Analysis of polymorphism in porcine MHC class I genes: alterations in signals recognized by human cytotoxic lymphocytes.

Elucidation of the mechanism of the immune response against transplanted porcine tissue is critical for the success of xenografting in humans. Both human T cells and NK cells recognize MHC Ags, and human receptors may bind to MHC Ags across species barriers. Molecular characterization of porcine MHC class I clones from two MHC class I loci (P1 and P14) obtained from homozygous inbred miniature swine of three haplotypes (aa, cc, and dd), revealed extensive conservation between loci, suggesting that the genes were products of duplication from a common ancestral sequence. The level of homology between loci was similar to that between the haplotypes at each locus, suggesting that intergenic exchange had limited divergence of these genes. Comparison of the alleles indicated that the polymorphism occurred in the alpha-1 and alpha-2 domains of the class I heavy chain, while the alpha-3 domain was highly conserved among the six genes analyzed. Amino acids in the alpha-2 and alpha-3 domains responsible for the binding of human CD8 to MHC class I were largely conserved in the porcine genes, but several critical residues were altered. Comparison of sequences recognized by human NK cell inhibitory receptors revealed that the residues critical for recognition by these receptors were altered in the porcine genes; thus, the porcine class I molecules would be unable to inhibit lysis by human NK clones characterized to date. This finding provides a likely explanation for the susceptibility of porcine cells to cytolysis by human NK cells.

Porcine repeat element DNA: in situ detection of xenotransplanted cells.

Using a digoxygenin-labelled DNA probe derived from the porcine repeat element PRE-1, we have developed a protocol for the detection of transplanted porcine islets and hepatocytes against a background of murine host tissue. Analysis of this probe by Southern blotting indicated that PRE-1 hybridizes to pig genomic DNA but not to human or mouse DNA. On tissue sections, hybridizing probe was detected using alkaline phosphatase-conjugated antidigoxygenin antibody visualized with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro-blue tetrazolium chloride (BCIP/NBT) substrate. We have demonstrated sensitive and highly specific staining of porcine nuclei in fixed, paraffin embedded tissue sections, and have applied the technique to detect porcine pancreatic islets and hepatocytes transplanted into murine kidney and spleen. Application of this technique include detection of transplanted cells or organs across the variety of xenogeneic barriers.

Biological activity of recombinant human myelin basic protein.

Using an inducible expression vector in Escherichia coli, we have expressed, purified, and biologically characterized recombinant human myelin basic protein (r-MBP). The recombinant protein binds in cation-exchange chromatography with similar affinity to purified, human MBP, and elutes as a single, 18.5-kDa species as judged by SDS-PAGE. The recombinant protein exhibits similar conformation to native MBP, as demonstrated by ELISA reactivity with a panel of six monoclonal antibodies directed against at least three different epitopes on human MBP. Additionally, recombinant MBP can trigger the proliferation of human T cell clones recognizing MBP, can induce experimental autoimmune encephalomyelitis (EAE) in the SJL mouse, and is capable of suppressing EAE following tolerization via oral administration. Most important, by using a novel method of purification, the recombinant protein preparation contains no detectable proteolytically derived fragments of MBP, a significant advantage over MBP purified from protease-rich central nervous system tissue. Purified recombinant MBP produced in E. coli will be useful as a biological reagent where highly purified protein devoid of degradation products is needed. Relevance to the study of antigen processing, interactions between MHC and the TCR, as well as for the investigation of antigen-driven immune tolerance are discussed.

Recombinant peptides as immunogens: a comparison of protocols for antisera production using the pGEX system.

Using an inducible vector system that directs high-level production and rapid purification of recombinant protein, we have immunized mice with peptides prepared by several methods: 1) samples fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) subsequently transferred to PVDF membrane and subcutaneously implanted in mice; 2) samples cut directly from SDS-PAGE gels and injected intraperitoneally; 3) injection of recombinant protein bound to agarose beads; and 4) injection of log-phase E. coli transformed with recombinant vector. All four strategies yielded specific antisera reacting with both the parental fusion protein and the recombinant fragment as determined by enzyme-linked immunosorbent assay and immunoblot analysis. Specific recognition of the recombinant fragment was demonstrated by a competitive inhibition assay in which the parental fusion protein abrogated reactivity of serum with the isolated recombinant fragment.

Chromosome mapping of the murine syndecan gene.

The chromosomal localization of the murine syndecan gene was determined by analysis of DNA from a panel of mouse-hamster cell hybrids containing various mouse chromosomes, detection of immunoreactive syndecan in culture medium of these cells, and linkage analysis of a mouse interspecific backcross. Southern analysis of the mouse-hamster cell hybrid DNA shows two distinct hybridizing sequences, one on mouse Chromosome 12 and the other on the X chromosome. Localization of the syndecan gene to mouse Chromosome 12 was determined by detection of immunoreactive syndecan in the culture medium of cell hybrids containing mouse Chromosome 12. Hybrids containing other mouse chromosomes were negative. Linkage analysis by Southern hybridization of DNA from a mouse interspecific backcross using a syndecan-specific probe localized the syndecan gene locus, Synd, to the proximal end of Chromosome 12, tightly linked to the Pomc-1 and Nmyc loci. The syndecan gene is likely on human Chromosome 2 because this region shows conservation of synteny between mouse and human chromosomes.

Type X collagen gene expression is transiently up-regulated by retinoic acid treatment in chick chondrocyte cultures.

During endochondral ossification, resting and proliferating chondrocytes mature into hypertrophic chondrocytes that initiate synthesis of type X collagen. The mechanisms regulating the differential expression of type X collagen gene were examined in confluent Day 12 secondary cultures of chick vertebral chondrocytes in monolayer treated with the vitamin A analog retinoic acid (RA). Preliminary results showed that major effects of RA on chondrocyte gene expression occurred between 24 and 48 h of treatment. Thus in subsequent experiments cultures were treated for 24, 30, 36, 42, 48, 72, 96, and 120 h. Total RNAs were isolated and analyzed by hybridization with 32P-labeled plasmid probes coding for five matrix macromolecules including type X collagen. We found that the steady-state levels of mRNAs for the large keratan sulfate/chondroitin sulfate proteoglycan (KS:CS-PG) core protein and type II collagen decreased several fold between 24 and 48 h of treatment compared to untreated cells, and remained low with further treatment. In sharp contrast, the level of type X collagen mRNA increased threefold by 42 h of treatment; thereafter it began to decrease and reached minimal levels by 72-120 h of treatment. The changes in steady-state mRNA levels during RA regimen paralleled similar changes in relative rates of protein synthesis. The transient up-regulation of type X collagen gene expression at 42 h of treatment was preceded by a five-fold increase in fibronectin gene expression, was followed by a several fold increase in type I collagen gene expression, and was accompanied by cell flattening and loss of the pericellular proteoglycan matrix. Thus, RA treatment leads to a unique biphasic modulation of type X collagen gene expression in maturing chondrocyte cultures. The underlying, RA-sensitive mechanisms effecting this modulation may reflect those normally regulating the differential expression of this collagen gene during endochondral ossification.

Stable phenotypic expression by chick chondroblasts in long-term suspension cultures as determined by proteoglycan analysis.

The influence of cell shape on phenotypic expression was studied in chick vertebral chondroblasts maintained for several weeks in suspension culture. To monitor phenotypic expression, synthesis of proteoglycans was studied in cultures of freshly-isolated 1-day-old chondroblasts and 1-to-6-week-old chondroblasts. The rate of proteoglycan synthesis was virtually identical in 1-week or older chondroblasts; however, this rate was 3- to 5-fold higher than in 1-day-old cells. When compared to the latter cells, the various populations of older chondroblasts synthesized monomers of the major cartilage proteoglycan (KS: CS-PG) of slightly lower molecular size and a lower level of unsubstituted N-acetylgalactosamine residues on their core protein but with similar chondroitin sulfate chains and levels of O-linked oligosaccharides. At no time of culture were changes in the proportions of the major vs the minor cartilage proteoglycans detected. The results suggest that in contrast to epithelioid chondroblasts in standard monolayer cultures studied previously, the round floating chondroblasts express very stable biosynthetic properties for a prolonged time in suspension. The distinct biosynthetic properties of 1-day-old chondroblasts are discussed in terms of an initial, transitory response to the culture condition and in relation to regulatory mechanisms for proteoglycan elaboration.

Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules.

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.