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Chronic pancreatitis (CP) is a major risk factor of pancreatic ductal adenocarcinoma (PDAC). How CP promotes pancreatic oncogenesis remains unclear. A characteristic feature of PDAC is its prominent desmoplasia in the tumor microenvironment, composed of activated fibroblasts and macrophages. Macrophages can be characterized as M1 or M2, with tumor-inhibiting or -promoting functions, respectively. We reported that Gremlin 1 (GREM1), a key pro-fibrogenic factor, is upregulated in the stroma of CP. The current study aimed to investigate the expression of GREM1 and correlation between GREM1 and macrophages within the pancreas during chronic inflammation and the development of PDAC. By mRNA in situ hybridization, we detected GREM1 mRNA expression within α-smooth muscle actin (SMA)-positive fibroblasts of the pancreatic stroma. These designated FibroblastsGrem1+ marginally increased from CP to pancreatic intraepithelial neoplasia (PanIN) and PDAC. Within PDAC, FibroblastsGrem1+ increased with higher pathological tumor stages and in a majority of PDAC subtypes screened. Additionally, FibroblastsGrem1+ positively correlated with total macrophages (MacCD68+) and M2 macrophages (M2CD163+) in PDAC. To begin exploring potential molecular links between FibroblastsGrem1+ and macrophages in PDAC, we examined the expression of macrophage migration inhibitory factor (MIF), an endogenous counteracting molecule of GREM1 and an M1 macrophage promoting factor. By IHC staining of MIF, we found MIF to be expressed by tumor cells, positively correlated with GREM1; by IHC co-staining, we found MIF to be negatively correlated with M2CD163+ expression. Our findings suggest that GREM1 expression by activated fibroblasts may promote PDAC development, and GREM1/MIF may play an important role in macrophage phenotype.

This document provides detailed captions for figures S1-S8.

This file contains Figures S1-S8. Figure S1 shows examples of representative images used to score p120 catenin expression in human TMAs and pancreatic histology of mice with loss of p120 catenin. Figure S2 depicts recruitment of inflammation and a unique stromal composition in KCiMist1; p120f/wt and KCiMist1; p120f/f pancreata. Figure S3 shows that prominent basal epithelial cell extrusion in pancreata of KCiMist1; p120f/f mice is not associated with incomplete EMT. Figure S4 shows that pancreatic loss of p120 catenin in a mouse model of acute pancreatitis delays regeneration and results in significant recruitment of inflammation, observations which are mediated at least in part through activation of NF-kB. Figure S5 illustrates that a subset of epithelial cells extruding apically in KCiMist1; p120wt/wt, KCiMist1; p120f/wt, and KCiMist1; p120f/f pancreata express cleaved Caspase-3, while epithelial cells extruding basally do not express cleaved Caspase-3. Figure S6 depicts an analysis of chromosome content using Feulgen stain, which showed abnormal DNA content and aneuploidy in a subset of basally extruded epithelial cells in KCiMist1; p120f/f pancreata. Figure S7 shows mislocalized p120 catenin expression in greater than 95% isolated epithelial cells in human PDA. Figure S8 illustrates IPA results of microarray performed on GFP+ pancreatic cells of KCiMist1G; p120wt/wt and KCiMist1G; p120f/f mice as well as IHC of select targets identified from IPA results.

This table provides a description of antibodies used in this study.

Aberrant regulation of cellular extrusion can promote invasion and metastasis. Here, we identify molecular requirements for early cellular invasion using a premalignant mouse model of pancreatic cancer with conditional knockout of p120 catenin (Ctnnd1). Mice with biallelic loss of p120 catenin progressively develop high-grade pancreatic intraepithelial neoplasia (PanIN) lesions and neoplasia accompanied by prominent acute and chronic inflammatory processes, which is mediated, in part, through NF-κB signaling. Loss of p120 catenin in the context of oncogenic Kras also promotes remarkable apical and basal epithelial cell extrusion. Abundant single epithelial cells exit PanIN epithelium basally, retain epithelial morphology, survive, and display features of malignancy. Similar extrusion defects are observed following p120 catenin knockdown in vitro, and these effects are completely abrogated by the activation of S1P/S1pr2 signaling. In the context of oncogenic Kras, p120 catenin loss significantly reduces expression of genes mediating S1P/S1pr2 signaling in vivo and in vitro, and this effect is mediated at least, in part, through activation of NF-κB. These results provide insight into mechanisms controlling early events in the metastatic process and suggest that p120 catenin and S1P/S1pr2 signaling enhance cancer progression by regulating epithelial cell invasion. Cancer Res; 76(11); 3351–63. ©2016 AACR.

We studied the expression of two hepatocyte nuclear factor 4 alpha (HNF4α) isoforms, p-STAT3. and c-Myc in 49 consecutive liver biopsies with nonalcoholic fatty liver disease (NAFLD) using immunohistochemistry. All 49 biopsies (100%) were positive for nuclear expression of P1-HNF4α. Twenty-eight (57%) cases were positive for P2-HNF4α, 6 (12%) were positive for p-STAT3 and 5 (10%) were positive for c-Myc. All 6 (100%) p-STAT3-positive cases were also positive for P2-HNF4α (

Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, and the most common primary liver malignancy to present in the clinic. With the exception of liver transplant, treatment options for advanced HCC are limited, but improved tumor stratification could open the door to new treatment options. Previously, we demonstrated that the circadian regulator Aryl Hydrocarbon-Like Receptor Like 1 (ARNTL, or Bmal1) and the liver-enriched nuclear factor 4 alpha (HNF4α) are robustly co-expressed in healthy liver but incompatible in the context of HCC. Faulty circadian expression of HNF4α– either by isoform switching, or loss of expression- results in an increased risk for HCC, while BMAL1 gain-of-function in HNF4α-positive HCC results in apoptosis and tumor regression. We hypothesize that the transcriptional programs of HNF4α and BMAL1 are antagonistic in liver disease and HCC. Here, we study this antagonism by generating a mouse model with inducible loss of hepatic HNF4α and BMAL1 expression. The results reveal that simultaneous loss of HNF4α and BMAL1 is protective against fatty liver and HCC in carcinogen-induced liver injury and in the “STAM” model of liver disease. Furthermore, our results suggest that targeting Bmal1 expression in the absence of HNF4α inhibits HCC growth and progression. Specifically, pharmacological suppression of Bmal1 in HNF4α-deficient, BMAL1-positive HCC with REV-ERB agonist SR9009 impairs tumor cell proliferation and migration in a REV-ERB-dependent manner, while having no effect on healthy hepatocytes. Collectively, our results suggest that stratification of HCC based on HNF4α and BMAL1 expression may provide a new perspective on HCC properties and potential targeted therapeutics.

BACKGROUND AND AIMS. Disease activity and severity of eosinophilic esophagitis (EoE) dictate therapeutic options and management, but the decision-making process for determining severity varies among practitioners. To reduce variability in practice patterns and help clinicians monitor the clinical course of the disease in an office setting, we aimed to create an international consensus severity scoring index for EoE. METHODS. A multidisciplinary international group of adult and pediatric EoE researchers and clinicians, as well as non-EoE allergy immunology and gastroenterology experts, formed 3 teams to review the existing literature on histology, endoscopy, and symptoms of EoE in the context of progression and severity. A steering committee convened a one-day virtual meeting to reach consensus on each team’s opinion on salient features of severity across key clinicopathologic domains and distill features that would allow providers to categorize disease severity. RESULTS. Symptom features and complications, and inflammatory and fibrostenotic features on both endoscopic and histologic examination, were collated into a simplified scoring system, the Index of Severity for Eosinophilic Esophagitis (I-SEE), that can be completed at routine clinic visits to assess disease severity using a point scale of 0–6 for mild, 7–14 for moderate, and ≥15 for severe EoE. CONCLUSIONS. A multidisciplinary team of experts iteratively created a clinically usable EoE severity scoring system denominated I-SEE to guide practitioners in EoE management by standardizing disease components reflecting disease severity beyond eosinophil counts. I-SEE should be validated and refined using data from future clinical trials and routine clinical practice to increase its utilization and functionality.

Inflammatory bowel disease (IBD) patients in apparent clinical remission who present with irritable bowel syndrome (IBS)-like symptoms pose a diagnostic and therapeutic dilemma that is called post-IBD IBS. When associated with a diarrheal IBS presentation, this clinical syndrome is known as post-IBD IBS-D.We review and describe the literature regarding the clinical overlap of IBD and IBS. We discuss prevalent theories regarding the pathophysiology of post-IBD IBS-D and whether this presentation represents coincident inherent IBS-D, IBS-D triggered by IBD, or an even more subtle level of IBD activity that is unrecognized by available laboratory modalities. We also discuss observations that post-IBD IBS-D patients harbor significantly increased colon mucosal eosinophils and appear to respond to a GI-hypoallergenic diet and budesonide therapy.The symptoms overlap between IBD and IBS complicates diagnosis and subsequent management of patients with post-IBD IBS-D. In addition to current theories regarding the pathophysiology of this condition such as alterations in mucosal inflammation, the microbiota, mucosal permeability, and gut-brain interactions. This new avenue of eosinophilic colopathy and therapy directed toward food-derived immune response in patients with post-IBD IBS-D deserves additional investigation.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Despite the high prevalence of Kras mutations in pancreatic cancer patients, murine models expressing the oncogenic mutant Kras (Krasmut) in mature pancreatic cells develop PDAC at a low frequency. Independent of cell of origin, a second genetic hit (loss of tumor suppressor TP53 or PTEN) is important for development of PDAC in mice. We hypothesized ectopic expression and elevated levels of oncogenic mutant Kras would promote PanIN arising in pancreatic ducts. To test our hypothesis, the significance of elevating levels of K-Ras and Ras activity has been explored by expression of a CAG driven LGSL-KrasG12V allele (cKras) in pancreatic ducts, which promotes ectopic Kras expression. We predicted expression of cKras in pancreatic ducts would generate neoplasia and PDAC. To test our hypothesis, we employed tamoxifen dependent CreERT2 mediated recombination. Hnf1b:CreERT2;KrasG12V (cKrasHnf1b/+) mice received 1 (Low), 5 (Mod) or 10 (High) mg per 20 g body weight to recombine cKras in low (cKrasLow), moderate (cKrasMod), and high (cKrasHigh) percentages of pancreatic ducts. Our histologic analysis revealed poorly differentiated aggressive tumors in cKrasHigh mice. cKrasMod mice had grades of Pancreatic Intraepithelial Neoplasia (PanIN), recapitulating early and advanced PanIN observed in human PDAC. Proteomics analysis revealed significant differences in PTEN/AKT and MAPK pathways between wild type, cKrasLow, cKrasMod, and cKrasHigh mice. In conclusion, in this study, we provide evidence that ectopic expression of oncogenic mutant K-Ras in pancreatic ducts generates early and late PanIN as well as PDAC. This Ras rheostat model provides evidence that AKT signaling is an important early driver of invasive ductal derived PDAC., Summary In this study, the authors provide evidence that ectopic expression of oncogenic mutant Kras in pancreatic ducts generates early and late (PanIN) and pancreatic ductal adenocarcinoma (PDAC) . They characterized this Ras rheostat model which reveals elevated Kras mutation frequency and loss of PTEN are important drivers of PanIN and invasive ductal derived PDAC.

To elucidate the reasons for the decreased effectiveness of Vedolizumab (VDZ) treatment in patients with Crohn's disease (CD) previously treated (CD-T) with anti-TNF-α biologics.Immunohistochemical staining was performed on sections of formalin-fixed paraffin-embedded ileocolonic biopsies using antibodies for the mucosal addressin molecule (MAdCAM-1) and Etrolizumab.The mean number of MAdCAM-1 positive capillaries (MAdCAM-1-C) was 3 in controls, 8.5 in CD, 5.37 in CD-T, 5.7 in ulcerative colitis (UC), and 3.1 in lymphocytic colitis (LC) (Our results suggest that treatment with anti-TNF-α reduces the number of MAdCAM-1-C in CD, even in biopsies with high HAS. This suggests that high inflammation in such cases obviously failed to respond to anti-TNF-α, may be less dependent on the migration of a4b7-lymphocytes to the inflamed mucosa, and therefore may not optimally respond to VDZ treatment.Presented in part at the Digestive Diseases Week meeting, San Diego, CA, May 2019. Supported by Takeda Pharmaceuticals.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition driven by diverse genetic and nongenetic programs that converge to disrupt immune homeostasis in the intestine. We have reported that, in murine intestinal epithelium with telomere dysfunction, DNA damage-induced activation of ataxia-telangiectasia mutated (ATM) results in ATM-mediated phosphorylation and activation of the YAP1 transcriptional coactivator, which in turn up-regulates pro-IL-18, a pivotal immune regulator in IBD pathogenesis. Moreover, individuals with germline defects in telomere maintenance genes experience increased occurrence of intestinal inflammation and show activation of the ATM/YAP1/pro-IL-18 pathway in the intestinal epithelium. Here, we sought to determine the relevance of the ATM/YAP1/pro-IL-18 pathway as a potential driver of IBD, particularly older-onset IBD. Analysis of intestinal biopsy specimens and organoids from older-onset IBD patients documented the presence of telomere dysfunction and activation of the ATM/YAP1/precursor of interleukin 18 (pro-IL-18) pathway in the intestinal epithelium. Employing intestinal organoids from healthy individuals, we demonstrated that experimental induction of telomere dysfunction activates this inflammatory pathway. In organoid models from ulcerative colitis and Crohn's disease patients, pharmacological interventions of telomerase reactivation, suppression of DNA damage signaling, or YAP1 inhibition reduced pro-IL-18 production. Together, these findings support a model wherein telomere dysfunction in the intestinal epithelium can initiate the inflammatory process in IBD, pointing to therapeutic interventions for this disease.

Non-small cell lung cancer remains a highly lethal malignancy. Using the tamoxifen inducible Hnf1b:CreERT2 (H) transgenic mouse crossed to the LsL-KrasG12D (K) transgenic mouse, we recently discovered that an Hnf1b positive cell type in the lung is sensitive to adenoma formation when expressing a mutant KrasG12D allele. In these mice, we observe adenoma formation over a time frame of three to six months. To study specificity of the inducible Hnf1b:CreERT2 in the lung, we employed lineage tracing using an mTmG (G) reporter allele. This technique revealed recombined, GFP+ cells were predominantly SPC+. We further employed this technique in HKG mice to determine Hnf1b+ cells give rise to adenomas that express SPC and TTF1. Review of murine lung tissue confirmed a diagnosis of adenoma and early adenocarcinoma, a pathologic subtype of non-small cell lung cancer. Our expanded mouse model revealed loss of Mst1/2 promotes aggressive lung adenocarcinoma and large-scale proteomic analysis revealed upregulation of PKM2 in the lungs of mice with genetic deletion of Mst1/2. PKM2 is a known metabolic regulator in proliferating cells and cancer. Using a human lung adenocarcinoma cell line, we show pharmacologic inhibition of Mst1/2 increases the abundance of PKM2, indicating genetic loss or pharmacologic inhibition of Mst1/2 directly modulates the abundance of PKM2. In conclusion, here we report a novel model of non-small cell lung cancer driven by a mutation in Kras and deletion of Mst1/2 kinases. Tumor development is restricted to a subset of alveolar type II cells expressing Hnf1b. Our data show loss of Mst1/2 regulates levels of a potent metabolic regulator, PKM2.

Unlike eosinophilic esophagitis (EoE), there is no consensus on the minimum number of intraepithelial lymphocytes (IEL) that is diagnostic of lymphocytic esophagitis (LyE). The aim of this study was to determine whether significant correlations exist between the numbers of intraepithelial lymphocytes (IEL) in esophageal biopsies and clinical and endoscopic manifestations usually associated with EoE.HE slides from esophageal biopsies from 330 patients were reviewed. The number of IEL and intraepithelial eosinophils (IEE) per mmAs expected, a higher number of IEE was significantly associated with food impaction (In esophageal biopsies, increased IEL has no significant correlation with food impaction or dysphagia or with esophageal stricture, rings, or furrows. There is significant variation in the number of IEL depending on the season when the biopsy is obtained, which has not been previously reported.

We have previously demonstrated that the pancreas can recover from chronic pancreatitis (CP) lesions in the cerulein-induced mouse model. To explore how pancreatic recovery is achieved at the molecular level, we used RNA-sequencing (seq) and profiled transcriptomes during CP transition to recovery. CP was induced by intraperitoneally injecting cerulein in C57BL/6 mice. Time-matched controls (CON) were given normal saline. Pancreata were harvested from mice 4 days after the final injections (designated as CP and CON) or 4 weeks after the final injections (designated as CP recovery (CPR) and control recovery (CONR)). Pancreatic RNAs were extracted for RNA-seq and quantitative (q) PCR validation. Using RNA-seq, we identified a total of 3,600 differentially expressed genes (DEGs) in CP versus CON and 166 DEGs in CPR versus CONR. There are 132 DEGs overlapped between CP and CPR and 34 DEGs unique to CPR. A number of selected pancreatic fibrosis-relevant DEGs were validated by qPCR. The top 20 gene sets enriched from DEGs shared between CP and CPR are relevant to extracellular matrix and cancer biology, whereas the top 10 gene sets enriched from DEGs specific to CPR are pertinent to DNA methylation and specific signaling pathways. In conclusion, we identified a distinct set of DEGs in association with extracellular matrix and cancer cell activities to contrast CP and CPR. Once during ongoing CP recovery, DEGs relevant to DNA methylation and specific signaling pathways were induced to express. The DEGs shared between CP and CPR and the DEGs specific to CPR may serve as the unique transcriptomic signatures and biomarkers for determining CP recovery and monitoring potential therapeutic responses at the molecular level to reflect pancreatic histological resolution.

Background A systemic evaluation of immune cell infiltration patterns in experimental acute pancreatitis (AP) is lacking. Using multi-dimensional flow cytometry, this study profiled infiltrating immune cell types in multiple AP mouse models. Methods Three AP models were generated in C57BL/6 mice via cerulein (CAE) injection, alcohol and palmitoleic acid (EtOH + POA) injection, and alcohol diet feeding and cerulein (EtOH + CAE) injection. Primary pancreatic cells and splenocytes were prepared, and multi-dimensional flow cytometry was performed and analyzed by manual gating and computerized PhenoGraph, followed by visualization with t-distributed stochastic neighbor embedding (t-SNE). Results CAE treatment induced a time-dependent increase of major innate immune cells and a decrease of follicular B cells, and TCD4+ cells and the subtypes in the pancreas, whereas elicited a reversed pattern in the spleen. EtOH + POA treatment resulted in weaker effects than CAE treatment. EtOH feeding enhanced CAE-induced amylase secretion, but unexpectedly attenuated CAE-induced immune cell regulation. In comparison with manual gating analysis, computerized analysis demonstrated a remarkable time efficiency and reproducibility on the innate immune cells and B cells. Conclusions The reverse pattern of increased innate and decreased adaptive immune cells was consistent in the pancreas in CAE and EtOH + POA treatments. Alcohol feeding opposed the CAE effect on immune cell regulation. Together, the immune profiling approach utilized in this study provides a better understanding of overall immune responses in AP, which may facilitate the identification of intervention windows and new therapeutic strategies. Computerized analysis is superior to manual gating by dramatically reducing analysis time.

Aquaporin-5 (AQP5) is a member of a family of water channel proteins involved in the bidirectional transfer of water across cell membranes. Lymphocytic colitis (LC) and collagenous colitis (CC) are clinically similar diseases characterized by chronic watery diarrhea in patients with usually unremarkable colonic mucosa on colonoscopy. The aim of this study was to determine whether AQP5 expression in colonic epithelium is altered in LC and CC.Sections of formalin-fixed and paraffin-embedded colorectal biopsies from three control patients (CTL), 8 patients with chronic non-bloody diarrhea with biopsies negative for active inflammation or significant distortion (CTL-D), 8 patients with LC, and 5 with CC were stained for AQP5 using immunohistochemistry. The staining intensity was scored as 3 (strong), 2 (intermediate), 1 (weak), or 0 (no staining). Statistical analysis was performed using Prism 7 Statistical Soft-ware.AQP5 was strongly expressed (score 3) in the epithelial cells in all three CTL cases and all 8 CTL-D cases. In the 5 cases of CC, 3(60%) had score 3 and 2(40%) had score 2, but none had a score of 1 or 0. Of the 8 LC cases, 2(25%) had score 3, 3 had score 2(37.5%), and 3 had score 1(37.5%) (Colorectal AQP5 expression is reduced in most cases of LC. Markedly reduced AQP5 expression in LC may identify a subset of patients with suboptimal response to enteric steroid treatment. Additional larger studies are needed to confirm these findings.This abstract was presented in part at Digestive Diseases Week in San Diego, CA, May 2019.

Autoimmune hepatitis (AIH) is a chronic inflammatory immune-mediated hepatic pathology of unclear etiology. The mechanisms initiating and driving autoimmune inflammation of the liver and the loss of hepatic tolerance are still elusive. Several studies have documented the involvement of genetic factors and triggering agents such as toxic injury, treatment with immune-modifying drugs, or previous viral infections with perhaps strongest evidence for the hepatitis C virus. Rarely, AIH has been reported to develop after hepatitis A virus infection. We describe a case of de novo AIH in a patient with a history of recent hepatitis A virus infection.

The caudal-related homeobox transcription factor 2 (CDX2) plays an important role in intestinal epithelial differentiation, proliferation, migration, and adhesion. It has been previously reported that TNF-α reduces CDX2 expression in cultured colon epithelial cells in a dose-dependent manner, and that this effect was reduced by adding the anti-TNF-α drug infliximab to the culture medium. The aim of this study was to determine whether CDX2 expression is reduced in biopsies from patients with Crohn's disease (CD), and whether treatment with anti-TNF-α drugs reverses CDX2 downregulation in these patients.Sections of ileocolonic biopsy tissues from patients with CD, CD treated with anti-TNF-α biologics (CD-T), and controls were stained for CDX2 and evaluated using OTMIAS digital image analysis.CDX2 expression in biopsies from patients with CD and CD-T was lower than in controls (Although CDX2 is downregulated in CD, it did not revert to normal in patients treated with anti-TNF-α biologics.

Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation., How telomere dysfunction is directly linked to inflammation in humans is currently unclear. Here the authors reveal that telomere dysfunction drives activation of the YAP1 transcription factor, up-regulating the pro inflammatory factor, pro-IL-18 thus revealing a link between telomere dysfunction and initiation of intestinal inflammation.

CRM1/XPO1 (CRM1) is a nuclear export chaperone that mediates the export of proteins essential to growth regulation and tumor suppression. Its overexpression in tumors was found to be associated with poor prognosis. Selective inhibitors of nuclear export are in phase I and II clinical trials for several tumor types. Our aim was to investigate CRM1 expression in pancreatic adenocarcinoma (PAC) and its relationship to survivin expression and the proliferative activity. Sections of tissue microarray containing 76 formalin fixed and paraffin embedded PAC were stained by immunohistochemistry (IHC) for CRM1, survivin, and Cyclin A. Expression levels of CRM1 and survivin and the proliferative activity, the S-phase fraction (SPF) in tumor cells, were determined using a quantitative digital image analysis solution (OTMIAS). Sixty-six of the 76 (86%) PAC showed positive staining for CRM1, and 10 (14%) were completely negative. The mean CRM1 expression levels ranged from 0.3 to 53 units and the median from 0.3 to 45 units. There was significant positive correlation between the mean and median expression levels of CRM1 in tumor cells and the mean and median levels of survivin (p

Raman spectroscopy provides diagnostic information by detecting disease-associated subtle biochemical changes in the tissue. Yet the physiological variations among normal subjects could confound data interpretation and thus compromise the sensitivity in disease discrimination. We analyzed Raman spectra acquired from colon tissue in vivo through an endoscopic Raman system and evaluated potential physiological factors affecting tissue biochemistry. Fifty-six healthy patients scheduled for colonoscopy screening were enrolled in the study. Intra-subject variability was evaluated via univariate analysis by comparing the intensities of major Raman bands from different anatomical locations. Inter-subject variability was investigated based on various physiological variables, such as age, gender, ethnicity (White/Caucasian, African American, and Hispanic), and body mass index (BMI). Both univariate analysis and principal component analysis-based multivariate analysis were implemented for the investigation of inter-subject variability. The differences among certain physiological variables were further analyzed after accounting for intra-subject variation by generalized estimating equation method due to its advantage in handling repeated measurements. The results showed that physiological factors including gender, ethnicity, age, BMI, and anatomical locations along the colon were significant sources of variability, resulting from different abundance in lipids and proteins. Further correlation analysis revealed that the variability from gender, ethnicity, and age is significantly associated with that from BMI, indicating that BMI might be the key contributor to the inter-subject variability in the spectra. This study suggested the importance of including normal variability, especially BMI and anatomical locations, into the interpretation of Raman spectra for in vivo application. Copyright © 2017 John Wiley & Sons, Ltd.

Context.—The cause of pancreatic acinar metaplasia (PAM) at the distal esophagus/esophagogastric junction is still controversial. Whereas some authors believe it is congenital, others believe it is acquired because of inflammation of the gastric cardia, and more recently it was proposed to be due to chronic proton pump inhibitor use based on a study in rats.Objective.—To determine whether there is correlation between chronic proton pump inhibitor use and PAM in humans. We also investigated the correlation between several clinical and pathologic factors and PAM.Design.—Four hundred forty-four consecutive biopsies from the distal esophagus/esophagogastric junction were reviewed for the presence of PAM, which was then correlated with several clinical and pathologic findings.Results.—Pancreatic acinar metaplasia was found in 71 patients (16%). Pancreatic acinar metaplasia was significantly associated with patient age younger than 51 years (P < .001), chronic carditis (P = .01), and chronic proton pump inhibitor use (P = .008). Surprisingly, we also found significant association between PAM and chronic nonsteroidal anti-inflammatory drug use (P < .001). These associations, including that with chronic nonsteroidal anti-inflammatory drug use, remained significant in multivariate analysis.Conclusions.—Our findings confirm the previous reports of significant association between PAM and chronic carditis and the findings from animal studies of association with chronic proton pump inhibitor use. The strong association with chronic nonsteroidal anti-inflammatory drug use has not been previously reported and warrants further studies.

CpG island methylator phenotype (CIMP)-positive colorectal cancers (CRC) and CRC with microsatellite instability (MSI) were reported to have a decreased expression of estrogen receptor, beta1 (ER-β1), and methylation accompanied by decreased expression for the caudal-related homeobox, transcription factor 2 (CDX2). While precursor lesions of these cancers, known as sessile serrated adenomas (SSA), were found to have decreased CDX2 expression, the status of ER-β1 expression in SSA is unknown. The aim of this study is to determine ER-β1 expression in SSA and its relation to CDX2 expression.Sections of formalin fixed and paraffin embedded tissue from 62 consecutive cases of SSA were stained by immunohistochemistry for ER-β1 and CDX2. SSA with ER-β1 or CDX2 expression similar to that of a normal colon were scored as 0, while those with a loss of expression in10% of SSA crypts as 1, 11-25% as 2, 26-50% as 3, 51-75% as 4, and CDX2 loss in75% of the SSA crypts scored as 5.There is a significant correlation between a loss of CDX2 and the loss of ER-β1 scores in SSA (Our findings show significant downregulation of both ER-β1 and CDX2 expression in SSA, especially in the right colon. These findings suggest that ER-β1 downregulation plays a significant role in the malignant progression of SSA.

The incidence of hepatocellular carcinoma (HCC) is on the rise worldwide. Although the incidence of HCC in males is considerably higher than in females, the projected rates of HCC incidence are increasing for both sexes. A recently appreciated risk factor for HCC is the growing problem of nonalcoholic fatty liver disease, which is usually associated with obesity and the metabolic syndrome. In this study, we showed that under conditions of fatty liver, female mice were more likely to develop HCC than expected from previous models. Using an inducible knockout model of the tumor-suppressive isoform of hepatocyte nuclear factor 4 alpha (“P1-HNF4α”) in the liver in combination with prolonged high fat (HF) diet, we found that HCC developed equally in male and female mice as early as 38 weeks of age. Similar sex-independent HCC occurred in the “STAM” model of mice, in which severe hyperglycemia and HF feeding results in rapid hepatic lipid deposition, fibrosis, and ultimately HCC. In both sexes, reduced P1-HNF4α activity, which also occurs under chronic HF diet feeding, increased hepatic lipid deposition and produced a greatly augmented circadian rhythm in IL6, a factor previously linked with higher HCC incidence in males. Loss of HNF4α combined with HF feeding induced epithelial–mesenchymal transition in an IL6-dependent manner. Collectively, these data provide a mechanism-based working hypothesis that could explain the rising incidence of aggressive HCC. Significance: This study provides a mechanism for the growing incidence of hepatocellular carcinoma in both men and women, which is linked to nonalcoholic fatty liver disease.

Pancreatic cancer remains a highly lethal malignancy. We have recently shown that simultaneous expression of Kras and mutant Tp53(R175H) promotes invasive ductal adenocarcinoma from pancreatic ductal cells. We hypothesized specific mutations in TP53 have divergent mechanisms of transforming ductal cells. In order to understand the role of mutant TP53 in transforming pancreatic ductal cells, we used a lentiviral system to express mutant TP53(R175H) and TP53(R273H), two of the most frequently mutated TP53 alleles in pancreatic cancer patients, in immortalized, but not transformed, pancreatic ductal epithelial cells carrying a KRAS mutation (HPNE:KRAS(G12D)). Mutant TP53 expression enhanced colony formation and an RPPA assay results revealed TP53(R175H) uniquely induced HSP70 expression in HPNE:KRAS(G12D) cells. In the context of TP53(R175H) expression; we observed nuclear localization of HSP70. We performed immunoprecipitation experiments to show mutant p53(R175H) binds to HSP70. We also provide evidence mutant p53(R175H) is important for HSP70 stability and, more importantly, HSP70 is required for mutant p53 stability. These data are critical in the context of events leading to cellular transformation in the pancreas.

The cerulein-induced mouse pancreatitis model is a well-established, commonly used representation of human chronic pancreatitis pathology. Although studies report sex-dependent differences in human chronic pancreatitis, there are no studies in this model directly comparing sex response to pancreatic injury and recovery. Therefore, we designed a study to investigate whether sex- dependent differences in chronic pancreatitis injury and recovery exist in the cerulein-induced pancreatitis model.Adult male and female C57BL/6 mice were administered cerulein (50 μg/kg, 5 hourly intraperitoneal injections/day, 3 days/week) for 4 weeks to induce chronic pancreatitis; control mice received normal saline injections. Pancreata and blood were harvested at 4 days (as injury group) or 4 weeks (as recovery group) after the last injection. Amylase secretion was measured from the serum. Acinar injury was scored on HE sections. Fibrosis was assessed by Sirius Red and collagen immunofluorescence staining.Compared to time-matched controls, injury group displayed decreased body and pancreas weight, and increased acinar injury and fibrosis, with no significant differences between males and females. Recovery group demonstrated recovery of body weight, partial recovery of pancreas weight, reversal of acinar injury, and partial reversal of fibrosis, with no significant differences between males and females. Amylase secretion/body weight was similar across all groups.Male and female mice of the cerulein-induced chronic pancreatitis demonstrate similar responses to chronic pancreatitis injury and recovery. Although this model may not sufficiently emulate sex-dependent responses in human chronic pancreatitis, our study supports that both sexes of mice from this model can be used for the study of chronic pancreatitis.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Despite the high prevalence of Kras mutations in pancreatic cancer patients, murine models expressing the oncogenic mutant Kras (Kras

Aberrant regulation of cellular extrusion can promote invasion and metastasis. Here, we identify molecular requirements for early cellular invasion using a premalignant mouse model of pancreatic cancer with conditional knockout of p120 catenin (Ctnnd1). Mice with biallelic loss of p120 catenin progressively develop high-grade pancreatic intraepithelial neoplasia (PanIN) lesions and neoplasia accompanied by prominent acute and chronic inflammatory processes, which is mediated, in part, through NF-κB signaling. Loss of p120 catenin in the context of oncogenic Kras also promotes remarkable apical and basal epithelial cell extrusion. Abundant single epithelial cells exit PanIN epithelium basally, retain epithelial morphology, survive, and display features of malignancy. Similar extrusion defects are observed following p120 catenin knockdown in vitro, and these effects are completely abrogated by the activation of S1P/S1pr2 signaling. In the context of oncogenic Kras, p120 catenin loss significantly reduces expression of genes mediating S1P/S1pr2 signaling in vivo and in vitro, and this effect is mediated at least, in part, through activation of NF-κB. These results provide insight into mechanisms controlling early events in the metastatic process and suggest that p120 catenin and S1P/S1pr2 signaling enhance cancer progression by regulating epithelial cell invasion. Cancer Res; 76(11); 3351–63. ©2016 AACR.

Introduction Inflammatory bowel disease (IBD) patients in remission who present with irritable bowel syndrome with diarrhea (IBS-D) like symptoms pose a diagnostic and therapeutic dilemma that is called post-IBD IBS-D. This syndrome was first reported by Isgar et al. who documented IBS-type symptoms in 33% of their patients with chronic ulcerative colitis (CUC) in remission in 1983. In their meta-analysis of 13 studies incorporating 1,703 patients, Halpin and Ford in 2012 calculated a pooled prevalence of 35% for IBS symptoms among IBD subjects in remission. The post-IBD IBS-D syndrome represents a source of considerable stress, incurs morbidity and impairs quality of life of these patients. The etiology of this syndrome is unknown and may be multifactorial such as environmental, psychological, GI motility disarray, genetic components and possibly by gut microbiome. Methods We conducted a retrospective, single-center study of patients with history of IBD and IBS-D like symptoms. The demographic characteristics, type of IBD, colonoscopy, biopsy findings and empirical medical management outcome were analyzed. Results We have found significantly increased mucosal eosinophils (>50 HPF in both side of the colon examined) in our patients with post-IBD IBS-D syndrome. In this study, 15 CUC patients and 20 Crohn’s disease patients with this syndrome were investigated and showed no clinical, serological, mucosal or microscopic IBD activity. These patients had non-bloody diarrhea (5–20 x daily), lower abdominal cramps (%90), mild weight loss (4–6 lbs) and fecal incontinence (%25) who were placed on the GI-hypoallergenic diet and budesonide therapy. Sixty seven percent of these 35 patients with post-IBD IBS-D responded well clinically to this management. Increased mucosal eosinophils in these patients with post-IBD IBS-D may have eosinophilic colopathy that may be related to intestinal permeability disarray. The epithelium in these IBD patients who are in remission may have the production of pro-inflammatory cytokines by the eosinophils, especially IL-23 and IL-33. This interesting area investigation is in progress by our research faculty. Conclusion In summary, this is an exciting new finding that significantly increased mucosal eosinophils in our patients with post-IBD IBS-D syndrome may have a new avenue, eosinophilic colopathy. Interestingly, these patients have responded to the GI-hypoallergenic diet and budesonide therapy. Obviously, we need more patients in the near future with longer follow-up and we hope that the other investigators may confirm our findings.

Limited studies have shown that some patients with pancreatic adenocarcinoma (PAC) may benefit from treatment with tamoxifen. PAC has been shown to be largely negative for estrogen receptor alpha (ER-alpha). The aim of this pilot study was to investigate ER-beta expression in human PAC. Sections of tissue microarray with 18 evaluable cases of human PAC were stained by immunohistochemistry (IHC) for ER-beta1, ER-beta2, ER-beta5, and Cyclin A. The levels of ER-beta isoform expression and the S-phase fraction (SPF) were determined using quantitative digital image analysis. Higher mean and median ER-beta2 levels correlated with male sex (p = 0.057 and p = 0.035, respectively), older age (p = 0.005 and p = 0.006, respectively), and lower pT stage (p = 0.008 and p = 0.009). Mean and median ER-beta5 levels correlated negatively with SPF (p = 0.021 and p = 0.047, respectively). Mean ER-beta1 expression did not correlate with any of the above mentioned clinicopathologic factors. The findings in this pilot study, although should be considered preliminary, suggest that some ER-beta isoforms may play a role in the biology of PAC. Additional larger studies are needed to confirm our findings, and to determine whether ER-beta may be considered for future targeted therapy.

Pancreatic cancer is one of the most lethal malignancies, with virtually all patients eventually succumbing to their disease. Mutations in p53 have been documented in >50% of pancreatic cancers. Owing to the high incidence of p53 mutations in PanIN 3 lesions and pancreatic tumors, we interrogated the comparative ability of adult pancreatic acinar and ductal cells to respond to oncogenic Kras and mutant Tp53(R172H) using Hnf1b:CreER(T2) and Mist1:CreER(T2) mice. These studies involved co-activation of a membrane-tethered GFP lineage label, allowing for direct visualization and isolation of cells undergoing Kras and mutant p53 activation. Kras activation in Mist1(+) adult acinar cells resulted in brisk PanIN formation, whereas no evidence of pancreatic neoplasia was observed for up to 6 months following Kras activation in Hnf1beta(+) adult ductal cells. In contrast to the lack of response to oncogenic Kras alone, simultaneous activation of Kras and mutant p53 in adult ductal epithelium generated invasive PDAC in 75% of mice as early as 2.5 months after tamoxifen administration. These data demonstrate that pancreatic ductal cells, whereas exhibiting relative resistance to oncogenic Kras alone, can serve as an effective cell of origin for pancreatic ductal adenocarcinoma in the setting of gain-of-function mutations in p53.

Increased esophageal cyclooxygenase-2 (COX-2) expression has been associated with Barrett’s esophagus (BE); however, it is unknown whether COX-2 expression varies among patient groups with different clinical or socio-demographic factors. We conducted a case–control study among eligible patients scheduled for elective esophagogastroduodenoscopy and patients eligible for screening colonoscopy recruited from primary clinics. We compared 39 BE tissue samples and 47 squamous tissue samples from BE cases and 240 squamous tissue samples from controls. Clinical and socio-demographic data were prospectively collected. Immunohistochemical staining for esophageal COX-2 was performed and scored. The median COX-2 score was significantly higher in BE tissue than squamous tissue from cases or controls (p

Question: A 48year-old Hispanic man developed melena after 25 days of hospitalization. He was initially brought to the emergency department after a motor vehicle accident that resulted in hemopneumothorax, multiple fractures, and blunt abdominal trauma. No past medical history was reported. After emergent endotracheal intubation and chest tubes placement, he underwent splenectomy, subtotal colectomy, and distal ileum resection. In the intensive care unit (ICU), he developed septic shock requiring broad-spectrum antibiotics, vassopressors, corticosteroids, and hemodialysis. Repeat laparotomy on day 9 revealed colocolostomy disruption, requiring additional bowel resection. Vassopressors and steroids were stopped after 5 days. His course was complicated by pulmonary embolism and persistent hyperglycemia. Multiple intra-abdominal washouts were required and abdominal cavity was closed on day 24. On day 25, melena was noted. He was tachycardic, afebrile, and normotensive. Surgical wound was clean, and abdominal examination revealed diffuse tenderness. Hemoglobin acutely dropped from 9 to 6.7 g/dL. Anticoagulation was stopped and an inferior vena cava filter placed. Upper endoscopy showed diffuse friable mucosa of the entire stomach. Large exudative ulceration was found in the gastric body (Figure A) from where biopsy specimens were taken. Other, scattered, clean-based ulcers were seen in the antrum and fundus (Figure B). The esophagus and duodenum were normal. A representative histopathology is provided (Figure C, D). What is the diagnosis? Look on page 1136 for the answer and see the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI.