Stepwise Release of Biologically Active HMGB1 during HSV-2 Infection

BACKGROUND: High mobility group box 1 protein (HMGB1) is a major endogenous danger signal that triggers inflammation and immunity during septic and aseptic stresses. HMGB1 recently emerged as a key soluble factor in the pathogenesis of various infectious diseases, but nothing is known of its behaviour during herpesvirus infection. We therefore investigated the dynamics and biological effects of HMGB1 during HSV-2 infection of epithelial HEC-1 cells. METHODOLOGY/PRINCIPAL FINDINGS: Despite a transcriptional shutdown of HMGB1 gene expression during infection, the intracellular pool of HMGB1 protein remained unaffected, indicating its remarkable stability. However, the dynamics of HMGB1 was deeply modified in infected cells. Whereas viral multiplication was concomitant with apoptosis and HMGB1 retention on chromatin, a subsequent release of HMGB1 was observed in response to HSV-2 mediated necrosis. Importantly, extracellular HMGB1 was biologically active. Indeed, HMGB1-containing supernatants from HSV-2 infected cells induced the migration of fibroblasts from murine or human origin, and reactivated HIV-1 from latently infected T lymphocytes. These effects were specifically linked to HMGB1 since they were blocked by glycyrrhizin or by a neutralizing anti-HMGB1 antibody, and were mediated through TLR2 and the receptor for Advanced Glycation End-products (RAGE). Finally, we show that genital HSV-2 active infections also promote HMGB1 release in vivo, strengthening the clinical relevance of our experimental data. CONCLUSIONS: These observations target HMGB1 as an important actor during HSV-2 genital infection, notably in the setting of HSV-HIV co-infection

A High-Sensitivity Method for Detection and Measurement of HMGB1 Protein Concentration by High-Affinity Binding to DNA Hemicatenanes

BACKGROUND: Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration. METHODOLOGY/PRINCIPAL FINDINGS: In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum. CONCLUSION: The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies

Contribution de deux agonistes de RACE (HMGB-1 et AGEs, dans la modulation des infections virales)

HMGB-1 (High-mobility group protein B1) est un composant non structural de la chromatine. Elle est aussi le chef de file des alarmines , un groupe de molécules impliquées dans la signalisation de stress d origine infectieuse ou non. Sécrétée par certaines cellules immunes activées ou libérée par les cellules nécrotiques, HMGB-1 agit seule ou en combinaison avec l ADN CpG, l ARN, le LPS ou l IL-1ß et module le comportement d un large spectre de cellules. Elle contribue notamment à signaler la présence d agents pathogènes variés, à coordonner la réponse innée et la réparation tissulaire en réponse à des dommages tissulaires d origine septique ou aseptique. Trois récepteurs pour HMGB-1 ont été décrits : TLR-2, TLR-4, et RAGE, un récepteur des protéines glyquées (AGEs). Nous nous sommes intéressés au rôle d HMGB-1 dans la modulation d infections virales d intérêt médical majeur. Plusieurs virus ont été pris comme modèles : le VIH, l herpes génital (HSV-2) et le virus Epstein-Barr (EBV). Au cours de cette thèse, nous avons ainsi étudié le comportement et les effets de la protéine HMGB-1 au cours de l infection par HSV-2, en nous intéressant tout particulièrement au rôle que pouvait avoir cette alarmine dans le contexte de la co-infection HSV-2/VIH. Nous avons ensuite analysé son impact sur des lignées lymphomateuses B infectées ou non par le virus Epstein-Barr (EBV). Enfin, nous nous sommes intéressés au rôle des AGEs ( Advanced Glycation End product ) un autre ligand de RAGE - dans l infection par le VIH-1. Ce projet s intègre dans le cadre d une question plus large portant sur le rôle des récepteurs d HMGB-1 dans les infections et, par voie de conséquence, de celui de leur(s) agoniste(s). Les résultats présentés dans ce travail démontrent le rôle central des interactions entre HMGB-1 ou AGE et les récepteurs TLR-2, TLR-4 et RAGE, au cours des infections viralesPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Drug-resistant and immune-escape HBV mutants in HIV-infected hosts.

International audienceHIV-HBV-coinfected patients require optimal control of viral replication in order to prevent the development of severe comorbidities, such as liver cirrhosis and hepatocellular carcinoma. The genetic diversity of HBV is a poorly investigated factor of such viral replication in HIV-infected hosts. HBV genome diversity can be differentiated into two major aspects: genotypic and phenotypic. Genotypic diversity is more related to the natural history of HBV infection and genotypes are mostly determined by geographical origin of the hosts. Phenotypic diversity arises from attempts to escape from host immune surveillance (that is, precore, core and basal core promoter mutants), selection resulting from the use of treatments with weak genetic barrier (that is, pol mutants), exposure to hepatitis B immunoglobulin (that is, 'immune-escape' S gene mutants) or treatment-induced mutations from overlapping genes (that is, pol mutants inducing 'vaccine-escape' S gene mutants). pol mutations typically lead to uncontrolled viral replication, whereas S gene mutations can significantly alter hepatitis B surface antigen synthesis and reduce binding to antibodies, which renders individuals who are vaccinated or cured of HBV infection susceptible to infection. For patients coinfected with HIV, hepatitis B treatment options that aim to reduce the risk of HBV mutations from emerging must be seriously considered, not only from clinical but also public health perspectives

Testing for Hepatitis B Virus Alone Does Not Increase Vaccine Coverage in Non-Immunized Persons

AIM: To determine whether hepatitis B virus (HBV)-testing could serve as a gateway to vaccinate non-immunized individuals in a low-prevalent country. METHODS: Non-immunized subjects participating in a multi-center, HBV-testing campaign in Paris, France were identified and contacted via telephone 3-9 mo after testing in order to determine vaccination status. Vaccination coverage was evaluated in per-protocol (for all respondents) and intent-to-treat analysis (assuming all non-responders did not vaccinate). RESULTS: In total, 1215/4924 (24.7%) enrolled subjects with complete HBV serology were identified as nonimmunized and eligible for analysis. There were 99/902 successfully contacted subjects who had initiated HBV vaccination after screening: per-protocol, 11.0% (95%CI: 9.0-13.2); intent-to-treat, 8.2% (95%CI: 6.7-9.8). In multivariable analysis, vaccination was more likely to be initiated in individuals originating from moderate or high HBV-endemic countries (P < 0.001), patients with limited healthcare coverage (P = 0.01) and men who have sex with men (P = 0.02). When asked about the reasons for not initiating HBV vaccination, the most frequent response was "will be vaccinated later" (33.4%), followed by "did not want to vaccinate" (29.8%), and "vaccination was not proposed by the physician" (21.5%). Sub-group analysis indicated a stark contrast in vaccination coverage across centers, ranging from 0%-56%. CONCLUSION: HBV-vaccination after HBV screening was very low in this study, which appeared largely attributed to physician-patient motivation towards vaccination. Increased vaccination coverage might be achieved by emphasizing its need at the organizational level

Cell Cycle Arrest in G(2) Induces Human Immunodeficiency Virus Type 1 Transcriptional Activation through Histone Acetylation and Recruitment of CBP, NF-κB, and c-Jun to the Long Terminal Repeat Promoter

In human immunodeficiency virus type 1 (HIV-1)-infected cells, a cell cycle arrest in G(2) increases viral expression and may represent a strategy for the virus to optimize its expression. In latently infected cells, balance between viral silencing and reactivation relies on the nucleosomal organization of the integrated long terminal repeat (LTR). It is shown here that nucleosome nuc-1, which is located downstream of the TATA box, is specifically modified when latently infected cells are arrested in G(2) by chemical inducers. Notably, histones H3 and H4 are hyperacetylated, and this modification is associated with an increased LTR-driven transcription. nuc-1 hyperacetylation is also associated with the recruitment of histone acetyltransferase CBP and transcription factors NF-κB and c-Jun. NF-κB and/or c-Jun binding to the LTR in G(2)-arrested cells appears to be required for CBP recruitment as well as for nuc-1 remodeling and viral reactivation

Testing for hepatitis B virus alone does not increase vaccine coverage in non-immunized persons

AIM: To determine whether hepatitis B virus (HBV)-testing could serve as a gateway to vaccinate non-immunized individuals in a low-prevalent country. METHODS: Non-immunized subjects participating in a multi-center, HBV-testing campaign in Paris, France were identified and contacted via telephone 3-9 mo after testing in order to determine vaccination status. Vaccination coverage was evaluated in per-protocol (for all respondents) and intent-to-treat analysis (assuming all non-responders did not vaccinate). RESULTS: In total, 1215/4924 (24.7%) enrolled subjects with complete HBV serology were identified as nonimmunized and eligible for analysis. There were 99/902 successfully contacted subjects who had initiated HBV vaccination after screening: per-protocol, 11.0% (95%CI: 9.0-13.2); intent-to-treat, 8.2% (95%CI: 6.7-9.8). In multivariable analysis, vaccination was more likely to be initiated in individuals originating from moderate or high HBV-endemic countries (P < 0.001), patients with limited healthcare coverage (P = 0.01) and men who have sex with men (P = 0.02). When asked about the reasons for not initiating HBV vaccination, the most frequent response was "will be vaccinated later" (33.4%), followed by "did not want to vaccinate" (29.8%), and "vaccination was not proposed by the physician" (21.5%). Sub-group analysis indicated a stark contrast in vaccination coverage across centers, ranging from 0%-56%. CONCLUSION: HBV-vaccination after HBV screening was very low in this study, which appeared largely attributed to physician-patient motivation towards vaccination. Increased vaccination coverage might be achieved by emphasizing its need at the organizational level

HMGB1 assay in serums from patients.

<p>(A) without PCA treatment, or (B) after sample purification by PCA treatment. On each gel the five samples on the left, prepared with known amounts of pure HMGB1, were used to calibrate the method. The 15 serums tested are labeled a-o, serum a originating from a healthy individual, serums b-o from randomly selected patients in intensive care unit (d,g,h,i,k,l,n: sepsis; b,c,e,f,j,m,o: septic shock). Note the lower sensitivity, higher background, and non-specific bands (arrowheads) obtained with crude serums, as compared with the strong increase in sensitivity and marked background decrease after treatment of serums with PCA. (C) Comparison of band-shift assay with ELISA. Concentrations measured by ELISA are plotted against band-shift assay data obtained after PCA treatment of serums, on a double logarithmic scale. Each circled letter on the diagram represents the corresponding serum as assayed in (B).</p