Autoimmunity Links Vinculin to the Pathophysiology of Chronic Functional Bowel Changes Following Campylobacter jejuni Infection in a Rat Model.

BACKGROUND: Acute gastroenteritis can precipitate irritable bowel syndrome (IBS) in humans. Cytolethal distending toxin is common to all pathogens causing gastroenteritis. Its active subunit, CdtB, is associated with post-infectious bowel changes in a rat model of Campylobacter jejuni infection, including small intestinal bacterial overgrowth (SIBO). AIM: To evaluate the role of host antibodies to CdtB in contributing to post-infectious functional sequelae in this rat model. METHODS: Ileal tissues from non-IBS human subjects, C. jejuni-infected and control rats were immunostained with antibodies to CdtB, c-Kit, S-100, PGP 9.5 and vinculin. Cytosolic and membrane proteins from mouse enteric neuronal cell lysates were immunoprecipitated with anti-CdtB and analyzed by mass spectrometry. ELISAs were performed on rat cardiac serum using CdtB or vinculin as antigens. RESULTS: Anti-CdtB antibodies bound to a cytosolic protein in interstitial cells of Cajal (ICC) and myenteric ganglia in C. jejuni-infected and naïve rats and human subjects. Mass spectrometry identified vinculin, confirmed by co-localization and ELISAs. Anti-CdtB antibodies were higher in C. jejuni-infected rats (1.27 ± 0.15) than controls (1.76 ± 0.12) (P < 0.05), and rats that developed SIBO (2.01 ± 0.18) vs. rats that did not (1.44 ± 0.11) (P = 0.019). Vinculin expression levels were reduced in C. jejuni-infected rats (0.058 ± 0.053) versus controls (0.087 ± 0.023) (P = 0.0001), with greater reductions in rats with two C. jejuni infections (P = 0.0001) and rats that developed SIBO (P = 0.001). CONCLUSIONS: Host anti-CdtB antibodies cross-react with vinculin in ICC and myenteric ganglia, required for normal gut motility. Circulating antibody levels and loss of vinculin expression correlate with number of C. jejuni exposures and SIBO, suggesting that effects on vinculin are important in the effects of C. jejuni infection on the host gut.

Intestinal Methanobrevibacter smithii but not total bacteria is related to diet-induced weight gain in rats.

UNLABELLED: It is increasingly understood that gastrointestinal (GI) methanogens, including Methanobrevibacter smithii, influence host metabolism. OBJECTIVE: Therefore, we compared M. smithii colonization and weight gain in a rat model under different dietary conditions. DESIGN AND METHODS: Sprague-Dawley rats were inoculated with M. smithii or vehicle (N = 10/group), fed normal chow until day 112 postinoculation, high-fat chow until day 182, then normal chow until day 253. Thereafter, five rats from each group were fed high-fat and normal chow until euthanasia. RESULTS: Both groups exhibited M. smithii colonization, which increased following inoculation only for the first 9 days. Change to high-fat chow correlated with significant increases in weight (P < 0.00001) and stool M. smithii (P < 0.01) in all rats, with stool M. smithi decreasing on return to normal chow. Rats switched back to high-fat on day 253 further increased weight (P < 0.001) and stool M. smithii (P = 0.039). Euthanasia revealed all animals had higher M. smithii, but not total bacteria, in the small intestine than in the colon. Rats switched back to high-fat chow had higher M. smithii levels in the duodenum, ileum, and cecum than those fed normal chow; total bacteria did not differ in any bowel segment. Rats which gained more weight had more bowel segments colonized, and the lowest weight recorded was in a rat on high-fat chow which had minimal M. smithii colonization. CONCLUSIONS: We conclude that M. smithii colonization occurs in the small bowel as well as in the colon, and that the level and extent of M. smithii colonization is predictive of degree of weight gain in this animal model.

Role of Cytolethal Distending Toxin in Altered Stool Form and Bowel Phenotypes in a Rat Model of Post-infectious Irritable Bowel Syndrome.

BACKGROUND/AIMS: Campylobacter jejuni infection is a leading cause of acute gastroenteritis, which is a trigger for post-infectious irritable bowel syndrome (PI-IBS). Cytolethal distending toxin (CDT) is expressed by enteric pathogens that cause PI-IBS. We used a rat model of PI-IBS to investigate the role of CDT in long-term altered stool form and bowel phenotypes. METHODS: Adult Sprague-Dawley rats were gavaged with wildtype C. jejuni (C+), a C. jejunicdtB knockout (CDT-) or saline vehicle (controls). Four months after gavage, stool from 3 consecutive days was assessed for stool form and percent wet weight. Rectal tissue was analyzed for intraepithelial lymphocytes, and small intestinal tissue was stained with anti-c-kit for deep muscular plexus interstitial cells of Cajal (DMP-ICC). RESULTS: All 3 groups showed similar colonization and clearance parameters. Average 3-day stool dry weights were similar in all 3 groups, but day-to-day variability in stool form and stool dry weight were significantly different in the C+ group vs both controls (P < 0.01) and the CDT- roup (P < 0.01), but were not different in the CDT- vs controls. Similarly, rectal lymphocytes were significantly higher after C. jejuni (C+) infection vs both controls (P < 0.01) and CDT-exposed rats (P < 0.05). The counts in the latter 2 groups were not significantly different. Finally, c-kit staining revealed that DMP-ICC were reduced only in rats exposed to wildtype C. jejuni. CONCLUSIONS: In this rat model of PI-IBS, CDT appears to play a role in the development of chronic altered bowel patterns, mild chronic rectal inflammation and reduction in DMP-ICC.

Effects of rifaximin treatment and retreatment in nonconstipated IBS subjects.

UNLABELLED: Recent evidence suggests a role for gut bacteria and antibiotics in the pathophysiology and treatment of irritable bowel syndrome (IBS), respectively. While the benefits of the antibiotic rifaximin have demonstrated efficacy and durable improvement in symptoms over 3 months, the long-term need for retreatment using this approach is mostly unknown. In this retrospective study, subjects with nonconstipated IBS who were retreated with rifaximin were examined. METHODS: Charts of patients who were seen at a tertiary care medical center between 2007 and 2011 were reviewed. After exclusion criteria were applied, subjects who had received rifaximin and were seen for retreatment were fully reviewed. During review, demographic information, duration of response, and success of treatment and retreatment were evaluated. RESULTS: A total of 522 charts were reviewed. Of these 522 charts, 71 subjects were nonconstipated IBS subjects who had received at least one retreatment. Of these, 48 had a second, 22 had a third, 7 had a fourth, and 4 had a fifth treatment. More than 75% of subjects who initially responded to rifaximin also responded to any further retreatment, with no significant reduction in benefit for successive retreatments. Furthermore, there was no change in the duration of benefit (median time between treatments) for successive retreatments. CONCLUSIONS: Retreatment with rifaximin for subjects with nonconstipated IBS in a real-world clinical practice was successful up to five times without decrease in duration or effect.

Acute and chronic histological changes of the small bowel secondary to C. jejuni infection in a rat model for post-infectious IBS.

BACKGROUND: Campylobacter jejuni has been implicated in the pathogenesis of post-infectious irritable bowel syndrome (PI-IBS) in humans, effects which may be because of cytolethal distending toxin (CDT). In this study, we characterized both acute and chronic-phase histological changes of the small bowel in rats exposed to wild-type C. jejuni 81-176, or a strain that does not produce CDT, by using a validated rat model of PI-IBS. METHODS: Sprague-Dawley rats were given 1.0 × 10(8) CFU of either wild-type C. jejuni 81-176 (C+, PI/C+) or the CDT-negative strain (CDT-), or vehicle alone (Control). Acute-phase rats (C+, CDT-) were euthanized on days 2, 4, 8, 16, and 32. Chronic-phase rats (PI/C+, Control) were euthanized 3 months after clearing the initial infection. Segments of duodenum, jejunum, and ileum were resected and the contents plated for C. jejuni culture, and tissue sections were stained for histology. RESULTS: We observed preferential infection of the ileum and jejunum by Campylobacter jejuni. Compared with controls, epithelial cell basal membrane ballooning, villous tip disruption, and reduced villous-to-crypt ratios were observed for both C+ and CDT- rats. Villous widening, the only result significantly different in C+ vs. CDT- rats, was greatest at day 4 (134.1 ± 21.12 µm vs. 109.9 ± 10.6 µm for CDT-, P < 0.01). Little or no cellular inflammatory changes were seen during acute C. jejuni infection. Three months after clearing the initial infection, no histological changes remained. CONCLUSION: Significant histological changes, with the absence of inflammatory cells, are seen in the duodenum, jejunum, and ileum of rats during acute infection with C. jejuni. These changes occurred irrespective of the presence or absence of the CDT toxin.

Antibiotic prophylaxis prevents the development of a post-infectious phenotype in a new rat model of post-infectious IBS.

BACKGROUND: A recent post-infectious rat model with Campylobacter jejuni 81-176 has replicated the events noted in humans with post-infectious irritable bowel syndrome (IBS). In this study, we test whether prophylactic treatment with the antibiotic rifaximin will prevent the development of long-term altered bowel function in this model. METHODS: Sprague-Dawley rats were divided into two groups. Both groups were gavaged with a 1 mL solution of 10(8) cfu/mL of C. jejuni. However, one group was also prophylactically gavaged with a solution of rifaximin 200 mg per day for 3 days (the day before gavage, the day of gavage, and the day after gavage with C. jejuni). Fresh stool was collected from rats daily until two consecutive stool cultures were negative for C. jejuni. The rats were then housed for 3 months. At the end of 3 months, fresh stool was collected on three consecutive days to determine stool % wet weight and stool consistency on a stool score. RESULTS: Rats that received rifaximin antibiotic prophylaxis had a greater rate of stool shedding of C. jejuni. However, the mean duration of colonization was shorter in the rifaximin-treated group (10.3 ± 7.1 days) compared to rats receiving no prophylaxis (12.6 ± 5.9 days) (P < 0.01). After 3 months, rats that did not receive rifaximin had a greater variability in stool % wet weight (P < 0.01). Furthermore, the average stool consistency over 3 days of measurement was closer to normal in the rifaximin-treated rats, with a consistency of 1.1 ± 0.3, compared to 1.5 ± 0.4 in rats receiving no prophylaxis (P < 0.00001). CONCLUSIONS: Prophylactic treatment of rats with the antibiotic rifaximin in a new animal model of post-infectious IBS with C. jejuni mitigated the development of long-term altered stool form and function.

ICC density predicts bacterial overgrowth in a rat model of post-infectious IBS.

AIM: To investigate the interstitial cells of Cajal (ICC) number using a new rat model. METHODS: Sprague-Dawley rats were assigned to two groups. The first group received gavage with Campylobacter jejuni (C. jejuni) 81-176. The second group was gavaged with placebo. Three months after clearance of Campylobacter from the stool, precise segments of duodenum, jejunum, and ileum were ligated in self-contained loops of bowel that were preserved in anaerobic bags. Deep muscular plexus ICC (DMP-ICC) were quantified by two blinded readers assessing the tissue in a random, coded order. The number of ICC per villus was compared among controls, Campylobacter recovered rats without small intestinal bacterial overgrowth (SIBO), and Campylobacter recovered rats with SIBO. RESULTS: Three months after recovery, 27% of rats gavaged with C. jejuni had SIBO. The rats with SIBO had a lower number of DMP-ICC than controls in the jejunum and ileum. Additionally there appeared to be a density threshold of 0.12 DMP-ICC/villus that was associated with SIBO. If ileal density of DMP-ICC was < 0.12 ICC/villus, 54% of rats had SIBO compared to 9% among ileal sections with > 0.12 (P < 0.05). If the density of ICC was < 0.12 DMP-ICC/villus in more than one location of the bowel, 88% of these had SIBO compared to 6% in those who did not (P < 0.001). CONCLUSION: In this post-infectious rat model, the development of SIBO appears to be associated with a reduction in DMP-ICC. Further study of this rat model might help understand the pathophysiology of post-infectious irritable bowel syndrome.

Gender distribution in irritable bowel syndrome is proportional to the severity of constipation relative to diarrhea.

BACKGROUND: A significant gender disparity has been observed in individuals with irritable bowel syndrome (IBS), with females outnumbering males, especially in constipation-predominant IBS (C-IBS). However, this observation is based on Rome criteria categorization and does not take into account the severity of constipation or diarrhea. OBJECTIVE: In a large prospective data set, gender differences across the severity of constipation and diarrhea were examined in patients with IBS. METHODS: Consecutive adult patients with Rome I positive IBS who were referred to a tertiary care medical center (Cedars-Sinai Medical Center, Los Angeles, California) in 1999-2003 were given a questionnaire. The questionnaire asked subjects to rate their constipation and diarrhea according to perceived severity using a scale from 0 (none) to 5 (very severe). C-IBS was determined to be present if the severity of constipation was greater than the severity of diarrhea (sevC > sevD). If sevD > sevC, subjects were considered to have diarrhea-predominant IBS (D-IBS). To further categorize their symptoms, subjects were then grouped by the difference between the severity of constipation and diarrhea, creating a range of values from -5 to +5. For each of these 11 constipation/diarrhea severity values, the female:male ratio was determined. The severity of constipation to diarrhea was compared by Spearman rank correlation. RESULTS: A total of 429 subjects with IBS (325 women: mean [SD] age, 42.5 [0.8] years; 104 men: mean age, 42.2 [1.7] years) completed the questionnaire. Constipation occurred more frequently in women (79.7%) compared with men (61.5%) (odds ratio [OR] = 2.49; 95% CI, 1.55-4.02). The prevalence of diarrhea was similar between the sexes. Whereas C-IBS was more common in women (31.8%) than in men (26.0%) (OR = 2.03; 95% CI, 1.24-3.30), D-IBS was more prevalent in men (62.5%) than in women (36.3%) (OR = 2.39; 95% CI, 1.53-3.75). When the female:male ratio was evaluated across the 11 severity score categories of constipation and diarrhea, the greater the sevC - sevD score, the higher the proportion of women (R = 0.80; P = 0.003). CONCLUSION: In this study of patients with IBS, the observation of the association of constipation and gender in IBS is extended to indicate that the female:male ratio significantly increases according to the severity of constipation relative to the severity of diarrhea.

Evaluating breath methane as a diagnostic test for constipation-predominant IBS.

Studies suggest that subjects with IBS have altered gut flora. Among these findings, methane production is more commonly associated with constipation-predominant symptoms. In this study, we prospectively evaluated the role of methane as a diagnostic test. Consecutive Rome I positive IBS patients referred for a lactulose breath test were eligible to participate. After exclusion criteria, subjects completed a symptom questionnaire grading bloating, diarrhea, and constipation on a VAS scale (0-100 mm). Once completed, a physician interviewed the subjects and rated the subject accordingly, and also determined whether the patient had C-IBS, D-IBS, or neither. Subjects and physicians were blinded to the results of the breath test. The presence of methane in the breath test was compared to the results of the scoring by subjects and physicians. A total of 56 Rome I positive IBS subjects were enrolled. During breath testing, 28 subjects produced methane. Good agreement between physician's evaluation and the patient's was seen (diarrhea = 0.69; constipation = 0.69; bloating = 0.62). The severity of constipation was noted to be greater in the methane group (49.3 +/- 28.7) than in the non-methane group (25.3 +/- 31.47) (P < 0.01). In contrast, diarrhea was less severe in the methane group (12.3 +/- 21.0) than the non-methane group (36.7 +/- 32.4) (P < 0.01). Out of the 56 patients, 23 C-IBS subjects were identified by the physician. When methane was used to predict the assignment of C-IBS compared to non-C-IBS, it had a sensitivity of 91.7% and a specificity of 81.3% (OR = 47.7, CI = 9.4-232, P < 0.00001). In conclusion, methane is a potential diagnostic test for the identification of C-IBS and may guide treatment.