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M M Simon

Publications and source records attributed to M M Simon.

At least 73 records · Page 4Linked to original sources
PubMed1994

From RNA to sequenced clones within three days: a complete protocol.

Detection of specific mRNA transcripts by the reverse transcription/polymerase chain reaction (RT/PCR) technique has become increasingly important. The technique is fast and has a very high resolution. Cloning of these PCR fragments into vectors is sometimes necessary for identification of alternative splicing products, for bacterial expression or for generation of a DNA probe. Here we present a complete protocol for RT/PCR, cloning and sequencing of PCR, cloning and sequencing of PCR products beginning with the total RNA and ending with the DNA sequence within three days. To illustrate the procedure as an example, a fragment of the human glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified from total RNA, cloned and partially sequenced. The protocol has been optimized for small scale to facilitate handling and to reduce costs.

Base Sequence↗
PubMed1994

A recombinant vaccine for Lyme disease.

We are studying processes leading to protective immunity in murine Lyme borreliosis in order to derive a suitable vaccine candidate for clinical use. Our work focuses on the role of the two main outer surface lipoproteins A and B (OspA and OspB) of the causative agent of Lyme disease, the spirochete Borrelia burgdorferi, as targets for its specific elimination. We could show that native and recombinant LipOspA induce monospecific immune sera able to passively transfer protection in SCID mice against experimental and tick-borne infection and disease. Recent results for phase I and II safety and efficacy trails are promising in demonstrating that the recombinant LipOspA vaccine candidate is safe and immunogenic and elicits borreliacidal antibodies.

Animals↗
PubMed1994

A 14,000 MW lipoprotein and a glycolipid-like structure of Borrelia burgdorferi induce proliferation and immunoglobulin production in mouse B cells at high frequencies.

Sonicated preparations of Borrelia burgdorferi are able to stimulate unselected resting BALB/c spleen cells to proliferate and to produce immunoglobulin in vitro. FACS analysis of target cells prestained with an integrated cell-surface marker as well as cell-depletion experiments demonstrate that the majority of responding lymphocytes are B cells. Limiting dilution analyses of resting B cells revealed high frequencies of cells producing IgM (F 1/11-1/62) or IgG (F 1/5-1/163) in response to B. burgdorferi sonicate (B.b. sonicate). These numbers were similar to those obtained with lipopolysaccharide (LPS) (IgM: F 1/20-1/84; IgG: F 1/14-1/85) or a synthetic lipopeptide of Braun's Escherichia coli lipoprotein (IgM: F 1/15, 1/19; IgG: F 1/148, 1/34). The mitogenic structure(s) expressed by B. burgdorferi is distinct from LPS, as similar proliferative responses were obtained with B cells from LPS-resistant (C57BL/10ScCr and C3H/HeJ) and LPS-susceptible (C57BL/10ScSn, C3H/HeN) mice. Furthermore, B-cell mitogenic properties were also found in two distinct fractions of a phenol-chloroform-petroleum ether extract of B. burgdorferi: they consisted of a lipoprotein distinct from the outer surface proteins (Osp) A and B and glycolipid-like structures, respectively. These data suggest that spirochetes express a multitude of distinct structures with mitogenic activity for B cells including various lipoproteins as well as glycolipid(s).

Animals↗
PubMed1993

A Saccharomyces cerevisiae upstream activating sequence mediates induction of peroxisome proliferation by fatty acids.

Peroxisome proliferation in Saccharomyces cerevisiae is induced by fatty acids via as yet unknown mechanisms. We have initiated a study of these mechanisms by identifying control sequences sufficient for fatty acid control of the CTA1 gene (encoding the peroxisomal catalase A). Promoter regions previously shown to be necessary for control were tested for their potential to mediate induction by oleic acid to a CYC1::lacZ fusion gene. A region previously demonstrated to control CTA1 via the ADR1 transcription activator (bp -156 to -184) does not mediate induction by oleic acid. In contrast, an adjacent sequence (-184 to -198) is sufficient for oleic acid induction, and a neighbouring element (-197 to -215) has marginal inducing activity. These two elements are characterized by a consensus sequence, 5'-CGGNNNTNA ('peroxisome box'), which is found in a number of S. cerevisiae peroxisomal protein-encoding genes. Mutation of either the CGG or the TNA block in the box has a dramatic down-regulating effect on the gene expression in oleic acid medium.

Base Sequence↗
PubMed1993

An ovalbumin peptide-specific cytotoxic T cell clone with antigen self-presentation capacity uses two distinct mechanisms to kill target cells.

Cloned 10BK.1 T cells with specificity for the ovalbumin peptide OVA257-264 are representative of a novel cell type within the CD8+ subset of T cells. In the presence and in the absence of added antigen presenting cells these T cells react toward antigen (Ag) by proliferation and lymphokine production. These data suggest self-presentation of the Ag by 10BK.1 cells. Here we present evidence that 10BK.1 cells exhibit cytotoxic activity that involves two different cytotoxic effector mechanisms. (i) One mechanism is fast killing activity, apparent within 4 hr. Constitutive mouse T cell-specific proteinase-1 (MTSP-1) activity, constitutive expression of MTSP-1 RNA, increased by Ag challenge, and Ag-inducible perforin RNA expression were observed. Electron microscopic dense granules of the CTL were oriented toward Ag-pulsed target cells. The fast form of cytotoxicity was triggered by Ag recognition and by contact with IL-2. (ii) The other mechanism is slow cytolytic activity, manifested within 2 days. This activity was contained in the supernatant of 10BK.1 cells after Ag activation. It was inhibited by monoclonal anti-TNF antibodies and therefore presumably represents TNF alpha/beta. Cytotoxic T cells capable of antigen self-presentation may be responsible for tissue damage during bacterial and viral infections.

Animals↗
PubMed1993

Distinct patterns of protective antibodies are generated against Borrelia burgdorferi in mice experimentally inoculated with high and low doses of antigen.

We have studied the development of clinical arthritis and the generation of protective antibodies in two normal, inbred strains of mice either infected by ticks or experimentally (subcutaneous) inoculated with increasing numbers of Borrelia burgdorferi organisms. AKR/N mice developed only mild and DBA/2 mice only marginal clinical arthritis irrespective of the route of infection or the numbers of spirochetes (10-10(8)) inoculated. In contrast, immunodeficient SCID mice developed severe chronic arthritis under similar conditions, but with a delayed onset at lower numbers of needle-inoculated spirochetes or after tick bite. AKR/N and DBA/2 mice inoculated with either 10(4) (and fewer) B. burgdorferi organisms or via experimentally infected ticks generated antibodies with specificities for a variety of B. burgdorferi antigens except those to the outer surface proteins A and B (OspA, OspB). In contrast, mice inoculated with more than 10(4) spirochetes (10(5)-10(8)) developed in addition antibodies to OspA and OspB. Most notably, all three types of immune sera taken from DBA/2 mice showed similar capacities to confer protection on SCID mice against subsequent challenge with viable B. burgdorferi organisms. The data not only demonstrate that the quality of humoral immune responses to B. burgdorferi in mice is determined by the antigenic load, they also indicate the existence of further protective antibodies with specificities distinct from OspA and OspB.

Animals↗
PubMed1993

Immune sera to individual Borrelia burgdorferi isolates or recombinant OspA thereof protect SCID mice against infection with homologous strains but only partially or not at all against those of different OspA/OspB genotype.

The outer surface proteins OspA and OspB of Borrelia burgdorferi have recently been demonstrated to be major target proteins for protective antibodies in mice against infection with the homologous spirochaetal strain. However, it has become clear from a variety of studies that B. burgdorferi isolates of different geographical origin and/or sources are heterogeneous and that they can be divided into at least six subgroups according to their distinct OspA/OspB genotypes. In order to analyse cross-protection between these subgroups we have now generated immune sera to various isolates of B. burgdorferi with different OspA/OspB genotypes. We show that passive immunization with antisera specific for whole spirochaetes or recombinant OspA of one spirochaetal isolate protects severe combined immunodeficiency mice against infection with strains of the corresponding OspA/OspB genotype but only partially or not at all against infection with isolates expressing distinct OspA/OspB genotypes. The incomplete protection mediated by individual antisera against independent isolates of B. burgdorferi suggests that an effective subunit vaccine against Lyme disease should consist of a mixture of OspA structures covering the heterogeneity of this protein within the species B. burgdorferi.

Animals↗
PubMed1993

Mode of inoculation of the Lyme disease agent Borrelia burgdorferi influences infection and immune responses in inbred strains of mice.

Mice were infected with Borrelia burgdorferi by infection via Ixodes ricinus and experimental inoculation to determine whether transmission rates of spirochetes and antibody responses are influenced. Mice infected by the natural route were substantially more infective for ticks; two- to sixfold more tick larvae were positive for B. burgdorferi than those fed on experimentally inoculated mice. In natural infection, spirochetemia may be greater or spirochetes may be more accessible for transmission. Thus, this form of xenodiagnosis could be used to determine levels of spirochetes in the vertebrate host. Similar levels of antibody were present in all mice; however, those infected by the natural route lacked antibodies to outer surface proteins (Osp) A and B. The small antigen dose given through a tick bite may not have been sufficient to induce rapid OspA or OspB antibodies, thereby allowing the later development of higher levels of spirochetemia.

Animals↗
PubMed1993

Alterations in gene expression associated with stepwise acquisition of malignancy in murine cytotoxic T cell lines.

We have isolated a series of variant cell lines from a murine CD8+ T cell clone representing distinct stages in stepwise acquisition of malignancy. A first type of variant has acquired independency of restimulation with MHC/Ag but has kept dependence on IL-2 for continuous growth in culture. A second type of variant has acquired, in addition, independency of IL-2. A third type of variant was isolated from tumors induced upon injection of IL-2 independent variants into syngeneic mice. Clonal relatedness between the cell line was ascertained by Southern blot and sequence analyses of their TCR beta chain genes. The cell lines were analyzed for their expression of genes typical for CD8+ T cells, using Northern blot hybridization, flow cytometry, and functional methods. Concentrating on the transition from IL-2 dependent to IL-2 independent cellular growth, we find the same triad of changes in two independently derived groups of variant cell lines: loss of expression of the CD8 alpha gene with concomitant loss of CD8 from the cell surface, a slight but significant overexpression of IL-2R alpha and beta chains with increased low affinity IL-2 binding sites, and constitutive overexpression of c-myc. Autocrine IL-2 dependent growth could be excluded. Expression of p56lck did not vary between the cell lines. We discuss the possibility that IL-2 independent growth may be associated with intracellular redistribution of p56lck from CD8 alpha to IL-2R beta, thus generating constitutively active IL-2R. Ex vivo established tumor variants differed from their parental culture cell lines by their constitutive secretion of IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Molecular and immunological characterization of a novel polymorphic lipoprotein of Borrelia burgdorferi.

We describe the cloning, expression, and molecular characterization of a novel polymorphic Borrelia burgdorferi lipoprotein recognized by monoclonal antibody LA7. Sequence analysis revealed an open reading frame encoding a 21,866-Da polypeptide (IpLA7). Comparison with other known proteins indicated sequence similarity between IpLA7 signal peptides and those of other prokaryotic lipoproteins, including the immunodominant B. burgdorferi outer surface proteins OspA, OspB, pC, and OspD. Both natural IpLA-7 and recombinant IpLA-7 could be biosynthetically labeled with [3H]palmitate. Upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114, IpLA7 was extracted together with other lipoproteins into the detergent phase. Indirect immunolabeling studies indicated that the epitope recognized by monoclonal antibody LA7 is mainly located in the periplasmic space. Two-dimensional gel electrophoresis and immunoblotting confirmed the calculated acidic pI of 5.7 for IpLA-7. The LA7 gene was shown to be species specific and to be located on the linear chromosome of B. burgdorferi. The analysis of 40 individual spirochetal isolates on the basis of restriction fragment length polymorphisms revealed considerable genotypic heterogeneity of LA7 corresponding to that previously found for ospA. Native IpLA-7 and recombinant IpLA-7 were recognized by immune sera from infected mice as well as some human sera derived from infected but healthy donors and may thus prove useful as an additional marker for the serodiagnosis of Lyme disease.

Amino Acid Sequence↗
PubMed1992

Differentially regulated malate synthase genes participate in carbon and nitrogen metabolism of S. cerevisiae.

We have isolated a second gene (MLS1), which in addition to DAL7, encodes malate synthase from S. cerevisiae. Expression of the two genes is specific for their physiological roles in carbon and nitrogen metabolism. Expression of MLS1, which participates in the utilization of non-fermentable carbon sources, is sensitive to carbon catabolite repression, but nearly insensitive to nitrogen catabolite repression. DAL7, which participates in catabolism of the nitrogenous compound allantoin, is insensitive to carbon catabolite repression, but highly sensitive to nitrogen catabolite repression. Results obtained with null mutations in these genes suggest that S. cerevisiae contains at least one and perhaps two additional malate synthase genes.

Amidine-Lyases↗
PubMed1992

Organization of the gene encoding the mouse T-cell-specific serine proteinase "granzyme A".

The mouse serine protease granzyme A is a member of a closely related family of T-cell-associated proteolytic enzymes, designated granzymes A-G. Previous studies have indicated that granzymes A and B are involved in various T-cell-mediated processes. Here we report the genomic organization of the granzyme A gene. We have cloned a 15-kb DNA fragment from a genomic library of a cloned CD8+ T-cell line and sequenced the exon-intron boundaries. The gene consists of five exons, and its genomic organization is very similar to that described for granzymes B, C, and F. In addition, we have sequenced 1.4 kb of the 5'-region and 1.1 kb of the 3'-region flanking the granzyme A gene. Putative promoter and enhancer elements were identified by sequence comparison with known consensus sequences. Some of these regulatory elements seem to be associated exclusively with granzyme A, whereas others are shared by members of the granzyme family.

Amino Acid Sequence↗
PubMed1992

Enhanced association of plasminogen/plasmin with lesional epidermis of bullous pemphigoid.

The distribution of plasminogen/plasmin, the central proteolytic component of the plasminogen activator/plasmin system was analysed in lesional skin of bullous pemphigoid by using monoclonal antibodies (MAbs) specific for distinct epitopes of the plasminogen/plasmin molecule. Four groups of MAbs were used: (i) MAbs HD-PG 1 and HD-PG 2, specific for epitopes associated with the lysine-binding sites I (kringle domain 1 + 2 + 3) and II (kringle domain 4) of plasminogen/plasmin, (ii) MAbs HD-PG 6 and HD-PG 7, specific for the lysine binding site I only, (iii) MAbs HD-PG 12 (formerly designated P 2) and HD-PG 18, specific for non-kringle domains of glu- and lys-plasminogen, and (iv) MAb HD-PG 13 which recognizes glu-plasminogen, only. The basal cell layers of normal skin consistently reacted with MAb HD-PG 12, whereas only faint staining was seen with the other MAbs in the same biopsies. In contrast, all anti-plasminogen/plasmin MAbs strongly stained lower and intermediate epidermal cell layers of fully developed bullous pemphigoid lesions.

Aged↗
PubMed1992

Coiling phagocytosis is the preferential phagocytic mechanism for Borrelia burgdorferi.

The uptake mechanism for the spirochete Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by electron microscopy for human and murine phagocytes. Spirochetes of both a low- and a high-passage strain were preferentially internalized by coiling rather than by conventional phagocytosis. The spirochetes engulfed by coiling phagocytosis were found to disintegrate in an organelle exclusion zone without evident participation of lysosomes. Preincubation of B. burgdorferi with monoclonal antibody to the spirochetal OspA enhanced phagocytosis in general but did not consistently influence the uptake mechanism. Quantitative and kinetic differences concerning the phagocytic rate and mechanism were evident between cells from different lineages, different human individuals, and mice and humans. In general, when few phagocytes participated in spirochete uptake, the active cells displayed a high ratio of coiling versus conventional phagocytosis. These results suggest that coiling phagocytosis of B. burgdorferi plays a critical role in the control of spirochetal infection. More detailed studies on the molecular basis of this phagocytic mechanism may lead to new insights into the pathogenesis of Lyme borreliosis, a disease which is frequently characterized by the host's inability to eliminate the pathogenic spirochete.

Adult↗
PubMed1992

Evaluation of genetic divergence among Borrelia burgdorferi isolates by use of OspA, fla, HSP60, and HSP70 gene probes.

In order to assess the genetic variation of immunologically relevant structures among isolates of the Lyme disease spirochete, Borrelia burgdorferi, three chromosomal genes encoding flagellin (fla) and the heat shock proteins HSP60 and HSP70, as well as the plasmid gene encoding outer surface protein A (OspA), from 55 different European and North American strains obtained from ticks and mammal hosts have been investigated by restriction fragment length polymorphisms (RFLPs). RFLPs of fla and the HSP60 and HSP70 genes revealed two distinct banding patterns (A and B) for each of the three genes and allowed the definition of four genomic groups [AAA, BBB, BBA, and B(A/B)A] for the three chromosomal genes. On the other hand, RFLPs of the OspA gene revealed six distinct banding patterns (types I to VI) making up six independent genomic groups for the plasmid-encoded gene. Furthermore, we have sequenced the chromosomal HSP60 gene from B. burgdorferi ZS7 and the plasmid-encoded OspA gene from two strains, ZQ1 and 19857. Alignment of the deduced HSP60 amino acid sequence from B. burgdorferi ZS7 (genomic group AAA) to a previously published HSP60 sequence derived from strain ACA-1, which according to the proposed classification is in a different genomic group (BBA), revealed a sequence identity of > 99%. Similar alignments of the OspA sequence of strain ZQ1 to those of other isolates that were published previously revealed sequence identities of between 70 and 94% among strains of distinct OspA genomic groups. These data indicate the existence of a restricted number of species-specific subgroups and clearly show that genotypic variation is much more pronounced for the OspA gene than for fla and the HSP60 and HSP70 genes. A phylogenetic tree constructed on the basis of distance matrix analyses of 12 OspA sequences supports the proposed classification of genomic groups of B. burgdorferi.

Amino Acid Sequence↗
PubMed1992

Cellular immune reactivity to recombinant OspA and flagellin from Borrelia burgdorferi in patients with Lyme borreliosis. Complexity of humoral and cellular immune responses.

Patients with Lyme borreliosis (LB) usually develop a vigorous T cell response against the causative pathogen Borrelia burgdorferi, but little is known about the antigens recognized in the cellular response. Therefore, T cell reactivities against whole bacteria, recombinant 31-kD (outer surface protein A, [OspA]), and 41-kD proteins (flagellin) from B. burgdorferi were studied in patients with LB, non-LB patients, and healthy donors. In parallel, specific antibodies were determined by Western blot analysis. Virtually all patients with LB exhibited marked cellular responses to whole B. burgdorferi, which were significantly elevated compared with the control groups in both early and late disease stages. However, analyses using the purified antigens OspA and flagellin revealed considerable heterogeneity in the cellular reactivities among individuals as well as variations during the course of infection. T cell responses to OspA were significantly increased in patients with early LB compared with both control groups whereas in late-stage disease responses only exceeded those of non-LB patients and were not different from normal donors. Cellular immune reactivities to flagellin were significantly higher only in early LB compared with both control groups. Reciprocally, several control subjects demonstrated marked cellular responses to OspA and flagellin, suggesting that reactions to these proteins may not always be related to LB. T cell reactivity did not correlate well with the presence of specific antibodies. Almost all seropositive patients in both early and late stage LB had serum antibodies against flagellin, but antibodies to OspA were detectable only in a subset of late LB sera. These data demonstrate the complexity of the humoral and the cellular immune responses to components of B. burgdorferi.

Adolescent↗