Characterization of the heat shock response and identification of heat shock protein antigens of Borrelia burgdorferi.

The heat shock response of Borrelia burgdorferi B31 cells was characterized with regard to the heat shock proteins (Hsps) produced. Five to seven Hsps were detected by sodium dodecyl sulfate-gel electrophoresis and fluorography of proteins from cells labeled with [35S]methionine after shifts from 33 degrees C to 37 or 40 degrees C or from 20 degrees C to 33, 37, or 40 degrees C. Analysis of [35S]methionine-labeled Hsps by two-dimensional electrophoresis and autoradiography revealed 12 Hsps. Western immunoblot analysis with antisera to highly conserved Escherichia coli and Mycobacterium tuberculosis Hsps revealed a single 72-kilodalton (kDa) protein band that reacted with antibodies to E. coli DnaK and with antibodies to the M. tuberculosis 71-kDa Hsp homolog of E. coli DnaK. Two proteins with apparent molecular masses of 66 and 60 kDa reacted with antibodies against the M. tuberculosis 65-kDa Hsp homolog of E. coli GroEL. Human immune sera collected from patients with Lyme disease reacted with both the 66-kDa Hsp and the 60-kDa Hsp but failed to react with the 72-kDa Hsp. These data are discussed with regard to the possibility that host recognition of highly conserved epitopes of GroEL homologs of B. burgdorferi may result in autoimmune reactions causing arthritis and other pathologies.     

Monoclonal antibodies to outer membrane antigens of Vibrio cholerae.

Hybridoma-derived monoclonal antibodies were prepared against outer membrane antigens of four strains of Vibrio cholerae that were cultivated under iron-limited conditions, and these antibodies were partially characterized. We established a library of 66 hybridomas which produced monoclonal antibodies defining 16 different V. cholerae antigens. Two antigens (molecular weights, 18,000 and 112,000) were heat modifiable, whereas the reacting epitope of a third antigen (40,000-dalton-18,000-dalton doublet) was completely destroyed when it was heated at 100 degrees C. The 112,000-dalton heat-modifiable protein was an iron-regulated outer membrane protein. This protein bound 59Fe in vitro when it was combined with the V. cholerae siderophore-iron complex 59Fe-vibriobactin; it was also found in in vivo grown V. cholerae, as were three other antigens. A total of 26 hybridomas produced antibody to V. cholerae lipopolysaccharide. Of these, 12 were cross-reactive with lipopolysaccharides of other gram-negative bacteria, including 2 which recognized lipid A. Several of these anti-lipopolysaccharide monoclonal antibodies appeared to be lipopolysaccharide region specific. Some membrane antigens were strain specific, whereas others were common to both O group 1 and non-O group 1 vibrios.     

Human immune response to Vibrio cholerae O1 whole cells and isolated outer membrane antigens.

The serum immunoglobulin G (IgG) and mucosal secretory IgA (SIgA) response of human volunteers challenged with Vibrio cholerae O1 was analyzed for reactivity to V. cholerae O1 antigens by the immunoblot technique. Components of both in vitro- and in vivo (rabbit ligated ileal loop)-grown V. cholerae O1 were separated by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Postchallenge serum IgG reacted uniquely with 15 antigens and with greater intensity than did prechallenge serum with at least 16 antigens. Serum IgG and SIgA reacted with antigens present in preparations from the homologous challenge strain of V. cholerae as well as antigens from strains of heterologous biotype or serotype. These heterologous antigens may represent antigens responsible for protection to rechallenge with a heterologous strain of V. cholerae. All the antigens detected by postchallenge jejunal fluid SIgA had an apparent molecular size of less than 25 kilodaltons. Serum IgG and jejunal fluid SIgA also reacted with antigens unique to in vivo-grown cells and several antigens in outer membrane preparations, suggesting that studies of protective immunity and V. cholerae O1 pathogenesis should include examination of both in vitro- and in vivo-grown V. cholerae O1 cellular antigens.     

Stimulatory effect of Vibrio cholerae neuraminidase on the antibody response to various antigens

Vibrio cholerae neuraminidase (VCN) splits off peripheral sialic acids from cell membranes. Treatment of tumour cells with VCN increases their immunogenicity and it has been suggested that VCN may unmask hidden tumour specific antigens. On the other hand, it has been shown that VCN can affect the cell-cell interactions of immune cells in vitro and it seems possible that the enzyme may have a direct effect on the immune response to antigen in vivo.

The present report describes the effect of VCN on the antibody response of mice to SRBC, to antigens which do not contain peripheral neuraminic acid such as certain bacterial vaccines and rubella virus, and to the soluble antigen BSA.

VCN injected i.m. or i.p., but not i.v., together with the antigens increases; (1) the PFC response to SRBC, (2) the antibody response to various bacteria (E. coli, V. cholerae, S. typhimurium), (3) the antibody response to rubella virus, and inhibits tolerance induction by aggregate free BSA.

The optimal dose required to stimulate the antibody response is between 0.5 and 50 units per animal. Possible mechanisms of the adjuvant activity of VCN are discussed.

     

Adaptive response of Vibrio cholerae and Escherichia coli to nitrofurantoin.

Pretreatment with sublethal doses of nitrofurantoin induced adaptive response in both Vibrio cholerae and Escherichia coli cells as indicated by their greater resistance to the subsequent challenging doses of the same drug. Adaptive response was maximum corresponding to pretreatment drug concentrations of 0.40 microgram/ml and 0.015 microgram/ml respectively for V. cholerae OGAWA 154 (wild type) and E. coli K-12 AB 2463 (recA-) cells. Adaptive response was inhibited by chloramphenicol (100 micrograms/ml) indicating the need of concomitant protein synthesis. Induction of adaptive response in recA deficient E. coli cells indicated that it was different from the conventional "SOS" response. Melting temperature of DNA of V. cholerae cells subjected to adaptive (0.4 microgram/ml for 1 hr) and challenging (120 micrograms/ml for 1 hr) doses of nitrofurantoin (76 degrees C) was closer to that of native DNA (75 degrees C) vis-a-vis DNA isolated from nonadapted and drug treated cells (77.5 degrees C). Also, DNA isolated from V. cholerae cells subjected to adaptive and challenging doses of the drug revealed the presence of fewer interstrand cross-links (16% reversible DNA) vis-a-vis DNA from nonadapted but drug treated cells (55% reversible DNA). Photomicrographic studies revealed that V. cholerae cells that were nonadapted but drug treated grew into long filamentous forms (4.25 +/- 2.97 micron) whereas those subjected to both adaptive and challenge doses of the drug exhibited much less filamentation (2.08 +/- 0.84 micron) vis-a-vis native cells (1.42 +/- 0.5 micron). Similar results on DNA melting temperature, cross-links in DNA, and filamentation of cells were obtained for E. coli AB 2463 (recA-) cells subjected to adaptive and challenging treatments with nitrofurantoin. Almost equal degree of resistance against nitrofurantoin could be induced in both V. cholerae OGAWA 154 (wild type) and E. coli strain PJ3 (AB 1157 ada-) when these cells were pretreated with nontoxic doses of hydrogen peroxide or nitrofurantoin. Evidence obtained in this work on the nature of the nitrofuratoin induced adaptive response with particular references to the oxidative and/or alkylating DNA damages were discussed. Nitrofuratoin induced adaptive response appeared similar to that elicited by furazolidone in V. cholerae cells and appeared to be directed towards oxidative and not alkylating adaptive repair pathway.     

Heat shock response of spirochetes.

We examined the heat shock response of the pathogenic spirochetes Treponema pallidum, Borrelia burgdorferi, and Leptospira interrogans and certain saprophytic spirochetes. Cellular proteins synthesized after shifts to higher temperatures were [35S]methionine labeled and analyzed by gel electrophoresis and fluorography. Only T. pallidum failed to exhibit an obvious heat shock response. GroEL and DnaK homologs were identified in the various species, although these proteins were not thermoinducible in T. pallidum or Treponema denticola. DNA hybridization studies indicate that spirochetal groEL and dnaK genes are highly conserved.     

Heat shock protein 60 specific T-cell response in chlamydial infections.

Heat shock proteins (HSPs) of most pathogens, including Chlamydia, are major immune targets of both humoral- and cell-mediated immune mechanisms. During the last decade, many investigators have focused their research to elucidate the complex relationship of chlamydial HSPs, especially chlamydial HSP60, and the host immune response. A central issue is whether the pathologic mechanisms in chronic chlamydial diseases are associated with an enhanced immune response to chlamydial HSP60 which can mediate tissue destruction through cytotoxic reactions, or whether they are related to the Th2 type of response that eventually leads to partial or temporary suppression of an effective antichlamydial response. Our review highlights the available knowledge between immune responses to chlamydial HSP60 and chronic chlamydial infections in human.     

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.     

Heat shock protein bystander antigens for peptide immunotherapy in autoimmune disease

Mucosal administration of an antigen eliciting bystander suppression at the site of inflammation results in effective antigen‐specific immunotherapy for autoimmune diseases. Heat shock proteins are bystander antigens that are effective in peptide‐specific immunotherapy in both experimental and human autoimmune disease. The efficacy of preventive peptide immunotherapy is increased by enhancing peptide‐specific immune responses with proinflammatory agents. Combining peptide‐specific immunotherapy with general suppression of inflammation may improve its therapeutic effect.     

Development of an enzyme-linked immunosorbent assay for studying Vibrio cholerae cell surface antigens.

An enzyme-linked immunosorbent assay for the measurement of antibodies directed against cell surface antigens of Vibrio cholerae (CSA ELISA) was developed. NaN3-killed whole cells of V. cholerae, adsorbed to polystyrene tubes, were used as immobilized antigens. The assay was capable of detecting antibodies directed against lipopolysaccharide and non-lipopolysaccharide surface antigens. In addition, the CSA ELISA was capable of detecting non-vibriocidal antibody. An antiserum raised in rabbits by immunization with live V. cholerae 1418 (Ogawa, El Tor) was capable of reacting with various heterologous strains of V. cholerae used as immobilized antigens. Therefore, common antigens shared by V. cholerae strains could be detected by using the CSA ELISA.     

Berberine inhibits intestinal secretory response of Vibrio cholerae and Escherichia coli enterotoxins.

Eight strains of laboratory mice were susceptible to subclinical infections with Cryptosporidium sp. at 1 to 4 days of age, but only a transient infection could be established at 21 days of age or older. Immunosuppression of 21-day-old mice failed to render them more susceptible to infection. Laboratory storage conditions for Cryptosporidium sp. were investigated by titration in 1- to 4-day-old mice. Storage by freezing with a variety of cryoprotectants was unsuccessful, but storage at 4 degrees C in phosphate-buffered saline or 2.5% potassium dichromate was possible for 4 to 6 months.     

Effect of chemical and heat inactivation on the antigenicity and immunogenicity of Vibrio cholerae.

The effects of heat and chemical inactivation on the antigenicity and immunogenicity of Vibrio cholerae 1418 in rabbits were studied. V. cholerae 1418 was inactivated with heat and chemical inactivants (phenol or Formalin) alone or in combination. Enzyme-linked immunoassay systems employing whole cells of V. cholerae 1418, lipopolysaccharide, or flagella as immobilized antigens were used to measure the antibody response (immunoglobulins G and M) after parenteral immunization of rabbits with various inactivated whole-cell preparations. The "classical" whole-cell vaccine, produced by phenol treatment, was found to be a comparatively poor immunogen. When Formalin was used instead of phenol, the antibody response to all three enzyme-linked immunosorbent assay antigens was greatly increased. Immunoglobulin G titers to intact V. cholerae cells were as much as 100-fold higher in rabbits immunized with the Formalin-inactivated preparation as compared to the classical phenol-inactivated vaccine. Furthermore, antibody produced against the Formalin-inactivated preparation was capable of recognizing antigenic determinants expressed on the cell surface of several heterologous strains of V. cholerae. These results indicate that the antigenicity and immunogenicity of V. cholerae are greatly affected by the inactivation conditions employed for vaccine production and that Formalin is much superior to phenol as an inactivant under the conditions employed in the present study.     

Vibrio cholerae Hcp, a secreted protein coregulated with HlyA.

Hcp is a 28-kDa secreted protein of Vibrio cholerae regulated coordinately with the hemolysin, HlyA. Both proteins show a dependence on HlyU for expression, suggesting that Hcp may be secreted by V. cholerae in vivo. We have identified and sequenced two genes for Hcp, designated hcpA and hcpB (hemolysin-coregulated protein). The genes encode identical amino acid sequences. Both express a 28-kDa protein, despite open reading frames with only a 19-kDa capacity, suggesting that the Hcp protein runs aberrantly on polyacrylamide gel electrophoresis. There is no cleavage involved in secretion of Hcp from the cell, suggesting a novel mechanism of secretion. An hcp null mutant was constructed, and this strain displayed no deficiency in virulence or colonization in the infant mouse cholera model. From sequence data and primer extension analysis, we predict that the hcp promoter is the sigma 54 type, with a candidate integration host factor binding site upstream. Although hcp and hlyA are coregulated by HlyU, there are no obvious similarities between their promoters. We predict that an intermediate regulator may be involved in the activation of hcp by HlyU. This raises the possibility that HlyU is part of a regulatory cascade.     

Different types of monoclonal antibodies to Ogawa-specific and group-specific antigens of Vibrio cholerae O1.

Nine monoclonal antibodies to Ogawa-specific antigenic determinants of Vibrio cholerae O1 and seven monoclonal antibodies to the Ogawa-Inaba common antigenic determinants were obtained. Specificities and reactivities were examined by slide or microdilution agglutination methods, along with enzyme-linked immunosorbent assays and immunoblotting analysis. In both the Ogawa-specific and Ogawa-Inaba common groups, it was revealed that there were two types of antibodies. One type showed strong agglutination with both live cells and heat-killed (100 degrees C for 30 min) cells in the microdilution agglutination test and in the slide agglutination test (type 1), while the other showed rather weak agglutination with heat-killed cells, especially in the slide agglutination test (type 2). Electronic measurement of agglutinates from these antibodies revealed that the size of aggregates varied. In the case of the type 2 antibodies, the size of aggregates obtained with heat-killed cells was smaller than those obtained with Formalin-killed cells. Results of enzyme-linked immunosorbent assays and immunoblotting analysis showed that all 16 antibodies were to the lipopolysaccharide of V. cholerae O1.     

General Transduction in Vibrio cholerae.

Evidence was obtained for general transduction in Vibrio cholerae. Transduction of three amino acid markers and three antibiotic resistance characters was demonstrated using strains of biotype eltor and biotype cholerae. Some of the genetic characters were transduced from a biotype eltor donor (and its mutant derivatives) to biotype cholerae and eltor recipients. For the genetic traits examined, the frequencies of transduction ranged between 10(-5) and 10(-8). Maximal frequencies were obtained with transducing phage lysates that were irradiated with ultraviolet light. The development of a system of general transduction will now aid in fine structure analysis and detailed mapping of the chromosome of V. cholerae.     

Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1.

Vibrio cholerae serogroup O139, now considered to be the second organism capable of causing epidemic severe dehydrating cholera, contains a capsular polysaccharide which makes it difficult for it to be used in the conventional vibriocidal antibody assay optimized for V. cholerae O1. After modification of the procedure, which involved the use of specific bacterial strains, a lower bacterial inoculum, and increased amounts of complement, the vibriocidal antibody responses to V. cholerae O139 were measured in acute- and convalescent-phase sera from 33 V. cholerae O139-infected and 18 V. cholerae O1-infected patients and in single serum samples from 20 healthy control subjects. The responses in these individuals to V. cholerae O1 strains were also determined. Significant elevations in the homologous antibody response were found only in the convalescent-phase sera from both groups of patients with cholera. These findings may explain the basis for the lack of heterologous protection between the two serogroups of V. cholerae. Healthy controls had higher background levels of vibriocidal antibody to V. cholerae O1 than to V. cholerae O139.     

Heat shock protein 88 and Aspergillus infection.

Immunoblotting was used to dissect the antibody responses in the sera of 50 patients with proven invasive aspergillosis, 28 patients with suspected invasive aspergillosis, 35 patients with allergic bronchopulmonary aspergillosis, and 10 patients with an aspergilloma. This demonstrated the immunodominance of antigenic bands at 88, 84, 51, and 40 kDa. Monoclonal antibodies against the heat shock protein 90 complexes of Candida albicans and the water mold Achlya ambisexualis identified these four antigenic bands as homologous proteins. Similar antigens have been described in humans, mice, Saccharomyces cerevisiae, chickens, and Drosophila species. The antibody against A. ambisexualis has previously been shown to cross-react with antigens belonging to the human heat shock protein 90 complex. Aspergillus heat shock protein 90 was extracted from the sera of patients with invasive aspergillosis by affinity chromatography. This was done with both a rabbit hyperimmune antiserum raised against an extract of Aspergillus fumigatus NCPF 2109 and a monoclonal antibody against the heat shock protein 90 of C. albicans. In vivo expression of the antigen was demonstrated in an aspergilloma surgically removed from a patient. The role of the antigen as an allergen in allergic bronchopulmonary aspergillosis is also discussed.