Isolation and identification of Helicobacter spp. from canine and feline gastric mucosa

It is known that virtually all healthy adult dogs and cats harbor spiral helicobacters in their gastric mucosa. Three species, Helicobacter felis, Helicobacter bizzozeronii, and Helicobacter salomonis have been isolated in vitro from the gastric mucosa of these animals. The aims of this study were to evaluate the efficacy of an isolation method for canine and feline gastric helicobacters that has been developed at the University of Helsinki; to estimate the prevalence and distribution of these taxa in the samples examined; and to assess the efficacy and validity of an extensive set of standardized conventional phenotypic tests, whole-cell protein profiling, and ultrastructural analysis in identifying the different species isolated from canine and feline gastric mucosa. We cultured 95 and 22 gastric mucosal biopsies from dogs and cats, respectively. Twenty-one H. bizzozeronii strains, 8 H. felis strains, 8 H. salomonis strains, 3 mixed cultures, 2 "Flexispira rappini"-like organisms, and 3 as yet uncharacterized strains were isolated from the dogs, and 3 H. felis strains were isolated from the cats. The methods used here yielded Helicobacter isolation rates of 51% from dogs and 13.6% from cats, which exceed those reported previously. The main difficulties were primary isolation, mixed cultures, and identification to the species level. In the species identification, a detailed morphological examination was found to yield important phenotypic characteristics. A large panel of biochemical and tolerance tests did not clearly differentiate the closely related species H. bizzozeronii, H. felis, and H. salomonis. Highly standardized whole-cell protein profiling was shown to be an excellent method for species identification. Improvements in culture conditions for these bacteria are still needed, especially for cats. A genetic identification method not requiring culture is needed for future studies of these very fastidious helicobacters, as the clinical significance and ecology of these species within the gastric mucosa of the domestic carnivores remain largely unknown.     

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.     

Detection and effects of helicobacters in healthy dogs and dogs with signs of gastritis

OBJECTIVES: To determine prevalence, colonization density, and distribution of helicobacters and gastric histologic findings in healthy dogs and dogs with signs of gastritis; to evaluate association of colonization density and gastric inflammation; and to compare the number of Helicobacter spp with degree of inflammation. DESIGN: Cross-sectional prevalence survey. ANIMALS: 25 healthy dogs and 21 dogs with signs of gastritis. PROCEDURE: During endoscopy, gastric mucosal biopsy specimens were obtained from healthy and affected client-owned dogs. Histologic and cytologic evaluation and results of a urease test were used for detecting helicobacters, which were identified definitively by use of transmission electron microscopy and bacterial culture. RESULTS: Helicobacters were detected in all 25 healthy and 20 of 21 affected dogs. Cytologic examination was a more sensitive method than histologic examination or the urease test. Helicobacters were found least frequently and in fewest number in the antrum in both groups of dogs. Gastric inflammation was evident in both groups of dogs and did not differ significantly between groups. A significant association was not detected between colonization density or the number of Helicobacter spp and degree of gastric inflammation. In both groups, H bizzozeronii, H felis, and H salomonis were cultured. CLINICAL IMPLICATIONS: Histologically verified chronic gastritis is common in dogs with signs of gastritis as well as in healthy dogs. Colonization density of helicobacters was not associated with degree of gastric inflammation in the dogs of our study. It remains to be determined whether certain strains of Helicobacter spp can induce gastritis in dogs.     

Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.     

Reactive oxygen species activity and lipid peroxidation in Helicobacter pylori associated gastritis: relation to gastric mucosal ascorbic acid concentrations and effect of H pylori eradication

BACKGROUND: Helicobacter pylori is an independent risk factor for gastric cancer, and this association may be due to the bacterium causing reactive oxygen species mediated damage to DNA in the gastric epithelium. High dietary ascorbic acid intake may protect against gastric cancer by scavenging reactive oxygen species. AIMS: To assess reactive oxygen species activity and damage in gastric mucosa in relation to gastric pathology and mucosal ascorbic acid level, and to determine the effect of H pylori eradication on these parameters. PATIENTS: Gastric biopsy specimens were obtained for analysis from 161 patients undergoing endoscopy for dyspepsia. METHODS: Reactive oxygen species activity and damage was assessed by luminol enhanced chemiluminescence and malondialdehyde equivalent estimation respectively. Ascorbic acid concentrations were measured using HPLC. RESULTS: Chemiluminescence and malondialdehyde levels in gastric mucosa were higher in patients with H pylori gastritis than in those with normal histology. Successful eradication of the bacterium led to decreases in both parameters four weeks after treatment was completed. Gastric mucosal ascorbic acid and total vitamin C concentrations were not related to mucosal histology, but correlated weakly with reactive oxygen species activity (chemiluminescence and malodialdehyde levels). CONCLUSIONS: Data suggest that reactive oxygen species play a pathological role in H pylori gastritis, but mucosal ascorbic acid is not depleted in this condition.     

Diagnosis of otitis media with effusion by acoustic reflectometry

Colonization of human gastric mucosa with Helicobacter pylori leads to chronic active gastritis and induces the occurrence of an acquired mucosa-associated lymphoid tissue (MALT) in the stomach. This remodelling of the gastric mucosa together with chronic antigen persistence may induce autoimmune reactions. The aim of this study was to investigate humoral autoimmune reactions to human gastric mucosa in H. pylori gastritis and their clinical relevance. Sera from patients with dyspeptic symptoms were tested for presence of IgG immunoglobulins against H. pylori. Gastric infection with H. pylori and alterations of gastric mucosa were demonstrated by histological examination of gastric biopsy specimens. All sera were tested for reactivity against human gastric mucosa by immunohistochemistry. Two different in-situ binding sites of antigastric autoantibodies were observed. Binding to canalicular structures within parietal cells was significantly correlated with antibodies to H. pylori, elevated basal gastrin levels and atrophy of gastric corpus glands. Our data indicate that autoimmune reactions to antigens in the human gastric mucosa occur in H. pylori gastritis and that they may play a role in the pathogenesis of the disease.     

Deciding not to resuscitate. Responsibilities of physicians and nurses--a proposal

Helicobacter pylori is a Gram negative bacteria that colonizes gastric epithelial cells. It has been associated with several gastric disease including chronic gastritis and peptic ulcer. Helicobacter pylori infection diagnosis can be done with invasive and non-invasive methods. In invasive methods an endoscopic gastric mucosa biopsy specimen is used. In our study we compare the sensitivity, specificity, costs and applicability of four invasive diagnostic tests: culture, urease ultra-rapid test, histology (Giemsa and Hematoxilineosin stain) and fuchsin stained mucosal slides. Urease test was the easiest, fastest diagnostic test, with sensitivity of 86% and specificity of 100%, being also the cheapest test. We concluded that it should be the test of choice for Helicobacter pylori infection diagnosis.     

Helicobacter pylori detection by polymerase chain reaction in gastric juice and its correlation with the histology (Giemsa)]

HP infection is involved in the pathogenesis of several gastroduodenal diseases, as type B chronic gastritis, duodenal and gastric ulcer, MALT lymphoma and gastric cancer. The recent availability of molecular techniques, specifically the PCR, allow us to detect very low amounts of the bacterium. The aim of the study is to evaluate the presence of HP in gastric juice by PCR technique and to correlate this findings with histology (Giemsa) of gastric mucosa. Gastric juice PCR positive findings were found in 10/31 (32.3%) HP positive patients at histology. We concluded that HP in gastric juice is possible to detect by molecular techniques. In our study 32.3% of the patients showed the presence of HP in gastric juice.     

Dental erosions in subjects living on a raw food diet

Helicobacter pylori infection of the gastric mucosa can result in gastritis and peptic ulcer disease. Although vaccination can induce protective immunity in animal models of Helicobacter infection, the mechanism(s) of protective immunity has not been fully elucidated. This study was designed to determine whether humoral immune responses are required for protective Helicobacter immunity. IgA-deficient or immunoglobulin-deficient mice were orally immunized against Helicobacter felis and then challenged with live H. felis. Both groups were protected at levels comparable to that of wild-type mice. Additionally, inflammation was equivalent in extent and character between wild-type and antibody-deficient mice. Therefore antibody-independent mechanisms of immunity can protect mice against gastric Helicobacter infection.     

Helicobacter pylori: the mouth, stomach, and gut axis

The aim of this study was to identify the natural reservoir and route of transmission of Helicobacter pylori infection. Two hundred eight (208) dyspeptic patients (114 males, 94 females; peak age of cohort, 50-59.9) were recruited. Specimens were collected from saliva, supra- and subgingival dental plaque, tongue scrapings, and oropharyngeal swabs. At subsequent endoscopy, gastric antral biopsy was performed for the rapid urease test (RUT), microbiological culture, and, in some patients, histology. Gastric juice samples were aspirated, and in 50 patients duodenal aspirate was collected. Polymerase chain reaction (PCR) with primers targeted to the 16S rRNA sequence of H. pylori was also employed for each of the specimens. In those patients where H. pylori was detected from multiple sites (dental plaque, gastric juice, gastric biopsy, and duodenal aspirate), restriction endonuclease digestion with Hae III was performed to determine if they were epidemiologically linked. The results indicated that 15/208 patients (7%) tested positively for H. pylori by PCR in dental plaque; only 2 samples were positive by culture. In none of the other oral sites sampled was H. pylori detected by any test used in the study. Gastric juice and gastric biopsy specimens from 36/ 208 patients (17%) and 114/208 patients (55%), respectively, were positive by PCR. Duodenal aspirate from 6/50 patients (12%) also tested positively by PCR. All specimens tested by restriction endonuclease digestion with Hae III (15/15 patients) were positive in both antral biopsy and gastric juice specimens, as well as 5 specimens from the duodenal aspirate. Four of the dental plaque strains had restriction patterns similar to those of the stomach and duodenal sites, providing evidence that these sites were infected with the same strain of H. pylori. In conclusion, the results suggest that H. pylori selects the gastric mucosa as its preferred site. The detection in dental plaque could indicate that the oral cavity may act as a reservoir or sanctuary for the organism. Whether H. pylori is a resident or transient oral microorganism is still unclear, although it is more likely to be transient in nature.     

Consistent improvement in sphincterotome orientation with manual grooming

AIMS: To determine the prevalence of lymphoid follicles in Helicobacter pylori positive and negative gastritis in antral and body type gastric mucosa in patients with non-ulcer dyspepsia (NUD), duodenal ulcer, or gastric ulcer; to correlate follicle presence with patient age; to evaluate the correlation between the prevalence of lymphoid follicles and active and inactive gastritis and its severity; and to assess the positive predictive value of lymphoid follicle prevalence with respect to H pylori infection. METHODS: Gastric biopsy specimens, graded according to the Sydney system, from 337 patients were studied. RESULTS: Lymphoid follicles occurred more often in antral mucosa (78%) than in body type mucosa (41%) and were observed in 85% of patients with H pylori positive gastritis. There was no significant difference between NUD and gastric and duodenal ulcer disease with regard to the presence of lymphoid follicles. The positive predictive value of the presence of lymphoid follicles in H pylori infection was 96%. Lymphoid follicles were more commonly observed in patients aged between 10 and 29 years. Lymphoid follicles were more frequently found in pangastritis of all subtypes than in antral gastritis and also in active gastritis than in inactive gastritis. The presence of lymphoid follicles correlated strongly with the degree and severity of gastritis. CONCLUSION: Lymphoid follicles are a constant morphological feature of H pylori associated gastritis.     

Regulation of ciprofloxacin uptake in human promyelocytic leukemia cells and polymorphonuclear leukocytes

BACKGROUND: It has recently been shown that humoral antigastric autoreactivities occur in a substantial number of Helicobacter pylori infected patients. AIMS: To analyse the relevance of such antigastric autoantibodies for histological and serological parameters of the infection as well as for the clinical course. METHODS: Gastric biopsy samples and sera from 126 patients with upper abdominal complaints were investigated for evidence of H pylori infection using histology and serology. Autoantibodies against epitopes in human gastric mucosa were detected by immunohistochemical techniques. Histological and clinical findings of all patients were then correlated with the detection of antigastric autoantibodies. RESULTS: H pylori infection was significantly associated with antigastric autoantibodies reactive with the luminal membrane of the foveolar epithelium and with canalicular structures within parietal cells. The presence of the latter autoantibodies was significantly correlated with the severity of body gastritis, gastric mucosa atrophy, elevated fasting gastrin concentrations, and a decreased ratio of serum pepsinogen I:II. Furthermore the presence of anticanalicular autoantibodies was associated with a greater than twofold reduced prevalence for duodenal ulcer. CONCLUSION: The data indicate that antigastric autoantibodies play a role in the pathogenesis and outcome of H pylori gastritis, in particular in the development of gastric mucosal atrophy.     

Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pylori-associated gastritis?

The aim of the present study was to correlate molecular evidence of the presence of Helicobacter pylori in gastric biopsy samples, based on analysis of 16S rDNA, vacuolating toxin (vacA), urease A (ureA) and cagA genes, with the clinical, histological and serological findings in patients with H. pylori-associated gastritis. Fresh biopsy samples were collected from the gastric antrum and corpus of 22 asymptomatic volunteers with or without H. pylori-associated gastritis. Total DNA was extracted from the biopsy material and subjected to 16S rDNA PCR amplification, Southern blotting and 16S rDNA sequence analysis of the PCR products. The vacA, ureA and cagA genes were characterised by PCR amplification and Southern blot analysis. Based on partial 16S rDNA sequence analysis, DNA belonging to the genus Helicobacter was detected in gastric biopsy samples from 20 of 22 subjects, including seven of nine histologically and serologically normal controls. Six of 20 partial 16S rDNA sequences revealed variations within variable regions V3 and V4 that deviated from those of the H. pylori type strain ATCC 4350T and, therefore, possibly represented other species of Helicobacter. VacA genes identical with those of the type strain were found predominantly in the subjects with H. pylori gastritis, and all the patients except one were found to be cagA-positive. There was no evidence of false positive PCR reactions. In conclusion, the PCR-based molecular typing methods used here were apparently too sensitive when applied to the detection of H. pylori in human gastric tissues. The lack of quantitative analysis makes them inappropriate as clinical tools for the diagnosis of H. pylori-associated gastritis, despite the fact that they provide a qualitative and sensitive tool for the detection and characterisation of H. pylori in the gastrointestinal tract.     

Beta1B subunit of voltage-dependent Ca2+ channels is predominant isoform expressed in human neuroblastoma cell line IMR32

BACKGROUND: In adults, Helicobacter pylori infection is always associated with gastritis or ulcer. However, very active gastritis and ulcers are rarely seen in children. The aim of the present work was to study the relationships between H. pylori and gastric mucosa in children. METHODS: Eighty infected children and adolescents including 48 (60%) neurologically impaired institutionalized patients, aged 2 months-22 years (mean 11.7 +/- 5.2 years) were studied retrospectively. All the patients underwent gastroscopy, and three antral and two fundic biopsy specimens were taken for histology and bacteriology. RESULTS: A normal gastric mucosa was found in 22 of 80 patients (27.5%), whereas the others had gastritis (n = 58, 72.5%). There were no statistical differences between patients with normal histology and those presenting with gastritis for age, sex, ethnic background, symptoms, and the degree of bacterial colonization. The macroscopic aspect of gastritis was less frequently found in children with a normal histology compared with those with histological gastritis (p < 0.001). CONCLUSIONS: These data show that H. pylori infection can be associated with a normal gastric histology in children.     

Gastric mucosal superoxide dismutases in Helicobacter pylori infection

BACKGROUND: The mucosal pathology of Helicobacter pylori infection may in part be due to excessive production of reactive oxygen metabolites (ROMs) by phagocytes. The influence of H pylori infection on mucosal superoxide dismutases, some major scavenger enzymes of ROM was investigated. In humans superoxidase dismutase is present in at least two forms-that is, mitochondrial manganese (Mn)-superoxide dismutase and cytoplasmic copper-zinc (CuZn)-superoxide dismutase. METHODS: The amount and activity of both superoxide dismutases were measured, respectively by enzyme linked immunosorbent assay (ELISA) and spectrophotometrical enzyme activity assay, in gastric biopsy homogenates of patients with normal mucosa (n = 39) and in patients with H pylori related gastritis (n = 71). Infection and gastritis were confirmed by a combination of culture, serology, and histology. RESULTS: The amount (p < 0.001) and activity (p < or = 0.05) of Mn-superoxide dismutase were increased by about twofold to three-fold, whereas the amount and activity of CuZn-superoxide dismutase showed a slight decrease in gastric mucosa of patients with H pylori gastritis, in both antrum and corpus, compared with normal mucosa of patients without H pylori infection. Mn-superoxide dismutase concentrations in biopsy specimens of histologically normal corpus from patients with an inflamed antrum were significantly higher (p < 0.01) than that of patients with a histologically normal antrum. CONCLUSION: H pylori infection has a differential effect on mitochondrial and cytoplasmic superoxide dismutase in the gastric mucosa, reflected by a pronounced increase in the cytokine inducible Mn-superoxide dismutase and a marginal decrease in the constitutive CuZn-superoxide dismutase.     

Hunterian peptic ulcers and Helicobacter pylori

Gastric spiral organisms were first described in man in 1939 and identified as Helicobacter pylori causing peptic ulcers in the early 1980s. Surgical specimens of gastric resections from 1939 showed H. pylori to be present. Full-thickness sections of gastric mucosa from gastric specimens from the eighteenth-century Hunterian Collection at The Royal College of Surgeons of England were examined by histology for the presence of H. pylori. Four gastric ulcers and a section from an oesophageal varix showed remarkable preservation of the overall architecture, but surface autolysis did not allow identification of the bacteria. However, the presence of lymphoid aggregates in the Hunterian specimens suggests that H. pylori may have been present before autolysis.     

Gastric mucosa after partial gastrectomy

A partial gastrectomy of Billroth I or II type was performed in a series of 146 patients with peptic ulcer. Gastric biopsy was carried out two years later and the histology of the specimens compared with that of the body mucosa at the time of operation. In 138 patients without body atrophic gastritis (AG) before operation this condition was found in 74 (54%) two years after (46% of DU patients and 73% of GU patients). Those with antral or pyloric canal ulcers were particularly liable to develop AG (81%). Apart from site of ulcer various other factors possibly associated with the development of AG were examined: no positive correlations were found with the possible exception of anaemia. Gastric parietal cell antibodies were not found in any patient with AG tested. The cause of gastritis after partial gastrectomy and its possible relationship with gastric carcinoma are discussed.     

Animal models of bacterial gastritis: the role of host, bacterial species and duration of infection on severity of gastritis

Gastric bacteria from cheetahs with gastritis were used to inoculate specific-pathogen free kittens and conventional mice. Helicobacter sp. and Gastrospirillum sp. colonized kittens, while only Gastrospirillum sp. colonized mice. In kittens, both bacterial species induced mild lymphofolliclar gastritis which did not change over the course of the 11 months observation period. In mice, Gastrospirillum sp. induced lymphoplasmacytic and follicular gastritis which increased in severity over 6 months and persisted for the 12 month observation period. Gastric ulcers and gastric mucosal hypertrophy were present in chronically infected mice. These results indicate that host but not bacterial factors influence the severity of gastritis, and that in mice, bacterial gastritis increases in severity with time and may lead to gastric ulceration in some individuals.     

High rate of chromosomal abnormalities in HTLV-I-infected T-cell colonies derived from prodromal phase of adult T-cell leukemia: a study of IL-2-stimulated colony formation in methylcellulose

An epizootic of subclinical lymphoplasmacytic gastritis occurred in cynomolgus monkeys maintained at our research facility. Gastric pathology data and histologic sections of 63 adolescent monkeys (2.5-3.5 years old) sacrificed during the epizootic were reviewed. Localized to multifocal reddening of the gastric mucosa was noted grossly in 7 of 44 (16%) monkeys harboring Helicobacter pylori, but not in any of 19 monkeys in which these bacteria were not seen. Gastritis, characterized by accentuation of lymphoplasmacytic infiltrates in antral and to a lesser degree cardiac mucosa, occurred in 42 of 63 (67%) monkeys evaluated and in 42 of 44 (93%) monkeys in which H. pylori was observed microscopically. Two monkeys with H. pylori infection had infiltrate scores that overlapped with the upper limit of scores of H. pylori-negative animals. Coincident with accentuated infiltrates were gastric gland epithelial hyperplasia, reduction in mucin content of surface and gland epithelia, and comparatively minor infiltrates of neutrophils in superficial lamina propria and gastric glands. Antral mucosa thickness often exceeded 1.5 to 2 times normal. Antral mucosal erosions occurred in 7 of 44 (16%) monkeys with H. pylori. Argyrophilic bacteria morphologically consistent with H. pylori were present in antral and less commonly cardiac mucosal glands. Intensity of bacterial colonization correlated with lymphoplasmacytic infiltrates (r = 0.754) and hyperplasia (r = 0.700), although responses were quite variable. These bacteria were not detected in fundic mucosa except in instances where parietal cells were substantially depleted in glands coincident with localized increases in lamina propria inflammatory cell infiltrates. Helicobacter heilmannii-like organisms (HHLOs) were present in fundic glands of all 63 monkeys; colonization was often pronounced. Scores for fundic mucosal inflammation did not correlate with presence or intensity of colonization with HHLOs (r = 0.005). Rather, fundic inflammation scores positively correlated with the antral inflammation scores (r = 0.548). Bacteria morphologically, biochemically, and genetically consistent with H. pylori were cultured from gastric mucosal specimens confirming bacterial identification. These findings demonstrate that adolescent cynomolgus monkeys are susceptible to natural infection with H. pylori and develop many morphologic hallmarks of H. pylori-related gastritis in humans.     

Interleukin-12 expression is focally enhanced in the gastric mucosa of pediatric patients with Crohn's disease

The stomach is frequently involved in children suffering from Crohn's disease (CD). Diagnosis of specific gastritis may be difficult when granulomas are absent. We have used in situ hybridization to examine the expression of interleukin (IL)-12, a key cytokine in the Th1 response. IL-12 p35 and p40 antisense probes were used to examine ileal specimens from 9 children with CD and gastric biopsies from 24 children (13 with CD, 6 with Helicobacter pylori chronic gastritis, and 5 with a normal gastric mucosa). In all patients with CD, many clusters of IL-12-positive cells were present in the lamina propria. This was the case in the ileal specimens as well as in gastric mucosa showing granulomatous gastritis or nongranulomatous gastritis. The same distribution patterns were found for the IL-12 p35 and p40. In three patients with Helicobacter pylori gastritis, few scattered IL-12-positive cells were found. No positive cells were found in the normal gastric mucosa. The focally enhanced IL-12 expression in the gastric mucosa of pediatric patients with CD, with or without specific lesions, suggests that both are indeed linked to the disease and supports the major part of IL-12 in initiating and maintaining of the cascade resulting in the Th1 responses.