Detection of E. coli colonies expressing the v-sis oncogene product with monoclonal antibodies made against synthetic peptides.

A method for immunodetection of individual epitopes on eukaryotic proteins synthesized in E. coli colonies is described. The system is developed using monoclonal antibodies produced against the Ha-ras protein produced in E. coli JM103. Monoclonal antibodies made against a synthetic peptide from the v-sis oncogene sequence are then used to identify bacterial colonies in which the v-sis protein is being produced. Production of the v-sis protein by these E. coli colonies was confirmed by immunoblot analysis. The assay utilizes peroxidase conjugated anti-mouse immunoglobulin and 4-chloro-1-naphthol to detect the positive colonies and can detect on the order of 200 pg antigen per E. coli colony.

Cytotoxic activity in broth-culture filtrates of Campylobacter pylori.

Broth-culture filtrates of Campylobacter pylori induced non-lethal cytopathic effects in vitro in 7 of 9 mammalian cell lines tested. Transmission electronmicroscopy revealed that the response consisted of intracellular vacuolisation. Intestine 407 cells were among the most responsive and were used for routine assay. About 55% of isolates of C. pylori tested, originating from four geographic regions worldwide, produced cytotoxic activity. The activity was neutralisable by specific antisera to broth-culture filtrates or to sonicated bacteria but not by antisera to other bacterial preparations. Cytotoxic activity was heat-labile (70 degrees C for 30 min), was protease-sensitive and ammonium-sulphate precipitable. It did not pass through an ultrafiltration membrane with a nominal mol.-wt limit of 100 X 10(3). It was concluded that C. pylori can produce a factor that alters cultured cells in vitro. The relevance of this factor to the pathogenesis of gastritis associated with C. pylori remains to be determined.

Manifestations of the local gastric immune response in gnotobiotic piglets infected with Helicobacter pylori.

Helicobacter pylori, a human gastric bacterial pathogen, was inoculated into gnotobiotic piglets and manifestations of the resultant gastric inflammation was analyzed by in situ immunochemistry and flow cytometric analysis of isolated lamina propria leukocytes (LPL) and peripheral blood leukocytes (PBL) recovered from infected and control piglets. Gastric mucosa tissue sections from uninfected control piglets were essentially negative for cluster differentiation- (CD-) positive leukocytes. Failure to isolate significant numbers of LPL from the gastric lamina propria confirmed this observation. A local and systemic immune response occurs in piglets after infection with H. pylori. This is manifest by the appearance of cells associated with a local immune response in gastric mucosa. In gastric tissue sections from H. pylori-infected piglets, CD4-positive leukocytes were sparse and closely associated with developing lymphoid follicles whereas the CD8-positive cellular phenotype was abundant. The latter formed a continuous band in the lamina propria just above the muscularis mucosa. Perivascular accumulations of lymphocytes in the outer muscular tunic(s) were strongly positive for expression of CD8 antigen. Class II-positive cells were prominent in CD8 lymphocytic infiltrates, developing follicles and vascular endothelia but were uniformly absent from gastric epithelia even in sites overlying areas of immunocyte proliferation and infiltration. Leukocytes possessing the monocyte and granulocyte markers were rare. Plasma cells containing IgA were common in the periphery of developing lymphoid follicles or distributed as discrete foci around individual gastric pits. Fewer numbers of IgG- and IgM-positive plasma cells were identified. When the LPL flow cytometry data were compared with the flow cytometry data obtained from PBL in these same H. pylori-infected piglets, leukocytes bearing the CD8 marker predominated in LPL whereas leukocytes bearing the CD4-reactive and MHC class II markers predominated in PBL. Finally, local ELISA antibody responses were measured in mucosal explant culture supernatants and compared with in vivo antibody levels in sera, bile, and gastric juice. Antibody activity, specific for H. pylori, was detected in supermatants and serum in all three isotypes in actively infected piglets whereas gastric juice lacked antibodies. Gastric explants prepared from piglets in which infection had been successfully eradicated failed to produce local antibody into supermatant fluids. These data support the concept that the gastric inflammation observed is mediated by local immunological events.

Production of a cytotoxin by Helicobacter pylori.

Helicobacter pylori produces a cytotoxin that was initially detected as the ability of broth culture filtrates of this bacterium to induce intracellular vacuolation of cultured cells. Fifty-three percent of more than 200 isolates of H. pylori tested produce the cytotoxin, which appears to be unique to H. pylori. Results of characterization studies suggest that the cytotoxin is a high-molecular-weight protein. Detection of serum antibody to the cytotoxin by neutralization or immunoblotting indicates that this toxin is produced in vivo. The cytotoxin is more frequently associated with cases of peptic ulceration than with cases of gastritis; this finding suggests that the toxin contributes to the severity of disease associated with H. pylori infection.

Physiological and metabolic alterations accompanying systemic candidiasis in mice.

Mice challenged intravenously with 10(6) viable Candida albicans died between 1 and 16 days after infection. Near the time of death, over 98% of the recoverable fungi came from the kidneys. Physiologically, animals were in renal failure near the time of death as evidenced by elevated blood urea nitrogen (BUN) and blood creatinine levels and a creatinine clearance rate which was about one-half normal. No abnormalities in liver glucogen and blood glucose levels were detectable. When mice were challenged with 4.5 X 10(6) viable C. albicans, they all died within 12 h. Near the time of death they had normal BUN values and were hyperglycemic. In mice receiving 4.5 X 10(6) heat-killed C. albicans, no deaths occurred and liver glycogen, blood glucose, and BUN levels all remained within a normal range and were different from responses to bacterial endotoxin. Cumulatively, the results demonstrate two distinct syndromes for the pathogenesis of experimental C. albicans infections. At the lower dose, mice were in renal failure associated with progressive renal infection. At the higher dose, renal failure was not observed. If a toxin was associated with death from the latter dose, it was not similar to bacterial endotoxin.

Association of type 1 pili with the ability of livers to clear Salmonella typhimurium.

The role of type 1 pili in the adherence of Salmonella typhimurium strain SR-11 to hepatic sinusoidal cells was investigated. An average of 66.7% of piliated organisms was cleared by perfused livers on a single pass. Mannose and alpha-methyl-D-mannoside inhibited such trapping in a dose-dependent manner. Preincubation of the bacteria, but not the liver, with either sugar also inhibited trapping, suggesting that the sugar binds to bacterial, not hepatic, receptors. Significant numbers of previously trapped bacteria could be eluted by adding mannose to the wash medium. Bacteria with reduced piliation, obtained either by growing bacteria on agar or by using a nonpiliated variant of the parent strain, were trapped to a significantly lesser extent than the parent strain. The liver appears to selectively trap heavily piliated organisms since reperfusion of bacteria through a second liver results in significantly less trapping than occurs with the first perfusion. In vivo, the nonpiliated variant strain was cleared much more slowly than the piliated parent strain. Mannose and alpha-methyl-D-mannoside, but not glucose, decreased clearance rates of piliated organisms. Cumulatively, the data suggest that type 1 pili are a major factor in hepatic clearance of S. typhimurium.

Comparison of isolates of Helicobacter pylori and Helicobacter mustelae.

On the basis of analysis of protein profiles, isolates of Helicobacter pylori and Helicobacter mustelae were less than 40% similar. Cytotoxin produced by H. pylori was not detected in isolates of H. mustelae. Both bacterial species agglutinated human erythrocytes. These results substantiate a taxonomic difference between H. pylori and H. mustelae.

Antimicrobial therapies for Helicobacter pylori infection in gnotobiotic piglets.

Gnotobiotic piglets infected with Helicobacter pylori were treated with various antimicrobials as monotherapy and dual therapy, and the results were compared to those for piglets treated with a triple-therapy regimen (bismuth subsalicyclate at 5.7 mg/kg of body weight, metronidazole at 4.4 mg/kg, and amoxicillin at 6.8 mg/kg four times a day [QID]). Clearance of infection was assessed after 7 days of treatment, and eradication was assessed following 7 days of treatment and a 14-day posttreatment observation interval. Monotherapy with amoxicillin, clarithromycin, and ciprofloxacin cleared and eradicated the organism from porcine stomachs; monotherapy with metronidazole cleared the infection and eradicated it from some piglets. Metronidazole-resistant microbes were recovered from treated piglets which cleared but did not eradicate the infection. Monotherapy with bismuth subsalicylate, erythromycin, nitrofurantoin, and tetracycline in the dosage range of 5.0 to 7.1 mg/kg QID was less than 100% effective in clearance and eradication, in that these drugs cleared and/or eradicated the infection from some of the piglets but did not eradicate the infection from all of the piglets. Monotherapy with an H-2 receptor antagonist (ranitidine) or a proton pump inhibitor (omeprazole) was ineffective at either clearance or eradication. In vivo dose titrations with several effective monotherapies were performed to determine the lowest effective in vivo dose of drug. In piglets, eradication was associated with a statistically significant decline in serum H. pylori-specific immunoglobulin M (IgM) antibodies; the titers of both IgA and IgG also declined, but the values were not statistically significant. For many antimicrobials, piglets are more sensitive indicators of clearance and eradication than humans. These data establish the H. pylori-infected gnotobiotic piglet as a useful model for the identification of novel antimicrobials for the treatment of this disease and for drug assessment during preclinical evaluations.

Establishment of gastric Campylobacter pylori infection in the neonatal gnotobiotic piglet.

Campylobacter pylori, a gram-negative microaerophilic bacterium, has been implicated in the genesis of human gastritis, dyspepsia, and gastroduodenal ulceration. Previous attempts to reproduce the diseases in conventional laboratory animal species have been unsuccessful. To determine if neonatal gnotobiotic piglets were susceptible to C. pylori, we orally challenged two litters (n = 17) with 10(9) CFU after pretreating them with cimetidine. Controls housed in separate units received nothing or peptone water alone. Piglets were examined 1, 2, 3, and 4 weeks after challenge. Colonization by the bacterium and inflammation of the gastric mucosa persisted throughout the study period. Organisms were revealed by Warthin-Starry silver stain to reside between the mucus layer and the gastric epithelium. Culturing of samples from sites along the gastrointestinal tract revealed that the bacterium colonized essentially only the gastric and proximal duodenal mucosae. Gross pathological changes were restricted to the stomachs of infected piglets and consisted of submucosal edema, increased gastric mucus production, and progressive development of mucosal lymphoid follicles. Microscopic lesions consisted of transient neutrophilic infiltrates followed by diffuse and follicular infiltrations of mononuclear leukocytes into the mucosa and submucosa. Alcian blue-periodic acid-Schiff stains suggested that the infection resulted in the depletion of mucopolysaccharide production by deep gastric glands. These data indicate that gnotobiotic piglets reproduce many of the features of diseases associated with C. pylori in humans.

Antibody to cytotoxin in infection by Helicobacter pylori.

Gastrointestinal disease and colonization by Helicobacter pylori were determined in 36 asymptomatic volunteers and 30 symptomatic individuals undergoing endoscopy and biopsy. Serum antibody immunoglobulin G (IgG) and IgA to H. pylori were measured by enzyme-linked immunosorbent assay. Serum antibody to a cytotoxin produced by H. pylori was detected with a neutralization assay. Serum IgG was 95% predictive of infection by H. pylori, and serum IgA was 88% predictive. Antibody to the cytotoxin was detected in 12 of 18 infected individuals. Antibody to the cytotoxin was a highly specific (96%), but not a very sensitive (67%), indicator of infection by H. pylori. The neutralization assay was 87% predictive of infection. These data confirm the diagnostic value of serum antibody to H. pylori for the detection of infection. The toxin-neutralizing activity of sera from individuals infected with H. pylori suggests that the cytotoxin is produced in vivo. It may therefore contribute to disease associated with H. pylori.

Characterization and therapy for experimental infection by Helicobacter mustelae in ferrets.

BACKGROUND: Numerous clinical trials evaluating the efficacy of various antimicrobial compounds against Helicobacter pylori infection have been performed in humans. A convenient animal model for Helicobacter infection would facilitate the evaluation of novel therapies. These experiments were performed to evaluate the use of ferrets as a model of Helicobacter infection. MATERIALS AND METHODS: Ferrets were infected experimentally with Helicobacter mustelae and subsequently treated with bismuth subsalicylate (BSS) triple therapy (BSS, metronidazole, and amoxicillin), or left untreated. The status of infection and serology was assessed during treatment and for 8 weeks posttreatment. Seven ferrets successfully treated with triple therapy were challenged with H. mustelae and monitored for infection for an additional 5 weeks. RESULTS: Infection of ferrets by H. mustelae was accompanied by gastritis and a specific antibody response. Treatment of H. mustelae-infected ferrets with BSS suppressed bacterial growth in four of nine animals but did not eradicate infection. Triple therapy eradicated infection in all nine ferrets with a reduction in gastric inflammation. No relapse of infection occurred up to 8 weeks posttherapy. Challenge with H. mustelae of ferrets successfully treated with triple therapy resulted in a 100% rate of reinfection. CONCLUSIONS: H. mustelae infection can be eliminated by triple therapy, but this does not result in protective immunity against reinfection by H. mustelae. This model, using a strain of Helicobacter indigenous to the host, may be useful for assessing therapeutic efficacy of novel therapies for the treatment of human infection by H. pylori.