PubMed Health⌕ Search

Biomedical subjects

M F Flajnik

Publications and source records attributed to M F Flajnik.

69 records · Page 4Linked to original sources
PubMed1990

Evolution of the MHC: antigenicity and unusual tissue distribution of Xenopus (frog) class II molecules.

Antibodies that recognize Xenopus class II molecules have been developed. Mouse monoclonal antibodies were prepared by immunizing BALB/c mice with frog MHC antigens that had been partially purified with alloantisera, and by immunizing mouse spleen cells in vitro with activated Xenopus T lymphocytes. In addition, five mouse monoclonal antibodies specific for human class II antigens were found to cross-react with Xenopus class II antigens. A.TH mice, which do not express E class II molecules, always produce immunoprecipitating antibodies reactive with frog class II molecules after immunization with frog lymphocytes; other mouse strains rarely produce such antibodies. Two of the monoclonal antibodies raised against frog class II molecules recognize the denatured class II beta chain on Western blots, and the other three appear to recognize only the class II heterodimeric complex. The antibodies display differential reactivity with the allelic class II products of Xenopus. The monoclonal antibodies react with all adult lymphocytes in the spleen and peripheral blood, T cells and B cells having equivalent levels of class II antigens per cell. Class II molecules are "differentiation antigens" on adult thymocytes as the expression is greatest on the mature medullary population. The number of class II molecules/lymphocyte increases after culturing in medium containing fetal bovine serum. Sequential immunoprecipitation and isoelectric focusing experiments have shown that cell surface class II molecules immunoprecipitated with the monoclonal antibodies are the same as those immunoprecipitated with the cross-reactive antiserum specific for DR antigens which was previously used to identify frog class II molecules.

Animals↗
PubMed1990

Expression of MHC class II antigens during Xenopus development.

Larval and adult forms of the amphibian Xenopus differ in their MHC class II expression. In tadpoles, class II epitopes can be detected by monoclonal antibodies only on B cells, macrophages (whatever their location), spleen reticulum, thymus epithelium, and the pharyngobuccal cavity. In contrast, all adult T cells express class II on their surface. The transitions in class II expression occur at metamorphosis and are accompanied by other changes. The skin is invaded by class II positive dendritic cells, and the skin glands differentiate and also express class II. The gut, which expressed class II in discrete areas of the embryonic tissue, becomes invaded with B cells, and its epithelium also becomes class II positive.

Animals↗
PubMed1989

Experimental analysis of ventral blood island hematopoiesis in Xenopus embryonic chimeras.

The frequencies and potentialities of hematopoietic stem cells from 20-hr-old Xenopus embryos were examined by transplanting cytogenetically distinct ventral blood island tissue from diploid to triploid embryos. Thirty-five-day-old larvae were examined for the presence of donor-derived cells in their erythrocyte, thymocyte, and B lymphocyte populations by analyzing DNA content using flow cytometry. These experiments demonstrated that B lymphocytes, as well as erythrocytes and thymocytes, were derived from the ventral blood island. Data obtained by transplanting graded sized pieces of ventral blood island suggested that restricted erythroid precursors were present within the region by 20 hr postfertilization. Differentiation of both B- and T-lymphoid precursors from small pieces of ventral blood island was markedly enhanced when this tissue was grafted onto peripheral areas within the blood island region. Analysis of these data using repopulation statistics suggested that circulating larval erythrocytes of ventral blood island origin were derived from six or seven precursors. Each lobe of the thymus was colonized by three precursors, one of which was ventral blood island derived.

Animals↗
PubMed1988

MHC class I antigens as surface markers of adult erythrocytes during the metamorphosis of Xenopus.

An alloantiserum produced against Xenopus MHC class I antigens has been used to distinguish different erythrocyte populations at metamorphosis. By analysis using a fluorescence-activated cell sorter (FACS) analyzer, tadpole (stage 55) and adult erythrocytes have distinct volume differences and tadpole cells have no MHC antigens on the cell surface. Both tadpole and adult erythrocytes express a "mature erythrocyte" antigen marker, recognized by its monoclonal antibody (F1F6). During metamorphosis, immature erythrocytes, at various stages of differentiation, which express adult levels of cell-surface MHC antigens by 12 days after tail resorption, are found in the bloodstream. These immature cells are biosynthetically active, produce adult hemoglobin, and mature by 60 days after the completion of metamorphosis. Percoll gradient-density fractionation has shown that all of the cells in the new erythrocyte series express adult levels of MHC antigens but there is only a gradual increase in the amount of "mature erythrocyte" antigen. Tadpole erythrocytes, which are biosynthetically active during larval stages, produce small amounts of surface MHC antigens before the metamorphic climax and then become metabolically inactive. They are completely cleared from the circulation by 60 days after metamorphosis. Erythrocytes from tadpoles arrested in their development for long periods of time express intermediate levels of MHC antigens, suggesting a "leaky" expression of these molecules in the tadpole cells. The most abundant erythrocyte cell-surface proteins from tadpoles and adults, as judged by two-dimensional gel electrophoresis, are very different.

Animals↗
PubMed1986

Major histocompatibility complex-encoded class I molecules are absent in immunologically competent Xenopus before metamorphosis.

The expression of class I and class II major histocompatibility complex (MHC)-encoded antigens has been examined at various stages of the development of the clawed frog, Xenopus. By immunoprecipitation with alloantisera or xenoantisera from radio-labeled spleen and thymus lysates, and by mixed lymphocyte reaction analysis, it was determined that the same class II molecules are expressed throughout ontogeny. In contrast, by fluorescence on frozen sections of tadpoles and by immunoprecipitation, the class I molecule is not detected in tadpoles, but appears on all tissues at the climax of metamorphosis. Animals maintained as tadpoles for long periods of time by chemical treatment do express class I antigens; thus, their expression can be independent of other biochemical and morphological changes that occur at metamorphosis. Immunofluorescence detects an otherwise uncharacterized MHC-linked alloantigen on tadpole thymic epithelium from the earliest stages of thymus differentiation.

Animals↗
PubMed1985

Immune responses of thymus/lymphocyte embryonic chimeras: studies on tolerance and major histocompatibility complex restriction in Xenopus.

A new model has been developed to address the question of whether T cells that traverse an allogeneic thymus during early and late life become restricted to interact, in vivo, with other leukocytes and target cells that display the major histocompatibility complex (MHC) antigens of the thymus haplotype. Chimeras were made microsurgically with pairs of 24-h-old Xenopus embryos such that the anterior region of an embryonic chimera contained the thymus anlagen and was of one MHC genotype, whereas the posterior region contained the anlagen of all hemopoietic cells and was of another genotype. Assays to determine the MHC haplotype restriction specificity of T cells in chimeras that had been reared through metamorphosis involved: specific antibody responses (IgM and IgG) to dinitrophenylated keyhole limpet hemocyanin; rejection of minor H locus disparate skin grafts that expressed the MHC antigens of either the thymus donor or the lymphocyte donor; and mixed leukocyte culture. MHC-mismatched chimeras displayed split tolerance since they accepted skin grafts of the thymus haplotype but had lymphocytes that proliferated in response to MHC antigens of the thymus donor strain as well as to MHC antigens of third-party donors. IgM responses of MHC-matched and MHC-mismatched chimeras and of nonchimeric controls did not differ. However, the IgG responses of MHC-mismatched thymus/lymphocyte chimeras peaked later than those of MHC-matched chimeras and normal controls. Data from skin grafting protocols were consistent with the proposition that there may be in vivo selection of T cells reactive to minor H antigens presented in association with the MHC antigens of the thymus rather than the MHC antigens of the lymphocytes themselves. These data suggest that although it is not absolute, there is thymic selection of the T cell repertoire in Xenopus.

Animals↗
PubMed1985

Xenopus MHC class II molecules. I. Identification and structural characterization.

Class II antigens from the Xenopus laevis MHC (f haplotype) were identified by using a rabbit antihuman class II beta-chain serum (anti-p29boost). This xenoantiserum inhibits bidirectional Xenopus MLR (but not PHA-stimulation), recognizes the same molecules as certain MHC-linked Xenopus alloantisera, and immunoprecipitates class II molecules from Xenopus cells consistent with the tissue distribution of mammalian class II molecules. The Xenopus class II molecules are composed of two different chains, both of which are 30 to 35kD transmembrane glycoproteins. The alpha-chains have some N-terminal sequence homology with mammalian class II alpha-chains (both I-E/DR and I-A/DC); the beta-chains are directly recognized by anti-p29boost and have a markedly increased SDS gel mobility under nonreducing conditions. During biosynthesis, they are noncovalently associated with a number of other chains, including ones at 25kD, 33kD, and 40 to 45kD. The alpha-chains bear three N-linked glycans (two Endo H insensitive in mature material) and the beta-chains bear two (one Endo H insensitive). Unlike most mammalian class II molecules, the deglycosylated beta-chains are significantly larger and more acidic than the alpha-chains.

Animals↗
PubMed1985

A third immunoglobulin class in amphibians.

A new class of immunoglobulin (IgX) has been found in the South African frog, Xenopus laevis, and other related species. IgX can be immunoprecipitated by monoclonal antibodies directed against determinants found on Xenopus light chain, or on variable regions of heavy chains. Reagents specific for the heavy chain of IgM or the amphibian IgG equivalent, IgY, failed to react with IgX. IgX, which exists in serum as a polymer, is composed of subunits of disulfide-bonded heavy chains of 80,000 daltons and light chains of 25,000 to 29,000 daltons. Like mu, the heavy chain of IgX carries a large amount of asparagine-linked carbohydrate, but the partial peptide maps of the two are different. Although the concentration of IgX varies greatly in the serum of individual frogs, it is always secreted in cultures of cells from the spleen and intestinal mucosae.

Animals↗
PubMed1985

Xenopus MHC class II molecules. II. Polymorphism as determined by two-dimensional gel electrophoresis.

The class II antigens from four inbred strains of Xenopus laevis (r, f, g, and j haplotypes) and six gynogenetic LG clones (two Xenopus laevis, two Xenopus gilli haplotypes) with functionally well-defined MHC types have been immunoprecipitated with the rabbit anti-human class II beta-chain serum anti-p29boost and analyzed by two-dimensional gel electrophoresis. The glycosylated material from 15-hr biosynthetically labeled cells runs as a broad fuzzy band around 33kD that, upon removal of N-linked glycans by Endo F, resolves into upper beta-chain bands and lower alpha-chain bands. Both the glycosylated and deglycosylated class II antigens give rise to multiple IEF spots in evenly spaced arrays (alpha-chain: two to eight spots in one to three arrays, beta-chain: two to 12 spots in one to five arrays). Both chains are polymorphic and both map to the functionally defined MHC. The large number of spots argues for multiple class II antigens; by radioactive N-terminal sequencing, two homologous alpha-chains and five beta-chains are present in the f haplotype. By comparison with MHC-linked alloantisera, anti-p29boost recognizes all major polymorphic class II molecules in Xenopus laevis. A selection of outbred animals were typed by using an IEF procedure requiring only a million PHA-stimulated blood cells.

Animals↗
PubMed1984

Identification of class I major histocompatibility complex encoded molecules in the amphibian Xenopus.

Class I-like molecules have been immunoprecipitated from Xenopus leukocytes and erythrocytes with alloantisera directed against major histocompatibility complex (MHC)-linked antigens. The heavy chains, depending on the allele examined, have molecular weights of 40 000-44 000 of which 3000 daltons are asparagine-linked carbohydrates, probably present as one N-linked glycan. The presumed analogue of beta 2-microglobulin has a molecular weight of 13 000 and bears no asparagine-linked glycans. Family studies show that the heavy chains are encoded by genes residing in or closely linked to the MHC.

Alleles↗
PubMed1984

A flow cytometric analysis of the embryonic origin of lymphocytes in diploid/triploid chimeric Xenopus laevis.

Quantitative flow cytometry was used to examine the embryonic origin of lymphocytes in Xenopus laevis. Reciprocal head/body transplants were made between diploid (2N) and triploid (3N) embryos of the same developmental stages ranging from neural plate to tail bud stages. Thymuses and spleens were removed from postmetamorphic chimeras. Cell suspensions were stained with the fluorescent DNA stain, mithramycin, and the ploidy (relative fluorescence intensity) of the cells was then determined by flow cytometry. All lymphocytes in the chimeras were derived from the posterior portion of the embryo. In other experiments, various regions of the lateral plate or ventral mesoderm were grafted from triploid to diploid embryos. Only transplants that included middorsal mesoderm gave rise to lymphocytes.

Animals↗
PubMedDate not supplied

MHC restriction of T-cell proliferative responses in Xenopus.

The MHC restriction of Xenopus allogeneic MHC- and antigen-specific T-cell proliferative responses was assessed. Xenopus MHC-specific monoclonal antibodies that recognize class I and class II molecules were tested for inhibitory effects on the generation of secondary T-cell proliferative responses. Antigen-specific T-cell lines were inhibited by anti-class II but not anti-class I monoclonal antibodies. Secondary alloantigen-specific proliferative responses also demonstrated MHC class II restriction. Allogeneic MHC- and antigen-specific T-cell lines demonstrated differential sensitivity to anti-class II monoclonal antibodies directed at discrete class II epitopes. These results indicate that Xenopus T cells interact with antigen-presenting cells similarly to mammals, and directly confirm previous data indicating that MHC class II restriction of proliferative responses is present in amphibians.

Animals↗