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Background: As a result of technological and analytical advances, genome-wide characterization of key epigenetic alterations is now feasible in complex diseases. We hypothesized that this may provide important insights into gene-environmental interactions in Crohn's disease (CD) and is especially pertinent to early onset disease., Methods: The Illumina 450K platform was applied to assess epigenome-wide methylation profiles in circulating leukocyte DNA in discovery and replication pediatric CD cohorts and controls. Data were corrected for differential leukocyte proportions. Targeted replication was performed in adults using pyrosequencing. Methylation changes were correlated with gene expression in blood and intestinal mucosa., Results: We identified 65 individual CpG sites with methylation alterations achieving epigenome-wide significance after Bonferroni correction (P < 1.1 × 10(-7)), and 19 differently methylated regions displaying unidirectional methylation change. There was a highly significant enrichment of methylation changes around GWAS single nucleotide polymorphisms (P = 3.7 × 10(-7)), notably the HLA region and MIR21. Two-locus discriminant analysis in the discovery cohort predicted disease in the pediatric replication cohort with high accuracy (area under the curve, 0.98). The findings strongly implicate the transcriptional start site of MIR21 as a region of extended epigenetic alteration, containing the most significant individual probes (P = 1.97 × 10(-15)) within a GWAS risk locus. In extension studies, we confirmed hypomethylation of MIR21 in adults (P = 6.6 × 10(-5), n = 172) and show increased mRNA expression in leukocytes (P < 0.005, n = 66) and in the inflamed intestine (P = 1.4 × 10(-6), n = 99)., Conclusions: We demonstrate highly significant and replicable differences in DNA methylation in CD, defining the disease-associated epigenome. The data strongly implicate known GWAS loci, with compelling evidence implicating MIR21 and the HLA region.

Background: An accurate indication of the changing incidence of pediatric inflammatory bowel disease (PIBD) within a population is useful in understanding concurrent etiological factors. We aimed to compare the current incidence and other demographic attributes of PIBD in the Scottish population to previous data., Methods: A national cohort of prospectively and retrospectively acquired incident cases of PIBD diagnosed less than 16 years old in pediatric services in Scotland was captured for the period 2003-2008; historical Scottish data were used for comparison (1990-1995). Age/sex-adjusted incidences were calculated and statistical comparisons made using Poisson regression., Results: During the 2003-2008 study period 436 patients were diagnosed with PIBD in Scotland, giving an adjusted incidence of 7.82/100,000/year. The incidence of Crohn's disease (CD) was 4.75/100,000/year, ulcerative colitis (UC) 2.06/100,000/year, and inflammatory bowel disease-unclassified (IBDU) 1.01/100,000/year. Compared with data from 1990-1995 when 260 IBD patients were diagnosed, significant rises in the incidence of IBD (from 4.45/100,000/year, P < 0.0001), CD (from 2.86/100,000/year, P < 0.0001), and UC (from 1.59/100,000/year, P = 0.023) were seen. There was also a significant reduction in the median age at IBD diagnosis from 12.7 years to 11.9 years between the periods (P = 0.003), with a continued male preponderance., Conclusions: The number of Scottish children diagnosed with IBD continues to rise, with a statistically significant 76% increase since the mid-1990 s. Furthermore, PIBD is now being diagnosed at a younger age. The reason for this continued rise is not yet clear; however, new hypotheses regarding disease pathogenesis and other population trends may provide further insights in future years., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)

Background: Germline variation in the 71 Crohn's disease (CD) loci implicated by genome-wide association studies (GWAS) only accounts for approximately 25% of estimated heritability. The contribution of epigenetic alterations to disease pathogenesis is emerging as a research priority., Materials and Methods: The methylation status of 27,578 CpG sites across the genome was analyzed using the Illumina Human Methylation27 assay in DNA extracted from whole blood samples from 40 adult females (21 ileal CD, 19 healthy controls) and 16 girls with childhood-onset CD, all nonsmokers. Our primary analysis compared methylation profiles in adult cases and controls., Results: Our data define a global methylation profile characteristic of ileal CD. In all, 1117 sites were differentially methylated (corrected P < 0.01); 50 showed significantly altered methylation in cases compared with controls (uncorrected P < 10(-6), corrected P < 0.0006), including genes altering immune activation: MAPK13, FASLG, PRF1, S100A13, RIPK3, and IL-21R. Gene ontology analyses implicated immunity-related pathways as targets of epigenetic modification (immune system processes [P = 1.3 × 10(-22)], immune response [P = 8.1 × 10(-16)], defense responses to bacteria [P = 1.8 × 10(-15)]). Ingenuity canonical pathway analyses implicated dendritic cell activity (P = 2.4 × 10(-8)) and differential regulation of cytokines by interleukin (IL)-17A and IL-17F (P = 5.8 × 10(-7)). We identified a significant enrichment of methylation changes within 50 kb of CD GWAS loci (8.6-fold [P = 0.021] in adults; 2.4-fold [P = 0.009] in adults and children combined), including IL-27, IL-19, TNF, MST1, and NOD2. Methylation status was predictive of disease status (sensitivity 0.71, specificity 0.83). Disease activity, drug therapy, NOD2 and DNMT3A genotypes were not associated with methylation changes., Conclusions: These data provide an important insight into the impact of epigenetic mechanisms in the pathogenesis of CD., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)

Background & Aims: The mechanisms by which specific mutations in NOD2/CARD15 increase the risk for Crohn's disease (CD) are unclear. We identified proteins that interact with NOD2 and investigated them by expression, genetic, and functional analyses., Methods: By using a yeast 2-hybrid screen of an intestinal epithelial library, we identified proteins that interact with NOD2 and confirmed the interactions in mammalian cells using co-immunoprecipitation. We used microarray analysis to analyze gene expression patterns in 302 intestinal biopsy samples (129 from patients with ulcerative colitis [UC], 106 with CD, and 67 controls). Eighty single-nucleotide polymorphisms within the genes that encoded 6 interacting proteins were genotyped in a discovery cohort (869 cases of inflammatory bowel disease [IBD], 885 controls) and a replication cohort (504 patients with IBD, 713 controls). We investigated interaction between transducin-like enhancer of split 1 (TLE1) and NOD2 in HEK293 cells., Results: We identified 6 NOD2-interacting proteins (TLE1, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 [GALNT2], HIV-1 Tat interactive protein [HTATIP], Vimentin, fission 1 (mitochondrial outer membrane) homolog [FIS1], and protein phosphatase 2, regulatory subunit B', epsilon isoform [PPP2R5E]). Of these, expression of GALNT2 (CD, P = .004) and vimentin (CD, P = .006; UC, P = .0025) was altered in patients with IBD compared with controls. Single-nucleotide polymorphisms within TLE1 were associated with susceptibility to CD, specifically with ileal disease (rs6559629, P = 3.1 × 10⁻⁵; odds ratio, 1.45). The TLE1 risk allele is required for susceptibility to CD in carriers of NOD2 mutations. In cells, TLE1 and NOD2 co-localized around the nuclear membrane and TLE1 inhibited activation of nuclear factor-κB by NOD2., Conclusions: Epistatic and biological interactions between TLE1 and NOD2 are involved in IBD pathogenesis. NOD2 might be involved in a series of pathways such as epigenetic regulation of expression (via TLE1 and HTATIP), biosynthesis of mucin (via GALNT2), apoptosis (via PPP2R5E and FIS1), and integrity of the intracellular cytoskeleton (vimentin)., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed approximately 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated approximately 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease-IRGM for Crohn's disease, HLA for Crohn's disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes-although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases.

Background: Pediatric inflammatory bowel disease (IBD) has a high prevalence of coexistent atopy. Filaggrin (FLG) loss-of-function variants (null-alleles) are associated with eczema and asthma in association with eczema. The aim was to assess the contribution of FLG null-alleles to pediatric IBD susceptibility and to coexistent atopy (eczema, asthma, allergic rhinitis, or food allergy)., Methods: FLG variants (R501X and 2282del4) were genotyped in 403 children with IBD, 683 parents, and 996 population controls., Results: In all, 11% of IBD patients carried at least 1 FLG null-allele compared to 11% of population controls (P > 0.4). Carriage of 1 or more null-alleles in patients with atopy (present in 52% of IBD patients) differed from IBD patients without atopy (14% versus 6%, P = 0.01; odds ratio [OR] 2.4, 95% confidence interval [CI] 1.2-5.1). The effect of FLG null-alleles was strongest for eczema (19% versus 7%, P = 0.0003; OR 3.3, 95% CI 1.7-6.6) and food allergy (28% versus 8%, P = 0.0001; OR 4.5, 95% CI 2.0-10.0). The presence of more than 1 atopic disease tended to increase the associated OR: eczema + asthma (23% versus 7%, P = 0.001; OR 3.9, 95% CI 1.6-9.1), eczema + asthma + allergic rhinitis (29% versus 7%, P = 0.0006; OR 5.4, 95% CI 1.9-15.4) and eczema + asthma + allergic rhinitis + food allergy (45% versus 6%, P < 10(-4); OR 12.2, 95% CI 3.2-46.3). Logistic regression analysis of IBD cases confirmed the association of carriage of an FLG null-allele with atopy (P = 0.01; OR 2.4, 95% CI 1.2-5.1) and co-occurrence of different forms of atopy (P = 0.003; OR 3.5, 95% CI 1.5-8.1)., Conclusions: Filaggrin null-alleles have no effect on IBD susceptibility but contribute to coexistent eczema and food allergy.

Background: We aimed to study fecal calprotectin in Scottish children with inflammatory bowel disease (IBD) and compare its diagnostic accuracy with blood parameters., Methods: Stool samples from 48 Scottish children (29 males, 19 females) had calprotectin measured at IBD diagnosis. The median age at diagnosis was 11.2 years (interquartile range [IQR] 8.7-13.0 years). There were 33 patients with Crohn's disease, 5 with ulcerative colitis, and 10 with IBD type unspecified. IBD was diagnosed by standard criteria. Calprotectin was measured using a commercially available kit (PhiCal Test) and 47/48 patients had comparative blood results available at diagnosis., Results: The fecal calprotectin concentrations were raised in 96% (46/48) of patients studied. The median calprotectin value was 750 microg/g (IQR 235.8-1251 microug/g). In comparison with standard blood tests, 32/45 (71.1%) had abnormal erythrocyte sedimentation rate, 19/38 (50.0%) had abnormal C-reactive protein, 29/46 (63.0%) had raised platelets, 12/45 (26.7%) had hypoalbuminemia, and 38/46 (82.6%) had abnormal hemoglobin. We identified 7/47 (14.9%) patients with raised calprotectin at diagnosis who did not have any abnormalities detected in the blood tests performed. All 48 patients (100%) had at least 1 abnormal blood test and/or raised calprotectin at diagnosis., Conclusions: Calprotectin is significantly more likely to be raised than any commonly employed blood tests at IBD diagnosis. When used in combination with these bloods tests an abnormality was demonstrated in 1 or both tests in all patients at diagnosis in this study. Fecal calprotectin measurement is a significant advance when used contemporaneously and in addition to a routine panel of blood tests in the diagnosis of pediatric IBD.

Background: Ulcerative colitis (UC) and Crohn's disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis., Methods and Findings: Using a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C-->G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09-1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1(+/lacZ) mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1(+/lacZ) mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model., Conclusions: HH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.

Background & Aims: Childhood-onset inflammatory bowel disease (IBD) might be etiologically different from adult-onset IBD. We analyzed disease phenotypes and progression of childhood-onset disease and compared them with characteristics of adult-onset disease in patients in Scotland., Methods: Anatomic locations and behaviors were assessed in 416 patients with childhood-onset (276 Crohn's disease [CD], 99 ulcerative colitis [UC], 41 IBD type unclassified [IBDU] diagnosed before seventeenth birthday) and 1297 patients with adult-onset (596 CD, 701 UC) IBD using the Montreal classification., Results: At the time of diagnosis in children, CD involved small bowel and colon (L3) in 51% (138/273), colon (L2) in 36%, and ileum (L1) in 6%; the upper gastrointestinal (GI) tract (L4) was also affected in 51%. In 39%, the anatomic extent increased within 2 years. Behavioral characteristics progressed; 24% of children developed stricturing or penetrating complications within 4 years (vs 9% at diagnosis; P < .0001; odds ratio [OR], 3.32; 95% confidence interval [CI], 1.86-5.92). Compared with adults, childhood-onset disease was characterized by a "panenteric" phenotype (ileocolonic plus upper GI [L3+L4]; 43% vs 3%; P < .0001; OR, 23.36; 95% CI, 13.45-40.59) with less isolated ileal (L1; 2% vs 31%; P < .0001; OR, 0.06; 95% CI, 0.03-0.12) or colonic disease (L2; 15% vs 36%; P < .0001; OR, 0.31; 95% CI, 0.21-0.46). UC was extensive in 82% of the children at diagnosis, versus 48% of adults (P < .0001; OR, 5.08; 95% CI, 2.73-9.45); 46% of the children progressed to develop extensive colitis during follow-up. Forty-six percent of children with CD and 35% with UC required immunomodulatory therapy within 12 months of diagnosis. The median time to first surgery was longer in childhood-onset than adult-onset patients with CD (13.7 vs 7.8 years; P < .001); the reverse was true for UC., Conclusions: Childhood-onset IBD is characterized by extensive intestinal involvement and rapid early progression.

The high incidence of Scottish Crohn's disease (CD) is not explained by the common three NOD2/CARD15 variants. We aimed to identify population-specific NOD2/CARD15 coding variants. A total of 1478 (320 inflammatory bowel disease patients <16 years, 343 adult CD patients, 542 parents and 273 controls). All NOD2/CARD15 exons were sequenced in 24 CD patients. Sequencing identified 18 single-nucleotide polymorphisms (SNPs) including 4 non-synonymous coding SNPs altering the structure of the Leucine-rich region--two were well established (1007-/C and 908G/R). Two other variants, valine955isoleucine (955V/I) and methionine863valine (863M/V), were genotyped in all subjects. 863M/V carriage was not significantly higher in CD patients vs controls (1.35 vs 0.37%, P=0.27). 955V/I carriage was no higher in CD or ulcerative colitis patients (12.8 and 15.8%, respectively) compared to controls (16.2%). Transmission disequilibrium test analysis was negative. 955V/I carriage was higher in indeterminate colitis patients (n=29) compared to controls (41.4 vs 16.2%, P=0.001, OR=3.6 (1.6-8.2)). Population-specific NOD2/CARD15 exonic variants do not account for the high-CD prevalence in Scotland.

Background: The rs2241880A/G variant of the ATG16L1 gene has been associated with susceptibility to ileal Crohn's disease (CD) in adults. Our aim was to assess whether germline variation of ATG16L1 acts as an independent determinant of susceptibility to childhood-onset CD in the high-incidence Scottish population., Methods: In all, 2195 subjects (361 children (inflammatory bowel disease [IBD] diagnosis <17 years), their parents (n = 634), 855 adult IBD patients, and 345 controls were genotyped. Case-control analysis was powered to detect effect sizes with an odds ratio (OR) >1.39 in pediatric CD. Case-control analysis, transmission disequilibrium testing (TDT), analysis of variance (ANOVA) of growth parameter z-scores, Kruskal-Wallis test (age at diagnosis), and multifactorial genotype-phenotype analysis (Montreal classification) were performed. 7.8% of pediatric CD patients and 37.2% of adult CD patients had pure ileal disease., Results: We confirmed the association of the rs2241880G-allele with adult-onset CD (60.7% versus controls 53.9%, P = 0.01, OR 1.32, 95% confidence interval [CI] 1.07-1.63) in contrast to childhood-onset CD (54.1% versus controls, P = 0.95, OR 1.01, 95% CI 0.80-1.26). TDT analysis was negative. Genotype-phenotype analysis demonstrated an association of pure ileal disease with the rs2241880G-allele (P = 0.02, OR 1.34, 95% CI 1.03-1.74). Using binary logistic regression analysis we confirmed the effect of rs2241880 genotype (GG) on ileal disease versus colonic disease (P = 0.03, OR 2.43, 95% CI 1.05-5.65). ATG16L1 genotype did not influence age at CD diagnosis. ANOVA of z-scores of height, weight, and body mass index (BMI) at CD diagnosis in children showed no association with genotype., Conclusions: The ATG16L1 variant is associated with susceptibility to adult CD in Scotland, but not early-onset disease. These contrasting effects are primarily driven by differences in disease location between early-onset and adult-onset disease.

Both NOD1/CARD4 and NOD2/CARD15 are intracellular pattern-recognition receptors involved in the innate immune response. Germline NOD2/CARD15 variation has a definite effect on susceptibility to Crohn's disease (CD) and phenotype, although this contribution is weak in Scotland and Scandinavia. The NOD1/CARD4 gene lies within the putative inflammatory bowel disease (IBD) locus at 7p14.3. We have assessed, in detail, the influence of germline NOD1/CARD4 variation on IBD susceptibility and phenotype in the Scottish population. Two thousand two hundred and ninety-six subjects, including 356 children with IBD, were involved in parallel case-control and family-based association studies. Nine tagging single-nucleotide polymorphisms (SNPs) were selected based on HapMap data spanning the whole of the NOD1/CARD4 gene. Our case-control SNP analysis was powered to detect an effect size with OR 1.5 for IBD and OR 1.6 for CD. No significant associations were observed between any of nine the NOD1/CARD4 SNPs studied and IBD, CD or ulcerative colitis (UC) (P > 0.05 for all). Haplotype case-control analysis was also negative (P > 0.05 in IBD, CD and UC). Multimarker transmission disequilibrium testing analysis was negative (P > 0.05 in IBD, CD and UC). NOD2/CARD15 variant carriage had no influence on NOD1/CARD4 effect on IBD susceptibility. This study represents the first application of a gene -wide haplotype-tagging approach to assess, in detail, the contribution of NOD1/CARD4 in IBD.

Background: NOD1/CARD4 and NOD2/CARD15 are both intracellular pattern-recognition receptors. The NOD1/CARD4 gene lies within a previously described inflammatory bowel disease (IBD) locus (7p14). An association has been suggested between the NOD1/CARD4+32656 deletion*1 variant of a complex deletion*1/insertion*2 polymorphism and IBD in 1 recent study in Europe. Our aim was to assess the influence of NOD1/CARD4+32656 on disease susceptibility and phenotype in the Scottish and Swedish IBD populations., Methods: A total of 3,962 individuals (1,791 IBD patients, 522 parents, 1,649 healthy controls) from 2 independent populations (Scotland and Sweden) were genotyped for NOD1/CARD4+32656 A/C by TaqMan and direct sequencing. Case-control, Transmission Disequilibrium Testing (TDT) and detailed genotype-phenotype (Montreal) analyses were performed. The case-control analysis had 80% power to detect an effect size of odds ratio (OR) 1.21 for IBD., Results: In case-control analyses in Scottish and Swedish patients, none of the genotypes studied in IBD, Crohn's disease (CD) or ulcerative colitis (UC), differed significantly from controls (deletion*1 allelic frequency 73.9%, 73.6%, 73.9%, and 73.6%, respectively: all P > 0.8). No epistatic interaction with NOD2/CARD15 was seen for CD susceptibility. TDT analysis in our Scottish early onset cohort was negative., Conclusions: This variant allele of NOD1/CARD4+32656 is not associated with a strong effect on susceptibility to IBD in children and adults in Northern Europe. A gene-wide haplotype-based approach may be preferable to analysis of individual variants to assess the contribution of the NOD1/CARD4 gene to IBD.

Introduction: Cigarette smoking affects susceptibility to ulcerative colitis (UC), but its effects on age at diagnosis, disease extent, and need for surgery are less well defined. We examined these parameters in a detailed retrospective analysis of a large cohort of well-characterized UC patients., Methods: 499 UC patients (254 male, median age 34.3 yr) were studied. Data were collected on smoking habits, smoking load (pack-years), age at recruitment, age at diagnosis, surgery, and disease extent. Colonoscopic and histological data at both diagnosis and follow-up (median follow-up time 4.6 yr) were available on 349 patients., Results: Ex-smokers were older at diagnosis than current or nonsmokers, (46.5 yr vs 31.1 or 29.4 yr, respectively, P < 0.001). Before diagnosis, ex-smokers had a higher smoking load than current smokers (13.0 vs 6.94 pack-years, P < 0.001). A Cox model for age at diagnosis, with smoking as a time-dependent covariate, showed that at any age, ex-smokers were significantly more likely to develop UC than current smokers (hazard ratio 1.8, 95% CI 1.41-2.44, P < 0.001). For current smokers at latest colonoscopy, those with extensive disease were the lightest smokers (median 0.320 pack-years), whereas those with healthy colons were the heaviest smokers (median 9.18 pack-years, P= 0.006). At 5 yr, regression of extensive disease was more frequent in current than ex-smokers or nonsmokers (30% current smokers vs 8% nonsmokers and 5% ex-smokers, chi(2)= 30.4, P < 0.001) but these differences were not maintained over a longer time period., Conclusions: Smoking habit influences the age at diagnosis and changes in disease extent in UC. Mechanisms are likely to be complex and require further investigation.

Objectives: The clinical subclassification of Crohn's disease by phenotype has recently been reevaluated. We have investigated the relationships between smoking habit, age at diagnosis, disease location, and progression to stricturing or penetrating complications using the Montreal classification., Methods: 408 patients (157 male, median age 29.4 yr) were assessed. Data were collected on smoking habit, age at diagnosis, anatomical distribution, and disease behavior. Follow-up data were available on all patients (median 10 yr)., Results: At diagnosis, ex-smokers (N = 53) were older than nonsmokers (N = 177) or current smokers (N = 178, medians 43.2 vs 28.3 or 28.9 yr, respectively, P < 0.001). Disease location differed according to smoking habit at diagnosis (chi(2)= 24.1, P= 0.02) as current smokers had less colonic (L2) disease than nonsmokers or ex-smokers (30%vs 45%, 50%, respectively). In univariate Kaplan-Meier survival analysis, smoking habit at diagnosis was not associated with time to development of stricturing disease, internal penetrating disease, perianal penetrating disease, or time to first surgery. Patients with isolated colonic (L2) disease were slower to develop strictures (P < 0.001) or internal penetrating disease (P= 0.001) and to require surgery (P < 0.001). Cox models with smoking habit as time-dependent covariates showed that, relative to ileal (L1) location of disease, progression to stricturing disease was less rapid for patients with colonic (L2) disease (HR 0.140, P < 0.001), but not independently affected by smoking habit. Progression to surgery was also slower for colonic (L2) than ileal (L1) disease location (HR 0.273, P < 0.001), but was independent of smoking habit., Conclusions: Smoking habit was associated with age at diagnosis and disease location in Crohn's disease, while disease location was associated with the rate of development of stricturing complications and requirement for surgery. The pathogenic basis of these observations needs to be explained.

Objective: To assess the contribution of the 113 G-->A missense mutation within the discs, large homolog 5 (DLG5) gene in childhood-onset inflammatory bowel disease (IBD) in Scotland., Study Design: Two-hundred and ninety-six children with IBD were studied. Parental DNA was also collected for transmission disequilibrium testing (TDT) analysis. Genotyping was performed by TaqMan. Genotype-phenotype analysis was also undertaken. Socioeconomic status was assigned using a deprivation category (DepCat) score 1 through 7 (1 = most affluent)., Results: TDT analysis demonstrated a significant association with IBD (P = .045). On unifactorial analysis, 113A carriage was associated with: (1) higher social class (DepCat 1 compared with 2-7, and 1-2 compared with 3-7) (66.7% vs 22.6%, P = .0005, OR 6.84 [1.99-23.55] and 37.2% vs 22.2%, P = .03, OR 2.08 [1.04-4.17], respectively); (2) higher height centile (>75th centile vs <75th centile) (42.9% vs 23.1%, P = .01, OR 2.50 [1.18-5.28]); and (3) male sex in Crohn's disease (CD) (29.3% vs 16.9%, P = .04, OR 2.04 [1.01-4.11]). Multifactorial analysis demonstrated that higher social class (DepCat 1) was independently associated with carriage of variants of 113A (P = .001, OR = 6.92 [2.24-21.33])., Conclusions: DLG5 113A is associated with increased susceptibility to IBD in Scottish children. The effect may be most marked for those children living in relative affluence.

Background and Aims: The OCTN1 (SLC22A4 1672C-->T) and OCTN2 (SLC22A5 -207G-->C) variants within the IBD5 locus have been associated with susceptibility to adult onset Crohn's disease (CD), but their contribution in children has not been examined., Methods: These OCTN1/2 variants and IBD5 marker single nucleotide polymorphisms (SNPs) (IGR2096a&#95;1, IGR2198a&#95;1, and IGR2230a&#95;1) were examined in 299 Scottish children (200 with CD, 74 with ulcerative colitis (UC), and 25 with indeterminate colitis (IC)), together with 502 parents (for transmission disequilibrium testing) and 256 controls., Results: All SNPs were in strong linkage disequilibrium (D' >0.94). TDT analysis showed association of the OCTN1 variant with inflammatory bowel disease (IBD) (p = 0.01) and CD (p = 0.04). Allele frequencies of the OCTN1/2 variants were significantly higher in IBD/CD cases (p<0.04). The homozygous mutant OCTN1/2 haplotype was increased in IBD (24.3% v 16.1%, p = 0.02) and UC (28.2% v 16.1%, p = 0.02) compared with controls. The OCTN1/2 variants were not independent of the background IBD5 risk haplotype in conferring disease susceptibility. Unifactorial analysis in CD patients showed that carriage of the TC haplotype was associated with lower weight, height, and BMI centile (<9(th) centile) at diagnosis (weight: 87.9% v 67.3% (p = 0.002), odds ratio (OR) = 3.52 (95% confidence interval, 1.51 to 8.22); height: 84.1% v 68.4% (p<0.05), OR = 2.44 (1.00 to 5.99); BMI: 79.6% v 61.1% (p = 0.02), OR = 2.49 (1.14 to 5.44)), and lower weight centile at follow up (87.5% v 64.6% (p = 0.03), OR = 3.83 (1.03 to 14.24)). Multifactorial binary logistic regression analysis confirmed association of the TC haplotype with lower weight centile at diagnosis (p = 0.02, OR = 3.41 (1.20 to 9.66))., Conclusions: These data implicate variants within the IBD5 haplotype, as determinants of disease susceptibility and growth indices in early onset IBD. The OCTN1/2 variants remain potential positional candidate genes, but require further analysis.

Introduction: The incidence of early-onset CD in Scotland is among the highest worldwide. Three single nucleotide polymorphisms (SNPs) R702W, G908R and Leu1007finsC in the NOD2/CARD15 gene predispose to adult CD. We investigated the contribution of these variants to disease susceptibility and phenotype in the Scottish early-onset IBD population., Patients and Methods: 906 individuals including 247 Scottish IBD patients aged <16 years at diagnosis, 414 parents and 245 controls were genotyped. Transmission disequilibrium testing (TDT), case-control analysis and detailed genotype-phenotype analysis were performed., Results: The Leu1007finsC variant was associated with susceptibility to CD by case-control (4.2% versus. 1.4%, P = 0.01) and TDT analysis (P = 0.006). The Population Attributable Risk (PAR) for the 3 NOD2/CARD15 mutations was 7.9%. Carriage of NOD2/CARD15 variants was associated with, at diagnosis: decreased albumin (31.0% versus. 9.0%, P = 0.001) and raised CRP (25% versus. 9.5%, P = 0.04) and at follow up: need for surgery (39.5% versus. 12.8%, P = 0.0002) jejunal involvement (50% versus. 18.4%, P = 0.01) jejunal and ileal involvement (50% versus. 10.7%, P = 0.009), raised CRP (57.1% and 12.8%, P = 0.0009), lower weight/height centile (75.0% versus. 20.2%, P = 0.03, 50.0% versus. 16.0%, P = 0.001 respectively) and stricturing disease (45.5% versus. 19.4%, P < 0.05). Multifactorial analysis demonstrated carriage was associated with need for surgery (P = 0.004, OR 4.9 [1.5-14.7])., Conclusions: These NOD2/CARD 15 variants in the Scottish early onset CD population have a definite, albeit relatively small contribution to CD susceptibility (PAR 7.9%) but a major impact on phenotype. In particular NOD2/CARD15 variants are strongly associated with several markers of disease severity in pediatric CD, notably need for surgery.

Background and Aims: Antibodies directed against the porin protein C of Escherichia coli (anti-OmpC) and Pseudomonas fluorescens (anti-I2) have recently been described in Crohn's disease (CD). Those directed against Saccharomyces cerevisiae (ASCA) and the perinuclear component of neutrophils (pANCA) have been more widely studied and may be of diagnostic importance. We aimed to assess the frequency of anti-OmpC, anti-I2, ASCA, and pANCA, in an independent Scottish CD cohort, establish phenotypic associations, and compare with a U.S. cohort., Methods: One hundred and forty-two well-characterized CD patients (76 females, median age 39 yr (17-88)) were studied. CD was classified by the Vienna classification. Sera were assayed for anti-OmpC, anti-I2, ASCA, and pANCA. Allele specific primers were used for NOD2/CARD15 genotyping., Results: Anti-OmpC, anti-I2, ASCA, and pANCA were present in sera from 37%, 52%, 39%, and 14% of CD patients, respectively. Multivariate analysis demonstrated independent associations of anti-OmpC to be progression of disease type (p= 0.005) and long disease duration (p= 0.002), and those of anti-I2 to be long disease duration (p= 0.002) and the need for surgery (p= 0.033). ASCA were associated with disease progression (p < 0.001). When the presence and magnitude of all antibody responses were considered, reactivity to microbial components was associated with long disease duration (p < 0.001), progression of disease type (p < 0.001), penetrating disease (p= 0.008), small bowel disease (p < 0.02), and the need for surgery (p < 0.001). There was no association of antibody status to NOD2/CARD15 genotype., Conclusion: Reactivity to microbial components is associated with severe CD characterized by small bowel involvement, frequent disease progression, longer disease duration, and greater need for intestinal surgery.

Background & Aims: The incidence of Crohn's disease in Scottish children has increased steadily over 30 years. Many studies have investigated genetic influence or possible links with childhood events. We aimed to study sociodemographic and/or geographic distribution of juvenile=onset Crohn's disease in Scotland., Methods: Using a previously established and validated database covering the entire Scottish population, 580 Scottish children (<16 years of age at symptom onset) with inflammatory bowel disease incident between 1981 and 1995 were identified. Postcodes of incident cases were classed for geographic location and material deprivation. Incidence rates (/100,000/year) were sex standardized to the 1991 census population. The effects of sex, geographic location, time, and deprivation category were estimated from a multifactorial Poisson regression model., Results: The incidence of juvenile-onset Crohn's disease was 2.3 (95% CI: 2.0-2.5) for the time period 1981 to 1995 and was significantly higher in northern (3.1, 95% CI: 2.6-3.8) than in southern Scotland (2.1, 95% CI: 1.9-2.4, P < 0.001). The incidence of juvenile-onset ulcerative colitis did not show north/south variation ( P = 0.677). The relative risks of developing CD were significantly lower in postcode areas with deprivation categories 2-7 as compared with deprivation score 1 (most affluent, P = 0.033). This pattern was not seen for UC., Conclusions: There was an increased incidence of juvenile-onset Crohn's disease in northern compared with southern Scotland. Children from more affluent areas had a higher relative risk of developing Crohn's disease. Juvenile onset ulcerative colitis did not show north/south variation in incidence or association with affluence.

Background: Crohn's disease (CD) is characterized by heterogeneity of phenotype. The Vienna classification can be used to classify CD, and recent data illustrate that behavior evolves over the course of the disease. Clinical and biological influences on disease progression remain unclear. We examined the associations of CD disease progression at diagnosis and for up to 20 years of follow-up., Methods: Two hundred thirty-one well-characterized CD patients were studied. Demographic, clinical, and NOD2/CARD15 data were collected. Disease behavior according to the Vienna classification was assessed at diagnosis and for up to 20 years following diagnosis., Results: At diagnosis, 70% of patients had inflammatory disease, 9% stricturing, and 21% penetrating. Early age at diagnosis was associated with ileocolonic and upper GI disease (p = 0.015), and positive anti-Saccharomyces cerevisiae antibody (ASCA) was associated with ileal involvement (p = 0.008). Smoking was relatively protective against colonic, rather than ileal involvement at diagnosis (p < 0.02). At 20 years, 92% had progressed to a more severe disease type. Patients who progress to a more severe disease type require more frequent surgery (p < 0.00001). Multivariate analysis found disease progression to be associated with ileal disease location (p = 0.001) and positive ASCA (p = 0.003). Variant NOD2/CARD15 alleles were protective against rapid progression of disease phenotype (p = 0.04). The presence of perianal disease was independent of intestinal penetrating disease., Conclusions: The progression of disease type in CD is associated with the need for more frequent surgery. Rapid progression is associated with ileal disease and positive ASCA, and delayed progression is associated with variant NOD2/CARD15 alleles. Consideration should be given to a separate Vienna classification for perianal disease., (Copyright 2004 Lippincott Williams & Wilkins)

NOD2/caspase recruitment domain (CARD)15 variants are identified in up to 50% of Crohn's disease (CD) patients. Functional variants of toll-like receptor-4 (TLR4) and CD14 genes may also be relevant to disease pathophysiology. We aimed to assess the contribution of NOD2/CARD15, TLR4 and CD14 variants in Scottish and Irish CD patients. In all, 612 patients with well-characterised inflammatory bowel disease (252 Scottish CD, 247 Scottish UC, 113 Irish CD) and 304 controls were genotyped for variants of NOD2/CARD15 (1007fsinsC, G908R, R702W, P268S), TLR4 (A299G) and CD14 (T-159C). Genotype-phenotype analyses were performed. Variant 1007fsinsC (P=0.003) and G908R (P=0.008) but not R702W (P=0.269) alleles were more prevalent in Scottish CD (4.7, 1.8 and 7.1%, respectively) than Scottish control (2.3, 0.3 and 5.4%). CD allelic frequencies were lower than the series from Europe (P<0.00001) and North America (P<0.00001) but not Scandinavia (P<0.7). Associations were identified with age at diagnosis (P=0.002), ileal disease (P<0.02), penetrating disease (P=0.04) and inflammatory joint disease (P<0.02). TLR4 and CD14 variants did not differ between CD and controls. In conclusion, we present compelling evidence for genetic heterogeneity within Europe. These NOD2/CARD15 variants have a minor contribution in Scottish and Irish CD patients, consistent with an emerging pattern from Northern Europe.

Anti-Saccharomyces cerevisiae antibodies (ASCAs) have been proposed as serological markers, which may differentiate Crohn's disease (CD) from ulcerative colitis (UC) and predict disease phenotype. Their importance in pathogenesis is unproven. We investigated the relationship between ASCAs, disease phenotype and NOD2/CARD15 genotype in CD and whether ASCAs were related to antibodies to other fungal proteins. Serum from 228 patients [143 CD, 75 UC, 10 with indeterminate colitis (IC)] and 78 healthy controls (HC) were assayed for ASCA. Antibodies (IgA, IgG) to other fungal proteins (Fusarium species ATC20334, Mycoprotein) were measured in the same samples using an in-house enzyme-linked immunosorbent assay (ELISA) assay. ASCAs were present in 57% of CD, 19% of UC, 30% of IC and 8% of HCs. ASCA-positive status was a predictor for CD with sensitivity of 57%, specificity of 87%, positive predictive value of 78% and negative predictive value of 68%. ASCA was associated with proximal (gastroduodenal and small bowel involvement) rather than purely colonic disease (P < 0.001) and with a more severe disease phenotype and requirement for surgery over a median follow-up time of 9 years (P < 0.0001). No associations with NOD2/CARD15 mutations were seen. There was no association between ASCA and antibodies to MP (IgA or IgG). These data implicate ASCA as a specific marker of disease location and progression in CD, emphasizing the heterogeneity within IBD.

Introduction: Smoking in patients with Crohn's disease is associated with more frequent relapse. The mechanism responsible is unknown but a direct pro-inflammatory action on intestinal mucosa has been postulated. Mucosal inflammation in clinically inactive Crohn's disease predicts forthcoming relapse. Whole gut lavage fluid obtained after bowel cleansing with a polyethylene glycol electrolyte solution is an assessment of gut inflammation and immunity., Aim: To assess whether whole gut lavage fluid interleukin-1beta and interleukin-8 differed between smokers and non-smokers with clinically inactive Crohn's disease., Methods: A total of 34 patients with inactive Crohn's disease (Crohn's disease activity index <150 and whole gut lavage fluid IgG concentration of <10 mg/ml) underwent whole gut lavage with interleukin-1beta and interleukin-8 analysed by enzyme-linked immunosorbent assay. Clinical details and blood markers of inflammation were collected., Results: In this series, 14 patients smoked (10 females, mean age 44.3+/-14.3 years), 20 did not (12 females, mean age 40.7+/-14.3). Surgical resection was more common in smokers (12/14 vs 8/20, p<0.008). Whole gut lavage fluid IgG was significantly lower in smokers (median 1.5 mg/ml (range 1.0-8.0 mg/ml) vs median 3.5 mg/ml (range 1.0-7.0 mg/ml), p<0.05). Whole gut lavage fluid interleukin-1beta was also lower in smokers [median 14.5 pg/ml (range 2-72 pg/ml) vs 26 pg/ml (range 7-1700 pg/ml)], p<0.03., Conclusion: Markers of mucosal inflammation in inactive Crohn's disease are lower in smokers than non-smokers. This is against the hypothesis that nicotine exerts a direct pro-inflammatory action via interleukin-1beta and interleukin-8. Further research is required to elucidate the exact mechanisms involved.

Objective: A previous study reported a three-fold rise in the incidence of juvenile-onset Crohn's disease in Scottish children and a marginal fall in ulcerative colitis between 1968 and 1983. The present study aimed to document the incidence of juvenile-onset inflammatory bowel disease between 1981 and 1995 and examine temporal trends between 1968 and 1995 in Scotland., Setting: Scotland (latitude 55-60 degrees N) has a total area of 77 837 km2 (30 405 square miles) and includes four urban centres each with a population of over 100,000., Participants: The Scottish hospital discharges linked database was used to identify 1002 patients less than 19 years old who were coded as having inflammatory bowel disease between 1981 and 1997. All case notes were reviewed and diagnoses verified. Incident cases were defined as those with symptom onset before or at 16 years of age between 1 January 1981 and 31 December 1995., Results: During the 15 year period 1981-1995, 438 incident cases of Crohn's disease and 227 of ulcerative colitis were identified, giving standardized incidences of 2.5 cases and 1.3 cases per 100,000 population per year for Crohn's disease and ulcerative colitis respectively. On 31 December 1995 there were 150 children < or = 16 years of age with Crohn's disease and 101 with ulcerative colitis, giving crude prevalences of 13.7 cases per 100,000 population for Crohn's disease and 9.2 for ulcerative colitis. The continuing rise in Crohn's disease incidence between 1981 and 1995 fits that predicted by linear trend analysis of the 1968-1983 data. The incidence of Crohn's disease in the 12-16 age range almost doubled between 1981 and 1995 and was greater for males than females. Ulcerative colitis incidence was thought to show a slight fall in the 1968-1983 data, but this is reversed in the 1981-1995 data., Conclusion: The incidence of juvenile-onset Crohn's disease continues to rise in Scotland and the prevalence has increased by 30% since 1983. Unlike the previous report from Scotland, the incidence of juvenile-onset ulcerative colitis also is apparently rising. Whether this represents a real rise in incidence, or merely the inclusion of milder cases which were not previously hospitalized remains uncertain.

The treatment goal in Crohn's disease is clinical remission, not complete mucosal healing. The incidence of mucosal inflammation in Crohn's disease patients in clinical remission is not known. Whole gut lavage is an objective method of assessing mucosal inflammation. We aimed to assess levels of mucosal inflammatory activity in a group of patients with clinically inactive Crohn's disease. We prospectively assessed 30 patients with inactive Crohn's disease and 28 controls. Inactive disease was defined as Crohn's disease activity index of less than 150. All underwent whole put lavage, with analysis of whole gut lavage fluid IgG, haemoglobin, interleukin-1 beta, interleukin-8 and granulocyte elastase. Serum inflammatory parameters were collected for comparison. Of the 30 patients with Crohn's disease, 10 (33%) had an abnormal immunoglobulin G, 21 (70%) had an elevated interleukin-1 beta 20 (66%) interleukin-8 and 10 (33%) granulocyte elastase in the whole gut lavage fluid. 58% of patients had either 1 or 2 abnormal results. In contrast only 10% had 1 or 2 abnormal serum results. Few abnormalities were present in lavage fluid or serum of the control population. We concluded that ongoing mucosal inflammation is detectable in whole gut lavage fluid of up to 2/3 of Crohn's disease patients in clinical remission.

Background: Clinical differences between small- and large-bowel Crohn's disease have been demonstrated. Neutrophil migration and degranulation are important effector mechanisms in gut damage. Granulocyte elastase, a neutrophil-bound enzyme, interleukin 8 and 1beta can be detected in whole-gut lavage fluid. We aimed to assess differences between large- and small-bowel Crohn's disease., Methods: A total of 167 patients with active inflammatory bowel disease (118 Crohn's disease, 49 ulcerative colitis) underwent whole-gut lavage with a polyethylene glycol electrolyte solution. Granulocyte elastase was assayed using an enzyme substrate reaction, IL-8 and IL-1beta by ELISA., Results: Twenty-seven of 36 patients with isolated colonic Crohn's disease had detectable granulocyte elastase (median 0.259 pKat/l, range < 0.039-2.742 microKat/l), whereas 3 of 15 with small-bowel involvement alone had detectable granulocyte elastase (median < 0.039 microKat/l, range < 0.039-0.266 microKat/l; P < 0.0001). Granulocyte elastase levels were significantly higher in patients with ileocolonic disease and post-ileocaecal resection compared with small-bowel disease alone. IL-8 (P< 0.0001) and IL-1beta (P < 0.04) levels differed between colonic and ileal distributions. No variations were seen in ulcerative colitis., Conclusions: Neutrophil migration to the gut lumen in Crohn's disease is a feature of colonic disease irrespective of associated ileal lesions. This suggests that bacterial-derived chemo-attractants may play a role. High levels of IL-8 in colonic disease are consistent with this hypothesis.

Trials of maintenance therapy in Crohn's disease are often underpowered, and there is need for objective markers of relapse. We assessed the relationship of whole gut lavage fluid cytokines to relapse in inactive Crohn's disease. Fifty-four patients with inactive Crohn's disease were prospectively assessed. Inactivity was determined as a Crohn's disease activity index of <150 and whole gut lavage fluid immunoglobulin G <10 microg/ml. All patients underwent whole gut lavage with analysis of IL-1beta and IL-8. Follow up was for one year. Patients with elevated whole gut lavage fluid IL-1beta (P < 0.004) and IL-8 (P < 0.02) had greater chance of relapse. Young age, short disease duration, and fistulating disease also relapsed more frequently. Multiple regression identified IL-1beta as an independent variable. In conclusion, an elevated whole gut lavage fluid IL-1beta in inactive Crohn's disease identifies patients at high risk of relapse.

Studies of intestinal mucosal immunity and inflammation are limited by the relative inaccessibility of most of the small intestine. Any new method of studying mucosal immunity and inflammation in patients should be minimally invasive, cost-effective, and provide information not readily available using current methods. Gaspari et al. (1) described gut lavage with 3 to 4 L of nonabsorbable, commercially available polyethylene glycol (PEG)-based bowel cleansing fluid as a method for analyzing human intestinal secretions for antibody content. Peroral gastrointestinal (GI) lavage is widely used to cleanse the GI tract prior to colonoscopy, barium enema examination, or colonic surgery. Whole gut lavage fluid (WGLF) therefore often becomes available without subjecting a patient to any additional investigation. This method is also often more acceptable to healthy volunteers than almost any other method of studying small intestinal secretions. Over the past decade, we have found that WGLFs from patients (both adults and children) suffering from a variety of intestinal diseases contain immunoglobulins (Igs) and antibodies, hemoglobin, plasma-derived proteins, cytokines, inflammatory cells, and their granule-derived proteins and growth factors (2,3). In this chapter, we describe our experience of the use of WGLF to study mucosal immunity and inflammation.

The use of pen-based electronic questionnaires and conventional paper questionnaires was compared in a randomized crossover study. Forty-six patients, aged 17-81 years, suffering from gastro-intestinal disorders, initially filled in a paper quality of life questionnaire for familiarization purposes, then on two subsequent visits completed electronic and paper questionnaires in randomized order. At the last visit they completed a preference survey. The results showed a high degree of acceptability of the electronic questionnaire, with 57% of patient preferring electronic and 13% preferring paper, while the remaining 30% expressed no preference. Neither age, gender nor familiarity with technology showed any marked association with patients' preferences. All patients found both paper and electronic questionnaires easy to use. Data were more complete on the electronic questionnaire (100%) than on the paper (99.1%). Data handling procedures were greatly simplified. These results show that major benefits in completeness of data, speed of data flow, and data handling workload can be obtained from the use of pen-based electronic questionnaires.

Indirect evidence of enteropathy has been sought in a group of 57 subjects with farmer's lung (FL) by clinical questionnaire, estimation of red cell folate levels, the presence of precipitating antibodies to various food antigens and detection of reticulin antibodies. One subject was found who had villous atrophy and responded clinically to gluten withdrawal. There were two subjects with low red cell folate levels and multiple food antibodies, and one subject with reticulin antibodies and multiple food antibodies. Enteropathy is infrequent in subjects diagnosed clinically as having farmer's lung but seems to be more common than would be expected by chance. The mechanism has yet to be elucidated.

Feeding ovalbumin over a wide range of doses is known to reduce subsequent systemic immune responses to parenteral immunization. In the present study, we have fed mice 2 mg and 25 mg ovalbumin (OVA) 2 weeks before systemic immunization and followed the resulting humoral antibody and cell-mediated immune (CMI) responses. The results indicate that while 25 mg OVA will reduce subsequent IgM, IgG and CMI responses to OVA, feeding 2 mg OVA will only suppress CMI responses and to a lesser extent the IgM response. Furthermore, the tolerant state induced by feeding 25 mg OVA was only partially prevented by 100 mg/kg cyclophosphamide (CY) while the suppressed CMI after feeding 2 mg OVA was completely blocked by CY pretreatment. These findings suggest that the humoral and cell-mediated limbs of the immune response may be controlled by different regulatory systems after feeding antigen, and that activation of these systems is dependent on the dose of oral antigen use. In addition, the results are in agreement with our previous finding that CY pretreatment will allow the development of CMI in the gut and gut-associated lymphoid tissue (GALT) after oral OVA and suggest that this phenomenon is related to breakdown of oral tolerance induction.

Mice fed ovalbumin develop specific systemic hyporesponsiveness. This oral tolerance is abrogated by cyclophosphamide pretreatment, and the mechanism of abrogation could be either via T suppressor cells or via damage to the gut epithelium. A serum transfer protocol was used to examine the site of action of cyclophosphamide in this system. Serum was collected from ovalbumin-fed mice and transferred into recipients which were then parenterally immunized with ovalbumin in Freund's complete adjuvant. Serum transfer suppressed the delayed-type hypersensitivity (DTH) responses but not the antibody responses of the recipients. Cyclophosphamide pretreatment (100 mg/kg) of recipients (but not of donors) abrogated this suppressor effect. Parenteral administration of ovalbumin in a range of doses did not induce immunological hyporesponsiveness. It is suggested that absorption across the gut mucosa leads to generation of fragments of ovalbumin that induce suppressor cells selective for DTH.