Quantitative measurements of crypts from advanced endoscopy imaging using deep learning-based segmentation.

Protein kinase C (PKC) activity was measured in the inflamed colonic mucosa of 24 patients with ulcerative colitis and in the normal colonic mucosa of 10 patients with other benign diseases. The particulate fraction activity in ulcerative colitis mucosa was significantly increased compared with that of normal mucosa (320 +/- 47 versus 200 +/- 30 pmol/min/mg protein; p less than 0.05). Inflamed ulcerative colitis mucosa also showed significantly increased total PKC activity in the particulate fractions compared with normal mucosa (147 +/- 26 versus 37 +/- 8 pmol/min/mg tissue; p less than 0.05). Mucosal samples from ulcerative colitis patients were divided into 12 with mild and 12 with severe inflammation by histologic examination. The particulate PKC activity of severely inflamed mucosa was significantly lower than that of mildly inflamed mucosa (p less than 0.05). These results indicate that colonic inflammation in ulcerative colitis may be associated with altered cellular PKC activity.

The distribution of binding sites for three lectins labeled with rhodamine was studied by using immunofluorescence microscopy in the colonic mucosa in ulcerative colitis.Binding sites for Dolichos biflorus agglutinin (DBA), a lectin specific to terminal α-N-acetylgalactosamine residue, and soy bean agglutinin (SBA), specific to teminal α or β-N-acetylgalactosamine residue of surface glycoprotein in the endoscopically uninvolved mucosa in ulcerative colitis were almost equal in distribution to those of normal human colon. These two lectins were markedly decreased in distribution in the endoscopically involved mucosa, whereas they were also decreased in the boundary zone which was considered as locating between the involved and the uninvolved portion. On the other hand, there was no definite difference in binding sites for concanavalin A (Con A), a lectin specific to terminal α-mannopyranoside, α-glucopyranoside, between normal colon and ulcerative colitis.These results suggest that a quantitative and/or qualitative alteration in the colonic mucin has an important role in the colonic mucosa in ulcerative colitis. And lectins may be developed into a powerful probe for the detection of biochemical change in the colonic mucosa in ulcerative colitis.

Inflammatory bowel diseases (IBD) are characterized by a sustained inflammatory cascade that gives rise to the release of mediators capable of degrading and modifying bowel wall structure. Our aims were (i) to measure the production of matrix metalloproteinase-3 (MMP-3), and its tissue inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), by inflamed and uninflamed colonic mucosa in IBD, and (ii) to correlate their production with that of proinflammatory cytokines and the anti-inflammatory cytokine, IL-10. Thirty-eight patients with IBD, including 25 with Crohn's disease and 13 with ulcerative colitis, were included. Ten controls were also studied. Biopsies were taken from inflamed and uninflamed regions and inflammation was graded both macroscopically and histologically. Organ cultures were performed for 18 h. Tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-1beta, IL-10, MMP-3 and TIMP-1 concentrations were measured using specific immunoassays. The production of both MMP-3 and the TIMP-1 were either undetectable or below the sensitivity of our immunoassay in the vast majority of uninflamed samples either from controls or from those with Crohn's disease or ulcerative colitis. In inflamed mucosa, the production of these mediators increased significantly both in Crohn's disease (P < 0.01 and 0.001, respectively) and ulcerative colitis (P < 0.001 and 0.001, respectively). Mediator production in both cases was significantly correlated with the production of proinflammatory cytokines and IL-10, as well as with the degree of macroscopic and microscopic inflammation. Inflamed mucosa of both Crohn's disease and ulcerative colitis show increased production of both MMP-3 and its tissue inhibitor, which correlates very well with production of IL-1beta, IL-6, TNF-alpha and IL-10.

We aimed to contrast the mucosal microbiota in Crohn's disease (CD) and ulcerative colitis (UC). We assessed the concept of localized dysbiosis by comparing the bacterial communities of inflamed and noninflamed mucosa of patients with inflammatory bowel disease (IBD) and by analysis of the microbiota composition at distinct gut compartments (ileum, cecum, mid-colon, and rectum). We performed 16S rDNA sequencing to analyze population structures. Quality control and operational taxonomic unit classification of reads were performed using mothur with statistical analyses executed in the R package, phyloseq. There was no variation in any phyla or genera comparing inflamed to noninflamed mucosa within CD (or UC) or when comparing different gut compartments within CD (or UC). There were differences between the inflamed and noninflamed mucosa between CD and UC: analysis of the inflamed IBD gut at the phylum level indicated that Bacteroidetes (P = 0.002) and Fusobacteria (P < 0.05) were detected more frequently in inflamed CD mucosa than in inflamed UC mucosa. Conversely, Proteobacteria and Firmicutes (P < 0.05) were more frequently observed in the inflamed UC mucosa. At the genus level, the abundance of Faecalibacterium (P ≤ 0.05), Bacteroides (P = 0.003), and Pseudomonas (P < 0.001) were significantly different between the inflamed CD and UC and the abundance of 13 genera were significantly different within the noninflamed mucosa. The noninflamed UC mucosa was the most different from non-IBD mucosa. Dramatic shifts of microbial communities were not observed between the noninflamed and inflamed mucosa within CD (or UC) although both the inflamed (and noninflamed) mucosa was different between CD and UC.

No available test objectively measures impairment of function of the inflamed colonic mucosa in ulcerative colitis. To study function we assessed rectal bicarbonate output by rectal dialysis in the presence of water and bacterial fatty acid (n-butyrate) in 21 controls, 18 patients with acute ulcerative colitis, 12 patients with ulcerative colitis in remission, and 12 patients with other forms of colitis. In acute ulcerative colitis, compared with controls, bicarbonate output and pH was reduced (p less than 0.001); stimulated bicarbonate output with bacterial fatty acid (incremental bicarbonate output) was reduced by 80% in acute ulcerative colitis (p less than 0.01). Results indicate that bicarbonate output is a useful and selective test of mucosal function in acute ulcerative colitis. A diminished incremental bicarbonate output with n-butyrate supports the view of inadequate oxidation of bacterial fatty acids in vivo by the mucosa in ulcerative colitis. Whether the test will prove to be an index of prognosis or will aid choice between medical or surgical therapy requires further study.

Ulcerative colitis with low-grade dysplasia

Aberrant expression of the cytidine DNA deaminase AID in enterocytes within inflamed mucosa in ulcerative colitis (UC) patients has been proposed to be involved in progression of UC to colitis-associated colorectal cancer (CA-CRC). Here, we followed the expression of AID and other nuclear cytidine DNA deaminases of the APOBEC family through several stages of progression of UC in a CA-CRC case (UC progressor) and in a cohort of UC progressors and non-progressors. In inflamed and high-grade dysplastic mucosa, AICDA and APOBEC1 mRNAs, but not APOBEC3-family enzyme mRNAs, were overexpressed compared to non-inflamed tissue. Immunohistochemical staining did not show expression of AID or APOBEC3 enzymes in the enterocytes, neither in specimens from the CA-CRC case nor in biopsies from the progressor and non-progressor cohort. APOBEC1 was highly expressed in the enterocytes throughout the colorectum from the CA-CRC case and in most biopsies examined from the progressor and non-progressor cohort. The expression was not correlated with the histology of the mucosa or the progression status of the patients. In the CA-CRC case, we identified AID- and APOBEC3-associated mutation signatures in inflamed, high-grade dysplasia and cancer genomes. In conclusion, our findings suggest that AID or other nuclear APOBECs are unlikely to drive the progression of UC to CA-CRC late during course of UC. However, we cannot exclude the possibility that bursts of AID and nuclear APOBEC3 activity may contribute to formation of genome instability in early phases of disease development.

P073 Ex vivo activation of triggering receptor expressed on myeloid cells 1 enhances the production of pro-inflammatory cytokines by inflammatory bowel disease mucosa

Mucosal addressin cell adhesion molecule 1 [MAdCAM-1] is upregulated in the vascular endothelium of the colonic mucosa in ulcerative colitis [UC]. Although the association between MAdCAM-1 expression and mucosal inflammation has been discussed, the association with the clinical course of UC patients has not been reported. In this study we investigated not only the association between mucosal MAdCAM-1 expression and mucosal inflammation, but also its association with subsequent relapse in UC patients with clinical remission. Eighty UC patients in remission who visited Kyoto Prefectural University of Medicine for follow-up for 2 years were included. Biopsy samples were collected during colonoscopy, and transcriptional expression levels of UC-related cytokines and MAdCAM-1 were quantified using real-time polymerase chain reaction. MAdCAM-1 mRNA expression and protein expression by immunohistochemistry were compared in patients who subsequently relapsed and those who remained in remission and were examined in relation to endoscopic findings, histological activity and cytokine expression. MAdCAM-1 expression was correlated with endoscopic severity, and significantly elevated in histologically active mucosa than inactive mucosa. Furthermore, MAdCAM-1 expression levels were closely correlated with those of several cytokines. MAdCAM-1 mRNA and protein expression were significantly higher in the relapse group than in the remission group, indicating that MAdCAM-1 expression in the mucosa is already elevated in UC patients in clinical remission who subsequently relapse. MAdCAM-1 expression in the colonic mucosa of UC patients is related to mucosal inflammation and subsequent relapse; it may serve as a marker for both relapse and therapeutic effectiveness in UC.

Telomere shortening occurs with human aging in many organs and tissues and is accelerated by rapid cell turnover and oxidative injury. To clarify the clinical importance of telomere shortening in colonic mucosa in ulcerative colitis (UC), we measured average telomere length using quantitative real-time PCR in non-neoplastic colonic mucosa in UC patients and assessed its relationship to various clinical subtypes. Relative telomere length in genomic DNA was measured in colonic biopsies obtained from rectal inflammatory mucosa from 86 UC patients as well as paired non-inflammatory proximal colonic mucosae from 10 patients. Data were correlated with various clinical phenotypes. In paired samples, average relative telomere length of rectal inflammatory mucosa was shortened compared to normal appearing proximal colon in eight out of ten cases (p=0.01). Telomere length shortening was significantly associated with more severe Mayo endoscopic subscore (p<0.0001) and cases needing surgery due to toxic megacolon or cancer occurrence (p=0.043). When the severe clinical phenotype was defined as having at least one of following phenotypes, more than two times of hospitalization, highest Mayo endoscopic subscore, steroid dependent, refractory, or needing operation, average relative telomere length was significantly shortened in the same phenotypes than the others (p=0.003). Telomere shortening is associated with more severe clinical phenotypes of UC, reflecting severe inflammatory state in the colonic mucosa.

In situ evidence of involvement of Schwann cells in ulcerative colitis: autocrine and paracrine signaling by A disintegrin and metalloprotease-17–mediated tumor necrosis factor α production

Crohn's disease (CD) is a condition characterized by excessive numbers of activated T cells in the mucosa. We investigated whether a defect in apoptosis could prolong T cell survival and contribute to their accumulation in the mucosa. Apoptotic, Bcl-2+, and Bax+ cells in tissue sections were detected by the TUNEL method and immunohistochemistry. T cell apoptosis was induced by IL-2 deprivation, Fas Ag ligation, and exposure to TNF-alpha and nitric oxide. TUNEL+ leukocytes were few in control, CD, and ulcerative colitis (UC) mucosa, with occasional CD68+ and myeloperoxidase+, but no CD45RO+, apoptotic cells. Compared with control and UC, CD T cells grew remarkably more in response to IL-2 and were significantly more resistant to IL-2 deprivation-induced apoptosis. CD T cells were also more resistant to Fas- and nitric oxide-mediated apoptosis, whereas TNF-alpha failed to induce cell death in all groups. Compared with control, CD mucosa contained similar numbers of Bcl-2+, but fewer Bax+, cells, while UC mucosa contained fewer Bcl-2+, but more Bax+, cells. Hence, the Bcl-2/Bax ratio was significantly higher in CD and lower in UC. These results indicate that CD may represent a disorder where the rate of T cell proliferation exceeds that of cell death. Insufficient T cell apoptosis may interfere with clonal deletion and maintenance of tolerance, and result in inappropriate T cell accumulation contributing to chronic inflammation.

We explored a Mask Region-based Convolutional Neural Network (Mask R-CNN) to detect macrophages in quantitative phase images, which were acquired by digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI). The Mask R-CNN deep learning architecture is capable to detect and segment single macrophage cells in quantitative phase images and allows to perform both tasks in a multi-stage process. Our results show that the combined detection and segmentation of cells through Mask R-CNN-based automated evaluation prospects a fast and robust screening in label-free high throughput microscopy.

Tu1311 Quantitative Proteomics in Ulcerative Colitis Reveals Mucosal Inflammation Reduces Levels of Keratins in the Insoluble Fraction of the Intermediate Filament Proteome

Fusion of Mask RCNN and attention mechanism for instance segmentation of apples under complex background