Colon Tissue Samples Research Articles

Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content

BackgroundHypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules’ (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect.MethodsLINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules’ in situ level (n = 40).ResultsSignificantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes—which met our analysing criteria—showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05).ConclusionOur results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

Differential diagnosis of acute and chronic colitis in mice by optical coherence tomography.

The differential diagnosis of acute and chronic colitis remains a common clinical problem. Optical coherence tomography (OCT) is a non-invasive, high-resolution imaging technique that can be used to measure morphological changes in the intestinal wall and estimate intestinal inflammation. We aimed to conduct an ex vivo experiment on a mouse model investigate the value of OCT as a tool for the differential diagnosis of acute and chronic colitis. Mice were administered dextran sulfate sodium salt (DSS) to construct acute and chronic colitis models. Acutely- and chronically-affected intestinal walls were scanned by OCT, and then the scanned colonic tissue samples were stained with hematoxylin and eosin (HE). Structural and morphological changes indicating inflammation in the intestinal wall were evaluated in the HE sections and OCT images using different parameters. The parameters were used in one-way analysis of variance (ANOVA) to screen for a differential diagnosis of acute or chronic colitis. For the HE sections, the angle of the mucosal folds, length of the basilar part, and submucosal height and area were statistically significant parameters in the comparisons between the mice with acute colitis and the control-group mice (P<0.05). In the comparisons between chronic colitis mice and control-group mice, the angle of the mucosal folds, length of the basilar part, submucosal height and area, muscularis thickness, submucosal height + muscularis thickness, and mucosal thickness were statistically significant parameters (P<0.05). Finally, in the comparisons between acute colitis mice and those with chronic colitis, the angle of the mucosal folds, submucosal height and area, muscularis thickness, submucosal height + muscularis thickness, and mucosal thickness were statistically significant parameters (P<0.05). For the OCT images, only the length of the basilar part and submucosal height + muscularis thickness were statistically significant parameters between the acute colitis mice and control-group mice (P<0.05). The length of the basilar part and submucosal height + muscularis thickness were statistically significant between chronic colitis mice and control-group mice (P<0.05). In the comparisons between acute colitis mice and those with chronic colitis, only submucosal height + muscularis thickness was a statistically significant parameter (P<0.05). Certain intestinal wall parameters in OCT can be used to make a differential diagnosis between acute and chronic colitis possible. This study contributes to constructing a potential diagnostic system for evaluating colorectal inflammation using OCT.

MS-HRM protocol: a simple and low-cost approach for technical validation of next-generation methylation sequencing data.

DNA methylation is a fundamental epigenetic process and have a critical role in many biological processes. The study of DNA methylation at a large scale ofgenomiclevels is widely conducted by several techniques that are next-generation sequencing (NGS)-based methods. Methylome data revealed by DNA methylation next-generation sequencing (mNGS), should be always verified by another technique which they usually have a high cost. In this study, we offered a low-cost approach to corroborate the mNGS data. In this regard, mNGS was performed on 6 colorectal cancer (case group) and 6 healthy individual colon tissue (control group) samples. An R-script detected differentially methylated regions (DMRs), was further validated by high resolution melting (MS-HRM) analysis. After analyzing the data, the algorithm found 194 DMRs. Two locations with the highest level of methylation difference were verified by MS-HRM, which their results were in accordance with the mNGS. Therefore, in the present study, we suggested MS-HRM as a simple, accurate and low-cost method, useful for confirming methylation sequencing results.

FuZhengHuaYuJiangZhuTongLuoFang Prescription Modulates Gut Microbiota and Gut-Derived Metabolites in UUO Rats.

BackgroundAlteration of intestinal flora and metabolites is closely related to chronic kidney disease (CKD) across early to advanced stages. FuZhengHuaYuJiangZhuTongLuoFang prescription (FZHY) is a Chinese herb that has been proven to effectively treat CKD, but the underlying mechanism is not clear.MethodsRats were subjected to intragastric treatment with FZHY 7, 14, and 21 days after unilateral ureteral obstruction (UUO) surgery, and kidney tissue, colon tissue, serum, and stool samples were collected.ResultsFZHY treatment effectively ameliorated UUO-induced renal function loss, renal injury and renal fibrosis, and colon tissue damage and fibrosis on day 7. The results of 16S flora analysis (day 7) showed that, compared with the UUO group, both the FZHY group and the sham group showed decreased levels of g_Monoglobus, g_Papillibacter, g_Eubacterium_nodatum, and g_Family_XIII_AD3011. Additionally, FZHY obviously induced the reduction of serum citrulline, glycoursodeoxycholic acid, 23-nordeoxycholic acid, 7-ketodeoxycholic acid, kahweol, lipoid B4, 4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-2-methyl-1,3-thiazole, taurolithocholic acid sodium salt, indoline-2-carboxylic acid, 5(S),15(S)-diHETE, and others and the increase of bilirubin, asparagine, and others, which were positively associated with the above four candidate bacteria. Moreover, FZHY increased the levels of ZO-1, occludin, and claudin-1 in the colonic mucosa and reduced the levels of CRP, TNF-α, IL-6, and IL-1 in the serum and LN, FN, Col-I, and Col-III in the tubulointerstitium of UUO rats on day 7.ConclusionOur study revealed that FZHY reduced kidney damage at the early stage of CKD by regulating the above four candidate bacteria biomarkers and gut-derived harmful metabolites, inhibiting the inflammation response and tubulointerstitial fibrosis, providing deep insight into CKD therapeutic strategy.

Diacerein attenuates 1,2‐dimethylhydrazine induced colon cancer progression in rats: Modulation of IL‐6/STAT3/ lncRNA HOTAIR pathway

Diacerein (DIA) is an FDA approved anti‐inflammatory drug. Over the past few years, several studies reported its anti‐proliferative action against different types of cancers. However, the anti‐tumor effect against colon cancer remains obscure. Colon cancer is one of the top solid tumors diagnosed around the world. The current study aims to investigate DIA‐associated anti‐tumor effect In vivo and invitro and to further elaborate the potential underling molecular mechanism. In vitro, human Caco‐2 colon cancer cell lines were treated with serial dilution of DIA and were evaluated for cell viability and apoptotic percentages via MTT and Annexin V assays, respectively. In vivo, subcutaneous injection of 1,2‐dimethylhydrazine (DMH) (20 mg/kg for 15 weeks) was used to induce colon carcinogenesis. DIA oral administration at dose of 50 mg/kg/day for additional of 8 weeks was tested. This therapeutic regimen showed significant anti‐tumor effect and improvement of colonic tissue structure as evident by histopathological examination. Mechanistically, colon tissue samples were collected for ELISA and immunohistochemical analyses. Data showed that DIA treatment reduced colonic inflammatory status as evident by reduction of TLR4, NF‐κB and TNF‐α protein levels. In addition, DIA treatment reduced angiogenesis levels as evident by downregulation of VEGF tissue expression levels. Furthermore, DIA treatment modulated Wnt/β‐catenin pathway, induced caspase‐3 expression and interrupted IL‐6/STAT3/lncRNA HOTAIR axis. Altogether, this study provides evidence that DIA treatment might be beneficial in controlling colon cancer progression via multi pathways targeting mechanism.

Intestinal epithelial specific AhR deficient mice exhibit increased severity of experimentally induced chronic colitis

Inflammatory Bowel Diseases (IBD), including Crohn’s Disease (CD) and ulcerative colitis (UC), are associated with chronic inflammation of the gastrointestinal tract. While it is a multifactorial disease, recently the aryl hydrocarbon receptor (AhR) has been identified as a susceptibility gene for IBD. AhR is a ubiquitously expressed transcription factor and its activation results in xenobiotic detoxification by CYP1A1 and CYP1B1, as well as induction of anti‐inflammatory pathways such as IL‐10 and IL‐22. In the gut, AhR is vital in maintaining immune cell populations, proliferation, and motility. Previous studies utilizing constitutive knock out of AhR in IECs showed exacerbated inflammation in acute DSS model. How intestinal epithelial cell (IEC) AhR deficiency impacts chronic colitis resembling human IBD is not yet known. Here, we investigated the effect of removing of IEC AhR in an inducible manner utilizing DSS induced chronic colitis (resembling Th1/Th17 and Th2 infiltration as seen in CD patients) and elucidated underlying mechanisms.MethodsAhRfl/fl x Villin‐CreERT mice received intraperitoneal injections of Tamoxifen (AhRΔIECKO) to induce knockout or the vehicle (WT) for 5 days. The mice were given water or 1.0% DSS for 5 weeks, alternating weekly with water. Distal colon tissue samples were taken for myeloperoxidase (MPO) assay, immunostaining, and histology to assess severity of inflammation. Additionally, distal colon mucosa samples were collected for mRNA and protein expression. Serum was used to assess levels of LPS through ELISA.ResultsAhRΔIECKOmice given DSS had increased severity of colitis as evidenced by i) a significant decrease in the colon length when compared to the WT given DSS; ii) an increase in mRNA expression of pro‐inflammatory cytokines, IFN‐ γ and IL‐1□; iii) an increase in MPO activity in the distal colon; iv) a significant increase in the spleen to body weight ratio; and v) a significant increase in histological scores. Interestingly, DSS treatment caused an increase in lipopolysaccharide (LPS) in the serum that was significantly higher in AhRΔIECKO as compared to WT indicative of increased paracellular permeability. There were no significant differences observed in the protein expression of Occludin, Claudin‐2, and ZO‐1 between AhRΔIECKO and WT DSS groups, however AhR KO mice given DSS had significantly increased phosphorylated ERK1/2 (pERK normalized to total ERK) levels which may contribute to the increased permeability and severity of inflammation.ConclusionDeletion of AhR from intestinal epithelial cells increases severity of chronic colitis associated with increased permeability possibly through ERK1/2 dependent mechanisms.

Colonic cryptococcosis presenting with chronic diarrhea in a person with advanced human immunodeficiency virus disease: a case report

Cryptococcus neoformans infection usually occurs in patients with advanced human immunodeficiency virus (HIV) infection or with a CD4 T lymphocyte count of &lt;100 cells/μL. Pulmonary and central nervous system infections are the most frequently encountered forms of cryptococcosis; however, colonic cryptococcosis is uncommon. We describe the case of a 41-year-old antiretroviral-naïve man with HIV infection diagnosed eight years prior and intermittent diarrhea for 4 months who presented to the emergency department with a 1-day history of low-grade fever and confusion. Brain magnetic resonance imaging and cerebrospinal fluid analysis revealed normal results; however, he was diagnosed with Pneumocystis jirovecii pneumonia based on chest computed tomography and bronchoalveolar lavage analysis. Trimethoprim-sulfamethoxazole administration was initiated followed by antiretroviral treatment. Although his condition gradually improved, he developed fever and abdominal discomfort, and the diarrhea worsened. Endoscopy revealed a small ulcer in the distal transverse colon. Histopathological examination of a colon tissue sample revealed cryptococcal infection. He improved substantially during liposomal amphotericin B and fluconazole treatment. We encountered a rare case of colonic cryptococcosis that caused chronic diarrhea in a patient with advanced HIV infection. Colonic cryptococcosis should be considered when patients with acquired immune deficiency syndrome present with gastrointestinal symptoms.

Possible Curative Effects of Boric Acid and Bacillus clausii Treatments on TNBS-Induced Ulcerative Colitis in Rats.

Crohn's disease (CD) and ulcerative colitis (UC) are two chronic relapsing inflammatory bowel diseases (IBD). Although there are several treatment options available to improve the symptoms of IBD patients, there is no effective treatment that provides a definitive solution. In the present study, we aim to investigate the antioxidative/anti-inflammatory effects of oral administration of boric acid and Bacillus clausii in a rat trinitrobenzenesulfonic acid (TNBS)-induced colitis model. The effects of boric acid and B. clausii were examined in serum and colon tissues with the help of some biochemical and histological analyses. Elevated inflammation and oxidative damage were found in the blood and colon tissue samples in the TNBS-induced group according to the complete blood count (CBC), tumor necrosis factor (TNF) alpha, interleukin-35 (IL-35), malondialdehyde (MDA), glutathione peroxidase (GPx), myeloperoxidase (MPO), nitric oxide (NO), and histological findings. Particularly, the highest IL-35 level (70.09 ± 12.62ng/mL) in the combined treatment group, highest catalase activity (5322 ± 668.1 U/mg protein) in the TNBS-induced group, and lower relative expression of inducible nitric oxide synthase in the TNBS-induced group than the control group were striking findings. According to our results, it can be concluded that boric acid showed more curative effects, even if B. clausii probiotics was partially ameliorative.

Deficiency in glutathione synthesis and reduction contributes to the pathogenesis of colitis-related liver injury.

Liver disease is the most common extra-intestinal manifestation of ulcerative colitis (UC), but the underlying pathogenesis is still not clarified. It is well accepted that the occurrence of UC-related liver disease has close correlation with immune activation, intestinal bacterial liver translocation, inflammatory cytokine storm, and the disturbance of bile acid circulation. The occurrence of UC-related liver disease makes the therapy difficult, therefor study on the pathogenesis of UC-related liver injury is of great significance for its prevention and treatment. Glutathione (GSH) shows multiple physiological activities, such as free radical scavenging, detoxification metabolism and immune defense. The synthesis and the oxidation-reduction all contribute to GSH antioxidant function. It is reported that the deficiency in hepatic GSH antioxidant function participates in multiple liver diseases, but whether it participates in the pathogenesis of UC-related liver injury is still not clear. This study aims to investigate the feature and underlying mechanism of GSH synthesis and oxidation-reduction function during the development of UC, which will provide useful information for the pathogenesis study on UC-related liver injury. UC model was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS)-ethanol solution (5 mg/0.8 mL per rat, 50% ethanol) via intra-colonic administration in rats, and the samples of serum, liver, and colon tissue of rats were collected at the 3rd, 5th, and 7th days post TNBS. The severity degree of colitis was evaluated by measuring the disease activity index, colonic myeloperoxidase activity, and histopathological score, and the degree of liver injury was evaluated by histopathological score and the serum content of alanine aminotransferase. Spearman correlation analysis was also conducted between the degree of colonic lesions and index of hepatic histopathological score as well as serum aspartate aminotransferase level to clarify the correlation between liver injury and colitis. To evaluate the hepatic antioxidant function of GSH in UC rats, hepatic GSH content, enzyme activity of GSH peroxidase (GSH-Px), and GSH reductase (GR) were determined in rats at the 3rd, 5th, and 7th days post TNBS, and the protein expressions of glutamine cysteine ligase (GCL), GSH synthase, GSH-Px, and GR in the liver of UC rats were also examined by Western blotting. Compared with the control, the disease activity index, colonic myeloperoxidase activity, and histopathological score were all significantly increased at the 3rd, 5th, and 7th days post TNBS (all P<0.01), the serum aspartate aminotransferase level and hepatic histopathologic score were also obviously elevated at the 7th day post TNBS (all P<0.05). There was a significant positive correlation between the degree of liver injury and the severity of colonic lesions (P=0.000 1). Moreover, compared with the control, hepatic GSH content and the activity of GSH-Px and GR were all significantly decreased at the 3rd and 5th days post TNBS (P<0.05 or P<0.01), and the protein expressions of GCL, GSH-Px, and GR were all obviously down-regulated at the 3rd, 5th, and 7th days post TNBS (P<0.05 or P<0.01). There is a significant positive correlation between the degree of liver injury and the severity of colonic lesions, and the occurrence of reduced hepatic GSH synthesis and decreased GSH reduction function is obviously earlier than that of the liver injury in UC rats. The reduced hepatic expression of enzymes that responsible for GSH synthesis and reduction may contribute to the deficiency of GSH synthesis and oxidation-reduction function, indicating that the deficiency in GSH antioxidant function may participate in the pathogenesis of UC related liver injury.

Pentosan Polysulfate Sodium augments the therapeutic effect of 5-Aminosalicylic Acid in DSS colitis model; the role of IL-35 expression

Ulcerative colitis (UC) primarily affects the mucosa of the distal colon. Dysregulated immune response in genetically-prone persons is claimed to be responsible for chronic intestinal inflammation. This study aimed to explore the efficacy and the hematological effects of pentosan polysulfate sodium (PPS) in a dextran sulfate sodium (DSS)-induced colitis model. Forty C57BL/6 female mice were equally divided into five groups: control group, DSS-colitis group, DSS-colitis treated with 5-aminosalicylic acid, DSS-colitis treated with PPS, and DSS-colitis treated with both drugs. Disease activity index (DAI) and colon length were calculated. Colonic IL-6 and IL-35 levels were assayed by ELISA. IL-35 gene expression was evaluated by qRT-PCR. Colon tissue samples were examined by H&E stain and immunohistochemistry (IHC) of Ki67. The colitis group subjected to combined treatment showed the best outcome with significant improvement of DAI and increased colon length. Colonic IL-6 was significantly lower in both PPS- and combination-treated groups accompanied by a significantly higher IL-35 level and its EBI3 subunit mRNA expression. However, the PPS-treated colitis group showed higher gene expression of IL-35 EBI3 subunit by 1.5-fold compared with the combined group. The colon mucosa and crypts were significantly preserved in mice treated with both drugs with the best Ki67 positive cell density. PPS is a safe and promising drug in the treatment of UC as it exerted the best positive effect on the anti-inflammatory IL-35 level and gene expression. However, superior improvement of DAI was seen when PPS was added to ASA with a greater mucosal proliferation and repair.

Indoleamine-2,3-dioxygenase-related anti-inflammatory effects of 3-aminobenzamide and infliximab in experimental colitis.

This study aimed to investigate the presence of indoleamine-2,3-dioxygenase and bacterial translocation after the administration of 3-aminobenzamide and infliximab in the TNBS model of rat colitis. The study group was divided into five categories as follows: group 1: (control), group 2: colitis+saline, group 3: colitis+3-aminobenzamide, group 4: colitis+infliximab, and group 5: colitis+3-aminobenzamide+infliximab. Intestinal mesenteric cultures were incubated on specific agar media plates under aerobic and anaerobic conditions, bacterial translocation was evaluated and assessed as colony-forming units per gram of tissue. Colonic tissue samples were evaluated by Western blotting method to detect the presence of indoleamine-2,3-dioxygenase. The results obtained were as follows: group 1: normal gut flora; group 2: eight of nine samples had bacterial translocation, of which six of them had positive indoleamine-2,3-dioxygenase protein; group 3: five of nine samples had bacterial translocation, of which seven of them had positive indoleamine-2,3-dioxygenase; group 4: three of nine samples had bacterial translocation, of which seven of them had positive indoleamine-2,3-dioxygenase; and group 5: only one sample had exact indoleamine-2,3-dioxygenase protein. Altered expression of indoleamine-2,3-dioxygenase results in a lower bacterial translocation via infliximab compared with 3-aminobenzamide treatment. Combined treatments emphasized different approaches for the new molecules related to indoleamine-2,3-dioxygenase.

Low expression of bestrophin-2 is associated with poor prognosis in colon cancer.

The purpose of this research was to confirm the prognostic value of bestrophin-2 (BEST2), one of the hub genes in colon cancer, via bioinformatics analysis and validation in public databases and immunohistochemistry detection. The GEO2R online tool and Venn diagram software were utilized to identify differentially expressed genes (DEGs) from expression profiles, including GSE20916, GSE44861 and GSE74602, from the Gene Expression Omnibus (GEO). The overall survival (OS) and disease-free survival (DFS) of colon cancer patients from The Cancer Genome Atlas (TCGA) were analyzed through Kaplan-Meier survival curves. Verification of the significance of BEST2 in colon cancer was based on TCGA, Genotype Tissue Expression (GTEx) and 10 datasets from GEO. BEST2 expression was detected with immunohistochemistry (IHC) in 330 colon tissue samples on microarrays including 165 colon cancerand 165 adjacent normal tissues. For further validation, comprehensive analysis from tissue microarrays and multiple datasets was performed by the summarizing of receiver operating characteristic (SROC) curves and the standard mean differences (SMDs). BEST2 expression in various kinds of colon cancer tissues and cell lines in the context of pancancer was obtained from the Expression Atlas database. The CBioPortal database was queried to identify BEST2 gene alterations and mutation status in colon cancer. Correlated genes (CEGs) with BEST2 and DEGs from public database data were assembled for functional and pathway enrichment analysis. We identified 85 DEGs from the three datasets and screened out BEST2 as a prognostic predictor via the TCGA database. Colon cancer patients with high expression of BEST2 had better survival than patients with low BEST2 (HR=0.5, P=0.006) as shown in Kaplan-Meier survival curves in GEPIA. In all, 1463 colon cancer tissues and 1023 colon normal tissues were gathered via public databases as well as in-house tissue microarrays. The comprehensiveexpression analysis suggested low-expression of BEST2 in colon cancer (SMD=-2.48, 95% CI [-3.15- -1.80]) and the notable efficacy of BEST2 expression in differentiating colon cancer from noncancer samples (AUC=0.97). Gene alteration status of BEST2 occurred in 5% of colon cancer cases, mostly missense mutations and deep deletions. Genes positively correlated with BEST2 and DEGs primarily aggregated in pathways such as anion absorption, digestive juice secretion, cAMP signaling and so on (P<0.05). Ampleevidencesupportsthe role of BEST2 in distinguishing colon cancer from normal tissues in this research. Low expression of BEST2 is correlated with a shorter OS, which implies that BEST2 can be employed as a potential biomarker and therapeutictarget in colon cancer.

A combination of immunohistochemical markers, MUC1, MUC5AC, PAX8 and growth pattern for characterization of mucinous neoplasm of the ovary

ObjectiveBecause mucinous carcinomas are rare tumors that affect several organ sites and are known to originate from different tissues, leading to frequent misdiagnoses, the objective was to characterize the differences...

Phase II trial of moderate dose omega-3 acid ethyl esters for colorectal cancer prevention in patients with lynch syndrome (COLYNE).

TPS209 Background: Lynch syndrome (LS) is the most common inherited colorectal cancer (CRC) syndrome and is responsible for about 3% of newly diagnosed CRC. It is caused by germline mutations in one of the DNA mismatch repair (MMR) genes, and patients with LS carry a lifetime risk of CRC ranging between 10% and 70%. The role of inflammation in driving this malignant transformation is now well established and retrospective studies have revealed a potential chemo-preventative role for omega-3 (ω-3) polyunsaturated fatty acids (PUFAs), possibly via inhibition of inflammatory pathways associated with the development of defective MMR CRC tumors. While patients with LS have the highest risk of developing CRC, the majority of chemoprevention trials are focused on sporadic CRC. Effective interventions to reduce the risk of developing CRC in this population are limited to close surveillance and surgical prophylaxis. There is an unmet need for safe, effective, and non-invasive chemo-preventive interventions in patients with LS. Methods: This pilot study is a single-arm, open-label, phase 2 clinical trial of omega-3 acid ethyl esters (generic Lovasa; 2 grams orally once daily) for adult patients (≥ 18 years of age) with confirmed LS (based on germline testing of the MMR genes panel: MLH1, MSH2, MSHS6, PMS2 or deletion in EPCAM gene). Patients who are not candidate for elective endoscopy and/or with prior history of right sided or pan-colectomy are excluded. Thirty-four patients are expected to enroll, with a primary objective to determine the feasibility (defined as 80% retention rate) of 12 months of treatment with 2 grams capsules of omega-3 acid ethyl esters daily. Secondary endpoints include safety and tolerability of the intervention. Correlative aims include pre and post treatment assessment of colon mucosal tissue proliferation (right sided colon specimens will be evaluated for markers of proliferation (Ki-67) and apoptosis (Caspase-3)), the effect of omega-3 acid ethyl esters on inflammatory markers in serum, urine and feces (PGE-2, COX-2, β-catenin levels, and EPA:AA ratios), and gene expression related to proliferation, apoptosis and cell survival in colon tissue (NF-κB/Wnt pathways). The impact of omega-3 acid ethyl esters on intestinal microbiota will also be assessed (16S rRNA-based profiling). Correlative Colon tissue, serum, urine and feces samples are collected at baseline and at 12 months. The study is actively enrolling with 20 patients enrolled at the time of submission. Clinical trial information: NCT03831698.

Natural Herbal Remedy Wumei Decoction Ameliorates Intestinal Mucosal Inflammation by Inhibiting Th1/Th17 Cell Differentiation and Maintaining Microbial Homeostasis.

Evidence has shown that the traditional Chinese herbal medicine Wumei decoction (WMD) has a protective effect on ulcerative colitis. Here, we studied the anti-inflammatory effects and potential mechanisms of WMD on chronic colitis in mice. A dextran sulfate sodium (DSS)-induced chronic colitis model and CD45RBhighCD4+ T cell transfer model were established in mice. Body weight, Disease Activity Index, and colon length were assessed, and histopathology was confirmed by hematoxylin and eosin staining. Colon tissue samples were collected to detect the frequencies of various immune cells, expression of cytokines, and tight junction-related proteins using flow cytometry, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. 16S ribosomal DNA sequencing was performed to distinguish differential microbiota of fecal samples. Severe chronic colitis was observed in mice after DSS exposure and in Rag1-/- mice reconstituted with CD45RBhighCD4+ T cells, as manifested by weight loss, hematochezia, and shortening and thickening of the colon, which were reversed by WMD treatment. WMD markedly suppressed intestinal mucosal CD4+ T cell differentiation and the secretion of proinflammatory cytokines (eg, tumor necrosis factor α, interleukin-1β, interferon γ, and IL-17A) by flow cytometry, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. Moreover, WMD promoted the expression of occludin, zonula occludens-1, and E-cadherin, thereby maintaining the epithelial barrier function. Additionally, 16S ribosomal DNA sequencing revealed that WMD regulated the dysbiosis of gut microbiota in CD45RBhighCD4+ T cell-reconstituted Rag1-/- mice, evidenced by an increase of Allobaculum and Bacteroides and a decrease of Ileibacterium. WMD ameliorates chronic colitis in mice induced by DSS or reconstituted with CD45RBhighCD4+ T cells through suppressing Th1/Th17 cell differentiation and the secretion of proinflammatory cytokines, maintaining epithelial barrier function, and improving the dysbiosis.

Three-dimensional morphological characterization of colorectal pits from label-free microscopy images.

The prognosis of colorectal cancer (CRC), one of the most prevalent pathologies worldwide, is linked to early detection. Kudo's pit pattern classification states morphological pit patterns of the Lieberkühn crypts by analyzing the superficial mucosa, predicting the histology of colorectal lesions. Its use as a highly accurate two-dimensional diagnostic criterion has increased, mostly involving expert endoscopists' judgment. The processing of autofluorescence images could allow the diagnostic, bypassing staining techniques and decreasing the biopsies, resources and times involved in the inspection. That criterion could be extended by data of the pit three-dimensional (3D) morphology. Thus, this work was aimed at obtaining 3D morphological information by quantifying geometrical and shape descriptors through software processing and analysis of widefield autofluorescence microscopy image stacks acquired by fresh colon tissue samples from a murine model of CRC. Statistical analyses included pits from control mice and from the second (2nd), fourth (4th), and eighth (8th) weeks of treatment. Statistically significant differences were found for almost all parameters between the pits from control and from the 4th treated week, stating that the major morphological changes begin after the 2nd week. In particular, pits from control or initial treatment time points were more tubular, straighter and less rough than the ones from later treatment points. Therefore, they may be more associated to normal or non-neoplastic crypt lumens than linked to adenomas or even cancer crypts. These preliminary outcomes could be considered an advance in 3D pit morphology characterization.

DOP86 Mirikizumab-induced upregulation of colonic transcripts correlates with improvements in stool frequency in a phase 2 study of patients with moderately to severely active Ulcerative Colitis

Abstract Background We have previously shown that treatment with mirikizumab (miri), a p19-directed IL-23 antibody, significantly downregulates inflammatory genes associated with disease activity and upregulates genes expressing epithelial transporter proteins in colonic tissue in patients with ulcerative colitis (UC). Here we explored the correlation between the expression of colonic mucosa genes and stool frequency (SF), a symptom reflective of disease activity, during the 12-week induction period of a Phase 2 study of patients with moderately to severely active UC (NCT02589665). Methods Patients were randomised 1:1:1:1 to receive intravenous placebo (PBO), miri 50mg or 200mg with possibility of exposure-based dose increases, or fixed miri 600mg every 4 weeks for 12 weeks. SF was reported daily by patients and transformed on a 4-level ordinal scale [0–3] representing increased SF above their normal or healthy baseline (BL). Patient colonic biopsies (PBO N=58, miri 50mg N=52, 200mg N=51, 600mg N=54) were collected at BL and Week (W)12, and gene expression measured using an Affymetrix HTA2.0 microarray workflow. BL and W12 gene expression or SF values were pooled and associations identified based on non-parametric Kendall’s tau. Pathway analysis (Hallmark and Reactome) of correlated genes was performed using over-representation analysis. p values of enrichment were determined by hypergeometric distribution test and adjusted for multi-testing with Benjamini-Hochberg procedure. Differential gene expression after miri treatment was determined by paired t-test comparing expression levels at BL and at W12 using data from the 200mg treatment group. Results A total of 267 genes were correlated with SF (|tau| &amp;gt;0.3 and qval &amp;lt;0.001). Of these, 212 were positively correlated (high expression associated with high SF) and 55 were negatively correlated (high expression associated with low SF). The 212 transcripts that were positively correlated with SF were uniformly and consistently downregulated with miri treatment, while the 55 transcripts that negatively correlated with SF, were consistently upregulated with miri treatment (Table 1). Biological pathways significantly associated with the miri-responsive transcripts that correlated with SF included inflammatory response, extracellular matrix dysregulation, neutrophil degranulation and cytokine signaling pathways, especially TNF and IL6 pathways (Table 2). Conclusion This is the first study to identify colon-based transcripts that correlate with a clinical disease activity measure, stool frequency, and it demonstrates that treatment with miri may upregulate genes associated with normalization of SF and down regulate genes associated with inflammation in colonic tissue samples of patients with UC.

P075 Neutrophils prevent rectal bleeding in Ulcerative Colitis by peptidyl-arginine deiminase-4-dependent immunothrombosis

Abstract Background Bleeding ulcers and erosions are hallmarks of active ulcerative colitis (UC). However, the mechanisms controlling bleeding and mucosal haemostasis remain elusive. Methods We used high resolution endoscopy and colon tissue samples of active UC (n = 36) as well as experimental models of physical and chemical mucosal damage in mice deficient for peptidyl-arginine deiminase-4 (PAD4), gnotobiotic mice and controls. We employed endoscopy, histochemistry, live-cell microscopy and flow cytometry to study eroded mucosal surfaces during mucosal haemostasis. Results Erosions and ulcerations in UC were covered by fresh blood, haematin or fibrin visible by endoscopy. Fibrin layers rather than fresh blood or haematin on erosions were inversely correlated with rectal bleeding in UC. Fibrin layers contained ample amounts of neutrophils co-aggregated with neutrophil extracellular traps (NETs) with detectable activity of peptidyl-arginine deiminases (PAD). Transcriptome analyses showed significantly elevated PAD4 expression in active UC. In experimentally inflicted wounds, we found that neutrophils underwent NET formation in a PAD4-dependent manner hours after formation of primary blood clots, and remodelled clots to immunothrombi containing citrullinated histones, even in the absence of microbiota. PAD4-deficient mice experienced an exacerbated course of DSS-induced colitis with markedly increased rectal bleeding (96 % vs 10 %) as compared to controls. PAD4-deficient mice failed to remodel blood clots on mucosal wounds eliciting impaired healing. Thus, NET-associated immunothrombi are protective in acute colitis, while insufficient immunothrombosis is associated with rectal bleeding. Conclusion Our findings uncover that neutrophils induce secondary immunothrombosis by PAD4-dependent mechanisms. Insufficient immunothrombosis may favor rectal bleeding in UC.

Expression of Toll-Like Receptors 2, 4 and 5 in Relation to Gut Microbiota in Colon Neoplasm Patients with and without Inflammatory Bowel Disease.

Background:Toll-Like Receptors (TLRs) are the critical mediators of inflammatory routs in the gut, which play an essential role in regulating the immune responses towards various ligands derived from pathogenic bacteria. Also, TLR signaling has been implicated in the development of Inflammatory Bowel Disease (IBD), Adenomatous Polyp (AP), and Colorectal Cancer (CRC). Here, we aimed to examine the expression of some TLRs concerning certain fecal bacteria in AP and CRC patients with and without IBD.Methods:This case-control study collected fecal and colonic tissue samples from 93 patients versus Normal Controls (NC) via colonoscopy. Fecal samples were used for DNA extraction, and the abundance of selected fecal bacteria was determined by absolute real-time PCR. Also, the gene expression of TLR2, 4, and 5 was analyzed using RT-PCR on the colonic tissues of participants.Results:Compared to NC individuals, in AP and CRC patients, the mRNA expressions of TLR4 and TLR2 were significantly increased while TLR5 was decreased. A meaningful association between TLRs mRNA expression levels and the abundance of some selected fecal bacteria was detected. Also, there was a significant relationship between participant’s food regimes, smoking habit and intestinal TLRs expression.Conclusion:Our study proposed the important role of TLRs during adenomatous and CRC formation. Alterations in TLRs expression associated with certain gut bacteria may contribute to disease development.

Comparison of the healing effects of mesazaline and Ganoderma lucidum in acetic acid-induced colitis in rats.

PurposeThe etiology and pathogenesis of distal colitis (DC) are poorly understood. Activation of intestinal inflammatory response may lead to intestinal tissue necrosis. Antioxidant and anti-inflammatory agents are among the treatment options. Our study aimed to compare the protective effects of mesalazine and Ganoderma lucidum in acetic acid (AA)-induced colitis in rats.MethodsTwenty-four rats were randomly grouped as colitis, mesalazine, G. lucidum, and combined (G. lucidum + mesalazine) groups. DC was induced by intrarectal administration of AA. Statistical comparisons were done by using parameters including colonic tissue IL-1, IL-6, TNF-α, and CRP levels. Histopathologic changes of the samples of colonic tissue were scored as mucosal damage score and inflammatory score. A P-value of <0.05 was considered significant.ResultsIntrarectal administration of AA leads to increased interleukin and CRP levels. High mucosal damage and inflammatory scores were noted in colitis group animals. Single mesalazine or G. lucidum treatment produced considerably decreased tissue interleukin and CRP levels. The lowest tissue interleukin and CRP levels were noted in the combined treatment group of animals. Mucosal damage and inflammatory scores were found to be significantly low in this group of animals.ConclusionThe intrarectal administration of AA results in an activation of intestinal inflammation and severe mucosal damage in colonic tissue. Single use of mesalazine and G. lucidum treatment decreases the severity of intestinal inflammatory response and mucosal damage. The healing effects of the combined treatment of mesalazine and G. lucidum seem to be more effective than that of separate use in the treatment of DC.