0000000000077092
AUTHOR
Evelyne Kohli
Inhibition of in vitro reconstitution of rotavirus transcriptionally active particles by anti-VP6 monoclonal antibodies
International audience; Six monoclonal antibodies specific for the major capsid protein of rotavirus, VP6, previously characterized, were tested in a biological assay for their capacity to block the transcriptase activity associated with the single-shelled particles. The results showed that two MAbs (RV-50 and RV-133), specific for distinct antigenic sites, were able to block the transcription when they were incubated with a purified baculovirus-expressed group A VP6, prior to the reconstitution of the single-shelled particles from the cores, suggesting that at least two domains are involved in active single-shelled particle reconstitution. The results obtained previously from immunochemist…
Different profile and distribution of antigen specific T cells induced by intranasal and intrarectal immunization with rotavirus 2/6-VLP with and without LT-R192G
International audience; In this study, we compared both the profile and distribution of antigen specific primed T cells after intrarectal (IR) and intranasal (IN) immunization with rotavirus (RV) 2/6-VLP, alone or in the presence of LT-R192G, in order to highlight the differences between the two routes and the impact of the adjuvant. Adult BALB/c mice were immunized once with 2/6-VLP with or without adjuvant and the T cell response was analyzed in lymphoid tissues after in vitro restimulation with the antigen. IN, but not IR, immunization of mice with 2/6-VLP alone induced antigen-specific IL-10 and IL-17 secreting T cells. IL-10-, in contrast to IL-17-, secreting T cells did not migrate to…
Les cercles de qualité médecins-pharmaciens, un mode de collaboration vertueux expérimenté en France
S’inspirant du modele suisse, l’unite mixte de developpement professionnel continu sante de l’universite de Bourgogne a mis en place, sur neuf sites, des cercles de qualite medecins-pharmaciens sur le territoire francais. Les resultats, attendus en 2021, permettront d’evaluer l’apport de ces cercles dans differentes organisations de soins primaires et mettront en avant les freins ainsi que les leviers rencontres avant le lancement d’une experimentation plus large.
GARP: A Key Target to Evaluate Tumor Immunosuppressive Microenvironment
Simple Summary Tumors are not only composed of cancer cells but also of various infiltrating cells constituting the tumor microenvironment (TME); all these cells produce growth factors which contribute to tumor progression and invasiveness. Among them, transforming growth factor-β1 (TGF-β1) has been shown to be a potent immunosuppressive cytokine favoring cell proliferation and invasion and to be associated with resistance to anticancer treatments. Glycoprotein-A repetition predominant (GARP) plays a critical role in the activation of TGF-β1 and has been shown to be expressed at the membrane of cancer cells and also of regulatory T cells and platelets in the TME. An increased GARP expressio…
Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant.
Background Highly publicised outbreaks of norovirus gastroenteritis in hospitals in the UK and Ireland and cruise ships in the USA sparked speculation about whether this reported activity was unusual. Methods We analysed data collected through a collaborative research and surveillance network of viral gastroenteritis in ten European countries (England and Wales were analysed as one region). We compiled data on total number of outbreaks by month, and compared genetic sequences from the isolated viruses. Data were compared with historic data from a systematic retrospective review of surveillance systems and with a central database of viral sequences. Findings Three regions (England and Wales,…
The GRP94 Inhibitor PU-WS13 Decreases M2-like Macrophages in Murine TNBC Tumors: A Pharmaco-Imaging Study with 99mTc-Tilmanocept SPECT
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancers and is not eligible for hormone and anti-HER2 therapies. Identifying therapeutic targets and associated biomarkers in TNBC is a clinical challenge to improve patients’ outcome and management. High infiltration of CD206+ M2-like macrophages in the tumor microenvironment (TME) indicates poor prognosis and survival in TNBC patients. As we previously showed that membrane expression of GRP94, an endoplasmic reticulum chaperone, was associated with the anti-inflammatory profile of human PBMC-derived M2 macrophages, we hypothesized that intra-tumoral CD206+ M2 macrophages expressing GRP94 may represent innovative…
Recombinant virus-like particles of a norovirus (genogroup II strain) administered intranasally and orally with mucosal adjuvants LT and LT(R192G) in BALB/c mice induce specific humoral and cellular Th1/Th2-like immune responses
We investigated the immune response induced by mucosal immunization of BALB/c mice with virus-like particles (VLPs) of a genogroup II norovirus, Dijon171/96 virus, produced in the baculovirus system. VLPs administered alone by the intranasal route induced a high serum antibody response as well as fecal IgA, which were enhanced when the heat-labile Escherichia coli toxin or its non toxic mutant LT(R192G) was coadministered. In these conditions, the oral route was also efficient. Cytokine production by cells from different lymphoid tissues was then assessed after in vitro restimulation. A Th1/Th2-like response was observed in cervical lymph node and Peyer's patch (PP) cell cultures from mice …
Virus diversity in a winter epidemic of acute diarrhea in France.
ABSTRACT In France, an epidemic peak of acute diarrhea is observed each winter. Previous results suggested a viral etiology for these winter epidemics. We investigated the role of enteric viruses in acute diarrhea and their molecular diversity. One hundred sixty-one patients with acute diarrhea and 45 healthy patients (controls) from the general population were given a standardized questionnaire between December 1998 and May 1999. Stool specimens were screened for group A and C rotaviruses, human caliciviruses, astroviruses, and adenovirus types 40 and 41 by reverse transcription-PCR and/or enzyme immunoassay. Virologic analysis was positive for 63 cases (39%). Caliciviruses and group A rot…
B subunits of cholera toxin and thermolabile enterotoxin of Escherichia coli have similar adjuvant effect as whole molecules on rotavirus 2/6-VLP specific antibody responses and induce a Th17-like response after intrarectal immunization.
The purpose of this study was to evaluate the adjuvant effect of the B subunits of cholera toxin (CT) and the thermolabile enterotoxin of Escherichia coli (LT) by the intrarectal route of immunization and compare them to the whole molecules CT and LT-R192G, a non toxic mutant of LT, using 2/6-VLP as an antigen, in mice. All molecules induced similar antigen specific antibody titers in serum and feces, whereas different T cell profiles were observed. CTB and LTB, conversely to CT and LT-R192G, did not induce detectable production of IL-2 by antigen specific T cells. Moreover, CTB, conversely to LT-R192G, CT and LTB, did not induce antigen specific CD4+CD25+Foxp3- and Foxp3+ T cells, thus sho…
Low Trough Plasma Concentrations of Nevirapine Associated with Virologic Rebounds in HIV-Infected Patients Who Switched from Protease Inhibitors
BACKGROUND:The substitution of a nonnucleoside reverse-transcriptase inhibitor (NNRTI) for protease inhibitors (PIs) has demonstrated its suitability to maintain virologic response. However, the switch from PIs to an NNRTI could fail for a number of reasons, including NNRTI-associated toxicity and emergence of NNRTI-resistant variants.OBJECTIVE:To describe the virologic failures among 74 HIV-infected patients who switched from PIs to nevirapine.METHODS:Virologic failure was defined as any rebound of the plasma HIV-RNA (pVL) levels >1000 copies/mL on one occasion or 2 consecutive intermittent viremia episodes defined as increases of the pVL >20 copies/mL but <1000 copies/mL. Virolog…
Oral delivery of homologous and heterologous strains of rotavirus to BALB/c mice induces the same profile of cytokine production by spleen cells.
Abstract In this work, we wanted to clarify if differences in antibody (Ab) and particularly in secretory immunoglobulin A (IgA) responses following homologous or heterologous rotavirus infection could be explained by different priming of specific T helper (Th) cells. We compared the Ab responses from suckling BALB/c mice orally inoculated with either a heterologous simian (SA11) or bovine (RF) rotavirus or a homologous murine rotavirus (EHP w ), as well as the profile of cytokines produced by spleen cells after in vitro restimulation. Oral inoculation of EHP w and SA11 induced a similar pattern of Ab with mucosal and serum IgA associated with serum IgG with equal levels of IgG1 and IgG2a, …
Endoplasmic Reticulum Chaperones in Viral Infection: Therapeutic Perspectives
SUMMARY Viruses are intracellular parasites that subvert the functions of their host cells to accomplish their infection cycle. The endoplasmic reticulum (ER)-residing chaperone proteins are central for the achievement of different steps of the viral cycle, from entry and replication to assembly and exit. The most abundant ER chaperones are GRP78 (78-kDa glucose-regulated protein), GRP94 (94-kDa glucose-regulated protein), the carbohydrate or lectin-like chaperones calnexin (CNX) and calreticulin (CRT), the protein disulfide isomerases (PDIs), and the DNAJ chaperones. This review will focus on the pleiotropic roles of ER chaperones during viral infection. We will cover their essential role …
Antibody inhibition of the transcriptase activity of the rotavirus DLP: a structural view.
On entering the host cell the rotavirus virion loses its outer shell to become a double-layered particle (DLP). The DLP then transcribes the 11 segments of its dsRNA genome using its own transcriptase complex, and the mature mRNA emerges along the 5-fold axis. In order to better understand the transcription mechanism and the role of VP6 in transcription we have studied three monoclonal antibodies against VP6: RV-238 which inhibits the transcriptase activity of the DLP; and RV-133 and RV-138 which have no effect on transcription. The structures obtained by cryo-electron microscopy of the DLP/Fab complexes and by X-ray crystallography of the VP6 trimer and the VP6/Fab-238 complex have been co…
Rotavirus 2/6 virus-like particles administered intranasally in mice, with or without the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin, induce a Th1/Th2-like immune response
ABSTRACTWe investigated the rotavirus-specific lymphocyte responses induced by intranasal immunization of adult BALB/c mice with rotavirus 2/6 virus-like particles (2/6-VLPs) of the bovine RF strain, by assessing the profile of cytokines produced after in vitro restimulation and serum and fecal antibody responses. The cytokines produced by splenic cells were first evaluated. Intranasal immunization with 50 μg of 2/6-VLPs induced a high serum antibody response, including immunoglobulin G1 (IgG1) and IgG2a, a weak fecal antibody response, and a mixed Th1/Th2-like profile of cytokines characterized by gamma interferon and interleukin 10 (IL-10) production and very low levels of IL-2, IL-4, and…
Distribution and phenotype of rotavirus-specific B cells induced during the antigen-driven primary response to 2/6 virus-like particles administered by the intrarectal and the intranasal routes.
AbstractSelection of mucosal sites is an important step in mucosal vaccine development. The intrarectal (IR) route represents an alternative to the oral route of immunization; nevertheless, immune responses induced by this route are not well defined. Here, we studied the early primary B cell response (induction, homing, and phenotype) induced by IR immunization with rotavirus (RV)-2/6 virus-like particles (VLP). Using flow cytometry, we traced RV-specific B cells in different lymphoid tissues and analyzed the expression of α4β7 and CCR9, which are important receptors for homing to the gut, as well as CD5, a marker expressed by B1-a cells, which are a major source of natural antibodies. We o…
The HSP90 inhibitor, 17AAG, protects the intestinal stem cell niche and inhibits graft versus host disease development.
IF 7.932; International audience; Graft versus host disease (GvHD), which is the primary complication of allogeneic bone marrow transplantation, can alter the intestinal barrier targeted by activated donor T-cells. Chemical inhibition of the stress protein HSP90 was demonstrated in vitro to inhibit T-cell activation and to modulate endoplasmic reticulum (ER) stress to which intestinal cells are highly susceptible. Since the HSP90 inhibitor 17-allylamino-demethoxygeldanamycin (17AAG) is developed in clinics, we explored here its ability to control intestinal acute GvHD in vivo in two mouse GvHD models (C57BL/6 -> BALB/c and FVB/N -> Lgr5-eGFP), ex vivo in intestine organoids and in vitro in …
Cholera-Like Enterotoxins and Regulatory T cells
Cholera toxin (CT) and the heat-labile enterotoxin of E. coli (LT), as well as their non toxic mutants, are potent mucosal adjuvants of immunization eliciting mucosal and systemic responses against unrelated co-administered antigens in experimental models and in humans (non toxic mutants). These enterotoxins are composed of two subunits, the A subunit, responsible for an ADP-ribosyl transferase activity and the B subunit, responsible for cell binding. Paradoxically, whereas the whole toxins have adjuvant properties, the B subunits of CT (CTB) and of LT (LTB) have been shown to induce antigen specific tolerance when administered mucosally with antigens in experimental models as well as, rece…
Crystallization and Preliminary X-Ray Analysis of Rotavirus Protein VP6
ABSTRACT As a first step to gain insight into the structure of the rotavirus virion at atomic resolution, we report here the expression, purification, and crystallization of recombinant rotavirus protein VP6. This protein has the property of polymerizing in the form of tubular structures in solution which have hindered crystallization thus far. Using a combination of electron microscopy and small-angle X-ray scattering, we found that addition of Ca 2+ at concentrations higher than 100 mM results in depolymerization of the tubes, leading to an essentially monodisperse solution of trimeric VP6 even at high protein concentrations (higher than 10 mg/ml), thereby enabling us to search for crysta…
Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion
The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple-layered viral capsid. Here we present the crystal structure of VP6 determined to 2 A resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal beta-barrel domain and a proximal alpha-helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping …
Unexpected Modulation of Recall B and T Cell Responses after Immunization with Rotavirus-like Particles in the Presence of LT-R192G
LT-R192G, a mutant of the thermolabile enterotoxin of E. coli, is a potent adjuvant of immunization. Immune responses are generally analyzed at the end of protocols including at least 2 administrations, but rarely after a prime. To investigate this point, we compared B and T cell responses in mice after one and two intrarectal immunizations with 2/6 rotavirus-like particles (2/6-VLP) and LT-R192G. After a boost, we found, an unexpected lower B cell expansion measured by flow cytometry, despite a secondary antibody response. We then analyzed CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) and CD4(+)CD25(+)Foxp3(-) helper T cells after in vitro (re)stimulation of mesenteric lymph node cells …
Voyage d’étude autour de la pratique officinale suisse
International audience; Twenty-seven students from the Dijon health sciences training and research unit in the 6th year of their course and specialising in the community setting had the opportunity to undertake a study trip to meet Swiss colleagues. They discovered an innovative vision of pharmacy practice. The results of a feedback questionnaire highlighted the benefit of this pedagogical approach for the construction of the professional identity of these future community pharmacists.; L’intégration d’un voyage d’étude dans le cursus pharmaceutique parcours “officine” a permis à 27 étudiants de 6e année de l’unité de formation et de recherche des sciences de santé de Dijon d’aller à la ren…
Implication des pharmaciens en maisons de santé, résultats d’une enquête
Resume Ces dernieres annees, de nombreux pharmaciens d’officine se sont engages dans des actions interprofessionnelles en soins de premier recours. Dans certains pays, ces dernieres ont fait l’objet d’une evaluation et sont reconnues en tant qu’actes pharmaceutiques pris en charge. En France, l’implication des pharmaciens dans ces actions etait jusqu’alors meconnue. Une enquete realisee aupres de professionnels œuvrant au sein de maisons de sante permet de dresser un premier bilan.
Molecular Cloning, Expression, Self-Assembly, Antigenicity, and Seroepidemiology of a Genogroup II Norovirus Isolated in France
ABSTRACT Virus-like particles of Dijon171/96 virus, a genogroup II norovirus, were expressed in a baculovirus system and were used for a seroepidemiological study of 1,078 age-stratified human sera collected in Dijon, France. The results showed a seroprevalence of 74.1%. Furthermore, we showed that murine antibodies generated against recombinant Dijon171/96 virus, and human antibodies recognized discontinuous epitopes on the particles.
In vitro reconstitution of rotavirus transcriptional activity using viral cores and recombinant baculovirus expressed VP 6
International audience; Purified baculovirus-expressed group A rotavirus VP6 polypeptide was shown to be active in the recovery of the transcriptase activity associated with the reconstitution of the single-shelled rotavirus particle. Recombinant VP6 polypeptide was able to restore the transcriptional activity in purified viral cores from both SA-11 and RF rotavirus strains. Recombinant group C VP 6 (Cowden strain) is capable of binding as a trimer to group A viral core particles but unable to restore the transcriptase activity, suggesting that the binding of the polypeptide to cores is not the only requirement to restore the transcriptase activity. The VP 6 group A polypeptide was shown to…
Detection and characterization of Human caliciviruses associated with sporadic acute diarrhea in adults in Djibouti (horn of Africa).
International audience; Recent advances in molecular diagnostics have allowed us to recognize Human caliciviruses (HuCVs) as important agents of acute diarrhea in industrialized countries. Their prevalence and genetic diversity in developing countries remains unknown. We report on the characterization of HuCVs among adults presenting acute diarrheas in Djibouti; 108 stool samples collected were screened by EIA, RTPCR, or cell cultures for the group A Rotaviruses, Adenoviruses, Astroviruses, and HuCVs, which were further characterized by genotyping. Among stool samples screened for HuCVs, 25.3% were positive. The other enteric viruses were less prevalent. The 11 HuCV strains sequenced reveal…
CCDC 1476682: Experimental Crystal Structure Determination
Related Article: Nicolas Sok, Isabelle Baglin, Christelle Basset, Fatima Fakkor, Evelyne Kohli, Yoann Rousselin, Claire Bernhard, Frédéric Boschetti, Christine Goze, Franck Denat|2017|RSC Advances|7|28291|doi:10.1039/C7RA04218C
CCDC 1476683: Experimental Crystal Structure Determination
Related Article: Nicolas Sok, Isabelle Baglin, Christelle Basset, Fatima Fakkor, Evelyne Kohli, Yoann Rousselin, Claire Bernhard, Frédéric Boschetti, Christine Goze, Franck Denat|2017|RSC Advances|7|28291|doi:10.1039/C7RA04218C
CCDC 1476681: Experimental Crystal Structure Determination
Related Article: Nicolas Sok, Isabelle Baglin, Christelle Basset, Fatima Fakkor, Evelyne Kohli, Yoann Rousselin, Claire Bernhard, Frédéric Boschetti, Christine Goze, Franck Denat|2017|RSC Advances|7|28291|doi:10.1039/C7RA04218C