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M D Kramer

Publications and source records attributed to M D Kramer.

At least 127 records · Page 7Linked to original sources

Characterization of Borrelia burgdorferi associated antigens by monoclonal antibodies.

In this paper, we present a series of murine mAb recognizing B. burgdorferi antigens. The antibodies were characterized by immuno-blotting and immuno-fluorescence studies using isolates of B. burgdorferi from North America and Europe, respectively. Moreover, reactivity of the antibodies with recombinant B. burgdorferi flagellin and OspA was studied. The results suggest these anti-B. burgdorferi mAb as valuable tools for the serological analysis of B. burgdorferi isolates and for affinity-purification of the respective proteins. Moreover, these mAb appear suitable to classify antigenic variants of B. burgdorferi and to study the protective capacity of antibodies in a murine model for B. burgdorferi infection.

Animals↗

Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice.

We have recently shown that viable Borrelia burgdorferi organisms induce a chronic infection associated with arthritis and carditis in severe combined immunodeficiency (scid) mice but not in immunocompetent mice. The disease is similar to that found in patients suffering from Lyme disease. We now show that B. burgdorferi-specific immune mouse sera as well as a monoclonal antibody to the spirochetal outer surface antigen A (31 kDa) but not monoclonal antibodies specific for the 41-kDa antigenic component of the periplasmic flagella are able to prevent (or mitigate) the development of the disease in scid mice when passively transferred at the time of the bacterial inoculation. The identification of a B. burgdorferi-associated protective antigen suggests that the corresponding spirochetal protein should be tested as a vaccine against Lyme disease.

Animals↗

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↗

Identification of herpes simplex virus type 1 glycoproteins interacting with the cell surface.

To investigate the interaction of herpes simplex virus type 1 (HSV-1) with the cell surface, we studied the formation of complexes by HSV-1 virion proteins with biotinylated cell membrane components. HSV-1 virion proteins reactive with surface components of HEp-2 and other cells were identified as gC, gB, and gD. Results from competition experiments suggested that binding of gC, gB, and gD occurred in a noncooperative way. The observed complex formation could be specifically blocked by monospecific rabbit antisera against gB and gD. The interaction of gD with the cell surface was also inhibited by monoclonal antibody IV3.4., whereas other gD-specific monoclonal antibodies, despite their high neutralizing activity, were not able to inhibit this interaction. Taken together, these data provide direct evidence that at least three of the seven known HSV-1 glycoproteins are able to form complexes with cellular surface structures.

Adsorption↗

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↗

[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↗

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↗

[Acute febrile neutrophilic dermatosis (Sweet syndrome). Case report and immunohistologic study of neutrophil-associated proteolytic enzymes and their inhibitors].

A 59-year-old female patient who developed fever, headache, arthralgia and painful, raised red cutaneous plaques located predominantly on the upper trunk is presented. On the basis of the clinical and histological findings acute febrile neutrophilic dermatosis (Sweet's syndrome) was diagnosed. In affected skin we applied immunohistochemical methods to examine the topographical organization of neutrophil-associated proteinase/proteinase inhibitor systems, i.e. the PMN-elastase/alpha 1-proteinase inhibitor and the cathepsin G/alpha 1-antichymotrypsin systems. Cathepsin G, PMN-elastase and alpha 1-antichymotrypsin were found in association with the polymorphonuclear infiltrating cells, whereas alpha 1-proteinase inhibitor was localized exclusively in the subepidermal oedema. In conclusion, our immunohistological findings support the view that free PMN-elastase is involved in the pathogenesis of cutaneous lesions in Sweet's syndrome.

Dermatitis↗

[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↗

Ataxic hemiparesis--localization by MRI.

Ataxic hemiparesis is a recognized lacunar syndrome that has been demonstrated previously by neuropathologic examination and computed tomography. In a subgroup of ataxic hemiparesis, pontine lesions have been localized by magnetic resonance imaging.

Cerebellar Ataxia↗

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↗

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↗

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↗

Internal mammary artery bypass grafting: influence on recurrent angina and survival in 2,100 patients.

Internal mammary artery (IMA) bypass grafting to the anterior descending coronary artery was performed in 2,100 patients between January 1978 and July 1986. The average number of additional saphenous vein grafts (SVGs) per patient was 1.8. During the same period, 1,753 patients underwent coronary artery bypass grafting using an SVG (average number of grafts per patient, 3.2). The average patient age was similar: 62.3 years for IMA grafts and 64.7 years for SVGs. Men constituted two thirds of each group. Left ventricular function was impaired (ejection fraction less than 45%) in 1,071 (51%) of IMA grafts and 847 (48.3%) of SVGs. Other aggregate risk factors, ie, elevated blood pressure, diabetes mellitus, previous myocardial infarction, and congestive heart failure, were similar in each group. Operative results and postoperative mortality of the IMA and SVG patients were comparable. However, the long-term probability of cumulative survival and occlusion-free survival were significantly greater and the probability of recurrent angina and reoperative coronary artery bypass grafting were significantly less in IMA graft patients (p less than 0.015). The relative risk of occlusion in an SVG was 4 to 5 times greater than that of the IMA graft. These data indicate that a patent IMA graft to the anterior descending coronary artery protects against recurrent angina and death from cardiac-related causes, and that the IMA should be the conduit of choice.

Adult↗

Internal mammary artery bypass: effect on longevity and recurrent angina pectoris in 2900 patients.

Internal mammary artery bypass (IMA) to the anterior descending coronary artery was performed in 2900 patients from January 1978 to December 1987. The average age of the patients was 64 years. Males accounted for 68% (1972 patients) and 82% (2378 patients) were in New York Heart Association (NYHA) class III. Left ventricular function was impaired in 51%. The average number of additional saphenous vein grafts per patient was 1.8. The operative mortality was 1.6%. Mediastinitis occurred in 29 patients (1%). Reoperation for bleeding was necessary in 32 patients (1.1%). Perioperative myocardial infarction (MI) was seen in 58 patients (2%) and neurological complications occurred in 32 patients (1.1%). Repeat coronary angiography was performed in 703 patients (25%) and demonstrated a patency rate of 96% in IMA grafts and 81% in saphenous vein grafts (SVG). Survival at 9 years was 90% from all causes and 95% when noncardiac deaths were excluded. Recurrence of angina occurred in 522 patients (18%) and reoperation was performed in 15 patients (0.5%). During the same time period, 1783 patients underwent coronary artery bypass utilizing a SVG. Survival at 9 years was 78% from all causes and 83% when noncardiac deaths were excluded. Recurrent angina was present in 546 patients (39%). These data suggest that a patent-IMA to the anterior descending protects against recurrent angina and death from cardiac causes and should be the conduit of choice.

Angina Pectoris↗

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↗