The effect of renin angiotensin system genetic variants in acute pancreatitis.

OBJECTIVES: We sought association of genetic variants in the renin-angiotensin system (RAS) and vitamin D system with acute pancreatitis (AP) development and severity. BACKGROUND: The endocrine RAS is involved in circulatory homeostasis through the pressor action of angiotensin II at its AT1 receptor. However, local RAS regulate growth and inflammation in diverse cells and tissues, and their activity may be suppressed by vitamin D. Intrapancreatic angiotensin II generation has been implicated in the development of AP. METHODS: Five hundred forty-four white patients with AP from 3 countries (United Kingdom, 22; Germany, 136; and The Netherlands 386) and 8487 control subjects (United Kingdom 7833, The Netherlands 717) were genotyped for 8 polymorphisms of the RAS/vitamin D systems, chosen on the basis of likely functionality. RESULTS: The angiotensin-converting enzyme I (rather than D) allele was significantly associated with alcohol-related AP when all cohorts were combined (P = 0.03). The renin rs5707 G (rather than A) allele was associated with AP (P = 0.002), infected necrosis (P = 0.025) and mortality (P = 0.046). CONCLUSIONS: The association of 2 RAS polymorphisms with AP suggests the need for further detailed analysis of the role of RAS/vitamin D in the genesis or severity of AP, particularly given the ready potential for pharmacological manipulation of this system using existing marketed agents. However, further replication studies will be required before any such association is considered robust, particularly given the significant heterogeneity of AP causation and clinical course.

Impact of global Fxr deficiency on experimental acute pancreatitis and genetic variation in the FXR locus in human acute pancreatitis.

BACKGROUND: Infectious complications often occur in acute pancreatitis, related to impaired intestinal barrier function, with prolonged disease course and even mortality as a result. The bile salt nuclear receptor farnesoid X receptor (FXR), which is expressed in the ileum, liver and other organs including the pancreas, exhibits anti-inflammatory effects by inhibiting NF-κB activation and is implicated in maintaining intestinal barrier integrity and preventing bacterial overgrowth and translocation. Here we explore, with the aid of complementary animal and human experiments, the potential role of FXR in acute pancreatitis. METHODS: Experimental acute pancreatitis was induced using the CCK-analogue cerulein in wild-type and Fxr-/- mice. Severity of acute pancreatitis was assessed using histology and a semi-quantitative scoring system. Ileal permeability was analyzed in vitro by Ussing chambers and an in vivo permeability assay. Gene expression of Fxr and Fxr target genes was studied by quantitative RT-PCR. Serum FGF19 levels were determined by ELISA in acute pancreatitis patients and healthy volunteers. A genetic association study in 387 acute pancreatitis patients and 853 controls was performed using 9 tagging single nucleotide polymorphisms (SNPs) covering the complete FXR gene and two additional functional SNPs. RESULTS: In wild-type mice with acute pancreatitis, ileal transepithelial resistance was reduced and ileal mRNA expression of Fxr target genes Fgf15, SHP, and IBABP was decreased. Nevertheless, Fxr-/- mice did not exhibit a more severe acute pancreatitis than wild-type mice. In patients with acute pancreatitis, FGF19 levels were lower than in controls. However, there were no associations of FXR SNPs or haplotypes with susceptibility to acute pancreatitis, or its course, outcome or etiology. CONCLUSION: We found no evidence for a major role of FXR in acute human or murine pancreatitis. The observed altered Fxr activity during the course of disease may be a secondary phenomenon.

Early angiopoietin-2 levels after onset predict the advent of severe pancreatitis, multiple organ failure, and infectious complications in patients with acute pancreatitis.

BACKGROUND: Acute pancreatitis is a severe condition that requires early identification of patients at risk of developing potentially lethal complications. Current clinical scoring systems and biochemical parameters are insufficient. In this study, we aimed to assess whether early plasma Angiopoietin-2 (Ang-2) is associated with adverse outcomes in patients with predicted severe acute pancreatitis (SAP). STUDY DESIGN: This analysis is a substudy of the PROPATRIA trial (probiotics vs placebo in patients with predicted SAP). The Ang-2 levels were measured prospectively in plasma in the first 5 days after admission in 115 patients. RESULTS: Early Ang-2 levels were higher in patients who developed SAP: 6.4 vs 3.1 µg/L (p < 0.001) and also were higher in patients who developed multiorgan failure in the first week (p = 0.001) and after the first week (p = 0.049). Furthermore, high Ang-2 levels were associated with infectious complications in the first week (p < 0.001) and after the first week (p < 0.001). Finally, plasma Ang-2 was significantly higher in patients who died (p < 0.001) and in patients who developed bowel ischemia (p < 0.001). As a predictor of adverse outcomes, plasma Ang-2 was superior to a number of current scores, such as the APACHE II score, the Imrie score, C-reactive protein, lipopolysaccharide binding protein, and procalcitonin. CONCLUSIONS: In the setting of this randomized controlled trial, early plasma Ang-2 was found to be an accurate predictor of SAP, multiorgan failure, and infectious complications. As a biomarker, it did outperform all of the investigated conventional predictors that are currently used in clinical practice.

Association analysis of genetic variants in the myosin IXB gene in acute pancreatitis.

INTRODUCTION: Impairment of the mucosal barrier plays an important role in the pathophysiology of acute pancreatitis. The myosin IXB (MYO9B) gene and the two tight-junction adaptor genes, PARD3 and MAGI2, have been linked to gastrointestinal permeability. Common variants of these genes are associated with celiac disease and inflammatory bowel disease, two other conditions in which intestinal permeability plays a role. We investigated genetic variation in MYO9B, PARD3 and MAGI2 for association with acute pancreatitis. METHODS: Five single nucleotide polymorphisms (SNPs) in MYO9B, two SNPs in PARD3, and three SNPs in MAGI2 were studied in a Dutch cohort of 387 patients with acute pancreatitis and over 800 controls, and in a German cohort of 235 patients and 250 controls. RESULTS: Association to MYO9B and PARD3 was observed in the Dutch cohort, but only one SNP in MYO9B and one in MAGI2 showed association in the German cohort (p < 0.05). Joint analysis of the combined cohorts showed that, after correcting for multiple testing, only two SNPs in MYO9B remained associated (rs7259292, p = 0.0031, odds ratio (OR) 1.94, 95% confidence interval (95% CI) 1.35-2.78; rs1545620, p = 0.0006, OR 1.33, 95% CI 1.16-1.53). SNP rs1545620 is a non-synonymous SNP previously suspected to impact on ulcerative colitis. None of the SNPs showed association to disease severity or etiology. CONCLUSION: Variants in MYO9B may be involved in acute pancreatitis, but we found no evidence for involvement of PARD3 or MAGI2.

Endoscopic transgastric vs surgical necrosectomy for infected necrotizing pancreatitis: a randomized trial.

CONTEXT: Most patients with infected necrotizing pancreatitis require necrosectomy. Surgical necrosectomy induces a proinflammatory response and is associated with a high complication rate. Endoscopic transgastric necrosectomy, a form of natural orifice transluminal endoscopic surgery, may reduce the proinflammatory response and reduce complications. OBJECTIVE: To compare the proinflammatory response and clinical outcome of endoscopic transgastric and surgical necrosectomy. DESIGN, SETTING, AND PATIENTS: Randomized controlled assessor-blinded clinical trial in 3 academic hospitals and 1 regional teaching hospital in The Netherlands between August 20, 2008, and March 3, 2010. Patients had signs of infected necrotizing pancreatitis and an indication for intervention. INTERVENTIONS: Random allocation to endoscopic transgastric or surgical necrosectomy. Endoscopic necrosectomy consisted of transgastric puncture, balloon dilatation, retroperitoneal drainage, and necrosectomy. Surgical necrosectomy consisted of video-assisted retroperitoneal debridement or, if not feasible, laparotomy. MAIN OUTCOME MEASURES: The primary end point was the postprocedural proinflammatory response as measured by serum interleukin 6 (IL-6) levels. Secondary clinical end points included a predefined composite end point of major complications (new-onset multiple organ failure, intra-abdominal bleeding, enterocutaneous fistula, or pancreatic fistula) or death. RESULTS: We randomized 22 patients, 2 of whom did not undergo necrosectomy following percutaneous catheter drainage and could not be analyzed for the primary end point. Endoscopic transgastric necrosectomy reduced the postprocedural IL-6 levels compared with surgical necrosectomy (P = .004). The composite clinical end point occurred less often after endoscopic necrosectomy (20% vs 80%; risk difference [RD], 0.60; 95% CI, 0.16-0.80; P = .03). Endoscopic necrosectomy did not cause new-onset multiple organ failure (0% vs 50%, RD, 0.50; 95% CI, 0.12-0.76; P = .03) and reduced the number of pancreatic fistulas (10% vs 70%; RD, 0.60; 95% CI, 0.17-0.81; P = .02). CONCLUSION: In patients with infected necrotizing pancreatitis, endoscopic necrosectomy reduced the proinflammatory response as well as the composite clinical end point compared with surgical necrosectomy. TRIAL REGISTRATION: isrctn.org Identifier: ISRCTN07091918.

Farnesoid X receptor (FXR) activation and FXR genetic variation in inflammatory bowel disease.

BACKGROUND: We previously showed that activation of the bile salt nuclear receptor Farnesoid X Receptor (FXR) protects against intestinal inflammation in mice. Reciprocally, these inflammatory mediators may decrease FXR activation. We investigated whether FXR activation is repressed in the ileum and colon of inflammatory bowel disease (IBD) patients in remission. Additionally, we evaluated whether genetic variation in FXR is associated with IBD. METHODS: mRNA expression of FXR and FXR target gene SHP was determined in ileal and colonic biopsies of patients with Crohn's colitis (n = 15) and ulcerative colitis (UC; n = 12), all in clinical remission, and healthy controls (n = 17). Seven common tagging SNPs and two functional SNPs in FXR were genotyped in 2355 Dutch IBD patients (1162 Crohn's disease (CD) and 1193 UC) and in 853 healthy controls. RESULTS: mRNA expression of SHP in the ileum is reduced in patients with Crohn's colitis but not in patients with UC compared to controls. mRNA expression of villus marker Villin was correlated with FXR and SHP in healthy controls, a correlation that was weaker in UC patients and absent in CD patients. None of the SNPs was associated with IBD, UC or CD, nor with clinical subgroups of CD. CONCLUSIONS: FXR activation in the ileum is decreased in patients with Crohn's colitis. This may be secondary to altered enterohepatic circulation of bile salts or transrepression by inflammatory signals but does not seem to be caused by the studied SNPs in FXR. Increasing FXR activity by synthetic FXR agonists may have benefit in CD patients.

Visceral endoderm induces specification of cardiomyocytes in mice.

The endoderm plays an inductive role in the formation of cardiomyocytes in many vertebrates. Here, we provide further evidence for this in the mouse and demonstrate enhanced cardiomyogenesis in mouse embryonic stem cells cultured in the presence of native visceral endoderm. Isolated mesoderm from late-primitive streak stage mouse embryos that still have an open proamniotic canal had a reduced capacity to form cardiomyocytes after 4 days in culture compared with mesoderm isolated from later stages but prior to cardiomyogenesis. Moreover, removal of the visceral endoderm but not the primitive streak reduced the formation of beating areas in embryo explants in culture. Coculture with the END2 cell line, which has visceral endoderm-like properties, restored the formation of beating areas. Immunohistochemical analysis showed that the expected candidate signaling pathways downstream of Wnts and bone morphogenetic proteins (BMPs) were active in the embryo at the appropriate time and place to be involved. Overall, the results show that, as in other vertebrates, the (visceral) endoderm plays an important role in the early events of mouse cardiomyogenesis.

Physical activity and endogenous sex hormone levels in postmenopausal women: a cross-sectional study in the Prospect-EPIC Cohort.

BACKGROUND: The protective effect of physical activity on breast cancer risk might be mediated by sex hormone levels. In this study, we examined the association between usual physical activity and plasma levels of estrogens, androgens, dehydroepiandrosterone sulphate (DHEAS), and sex hormone binding globulin (SHBG) in postmenopausal women. METHODS: We conducted a cross-sectional study among 806 postmenopausal women participating in the Prospect-EPIC study. Usual physical activity was assessed using a short questionnaire and summarized into a simple, validated four-level index. This index combines occupational physical activity with time spent on cycling and sporting. Levels of estrone, estradiol, androstenedione, DHEAS, testosterone, and SHBG were measured in plasma. General linear models were used to examine the association between usual physical activity and sex hormone levels, adjusted for confounders. RESULTS: We observed an inverse association between physical activity and estradiol levels (free: inactive, 0.26 pg/mL; active, 0.23 pg/mL; P-trend = 0.045; total: inactive, 8.8 pg/mL; active, 8.0 pg/mL; P-trend = 0.08) and a positive association between physical activity and SHBG (inactive, 15.1 nmol/L; active, 19.3 nmol/L; P-trend = 0.05). These associations could largely be explained by the effects of sporting. We also observed a positive association between physical activity and DHEAS (inactive, 352.4 ng/mL; active, 460.3 ng/mL; P-trend = 0.01). CONCLUSIONS: Our results are in accordance with the hypothesis that usual physical activity decreases estradiol levels and increases SHBG. We also found that high levels of physical activity are associated with high levels of DHEAS. Furthermore, our results suggest that vigorous forms of physical activity influence sex hormone levels most.

Probiotics prevent intestinal barrier dysfunction in acute pancreatitis in rats via induction of ileal mucosal glutathione biosynthesis.

BACKGROUND: During acute pancreatitis (AP), oxidative stress contributes to intestinal barrier failure. We studied actions of multispecies probiotics on barrier dysfunction and oxidative stress in experimental AP. METHODOLOGY/PRINCIPAL FINDINGS: Fifty-three male Spraque-Dawley rats were randomly allocated into five groups: 1) controls, non-operated, 2) sham-operated, 3) AP, 4) AP and probiotics and 5) AP and placebo. AP was induced by intraductal glycodeoxycholate infusion and intravenous cerulein (6 h). Daily probiotics or placebo were administered intragastrically, starting five days prior to AP. After cerulein infusion, ileal mucosa was collected for measurements of E. coli K12 and (51)Cr-EDTA passage in Ussing chambers. Tight junction proteins were investigated by confocal immunofluorescence imaging. Ileal mucosal apoptosis, lipid peroxidation, and glutathione levels were determined and glutamate-cysteine-ligase activity and expression were quantified. AP-induced barrier dysfunction was characterized by epithelial cell apoptosis and alterations of tight junction proteins (i.e. disruption of occludin and claudin-1 and up-regulation of claudin-2) and correlated with lipid peroxidation (r>0.8). Probiotic pre-treatment diminished the AP-induced increase in E. coli passage (probiotics 57.4+/-33.5 vs. placebo 223.7+/-93.7 a.u.; P<0.001), (51)Cr-EDTA flux (16.7+/-10.1 vs. 32.1+/-10.0 cm/s10(-6); P<0.005), apoptosis, lipid peroxidation (0.42+/-0.13 vs. 1.62+/-0.53 pmol MDA/mg protein; P<0.001), and prevented tight junction protein disruption. AP-induced decline in glutathione was not only prevented (14.33+/-1.47 vs. 8.82+/-1.30 nmol/mg protein, P<0.001), but probiotics even increased mucosal glutathione compared with sham rats (14.33+/-1.47 vs. 10.70+/-1.74 nmol/mg protein, P<0.001). Glutamate-cysteine-ligase activity, which is rate-limiting in glutathione biosynthesis, was enhanced in probiotic pre-treated animals (probiotics 2.88+/-1.21 vs. placebo 1.94+/-0.55 nmol/min/mg protein; P<0.05) coinciding with an increase in mRNA expression of glutamate-cysteine-ligase catalytic (GCLc) and modifier (GCLm) subunits. CONCLUSIONS: Probiotic pre-treatment diminished AP-induced intestinal barrier dysfunction and prevented oxidative stress via mechanisms mainly involving mucosal glutathione biosynthesis.

Genetic analysis of innate immunity in Crohn's disease and ulcerative colitis identifies two susceptibility loci harboring CARD9 and IL18RAP.

The two main phenotypes of inflammatory bowel disease (IBD)--Crohn's disease (CD) and ulcerative colitis (UC)--are chronic intestinal inflammatory disorders with a complex genetic background. Using a three-stage design, we performed a functional candidate-gene analysis of innate immune pathway in IBD. In phase I, we typed 354 SNPs from 85 innate immunity genes in 520 Dutch IBD patients (284 CD, 236 UC) and 808 controls. In phase II, ten autosomal SNPs showing association at p < 0.006 in phase I were replicated in a second cohort of 545 IBD patients (326 CD, 219 UC) and 360 controls. In phase III, four SNPs with p < 0.01 in the combined phase I and phase II analysis were genotyped in an additional 786 IBD samples (452 CD, 334 UC) and 768 independent controls. Joint analysis of 1851 IBD patients (1062 CD, 789 UC) and 1936 controls demonstrated strong association to the IL18RAP rs917997 SNP for both CD and UC (p(IBD) 1.9 x 10(-8); OR 1.35). Association in CD is independently supported by the Crohn's disease dataset of the Wellcome Trust Case Control Consortium (imputed SNP rs917997, p = 9.19 x 10(-4)). In addition, an association of the CARD9 rs10870077 SNP to CD and UC was observed (p(IBD) = 3.25 x 10(-5); OR 1.21). Both genes are located in extended haplotype blocks on 2q11-2q12 and 9q34.3, respectively. Our results indicate two IBD loci and further support the importance of the innate immune system in the predisposition to both CD and UC.