Valentina Gentili

Human herpesvirus 7, Epstein-Barr virus and human cytomegalovirus in periodontal tissues of periodontally diseased and healthy subjects.

AIMS To evaluate (i) the presence of human herpesvirus 7 (HHV-7), Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV), and (ii) the transcription pattern of HHV-7 in gingival biopsies from patients affected by periodontitis (P) and periodontally healthy subjects (H). MATERIAL AND METHODS Thirty-seven subjects (P: n=24; H: n=13) were included. Each P patient contributed two gingival biopsies (representative of a clinically affected and non-affected site) and each H subject contributed one gingival biopsy. After DNA extraction, nested polymerase chain reaction was used to identify the viruses. RESULTS HHV-7 was detected in 91.7% of P patients and in 61.5% of H subjects (p=0.02), EBV in 50.0% samples of P patients and 7.7% of H subjects (p=0.005) and HCMV only in one sample from H group. EBV was more frequently detected in biopsies from affected sites (50.0%) than from non-affected sites (16.7%) (p=0.008). HHV-7 transcription was detected in 15.4% of affected and 15.4% of non-affected sites. CONCLUSIONS The results indicate that (i) gingival tissues can be considered a potential reservoir for HHV-7; (ii) when present, HHV-7 persists in a latent state in the majority of cases; (iii) the presence of EBV seems to be associated with the diseased state of the patient and site.

Altered natural killer cells' response to herpes virus infection in multiple sclerosis involves KIR2DL2 expression

The role of herpes viruses as potential triggers of multiple sclerosis (MS) is still debated. Peripheral blood mononuclear cells from MS patients and controls were treated with CpG sequences and infected in vitro with HSV-1. Samples were analyzed for viral yield, TLR9 pathways, cytokine secretion, NK cell activation and killer immunoglobulin-like receptor (KIR) expression. CpG treatment promoted an unexpected sensitivity to herpes virus infection in a subset of MS patients: TLR9 pathways did not show defects while NK cells presented decreased degranulation and cytotoxicity and up-regulated the inhibitory KIR2DL2 receptor. CpG treatment of purified NK cells affected directly KIR2DL2 modulation and cell activation. These data suggest potential implications for viral pathogenesis of MS.

Detection of HHV-6-specific mRNA and antigens in PBMCs of individuals with chromosomally integrated HHV-6 (ciHHV-6).

After inheritance of chromosomally integrated HHV-6 (ciHHV-6), viral DNA is found in every nucleated cell. The prevalence of ciHHV-6 is estimated to be 0.2-5% of humans. There are conflicting data on the potential for replication, possibly leading to clinical implications. We analysed peripheral blood mononuclear cells (PBMCs) from individuals with ciHHV-6 proven by fluorescence in situ hybridization (FISH) for HHV-6-specific mRNA (U94, U42, U22) and antigens by means of reverse transcription PCR and an indirect immunoperoxidase staining. U94 transcripts indicative of latent infection were detected in six (54.5%) out of 11 individuals at least once. Transcripts indicative of lytic infection (i.e. U42 and U22) were detected in four (36.4%) out of 11 individuals at least once. HHV-6 antigen was detected in seven (70%) out of 10 individuals at least once. The presence of viral mRNA and proteins supports virus gene expression from ciHHV-6, which may lead to virus replication. Considering the properties of active HHV-6 infection together with obvious replicative activity in individuals with ciHHV-6, pathophysiological effects leading to clinical consequences of chromosomally integrated viral DNA might be considered.

Panbacterial real-time PCR to evaluate bacterial burden in chronic wounds treated with Cutimed™ Sorbact™

The impact of polymicrobial bacterial infection on chronic wounds has been studied extensively, but standard bacteriological analysis is not always sensitive enough. Molecular approaches represent a promising alternative to the standard bacteriological analysis. This work aimed to assess the usefulness of a panbacterial quantitative real-time PCR reaction to quantitate the total bacterial load in chronic wounds treated with Cutimed™ Sorbact™, a novel therapeutic approach based on hydrophobic binding of bacteria to a membrane. The results obtained by panbacterial real-time PCR on conserved sequences of the bacterial 16S gene show that the bacterial burden significantly decreased in 10 out of 15 healing chronic wounds, and did not change in 5 out of 5 non-healing chronic wounds. On the contrary, classical culture for S. aureus and P. aeruginosa, and real-time PCR for Bacteroides and Fusobacterium did not show any correlation with the clinical outcome. Our study also shows that quantification of chronic wounds by panbacterial real-time PCR is to be performed on biopsies and not on swabs. These results show that panbacterial real-time PCR is a promising and quick method of determining the total bacterial load in chronic wounds, and suggest that it might be an important biomarker for the prognosis of chronic wounds under treatment.

Presence of HHV-6A in Endometrial Epithelial Cells from Women with Primary Unexplained Infertility

To elucidate the roles of human herpesvirus (HHV)-6 primary unexplained infertile women, a prospective randomized study was conducted on a cohort of primary unexplained infertile women and a cohort of control women, with at least one successful pregnancy. HHV-6 DNA was analyzed and the percentage and immune-phenotype of resident endometrial Natural Killer (NK) cells, as the first line of defense towards viral infections, was evaluated in endometrial biopsies. Cytokine levels in uterine flushing samples were analyzed. HHV-6A DNA was found in 43% of endometrial biopsies from primary unexplained infertile women, but not in control women. On the contrary, HHV-6B DNA was absent in endometrial biopsies, but present in PBMCs of both cohorts. Endometrial NK cells presented a different distribution in infertile women with HHV6-A infection compared with infertile women without HHV6-A infection. Notably, we observed a lower percentage of endometrial specific CD56brightCD16- NK cells. We observed an enhanced HHV-6A-specific endometrial NK cell response in HHV-6A positive infertile women, with a marked increase in the number of endometrial NK cells activating towards HHV-6A infected cells. The analysis of uterine flushing samples showed an increase in IL-10 levels and a decrease of IFN-gamma concentrations in infertile women with HHV6-A infection. Our study indicates, for the first time, that HHV-6A infection might be an important factor in female unexplained infertility development, with a possible role in modifying endometrial NK cells immune profile and ability to sustain a successful pregnancy.

Genome sequences of segmented filamentous bacteria in animals: implications for human research.

Experimental studies indicate that segmented filamentous bacteria (SFB) can recapitulate fundamental immune responses, particularly in reference to Th 17 and regulatory T-cell activity. The recent description of whole genomic DNA sequences of mouse and rat SFBs and the comparison between these sequences opens new important perspectives. In particular this knowledge allows to perform quali-quantitative studies in human beings regarding these yet unculturable anaerobic bacteria. The comparative analysis of rat and mouse SFB 16S rRNA sequences suggests the existence of highly conserved regions that could represent SFB-specific molecular targets potentially useful to develop PCR probes for study SFB in humans. These molecular tools may be of inestimable value to evaluate the possible role played by SFB in the immune system physiology, as well as its potential involvement in human chronic inflammatory and autoimmune diseases.

Cerebrospinal fluid amounts of HLA-G in dimeric form are strongly associated to patients with MRI inactive multiple sclerosis

Background: The relevance of human leukocyte antigen (HLA)-G in dimeric form in multiple sclerosis (MS) is still unknown. Objective: To investigate the contribution of cerebrospinal fluid (CSF) HLA-G dimers in MS pathogenesis. Methods: CSF amounts of 78-kDa HLA-G dimers were measured by western blot analysis in 80 MS relapsing–remitting MS (RRMS) patients and in 81 inflammatory and 70 non-inflammatory controls. Results: CSF amounts of 78kDa HLA-G dimers were more frequent in RRMS than in inflammatory (p<0.01) and non-inflammatory controls (p<0.001) and in magnetic resonance imaging (MRI) inactive than in MRI active RRMS (p<0.00001). Conclusion: Our findings suggest that HLA-G dimers may be implicated in termination of inflammatory response occurring in MS.

Impact of HLA-G analysis in prevention, diagnosis and treatment of pathological conditions

Human leukocyte antigen-G (HLA-G) is a non-classical HLA class I molecule that differs from classical HLA class I molecules by low polymorphism and tissue distribution. HLA-G is a tolerogenic molecule with an immune-modulatory and anti-inflammatory function on both innate and adaptative immunity. This peculiar characteristic of HLA-G has led to investigations of its role in pathological conditions in order to define possible uses in diagnosis, prevention and treatment. In recent years, HLA-G has been shown to have an important implication in different inflammatory and autoimmune diseases, pregnancy complications, tumor development and aggressiveness, and susceptibility to viral infections. In fact, HLA-G molecules have been reported to alternate at both genetic and protein level in different disease situations, supporting its crucial role in pathological conditions. Specific pathologies show altered levels of soluble (s)HLA-G and different HLA-G gene polymorphisms seem to correlate with disease. This review aims to update scientific knowledge on the contribution of HLA-G in managing pathological conditions.

Impact of Soluble HLA-G Levels and Endometrial NK Cells in Uterine Flushing Samples from Primary and Secondary Unexplained Infertile Women

The aim of this research was to determine the levels of human leukocyte antigen G (HLA-G) and endometrial Natural Killer ((e)NK) cell percentages in uterine flushing samples from primary and secondary infertile women. sHLA-G levels were lower in the uterine flushing samples from primary infertile women in comparison with women with secondary infertility. Lower CD56+KIR2DL4+ (e)NK cell percentages were detected in primary infertile women compared with secondary infertile women. This is the first study demonstrating that primary and secondary unexplained infertilities are characterized by different basal sHLA-G levels and CD56+KIR2DL4+ (e)NK cell percentages.

KIR2DL2 inhibitory pathway enhances Th17 cytokine secretion by NK cells in response to herpesvirus infection in multiple sclerosis patients

We have previously demonstrated that multiple sclerosis (MS) patients with KIR2DL2 expression on Natural killer (NK) cells are more susceptible to herpes simplex virus 1 (HSV-1) infection. We explored cytokine expression by NK cells during HSV-1 infection in association with KIR2DL2 expression. MS KIR2DL2(+) NK cells failed to control HSV-1 infection and secreted high levels of Th17 cytokines, while MS KIR2DL2(-) NK cells released Th1 cytokines, mainly IFN-gamma. Our data showed, for the first time, a peculiar Th17 cytokine secretion by MS KIR2DL2(+) NK cells in the presence of HSV-1 infection, that could be implicated in MS pathogenesis.