Colonic Biopsies Research Articles

Non-invasive assessment of ulcerative colitis activity when choosing a treatment regimen

In recent decades, there has been a steady increase in the incidence of ulcerative colitis worldwide. The purpose of the work was to analyze the literature data on modern features of the treatment of ulcerative colitis, as well as to present our own results and cases from practice. Mesalazine remains the mainstay of remission and often its induction in patients with ulcerative colitis. Currently, the nature of the treatment of ulcerative colitis is determined by the target level of remission. The fact of the onset of endoscopic remission is known to occur much later than subjective clinical improvement. In recent years, this provision has been supplemented by evidence of a delay in histological, laboratory (fecal calprotectin) and transmural remission from endoscopic. There is increasing evidence that the duration and quality of remission depends on the depth of remission. When using ultrasound, it is not difficult to urgently assess the activity and prevalence of inflammation by the parameters of the intestinal wall. Together with the level of fecal calprotectin, this information may be key to the choice of induction, escalation, maintenance or de-escalation treatment options. Of course, endoscopic examination with colon biopsy remains a necessary planned component of the management of a patient with ulcerative colitis. There are 3 cases from practice in which the achievement of transmural remission was carried out during the treatment with mesalazine. The possibility of monitoring the activity of the inflammatory process and its prevalence in the colon with the help of ultrasound examination of the intestinal wall has been clearly demonstrated. According to the results of our study, it was found that with a high activity of the disease according to the scale of Doppler mapping of the colon wall (Limberg 4), an erosive-ulcerative process was recorded in all patients according to the results of endoscopic examination (Mayo 3). Detection of the normal intestinal wall on the echogram in all cases was accompanied by the absence of endoscopic activity (Mayo 0) or its minimal manifestations (Mayo 1).

Deep learning for multi-class semantic segmentation enables colorectal cancer detection and classification in digital pathology images

In colorectal cancer (CRC), artificial intelligence (AI) can alleviate the laborious task of characterization and reporting on resected biopsies, including polyps, the numbers of which are increasing as a result of CRC population screening programs ongoing in many countries all around the globe. Here, we present an approach to address two major challenges in the automated assessment of CRC histopathology whole-slide images. We present an AI-based method to segment multiple (n=14) tissue compartments in the H &E-stained whole-slide image, which provides a different, more perceptible picture of tissue morphology and composition. We test and compare a panel of state-of-the-art loss functions available for segmentation models, and provide indications about their use in histopathology image segmentation, based on the analysis of (a) a multi-centric cohort of CRC cases from five medical centers in the Netherlands and Germany, and (b) two publicly available datasets on segmentation in CRC. We used the best performing AI model as the basis for a computer-aided diagnosis system that classifies colon biopsies into four main categories that are relevant pathologically. We report the performance of this system on an independent cohort of more than 1000 patients. The results show that with a good segmentation network as a base, a tool can be developed which can support pathologists in the risk stratification of colorectal cancer patients, among other possible uses. We have made the segmentation model available for research use on https://grand-challenge.org/algorithms/colon-tissue-segmentation/.

Quantitative Evaluation of NOD2 Promoter Methylation Profiling in Colon Biopsy Samples from Ulcerative Colitis Patients and Non-Colitis Controls

Background: Ulcerative colitis (UC) is an idiopathic chronic inflammatory disease of the colon with evidence addressing the role of epigenetic changes. The intention of this study was to detect the association of DNA methylation levels of NOD2 gene with UC and to evaluate whether any of these changes might be a useful biomarker for detecting patients with UC.
 Methods: The methylation status of the promoter CpG islands (CGIs) of NOD2 gene was examined in the colonic mucosae of 15 cancer-free patients with UC and 15 age- and sex-matched healthy controls by the real-time quantitative multiplex-methylation specific PCR (QM-MSP) assay. Methylation-specific melting curve analysis (MS-MCA) were used to analyze the data.
 Results: The median unmethylated DNA index was significantly higher in cases compared to controls, and hypormethylation of NOD2 gene was significantly correlated with UC (Unmethylated DNA in UC vs. HC; 0.102±0.055 vs. 0.025±0.016, P = 0.000).
 Conclusion: The NOD2 gene that was differentially methylated in UC patients, providing new insights into the pathogenesis of UC, with a view to making steps toward the development of accurate biomarkers for diagnostic tools in UC.

Antibiotics and Probiotics for Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a disorder of a gut-brain interaction characterised by abdominal pain and a change in stool form or frequency. Current symptom-based definitions and the classification of IBS promote heterogeneity amongst patients, meaning that there may be several different pathophysiological abnormalities leading to similar symptoms. Although our understanding of IBS is incomplete, there are several indicators that the microbiome may be involved in a subset of patients. Techniques including a faecal sample analysis, colonic biopsies, duodenal aspirates or surrogate markers, such as breath testing, have been used to examine the gut microbiota in individuals with IBS. Because of a lack of a clear definition of what constitutes a healthy gut microbiota, and the fact that alterations in gut microbiota have only been shown to be associated with IBS, a causal relationship is yet to be established. We discuss several hypotheses as to how dysbiosis may be responsible for IBS symptoms, as well as potential treatment strategies. We review the current evidence for the use of antibiotics and probiotics to alter the microbiome in an attempt to improve IBS symptoms. Rifaximin, a non-absorbable antibiotic, is the most studied antibiotic and has now been licensed for use in IBS with diarrhoea in the USA, but the drug remains unavailable in many countries for this indication. Current evidence also suggests that certain probiotics, including Lactobacillus plantarum DSM 9843 and Bifidobacterium bifidum MIMBb75, may be efficacious in some patients with IBS. Finally, we describe the future challenges facing us in our attempt to modulate the microbiome to treat IBS.

Round-Robin test for the histological diagnosis of acute colonic Graft-versus-Host disease validating established histological criteria and grading systems

Histomorpholgy is one of the mainstays of acute Graft-versus-host disease (GvHD) diagnosis. However, concerns about reproducibility and the most appropriate grading system question its usefulness. Our aim was to assess histomorphological parameters and previously reported grading systems for GvHD regarding reproducibility and validity. Moreover, we propose that sum scores, derived by combining separately scored morphological parameters into a total score, might provide a simplified but equally effective means to grade GvHD. A total of 123 colon biopsies were assessed across four pathologists for intestinal GvHD using a Round-Robin test and results were correlated with clinical findings. Interobserver reproducibility was high for histological parameters that were evaluated as indicators of acute GvHD. Published grading systems were moderately reproducible (ICC 0.679–0.769) while simplified sum scores, in comparison, showed better interrater reliability (ICC 0.818–0.896). All grading systems and sum scores were associated with clinical signs of GvHD and in part with therapy response and survival. However, they were not able to stratify patients according to the clinical severity of GvHD. In a hot-spot analysis 1 crypt apoptotic body (CAB) in 10 crypts was a reasonable cut-off value for minimal diagnostic criteria of GvHD. In conclusion, histology can contribute to the diagnosis of GvHD and is reproducible. Published grading systems are able to reflect clinical findings as are simplified sum scores, which showed improved reproducibility and might be easier to handle as they are based on adding up histological parameters rather than transferring histological findings into a separate grading system. Sum scores will have to be further tested in a prospective setting.

Multi-omic analysis in normal colon organoids highlights MSH4 as a novel marker of defective mismatch repair in Lynch syndrome and microsatellite instability.

Lynch syndrome (LS) is a hereditary condition that increases the risk of colorectal (CRC) and extracolonic cancers that exhibit microsatellite instability (MSI-H). MSI-H is driven by defective mismatch repair (dMMR), and approximately 15% of nonhereditary CRCs also exhibit MSI-H. Here, we aimed to better define mechanisms underlying tumor initiation in LS and MSI-H cancers through multi-omic analyses of LS normal colon organoids and MSI-H tumors. Right (n = 35) and left (n = 23) colon organoids generated from normal colon biopsies at routine colonoscopy of LS and healthy individuals were subjected to Illumina EPIC array. Differentially methylated region (DMR) analysis was performed by DMRcate. RNA-sequencing (n = 16) and bisulfite-sequencing (n = 15) were performed on a subset of right colon organoids. CRISPR-cas9-mediated editing of MMR genes in colon organoids of healthy individuals was followed by quantitative PCR of MSH4. The relationship between MSH4 expression and tumor mutational burden was further explored in three independent tumor data sets. We identified a hypermethylated region of MSH4 in both the right and left colon organoids of LS versus healthy controls, which we validated using bisulfite-sequencing. DMR analysis in three gastrointestinal and one endometrial data set revealed that this region was also hypermethylated in MSI-H versus microsatellite stable (MSS) tumors. MSH4 expression was increased in colon organoids of LS versus healthy subjects and in publicly available MSI-H versus MSS tumors across four RNA-seq and four microarray data sets. CRISPR-cas9 editing of MLH1 and MSH2, but not MSH6, in normal colon organoids significantly increased MSH4 expression. MSH4 expression was significantly associated with tumor mutational burden in three publicly available data sets. Our findings implicate DNA methylation and gene expression differences of MSH4 as a marker of dMMR and as a potential novel biomarker of LS. Our study of LS colon organoids supports the hypothesis that dMMR exists in the colons of LS subjects prior to CRC.

Diagnostic yield of random colon biopsy sampling in patients with chronic diarrhea and normal colonoscopy: a systematic review and meta-analysis

Background and AimsMost colonoscopy examinations in patients with chronic unexplained diarrhea do not reveal endoscopic abnormalities, and routine random colon biopsy specimens are taken for further diagnostic assessment. In this study, we aimed to perform a systematic review and meta-analysis to study the pooled diagnostic value of random colon biopsy sampling in this patient population.MethodsMultiple databases (MEDLINE, Scopus, Embase) were searched in May 2022 for studies evaluating the diagnostic yield of random biopsy sampling in patients with chronic diarrhea with normal colonoscopy examinations. Standard meta-analysis methods used the random-effects model, and heterogeneity was assessed using the I2 statistics.ResultsTwenty-one studies were included in the study (3322 patients). The pooled rate of normal colon biopsy sampling was 48% (95% confidence interval [CI], 31-65.2; I2 = 98%), and normal ileal biopsy sampling was 88.5% (95% CI, 51.3-98.3; I2 = 93%). The pooled rate of significant histopathologic change was 30% (95% CI, 20-41; I2 = 96%). The pooled rates of specific diagnoses based on random colon biopsy sampling were as follows: microscopic colitis, 15% (95% CI, 10-21; I2 = 92%); lymphocytic colitis, 9% (95% CI, 6 -13; I2 = 85%); collagenous colitis, 3.5% (95% CI, 2.4-5.7; I2 = 80%); nonspecific colitis, 20% (95% CI, 11-32.8; I2 = 97%); inflammatory bowel disease, 1.7% (95% CI, .8-3.5; I2 = 54%); and eosinophilic colitis, 2.3% (95% CI, 1.1-4.5; I2 = 69%).ConclusionsThis meta-analysis of 21 studies demonstrated significant histopathologic change in 30% and microscopic colitis in 15%. Normal colon biopsy sampling was noted in 48% and normal ileum biopsy sampling in 88.5%. Ileal biopsy data were based on limited data and studies (only 3 included). In studies with a mean patient age <49 years, 40% demonstrated significant histopathologic changes. Most colonoscopy examinations in patients with chronic unexplained diarrhea do not reveal endoscopic abnormalities, and routine random colon biopsy specimens are taken for further diagnostic assessment. In this study, we aimed to perform a systematic review and meta-analysis to study the pooled diagnostic value of random colon biopsy sampling in this patient population. Multiple databases (MEDLINE, Scopus, Embase) were searched in May 2022 for studies evaluating the diagnostic yield of random biopsy sampling in patients with chronic diarrhea with normal colonoscopy examinations. Standard meta-analysis methods used the random-effects model, and heterogeneity was assessed using the I2 statistics. Twenty-one studies were included in the study (3322 patients). The pooled rate of normal colon biopsy sampling was 48% (95% confidence interval [CI], 31-65.2; I2 = 98%), and normal ileal biopsy sampling was 88.5% (95% CI, 51.3-98.3; I2 = 93%). The pooled rate of significant histopathologic change was 30% (95% CI, 20-41; I2 = 96%). The pooled rates of specific diagnoses based on random colon biopsy sampling were as follows: microscopic colitis, 15% (95% CI, 10-21; I2 = 92%); lymphocytic colitis, 9% (95% CI, 6 -13; I2 = 85%); collagenous colitis, 3.5% (95% CI, 2.4-5.7; I2 = 80%); nonspecific colitis, 20% (95% CI, 11-32.8; I2 = 97%); inflammatory bowel disease, 1.7% (95% CI, .8-3.5; I2 = 54%); and eosinophilic colitis, 2.3% (95% CI, 1.1-4.5; I2 = 69%). This meta-analysis of 21 studies demonstrated significant histopathologic change in 30% and microscopic colitis in 15%. Normal colon biopsy sampling was noted in 48% and normal ileum biopsy sampling in 88.5%. Ileal biopsy data were based on limited data and studies (only 3 included). In studies with a mean patient age <49 years, 40% demonstrated significant histopathologic changes.

Altered mucosal and plasma polyunsaturated fatty acids, oxylipins, and endocannabinoids profiles in Crohn’s disease

Selected mucosal and plasma polyunsaturated fatty acids (PUFAs) and related oxylipins and endocannabinoids were determined in 28 Crohn’s disease (CD) patients and 39 controls. Fasting blood and colonic biopsies were collected in all participants, during a disease flare for the patients. Thirty-two lipid mediators including PUFAs, oxylipins, and endocannabinoids were assessed by LC-MS/MS. The pattern of lipid mediators in CD patients is characterized by an increase in arachidonic acid-derived oxylipins and endocannabinoids and a decrease in n-3 PUFAs and related endocannabinoids. A model combining increased 6-epi-lipoxin A4 and 2-arachidonyl glycerol with decreased docoasapentaenoic acid in plasma fairly discriminates patients from controls and may represent a lipidomic signature for CD flare. The study findings suggest that lipid mediators are involved in CD pathophysiology and may serve as biomarkers for disease flare. Further research is required to confirm the role of these bioactive lipids and test their therapeutic potential in CD.

A Young Woman with High-Grade Rectosigmoid Adenocarcinoma, no Other Specified, T2n2aM0: A Case Report and Literature Review

BACKGROUND: Colorectal cancer (CRC) is the fourth most common cancer in which the majority of CRCs are diagnosed between the ages of 50 and 70 years. Based on current trends, the incidence of colon and rectal cancers can increase by 124% in people aged 35–49 years and at the age of 20–34 will increase by 28% and 46%. OBJECTIVES: To report a case of a 24-year-old female patient with CRC. CASE REPORT: A 24-year-old female patient came to the Emergency Room at Bendan Hospital, Pekalongan City, with complaints of bleeding since 1 year ago. There are complaints in the past 6 months; the patient’s weight has decreased by up to 20 kg. Rectal toucher examination revealed normal (+) anal sphincter tone, a palpable mass in the anal canal, and was fragile; there were feces and blood in gloves. Abdominal ultrasonography found a solid tumor mass in the rectum area with a volume of 181 cc, suggesting a malignancy of the rectum. During a colonoscopy, a circular, fragile, and easily bleeding tumor was found, approximately 1 cm from the anal verge, and then biopsy was performed. Histopathological examination of the colon biopsy suggests a well-differentiated, rectosigmoid, NOS adenocarcinoma. The patient then underwent surgery using the Miles procedure technique. The patient’s condition after the operation improved, so after a week of treatment, the patient was able to undergo outpatient treatment. CONCLUSION: A complete history, physical examination, and supporting investigation are very useful in early detection of colorectal carcinoma, especially in young patients.

Interleukin 36 receptor-inducible matrix metalloproteinase 13 mediates intestinal fibrosis.

Fibrostenotic disease is a common complication in Crohn's disease (CD) patients hallmarked by transmural extracellular matrix (ECM) accumulation in the intestinal wall. The prevention and medical therapy of fibrostenotic CD is an unmet high clinical need. Although targeting IL36R signaling is a promising therapy option, downstream mediators of IL36 during inflammation and fibrosis have been incompletely understood. Candidate molecules include matrix metalloproteinases which mediate ECM turnover and are thereby potential targets for anti-fibrotic treatment. Here, we have focused on understanding the role of MMP13 during intestinal fibrosis. We performed bulk RNA sequencing of paired colon biopsies taken from non-stenotic and stenotic areas of patients with CD. Corresponding tissue samples from healthy controls and CD patients with stenosis were used for immunofluorescent (IF) staining. MMP13 gene expression was analyzed in cDNA of intestinal biopsies from healthy controls and in subpopulations of patients with CD in the IBDome cohort. In addition, gene regulation on RNA and protein level was studied in colon tissue and primary intestinal fibroblasts from mice upon IL36R activation or blockade. Finally, in vivo studies were performed with MMP13 deficient mice and littermate controls in an experimental model of intestinal fibrosis. Ex vivo tissue analysis included Masson's Trichrome and Sirius Red staining as well as evaluation of immune cells, fibroblasts and collagen VI by IF analysis. Bulk RNA sequencing revealed high upregulation of MMP13 in colon biopsies from stenotic areas, as compared to non-stenotic regions of patients with CD. IF analysis confirmed higher levels of MMP13 in stenotic tissue sections of CD patients and demonstrated αSMA+ and Pdpn+ fibroblasts as a major source. Mechanistic experiments demonstrated that MMP13 expression was regulated by IL36R signaling. Finally, MMP13 deficient mice, as compared to littermate controls, developed less fibrosis in the chronic DSS model and showed reduced numbers of αSMA+ fibroblasts. These findings are consistent with a model suggesting a molecular axis involving IL36R activation in gut resident fibroblasts and MMP13 expression during the pathogenesis of intestinal fibrosis. Targeting IL36R-inducible MMP13 could evolve as a promising approach to interfere with the development and progression of intestinal fibrosis.

Melanosis Coli: A Case Report of a Rare and Harmless Entity

Melanosis coli (MC) is a non-inflammatory, rare, benign, and reversible colonic disorder. That refers to pigmentation of the colonic mucosa, shown by colonoscopy, and Confirmed by the anatomopathological study of colon biopsies. It results from excessive and prolonged use of anthraquinone laxatives, and usually resolves within a few months after discontinuing the laxatives. We present the case of a 60 year old woman presenting with chronic abdominal pain constipation, and revealing Melanosis Coli following prolonged use of laxatives.

Is the Routine Histopathologic Diagnosis of Ulcerative Colitis Based on Cross-cut Sections or on Well-oriented Sections?

For many years, it was empirically estimated that the majority of the routine colon biopsies in Swedish patients with ulcerative colitis (UC), exhibited cross-cut crypts. The aim of the present study was to assess the frequency of cross-cut crypts (CCC) and well-oriented crypts in routine colon biopsies in German patients with UC. In total, 447 colon biopsies: 376 with UC and 71 controls were investigated. Out of 376 colon biopsies with UC, 73% exhibited ≥60% CCC. Out of the 237 biopsies showing ≥80% CCC, as many as 71% exhibited 100% CCC in individual biopsies. Similar percentages were found in control biopsies. The majority of the routine colon biopsies with UC, as well as control biopsies in German patients displayed CCC. Thus, an unnoticed, consequent, and systematic cutting technical hitch was introduced during the laboratory processing of colon biopsies. The reason(s) behind the similar histologic processing mode of colon biopsies at the two geographically disparate laboratories (Sweden and Germany) remains elusive. The cross-cutting mode influenced the narrative of biopsies in UC, inasmuch as some histological parameters listed among well-oriented colon sections were not present in sections displaying CCC.

Prediction and Verification of Potential Therapeutic Targets for Non-Responders to Infliximab in Ulcerative Colitis.

Infliximab (IFX) has been widely used in ulcerative colitis (UC) patients. However, the subsequent effective treatment of IFX non-response in UC patients remains a challenge. This study aims to predict potential therapeutic targets for non-responders by performing a bioinformatic analysis of the data in the Gene Expression Omnibus (GEO) database and validation by biopsies. Colonic mucosal biopsies expression profiles of IFX-treated UC patients (GSE73661, GSE16879) were utilized to predict potential therapeutic targets. Bioinformatics analyses were used to explore potential biological mechanisms. CytoHubba was performed to screen hub genes. We used a validation dataset and colonic mucosal biopsies of UC patients to validate hub genes. A total of 147 DEGs were identified (119 upregulated genes and 28 downregulated genes). GSEA showed that DEGs in GSE73661 were enriched in the pathways of the cytokine-cytokine receptor, the chemokine, and the adhesion molecules system. Based on the PPI network analysis, we identified four hub genes (and the transcription factor NF-κB). Then, we validate the expression of hub genes by reverse transcription-polymerase chain reaction (RT-PCR). We found higher expression of IL-6, IL1B, CXCL8, and CCL2 in non-responders compared to responders. In summary, four potential targets (IL-6, IL1B, CXCL8, and CCL2) were finally identified by performing a bioinformatics analysis of the datasets in the GEO database. Their expression was confirmed in colonic mucosal biopsies of patients with UC. These results can help to further explore the mechanism of non-responders to IFX in UC and to provide potential targets for their subsequent treatment.

Crypt-rings in tandem detected in infectious colitis, in IBD and in IBD-associated noninvasive neoplasia.

Recently, eight novel histologic structures in colon mucosa with inflammation were described. Here, we assessed the frequency of one of them: crypt rings in tandem (CRT), in patients with infectious colitis (IC), IBD (ulcerative colitis; UC or Crohn colitis; CrC) and UC in remission (UCR). In addition, the frequency of dysplastic CRT (DCRT) in IBD-associated noninvasive neoplasia (IBDNIN) were also calculated. Colon biopsies in 578 cases were reviewed: 42 cases with IC, 280 with IBD (180 UC and 100 CrC), 100 UCR and the remaining 156, IBDNIN. The proportions of CRT in IC was 16.7%, in IBD 14.3% %, in UCR 3%, and of DCRT in IBDNIN, 20%. No differences were recorded between the proportions of CRT in IC, UC and CrC. Conversely, the difference in CRT frequency between UC and UCR, and between CRT and DCRT were significant (P=0.006, and p=0.05, respectively). CRT evolved in IC and in IBD. The finding of CRT in IC strongly suggest that those characteristic crypts were shaped at the early stages of mucosal inflammation. CRT persisted in IBD with protracted inflammation but plummeted in UCR, that is when the mucosal inflammation waned. The proportion of DCRT was significantly higher than that of CRT. It is submitted that DCRT might had developed in IBDNIN using CRT as scaffolds. This is the first study in which a characteristic pathologic aberration of cryptogenesis was tracked in colon biopsies from patients with IBD and with IBD-associated neoplastic transformation.

Heavy metal-laden particulate dust disrupts intestinal epithelial stem cells and lineage differentiation

Chronic heavy metals exposure has been associated with intestinal inflammation and higher incidences of colorectal cancer, however there is no molecular evidence that metal laden-particulates can cause injury to the gut epithelia. Communities that live adjacent to abandoned uranium mines have higher incidence of metabolic and digestive diseases; however, the cause is unknown. The goal of this study is to characterize particulate dust containing non-fissile uranium as an environmental toxicant that damages intestinal epithelia and characterize the subsequent transcriptomic changes. We hypothesized that non-fissile uranium will alter the differentiation pattern of progenitor cells in the crypt biasing them towards the secretory lineage. Human colonoids derived from healthy adult colonic biopsies (UNM IRB approved study 18-626 and 18-171) were established and shown to physiologically model the intestinal epithelia. Colonoids were acutely exposed (24h) to non-fissile uranium dust obtained near the Jackpile uranium mine in New Mexico, one of the largest open pit uranium mines in the US, located on the Laguna Pueblo. Control and dust-exposed colonoids (n=3 unique donors) were dissociated into single cells and processed for droplet-based single cell sequencing (scRNAseq). scRNAseq identified significant changes in the secretory lineage in uranium dust-exposed colonoids. Specifically, there was a four-fold expansion of enteroendocrine cells (EEC) with increases in EEC hormones serotonin and PYY in all colonoid lines. Proliferative pathways, such as Wnt signaling and negative regulation of apoptosis were upregulated in these EECs. These transcriptomic changes in EECs and proliferative cells and increased basolateral secretion of secretory granules and serotonin was validated in colonoids (˃5 biologically unique human donors) via RNA FISH, immunofluorescence and ELISAs. Our results demonstrate that acute uranium dust exposure directly induces transcriptomic and cellular changes in intestinal epithelia independently of microbiota, stroma, and immune cells. These results indicate that adult human colonoids are a relevant model for investigating environmental toxicants on intestinal epithelia and provide an improved understanding of uranium dust as a model of intestinal injury. This study was funded by NIDDK (K01DK106323), NIEHS (R56ES034400, P42ES025589), and NIGMS (P20GM121176, P20GM130422). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel method for isolation and flow cytometry analysis of intraepithelial lymphocytes from colon biopsies

Investigating the immune responses of the intestine in response to different insults is predominantly limited to indirect methods such as circulating markers of intestinal health or gene expression from dissections. We describe here a validated protocol for the isolation and subsequent flow cytometry analysis of intestinal intraepithelial lymphocytes (IEL) from colonic biopsy samples. Colon biopsy samples were collected with endoscopy forceps from Holstein dairy bull calves at d 2, 28, and 42 of life. The biopsies were put into an isolation solution of Hanks' balanced salt solution, and fetal bovine serum followed by digestion solution. The solution was filtered and the flow-through, containing IEL, was stained with fluorescent antibodies for flow cytometry analysis. Density gradient separation of the isolate yielded higher viability and cleaner samples for flow cytometry analysis. Anti-bovine γ chain of the T cell receptor was used to identify populations of gamma delta (γδ) T cells via flow cytometry. In addition, γδ T cell subsets were identified using an anti-bovine antibody against the coreceptor workshop cluster 1. This method allowed for the precise identification of lymphocyte populations and evaluation of the proportion of different subsets of γδ T cells from intestinal IEL over time. The technique described here will allow the research community to characterize intestinal immune function over time and improve our understanding of how different management and nutritional strategies affect intestinal health.

Characterization of intestinal fibrosis in cats with chronic inflammatory enteropathy.

Intestinal fibrosis (IF) is commonly identified on histopathology of intestinal biopsy specimens (IBSp) from cats with chronic inflammatory enteropathy (CIE) however, its clinical relevance is unknown. Characterize and determine the clinical relevance of IF in cats with CIE. Sixty-five client-owned cats diagnosed with CIE after gastrointestinal histopathology from a single referral hospital in the United Kingdom. Medical records were retrospectively searched for cases of CIE on the basis of histopathology of IBSp. The IBSp from eligible cats were re-reviewed by a single board-certified veterinary pathologist for inclusion. Masson's trichrome (MT) stain and immunohistochemical labeling using antivimentin and anticollagen I antibodies to identify IF. For each case, various variables at the time of diagnostic investigation were recorded and referring veterinarians were contacted for follow-up information. Mucosal fibrosis was identified in 51% of duodenal and 76% of colonic hematoxylin and eosin (HE)-stained IBSp. Vimentin labeling and MT staining identified additional cases of IF in 65% and 58% of the duodenal biopsy specimens, respectively. Vimentin labeling detected IF in 79% of the colonic biopsy specimens. Positive vimentin labeling and MT staining of the colonic mucosa were associated with decreased likelihood of attaining clinical remission and increased risk of death because of CIE (P < .05). Additional stains at initial histopathologic examination of IBSp allow for better identification of IF compared to routine HE staining. Identification of IF in colonic biopsy specimens by vimentin immunolabeling and MT staining may provide prognostic information in cats with CIE.

Microscopic colitis - state of current knowledge on pathogenesis, diagnostics and treatment

Introduction and purpose Microscopic colitis is multicausal inflammatory bowel disease, which affects mostly elderly, particularly women. It is a cause of chronic, watery diarrhea. MC usually presents macroscopically normal, thus diagnosis requires colonoscopy and histological examination of colonic biopsies. MC doesn’t increase risk of colon cancer. However, it leads to impaired patients’ quality of life and reduced living activity. This review aims to sum up the current state of knowledge about symptoms, diagnosis and treatment of microscopic colitis. Material and methods This review was based on available data collected in the PubMed database, using the following keywords: „microscopic colitis”, “microscopic colitis treatment”, „microscopic colitis pathogenesis”, and “ microscopic colitis diagnosis”. ConclusionsDiagnosis of MC is often delayed due to similarity of clinical manifestation with other inflammatory bowel diseases, like IBS or IBD. Raising awareness about MC as a potential cause of chronic diarrhea can lead to earlier diagnosis and increased patients’ quality of life.

Predicting Crohn's disease severity in the colon using mixed cell nucleus density from pseudo labels.

Crohn's disease (CD) is a debilitating inflammatory bowel disease with no known cure. Computational analysis of hematoxylin and eosin (H&E) stained colon biopsy whole slide images (WSIs) from CD patients provides the opportunity to discover unknown and complex relationships between tissue cellular features and disease severity. While there have been works using cell nuclei-derived features for predicting slide-level traits, this has not been performed on CD H&E WSIs for classifying normal tissue from CD patients vs active CD and assessing slide label-predictive performance while using both separate and combined information from pseudo-segmentation labels of nuclei from neutrophils, eosinophils, epithelial cells, lymphocytes, plasma cells, and connective cells. We used 413 WSIs of CD patient biopsies and calculated normalized histograms of nucleus density for the six cell classes for each WSI. We used a support vector machine to classify the truncated singular value decomposition representations of the normalized histograms as normal or active CD with four-fold cross-validation in rounds where nucleus types were first compared individually, the best was selected, and further types were added each round. We found that neutrophils were the most predictive individual nucleus type, with an AUC of 0.92 ± 0.0003 on the withheld test set. Adding information improved cross-validation performance for the first two rounds and on the withheld test set for the first three rounds, though performance metrics did not increase substantially beyond when neutrophils were used alone.

Abstract 5299: Impact of gut microbial metabolites on colon epithelium

Abstract Inflammatory bowel disease (IBD) is a major risk factor for colorectal cancer (CRC) development. Dysbiosis of the gut microbiome has been implicated in the pathogenesis of IBD and CRC. The gut microbiome and its interaction with the colon may play a significant role in enabling tumor permissive state in an inflammatory environment. However, the functional impact of colon microbiome on epithelial cells that enables neoplastic transformation under inflammatory condition is not clear. This study seeks to examine the impact of mucosa-adherent microbiota on colon epithelial cells. We used in vitro mini-bioreactor arrays to cultivate mucosa-adherent microbiota from colon biopsies, including biopsies from IBD patients with dysplasia (progressors) and without dysplasia (non-progressors). Paired dysplastic and non-dysplastic tissue from progressors was included in the study. HCT116 cells were exposed to the metabolites derived from the cultivation of microbial communities. Cell lysate and secretome were collected after the treatment of microbial metabolites and analyzed by LC-MS/MS. The differentially expressed proteins were identified using Limma. Over-representation analysis was performed using Reactome in Grofiler. Pathways relating to cell division, mitosis and cell proliferation were enriched in cells treated with microbiome metabolites from progressors. Pathways reflecting a proliferative phenotype were also found enriched in the cell secretomes after treatment with microbiome metabolites from progressors. We functionally verified the proliferative phenotype in cells using BRDU incorporation assay. Microbial metabolites from non-dysplastic biopsy from progressor showed significantly higher cell proliferation compared to non-dysplastic biopsy from non-progressors (p&amp;lt;0.05). Furthermore, microbial metabolites from dysplastic tissue showed a significantly higher proliferation compared to non-dysplastic tissue from the same patient (p&amp;lt;0.01) and patients without dysplasia (p&amp;lt;0.0001). A significantly higher degree of DNA damage was induced by microbial metabolites derived from IBD progressors than non-progressors (p&amp;lt;0.05). Together, our findings suggest that microbial metabolites in the dysplastic tissue alter the colon microenvironment to induce higher cellular proliferation and DNA damage, which may contribute to colon neoplastic progression in the IBD patients. Citation Format: Nobel Bhasin, Lakmini Herath Senavirathna, Madeline Bresson, Lyu Zhe, Victoria B. Poplaski, Zahraa A. Lami, Teresa A. Brentnall, Antone R. Opekun, John F. Valentine, Britton A. Britton, Pan Sheng, Ru Chen. Impact of gut microbial metabolites on colon epithelium. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5299.