Colonic Mucosal Biopsy Specimens Research Articles

Integrated single-cell and bulk RNA sequencing reveals CREM is involved in the pathogenesis of ulcerative colitis

BackgroundUlcerative colitis (UC) is an inflammatory bowel disease characterized by persistent colonic inflammation. Here, we performed a systematic analysis to gain better insights into UC pathogenesis. MethodsWe analyzed two UC-related datasets extracted from the gene expression omnibus database using several bioinformatics tools. The primary cell types and key subgroups of primary cells associated with UC and differentially expressed genes (DEGs) between UC and control samples were identified. The molecular regulation of the key genes was also predicted. The gene ontology and Kyoto encyclopedia of genes and genomes enrichment analyses of marker genes of key cell subgroups and model genes were performed. The expression of key enriched genes was validated in 10 clinical samples using real-time quantitative polymerase chain reaction (RT-qPCR). ResultsMonocytes were identified as the major cell type. Ten differentially expressed marker genes were obtained by intersecting the 3121 DEGs, 38 marker genes in major cell types, and 104 marker genes in key cell subgroups. Four essential genes, associated with immune response, were obtained using support vector machine recursive feature elimination and least absolute shrinkage and selection operator analyses. The four essential genes were highly expressed in Cluster 0 during differentiation. Validation of the four key genes in colonic mucosal biopsy specimens from 10 normal and 10 UC patients revealed that CREM was highly expressed in both the lesion-free sites and lesion sites colonic mucosa of UC patients compared with normal adults. ConclusionsWe identified CREM involved in UC pathogenesis, which is expected to provide a new therapeutic target for UC.

Variation of histopathological features in colonic mucosal biopsy with clinical diagnosis of suspicious inflammatory bowel disease in Dr. Soetomo General Academic Hospital, Surabaya period 2015 - 2019

Inflammatory bowel diseases (IBDs) are chronic inflammatory diseases that often relapse and divided into two types, Crohn’s disease (CD) and ulcerative colitis (UC). Histopathological findings in colonic mucosal biopsy with clinical diagnosis suspicious IBD can vary and overlap. Therefore, criteria and guidelines have been created to improve the diagnostic accuracy. This descriptive observational study was performed retrospectively with cross sectional approach. 122 samples of colonic mucosal biopsies with clinical diagnosis of suspicious IBD were retrieved from histopathological archives in the Anatomical Pathology Laboratorium of Dr. Soetomo Hospital, Surabaya during period 1st January 2015 - 31st December 2019. The most common histopathological feature found in colonic mucosal biopsies with clinical diagnosis of suspicious IBD was crypt distortion (97/79.50% samples), and the least was irregularity of surface epithelium (30/24.59% samples). 10 of 122 samples was concordant with the final diagnosis of IBD. Knowledges regarding the variations of histopathological features in colonic mucosal biopsy specimens with clinical diagnosis of suspicious IBD, can improve the diagnostic accuracy.

Vascular E-selectin Expression Detected in Formalin-fixed, Paraffin-embedded Sections With an E-selectin Monoclonal Antibody Correlates With Ulcerative Colitis Activity.

It is widely accepted that E-selectin, an inducible endothelial cell adhesion molecule, plays a critical role in the initial step of neutrophil recruitment to sites of acute inflammation. However, immunohistological analysis of E-selectin has been hampered by lack of E-selectin-specific monoclonal antibodies that can stain formalin-fixed, paraffin-embedded (FFPE) tissue sections. Here, we employed E-selectin•IgM (a soluble form of E-selectin) as immunogen, and then, after negative selection with L-selectin•IgM and P-selectin•IgM and screening of FFPE sections of both COS-1 cells overexpressing E-selectin and acute appendicitis tissues, we successfully generated an E-selectin-specific monoclonal antibody capable of staining FFPE tissue sections. We used this antibody, designated U12-12, to perform quantitative immunohistological analysis of 390 colonic mucosal biopsy specimens representing ulcerative colitis. We found that the higher the histological disease activity, the greater the number of vessels expressing E-selectin, an observation consistent with previous analyses of frozen tissue sections. Furthermore, in active ulcerative colitis, E-selectin-expressing vessels contained neutrophils attached to endothelial cells, presumably in the process of extravasation, which eventually could cause epithelial damage. These results overall indicate that U12-12 is effective for E-selectin immunohistochemistry in archived FFPE samples representing various human diseases.

Impact of specimen type on findings for bacterial composition within the intestinal tract of dogs and cats with and without chronic enteropathy.

To compare bacterial diversity and community composition among fecal, rectal swab, and colonic mucosal biopsy specimens from dogs and cats with and without chronic enteropathy (CE). 9 healthy dogs, 8 dogs with CE, 8 healthy cats, and 9 cats with CE. In a cross-sectional study design, fecal, rectal swab, and colonic mucosal biopsy specimens were obtained by colonoscopy from healthy dogs and dogs and cats with CE. Fecal and rectal swab specimens were collected from healthy cats. Genomic DNA was extracted, the 16S rRNA V4 gene region was amplified, and sequencing was performed by use of primers 515F to 806R on a paired-end platform. For healthy dogs and dogs and cats with CE, bacterial diversity based on the Chao1 estimate of total species richness was higher for colonic mucosal biopsy specimens than for fecal specimens. Analysis of similarities by use of the Bray-Curtis dissimilarity index revealed that the bacterial communities captured in rectal swab specimens were similar to those captured in fecal specimens for healthy dogs and dogs with CE and similar to those captured in colonic mucosal biopsy specimens for both dog groups and cats with CE. Rectal swab and colonic biopsy specimens were successfully used to characterize the bacteriome of the intestinal tract in dogs and cats by 16S rRNA gene sequencing. Although the specimen types evaluated in this study were not interchangeable in results, rectal swab specimens were practical to collect from dogs and cats to study bacterial composition within the intestinal tract and may provide an alternative to colonic mucosal biopsy and fecal specimens.

Genetic Effects on Transcriptome Profiles in Colon Epithelium Provide Functional Insights for Genetic Risk Loci.

Background & AimsThe association of genetic variation with tissue-specific gene expression and alternative splicing guides functional characterization of complex trait-associated loci and may suggest novel genes implicated in disease. Here, our aims were as follows: (1) to generate reference profiles of colon mucosa gene expression and alternative splicing and compare them across colon subsites (ascending, transverse, and descending), (2) to identify expression and splicing quantitative trait loci (QTLs), (3) to find traits for which identified QTLs contribute to single-nucleotide polymorphism (SNP)-based heritability, (4) to propose candidate effector genes, and (5) to provide a web-based visualization resource.MethodsWe collected colonic mucosal biopsy specimens from 485 healthy adults and performed bulk RNA sequencing. We performed genome-wide SNP genotyping from blood leukocytes. Statistical approaches and bioinformatics software were used for QTL identification and downstream analyses.ResultsWe provided a complete quantification of gene expression and alternative splicing across colon subsites and described their differences. We identified thousands of expression and splicing QTLs and defined their enrichment at genome-wide regulatory regions. We found that part of the SNP-based heritability of diseases affecting colon tissue, such as colorectal cancer and inflammatory bowel disease, but also of diseases affecting other tissues, such as psychiatric conditions, can be explained by the identified QTLs. We provided candidate effector genes for multiple phenotypes. Finally, we provided the Colon Transcriptome Explorer web application.ConclusionsWe provide a large characterization of gene expression and splicing across colon subsites. Our findings provide greater etiologic insight into complex traits and diseases influenced by transcriptomic changes in colon tissue.

Clinicopathological features of intestinal mucosal lesions in AIDS patients and their relation with CD4+ T lymphocytes

Objective: To observe the histopathological features of different opportunistic infections and tumors of the intestinal mucosa in AIDS patients, and to explore the correlation between different lesions and CD4+ T lymphocyte levels. Methods: Colonic mucosal biopsy specimens of 263 patients with clinically diagnosed AIDS and abdominal pain, diarrhea, blood in the stool and other gastrointestinal symptoms were collected from Beijing Ditan Hospital from 2010 to 2018. There were 232 males and 31 females, with age range 10-81 (mean 40±13) years. HE staining, histochemical special staining, immunohistochemical staining, and in-situ hybridization were used to detect the expression of different opportunistic infection pathogens, tumors and CD4+ T lymphocytes. Peripheral blood was also taken for CD4+ T lymphocytes, CD8+ T lymphocytes, HIV viral load and routine indicators. Results: The cohort included 263 intestinal mucosal biopsy specimens. There were 175 cases (66.5%) of non-specific inflammation, and pathogens were detected in 41 cases (15.6%), including 20 cases(7.6%) of cytomegalovirus (CMV) infection, 12 cases (4.6%) of mycobacterial infection, eight cases (3.0%) of amoeba infestation, and one case (0.3%) of talaromycesmarneffei infection; there were also 41 (15.6%) neoplastic lesions including 25 cases (9.5%) of intraepithelial neoplasia, 10 cases (3.8%) of adenocarcinoma and squamous cell carcinoma, six cases (2.3%) of lymphoma; and six cases (2.3%) of ulcerative colitis. The peripheral blood CD4+T lymphocyte levels of patients with CMV, mycobacteria and talaromycesmarneffei were less than 200/μL; the peripheral blood CD4+ T lymphocyte level (P<0.01) and intestinal mucosa CD4+T lymphocytes (P<0.01) were all significantly lower than those in patients with non-specific inflammation. The peripheral red blood cells and hemoglobin levels of patients with CMV and mycobacterial infection (P<0.01), adenocarcinoma and squamous cell carcinoma (P<0.05) were significantly lower than those of non-specific inflammation patients. Conclusions: Pathologic examination of intestinal mucosa can identify specific infections and neoplastic lesions in AIDS patients; the most common lesions are non-specific inflammation, and CMV infection is the most common opportunistic infections; CMV, mycobacteria and talaromycesmarneffei infections are associated with decreased levels of CD4+ T lymphocytes in peripheral blood and intestinal mucosa; entamoeba histolytica infestation and non-HIV-related neoplastic lesions such as intraepithelial neoplasia, adenocarcinoma and squamous cell carcinoma are not associated with changes in AIDS immune function.

Colonic Diverticula Are Not Associated With Mucosal Inflammation or Chronic Gastrointestinal Symptoms

Colonic diverticulosis has been reported to be associated with low-grade mucosal inflammation, possibly leading to chronic gastrointestinal symptoms. However, there is poor evidence for this association. We aimed to determine mucosal inflammation and whether diverticula are associated with chronic gastrointestinal symptoms. We explored whether inflammation was present among symptomatic participants with and without diverticula. We analyzed data from a prospective study of 619 patients undergoing a screening colonoscopy from 2013 through 2015 at the University of North Carolina Hospital in Chapel Hill, North Carolina. Among our participants, 255 (41%) had colonic diverticula. Colonic mucosal biopsy specimens were analyzed for levels of interleukin 6 (IL6), IL10, and tumor necrosis factor messenger RNAs by quantitative reverse-transcriptase polymerase chain reaction, and numbers of immune cells (CD4+, CD8+, CD57+, and mast cell tryptase) by immunohistochemistry. Gastrointestinal symptoms were assessed using Rome III criteria. Proportional odds models were used to estimate odds ratios (ORs) and 95% confidence interval (CIs). After adjustment for potential confounders, there was no association between diverticulosis and tumor necrosis factor (OR, 0.85; 95% CI, 0.63-1.16), and no association with CD4+ cells (OR, 1.18; 95% CI, 0.87-1.60), CD8+ cells (OR, 0.97; 95% CI, 0.71-1.32), or CD57+ cells (OR, 0.80; 95% CI, 0.59-1.09). Compared with controls without diverticulosis, biopsy specimens from individuals with diverticulosis were less likely to express the inflammatory cytokine IL6 (OR, 0.59; 95% CI, 0.36-0.96). There was no association between diverticulosis and irritable bowel syndrome (OR, 0.53; 95% CI, 0.26-1.05) or chronic abdominal pain (OR, 0.68; 95% CI, 0.38-1.23). There was no evidence for inflammation in patients with symptoms when patients with vs without diverticulosis were compared. We found no evidence that colonic diverticulosis is associated with mucosal inflammation or gastrointestinal symptoms. Among patients with symptoms and diverticula, we found no mucosal inflammation.

Colonic cytokines for severe ulcerative colitis

Aim of investigation. To assess colonic cytokines profile at severe ulcerative colitis (UC). Material and methods. Overall 48 biopsy specimens of colonic mucosa obtained at resection operations in 7 patients with total form of UC were investigated. All patients underwent total colectomy for severe treatment-resistant UC. Activity of inflammatory process was determined by severity of erosive and ulcerative lesions of the colon. Cytokine concentration was evaluated in supernatant fluid after centrifugal separation of 10 mg biopsy specimen homogenate by the protein analyzer «Bio-Plex» («Bio-Rad», USA). Colonic mucosa biopsy specimens of unaffected parts of the large intestine resected in 10 patients with colorectal cancer were used as controls. Results. Tissue levels of IL-1β, IL-8, IL-6, TNF-α (tumor necrosis factor-α), IFN-γ (interferon-γ), IL-17 and IL-12 in biopsy specimens the large intestine of UC patients were significantly elevated. The most significant elevation was found for IL-8 and IL-1β, with the ratio of 3:1 (in the control group 0,35:1). The cytokine content in biopsy specimens of the large intestine has been increased in comparison to terminal portion of the small intestine, and the cytokine level in the left and transverse part of the colon was higher, than in cecum and ascending colon. At severe inflammation as compared to moderate inflammation the most significant increase was found for IL-8, IL-6 and TNF-α. Cytokine profile with the prevalence of IL-1β (IL-1β>IL-8>IL-6 (IFN-γ)>TNF-α) was observed in 14 (77,7%) of 18 biopsy specimens with moderate inflammation while similar cytokine pattern was observed in 9 (47,3%) of 19 biopsy specimens with severe inflammation (p=0,05). Domination of IL-8 (IL-8> IL-1β> IL-6 (TNF-α)> TNF-α (IL-6)) in cytokine profile was found in 3 of 18 (16,6%) biopsy specimens with moderate and in 9 of 19 (47,3%) biopsy specimens with a severe inflammation (p<0,05). Cytokines IL-2, IL-4, IL-5, IL-7, IL-10 and IL-13 when undetected neither in UC patients, nor in control group. Conclusions. Significant increase in proinflammatory cytokine level with prevalence of IL-1β and IL-8 at moderate and severe erosive - ulcerative involvement respectively was observed in colonic mucosa biopsies in comparison to terminal ileum of UC patients and control group; in the left part and transverse colon versus ascending colon and caecum of UC patients. Elevation of IFN-γ and IL-17 content at severe inflammation indicates presence of common mechanisms in ulcerative colitis disease and Crohn's disease pathogenesis.

Pseudomembranous colitis: Not always Clostridium difficile.

Although Clostridium difficile infection is the cause of most cases of pseudomembranous colitis, clinicians should consider less common causes, especially if pseudomembranes are seen on endoscopy but testing remains negative for C difficile or if presumed C difficile infection does not respond to treatment. Histologic review of colonic mucosal biopsy specimens can provide clues to the underlying cause.

Expression of Toll-like receptors in the mucosa of patients with ulcerative colitis

Patients with ulcerative colitis (UC) have a high risk of developing colorectal cancer. The aim of the present study was to evaluate the expression pattern of Toll-like receptors (TLRs) in the colonic mucosa of patients with UC. Colonic mucosal biopsy specimens were collected during colonoscopy from 30 patients with UC and 30 patients with normal findings as controls. The protein and mRNA expression levels of TLRs 1-4 and TLR9 were measured by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction analysis, respectively. The results showed that the mRNA and protein expression of TLR2, TLR4 and TLR9, but not TLR1 and TLR3, was significantly increased in the colonic mucosa of patients with UC compared with that in the normal controls. TLR (TLR2, TLR4 and TLR9) immunoreactivity was found in the cytoplasm of epithelial cells in the mucosa, and occasionally in the endothelium of small vessels of the stromal tissues. In conclusion, TLR2, TLR4 and TLR9 expression may be important in the biological pathogenesis of UC. TLR alterations in the innate response system may contribute to the pathogenesis of UC.

Chronic non-bloody diarrhoea: a prospective study in Malmö, Sweden, with focus on microscopic colitis.

BackgroundChronic non-bloody diarrhoea affects up to 5% of the population. Microscopic colitis is one of the most common causes, encompassing the subtypes collagenous colitis and lymphocytic colitis. The diagnosis of microscopic colitis is made by histological examination of colonic mucosal biopsy specimens. The aim of this investigation was to determine whether laboratory parameters or questions about disease history or concomitant disease could be helpful in discriminating patients with MC from those with a histologically normal colonic mucosa.FindingsPatients admitted for colonoscopy because of chronic non-bloody diarrhoea (>2 loose stools for >3 weeks) at endoscopy units in Malmö during 2007 and 2009, were enrolled. A total number of 78 patients were included (60 women, 18 men, median age 59, IQR 45–69 years). Out of these 78, 15 patients (19%) had microscopic colitis (CC; n = 10, LC; n = 5). MC was especially prevalent in patients above the age of 50 (25%). No differences were found between those with normal histology and MC in laboratory analyses (inflammatory and liver parameters). Neither were differences shown in questions regarding symptoms, environmental factors or concomitant diseases except for an association with celiac disease (p = 0.019) and a trend maybe indicating an inverse association with appendectomy (p = 0.057).ConclusionsMicroscopic colitis is associated with female gender, celiac disease and consumption of NSAIDs. Trends were observed indicating that age above 50 years, acute onset and absence of appendectomy may be associated with MC. No associations were observed with other symptoms, calprotectin levels or liver parameters.

Antibody to Tropomyosin Isoform 5 and Complement Induce the Lysis of Colonocytes in Ulcerative Colitis

Tropomyosins (TMs) are cytoskeletal microfilament proteins present in all eukaryotic cells. Human TM isoform 5 (hTM5) is the predominant isoform in colonic epithelial cells. Antibodies against hTM5 are found both in the sera and in the mucosa of patients with ulcerative colitis (UC) but not Crohn's disease (CD). We investigated whether anti-hTM5 autoantibodies are pathogenic. Normal-appearing colonic mucosal biopsy specimens were incubated with autologous serum. After 45 min, deposition of the complement component C3b was identified by indirect immunofluorescence assay (IFA). Additional specimens were incubated with autologous serum fixed in formalin, and their architecture was examined by hematoxylin and eosin (H&E) staining. For 79% of UC patients, autologous serum caused C3b staining along the colonic epithelium. Recombinant hTM5 or anti-hTM5 monoclonal antibody blocked serum-induced C3b deposition. Immunoglobulin G (IgG) antibody and affinity-purified anti-hTM5 IgG antibody from UC sera with complement caused C3b deposition, indicating specificity of hTM5 as an autoantigen. When analyzed by H&E staining, sera obtained from 71% of UC patients caused a significant loss of epithelium. This process was inhibited by Fc fragments, indicating that it is complement mediated. With medium, normal, or CD serum, there was no C3b deposition or morphological changes of the colonic epithelium, indicating disease specificity. The ileal mucosa was not affected by UC sera, suggesting specificity for the colon. In UC mucosa, expression of hTM5 increased. hTM5 acts as an autoantigen in UC. hTM5-specific IgG autoantibody in sera from UC patients induces C3b deposition and destruction of colonic epithelial cells, suggesting a direct pathogenic effect. If used as a diagnostic test to distinguish UC from CD, IFA would have 79% sensitivity and 100% specificity. Development of blocking antibodies may lead to novel therapies.

W1640 Detection of Tropheryma Whipplei DNA in Intestinal Biopsy Specimens with a Novel Real-Time PCR

Introduction Whipple's disease (WD) is a rare, multisystemic infectious disease caused by the bacterium Tropheryma whipplei (Tw). Periodic acid Schiff (PAS) stain is usually performed to diagnose the disease. PCR is currently used to confirm the presence of Tw DNA. However, conventional PCR is not specific, whereas real-time PCR (rtPCR) based on the 16S rRNA gene is insensitive since only one copy of the gene is present in Tw genome. Recently, we developed a novel, more sensitive and specific rtPCR to detect the presence of Tw DNA. Aims To determine prevalence of Tw in duodenal and colonic biopsy specimens with rtPCR in a random population of patients undergoing routine gastroscopy or colonoscopy. Materials and methods Mucosal biopsy specimens from the duodenum or sigmoid were harvested from subsequent patients undergoing upper or lower endoscopy. Most frequent indications for gastroscopy were dyspepsia, pyrosis, and follow-up of celiac disease. Common indications for colonoscopy were colorectal cancer screening and surveillance, and abdominal pain. Specimens were analyzed by rtPCR targeting an exclusive, repetitive sequence which is seven times present in the Tw genome. Results In total, 48 duodenal and 90 colonic mucosal biopsy specimens of 118 patients were analyzed. Of these patients, 40% were men, mean age 54y (range 18-84y). Normal appearance of duodenal and colonic mucosa was found in 87% and 83%, respectively. Main abnormalities found in the duodenum were signs of celiac disease (CD). Abnormalities of colonic specimens included signs of IBD and pseudomelanosis coli. Of the duodenal and colonic specimens 2.1% and 6.7% were positive in the Tw rtPCR, respectively (mean age 43y, male/female: 2/5). One showed CD at microscopy, whereas all others were normal. In another patient, signs and symptoms of WD were recorded namely arthralgia, weight loss and peripheral neuropathy. The Cycle threshold-value of the PCR was considerably lower in this patient, consistent with a high load of Tw DNA. Discussion/ Conclusion With a novel rtPCR, Tw DNA is present in duodenal or colonic biopsy specimens in a proportion of patients without signs or symptoms of WD. Therefore, additional factors, other than merely the presence of Tw, play a role in development of disease. Initiation of antibiotic therapy against Tw may not exclusively depend on positive PCR findings, but should additionally be based on PAS stain and signs and symptoms ofWD. Clinical significant WD appears to be associated with a higher Tw load.

Alterations in the Immunohistochemical Expression of Das-1 and CG-3 in Colonic Mucosal Biopsy Specimens Helps Distinguish Ulcerative Colitis From Crohn Disease and From Other Forms of Colitis

Distinction between ulcerative colitis (UC) and Crohn disease (CD) in mucosal biopsies is often difficult. Das-1 and CG-3 are monoclonal antibodies directed against an unknown colonic epithelial protein and human tropomyosin isoform-5, respectively, both show altered expression in patients with UC. In this study, we evaluated the utility of Das-1 and CG-3 in distinguishing UC from CD and from other types of colitis. One colonic biopsy specimen from each of 85 patients with confirmed UC (n=25), CD (n=15), lymphocytic (n=15), collagenous (n=15), and ischemic (n=15) colitis, and also 10 samples from normal controls, were stained for Das-1 and CG-3 using standard techniques. Reactivity for Das-1 and CG-3 was noted to be absent or present, and the location (ie, surface+/-crypt epithelium) and degree (weak or strong) of CG-3 staining was recorded. Loss of Das-1 staining occurred more frequently in UC (96%) compared with CD (20%), lymphocytic (20%), collagenous (13%), and ischemic colitis (0%) cases, as well as controls (10%, P<0.001 for all comparisons). CG-3 positivity in crypt epithelium was significantly more common in UC (52%) compared with the other groups (P< or =0.02 for all comparisons). The combination of strong crypt CG-3 staining and loss of Das-1 staining was noted in 44% of UC cases, but not in any other type of colitis (P=0.003 for all comparisons). We conclude that the patterns of Das-1 and CG-3 staining in colonic mucosal biopsies may be clinically useful in distinguishing UC from CD and from other colitidies.

COX‐2 expression in sporadic colorectal adenomatous polyps is linked to adenoma characteristics

To assess cyclooxygenase-2 (COX-2) expression in sporadic colonic adenomas and to explore the association of COX-2 positivity with adenoma characteristics linked to increased risk of malignant transformation. COX-2 expression and localization were assessed in 64 colorectal adenomas and 35 paired adjacent normal colonic mucosal biopsy specimens. The number of adenoma specimens was then extended to include polyps exhibiting an increasing degree of epithelial dysplasia. Forty colonic hyperplastic polyps were also identified from the pathology diagnostic database and included in the analysis. Immunohistochemistry was performed with the Envision+ peroxidase-linked biotin-free system incorporating a signal amplification step. There was a statistically significant increase in COX-2 expression in colonic polyps compared with paired adjacent normal mucosa, chi(2) = 40.1, P = 0.001. The probability of COX-2 expression increased along with increasing adenoma size and increasing degree of epithelial dysplasia. Fifty-five per cent of the hyperplastic polyp specimens expressed COX-2. This study associates COX-2 epithelial expression with a number of adenoma characteristics that convey an increased risk of malignant transformation. This is in keeping with a positive role for COX-2 in early colorectal carcinogenesis.

Multigene Analysis Can Discriminate Between Ulcerative Colitis, Crohn's Disease, and Irritable Bowel Syndrome

Inflammatory bowel diseases (IBDs) and the irritable bowel syndrome (IBS) are heterogeneous disorders of the gastrointestinal tract and can profoundly affect the quality of life. Because many of the symptoms of IBD are similar to those of IBS, the former may be misdiagnosed. In addition, the 2 major forms of IBD, ulcerative colitis (UC) and Crohn's disease (CD), have overlapping nonspecific, pathologic features leading to difficulties in assessing colonic inflammation and hence the term IBD unclassified has been proposed. The aim of this study was to identify and assess the utility of a certain set of marker genes that could help to distinguish IBS from IBD, and further to discriminate between UC and CD. Subtractive suppression hybridization was used to identify IBD-specific genes in colonic mucosal biopsy specimens. In quantitative polymerase chain reaction experiments, the differential expressions of identified genes then were analyzed using a classification algorithm and the possible clinical value of these marker genes was evaluated in a total of 301 patients in 3 stepwise studies. Seven marker genes were identified as differentially expressed in IBD, making it possible to discriminate between patients suffering from UC, CD, or IBS with area under the receiver-operating characteristic curves ranging from 0.915 to 0.999 (P < .0001) using the clinical diagnosis as gold standard. Expression profiling of relevant marker genes in colonic biopsy specimens from patients with IBD/IBS-like symptoms may enable swift and reliable determination of diagnosis, ultimately improving disease management.

Contribution of TIA-1+ and granzyme B+ cytotoxic T lymphocytes to cryptal apoptosis and ulceration in active inflammatory bowel disease

In spite of the clinicopathological differences between Crohn's disease (CD) and ulcerative colitis (UC), they share the fundamental feature of destructive inflammatory processes involving the intestinal wall. The aim of the present study was to investigate the contribution of cell-mediated cytotoxicity to mucosal damage in CD and UC. Colonic mucosal biopsy specimens from patients with active CD ( n=25) and UC ( n=26) and normal controls ( n=12) were immunohistochemically analyzed for the expression of CD3, CD4, CD8, and T cell-restricted intracellular antigen (TIA)-1, which promotes apoptosis by alternative splicing of pre-messenger RNA of the Fas receptor, and granzyme B (GrB), which leads to apoptosis through induction of perforin. Histological scores for cryptal apoptosis and ulceration were assessed in hematoxylin- and eosin-stained sections. In patients with CD and UC, CD3+( P<0.001), CD4+( P<0.001), CD8+( P<0.01), TIA-1+(CD, P<0.01; UC, P<0.001), and GrB+(CD, P<0.01; UC, P<0.001) intraepithelial lymphocytes (IELs) were significantly increased as compared with controls. Positive relationships were found between the histological scores for apoptosis or ulceration and the numbers of CD8+or TIA-1+IELs. In conclusion, cytotoxic T lymphocytes are present in increased numbers in the mucosa of patients with active CD and UC, and local activation of IELs may contribute to mucosal damage with these diseases.

Intestinal Spirochetosis: A Cause of Symptoms Resembling Irritable Bowel Syndrome

Purpose: Intestinal spirochetosis (IS) is an uncommon, but treatable cause of gastrointestinal symptoms that often resemble irritable bowel syndrome (IBS). We report a case of IS in a homosexual male presenting with chronic watery diarrhea. A 25 year old homosexual male of Asian origin presented with an eight-year history of watery diarrhea. He denied rectal bleeding, abdominal pain and weight loss. Physical examination was unremarkable. Biochemical and hematological parameters were normal. Stool examination did not reveal conventional pathogens. No gross abnormalities were noted on colonoscopy. Hematoxylin and eosin (H &E) stained slides of colonic mucosal biopsy specimens revealed a focally prominent surface epithelial brush border with basophilic microorganisms adherent to the epithelial surface, suggestive of IS. The diagnosis was confirmed with Steiner silver stain, which highlighted surface microorganisms. A seven day course of oral metronidazole was offered as therapy for IS. IS is an uncommon clinical entity in North America, with an estimated prevalence of 2.5% to 6.5%. Higher prevalence rates (11.4 to 64.3%) have been observed in developing countries, in homosexuals and HIV-infected individuals. Colonization of the colonic mucosa by various species of spirochetes, including Brachyspira aalborgi and Serpulina pilosicoli, is almost an incidental finding in an asymptomatic patient. Spirochetal invasion of the colonic epithelium, resulting in epithelial changes and inflammatory infiltrate in the lamina propria, is associated with symptoms. However, in homosexual men, regardless of HIV status, symptoms may be present in the absence of epithelial invasion. Persistent diarrhea, abdominal pain or constipation occur either alone or in combination, often resembling IBS. The diagnosis of IS is suspected on H & E staining of colonic mucosal specimens, and confirmed by silver stains, immunohistochemistry or electron microscopy. Due to patchy distribution of spirochetes, multiple site colonic biopsies are recommended for a definitive diagnosis. Of the several regimens proposed, metronidazole seems to be superior in eradicating spirochetes from the intestine. In conclusion, IS should be considered as a differential diagnosis in patients with IBS-like symptoms, with an otherwise grossly normal-appearing colonic mucosa. Metronidazole should be offered as first-line therapy for the treatment of IS.

A Functional Role of Flip in Conferring Resistance of Crohn’s Disease Lamina Propria Lymphocytes to FAS-Mediated Apoptosis

There is evidence that, in Crohn's disease (CD), lamina propria T lymphocytes (LPLs) are resistant to FAS-mediated apoptosis and that this defect contributes to the mucosal T-cell accumulation. In this study we examined the functional role of Flip, a Flice inhibitor protein, in the resistance of CD LPL to FAS-mediated apoptosis. Biopsy specimens and LPLs were taken from CD and ulcerative colitis (UC) patients and normal controls and analyzed for Flip by Western blotting. We also examined whether inhibition of Flip by antisense oligonucleotide restored the susceptibility of CD LPLs to FAS-induced apoptosis. LPL apoptosis was assessed by flow cytometry. After FAS stimulation, the rate of apoptosis of CD3+ LPLs was higher in normal controls and patients with UC than in patients with CD. Enhanced expression of both long and short Flip isoforms was seen in biopsy specimens and purified CD3+ and CD45RO+ LPLs of CD patients in comparison with UC patients and normal controls. No increase in Flip was documented in untreated celiac disease mucosa, thus suggesting the possibility that induction of Flip in the gut does not simply rely on the ongoing inflammation. Finally, we showed that inhibition of Flip by antisense oligonucleotide reverted the resistance of CD LPLs to FAS-induced apoptosis. Data suggest a role for Flip in the resistance of CD LPLs to FAS-mediated apoptosis.

Correlation betweenSaccharomyces cerevisiaeDNA in intestinal mucosal samples and anti-Saccharomyces cerevisiaeantibodies in serum of patients with IBD

To investigate the correlation between ASCA and presence of mucosal S. cerevisiae DNA in a population of CD, ulcerative colitis (UC) patients and controls. S. cerevisiae-specific primers and a fluorescent probe were designed for a 5' exonuclease real time PCR (TaqMan) assay, which is a homogenous system using a fluorescent-labelled probe for the detection of PCR product in real time. We analyzed the relation of the PCR results with the ASCA findings in a group of 76 inflammatory bowel disease (IBD) patients (31 CD, 45 UC) and 22 healthy controls (HC). ASCA (IgA or IgG) were positive in 19 (61%) patients with CD, 12 (27%) with UC and none of the HC. PCR amplification was inhibited and excluded from the final results in 10 (22%) UC patients, 7 (22%) CD patients, and 6 (30%) HC. In only 15 of the mucosal samples, S. cerevisiae DNA was detected by real time PCR, including 7 (29%) in CD, 7 (19%) in UC, 1 (6%) in HC. In 4 CD and in 4 UC patients, ASCA and mucosal S. cerevisiae were positive. Mucosal S. cerevisiae was present in combination with negative ASCA IgA and IgG in 3 UC, and 3 CD patients. We conclude that since the presence of S. cerevisiae in colonic mucosal biopsy specimens is very rare, ASCA is unlikely to be explained by continuous exposure to S. cerevisiae in the mucosa. Therefore, ASCA formation must occur earlier in life and levels remain relatively stable thereafter in immunological susceptible persons.