immediately upon removal from Ussing Chamber and stored at 280uC until assay. Tryptase Assay of Ileal Culture Supernatant Ileal segments weighing 0.05 g were dissected into small fragments, weighed, and placed into 24 well culture plates with cRPMI +0.5% gentamicin. Tissues were incubated with CRF receptor agonist treatments for 180 minutes before supernatant was removed, centrifuged, and stored at 220uC until assay. Supernatant samples were analyzed for tryptase content using Thiobenzyl Ester Substrate assay as previously described [51]. Immunofluorescence and Confocal Analysis Porcine ileal sections were stained with the primary antibody as indicated by the manufacturer’s instructions. Confocal images were obtained with a 3-Laser Nikon Confocal Laser Scanning Instrument (Nikon Instruments). Images were obtained using EZ- C1 Nikon software (Silver Version 2.01). Threshold values were determined using the appropriate isotype-matched controls. A channel series approach was used to ensure no spectral overlap References 1. Blikslager AT, Moeser AJ, Gookin JL, Jones SL, Odle J (2007) Restoration of barrier function in injured intestinal mucosa. Physiol Rev 87: 545–564. 2. Hart A, Kamm MA (2002) Review article: mechanisms of initiation and perpetuation of gut inflammation by stress. Aliment Pharmacol Ther 16: 2017– 2028. 3. Zhao J, Wang J, Dong L, Shi H, Wang Z, et al. (2011) A protease inhibitor against acute stress-induced visceral hypersensitivity and paracellular perme- ability in rats. Eur J Pharmacol 654: 289–294. 4. Ait-Belgnaoui A, Bradesi S, Fioramonti J, Theodorou V, Bueno L (2005) Acute stress-induced hypersensitivity to colonic distension depends upon increase in paracellular permeability: role of myosin light chain kinase. Pain 113: 141–147. 5. Smith F, Clark JE, Overman BL, Tozel CC, Huang JH, et al. (2010) Early weaning stress impairs development of mucosal barrier function in the porcine intestine. Am J Physiol Gastrointest Liver Physiol 298: G352–363. 6. Santos J, Yates D, Guilarte M, Vicario M, Alonso C, et al. (2008) Stress neuropeptides evoke epithelial responses via mast cell activation in the rat colon. Psychoneuroendocrinology 33: 1248–1256. 7. Aggarwal A, Cutts TF, Abell TL, Cardoso S, Familoni B, et al. (1994) Predominant symptoms in irritable bowel syndrome correlate with specific autonomic nervous system abnormalities. Gastroenterology 106: 945–950. 8. Bennett EJ, Tennant CC, Piesse C, Badcock CA, Kellow JE (1998) Level of chronic life stress predicts clinical outcome in irritable bowel syndrome. Gut 43: 256–261. 9. Delvaux MM (1999) Stress and visceral perception. Can J Gastroenterol 13 Suppl A: 32A–36A. 10. Farhadi A, Keshavarzian A, Van de Kar LD, Jakate S, Domm A, et al. (2005) Heightened responses to stressors in patients with inflammatory bowel disease. Am J Gastroenterol 100: 1796–1804. 11. Teitelbaum AA, Gareau MG, Jury J, Yang PC, Perdue MH (2008) Chronic peripheral administration of corticotropin-releasing factor causes colonic barrier dysfunction similar to psychological stress. Am J Physiol Gastrointest Liver Physiol 295: G452–459. 12. Tache Y, Perdue MH (2004) Role of peripheral CRF signalling pathways in stress-related alterations of gut motility and mucosal function. Neurogastroen- terol Motil 16 Suppl 1: 137–142. 13. Gue M, Del Rio-Lacheze C, Eutamene H, Theodorou V, Fioramonti J, et al. (1997) Stress-induced visceral hypersensitivity to rectal distension in rats: role of CRF and mast cells. Neurogastroenterol Motil 9: 271–279. 14. Nozu T, Kudaira M (2006) Corticotropin-releasing factor induces rectal hypersensitivity after repetitive painful rectal distention in healthy humans. J Gastroenterol 41: 740–744. 15. Chrousos GP (1995) The hypothalamic-pituitary-adrenal axis and immune- mediated inflammation. N Engl J Med 332: 1351–1362. 16. Theoharides TC, Donelan JM, Papadopoulou N, Cao J, Kempuraj D, et al. (2004) Mast cells as targets of corticotropin-releasing factor and related peptides. Trends Pharmacol Sci 25: 563–568. 17. Wallon C, Yang PC, Keita AV, Ericson AC, McKay DM, et al. (2008) Corticotropin-releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro. Gut 57: 50–58. 18. Abraham SN, St John AL (2010). Mast cell-orchestrated immunity to pathogens. Nat Rev Immunol 10: 440–452. 19. Keita AV, Soderholm JD, Ericson AC (2010) Stress-induced barrier disruption of rat follicle-associated epithelium involves corticotropin-releasing hormone, Mechanisms of CRF-Induced Intestinal Dysfunction between fluorescent signals. Primary antibodies include occluding (mouse-anti-occludin, Invitrogen), 1:1000. Secondary antibodies used include Fab-antibodies, Jackson Labs Inc (donkey-anti-mouse FITC), 1:1000. Nuclei were visualized with To-Pro-3 iodide 642/ 661 (Invitrogen) 1:1000. Statistical Analyses Data are reported as means 6 SE based on the experimental number (n). Data were analyzed by using a standard one-way ANOVA (Sigmastat, Jandel Scientific, San Rafael, CA). A post hoc Fisher’s least significant difference (LSD) test was used to determine differences between treatments following ANOVA. MC counts were analyzed using Mann-Whitney test, and differences considered significant if p,0.05. Author Contributions Conceived and designed the experiments: AJM. Performed the experi- ments: ELO. Analyzed the data: ELO AJM. Contributed reagents/ materials/analysis tools: JER. Wrote the paper: ELO JER AJM. acetylcholine, substance P, and mast cells. Neurogastroenterol Motil 22: 770– 778, e221–772. 20. Tjong YW, Ip SP, Lao L, Wu J, Fong HH, et al. (2010) Neonatal maternal separation elevates thalamic corticotrophin releasing factor type 1 receptor expression response to colonic distension in rat. Neuro Endocrinol Lett 31: 215– 220. 21. Gareau MG, Jury J, Perdue MH (2007) Neonatal maternal separation of rat pups results in abnormal cholinergic regulation of epithelial permeability. Am J Physiol Gastrointest Liver Physiol 293: G198–203. 22. Gareau MG, Jury J, Yang PC, MacQueen G, Perdue MH (2006) Neonatal maternal separation causes colonic dysfunction in rat pups including impaired host resistance. Pediatr Res 59: 83–88. 23. Moeser AJ, Klok CV, Ryan KA, Wooten JG, Little D, et al. (2007) Stress signaling pathways activated by weaning mediate intestinal dysfunction in the pig. Am J Physiol Gastrointest Liver Physiol 292: G173–181. 24. Saunders PR, Santos J, Hanssen NP, Yates D, Groot JA, et al. (2002) Physical and psychological stress in rats enhances colonic epithelial permeability via peripheral CRH. Dig Dis Sci 47: 208–215. 25. Tache Y, Martinez V, Wang L, Million M (2004) CRF1 receptor signaling pathways are involved in stress-related alterations of colonic function and viscerosensitivity: implications for irritable bowel syndrome. Br J Pharmacol 141: 1321–1330. 26. Santos J, Benjamin M, Yang PC, Prior T, Perdue MH (2000) Chronic stress impairs rat growth and jejunal epithelial barrier function: role of mast cells. Am J Physiol Gastrointest Liver Physiol 278: G847–854. 27. Santos J, Yang PC, Soderholm JD, Benjamin M, Perdue MH (2001) Role of mast cells in chronic stress induced colonic epithelial barrier dysfunction in the rat. Gut 48: 630–636. 28. Ma TY, Iwamoto GK, Hoa NT, Akotia V, Pedram A, et al. (2004) TNF-alpha- induced increase in intestinal epithelial tight junction permeability requires NF- kappa B activation. Am J Physiol Gastrointest Liver Physiol 286: G367–376. 29. Weber CR, Raleigh DR, Su L, Shen L, Sullivan EA, et al. (2010) Epithelial myosin light chain kinase activation induces mucosal interleukin-13 expression to alter tight junction ion selectivity. J Biol Chem 285: 12037–12046. 30. Marchiando AM, Shen L, Graham WV, Weber CR, Schwarz BT, et al (2010). Caveolin-1-dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo. J Cell Biol 189: 111–126. 31. Jacob C, Yang PC, Darmoul D, Amadesi S, Saito T, et al. (2005) Mast cell tryptase controls paracellular permeability of the intestine. Role of protease- activated receptor 2 and beta-arrestins. J Biol Chem 280: 31936–31948. 32. Gebhardt T, Gerhard R, Bedoui S, Erpenbeck VJ, Hoffmann MW, et al. (2005) beta2-Adrenoceptor-mediated suppression of human intestinal mast cell functions is caused by disruption of filamentous actin dynamics. Eur J Immunol 35: 1124–1132. 33. Groschwitz KR, Ahrens R, Osterfeld H, Gurish MF, Han XN, et al. (2009) Mast cells regulate homeostatic intestinal epithelial migration and barrier function by a chymase/Mcpt4-dependent mechanism. Proceedings of the National Acad- emy of Sciences of the United States of America 106: 22381–22386. 34. Shen L, Weber CR, Raleigh DR, Yu D, Turner JR (2011). Tight junction pore and leak pathways: a dynamic duo. Annu Rev Physiol 73: 283–309. 35. Buhner S, Schemann M (2011) Mast cell-nerve axis with a focus on the human gut. Biochim Biophys Acta. PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39935