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Urea entry into the gastrointestinal tract is facilitated by the urea transporter UT-B1 (Stewart 2011). This process is crucial to our intestinal wellbeing as it is the first step in the urea nitrogen salvaging mechanism, which is necessary for maintaining healthy gut bacterial populations. In a previous study using a bovine UT-B antibody, a 35kDa human UT-B1 protein was found to be highly expressed in the ascending colon (Collins et al., 2010). The aim of this study was to produce a reliable human UT-B antibody to be used for further investigations of human UT-B expression. Initial PCR analysis using primers designed along the UT-B gene confirmed UT-B1 as the major isoform expressed at the RNA level in all human gastrointestinal tissues. Three polyclonal human UT-B antibodies - one designed against the N-terminal (UT-B#N) and two against the C-terminal (UT-B#C and UT-Bc19) - were made according to the UT-B1 amino acid sequence and extensive characterization was carried out using Western blot analysis. Wh...

1. This study examined the effects of chronic exposure of rats to 3,4-methylenedioxymethamphetamine (MDMA) on [(3)H]5-hydroxytryptamine ([(3)H]5-HT) re-uptake into purified rat brain synaptosomes, 5-HT-induced isometric contraction of aortic rings and [(3)H]5-HT re-uptake into rat aorta. 2. Rats were administered MDMA (20 mg kg(-1) i.p.) twice daily over 4 days. One, 7, 14 or 21 days post treatment, whole brain synaptosomes and descending thoracic aortic rings were prepared for investigation. 3. Chronic MDMA treatment significantly reduced the maximum rate (V(max)) of specific high-affinity [(3)H]5-HT re-uptake 1 day after treatment and for up to 21 days post-final administration of MDMA. Direct application of MDMA (100 microM) abolished synaptosomal re-uptake of [(3)H]5-HT in vitro. 4. Chronic MDMA administration significantly reduced the maximum contraction (E(max)) to 5-HT at 1 and 7 days after treatment, but not at 14 or 21 days. 5. Chronic MDMA administration had no effect on sodium-dependent [(3)H]5-HT re-uptake into aorta 1 day after treatment, nor did 100 microM MDMA have any direct effect on [(3)H]5-HT uptake into aortic rings in vitro. 6. These results show, for the first time, an altered responsiveness of vascular tissue to MDMA after chronic administration. In addition, they demonstrate a difference in the sensitivity of central and peripheral 5-HT uptake systems to chronic MDMA exposure, and suggest that the action of MDMA in the cardiovascular system does not arise from a direct effect of MDMA on peripheral 5-HT transport.

OBJECTIVES: Tachykinins have been implicated in the pathogenesis of colonic dysmotility. The sources of activator calcium for neurokinin A (NKA)–induced contraction of human colonic smooth muscle have not been assessed. We evaluated the contribution of extracellular and intracellular Ca2+ to NKA-induced contractions. METHODS: Circular smooth muscle strips of human colon were suspended under 1 g of tension in organ baths containing Krebs solution at 37°C gased with 95% O2/5% CO2. Contractile activity was recorded isometrically. RESULTS: Cumulatively applied NKA (0.1 nmol/L–0.3 μmol/L), produced concentration-dependent contractions of human colonic smooth muscle strips that were not affected by tetrodotoxin (1 μmol/L). The contractile response to NKA was abolished in a Ca2+-free medium containing ethylenediaminetetraacetate (EDTA) (1 mmol/L). Pretreatment of muscle strips with nifedipine (1 μmol/L), an L-type voltage-operated Ca2+ channel antagonist, abolished the contractile responses to NKA. Pretreatment with SK&F 96365 (10 μmol/L and 30 μmol/L), a putative receptor-activated and voltage-operated Ca2+ channel antagonist, attenuated the contractile responses. Depletion of intracellular Ca2+ stores with thapsigargin (1 μmol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ ATP-ase, had no effect on NKA-induced contractions. CONCLUSIONS: NKA-mediated contraction of human colonic smooth muscle is dependent on an influx of extracellular Ca2+ through L-type voltage-operated Ca2+ channels. Intracellular Ca2+ release seems to have little role to play in NKA-mediated contractions.

Objective and design: This study aimed to determine if mucosal expression of the chemokines IL-8, RANTES and MCP-1 and the pro-inflammatory cytokines TNFα and IL-6 are elevated in patients with inflammatory bowel disease.¶Materials and subjects: Intestinal mucosa samples were obtained at the time of surgical resection, n=16 from each of the following groups: normal/control, CD and UC.¶Methods: An homogenate was prepared of each tissue sample and cytokines measured by ELISA.¶Results: IL-8 was significantly increased in both disease groups compared to controls Similarly, RANTES levels were also significantly increased. MCP-1 levels were increased in both disease groups, this increase was statistically significant in the UC group only. TNFα and IL-6 were significantly increased in the CD group only.¶Conclusions: Chemokines, together with key cytokines that promote their release are elevated in mucosal tissues from patients with IBD. It is likely that these chemokines play an important role in the perpetuation of tissue destructive inflammatory processes.

Entrapment of antigens in biodegradable particles for mucosal immunisation has given successful outcomes in animals, but not as yet in man. Formulations using genuinely stable biocompatible nanoparticles with co-entrapped mucosal adjuvants and/or with surface-conjugated human M-cell-targeting ligands may lead to better uptake of intact antigen by Peyer's patch M cells and delivery to antigen-presenting cells.

This in vitro study was designed to determine the potential use of the NK1 antagonist, SR140333 as an anti-diarrhoeal treatment for food allergy or inflammatory bowel disease. The effect of various immune and neuronal stimuli on human colonic substance P (SP) release and the effect of SR140333 on subsequently stimulated mucosal ion transport was investigated. Submucosal and sensory nerve fibre stimulation using electrical field stimulation (1 ms/7 Hz/7 V) and capsaicin (50 μM) respectively, mast cell activation by anti-IgE (1/250 dilution) and granulocyte stimulation using fMLP (50 μM) each released SP and evoked a secretory response. SP and the NK1 selective agonist, Sar-SP (0.1–1000 nM) stimulated an increase in colonic secretion which was antagonized by SR140333 (pD′2=6.7 and 7.25 versus SP and Sar-SP respectively). SR140333, at a concentration that blocked NK1-mediated secretion (500 nM), also reduced the secretory response to both αIgE and capsaicin. This suggests a pathophysiologic role for NK1 receptors. Capsaicin evoked SP release was increased in tissue taken from Crohn's disease but not ulcerative colitis patients. The response to SP was however reduced by 70 and 89% respectively. Mast cells and sensory afferents contribute to allergic diarrhoea. Since SR140333 reduced the secretory response to mast cell and afferent stimulation this compound may be particularly useful in reducing the symptoms of food allergy. British Journal of Pharmacology (2001) 133, 1346–1354; doi:10.1038/sj.bjp.0704194

Clostridium difficile is the most common cause of diarrhoea in hospitalised patients. Bacterial adherence to gut epithelial cells is a likely prerequisite to infection and toxin production. A novel flow cytometric method was developed for detecting adherence of C. difficile to human colonic and small intestinal epithelial cells (EC) and human intestinal cell lines. Small intestinal and colonic EC were isolated from biopsy specimens with mucolytic and chelating agents. Adherence of fluorochrome-labelled C. difficile to EC was measured by flow cytometry and was calculated as increase in median fluorescent intensity (deltaMFI). Cells with bacteria attached could be distinguished easily from cells alone or cells with unlabelled bacteria attached. Toxin-positive C. difficile adhered to colonic and small intestinal EC (deltaMFI mean 21.2 SD 16.7, n = 33 and 16.5 SD 20.7, n = 19 respectively). The toxin-negative strain also adhered to both epithelial cell types (deltaMFI 26.1 SD 32.5, n = 17 and 18.3 SD 31.3, n = 16). Adherence of toxin-positive C. difficile to the intestinal cell lines Caco-2 (deltaMFI 9.4 SD 4.4, n = 14) and HT29 (deltaMFI 8.1 SD 3.1, n = 12) was quantifiable, although at a significantly lower level than with primary colonic epithelial cells. Adherence of the toxin-negative strain was slightly lower, deltaMFI 6.5 SD 1.8, n = 9 with Caco-2 cells and deltaMFI 6.0 SD 2.0, n = 10 with HT29 cells. Adherence of C. difficile to epithelial cell lines was blocked with C. difficile antiserum, confirming specificity of adherence. In conclusion, flow cytometry is a useful approach to quantifying adherence of C. difficile to human colonic and small intestinal epithelial cells. Binding of toxin-negative as well as toxin-positive bacteria was detectable by this approach. Analysis of C. difficile adherence to target cells may have important implications for the understanding of the pathogenesis of C. difficile-related disease.

Background: The efficacy of cyclosporin in the management of ulcerative colitis is recognized. Not all patients respond to this treatment. Existing clinical and laboratory parameters are of little use in identifying those most likely to respond. Aims: To determine whether in-vitro sensitivity to cyclosporin as measured by a lymphocyte proliferation assay is predictive of in-vivo response to therapy. Methods: The study comprised seven responders with ulcerative colitis, seven non-responders, and 14 healthy matched controls. A lymphocyte proliferation assay was carried out in the presence of a range of concentrations of cyclosporin and a dose–response curve constructed for each subject. The IC50 value, the concentration of cyclosporin that resulted in 50% inhibition of proliferation, was calculated for each subject. IC50 values for responders, non-responders and controls were compared using a Mann–Whitney test. Results: There was a wide range of values obtained for the study group as a whole. IC50 values for non-responders were significantly higher than those of responders (P

Background/Aims: Activation of cells within the lamina propria can cause electrogenic chloride secretion across intestinal epithelia by release and/or synthesis of mediator molecules, including reactive oxygen metabolites (ROMs). In this investigation we examined whether indirect (immune) stimulation of ion transport across rat colon was ROM-mediated. Methods: Paired segments of rat colon, stripped of underlying smooth muscle, were mounted on Ussing chambers in order to measure electrogenic ion transport. Changes in short circuit current (SCC) were used as a measure of net electrogenic ion transport measured in response to the bacterial tri-peptide formyl-Met-Leu-Phe (fMLP) which was used to activate lamina propria neutrophils. The effect of the established anti-oxidants catalase and diethyldithiocarbamate (DDTC) and the putative anti-oxidant taurine upon immune-stimulated ion transport was examined. Results: The anti-oxidant DDTC but not catalase significantly attenuated ion transport responses to fMLP. Taurine applied basolaterally reduced ion transport response to fMLP but not to the directly acting secretagogues forskolin. Taurine applied apically enhanced ion transport responses to fMLP. Conclusion: Anti-oxidants, including taurine, may be useful in treatment of colitis. The enhancement of effect seen when taurine was applied apically may have negative implications regarding the therapeutic usefulness of taurine administration to the lumenal compartment.

Purpose. To assess the Caco-2 monolayer as a model for iontophoresis of drugs across a model epithelium.

The effects of berberine on ion transport in both human colonic mucosal epithelia and an intestinal epithelial cell line (T84) were examined. Berberine (concentration range 0-500 microM) reduced both basal and stimulated ion transport responses in human colonic mucosae in a manner which was non-specific for Ca2+ -or cAMP-mediated signals. Similarly, in cultured intestinal epithelial monolayers, berberine inhibited Ca2+ -and cAMP-mediated responses indicating an inhibitory activity directly at the level of the epithelium rather than an indirect effect through other mucosal element(s). Berberine did not alter the rate of generation of cAMP by adenylyl cyclase or the activity of protein kinase A, the effector enzyme of the cAMP pathway. Berberine inhibited carbachol-stimulated 86Rb+ efflux from T84 monolayers. Berberine also inhibited K+ conductance in apically-permeabilised re-sected mucosae. These results indicate i) that berberine exerts an anti-secretory action directly upon epithelial cells and ii) the mechanism of action may be at the level of blockade of K+ channels.

The intestinal epithelium serves as a dynamic barrier which, in the course of its normal function, maintains regulated uptake of nutrients and water at the same time as excluding potential pathogens. Enteropathies, including inflammatory bowel disease (IBD) result in, or perhaps even from, perturbed epithelial function. Despite the idiopathic nature of IBD, it is apparent that much of the pathophysiology, tissue damage and symptomatology of these disorders are due to inappropriate or exaggerated immune reactions.1 2 Cytokines are, inter alia, regulators of both innate and acquired immunity, so therefore it is not surprising that exaggerated effects of cytokines in acute or chronic inflammatory states may be due to unregulated production of pro-inflammatory cytokines or inadequate synthesis of anti-inflammatory cytokines. Indeed, increases in cytokine concentrations in models of enteric inflammation or IBD are well documented.3 Here, we provide an overview of cytokine regulation of epithelial ion transport and permeability, highlighting, where appropriate, the potential of the epithelium to modulate mucosal immune reactions and its own function. Relatively few studies have demonstrated cytokine effects on epithelial function in vivo or by examining intestinal tissue responses ex vivo.4 5 Several features contribute to difficulties with interpretation of such studies. Firstly, cytokines act in a coordinated interplay of often redundant, pleiotropic and occasionally opposing actions.6 Secondly, given the complex intercellular signalling that occurs within the gut,7cytokine effects may involve other, non-cytokine mediators.8-10 Finally, virtually every putative mediator of inflammation has been implicated in diseases of the intestine, representing at one time the academic pharmacologist’s dream and the therapist’s nightmare. The advent of epithelial cell lines, principally the tumour derived T84, Caco-2 and HT-29 cell lines, has offered opportunities to study influences of cytokines on epithelial function by taking a reductionist approach. These cell lines, when grown on …

BACKGROUND: Elements of the mucosal immune system may play an important part in regulating epithelial barrier function in the intestinal tract. Intraepithelial lymphocytes (IELs) represent a subtype of immunocyte which is strategically placed to regulate epithelial function at most mucosal sites. AIMS AND METHODS: An IEL derived cell line (SC1) was used to examine its effects on the model epithelium T84--a tumour derived cell line which retains the phenotype of colonic crypt cells. Transepithelial electrical resistance (TER) was used as a marker of epithelial integrity. RESULTS: Coculture of T84 cells with SC1 produced a significant fall in TER as did exposure of T84 monolayers to IEL derived supernatant. Recombinant interferon-gamma (rIFN gamma) also reduced TER in T84 monolayers. Cycloheximide prevented the effects of IEL supernatant and of rIFN gamma on TER. The fall in TER in response to rIFN gamma was attenuated by blocking antibodies, which did not alter the fall in resistance induced by IEL supernatant. Fractions of IEL supernatant, separated on the basis of size, evoked temporally distinct changes in TER. Ultrastructural studies support the hypothesis that the slow onset but severe fall in TER indicates catastrophic effects on the monolayer. The more rapid onset fall in TER was not associated with gross changes in monolayer morphology. Reduction of TER by IEL supernatant was not influenced by inhibitors of tyrosine phosphatase or of protein kinase C. Although herbimycin did reduce the rapid onset change in TER, the tyrosine kinase inhibitor genistein did not alter responses to IEL supernatant. CONCLUSIONS: Mucosal T cells may influence barrier function by a process involving new protein synthesis by epithelial cells. This model may have relevance in some inflammatory conditions of the gastrointestinal tract.

This review examines the anti-diarrhoeal actions of loperamide and berberine and shows evidence that there is much in common between the two agents in terms of their pharmacokinetics, an anti-motility action on intestinal smooth muscle and an anti-secretory effect on intestinal epithelia. Novel combined in vitro flux and short-circuit current data is presented illustrating that in normal rat intestinal epithelial tissue both agents require access to the basolateral side of the intestinal epithelium in order to inhibit effect on stimulated electrogenic chloride secretion, an effect most likely mediated by interaction with a potassium channel. Permeation and associated anti-secretory activity of luminally-applied berberine and loperamide, although normally poor, was permitted by the tight junction opener, cytochalasin D. There is a possibility that increased epithelial cell permeability, resulting from the loss of cell integrity occurring in conditions of secretory diarrhoea, can contribute to luminal effectiveness of anti-secretory/anti-motility agents.

Consumption of Vibrio parahaemolyticus via contaminated shellfish results in inflammatory gastroenteritis characterised by severe diarrhoea, nausea and stomach cramps. This study investigated the translocation of V. parahaemolyticus across a Peyer's patch M cell-like Caco-2/Raji B co-culture model system, as M cells represent a primary site of infection for many pathogenic bacteria. Vibrio parahaemolyticus translocated across co-culture monolayers in higher numbers as compared to Caco-2 monolayers. Moreover, the bacteria induced a greater disruption of the transepithelial resistance in M cell-like co-cultures than in Caco-2 monocultures. Virulence factors associated with this pathogen include two type three secretion systems (TTSS-1 and TTSS-2). TTSS-1 had no effect on translocation efficiency, with TTSS-2 exhibiting a modest enhancing effect. ERK activity was required for optimal translocation 1 h postinfection, however, neither ERK nor the JNK and p38 MAPK were required at 2 h pi. Additionally, TER disruption in response to bacterial infection occurred independently of the TTSS and MAPK activation. It was concluded that V. parahaemolyticus causes TER disruption of M cell-like co-cultures and translocates in high numbers across the M cell-like co-culture monolayer. These data implicate M cells as important sites for V. parahaemolyticus invasion across the intestinal epithelium during infection.

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.

Whey protein hydrolysates (WPHs) represent novel antidiabetic agents that affect glycemia in animals and humans, but little is known about their insulinotropic effects. The effects of a WPH were analyzed in vitro on acute glucose-induced insulin secretion in pancreatic BRIN-BD11 β cells. WPH permeability across Caco-2 cell monolayers was determined in a 2-tiered intestinal model. WPH effects on insulin resistance were studied in vivo following an 8-wk oral ingestion (100 mg/kg body weight) by ob/ob (OB-WPH) and wild-type mice (WT-WPH) compared with vehicle control (OB and WT groups) using a 2 × 2 factorial design, genotype × treatment. BRIN-BD11 cells showed a robust and reproducible dose-dependent insulinotropic effect of WPH (from 0.01 to 5.00 g/L). WPH bioactive constituents were permeable across Caco-2 cell monolayers. In the OB-WPH and WT-WPH groups, WPH administration improved glucose clearance after a glucose challenge (2 g/kg body weight), as indicated by differences in the area under curves (AUCs) (P ≤ 0.05). The basal plasma glucose concentration was not affected by WPH treatment in either genotype. The plasma insulin concentration was lower in the OB-WPH than in the OB group (P ≤ 0.005) but was similar between the WT and WT-WPH groups; the interaction genotype × treatment was significant (P ≤ 0.005). Insulin release from pancreatic islets isolated from the OB-WPH group was greater (P ≤ 0.005) than that from the OB group but did not differ between the WT-WPH and WT groups; the interaction genotype × treatment was not significant. In conclusion, an 8-wk oral administration of WPH improved blood glucose clearance, reduced hyperinsulinemia, and restored the pancreatic islet capacity to secrete insulin in response to glucose in ob/ob mice. Hence, it may be useful in diabetes management.

Author has checked copyright SB. 13/6/2013

1. The effects of the alkaloid berberine on basal and stimulated ion transport were investigated in voltage-clamped rat colonic epithelia. 2. Berberine (100-500 microM) reduced basal short circuit current (SCC) when applied basolaterally but not when applied apically. 3. SCC responses to mast cell activation by anti-rat IgE were significantly attenuated in the presence of berberine. 4. Berberine, applied to the basolateral bathing solution, also reduced SCC responses to the following agents which stimulate chloride secretion in rat colon: carbachol, forskolin, sodium nitroprusside, dibutyryl cyclic-AMP, heat-stable E. coli enterotoxin, 8-bromo-cyclic GMP and thapsigargin. Calcium mediated ion transport responses appear to be more sensitive to berberine inhibition than those which are cyclic GMP-mediated, which in turn are more sensitive than cyclic AMP-mediated responses. 5. Berberine added apically was without effect upon forskolin-stimulated ion transport. Cytochalasin D treatment of the lumenal surface of rat colon conferred apical-side sensitivity to berberine. 6. Berberine (at concentrations up to 500 microM) was without effect on generation of cyclic AMP by forskolin or on generation of cyclic GMP by sodium nitroprusside in isolated mucosal segments. Protein kinase A activity stimulated by dibutyryl cyclic AMP was unaffected by berberine (at concentrations up to 500 microM). 7. The precise mechanism of action of berberine remains to be elucidated. However, its site of action appears to be distal to second messenger production and may be at a level common to all stimuli of colonic chloride secretion.

Histamine, added to the basolateral side of voltage clamped human colon in vitro, induced a rapid onset, transient inward short circuit current which was concentration dependent over the range 0.01-3 mM. This response was largely due to electrogenic chloride section since it was virtually abolished by bumetanide or by chloride replacement in the bathing solutions. Responses were unaffected by amiloride or acetazolamide. Neither the histamine H2 receptor agonist dimaprit (1 mM) nor the histamine H3 receptor agonist S-(+)-alpha-methyl histamine (1 mM) altered short circuit current. Responses to histamine were significantly reduced by the histamine H1 receptor antagonist mepyramine (1-10 microM) but not altered by the histamine H2 receptor antagonist cimetidine (100 microM) or by the histamine H3 receptor antagonist thioperamide (1 microM). Short circuit current responses to histamine were not altered by tetrodotoxin (1 microM). Piroxicam (10 microM) and nordihydroguaiaretic acid (100 microM) were without effect when used individually but significantly reduced responses to histamine when used simultaneously. These results indicate that histamine stimulates chloride secretion across human colonic epithelium by a mechanism which is mediated exclusively via histamine H1 receptors. This action does not involve intrinsic nerves but appears to be dependent upon eicosanoid synthesis.

Acetylcholine receptor agonists and antagonists were used in a pharmacological analysis to identify which muscarinic receptor(s) may be involved in cholinergic regulation of Cl- secretion across rat colonic mucosa in vitro. A comparative ligand binding analysis for each of the antagonists was carried out in parallel. Both studies elicited identical rank order potencies (atropine > or = 4-diphenyl-acetoxy-N-piperidine methiodide (4-DAMP) > pirenzepine > 11-[[2[(diethylamino)methyl]-1-pipiridinyl]acetyl[5,11- dihydro-6H-pyrido[2,3-b]]1,4]benzodiazepine-6-one (AF-DX 116). Cholinomimetic-induced Cl- secretion was predominantly mediated by activation of muscarinic receptors in rat isolated colonic mucosa, with only a modest contribution from nicotinic receptors. Short circuit current responses evoked by the selective muscarinic M1 receptor agonist 4-[[(3-chlorophenyl)amino]carbonyl]-N,N,N-trimethyl-2-butyn-1-a minium chloride (McN-A-343) suggest that this receptor subtype, which is thought to be neuronally sited, also plays a minor role in regulation of intestinal ion transport. The principal epithelial cell receptors responsible for acetylcholine receptor-mediated Cl- secretion appear to belong to the M3 class.

Mucus within the cervical canal represents a hormonally regulated barrier that reconciles the need to exclude the vaginal microflora from the uterus during progesterone dominance, while permitting sperm transport at estrus. Its characteristics change during the estrous cycle to facilitate these competing functional requirements. Hydrated mucin glycoproteins synthesized by the endocervical epithelium form the molecular scaffold of this mucus. This study uses the bovine cervix as a model to examine functional groups of genes related to mucin biosynthesis and mucus production over the periestrous period when functional changes in cervical barrier function are most prominent. Cervical tissue samples were collected from 30 estrus synchronized beef heifers. Animals were slaughtered in groups starting 12 h after the withdrawal of intravaginal progesterone releasing devices (controlled internal drug releases) until 7 days postonset of estrus (luteal phase). Subsequent groupings represented proestrus, early estrus, late estrus, metestrus, and finally the early luteal phase. Tissues were submitted to next generation RNA-seq transcriptome analysis. We identified 114 genes associated with biosynthesis and intracellular transport of mucins, and postsecretory modifications of cervical; 53 of these genes showed at least a twofold change in one or more experimental group in relation to onset of estrus, and the differences between groups were significant ( P < 0.05). The majority of these genes showed the greatest alteration in their expression in the 48 h postestrus and luteal phase groups.

Emerging drug discovery technologies are helping us to break from the traditional simplistic cycle of animal experimentation for human applications with “spin off” benefits for veterinary medicine. A more coordinated effort can develop synergies. In this chapter we attempt to profile how those technologies harnessed independently for human or veterinary medicine have related features. We discuss shared approaches and requirements that are reaping benefits for both human and veterinary patients.

Zinc's usefulness in the treatment of diarrhoea is well established as an addition to oral rehydration. Mechanisms of action of zinc have been explored in intestinal epithelia from rodents and in cell lines. The aim was to examine how zinc alters ion transport and signal transduction in human colon in vitro. Voltage clamped colonic sheets obtained at the time of surgical resection were used to quantify ion transport responses to established secretagogues. Nystatin permeabilisation was used to study basolaterally-sited ion channels. Direct actions of zinc were determined using preparations of colonic crypts isolated from human mucosal sheets. Electrophysiological measurements revealed zinc to be an inhibitor of electrogenic ion transport stimulated by forskolin, PGE(2), histamine and carbachol in isolated human colonic epithelium. Basolateral addition of zinc sulphate had no direct effect on the epithelium. To further outline the mechanism of action, levels of secondary intracellular messengers (3', 5'-cyclic adenosine monophosphate; cAMP) were determined in isolated colonic crypts, and were found to be reduced by zinc sulphate. Finally, indirect evidence from nystatin-permeabilised mucosae further suggested that zinc inhibits basolateral K(+) channels, which are critical for transepithelial Cl(-) secretion linked to water flux. Anti-secretory, and therefore anti-diarrhoeal, actions of exogenous zinc are due, at least in part, to direct basolateral epithelial K(+) channel inhibition.

The gastrointestinal lumen is directly exposed to dietary contaminants, including patulin, a mycotoxin produced by moulds. Patulin is known to increase permeability across intestinal Caco-2 monolayers. This study aimed to determine the effect of patulin on permeability, ion transport and morphology in isolated rat colonic mucosae. Mucosal sheets were mounted in Ussing chambers and voltage clamped. Apical addition of patulin (100–500 μM) rapidly reduced transepithelial electrical resistance (TEER) and increased permeability to [ 14 C] mannitol (2.9-fold). Patulin also inhibited carbachol-induced electrogenic chloride secretion and histological evidence of mucosal damage was observed. To examine potential mechanisms of action of patulin on colonic epithelial cells, high-content analysis of Caco-2 cells was performed and this novel, quantitative fluorescence-based approach confirmed its cytotoxic effects. With regard to time course, the cytotoxicity determined by high content analysis took longer than the almost immediate reduction of electrical resistance in isolated mucosal sheets. These data indicate patulin is not only cytotoxic to enterocytes but also has the capacity to directly alter permeability and ion transport in intact intestinal mucosae. These data corroborate and extend findings in intestinal cell culture monolayers, and further suggest that safety limits on consumption of patulin may be warranted.

Nuclear receptors are of integral importance in carcinogenesis. Manipulation of classic ligand-activated nuclear receptors, such as estrogen receptor blockade in breast cancer, is an important established cancer therapy. Orphan nuclear receptors, such as nuclear family 4 subgroup A (NR4A) receptors, have no known natural ligand(s). These elusive receptors are increasingly recognized as molecular switches in cell survival and a molecular link between inflammation and cancer. NR4A receptors act as transcription factors, altering expression of downstream genes in apoptosis (Fas-ligand, TRAIL), proliferation, DNA repair, metabolism, cell migration, inflammation (interleukin-8), and angiogenesis (VEGF). NR4A receptors are modulated by multiple cell-signaling pathways, including protein kinase A/CREB, NF-κB, phosphoinositide 3-kinase/AKT, c-jun-NH2-kinase, Wnt, and mitogen-activated protein kinase pathways. NR4A receptor effects are context and tissue specific, influenced by their levels of expression, posttranslational modification, and interaction with other transcription factors (RXR, PPAR-ϒ). The subcellular location of NR4A “nuclear receptors” is also important functionally; novel roles have been described in the cytoplasm where NR4A proteins act both indirectly and directly on the mitochondria to promote apoptosis via Bcl-2. NR4A receptors are implicated in a wide variety of malignancies, including breast, lung, colon, bladder, and prostate cancer; glioblastoma multiforme; sarcoma; and acute and/or chronic myeloid leukemia. NR4A receptors modulate response to conventional chemotherapy and represent an exciting frontier for chemotherapeutic intervention, as novel agents targeting NR4A receptors have now been developed. This review provides a concise clinical overview of current knowledge of NR4A signaling in cancer and the potential for therapeutic manipulation. Clin Cancer Res; 18(12); 3223–8. ©2012 AACR.

Objectives Zinc is a useful addition to oral rehydration therapy for acute diarrhoea. We have assessed the mechanism of its epithelial antisecretory action when intestinal epithelial tight junctions were pharmacologically opened. Methods Rat isolated ileal and colonic mucosae were mounted in Ussing chambers and exposed to ZnSO4 (Zn2+) in the presence of secretagogues and inhibition of short circuit current (Isc) was measured. Key findings Pre-incubation with basolateral but not apical Zn2+ reduced Isc stimulated by forskolin, carbachol and A23187. In the presence of the tight junction-opener, cytochalasin D, antisecretory effects of apically-applied Zn2+ were enabled in colon and ileum. The apparent permeability coefficient (Papp) of Zn2+ was increased 1.4- and 2.4-fold across rat ileum and colon, respectively, by cytochalasin D. Basolateral addition of Zn2+ also reduced the Isc stimulated by nystatin in rat colon, confirming K channel inhibition. In comparison with other inhibitors, Zn2+ was a relatively weak blocker of basolateral KATP and K Ca2+ channels. Exposure of ileum and colon to Zn2+ for 60 min had minimal effects on epithelial histology. Conclusions Antisecretory effects of Zn2+ on intestinal epithelia arose in part through nonselective blockade of basolateral K channels, which was enabled when tight junctions were open.

A number of procedures or interventions which activate electrogenic ion transport in mammalian intestine are reviewed. Using in vitro models it is possible to demonstrate direct or indirect pathways to stimulate chloride secretion. Such activation, in vivo, would change the gut from a state of net water absorption to one of fluid secretion. The movement of water is driven by electrical and osmotic gradients set up as a consequence of opening regulated ion channels in epithelial cells. Secretagogues may govern epithelial intracellular second messenger pathways to regulate ion channel activity directly or by activation of membrane bound receptors on the surface of epithelial cells. In health or disease secretagogues may be derived from cells within the attendant lamina propria of the intestinal mucosa. Pharmacological techniques may be employed to determine which mediators contribute to indirect stimulation of electrogenic ion transport by activation of neurons or of immunocytes (mast cells or phagocytes). Dissection and reconstruction of models of intestinal hypersensitivity reactions show that neuro-immune networks which regulate intestinal ion transport appear to be complex, functionally integrated systems. Analysis of such interactions may identify cellular or humoral targets with which to examine novel diagnostic, preventative or therapeutic strategies with regard to intestinal diseases.

The intestinal epithelial layer can switch from a net absorptive state to one of net secretion in the presence of luminal toxins and pathogens. This suggests an innate defence role for regulated secretion of mucus, electrolytes and water. We hypothesised that chloride-led fluid secretion across the mucus-covered intestinal epithelium alters barrier properties by influencing the overlying mucous-gel layer.We demonstrated that chloride-led disruption of the epithelial-associated mucus-gel covering HT29-MTX-E12 (E12) human colonic epithelial monolayers, a goblet-cell like line derived from parental HT29 cells, resulted in reduction of associated mucus as well as a reduction in mucous-gel density and barrier properties. Changes in epithelial secretory state were accompanied by increased water transport, and the resulting loss of gel integrity reduced Salmonella typhimurium invasion of epithelia in both E12 monolayers and of isolated rat ileal mucosae. However, neither chloride secretion nor mucus disruption altered numbers of adhering bacteria.These data suggest a role for chloride led fluid secretion in the shedding of the adherent mucous-gel layer, possibly as a rate-limiting innate defence mechanism, and offer evidence for "enteric tears" in intestinal host defence.

Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development.Wedescribe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic-helical peptide, melittin, and selected structural analogs. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity. Irish Research Council for Science, Engineering and Technology Science Foundation Ireland Merrion Pharmaceuticals NYP: http://www.sciencedirect.com/science/article/pii/S0196978111002300 - AV 11/07/2011

The calcium-sensing receptor (CaSR) is expressed abundantly in normal colonic epithelium and lost in colon cancer, but its exact role on a molecular level and within the carcinogenesis pathway is yet to be described. Epidemiologic studies show that inadequate dietary calcium predisposes to colon cancer; this may be due to the ability of calcium to bind and upregulate the CaSR. Loss of CaSR expression does not seem to be an early event in carcinogenesis; indeed it is associated with late stage, poorly differentiated, chemo-resistant tumors. Induction of CaSR expression in neoplastic colonocytes arrests tumor progression and deems tumors more sensitive to chemotherapy; hence CaSR may be an important target in colon cancer treatment. The CaSR has a complex role in colon cancer; however, more investigation is required on a molecular level to clarify its exact function in carcinogenesis. This review describes the mechanisms by which the CaSR is currently implicated in colon cancer and identifies areas where further study is needed.

Background 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. Results 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. Conclusions 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.

The process of urea nitrogen salvaging plays a vital role in the symbiotic relationship between mammals and their intestinal bacteria. The first step in this process requires the movement of urea from the mammalian bloodstream into the gastrointestinal tract lumen via specialized proteins known as facilitative urea transporters. In this study, we examined both transepithelial urea fluxes and urea transporter protein abundance along the length of the rat gastrointestinal tract. Urea flux experiments that used rat gastrointestinal tissues showed significantly higher transepithelial urea transport was present in caecum and proximal colon (P < 0.01, n = 8, analysis of variance [ANOVA]). This large urea flux was significantly inhibited by 1,3,dimethylurea (P < 0.001, n = 8, ANOVA) and thiourea (P < 0.05, n = 6, unpaired t-test), both known blockers of facilitative urea transporters. Immunoblotting analysis failed to detect any UT-A protein within rat gastrointestinal tissue protein samples. In contrast, a 30-kDa UT-B1 protein was strongly detected in both caecum and proximal colon samples at significantly higher levels compared to the rest of the gastrointestinal tract (P < 0.01, n = 4, ANOVA). We therefore concluded that UT-B1 mediates the transepithelial movement of urea that occurs in specific distal regions of the rat gastrointestinal tract.

Delivery of biologically active agents to animals is often perceived to be the poor relation of human drug delivery. Yet this field has a long and successful history of species-specific device and formulation development, ranging from simple approaches and devices used in production animals to more sophisticated formulations and approaches for a wide range of species. While several technologies using biodegradable polymers have been successfully marketed in a range of veterinary and human products, the transfer of delivery technologies has not been similarly applied across species. This may be due to a combination of specific technical requirements for use of devices in different species, inter-species pharmacokinetic, pharmacodynamic and physiological differences, and distinct market drivers for drug classes used in companion and food-producing animals. This chapter reviews selected commercialised and research-based parenteral and non-parenteral veterinary drug delivery technologies in selected domestic species. Emphasis is also placed on the impact of endogenous drug transporters on drug distribution characteristics in different species. In vitro models used to investigate carrier-dependent transport are reviewed. Species-specific expression of transporters in several tissues can account for inter-animal or inter-species pharmacokinetic variability, lack of predictability of drug efficacy, and potential drug-drug interactions.

Delivery of biologically active agents to animals is often perceived to be the poor relation of human drug delivery. Yet this field has a long and successful history of species-specific device and formulation development, ranging from simple approaches and devices used in production animals to more sophisticated formulations and approaches for a wide range of species. While several technologies using biodegradable polymers have been successfully marketed in a range of veterinary and human products, the transfer of delivery technologies has not been similarly applied across species. This may be due to a combination of specific technical requirements for use of devices in different species, inter-species pharmacokinetic, pharmacodynamic and physiological differences, and distinct market drivers for drug classes used in companion and food-producing animals. This chapter reviews selected commercialised and research-based parenteral and non-parenteral veterinary drug delivery technologies in selected domestic species. Emphasis is also placed on the impact of endogenous drug transporters on drug distribution characteristics in different species. In vitro models used to investigate carrier-dependent transport are reviewed. Species-specific expression of transporters in several tissues can account for inter-animal or inter-species pharmacokinetic variability, lack of predictability of drug efficacy, and potential drug–drug interactions.

Facilitative UT-B urea transporters enable the passage of urea across cell membranes. Gastrointestinal urea transporters are thought to play a significant role in the urea nitrogen salvaging process that occurs between mammalian hosts and their gut bacteria. This study investigated the expression of UT-B urea transporters in different segments of human colon. Immunoblot analysis showed that human colon expressed a 35-kDa glycosylated UT-B protein in the colonic mucosa. The 35-kDa UT-B transporter was predominantly located in plasma membrane-enriched samples ( P < 0.001; n = 6), and its expression was greater in the ascending colon compared with the descending colon ( P < 0.01; n = 3). At the cellular level, UT-B transporters were located throughout colonocytes situated in the upper portion of the colonic crypts. Bidirectional trans-epithelial urea transport was significantly greater in the ascending colon than the descending colon ( P < 0.05; n = 6). In addition, the facilitative urea transporter inhibitor 1,3,dimethylurea significantly reduced urea transport in the ascending colon ( P < 0.05; n = 6) but had no effect in the descending colon (NS; n = 6). These results illustrate differential protein abundance of functional UT-B protein in different sections of the human colon, strongly correlating to regions that contain the largest populations of intestinal bacteria. This study suggests an important role for UT-B urea transporters in maintaining the symbiotic relationship between humans and their gut bacteria.

The aim of this study is to evaluate the many practical formats that support the first-year veterinary curriculum. These practical classes are diverse in content and style. They include laboratory-based formats, classes involving live animals and cadavers, classes conducted using computer-aided learning tools, study groups, and information technology training. This preliminary study examines ratings for these practical classes, but also relates these ratings to students’ approaches to study with the aim of understanding how a deep learning approach manifests itself in the practical setting. The diverse behaviors and attitudes to practical classes are also evaluated in the light of the approaches to study. A questionnaire that evaluated (1) a total of 24 practical classes, (2) the 52-item Approaches to Study Inventory, and (3) 13 behaviors within and attitudes to practical classes was distributed to 69 first-year veterinary students in their final term. Practical classes that involved live animals and cadavers were rated most positively by this group of students. These ratings, however, did not correlate significantly with the deep or surface learning score. The majority of practical classes where the ratings were found to be associated with deep and surface learning were laboratory-based, although overall these practical classes tended to be rated lower than those involving animals. Ratings did not correlate significantly with the strategic approach. A number of behaviors and attitudes to practical classes were also found to be positively and significantly (p = 0.0001) associated with the deep learning approach. This preliminary study indicates that this cohort of veterinary students has an overall positive perception of practical classes that permit contact with live animals or cadavers. Although the perception of laboratory-type practical classes was lower overall, the ratings for these practical classes appeared to be influenced by their deep and surface learning scores. We hypothesize that these approaches influence student engagement with and appreciation of laboratory-type classes, but not of classes involving live animals or cadavers. This would suggest that a different “type” of learning is taking place in these different contexts.

International audience; The concept that E2 exerts an effect on the gastrointestinal tract is not new and its actions on intestinal mucosa have been investigated for at least three decades. An attempt to consolidate results of these investigations generates more questions than answers, thus suggesting that many unexplored avenues remain and that the full capabilities of this steroid hormone are far from understood. Evidence of its role in esophageal, gastric and gallbladder cancers is confusing and often equivocal. The most compelling evidence regards the protective role conferred by estrogen (or perhaps ERβ) against the development and proliferation of colon cancer. Not only has the effect been described but many mechanisms of action have been explored. It is likely that, along with surgery, chemotherapy and radiotherapy, hormonal manipulation will play an integral role in colon cancer management in the very near future.