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Didier Hober - One of the best experts on this subject based on the ideXlab platform.
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Coxsackievirus B4 infection of human primary pancreatic ductal cell cultures results in impairment of differentiation into insulin producing cells
Viruses, 2019Co-Authors: Antoine Bertin, Famara Sane, Valery Gmyr, Delphine Lobert, Arthur Dechaumes, Julie Kerrconte, Francois Pattou, Didier HoberAbstract:Coxsackievirus-B4 (CV-B4) E2 can persist in the pancreatic ductal-like cells (Panc-1 cell line), which results in an impaired differentiation of these cells into islet-like cell aggregates (ICA). In this study, primary pancreatic ductal cells obtained as a by-product of islet isolation from the pancreas of seven brain-dead adults were inoculated with CV-B4 E2, followed-up for 29 days, and the impact was investigated. Viral titers in culture supernatants were analyzed throughout the culture. Intracellular viral RNA was detected by RT-PCR. Levels of ductal cell marker CK19 mRNA and of insulin mRNA were evaluated by qRT-PCR. The concentration of c-peptide in supernatants was determined by ELISA. Ductal cells exposed to trypsin and serum-free medium formed ICA and resulted in an increased insulin secretion. Ductal cells from five brain-dead donors were severely damaged by CV-B4 E2, whereas the virus persisted in cultures of cells obtained from the other two. The ICAs whose formation was induced on day 14 post-inoculation were scarce and appeared tiny in infected cultures. Also, insulin mRNA expression and c-peptide levels were strongly reduced compared to the controls. In conclusion, CV-B4 E2 lysed human primary pancreatic ductal cells or persisted in these cells, which resulted in the impairment of differentiation into insulin-producing cells.
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emergence of fluoxetine resistant variants during treatment of human pancreatic cell cultures persistently infected with Coxsackievirus B4
Viruses, 2019Co-Authors: Enagnon Kazali Alidjinou, Famara Sane, Delphine Caloone, Ilka Engelmann, Antoine Bertin, Didier HoberAbstract:This study reports the antiviral activity of the drug fluoxetine against some enteroviruses (EV). We had previously established a model of persistent Coxsackievirus B4 (CVB4) infection in pancreatic cell cultures and demonstrated that fluoxetine could clear the virus from these cultures. We further report the emergence of resistant variants during the treatment with fluoxetine in this model. Four independent persistent CVB4 infections in Panc-1 cells were treated with fluoxetine. The resistance to fluoxetine was investigated in an acute infection model. The 2C region, the putative target of fluoxetine antiviral activity, was sequenced. However, Fluoxetine treatment failed to clear CVB4 in two persistent infections. The resistance to fluoxetine was later confirmed in HEp-2 cells. The decrease in viral titer was significantly lower when cells were inoculated with the virus obtained from persistently infected cultures treated with fluoxetine than those from susceptible mock-treated cultures (0.6 log TCID50/mL versus 4.2 log TCID50/mL, p < 0.0001). Some previously described mutations and additional ones within the 2C protein were found in the fluoxetine-resistant isolates. The model of persistent infection is an interesting tool for assessing the emergence of variants resistant to anti-EV molecules. The resistance of EV strains to fluoxetine and its mechanisms require further investigation.
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inhibition of Coxsackievirus B4 by lactobacillus plantarum
Microbiological Research, 2018Co-Authors: Mattia Pia Arena, Famara Sane, Firas Elmastour, Djamel Drider, Daniela Fiocco, Giuseppe Spano, Didier HoberAbstract:Abstract The enterovirus Coxsackievirus B4 (CV-B4) can infect different human tissues and provoke abnormal function or destruction of various organs and cells. Moreover, its infections have been linked to the onset of type 1 diabetes. Coxsackievirus B4 is classified as a “challenging virus”, due to the intense yet vain efforts to find effective prevention and therapeutic agents, especially within biological compounds. Lactobacillus plantarum is a lactic acid bacterium that is endowed with probiotic properties, and holds great potential for applications in medical and food industry sectors. Several compounds produced by this microorganism have been associated with various benefits including antimicrobial activity. In this work, we investigated the possible antiviral abilities of two Lb. plantarum strains and their derivatives against CV-B4. The different assays carried out (e.g. pre-incubation, competition and post-infection, using HEp-2 cells as human cell model) suggest that the tested microorganisms and their derivatives have an in vitro inhibiting activity against CV-B4. This is the first report showing the anti-CVB4 activity of Lb. plantarum strains and their derivatives.
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bifidobacteria derived lipoproteins inhibit infection with Coxsackievirus B4 in vitro
International Journal of Antimicrobial Agents, 2017Co-Authors: Enagnon Kazali Alidjinou, Famara Sane, Ilka Engelmann, Khalil Antoine El Kfoury, Mariebenedicte Romond, Angelo Scuotto, Fouad Dabboussi, Monzer Hamze, Didier HoberAbstract:The aim of the present study was to investigate the potential of bifidobacteria in protecting cells from Coxsackievirus B4 (CV-B4) infection. Bifidobacterial screening identified two of five strains that protected human epithelial type 2 (HEp-2) cell viability when bifidobacteria were incubated with viral particles prior to inoculation. In contrast, no effect was shown by incubating HEp-2 cells with bifidobacteria prior to CV-B4 inoculation. Cell wall lipoprotein aggregates (LpAs) secreted by the selected strains were assayed for their antiviral activity. The two LpAs exhibited antiviral activity when they were incubated with viral particles prior to inoculation of HEp-2 cells. Recombinant LpA-derived protein exhibited identical antiviral activity. To identify the peptide sequences interacting with the virus particles, LpA proteins were aligned with the peptide sequences of the north canyon rim and puff footprint onto Coxsackievirus and adenovirus receptor (CAR). The in silico molecular docking study using CV-B3 as template showed low-energy binding, indicating a stable system for the selected peptides and consequently a likely binding interaction with CV-B. Bifidobacterium longum and Bifidobacterium breve peptides homologous to the viral north rim footprint onto CAR sequence formed hydrogen bonds with several viral residues in the north rim of the canyon, which were already predicted as interacting with CAR. In conclusion, proteins from bifidobacterial LpAs can inhibit infection with CV-B4, likely through binding to the capsid amino acids that interact with CAR.
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Persistent Coxsackievirus B4 infection induces microRNA dysregulation in human pancreatic cells.
Cellular and Molecular Life Sciences, 2017Co-Authors: Ilka Engelmann, Enagnon Kazali Alidjinou, Famara Sane, Antoine Bertin, Johann Bossu, Céline Villenet, Martin Figeac, Didier HoberAbstract:Enterovirus infections are implicated in the development of type 1 diabetes (T1D). MicroRNAs as regulators of gene expression are involved in many physiological and pathological processes. Given that viral infections dysregulate cellular microRNAs, we investigated the impact of persistent Coxsackievirus B4 infection on microRNA expression of human pancreatic cells. Next-generation sequencing was used to determine microRNA expression in PANC-1 cells persistently infected (for several weeks) with Coxsackievirus B4 and uninfected control cells. Target prediction restricted to T1D risk genes was performed with miRWalk2.0. Functional annotation analysis was performed with DAVID6.7. Expression of selected microRNAs and T1D risk genes was measured by quantitative reverse-transcription polymerase chain reaction. Eighty-one microRNAs were dysregulated in persistently infected PANC-1 cells. Forty-nine of the known fifty-five T1D risk genes were predicted as putative targets of at least one of the dysregulated microRNAs. Most functional annotation terms that were enriched in these 49 putative target genes were related to the immune response or autoimmunity. mRNA levels of AFF3, BACH2, and IL7R differed significantly between persistently infected cells and uninfected cells. This is the first characterization of the microRNA expression profile changes induced by persistent Coxsackievirus B4 infection in pancreatic cells. The predicted targeting of genes involved in the immune response and autoimmunity by the dysregulated microRNAs as well as the dysregulated expression of diabetes risk genes shows that persistent Coxsackievirus B4 infection profoundly impacts the host cell. These data support the hypothesis of a possible link between persistent Coxsackievirus B4 infection and the development of T1D.
Arlene I Ramsingh - One of the best experts on this subject based on the ideXlab platform.
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dynamics of molecular responses to Coxsackievirus B4 infection differentiate between resolution and progression of acute pancreatitis
Virology, 2012Co-Authors: Rui Gu, Anae Shampang, Andrew A Reilly, Dusti Fisher, William G Glass, Arlene I RamsinghAbstract:A Coxsackievirus B4 induces acute pancreatitis with different outcomes. The study utilized a systems biology approach to identify molecular immune responses that differentiate between disease resolution and disease progression. The data establish a temporal pattern of host responses that differentiate the resolution of acute pancreatitis from the progression to chronic pancreatitis. A group of twenty-five genes exhibited characteristic expression profiles that were observed during the development of chronic pancreatitis but not during the resolution of disease. We postulate that the temporal dynamics of the twenty-five genes influence the development of pathogenic immune responses associated with chronic pancreatitis. Furthermore, a subset of eleven genes exhibited increased expression as viral titers waned. Of the eleven gene products, five are secreted molecules, TNF-α, IFN-γ, CXCL10, IL-10, and IL-22b, and represent novel potential therapeutic targets since they can be readily modulated with antibodies against the specific cytokine/chemokine or with antibodies against the corresponding receptors.
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il 10 is pathogenic during the development of Coxsackievirus B4 induced chronic pancreatitis
Virology, 2009Co-Authors: Rui Gu, Anae Shampang, Andrew A Reilly, Dusti Fisher, William G Glass, Arlene I RamsinghAbstract:Abstract Using a mouse model of Coxsackievirus B4 (CVB4-V)-induced chronic pancreatitis, we investigated whether cytokines are involved in the progression of acute disease to chronic inflammatory disease. We show that IL-10 contributed to the development of chronic pancreatitis since acute disease resolved when IL-10 was absent or when IL-10 signaling was disrupted. We explored the underlying mechanisms by which IL-10 affected disease progression, using a novel approach to assess immunological events occurring in situ . Multiple markers that define functional innate immune responses and functional T cell responses were monitored over the course of CVB4-V infection of wild-type and IL-10 knockout mice, using a multiplex transcriptional profiling approach. We show that high levels of IL-10 early during infection were associated with delayed innate and T cell responses. Furthermore, high IL-10 production correlated with altered kinetics of T regulatory responses indicating a disruption in the balance between effector and regulatory T cell responses.
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progression or resolution of Coxsackievirus B4 induced pancreatitis a genomic analysis
Journal of Virology, 2004Co-Authors: Stephanie E Ostrowski, Andrew A Reilly, Doris N Collins, Arlene I RamsinghAbstract:Group B Coxsackieviruses are associated with chronic inflammatory diseases of the pancreas, heart, and central nervous system. Chronic pancreatitis, which can develop from acute pancreatitis, is considered a premalignant disorder because it is a major risk factor for pancreatic cancer. To explore the genetic events underlying the progression of acute to chronic disease, a comparative analysis of global gene expression during Coxsackievirus B4-induced acute and chronic pancreatitis was undertaken. A key feature of acute pancreatitis that resolved was tissue regeneration, which was accompanied by increased expression of genes involved in cell growth, inhibition of apoptosis, and embryogenesis and by increased division of acinar cells. Acute pancreatitis that progressed to chronic pancreatitis was characterized by lack of tissue repair, and the expression map highlighted genes involved in apoptosis, acinoductular metaplasia, remodeling of the extracellular matrix, and fibrosis. Furthermore, immune responses appeared skewed toward development of alternatively activated (M2) macrophages and T helper 2 (Th2) cells during disease that resolved and toward classically activated (M1) macrophages and Th1 cells during disease that progressed. Our hypothesis is that growth and differentiation signals coupled with the M2/Th2 milieu favor acinar cell proliferation, while diminished growth signals and the M1/Th1 milieu favor apoptosis of acinar cells and remodeling/proliferation of the extracellular matrix, resulting in fibrosis.
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exogenous interleukin 12 protects against lethal infection with Coxsackievirus B4
Journal of Virology, 2003Co-Authors: Daniel M Potvin, Arlene I Ramsingh, Doris N Collins, Dennis W MetzgerAbstract:The group B Coxsackieviruses are enteroviruses belonging to the Picornaviridae family. In humans, most infections with the group B viruses are asymptomatic (25, 30). Generally, symptomatic infections result in mild illnesses of the upper respiratory tract, the gastrointestinal tract, and the skin. Occasionally, the group B Coxsackieviruses cause chronic inflammatory disease of the pancreas (insulin-dependent type I diabetes mellitus, idiopathic chronic pancreatitis), heart (dilated cardiomyopathy), and central nervous system (7). The mechanism by which the group B viruses cause chronic disease is a topic of ongoing debate. One view is that Coxsackieviruses cause chronic disease via viral persistence (18, 19, 38, 40). Viral persistence may be due to the prolonged presence of low levels of infectious virus that continue to cause tissue injury. Alternatively, viral persistence may be due to the prolonged presence of viral RNA in the absence of infectious virus. The implication is that a low level of translation results in the synthesis of viral proteins that continue to stimulate immune responses, which cause prolonged tissue destruction. Another view is that Coxsackieviruses cause chronic disease via immune-mediated mechanisms directed against self-antigens. In mouse models, extensive evidence indicates that immunopathological mechanisms contribute to Coxsackievirus-induced chronic disease (11, 15, 16, 32, 35, 36). We have developed a mouse model of Coxsackievirus B4-induced disease by using two serologically indistinguishable variants of the B4 serotype, CVB4-P and CVB4-V (5). The avirulent CVB4-P variant induces a mild, transient inflammation of the pancreas (pancreatitis) with full recovery of the tissues by 10 days of infection. The virulent CVB4-V variant induces a severe pancreatitis, which can lead to mortality or progress to chronic disease (chronic pancreatitis). We have shown that the severity of CVB4-V-induced chronic disease is influenced by the major histocompatibility complex (31), cytokines (33), and T cells (33). Chronic pancreatitis, in this model, is due to immunopathological mechanisms. Given that CVB4-V-induced disease is due to immunopathological mechanisms, we addressed whether the outcome of CVB4-V infection could be altered by treatment with cytokines. The cytokine chosen for the present study was interleukin-12 (IL-12), because several studies have shown that administration of IL-12 can be beneficial during infection with a variety of viruses (21). In addition, IL-12 is made early in infection (34) and has a broad range of effects on both innate and adaptive immunity (34, 39). Our reasoning was that administration of IL-12 early in the course of CVB4-V infection could alter the development of immunopathology. In the present study, we show that (i) mice treated with IL-12 survive infection with the lethal CVB4-V variant, (ii) the beneficial effect of IL-12 is mediated via gamma interferon (IFN-γ), (iii) IFN-γ is produced by NK and NKT cells after IL-12 treatment, and (iv) the beneficial effect of IL-12 is not due to a decreased viral load in the target organ.
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a point mutation in vp1 of Coxsackievirus B4 alters antigenicity
Virology, 2000Co-Authors: Sadia S Halim, Arlene I RamsinghAbstract:Abstract While Coxsackievirus infections have been linked to several autoimmune diseases, very little is known about the immunogenicity of the Coxsackieviruses. Using two genetically related variants of Coxsackievirus B4, CB4-P and CB4-V, the relationship between virulence and antigenicity was examined. The virulent variant, CB4-V, was shown to be more antigenic than the avirulent CB4-P variant. The increased antigenicity of CB4-V was due to a single amino acid substitution in the VP1 capsid protein (a threonine residue at amino acid position 129), a site that had been previously identified as a major determinant of viral virulence. Thr-129 of VP1 is predicted to lie within a conformational B cell epitope. In addition, a nearby linear B cell epitope spanning residues 68 to 82 of VP1 was identified as a potential serotype-specific, neutralization antigenic site. The linear and conformational B cell epitopes of Coxsackievirus B4 may be analogous to antigenic sites 1 and 1B of poliovirus. To address whether the increased antigenicity of CB4-V influenced the severity of disease, mouse strains that differ in their outcome to viral infection were analyzed. Mice that developed the most severe disease and succumbed to infection were more immunoresponsive than mice that survived infection with CB4-V. The data suggest that immune-mediated mechanisms play a role in the severity of CB4-V induced disease.
Mark Peakman - One of the best experts on this subject based on the ideXlab platform.
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characterization of the t cell response to Coxsackievirus B4 evidence that effector memory cells predominate in patients with type 1 diabetes
Diabetes, 2002Co-Authors: Ruben Varelacalvino, Richard Ellis, Gianluca Sgarbi, Colin M Dayan, Mark PeakmanAbstract:Most of the evidence linking enterovirus (EV) infection with the development and/or acceleration of type 1 diabetes is indirect. Few studies have examined T-cell responses to these viruses, and therefore the nature of the viral targets and the immune cells involved in antiviral responses remain unclear. In the present study, we examined the characteristics of the T-cell response to the EV Coxsackievirus B4 (CVB4) in patients with type 1 diabetes and healthy control subjects. We find that CVB4-specific T-cells preferentially target the envelope proteins VP1, VP2, and VP3, and that the response to these and other CVB4 proteins differs markedly in type 1 diabetic patients compared with nondiabetic control subjects. The frequency of T-cell proliferative responses against VP2 was significantly reduced in type 1 diabetic patients compared with control subjects, especially in patients tested near to diagnosis ( P P P P P
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t cell activation by Coxsackievirus B4 antigens in type 1 diabetes mellitus evidence for selective tcr vβ usage without superantigenic activity
Journal of Immunology, 2001Co-Authors: Ruben Varelacalvino, Gianluca Sgarbi, Colin M Dayan, Lucy R Wedderburn, Jenny Tremble, Mark PeakmanAbstract:Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vβ chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vβ chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vβ chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vβ8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vβ chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vβ chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vβ chain usage which is driven by viral Ags rather than a superantigen.
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t cell reactivity to the p2c nonstructural protein of a diabetogenic strain of Coxsackievirus B4
Virology, 2000Co-Authors: Ruben Varelacalvino, Gianluca Sgarbi, Sefina Arif, Mark PeakmanAbstract:Abstract Enteroviruses are proposed as initiating factors in the etiology of Type 1 diabetes mellitus (Type 1 DM). Molecular mimicry between the autoantigen glutamic acid decarboxylase 65 (GAD65) and the Coxsackievirus B4 (CVB4) nonstructural protein P2C is frequently cited as a mechanism by which this virus triggers the disease, but little is known about the immunogenicity of this viral protein in humans, mainly due to the problem of obtaining highly pure preparations of P2C. We generated large amounts of highly pure, soluble P2C protein, coupled to the fusion partner maltose binding protein (MBP–P2C) using the PMAL-c2 bacterial expression plasmid and a two-step purification system comprising amylose resin and ion exchange. Using purified viral protein we show that specific T-cell responses against P2C are detected in the blood of healthy donors and Type 1 DM patients. Proliferation responses to P2C were detected only in subjects also demonstrating T-cell proliferation to CVB4 Vero cell lysates. However, in additional cases T-cell responses to P2C were detectable through the release of interferon-γ or interleukin-4 in individuals who did not make proliferative responses. Taken together, our data show that the P2C nonstructural protein of CVB4 is targeted by T cells during the antiviral immune response and may trigger the production of T helper 1 and T helper 2 cytokines. The availability of pure, immunogenic P2C should allow the putative role of antiviral responses in the development of autoimmune diabetes to be investigated.
Ruben Varelacalvino - One of the best experts on this subject based on the ideXlab platform.
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characterization of the t cell response to Coxsackievirus B4 evidence that effector memory cells predominate in patients with type 1 diabetes
Diabetes, 2002Co-Authors: Ruben Varelacalvino, Richard Ellis, Gianluca Sgarbi, Colin M Dayan, Mark PeakmanAbstract:Most of the evidence linking enterovirus (EV) infection with the development and/or acceleration of type 1 diabetes is indirect. Few studies have examined T-cell responses to these viruses, and therefore the nature of the viral targets and the immune cells involved in antiviral responses remain unclear. In the present study, we examined the characteristics of the T-cell response to the EV Coxsackievirus B4 (CVB4) in patients with type 1 diabetes and healthy control subjects. We find that CVB4-specific T-cells preferentially target the envelope proteins VP1, VP2, and VP3, and that the response to these and other CVB4 proteins differs markedly in type 1 diabetic patients compared with nondiabetic control subjects. The frequency of T-cell proliferative responses against VP2 was significantly reduced in type 1 diabetic patients compared with control subjects, especially in patients tested near to diagnosis ( P P P P P
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t cell activation by Coxsackievirus B4 antigens in type 1 diabetes mellitus evidence for selective tcr vβ usage without superantigenic activity
Journal of Immunology, 2001Co-Authors: Ruben Varelacalvino, Gianluca Sgarbi, Colin M Dayan, Lucy R Wedderburn, Jenny Tremble, Mark PeakmanAbstract:Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vβ chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vβ chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vβ chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vβ8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vβ chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vβ chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vβ chain usage which is driven by viral Ags rather than a superantigen.
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t cell reactivity to the p2c nonstructural protein of a diabetogenic strain of Coxsackievirus B4
Virology, 2000Co-Authors: Ruben Varelacalvino, Gianluca Sgarbi, Sefina Arif, Mark PeakmanAbstract:Abstract Enteroviruses are proposed as initiating factors in the etiology of Type 1 diabetes mellitus (Type 1 DM). Molecular mimicry between the autoantigen glutamic acid decarboxylase 65 (GAD65) and the Coxsackievirus B4 (CVB4) nonstructural protein P2C is frequently cited as a mechanism by which this virus triggers the disease, but little is known about the immunogenicity of this viral protein in humans, mainly due to the problem of obtaining highly pure preparations of P2C. We generated large amounts of highly pure, soluble P2C protein, coupled to the fusion partner maltose binding protein (MBP–P2C) using the PMAL-c2 bacterial expression plasmid and a two-step purification system comprising amylose resin and ion exchange. Using purified viral protein we show that specific T-cell responses against P2C are detected in the blood of healthy donors and Type 1 DM patients. Proliferation responses to P2C were detected only in subjects also demonstrating T-cell proliferation to CVB4 Vero cell lysates. However, in additional cases T-cell responses to P2C were detectable through the release of interferon-γ or interleukin-4 in individuals who did not make proliferative responses. Taken together, our data show that the P2C nonstructural protein of CVB4 is targeted by T cells during the antiviral immune response and may trigger the production of T helper 1 and T helper 2 cytokines. The availability of pure, immunogenic P2C should allow the putative role of antiviral responses in the development of autoimmune diabetes to be investigated.
Fabienne Brilot - One of the best experts on this subject based on the ideXlab platform.
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Demonstration and immunologic effect of an infection of the human thymus by the diabetogenic human Coxsackievirus B4
Bulletin et mémoires de l'Académie royale de médecine de Belgique, 2020Co-Authors: Fabienne BrilotAbstract:: Thymus exerts a prominent role in the establishment os central T-cell tolerance, as well as in the development of self major histocompatibility complex (MHC)-restricted T lymphocytes. Like others autoimmune diseases, type 1 diabetes emergence implies central or peripheric self tolerance breakdown. Environmental factors, especially enterovirus infections, are supposed to be involved in diabetes pathophysiology. Epidemiological studies have highlighted a frequent association between enterovirus Coxsackievirus B4 (CVB4) and type 1 diabetes. The aim of our work was to study whether a thymus infection by CVB4 could induce modifications of thymic function. In primary cultures of thymic epithelial cells (TEC), we detected viral proteins, positive- and negative- strand RNA, and infectious virus in the supernatants, meaning that TEC cultures were susceptible to CVB4 infection and that CVB4 induced a persistent infection in those cells. CVB4 also modulated TEC proliferation and cytokine, such as IL-6, GM-CSF and LIF secretions. Studies using fetal organ thymus culture (FTOC) showed that CVB4 induced a marked and progressive thymocytes depletion, in particular double positive (DP) and CD4+ cells. CVB4 replicated in those subpopulations, indeed positive- and negative-atrand RNA were detected. CVB4 also upregulated MHC class I expression on DP thymocytes. The upregulation of MHC expression required viral infection in DP cells. IL-6 and GM-CSF secretions were also involved in this phenomenom, but IFN-alpha was shown not to be involved. Taken together, our results showed the susceptibility of the human thymus to CVB4 infection, and an important thymic dysfuntion due to this infection. Our work is a novel approach in the understanding of the mechanisms of CVB4-induced type 1 diabetes.
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Coxsackievirus B4 infection of murine foetal thymus organ cultures
Journal of Medical Virology, 2008Co-Authors: Hela Jaidane, Vincent Geenen, Fabienne Brilot, Didier HoberAbstract:The infection of foetal thymus with Coxsackievirus B4 (CV-B4) E2 has been studied ex vivo by using CD-1 mice on foetal day 14, as a ready source of organs for experimentation to investigate the hypothesis of the role of thymic viral infections in the pathogenesis of type 1 diabetes. The replication of CV-B4 E2 in murine foetal thymus organ cultures has been demonstrated by evaluating the levels of positive- and negative-stranded viral RNA in cells by using a real-time quantitative RT-PCR method and by determining titres of infectious viral particles in culture supernatants for 7 days post-infection (p.i.). Staining of tissue sections with an anti-cytokeratin antibody and haematoxylin–eosin showed that CV-B4 infection had no visible effect on cell survival and organ integrity. Cell counts in mock- and virus-infected foetal thymus organ cultures increased from day 1 through day 7, and live cell numbers were comparable in both conditions as shown by Trypan blue exclusion test and 7-amino-actinomycin D staining of thymocytes. Compared with controls on day 7 p.i., cytofluorometric analyses on cells from CV-B4 E2-infected foetal thymus organ cultures displayed a marked increase in the percentage of the most immature CD3−CD4−CD8− thymocytes, and a decrease in the percentage of immature CD3−CD4+CD8+ cells, together with an increase in the percentage of mature CD3+CD4+ and CD3+CD8+ cells. These data show that CV-B4 E2 disturbs T-cell maturation and differentiation processes in infected murine foetal thymus organ cultures and provide evidence of a suitable system to investigate the effect of viruses in T-cell differentiation. J. Med. Virol. 80:659–666, 2008. © 2008 Wiley-Liss, Inc.
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prolonged viral rna detection in blood and lymphoid tissues from Coxsackievirus B4 e2 orally inoculated swiss mice
Microbiology and Immunology, 2006Co-Authors: Hela Jaidane, Pierre-emmanuel Lobert, Mahjoub Aouni, Vincent Geenen, Fabienne Brilot, Jawhar Gharbi, Bernadette Lucas, Raida El Hiar, Manel Ben Mhadheb, Didier HoberAbstract:The spreading of viral RNA within Swiss Albino mice orally inoculated with Coxsackievirus B4 E2 strain (CVB4 E2) was studied by using RT-PCR and semi-nested-RT-PCR methods. Viral RNA was detected in various organs: pancreas, heart, small intestine, spleen, thymus, and blood at various postinfectious (p.i.) times ranging from 8 hr to 150 days. Our results show that (i) outbred mice can be infected with CVB4 E2 following an oral inoculation, which results in systemic spreading of viral RNA, (ii) CVB4 E2 infection can be associated with a prolonged detection of viral RNA in spleen, thymus and blood, up to 70 days p.i. and further in other organ tissues.
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Coxsackievirus B4 infection of human fetal thymus cells
Journal of Virology, 2004Co-Authors: Fabienne Brilot, Didier Hober, Vincent Geenen, Cheryl A StoddartAbstract:The infection of human fetal thymus organ cultures (FTOC) with Coxsackievirus B4 E2 (CVB4 E2) was investigated. Both positive- and negative-strand viral RNA were detected by real-time quantitative reverse transcription-PCR (RT-PCR) in CVB4 E2-infected FTOC, which supported high yields of virus production (10 6 50% tissue culture infective doses/ml), and in flow-sorted thymocyte populations for 7 days after inoculation. Cortical CD4 CD8 thymocytes were found to be the principal targets of infection. Inoculation of human FTOC with CVB4 E2 led to a marked and progressive depletion of immature thymocytes (CD4 CD8 cells) with no enhancement of Annexin V-positive cells. CVB4 E2 replication caused significant major histocompatibility complex (MHC) class I upregulation on these cells. MHC class I upregulation was correlated with positive- and negative-strand RNA quantitative detection and the release of infectious particles. In addition, chloroquine treatment of FTOC and single-thymocyte suspensions suggested that MHC class I upregulation on thymocytes was the result of direct infection rather than caused by production of soluble factors such as alpha interferon. Thus, CVB4 E2 can infect human fetal thymocytes, which subsequently results in quantitative and qualitative abnormalities of these cells.
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persistent infection of human thymic epithelial cells by Coxsackievirus B4
Journal of Virology, 2002Co-Authors: Fabienne Brilot, Henri Martens, Vincent Geenen, Wassim Chehadeh, Chantal Charletrenard, Didier HoberAbstract:Persistent replication of Coxsackievirus B4 (CVB4) E2 (diabetogenic) and CVB4 JBV (nondiabetogenic) strains in thymic epithelial cell (TEC)-enriched cultures (≥95%) was proved by detection of positive- and negative-strand viral RNA by reverse transcription-PCR in extracted RNA from cell cultures, VP1 capsid protein detection by immunofluorescence (IF) staining, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double-IF staining, cytokeratin-containing cells were shown to be susceptible to CVB4. The persistence of CVB4 was associated with a significantly increased rate of TEC proliferation (up to 70%) after 20 days of culture and a significantly increased chronic production of immunoreactive interleukin-6 (IL-6), leukemia inhibitory factor, and granulocyte-macrophage colony-stimulating factor in supernatant after 3 days of culture. The CVB4 replication and the release of cytokines were not restricted to the CVB4 E2 diabetogenic strain and did not depend on the genetic background of the host; however, TEC were more responsive to CVB4 E2 than CVB4 JBV as far as the production of cytokines.
