and was partially recovered by dbcGMP. Conclusion: These results suggest that NO contributes to gastric mucosal restitution at least in part by stimulating basolateral K ÷ channels of the migrating epithelial cells.

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Epithelial Migration: A Novel Role Of Nuclear Factor-KB (NF-KB) Eric D. Strauch, Jian-Ying Wang, Li Li, Rag N. Jaladanki, Jennifer A. Vann, Barbara L. Bass, Univ of Maryland and VA Medical Ctr, Baltimore, MD

Restitution, the migration of adjacent epithelial cells to seal an injury, is an important mecha- nism of gastrointestinal mucosal repair. Physiological concentrations of bile salts stimulate migration. The role of NF-KB, a ubiquitous multifactorial transcription factor, in epithelial migration has not been studied. We hypothesized that the process of epithelial repair could involve the NF-KB pathway and studied NF-KB binding, and the effect of NF-KB inactivation on epithelial migration after wounding. Method~. IEC-6 enterocytes were grown on Metrigel coated plastic dishes. Monolayers were wounded and cell migration was examined using an in vitro model that mimics the early cell division-independent stages of epithelial restitution. Migration was measured in cells with control media, media with .05 mM taurodeoxycholic acid, and media with the NF-KB inhibitor MG-132 (1 and 10 p.M). NF-KB sequence specific binding was measured by gel shift analysis of nuclear protein extracts. Resu/t~. NF-KB binding was increased in all migrating cells. NF-KB binding occurred at 2 h and peaked between 4 and 6 hours after wounding. Inhibition of NF-KB binding activity by the inhibitor MG-132 inhibited cell migration after wounding. Bile salt further stimulated migration and increased NF-KB binding activity. Conc/usion~. 1. Migrating enterocytes show increased NF-KB binding activity; 2) inhibition of NF-K6 by MG-132 blocks cell migration; 3) bile salt stimulation of migration involves activation of the NF-KB pathway. These data support a previously unrecog- nized role for the NF-KB pathway in epithelial repair and maintenance of intestinal integrity.

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Targetted Gene Deletion Of Murine SP/I"FF2 Results In Decreased Gastric Proliferation, Increased Basal Acid Secretion And increased Susceptibility To NSAID Injury James J. Farrell, Doug Taupin, Theodore Koh, MA Gen Hosp and Harvard Medical Sch, Boston, MA; Daniel K. Podolsky, Timothy C. Wang, MA Gen Hosp, Boston, MA

Background: Spasmolytic polypeptide (SP / TFF2) is a member of the trefoil peptide family, expressed predominantly in the mucous cells in the stomach. While it is thought to function in mucosal protection and restitution, its precise physiological role has not yet been elucidated. We further determined the function of SP/TFF2 by inactivating the mSP/TTF2 gene. Method: SP/TFF2-dedcient mice were generated through targeted gene disruption. Gastric tissue from sP/rFF2 deficient and wild-type mice of equivalent age and genetic background was used for the purposes of histological and electron micrograph comparison. Glandular height, number of parietal cells per gland and gastric proliferation rates (BrdU Labelling Index) were calculated. Multiple random low powered electron micrographs were obtained (3OOOx) and the percentages of activated parietal cells were calculated. Activated parietal cell count and total acid output was determined in both basal acid secretion (in the fed state) and maximal acid secredon (after 4 hours of pyloric ligedon). Gastric ulceration was induced using intraperitoneal indo- methacin at different doses and times and the mean number of ulcers per histological section was calculated for both TFF2 deficient and wild-type mice. All results are expressed as mean + / - SEM. Results: Homozygous mutant mice were viable and fertile without obvious gastrointestinal abnormalities. Quantitative measurements revealed an 11.5% decrease in gastric mucosal thickness (p < 0.0005) and a 33.6% reduction in gastric mucosal proliferation rates (p<0.004). In addition, there was a two-fold increase in activated parietal cells (34% vs 62%, p<0.02) resulting in a two-fold increase in basal gastric acid output (1.12 + / - 0.23 vs 2.06 + / - 0.39 uEq H*, p<0.05). There was significantly more ulceration in TFF2 deficient mice given indomethacin 40 mg/kg when compared with wild-type mice (p<0.05). Conclusion: These results suggest a physiologic role for TFF2/SP to promote mucosal healing through the stimulation of proliferation and downreguledon of gastric acid secretion.

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Molecular Mechanisms of Transcriptional Repression Shared by the Growth Regulatory Spl-like Proteins and the Tumor Suppressor Mad1 Jin-San Zhang, Martin Moncrieffe, Joanna Kaczynski, Franklyn Prendergast, Raul Urrutia, Mayo Clin, Rochester, MN

Our laboratory studies the role of Spl-like zinc finger proteins in the regulation of gene expression as well as in the control of normal and neoplastic cell growth in the exocrine pancreas. Studies from our lab and others indicate that the Spl-like transcriptional repressors TIEGs and BTEBs suppress cell growth and induce cell differentiation, respectively. In the present study, we report that an important feature of these proteins is the presence of a conserved repression domain within their N-termini, which is responsible for the interaction between these proteins and the co-repressor mSin3A. Sin3A functions as a large protein scaffold capable of supporting multiple protein-protein interactions during the process of gene silencing. Based on both circular dichroism and molecular modeling studies we find that the Sin3 Interaction Domain (SID) of both the TIEG and the BTEB proteins forms an helical motif that is highly similar to the SID repression domain of the tumor suppressor protein Mad1. Mad1 is a Myc antagonist that heterodimerizes with Max and functions as a Sin3A-dependent transcription repressor to suppress neoplastic transformation. Proline substitution within this conserved helical domain of Mad1, TIEG, and BTEB completely abol- ishes their ability to bind mSin3A. Interestingly, these SID domains display a conserved consensus sequence, AA/VXXL, which differs slightly from both the LXXL and the LXX I/H I XXX I/L of-helical domains of steroid receptors. These two distinct ~helical domains differen- tially mediate the interaction of steroid receptors/ligand complexes with either their respective coactivators or corepressors. In conclusion, our results reveal that the SID-Sin3A interaction is not exclusive to the transcriptional regulatory function of the tumor suppressor Mad1 but

rather represents a widespread mechanism of repression utilized by different families of transcription factors. Because the transcription regulatory activity of these proteins is necessary for their growth suppressive properties, this novel mechanism is also relevant to the regulation of cell growth. This work supported by the Mayo Cancer Center, NIH Grant DK52913 (RU) and NIH Training Grant CA75926 to the Tumor Biology Program (JK),

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Transcription Factor ZBP-89 Inhibits Cell Growth and Promotes Apoptosts by Stabilizing Tumor Suppressor Protein p53 Longchuan Bat, Juanita L. Merchant, Univ of Michigan, Ann Arbor, MI

BACKGROUND: ZBP-89 is a widely expressed four-zinc finger transcription factor that binds to GC-rich DNA elements in a variety of promoters involved in growth regulation, e.g., gastrin and ornithine decarboxylase. We have recently shown that ZBP-89 stimulates p21 .~tl in a p53-independent manner that requires histone daacetylase inhibition and the coactivator p30O. In addition, transient expression of ZBP-89 arrests cell growth in G1. Since the tumor suppressor p53 also targets p21 and is involved in growth arrest, we investigated whether the mechanism of ZBP-89 regulation may be through p53 activation. METHODS: A ZBP-89 adenoviral expression vector (Ad-ZBP-89) or the empty vector was used to transiently express the protein in either gastric (AGS) or colonic (HCT116) adenocarcinoma cell lines. Row cytometry was used to analyze the cell cycle of infected cells. Apoptosis was verified by TUNEL assay, flow cytometry and changes in pro-caspasa3 protein. Changes in p53 protein with Ad-ZBP-89 expression were documented by immunofluorescence and pulse chase analysis. In vivo and in vitro protein-protein interactions were documented by coprecipitation and either immunoblotting or autoradiography. RESULTS: ZBP-89 overexpression arrested both AGS and HCT116 cells in G1 and G2. The number of cells in S phase was reduced by 80%. The number of cells in sub-G1 increased 5-fold suggesting the presence of apoptotic cells. Apoptosis was confirmed by fluorescent detection of DNA fragments using the TUNEL assay and a~vation of the pro-apoptotic enzyme pm-caspase3. Immunoblot analysis revealed that both endogenous p53 and p2f protein levels increased about 5 and 2-fold respectively with ZBP-89 expression. To determine if ZBP-89 increased p53 protein by transcriptional activation, Northern blots were performed and revealed no increase in p53 mRNA. This result suggested that ZBP-89 has no effect on p53 transcription but might regulate the protein by stabilization. To demonstrate that ZBP-89 stabilizes p53, pulse chase was performed. The half-life of p53 was 84 min in the presence of ZBP-89 and 30 min in mock or vector infected cells. To show direct protein interactions, p53 antibody coprecipiteted trans~ected Z£,P-89 1rum AGS ceils. Further, in vitro translated ZBP-89 was coprecipitated with p53 antibody and vice versa. CONCLUSIONS: The results show that ZBP-89 overexpression inhibits proliferation and pro- motes apoptnsis through direct binding to p53 with subsequent stabilization.

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The Homeodomain Tmnssription Factor Cdxl Reduces the Growth of Human Colon Cancer Cells by Reducing 61 cdk Kinase Activity and Cyclin D1 Expression. John P. Lynch, Matthew S. Keller, Eun Ran Suh, Univ of Pennsylvania, Philadelphia, PA; Peter G. Traher

INTRODUCTION: Cdxl is a homeodomain transcription factor that regulates intestine-specific gene expression. Studies of human colonic neoplasms and colon cancer cell lines have found that CDX1 expression is diminished or absent. We have previously demonstrated that expression of Cdxl in rat intestinal IEC6 cells or the human colon cancer DLDlcells led to a dramatic reduction in pmlifaration and DNA synthesis which was associated with a hypophosphorylated retinoblastoma protein (pRb). Herein, we sought to elucidate the mecha- nism by which Cdxl regulates proliferation of human colon cancer cells. METHODS: We utilized a Cdxl edenovirus expression system and transient transfections of OLD1 or HCT116 cells in our studies. Flow cytometry, cdk kinase assays, and cyclin D1 mRNA stability studies were then carried out in Cdxl expressing and control cells. RESULTS: pRb is phosphorylated in G1 by the cyclin dependent kinases (cdk) 2, 4, and 6. Cdxl expression led to a reduction in Cyclin D associated cdk kinase activity (cdk4 and cdk6). Cdk4 kinase activity was reduced by 40%. More significantly, a 3 to 4-fold reduction in cdk6 kinase activity was noted. Cyclin A, B, or E dependent cdk2 kinase activity was reduced 3-fold in DLD1 cells expressing Cdxl. We have observed that cyclin D1 mRNA and protein levels are reduced by Cdxl expression, and this may in turn explain the reduction in cdk 4 and cdk6 kinase activity. It is not known if this is a transcriptional or post-transcriptional effect. We report here that preliminary studies by us have not found that cyclin Df mRNA stability is altered by Cdxl expression. Finally, ongoing studies with transiently transfected cells suggest the N-terminal domain of Cdxl is essential for the antipmtiferative effect. Transfection of full-length and C-terminal truncations of Cdxl induce a G1 accumulation of cells that is not seen with the N-terminal Cdxl truncation. CONCLUSIONS: Cdxl expression inhibits the proliferation of DLD1 cells by a reduction in G1 cdk kinase activity, in particular activity associated with cyclin DI. Cyclin D1 mRNA levels are likely reduced by Cdxl expression at the level of cyciin D1 gene transcription. The antiproliferative effect of Cdxl appears to require the N-tarminus but not the C-terminus of Cdxl. Our data suggest that Cdxl may be an important negative regulator of cyclin D1 levels and G1 cdk kioase activity. Therefore, loss of Cdxl expression in colorectal cancers may contribute to their unregulated growth.

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Gastric IMestinal Metaplasia Is Induced by Estopic Expression of Cdx2 Debra G. Silberg, Jessica Sullivan, Eugene Kang, Jennifer Moffett, Newman J. Sund, Sara D. Sackett, Klaus H. Kaestner, Univ of Pennsylvania Sch of Medicine, Philadelphia, PA

Gastric cancer is a common malignancy and leading cause of cancer related death worldwide. The more frequent intestinal-type gastric cancer occurs through a progression from chronic gastritis to intestinal metaplasia and dysplasia. The genes that induce the human premalignant lesion of intestinal metaplasia are unknown but of clear clinical relevance. Cdx2, an intestine specific homeobox transcription factor, although not expressed in the normal stomach, is

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expressed in human intestinal metaplasia of the gastric mucosa. Cdx2 is known to induce the differentiation of intestinal epithelial cells in vitro, therefore we hypothesized that ectopic expression of Cdx2 in the gastric mucosa could induce the transdifferentiation process that occurs in intestinal metaplasia. Methods: Cdx2 was expressed ectopically in the gastric epithelium of transgenic mice using a construct in which the mouse Cdx2 cDNA was placed under the control of the cis-regulatory elements of the winged helix transcription factor Hnf3.y. The transgenic mice generated were studied to determine if Cdx2 expression could induce intestinal type differentiation or gene expression in the gastric epithelium indicative of intestinal metaplasia. Results: By gross morphology, the stomachs of Hnf3~//Cdx2 transgenic mice were smaller and deformed as compared to their wild-type littermates. This correlated microscopi- cally with a decrease in the depth of the gastric mucosa in the transgenic mice. Additional histologic examination revealed the presence of alcian blue positive, intestinal-type goblet cells in the stomachs of the transgenic mice, which is a hallmark of intestinal metaplasia. Intestinal gene expression was analyzed by Northern analysis from paired RNA samples isolated from the stomachs of transgenic and wild-type mice. These studies demonstrated that expression of intestine specific genes such as villin, guanylyl cyclase C, and intestinal trefoil factor were induced by the ectopic expression of Cdx2 in the gastric epithelium of transgenic mice. Conclusion: Ectopic expression of Cdx2 alone can induce intestinal metablasia in the gastric mucosa of transgenic mice characterized by the presence of intestinal type goblet cells and intestine specific oene expression. These mica represent the first genetic model of intestinal metaplasia and provide a powerful tool to investigate the molecular mechanism underlying intestinal metaplasia.

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Functional Disruption of IEX-1 by Hammerhead-Ribozymes Alters Grewtk Pvopedlu and Survival of 293 Cells Olaf Grobe, Alexander Ant, UKK, Kiel Germany; Guido Krupp, Dept of Haematopathology, UKK, Kiel Germany; Henddk Ungefroren, Dept of Gen Surg, UKK, Kiel Germany; Wolfgang E. Schmidt, Dept of Medicine 1, UK St Josef Hosp, Bochum Germany; Ulrich R. Foelsch, Heiner Schaefer, UKK, Kiel Germany

The role of the early response gene IEX-1 (PRG1/DIF-2) in apoptotic signalling and cell cycle control is a controversial issue. On the one hand, IEX-1 has been shown to be involved in cellular responses initiated by the tumorsuppressor p53, thus qualifying IEX-1 as a gene contributing to growth control and apoptosis. On the other hand, IEX-1 is a target gane for NFKB and could be regarded as a potential antiapoptotic gene involved in NFKB mediated resistance to apoptosis. In the present study, the action of IEX-1 in 293-cells was disrupted by hammerhead ribozymes specifically targeted to IEX-1 mRNA. Upon in vitro testing of various single ribozymes that cleave IEX-1 mRNA at various positions, a highly effective concatameric ribozyme construct was generated composed of four antisense ripozymes. This construct was cloned into the retroviral expression vector pBabe-Puro that was used for stable transfection of 293 cells. As shown by northern- and westemblotting, respectively, 293 cells expressing the concatameric ribozyme contain strongly reduced levels of 119(-1 mRNA as well as IEX-1 protein. Compared to cells transfected with a nonfunctional-dboz'p~e construct or to mock-transfected cells, 293 cell lines with abolished IEX-1 expression exhibit a reduced cell cycle progression, as shown by PI-cell cycle analysis. These 298-ceils grew significantly slower compared to control cells, as shown by the colorimetric MTS-1 assay. Furthermore, 293 cells became much less sensitive to apoptosis induced by an activating Fes/CD95 antibody or by TNF,~ if expressing the concatameric ribozyme. Our data indicate that IEX-1 is involved in the modulation of the cell cycle. Under conditions that favour cell growth, IEX-1 might be part of a growth signal, as demonstrated by previous work. However, under unfavourable conditions, i.e. death receptor activation, IEX-1 facilitates apoptosis. This may be due to a signal conflict that emerges if an unappropriate cell cycle progression occurs, as already described for other cell cycle promoting mediators. (Supported by the German Research Society DFG; SFB-415/C3)

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Cyclic GMP Phosphodiesterese (cG PDE): Overexpression in Human Pancreatic Carcinomas and a Target for Selective Apoptofic Aofineoplactlc Dregs Gary A. Piazza, Gerhard Sped, Clark M. Whitehead, Songmei Xu, Cell Pathways Inc, Horsham, PA; Andres J. Klein-Szanto, Fox Chase Cancer Ctr, Philadelphia, PA; Michele T. Yip-Sneider, Christopher J. Sweeney, Indiana Univ, Indianapolis, IN; Martha Lloyd, Li Uu, John Fetter, Rifat Pamukcu, William J. Thompson, Cell Pathways Inc, Horsham, PA

Background: CP461 is a benzylamide of the pyddine analogue of exisulind (sulindac sulfone) and is in clinical trials for chemotherapy. Like exisulind, CP461 induces apoptosis of colon tumor cells by inhibition of cG PDEs (PDE2 and PDE5), cG elevation, and PKG activation, but is approximately lOOx more potent. CP461 also inhibits angiogenesis in experimental models. To assess potential applications of CP461 for pancreatic cancer, we studied the expression of a cG PDE isozyme, PDE5, in human pancreatic carcinoma and measured the sensitivity of human pancreatic tumor cells to CP46t. Methods: PDE5 expression was deter- mined using immunohistochemistry with affinity purified anti-PDE5 IgG. Antibody specificity was confirmed using Western blots and antigen blockade of labeling. Labeling intensity (LI) was graded as: 0 = none, 1 = moderate, 2 = strong, 3 = very strong. Ras mutational status and COX-2 expression were determined as described previously (Carcinogenesis. 21, 139, 2000). The effect of CP461 on the growth of pancreatic tumor cell lines was determined by XTI and SRB assays. Apoptosis was measured by DNA fragmentation, caspase cleavage of cytokeratin 18, and morphology (DAPI). The subcellular localization of PDE5 was studied using anti-PDE5 IgG and confocal immunofluorescence microscopy. Results: PDE5 was stron- gly expressed in 10 out of 13 (73%) pancreatic carcinomas with a mean LI = 2.0 _+ 0.2. By contrast, none to moderate labeling occurred in normal pancreatic acini and islets (LI = 0,5 _+ 0.3; p -< 0.01) and ductal epithelium (LI = 1.2 _+ 0.2; p -< 0.05). Most of the tumors (70%) were confirmed to have ras mutations. There was no correlation between PDE5 and COX-2 expression, nor COX-2 expression and ras mutational status. CP461 inhibits PDE5 and PDE2 with IC5o = 3 and 14/~M, respectively, and was used to assess the role of the cG pathway in pancreatic tumor growth. CP461 treatment of human pancreatic cell lines, PaCa-2 and BxPC-3, inhibited growth with IC5o = 1.2 and 1.0 /LM and strongly induced

apoptosis with ECso = 0.8 and 1.4 #M, respectively PDE5 was localized to discrete perinuclear foci in both cell lines. Conclusions: Pancreatic adenocarcinomas overexpress PDE5 compared with normal acini, islets, and ductal epithelium. The cG pathway may be involved in pancreatic tumorigenesis as 0P461 had pronounced effects on growth and apoptosis, cG POE is a promising new chemotherapeutic target and clinical trials of CP461 in pancreatic patients should be considered.

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Subclinical IofosUnal Inflammation and Immune Abnormalities in Healthy Relatives of Crohn's Disease (CO) Patients Jeffry A. KCtz, Joseph E. Willis, Gregory S. Cooper, Manijeh Phillips, Laura Veri, Case Western Reserve Univ, Cleveland, OH; Lloyd R. Suthedand, Jon B. Meddings, Univ of Calgary, Calgary Canada; Claudio Fiocchi, Case Western Reserve Unlv, Cleveland, OH

BACKGROUND: Up to 15% of healthy first-degree relatives of CD patients have increased intestinal permeability. We investigated whether in this population intestinal permeability correlates with obieetlve evidence of intestinal inflammation as determined by endoscopic appearance, histologic assessment, and measurement of mucosal cytokine levels. METHODS: 28 symptom-free first-degree relatives and 12 control subjects (healthy subjects undergoing colon cancer screening) were studied. Intestinal permeability was measured by the lactulose- mannitol ratio. Complete ileocolonoscopy was performed and mucosal biopsies obtained from the terminal ileum (11), ascending and descending colon, and rectum for histologic and cytokine determination. IL-6, IL-8, IL-12, and TNF-a were assayed by ELISA in medium of mucosal organ cultures. Results were analyzed using Wilcoxon rank sum. RESULTS: Among relatives, 2/28 (7%) had elevated permeability, 2/28 (7%) had endoscopic ileitis, and 3/28 (11%) had hictolngic ileitis and/or colitis. Compared to controls, mucosal biopsies from relatives generated significantly higher levels of IL-6 (.25-+.3 vs..08-+.05 pg/ug; p=O.02) and IL-8 (25.4-+54.5 vs. 5.3_+2.6 pg/ug; p = 0.005). IL-12 was detected in 9 (32%) relatives but in only 2 (17%) controls; differences were not significant (p = 0.08). TNF-a was undetectable in all samples. 3 relatives with hietologic inflammation had levels of IL-8 significantly greater (p=O.04) than those of inflammation-free relatives. 2 relatives with endoscopic ileitis had significantly greater IL-6 levels (p = 0.04) than the other relatives. One of the 2 relatives with elevated permeability had IL-6 levels (280.7 pg/ug) >10 times the mean level of the other relatives combined. No direct correlation was observed between permeability and the presence/ absence of inflammation or cytokine levels. CONCLUSION: Healthy first-degree relatives of CD patients produce abnormally high levels of inflammatory cytokines compared to normal controls. Some of them also display active intestinal inflammation. The apparent lack of correlation between abnormal permeability, inflammation, or elevated cytokine production may be due to small sample size. This preliminary report demonstrates, for the first time, the objective evidence of intestinal inflammation and immune abnormalities in healthy relatives of CD patients and suggests that subclinical CD may be more common than previously suspected. Supported by a grant from CCFA

Siblings Iofldoca Risk of Crehn's Disease Supporting an Infectious Aofioiogy Scott M. Montgomery, Karolinska Sjukhuset, Stockholm Sweden; Mats Lambe, Karolinska Inst, Stockholm Sweden; Andrew J. Wakefield, Roy E. Pounder, Royal Free and Univ Coil Medical Schools, London United Kingdom; Anders Ekbom, Karolinska Inst, Stockholm Sweden BACKGROUND It has been suggested that lower dose or delayed antigenic exposures increase the risk of Crohn's disease. Previous studies have used siblings as a marker for antigenic exposures as their presence increases the risk of a higher dose infectious exposure at a younger age. These studies have reported a protective effect for siblings, but have lacked sufficient statistical power to comprehensively test the hypothesis and rule out the possibilities of in utero programming or a genetic factor that influences both the risk of Crohn's disease and fertility in the parents. METHODS All patients on the Swedish inpatient register up to December 1998 with Crohn's disease born between 1935 and 1994 were identified (n = 13,988) and these data were linked with information about family structure using general population registers. Similar data were identified for general population controls matched for age, sex and region (n = 65,412).Multiple logistic regression was used, with adjustment for all results for period of birth, modelled as a series of dummy vadables each representing ten year periods. RESULTS A larger number of siblings was associated with a lower risk of Cmhn's disease. Compared with no siblings, the relative odds, RO, (with 95% confidence intervals) for Crohn's disease of one or at least two siblings are 0.78 (0.74-0.82) and 0.74 (0.70-0.77), respectively, p<O.O001. Those with any older siblings were excluded to investigate the relationship with younger siblings, reducing the number analysed to 40,992. Compared with those without younger siblings the protective effect of exclusively younger siblings has RO of 0.76 (0.72-0.80) and 0.72 (0.68-0.77) among those with one or two plus siblings, respectively. Younger siblings born more than 11 years after the study members had a lesser protective association than those born 6-10 or 0-5 years later, with RO 0.95 (0.86-1.04) 0.90 (0.84- 0.98) and 0.77 (0.73-0.81), respectively. CONCLUSIONS Novel findings of this study include confirmation that in utero programming does not explain the phenomenon as younger siblings independently influence risk. The differentially graded effect of duration between subsequent births, points to infectious exposures operating during a developmental window in early life and not a genetic factor that influences both risk of Crohn's disease and mothers' fertility.

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