Axel von Herbay

Sequencing and analysis of the genome of the Whipple's disease bacterium Tropheryma whipplei

BACKGROUND Whipples disease is a rare multisystem chronic infection, involving the intestinal tract as well as various other organs. The causative agent, Tropheryma whipplei, is a Gram-positive bacterium about which little is known. Our aim was to investigate the biology of this organism by generating and analysing the complete DNA sequence of its genome. METHODS We isolated and propagated T whipplei strain TW08/27 from the cerebrospinal fluid of a patient diagnosed with Whipples disease. We generated the complete sequence of the genome by the whole genome shotgun method, and analysed it with a combination of automatic and manual bioinformatic techniques. FINDINGS Sequencing revealed a condensed 925938 bp genome with a lack of key biosynthetic pathways and a reduced capacity for energy metabolism. A family of large surface proteins was identified, some associated with large amounts of non-coding repetitive DNA, and an unexpected degree of sequence variation. INTERPRETATION The genome reduction and lack of metabolic capabilities point to a host-restricted lifestyle for the organism. The sequence variation indicates both known and novel mechanisms for the elaboration and variation of surface structures, and suggests that immune evasion and host interaction play an important part in the lifestyle of this persistent bacterial pathogen.

Pulmonary tumor thrombotic microangiopathy with pulmonary hypertension

Pulmonary tumor thrombotic microangiopathy is characterized by fibrocellular intimal proliferation of small pulmonary arteries and arterioles in patients with metastastic carcinoma. Its morphologic features, including precursor lesions, were studied in 21 patients diagnosed in 630 consecutive autopsy cases with carcinoma (3.3%). Nineteen of 21 patients had adenocarcinoma and 11 of these 19 patients had gastric carcinoma. The pathogenetic events start with microscopic tumor cell embolism. Tumor emboli do not occlude affected vessels but induce both local activation of coagulation and fibrocellular intimal proliferation, which lead into stenosis or occlusion. Hemodynamically, an increase in vascular resistance results in pulmonary hypertension. In three patients, metastatic carcinoma was unknown before death, and the condition was diagnosed as pulmonary hypertension of unknown origin. Thus, pulmonary tumor thrombotic microangiopathy should be considered in the differential diagnosis of primary pulmonary hypertension, particularly in patients with well‐known carcinoma who develop acute or subacute cor pulmonale.

Serum Antibodies Against Helicobacter pylori Proteins VacA and CagA Are Associated with Increased Risk for Gastric Adenocarcinoma

Infection with Helicobacter pylori is associatedwith the development of gastric cancer. To study whetherthe infection with H. pylori strains expressing thevacuolating cytotoxin (VacA) and/or thecytotoxin-associated protein (CagA) is associated with an increasedrisk of developing gastric adenocarcinoma, sera of 90patients with gastric cancer and 90 matched controlswith cardiovascular diseases were investigated for the presence of antibodies to VacA and CagA byimmunoblot. Although no significant difference in theoverall H. pylori seropositivity was found betweencancer patients and controls, antibodies against VacA or CagA were significantly more frequent incancer patients than in control subjects. Seventyfive(97.4%) of 77 H. pylori-positive patients in the cancergroup, but only 60 (84.5%) of 71 H. pylori-positive control patients had antibodies against eitherVacA or CagA (χ2 6.63; relative risk,2.00; 95% confidence interval, 1.18–3.39; P =0.01). The presence of antibodies against VacA or CagAalone was also associated with an increased cancer risk (92.2%vs 80.3%; χ2 = 5.30; relative risk, 1.74;95% confidence interval, 1.08–2.78; P = 0.021, forVacA; and 87.0% vs 74.6%; χ2 4.90;relative risk, 1.61; 95% confidence interval, 1.06–2.45; P =0.037, for CagA). The relative risk for gastric cancerwas mainly elevated in patients under 65 years, but notin patients at or over 65 years. There is evidence that infection with VacA- or CagA-producing H.pylori strains increases the risk of developing gastriccancer, especially in younger patients.

Genetics of adenocarcinomas of the small intestine: frequent deletions at chromosome 18q and mutations of the SMAD4 gene.

The small intestinal mucosa makes up about 90% of the total surface of the gastrointestinal tract. However, adenocarcinomas arise rarely in this location. To elucidate genetic alterations underlying tumour development in the small intestine we investigated 17 sporadic adenocarcinomas. By comparative genomic hybridization recurrent gains of chromosomal material were found at chromosomes 7, 8, 13q, and 20 (5/17, each), while non-random losses were seen at 8p, 17p (4/17, each), and 18 (8/17 cases). Deletions at 5q, the location of the APC tumour suppressor gene, were seen in three cases. Microsatellite analysis with markers on chromosomal arms 1p, 5q, 8p, 17p, 18q, 19p, and 22q revealed a microsatellite instable phenotype in two cases and a high frequency of loss at 18q21-q22 (80%). Given the high incidence of 18q21-q22 deletions, we performed sequencing analysis of SMAD4, a downstream component of the TGFβ-pathway, located at 18q21. Four tumours displayed mutations in highly conserved domains of the gene indicating disruption of TGFβ-signalling. Our data reveal complex genetic alterations in sporadic small intestinal carcinomas. However, most tumours share deletions of 18q21-q22, which frequently target SMAD4. This indicates that disruption of TGFβ-signalling plays a critical role in small intestinal tumorigenesis.

Reduction of chemotherapy-induced side-effects by parenteral glutamine supplementation in patients with metastatic colorectal cancer.

In animal studies, glutamine (Gln) reduces chemotherapy-associated mucositis and mucosal atrophy. Therefore, this study examined the protective effects of a parenteral Gln supplementation in patients with metastatic colorectal carcinoma receiving 5-fluorouracil (5-FU)/calcium-folinate (CF) chemotherapy. In a prospective study, a total of 24 patients underwent three courses of 5-FU/CF chemotherapy and were randomised with (n = 12) or without (n = 12) glycyl-L-glutamine. Effects on gastrointestinal mucosa were assessed by endoscopic examinations and histomorphometric measurements. Clinical side-effects were documented according to the World Health Organisation grading. In the Gln group, a significant reduction in mucositis and ulcerations of the gastric (P < 0.01) and duodenal mucosa (P < 0.05) was documented after the third course of chemotherapy. In the same group, the villus height/crypt depth ratio was significantly higher after therapy than in the unsupplemented group (1st course P < 0.01; 3rd course P < 0.05). However, there were no significant differences in the incidence and severity of clinical side-effects. The results suggest that parenteral Gln supplementation protects the gastrointestinal mucosa against 5-FU/CF chemotherapy-induced damage.

Treatment of Refractory Whipple Disease with Interferon-γ

Whipple disease is an infectious, chronic multisystem disorder characterized by diarrhea, malabsorption, arthralgias, and (in later stages) involvement of the central nervous system [1]. The infection is caused by an intracellular bacterium for which the name Tropheryma whippelii has been proposed [2]. The pathogen has only recently been successfully isolated and propagated by using interleukin-4-deactivated macrophages [3]. Before the use of antibiotics, Whipple disease was fatal [4]; even with antimicrobial therapy, some patients have severe relapse [5-7]. The pathogenesis of T. whippelii infection is poorly understood; immune defects involving T cells and macrophages have been described [8-12]. Recently, reduced interleukin-12 and interferon- production in patients with Whipple disease has been detected [13]. These cytokines may be important in this context because of their ability to contain and clear intracellular bacteria [14, 15]. Therefore, we tested the usefulness of antibiotic therapy supplemented by interferon- in treating a patient with a 10-year history of antibiotic-resistant Whipple disease. Case Report In 1985, Whipple disease was diagnosed by periodic acid-Schiff (PAS)-positive macrophages in the duodenal biopsy specimen of a 66-year-old patient. The patient had a 10-year history of diarrhea and weight loss and was continuously treated with antibiotics to eradicate the bacterium. However, the patient had seven relapses despite this therapy. Duodenal biopsy results remained positive on testing for T. whippelii-specific DNA, PAS staining, and electron microscopy. In 1995, although the patient had no neurologic symptoms, involvement of the central nervous system was documented by the presence of T. whippelii-specific DNA and PAS-positive cells in the cerebrospinal fluid (Table 1). At this time, both in vivo and in vitro evidence of deficient T-cell function was detected. Testing of cutaneous delayed hypersensitivity with seven recall antigens (Multitest Merieux, Institut Merieux GmbH, Leimen, Germany) yielded abnormal results; only two responses were observed, and the combined diameter was 8 mm (normal test results yield at least 3 responses with a combined score >10 mm in diameter). The patient had no serologic evidence of HIV or cytomegalovirus infection, and repeated stool tests were negative for pathogenic bacteria and parasites. Table 1. Characteristics of the Patient before and after Treatment with Combination Therapy (Interferon- and Trimethoprim-Sulfamethoxazole)* Because of central nervous system involvement and the refractory course of the disease, the patient was treated with ceftriaxone and chloramphenicol (Table 1) and trimethoprim-sulfamethoxazole supplemented with recombinant human interferon- (Polyferon, Rentschler, Laupheim, Germany) (100 g subcutaneously three times per week). The patients clinical state indicated severe Whipple disease: weight loss, diarrhea, and reduced serum -carotene level (one of the best markers for malabsorption in Whipple disease [1]). Three weeks after initiation of therapy with interferon-, the patient recovered from clinical symptoms (Figure 1). Six months later, T. whippelii-specific DNA and PAS-positive cells were no longer found in the cerebrospinal fluid. However, duodenal biopsy continued to show PAS-positive cells and T. whippelii-specific DNA (Table 1). Figure 1. Effects of interferon- therapy. To deal with persistent subclinical infection, the dose of interferon- was increased to 150 g three times per week. Four months after initiation of high-dose interferon- therapy, cutaneous hypersensitivity testing showed normal responses to three antigens (with a score of 28 mm in diameter); electron microscopy of duodenal mucosa showed no intact bacteria in the remaining PAS-positive macrophages. For the first time, polymerase chain reaction analysis of duodenal biopsy was negative for T. whippelii-specific DNA. Combination therapy was discontinued after 16 months, and the patient remained symptom-free. When he was reevaluated in July 1997, a normal duodenal mucosa was seen during endoscopy, no cells containing sickleform particles were seen in the biopsy specimens, and results of polymerase chain reaction assay remained negative. In December 1997, the dose of trimethoprim-sulfamethoxazole was reduced to one application daily. In March 1998, the patient had no clinical symptoms. Methods Details of the use of the polymerase chain reaction assay to detect T. whippelii-specific DNA have been described previously [16, 17]. T-cell subpopulations were analyzed by flow cytometry, and interferon- production was studied in cultures of phytohemagglutinin-stimulated mononuclear cells from peripheral blood after 48 hours by a commercially available enzyme-linked immunosorbent assay (Laboserv, Staufenberg, Germany). The peripheral CD4+ T-cell count was as low as 126 cells/L before treatment (control [n = 5]: median, 1006 cells/L [range, 620 to 1295 cells/L]). Nine months after initiation of interferon- therapy, the CD4+ T-cell count was 616 cells/L, and 1 year after stopping interferon- therapy, the CD4+ T-cell count increased to 1003 cells/L (Figure 1). Interferon- production increased from 329 ng/L per 105 cells (control [n = 5]: median, 1595 ng/L per 105 cells [range, 817 to 2235 ng/L per 105 cells]) to 1865 ng/L per 105 cells at 9 months. In March 1998, interferon- production still exceeded pretreatment levels (Figure 1). Role of Funding Source The funding source had no role in gathering, analyzing, or interpreting the data or in deciding to submit the paper for publication. Discussion Our findings provide evidence that immunomodulatory therapy with interferon- in addition to antibiotics is beneficial in treating patients infected with the intracellular pathogen T. whippelii. Our patient had chronic Whipple disease that involved the central nervous system and was refractory to therapy with several usually effective antibiotics, including those that cross the blood-brain barrier. Final eradication of the bacterium occurred after the patient received combination therapy and has lasted for 1 year after interferon- therapy was stopped. It is unlikely that the eradication resulted from initial ceftriaxone-chloramphenicol treatment because the patient did not have a clinical response until 3 weeks after initiation of interferon- therapy. In addition, clearance of infection required a course of high-dose interferon-. The decision to include interferon- in this patients therapy was influenced by studies that showed the important role of this cytokine in controlling intracellular infections under experimental conditions and its success in treating persistent intracellular infections [15]. In addition, immunologic studies have shown reduced production of interleukin-12 and interferon- in patients with Whipple disease [13]. Of interest, the patients immunologic status improved during and after interferon- therapy. However, our study does not allow us to determine whether the immunoregulatory defect is a primary or secondary result of the infection. The addition of interferon- may be an option in the treatment of patients with refractory Whipple disease, but further studies are needed to clarify the clinical benefit of this therapy.

Altered Content and Distribution of Tenascin in Colitis, Colon Adenoma, and Colorectal Carcinoma

Tenascin is a fibroblast product and extracellular matrix protein probably excerting a fibronectin-antagonizing role. Tenascin is broadly distributed interstitially during embryogenesis but restricted to a small range of structures in normal adult tissues. Using tenascin antibodies and an indirect immunoperoxidase method, normal colon, colitis, colon adenomas, and colorectal carcinomas were examined for tissue distribution of tenascin. Normal mucosa displayed a sparce meshwork of microfibrillar tenascin in the lamina propria. The basement membrane was tenascin negative at the bottom of the crypt and developed into a positive band steadily broadening towards the mucosal surface. In colitis, this polarity was effaced; the basement membrane was a broad tenascin-positive band nearly throughout while interstitial tenascin was moderately increased. Loss of polarity in tenascin content of the basement membrane was a constant feature of adenomas, inconsistently paralleled by structural alterations in surface qualities and continuity of tenascin pattern of the basement membrane. These were most pronounced in carcinomas, where this interface was often discontinuous and had a rough surface; in addition, interstitial tenascin was considerably increased. In carcinomas, the rough surface aspect of the tenascin pattern of the basement membrane was correlated with presence of lymph node metastases (P = 0.04). It is concluded that alterations in tenascin pattern and content reflect complex disturbances in the interaction of inflamed/neoplastic colon epithelium and underlying matrix, leading to an organoid induction of tenascin in the inflammatory context and to induction together with structural abnormalities in neoplasia.

Tropheryma whippelii DNA Is Rare in the Intestinal Mucosa of Patients without Other Evidence of Whipple Disease

Whipple disease is a systemic condition characterized by the presence of uniform rod-shaped bacteria in affected tissues and by macrophage inclusions that are positive on periodic acidSchiff (PAS) staining. The classic clinical features of the disease include diarrhea with malabsorptive findings, abdominal pain, weight loss, arthralgias, lymphadenopathy, and occasionally, neurologic abnormalities. Numerous attempts to cultivate the causative bacterium in the past have failed. Molecular methods have revealed this organism, Tropheryma whippelii, to be a novel actinomycete (1, 2). Recently, propagation in human fibroblasts has been reported (3), but this finding has not yet been reproduced by other investigators. Little is known about the pathogenesis of Whipple disease. Abnormalities of immune functions have been described (4) and are presumed to play a role as predisposing factors, but precisely defined preexisting immunologic defects have not been identified. Neither the natural habitats of the bacterium nor the route of infection are fully understood. Because of the frequency of intestinal manifestations, an oral route of acquisition is suspected (5, 6). The 16S ribosomal DNA (rRNA gene) sequence of the bacterium has been detected in a polymicrobial community of sewage effluent, suggesting a possible environmental habitat and source of infection (7). This possibility would be in accordance with the phylogeny of the bacterium (8) and with the epidemiologic features of the disease (5, 9). Two recent reports have described polymerase chain reaction (PCR)based detection of T. whippelii DNA in specimens of persons without the classic clinical or histologic features of Whipple disease. In one series, positive PCR results were obtained from saliva samples of 14 of 40 (35%) apparently healthy persons (10). In another series, positive PCR results were reported from intestinal biopsy or gastric juice samples of 14 of 105 (13%) patients undergoing elective endoscopy for reasons other than suspicion of Whipple disease (11). These investigators have speculated that T. whippelii is a commensal of the human gastrointestinal tract (10, 11). At the same time, several published series on diagnostic PCR have found no evidence of T. whippelii DNA in the intestinal biopsy specimens of controls (12-14). These conflicting and confusing data and the treatable nature of this disease emphasize the need for additional information on the prevalence of T. whippelii in the intestinal mucosa of persons without evidence of Whipple disease by classic diagnostic methods, such as PAS staining or electron microscopy. Such information would provide further insights into the epidemiology and pathogenesis of the disease and would be crucial information with which to judge the indications for and interpretations of diagnostic PCR results. Thus, we examined intestinal biopsy specimens from three groups of patients. Two of the groups were prospectively examined: Patients underwent endoscopy for reasons other than Whipple disease in the first group and for work-up of malabsorption in the second group. In addition, we evaluated a third groupcases without histologic evidence for Whipple disease among intestinal biopsy specimens submitted to Stanford University (Stanford, California), the Mayo Clinic (Rochester, Minnesota), or the University of Heidelberg (Heidelberg, Germany) for diagnostic PCR testing for T. whippelii. Methods Patients and Samples Endoscopic biopsy specimens of the small intestine in patients from three categories were examined by using routine histologic methods, including PAS staining, and PCR testing for T. whippelii. Group A consisted of 173 patients who underwent upper gastrointestinal endoscopy for evaluation of dyspepsia, abdominal pain, or possible gastroesophageal reflux or peptic ulcer disease, without signs of malabsorption or consideration of Whipple disease. Group B consisted of 37 patients who underwent endoscopy in the work-up for malabsorption (endoscopy was prompted by unexplained weight loss or chronic diarrhea, for example, or by the referring gastroenterologists concern about protein or fat malabsorption). Biopsy specimens from groups A and B were obtained prospectively at the Mayo Clinic. Patients gave informed consent to allow additional specimens to be obtained for PCR testing for T. whippelii. Group C consisted of 132 patients who underwent endoscopy for clinical suspicion of Whipple disease, based on intestinal or extraintestinal findings or both. The patients in group C were selected only if histologic studies (including PAS staining) of intestinal biopsy specimens gave no evidence of Whipple disease. The specimens in group C were submitted to the University of Heidelberg (70 specimens), Stanford University (30 specimens), and the Mayo Clinic (32 specimens). All biopsy specimens used in PCR analysis from groups A and B, as well as those from group C that were studied at the Mayo Clinic, were fresh-frozen samples; those from group C submitted to the University of Heidelberg and Stanford University were either fresh-frozen or formalin-fixed, paraffin-embedded specimens. PCR Testing The PCR assays performed at our institutions have been described previously in detail (2, 12, 13, 15). Briefly, assays were performed by using nonnested PCR protocols with an analytic sensitivity of 10 to 100 spiked copies of cloned T. whippelii 16S rDNA per reaction (12, 13, 15). The specificity of these assays has been verified by testing on a wide variety of other bacteria, including many closely related organisms and on control tissues from patients with other diseases (12, 13). Positive PCR results were confirmed by oligonucleotide hybridization or direct sequencing (12, 13, 15). In terms of diagnostic sensitivity, PCR testing gave positive results with histologically positive samples in 95 of 96 patients tested at our institutions. This series comprises 60 previously described patients (12, 13), as well as 36 previously unreported cases. Different rooms were used for pre- and post-PCR procedures at each of the three institutions. To assess the adequacy of DNA extraction and the presence of PCR inhibitors, all samples were tested by using PCR for human DNA (-globin or c- myc), as described elsewhere (12, 13). Results None of the intestinal biopsy specimens in groups A, B, and C showed histologic evidence of Whipple disease, as defined by previous histologic descriptions and criteria (5, 16). All specimens from the 173 patients in group A and the 37 patients in group B yielded negative PCR results for T. whippelii DNA (Table) and positive results for human DNA. In group C, all intestinal biopsy specimens from 70 patients tested at the University of Heidelberg, 30 patients tested at Stanford University, and 32 patients tested at the Mayo Clinic were negative for T. whippelii DNA (Table), and all were positive for human DNA. Symptoms in this group included diarrhea; arthritis; unexplained weight loss; and central nervous system signs, including cerebellar ataxia and features of hypothalamic disease. Included in this group was one patient with cerebellar ataxia who had PCR-positive cerebrospinal fluid. Table. Results of Polymerase Chain Reaction Analysis for Tropheryma whippelii in Small-Intestinal Biopsy Samples Discussion The distribution and abundance of the Whipple disease bacterium, T. whippelii, in nature remain poorly defined. Dependence on clinical recognition of this disease, and hence on its typical clinical features, may have led to biased and limited surveillance for this organism. Our data address this problem to some degree and allow an important comparison to be made with findings recently published by two groups that suggest a surprisingly high prevalence of T. whippelii DNA in the upper alimentary tract of persons without findings typical of Whipple disease (10, 11). We examined duodenal biopsy specimens from three different sets of patients. Group A represents patients with normal duodenal histologic findings who underwent endoscopy for reasons other than suspicion of Whipple disease and whose probability of harboring T. whippelii might be considered at most only slightly higher than that of the normal population. Groups B and C might be considered to have had a higher pretest probability of harboring T. whippelii because symptoms and clinical findings consisted of malabsorption, one of the cardinal symptoms of Whipple disease, or otherwise led to the inclusion of Whipple disease in the differential diagnosis. No evidence of T. whippelii DNA was found in any of the intestinal biopsy specimens of the three groups in this study. It should be mentioned that a previously tested intestinal biopsy sample from a patient with negative intestinal histologic findings examined at Stanford University yielded a positive PCR result for T. whippelii DNA, as reported elsewhere (15). This patient had uveitis, and positive results were also obtained at the extraintestinal site (ocular vitreous fluid) by PAS staining, electron microscopy, and PCR. In addition, two other previously examined patients with suspected Whipple disease and negative intestinal histologic findings had PCR results positive for T. whippelii (13). One of these patients had suggestive histologic findings in a lymph node. Thus, the results of this study and previous work performed at our institutions offer several insights. First, T. whippelii DNA was not detected in the histologically negative intestinal mucosa of patients for whom Whipple disease was not considered (group A). Second, Whipple disease was not a common diagnosis in patients with malabsorption (group B), despite the common occurrence of malabsorption in this disease and the importance of including Whipple disease in the differential diagnosis. Third, T. whippelii DNA may be detected in intestinal mucosa in the absence of histopathologic evidence, but the rate at which this was observed is very low,

Cultivation of Tropheryma whipplei from Cerebrospinal Fluid

Whipple disease (WD) is a systemic disorder caused by the bacterium Tropheryma whipplei. Since the recognition of a bacterial etiology in 1961, many attempts have been made to cultivate this bacterium in vitro. It was eventually isolated, in 2000, from an infected heart valve, in coculture with human fibroblasts. Here we report the isolation of 2 new strains of T. whipplei from cerebrospinal fluid (CSF) of 2 patients with intestinal WD but no neurological signs or symptoms. One culture-positive specimen was obtained before treatment; the other was obtained 12 months after discontinuation of therapy, at a time of intestinal remission. In both cases, 15 passages of the cultures were completed over 17 months. Bacterial growth was measured by quantitative polymerase chain reaction, which suggested a generation time of 4 days. Staining with YO-PRO nucleic-acid dye showed characteristic rod-shaped bacteria arranged in chains. Fluorescent in situ hybridization with a T. whipplei-specific oligonucleotide probe, a broad-range bacterial probe, and a nonspecific nucleic-acid stain indicated that all visible bacteria were T. whipplei. Scanning electron microscopy and transmission electron microscopy showed both intracellular and extracellular bacteria. This first isolation of T. whipplei from CSF provides clear evidence of viable bacteria in the central nervous system in individuals with WD, even after prolonged antibiotic therapy.

Role of apoptosis in gastric epithelial turnover.

Gastric epithelial turnover is a dynamic process. It is characterized by continous cell proliferation, which is counterbalanced by cell loss. The biological principle that mediates the homeostasis of epithelium is programmed cell death, or apoptosis. Currently, several subtypes of apoptosis are distinguished, which are mediated by different mechanisms. Various subtypes of apoptosis also occur in the gastric epithelium under various conditions. In the normal stomach, apoptosis due to cell isolation (anoikis) mediates the physiological epithelial turnover. Albeit rarely seen in routine histology, approximately 2% of epithelial cells in the normal stomach are apoptotic. In Helicobacter pylori‐induced gastritis, apoptosis and epithelial proliferation are moderately increased, with approximately 8% apoptotic epithelial cells. In gastritis, factors such as CD95 ligand or tumor necrosis factor (TNF) alpha act as death factors. They bind to specific receptors, CD95 and TNF‐R, which are induced either by other cytokines, such as interferon gamma, or by Helicobacter pylori itself. In addition to CD95, H.pylorican also induce upregulation of CD95 ligand expression. Taken together, the upregulated expression of CD95, and the presence of CD95L in the close proximity to apoptotic gastric epithelial cells suggest a functional role of the CD95‐CD95L system in the induction of apoptosis in H.pylori‐gastritis. The role of other pathways to apoptosis is currently under study. Apart from being a biological phenomenon, apoptosis in the stomach may also have direct clinical consequences. An extreme example is given in gastric graft‐vs.‐host disease when epithelial denudement occurs. Microsc. Res. Tech. 48:303–311, 2000