; JC LOPEZ BERNALDO DE, Quiros; E., Martinez; P., Cinque; V., Borghi; Bedini, Andrea; P., Domingo; P., Cahn; P., Bossi; A., DE LUCA; A., D'ARMINIO MONFORTE; M., Nelson; N., Nwokolo; S., Helou; R., Neuroni; G., Jacchetti; S., Antinori; A., Lazzarin; Cossarizza, Andrea; Esposito, Roberto; A., Antinori; Ja, Aberg
abstract

We conducted a retrospective, multicenter study evaluating the safety of discontinuing maintenance therapy for cryptococcal meningitis after immune reconstitution. Inclusion criteria were a previous definitive diagnosis of cryptococcal meningitis, a CD4 cell count of &gt;100 cells/muL while receiving highly active antiretroviral therapy (HAART), and the subsequent discontinuation of maintenance therapy for cryptococcal meningitis. The primary end point was relapse of cryptococcal disease. As of July 2002, 100 patients were enrolled. When maintenance therapy was discontinued, the median CD4 cell count was 259 cells/muL and the median plasma virus load was &lt;2.30 log(10) copies/mL, and serum cryptococcal antigen was undetectable in 56 patients. During a median follow-up period of 28.4 months (range, 6.7-64.5; 262 person-years), 4 events were observed (incidence, 1.53 events per 100 person-years; 95% confidence interval, 0.42-3.92). Three of these patients had a CD4 cell count of &gt;100 cells/mL and a positive serum cryptococcal antigen test result during the recurrent episode. In conclusion, discontinuation of maintenance therapy for cryptococcal meningitis is safe if the CD4 cell count increases to &gt;100 cells/mL while receiving HAART. Recurrent cryptococcal infection should be suspected in patients whose serum cryptococcal antigen test results revert back to positive after discontinuation of maintenance therapy.

2004 - Functional changes during apoptosis revealed by the simultaneous detection of mitochondrial membrane potential and DNA content by JC-1 and Hoechst 33342 [Abstract in Atti di Convegno]
Troiano, Leonarda; Lugli, E; Ferraresi, Roberta; Ost, V; Gualdi, E; Nemes, Elisa; Rossi, D; Cossarizza, Andrea
abstract

ApopThe study of functional changes that occur in intracellular organelles such asmitochondria during cell death/apoptosis are of consistent interest since severalyears. Probes exist that allow the analysis of a variety of mitochondrial (mt)parameters, including mt mass and mt membrane potential (MMP), along withthose that stain DNA and reveal its modifications. The dye JC-1, excited by theargon laser present in almost all flow cytometers, is considered the gold standardfor detecting MMP (Current Protocols in Cytometry, 9.14.1-9.14.7, 2000), andemits in FL-1 (monomers) and FL-2 (aggregates, whose formation is due by ahigh MMP). However, its spectrum of emission does not allow the use of otherdyes excitable by such laser since there is a relevant signal also in FL-3. Thus,until now, the simultaneous study of changes in DNA content and MMP duringapoptosis have been difficult if not impossible, and all data, including ours(Cytometry, 40: 189-197, 2000) have been obtained by indirect measures. In orderto investigate such parameters in the same cell, we took advantage of a novelinstrument, i.e. CyFlow ML by Partec (Germany), equipped with an air-cooledargon ion laser (488 nm, 200 mW), a UV Mercury lamp HBO (100 long life, 100W), a red diode laser (635 nm, 25 mW), a Nd:YAG laser (532 nm, 50 mW) and aCCD camera. Such configuration allowed us to use JC-1, excited by the Ar laser,along with Hoechst 33342 excited by the UV lamp. Apoptosis was induced inU937 or in CEM cell lines by a 48 hour incubation with 50 μM quercetin. Theanalysis of MMP allowed us to identify 3 different populations, which correspondedto three different stages of cell death. The first (about 50% of cells) hada normal MMP (high red and intermediate green fluorescence by JC-1), normalDNA content, FSC and SSC. In the second population (about 20%), MMP waschanging (high red and high green fluorescence), DNA content was normal whileFSC and SSC were those of apoptotic cells. In the third population (about 30%),MMP was consistently decreased (low red and high green fluorescence), DNAcontent was decreased (with the presence of the typical hypodiploic peak), FSCand SSC were altered. Thus, our data indicate that, in this model of apoptosis,cells start to loose MMP (as JC-1 aggregates begin to become monomers, causingan increase in green fluorescence) and change their physical parameter beforethe beginning of loss of nuclear DNA and the appearance of the hypodiploic peak.Studies are in course for the simultaneous detection of changes in mt mass (measuredby Mitotracker Deep Red) or other parameters (such as plasma membranepermeability or phosphatidylserine exposure) with dyes excited by the red laser.

2004 - HIV: no PUMA no death? [Articolo su rivista]
P., Salomoni; Cossarizza, Andrea
abstract

The presence of syncytia-inducing (SI) virus, and theformation of multinucleated cells (syncytia) is supposed toplay a major role in the progression of infection with the humanimmunodeficiency virus (HIV). SI viruses, which use thechemokine receptor CXCR4 to infect cells expressing theCD4 molecule (the main receptor for HIV, present on T-helperlymphocytes, monocytes and macrophages), are found morefrequently in patients with an advanced stage of the diseasethan the non-syncytia-inducing (NSI) type, which are usuallyisolated in the asymptomatic period of the infection. SI HIVisolates have a greater capacity to kill infected cells in culturethan do NSI strains, and the switch from NSI to SI isconsidered a marker of disease progression and an unfavourableprognostic sign. Formation of syncytia causes cell deathwith features of either apoptosis or necrosis. As far aslymphocytes or monocytes are concerned, syncytia areformed by the fusion of infected with uninfected CD4þ cells:in culture, their formation is one of the first signs of HIVinfection of peripheral blood mononuclear cells (PBMC),appearing 2–3 days after adding viral particles or infectedbiological material (such as cells or plasma). In T-cell linessuch as MT-2, the formation of syncytia can occur as early as2 h after the infection.1 Cell fusion is temperature-dependentand does not require DNA, RNA or protein synthesis. Itinvolves carbohydrates and lipids present on the cellmembrane and depends on CD4 and the HIV gp120 andgp41 envelope proteins.2As far as the progressive loss of CD4þ cells occurring inHIVþ patients is concerned, the real role of syncytiaformation is still a matter of debate. Indeed, evidence for thepresence of multinucleated cells in vivo is lacking except in thebrain.3 HIV infects and induces syncytium formation inmicroglia cells from the central nervous system (CNS).Macrophages/microglia cells are the main reservoir for HIVin the CNS, and multinucleated giant cells, the result of fusionof HIV-infected microglia and brain macrophages, are theneuropathologic hallmark of HIV dementia.The formation of a syncytium leads to a cytopathic effectwith a typical balloon degeneration of the cells, which iscaused by a variety of mechanisms. Among these, thoserelated to triggering of apoptotic pathways play a major role.The growing understanding of p53 function helped to establishits fundamental role in the regulation of apoptosis in differentcontexts. Many viruses encode proteins that can specificallyescape p53-mediated apoptotic programmes (i.e. adenovirus,human papilloma viruses, SV40, etc.).4 In contrast, HIV is ableto hijack the apoptotic machinery of the infected cell to induceits demise, possibly to facilitate virus spreading and counteracteither the innate or acquired immune response of thehost.5 Recent reports demonstrated that p53 and its targetgene Bax are involved in the induction of apoptosis triggeredby HIV in infected primary lymphocytes.6,7 The role of p53 inHIV-induced cell death has now been more firmly validated bya recent report by Perfettini et al.8 in the Journal ofExperimental Medicine. The same group had previouslydemonstrated the existence of a sequence of events duringsyncytia formation:9 (i) activation of mammalian target ofrapamycin (mTOR); (ii) mTOR-dependent phosphorylation ofp53 at serine 15 and the induction of its target gene Bax; (iii)activation of a mitochondrial death pathway. This process alsoresults in deregulated mitosis through the induction of cyclinB1. However, the exact mechanisms regulating these eventsare still unclear. To dissect the pathways involved in syncytiaformation, the authors analyse the transcriptional activities ofseveral transcription factors upon cell fusion. The activities ofp53, NF-kB and AP-1 are strongly enhanced by cell fusion,thus suggesting that these factors are functionally involved inthis process. Strikingly, AP-1 activity was abrogated upontransfecti

2004 - Highly active antiretroviral therapy restores CD4(+) V beta T-cell repertoire in patients with primary acute HIV infection but not in treatment-naive HIV+ patients with severe chronic infection [Articolo su rivista]
Cossarizza, Andrea; F., Poccia; C., Agrati; G., D'Offizi; R., Bugarini; Pinti, Marcello; V., Borghi; Mussini, Cristina; Esposito, Roberto; G., Ippolito; P., Narciso
abstract

In drug-naive HIV+ patients, we analyzed the effects of highly active antiretroviral therapy (HAART) on the reconstitution of the T-cell receptor (TCR) repertoire. We followed 2 groups of patients for 1 year: 18 individuals who experienced acute HlV infection and 24 patients who had HIV infection for many years but never took HAART. They were compared with 10 healthy controls who were longitudinally analyzed for the same period. We performed cytofluorometric analysis of the Vbeta TCR repertoire and detected the clonality of different Vbeta families by the spectratyping method. A new statistical approach based on the use of mixed models was then employed to analyze the data. Before the beginning of therapy, the repertoire of patients with acute or chronic infection was significantly different from that of healthy controls. After therapy, patients with acute HIV infection showed an improvement of the repertoire among either CD4(+) or CD8(+) T lymphocytes. Conversely, patients with chronic infection were capable of changing their repertoire among CD8(+) but not CD4(+) T lymphocytes. Our results indicate that HAART can restore the T-cell repertoire in individuals whose immune system is not severely compromised by the infection.

2004 - Mitochondria analysis for investigating toxicity of antiretroviral drugs. [Abstract in Atti di Convegno]
Troiano, Leonarda; Ferraresi, Roberta; Nemes, Elisa; Lugli, E; Rossi, D; Gualdi, E; Nasi, Milena; Galluzzi, Lorenzo; Prada, Nicole; Maffei, Stefania; Pinti, Marcello; Cossarizza, Andrea
abstract

Mitochondria are the key organelles in intracellular energy production, but performseveral other biologic functions and carry factors involved in cell apoptosis.Both HIV infection and antiretroviral nucleoside analogues (NRTI) can affectmitochondrial (mt) function and DNA content. Several side effects occurring inindividuals with HIV infection who are on antiretroviral therapy have been linkedto mitochondrial injury and dysfunction. Clinical studies of NRTIs, while collectingadverse event data, have not specifically evaluated mitochondrial toxicityin a systematic way. In vitro studies have demonstrated that NRTIs may differ intheir effects on mitochondria activities and may affect mtDNA content in differentcell lines in different ways, and several models are actually employed to betterunderstand not only the mechanism(s) of action of antiretroviral compounds,but also their possible side effects. Accordingly, it has been hypothesized thatclinical syndromes associated with toxicity to these agents are likely caused, atleast in part, by drug-related mitochondria alterations. Dideoxy-NRTIs have thegreatest affinity for mtDNA polymerase-γ, the enzyme responsible for mtDNAreplication, whereas other nucleoside analogues may influence mitochondrialfunction also through other mechanisms. By using cytometric assays to analyzemt or cell functionality (mt membrane potential by JC-1, mt mass by Mitotrackergreen, cell viability and apoptosis by classical stainings) along with originalmolecular biology techniques based upon real time PCR for determining mtDNAcontent, we have investigated the effects of different drugs commonly employedin anti-HIV therapy. We have used several human cell lines of different origin(lymphocytic such as CEM; monocytic, U937 or hepatocytic, HepG2). We foundthat the aforementioned parameters can be altered by antiretroviral drugs, with adifferent extent in the cell lines we studied. In general, stavudine seemed morepotent than zidovudine or didanosine in inducing damages to mt function ormtDNA. Antiretroviral drugs of the protease inhibitor category (such as indinavir)had no effects on any mt parameter we analyzed. From our experience, it can beconcluded that the choice of adequate molecular or cellular techniques and modelsis important in evaluating differences in drug activity that are related to theimpact of antiretroviral agents on mitochondria, as well as in understanding theside effects of such drugs on different cells, organs or systems.

2004 - Mitochondrial membrane potential and nucleosidic inhibitors of HIV reverse transcriptase: a cytometric approach. [Articolo su rivista]
Ferraresi, R; Troiano, L; Rossi, D; Gualdi, E; Lugli, E; Mussini, Cristina; Cossarizza, Andrea
abstract

Mitochondrial toxicity is a relevant side effect of anti-HIV antiretroviral therapy. Adequate experimental models and advanced technologies are crucial to investigate properly mitochondrial toxicity. Functional flow cytometry allows a rapid and sensitive evaluation of several parameters on single cells, and is an excellent tool to investigate the impact of antiviral drug on mitochondrial activity. We used such approach to analyze in vitro mitochondrial toxicity induced by stavudine and zidovudine on cell lines of different origin (hemopoietic: A301, U937, CEM, K562; hepatic: HepG2), and found that the cell lines had a different sensitivity to the action of the drugs.

2004 - Mutant PML proteins form novel cytoplasmic bodies. [Abstract in Atti di Convegno]
Bellodi, C; Kindle, K; Bernassola, F; Dinsdale, D; Melino, G; Cossarizza, Andrea; Heery, D; Salomoni, P.
abstract

The promyelocytic leukaemia protein PML is a tumor suppressor inactivated in acute promyelocytic leukaemia (APL). PML is essential for multiple apoptotic pathways and controls senescence induced by oncogenic activation. PML transcript undergoes alternative splicing to produce nuclear and cytoplasmic isoforms. The PML nuclear isoform IV interacts with p53 and potentiates its proapoptotic and growth suppressive functions. By contrast, the function of PML cytoplasmic isoforms is presently unknown. Two PML mutants have been identified in APL, which lack the nuclear localization signal and the p53 interaction domain. We found that both the wild type and mutant forms of cytoplasmic PML reside in cytoplasmic bodies (PML-CB), which rely on the formation of PML oligomers. Mutant PML specifically delocalizes the acetyl-transferase CBP into the PML-CB, while it does not affect the localization of other PML-NB components such as DAXX and Sp100. PML-RAR itself co-localizes and interacts with mutant PML and causes the delocalization of CBP into the PML-CB. Remarkably, mutant PML, like PML-RAR, inhibits RA-dependent transcription. Overall, these findings uncover a novel function of PML in the cytoplasm and shed new light on the molecular mechanisms of APL.

2004 - Studio comparativo di innesti autologhi da siti di prelievo diversi. [Abstract in Rivista]
Bertoldi, Carlo; Consolo, Ugo; Zaffe, Davide; Cossarizza, Andrea; Ceccherelli, G.
abstract

Scopo dello studio prospettico era la valutazione dello stato anatomo-funzionale e del comportamento degli innesti ossei prelevati da metafisi tibiale, cresta iliaca, sinfisi mentoniera e calvaria in rapporto alla tipologia degli interventi sui mascellari nell’ambito della chirurgia pre-protesica. Si sono scelte, a questo scopo, 2 tipologie di innesto: ad “onlay “ sul mascellare superiore e “particolato” per sinus lift. Al fine di inquadrare correttamente l’età ossea di riferimento per lo studio si è compiuta una valutazione preliminare su ossa di cadaveri utilizzando gli archivi del Dipartimento di Anatomia dell’Università di Modena e Reggio Emilia. Da tale studio si è evidenziata una grossa differenza nella strutturazione ossea tra l’individuo giovane e l’anziano (tranne che per la cresta iliaca in soggetti sani) per cui si è stabilito di restringere lo studio su soggetti sani in una fasca di età compresa tra i 20 ed i 65 anni. Valutazioni cliniche, radiologiche, istologiche e mediante tecniche biomolecolari (individuazione dei mRNA per le BMP-2 e BMP-7 mediante Real Time PCR) sono state compiute sugli innesti subito dopo il prelievo ed in fase di riapertura a scopo implantologico (circa 4 mesi dopo). Per quanto riguarda gli innesti ad “onlay” dal punto di vista istologico, istomorfometrico e biomolecolare pare regnare una situazione di rimodellamento osseo assai ridotta in tutti i casi e la produzione delle BMPs esaminate non pare essere significatamene differente tra i campioni neppure con stimolo mediante PRP. Dal punto di vista del Trabecular Bone Volume (TBV), invece, si distingue dagli altri l’innesto di origine mentoniera che appare significativamente più denso. I migliori risultati clinici che, secondo diversi studi, sono propri della calvaria in rapporto ad un suo migliore rimodellamento non possono essare ascritti a situazioni anatomiche di partenza né a differente attività di rimodellamento (quantitativamente parlando) o a una diversa situazione biomolecolare in rapporto alle BMP-2 & BMP-7.

2004 - The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans [Articolo su rivista]
Blasi, Elisabetta; Colombari, Bruna; Orsi, Carlotta Francesca; Pinti, Marcello; Troiano, L.; Cossarizza, Andrea; Esposito, Roberto; Peppoloni, Samuele; Mussini, Cristina; Neglia, Rachele Giovanna
abstract

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

2003 - Apoptosis-resistant phenotype in HL-60-derived cells HCW-2 is related to changes in expression of stress-induced proteins that impact on redox status and mitochondrial metabolism [Articolo su rivista]
S., Salvioli; G., Storci; Pinti, Marcello; Quaglino, Daniela; L., Moretti; M., Merlo Pich; G., Lenaz; S., Filosa; A., Fico; M., Bonafe; D., Monti; Troiano, Leonarda; Nasi, Milena; Cossarizza, Andrea; C., Franceschi
abstract

The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapic drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.

2003 - CD4-Guided treatment interruptions: a new possibile therapeutic strategy [Abstract in Atti di Convegno]
Mussini, Cristina; Cozzi Lepri, A.; Borghi, V.; D’Arminio Monforte, A.; De Luca, A.; Sighinolfi, L.; Ortolani, P.; Barchi, E.; Guaraldi, Giovanni; Bedini, A.; Bratt, G.; Erikson, L.; Antinori, A.; Cossarizza, Andrea; Esposito, Roberto
abstract

Aim of this study is to evaluate the safety of CD4 guided treatment interruptions.

2003 - Development of real time PCR assays for the quantification of Fas and FasL mRNA levels in lymphocytes: studies on centenarians [Articolo su rivista]
Pinti, Marcello; Troiano, Leonarda; Nasi, Milena; Monterastelli, Elena; L., Moretti; C., Bellodi; A., Mazzacani; Mussi, Chiara; Salvioli, Gianfranco; Cossarizza, Andrea
abstract

Apoptosis plays a central role in the homeostasis of the immune system. During aging, there is an altered regulation of pivotal molecules that are responsible for the regulation of this type of cell death, such as those of the Fas/FasL system. Understanding the regulation of these genes can help to clarify, at least in part, the age-related changes that occur in immune cells. We have developed an original real time PCR assay for quantification of mRNA for Fas and FasL, and have studied a group of young donors, middle aged subjects and centenarians. We have found that the production of Fas mRNA is greatly increased in resting lymphocytes from centenarians; such an increase follows an age-related trend. On the contrary, the production of mRNA for the molecule, which is the natural ligand of Fas, i.e. FasL, is consistently reduced. Our preliminary results suggest that during aging a subtle balance in the production of molecules that cause apoptosis could exist, and that, in order to avoid an excessive death of immune cells, a still unknown mechanism could compensate the increase of Fas with the reduction of FasL. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

2003 - Different sensitivity to apoptosis in cells of monocytic or lymphocytic origin chronically infected with human immunodeficiency virus type-1 [Articolo su rivista]
Pinti, Marcello; Biswas, P; Troiano, L; Nasi, Milena; Ferraresi, Roberta; Mussini, Cristina; Vecchiet, J; Esposito, Roberto; Paganelli, R; Cossarizza, Andrea
abstract

Apoptotic death of CD4+ T lymphocytes is a major cause of the immunodeficiency caused by human immunodeficiency virus (HIV), but it is still unclear how this process precisely occurs. To characterize a potentially useful cellular model, we have analyzed the tendency of chronically HIV-infected CD4+ human cell lines of different origin to undergo apoptosis. We studied ACH-2 and U1 lines, derived from the CD4+ T-cell A301 and the promonocytic U937 cell lines, respectively, and induced apoptosis via several stimuli that trigger different pathways. Their capacity to regulate plasma membrane CD95 expression and to produce soluble CD95 was also analyzed. Using staurosporine, TNF-alpha plus cycloheximide, and gamma-radiations, we observed that ACH-2 were more sensitive to programmed cell death than A301, while U1 were less sensitive than U937. Both infected cell types had a lower sensitivity to CD95-induced apoptosis; the analysis of changes in mitochondrial membrane potential corroborated these observations. Plasma membrane CD95 was similarly regulated in all cell types, which, however, presented a different capacity to produce soluble CD95 molecules. Our in vitro results may offer a new perspective for developing further studies on the pathogenesis of HIV infection. A chronically infected cell line of lymphocytic origin is more susceptible to apoptosis than its parental cell type, while infected monocytic cells are less sensitive than their uninfected counterpart. Thus, it is possible to hypothesize that one of the reasons by which circulating monocytes survive and represent a viral reservoir is the capacity of HIV to decrease the sensitivity to apoptosis of this cell type. However, further studies on ex-vivo collected fresh cells, as well as on other cell lines, are urgently needed to confirm such hypothesis.

2003 - Discontinuation of secondary prophylaxis for Pneumocystis carinii pneumonia in human immunodeficiency virus-infected patients: A randomized trial by the CIOP Study Group [Articolo su rivista]
Mussini, Cristina; Pezzotti, P; Antinori, A; Borghi, V; Monforte, A; Govoni, A; De Luca, A; Ammassari, A; Mongiardo, N; Cerri, Mc; Bedini, Andrea; Beltrami, C; Ursitti, Ma; Bini, T; Cossarizza, Andrea; Esposito, R.
abstract

This subgroup analysis assessing secondary prophylaxis for Pneumocystis carinii pneumonia (PCP) describes a multicenter, open-labeled, randomized, controlled trial evaluating the discontinuation of PCP prophylaxis. The main inclusion criterion was a history of PCP and an increase in the CD4 cell count to >200 cells/microL associated with receipt of highly active antiretroviral therapy for >or=3 months. The primary end point was the development of definitive or presumptive PCP. A total of 146 patients were enrolled (77 in the treatment discontinuation arm). After >2 years, 1 definitive and 1 presumptive case of PCP were observed, both of which occurred in patients who discontinued therapy. In most patients, secondary prophylaxis for PCP can be safely discontinued after potent antiretroviral therapy is initiated, but the threshold of >200 CD4 cells/microL may not be considered absolutely safe. Patients who present with symptoms after discontinuation of secondary prophylaxis should be evaluated for PCP despite high CD4 count and complete virus suppression.

2003 - Hepatoma HepG2 cells as a model for in vitro studies on mitochondrial toxicity of antiviral drugs: which correlation with the patient? [Articolo su rivista]
Pinti, Marcello; L., Troiano; Nasi, Milena; R., Ferraresi; J., Dobrucki; Cossarizza, Andrea
abstract

Currently, drugs have been synthesised that can significantly delay the course of several viral infections, including those provoked by HBV, HCV or HIV, but that display consistent side effects, including toxicity for organelles such as mitochondria. Several in vitro models and techniques have been developed to analyse the effects of such compounds. HepG2 cells (from human hepatoma) are an excellent model to investigate mitochondrial (mt) toxicity because of their high content of organelles and mtDNA, and actually different investigators are indeed using such cells. Studies in vitro on cell lines are relatively easy, but it is necessary to be careful. in the interpretation of data, which are usually obtained on continuously growing, tumour cells, quite different from normal, resting, non-neoplastic cells collected from a patient. Direct analysis of drug-induced mt damage in patients is extremely more complex than that performed using in vitro models because of the difficulty to obtain adequate cells or to have discrete amounts of biological material, the status of the patient at the moment of cell collection, the use of an adequate assay and its correct execution, and finally the possibility to find sex- and age-matched healthy controls as source of reference parameters.

2003 - Increased mitochondrial DNA content in peripheral blood lymphocytes from HIV-infected patients with lipodystrophy. [Articolo su rivista]
Cossarizza, Andrea; Riva, A; Pinti, Marcello; Ammannato, S; Fedeli, P; Mussini, Cristina; Esposito, R; Galli, M.
abstract

We have evaluated mitochondrial (mt) DNA content in CD4 and CD8 peripheral blood lymphocytes (PBLs) from HIV-infected patients taking highly active antiretroviral therapy (HAART) who display different types of adipose tissue alterations. A cross-sectional study was performed in a total of 23 patients with lipodystrophy (LD): nine patients with fat accumulation, six patients with fat loss, eight patients with combined form, who were compared to 11 individuals infected by HIV without LD (HIV-positive) and 10 seronegative controls (CTRL). PBLs were obtained by standard methods, that is, gradient density centrifugation on Ficoll, and CD4 or CD8 cells were positively isolated by magnetic sorting to eliminate the contamination of platelets. mtDNA content was then measured by an original assay based upon real-time PCR. mtDNA content was significantly increased in CD4 T cells from patients with LD, while no differences were present between CD4 and CD8 cells from HIV-positive and CTRL individuals. Nor were any differences found when comparing LD or HIV-positive patients treated with stavudine or zidovudine, or taking D-drugs or non D-drugs. Patients with fat accumulation had significantly higher mtDNA content compared to HIV-positive and CTRL, this phenomenon regarding both CD4 and CD8 PBLs. Considering all HIV-positive patients (including LD), mtDNA content showed a significant, positive correlation with cholesterolaemia but not with triglyceridaemia and glycaemia. Relatively high mtDNA content in LD patients, as well as the correlation between mtDNA content and cholesterol in all HIV-positive subjects, suggest the involvement of mitochondria in such a pathology. However, further studies are needed to confirm these initial observations and ascertain whether the quantification of mtDNA in PBL is a useful and reliable marker to investigate and monitor HAART-related changes in fat distribution.

2003 - Le tecniche per il dosaggio del DNA mitocondriale. [Articolo su rivista]
Cossarizza, Andrea; Pinti, Marcello
abstract

Nessun abstract

2003 - MDR1 C3435T genetic polymorphism does not influence the response to antiretroviral therapy in drug-naive HIV-positive patients [Articolo su rivista]
Nasi, Milena; V., Borghi; Pinti, Marcello; C., Bellodi; E., Lugli; S., Maffei; L., Troiano; Richeldi, Luca; Mussini, Cristina; Esposito, Roberto; Cossarizza, Andrea
abstract

P-glycoprotein, a membrane-localized protein transporter, codified by the MDR1 gene, influences the response to pharmacological treatments, including antiretroviral drugs. MDR1 polymorphism C3435T is correlated with the functionality of the protein. We investigated the influence of this polymorphism in the reconstitution of the peripheral C134 T cell pool in 149 drug-naive HIV-positive patients starting highly active antiretroviral therapy. The MDR1 C3435T polymorphism did not influence response to therapy, suggesting no disadvantages for individuals with a different genotype.

2003 - Placenta and cord blood mitochondrial DNA toxicity in HIV-infected women receiving nucleoside reverse transcriptase inhibitors during pregnancy. [Articolo su rivista]
B., Shiramizu; Km, Shikuma; L., Kamemoto; M., Gerschenson; G., Erdem; Pinti, Marcello; Cossarizza, Andrea; C., Shikuma
abstract

Recent studies of pregnant women and animal models have raised concerns regarding potentially serious mitochondrial toxicity-related side effects in infants born to mothers who received nucleoside reverse transcriptase inhibitors (NRTIs) during their pregnancy to prevent HIV-1 perinatal transmission. The aim of this study was to assess mitochondrial DNA (mtDNA) content of cord blood and placenta in HIV-infected pregnant women receiving NRTI compared with HIV-negative women, hypothesizing that placenta and cord blood mtDNA copies per cell would be decreased in women on NRTI therapy. Immediately following delivery, placenta and cord blood were obtained from eight HIV-infected pregnant women on NRTIs and five HIV-negative women. Assessment of mtDNA copies per cell was accomplished by quantitative real-time PCR. The mean mtDNA copies per cell from the placenta of the HIV-infected women compared with HIV-negative women was 152 +/- 119 and 880 +/- 136 ( =.0016), respectively. Similarly, the mean mtDNA copies per cell from the cord blood of the HIV-positive women compared with HIV-negative women was 144 +/- 101 and 865 +/- 331 ( =.0026), respectively. There was a statistically significant decrease in mtDNA copies per cell in placenta and cord blood between the HIV-infected women on NRTIs compared with HIV-negative women. Further studies are needed to better understand the morbidity to infants and mothers treated with NRTI to prevent vertical transmission of HIV.

2003 - Tests for mitochondrial function and DNA: potentials and pitfalls [Articolo su rivista]
Cossarizza, Andrea
abstract

In the past few years, mitochondria have been carefully studied to ascertain whether and how in patients affected by HIV antiretroviral therapy is able to alter their functionality and exert a toxic effect on immune cells, as well as on cells present in other districts. Recent findings. A variety of in-vivo and ex-vivo models have been developed to investigate the functionality of mitochondria and DNA during a variety of physiopathological situations, including HIV infection and its treatment. Numerous technologies are available to study at the single-cell or at the single-organelle level a variety of parameters, such as membrane potential, the activity of respiratory chain enzymes, and DNA content or its sequence. As far as in-vitro studies are concerned, a substantial homogeneity of data exists, and several changes in different mitochondrial parameters have been described that depend upon the drug used, the cell model and the parameter investigated. On the other hand, different results have been reported on biological material collected from HIV-positive patients and immediately analysed. Ex-vivo studies showed that changes in mitochondrial DNA content or in the functionality of the organelle exist in some tissues or cells, but not in others. Summary. One of the possible causes of the discrepancies is the technologies used to investigate mitochondria, and this paper summarizes some of the pros and cons of the main methods used to study mitochondrial function or DNA.

2002 - Age- and gender-related values of lymphocyte subsets in subjects from northern and southern Italy [Articolo su rivista]
G., Colonna Romano; Cossarizza, Andrea; A., Aquino; G., Scialabba; M., Bulati; D., Lio; G., Candore; G., Di Lorenzo; G., Frada; C., Caruso
abstract

Changes in the T-lymphocyte (identified by CD3 surface marker) compartment represent the most critical component of immunological aging. Many studies have demonstrated that the age-related decline in T-cell mediated immunity is accompanied by a reduced number of lymphocytes. Moreover, the enumeration of lymphocyte subsets is extensively used in the diagnosis and monitoring of various disease states. The use of flow cytometric technology and whole blood analysis by fluorochrome-labeled monoclonal antibodies is successfully applied for these purposes. It is so important to establish reference intervals in normal, healthy individuals of different ages using this technique. The present study was undertaken in order to establish age-related differences in lymphocyte subpopulations by simultaneously measuring common surface antigens in healthy individuals from several ages including very olds, taking into account the gender of the studied subjects. In particular, we analyzed 274 subjects separated in male and female groups, for the T helper (identified by CD4 surface marker) and cytotoxic cells (CD8), B (CD19) and NK (CD16) lymphocytes. In 138 individuals of different age and gender groups, we also evaluated the number of lymphocytes bearing the T-cell receptor gamma delta. Our data confirm the literary data on lymphocyte subpopulations and show that the reported differences in the number of CD16 and CD19 lymphocytes is not sex-related. The analysis was performed on the whole blood obtained from subjects from Northern and Southern Italy.

2002 - An observational study on patients treated or not for acute HIV infection. [Articolo su rivista]
Mussini, Cristina; Cozzi Lepri, A; Bedini, A; Borghi, V; Mongiardo, N; Cossarizza, Andrea; Esposito, R.
abstract

BACKGROUND: A vast majority of HIV-infected subjects who experience HIV acute seroconversion actually receive treatment. Open questions are how can we identify patients who will be slow progressors or long-term non progressors, and, as a consequence, do not require treatment. METHODS: An observational retrospective study on patients who experienced acute HIV seroconversion from August 1995 to June 2001, who are still alive and followed as outpatients at the Clinic of Infectious Diseases of Modena, Italy. RESULTS: Twelve patients were studied. Five patients (45.4%) were treated during acute seroconversion, while 7 were not treated. Two of these seven subjects received antiretroviral treatment 12 and 26 weeks after acute seroconversion. All the untreated patients were in good viro-immunological condition 6 months after seroconversion, and 2 of them also after 3 and 7 years. Patients who were treated showed a significant daily increase in CD4/CD8 T cell ratio with longer time spent on therapy (0.04% increase per day longer on antiretroviral therapy, p=0.02). CONCLUSIONS: This study suggests that treatment during primary HIV infection should not be considered in all patients. Randomized clinical trials enrolling patients with an asymptomatic primary HIV infection, with a high CD4 count and low HIV plasma viremia are needed to evaluate the indications for treatment in this subgroup of patients. On the other hand, this study confirms the good viro-immunological response obtained after treating patients during primary HIV infection.

2002 - CCF, an invertebrate analogue of TNF, is not related to the other lytic components from Eisenia foetida earthworm - Reply [Articolo su rivista]
Cooper, El; Kauschke, E; Cossarizza, Andrea
abstract

2002 - Centenarians in good health conditions [Articolo su rivista]
Motta, M.; Maugeri, D.; Malaguarnera, M.; Capurso, A.; Colacicco, A. M.; Solfrizzi, V.; Bonafe', M.; Barbi, C.; Gaddi, A.; D'Addato, S.; Sangiorgi, Z.; Trabucchi, M.; Boffelli, S.; Rozzini, R.; Rapisarda, R.; Tomasello, F. B.; Bennati, E.; Ferito, L.; Frantone, A.; Zoccolo, A.; Perticone, F.; Nardi, L.; Berardelli, M.; De Benedictis, G.; Falcone, E.; De Luca, M.; Casotti, G.; Monti, D.; Petruzzi, E.; Sorbi, S.; Grassi, E.; Latorraca, S.; Bertolini, S.; Agretti, M.; Costelli, P.; Nicita Mauro, V.; Basile, G.; Mari, D.; Duca, F.; Terrazzi, P.; Bosi, E.; Manzoni, M.; Salvioli, G.; Baldeli, M. V.; Neri, M.; Cossarizza, A.; Troiano, L.; Pini, G.; Varricchio, M.; Gambardella, A.; Prolisso, G.; Frada', G.; Barbagallo, M.; Pollina, R.; Passeri, M.; Sansoni, P.; Lavagetto, G.; Ferrari, E.; Battegazzore, C.; Molla, G.; Senin, U.; Cherubini, A.; Polidori, M. C.; Marigliano, V.; Bauco, C.; Borriello, C.; Deiana, L.; Carru, C.; Pes, G. M.; Baggio, G.; Forconi, S.; Boschi, S.; Guerrini, M.; Fabris, F.; Cappa, G.; Ferrario, E.
abstract

The increased survival of the population represents the most relevant demographic phenomenon during the end of the 20th century. Aging of the population is correlated with two factors: (i) reduction of the birth rate, and (ii) decrease of mortality rate. The progressive increase of the advanced age suggested the necessity of analyzing absolutely new aspects and facts, namely the properties of centenarians. The true centenarian subjects are in good clinical conditions, therefore, we can call them healthy centenarians. This is the reason why we can classify the centenarians in 3 groups on the basis of the criteria evaluating the clinical and physical conditions of them (groups A, B, C, being in good, moderate or poor clinical conditions, respectively). The healthy centenarian subject had overcome the negative environmental factors, and therefore, his/her survival is accompanied by a slow and gradual loss of the reserves of functional capacities, i.e., the exhaustion of the factors being the true determinants of the longevity.

2002 - Digging for innate immunity since Darwin and Metchnikoff [Articolo su rivista]
El, Cooper; E., Kauschke; Cossarizza, Andrea
abstract

Immune systems are, increasingly, being studied from comparative perspectives. The analysis of the immunedefense systems of invertebrates, such as fruit flies and earthworms, is an important part of this effort. These systems are innate, natural non-specific, non-anticipatory and non-clonal. This is in contrast to the macrophage T and B systems that characterize vertebrate adaptive immunity whose properties can be categorized as adaptive, induced, specific, anticipatory, and clonal. In this review, we will focus on the earthworm system. Earthworms, like other complex invertebrates, possess several leukocyte types and synthesize and secrete: a variety of immunoprotective molecules. The system as a whole effects phagocytosis, encapsulation, agglutination, opsonization, clotting and lysis of foreign components. At least two major leukocytes, small coelomocytes, and large coelomocytes mediate lytic reactions against several targets. Destruction of tumor cells in vitro shows that phagocytosis and natural killer cell responses are distinct properties of coelomocytes. A third type, the chlorogogen cell, synthesizes and sheds effector lytic molecules. Among the lytic molecules, three have been identified and sequenced (fetidins, CCF-1, lysenin) and another has been discovered (elseniapore), while three other molecules, H-1, H-2 H-3, share agglutinating and lysing functions. In contrast to these, Lumbricin I is the only known molecule of the earthworm system that is antimicrobial but non-lytic. Altogether the cellular and humoral components of the earthworm system function to distinguish between self and not self, dispose of internal (cancer?), damaged components and external antigens (microbes). The evolutionary context of the earthworm innate immune system is discussed at the end of this article.

2002 - Early changes in intramitochondrial cardiolipin distribution during apoptosis. [Articolo su rivista]
Garcia Fernandez, M.; Troiano, L.; Moretti, L.; Nasi, Milena; Pinti, Marcello; Salvioli, S.; Dobrucki, J.; Cossarizza, Andrea
abstract

Cardiolipin (CL) is essential for the functionality of several mitochondrial proteins. Its distribution between the inner and outer leaflet of the mitochondrial internal membrane is crucial for ATP synthesis. We have investigated alterations in CL distribution during the early phases of apoptosis. Using two classical models (staurosporine-treated HL-60 cells and tumor necrosis factor alpha-treated U937 cells), we found that in apoptotic cells CL moves to the outer leaflet of mitochondrial inner membrane in a time-dependent manner. This occurs before the appearance of apoptosis markers such as plasma-membrane exposure of phosphatidylserine, changes in mitochondrial membrane potential, DNA fragmentation, but after the production of reactive oxygen species. The exposure of a phospholipid on the outer surface during apoptosis thus occurs not only at the plasma membrane level but also in mitochondria, reinforcing the hypothesis of mitoptosis as a crucial regulating system for programmed cell death, also occurring in cancer cells after treatment with antineoplastic agents.

2002 - Efficacia del PRP nella riparazione dei difetti ossei. [Abstract in Rivista]
Consolo, Ugo; Bertoldi, Carlo; Ceccherelli, G.; Cossarizza, Andrea; Zaffe, Davide
abstract

L’attivita del platelet-rich plasma (PRP) sulla rigenerazione ossea è stata valutata su colture cellulari “in vitro” ed “in vivo”. Una volta compiuto un espianto osseo, parte di questo materiale, con il preventivo consenso informato del paziente, era posto in coltura cellulare con 2 modalità: coltivando l’intero trabecolato, senza estrarne la cellularità e coltivando la cellularità, estratta con metodi fisici dall’osso raccolto. In entrambi i casi si sviluppavano colture in terreni arricchiti o meno con PRP. Su tali espianti si eseguono valutazioni morfologiche, micromorfologiche, antigeniche, per mezzo della citofluorimetria, e biomolecolari, andando ad analizzare la produzione di mRNA per le BMP2 e 7 tramite la REAL TIME PCR. In vivo, il PRP era appilicato su una popolazione di 30 difetti cistici odontogeni apicali (resistenti alla terapia endodontica e di diametro * di 2cm) e di 12 siti atrofici mascellari bilaterali (grado IV-VI secondo Cawood e Howell) sovrapponibili. In alcuni difetti cistici il PRP è testato da solo come gel di riempimento, , addizionato ad osso autologo in altri difetti cistici ed in uno dei due seni mascellari. Tali interventi vengono confrontati tra loro ed in rapporto al solo osso autologo, rispettivamente, impiegato come riempitivo in altri difetti cistici e nei sinus lift nel seno mascellare controlaterale a quello in cui l’osso era utilizzato con PRP. I confronti, in questo campo, avvengono su base clinica, radiologica (completa di analisi densitometrica) ed istologica Le colture presentano risultati morfologici contrastanti nei due diversi casi in cui nelle fiasche si coltivi l’intero trabecolato o le sole cellule estratte dall’espianto osseo. Nel primo caso il PRP parrebbe favorire la formazione di tessuto calcifico; non così nel secondo caso in cui le colture hanno un aspetto del tutto sovrapponibile. I risultari micromorfologici ed antigenici citofluorimentrici parrebbero indicare la presenza di una popolazione cellulare attivata pro-osteogenetica nelle colture trattate con PRP. I risultati derivanti dalla REAL TIME PCR rilevano capacità di induzione delle BMPs studiate, da parte del PRP, presente ma non eclatante. I risultati “in vivo” sembrerebbero confermare la capacità del PRP di causare una accelerazione dei processi di guarigione e di osteogenesi nel caso in cui l’ambiente sia “condizionato” dalla presenza di tessuto osseo. I limiti temporali, tuttavia parrebbero essere molto importanti e l’azione del PRP sembrerebbe vantaggiosa fino a non oltre i 4/5 mesi dall’intervento di innesto. Tali risultati, che potrebbero essere contigui a quelli ottenuti “in vitro”, necessitano tuttavia di ulteriori studi e conferme.

2002 - Erratum: Digging for innate immunity since Darwin and Metchnikoff (Bioessays (2002) 24 (319-333)) [Articolo su rivista]
Cooper, E. L.; Kauschke, E.; Cossarizza, A.
abstract

2002 - Features of 'CD4-exploders', HIV-positive patients with an optimal immune reconstitution after potent antiretroviral therapy [Articolo su rivista]
Mussini, Cristina; Pinti, Marcello; Borghi, V; Nasi, Milena; Amorico, G; Monterastelli, E; Moretti, L; Troiano, Leonarda; Esposito, Roberto; Cossarizza, Andrea
abstract

Objective: To identify crucial immunological characteristics of a group of patients, defined ´CD4-exploders´, who were able to fully reconstitute their immune system after receiving highly active antiretroviral therapy (HAART). Patients: Among a population of 540 HIV-positive patients treated with HAART, six individuals were identified who experienced a nadir of less than 85 X 10(6) CD4+ cells/l, had major opportunistic infections (four out of six), started HAART in 1996 or 1997, and showed a rapid and relevant CD4+ lymphocyte increase (mainly due to virgin cells), in some cases regardless of virological control. Methods: Enzyme-linked immunosorbent assay for the determination of interleukin (IL)-7 plasma levels; flow cytometry to analyse surface antigens and CD127 (IL-7 receptor alpha-chain) expression; quantitative-competitive (QC) PCR for detecting cells containing T-cell receptor rearrangement excision circles (TREC) chest-computed tomography (CT) to analyse thymus volume and content. Results: In ´CD4-exploders´, high levels of TREC+ lymphocytes were found among CD4+ T cells, which also contained a high percentage of naive cells. However, CT revealed a dramatic depletion of intrathymic lymphoid tissue. Plasma levels of IL-7 were significantly high. Most CD4+ and CD8+ T lymphocytes expressed CD127, whose level was similar to that of healthy donors. CD127 expression on CD8+ lymphocytes was markedly higher than in HIV-positive individuals treated with the same therapy or in patients with AIDS. Conclusion: In ´CD4-exploders´, HAART-incluced reconstitution of the T-cell compartment is independent from thymus volume, and is favoured by the upregulation of the IL-7/IL-7 receptor system.

2002 - Genetic polymorphisms of Fas (CD95) and FasL (CD178) in human longevity: studies on centenarians [Articolo su rivista]
Pinti, Marcello; Troiano, Leonarda; Nasi, Milena; L., Moretti; E., Monterastelli; A., Mazzacani; Mussi, Chiara; Ventura, Paolo; F., Olivieri; C., Franceschi; Salvioli, Gianfranco; Cossarizza, Andrea
abstract

Apoptosis plays a crucial role in immunosenescence, as also evidenced by the increased expression of Fas in lymphocytes from aged people. However, little is known about the genetic regulation of Fas and its ligand, FasL. We have studied their polymorphisms in 50 centenarians and 86 young donors living in Northern Italy. The first Fas polymorphism; at position -670, has in Caucasian a heterozigosity of 51%; the second, at -1377 position, has the wild type allele (G) with a very high frequency (83%) respect to the mutant allele. Genotype and allele distribution for both polymorphisms were similar in controls and centenarians. Similar results were found as far as two FasL polymorphisms (IVS2nt-124 and IVS3nt169) are concerned. On the whole, our data suggest that Fas and FasL polymorphisms, as well as their haplotypes, are unlikely to be associated with successful human longevity.

2002 - Kinetics of CD4 cells after discontinuation of antiretroviral therapy in patients with virological failure and a CD4 cell count grater than 500 cells [Articolo su rivista]
Mussini, Cristina; Bugarini, R.; Perno, C. F.; Antinori, A.; Borghi, V.; Bertoli, A.; D`arrigo, R.; Bedini, A.; Cossarizza, Andrea; Esposito, Roberto
abstract

After a median of 37 months on antiretroviral therapy, 16 patients were asked to discontinue treatment instead of changing it. After a median observation time of 10.5 months, most patients experienced a rapid and progressive decrease in their CD4 cell count, even without a high viral load rebound. This decline was unrelated to the CD4 cell count and HIV-RNA values at interruption, but was more profound in patients in whom the M184V mutation had disappeared after lamivudine discontinuation.

2002 - Kinetics of CD4 cells after discontinuation of antiretroviral therapy in patients with virological failure and a CD4 cell count greater than 500 cells/μl [Articolo su rivista]
Mussini, Cristina; Bugarini, R; Perno, Cf; Antinori, A; Borghi, V; Bertoli, A; D'Arrigo, R; Bedini, A; Mongiardo, N; Cossarizza, Andrea; Esposito, R.
abstract

After a median of 37 months on antiretroviral therapy, 16 patients were asked to discontinue treatment instead of changing it. After a median observation time of 10.5 months, most patients experienced a rapid and progressive decrease in their CD4 cell count, even without a high viral load rebound. This decline was unrelated to the CD4 cell count and HIV-RNA values at interruption, but was more profound in patients in whom the M184V mutation had disappeared after lamivudine discontinuation.

2002 - Mitochondria and HIV infection: the first decade. [Articolo su rivista]
Cossarizza, Andrea; Troiano, Leonarda; Mussini, Cristina
abstract

In the last few years, the interactions between mitochondria and infection with the human immunodeficiency virus (HIV) have received careful attention. Starting from the first studies regarding the presence of mitochondrial damage in cardiac tissue from patients who died of AIDS, researchers have investigated different aspects of the interactions between the virus and mitochondria, from acute primary infection to the final stages of the disease. Only recently a significant impulse to this type of research has come from the observation that the so called "highly active antiretroviral therapy" (HAART), a combination of potent antiretroviral drugs such as viral protease inhibitors or nucleoside-analogue reverse-transcriptase inhibitors, is capable of damaging these organelles and cause a clinical syndrome called lipodystrophy. There is still an open debate concerning the exact responsibility of HAART as well as on metabolic pathways and mechanisms that are involved in the onset of lipodystrophy. The hypothesis that drug-induced damage to mitochondrial (mt) DNA is able to alter mitochondria functionality to a similar extent as that occurring in genetic disease affecting mtDNA suggests that mitochondria plays a crucial role in the pathogenesis of this syndrome. In this paper, data concerning the interactions between mitochondria and HIV infection will be reviewed.

2002 - Mitochondrial functionality and mitochondrial DNA content in lymphocytes of vertically infected human immunodeficiency virus-positive children with highly active antiretroviral therapy-related lipodystrophy. [Articolo su rivista]
Cossarizza, Andrea; Pinti, Marcello; Moretti, L; Bricalli, D; Bianchi, R; Troiano, Leonarda; Fernandez, Mg; Balli, Fiorella; Brambilla, P; Mussini, Cristina; Viganò, A.
abstract

Mitochondria functionality and apoptosis were studied in peripheral blood lymphocytes (PBL) of human immunodeficiency virus type 1-infected children, with or without lipodystrophy (LD), who were receiving highly active antiretroviral therapy (HAART) and in PBL of healthy control subjects (HCs). By flow cytometry, mitochondrial (mt) membrane potential, mt mass, intra-mt cardiolipin distribution, and early and late apoptosis in fresh PBL or in PBL cultured with different stimuli were assessed. mtDNA content was evaluated in fresh PBL by an original double-competitive quantitative polymerase chain reaction method, which enabled direct quantification of the number of mtDNA copies present in human lymphocytes. PBL from LD-positive and LD-negative children and from HCs were similar in mt functionality and in their tendency to undergo apoptosis. mtDNA content was also similar in PBL of LD-positive children and HCs, suggesting that normal mt functionality and normal tendency to undergo apoptosis are present in PBL of children with HAART-associated LD.

2002 - Mitochondrial functionality and mitochondrial DNA content in lymphocytes of vertically-infected HIV+ children with HAART-related lipodystrophy [Articolo su rivista]
Cossarizza, Andrea; Pinti, Marcello; Moretti, L.; Bricalli, D.; Bianchi, R.; Troiano, L.; Garcia Fernandez, M.; Balli, Fiorella; Brambilla, P.; Mussini, Cristina; Viganò, A.
abstract

Mitochondria functionality and apoptosis were studied in peripheral blood lymphocytes (PBL) of human immunodeficiency virus type 1-infected children, with or without lipodystrophy (LD), who were receiving highly active antiretroviral therapy (HAART) and in PBL of healthy control subjects (HCs). By flow cytometry, mitochondrial (mt) membrane potential, mt mass, intra-mt cardiolipin distribution, and early and late apoptosis in fresh PBL or in PBL cultured with different stimuli were assessed. mtDNA content was evaluated in fresh PBL by an original double-competitive quantitative polymerase chain reaction method, which enabled direct quantification of the number of mtDNA copies present in human lymphocytes. PBL from LD-positive and LD-negative children and from HCs were similar in mt functionality and in their tendency to undergo apoptosis. mtDNA content was also similar in PBL of LD-positive children and HCs, suggesting that normal mt functionality and normal tendency to undergo apoptosis are present in PBL of children with HAART-associated LD.

2002 - Skewed T-cell receptor repertoire, decreased thymic output, and predominance of terminally differentiated T cells in ataxia telangiectasia [Articolo su rivista]
Giovannetti, A.; Mazzetta, F.; Caprini, E.; Aiuti, A.; Marziali, M.; Pierdominici, M.; Cossarizza, Andrea; Chessa, L.; Scala, E.; Quinti, L.; Russo, G.; Fiorilli, M.
abstract

Ataxia telangiectasia (A-T), a genetic disorder caused by the homozygous mutation of the ATM gene, frequently associates with variable degrees of cellular and humoral immunodeficiency. However, the immune defects occurring in patients with A-T are still poorly characterized. Here we show that the T-cell receptor (TCR) variable beta (BV)-chain repertoire of 9 A-T patients was restricted by diffuse expansions of some variable genes prevalently occurring within the CD4 subset and clustering to certain TCRBV genes (eg, 5.1, 11, 14, and 23). In addition, the study of the third complementarity-determining region (CDR3) showed, in all patients, significantly altered profiles in most BV genes examined suggesting diffuse oligoclonal expansions. The sequencing of TCR CDR3 regions revealed completely normal V(D)J coding joints and confirmed a reduced diversity of the antigen-receptor repertoire. The B-cell repertoire was similarly restricted and skewed by diffuse oligoclonal expansions with normal V(D)J joints. Thymic output, evaluated by measuring TCR rearrangement excision circles, was extremely low. The majority of peripheral T cells had the phenotype and the function of effector memory cells, indicating that in vivo they are able to respond normally by terminal differentiation to antigenic stimulation. These results indicate that ATM mutation limits the generation of a wide repertoire of normally functioning T and B cells.

2002 - The evolution of cell killing: when a target cell became “invited” to choose how to die. [Relazione in Atti di Convegno]
Nasi, Milena; Pinti, Marcello; Troiano, Leonarda; Moretti, L.; Cooper, E. L.; Cossarizza, Andrea
abstract

During evolution, in the world of cytotoxic reactions a new mechanism has emerged that was based upon the direct "responsibility" of targets in their death. In other words, effector cells thought targets to use their own genetic material to trigger their suicide. To investigate this hypothesis, we have used an experimental model represented by Eisenia foetida coelomocytes that kill human cells, and asked whether coelomocytes can trigger apoptosis, or are able to provoke their death via necrosis, or even both, Using a strategy based upon a technique we have developed, that evaluates the expression of different forms of Fas (CD95/APO-1, i.e. the proapoptotic membrane form and the anti-apoptotic soluble form) mRNA in target cells, we tested the capacity of either coelomocytes from E. foetida or supernatant from E. foetida coelomocyte cultures to kill human cell lines of different origin. When target cells were incubated with coelomocytes, an upregulation of the membrane form of Fas occurred, along with an increase of the total form. On the contrary, cells treated with supernatant (containing cytotoxic molecules) had a significant reduction of the production of both forms of Fas mRNA, suggesting that mechanisms devoted to triggering of apoptosis were downregulated. Our data suggest that the production of soluble mediators, that could be considered the ancestors of humoral immunity, causes death of the foreign cell because of an aspecific activity of lytic molecules, that likely bind target membrane for physicochemical reasons, and determine necrosis. The development of cellular mechanisms to kill targets, i.e. likely representing the onset of cellular immunity, could act by modulating the expression of genes involved in apoptosis, determining an increase of total Fas expression.

2002 - Ultraviolet B radiation induces activation of neutral and acidic sphingomyelinases and ceramide generation in cultured normal human keratinocytes [Articolo su rivista]
Magnoni, Cristina; Euclidi, Emanuela; Benassi, Luisa; Bertazzoni, Giorgia; Cossarizza, Andrea; Seidenari, Stefania; Giannetti, Alberto
abstract

The sphingomyelin pathway is an ubiquitous, evolutionary conserved signaling system which transduces an extracellular signal into the cell. During the past few years increasing evidence has shown that the sphingolipid ceramide may play a role as a second messenger in intracellular signal transduction. The ceramide generation via sphingomyelinase (SMase) is followed by three major cellular responses: cell growth arrest, induction of cell differentiation and/or induction of programmed cell death or apoptosis. The aim of this study is to investigate whether activation of SMases and generation of ceramide can be induced by UVB radiation in normal human keratinocytes. The present data show that exposure to UVB radiation results in rapid generation of ceramide. The ceramide accumulation starts 15 min after UV exposure and progressively increases up to 24 h. In vitro measurement of SMase activity following exposure to UVB evidences an activation of both neutral and acidic SMases. Moreover, UVB induces apoptosis in normal human keratinocytes as shown by TUNEL technique and FACS analysis. These data indicate that UVB induced ceramide generation and activation of both neutral and acidic SMases, suggesting that sphingolipids metabolism may be involved in the UVB signaling pathway. (C) 2002 Elsevier Science Ltd. All rights reserved.

2001 - Analysis of mitochondria during cell death [Articolo su rivista]
Cossarizza, Andrea; Salvioli, S.
abstract

2001 - Annelid humoral immunity: Cell lysis in earthworms [Capitolo/Saggio]
Cooper, E. L.; Kauschke, E.; Cossarizza, A.
abstract

2001 - Chemokines, sTNF-Rs and sCD30 serum levels in healthy aged people and centenarians [Articolo su rivista]
R., Gerli; D., Monti; O., Bistoni; Am, Mazzone; G., Peri; Cossarizza, Andrea; M., Di Gioacchino; Mef, Cesarotti; A., Doni; A., Mantovani; C., Franceschi; R., Paganelli
abstract

Several lines of evidence point to a profound remodelling of the cytokine network in healthy elderly subjects, with decreased type-1 cytokine production (IL 2) and a shift to type 0 and 2. We have also observed an increase of proinflammatory cytokines (IL-1, IL-6, TNF-alpha) in vitro, and an increase of circulating stem cell factor in vivo. In this setting, we studied changes of chemokines (MCP-I and RANTES) with aging, as well as other molecules, namely, sTNF-RI and sTNF-RII, and the soluble form of the CD30 molecule (sCD30), involved in the pro- and antiinflammatory cytokine balance. The subjects enrolled in the study belonged to three different selected healthy groups of young, aged and centenarians. The presence of rheumatoid factor (RF) and antinuclear antibodies (ANA) was simultaneously assessed. The results show that MCP-1 serum levels were higher in the healthy aged and lowest in the young, while RANTES increased exclusively in centenarians. Only centenarians had autoantibodies (ANA and RF). sTNF-RI and sTNF-RII were significantly elevated in healthy old subjects compared to the young, and even higher in selected centenarians compared to the other age groups, sCD30 serum levels were significantly raised in centenarians compared to the young, despite absence of circulating CD30 + cells in the peripheral blood of the whole study population. No relationship among serum values of these different members of the TNF-R family was found, despite a strong correlation for sTNF-RI and sTNF-RII in all groups. We hypothesize that the increased chemokine levels in aged people, and raised sCD30 levels in centenarians, may reflect a general shift towards type 0/2 cytokines in normal aging, which may be responsible, at least in part, for the appearance of circulating autoantibodies without definite clinical consequences at advanced age. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

2001 - Decreased susceptibility to oxidative stress-induced apoptosis of peripheral blood mononuclear cells from healthy elderly and centenarians [Articolo su rivista]
Monti, D; Salvioli, S; Capri, M; Malorni, W; Straface, E; Cossarizza, Andrea; Botti, B; Piacentini, M; Baggio, Giosuè Gabriele; Barbi, C; Valensin, S; Bonafe, M; Franceschi, C.
abstract

The susceptibility to undergo apoptosis of fresh human peripheral blood mononuclear cells (PBMCs) from three groups of healthy donors of different ages: young people (19-40 years), old people (65-85 years) and centenarians was assessed. Apoptosis was induced by 2-deoxy-D-ribose (dRib), an agent which induces apoptosis in quiescent PBMCs by interfering with cell redox status and mitochondrial membrane potential (MMP). Our major finding is that an inverse correlation emerged between the age of the donors and the propensity of their PBMCs to undergo dRib-induced apoptosis. PBMCs from old people and centenarians also showed an increased resistance to dRib-induced glutathione depletion and a decreased tendency to lose MMP. The anti-apoptotic molecule Bcl-2 was similarly expressed in PBMCs from the three age groups. Moreover, the plasma level of the stable product of transglutaminase, epsilon(gamma -glutamyl)lysine isodipeptide, a marker of total body apoptotic rate, was decreased in centenarians compared to young and elderly people. On the whole, these findings suggest that physiological aging is characterised by a decreased tendency to undergo apoptosis, a phenomenon likely resulting from adaptation to lifelong exposure to damaging agents, such as reactive oxygen species, and may contribute to one of the major phenomena of immunosenescence, i.e. the progressive accumulation of memory/effector T cells. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

2001 - Direct analysis of mitochondrial toxicity of antiretroviral drugs [Articolo su rivista]
Foli, A.; Benvenuto, F.; Piccinini, G.; Bareggi, A.; Cossarizza, A.; Lisziewicz, J.; Lori, F.
abstract

Objectives: Mitochondrial toxicity is a serious side-effect of antiretroviral drugs, especially nucleoside reverse transcriptase inhibitors (NRTI). An in vitro assay to predict mithocondrial toxicity of in-use and developmental NRTI would be invaluable. To test the ability of a cytofluorimetric technique to predict the mitochondrial-dependent pancreatic and hepatic toxicity we used didanosine (ddl) alone or in combination with hydroxyurea (HU). Methods: The technique is based on the ability of the lipophilic cation JC-1 to enter selectively into mitochondria and change its colour as the membrane potential changes due to toxicity. Mitochondrial toxicity by HU and ddl was evaluated in pancreatic and hepatic human cell lines. The results were expressed as mitochondrial toxicity index (MTI), ranging from 0 to 100: the negative control was 0, and 100 indicating maximal toxicity. Results: Dose-dependent pancreatic toxicity of ddl was evident after 14 days of culture (MTI 34 ± 4 at 100 μM, 10 ± 4 at 10 μM, 2 ± 3 at 1 μM ddl). HU alone was not toxic (MTI 7 ± 10 at 100 μM, 2 ± 2 at 50 μM and 2 ± 4 at 10 μM HU); however, HU increased the toxicity of high, but not low, concentrations of ddl For example, the MTI of 10 μM ddl plus 50 μM HU was 54 ± 9. Negligible mitochondrial toxicity was observed in the hepatic cell line exposed to ddl alone or in combination with HU. Conclusions: This in vitro assay might have in vivo relevance. First, ddl-related pancreatitis is dose dependent, and is reported more frequently than hepatic failure, consistent with our in vitro results. Second, patients who developed pancreatitis during randomized, controlled trials were treated with HU in combination with 400 mg ddl once daily (high peak concentration of ddl in the blood). In contrast, no pancreatitis was observed when HU was combined with 200 mg ddl twice daily (low peak concentration of ddl). These in vivo results are consistent with our in vitro observation that HU increases pancreatic cell toxicity in the presence of high concentrations of ddl. The in vitro assay described here might be used to predict the mitochondrial toxicity of other NRTI, alone or in combination. © 2001 Lippincott Williams &amp; Wilkins.

2001 - Discontinuation of maintenance therapy for cryptococcal meningitis in HIV-1-infected persons receiving highly active antiretroviral therapy [Articolo su rivista]
Cossarizza, Andrea
abstract

Background. Little is known on whether HIV-infected persons with cryptococcal meningitis can discontinue maintenance therapy following HAART-induced immune-reconstitution. The present study is a longitudinal multicentre observational study which aims to determine the incidence of relapses of cryptococcal meningitis among persons undergoing HAART. Methods. The study was conducted by reviewing the clinical charts of HIV-positive adults who had ever attended, as consecutive outpatients, 10 hospital-based infectious-disease clinics located throughout Italy. We included all of the persons who had recovered from at least one episode of cryptococcal meningitis, and who were currently undergoing HAART. The study group consisted of those persons who had discontinued maintenance therapy for cryptococcal meningitis; the, control group consisted of those persons who were still undergoing maintenance therapy. Of the 45 study participants, 24 had discontinued maintenance therapy and 21 had continued. At the time of discontinuation, for the discontinuing participants, the mean CD4 cell count was 113.5 cells/mL, although 12 (50.0%) of them had less than 100 cells/mL; the mean plasma viral load was 2.48 log10 copies/mL; and 12 of them (50.0%) were seropositive for cryptococcal antigen. Results. At the end of the observation period, for the discontinuing participants, the mean CD4 count was 330 cells/mL (compared to 240 cells/mL for the continuing participants); the mean plasma viral load was 2.34 log10 copies/mL (compared to &lt;1.90 log10 copies/mL for the continuing participants); and 2 of the discontinuing participants were seropositive for cryptococcal antigen (compared to 13 continuing participants) (p&lt;0.01). After a mean observation time of 27.5 months for the discontinuing participants (57.9 person-years), no relapse of cryptococcal meningitis was observed or even suspected. Conclusions. This study suggests that persons receiving HAART could consider safety discontinuing maintenance therapy for cryptococcal meningitis, as has been previously described for other opportunistic infections.

2001 - Flow cytometric analysis of mitochondrial membrane potential using JC-1 [Capitolo/Saggio]
Cossarizza, Andrea
abstract

Changes in membrane potential have long been known to represent early activation events. This unit presents very recent developments in both fluorescent probes and functional applications and demonstrates the use of the JC-1 probe for measuring mitochondrial membrane potential by flow cytometry. A valuable component of this measurement system is the possibility of making quantitative measurements of changes in membrane potential.

2001 - Markers of cell death-activation in lymphocytes of vertically HIV-infected children naive to highly active antiretroviral therapy: the role of age. [Articolo su rivista]
Viganò, A; Pinti, Marcello; Nasi, Milena; Moretti, L; Balli, Fiorella; Mussini, Cristina; Bricalli, D; Sala, N; Bugarini, R; Vella, S; Principi, N; Cossarizza, Andrea
abstract

BACKGROUND: Apoptosis plays a major role in depleting CD4(+) lymphocytes during infection with HIV-1. Few data exist on its role during HIV infection of children. Sensitivity of peripheral blood lymphocytes (PBLs) to apoptotic stimuli and the importance of the patient's age remain unclear. OBJECTIVES: We sought to analyze the following: (1) markers of cell death-activation (CD95, CD45 isoforms, and CD28) in PBLs from vertically HIV-infected children of different ages before highly active antiretroviral therapy; (2) changes in other PBL populations; (3) PBL sensitivity to cell death and mitochondrial damages; and (4) role of age during progression of infection. METHODS: Cell culture techniques and flow cytometry were used to analyze surface antigens, PBL susceptibility to apoptosis, or PBL susceptibility to change of mitochondrial membrane potential. RESULTS: Donor age had a strong negative correlation with numbers of CD4(+) and CD8(+) T cells. Virgin T lymphocyte (CD45RA(+), CD95(-)) levels and those of CD95(+) cells showed no correlation with the children's clinical status but did show a correlation with patient age. CD28(-) T lymphocytes were markedly augmented in HIV-infected children but were unrelated to stage of infection or age. A relevant decrease in B lymphocytes and an increase in natural killer cells were also found. Finally, PBLs from HIV-positive children had a marked tendency to undergo apoptosis and mitochondrial damage. CONCLUSION: Changes in PBL phenotype, increased expression of CD95, and high sensitivity to apoptosis suggest that a precocious aging of the immune system occurs in HIV-infected children.

2001 - Mitochondria in the pathogenesis of lipodystrophy induced by anti-HIV antiretroviral drugs: actors or bystanders? [Articolo su rivista]
Cossarizza, Andrea; Mussini, Cristina; Viganò, A.
abstract

Effective therapies are now available that can stop the progression of HIV infection and significantly delay the onset of AIDS. The "highly active antiretroviral therapy" (HAART) is a combination of potent antiretroviral drugs such as viral protease inhibitors or nucleoside-analogue reverse-transcriptase inhibitors, that has a variety of serious side effects, including lipodystrophy, a pathology characterized by accumulation of visceral fat, breast adiposity, cervical fat-pads, hyperlipidemia, insulin resistance as well as fat wasting in face and limbs. There is still an open debate that concerns the precise responsibility of HAART as well as metabolic pathways and mechanisms that are involved in the onset of lipodystrophy. The similarities with multiple symmetric lipomatosis (MSL), in which mitochondria impairment plays a crucial role, lead to the hypothesis that drug-induced damages to mitochondrial DNA are able to alter mitochondria functionality to an extent that is similar to what occurs in MSL. In addition, several evidences indicate that HAART is also linked to a deregulated production of tumour necrosis factor-alpha, which uses mitochondria as intracellular targets. In this paper, we review data concerning the role of mitochondria in the pathogenesis of lipodystrophy, and advance a unifying hypothesis involving either direct or indirect effects of the drugs employed during HAART.

2001 - Nramp1 gene affects selective early steps in macrophage-mediated anti-cryptococcal defense [Articolo su rivista]
Blasi, Elisabetta; Colombari, Bruna; Mucci, Anna; Cossarizza, Andrea; Radzioch, D; Boelaert, Jr; Neglia, Rachele Giovanna
abstract

Cryptococcus neoformans is an opportunistic fungus responsible for severe and often recurrent meningoencephalitis in immunodepressed patients. Initial evidence suggests that C. neoformans is a facultative intracellular pathogen; however, the strategies by which C. neoformans undergoes survival and eventually proliferation have not been elucidated. We investigated the role of Nrampl gene in macrophage-mediated anti-cryptococcal defense. Using cell lines expressing the functional, mutated or knockout gene, it was established that Nramp1 (1) is not involved in the phagocytic event, (2) influences anti-cryptococcal activity in the early steps but not at later times, and (3) is unrelated to the biomolecular pathways through which C. neoformans impairs macrophage secretory response. Although the functional role of Nramp1 is still far from being elucidated, the present data add insight into its involvement in macrophage-mediated antimicrobial defense, particularly in the initial steps allowing C. neoformans growth inhibition

2001 - PRP nel sinus lifting e nel trattamento dei difetti cistici. [Abstract in Atti di Convegno]
Consolo, Ugo; Bertoldi, Carlo; Ceccherelli, G.; Cossarizza, Andrea; Zaffe, Davide
abstract

Gli autori hanno studiato una popolazione di pazienti che sarebbero stati sottoposti a procedura di enucleazione cistica (cisti odontogene di tipo flogistico apicale, con diametro *2cm e resistenti alla sola terapia endodontica) ed una popolazione selezionata di soggetti che avrebbero dovuto sottoporsi a sinus lift bilaterale per sanare situazioni bilateralmente sovrapponibili di atrofia di 4-6 grado secondo Cawood e Howell. Una tipologia di sperimentazione avveniva in un ambito favorevole alla genesi ossea (lesione cistica) mentre l’altra tipologia di intervento veniva eseguita in una posizione sfavorevole. Nello stesso tempo la bilateralità dell’intervento di quest’ultima serie (sinus lift) dava la possibilità di confrontare sullo stesso soggetto, quindi con una forte epicriticità di osservazione, le differenze indotte dal Platelet-rich Plasma (PRP). Infatti la procedura di sinus lift sarebbe stata eseguita da un lato con solo innesto osseo autologo mentre, nel controlaterale, l’innesto osseo sarebbe stato addizionato con PRP. Scopo del presente lavoro era analizzare la capacità del PRP di indurre tessuto osseo e di stabilizzarlo in sede. I nostri risultati, sebbene preliminari, sembrerebbero indicare un’accelerazione, indotta dal PRP, dei processi di rigenerazione tissutale. Tale costituente sarebbe in grado di agire in modo generalizzato su tutti i tessuti di tipo connettivale, almeno, stimolandone la crescita. Codice: IT\ICCU\BVE\0248681

2001 - Sinus lifting and cystic bone defects treated with platelet-rich plasma. [Abstract in Rivista]
Consolo, Ugo; Bertoldi, Carlo; Zaffe, Davide; Cossarizza, Andrea; Ceccherelli, G.; Bellini, Pierantonio
abstract

Vedi allegato (in Inglese) Riassunto italiano: Da una popolazione di pazienti che si sottopone a procedure di innesto osseo, previo consenso informato, vengono prelevati alcuni cc aggiuntivi di osso. Tali campioni vengono ad essere coltivati in vitro in presenza o, rispettivamente, in assenza di Platelet-rich Plasma. La cellularità ossea, tuttavia, non veniva separata dalle rispettive trabecole con cui, invece, veniva incubata. Le diverse colture, una volta giunte a confluenza, vengono ad essere analizzate e, quindi, distaccate dalle fiasche di coltura. La cellularità ottenuta è, quindi, marcata con appositi anticorpi ed è analizzata al citofluorimetro per ottenerne dati micromorfologici ed immunoistochimici. Dal punto di vista clinico, il PRP viene testato in rapporto a difetti cistici odontogeni apicali (resistenti alla terapia endodontica e di diametro * di 2cm) e di siti atrofici mascellari bilaterali (grado IV-VI secondo Cawood e Howell) sovrapponibili. Il PRP è testato da solo come gel di riempimento, in alcuni difetti cistici, addizionato ad osso autologo, in altri difetti cistici ed in uno dei due seni mascellari. Tali interventi vengono confrontati tra loro ed in rapporto al solo osso autologo, rispettivamente, impiegato come riempitivo in altri difetti cistici e nei sinus lift nel seno mascellare controlaterale a quello in cui l’osso era utilizzato con PRP. Scopo del lavoro era precisare gli effetti del PRP in rapporto alla rigenerazione ossea. I risultati ottenuti dimostrano la capacità, in vitro, del PRP di generare colture complete di tessuto calcifico ove, diversamente, si ottengono quasi esclusivamente fibroblasti. Micromorfologicamente, la cellularità coltivata con PRP presenta una volumetria ed una densità cellulare comparabile a quella osteoblastica ed anche il mosaico antigenico rilevato, ed in particolare la glicoproteina CD38, fanno pensare ad un’attivazione cellulare probabilmente in tal senso. I rilievi clinico/radiologici (anche densitometrici) ed istologici fanno pensare che il PRP possa supportare, prevalentemente processi di accelerazione della neorigenerazione tissutale. Codici: ISSN: 0177-3348 (Print) - 0177-3348 (Linking) – Altre ID: (DNLM)SR0058226(s) - (OCoLC)28226223 NLM ID: 9426456 [Serial]

2001 - T cell responses to highly active antiretroviral therapy defined by chemokine receptors expression, cytokine production, T cell receptor repertoire and anti-HIV T-lymphocyte activity [Articolo su rivista]
A., Giovannetti; M., Pierdominici; F., Mazzetta; S., Salemi; M., Marziali; D., Kuonene; F., Iebba; Ea, Lusi; Cossarizza, Andrea; F., Aiuti
abstract

The immunological correlates of highly active antiretroviral therapy (HAART)-induced suppression of human immunodeficiency virus type 1 (HIV-1) replication have been investigated. 20 HIV-1-infected patients with mean CD4(+) T cell count of 298/mul, plasma viral load of 4.7 log(10) copies/ml and naive for protease inhibitors (PI) were studied during 12 months of HAART. An increased number of both CD4(+) and CD8(+) naive T cells and a normalization of the frequency of CCR5- and CXCR4-expressing CD4(+) T cells were readily observed after starting therapy. Single cell analysis of cytokine production after 12 months of HAART showed an increased number of interleukin (IL)-2-, but not IIL-4- and (IFN)-gamma-, producing T cells and a decreased percentage of CD8+ TFN-gamma + cells. A correlation between the frequency of IFN-gamma-producing T cells and that of memory, CCR5(+) and CD95(+) T cells was demonstrated in both CD4(+) and CD8(+) subsets. The diversity of T cell receptor (TCR) variable beta (BV) chain repertoire significantly increased after 12 months of HAART within the CD4(+) but not the CD8(+) T cell subset. However, the level of perturbation of the third complementarity-determining region (CDR3), was not significantly modified by effective therapy. The number of anti-HIV Gag and Pol cytotoxic T lymphocytes precursors (CTLp) decreased during HAART and highly correlated with the CD8 IFN-gamma response. Ameliorated clinical conditions were observed in all patients in absence of any opportunistic infections during all the study period. These observations indicate that a better restoration of immunity may be obtained in patients starting HAART at less advanced stages of the disease.

2000 - A cytofluorimetric study of T lymphocyte subsets in rat lymphoid tissues (thymus, lymph nodes) and peripheral blood: a continuous remodelling during the first year of life [Articolo su rivista]
M., Capri; Quaglino, Daniela; G., Verzella; D., Monti; M., Bonafe; Cossarizza, Andrea; Troiano, Leonarda; L., Zecca; Ronchetti, Ivonne; C., Franceschi
abstract

We previously demonstrated that the rat thymus undergoes a progressive remodelling long before the appearance of typical signs of involution [Quaglino, D., Capri, M., Bergamini, G., Euclidi, E., Zecca, L., Franceschi, C., Pasquali Ronchetti, I., 1998. Age-dependent remodelling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age. Eur. J. Cell. Biol. 76, 156-166]. To focus better on the complex remodelling that occurs in the rat immune system during the first year of life, we analysed the phenotype profile of thymocytes, and T lymphocytes from mesenteric lymph nodes and peripheral blood of the same animals by flow cytometry. Two experimental sets were performed simultaneously using the same animal strain, but starting and ending the study at different ages (15 days up to 300 days in the first experimental set, and 90 days up to 360 days of life in the second). In the rat these ages appear to be crucial not only for developmental, maturative and early involutional processes of the thymus, but also of the entire immune system. The main findings were the following: (1) in the thymus, CD8(-)CD3(-) cells increased, CD5(+) alpha beta TCR- and CD8(+)CD4(+) thymocytes decreased, while the most mature cell subset appeared well preserved with ageing; (2) in the lymph nodes, T helper and T cytotoxic lymphocytes decreased in the most aged animals. Memory/activated CD4(+)CD45RC(-) T cells decreased, while naive/resting CD4(+)CD45RC(+) cells increased in the youngest animals and decreased in the oldest. CD8(+)CD45RC(-) and CD8(+)CD45RC(+) lymphocytes showed a complex age-dependent trend, and (3) in peripheral blood, minor modifications were evident, such as an age-dependent increase in the alpha beta TCR(+)CD25(+) cell subset. Some of these changes were related to the developmental process, while others could likely be interpreted as early signs of immunosenescence. The role of these modifications in immune system is discussed within the framework of the remodelling hypothesis of immunosenescence. The age-dependent changes in these three lymphoid compartments, however, appear to be different and only partially overlapping, thus suggesting that the maturational, developmental and ageing processes have distinct characteristics in the central and peripheral lymphoid organs. (C) 2000 Elsevier Science Inc. All rights reserved.

2000 - Ceramide 2 (N-acetyl sphingosine) is associated with reduction in Bcl-2 protein levels by Western blotting and with apoptosis in cultured human keratinocytes [Articolo su rivista]
A., Di Nardo; Benassi, Luisa; Magnoni, Cristina; Cossarizza, Andrea; Seidenari, Stefania; Giannetti, Alberto
abstract

Background Ceramides produced by sphingomyelin hydrolysis activate a cycle that is followed by three different major cellular responses: downregulation of cell proliferation, induction of cell differentiation and apoptosis. In the skin, the generation of intracellular ceramide may also provide a link between an extracellular signal and the induction of the apoptosis programme for the elimination of damaged cells. Objectives We investigated the effect of ceramides capable of entering cells on cultured keratinocytes. Methods Human keratinocytes from neonatal skin were cultured in serum-free medium with or without increasing concentrations of ceramide 2 (CER-2; N-acetyl sphingosine) (5, 10, 20 and 40 mu mol L-1). Proliferative effects were studied either by cell counts or by H-3-thymidine incorporation and flow cytometric analysis. Apoptosis was studied by TUNEL staining and Western blot analysis of Bcl-2 protein. Results Cell counts and DNA synthesis were reduced in a dose-dependent manner following CER-2 treatment. TUNEL staining showed CER-2-induced apoptosis at 48, 72 and 96 h. Western blot analysis showed that CER-2 induces downregulation of Bcl-2, at 24-96 h. Conclusions These results demonstrate that CER-2 inhibits cell proliferation and induces apoptosis, possibly via a Bcl-2-dependent mechanism.

2000 - Changes in intramitochondrial cardiolipin distribution in apoptosis-resistant HCW-2 cells, derived from the human promyelocytic leukemia HL-60. [Articolo su rivista]
Garcia Fernandez, M.; Troiano, L.; Moretti, L.; Pedrazzi, J.; Salvioli, S.; Castilla Cortazar, I.; Cossarizza, Andrea
abstract

Using a cytofluorimetric approach, we studied intramitochondrial cardiolipin (CL) distribution in HCW-2 cells, an apoptosis-resistant clone of human HL-60 cells. In HL-60, about 50% of total CL is distributed in the outer leaflet of mitochondrial inner membrane, while in HCW-2 a significantly higher amount of CL (about 65%) is in that site. In basal conditions, HSW-2 cells also show a reduced mitochondrial membrane potential even if they are able to proliferate as the parental line. Taking into account the complex functions that CL plays in the regulation of mitochondrial activity, it is likely that HCW-2 could produce ATP utilizing more glycolytic pathways rather than mitochondrial respiratory chain.

2000 - Deregulation of the CD95/CD95L system in lymphocytes from patients with primary acute HIV infection [Articolo su rivista]
Cossarizza, Andrea; Stent, G; Mussini, Cristina; Paganelli, R; Borghi, V; Nuzzo, C; Pinti, Marcello; Pedrazzi, J; Benatti, F; Esposito, Roberto; Røsok, B; Nagata, S; Vella, S; Franceschi, C; DE RIENZO, Bruno
abstract

OBJECTIVE: To analyze the role of CD95/CD95 ligand (CD95L) expression and functionality in peripheral blood lymphocytes (PBL) during primary, acute HIV syndrome (AHS) and in the subsequent period. PATIENTS: Twelve patients were studied during the acute phase of the viral infection and most were followed for some months. METHODS: Cell culture and cytotoxicity assays based upon 51Cr release and flow cytometry were used to evaluate cell killing via CD95 molecule, flow cytometry to assess surface antigens, enzyme-linked immunosorbent assay (ELISA) for the determination of soluble CD95 and CD95L plasma levels, quantitative competitive (QC) reverse transcription polymerase chain reaction (RT-PCR) with an original RNA competitor for the analysis of CD95L mRNA expression and QC RT-PCR for determining plasma viral load. RESULTS: The analysis of PBL during this phase revealed that almost all cells, including CD8 T cells with a virgin phenotype, B lymphocytes and natural killer cells displayed CD95 molecules on the plasma membrane. Activation of CD95 on the surface of isolated lymphocytes by anti-CD95 monoclonal antibodies or binding to CD95L induced rapid apoptosis. However, CD95L mRNA was not expressed in PBL from these patients and was poorly inducible. Soluble CD95 was found in the plasma of all patients, but only in a few at high levels, even some months after seroconversion. In contrast, soluble CD95L was detected in only one patient, this occurring after the symptomatic period. For 10 of the 12 patients, expression of CD95 on the cell membrane or in the plasma did not correlate with the plasma viral load, which varied widely from patient to patient. Further, plasma levels of soluble CD95 were not altered by decreased lymphocyte activation or by efficient antiretroviral therapy. CONCLUSIONS: In patients experiencing an acute, primary HIV infection, a prolonged deregulation of the CD95/CD95L system may exist, which is probably not entirely related to virus production but may contribute to the pathogenesis of the disease. The hypothesis can be put forward that a complex balance exists between proapoptotic events (increase in CD95 expression), probably triggered by the host as a method to limit viral production, and antiapoptotic events (decrease in CD95L expression) probably triggered by the virus as a way to increase its production and survival.

2000 - Discontinuation of primary prophylaxis for Pneumocystis carinii pneumonia and toxoplasmic encephalitis in human immunodeficiency virus type I-infected patients: The changes in opportunistic prophylaxis study [Articolo su rivista]
Mussini, Cristina; P., Pezzotti; A., Govoni; V., Borghi; A., Antinori; A. D., Monforte; A., De Luca; N., Mongiardo; M. C., Cerri; F., Chiodo; E., Concia; L., Bonazzi; M., Moroni; L., Ortona; Esposito, Roberto; Cossarizza, Andrea; B., De Rienzo
abstract

A multicenter open, randomized, controlled trial was conducted to determine whether primary proyhylaxis for Pneumocystis carinii pneumonia and toxoplasmic encephalitis can be discontinued in patients infected with human immunodeficiency virus type 1 (HIV-1) whose CD4(+) T cell counts have increased to &gt;200 cells/mm(3) (and who have remained at this level for at least 3 months) as a result of highly active antiretroviral therapy (HAART), Patients were randomized to either the discontinuation arm (i.e., those who discontinued prophylaxis; 355) or to the continuation arm (n = 353); the 2 arms of the study were similar in terms of demographic, clinical, and immunovirologic characteristics. During the median follow-ups of 6.4 months (discontinuation arm) and 6.1 months (continuation arm) and with a total of 419 patient-years, no patient developed P. carinii pneumonia or toxoplasmic encephalitis. The results of this study strongly indicate that primary prophylaxis for P. carinii pneumonia and toxoplasmic encephalitis can be safely discontinued in patients whose CD4(+) T cell counts increase to &gt;200 cells/mm(3) during HAART.

2000 - Do men and women follow different trajectories to reach extreme longevity? [Articolo su rivista]
Franceschi, C.; Motta, L.; Valensin, S.; Rapisarda, R.; Franzone, A.; Berardelli, M.; Motta, M.; Monti, D.; Bonafe, M.; Ferrucci, L.; Deiana, L.; Pes, G. M.; Carru, C.; Desole, M. S.; Barbi, C.; Sartoni, G.; Gemelli, C.; Lescai, F.; Olivieri, F.; Marchegiani, F.; Cardelli, M.; Cavallone, L.; Gueresi, P.; Cossarizza, A.; Troiano, L.; Pini, G.; Sansoni, P.; Passeri, G.; Lisa, R.; Spazzafumo, L.; Amadio, L.; Giunta, S.; Stecconi, R.; Morresi, R.; Viticchi, C.; Mattace, R.; De Benedictis, G.; Baggio, G.
abstract

Gender accounts for important differences in the incidence and prevalence of a variety of age-related diseases. Considering people of far advanced age, demographic data document a clear-cut prevalence of females compared to males, suggesting that sex-specific mortality rates follow different trajectories during aging. In the present investigation, we report data from a nationwide study on Italian centenarians (a total of 1162 subjects), and from two studies on centenarians living in two distinct zones of Italy, i.e., the island of Sardinia (a total of 222 subjects) and the Mantova province (Northern Italy) (a total of 43 subjects). The female/male ratio was about 2:1 in Sardinia, 4:1 in the whole of Italy, and about 7:1 in the Mantova province. Thus, a complex interaction of environmental, historical and genetic factors, differently characterizing the various parts of Italy, likely plays an important role in determining the gender-specific probability of achieving longevity. Gender differences in the health status of centenarians are also reported, and an innovative score method to classify long-lived people in different health categories, according to clinical and functional parameters, is proposed. Our data indicate that not only is this selected group of people, as a whole, highly heterogeneous, but also that a marked gender difference exists, since male centenarians are less heterogeneous and more healthy than female centenarians. Immunological factors regarding the age-related increase in proinflammatory status, and the frequency of HLA ancestral haplotypes also show gender differences that likely contribute to the different strategies that men and women seem to follow to achieve longevity. Concerning the different impact of genetic factors on the probability of reaching the extreme limits of the human lifespan, emerging evidence (regarding mtDNA haplogroups, Thyrosine Hydroxilase, and IL-6 genes) suggests that female longevity is less dependent on genetics than male longevity, and that female centenarians likely exploited a healthier life-style and more favorable environmental conditions, owing to gender-specific cultural and anthropological characteristics of the Italian society in the last 100 years. (C) 2000, Editrice Kurtis.

2000 - Hemopoiesis in healthy old people and centenarians: Well-maintained responsiveness of CD34+ cells to hemopoietic growth factors and remodeling of cytokine network [Articolo su rivista]
Bagnara, G. P.; Bonsi, L.; Strippoli, P.; Bonifazi, F.; Tonelli, R.; D'Addato, S.; Paganelli, R.; Scala, E.; Fagiolo, U.; Monti, D.; Cossarizza, A.; Bonafe, M.; Franceschi, C.
abstract

In vitro hemopoiesis and hemopoietic cytokines production were evaluated in 9 centenarians (median age 100.5 years, age range: 100-104 years), 10 old people (median age: 71 years, age range: 66-73 years), and 10 young people (median age: 35 years, age range: 30-45 years), all carefully selected for their healthy status. The main findings were the following: (i) a trend towards a decreased absolute number of CD34+ progenitor cells in the peripheral blood of old people and centenarians, in comparison to young subjects; (ii) a well-preserved capability of CD34+ cells from old people and centenarians to respond to hemopoietic cytokines, and to form erythroid (BFU- E), granulocyte-macrophagic (CFU-GM), and mixed colonies (CFU-GEMM) in a way (number, size, and morphology) indistinguishable from that of young subjects; (iii) an age-related decreased in vitro production of granulocyte-macrophagic colony-stimulating factor (GM-CSF) and a decreased production of interleukin- 3 (IL-3) in centenarians by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC); (iv) a linear increase of the serum level of stem cell factor (SCF), measured in the above-mentioned subjects and in 65 additional subjects, including 4 centenarians. These data suggest that basal hematopoietic potential is well preserved in healthy centenarians, and that the hemopoietic cytokine network undergoes a complex remodeling with age.

2000 - Mitochondria and apoptosis: Functional studies on membrane potential (Δψ) [Articolo su rivista]
Cossarizza, A.
abstract

Apoptosis is a phenomenon of crucial importance in all multicellular, living organisms: it is involved in several physiological and pathological phenomena which incluse embryogenesis, differentiation, development of the immune system, as well as control of cell growth, cancerogenesis, viral production. The assumption that apoptosis is an active process led to study mitochondria, the main intracellular source of energy, which is produced in form of ATP. It is reasonable to hypothesize that, at least in early apoptotic phases, cells have to maintain a good driving force for ATP synthesis, and, consequently, a high mitochondrial membrane potential (Δψ), that becomes a reliable mirror of the capacity to synthesize ATP and, in general, of mitochondrial membrane functionality. In this article, the use of different fluorescent probes for the analysis of Δψ is discussed paying a particular attention for JC-1, that is considered the golden standard for this type of measure.

2000 - Mitochondrial heterogeneity during staurosporine-induced apoptosis in HL60 cells: Analysis at the single cell and single organelle level [Articolo su rivista]
Salvioli, S.; Dobrucki, J.; Moretti, L.; Troiano, L.; Garcia Fernandez, M.; Pinti, Marcello; Pedrazzi, J.; Franceschi, C.; Cossarizza, Andrea
abstract

Background: Apoptosis is a complex phenomenon during which several events occur. A growing interest exists on the role and functionality of mitochondria during this type of cell death. The responsibility of modifications in mitochondrial membrane potential (Delta Psi) in triggering apoptosis is under investigation. Methods: We evaluated Delta Psi changes in HL60 cells treated with staurosporine (STS). Flow cytometry and confocal microscopy have been used to analyze samples stained with two Delta Psi-sensitive probes, JC1 and MitoTracker(TM) Red CMXRos. Results: At the cellular level, we found heterogeneic behavior. Indeed, after STS treatment, some cells displayed typical markers of apoptosis and a collapse in Delta Psi. Others were apoptotic with no changes in Delta Psi, others changed Delta Psi without being apoptotic, and others were healthy. The same heterogeneic response to STS was found at the single organelle level. In a given cell, some mitochondria were depolarized whereas others were not. Conclusion: In this model of apoptosis, changes in Delta Psi can be different among cells of the same type and among different organelles of the same cell. The collapse in Delta Psi is thus a heterogeneic phenomenon that seems to be an ancillary event following the irreversible phase of the apoptotic process.

2000 - Opposite role of changes in mitochondrial membrane potential in different apoptotic processes [Articolo su rivista]
S., Salvioli; C., Barbi; J., Dobrucki; L., Moretti; Troiano, L.; Pinti, Marcello; J., Pedrazzi; T. L., Pazienza; V., Bobyleva; Franceschi, C.; Cossarizza, Andrea
abstract

We have studied the role of changes in mitochondrial membrane potential (Delta Psi) in two widely-used models of apoptosis, such as dexamethasone-treated rat thymocytes and U937 human cells treated with tumor necrosis factor-alpha and cycloheximide. To dissipate Delta Psi, we used low concentrations of valinomycin, unable per se to induce apoptosis, and demonstrated that the decline in Delta Psi exerts opposite effects in the two models. Indeed, in U937 cells, depolarization of mitochondria increased apoptosis, which decreased in rat thymocytes. This leads to the suggestion that disruption of Delta Psi plays opposite roles depending on the experimental model. In U937 cells, the drop of Delta Psi is a possible contributory cause for the apoptotic process; in rat thymocytes, it could be a limiting factor. We propose that these opposite effects could be due to the different ATP requirement of each apoptotic pathway. (C) 2000 Federation of European Biochemical Societies.

2000 - Potenziali rischi per un eccesso di igiene in casa.Potential risks from an excess of hygienic cleaning in the domestic setting [Articolo su rivista]
Borella, Paola; Aggazzotti, Gabriella; Angelillo, I. F.; Balli, Fiorella; Barbone, F.; Carpenè, E.; Cossarizza, Andrea; Chiericozzi, M.; Gobba, Fabriziomaria; Seidenari, Stefania; Triassi, M.; Vercilli, F.
abstract

The most relevant and recent risks associated with an excessive hygiene are here presented. Besides general observations on the relationship between hygiene and immune system, we discuss possible acute and chronic effects due to home exposure to detergents. The specific problem of hygienic cleaning introduced on a vaste scale at home requires the comparison with similar experiences in the hospitals,, evaluation of efficacy, comprhension of risks such as the selection of resistant micro-organisms.

2000 - Quantitation of CD95 and CD95L mRNA expression in chronic and acute HIV-1 infection by competitive RT-PCR [Articolo su rivista]
Pinti, Marcello; Nasi, Milena; Moretti, L; Mussini, Cristina; Petrusca, D; Esposito, Roberto; Cossarizza, Andrea
abstract

Human immunodeficiency virus-type 1 (HIV-1) infection is characterized by increased immune cell apoptosis. Apoptosis can be triggered by signals that arise from within the cell, or by signals that are elicited by binding of extracellular death ligands to their death receptors, most of which belong to the tumor necrosis factor (TNF)-receptor family, such as CD95 (Fas/Apo-1). In immune cells the oligomerization of CD95, induced by its ligand CD95L, and the recruitment of different intracytoplasmic molecules that in turn activate FLICE/caspase 8 are crucial. To study the role of CD95/CD95L interactions during HIV-1 infection, we developed an original method based upon quantitative-competitive (QC) RT-PCR that allowed us to quantify the amounts of mRNA coding for the total (tCD95) and membrane (mCD95) forms of CD95. We first studied the expression of different forms of CD95 mRNA in a classical model of chronic HIV infection using two infected cell lines of different origin-lymphocytic (ACH-2) or monocytic (U1). We have shown that infected cells of monocytic origin preferentially produce the protective (soluble) form of CD95, and no detectable CD95L mRNA, while lymphoid cells produce more mRNA for the membrane form of CD95 (which triggers apoptosis) along with low but detectable amounts of CD95L mRNA. One can hypothesize that a complex balance exists between pro-apoptotic events, perhaps triggered by the host to limit viral production, and anti-apoptotic events likely triggered by the virus to increase its production and survival. In cells of monocytic origin, which act as a reservoir for the virus, the anti-apoptotic molecules are favored; in cells of lymphocytic origin, molecules with an apoptotic meaning are prevalent.

2000 - Studio preliminare dell’utilizzo del gel di piastrine autologo in chirurgia maxillo-facciale. [Abstract in Rivista]
Ceccherelli, G. B.; Bertoldi, Carlo; Bellini, Pierantonio; Zaffe, Davide; Cossarizza, Andrea; De Palma, M.; Consolo, Ugo
abstract

Vedasi l'allegato

1999 - Apoptotic features of peripheral blood granulocytes and monocytes during primary, acute HIV infection. [Articolo su rivista]
Cossarizza, Andrea; Mussini, Cristina; Borghi, V; Mongiardo, N; Nuzzo, C; Pedrazzi, J; Benatti, F; Moretti, L; Pinti, Marcello; Paganelli, R; Franceschi, Claudio; DE RIENZO, Bruno
abstract

Apoptosis plays a major role during HIV infection, including the primary, acute HIV syndrome (AHS), during which such phenomenon is massive. We asked whether apoptosis involved not only peripheral blood lymphocytes, but also monocytes (PBM) and granulocytes (PBG). Thus, we studied cells from different patients during the acute phase of the viral syndrome. The CD95 molecule was expressed at high density on the PBM and PBG surface during AHS. Culturing PBG for a few hours resulted in a significant membrane expression of phosphatidylserine, consistent with apoptosis. However, cells maintained for hours plasma membrane integrity and showed no relevant changes in mitochondrial membrane potential. The overexpression of CD95 was not associated with high plasmatic levels of sCD95 and, together with apoptosis and its related markers decreased after a few weeks of highly active antiretroviral therapy. During AHS, a deregulation of the CD95 system occurs in monocytes and granulocytes, is related to a high propensity of PBG to undergo apoptosis, and may contribute to the pathogenesis of the disease. Antiretroviral treatment resulted not only in a decrease of virus production, but also in a reduced PBG tendency to undergo spontaneous apoptosis. Even if the mechanism(s) responsible for this phenomenon remains to be elucidated, our data suggest a possible (indirect?) action of antiretroviral therapies on PBG and PBM which could explain, at least partially, the rescue of natural immunity and the reduced use of granulocyte-colony stimulating factor during such treatments.

1999 - Cell adhesion and the immune system: A case study using earthworms [Articolo su rivista]
Cooper, El; Cossarizza, Andrea; Kauschke, E; Franceschi, C.
abstract

In the earthworm´s immune system, cell adhesion, which occurs by putative receptors on leukocytes, is essential after recognition of self vs. non-self. Confrontation with foreign antigens is a normal event in the environment, replete with microbial pathogens that pose a threat to survival. To better understand what happens when an effector cell first recognizes a foreign target followed by its adhesion to it, isolated leukocytes, in sufficient quantities to be subjected to various analyses, have been extremely beneficial. In vitro approaches when accompanied by biochemical, immunological, and molecular technologies, have opened up new vistas concerning the immune response of earthworms and other invertebrates. The most recent discovery includes the preliminary identification of cell differentiation (CD) markers that play vital roles in recognitive and adhesive events. Certain leukocyte effecters show characteristics of natural killer (NK) cells that may act differently depending upon their source, whether autogeneic, allogeneic, xenogeneic, or expressed under normal or varying environmental conditions including exposure to xenobiotics. At the level of earthworm evolution, there is apparently a dissociation of phagocytosis from the process of killing by NK-like effecters. There are at least three future challenges. First, it is essential to determine the precise nature of the CD markers with respect to their molecular structure. Second, once their molecular and biochemical characteristics have been defined, the role of these markers in cellular and humoral mechanisms must be clarified in order to define effector cell products and resulting immune responses. Third, there is a need to differentiate between the several lytic factors that have been found in earthworms with respect to molecular structure, and biochemical and functional characterization. Microsc. Res. Tech. 44:237-253, 1999.

1999 - Differential down-regulation of CD95 or CD95L in chronically HIV-infected cells of monocytic or lymphocytic origin: cellular studies and molecular analysis by quantitative competitive RT-PCR. [Articolo su rivista]
Pinti, Marcello; Pedrazzi, J; Benatti, F; Sorrentino, V; Nuzzo, C; Cavazzuti, V; Biswas, P; Petrusca, Dn; Mussini, Cristina; DE RIENZO, Bruno; Cossarizza, Andrea
abstract

We analysed the expression of CD95/CD95L in two widely used models for studying the cellular effects of chronic infection with human immunodeficiency virus type 1 (HIV-1), i.e. ACH-2 cells, derived from the lymphocytic cell line A301, and U1, derived from monocytic U937 cells. A301 and ACH-2 mounted the same amount of plasma membrane CD95, while U1 had a consistent decrease in CD95 expression. Using different antibodies, we failed to detect the plasma membrane form of its ligand, CD95L, but we could see the intracellular presence of that molecule in A301 cells and, to a lesser extent, in ACH-2 cells, but not in U937 or U1 cells. To confirm the cytofluorimetric data and quantify the expression of CD95L at the RNA level, we developed a quantitative competitive RT-PCR assay. The HUT78 cell line had about 50,000 copies mRNA/1000 cells, three times more after induction with a phorbol ester and ionomycin. ACH-2 expressed about 400- (basal) or 10- (induced) fold less CD95L mRNA than the parental cell line A301; U937 and U1 were below the limit of detection. In cells of lymphoid origin (ACH-2) chronic HIV infection inhibits the expression of CD95L, the phenomenon occurring at the transcriptional level. In cells of monocytic origin (U1) the infection decreases the plasma membrane expression of CD95. This suggests that HIV could trigger different anti-apoptotic strategies which likely depend upon the cell line which is infected. In monocytic cells which act as a viral reservoir, the expression of the molecule whose binding triggers apoptosis decreases, while in lymphoid cells, capable of exerting cytotoxicity, the expression of a molecule which induces apoptosis is reduced.

1999 - Long-term immunologic effects of thymectomy in patients with myasthenia gravis [Articolo su rivista]
Gerli, R; Paganelli, R; Cossarizza, Andrea; Muscat, C; Piccolo, G; Barbieri, D; Mariotti, S; Monti, D; Bistoni, O; Raiola, E; Venanzi, Fm; Bertotto, A; Franceschi, C.
abstract

Background: Thymectomy (Tx) is a common therapeutic option to treat myasthenia gravis (MG), but its effects on the immune system are still obscure in humans. Objective: We sought to evaluate long-term immunologic effects of therapeutic Tx in patients with MG. Methods: T- and B-cell subsets and T-cell repertoire were analyzed in 35 patients with MG, 16 with previous Tx (at least 8 years before), 6 with recent (<1 year) Tx, and 13 without Tx, as well as in 32 healthy subjects used as normal control subjects. Serum immunoglobulins and a variety of autoantibodies were also measured. A subsequent 3-gear clinical follow-up was performed to verify the possible appearance of systemic autoimmune diseases. Results: The long-term thymectomized (Txd) patients had mild T-cell lymphopenia and an expansion of some V beta families among circulating CD4+ and CD8+ T cells. They displayed a normal number of total B and CD5+ B-circulating lymphocytes, but they also displayed a polyclonal increase in serum IgM and IgG associated with the presence of high levels of a variety of organ- and nonorgan-specific autoantibodies, including anti-dsDNA and anticardiolipin, without clinical evidence of autoimmune disease. These serologic abnormalities mere not detectable in both non-Txd and recently Txd patients. After 3 years, 2 long-term Txd patients had systemic lupus erythematosus and an undifferentiated connective tissue disease. Conclusions: The association between MG and laboratory findings of systemic autoimmune disease may be in part related to Tx rather than to MG. Tx may represent a risk for the development of systemic autoimmune disorders over years in patients with MG.

1998 - Age-related modifications of the human alpha beta T cell repertoire due to different clonal expansions in the CD4(+) and CD8(+) subsets [Articolo su rivista]
A., Wack; Cossarizza, Andrea; S., Heltai; D., Barbieri; S., D'Addato; C., Fransceschi; P., Dellabona; G., Casorati
abstract

We have studied the effects of a life-long antigen stimulation on the clonal heterogeneity of human peripheral T cell subsets, as defined by their CD45 isoform expression. CD4(+) or CD8(+) T cells were obtained from healthy donors ranging in age from 20 to 100 years, and sorted into CD45RA(+) and CD45RO(+) populations. A modified PCR-heteroduplex analysis was then used to directly compare the TCR Vp clonal make up of either compartment pair. We find that the CD4(+) T cell repertoire remains largely polyclonal throughout life, since CD4(+) expanded clones are rare and accumulate predominantly in the CD45RO(+) compartment of exceptionally old donors (100 years old). In contrast, the CD8(+) T cell subset contains expanded clones which are already detectable in young adults and become very frequent in 70- to 75-year-old donors in both CD45RA(+) and CD45RO(+) compartments analyzed. Interestingly, some expanded clones are detectable in the CD45RA(+) or in both CD45RA(+) and CD45RO(+) compartments of either CD4(+) or CD8(+) T cells. These results indicate that the age-dependent accumulation of expanded clones starts earlier and is more pronounced in the CD8(+) than in the CD4(+) T cell subset, reinforcing the concept that clonal expansion in the two subsets is controlled by substantially different mechanisms. Furthermore, whereas the finding of expanded CD45RO(+) T cell clones is explained by antigen-driven proliferation, the detection of expanded clones in the CD45RA(+) or in both CD45RA(+) and CD45RO(+) compartments would support the hypothesis of reversion from the CD45RO(+) to the CD45RA(+) phenotype after antigen encounter.

1998 - Apoptosis by 2-chloro-2 '-deoxy-adenosine and 2-chloro-adenosine in human peripheral blood mononuclear cells [Articolo su rivista]
Barbieri, D; Abbracchio, Mp; Salvioli, S; Monti, D; Cossarizza, Andrea; Ceruti, S; Brambilla, R; Cattabeni, F; Jacobson, Ka; Franceschi, C.
abstract

Adenosine has profound effects on immune cells and has been implicated in the intrathymic apoptotic deletion of T-cells during development. In order to characterize adenosine effects on quiescent peripheral blood mononuclear cells (PBMC), we have evaluated the ability of the previously characterized adenosine receptor agonist 2-chloro-adenosine (2CA; Ceruti, Barbieri et al., 1997) and of the antineoplastic drug 2-chloro-2'-deoxy-adenosine (2CdA, cladribine) to trigger apoptosis of PBMC. Apoptosis was assessed by morphological changes, DNA fragmentation by agarose gel electrophoresis and appearance of hypodiploid DNA peak by flow cytometry. 2CA (10 mu M) and 2CdA (1 mu M) induced apoptosis in human PBMC, which are relatively insensitive to apoptosis. For both agents, the effect was concentration- and time-dependent, although 2CdA induced apoptosis more potently than 2CA. Evaluation of mitochondrial function in parallel samples using the mitochondrial membrane-potential-specific dye JC-1 showed that mitochondrial damage followed the same kinetics as apoptosis, hence an early damage of mitochondria is likely not responsible for adenosine-induced death of PBMC. The effect of 2CA was partially prevented by addition of dipyridamole (DP), a nucleoside transport inhibitor, hence some of the apoptotic effect of this nucleoside is, at least in part, due to intracellular action. Alternatively, DP did not affect 2CdA-induced apoptosis, suggesting that 2CdA may enter cells via a DP-insensitive transporter. 5-Iodotubercidin (5-Itu), a nucleoside kinase inhibitor, was also able to partially prevent the action of 2CA and was not able to affect 2CdA-induced apoptosis, suggesting a different role for phosphorylation in 2CA- vs 2CdA-induced apoptosis. To test the role of P1 receptors, agonists and antagonists selective at various P1 receptor subtypes were used. Data suggest that, for 2CA, apoptosis is partially sustained by activation of the A,, receptor subtype, whereas no role is exerted by P1 receptors in 2CdA-dependent apoptosis. Moreover, in these cells, apoptosis could also be triggered through intense activation of the A(3) receptor via selective agonists such as 2-chloro-N-6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA), but this mechanism plays no role in either 2CA- or 2CdA-induced apoptosis. On the whole, our results suggest that 2CA and 2CdA follow different pathways in inducing apoptosis of immune cells. Moreover, our data also suggest that there are at least three different ways by which adenosine derivatives may induce apoptosis of human PBMC: (i) through an A(2A)-like extracellular membrane receptor; (ii) through entry of nucleosides into cells and direct activation of intracellular events involved in the apoptotic process; or (iii) through activation of the A(3) receptor. (C) 1998 Elsevier Science Ltd. All rights reserved.

1998 - Apoptosis-like, reversible changes in plasma membrane asymmetry and permeability, and transient modifications in mitochondrial membrane potential induced by curcumin in rat thymocytes [Articolo su rivista]
Jaruga, E; Salvioli, S; Dobrucki, J; Chrul, S; Bandorowicz Pikula, J; Sikora, E; Franceschi, C; Cossarizza, Andrea; Bartosz, G.
abstract

Curcumin (diferuoylmethane) is a natural compound with anticarcinogenic activities which is able to exert either proapoptotic or antiapoptotic effects in different cell types, This paper focuses on the sequence and extent of primary events induced by curcumin, in comparison with those occurring during dexamethasone-induced apoptosis in rat thymocytes, It also presents annexin VI-FITC as a new probe for studying membrane asymmetry. Curcumin readily penetrates into the cytoplasm, and is able to accumulate in membranous structures such as plasma membrane, endoplasmic reticulum and nuclear envelope. Curcumin-treated cells exhibit typical features of apoptotic cell death, including shrinkage, transient phosphatidylserine exposure, increased membrane permeability and decrease in mitochondrial membrane potential. However, nuclei morphology, DNA fragmentation, the extent and time-course of membrane changes are different from those observed during dexamethasone-induced apoptosis, suggesting that, despite many similarities, the mode of action and the events triggered by curcumin are different from those occurring during typical apoptosis, (C) 1998 Federation of European Biochemical Societies.

1998 - Assessment of sense of taste in Italian centenarians [Abstract in Rivista]
Baggio, G.; Dalla Vestra, M.; Donazzan, S.; Barbagallo Sangiorgi, G.; Barbagallo, M.; Frada, G.; Bertolini, S.; Agretti, M.; Costelli, P.; Bosi, E.; Manzoni, M.; Tomasello, F. B.; Capurso, A.; Colacicco, A. M.; Solfrizzi, V.; Fabris, F.; Cappa, G.; Ferrario, E.; Forconi, S.; Guerrini, M.; Boschi, S.; Franceschi, C.; Cossarizza, A.; Monti, D.; Gaddi, A.; D'Addato, S.; Galletti, C.; Giarelli, L.; Cavalieri, F.; Stanta, G.; Mari, D.; Duca, F.; Ferrazzi, P.; Marigliano, V.; Bauco, C.; Cacciafesta, M.; Masotti, G.; Marchionni, N.; Petruzzi, E.; Mattace, R.; Motta, M.; Pansini, L.; Motta, L.; Rapisarda, R.; Receputo, G.; Passeri, M.; Fagnoni, F.; Sansoni, P.; Salvioli, G.; Baldelli, M. V.; Neri, M.; Senin, U.; Cherubini, A.; Polidori, M. C.; Trabucchi, M.; Boffelli, S.; Rozzini, R.; Varricchio, M.; Gambardella, A.; Paolisso, G.
abstract

The Italian multicentric study on centenarians (IMSC) was aimed at assessing the level of preservation of the sense of taste, and at estimating to what extent the recognition of various gustative stimuli can give satisfaction and information regarding the surrounding environment for the centenarians. Taste sensitivity has been qualitatively established in a group of 126 Italian centenarians (mean age 101.9 ± 1.4 years) and compared to that of a group of 100 elderly subjects (mean age 70.5 ± 5.0 years). All the individuals included in the study groups had a mini mental state examination (MMSE) score larger than 19. The results revealed that taste sensitivity is significantly reduced in the centenarians; as a matter of fact, the elderly control subjects are able to perceive taste stimuli in 84.25 ± 3.40% of the testing, while the centenarians were successful only in 74.23 ± 6.19% of the experiments (P &lt; 0.001). Furthermore, elderly subjects can correctly recognize taste stimuli in 70% of the testings, while correct recognition amounted only to 49.25% in cases of centenarians assessed. In spite of these differences between the elderly and centenarians, the latter are still able to perceive and recognize taste stimuli adequately, and as a consequence, are able to obtain information on tastes and receive a sufficient sensory stimulation through the tasting pathway.

1998 - Decrease in mitochondrial energy couplings by thyroid hormones: a physiological effect rather than a pathological hyperthyroidism consequence [Articolo su rivista]
Bobyleva, V; Pazienza, Tl; Maseroli, R; Tomasi, Aldo; Salvioli, S; Cossarizza, Andrea; Franceschi, C; Skulachev, Vp
abstract

The effect of the in vivo thyroid status on mitochondrial membrane potential (Delta Psi(m)) in isolated rat hepatocytes was studies by means of a cytofluorimetric technique and the Delta Psi(m)-specific probe JC-1. It is shown that the Delta Psi(m) level decreases in the order hypothyroid > euthyroid > hyperthyroid, Polarographic measurement of the hepatocyte respiratory rates revealed an opposite trend of values: the highest respiratory rate in hepatocytes from hyperthyroid animals, the lowest in those from hypothyroid ones. This means that mitochondrial energy coupling is highest in hypothyroid hepatocytes and lowest in hyperthyroid hepatocytes. 6-Ketocholestanol added to hepatocytes failed to counterbalance the uncoupling effect of thyroid hormones on Delta Psi(m) and respiration rate. Under the same conditions, 6-ketocholestanol appeared to be effective in recoupling of respiration uncoupled by low concentrations of the artificial protonophore FCCP, The mechanism and possible physiological functions of the thyroid hormone-induced decrease in mitochondrial energy coupling are discussed. (C) 1998 Federation of European Biochemical Societies.

1998 - Mitochondrial membrane potential and DNA stainability in human sperm cells: A flow cytometry analysis with implications for male infertility [Articolo su rivista]
L., Troiano; Arm, Granata; Cossarizza, Andrea; G., Kalashnikova; R., Bianchi; G., Pini; F., Tropea; Carani, Cesare; C., Franceschi
abstract

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-l and DNA stainability with propidium iodide (PI). JC-l staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-l staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.

1998 - Toxin-induced activation of Rho GTP-binding protein increases Bcl-2 expression and influences mitochondrial homeostasis [Articolo su rivista]
Fiorentini, C; Matarrese, P; Straface, E; Falzano, L; Fabbri, A; Donelli, G; Cossarizza, Andrea; Boquet, P; Malorni, W.
abstract

It is now well established that apoptosis plays a pivotal role in several physiological and pathological situations. Consequently, the mechanisms controlling the cell fate are currently the subject of intense investigation. In this work, we report that an Escherichia coli protein toxin (Cytotoxic Necrotizing Factor 1, CNF1) which activates the Rho GTP-binding protein and prevent apoptosis in epithelial cells was able to: (i) influence the mitochondrial homeostasis and (ii) modulate the expression of proteins belonging to the Bcl-2 family. In particular, the content of the antiapoptotic products Bcl-2 and Bcl-X-L resulted to be increased in treated cells, whereas the expression of the proapoptotic protein Fax remained unaltered. CNF1 induces cell spreading via activation of Rho and cell spreading has been reported to promote cell survival. Cytochalasin B, which provokes most of the morphological changes typical of CNF1, including cell spreading, but without the involvement of Rho, was unable to counteract apoptosis. Altogether our results suggest a link between the Rho GTP-binding protein and the regulation of the mitochondrial homeostasis via an effect on the antiapoptotic proteins of the Bcl-2 family.

1998 - Use of flow cytometry as a tool to study mitochondrial membrane potential in isolated, living hepatocytes [Articolo su rivista]
Salvioli, S; Maseroli, R; Pazienza, Tl; Bobyleva, V; Cossarizza, Andrea
abstract

The present paper describes the possibility of determination of mitochondrial membrane potential (Delta omega) in isolated hepatocytes making use of a Delta psi-sensitive dye, i.e., the lipophilic cationic probe 5,5'6,6'-tetrachloro-1,1'3,3 '-tetraethylbenzimidazolcarbocyanine iodide (JC-1) and of cytofluorimetry. The validity of the method was proved by treating hepatocytes with FCCP (decrease of Delta psi) and subsequent addition of 6-ketocholestanol (increase of Delta psi). The results indicate that the proposed method may be used in laboratory practice.

1997 - Cytometric analysis of immunosenescence [Articolo su rivista]
Cossarizza, Andrea; Ortolani, C; Monti, D; Franceschi, C.
abstract

We have been studying the immune system of healthy centenarians for many years, and they provide the best example of successful aging, They are people who have escaped major age-related diseases and reached the extreme limit of human life in good clinical condition, in most cases, histories of centenarians reveal them to be free of cancer, dementia, diabetes, cardiovascular diseases, and cataracts. Moreover, in order to reach such an advanced age, they should be equipped with well preserved and efficient immuno- and defense mechanisms, and optimal combinations of an appropriate lifestyle and genetic background. Using this approach, several paradoxes emerged as far as the immune system of centenarians is concerned, regarding: i) humoral immunity increase in plasma immunoglobulins and nonorgan-specific autoantibodies, decrease in B cell number and lack of organ-specific autoantibodies); ii) cellular immunity (cell preserved number of ´´virgin´´ T cells, a relatively intact T cell repertoire despite a thymus involuting since puberty, increased number of cells with markers of NK activity); iii) decreased peripheral blood lymphocyte tendency to programmed cell death, associated with a well preserved mitochondria functionality and intracellular bcl-2 levels, An age-related increase in the levels of adhesion molecule present on lymphocyte plasma-membrane, accompanied by a complex reshaping of the cytokine network, must be added to this scenario, All our data fit the hypothesis that a complex, unpredicted remodelling of the immune system occurs with age. In the present review it is underlined how flow cytometry has been used to study most of the above mentioned aspects of immunosenescence, and to establish new age-related reference values. Cytometry 27:297-313, 1997.

1997 - Different pathways of apoptosis revealed by 2-chloro-adenosine and deoxy-D-ribose in mammalian astroglial cells [Articolo su rivista]
Ceruti, S; Barbieri, D; Veronese, E; Cattabeni, F; Cossarizza, Andrea; Giammarioli, Am; Malorni, W; Franceschi, C; Abbracchio, Mp
abstract

Both the adenosine analogue 2-chloro-adenosine (2-CA) and the reducing sugar deoxy-D-ribose (dRib) induce apoptosis of astroglial cells in rat brain primary cultures (Abbracchio et al.: Biochem Biophys Res Commun 213:908-915, 1995), The present study was undertaken to elucidate by both morphological and cytofluorimetric analyses the intracellular mechanism(s) involved in induction of apoptosis by these two agents, The poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide did not prevent either 2-CA- or dRib-induced cell death, suggesting that activation of PARP is not critically important for induction of apoptosis in astrocytes. The radical scavenger N-acetyl-cysteine (NAG) strongly inhibited dRib- but not 2-CA-induced cell death, suggesting a differential role for radical formation in apoptosis by these two agents, A time-dependent increase of cells with depolarized mitochondria was observed in dRib-, and to a lesser extent, in 2-CA-treated cultures, NAC also prevented dRib- but not 2-CA-induced mitochondrial changes, We conclude that, in mammalian astrocytes, apoptosis can proceed through diverse and multiple pathways, depending upon the apoptotic stimulus, For dRib, apoptosis likely proceeds through generation of radicals and mitochondrial involvement, An adenosine extracellular receptor linked to an as yet unidentified signaling pathway may instead mediate 2-CA-induced cell death, which may have intriguing implications for both nervous system development and brain response to trauma and ischemia.

1997 - JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨ changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis [Articolo su rivista]
Salvioli, S.; Ardizzoni, A.; Franceschi, C.; Cossarizza, A.
abstract

The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (ΔΨ) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC-1, DiOC6(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing ΔΨ. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC6(3) - but not of R123 - only cells stained with JC-1 responded to valinomycin. On the whole, our data indicate that JC-1 is a reliable probe for analyzing ΔΨ changes with flow cytometry, while the others shown a lower sensitivity (R123), or a non-coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC6(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to ΔΨ, hypothesized that the fall in ΔΨ is one of the early events, if not one of the main causes, of apoptosis.

1997 - Lymphocytes [Capitolo/Saggio]
Richeldi, Luca; A., Franchi; E., Rovatti; Cossarizza, Andrea; R. M., Dubois; C., Saltini
abstract

Non disponibile

1997 - Mitochondria alterations and dramatic tendency to undergo apoptosis in peripheral blood lymphocytes during acute HIV syndrome. [Articolo su rivista]
Cossarizza, Andrea; Mussini, Cristina; Mongiardo, N; Borghi, V; Sabbatini, A; DE RIENZO, Bruno; Franceschi, Claudio
abstract

OBJECTIVE: To study alterations of mitochondrial membrane potential (delta psi) and the propensity to undergo apoptosis in peripheral blood lymphocytes (PBL) from subjects with acute HIV syndrome; and to evaluate possible modulations of these phenomena by antioxidants that can be used in therapy, such as N-acetyl-cysteine (NAC), nicotinamide (NAM), or L-acetyl-carnitine (LAC). METHODS: Mitochondrial function and the tendency of PBL to undergo spontaneous apoptosis were studied on freshly collected PBL from patients with symptomatic, acute HIV-1 primary infection, which were cultured for different durations in the presence of absence of NAC. NAM or LAC. By a cytofluorimetric method allowing analysis of delta psi in intact cells, we studied the function of these organelles under the different conditions. PBL apoptosis was evaluated by the classic cytofluorimetric method of propidium iodide staining, capable of revealing the typical DNA hypodiploid peak. RESULTS: Significant delta psi alterations and tendency to undergo apoptosis were present in PBL from the subjects we studied. Indeed, when cultured even for a few hours in the absence of any stimulus, a consistent number of cells died. However, the presence of even different levels of NAC, NAM or LAC was able to rescue most of them from apoptosis. Both a fall in delta psi and apoptosis were evident in PBL collected in the earliest phases of the syndrome (before seroconversion), and changed significantly after a few days. A significant correlation was found between spontaneous apoptosis and tumour necrosis factor (TNF)-alpha or p24 plasma levels, as well as between apoptosis and the percentages of circulating CD4+ or CD8+ T cells. CONCLUSIONS: PBL from patients with acute HIV syndrome are characterized by both significant mitochondrial alterations and a dramatic tendency to undergo apoptosis. The use of NAC, NAM or LAC seems to rescue cells through a protective effect on mitochondria, a well-known target for the action of TNF-alpha and for reactive oxygen species, the production of which is strongly induced by this cytokine. Thus, our data could provide the rationale for the use of such agents in addition to antiviral drugs in primary infection.

1997 - Phenotypic characteristics and tendency to apoptosis of peripheral blood mononuclear cells from HIV+ long term non progressors [Articolo su rivista]
Franceschi, C; Franceschini, Mg; Boschini, A; Trenti, T; Nuzzo, C; Castellani, G; Smacchia, C; Derienzo, B; Roncaglia, R; Portolani, Marinella; Pietrosemoli, P; Meacci, M; Pecorari, M; Sabbatini, Anna Maria Teresa; Malorni, W; Cossarizza, Andrea
abstract

The aim of this study was to analyze (i) phenotype, (ii) in vitro spontaneous and induced apoptosis, (iii) glutathione (GSH) intracellular content and (iv) inhibitors of apoptosis of potential therapeutical use in peripheral blood mononuclear cells (PBMC) from HIV+ long term non progressors (LTNP), in comparison with progressors (HIV+P) and seronegative controls (HIV-). Three groups of subjects were studied: 15 HIV+P (patients losing &gt; 150 CD4+/year), 9 LTNP (subjects infected by HIV for at least 7 years without clinical and immunological signs of progression, with a mean of 898 CD4+/mu L) and 18 HIV-. All subjects were living in a large community for former drug addicts, and were matched for age and sex. We used flow cytometry for analyzing PBMC phenotype ansi apoptosis; high performance liquid chromatography for measuring intracellular GSH content. PBMC phenotype of LTNP shared characteristics with those of both HIV- and HIV+P. Indeed, LTNP showed a normal number CD4+ cells (an inclusion criteria), butsignificantly increased numbers of CD8+ lymphocytes, activated T cells, CD19+, CD5+ B lymphocytes and CD57+ cells, as well as a decrease in CD19+, CD5- B lymphocytes and CD16+ cells. In LTNP, spontaneous apoptosis was similar to that of HIV- and significantly lower than that of HIV+P. Adding interleukin-2 (IL-2) or nicotinamide (NAM) significantly decreased spontaneous apoptosis in LTNP and HIV+P. Pokeweed mitogen-induced apoptosis was also similar in LTNP and HIV-, but significantly lower than that of HIV+P. In HIV+P, but also in LTNP, spontaneous apoptosis was inversely correlated to the absolute number and percentage of CD4+ cells and directly correlated to the number and percentage of activated T cells present in peripheral blood. GSH intracellular content was greatly decreased in PBMC from HIV+P and slightly, but significantly, reduced in LTNP. Adding 2-deoxy-D-ribose, an agent provoking apoptosis through GSH depletion, to quiescent PBMC resulted in similar levels of massive cell death in the three groups. This phenomenon was equally prevented in the three groups by N-acetyl-cysteine but not by IL-2. A complex immunological situation seems to occur in LTNP. Indeed, PBMC from LTNP are characterized by a normal in vitro tendency to undergo apoptosis despite the presence activation of their immune system, unexpectedly similar to that of HIV+P. Our data suggest that NAM and IL-2 are possible candidates for reducing spontaneous apoptosis in HIV infection.

1997 - Relevance of clinical and laboratory findings in the diagnosis of cytomegalovirus encephalitis in patients with AIDS. [Articolo su rivista]
Mussini, Cristina; Mongiardo, N; Manicardi, G; Trenti, F; Alessandrì, A; Paolillo, F; Catania, A; Portolani, Marinella; Pecorari, M; Borghi, V; Ficarra, G; Cossarizza, Andrea; DE RIENZO, Bruno
abstract

A retrospective evaluation was conducted in patients with AIDS and an autopsy diagnosis of cytomegalovirus (CMV) encephalitis to determine the relevance of clinical and laboratory findings in establishing a diagnosis. On autopsy of 100 patients, CMV encephalitis was diagnosed in 13 patients; eight had periventricular CMV encephalitis, four micronodular CMV encephalitis, and one both conditions. Seven patients had had a CMV infection previously (6 cases of retinitis, 1 case of colitis), and at the onset of encephalitis all of them were receiving a maintenance dose of ganciclovir. Examination of the CSF showed specific changes in patients with periventricular encephalitis. CT revealed no characteristic findings, while MRI showed an increased signal intensity on T2 weighted images. CMV DNA amplification by nested PCR was performed in nine patients with CMV encephalitis; PCR was positive in eight patients whose CSF was collected during CMV encephalitis, and negative in one patient whose CSF was collected six months before death. In conclusion, some clinical findings suggest a presumptive diagnosis, especially of periventricular encephalitis, and nested PCR appears to be a reliable and rapid technique for making an antemortem diagnosis.

1997 - Resistance to apoptosis in Fanconi's anaemia - An ex vivo study in peripheral blood mononuclear cells [Articolo su rivista]
Monti, D; Macchioni, S; Guido, M; Pagano, G; Zatterale, A; Calzone, R; Cossarizza, Andrea; Straface, E; Malorni, W; Franceschi, C.
abstract

Fanconi's anaemia (FA) is a rare autosomal recessive disease characterised by progressive pancytopoenia, a diverse assortment of congenital malformations, an increased sensitivity to reactive oxygen species and a predisposition to the development of malignancies. In the present study, me assessed the propensity to undergo apoptosis of peripheral blood mononuclear cells (PBMC) from Italian FA patients, Cells were challenged by 2-deoxy-D-ribose (dRib) or TNF-alpha plus cycloheximide as agents that induce apoptosis by interfering with cell redox status and mitochondrial membrane potential (MMP), and PBMC from FA patients resulted to be less prone to die than those from healthy subjects. The decreased susceptibility of FA cells to undergo apoptosis was also evident when another parameter highly correlated with the apoptotic process, i.e. MMP, was measured. Moreover, when N-acetylcysteine was added to dRib-treated PBMC, a strong protection was evident either in PBMC from control subjects or from FA patients, These data indicate that an alteration of unknown nature of the mechanisms favouring apoptosis is present in freshly collected cells from FA patients, and that such alteration could contribute to the pathogenesis of the disease, and particularly to the increased susceptibility to cancer. (C) 1997 Federation of European Biochemical Societies.

1997 - Self gamma 2a(b) protein is presented in vivo by gamma 2a(b) B cells but not by dendritic cells [Articolo su rivista]
Granucci, F; Foti, M; Cossarizza, Andrea; Ricciardicastagnoli, P.
abstract

We have previously shown that IgG2a(b) Ig does not induce tolerance in MHC class II restricted CD4 T cells in a TCR transgenic model and that anti-IgC2a(b) transgenic T cells specific for peptide 435-451 are indeed present in the periphery where they interact with gamma 2a(b)-positive B cells, We also observed that because T cell tolerance depends on the presentation of self peptides, it was probable that IgG2a(b) was not easily processed and presented in vivo, In this study, we have investigated the presentation of naturally processed gamma 2a(b) (435-451) determinants to specific T cells. Dendritic cells, macrophages, and B cells were purified from the spleens of Igh-1b mice, These cells were then functionally tested for the presence of specific peptide-MHC complexes, The results showed that, in vivo, gamma 2a(b)-producing B cells, but not dendritic cells, are the only APCs able to present this self peptide, This indicates that recognition of the IgG2a(b)-self peptide is exclusively mediated by T-B cell interaction.

1997 - Senescence and immune system: The example of centenarians [Abstract in Rivista]
Cossarizza, Andrea
abstract

Healthy centenarians are the best example of successful ageing, as they have escaped the major age-associated diseases, and most of them are in good mental and physical condition. In this review, it is discussed how the study of their immune systems reveals that several immune parameters are still well conserved, suggesting that a complex remodelling of most immune parameters occurs with age, rather than a unidirectional deterioration.

1997 - T lymphocyte proliferative capability to defined stimuli and costimulatory CD28 pathway is not impaired in healthy centenarians [Articolo su rivista]
Sansoni, P; Fagnoni, F; Vescovini, R; Mazzola, M; Brianti, V; Bologna, G; Nigro, E; Lavagetto, G; Cossarizza, Andrea; Monti, D; Franceschi, C; Passeri, M.
abstract

It is generally assumed that T cell proliferation is impaired in aged individuals. We report data on the proliferative capability of peripheral blood mononuclear cells (PBMC) and T lymphocytes from 40 healthy people of different ages, (19-107 years), including 14 centenarians, to defined mitogenic stimuli. We observed no age-related proliferative impairment both in PBMC and in purified T cells stimulated by anti-CD3 mAb or phorbol myristate acetate (PMA). Furthermore, T cells stimulated by anti-CD3 mAb or PMA and costimulated by CD28 mAb did not proliferate differently among young, middle aged subjects and centenarians. Thus, short term T cell proliferation is not affected even at extreme age when well defined stimuli are used on cells deriving from carefully selected healthy subjects.

1997 - T-cell repertoire and HIV infection: Facts and perspectives [Articolo su rivista]
Cossarizza, Andrea
abstract

1996 - A shift to Th0 cytokine production by CD4(+) cells in human longevity: Studies on two healthy centenarians [Articolo su rivista]
Paganelli, R; Scala, E; Rosso, R; Cossarizza, Andrea; Bertollo, L; Barbieri, D; Fabrizi, A; Lusi, Ea; Fagiolo, U; Franceschi, C.
abstract

Centenarians, particularly healthy centenarians, constitute the example of successful aging and the study of their immune status can help to define the endpoint of the changes occurring throughout life. We characterized T cell clones (TCC) of two healthy centenarians, studying their phenotypes and production of representative Th1 and Th2 cytokines (IFN-gamma and IL-4) and compared them with TCC obtained by three young normal subjects; in all 180 TCC were analyzed, In young donors, 35 TCC were CD4(+), 56 CD8(+) and 2 were alpha beta(+)CD4(-)CD8(-) (double negative). In centenarians, we obtained 46 CD4(+) TCC, 38 CD8(+), 2 CD4(+)CD8(+) (double positive) and 1 gamma delta(+) double negative. Of the young subjects' TCC, 71% produced IFN-gamma but no IL-4 (Th1 pattern) and this prevalence decreased to 39% in TCC from the centenarians. The number of clones showing the opposite Th2 pattern was similar in young and aged donors (3 out of 93 TCC and 2 out of 87 TCC, respectively). The intermediate profile of TCC producing both IL-4 and IFN-gamma (Th0) was found in 25.8% of clones from young people, but it almost doubled to 58.6% in centenarians. The analysis shows that the Th profiles of CD8(+) TCC is nearly superimposable in the two groups, whereas a major shift from a Th1 to a Th0 pattern is presented by CD4(+) TCC. The balance provided by a majority of CD4(+) TCC showing a Th0 pattern may ensure both humoral and cell-mediated defences. In CD8(+) TCC, however, a Th1 pattern still is present, possibly for efficient generation of cytotoxic responses. These findings should be extended by studying other centenarians and elderly subjects.

1996 - CD45 isoforms expression on CD4(+) and CD8(+) T cells throughout life, from newborns to centenarians: Implications for T cell memory [Articolo su rivista]
Cossarizza, Andrea; Ortolani, C; Paganelli, R; Barbieri, D; Monti, D; Sansoni, P; Fagiolo, U; Castellani, G; Bersani, F; Londei, M; Franceschi, C.
abstract

CD4(+) and CD8(+) peripheral blood T lymphocytes show mutually exclusive expression of CD45RA or CD45R0, two isoforms of the common leukocyte antigen that seem to recognize so-called virgin/unprimed and memory/activated T cells. The expression of these isoforms has been studied by three colour cytofluorimetric analysis on CD4(+) or CD8(+) peripheral blood CD3(+) cells from 22 healthy centenarians, analyzed in a context of 202 healthy donors 0-110 years old. An age-related unbalance of virgin and memory cells was found between CD4(+) and CD8(+) subsets. As expected, at birth 95-99% of the CD3(+) lymphocytes expressed the CD45RA isoform. A rapid increase of CD45R0(+) cells was observed in the first 2-3 decades of life, this phenomenon being much more pronounced on CD4(+) cells. Subsequently, the increase of the 'memory' compartment was much less rapid, so that in centenarians a consistent reservoire of CD45RA(+) among CD4(+) cells was still present (about 20%). In these exceptional individuals the percentage of CD45RA(+) cells among CD8(+) T lymphocytes was even higher (about 50%), and only slightly lower than that of young donors (about 55-60%). Thus, the main changes occurred at a different rate in CD4(+) (about 20%). In these exceptional individuals the percentage of CD45RA(+) cells among CD8(+) T lymphocytes was even higher (about 50%), and only slightly lower than that of young donors (about 55-60%). Thus, the main changes occurred at a different rate in CD4(+) and in CD8(+) T cells, at an age of between 0 and 30 years, when the thymus is still functionally active. Interestingly, no difference in the usage of CD45 isoforms was observed within T cells bearing four different VP-T cell receptor (TCR). The significance of this age-related unbalance is unknown. However, the presence of a great number of CD45RA(+) T lymphocytes within the CD4(+) and the CD8(+) T cell subsets even in the peripheral blood of centenarians poses the problem of their origin (thymus? extrathymic sites?), of their functional role and of their lifespan. Moreover, the data on centenarians suggest that they may represent a very selected population where a slowing of immunosenescence occurs.

1996 - Control of apoptosis by the cellular ATP level [Articolo su rivista]
Christoph, Richter; Matthias, Schweizer; Cossarizza, Andrea; Franceschi, Claudio
abstract

Apoptosis is a physiological form of cell death, Its causes and execution mechanisms are not clearly understood, Oxidative stress, nitric oxide and its congeners, Ca2+, proteases, nucleases, and mitochondria are considered mediators of apoptosis, At present their importance and exact role are elusive but it is clear that mitochondria are both the target and the source of oxidative stress, nitric oxide, and Ca2+, The mitochondrial membrane potential(Delta psi), which is the driving force for mitochondrial ATP synthesis, declines during apoptosis, and maintenance of Delta psi prevents apoptosis, Since apoptosis is highly regulated and involves the activity of hydrolytic enzymes, chromatin condensation and vesicle formation apoptosis is likely to have a high energy demand, We propose that the cellular ATP level is an important determinant for cell death, This hypothesis is supported by circumstantial evidence, is consistent with the available data, has a corrolary in aging, and is amenable to direct experimental testing particularly with flow cytometry as a promising tool.

1996 - Earthworm coelomocytes in vitro: Cellular features and ''granuloma'' formation during cytotoxic activity against the mammalian tumor cell target K562 [Articolo su rivista]
Quaglino, Daniela; Cooper, El; Salvioli, S; Capri, M; Suzuki, Mm; Ronchetti, Ivonne; Franceschi, C; Cossarizza, Andrea
abstract

Earthworms possess specific, adaptive, cellular immunodefense as well as non-specific responses found in other complex metazoans. Here we characterized coelomocytes from the earthworm Eisenia foetida by electron microscopy and cytofluorimetric analyses, and investigated structural changes that occur when effector coelomocytes and target K562 erythromyeloid human tumor cells interact during cytotoxic activity. In in vitro cultures 1) the two earthworm cell types (i.e. small and large coelomocytes) retained their morphological features; 2) their DNA content was significantly less than that of human lymphocytes and the erythromyeloid human tumor cell line K562; 3) significant percentages of coelomocytes were found to be in S or G2/M phases of the cell cycle. When cultivated alone for up to 3 h, coelomocytes formed no aggregates. However, when mixed with K562, coelomocytes spontaneously killed tumor cells, and cytotoxic reactivity was accompanied by the formation of multiple aggregates similar to granulomas. These results are the first to describe this type of earthworm non-specific ''inflammatory'' response in vitro against tumor cells.

1996 - Earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines [Articolo su rivista]
Cossarizza, Andrea; Cooper, El; Suzuki, Mm; Salvioli, S; Capri, M; Gri, G; Quaglino, Daniela; Franceschi, C.
abstract

Earthworm coelomocytes (leukocytes) effect cytotoxicity at significantly high levels against the NK-sensitive, human tumor cell line, K562, and the NK-resistant targets (U937, BSM, GEM). By cytofluorimetric analyses using mouse anti-human monoclonal antibodies and by morphological evaluations, two types of coelomocytes were identified: (1) small (8-11 mu m) electron-dense cells (SC): CD11a+, CD45RA+, CD45RO+, CDw49b+, CD54+, beta(2)-m+, and Thy-1+; (2) large (12-15 mu m) electron-lucent cells (LC) that are negative for these markers. Both cell types were negative for other CD and MHC class I and class II markers. SC were active during recognition, rapidly binding to targets; LC were phagocytic. Release of Cr-51 revealed rapid, significant, and equal levels of killing at 4 degrees, 20 degrees, and 37 degrees C. We propose that primitive NK-like activity appeared early in evolution.

1996 - Erratum: Protective effect of N-acetylcysteine in tumor necrosis factor- α-induced apoptosis in U937 cells: The role of mitochondria (Experimental Cell Research (1995) 220:1 (232-240)) [Abstract in Rivista]
Cossarizza, Andrea
abstract

1996 - Functional heterogeneity of an isolated mitochondrial population revealed by cytofluorometric analysis at the single organelle level [Articolo su rivista]
Cossarizza, Andrea; Ceccarelli, Daniela; Masini, Alberto
abstract

Isolated rat Liver mitochondria were incubated under various metabolic conditions to determine their membrane potential (MMP) as measured continuously by a tetraphenylphosphonium (TPP+)-selective electrode, By how cytometry, a parallel analysis of fluorescence emissions observing single mitochondria stained with the lipophilic cation 5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethylbenzimidazolcarbocyanine iodide (JC-l) revealed linear correlation between the median orange fluorescence (FL2) due to J-aggregate formations and MMP values measured by TPP+. No correlation was detected with the green fluorescence (FL1) emission. A significantly higher correlation appeared between the FL2/FL1 ratio and MMP values. Within the same mitochondrial population, cytofluorimetric analysis revealed the presence of various classes of organelles with different MMP, whose distribution was dependent on metabolic condition. The highest functional heterogeneity was found in deenergized mitochondria, while the highest homogeneity was observed during the first phase of the phosphorylative process. Thus, these data suggest that the cytofluorimetric use of JC-l provides direct experimental evidence for the hypothesis of functional mitochondrial heterogeneity, at least with respect to their membrane potential.

1996 - Mitochondria are selective targets for the protective effects of heat shock against oxidative injury [Articolo su rivista]
Bs, Polla; S., Kantengwa; D., Francois; S., Salvioli; C., Franceschi; C., Marsac; Cossarizza, Andrea
abstract

Heat shock (HS) proteins (HSPs) induce protection against a number of stresses distinct from HS, including reactive oxygen species, In the human premonocytic line U937, we investigated in whole cells the effects of preexposure to HS and exposure to hydrogen peroxide (H2O2) on mitochondrial membrane potential, mass, and ultrastructure. IIS prevented H2O2-induced alterations in mitochondrial membrane potential and cristae formation while increasing expression of HSPs and the protein product of bcl-2. Protection correlated best with the expression of the 70-kDa HSP, hsp70. We propose that mitochondria represent a selective target for HS-mediated protection against oxidative injury.

1996 - Prolactin increases the susceptibility of primary leukemia cells to NK and LAK effectors [Articolo su rivista]
Oberholtzer, E; Contarini, M; Veglia, F; Cossarizza, Andrea; Franceschi, C; Geuna, M; Provinciali, M; Distefano, G; Sissom, J; Brizzi, Mf; Pegoraro, L; Matera, L.
abstract

Our previous studies have shown that prolactin (PRL), a pituitary and lymphocyte hormone and a ligand of the cytokine/hemopoietin receptors (R) superfamily, acts synergistically with interleukin (IL)-2 on the development of lymphokine activated killer (LAK) cells and enhances the effects of GM-CSF and IL-3 on myeloid progenitors´ proliferation and differentiation. More recently, we have demonstrated that GM-CSF and IL-3 increase the sensitivity of acute myeloid leukemic (AML) cells to LAK activity. Together, these findings have prompted us to study the role of PRL on the target arm of the LAK response. We show here that CD33+ blasts from AML patients express membrane PRL-R and that the PRL/PRL-R interaction is followed by increased susceptibility to natural killer (NK) (p < 0.02) and LAK (p < 0.001) cells. As predicted from the dimerization model of PRL-R and in agreement with previous reports, the response of AML blasts to PRL was bell-shaped with a trend peak at 25 ng/ml. Although enhanced lysis occurred at the target recognition level, it was not accompanied by changes in the MHC class I, cellular adhesion molecules, or myeloid differentiation antigens. Cell cycle recruitment and lysis increased concurrently in three cases studied, suggesting a modulatory action of PRL on the expression of putative cycle-related NK/LAK-target structures. Together, these data strengthen the role of PRL in the LAK response. Copyright

1996 - Successful immunosenescence and the remodelling of immune responses with ageing [Articolo su rivista]
Franceschi, C; Monti, D; Barbieri, D; Salvioli, S; Grassilli, E; Capri, M; Troiano, L; Guido, M; Bonafe, M; Tropea, F; Salomoni, P; Benatti, F; Bellesia, E; Macchioni, S; Anderlini, R; Sansoni, P; Mariotti, S; Wratten, Ml; Tetta, C; Cossarizza, Andrea
abstract

In recent decades, major theoretical and technological advances have been achieved in the field of immunology. These have allowed the scientific community to analyse the immune system in a much more sophisticated manner than was possible even 20 years ago. Moreover, great theoretical changes have also occurred in gerontology - in particular, the hypothesis has been put forward that ageing and diseases are two different phenomena, and that successful ageing, i.e. ageing in good psychophysical conditions, is really possible for most humans and animals. Immunosenescence was then carefully investigated, either in selected healthy people of advanced age or in the oldest old people, such as healthy centenarians. The main results showed that most immune parameters are indeed well preserved even at this far advanced age. This paper deals with some of the most important theoretical problems of immunosenescence. An immunological tenet was that the most important phenomenon of immunosenescence is the involution of the thymus. In most textbooks and papers it is taken for granted that the thymus starts its involution immediately after puberty. When people aged 60-65 were considered old, it was not difficult to think that they could live for the rest of their life with a fully involuted thymus. The findings on centenarians challenge this tenet, as they have only a small reduction of T lymphocytes, and a relatively normal number of virgin and memory T cells, together with a functional T cell repertoire. Other observations reported here on centenarians, concerning the activity of B lymphocytes and the cytokine network, as well as those on the well-preserved innate immunity and the cells' capability of undergoing proliferation after appropriate stimuli, suggest that complex immune changes occur with age, but also indicate that we have to modify our attitude, to grasp the new scenario which is emerging. Immunosenescence can no longer be considered as a unidirectional deterioration, and this complex phenomenon is much better described by terms such as 'remodelling', 'reshaping' or 'retuning'.

1996 - TNF alpha alters mitochondrial membrane potential in L929 but not in TNF alpha-resistant L929.12 cells: Relationship with the expression of stress proteins, annexin 1 and superoxide dismutase activity [Articolo su rivista]
Polla, Bs; Jacquiersarlin, Mr; Kantengwa, S; Mariethoz, E; Hennet, T; Russomarie, F; Cossarizza, Andrea
abstract

Tumour necrosis factor alpha (TNF alpha) cytotoxicity is mediated, at least in part, by oxidative stress and phospholipase A2 activation. The first post-receptor events to be observed in TNF alpha-sensitive lines are the generation of superoxide anion (O-2(-)) within the mitochondria and the activation of phospholipase A2. Using the lipophilic dye JC-1 to determine mitochondrial membrane potential, we showed that TNF alpha induces time-dependent alterations in mitochondrial membrane potential in L929 cells but not in the TNF alpha-resistant L929.12 subclone. Heat shock (HS) proteins (HSP) and superoxide dismutase (SOD) have been shown to protect cells from TNF alpha cytotoxicity, while glucose regulated proteins (GRP) and annexins might also be involved in cellular protection. We thus compared the expression of HSP, grp78 and annexin 1 as well as SOD activity in TNF alpha sensitive and resistant lines. We found no difference in the expression of HSP, grp78 or annexin 1, but an increase in the constitutive activity of SOD in the L929.12 cells as compared to L929. Furthermore, SOD was inducible by TNF alpha in L929 cells, but not in L929.12 cells. These data suggest that in TNF alpha-resistant Lines, mitochondrial damage by TNF alpha is prevented by an increase in SOD rather than in overexpression of stress proteins or annexins.

1996 - Use of flameless atomic absorption spectroscopy in immune cytolysis for non radioactive determination of killer cell activity [Articolo su rivista]
Borella, Paola; Bargellini, Annalisa; S., Salvioli; Cossarizza, Andrea
abstract

We describe here a novel method to evaluate natural killer (NK) cytolytic activity by use of nameless atomic absorption spectroscopy (GF-AAS). This technique may be adopted for use in laboratories equipped with electrothermal atomic absorption spectrometers. Nonradioactive Cr as Na2CrO4 was used to label target cells (K562), and cell lysis was evaluated by measuring Cr released after 4 h of incubation with the effecters. We selected 520 mu g/L as the optimal dose for labeling targets, between 12 and 20 h as the optimal incubation time, and 10(4) cells as the optimal target size. Advantages of this method include: (a) exclusion of radioactive tracer, with no risk for workers; (b) limited costs; (c) high sensitivity and reproducibility; (d) possibility to store samples; and (e) better control of Cr used for labeling cells due to well-determined, fixed Cr concentrations in the range of nontoxic and linear cellular uptake. Comparison with data obtained by conventional Cr-51 labeling of targets killed by the same effecters was excellent, yielding comparable results and corroborating the method.

1995 - 3-AMINOBENZAMIDE PROTECTS CELLS FROM UV-B-INDUCED APOPTOSIS BY ACTING ON CYTOSKELETON AND SUBSTRATE ADHESION [Articolo su rivista]
Malorni, W; Rivabene, R; Straface, E; Rainaldi, G; Monti, D; Salvioli, S; Cossarizza, Andrea; Franceschi, C.
abstract

3-aminobenzamide (3-ABA) is an inhibitor of poly-(ADP-ribose)-polymerase, an enzyme involved in numerous subcellular processes, including cell death. Recently, a target effect of the drug on some cytoskeletal elements has also been described (Malorni et al., Biochem. Biophys. Res. Commun. 202: 915-922, 1994). In this study we evaluated the ability of 3-ABA to interfere with UV-B ray-induced apoptosis in cells selected for their cytoskeletal features and their different capability to adhere to the substrate. Human melanoma (M14) and epithelial (A431) cell lines and murine primary fibroblastic cultures (MFC) were studied. Our results indicate that cytoskeleton is indeed an important cellular target of 3-ABA, which can prevent apoptotic cell death by UV-B through a specific effect on the adhesion properties of the cells. indeed, an inverse correlation was observed between sensitivity to UV-B-induced apoptosis (M14&gt;A431&gt;MFC) and substrate adhesion (MFC&gt;A431&gt;M14). The potential relevance of these observations to understand the possible relationships among apoptosis, cytoskeletal functions and substrate adhesion is discussed.

1995 - AUTOGENEIC BUT NOT ALLOGENEIC EARTHWORM EFFECTOR CELOMOCYTES KILL THE MAMMALIAN TUMOR-CELL TARGET K562 [Articolo su rivista]
Cooper, El; Cossarizza, Andrea; Suzuki, Mm; Salvioli, S; Capri, M; Quaglino, Daniela; Franceschi, C.
abstract

Earthworm coelomocytes have been used as effector cells against the human tumor target, K562. To first assess the viability of effecters, incorporation of [H-3]-thymidine was tested and was higher in autogeneic (A double left right arrow A, self) than in allogeneic (A double left right arrow B, nonself) coelomocytes, A double left right arrow A showed significantly greater numbers in S, G2, or M phases than A double left right arrow B coelomocytes. When A double left right arrow A or A double left right arrow B were cultured, no significant cell killing occurred in either, as measured in a 4-hr Cr-51 release assay, A double left right arrow A but not A double left right arrow B killed K562 target cells, Cytotoxicity was dependent upon membrane binding between small, electron-dense coelomocytes and targets; it was enhanced by adding PHA. The heat labile supernatant from A double left right arrow A but not from A double left right arrow B killed K562 targets after cultivation for 10 min at 22 degrees C, but not immediately after washing, Recognition of, binding to, and killing of foreign cells in a natural killer cell-like reaction may reflect natural immunity in earthworms.

1995 - Apoptosis and immunosenescence [Articolo su rivista]
Monti, D; Cossarizza, Andrea; Salvioli, S; Barbieri, D; Macchioni, S; Guido, M; Baggio, Giosuè Gabriele; Franceschi, C.
abstract

Apoptosis and immunosenescene

1995 - Evidence for dissimilar mechanisms of enhancement of inorganic and organic hydroperoxide cytotoxicity by L-histidine [Articolo su rivista]
Guidarelli, A; Sestili, P; Cossarizza, Andrea; Franceschi, C; Cattabeni, F; Cantoni, O.
abstract

L-Histidine markedly increases inorganic and organic hydroperoxide-induced cytotoxicity and DNA single-strand breaks (SSBs) in Chinese hamster ovary cells. These effects were prevented by the iron chelator o-phenanthroline and were insensitive to the antioxidant N,N'-diphenyl-1,4-phenylenediamine, An excess of L-glutamine, a competitive inhibitor of L-histidine uptake, prevented the L-histidine-mediated enhancement of cytotoxicity induced by both inorganic and organic peroxides. L-Glutamine did not affect the level of DNA SSBs produced by H2O2/L-histidine, although it abolished the enhancement of SSB formation triggered by L-histidine in cells exposed to the organic peroxides. DNA SSBs generated by the organic hydroperoxides either alone or associated with L-histidine were removed with superimposable kinetics, whereas those produced by H2O2 in the presence of the amino acid were repaired more slowly than SSBs produced by the oxidant alone. DNA double-strand breaks, which are considered to be highly cytotoxic, were detected only in cells treated with H2O2 and L-histidine. Finally, L-histidine was shown to markedly increase the extent of mitochondrial damage produced by organic but not by inorganic hydroperoxides.

1995 - FUNCTIONAL INTERCELLULAR-ADHESION MOLECULE-3 IS EXPRESSED BY FRESHLY ISOLATED EPIDERMAL LANGERHANS CELLS AND IS NOT REGULATED DURING CULTURE [Articolo su rivista]
Zambruno, G; Cossarizza, Andrea; Zacchi, V; Ottani, D; Luppi, Am; Giannetti, Alberto; Girolomoni, G.
abstract

Activation of T lymphocytes by antigen-presenting cells requires the interaction of major histocompatibility complex/antigen complexes with the T-cell receptor as well as the binding of co-stimulatory molecules to receptors on T cells, Freshly isolated epidermal Langerhans cells (LC) do not display a significant number of co-stimulatory molecules, After short-term culture, LC express and then upregulate intercellular adhesion molecule-1 (ICAM-1) (CD54), leukocyte function-associated antigen (LFA)-3 (CD58), and B7-1 (CD80) accessory molecules and exhibit an enhanced antigen-presenting function. The present study examined the presence on human LC of the LFA-1 ligands ICAM-2 (CD102) and ICAM-3 (CD50) and their functional role in the activation of allogeneic T cells; Immunohistochemistry of skin sections and flow-cytometry analysis of freshly procured epidermal cell suspensions showed that LC (CD1a(+) or HLA-DR(+)) expressed ICAM-3 but not ICAM-2. After 48-72-h culture in the presence of granulocyte/macrophage colony-stimulating factor, LC did not stain for ICAM-2 but expressed ICAM-3 at the same level as fresh cells, Incubation of both freshly isolated and cultured LC with monoclonal antibodies directed against ICAM-3 reduced T-cell proliferation (25-75% inhibition) in the primary allogeneic mixed leukocyte reaction assay; incubation of cultured LC with anti-ICAM-1 and anti-ICAM-3 synergistically reduced T-cell response, The results indicate that ICAM-3 is constitutively expressed and represents an important costimulatory molecule on freshly isolated LC but, in contrast to other accessory molecules, is not subjected to regulation during LC culture.

1995 - Human natural killer cytotoxic activity is not affected by in vitro exposure to 50-Hz sinusoidal magnetic fields [Articolo su rivista]
Ramoni, C; Dupuis, Ml; Vecchia, P; Polichetti, A; Petrini, C; Bersani, F; Capri, M; Cossarizza, Andrea; Franceschi, C; Grandolfo, M.
abstract

Epidemiological studies have suggested, but not demonstrated, a role of exposure to 50/60-Hz magnetic fields in increasing cancer risk in man (workers and the general population). A possible target of magnetic fields is the immune system. In particular, it is known that an important defence against cancer is represented by natural killer (NK) cells capable of killing cancer cell targets. To test this hypothesis, human NK cells, stimulated or not with phytohaemagglutinin or interleukin 2, were exposed to 50-Hz sinusoidal magnetic fields before or during the cytotoxicity test, and then mixed with a variety of target cancer cell lines (Daudi, Raji, U937, H14, IGROV, SW626, K562, HL60). The experiments were performed in two laboratories (Rome and Modena) by means of two different exposure systems. The results of both laboratories suggest that 50-Hz sinusoidal magnetic fields with flux densities up to 10 mT do not affect the cytotoxic activity of human NK cells.

1995 - INVOLVEMENT OF CD45 IN DEXAMETHASONE-INDUCED AND HEAT-SHOCK-INDUCED APOPTOSIS OF RAT THYMOCYTES [Articolo su rivista]
Troiano, L; Monti, D; Cossarizza, Andrea; Lovato, E; Tropea, F; Barbieri, D; Morale, Mc; Gallo, F; Marchetti, B; Franceschi, C.
abstract

CD45 is a transmembrane tyrosine-specific phosphatase which participates in lymphoid cell signal transduction during T cell activation, as well as in intrathymic negative and positive selection. In mammals, this molecule exhibits a variety of isoforms of different molecular weight, whose roles have still to be fully elucidated. We report here that apoptosis of rat thymocytes after in vitro dexamethasone and heat shock treatment was accompanied by an early significative increase of cells expressing CD45RC, the high molecular weight isoform of CD45 molecule. The same phenomenon was observed in thymocytes derived from irt vivo dexamethasone-treated rats. However, the increase of CD45RC(+) cells was not apparently characteristic of cells undergoing apoptosis, as the same phenomenon was also observed in rat thymocytes induced to proliferate by Concanavalin A. On the whole, these results suggest that CD45 modulation can be added to the list of early molecular events, such as the increased expression of genes (ornithine decarboxylase), proto-oncogenes (c-fos, c-jun, c-myc) and activation of transcription factors (AP-I, NFkB), we previously demonstrated in the same experimental model to occur and to be shared by these two apparently opposite biological processes, i.e.,cell proliferation and apoptosis, both likely depending on a complex balance of protein phosphorylation and dephosphorylation.

1995 - Immunosenescence in humans: Deterioration or remodelling¿? [Articolo su rivista]
Cossarizza, Andrea
abstract

1995 - Introduction: The reshaping of the immune system with age [Articolo su rivista]
Cossarizza, Andrea
abstract

1995 - Lack of selective V beta deletion in CD4+ or CD8+ T lymphocytes and functional integrity of T-cell repertoire during acute HIV syndrome [Articolo su rivista]
Cossarizza, Andrea; C., Ortolani; Mussini, Cristina; Guaraldi, Giovanni; N., Mongiardo; V., Borghi; D., Barbieri; E., Bellesia; Mg, Franceschini; B., Derienzo; C., Franceschi
abstract

Objective: To study the V beta T-cell repertoire in peripheral blood lymphocytes (PBL) during acute HIV syndrome by using several anti-V beta monoclonal antibodies (MAb) and to analyse its functionality by stimulating PBL with superantigens (SAg) such as Staphylococcus aureus enterotoxins. Methods: Cytofluorimetric analysis of V beta T-cell-receptor expression was performed on PBL from eight patients with symptomatic, acute HIV-1 primary infection, showing a dramatic decrease of CD4+ PBL accompanied by a marked increase in activated/memory CD8+ T cells, and on 12 age- and sex-matched healthy controls. PBL were then isolated, stimulated with different SAg, anti-CD3 MAb or phytohaemagglutinin and cultured for 3 days. PBL capability to progress through cell cycle was studied by the classic cytofluorimetric method of bromodeoxyuridine incorporation and DNA staining with propidium iodide. Results: Despite the presence of a few expansions of some V beta families among CD8+ T lymphocytes, no gross alterations in T-cell repertoire were present in patients with acute HIV syndrome. Its functionality was maintained overall, as PBL responsiveness to SAg was well preserved. Interestingly, all CD8+ T cells, although bearing different V beta T-cell receptors, expressed marked signs of activation, i.e., CD45R0, CD38 and major histocompatibility complex class II molecules, and also high amounts of CD11a and CD18. Conclusions: Our data suggest, at least in the early phases and in the acute form of the infection, that HIV is not likely to act as a SAg. However, further studies are needed to analyse other sites, such as lymph nodes, where HIV could exert other, significant effects, and to study the expression of other V beta families than those investigated here.

1995 - MITOCHONDRIA AND APOPTOSIS - A CYTOFLUOROMETRIC APPROACH [Articolo su rivista]
Cossarizza, Andrea; Salvioli, S; Franceschini, Mg; Kalashnikova, G; Barbieri, D; Monti, D; Grassilli, E; Tropea, F; Troiano, L; Franceschi, C.
abstract

no abstract available

1995 - MITOCHONDRIAL MASS AND MEMBRANE-POTENTIAL IN CELOMOCYTES FROM THE EARTHWORM EISENIA-FOETIDA - STUDIES WITH FLUORESCENT-PROBES IN SINGLE INTACT-CELLS [Articolo su rivista]
Cossarizza, Andrea; Cooper, El; Quaglino, Daniela; Salvioli, S; Kalachnikova, G; Franceschi, C.
abstract

Earthworm coelomocytes exist in two forms, i.e., small (SC) and large (LC) cells, as demonstrated by velocity sedimentation, electron microscopy, and FCM. However, we know little concerning the functional activities of various, important organelles, such as mitochondria. In comparison with SC, LC from Eisenia foetida have a higher number of mitochondria, and, accordingly, showed a greater fluorescence intensity when mitochondrial mass was measured by nonyl acridine orange and FCM. To measure MMP we used both the lipophilic cationic probe JC-1 and Rh123. The intracellular localization of JC-1 in SC and LC was observed by fluorescence microscopy. Using JC-1, MMP was analyzed separately on SC and LC by FCM, and significant percentages of coelomocytes (>95% of SC and about 90% of LC) displayed a high MMP. Adding 0.1 mu M VAL caused most SC to depolarize, while this occurred in only a few LC. Rh123 gave different results: no effects of VAL were observed either in SC or in LC. In coelomocytes there may be several energy-independent Rh123-binding sites whose role must still be elucidated. On the whole, these data indicate that it is possible to analyze mitochondrial parameters by FCM in intact invertebrate coelomocytes, and that the type of cell and the probe used have a critical importance.

1995 - Massive activation of immune cells with an intact T cell repertoire in acute human immunodeficiency virus syndrome [Articolo su rivista]
Cossarizza, Andrea; C., Ortolani; Mussini, Cristina; V., Borghi; Guaraldi, Giovanni; N., Mongiardo; E., Bellesia; Mg, Franceschini; B., Derienzo; C., Franceschi
abstract

In 8 patients with symptomatic, acute primary infection with human immunodeficiency virus (HIV), a dramatic and persistent decrease in CD4(+) lymphocytes was seen, accompanied by a marked increase in activated/memory CD8(+) T cells (CD38(+), CD45R0(+), HLA-DR(+), with high amounts of cell adhesion molecules), which represented most circulating lymphocytes, but no gross alterations in vp T cell repertoire. Extremely high plasma levels of proinflammatory cytokines were observed. Three patients were followed for 2-3 years: The number of CD4(+) cells, extremely low at first, increased significantly in a few months but decreased rapidly after a short stable period. Cytotoxic T lymphocytes bearing markers of immunologic activation/memory could play an important role in the earliest phases of the disease. It remains to be established how such a dramatic onset could determine the rapid progression of the infection that seems characteristic of patients with acute HIV syndrome.

1995 - PROTECTIVE EFFECT OF N-ACETYLCYSTEINE IN TUMOR NECROSIS FACTOR-ALPHA-INDUCED APOPTOSIS IN U937 CELLS - THE ROLE OF MITOCHONDRIA [Articolo su rivista]
COSSARIZZA, Andrea; FRANCESCHI, C; MONTI, D; SALVIOLI, S; BELLESIA, E; RIVABENE, R; BIONDO, L; RAINALDI, G; TINARI, A; MALORNI, W.
abstract

The existence of two different pathways for cell death has been postulated. In addition to the passive and traumatic process leading to necrosis, an active program characterized by organelle integrity and called apoptosis has been described. A positive correlation between the apoptotic cell death process and oxidative imbalance has been demonstrated. In fact, the antioxidant N-acetylcysteine (NAG) seems to be capable of impairing the apoptotic program, replenishing intracellular reduced glutathione content in cells exposed to tumor necrosis factor-alpha (TNF) as apoptotic inducer. Moreover, protein synthesis inhibitors such as cycloheximide (CHX) can facilitate apoptotic triggering by TNF, and mitochondrial function was suggested to be essential in the TNF-mediated apoptotic process. With this in mind, a specific analysis using the JC-1 probe, a fluorescent dye which is capable of indicating mitochondrial membrane potential (Delta Psi m) changes, was carried out. Our results show that TNF exposure is capable of altering the mitochondria and that NAC protection from CHX f TNF-induced apoptosis could be due to a direct effect of the drug on mitochondrial integrity and function.

1995 - Reply from Franceschi et al [Abstract in Rivista]
Franceschi, C.; Monti, D.; Cossarizza, A.; Sansoni, P.
abstract

1995 - SENDAI VIRUS AND HERPES-VIRUS TYPE-1 INDUCE APOPTOSIS IN HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS [Articolo su rivista]
Tropea, F; Troiano, L; Monti, D; Lovato, E; Malorni, W; Rainaldi, G; Mattana, P; Viscomi, G; Ingletti, Mc; Portolani, Marinella; Cermelli, Claudio; Cossarizza, Andrea; Franceschi, C.
abstract

Recent reports suggest that several viruses, besides human immunodeficiency virus, induce apoptosis in infected cells, We report here that Sendai virus or Herpes simplex virus type 1 (HSV-1), two potent inducers of interferon-alpha, caused cell death in a consistent number of human peripheral blood mononuclear cells, A careful analysis of infected cells by different techniques, such as optical and electron microscopy, DNA agarose gel electrophoresis, and cytofluorimetric analysis of DNA content, showed that cell death was of apoptotic type, Sendai virus was more apoptogenic than HSV-1, and it was further studied to understand the mechanism(s) by which it induced apoptosis, Physical (uv and heat) and chemical (beta-propiolactone) inactivation reduced or abolished the apoptogenic power of Sendai virus, The use of a novel technique, which allows the study of mitochondrial membrane potential (MMP) in intact cells by flow cytometry, showed that a decrease of MMP is concomitant with the appearance of the hypodiploid peak. These results suggest that Sendai virus and HSV-1 can be added to the list of viruses causing apoptosis, which appears to be a general mechanism occurring during viral infection.

1995 - T cell repertoire usage in humans, from newborns to centenarians [Articolo su rivista]
Cossarizza, Andrea
abstract

1995 - THE AGING THYROID [Articolo su rivista]
Mariotti, S; Franceschi, C; Cossarizza, Andrea; Pinchera, A.
abstract

no abstract available

1995 - THE USE OF NONRADIOACTIVE CHROMIUM AS AN ALTERNATIVE TO CR-51 IN NK ASSAY [Articolo su rivista]
Borella, Paola; Bargellini, Annalisa; Salvioli, S; INCERTI MEDICI, C; Cossarizza, Andrea
abstract

A novel method to measure target cell cytolysis based on the use of 'cold', non-radioactive chromium and on the determination of metal release by graphite furnace atomic absorption spectroscopy (FAAS) is proposed. Natural killer (NK) assays were performed by labelling target cells with chromium as Na2CrO4, and results were compared with those obtained by conventional overnight labelling with Cr-51 of targets killed by the same effecters. The cytotoxic capacity of peripheral blood lymphocytes from healthy subjects was evaluated, and NK activity measured with both methods showed a good agreement at each of the tested effector to target cell ratios (between 100:1 and 1:1), with a high and significant coefficient of correlation (r = 0.931, p < 0.0001). The selection of the appropriate Cr concentrations for labelling target cells took into account both the sensitivity of our instrumentation and the possible toxic effects of the metal. A study of the effects of Cr on the cell line (K562) which is usually employed as a target in NK tests showed that Cr could have a detrimental effect on cellular function, with significant numbers of cells with depolarised mitochondria and reduced DNA synthesis after 24 h incubation using Cr levels higher than 15 mu mol/l (780 mu g/l). The method proposed here has a number of advantages, including the use of a non-radioactive tracer, limited costs, high sensitivity and reproducibility, and the possibility of storing samples. In addition, the technique uses a fixed Cr concentration which is known to be non toxic.

1995 - THYROID AUTOIMMUNITY AND AGING [Articolo su rivista]
Pinchera, A; Mariotti, S; Barbesino, G; Bechi, R; Sansoni, P; Fagiolo, U; Cossarizza, Andrea; Franceschi, C.
abstract

Ageing is associated with the appearance of several serum autoantibodies, including thyroid autoantibodies. The biological and clinical significance of this phenomenon is still unknown, since, with the exception of primary myxedema, the prevalence of clinically overt thyroid autoimmune diseases is not increased in the elderly. The peculiar link between autoimmune thyroid failure and ageing is also underscored by the high prevalence of subclinical hypothyroidism in elderly subjects with positive serum thyroid autoantibodies, and could be the consequence of preferential age-dependent expression of destructive effector mechanisms and/or increased target gland susceptibility. Thyroid autoimmunity and subclinical hypothyroidism have also been implicated in the pathogenesis of other age-associated disorders, in particular coronary heart disease. Interestingly, recent data from our laboratories showed that thyroid autoantibodies are rare in healthy centenarians and in other highly selected aged populations, while they are frequently observed in unselected or hospitalized elderly. Taken together, these data suggest that thyroid autoimmune phenomena are not the consequence of the ageing process itself, but rather might be related to age-associated disease.

1995 - The immunology of exceptional individuals: the lesson of centenarians [Articolo su rivista]
C., Franceschi; D., Monti; P., Sansoni; Cossarizza, Andrea
abstract

Centenarians are the best example of successful ageing, since they have escaped the major age-associated diseases, and most are in good mental and physical condition. Here, Claudio Franceschi and colleagues discuss how the study of their immune systems reveals that several immune parameters are well conserved, suggesting that a complex remodelling of most immune parameters occurs with age, rather than a unidirectional deterioration.

1994 - 3-AMINOBENZAMIDE INDUCES CYTOSKELETON REARRANGEMENT IN M14 MELANOMA-CELLS [Articolo su rivista]
Malorni, W; Rainaldi, G; Straface, E; Rivabene, R; Cossarizza, Andrea; Capri, M; Monti, D; Franceschi, C.
abstract

3-aminobenzamide (3-ABA) is an inhibitor of poly-ADP-ribose-polymerase, an enzyme involved in numerous subcellular processes including cell death. With the aim of contributing to clarify the mode of action of the drug, which is still poorly understood, its effects on cultured melanoma cells M14 have been assessed. in particular, an impairment of cell growth accompanied by the formation of long and numerous dendritic-like protrusions has been detected. This finding appears to be due to a specific effect of the drug on some cytoskeletal elements and could be associated with its differentiating capability. This finding, together with previous data from our group - Biochem. Biophys. Res. Commun., (1994) 199, 525-530 and 1250-1255 - suggests that cytoskeleton is an important target of 9-ABA.

1994 - CELL-DEATH PROTECTION BY 3-AMINOBENZAMIDE - IMPAIRMENT OF CYTOSKELETON FUNCTION IN HUMAN NK CELL-MEDIATED KILLING [Articolo su rivista]
Malorni, W; Rainaldi, G; Straface, E; Rivabene, R; Cossarizza, Andrea; Salvioli, S; Monti, D; Franceschi, C.
abstract

3-aminobenzamide, an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase, is capable of interfering with the tumor cell lysis induced by specialized cells from the immune system, i.e., natural killer (NK) cells. In this report we suggest that the mechanism by which the drug can exert its protective effects on target cell killing by NK effectors can also be due to its ability to impaire cell-to-cell conjugate formation (binding), without affecting either the expression of cell adhesion molecules nor the features of effector-target cell contact. The mechanism of this inhibition seems to be associated with an alteration of cytoskeletal elements involved during conjugate formation, i.e. with the integrity and function of the microfilament system.

1994 - CELL-DEATH PROTECTION BY 3-AMINOBENZAMIDE AND OTHER POLY(ADP-RIBOSE)POLYMERASE INHIBITORS - DIFFERENT EFFECTS ON HUMAN NATURAL-KILLER AND LYMPHOKINE-ACTIVATED KILLER-CELL ACTIVITIES [Articolo su rivista]
MONTI, D; COSSARIZZA, Andrea; SALVIOLI, S; FRANCESCHI, C; RAINALDI, G; STRAFACE, E; RIVABENE, R; MALORNI, W.
abstract

The death of target cells by cytotoxic effector cells is a relevant biological phenomenon, where cells are activated and a very quick apoptotic program occurs. In order to test the hypothesis that the nuclear enzyme poly(AD P-ribose)polymerase (PADPRP) plays a role in such a process, a variety of PADPRP inhibitors such as 3-aminobenzamide, nicotinamide, 4-aminobenzamide and luminol were used. All of them were able to strongly inhibit K562 target cell killing by human effector natural killer cells (NK) in a 4hr Cr-51 release assay. PADPRP inhibitors were much less effective in protecting target cells when lymphokine activated killer cells (LAK) were used as effecters. These substances were active only when both target and effector cells were mixed, being ineffective on target or effector cells alone. On the whole, these data indicate that PADPRP is involved in the death of target cells. Moreover, the different sensitivity of NK and LAK activities to PADPRP inhibitors suggests that the molecular mechanisms underlying these two types of cytotoxicity are at least partially different.

1994 - D-ribose and deoxy-D-ribose induce apoptosis in human quiescent peripheral blood mononuclear cells. [Articolo su rivista]
Barbieri, D.; Grassilli, E.; Monti, D.; Salvioli, S.; Franceschini, Mg.; Franchini, A.; Bellesia, E.; Salomoni, P.; Negro, P.; Capri, M.; Et, Al.; Cossarizza, Andrea
abstract

In previous papers we reported that D(-)-ribose and 2-deoxy-D-ribose, which rank at the top among reducing sugars, kill a variety of human and animal cells and cell lines. Here we demonstrate that these two sugars induce apoptosis in human quiescent peripheral blood mononuclear cells which are relatively insensitive to apoptosis. Apoptosis was assessed by morphological changes, DNA fragmentation by agarose gel electrophoresis and the appearance of an hypodiploid peak by flow cytometry. 2-deoxy-D-ribose was more potent than D(-)-ribose and apoptosis was evident from 48 hours of culture onwards. 2-deoxy-D-ribose-induced apoptosis was inhibited by N-acetyl-L-cysteine, suggesting that glutathione metabolism and/or oxidative stress are involved in this type of apoptosis. Thus, D(-)-ribose and 2-deoxy-D-ribose can be useful tools to study the cellular and molecular events of apoptosis in human quiescent lymphocytes.

1994 - Expression And Function Of Nerve Growth-Factor And Nerve Growth-Factor Receptor On Cultured Keratinocytes. [Articolo su rivista]
Pincelli, Carlo; C., Sevignani; Manfredini, Rossella; Grande, Alexis; F., Fantini; L., Bracci Laudiero; L., Aloe; Ferrari, Sergio; Cossarizza, Andrea; Giannetti, Alberto
abstract

Keratinocytes, a key cellular component both for homeostasis and pathophysiologic processes of the skin, secrete a number of cytokines and are stimulated by several growth factors. Nerve growth factor (NGF) is synthesized in the skin and basal keratinocytes express the low-affinity nerve growth factor receptor (NGF-R). We present evidence that normal human keratinocytes in culture express the low- and the high-affinity NGF-R both at the mRNA level, as determined by reverse-transcription polymerase chain reaction and at the protein level, as shown by cytofluorimetric analysis. NGF significantly stimulates the proliferation of normal human keratinocytes in culture in a dose-dependent manner. This effect can be prevented by the addition of both an anti-NGF neutralizing antibody and a high-affinity NGF-R (trk) specific inhibitor, the natural alkaloid K252a. By contrast, keratinocyte proliferation is not inhibited by an anti - low-affinity NGF-R monoclonal antibody, thus suggesting that NGF effect on human keratinocytes is mediated by the high-affinity NGF-R. Moreover, NGF mRNA is expressed in normal human keratinocytes and NGF is secreted by keratinocytes in increasing amounts during growth, as detected by enzyme-linked immunosorbent assay. These results suggest that NGF could act as a cytokine in human skin and take part in disorders of keratinocyte proliferation.

1994 - Expression of B7 costimulatory molecule in cultured human epidermal Langerhans cells is regulated at the mRNA level. [Articolo su rivista]
Girolomoni, G; Zambruno, G; Manfredini, Rossella; Zacchi, V; Ferrari, Sergio; Cossarizza, Andrea; Giannetti, A.
abstract

Langerhans cells (LC) belong to the dendritic cell lineage and are the principal antigen-presenting cells of squamous epithelia. Short-term cultured LC (cLC) exhibit a marked augmented capacity to stimulate allogeneic T cells and acquire the ability to activate naive T cells, probably in relation to enhanced expression of accessory signals. In this study, we evaluated the expression of B7 costimulatory molecule (CD80) in human freshly isolated (fLC) and cLC at both the protein and mRNA level. Staining of frozen skin sections did not reveal any epidermal dendritic cell reactive with either of two different anti-B7 monoclonal antibodies. fLC in suspension did not exhibit any B7 staining as evaluated by two-color flow-cytometry analysis and immunoelectron microscopy. In contrast, LC that were cultured for 24-72 h displayed strong surface B7 reactivity with a characteristic patchy pattern. Treatment with dispase and trypsin did not reduce B7 staining of cLC. Following warming to 37°C, cLC tagged with anti-B7 monoclonal antibody and gold-conjugated secondary antibody could internalize surface B7 by using the organelles of receptor-mediated endocytosis. B7 mRNA, detected by the reverse-transcriptase polymerase chain reaction technique, was expressed at a low level in purified (&gt; 90% HLA-DR+) fLC but not in LC-depleted epidermal cells, and was markedly upregulated in purified cLC. The results indicate that 1) fLC do not express B7 protein on their surface, but acquire B7 during culture, 2) surface B7 is not sensitive to trypsin, 3) B7 expression is regulated primarily at the mRNA level, and 4) membrane B7 can be internalized within cLC. B7 molecule on CLC may be relevant to their increased antigen-presenting cell potency and ability to stimulate naive T lymphocytes.

1994 - G2.1 Earthworm leukocytes (CD11a+, CD45RA+, ß2-m+, Thy-1+) can kill human K562 tumor cell targets [Relazione in Atti di Convegno]
Cooper, E. L.; Suzuki, M. M.; Cossarizza, A.; Salvioli, S.; Capri, M.; Quaglino, D.; Pasquali-Ronchetti, I.; Franceschi, C.
abstract

1994 - MITOCHONDRIAL MODIFICATIONS DURING RAT THYMOCYTE APOPTOSIS - A STUDY AT THE SINGLE-CELL LEVEL [Articolo su rivista]
Cossarizza, Andrea; Kalashnikova, G; Grassilli, E; Chiappelli, F; Salvioli, S; Capri, M; Barbieri, D; Troiano, L; Monti, D; Franceschi, C.
abstract

Apoptosis is an active type of cell death, occurring under several physiological and pathological conditions, The role of cellular organelles such as mitochondria in this process is still an open question. We recently described a new method to measure mitochondrial membrane potential in intact cells using dow cytometry. Using this method we studied alterations of mitochondrial membrane potential in a classical model of apoptosis, i.e., dexamethasone-treated rat thymocytes. Moreover, apoptosis induced by heat shock was also studied in the same cells. Mitochondria are functionally intact during the early phases of apoptosis, when DNA fragmentation occurs, whereas early alteration in their potential and mass takes place after DNA damage. According to flow-cytometric analysis, the presence of a hypodiploid peak, an index of nuclear DNA loss, predates depolarization of mitochondrial membrane and decrease of mitochondrial mass. Loss of plasma membrane integrity, which indicates cell death and is revealed by the permeability to propidium iodide, eventually follows. All these phenomena are more evident in dexamethasone-treated cells than in heat-shocked cells, Thus, in these types of apoptosis the involvement of mitochondria is apparently not a primary event.

1994 - RELATIONSHIP BETWEEN CORTICOTROPIN-RELEASING FACTOR AND INTERLEUKIN-2 - EVOLUTIONARY EVIDENCE [Articolo su rivista]
Ottaviani, Enzo; Franchini, Antonella; Caselgrandi, E; Cossarizza, Andrea; Franceschi, C.
abstract

The addition of corticotropin-releasing factor (CRF) to molluscan hemocytes induces the release of biogenic amines (norepinephrine, epinephrine, dopamine), a phenomenon we have considered as an ancestral type of stress response [(1992) Gen. Comp. Endocrinol. 87, 354-360]. A similar but less significant response was observed following the addition of interleukin-2 (IL-2). Pre-incubation of hemocytes with IL-2 or anti-IL-2 monoclonal antibody significantly reduced or completely eliminated the CRF-induced release of biogenic amines. Further direct evidence of competition between CRF and IL-2 was revealed by immunocytochemical and cytofluorimetric analysis. The data are compatible with the presence of a unique (ancestral?) receptor on molluscan hemocytes, capable of binding both CRF and IL-2, two key molecules of the neuroendocrine and immune system, respectively.

1994 - Steroid regulation of cytokines. Relevance for TH1-to-TH2 shift? [Articolo su rivista]
Chiappelli, F.; Manfrini, E.; Franceschi, C.; Cossarizza, A.; Black, K. L.
abstract

1994 - UNEXPECTED PHENOTYPES AND PREDOMINANT TCR-V GENE USAGE IN RANDOM SELECTED SUBJECTS [Articolo su rivista]
Ortolani, C; Dandrea, E; Cibin, R; Radin, E; Cossarizza, Andrea
abstract

The presence of a subset of T lymphocytes with an irregular phenotype has been identified in the peripheral blood of 22 subjects, selected among more than 5000 individuals evaluated for peripheral blood lymphocyte subset distribution as part of a routine procedure. By Southern Blot Analysis 13 out of 14 of the analysed samples presented additional non-germline bands, indicative of monoclonal or oligoclonal T cell expansions. Moreover, the cytometric analysis showed that 7 out of 19 analysed samples were restricted for a subfamily of the TCR variable regions. Thus, lymphocyte subsets with phenotypic irregularities could represent the clonally driven expansion of otherwise normally subpopulations, which may be present in peripheral blood below the limit of detection of routine phenotypic analyses. Such clonal populations could exert a regulatory activity either on the pathogenetic mechanisms of the disease or in maintaining homeostasis in healthy people.

1994 - WHEN DID COMMUNICATION IN THE IMMUNE-SYSTEM BEGIN [Articolo su rivista]
Cooper, El; Zhang, Z; Raftos, Da; Habicht, Gs; Beck, G; Connors, V; Cossarizza, Andrea; Franceschi, C; Ottaviani, Enzo; Scapigliati, G; Parrinello, N.
abstract

Cytokines are a family of regulatory molecules that facilitate the communication between cells, most especially those of the hemopoietic system and there is evidence that the neuroendocrine system may benefit by this collaboration as well, Evolutionary evidence indicates that the beginnings of communication between cells with the protozoans as pheromone molecules. These underwent modification to become more akin to the cytokines of multicellular forms. Since the activities of invertebrate (ciliate pheromones) cytokines of multicellular forms. Since the activities of invertebrate (ciliate pheromones) cytokines are easily assayed in vertebrate systems, this suggests that the structure-function relationships of these molecules have been conserved. Cytokines have been present for millions of years in animals and as such, are important, ancient, and functionally conserved host defense molecules.

1993 - 50 HZ AC SINUSOIDAL ELECTRIC-FIELDS DO NOT EXERT GENOTOXIC EFFECTS (MICRONUCLEUS FORMATION) IN HUMAN-LYMPHOCYTES [Articolo su rivista]
Scarfi, Mr; Bersani, F; Cossarizza, Andrea; Monti, D; Zeni, O; Lioi, Mb; Franceschetti, G; Capri, M; Franceschi, C.
abstract

Electromagnetic fields produce a variety of effects in several biological systems, including human peripheral blood lymphocytes. A great concern exists regarding possible carcinogenic effects in subjects exposed environmentally to such fields in the vicinity of power lines and electric domestic appliances. Using human lymphocytes from 33 healthy donors and a sensitive cytogenetic method, the cytokinesis-block micronucleus assay, we have demonstrated that the exposure to 50-Hz AC sinusoidal electric fields over a wide range of intensities (0.5-10 kV/m in air) does not increase the spontaneous frequency of micronuclei. Moreover, these fields did not affect the mitomycin-C-induced micronucleus formation, suggesting that they did not exert any synergistic or antagonistic effect.

1993 - A new method for the cytofluorometric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzim idazolcarbocyanine iodide (JC-1) [Articolo su rivista]
Cossarizza, A.; Baccarani-Contri, M.; Kalashnikova, G.; Franceschi, C.
abstract

A new method for the cytofluorimetric analysis of mitochondrial membrane potential in intact cells has been developed by using the lipophilic cationic probe 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzim idazolcarbocyanine iodide (JC-1), whose monomer emits at 527 nm after excitation at 490 nm. Depending on the membrane potential, JC-1 is able of forming J-aggregates that are associated with a large shift in emission (590 nm). The color of the dye changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarized. In two human cell lines (K562 and U937), we have studied by flow cytometry the changes in membrane potential provoked by the K+ ionophor valinomycin, a drug known to affect mitochondrial membrane potential, while the K+/H+ ionophor nigericin, known to affect intracellular pH but not mitochondrial membrane potential, was used as control. The incubation with valinomycin for 10 min. at 37°C in a low K+ medium provoked a marked and dose-dependent reduction in JC-1 greenish orange fluorescence, while nigericin had no effect. © 1993 Academic Press, Inc.

1993 - CYTOFLUOROMETRIC IDENTIFICATION OF 2 POPULATIONS OF DOUBLE POSITIVE (CD4+,CD8+) T-LYMPHOCYTES IN HUMAN PERIPHERAL-BLOOD [Articolo su rivista]
Ortolani, C; Forti, E; Radin, E; Cibin, R; Cossarizza, Andrea
abstract

Two different subsets of CD4+,CD8+ T lymphocytes have been identified in peripheral blood collected from normal subjects and from patients with different diseases. The subpopulations differed in the degree of CD4 and CD8 antigen expression. Hence, it was possible to distinguish by cytofluorimetric analysis cells with a low (dim) or with a high (bright) fluorescence intensity after the staining with anti-CD4 or anti-CD8 mAbs. CD4+dim,CD8+bright lymphocytes were found in patients with EBV-infectious mononucleosis and were present for less than a month. CD4+bright,CD8+dim T cells were observed in neoplastic patients as well as in healthy subjects and were continuously present in similar percentages over a long period of time (at the moment, about 3 years). Both the subpopulations expressed CD2, CD3, CD5 antigens and had an alpha beta-TCR, but did not express CD1a or CD7. Only CD4+dim,CD8+bright cells expressed HLA-DR antigen and the activation marker CD38, while only CD4+bright,CD8+dim lymphocytes expressed CD56 and CD57 molecules. The hypothesis may be put forward that these two subsets represent an effort of the immune system to cope with different requirements, i.e., of viral or neoplastic origin, while it is not clear the meaning of these cells in healthy subjects.

1993 - Corticotropin-releasing-hormone modulates cytokines release in cultured human peripheral blood mononuclear cells. [Articolo su rivista]
Angioni, S; Petraglia, F; Gallinelli, A; Cossarizza, Andrea; Franceschi, C; Muscettola, M; Genazzani, Alessandro; Surico, N; Genazzani, Ar
abstract

..

1993 - EXPOSURE TO LOW-FREQUENCY PULSED ELECTROMAGNETIC-FIELDS INCREASES INTERLEUKIN-1 AND INTERLEUKIN-6 PRODUCTION BY HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS [Articolo su rivista]
Cossarizza, Andrea; Angioni, S; Petraglia, Felice; Genazzani, Andrea Riccardo; Monti, D; Capri, M; Bersani, F; Cadossi, R; Franceschi, Claudio
abstract

..

1993 - INCREASED CYTOKINE PRODUCTION IN MONONUCLEAR-CELLS OF HEALTHY ELDERLY PEOPLE [Articolo su rivista]
Fagiolo, U; Cossarizza, Andrea; Scala, E; Fanalesbelasio, E; Ortolani, C; Cozzi, E; Monti, D; Franceschi, C; Paganelli, R.
abstract

The production of cytokines during aging, except interleukin (IL)-2, has been neglected in humans.We measured the in vitro production of IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and IL-1beta by peripheral mononuclear cells from selected healthy young (mean age 26.8 years) and aged (mean age 80.2 years) subjects. Significant increases of IL-6, TNF-alpha and IL-1beta levels were found in mitogen-stimulated cultures from aged donors, occurring at 24 to 72 h after stimulation. No significant differences were observed for IFN-gamma production. Proliferative capability of cells stimulated with PHA was not impaired in aged subjects. Since the amounts of all cytokines studied were similar in unstimulated cultures from young and aged subjects, and also serum levels of TNF-alpha did not differ, these data indicate that the cellular machinery for the production of these cytokines is well preserved in aging, and also that cells from old people are able to up-regulate their production in response to appropriate stimuli. The increases in cytokine synthesis were not dependent on changes in the number of monocytes, nor were they related to the significant rise of CD45RO+, and the concomitant decrease of CD45RA+, occurring in both CD4+ and CD8+ lymphocytes from aged subjects. The increased production of pro-inflammatory cytokines by stimulated mononuclear cells of healthy aged subjects may be relevant to several aspects of age-associated pathological events, including atherosclerosis, osteoporosis, fibrosis and dementia.

1993 - LYMPHOCYTE SUBSETS AND NATURAL-KILLER-CELL ACTIVITY IN HEALTHY OLD-PEOPLE AND CENTENARIANS [Articolo su rivista]
Sansoni, P; Cossarizza, Andrea; Brianti, V; Fagnoni, F; Snelli, G; Monti, D; Marcato, A; Passeri, G; Ortolani, C; Forti, E; Fagiolo, U; Passeri, M; Franceschi, C.
abstract

The contribution of the immune system to healthy aging and longevity is still an open question. For this reason, several immune parameters (T, B, and natural killer [NK] cell subsets; non-major histocompatibility complex [MHC]-restricted cytotoxic activities, ie, natural and redirected killing [RDK] activities) were studied in a total of 138 healthy subjects of different ages, from 4 to 106 years of age, including 26 centenarians. The major age-related modifications were the following: (1) a decrease in the absolute number of T lymphocytes (CD3+), involving both CD4+ and CD8+ subsets, accompanied by a marked concomitant increase in the number of activated T cells (CD3+, HLA-DR+); (2) a marked decrease in the number of B lymphocytes (CD19+); and (3) an increase in the number of cells with markers of NK activity and of T lymphocytes able to mediate non-MHC-restricted cytotoxicity. These modifications linearly progressed with age and centenarians followed the trend, suggesting that their immune system did not escape the aging process. However, other immunohematologic parameters (number of red blood cells, platelets, and leukocytes) and important immune functions, such as cytotoxic activities (NK and RDK cell activities), were well preserved throughout life until the last decades of life. Unexpectedly, in apparently healthy middle-aged subjects, a decrease of cytotoxic activities was observed in comparison with those of both young controls and centenarians. In conclusions, our data suggest that in centenarians some immune responses are kept at a high level of efficiency, likely contributing to their successful aging. However, this selected group of people does not escape the aging process, as shown by the progressive derangement of a variety of immune parameters.

1993 - POLYMERASE CHAIN-REACTION IN THE EARLY DIAGNOSIS OF HIV-1 INFECTION IN HIGH-RISK SUBJECTS [Articolo su rivista]
Borghi, V; Lami, G; Frigieri, G; Cecchi, Mt; Zoboli, G; Tavio, M; Pecorari, M; Mongiardo, N; Cossarizza, Andrea; Derienzo, B.
abstract

We investigated the presence of the HIV-1 infection using the polymerase chain reaction (PCR) test in seronegative sexual partners of HIV-infected subjects and in children born to seropositive mothers. By using PCR assay, no HIV-1 DNA was detected in 32 female partners of HIV positive patients including three pregnant women who were also studied during pregnancy and after delivery. HIV-1 DNA was found in 12 out of 38 children born to seropositive mothers; five of them also had detectable serum HIV-1 p24 Ag levels. On the whole, our data stress the importance of using a very sensitive technique, i.e. PCR, for the early diagnosis of HIV-1 infection.

1992 - ACTH-LIKE MOLECULES IN LYMPHOCYTES - A STUDY IN DIFFERENT VERTEBRATE CLASSES [Articolo su rivista]
Ottaviani, Enzo; Franchini, Antonella; Cossarizza, Andrea; Frenceschi, C.
abstract

Immunocytochemical and cytofluorimetric studies were performed in several species of different classes (Pisces, Amphibia, Reptilia and Aves), in order to ascertain the presence of ACTH-like molecules in blood cells. Using monoclonal and polyclonal antibodies, we demonstrated that lymphocytes containing ACTH-like molecules are first detectable in anuran amphibia, and are present in lymphocytes of reptilia and aves. Taking into account previous results demonstrating that ACTH-like molecules are present in and modulate macrophage functions from molluscs to man, it is argued that a new biological role has probably been acquired by ACTH in higher vertebrates, where it is also expressed in cells belonging to the lymphocyte lineage, possibly exerting a regulatory role on antibody response.

1992 - AGE-RELATED IMBALANCE OF VIRGIN (CD45RA+) AND MEMORY (CD45R0+) CELLS BETWEEN CD4+ AND CD8+ LYMPHOCYTES-T IN HUMANS - A STUDY FROM NEWBORNS TO CENTENARIANS [Articolo su rivista]
Cossarizza, Andrea; Ortolani, C; Paganelli, R; Monti, D; Barbieri, D; Sansoni, P; Fagiolo, U; Forti, E; Londei, M; Franceschi, C.
abstract

The expression of CD45RA and CD45R0 isoforms of the leukocyte common antigen - characteristic of virgin and memory T cells, respectively - has been studied by three colour cytofluorimetric analysis on CD4+ or CD8+ peripheral blood CD3+ cells from 131 healthy donors 0-106 years old. As expected, at birth 95-99% of the CD3+ lymphocytes expressed CD45RA isoform. A rapid increase of CD45R0+ cells was observed in the first 2-3 decades of life, this phenomenon being much more pronounced on CD4+ cells. Subsequently, the increase of the memory compartment was much less rapid. After the tenth decade of life a consistent reservoire of CD45RA+ among CD4+ cells was still present (about 20%). At this advanced age the percentage of virgin cells among CD8+ T lymphocytes was even higher (about 50%), and only slightly lower than that of young donors (about 55-60%). The significance of the age-related unbalance in the proportion of virgin and memory cells between CD4+ and CD8+ T lymphocytes is unknown. The presence of a great number of virgin T lymphocytes within the CD4+ and the CD8+ T cell subsets even in the peripheral blood of centenarians poses the problem of their origin (thymus? extrathymic sites?) and their functional role.

1992 - APOPTOSIS - PROGRAMMED CELL-DEATH - A ROLE IN THE AGING PROCESS [Articolo su rivista]
Monti, D; Troiano, L; Tropea, F; Grassilli, E; Cossarizza, Andrea; Barozzi, D; Pelloni, Mc; Tamassia, Mg; Bellomo, G; Franceschi, C.
abstract

Cells continuously exposed to genotoxic agents, such as oxygen free radicals (OFRs), deeply involved in the aging process use a variety of cellular defense mechanisms. These defense mechanisms include DNA repair enzymes, antioxidants, poly(ADP-ribosyl)polymerase (pADPRP), and stress proteins and they constitute an integrated network. An age-related failure of the efficiency of this network can affect cell proliferation and cell death, two phenomena tightly linked and regulated. Recent data from our laboratory on the role of DNA damage and pADPRP activation and on the type of cell death induced by OFRs in human lymphocytes are reviewed. In vitro and in vivo data on possible strategies to reduce oxidative stress in lymphocytes from normal and Down syndrome subjects, by using natural compounds and trace elements, are presented. They indicate that nicotinamide and L-carnitine protect human cells from OFR-induced damage and suggest that they are possible candidates as antiaging substances.

1992 - CELL-PROLIFERATION AND CELL-DEATH IN IMMUNOSENESCENCE [Articolo su rivista]
Monti, D; Troiano, L; Grassilli, E; Agnesini, C; Tropea, F; Barbieri, D; Capri, M; Cristofalo, Ea; Salvioli, S; Ronchetti, Ivonne; Bellomo, G; Cossarizza, Andrea; Franceschi, C.
abstract

1992 - CHANGES IN CIRCULATING B-CELLS AND IMMUNOGLOBULIN CLASSES AND SUBCLASSES IN A HEALTHY AGED POPULATION [Articolo su rivista]
Paganelli, R; Quinti, I; Fagiolo, U; Cossarizza, Andrea; Ortolani, C; Guerra, E; Sansoni, P; Pucillo, Lp; Scala, E; Cozzi, E; Bertollo, L; Monti, D; Franceschi, C.
abstract

The study of 87 adults of different ages, including 15 centenarians, selected for their healthy status, showed that profound changes of humoral immunity occur throughout life. In particular, a statistically significant age-related increase of the serum level of immunoglobulin classes (IgG and IgA but not IgM) and IgG subclasses (IgG1, 2 and 3, but not IgG4) was detected. A parallel age-related decrease of circulating B cells was also observed. The hypothesis of a complex derangement of B cell function and/or compartmentalization with age is put forward, together with the proposal that healthy centenarians (as representative of successful ageing) may be helpful in identifying the physiological age-related modifications of the immune system.

1992 - CYTOTOXICITY AND CELL-DEATH - STUDIES ON MOLLUSCAN CELLS AND EVOLUTIONARY CONSIDERATIONS [Articolo su rivista]
Monti, D; Salvioli, S; Cossarizza, Andrea; Franceschi, C; Ottaviani, Enzo
abstract

In a previous study we demonstrated the presence of NK-like activity in the mollusc Planorbarius corneus. This activity ran be ascribed to round hemocytes that have a morphology similar to that of vertebrate lymphocytes. We show here that this NK-like cytotoxicity is evident in serum-free culture medium. Moreover, the type of death induced by molluscan effector cells on their targets is probably similar to that induced by vertebrate effector cells, i.e. apoptosis or programmed cell death, as assessed by the protective effect exerted by 3-aminobenzamide when both types of effector cells were used.

1992 - Erratum: ACTH-like molecules in gastropod molluscs: A possible role in ancestral immune response and stress (Proceedings of the Royal Society of London - Biological Sciences (1991) B 245 (215-218)) [Abstract in Rivista]
Cossarizza, Andrea
abstract

1992 - GENOMIC INSTABILITY AND AGING - STUDIES IN CENTENARIANS (SUCCESSFUL AGING) AND IN PATIENTS WITH DOWNS-SYNDROME (ACCELERATED AGING) [Articolo su rivista]
Franceschi, C; Monti, D; Scarfi, Mr; Zeni, O; Temperani, Paola; Emilia, G; Sansoni, P; Lioi, Mb; Troiano, L; Agnesini, C; Salvioli, S; Cossarizza, Andrea
abstract

1992 - HLA ANTIGENS AND AGING [Articolo su rivista]
Morellini, M; Trabace, S; Lulli, P; Cappellacci, S; Brioli, G; Orru, D; Pennesi, G; Monti, D; Cossarizza, Andrea; Lambertgardini, S; Sansoni, P; Franceschi, C.
abstract

no abstract available

1992 - INCREASED CYTOKINE PRODUCTION BY PERIPHERAL-BLOOD MONONUCLEAR-CELLS FROM HEALTHY ELDERLY PEOPLE [Articolo su rivista]
Fagiolo, U; Cossarizza, Andrea; Santacaterina, S; Ortolani, C; Monti, D; Paganelli, R; Franceschi, C.
abstract

no abstract available

1992 - INHIBITION OF APOPTOSIS BY ZINC - A REAPPRAISAL [Articolo su rivista]
Barbieri, D; Troiano, L; Grassilli, E; Agnesini, C; Cristofalo, Ea; Monti, D; Capri, M; Cossarizza, Andrea; Franceschi, C.
abstract

no abstract available

1992 - LYMPHOCYTES AND LOW-FREQUENCY ELECTROMAGNETIC-FIELDS [Articolo su rivista]
Cadossi, R; Bersani, F; Cossarizza, Andrea; Zucchini, P; Emilia, G; Torelli, Giuseppe; Franceschi, C.
abstract

Human lymphocytes have been used by several researchers to investigate the biological effect of electromagnetic fields (EMF). EMF modulate the response by lymphocytes to lectin stimulation. The size and direction of the effect depends both on the lymphocyte physiology and on the physical parameters characterizing the EMF. Lymphocytes have also been used to investigate the genotoxicity of EMP exposure.

1992 - NK CELL-ACTIVITY AND LYMPHOCYTE-T PROLIFERATION IN HEALTHY CENTENARIANS [Articolo su rivista]
Sansoni, P; Brianti, V; Fagnoni, F; Snelli, G; Marcato, A; Passeri, G; Monti, D; Cossarizza, Andrea; Franceschi, C.
abstract

no abstract available

1992 - SELECTIVE DEFICIENCY OF CD4+/CD45RA+ LYMPHOCYTES IN PATIENTS WITH ATAXIA-TELANGIECTASIA [Articolo su rivista]
Paganelli, R; Scala, E; Scarselli, E; Ortolani, C; Cossarizza, Andrea; Carmini, D; Aiuti, F; Fiorilli, M.
abstract

Several immunological abnormalities have been observed in ataxia-telangiectasia (AT), the most consistent being defects of immunoglobulin isotypes, decreased T-cell numbers, and reduced proliferative responses to mitogens. We examined the distribution of T lymphocytes expressing distinctive surface Ag characteristic of naive (CD45RA+) and memory (CD29+, CD45RO+) T cells, in both CD4+ and CD8+ (bright and dim) lymphocytes from 13 AT patients, compared with healthy age-matched controls. We found that, irrespective of age, patients with AT had a severe deficiency of CD4+/CD45RA+ lymphocytes. This decrease accounted for the reduction of total CD4+ cells, since the absolute numbers of memory CD4+ cells were not significantly different in AT and in controls. Functional tests revealed poor proliferative responses to phytohemagglutinin and normal responses to soluble Ag (tetanus toxoid) in AT patients. These data fit with the distribution of naive and memory cells, which are known to respond predominantly to mitogens or to recall Ag, respectively. CD45RA molecules were normally expressed on CD8+ lymphocytes. This rules out a generalized defect of regulation or differential splicing as the cause of defective expression of CD45RA on CD4+ cells. The selective deficiency of CD4 + CD45RA+ may provide a cellular basis for some functional T-cell abnormalities of AT patients. Furthermore, it might practically serve for an early, or even prenatal, diagnosis of this disease.

1992 - SENESCENCE, IMMORTALIZATION, AND APOPTOSIS - AN INTRIGUING RELATIONSHIP [Articolo su rivista]
D., Monti; E., Grassilli; L., Troiano; Cossarizza, Andrea; S., Salvioli; D., Barbieri; C., Agnesini; S., Bettuzzi; Mc, Ingletti; Corti, Arnaldo; C., Franceschi
abstract

no abstract available

1992 - SENSITIVITY TO GENOTOXIC AGENTS AND IMMUNOLOGICAL STATUS IN A CASE OF WERNER SYNDROME [Articolo su rivista]
Monti, D; Cossarizza, Andrea; Chiricolo, M; Vannini, P; Ciavarella, A; Franceschi, C.
abstract

no abstract available

1992 - THYROID AND OTHER ORGAN-SPECIFIC AUTOANTIBODIES IN HEALTHY CENTENARIANS [Articolo su rivista]
Mariotti, S; Sansoni, P; Barbesino, G; Caturegli, P; Monti, D; Cossarizza, Andrea; Giacomelli, T; Passeri, G; Fagiolo, U; Pinchera, A; Franceschi, C.
abstract

To investigate the prevalence of thyroid autoantibodies in very old subjects, we assayed sera from 34 healthy centenarians (7 men, 27 women; age range 100-108 years) for these antibodies. There was a clear age-dependent increase in prevalence of thyroid autoantibodies in sera from 549 control subjects aged 7-85 years, prevalence in 40 subjects aged 70-85 being significantly greater (p<0.001, chi-2) than that in 436 subjects aged less than 50. By contrast, prevalence of thyroid autoantibodies in centenarians was not significantly different from that in controls aged less than 50. Cytofluorimetric analysis of peripheral blood lymphocytes showed a striking age-dependent decrease in total and CD5+ B cells (without changes in their ratio), which reached its nadir in centenarians. The age-dependent increase in prevalence of thyroid autoantibodies in the elderly is not seen after the ninth decade of life. What relation this characteristic has to derangement of circulating B cells is unknown.

1991 - ACTH-like molecules in gastropod molluscs: A possible role in ancestral immune response and stress [Articolo su rivista]
Ottaviani, E.; Cossarizza, A.; Ortolani, C.; Monti, D.; Franceschi, C.
abstract

The presence of immunoreactive ACTH molecules on phagocytic cells from the freshwater snails Planorbarius corneus and Lymnaea stagnalis was shown by cytofluorimetric analysis. The role of ACTH in phagocytosis and in the release of biogenic amines, two biological responses that may be taken as ancestral types of immune response and stress, respectively, has also been evaluated. ACTH markedly increased the phagocytosis of Staphylococcus aureus by P. corneus haemocytes and caused the release of biogenic amines from such cells into the serum. These data, as well as tracing the ancestral physiological role of ACTH, favour the hypothesis that immune and neuroendocrine systems share a common evolutionary origin.

1991 - AGE-RELATED EXPANSION OF FUNCTIONALLY INEFFICIENT CELLS WITH MARKERS OF NATURAL-KILLER ACTIVITY IN DOWNS-SYNDROME [Articolo su rivista]
Cossarizza, Andrea; Ortolani, C; Forti, E; Montagnani, G; Paganelli, R; Zannotti, M; Marini, M; Monti, D; Franceschi, C.
abstract

1991 - AGING, LONGEVITY, AND CANCER - STUDIES IN DOWNS-SYNDROME AND CENTENARIANS [Articolo su rivista]
Franceschi, C; Monti, D; Cossarizza, Andrea; Fagnoni, F; Passeri, G; Sansoni, P.
abstract

no abstract available

1991 - AN INVITRO MODEL FOR STUDYING OXIDATIVE DAMAGE AND PROTECTIVE SUBSTANCES IN HUMAN-CELLS [Articolo su rivista]
Monti, D; Barozzi, D; Buttafoco, P; Troiano, L; Tropea, F; Cossarizza, Andrea; Grassilli, E; Pelioni, Mc; Marini, M; Tiozzo, Roberta; Franceschi, C.
abstract

An in vitro experimental system comprising human peripheral blood lymphocytes or rat splenocytes exposed to oxygen free radicals (OFR) generated extracellularly by a classic enzymatic system, i.e. xanthine oxidase (XOD) plus hypoxanthine (HYP), is proposed as a model for the study of OFR-induced damage, for investigating the mechanisms responsible for cell injury and for identifying possible protective substances. We show that rat splenocytes are much more sensitive than human lymphocytes to oxidative stress. The protective effects exerted on OFR-exposed human lymphocytes by inhibitors of poly(ADP-ribosyl)polymerase (ADPRP), such as 3-aminobenzamide, 3-methoxybenzamide and nicotinamide, suggest that the activation of ADPRP is a critical step in the metabolic pathways which mediate the toxic effect of OFR in human cells.

1991 - CYTOTOXICITY AND IMMUNOCYTE MARKERS IN CELLS FROM THE FRESH-WATER SNAIL PLANORBARIUS-CORNEUS (L) (GASTROPODA-PULMONATA) - IMPLICATIONS FOR THE EVOLUTION OF NATURAL-KILLER-CELLS [Articolo su rivista]
Franceschi, C; Cossarizza, Andrea; Monti, D; Ottaviani, Enzo
abstract

The hypothesis that natural killer (NK) cells represent an important form of cell recognition and cytotoxicity leads to the prediction that NK-like activity should be preserved throughout phylogenetic development. This was tested in the invertebrate Planorbarius corneus. Two types of cells can be identified and separated from the hemolymph of this mollusc, i.e. glass-adherent macrophage-like spreading hemocytes (SH) and nonadherent round hemocytes (RH). Only RH are able to lyse the K-562 human target cell line in a short-term NK cytotoxicity test. This NK-like activity, severely reduced after 18 h incubation at 24-degrees-C, is preserved by human recombinant interleukin 2. A further analysis of P. corneus hemocytes has been performed by using several mouse anti-human monoclonal antibodies and cytofluorimetric analysis. Unexpectedly, both SH and RH react with several monoclonal antibodies, including those directed against epitopes typical of mammalian NK cells and cell adhesion molecules. On the whole, these data support the hypothesis that a primitive NK-like activity appeared early in evolution and is not shared by phagocytic cells.

1991 - Caloric restriction modulates aging rate and sensitivity to oxygen free radical damage in rats [Articolo su rivista]
Cossarizza, Andrea
abstract

1991 - DNA AND CELL-DEATH [Articolo su rivista]
Monti, D; Troiano, L; Tropea, F; Grassilli, E; Cossarizza, Andrea; Barozzi, D; Bettuzzi, S; Corti, Arnaldo; Franceschi, C.
abstract

The type of DNA damage and the role of poly(ADP-ribosyl)polymerase (ADPRP) and sulphated glyprotein 2 (SGP-2) in programmed cell death (apoptosis) was investigated in the following model systems: i) rat thymocytes treated with dexamethasone (DEX) either in vitro or in vivo; ii) human perypheral blood mononuclear cells (hPBMCs) exposed to oxygen free radicals (OR); iii) K562 cell line killed by hPBMCs during spontaneous (NK) or interleukin-2 (IL-2)-induced (LAK) cytotoxic activity. The results suggest that ADPRP and SGP-2 are involved in the apoptotic process, but their role probably differs according to the type of cell and the inducing damage/stimulus. Moreover, no simple correlation appears to exist between the extent of DNA damage and cell survival or cell death.

1991 - EFFECT OF LOW-FREQUENCY LOW-ENERGY PULSING ELECTROMAGNETIC-FIELDS ON MICE INJECTED WITH CYCLOPHOSPHAMIDE [Articolo su rivista]
Cadossi, R; Zucchini, P; Emilia, G; Franceschi, C; Cossarizza, Andrea; Santantonio, M; Mandolini, G; Torelli, Giuseppe
abstract

C3H mice have been used to investigate the effect of a combination of cyclophosphamide (CY) and electromagnetic fields (PEMF). Mice were injected i.p. with a single dose of 200 mg/kg body weight of CY and then exposed to PEMF 24 h per day. In an initial series of experiments immediately after CY injection mice were exposed to PEMF until sacrifice. WBC counts in the peripheral blood demonstrated a quicker decline in WBC at days 1 and 2 in mice exposed to PEMF. Groups of mice were sacrificed at days 1, 4, 6, 8, and 10 after CY injection. In mice exposed to PEMF the spleen weight was less than in controls at days 6, 8, and 10. Autoradiographic studies demonstrated that the labeling index of bone marrow smears did not significantly differ between controls and experimental mice exposed to PEMF, whereas the spleen labeling index proved to be higher among control mice versus mice exposed to PEMF at day 6, and higher among mice exposed to PEMF versus controls at day 8. In a second series of experiments mice were exposed to PEMF only over the 24 h following CY injection. We found that the spleens of mice exposed to PEMF weighed less than those of controls at days 6 and 8. The labeling index of bone marrow did evidence a slight decrease among mice exposed to PEMF at days 8 and 10 after CY injection versus control mice. The spleen labeling index proved to be lower in experimental mice exposed to PEMF than in controls at days 4, 6, and 8. Mice were then injected with CY, half were exposed to PEMF, and 24 h later bone marrow was recovered from both groups of animals. The same number of bone marrow cells was injected via the tail vein into recipient mice irradiated to 8.5 Gy. The grafting efficiency of the bone marrow was evaluated by examining the number of spleen colonies and the spleen and bone marrow labeling indices at day 8; all parameters proved to be significantly lower among mice grafted with the bone marrow of mice injected with CY and exposed to PEMF than among controls injected with CY only. Finally, we found that the effect of PEMF is evident only if mice are exposed during the 24 h following CY injection. The data reported here indicates that PEMF exposure after CY injection increases the damage induced in mice by CY.

1991 - Exposure to low-frequency pulsed electromagnetic fields increases mitogen- induced lymphocyte proliferation in Down’s syndrome [Articolo su rivista]
Cossarizza, Andrea
abstract

We previously reported that exposure of human mitogen-stimulated peripheral blood lymphocytes (PBL) to extremely low frequency pulsed electromagnetic fields (PEMFs) could restore the defective proliferative capability of PBL from aged subjects. The effects of exposure to PEMFs were studied in PBL from 25 patients with Down’s syndrome (DS), a syndrome of premature aging characterized by precocious immune system derangement, including age-related defective PBL proliferative capability. PBL were stimulated with different doses of phytohemagglutinin, and cell proliferation was assessed by measuring the incorporation of tritiated thymidine. After PEMF-exposure, a significant increase in cell proliferation was observed in cells from DS children and young adults, but it was much more evident in PBL from relatively aged DS patients. The age-related effect of PEMFs on DS lymphocytes demonstrates that age must be considered a major variable when studies on DS are performed, and confirms that DS must be regarded as a syndrome of accelerated aging. (Aging 3: 241–246, 1991) © 1991, Editrice Kurtis s.r.l.. All rights reserved.

1991 - IMMUNODEFICIENCY AS A MODEL TO STUDY NEUROIMMUNE DEVELOPMENT - THE CASE OF ATAXIA TELANGIECTASIA [Articolo su rivista]
Paganelli, R; Scala, E; Ortolani, C; Cossarizza, Andrea; Fiorilli, M.
abstract

no abstract available

1991 - INHIBIN AND ACTIVIN MODULATE HUMAN MONOCYTE CHEMOTAXIS AND HUMAN LYMPHOCYTE INTERFERON-GAMMA PRODUCTION [Articolo su rivista]
Petraglia, F; Sacerdote, P; Cossarizza, Andrea; Angioni, S; Genazzani, Alessandro; Franceschi, C; Muscettola, M; Grasso, G.
abstract

Inhibin and activin are referred to as gonadal glycoprotein hormones whose function is the control of FSH release from the pituitary gland. However, several observations indicate that inhibin and activin are produced in various organs and serve multiple functions. Because bone marrow and spleen produce inhibin and activin, our aim was to evaluate their possible effect on cell-mediated immune function. For this reason we studied 1) monocyte chemotaxis, 2) lymphocyte interferon-gamma production, 3) phytohemagglutinin-induced lymphocyte proliferation, and 4) nonmajor histocompatibility complex-restricted and lymphokine-activated lymphocyte cytotoxicity. All studies were performed on human peripheral blood cells in the absence or presence of various doses of inhibin, activin, or inhibin plus activin. A significant dose-related increase in monocyte chemotaxis was induced by inhibin. Activin increased the migrational activity of monocytes, but via random, not directed, migration. Inhibin significantly decreased interferon-gamma production, and its effect was reversed by activin. Inhibin and/or activin had no significant effect on either phytohemagglutinin-induced lymphocyte proliferation or lymphocyte cytotoxic capability. The present demonstration that inhibin and activin may affect some immune parameters suggests a possible involvement of these hormones in regulating cell-mediated immune function.

1991 - NATURAL CYTOTOXICITY IN A FRESH-WATER PULMONATE MOLLUSK - AN UNORTHODOX COMPARATIVE APPROACH [Articolo su rivista]
Franceschi, C; Cossarizza, Andrea; Ortolani, C; Monti, D; Ottaviani, Enzo
abstract

1991 - Preferential expression of Vβ6.7 domain on human peripheral CD4+ T cells. Implication for positive selection of T cells in man [Articolo su rivista]
Cossarizza, A.; Kahan, M.; Ortolani, C.; Franceschi, C.; Londei, M.
abstract

The peripheral T cell receptor repertoire is mainly controlled by the processes of positive and negative selection occurring in the thymus. Studies in normal or transgenic mice have provided compelling evidence for both negative and positive selection. Negative selection is characterized by partial or total disappearance from the periphery of T cells expressing certain Cβ regions, which are normally present in the thymus. Positive selection is chiefly characterized, in the periphery, by an imbalanced ratio between CD4+ and CD8+ T cells expressing a given V domain. To date little information concerning positive and negative selection has been available in man. We studied the distribution of 4 Vβ domains on CD4+ and CD8+ peripheral T cells of 34 healthy individuals with a wide age‐range (0–96 years). One of the Vβ domain studied, Vβ6.7, is preferentially expressed on CD4+ compared to CD8+ T cells (p &gt; 0.001). No significant differences were observed using the other Vβ domain‐specific monoclonal antibodies (Vβ5.2–5.3, 8 or 12). These results provide evidence that a process of thymic education, similar to that described in murine animal models, may also take place in man. Copyright © 1991 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim

1991 - SPONTANEOUS AND MITOMYCIN-C-INDUCED MICRONUCLEI IN HUMAN-LYMPHOCYTES EXPOSED TO EXTREMELY LOW-FREQUENCY PULSED MAGNETIC-FIELDS [Articolo su rivista]
Scarfi, Mr; Bersani, F; Cossarizza, Andrea; Monti, D; Castellani, G; Cadossi, R; Franceschetti, G; Franceschi, C.
abstract

Peripheral blood lymphocytes from 7 patients with Down's syndrome (DS; trisomy 21) and 14 healthy age-matched controls were studied for the induction of micronuclei (MN) by the cytokinesis-block method. The spontaneous incidence of MN in lymphocytes from DS subjects was lower than that of control cultures. When lymphocytes were treated with mitomycin C (MMC) at the beginning of the culture period, an increase in MN formation was found in cells from both DS and control subjects. In DS subjects this increase was much more marked than in control donors. This effect had to be ascribed to cells from older DS subjects (37-55 years old), which showed an MMC-induced MN formation that was markedly and significantly higher than that observed in cells from younger (9-16 years old) DS subjects. These data indicate that age has to be considered a major variable when studies on the genetic instability of DS subjects are performed.

1991 - The «immune-mobile brain»: evolutionaryevidence. [Articolo su rivista]
Ottaviani, Enzo; Caselgrandi, Eva; Bondi, Moreno; Cossarizza, Andrea; Monti, Daniela; Franceschi, Claudio
abstract

One of the possible strategy to study the relationships between the immune and the neuroendocrine system is to trace their evolutionary origin. In a previous study we showed that immunoreactive ACTH-and beta-endorphin (BE)-like molecules were present in the serum and in the phagocytic cells, i.e., spreading hemocites (sh).We hypothesized a possible biological role of ACTH and BE in chemotaxis and phagocytosis, and in some sort of stress reaction.

1990 - AGE-RELATED INCREASE OF MITOMYCIN-C-INDUCED MICRONUCLEI IN LYMPHOCYTES FROM DOWNS-SYNDROME SUBJECTS [Articolo su rivista]
Scarfi, Mr; Cossarizza, Andrea; Monti, D; Bersani, F; Zannotti, M; Lioi, Mb; Franceschi, C.
abstract

Peripheral blood lymphocytes from 7 patients with Down's syndrome (DS; trisomy 21) and 14 healthy age-matched controls were studied for the induction of micronuclei (MN) by the cytokinesis-block method. The spontaneous incidence of MN in lymphocytes from DS subjects was lower than that of control cultures. When lymphocytes were treated with mitomycin C (MMC) at the beginning of the culture period, an increase in MN formation was found in cells from both DS and control subjects. In DS subjects this increase was much more marked than in control donors. This effect had to be ascribed to cells from older DS subjects (37-55 years old), which showed an MMC-induced MN formation that was markedly and significantly higher than that observed in cells from younger (9-16 years old) DS subjects. These data indicate that age has to be considered a major variable when studies on the genetic instability of DS subjects are performed.

1990 - Immunocytochemical evidence of vertebrate bioactive peptide-like molecules in the immuno cell types of the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata) [Articolo su rivista]
Ottaviani, Enzo; Cossarizza, Andrea
abstract

An immunocytochemical investigation was carried out on round and spreading hemocytes of Planorbarius corneus by using 20 antisera to vertebrate bioactive peptides. The immunotests showed the presence of alpha 1-antichymotrypsin-bombesin-, calcitonin-, CCK-8 (INC)-, CCK-39-, gastrin-, glucagon-, Met-enkephalin-, neurotensin-, oxytocin-, somatostatin-, substance P-, VIP-, and vasopressin-immunoreactive molecules in the spreading hemocytes. The round hemocytes were only positive to anti-bombesin, anticalcitonin, anti-CCK-8 (INC), anti-CCK-39, anti-neurotensin, anti-oxytocin, anti-substance P and anti-vasopressin antibodies. No immunostaining was observed with anti-CCK-8 (Peninsula), anti-insulin, anti-prolactin, anti-thyroglobulin and anti-thyroxin (T4) antibodies. As probably in vertebrates, these bioactive peptides may modulate immuno cell function.

1990 - Immunological parameters in aging: Studies on natural immunomodulatory and immunoprotective substances [Articolo su rivista]
Franceschi, C.; Cossarizza, A.; Troiano, L.; Salati, R.; Monti, D.
abstract

Several immune parameters--particularly T-cell dependent immune responses--are altered in aged subjects. To test the hypothesis that they may be the consequence of more general age-related lymphocyte biochemical alterations, and particularly of the energy producing system, the effect of L-carnitine and acetyl-L-carnitine on cell proliferation was studied in peripheral blood lymphocytes from donors of different ages. The results showed that phytohaemagglutinin-induced peripheral blood lymphocyte proliferation was markedly increased in L-carnitine- or acetyl-L-carnitine-preloaded lymphocytes from young and especially from old subjects. Cells from aged subjects considerably improved their defective proliferative capability. Preliminary observations suggest that L-carnitine-preloading also protected peripheral blood lymphocytes from old donors when such cells were exposed to an oxidative stress.

1990 - LAK ACTIVITY IS INDUCIBLE IN BLOOD MONONUCLEAR-CELLS FROM HUMAN FETUS [Articolo su rivista]
Cossarizza, Andrea; Monti, D; Dagnabricarelli, F; Montagnani, G; Franceschi, C.
abstract

Mitochondria functionality and apoptosis were studied in peripheral blood lymphocytes (PBL) of human immunodeficiency virus type 1-infected children, with or without lipodystrophy (LD), who were receiving highly active antiretroviral therapy (HAART) and in PBL of healthy control subjects (HCs). By flow cytometry, mitochondrial (mt) membrane potential, mt mass, intra-mt cardiolipin distribution, and early and late apoptosis in fresh PBL or in PBL cultured with different stimuli were assessed. mtDNA content was evaluated in fresh PBL by an original double-competitive quantitative polymerase chain reaction method, which enabled direct quantification of the number of mtDNA copies present in human lymphocytes. PBL from LD-positive and LD-negative children and from HCs were similar in mt functionality and in their tendency to undergo apoptosis. mtDNA content was also similar in PBL of LD-positive children and HCs, suggesting that normal mt functionality and normal tendency to undergo apoptosis are present in PBL of children with HAART-associated LD.

1990 - Oxidative stress, poly(ADP)ribosylation and aging: In vitro studies on lymphocytes from normal and Down's syndrome subjects of different age and from patients with Alzheimer's dementia [Relazione in Atti di Convegno]
Franceschi, C.; Monti, D.; Cossarizza, A.; Tomasi, A.; Sola, P.; Zannotti, M.
abstract

1990 - PHAGOCYTOSIS AND ACTH-LIKE AND BETA-ENDORPHIN-LIKE MOLECULES IN INVERTEBRATE (MOLLUSCAN) AND IN VERTEBRATE (HUMAN) CELLS - POSSIBLE SIGNIFICANCE FOR THE EVOLUTION OF THE IMMUNONEUROENDOCRINE SYSTEM [Articolo su rivista]
Ottaviani, Enzo; Petraglia, F; Genedani, Susanna; Bernardi, M; Bertolini, Alfio; Cossarizza, Andrea; Monti, D; Franceschi, C.
abstract

1990 - PRECOCIOUS AGING OF THE IMMUNE-SYSTEM IN DOWN-SYNDROME - ALTERATION OF LYMPHOCYTES-B LYMPHOCYTE-T SUBSETS, AND CELLS WITH NATURAL-KILLER MARKERS [Articolo su rivista]
Cossarizza, Andrea; Monti, D; Montagnani, G; Ortolani, C; Masi, M; Zannotti, M; Franceschi, C.
abstract

Phenotype and proliferative ability of peripheral blood lymphocytes from 15 noninstitutionalized children affected with Down Syndrome (DS), in apparently good health, were studied and compared with those of 16 healthy control children of the same age. A complex derangement of all the major peripheral blood cell subsets, i.e., B cells, T cells, and natural killer (NK) cells, was present in DS children. A significant decrease of the absolute number of circulating lymphocytes, a marked and significant decrease of B lymphocytes, a marked and significant decrease of B lymphocyte absolute number and percentage, and dramatic modifications of the T-cell subsets were observed. The absolute number of CD4+ cells was significantly decreased, whereas CD8+ cells increased significantly in percentage but not in absolute number. A derangement of cells bearing markers associated with NK activity, such as CD57, CD16, and CD56, was observed. Among the most important alterations, the presence of a high number of CD57+, CD16- cells, of CD57+, CD8+ lymphocytes, and of CD3+, CD56+ lymphocytes was seen. Many of these alterations are similar to those characteristic of chromosomally normal subjects of advanced age. The hypothesis that the reduced thymic endocrine activity and the zinc deficiency characteristic of DS are responsible for the derangement of T and NK subsets is discussed.

1990 - PRESENCE OF ACTH AND BETA-ENDORPHIN IMMUNOREACTIVE MOLECULES IN THE FRESH-WATER SNAIL PLANORBARIUS-CORNEUS (L) (GASTROPODA, PULMONATA) AND THEIR POSSIBLE ROLE IN PHAGOCYTOSIS [Articolo su rivista]
Ottaviani, Enzo; Petraglia, F; Montagnani, G; Cossarizza, Andrea; Monti, D; Franceschi, C.
abstract

1990 - Precocious aging of the immune system in Down syndrome: Alteration of B lymphocytes, T-lymphocyte subsets, and cells with natural killer markers [Articolo su rivista]
Cossarizza, A.; Monti, D.; Montagnani, G.; Ortolani, C.; Masi, M.; Zannotti, M.; Franceschi, C.
abstract

Phenotype and proliferative ability of peripheral blood lymphocytes from 15 noninstitutionalized children affected with Down Syndrome (DS), in apparently good health, were studied and compared with those of 16 healthy control children of the same age. A complex derangement of all the major peripheral blood cell subsets, i.e., B cells, T cells, and natural killer (NK) cells, was present in DS children. A significant decrease of the absolute number of circulating lymphocytes, a marked and significant decrease of B lymphocyte absolute number and percentage, and dramatic modifications of the T-cell subsets were observed. The absolute number of CD4+ cells was significantly decreased, whereas CD8+ cells increased significantly in percentage but not in absolute number. A derangement of cells bearing markers associated with NK activity, such as CD57, CD16, and CD56, was observed. Among the most important alterations, the presence of a high number of CD57+, CD16- cells, of CD57+, CD8+ lymphocytes, and of CD3+, CD56+ lymphocytes was seen. Many of these alterations are similar to those characteristic of chromosomally normal subjects of advanced age. The hypothesis that the reduced thymic endocrine activity and the zinc deficiency characteristic of DS are responsible for the derangement of T and NK subsets is discussed.

1990 - Recovery of human lymphocytes damaged with γradiation or enzymatically produced oxygen radicals: Different effects of poly(ADP-ribosyl)polymerase inhibitors [Articolo su rivista]
Marini, M.; Zunica, G.; Tamba, M.; Cossarizza, A.; Monti, D.; Franceschi, C.
abstract

Quiescent human lymphocytes were damaged in two different ways, both producing toxic oxygen radicals: xanthine oxidase plus hypoxanthine (XOD/HYP), or γrays. Under conditions where DNA synthesis was reduced to 10-20% of control, inhibitors of poly(ADP-ribosyl)polymerase (ADPRP, an enzyme that becomes activated in the presence of DNA strand breaks) allowed lymphocytes to recover completely when the damage was caused by XOD/HYP, but they did not affect DNA synthesis of irradiated cells. However, a protective effect of ADPRP inhibitors was observed with irradiated lymphocytes receiving doses ≥ 50 Gy. Unscheduled DNA synthesis was significantly increased when lymphocytes were damaged by high radiation doses in the presence of ADPRP inhibitors. We suggest that ionizing radiation does not stimulate poly(ADP-ribose) synthesis in lymphocytes at doses that impair lymphocyte DNA synthesis by 80-90%, while ADPRP may be involved in the repair of DNA lesions occurring at higher radiation doses. © 1990 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1989 - DNA repair after γ irradiation in lymphocytes exposed to low-frequency pulsed electromagnetic fields [Articolo su rivista]
Cossarizza, A.; Monti, D.; Sola, P.; Moschini, G.; Cadossi, R.; Bersani, F.; Franceschi, C.
abstract

The effect of exposure to extremely low-frequency pulsed electromagnetic fields (EMFs) on DNA repair capability and on cell survival in human lymphocytes damaged in vitro with γ rays was studied by two different micromethods. In the first assay, which measures DNA repair synthesis (unscheduled DNA synthesis, UDS), lymphocyte cultures were stimulated with phytohemagglutinin (PHA) for 66 h and then treated with hydroxyurea (which blocks DNA replication), irradiated with 100 Gy of 60Co, pulsed with [3H]thymidine ([3H]TdR), and then exposed to pulsed EMFs for 6 h (the period in which cells repaired DNA damage). In the second assay, which measures cell survival after radiation or chemical damage, lymphocytes were first irradiated with graded doses of γ rays or treated with diverse antiproliferative agents, and then stimulated with PHA, cultured for 72 h, and pulsed with [3H]TdR for the last 6 h of culture. In this case, immediately after the damage induced by either the radiation or chemicals, cultures were exposed to pulsed EMFs for 72 h, during which cell proliferation took place. Exposure to pulsed EMFs did not affect either UDS or cell survival, suggesting that this type of nonionizing radiation - to which humans may be exposed in the environment, and which is used for both diagnostic and therapeutic purposes - does not affect DNA repair mechanisms.

1989 - DNA repair, sensitivity to gamma radiation and to heat shock in lymphocytes from acute, untreated multiple sclerosis patients [Articolo su rivista]
Sola, P.; Merelli, E.; Faglioni, P.; Monti, D.; Cossarizza, A.; Franceschi, C.
abstract

Increases in spontaneous sister chromatid exchange (SCE) and gamma radiation-induced chromosome aberrations have been reported in peripheral blood lymphocytes (PBL) from multiple sclerosis (MS) patients, suggesting the presence of an abnormality in repair in this disease. We tested this hypothesis by measuring the ability to repair DNA and survival, after exposure to low (2-12 Gy) and high (100 Gy) gamma ray doses or to a high temperature (37-45°C), of freshly isolated PBL from 15 patients affected by definite MS and 15 healthy subjects. The MS patients were untreated and in the acute phase of the disease. No significant difference was found between the two groups. We suggest that the previously reported genomic instability may be of viral origin and not due to a genetic defect in repair of DNA in these patients. © 1989.

1989 - Diverse effects of three furocoumarins on human lymphocyte proliferation [Articolo su rivista]
Arslan, P.; Cantini, M.; Cossarizza, A.; Franceschi, C.; Dall'Acqua, F.
abstract

The biological effects of three furocoumarins on the proliferation of human normal peripheral blood lymphocytes have been investigated. Mitogen-stimulated human lymphocytes were assayed "in vitro" by measuring 3H-thymidine (3H-TdR) incorporation in the presence and in the absence of 15-30 μM 3-carbethoxypsoralen (3-CPs), trimethylangelicin (TMA) and psoralen (PSR) with and without UV-A irradiation (365 nm). The three furocoumarins differ in their ability to form mono- and bi-functional adducts with DNA pyrimidine bases and in producing reactive species of oxygen. At low furocoumarin doses and short times of UV-A irradiation (15-30 sec) used in the present study, 3-CPs did not affect 3H-TdR incorporation in PHA-stimulated human lymphocytes, TMA strongly inhibited 3H-TdR incorporation, while unexpectedly, PSR increased 3H-TdR incorporation in the absence of irradiation, likely acting, under these experimental conditions, as a co-mitogen. © 1989.

1989 - EFFECTS OF PULSED ELECTROMAGNETIC-FIELDS ON THE PROLIFERATION OF LYMPHOCYTES FROM AIDS PATIENTS, HIV-SEROPOSITIVE SUBJECTS, AND SERONEGATIVE DRUG-USERS [Articolo su rivista]
Cossarizza, Andrea; Borghi, V; Bersani, F; Cantini, M; Derienzo, B.
abstract

.

1989 - Effect of low frequency low energy pulsing electromagnetic field (PEMF) on x-ray-irradiated mice [Articolo su rivista]
Cadossi, R.; Hentz, V. R.; Kipp, J.; Eiverson, R.; Ceccherelli, G.; Zucchini, P.; Emilia, G.; Torelli, G.; Franceschi, C.; Cossarizza, A.
abstract

C3H/Km flora-defined mice were used to investigate the effect of exposure to pulsing electromagnetic field (PEMF) after total body x-ray irradiation. Prolonged exposure to PEMF had no effect on normal nonirradiated mice. When mice irradiated with different doses of x-ray (8.5 Gy, 6.8 Gy, and 6.3 Gy) were exposed to PEMF 24 h a day, we observed a more rapid decline in white blood cells (WBC) in the peripheral blood of mice exposed to PEMF at all the x-ray dosages used. No effect of exposure to PEMF was observed on the survival of the mice irradiated with 6.3 Gy and 8.5 Gy; in mice irradiated with 6.8 Gy, 2 out of 12 survived when exposed to PEMF as compared to 10 out of 12 control mice that were irradiated only. At day 4 after irradiation autoradiographic studies performed on bone marrow and spleen of 8.5-Gy-irradiated mice showed no difference between controls and mice exposed to PEMF, whereas on 6.8-Gy mice the bone marrow labeling index was lower in mice exposed to PEMF. In mice irradiated to 6.3 Gy we observed that the recovery of WBC in the peripheral blood was slowed in the mice exposed to PEMF and their body weight was significantly lower than in control mice that were irradiated only. The spleen and bone marrow of the mice irradiated to 6.3 Gy and sacrificed at days 4, 14, 20, and 25 after irradiation were analyzed by autoradiography to evaluate the labeling index. Half of the spleens from mice sacrificed at day 25 after irradiation were used to evaluate the RNA content. Autoradiography showed that in the spleen and bone marrow of control mice, there were more cells labeled with [3H]thymidine at days 4 and 14 and less at days 20 and 25 after irradiation in comparison with mice irradiated and exposed to PEMF. The Northern blot analysis of histone H3 and p53 protein RNAs extracted from the spleens at day 25 after irradiation showed a slight increase in cycling cells among spleens of mice exposed to PEMF. We suggest that the exposure of PEMF immediately after x-ray irradiation results in increased damage.

1989 - Effects of extremely low frequency (ELF) pulsed electromagnetic fields (PEMFs) on immunocompetent cells [Abstract in Rivista]
Bersani, Ferdinando; Cossarizza, Andrea; Franceschi, Claudio
abstract

The effects of the exposure of mitogen-stimulated human lymphocytes from young and aged subjects to low-frequency pulsed electromagnetic fields (PEMFs) were studied, by measuring cell proliferation, production and utilization of interleukin-2 (IL-2) and the expression of IL-2 receptors. Moreover, data are presented about DNA repair, cell survival and genotoxic effects in cultures exposed to PEMFs. PEMFs increased DNA synthesis, and the effect was more pronunced in the cells from very old subjects. Moreover a higher percentage of IL-2 receptor-positive cells and T-activated lymphocytes was found in the exposed cultures. No effects were found as far as DNA repair and genotoxic effects are concerned.

1989 - Effects of pulsed electromagnetic fields on the proliferation of lymphocytes from aids patients, HIV-seropositive subjects, and seronegative drug users [Articolo su rivista]
Cossarizza, A.; Borghi, V.; Bersani, F.; Cantini, M.; Rienzo, B. D.; Zucchini, P.; Montagnani, G.; Mussini, D.; Troiano, L.; Tropea, F.; Grassilli, E.; Monti-Biasi, D.; Franceschi, C.
abstract

The effect of the exposure of mitogen-stimulated lymphocytes from subjects infected by human immunodeficiency virus to extremely low frequency pulsed electromagnetic fields (PEMFs) was studied, by evaluating the incorporation of tritiated thymidine, the expression of IL-2 receptor, and the amount of activated T lymphocytes. Four groups of subjects were considered patients with acquired immunodeficiency syndrome (AIDS), asymptomatic seropositive subjects, seronegative drug users, and young healthy controls. PEMFs increased cell proliferation only in the group of healthy controls, as measured at the 72nd hour of culture, but an increase in the number of activated T lymphocytes was observed by cytofluorimetric analysis after 18 hrs of PEMF exposure in cultures from AIDS patients. © 1989 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1989 - Extremely low frequency pulsed electromagnetic fields increase cell proliferation in lymphocytes from young and aged subjects. [Articolo su rivista]
Cossarizza, Andrea; Monti, Daniela; Bersani, F; Cantini, M; Cadossi, R; Sacchi, Stefano; Franceschi, Claudio
abstract

The effect of the in vitro exposure to extremely low frequency pulsed electromagnetic fields (PEMFs) on the proliferation of human lymphocytes from 24 young and 24 old subjects was studied. The exposure to PEMFs during a 3-days culture period or during the first 24 hours was able to increase phytohaemagglutinin-induced lymphocyte proliferation in both groups. Such effect was greater in lymphocytes from old people which showed a markedly reduced proliferative capability and, after PEMF exposure, reached values of 3H-TdR incorporation similar to those of young subjects. The relevance of these data for the understanding and the reversibility of the proliferative defects in cells from aged subjects and for the assessment of risk related to the environmental exposure to PEMFs has to be considered.

1989 - Extremely low frequency pulsed electromagnetic fields increase interleukin-2 (IL-2) utilization and IL-2 receptor expression in mitogen-stimulated human lymphocytes from old subjects [Articolo su rivista]
Cossarizza, A.; Monti, D.; Bersani, F.; Paganelli, R.; Montagnani, G.; Cadossi, R.; Cantini, M.; Franceschi, C.
abstract

The effects of the exposure of mitogen-stimulated human lymphocytes from aged subjects to low-frequency pulsed electromagnetic fields (PEMFs) were studied by measuring the production of interleukin-2 (IL-2) and the expression of IL-2 receptor. PEMF-exposed cultures that presented increased [3H]thymidine incorporation showed lower amounts of IL-2 in their supernatants, but higher percentages of IL-2 receptor-positive cells and of T-activated lymphocytes. Taken together, these data suggest that PEMFs were able to modulate mitogen-induced lymphocyte proliferation by provoking an increase in utilization of IL-2, most likely acting on the expression of its receptor on the plasma membrane. © 1989.

1989 - FETAL THYMIC DIFFERENTIATION IN DOWNS-SYNDROME [Articolo su rivista]
Cossarizza, Andrea; Monti, D; Montagnani, G; Forabosco, Antonino; Dagnabricarelli, F; Franceschi, C.
abstract

Phenotypic features and proliferative ability of thymocytes, splenocytes and peripheral blood lymphocytes of 20-22 weeks human fetuses affected by Down's syndrome (DS) were studied and compared to those of fetuses of the same gestational age with a normal karyotype. In the thymus of both DS and normal fetuses, the great majority of cells was CD1+, CD2+, CD5+, CD4+, CD8+; using double fluorescence analysis, these markers could be detected on the same cell. About 50-60% showed CD3 antigen and about 40-50% presented the alpha beta T cell receptor. Thymocytes with NK markers (CD16, CD57, CD56) were not found. After stimulation with phytohemagglutinin, thymocytes showed a low but detectable proliferative capability, while splenocytes and peripheral blood lymphocytes showed a high responsiveness to the mitogen. These data show that the impaired immune system in DS is not associated with gross abnormalities of phenotypic T cell development in the fetal thymus or with an inability of such fetal cells to proliferate after a mitogenic stimulus.

1989 - Immunomodulatory properties of L-acetylcarnitine on lymphocytes from young and old humans [Relazione in Atti di Convegno]
Monti, D.; Cossarizza, A.; Troiano, L.; Arrigoni-Martelli, E.; Franceschi, C.
abstract

1989 - Inhibition of poly(ADP-ribosyl)ation does not prevent lymphocyte entry into the cell cycle [Articolo su rivista]
Marini, M.; Zunica, G.; Monti, D.; Cossarizza, A.; Ortolani, C.; Franceschi, C.
abstract

The enzyme poly(ADP-ribosyl)transferase (ADPRT) becomes activated soon after a mitogenic stimulus is applied to lymphocyte cultures. It has also been reported that ADPRT inhibitors prevent cell proliferation when added to cultures at the same time as the mitogen. While this has been ascribed to the need to seal physiologically present DNA strand breaks before cells enter S phase, the presence of DNA strand breaks in quiescent human lymphocytes has been recently questioned. We demonstrate here that non-toxic concentrations of ADPRT inhibitors do not affect lymphocyte blastization and proliferation, as measured by thymidine incorporation and cytofluorimetry. We therefore suggest that ADPRT activation is required for late functions which are not needed for cell cycle progression. © 1989.

1988 - DNA repair in lymphocytes from humans and rats with chronic iron overload. [Articolo su rivista]
Pietrangelo, Antonello; Cossarizza, Andrea; Monti, D; Ventura, Ezio; Franceschi, C.
abstract

A marked reduction of the proliferative capability after a mitogenic stimulus and a dramatic decrease of the capacity to repair DNA damages were found in lymphocytes from iron overloaded rats. These immunological parameters were not significantly different from controls in peripheral blood lymphocytes from patients with primary iron overload: hereditary hemochromatosis and porphyria cutanea tarda. This discrepancy could be due to the accelerated modality of iron overload in the rat model and to the fact that rat lymphocytes were obtained from an highly iron repleted microenvironment (i.e. spleen). Our data indicate that iron overload can affect the structure and/or the function of cellular DNA thus offering new insights on the close association of iron overload conditions and cancer.

1988 - EFFECT OF ADP-RIBOSYL TRANSFERASE INHIBITORS ON THE SURVIVAL OF HUMAN-LYMPHOCYTES AFTER EXPOSURE TO DIFFERENT DNA-DAMAGING AGENTS [Articolo su rivista]
Franceschi, C; Marini, M; Zunica, G; Monti, D; Cossarizza, Andrea; Bologni, A; Gatti, C; Brunelli, Ma
abstract

no abstract available

1988 - Effects of extremely low frequency pulsed electromagnetic fields (ELF) on human lymphocyte proliferation in vitro and DNA repair [Relazione in Atti di Convegno]
Bersani, F.; Franceschi, C.; Monti, D.; Bologni, A.; Gatti, C.; Cadossi, R.; Nichelatti, M.; Moschini, G.; Cossarizza, A.
abstract

The effect of extremely-low-frequency electromagnetic fields on in-vitro mitogen-induced lymphocyte proliferation was studied in two groups of healthy subjects (23 and 85 years, mean ages). An increase of cell proliferation was observed to be much higher in the group of aged subjects. The effects depended on the mitogen dose and involved interleukin-2 production and/or utilization. The DNA repair capability and cell survival in the exposed cultures after gamma irradiation was also evaluated, and no genotoxic effect was found.

1988 - Genetic and environmental influences on serum levels of immunoglobulins and complement components in monozygotic and dizygotic twins [Articolo su rivista]
Di Franco, P.; Brai, M.; Misiano, G.; Piazza, A. M.; Giorgi, G.; Cossarizza, A.; Franceschi, C.
abstract

The influence of hereditary and environmental factors in the regulation of the serum levels of IgM, IgG and IgA and of the complement components C3, C4 and factor B has been studied. For this purpose, sera from 9 monozygotic twin pairs, from 10 dizygotic twin pairs and from two control groups were analyzed. The first control group consisted of three healthy donors analyzed once a week for three weeks, as a genetic identity experimental control, while the second one was constituted by nine subjects randomly selected as a genetic heterogeneity control. The results indicate that the serum proteins with immunological functions can be subdivided into three groups. The first, represented by IgM and IgG, appears to be under strict genetic control; the second, represented by IgA and C3, appears to be influenced either by genetic or environmental factors; the third one, including C4 and factor B, is strongly influenced by environmental factors.

1988 - Kinetic control in healthy volunteers of low molecular weight heparin antithrombotic activity [Articolo su rivista]
Cossarizza, Andrea
abstract

1988 - Low molecular weight heparins for long-term therapy of peripheral vascular disease. Results of a controlled study [Articolo su rivista]
Di Stefano, F.; Giglio, A.; Vinci, M.; Romano, M.; Salanitri, G.; Cossarizza, A.; Iani, P.; Di Stefano, V. A.
abstract

The effects of long-term therapy (7 months) with a new low molecular weight heparin (LMWH) on 27 patients with peripheral vascular disease were compared with the therapeutic effects of traditional calcium heparin (Ca-He) on 28 patients with the same pathologies. The drugs were administered subcutaneously and the results were checked after one and three months and after the end of therapy. Good improvement in the clinical symptoms in the two groups of subjects (pain, cramps, formications, pangs, edemas, or hypoesthesia) was already observed at the first control and was similar for the two therapies. LMWH was able to exert a greater separation between its antithrombotic and anticoagulant actions, as demonstrated by the laboratory controls (factor Xa, activated partial thromboplastin time, fibrinogen). The local tolerability of LMWH was higher, as demonstrated by the lower number of side effects, such as pain, hematomas, and burning at injection site. No patient in either group presented any problem of general tolerance.

1988 - The effects of low frequency pulsing electromagnetic fields on the response of human lymphocytes to lectins. Changes at different values of induced electrical tension [Abstract in Rivista]
Cadossi, R.; Ceccherelli, G. B.; Emilia, G.; Torelli, G.; Ruggieri, M. P.; Bersani, F.; Cossarizza, A.; Franceschi, C.
abstract

Lymphocytes obtained from the periferal blood of normal donors and patients suffering from B-chronic lymphocytic leukemia (B-CLL) were cultured. Lymphocyte cultures containing phytohemagglutinin (PHA) were exposed to low frequency pulsing electromagnetic fields (PEMFs). An increase in cell proliferation was observed when relatively "low" amplitude electromagnetic fields were used. By increasing the amplitude of the electromagnetic field, a reversal effect was obtained and inhibition of lymphocyte proliferation was observed. Lymphocyte cultures exposed to low amplitude PEMFs showed increased cell proliferation also when cultured in the presence of sub-optimum concentrations of PHA. Data are presented showing that in consequence of PEMF exposure more B-cell soluble growth factors are found in the culture medium. © 1988.

1988 - The effects of low frequency pulsing electromagnetic fields on the response of human lymphocytes to lectins. Changes at different values of induced electrical tension [Abstract in Rivista]
Cadossi, R.; Ceccherelli, G. B.; Emilia, G.; Torelli, G.; Ruggieri, M. P.; Bersani, F.; Cossarizza, A.; Franceschi, C.
abstract

Lymphocytes obtained from the periferal blood of normal donors and patients suffering from B-chronic lymphocytic leukemia (B-CLL) were cultured. Lymphocyte cultures containing phytohemagglutinin (PHA) were exposed to low frequency pulsing electromagnetic fields (PEMFs). An increase in cell proliferation was observed when relatively "low" amplitude electromagnetic fields were used. By increasing the amplitude of the electromagnetic field, a reversal effect was obtained and inhibition of lymphocyte proliferation was observed. Lymphocyte cultures exposed to low amplitude PEMFs showed increased cell proliferation also when cultured in the presence of sub-optimum concentrations of PHA. Data are presented showing that in consequence of PEMF exposure more B-cell soluble growth factors are found in the culture medium. © 1988.

1987 - Effect of naproxen on some immunologic and metabolic parameters in man [Articolo su rivista]
Cossarizza, Andrea
abstract

1986 - Enhancing effect of low frequency pulsed electromagnetic fields on lectin-induced human lymphocyte proliferation [Articolo su rivista]
Cantini, M.; Cossarizza, A.; Bersani, F.; Cadossi, R.; Ceccherelli, G.; Tenconi, R.; Gatti, C.; Franceschi, C.
abstract

The effect on cell proliferation of continuous exposure of human peripheral lymphocytes from a total of 19 subjects (14 healthy donors, 1 patient with muscular dystrophy (M.D.) and his 4 siblings) to pulsed extremely-low-frequency, low-intensity electromagnetic fields (PEMFs) was studied. PEWS were not mitogenic by themselves, and a slightly reduced 3H-thymidine (3H-TdR) incorporation in unstimulated lymphocyte cultures was observed. In phytohemagglutinin-stimulated lymphocyte cultures, increased 3H-TdR incorporation was observed in all subjects. The increase was statistically significant at optimal and supraoptimal phytohemagglutinin doses. The subjects belonging to the M.D. family exhibited a PEMF-induced increase of 3H-TdR incorporation higher than that found in healthy donors. The variables that appeared to be important ere the characteristics of the field, the geometry of the microculture system, the dose of the mitogen employed, and the immune status of the lymphocyte donors. The hypothesis that PEWS may affect lymphocyte proliferation by decreasing cell membrane fluidity is suggested. © 1986 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.