Colonic Submucosa Research Articles

A7 FOXL1+ TELOCYTES IN MOUSE COLON ORCHESTRATE ECM BIODYNAMICS AND WOUND REPAIR RESOLUTION

Abstract Background The extracellular matrix (ECM) is a complex assembly of proteins that provide mechanical and biochemical stimuli to the epithelial and mesenchymal cells of the GI mucosa. Deficiencies in ECM assembly, protein production or excessive accumulation can lead to multiples pathologies including fibrosis and cancer. FoxL1+-Telocytes (TCFoxL1+) are subepithelial cells that form a network underneath the epithelium, contributing to the microenvironment that supports epithelial and immune cell homeostasis. We have previously shown that BMPR1A signaling deletion in TCFoxL1+ influences the microenvironment via stromagenesis, immune infiltration and colonic dysplasia in mouse model of GI diseases. However, the precise molecular and mechanical events that contributes to the onset of this state have yet to be elucidated. Aims Characterize the modulations in ECM biodynamics induced by BmpR1a-deficient TC FoxL1+ ( BmpR1a△FoxL1+) in mouse colon submucosa. Methods Matrisomics was performed to determine the inventory of ECM proteins expressed solely in the GI stromal compartment following tissue deconstruction of control and BmpR1a△FoxL1+ mice colons. Histological and biochemical methods were used to further characterize the collagen network and matrisome-associated modulations. Fluorescence In Situ Hybridization (FISH) was performed to study the bacterial presence in the mucosa. Results The set of identified proteins shows an enrichment for proteins involved in collagen network regulation, wound repair homeostasis and immune regulation such as Col1a2, Col3a1, Col6a4 and Coll14a1, as well as SerpinH1, MFAP4, ANXA1 and S100A9. Collagen network is affected with increased deposition and reorganization of fiber alignment. Unfolded collagen content was also increased in dysplastic areas of BmpR1a△FoxL1+ mouse colon with a concomitant increase in the collagen-chaperone SerpinH1. Validations of other targets indicate that BmpR1a△FoxL1+ mice deals with some type of tissue micro-injury and inflammation that is unresolved, creating a unfavorable microenvironment for tissue homeostasis. Conclusions Taken together, these results suggest that Bmp-signaling deficient TCFoxL1+ significantly contribute to the collagen network biodynamics through increased collagen deposition, fiber alignment reorganization and regulation of the collagen triple-helix assembly. Other matrisome modulations suggest a state of unresolved wound healing due to tissue injury, that could be the etiology of GI pathology and lead to more severe conditions upon various environmental triggers. Funding Agencies CIHR

Ultrasonographic Evaluation of Bowel Wall Thickness and Intramural Blood Flow in Ulcerative Colitis

Abstract Background Ulcerative colitis, a type of inflammatory bowel disease that merely affects the mucosa and submucosa of colon in the form of inflammatory ulcers. Colonoscopy is the gold standard for its diagnosis. For optimal monitoring of disease activity in UC patients, colonoscopy should be performed on a regular basis. However, repeated colonoscopies represent a logistic and economic challenge, as well as significant burden for the patients. Objectives Our study aimed to provide an extensive overview of the main pathologic features of gut wall vessels and bowel wall thickness at US examination of UC. Patients and Methods This prospective case control study was done on 40 patients confirmed to have UC attending to Outpatient Clinics of Internal Medicine and Gastroenterology Department – Ain-Shams University from October 2018 to Augost 2019. They were divided into two groups: Relapse group: Include 20 patients with active UC disease. Remission group: Include 20 patients with inactive UC disease (in remission state). These two groups were matched with 20 healthy individuals, matched for age and gender and considered to be a control group. Disease activity was categorized according to the endoscopic Mayo score.Ultrasound and endoscopic findings were compared for each colon segment except for the rectum. Results The peak incidence of affected patients was 30–40 years of age. Female predominance compared to male with a ratio of 2.6:1. 20% of remission patients complaining from 1-2 bowel movement while 45% and 50% of relapsing patients suffer from 3-4 and 5 bowel movement respectively. 100%, 100%, 20% and 15% of relapsing patients suffer from bleeding per rectum, abdominal pain, tenesmus and urgency. Higher ESR and CRP and lower hemoglobin in relapsing compared to remission group. Furthermore, The last group has higher value of ESR and CRP and lower value of hemoglobin compared to control group. BWT was significantly thicker in relapse group (4.8±0.7 mm) than of remission (3.55±0.5 mm) compared to control group (1.6±0.5) (p value <0.001). BWT at a cut-offs > 4 mm discriminating between cases with relapse from those with remission and at a cut-offs >4 mm discriminating between mild endoscopic severity from moderate and severe UC. Furthermore, BWT at a cut-offs >4.6 mm discriminating between mild and moderate endoscopic severity from severe UC. Vascular signal number at a cut-offs >1 discriminating between cases with relapse from those with remission and at a cut-offs >2 discriminating between mild and moderate endoscopic severity of UC. Conclusion Abdominal ultrasound is a widely available non-invasive method for imaging of UC. It provides a high sensitivity, specificity and accuracy in diagnosis and monitoring of UC activity.

Granulomatous Variant of Food Protein-Induced Allergic Proctocolitis.

A 7-month-old infant on commercial cow milk formula presented with a 4-week history of bloody stools without vomiting, diarrhea, fever, or skin rash. The patient did not appear ill, had a normal physical examination, and took his feeds well. Laboratory results showed a leukocyte count of 17,400/mm3 (reference range, 4400–19,100/mm3) with 12% eosinophils (0.0%–5.0%), hemoglobin level of 12.7 g/dL (10.1–14.2 g/dL), and albumin level of 4.3 g/dL (3.2–4.8. g/dL). Stool cultures were unremarkable. A stool smear showed a large number of eosinophils. Serum IgE, IgG, IgM, and IgA levels were unremarkable, and radioallergosorbent test, performed to determine the presence of specific IgE against cow milk, α-lactalbumin, or casein were all negative. Rectosigmoidoscopy revealed patchy erythema with congestion of the rectal and sigmoid colon mucosa (Fig. 1). Biopsy specimens revealed numerous eosinophils and degranulation in the mucosa (Fig. 2), and notably, granulomatous infiltrates composed of multinucleate giant cells with prominent eosinophils and degranulation in the submucosa (Fig. 3).FIGURE 1.: Rectosigmoidoscopy results showing patchy erythema with congestion of the mucosa in the rectum and sigmoid colon.FIGURE 2.: Biopsy specimens showing numerous eosinophils and degranulation in the mucosa of the sigmoid colon (hematoxylin and eosin staining; original magnification, 200X).FIGURE 3.: Biopsy specimens showing granulomatous infiltrates composed of multinucleate giant cells with prominent eosinophils and degranulation in the submucosa of the sigmoid colon (arrow: multinucleate giant cell, hematoxylin and eosin staining; original magnification, 200X).Immunodeficiency was considered, but history of unusual, severe, or recurrent infections was negative, and serum immunoglobulin levels were normal. Although presentation with granulomas is atypical, the patient was diagnosed with food protein-induced allergic proctocolitis (FPIAP) based on the clinical and histological findings. The commercial cow milk formula was replaced with an extensively hydrolyzed formula, resulting in a dramatic improvement in the condition of the patient. The patient has remained on an extensively hydrolyzed formula and has been consuming a variety of solid foods. No relapse of symptoms has been observed, 5 months after the diagnosis. FPIAP is a common cause of rectal bleeding in an otherwise healthy young infant (1). In most cases of FPIAP, biopsy specimens show prominent eosinophilic infiltration of the rectosigmoid mucosa (2,3). Occurrence of a granulomatous component, as seen in this patient, is a distinctly rare phenomenon in this disease. To date, only two studies have shown the presence of granulomas in FPIAP (4,5). The presence of a granulomatous infiltrate may indicate a severe form of FPIAP (4). Generally, the prognosis of FPIAP is favorable, and tolerance is acquired by around the 1-year of age (6,7). The presence of granulomas in FPIAP should not lead to a diagnosis of any other disease that presents with granulomas based on histological findings, such as early-onset inflammatory bowel disease, primary immunodeficiency, or various infectious diseases (8). Awareness of such unusual and misleading features in FPIAP is, therefore, necessary to avoid unfortunate misdiagnosis as another disease. ACKNOWLEDGMENTS We would like to thank Editage (www.editage.com) for English language editing.

Schistosomiasis with Duodenal Involvement Presenting as Irritable Bowel Syndrome in a 9-Year-Old Patient

Abstract Introduction/Objective Schistosomiasis is extremely rare in the United States. Most patients diagnosed in the US have an international travel history or recently immigrated from endemic areas. We report a case of a 9-year-old boy who presented with a three-month history of daily abdominal pain, decreased appetite, weight loss, and microcytic anemia. The patient had alternating symptoms of constipation and diarrhea mimicking Irritable Bowel Syndrome (IBS). Methods CT scans showed distal sigmoid colon with wall thickening and hyperenhancement. Stool Helicobacter pylori antigen test was positive and hemoglobin levels were low (11 g/dL). The blood eosinophil count was initially within the normal range but was significantly elevated three months later (2,115 cells/µL). Esophagogastroduodenoscopy and colonoscopy were performed, which showed patchy areas of erythema and punctate bleeding within the descending and sigmoid colon. Results Gastrointestinal tract biopsies revealed focal, prominent eosinophilic infiltrates in the lamina propria. There were multifocal granulomas and fibrosis surrounding Schistosoma ova in the lamina propria and submucosa of duodenum, cecum, colon, and rectum. Acid-fast stain highlights the shell and spine of the ova. A retrospective chart review revealed that the patient had traveled to Yemen one month before the onset of symptoms. Schistosomiasis is among the top differentials for marked, sustained eosinophilia, especially with relevant travel history. Distortions caused by processing and sectioning can make speciation difficult on tissue sections. Acid-fast staining of the shell would favor S. mansoni over S. intercalatum; however, the S. intercalatum-S. hematobium hybrid also has an acid fast–positive shell. The duodenum is an uncommon location to find Schistosoma ova, which are usually found in the rectum and sigmoid colon. Conclusion The symptoms of gastrointestinal schistosomiasis may mimic IBS, and eosinophilia may initially be not evident as in this case. However, a high degree of suspicion is required in diagnosing schistosomiasis in patients from endemic areas.

Diverticulosis, Diverticulitis, and Appendicitis

Colonic diverticula are herniations of colonic mucosa and submucosa through the muscularis propria. They occur where perforating arteries traverse the circular muscle layer, in parallel rows between the mesenteric and antimesenteric taenia. Colonic diverticular disease may present as diverticulosis, diverticulitis, or diverticular bleeding. Of patients with known diverticulosis, only 10% to 20% will develop diverticulitis. Diverticulitis varies in presentation and severity. This chapter discusses the diagnosis, differential diagnosis, and management of diverticulitis and its complications. Appendicitis is generally caused by obstruction of the lumen of the appendix, followed by infection. In the United States, the lifetime risk of appendicitis is about 9% for males and 7% for females. This chapter also discusses the diagnosis of appendicitis (including typical and atypical presentations and appendicitis as it presents in special groups of patients) and its management. This review contains 3 figures, 21 tables, and 76 references. Keywords: Diverticulosis, diverticulitis, gastrointestinal bleeding, appendicitis, abdominal infection, appendiceal perforation, antibiotics, appendectomy, colectomy, laparoscopy

Expression of CA2 and CA9 carbonic anhydrases in ulcerative colitis and ulcerative colitis-associated colorectal cancer.

Ulcerative colitis (UC) is characterized by chronic inflammation in the colonic mucosa and submucosa with repeating relapse and remission, but the pathogenesis is unknown. Patients with long-standing UC are at high risk of neoplasm development. The aim of the present study was to identify molecules whose expression is associated with UC and UC-associated colorectal cancer (UCCA). Biopsy specimens from UC and normal colonic mucosae were analyzed using a proteomics approach, in which carbonic anhydrase 2 (CA2) was identified as a molecule downregulated in UC mucosae. Immunohistochemically, CA2 expression was detected in normal and diverticulitis mucosal epithelia, and its expression decreased as UC activity increased. CA2 expression was almost undetectable in UCCA. We also analyzed the expression of another carbonic anhydrase, CA9, and its upstream molecule, hypoxia-inducible factor-1α (HIF-1α), both of which are induced under hypoxic conditions. It was revealed that CA9 expression was relatively low in normal, diverticulitis and UC mucosae, and was upregulated in UCCA. HIF-1α expression was consistently low in all tissue types examined. In conclusion, these results suggest that CA2 and CA9 may be possible indicators of UC activity and UCCA development, respectively.

Time-Dependent Indirect Antioxidative Effects of Oat Beta-Glucans on Peripheral Blood Parameters in the Animal Model of Colon Inflammation.

Background: Oat beta-glucans are polysaccharides, belonging to soluble fiber fraction, that show a wide spectrum of biological activity. The aim of this study was to evaluate the time-dependent antioxidative effect of chemically pure oat beta-glucan fractions, characterized by different molar mass, which were fed to animals with early stage of 2,4,6-trinitrobenzene sulfonic acid (TNBS) - induced colitis. Methods: The study was conducted on 150 adult male Sprague Dawley rats assigned to two groups—healthy control (H) and colitis (C) with colon inflammation induced by per rectum administration of TNBS. The animals from both groups were divided into 3 nutritional subgroups, receiving for 3, 7 or 21 days AIN-93M feed without beta-glucan (βG−) or with 1% (w/w) low molar mass oat beta-glucan (βGl+) or 1% (w/w) high molar mass oat beta-glucan (βGh+). After 3, 7 and 21 days, the animals were euthanized, peripheral blood was collected from the heart for further analysis. Results: The results of analyses performed on blood samples showed small changes in lymphocytes count and red blood cell parameters such as the number of red blood cell, mean corpuscular hemoglobin concentration and mean corpuscular volume (RBC, MCHC, MCV respectively) as well as normalization of antioxidant potential accompanying moderate inflammatory state of colon mucosa and submucosa. Conclusion: Oat beta-glucans exert an indirect antioxidant effect in animals with TNBS-induced colitis, with greater effectiveness in removing systemic effects of colon inflammation found for low molar mass oat beta-glucan.

Anti-Ulcerative Effect of Curcumin-Galactomannoside Complex on Acetic Acid-Induced Experimental Model by Inhibiting Inflammation and Oxidative Stress.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that affects the mucosa and submucosa of colon. The pathogenesis of ulcerative colitis (UC) is related to reduced antioxidant capacity and increased inflammatory processes. Reactive oxygen metabolites are the potent inflammatory mediators that may be involved in tissue injury in inflammatory bowel disease. Conventional drug therapies for UC come with a myriad of side effects which further raise the need for natural bioactive agents. Curcumin has proven to be beneficial in the prevention and treatment of a number of inflammatory diseases, but due its poor bioavailability, thetherapeutic applications are limited. Thus, to enhance its bioavailability, a new formulation- curcumin-galactomannoside (CGM)- was made by complexing curcumin with galactomannans derived from fenugreek. The present study aims to evaluate the effects of CGM on experimental UC model. Adult male Wistar rats were divided into 5 groups: normal control rats (NC); ulcerative colitis control rats (UC); UC + sulfasalazine (SS) treated; UC + curcumin (CM) treated; and UC + CGM supplemented for 21days. The colonic mucosal injury was assessed by macroscopic and histological examination, along with evaluation of antioxidant status, inflammatory mediators, and gene expressions. Administration of CGM significantly enhanced antioxidant activities and decreased the level of inflammatory mediators and also suppressed the expression of inflammatory markers as compared with other groups. In conclusion, findings from these results reveal that CGM exerts marked curative effects on acute experimental colitis, possibly by regulating the antioxidant status and modulating inflammatory cascade.

A118 GUT MICROBIOTA TRANSPLANTATION FROM A PATIENT WITH SEVERE CONSTIPATION INDUCED CHANGES IN COLONIC FUNCTION AND STRUCTUR OF GNOTOBIOTIC MICE

Abstract Background Increasing evidence suggests that gut microbiota play a key role in gastrointestinal (GI) tract function. We have previously shown that fecal microbiota transplantation diarrhea predominant IBS patients into germ-free mice induces faster GI transit, increased permeability and innate immune activation. However, it is unknown whether gut dysfunction is induced by microbiota from patients with chronic constipation. Aims Here, we investigated the role of the intestinal microbiota in the expression of severe slow transit constipation in a patient with previous C difficile infection and extensive antibiotic exposure. Methods Germ-free (GF) mice (14 weeks old) were gavaged with diluted fecal content from the patient with constipation (PA) or a sex and age-matched healthy control (HC). 12 weeks later, we assessed gut motility and GI transit using videofluoroscopy and a bead expulsion test.. We then investigated intestinal and colonic smooth muscle isometric contraction in vitro using electric field stimulation (EFS), and acetylcholine (Ach) release was assessed by superfusion using [3H] choline. Histological changes were evaluated by H&E and immunohistochemistry. Results Mice with PA microbiota had faster whole GI transit (score 18.9 ± 0.9 (N=9) than mice with HC microbiota (15.4 ± 1.0, N=10, p=0.032), with markers located mainly in the distal small bowel and cecum. However, bead expulsion from the colon was significantly longer in PA mice (420.8 s ± 124.6 s, N=9) than in HC mice (82.6 s ± 20.0 s, N=10, p=0.026). This delayed colonic transit was likely due to colonic retroperistalsis visualized videofluoroscopically by retrograde flow of barium in the right colon of PA mice. There was no difference between the two groups in small intestinal or colonic tissues in Ach release or contractility induced by carbachol or KCl,. EFS caused transient biphasic relaxation and contraction in small intestine and colon, with the colonic contraction being stronger in the PA group. Microscopic tissue analysis showed disruption of the interstitial cells of Cajal (ICC) network and increased lymphocyte infiltration in colonic mucosa and submucosa in PA mice. Conclusions These results indicate that the microbiota is a driver of delayed colonic transit in a patient whose constipation started following extensive antibiotic exposure for C. difficile infection. The observed dysmotility pattern was not due to lower muscle contractility but likely caused by immune mediated changes in the ICC network. Funding Agencies CIHR

P016 Efficiency of evaluating the expression of Toll-like receptors 2, 4, 6 and proteins proteomic profiling as integral indicators for predicting the risk of early relapse of ulcerative colitis

Abstract Background Success was made in the study of TLR in congenital and adaptive immunity, which determined a new look at immune processes at ulcerative colitis (UC). Modern achievements of proteomic methods of analysis research allow to define molecular characteristics of the inflammation in colon mucosa of patients with UC. Methods The study included 86 patients with UC, an average age of 39.0 ± 1.4 years. Groups: 1–15 (17.4%) patients with distal form of UC, 2–42 (48%) left-sided form, 3–29 (33.7%) patients with total UC. The expression of TLR on peripheral blood monocytes was determined in the immunofluorescence test. Two-colour analysis was performed on a flowthrough laser cytofluorimeter (Cytomics FC500, Beckman Coulte). The percentage of monocytes (CD14 + cells) carrying TLR2, TLR4, and TLR6 on their surface was assessed. The separation of proteins of colon mucosa was based on technologies of IEF, SDS–PAGE, 2DPAGE, by standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, Bruker, USA). The getting of mass spectrogram was determined by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, Bruker, USA). Statistical analysis was performed using the software Statistica 10.0 (Statsoft). Results The direct average relationship was established between the number of monocytes expressing CD14 + CD282 +, CD14 + CD284 +, CD14 + CD286 + and the area of inflammation (r = 0.49, r = 0.55, r = 0.42, p < 0.05). The nonlinear regression equation was used. Calculation example: the risk of recurrence UC = exp (−26.1 + (0.4) × TLR 2)/(1 + exp (−26.1 + (0.4) × TLR 2)), χ2 = 130, 59, p < 0.0001. Thus, when the number of monocytes expressing TLR2 is not more than 60%, the risk of recurrence of the UC is not more than 11%, with values above 70%, the probability of recurrence exceeds 80%. We identified potentially new molecular markers of the early relapse of ulcerative colitis: SMAD2 activates the transcription of TFG1β and leads to development of fibrosis in colon submucosa in patients with UC; significant decrease of the expression of PPARγ promotes the activation of STAT and AP-1 signalling pathways that promotes the increase of the synthesis of IL-2, 6, 8, 12, TNFα, the activity of immune and inflammation processes in colon mucosa; the reduction of expression of β-defensin-1 in cells of colon mucosa is accompanied with increased expression of CCR6 that promotes the formation of inflammatory infiltrates in colonic submucosa in UC. Conclusion Expression of TLR 2, 4, 6 in blood monocytes (the risk of recurrence UC ratio) and new protein molecular markers of colon mucosa (SMAD2, PPARγ, and apoС-III) can be used as a tool for prediction of early relapse UC.

P078 Efficiency of evaluating the expression of Toll-like receptors 2, 4, 6 and proteins proteomic profiling as integral indicators for predicting the risk of early relapse of ulcerative colitis

Abstract Background Success was made in the study of TLR in congenital and adaptive immunity, which determined a new look at immune processes at ulcerative colitis (UC). Modern achievements of proteomic methods of analysis research allow to define molecular characteristics of the inflammation in colon mucosa of patients with UC. Methods The study included 86 patients with UC, an average age of 39.0 ± 1.4 years. Groups: 1–15 (17.4%) patients with distal form of UC, 2–42 (48%) left-sided form, 3–29 (33.7%) patients with total UC. The expression of TLR on peripheral blood monocytes was determined in the immunofluorescence test. Two-colour analysis was performed on a flow-through laser cytofluorimeter (Cytomics FC500, Beckman Coulte). The percentage of monocytes (CD14 + cells) carrying TLR2, TLR4, TLR6 on their surface was assessed. The separation of proteins of colon mucosa was based on technologies of IEF, SDS–PAGE, 2DPAGE (Bruker, USA). The getting of mass spectrogram was determined by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, Bruker, USA). Statistical analysis was performed using the software Statistica 10.0 (Statsoft). Results The direct average relationship was established between the number of monocytes expressing CD14 + CD282 +, CD14 + CD284 +, CD14 + CD286 + and the area of inflammation (r = 0.49, r = 0.55, r = 0.42, p < 0.05). The nonlinear regression equation was used. Calculation example: the risk of recurrence UC = exp (−26.1 + (0.4) × TLR 2)/(1 + exp (−26.1 + (0.4) × TLR 2)), χ2 = 130, 59, p < 0.0001. Thus, when the number of monocytes expressing TLR2 is not more than 60%, the risk of recurrence of the UC is not more than 11%, with values above 70%, the probability of recurrence exceeds 80%. We identified potentially new molecular markers of the early relapse of ulcerative colitis: SMAD2 activates the transcription of TFG1β and leads to development of fibrosis in colon submucosa in patients with UC; significant decrease of the expression of PPARγ promotes the activation of STAT and AP-1 signalling pathways that promotes the increase of the synthesis of IL-2,6,8,12,TNFα, the activity of immune and inflammation processes in colon mucosa; the reduction of expression of β-defensin-1 in cells of colon mucosa are accompanied with increased expression of CCR6, that promotes the formation of inflammatory infiltrates in colonic submucosa in UC. Conclusion Expression of TLR 2,4,6 in blood monocytes (the risk of recurrence UC ratio) and new protein molecular markers of colon mucosa (SMAD2, PPARγ and apoС-III) can be used as a tool for prediction of early relapse UC.

P073 Compartmentalised human gut immunity: studies of innate and adaptive lymphocyte distribution in the epithelium, lamina propria and GALT of healthy gut mucosa by flow cytometry

Abstract Background The human gut mucosal immune system is compartmentalised in distinct and specialised immune niches. The epithelium and the lamina propria have been proposed as effector sites, while gut-associated lymphoid tissues (GALTs) constitute inductive immune niches. The major mucosal GALTs are the Peyer’s patches in the ileum and the colonic isolated lymphoid follicles (ILFs), scattered in the submucosa of the colon. The majority of studies of human gut immune function in health and disease have analysed unfractionated mucosal tissue samples. Hence, in contrast to mice, little is known about compartmentalised immune cell specialisation in the human gut. The aim of this study was to use novel dissection methods to analyse separate human gut immune niches. Methods Macroscopically healthy margins from colorectal cancer colectomies were obtained at a minimum distance of 10 cm from the tumour border. After faeces, mucus, fat and muscle removal, Peyer’s patches were identified and dissected using a stereomicroscope (based on Keita et al., Lab Invest, 2006). Colonic mucosa and submucosa (containing ILFs) fractions were mechanically separated by forceps (based on the method developed by Fenton et al., Immunity, under revision). Isolation of epithelial and lamina propria fractions from the mucosal compartment was performed by calcium chelation (DTT and EDTA) and enzymatic digestion (Collagenase II and DNAse), respectively. Cell suspensions from each fraction were analysed by flow cytometry (BD LSR-Fortessa and BD FACSymphony). Results As expected, mucosal GALTs were characterised by an enrichment of germinal centre B cells (CD19+CD20+CD38+), lymphoid tissue-like innate lymphoid cells (Lin−CD127+CD117+Nrp1+) and a higher CD4+/CD8+ T-cell ratio vs. mucosa, whereas the mucosal fraction was enriched for plasma cells (CD19+CD20−CD38high) and distinguished by a decreased CD4+/CD8+ T-cell ratio as compared with the GALT in both ileum and colon. CD19+/CD3+ ratios were only higher in Peyer’s patches but not in colonic submucosa enriched with ILFs, possibly due to the smaller size of the B-cell follicles in the latter. The intraepithelial compartment lacked B cells and contained more γδ-T cells as compared with the GALT and lamina propria. Conclusion We have used novel dissection methods in human intestinal tissues that reveal a compartmentalised immune cell specialisation that is in line with what has previously been described in mice. The method will allow for future deeper analysis of the human gut immune niches in health and disease, such as in inflammatory bowel disease.

Identification of spinal afferent nerve endings in the colonic mucosa and submucosa that communicate directly with the spinal cord: The gut-brain axis.

The major sensory nerve pathway between the colon and central nervous system (spinal cord and brain) that underlies the gut-brain axis, is via spinal afferent neurons, with cell bodies in dorsal root ganglia (DRG). Our aim was to identify the sensory nerve endings in the colon that arise from single colorectal-projecting DRG neurons. C57BL/6 mice were anesthetized and lumbosacral L6-S1 DRG injected with dextran biotin. Mice recovered for 7 days. The whole colon was then removed and stained to visualize single axons and nerve endings immunoreactive to calcitonin gene-related peptide (CGRP). Single axons arising from DRG were identified in the distal colon and their morphological features and CGRP immunoreactivity characterized. After entering the colon, single axons ramified rostrally or caudally along many rows of myenteric ganglia with little circumferential displacement, giving off varicose endings in multiple ganglia. Nerve endings arising from two classes of colorectal-projecting DRG neuron were identified. One class was peptidergic neurons that had nerve endings in circular muscle, myenteric ganglia, and submucosa. Another class of nonpeptidergic neurons innervated mucosal crypts, myenteric ganglia, and submucosa. Different morphological types of nerve endings which innervate different anatomical layers of colon can arise from the same axon and sensory neuron in DRG. These findings suggest single peptidergic and nonpeptidergic sensory neurons in DRG are potentially capable of detecting sensory stimuli from different anatomical layers of the colon, via different types of nerve endings.

Beneficial Effects of Oat Beta-Glucan Dietary Supplementation in Colitis Depend on its Molecular Weight.

Background: Inflammatory bowel diseases are an important health problem. Therefore, the aim of the present study was to compare the impact of isolated oat beta-glucan fractions of low and high molecular weight, taken as dietary supplementation, on inflammatory markers in the colitis model. Methods: Two groups of Sprague–Dawley rats—control and with experimentally induced colitis—were subsequently divided into three subgroups and fed over 21 days feed supplemented with 1% of low (βGl) or high (βGh) molecular weight oat beta-glucan fraction or feed without supplementation. The level of colon inflammatory markers, cytokines, and their receptors’ genes expressions and immune cells numbers were measured by ELISA, RT-PCR, and by flow cytometry methods, respectively. Results: The results showed moderate inflammation affecting the colon mucosa and submucosa, with significant changes in the number of lymphocytes in the colon tissue, elevated cytokines and eicosanoid levels, as well as disruption of the main cytokine and chemokine cell signaling pathways in colitis rats. Beta-glucans supplementation caused a reverse in the percentage of lymphocytes with stronger effects of βGh and reduction of the levels of the inflammatory markers, and improvement of cytokine and chemokine signaling pathways with stronger effects of βGl supplementation. Conclusions: The results indicate the therapeutic effect of dietary oat beta-glucan supplementation in the colitis in evident relation to the molecular weight of polymer.

2019 Lower Gastrointestinal Bleeding Due to Sevelamer-Induced Colitis

INTRODUCTION: Acute lower gastrointestinal (GI) bleeding is uncommonly due to medication induced-colitis, reported in less than 6% of all lower GI bleeds. Identifying medications that may cause GI bleeding is particularly important in chronically ill patients at high risk for morbidity and mortality. A commonly used medication in patients with advanced renal disease is sevelamer, a phosphate binder in renal failure patients. CASE DESCRIPTION/METHODS: A 53-year-old female with end-stage renal disease presented with hematochezia for 2 days. She notes dark black stools for two weeks followed by rectal bleeding. Her past history is notable for a renal transplantation in 2008 with graft failure requiring hemodialysis. She has no history of NSAID use and her prior screening colonoscopy showed no evidence of colitis. Her medication list was extensive and included sevelamer and cinacalcet. The patient was hemodynamically stable. Initial labs showed a hemoglobin of 6.0 g/dl, baseline was 8.0-9.0 g/dl. She was resuscitated with intravenous fluids and received 2 units of blood. Next, the patient’s upper endoscopy showed gastric erythema only with no stigmata of recent bleeding. Her colonoscopy showed a macroscopically normal appearing colon however random biopsies revealed expansion of the lamina propria with significant population of increased eosinophils. Several crystals were embedded within the colonic mucosa and submucosa resembling sevelamer crystals. Based on the pathology, she was diagnosed with sevelamer-induced colitis and this medication was discontinued. Follow-up at 3 months showed the patient remained asymptomatic without further GI bleeding. DISCUSSION: Sevelamer is a common medication used in renal failure patients. It is a resin that binds and prevents absorption of phosphate within the GI tract and has been shown to cause GI issues such as nausea, vomiting and diarrhea. Rarely it can crystalize, become embedded in the GI tract and ulcerate and cause bleeding. A few case reports have described rectosigmoid ulceration and pseudotumor presentation of sevelamer induced colitis.1,2 Understanding and recognizing the potential for sevelamer-induced colitis is important with such a widely used medication.

Mouse models for human colorectal cancer with liver metastasis

Objective: To established Apc(loxp/loxp)+Kras(LSL-G12D/-)+Smad4(loxp/loxp) transgenic mouse model that mimick the occurrence and development of human sporadic colorectal cancer(CRC) and its liver metastasis. Methods: C57BL/6-Apc(tm1Tyj)/J(Apc(loxP)), B6.129S4-Kras(tm4Tyj)/J(Kras(LSL-G12D)), 129S6-Smad4(tm2.1Cxd)/J(Smad4(loxP)) and C57BL/6J mice were crossed, and genotype with Apc(loxP/loxP)+Kras(LSL-G12D/-)and Apc(loxP/loxP)+Kras(LSL-G12D/-)+Smad4(loxP/loxP)were generated. Genotypes of the mice were identified by PCR and real-time quantitative PCR. The mice were divided into Apc(loxP/loxP)+Kras(LSL-G12D/-)+Smad4(loxP/loxP) group (n=20) and Apc(loxP/loxP)+Kras(LSL-G12D/-)group(n=24). Lentivirus expressing Cre enzyme and IRES-luciferase were injected into the submucosa of colon or rectum of the transgenic mice under colonoscopy. Intraabdominal injection of D-luciferase into mice every 4 weeks, imaging with small animal in vivo imaging system(IVIS). The tumor size, tumorigenesis rate and metastasis ratio were analyzed. At the end of the 20th week, the colorectal lesions and metastatic tissues of mice were stained with hematoxylin-eosin(HE) and the pathological changes were observed under microscope. Results: Apc(loxp/loxp)+Kras(LSL-G12D/-)+Smad4(loxp/loxp) and Apc(loxp/loxp)+Kras(LSL-G12D/-)transgenic mice were successfully bred. The colorectal stem cells of the transgenic mouse mutated leading tumor lesion and liver metastatic under the induction of Lentivirus(Cre-IRES-luciferase). The primary and metastatic foci of colorectal carcinoma and liver metastasis in mice were proved to be adenocarcinoma and liver metastatic carcinoma by histopathological examination. The primary tumor size inApc(loxP/loxP)+Kras(LSL-G12D/-)+Smad4(loxP/loxP) group and Apc(loxP/loxP)+Kras(LSL-G12D/-)group was(3.52±0.26) and(3.45±0.20)mm, respectively,without significant difference(t=0.872, P=0.388).The tumorigenesis rate was 70.0% and 50.0% respectively, and there was no significant difference between the two groups(χ(2)=0.440, P=0.507). The metastasis rate of two groups were 58.3% and 8.3%respectively(Fisher's exact test, P=0.027). Conclusions: In this study, the colorectal carcinogenesis and its spontaneously metastasis to the liver of CRC were induced by Lentivirus(Cre-IRES-luciferase) in our established transgenic mice,which successfully simulated the occurrence and development of human sporadic CRC and its liver metastasis.

Oral Supplementation of Tocotrienol-Rich Fraction Alleviates Severity of Ulcerative Colitis in Mice.

Ulcerative colitis (UC) is characterized by damaged colonic mucosa and submucosa layers that are caused by excessive inflammatory reactions and oxidative stress. This study aimed to examine the use of tocotrienol-rich fraction (TRF) in mitigating damages caused by UC on the colon epithelium. Dextran sulfate sodium (DSS)-induced UC mice were treated with vehicle control, TRF, alpha-tocopherol (αTP) and 5-aminosalicylic acid (5-ASA). Observable clinical signs, quality of stool, histopathological scoring, inflammatory and oxidative markers were assessed. Vitamin E levels of colons and plasma were quantified. Oral supplementation of TRF significantly reduced the severity of DSS-induced UC by lowering the disease activity index (DAI) and histopathological inflammatory scoring. TRF also attenuated the DSS-induced enlargement of spleen and shortening of the colon. TRF has demonstrated marked anti-inflammatory and antioxidative properties indicated by the attenuation of DSS-induced upregulation of inflammation and oxidative stress markers including interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), nitric oxide (NO), malondialdehyde (MDA) and pNF-κB. These improvements were similar to that of 5-aminosalicylic acid (5-ASA) treatment. In contrast, αTP did not demonstrate evident clinical and histopathological improvements. The superior protective effect of TRF may be ascribed to the preferential absorption of TRF by the gut mucosa. TRF alleviated the signs and symptoms of acute UC in murine model via the reduction of local inflammatory reactions and oxidative stress. These effects suggested that TRF could serve as a gut health supplement for preventive measures for UC condition in patients.

Current and Evolving Concepts on the Pathogenesis of Diverticular Disease.

Diverticulosis of the colon is the most common anatomic alteration of the human colon, and it is characterized by the out-pouching of the colonic mucosa and submucosa through the muscular layer. Recurrent abdominal pain is experienced by about 20% of patients with diverticulosis, and inflammation of diverticula may lead to acute diverticulitis. In the past few years, several studies have investigated the factors predisposing or triggering diverticular disease (DD) occurrence. Moreover, new physiopathological knowledge has been acquired. The aim of this study was to review current knowledge regarding the pathogenesis of DD. A search of PubMed and EMBASE database was performed to identify articles relevant to the pathogenesis of DD. Several papers have shown that genetic predisposition, environmental factors, and colonic dysmotility are implicated in the pathogenesis of DD. More recent studies have associated specific host immune responses, gut microbiota imbalance and therefore low-grade inflammation as contributors to symptom occurrence in DD and diverticulitis. Current and evolving evidence highlighted the role of genetic susceptibility, environment, colonic motility, visceral sensitivity, immune response, and microbiota in the pathogenesis of this disease. Further studies are required to identify potential targets for medical or surgical decision-making.

Coinfection with Entamoeba polecki and Brachyspira hyodysenteriae in a pig with severe diarrhea

Enteric disease in pigs is usually of multifactorial etiology, including infectious and non-infectious factors. In many cases of endemic diarrhea in weaner-to-finisher pigs, the combination of 2 or more microorganisms leads to aggravation of intestinal lesions and, consequently, clinical signs. We autopsied a 4-mo-old fattening pig with diarrhea and diagnosed severe fibrinonecrotizing typhlocolitis. Numerous spiral-shaped bacteria and amoeba-like PAS-positive protozoa were observed in the cecal and colonic mucosa and submucosa. Brachyspira hyodysenteriae was detected by PCR from colonic content. By in situ hybridization, large numbers of Entamoeba polecki were found within the lamina propria and submucosa; moderate numbers of Blastocystis sp. and scattered trichomonads were present in intestinal content. In addition, Entamoeba polecki, Balantidium spp., Blastocystis sp., and Trichomonas sp. were also detected by PCR.

Enteric peripheral neuroblastoma in a calf

An 11-month-old female Japanese Black calf had showed chronic intestinal symptoms. A large mass surrounding the colon wall that was continuous with the colon submucosa was surgically removed. After recurrence and euthanasia, a large mass in the colon region and metastatic masses in the omentum, liver, and lung were revealed at necropsy. Pleomorphic small cells proliferated in the mass and muscular layer of the colon. The cells were positively stained with anti-doublecortin (DCX), PGP9.5, nestin, and neuron specific enolase (NSE). Thus, the diagnosis of peripheral neuroblastoma was made. This is the first report of enteric peripheral neuroblastoma in animals. Also, clear DCX staining signal suggested usefulness of DCX immunohistochemistry to differentiate the neuroblastoma from other small cell tumors in cattle.