Elongation of the cytoplasmic domain,due to a point deletion at exon 7, results in an HLA-C null allele,Cw*0409 N

The development of molecular techniques for HLA typing has allowed the identification of genes previously assigned as serologic blank alleles. Lack or poor cell surface expression has been found for molecules coded by HLA-A, -B, -DRB4, -DRB5, and -DPB1 genes. In this report we describe the first HLA-C gene encoding for a null cell surface molecule. HLA-Cw*0409 N shows a point deletion at position 1095 within exon 7. This mutation provokes a codon reading shift, generating a new translation stop codon 97 bp downstream to that described in alleles normally expressed. This new stop codon location implies the presence of 32 extra amino acid residues in the cytoplasmic domain. Transfection experiments suggest that elongation of the cytoplasmic domain in Cw*0409 N would be the cause of cell surface expression failure, although Cw*0409 N heavy chain is able to create stable complexes with beta2-microglobulin. HLA-C fragment length analysis in a small selected group of samples with B44-Cblk haplotypic associations allowed us to identify two additional subjects showing both a serologic silent Cw*04 allele and a point base deletion at the 3' end of the HLA-C gene. This finding indicates that the allele frequency of Cw*0409 N within serologic C blank alleles would be appreciable, although basically restricted to the (A23)-Cw*0409 N-B*4403-DR7-DQ2 haplotype.     

HLA-B44 subtyping in a Spanish population: further evidence of Caucasian population diversity

HLA-B44 is the most frequent HLA-B allele in Caucasian populations. Several B44 subtypes, B*4402-B*4406, have been identified in individuals with this ethnic origin. Mismatches among B44 subtypes have been described as major targets for allogeneic responses in bone marrow transplantation. We have developed a PCR-SSO method, based on a B12- specific DNA amplification of exon 2 through exon 3 and subsequent non radioactive hybridization with eight probes, which allow us to discriminate all B12 homozygous combinations. We applied this method to determine the frequency of B44 subtypes in a Spanish population, as well as their HLA-A.-C.-DRB1,-DRB3/DRB4/DRB5.-DQA1 and -DQB1 associated haplotypes. A total of 141 healthy unrelated Spanish individuals and 31 B44-bearing haplotypes were investigated. Four B44 alleles were identified, B*4402 (33%), B*4403 (66%), B*4404 (0.7%), and B*4405 (0.7%). Haplotype analysis showed a clear differentiated distribution pattern for the two major B44 subtypes. B*4402 is associated with Cw5 (11/13) and A2 antigens (10/13). In contrast, B*4403 is mainly found together with DRB1*0701 (14/16). An inverted B*4402/B*4403 frequency in comparison with other European and North American Caucasian populations, revealed the existence of an extended haplotype diversity between populations of the same ethnic origin. Apart from anthropological studies, high resolution typing for HLA class I antigens presenting molecular polymorphism will be of great relevance in unrelated bone marrow transplantation.     

Four new HLA class I alleles in Cauacasoids

Four new HLA classical class I alleles in the three loci are described in Caucasian individuals. A*3012 was first suspected by an abnormal serologic pattern that would be explained by the single amino acid substitution at the A30-specific Ser17. B*270505 differs from B*270502 in a silent substitution at an up to now constant position in the B locus. B*3541 encodes for a new Cys at position 118 that has not been encountered in neither human nor primate alleles. Cw*0716 seems to be originated by a large-scale interallelic recombination event between Cw*0701/*0706/*0718 and Cw*020202, giving rise to a new antigen-binding cleft conformation.     

Testicular sperm retrieval: What should we expect from the fresh and subsequent cryopreserved sperm injection?

We sought to compare ICSI outcomes of cycle using fresh versus thawed TESE spermatozoa obtained during the previous fresh TESE. All consecutive couples undergoing ICSI cycles using fresh TESE spermatozoa, followed by ICSI cycle using cryopreserved sperm remaining from the previous fresh TESE procedure were included. Ovarian stimulation (OS)/laboratory variables and cycle outcome were assessed and compared between those utilising fresh versus thawed TESE spermatozoa. Seventy-five couples were evaluated, with no in-between groups differences in OS nor embryological variables. While implantation and LBR per embryo transfer were nonsignificantly higher in the frozen as compared to the fresh TESE, there was a trend towards higher LBRs per patient in the frozen TESE group. The cumulative miscarriage rate (4% versus 14.7%, p < .022 respectively) was significantly lower and the cumulative LBR (34.7% versus 16%, p < .007 respectively) was significantly higher using frozen TESE spermatozoa. Moreover, significantly higher proportion of frozen TESE sperm samples used pentoxifylline to enhance sperm motility. In conclusion, the results of ICSI cycles using frozen TESE spermatozoa are as good, or even better than using fresh TESE spermatozoa. Further studies are required to explore the factors responsible for the improved ICSI outcome, while using frozen versus fresh TESE sperm samples.     

The major T cell epitope on type II collagen is glycosylated in normal cartilage but modified by arthritis in both rats and humans

Type II collagen (CII) is a target for autoreactive T cells in both rheumatoid arthritis and the murine model collagen-induced arthritis. The determinant core of CII has been identified as CII260-270, and the alteration of this T cell epitope by posttranslational modifications is known to be critical for development of arthritis in mice. Using CII-specific T cell hybridomas we have now shown that the immunodominant T cell epitope in the normal (healthy) human and rat joint cartilage is O-glycosylated at the critical T cell receptor recognition position 264 with a mono- or di-saccharide attached to a hydroxylysine. In contrast, in the arthritic human and rat joint cartilage there are both glycosylated and non-glycosylated CII forms. Glycosylated CII from normal cartilage could not be recognized by T cells reactive to peptides having only lysine or hydroxylysine at position 264, showing that antigen-presenting cells could not degrade the O-linked carbohydrate. Thus, the variable forms of the glycosylated epitope are determined by the structures present in cartilage, and these vary during the disease course. We conclude that the chondrocyte determines the structures presented to the immune system and that these structures are different in normal versus arthritic states.     

Toll-like receptors and their ligands control mesenchymal stem cell functions.

Mesenchymal stem cells (MSCs) are widespread in adult organisms and may be involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. Thus, it is important to discover the factors controlling MSC renewal and differentiation. Here we report that adult MSCs express functional Toll-like receptors (TLRs), confirmed by the responses of MSCs to TLR ligands. Pam3Cys, a prototypic TLR-2 ligand, augmented interleukin-6 secretion by MSC, induced nuclear factor kappa B (NF-kappaB) translocation, reduced MSC basal motility, and increased MSC proliferation. The hallmark of MSC function is the capacity to differentiate into several mesodermal lineages. We show herein that Pam3Cys inhibited MSC differentiation into osteogenic, adipogenic, and chondrogenic cells while sparing their immunosuppressive effect. Our study therefore shows that a TLR ligand can antagonize MSC differentiation triggered by exogenous mediators and consequently maintains the cells in an undifferentiated and proliferating state in vitro. Moreover, MSCs derived from myeloid factor 88 (MyD88)-deficient mice lacked the capacity to differentiate effectively into osteogenic and chondrogenic cells. It appears that TLRs and their ligands can serve as regulators of MSC proliferation and differentiation and might affect the maintenance of MSC multipotency.