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Biomedical subjects

R A Flavell

Publications and source records attributed to R A Flavell.

At least 307 records · Page 17Linked to original sources
PubMed1991

Protection of mice from Lyme borreliosis by oral vaccination with Escherichia coli expressing OspA.

Mice immunized with recombinant outer surface protein A (OspA) in Freund's adjuvant or with intraperitoneal injections of live Escherichia coli expressing OspA have been shown to be protected from infection with Borrelia burgdorferi. To investigate the efficacy of oral vaccination, C3H/He mice were inoculated with 10(8) live E. coli expressing recombinant OspA by gavage and boosted in a similar manner on days 10, 20, 30, and 40. The animals developed serum IgG antibodies to OspA by immunoblot and were protected from infection when challenged with 10(4) B. burgdorferi intradermally 14 days after the last boost. Control mice did not develop antibodies to OspA and were not protected against challenge infection. These results suggest that an oral preparation of recombinant OspA could potentially be used for vaccination.

Animals↗
PubMed1991

Molecular characterization of the humoral response to the 41-kilodalton flagellar antigen of Borrelia burgdorferi, the Lyme disease agent.

The earliest humoral response in patients infected with Borrelia burgdorferi, the agent of Lyme disease, is directed against the spirochete's 41-kDa flagellar antigen. In order to map the epitopes recognized on this antigen, 11 overlapping fragments spanning the flagellin gene were cloned by polymerase chain reaction and inserted into an Escherichia coli expression vector which directed their expression as fusion proteins containing glutathione S-transferase at the N terminus and a flagellin fragment at the C terminus. Affinity-purified fusion proteins were assayed for reactivity on Western blots (immunoblots) with sera from patients with late-stage Lyme disease. The same immunodominant domain was bound by sera from 17 of 18 patients. This domain (comprising amino acids 197 to 241) does not share significant homology with other bacterial flagellins and therefore may be useful in serological testing for Lyme disease.

Amino Acid Sequence↗
PubMed1990

Expression of epidermal-growth-factor receptor in the K562 cell line by transfection. Altered receptor biochemistry.

The epidermal-growth-factor (EGF) receptor was expressed in the human erythroleukaemic cell line K562 by transfection of the receptor cDNA. EGF-receptor biochemistry appears altered in the K562 transfectants. Autophosphorylation of the K562 receptor is not stimulated substantially by EGF. Tyrosine kinase activity of the receptor is high in the absence of EGF, whereas receptor affinity for EGF is low. K562 cells are shown to lack mRNA for transforming growth factor alpha (TGF alpha). Therefore autocrine stimulation of the K562 receptor, at least by TGF alpha, does not explain the observed receptor biochemistry. The K562 receptor is phosphorylated at a single major site in intact cells, a threonine residue that may be Thr-669. Possible mechanisms of regulation of the EGF receptor in the K562 transfectants are discussed.

DNA↗
PubMed1990

Protection of mice against the Lyme disease agent by immunizing with recombinant OspA.

Lyme borreliosis is a tick-borne illness caused by Borrelia burgdorferi. The gene for outer surface protein A (OspA) from B. burgdorferi strain N40 was cloned into an expression vector and expressed in Escherichia coli. C3H/HeJ mice actively immunized with live transformed E. coli or purified recombinant OspA protein produced antibodies to OspA and were protected from challenge with several strains of B. burgdorferi. Recombinant OspA is a candidate for a vaccine for Lyme borreliosis.

Animals↗
PubMed1990

Tolerance in transgenic mice expressing major histocompatibility molecules extrathymically on pancreatic cells.

Transgenic mice with defined expression of major histocompatibility complex (MHC) proteins provide novel systems for understanding the fundamental question of T cell tolerance to nonlymphoid self components. The MHC class II I-E and I-A and class I H-2K molecules expressed specifically on pancreatic islet or acinar cells serve as model self antigens. In these systems, transgenic proteins are not detected in the thymus or other lymphoid tissues. Yet mice are tolerant to the pancreatic MHC products in vivo; this tolerance is not induced by clonal deletion. These studies have been aided by monoclonal antibodies specific for I-E-reactive T cells and indicate that clonal anergy may be an important mechanism of tolerance to peripheral proteins.

Animals↗
PubMed1990

Infertility in male transgenic mice: disruption of sperm development by HSV-tk expression in postmeiotic germ cells.

Previous experiments revealed that male transgenic mice bearing a cosmid that included the Class II E alpha gene, about 35 kb of 5' flanking DNA, and the cosmid vector sequences were sterile. To ascertain the cause of the sterility, various subfragments of the cosmid were tested in transgenic mice. Only those pieces of DNA that included some of the E alpha flanking chromosomal DNA and the herpes simplex virus (HSV)-thymidine kinase (tk) gene that was in the vector resulted in male sterility. Histological analysis revealed abnormalities in nuclear morphology of elongating spermatids and retention of mature spermatids within the seminiferous epithelium. Immunocytochemical studies showed that the HSV-tk gene was expressed at low levels in postmeiotic round spermatids and at higher levels in more mature elongating spermatids. To determine whether expression of HSV-tk in spermatids might be responsible for the sterility, the protamine gene promoter was used to direct the expression of HSV-tk to postmeiotic germ cells. Since the mice so treated were also sterile, the data suggest that expression of this enzyme in spermatids is responsible for the sterility phenotype.

Animals↗
PubMed1989

T-cell tolerance by clonal anergy in transgenic mice with nonlymphoid expression of MHC class II I-E.

T-cell reactivity to the class II major histocompatibility complex I-E antigen is associated with T-cell antigen receptors containing the V beta gene segments V beta 17a and V beta 5. Mice expressing I-E with the normal tissue distribution (on B cells, macrophages, dendritic cells and thymic epithelium) induce tolerance to self I-E by clonal deletion in the thymus. By contrast, we find that transgenic INS-I-E mice that express I-E on pancreatic beta-cells, but not in the thymus or peripheral lymphoid organs, are tolerant to I-E but have not deleted V beta 5- and V beta 17a-bearing T cells. Moreover, whereas T-cell populations from nontransgenic mice proliferate in response to receptor crosslinking with V beta 5- and V beta 17a-specific antibodies, T cells from INS-I-E mice do not. Thus, our experiments provide direct evidence that T-cell tolerance by clonal paralysis does occur during normal T-cell development in vivo.

Animals↗
PubMed1989

Specific molecular interaction between the insulin receptor and a D product of MHC class I.

The density of MHC class I was determined on a murine thymoma cell line (R1), an H-2 negative variant (R1E), and R1E-derived cell lines in which H-2 expression was restored by transfection of various MHC class I genes (Db, Kb, and truncated Db) and/or a beta-2-microglobulin gene (beta 2-m; B2). Appreciable MHC class I expression was found on R1 cells and on the variants in which MHC class I expression was restored by transfection of Db/beta 2-m or Kb/beta 2-m genes. Only approximately 20% difference was observed between the number of Db molecules and Kb molecules on the R1E/B2/Db and on R1E/B2/Kb, respectively. However, specific insulin binding was significantly different between these lines. By using a computer assisted curve fitting program, the insulin binding data for R1 and R1E/B2/Db cell lines best fitted a two-site model (K approximately 6 x 10(-9) M for high-affinity sites and a 2 to 3 x 10(-7) M for low-affinity sites), whereas all other lines only expressed one type of insulin binding site. These sites were unrelated to IGF-I and IGF-II receptors. Cross-linking of 125I-labeled insulin demonstrated specific binding of the ligand to a Mr approximately 130,000 dalton band in all lines. In the R1E/B2/Db cells, insulin also cross-linked to cell membrane molecules with Mr approximately 48,000 and approximately 60,000 Da, which were identified by immunoprecipitation to be the H chain of MHC class I and the heavy chain of MHC class I plus beta 2-m, respectively. It is concluded that the insulin receptors in the cell membrane interact specifically with D-products of MHC class I and that class I molecules of MHC may have a crucial role in insulin receptor expression. This may reflect a more general nonimmunologic role of MHC class I.

Animals↗
PubMed1989

Tolerance in transgenic mice expressing class II major histocompatibility complex on pancreatic acinar cells.

To study the nature of tolerance to antigens not expressed by cells of the lymphoid system, expression of class II MHC I-E was targeted to the acinar cells of the exocrine pancreas in transgenic mice (elastase [EL]-I-E). Despite the absence of detectable I-E in the thymus of EL-I-E transgenic mice, both thymocytes and peripheral T lymphocytes were tolerant to I-E, and the pancreas was free of autoimmune infiltrates. Nontolerant T cells adoptively transferred into irradiated or T-depleted transgenic mice rapidly destroy the I-E+ components of the pancreas; however, adoptive transfer of nontolerant T lymphocytes into nonirradiated transgenic mice do not. These results suggest that tolerance in transgenic mice is maintained by some peripheral tolerance mechanism. However, further studies indicate that tolerance in transgenic mice is not maintained by specific Ts cells. For example, cell mixing experiments both in vitro and in vivo fail to reveal dominant unresponsiveness. Furthermore, nontolerant T cells injected into otherwise unmanipulated EL-I-E mice can be primed in situ (by injections of I-E+ spleen cells) to destroy the I-E+ acinar cells.

Animals↗
PubMed1989

A novel MHC class II epitope expressed in thymic medulla but not cortex.

The repertoire of receptors expressed by peripheral T cells is the result of two selective events that occur during intrathymic development. Positive selection expands cells able to recognize foreign peptides presented by self MHC molecules, and negative selection eliminates cells reactive to self MHC molecules and associated self peptides. Chimaera studies suggest that, at least in the case of T cells recognizing MHC class II, interaction with thymic cortical epithelial cells is responsible for the former, whereas thymic medullary cells, of bone marrow origin, mediate the latter. This view of thymic development is supported by recent morphometric analyses, showing that autoreactive cells are found in thymic cortex but not medulla. Although numerous studies have shown that MHC class II molecules are expressed in both sites, none provides any explanation for the differential selection of T cells that is observed. Here, we describe a novel MHC class II epitope which is found on cells in thymic medulla but not cortex. The antibody to this epitope reacts with about 10% of class II molecules on B cells and may be recognizing a self peptide-MHC complex. These results provide the first evidence for differential expression of class II epitopes in different tissues and are compatible with the hypothesis that different ligands, rather than different affinity thresholds for the same ligand, are involved in positive and negative selection of the T-cell repertoire.

Animals↗
PubMed1989

Role of beta 2-microglobulin in the intracellular transport and surface expression of murine class I histocompatibility molecules.

We have examined the requirement for beta 2-microglobulin (beta 2m) in the intracellular transport of murine class I histocompatibility molecules to the cell surface. R1E cells that are defective in the synthesis of beta 2m were transfected with either the class I H-2Kb or H-2Db genes alone, or together with the beta 2m gene. Kb or Db heavy chains synthesized in the presence of beta 2m were transported rapidly through the cell and expressed efficiently at the cell surface. In the absence of beta 2m, no "free" Kb heavy chains were detectable at the cell surface and their intracellular transport was blocked at an early stage. In contrast, a significant quantity of "free" Db heavy chains could be detected at the cell surface as we have reported previously. However, we have shown here that defects in intracellular transport were apparent in that the majority (approximately 70%) of newly synthesized Db heavy chains accumulated intracellularly and were degraded. Therefore, although Kb and Db heavy chains differ in their abilities to be expressed at the cell surface in the absence of beta 2m they both require association with beta 2m for efficient intracellular transport. In addition, R1E cells transfected with a deletion construct of the Kb gene expressed a truncated molecule lacking the alpha 3 extracellular domain (Kb-3) at the cell surface, but, like free Db, most newly synthesized Kb-3 molecules accumulated intracellularly. The free Kb, Kb-3, and Db heavy chains were not recognized by most mAb specific for Kb and Db, respectively. Therefore, even the transported forms of free Db and Kb-3 were not native in conformation, which is surprising given the current view that correct folding is essential for intracellular transport. Interestingly, the free Db and Kb-3 heavy chains that reached the cell surface differed in their detergent binding properties from those retained within the cell. This suggests that the transported heavy chains may have folded differently thus allowing their export to the cell surface.

Animals↗
PubMed1989

Selective expression of class II E alpha d gene in transgenic mice.

Class II genes of the MHC must be expressed by APC for activation of CD4+ T cells and efficient delivery of T cell help to B lymphocytes. Class II genes have restricted tissue expression and are under complex regulation. By using various deletion constructs of the class II E alpha d gene in transgenic mice we have mapped different 5' flanking regions which control E alpha d gene expression in distinct cell types. We demonstrate dissociate expression of E alpha d within the macrophage lineage as well as within the B cell lineage, and present evidence for a repressive element operative in B cells and macrophages. We describe the generation of novel transgenic lines with limited constitutive and inducible E alpha mRNA and I-E protein.

Animals↗
PubMed1989

A gamma-interferon-induced factor that binds the interferon response sequence of the MHC class I gene, H-2Kb.

Transcription of class I genes of the major histocompatibility complex (MHC) can be induced by interferons. Treatment of HeLa cells with interferon-gamma induces a DNA-binding factor, IBP-1, specific for a site within the interferon response sequence (IRS) of the H-2Kb promoter. The mol. wt of IBP-1, as estimated by photoactivated protein-DNA crosslinking analysis, is approximately 59 kd. Point-mutation of this binding site abolishes IBP-1 interaction and the ability of the MHC promoter to respond to interferon. Induction of this binding activity is rapid and closely parallels the previously reported time course of transcriptional activation of endogenous MHC class I genes. Treatment of cells with cycloheximide, a protein synthesis inhibitor, blocked the induction of the DNA-binding activity. An oligonucleotide derived from the virus- and double-stranded RNA-inducible promoter of the interferon-beta 1 gene is able to bind IBP-1. Sequences similar to the IBP-1 binding site are found upstream of many interferon-responsive genes.

Animals↗
PubMed1989

Correlation of fetal kidney and testis congenic graft survival with reduced major histocompatibility complex burden.

Prolonged survival was enjoyed by fetal and postnatal testis and midgestational renal grafts transplanted beneath the renal capsule of adult congenic mice, confirming previous findings in nonimmunosuppressed outbred rats (3,5). The strategies that enable immature tissues to escape rejection in a graft survival assay were studied by comparing expression of major histocompatibility class I and class II protein and messenger ribonucleic acid (mRNA) in each tissue at different ages. In general, graft survival was best when class I and II expression was low. After transplantation, surviving kidney and testis grafts both showed marked induction of class I and II mRNA measured using donor and recipient-specific oligonucleotide probes. Immunohistochemically detected protein of both classes, however, could not be found in the kidney and was minimal in the testis. Fetal tissues appear to express lower levels of protein and mRNA--and, although invading lymphocytes may induce expression of class I and II mRNA after transplantation, protein was not inducible. The failure of these tissues to express significant levels of transplantation antigens may explain the prolonged survival of these immature grafts.

Animals↗