Non-TGFß profibrotic signaling in ulcerative colitis after in vivo experimental intestinal injury in humans.

Although impaired regeneration is important in many gastrointestinal diseases including ulcerative colitis (UC), the dynamics of mucosal regeneration in humans are poorly investigated. We have developed a model to study these processes in vivo in humans. Epithelial restitution (ER) and extracellular matrix (ECM) regulation after an experimental injury of the sigmoid colonic mucosa was assessed by repeated high-resolution endoscopic imaging, histological assessment, RNA sequencing, deconvolution analysis, and 16S rDNA sequencing of the injury niche microbiome of 19 patients with UC in remission and 20 control subjects. Human ER had a 48-h lag before induction of regenerative epithelial cells [wound-associated epithelial (WAE) and transit amplifying (TA) cells] along with the increase of fibroblast-derived stem cell growth factor gremlin 1 mRNA (GREM1). However, UC deconvolution data showed rapid induction of inflammatory fibroblasts and upregulation of major structural ECM collagen mRNAs along with tissue inhibitor of metalloproteinase 1 (TIMP1), suggesting increased profibrotic ECM deposition. No change was seen in transforming growth factor ß (TGFß) mRNA, whereas the profibrotic cytokines interleukin 13 (IL13) and IL11 were upregulated in UC, suggesting that human postinjury responses could be TGFß-independent. In conclusion, we found distinct regulatory layers of regeneration in the normal human colon and a potential targetable profibrotic dysregulation in UC that could lead to long-term end-organ failure, i.e., intestinal damage.NEW & NOTEWORTHY The study reveals the regulatory dynamics of epithelial regeneration and extracellular matrix remodeling after experimental injury of the human colon in vivo and shows that human intestinal regeneration is different from data obtained from animals. A lag phase in epithelial restitution is associated with induction of stromal cell-derived epithelial growth factors. Postinjury regeneration is transforming growth factor ß-independent, and we find a profibrotic response in patients with ulcerative colitis despite being in remission.

Dose-response relationships of intestinal organs and excessive mucus discharge after gynaecological radiotherapy.

BACKGROUND: The study aims to determine possible dose-volume response relationships between the rectum, sigmoid colon and small intestine and the 'excessive mucus discharge' syndrome after pelvic radiotherapy for gynaecological cancer. METHODS AND MATERIALS: From a larger cohort, 98 gynaecological cancer survivors were included in this study. These survivors, who were followed for 2 to 14 years, received external beam radiation therapy but not brachytherapy and not did not have stoma. Thirteen of the 98 developed excessive mucus discharge syndrome. Three self-assessed symptoms were weighted together to produce a score interpreted as 'excessive mucus discharge' syndrome based on the factor loadings from factor analysis. The dose-volume histograms (DVHs) for rectum, sigmoid colon, small intestine for each survivor were exported from the treatment planning systems. The dose-volume response relationships for excessive mucus discharge and each organ at risk were estimated by fitting the data to the Probit, RS, LKB and gEUD models. RESULTS: The small intestine was found to have steep dose-response curves, having estimated dose-response parameters: γ50: 1.28, 1.23, 1.32, D50: 61.6, 63.1, 60.2 for Probit, RS and LKB respectively. The sigmoid colon (AUC: 0.68) and the small intestine (AUC: 0.65) had the highest AUC values. For the small intestine, the DVHs for survivors with and without excessive mucus discharge were well separated for low to intermediate doses; this was not true for the sigmoid colon. Based on all results, we interpret the results for the small intestine to reflect a relevant link. CONCLUSION: An association was found between the mean dose to the small intestine and the occurrence of 'excessive mucus discharge'. When trying to reduce and even eliminate the incidence of 'excessive mucus discharge', it would be useful and important to separately delineate the small intestine and implement the dose-response estimations reported in the study.

Dissecting Common and Unique Effects of Anti-α4ß7 and Anti-Tumor Necrosis Factor Treatment in Ulcerative Colitis.

BACKGROUND AND AIMS: Vedolizumab is an anti-α4ß7 antibody approved for the treatment of ulcerative colitis [UC]. Although it is assumed that vedolizumab blocks intestinal homing of lymphocytes, its effects on different intestinal cell populations are not fully stablished. In order to establish the unique mechanisms of action of vedolizumab in UC patients, we compared its effects to those induced by anti-tumour necrosis factor [TNF]. METHODS: Patients with active UC [endoscopic Mayo score >1] starting vedolizumab [n = 33] or anti-TNF [n = 45] and controls [n = 22] were included. Colon biopsies [at weeks 0, 14 and 46] and blood samples [at weeks 0, 2, 6, 14, 30 and 46] were used for cell phenotyping, transcriptional analysis [qPCR], and to measure receptor occupancy. RESULTS: Vedolizumab, in contrast to anti-TNF, significantly reduced the proportion of α4ß7+ cells within intestinal T subsets while preserving the percentage of α4ß7+ plasma cells. The marked decrease in α4ß7 did not change the percentage of colonic αEß7+ cells [at 46 weeks]. Both vedolizumab and anti-TNF significantly downregulated inflammation-related genes in the colon of responders [Mayo score < 2]. Moreover, both treatments significantly decreased the percentage of intestinal, but not blood, total lymphocytes [T and plasma cells], as well as the proportion of α4ß1+ cells within intestinal T lymphocytes. CONCLUSIONS: Our data show that while vedolizumab and anti-TNF block two unrelated targets, they induce remarkably similar effects. On the other hand, vedolizumab's unique mechanism of action relies on blocking intestinal trafficking of α4ß7 T cells, despite effectively binding to B and plasma cells that express α4ß7.

Intrinsic cholinergic innervation in the human sigmoid colon revealed using CLARITY, three-dimensional (3D) imaging, and a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum.

BACKGROUND: We previously reported the specificity of a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum for immunostaining of cholinergic neuronal cell bodies and fibers in the human colon. In this study, we investigate 3D architecture of intrinsic cholinergic innervation in the human sigmoid colon and the relationship with nitrergic neurons in the enteric plexus. METHODS: We developed a modified CLARITY tissue technique applicable for clearing human sigmoid colon specimens and immunostaining with hpChAT antiserum and co-labeling with neuronal nitric oxide synthase (nNOS) antibody. The Z-stack confocal images were processed for 3D reconstruction/segmentation/digital tracing and computational quantitation by Imaris 9.2 and 9.5. KEY RESULTS: In the mucosa, a local micro-neuronal network formed of hpChAT-ir fibers and a few neuronal cell bodies were digitally assembled. Three layers of submucosal plexuses were displayed in 3D structure that were interconnected by hpChAT-ir fiber bundles and hpChAT-ir neurons were rarely co-labeled by nNOS. In the myenteric plexus, 30.1% of hpChAT-ir somas including Dogiel type I and II were co-labeled by nNOS and 3 classes of hpChAT-ir nerve fiber strands were visualized in 3D images and videos. The density and intensity values of hpChAT-ir fibers in 3D structure were significantly higher in the circular than in the longitudinal layer. CONCLUSIONS AND INFERENCES: The intrinsic cholinergic innervation in the human sigmoid colon was demonstrated layer by layer for the first time in 3D microstructures. This may open a new venue to assess the structure-function relationships and pathological alterations in colonic diseases.

Transcription Factors That Regulate the Pathogenesis of Ulcerative Colitis.

Ulcerative colitis (UC) is one of the inflammatory bowel diseases (IBD) characterized by occurrence in the rectum and sigmoid colon of young adults. However, the functional roles of transcription factors (TFs) and their regulating target genes and pathways are not fully known in ulcerative colitis (UC). In this study, we collected gene expression data to identify differentially expressed TFs (DETFs). We found that differentially expressed genes (DEGs) were significantly enriched in the target genes of HOXA2, IKZF1, KLF2, XBP1, EGR2, ETV7, BACH2, CBFA2T3, HLF, and NFE2. TFs including BACH2, CBFA2T3, EGR2, ETV7, NFE2, and XBP1, and their target genes were significantly enriched in signaling by interleukins. BACH2 target genes were enriched in estrogen receptor- (ESR-) mediated signaling and nongenomic estrogen signaling. Furthermore, to clarify the functional roles of immune cells on the UC pathogenesis, we estimated the immune cell proportions in all the samples. The accumulated effector CD8 and reduced proportion of naïve CD4 might be responsible for the adaptive immune response in UC. The accumulation of plasma in UC might be associated with increased gut permeability. In summary, we present a systematic study of the TFs by analyzing the DETFs, their regulating target genes and pathways, and immune cells. These findings might improve our understanding of the TFs in the pathogenesis of UC.

Identification of the HT-29 cell line as a model for investigating MCT1 transporters in sigmoid colon adenocarcinoma.

MCT1 transporters play a crucial role in the symbiotic relationship between humans and their colonic microbiome by facilitating the transport of bacteria-derived short chain fatty acids. Expression of colonic MCT1 transporters, localized in surface epithelial cells, is regulated by luminal butyrate levels. However, MCT1 also transports lactate and can be used by cancer cells to facilitate anaerobic glycolysis. Using immunolocalization techniques, this study investigated whether changes in MCT1 during cancer varied between different colonic regions. Whilst MCT1 abundance did not significantly change in transverse colon adenocarcinoma (P = 0.363, N = 6, paired T-Test), there was an increase in MCT1 in sigmoid colon adenocarcinoma (P = 0.010, N = 21, paired T-test). Using RT-PCR and western blotting, three human intestinal cell lines were tested for their suitability as a MCT1 cancer cell model. Experiments with Caco-2 cells confirmed that they modelled normal cells, with MCT1 only expressed after exposure to butyrate. In contrast, MCT1 was expressed in the absence of butyrate in both HCT-8 and HT-29 cell lines, with consistently high levels of MCT1 protein being present in HT-29 cells. Furthermore, butyrate treatment of HT-29 cells significantly decreased both MCT1 protein abundance (P < 0.001, N = 4, unpaired T-test) and glycosylation of its' chaperone protein, CD147 (P < 0.001, N = 4, unpaired T-test). These data suggest that (i) MCT1 transporter abundance increases in sigmoid colon adenocarcinoma, and (ii) HT-29 cells are an appropriate cell model with which to investigate MCT1 function in this disease.

Mucosal concentrations of N-acetyl-5-aminosalicylic acid related to endoscopic activity in ulcerative colitis patients with mesalamine.

BACKGROUND AND AIM: 5-Aminosalicylic acid (5-ASA) is a fundamental treatment for mild-to-moderate ulcerative colitis (UC). 5-ASA is taken up into the colonic mucosa and metabolized to N-acetyl-5-ASA (Ac-5-ASA). Few studies have assessed whether mucosal 5-ASA and Ac-5-ASA concentrations are associated with endoscopic remission. This study aimed to investigate differences in 5-ASA and Ac-5-ASA concentrations according to endoscopic activity. METHODS: This single-center, prospective, cross-sectional study was conducted between March 2018 and February 2019. UC patients who were administered with 5-ASA medication for at least 8 weeks before sigmoidoscopy were enrolled. Mucosal 5-ASA and Ac-5-ASA concentrations were measured using liquid chromatography with tandem mass spectrometry. The primary endpoint was defined as the difference in mucosal concentrations of 5-ASA and Ac-5-ASA, according to the Mayo endoscopic subscore (MES). RESULTS: Mucosal concentrations were analyzed in 50 patients. In the sigmoid colon, the median 5-ASA concentration in patients with MES of 0 (17.3 ng/mg) was significantly higher than MES ≥ 1 (6.4 ng/mg) (P = 0.019). The median 5-ASA concentrations in patients with Ulcerative Colitis Endoscopic Index of Severity ≤ 1 (16.4 ng/mg) were also significantly higher than in patients with Ulcerative Colitis Endoscopic Index of Severity ≥ 2 (4.63 ng/mg) (P = 0.047). In the sigmoid colon, the concentration of Ac-5-ASA was higher in patients with MES of 0 (21.2 ng/mg) than in patients with MES ≥ 1 (5.81 ng/mg) (P = 0.022). CONCLUSIONS: The present study showed that mucosal Ac-5-ASA concentrations, as well as 5-ASA concentrations, are higher in UC patients with endoscopic remission. Ac-5-ASA may be useful for a biomarker of 5-ASA efficacy.

Sialylation and fucosylation modulate inflammasome-activating eIF2 Signaling and microbial translocation during HIV infection.

An emerging paradigm suggests that gut glycosylation is a key force in maintaining the homeostatic relationship between the gut and its microbiota. Nevertheless, it is unclear how gut glycosylation contributes to the HIV-associated microbial translocation and inflammation that persist despite viral suppression and contribute to the development of several comorbidities. We examined terminal ileum, right colon, and sigmoid colon biopsies from HIV-infected virally-suppressed individuals and found that gut glycomic patterns are associated with distinct microbial compositions and differential levels of chronic inflammation and HIV persistence. In particular, high levels of the pro-inflammatory hypo-sialylated T-antigen glycans and low levels of the anti-inflammatory fucosylated glycans were associated with higher abundance of glycan-degrading microbial species (in particular, Bacteroides vulgatus), a less diverse microbiome, higher levels of inflammation, and higher levels of ileum-associated HIV DNA. These findings are linked to the activation of the inflammasome-mediating eIF2 signaling pathway. Our study thus provides the first proof-of-concept evidence that a previously unappreciated factor, gut glycosylation, is a force that may impact the vicious cycle between HIV infection, microbial translocation, and chronic inflammation.

Colorectal adenocarcinoma with enteroblastic differentiation: a clinicopathological study of five cases.

AIMS: Colorectal adenocarcinoma with enteroblastic differentiation (CAED) is a rare malignancy, and its clinicopathological characteristics have not yet been fully elucidated. This study aimed to elucidate the clinicopathological features of CAED through immunostaining of enteroblastic lineage markers alpha-fetoprotein (AFP), glypican-3 (GPC3), and spalt-like transcription factor 4 (SALL4). METHODS AND RESULTS: We identified five CAED cases (0.3%) from 1666 colorectal carcinomas, analysed the clinicopathological characteristics and performed immunostaining for AFP, GPC3 and SALL4. Three patients were male and two were female. All cases were located in the sigmoid colon or rectum. Histologically, all cases showed adenocarcinoma composed of cuboidal or columnar cells, with clear cytoplasm resembling the primitive gut; one case exhibited a partial hepatoid pattern. The depth of invasion was T2 and T3 in two and three cases, respectively. Lymphatic/venous invasion was found in all cases (100%), lymph node metastases in four of five cases (80%) and distant metastases in three of five cases (60%) (liver, two cases; lung, one case). Two patients died as a result of their disease during follow-up. Immunohistochemically, SALL4 and GPC3 were each positive in four of five cases, whereas one case with a hepatoid component was positive for AFP. All three CAED cases with distant metastases were GPC3-positive. CONCLUSIONS: CAED was frequently located in the sigmoid colon or rectum, showed aggressive behaviour, such as lymph node metastasis and distant metastasis, and had a dismal prognosis. In addition, CAED was immunoreactive to AFP, GPC3 or SALL4, indicating that these markers may be characteristic of CAED.

Alterations in enteric calcitonin gene-related peptide in patients with colonic diverticular disease: CGRP in diverticular disease.

Diverticular disease (DD) is one of the most prevalent diseases of the large bowel. Lately, imbalance of neuro-muscular transmission has been recognized as a major etiological factor for DD. Neuronal calcitonin gene-related peptide (CGRP) is a potent gastrointestinal smooth muscle relaxant shown to have a widespread effect within the alimentary tract. Nevertheless, CGRPergic innervation of the enteric ganglia has never been considered in the context of motility impairment observed in DD patients. Changes in CGRP and calcitonin receptor-like receptor (CRLR) abundance within enteric ganglia were investigated in sigmoid samples from symptomatic and asymptomatic DD patients using quantitative fluorescence microscopy. CGRP effect on gastrointestinal smooth muscle was investigated using organ bath technique. We found CGRP levels within the enteric ganglia to be declined by up to 52% in symptomatic DD patients. Conversely, CRLR within the enteric ganglia was upregulated by 41% in symptomatic DD. Longitudinal smooth muscle displayed an elevated (+10.5%) relaxant effect to the exogenous application of CGRP in colonic strips from symptomatic DD patients. Samples from asymptomatic DD patients consistently showed intermediate values across different experiments. In conclusion, the present study demonstrates that CGRPergic signaling is subject to alteration in DD. Our results suggest that a hypersensitization mechanism to gradually decreasing levels of CGRP-IR nerve fibers takes place during DD progression. Alterations to CGRPergic signaling in DD disease may have implications for physiological abnormalities associated with colonic DD.

Colonic neuropathology is not associated with autonomic dysfunction in Parkinson's disease.

INTRODUCTION: Dysautonomia in Parkinson's disease (PD) has been shown to be associated with disease severity and especially with the occurrence of dementia. One proposed explanation for this finding is that phosphorylated alpha-synuclein histopathology (PASH), the characteristic pathological feature of PD is more diffuse in dysautonomia-associated PD than in disease without dysautonomia, not only in the central nervous system but also in peripheral autonomic networks. The aim of this study was therefore to determine if colonic alpha-synuclein histopathology is associated with dysautonomia in PD. METHODS: A total of 43 PD patients participated in this study. For each patient, two biopsies were taken in the sigmoid colon and analyzed by immunohistochemistry with antibodies against phosphorylated alpha-synuclein and PGP 9.5. All patients had a complete neuropsychological and neurological assessment along with a comprehensive evaluation of dysautonomia with questionnaires (SCOPA-Aut, NMS-Quest, Rome III constipation criteria and dry eye symptoms) and functional tests (pupillometry, Saxon and Schirmer's tests, heart rate variability, orthostatic blood pressure measure and sympathetic skin response). RESULTS: Colonic PASH was observed in 20/43 PD patients. No differences were observed in autonomic symptoms and testing between patients with and without PASH. CONCLUSIONS: Although frequent in PD, autonomic dysfunction is not related to colonic PASH. In addition to the existing literature, our findings further suggest that each dysautonomic symptom in PD might not be associated with a more severe or diffuse PASH not only in the central nervous system but also in the peripheral autonomic nervous systems.

Sigmoid colon mucosal gene expression supports alterations of neuronal signaling in irritable bowel syndrome with constipation.

Peripheral factors likely play a role in at least a subset of irritable bowel syndrome (IBS) patients. Few studies have investigated mucosal gene expression using an unbiased approach. Here, we performed mucosal gene profiling in a sex-balanced sample to identify relevant signaling pathways and gene networks and compare with publicly available profiling data from additional cohorts. Twenty Rome III+ IBS patients [10 IBS with constipation (IBS-C), 10 IBS with diarrhea (IBS-D), 5 men/women each), and 10 age-/sex-matched healthy controls (HCs)] underwent sigmoidoscopy with biopsy for gene microarray analysis, including differential expression, weighted gene coexpression network analysis (WGCNA), gene set enrichment analysis, and comparison with publicly available data. Expression levels of 67 genes were validated in an expanded cohort, including the above samples and 18 additional participants (6 each of IBS-C, IBS-D, HCs) using NanoString nCounter technology. There were 1,270 differentially expressed genes (FDR < 0.05) in IBS-C vs. HCs but none in IBS or IBS-D vs. HCs. WGNCA analysis identified activation of the cAMP/protein kinase A signaling pathway. Nine of 67 genes were validated by the NanoString nCounter technology (FDR < 0.05) in the expanded sample. Comparison with publicly available microarray data from the Mayo Clinic and University of Nottingham supports the reproducibility of 17 genes from the microarray analysis and three of nine genes validated by nCounter in IBS-C vs. HCs. This study supports the involvement of peripheral mechanisms in IBS-C, particularly pathways mediating neuronal signaling. NEW & NOTEWORTHY Peripheral factors play a role in the pathophysiology of irritable bowel syndrome (IBS), which, to date, has been mostly evident in IBS with diarrhea. Here, we show that sigmoid colon mucosal gene expression profiles differentiate IBS with constipation from healthy controls. These profiling data and analysis of additional cohorts also support the concept that peripheral neuronal pathways contribute to IBS pathophysiology.

DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells From Pediatric Patients With Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate With Outcome.

BACKGROUND & AIMS: We analyzed DNA methylation patterns and transcriptomes of primary intestinal epithelial cells (IEC) of children newly diagnosed with inflammatory bowel diseases (IBD) to learn more about pathogenesis. METHODS: We obtained mucosal biopsies (N = 236) collected from terminal ileum and ascending and sigmoid colons of children (median age 13 years) newly diagnosed with IBD (43 with Crohn's disease [CD], 23 with ulcerative colitis [UC]), and 30 children without IBD (controls). Patients were recruited and managed at a hospital in the United Kingdom from 2013 through 2016. We also obtained biopsies collected at later stages from a subset of patients. IECs were purified and analyzed for genome-wide DNA methylation patterns and gene expression profiles. Adjacent microbiota were isolated from biopsies and analyzed by 16S gene sequencing. We generated intestinal organoid cultures from a subset of samples and genome-wide DNA methylation analysis was performed. RESULTS: We found gut segment-specific differences in DNA methylation and transcription profiles of IECs from children with IBD vs controls; some were independent of mucosal inflammation. Changes in gut microbiota between IBD and control groups were not as large and were difficult to assess because of large amounts of intra-individual variation. Only IECs from patients with CD had changes in DNA methylation and transcription patterns in terminal ileum epithelium, compared with controls. Colon epithelium from patients with CD and from patients with ulcerative colitis had distinct changes in DNA methylation and transcription patterns, compared with controls. In IECs from patients with IBD, changes in DNA methylation, compared with controls, were stable over time and were partially retained in ex-vivo organoid cultures. Statistical analyses of epithelial cell profiles allowed us to distinguish children with CD or UC from controls; profiles correlated with disease outcome parameters, such as the requirement for treatment with biologic agents. CONCLUSIONS: We identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes.

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

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

Multicenter Assessment of Immunohistochemical Methods for Pathological Alpha-Synuclein in Sigmoid Colon of Autopsied Parkinson's Disease and Control Subjects.

BACKGROUND: Conflicting results from studies of Lewy-type α-synucleinopathy (LTS) in colonic biopsies of subjects with Parkinson's disease (PD) prompted a two-part multicenter assessment. The first assessment, now published (Acta Neuropathol Commun 4 : 35, 2016), examined archived colonic biopsies and found that none of the tested methods was adequately sensitive or specific. OBJECTIVE: As the amount of nervous tissue in typical colonic biopsies may be insufficient, and the clinical diagnosis of PD not completely accurate, the objective of the current study was to use instead full-thickness sections of sigmoid colon from autopsy-proven PD and normal subjects. METHODS: Seven different immunohistochemical (IHC) methods were used, employing five different primary antibodies and four different combinations of epitope exposure and signal development protocols. Specific staining was defined as being restricted to morphological features consistent with neuronal elements. Stained slides from each subject were independently categorized as being positive or negative for LTS, and their density semi-quantitatively graded, by four raters blinded to diagnosis. RESULTS: Agreement and mean diagnostic performance varied markedly between raters. With the two most accurate raters, 5 methods achieved diagnostic accuracies of 70% or greater; one method had 100% accuracy and 100% inter-rater agreement. The submucosa had the highest prevalence of pathological LTS staining, followed by the muscularis and mucosa. CONCLUSIONS: The major conclusion of this study is that, when sufficient submucosa and LTS is present, and when specific staining is defined as being consistent with neuronal morphology, adequately-trained raters may reliably distinguish PD colon from control using suitable IHC methods.

Comprehensive site-specific whole genome profiling of stromal and epithelial colonic gene signatures in human sigmoid colon and rectal tissue.

The strength of associations between various exposures (e.g., diet, tobacco, chemopreventive agents) and colorectal cancer risk may partially depend on the complex interaction between epithelium and stroma across anatomic subsites. Currently, baseline data describing genome-wide coding and long noncoding gene expression profiles in the healthy colon specific to tissue type and location are lacking. Therefore, colonic mucosal biopsies from 10 healthy participants who were enrolled in a clinical study to evaluate effects of lignan supplementation on gut resiliency were used to characterize the site-specific global gene expression signatures associated with stromal vs. epithelial cells in the sigmoid colon and rectum. Using RNA-seq, we demonstrate that tissue type and location patterns of gene expression and upstream regulatory pathways are distinct. For example, consistent with a key role of stroma in the crypt niche, mRNAs associated with immunoregulatory and inflammatory processes (i.e., CXCL14, ANTXR1), smooth muscle contraction (CALD1), proliferation and apoptosis (GLP2R, IGFBP3), and modulation of extracellular matrix (MMP2, COL3A1, MFAP4) were all highly expressed in the stroma. In comparison, HOX genes (HOXA3, HOXD9, HOXD10, HOXD11, and HOXD-AS2, a HOXD cluster antisense RNA 2), and WNT5B expression were also significantly higher in sigmoid colon compared with the rectum. These findings provide strong impetus for considering colorectal tissue subtypes and location in future observational studies and clinical trials designed to evaluate the effects of exposures on colonic health.

P2Y1, P2Y2, and TRPV1 Receptors Are Increased in Diarrhea-Predominant Irritable Bowel Syndrome and P2Y2 Correlates with Abdominal Pain.

BACKGROUND: Previous studies indicated that P2Y1 and P2Y2 receptors, which are widely distributed in the enteric nervous system, are related to pain, while TRPV1 may contribute to visceral pain and hypersensitivity states in irritable bowel syndrome (IBS). Other studies showed that ATP activates the capsaicin-sensitive TRPV1 channel via P2Y receptors. AIM: To detect the expression of P2Y1, P2Y2, and TRPV1 receptors in diarrhea-predominant IBS (IBS-D) patients and analyze any correlations with abdominal pain and to investigate interactions between P2Y receptors and the TRPV1 receptor in IBS-D patients. METHODS: Rectosigmoid biopsies were collected from patients with IBS-D (n = 36) and healthy controls (n = 15). Abdominal pain was scored using a 10-cm visual analogue scale. Expression levels of P2Y1, P2Y2, and TRPV1 receptors in rectosigmoid biopsies were determined by real-time PCR and double-labeling immunofluorescence with specific antibodies. RESULTS: Both mRNA and protein expression levels of P2Y1, P2Y2, and TRPV1 receptors were increased in IBS-D compared with controls. Of these receptors, P2Y2 expression correlated with the maximum pain scores (p = 0.02, r = 0.63, Spearman correlation) in IBS-D patients. However, no relationships were detected between P2Y receptors and the TRPV1 receptor. CONCLUSION: In the present study, we identified an increased expression of P2Y1 and P2Y2 receptors in the rectosigmoid mucosa of IBS-D patients, and P2Y2 correlated with abdominal pain. Furthermore, we identified an increase in TRPV1 expression; however, there were no correlations found between P2Y receptors and the TRPV1 receptor.

Global Cytokine Profiles and Association With Clinical Characteristics in Patients With Irritable Bowel Syndrome.

OBJECTIVES: Evidence suggests that patients with irritable bowel syndrome (IBS) have an altered cytokine profile, although it is unclear whether cytokines are linked with symptom severity. We aimed to determine whether global serum and mucosal cytokine profiles differ between IBS patients and healthy subjects and whether cytokines are associated with IBS symptoms. METHODS: Serum from 144 IBS patients and 42 healthy subjects was analyzed for cytokine levels of interleukin (IL)-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, interferon (IFN)-γ, and tumor necrosis factor (TNF) by MSD MULTI-ARRAY. In total, 109 IBS and 36 healthy sigmoid colon biopsies were analyzed for mRNA expression of IL-8, IL-10, TNF, and FOXP3 by quantitative reverse transcription PCR. Multivariate discrimination analysis evaluated global cytokine profiles. Rectal sensitivity, oroanal transit time, and psychological and gastrointestinal symptom severity were also assessed. RESULTS: Global cytokine profiles of IBS patients and healthy subjects overlapped, but cytokine levels varied more in IBS patients. Serum levels of IL-6 and IL-8 tended to be increased and levels of IFN-γ tended to be decreased in IBS patients. Mucosal mRNA expression of IL-10 and FOXP3 tended to be decreased in IBS patients. Within both the full study cohort and IBS patients alone, serum level of TNF was associated with looser stool pattern, while subjects with more widespread somatic symptoms had increased serum levels of IL-6. Although neither IBS bowel habit subgroups nor patients with possible post-infectious IBS were associated with distinct cytokine profiles, a small cluster of IBS patients with comparatively elevated immune markers was identified. CONCLUSIONS: Global cytokine profiles did not discriminate IBS patients from healthy subjects, but cytokine profiles were more varied among IBS patients than among healthy subjects, and a small subgroup of patients with enhanced immune activity was identified. Also, association of inflammatory cytokines with some clinical symptoms suggests that immune activation may be of importance in a subset of IBS patients.

Short-term oxycodone treatment does not affect electrogenic ion transport in isolated mucosa from the human rectosigmoid colon.

OBJECTIVE: Opioid therapy is associated with altered secretion and motility of the gut. The relative contribution of decreased secretion to the development of opioid-induced constipation remains unknown. MATERIALS AND METHODS: Twenty-five healthy males were treated with oxycodone for 5 d in a placebo-controlled, randomised cross-over design. Gastrointestinal adverse effects were assessed with validated questionnaires (bowel function index and gastrointestinal symptom rating scale). Rectosigmoid mucosal biopsies were taken at baseline and on day 5 during both treatments and mounted in Ussing chambers. Electrogenic ion transport parameters (short circuit current (SCC) and slope conductance) were measured after addition of secretagogues (prostaglandin E2 (PGE2) (6 µm), theophylline (400 µm)), and an inhibitor (ouabain (200 µm)). Additionally, morphine (50 µm) was added to investigate the direct opioid effect on colonic mucosa. RESULTS: Questionnaires showed pronounced bowel symptoms, including constipation during oxycodone treatment (eight-fold increase in bowel function index score from day 1 to day 5 (p < 0.001) while no significant change occurred during placebo treatment (p = 0.47). Basal SCC and slope conductance did not differ between treatments (all p > 0.05) and application with PGE2, theophylline, and ouabain yielded comparable results on all examinations (all p > 0.05). Morphine application consistently did not evoke a change in ion transport. CONCLUSION: Compared to placebo, epithelial electrogenic ion transport is not altered in mucosal biopsies from the rectosigmoid colon following 5-d oxycodone treatment. The secretory mechanisms in isolated mucosa appear to play a negligible role in the development of opioid-induced constipation.

Comparative study of collagen deposition in the colon wall of patients operated for sigmoid diverticular disease.

PURPOSE: To investigate the deposition of collagen in the colon wall of patients with sigmoid diverticulitis. METHODS: Samples of sigmoid tissue from 15 patients (disease group), seven men and eight women aged 37-77 years who underwent surgery for the treatment of diverticulitis, were selected. For the control group, specimens from five patients, three men and two women aged 19-58 years undergoing emergency surgery for sigmoid trauma were selected. These subjects had no associated diseases. The histological study of the surgical specimens was performed by staining with hematoxylin-eosin and picrosirius and using a histochemical method for collagen quantification. RESULTS: Collagen deposition in the colon wall in terms of area (F), glandular epithelium (E) and total area was significantly higher in the disease group compared to control (p=0.003, p=0.026 and p=0.010, respectively). The collagen volume fraction (F fraction) and muscle tissue (M fraction) were also significantly higher compared to control (p=0.044 and p=0.026, respectively). The muscle (M area) and volume fraction of glandular epithelium (E fraction) did not differ significantly between the two groups, (p=0.074 and p=1.000, respectively). CONCLUSION: In this study, collagen deposition in the colon wall of the patients operated for sigmoid diverticulitis was higher compared to patients without the disease.