Biomedical subjects

R A Flavell

Publications and source records attributed to R A Flavell.

At least 289 records · Page 16Linked to original sources

PubMed1993

A monoclonal antibody to OspA inhibits association of Borrelia burgdorferi with human endothelial cells.

Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells. In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B. burgdorferi to HUVE cells by 73%. Subsequently, MAbs were generated for use in determining whether or how B. burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B. burgdorferi. Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa. Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells. None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5%. This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer). Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence. MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 [corrected] of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells. Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA. MAb 9B3D promises to be a valuable tool for elucidating the domain or domains of OspA involved in the endothelial cell cytadherence of North American strains of B. burgdorferi.

Animals↗

PubMed1993

OspA vaccination of mice with established Borrelia burgdorferi infection alters disease but not infection.

C3H mice were actively immunized with outer surface protein A (OspA) at different intervals after infection with Borrelia burgdorferi to determine the effect of postexposure vaccination on the course of murine Lyme borreliosis. Mice were vaccinated with an OspA-glutathione transferase fusion protein or glutathione transferase (control) in complete Freund's adjuvant; vaccination was followed by two weekly booster injections in incomplete adjuvant. Two weeks after the final booster injection, organs were cultured for B. burgdorferi (blood, spleen, skin, and bladder) and examined for histopathology (joints and hearts). When vaccination was commenced in the early stages (5 to 14 days) of infection, active immunization with OspA partially cleared spirochetes from the bloodstream but did not eliminate them from other tissues or alter the course of joint or heart disease. Commencement of vaccination at 60 days after infection (at which time joint or heart disease is resolving), however, reduced both the number of mice and individual joints with arthritis, a result suggesting an acceleration of the resolution phase of the disease. Postexposure immunization with OspA may partially alter the course of murine Lyme arthritis but does not eliminate infection.

Animals↗

PubMed1993

Serologic analysis of dogs, horses, and cottontail rabbits for antibodies to an antigenic flagellar epitope of Borrelia burgdorferi.

Enzyme-linked immunosorbent assays (ELISA) and immunoblots using either whole-cell lysates of Borrelia burgdorferi or an antigenic region of flagellin (41-G) as the antigen were performed, and the abilities of the two assays to detect antibodies to this spirochete in dog, cottontail rabbit, and horse sera were compared. Assays using whole-cell B. burgdorferi lysates as the antigen were more sensitive for detecting antibodies. ELISA with 41-G as the antigen were specific for Borrelia antibodies but were not as sensitive as the assays with whole-cell lysates coated to the solid phase. Use of recombinant full-length flagellin, rather than 41-G, as the antigen in immunoblots increased the sensitivity of each assay. However, antibodies to other bacterial antigens cross-react with whole flagellin and may account for false-positive results. Antibodies to B. burgdorferi outer surface protein A or B were usually undetected when the sera were tested by immunoblotting methods. Borrelia lysates or the 41-G antigen may be used in ELISA or immunoblots to document host exposure to this spirochete. The use of 41-G as the antigen may increase the specificity of an assay or help confirm the serologic diagnosis of Lyme borreliosis in dogs, horses, and cottontail rabbits.

Animals↗

PubMed1992

Insulitis in transgenic mice expressing tumor necrosis factor beta (lymphotoxin) in the pancreas.

Tumor necrosis factor beta (TNF-beta) (lymphotoxin) may play an important role in the immune response and pathologic inflammatory diseases. Insulitis is an important early step in the development of insulin-dependent diabetes mellitus. To understand better the role of TNF-beta in the regulation of inflammation and type 1 diabetes, we produced transgenic mice in which the murine TNF-beta gene was regulated by the rat insulin II promoter. The transgene was expressed in the pancreas, kidney, and skin of transgenic mice. The expression of TNF-beta in the pancreas of transgenic mice resulted in a leukocytic inflammatory infiltrate consisting primarily of B220+ IgM+ B cells and CD4+ and CD8+ T cells. The insulitis is reminiscent of the early stages of diabetes, though the mice did not progress to diabetes.

Animals↗

PubMed1992

Reactivation of a major histocompatibility complex class II gene in mouse plasmacytoma cells and mouse T cells.

Terminally differentiated plasma cells and mouse T cells do not express major histocompatibility complex (MHC) class II genes although class II gene expression is observed in pre-B and mature B cells as well as in activated human T cells. Transient heterokaryons were prepared and analyzed to investigate the mechanisms of inactivation of MHC class II gene in mouse plasmacytoma cells and mouse T cells. The endogenous MHC class II genes in both mouse plasmacytoma cells and mouse T cells can be reactivated by factors present in B cells. This reactivation of class II gene is also observed by fusion with a human T cell line which expresses MHC class II genes, but not with a class II negative human T cell line. It appears that the loss of MHC class II gene expression during the terminal differentiation of B cells or T cell lineage is due to absence of positive regulatory factor(s) necessary for class II transcription.

Animals↗

PubMed1992

Elimination of Borrelia burgdorferi from vector ticks feeding on OspA-immunized mice.

Although recombinant outer surface protein A (OspA) of Borrelia burgdorferi protects mice against injected Lyme disease spirochetes, the mode of protection has not yet been explored. Indeed, the efficacy of vaccine-induced immunity against a realistic vector-mediated challenge remains unexplored. Accordingly, we determined whether this immunogen protects mice against spirochetes delivered by nymphal Ixodes dammini ticks. Following challenge by tick bite, no spirochetes could be cultured from immunized mice, and no characteristic histopathology was found. The spirochete was not detected in ticks that fed on immunized animals and was present in virtually all ticks that fed on nonimmunized mice. We conclude that OspA-immunized mice are protected from spirochetal infection, at least in part, because the spirochete is destroyed in the infecting tick.

Animals↗

PubMed1992

T-cell responsiveness to an oncogenic peripheral protein and spontaneous autoimmunity in transgenic mice.

Why T cells develop autoimmune reactivity to some antigens and tolerance to others is unknown. Various mechanisms can provide for T-cell tolerance. These include deletion in the thymus, exhaustive differentiation in the periphery, T-cell receptor and coreceptor downregulation, and anergy. Which mechanisms normally provide for tolerance to antigens expressed on specific tissues and why they sometimes fail is unclear. To understand this, we analyzed how a tissue-specific protein with defined timing and location of expression is recognized by T cells so as to induce tolerance or autoimmunity. We crossed mice expressing the simian virus 40 large tumor antigen on pancreatic acini beginning 4-25 days after birth with mice transgenic for a rearranged T-cell receptor that recognizes this antigen presented by the class I major histocompatibility complex molecule H-2Kk. No T-cell tolerance was found; rather, T-cell reactivity accompanied lymphocytic infiltration and pancreatic acinar destruction. This result argues that T cells may become spontaneously autoreactive to certain postnatally expressed peripheral proteins and that this reactivity may lead to autoimmune disease.

Age Factors↗

PubMed1992

Borrelia burgdorferi strain 25015: characterization of outer surface protein A and vaccination against infection.

Mice vaccinated with outer surface protein A (OspA) from Borrelia burgdorferi strain N40 are protected from challenge with an intradermal syringe inoculum of B. burgdorferi strains N40, B31, and CD16. Vaccination experiments were done to determine if protection extended to strains 297 and 25015. We now show that OspA-N40 immunized mice are protected against challenge with strain 297, isolated from the cerebrospinal fluid of a patient with neuroborreliosis, but not against challenge with strain 25015, isolated from a tick in Millbrook, NY. The OspA gene from strain 25015 was therefore cloned and sequenced. The deduced OspA-25015 protein sequence differs from OspA-N40 at 40 of 273 amino acids. Furthermore, mice vaccinated with rOspA-25015 are protected from challenge with strain 25015 but not against strain N40. The results extend the usefulness of OspA as a vaccine candidate, but indicate that OspA can vary among strains of B. burgdorferi and that vaccination of mice with OspA-N40 does not protect against intradermal challenge with an inoculum of 10(4) strain 25015 spirochetes.

Amino Acid Sequence↗

PubMed1992

Dissection of the interferon gamma-MHC class II signal transduction pathway reveals that type I and type II interferon systems share common signalling component(s).

We have used a herpes virus thymidine kinase (HSV-TK) based metabolic selection system to isolate mutants defective in the interferon gamma mediated induction of the MHC class II promoter. All the mutations act in trans and result in no detectable induction of MHC and invariant chain (Ii) gene expression. Scatchard analysis indicates that the mutants have a normal number of surface IFN gamma receptors with the same affinity constant. The mutants fall into two broad categories. One class of mutants is still able to induce MHC class I, IRF-1, 9-27, 1-8 and GBP genes by IFN gamma. A second class of mutants is defective for the IFN gamma induction of all the genes tested; surprisingly, the IFN alpha/beta induction of MHC class I, 9-27, ISG54 and ISG15 genes is also defective in these mutants, although different members of this class can be discriminated by the response of the GBP and IRF-1 genes to type I interferons. These data demonstrate that the signalling pathways of both type I and type II interferon systems share common signal transduction component(s). These mutants will be useful for the study of IFN gamma regulation of class II genes and Ii chain, and to elucidate molecular components of type I and type II interferon signal transduction.

Base Sequence↗

PubMed1992

The activation of major histocompatibility complex class I genes by interferon regulatory factor-1 (IRF-1).

We have investigated the role of interferon regulatory factor-1 (IRF-1), an interferon-gamma (IFN-gamma) inducible transcriptional activator, on major histocompatibility complex (MHC) class I gene transcription. IRF-1 alone is sufficient to trans-activate both transfected and endogenous class I genes and the effect of IRF-1 appears to be direct and sequence-specific. These data suggest that IRF-1 is involved in the IFN-gamma mediated induction of MHC class I expression.

Base Sequence↗

PubMed1992

Nonspecific proliferative responses of murine lymphocytes to Borrelia burgdorferi antigens.

Proliferative responses of naive splenocytes to Borrelia burgdorferi antigens from mice susceptible (C3H) and resistant (BALB) to Lyme borreliosis were investigated. B. burgdorferi spirochetes and recombinant outer surface proteins, OspA and OspB, were found to induce nonspecific proliferation of naive splenocytes from both strains of mice. Cell purification studies localized nonspecific proliferation to the B cell-enriched fraction. B. burgdorferi, OspA, and OspB were found to induce IgM and IgG synthesis in vitro. The mitogenic effect of B. burgdorferi was dissimilar to that of lipopolysaccharide (LPS), in that B cells from C3H/HeJ mice (LPS-unresponsive) responded at levels comparable to those from C3H/HeNCrlBr mice. These results emphasize the need for caution in the study of antigen-specific proliferation for B. burgdorferi.

Animals↗

PubMed1992

Serologic diagnosis of Lyme disease using recombinant outer surface proteins A and B and flagellin.

Recombinant (r) outer surface proteins (Osp) A and B, flagellin, and an immunogenic region of flagellin (41-G) from Borrelia burgdorferi were evaluated using immunoblot and ELISA for usefulness as substrates in diagnostic testing for Lyme disease. Antibodies to rOspA, rOspB, and r-flagellin were detected by immunoblot and ELISA using the recombinant proteins. Patients with late disease responded to rOspA, rOspB, and r-flagellin, or only to r-flagellin, whereas patients with early disease showed no response or responded only to r-flagellin. Patients who developed antibodies to r-flagellin also responded to 41-G. The data suggest that recombinant B. burgdorferi antigens can serve as substrates for immunoblot and ELISA, which may be helpful in diagnosing Lyme disease.

Antibodies, Bacterial↗

PubMed1992

Roles of OspA, OspB, and flagellin in protective immunity to Lyme borreliosis in laboratory mice.

Vaccination with recombinant outer surface protein A (OspA) has been shown to protect mice from infection with Borrelia burgdorferi, the Lyme disease agent. To determine whether antibodies to B. burgdorferi proteins other than OspA are involved in protective immunity, antibodies to OspA were removed from protective anti-B. burgdorferi serum; the residual serum was still protective. Absorption of OspA and OspB antibodies from anti-B. burgdorferi serum eliminated the protective effect. Therefore, active immunization experiments were performed to determine the roles of OspB and flagellin in protective immunity and to determine whether protective immunity induced by OspA is dose dependent. Active immunization with recombinant OspA protected mice from infection with an inoculum of 10(4) spirochetes, but this protection could be overcome with a challenge of 10(7) spirochetes; OspB protected mice from infection with an inoculum of 10(3) spirochetes but was insufficient to fully protect against 10(4) organisms; and immunization with flagellin had no protective effect. These studies suggest that OspA and OspB, but not flagellin, play roles in protective immunity to spirochete infection.

Animals↗

PubMed1992

Long-term protection of mice from Lyme disease by vaccination with OspA.

Mice vaccinated with recombinant outer surface protein A (OspA) have been shown to be protected from infection with Borrelia burgdorferi, the agent of Lyme disease, when sacrificed 14 days after challenge with an intradermal inoculum of the spirochete. To determine whether infection was not merely delayed and that protection was long-lasting, we sacrificed vaccinated mice 60, 120, and 180 days after challenge; and to determine whether vaccinated mice retained their immune state over long periods, we challenged mice with B. burgdorferi 60, 90, 120, and 150 days after vaccination. The results of both groups of experiments show that the mice remained free from infection and disease and extend the usefulness of OspA as a vaccine candidate for Lyme borreliosis.

Animals↗

PubMed1992

Comparison of whole-cell antibodies and an antigenic flagellar epitope of Borrelia burgdorferi in serologic tests for diagnosis of Lyme borreliosis.

A recombinant protein (p41-G) of an antigenic region of flagellin was used in a standard and amplified enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis. Comparable sensitivities (88 to 94%) were noted when sera from 17 persons who had erythema migrans and antibodies to whole-cell B. burgdorferi were tested against the p41-G antigen. In tests of a second study group of 36 persons who had erythema migrans but no detectable antibodies to whole-cell B. burgdorferi, 3 (8%) were positive when the p41-G antigen was used. Assay specificity likewise increased when the p41-G fragment was included in an ELISA with human sera containing treponemal antibodies. Recombinant flagellar proteins of B. burgdorferi, such as the p41-G antigen, can be used in an ELISA and may help confirm Lyme borreliosis during early stages of infection and improve specificity.

Antibodies, Bacterial↗

PubMed1991

Molecular mapping of Osp-A mediated immunity against Borrelia burgdorferi, the agent of Lyme disease.

It is paradoxical that although antibodies to the outer surface protein (Osp) A of Borrelia burgdorferi protect mice against infection and that immunization of uninfected mice with Osp-A is protective, antibodies to Osp-A induced early in natural infection of mice are not curative. A region recognized by a neutralizing mAb is also recognized by sera from chronically infected or immunized mice but is not bound by sera from mice infected for 15 days. Infection in mice, despite the presence of early Osp-A antibody, may therefore be explained in part by the lack of response to this epitope. Sera from infected humans recognizes this region, although in this case the immune response to Osp-A occurs only late in infection. Nonetheless, the fact that both human sera and sera from mice immunized with Osp-A bind a conformationally dependent epitope that localizes to the same region tentatively suggests that humans are able to respond to a protective epitope and that an Osp-A-based vaccine may elicit protective immunity in humans.

Animals↗

PubMed1991

The use of the polymerase chain reaction to map CD4+ T cell epitopes.

CD4+ T cells recognize processed exogenous antigen in the form of peptides bound to syngeneic major histocompatibility complex class II molecules on antigen-presenting cells. We have developed a novel and convenient method to synthesize and map CD4+ T cell epitopes of cloned antigens using polymerase chain reaction (PCR)-directed construction of genes expressing recombinant protein fragments. Unique restriction sites incorporated into the PCR primers were employed for the unidirectional cloning of gene fragments into a bacterial expression vector that can be induced to high-level expression. The bacterial lysate could be used directly in T cell proliferation assays. Overlapping recombinant fragments spanning the entire protein were generated and tested. The length of the sequence containing the epitope was further reduced by utilizing PCR to generate 3' truncations. Finally, a small number of overlapping peptides spanning a sequence of 39 amino acids were synthesized to identify a thirteen-amino acid peptide epitope within chicken transferrin that stimulates the T helper cell clone D10.G4.1. PCR-directed construction of fragments of antigen allows for optimal design of strategies for the mapping and analysis of CD4+ T cell epitopes.

Amino Acid Sequence↗

PubMed1991

The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation.

Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation.

Amino Acid Sequence↗