Human Colon Tissue Research Articles

S0832 Characterization of Intestinal Subepithelial PDGFRα(high), DLL1+, F3+ Cells in Ulcerative Colitis

INTRODUCTION: A deeper understanding of the cellular features of disease represents an important step in unlocking the pathogenesis of ulcerative colitis (UC). We recently characterized a novel cell type in the human fetal gut located in the subepithelial compartment adjacent to the epithelium expressing a unique combination of genes and niche factors. Due to their gene expression, we refer to these cells as intestinal, Subepithelial, PDGFR-αHI, DLL1+, F3+ (iSPαD3). Data suggests iSPαD3 cells are involved in intestinal development and maintenance of the stem cell niche. We aimed to determine if these cells are present in the adult colon, and if they play a role in the pathogenesis of inflammation in UC. METHODS: Primary human colonic tissues from non-IBD control and UC patients were obtained via surgical resection. Patients provided informed consent and this study was approved by the University of Michigan Institutional Review Board. Tissues were formalin fixed and paraffin embedded. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed. Further, we analyzed publicly available single cell RNA sequencing data and performed differential expression analysis on an iSPαD3 population. Data were queried for gene ontology term enrichment using the DAVID database. RESULTS: A population analogous to human fetal small intestinal iSPαD3 cells are present in the adult colon. In health iSPαD3 cells encircle crypts with a high degree of proximity and continuity. There are fewer cells that exhibit morphological changes in inflammation (Figures 1 and 2). In healthy colon iSPαD3 cells are enriched for genes involved in Wnt and NOTCH signaling, immune response to bacteria, B-cell regulation, TGFβ signaling, and cell growth. In inflammation they are enriched for genes related to apoptotic pathways, neutrophil chemotaxis, extracellular matrix, and regulation of IL-6, tumor necrosis factor, and JAK-STAT (Figure 3). CONCLUSION: iSPαD3 cells appear to have an important supportive role in the intestinal epithelium and are involved in the pathogenesis of inflammation in UC. Our study suggests that iSPαD3 cells function in homeostatic maintenance and containment of luminal microbiota in health. In contrast, inflammation is associated with loss of iSPαD3 homeostatic functions and in favor of apoptotic and inflammatory pathways, implicating these cells as important modulators in colonic inflammation from UC. More work is needed to elucidate a potential role for iSPαD3 cells in inflammatory bowel disease.Figure 1Figure 2Figure 3

Automated classification of protein subcellular localization in immunohistochemistry images to reveal biomarkers in colon cancer

BackgroundProtein biomarkers play important roles in cancer diagnosis. Many efforts have been made on measuring abnormal expression intensity in biological samples to identity cancer types and stages. However, the change of subcellular location of proteins, which is also critical for understanding and detecting diseases, has been rarely studied.ResultsIn this work, we developed a machine learning model to classify protein subcellular locations based on immunohistochemistry images of human colon tissues, and validated the ability of the model to detect subcellular location changes of biomarker proteins related to colon cancer. The model uses representative image patches as inputs, and integrates feature engineering and deep learning methods. It achieves 92.69% accuracy in classification of new proteins. Two validation datasets of colon cancer biomarkers derived from published literatures and the human protein atlas database respectively are employed. It turns out that 81.82 and 65.66% of the biomarker proteins can be identified to change locations.ConclusionsOur results demonstrate that using image patches and combining predefined and deep features can improve the performance of protein subcellular localization, and our model can effectively detect biomarkers based on protein subcellular translocations. This study is anticipated to be useful in annotating unknown subcellular localization for proteins and discovering new potential location biomarkers.

Tissue Rheology as a Possible Complementary Procedure to Advance Histological Diagnosis of Colon Cancer.

In recent years, rheological measurements of cells and tissues at physiological and pathological stages have become an essential method to determine how forces and changes in mechanical properties contribute to disease development and progression, but there is no standardization of this procedure so far. In this study, we evaluate the potential of nanoscale atomic force microscopy (AFM) and macroscopic shear rheometry to assess the mechanical properties of healthy and cancerous human colon tissues. The direct comparison of tissue mechanical behavior under uniaxial and shear deformation shows that cancerous tissues not only are stiffer compared to healthy tissue but also respond differently when shear and compressive stresses are applied. These results suggest that rheological parameters can be useful measures of colon cancer mechanopathology. Additionally, we extend the list of biological materials exhibiting compressional stiffening and shear weakening effects to human colon tumors. These mechanical responses might be promising mechanomarkers and become part of the new procedures in colon cancer diagnosis. Enrichment of histopathological grading with rheological assessment of tissue mechanical properties will potentially allow more accurate colon cancer diagnosis and improve prognosis.

Abstract CT113: A novel laser emission microscopy for early detection of colon cancer: Analysis of at-risk human colon from an interventional trial (research in progress)

Abstract Introduction: Accurate identification of neoplastic or pre-neoplastic lesions is traditionally dependent on microscopic identification of nuclear abnormalities. It is both time-consuming and challenging in early-stage lesions and difficult to detect. Laser-emission microscopy (LEM) that uses laser emission (not fluorescence) is an emerging technology that takes advantage of the finding that highly-proliferating cells like those in pre-neoplastic lesions can be distinguished from less proliferative cells by a lower laser emission dot density (LDD) when stained with a nuclear dye. We have successfully used this technique to detect (early) nuclear changes in a high-fat induced colon cancer model. LEM has also been used successfully to differentiate healthy tissue from cancerous tissue in human patients. Methods: This LEM system is capable of rapidly scanning tissue as large as 2 cm×2 cm in ~10 min. Both laser emission and regular fluorescence are imaged simultaneously, thus providing information regarding the lasing spot density and location within the human colon tissues. In this study, we employed colon biopsies from 12 human subjects (at risk for colorectal cancer). These subjects participated in a 90-day intervention, placebo-controlled, randomized trial. 8 subjects ingested known chemopreventives and 4 subjects took a placebo. Colon biopsies were taken from the sigmoid colon at baseline and after 90 days. The formalin-fixed, paraffin-embedded tissues were subjected to LEM microscopic assessment. For this, the tissues were sandwiched between two mirrors to form a laser cavity and laser excitation was applied. By comparing the laser emission spectra, highly proliferative tissue could be differentiated from less proliferative tissue. Post-treatment biopsies were compared to their baseline status. These biopsies were also stained with Ki67 to obtain their proliferation index to compare with the LEM results. Results: On average, there was a 20% increase in the LDD among the 8 subjects on chemopreventives while no change was seen in Ki67 positivity. In placebo group, Ki67 was decreased by 14% and LDD was 12% increased. On individual comparison, LDD and Ki67 data were correlated positively in 6 subjects. Conclusion: The preliminary data suggest that in histologically-normal but “at-risk” human colon LEM may pick early nuclear changes that may not be detected by a proliferation marker. Table.A comparison of lasing dot density (LDD) data to Ki67 positivity (Whole slide analysis*)Mean LDDPercent Positive NucleiChange RatioSubject IDPrePostPrePostLDDKi67Data correlated?Placebo 1491336504.75.40.741.15NoPlacebo 2851078159.28.70.920.95YesPlacebo 3573183558.96.01.460.67NoPlacebo 4659288469.76.71.340.69NoChemopreventive#1 1296733436.513.61.132.10YesChemopreventive#1 2528261406.57.71.161.20YesChemopreventive#1 3468453429.67.01.140.73NoChemopreventive#1 4758173358.25.60.970.68YesChemopreventive#2 1323046255.34.91.430.91NoChemopreventive#2 2508078408.29.11.541.11YesChemopreventive#2 35587400110.46.80.720.66YesChemopreventive#2 4491373226.94.91.490.71NoPlacebo combined1.12±0.340.86±0.23Chemopreventive combined1.20±0.281.01±0.49*Both mucosa and submucosa were involved in the analysis Citation Format: Muhammad N. Aslam, Yun-Lu Sun, Ingrid Bergin, Mohamed Ali H. Jawad-Makki, Karsten Knuver, Danielle (Kim) Turgeon, James Varani, Xudong Fan. A novel laser emission microscopy for early detection of colon cancer: Analysis of at-risk human colon from an interventional trial (research in progress) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT113.

Abstract 4690: ARD1-mediated NRF2 acetylation promotes human colon cancer cell proliferation

Abstract The transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2) is regarded as one of the main orchestrators of the cellular antioxidant and carcinogen-detoxifying response. NRF2 plays a pivotal role in protecting normal cells from carcinogenic insults, but it may also accelerate the proliferation and progression of cancer cells. Abnormally elevated expression or activation of NRF2 has been attributed to mutation, especially in the sequences involved in interaction with its negative regulator KEAP1. However, other mechanisms responsible for NRF2 overactivation/overexpression are less well understood. Arrest defective (ARD1), an N-terminal acetyltransferase that catalyzes N-terminal acetylation of target proteins, has lysine acetyltransferase activity and is involved in mediating various (patho)physiological processes, such as proliferation, apoptosis, autophagy, and differentiation. Aberrant overexpression of ARD1 is correlated with metastasis and poor prognosis in several types of cancer, suggesting that it may act as a tumor promoter. In the present study, we found that NRF2, acteyl-NRF2, and ARD1 were highly expressed in some human colon cancer cell lines (HCT-116, HCT-15, DLD1) and tissues obtained from colorectal cancer patients. Furthermore, overexpression of NRF2 and ARD1 was also verified by quantitative immunofluorescence analysis of colorectal tissue microarray. We noticed that silencing of NRF2 and ARD1 individually in human colon cancer cells (HCT-116) led to the decreases in viability, anchorage-independent growth, and migration capacity of HCT-116 cells. While there was no change in the protein level of ARD1 when NRF2 expression was knock down by siRNA, protein expression of Nrf2 and acteyl-NRF2 was markedly reduced by silencing of the ARD1 gene. This prompted us to explore the association between ARD1 and NRF2 in the human colon cancer cell proliferation and progression. Co-immunoprecipitation and immunocytochemical analyses showed that two proteins colocalized in the cytosol, and ARD1 directly bound to NRF2. Taken together, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer cells by acetylating and stabilizing this transcription factor. This study was supported by the Global Core Research Center (GCRC) Grant (No. 2011-003-0001) from the National Research Foundation (NRF) of Republic of Korea. Citation Format: Xizhu Fang, Yeon-Hwa Lee, Jie Zheng, Seong Hoon Kim, Jeong-Hoon Jang, Do-Hee Kim, Young-Joon Surh. ARD1-mediated NRF2 acetylation promotes human colon cancer cell proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4690.

Translation Initiation Factor eIF2Bε Promotes Stemness of Intestinal Epithelial Cells

Modulation of global mRNA translation is essential for intestinal stem cell function. In colorectal cancer, proliferating stem-like cells are defined by high WNT signaling and display dysregulated translational control. It is currently unclear how factors that coordinate global translation affect stem cell behavior. Here we identified nucleotide exchange factor eIF2Be as a translation initiation factor involved in intestinal epithelial stemness. In eIF2Be Arg191His mice, which have a homozygous point mutation that intrinsically impairs eIF2Be’s enzymatic function, we demonstrate that dysfunctional eIF2Be affects small intestinal crypt formation, proliferation and stemness marker expression. By hyperactivating stem cell activity in ex vivo organoids, using a GSK3β inhibitor to mimic loss of tumor suppressor Apc , we demonstrate that eIF2Be is essential for clonogenic potential and associated with an increase in global translation. Finally, we observe high eIF2Be expression in human colonic adenoma tissues, exposing eIF2Be as a potential target in modulation of stemness.

Zinc Phthalocyanine Photochemistry by Raman Imaging, Fluorescence Spectroscopy and Femtosecond Spectroscopy in Normal and Cancerous Human Colon Tissues and Single Cells.

Photodynamic therapy is a clinically approved alternative method for cancer treatment in which a combination of nontoxic drugs known as photosensitizers and oxygen is used. Despite intensive investigations and encouraging results, zinc phthalocyanines (ZnPcs) have not yet been approved as photosensitizers for clinical use. Label-free Raman imaging of nonfixed and unstained normal and cancerous colon human tissues and normal human CCD18-Co and cancerous CaCo-2 cell lines, without and after adding ZnPcS4 photosensitizer, was analyzed. The biochemical composition of normal and cancerous colon tissues and colon cells without and after adding ZnPcS4 at the subcellular level was determined. Analyzing the fluorescence/Raman signals of ZnPcS4, we found that in normal human colon tissue samples, in contrast to cancerous ones, there is a lower affinity to ZnPcS4 phthalocyanine. Moreover, a higher concentration in cancerous tissue was concomitant with a blue shift of the maximum peak position specific for the photosensitizer from 691–695 nm to 689 nm. Simultaneously for both types of samples, the signal was observed in the monomer region, confirming the excellent properties of ZnPcS4 for photo therapy (PDT). For colon cell experiments with a lower concentration of ZnPcS4 photosensitizer, c = 1 × 10−6 M, the phthalocyanine was localized in mitochondria/lipid structures; for a higher concentration, c = 9 × 10−6 M, localization inside the nucleus was predominant. Based on time-resolved experiments, we found that ZnPcS4 in the presence of biological interfaces features longer excited-state lifetime photosensitizers compared to the aqueous solution and bare ZnPcS4 film on CaF2 substrate, which is beneficial for application in PDT.

Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients.

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders, where epithelial defects drive, at least in part, some of the pathology. We reconstituted human intestinal epithelial organ, by using three-dimension culture of human colon organoids. Our aim was to characterize morphological and functional phenotypes of control (non-IBD) organoids, compared to inflamed organoids from IBD patients. The results generated describe the epithelial defects associated with IBD in primary organoid cultures, and evaluate the use of this model for pharmacological testing of anti-inflammatory approaches. Human colonic tissues were obtained from either surgical resections or biopsies, all harvested in non-inflammatory zones. Crypts were isolated from controls (non-IBD) and IBD patients and were cultured up to 12-days. Morphological (size, budding formation, polarization, luminal content), cell composition (proliferation, differentiation, immaturity markers expression), and functional (chemokine and tight junction protein expression) parameters were measured by immunohistochemistry, RT-qPCR or western-blot. The effects of inflammatory cocktail or anti-inflammatory treatments were studied in controls and IBD organoid cultures respectively. Organoid cultures from controls or IBD patients had the same cell composition after 10 to 12-days of culture, but IBD organoid cultures showed an inflammatory phenotype with decreased size and budding capacity, increased cell death, luminal debris, and inverted polarization. Tight junction proteins were also significantly decreased in IBD organoid cultures. Inflammatory cytokine cocktail reproduced this inflammatory phenotype in non-IBD organoids. Clinically used treatments (5-ASA, glucocorticoids, anti-TNF) reduced some, but not all parameters. Inflammatory phenotype is associated with IBD epithelium, and can be studied in organoid cultures. This model constitutes a reliable human pre-clinical model to investigate new strategies targeting epithelial repair.

Integrin-Linked-Kinase Overexpression Is Implicated in Mechanisms of Genomic Instability in Human Colorectal Cancer.

Genomic instability is a hallmark of cancer cells contributing to tumor development and progression. Integrin-linked kinase (ILK) is a focal adhesion protein with well-established role in carcinogenesis. We have previously shown that ILK overexpression is critically implicated in human colorectal cancer (CRC) progression. In light of the recent findings that ILK regulates centrosomes and mitotic spindle formation, we aimed to determine its implication in mechanisms of genomic instability in human CRC. Association of ILK expression with markers of genomic instability (micronuclei formation, nucleus size, and intensity) was investigated in diploid human colon cancer cells HCT116 upon ectopic ILK overexpression, by immunofluorescence and in human CRC samples by Feulgen staining. We also evaluated the role of ILK in mitotic spindle formation, by immunofluorescence, in HCT116 cells upon inhibition and overexpression of ILK. Finally, we evaluated association of ILK overexpression with markers of DNA damage (p-H2AX, p-ATM/ATR) in human CRC tissue samples by immunohistochemistry and in ILK-overexpressing cells by immunofluorescence. We showed that ILK overexpression is associated with genomic instability markers in human colon cancer cells and tissues samples. Aberrant mitotic spindles were observed in cells treated with specific ILK inhibitor (QLT0267), while ILK-overexpressing cells failed to undergo nocodazole-induced mitotic arrest. ILK overexpression was also associated with markers of DNA damage in HCT116 cells and human CRC tissue samples. The above findings indicate that overexpression of ILK is implicated in mechanisms of genomic instability in CRC suggesting a novel role of this protein in cancer.

Acute Effects of Butyrate on Induced Hyperpermeability and Tight Junction Protein Expression in Human Colonic Tissues.

Intact intestinal barrier function is essential for maintaining intestinal homeostasis. A dysfunctional intestinal barrier can lead to local and systemic inflammation through translocation of luminal antigens and has been associated with a range of health disorders. Butyrate, a short-chain fatty acid derived from microbial fermentation of dietary fibers in the colon, has been described as an intestinal barrier-strengthening agent, although mainly by using in vitro and animal models. This study aimed to investigate butyrate’s ability to prevent intestinal hyperpermeability, induced by the mast cell degranulator Compound 48/80 (C48/80), in human colonic tissues. Colonic biopsies were collected from 16 healthy subjects and intestinal permeability was assessed by Ussing chamber experiments. Furthermore, the expression levels of tight junction-related proteins were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Pre-treatment with 5 mM butyrate or 25 mM butyrate did not protect the colonic tissue against induced paracellular or transcellular hyperpermeability, measured by FITC-dextran and horseradish peroxidase passage, respectively. Biopsies treated with 25 mM butyrate prior to stimulation with C48/80 showed a reduced expression of claudin 1. In conclusion, this translational ex vivo study did not demonstrate an acute protective effect of butyrate against a chemical insult to the intestinal barrier in healthy humans.

OR16-06 Age-Associated Local GH Promotes Colon Neoplasia

Colon polyp and cancer frequency increase with age, yet little is known about age-related mechanisms underlying development of these neoplasms. Defective DNA damage response and accumulation of unrepaired DNA damage can trigger genomic instability and cellular transformation.Patients with acromegaly have a higher prevalence of colon polyps and, arguably, colon adenocarcinoma, while those with GH signaling deficiency do not develop cancer. We showed that APC+/- mice that all develop colon adenomas exhibit a significant decrease in the number and volume of colon tumors with deletion of the GH transcription factor Prop1. Further, DNA damage response triggered GH expression in colon cells, and GH, in turn, altered DNA damage repair, resulting in DNA damage accumulation and cell transformation both in vitro and in vivo. These findings prompted us to hypothesize that accumulated DNA damage in aging colon induces local GH, suppressing DNA damage repair and creating a milieu consistent with genomic instability favoring neoplastic development.In human colon tissue we now show increased expression of γH2AX, a marker of DNA breaks, associated with increased GH transcription (detected by RNA scope) and translation (assessed by immunohistochemistry) as well as GH induction in both human and murine colon after DNA damaging radiotherapy. In vitro studies support these results, showing GH induction in normal human colon epithelial cells, fibroblasts, and 3D human intestinal organoids after DNA damage. Of note, local GH was secreted in the medium, indicating a paracrine effect. Paracrine/autocrine GH expression in these cellular models resulted in suppression of p53, induction of EMT, and attenuation of DNA damage response, and accumulated unrepaired DNA damage in human colon cells and in human intestinal organoids. In vivo, colon cells infected with lentivirus expressing GH generated more metastases than did cells expressing control vector. Co-culturing of human normal colon fibroblasts expressing GH together with normal human colon cells led to increased motility and accumulation of DNA damage as well as increased proliferation of epithelial cells on a gut-on-a-chip microfluidic device, confirming paracrine GH effects. In an in vitro model of aging, culturing human intestinal organoids for up to 2 months resulted in decreased telomere length and increased GH mRNA and protein expression associated with suppressed DNA damage response evident by decreased phosphorylation of ATM and DNA-PKcs, both kinases involved in DNA repair, and DNA damage accumulation assessed by Comet assay. Suppression of GH in these aging organoids led to increased phospho-p53 and reduced DNA damage. Although somatotroph axis endocrine activity decreases with age, local GH induced in response to age-related DNA damage may trigger a “field change,” creating a milieu favorable for colon neoplastic development.

High-speed chemical imaging of dynamic and histological samples with stimulated Raman micro-spectroscopy.

We report a shot noise limited high-speed stimulated Raman microscopy platform allowing to acquire molecular vibrational spectra over 200 cm-1 in 12 µs at a scan rate of 40kHz. Using spectral focusing together with optimized acousto-optics programmable dispersive filters, the designed low noise imaging platform performs chemical imaging of dynamical processes such as Mannitol crystal hydration and reaches a signal to noise ratio sufficient to perform label free histological imaging on frozen human colon tissue slides.

Ileo-colonic delivery of conjugated bile acids improves glucose homeostasis via colonic GLP-1-producing enteroendocrine cells in human obesity and diabetes

BackgroundThe bile acid (BA) pathway plays a role in regulation of food intake and glucose metabolism, based mainly on findings in animal models. Our aim was to determine whether the BA pathway is altered and correctable in human obesity and diabetes. MethodsWe conducted 3 investigations: 1) BA receptor pathways were studied in NCI-H716 enteroendocrine cell (EEC) line, whole human colonic mucosal tissue and in human colonic EEC isolated by Fluorescence-activated Cell Sorting (ex vivo) from endoscopically-obtained biopsies colon mucosa; 2) We characterized the BA pathway in 307 participants by measuring during fasting and postprandial levels of FGF19, 7αC4 and serum BA; 3) In a placebo-controlled, double-blind, randomised, 28-day trial, we studied the effect of ileo-colonic delivery of conjugated BAs (IC-CBAS) on glucose metabolism, incretins, and lipids, in participants with obesity and diabetes. FindingsHuman colonic GLP-1-producing EECs express TGR5, and upon treatment with bile acids in vitro, human EEC differentially expressed GLP-1 at the protein and mRNA level. In Ussing Chamber, GLP-1 release was stimulated by Taurocholic acid in either the apical or basolateral compartment. FGF19 was decreased in obesity and diabetes compared to controls. When compared to placebo, IC-CBAS significantly decreased postprandial glucose, fructosamine, fasting insulin, fasting LDL, and postprandial FGF19 and increased postprandial GLP-1 and C-peptide. Increase in faecal BA was associated with weight loss and with decreased fructosamine. InterpretationsIn humans, BA signalling machinery is expressed in colonic EECs, deficient in obesity and diabetes, and when stimulated with IC-CBAS, improved glucose homeostasis. ClinicalTrials.gov number, NCT02871882, NCT02033876. FundingResearch support and drug was provided by Satiogen Pharmaceuticals (San Diego, CA). AA, MC, and NFL report grants (AA- C-Sig P30DK84567, K23 DK114460; MC- NIH R01 DK67071; NFL- R01 DK057993) from the NIH. JR was supported by an Early Career Grant from Society for Endocrinology.

MicroRNA Expression Profiling of Normal and Malignant Human Colonic Stem Cells Identifies miRNA92a as a Regulator of the LRIG1 Stem Cell Gene.

MicroRNAs (miRNAs) have a critical role in regulating stem cells (SCs) during development, and because aberrant expression of miRNAs occurs in various cancers, our goal was to determine if dysregulation of miRNAs is involved in the SC origin of colorectal cancer (CRC). We previously reported that aldehyde dehydrogenase (ALDH) is a marker for normal and malignant human colonic SCs and tracks SC overpopulation during colon tumorigenesis. MicroRNA expression was studied in ALDH-positive SCs from normal and malignant human colon tissues by Nanostring miRNA profiling. Our findings show that: (1) A unique miRNA signature distinguishes ALDH-positive CRC cells from ALDH-positive normal colonic epithelial cells, (2) Expression of four miRNAs (miRNA200c, miRNA92a, miRNA20a, miRNA93) are significantly altered in CRC SCs compared to normal colonic SCs, (3) miRNA92a expression is also upregulated in ALDH-positive HT29 CRC SCs as compared to ALDH-negative SCs, (4) miRNA92a targets the 3′UTR of LRIG1 SC gene, and (5) miRNA92a modulates proliferation of HT29 CRC cells. Thus, our findings indicate that overexpression of miRNA92a contributes to the SC origin of CRC. Strategies designed to modulate miRNA expression, such as miRNA92a, may provide ways to target malignant SCs and to develop more effective therapies against CRC.

High circulating elafin levels are associated with Crohn's disease-associated intestinal strictures.

Antimicrobial peptide expression is associated with disease activity in inflammatory bowel disease (IBD) patients. IBD patients have abnormal expression of elafin, a human elastase-specific protease inhibitor and antimicrobial peptide. We determined elafin expression in blood, intestine, and mesenteric fat of IBD and non-IBD patients. Serum samples from normal and IBD patients were collected from two UCLA cohorts. Surgical resection samples of human colonic and mesenteric fat tissues from IBD and non-IBD (colon cancer) patients were collected from Cedars-Sinai Medical Center. High serum elafin levels were associated with a significantly elevated risk of intestinal stricture in Crohn's disease (CD) patients. Microsoft Azure Machine learning algorithm using serum elafin levels and clinical data identified stricturing CD patients with high accuracy. Serum elafin levels had weak positive correlations with clinical disease activity (Partial Mayo Score and Harvey Bradshaw Index), but not endoscopic disease activity (Mayo Endoscopic Subscore and Simple Endoscopic Index for CD) in IBD patients. Ulcerative colitis (UC) patients had high serum elafin levels. Colonic elafin mRNA and protein expression were not associated with clinical disease activity and histological injury in IBD patients, but stricturing CD patients had lower colonic elafin expression than non-stricturing CD patients. Mesenteric fat in stricturing CD patients had significantly increased elafin mRNA and protein expression, which may contribute to high circulating elafin levels. Human mesenteric fat adipocytes secrete elafin protein. High circulating elafin levels are associated with the presence of stricture in CD patients. Serum elafin levels may help identify intestinal strictures in CD patients.

Association of lncRNA-p53 regulatory network (lincRNA-p21, lincRNA-ROR and MALAT1) and p53 with the clinicopathological features of colorectal primary lesions and tumors.

Colorectal cancer (CRC) is a common intestinal cancer with a high mortality rate. Early detection of this type of cancer is fundamental to the prevention of the disease, which results in improved survival rates. In the human colon tissue, transition from normal epithelium to adenoma is considered to be caused by unknown molecular incidents occurring over 5–10 years. The detection of CRC has proved problematic when in the early stages of disease. In addition, identifying suitable biomarkers for the detection of CRC progress in patients remains one of the most significant challenges. Long non-coding RNAs have been demonstrated to contribute to the promotion of CRC. The aim of the present study was to investigate the clinical and biological significance of long intergenic non-coding (linc)RNA-p21, lincRNA-regulator of reprogramming (ROR) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the colon tumor and polyp tissue, and the association that these have with the expression of p53 at the mRNA level. Neoplastic and paired adjacent normal tissue samples were obtained from 72 patients (46 polyps and 26 tumors). Reverse transcription-quantitative PCR was performed to determine the relative fold changes in the expression of lincRNA-p21, lincRNA-RoR, MALAT1 and p53 in the samples. A significant association was observed between the levels of MALAT1 and p53 in neoplasm tissues (R=0.073; P<0.05). The relative expression of the MALAT1 gene revealed a statistically significant difference between the different polyp types and number of polyps (P=0.0028 and 0.022, respectively). Adjuvant therapy in patients with tumors revealed an association between the levels of lincRNA-ROR and lincRNA-p21 expression (P=0.015 and 0.038, respectively). MALAT1 may be selected as an early detection biomarker for CRC. Furthermore, lincRNA-ROR and lincRNA-p21 may serve as prognostic and therapeutic biomarkers in patients with CRC.

Intestinal TMEM16A function as a luminal chloride channel

There has been a high level of uncertainty whether TMEM16A channels are truly expressed on the apical membranes of enterocytes and contribute to luminal Cl− secretion. Here, we describe a series of independent experimental approaches including qPCR, western blot, immuno‐staining and electrophysiology using Tmem 16a+/− mouse intestinal tissue, human colonic tissue and T84 cells to characterize the expression, localization and physiological function of TMEM16A and its regulation by the multi‐PDZ domain‐containing protein NHERF1.qPCR analysis revealed that Tmem16a transcripts are differentially expressed across the intestinal epithelial tissue with highest expression in the colon. Tmem16a mRNA expression did not correlate with TMEM16A protein expression. Western blot analysis of TMEM16A in mouse intestinal tissue revealed that one (Boster Bio PA2290) out of five commercially available polyclonal antibodies appreciably recognized a band at the expected size of below 100 kDa monomeric protein in jejunum, ileum, proximal and distal colon, which was absent in Tmem16a+/− mice. Ussing chamber experiments confirmed that TMEM16A protein abundance correlated with Cl− secretion in different regions of native intestine activated by the Ca2+‐dependent agonist carbachol (CCH). In Tmem16a+/− colon, basal and CCH stimulated Isc was largely reduced. Immunostaining of intestinal tissue with PA2290 antibody confirmed luminal expression of TMEM16A proteins compared to Tmem16a+/− mice. Luminal but not serosal application of MONNA, a TMEM16A specific inhibitor, resulted in significant reduction of CCH stimulated Isc, indicating that TMEM16A is primarily expressed and functions as a luminal Cl− channel. Luminal expression of TMEM16A in human colon was confirmed by confocal microscopy. Localization of TMEM16A to the luminal membrane may be facilitated through interactions with the scaffold protein NHERF1 which we found immunoprecipitated TMEM16A. Our results indicate that TMEM16A is prominently expressed in different segments of native mouse intestine as a luminal membrane protein and in human colonic tissue. TMEM16A also interacts with NHERF1. These data are consistent with TMEM16A localizing to the luminal membrane via the actin cytoskeleton and NHERF1 to influence CCH stimulated luminal Clsecretion.Support or Funding InformationUniversity Grant Commission, and Indian Council of Medical Research, Govt. of India

Protective properties of grape-seed proanthocyanidins in human ex vivo acute colonic dysfunction induced by dextran sodium sulfate.

Anti-inflammatory and barrier-protective properties have been attributed to proanthocyanidins in the context of intestinal dysfunction, however little information is available about the impact of these phytochemicals on intestinal barrier integrity and immune response in the human. Here we assessed the putative protective properties of a grape-seed proanthocyanidin extract (GSPE) against dextran sodium sulfate (DSS)-induced acute dysfunction of the human colon in an Ussing chamber system. Human proximal and distal colon tissues from colectomized patients were submitted ex vivo for a 30-min preventive GSPE treatment (50 or 200µgmL-1) followed by 1-h incubation with DSS (12%wv-1). Transepithelial electrical resistance (TEER), permeation of a fluorescently-labeled dextran (FD4) and proinflammatory cytokine release [tumor necrosis factor (TNF)-α and interleukin (IL)-1β] of colonic tissues were determined. DSS reduced TEER (45-52%) in both the proximal and distal colon; however, significant increments in FD4 permeation (fourfold) and TNF-α release (61%) were observed only in the proximal colon. The preventive GSPE treatment decreased DSS-induced TEER loss (20-32%), FD4 permeation (66-73%) and TNF-α release (22-33%) of the proximal colon dose-dependently. The distal colon was not responsive to the preventive treatment but showed a reduction in IL-1β release below basal levels with the highest GSPE concentration. Our results demonstrate potential preventive effects of GSPE on human colon dysfunction. Further studies are required to test whether administering GSPE could be a complementary therapeutic approach in colonic dysfunction associated with metabolic disorders and inflammatory bowel disease.

Differentiation of human colon tissue in culture: Effects of calcium on trans-epithelial electrical resistance and tissue cohesive properties.

Background and aimsHuman colonoid cultures maintained under low-calcium (0.25 mM) conditions undergo differentiation spontaneously and, concomitantly, express a high level of tight junction proteins, but not desmosomal proteins. When calcium is included to a final concentration of 1.5–3.0 mM (provided either as a single agent or as a combination of calcium and additional minerals), there is little change in tight junction protein expression but a strong up-regulation of desmosomal proteins and an increase in desmosome formation. The aim of this study was to assess the functional consequences of calcium-mediated differences in barrier protein expression.MethodsHuman colonoid-derived epithelial cells were interrogated in transwell culture under low- or high-calcium conditions for monolayer integrity and ion permeability by measuring trans-epithelial electrical resistance (TEER) across the confluent monolayer. Colonoid cohesiveness was assessed in parallel.ResultsTEER values were high in the low-calcium environment but increased in response to calcium. In addition, colonoid cohesiveness increased substantially with calcium supplementation. In both assays, the response to multi-mineral intervention was greater than the response to calcium alone. Consistent with these findings, several components of tight junctions were expressed at 0.25 mM calcium but these did not increase substantially with supplementation. Cadherin-17 and desmoglein-2, in contrast, were weakly-expressed under low calcium conditions but increased with intervention.ConclusionsThese findings indicate that low ambient calcium levels are sufficient to support the formation of a permeability barrier in the colonic epithelium. Higher calcium levels promote tissue cohesion and enhance barrier function. These findings may help explain how an adequate calcium intake contributes to colonic health by improving barrier function, even though there is little change in colonic histological features over a wide range of calcium intake levels.

Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning

Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions. Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions. We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4× 5 mm2. Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm3 with 38,706 ± 5763 neuron/mm3 and 129,321 ± 25,356 glia/mm3. Images of large areas provided insight into why published values of ENS density vary up to 150-fold-ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions. We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well.