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Biomedical subjects

J Holmgren

Publications and source records attributed to J Holmgren.

At least 163 records · Page 9Linked to original sources
PubMed1990

Purification of El Tor cholera enterotoxins and comparisons with classical toxin.

In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml-1 and 1 ng ml-1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 micrograms ml-1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.

Cholera Toxin↗
PubMed1990

Roles of different coli surface antigens of colonization factor antigen II in colonization by and protective immunogenicity of enterotoxigenic Escherichia coli in rabbits.

The roles of the subcomponents of colonization factor antigen II, the coli surface antigens CS1, CS2, and CS3, as colonization factors and protective antigens was studied in a nonligated rabbit intestine model (RITARD). Infection with enterotoxigenic Escherichia coli (ETEC) carrying CS3 alone or CS1 plus CS3 induced diarrhea in most (80%) of the rabbits, whereas nonenterotoxigenic strains expressing CS1 or CS2 rarely induced diarrhea. Strains carrying CS1, CS2, or CS3 alone were all shed in stools for a significantly longer period than normal fecal flora-type E. coli. Initial infection with ETEC positive for CS1 plus CS3 induced significant protection against disease caused by reinfection with a highly diarrheagenic dose of the homologous strain; rabbits previously infected with serotype-heterologous, nontoxigenic bacteria carrying CS1 only were also protected against this challenge, whereas no such protection was induced by serogroup-homologous E. coli carrying CS2 only. Animals previously infected with CS1-, CS3-, or CS1-plus-CS3-positive bacteria excreted the CS1-plus-CS3 challenge strain for a significantly shorter period than did "nonimmunized" rabbits, whereas initial infection with bacteria carrying CS2 only did not result in such reduced shedding. Monoclonal antibodies against CS1, CS2, or CS3 all protected against experimental infection with ETEC carrying the corresponding CS factor. These results suggest that all the subcomponents of colonization factor antigen II are colonization factors and may induce anticolonization immunity.

Animals↗
PubMed1990

Interferon production by Shigella flexneri-infected fibroblasts depends upon intracellular bacterial metabolism.

The role of bacterial invasion and subsequent intracellular metabolism or replication, or both, in the induction of interferon (IFN) production in primary cultures of murine embryo fibroblasts (MEFs) was examined. IFN production appeared to be dependent upon bacterial invasion. MEFs that were challenged with Shigella flexneri cultured at 30 degrees C to inhibit the temperature-dependent virulence gene expression that is essential for invasion failed to produce IFN. Furthermore, inhibition of S. flexneri invasion by pretreatment of MEFs with cytochalasin B resulted in a reduction in IFN production. Intracellular bacterial residence alone, however, was not sufficient for the induction of IFN production since an avirulent isogenic variant of S. flexneri which invades but fails to grow intracellularly did not induce IFN production. In fact, the blocking of bacterial RNA synthesis immediately after cellular uptake of S. flexneri by rifampin inhibited IFN production by MEFs. Transfer of the invasion-encoding plasmid to a noninvasive Escherichia coli strain conferred upon the bacteria the ability to invade MEFs and induce IFN production. These results suggest that the induction of IFN production in S. flexneri-infected fibroblasts requires bacterial invasion and intracellular bacterial metabolism or replication, or both.

Animals↗
PubMed1990

Recombinant fusion protein for simple detection of Escherichia coli heat-stable enterotoxin by GM1 enzyme-linked immunosorbent assay.

A recombinant gene fusion protein composed of an Escherichia coli heat-stable enterotoxin (STa) peptide epitope fused to the amino end of the cholera toxin B subunit was used to detect STa produced by clinical isolates of enterotoxigenic E. coli (STa-ETEC) by a single monoclonal antibody-based inhibition GM1 enzyme-linked immunosorbent assay. In this test, 100% sensitivity and 100% specificity were observed for use of the recombinant protein in either its purified form or as crude Vibrio cholerae culture supernatants in detection of STa-ETEC.

Amino Acid Sequence↗
PubMed1990

Development of oral vaccines against cholera and enterotoxinogenic Escherichia coli diarrhea.

An oral cholera vaccine consisting of the immunogenic but completely nontoxic B subunit of cholera toxin in combination with heat- and formalin-killed cholera vibrios has been developed. This vaccine, which was designed to evoke antitoxic as well as antibacterial intestinal immunity, has in extensive clinical trials including a large field trial been shown to confer, without any side-effects, protection against cholera lasting for at least 3 years. The vaccine also induced protection of shorter duration against diarrhea caused by enterotoxinogenic Escherichia coli (ETEC). Significant progress has also been made recently using recombinant DNA techniques towards development of a live attenuated oral cholera vaccine. Furthermore, new knowledge about virulence factors and protective antigens of ETEC has given promise that an effective oral ETEC vaccine may soon be developed combining a B subunit toxoid with inactivated ETEC expressing the most important colonization fimbrial antigens.

Administration, Oral↗
PubMed1990

New vaccines against bacterial enteric infections.

During the last 10 years, rapid progress in basic research and biotechnology related to enteric infection has now begun to have a substantial impact on vaccine development against these infections. Two new typhoid vaccines, one for oral administration and the other for injection, which have much fewer and milder side effects than previous whole-cell parenteral vaccines, have become available. An oral cholera vaccine has been developed which, without any adverse reactions at all, confers long-lasting protection against cholera. The new cholera vaccine also, through antitoxic immunologic cross-reactivity, protects significantly against diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Notable progress has also been made towards the development of an oral killed vaccine against ETEC diarrhoea as well as live attenuated vaccines against rotaviruses, cholera and shigellosis.

Bacterial Vaccines↗
PubMed1989

Immunohistological detection of fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies.

With the aid of a highly specific murine monoclonal antibody, F12, an immunofluorescence method was elaborated that allowed sensitive and specific detection of the ganglioside antigen fucosyl-GM1 (IV2FucII3NeuAcGgOse4Cer) in different types of human lung cancer and normal tissues. Nineteen of 21 cases of small cell lung cancer were positive with the F12 immunofluorescence method as compared to 2 of 10 squamous epithelial cell lung cancers and 1 of 5 large cell lung cancer specimens. Specimens of lung adenocarcinoma (8 cases) and bronchial carcinoid (3 cases) were all negative, as were 2 examined cases of neuroblastoma. No fucosyl-GM1 could be detected in normal lung and bronchus. However, in thymus, spleen, and lamina propria of the small intestine sparsely distributed clusters of small round cells were stained as well as intramural ganglionic cells of the small intestine and islet cells of the pancreas. All other normal tissues tested were negative. Results obtained with immunofluorescence closely agreed with immunochemical determination of fucosyl-GM1 in lipid extracts of tissues. Our findings suggest that fucosyl-GM1 is strongly associated with small cell cancer of the lung and demonstrate that this tumor-associated antigen can be detected with high sensitivity and specificity with an immunofluorescence method based on the use of the F12 monoclonal antibody.

Animals↗
PubMed1989

Cellular basis of immunomodulation by cholera toxin in vitro with possible association to the adjuvant function in vivo.

Cholera toxin (CT) is a potent oral immunogen that also acts as a strong mucosal adjuvant for immune responses to related as well as unrelated Ag. To elucidate the immunomodulating effects of CT at the cellular level we have examined interactions of CT with APC and with B and T lymphocytes in vitro. CT markedly stimulated the production of IL-1 from APC (mouse peritoneal macrophages or macrophage cell line P388D1) but did not induce Ia-Ag and had marginal, if any, effect in potentiating Ia Ag expression stimulated by rIFN-gamma on these cells. CT had differential effect on T cell proliferation in vitro, usually strongly inhibitory but on Con A-stimulated spleen cells during prolonged (greater than or equal to 5 days) culture or when added on day 4 or later to these cultures up to a two- to three-fold enhancement of proliferation was seen. CT-induced inhibition of T cell proliferation was associated with decreased production of IL-2 and anergy to exogenously added IL-2 despite apparently normal expression of IL-2R. Similar to what was found with T cells LPS-stimulated spleen B cells demonstrated both inhibition and enhancement of proliferation in the presence of CT: in high concentrations (greater than or equal to 10(-8) M) and early in culture (day 3) CT had a strong inhibitory effect on the proliferation of B cells, whereas later (day 6) and/or at lower CT concentrations (10(-9) to 10(-11) M) the proliferation was increased up to 10-fold. The net effect of CT treatment on Ig-production by LPS-stimulated spleen B cells was seen as an enhanced level of IgA and IgG but not IgM in culture supernatants. The differential effects of CT on the cells of the immune system observed in vitro may, singly or in combination, explain the immunostimulatory function of CT.

Adjuvants, Immunologic↗
PubMed1989

New cholera vaccines.

Development of improved cholera vaccines has progressed rapidly in recent years. An oral killed vaccine, designed to evoke antibacterial as well as antitoxic intestinal immunity, has proved to be completely safe and to protect against cholera for at least 3 years. This vaccine also confers substantial though more short-lasting immunity against diarrhoea caused by enterotoxigenic Escherichia coli. Significant progress has also been made towards developing a live attenuated cholera vaccine using recombinant DNA techniques. The advent of effective oral cholera vaccines gives hope for improved control of cholera in the Third World.

Administration, Oral↗
PubMed1989

Development of oral vaccines against enterotoxinogenic Escherichia coli diarrhoea.

Even though enterotoxin-producing Escherichia coli (ETEC) is the most important cause of diarrhoea in developing countries and among travellers, no vaccine for use in humans is yet available. New knowledge about virulence factors and protective antigens of ETEC, however, suggests that development of a useful vaccine may soon become possible. Such a vaccine should be given orally and ideally evoke both anticolonization and antitoxic immune responses in the gut. An oral cholera vaccine, containing a component (B subunit) which crossreacts immunologically with the major, heat-labile enterotoxin (LT) of ETEC, has been shown to afford significant protection against diarrhoea caused by LT-producing ETEC. Promising prototype oral ETEC vaccines combining B subunit toxoid with inactivated ETEC bacteria expressing the most prevalent colonization factor antigens (CFAs) have been developed, and work is in progress to find means for adding to this CFA-toxoid vaccine a component that could also provide immunity against heat-stable enterotoxin.

Antigens, Bacterial↗
PubMed1989

Recombinant system for overexpression of cholera toxin B subunit in Vibrio cholerae as a basis for vaccine development.

We have constructed an overexpression system in which the gene encoding the B subunit of cholera toxin (CTB) was placed under the control of the strong tacP promoter in a wide host range plasmid. Recombinant nontoxigenic classical and E1 Tor Vibrio cholerae strains of different serotypes harboring this plasmid excreted 10- to 100-fold higher amounts of CTB than any other wild-type or recombinant strain tested and may therefore be useful killed oral vaccine strains. The manipulations to place the CTB gene under tacP also included, by design, the introduction of single enzyme restriction sites for gene fusions to the CTB amino terminus. Cloning into these sites allows construction of CTB-derived hybrid proteins carrying various putative vaccine peptide antigens.

Animals↗
PubMed1989

Gamma-interferon-mediated down-regulation of electrolyte secretion by intestinal epithelial cells: a local immune mechanism?

Active chloride (Cl-) secretion by intestinal crypt enterocytes is the central pathophysiological disturbance in most cases of acute diarrhoea. We examined monolayers of the human intestinal cell line T84 mounted in Ussing chambers to see whether the T-cell lymphokine gamma interferon (IFN-gamma) might affect the Cl- secretory properties of these cells, which morphologically and functionally resemble native crypt enterocytes. Pretreatment of T84 cell layers with IFN-gamma for 24 h (but not for 3 h) markedly decreased the Cl- secretory response to vaso-active intestinal polypeptide (VIP) and to cholera toxin and carbachol without appreciably affecting the overall morphology, electrical resistance, or cyclic AMP response of the T84 cell monolayer. The IFN-gamma treatment, however, did induce subtle changes in the T84 cell membrane protein composition which might have affected ion channels regulating Cl- secretion. Our results may indicate a possible novel 'cell-mediated' immune mechanism through which activated gut T cells could modulate the extent of intestinal electrolyte and fluid secretion in, for example, enteric infections.

Carbachol↗
PubMed1989

Fusion of genes encoding Escherichia coli heat-stable enterotoxin and outer membrane protein OmpC.

The OmpC outer membrane protein of Escherichia coli was used as a carrier molecule for the nonimmunogenic heat-stable enterotoxin STa. Two fragments of different lengths of the gene encoding STa were fused in vitro to the 3' terminus of the truncated ompC gene. The resulting OmpC-STa hybrid proteins could be detected by L-[35S]cysteine labeling, and they were processed and thus exported. All synthesized hybrid protein remained cell bound and was found by fractionation mainly in the periplasm. Immunoblot analysis showed that the hybrid proteins reacted in vitro both with anti-OmpC and anti-STa antibodies, and immunization of rabbits evoked an antibody response to either of these proteins.

Antibodies, Bacterial↗
PubMed1989

Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary glands and extramucosal tissues.

Generation of local and systemic immune responses by the oral administration of antigens is frequently inefficient, requiring large quantities of immunogens and yielding only modest antibody responses. In this study, we have demonstrated that oral administration of microgram amounts of Streptococcus mutans protein antigen I/II covalently coupled to the B subunit of cholera toxin elicits vigorous mucosal as well as extramucosal immunoglobulin A and G antistreptococcal antibody responses in mice. These responses were manifested by the presence of large numbers of antibody-secreting cells in salivary glands, mesenteric lymph nodes, and spleens and by the development of high levels of circulating antibodies. This novel immunization strategy may find broad application in the construction of oral vaccines for the control of infectious diseases caused by pathogens encountered at mucosal and extramucosal sites.

Administration, Oral↗
PubMed1989

Adoptive transfer of gut mucosal antitoxin memory by isolated B cells 1 year after oral immunization with cholera toxin.

A protocol was elaborated for the adoptive transfer of lymphocytes from mice which were orally immunized with cholera toxin (CT) to enable the study of long-term gut mucosal immunological memory at the single-cell level. Mesenteric lymph node (MLN) cells were transferred 1 year after priming immunizations, and recipient animals were challenged perorally on days 1 and 2 with CT before sacrifice on day 6 to 7 following transfer of cells. Strong antitoxin ELISPOT spot-forming cell (SFC) responses were recorded in spleens, MLN, and laminae propriae (LP) of recipient mice. In contrast, no SFC were found in Peyer's patches. The magnitude of the response equaled that of the acute response seen after optimal oral CT immunization and was directly dependent on the number of transferred cells. The memory antitoxin response in MLN and LP required oral challenge with CT as opposed to the spleen SFC response, which could also be triggered by intravenous challenge with antigen. Spleen cells from mice immunized perorally with CT were as effective as MLN cells in transferring immunological memory detectable in the gut immune system. Irrespective of the tissue source of transferring immunological memory detectable in the gut immune system. Irrespective of the tissue source of the memory cells, the isotype distribution of the antitoxin SFC response in recipient mice was similar with predominantly immunoglobulin A (96%) in LP and immunoglobulin G (66%) in MLN and spleen. Transfer of antitoxic memory was completely abrogated by treatment of the cells with J11d monoclonal antibody and complement prior to their injection into recipient mice by was unaffected by treatment with anti-Thy-1.2 antibody and complement, suggesting that long-term gut mucosal memory is carried by B cells. Antitoxin B memory cells might help explain the long-term protection against recurrent disease seen in convalescents from cholera in cholera-endemic areas.

Administration, Oral↗
PubMed1989

Vibrio cholerae expresses cell surface antigens during intestinal infection which are not expressed during in vitro culture.

Vibrio cholerae O1 bacteria harvested directly from ligated or nonligated intestines of rabbits with experimental cholera expressed at least 7 to 8 novel, in vivo-specific cell envelope (env) proteins that were not found on vibrios after in vitro culture in various ordinary liquid media. At the same time, several of the env proteins ordinarily expressed in vitro had disappeared or become much reduced. The infection-induced novel env protein were immunogenic. In immunoblot analyses, antisera raised against in vivo-grown vibrios and then absorbed with in vitro-grown bacteria of the same strain specifically stained at least eight infection-induced antigens ranging from 62 to approximately 200 kilodaltons; absorption with washed in vivo-grown bacteria, on the other hand, removed the antibodies reacting with these antigens, indicating that the antigens were present on the bacterial cell surface. Conversely, antiserum against in vitro-grown bacteria reacted with several env antigens in in vitro-grown bacteria that were missing in the infection-derived vibrios. These adaptational changes were strikingly similar for different strains of cholera vibrios of both classical and El Tor biotypes. Most of the in vivo-specific proteins (with apparent molecular masses of approximately 200, approximately 150, approximately 140, 92, 68, 62, 43, and 29 kilodaltons) were not induced during cultivation of bacteria in iron-depleted medium and are probably not related to the iron-regulated env proteins known to be involved in iron transport systems.

Animals↗