Biomedical subjects

R A Flavell

Publications and source records attributed to R A Flavell.

At least 235 records · Page 13Linked to original sources

PubMed1995

Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis.

Using gene targeting in embryonic stem cells, we have derived mice with a null mutation in a DNA mismatch repair gene homolog, PMS2. We observed microsatellite instability in the male germline, in tail, and in tumor DNA of PMS2-deficient animals. We therefore conclude that PMS2 is involved in DNA mismatch repair in a variety of tissues. PMS2-deficient animals appear prone to sarcomas and lymphomas. PMS2-deficient males are infertile, producing only abnormal spermatozoa. Analysis of axial element and synaptonemal complex formation during prophase of meiosis I indicates abnormalities in chromosome synapsis. These observations suggest links among mismatch repair, genetic recombination, and chromosome synapsis in meiosis.

Adenosine Triphosphatases↗

PubMed1995

B7-1 expression by a non-antigen presenting cell-derived tumor.

The existence of a naturally occurring immunosurveillance against neoplastic cells is controversial. A difficulty with this concept is that tumor-specific antigen-reactive T cells would not be expected to become activated after encountering tumor cells, since T cells that bind to antigen in the absence of the costimulation provided by antigen-presenting cells may be inactivated. We studied a transgenic model of tumorigenesis where T cells reactive to a particular tumor-specific antigen are lost prior to the development of non-antigen-presenting cell-derived tumors; therefore, the tumors that develop are not subjected to immunosurveillance. We found that a tumor cell line derived from one such tumor expresses the T-cell costimulatory molecule B7-1, the expression of which is normally restricted to antigen-presenting cells. In addition, we found that several immortalized cell lines, which are nontumorigenic and thus have suffered only early genetic events in the tumorigenesis process, express B7. This suggests that a host cell can be induced to express surface B7-1 molecules after suffering an oncogenic insult, which might possibly be a primary mechanism of immunosurveillance against tumors.

3T3 Cells↗

PubMed1995

Borrelia burgdorferi genes selectively expressed in the infected host.

An immunological screening strategy was used to select microbial genes expressed only in the host. Differential screening of a Borrelia burgdorferi (the Lyme disease agent) expression library identified a gene (p21) encoding a 20.7-kDa antigen that reacted with antibodies in serum from actively infected mice but not serum from mice immunized with heat-killed B. burgdorferi. Selective expression of p21 in the infected host was confirmed by Northern blot analysis and RNA PCR. Further differential screening of the expression library identified at least five additional B. burgdorferi genes are selectively expressed in vivo. This screening method can be used to identify genes induced in vivo in a wide variety of pathogenic microorganisms for which a gene transfer system is not currently available.

Amino Acid Sequence↗

PubMed1995

Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme.

The interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the inactive IL-1 beta precursor to the proinflammatory cytokine. Adherent monocytes from mice harboring a disrupted ICE gene (ICE-/-) did not export IL-1 beta or interleukin-1 alpha (IL-1 alpha) after stimulation with lipopolysaccharide. Export of tumor necrosis factor-alpha and interleukin-6 (IL-6) from these cells was also diminished. Thymocytes from ICE-/- mice were sensitive to apoptosis induced by dexamethasone or ionizing radiation, but were resistant to apoptosis induced by Fas antibody. Despite this defect in apoptosis, ICE-/- mice proceed normally through development.

Amino Acid Sequence↗

PubMed1995

Class II transactivator regulates the expression of multiple genes involved in antigen presentation.

CIITA (a major histocompatibility complex [MHC] class II transactivator) has been shown to be required for the expression of MHC class II genes in both B cells and interferon gamma-inducible cells. Here we demonstrate that CIITA not only activates MHC class II genes but also genes required for antigen presentation. Mutant HeLa cells, defective in the expression of classic MHC class II genes, invariant chain, and the newly described human histocompatibility leukocyte antigen-DM genes, were used to study the role of CIITA in the regulation of these genes. Upon transfection with CIITA cDNA, the mutant cells expressed all three genes, suggesting that CIITA is a global regulator for the expression of genes involved in antigen presentation.

Antigen Presentation↗

PubMed1995

Vaccination against Lyme disease caused by diverse Borrelia burgdorferi.

Diversity and mutations in the genes for outer surface proteins (Osps) A and B of Borrelia burgdorferi sensu lato (B. burgdorferi), the spirochetal agent of Lyme disease, suggests that a monovalent OspA or OspB vaccine may not provide protection against antigenically variable naturally occurring B. burgdorferi. We now show that OspA or OspB immunizations protect mice from tick-borne infection with heterogeneous B. burgdorferi from different geographic regions. This result is in distinct contrast to in vitro killing analyses and in vivo protection studies using syringe injections of B. burgdorferi as the challenge inoculum. Evaluations of vaccine efficacy against Lyme disease and other vector-borne infections should use the natural mode of transmission and not be predicated on classification systems or assays that do not rely upon the vector to transmit infection.

Animals↗

PubMed1995

Evaluation of a Lyme disease enzyme immunoassay using the 41-G fragment of flagellin.

An immunogenic region of the Borrelia burgdorferi flagellin encompassing amino acids 197-273 and designated 41-G was evaluated as an antigen in an enzyme immunoassay (EIA) for Lyme disease on a routine basis in a reference laboratory. Sera that tested positive for Lyme disease by EIA using 41-G or the whole-cell Borrelia burgdorferi lysate as the antigen were also evaluated by immunoblot for reactivity with Borrelia burgdorferi, and the patient's clinical history was determined retrospectively by a questionnaire distributed to the referring physician. The sensitivity of the 41-G based EIA for the serologic diagnosis of Lyme disease, when compared with that of the Borrelia burgdorferi lysate EIA, was 70% (35 of 50). These data demonstrate that 41-G has utility as an antigen in EIA, although the sensitivity is at present less than that of the assay employing the Borrelia burgdorferi whole-cell lysate.

Antigens, Bacterial↗

PubMed1995

In the absence of a CD40 signal, B cells are tolerogenic.

When B cells are deprived of signaling through CD40, they exhibit the ability to induce T cell tolerance. The in vivo administration of anti-gp39 and allogeneic B cells diminished the ability of mice to mount an allogeneic response. Tolerance induction was specific for the haplotype expressed on the allogeneic B cells. Selective allospecific unresponsiveness was induced in the CD8 and CD4 compartments by the administration of anti-gp39 and class II-deficient B cells or class I-deficient B cells, respectively. As predicted by studies with anti-gp39 treatment, diminished allospecific responsiveness was induced by the administration of B cells to mice genetically deficient in gp39. Taken together, these data are consistent with the premise that deprivation of CD40 signaling engenders B cells with enhanced tolerogenicity. These studies provide insights into the tolerogenic capacity of resting B cells and outlines a practical approach to exploit this function.

Animals↗

PubMed1995

Coexpression of B7-1 and viral ("self") transgenes in pancreatic beta cells can break peripheral ignorance and lead to spontaneous autoimmune diabetes.

We evaluated the role of the costimulatory molecule B7-1 in overcoming peripheral ignorance in transgenic mice, which expressed the glycoprotein (GP) or nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) as the self-antigen in pancreatic beta cells. The viral transgenes or B7-1 alone did not induce autoimmune diabetes (IDDM). However, in bigenic mice expressing B7-1 and LCMV-GP, anti-self (viral) cytotoxic T lymphocytes (CTL) were activated without viral infection and spontaneous IDDM occurred. In contrast, bigenic RIP-B7-1 x RIP-NP mice with thymic expression of the self (viral-NP) antigen deleted the majority of their autoreactive CTL and did not develop spontaneous IDDM. However, these mice developed fast-onset IDDM 14 days after LCMV infection, whereas single-transgenic RIP-NP littermates developed IDDM only within 4-5 months. Rapid IDDM was associated with increased numbers of anti-self CTL and a predominance of IFN gamma produced by islet-infiltrating lymphocytes, whereas single transgenic RIP-NP littermates with slow-onset IDDM displayed less anti-self CTL and more IL-4- and IL-10-producing T lymphocytes in pancreatic infiltrates.

Animals↗

PubMed1995

Reconstitution of invariant chain function in transgenic mice in vivo by individual p31 and p41 isoforms.

MHC class II molecules associate with invariant chain (li) during biosynthesis. If facilitates folding of class II molecules, interferes with their association with peptides, and is involved in their transport. The murine Ii gene encodes two chains, p31 and p41. The role of these isoforms has been studied in vitro only in inappropriate antigen-presenting cells. To circumvent this problem, we have generated invariant chain-deficient mice (delta Ii), which express exclusively the p31 and p41 isoforms. Low level expression of p31 or p41 is not sufficient for rescuing high levels of cell surface class II expression. However, low levels of the typical compact dimer conformation indicative of tight peptide binding are observed. Thus, both isoforms participate in class II folding and assembly. Furthermore, p31 and p41 retrieve the CD4+ T cell population, which is reduced in the (delta Ii) mice. Moreover, the immune response to protein antigen is restored by both isoforms.

Amino Acid Sequence↗

PubMed1995

Direct physical interaction involving CD40 ligand on T cells and CD40 on B cells is required to propagate MMTV.

The propagation of mouse mammary tumor virus (MMTV) has been analyzed in mice defective for expression of CD40 ligand (CD40L). Mice with endogenous viral superantigen (SAG) delete T cells with cognate V beta independent of CD40L expression. Nevertheless, CD40L-mice do not show deletion of cognate T cells after being exposed to infectious MMTV and have greatly diminished viral replication. The response of CD40L- T cells to SAG in vitro is also impaired, but can be reconstituted by adding B cells activated by recombinant CD40L to express costimulatory molecules. Thus, direct CD40L-dependent B cell activation appears to be a critical step in the life cycle of MMTV. The initial step in SAG-dependent T cell activation, and hence the MMTV life cycle, may be mediated by non-B cells, because splenocytes from B cell-deficient SAG-transgenic mice are able to activate cognate T cells.

Animals↗

PubMed1995

Efficacy of human Lyme disease vaccine formulations in a mouse model.

Although immunization with recombinant outer surface protein A (OspA) appears to protect mice against infection by the agent of Lyme disease, all reported experiments have involved formulations that would not be suitable for use in humans or have not used realistic challenges. This study was designed to determine whether vaccines prepared and used in a phase I human trial, including one currently being used for a phase II trial in sites with endemic Borrelia burgdorferi, conferred protection in the C3H/HeJ mouse model. The challenge was ticks collected from a major site of the trial. None of the vaccinated mice became infected or developed disease, whereas 60% of unvaccinated mice became infected. Spirochetes were destroyed within the guts of virtually all recovered challenge ticks. These preparations of recombinant OspA effectively induced immunity to protect mice from Lyme disease when bitten by ticks collected from a field trial site.

Adjuvants, Immunologic↗

PubMed1995

Mechanisms of immune tolerance induction through the thymic expression of a peripheral tissue-specific protein.

A major process through which the immune system becomes tolerant to self proteins involves the deletion of self reactive cells in the thymus. However, T cells reactive to peripheral tissue-specific proteins can escape this deletion and become tolerized in the periphery by a variety of mechanisms. We report here, contrary to expectation, that the pancreas-specific protein, elastase I, is also expressed at a low level in the thymus, and that this thymic expression contributes to tolerance induction. To study the mechanism of this tolerance induction, we utilized a double transgenic mouse model. In these mice the expression of a model protein, SV40 T antigen, is directed by the elastase I promoter and hence parallels elastase I expression in the pancreas and thymus. These mice were crossed with mice transgenic for a TCR specific for T antigen, so the majority of thymocytes and T cells in these mice express the transgene. In double transgenic mice we find that thymic expression of T antigen results in anergic thymocytes which also show a reduction of Th1 activity with no decrease in Th2 activity. These functional characteristics persist in peripheral T cells, but there is also a depletion in the number of T antigen reactive T cells in lymph nodes. Chimeras were constructed which directly demonstrated that the thymus is the site of tolerance induction and that the tolerizing element is thymic epithelium. We propose that the loss of Th1 activity as a consequence of the thymic epithelium being encountered by tissue-specific proteins results in the functional tolerization of CTL in vivo, despite the fact that CTL are fully functional in vitro. In this way autoimmune destruction is contained. Thymic expression of peripheral proteins may therefore be an additional way in which tolerance to peripheral proteins can be achieved.

Animals↗

PubMed1995

CIITA activates the expression of MHC class II genes in mouse T cells.

It has long been a puzzle that MHC class II molecules are expressed in human T cells after activation but not in mouse T cells; this expression is believed to play a role in the cell mediated immune response. Recently the MHC class II transactivator (CIITA) has been reported to be a major regulatory factor for both the constitutive and IFN inducible expression of MHC class II genes. Here we show that human T cells expressing MHC class II have CIITA transcripts while MHC class II-negative human T cells and mouse T cells do not. The expression of MHC class II genes in mouse T cells can be reconstituted upon transfection with the human CIITA cDNA. These data indicate that the expression of CIITA explains the expression or lack of expression of MHC class II in human and mouse T cells respectively.

Animals↗

PubMed1995

The hook protein of Borrelia burgdorferi, encoded by the flgE gene, is serologically recognized in Lyme disease.

The periplasmic flagellum of Borrelia burgdorferi consists of a unipeptide flagellar filament, a hook, and a basal body. Here, we report the cloning and expression of the hook gene, flgE, of B. burgdorferi N40. The flgE gene is 1,119 nucleotides long and is located on the 950-kb linear chromosome of B. burgdorferi. The primary protein sequence of FlgE shows 73% similarity to the FlgE protein of Treponema phagedenis and approximately 50% similarity to the FlgG proteins of both gram-positive and gram-negative bacteria. The flgE gene was cloned into an Escherichia coli expression plasmid, pMX, to produce FlgE protein. Subsequently, FlgE murine antiserum was prepared by immunizing mice with the partially purified B. burgdorferi FlgE protein. By Western blot (immunoblot) analysis, the antiserum was found to react with a 40-kDa peptide in the whole-cell lysates, confirming the expression of the flgE gene in B. burgdorferi. Additionally, antibodies to FlgE were found in serum specimens from 19 of 42 patients with Lyme disease. Moreover, when other antigens, including 41G (the immunodominant domain of flagellin), OspE, OspF, and p22, were used to test for the development of corresponding antibodies in these patients, 67% of these patients (28 of 42) reacted to at least one of these five antigens, suggesting that a combination of FlgE with other available B. burgdorferi recombinant proteins is a good candidate for substrates in assays to aid in the diagnosis of Lyme disease.

Amino Acid Sequence↗

PubMed1995

Oral vaccination with an attenuated Salmonella typhimurium strain expressing Borrelia burgdorferi OspA prevents murine Lyme borreliosis.

Borrelia burgdorferi is the causative agent of Lyme disease. In the mouse model, protection is correlated with the development of antibodies to a major outer surface protein, OspA. In this study, we expressed OspA in an attenuated strain of Salmonella typhimurium and tested the efficacy of the transformed strain in protecting against disease. We show that mice inoculated by gavage developed high titers of anti-OspA antibodies and were protected against an intradermal challenge with the spirochete.

Administration, Oral↗

PubMed1995

Selection of variant Borrelia burgdorferi isolates from mice immunized with outer surface protein A or B.

A nonclonal population of Borrelia burgdorferi N40 (passage 3) that survived protective immunity following challenge inoculation of outer surface protein (Osp) A- or B-hyperimmunized mice were characterized for the molecular basis of evasion of immunity. Two of six B. burgdorferi isolates, cultured from OspA-immunized mice, had antigenic diversity in the carboxyl terminus of OspA and did not bind to the protective OspA monoclonal antibody designated IXDII. However, OspA-immunized mice challenged with these variants were fully protected. Moreover, B. burgdorferi isolates with a point mutation in ospB, which results in a truncated OspB that does not bind to protective OspB monoclonal antibody 7E6C, were frequently enriched after infection of OspB-immunized mice. These studies suggest that the incomplete efficacy of an OspA- or OspB-based vaccine may be partly due to immunomediated in vivo selective pressure, resulting in the persistence of some spirochetes that do not bind to protective antibodies.

Animals↗

PubMed1995

A 55-kilodalton antigen encoded by a gene on a Borrelia burgdorferi 49-kilobase plasmid is recognized by antibodies in sera from patients with Lyme disease.

We have identified a 55-kDa antigen encoded by a gene on a 49-kb plasmid of Borrelia burgdorferi. The screening of a B. burgdorferi DNA expression library (N40 strain) with rabbit anti-B. burgdorferi serum and then with serum from a patient with Lyme disease arthritis revealed a clone that synthesized an antigen that was reactive with both sera. DNA sequence analysis identified an operon with two genes, s1 and s2 (1,254 and 780 nucleotides), that expressed antigens with the predicted molecular masses of 55 and 29 kDa, respectively. Pulsed-field gel electrophoresis showed that the s1-s2 operon was located on the 49-kb plasmid. Recombinant S1 was synthesized as a glutathione S-transferase fusion protein in Escherichia coli. Antibodies to recombinant S1 bound to a 55-kDa protein in lysates of B. burgdorferi, indicating that cultured spirochetes synthesized S1. Thirty-one of 100 Lyme disease patients had immunoglobulin G (IgG) and/or IgM antibodies to S1. IgG antibodies to S1 were detected by enzyme-linked immunosorbent assay and immunoblots in the sera of 21 (21%) of 100 patients with Lyme disease; 11 (27.5%) of the S1-positive samples were from patients (40) with early-stage Lyme disease, and 10 (16.7%) were from patients (60) with late-stage Lyme disease. Fifteen (38.5%) of 40 serum samples from patients with early-stage Lyme disease had IgM antibodies to S1. These data suggest that the S1 antigen encoded by a gene on the 49-kb plasmid is recognized serologically by a subset of patients with early- or late-stage Lyme disease.

Amino Acid Sequence↗