Inflamed Mucosa Of Ulcerative Colitis Research Articles

DOP38 A rare KRT17+ population emerges from the epithelium in the inflamed intestinal mucosa of patients with Ulcerative Colitis

Abstract Background KRT17 is an epithelial "alarmin" that contributes to the early host innate immune response to insults. We previously found that KRT17 transcription, not detectable in the healthy colon, is up-regulated in the intestinal mucosa of patients with active ulcerative colitis (UC). Here, we aimed to characterize the epithelial cells expressing KRT17 in UC mucosa and explore the contribution of the intestinal environment in promoting the emergence of this cell population. Methods We conducted single-cell RNAseq (scRNA-seq) on the epithelial compartment of intestinal biopsies from a cohort of non-inflammatory bowel disease (IBD) controls and patients with active UC. Analysis was performed using our own-curated pipeline in Seurat1. Spatial protein localization was analysed by immunofluorescence on fixed intestinal samples. To measure the modulation of KRT17 and associated markers, ex vivo air-liquid interface (ALI) epithelial cultures derived from human non-IBD organoids were used. The signatures of UC-derived organoids2 were mapped onto the scRNA-seq dataset. Results ScRNA-seq analysis identified a rare KRT17+ epithelial cell population emerging in active UC characterized by the expression of KRT17 (Fig. 1A) and other alarmins (KRT6A, KRT16, S100A8), metalloproteinases (MMP1, MMP10), and inflammatory markers (SAA1, DUOX2…). KRT17 protein, absent in non-IBD colon, was expressed in the epithelium of the inflamed UC mucosa exhibiting a patchy pattern, and marked epithelial clusters with both columnar and squamous-like morphology (Fig. 1B). The ALI culture closely recapitulated the differentiated intestinal epithelium in homeostasis by showing a columnar aspect, high expression of differentiation markers, and low or undetectable expression of stem/proliferation or KRT17+-cell markers. Exposure of the ALI culture to hypoxic stress (by re-submersion in expansion medium) induced the expression of KRT17+ cell-specific and inflammation markers (Fig. 1C), and the acquisition of a squamous morphology (Fig. 1D). IBD-related IFN-ɣ and IL-22 cytokines could not fully replicate this phenotype (Fig. 1E). Lastly, we observed that genes up-regulated in UC, compared to non-IBD organoids, were predominantly expressed within the KRT17+ population (Fig. 1F). Conclusion Our results suggest that a novel KRT17+ epithelial population may arise from the intestinal cells of inflamed UC mucosa under the influence of specific environmental factors. This population could contribute to the persistence of the disease over time.

Characteristics of flat-type ulcerative colitis-associated neoplasia on chromoendoscopic imaging with indigo carmine dye spraying.

Despite recent advances in endoscopic equipment and diagnostic techniques, early detection of ulcerative colitis-associated neoplasia (UCAN) remains difficult because of the complex background of the inflamed mucosa of ulcerative colitis and the morphologic diversity of the lesions. We aimed to describe the main diagnostic patterns for UCAN in our cohort, including lateral extension surrounding flat lesions. Sixty-three lesions in 61 patients with flat-type dysplasia that were imaged with dye chromoendoscopy (DCE) were included in this analysis. These DCE images were analyzed to clarify the dye-chromoendoscopic imaging characteristics of flat dysplasia, and the lesions were broadly classified into dysplastic and nondysplastic mucosal patterns. Dysplastic mucosal patterns were classified into two types: small round patterns with round to roundish structures, and mesh patterns with intricate mesh-like structures. Lesions with a nondysplastic mucosal pattern were divided into two major types: a ripple-like type and a gyrus-like type. Of note, 35 lesions (55.6%) had a small round pattern, and 51 lesions (80.9%) had some type of mesh pattern. About 70% of lesions with small round patterns and 49% of lesions with mesh patterns were diagnosed as high-grade dysplasia or carcinoma, while about 30% of lesions with small round patterns and 51% of lesions with mesh patterns were diagnosed as low-grade dysplasia. When a characteristic mucosal pattern, such as a small round or mesh pattern, is found by DCE, the possibility of UCAN should be considered.

Enhanced oxidative phosphorylation of IgG plasma cells can contribute to hypoxia in the mucosa of active ulcerative colitis.

Mucosal hypoxia is detected in the mucosa of ulcerative colitis (UC), however the mechanism and the cause of hypoxia is not fully understood, while a dense infiltration of plasma cells is observed in the inflamed mucosa of UC. When differentiating from a B cell to a plasma cell, the energy metabolism dramatically shifts from glycolysis to oxidative phosphorylation, which results in a large amount of oxygen consumption of the plasma cell. We hypothesized that the plasma cell infiltration into the inflamed mucosa contributes to the mucosal hypoxia in UC in part. We examined the association between mucosal hypoxia and plasma cell infiltration in UC. More IgG plasma cells (but not IgA plasma cells) were distributed, and the nuclear and cell sizes were enlarged in hypoxic mucosa compared to normoxic mucosa in UC. Oxidative phosphorylation signature genes of these IgG plasma cells were markedly upregulated compared to those of other lymphoid cells infiltrating the lamina propria of inflamed mucosa of UC. Enlarged IgG plasma cells, which increase in number in the inflamed mucosa of UC, can be related to the hypoxic state of the inflamed mucosa of UC.

Protein Kinase CK2 Maintains Reciprocal Balance Between Th17 and Treg Cells in the Pathogenesis of UC.

T helper 17 and regulatory T cells balance have crucial effects on the development of ulcerative colitis (UC). Currently, how to break this balance has not yet been found. Protein kinase CK2 is involved in the pathogenesis of immune-related disorders. However, its effects on the development of UC are obscure. The level of CK2 in the colonic tissues of UC patients was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and immune-histochemistry. Peripheral blood CD4+ T cells were treated with CK2 inhibitor CX4945 or transfected with Csnk2-interfering lentivirus; the mRNA expression and protein levels of inflammatory cytokines were detected by qRT-PCR, enzyme-linked immunosorbent assay, and flow cytometry. Moreover, CX4945 was administered to trinitrobenzene sulfonic acid (TNBS)-induced colitis mice model for determining the function of CK2 on the regulation of intestinal inflammation. The CK2 level was markedly increased in inflamed mucosa of UC and highly expressed in CD4+ T cells. Blockade of CK2 by CX4945 inhibited Th17 but promoted regulatory T-cell (Treg) immune responses in CD4+ T cells from patients with UC. Moreover, CK2 blockade alleviated TNBS-induced colitis in mice. Inhibition of CK2 suppressed Th17 but promoted Treg differentiation by decreasing the phosphorylation level of signal transducer and activator of transcription (STAT) 3 and increasing the phosphorylation level of STAT5. The RNA-Seq and co-immunoprecipitation analysis further showed that CK2 could interact with Sirtuin 1 (SIRT1) and downregulate SIRT1 expression, which participated in Th17 inhibition but promoted Treg differentiation. Sirtuin 1 upregulation ameliorated TNBS-induced colitis, whereas SIRT1 blockade aggravated TNBS-induced colitis in mice. CK2 have crucial effects on the development of UC by maintaining reciprocal balance between Th17 and Treg cells. Protein kinase CK2 blockade might be considered as a new therapeutic approach for UC treatment.

P066 Exploration of critical molecules for optimizing treatment by analyzing in flammatory cytokines in the intestinal mucosa of patients with Inflammatory Bowel Disease

Abstract Background Inflammatory cytokines initiate and sustain the perpetuation of processes leading to chronic inflammatory conditions such as inflammatory bowel diseases (IBD). Research into the cytokine biology of IBD has nurtured the dogmatic view that distinct immunotypes characterize different IBD subtypes. Recently, many biologics have emerged as the treatment options for IBD patients. Still, it is challenging to establish biomarkers for predicting their effectiveness because the cytokines that determine the IBD clinical phenotype vary in individual patients. Therefore, this study aims to explore critical molecules for optimizing treatment by analyzing inflammatory cytokines in the intestinal mucosa of IBD patients. Methods From September 2019, to February 2020, patients with ulcerative colitis (UC) and Crohn’s disease (CD) who underwent colonoscopy or flexible sigmoidoscopy, and the control group, patients who received colorectal polypectomy at our institution, were enrolled in the study. We took biopsies from inflamed ileal or colonic mucosa. We analyzed the gene expression of various inflammatory cytokines by real-time PCR using RT2 Profiler PCR Array (96-Well Format) Human Inflammatory Cytokines & Receptors (QIAGEN). Cytokines not included in this array were subjected to real-time PCR separately. Based on these data, we investigated the relationship between mucosal cytokines and clinical characteristics. Results The total number of patients was 23 (control 5, UC 12, CD 6) cases, the median age was 51 years (18–78), genders were fifteen males and eight females. Disease phenotype was divided into extensive colitis/ left-sided colitis/proctitis (10/1/1) in UC patients, and A1/A2/A3 (2/3/1), L1/L3 (2/4), B1/B1+p/B2/B3 (3/1/1/1) in CD patients. Seven patients were steroid-dependent, and six patients were refractory to the anti-TNFα agent. The higher gene expressions of CCR2/CXCL1/CXCL3/CXCL13, IL-8, IL-33, and SPP1 in inflamed mucosa of UC compared to those of CD (p<0.05). The positive correlation between Mayo score and IL-1R1 expression in intestinal mucosa was observed (r=0.68, p=0.028). The expression of IL-1β, IL-6, IL-8, and oncostatin M (OSM) was higher in steroid-dependent UC patients, and the primary non-responders to anti-TNFα therapies had higher expressions of IL-7 in colonic mucosa than the secondary non-responders in UC. Conclusion Our results suggest that UC exhibits different cytokine patterns in the intestinal mucosa from CD as reported previously. Exploration of cytokine expressions in the intestinal mucosa may lead to predicting refractory IBD. The identification of molecules that can predict therapeutic effectiveness helps to optimize IBD treatment in the future.

Difference in the clinical characteristic and prognosis of colitis-associated cancer and sporadic neoplasia in ulcerative colitis patients

BackgroundAlthough various studies have been conducted on colitis-associated cancer (CAC), few have assessed the differences in the clinical and endoscopic features, treatment, and prognosis of CAC and sporadic neoplasia (SN) in the inflamed mucosa of ulcerative colitis (UC) patients. AimsTo compare the characteristics of CAC and SN within the previously or currently inflamed mucosa. MethodsBetween 1997 and 2017, we retrospectively analyzed the endoscopic chart data of 348 colonic lesions from 266 UC patients. Non-dysplastic lesions and lesions located outside the inflamed mucosa were excluded. The diagnosis of CAC or SN was confirmed by conventional histopathological and immunohistochemical evaluation of p53 and Ki67. ResultsIn total, 74 patients with CAC (97 lesions) and 46 with SN (58) were enrolled. The proportions of patients with a younger age of onset of UC, with chronic persistent UC, and with severe inflamed mucosa were significantly higher in the CAC group. In the SN group, no flat lesions were found, whereas 26% of the lesions in the CAC group were flat. Sixteen patients died during a median follow-up of 6.1 years (interquartile range (IQR) 1.8–11.1)in the CAC group, whereas 1 patient died during a median follow-up 3.2 years(IQR 1.4–4.6) in the SN group. Mortality from colorectal cancer was significantly higher (P = 0.015) in the CAC group (12/68; 17.6%) than in the SN group (1/44; 2.3%). The 5-year survival rate was 100% in the SN group and 97% in the CAC group for lesions located in the mucosa or submucosa. ConclusionRecognizing differences in the characteristics of CAC and SN within the inflamed mucosa is critical to avoid unnecessary total colectomy in patients with SN.

MicroRNA-31 and MicroRNA-155 Are Overexpressed in Ulcerative Colitis and Regulate IL-13 Signaling by Targeting Interleukin 13 Receptor α-1.

Interleukin-13 (IL-13) is an important Type 2 T helper (Th2) cytokine, controlling biological functions in epithelium and has been linked to asthma, atopic dermatitis and ulcerative colitis (UC). Interleukin-13 signals through IL-13 receptor α-1 (IL13RA1 (gene) and IL13Rα1 (protein)), a receptor that can be regulated by microRNAs (miRs). MicroRNAs are small non-coding single-stranded RNAs with a role in several pathologies. However, their relevance in the pathophysiology of UC, a chronic inflammatory condition of the colonic mucosa, is poorly characterised. Here, we determined the expression of IL13Rα1 in UC, its potential regulation by miRs and the subsequent effect on IL-13 signalling. Inflamed mucosa of UC patients showed decreased mRNA and protein expression of IL13RA1 when compared to healthy controls. We show that miR-31 and miR-155 are upregulated in inflamed UC mucosa and that both directly target the 3′ untranslated region of IL13RA1 mRNA. Transfection of miR-31 and miR-155 mimics reduced the expression of IL13RA1 mRNA and protein, and blocked IL-13-dependent phosphorylation of signal transducer and activator of transcription 6 (STAT6) in HT-29 cells, a gut epithelium cell line. Interleukin-13 activation of suppressor of cytokine signaling 1 (SOCS1) and eotaxin-3 (CCL26) expression was also diminished. MicroRNA-31/microRNA-155 mimics also downregulated IL13RA1 in ex vivo human inflamed UC biopsies. We propose that miR-31 and miR-155 have an important role in limiting IL-13 signalling in UC disease.

Expression of human cathelicidin peptide LL-37 in inflammatory bowel disease.

Cathelicidin peptide LL-37 plays an important role in the early host response against invading pathogens via its broad-spectrum anti-microbial activity. In this study, we investigated LL-37 expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, the regulatory mechanism of LL-37 induction was investigated in human colonic subepithelial myofibroblasts (SEMFs). LL-37 mRNA expression and protein secretion were analysed using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Intracellular signalling pathways were analysed using immunoblotting and specific small interference RNA (siRNA). The expression of LL-37 mRNA was increased significantly in the inflamed mucosa of ulcerative colitis and Crohn's disease. The Toll-like receptor (TLR)-3 ligand, polyinosinic-polycytidylic acid (poly(I:C), induced LL-37 mRNA expression and stimulated LL-37 secretion in colonic SEMFs. The transfection of siRNAs specific for intracellular signalling proteins [Toll/IL-1R domain-containing adaptor-inducing interferon (IFN) (TRIF), tumour necrosis factor receptor-associated factor (TRAF)6, transforming growth factor β-activated kinase (TAK)1] suppressed the poly(I:C)-induced LL-37 mRNA expression significantly. Poly(I:C)-induced phosphorylation of mitogen-activated protein kinases (MAPKs) and activated nuclear factor kappa B (NF-κB) and activating factor protein (AP)-1. siRNAs specific for NF-κB and c-Jun inhibited poly(I:C)-induced LL-37 mRNA expression. LL-37 suppressed lipopolysaccharide (LPS)-induced interleukin (IL)-6 and IL-8 expression significantly in colonic SEMFs. The expression of LL-37 was up-regulated in the inflamed mucosa of IBD patients. LL-37 was induced by TLR-3 stimulation and exhibited an anti-microbial effect via interaction with lipopolysaccharide (LPS).

Endoscopic morphologic features of ulcerative colitis–associated dysplasia classified according to the SCENIC consensus statement

Recent advances in endoscopic equipment and diagnostic techniques have made possible the detection of early dysplasia in the inflamed mucosa of ulcerative colitis (UC). The SCENIC consensus statement recommends the use of unified terminology for the morphology of dysplasia. In this study, we investigated the endoscopic features of high-grade dysplasia (HGD) in a clinical setting. We retrospectively identified 62 patients with UC who were diagnosed with colitis-associated cancer or HGD between 1997 and 2015. A total of 39 lesions of HGD detected by targeted biopsy sampling in 31 patients were reviewed, and the endoscopic morphology was classified according to the SCENIC guidelines. In total, 0 (0%), 6 (15.4%), 19 (48.7%), 12 (30.8%), and 2 (5.1%) lesions with HGD were classified as pedunculated, sessile, superficial elevated, flat, and depressed, respectively. Nearly 80% of the lesions were located in the rectum or sigmoid colon. All flat and depressed lesions were red in color. Typically, sessile/superficial elevated lesions accompanied a flat area (Is+IIb/IIa+IIb). Ulceration was observed in 2 depressed lesions (5.1%). Although the borders were indistinct in 21 lesions (53.8%) without the use of magnifying colonoscopy, all lesions could be distinguished from the surrounding mucosa using magnifying endoscopy. This is the first study to classify the morphologic features of HGD using the SCENIC guidelines in a clinical setting. Based on our findings, endoscopists should recognize that HGD is frequently associated with a flat/superficial elevated area and red discoloration and should inspect particularly carefully in the rectum and sigmoid colon. The findings of chromoendoscopy and magnifying colonoscopy may also be useful in distinguishing lesions from the surrounding mucosa.

MAIT cells are activated and accumulated in the inflamed mucosa of ulcerative colitis.

Ulcerative colitis (UC) is a chronic, relapsing and remitting, inflammatory disorder of the large intestine. Mucosal associated invariant T (MAIT) cells are a member of innate-like lymphocytes found abundantly in the mucosal tissue. The contribution of MAIT cells in the pathogenesis of UC is still unclear; therefore, this study aimed at investigating the role of these cells in patients with UC. The frequency of MAIT cells, as well as the production of cytokines and expression levels of activation markers by these cells in the peripheral blood of UC patients and healthy controls, was analyzed by flow cytometry. MAIT cells were also quantified in colon biopsies of UC patients using a confocal microscope. There was a significant reduction in MAIT cell frequency in the peripheral blood of UC patients compared with healthy controls (P < 0.0001). MAIT cells from UC patients secreted more interleukin (IL)-17 than healthy controls (P < 0.05). The expression levels of CD69 on these cells were correlated with disease activity and endoscopic scores and plasma levels of IL-18. Furthermore, MAIT cells increased in the inflamed mucosa, and their frequency was correlated with clinical and endoscopic disease activity in UC patients. The findings from this study indicate that MAIT cells could be associated with UC and may serve as potential biomarkers or therapeutic targets in UC.

Integrated miRNA and mRNA expression profiling in inflamed colon of patients with ulcerative colitis.

BackgroundUlcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.MethodologyColonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.ResultsWhen comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.ConclusionDifferential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.

Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

PTU-114 Expression of toll-like receptor (TLR)-2 and -4 in the intestinal crypt epithelial cells in inflammatory bowel disease (IBD)

IntroductionTLRs are pattern recognition receptors which detect conserved molecular patterns on commensal and pathogenic bacteria. Studies in mice have shown that TLR signalling protects against the development of colitis but...

Tu1850 Expression of Toll-Like Receptor (TLR)-2 and −4 in the Intestinal Crypt Epithelial Cells in Inflammatory Bowel Disease (IBD)

Introduction TLRs are pattern recognition receptors which detect conserved molecular patterns on commensal and pathogenic bacteria. Studies in mice have shown that TLR signalling protects against the development of colitis but may exacerbate established colitis. Epithelial stem cells and their progeny are located in crypts of the small and large intestine. We have investigated TLR2 and TLR4 expression in ulcerative colitis (UC) and Crohn9s disease (CD) crypt intestinal epithelial cells (IEC). Methods Ileal and colonic mucosal samples were obtained from operation resection specimens and were histologically normal (>5 cm from cancer; normal controls) or inflamed (CD or UC). Additionally, paired samples from histologically normal (proximal colon) and inflamed (distal colon) mucosa were obtained from five colectomy specimens with isolated left-sided UC. Crypt IECs were isolated using EDTA and pancreatin. Expression of mRNA transcripts was studied by conventional and real-time RT-PCR. Cell surface TLR protein was studied by flow cytometry (expressed as median fluorescent intensity, MFI). TLR expression was also studied by immunofluorescent (IF) staining of tissue sections. Data are expressed as median (IQR). Results Relative to normal controls, TLR2 mRNA expression in IEC was significantly elevated in inflamed colonic UC [3.18-fold (1.03–10.40), p=0.003] and inflamed colonic CD [3.45 (0.80–5.40), p=0.012]. TLR4 mRNA was significantly elevated in normal colonic UC [1.90 (1.69–4.31], p=0.017), inflamed colonic UC [2.33 (1.15–4.45), p=0.024], inflamed colonic CD [1.71 (1.00–4.32), p=0.042] and inflamed ileal CD [1.84 (1.43–4.66), p=0.03]. In paired analyses, IEC TLR2 and TLR4 mRNA expression was not significantly different in normal UC mucosa compared to inflamed UC mucosa. Compared to IEC from normal control colon, IEC from colonic CD showed significantly greater expression of cell surface TLR2 protein [MFI 10.1 (2.4–29.0) vs 33.1 (17.7–107.5), p=0.05] and TLR4 protein [MFI 12.1 (4.9–25.3) vs 40.9 (23.2–103.5), p=0.04]. IF staining confirmed TLR2 and TLR4 protein expression on the luminal surface of crypt IEC. Conclusion IEC expression of TLR4 mRNA was up-regulated in inflamed and un-inflamed colonic and ileal IBD. TLR2 mRNA was up-regulated in inflamed colonic IBD. Interestingly, TLR2 and TLR4 mRNA expression was similar in IECs isolated from un-inflamed, histologically normal mucosa as it was in IECs isolated from inflamed mucosa in UC. This may suggest TLR2 and TLR4 up-regulation is a primary rather than secondary event in UC. Moreover, TLR2 and TLR4 protein was expressed in greater amounts on the surface of IECs in CD. Competing interests None declared.

Peri-Appendiceal Red Patch and Pathogenesis of the Appendix in Ulcerative Colitis

To the Editor,We read with interest the article by Rubin and Roth [1]on the peri-appendiceal red patch (PARP) in ulcerativecolitis (UC). In 367 UC patients with distal colitis, 29(7.9%) patients had PARP. In the 29 patients, 23 (79%)were male, none had prior appendectomy, 20 of 30 (67%)biopsy findings showed that the parallel histologic activityin the PARP and the distal colitis, and 11 of 21 patients(52%) with endoscopic follow-up progressed to moreextensive disease. We believe that the pathogenesis of theappendix in UC patients should be highlighted.Although UC is characterized by continuous and diffuseinflammation extending proximally from the rectum, PARPhas been increasingly recognized in 48–86% patients withdistal UC [2–4], as in the study of Rubin and Rothe [1].Patients with PARP experience a more aggressive andrelapsing disease courses compared with patients withoutPARP [3]. Many case–control studies suggest that previousappendectomy is rare in UC patients [2, 3]. Patients withprevious appendectomy have a delayed onset of UC, areduced need for immunomodulators and proctocolectomy,and a reduced relapse rate and extent of UC [5]. Moreover,we and several investigators have reported the improve-ment of UC after appendectomy, especially in youngpatients with PARP [2, 6].The pathogenesis of UC has not been determined, but anabnormal mucosal immune response plays a major role inthe occurrence and pathophysiology of UC [2, 4]. Extensiveinfiltration of lymphocytes, especially CD4? T cells, hasbeen observed in the inflamed mucosa of UC patients.Activated CD4? T cells exhibit increased cytotoxic activityand secrete cytokines that enhance the inflammatory state,resulting in tissue injury. We have disclosed that the pro-portion of CD4? early-but-not-mature-activated T cells issignificantly increased in the appendix of UC patients [4, 7],and suspect that the appendix may be a priming site in theoccurrence of UC. We therefore believe that the appendixshould no longer be considered an evolutionary redundancy,especially in UC patients with PARP.References

Proteomic profiling of mucosal and submucosal colonic tissues yields protein signatures that differentiate the inflammatory colitides

Differentiating ulcerative colitis (UC) from Crohn's colitis (CC) can be difficult and may lead to inaccurate diagnoses in up to 30% of inflammatory bowel disease (IBD) patients. Much of the diagnostic uncertainty arises from the overlap of clinical and histologic features. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) permits a histology-directed cellular protein analysis of tissues. As a pilot study, we evaluated the ability of histology-directed MALDI-MS to determine the proteomic patterns for potential differences between CC and UC specimens. Mucosal and submucosal layers of CC and UC colon resection samples were analyzed after histologic assessment. To determine whether MALDI-MS would distinguish inflammation, the uninflamed (n = 21) versus inflamed submucosa (n = 22) were compared in UC and the uninflamed (n = 17) versus inflamed submucosa (n = 20) in CC. To determine whether there were proteomic differences between the colitides, the uninflamed UC submucosa (n = 21) was compared versus the uninflamed CC submucosa (n = 17), the inflamed UC submucosa (n = 22) was compared versus the inflamed CC submucosa (n = 20), and inflamed UC mucosa versus inflamed CC mucosa. Pairwise statistics comparisons of the subsets were performed. Pairwise comparative analyses of the clinical groups allowed identifying subsets of features important for classification. Comparison of inflamed versus uninflamed CC submucosa showed two significant peaks: m/z 6445 (P = 0.0003) and 12692 (P = 0.003). In the case of inflamed versus uninflamed UC submucosa, several significant differentiating peaks were found, but classification was worse. Comparisons of the proteomic spectra of inflamed submucosa between UC and CC identified two discrete significant peaks: m/z 8773 (P = 0.006) and 9245 (P = 0.0009). Comparisons of the proteomic spectra of uninflamed submucosa between UC and CC identified three discrete significant peaks: m/z 2778 (P = 0.005), 9232 (P = 0.005), and 9519 (P = 0.005). No significantly different features were found between UC and CC inflamed mucosa. MALDI-MS was able to distinguish CC and UC specimens while profiling the colonic submucosa. Further analyses and protein identification of the differential protein peaks may aid in accurately diagnosing IBD and developing appropriate personalized therapies.

Aberrant methylation of DAPK in long-standing ulcerative colitis and ulcerative colitis-associated carcinoma

Death-associated protein kinase (DAPK) has pro-apoptotic functions and participates in various apoptotic systems. DAPK acts as a tumor suppressor, and its inactivation by promoter hypermethylation has been frequently observed in various human cancers. As alterations of pro-apoptotic genes might cause instability in the balance of cell-turnover during chronic inflammatory processes, epigenetic silencing of DAPK might be involved in the carcinogenesis of ulcerative colitis-associated carcinoma (UCC). To evaluate the role of DAPK in the inflammation-driven carcinogenesis of ulcerative colitis (UC), we analyzed promoter hypermethylation and protein expression of DAPK using methylation-specific PCR and immunohistochemistry in 43 UCCs and paired UC-background mucosa, as well as in UC-background mucosa of 50 patients without UCC. The frequency of methylation of DAPK in UCCs was low (27.6%) compared to overall non-neoplastic UC-background mucosa (48.3%; p = 0.02) and sporadic colorectal carcinoma (57.4%, p = 0.019). The difference in the methylation frequency in UC-background mucosa in patients without UCC (54.2%), compared to those with UCC (40.0%), was not significant ( p = 0.141). Promoter methylation correlated significantly with decreased DAPK protein expression ( p < 0.001) and severity of inflammatory activity ( p = 0.024). In unmethylated UC-background mucosa, DAPK protein expression increased with activity of UC-associated inflammation, suggesting a protective role of the pro-apoptotic DAPK during the chronic inflammatory process of UC. Thus, inactivation of DAPK by promoter hypermethylation might be crucial for accumulation of DNA damage in inflamed mucosa of UC, and might therefore contribute to the initiation of the neoplastic process and development of UC-associated carcinoma.

Interleukin-33 expression is specifically enhanced in inflamed mucosa of ulcerative colitis

Interleukin (IL)-33 is a cytokine belonging to the IL-1 family. IL-33 has been shown to elicit a Th2-like cytokine response in immune cells. In this study, we investigated IL-33 expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for IL-33 expression in human colonic subepithelial myofibroblasts (SEMFs). IL-33 mRNA expression was determined by real-time polymerase chain reaction (PCR). IL-33 expression in the IBD mucosa was evaluated by immunohistochemical methods. IL-33 mRNA expression was significantly elevated in active lesions from patients with ulcerative colitis (UC), but was not detected in inactive lesions from UC patients or in lesions from patients with either active or inactive Crohn's disease. Colonic SEMFs were identified as a major source of IL-33 in the mucosa. IL-1β and tumor necrosis factor-α (TNF-α) significantly enhanced IL-33 mRNA and protein expression in isolated colonic SEMFs. IL-1β and TNF-α did not affect IL-33 expression in intestinal epithelial cell lines (HT-29 and Caco-2 cells). This IL-1β- and TNF-α-induced IL-33 mRNA expression was mediated by p42/44 mitogen activated protein kinase (MAPK) pathway-dependent activation of nuclear factor (NF)-κB and activator protein (AP)-1. IL-33, derived from colonic SEMFs, may play an important role in the pathophysiology of UC.

Altered expression of MUTYH and an increase in 8‐hydroxydeoxyguanosine are early events in ulcerative colitis‐associated carcinogenesis

8-Hydroxy-guanine (8-OH-G) mismatches readily with adenine residues, leading to a G : C to T : A transversion mutation. The human mutY homologue (MUTYH) excises adenine misincorporated opposite 8-OH-G during replication and suppresses mutations caused by reactive oxygen species. We defined the expression of 8-hydroxydeoxyguanosine (8-OHdG) and MUTYH in ulcerative colitis (UC)-associated neoplasia by immunohistochemistry and compared this with expression in UC patients without neoplasia and patients unaffected by UC. We also performed mutation analyses for MUTYH and K-ras. 8-OHdG was expressed more intensely in the mucosa of UC-associated neoplasia and UC without neoplasm than in the mucosa unaffected by UC. Immunohistochemistry with two different types of MUTYH antibody showed that UC-associated neoplasia and UC without neoplasia exhibited strong cytoplasmic expression and attenuated nuclear expression of MUTYH when compared with patients unaffected by UC. No pathological MUTYH mutations were detected in any of the UC-associated neoplasia cases. However, K-ras mutation was detected in two cases, one of which showed G : C to T : A transversion mutation and attenuated nuclear staining of MUTYH. In conclusion, inflamed mucosa of UC is exposed to oxidative damage. An increase in cytoplasmic MUTYH, rather than its mutation, may contribute to the promotion of carcinogenesis in UC.

Proliferation of immature plasma cells in pouchitis mucosa in patients with ulcerative colitis

Pouchitis is the most common complication of restorative proctocolectomy in patients with ulcerative colitis (UC). The etiology of pouchitis is not known. We have previously reported the specific and significant proliferation of immature plasma lineage cells in the ulcer bases and inflamed mucosa of UC. In the present study we report the results of a phenotypic study of ileal pouch mucosa. Biopsy samples were taken from the ileal pouch of 22 patients with UC (12 with pouchitis, 10 with a normal pouch) and 5 patients with familial adenomatous polyposis (FAP) (with a normal pouch) who underwent restorative proctocolectomy, and normal ileum of 10 patients with UC yet to undergo pouch surgery. Frozen sections were cut from fixed samples and reacted with various lymphocyte markers and anti-Ki-67 antibodies. Ki-67+ cells, CD19+ cells, and CD138+ cells were significantly increased in the pouchitis mucosa of patients with UC. Immunological double staining revealed significantly increased numbers of CD19+Ki-67+ cells and CD138+Ki-67+ cells in the pouchitis mucosa of patients with UC compared to noninflamed UC pouch, FAP pouch, and normal ileum of UC patients. The number of CD19+CD138+ cells was significantly increased in inflamed pouch mucosa. The increased number of CD19+CD138+ cells we observed represents proliferation of immature plasma cells. Moreover, the increase in labeling for Ki-67 among CD19 cells and CD138 cells suggests proliferative activity of these cells, consistent with their immaturity. Proliferation of these immature plasma cells suggests the possibility of involvement of UC-derived abnormality in the pathogenesis of pouchitis.