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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

: 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.

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

: 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.

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 - 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

: 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 - 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.

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

: 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

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.

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

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.

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

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 - 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

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 - Differential Expression of Lonp1 Isoforms in Cancer Cells [Articolo su rivista]
Zanini, Giada; Selleri, Valentina; De Gaetano, Anna; Gibellini, Lara; Malerba, Mara; Mattioli, Anna Vittoria; Nasi, Milena; Apostolova, Nadezda; Pinti, Marcello

Lonp1 is a mitochondrial protease that degrades oxidized and damaged proteins, assists protein folding, and contributes to the maintenance of mitochondrial DNA. A higher expression of LonP1 has been associated with higher tumour aggressiveness. Besides the full-length isoform (ISO1), we identified two other isoforms of Lonp1 in humans, resulting from alternative splicing: Isoform-2 (ISO2) lacking aa 42-105 and isoform-3 (ISO3) lacking aa 1-196. An inspection of the public database TSVdb showed that ISO1 was upregulated in lung, bladder, prostate, and breast cancer, ISO2 in all the cancers analysed (including rectum, colon, cervical, bladder, prostate, breast, head, and neck), ISO3 did not show significant changes between cancer and normal tissue. We overexpressed ISO1, ISO2, and ISO3 in SW620 cells and found that the ISO1 isoform was exclusively mitochondrial, ISO2 was present in the organelle and in the cytoplasm, and ISO3 was exclusively cytoplasmatic. The overexpression of ISO1 and, at a letter extent, of ISO2 enhanced basal, ATP-linked, and maximal respiration without altering the mitochondria number or network, mtDNA amount. or mitochondrial dynamics. A higher extracellular acidification rate was observed in ISO1 and ISO2, overexpressing cells, suggesting an increase in glycolysis. Cells overexpressing the different isoforms did not show a difference in the proliferation rate but showed a great increase in anchorage-independent growth. ISO1 and ISO2, but not ISO3, determined an upregulation of EMT-related proteins, which appeared unrelated to higher mitochondrial ROS production, nor due to the activation of the MEK ERK pathway, but rather to global metabolic reprogramming of cells.

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.

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 - 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

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 - 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

: 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.

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.

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.

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.

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 - 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.

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.

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.

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 - 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.

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

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 - 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.

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.

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 - 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

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 - 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.

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.

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.

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 - 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.

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.

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

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

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 - 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.

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

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

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 - Programmed cell death in health and disease [Articolo su rivista]
Gibellini, L.; Moro, L.

: Programmed cell death is a conserved evolutionary process of cell suicide that is central to the development and integrity of eukaryotic organisms [...].

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.

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 - 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

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 - 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

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

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.

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

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

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 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.

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 - 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.

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.

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

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 - 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

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 - 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.

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 - 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.

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 - 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

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

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 - 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,

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 - 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.

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 - 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

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 - 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.

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.

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.

2018 - Core-rod myopathy due to a novel mutation in BTB/POZ domain of KBTBD13 manifesting as late onset LGMD [Articolo su rivista]
Garibaldi, Matteo; Fattori, Fabiana; Bortolotti, Carlo Augusto; Brochier, Guy; Labasse, Clemence; Verardo, Margherita; Servian-Morilla, Emilia; Gibellini, Lara; Pinti, Marcello; Di Rocco, Giulia; Raffa, Salvatore; Pennisi Elena, Maria; Bertini Enrico, Silvio; Paradas, Carmen; Romero Norma, Beatriz; Antonini, Giovanni.

Few genes (RYR1, NEB, ACTA1, CFL2, KBTBD13) have been associated with core-rod congenital myopathies [7]. KBTBD13 belongs to the Kelch-repeat super-family of proteins and is implicated in the ubiquitination pathway. Dominant mutations in KBTBD13 have been associated with a peculiar form of core-rod myopathy (NEM6) so far [10]. Childhood onset, slowly progressive proximal muscle weakness with characteristic slowness of movements and combination of nemaline rods, irregular shaped cores and unusual type2 fibres hypotrophy at muscle biopsy, were the main characteristics shared in all the affected members of the four KBTBD13 families reported in the literature [12]. We report on a 65 years old patient, of Sardinian origin, with atypical clinical and morphological presentation of NEM6 due to a novel mutation in KBTBD13 gene.

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

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 - 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

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

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 - 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.

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.

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

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

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 - 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

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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

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 - Lon protease: a novel mitochondrial matrix protein in the interconnection between drug-induced mitochondrial dysfunction and ER stress. [Articolo su rivista]
Miriam, Polo; Fernando, Alegre; Moragrega, Angela; Gibellini, Lara; Alberto, Marti rodrigo; Ana, Blas garcia; Esplugues, Juan; Apostolova, Nadezda

BACKGROUND AND PURPOSE Mitochondria-associated membranes (MAMs) are specific endoplasmic reticulum (ER) domains that enable it to interact directly with mitochondria and mediate metabolic flow and Ca2+ transfer. A growing list of proteins have been identified as MAMs components, but how they are recruited and function during complex cell stress situations is still not understood, while the participation of mitochondrial matrix proteins is largely unrecognized. EXPERIMENTAL APPROACH This work compares mitochondrial/ER contact during combined ER stress/mitochondrial dysfunction using a model of human hepatoma cells (Hep3B cell line) treated for 24 h with classic pharmacological inducers of ER stress (thapsigargin), mitochondrial dysfunction (carbonyl cyanide m-chlorophenyl hydrazone or rotenone) or both (the antiretroviral drug efavirenz used at clinically relevant concentrations). KEY RESULTS Markers of mitochondrial dynamics (dynamin-related protein 1, optic atrophy 1 and mitofusin 2) were expressed differently with these stimuli, pointing to a specificity of combined ER/mitochondrial stress. Lon, a matrix protease involved in protein and mtDNA quality control, was up-regulated at mRNA and protein levels under all conditions. However, only efavirenz decreased the mito- chondrial content of Lon while increasing its extramitochondrial presence and its localization to MAMs. This latter effect resulted in an enhanced mitochondria/ER interaction, as shown by co-immunoprecipitation experiments of MAMs protein partners and confocal microscopy imaging. CONCLUSION AND IMPLICATIONS A specific dual drug-induced mitochondria-ER effect enhances the MAMs content of Lon and its extramitochondrial expression. This is the first report of this phenomenon and suggests a novel MAMs-linked function of Lon protease.

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

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 - 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

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 - Effect of diets supplemented with different conjugated linoleic acid (CLA) isomers on protein expression in C57/BL6 mice [Articolo su rivista]
DELLA CASA, Lara; Rossi, Elena; Romanelli, Claudia; Gibellini, Lara; Iannone, Anna

The individual genetic variations, as a response to diet, have recently caught the attention of several researchers. In addition, there is also a trend to assume food containing beneficial substances, or to supplement food with specific compounds. Among these, there is the conjugated linoleic acid (CLA), which has been demonstrated to reduce fat mass and to increase lean mass, even though its mechanism of action is still not known. We investigated the effect of CLA isomers (CLA c9,t11 and CLA t10,c12) on the proteomic profile of liver, adipose tissue, and muscle of mouse, with the aim of verifying the presence of a modification in fat and lean mass, and to explore the mechanism of action.

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

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,

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

Guidelines for studying autophagy

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

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

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

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 - 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

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

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

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

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 - Human dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations [Articolo su rivista]
Pisciotta, Alessandra; Carnevale, Gianluca; Meloni, Simona; Riccio, Massimo; De Biasi, Sara; Gibellini, Lara; Ferrari, Adriano; Bruzzesi, Giacomo; De Pol, Anto

Human dental pulp represents a suitable alternative source of stem cells for the purpose of cell-based therapies in regenerative medicine, because it is relatively easy to obtain it, using low invasive procedures. This study characterized and compared two subpopulations of adult stem cells derived from human dental pulp (hDPSCs). Human DPSCs, formerly immune-selected for STRO-1 and c-Kit, were separated for negativity and positivity to CD34 expression respectively, and evaluated for cell proliferation, stemness maintenance, cell senescence and multipotency.

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

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

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

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

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 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 - 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

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 - Stem cells isolated from human dental pulp and amniotic fluid improve skeletal muscle histopathology in mdx/SCID mice [Articolo su rivista]
Pisciotta, Alessandra; Riccio, Massimo; Carnevale, Gianluca; Lu, Aiping; DE BIASI, Sara; Gibellini, Lara; LA SALA, Giovanni Battista; Bruzzesi, Giacomo; Ferrari, Adriano; Huard, Johnny; DE POL, Anto

Introduction: Duchenne muscular dystrophy (DMD), caused by a lack of the functional structural protein dystrophin, leads to severe muscle degeneration where the patients are typically wheelchair-bound and die in their mid-twenties from cardiac or respiratory failure or both. The aim of this study was to investigate the potential of human dental pulp stem cells (hDPSCs) and human amniotic fluid stem cells (hAFSCs) to differentiate toward a skeletal myogenic lineage using several different protocols in order to determine the optimal conditions for achieving myogenic commitment and to subsequently evaluate their contribution in the improvement of the pathological features associated with dystrophic skeletal muscle when intramuscularly injected into mdx/SCID mice, an immune-compromised animal model of DMD. Methods: Human DPSCs and AFSCs were differentiated toward myogenic lineage in vitro through the direct co-culture with a myogenic cell line (C2C12 cells) and through a preliminary demethylation treatment with 5-Aza-2′-deoxycytidine (5-Aza), respectively. The commitment and differentiation of both hDPSCs and hAFSCs were evaluated by immunofluorescence and Western blot analysis. Subsequently, hDPSCs and hAFSCs, preliminarily demethylated and pre-differentiated toward a myogenic lineage for 2 weeks, were injected into the dystrophic gastrocnemius muscles of mdx/SCID mice. After 1, 2, and 4 weeks, the gastrocnemius muscles were taken for immunofluorescence and histological analyses. Results: Both populations of cells engrafted within the host muscle of mdx/SCID mice and through a paracrine effect promoted angiogenesis and reduced fibrosis, which eventually led to an improvement of the histopathology of the dystrophic muscle. Conclusion: This study shows that hAFSCs and hDPSCs represent potential sources of stem cells for translational strategies to improve the histopathology and potentially alleviate the muscle weakness in patients with DMD.

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

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 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

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

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 - 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.

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 - 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

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

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 - 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

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 - 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

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 - 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

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 - 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.

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

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 >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.

2012 - Human serum promotes osteogenic differentiation of Human Dental Pulp Stem Cells in vitro and in vivo. [Articolo su rivista]
Pisciotta, Alessandra; Riccio, Massimo; Carnevale, Gianluca; Beretti, Francesca; Gibellini, Lara; Maraldi, Tullia; Cavallini, Gian Maria; Ferrari, Adriano; Bruzzesi, G; DE POL, Anto

Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS) is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

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

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 - 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

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 >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 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

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.

Cossarizza, Andrea; Cevenini, E.; Stazi, M. A.; Cotichini, R.; Monti, Daniela; Nasi, Milena; Gibellini, Lara; DE BIASI, Sara; Benatti, Stefania; Pinti, Marcello; Franceschi, Claudio

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

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

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.

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

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

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 - 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

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 - 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: 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 - 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

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

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

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.

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

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.

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.

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 - 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

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 - 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

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.

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 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.