Adherent-invasive Escherichia coli in inflammatory bowel disease

Increased numbers of mucosa-associated Escherichia coli are observed in both major inflammatory bowel diseases, Crohn's disease (CD) and ulcerative colitis (UC). With the identification of mutations in the NOD2-encoding gene in patients with CD and given the intracellular location of NOD2, the presence of pathogenic invasive bacteria could be the link between innate immune response to invasive bacteria and the development of the inflammation. Adherent-invasive E. coli (AIEC) are isolated from ileal biopsies of 36.4% of patients with ileal involvement of CD. These pathogenic E. coli colonize the intestinal mucosa by adhering to intestinal epithelial cells and are also true invasive pathogens, able to invade intestinal epithelial cells and to replicate intracellularly. AIEC strains also survive and replicate extensively within macrophages without inducing host cell death, and their high replication rates induce the secretion of large amounts of tumor necrosis factor alpha (TNF-alpha). There is also evidence suggesting that AIEC is involved in the formation of granulomas. The presence of AIEC is restricted to CD patients. Mucosa-associated E. coli in patients with UC can adhere to intestinal epithelial cells and induce the secretion of IL-8, but there is no evidence that these E. coli strains are invasive.     

Are NOD2 polymorphisms linked to a specific disease endophenotype of Crohn's disease?

The complex and yet unknown etiology of Crohn's disease (CD) might consist of various disease endophenotypes, each of which represent their own pathogenesis. This review focuses on the disease endophenotype linked to polymorphisms in the nucleotide-binding oligomerization domain containing 2 (NOD2) protein and on the importance of established adherent-invasive E. coli (AIEC) in ileal mucosa. To date, there are several reports pointing to the implications of NOD2 polymorphisms in epithelial and immunological responses against microbes, but the pathological significance of NOD2 mutations in CD is not yet clarified. The enhanced number of pathogenic E. coli in the ileal mucosa of CD as compared to healthy controls may result from a genetically based failure in one of the intestinal bacteria sensing systems, like NOD2, making the ileal epithelium more prone to colonization with microbes harboring specific properties such as AIEC. Increasing the focus on defining subgroups of patients with similar disease initiations, mechanisms of action, and manifestations in CD may be pivotal for the development and implementation of future individualized treatment strategies of benefit for the single patient at an early stage.     

Role of bacteria in the etiopathogenesis of inflammatory bowel disease

Increased numbers of mucosa-associated Escherichia coli are observed in both of the major inflammatory bowel diseases, Crohn＇s disease （CD） and ulcerative colitis （UC）. A potential pathophysiological link between the presence of pathogenic invasive bacteria and genetic host susceptibility of patients with ileal CD is suspected. In CD patients, with increased ileal expression of the CEACAM6 molecule acting as a receptor recognized by type 1 pilus bacterial adhesin, and with the identification of mutations in the NOD2-encoding gene, the presence of pathogenic invasive bacteria could be the link between abnormal ileal bacterial colonization and innate immune responses to invasive bacteria. In a susceptible host, the sequential etiological steps of the disease induced by adherent-invasive E. coli （AIEC） are： （1） abnormal colonization via binding to the CEACAM6 receptor, which is overexpressed in the ileal mucosa of CD patients; （2） ability to adhere to and to invade intestinal epithelial cells, which allows bacteria to cross the mucosal barrier; （3） survival and replication within infected macrophages in the lamina propria; and （4） induction of tumor necrosis factor-α secretion and granuloma formation.     

Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

ABSTRACT

Adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of Crohn’s disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic E. coli K12 strain (Exo-K12) or the commensal E. coli HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred via exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.     

Mucosal and Invading Bacteria in Patients with Inflammatory Bowel Disease Compared with Controls

Background: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa. Methods: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis. Results: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls ( P < 0.05). Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls. Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria , the Enterobacteriaceae , the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups. Conclusion: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon.     

Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls

BACKGROUND: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa. METHODS: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis. RESULTS: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls (P < 0.05). Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls. Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria, the Enterobacteriaceae, the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups. CONCLUSION: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon.     

Genetically related Escherichia coli strains associated with Crohn's disease

Escherichia coli strains isolated from patients with Crohn's disease (CD) with chronic ileal lesions (n=14), early endoscopic recurrent lesions (n=20), without endoscopic recurrence (n=7), and controls (n=21) were compared by ribotyping. The dendrogram generated by 50 ribotype profile analysis revealed a large cluster of genetically linked E coli strains isolated significantly more frequently from patients with chronic and recurrent CD (24/33 patients) than from controls (9/21) (p<0.05). Most patients operated on for chronic ileal lesions (78.5%) harboured E coli strains belonging to cluster A (p<0.002 v controls). The prevalence of patients with early recurrent lesions harbouring E coli strains belonging to this cluster was high but not significant, although 16 strains isolated from eight patients presented the same ribotype profile. In this cluster, 21 of 26 strains isolated from patients with active CD demonstrated adherent ability to differentiated Caco-2 cells, indicating that most of the genetically related strains share a common virulence trait. Comparison of E coli strains recovered from ulcerated and healthy mucosa of patients operated on for CD demonstrated in each patient that a single strain colonised the intestinal mucosa. Our results suggest that although a single E coli isolate was not found in Crohn's ileal mucosa, some genotypes were more likely than others to be associated with chronic or early recurrent ileal lesions.     

Deposits of terminal complement complex (TCC) in muscularis mucosae and submucosal vessels in ulcerative colitis and Crohn's disease of the colon.

Extensively washed, ethanol fixed and paraffin embedded colonic specimens from 15 patients with ulcerative colitis (UC) and nine patients with Crohn's disease (CD) of the colon, ileal specimens from six patients with CD of the ileum, and histologically normal control specimens obtained from 10 patients operated for colonic carcinoma, were examined by immunohistochemistry with a monoclonal antibody specific for a neoepitope in the C9 part of the terminal complement complex (TCC). The submucosal blood vessels in inflammatory bowel disease (IBD) showed significantly more TCC positivity than the controls, and vascular TCC deposition was statistically related (p less than 0.001) to degree of inflammation. Five of the six ileal CD specimens contained likewise vascular TCC deposits. In addition, five UC specimens and one colonic CD specimen contained TCC-positive fibrils in the muscularis mucosae or submucosa. There was no significant difference in vascular TCC deposits between UC and CD. The results suggested that terminal complement activation takes place in the intestinal lesions of IBD.     

Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease

Background & Aims: Infectious agents are suspected of being involved in the pathogenesis of Crohn's disease. This study was designed to look for the presence of virulent Escherichia coli strains associated with the ileal mucosa of patients with Crohn's disease. Methods:E. coli strains were recovered from resected chronic ileal lesions (n = 20), neoterminal ileum after surgery from patients with (n = 19) and without (n = 11) endoscopic recurrence, and controls (n = 13). Bacterial adhesion was determined in vitro using intestinal cell lines; other associated virulence factors were assessed by DNA hybridization and polymerase chain reaction experiments. Results: None of the strains harbored any of the virulence factor–encoding genes of E. coli involved in acute enteric diseases. However, mannose-resistant adhesion to differentiated Caco-2 cells was found for 84.6% and 78.9% of the E. coli strains isolated from chronic and early recurrent lesions, respectively, compared with 33% of controls (P < 0.02). In addition, 21.8% of the strains induced a cytolytic effect by synthesis of an α-hemolysin. Conclusions:E. coli strains isolated from the ileal mucosa of patients with Crohn's disease adhere to differentiated intestinal cells and may disrupt the intestinal barrier by synthesizing an α-hemolysin.GASTROENTEROLOGY 1998;115:1405-1413     

PGC-1α在炎症性肠病肠黏膜组织中的表达及临床意义

目的:检测过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)在炎症性肠病(inflammatory bowel disease,IBD)患者肠黏膜的表达水平,探讨其在IBD患者肠黏膜组织中的作用.方法:收集15例溃疡性结肠炎(Ulcerative colitis,UC)患者、17例克罗恩病(Crohn’s disease,CD)患者炎性肠黏膜活检标本及14例正常对照者内镜肠黏膜标本,采用免疫组织化学染色技术分析PGC-1α蛋白在肠黏膜中的原位表达,荧光定量PCR技术检测肠黏膜内PGC-1αmRNA的表达水平.结果:免疫组织化学分析显示:PGC-1α蛋白在正常肠黏膜上皮细胞内表达较多,黏膜固有层细胞内表达较少.与正常对照组相比,PGC-1α蛋白在UC患者肠黏膜上皮细胞内表达量明显减少,而肠黏膜固有层细胞内表达增加,PGC-1α蛋白在CD患者肠黏膜组织内表达量无明显差异.荧光定量PCR分析显示:UC患者炎症肠黏膜组织内PGC-1αmRNA表达水平显著低于正常对照组(0.48±0.15vs1.59±0.38,P<0.05),CD患者炎症肠黏膜组织内PGC-1αmRNA表达水平与正常对照组相比差异无统计学意义(1.55±0.47vs1.59±0.38,P>0.05).结论:与正常对照组相比,PGC-1α在UC炎症肠黏膜组织内表达水平降低,而在CD炎症肠黏膜中表达无明显差异,提示PGC-1α可能参与了UC发生发展过程.     

骨桥蛋白在炎症性肠病患者结肠组织中的表达

背景：骨桥蛋白（OPN）是一种分泌型磷酸化糖蛋白,参与细胞信号转导,促进细胞黏附和迁移。近年研究发现炎症性肠病（IBD）患者血浆OPN水平升高,患者结肠黏膜中可检出OPN表达。目的：了解OPN在IBD患者结肠黏膜组织中的表达情况,探讨其在IBD发病机制中的作用。方法：以免疫组化半定量方法检测53例溃疡性结肠炎（UC）、62例克罗恩病（CD）和15例源自结肠腺瘤患者的正常结肠组织标本中OPN的表达。结果：OPN广泛表达于结肠黏膜上皮细胞和固有层渗出细胞。UC组和CD组结肠上皮细胞的OPN平均光密度值显著低于对照组（11．84±0．98和10．04±1．37对12．64±1．45,P〈0．05）,结肠黏膜固有层渗出细胞的OPN阳性细胞百分率则显著高于对照组（20．31％±4．50％和12．69％±3．06％对3．85％±0．66％,P〈0．001）。UC和CD患者的病情严重程度与结肠上皮细胞和固有层渗出细胞中的OPN表达量均无相关性。结论：OPN在IBD患者结肠黏膜上皮和同有层中的表达不一致,结肠上皮细胞中OPN表达下调可能与肠黏膜屏障功能受损有关,而黏膜固有层渗出细胞中OPN表达上调可能与免疫反应有关。     

Crohn disease-associated Escherichia coli promote gastrointestinal inflammatory disorders by activation of HIF-dependent responses

Crohn disease (CD) ileal lesions are colonized by adherent-invasive E. coli (AIEC) that locally induce inflammation. Hypoxia inducible factor (HIF)-1alpha protein is expressed in acute and chronically inflamed site; however the molecular basis of this expression is not fully understood. The aim of the study was to access whether AIEC induce HIF-1α expression and to study the consequence of HIF-1α expression on the onset of Crohn disease pathogenesis. We show that HIF-1α is maximally expressed in inflamed ileal epithelium of CD-patients. CEACAM6, a protein that acts as a receptor of AIEC, is expressed in this particular condition. Using CEABAC 10 transgenic mice that express CEACAM6, we show that AIEC bacteria, but not non-pathogenic E. coli K12, induce the production of HIF-1alpha protein and the activation of VEGF/VEGFR signaling. Downstream analyses on human intestinal epithelial cells silenced for hif- 1α, highlight the crucial role of this protein in production of pro-angiogenic factors. This study highlights the crucial role of AIEC bacteria as promoter of inflammatory disorders of the gastrointestinal tract and provides clear evidence that HIF-1α protein plays a major role in mediating this effect.     

Expression of NOD2 in Paneth cells: a possible link to Crohn's ileitis

BACKGROUND AND AIMS: Genetic variation in NOD2 has been associated with susceptibility to Crohn's disease (CD) and specifically with ileal involvement. The reason for the unique association of NOD2 mutations with ileal disease is unclear. To identify a possible link, we tested expression of NOD2 in intestinal tissue of CD patients and controls. PATIENTS AND METHODS: Fifty five specimens of ileum or colon from 21 CD patients, seven ulcerative colitis (UC) patients, and five controls with pathology other than CD or UC were stained for NOD2 using an immunoperoxidase method. RESULTS: Using a monoclonal antibody against NOD2 developed in our laboratory, we detected uniform expression of NOD2 in terminal ileum Paneth cells from controls and patients as well as in metaplastic Paneth cells in the colon. Mechanical purification showed enriched expression of NOD2 mRNA in ileal crypts. In Paneth cells, NOD2 was located in the cytosol in close proximity to the granules that contain antimicrobial peptides. We detected minimal NOD2 in the villous epithelium of the ileum or in the colonic epithelium from both CD patients and controls. CONCLUSIONS: These results suggest a role for NOD2 in the regulation of Paneth cell mediated responses against intestinal bacteria and a plausible mechanism to explain the selective association of NOD2 mutations with ileal disease. The impaired capacity of CD associated mutations to sense luminal bacteria may result in increased susceptibility to certain gut microbes.     

白细胞介素-25在炎症性肠病患者中的表达及临床意义

目的 通过检测白细胞介素(IL)-25在炎症性肠病(IBD)患者肠黏膜及血清中的表达水平,探讨其在IBD发病过程中的作用及意义.方法 收集12例溃疡性结肠炎(UC)患者、16例克罗恩病(CD)患者及13例对照者的内镜肠黏膜活检标本,采用荧光定量PCR技术检测肠黏膜内IL-25 mRNA的表达情况,免疫组化技术分析IL-25在肠黏膜中的原位表达;同期收集20例UC、24例CD患者及20名健康对照者血清标本,采用酶联免疫吸附测定(ELISA)检测血清中IL-25水平.结果 与健康对照组相比,UC及CD患者肠黏膜组织内IL-25 mRNA表达显著降低(P＜0.05),UC及CD组间的表达量差异无统计学意义(P＞0.05).免疫组化分析显示IL-25阳性细胞在正常肠黏膜固有层内有较多表达,同时黏膜内的肠上皮细胞也存在IL-25低表达,UC及CD患者肠黏膜IL-25蛋白表达量显著降低(P＜0.05),UC及CD组间的表达量差异无统计学意义(P＞0.05).ELISA显示UC及CD患者血清中IL-25表达量显著低于健康对照组(P＜0.05).结论 IL-25在IBD患者肠黏膜及血清中表达显著降低,提示IL-25表达缺陷与IBD的发生发展密切相关,IL-25有可能成为IBD治疗的新靶点.     

Mucosal Expression of Interleukin-6 and Interleukin-8 Messenger RNA in Ulcerative Colitis and in Crohn's Disease

To elucidate the possible role ofproinflammatory cytokines in inflammatory bowel disease,the expression and localization of interleukin (IL)-6and IL-8 mRNAs were examined in colonic biopsy specimens obtained from 10 patients with activeulcerative colitis (UC), 5 with inactive UC, 6 withCrohn's disease (CD), and 5 normal controls. In situhybridization with digoxigenin-labeled probes andimmunohistochemistry for both cytokines were performed. The IL-6mRNA expression was enhanced in the inflamed mucosa in4 of 6 CD patients, while that of UC patients stayed atbaseline. In contrast, IL-8 mRNA expression was apparently augmented (P = 0.044) in 7 of 10active UC and 3 of 6 CD patients (NS). The cell countpositive for IL-8 mRNA per unit area was definitelyincreased in moderate/severe UC when compared to mild UC (53.1 ± 14.4/mm² vs 9.0± 5.1/mm², P = 0.028) according to thedegree of inflammation. IL-6 mRNA positive cells in CDwere preferentially located in deeper lamina propriathan IL-8 mRNA positive cells in UC. Interestingly, IL-8 mRNA wasexpressed in the mucosal epithelial cells in one UCpatient. The patients treated by corticosteroids tendedto show suppressed expression of each mRNA, except one patient with intractable UC. Our data suggestenhanced expression of mucosal IL-6 mRNA in CD and ofIL-8 mRNA in UC by infiltrating mononuclear cells,indicating the distinct participation of each cytokine in the pathogenesis of UC and CD. Moreover,intestinal epithelial cells in UC occasionally exhibitIL-8 mRNA.     

Ileal lesions in patients with ulcerative colitis after ileo-rectal anastomosis：Relationship with colonic metaplasia

AIM：To assess whether in ulcerative colitis （UC） patients with ileo-rectal anastomosis （IRA）, ileal lesions may develop in the neo-terminal-ileum and their possible relation with phenotypic changes towards colonic epithelium. METHODS： A total of 19 patients with IRA under regular follow up were enrolled, including 11 UC and 8 controls （6 Crohn’s disease, CD; 1 familial adenomatous polyposis, FAP; 1 colon cancer, colon K）. Ileal lesions were identifi ed by ileoscopy with biopsies taken from the ileum （involved and uninvolved） and from the rectal stump. Staining included HE and immunohistochemistry using monoclonal antibodies against colonic epithelial protein CEP （Das-1） and human tropomyosin isoform 5, hTM5 （CG3）. Possible relation between development of colonic metaplasia and ileal lesions was investigated.RESULTS： Stenosing adenocarcinoma of the rectal stump was detected in 1 UC patient. The neo-terminal ileum was therefore investigated in 10/11 UC patients. Ileal ulcers were detected in 7/10 UC, associated with colonic metaplasia in 4/7 （57.1%） and Das-1 and CG3 reactivity in 3/4 UC. In controls, recurrence occurred in 4/6 CD, associated with colonic metaplasia in 3/4 and reactivity with Das-1 and CG3 in 2/3. CONCLUSION： Present fi ndings suggest that in UC, ileal lesions associated with changes towards colonic epithelium may develop also after IRA. Changes of the ileal content after colectomy may contribute to the development of colonic metaplasia, leading to ileal lesions both in the pouch and in the neo-terminal ileum after IRA.     

Antibodies to colonic epithelial cells from the serum and colonic mucosal washings in ulcerative colitis.

It has been suggested that antibodies to a colonocyte protein of 40 kD (an intestinal isoform of tropomyosin) are specifically found in the serum and mucosa of patients with ulcerative colitis, which has important pathogenic implications. This study isolated and purified tropomyosin from the colonic mucosa, but no specific binding to this protein has been detected in serum samples or immunoglobulins isolated from mucosal washings of 20 ulcerative colitis (UC) patients by enzyme linked immunosorbent assay (ELISA) compared with 21 controls or 17 Crohn's disease (CD) patients. Samples from a further 12 patients with UC and primary sclerosing cholangitis (it is proposed that cross reactivity against the intestinal tropomyosin isoform accounts for the extraintestinal disease) also did not show binding to tropomyosin, whereas monoclonal antitropomyosin antisera bound both ELISAs and western blots. This study also examined the proteins in the normal colonic biopsy specimens on western blots that are bound by both serum samples and mucosal immunoglobulin preparations from these patients groups; there was no specific IgG or IgA binding to patients with UC or UC/primary sclerosing cholangitis, whereas binding to mitochondrial proteins of 70,000 and 45,000 was seen in samples from 12 primary biliary cirrhosis positive controls. This work does not support the hypothesis that autoimmune activity against the intestinal isoform or tropomyosin is important in the pathogenesis of ulcerative colitis.     

Characterization of structures with T-lymphocyte aggregates in ileal villi of Crohn's disease

OBJECTIVES: Epithelioid granulomas with transmural inflammation are a characteristic histological feature in Crohn's disease (CD). However, these are not frequently detected by histopathological examination in the biopsy specimens. Here, we demonstrated unique structures with T-lymphocyte aggregates (TLAs) that were specifically found in ileal villi of CD. We characterized the histological and phenotypical features of these structures and assessed the diagnostic value of TLAs for CD. METHODS: Tissue samples were obtained from the inflamed and uninflamed areas of ileal and colonic mucosa of 32 patients with CD. For controls, mucosal samples were obtained from unaffected areas of 18 patients with colon cancer and inflamed and uninflamed areas of 12 patients with ulcerative colitis (UC). RESULTS: 1) In 21 of 32 cases of CD (66%), we found unique structures with lymphoid cell aggregates that were localized in villi of ileum. These structures were not detected in any normal or UC intestine. 2) These structures were composed mainly of T cells, and CD4+ CD45RO+ cells dominated. Neither B cells nor c-kit positive immature lymphocytes were found. Moreover, CD68 positive macrophages were demonstrated in these aggregates, and cells positive for interleukin 18, a pivotal cytokine for Th1 differentiation, were expressed. 3) The aggregates were detected in seven of 13 patients with CD in whom granuloma was not detected by precise histopathological examination. Furthermore, we detected TLAs even in the ilea of two of four CD patients (50%) whose affected lesions were limited to the colon. CONCLUSIONS: We demonstrated TLAs in intestinal villi that may contribute to the pathogenesis of CD. Detection of these lymphocyte aggregates is helpful for diagnosis of CD when granuloma is not found.