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Crohn's disease (CD) is a chronic inflammatory disease with increasing incidence in children. Current medications have potentially serious side effects, hence increasing interest in alternative therapies. We previously developed an herbal formula, FAHF-2, based on a classical traditional Chinese herbal formula Wu Mei Wan that has long been used in China to treat colitis. We investigated FAHF-2's potential anti-inflammatory effects.FAHF-2 efficacy was tested in vivo in the CD45RbRAG1 transfer colitis model. Weight loss, colonic histology, and cytokine production from mesenteric lymph nodes were assessed. Human peripheral blood mononuclear cells (PBMCs) and colonic biopsies were obtained from children newly diagnosed with CD and controls and cultured with or without FAHF-2. Cytokine levels were measured by multiplex immunoassay. The effect of FAHF-2 on TNF-α-producing cells was determined by flow cytometry. NF-κB signaling was investigated in human lamina propria mononuclear cells upon FAHF-2 treatment by In-Cell Western.FAHF-2-treated mice had decreased weight loss, improved histology, and reduced TNF-α, IL-17, IL-6, and IFN-γ production. In vitro treated PBMCs produced less TNF-α, IFN-γ, and IL-12. FAHF-2 reduced the TNF-α-producing monocytes and T cells. Inflamed CD biopsies produced less TNF-α, IL-17, IL-6, and IL-1β. These effects are because of decreased NF-κB activation.FAHF-2 inhibited both adaptive and innate immune proinflammatory cytokine responses in PBMCs and inflamed CD mucosa due in part to blockage of NF-κB activation. FAHF-2 was effective in halting progression of colitis in a murine model. This study shows that FAHF-2 has potential as a novel treatment of CD.

Background & Aims Epithelial cancers can be initiated by activating mutations in components of the mitogen-activated protein kinase signaling pathway such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR). Human intestinal serrated polyps are a heterogeneous group of benign lesions, but some progress to colorectal cancer. Tumors that arise from these polyps frequently contain activating mutations in BRAF or KRAS, but little is known about the role of EGFR activation in their development. Methods Polyp samples were obtained from adults during screening colonoscopies at Mount Sinai Hospital in New York. We measured levels of EGFR protein and phosphorylation in human serrated polyps by immunohistochemical and immunoblot analyses. We generated transgenic mice that express the ligand for EGFR, Heparin-binding EGF-like growth factor (HB-EGF), in the intestine. Results EGFR and the extracellular-regulated kinases (ERK)1/2 were phosphorylated in serrated areas of human hyperplastic polyps (HPPs), sessile serrated adenomas, and traditional serrated adenomas. EGFR and ERK1/2 were phosphorylated in the absence of KRAS or BRAF activating mutations in a subset of HPP. Transgenic expression of the EGFR ligand HB-EGF in the intestines of mice promoted development of small cecal serrated polyps. Mice that expressed a combination of HB-EGF and US28 (a constitutively active, G-protein–coupled receptor that increases processing of HB-EGF from the membrane) rapidly developed large cecal serrated polyps. These polyps were similar to HPPs and had increased phosphorylation of EGFR and ERK1/2 within the serrated epithelium. Administration of pharmacologic inhibitors of EGFR or MAPK to these transgenic mice significantly reduced polyp development. Conclusions Activation of EGFR signaling in the intestine of mice promotes development of serrated polyps. EGFR signaling also is activated in human HPPs, sessile serrated adenomas, and traditional serrated adenomas.

Background & Aims Several studies have shown that signaling via the interleukin 23 (IL23) receptor is required for development of colitis. We studied the roles of IL23, dietary factors, alterations to the microbiota, and T cells in the development and progression of colitis in mice. Methods All mice were maintained on laboratory diet 5053, unless otherwise noted. We generated mice that express IL23 in CX3CR1-positive myeloid cells (R23FR mice) upon cyclic administration of tamoxifen dissolved in diet 2019. Diets 2019 and 5053 have minor differences in the overall composition of protein, fat, fiber, minerals, and vitamins. CX3CR1CreER mice (FR mice) were used as controls. Some mice were given antibiotics, and others were raised in a germ-free environment. Intestinal tissues were collected and analyzed by histology and flow cytometry. Feces were collected and analyzed by 16S rDNA sequencing. Feces from C57/Bl6, R23FR, or FR mice were fed to FR and R23FR germ-free mice in microbiota transplant experiments. We also performed studies with R23FR/Rag–/–, R23FR/Mu–/–, and R23FR/Tcrd–/– mice. R23FR mice were given injections of antibodies against CD4 or CD8 to deplete T cells. Mesenteric lymph nodes and large intestine CD4+ cells from R23FR or FR mice in remission from colitis were transferred into Rag–/– mice. CD4+ cells were isolated from donor R23FR mice and recipient Rag–/– mice, and T-cell receptor sequences were determined. Results Expression of IL23 led to development of a relapsing–remitting colitis that was dependent on the microbiota and CD4+ T cells. The relapses were caused by switching from the conventional diet used in our facility (diet 5053) to the diet 2019 and were not dependent on tamoxifen after the first cycle. The switch in the diet modified the microbiota but did not alter levels of IL23 in intestinal tissues compared with mice that remained on the conventional diet. Mesenteric lymph nodes and large intestine CD4+ cells from R23FR mice in remission, but not from FR mice, induced colitis after transfer into Rag–/– mice, but only when these mice were placed on the diet 2019. The CD4+ T-cell receptor repertoire of Rag–/– mice with colitis (fed the 2019 diet) was less diverse than that from donor mice and Rag–/– mice without colitis (fed the 5053 diet) because of expansion of dominant T-cell clones. Conclusions We developed mice that express IL23 in CX3CR1-positive myeloid cells (R23FR mice) and found that they are more susceptible to diet-induced colitis than mice that do not express IL23. The R23FR mice have a population of CD4+ T cells that becomes activated in response to dietary changes and alterations to the intestinal microbiota. The results indicate that alterations in the diet, intestinal microbiota, and IL23 signaling can contribute to pathogenesis of inflammatory bowel disease.

Catecholamines stimulate epithelial proliferation, but the role of sympathetic nerve signaling in pancreatic ductal adenocarcinoma (PDAC) is poorly understood. Catecholamines promoted ADRB2-dependent PDAC development, nerve growth factor (NGF) secretion, and pancreatic nerve density. Pancreatic Ngf overexpression accelerated tumor development in LSL-Kras+/G12D;Pdx1-Cre (KC) mice. ADRB2 blockade together with gemcitabine reduced NGF expression and nerve density, and increased survival of LSL-Kras+/G12D;LSL-Trp53+/R172H;Pdx1-Cre (KPC) mice. Therapy with a Trk inhibitor together with gemcitabine also increased survival of KPC mice. Analysis of PDAC patient cohorts revealed a correlation between brain-derived neurotrophic factor (BDNF) expression, nerve density, and increased survival of patients on nonselective β-blockers. These findings suggest that catecholamines drive a feedforward loop, whereby upregulation of neurotrophins increases sympathetic innervation and local norepinephrine accumulation. Renz et al. show that catecholamines promote ADRB2-dependent pancreatic ductal adenocarcinoma development and secretion of neurotrophins (NT), which in turn promote tumor innervation leading to increased NE and tumor growth. Blockade of ADRB2 or NT receptors improves gemcitabine's therapeutic effect.

Cases of small pericardial effusion have been reported in association with dengue fever (DF), largely with dengue hemorrhagic fever during epidemic outbreaks. However, cardiac tamponade developed by a patient with DF has not yet been reported in the English literature. We report a case of cardiac tamponade in a patient with DF and lupus nephritis. We describe the characteristic features to differentiate pericardial effusion of lupus origin from that of viral etiology. A 59-year-old Hispanic woman presented to the emergency department with complaints of 5 to 6 days of fever, myalgia, headache, and retro-orbital pain. Her symptoms started 3 days after returning from the Dominican Republic, where a dengue outbreak was reported. Her past medical history was significant for hypertension and lupus nephritis diagnosed 3 months earlier. On day 2, patient developed a large pericardial effusion that progressed to tamponade over the next 2 days, requiring surgical drainage. Subsequently, the patient improved; however, serological analysis did not suggest any lupus flare-up. Pericardial fluid analysis showed hypocellularity without lupus erythematosus cell and biopsy revealed only reactive mesothelial cells suggestive of viral etiology. Dengue serology was reported as markedly elevated, supporting a diagnosis of classic DF (both immunoglobulin M [IgM] titer 2.93 and IgG titer 12.13 by enzyme-linked immunosorbent assay [ELISA]; reference range

Down's syndrome is caused by an extra chromosome; somehow a 1.5-fold increase in the dosage of a gene or genes on chromosome 21 causes the wide-reaching effects associated with the condition. A study using ‘knockout’ mice now identifies two genes as candidates for involvement. A 1.5-fold increase in dosage of DSCR1 and DYRK1a destabilizes the regulation of signalling pathways involving the NFAT transcription factor. The discovery follows the surprise finding that NFATc1-4 and calcineurin mutant mice demonstrate nearly all the characteristics of Down's syndrome. In an unrelated paper, a genome-wide RNAi screen reveals conserved regulators of NFAT in Drosophila. NFAT is a purely vertebrate transcription factor, but this work breaks new ground by using Drosophila cells to study the function of a protein artificially introduced from a mammalian species. Pathways regulating the subcellular localization of NFAT proteins are strongly conserved across species and this new approach can identify new regulators of a transcription factor normally expressed in vertebrates. Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1–4) is essential for vertebrate development and function1. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus1. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)1,2,3,4,5. Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila6,7 to identify additional regulators of the signalling pathway leading from Ca2+–calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+–calmodulin–calcineurin signalling and NFAT kinases) are conserved across species8,9, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.

While controversy over the existence of adult pancreatic stem cells persists, it is now appreciated that the acinar compartment of the pancreas harbors heterogeneous progenitors. Recent single-cell analysis also demonstrated the presence of molecularly distinct, albeit morphologically identical, acinar cell sub-lineages. Previously, using lineage-tracing approach, we reported the Dclk1+ facultative progenitors that are critical for pancreatic regeneration. Here, we identified a different pancreatic progenitor-like subpopulation which is labelled by trefoil factor 2 (Tff2), a known progenitor marker and capable of tracing multiple cell lineages in the stomach. In addition, Tff2 molecules have been shown to play a suppressive role in PDAC progression. We utilized constitutive Tff2Cre and inducible Tff2CreERT2-DTR mice which were generated through modification of a BAC allele. We crossed Tff2CreERT2-DTR with reporter mice (R26R-mTmG, -tdTomato) to trace Tff2 labeled cells, and found that Tff2 labels ~2 % of the overall population in the adult acinar compartment, which showed slow proliferation (1 year, descendants Citation Format: Zhengyu Jiang, Bernhard W. Renz, Marina Macchini, Tanaka Takayuki, Ryota Takahashi, Giovanni Valenti, Woosook Kim, Wenju Chang, Yoku Hayakawa, Kosuke Sakitani, Moritz Middelhoff, Zinaida Dubeykovskaya, Timothy Chu, Karan Nagar, Yagnesh Tailor, Chythra R. Chandregowda, Akanksha Anand, Samuel Asfaha, Alina C. Iuga, Timothy C. Wang. Tff2 labels pancreatic progenitors that lack proliferative potential during tissue regeneration but can serve as the origin of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-144. doi:10.1158/1538-7445.AM2017-LB-144

The Notch-1 signaling pathway is responsible for homeostatic tight junction expression in vitro, and promotes barrier function in vivo in the RAG1-adoptive transfer model of colitis. In this study, we sought to determine the role of colonic Notch-1 in the lymphoepithelial crosstalk in health and disease. We utilized in vivo and in vitro knockdown to target the expression of Notch-1. We identified that epithelial Notch-1 is required for appropriate activation of intestinal epithelial cells at steady state and upon inflammatory stimulus. Notch-1 expression modulates mucosal chemokine and cytokine secretion, and FoxP3 and effector T-cell responses. We showed that epithelial Notch-1 controls the immune function of the epithelium through crosstalk with the nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathways that, in turn, elicits T-cell responses. Overall, epithelial Notch-1 bridges innate and adaptive immunity in the gut. Our findings highlight an indispensable role for Notch-1-mediated signaling in the intricate epithelial-immune crosstalk, and validate that epithelial Notch-1 is necessary and sufficient to support protective epithelial proinflammatory responses.

Introduction and objectives: There is increasing evidence that chronic activation of sympathetic neurons can lead to increased noradrenaline levels in the tumor microenvironment (TME) in PDAC. However, the mechanisms by which adrenergic neurotransmitters are delivered to the TME are not well understood. In this study we aimed to investigate the effect of locally and systemically delivered catecholamines into the TME in two well established genetically engineered mouse models of PDAC (Pdx1-Cre/KRasG12D (KC) and Pdx1-Cre/KRasG12D/Trp53R172H (KPC)). Methods: Adrenergic signaling was induced or inhibited in KC or KPC mice and in pancreatic organoids using various pharmacological compounds. Adrenergic receptor expression was assessed by RT-PCR, WB and IHC. Downstream pathways were identified by phosphorylation assays and WB. Adrenergic signaling was modeled in vivo applying restraint stress. To elucidate the crosstalk between nerves and cancer cells, pancreatic spheres and PDAC cells were co-cultured with DRGs and neuronal plasticity was quantified. NGF secretion was measured by RT-PCR and ELISA. Sympathetic denervation of the pancreas and blockage of the 2-receptor was employed as a treatment strategy in KPC mice. Overexpression of NGF was targeted to the mouse pancreas using a novel NGF knockin mouse, which was crossed to KC and KPC mice. Results: ADRB2 was upregulated during pancreatic cancer development. Furthermore, in vitro stimulation of cells harboring an oncogenic KRas mutation with catecholamines resulted in significantly increased sphere forming efficiency. The non-specific -blocker propranolol and the selective 2-blocker ICI 118,551 inhibited this effect. Selective blockade of 2-adrenergic signaling suppressed pancreatic sphere formation caused by co-cultures with DRGs. NGF secretion was stimulated through beta2-adrenergic signaling. Furthermore, restraint stress accelerated PDAC development in KC mice. The effects of stress were prevented by treatment with the selective ADRB2 antagonist ICI 118,551. Specific β2-blocker treatment as well as sympathetic denervation in addition to GEM significantly increased the survival of mice with 3-5 mm tumors in comparison to controls treated by GEM alone. Targeted overexpression of NGF in the mouse pancreas using a novel NGF knockin mouse resulted in marked acceleration of PanIN lesions in the KC mouse and shortened overall survival in KPC mice significantly. Finally, retrospective analysis of a PDAC patient cohort revealed a extension of overall survival in PDAC patients on non-selective -blockers. Conclusions: Taken together, these studies suggest that increased catecholamines can engender a feed forward loop, whereby upregulation of NGF can lead to increased innervation and local NE accumulation, thereby enhancing tumor growth. Therapies targeting these adrenergic and NGF pathways may prove useful in the treatment and prevention of pancreatic cancer. Citation Format: Bernhard W. Renz, Marina Macchini, Yoku Hayakawa, Xiaowei Chen, Zhengyu Jiang, Takayuki Tanaka, C. Benedikt Westphalen, Yagnesh Tailor, Jens Werner, Axel Kleespies, Alina C. Iuga, Daniel L. Worthley, Kenneth P. Olive, Timothy C. Wang. A novel β 2 adrenergic-nerve growth factor feed forward loop promotes pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5111.

Background/Aims: The parasympathetic nervous system plays an important role in the regulation of epithelial homeostasis and has also been postulated to play a role in tumorigenesis. However, the exact role of the vagus nerve in pancreatic carcinogenesis is not well understood. Here we study the effects of muscarinic signaling on pancreatic tumorigenesis in a genetically engineered mouse model of pancreatic cancer (PDx1-Cre/KRasG12D (KC). Methods: Mice were either vagotomized at 8 weeks or treated with a cholinergic agonist. Pancreatic tissue was collected and analyzed by immunohistochemistry and RT-PCR at 20 weeks of age; cells were isolated and assayed for colony and sphere forming assays. Different human (AsPC-1, BxPC-3, Mia PaCa-2, Panc-1) and murine (K-2548 and K-8282) pancreatic cancer cell lines were subjected to cholinergic and anti-cholinergic drugs and assayed by RT-PCR, Western blot and flow cytometry. Results: In pancreatic organoid cultures derived from pancreata harboring an oncogenic KRas mutation, cholinergic agonists suppressed sphere formation significantly. Furthermore, pharmacological inhibition or genetic knockout of the muscarinic M3 receptor abolished this effect in vitro. In human and murine pancreatic cancer cells, anchorage independent growth and tumor sphere forming capacity were reduced by pretreatment with cholinergic agonists. Further evaluation revealed that parasympathetic agonists decrease the CD44+CD24+EpCAM+ proved CSC's population. Vagotomy, when performed in KC mice at 8 weeks resulted in pancreatic cancer development in 20% of the animals at 20 week of age. Treatment with the direct muscarinic agonist bethanechol caused a significant delay of PanIN progression in KC mice. Conclusions: Taken together, our findings suggest that vagal innervation has a regulatory role in pancreatic tumorigenesis via M3 receptor-mediated suppression of CSCs. Since current surgical approaches to resection of PDAC from the head of the pancreas are necessarily associated with a parasympathetic denervation of the pancreas, and thereby a loss of its suppressive effect, this fact may might partly explain the high local recurrence rate of this dismal disease. Additional treatment with cholinergic agonists should be considered in future regimen. Citation Format: Bernhard W. Renz, Marina Macchini, Yoku Hayakawa, C. Benedikt Westphalen, Michael Churchill, Suchetak Kar, Xiaowei Chen, Karan Nagar, Yagnesh Tailor, Daniel L. Worthley, Alina C. Iuga, Ken P. Olive, Timothy C. Wang. Parasympathetic signaling suppresses pancreatic cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5079. doi:10.1158/1538-7445.AM2015-5079

Introduction Anti-tumor necrosis factor (TNF) therapies are currently being used to treat fistulising perianal Crohn's disease (CD). We evaluated the clinical and radiological outcomes of patients with perianal Crohn's fistulas who have been treated with immunomodulators alone as compared with those treated with anti-TNF therapies. Methods A local database of 270 consecutive patients with CD anal fistulas treated at our institution between 2000 and 2014 was established. Demographic and clinical data were recorded in addtion to radiological reports. Results Ninety patients were in the non-anti-TNF group and 180 were treated with anti-TNF therapy (Infliximab or Adalimumab). Clinical response rates were significantly higher in the anti-TNF group (74% vs 62%, p=0.04). Similarly, radiological response rates were higher in the anti-TNF group (56% vs 28%, p

Objectives: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. CA19-9 is a glycoprotein that predicts poor prognosis in pancreatic and biliary malignancies. We evaluated it as a prognostic biomarker for patients with HCC. Methods: We prospectively enrolled 145 patients with HCC, diagnosed using American Association for Study of Liver Diseases criteria, between October 2008 and November 2012. We examined whether baseline serum CA19-9 levels predicted overall survival. We also examined immunostains of hepatic resections and explants of patients with elevated and normal serum CA19-9. Results: In a cohort of predominantly hepatitis C and B patients, CA19-9 ≥100 U/ml was associated with a 2.7-fold increased mortality (hazard ratio (HR): 2.72; 95% confidence interval (CI): 1.52–4.88, P

BACKGROUND/AIMS: We previously reported that inhibition of Bone Morphogenetic Protein (BMP) signaling in vivo, leads to enhancement of Helicobacter-induced gastric inflammation suggesting that the BMPs exert anti-inflammatory actions in the gut. The aim of this study was to examine the expression of BMP-4 and the localization of BMP-generated signals during gastrointestinal (GI) inflammation. METHODS: 3 month-old transgenic (TG) mice expressing the β-galactosidase (gal) gene under the control of a BMP responsive element (BRE) and C57BL/6-BMP-4β-gal/+ mice were either infected with H. felis (HF) or given 3% DSS-containing water for 4 days, to induce gastric and colonic inflammation, respectively. Animals were analyzed 2 months after HF infection and 3 days after DSS administration. Expression of BMP-4, BRE-β-gal and BMP Receptor 1A (BMPR1A) were assessed by X-Gal staining, IHC with anti-β-gal antibodies (abs) and QRT-PCR. Morphology of the GI mucosa was examined in sections stained with H&E. Phosphorylation of the BMP-signal transducers Smad1-5-8 was determined by IHC with anti-p-Smad1-5-8 abs. Identification of myofibroblasts, B and T lymphocytes, neutrophils and macrophages was achieved by IHC with antiα smooth muscle actin (SMA), -CD19, -CD3, -MPO, and -F4/80 abs, respectively. Biopsies were obtained from the colonic mucosa of normal (16 samples from 4 patients) and Inflammatory Bowel Disease (IBD) individuals (12 samples from 4 patients) with severe inflammation. RESULTS: BMP-4 is expressed in the mesenchymal layers of the GI mucosa while BMP signaling targets cells of the GI epithelium. BMP-4 staining was seen in α-SMA-positive cells of both the gastric and the colonic mucosa. HF-induced inflammation led to decreased BMP4 expression and signaling. No BMP-4 staining was seen in CD19-, CD3-, F4/80and MPOpositive cells of the gastric mucosa. Similarly, no p-Smad1-5-8 signals were seen in cells of the inflammatory infiltrates. 3% DSS caused inflammatory infiltrates, stromal expansion and loss of glands. The inflamed areas exhibited decreased BMP-4 expression and signaling and reduced BMPR1A mRNA expression. BMP-4 and BMPR1A mRNAs were also decreased in biopsies from patients with severe colonic IBD. CONCLUSIONS: BMP-4 is expressed in αSMA-positive cells of the GI mucosa but not in immune cells. BMP signals target epithelial cells but not cells of the inflammatory infiltrates. Inflammation reduces both the expression of BMP-4 and the intensity of BMP signaling in mouse models of gastric and colonic inflammation and in biopsies from IBD patients. Thus, BMP signaling, a pathway that exerts anti-inflammatory effects, is reduced during severe GI inflammation. Induction and reactivation of this mechanism might therefore represent a possible, novel modality for the treatment of inflammatory diseases of the gut.