Francesca Elia

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.

Search for genomic sequences of microbial agents in atherosclerotic plaques.

Atherosclerosis is a complex, multifactorial disease. Several studies have reported a possible association between infection with microbial agents and atherogenesis. Chlamydia pneumoniae (C. pneumoniae), Herpes Simplex Virus 1 (HSV1), Human Cytomegalovirus (HCMV), and Epstein Barr Virus (EBV) have been widely investigated for their possible role in atherosclerosis development, but the results obtained to date are contradictory. The aim of our study is to search DNA of the aforementioned infectious agents by means of Quantitative Real Time PCR in atherosclerotic plaques from carotid arteries obtained from 17 patients. Genomic sequences of C. pneumoniae, HSV1, HCMV were not found in any atherosclerotic lesion. Therefore, our results do not support the hypothesis of an association between these infectious agents and atherosclerosis. Conversely, three patients were found to be positive for EBV DNA, thus indicating that, at least in a limited number of patients, EBV could play a role in atherogenesis.

Epstein-Barr Virus Specific Antibody Response in Multiple Sclerosis Patients during 21 Months of Natalizumab Treatment

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. Natalizumab, a humanized anti-α4 integrin monoclonal antibody, is a highly effective treatment approved for MS. An association between MS and an exposure to Epstein-Barr Virus (EBV) sustained by the levels of antiviral capsid antigen (VCA) and anti-Epstein-Barr nuclear antigen-1 (EBNA-1) IgG has been described. Our goal was to verify the utility of EBV-specific IgG as a marker in Natalizumab treated MS. Twenty patients (17 female and 3 male) in treatment with Natalizumab were enrolled. Serum levels of anti-VCA and anti-EBNA-1 IgG were determined and expressed as arbitrary units (AU) before treatment and every three months for 21 months of therapy. Anti-VCA IgG levels were increased at the 15th month (235410 ± 196712 AU) comparing with the 3rd (98146 ± 47145 AU) and the 6th (109866 ± 52270 AU) months of therapy (p < 0.05). No significant differences were found for serum anti-EBNA-1 IgG levels. Our data indicate that a transient, self-limited, EBV reactivation can occur in MS during Natalizumab therapy but our results do not support the use of serum EBV-specific antibody levels as biomarkers for monitoring therapeutic response to Natalizumab in the course of MS.

Identification of Lymphotropic Polyomavirus in Peripheral Blood from Patients with Leukoencephalopathies and Healthy Individuals

Objective: Epstein-Barr virus (EBV), a ubiquitous virus that becomes latent in human B cells, has been considered a risk factor for the development of multiple sclerosis (MS). We searched for EBV in MS brain and in B-lymphocytes and plasma cells of MS cerebrospinal fluid (CSF), and for evidence of an intrathecal anti-EBV humoral immune response in MS CSF. Methods: Nested non-quantitative real-time PCR was used to detect endogenous and EBV-specific transcripts in MS brain and in single B-lymphocytes and plasma cells of MS CSF. Immunocytochemistry and immunoblotting were used to detect the ability of MS CSF as well as recombinant antibodies (rAbs) prepared from clonally expanded plasma cells in MS CSF to bind to EBV-infected cells. Intrathecal anti-EBV antibody synthesis was measured by an enzyme-linked immunosorbent assay (ELISA). Results: EBV-specific transcripts were not found in perivascular B cell infiltrates of active MS plaques from subjects with relapsing remitting MS or with secondary progressive MS, or in B-lymphocytes and plasma cells in MS CSF. The frequency of intrathecal anti-EBV antibody synthesis in MS patients did not differ from that found in patients with other inflammatory diseases of the brain. While the CSF of all MS patients bound EBV antigens, rAbs generated from clonally expanded plasma cells of the same patients did not react with EBV. Conclusions: EBV-infected B-lymphocytes and plasma cells were not detected in MS brain plaques or CSF. rAbs prepared from clonally expanded plasma cells in MS CSF were not directed against EBV. Intrathecal synthesis of anti-EBV antibodies was not exclusive to MS patients. Overall, our studies do not reveal the presence of EBV in MS brain or an intrathecal EBV-specific antibody response in patients with MS. P176 JC Virus Small Tumor Antigen Promotes Cell Cycle Progression and Induces Phosphorylation of Akt and Gsk3-Beta Ilker K Sariyer, Ahmet Ozdemir, Asif Chowdhury, Kamel Khalilli, and Mahmut Safak Department of Neuroscience, Laboratory of Molecular Neurovirology, Temple University School of Medicine, 1900 North 12th Street, Philadelphia, PA 19122 JC virus (JCV) is a human DNA tumor virus within the polyomavirus family. Oncogenic potential of JCV has been demonstrated in experimental animals including monkeys, hamsters and mice. Recent data indicate that JCV genome was also detected in a variety of human tumors suggesting that it may also be involved in the induction of some of the human tumors. Cell transformation studies using the polyomaviruses as model systems have provided many insights into the pathways involved in spontaneously arising cancers. JCV encodes two oncoproteins, the large T antigen (LT-Ag) and small t antigen (Sm t-Ag) and both of which play critical roles in tumor induction. LT-Ag has been previously shown to target a number of host regulatory pathways including pRb and p53; however the mechanism by which JCV Sm t-Ag contributes to such a process remains largely unknown. Several studies with SV40 have demonstrated that LT-Ag and Sm t-Ag, when together, sporadically induce tumors in a 9th International Symposium on NeuroVirology

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.

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.

Serum Gelatinases Levels in Multiple Sclerosis Patients during 21 Months of Natalizumab Therapy

Background. Natalizumab is a highly effective treatment approved for multiple sclerosis (MS). The opening of the blood-brain barrier mediated by matrix metalloproteinases (MMPs) is considered a crucial step in MS pathogenesis. Our goal was to verify the utility of serum levels of active MMP-2 and MMP-9 as biomarkers in twenty MS patients treated with Natalizumab. Methods. Serum levels of active MMP-2 and MMP-9 and of specific tissue inhibitors TIMP-1 and TIMP-2 were determined before treatment and for 21 months of therapy. Results. Serum levels of active MMP-2 and MMP-9 and of TIMP-1 and TIMP-2 did not differ during the treatment. The ratio between MMP-9 and MMP-2 was increased at the 15th month compared with the 3rd, 6th, and 9th months, greater at the 18th month than at the 3rd and 6th months, and higher at the 21st than at the 3rd and 6th months. Discussion. Our data indicate that an imbalance between active MMP-9 and active MMP-2 can occur in MS patients after 15 months of Natalizumab therapy; however, they do not support the use of serum active MMP-2 and active MMP-9 and TIMP-1 and TIMP-2 levels as biomarkers for monitoring therapeutic response to Natalizumab.