Karen R. Pratt

Class II genes of miniature swine

Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human α probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human β probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLAc haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique α genes were obtained which showed no evidence of cross-hybridization, while β genes showed extensive cross-hybridization and were frequently detected in the library by more than one human β gene probe. These data are consistent with early evolutionary divergence of a genes, prior to mammalian speciation, and with continuing evolution of β genes, with possible shared usage of these genes by different a loci. The data also imply that α genes can readily be assigned to loci homologous to their human counterparts, but that β genes will require further mapping and/or sequence analysis to confirm assignments.

Class II genes of miniature swine. III. Characterization of an expressed pig class II gene homologous to HLA-DQA.

This report presents an analysis of the SLA-DQA gene organization, the isolation of two SLA-DQA allelic cDNA clones, as well as sequence comparisons between the SLA-DQA clones and other DQA-related genes

Class II genes of miniature swine. II. Molecular identification and characterization of B (beta) genes from the SLAc haplotype.

Genomic clones corresponding to class II β genes of theSLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding theDRB andDQB genes were identified on the basis of hybridization with locus-specific 3′ untranslated cDNA probes. Cluster 4 contained exons of bothDOB andDQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containingDP, DR, andDO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.

PLAQUE FORMING CELL ASSAY FOR THE MEASUREMENT OF POLYCLONAL ACTIVATION OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES. CELLULAR REQUIREMENTS AND INTERACTIONS

Publisher Summary This chapter explores the plaque-forming cell assay for the measurement of polyclonal activation of human peripheral blood lymphocytes. In a study described in the chapter, heparinized venous blood was drawn from 20 normal adults and mononuclear cells were obtained by Hypaque–Ficoll density centrifugation. Purified lymphocyte suspensions depleted of monocytes were obtained by the glass plate adherence method. T-cell enriched and T-cell depleted mononuclear cell suspensions were obtained by differential centrifugation over a Hypaque–Ficoll gradient. Cells were cultured in 12 × 75 mm round bottom plastic tubes at a density of 2 × 106 in 1 ml of RPMI-1640 media containing 10% pooled human AB serum (absorbed with SRBC) in the absence or presence of various concentrations of pokeweed mitogen. Cultures were incubated in 5% CO2 in air at 100% humidity for 1 to 10 days on a rocker platform. The assay method employed was a combination monolayer-gel technique using a drop of agar-cell suspension-complement mixture beneath a glass coverslip. Plaque-forming cell (PFCs) were not detected until day 3 to 4, with a sharp peak by day 6 to 7. Background PFC was almost always 0. The number of PFC varied considerably from person to person with a mean (±SEM) of 175 (±76) PFC per 106 lymphocytes.

Cytotoxic Effector Capacity of Bone Marrow Mononuclear Cells

Purified mononuclear cells from guinea pig bone marrow possess highly efficient cytotoxic effector cell capabilities in the phytohemagglutinin-induced cellular cytotoxity and antibody-dependent cellular cytotoxicity and antibody-dependent cellular cytotoxicity assays against chicken red blood cell targets. This cytotoxic activity is not removed by depletion of adherent cells by passage through rayon wool columns. Thus bone marrow lymphocytes themselves, depleted of adherent monocytes, possess this killer cell capacity. These effector cells may either arise de novo within the bone marrow parenchyma or arrive there as part of the recirculating lymphocyte pool. These studies lend further understanding of the development of functional capabilities of bone marrow lymphoid cells.