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Biomedical subjects

D G Newell

Publications and source records attributed to D G Newell.

At least 55 records · Page 3Linked to original sources

Differential flagellin expression in a flaA flaB+ mutant of Campylobacter jejuni.

Campylobacter jejuni 81116 has two genes coding for flagellin, flaA and flaB. Fully motile wild-type C. jejuni bacteria express the flaA gene, with no flaB message being detected. A nonmotile flaA flaB+ mutant, R1, produced detectable levels of flagellin B which was incorporated into truncated flagella. After R1 had invaded INT-407 cells, a variant with increased motility, R1-V2, was isolated. R1-V2 produced full-length flagella and an increased amount of flagellin B. Transcriptional analysis showed that R1-V2 contained more flaB mRNA than its parental strain, R1. The flaB gene promoter sequence and primer extension experiments confirmed that transcription of the flaB gene is initiated from a sigma 54 promoter. Neither the promoter sequence nor the coding sequence of flaB had changed in R1-V2. In contrast to R1, R1-V2 no longer produced (truncated) flaA mRNA. The sigma 28 flaA promoter sequence was not changed in R1-V2. We propose that expression of the two flagellin genes in C. jejuni 81116 is regulated at the transcriptional level, in such a way that predominantly one gene at a time is transcribed. We compared the levels of invasiveness of the wild-type strain, R1, and R1-V2 for INT-407 cells. The shift in expression from flaA to flaB occurred not only during invasion assays but also under different conditions in the absence of eukaryotic cells.

Bacterial Proteins↗

Colonization of chicks by motility mutants of Campylobacter jejuni demonstrates the importance of flagellin A expression.

Campylobacter jejuni strain 81116 contains two flagellin genes, flaA and flaB. Wild-type (WT) bacteria express flaA only, but flaB can be expressed under certain conditions. We have determined the importance of flagella for colonization of the avian caecum, which appears to be the natural environment for these bacteria. Mutants in which flaA or flaB, or both had been inactivated, and motility variants, were investigated. Flagella are not a requisite for colonization, but mutants lacking both flagellin genes colonized less efficiently than WT. Inactivation of the flaB gene, which had no effect on bacterial motility, enhanced chicken caecal colonization 1000-fold compared to WT. A variant (SF-1) with flagella composed of flagellin A, but with poor motility, also colonized better than WT. Conversely, mutants with an inactivated flaA gene colonized 100- to 1000-fold less efficiently than WT, regardless of their motility conferred by truncated or full-length flagellin B flagella. These results suggest that the presence of flagellin A, rather than motility, is essential for optimal bacterial colonization of chicken caeca.

Animals↗

Seroepidemiology of Helicobacter pylori infection in vegans and meat-eaters.

An enzyme-linked immunosorbent assay has been used to diagnose serologically the prevalence of Helicobacter pylori infection in Asian life-long vegans. There was no difference in the seropositivity between these individuals and a group of age- and sex-matched Asian meat-eaters, indicating the meat consumption is not a risk factor for H. pylori infection. However, both Asian groups had a higher prevalence of infection than age- and sex-matched Caucasian meat-eaters. Additionally, the Asian individuals had a wider range of specific IgG antibody concentrations than the Caucasians. This did not appear to be due to antigenic cross-reactivity between H. pylori and Campylobacter jejuni. The significance of these observations to the establishment of cut-off levels for the serodiagnosis of certain ethnic groups is discussed.

Adolescent↗

Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation.

OBJECTIVE: To investigate the association between gastric cancer and prior infection with Helicobacter pylori. DESIGN: Case-control comparison of prevalence of IgG antibodies to H pylori in blood samples collected prospectively, before diagnosis of gastric cancer in the cases. Presence of H pylori antibody (greater than 10 micrograms IgG/ml) determined by enzyme linked immunosorbent assay (ELISA). SUBJECTS: 29 men with a subsequent diagnosis of gastric cancer and 116 aged matched controls selected from over 22,000 middle aged men participating in two ongoing cohort studies (the British United Provident Association study and the Caerphilly collaborative heart disease study), who had provided blood samples during 1975-1982. RESULTS: 20 of the 29 cases (69%) and 54 of the 116 controls (47%) were positive for H pylori specific antibody. The median specific IgG concentration was significantly higher in the cases than controls (90 micrograms/ml v 3.6 micrograms/ml, p less than 0.01). The estimated odds ratio for the risk of gastric cancer in those with a history of infection with H pylori was 2.77 (95% confidence interval 1.04 to 7.97, 2p = 0.039). CONCLUSIONS: H pylori infection may be an important cause of gastric cancer; between 35% and 55% of all cases may be associated with such an infection.

Adult↗

The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy.

Fast protein liquid chromatography and SDS-PAGE have been used to isolate and purify Helicobacter pylori urease. A nickel component of the urease was detected in the purified proteins by atomic absorption spectroscopy. The nickel was present only in the 61 kDa polypeptide and in the ratio of between five and six atoms to one molecule of urease, suggesting a hexameric structure. These results are discussed in relation to other bacterial ureases and urease activity at low pH.

Chromatography, Liquid↗

Gastric injury and invasion of parietal cells by spiral bacteria in rhesus monkeys. Are gastritis and hyperchlorhydria infectious diseases?

The possibility of using the rhesus monkey as a model for studying gastric function in the presence of infection with spiral bacteria was studied. Endoscopic evaluation of the gastric mucosa was performed under general anesthesia in 29 colony-bred rhesus monkeys, and gastric pinch biopsy specimens were obtained from each animal. On a separate day, gastric emptying and acid output were determined using a 99mTc dilution technique. Biopsy samples were fixed for light microscopy (H&E, Gram, and Warthin-Starry stains) and for transmission electron microscopy. The presence of spiral bacteria and gastritis was assessed and rated on coded slides. In 8 of 29 monkeys, Helicobacter pylori-like organisms were observed in close proximity to the mucosal epithelial cells or in the lumen of the gastric pits. In 14 other monkeys, "Gastrospirillum hominis"-like organisms were observed in the mucus covering the surface of epithelial cells, in the lumina of the gastric glands, and overlying parietal cells. Gastritis was present in 8 of 8 animals positive for H. pylori-like organisms, in 2 of 14 animals positive for "G. hominis"-like organisms, and in none of the uninfected monkeys, and the mean gastritis index was significantly greater in animals positive for H. pylori-like organisms. Moreover, acid output was significantly higher in monkeys positive for "G. hominis"-like organisms than in controls or animals positive for H. pylori-like organisms. Gastric emptying was not significantly different in the three groups. In conclusion, (a) H. pylori-like, but not "G. hominis"-like, organisms cause gastritis while not modifying acid output; (b) "G. hominis"-like, but not H. pylori-like organisms, invade and on occasion damage parietal cells while apparently causing hyperchlorhydria; and (c) the rhesus monkey appears to be a good model for the study of gastric infection with spiral bacteria.

Analysis of Variance↗

Localization of immunogenic regions on the flagellin proteins of Campylobacter jejuni 81116.

The purpose of this study was to localize antigenic regions on the flagellin protein of Campylobacter jejuni 81116. This strain has two flagellin genes, flaA and flaB, which are 95% identical; only flaA seems to be expressed in motile C. jejuni 81116 cells. Fragments of flaA and flaB were cloned in the bacterial expression vector pEX, and the expression products were incubated with flagellin-specific antibodies. Monoclonal antibodies to broadly cross-reactive epitopes recognized fragments that are located in the termini (CF16 and CF17) and in the center (CF15) of both flagellin A and B proteins. Most of the serotype-specific monoclonal antibodies (CF1, CF2, CD3, CF4, and CF13) reacted with only the center of flagellin A in an area where flagellin A and B differ in 6 amino acid residues. The epitopes in this area were further characterized by competitive binding experiments. The charge and molecular weight microheterogeneity of flagellin, as determined by two-dimensional gel electrophoresis, were unrelated to the expression of both flagellin genes or parts thereof.

Amino Acid Sequence↗

Serodiagnosis of Helicobacter pylori: comparison of enzyme-linked immunosorbent assays.

Enzyme-linked immunosorbent assays (ELISAs) have been developed to diagnose Helicobacter pylori infection. However, the methods are not standardized. We therefore prospectively evaluated the sensitivities and specificities of ELISAs developed in the United States and the United Kingdom in a study population comprising 41 consecutive symptomatic outpatients and 35 volunteers. At endoscopy, multiple biopsies were obtained for histology and culture and stained sections were graded for chronic gastritis, active chronic gastritis, and density of H. pylori. Serum samples were analyzed for H. pylori by ELISA. The first set of assays for immunoglobulin G (IgG) and IgA used a pool of sonicated isolates of H. pylori from five patients in the United States (antigen A). The second set of assays, developed in the United Kingdom, used three different antigens: antigen 1, an acid-extractable surface antigen; antigen 2, an acid-extractable antigen from an aflagellate variant; and antigen 3, a urease-containing fraction. Cutoff scores for positive results were determined a priori on the basis of previous serological studies. There was close agreement between histology and culture. In the study population, 36% of the individuals were H. pylori positive. The diagnostic value of the different ELISAs were highly comparable, and the crude antigens performed as well as the more purified antigens. The antigen A IgG had a sensitivity and specificity of 96 and 94%, respectively; the values for antigen 1 were 93 and 96%, respectively. The antigen A IgA and antigen 3 assays were the least sensitive tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

Estimation of prevalence of Helicobacter pylori infection in an asymptomatic elderly population comparing [14C] urea breath test and serology.

A non-invasive serological assay devised in this laboratory had a sensitivity and specificity of 100% as determined by culture and confirmed by histology in a group of 47 patients who had undergone endoscopy. The correlation between serology and the non-invasive [14C] breath test was very good. Only one of 24 culture positive patients was, while all 23 culture negative patients were, breath test negative. In a group of 46 healthy elderly persons, however, significant anomalies between serology and breath test were observed. Only 83% of the breath test negative persons were seronegative, while only 68% of the breath test positive persons were seropositive. These results can be explained in terms of age related atrophic gastritis and immune incompetence, causing reduced colonisation and decreased antibody production, respectively. These investigations suggest that non-invasive tests for H pylori infection may not be reliable in the elderly.

Adult↗

Virulence factors of Helicobacter pylori.

Helicobacter pylori can be considered a very successful organism effectively colonizing the majority of the world's population. Although various disease states associated with this infection have been described, the mechanisms of pathogenicity remain unknown. The easiest virulence factors to identify are those enabling the organism to colonize the hazardous microenvironment of the gastric epithelium, survive at this site, and multiply sufficiently for transmission to a new host. The factors identified to date include the bacterial enzymes urease and catalase, flagella, and lectin-like adhesins. In addition, it is proposed that the organism has evolved mechanisms to avoid the local antibody responses of the host. Several putative virulence factors that could directly cause gastroduodenal damage have also been identified. These include the direct tissue damage by cytotoxins or the products of urease activity and the indirect tissue damage due to disruption of mucin integrity. Such mechanisms may contribute to peptic ulcer formation; however, the chronic superficial gastritis most frequently associated with this infection is probably caused by immunopathologic events mediated by the host in response to the continued antigen load on the gastric mucosa.

Animals↗

Geographic association of Helicobacter pylori antibody prevalence and gastric cancer mortality in rural China.

To examine the geographic association between Helicobacter pylori infection and gastric cancer, we have assessed the prevalence of IgG antibodies to H. pylori in plasma samples taken in 1983 from 1882 men, aged 35-64 years, in 46 rural counties of the People's Republic of China. The gastric cancer mortality rates in these countries in 1973-75 varied from 3 per 1,000 (cumulative rate, 0-64 years) to 69 per 1,000, while the proportions of the population positive for H. pylori antibodies (based on an average of about 41 men per county) varied from 28% to 96%. After correction for the limited number of blood samples per county, the estimated correlation between H. pylori antibody prevalence and gastric cancer mortality was 40% (p = 0.02). No other type of cancer showed a significant association with H. pylori.

Adult↗

Antigenicity of fractions of Helicobacter pylori prepared by fast protein liquid chromatography and urease captured by monoclonal antibodies.

The antigenicity of Helicobacter pylori protein fractions separated by fast protein liquid chromatography size exclusion was investigated by EIA with sera from patients of well defined Helicobacter pylori status. The antigenic material of Helicobacter pylori was confined to fractions 8 and 14 to 21. Urease containing fractions (14/15) and flagella containing fractions (17/18) were identified. Fraction 8 non-specifically bound human immunoglobulin as demonstrated by the binding of Helicobacter pylori negative sera. The remaining fractions 14 to 21 when used individually as EIA antigens were 91-100% specific, however fractions 16 to 19 showed a reduced sensitivity (78%) compared with the acid extract (95%). The urease fractions were 91% sensitive. Purified urease antigen captured by antiurease monoclonal antibodies was 83% sensitive and 93.3% specific.

Adult↗

Investigation of the structure and localization of the urease of Helicobacter pylori using monoclonal antibodies.

The urease of Helicobacter pylori (formerly Campylobacter pylori) has been partly purified by fast protein liquid chromatography. This material contained 10 nm doughnut-like structures when examined by electron microscopy and comprised three major polypeptides (61 kDa, 56 kDa and 28 kDa). Only two of these polypeptides (61 kDa and 28 kDa) were observed in urease-containing material isolated by preparative non-denatured PAGE. Monoclonal antibodies (mAbs) were produced which were directed against two of these polypeptides (56 kDa and 28 kDa). Only mAbs directed against the 28 kDa polypeptide inhibited or captured urease activity. These results suggest that the 56 kDa polypeptide is not essential for enzyme activity. Anti-urease mAbs were used in an indirect immunogold technique to localize the enzyme at the ultrastructural level. In both prefixed bacteria and ultrathin cryosectioned bacteria the enzyme was located on the cell surface and in material apparently shed from that surface.

Antibodies, Monoclonal↗

Production and characterization of monoclonal antibodies directed against guinea pig IgG subclasses.

Monoclonal antibodies directed against guinea pig IgG subclass-specific epitopes have been generated and characterized. Three monoclonal antibodies designated GP1, GP2 and GP3 have been compared to rabbit polyclonal reagents for specificity against purified IgG subclass antigens and for the detection of herpes simplex-specific IgG subclasses, from infected guinea pig, by an indirect ELISA. The monoclonal antibodies were shown to have greater specificity than the corresponding polyclonal reagents.

Animals↗

Systemic and mucosal cell mediated immune responses against herpes simplex virus in vaccinated guinea-pigs detected by lymphocyte proliferation and monoclonal antibodies against macrophages and T cells.

Peripheral blood lymphocytes from guinea-pigs immunized with a herpes simplex virus (HSV) sub-unit vaccine (Skinner vaccine) were shown to produce an HSV antigen-specific lymphocyte proliferative response post vaccination. This response was associated with a reduction, compared with mock vaccinated and unvaccinated animals, in the clinical disease scores post intravaginal challenge. This reduction was not associated with the presence of macrophage or T cell infiltration in the vaginal mucosa postchallenge. In contrast mock vaccinated and unvaccinated guinea-pigs did not produce an HSV antigen-specific response post-vaccination but did demonstrate HSV antigen-specific lymphocyte proliferative responses post intra-vaginal challenge. The mock and unvaccinated animals had higher clinical disease scores postchallenge compared with sub-unit vaccinated animals and this was associated with the presence of cellular infiltrates in the vaginal mucosa. These results are discussed in relation to the immunopathology of the disease.

Animals↗

Antigenic conservation of the ureases of spiral- and helical-shaped bacteria colonising the stomachs of man and animals.

A monoclonal antibody, CP11, has been produced which is directed against the ureas of Campylobacter pylori. This antibody has been used to look for antigenic cross-reactivity, in other ureolytic and non-ureolytic campylobacters, by immunohistological techniques. It has also been used to investigate the helical-shaped organisms found in the stomach of the human, monkey and cat (CS1) and the ileum of the rat (ST1). Interestingly the antibody cross-reacted with the gastric helical organisms from the human, monkey and cat but not with the rat helical organism. No cross-reactivity was observed with C. mustelae or the other ureolytic campylobacters, C. nitrofigilis and the urease positive thermophilic campylobacters. These results are discussed in relation to the phylotaxonomy of these organisms.

Animals↗

Campylobacter infection in a closed dog breeding colony.

Most dogs in a closed breeding unit were shown to be asymptomatic excretors of campylobacter organisms by eight weeks old. Increasing serum antibody levels, which were correlated with the excretion of organisms, were demonstrated in the puppies and serum antibodies were also demonstrated in adult dogs. The significance of these findings with respect to the pathogenicity of campylobacter in dogs and their zoonotic implications are discussed.

Animals↗