Due to the persisting threat of development of new highly pathogenic influenza A subtypes, a mucosal vaccination which would induce a potent and cross-protective reaction is desirable. We succeeded in mucosal immunization of mice with an inactivated influenza A virus by using delipidated Bacillus firmus (DBF) as adjuvant. The mechanism of adjuvant effect was followed in NALT by comparing the response after intranasal immunization by inactivated influenza virus type A (H1N1) alone, adjuvant alone (DBF), or by a mixture of virus+DBF. Expression of selected gene groups was tested via qPCR at 7 different time points: cytokines (IL-2, IFN-gamma, IL-4, IL-6,& IL-10), type I interferons (IFN-alpha4, IFN-alpha11, IFN-alpha12,& IFN-beta), toll-like receptors (TLR2, TLR3, TLR7,& TLR9), iNOS and CCR7. Intranasally administered DBF and the mixture of virus+DBF induced an elevated expression of IFN-gamma, IL-6 and IL-10 cytokines, type I interferons, iNOS, and pDC markers in NALT. Multimarker qPCR data was analyzed by relative quantification and by principal component analysis. DBF has been shown to be a very efficient adjuvant for the stimulation of innate immunity after IN immunization. DBF accelerated, increased, and prolonged the antiviral response.

We have previously generated a transgenic mouse strain (LSL-TbetaRI(CA)) containing a Cre-inducible constitutively active TGFbeta type I receptor (Bartholin L. et al. 2008. Generation of mice with conditionally activated transforming growth factor beta signaling through the TbetaRI/ALK5 receptor, GENESIS). Transgene expression depends on the excision of a floxed-transcriptional STOP (LSL, Lox-STOP-Lox) located upstream the TbetaRI(CA) coding sequence. In order to evaluate the correct excision of the STOP signal in the presence of Cre-recombinase, we developed a rapid screening based on an original PCR genotyping strategy. More precisely, we designed a set of primers flanking the LSL containing region. The size of the amplified products will differ according to recombination status of the LSL-TbetaRI(CA) allele. Indeed, the size of the STOP containing PCR product is 1.93 kb, but is reduced to 0.35 kb when the STOP signal is removed after Cre-mediated recombination. We validated excision in several compartments, including pancreas, liver, T lymphocytes and embryos using different Cre expressing transgenic mouse strains. This represents a simple and efficient way of monitoring the tissue specific recombination of the LSL-TbetaRI(CA) allele.(c) 2010 Wiley-Liss, Inc.

Abstract Dog fleas collected in New Caledonia harbored flea-borne pathogens Rickettsia felis and Bartonella clarridgeiae in 81% and 5%, respectively.

Abstract A precise assessment of the epidemiological extent of equine Lyme disease is not well established in metropolitan France, French Guiana, and Africa (Chad, Djibouti, Ivory Coast, Gabon, and Democratic Republic of Congo). Blood samples were obtained from 570 horses. The samples were tested for Borrelia burgdorferi infection by a commercial ELISA Dot-Blot method (SNAP((R)) 4 Dx; IDEXX S. Laboratory). Lyme disease antibodies were only detected in metropolitan France, specifically in the eastern and center-western regions (48% and 31%). The geographical distribution of the disease follows the distribution of the vector.

Oropharyngeal dysphagia is frequent in stroke patients and increases mortality, mainly because of pulmonary complications. We hypothesized that sensitive transcutaneous electrical stimulation applied submentally during swallowing could help rehabilitate post-stroke oropharyngeal dysphagia by improving cortical sensory motor circuits. Eleven patients were recruited for the study (5 females, 68 +/- 11 years). They all suffered from recent oropharyngeal dysphagia (>eight weeks) induced by a hemispheric (n = 7) or brainstem (n = 4) stroke, with pharyngeal residue and/or laryngeal aspiration diagnosed by videofluoroscopy. Submental electrical stimulations were performed for 1 h every day for 5 days (electrical trains: 5 s every minute, 80 Hz, under motor threshold). During the electrical stimulations, the patients were asked to swallow one teaspoon of paste or liquid. Swallowing was evaluated before and after the week of stimulations using a dysphagia handicap index questionnaire, videofluoroscopy, and cortical mapping of pharyngeal muscles. The results of the questionnaire showed that oropharyngeal dysphagia symptoms had improved (p < 0.05), while the videofluoroscopy measurements showed that laryngeal aspiration (p < 0.05) and pharyngeal residue (p < 0.05) had decreased and that swallowing reaction time (p < 0.05) had improved. In addition, oropharyngeal transit time, pharyngeal transit time, laryngeal closure duration, and cortical pharyngeal muscle mapping after the task had not changed. These results indicated that sensitive submental electrical stimulations during swallowing tasks could help to rehabilitate post-stroke swallowing dysphagia by improving swallowing coordination. Plasticity of the sensory swallowing cortex is suspected.

Three homology models of the human Ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of Rhodopsin (RHO model), Opsin (OPS model) and Beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom Normal Modes Analysis (NMA). A low frequency normal mode (mode 16) perfectly reproduced the intra-cellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures get closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the 7TM segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (CWLP) residue, some features related to GPCRs activation. According to our model, the His280 residue was proposed to stabilize the Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants which were fully functional but sharing an important decrease of their basal activities. Docking performed with short Ghrelin derivatives Gly-Ser-Ser([octa])-Phe-NH(2) and Gly-Ser-Ser([octa])-Phe-Leu-NH(2) further allowed to identify a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 Ghrelin receptor ligands based on the 1,2,4-triazole scaffold. Finally, NMA performed on the obtained peptide:receptor complex suggested a stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along the mode 16 computed for the unbound receptor. Our results show that NMA can be a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.

The routes of transmission of hepatitis E virus (HEV) in industrialised countries are largely unknown, but several studies suggest that HEV can be a porcine zoonosis. The aim of the present study was to determine the prevalence of HEV in the wild boar (Sus scrofa) and to determine the genetic relationships between HEV sequences recovered from wild boars and from domestic pigs and humans. HEV RNA was detected by real time reverse transcriptase PCR in 7/285 (2.5%) liver samples from wild boars hunted in South-Eastern France. HEV sequences were recovered from five wild boars and belonged to genotype 3f. These sequences shared 89-100% nucleotide identity with each other and were genetically close to HEV sequences recovered from humans in Southern France. Wild boars in South-Eastern France may be a source of HEV transmission to humans.

Leishmania infantum leishmaniasis is endemic in south-east of France. The main goal of our study was to evaluate the real prevalence of asymptomatic carriage in dogs by means of real time quantitative PCR (qPCR) and serology. We included prospectively 140 military dogs wearing deltamethrine-impregnated collars. Parasitaemia levels were then measured by means of quantitative real time PCR targeting kinetoplast DNA with TaqMan((R)) chemistry. ELISA and western blotting (WB) were used for serological screening. The number of dogs working in three areas was the following: Var (n=48), Bouches-du-Rhône (n=61) and Corsica (n=31). Prevalence of symptomatic dogs was 0.7%(n=1). ELISA and WB were positive in one (0.71%) and 19 (14%) dogs, respectively. Fifty-eight dogs (41.4%) had a positive parasitaemia. Global prevalence (positive WB and/or positive qPCR) was 50%(n=70). Mean parasitaemia was 0.018parasites/mL in the global population and 0.043parasites/mL in positive dogs [min: 0.0002 to max: 2]. The concordance percent for WB and qPCR results was 55%(n=77). Regarding the prevalence of positive parasitaemia, a significant difference was noticed between dogs living in the Var region and those coming from the two other areas. Parasitaemia was rapidly positive within the first semester of stay in an enzootic area. Despite the use of deltamethrine-impregnated collars, the proportion of dogs with low parasitaemia is important. Thus, it may be relevant to evaluate the effect of screening and treating asymptomatic canine reservoirs on human infection by performing further studies comparing both populations.

[This corrects the article on p. e1000575 in vol. 5, PMID: 19629168.].