Genetic control of peripheral TCRAV usage by representation in the preselection repertoire and MHC allele-specific overselection.

TCRAV segments contribute significantly to MHC restriction as illustrated by their general preference for either the CD4 or CD8 T cell subset and additional, MHC allele-specific overselection during T cell differentiation. The 10-fold over-representation of the TCRAV8S2 (VA8S2) segment in CD8 over CD4 T cells by the RT1(f) haplotype of LEW.1F rats provides the most striking example of MHC allele-specific overselection of a VA segment reported so far. Also in alloreactivity, VA8S2(+) CD8 cells from RT1(f-) rats are preferentially expanded by RT1(f+) stimulators. We have identified the class I molecule, A(f), mediating VA8S2 overselection and report that it differs only in four amino acids at the MHC-TCR interface from the class I molecule A(a), which is neutral with regard to selection of VA8S2. We also provide an extensive survey of the TCRAV8 family and show that among 14 functional VA8 segments in LEW rats, the dramatic A(f)-dependent overselection is unique for VA8S2. Surprisingly, VA8S2 expression in CD8 T cells of RT1(f+) rats derived from a Sprague-Dawley stock was only 3% as compared to the 12% observed in LEW.1F. The VA8S2 segment of Sprague-Dawley (VA8S2(SD)) differs from VA8S2 of the LEW background (VA8S2(l)) in only two amino acids, one of which is located in CDR2 and could thus participate in allele-specific recognition of A(f). However, analysis of the pre- and postselection thymic repertoires of Sprague-Dawley and LEW.1F rats and of the repertoire of CD8 cells from both strains expanded in the alloreactive response to RT1(f) revealed that the difference in VA8S2 representation between the two backgrounds is explained by differential availability in the preselection repertoires and not by a difference in overselection. Sequence comparisons of A(f) and A(a) and of both VA8S2 segments suggest a predominant role of CDR1 in hyper-reactivity to A(f). Thus, the VA composition of the mature TCR repertoire is influenced by TCRA: locus polymorphisms at two levels: the regulation of VA usage in the preselection repertoire and the composition of structural elements which contribute to specific VA-MHC interactions during thymic selection.

Thymic development and repertoire selection: the rat perspective.

This review summarizes our current knowledge of T-cell maturation and repertoire selection in the rat thymus. Some unique features of early thymocyte development and of CD4/CD8 lineage decision are described. A detailed analysis of lineage progression through the CD4, CD8 "double positive" compartment and T-cell receptor-induced CD8 T-cell maturation in cell culture is provided. A second emphasis is placed on interactions between germline-encoded T-cell receptor elements with MHC molecules in thymic repertoire selection and alloreactivity

Co-evolution of rat TAP transporters and MHC class I RT1-A molecules.

The genes for rat major histocompatibility complex (MHC) class I molecules are associated either with those for the A allele of the transporter associated with antigen processing (TAP-A), which can transport peptides with basic carboxy-terminal residues, or with those for TAP-B, which cannot [1-5]. To explore whether these associations have a functional basis, we compared the sequences of 13 rat MHC class la RT1-A cDNAs from nine MHC haplotypes. Of seven TAP-A- linked RT1-A molecules, six possess strongly acidic F pockets, and these bind a high proportion of peptides with basic carboxy-terminal residues. The F pockets of TAP-B-linked molecules, by contrast, were more basic. Furthermore, we identified six positions at the 'righthand end' of the peptide-binding groove, at which a majority of TAP-B-linked molecules diverge from the consensus sequence for class la molecules whereas, at these positions, all the TAP-A-linked molecules reflect the consensus sequence. Our results suggest that the linked rat class la and TAP genes have co-evolved to maximize the supply of appropriate peptides to the presenting molecules.

Mutagenic activity of ambient oxygen and mitomycin C in Fanconi's anaemia cells.

Cellular evidence suggests that Fanconi's anaemia (FA) might be a condition of increased oxygen sensitivity. In order to test this hypothesis, a common shuttle vector assay with the plasmid pZ189 was utilized. We transfected intact, circular plasmid into FA and control lymphoblast and fibroblast host cells maintained at 5 and 20% O2 (v/v). In parallel experiments, host cells were exposed to different concentrations of mitomycin C (MMC), a cross-linking agent towards which FA cells are known to be hypersensitive. Baseline mutation frequencies at 20% oxygen were significantly higher in plasmids passaged through FA lymphoblasts or FA fibroblasts in comparison with passage through the corresponding control cells. Lowering the oxygen concentration during the 48 h transfection period to 5% resulted in a significant decrease of mutation frequencies in plasmids passaged through FA cells. Sequence analysis of plasmids recovered from FA lymphoblasts revealed a mutation hot spot (22% of point mutations with G:C to A:T base substitutions) at base 117 of the supF tRNA gene. This hot spot was present only at 20% oxygen. 59% of the base changes at the hot spot and 39% of the changes elsewhere in the supF gene were C to T transitions (the corresponding figures are 0 and 27% at 5% oxygen), the most common type of base change induced by oxygen. The mutation spectrum observed suggests a role for 8-hydroxydeoxyguanosine in G:C to A:T base substitutions: at 20% oxygen, FA cells displayed 4 times as many G:C to T:A transversions than FA cells kept at 5% O2. In MMC treated cells the decrease in plasmid survival is dose dependent and more pronounced in FA than control cells. Mutation analysis shows similar rates of deletions for both control and FA cells. However, FA cells generate a specific type of deletion whose breakpoint involves an indirect repeat that corresponds to a heptamer signal sequence commonly seen at recombination sites. Together our data provide compelling evidence that the genetic defect in FA causes oxygen sensitivity and recombinational types of DNA lesions following exposure to MMC.

Heparin-induced endothelial cell cytoskeletal reorganization: a potential mechanism for vascular relaxation.

We have previously shown that heparin given subcutaneously on a daily basis lowers blood pressure in hypertensive rat models, and that this blood pressure lowering effect is endothelium-dependent. The present study describes the effects of heparin on endothelial cell (EC) apical surface structures and cytoskeletal elements, namely, actin and vimentin as well as EC proliferative activity. The EC line (CRL 1998) was cultured, treated with different concentrations of heparin (0, 50, 100, 500 U/ml) for 4, 24 or 48 hours, and fixed for scanning electron microscopy (SEM), and immunofluorescence microscopy (IFM) studies. Enzyme-linked immunosorbent assays (ELISA) and flow cytometric analysis were performed on EC monolayers treated with different concentrations of heparin for quantitative detection of actin and vimentin. By SEM study the cell surface showed generalized smoothing as a result of blunting of surface microvilli with increasing time of exposure and dosage of heparin. By IFM study, the detectable actin signal within ECs became progressively reduced in both its cellular distribution and the apparent number of cells that remained reactive. By 48 hr/500 U heparin, the actin signal was almost undetectable. Vimentin showed a moderate reduction in the cellular distribution of labeling. Quantitatively, actin was significantly reduced after the 24 hour treatment with a higher dose of heparin (500 U/ml), from a baseline optical density (OD) of 1.12 +/- 0.060 to 0.866 +/- 0.008 (P < 0.0027). After 48 hours of treatment at both 100 U/ml and 500 U/ml heparin, actin was significantly reduced from a baseline OD of 1.347 +/- 0.063 to 1.090 +/- 0.039 (P < 0.0039) and 0.844 +/- 0.074 (P < 0.008), respectively. However, vimentin was significantly reduced only after 48 hours of treatment with a high dose of heparin (500 U/ml), from baseline OD 1.82 +/- 0.052 to 1.41 +/- 0.004 (P < 0.002). The flow cytometric findings were virtually identical to the ELISA data for actin and vimentin. These qualitative and quantitative changes in actin and vimentin are consistent with apparent smoothing and relaxation of the EC's apical surface. Labeling with the cell cycle marker MIB-1 (monoclonal antibody Ki-67), showed a progressive reduction in the observed intensity in heparin treated cells with substantially fewer cells being positive. After a 48 hour treatment with heparin (500 U/ml), most ECs displayed only dim labeling of the nucleolus. This finding is consistent with an antiproliferative effect. Overall, these findings are additive to our previous observations, and demonstrate that heparin causes EC cytoskeletal reorganization which is a potential mechanism for vascular relaxation.

The use of a zinc based bioceramic as an osteoconductive agent in the rat model.

The many complications and risks of autograft and allograft bone have resulted in the search for an effective synthetic bone graft substitute [1,2]. The purpose of this study is to determine the effectiveness of ZCAP (zinc calcium phosphate) ceramic organic acid composite as an osteoconductive agent, including its possible benefits or risks for patients requiring bone grafting. A solid control model has been developed with an isolated mid-diaphyseal femur fracture immobilized by intramedullary threaded K-wire fixation. Two trials were performed in which two formulations of ZCAP composite ceramic were tested. The first, used in eighteen rats will be referred to as ZCAP1, the second, used in six rats will be referred to as ZCAP2. In the first trial rat femurs were augmented with 0.1 g ZCAP1 composite ceramic which varied in pore size. One pore size performed better than the others, so that pore size along with some minor changes in the composite design to increase resistance to the stresses inherent in our weight bearing model resulted in ZCAP2, which was tested in the second trial. The rats were sacrificed at ten weeks, during which time all rats remained viable. Blood work was done prior to sacrifice and radiography was done on all femurs. After sacrifice, gross morphological exam showed filling of the defect. Sections of the femurs from each group were analyzed by decalcified histological examination, electron microscopy, and tensile strength testing. The ZCAP composite ceramic had no significant effect on blood chemistry or body weight. Initial histological studies were consistent with endochondral ossification which was confirmed by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)