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sin the active intervention arm, with some reversals after six months off drugs. The finalreport to be presented will include off study colonoscopies and adverse events in subjectswho were still on study at the time of the second interim analysis. Larger trials will beneeded to determine the absolute risk of cardiovascular events and audiologic changes fromthis regimen. Longer term studies will be needed to determine whether the absolute endpointof colorectal cancer incidence can be reduced in either patients with low stage prior colorectalcancers or in very high risk individuals.

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The Polyp Prevention Trial Continued Follow-Up Study (Ppt-Cfs): OngoingColorectal Cancer Risk Despite Surveillance ColonoscopyKeith Leung, Paul Pinsky, Adeyinka O. Laiyemo, Elaine Lanza, Arthur Schatzkin, RobertE. Schoen

INTRODUCTION: Despite regular colonoscopy, interval colorectal cancer (CRC) may occur.In the dietary Polyp Prevention Trial (PPT), 13 cancers occurred over 5,810 person yearsof observation (PYO) (2.2/1000 PYO) despite participants undergoing a mean of 3.1 colonos-copies, including at study entry, one and four years after enrollment. We examined the rateof interval CRC in the PPT-Continued Follow-up Study (PPT-CFS), a subsequent observa-tional study of participants in the PPT trial, to determine if the cancer risk persisted.METHODS: The dietary PPT was a multicenter, randomized controlled trial to examine theeffects of a low fat, high fiber, high fruit and vegetable diet on the recurrence of colorectaladenomas. For the PPT-CFS, participants provided health information via annual question-naires. Medical records of colonoscopies, cancer diagnosis and treatment were collected andreviewed. RESULTS: Among 2,079 PPT participants, 1,297 subjects completed the PPT trialand agreed to participate in the PPT-CFS. They were followed for a median of 6.2 years.PPT-CFS participants were more likely male (67 vs. 59%, p<.001), more educated (78%vs. 68% post high school education, p<.001), and more likely to have a family history ofCRC (28 vs. 24%, p=.03) than non PPT-CFS participants. In the PPT-CFS, 10 cases of CRCwere diagnosed over 7,626 PYO, for an incidence rate of 0.77% or 1.3/1000 PYO. The ratioof cancers observed compared to that expected by SEER was 0.71 (95% CI 0.29 - 1.14).For all cancers diagnosed since the start of the PPT trial, the ratio of cancers observedcompared to that expected by SEER was 0.78 (95% CI 0.48-1.12). In the PPT-CFS, themean interval between the last cancer free colonoscopy to cancer diagnosis was 41 ± 26months and mean tumor size was 3 cm. CONCLUSIONS: Despite frequent colonoscopyduring the PPT trial, after the trial ended, subjects had an ongoing risk for colorectal cancerthat was similar to, and not statistically different from, the absolute rate expected by SEER.Subjects with a history of adenomatous polyps continue to have a risk for cancer and ongoingsurveillance is required.

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Lgr5/Gpr49: A Stem Cell Marker in the Native Human Colonic EpitheliumAmy Reynolds, Alyson Parris, Natalia Scobioala-laker, Richard Tighe, Michael P. Lewis,Mark R. Williams

The human intestinal epithelium is the most rapidly self-renewing tissue in the body. Tenbillion or so cells are shed into the intestinal lumen each day and are replaced by stem cellprogeny. In the absence of a molecular marker, the physiological status of intestinal stemcells in human health and disease has remained an enigma. An operational definition ofstem cells has been based on analyses of mouse chimaeras and mutagen-induced somaticclones; while thymidine pulse-chase experiments indicated that the stem cell niche waslocated towards the crypt base. In a landmark discovery, Clevers and colleagues demonstratedrecently that the Wnt target gene Lgr5/Gpr49 is a molecular marker of mouse intestinalstem cells (Barker et al., Nature 2007). AIMS: To investigate the expression of Lgr5/Gpr49and other candidate stem cell markers in human colonic crypts. METHODS: Colonic cryptswere isolated from tissue biopsies obtained at sigmoidoscopy from healthy-subjects (Ethicalapproval). Isolated crypts were either fixed immediately or following a tissue culture periodof 24-48 hours in the presence or absence of recombinant human Wnt-1 ligand (50ng/ml).Crypt cell proliferation was assessed by BrDU incorporation/Ki67 labelling and time-lapsedigital video microscopy. Nuclear beta-catenin levels along the crypt-axis were detected byimmunolabelling. Candidate stem cell markers Lgr5/Gpr49, CD133 and musashi were probedfor by mRNA in situ hybridisation and/or immunocytochemistry. Immunolabelling of DigRNA probes and primary antibodies was visualised in three dimensions by fluorescenceconfocal microscopy. RESULTS: Wnt-1 treatment stimulated nuclear translocation of beta-catenin in all cells located at the crypt base, and promoted an increase of more than 30%in the number of BrDU/Ki67 positive cells (P<0.001). Accordingly, a dramatic increase inmitotic activity at the crypt-base was evident from time-lapse microscopy. Between 6-10Lgr5/Gpr49 mRNA positive cells were located within four cell positions from the crypt-baseduring the short-term culture period. CD133 and musashi labelling was diffuse throughoutthe lower half of the crypt. CONCLUSIONS: Discrete labelling of the Wnt target gene Lgr5/Gpr49 is consistent with the presence of 6-10 stem cells located at the human colonic cryptbase. Canonical Wnt signalling is active in this region and likely supports stem cell prolifera-tion and Lgr5/Gpr49 expression. Future studies will investigate the molecular signature andphysiological status of intestinal stem cells in human health and disease.

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Repetitive Colorectal Distension in Neonatal Rats Induces Colonic Mucosaland Muscular Dysfunction in AdulthoodArchana Rao, Elie D. Al-Chaer, Beverley Greenwood-Van Meerveld

Background: Early life stress (ELS) is a predisposing factor for irritable bowel syndrome(IBS). Previous studies have shown that ELS induced by maternal separation (MS) or colonicirritation (CI) leads to hypersensitivity to colorectal distension (CRD) in adulthood. AlthoughELS from MS increases colonic permeability, the effect of neonatal CI on the colonic mucosaland musculature requires further investigation. Our goal was to test the hypothesis that

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repetitive CRD in neonates leads to abnormalities in colonic permeability and changes insmooth muscle function in the adult rat. Methods: In neonatal rats (n=14) repetitive CRDwas performed on days 8, 10 and 12. The control group (n=7) did not receive colonicmanipulation. As adults (3 months of age) stool consistency was graded from 0 (formedstool) to 3 (liquid stool). Colonic tissue was isolated for histology and myeloperoxidase(MPO) levels. The colonic mucosa was placed in modified Ussing chambers for analysis ofelectrophysiological measurements of permeability, and short circuit current (Isc) responsesto forskolin, histamine, electrical field stimulation (EFS) and carbachol (CCh). Segments ofcolonic musculature from treated and control rats were placed in organ baths and contractileresponses to KCl, EFS and CCh were determined. Results: In adult rats no significantchanges in colonic histology or MPO activity were observed between the treated or controlgroups. However, the stool consistency score was significantly increased in rats that under-went CRD as neonates (0.3 ± 0.05 to 1.5 ± 0.05, p<0.05). Mucosal permeability, measuredas an increase in basal conductance, was significantly increased in the treatment group (22± 3 to 33 ± 5 ms/cm2, p<0.05). No changes in Isc responses to forskolin, histamine, EFSor CCh were observed between the treated and control groups. As adults, rats that underwentCRD as neonates exhibited a significantly elevated smooth muscle contractile response toKCl, but no changes in contractility in response to EFS or CCh. Summary and Conclusion:Repetitive CRD in neonatal rats, shown previously to produce colonic hypersensitivity inthe absence of colon inflammation in adulthood, also leads to significant alterations incolonic mucosal and smooth muscle function characterized by loose stools, increased mucosalpermeability and increased smooth muscle contractility in adult rats.

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Direct Medical Costs Associated with Constipation from Childhood to EarlyAdulthood: A Birth Cohort StudyDenesh K. Chitkara, Nicholas J. Talley, Nilay Shah, Amy Weaver, Slavica Katusic, MirandaA. Van Tilburg, William E. Whitehead, G. Richard Locke

Background: Constipation affects 9% of children and 12-17% of the adults. Although directmedical costs for constipation related medical visits are thought to be high, to date therehave been no studies examining if longitudinal resource utilization is persistently elevatedin individuals with constipation from childhood to early adulthood. Aim: To estimate theincremental direct medical costs and type of health care associated with constipation fromchildhood to early adulthood. Methods: This is a nested case-control retrospective study.The sample consists of all mothers of 5,718 children in the population-based birth cohortswho were born during 1976-1982 in Rochester, MN and did not migrate from the areaprior to 5 years of age. Eighty percent of subjects remained in the area until 18 years ofage. All medical visits for providers were available for research review through the RochesterEpidemiology Project. The cases included individuals who presented to Olmsted countymedical facilities with constipation. Controls were randomly selected among all non-casesin the sample (i.e. no diagnosis for constipation). The controls were matched to cases in a2:1 ratio, matching on gender, DOB, and years of follow-up. Direct medical costs for casesand controls were collected from the time subjects were between 5-18 years of age or untilthe subject emigrated from the community. The medical costs and utilization were obtainedfrom administrative data (excluding outpatient pharmaceutical costs). Results: We identified229 among the birth-cohort members with a diagnosis of constipation. We observed amedian follow-up of 6.7 years for the cases and 6.7 years for the controls. The meanpredicted annual total costs for cases were higher for cases of constipation for every year(age 5-18 years) compared to controls. The median annual direct medical costs for caseswere twice those of controls ($2586 vs. $1209; p=0.005). The median annual inpatientcosts for cases were $910 compared to $499 for controls (p=0.14), while the medianoutpatient costs for cases were $1647 compared to $696 for control (p=0.0002). The meanannual number emergency department visits for cases were 1.2 compared to 0.4 for controls(p<0.0001). Conclusion: Individuals with constipation consistently experience higher longit-udinal medical care utilization, higher outpatient costs and more ED visits from childhoodto early adulthood.

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The Economic Burden of Childhood Constipation in the United StatesOlivia Liem, Jeffrey S. Harman, Hayat M. Mousa, Kelly J. Kelleher, Marc A. Benninga,Carlo Di Lorenzo

BACKGROUND: Constipation is a condition that affects a substantial proportion of thechildhood population. It is often chronic in nature and is associated with impaired qualityof life. The economical impact and healthcare utilization related to childhood constipationhave not yet been analyzed. METHODS: We estimated the use and cost of health careservices using the 2003-2004 Medical Expenditure Panel Survey, a nationally representativehousehold survey. We identified anyone whose parent either reported constipation orreceived a prescription for laxatives in a given year. Individuals were counted only onceover the 2 year period. RESULTS: There were 21,778 children aged 0-18 years in the MEPSdatabase, representing 158 million children nationally. It was estimated that 1.1% (1.7million) of US children were constipated in the 2-year period. No differences were foundwith respect to age, gender, race and socioeconomic status between constipated childrenand children without constipation. Constipated children used more health services thanchildren without constipation resulting in significantly higher costs: $3,362 (3,209 - 3,516)/year vs. $1,095 (1,083- 1,108)/year. This amounts to an additional cost for children withconstipation of 3,9 billion US dollars/year. CONCLUSION: Childhood constipation has asignificant impact on the use and cost of medical care services among US children. Theestimated cost per yr is 3 times higher compared to children without constipation andprobably underestimates the real burden of childhood constipation since it does not accountfor loss of quality of life, undiagnosed constipation and unaccounted costs like extra under-wear and mattresses.