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Leonarda TROIANO

Personale tecnico amministrativo
Servizio Prevenzione e Protezione

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Pubblicazioni

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

2004 - 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.

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.

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

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

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

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

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

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

2002 - 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.

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

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

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

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

1990 - Immunological parameters in aging: Studies on natural immunomodulatory and immunoprotective substances [Articolo su rivista]
Franceschi, C.; Cossarizza, A.; Troiano, L.; Salati, R.; Monti, D.
abstract

Several immune parameters--particularly T-cell dependent immune responses--are altered in aged subjects. To test the hypothesis that they may be the consequence of more general age-related lymphocyte biochemical alterations, and particularly of the energy producing system, the effect of L-carnitine and acetyl-L-carnitine on cell proliferation was studied in peripheral blood lymphocytes from donors of different ages. The results showed that phytohaemagglutinin-induced peripheral blood lymphocyte proliferation was markedly increased in L-carnitine- or acetyl-L-carnitine-preloaded lymphocytes from young and especially from old subjects. Cells from aged subjects considerably improved their defective proliferative capability. Preliminary observations suggest that L-carnitine-preloading also protected peripheral blood lymphocytes from old donors when such cells were exposed to an oxidative stress.

1989 - Effects of pulsed electromagnetic fields on the proliferation of lymphocytes from aids patients, HIV-seropositive subjects, and seronegative drug users [Articolo su rivista]
Cossarizza, A.; Borghi, V.; Bersani, F.; Cantini, M.; Rienzo, B. D.; Zucchini, P.; Montagnani, G.; Mussini, D.; Troiano, L.; Tropea, F.; Grassilli, E.; Monti-Biasi, D.; Franceschi, C.
abstract

The effect of the exposure of mitogen-stimulated lymphocytes from subjects infected by human immunodeficiency virus to extremely low frequency pulsed electromagnetic fields (PEMFs) was studied, by evaluating the incorporation of tritiated thymidine, the expression of IL-2 receptor, and the amount of activated T lymphocytes. Four groups of subjects were considered patients with acquired immunodeficiency syndrome (AIDS), asymptomatic seropositive subjects, seronegative drug users, and young healthy controls. PEMFs increased cell proliferation only in the group of healthy controls, as measured at the 72nd hour of culture, but an increase in the number of activated T lymphocytes was observed by cytofluorimetric analysis after 18 hrs of PEMF exposure in cultures from AIDS patients. © 1989 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1989 - Immunomodulatory properties of L-acetylcarnitine on lymphocytes from young and old humans [Relazione in Atti di Convegno]
Monti, D.; Cossarizza, A.; Troiano, L.; Arrigoni-Martelli, E.; Franceschi, C.
abstract