Antigen-presenting cell-T cell interaction in the chicken is MHC class II antigen restricted

The involvement of the MHC in the recognition of Ag by avian T lymphocytes was analyzed. PBL from chickens primed with keyhole limpet hemocyanin in vivo were induced to synthesize DNA in an in vitro response to specific Ag. Responding cells were T cells as judged by immunofluorescence staining. In vivo Ag-primed PBL were stimulated in vitro with specific Ag and further propagated in the presence of IL-2. Subsequent Ag-specific T cell proliferation required the presence of Ag-pulsed peripheral blood adherent cells (APC). T cell responses were restricted by the MHC of the APC; Ag presented by allogeneic APC did not support T cell proliferation. By using MHC-recombinant chicken lines, the gene products controlled by MHC class II loci were shown to restrict the T cell-APC interaction. This conclusion was substantiated by the inhibition of the Ag-specific T cell response by a mAb against chicken MHC class II gene products but not by a mAb against chicken MHC class I gene products.     

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Mapping the idiotopes of a monoclonal anti-myoglobin antibody with syngeneic monoclonal anti-idiotypic antibodies: detection of a common idiotope

A panel of syngeneic monoclonal anti-idiotypic antibodies was prepared by immunizing A.SW mice with keyhole limpet hemocyanin-coupled A.SW monoclonal anti-myoglobin (HAL 19, IgG1) and screening the cloned hybridomas for production of IgG2 binding to idiotype but not to certain other anti-myoglobin antibodies of the same subclass in an ELISA. With these antibodies, we identified three nonoverlapping idiotopes, based on three clusters of monoclonal anti-idiotopes that mutually inhibit within each cluster, but not between clusters (Cluster I: S2, S6, S8; Cluster II: S5, S7; Cluster III: S9). Only Cluster II antibodies block the binding of myoglobin to HAL 19 and so identify a binding site-related idiotope(s). Binding of both Cluster II monoclonals (S5 and S7) to Hal 19 is inhibited by a rabbit anti-idiotype that we previously reported detects a common cross-reactive anti-myoglobin idiotope in immune sera. However, only one of these, S7, and not S5, identifies an idiotope that is present on 20 to 30% of A.SW anti-myoglobin antibodies in immune sera and ascites. The panel of syngeneic monoclonal anti-idiotype antibodies also detects new idiotopes not detected by the rabbit anti-idiotype. The development of a panel of syngeneic monoclonal anti-idiotypic antibodies to different clusters of idiotopes on the same antibody molecule, including one that identifies a major common idiotope in immune sera, should allow the analysis of possible idiotype network regulation in vivo and in vitro in a completely syngeneic system.     

Hydrolysis of phospholipid monolayers by human spermatozoa. Inhibition by male contraceptive gossypol

Monomolecular films of phospholipid were used to study the interaction of intact human spermatozoa with model membranes. Exclusively with negatively charged phosphatidylglycerol monolayers rapid penetration of spermatozoa into the monolayer with subsequent hydrolysis of the lipid was triggered by the addition of 5 mM calcium into the medium. The results suggest the localization of a calcium-dependent phospholipase A2 at the outer acrosomal or plasma membrane of human spermatozoa with its active site exposed to the external environment. Preincubation of the cells with 100 microM gossypol completely abolished the ability of human spermatozoa to hydrolyze or penetrate monolayers of phosphatidylglycerol. The inhibition of the phospholipase activity by gossypol may contribute to the unknown contraceptive mechanisms of this non-steroidal male antifertility agent.     

Increased endogenous retroviral gene expression is a consequence of lymphocyte activation

Plasma cells of line 151(5) chickens have been shown to express elevated levels of endogenous retroviral envelope glycoprotein (VEG), measured relative to levels expressed by both immature B cells and resting peripheral B lymphocytes. In this study we analyzed the relationship between peripheral blood lymphocyte (PBL) maturation and the level of VEG expression. A culture system was developed that would support maturation of pokeweed mitogen-activated peripheral B lymphocytes. As analyzed by cytofluorometry, both Ig+ and Ig- lymphoblasts present in the pokeweed mitogen-stimulated cultures expressed detectable levels of VEG in contrast to bursacytes and PBL. Similarly, Ig- blasts, which were present in concanavalin A-stimulated cultures of PBL and presumed to represent activated T cells, were also positive for the expression of VEG. Immature T cells, i.e., thymocytes, although negative by immunofluorescence analysis, expressed VEG at levels that were detectable by radioimmunochemical techniques. These results indicate that T cells as well as B cells constitutively express VEG, and that mitogenic activation of the resting lymphocyte induces an increase in VEG expression.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.