Multivariate analysis of the immune response to different rabies vaccines.

In a previous study, we proposed as an alternative to the use of animals in infectious challenge studies, a new approach describing the vaccine-induced immune response through the multivariate analysis of a defined set of immune parameters characterizing the B and T immune responses. This multivariate analysis, i.e. immune fingerprint, was evaluated first to assess the impact of minor changes in well characterized vaccines. The approach showed promising results in the assessment of the compatibility between two licensed vaccines. In the present study, the immune fingerprint was used to compare adjuvants with the various immunological parameters of the immune fingerprint as well as to assess the ability of this approach to discriminate different Rabies vaccine formulations in dogs. RABISIN® was the reference vaccine, adjuvanted with aluminum hydroxide. An exploratory factor analysis was used to analyse the covariance structure of the immunological data. Significant differences were observed between groups. RABISIN and a linear polyacrylate (SPA09) adjuvanted vaccine performed better than chitosan adjuvanted ones, both for humoral and cell immune responses. This study showed that the immune fingerprint approach can be used to screen vaccine formulations. It provides additional information compared to classical vaccination and infectious challenge efficacy study.

A canine vaccine against Leptospira serovars Icterohaemorrhagiae, Canicola and Grippotyphosa provides cross protection against Leptospira serovar Copenhageni.

Efficacy of the Leptospira components of multivalent vaccine DAPPi-L was previously demonstrated against virulent challenge with three serovars of Leptospira interrogans (Canicola, Icterohaemorrhagiae and Grippotyphosa) carried out 14 days after primary vaccination. In this study we demonstrate that this vaccine provides, two weeks after vaccination, an additional protection (prevention of mortality, clinical signs, renal infection, bacterial excretion, renal carriage and renal lesions) against fatal leptospirosis due to Leptospira interrogans serovar Copenhageni (serovar of major medical importance).

Compatibility between a rabies vaccine and a combined vaccine against canine distemper, adenovirosis, parvovirosis, parainfluenza virus and leptospirosis.

In many cicumstances, veterinarians are requiring to be able to administer rabies vaccine in dogs at the same time as vaccinating against canine distemper, adenovirus, parvovirus, parainfluenza virus and leptospirosis. The aim of this study was to assess the compatibility between a multivalent vaccine and a rabies vaccine when injected at two separate sites. Lack of interference was assessed by comparing serological response to viral components during one year following primary vaccination with vaccines administered alone or concomitantly. Antibody response to all tested components was comparable, irrespective of whether vaccines were administered individually or concurrently. Notably, the rabies vaccine induced very strong and protective seroconversion in dogs, whether it was administered concomitantly with the combo vaccine or not. This facilitates administration of rabies vaccine, which is a key factor for controlling the disease.

Use of inactivated bluetongue virus serotype 8 vaccine against virulent challenge in sheep and cattle.

The immunisation properties of an inactivated bluetongue virus serotype 8 (BTV-8) vaccine were evaluated in sheep and cattle. Five sheep were vaccinated with one dose of vaccine and five cattle were vaccinated with two doses 28 days apart. Six sheep and five cattle served as unvaccinated controls. All animals were subjected to a virulent BTV-8 challenge, and safety and antibody responses were monitored. All control animals developed disease and viraemia, while vaccinated animals were clinically protected and viraemia was completely prevented.

Protective duration of immunity of an inactivated bluetongue (BTV) serotype 2 vaccine against a virulent BTV serotype 2 challenge in sheep.

The protective properties of an inactivated bluetongue virus serotype 2 (BTV-2) vaccine were evaluated in sheep. Sheep (two groups of seven), vaccinated with either one or two doses of the vaccine, were monitored for antibody response over one year. All sheep developed high titres of neutralizing antibodies by 35 days after first vaccination and titres were maintained over one year. Control sheep (n=7) remained seronegative until challenge. One year after vaccination, all sheep were inoculated with a virulent BTV-2. All controls developed pyrexia, clinical signs and viraemia. In contrast, the sheep vaccinated with one or two doses of inactivated BTV-2 vaccine were protected from clinical disease and viraemia was completely prevented. These data show that a single dose of the BTV-2 vaccine given to sheep induces a strong immunity which confers protection for at least one year.

Onset and duration of protective immunity against clinical disease and renal carriage in dogs provided by a bi-valent inactivated leptospirosis vaccine.

Protection against clinical disease and prevention of the renal carrier state remain the key objectives of vaccination against leptospirosis in the dog. In the present paper, groups of dogs were vaccinated twice with a commercial bacterin (EURICAN L) containing Leptospira interrogans serovars icterohaemorrhagiae and canicola and challenged with heterologous representatives of both serovars at 2 weeks (onset of immunity) or 14 months (duration of immunity) after the second vaccination. Control dogs were not vaccinated against leptospirosis and kept with the vaccinated dogs. The challenges, irrespective of the serovar, reliably produced clinical signs consistent with Leptospira infection in the control pups with up to 60% mortality. As expected clinical disease in the adult controls was less severe, but we were able to induce morbidity and mortality as well. Under these extreme challenge conditions, clinical signs in the vaccinated dogs were rare, and when observed, mild and transient in nature. Following experimental infection, 100% of the control pups and 83% of the adult controls became renal carriers. Despite the heavy challenges, none of the 18 vaccinated puppies (onset of immunity studies) and only 2 out of the 16 vaccinated adult dogs (duration of immunity studies) developed a renal carrier state. These results show that a primary course of two doses of EURICAN L provided quick onset and long-term protection against both clinical leptospirosis and the renal carrier stage. This vaccine should provide veterinarians with a powerful tool to prevent clinical disease in dogs and zoonotic transmission of leptospirosis to humans.

Comparison of antibody responses after vaccination with two inactivated rabies vaccines.

Thirty laboratory dogs were randomly assigned to two groups (A and B) of 15 dogs and subcutaneously vaccinated with a single dose of one of two commercially available monovalent inactivated rabies vaccines: RABISIN (Merial, France) (group A) and NOBIVAC Rabies (Intervet International) (group B). Rabies antibodies were measured over a period of 4 months using the fluorescent antibody virus neutralization (FAVN) test. The two vaccines performed differently in terms of magnitude and persistence of rabies antibodies titers in dogs. Two weeks after vaccination, average rabies antibody titers peaked at 2.53 IU/mL (range, 0.17-13.77 IU/mL) and 1.26 IU/mL (range, 0.50-4.56 IU/mL) in groups A and B dogs, respectively. The average FAVN antibody titres against rabies on D28, D56, D84, D112 and D120 were significantly higher in group A than in group B. Although all dogs from group B serologically responded to vaccination, the proportion of dogs with antibody titres >or=0.5 IU/mL dropped significantly after D28 and was statistically significantly lower on D56, D84 and D112 compared to group A dogs. In conclusion, in the context of international trade, the choice of the vaccine and the timing of blood tests are critical factors in achieving successful serological test results after rabies vaccination. RABISIN induces high and sustained antibody titres against rabies, increasing the flexibility for the time of blood sampling after primo-vaccination.

[Prevalence of obesity in children: study in the primary public Parisian schools]. / Prévalence de l'obésité chez l'enfant: étude dans les écoles primaires publiques parisiennes.

Obesity is an important risk factor in public health. In Paris, few statistical data are available in this area. The purpose of the present study is to evaluate the prevalence of overweight and obesity in 10 years-old children attending Paris elementary schools (cours moyen deuxième année--CM2--last level of the elementary school). 148 classes were randomly selected, gathering 3,621 schoolchildren 10 years 6 months old. 66 doctors in charge of health at school participated in the study, doing the measurements of weight, size and collecting also the weight and size at birth and at the "grande section-GS-level" (last level of the infant school, 5 years-old children) from the individual health file of the schoolchildren. The statistical analysis was based on the study of distributions of the observed Quetelet index (Q0) at the different ages, compared to French reference curves. A logistic regression analysis was performed to determine whether birth weight and GS weight predict obesity in CM2. In GS and in CM2, observed Quetelet indices are over expected values: in CM2, 22.8% of boys and 25.6% of girls exceed the reference value Q90; the prevalence of obesity (Q0 > or = Q97) is 13.4% in boys and 13.5% in girls. Among the variables "term", "weight at birth", weight in GS level and "gender", the weight in GS level is the only predictive factor of obesity in CM2 level. The situation in Paris appears to be serious. Preventive actions are needed at early stages to try to stop and, if possible, to reverse the present increase of overweight. In this context, school doctors have to play a prominent role.

Characterization of the epstein-barr virus BRRF1 gene, located between early genes BZLF1 and BRLF1.

The switch from latency to a productive cycle in Epstein-Barr virus (EBV)-infected B cells proliferating in vitro is thought to be due to the transcriptional activation of two viral genes, BZLF1 and BRLF1, encoding two transcription factors called EB1 and R respectively. However, a third gene, BRRF1 is contained in the BZLF1/BRLF1 locus, overlapping with BRLF1 but in inverse orientation. We have characterized the 5' end of the BRRF1 mRNA and the promoter, PNa, at which BRRF1 pre-mRNA is initiated. We show that although a single BRRF1 mRNA species is induced by 12-O-tetradecanoylphorbol 13-acetate/sodium butyrate in several EBV-infected B cell lines, in Akata cells treated with anti-IgG two BRRF1 mRNAs can be detected. Transcription initiated at the BRRF1 promoter was activated by EB1 but not by R, and EB1-binding sites which contribute to the EB1-activated transcription have been mapped to between positions -469 and +1. A 34 kDa protein could be translated from the BRRF1 mRNA both in vitro and in vivo, and was found predominantly in the nucleus of HeLa cells transfected with a BRRF1 expression vector. Thus there are three promoters in the region of the EBV chromatin containing the BZLF1/BRLF1 genes, two of which, PZ and PNa, potentially share regulatory elements.

Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/AKT signaling pathway.

The RET gene codes for a receptor tyrosine kinase that plays a crucial role during the development of both the enteric nervous system and the kidney. Germ line missense mutations at one of six codons specifying extracytoplasmic cysteines are responsible for two related cancer disorders as follows: multiple endocrine neoplasia type2A (MEN2A) and familial medullary thyroid carcinoma (FMTC). MEN2A and FMTC mutations result in a constitutive catalytic activity and as a consequence convert RET into a dominantly acting transforming gene. Although it has been shown that RET-MEN2 mutants activate several transduction pathways, their respective contribution to the neoplastic phenotype remains poorly understood. Over the past few years, it has become increasingly clear that the transforming ability of several viral and cellular oncoproteins depends on their capacity to activate phosphatidylinositol 3-kinase (PI3K). We now report that RET carrying a representative MEN2A mutation at Cys-634 (termed RET-MEN2A) activates PI3K and its downstream effector, the serine/threonine kinase AKT/protein kinase B. Previous studies have demonstrated that mutation of Tyr-1062, which is the intracellular docking site for Shc and Enigma on RET, abolishes the RET-MEN2A transforming activity. We provide evidence that mutation of Tyr-1062 abrogates the binding of the p85 regulatory subunit of PI3K to RET-MEN2A and the subsequent stimulation of the PI3K/AKT pathway. Furthermore, infection of rat fibroblasts with a retrovirus expressing a dominant-interfering form of PI3K suppresses RET-MEN2A-dependent transformation, whereas overexpression of AKT enhances the RET-MEN2A oncogenic potential. In summary, these data are consistent with the notion that RET-mediated cell-transforming effect is critically dependent on the activation of the PI3K/AKT pathway.

Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines.

The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations affecting one of five cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and affected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disulfide homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent fibroblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These findings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating effects.