Epstein Barr virus shedding in multiple sclerosis: similar frequencies of EBV in saliva across separate patient cohorts

Background: Epstein Barr Virus (EBV) infection is closely associated with multiple sclerosis (MS), but the relationship between viral load and disease activity is unclear. This study tested the observed levels of salivary EBV in MS, as a first step in investigating this relationship. Methods: Real-time quantitative PCR (qPCR) was used to measure EBV DNA levels in saliva samples from three separate Multiple Sclerosis (MS) patient cohorts. Results: The qPCR assay was used to delineate EBV shedding, defined here as a reliably detectable level of extracellular EBV DNA in saliva. Frequency of EBV shedding was found to be similar across the groups, with 20–25% of subjects releasing virus on any given sampling date. Diurnal variation in EBV count was tested in one of the cohorts, in which 26% of subjects showed more than a 10-fold difference between the highest and lowest EBV levels on a single day. In the same cohort, elevated viral levels at one time point did not predict elevated viral levels at a subsequent time point. Conclusions: These results indicate that EBV lytic activity in a subject cannot be inferred from a single measure of EBV in saliva. Also, subjects do not appear to be behave constantly as “EBV shedders” or “non-shedders”. The assay is useful in giving a clear indication of salivary gland EBV lytic activity across a patient cohort – for example, in testing anti-viral drugs in M

Incomplete Cd8+ T Lymphocyte Differentiation as a Mechanism for Subdominant Cytotoxic T Lymphocyte Responses to a Viral Antigen

CD8+ cytotoxic T lymphocytes (CTLs) recognize antigen in the context of major histocompatibility complex (MHC) class I molecules. Class I epitopes have been classified as dominant or subdominant depending on the magnitude of the CTL response to the epitope. In this report, we have examined the in vitro memory CTL response of H-2d haplotype murine CD8+ T lymphocytes specific for a dominant and subdominant epitope of influenza hemagglutinin using activation marker expression and staining with soluble tetrameric MHC–peptide complexes. Immune CD8+ T lymphocytes specific for the dominant HA204-210 epitope give rise to CTL effectors that display activation markers, stain with the HA204 tetramer, and exhibit effector functions (i.e., cytolytic activity and cytokine synthesis). In contrast, stimulation of memory CD8+ T lymphocytes directed to the subdominant HA210-219 epitope results in the generation of a large population of activated CD8+ T cells that exhibit weak cytolytic activity and fail to stain with the HA210 tetramer. After additional rounds of restimulation with antigen, the HA210-219–specific subdominant CD8+ T lymphocytes give rise to daughter cells that acquire antigen-specific CTL effector activity and transition from a HA210 tetramer–negative to a tetramer-positive phenotype. These results suggest a novel mechanism to account for weak CD8+ CTL responses to subdominant epitopes at the level of CD8+ T lymphocyte differentiation into effector CTL. The implications of these findings for CD8+ T lymphocyte activation are discussed

Inhibition of p53-Dependent, but Not p53-Independent, Cell Death by U19 Protein from Human Herpesvirus 6B

Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein

Partially circumventing peripheral tolerance for oncogene-specific prostate cancer immunotherapy

BACKGROUND Failure of cancer immunotherapy is essentially due to immunological tolerance to tumor-associated antigens (TAAs), as these antigens are also expressed in healthy tissues. METHODS Here, we used transgenic adenocarcinoma of mouse prostate (TRAMP) mice, which develop lethal prostate cancer due to prostate-specific expression of SV40 T antigen (Tag), to evaluate effects of prostatic transformation on oncogene TAA-specific tolerance and to test the possibility of breaking such tolerance using a modified recombinant vaccinia virus. RESULTS We showed that Tag expression in TRAMP mice is uniquely extra-thymic, and levels of prostatic Tag expression positively correlate with malignant transformation of the prostate. Yet, young tumor-free TRAMP mice were tolerant to Tag antigen. We therefore attempted overcoming such peripheral oncogene-specific T cell tolerance through immunization with a vaccinia construct encoding Tag immunogenic epitopes. This vaccination modality showed that oncogene-specific tolerance was successfully overcome by effective in vivo priming of Tag-specific cytotoxic T cells (CTLs). However, this was restricted to young TRAMP mice. Tag-specific CTL from “tumor naÏve” young TRAMP mice showed significant anti-tumor efficacy in vivo by eliminating established heterotopic prostate tumors and prolonging survival in SCID mice harboring Tag-expressing tumors. In contrast, older TRAMP mice with established prostate tumors exhibited oncogene-specific tolerance as evidenced by failure to generate Tag-specific CTL following Tag-specific immunization. CONCLUSIONS Peripheral tolerance can be overcome for effective anti-tumor therapy following oncogene-specific immunization. However, this ability to elicit oncogene-specific CTL is impeded in the tumor-bearing host, in the context of increased oncogene expression associated with tumor progression. Prostate 68: 715–727, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58560/1/20689_ftp.pd

HLA-A*02:07 Is a Protective Allele for EBV Negative and a Susceptibility Allele for EBV Positive Classical Hodgkin Lymphoma in China

HLA-A2 protects from EBV+ classical Hodgkin lymphoma (cHL) in Western Europe, but it is unknown whether this protective effect also exists in the Chinese population. We investigated the association of HLA-A2 and specific common and well documented HLA-A2 subtypes with EBV stratified cHL patients (n = 161) from the northern part of China. Quantitative-PCR and sequence-based subtyping was performed to identify HLA-A2 positive samples and their subtypes. 67 (42%) of the cHL patients were EBV+. There were no significant differences in percentages of HLA-A2 positivity between cHL and controls (65% vs 66%) and between EBV+ and EBV− cHL patients (70% vs 61%). The frequency distribution of HLA-A2 subtypes was significantly different between EBV stratified cHL subgroups and controls. This difference was most striking for the HLA-A*02:07 type with a frequency of 38% in EBV+ cHL, 8% in EBV− cHL and 20% in controls. Significant differences were also observed for the HLA-A*02:07, HLA-A2 (non-02:07) and the A2-negative typings between EBV+ cHL vs controls (p = 0.028), EBV− cHL vs controls (p = 0.045) and EBV+ vs EBV− cHL cases (p = 2×10−5). In conclusion, HLA-A*02:07 is a predisposing allele for EBV+ cHL and a protective allele for EBV− cHL in the northern Chinese population

Molecular Mimicry of Human Cytochrome P450 by Hepatitis C Virus at the Level of Cytotoxic T Cell Recognition

Hepatitis C virus (HCV) is thought to be involved in the pathogenesis of autoimmune hepatitis (AIH) type 2, which is defined by the presence of type I antiliver kidney microsome autoantibodies directed mainly against cytochrome P450 (CYP)2D6 and by autoreactive liver infiltrating T cells. Virus-specific CD8+ cytotoxic T lymphocytes (CTLs) that recognize infected cells and contribute to viral clearance and tissue injury during HCV infection could be involved in the induction of AIH. To explore whether the antiviral cellular immunity may turn against self-antigens, we characterized the primary CTL response against an HLA-A*0201–restricted HCV-derived epitope, i.e., HCV core 178–187, which shows sequence homology with human CYP2A6 and CYP2A7 8–17. To determine the relevance of these homologies for the pathogenesis of HCV-associated AIH, we used synthetic peptides to induce primary CTL responses in peripheral blood mononuclear cells of healthy blood donors and patients with chronic HCV infection. We found that the naive CTL repertoire of both groups contains cross-reactive CTLs inducible by the HCV peptide recognizing both CYP2A6 and CYP2A7 peptides as well as endogenously processed CYP2A6 protein. Importantly, we failed to induce CTLs with the CYP-derived peptides that showed a lower capacity to form stable complexes with the HLA-A2 molecule. These findings demonstrate the potential of HCV to induce autoreactive CD8+ CTLs by molecular mimicry, possibly contributing to virus-associated autoimmunity

HLA Alleles Associated with Delayed Progression to AIDS Contribute Strongly to the Initial CD8(+) T Cell Response against HIV-1

BACKGROUND: Very little is known about the immunodominance patterns of HIV-1-specific T cell responses during primary HIV-1 infection and the reasons for human lymphocyte antigen (HLA) modulation of disease progression. METHODS AND FINDINGS: In a cohort of 104 individuals with primary HIV-1 infection, we demonstrate that a subset of CD8(+) T cell epitopes within HIV-1 are consistently targeted early after infection, while other epitopes subsequently targeted through the same HLA class I alleles are rarely recognized. Certain HLA alleles consistently contributed more than others to the total virus-specific CD8(+) T cell response during primary infection, and also reduced the absolute magnitude of responses restricted by other alleles if coexpressed in the same individual, consistent with immunodomination. Furthermore, individual HLA class I alleles that have been associated with slower HIV-1 disease progression contributed strongly to the total HIV-1-specific CD8(+) T cell response during primary infection. CONCLUSIONS: These data demonstrate consistent immunodominance patterns of HIV-1-specific CD8(+) T cell responses during primary infection and provide a mechanistic explanation for the protective effect of specific HLA class I alleles on HIV-1 disease progression

Chemoproteomics reveals Toll-like receptor fatty acylation

Partial funding for Open Access provided by The Ohio State University Open Access Fund.Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells. Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.This work was supported by funding from the NIH/NIAID (grant R00AI095348 to J.S.Y.), the NIH/NIGMS (R01GM087544 to HCH), and the Ohio State University Public Health Preparedness for Infectious Diseases (PHPID) program. NMC is supported by the Ohio State University Systems and Integrative Biology Training Program (NIH/NIGMS grant T32GM068412). BWZ is a fellow of the National Science Foundation Graduate Research Fellowship Program (DGE-0937362)

Expansion in CD39(+) CD4(+) Immunoregulatory T Cells and Rarity of Th17 Cells in HTLV-1 Infected Patients Is Associated with Neurological Complications

HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develops HAM/TSP. CD4(+) T cells are the main target of infection and play a pivotal role in regulating immunity to HTLV and are hypothesized to participate in the pathogenesis of HAM/TSP. the CD39 ectonucleotidase receptor is expressed on CD4(+) T cells and based on co-expression with CD25, marks T cells with distinct regulatory (CD39(+)CD25(+)) and effector (CD39(+)CD25(-)) function. Here, we investigated the expression of CD39 on CD4(+) T cells from a cohort of HAM/TSP patients, HTLV-1 asymptomatic carriers (AC), and matched uninfected controls. the frequency of CD39(+)CD4(+) T cells was increased in HTLV-1 infected patients, regardless of clinical status. More importantly, the proportion of the immunostimulatory CD39(+)CD25(-) CD4+ T-cell subset was significantly elevated in HAM/TSP patients as compared to AC and phenotypically had lower levels of the immunoinhibitory receptor, PD-1. We saw no difference in the frequency of CD39(+)CD25(+) regulatory (Treg) cells between AC and HAM/TSP patients. However, these cells transition from being anergic to displaying a polyfunctional cytokine response following HTLV-1 infection. CD39(-)CD25(+) T cell subsets predominantly secreted the inflammatory cytokine IL-17. We found that HAM/TSP patients had significantly fewer numbers of IL-17 secreting CD4(+) T cells compared to uninfected controls. Taken together, we show that the expression of CD39 is upregulated on CD4(+) T cells HAM/TSP patients. This upregulation may play a role in the development of the proinflammatory milieu through pathways both distinct and separate among the different CD39 T cell subsets. CD39 upregulation may therefore serve as a surrogate diagnostic marker of progression and could potentially be a target for interventions to reduce the development of HAM/TSP.National Institute of Allergies and Infectious DiseasesNational Institutes of HealthUniversity of CaliforniaSan Francisco-Gladstone Institute of Virology & Immunology Center for AIDS ResearchFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)John E. Fogarty International CenterNational Center for Research ResourcesNational Institute of General Medical Sciences from the National Institutes of HealthUniv Calif San Francisco, Dept Med, Div Expt Med, San Francisco, CA 94143 USAUniv Hawaii, John A Burns Sch Med, Dept Trop Med, Hawaii Ctr AIDS, Honolulu, HI 96822 USAUniv São Paulo, Sch Med, Deparment Infect Dis, São Paulo, BrazilUniv São Paulo, Sch Med, Div Clin Immunol & Allergy, São Paulo, BrazilFuncacao Prosangue, Hemoctr São Paulo, Mol Biol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilSan Francisco-Gladstone Institute of Virology & Immunology Center for AIDS Research: P30 AI027763FAPESP: 04/15856-9/KallasFAPESP: 2010/05845-0/KallasFAPESP: 11/12297-2/SanabaniJohn E. Fogarty International Center: D43 TW00003National Center for Research Resources: 5P20RR016467-11National Institute of General Medical Sciences from the National Institutes of Health: 8P20GM103466-11Web of Scienc

HTLV-1 Tax Mediated Downregulation of miRNAs Associated with Chromatin Remodeling Factors in T Cells with Stably Integrated Viral Promoter

RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type