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Nonalcoholic fatty liver disease (NAFLD) commonly coexists with Crohn's disease (CD); however, it remains unclear if it is more prevalent than would be expected as ultrasound surveys of CD patients report a very wide range of prevalence (9%-40%).1-3 To address this uncertainty, we performed a prospective, cross-sectional survey of NAFLD in CD patients by generating magnetic resonance proton density fat fraction (MR-PDFF) maps as compared with 2 control populations. MR-PDFF provides a quantitative, sensitive and specific (97% and 100%, respectively) radiographic surrogate for liver fat.4.

We have previously identified NOD2 genotype and inflammatory bowel diseases (IBD) phenotype, as associated with shifts in the ileal microbiome ("dysbiosis") in a patient cohort. Here we report an integrative analysis of an expanded number of Crohn's disease (CD) related genetic defects in innate immune function (NOD2, ATG16L1, IRGM, CARD9, XBP1, ORMDL3) and composition of the ileal microbiome by combining the initial patient cohort (Batch 1, 2005-2010, n = 165) with a second consecutive patient cohort (Batch 2, 2010-2012, n = 118). These combined patient cohorts were composed of three non-overlapping phenotypes: 1.) 106 ileal CD subjects undergoing initial ileocolic resection for diseased ileum, 2.) 88 IBD colitis subjects without ileal disease (predominantly ulcerative colitis but also Crohn's colitis and indeterminate colitis, and 3.) 89 non-IBD subjects. Significant differences (FDR < 0.05) in microbiota were observed between macroscopically disease unaffected and affected regions of resected ileum in ileal CD patients. Accordingly, analysis of the effects of genetic and clinical factors were restricted to disease unaffected regions of the ileum. Beta-diversity differed across the three disease categories by PERMANOVA (p < 0.001), whereas no significant differences in alpha diversity were noted. Using negative binomial models, we confirmed significant effects of IBD phenotype, C. difficile infection, and NOD2 genotype on ileal dysbiosis in the expanded analysis. The relative abundance of the Proteobacteria phylum was positively associated with ileal CD and colitis phenotypes, but negatively associated with NOD2R genotype. Additional associations with ORMDL3 and XBP1 were detected at the phylum/subphylum level. IBD medications, such as immunomodulators and anti-TNFα agents, may have a beneficial effect on reversing dysbiosis associated with the IBD phenotype. Exploratory analysis comparing microbial composition of the disease unaffected region of the resected ileum between 27 ileal CD patients who subsequently developed endoscopic recurrence within 6-12 months versus 34 patients who did not, suggested that microbial biomarkers in the resected specimen helped stratify patients with respect to risk of post-surgical recurrence.

The tryptophan-metabolizing enzyme indoleamine 2,3 dioxygenase 1 (IDO1) is frequently overexpressed in epithelial-derived malignancies, where it plays a recognized role in promoting tumor immune tolerance. We previously demonstrated that the IDO1–kynurenine pathway (KP) also directly supports colorectal cancer growth by promoting activation of β-catenin and driving neoplastic growth in mice lacking intact adaptive immunity. In this study, we sought to delineate the specific role of epithelial IDO1 in colon tumorigenesis and define how IDO1 and KP metabolites interact with pivotal neoplastic signaling pathways of the colon epithelium. We generated a novel intestinal epithelial–specific IDO1 knockout mouse and utilized established colorectal cancer cell lines containing β-catenin–stabilizing mutations, human colorectal cancer samples, and human-derived epithelial organoids (colonoids and tumoroids). Mice with intestinal epithelial–specific knockout of IDO1 developed fewer and smaller tumors than wild-type littermates in a model of inflammation-driven colon tumorigenesis. Moreover, their tumors exhibited reduced nuclear β-catenin and neoplastic proliferation but increased apoptosis. Mechanistically, KP metabolites (except kynurenic acid) rapidly activated PI3K-Akt signaling in the neoplastic epithelium to promote nuclear translocation of β-catenin, cellular proliferation, and resistance to apoptosis. Together, these data define a novel cell-autonomous function and mechanism by which IDO1 activity promotes colorectal cancer progression. These findings may have implications for the rational design of new clinical trials that exploit a synergy of IDO1 inhibitors with conventional cancer therapies for which Akt activation provides resistance such as radiation. Significance: This study identifies a new mechanistic link between IDO1 activity and PI3K/AKT signaling, both of which are important pathways involved in cancer growth and resistance to cancer therapy.

TPS2070 Background: Recurrent glioblastoma (rGBM) after chemoradiotherapy has a dismal outcome with very limited treatment options. Addition of reirradiation to bevacizumab appears to improve progression-free survival (PFS) but does not improve overall survival (OS). Immune checkpoint inhibitors of programmed cell death-1 (PD-1) pathway appear to have limited single-agent activity for rGBM due to its immunesuppressive microenvironment. Indoleamine 2,3 dioxygenase 1 (IDO1) is an inducible and rate-limiting enzyme that catabolizes tryptophan (Trp) into kynurenine (Kyn). IDO1 is over-expressed in 50̃90% of GBM patients, and high IDO1 levels correlate with reduced OS. Epacadostat is a highly potent and selective oral inhibitor of IDO1 and may increase tumor sensitivity to anti-PD-1 blockade. Retifanlimab is a humanized anti-PD-1 monoclonal antibody directed against PD-1. The purpose of this study is to evaluate the safety and efficacy of combining retifanlimab plus or minus epacadostat with reirradiation and bevacizumab for rGBM patients. Methods: This is an open-label nonrandomized phase II study of two sequential cohorts for bevacizumab-naïve adults with rGBM: retifanlimab + bevacizumab+ radiation (cohort A), and retifanlimab + epacadostat + bevacizumab + radiation (cohort B). Each cohort will enroll 24 evaluable patients. Key eligibility criteria include candidates for reirradiation and bevacizumab, age ≥ 18 years, Karnofsky performance status ≥ 60%, and dexamethasone dose ≤ 4 mg/day. The primary endpoint is OS. Secondary endpoints include PFS, neurologic functions, and toxicity. The correlative endpoints include studies assess the anti-glioma immune response, serum Kyn/Trp ratio, and RNA expression of IDO1 and PD-L1 from available tissue. The trial is actively enrolling. At the time of abstract submission, 16 of the planned 24 patients in Cohort A have been enrolled. Clinical trial information: NCT03532295. [Table: see text]

Background Ulcerative Colitis (UC) is characterized by intestinal inflammation with chronic inflammatory features. Along with the typical neutrophil-predominant inflammation, there has been growing understanding of the role of eosinophils in UC and the potential therapeutic benefit of Vedolizumab (VDZ) in those cases. VDZ blocks gastrointestinal homing of T lymphocytes and eosinophil recruitment to the intestinal mucosa via blocking α4β7 which plays a major role in eosinophils localization to the intestinal mucosa. We aimed to assess whether mucosal eosinophilia can predict response to VDZ. Methods A retrospective study in a single IBD center. Active UC patients who were initiated on VDZ were identified. All patients had at least one documented encounter of VDZ infusion, colonoscopy prior to initiation of VDZ and afterwards with mucosal biopsies at both time points. The endoscopic activity was scored using Mayo endoscopic subscore (MES) with remission as MES 0 or 1. Clinical response was assessed using Physician’s Global Assessment (PGA) as no response, partial or complete clinical response and clinical remission. Histology was reported by an expert GI pathologist and the response was assessed as none, partial or complete. Mucosal eosinophilia was graded at initial and follow-up biopsies as not conspicuous vs. conspicuous with predominance of eosinophils (>100 eosinophils in at least 2 hpf). Eosinophil activity defined as presence of at least 1 of the 3 histologic features: eosinophilic microabscesses, eosinophilic cryptitis, or degranulated eosinophils Results 107 UC patients were included in the study with a mean age of 44.9 years, majority were males (N = 61, 55.5%), Caucasians (N = 96, 87.3%) and with pancolonic location (N = 100, 90.9%). Mean disease duration was 11.3 yrs (SD, 8.6). 55.5% of patients had prior aTNF exposure. Clinical remission was observed in (N = 63, 57.3%) and endoscopic remission (N = 73, 66.4%) after initiating VDZ. Complete histological response was observed in (N=50, 46.7%). Baseline characteristics based on endoscopic remission is shown where male sex, disease location and the proportion of patients with conspicuous eosinophils with abnormal activity in mucosal biopsies as well as no improvement in eosinophil activity compared to pre-treatment biopsies is greater among those not in endoscopic remission. In a stepwise backward logistic regression model including sex and disease location, improvement in abnormal eosinophilic activity was independently associated with increased odds of endoscopic remission after initiating vedolizumab (OR 4.3; CI, 1.1 - 16.4, p = 0.03). Conclusion In ulcerative colitis, change in mucosal eosinophilia after initiating VDZ was an independent predictor of endoscopic remission with VDZ.

Background RNA-binding motif protein 47 (RBM47) is a requisite cofactor in APOBEC1-dependent C-to-U RNA editing whose RNA targets are unknown. Aims Because Apobec-1 knockout mice are protected against polyposis in the ApcMin background, we asked if intestine-specific Rbm47 knockout mice (Rbm47IKO) exhibit altered tumor susceptibility, either spontaneously or in different murine cancer models. Results 86% (6/7) of 12-month old chow-fed Rbm47IKOmice developed spontaneous intestinal and colonic polyps (Fig 1A), compared to 10% (1/10) aged Rbm47floxcontrols. RNA-seq showed upregulated proliferation, glutathione metabolism, and anti-oxidative pathways in Rbm47 IKOintestine. QPCR revealed reduced abundance of the long Tjp1 mRNA isoform (Tjp1+) in Rbm47 IKOmice (Fig 1B), which was further reduced in polyp vs uninvolved tissue (11.8±0.2 vs 15.1±1.2, P=.03) in aged Rbm47 IKOmice. In support, we observed decreased Rbm47 mRNA (–DCT 0.97±2.79 vs 3.47±2.58, P Conclusion Rbm47IKOmice demonstrate spontaneous polyposis through upregulating proliferation and modifying Tjp1 alternative RNA splicing, with increased proximal intestinal polyposis in the ApcMin/+ background. By contrast, Rbm47IKOmice are protected against AOM-DSS-driven colitis-associated colon tumorogenesis by augmenting antioxidant capacity, over-expression of Il-33, and decreasing inflammatory activity. These contrasting phenotypes suggest that RBM47 exerts pathway-specific effects on intestinal tumorigenesis as a genetic modifier of growth, proliferation and inflammatory pathways. Download : Download high-res image (81KB) Download : Download full-size image Figure 1 . A: dissecting microscope images (left) and hematoxylin and eosin staining sections (right) of spontaneous polypogenesis in small intestine and colon of 12-month old chowfed Rbm47 IKOmice. B: Relative abundance of long Tjp1 mRNA isoform (Tjp1+) by quantitative RT-PCR in young adult mice jejunum. C: Rbm47 mRNA expression and relative abundance of Tjp1+ isoform by quantitative RT-PCR in human colorectal cancer tissue vs paired normal tissue. D: Comparison of overall survival among colorectal cancer patients with different levels of Rbm47 mRNA expression in tumor tissue using the cancer genome atlas (TCGA) database. Download : Download high-res image (71KB) Download : Download full-size image Figure 2 . A: Macroscopic illustration of colitis-associated colon tumors in mice treated with AOM)-DSS. B: left; the HE stained microscopic sections illustrate abundance of crypt abscess formation (yellow arrowheads) within dysplastic lesions of colon in AOM-DSS treated mice. Middle; F4/80 immunohistochemical staining of dysplastic lesions of colon in AOM-DSS treated mice. Right: Relative mRNA expression of inflammatory genes by quantitative RTPCR in dysplastic lesions of AOM-DSS treated mice. C: Relative mRNA expression of genes related to interleukin 33 by quantitative RT-PCR in dysplastic lesions of AOM-DSS treated mice. D: Macroscopic illustration of proximal small intestine tumors in mice with Apc Min/+background.

Introduction Coronavirus Disease 2019 (COVID-19) is an ongoing public health crisis that has sickened or precipitated death in millions. The etiologic agent of COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infects the intestinal epithelium, with viral RNA shed in the stool, and can induce GI symptoms similar to the human inflammatory bowel diseases (IBD). An international surveillance epidemiology study, SECURE-IBD, reported that the standardized mortality ratio trends higher in IBD patients (1.5–1.8) and that 5-aminosalicylic acid (5-ASA) therapy correlates with poor outcome. Together these data indicate patients with IBD may represent a particularly vulnerable population during this COVID-19 pandemic. Methods Published datasets GSE75214 and GSE16879 were downloaded and expression levels of select genes were querried using RStudio. Primary human ileal spheroids (enteroids), derived from healthy donors and patients with Crohn’s disease (CD), were grown on 2D transwells until confluent. Cells were then differentiated for 3d before infection with a modified vesicular stomatitis virus expressing the SARS-CoV-2 spike protein (VSV-SARS-CoV-2) and green fluorescent protein (GFP) for 1 h at a multiplicity of infection (MOI) of ~0.5. Healthy enteroids were treated with 10 ng/ml of human Tumor Necrosis Factor alpha (TNF-α) for 24h before infection via the basolateral reservoir or 5-ASA 5h before infection via the apical reservoir. 24h after infection, cells were processed for immunofluorescence or RNA expression of select genes by qRT-PCR. Results VSV-SARS-CoV-2 was able to infect both healthy and CD enteroids as determined by co-staining of GFP, indicative of virus infection, and the viral receptor ACE2. However, levels of GFP fluorescence did not correlate with ACE2 expression in CD enteroids. A subset of CD enteroids exhibited enhanced protease expression (TMPRSS2, TMPRSS4, CTSL), each of which correlated with higher viral RNA levels (P=0.04, P=0.002, P=0.006, respectively). In Vero E6 cells, 5-ASA inhibited the replication of a clinical isolate of SARS-CoV-2 in a concentration-dependent manner. Treating healthy enteroids with 5-ASA did not have any effect on viral proliferation, while TNF-α pretreatment reduced viral RNA. 5-ASA treatment caused a reduction of ACE2 and an increase in CTSL expression. Conclusions Host proteases, particularly the lysosomal protease CTSL, contribute to the infection of CD enteroids and may represent novel therapeutic targets in patients with IBD and COVID-19. 5-ASA modulates the expression of several epithelial genes relevant to SARS-CoV-2 infection, yet does not alter viral replication in healthy enteroids.

Sleep disturbances are common, and perhaps are even more prevalent in irritable bowel syndrome (IBS).To determine the effect of measured sleep on IBS symptoms the following day, IBS-specific quality of life (IBS-QOL) and non-GI pain symptoms.IBS patients' sleep patterns were compared to healthy individuals via wrist-mounted actigraphy over 7 days. Daily bowel pain logs (severity, distress; 10-point Likert) stool pattern (Bristol scale) and supporting symptoms (e.g. bloating, urgency; 5-point Likert) were kept. Validated measures, including the GI Symptom Rating Scale-IBS, Visceral Sensitivity Index, Pittsburgh Sleep Quality Index and the IBS-Quality of Life were collected. Mediation analysis explored the relationship between sleep, mood and bowel symptoms.Fifty subjects (38.6 ± 1.0 years old, 44 female; 24 IBS and 26 healthy controls) completed sleep monitoring. IBS patients slept more hours per day (7.7 ± 0.2 vs. 7.1 ± 0.1, P = 0.008), but felt less well-rested. IBS patients demonstrated more waking episodes during sleep (waking episodes; 12.1 vs. 9.3, P0.001). Waking episodes predicted worse abdominal pain (P ≤ 0.01) and GI distress (P0.001), but not bowel pattern or accessory IBS symptoms (P0.3 for each). Waking episodes negatively correlated with general- and IBS-specific QOL in IBS (r = -0.58 and -0.52, P0.001 for each). Disturbed sleep effects on abdominal pain were partially explained by mood as an intermediate.Sleep disturbances are more common in irritable bowel syndrome, and correlate with IBS-related pain, distress and poorer irritable bowel syndrome-related quality of life. Disturbed sleep effects extend beyond the bowel, leading to worse mood and greater somatic pain in patients with the irritable bowel syndrome.

Ulcerative colitis (UC) is an immune-mediated disease of the colon that is character- ized by diffuse and continuous inflammation contiguous from the rectum. Half of UC patients have inflammation limited to the distal colon (proctitis or proctosigmoiditis) that primarily causes symptoms of bloody diarrhea and urgency. Mild-to-moderate distal UC can be effectively treated with topical formulations (rectal suppositories, enemas, or foam) of mesalamine or steroids to reduce mucosal inflammation and alleviate symptoms. Enemas or foam formulations adequately reach up to the splenic flexure, have a minimal side-effect profile, and induce remis- sion alone or in combination with systemic immunosuppressive therapy. Herein, we compare the efficacy, cost, patient tolerance, and side-effect profiles of steroid and mesalamine rectal formulations in distal UC. Patients with distal mild-to-moderate UC have a remission rate of approximately 75% (NNT =2) after treatment for 6 weeks with mesalamine enemas. Rectal budesonide foam induces remission in 41.2% of patients with mild-to-moderate active distal UC compared to 24% of patient treated with placebo (NNT =5). However, rectal budesonide has better patient tolerance profile compared to enema formulations. Despite its favorable efficacy, safety, and cost profiles, patients and physicians significantly underuse topical treat- ments for treating distal colitis. This necessitates improved patient education and physician familiarity regarding the indications, effectiveness, and potential financial and tolerability barriers in using rectal formulations.

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death in the United States. Cytotoxic therapies cause significant side effects for most patients and do not offer cure in many advanced cases of CRC. Immunotherapies are a promising new approach to harness the body’s own immune system and inflammatory response to attack and clear the cancer. Tryptophan metabolism along the kynurenine pathway is a particularly promising target for immunotherapy. Indoleamine 2,3 dioxygenase 1 (IDO1) is the most well studied of the enzymes that initiate this pathway and it is commonly overexpressed in CRC. Herein, we provide an in-depth review of how tryptophan metabolism and kynurenine pathway metabolites shape factors important to CRC pathogenesis including the host mucosal immune system, pivotal transcriptional pathways of neoplastic growth and luminal microbiota. This pathway’s role in other gastrointestinal malignancies such as gastric, pancreatic, esophageal and gastrointestinal stromal tumors (GIST) is also discussed. Finally, we highlight how currently available small molecule inhibitors and emerging methods for therapeutic targeting of IDO1 might be applied to colon, rectal and colitis associated cancer.

Background: Epacadostat is an orally active, potent and selective inhibitor of indoleamine 2, 3-dioxygenase 1 (IDO1). IDO1 inhibition enhances cytotoxic T cell activation by dendritic cells, and significantly decreases regulatory T cell proliferation. We developed preclinical data supporting combination of epacadostat with radiation therapy in rectal cancer. In rectal cancer samples obtained from patients who received hypofractionated radiation, IDO1 was overexpressed in tumor tissue as compensatory response after radiation in both microsatellite stable and unstable cell lines. IDO1 inhibition with epacadostat selectively reduced survival of cancer cells and enhanced radiosensitivity without impacting normal epithelial cells. Epacadostat in addition to radiation showed both increased tumor cytotoxicity and enhanced immune activation in tumor microenvironment in a syngeneic mouse model of colorectal cancer. Literature also supports the choice of high dose, hypofractionated radiation to induce a more favorable anti-tumor immune response. Therefore, we hypothesized that IDO1 expression is a mechanism of radioresistance in rectal cancer and IDO1 inhibition is a safe and well-tolerated combination therapy to enhance tumor radiosensitivity. Methods: Patients with locally advanced rectal cancer who are candidates for neoadjuvant therapy using short-course radiation and chemotherapy are included in this study. Primary objective of the study is to determine the recommended phase II dose (RP2D) of epacadostat for combination with short course radiation and chemotherapy in preoperative treatment of locally advanced rectal cancer. This study includes dose-escalation part of patients receiving epacadostat with radiation and chemotherapy (n=6-18) followed by dose-expansion (n=up to 27 including those treated at RP2D during escalation). Two dose levels of epacadostat (300mg and 600mg orally twice daily) will be explored. Epacadostat will be combined with short-course radiation (5Gy x 5 fractions) followed by 6 cycles of CAPOX chemotherapy, until the day of surgery for a total of approximately 24 weeks of therapy. Research biopsies of tumor and adjacent non-tumor tissue along with blood sample collection will be performed before and after the radiation, and at the time of surgery. Tryptophan pathway metabolites, immune checkpoint biomarkers, markers of cell death, proliferation and potentially prognostic molecular biomarkers will be measured in tumor tissues before and after radiation therapy. In addition, patient-derived organoid and xenograft models from rectal biopsy samples will be used to determine the success rate of organoid generation, to evaluate treatment response and to characterize molecular changes to identify potential predictors of response and mechanisms of resistance. Enrollment began in November 2019. NCT03516708 Citation Format: Haeseong Park, Ebunoluwa Otegbeye, Hyun Kim, Matthew Mutch, Katrina Pedersen, Manik Amin, Benjamin Tan, Nikolaos Trikalinos, Kian-Huat Lim, Olivia Aranha, Rama Suresh, Shahed Badiyan, Matthew Silviera, Lauren Henke, Paul Wise, Steven Hunt, Jonathan Mitchem, Esther Lu, Andrea Wang-Gillam, Matthew Ciorba. Phase I study of epacadostat added to preoperative chemoradiation in patients with locally advanced rectal cancer [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 CT234.

Background Disease location, behavior, and average activity over time vary significantly amongst patients with inflammatory bowel disease (IBD). Despite this heterogeneity patients are often subjected to a “one size fits all” treatment plan. Clinical and multi-omic profiles which predict responsiveness to treatment are needed given the expanding range of therapeutic options for IBD. Methods The Study of Prospective Adult Research Cohort of IBD (SPARC IBD) is a prospective, longitudinal cohort study of patients with IBD ages 18 and older managed according to local standards at 17 academic medical centers in the US. Demographic data, disease-related data, and patient-reported outcomes are collected at baseline, during routine GI office visits, and quarterly by data abstraction from the electronic health record, patient surveys, and highly structured electronic case report forms (eCRF) created with Epic Systems (IBD SmartForm). Biosamples are collected at baseline (blood and stool), at the time colonoscopy (stool and tissue), and after key medication changes (blood and stool). Clinical data is transferred from sites on a periodic basis and stored in the Crohn’s & Colitis Foundation’s exchange platform called IBD Plexus. A portion of the biosamples are analyzed and the rest are banked for future use. Results Between November 2016 and October 2019, 2582 patients have enrolled in SPARC IBD (66% Crohn’s disease [CD], 33% ulcerative colitis (UC), and 1% IBD-U). Females comprise 52.5% of the cohort. Median age at enrollment is 37 years (range18-85). The majority of patients are white (80.5%) and non-Hispanic or Latino (84.5%). Median disease duration is 12 years (range 0–53). Distribution of disease location in the CD sub-group is 44% with small bowel and colonic disease, 28% with isolated small bowel disease, and 15% with isolated colonic disease. In the UC sub-group, 50.5% of patients have extensive colitis, followed by 23.5% with left-sided colitis, and 8.8% with proctitis. Among CD patients, 17.6% have stricturing disease, 12.5% have penetrating disease, 5.8% have both stricturing and penetrating disease and 16.2% have perianal involvement. At time of study enrollment 60% of CD patients, 41% of UC patients and 37% of IBD-U patients were on a biologic. The majority of patients with CD (88%) and UC (89%) were in remission. Conclusions SPARC IBD is a prospective cohort study of adult IBD patients which combines high-resolution phenotyping with biosample collection at multiple points in time. The unique aspects of this study including the multiple modalities for clinical data collection, the geographic diversity of patients enrolled, as well as the biosample procurement at multiple time points position SPARC IBD to aid in the discovery of novel biomarkers which can predict therapeutic responsiveness.

Hyaluronic acid (HA), a constituent of the extracellular matrix, promotes colorectal cancer growth. CD44 is a relevant HA receptor in this context. However, HA is also a ligand for TLR4, a receptor of significance in colorectal cancer. In this study, we examine the relative contribution of HA interactions with CD44 and TLR4 in colon tumorigenesis. Colorectal cancer models included ApcMin/+ mice, azoxymethane/dextran sodium sulfate (AOM-DSS), and CT26 tumor isografts. We used knockout mice and CT26 colorectal cancer cells with CRISPR knockdown of CD44 and TLR4. HA activity was modulated by PEP1 (a 12-mer peptide that blocks HA from binding its receptors), hyaluronidase (which promotes HA degradation), or 4-MU (HA synthesis inhibitor). Blockade of HA binding via PEP1 decreased growth in all colorectal cancer models and in cell culture. The effects were significant in WT and with CD44 deletion, but not with TLR4 deletion. In the AOM-DSS model, mice deficient in CD44 or TLR4 had fewer tumors. CD44- and TLR4-deficient CT26 isografts grew more slowly, exhibiting decreased tumor cell proliferation and increased apoptosis. In vitro, endogenous HA blocked LPS binding to TLR4 suggesting that HA is a relevant TLR4 ligand in colon cancer. Finally, PEP1 enhanced tumor radiation sensitivity in the isograft model. Together, these results indicate that HA binding to TLR4, as well as CD44, plays a key role in colon tumorigenesis. These findings also raise the possibility that an agent that blocks HA binding, such as PEP1, may be useful as an adjuvant therapy in colon cancer.

ObjectiveLactobacillus rhamnosusGG (LGG), a probiotic, given by gavage is radioprotective of the mouse intestine. LGG-induced radioprotection is toll-like receptor 2 (TLR2) and cyclooxygenase-2 (COX-2)-dependent and is associated with the migration of COX-2+mesenchymal stem cells (MSCs) from the lamina propria of the villus to the lamina propria near the crypt epithelial stem cells. Our goals were to define the mechanism of LGG radioprotection including identification of the TLR2 agonist, and the mechanism of the MSC migration and to determine the safety and efficacy of this approach in models relevant to clinical radiation therapy.DesignIntestinal radioprotection was modelled in vitro with cell lines and enteroids as well as in vivo by assaying clinical outcomes and crypt survival. Fractionated abdominal and single dose radiation were used along with syngeneic CT26 colon tumour grafts to assess tumour radioprotection.ResultsLGG with a mutation in the processing of lipoteichoic acid (LTA), a TLR2 agonist, was not radioprotective, while LTA agonist and native LGG were. An agonist of CXCR4 blocked LGG-induced MSC migration and LGG-induced radioprotection. LGG given by gavage induced expression of CXCL12, a CXCR4 agonist, in pericryptal macrophages and depletion of macrophages by clodronate liposomes blocked LGG-induced MSC migration and radioprotection. LTA effectively protected the normal intestinal crypt, but not tumours in fractionated radiation regimens.ConclusionsLGG acts as a ‘time-release capsule’ releasing radioprotective LTA. LTA then primes the epithelial stem cell niche to protect epithelial stem cells by triggering a multicellular, adaptive immune signalling cascade involving macrophages and PGE2 secreting MSCs.Trial registration numberNCT01790035; Pre-results.

Purpose of review Strong preclinical data support prophylactic probiotics as an effective preventive strategy for diarrhoea secondary to anticancer therapies. To determine the composite evidence that this approach translates to the clinic, we performed a meta-analysis of randomized controlled trials (RCTs) of prophylactic probiotics for the prevention of cancer therapy induced diarrhoea. Recent findings A three-step search strategy was used to identify relevant studies (1 June 2000–1 June 2017) investigating probiotic intervention for diarrhoea secondary to any cancer therapy (cytotoxic, targeted and immunotherapies). RCTs across PubMed, Embase, CINAHL and CENTRAL were assessed for eligibility and assessed using RevMan 5.3 (The Cochrane Collaboration). Seven trials with a total of 1091 patients were included in this meta-analysis. Compared with placebo, prophylactic probiotics did not prevent or reduce the overall incidence of diarrhoea or severe CTCAE Grade at least 3 diarrhoea [relative risk (RR) = 0.81, 95% confidence interval (95% CI) = 0.60–1.09, Z = 1.41, P = 0.16; RR = 0.54, 95% CI = 0.25–1.16, Z = 1.58, P = 0.11], nor did it influence the use of rescue medication (RR = 0.93, 95% CI = 0.53–1.65, Z = 0.24, P = 0.81). Summary Current evidence does not support widespread implementation of probiotics for diarrhoea secondary to cytotoxic therapy and the tyrosine kinase inhibitor, dacomitinib. Research efforts should be diverted to pair specific forms of gastrointestinal toxicity and their unique microbial phenotype to develop the ideal microbial protectant. Refereed/Peer-reviewed

Hyaluronic acid, a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously addressed the role of hyaluronic acid in small intestinal and colonic growth in mice. We addressed the role of exogenous hyaluronic acid by giving hyaluronic acid intraperitoneally and the role of endogenous hyaluronic acid by giving PEP-1, a peptide that blocks hyaluronic acid binding to its receptors. Exogenous hyaluronic acid increased epithelial proliferation but had no effect on intestinal length. PEP-1 resulted in a shortened small intestine and colon and diminished epithelial proliferation. In the current study, we sought to determine whether the effects of hyaluronic acid on growth were mediated by signaling through CD44 or TLR4 by giving exogenous hyaluronic acid or PEP-1 twice a week from 3–8 wk of age to wild-type, CD44−/−, and TLR4−/− mice. These studies demonstrated that signaling through both CD44 and TLR4 were important in mediating the effects of hyaluronic acid on growth in the small intestine and colon. Extending our studies to early postnatal life, we assessed the effects of exogenous hyaluronic acid and PEP-1 on Lgr5+ stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5+ reporter mice from postnatal day 7 to day 14 decreased Lgr5+ cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5+ stem cell proliferation, crypt fission, and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4.

There is currently an unmet need for agents that can prevent the gastrointestinal toxicity (mucositis and enteritis) associated with chemotherapy and radiation therapy of abdominal and pelvic cancers. Herein we provide an overview of how manipulation of the gut microbiota by probiotic administration affects these gastrointestinal symptoms. We focus this review on published human trials and also provide suggestions on how the field can move forward.Several clinical trials of varying design, patient populations and probiotic products have been reported. Lactobacillus probiotics of adequate dosage demonstrate a potential to reduce gastrointestinal toxicity when administered prophylactically. Common study limitations prevent the widespread adoption of this practice at this point but are informative for rational design of future trials.No single probiotic strain or product has emerged from human clinical trials for this indication. Further human studies are required to address limitations in the current literature. Preclinical model data should be used to inform the rational design of these new clinical trials to adequately address this important question.

Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries, where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, which may involve strain-specific virulence features as well as host factors, has not been elucidated. We demonstrate that, when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete the EtpA adhesin molecule. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and noncanonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A-specific lectin/hemagglutinin. Importantly, we have also shown that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and effective delivery of both the heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.

Background & Aims Crohn disease (CD) presents as chronic and often progressive intestinal inflammation, but the contributing pathogenic mechanisms are unclear. We aimed to identify alterations in intestinal cells that could contribute to the chronic and progressive course of CD. Methods We took an unbiased system-wide approach by performing sequence analysis of RNA extracted from formalin-fixed paraffin-embedded ileal tissue sections from patients with CD (n = 36) and without CD (controls; n = 32). We selected relatively uninflamed samples, based on histology, before gene expression profiling; validation studies were performed using adjacent serial tissue sections. A separate set of samples (3 control and 4 CD samples) was analyzed by transmission electron microscopy. We developed methods to visualize an overlapping modular network of genes dysregulated in the CD samples. We validated our findings using biopsy samples (110 CD samples for gene expression analysis and 54 for histologic analysis) from the UNITI-2 phase 3 trial of ustekinumab for patients with CD and healthy individuals (26 samples used in gene expression analysis). Results We identified gene clusters that were altered in nearly all CD samples. One cluster encoded genes associated with the enterocyte brush border, leading us to investigate microvilli. In ileal tissues from patients with CD, the microvilli were of decreased length and had ultrastructural defects compared with tissues from controls. Microvilli length correlated with expression of genes that regulate microvilli structure and function. Network analysis linked the microvilli cluster to several other down-regulated clusters associated with altered intracellular trafficking and cellular metabolism. Enrichment of a core microvilli gene set also was lower in the UNITI-2 trial CD samples compared with controls; expression of microvilli genes was correlated with microvilli length and endoscopy score and was associated with response to treatment. Conclusions In a transcriptome analysis of formalin-fixed and paraffin-embedded ileal tissues from patients with CD and controls, we associated transcriptional alterations with histologic alterations, such as differences in microvilli length. Decreased microvilli length and decreased expression of the microvilli gene set might contribute to epithelial malfunction and the chronic and progressive disease course in patients with CD.

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens., Author summary Enterotoxigenic Escherichia coli (ETEC) infections contribute substantially to death and morbidity due to diarrheal illness and are associated with serious sequelae including malnutrition, stunted growth, and intellectual impairment among young children in developing countries. Effective engagement of intestinal epithelial cells is essential for ETEC pathogenicity. Consequently, pathovar specific plasmid-encoded adhesin structures known as colonization factors (CFs) have been a principal target for vaccines. However, tremendous inter-strain variation in the carriage of gene clusters encoding different CFs and significant antigenic diversity of the CF adhesins has posed a challenge to vaccine development. In contrast, type 1 pili are encoded by the fim operon located in the chromosome of most ETEC strains and are highly conserved. While type 1 pili are known to play a critical role in virulence of extraintestinal pathogenic E. coli, the present studies represent the first detailed examination of the contribution of these pili in ETEC pathogenesis. Here we demonstrate that ETEC type 1 pili are essential for optimal interactions with intestinal epithelia and that they play a critical role in virulence. These data may inform additional approaches toward development of broadly protective vaccines for these pathogens of global importance.

Gut microbiota and cancer therapy share an intimate and dynamically connected relationship. The mucosa lining the gastrointestinal (GI) tract forms a selective barrier between ourselves and the external environment. From mouth to rectum, cytotoxic cancer therapy (radiation and chemotherapy) can lead to dysfunction and disruption of this mucosal lining with a pathophysiology involving epithelial and endothelial cell death as well as activation of the mucosal immune system. The symptoms of mucositis, the clinical term for this condition include abdominal pain, nausea, vomiting, and diarrhea. Radiation and chemotherapy-induced mucositis is a frequent and major dose limiting side effect of cancer therapy. In practice, there is currently no therapy that successfully prevents or treats mucositis. However, therapeutic manipulation of gut microbiota or harnessing the biologic effects of microbial products may hold the key to mucositis prevention and treatment. Recent advances also indicate that beneficial commensals and probiotic bacteria have the potential to dramatically enhance the antitumor effects of cytotoxic therapies as well as immune checkpoint inhibitors.