Håkan Sjunnesson

Severe gastritis in guinea-pigs infected with Helicobacter pylori

An appropriate animal model is essential to study Helicobacter pylori infection. The aim of this study was to investigate if H. pylori can colonise the guinea-pig stomach and whether the infection causes gastritis and a serological response similar to that observed in man. Guinea-pigs were infected either with fresh H. pylori isolates from human gastric biopsies or with a guinea-pig passaged strain. When the animals were killed, 3 and 7 weeks after inoculation, samples were taken for culture, histopathology and serology. H. pylori was cultured from 22 of 29 challenged animals. All culture-positive animals exhibited a specific immune response against H. pylori antigens in Western blotting and gastritis in histopathological examination. Antibody titres in enzyme immunoassay were elevated among animals challenged with H. pylori. The inflammatory response was graded as severe in most animals and consisted of both polymorphonuclear leucocytes and lymphocytes. Erosion of the gastric epithelium was found in infected animals. These results suggest that the guinea-pig is suitable for studying H. pylori-associated diseases. Moreover, guinea-pigs are probably more similar to man than any other small laboratory animal as regards gastric anatomy and physiology.

Proximity of brain infarcts to regions of endogenous neurogenesis and involvement of striatum in ischaemic stroke.

Clinical stroke trials with stem cell‐based approaches aiming for trophic actions, modulation of inflammation and neuroprotection are ongoing. However, experimental studies also suggest that neuronal replacement by grafted neural stem cells (NSCs) and possibly by endogenous NSCs from the subventricular zone (SVZ) may restore function in the stroke‐damaged striatum. To evaluate the potential clinical impact of these findings, we analyzed the spatial relationship of infarcts to the SVZ and the proportion of individuals with striatal lesions in a consecutive series of ischaemic stroke patients.

Lectin histochemistry of the gastric mucosa in normal and Helicobacter pylori infected guinea-pigs.

Helicobacter pylori attaches via lectins, carbohydrate binding proteins, to the carbohydrate residues of gastric mucins. Guinea-pigs are a suitable model for a H. pylori infection and thus the carbohydrate composition of normal and H. pylori infected gastric mucosa was investigated by lectin histochemistry. The stomach of all infected animals showed signs of an active chronic gastritis in their mucosa, whereas no inflammation was present in the control animals. The corpus–fundus regions of the controls showed heterogeneous WGA, SNA-I, UEA-I and HPA binding in almost all parts of the gastric glands. While these lectins labelled the superficial mucous cells and chief cells heterogeneously, the staining of the parietal cells was limited to WGA and PHA-L. Mucous neck cells reacted heterogeneously with UEA-I, HPA, WGA and PHA-L. In the antrum, the superficial mucous cells and glands were stained by WGA, UEA-I, HPA, SNA-I or PHA-L. WGA, UEA-I, SNA-I and HPA labelled the surface lining cells strongly. The mucoid glands reacted heterogeneously with WGA, UEA-I, HPA, SNA-I and PHA-L. In both regions, the H. pylori infected animals showed similar lectin binding pattern as the controls. No significant differences in the lectin binding pattern and thus in the carbohydrate composition between normal and H. pylori infected mucosa could be detected, hence H. pylori does not induce any changes in the glycosylation of the mucosa of the guinea-pig. This unaltered glycosylation is of particular relevance for the sialic acid binding lectin SNA-I as H. pylori uses sialic acid binding adhesin for its attachment to the mucosa. As sialic acid binding sites are already expressed in the normal mucosa H. pylori can immediately attach via its sialic acid binding adhesin to the mucosa making the guinea-pig particularly useful as a model organism.

Dietary factors influence the recovery rates of Helicobacter pylori in a BALB/cA mouse model

The aim of this study was to assess the ability of different mouse diets to sustain an H. pylori infection in BALB/cA mice. Four commercially available mouse diets were compared. Experiment 1: Mice were fed the four diets for seven days before infection, infected three times at two-day intervals with 0.1 ml of 10(9) colony-forming units/ml H. pylori cells. H. pylori strains (n = 4) were cultured on GAB-Camp agar for 2 days, harvested and suspended in PBS. All animals were sacrificed at 2 and 4 weeks post inoculation. Experiment 2: Mice infected for 8 weeks were fed RM2, changed to the different diets for 10 days and sacrificed. Stomachs were collected, cultured on GAB-Camp agar to estimate H. pylori growth and stomach biopsies were analyzed by PCR. There were significant differences between diets in their ability to sustain growth of H. pylori. The range was from a few hundred colonies to no growth at all on the GAB-Camp agar. PCR signals showed good correlation with the culture results. All H. pylori-infected mice gave a significantly higher inflammation score compared to non-infected mice. The diet RM2, having the highest number of culturable H. pylori in the mouse stomach, also showed the highest inflammation. These results suggest that the dietary factors affect the amounts of H. pylori in an infection of BALB/cA mice.

PCR-denaturing gradient gel electrophoresis and two feces antigen tests for detection of Helicobacter pylori in mice

PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57B1/6 mouse model of H. pylori infection. PCR-DGGE detected only Helicobacter ganmani in feces from H. pylori-infected as well as control animals, whereas in stomach specimens it demonstrated the presence of H. pylori in challenged and H. ganmani in control animals. Hence, the method detected DNA only of the predominant Helicobacter spp., which was also shown in cell dilution experiments. The Amplified IDEIA Hp StAR feces antigen test detected H. pylori in feces from all infected animals and generated no false-positive results, whereas the Premier Platinum HpSA-test also detected H. pylori in all infected animals but generated false-positive or equivocal results in 50% of the control animals. Premier Platinum HpSA, as opposed to Hp StAR, cross-reacted with non-pylori Helicobacter spp. in vitro.

Helicobacter species in human colon biopsies

Sirs, We read with interest the study by Bell et al., who failed to detect DNA of the Helicobacter genus in colon biopsies from subjects with or without inflammatory bowel disease. At the XVIth International Workshop on Gastrointestinal Pathology and Helicobacter (Stockholm, September 3–6, 2003), we presented a similar study that came to a partly different conclusion. Twenty patients undergoing colonoscopy at Lund University Hospital for clinical reasons were enrolled in our study (15 females and five males; age range, 17–66 years; mean age, 43 years). Of the 20 patients, five had ulcerative colitis, four had Crohn s disease and 11 had other ailments. The study was approved by the Lund University Ethical Committee (permit: LU 345-02) and signed informed consent was obtained from all patients. Biopsies from the proximal and distal colon were collected from each patient in a transport medium (tryptone soy broth, 10% horse serum, 20% glycerol) and transported to the laboratory, where DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) within 1–2 h. Detection of the Helicobacter genus was performed by a nested 16S rDNA Helicobacter genus-specific polymerase chain reaction (PCR) assay using primers C97, C05 for step one and N16S 1F, N16S 2R for step two. Positive products, as determined by visualization in gel electrophoresis, were analysed by denaturing gradient gel electrophoresis and sequencing as described elsewhere. Positive samples were re-amplified to obtain 400-bp or 800-bp amplicons for sequencing, followed by comparison with known DNA sequences using BLASTN 2.2.1. PCR displayed a Helicobacter-specific product in five patients (Table 1). One 771-bp sequence clustered with Helicobacter cholecystus (two mismatches, both n in Genbank U46129), Helicobacter pametensis (eight mismatches) and Helicobacter sp. B9A Seymour (12 mismatches). One sequence clustered with Helicobacter muridarum (784of 784-bp, Genbank AF302104) and the closest other sequence was Helicobacter typhlonius (16 mismatches). At least three different species of Helicobacter were detected in our study, two of which (H. cholecystus and H. muridarum), to our knowledge, have not been described previously in humans. Our investigation has so far not found any differences in Helicobacter detection in the two studied groups (inflammatory bowel disease vs. other ailments). Culture showed no growth of Helicobacter spp.; however, culture from biopsies from one patient displayed massive growth of Campylobacter concisus. This isolate came from a male patient (46 years of age) with abdominal

Five month persistence of Helicobacter pylori infection in guinea pigs.

Seven Dunkin‐Hartley guinea pigs were infected with the Sydney strain of H. pylori (SS1). Gastric histopathology was evaluated and serum antibody response to H. pylori cell‐surface proteins was analysed by enzyme immunoassay (EIA) and immunoblot. Tissue and faecal samples from five control animals were analysed for the presence of naturally occurring Helicobacter spp. infection by culture and Helicobacter genus‐specific PCR. The H. pylori infection persisted for 5 months, in most animals accompanied by a histologically severe antral gastritis, exhibiting focal degeneration and necrosis of gastric crypt epithelium. Increased numbers of mitotic figures were observed in the gastric epithelium, indicating a regenerative process. Infected animals displayed specific antibodies towards H. pylori cell‐surface proteins in immunoblot, whereas EIA was of dubious value creating false‐positive results. Serum complement C3 and cholesterol levels appeared to be elevated in infected animals. Helicobacter spp. infection was not detected in the control animals. The persistent infection, accompanied by severe gastritis and a prominent serum antibody response, and the apparent absence of a natural Helicobacter spp. infection makes the guinea pig model useful in H. pylori research.