L’avvento della terapia antiretrovirale ha trasformato l’infezione da HIV in una patologia di tipo cronico, rendendo possibile la manifestazione di comorbidità HIV-relate. Particolare importanza rivestono le lesioni a carico del compartimento osseo, infatti i soggetti sieropositivi hanno un rischio maggiore di sviluppare osteopenia e osteoporosi, rispetto alla popolazione sana. Numerosi lavori hanno dimostrato come sia il virus HIV che la terapia antivirale siano in grado di determinare l’alterazione di questo bilancia, attraverso un’azione negativa diretta sulle cellule della linea osteoblastica. Per quanto riguarda il compartimento osteoclastico, invece, le indicazioni presenti in letteratura sono poche e spesso contrastanti. La prima parte di questo elaborato si è quindi focalizzata sullo studio dell’effetto indotto dal virus e dalla terapia antiretrovirale sulle cellule della linea osteoclastica in un modello sperimentale in vitro. I risultati ottenuti mostrano che in presenza del virus e della terapia si osserva un’intensificazione dell’attività osteoclastica, con conseguente riduzione della densità di massa ossea. Tuttavia, nonostante gli indubbi successi della cART la terapia non si è dimostrata in grado di eradicare l’infezione. Questo scenario ha aperto la strada allo sviluppo di nuove molecole ad azione antiretrovirale in grado di bloccare l’ingresso del virus attraverso il contatto sessuale: in particolare, alcuni studi si sono focalizzati sull’utilizzo di composti da impiegare a livello delle mucose genitali, con lo scopo di contrastare, o almeno ridurre, la trasmissione di HIV-1. In tal senso si è indirizzata la seconda parte della tesi, volta a valutare l’efficacia di una molecola di nuova concezione, un mini CD4 chiamato M48U1, da solo o in combinazione con farmaci tradizionali, nel ridurre o prevenire l’infezione di HIV-1. Dai dati emersi è possibile osservare una consistente riduzione della trasmissione dell’infezione sia su un modello in vitro che su un modello ex vivo. / Several studies have shown that HIV infection and treatment with antiretroviral drugs can induce a significant increase in cases of osteopenia / osteoporosis in HIV-infected patients. The loss of bone mass density (BMD) is related to the functional loss of balance of bone forming unit (BFU) that is characterized by fine adjustment that normally elapses between the trophic action of osteoblasts and bone resorption action of osteoclasts. In the first part of this work we evaluate the effect of HIV, specific viral proteins and some conditioned medium on the differentiation of osteoclasts. The achieved results show the contribute of the therapy and the viral protein on the osteoclast differentiation, with a consequent higher bone resorption and a decrease of Bone Mass Density. In the second part of this work, we analysed the antiviral activity of mini CD4 M48U1 peptide formulated in HEC on activated peripheral blood mononuclear cells (PBMCs) infected by R5 and X4–tropic HIV strains. The results demonstrated that M48U1 prevented the HIV-1BaL and HIV-1IIIB infection in activated PBMCs with the half maximal effective concentration (EC50) determined at 30 and 20.3 nM, respectively. In addition, M48U1 was tested in association with Tenofovir in HEC and these two antiretroviral drugs synergized for the HIV inhibition. It is noteworthy that M48U1 alone or in combination with Tenofovir in HEC hydrogel strongly inhibited HIV replication in an organ-like structure of human cervico-vaginal tissue without any induction of significant tissue toxicity.

Microvesicles (MVs) are small extracellular vesicles divided in exosomes (40-100nm), shedding microvesicles (100nm–1µm), and apoptotic bodies (1-5µm). MVs contain bioactive molecules, nucleic acids and proteins and are physiologically secreted by cells in order to guarantee cell-to-cell communication. The presence of viral miRNA in exosomes and viral DNA in apoptotic bodies was demonstrated. Viral DNA sequences like EBV and HPV have been identified in numerous cancers tissues, like in breast cancer, in the absence of a primary infection. Two meta-analyses studies analyzed the presence of HPV and EBV sequences in breast cancer tissues worldwide, and identified the viral nucleic acids in the 23% and 29% of the samples, respectively. However, the mechanisms of viral DNA acquisition in this cancer have not been investigated. This paper demonstrates the presence of viral nucleic acids in MVs coming from cell line models of onco-viral dependent transformation, including cervical carcinoma (HPV) and lymphoma (EBV), and their transfer through MVs to viral negative epithelial cells and fibroblasts. The presence and transfer of murine mitochondrial DNA in MVs coming from murine cancer-associated fibroblasts is also demonstrated, as well as its transfer to human recipient cells. This study also shows the presence of viral HPV16/18/31 DNA in the circulating MVs from two different cohorts: one of newly diagnosed breast cancer patients, collected in Bologna, Italy, and the other one of patients affected by hormonal therapy-resistant breast cancer, collected in New York, USA. The presence of DNaseI-resistant DNA in the RNA fraction prompted us to study the physical-chemical status of the viral DNA sequences packaged in the MVs coming from viral positive cell lines; the DNA is found in the form of DNA:RNA hybrids (R-loops), which are known to be more stable structures, mostly synthesized in hypoxic conditions.

West Nile virus (WNV) is a neurotropic flavivirus that is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in humans and other vertebrate species. Most of WNV infections in humans are asymptomatic, but approximately 20% of infected people develop clinical symptoms, although severe neurological diseases are observed in less than 1% of them. WNV is the most widely distributed arbovirus in the world and has been recently associated with outbreaks of meningo-encephalitis in Europe, including Italy, caused by different viral strains belonging to distinct lineages 1 and 2. The hypothesis is that genetic divergence among viral strains currently circulating in Italy might reflect on their pathogenic potential and that the rapid spread of WNV with increased pathogenicity within naïve population suggest that epidemic forms of the virus may encode mechanisms to evade host immunity. Infection with WNV triggers a delayed host response that includes a delay in the production of interferon-α (IFN-α). IFNs are a family of immuno-modulatory cytokines that are produced in response to virus infection and serve as integral signal initiators of host intracellular defenses. The increased number of human cases and the lack of data about virulence of European WNV isolates highlight the importance to achieve a better knowledge on this emerging viral infection. In the present study, we investigate the phenotypic and IFN-α-regulatory properties of different WNV lineage 1 and 2 strains that are circulating in Europe/Italy in two cell lines: Vero and 1321N1. We demonstrate that: Vero and 1321N1 cells are capable of supporting WNV replication where different WNV strains show similar growth kinetics; WNV lineage 2 strain replicated in Vero and 1321N1 cells as efficiently as WNV lineage 1 strains; and both lineages 1 and 2 were highly susceptible to the antiviral actions of IFN-α.

In 51 (33 adult-18 paediatric) allogeneic hematopoietic stem cell transplant recipients we aimed to evaluate: i) the incidence of EBV infection and potential risk factors; ii) the suitability of whole blood (WB) as clinical specimen to monitor the risk of patients to develop EBV-related post-transplant lymphoproliferative disorder (EBV-PTLD); iii) the clinical utility of combined virological-immunological monitoring; iv) the management of infection and the incidence of EBV-PTLD. Quantitative real-time PCR assay was performed on WB samples for all patients. EBV-DNA quantification also in peripheral blood mononuclear cells (PBMCs) samples was adopted for actively EBV infected patients. Immunological monitoring of infection was performed by Enzyme-linked ImmunoSPOT assay evaluating the EBV-specific cell-mediated immunity (CMI). The incidence of EBV infection was 51% and the frequency of EBV-PTLD was 3.9%. Reduced-intensity conditioning (RIC) in combination with in vivo T-cell depletion was associated with higher frequencies of infection (P=0.036). A significant correlation (P<0.001) between EBV-DNA levels in WB and PBMC samples was obtained in adult (r=0.787) and paediatric (r=0.976) patients. A similar kinetics of EBV-DNA in blood compartments was observed. Clinically, both specimen types appeared to be equally informative. The lack of EBV-specific CMI during/after active infection was associated with a higher median peak level of EBV-DNA in WB (P=0.013) and a greater severity of infection. The 54.5% of the patients without EBV-specific CMI needed anti-CD20 therapy and the 27.2% developed EBV-related complications, including a lethal PTLD. All patients with EBV-specific CMI controlled EBV replication and were asymptomatic. WB proved to be a suitable clinical specimen to monitor the risk of patients to develop EBV-related complications. RIC combined with in vivo T-cell depletion is a risk factor for the development of infection. EBV-specific CMI is a critical determinant in controlling the infection and consequently the EBV-related complications. Combined virological-immunological monitoring could improve the management of infection.

No description available.

No description available.

Introduzione: Il cervico-carcinoma è la seconda neoplasia maligna per incidenza e mortalità nelle donne in tutto il mondo dopo il cancro al seno. L’infezione persistente da Papillomavirus Umani (HPV) è causa necessaria dell’insorgenza del cervico-carcinoma e delle sue lesioni pre-cancerose. L’infezione da HPV si associa anche ad altri carcinomi del distretto ano-genitale (a livello anale, vulvare, vaginale e del pene) e a circa il 25% dei carcinomi squamosi dell’orofaringe. I circa 40 genotipi di HPV che infettano la mucosa genitale vengono suddivisi in alto rischio (HR-HPV) e basso rischio (LR-HPV) oncogeno a seconda della alta e bassa associazione con la neoplasia cervicale. I 13 genotipi a più alto rischio oncogeno sono 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 e 68. Di questi, otto (16, 18, 31, 33, 35, 45, 52 e 58) sono associati alla maggior parte dei carcinomi cervicali (circa 89%) e i genotipi 16 e 18 da soli sono riscontrati nel 70% circa delle neoplasie. L’insorgenza e progressione delle lesioni preneoplastiche cervicali è, però, associata non solo alla presenza di HPV ad alto rischio, ma, soprattutto, alla persistenza virale (> 18 mesi), alla capacità di integrazione degli HPV ad alto rischio e alla conseguente sovraespressione delle oncoproteine E6/E7. Inoltre, l’integrazione è spesso favorita da un’alta carica virale soprattutto per quanto riguarda alcuni genotipi (HPV16 e 18). L’infezione da HPV non interessa solo la cervice uterina ma tutto il distretto ano-genitale e quello testa-collo. L’HPV, in particolare il genotipo 16, è implicato, infatti, nell’insorgenza delle lesioni preneoplastiche della vulva (VIN) classificate come VIN classiche e nei carcinomi ad esse associati. L’incidenza del carcinoma vulvare in Europa è di 1.5/100.000 di cui circa il 45% è dovuto a HR-HPV (80% ad HPV16). Nonostante l’associazione tra HPV16 e carcinoma vulvare sia alta, ancora poco si conosce sul ruolo della carica virale e dell’integrazione in tali lesioni. Le lesioni che possono presentarsi nella regione testa-collo possono essere sia di natura benigna che maligna. I genotipi più frequentemente riscontrati in associazione a lesioni benigne (papillomi) sono HPV 6 e 11, quelli associati a forme tumorali (HNSCC) sono il genotipo 18 ma soprattutto il 16. Molti aspetti del coinvolgimento di HPV in queste patologie non sono ancora perfettamente conosciuti e spesso studi su tale argomento hanno mostrato risultati contraddittori, soprattutto perché vengono utilizzate metodiche con gradi diversi di sensibilità e specificità. Recenti dati di letteratura hanno tuttavia messo in evidenza che i pazienti affetti da HNSCC positivi ad HPV hanno una elevata risposta al trattamento chemioradioterapico rispetto ai pazienti HPV-negativi con un notevole impatto sul controllo locale e sulla sopravvivenza ma soprattutto sulla qualità di vita di tali pazienti, evitando di sottoporli a chirurgia sicuramente demolitiva. Scopo del lavoro: Sulla base di queste premesse, scopo di questo lavoro è stato quello di valutare l’importanza di marker quali la presenza/persistenza di HPV, la carica virale, la valutazione dello stato fisico del genoma virale e l’espressione degli mRNA oncogeni nella gestione di pazienti con lesioni preneoplastiche e neoplastiche di diverso grado, associate a papillomavirus mucosi. Per la valutazione dei markers virologici di progressione neoplastica abbiamo sviluppato dei saggi di real time PCR qualitativi e quantitativi studiati in modo da poter fornire, contemporaneamente e a seconda delle esigenze, risposte specifiche non solo sulla presenza e persistenza dei diversi genotipi di HPV, ma anche sul rischio di insorgenza, progressione e recidiva delle lesioni mediante lo studio di markers virologici quali carica virale, integrazione ed espressione degli mRNA. Abbiamo pertanto indirizzato la nostra attenzione verso tre popolazioni specifiche di pazienti: - donne con lesioni vulvari preneoplastiche (VIN) e neoplastiche, allo scopo di comprendere i complessi meccanismi patogenetici di tali patologie non sempre associate ad infezione da HPV; - pazienti con lesioni maligne a livello della regione testa-collo allo scopo di fornire informazioni utili all’elaborazione di un percorso terapeutico mirato (radiochemioterapico o chirurgico) a seconda o meno della presenza di infezione virale; - donne con lesioni cervicali di alto grado, trattate chirurgicamente per la rimozione delle lesioni e seguite nel follow-up, per stabilire l’importanza di tali marker nella valutazione della persistenza virale al fine di prevenire recidive di malattia.

No description available.

No description available.

As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon attivation, its analysis, in addition to RNA viral load, could be considered a useful marker during the follow-up of infected individuals, to evaluate reservoir status, especially in HAART-treated patients when RNA viral load is undetectable by current techniques and the antiretroviral efficacy of new, more potent therapeutic regimens. Standardized methods for the measurement of the two most significant forms of proviral DNA, total and non-integrated, are currently lacking, despite the widespread of molecular biology techniques. In this study, total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load, CD4 cell count and serological parameters, were determined by quantitative analysis in peripheral blood mononuclear cells (PBMC) in naïve or subsequently HAART-treated patients with acute HIV-1 infection in order to establish the role of these two DNA proviral forms in the course of HIV infection. The study demonstrated that HAART-treated individuals show a significant decrease in both total and 2-LTR circular HIV-1 DNA proviral load compared with naïve patients: these findings confirm that HIV-1 reservoir decay correlates with therapeutic effectiveness. The persistence of small amounts of 2-LTR HIV-1 DNA form, which is considered to be a molecular determinant of infectivity, in PBMC from some patients demonstrates that a small rate of replication is retained even when HAART is substantially effective: HAART could not eradicate completely the infection because HIV is able to replicate at low levels. Plasma-based viral RNA assays may fail to demonstrate the full extent of viral activity. In conclusion, the availability of a new standardized assay to determine DNA proviral load will be important in assessing the true extent of virological suppression suggesting that its quantification may be an important parameter in monitoring HIV infection.