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Flagellin Stimulates IL-8 Production in Native Human Colon and Enhances Ongoing Colonic Inflammation in Mice
September 2005
3 A Methodological Approach to Adjust for Channeling Bias in Nonrandomized Observational Studies: Application to Cyclooxygenase-2 (COX-2) Specific Inhibitors Dan Pettitt, J.S. Schwartz, Fleur Lee, Glenn M. Eisen, and Jay L. Goldstein Background: Channeling bias may confound the interpretation of results of nonrandomized observational studies. We undertook this analysis to develop a methodology for examining potential channeling bias in the prescription of cyclooxygenase-2 specific inhibitors (COX-2 SIs) and to determine the effect of such bias on measured estimates of drug safety. Methods: The Protocare Sciences Propriety Research database, a managed care claims database that has integrated information on patient eligibility and claims, was used as the data source. Patients were identified based on dispensation of either COX-2 SIs (n ⫽ 43,653) or nonspecific (ns) NSAIDs (n ⫽ 492,831) between January 1, 1999 and December 31, 2001. Odds ratios were calculated to determine the association of health status and gastrointestinal (GI) history with COX-2 SI and ns NSAID dispensation. Propensity scores were derived from multivariate logistic regression analyses to adjust for risk factors associated with COX-2 SI and ns NSAID dispensation. Poisson regression accounting for duration of exposure was used in conjunction with propensity scores to examine the incidence of NSAID-related and ulcer specific hospitalizations during therapy. Results: Among patients with a history of GI hospitalization, the crude incidence rates were approximately 4-fold higher in the ns NSAID compared with the COX-2 SI cohort (incidence rate/100 pt-years: 4.06, 95% CI 3.10 –5.32 and 0.88, 95% CI 0.33–2.34, respectively). Disease state severity based on advancing age and history of GI healthcare resource utilization or NSAID intolerance were strongly associated with COX-2 SI dispensation. After adjusting for these predictors of COX-2 prescribing or factors associated with selective COX-2 prescribing (channeling bias), the relative risk (RR) of both NSAID-related and ulcer-specific GI hospitalizations was reduced among COX-2 SI users compared with ns NSAID users (RR 0.61, 95% CI 0.48 – 0.78 and 0.68, 95% CI 0.48 – 0.96, respectively). Similar results were obtained regardless of exposure to gastroprotective agents (H2RAs, PPIs, or misoprostol) (RR 0.55, 95% CI 0.40 – 0.77 and 0.52, 95% CI 0.31– 0.87 for NSAID-related and ulcer-specific hospitalizations, respectively). Conclusions: Managed care patients with a history of GI problems or ns NSAID intolerance are being channeled to COX-2 SIs. After adjusting for channeling bias, COX-2 SI therapy was associated with fewer GI events, even though these patients had a higher background risk of GI complications.
AMERICAN GASTROENTEROLOGICAL ASSOCIATION
1105
4 Flagellin Stimulates IL-8 Production in Native Human Colon and Enhances Ongoing Colonic Inflammation in Mice Sang Hoon Rhee, Martin Riegler, and Charalabos Pothoulakis Background & Objectives: Flagellin from commensal or enteroinvasive bacteria stimulates Toll-like receptor 5(TLR5) leading to proinflammatory cytokine production. However, whether flagellin participates in the pathophysiology of colitis has not been determined. Our goal was to examine whether flagellin (1) stimulates chemokine production in native human colon; (2) generates colitis; or (3) affects ongoing colonic inflammation in mice. Methods: Human colonic mucosal strips mounted in Ussing chambers were exposed either apically or basolaterally to flagellin (100 ng/ mL), and IL-8 production (ELISA) was measured in the respective supernatants. CD-1 and LPS-resistant C3H/HeJ mice received water alone or water containing 2.5% dextran sulfate sodium (DSS) and, after 4 days, were injected intracolonically with flagellin (0.8 mg)-containing enema (n ⫽ 24 –32 mice/ group), everyday, for 14 days. Severity of colitis and activation of MEK1/2 was examined in colonic tissue sections by histologic and immunofluorescence analysis, respectively, while apoptosis in colonic mucosa was studied by TUNEL assay. Total protein from colon tissues was also analyzed by Western blots to confirm flagellin-induced MEK1/2 activation and apoptosis. Results: Basolateral, but not apical flagellin exposure increased IL-8 release at 2 (by 3-fold, P ⬍ .05) and 4 hours (by 4-fold, P ⬍ .01), implying the presence of basolaterally restricted TLR5 in native colonic mucosa. Both strains of mice gave similar results. Mice that received water alone or water plus flagellin, in the absence of DSS, showed no sign of disease, and no evidence of histologic damage. Mice treated with DSS alone had clinical and histological signs of mild disease and inflammation. In contrast, flagellin together with DSS resulted in substantially reduced survival rates (P ⬍ .001), profoundly less stool consistency (P ⬍ .005), marked weight loss (P ⬍ .001), increased rectal bleeding (P ⬍ .005), and significantly increased erosive lesions and mucosal inflammation (P ⬍ .005–.05) compared with DSS alone. Flagellin caused in vivo activation of MEK1/2 in intestinal epithelial cells and apoptosis in the colonic mucosa of DSS-treated mice. Flagellin exposure to colonic explants from DSS-treated mice up-regulated IL-6, TNF␣, and KC production (n ⫽ 4, P ⬍ .05). Conclusion: Flagellin participates in the pathophysiology of colonic inflammation in animals and humans by interacting with basolateral TLR5 in colonocytes. Supported by a Research Fellowship Award from the CCFA.
3 A Methodological Approach to Adjust for Channeling Bias in Nonrandomized Observational Studies: Application to Cyclooxygenase-2 (COX-2) Specific Inhibitors Dan Pettitt, J.S. Schwartz, Fleur Lee, Glenn M. Eisen, and Jay L. Goldstein Background: Channeling bias may confound the interpretation of results of nonrandomized observational studies. We undertook this analysis to develop a methodology for examining potential channeling bias in the prescription of cyclooxygenase-2 specific inhibitors (COX-2 SIs) and to determine the effect of such bias on measured estimates of drug safety. Methods: The Protocare Sciences Propriety Research database, a managed care claims database that has integrated information on patient eligibility and claims, was used as the data source. Patients were identified based on dispensation of either COX-2 SIs (n ⫽ 43,653) or nonspecific (ns) NSAIDs (n ⫽ 492,831) between January 1, 1999 and December 31, 2001. Odds ratios were calculated to determine the association of health status and gastrointestinal (GI) history with COX-2 SI and ns NSAID dispensation. Propensity scores were derived from multivariate logistic regression analyses to adjust for risk factors associated with COX-2 SI and ns NSAID dispensation. Poisson regression accounting for duration of exposure was used in conjunction with propensity scores to examine the incidence of NSAID-related and ulcer specific hospitalizations during therapy. Results: Among patients with a history of GI hospitalization, the crude incidence rates were approximately 4-fold higher in the ns NSAID compared with the COX-2 SI cohort (incidence rate/100 pt-years: 4.06, 95% CI 3.10 –5.32 and 0.88, 95% CI 0.33–2.34, respectively). Disease state severity based on advancing age and history of GI healthcare resource utilization or NSAID intolerance were strongly associated with COX-2 SI dispensation. After adjusting for these predictors of COX-2 prescribing or factors associated with selective COX-2 prescribing (channeling bias), the relative risk (RR) of both NSAID-related and ulcer-specific GI hospitalizations was reduced among COX-2 SI users compared with ns NSAID users (RR 0.61, 95% CI 0.48 – 0.78 and 0.68, 95% CI 0.48 – 0.96, respectively). Similar results were obtained regardless of exposure to gastroprotective agents (H2RAs, PPIs, or misoprostol) (RR 0.55, 95% CI 0.40 – 0.77 and 0.52, 95% CI 0.31– 0.87 for NSAID-related and ulcer-specific hospitalizations, respectively). Conclusions: Managed care patients with a history of GI problems or ns NSAID intolerance are being channeled to COX-2 SIs. After adjusting for channeling bias, COX-2 SI therapy was associated with fewer GI events, even though these patients had a higher background risk of GI complications.
AMERICAN GASTROENTEROLOGICAL ASSOCIATION
1105
4 Flagellin Stimulates IL-8 Production in Native Human Colon and Enhances Ongoing Colonic Inflammation in Mice Sang Hoon Rhee, Martin Riegler, and Charalabos Pothoulakis Background & Objectives: Flagellin from commensal or enteroinvasive bacteria stimulates Toll-like receptor 5(TLR5) leading to proinflammatory cytokine production. However, whether flagellin participates in the pathophysiology of colitis has not been determined. Our goal was to examine whether flagellin (1) stimulates chemokine production in native human colon; (2) generates colitis; or (3) affects ongoing colonic inflammation in mice. Methods: Human colonic mucosal strips mounted in Ussing chambers were exposed either apically or basolaterally to flagellin (100 ng/ mL), and IL-8 production (ELISA) was measured in the respective supernatants. CD-1 and LPS-resistant C3H/HeJ mice received water alone or water containing 2.5% dextran sulfate sodium (DSS) and, after 4 days, were injected intracolonically with flagellin (0.8 mg)-containing enema (n ⫽ 24 –32 mice/ group), everyday, for 14 days. Severity of colitis and activation of MEK1/2 was examined in colonic tissue sections by histologic and immunofluorescence analysis, respectively, while apoptosis in colonic mucosa was studied by TUNEL assay. Total protein from colon tissues was also analyzed by Western blots to confirm flagellin-induced MEK1/2 activation and apoptosis. Results: Basolateral, but not apical flagellin exposure increased IL-8 release at 2 (by 3-fold, P ⬍ .05) and 4 hours (by 4-fold, P ⬍ .01), implying the presence of basolaterally restricted TLR5 in native colonic mucosa. Both strains of mice gave similar results. Mice that received water alone or water plus flagellin, in the absence of DSS, showed no sign of disease, and no evidence of histologic damage. Mice treated with DSS alone had clinical and histological signs of mild disease and inflammation. In contrast, flagellin together with DSS resulted in substantially reduced survival rates (P ⬍ .001), profoundly less stool consistency (P ⬍ .005), marked weight loss (P ⬍ .001), increased rectal bleeding (P ⬍ .005), and significantly increased erosive lesions and mucosal inflammation (P ⬍ .005–.05) compared with DSS alone. Flagellin caused in vivo activation of MEK1/2 in intestinal epithelial cells and apoptosis in the colonic mucosa of DSS-treated mice. Flagellin exposure to colonic explants from DSS-treated mice up-regulated IL-6, TNF␣, and KC production (n ⫽ 4, P ⬍ .05). Conclusion: Flagellin participates in the pathophysiology of colonic inflammation in animals and humans by interacting with basolateral TLR5 in colonocytes. Supported by a Research Fellowship Award from the CCFA.