Targeted identification of infection-related HLA class I-presented epitopes by stable isotope tagging of epitopes (SITE).

Identification of peptides presented in human leukocyte antigen (HLA) class I molecules after viral infection is of strategic importance for immunology and vaccine development. A powerful strategy aimed at the rapid, unambiguous identification of naturally processed HLA class I-associated peptides, which are induced by viral infection, is presented here. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues for the human leukocyte antigen allele of interest. Subsequently, these cells are mixed with an equal number of noninfected cells, which are cultured in normal medium. Finally, peptides are acid-eluted from immunoprecipitated HLA molecules and subjected to two-dimensional nanoscale liquid chromatography-mass spectrometry analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules.

Bovine respiratory syncytial virus infection influences the impact of alpha- and beta-integrin-mediated adhesion of peripheral blood neutrophils.

Neutrophil migration into the airways and pulmonary tissue is a common finding in bovine respiratory syncytial virus (BRSV) infections. Although neutrophil trans-endothelial migration in general depends on beta2-integrins, alternative integrins such as the alpha4-integrins have been implicated. In this study, rolling and firm adhesion of peripheral blood neutrophils isolated from healthy and BRSV-infected calves to tumour necrosis factor (TNF)-alpha activated pulmonary endothelium was investigated under flow conditions in vitro. For neutrophils obtained from healthy animals, inhibition of the beta2-integrin reduced firm adhesion to 63% and inhibition of alpha4-integrin to 73% compared with untreated controls. Inhibition of both integrins reduced firm adhesion to 25%. Rolling velocity, which is used as a parameter for integrin involvement in neutrophil rolling, increased 1.7-fold by blocking beta2-integrin and was significantly augmented to 2.5-fold by blocking both alpha4- and beta2-integrins. For neutrophils obtained from BRSV-infected animals, however, rolling velocities at 10 days after infection (p.i.) were not influenced by blocking adhesion of alpha4- and beta2-integrins, indicating that these integrins did not support neutrophil rolling. In addition, the inhibition of firm adhesion by blocking both alpha4- and beta2-integrins was reduced significantly 9 days post-infection, resulting in a residual 68% neutrophil binding at 9 days p.i. Non-blocked firm adherence was not reduced, indicating that binding was achieved by other mechanisms than through alpha4- and beta2-integrins. These results demonstrate an important function for alpha4- and beta2-integrins in rolling and firm adherence of bovine neutrophils, to TNF-alpha-activated endothelium and show the dynamic use of these integrins for adhesion and migration by neutrophils in the course of BRSV infection.

Identification and phenotyping of leukocytes in bovine bronchoalveolar lavage fluid.

A method is proposed to identify leukocyte subpopulations in bovine bronchoalveolar lavage fluid by dual-laser flow cytometry. The technique uses several parameters, i.e., exclusion of highly autofluorescent alveolar macrophages and inclusion of leukocytes on the basis of labeling by specific antibodies and light scatter characteristics.

A direct StyI polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) test for the myophosphorylase mutation in cattle.

Myophosphorylase deficiency in cattle is a muscle disease induced by a C-->T point mutation in codon 489 of the myophosphorylase gene, which until now has only been diagnosed in the Charolais breed. The disease seems to be inherited in an autosomal monogenic recessive manner. A calf of double muscled phenotype was suspected of suffering from myophosphorylase deficiency based on typical symptoms, i.e. brown-coloured, transparent urine, occurring after exercise; exercise intolerance; symptoms of pain; and an elevated level of plasma creatine kinase. The presence of the previously described mutation was excluded using a newly developed, improved polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure to identify easily heterozygous carriers and homozygous affected animals.