Lucia Signorini

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.

High frequency of Merkel cell polyomavirus DNA in the urine of kidney transplant recipients and healthy controls

BACKGROUND Polyomavirus (PyV) infection is common, ranging from 60% to 100% depending on the virus. The urinary excretion rates of JC virus (JCV) and BK virus (BKV) have been extensively studied, but less is known about the more recently discovered PyVs. OBJECTIVES To investigate the urinary excretion of Merkel cell PyV (MCPyV), which is associated with Merkel cell carcinoma (MCC), in kidney transplant recipients and healthy subjects, as well as those of lymphotropic polyomavirus (LPV), which was isolated from the B-cells of African green monkeys but has also been found in human blood, JCV and BKV. STUDY DESIGN Urine samples were collected from 62 adult (ATP) and 46 pediatric (PTP) kidney transplant recipients and from 67 adult (AHC) and 40 pediatric (PHC) healthy controls. DNA was isolated and analyzed using real-time PCR (Q-PCR) to determine the viral loads of MCPyV, LPV, JCV and BKV. RESULTS MCPyV DNA was more frequently detected (p<0.05) in the PTP (36.9%) and PHC (30.0%) groups compared to JCV (8.7% in PTP, 12.5% in PHC), BKV (6.5% in PTP, 2.5% in PHC), and LPV (2.2% in PTP, 5% in PHC) and in the AHC (47.8%) group compared to BKV (13.4%) and LPV (0%). CONCLUSIONS Based on the results, it could be concluded that: (a) Despite the rarity of MCC, MCPyV is a common infection; (b) MCPyV demonstrates an excretion pattern similar to those of JCV and BKV, persisting in the kidney and infecting skin cells upon reactivation, with subsequent integration and transformation.

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 6 DNA in the cerebrospinal fluid of an HIV-positive patient with leukoencephalopathy

BACKGROUND Leukoencephalopathies in HAART-treated, HIV-positive patients include progressive multifocal leukoencephalopathy (PML), a result of lytic infection oligodendrocytes by JC polyomavirus (JCV), and another form characterized by the absence of JCV genome in cerebrospinal fluid (CSF). OBJECTIVES To test the potential viral etiology of JCV-negative leukoencephalopathy. STUDY DESIGN CSF was collected from 43 HIV-positive patients with MRI suggestive of leukoencephalopathies. DNA was isolated and real-time PCR assays for neurotropic viruses (Herpes Simplex Viruses 1/2, Varicella Zoster Virus, Epstein Barr Virus, Human Cytomegalovirus, Human Herpesvirus 6, JCV and HIV) were conducted. CSF from 14 non-reactive cases were subjected to random nucleic acid amplification, deep sequencing, and in silico search for viral sequences. RESULTS JCV genome was detected in the CSF of 19/43 PML patients, HIV genome in the CSF of 5 PML patients including 2 JCV negative patients, and no viruses were detected in 22 patients. Human Polyomavirus 6 (HPyV6) DNA was detected by deep sequencing in one JCV-negative leukoencephalopathy CSF sample. CONCLUSIONS HPyV6 DNA was detected in CSF of a case of demyelinating disease. HPyV6 has not been previously reported in CSF or associated with any disease. Demonstrating a causative role will require further studies.

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.

Review on the immunotherapy strategies against metastatic colorectal carcinoma

Colorectal cancer (CRC) is one of the most common malignancies throughout the world and the leading cause of cancer-related mortality in Western countries. Recent progress in CRC treatment options, such as surgery, chemotherapy, radiotherapy and target therapy, has improved the prognosis, but advanced disease with recurrence or distant metastasis is usually incurable and has an unfavorable prognosis. The introduction of immunotherapy-associated strategies, both active and passive, to the treatment of CRC aims to overcome the limits of classical treatments. We review the state of the art for CRC with respect to different immunotherapeutic approaches, such as the use of cancer vaccines and/or adoptive cellular therapy, their most current advances and limitations and perspectives for further improvements.

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.

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.

Do the Human Endogenous Retroviruses Play a Role in Colon Cancer

Long terminal repeat retroelements comprise about 8% of the human genome and include the human endogenous retroviruses (HERVs). Earllier it was suspected that HERVs can become active and be involved in the process of transformation of cells, through several oncogenic mechanisms. Abnormal expression of HERVs proteins has been reported for various types of cancer, such as melanoma, breast, prostate, and germ cell cancer, in which encoded transcripts or proteins are overexpressed in the tumor tissues. However, less is known about the association between the HERVs and the colon cancer development. We review the state of the art for colon cancer with respect to the HERVs that can be considered as an open area of investigation, potentially leading to future innovative diagnostic and therapeutic approaches.