Iron-sulfur cluster loss in mitochondrial CISD1 mediates PINK1 loss-of-function phenotypes.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Familial cases of PD are often caused by mutations of PTEN-induced kinase 1 (PINK1) and the ubiquitin ligase Parkin, both pivotal in maintaining mitochondrial quality control. CISD1, a homodimeric mitochondrial iron-sulfur-binding protein, is a major target of Parkin-mediated ubiquitination. We here discovered a heightened propensity of CISD1 to form dimers in Pink1 mutant flies and in dopaminergic neurons from PINK1 mutation patients. The dimer consists of two monomers that are covalently linked by a disulfide bridge. In this conformation CISD1 cannot coordinate the iron-sulfur cofactor. Overexpressing Cisd, the Drosophila orthologue of CISD1, and a mutant Cisd incapable of binding the iron-sulfur cluster in Drosophila reduced climbing ability and lifespan. This was more pronounced with mutant Cisd and aggravated in Pink1 mutant flies. Complete loss of Cisd, in contrast, rescued all detrimental effects of Pink1 mutation on climbing ability, wing posture, dopamine levels, lifespan, and mitochondrial ultrastructure. Our results suggest that Cisd, probably iron-depleted Cisd, operates downstream of Pink1 shedding light on PD pathophysiology and implicating CISD1 as a potential therapeutic target.

Multiomic analysis reveals cellular and epigenetic plasticity in intestinal pouches of ulcerative colitis patients.

Background & Aims: Total proctocolectomy with ileal pouch anal anastomosis (IPAA) is the standard of care for patients with severe treatment resistant ulcerative colitis (UC). Despite improvements in patient outcomes, about 50% of patients will develop inflammation of the pouch within 1-2 years following surgery. Establishment of UC pouches is associated with profound histological changes of the mucosa. A detailed characterization of these changes on a cellular and molecular level is crucial for an improved understanding of pouch physiology and diseases management. Methods: We generated cell-type-resolved transcriptional and epigenetic atlases of UC pouches using scRNA-seq and scATAC-seq data from paired biopsy samples from the ileal pouch and ileal segment above the pouch (pre-pouch) of UC-IPAA patients (n=6, female=2) without symptoms. We also collected data from paired biopsies of the terminal ileum (TI) and ascending colon (AC) from healthy controls (n=6, female=3). Results: We identified novel populations of colon-like absorptive and secretory epithelial cells, constituting a significant proportion of the epithelial cell fraction in the pouch but not in matched pre-pouch samples. Pouch-specific enterocytes expressed colon-specific genes, including CEACAM5, CA2. However, in contrast to normal colonic epithelium, these cells also expressed a range of inflammatory and secretory genes, similar to previously detected gene expression signatures in IBD patients. Comparison to longitudinal bulk RNA-seq data from UC pouches demonstrated that colon-like epithelial cells are present early after pouch functionalization and independently of subsequent pouchitis. Finally, single cell chromatin accessibility revealed activation colonic transcriptional regulators, including CDX1, NFIA, and EHF. Conclusion: UC pouches are characterized by partial colonic metaplasia of the epithelium. These data constitute a resource of transcriptomic and epigenetic signatures of cell populations in the pouch and provide an anchor for understanding the underlying molecular mechanisms of pouchitis.

Epithelial IFNγ signalling and compartmentalized antigen presentation orchestrate gut immunity.

All nucleated cells express major histocompatibility complex I and interferon-γ (IFNγ) receptor1, but an epithelial cell-specific function of IFNγ signalling or antigen presentation by means of major histocompatibility complex I has not been explored. We show here that on sensing IFNγ, colonic epithelial cells productively present pathogen and self-derived antigens to cognate intra-epithelial T cells, which are critically located at the epithelial barrier. Antigen presentation by the epithelial cells confers extracellular ATPase expression in cognate intra-epithelial T cells, which limits the accumulation of extracellular adenosine triphosphate and consequent activation of the NLRP3 inflammasome in tissue macrophages. By contrast, antigen presentation by the tissue macrophages alongside inflammasome-associated interleukin-1α and interleukin-1ß production promotes a pathogenic transformation of CD4+ T cells into granulocyte-macrophage colony-stimulating-factor (GM-CSF)-producing T cells in vivo, which promotes colitis and colorectal cancer. Taken together, our study unravels critical checkpoints requiring IFNγ sensing and antigen presentation by epithelial cells that control the development of pathogenic CD4+ T cell responses in vivo.

Ozanimod-Exposed Patients with Ulcerative Colitis Undergoing Total Colectomy Exhibit Unique Lymph Node Histologic Changes.

INTRODUCTION: Ozanimod regulates lymphocyte egress from the spleen and lymph nodes into the systemic circulation. The histologic changes which occur in the lymph nodes of patients on ozanimod is unknown. MATERIALS AND METHODS: This retrospective study included patients with UC undergoing total colectomy for treatment-refractory disease who received ozanimod and a cohort of patients with UC undergoing colectomy who did not have ozanimod exposure. Histology of the lymph nodes from the mesentery of colectomy specimens was reviewed and multiple features were scored by experienced pathologists. RESULTS: Six (13%) ozanimod-treated patients with UC required surgery for treatment-refractory disease. Colectomy specimen data were available for 5 patients (1 patient had surgery at an outside center). Lymph node specimens from 6 control patients with UC who had colectomy were examined. Histologic examination of lymph nodes showed that patients treated with ozanimod had significantly greater extent of dilated sinuses (p=0.03) and greater degrees of sinus histiocytosis (p=0.03) compared with control patients. In addition, there was a trend towards more Castleman-like angiotrophic hyperplasia, plasma cell infiltration and subcortical interfollicular expansion in ozanimod treated patients. CONCLUSION: This study identifies unique histologic changes in the lymph nodes of patients with UC treated with ozanimod. The presence of sinus histiocytosis and dilated sinuses is in keeping with the known mechanism of action of ozanimod and suggests that blocking lymphocyte egression from lymph nodes was insufficient to ameliorate disease severity in these patients. The possibility of Castleman-like features identified in several of the cases, needs to be further investigated.

A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5 -year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, (E)-19-[(1'-benzoyloxy-1'-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and used human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo. Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 hr and ~8,000 DEGs at 12 hr. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Thus, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.

Pancreatic cancer is a major cause of cancer-related deaths worldwide, with only 12% of patients surviving for five years after diagnosis. Individuals generally experience few symptoms of the disease in the early stages and are often diagnosed once the cancer has already spread to other parts of the body. By this point, options for treatment are limited. A molecule known as triptolide has been shown to kill breast, lung, pancreatic and other types of cancer cells. However, triptolide is toxic to humans and other animals, making it unsuitable for use in patients. One way to make drugs safer without compromising their beneficial effects is to modify their molecular structure. By formulating triptolide into an emulsion ­ a mixture of liquids allowing it to dissolve ­ Tian, Zhang et al. synthesized a new analogue called CK21. Experiments showed that CK21 inhibited the growth of human pancreatic cancer cells grown in a laboratory including cells grown in artificial organs similar to the pancreas, known as pancreatic tumor organoids. Furthermore, CK21 killed large tumors in mice pancreases with very few side effects, suggesting the structural modification of triptolide increased safety of the drug. To better understand how CK21 works, Tian, Zhang et al. examined the genes that were induced in the pancreatic tumor organoids at various time points after treatment with the drug. This revealed that CK21 switched off genes involved in the NF-κB cell signaling pathway, which regulates how cells grow and respond to stress. In turn, it triggered programmed cell death, killing the tumor cells in a controlled manner. The findings suggest that CK21 could be a promising candidate for treating pancreatic cancer. In the future, clinical trials will be required to establish whether CK21 is a safe and effective therapy for humans.

Inflammation in the proximal colon is a risk factor for the development of colorectal neoplasia in inflammatory bowel disease patients with primary sclerosing cholangitis.

Background: Patients with primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) have an increased risk of developing colorectal neoplasia (CRN) in the proximal colon. Objectives: To evaluate whether duration and severity of inflammation are linked to the development of CRN in this population. Design: Retrospective, case-control chart review of patients with PSC and IBD at a tertiary care center. Methods: Disease activity was scored per colonic segment at each colonoscopy prior to the first instance of observed CRN using a modified Mayo endoscopic sub-score and histologic assessment. Patients in the CRN-positive group were compared to controls that did not. Results: In all, 72 PSC-IBD patients with no history of CRN were identified, 13 of whom developed CRN after at least one colonoscopy at our institution. Patients in the CRN-positive group had significantly more endoscopic (p < 0.01) and histologic (p < 0.01) inflammation in the right compared to the control group prior to the development of dysplasia. There was significantly greater endoscopic inflammation in the segment of the colon with a dysplastic lesion than other segments of the colon (p = 0.018). Patients with moderate/severe lifetime endoscopic (p = 0.02) or histologic inflammation (p = 0.04) score had a lower probability of remaining free of dysplasia during follow-up. Nearly half of the patients with dysplasia had invisible lesions found on random biopsy. Conclusions: Endoscopic and histologic inflammation in the proximal colon are risk factors for CRN in patients with PSC-IBD. PSC-IBD patients frequently have subclinical inflammation, and these findings support the practice of regular assessment of disease activity and random biopsy of inflamed and uninflamed areas in patients with PSC with the goal of reducing inflammation to prevent the development of CRN.

Patients with PSC and IBD have not been examined as a cohort to assess for risk factors for CRN. We found that severe inflammation in the proximal colon is the main risk factor for CRN in these patients.

Antigen-driven colonic inflammation is associated with development of dysplasia in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an immune-mediated disease of the bile ducts that co-occurs with inflammatory bowel disease (IBD) in almost 90% of cases. Colorectal cancer is a major complication of patients with PSC and IBD, and these patients are at a much greater risk compared to patients with IBD without concomitant PSC. Combining flow cytometry, bulk and single-cell transcriptomics, and T and B cell receptor repertoire analysis of right colon tissue from 65 patients with PSC, 108 patients with IBD and 48 healthy individuals we identified a unique adaptive inflammatory transcriptional signature associated with greater risk and shorter time to dysplasia in patients with PSC. This inflammatory signature is characterized by antigen-driven interleukin-17A (IL-17A)+ forkhead box P3 (FOXP3)+ CD4 T cells that express a pathogenic IL-17 signature, as well as an expansion of IgG-secreting plasma cells. These results suggest that the mechanisms that drive the emergence of dysplasia in PSC and IBD are distinct and provide molecular insights that could guide prevention of colorectal cancer in individuals with PSC.

Pathways Related to Colon Inflammation Are Associated with Colorectal Carcinoma: A Transcriptome- and Methylome-Wide Study.

The association of chronic inflammation with colorectal carcinoma (CRC) development is well known in ulcerative colitis (UC). However, the role of inflammatory changes in sporadic CRC pathogenesis is less widely appreciated. In this study, in the first step using RNA-seq, we identified gene-pathway-level changes in UC-associated CRC (UC CRC, n = 10) and used the changes as a proxy for inflammation in human colon to ask if there were associations of inflammatory pathway dysregulations in sporadic CRC pathogenesis (n = 8). We found down-regulations of several inflammation-related metabolic pathways (nitrogen metabolism, sulfur metabolism) and other pathways (bile secretion, fatty acid degradation) in sporadic CRC. Non-inflammation-related changes included up-regulation of the proteasome pathway. In the next step, from a larger number of paired samples from sporadic CRC patients (n = 71) from a geographically and ethnically different population and using a different platform (microarray), we asked if the inflammation-CRC association could be replicated. The associations were significant even after stratification by sex, tumor stage, grade, MSI status, and KRAS mutation status. Our findings have important implications to widen our understanding of inflammatory pathogenesis of sporadic CRC. Furthermore, targeting of several of these dysregulated pathways could provide the basis for improved therapies for CRC.

Pancreatic tumors exhibit myeloid-driven amino acid stress and upregulate arginine biosynthesis.

Nutrient stress in the tumor microenvironment requires cancer cells to adopt adaptive metabolic programs for survival and proliferation. Therefore, knowledge of microenvironmental nutrient levels and how cancer cells cope with such nutrition is critical to understand the metabolism underpinning cancer cell biology. Previously, we performed quantitative metabolomics of the interstitial fluid (the local perfusate) of murine pancreatic ductal adenocarcinoma (PDAC) tumors to comprehensively characterize nutrient availability in the microenvironment of these tumors. Here, we develop Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains nutrient levels representative of the PDAC microenvironment, enabling us to study PDAC metabolism ex vivo under physiological nutrient conditions. We show that PDAC cells cultured in TIFM adopt a cellular state closer to that of PDAC cells present in tumors compared to standard culture models. Further, using the TIFM model, we found arginine biosynthesis is active in PDAC and allows PDAC cells to maintain levels of this amino acid despite microenvironmental arginine depletion. We also show that myeloid derived arginase activity is largely responsible for the low levels of arginine in PDAC tumors. Altogether, these data indicate that nutrient availability in tumors is an important determinant of cancer cell metabolism and behavior, and cell culture models that incorporate physiological nutrient availability have improved fidelity to in vivo systems and enable the discovery of novel cancer metabolic phenotypes.

OrganoID: A versatile deep learning platform for tracking and analysis of single-organoid dynamics.

Organoids have immense potential as ex vivo disease models for drug discovery and personalized drug screening. Dynamic changes in individual organoid morphology, number, and size can indicate important drug responses. However, these metrics are difficult and labor-intensive to obtain for high-throughput image datasets. Here, we present OrganoID, a robust image analysis platform that automatically recognizes, labels, and tracks single organoids, pixel-by-pixel, in brightfield and phase-contrast microscopy experiments. The platform was trained on images of pancreatic cancer organoids and validated on separate images of pancreatic, lung, colon, and adenoid cystic carcinoma organoids, which showed excellent agreement with manual measurements of organoid count (95%) and size (97%) without any parameter adjustments. Single-organoid tracking accuracy remained above 89% over a four-day time-lapse microscopy study. Automated single-organoid morphology analysis of a chemotherapy dose-response experiment identified strong dose effect sizes on organoid circularity, solidity, and eccentricity. OrganoID enables straightforward, detailed, and accurate image analysis to accelerate the use of organoids in high-throughput, data-intensive biomedical applications.

AL101, a gamma-secretase inhibitor, has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling.

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.

The Interaction of SWI/SNF with the Ribosome Regulates Translation and Confers Sensitivity to Translation Pathway Inhibitors in Cancers with Complex Perturbations.

Subunits from the chromatin remodelers mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) are mutated, deleted, or amplified in more than 40% of cancers. Understanding their functions in normal cells and the consequences of cancerous alterations will provide insight into developing new targeted therapies. Here we examined whether mSWI/SNF mutations increase cellular sensitivity to specific drugs. Taking advantage of the DepMap studies, we demonstrate that cancer cells harboring mutations of specific mSWI/SNF subunits exhibit a genetic dependency on translation factors and are sensitive to translation pathway inhibitors. Furthermore, mSWI/SNF subunits were present in the cytoplasm and interacted with the translation initiation machinery, and short-term inhibition and depletion of specific subunits decreased global translation, implicating a direct role for these factors in translation. Depletion of specific mSWI/SNF subunits also increased sensitivity to mTOR-PI3K inhibitors. In patient-derived breast cancer samples, mSWI/SNF subunits expression in both the nucleus and the cytoplasm was substantially altered. In conclusion, an unexpected cytoplasmic role for mSWI/SNF complexes in translation suggests potential new therapeutic opportunities for patients afflicted by cancers demonstrating alterations in their subunits. SIGNIFICANCE: This work establishes direct functions for mSWI/SNF in translation and demonstrates that alterations in mSWI/SNF confer a therapeutic vulnerability to translation pathway inhibitors in cancer cells.

Histopathology of Colectomy Specimens Predicts Endoscopic Pouch Phenotype in Patients with Ulcerative Colitis.

BACKGROUND: The endoscopic appearance in patients with "pouchitis" after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) can be quite heterogenous. Patients with an endoscopic phenotype resembling Crohn's disease (CD) are at high risk of pouch loss. AIMS: We aimed to assess how the histopathology of colectomy specimens predicts endoscopic pouch phenotypes in UC. METHODS: We retrospectively assessed pouchoscopies from patients with UC who underwent IPAA and classified pouch findings into 7 main phenotypes: (1) normal, (2) afferent limb involvement, (3) inlet involvement, (4) diffuse, (5) focal inflammation of the pouch body, (6) cuffitis, and (7) pouch with fistulas noted ≥ 6 months from ileostomy takedown. We assessed the clinical and pathological data including deep, focal inflammation, granulomas, and terminal ileal involvement in the colectomy specimens. Logistic regression analysis was performed to identify contributing factors to each phenotype. RESULTS: This study included 1,203 pouchoscopies from 382 patients with UC. On multivariable analysis, deep inflammation was significantly associated with pouch fistulas (Odds ratio 3.27; 95% confidence interval 1.65-6.47; P = 0.0007). Of the 75 patients with deep inflammation, only two patients (2.7%) were diagnosed with CD based on pathology review. Terminal ileal involvement significantly increased the risk of afferent limb involvement (Odds ratio 2.96; 95% confidence interval 1.04-8.47; P = 0.04). There were no significant associations between other microscopic features and phenotypes. CONCLUSIONS: We identify histologic features of colectomy specimens in UC that predict subsequent pouch phenotypes. Particularly, deep inflammation in the resected colon was significantly associated with pouch fistulas, a pouch phenotype with poor prognosis.

Epigenetic Dysregulation of 5-hydroxymethylcytosine in Well-Differentiated Pancreatic Neuroendocrine Tumors.

Dysregulation of epigenetic mechanisms, reflected by loss of expression of 5-hydroxymethylcytosine (5-hmC) is being increasingly recognized as a marker of aggressive behavior in several neoplasms; however, the role of such epigenetic modifiers in pancreatic neuroendocrine tumors (PanNETs) has not been studied. Annotated cohort of 60 PanNETs was evaluated for 5-hmC expression using immunohistochemistry. Univariable and multivariable analyses were performed. To determine intratumor heterogeneity of 5-hmC expression, 26 additional synchronous metastatic deposits of PanNETs from 8 patients were evaluated for 5-hmC expression. 5-hmC level showed significant association with the presence of distant metastases (P=0.02), female sex (P=0.04), and Ki-67 proliferation index (P=0.002). A multivariate model created using the stepwise logistic regression analysis showed the presence of nodal metastases (odds ratio=6.15), lymphovascular invasion (odds ratio=4.07) and lack of 5-hmC expression (odds ratio=5.34) were predictive of the risk of distant metastasis in PanNETs with a c-statistic of 0.845. Epigenetic intratumoral heterogeneity of 5-hmC expression was seen in 37.5% cases (3/8). Our work provides evidence that epigenetic regulators are involved in the pathobiology of PanNETs and immunohistochemical analysis of 5-hmC may be able to refine prognostic evaluation of these tumors.

T cell protein tyrosine phosphatase protects intestinal barrier function by restricting epithelial tight junction remodeling.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.

mSWI/SNF promotes Polycomb repression both directly and through genome-wide redistribution.

The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.

Wnt-ß-catenin activation epigenetically reprograms Treg cells in inflammatory bowel disease and dysplastic progression.

The diversity of regulatory T (Treg) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt+ Treg cells with elevated expression of ß-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt+ Treg cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt-ß-catenin signaling in human and murine Treg cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt+ Treg cells coexpressing multiple pro-inflammatory cytokines. Binding of the ß-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt-ß-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Treg cells. Activation of ß-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.

Objective Visual Analog Scale for Biopsy Diagnosis of Helicobacter pylori Infection in Clinical Practice.

Historic and current pathology society guidelines recommend using visual gestalt to identify substantial inflammatory cell infiltrate in Helicobacter pylori gastritis, but these scales were subjectively designed. This study aims to objectively investigate the density of inflammation that justifies additional workup for H. pylori infection. We retrospectively identified 2 patient cohorts who had undergone endoscopy with gastric biopsies; 1 with H. pylori infection (n=66), confirmed with a positive stool antigen test and/or Campylobacter-like organism test, and 1 without infection (n=81). Antral and body biopsies were selected from each case, if available, and stained with MUM-1 to highlight mucosal plasma cells. Digital analysis was performed to calculate the number of plasma cells/mm2, termed the "inflammatory score" (IS). Patients with H. pylori infection had an average of 1289 plasma cells/mm2 in the antrum and 835 plasma cells/mm2 in the body, compared with 346 plasma cells/mm2 in the antrum and 178 plasma cells/mm2 in the body in patients without infection. IS cut-off values for a positive infection were 714 plasma cells/mm2 in the antrum and 316 plasma cells/mm2 in the body, with high sensitivities and specificities in both the antrum (92%, 92%) and body (85%, 84%), respectively. A visual analog scale was created to provide a histologic correlate of the observed IS ranges and cut-offs. This practical and objective scale is associated with a high sensitivity and specificity for diagnosing H. pylori infection and justifies moving away from upfront universal H. pylori testing in routine clinical practice.

Cumulative Histologic Inflammation Predicts Colorectal Neoplasia in Ulcerative Colitis: A Validation Study.

BACKGROUND: Chronic inflammation in ulcerative colitis (UC) is associated with the development of colorectal neoplasia (CRN). A group at St. Mark's Hospital reported a novel cumulative inflammatory index that predicted the development of CRN in UC patients that we validated with an independent, well-described, matched, case-controlled cohort from the University of Chicago. METHODS: Cumulative inflammatory burden (CIB) was calculated by summing each histological inflammatory activity (HIA) score and multiplying it by the length of the surveillance interval. Persistency was defined by the number of surveillance episodes (with a severity score >2) divided by the total number of surveillance procedures. T tests compared the mean and maximum HIA scores, assessing mean and maximum severity, CIB, and persistency. RESULTS: Sixty-two UC patients (26 patients with CRN, 36 control patients without CRN) were analyzed. Fifty-five percent were men, mean disease duration was 20.6 years, and mean age at CRN diagnosis was 43.9. Of the CRN patients, 6 (23%) had colorectal cancer, 16 (62%) had low-grade dysplasia, and 4 (15%) had indefinite dysplasia. Using mean HIA scores, we found CIB to be statistically greater in CRN patients (P = 0.04). Using maximum HIA scores, we found CIB (P = 0.02), mean severity (P = 0.03), and persistency (P = 0.01) to be significantly greater in CRN patients. Maximum severity was numerically greater for mean and maximum HIA scores but did not reach significance. CONCLUSION: Cumulative histologic inflammation is significantly associated with the development of CRN in UC patients. This suggests a management strategy of controlling inflammation to reduce the risk of CRN and may influence the selection of surveillance intervals.

Histologic changes caused by injection of a novel submucosal lifting agent for endoscopic resection in GI lesions.

BACKGROUND AND AIMS: ORISE Gel is a recently introduced, U.S. Food and Drug Administration-approved submucosal lifting agent used in endoscopic resection of GI lesions. Histologically evident gel deposits in resected specimens may pose a potential diagnostic pitfall. To aid in recognition of this procedure-related artifact, we report the largest histologic series of ORISE Gel in endoscopic and surgical resection specimens to date. METHODS: Fifty-eight EMR/endoscopic submucosal dissection (ESD) specimens with ORISE Gel injection and 5 interval surgical resection specimens with previous ORISE Gel injection were included. Patient demographics and endoscopy reports were obtained. Histologic slides from all cases were reviewed. Histochemical stains were performed on select cases. RESULTS: Fifty-one EMR and 7 ESD specimens were identified. In 51 of 58 (88%) endoscopic resection specimens, amorphous, pale blue-gray, finely granular material was evident in the submucosa, as well as focally within the mucosa in 4 cases. Most cases showed homogeneous near-complete filling of the submucosa with this material, whereas a few demonstrated areas of condensation and retraction. Mucicarmine and periodic acid-Schiff stains were negative for mucin. Interval surgical resection specimens revealed extensive deposition of dense, eosinophilic material with associated multinucleated giant cells in the submucosa in all cases, with transmural extension in 3 cases. CONCLUSION: ORISE Gel injection during endoscopic resection of GI lesions results in deposition of amorphous, blue-gray material seen in histologic sections, whereas interval surgical resection specimens demonstrate dense, eosinophilic material with an associated giant cell reaction. Awareness of these artifacts will help avoid misinterpretation of their presence as pathologic findings.