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Biomedical subjects

M M Simon

Publications and source records attributed to M M Simon.

At least 109 records · Page 6Linked to original sources
PubMed1990

The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene.

Monoclonal antibodies directed against the major Borrelia burgdorferi flagellar protein, the 41-kilodalton (kDa) protein flagellin, were used to monitor cloning and expression of the flagellin gene from a Borrelia burgdorferi genomic library. The structure of the gene was analyzed, and recombinant nonfusion flagellin was produced in Escherichia coli. A DNA sequence analysis of the 41-kDa flagellin gene revealed the presence of an open reading frame that encoded a protein having 336 amino acid residues and a calculated molecular mass of 35.8 kDa, indicating that there was posttranslational modification of the natural 41-kDa flagellin protein. Upstream from the AUG start codon sequence we identified motifs corresponding to consensus procaryotic promoter elements which could be utilized by the cloned flagellin gene when it was expressed in E. coli MC1061. The deduced flagellin protein sequence exhibited high levels of homology to sequences of flagellin proteins from Bacillus subtilis and Salmonella typhimurium. The levels of sequence similarity for the amino- and carboxy-terminal portions were about 65 and 56%, respectively. DNA sequence information on the flagellin gene was used to design oligonucleotides for gene amplification by the polymerase chain reaction method, and by using this method 0.01 pg of Borrelia burgdorferi DNA could be detected. Our results provide a basis for further biochemical analysis of the 41-kDa flagellin protein, investigation of the role of this protein in host-pathogen interactions, and development of a standardized reagent for diagnostic systems for Borrelia burgdorferi infections.

Amino Acid Sequence↗
PubMed1990

Regulation of mouse T cell associated serine proteinase-1 (MTSP-1) by proteinase inhibitors and sulfated polysaccharides.

Mouse T cell associated serine proteinase-1 (MTSP-1) is expressed by activated T cells in vitro and in vivo, stored in cytoplasmic granules and secreted upon their specific restimulation. The aim of this study was to identify those structures which may control proteolysis by MTSP-1 in vivo in the vascular and extravascular systems. Here we show, that MTSP-1 was effectively inhibited by vascular and extravascular serpins such as antithrombin III and Cl-esterase inhibitor, as well as by aprotinin and alpha 2-macroglobulin. On the other hand, interaction of MTSP-1 with sulfated glycosaminoglycans, i.e., heparin and chondroitin sulfate, led to increased enzymatic activity and an altered fine specificity of MTSP-1 for peptide substrates. These results suggest that the level of MTSP-1 activity as well as its specificity can be regulated by constituents of the extracellular environments.

Animals↗
PubMed1990

[Borrelia burgdorferi infection. I. Aspects of basic research, current approach for diagnosis and therapy].

In this review article the current concepts on the infection caused by Borrelia burgdorferi are presented. The problems associated with the diagnosis and therapy of the Borrelia burgdorferi infection are pointed out. A series of monoclonal antibodies recognizing Borrelia burgdorferi-associated antigens is presented. In collaborative research projects these antibodies were used for molecular biological (cloning and sequencing of Borrelia burgdorferi antigens) and immunological (protection experiments by passive transfer of monoclonal antibodies in an animal model) experiments. Taken together, these studies disclosed new aspects of the diagnosis and therapy of Borrelia burgdorferi infection. In the first part of the review article the immunochemical characterization of the aforementioned antibodies and their use for antigen detection by immunohistochemistry is described.

Algorithms↗
PubMed1990

Lyme borreliosis in the severe combined immunodeficiency (scid) mouse manifests predominantly in the joints, heart, and liver.

The authors describe the histopathologic evolution of Lyme disease in severe combined immunodeficiency (scid) and normal C.B-17 and C57BL/6 mice inoculated with Borrelia burgdorferi. Starting on day 7 after inoculation, all scid mice infected subcutaneously in the tail with a low-passage European tick isolate of B. burgdorferi had clinical evidence of arthritis characterized by reddening and swelling of tibiotarsal joints. Later on, other joints, ie, metatarsal and ulnacarpal joints were also affected. The infection of scid mice resulted in a persistent spirochetemia and the development of a multisystem disease with chronic progressive inflammation of joints, heart, and liver. Major histopathologic alterations included 1) severe joint lesions, characterized by the presence of hyperplastic inflamed synovial lining cells associated with the erosion and destruction of cartilage and/or bone; 2) pancarditis with infiltrations of mononuclear cells in the endocardium, myocardium, and pericardium; and 3) hepatitis with mononuclear cell infiltrations confined to the portal field and central vein, granulomatous reactions, and eventually the development of liver fibrosis. In addition, smaller more confined lesions were found in kidneys, lung, brain, and striated muscle. The inflammatory infiltrates in the various organs were associated mostly with Mac-1+ cells, largely monocytes and macrophages, as well as some polymorphonuclear leukocytes, but not B and T lymphocytes. Infective spirochetes could be readily isolated from blood and joints and were found at the site of inoculum and the myocardium. In contrast, subcutaneous inoculation of normal C.B-17 or C57BL/6 mice with spirochetes in general did not result in clinical signs of arthritis. Only 10% to 20% of the C57BL/6 mice, but none of the C.B-17 mice, showed clinical evidence of oligoarthritis, which appeared not before day 36 after inoculation. In general, the infection of normal mice resulted in minimal lesions in various organs, and no spirochetes could be visualized or reisolated from their tissues. The data demonstrate that Lyme borreliosis may develop in mice in the absence of detectable specific B and T cells and thus suggest an immunologic control of the disease in this species. The scid mouse model therefore can be used to define the components of the immune system responsible for the suppression and/or the progression of the disease.

Acquired Immunodeficiency Syndrome↗
PubMed1990

[Polymerase chain reaction in the demonstration of Borrelia burgdorferi DNA].

Borrelia burgdorferi is the etiological agent of Lyme disease. Certain diagnostic problems associated with Lyme disease could be solved if a sensitive detection method were available for the pathogen: the polymerase chain reaction for the sensitive detection of Borrelia burgdorferi is a possible candidate. The latest methods for the amplification of Borrelia burgdorferi DNA are discussed. In particular, a method for the amplification of a Borrelia burgdorferi flagellin (41 kDa antigen) gene segment by the polymerase chain reaction is presented. Owing to its high degree of conservation between different Borrelia burgdorferi isolates, the flagellin gene is a suitable target sequence for gene amplification. In conclusion, the polymerase chain reaction is now ready to be used on clinical specimens. This technique will allow investigation of aspects concerning latency and recurrency of Borrelia burgdorferi in infected individuals.

Borrelia burgdorferi Group↗
PubMed1989

The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis.

We report that the spirochete B. burgdorferi induces progressive polyarthritis and carditis in mice with severe combined immunodeficiency syndrome (scid) but not in normal C.B-17 mice. The onset and severity of the disease were dependent on (a) the viability; (b) the infectivity; and (c) the dose of inoculated B. burgdorferi organisms. Infective spirochetes were isolated from both blood and joints of inoculated scid mice. These findings suggest that B. burgdorferi-induced chronic arthritis and carditis in mice develops independently of lymphocyte function and makes the scid mouse an attractive laboratory model to study the role of the immune system in experimental Lyme Borreliosis.

Animals↗
PubMed1989

Protease nexin-1 complexes and inhibits T cell serine proteinase-1.

The T cell serine proteinase-1 (TSP-1) which most probably is involved in cell killing by cytotoxic T cells is inhibited by protease nexin-1 (PN-1), an extravascular serine protease inhibitor. The inhibition is irreversible and correlates with formation of SDS-stable complexes between the two proteins. Two distinct species of complexes (91 and 122 kDa) are observed upon SDS-PAGE analysis of the reacted proteins, indicating that PN-1 is capable of complexing and inhibiting both subunits of the homodimeric TSP-1 molecule. Heparin (2 micrograms/ml) increases the association rate constant from 4.2 x 10(4) M-1 sec-1 to 4.8 x 10(5) M-1 sec-1. These observations suggest that PN-1 may function as a major extravascular inhibitor of TSP-1 released from cytotoxic T lymphocytes.

Amyloid beta-Protein Precursor↗
PubMed1989

Expression of cytoplasmic granules with T cell-associated serine proteinase-1 activity in Ly-2+(CD8+) T lymphocytes responding to lymphocytic choriomeningitis virus in vivo.

Monoclonal antibodies (mAb) with specificity for the T cell-associated serine proteinase-1 of the mouse (MTSP-1) were used to study expression and storage of this enzyme in T lymphocytes during lymphocytic choriomeningitis (LCM) virus infection in vivo. Immunohistochemical analysis of splenic tissue at the peak of LCM virus-specific T cell-mediated cytolytic responses, i.e., at day 7 post infection, revealed high numbers of MTSP-1+ T lymphocytes in the interfollicular T cell-dependent area of the spleen. More than 50% of Ly-2+(CD8+) cells but only low numbers of Ly-2-(CD8-) cells, previously enriched by flow cytofluorometry, contained large amounts of cytoplasmic granules which stained both with MTSP-1-specific mAb and the esterase substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. These data demonstrate that in vivo generated LCM virus-induced cytolytic T lymphocytes develop cytoplasmic storage granules containing MTSP-1 and suggest that the mechanism of granule exocytosis is operative in vivo; possibly MTSP-1 is one effector molecule participating in the Ly-2+(CD8+) T lymphocyte-mediated control of virus infection.

Animals↗
PubMed1989

An inhibitor specific for the mouse T-cell associated serine proteinase 1 (TSP-1) inhibits the cytolytic potential of cytoplasmic granules but not of intact cytolytic T cells.

We have investigated a proteinase inhibitor, designed according to the preferred amino acid sequence that is cleaved by the murine T-cell specific serine proteinase 1 (TSP-1) for its effect on the cytolytic potential of cloned cytotoxic T-cell lines (CTLL) and of cytoplasmic granules, derived from these cells. Pretreatment of effector cells with H-D-Pro-Phe-Arg-chloromethyl-ketone (PFR-CK) prior to the cytotoxicity assay did not result in inhibition of cytolytic activity of three independent CTLL and did not effect their granule-associated TSP-1 activity after extraction with Triton X-100. Furthermore, PFR-CK did not interfere with cytolysis of target cells by CTLL when present for the entire incubation period. In contrast, PFR-CK inhibited in a dose-dependent manner both TSP-1 activity and the hemolytic/cytolytic potential of isolated cytoplasmic granules after their pretreatment with high-salt concentration. We interpret these results to mean that cytolysis of target cells by CTLL involves the granule-associated proteinase TSP-1, which probably becomes active upon exocytosis following effector-target cell interactions.

Animals↗
PubMed1989

Demonstration of antigen-specific T cells and histopathological alterations in mice experimentally inoculated with Borrelia burgdorferi.

Antigen-specific T-cell responses and histopathological changes were studied in mice experimentally inoculated with Borrelia burgdorferi B31. Inbred mice with different H-2 haplotypes and/or different genetic backgrounds were inoculated with B. burgdorferi organisms and tested for antigen-specific T-cell responses in vivo (delayed-type hypersensitivity [DTH]) and in vitro (T-cell proliferation). Comparable DTH responses were found after inoculation with either inactivated (in the presence of adjuvants) or viable microorganisms in all mouse strains, except BALB/c, irrespective of the H-2 haplotype (b, d, k, or s) tested and the sex of the animals. Moreover, in mice presensitized to B. burgdorferi, DTH responses could be induced only with antigen preparations derived from the corresponding strain but not with those obtained from either related spirochetes such as Treponema phagedenis and Leptospira interrogans or unrelated bacteria such as Mycobacterium tuberculosis. T cells isolated from lymph nodes or spleens of mice previously sensitized to B. burgdorferi but not those from naïve mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation. Histopathological examination of mice inoculated with viable B. burgdorferi organisms revealed significant perivascular infiltrates consisting mainly of mononuclear and a few polymorphonuclear leukocytes in different organs (brain, heart, lungs, liver, and kidneys) and the appearance of giant multinucleated cells within the spleen similar to those found in human skin specimens of patients suffering from cutaneous manifestations of Lyme disease. Our findings suggest that mice are a suitable animal model with which to study the immune response to B. burgdorferi and the pathogenesis of Lyme disease.

Animals↗
PubMed1988

Human T cell specific proteinase (HuTSP) is encoded by the T cell and natural killer cell specific human Hanukah factor (HuHF) gene.

HuTSP, a serine proteinase which is specifically associated with activated t lymphocytes, was purified to homogeneity and characterized. N-terminal amino acid sequencing revealed identity with a sequence predicted from the human Hanukah factor (HF) gene, which was isolated from a human T cell cDNA library. The dimeric structure of HuTSP, together with its extensive sequence homologies with the murine T cell specific proteinase, MTSP-1, suggests phylogenetic conservation of this serine proteinase family.

Amino Acid Sequence↗
PubMed1988

Determination of frequency of T cells expressing the T cell-specific serine proteinase 1 (TSP-1) reveals two types of L3T4+ T lymphocytes.

TSP-1 is a murine T cell-specific serine proteinase which is exclusively expressed in activated but not in resting T lymphocytes. Among T lymphocyte clones tested so far the enzyme was found to be associated with all Ly-2+ but only with a fraction of L3T4+ lines. Here we have applied a limiting dilution system to determine the frequency of precursor cells of resting L3T4+ and Ly-2+ lymphocytes which can be induced in vitro by antigen/lectin to express TSP-1. T cell subsets were either positively enriched by flow cytofluorometry cell sorting or by negative selection using monoclonal antibodies and complement. Following stimulation of lymphocytes in vitro, individual microwells were tested for growth by visual examination and for the TSP-1 protein/enzyme by analyzing cell lysates using either a specific rabbit anti-TSP-1 antiserum and/or the chromogenic model peptide substrate H-D-Pro-Phe-Arg-p-nitroanilide. Moreover, a large panel of L3T4+ and Ly-2+ T lymphocyte clones generated from primary cultures were similarly investigated. In some cell cultures the presence of TSP-1 was also tested on the mRNA level using a TSP-1-specific oligonucleotide probe. The data show that the majority, if not all, of antigen/lectin-induced-Ly-2+ T cells expressed TSP-1. In contrast, only 12%-27% of the growing lectin or antigen-reactive L3T4+ lymphocytes were positive for the enzyme. Studies performed in parallel with L3T4+ and Ly-2+ lymphocyte populations sensitized in bulk culture showed that under these conditions similar levels of TSP-1-specific mRNA and enzyme activity are detected in both subsets. The finding of primary L3T4+ T lymphocyte clones with distinct patterns of TSP-1 production provides evidence for the existence of two types of L3T4+ effector cells with different functional capacities. The data also suggest a cooperation between distinct L3T4+ lymphocytes for induction of optimal TSP-1 activity in L3T4+ T cells.

Animals↗
PubMed1988

Induction of T cell serine proteinase 1 (TSP-1)-specific mRNA in mouse T lymphocytes.

An oligonucleotide probe corresponding to nucleotides of a cDNA encoding the T cell-associated proteinase 1 (TSP-1) was chosen to study the induction and expression of TSP-1-specific transcripts in mouse T lymphocytes and tissues. We demonstrate that TSP-1 mRNA is only expressed in activated T lymphocytes and is absent from all mouse tissues tested including those containing resting mature T lymphocytes. Expression of the TSP-1 gene was observed in T lymphocytes in vitro in response to either phorbolester (phorbol 12-myristate 13-acetate), Ca2+ ionophore (A23187), lectin or alloantigen. In general, TSP-1 mRNA appeared and peaked later compared to interleukin 2 transcripts. Furthermore, TSP-1 mRNA was inducible in vitro in both Ly-2+ and L3T4+ lymphocyte populations treated with alloantigen and/or lectin. The transcription of the TSP-1 gene was always accompanied by the expression of proteinase activity. High expression of TSP-1 transcripts was also observed in in vivo derived T effector cells specific for lymphocytic choriomeningitis virus. However, TSP-1 mRNA was predominantly associated with virus-specific Ly-2+ T cells and correlated with their proteinase and cytolytic activities. The data suggest that TSP-1 gene transcription is a useful marker to characterize T effector cells in vitro and in vivo.

Amino Acid Sequence↗
PubMed1988

Esterase-18 (ES-18) of the house mouse (Mus musculus): biochemical characterization and genetics of an allozyme system linked to chromosome 19.

This study describes the biochemical characterization, genetic variation, and linkage of a codominantly inherited murine esterase, termed ES-18. The enzyme was identified by isoelectric focusing of supernatants obtained after centrifugation of tissue homogenates and subsequent staining for esterase using either alpha-naphthyl acetate or 4-methylumbelliferyl elaidate as substrate. ES-18 exhibited an organ-specific variation of the intensity pattern of bands as seen in kidney, spleen, and macrophages, respectively. Its activity was highly sensitive to inhibition by 1 mmol.liter-1 p-chloromercuriphenylsulfonate but was resistant to bis-p-nitrophenyl phosphate. Four allozymes could be distinguished in kidney supernatants obtained from the inbred strains C57BL/10Sn (ES-18A), MOLF/Ei (ES-18B), WLL/BrA (ES-18C), and CAST/Ei (ES-18D). The enzyme is shown to be controlled by a structural locus, Es-18, which resides on chromosome 19. The gene order Ly-1 - Got-1 - 4.7 +/- 1.6 - Es-18 is suggested.

Animals↗
PubMed1988

Alloreactive immune responses of transgenic mice expressing a foreign transplantation antigen in a soluble form.

Transfection of cells with the H-2Kk gene lacking the transmembrane and cytoplasmic segments resulted in secretion of the H-2Kk protein, as determined by immunoprecipitation with monoclonal anti-H-2Kk antibodies. Transgenic (H-2b X H-2d)F1 mice were established carrying integrated copies of the modified H-2Kk gene. Expression of the soluble H-2Kk antigen in the transgenic mice was demonstrated in cell supernatants of biosynthetically labeled splenic and thymic Con A blasts as well as bone marrow-derived macrophages. Soluble H-2Kk molecules were also present in the sera of the transgenic animals. No cell-surface expression of the H-2Kk antigen could be observed. In spite of the presence of the soluble H-2Kk molecules in the transgenic mice, the animals were able to generate H-2Kk-specific cytolytic T cells as well as antibody responses when stimulated with cell-surface-bound H-2Kk antigens. These responses were indistinguishable from those of the nontransgenic littermates. Possible explanations for the observed lack of tolerance are discussed.

Animals↗