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1.  Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis 
Mucosal immunology  2013;7(1):10.1038/mi.2013.29.
Pharmacological stabilization of hypoxia-inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition limits mucosal damage associated with models of murine colitis. However, little is known about how PHD inhibitors (PHDi) influence systemic immune function during mucosal inflammation or the relative importance of immunological changes to mucosal protection. We hypothesized that PHDi enhances systemic innate immune responses to colitis-associated bacteremia. Mice with colitis induced by TNBS were treated with AKB-4924, a new HIF-1 isoform-predominant PHDi and clinical, immunological and biochemical endpoints were assessed. Administration of AKB-4924 led to significantly reduced weight loss and disease activity compared to vehicle controls. Treated groups were pyrexic, but did not become subsequently hypothermic. PHDi treatment augmented epithelial barrier function and led to an approximately 50-fold reduction in serum endotoxin during colitis. AKB-4924 also decreased cytokines involved in pyrogenesis and hypothermia, significantly reducing serum levels of IL-1β, IL-6 and TNF-α, while increasing IL-10. Treatment offered no protection against colitis in epithelial-specific HIF-1α deficient mice, strongly implicating epithelial HIF-1α as the tissue target for AKB-4924-mediated protection. Taken together, these results indicate that inhibition of prolyl hydroxylase with AKB-4924 enhances innate immunity and identifies the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis.
PMCID: PMC3772994  PMID: 23695513
2.  Activation of AMPK Inhibits Cholera Toxin Stimulated Chloride Secretion in Human and Murine Intestine 
PLoS ONE  2013;8(7):e69050.
Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK), can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX) mediated diarrhea.
Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK). In order to substantiate our findings on the whole tissue level, short-circuit current (SCC) was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops.
CTX and forskolin (FSK) significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments.
The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.
PMCID: PMC3728293  PMID: 23935921
3.  The Vibrio parahaemolyticus Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis 
BMC Microbiology  2010;10:329.
Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined.
V. parahaemolyticus deployed its TTSS1 to induce activation of the JNK, p38 and ERK MAPK in human epithelial cells. VP1680 was identified as the TTSS1 effector protein responsible for MAPK activation in Caco-2 cells and the activation of JNK and ERK by this protein was important in induction of host cell death. V. parahaemolyticus actively induced IL-8 secretion in a response mediated by TTSS1. A role for VP1680 and for the ERK signalling pathway in the stimulation of IL-8 production in epithelial cells by V. parahaemolyticus was established. Interestingly, TTSS2 inhibited IL-8 mRNA transcription at early stages of interaction between the bacterium and the cell.
This study demonstrated that V. parahaemolyticus activates the three major MAPK signalling pathways in intestinal epithelial cells in a TTSS1-dependent manner that involves the TTSS1 effector VP1680. Furthermore VP1680 and JNK and ERK activation were needed for maximal cytotoxicity of the bacterium. It was shown that V. parahaemolyticus is a strong inducer of IL-8 secretion and that induction reflects a balance between the effects of TTSS1 and TTSS2. Increases in IL-8 secretion were mediated by TTSS1 and VP1680, and augmented by ERK activation. These results shed light on the mechanisms of bacterial pathogenesis mediated by TTSS and suggest significant roles for MAPK signalling during infection with V. parahaemolyticus.
PMCID: PMC3022711  PMID: 21192810
4.  Constitutive basal and stimulated human small bowel contractility is enhanced in obesity 
Small bowel contractility may be more prominent in obese subjects, such that there is enhanced nutrient absorption and hunger stimulation. However, there is little evidence to support this. This study examined in vitro small bowel contractility in obese patients versus non-obese patients.
Samples of histologically normal small bowel were obtained at laparoscopic Roux-en-Y gastric bypass from obese patients. Control specimens were taken from non-obese patients undergoing small bowel resection for benign disease or formation of an ileal pouch-anal anastamosis. Samples were transported in a pre-oxygenated Krebs solution. Microdissected circular smooth muscle strips were suspended under 1 g of tension in organ baths containing Krebs solution oxygenated with 95% O2/5% CO2 at 37°C. Contractile activity was recorded using isometric transducers at baseline and in response to receptor-mediated contractility using prostaglandin F2a, a nitric oxide donor and substance P under both equivocal and non-adreneregic, non-cholinergic conditions (guanethidine and atropine).
Following equilibration, the initial response to the cholinergic agonist carbachol (0.1 mmol/L) was significantly increased in the obese group (n = 63) versus the lean group (n = 61) with a mean maximum response: weight ratio of 4.58 ± 0.89 vs 3.53 ± 0.74; (p = 0.032). Following washout and re-calibration, cumulative application of substance P and prostaglandin F2a produced concentration-dependent contractions of human small bowel smooth muscle strips. Contractile responses of obese small bowel under equivocal conditions were significantly increased compared with non-obese small bowel (p < 0.05 for all agonists). However, no significant differences were shown between the groups when the experiments were performed under NANC conditions. There were no significant differences found between the groups when challenged with nitric oxide, under either equivocal or NANC conditions.
Stimulated human small bowel contractility is increased in obese patients suggesting faster enteric emptying and more rapid intestinal transit. This may translate into enhanced appetite and reduced satiety.
PMCID: PMC2673225  PMID: 19379492
5.  Extremophiles and their application to veterinary medicine 
Irish Veterinary Journal  2004;57(6):348-354.
Extremophiles are organisms that can grow and thrive in harsh conditions, e.g., extremes of temperature, pH, salinity, radiation, pressure and oxygen tension. Thermophilic, halophilic and radiation-resistant organisms are all microbes, some of which are able to withstand multiple extremes. Psychrophiles, or cold-loving organisms, include not only microbes, but fish that live in polar waters and animals that can withstand freezing. Extremophiles are structurally adapted at a molecular level to withstand these conditions. Thermophiles have particularly stable proteins and cell membranes, psychrophiles have flexible cellular proteins and membranes and/or antifreeze proteins, salt-resistant halophiles contain compatible solutes or high concentrations of inorganic ions, and acidophiles and alkaliphiles are able to pump ions to keep their internal pH close to neutrality. Their interest to veterinary medicine resides in their capacity to be pathogenic, and as sources of enzymes and other molecules for diagnostic and pharmaceutical purposes. In particular, thermostable DNA polymerases are a mainstay of PCR-based diagnostics.
PMCID: PMC3113819  PMID: 21851659
Extremophiles; Adaptation; Thermophiles; Extremozymes; Diagnostics; Polymerase chain reaction

Results 1-5 (5)