Serum of rabbits infected with Treponema pallidum (Nichols) inhibits in vitro transformation of normal rabbit lymphocytes.
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Animals↗
Biomedical subjects
J B Baseman
Publications and source records attributed to J B Baseman.
Capacity of virulent Treponema pallidum (Nichols) for deoxyribonucleic acid synthesis.
Treponema pallidum (Nichols) was extracted from infected rabbit tissue, and cell lysates were prepared for monitoring thymidine kinase and deoxyribonucleic acid polymerase activities. No thymidine kinase could be demonstrated in preparations of T. pallidum or the cultivable T. phagedenis biotype Reiter. Significant levels of deoxyribonucleic acid polymerase were detected in both treponemal samples. Interestingly, comparisons of polymerase activity among a spectrum of bacterial genera revealed a direct correlation between enzyme concentrations and estimated generation time. Incorporation of [3H]uridine and [3H]thymidine into macromolecules by intact T. pallidum and the Reiter treponeme was examined. Selective ribonuclease-deoxyribonuclease digestion and cesium chloride gradient banding demonstrated that T. pallidum, independent of the host, and T. phagedenis were capable of synthesizing deoxyribonucleic acid only from the [3H]-uridine precursor.
DNA, Bacterial↗
Isolation of mutants of Mycoplasma pneumoniae defective in hemadsorption.
Mutants of Mycoplasma pneumoniae incapable of hemadsorption were isolated by means of chemical mutagenesis. These hemadsorption-negative mutants did not attach to hamster tracheal rings in vitro at as high a frequency as that exhibited by the wild-type parent strain.
Bacterial Proteins↗
Two-dimensional gel electrophoretic comparison of proteins from virulent and avirulent strains of Mycoplasma pneumoniae.
The protein composition of the virulent M129 strain of Mycoplasma pneumoniae was compared to that of its homologous avirulent strain by the use of standard one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Forty-nine individual M. pneumoniae cell proteins were resolved by this method, and the virulent strain was shown to possess a single high-molecular-weight protein not present in avirulent cells. Variability in the resolution of this particular protein in one-dimensional gels prompted the application of two-dimensional gel electrophoresis to the analysis of M. pneumoniae cell proteins. The sequential use of isoelectric focusing in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension permitted the resolution of a least 142 individual M. pneumoniae cell proteins. Application of nonequilibrium pH gradient electrophoresis in the first dimension achieved the resolution of at least 20 additional basic proteins. Three proteins which are synthesized only by cells of the virulent strain, and not by the homologous avirulent strain, were identified by these two-dimensional gel electrophoresis techniques.
Bacterial Proteins↗
Surface-associated host proteins on virulent Treponema pallidum.
A surface coat of host serum proteins was detected on virulent Treponema pallidum by sodium dodecyl sulfate-gel electrophoresis. The loosely associated serum proteins could be removed by repeated washings in a protein-free medium. Washed T. pallidum retained the ability to readsorb numerous host proteins from rabbit serum as well as iodinated rabbit or human albumin. In addition, various avidly associated host serum proteins including albumin, alpha(2)-macroglobulin, transferrin, ceruloplasmin, immunoglobulin G, immunoglobulin M, and C3 were identified on the outer envelope of washed treponemes by an immunoadsorbent technique with protein A-bearing staphylococcus. Hyaluronidase treatment did not remove the avidly associated host proteins from the surface of washed treponemes, whereas trypsin treatment resulted in decreased levels of agglutination. Electrophoretic patterns of trypsin-treated treponemes showed that treponemal proteins as well as adsorbed host proteins were released concurrently by protease digestion. Reacquisition studies involving alpha(2)-macroglobulin and transferrin suggested the presence of noncompetitive binding sites for serum proteins on the treponemal outer envelope. Finally, differences among the T. pallidum preparations from individual rabbits with respect to incorporation of [(35)S]methionine, extent of agglutination with antisera, and length of time required for removal of avidly associated host proteins by trypsin treatment indicated biological variability among the treponemal populations.
Adsorption↗
Ribosomal ribonucleic acid synthesis by virulent Treponema pallidum.
Ribosomal ribonucleic acid (rRNA) synthesis by virulent Treponema pallidum was monitored by incorporation of [(3)H]uridine into trichloroacetic acid-precipitable counts and examination of radiolabeled rRNA on polyacrylamide gels. Verification that rRNA synthesis originated with T. pallidum was based upon co-electrophoresis with Escherichia coli rRNA, proportionate reductions in the amount of rRNA synthesized when numbers of treponemes were decreased, and inclusion of appropriate animal cell controls. The rate of treponemal rRNA synthesis was greater at temperatures of 37 and 39 degrees C than at 33 degrees C; rRNA synthesis was inhibited at 4 and 42 degrees C and was effectively inhibited by actinomycin D. Kinetic experiments indicated that the majority of rRNA synthesis occurred early after extraction of treponemes from infected rabbit testicular tissue. Polyacrylamide gel profiles demonstrated the capacity of virulent T. pallidum to synthesize and process RNA to 23s, 16s, and 4 to 5s classes. Although motility of T. pallidum appeared unaffected during longer periods of incubation, pulselabeling experiments confirmed significant reductions in the rate of rRNA synthesis. When the effect of various environmental conditions upon rRNA synthesis was investigated, optimal synthesis was found to occur in an atmosphere of 20% oxygen whereas virtually no synthesis was observed under anaerobic or low-oxygen conditions.
Animals↗
Cell-mediated immunity during syphilis.
Evidence is presented which reinforces the complexity of the host-parasite interaction during the course of syphilis. Infection with Treponema pallidum evokes a complicated antibody response and an assortment of cell-mediated immune reactions in the host. It appears that humoral immunity plays a minor role towards the complete elimination of syphilitic infection while the cellular limb of the immune response may be an important host defence mechanism. Information now available indicates that a state of anergy, or immunosuppression, exists in the early stages of human and experimental rabbit syphilis based upon negative skin reactions to T. pallidum antigen(s), the abnormal histological appearance of lymphoid organs, and impaired in vitro lymphocyte reactivity. It is also evident that in the later stages of the disease cellular immunity becomes activated as delayed type skin reactions can normally be elicited in tertiary syphilitics and lymphocyte behaviour in cell culture appears normal. Several mechanisms have been invoked to explain the delay in an effective immune response against syphilitic infection and the duration of the disease: (1) a capsule-like substance on the outer surface of virulant T. pallidum may act as a barrier against treponemicidal antibody; (2) this material and other biological properties of virulent treponemes could enable spirochaetes to escape being engulfed by macrophages and other phagocytic cells; (3) antigenic competition among different treponemal antigens causing partial tolerance; (4) T. pallidum infection may bring about the elaboration of immunosuppressive substances of host or treponemal origin which inhibit the proper function of lymphocytes, macrophages, and other cell types.
Animals↗
Surface parasitism by Mycoplasma pneumoniae of respiratory epithelium.
Identification of the attachment factor on virulent Mycoplasma pneumoniae organisms which permits surface parasitism of respiratory epithelium was attempted. Brief pretreatment of M. pneumoniae monolayers with protease prevented mycoplasma attachment ot sensitive host cells without reducing viability of the microorganisms. Gel electrophoretic analysis of mycoplasma proteins before and after exposure of intact mycoplasmas to protease revealed the absence of a major protein species (P1) in enzyme-treated preparations while other protein bands with the exception of P2 were virtually unaffected. The absence of P1 correlated with the failure of enzyme-treated mycoplasmas to attach to tracheal explants. P1 regeneration after protease treatment of mycoplasma monolayers was directly associated with reattachment capabilities in M. pneumoniae. Erythromycin inhibited P1 resynthesis, thus preventing resumed attachment activity by mycoplasmas. Lactoperoxidase-catalyzed iodination of intact M. pneumoniae organisms further confirmed that P1 was an external membrane protein and suggested that his surface component was required for the successful membrane-membrane interaction between host and parasite.
Animals↗
Summary of the workshop on the biology of Treponema pallidum: cultivation and vaccine development.
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Animals↗
Scanning electron microscopic study of hamster tracheal organ cultures infected with Bordetella pertussis.
Hamster tracheal organculture was employed as a model for the study of the pathogenesis of infection due to bordetella pertusis. Scanning electron microscopy provided a three-dimensional view of the surface infection of the tracheal explants. Phase I B. pertussis attached only to the ciliated epithelial cells, and a sequence of events involving the injury, expulsion, and destruction of these differentiated cells occurred. This in vitro model provides insights into the mechanisms by which B. pertussis mediates host cell injury at the site of infection.
Animals↗
Pathogenesis of infection with Bordetella pertussis in hamster tracheal organ culture.
Hamster tracheal organ culture was employed as a model for study of the pathogenesis of infection due to Bordetella pertussis. Infected tracheal explants were examined with light, immunofluorescence, and electrom microscopy. B. pertussis organisms preferentially attached to the ciliated cells, producing ciliostasis and marked destruction of the subcellular organelles followed by expulsion of these cells from the epithelial layer. Other nonciliated respiratory epithelial cells appeared to be unaffected. Metabolic studies on infected tracheal cultrues indicated that significant deficiencies in syntheiss of host cell protein accompanied early cytopathology. Similarities and differences in host cell and parasite interaction were noted between B. pertussis and other pathogenic agents studied in this system.
Amino Acids↗
Selective response of lymphocytes from Treponema pallidum-infected rabbits to mitogens and Treponema reiteri.
The in vitro response of peripheral blood lymphocytes from rabbit infected with Treponema pallidum was examined using various mitogens and avirulent Treponema reiteri. For the first 4 weeks after treponemal infection, the response of lymphocytes from syphilitic rabbits to phytohemagglutinin and pokeweed mitogen was markedly reduced in comparison to uninfected controls. Lymphocytes from both groups of rabbits responded normally to class-specific immunoglobulin anti-sera (anti-immunoglobulin M and anti-immunoglobulin G) and T. reiteri.
Animals↗
Parasitism by virulent Treponema pallidum of host cell surfaces.
The interaction between virulent Treponema pallidum extracted from infected rabbit testes and animal cells in culture was examined. The extent of treponemal attachment to monolayers of normal rabbit testicular and HEp-2 cells was dependent upon the incubation temperature and retained motility of the spirochetes. The specific orientation of treponemes to host cell surfaces was demonstrated by dark-field microscopic examination of wet-mount preparations and scanning and transmission electron microscopy. Once attached, T. pallidum organisms remained actively motile yet anchored in place by their terminal tapered structures. After several hours of co-incubation, maximal attachment was attained, and the degree of parasitism seemed regulated not only by available surface sites on individual host cells but also by the proposed membrane response of parasitized cells to continued exposure to treponemes. The avirulent strain, Treponema phagedenis biotype Reiter, did not adhere to monolayer cultures. Characterization of host cell determinants that permitted surface colonization by T. pallidum was attempted. Also, properties of virulent treponemes that enabled surface parasitism were monitored by measuring the effects of enzymes, detergents, and metabolic inhibitors on the host-parasite interaction. Results reinforced the specific nature of the treponemal attachment mechanism. Furthermore, the ability of convalescent rabbit sera to reduce attachment of treponemes to host cells suggested that surface structures on T. pallidum could be masked or inactivated by host components, thus providing a potentially effective research approach for investigating the pathogenesis of syphilis and screening appropriate vaccine candidates.
Antibodies, Bacterial↗
Development of of macrophage migration inhibition in rabbits infected with virulent Treponema pallidum.
Peritoneal exudate cells from rabbits infected with Treponema pallidum Nichols were used as indicators of macrophage migration inhibitory factor activity. Between 3 and 15 weeks after infection, the migration of peritoneal exudate cells was inhibited in the presence of 3 to 25 microgram of T. phagedenis biovar Reiter protein per ml. Before this period, the migration patterns of peritoneal exudate cells from infected animals were uninhibited and similar to those from noninfected control rabbits. These observations were correlated with the development of active cell-mediated immunity during experimental T. pallidum infection.
Animals↗
Selective in vitro response of thymus-derived lymphocytes from Treponema pallidum-infected rabbits.
The blastogenic response of nylon wool-separated peripheral-blood lymphocytes from Treponema pallidum-infected rabbits was tested in vitro with mitogens and T. pallidum antigens. The mitogenic response of the enriched T-cell population to concanavalin A and phytohemagglutinin was depressed during the first 3 to 4 weeks of infection, similar to the pattern observed with unfractionated cells. Shortly thereafter, levels of blastogenesis returned to values of uninfected cultures. Enhanced blast transformation was seen immediately when purified T-cells from infected rabbits were exposed in vitro to T. pallidum antigens. Although these relatively high levels of blastogenesis were maintained for the duration of the experiment, cultures of unfractionated lymphocytes from infected rabbits did not exhibit an increased blastogenic response to the same antigen preparation until 3 to 4 weeks after infection. Autologous serum from infected rabbits decreased the lymphocyte response to T. pallidum antigen. The stimulatory effects of anti-immunoglobulin G and lipopolysaccharide on nylon wool-fractionated or unfractionated lymphocytes from both infected and control rabbits were similar throughout the course of infection. During the first 6 weeks of experimental disease, there was a 25 to 31% increase in the number of lymphocytes circulating in the peripheral blood of T. pallidum-infected rabbits.
Animals↗
Anabolic potential of virulent Treponema pallidum.
Acrylamide gel autoradiography of 3H-labeled proteins from Treponema pallidum demonstrates that virulent treponemes incubated in vitro synthesize a spectrum of high-molecular-weight proteins. A comparison of the protein profiles of T. pallidum with the Reiter treponeme shows that T. pallidum possesses significant anabolic competence.
Autoradiography↗
Serum can initiate DNA synthesis in cells rendered unresponsive to insulin and somatomedin.
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Animals↗
Virulent Treponema pallidum: aerobe or anaerobe.
Substrate degradation and protein synthesis served as indicators of metabolism in virulent Treponema pallidum. Opitmal metabolic activity in these spirochetes was observed at 10 to 20% O2 concentrations, with markedly reduced activity at higher or lower O2 levels or under anaerobiosis; alternate functioning electron acceptors that might substitute for O2 were not found. Carbon monoxide and cyanide at concentrations that inactivate cytochrome oxidase were not effective metabolic poisons for T. pallidum, although Micrococcus lutea, a strict aerobe with cytochrome-dependent respiration, was inhibited under similar experimental conditions. Motility of virulent T. pallidum was vigorous in the presence of O2 and sluggish or inhibited in its absence, reinforcing the role of O2 in T. pallidum metabolism.
Aerobiosis↗