The HLA-DQ7 and -DQ8 associations in DR4-positive rheumatoid arthritis patients A combined analysis of data available in the literature

Several different lines of evidence have demonstrated that inherited susceptibility to rheumatoid arthritis (RA) is associated with the DRB1 genes encoding the HLA-DR4 and HLA-DR1 molecules. A contrasting hypothesis has recently been proposed, suggesting that, in general, the DRB1 locus is associated with protection to RA and that the RA-associated DRB1 alleles are not responsible for the primary disease association but merely permissive for the susceptibility conferred by the HLA-DQ alleles with which they are in linkage disequilibrium. We have performed a critical review of the literature on the HLA association in RA with special emphasis on studies in which both an HLA-DR and -DQ association has been investigated. Our analyses provide strong evidence against the hypothesis that HLA-DQ molecules play a major role in the general susceptibility to RA. Thus, the strongest association in rheumatoid arthritis is with DRB1 genes rather than DQB1 genes.     

Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection

The world's first deliveries of normal babies after use of flow cytometric separated human sperm cells (MicroSort) for preconception gender selection are reported. Offspring were of the desired female gender in 92.9% of the pregnancies. Most of these pregnancies and births were achieved after simple intrauterine insemination.      

Preliminary study of the incidence of disomy in sperm fractions after MicroSort flow cytometry

Using triple colour fluorescent in-situ hybridization (FISH) we have evaluated, on a blind basis, the disomy level for chromosome 21 and the sex chromosomes in flow cytometric sorted sperm samples. There were no statistically significant differences in the disomy rates when comparing the sorted samples (either for X- or Y-bearing spermatozoa) with non-sorted samples. There were no diploid spermatozoa observed in any sample group after MicroSort processing.     

Expression of HLA-DR4 and human CD4 transgenes in mice determines the variable region beta-chain T-cell repertoire and mediates an HLA-DR-restricted immune response.

Inherited susceptibility to rheumatoid arthritis is associated with genes encoding the human major histocompatibility complex class II molecule HLA-DR4. To study the immune function of HLA-DR4 and attempt to generate a murine model of rheumatoid arthritis we have produced triple transgenic mice expressing HLA-DRA*0101, -DRB1*0401, and human CD4. The expression of the HLA transgenes is driven by the promoter of the murine major histocompatibility complex class II I-E alpha gene and was found on murine cells that normally display major histocompatibility complex class II molecules. The expression of the human CD4 transgene is driven by the murine CD3 delta-promoter, and therefore its gene product was found on cells that express murine CD3. In contrast to other HLA-DR and HLA-DQ transgenic mouse lines, the transgenes are functional in our mice. In H-2 I-E-negative transgenic mice, T cells expressing variable region beta chain (V beta) 3, 5, 6, 7, 9, 11, 12, or 13 were either absent or significantly reduced, in contrast to H-2 I-E-negative nontransgenic littermates. In addition, the peptide antigen influenza A virus hemagglutinin 307-319, which binds to the HLA-DRA*0101/-DRB1*0401 heterodimer with high affinity and induces an HLA-DR-restricted and CD4+ T-cell response in humans, also induced a T-cell response in the triple transgenic mice but not in nontransgenic littermates. Thus, these transgenic mice should permit extensive testing of the antigen-presentation capabilities of the HLA-DRA*0101/-DRB1*0401 molecule.     

The nature of molecular recognition by T cells

Considerable progress has been made in characterizing four key sets of interactions controlling antigen responsiveness in T cells, involving the following: the T cell antigen receptor, its coreceptors CD4 and CD8, the costimulatory receptors CD28 and CTLA-4, and the accessory molecule CD2. Complementary work has defined the general biophysical properties of interactions between cell surface molecules. Among the major conclusions are that these interactions are structurally heterogeneous, often reflecting clear-cut functional constraints, and that, although they all interact relatively weakly, hierarchical differences in the stabilities of the signaling complexes formed by these molecules may influence the sequence of steps leading to T cell activation. Here we review these developments and highlight the major challenges remaining as the field moves toward formulating quantitative models of T cell recognition.     

Predominant selection of T cells specific for the glycosylated collagen type II epitope (263-270) in humanized transgenic mice and in rheumatoid arthritis

Rheumatoid arthritis (RA) is associated with certain MHC class II alleles and is characterized by a chronic autoimmune response in the joints. Using transgenic mice expressing human DR4 (DRB1*0401) and human CD4, but lacking endogenous MHC class II, we show that posttranslational glycosylation of type II collagen (CII) influences the level of T cell tolerance to this candidate cartilage-specific autoantigen. In such mice, the expression of human CII resulted in a tolerized murine T cell response to human CII. However, tolerance induction remained incomplete, preferentially deleting responses to the nonmodified CII 263-270 epitope, whereas T cell recognition of a glycosylated variant of this epitope was affected to a lesser degree. A similar dominance of T cell responses to CII-glycopeptides was recorded in a cohort of severely affected RA-patients (n = 14). Thus, RA T cells predominantly recognize the immunodominant CII peptide in its glycosylated form and may explain why previously it has been difficult to detect T cell responses to CII in RA patients.     

Reliable gender screening for human preimplantation embryos, using multiple DNA target-sequences.

Dependable methods were developed for preimplantation sexing of human IVF embryos, for use in clinical settings where prospective parents are at high risk for transmission of X-linked diseases. Using single cultured cells and blastomeres from human embryos as model systems, a multiplex protocol was developed for rapid analysis via nested polymerase chain reaction (PCR). Reliability was enhanced by co-amplification of conserved amelogenin gene segments from both X and Y chromosomes, as well as Y-linked DYZ1 repetitive elements. Each cell was manually isolated and individually washed to avoid potential contaminants. Multiplex amplification allowed recognition of spurious amplification failures specific to particular amelogenin single-copy targets. The X-linked internal control and multiple Y-linked markers allowed recognition and exclusion of most aberrant samples, thus averting potential misdiagnosis. The optimized single-cell protocol reduced experimental sexing errors to < 2% (1/60), but also revealed potential pitfalls of single-cell analysis. With human triploid embryos, separate sampling of individual blastomeres provided concordant female or male signals. Slight modification adapted the procedure for diagnosis of biopsy material from blastocyst stage embryos, allowing separate analysis of multiple tubes containing multiple cells for improved reliability.