Sigmoid Mucosa Research Articles

P011 Altered Expression and Enzymatic Activity of Xanthine Oxidase in Inflammatory Bowel Disease

Abstract Background Xanthine oxidase (XO) is involved in the last steps of purine metabolism; indeed, it catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid (UA) with the production of superoxide anion. The accumulation of UA has been shown to initiate the inflammatory process through NLRP3 inflammasome and the production of reactive oxygen species (ROS) contributes to inflammation-related tissue damage. In murine models of colitis, XO inhibitors, allopurinol or febuxostat, reduced the expression of proinflammatory cytokines supporting the involvement of XO in the intestinal inflammatory process. This study aims to conduct a thorough investigation of the role XO plays in IBD pathogenesis and to explore the potential of XO inhibition as a therapeutic strategy. Methods Biopsies from the colon of patients with ulcerative colitis (UC) and Crohn’s disease (CD) were collected at University Hospital of Cagliari. Disease activity was assessed using Mayo and SES-CD scores. XO expression levels were evaluated using Real-Time PCR, validated by GEO database (GSE11223, GSE117993). XO enzymatic activity was quantified spectrophotometrically in protein extracts from the biopsies. XO and NLRP3 inflammasome activation pathway, including NLRP3, ASC, and CASPASE1, was investigated using immunohistochemistry (IHC). In vitro, Caco2, HT29, SW480 cells were treated with xanthine, XO inhibitors (allopurinol, febuxostat) to assess XO inhibition on NLRP3 activation, analyzed by immunocytochemistry (ICC). Results Biopsies from the sigmoid colon of 15 HC, 20 UC, and 15 CD patients, as well as from the ileum of 10 HC and 10 CD, were analyzed. Increased XO expression was found in UC and CD sigmoid mucosa (Relative Quantification [RQ]: 1.93 vs. 0.99, p=0.004), and CD ileum (RQ: 2.72 vs. 1.26, p=0.004), confirmed by GEO data. XO activity was higher in UC and CD sigmoid (3.86 nmol UA/min/mg in UC, 4.82 in CD vs. 1.03 in HC, p<0.001) and CD ileum (8.41 vs. 1.83 in HC, p<0.001). IHC confirmed that XO and the NLRP3 inflammasome pathway are more highly expressed in IBD patients than HC, predominantly within epithelial cells. In vitro studies on Caco2, HT29, and SW480 cells demonstrated that suppressing XO activity with XO inhibitors leads to the inhibition of the NLRP3 pathway. Conclusion XO expression was higher in IBD pts than in HC individuals. Alongside this increase in XO activity, there was a concurrent hyperactivation of the NLRP3 inflammasome. The inhibition of XO activity in cell lines resulted in reduced activation of the NLRP3 inflammasome pathway. These findings support the hypothesis that XO plays a critical role in the pathogenesis of IBD and highlight its potential as a therapeutic target.

Colonic mucosal biopsy location can not affect the results of mucosal metabolomics and mucosal microbiota analysis in IBS.

To compare and analyze the mucosal metabolites and mucosal microbiota of different parts of colon in patients with IBS. A total of 10 patients with IBS-D and six healthy controls (HC) were enrolled. All enrolled participants underwent two biopsies of the ileocecal and sigmoid colon during colonoscopy. Metabolomic profiling of one piece of tissue was conducted using desorption electrospray ionization-mass spectrometry (DESI-MS), and the gut flora of the other piece was examined using 16S rRNA sequencing. The metabolic profiles and flora of the ileocecal and sigmoid colonic mucosa in each group were further analyzed in this study. (1) Principal components analysis (PCA) indicated that mucosal metabolites did not differ in different parts of the colon in either the IBS-D or HC groups. (2) In the mucosal microbiome analyses, no differences between the microbiota of the two parts of the colon were found by using Principal Co-ordinates Analysis (PCoA). In IBS group, comparing with sigmoid mucosa, the chao1 richness indice was higher and the Shannon index was lower in the ileocecal mucosa (p = 0.40, p = 0.22). However, in the HC group, microbiome analysis of the ileocecal mucosa showed lower values for Chao 1 and Shannon indices than those of the sigmoid colon mucosa (p = 0.06, p = 0.86). (3) Compared with the HC group, 1,113 metabolic signal peaks were upregulated, whereas 594 metabolites were downregulated in the IBS-D samples. Moreover, the PCA of the metabolites showed significant separation between the IBS-D and HC groups. (4) Chao1 expression was significantly higher in the mucosal microbiota with IBS-D than in the HC (p = 0.03). The Shannon index was lower in IBS-D, but the difference was not statistically significant (p = 0.53). PCoA revealed a significant difference in the microflora structure between the IBS-D and HC groups. The mucosal metabolic profile and mucosal flora structure of the colon were similar, despite different locations in IBS and healthy subjects. IBS had abnormal colonic mucosal metabolism and flora disturbances.

The alteration of intestinal mucosal α-synuclein expression and mucosal microbiota in Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease but still lacks a preclinical strategy to identify it. The diagnostic value of intestinal mucosal α-synuclein (αSyn) in PD has not drawn a uniform conclusion. The relationship between the alteration of intestinal mucosal αSyn expression and mucosal microbiota is unclear. Nineteen PD patients and twenty-two healthy controls were enrolled in our study from whom were collected, using gastrointestinal endoscopes, duodenal and sigmoid mucosal samples for biopsy. Multiplex immunohistochemistry was performed to detect total, phosphorylate, and oligomer α-synuclein. Next-generation 16S rRNA amplicon sequencing was applied for taxonomic analysis. The results implied that oligomer α-synuclein (OSyn) in sigmoid mucosa of PD patients was transferred from the intestinal epithelial cell membrane to the cytoplasm, acinar lumen, and stroma. Its distribution feature was significantly different between the two groups, especially the ratio of OSyn/αSyn. The microbiota composition in mucosa also differed. The relative abundances of Kiloniellales, Flavobacteriaceae, and CAG56 were lower, while those of Proteobacteria, Gammaproteobacteria, Burkholderiales, Burkholdriaceae, Oxalobacteraceae, Ralstonia, Massilla, and Lactoccus were higher in duodenal mucosa of PD patients. The relative abundances of Thermoactinomycetales and Thermoactinomycetaceae were lower, while those of Prevotellaceae and Bifidobacterium longum were higher in patients’ sigmoid mucosa. Further, the OSyn/αSyn level was positively correlated with the relative abundances of Proteobacteria, Gammaproteobacteria, Burkholderiales, Pseudomonadales, Burkholderiaceae, and Ralstonia in the duodenal mucosa, while it was negatively correlated with the Chao1 index and observed operational taxonomic units of microbiota in sigmoid mucosa. The intestinal mucosal microbiota composition of PD patients altered with the relative abundances of proinflammatory bacteria in the duodenal mucosa increased. The ratio of the OSyn/αSyn level in the sigmoid mucosa indicated a potential diagnostic value for PD, which also correlated with mucosal microbiota diversity and composition.Key points• The distribution of OSyn in sigmoid mucosa differed between PD patients and healthy controls.• Significant alterations in the microbiome were found in PD patients’ gut mucosa.• OSyn/αSyn level in sigmoid mucosa indicated a potential diagnostic value for PD.

Long Noncoding RNA FBXL19-AS1-Mediated Ulcerative Colitis-Associated Intestinal Epithelial Barrier Defect.

This study commenced to uncover the role of long non-coding RNA FBXL19 antisense RNA 1 (FBXL19-AS1) in the development of ulcerative colitis (UC) and its possible mechanism. FBXL19-AS1 expression in the colonic sigmoid mucosa of UC patients was detected. A colitis model was induced in mice using 5% dextran sodium sulfate. Hematoxylin-eosin staining was performed for histopathological examination. Apoptosis was detected by Tunel staining and tissue fibrosis was detected by immunohistochemistry. Also, intestinal permeability was examined. The concentrations of inflammatory factors IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay. The relationship between FBXL19-AS1, miR-339-3p and RHOB was verified by RNA immunoprecipitation assay and dual luciferase reporter assay. The expression of FBXL19-AS1 was increased in dextran sodium sulfate (DSS)-induced colitis mouse model. FBXL19-AS1 interference or miR-339-3p overexpression inhibited DSS-induced colonic epithelial cell apoptosis and inflammatory response, and improved intestinal epithelial barrier defects, thereby ameliorating DSS-induced colitis injury in mice. FBXL19-AS1 sponged miR-339-3p while miR-339-3p targeted RHOB. Overexpression of RHOB reversed the protective effect of inhibition of FBXL19-AS1 on DSS-induced colitis in mice. FBXL19-AS1 reduces miR-339-3p-mediated targeting of RHOB and aggravates intestinal epithelial barrier defect in DSS-induced colitis in mice.

Practical Applications of Prolonged Private Equity Holding Periods: Six Years is the New Normal

AIM--To determine whether there are characteristic immunohistological changes in the colonic mucosa in acute graft versus host disease (GvHD). METHODS--Consecutive allogeneic (n = 11) and autologous (n = 11) bone marrow transplant recipients underwent endoscopic biopsy of sigmoid mucosa before transplant and on day 30 post-transplant. Immunohistochemical staining and quantitation of intraepithelial and lamina propria mononuclear cells were undertaken using a panel of monoclonal antibodies and a Streptavidin-biotin alkaline phosphatase staining technique. RESULTS--In the allogeneic group (nine of whom had clinical acute GvHD) there was a fivefold increase in lamina propria CD16+ mononuclear cells (3.1 +/- 4.3 to 17.0 +/- 12.2 per 100 lamina propria nucleated cells), compared with autologous transplant recipients in whom this rise was twofold (5.5 +/- 4.6 to 10.6 +/- 7.1 per 100 lamina propria nucleated cells). The CD16+ mononuclear cells had morphological appearances of tissue macrophages, but in neither the allogeneic nor autologous groups was there an increase in total macrophage numbers (CD14+). In patients with acute GvHD the lamina propria CD4+:CD8+ lymphocyte ratio fell (1.97 +/- 1.12 to 1.07 +/- 1.01), primarily because of a fall in the number of lamina propria CD4+ lymphocytes. In both allogeneic and autologous groups there was a fall in intraepithelial lymphocyte numbers, but there was no change in CD19+ (B cell), CD25+ (interleukin-2 receptor positive) or CD56+ (natural killer) cell numbers. CONCLUSION--Following bone marrow transplantation, there appears to be upregulation of lamina propria tissue macrophage CD16 (an Fc receptor for IgG), a change which is more noticeable after allogeneic transplantation and which may be related to the development of acute GvHD. In patients with acute GvHD there was a fall in the lamina propria CD4+:CD8+ lymphocyte ratio. If these changes are functionally important, they may have significant implications for understanding the pathogenesis of GvHD.

S1960 Acute Pseudomembranous Colitis, Microangiopathic Hemolytic Anemia, and Renal Injury After Pericardiectomy

Introduction: Acute colonic ischemia following surgery is usually a result of colonic hypoperfusion, particularly in watershed regions; however, it is critical to maintain a broad differential diagnosis for alternate etiologies. Case description/methods: A 57-year-old man with lupus on hydroxychloroquine and prednisone underwent elective pericardiectomy for medically refractory constrictive pericarditis. On post-operative day (POD) 3, he developed abdominal pain, nausea, and non-bloody diarrhea. Hematochezia started on POD 4. Computed tomography showed rectosigmoid thickening and perirectal fat stranding (Figure 1A). Sigmoidoscopy revealed edematous, ulcerated, and discolored mucosa from the anal verge to 30 cm proximally that abruptly normalized thereafter (Figure 1B). Sigmoid histology showed lamina propria hemorrhage with crypt withering and focal pseudomembranes (Figure 1C). Clostridioides difficile testing was negative. Colonic ischemia was favored. Given persistent leukocytosis and low fevers, antibiotics were started. From POD 4 to 8, platelets dropped from 142 to 11 × 109/L, and creatinine rose from 1.1 to 2.0 mg/dL (Figure 2). On POD 6, labs showed lactate dehydrogenase 1,107 U/L, indirect bilirubin 2.7 mg/dL, and undetectable haptoglobin, with hemoglobin dropping from 14.0 to 12.9 g/dL from POD 7 to 8. Peripheral smear on POD 8 showed schistocytes (Figure 3). Serum ADAMTS-13, complement levels, and heparin-induced thrombocytopenia panels were normal. Stool PCR detected shiga-toxin producing E. coli and shiga toxin 2, and stool cultures did not grow the 0157:H7 E. coli strain. Antibiotics were stopped due to the diagnosis of shiga-toxin induced hemolytic uremic syndrome (ST-HUS). The hemolysis, thrombocytopenia, and renal injury resolved after one week of supportive measures. Discussion: Infection by shiga-toxin producing E. coli (STEC), especially with shiga toxin 2 (which disrupts protein synthesis, causing apoptosis), presents with hemorrhagic colitis in 80% of cases and ST-HUS in up to 15% of cases. STEC is often acquired from contaminated food/ water. In this case, a hamburger from an external vendor was the suspected source. After a 3-day incubation period, STEC classically presents with abdominal pain and non-bloody diarrhea, followed by bloody diarrhea 1-3 days later. If it occurs, ST-HUS presents 1-2 weeks later from thrombotic microangiopathy. Antibiotics are linked to shiga toxin production by the encoding bacteriophage in the STEC chromosome.Figure 1.: Acute pseudomembranous colitis. A) Rectosigmoid wall thickening (green arrow) and perirectal fat stranding. B) Rectosigmoid colon was edematous, cyanotic, ulcerated, with luminal narrowing by flexible sigmoidoscopy. There was an abrupt transition to normal sigmoid mucosa at 30 cm from anal verge (not shown). C) Pathologic specimens from sigmoid colon showed crypt withering (blue arrow), lamina propria hemorrhage (orange arrow), and pseudomembrane (yellow arrow).Figure 2.: Clinical course of shiga-toxin induced hemolytic uremic syndrome (ST-HUS). Non-bloody diarrhea started on POD 3, followed by bloody diarrhea on POD 4. The patient’s thrombotic microangiopathy, signified by reduction in platelets and renal injury, began 3-4 days later, illustrating the characteristic delay from diarrhea onset and thrombotic microangiopathy in ST-HUS.Figure 3.: Peripheral smear. Peripheral smear was notable for schistocytes (blue arrow), suggestive of hemolysis, and markedly diminished number of platelets (green arrow). The combination of these features was consistent with thrombotic microangiopathy.

Dosimetric comparison of two different applicators and rectal retraction methods used in inverse optimization-based intracavitary brachytherapy for cervical cancer.

PurposeThe purpose of this study was to evaluate the dosimetric differences between two different applicators and rectal-retraction methods used in image-guided brachytherapy (IGBT) for locally advanced cervical cancer (LACC).Material and methodsTen patients with LACC treated with definitive chemoradiotherapy and inverse optimization-based IGBT were included in this study. In each patient, at least one fraction of IGBT was performed using tandem-ovoids (TO) with vaginal gauze packing (VGP) or tandem-ring (TR) with rectal-retractor (RR). High-risk clinical target volume (CTVHR) and intermediate-risk CTV (CTVIR) were defined as CTVs, and bladder, rectum, sigmoid, small bowel, urethra, and vaginal mucosa were defined as organs at risk (OARs). All patients received 50.4 Gy external beam radiotherapy (EBRT) in 28 fractions. After EBRT, 28 Gy high-dose-rate (HDR) IGBT in 4 fractions was delivered to central disease. A plan comparison was performed using dose-volume histogram (DVH) and treatment planning parameters for CTVs and OARs.ResultsThere were no significant differences in D90 values of CTVHR. In terms of rectum dose, TR with RR was found to be significantly better than TO with VGP (p < 0.0001 for D2cm3 and p < 0.013 for V5Gy). Although, there were no statistically significant differences in D2cm3 value of bladder, sigmoid, small bowel, upper vaginal mucosa, and urethra, mean value of D2cm3 for all defined OARs were found lower in TR than in TO. Bladder V7Gy, upper vaginal mucosa V7Gy, middle and lower vaginal mucosa D2cm3 values were all found to be significantly lower for TR than for TO (p < 0.035). CTVHR and CTVIR volumes contoured in TR were approximately 11% and 9% smaller than TO, respectively.ConclusionsThe results showed that there were no statistically differences in D90 value of CTVHR and CTVIR. However, all DVH parameters for OARs in TR with RR were found to be better than in TO with VGP.

The Potential Role of Gut-Derived Inflammation in Multiple System Atrophy.

Recent evidence suggests that Parkinson's disease (PD) is associated with intestinal microbiota dysbiosis, abnormal intestinal permeability, and intestinal inflammation. Our study aimed to determine if these gut abnormalities are present in another synucleinopathy, multiple system atrophy (MSA). In six MSA and 11 healthy control subjects, we performed immunohistochemistry studies of colonic sigmoid mucosa to evaluate the intestinal barrier marker Zonula Occludens-1 and the endotoxin-related inflammation marker Toll-like-receptor-4 expression. We also assessed colonic sigmoid mucosal and fecal microbiota compositions using high-throughput 16S ribosomal RNA gene amplicon sequencing. MSA subjects showed disrupted tight junction protein Zonula Occludens-1 structure in sigmoid mucosa tissue suggesting intestinal barrier dysfunction. The lipopolysaccharide specific inflammatory receptor Toll-like-receptor-4 was significantly higher in the colonic sigmoid mucosa in MSA relative to healthy controls. Microbiota analysis suggested high relative abundance of gram-negative, putative "pro-inflammatory" bacteria in various family and genus level taxa, from the phylum Bacteroidetes and Proteobacteria, in MSA feces and mucosa. At the taxonomic level of genus, putative "anti-inflammatory" butyrate-producing bacteria were less abundant in MSA feces. Predictive functional analysis indicated that the relative abundance of a number of genes involved in metabolism were lower in MSA feces, whereas the relative abundance of genes involved in lipopolysaccharide biosynthesis were higher in both MSA feces and mucosa compared to healthy controls. This proof-of-concept study provides preliminary evidence that like PD, MSA subjects display evidence of disrupted intestinal barrier integrity, increased marker of endotoxin-related intestinal inflammation, and pro-inflammatory colonic microbiota.

Sigmoid Cavernous Hemangiomas: Overlooked and Misdiagnosed

Gastrointestinal hemangiomas and vascular malformations such as cavernous hemangiomas are benign and rare tumors. To date, approximately 100 cases have described recto-sigmoidal hemangiomas. These entities are usually misdiagnosed and have the potential to produce massive hemorrhage, with nearly 40% mortality as previously seen cases remaining untreated. A 44-year- old African American female presented with altered bowel habits for over a year, associated with a sensation of rectal itching and burning, and intermittent rectal bleeding for the past 5 years, which she attributed to hemorrhoids. Previously, she had a recto-sigmoidectomy with end colostomy for drainage of an intra-abdominal abscess. Physical examination revealed a large ventral hernia and conjunctival pallor. Laboratory analysis included a hemoglobin level of 10.2 mg/dL, MCV 80 fL, and MCHC of 30 g/dL. CT with and without contrast revealed multiple ventral hernias. The patient underwent colonoscopy and it revealed a large, 5 cm pedunculated polyp with a 3 cm long 1.5 cm wide stalk, in the sigmoid colon. Epinephrine was injected at the base of the polyp and an application of an endoloop. Hot snaring polypectomy technique achieved complete removal of the polyp, followed by the application of two endoclips. Polyp histology revealed sigmoid mucosa and submucosa demonstrating a submucosal cavernous hemangioma without evidence of epithelial dysplasia or malignancy. Inked margins of resection were benign. To date, nearly 100 cases have been described recto-sigmoidal hemangiomas. An estimated 80% of patients present with symptoms, of which nearly 60% include intraluminal rectal bleeding, 50% with chronic iron deficiency anemia, and 17% with obstructive symptoms. Unfortunately, 80% of cases undergo a minimum of one unnecessary surgical intervention as a result of misdiagnosis, as the average delay to diagnosis is approximately 19 years. Unlike our patient, some patients endorse symptoms stemming from a possible compression or invasion of adjacent structures, such as lumbar or perianal pain, hematuria, or metrorrhagia. Although CT, MRI and endorectal ultrasound may aid in the diagnosis, colonoscopy remains the procedure of choice for detection with subsequent eradication and complete resection. Clinicians must keep the differential of recurrent painless bleeding and iron deficiency anemia in the younger population broad, as entities such as sigmoidcavernous hemangiomas may remain undiagnosed for years.Figure 1Figure 2

Does the Diversity of the Microbiome Reflect Possible Colonic Polyps in a Multi-ethnic Population?

Introduction: Colorectal cancer (CRC) is the third most common cause of malignancy-related death in the USA. The etiology of colon cancer is multifactorial; however, linkage to alterations in the gut microbiome has been reported. Hypotheses for oncogenesis include the induction of a chronic inflammatory state, cell cycle dysregulation and exposure to carcinogenic compounds, any of which might reflect a disordered microbiome. In this pilot study, we aimed to assess colonic and fecal microbiota with respect to presence of colonic polyps in a multiethnic population. Methods: Asymptomatic patients at average risk for CRC referred for screening colonoscopy were prospectively enrolled and consented. A detailed history was obtained via a standardized validated questionnaire. Stool was collected prior to (baseline, pre-preparation) and 3 weeks after colonoscopy. Biopsies were obtained from normal-appearing sigmoid mucosa in all subjects and normal-appearing mucosa within 5 cm of any polyp. DNA was purified from stool and tissue using standard extraction methods and bacterial sequencing and taxa identified using high throughput (Ilumina) 16S rRNA gene (V4 region) sequencing and analysis using the Qiime pipeline. Results: Demographics are presented in Table 1. In the 17 non-polyp controls, α and β diversity were similar in the pre-colonoscopy and the follow-up samples, 3 weeks later, indicating that the microbiota had normalized after the procedure. Analysis of gut microbiota showed lower α-diversity at baseline in subjects with polyps compared to controls, by the PD- whole-tree (p=0.014) metric. In the second sample, after polyp removal, α-diversity trended toward normalization but differences were not significant (p=0.6). Healthy colonic tissue showed higher α-diversity in subjects with adenomatous polyps compared with those with hyperplastic or no polyps (p=0.025). In subjects with polyps, α-diversity was higher in healthy tissue compared to tissue obtained within proximity of a polyp (p=0.02). There were no significant differences in β-diversity between subjects with or without polyps at any time.Table 1: Demographics and clinical features of 34 study subjectsConclusion: In our population, the lowered α-diversity (richness) prior to colonoscopy in the subjects with polyps and in tissue near a polyp suggests that the microbiota changes might be secondary to the neoplastic lesion. Larger studies are needed to question this finding in other multi-ethnic populations. Supported in part by 1R24OD01A340-01 from NIH.

Successful conservative management of diffuse cavernous hemangioma of the rectum

Diffuse cavernous hemangioma of the rectum (DCHR) is a relatively rare disease. A 40-year-old man presented with long-standing lower abdominal discomfort and hematuria. At the time of hospitalization, his vital signs and hemoglobin level were normal. Colonoscopy showed markedly dilated blood vessels in the sigmoid mucosa, which was confirmed on magnetic resonance imaging and computed tomography as cavernous hemangioma. Without surgery, there have been no signs of progression of DCHR during a 3-year follow-up period.

Associations of sigmoid colon mucosal mast cells with bowel symptoms and psychological status in patients with irritable bowel syndrome with diarrhea

To investigate the bowel symptoms and psychological status of patients with irritable bowel syndrome (IBS) with diarrhea (IBS-D), and to verify whether sigmoid colon mucosal mast cells (MCs) and their activation have effect on the symptoms and psychological status of IBS-D patients. Patients meeting Rome Ⅲ diagnostic and subtyping criteria of IBS-D who visited the outpatient clinic of gastroenterology of Peking Union Medical College Hospital were consecutively enrolled between July 2009 and June 2012. IBS symptoms questionnaire was completed using face-to-face interview, and Hamilton Anxiety Scale (HAMA)/ Hamilton Depression Scale (HAMD) were administrated to evaluate psychological status, both by well-trained investigators. Mast cell tryptase monoclonal antibody was used for immunohistochemical staining to detect MCs and degranulated MCs in mucosal biopsy of sigmoid colon. MCs and degranulated MCs were blindly counted by a senior pathologist, and presented as number of cells in high power field (HPF) and percentage of activated MCs. Correlation analysis was performed using Spearman rank correlation analysis. Ninety-seven patients with IBS-D were enrolled in this study, with mean age of (44±11) years. 70.10%(68 cases) of the IBS-D patients had comorbid anxiety and/or depression. The median total numbers of MCs, activated MCs, and percentage of activated MCs in sigmoid mucosa were 11.60 (7.09)/HPF, 2.00 (1.40) /HPF, and 17.50% (10.90%), respectively. Patients having abdominal pain/discomfort before bowel movement "every day with intermediate to high severity" had significantly larger numbers of total MCs in sigmoid colon compared with those with pain or discomfort "not every day and mild" [13.80(4.85)vs 7.60(5.90)/HPF, P=0.019]; the patient having "frequent" urge to have a bowel movement and mushy stools showed significantly higher percentage of activated MCs in sigmoid colon mucosa compared to those having the symptoms "some of the time" [18.75%(9.12%) vs 14.50%(13.14%), P=0.031; 21.33%(7.43%)vs 11.51%(10.65%)vs 18.42%(8.61%), P=0.030]. There was a positive correlation between the bowel movement during IBS-D onset and the percentage of activated MCs (r=0.221, P=0.030). There were no statistically significant differences in the total number of MCs and percentage of activated MCs between the patients with anxiety/depression and those without anxiety/depression (P=0.255, P=0.315). Scores of HAMA and HAMD were found not correlated with either total MCs number or percentage of activated MCs in sigmoid colon mucosa(all P>0.05). The majority of IBS-D patients had comorbid anxiety and/or depression. The total number and activation status of MCs in sigmoid colon mucosa might be related with some intestinal symptoms in IBS-D patients. Psychological disorders might influence the pathogenesis and regression of IBS-D through brain-gut axis other than MCs in sigmoid colon mucosa.

Lower Neighborhood Socioeconomic Status Associated with Reduced Diversity of the Colonic Microbiota in Healthy Adults.

In the United States, there are persistent and widening socioeconomic gaps in morbidity and mortality from chronic diseases. Although most disparities research focuses on person-level socioeconomic-status, mounting evidence suggest that chronic diseases also pattern by the demographic characteristics of neighborhoods. Yet the biological mechanisms underlying these associations are poorly understood. There is increasing recognition that chronic diseases share common pathogenic features, some of which involve alterations in the composition, diversity, and functioning of the gut microbiota. This study examined whether socioeconomic-status was associated with alpha-diversity of the colonic microbiota. Forty-four healthy adults underwent un-prepped sigmoidoscopy, during which mucosal biopsies and fecal samples were collected. Subjects’ zip codes were geocoded, and census data was used to form a composite indicator of neighborhood socioeconomic-status, reflecting household income, educational attainment, employment status, and home value. In unadjusted analyses, neighborhood socioeconomic-status explained 12–18 percent of the variability in alpha-diversity of colonic microbiota. The direction of these associations was positive, meaning that as neighborhood socioeconomic-status increased, so did alpha-diversity of both the colonic sigmoid mucosa and fecal microbiota. The strength of these associations persisted when models were expanded to include covariates reflecting potential demographic (age, gender, race/ethnicity) and lifestyle (adiposity, alcohol use, smoking) confounds. In these models neighborhood socioeconomic-status continued to explain 11–22 percent of the variability in diversity indicators. Further analyses suggested these patterns reflected socioeconomic variations in evenness, but not richness, of microbial communities residing in the sigmoid. We also found indications that residence in neighborhoods of higher socioeconomic-status was associated with a greater abundance of Bacteroides and a lower abundance of Prevotella, suggesting that diet potentially underlies differences in microbiota composition. These findings suggest the presence of socioeconomic variations in colonic microbiota diversity. Future research should explore whether these variations contribute to disparities in chronic disease outcomes.

Comparative effect of electroacupuncture and moxibustion on the expression of substance P and vasoactive intestinal peptide in patients with irritable bowel syndrome

ObjectiveTo compare the impacts of electroacupuncture (EA) and moxibustion (Mox) on the primary gastrointestinal symptoms and the expressions of colonic mucosa-associated neuropeptide substance P (SP) and vasoactive intestinal peptide (VIP) in patients with either diarrhea-predominant or constipation-predominant irritable bowel syndrome (IBS-D and IBS-C, respectively). MethodsEighty-five IBS patients were randomly allocated to the EA and Mox groups. Zusanli (ST 36) and Shangjuxu (ST 37) were selected as acupoints for electroacupuncture or warm moxibustion treatment once a day for 14 consecutive days. Before and after the treatment sessions, a Visual Analog Pain Scale and the Bristol Stool Form Scale were used to evaluate gastrointestinal symptoms. There were four dropout cases, leaving 81 participants (41 with IBS-D and 40 with IBS-C) who volunteered to undergo colonoscopy before and after the treatment sessions. During colonoscopy, sigmoid mucosa were collected to detect SP and VIP expression using immunohistochemistry assay. ResultsBoth EA and Mox treatments were effective at relieving abdominal pain in IBS-D and IBS-C patients. However, Mox was more effective at reducing diarrhea in IBS-D patients, whereas EA was more effective at improving constipation in IBS-C patients. EA and Mox treatments both down-regulated the abnormally increased SP and VIP expression in the colonic mucosa, with no significant difference shown between the two treatments. ConclusionBoth EA and Mox treatments are effective at ameliorating gastrointestinal symptoms by reducing SP and VIP expression in the colonic mucosa of IBS patients.

Colonic bacterial composition in Parkinson's disease.

We showed that Parkinson's disease (PD) patients have alpha-synuclein (α-Syn) aggregation in their colon with evidence of colonic inflammation. If PD patients have altered colonic microbiota, dysbiosis might be the mechanism of neuroinflammation that leads to α-Syn misfolding and PD pathology. Sixty-six sigmoid mucosal biopsies and 65 fecal samples were collected from 38 PD patients and 34 healthy controls. Mucosal-associated and feces microbiota compositions were characterized using high-throughput ribosomal RNA gene amplicon sequencing. Data were correlated with clinical measures of PD, and a predictive assessment of microbial community functional potential was used to identify microbial functions. The mucosal and fecal microbial community of PD patients was significantly different than control subjects, with the fecal samples showing more marked differences than the sigmoid mucosa. At the taxonomic level of genus, putative, "anti-inflammatory" butyrate-producing bacteria from the genera Blautia, Coprococcus, and Roseburia were significantly more abundant in feces of controls than PD patients. Bacteria from the genus Faecalibacterium were significantly more abundant in the mucosa of controls than PD. Putative, "proinflammatory" Proteobacteria of the genus Ralstonia were significantly more abundant in mucosa of PD than controls. Predictive metagenomics indicated that a large number of genes involved in metabolism were significantly lower in the PD fecal microbiome, whereas genes involved in lipopolysaccharide biosynthesis and type III bacterial secretion systems were significantly higher in PD patients. This report provides evidence that proinflammatory dysbiosis is present in PD patients and could trigger inflammation-induced misfolding of α-Syn and development of PD pathology.

PTH-074 Drug therapies in ulcerative colitis influence the expression of micrornas and cytokines in the sigmoid mucosa in anex vivomodel

<h3>Introduction</h3> UC is thought to be characterised by a TH-2 predominant immune response. MicroRNAs (miRNA) are short 19–23 nucleotide long strands of single-stranded RNA which modify post transcriptional gene expression by degrading or inhibiting translation of mRNA. miR31 and miR155 are miRNA that directly and indirectly target genes in the IL-13 dependant pathway. Confounding factors in current IBD miRNA studies include disease activity and medication. The specific function of miRNAs in UC, how they might be influenced by medications, and potential clinical utility is yet to be investigated. In this study we aim to investigate how the expression of a number of miRNA and mRNA is influenced by medications and then explore these findings further using a human ex-vivo culture system. <h3>Method</h3> Sigmoid biopsies from 40 patients with untreated active and inactive UC, normal controls, and patients treated with 5-ASA, Azathioprine, Infliximab and Adalimumab were frozen in liquid nitrogen. RT-qPCR was performed to analyse the relative expression of miRNA and mRNA. An <i>ex vivo</i>model was designed. 6 biopsies were taken from 8 patients with active untreated UC, with a Mayo score of 6 or greater. Biopsies were placed in tissue culture media in 6 separate wells with either no treatment, 5-ASA, 6-TG, Infliximab, or Adalimumab, were incubated for 24hrs at 37^oC. RT-qPCR was performed to assess expression of miR31 and miR155 and mRNA of CCL18 and SOCS1 that are important in the TH-2 pathway. <h3>Results</h3> miR31 and miR155 expression was increased in patients with untreated active and inactive UC compared to normal mucosa. CCL18 expression was also increased in untreated active disease compared to inactive and normal mucosa. Azathioprine reduced miR31, miR155 and CCL18 expression to levels seen in quiescent disease. Infliximab treatment reduced miR155, and CCL18 expression to that of inactive disease, which was not seen in Adalimumab. Anti-TNFs did not reduce miR31 expression to the level of untreated inactive disease. 5ASAs had no impact on expression of the miRNA nor mRNA studied. <i>Ex vivo</i>treated samples confirmed down regulation of miR31 was confined to 6-TG and not seen in anti-TNF treatment, and that CCL18 expression is also reduced by 6-TG and Infliximab, but not Adalimumab. <h3>Conclusion</h3> Our data confirmed that miR31 miR155 and CCL18 are increased in active UC compared to normal mucosa and inactive disease. We demonstrate that thiopurine therapy reduces expression of miR31 and CCl18 in contrast to anti-TNF agents. This model may have utility in increasing understanding of the mechanisms of action of IBD medications. It highlights the importance of controlling for medication when analysing miRNA expression in whole tissue biopsies. <h3>Disclosure of interest</h3> None Declared.

T lymphocyte density and distribution in human colorectal mucosa, and inefficiency of current cell isolation protocols.

Mucosal tissues are critical immune effector sites containing complex populations of leukocytes in a tissue microenvironment that remains incompletely understood. We identify and quantify in human distal colorectal tissue absolute mucosal CD3+ lymphocytes, including CD4+ and CD8+ subsets, by direct visualization using immunohistochemistry (IHC), immunofluorescence (IF), and an automated counting protocol (r2=0.90). Sigmoid and rectal mucosal tissues are both densely packed with T lymphocytes in the mucosal compartment. Both compartments had similar densities of CD3+ T lymphocytes with 37,400 ± 2,801 cells/mm3 and 33,700 ± 4,324 cell/mm3, respectively. Sigmoid mucosa contained 57% CD3+CD4+ and 40% CD3+CD8+ T lymphocytes which calculates to 21,300 ± 1,476/mm3 and 15,000 ± 275/mm3 T lymphocytes, respectively. Rectal mucosa had 57% CD3+CD4+ and 42% CD3+CD8+ or 21,577 ± 332, and 17,090 ± 1,206 cells/mm3, respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm3 and 2,708 ± 245/mm3 CD4+ and CD8+ T lymphocytes. These data suggest only ~20.7% recovery compared to IHC results for these markers. Further studies will determine if this reflects a selective bias in only CD3+, CD4+ and CD8+ T cells or can be generalized to all flow-analyzed cells from mucosal tissues for phenotyping and functional testing.

Mo1870 Measuring Anti-Vinculin and Anti-CdtB Antibodies in Two Animal Models of Post-Infectious IBS

Diverticulosis is a common GI condition among older adults in the United States. However, limited information exists on the etiologic risk factors for diverticulosis. We hypothesized that the gut microbiome and chronic inflammation contribute to the development of diverticulosis.We evaluated the association between gut bacteria, mucosal inflammation and diverticulosis in a case-control study. Methods:Participants (n=196) were consented patients who underwent screening colonoscopy at UNC Hospitals. Subjects were classified as case if diverticula were present or control if no diverticula were present. The abundance of specific bacteria in the sigmoid mucosa was determined by qPCR based on the bacterial 16s rRNA gene for Bacteroides, Bifidobacterium, Clostridium, E. coli, Lactobacillus, and Eubacteria.Mucosal mRNA gene expression of cytokines of IL-6, IL-10, IL-17, and TNF-α and housekeeping gene HMBS were assessed by qRT-PCR. Immune cells in the lamina propria were stained for CD4, CD8, CD57 and Mast cell tryptase (MCT) by immunohistochemistry (IHC). The IHC stained sections were digitally imaged using the Aperio ScanScope XT. Analysis of biomarkers was performed using Definiens Tissue Studio software with the Composer_ Nuclei_and_Simulated_Cells algorithm. The percent cells with strong (+3), medium (+2) and weak (+1) positive signal, was used to compare biomarker levels among tissue samples. Statistical analysis included t-test and logistic regression to compare cases and controls as well as Spearman correlation to assess association between bacteria and inflammation. Results:We observed a higher relative abundance of Clostridium, Bacteroides, Bifidobacterium, E. coli, and Eubacteria in cases compared to controls. Cases had lower density of CD8 compared to controls. Although the cytokine gene expression levels of IL-6, IL-10, IL-17, and TNF-α did not differ between cases and controls, there was a significant positive correlation between CD57 expression and IL-6 (r=0.23, p=0.03), CD57 and TNFα (r0.33, p=0.001) and CD57 and IL-10 (r=0.22, p= 0.03) among cases. The association between bacterial abundance and immune cell density also varied depending on case-control status. Among cases, MCT expression showed positive correlations with Bacteroides (r=0.21, p= 0.05), Clostridium (r=0.18, p=0.08) and inverse correlation with Bifidobacteria (r=-0.26, p= 0.01). Among controls, CD57 expression was inversely correlated with Lactobacillus (r=0.19, p=0.06). Conclusions: Our findings suggest that gut bacteria and inflammation are associated with diverticulosis. This data combined with further research could have clinical implications for the prevention and treatment of diverticulosis.

Mo1727 Drug Therapies in Ulcerative Colitis Influence the Expression of MicroRNAs and Cytokines in the Sigmoid Mucosa in an Ex Vivo Model

Introduction UC is thought to be characterised by a TH-2 predominant immune response. MicroRNAs (miRNA) are short 19–23 nucleotide long strands of single-stranded RNA which modify post transcriptional gene expression by degrading or inhibiting translation of mRNA. miR31 and miR155 are miRNA that directly and indirectly target genes in the IL-13 dependant pathway. Confounding factors in current IBD miRNA studies include disease activity and medication. The specific function of miRNAs in UC, how they might be influenced by medications, and potential clinical utility is yet to be investigated. In this study we aim to investigate how the expression of a number of miRNA and mRNA is influenced by medications and then explore these findings further using a human ex-vivo culture system. Method Sigmoid biopsies from 40 patients with untreated active and inactive UC, normal controls, and patients treated with 5-ASA, Azathioprine, Infliximab and Adalimumab were frozen in liquid nitrogen. RT-qPCR was performed to analyse the relative expression of miRNA and mRNA. An ex vivo model was designed. 6 biopsies were taken from 8 patients with active untreated UC, with a Mayo score of 6 or greater. Biopsies were placed in tissue culture media in 6 separate wells with either no treatment, 5-ASA, 6-TG, Infliximab, or Adalimumab, were incubated for 24hrs at 37 o C. RT-qPCR was performed to assess expression of miR31 and miR155 and mRNA of CCL18 and SOCS1 that are important in the TH-2 pathway. Results miR31 and miR155 expression was increased in patients with untreated active and inactive UC compared to normal mucosa. CCL18 expression was also increased in untreated active disease compared to inactive and normal mucosa. Azathioprine reduced miR31, miR155 and CCL18 expression to levels seen in quiescent disease. Infliximab treatment reduced miR155, and CCL18 expression to that of inactive disease, which was not seen in Adalimumab. Anti-TNFs did not reduce miR31 expression to the level of untreated inactive disease. 5ASAs had no impact on expression of the miRNA nor mRNA studied. Ex vivo treated samples confirmed down regulation of miR31 was confined to 6-TG and not seen in anti-TNF treatment, and that CCL18 expression is also reduced by 6-TG and Infliximab, but not Adalimumab. Conclusion Our data confirmed that miR31 miR155 and CCL18 are increased in active UC compared to normal mucosa and inactive disease. We demonstrate that thiopurine therapy reduces expression of miR31 and CCl18 in contrast to anti-TNF agents. This model may have utility in increasing understanding of the mechanisms of action of IBD medications. It highlights the importance of controlling for medication when analysing miRNA expression in whole tissue biopsies. Disclosure of interest None Declared.

Mo1869 Gut Bacteria and Inflammation Are Associated With Diverticulosis

Diverticulosis is a common GI condition among older adults in the United States. However, limited information exists on the etiologic risk factors for diverticulosis. We hypothesized that the gut microbiome and chronic inflammation contribute to the development of diverticulosis.We evaluated the association between gut bacteria, mucosal inflammation and diverticulosis in a case-control study. Methods:Participants (n=196) were consented patients who underwent screening colonoscopy at UNC Hospitals. Subjects were classified as case if diverticula were present or control if no diverticula were present. The abundance of specific bacteria in the sigmoid mucosa was determined by qPCR based on the bacterial 16s rRNA gene for Bacteroides, Bifidobacterium, Clostridium, E. coli, Lactobacillus, and Eubacteria.Mucosal mRNA gene expression of cytokines of IL-6, IL-10, IL-17, and TNF-α and housekeeping gene HMBS were assessed by qRT-PCR. Immune cells in the lamina propria were stained for CD4, CD8, CD57 and Mast cell tryptase (MCT) by immunohistochemistry (IHC). The IHC stained sections were digitally imaged using the Aperio ScanScope XT. Analysis of biomarkers was performed using Definiens Tissue Studio software with the Composer_ Nuclei_and_Simulated_Cells algorithm. The percent cells with strong (+3), medium (+2) and weak (+1) positive signal, was used to compare biomarker levels among tissue samples. Statistical analysis included t-test and logistic regression to compare cases and controls as well as Spearman correlation to assess association between bacteria and inflammation. Results:We observed a higher relative abundance of Clostridium, Bacteroides, Bifidobacterium, E. coli, and Eubacteria in cases compared to controls. Cases had lower density of CD8 compared to controls. Although the cytokine gene expression levels of IL-6, IL-10, IL-17, and TNF-α did not differ between cases and controls, there was a significant positive correlation between CD57 expression and IL-6 (r=0.23, p=0.03), CD57 and TNFα (r0.33, p=0.001) and CD57 and IL-10 (r=0.22, p= 0.03) among cases. The association between bacterial abundance and immune cell density also varied depending on case-control status. Among cases, MCT expression showed positive correlations with Bacteroides (r=0.21, p= 0.05), Clostridium (r=0.18, p=0.08) and inverse correlation with Bifidobacteria (r=-0.26, p= 0.01). Among controls, CD57 expression was inversely correlated with Lactobacillus (r=0.19, p=0.06). Conclusions: Our findings suggest that gut bacteria and inflammation are associated with diverticulosis. This data combined with further research could have clinical implications for the prevention and treatment of diverticulosis.