DETERMINANTS OF HUMAN IMMUNODEFICIENCY VIRUS-1TRANSMISSION TO THE FEMALE GENITAL MUCOSA:ROLE OF CO-INFECTING PATHOGENS AND CYTOKINES IN SEMEN

Background. Nel seme Human immunodeficiency virus (HIV)-1 e co-patogeni sono presenti in una complessa rete di citochine che influenza la replicazione locale e l\u2019infettivita\u2019 dei patogeni sessualmente trasmissibili, e, allo stesso tempo, \ue8 influenzata dalle infezioni del tratto genitale maschile (MGT). La comprensione di queste interazioni richiede un'analisi approfondita dei co-patogeni che infettano il MGT e delle citochine presenti nel liquido seminale, e delle relazioni intercorrenti tra questi e HIV-1. Metodi. La carica virale di 6 herpesvirus e HIV-1 e\u2019 stata valutata nel plasma seminale e sanguigno di 83 soggetti HIV-1 infetti cronici non in terapia, e di 33 soggetti non infetti mediante real-time PCR. Un saggio multiplex basato su beads \ue8 stato utilizzato per misurare i livelli di 21 citochine. I rapporti tra citochine sono stati definiti mediante il calcolo del coefficiente di correlazione di Spearman per tutte le possibili coppie di citochine. Blocchi di tessuto cervico-vaginale e linfoide umano sono stati inoculati con HIV-1 e trattati con interleuchina (IL)-7. La replicazione di varianti HIV-1 R5 o X4 \ue8 stata monitorata mediante misurazione della produzione dell\u2019antigene p24gag. Cellule isolate da blocchi di tessuto al giorno 6 e 9 postinfezione sono state caratterizzate per l'espressione dei marcatori CD3, CD4, CD8,e HIV-1 p24gag, e l\u2019apoptosi \ue8 stata valutata misurando l'espressione delle proteine APO2.7 e Bcl-2 mediante citofluorimetria a flusso. Risultati. La maggioranza dei campioni di seme, ma non di sangue, dei soggetti HIV-1-infetti e\u2019 risultata positiva per EBV e CMV (56% e 70%). Sangue e seme sono compartimenti immunologici separati e con l\u2019infezione da HIV-1 il seme e\u2019 il compartimento interessato dai maggiori cambiamenti nella composizione citochinica (16 vs 9 citochine alterate nel sangue). La riattivazione di CMV nel MGT \ue8 associata ad un aumento dei livelli delle citochine CCL5, CCL11 e CXCL9 nel seme. L\u2019analisi dei rapporti tra citochine ha rivelato un numero maggiore di correlazioni e un aumento dell\u2019intensita\u2019 di correlazione per la maggior parte delle citochine sia nel seme che nel sangue dei soggetti HIV-1-infetti, rispetto ai non infetti. IL-7 e\u2019 risultata essere una tra le citochine pi\uf9 concentrate nel seme e l\u2019infezione da HIV-1 ne aumenta ulteriormente i livelli seminali. Abbiamo dunque utilizzato un sistema di infezione ex vivo di tessuti umani per studiare il ruolo di IL-7 nella trasmissione di HIV-1. IL-7 e\u2019 risultata promuovere la replicazione di varianti R5 e X4 di HIV-1 in modalita\u2019 dose e tempo-dipendente. In blocchi di tessuto trattati con 25 ng/mL di IL-7 e\u2019 stato osservato un numero maggiore di cellule T CD4+ infette e una riduzione della deplezione cellulare, rispetto a blocchi di tessuto non trattati con IL-7. Il trattamento con IL-7 e\u2019 risultato inoltre in una riduzione della frazione di cellule T CD4+ infette esprimenti il marcatore apoptotico APO2.7 e in un aumento dei livelli di espressione del fattore anti-apoptotico Bcl-2. Conclusioni. HIV-1 \ue8 associato ad una alterazione generale dello spettro citochinico nel seme e alla riattivazione locale di CMV e EBV nel MGT, due fenomeni che sembrano essere correlati e potrebbero dunque influenzarsi reciprocamente. La ridotta flessibilita\u2019 dei rapporti tra citochine potrebbe ulteriormente contribuire alla riattivazione di tali infezioni latenti. Tra le citochine nel seme, IL-7 esercita un effetto protettivo sulle cellule T CD4+ infette, favorendo lo stabilirisi di una infezione produttiva nel tratto genitale femminile. La comprensione di come il complesso di citochine, di concerto con i co-patogeni presenti nel seme, influenzano la trasmissione sessuale di HIV-1 sar\ue0 oggetto di ulteriori indagini.Background. In semen Human immunodeficiency virus (HIV)-1 and co-infecting pathogens are immersed in a complex network of cytokines that affects the replication and infectivity of sexually transmitted pathogens, and, at the same time, is affected by local infections in the male genital tract (MGT). Understanding the mechanisms of these interactions requires a comprehensive analysis of coinfecting pathogens and cytokines in semen, and of the relations with HIV-1. Methods. Load of HIV-1 and 6 herpesviruses in semen and blood plasma of 83 HIV-1 chronically infected individuals na\uefve to therapy and 33 HIV-uninfected individuals was evaluated by real-time PCR. A multiplex beads-based assay was used to measure the levels of 21 cytokines. The cytokine network was defined calculating the Spearman`s rank correlation coefficient for all pairwise combinations of cytokines. Human cervico-vaginal and lymphoid tissue blocks were inoculated with HIV-1 and treated with interleukin (IL)-7. Replication of R5 or X4 HIV-1 variants was monitored over a period of 12 days measuring HIV-1 p24gag antigen by a beads-based assay. Cells isolated from tissue blocks at day 6 and 9 post-infection were characterized for the expression of the markers CD3, CD4, CD8, and HIV-1 p24gag antigen, and apoptosis was evaluated measuring the expression of the proteins APO2.7 and Bcl-2 by multicolor flow cytometry. Results. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA was found in semen of the majority of HIV-1-infecetd individuals (56% and 70%), but not in their blood. Blood and semen are separated immunological compartments, and with HIV-1 infection the major changes in cytokine composition occur in semen (16 vs 9 cytokine levels altered in blood). CMV reactivation in the MGT was associated with increased levels of the cytokines CCL5, CCL11 and CXCL9. Analysis of the cytokines network revealed a higher number of correlations and increase in correlation strength for most of the cytokines in semen and blood of HIV-1-infected compared to HIV-uninfected individuals. Of note, interleukin (IL)-7 resulted to be one of the most concentrated cytokines in semen and HIV-1 infection further increased seminal IL-7. Thus we employed our system of human tissues ex vivo to study the effect of IL-7 on HIV-1 replication. IL-7 enhanced the replication of R5 and X4 HIV-1 variants in dose and time-dependent manner. In tissue blocks treated with 25 ng/mL of IL-7 we observed higher number of HIV-1 infected CD4+ T cells and reduced CD4+ T cell depletion, compared with tissue blocks infected and maintained in the absence of IL-7. The fraction of HIV-1 infected CD4+ T cells expressing the apoptotic marker APO2.7 was reduced and the levels of the antiapoptotic factor Bcl-2 in infected cells were increased in the presence of IL-7. Conclusions. HIV-1 infection is associated with a general alteration of the cytokine spectrum in semen, and with the local reactivation of CMV and EBV in the MGT, two phenomena that appear to be related and may affect each other. The reduced flexibility of the cytokine network may favors the reactivation of such latent infections. Among seminal cytokines, IL-7 exerts a protective effect on HIV-1-infected CD4+ T cells in the early stages of infection, preventing their death and thus promoting the establishment of a productive infection in the female genital tract. Understanding how other cytokines in concert with co-infecting pathogens found in semen affects HIV-1 sexual transmission will be object of further investigations

ROLE OF HHV-8 VIRAL PARAMETERS AND KIR/HLA COMPLEXES ON THE DEVELOPMENT OF CLASSIC AND EPIDEMIC KAPOSI'S SARCOMA.

Kaposi\u2019s sarcoma (KS) is a rare vascular tumor of the skin related to Human Herpesvirus 8 (HHV-8) infection. Based on clinical and pathological characteristics, KS is divided in four different forms: classic KS (cKS), epidemic (or AIDSassociated) KS, endemic KS and iatrogenic KS. Although the link between HHV-8 and disease has been demonstrated, it is important to note that the HHV-8 infection is necessary but not sufficient to develop the disease: many important aspects remain to be elucidated, regarding both viral factors, as presence of antibody anti-HHV-8, viral transmission and pathogenic potential of different viral genotypes, and host predisposition to tumor development, different from person to person. Natural killer cells are central components of the innate immune response against viral infection and tumor growth: the modulation of their activity is a complex and multi factorial phenomenon triggered by the binding of inhibitory or activating killer cell immunoglobulin-like receptors (KIRs) to class I human leukocyte antigens (HLAs). For these reasons, the objective of this thesis is to verify whether the development of cKS and epidemic KS is related with particular HHV-8 viral parameters (viral load, genotype) or with particular host KIR/HLA receptor/ligand genotype, comparing cKS and epidemic KS patients with the appropriate control groups. Regarding the viral parameters, anti-HHV-8 antibodies were detected in all (100%) cKS patients, and in 80% of epidemic KS patients, whereas they are present in 50% of HIV+ healthy individuals and in 10% of HIVhealthy donors only (p<0.0001). Moreover, when the HHV-8 genotypes were analyzed, A subtype was significantly more frequently isolated in cKS patients with fast progression of the disease (63.6% vs 23.1%), whereas C subtype in individuals with slow progression (76.9% vs 36.4%; p=0.003). Regarding the host genetic aspects, activating KIRs, toghether with their HLA ligands, were more frequent in cKS and epidemic KS compared to non-KS subjects, regardless of the presence or absence of HHV-8 or HIV infection. Considering the activating KIR one by one, the KIR2DS2 gene was significantly prevalent in both cKS and epidemic KS patients (p=0.02 for both), whereas KIR2DS1 and KIR3DS1 genes were more prevalent in cKS patients (p=0.02 and p=0.04 respectively). Moreover, considering the KIR/HLA complexes, the activating KIR2DS1/C2 genotype was positively associated with cKS development (p=0.01). Finally, the inhibitory KIR distribution, as well as the HLA ligand distribution alone, did not reveal any statistical association with cKS or epidemic KS. In conclusion, this study shows that 1) the A genotype of HHV-8 is associated with worst clinical parameters of KS, including faster progression, 2) that a KIR/HLA \u201cactivating milieu\u201d is present in cKS and epidemic KS and that such milieu may be a risk factor for the development of the tumor

EVALUATION OF ANTI-INFECTIVE ACTIVITY OF GLYCOMIMETIC DC-SIGN LIGANDS

Background. HIV remains one of the leading causes of mortality and morbidity worldwide. As the vast majority of HIV infections occurs via unprotected sexual intercourses, the development of topical microbicides is a new and promising approach to prevent HIV sexual transmission. DC-SIGN, a C-type Lectin Receptor (CLR), participates in the initial stages of sexually transmitted HIV infection by recognizing highly mannosylated structures displayed in multiple copies on HIV gp120. Dendritic cells located in genital mucosae internalize HIV through DC-SIGN and transmit the virus in trans to CD4 T lymphocytes, promoting virus dissemination. Furthermore, binding of HIV to DC-SIGN activates signaling pathways that induce immunosuppression and promote HIV replication and transmission. Thus, inhibition of HIV interaction with DC-SIGN represents a potential therapeutic approach to prevent HIV infection at the mucosal level. Aim of this study was to evaluate the efficacy in inhibiting HIV infection and the potential toxicity of a multimeric glycomimetic DC-SIGN ligand (dendron 12). Methods. In the initial phase of this study, the ability of dendron 12 to block laboratory and primary HIV-1 strains transmission to CD4 T cells was assessed using a trans infection assay in vitro. Owing the results obtained, the efficacy of dendron 12 in inhibiting HIV-1 infection of mucosal tissue taken from human uterine cervix was evaluated. Cervical explants were treated with the dendron 12 and then exposed to different HIV-1 strains in a non polarized manner, mimicking a condition of compromised epithelial integrity. Infection was determined by measuring p24 HIV-1 core protein concentration in supernatants of cell and explant cultures. \u3b2 chemokines production following stimulation of monocyte-derived DCs was also analyzed. The selectivity of dendron 12 towards DC-SIGN and Langerin, another CLR that recognize high mannosylated structures, was evaluated by Surface Plasmon Resonance (SPR) studies. Toxicity of the compound was evaluated in both cellular and tissue models. Results. Dendron 12 prevented trans infection of CD4 T lymphocytes and infection of human cervical tissue by multiple clades of R5 and X4 tropic HIV-1 strains, even in presence of elevated viral loads. The compound displayed a prolonged activity and absence of toxicity at the highest concentration tested in infection assays. Treatment with dendron 12 did not interfere with the activity of Langerin that, in contrast to DC-SIGN, prevents HIV transmission promoting the degradation of the virus. Moreover dendron 12 significantly elicited the production of the \u3b2 chemokines MIP-1\u3b1, MIP-1\u3b2 and RANTES. The dendron 12 was found to be soluble in physiological media and stable at both neutral and acid pH. Conclusion. Dendron 12 inhibits HIV-1 infection by competition with binding of HIV to DC-SIGN and stimulation of \u3b2 chemokines production. Furthermore dendron 12 is highly soluble in physiological media, stable at acidic vaginal pH, no toxic and endowed with a long-lasting effect. Thus, dendron 12 represents a promising lead compound for the development of anti-HIV topical microbicides

DENDRITIC CELLS IN A MURINE MODEL OF BREAST CANCER: FUNCTIONAL AND PHENOTYPIC EVALUATION USING NANOPARTICLES

Dendritic cells play a pivotal role in antigen presentation and adaptive immune response activation. Studies on mice models reported that tumoral antigens-loaded DCs can induce therapeutic and protective anti-tumour immune responses, both in vitro and in vivo. These results provide an evidence of the possible use of DCs as vaccines. However, the efficacy of such therapeutic vaccination was recently questioned due to the limited degree of actual tumour regression in some clinical trials. Thus, it is necessary to better characterize immunological and clinical parameters of DCs immunization to increase the therapeutic efficay of the vaccines. In vivo imaging tecniques, as magnetic resonance imaging (MRI) and single photon emission tomography (SPET), are used to study cell trafficking mechanisms in vivo and state anti-tumoral treatments efficacy, allowing an accurate assessment. In this work we initially set the optimal culture conditions to obtain functional immature DCs. Evaluations based on DCs number, cell viability and immature DCs percentage led to establish day 6 as the one in which the highest number of functionally active immature DCs can be achieved. We then set a DCs labelling protocol with commercial SPIO (Endorem\uae) evaluating Iron content using Perl\u2019s staining and relassometry. Time and dose-response kinetic evaluations allowed to define as optimal labelling conditions the use of 200 \u3bcg Iron of Endorem\uae/ ml of culture for 16 hours. Cell viability and phenotypic markers analysis proved that SPIO labelling had no influence on immature DCs viability or activation status. Images acquired by transmission electron microscopy showed that Endorem\uae were inside cells, in particular inside lysosomes, and were taken up by endocytosis mechanisms. We then focused on DCs antigens loading. Tumoral antigens were obtained lysing neoplastic masses extracted from a mammary tumour mouse model (MMTV-Ha-Ras). DCs pulsed with different antigen concentrations resulted mature by means of high levels of activation and maturation markers. Moreover, such cells were able to stimulate T lymphocytes extracted from MMTV-Ha-Ras spleens. 3H-thymidine and CFSE proliferation assays, together with IFN-\u3b3 production measured by ELISA, showed an increase in T lymphocytes activation when co-cultured with antigen-pulsed DCs. Cytokines production assessed by flow cytometry and ELISA allowed us to verify that tumoral antigen-pulsed DCs were able to elicit a specific T-lymphocyte response toward a Th1 type. Indeed, high amounts of IFN-\u3b3 were produced by T lymphocytes, high levels of IL-12 were produced by DCs and low levels of IL-10 were detectable. In vitro migration showed that tumoral antigen-pulsed DCs displayed migratory abilities toward MIP3\u3b2 and 6Ckine chemockines, responsible in vivo of mature DCs attraction. Flow cytometric analysis confirmed that migrated cells displayed a mature phenotype and expressed high levels of CCR7. We then performed in vivo experiments in MMTV-Ha-Ras mice bearing neoplastic lesions and in control mice without tumours. MRI showed that Endorem\uae-labelled DCs pulsed with tumour antigens are able to migrate to the draining lymph site in vivo. Histological analysis on ex vivo lymph nodes showed the presence of Iron-labelled cells in the cortical area. Immunohistochemistry reveals co-localization of Iron-containing cells with CD208-labelled DCs, but not with macrophages. In vivo SPET migration of antigen-pulsed DCs labelled with 111In-oxine showed specific radiotracer presence in the lymph nodes omolateral to injection site; \u3b3-counter analysis of ex-vivo organs confirmed such results. Taken together, these results suggest that this protocol could candidate for experimental use on animal models for in vivo tumour immunotherapy efficacy imaging

PROGENITORI ENDOTELIALI CIRCOLANTI: CARATTERIZZAZIONE IN SOGGETTI CON SARCOMA DI KAPOSI CLASSICO AD EFFETTI DELL'INFEZIONE IN VITRO DA HHV-8

Background Kaposi\u2019s sarcoma (KS) is an angioproliferative disorder associated with human herpesvirus-8 (HHV-8) infection. Spindle cells are the predominant cell type in the KS lesions and are endothelial in origin. We previously demonstrated that endothelial progenitor cells (EPCs) cultured ex vivo from KS classic (cKS) patients as late-EPCs are HHV-8-infected and support viral productive replication. In this study we investigated whether late-EPCs from KS patients showed a different in vitro behaviour compared to late-EPCs from healthy controls, in terms of efficiency of ex vivo isolation, and biological features. Moreover we infected in vitro control late-EPCs to investigate whether late EPCs can be infectable with HHV-8 and whether they support viral replication. We also analyzed the effects of HHV-8 infection on multiple aspects of late-EPCs biology. Methods Late-EPC were cultured from peripheral blood mononuclear cells (PBMCs) of 69 cKS patients (cKS-late-EPC) and 40 sex and age-matched healthy HHV-8 seronegative controls (ctr-late-EPC). Briefly, PBMCs were seeded on collagen-coated or fibronectin-coated plates in EGM-2 and late-EPCs were identified as cobblestone-like colonies. Cultures from all subjects were observed for initial late-EPCs colony appearance and number of colonies, cell viability, cell proliferation and cytokine production. ctr-late-EPC was infected in vitro with HHV-8 and the effects of HHV-8 infection on late-EPC biology were investigated by analyzing the following parameters: cell morphology, proliferation, immunophenotype and angiogenesis. Results In our study we demonstrated that the frequency of late EPCs isolation depends on substrate used to seed PBMCs. The average time of initial late-EPCs colony appearance was significantly lower in cKS-late-EPC than ctr-late-EPC (P<0.001). The number of late-EPC colonies per 20x106 seeded PBMCs was significantly higher in cKS-late-EPC than ctr-late-EPC (P<0.001). Senescence appear later time in cKS-late-EPC than ctr-late-EPC. Morover, cell proliferation and IL-6 production was higher in cKS patients than healthy controls. This data suggest that the presence of HHV-8 infection in cKS-late-EPC may induce late-EPCs to acquire several feature that resemble spindle cells. HHV-8 in vitro infection induced ctr-late-EPC to acquire a spindle shape similar to the morphology of KS spindle cells. HHV-8 induced ctr-late-EPC to reduce proliferation and promoted the activation of late-EPCs inducing the up-regulation of VCAM-1, ICAM-1, e-selectin. HHV-8-infection also induced the up-regulation of CD49c and \u3b1v\u3b23 integrins. Conclusions In this study we provide evidence that late-EPCs obtained from patients with cKS are characterized by strikingly higher ex vivo expansion and proliferation rate than late-EPCs from healthy controls. Moreover, upon HHV-8 in vitro infection, EPCs support persistent viral productive replication and acquire morphologic and functional features of KS spindle cells. These novel findings may be particularly relevant to the comprehension of KS pathogenesis, because late-EPCs may represent putative precursors of spindle cells that may home to permissive sites and propagate to produce KS lesions

IMMUNOMODULATORY EFFECTS OF NITAZOXANIDE AND RELATED MOLECULES

Background: Nitazoxanide (Alinia\uae, NTZ) and its active circulating metabolite tizoxanide (TIZ) belong to a new class of anti-infective agents active against parasites, anaerobic bacteria, and viruses. Nowadays, NTZ is licensed in the United States for the treatment of diarrhea caused by Cryptosporidium parvum and Giardia lamblia in adults and children older than twelve months of age. The amplitude of the spectrum of pathogens targeted by NTZ and new-generation non-nitro thiazolides, makes it very unlikely that the action of these compounds is mediated by a pathogen-specific mechanism(s), suggesting instead that thiazolides act as immunomodulants. To date, the potential effect of these compounds on immune responses has nevertheless not been analysed. In particular, because innate immunity and type 1 interferons are pivotal in early and effective antiviral immune responses that are not antigen-restricted, it is plausible to hypothesize that thiazolides could potentiate this arm of the immune system. To verify this possibility, we performed extensive in vitro analyses on the immunomodulatory effects of TIZ and the second generation non-nitro thiazolide RM4848 using two different models of viral infection: Influenza and HIV-1. Methods: Peripheral blood mononuclear cells (PBMCs) from 20 healthy donors were stimulated with influenza virus antigen (FLU) or infected with HIV-1BaL strain and cultured in presence/absence of TIZ or RM4848. Thiazolide effects on innate immunity were examined by evaluating TLRs expression on monocytes, IFN-secretion by dendritic cells, cytokine and chemokine production, mRNA expression of multiple genes involved in TLR, type I IFN and in cholesterol metabolism pathways. Results: Thiazolides are associated with strong immunomodulatory effects. Notably, these compounds, both in FLU-stimulated and in HIV-1-infected cells, significantly increase: 1) TLR-expression on monocytes, 2) IFN production, 3) chemokine and cytokine production, 4) mRNA expression of different genes operating in the TLR and type I IFN pathways, 5) genes involved in cholesterol metabolism and efflux. Conclusions: Data herein show that thiazolides are potent type I IFN inducers, triggering a selective activation of several IFN-stimulated gene (ISG) pathway. Thus, increased expression of innate antiviral factors and the different modulation of genes involved in cholesterol metabolism and efflux suggest a new mechanism of action mediated by thiazolides

ALTERED HOMEOSTASIS OF PERIPHERAL BLOOD B CELLS IN PATIENTS WITH CHRONIC HUMAN HERPESVIRUS-8 INFECTION AND KAPOSI\ubfS SARCOMA: IMPLICATION FOR INFLUENZA VACCINATION

Background: Human herpesvirus-8 (HHV-8) is the etiological agent of classic Kaposi\u2019s sarcoma (cKS), a lymphoangioproliferative disease found mainly in older men of Eastern European and Mediterranean origin. Due to the dual role of B cells in HHV-8 infection, virus reservoir as well as agents of humoral immune control, we analyzed the subset distribution and the functional state of peripheral blood B cells in HHV-8- infected individuals with and without cKS. Furthermore, in order to investigate whether the chronic HHV-8 infection in B cells could alter the functionality of these cells and particularly may impact on humoral responses to antigenic stimulation, we investigated cKS patients\u2019 response to influenza vaccination, in terms of clinical efficacy, antibody production and safety. Methodology: Circulating B cells and their subsets were analyzed by 6-color flow cytometry in the following groups: 1- patients HHV-8 positive with classic KS (cKS); 2- subjects HHV-8 positive and cKS negative (HSP); 3- healthy controls, HHV-8 negative and cKS negative. Adjuvated trivalent influenza vaccine was administered to cKS patients and age- and sex-matched healthy controls. Influenza symptoms and side effects were recorded by daily diary cards supplied to the patients. Blood analysis and measurement of serum antibodies against vaccine antigens (H1N1, H3N2 and B) were performed before, 1 and 3 months post vaccination. Principal Finding: The number of B cells belonging to the preimmune/natural effector compartment, including transitional, pre-na\u131\ua8ve, na\u131\ua8ve and MZ-like subsets, was significantly higher among HHV-8 positive subjects, with or without cKS, while was comparable to healthy controls in the antigen experienced T-cell dependent compartment. The increased number of preimmune/natural effector B cells was associated with increased resistance to spontaneous apoptosis, while did not correlate with HHV-8 viral load. The clinical efficacy of vaccination was similar in cKS patients and controls. Seroconversion and seroprotection occurred equally in both groups. A mild increase in HHV-8 viremia was observed in a proportion of cKS patients after vaccination, without concomitant worsening of cKS lesions. The safety of vaccination did not differ between cKS patients and controls. The frequency of B cell subpopulations was evaluated and did not change after vaccination both in cKS patients and in healthy controls. Conclusions: For the first time to our knowledge, in this study we report that HHV-8 chronic infection promotes a perturbation of peripheral B cell homeostasis characterized by expansion of B cells of the preimmune/natural effector compartment, in patients with or without cKS. The alterations observed in cKS patients did not lead to an altered response to influenza vaccination that resulted safe and immunogenic in cKS patients as well as in age- and sex-matched controls. This observation may broaden our understanding of the complex interplay between viral and immune factors leading HHV-8-infected individuals to develop HHV-8-associated malignancies. Furthermore, our results have clinical importance because annual influenza vaccination may be particularly recommended for cKS patients considering their advanced age and comorbidity

IMMUNE MECHANISMS ASSOCIATED TO NEUROINFLAMMATION IN ALZHEIMER&#191;S DISEASE

La patogenesi dell' Alzheimer (AD) non \ue8 nota, tuttavia \ue8 sempre pi\uf9 chiaro che l\u2019 infiammazione, processo associato all\u2019insorgenza di numerose malattie neurodegenerative del sistema nervoso centrale, svolga un ruolo in tale patologia. L\u2019infiammazione \ue8 una componente chiave della risposta immunitaria innata. L\u2019immunit\ue0 innata \ue8 un sistema altamente conservato che protegge l\u2019ospite dalle infezioni in maniera aspecifica. Sebbene questo sistema rappresenti una risposta efficace e potente agli stimoli acuti \ue8 necessario che sia finemente regolato: una deregolazione o un\u2019attivazione cronica possono infatti avere effetti dannosi per l\u2019ospite. L\u2019infiammazione cronica \ue8 stata implicata non solo in malattie periferiche ma anche in malattie neurodegenerative del sistema nervoso centrale come l\u2019Alzheimer. L\u2019ipotesi di questo lavoro \ue8 stata che l\u2019infiammazione rappresenti un fattore negativo per la malattia di Alzheimer e che i meccanismi che concorrono a regolare la risposta infiammatoria siano quantitativamente e funzionalmente compromessi negli AD rispetto agli MCI e HC. Uno dei meccanismi di regolazione della tolleranza immunologica \ue8 rappresentato dai linfociti Treg. I risultati presentati indicano che lo sviluppo della patologia di AD \ue8 associato ad una diminuzione del numero di Treg circolanti e in particolare della percentuale di Treg naive. Quest\u2019alterazione quantitativa \ue8 associata ad un\u2019alterazione qualitativa quale un\u2019aumentata proliferazione amiloide- specifica e ad una ridotta capacit\ue0 dei Treg di sopprimere tale proliferazione. L\u2019analisi del pathway PD1-PDL1, in grado di controllare la risposta infiammatoria mediante produzione di IL-10 e induzione di apoptosi antigene-specifica, ha mostrato una diminuzione dell\u2019espressione di PD-1 sui linfociti T CD4+ dei pazienti AD e MCI rispetto ai controlli sani. I risultati mostrano inoltre una diminuzione significativa della produzione di IL-10 da parte di CD14+PD-L1+. La down-regolazione di questi meccanismi osservata nei pazienti AD e MCI risulta in un aumento della proliferazione dei linfociti T stimolati alla \u3b2A. Il ruolo chiave svolto dall\u2019interazione PD-1/PD-L1 nell\u2019indurre l\u2019apoptosi dei linfociti T CD4+ specifici per \u3b2A \ue8 confermato dall\u2019osservazione che l\u2019apoptosi \ue8 bloccata preincubando queste cellule con un anticorpo bloccante anti- PD-L1. Lo studio delle sottopopolazioni linfocitarie nelle forme di AD rispetto alla forma MCI e agli HC ha inoltre evidenziato che nei pazienti AD non solo vi \ue8 un\u2019alterazione nei meccanismi di tolleranza immunologica ma anche uno shift nel differenziamento del linfociti T verso un fenotipo infiammatorio di tipo Th-17 e Th-9. I risultati hanno, infatti, mostrato un aumento della produzione delle citochine infiammatorie (IL-21, IL-23, IL-6) e dei fattori di trascrizione (RORc/gt) coinvolti nel differenziamento dei Th-17 cos\uec come delle citochine effettrici (IL-21 e IL-22) prodotte da tali cellule nei pazienti AD rispetto agli MCI e agli HC. In particolare, IL-9, la citochina effettrice prodotta dalle Th-9, \ue8 significativamente aumentata nei pazienti AD, indicando che oltre ai Th-17 anche i Th-9 specifici per la \u3b2A sono upregolati negli AD e Th-9 (IL-21 e IL-22). In conclusione la compromissione della risposta immunitaria, con una profonda inclinazione a favore di risposte effettrici e infiammatorie, sembra svolgere un ruolo chiave in questa patologia.The etiology of Alzheimer\u2019 s disease (AD) is still unresolved, even if it is becoming clearer that inflammation, a process associated to the onset of several neurodegenerative disorders, plays a central role in this disease. Inflammation is a key component of innate immune system. Innate immunity is a very highly conserved system that protects the host from infections in a non-specific manner. Even if this system provides a powerful response to a range of insults it must be tightly regulated: deregulation and chronic activation can have detrimental effects on the host. Chronic inflammation has been involved not only in peripheral diseases but also in neurodegenerative diseases of the central nervous system, like Alzheimer\u2019s disease. Our working hypothesis is that inflammation plays a negative role in this pathology and that the mechanisms regulating the inflammatory responses are functional compromised in AD patients compared to Mild cognitive impairment (MCI) and to healthy controls (HC). One of the main way in which immunologic tolerance is modulated is through T regulatory cells (Treg). Our results indicate that the development of AD is associated with a reduction of circulating T reg na\uefve cells, the subpopulation of Treg cells endowed with the strongest suppressive ability. These quantitative changes are associated with qualitative changes, summarized as an increase of A\u3b2-specific proliferation and a reduced ability of Treg to suppress such proliferation. The analysis of the PD-1/PD-L1 pathway, which modulates the balance between inflammation and tolerance by inducing IL-10 production and apoptosis of antigen-specific cells, shows a decrease of PD-1 expressing CD4+ T cells in AD and MCI compared to HC as well as a decrease of PD-L1- expressing and IL-10-producing CD14+ cells. The impairment of the PD-1/PD-L1 pathway in AD patients results in reduced IL-10 production and diminished apoptosis of A\u3b2-specific CD4+ T lymphocytes. The central role performed by PD-1/PD-L1 pathway in inducing the apoptosis of Abeta-specific T cells is confirmed by the observation that apoptosis is inhibited pre-incubating lymphocytes with a PD-L1-specific blocking antibody. The analysis of lymphocytes subpopulations in AD and MCI compared to controls highlight that in AD patients not only an alteration of immunological tolerance is present but also a shift in the differentiation of T lymphocytes towards an inflammatory phenotype Th-9 and Th-17. Our results showed indeed that cytokines (IL-21, IL-23, IL-6) and transcription factor (RORc/gt) involved in the differentiation of Th-17, as well as cytokines (IL-21, IL-22) produced by these cells are all augmented in AD compared to MCI and HC. Notably, IL-9, the effector cytokine produced by Th-9 cells, was significantly increased as well in AD patients, indicating that, beside Th-17, A\u3b2- specific Th-9 lymphocytes are upregulated in AD. In conclusion the impairment of the immune response, with a profound skewing favoring inflammatory and effector responses, seem to play a pivotal role in this pathology

EVALUATION OF IMMUNE MECHANISMS INVOLVED IN THE LACK OF CD4+ T CELL RECOVERY IN HIV-INFECTED PATIENTS NON RESPONDER TO ANTIRETROVIRAL THERAPY

INTRODUCTION HIV infection is characterized by a profound impairment of CD4+ T cell functionality with an immunological unbalance toward Th2 response during progression to AIDS. Antiretroviral therapy HAART suppresses viral replication and leads to the recovery of CD4+ T lymphocytes. 15-30% of HIV-infected HAART-treated patients are immunological non responder (INR) to therapy. Several factors are involved in this lack in CD4+ T cell recovery, our attention was focused on the excessive depletion of CD4+ T cells. Recently, hydroxychloroquine (HCQ), an anti-malarian drug, demonstrates to have antiviral and immune modulating effects. Aim of this study was to identify immune mechanisms involved in the lack of CD4+ T cell recovery observed in INR patients and to verify if HCQ might reduce immune activation typical of HIV infection thus leading to a recovery in CD4+ T cells. MATERIAL AND METHODS In the first part of this study, 67 HAART-treated HIV-infected patients were enrolled and stratified into two groups of the basis of CD4+ T cell counts (500cells/\ub5l) while in the second part of this study, analyses were conducted on 20 HAART-treated HIV-infected patients with an absolute CD4 count less than 200 cells/\ub5l during the last 12 months of therapy. Patients were treated with 400mg/die of HCQ and analyses were conducted at baseline, 6 months of therapy and 2 months after HCQ suspension. Immune suppression, immune activation and apoptosis were evaluated in flow cytometry. To identify microbial translocation, plasma LPS was measured with immunoenzymatic assay and TLR signaling pathway was analyzed in Real-Time PCR. Analyses were conducted in both unstimulated and stimulated (HIV-, CMV-, LPS-, ssRNA-specific) conditions. RESULTS AND DISCUSSION Patients characterized by CD4+ cell counts less than 500cells/\ub5l compared with individuals characterized by CD4+ cell counts more than 500cells/\ub5l showed: 1) increase percentage of Treg cells subpopulations and 2) apoptosis, 3) higher percentage of immune suppressive cytokine-expressing CD4+ T cells, 4) a higher percentage of KI67+ CD4+ T cells in both unstimulated and stimulated conditions. 5) an increased percentage of TLR expression on Treg cells and 6) a higher concentrations in plasma LPS. Data herein indicate that defective CD4+ cell counts recovery observed in patients characterized by CD4+ cell counts less than 500cells/\ub5l is associated with lower CD4+ nadir, gut microbial translocation and immune activation, augmented percentage and activity of Treg cells, and higher susceptibility to apoptosis. In the second part of the study, data showed a reduction in immune activation of CD4+ T cells, CD8+ T cells and monocytes. which was coupled with a reduction in plasma LPS concentration and of IL6 pro-inflammatory cytokines production. Moreover it has been shown the pivotal role played by plasmacytoid dendritic cells in the control of infection. Immune activation and immune suppression are strictly correlated, presence of immune suppressor state was well studied thanks to the analyses of both na\uefve and activated Treg lymphocytes, and TLR-expressing Treg cells: these populations were increased in percentage after 6 months of therapy. Several studies put in evidence HCQ effectiveness on TLR signaling pathway which influences immune activation. Flow cytometric analyses elucidate the reduction in TLR expressions by monocytes even in the presence of TLR4 and TLR7/8 agonists (LPS and ssRNA respectively). TLR signaling pathway was studied following PBMC stimulation with specific TLR agonists by Real-Time PCR: 6 months of HCQ treatment resulted in a reduction of TLR responsiveness and the effect was maintained also 2 months after HCQ suspension. CONCLUSIONS The lack in the recovery of CD4+ T lymphocytes characterizing immunological non responder patients to HAART is associated with a low CD4+ nadir, with immune activation due to an increase in microbial translocation and with immune suppression conducted by Treg cells through the induction of apoptosis mechanism and immune suppressor environment. The augment in Treg population might play a pivotal role in reducing immune activation and the increase of TLR-expressing Treg cells supports the effectiveness of a strong immune suppressive activity in controlling immune activation. Preliminary data suggest that HCQ plays a positive effect on CD4+ T cell recovery in immunological non responder patients to HAART and a control of immune activation, sustaining an application of this drug as immune modulating agent, in the treatment of HIV infection