IRES complexity before IFN-alpha treatment and evolution of the viral load at the early stage of treatment in peripheral blood mononuclear cells from chronic hepatitis C patients.

In the proof, we asked to correct the name of one Author, and the adddess; this was acknowledged by the Editor, but was omitted in the final published version!International audienceAt the early stage of treatment, IFN alpha-2a induces inhibition of HCV replication. The viral load reflects mainly the degradation rate of the viruses. However, differences in the behavior of the viral population depend on changes, which occurred in the HCV-IRES genome. In this study, cloning and sequencing strategies permitted the generation of a large number of IRES sequences from the PBMCs of 18 patients (5 women, 13 men) with chronic hepatitis C. The HCV IRES appeared to be highly conserved structurally. However, some variability was found between the different isolates obtained: 467 substitutions with a median of 7 variants/patients. No relationship was observed between pre-treatment IRES complexity and the viral load at the beginning. However, on review of the evolution of viral load in the PBMCs during the first 3 days of IFN alpha-2a treatment, patients could be classified into two groups: Group 1, in which the viral population continued to replicate and Group 2, in which the viral load decreased significantly (P = 0.01727). Positioning of the mutations on the predicted IRES secondary structure showed that the distribution of the mutations and their apparition frequency were different between the two groups. At the early stage of treatment, IFN alpha-2a was efficient in reducing the viral replication in a significant number of patients; mechanisms of response might affect the virus directly. However, pre-treatment genomic variations observed in the 5'NCR of HCV were not a parameter of a later response to antiviral therapy in chronic hepatitis C patients. (244

Features and distribution of CD8 T cells with human leukocyte antigen class I-specific receptor expression in chronic hepatitis C.: NKRs+ CD8 T cells in chronic Hepatitis C.

CD8(+) T cells represent a sizable component of the liver inflammatory infiltrate in chronic hepatitis C and are thought to contribute to immune-mediated tissue injury. Because chronic stimulation may promote the expression by CD8(+) T cells of distinct human leukocyte antigen class I-specific natural killer cell receptors (NKRs) susceptible to both inhibiting effector functions and promoting cell survival, we examined the distribution and characteristics of CD8(+) T cells with such receptors in chronic hepatitis C patients. NKR CD8(+) T cells were detectable in liver and peripheral blood from hepatitis C virus (HCV)-infected patients but were not major subsets. However, the frequency of NKG2A(+) CD8(+) in the liver and in a lesser extent in the peripheral blood was positively correlated to histological activity in HCV-infected patients. No such correlation was found with KIR(+) T cells in liver in HCV-infected patients and with the both NKR CD8(+) T cells in hepatitis B virus (HBV) infected patients. Circulating CD8(+) T cells expressing KIRs exhibited phenotypic features of memory T cells with exacerbated expression of the senescence marker CD57 in patients. NKG2A(+)CD8(+) T cells were committed T cells that appeared less differentiated than KIR(+)CD8(+) T cells. In HCV-infected patients, their content in perforin was low and similar to that observed in NKG2A(-)CD8(+) T cells; this scenario was not observed in healthy subjects and HBV-infected patients. Both NKG2A and KIRs could inhibit the response of HCV-specific CD8(+) T cells ex vivo. CONCLUSION: These results support the concept that an accumulation in the liver parenchyma of NKR(+)CD8(+) T cells that have functional alterations could be responsible for liver lesions. They provide novel insights into the complexity of liver-infiltrating CD8(+) T cells in chronic hepatitis C and reveal that distinct subsets of antigen-experienced CD8(+) T cells are differentially sensitive to the pervasive influence of HCV

Peptide-protein microarrays and surface plasmon resonance detection: biosensors for versatile biomolecular interaction analysis.

International audienceBiosensors in microarray format provide promising tools for high-throughput analyses of complex samples. Although they are able to detect, quantify and characterize a multitude of compounds, most of the available devices are specialized in the analysis of one type of interaction, limiting their application to a define area. The aim of our work was to develop and characterize versatile protein (or peptide) microarrays suitable for the simultaneous analysis of a large panel of biological interactions. Our system involved a simple procedure to immobilized proteins or peptides, based on pyrrole electropolymerization, and ligand binding was detected by imaging the surface plasmon resonance. We demonstrated its suitability in three different contexts, i.e. humoral response characterization, ion binding analysis and cell detection. This work evidences the potentiality of this approach which allows multiparametric, high-throughput and label-free analysis of biological samples suitable for the detection of compounds as various as proteins, ions or cells and the characterization of their interaction with peptides or proteins

Relationship between humoral response against hepatitis C virus and disease overcome

International audienceConclusionHumoral response against hepatitis C virus linear epitopes is partly modified according to the disease state. This study highlights the importance of considering relative quantities of antibodies with different specificities rather than the amount of each antibody.Hepatitis C virus infection leads to liver disease whose severity can range from mild to serious lifelong illness. However the parameters involved in the evolution of the disease are still unknown. Among other factors, the virus-elicited antibody profile is suspected to play a role in the outcome of the disease. Analysis of the relationship between anti-virus antibodies and disease state requires the analysis of a large number of serums from patients (hepatitis C virus+) and of epitopes from the viral proteins. Such a study would benefit from microarray-based screening systems that are appropriate for high-throughput assays.We used a method combining peptide chips and surface plasmon resonance imaging previously shown to be suitable for analyzing complex mediums and detecting peptide-protein interactions. 56 peptides covering the entire viral proteome were grafted on chips and their interaction with antibodies present in the 68 injected serums from infected and non-infected donors was measured. Statistical analyses were conducted to determine a possible relationship between antibodies (specificity and amount) and disease states.A good discrimination between infected and non-infected donors validated our approach, and several correlations between antibodies profiles and clinical parameters have been identified. In particular, we demonstrated that ratios between particular antibodies levels allow for accurate discrimination of patients according to their pathologic states

Etude comparative de critères d'évaluation de la segmentation

- Nous présentons dans cet article une étude comparative de plusieurs critères d'évaluation de résultats de segmentation d'image dans un contexte non supervisé. Nous avons utilisé une base de cent images synthétiques composées de régions uniformes et texturées. La valeur des sept critères analysés est comparée à la mesure de Vinet (correspondant au taux de classification) sur une base de trois cent résultats de segmentation

Inflammatory response of endothelial cells to a human endogenous retrovirus associated with multiple sclerosis is mediated by TLR4.

The MSRV (multiple sclerosis-associated retrovirus) belongs to the human endogenous retrovirus HERV-W family. The envelope protein originating from the MSRV has been found in most patients with multiple sclerosis (MS). This protein (Env-ms) has pro-inflammatory properties for several types of immune cells and could therefore play a role in MS pathogenesis by promoting the leukocyte diapedesis observed in the central nervous system of patients. Our study aims to analyze the effects of Env-ms on the blood-brain barrier (BBB) at a molecular and functional level. We demonstrate that the recombinant MSRV envelope is able to stimulate several inflammatory parameters in a human BBB in vitro model, the HCMEC/D3 brain endothelial cell line. Indeed, Env-ms induces over-expression of ICAM-1, a major mediator of leukocyte adhesion to endothelial cells, in a dose-dependent manner as well as a strong dose-dependent production of the pro-inflammatory cytokines IL-6 and IL-8. Furthermore, using a silencing approach with siRNAs, we show that Env-ms is recognized via the Toll-like receptor 4 receptor, a pattern recognition receptor of innate immunity present on endothelial cells. We also show, using functional assays, that treatment of brain endothelial cells with Env-ms significantly stimulated the adhesion and the transmigration of activated immune cells through a monolayer of endothelial cells. These findings support the hypothesis that MSRV could be involved in the pathogenesis of MS disease or at least in maintenance of inflammatory conditions, thus fueling the auto-immune disorder. MSRV could also play a role in other chronic inflammatory diseases

Mouse and human neutrophils induce anaphylaxis

International audienceAnaphylaxis is a life-threatening hyperacute immediate hypersensitivity reaction. Classically, it depends on IgE, FcεRI, mast cells, and histamine. However, anaphylaxis can also be induced by IgG antibodies, and an IgG1-induced passive type of systemic anaphylaxis has been reported to depend on basophils. In addition, it was found that neither mast cells nor basophils were required in mouse models of active systemic anaphylaxis. Therefore, we investigated what antibodies, receptors, and cells are involved in active systemic anaphylaxis in mice. We found that IgG antibodies, FcγRIIIA and FcγRIV, platelet-activating factor, neutrophils, and, to a lesser extent, basophils were involved. Neutrophil activation could be monitored in vivo during anaphylaxis. Neutrophil depletion inhibited active, and also passive, systemic anaphylaxis. Importantly, mouse and human neutrophils each restored anaphylaxis in anaphylaxis-resistant mice, demonstrating that neutrophils are sufficient to induce anaphylaxis in mice and suggesting that neutrophils can contribute to anaphylaxis in humans. Our results therefore reveal an unexpected role for IgG, IgG receptors, and neutrophils in anaphylaxis in mice. These molecules and cells could be potential new targets for the development of anaphylaxis therapeutics if the same mechanism is responsible for anaphylaxis in humans