[Characterisation of viral agents with potential to cause diarrhea in Djibouti]. / Caractérisation des virus agents potentiels des diarrhées chez l'adulte à Djibouti.

Due to limited laboratory facilities in the tropics, the exact role of enteric viruses in causing diarrhea among adults in the tropics is unknown. The purpose of this report is to describe a multicenter study undertaken in Djibouti to determine the prevalence of a large panel of enteric viruses using immunochromatography; antigenic detection by ELISA, RT-PCR cellular inoculation, sequence analysis; and indirect serology. Study samples were collected from 108 patients presenting acute and sporadic diarrhea. Although they are well known causes of diarrhea in children, rotavirus and adenovirus were identified in only 2 and 5% of adults respectively. In contrast human caliciviruses (HuCVs) and enterovirus were identified in 25 and 42% of adult cases respectively. Uncommon genotypes of HuCVs and recombinant forms (junction pol/l cap) as well as a significant number of sapovirus (30%) were identified. Further study is needed to clarify the role of enterovirus (echovirus) in the etiology of acute diarrhea in adults. No polivirus was identified. These new data from the Horn of Africa increase our knowledge about the epidemiology of acute infectious diarrhea that is a major public health problem and potential danger for travelers.

[Investigation of an outbreak of norovirus gastroenteritis in a geriatric hospital]. / Investigation d'une épidémie de gastro-entérite à norovirus dans un hôpital gériatrique.

In aged-care facilities, gastroenteritis outbreaks are responsible for big trouble in the management of cares to the elderly. In November 2002, a gastroenteritis outbreak was observed in 5 of the 6 wards of the geriatric hospital La Charité, University Hospital of Saint-Etienne, France, with an attack rate of 38.5% in the elderly (70 infected from 182 patients) and of 26.0% in the nursing staff (40 infected from 154 agents). The outbreak lasted 30 days with a peak corresponding to 79.8% of the cases between the 11(th) and the 20(th) of November. The first cases were observed in the two short-term-care wards; then, the outbreak spread rapidly to 3 of the 4 long-term care units. Health care workers were contaminated later than the elderly (P < 0.001 by Kruskal-Wallis test). A self-administered questionnaire was documented by most of the nursing staff; the most frequently observed clinical symptoms in this population were nausea (82.5%), abdominal pain (80.0%), diarrhoea (70.5%), asthenia (67.5%) and vomiting (62.5%). Thirty-five percent of the health care workers ceased their work. The causative agent of the gastroenteritis was identified by RT-PCR in the stools of 5 aged persons as a norovirus close to the Lordsdale strain (genogroup II). These findings illustrate the respective role of elderly and health care workers in the spread of the gastroenteritis outbreak inside the geriatric hospital.

The HSP90 inhibitor, 17AAG, protects the intestinal stem cell niche and inhibits graft versus host disease development.

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 intestinal epithelial cultures. We show that 17AAG decreases GvHD-associated mortality without impairing graft versus leukemia effect. While 17AAG effect in T-cell activation is just moderate at the dose used in vivo, we observe a striking intestinal integrity protection. At the intestine level, the drug promotes the splicing of the transcription factor X-box binding protein 1 (XBP1), which is a key component of the ER stress. This effect is associated with a decrease in intestinal damage and an increase in Lgr5(+) stem cells, Paneth cells and defensins production. The importance of XBP1 splicing control is further confirmed in cultured cells and organoids of primary intestinal epithelium where XBP1 is either shRNA depleted or inhibited with toyocamycin. In conclusion, 17AAG has a protective effect on the epithelial intestinal barrier in mouse models of acute GvHD. This compound deserves to be tested in the therapeutic control of acute GvHD.

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 combined to give pseudo-atomic structures. Steric hindrance between the Fabs results in limited Fab occupancy. In particular, there are on average only three of a possible five Fabs-238 which point towards the 5-fold axis. Thus, Fabs-238 are not in a position to block the exiting mRNA, nor is there any visible conformational change in VP6 on antibody binding at a resolution of 23 A. However, the epitope of the inhibiting antibody involves two VP6 monomers, whereas, those of the non-inhibiting antibodies have an epitope on only one VP6. Thus, the inhibition of transcription may be a result of inhibition of a possible change in the VP6 conformation associated with the transcription of mRNA.

Microsomal acetoxy drug: protein transacetylase of placenta: part 1. Characterization of DAMC: GST transacetylase.

We have earlier established in tissues of several mammalian animal species the existence of a novel membrane bound enzyme termed 7,8-diacetoxy-4-methylcoumarin (DAMC): protein transacetylase (TAase) that possibly transfers acetyl groups from the model acetoxy drug (DAMC) to certain enzyme protein viz. glutathione S-transferase (GST), cytochrome P-450 and NADPH cytochrome C reductase leading to the drastic modulation of their catalytic activities. We have in this report extended the studies to human tissue and characterized TAase from placenta. For this purpose placental microsomes were preincubated with DAMC along with the receptor protein (cytosolic GST) followed by the addition of the substrates of GST in order to quantify the catalytic activity of GST, the extent of inhibition of GST served as a measure of TAase. Placental TAase was also found to irreversibly activate NADPH cytochrome C reductase by DAMC. Placental enzyme activated the reductase even at very low concentration of DAMC. Iodoacetamide nearly abolished the placental TAase suggesting the presence of active thiol group in the enzyme and the TAase demonstrated hyperbolic kinetics. Kinetic constants obtained by varying the concentrations of either of the substrates DAMC or cytosolic GST characterized TAase catalysed reaction as the bimolecular reaction. Further studies are in progress to delineate the physiological importance of TAase in placenta.

Acetoxy-4-methylcoumarins confer differential protection from aflatoxin B(1)-induced micronuclei and apoptosis in lung and bone marrow cells.

The ability of various acetoxy derivatives of 4-methylcoumarins to inhibit the genotoxic changes due to aflatoxin B(1) (AFB(1)) is reported here. Several 4-methylcoumarins (test compounds), such as 7,8-diacetoxy-4-methylcoumarin (DAMC), monoacetoxy-4-methylcoumarin (MAC), 5-N-acetyl-6-acetoxy-4-methylcoumarin (NAMC) and 7,8-dihydroxy-4-methylcoumarin (DHMC) were separately administered intraperitoneally (i.p.) to male wistar rats followed by AFB(1) administration i.p. or intratracheally (i.t.) (2-8 mg/kg b.wt.) and another dose of the test compound. The animals were sacrificed 26h after AFB(1) administration. From animals receiving AFB(1) i.p., bone marrow (BM) cells were isolated and stained with Mayer's haematoxylin and eosin. Micronuclei (MN) in BM were scored by light microscopy. From animals receiving AFB(1) i.t., bronchoalveolar lavage (BAL) was obtained, lung cells (LG) were isolated and stained with fluorochrome 6-diamidino-2-phenylindole (DAPI) for the analysis of MN, apoptotic bodies (AP) and cell cycle variations. Rats were separately treated with the vehicle DMSO to serve as the proper control. AFB(1) caused significant dose-dependent induction of MN in BM as well as LG. AP were observed in LG of rats receiving AFB(1) and was found to correlate with MN induction. DAMC injection caused significant decrease in AP due to AFB(1) in LG and MN in both BM and LG. The effectiveness of MAC was approximately half that of DAMC, thereby indicating that number of acetoxy groups on the coumarin molecule determine the efficacy. The fact that NAMC had no effect either on MN or AP indicate that neither acetoxy group at C-6 nor the N-acetyl group at C-5 facilitate the transfer of acetyl group to P-450 required for inhibition of AFB(1)-epoxidation. DHMC, the deacetylated product of DAMC had no normalizing effect on the induction of MN and AP. These findings confirm our earlier hypothesis that DAMC-mediated acetylation of microsomal P-450 (catalysing epoxidation of AFB(1)) through the action of microsomal transacetylase is responsible for the protective action of DAMC. The relative number and position of acetoxy groups on the coumarin nucleus determine the specificity to the transacetylase necessary for the chemopreventive action.

Norovirus foodborne outbreaks associated with the consumption of oysters from the Etang de Thau, France, December 2002.

In January 2003, the Institut de Veille Sanitaire received notification of clusters of gastroenteritis (GE) thought to be associated with consumption of oysters harvested from Etang de Thau in the south of France. At the same time Italy reported an outbreak (200+ cases) associated with oysters from the Etang de Thau. An investigation was carried out to determine the source and vehicle of the outbreaks. Descriptive analysis of reported clusters in France, microbiological analysis of stool and oyster samples, genotyping of noroviruses and an environmental investigation of the Etang de Thau were carried out. A retrospective cohort study was also undertaken among those attending a number of family meals in Paris. Thirteen family clusters in four districts of France (69 cases) could be attributed to the consumption of Thau oysters based on descriptive evidence. Oysters distributed at an office in Paris and consumed at fourteen family meals between 19 and 24 December led to a further outbreak. In this outbreak the attack rate was 21/36 (58%) for Thau oyster consumers and 0/22 for non-consumers (p=0.00002). Noroviruses (genogroups I and II) were found in stool samples from four clusters and oysters from three clusters (including Paris). Environmental investigations revealed heavy rainfall, an overflow of a water purification station and faecal contamination of the Etang de Thau in December. Oysters from the Etang de Thau were responsible for a number of clusters of norovirus GE in winter 2002 in France and also in Italy. High Escherichia Coli levels in Thau water and shellfish led to an official request, mid-December, for oyster purification before distribution. This was not possible, due to lack of purification facilities. This investigation has contributed to a change in the way that shellfish harvesting areas are classified in France.

[Fosfomycin-cefotaxime combination in severe staphylococcal infections in newborn infants]. / L'association fosfomycine-céfotaxime dans les infections sévères staphylococciques du nouveau-né.

Clinical and bacteriological effectiveness of the fosfomycin-cefotaxime combination is reported in four cases of serious staphylococcal infections in neonates (1 meningitis, 2 osteomyelitis, 1 superinfection of congenital varicella). Owing to the strong synergistic effect of this combination on methicillin-resistant staphylococcal strains, the authors suggest that the fosfomycin-cefotaxime combination should be considered for anti-staphylococcal therapy in neonates with deep tissue and/or methicillin-resistant infections.

Quantifying Gp96/Grp94 complexes preparations for vaccines: a key step often inaccurate.

Glycoprotein 96 (Gp96)-peptide complexes are highly investigated for vaccines preparation, particularly in cancer therapy. Gp96, formerly called tumor rejection antigen (TRA)-1, is able to elicit an immune response that can protect mice against tumors, when the mice share the same haplotype than those bearing the tumors from which Gp96 was purified. Secreted Gp96-peptide complexes act as danger signals thereby stimulating the innate immunity regardless of the chaperoned peptides. In contrast, the tumor rejection antigen role of Gp96-peptide complexes is held by the chaperoned peptides in a dose-dependent manner. The purification step is crucial both for insuring the quality and the quantity of Gp96-peptide complexes. To this aim, different methods have been described but they are often suboptimal for the quantification of these complexes. In this review, we discuss a hot topic: the use of the chaperone Gp96 for vaccination purposes in cancer, and also detail the current methods for quantifying Gp96-peptide preparations.

Characterization of new recombinant noroviruses.

Noroviruses are important etiologic agents of acute gastroenteritis and show great genetic diversity. To characterize more fully previously detected strains that could not be assigned unequivocally to one particular genotype based on the RNA polymerase, we have sequenced a region in the capsid gene and, in some cases, in the junction between open reading frame 1 (ORF 1) and ORF 2. The results allowed us to identify several recombinant noroviruses: GGIIb viruses were detected for the first time in France in August 2000 and then spread through France and to Europe during the following winter. Here we present the characterization of three other probable GII recombinants which showed different phylogenetic positions depending on their ORF 1 and ORF 2 sequences. Analysis of the region located between ORF 1 and ORF 2 by a nucleotide identity window search showed a sudden shift in similarities. Moreover, recombination breakpoints were identified upstream and downstream of the beginning of ORF 2 by using a statistical test, thus confirming the involvement of this region in recombination. Unlike GGIIb, the three recombinants described here do not seem to have diffused widely in the community: one was found in a waterborne outbreak, and the other two were found in sporadic cases. Recombination is important for the evolution of RNA viruses and has already been described for noroviruses. Our results suggest that recombination is not a rare phenomenon among noroviruses, but not all these presumed recombinants that formed during RNA replication are able to spread widely.

Molecular epidemiology of caliciviruses detected in sporadic and outbreak cases of gastroenteritis in France from December 1998 to February 2004.

We compiled sequence and epidemiological data from 172 caliciviruses detected in France from December 1998 to February 2004 in sporadic and outbreak cases. The results showed a cocirculation of strains with a majority of genogroup II (GII) noroviruses. Three groups of noroviruses, not detected before in our laboratory, emerged and spread during the period: the recombinant GGIIb and Norwalk-related strains not amplified in the polymerase gene in 2000 and a new Lordsdale variant in 2002. We observed that (i) GII-4 noroviruses were predominant in nursing home and hospital outbreaks but rare in oyster- and water-related outbreaks despite continuous circulation in the population; (ii) at the opposite, genogroup I strains were detected in the majority of environmental outbreaks; (iii) several strains were frequently found in oyster- and water-linked outbreaks (up to seven), whereas one single strain was detected when transmission was from person to person; and (iv) whereas GII noroviruses were predominant in sporadic cases where patients were under 15 years of age, GI strains were more frequent in outbreaks occurring in this age group. Finally, from a methodology point of view, this compilation shows that detection and characterization in the polymerase gene are not adequate in a significant number of cases and should be completed by amplification and sequencing in the capsid gene.

Multicentre evaluation of a new commercial latex agglutination test using a monoclonal antibody for rotavirus detection.

A new commercial latex-agglutination test using a monoclonal antibody for detection of rotavirus (Slidex Rota-Kit 2) was compared with three other tests (Slidex Rota-Kit, Rotalex, Rotazyme II) using immunoelectron microscopy and a monoclonal enzyme immunoassay as reference tests. Slidex Rota-Kit 2 was more sensitive and specific than the other tests, and would thus appear to be a practical and accurate rotavirus assay for use in routine laboratory work.

Identification of T-cell epitopes adjacent to neutralizing antigenic domains on the fusion protein of respiratory syncytial virus.

F glycoprotein has been identified as an important target structure in the immunological response following respiratory syncytial virus (RSV) infection. Two sequential B epitopes corresponding to amino acids 200 to 225 and 255 to 278 have already been defined with anti-RSV rabbit serum. The T helper response to peptides which belong to these sequences was investigated in this study. Proliferative T-cell responses to these peptides were analysed in BALB/c mice (H-2d) and others strains: SJL (H-2s), C3H/He (H-2k), B10.BR (H-2k) and C57BL/6 (H-2b). By using various strategies, two T-cell epitopes were identified in the amino acid 200 to 225 and 255 to 278 regions, close to a neutralizing epitope. The T-cell responses to these peptides were H-2- restricted. In addition, the peptide 255-278 was able to stimulate T cells that responded to a subsequent immunization with F glycoprotein in vitro.

Epitope mapping of the major inner capsid protein of group A rotavirus using peptide synthesis.

Three hundred and ninety-one consecutive heptapeptides derived from the VP6 protein of bovine rotavirus (397 AA) were synthesized using the "pepscan" method and were assayed on the synthesis pins with monoclonal antibodies to VP6. Heptapeptides reactive with MAbs were located in four main regions: regions AA 32-64, AA 155-167, AA 208-274, and a fourth region at the C-terminal, from AA 380 to AA 397. Among these regions, two sequences were also reactive with the MAbs when longer peptides were assayed. The sequence located between AA 58 and AA 62 (NWNFD), recognized by MAbs RV-1026, RV-50, and RV-443, was previously reported. A new site was defined in the region essential for trimerization, between AA 159 and AA 165 (PYSASFT), which was recognized by MAbs RV-133 and RV-138.

[Combination between fosfomycin and oxacillin or cefotaxime against methicillin-resistant Staphylococci and Enterococci]. / Associations entre la fosfomycine et l'oxacilline ou le céfotaxime chez les staphylocoques méthicilline-résistants et les entérocoques.

The resistance of Gram positive cocci to oxacillin and cephalosporins do not appear to be in relation with the synthesis of a beta-lactamase, thus fosfomycin (FOS) could enhance the action of beta-lactam antibiotics by blocking another stage of the peptidoglycan synthesis. When FOS is combined with oxacillin (OXA) or cefotaxime (CTX) against fosfomycin sensitive strains (25 S. aureus 4-16 mg/l, 20 S. epidermidis 2-32 mg/l and 20 Enterococci 16-64 mg/l) one can see a dramatic synergistic effect of these two combinations. Respectively with S. aureus, S. epidermidis and Enterococci, FIC indices are 0.17-0.39 and 0.43 for FOS-OXA and 0.29-0.47 and 0.29 for FOS-CTX. If one considers the CSF concentrations of these three antibiotics, the combination of fosfomycin with oxacillin or cefotaxime may be used in the treatment of meningitis due to methicillin resistant Staphylococci.

[In vitro evaluation of moxalactam on enterobacteria producing beta-lactamases]. / Evaluation in vitro de moxalactam sur les entérobactéries productrices de bêta-lactamases.

We have studied strains of enterobacteria which are resistant to first generation cephalosporins, in relation to their cephalosporinase production. Therefore, this is not an epidemiological study. For these selected strains, if a difference in MIC greater than or equal to 2 base 2 logarithms is considered to be significant, Moxalactam appears as more potent than cefotaxim. An appropriate study shows that these findings are related to the production of cephalosporinase. If very little cephalosporinase is produced, cefotaxim is more potent, implying better accession to the lethal target of the bacteria. Conversely, when cephalosporinase production is higher, Moxalactam is more potent. Moxalactam can be shown to be more resistant to hydrolysis by cephalosporinases.

Oral delivery of homologous and heterologous strains of rotavirus to BALB/c mice induces the same profile of cytokine production by spleen cells.

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 (EHPw), as well as the profile of cytokines produced by spleen cells after in vitro restimulation. Oral inoculation of EHPw and SA11 induced a similar pattern of Ab with mucosal and serum IgA associated with serum IgG with equal levels of IgG1 and IgG2a, whereas RF elicited a weak humoral response. We found that these strains induced the same mixed Th1/Th2 pattern of cytokine production by spleen cells with IFN-gamma and IL-5 as well as IL-10, but not IL-2 or IL-4. These findings suggest that the induction of immune response is probably not different between these strains. Other factors such as the amount of antigen, strain immunogenicity, and other cytokines, particularly produced in effector sites, remain to be considered in order to better explain the differences in secretory IgA following homologous or heterologous rotavirus infection.

Bactericidal activity of cefotaxime and fosfomycin in cerebrospinal fluid during the treatment of rabbit meningitis experimentally induced by methicillin-resistant Staphylococcus aureus.

The purpose of this study was to evaluate the therapeutic effect of cefotaxime (CTX) and fosfomycin (FOS), alone or in combination, in an experimental meningitis, with cerebrospinal fluid (CSF) concentrations of the two antibiotics reproducing those obtained in human CSF during bacterial meningitis. With a dose of 50 mg/kg of CTX and 100 mg/kg of FOS injected i.v. (CTX over 0.5 h and FOS over 3 h), CSF concentrations were comparable to those observed in man. In a series of five rabbits per treatment group, the bacterial population was counted before and after treatment (two doses with a six-hour interval) with CTX, FOS or CTX + FOS (CTX over 0.5 h before the end of FOS infusion). By the 12th hour of treatment, the percentage of bacteria surviving in CSF compared to the initial population was 4.35% for CTX, 0.20% for FOS and 0.19% for CTX + FOS. Thus, it seemed that CTX + FOS was not more active than FOS alone. In another series of four rabbits per group, the bactericidal effect was followed at T0, T6, T12, T24 and T48 after treatment (two doses with a six-hour with a six-hour interval). With CTX, a variable drop in bacterial count from one rabbit to the other occurred during the first 12 h, and then a bacteriostasis followed. With FOS, a quick bactericidal effect was observed during the first 12 h, becoming slower during the following 36 h (0.03% of bacteria surviving at the 48th hour). With CTX and FOS in combination, a quick bactericidal effect was achieved, remaining steady over a 48-hour period (0.001% of bacteria surviving at the 48th hour).