Early development of the digestive tract (pharynx and gut) in the embryos and pre-larvae of the European sea bass Dicentrarchus labrax.

The European sea bass Dicentrarchus labrax is a marine teleost important in Mediterranean aquaculture. The development of the entire digestive tract of D. labrax, including the pharynx, was investigated from early embryonic development to day 5 post hatching (dph), when the mouth opens. The digestive tract is initialized at stage 12 somites independently from two distinct infoldings of the endodermal sheet. In the pharyngeal region, the anterior infolding forms the pharynx and the first gill slits at stage 25 somites. The other three gill arches and slits are formed between 1 and 5 dph. Posteriorly, in the gut tube region, a posterior infolding forms the foregut, midgut and hindgut. The anus opens before hatching, at stage 28 somites. Associated organs (liver, pancreas and gall bladder) are all discernable from 3 dph. Some aspects of the development of the two independent initial infoldings seem original compared with data in the literature. These results are discussed and compared with embryonic and post-embryonic development patterns in other teleosts.

HLA-DMA alleles are possible new markers of rheumatoid arthritis: study of a Corsican group.

The HLA-DMA gene, along with the HLA-DMB gene, encodes the not classical class II molecule. This molecule catalyzes the class-II-associated invariant-chain peptide (CLIP)-antigen peptide exchange in classical class II molecule peptide-binding groove. As such, the DM heterodimer is an antigen presentation regulator and may be linked to immune system deficiencies such as those observed in autoimmune diseases. The study of DMA gene polymorphism seems be a reasonable approach to provide an answer to this question. Thanks to PCR-derived methods, the relationship between DMA gene polymorphism and rheumatoid arthritis (RA) was demonstrated in the present study. The DMA*0101 allele was observed to confer a significant predisposition to RA while the DMA*0102 allele significantly protected from this disease. Polymorphism experiments with the HLA-DRB1 gene revealed that this relationship between DMA polymorphism and RA is not a consequence of a linkage disequilibrium with the HLA-DRB1 alleles implicated in this pathology. The study of the DMA gene could therefore prove to be very useful in the early diagnosis of RA.

Analysis of the polymorphism of the tumour necrosis factor (TNF) gene and promoter and of circulating TNF-alpha levels in heart-transplant patients suffering or not suffering from severe rejection.

Plasma TNF-alpha levels are generally higher in heart-graft patients who experience a rejection episode than in those who do not. Because the TNF gene and its promoter are polymorphic, we studied the relationships between genetic variability at the TNF locus, the occurrence of graft rejection and TNF-alpha plasma levels in 62 heart-transplant patients in order to investigate inter-individual differences in plasma TNF-alpha levels after allogeneic stimulation. TNF-alpha was immunoenzymatically measured in blood specimens collected on the same day as endomyocardial biopsy. After PCR amplification of DNA, NcoI and AspHI polymorphisms were characterized by their restriction profiles, TNFa microsatellites by electrophoretic separation on acrylamide and the promoter region by sequencing. Plasma levels and molecular genetic results were compared to the grade of heart graft rejection established according to pathological criteria. In our study, allograft rejection was associated neither with NcoI or AspHI polymorphism nor with nucleotide changes in the TNF-A promote. We observed low TNF-alpha levels in n1/n1 homozygous patients and in subjects with G-->A at position--308 of the promoter sequence. Concerning the polymorphism of the TNFa microsatellite, our results might suggest an association with graft rejection but we have to be very careful in drawing conclusions because of the small size of the sample.

Implication of HLA-DMA alleles in corsican IDDM.

The HLA-DM molecule catalyses the CLIP/antigen peptide exchange in the classical class II peptide-binding groove. As such, DM is an antigen presentation regulator and may be linked to autoimmune diseases. Using PCR derived methods, a relationship was revealed between DM gene polymorphism and IDDM, in a Corsican population. The DMA*0101 allele was observed to confer a significant predisposition to this autoimmune disease while the DMA*0102 allele protected significantly. Experiments examining polymorphism of the HLA-DRB1 gene established that these relationships are not a consequence of linkage disequilibrium with HLA-DRB1 alleles implicated in this pathology. The study of the DMA gene could therefore be an additional tool for early IDDM diagnosis in the Corsican population.

[HLA-DQ genotyping using a modified technique of PCR-RFLP: application to HLA-DQA1 gene]. / Génotypage HLA-DQ par une technique modifiée de RFLP-PCR: application au gène HLA-DQA1.

Since 1989, several HLA-DQA1 PCR-RFLP genotyping protocols have been published. These methods require complete digestion of the PCR products and determination of the restriction fragments length. The HLA-DQA1 PCR-RFLP genotyping protocol describe here uses one amplification step through PCR, digestion of the PCR-products with 8 restriction endonucleases, and determination of the fragments size after polyacrylamide gel electrophoresis. Five of the enzymes, having no more than one restriction site in each allele (ApaLI, HphI, BsaJI, FokI and MboII), allow distribution of all the genotypes in 19 allelic-combination groups on the base of the digestion pattern: cut/not cut. Three additional enzymes (MnlI, DdeI and RsaI), having at least 2 restriction sites in each allele, are used to assign the genotype in each allelic-combination group on the base of the restriction fragments length observed. Eight of the 13 alleles, 36 of the 91 HLA-DQA1 genotypes could be characterized. Four to 8 samples could be characterized each day, including DNA extraction. The number of endonucleases used could act as internal control of enzymatic activities and the genotyping protocol can include new HLA-DQA1 alleles without modification of the experimental steps. This protocol can be applied easily in a laboratory without specific technical training or specific equipment.