Comparison of endoscopic ultrasound tissue acquisition methods for genomic analysis of pancreatic cancer.

BACKGROUND AND AIM: Tumor genotyping may allow for improved prognostication and targeted therapy for pancreatic ductal adenocarcinoma (PDAC). We aimed to compare endoscopic ultrasonography (EUS) with fine needle aspiration (FNA) to fine needle biopsy (FNB) for obtaining sufficient tissue for genomic analysis and theranostic potential. METHODS: A retrospective cohort study of patients that underwent EUS-FNA or EUS-FNB with either positive or suspicious cytology for PDAC between March 2016 and December 2017. Demographic, procedural, and cytology data were recorded. Genetic alterations were recorded, and Kaplan-Meier survival curves were calculated. RESULTS: The study included 167 patients: 145 patients had FNA and 22 patients underwent FNB. Overall, 117 samples (70.1%) were sufficient for targeted next-generation sequencing. FNB resulted in a higher proportion of patients with sufficient samples compared with FNA (90.9% vs 66.9%; P = 0.02). In multivariable modeling, only FNB (odds ratio 4.95, 95% confidence interval 1.11-22.05, P = 0.04) was associated with sufficient sampling for genomic testing. FNB was more likely to obtain sufficient tissue from tumors ≤ 3 cm (100% vs 68.4%, P = 0.017) and tumors located in the head/neck of the pancreas (100% vs 63.1%, P = 0.03) compared with FNA. The most commonly identified alterations were in KRAS (88%), TP53 (68%), and SMAD4 (16%). CONCLUSIONS: Endoscopic ultrasonography can reliably obtain sufficient tissue from PDAC for targeted genomic sequencing for prognostication and theranostics. FNB should be considered when tumor genotyping is requested, especially for tumors ≤ 3 cm or tumors located in the head/neck of the pancreas.

Family history in colonoscopy patients: feasibility and performance of electronic and paper-based surveys for colorectal cancer risk assessment in the outpatient setting.

BACKGROUND AND AIMS: Family history is crucial in stratifying patients' risk for colorectal cancer (CRC). Previous risk assessment tools developed for use in clinic or endoscopy settings have demonstrated suboptimal specificity for identifying patients with hereditary cancer syndromes. Our aim was to test the feasibility and performance of 2 family history surveys (paper and electronic) in individuals presenting for outpatient colonoscopy. METHODS: Patients presenting for outpatient colonoscopy at a tertiary care center were asked to complete a 5-question paper risk assessment survey (short paper survey) either alone or in conjunction with a second, comprehensive electronic family risk assessment survey (comprehensive tablet survey). Each subject's survey results, along with the electronic medical record, were reviewed, and 10 high-risk criteria and PREMM1,2,6 model scores (a predictive model for carrying a Lynch syndrome-associated gene mutation) were used to identify patients warranting genetic evaluation for suspected hereditary cancer syndromes. RESULTS: Six hundred patients completed the short paper survey (cohort 1), with an additional 100 patients completing both the short paper and comprehensive tablet survey (cohort 2). Using 10 high-risk criteria and/or a PREMM1,2,6 score ≥5%, we identified 10% and 9% of patients as high risk for CRC in cohorts 1 and 2, respectively. Of the 69 high-risk subjects, 23 (33%) underwent genetic evaluations and 7 (10%) carried germline mutations associated with cancer predisposition. Both patients and endoscopists reported the tools were user-friendly and helpful for CRC risk stratification. CONCLUSIONS: Systematic assessment of family history in colonoscopy patients is feasible and can help endoscopists identify high-risk patients who would benefit from genetic evaluation.

Therapeutic Endoscopy for the Control of Nonvariceal Upper Gastrointestinal Bleeding in Children: A Case Series.

OBJECTIVES: Gastrointestinal bleeding is one of the most common indications for urgent endoscopy in the pediatric setting. The majority of these procedures are performed for control of variceal bleeding, with few performed for nonvariceal upper gastrointestinal (NVUGI) bleeding. The data on therapeutic endoscopy for NVUGI are sparse. The aims of our study were to review our experience with NVUGI bleeding, describe technical aspects and outcomes of therapeutic endoscopy, and determine gastroenterology fellows' training opportunities according to the national training guidelines. METHODS: We performed a retrospective review of endoscopy database (Endoworks, Olympus Inc, Center Valley, PA) from January 2009 to December 2014. The search used the following keywords: bleeding, hematemesis, melena, injection, epinephrine, cautery, clip, and argon plasma coagulation. The collected data included demographics, description of bleeding lesion and medical/endoscopic therapy, rate of rebleeding, relevant laboratories, physical examination, and need for transfusion and surgery. The study was approved by the institutional review board. RESULTS: During the study period 12,737 upper endoscopies (esophagogastroduodenoscopies) were performed. A total of 15 patients underwent 17 esophagogastroduodenoscopies that required therapeutic intervention to control bleeding (1:750 procedures). The mean ±â€Šstandard deviation (median) age of patients who required endoscopic intervention was 11.6 ±â€Š6.0 years (14.0 years). Seven out of 17 patients received dual therapy to control the bleeding lesions. All but 3 patients received medical therapy with intravenous proton pump inhibitor, and 3 received octreotide infusions. Six of the patients experienced rebleeding (40%), with 4 out of 6 initially only receiving single modality therapy. Two of these patients eventually required surgical intervention to control bleeding and both patients presented with bleeding duodenal ulcers. There were no cases of aspiration, perforation, or deaths. There were a total of 24 fellows trained in our program during the study period. Less than 1 therapeutic endoscopy per fellow for NVUGI bleeding was performed. CONCLUSIONS: NVUGI bleeding requiring therapeutic endoscopic intervention is rare in pediatrics. A high rate (40%) of rebleeding was noted with a large proportion (66%) of patients receiving single modality therapy. Two patients required surgical intervention to control bleeding and both presented with bleeding duodenal ulcers. An insufficient number of therapeutic procedures is available for adequate fellow training requiring supplemental simulator and hands-on animal model, or adult endoscopy unit training.

Secretion of the adipocyte-specific secretory protein adiponectin critically depends on thiol-mediated protein retention.

Adiponectin is a secretory protein abundantly secreted from adipocytes. It assembles into a number of different higher-order complexes. Adipocytes maintain tight control over circulating plasma levels, suggesting the existence of a complex, highly regulated biosynthetic pathway. However, the critical mediators of adiponectin maturation within the secretory pathway have not been elucidated. Previously, we found that a significant portion of de novo-synthesized adiponectin is not secreted and retained in adipocytes. Here, we show that there is an abundant pool of properly folded adiponectin in the secretory pathway that is retained through thiol-mediated retention, as judged by the release of adiponectin in response to treatment of adipocytes with reducing agents. Adiponectin is covalently bound to the ER chaperone ERp44. An adiponectin mutant lacking cysteine 39 fails to stably interact with ERp44, demonstrating that this residue is the primary site mediating the covalent interaction. Another ER chaperone, Ero1-Lalpha, plays a critical role in the release of adiponectin from ERp44. Levels of both of these proteins are highly regulated in adipocytes and are influenced by the metabolic state of the cell. While less critical for the secretion of trimers, these chaperones play a major role in the assembly of higher-order adiponectin complexes. Our data highlight the importance of posttranslational events controlling adiponectin levels and the release of adiponectin from adipocytes. One mechanism for increasing circulating levels of specific adiponectin complexes by peroxisome proliferator-activated receptor gamma agonists may be selective upregulation of rate-limiting chaperones.

Atraumatic Splenic Hemorrhage as a Rare Complication of Pancreatitis: Case Report and Literature Review.

Splenic hemorrhage (hematoma and rupture) is a rare complication of pancreatitis. In this article, we present a rare case of spontaneous splenic rupture as a complication of acute pancreatitis. A literature review was also completed to describe the patient characteristics, associated pancreatitis etiology, clinical presentations, risk factors, diagnostic and treatment modalities, and outcomes.

Paraventricular, subparaventricular and periventricular hypothalamic IRS4-expressing neurons are required for normal energy balance.

Understanding the neural components modulating feeding-related behavior and energy expenditure is crucial to combating obesity and its comorbidities. Neurons within the paraventricular nucleus of the hypothalamus (PVH) are a key component of the satiety response; activation of the PVH decreases feeding and increases energy expenditure, thereby promoting negative energy balance. In contrast, PVH ablation or silencing in both rodents and humans leads to substantial obesity. Recent studies have identified genetically-defined PVH subpopulations that control discrete aspects of energy balance (e.g. oxytocin (OXT), neuronal nitric oxide synthase 1 (NOS1), melanocortin 4-receptor (MC4R), prodynorphin (PDYN)). We previously demonstrated that non-OXT NOS1PVH neurons contribute to PVH-mediated feeding suppression. Here, we identify and characterize a non-OXT, non-NOS1 subpopulation of PVH and peri-PVH neurons expressing insulin-receptor substrate 4 (IRS4PVH) involved in energy balance control. Using Cre-dependent viral tools to activate, trace and silence these neurons, we highlight the sufficiency and necessity of IRS4PVH neurons in normal feeding and energy expenditure regulation. Furthermore, we demonstrate that IRS4PVH neurons lie within a complex hypothalamic circuitry that engages distinct hindbrain regions and is innervated by discrete upstream hypothalamic sites. Overall, we reveal a requisite role for IRS4PVH neurons in PVH-mediated energy balance which raises the possibility of developing novel approaches targeting IRS4PVH neurons for anti-obesity therapies.

Rapid Crypt Cell Remodeling Regenerates the Intestinal Stem Cell Niche after Notch Inhibition.

Intestinal crypts have great capacity for repair and regeneration after intestinal stem cell (ISC) injury. Here, we define the cellular remodeling process resulting from ISC niche interruption by transient Notch pathway inhibition in adult mice. Although ISCs were retained, lineage tracing demonstrated a marked reduction in ISC function after Notch disruption. Surprisingly, Notch ligand-expressing Paneth cells were rapidly lost by apoptotic cell death. The ISC-Paneth cell changes were followed by a regenerative response, characterized by expansion of cells expressing Notch ligands Dll1 and Dll4, enhanced Notch signaling, and a proliferative surge. Lineage tracing and organoid studies showed that Dll1-expressing cells were activated to function as multipotential progenitors, generating both absorptive and secretory cells and replenishing the vacant Paneth cell pool. Our analysis uncovered a dynamic, multicellular remodeling response to acute Notch inhibition to repair the niche and restore homeostasis. Notably, this crypt regenerative response did not require ISC loss.

Cellular Plasticity of Defa4Cre-Expressing Paneth Cells in Response to Notch Activation and Intestinal Injury.

BACKGROUND & AIMS: Loss of leucine-rich repeat-containing G-protein-coupled receptor 5-positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury. METHODS: Small intestine and enteroids from Defa4Cre;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4Cre;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4Cre;Rosa26reverse tetracycline transactivator-ires Enhanced Green Fluorescent Protein (EGFP);TetONICD), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4Cre;ADAM10flox/flox), and Adenomatous polyposis coli (APC) inactivation (Defa4Cre;APCflox/flox) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo. RESULTS: Defa4Cre-expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter-sorted Defa4Cre-marked cells showed clonogenic enteroid growth. Notch activation in Defa4Cre-expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4Cre-expressing cells contributed to regeneration in an ADAM10-Notch-dependent manner. CONCLUSIONS: Our studies have shown that Defa4Cre-expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model.

Loss of Paneth Cell Autophagy Causes Acute Susceptibility to Toxoplasma gondii-Mediated Inflammation.

The protozoan parasite Toxoplasma gondii triggers severe small intestinal immunopathology characterized by IFN-γ- and intestinal microbiota-mediated inflammation, Paneth cell loss, and bacterial dysbiosis. Paneth cells are a prominent secretory epithelial cell type that resides at the base of intestinal crypts and releases antimicrobial peptides. We demonstrate that the microbiota triggers basal Paneth cell-specific autophagy via induction of IFN-γ, a known trigger of autophagy, to maintain intestinal homeostasis. Deletion of the autophagy protein Atg5 specifically in Paneth cells results in exaggerated intestinal inflammation characterized by complete destruction of the intestinal crypts resembling that seen in pan-epithelial Atg5-deficient mice. Additionally, lack of functional autophagy in Paneth cells within intestinal organoids and T. gondii-infected mice causes increased sensitivity to the proinflammatory cytokine TNF along with increased intestinal permeability, leading to exaggerated microbiota- and IFN-γ-dependent intestinal immunopathology. Thus, Atg5 expression in Paneth cells is essential for tissue protection against cytokine-mediated immunopathology during acute gastrointestinal infection.

Interaction between smoking and ATG16L1T300A triggers Paneth cell defects in Crohn's disease.

It is suggested that subtyping of complex inflammatory diseases can be based on genetic susceptibility and relevant environmental exposure (G+E). We propose that using matched cellular phenotypes in human subjects and corresponding preclinical models with the same G+E combinations is useful to this end. As an example, defective Paneth cells can subtype Crohn's disease (CD) subjects; Paneth cell defects have been linked to multiple CD susceptibility genes and are associated with poor outcome. We hypothesized that CD susceptibility genes interact with cigarette smoking, a major CD environmental risk factor, to trigger Paneth cell defects. We found that both CD subjects and mice with ATG16L1T300A (T300A; a prevalent CD susceptibility allele) developed Paneth cell defects triggered by tobacco smoke. Transcriptional analysis of full-thickness ileum and Paneth cell-enriched crypt base cells showed the T300A-smoking combination altered distinct pathways, including proapoptosis, metabolic dysregulation, and selective downregulation of the PPARγ pathway. Pharmacologic intervention by either apoptosis inhibitor or PPARγ agonist rosiglitazone prevented smoking-induced crypt apoptosis and Paneth cell defects in T300A mice and mice with conditional Paneth cell-specific knockout of Atg16l1. This study demonstrates how explicit G+E can drive disease-relevant phenotype and provides rational strategies for identifying actionable targets.

Adipose-derived resistin and gut-derived resistin-like molecule-beta selectively impair insulin action on glucose production.

The adipose-derived hormone resistin is postulated to link obesity to insulin resistance and diabetes. Here, the infusion of either resistin or the resistin-like molecule-beta (RELMbeta) rapidly induced severe hepatic but not peripheral insulin resistance. In the presence of physiologic hyperinsulinemia, the infusion of purified recombinant resistin, increasing circulating resistin levels by approximately twofold to 15-fold, inhibited glucose metabolism such that lower rates of glucose infusion were required to maintain the plasma glucose concentration at basal levels. The effects of resistin and RELMbeta on in vivo insulin action were completely accounted for by a marked increase in the rate of glucose production. These results support the notion that a novel family of fat- and gut-derived circulating proteins modulates hepatic insulin action.

Role of resistin in diet-induced hepatic insulin resistance.

Resistin is an adipose-derived hormone postulated to link adiposity to insulin resistance. To determine whether resistin plays a causative role in the development of diet-induced insulin resistance, we lowered circulating resistin levels in mice by use of a specific antisense oligodeoxynucleotide (ASO) directed against resistin mRNA and assessed in vivo insulin action by the insulin-clamp technique. After 3 weeks on a high-fat (HF) diet, mice displayed severe insulin resistance associated with an approximately 80% increase in plasma resistin levels. In particular, the rate of endogenous glucose production (GP) increased more than twofold compared with that in mice fed a standard chow. Treatment with the resistin ASO for 1 week normalized the plasma resistin levels and completely reversed the hepatic insulin resistance. Importantly, in this group of mice, the acute infusion of purified recombinant mouse resistin, designed to acutely elevate the levels of circulating resistin up to those observed in the HF-fed mice, was sufficient to reconstitute hepatic insulin resistance. These results provide strong support for a physiological role of resistin in the development of hepatic insulin resistance in this model.

Optimization of protein production in mammalian cells with a coexpressed fluorescent marker.

The expression of mammalian proteins in sufficient abundance and quality for structural studies often presents formidable challenges. Many express poorly in bacterial systems, whereas it can be time consuming and expensive to produce them from cells of higher organisms. Here we describe a procedure for the direct selection of stable mammalian cell lines that express proteins of interest in high yield. Coexpression of a marker protein, such as green fluorescent protein, is linked to that of the desired protein through an internal ribosome entry site in the vector that is transfected into cells in culture. The coexpressed marker is used to select for highly expressing clonal cell lines. Applications are described to a membrane protein, the 5HT2c serotonin receptor, and to a secreted cysteine-rich protein, resistin. Besides providing an expeditious means for producing mammalian proteins for structural work, the resulting cell lines also readily support tests of functional properties and structure-inspired hypotheses.

Regulation of resistin expression and circulating levels in obesity, diabetes, and fasting.

Resistin was originally reported as an adipose tissue-specific hormone that provided a link between obesity and diabetes. Resistin protein level was elevated in obese mice and decreased by insulin-sensitizing thiazolidinediones. Immunoneutralization of resistin improved insulin sensitivity in diet-induced obese mice, while the administration of exogenous resistin induced insulin resistance. More recently, we have shown that ablation of the resistin gene in mice decreased fasting glucose through impairment of gluconeogenesis, while resistin treatment in these knockout mice increased hepatic glucose production. However, the link between resistin and glucose homeostasis has been questioned by studies demonstrating reduced, rather than increased, resistin mRNA expression in obese and diabetic mice. To better understand the regulation of resistin, we developed a sensitive and specific RIA resistin that could accurately measure serum resistin levels in several mouse models. We show that while resistin mRNA is indeed suppressed in obese mice, the circulating resistin level is significantly elevated and positively correlated with insulin, glucose, and lipids. Both resistin mRNA expression and protein levels in Lep(ob/ob) mice are suppressed by leptin treatment in parallel with reductions in glucose and insulin. In wild-type mice, serum resistin increases after nocturnal feeding, concordant with rising levels of insulin. Resistin mRNA and protein levels decline in parallel with glucose and insulin during fasting and are restored after refeeding. We performed clamp studies to determine whether resistin is causally related to insulin and glucose. Adipose resistin expression and serum resistin increased in response to hyperinsulinemia and further in response to hyperglycemia. Taken together, these findings suggest that the nutritional regulation of resistin and changes in resistin gene expression and circulating levels in obesity are mediated, at least in part, through insulin and glucose.

Removal of visceral fat prevents insulin resistance and glucose intolerance of aging: an adipokine-mediated process?

Age-dependent changes in insulin action and body fat distribution are risk factors for the development of type 2 diabetes. To examine whether the accumulation of visceral fat (VF) could play a direct role in the pathophysiology of insulin resistance and type 2 diabetes, we monitored insulin action, glucose tolerance, and the expression of adipo-derived peptides after surgical removal of VF in aging (20-month-old) F344/Brown Norway (FBN) and in Zucker Diabetic Fatty (ZDF) rats. As expected, peripheral and hepatic insulin action were markedly impaired in aging FBN rats, and extraction of VF (accounting for approximately 18% of their total body fat) was sufficient to restore peripheral and hepatic insulin action to the levels of young rats. When examined at the mechanistic level, removal of VF in ZDF rats prevented the progressive decrease in insulin action and delayed the onset of diabetes, but VF extraction did not alter plasma free fatty acid levels. However, the expression of tumor necrosis factor-alpha and leptin in subcutaneous (SC) adipose tissue were markedly decreased after VF removal (by approximately three- and twofold, respectively). Finally, extracted VF retained approximately 15-fold higher resistin mRNA compared with SC fat. Our data suggest that insulin resistance and the development of diabetes can be significantly reduced in aging rats by preventing the age-dependent accumulation of VF. This study documents a cause-and-effect relationship between VF and major components of the metabolic syndrome.

Sexual differentiation, pregnancy, calorie restriction, and aging affect the adipocyte-specific secretory protein adiponectin.

Adiponectin or adipocyte complement-related protein of 30 kDa (Acrp30) is a circulating protein produced exclusively in adipocytes. Circulating Acrp30 levels have been associated with insulin sensitivity in adult mice and humans, yet the Acrp30 profile over the lifespan and its hormonal regulation in vivo have not been previously described. Hence, we set forth to determine whether hormonal and metabolic changes associated with sexual maturation, reproduction, aging, and calorie restriction affect Acrp30. In mice, Acrp30 levels increase during sexual maturation by 4-fold in males and 10-fold in females. Neonatal castration (CX) allows Acrp30 of adults to reach female levels. CX in adults does not lead to female Acrp30 levels unless glucocorticoid exposure is elevated simultaneously by implant. Ovariectomy of infant mice does not interfere with the pubertal rise of Acrp30. However, ovariectomy in adults increases Acrp30. Estrogen suppressed Acrp30 in mice and 3T3-L1 adipocytes. In parallel to changes in estrogen action, Acrp30 decreased in late gestation but increased in both calorie-restricted and old (anovulatory) mice. The reduction of Acrp30 in lactating dams is consistent with a suppressive effect of prolactin and a stimulating effect of bromocriptine. In summary, Acrp30 levels in serum are under complex hormonal control and may play a key role in determining systemic insulin sensitivity under the respective conditions.

Cell type-specific expression and coregulation of murine resistin and resistin-like molecule-alpha in adipose tissue.

Adipocytes are the exclusive or predominant source of several secreted proteins that exert profound effects on systemic carbohydrate and lipid metabolism. Resistin, a 10-kDa adipose tissue specific secretory protein, has recently been implicated in exerting a negative effect on systemic insulin sensitivity. It is, however, not known how resistin mediates this insulin-desensitizing effect or what regulatory mechanisms control resistin expression. Resistin-like molecule-alpha (RELMalpha), a homolog of resistin originally identified by its upregulation in asthmatic lung, is another secreted protein expressed in adipose tissue. The regulation of RELMalpha in adipose tissue and its relationship to resistin expression has not been addressed so far. Here, we demonstrate that the expression of resistin and RELMalpha are similarly regulated in adipose tissue despite the fact that RELMalpha is exclusively expressed in the stromal vascular fraction of adipose tissue and not in adipocytes. Interestingly, this coregulation is limited to adipose tissue as the expression of RELMalpha in lung is independent of metabolic regulation. Additionally, we show that resistin and RELMalpha levels are not subject to regulation by proinflammatory stimuli. Finally, acute hyperglycemia leads to up-regulation of resistin and RELMalpha transcription in various adipose depots.

Tips and Tricks on How to Optimally Manage Patients with Upper Gastrointestinal Bleeding.

Effective endoscopic therapy for upper gastrointestinal (GI) bleeding has been shown to reduce rebleeding, need for surgery, and mortality. Effective endoscopic management of acute upper GI bleeding can be challenging and worrying. This article provides advice that is complementary to the in-depth reviews that accompany it in this issue. Topics include initial management, resuscitation, when and where to scope, benefits and limitations of devices, device selection based on lesion characteristics, improving visualization to localize the lesion, and tips on how to reduce the endoscopist's trepidation about managing these cases.