EFFICIENT PROPAGATION OF ARCHETYPE JC POLYOMAVIRUS IN COS-7 CELLS: EVALUATION OF REARRANGEMENTS WITHIN NCCR STRUCTURAL ORGANIZATION DURING TRANSFECTION.

John Cunningham virus (JCPyV) is an ubiqui-tous human pathogen that causes disease in immunocom-promised patients. The JCPyV genome is composed of an early region and a late region, which are physically sepa-rated by the non-coding control region (NCCR). The DNA sequence of the NCCR distinguishes two forms of JCPyV, the designated archetype and the prototype, which resulted from a rearrangement of the archetype sequence. To date, the cell culture systems for propagating JCPyV archetype have been very limited in their availability and robust-ness. Prior to this study, it was demonstrated that JCPyV archetype DNA replicates in COS-7 simian kidney cells expressing SV40 TAg and COS-7 cells expressing HIV-1 Tat. Based on these observations, the present study was conducted to reproduce an in vitro model in COS-7 cells transfected with the JCPyV archetype strain in order to study JCPyV DNA replication and analyze NCCR rear-rangements during the viral life cycle. The efficiency of JCPyV replication was evaluated by quantitative PCR (Q-PCR) and by hemagglutination (HA) assay after trans-fection. In parallel, sequence analysis of JCPyV NCCR was performed. JCPyV efficiently replicated in kidney-derived COS-7 cells, as demonstrated by a progressive increase in viral load and virion particle production after transfection. The archetypal structure of NCCR was maintained during the viral cycle, but two characteristic point mutations were detected 28 days after transfection. This model is a useful tool for analyzing NCCR rearrangements during in vitroreplication in cells that are sites of viral persistence, such as tubular epithelial cells of the kidne

Results, questions, perspectives of a study on human polyomavirus BK and molecular actors in prostate cancer development

Background: Prostate cancer (PC) is a common tumor in Western countries. Several risk factors play significant roles. MYC, BIRC5/survivin, CDC25 and P53 may contribute to PC risk. As demonstrated, human Polyomavirus BK (BKV) could affect cellular homeostasis contributing to PC pathogenesis. Materials and Methods: Biological samples were collected from PC patients. Viral RNA was searched using quantitative polymerase chain reaction (PCR), whereas a qualitative PCR was employed to find particular viral sequences. Proper size amplicons were analyzed. Single nucleotide polymorphisms (SNPs) were detected in p53 coding regions by means of a specific PCR. C-MYC, BIRC5/survivin and CDC25 gene expression was investigated using a Retro Transcriptional Quantitative PCR. Results: Viral DNA copy number was higher in cancer tissues taken from Gleason score 9 patients with Gleason score 7. Different p53 mutated compared to patients exons were found according to tumor advanced stage and a statistical significant correlation was found between Gleason score and p53 mutational rate. C-MYC, BIRC5/survivin and CDC25 expression was de-regulated according to the literature. Conclusion: The presence of BKV and its variants in transformed cells does not exclude viral pressure in cell immortalization. Expression of other target genes evidenced a significant change in their regulation, useful for cancer drug discovery and therapies

High frequency of JCV DNA detection in prostate cancer tissues

BACKGROUND: Prostate cancer (PC) represents the most frequently diagnosed cancer in men. Exposure to infectious agents has been considered to induce prostatic inflammation and cancerous transformation. Controversial data exist concerning the role of the human polyomaviruses BK (BKV) and JC (JCV) in PC etiology. Therefore, a possible association between these polyomaviruses and PC was investigated. MATERIALS AND METHODS: Urine, blood and fresh prostatic tissue specimens were collected from 26 patients with PC. The presence of BKV and JCV, the possible non-coding control region (NCCR) variations and the genotyping analysis of viral protein 1 (VP1) of both viruses were assessed. RESULTS: Data showed a preferential viral re-activation in the urinary compartment and a statistically significant prevalence of JC viruria and of BKV in PC tissues. A BKV DDP-like NCCR sequence was isolated in two patients, whereas JCV NCCR was consistently of an archetypal structural organization. A prevalence of the European genotypes was observed for both viruses. CONCLUSION: Our data demonstrated the presence of JCV DNA in 14/24 (58.3%) cancerous prostatic tissue specimens, confirming the results obtained in a previous study, in which JCV has been defined as common inhabitant of the prostate, and opening the discussion about its potential role in PC

Increased prevalence of Human Polyomavirus JC viruria in Chronic Inflammatory Rheumatic Diseases patients in treatment with anti-TNF α: a 18 month follow-up study.

Chronic inflammatory rheumatic diseases (CIRDs) are immune-mediated pathologies involving joints. To date, TNFα-blocking agents administration is the most promising therapy, although these treatments are associated with an increased Polyomavirus JC (JCPyV) reactivation, the etiological agent of the Progressive Multifocal Leukoencephalopathy (PML). The aim of this study was the recruitment and the analysis of a CIRDs cohort in order to investigate a possible correlation between JCPyV presence and the influence of anti-TNF-α agents on viral loads. Blood and urine samples were collected from 34 CIRDs subjects prior the first anti-TNF-α infusion (T0) and after 3 (T3), 6 (T6), 12 (T12), and 18 (T18) months. Results showed persistent JC viruria significantly higher than JC viremia throughout the 18 month follow-up study (p=0.002). In JCPyV positive samples, the non-coding control region (NCCR) was analyzed. Results evidenced archetypal structures (type II-S) in all isolates with the exception of a sequence isolated from a plasma sample, that corresponds to the type II-R found in PML subjects. Finally, the viral protein 1 (VP1) genotyping was performed and results showed the prevalence of the European genotypes 1A, 1B, and 4. Since only few studies have been carried out to understand whether there is a PML risk in CIRDs population infected by JCPyV, this study contributes to enrich literature insight on JCPyV biology in this cluster. Further investigations are necessary in order to recognize the real impact of biologics on JCPyV life cycle and to identify possible and specific viral variants related to increased virulence in CIRDs patient

Natalizumab affects T-cell phenotype in multiple sclerosis: implications for JCV reactivation

The anti-CD49d monoclonal antibody natalizumab is currently an effective therapy against the relapsing-remitting form of multiple sclerosis (RRMS). Natalizumab therapeutic efficacy is limited by the reactivation of the John Cunningham polyomavirus (JCV) and development of progressive multifocal leukoencephalopathy (PML). To correlate natalizumab-induced phenotypic modifications of peripheral blood T-lymphocytes with JCV reactivation, JCV-specific antibodies (serum), JCV-DNA (blood and urine), CD49d expression and relative abundance of peripheral blood T-lymphocyte subsets were longitudinally assessed in 26 natalizumab-treated RRMS patients. Statistical analyses were performed using GraphPad Prism and R. Natalizumab treatment reduced CD49d expression on memory and effector subsets of peripheral blood T-lymphocytes. Moreover, accumulation of peripheral blood CD8+ memory and effector cells was observed after 12 and 24 months of treatment. CD4+ and CD8+ T-lymphocyte immune-activation was increased after 24 months of treatment. Higher percentages of CD8+ effectors were observed in subjects with detectable JCV-DNA. Natalizumab reduces CD49d expression on CD8+ T-lymphocyte memory and effector subsets, limiting their migration to the central nervous system and determining their accumulation in peripheral blood. Impairment of central nervous system immune surveillance and reactivation of latent JCV, can explain the increased risk of PML development in natalizumab-treated RRMS subjects

Epidemiology of intracerebral haemorrhage in Livorno district

BACKGROUND: Intracranial haemorrhage represents the most feared stroke subtype. AIM: To evaluate the burden of intracranial haemorrhage in Tuscany hospitals with special reference to Livorno district. MATERIALS AND METHODS: Data of patients discharged in 2009 from Tuscan and Livorno hospitals with codes ICD-9-CM related to any type of spontaneous intracranial haemorrhage were selected and analyzed. RESULTS: 3,472 patients were discharged from Tuscan hospitals with these diagnoses. Overall mortality was 24.3%. 50% of patients were admitted in Internal Medicine wards. Incidence of intracranial haemorrhage and intracerebral haemorrhage (ICH) in population of Livorno district was 64 and 45/100,000 inhabitants/year with related mortality of 36.5% and 39.4%respectively. Intra-hospital mortality of patients admitted in Livorno hospitals for intracranial haemorrhage were 36.7%. 40% of deaths occurred in the first 48 hours. 69.6% of intracranial haemorrhage were ICHs, 16.8% subaracnoideal. Intra-hospital mortality, admissions for intracranial haemorrhage in respect of total admissions and mortality for intracranial haemorrhage in respect to total mortality increased in the last decade. 23% of patients with intracranial haemorrhage and 16% of patients with ICH underwent to surgical procedures. ICHs related to antithrombotic treatment significantly increased in the last years. Mortality in patients on antithrombotic drugs was three times over compared to that in patients not undergone these drugs (43.7% vs 12.8%, p < 0.01). CONCLUSION: There is an increasing trend in frequency, mortality and hospital burden of intracranial haemorrhage and ICH. Efforts aimed at reducing the burden and consequences of this devasting disease are warranted

Outcomes of pregnancies after kidney transplantation: lessons learned from CKD. A comparison of transplanted, nontransplanted chronic kidney disease patients and low-risk pregnancies: a multicenter nationwide analysis.

BACKGROUND: Kidney transplantation (KT) may restore fertility in CKD. The reasons why materno-foetal outcomes are still inferior to the overall population are only partially known. Comparison with the CKD population may offer some useful insights for management and counselling.Aim of this study was to analyse the outcomes of pregnancy after KT, compared with a large population of non-transplanted CKD patients and with low-risk control pregnancies, observed in Italy the new millennium. METHODS: We selected 121 live-born singletons after KT (Italian study group of kidney in pregnancy, national coverage about 75%), 610 live-born singletons in CKD and 1418 low-risk controls recruited in 2 large Italian Units, in the same period (2000-2014). The following outcomes were considered: maternal and foetal death; malformations; preterm delivery; small for gestational age baby (SGA); need for the neonatal intensive care unit (NICU); doubling of serum creatinine or increase in CKD stage. Data were analysed according to kidney diseases, renal function (staging according to CKD-EPI), hypertension, maternal age, partity, ethnicity. RESULTS: Materno-foetal outcomes are less favourable in CKD and KT as compared with the low-risk population. CKD stage and hypertension are important determinants of results. KT patients with e-GFR >90 have worse outcomes compared with CKD stage 1 patients; the differences level off when only CKD patients affected by glomerulonephritis or systemic diseases ('progressive CKD') are compared with KT. In the multivariate analysis, risk for preterm and early-preterm delivery was linked to CKD stage (2-5 versus 1: RR 3.42 and 3.78) and hypertension (RR 3.68 and 3.16) while no difference was associated with being a KT or a CKD patient. CONCLUSIONS: The materno-foetal outcomes in patients with kidney transplantation are comparable with those of nontransplanted CKD patients with similar levels of kidney function impairment and progressive and/or immunologic kidney diseas