Tesi di dottorato di ricerca
HHV-6 IN MULTIPLE SCLEROSIS: A POSSIBLE TRIGGER OF AUTOIMMUNE DISEASE
Settore scientifico disciplinare
Corso di studi
FISIOPATOLOGIA CLINICA E SCIENZE DEL FARMACO
tutor Prof. Ceccherini Nelli, Luca
- MULTIPLE SCLEROSIS
Data inizio appello
Data di rilascio
Background. Multiple sclerosis (MS) is predominantly regarded as an autoimmune disease in which viral infections could represent a trigger in genetically susceptible individuals. There is a growing evidence about an involvement of Epstein-Barr virus (EBV) and human herpesvirus-6 (HHV-6) in MS aethiopatogenesis. Previous studies failed to give a definitive answer to this question mainly because they fell short in patient’s selection, timing, volume and source (blood and/or CFS) of the biological sample collected for the analysis. In addition, none of them utilized a fully controlled system of highly sensitive calibrated quantitative real-time PCR (cQPCR) applied separately to both biological fluids (plasma and CFS) and their cellular component (PBMC and CSF-derived cellular pellet) to discriminate between active or latent infection and therefore between a direct or indirect role of these viruses in the MS.<br>Objective. To elucidate the role of HHV-6 and EBV in MS <br>Methods. Paired samples of plasma and cerebral spinal fluid (CSF) from patients with clinically active MS (n=55) and from patients with heterogeneous set of inflammatory and non inflammatory neurological diseases (OND/OIND) were examined for HHV-6 and EBV DNA load; a control group of was examined. Furthermore, in a subgroup of 11 patients affected from active MS at disease, the presence of HHV-6 and EBV DNA was measured in the fluidic fraction (CSF supernatant and centrifuged plasma) and in their cellular counterpart (CSF-derived cellular pellets and PBMCs). Finally, plasma viremia was also analyzed in samples derived from four cohorts of healthy (n=111) and patients controls including subjects affected by Autoimmune connective tissue diseases (ACTD, n=115), patients suffering of Chronic Fatigue Syndrome (CFS, n=63), and HIV-seropositive subjects. By a complete panel of calibrated quantitative real-time PCR (cQPCR) assays for plasma viral load measurements and of the parallel QPCR assay for the determination of human genomic DNA content (CCR5 assay) were excluded potential errors introduced by contamination of plasma by bystander cell lysis and/or by the presence of chromosomally integrated HHV-6 (CIHHV-6).<br>Results. Overall, 25/55 MS CSFs (45%) contained HHV-6 (20 samples; 36%) or EBV DNA (5 samples; 9%); by contrast, only 2/33 OND/OIND CSFs (6%) resulted positive for HHV-6 and none for EBV-DNA. Plasmatic levels of HHV-6 and EBV DNA resulted<br>significantly increased in patients affected by MS when compared to a heterogeneous set of OID/OIND. Both findings are not easily explainable with an accidental contamination due to breakage of latently infected cells or by the presence of ciHHV-6 carriers. <br>Therefore, the presence of HHV-6 DNA in CSF and in plasma of MS patients is most likely due to viral reactivation or to a destruction of infected cells occurring in the brain tissue.<br>The significant increase in HHV-6 detection and viral load observed in plasma of a relevant proportion of OND/OIND (33%) and (ACTD, 27%) patients deserve further investigation. Interestingly, the presence of HHV-6 DNA was detected in 5/11 clarified CSF supernatants. In one instances CSF viremia was accompanied also by cell-associated DNAemia in the absence of both plasma and PBMCs detectable viral load, suggesting the occurrence of a selective accumulation in the brain of HHV-6 infected cell. In other 4 patients the concomitant finding of HHV-6 DNA load in clarified CSF and plasma cell-free samples in the absence of detectable PBMCs (except for one with only 20 GEq/106 cells) and CSF-cellular load suggests the possibility of a sistemic infection as pathogenetic mechanism. Conversely, the concomitant presence of EBV in CSF derived cellular pellets associated to absence of plasma viremia suggest the EBV bystander damage hypothesis to CNS.<br>Conclusions. These data are compatible with the hypothesis that HHV-6 may act as a pathogenic factor predisposing patients to the development of MS.
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