Antonella Rotola

Latent BK virus infection and Kaposi's sarcoma pathogenesis

We have analyzed by PCR skin lesions from classic, endemic and AIDS‐related Kaposis sarcoma (KS), as well as from KS‐derived cell lines, the presence of ubiquitous transforming viruses. BK virus (BKV), a transforming human papovavirus which has been associated with human tumors, was detected in 100% of KS skin lesions and 75% of KS cell lines. KS specimens contained a full‐length, intact BKV early region, but minor rearrangements were observed in some tumors. BKV was also detected with a high prevalence (57–67%) in genital tissues and sperm, thus fulfilling the role of a sexually transmitted agent in KS. The closely related JC virus (JCV), which has never been associated with human malignancies, was present in 11–20% of KS specimens and was detected with a low prevalence (0–21%) in genital tissues and sperm. Simian virus 40 (SV40) was not detected in any KS lesions. Herpes simplex virus (HSV) DNA sequences were detected in 20–25% of KS lesions. Malignant human papillomavirus (HPV) types 16 and 11 were detected in KS specimens with a similar prevalence of 11–83%, suggesting that the presence of HPV‐transforming sequences is not a specific trait of HPV interaction with KS tissue. Furthermore, JCV, SV40, HSV and HPV DNA sequences were not detected in KS cell lines, suggesting that these viruses are not associated to KS neoplastic cells in KS tissue. KS cell lines were also negative for DNA sequences of KS‐HV, the novel herpesvirus detected in primary KS lesions. The constant association of BKV DNA with KS lesions and KS cell lines suggests that BKV‐transforming functions may participate in the development of KS.

Human herpesvirus 6 infects the central nervous system of multiple sclerosis patients in the early stages of the disease

The presence and the replicative state of human herpesvirus 6 (HHV-6) were evaluated in clinical samples from multiple sclerosis (MS) patients at the first time of MS diagnosis. HHV-6 variant B was present in peripheral blood mononuclear cells of 5/32 (15%) patients, but persisted with a latent infection. Viral sequences were present also in cerebrospinal fluid (CSF), both free in the liquid (7/32, 22%) and latent in the cellular fraction (3/32, 9%), as shown by analysis of viral transcription. In these cases, variant A was detected. HHV-6 DNA sequences present in the CSF were associated to mature viral particles. In fact, in vitro infectious assays of CSF showed the presence of replication-competent virions. These results show that about 20% of MS patients have active foci of HHV-6 variant A infection in the early stages of the disease and suggest that viral replication takes place within the central nervous system.

Human papillomavirus type 16 DNA in genital tumours: a pathological and molecular analysis.

The presence of human papillomavirus type 16 (HPV16) DNA in 34 genital tract tumours of Italian female patients was investigated by Southern blot hybridization in high stringency conditions. HPV16 DNA was detected in 16 neoplasias, including cervical invasive and intraepithelial lesions as well as vulvar intraepithelial neoplasias and, to a lesser extent, vulvar invasive carcinomas. Appropriate control tissues included in the study were negative. The data suggest that integration of viral DNA had occurred in most tumours, both in invasive and in intraepithelial lesions. HPV16 variants or defective genomes, lacking the BamHI restriction site, were detected in three tumours.

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.

HLA-G may predict the disease course in patients with early rheumatoid arthritis.

The current management of early rheumatoid arthritis (ERA) is to start an intensive treatment as soon as possible. To avoid under/overtreatment, it is important to identify reliable ERA evolution biomarkers. HLA-G molecules has been associated with rheumatoid arthritis, suggesting a role in disease regulation. HLA-G antigens are expressed as membrane bound and soluble isoforms (mHLA-G, sHLA-G) that act as ligand for immune-inhibitory receptors (ILT2, ILT4, KIR2DL4). Expression of HLA-G is influenced by a 14 bp insertion/deletion polymorphism in exon 8 of the gene, where the deletion is associated with mRNA stability. We analyzed 23 ERA patients during a 12 months follow-up disease treatment for sHLA-G, IL-1beta, IL-6, IL-10 and TNF-alpha levels in plasma samples by ELISA, mHLA-G and ILT2 expression on peripheral blood CD14 positive cells by flow cytometry and typed HLA-G 14 bp deletion/insertion polymorphism by Real-Time PCR. Disease status (DAS28), ultrasonography with power Doppler and laboratory data were checked. Cytokine levels confirmed the anti-inflammatory effect of the treatment. sHLA-G, mHLA-G and ILT2 expression inversely correlated with DAS28 disease scores. The frequency of 14 bp deletion allele increased in patients with disease remission. Based on these results, HLA-G may be a candidate biomarker to evaluate early prognosis and disease activity in ERA patients.

Matrix metalloproteinase-2 (MMP-2) generates soluble HLA-G1 by cell surface proteolytic shedding

Human leukocyte antigen-G (HLA-G) molecules are non-classical HLA class I antigens with an important role in pregnancy immune regulation and inflammation control. Soluble HLA-G proteins can be generated through two mechanisms: alternative splicing and proteolytic release, which is known to be metalloprotease mediated. Among this class of enzymes, matrix metalloproteinases (MMPs) might be involved in the HLA-G1 membrane cleavage. Of particular interest are MMP-2 and MMP-9, which regulate the inflammatory process by cytokine and chemokine modulation. We evaluated the effect of MMP-9 and MMP-2 on HLA-G1 membrane shedding. In particular, we analyzed the in vitro effect of these two gelatinases on the secretion of HLA-G1 via proteolytic cleavage in 221-G1-transfected cell line, in JEG3 cell line, and in peripheral blood mononuclear cells. The results obtained by both cell lines showed the role of MMP-2 in HLA-G1 shedding. On the contrary, MMP-9 was not involved in this process. In addition, we identified three possible highly specific cleavage sites for MMP-2, whereas none were detected for MMP-9. This study suggests an effective link between MMP-2 and HLA-G1 shedding, increasing our knowledge on the regulatory machinery beyond HLA-G regulation in physiological and pathological conditions.

Detection of Antibodies Directed against Human Herpesvirus 6 U94/REP in Sera of Patients Affected by Multiple Sclerosis

ABSTRACT The association between human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) is controversial. In fact, it is difficult to establish a causative role of HHV-6, due to the high prevalence of latently infected individuals in the healthy population. Therefore, the presence of virus sequences in tissue biopsy does not support a viral role, and serological assays do not show significant differences between MS patients and control populations. The only viral gene expressed during latency is U94/rep. Therefore, we have developed a serological assay for the detection of antibodies specifically directed against U94/REP protein. Different populations were analyzed by enzyme-linked immunosorbent assay, including healthy controls, MS patients, and subjects with diseases unrelated to HHV-6 infection, including other neurological diseases. The results show statistically significant differences (P > 0.01) between MS patients and control groups, both in antibody prevalence (87 and 43.9%, respectively) and in geometric mean titer (1:515 and 1:190, respectively). The detection of antibodies specific for HHV-6 U94/REP shows that the immune system is exposed to this antigen during natural infection. The higher prevalence and higher titers of antibodies to U94/REP suggest that MS patients and control groups might experience different exposures to HHV-6.

U94 of human herpesvirus 6 inhibits in vitro angiogenesis and lymphangiogenesis.

Human herpesvirus 6 (HHV-6) is a lymphotropic virus, but recent observations showed that also vascular endothelial cells (ECs) are susceptible to infection, both in vivo and in vitro. The observation that lymph nodes are a site of viral persistence suggests that lymphatic ECs (LECs) might be even more relevant for HHV-6 biology than vascular ECs. Here, we provide evidence that HHV-6 can infect LECs in vitro and establish a latent infection. Thus HHV-6 infection induces the loss of angiogenic properties both in LECs and in vascular ECs, as shown by the inability to form capillary-like structures and to seal wound scratches. The antiangiogenic effects observed in infected cells are associated to the expression of HHV-6 U94/rep, a latency-associated gene. In fact, transfection of U94/rep or addition of recombinant U94/REP protein to ECs inhibits the formation of in vitro capillary-like structures, reduces migration of ECs, and blocks angiogenesis, rendering rat aortic rings insensitive to VEGF-induced vasculogenetic activity. The ability of U94/rep to block different angiogenetic steps may lead to approaches in the potential control of the proliferation of blood and lymphatic vessels.

Human herpesvirus 6 is latent in peripheral blood of patients with relapsing-remitting multiple sclerosis

Studies of the association between HHV-6 and multiple sclerosis are hindered by the difficulty in discriminating between latent and active infection. A follow up study was undertaken of patients with multiple sclerosis, searching peripheral blood mononuclear cells for molecular markers associated with HHV-6 latency and lytic replication. The results show that HHV-6 is latent and did not support systemic infection in patients with multiple sclerosis. Likewise, patients with multiple sclerosis did not show any evidence of active infection with other human herpesviruses HHV-7 and HHV-8.

Detecting recombination in TT virus: a phylogenetic approach.

TT virus (TTV) has a remarkable genetic heterogeneity. To study TTV evolution, phylogenetic analyses were performed on 739 DNA sequences mapping in the N22 region of ORF1. Analysis of neighbor-joining consensus trees shows significant differences between DNA and protein phylogeny. Median joining networks phylogenetic clustering indicates that DNA sequence analysis is biased by homoplasy (i.e., genetic variability not originated by descent), indicative of either hypermutation or recombination. Statistical analysis shows that the significant excess of homoplasy is due to frequent recombination among closely related strains. Recombination events imply that the transmission of TTV is not clonal and provide the necessary basis to explain (i) the high degree of genetic divergence between TTV isolates, (ii) the lack of population structure on a world scale, and (iii) the number of highly divergent strains that seems typical of this virus. We show that recombination phenomena can be detected by phylogenetic analyses in very short sequences when a sufficiently large data set is available.