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K A Staines

Publications and source records attributed to K A Staines.

5 recordsLinked to original sources
PubMed1999

Variation in the number of expressed MHC genes in different cattle class I haplotypes.

Analysis of cattle major histocompatibility complex (MHC) (BoLA) class I gene expression using serological and biochemical methods has demonstrated a high level of polymorphism. However, analysis of class I cDNA sequences has failed to produce conclusive evidence concerning the number and nature of expressed genes. Such information is essential for detailed studies of cattle immune responses, and to increase our understanding of the mechanisms of MHC evolution. In this study a selective breeding programme has been used to generate a number of MHC homozygous cattle expressing common serologically defined class I specificities. Detailed analysis of five class I haplotypes was carried out, with transcribed class I genes identified and characterized by cDNA cloning, sequence analysis, and transfection/expression studies. Surface expression of the gene products (on lymphocytes) was confirmed using monoclonal antibodies of defined BoLA specificity. Phylogenetic analysis of available transcribed cattle MHC class I sequences revealed complex evolutionary relationships including possible evidence for recombination. The study of individual haplotypes suggests that certain groupings of related sequences may correlate with loci, but overall it was not possible to define the origin of individual alleles using this approach. The most striking finding of this study is that none of the cattle class I genes is consistently expressed, and that in contrast to human, haplotypes differ from one another in both the number and composition of expressed classical class I genes.

Amino Acid Sequence↗
PubMed1998

DNA typing for BoLA class I using sequence-specific primers (PCR-SSP).

The analysis of cattle MHC (BoLA) class I gene expression is an essential component of studies on immune responses and susceptibility to disease. International BoLA workshops have generated data and reagents that allow discrimination of class I molecules at the haplotype level, but progress has been limited by difficulties encountered in defining single alleles. Our aim in this study was to develop a DNA-based system for improved identification of expressed class I alleles, utilizing available cDNA sequences derived from cattle carrying a series of serologically defined class I specificities. This method has allowed more accurate typing of animals for expression of the class I genes present within a small number of haplotypes. The method has also reliably differentiated between allelic variants (identified by prior sequence analysis) and has split existing serological specificities. The data show that MHC class I genes in cattle are more polymorphic than demonstrated by serology and biochemical analysis.

Alleles↗
PubMed1996

Identification of potential CTL epitopes of bovine RSV using allele-specific peptide motifs from bovine MHC class I molecules.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young infants and housed calves. Depletion of CD8+ lymphocytes from calves inhibited their ability to clear the virus from the nasopharynx and lungs. To study these cells further, a cytotoxic T lymphocyte (CTL) assay was established. CTL could be demonstrated in the peripheral blood of gnotobiotic calves 7-10 days post infection (p.i.) with RSV and in lungs 10 days p.i. This response was both MHC-restricted and virus-specific. Following separation of the lung lymphocytes by magnetic activated cell sorting, it was shown that the cytolytic activity was mediated by cells of the CD8+ phenotype. To identify epitopes recognised by bovine CTL, the consensus motifs from MHC class I alleles found in the herd at Compton were identified. cDNA libraries were constructed and screened for full length class I sequences. The isolated cDNA clones were then transfected into mouse P815 cells and the expressed product immunoprecipitated and matched with a serological specificity. The bovine MHC class I molecules were isolated from lysed transfected cells by affinity chromatography, using a monoclonal antibody specific for bovine MHC class I, and bound peptides were separated by reverse-phase HPLC. Analysis of the protein sequences of bovine RSV for the defined motifs has identified potential CTL epitopes.

Alleles↗