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Marcello PINTI

Professore Associato
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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Pubblicazioni

2024 - Vascular "Long COVID": A New Vessel Disease? [Articolo su rivista]
Zanini, Giada; Selleri, Valentina; Roncati, Luca; Coppi, Francesca; Nasi, Milena; Farinetti, Alberto; Manenti, Antonio; Pinti, Marcello; Mattioli, Anna Vittoria
abstract

: Vascular sequelae following (SARS-CoV-2 coronavirus disease) (COVID)-19 infection are considered as "Long Covid (LC)" disease, when occurring 12 weeks after the original infection. The paucity of specific data can be obviated by translating pathophysiological elements from the original Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) infection (In a microcirculatory system, a first "endotheliitis," is often followed by production of "Neutrophil Extracellular Trap," and can evolve into a more complex leukocytoklastic-like and hyperimmune vasculitis. In medium/large-sized vessels, this corresponds to endothelial dysfunction, leading to an accelerated progression of pre-existing atherosclerotic plaques through an increased deposition of platelets, circulating inflammatory cells and proteins. Associated dysregulated immune and pro-coagulant conditions can directly cause thrombo-embolic arterial or venous complications. In order to implement appropriate treatment, physicians need to consider vascular pathologies observed after SARS-Cov-2 infections as possible "LC" disease.

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

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

2023 - Cardiovascular prevention in women: an update by the Italian Society of Cardiology working group on 'Prevention, hypertension and peripheral disease' [Articolo su rivista]
Mattioli, Anna Vittoria; Moscucci, Federica; Sciomer, Susanna; Maffei, Silvia; Nasi, Milena; Pinti, Marcello; Bucciarelli, Valentina; Dei Cas, Alessandra; Parati, Gianfranco; Ciccone, Marco Matteo; Palmiero, Pasquale; Maiello, Maria; Pedrinelli, Roberto; Pizzi, Carmine; Barillà, Francesco; Gallina, Sabina
abstract

: The recent pandemic has substantially changed the approach to the prevention of cardiovascular diseases in women. Women have been significantly impacted by the changes that occurred during the pandemic and the quarantine adopted to prevent the spread of the disease. Changes involved prevention both through the reduction of visits and preventive screening and through social and economic changes. It is necessary to adopt new cardiovascular prevention approaches focused on returning to healthy lifestyles, reducing stress and depression also using modern tools such as telemedicine, mobile phone applications and the web. These tools convey messages in a persuasive way especially in young and adult women. There is less impact of these new tools on older women towards whom it is important to adopt a more traditional approach. This review focuses on the new approach to cardiovascular prevention in women in light of the lifestyle changes recorded during the pandemic and which led to an increase in obesity examines the effects on the cardiovascular system induced by stress and depression and analyses the new high blood pressure guidelines and indications that are specific to women.

2023 - Long COVID: A New Challenge for Prevention of Obesity in Women [Articolo su rivista]
Mattioli, A. V.; Coppi, F.; Nasi, M.; Pinti, M.; Gallina, S.
abstract

2023 - Mitochondrial DNA as inflammatory DAMP: a warning of an aging immune system? [Articolo su rivista]
Zanini, Giada; Selleri, Valentina; Lopez Domenech, Sandra; Malerba, Mara; Nasi, Milena; Mattioli, Anna Vittoria; Pinti, Marcello
abstract

Senescence of the immune system is characterized by a state of chronic, subclinical, low-grade inflammation termed 'inflammaging', with increased levels of proinflammatory cytokines, both at the tissue and systemic levels. Age-related inflammation can be mainly driven by self-molecules with immunostimulant properties, named Damage/death Associated Molecular Patterns (DAMPs), released by dead, dying, injured cells or aged cells. Mitochondria are an important source of DAMPs, including mitochondrial DNA - the small, circular, double-stranded DNA molecule found in multiple copies in the organelle. mtDNA can be sensed by at least three molecules: the Toll-like receptor 9, the NLRP3 inflammasomes, and the cyclic GMP-AMP synthase (cGAS). All these sensors can lead to the release of proinflammatory cytokines when engaged. The release of mtDNA by damaged or necrotic cells has been observed in several pathological conditions, often aggravating the course of the disease. Several lines of evidence indicate that the impairment of mtDNA quality control and of the organelle homeostasis associated with aging determines an increase in the leakage of mtDNA from the organelle to the cytosol, from the cell to the extracellular space, and into plasma. This phenomenon, mirrored by an increase in mtDNA circulating levels in elderly people, can lead to the activation of different innate immune cell types, sustaining the chronic inflammatory status that is characteristic of aging.

2023 - Physical Activity and Diet in Older Women: A Narrative Review [Articolo su rivista]
Mattioli, Anna Vittoria; Selleri, Valentina; Zanini, Giada; Nasi, Milena; Pinti, Marcello; Stefanelli, Claudio; Fedele, Francesco; Gallina, Sabina
abstract

2023 - Predictors for progression in amyotrophic lateral sclerosis associated to SOD1 mutation: insight from two population-based registries [Articolo su rivista]
Martinelli, Ilaria; Ghezzi, Andrea; Zucchi, Elisabetta; Gianferrari, Giulia; Ferri, Laura; Moglia, Cristina; Manera, Umberto; Solero, Luca; Vasta, Rosario; Canosa, Antonio; Grassano, Maurizio; Brunetti, Maura; Mazzini, Letizia; De Marchi, Fabiola; Simonini, Cecilia; Fini, Nicola; Vinceti, Marco; Pinti, Marcello; Chiò, Adriano; Calvo, Andrea; Mandrioli, Jessica
abstract

BackgroundUncovering distinct features and trajectories of amyotrophic lateral sclerosis (ALS) associated with SOD1 mutations (SOD1-ALS) can provide valuable insights for patient' counseling and stratification for trials, and interventions timing. Our study aims to pinpoint distinct clinical characteristics of SOD1-ALS by delving into genotype-phenotype correlations and factors that potentially impact disease progression.MethodsThis is a retrospective observational study of a SOD1-ALS cohort from two Italian registers situated in the regions of Emilia-Romagna, Piedmont and Valle d'Aosta.ResultsOut of 2204 genotyped ALS patients, 2.5% carried SOD1 mutations, with a M:F ratio of 0.83. SOD1-ALS patients were younger, and more frequently reported a family history of ALS and/or FTD. SOD1-ALS had a longer survival compared to patients without ALS-associated gene mutations. However, here was considerable variability in survival across distinct SOD1 mutations, with an average survival of less than a year for the L39V, G42S, G73S, D91N mutations. Among SOD1-ALS, multivariate analysis showed that, alongside established clinical prognostic factors such as advanced age at onset and high progression rate at diagnosis, mutations located in exon 2 or within highly conserved gene positions predicted worse survival. Conversely, among comorbidities, cancer history was independently associated with longer survival.InterpretationWithin the context of an overall slower disease, SOD1-ALS exhibits some degree of heterogeneity linked to the considerable genetic diversity arising from the multitude of potential mutations sites and specific clinical prognostic factors, including cancer history. Revealing the factors that modulate the phenotypic heterogeneity of SOD1-ALS could prove advantageous in improving the efficacy of upcoming therapeutic approaches.

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

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

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

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

2023 - Reply to: Letter on the Recent Paper "Vascular 'Long COVID': A New Vessel Disease?" [Articolo su rivista]
Zanini, Giada; Selleri, Valentina; Roncati, Luca; Coppi, Francesca; Nasi, Milena; Farinetti, Alberto; Manenti, Antonio; Pinti, Marcello; Mattioli, Anna Vittoria
abstract

2023 - Sex Related Differences in the Complex Relationship between Coffee, Caffeine and Atrial Fibrillation [Articolo su rivista]
Coppi, F; Bucciarelli, V; Sinigaglia, G; Zanini, G; Selleri, V; Nasi, M; Pinti, M; Gallina, S; Mattioli, Av
abstract

: This literature review aims to explore the data of articles published on the association between coffee, caffeine and atrial fibrillation and to analyze any differences between the two sexes. Several factors influence this complex relationship; genetic, environmental and psychosocial factors come into play in the pathophysiology of atrial fibrillation. These factors are expressed differently in women and men. However, the analysis of the literature has shown that comparison works between the two sexes are extremely rare. Most population-based and prospective studies either analyze aggregated data or focus on exclusively male or female populations. This results in a lack of information that could be useful in the prevention of and treatment approach to atrial fibrillation. It is necessary to deepen this issue with dedicated studies.

2023 - Sex and Gender Differences in Medical Education: The Impact on Scientific Reports [Articolo su rivista]
Mattioli, A. V.; Coppi, F.; Bucciarelli, V.; Nasi, M.; Pinti, M.; Palumbo, C.; Gallina, S.
abstract

This commentary explores the reasons why sex and gender differences must be included in medical education and the impact on healthcare outcomes for patients. Understanding sex and gender differences could be useful in making more accurate diagnoses and to develop more effective treatment plans. Sex and gender medicine take into consideration both the genetic basis and the effects of exposure to environmental and socio-economic factors.

2023 - The Role of Lonp1 on Mitochondrial Functions during Cardiovascular and Muscular Diseases [Articolo su rivista]
Zanini, G.; Selleri, V.; Malerba, M.; Solodka, K.; Sinigaglia, G.; Nasi, M.; Mattioli, A. V.; Pinti, M.
abstract

The mitochondrial protease Lonp1 is a multifunctional enzyme that regulates crucial mitochondrial functions, including the degradation of oxidized proteins, folding of imported proteins and maintenance the correct number of copies of mitochondrial DNA. A series of recent studies has put Lonp1 at the center of the stage in the homeostasis of cardiomyocytes and muscle skeletal cells. During heart development, Lonp1 allows the metabolic shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation. Knock out of Lonp1 arrests heart development and determines cardiomyocyte apoptosis. In adults, Lonp1 acts as a cardioprotective protein, as its upregulation mitigates cardiac injury by preventing the oxidative damage of proteins and lipids, and by preserving mitochondrial redox balance. In skeletal muscle, Lonp1 is crucial for cell development, as it mediates the activation of PINK1/Parkin pathway needed for proper myoblast differentiation. Skeletal muscle-specific ablation of Lonp1 in mice causes reduced muscle fiber size and strength due to the accumulation of mitochondrial-retained protein in muscle. Lonp1 expression and activity decline with age in different tissues, including skeletal muscle, and are associated with a functional decline and structural impairment of muscle fibers. Aerobic exercise increases unfolded protein response markers including Lonp1 in the skeletal muscle of aged animals and is associated with muscle functional recovery. Finally, mutations of Lonp1 cause a syndrome named CODAS (Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies) characterized by the impaired development of multiple organs and tissues, including myocytes. CODAS patients show hypotonia and ptosis, indicative of skeletal muscle reduced performance. Overall, this body of observations points Lonp1 as a crucial regulator of mitochondrial functions in the heart and in skeletal muscle.

2023 - The landscape of cognitive impairment in superoxide dismutase 1-amyotrophic lateral sclerosis [Articolo su rivista]
Martinelli, I.; Zucchi, E.; Simonini, C.; Gianferrari, G.; Zamboni, G.; Pinti, M.; Mandrioli, J.
abstract

Although mutations in the superoxide dismutase 1 gene account for only a minority of total amyotrophic lateral sclerosis cases, the discovery of this gene has been crucial for amyotrophic lateral sclerosis research. Since the identification of superoxide dismutase 1 in 1993, the field of amyotrophic lateral sclerosis genetics has considerably widened, improving our understanding of the diverse pathogenic basis of amyotrophic lateral sclerosis. In this review, we focus on cognitive impairment in superoxide dismutase 1-amyotrophic lateral sclerosis patients. Literature has mostly reported that cognition remains intact in superoxide dismutase 1-amyotrophic lateral sclerosis patients, but recent reports highlight frontal lobe function frailty in patients carrying different superoxide dismutase 1-amyotrophic lateral sclerosis mutations. We thoroughly reviewed all the various mutations reported in the literature to contribute to a comprehensive database of superoxide dismutase 1-amyotrophic lateral sclerosis genotype-phenotype correlation. Such a resource could ultimately improve our mechanistic understanding of amyotrophic lateral sclerosis, enabling a more robust assessment of how the amyotrophic lateral sclerosis phenotype responds to different variants across genes, which is important for the therapeutic strategy targeting genetic mutations. Cognition in superoxide dismutase 1-amyotrophic lateral sclerosis deserves further longitudinal research since this peculiar frailty in patients with similar mutations can be conditioned by external factors, including environment and other unidentified agents including modifier genes.

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

2022 - Detection of Neurofilament Light Chain with Label-Free Electrolyte-Gated Organic Field-Effect Transistors [Articolo su rivista]
Solodka, K.; Berto, M.; Ferraro, D.; Menozzi, C.; Borsari, M.; Bortolotti, C. A.; Biscarini, F.; Pinti, M.
abstract

Neurofilaments are structural scaffolding proteins of the neuronal cytoskeleton. Upon axonal injury, the neurofilament light chain (NF-L) is released into the interstitial fluid and eventually reaches the cerebrospinal fluid and blood. Therefore, NF-L is emerging as a biomarker of neurological disorders, including neurodegenerative dementia, Parkinson's disease, and multiple sclerosis. It is challenging to quantify NF-L in bodily fluids due to its low levels. This work reports the detection of NF-L in aqueous solutions with an organic electronic device. The biosensor is based on the electrolyte-gated organic field-effect transistor (EGOFET) architecture and can quantify NF-L down to sub-pM levels; thanks to modification of the device gate with anti-NF-L antibodies imparted with potentially controlled orientation. The response is fitted to the Guggenheim–Anderson–De Boer adsorption model to describe NF-L adsorption at the gate/electrolyte interface, to consider the formation of a strongly adsorbed protein layer bound to the antibody and the formation of weakly bound NF-L multilayers, an interpretation which is also backed up by morphological characterization via atomic force microscopy. The label-free, selective, and rapid response makes this EGOFET biosensor a promising tool for the diagnosis and monitoring of neuronal damages through the detection of NF-L in physio-pathological ranges.

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
abstract

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.
abstract

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

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

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

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

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

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

2022 - Insegnare le differenze di genere alla facoltà di medicina: uno strumento per migliorare la sicurezza e l'efficacia della prescrizione dell’attività fisica personalizzata [Articolo su rivista]
Pinti, Marcello; Mattioli, Anna Vittoria; Nasi, Milena; Selleri, Valentina; Palumbo, Carla
abstract

ender medicine is defined by the World Health Organization (WHO) as the study of the influence of biological differences, defined by sex, and socioeconomic and cultural differences, defined by gender, on each person's health and disease status. Since gender bias is still strongly present and entrenched in medicine, recent studies indicate how crucial the teaching of gender medicine is in the training of doctors and health professionals to create not only a positive learning environment, but also a more equitable and organized healthcare system. Therefore, the aim of the following article is to highlight the importance of systematic teaching of gender medicine to improve the safety and effectiveness of prescribing personalized physical activity and sports.

2022 - Macrophages Modulate Hepatic Injury Involving NLRP3 Inflammasome: The Example of Efavirenz [Articolo su rivista]
Alegre, Fernando; Martí-Rodrigo, Alberto; Polo, Miriam; Ortiz-Masiá, Dolores; Bañuls, Celia; Pinti, Marcello; Álvarez, Ángeles; Apostolova, Nadezda; Esplugues, Juan V; Blas-García, Ana
abstract

Drug-induced liver injury (DILI) constitutes a clinical challenge due to the incomplete characterization of the mechanisms involved and potential risk factors. Efavirenz, an anti-HIV drug, induces deleterious actions in hepatocytes that could underlie induction of the NLRP3 inflammasome, an important regulator of inflammatory responses during liver injury. We assessed the potential of efavirenz to modulate the inflammatory and fibrogenic responses of major liver cell types involved in DILI. The effects of efavirenz were evaluated both in vitro and in vivo. Efavirenz triggered inflammation in hepatocytes, in a process that involved NF-kappa B and the NLRP3 inflammasome, and activated hepatic stellate cells (HSCs), thereby enhancing expression of inflammatory and fibrogenic markers. The NLRP3 inflammasome was not altered in efavirenz-treated macrophages, but these cells polarized towards the anti-inflammatory M2 phenotype and displayed upregulated anti-inflammatory mediators. Conversely, no evidence of damage was observed in efavirenz-treated animals, except when macrophages were depleted, which resulted in the in vivo manifestation of the deleterious effects detected in hepatocytes and HSCs. Efavirenz elicits a cell-specific activation of the NLRP3 inflammasome in hepatocytes and HSCs, but macrophages appear to counteract efavirenz-induced liver injury. Our results highlight the dynamic nature of the interaction among liver cell populations and emphasize the potential of targeting macrophage polarization as a strategy to treat NLRP3 inflammasome-induced liver injury.

2022 - Mitochondrial and Endoplasmic Reticulum Alterations in a Case of Amyotrophic Lateral Sclerosis Caused by TDP-43 A382T Mutation [Articolo su rivista]
Zanini, Giada; Selleri, Valentina; Nasi, Milena; De Gaetano, Anna; Martinelli, Ilaria; Gianferrari, Giulia; Lofaro, Francesco Demetrio; Boraldi, Federica; Mandrioli, Jessica; Pinti, Marcello
abstract

Amyotrophic lateral sclerosis is the most common form of motor neuron disease. Mutations in TARDBP, the gene encoding the RNA-binding protein TDP-43, are responsible for about 5% of familial ALS. Here we report the clinical and biological features of an ALS patients with pA382T mutation in TPD-43 protein. Disease began with right hand muscles weakness, and equally involved upper and lower motor neuron with a classic phenotype, without cognitive impairment. While a family history of neurological diseases was reported, there was no evidence of familial frontotemporal dementia. Cultured fibroblasts from the patient were characterized by profound alterations of cell proteome, which impacts particularly the mitochondrial metabolic pathways and the endoplasmic reticulum. TDP-43 levels were similar to control, healthy fibroblasts, but a higher fraction localized in mitochondria. Mitochondrial network appeared fragmented, and the organelles smaller and more spheric. In agreement with impaired proteome and morphology of mitochondria, basal cell respiration was reduced. Mitochondrial DNA levels appeared normal. However, a higher amount of mitochondrial DNA was present in the cytosol, suggesting a pronounced mitochondrial DNA misplacement which can promote a pro-inflammatory response mediating by cGAS/STING. Thus, this case report further expands the clinical and pathological phenotype of A382T mutation.

2022 - Teaching Gender Differences at Medical School Could Improve the Safety and Efficacy of Personalized Physical Activity Prescription [Articolo su rivista]
Mattioli, Anna Vittoria; Nasi, Milena; Pinti, Marcello; Palumbo, Carla
abstract

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

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

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

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

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

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

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

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

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

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

2021 - Letter: Does obesity affect the severity of exercise-induced muscle injury? (j obes metab syndr 2021;30:132-40) [Articolo su rivista]
Mattioli, A. V.; Coppi, F.; Nasi, M.; Pinti, M.
abstract

2021 - Microglia activation: A role for mitochondrial DNA? [Articolo su rivista]
Pinti, M.; Ferraro, D.; Nasi, M.
abstract

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

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

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

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

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

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

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

2021 - Molecular mechanisms of mtdna-mediated inflammation [Articolo su rivista]
De Gaetano, A.; Solodka, K.; Zanini, G.; Selleri, V.; Mattioli, A. V.; Nasi, M.; Pinti, M.
abstract

Besides their role in cell metabolism, mitochondria display many other functions. Mitochondrial DNA (mtDNA), the own genome of the organelle, plays an important role in modulating the inflammatory immune response. When released from the mitochondrion to the cytosol, mtDNA is recognized by cGAS, a cGAMP which activates a pathway leading to enhanced expression of type I interferons, and by NLRP3 inflammasome, which promotes the activation of pro-inflammatory cytokines Interleukin-1beta and Interleukin-18. Furthermore, mtDNA can be bound by Toll-like receptor 9 in the endosome and activate a pathway that ultimately leads to the expression of pro-inflammatory cytokines. mtDNA is released in the extracellular space in different forms (free DNA, protein-bound DNA fragments) either as free circulating molecules or encapsulated in extracellular vesicles. In this review, we discussed the latest findings concerning the molecular mechanisms that regulate the release of mtDNA from mitochondria, and the mechanisms that connect mtDNA misplacement to the activation of inflammation in different pathophysiological conditions.

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

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

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

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

2020 - Cerebrospinal fluid kappa and lambda free light chains in oligoclonal band‐negative patients with suspected multiple sclerosis [Articolo su rivista]
Ferraro, Diana; Trovati, Alice; Bedin, Roberta; Natali, Patrizia; Franciotta, Diego; Santangelo, Mario; Camera, Valentina; Vitetta, Francesca; Varani, Manuela; Trenti, Tommaso; Gastaldi, Matteo; De Biasi, Sara; Nasi, Milena; Pinti, Marcello; Meletti, Stefano; Sola, Patrizia
abstract

Cerebrospinal fluid (CSF) free light kappa chains (kappa FLC) may be a more sensitive marker of intrathecal IgG synthesis compared to oligoclonal bands (OCBs). Our aim was to retrospectively determine the additional value of the kappa and lambda index (CSF FLC/serum FLC)/(CSF albumin/serum albumin) in predicting a Multiple Sclerosis (MS) diagnosis in a group of OCB-negative patients with suspected MS.

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

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

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

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

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

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

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

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

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

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

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

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

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

2020 - Obesity risk during collective quarantine for the COVID-19 epidemic [Articolo su rivista]
Mattioli, Anna Vittoria; Pinti, Marcello; Farinetti, Alberto; Nasi, Milena
abstract

In March 2020, when COVID-19 epidemics involved several countries, the WHO defined the infection as a pandemic. Government adopted measures to prevent the diffusion of infection; i.e. quarantine and isolation. One of the consequences of quarantine-induced stress is a change in lifestyle and eating habits leading to obesity. The present commentary briefly analyzes the effects of quarantine on obesity.

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

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

2020 - Plasma neurofilaments correlate with disability in progressive multiple sclerosis patients [Articolo su rivista]
Ferraro, Diana; Guicciardi, Claudio; De Biasi, Sara; Pinti, Marcello; Bedin, Roberta; Camera, Valentina; Vitetta, Francesca; Nasi, Milena; Meletti, Stefano; Sola, Patrizia
abstract

Cerebrospinal fluid (CSF) and blood neurofilaments (NFLs) are markers of axonal damage and are being investigated, mostly in relapsing-remitting (RR) MS, as a marker of disease activity and of response to treatment, while there are less data in progressive MS patients. Primary aim was to measure NFL in plasma samples of untreated patients with primary (PP) and secondary (SP) progressive MS and to correlate them with disability, disease severity, and prior/subsequent disability progression.

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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
abstract

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

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

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

2017 - Label-free detection of interleukin-6 using electrolyte gated organic field effect transistors [Articolo su rivista]
Diacci, Chiara; Berto, Marcello; Di Lauro, Michele; Bianchini, Elena; Pinti, Marcello; Simon, Daniel T.; Biscarini, Fabio; Bortolotti, Carlo A.
abstract

Cytokines are small proteins that play fundamental roles in inflammatory processes in the human body. In particular, interleukin (IL)-6 is a multifunctional cytokine, whose increased levels are associated with infection, cancer, and inflammation. The quantification of IL-6 is therefore of primary importance in early stages of inflammation and in chronic diseases, but standard techniques are expensive, time-consuming, and usually rely on fluorescent or radioactive labels. Organic electronic devices and, in particular, organic field-effect transistors (OFETs) have been proposed in the recent years as novel platforms for label-free protein detection, exploiting as sensing unit surface-immobilized antibodies or aptamers. Here, the authors report two electrolyte-gated OFETs biosensors for IL-6 detection, featuring monoclonal antibodies and peptide aptamers adsorbed at the gate. Both strategies yield biosensors that can work on a wide range of IL-6 concentrations and exhibit a remarkable limit of detection of 1 pM. Eventually, electrolyte gated OFETs responses have been used to extract and compare the binding thermodynamics between the sensing moiety, immobilized at the gate electrode, and IL-6.

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

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

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

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

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

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

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

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

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

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

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

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

Guidelines for studying autophagy

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

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

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

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

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

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

2016 - TRPA1 Is Expressed in Central But Not in Peripheral Glia [Articolo su rivista]
Vellani, Vittorio; Gomis-Perez, Carolina; Pinti, Marcello; Prandini, Massimiliano; Pavesi, Giorgia; Giacomoni, Chiara; Caprini, Marco
abstract

TRPA1 are cation channels expressed in sensory neurons and in several other cell types. This channel is specifically activated by ally isothiocyanate (AITC), the pungent component of mustard oil, as well as by other electrophilic compounds. Although TRPA1 expression in central glia has been reported, its subcellular localization and its expression in peripheral glia have not been investigated before. In this paper we report the molecular and functional expression of TRPA1 in rat cortical astrocytes. Real-time RT-PCR identified low but significant amounts of TRPA1 mRNA in cortical astrocytes while no signal was seen in peripheral glia isolated from dorsal root ganglia (DRG) or in a glial cell line (DITNC-1). Calcium imaging showed AITC-induced signals in astro-cytes while no response in peripheral glia. AITC induced calcium signals in astrocytes in the presence and in the absence of extracellular calcium, suggesting an intracellular localization of TRPA1 channels. Whole cell electrophysiological recordings were performed in astrocytes, in peripheral glia and in DITNC-1 cells transfected with TRPA1 during AITC application. In TRPA1-transfected DITNC-1 cells typical TRPA1 currents were recorded with a reversal potential near 0 mV, consistent with the opening of a non-selective cation channel. No such currents were recorded in untransfected DITNC-1 cells, in astrocytes and in peripheral glial cells, where even high concentrations of AITC (up to 10 mM) induced no significant outward current. In astrocytes AITC transiently induced an outward rectifying current with the reversal potential near ?90 mV, consistent with K channel activation, likely activated by intracellular release of calcium. Our results suggest that TRPA1 channels are molecularly and functionally expressed in calcium-containing organelles of rat cortical astrocytes, with no expression in the plasma membrane.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

No abstract available

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

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

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

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

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

About 2-5% of HIV-1-infected subjects, defined as long-term non-progressors (LTNPs), remain immunologically stable for a long time without treatment. The factors governing this condition are known only in part, and include genetic factors. Thus, we studied 20 polymorphisms of 15 genes encoding proinflammatory and immunoregulatory cytokines, chemokines and their receptors, genes involved in apoptosis, and the gene HCP5. METHODS: We analyzed 47 Caucasian LTNPs infected for >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 - Drosophila Helical factor is an inducible protein acting as an immune-regulated cytokine in S2 cells. [Articolo su rivista]
Malagoli, Davide; Accorsi, Alice; Sacchi, Sandro; Basile, Valentina; Mandrioli, Mauro; Pinti, Marcello; D., Conklin; Ottaviani, Enzo
abstract

The innate immunity of Drosophila melanogaster is based on cellular and humoral components. Drosophila Helical factor (Hf), is a molecule previously discovered using an in silico approach and whose expression is controlled by the immune deficiency (Imd) pathway. Here we present evidence demonstrating that Hf is an inducible protein constitutively produced by the S2 hemocyte-derived cell line. Hf expression is stimulated by bacterial extracts that specifically trigger the Imd pathway. In absence of any bacterial challenge, the recombinant form of Hf can influence the expression of the antimicrobial peptides (AMPs) defensin but not drosomycin. These data suggest that in vitro Hf is an inducible and immune-regulated factor, with functions comparable to those of secreted vertebrate cytokines

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

2011 - In vitro characterization of the cytokine Drosophila Helical factor [Abstract in Rivista]
Malagoli, Davide; A., Accorsi; D., Conklin; Filaferro, Monica; Mandrioli, Mauro; Pinti, Marcello; S., Sacchi; Ottaviani, Enzo
abstract

Drosophila Helical factor (Hf) is a protein discovered through the QT method, an algorithm specifically designed for finding helical cytokines. Since in vivo experiments suggested the involvement of Hf in Drosophila melanogaster immunity, we have proceeded with the characterization of Hf functions in the macrophage-like Drosophila embryonic hemocytes, SL2 cell line. qPCR results demonstrated that Hf gene is induced in the SL2 cell line, after either 6 or 24 h incubation with Escherichia coli-purified peptidoglycan. The silencing of Hf expression through RNAi resulted in the reduced capability of synthesizing antimicrobial peptides (AMP) after exposure to heat-inactivated E. coli. The effects of the recombinant peptide rHf have also been tested in the SL2 cell line. rHf promotes the expression and triggers the release of Hf from the hemocytes, and stimulates the synthesis of the antimicrobial peptides (AMP) Defensin and Drosomycin, without any further immune stimulation. Consistent with the output of the QT method, which predicts Hf as a secreted protein, chromatin immune-precipitation experiments confirmed that Hf does not bind DNA, excluding that it acts as an immune-regulated transcription factor. Finally, rHf neither exerts chemotactic action nor triggers bacterial phagocytosis in SL2 cells.Altogether, our data supports the prediction that Hf is an helical cytokine produced and secreted by the hemocytes and it is mainly involved in the regulation of the humoral component of the immune response of D. melanogaster.

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

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

2011 - Mitochondrial liaisons of p53 [Articolo su rivista]
Galluzzi, Lorenzo; Morselli, Eugenia; O., Kepp; I., Vitale; Pinti, Marcello; G., Kroemer
abstract

Mitochondria play a central role in cell survival and cell death. While producing the bulk of intracellular ATP, mitochondrial respiration represents the most prominent source of harmful reactive oxygen species. Mitochondria participate in many anabolic pathways, including cholesterol and nucleotide biosynthesis, yet also control multiple biochemical cascades that contribute to the programmed demise of cells. The tumor suppressor protein p53 is best known for its ability to orchestrate a transcriptional response to stress that can have multiple outcomes, including cell cycle arrest and cell death. p53-mediated tumor suppression, however, also involves transcription-independent mechanisms. Cytoplasmic p53 can physically interact with members of the BCL-2 protein family, thereby promoting mitochondrial membrane permeabilization. Moreover, extranuclear p53 can suppress autophagy, a major prosurvival mechanism that is activated in response to multiple stress conditions. Thirty years have passed since its discovery, and p53 has been ascribed with an ever-increasing number of functions. For instance, p53 has turned out to influence the cell's redox status, by transactivating either anti- or pro-oxidant factors, and to regulate the metabolic switch between glycolysis and aerobic respiration. In this review, we will analyze the mechanisms by which p53 affects the balance between the vital and lethal functions of mitochondria.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

2010 - Viral strategies for the evasion of immunogenic cell death [Relazione in Atti di Convegno]
Galluzzi, Lorenzo; Kepp, O; Morselli, Eugenia; Vitale, I; Senovilla, L; Pinti, Marcello; Zitvogel, L; Kroemer, G.
abstract

Driven by co-evolutionary forces, viruses have refined a wide arsenal of strategies to interfere with the host defences. On one hand, viruses can block/retard programmed cell death in infected cells, thereby suppressing one of the most ancient mechanisms against viral dissemination. On the other hand, multiple viral factors can efficiently trigger the death of infected cells and uninfected cells from the immune system, which favours viral spreading and prevents/limits an active antiviral response, respectively. Moreover, several viruses are able to inhibit the molecular machinery that drives the translocation of calreticulin to the surface of dying cells. Thereby, viruses block the exposure of an engulfment signal that is required for the efficient uptake of dying cells by dendritic cells and for the induction of the immune response. In this review, we discuss a variety of mechanisms by which viruses interfere with the cell death machinery and, in particular, by which they subvert immunogenic cell death.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Vedi allegato

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Nessun abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

1999 - Identification of the Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase regulatory gene (touR) and of its cognate promoter [Articolo su rivista]
Arenghi, Fl; Pinti, Marcello; Galli, E; Barbieri, P.
abstract

Toluene-o-xylene monooxygenase is an enzymatic complex, encoded by the touABCDEF genes, responsible for the early stages of toluene and o-xylene degradation in Pseudomonas stutzeri OX1. In order to identify the loci involved in the transcriptional regulation of the tou gene cluster, deletion analysis and complementation studies were carried out with Pseudomonas putida PaW340 as a heterologous host harboring pFB1112, a plasmid that allowed regulated expression, inducible by toluene and o-xylene and their corresponding phenols, of the toluene-o-xylene monooxygenase. A locus encoding a positive regulator, designated touR, was mapped downstream from the tou gene cluster. TouR was found to be similar to transcriptional activators of aromatic compound catabolic pathways belonging to the NtrC family and, in particular, to DmpR (83% similarity), which controls phenol catabolism. By using a touA-C2,3O fusion reporter system and by primer extension analysis, a TouR cognate promoter (P(ToMO)) was mapped, which showed the typical -24 TGGC, -12 TTGC sequences characteristic of sigma(54)-dependent promoters and putative upstream activating sequences. By using the reporter system described, we found that TouR responds to mono- and dimethylphenols, but not the corresponding methylbenzenes. In this respect, the regulation of the P. stutzeri system differs from that of other toluene or xylene catabolic systems, in which the hydrocarbons themselves function as effectors. Northern analyses indicated low transcription levels of tou structural genes in the absence of inducers. Basal toluene-o-xylene monooxygenase activity may thus transform these compounds to phenols, which then trigger the TouR-mediated response.