Silvia Carluccio

A Review on JC Virus Infection in Kidney Transplant Recipients

The polyomavirus (PyV), JC virus (JCV), is a small nonenveloped DNA virus that asymptomatically infects about 80% of healthy adults and establishes latency in the kidney tissue. In case of immunodeficient hosts, JCV can lytically infect the oligodendrocytes, causing a fatal demyelinating disease, known as progressive multifocal leukoencephalopathy (PML). Although the reactivation of another human PyV, BK virus (BKV), is relatively common and its association with the polyomavirus associated nephropathy (PyVAN) following renal transplantation is proven, JCV replication and its impact on graft function and survival are less well studied. Here we describe the biology of JCV and its pathological features and we review the literature regarding the JCV infection analyzed in the setting of transplantations.

JC virus load in cerebrospinal fluid and transcriptional control region rearrangements may predict the clinical course of progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a severe disease of the central nervous system (CNS), caused by infection with the Polyomavirus JC virus (JCV). Because there are no known treatments or prognostic factors, we performed a long‐term study focusing mainly on cerebrospinal fluid (CSF) samples from PML patients to describe the virological features akin to the different forms of the disease. Twenty‐eight PML patients were enrolled: 10 HIV‐1+ patients with classical PML (CPML), 9 HIV‐1+ patients with slowly progressing or stable neurological symptoms (benign PML), 3 HIV‐1+ asymptomatic patients, and 6 HIV‐1‐negative patients. CSF, urine, and blood samples were collected at the enrollment (baseline) and every 6 months afterwards when possible. The JCV DNA and HIV‐1 RNA loads were determined, and the JCV strains were characterized. At baseline, the mean CSF JCV load was log 6.0 ± 1.2 copies/ml for CPML patients, log 4.0 ± 1.0 copies/ml for benign PML patients, log 4.2 ± 0.5 copies/ml for asymptomatic PML patients, and log 5.8 ± 1.3 copies/ml for HIV‐1‐negative PML patients (CPML vs. benign: P < 0.01; CPML vs. asymptomatic: P < 0.05; HIV‐1 negative vs. benign: P < 0.01). Organization of the JCV transcriptional control region (TCR) showed unusual archetype structures in two long‐term survival patients; the NF1 sequence was found most commonly, whereas the Sp1 binding site was the most common for both CPML patients and HIV‐1 negative patients. Our results suggest that the JCV load in the CSF and the organization of the TCR should be considered as indicators of PML clinical outcome. J. Cell. Physiol. 227: 3511–3517, 2012.

Investigation of polyomaviruses replication in pediatric patients with nephropathy receiving rituximab.

Rituximab is a chimeric monoclonal antibody reacting with the CD20 antigen on B cells. It has been proposed as treatment for the idiopathic nephrotic syndrome, recurrent idiopathic nephropathy, and focal segmental glomerulosclerosis refractory to steroids. Rituximab influences T‐cell immunity and may predispose the patients to opportunistic infections, such as progressive multifocal leukoencephalopathy caused by the polyomavirus JC (JCV). The risk of latent viruses infections/reactivations in pediatric patients receiving monoclonal antibodies is not well known yet. In this longitudinal 6‐month study, the effects of rituximab on JCV and BK virus (BKV) replication have been investigated. Blood, serum, and urine samples have been collected monthly from 11 pediatric patients (mean age: 11 years) with the idiopathic nephrotic syndrome and recurrent idiopathic nephropathy, under rituximab therapy. JCV and BKV real‐time PCRs and sequencing of the viral protein 1 and the non‐coding control region have been conducted. The same investigations have been undertaken on samples collected from eight pediatric patients (controls, mean age: 6 years), with idiopathic nephrotic syndrome or focal segmental glomerulosclerosis, treated with conventional chemotherapy. JCV was detected in the urine of one patient (9%), and one control (12.5%); BKV was found in the urine of 7/11 patients (63.6%) and 2/8 controls (25%) and in blood samples from four patients. No significant difference was found in the mean viral loads and in the viral molecular characterizations between the two groups. The polyomaviruses replication was not associated with rituximab therapy in children. J. Med. Virol. 84:1464–1470, 2012.

Human Polyomavirus JC monitoring and noncoding control region analysis in dynamic cohorts of individuals affected by immune-mediated diseases under treatment with biologics: an observational study

BackgroundProgressive multifocal leukoencephalopathy (PML) onset, caused by Polyomavirus JC (JCPyV) in patients affected by immune-mediated diseases during biological treatment, raised concerns about the safety profile of these agents. Therefore, the aims of this study were the JCPyV reactivation monitoring and the noncoding control region (NCCR) and viral protein 1 (VP1) analysis in patients affected by different immune-mediated diseases and treated with biologics.MethodsWe performed JCPyV-specific quantitative PCR of biological samples collected at moment of recruitment (t0) and every 4 months (t1, t2, t3, t4). Subsequently, rearrangements’ analysis of NCCR and VP1 was carried out. Data were analyzed using χ2 test.ResultsResults showed that at t0 patients with chronic inflammatory rheumatic diseases presented a JCPyV load in the urine significantly higher (p≤0.05) than in patients with multiple sclerosis (MS) and Crohn’s disease (CD). It can also be observed a significant association between JC viruria and JCPyV antibodies after 1 year of natalizumab (p=0.04) in MS patients. Finally, NCCR analysis showed the presence of an archetype-like sequence in all urine samples, whereas a rearranged NCCR Type IR was found in colon-rectal biopsies collected from 2 CD patients after 16 months of infliximab. Furthermore, sequences isolated from peripheral blood mononuclear cells (PBMCs) of 2 MS patients with JCPyV antibody at t0 and t3, showed a NCCR Type IIR with a duplication of a 98 bp unit and a 66 bp insert, resulting in a boxB deletion and 37 T to G transversion into the Spi-B binding site. In all patients, a prevalence of genotypes 1A and 1B, the predominant JCPyV genotypes in Europe, was observed.ConclusionsIt has been important to understand whether the specific inflammatory scenario in different immune-mediated diseases could affect JCPyV reactivation from latency, in particular from kidneys. Moreover, for a more accurate PML risk stratification, testing JC viruria seems to be useful to identify patients who harbor JCPyV but with an undetectable JCPyV-specific humoral immune response. In these patients, it may also be important to study the JCPyV NCCR rearrangement: in particular, Spi-B expression in PBMCs could play a crucial role in JCPyV replication and NCCR rearrangement.

Generation of tumor-specific cytotoxic T-lymphocytes from the peripheral blood of colorectal cancer patients for adoptive T-cell transfer

This study designs a strategy for an adoptive cellular therapy (ACT) protocol based on the ex‐vivo selection of autologous peripheral blood‐derived CD8‐enriched T‐cells, stimulated with dendritic cells (DCs) that had been pulsed with apoptotic tumor cells to generate cytotoxic T lymphocytes (CTLs) with anti‐tumor activity. Seventy‐eight colorectal cancer (CRC) patients were enrolled in this study. Tumor tissues and peripheral blood (PB) were obtained at surgery. Tissues were mechanically dissociated and cultured to obtain a primary tumor cell line from each patient. DCs were derived from peripheral blood mononuclear cells (PBMCs) using magnetic positive selection of CD14+ monocytes. Anti‐tumor CTLs were elicited in co‐/micro‐cultures using DCs as antigen‐presenting cells, autologous apoptotic tumor cells as a source of antigens, and CD8+ T lymphocytes as effectors. Interferon‐γ (IFN‐γ) secretion was assessed by ELISpot assays to evaluate the activation of the CTLs against the autologous tumor cells. Primary tumor cell lines were obtained from 20 of 78 patients (25.6%). DCs were generated from 26 patients, and of them, corresponding tumor cell lines were derived from six patients. ELISpot results showed that significant IFN‐γ secretion was detected after different numbers of stimulations for two patients, whereas weak secretion was observed for three patients. Despite difficulties due to contamination of several primary tumor cell lines with gut intestinal flora, the results suggest that the generation of tumor‐specific CTLs is feasible from patients with CRC, and could be useful for supporting an ACT approach in CRC. J. Cell. Physiol. 230: 1457–1465, 2015.

Interaction between Human Polyomavirus BK and Hypoxia inducible factor‐1 alpha

BK polyomavirus (BKV) has a worldwide seroprevalence of approximately 90%. After primary infection, BKV establishes a life‐long latency within the urogenital tract. The severe immunological impairment occurring in renal transplant recipients leads to BKV reactivation, which may result in polyomavirus associated nephropathy (PVAN). While the transplanted kidney is transiently unperfused, Hypoxia Inducible Factors (HIFs) mediate the cellular response to hypoxia. The α‐subunit of HIF isoform 1 (HIF‐1α) may interact with several viruses, but until now, there has been no information regarding the interaction between BKV and HIF‐1α. The aim of this study is to investigate the possible interaction between HIF‐1α and BKV and its potential effect on the pathogenesis of PVAN. Screening of 17 kidney tissue samples revealed that HIF‐1α expression was 13.6‐fold higher in PVAN tissues compared to control tissues. A luminometric assay in co‐transfected African green monkey kidney cells (VERO) demonstrated BKV promoter activation ranging from two to sixfold (P < 0.05) when HIF‐1α was over‐expressed. A Chromatin ImmunoPrecipitation (ChIP) assay showed structural binding between the BKV promoter and HIF‐1α. The amount of BKV DNA increased by threefold in VERO infected cells that were exposed to simulated hypoxia, compared to the cells not subjected to hypoxia. Both ex vivo and in vitro interactions between HIF‐1α and BKV were observed, suggesting that HIF‐1α, stabilized during transplantation, may be able to bind the BKV promoter and enhance BKV replication. Thus, hypoxia should be considered a risk factor for the development of PVAN in kidney transplant recipients. J. Cell. Physiol. 231: 1343–1349, 2016.

A Potential Linkage Between the JC and BK Polyomaviruses and Brain and Urinary Tract Tumors: A Review of the Literature

JC virus (JCV) and BK virus (BKV) belong to the Polyomaviridae family and were the first human polyomaviruses (PyVs) discovered. Both JCV and BKV remain latent in the kidneys and can reactivate in immunocompromised hosts, causing progressive multifocal leukoencephalopathy (PML) and nephropathy, respectively. PyVs induce cell transformation in vitro and in animal models, where they infect non-permissive cells. The molecular mechanisms of tumor formation are based on the large tumor antigen (T Ag) protein, which is able to bind and inactivate pRb and p53, inducing uncontrolled cell cycle progression. Many studies on clinical samples also indicate a positive association between JCV and BKV and human solid tumors; however no direct involvement of PyVs in the development of human cancers has been demonstrated. In this review we focused on the potential association between JCV and BKV with brain and urinary tract tumors, respectively.

Hemozoin impairs cell cycle progression and promotes chemokine release in human microvascular endothelial cells

Background Cerebral malaria (CM) is a fatal complication of P. falciparum infection caused by the cytoadherence of infected erythrocytes to brain endothelial cells followed by microcirculatory obstruction, blood-brain barrier (BBB) damage, ring hemorrhages, inflammatory response and neurological sequelae. The combination of both parasite and host factors are involved in the pathogenesis of CM. In particular, malarial pigment, hemozoin (HZ) was shown to interfere with monocytes and endothelial cell functions. Recently our group demonstrated that HZ enhanced total gelatinolytic activity in endothelial cells by inducing ex novo matrix metalloproteinases-9 (MMP9) and promoting proMMP-9 protein expression (Prato et al., 2011).

Upregulation of integrin expression on monocytes in multiple sclerosis patients treated with natalizumab

Natalizumab is a humanized monoclonal antibody against the α4 subunit of VLA-4 integrin that is used to treat conditions such as multiple sclerosis (MS). Although its effects on lymphocytes have been widely described, little is known about its effects on monocytes. Here we described the effects of natalizumab treatment on peripheral blood monocytes from a small cohort of MS patients in terms of relative frequencies and surface integrin (CD49d and CD18) expression. We showed that natalizumab treatment altered the surface integrin expression on monocyte subsets in the peripheral compartment, suggesting a role for them as mediators of natalizumab effects.