Faculty Bibliography

We sought to determine the prevalence of cytotoxic activity in fecal filtrates from persons with C. jejuni or C. coli enteritis. Stool specimens were collected from 20 persons with C. jejuni or C. coli enteritis, 20 persons with acute diarrheal illnesses of other causes, and 9 healthy, asymptomatic persons. Fecal filtrates were then incubated with Chinese hamster ovary (CHO) or HeLa cells. The fecal filtrate from 1 of the 20 (5%) persons with Campylobacter enteritis was cytotoxic for HeLa cells at a titer of 1:40, and 10 (50%) were cytotoxic for CHO cells at maximum titers of 1:20. Cytotoxic activity for CHO cells at a median titer of 1:20 was also present in 40% of the fecal filtrates from persons with diarrhea due to causes other than Campylobacter enteritis, and in 33% of filtrates from healthy, asymptomatic persons. The observed low level of cytotoxicity in fecal filtrates from all patient groups studied likely resulted from non-specific factors, unrelated to the pathogenesis of Campylobacter enteritis

Urease was purified 112-fold to homogeneity from the microaerophilic human gastric bacterium, Helicobacter pylori. The urease isolation procedure included a water extraction step, size exclusion chromatography, and anion exchange chromatography. The purified enzyme exhibited a Km of 0.3 +/- 0.1 mM and a Vmax of 1,100 +/- 200 mumols of urea hydrolyzed/min/mg of protein at 22 degrees C in 31 mM Tris-HCl, pH 8.0. The isoelectric point was 5.99 +/- 0.03. Molecular mass estimated for the native enzyme was 380,000 +/- 30,000 daltons, whereas subunit values of 62,000 +/- 2,000 and 30,000 +/- 1,000 were determined. The partial amino-terminal sequence (17 residues) of the large subunit of H. pylori urease (Mr = 62,000) was 76% homologous with an internal sequence of the homohexameric jack bean urease subunit (Mr = 90,770; Takashima, K., Suga, T., and Mamiya, G. (1988) Eur. J. Biochem. 175, 151-165) and was 65% homologous with amino-terminal sequences of the large subunits of heteropolymeric ureases from Proteus mirabilis (Mr = 73,000) and from Klebsiella aerogenes (Mr = 72,000; Mobley, H. L. T., and Hausinger, R. P. (1989) Microbiol. Rev. 53, 85-108). The amino-terminal sequence (20 residues) of the small subunit of H. pylori urease (Mr = 30,000) was 65 and 60% homologous with the amino-terminal sequences of the subunit of jack bean urease and with the Mr = 11,000 subunit of P. mirabilis urease (Jones, B. D., and Mobley, H. L. T. (1989) J. Bacteriol. 171, 6414-6422), respectively. Thus, the urease of H. pylori shows similarities to ureases found in plants and other bacteria. When used as antigens in an enzyme-linked immunosorbent assay, neither purified urease nor an Mr = 54,000 protein that co-purified with urease by size exclusion chromatography was as effective as crude preparations of H. pylori proteins at distinguishing sera from persons known either to be infected with H. pylori or not

Serologic studies in developed countries indicate that Helicobacter (formerly Campylobacter) pylori infection is uncommon until the third decade of life and achieves a peak prevalence of 50% in the seventh decade. In developing countries the epidemiology of H. pylori has not well been described. A sensitive and specific serologic assay for H. pylori infection was validated in Thai patients also studied by culture and histologic examination of biopsy specimens. The prevalence of H. pylori antibodies in persons from a rural Thai community began early (17.5% of children 5-9 years old), increased to 55% during the third decade of life, and peaked (75%) in the 30- to 49-year age group. At a Bangkok orphanage where enteric infections are hyperendemic, 74% of children 1-4 years old were seropositive. This study shows that the prevalence of H. pylori infection in Thailand is higher than in industrialized countries. The high infection rate at the orphanage suggests that person-to-person transmission of H. pylori may be occurring

Colonization of the gastric antrum by Helicobacter pylori (formerly Campylobacter pylori) has been associated with primary gastritis. We determined the frequency of colonization by H. pylori in gastric-antrum biopsy specimens from 93 children undergoing gastroscopy for the evaluation of upper gastrointestinal symptoms. We also determined H. pylori IgG antibody levels by enzyme-linked immunosorbent assay in coded serum samples from these children, family members, and control subjects of comparable ages. Among 27 children with primary, or unexplained, gastritis, H. pylori was identified by silver staining in 24 biopsy specimens and by culture in 22; specific antibodies were present in 23 children (96 percent). Three children with unexplained gastritis had no evidence of H. pylori in the antrum, nor did any of 13 children with secondary gastritis or any of 53 children with normal antral histologic features; specific antibodies were present in only 1 of these 69 children. H. pylori antibody was detected in 25 of 34 parents of colonized children, but in only 8 of 33 parents of noncolonized children (P less than 0.001). Of 22 siblings of children colonized by H. pylori, 18 had specific antibodies, as compared with only 5 of 37 controls (P less than 0.001). We conclude that H. pylori-specific IgG antibodies are associated with bacterial colonization of the gastric antrum by this organism. The intrafamilial clustering of H. pylori infection suggests that there may be person-to-person spread of these bacteria

We estimated the prevalences of Helicobacter pylori (formerly called Campylobacter pylori) infection and histologic gastritis in 113 asymptomatic persons, using endoscopic biopsy of the gastric antrum and corpus. Unsuspected lesions, mainly mucosal erosions, were revealed at endoscopy in 16 subjects (14 percent). Gastritis was found in 42 subjects (37 percent), of whom 36 (32 percent of the total) were found to be infected with H. pylori on the basis of hematoxylin-eosin staining. H. pylori was not found in any of the 71 subjects with normal histologic features. Gastritis and H. pylori were noted in both the antrum and corpus in 75 percent of those infected (n = 27). The prevalence of H. pylori infection increased from 10 percent (2 of 20 subjects) in those between the ages of 18 and 29, to 47 percent (7 of 15) in those between the ages of 60 and 69, but the effect of age did not reach statistical significance. The prevalence of gastritis increased significantly with advancing age. Stepwise logistic regression analysis revealed that the relative risk for H. pylori infection associated with recent (within six months) antibiotic use was 5.8 (95 percent confidence interval, 1.5 to 22.1), whereas the relative risk was 6.5 (95 percent confidence interval, 1.4 to 29.2) for those who had never used bismuth compounds. We conclude that histologic gastritis and H. pylori infection commonly occur in the stomach of apparently normal persons and increase in prevalence with advancing age. All the subjects with H. pylori infection had gastritis, suggesting a possible etiologic role for the bacterium in the histologic lesion

The humoral immune response to both Campylobacter jejuni cell surface antigens and to potential toxins of the organism was studied in 64 adults with inflammatory diarrhea. In an enzyme-linked immunosorbent assay (ELISA) for surface antigens, 17 (71%) of 24 persons with Campylobacter enteritis showed seroconversion in more than one immunoglobulin class, versus only 2 (5%) of 40 patients with non-Campylobacter enteritis. In a GM1, ganglioside-based ELISA for detecting serum IgG to cholera-like enterotoxin, only one patient studied showed seroconversion to the enterotoxin. Of 22 Campylobacter isolates studied for production of cholera-like toxin, none of the supernatants from the Campylobacter strains were positive. Supernatants were also tested for enterotoxin and cytotoxic activity on Chinese hamster ovary cells; all isolates were negative for enterotoxin activity. In contrast, cytotoxin was produced by 7 (32%) isolates but was usually low-level and was not neutralized by patient's serum. These findings indicate that production of cholera-like toxin and cytotoxin by Campylobacter strains in the United States occurs in few strains and that host immune response is absent; their biologic significance in the pathogenesis of Campylobacter infections remains unclear

Whole-cell, outer-membrane protein, flagellum-associated antigens and partially purified urease of Campylobacter pylori were analyzed by two-dimensional gel electrophoresis. C. pylori strains were readily distinguished from strains of Campylobacter jejuni, C. coli, and C. fetus by absence of major outer membrane proteins with Mrs of 41,000 to 45,000. C. pylori strains also lacked the acidic surface-array proteins at Mr 100,000 to 149,000 identified previously in serum-resistant strains of C. fetus. Surface labeling of intact C. pylori cells with 125I revealed two common major proteins, which we have designated protein 2 (pI 5.6 to 5.8, Mr 66,000) and protein 3 (pI 5.2 to 5.5, Mr 63,000). Proteins 2 and 3 were also the major components (subunits) observed in partially purified urease. Partially purified preparations of flagella consistently contained proteins 2 and 3. Thus, urease appears to be associated with both outer membranes and flagella of C. pylori. C. pylori strains also possessed an antigen at Mr 59,000 which was cross-reactive with antiserum against flagella of C. jejuni. However, the antigen did not appear to be associated with flagella per se in C. pylori. Protein 2 was unique to C. pylori among the Campylobacter species studied. It was not recognized by antibody against whole cells of C. jejuni or C. fetus or flagella of C. jejuni. Protein 3 was cross-reactive with antiserum against whole cells of C. jejuni and C. fetus, as were several other major protein antigens. Because protein 2 is a major outer membrane protein that is apparently unique to C. pylori, development of monospecific antibodies against this antigen may be useful for the identification of C. pylori in tissues, and purified antigen may be useful for serologic tests for specific diagnosis of C. pylori infections

STUDY OBJECTIVE: To determine the diagnostic value of assays to measure serum antibodies to Campylobacter pylori, and to use these assays to determine the prevalence of C. pylori infection in a healthy population. DESIGN: A survey of patients having endoscopies for upper gastrointestinal symptoms, patients with other gastrointestinal illnesses, and healthy controls. SETTING: Outpatients attending endoscopy suites in two university-affiliated medical centers. PATIENTS: One hundred and twenty patients who had gastroduodenoscopies, 61 patients with lower intestinal illnesses, and 166 healthy controls. INTERVENTION: Assay to detect serum IgA, IgG, and IgM antibodies specific for C. pylori. MEASUREMENTS AND MAIN RESULTS: Absorption with other gram-negative pathogens showed that IgG and IgA assays, but not IgM assays, were specific for C. pylori. In patients in whom C. pylori had been isolated and who had gastritis diagnosed by histologic methods, significantly higher mean IgA and IgG levels were seen compared with patients without demonstrable C. pylori or gastritis. The sensitivity and specificity of a positive value in both IgA and IgG assays were more than 93%. Among healthy persons, IgG and IgA antibodies were rarely seen in patients less than 20 years old, but antibody prevalence progressed with age, reaching 50% in patients more than 60 years old. High IgA and IgG levels to C. pylori in five persons tested remained stable for more than 1 year, suggesting the organism persists for at least that period. In 61 patients with acute bacterial enteritis, acute pancreatitis, Crohn disease, or ulcerative colitis, prevalence of antibodies to C. pylori was consistent with age and unrelated to current disease. CONCLUSIONS: Campylobacter pylori infection, which is highly associated with active gastritis, may be diagnosed by serologic assay. Acquisition of infection begins in adult life, and prevalence increases with age

Cell lysates and culture supernatants of 36 Campylobacter isolates from patients with enteritis were tested for cytotoxic activity on HeLa cells. Cytotoxic activity was considered Shiga-like if neutralized by monoclonal antibody to the B subunit of Shiga-like toxin I of Escherichia coli and rabbit anti-Shiga toxin. Fifteen of the Campylobacter isolates produced no detectable cytotoxin, 10 produced a non-neutralizable cytotoxin, and 11 produced low levels of a cell-associated SLT. However, under low stringency conditions no hybridization was observed between a DNA fragment containing cloned SLT-I genes and restriction enzyme-digested total DNA from a Campylobacter strain that produced low levels of a Shiga-like toxin I. The Shiga-like toxin neutralizing titers in sera from 15 patients with C. jejuni infections, 5 patients infected with S. sonnei, and 20 healthy persons were then determined. No rise in neutralizing titer between acute and convalescent sera of patients with C. jejuni infection or S. sonnei infection was observed, although 27% of C. jejuni-infected patients, 40% of S. sonnei-infected patients, and 30% of the healthy controls had neutralizing activity in their sera. These data indicate that low levels of Shiga-like toxin are produced by some Campylobacter isolates but that SLT is genetically distinct from the SLT-I toxin produced at high levels by certain E. coli. The findings also suggest that exposure to SLTs is common in the adult population but not as a consequence of infection with C. jejuni or S. sonnei