Altered Gut Microbiome Composition and Function Are Associated With Gut Barrier Dysfunction in Healthy Relatives of Patients With Crohn's Disease.

BACKGROUND & AIMS: The gut microbiome has been suggested to play a role in gut barrier hemostasis, but data are scarce and limited to animal studies. We therefore aimed to assess whether alterations in gut microbial composition and functional pathways are associated with gut barrier function in a cohort of healthy first-degree relatives of patients with Crohn's disease. METHODS: We used the Crohn's and Colitis Canada Genetic Environmental Microbial (CCC-GEM) cohort of healthy first-degree relatives of patients with Crohn's disease. Gut barrier function was assessed using the urinary fractional excretion of lactulose-to-mannitol ratio (LMR). Microbiome composition was assessed by sequencing fecal 16S ribosomal RNA. The cohort was divided into a discovery cohort (n = 2472) and a validation cohort (n = 655). A regression model was used to assess microbial associations with the LMR. A random forest classifier algorithm was performed to assess microbial community contribution to barrier function. RESULTS: Individuals with impaired barrier function (LMR >0.025) had reduced alpha-diversity (Chao1 index, P = 4.0e-4) and altered beta-diversity (Bray-Curtis dissimilarity index, R2 = 0.001, P = 1.0e-3) compared with individuals with an LMR ≤0.025. When taxa were assessed individually, we identified 8 genera and 52 microbial pathways associated with an LMR >0.025 (q < 0.05). Four genera (decreased prevalence of Adlercreutzia, Clostridia UCG 014, and Clostridium sensu stricto 1 and increased abundance of Colidextribacter) and 8 pathways (including decreased biosynthesis of glutamate, tryptophan, and threonine) were replicated in the validation cohort. The random forest approach revealed that the bacterial community is associated with gut barrier function (area under the curve, 0.63; P = 1.4e-6). CONCLUSIONS: The gut microbiome community and pathways are associated with changes in gut barrier function. These findings may identify potential microbial targets to modulate gut barrier.

Increased Intestinal Permeability Is Associated With Later Development of Crohn's Disease.

BACKGROUND & AIMS: Increased intestinal permeability has been associated with Crohn's disease (CD), but it is not clear whether it is a cause or result of the disease. We performed a prospective study to determine whether increased intestinal permeability is associated with future development of CD. METHODS: We assessed the intestinal permeability, measured by the urinary fractional excretion of lactulose-to-mannitol ratio (LMR) at recruitment in 1420 asymptomatic first-degree relatives (6-35 years old) of patients with CD (collected from 2008 through 2015). Participants were then followed up for a diagnosis of CD from 2008 to 2017, with a median follow-up time of 7.8 years. We analyzed data from 50 participants who developed CD after a median of 2.7 years during the study period, along with 1370 individuals who remained asymptomatic until October 2017. We used the Cox proportional hazards model to evaluate time-related risk of CD based on the baseline LMR. RESULTS: An abnormal LMR (>0.03) was associated with a diagnosis of CD during the follow-up period (hazard ratio, 3.03; 95% CI, 1.64-5.63; P = 3.97 × 10-4). This association remained significant even when the test was performed more than 3 years before the diagnosis of CD (hazard ratio, 1.62; 95% CI, 1.051-2.50; P = .029). CONCLUSIONS: Increased intestinal permeability is associated with later development of CD; these findings support a model in which altered intestinal barrier function contributes to pathogenesis. Abnormal gut barrier function might serve as a biomarker for risk of CD onset.

The intestinal barrier in multiple sclerosis: implications for pathophysiology and therapeutics.

Biological barriers are essential for the maintenance of homeostasis in health and disease. Breakdown of the intestinal barrier is an essential aspect of the pathophysiology of gastrointestinal inflammatory diseases, such as inflammatory bowel disease. A wealth of recent studies has shown that the intestinal microbiome, part of the brain-gut axis, could play a role in the pathophysiology of multiple sclerosis. However, an essential component of this axis, the intestinal barrier, has received much less attention. In this review, we describe the intestinal barrier as the physical and functional zone of interaction between the luminal microbiome and the host. Besides its essential role in the regulation of homeostatic processes, the intestinal barrier contains the gut mucosal immune system, a guardian of the integrity of the intestinal tract and the whole organism. Gastrointestinal disorders with intestinal barrier breakdown show evidence of CNS demyelination, and content of the intestinal microbiome entering into the circulation can impact the functions of CNS microglia. We highlight currently available studies suggesting that there is intestinal barrier dysfunction in multiple sclerosis. Finally, we address the mechanisms by which commonly used disease-modifying drugs in multiple sclerosis could alter the intestinal barrier and the microbiome, and we discuss the potential of barrier-stabilizing strategies, including probiotics and stabilization of tight junctions, as novel therapeutic avenues in multiple sclerosis.

The primary defect in experimental ileitis originates from a nonhematopoietic source.

The initiating etiologic factor in Crohn's disease (CD) remains unclear. SAMP1/YitFc (SAMP) mice develop chronic ileitis similar to human CD. We used bone marrow chimeras to determine if SAMP ileitis results from a primary immunological defect or from dysregulated mucosal immunity secondary to intrinsic, nonhematopoietic (e.g., epithelial) dysfunction. SAMP mice receiving wild-type (AKR) BM developed severe ileitis, whereas SAMP BM did not confer ileitis to WT recipients. WT lymphocytes from reconstituted SAMP mice resembled native SAMP populations in regard to surface phenotype and cytokine production. Ilea from native SAMP mice and SAMP recipients of wild-type BM displayed decreased epithelial barrier resistance ex vivo and increased epithelial permeability in vivo compared to native WT mice and AKR recipients of SAMP BM. This permeability defect preceded the development of ileal inflammation, was present in the absence of commensal bacteria, and was accompanied by altered ileal mRNA expression of the tight junction proteins claudin-2 and occludin. Our results provide evidence that the primary defect conferring ileitis in SAMP mice originates from a nonhematopoietic source. Generation of pathogenic lymphocytes is a consequence of this defect and does not reflect intrinsic proinflammatory leukocyte properties. Decreased barrier function suggests that defects in the epithelium may represent the primary source of SAMP ileitis susceptibility.

Spontaneous, immune-mediated gastric inflammation in SAMP1/YitFc mice, a model of Crohn's-like gastritis.

BACKGROUND & AIMS: Crohn's disease (CD) can develop in any region of the gastrointestinal tract, including the stomach. The etiology and pathogenesis of Crohn's gastritis are poorly understood, treatment approaches are limited, and there are not many suitable animal models for study. We characterized the features and mechanisms of chronic gastritis in SAMP1/YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, along with possible therapeutic approaches. METHODS: Stomachs from specific pathogen-free and germ-free SAMP and AKR mice (controls) were evaluated histologically; the presence of Helicobacter spp was tested in fecal pellets by polymerase chain reaction analysis. In vivo gastric permeability was quantified by fractional excretion of sucrose, and epithelial tight junction protein expression was measured by quantitative reverse-transcription polymerase chain reaction analysis. The effects of a proton pump inhibitor (PPI) or corticosteroids were measured, and the ability of pathogenic immune cells to mediate gastritis was assessed in adoptive transfer experiments. RESULTS: SAMP mice developed Helicobacter-negative gastritis, characterized by aggregates of mononuclear cells, diffuse accumulation of neutrophils, and disruption of epithelial architecture; SAMP mice also had increased gastric permeability compared with controls, without alterations in expression of tight junction proteins. The gastritis and associated permeability defect observed in SAMP mice were independent of bacterial colonization and reduced by administration of corticosteroids but not a PPI. CD4(+) T cells isolated from draining mesenteric lymph nodes of SAMP mice were sufficient to induce gastritis in recipient SCID mice. CONCLUSIONS: In SAMP mice, gastritis develops spontaneously and has many features of CD-like ileitis. These mice are a useful model to study Helicobacter-negative, immune-mediated Crohn's gastritis.

The rate of bloodstream infection is high in infants with short bowel syndrome: relationship with small bowel bacterial overgrowth, enteral feeding, and inflammatory and immune responses.

OBJECTIVE: This pilot study in parenteral nutrition-dependent infants with short bowel syndrome (SBS) evaluated the impact of feeding route and intestinal permeability on bloodstream infection (BSI), small bowel bacterial overgrowth (SBBO), and systemic immune responses, as well as fecal calprotectin as a biomarker for SBBO. STUDY DESIGN: Ten infants (ages 4.2-15.4 months) with SBS caused by necrotizing enterocolitis were evaluated. Nutritional assessment, breath hydrogen testing, intestinal permeability, fecal calprotectin, serum flagellin- and lipopolysaccharide-specific antibody titers, and proinflammatory cytokine concentrations (tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta, -6, and -8) were performed at baseline and at 60 and 120 days. Healthy, age-matched control subjects (n = 5) were recruited. RESULTS: BSI incidence was high (80%), and SBBO was common (50%). SBBO increased the odds for BSI (>7-fold; P = .009). Calprotectin levels were higher in children with SBS and SBBO versus those without SBBO and healthy control subjects (P < .05). Serum TNF-alpha, was elevated at baseline versus controls. Serum TNF-alpha and interleukin-1 beta, -6, and -8 levels diminished with increased enteral nutrition. Anti-flagellin and anti-lipopolysaccharide immunoglobulin G levels in children with SBS were lower versus control subjects and rose over time. CONCLUSION: In children with SBS, SBBO increases the risk for BSI, and systemic proinflammatory response decreases with increasing enteral feeding and weaning parenteral nutrition.

Low-dose aspirin affects the small bowel mucosa: results of a pilot study with a multidimensional assessment.

BACKGROUND & AIMS: Whether low-dose aspirin (acetylsalicylic acid [ASA]) produces intestinal damage is controversial. Our aim was to determine whether the small bowel is damaged by low-dose ASA on a short-term basis. METHODS: Twenty healthy volunteers (age range, 19-64 years) underwent video capsule endoscopy (VCE), fecal calprotectin, and permeability tests (sucrose and lactulose/mannitol [lac/man] ratio) before and after ingestion of 100 mg of enteric-coated ASA daily for 14 days. Video capsule images were assessed by 2 independent expert endoscopists, fully blinded to the treatment group, by using an endoscopic scale. RESULTS: Post-ASA VCE detected 10 cases (50%) with mucosal damage not apparent in baseline studies (6 cases had petechiae, 3 had erosions, and 1 had bleeding stigmata in 2 ulcers). The median baseline lac/man ratio (0.021; range, 0.011-0.045) increased after ASA use (0.036; range, 0.007-0.258; P = .08), and the post-ASA lac/man ratio was above the upper end of normal (>0.025) in 10 of 20 volunteers (vs baseline, P < .02). The median baseline fecal calprotectin concentration (6.05 microg/g; range, 1.9-79.2) also increased significantly after ASA use (23.9 microg/g; range, 3.1-75.3; P < .0005), with 3 patients having values above the cutoff (>50 microg/g). Five of 10 subjects with abnormal findings at VCE also had lac/man ratios above the cutoff. Median baseline sucrose urinary excretion (70.0 mg; range, 11.8-151.3) increased significantly after ASA administration (107.0 mg; range, 22.9-411.3; P < .05). CONCLUSIONS: The short-term administration of low-dose ASA is associated with mucosal abnormalities of the small bowel mucosa, which might have implications in clinical practice.

Analysis of Genetic Association of Intestinal Permeability in Healthy First-degree Relatives of Patients with Crohn's Disease.

Excessive intestinal permeability or intestinal barrier dysfunction as measured by various assays has been observed in various diseases. However, little is known about the factors contributing to altered gut permeability in these diseases. Our objective was to determine the genetic determinants of altered gut permeability as measured by the lactulose mannitol fractional excretion ratio (LacMan ratio) in 1075 healthy first-degree relatives of patients with Crohn's disease (CD). In a targeted analysis of single nucleotide polymorphisms (SNPs) located in genes associated with intestinal barrier function related or not to inflammatory bowel disease, we did not find a significant association with intestinal permeability. In an untargeted genome-wide association analysis, the top 100 associations were located in 22 genomic loci, although they were not statistically significant after correction for multiple testing (raw P values [1.8 × 10-7 - 1.4 × 10-5]. The lowest P value was obtained for rs9616637 (22q13.33, C22orf34), for which the minor allele A was associated with a decreased LacMan ratio. These results suggest that host genetic background has limited contribution toward intestinal permeability. Despite this, our study is currently the largest of its kind assessing gut permeability in vivo. It remains possible that smaller genetic effect sizes on LacMan ratio are not detectable in this sized cohort. Larger studies are warranted to identify the potential genetic contribution to intestinal permeability.

Alpha1-adrenoceptors down-regulate ClC-2 chloride channels in epithelial cells from the acutely denervated jejunum.

Acute sympathetic denervation of the small intestine up-regulates alpha1-adrenoceptors on villus enterocytes and activation of these alpha1-adrenoceptors inhibits chloride secretion. We tested whether alpha1-adrenoceptor-mediated inhibition of chloride secretion was the result of reduced ClC-2 chloride channel expression. Phorbol myristate acetate (PMA) (a protein kinase C (PKC) activator) had no effect on ClC-2 levels. In contrast, alpha1-adrenoceptor activation significantly decreased ClC-2 protein levels in both the villus (1.58+/-0.19 to 0.75+/-0.19 arbitrary units) and crypt (1.69+/-0.15 to 0.37+/-0.23 arbitrary units) epithelial cells from the acutely denervated jejunum but not innervated controls. These data suggest that inhibition of chloride secretion following alpha1-adrenoceptor activation in the acutely denervated small intestine may be through ClC-2 down-regulation.

Evidence for a genetic defect in oral tolerance induction in inflammatory bowel disease.

BACKGROUND: Previous studies have suggested that there may be a defect in the control of immune responses locally in the intestines of patients with inflammatory bowel disease (IBD). Recently, we documented a failure to induce oral tolerance to a fed soluble protein antigen, keyhole limpet hemocyanin (KLH), in IBD patients. Both Crohn's disease (CD) and ulcerative colitis (UC) appear to be multigenic disorders with evidence of familial segregation. In this study, we analyzed multiplex IBD families to determine whether the defect in oral tolerance induction is genetically regulated. METHODS: Patients and first-degree relatives from 6 multiplex families were fed KLH 50 mg on days 0 to 5 and 10 to 15, followed by subcutaneous immunizations on days 26 and 35. Blood was obtained and analyzed for KLH-specific T cell proliferative responses and cytokine production. Intestinal permeability was also assessed. RESULTS: In contrast to normal controls, all IBD patients, save 1 (10 patients out of 11 tested P<.0001 versus normal controls), failed to develop oral tolerance to KLH. Furthermore, in 3 of the 4 CD families, at least 1 unaffected family member (total of 5/14 unaffected individuals, P=.002 versus normal controls) also failed to tolerize. This is in sharp contrast to unaffected individuals with no family history of IBD (1/31 tested to date). CONCLUSIONS: This failure of tolerance induction could not be attributed to increased intestinal permeability. In the UC families, the defect in tolerance segregated with disease. These data support a genetic defect in tolerance induction in CD.

Increasing small intestinal permeability worsens colitis in the IL-10-/- mouse and prevents the induction of oral tolerance to ovalbumin.

BACKGROUND: Increased intestinal permeability is found in noninflamed portions of the gut of inflammatory bowel disease patients and in their first-degree relatives, suggesting that it is not a consequence of inflammation. Additionally, increased small intestinal permeability precedes colonic disease in animal models of inflammatory bowel disease. However, it is not known how small intestinal permeability modulates disease in the colon. The aim of this study was to determine if increasing small intestinal permeability modulates colonic inflammation in interleukin (IL)-10 mice and if an increase in permeability is sufficient to prevent oral tolerance to a dietary antigen. METHODS: IL-10 mice were treated with the zonula occludens toxin pathway agonist AT-1002 for 8 weeks, and colitis severity was measured at 12 weeks of age. Wild-type mice were also treated with AT-1002 and fed ovalbumin (OVA) to determine the local and systemic immune response to this antigen under increased small intestinal permeability conditions. RESULTS: IL-10 mice treated with AT-1002 showed exacerbated colitis at 12 weeks of age. AT-1002 also induced a significant OVA-specific humoral response compared with mice that received OVA alone. In addition, the intestinal production of IL-10 and TGF-ß in response to oral OVA was prevented when OVA was given with AT-1002. CONCLUSIONS: Increasing small intestinal permeability worsens colitis in IL-10 mice, and it prevents the development of oral tolerance to OVA in wild-type mice. This study suggests that small intestinal permeability is not merely a consequence of inflammation but a condition that leads to two of the main pathological features of inflammatory bowel disease.

3-0 methylglucose absorption in vivo correlates with nutrient absorption and intestinal surface area in experimental short bowel syndrome.

BACKGROUND: Inert carbohydrate probes are widely used to study intestinal permeability and examine the passive uptake of markers. This study examined the use of quantifying 3-0 methylglucose (3-0 MG) absorption as a marker of intestinal surface area and active nutrient transport capability. METHODS: Using a rat model, varying degrees of short bowel syndrome (SBS) were induced: sham operation (intestinal transection only), 50% resection (R50), 75% resection (R75), and 90% resection (R90; n = 6 to 8 in each group). Animals were pair fed, and over days 5 and 6 postoperation, a balance study was done to quantify absorption of dietary fat, protein, and carbohydrate. On day 7, animals were gavaged with the test solution containing 3-0 MG, lactulose, and mannitol, followed by a 16-hour urine collection. Urine recovery of probes was quantified using high-performance liquid chromotography. Animals were then killed, and the gross and microscopic intestinal morphology was determined. RESULTS: As expected, increased resection resulted in reduced absorption of dietary nutrients and 3-0 MG, which reached significance in the R90 resected animals. 3-0 MG absorption was significantly correlated with intestinal surface area and the absorption of dietary protein and fat (p < .01 for all comparisons). Interestingly, 3-0 MG absorption was not significantly correlated with the absorption of dietary carbohydrate. CONCLUSIONS: 3-0 MG absorption is a useful marker of functional intestinal surface area and of the absorption of dietary fat and protein in experimental SBS. 3-0 MG may be a useful marker of nutrient absorptive capacity in patients with SBS; further study is indicated.

Studies on bacterial endotoxin and intestinal absorption function in patients with chronic heart failure.

BACKGROUND: Small intestinal function may be altered in decompensated chronic heart failure (CHF) and translocating LPS may contribute to systemic inflammation observed in CHF. METHODS: We measured intestinal permeability (melibiose and rhamnose), active (3-O-methyl-d-glucose (3-OMG)) and passive (d-xylose) carrier-mediated absorption in 20 CHF patients (12 edematous and 8 non-edematous) and 8 controls by saccharide absorption technique assessing urinary recovery of orally administered sugars. We additionally measured LPS concentrations in 42 patients with decompensated heart failure and after recompensation. RESULTS: CHF patients had a 54% reduction of active carrier-mediated intestinal transport compared to controls (p<0.0001). This reduction was strongest in edematous compared to non-edematous patients and controls (recovery in urine: 13.2±2.0% vs. 20.8±2.4% vs. 36.0 ± 3.7%, all p ≤ 0.05). Patients showed a 34% reduction of passive carrier-mediated transport, strongest in edematous patients (p=0.006). A greater impairment of active carrier-mediated transport remained significant after adjustment for non-mucosal factors in CHF (p=0.0004). Non carrier-mediated intestinal permeability was not altered. Data from 42 decompensated patients showed a decrease in LPS after recompensation (p=0.004). Edematous patients had highest blood concentrations of LPS, TNF and sTNF-R1 (p<0.04). CHF patients with abnormal LPS concentrations >0.50EU/mL (n=7) had the highest concentrations of TNF (7.0 ± 1.6 vs. 3.1 ± 0.3pg/mL, p<0.02), and sTNF-R1 (3499 ± 52 vs. 1599±219 pg/mL, p=0.02). CONCLUSION: Active carrier-mediated intestinal transport is reduced in decompensated CHF indicating epithelial dysfunction possibly as a consequence of intestinal ischemia. Higher LPS concentrations in edematous CHF relate to inflammation. LPS decreased after recompensation. This suggests a cause/effect relationship between edematous gut wall, epithelial dysfunction and translocating LPS.

Epithelial distribution of neural receptors in the guinea pig small intestine.

Neural and paracrine agents, such as dopamine, epinephrine, and histamine, affect intestinal epithelial function, but it is unclear if these agents act on receptors directly at the enterocyte level. The cellular localization and villus-crypt distribution of adrenergic, dopamine, and histamine receptors within the intestinal epithelium is obscure and needs to be identified. Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs. Enterocytes were separated from intraepithelial lymphocytes by flow cytometry and specific binding was determined using fluorescent probes. Alpha1-adrenergic receptors were located on villus and crypt intraepithelial lymphocytes and enterocytes. Beta-adrenergic receptors were found on villus and crypt enterocytes. Dopamine receptors were found on all cell types examined, whereas histamine receptors were not detected (<10% for each cell population). These studies demonstrated that (1) receptors for epinephrine and dopamine exist on epithelial cells of the guinea pig jejunum, (2) beta-adrenergic receptors are found primarily on villus and crypt enterocytes and (3) intraepithelial lymphocytes contain alpha1-adrenergic, but have few beta-adrenergic, receptors. The presence of neural receptors suggests that these agents are acting, at least in part, at the enterocyte or intraepithelial lymphocyte levels to modulate intestinal and immune function.

Helicobacter pylori activates myosin light-chain kinase to disrupt claudin-4 and claudin-5 and increase epithelial permeability.

Helicobacter pylori is a spiral, gram-negative bacterium that specifically and persistently infects the human stomach. In some individuals, H. pylori-induced chronic gastritis may progress to gastroduodenal ulcers and gastric cancer. Currently, the host-microbe interactions that determine the clinical outcome of infection are not well defined. H. pylori strains capable of disrupting the gastric epithelial barrier may increase the likelihood of developing serious disease. In this study, H. pylori strain SS1 increased gastric, but not small intestinal, permeability in C57BL/6 mice. H. pylori strain SS1 was able to directly increase paracellular permeability, in the absence of host inflammatory cells, by disrupting the tight-junctional proteins occludin, claudin-4, and claudin-5 in confluent nontransformed epithelial cells. H. pylori SS1 also reduced claudin-4 protein levels in human gastric AGS cells. The ability of H. pylori SS1 to increase permeability appeared to be independent of the well-characterized virulence factors vacuolating cytotoxin and CagA protein. H. pylori activated myosin light-chain kinase in epithelial cells to phosphorylate myosin light chain and increase permeability by disrupting claudin-4 and claudin-5. The bacterial factor responsible for increasing epithelial permeability was heat sensitive, membrane bound, and required apical contact with monolayers. In conclusion, disruptions of the tight junctions observed in this study implicate host cell signaling pathways, including the phosphorylation of myosin light chain and the regulation of tight-junctional proteins claudin-4 and claudin-5, in the pathogenesis of H. pylori infection.

A pilot study of the use of epidermal growth factor in pediatric short bowel syndrome.

BACKGROUND: This study examined the effects of enterally administered epidermal growth factor (EGF) on nutrient absorption and tolerance of enteral feeds in pediatric patients with short bowel syndrome (SBS). METHODS: Patients identified with severe SBS (<25% bowel length predicted for age) were prospectively enrolled in treatment using human recombinant EGF (1-53); 100 microg/kg per day given mixed with enteral feeds and patients were treated for 6 weeks. End points followed were patient weight, tolerance of enteral feeds, nutrient absorption, and intestinal permeability as determined using carbohydrate probes and hematologic values for liver function parameters. RESULTS: Five patients were treated with EGF; all showed a significant improvement in carbohydrate absorption (3-0 methylglucose): absorption 24.7% +/- 9.7% pretreatment vs 34.1% +/- 13.8% posttreatment and improved tolerance of enteral feeds (enteral energy as % of total energy, 25% +/- 28% pretreatment vs 36% +/- 24% posttreatment; mean +/- SD; P < .05 by Wilcoxon's signed rank test). Epidermal growth factor treatment was not associated with significant changes in intestinal permeability, the rate of weight gain, or liver function tests. During the treatment phase, no patients developed episodes of sepsis; however, within 2 weeks of discontinuation of EGF treatment, 3 patients developed septic episodes. No adverse effects of EGF administration were noted. CONCLUSIONS: These results suggest that enteral treatment with EGF in pediatric SBS improves nutrient absorption, increases tolerance with enteral feeds, and may improve the infection rate. Further studies exploring treatment strategies including the timing and duration of EGF administration are indicated.

Subclinical intestinal inflammation: an inherited abnormality in Crohn's disease relatives?

BACKGROUND & AIMS: One approach to unraveling the genetics of complex inherited disease, such as Crohn's disease, is to search for subclinical disease markers among unaffected family members. We assessed the possible presence, prevalence, and inheritance pattern of subclinical intestinal inflammation in apparently healthy relatives of patients with Crohn's disease. METHODS: A total of 49 patients with Crohn's disease, 16 spouses, and 151 (58%) of 260 available first-degree relatives underwent a test for intestinal inflammation (fecal calprotectin concentration). The mode of inheritance was assessed from 36 index patients (by variance component analysis) when more than 50% of relatives were studied. RESULTS: Fecal calprotectin concentrations in patients with Crohn's disease (47 mg/L; confidence interval [CI], 27-95 mg/L) and relatives (11 mg/L; CI, 9-14 mg/L) differed significantly (P < 0.0001) from controls (4 mg/L; CI, 3-5 mg/L), whereas that of the spouses did not (4 mg/L; CI, 3-6 mg/L; P > 0.5). Fecal calprotectin concentration was increased in 49% of all relatives studied. The increased fecal calprotectin concentration among the relatives of the 36 index patients had an inheritance pattern that was most consistent with an additive inheritance pattern. CONCLUSIONS: There is a high prevalence of subclinical intestinal inflammation in first-degree relatives of patients with Crohn's disease that conforms best to an additive inheritance pattern. The genetic basis for this abnormality may represent a risk factor for Crohn's disease.