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A novel proprietary formulation, ViruSAL, has previously been demonstrated to inhibit diverse enveloped viral infections in vitro and in vivo. We evaluated the ability of ViruSAL to inhibit SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infectivity, using physiologically relevant models of the human bronchial epithelium, to model early infection of the upper respiratory tract. ViruSAL potently inhibited SARS-CoV-2 infection of human bronchial epithelial cells cultured as an air–liquid interface (ALI) model, in a concentration- and time-dependent manner. Viral infection was completely inhibited when ViruSAL was added to bronchial airway models prior to infection. Importantly, ViruSAL also inhibited viral infection when added to ALI models post-infection. No evidence of cellular toxicity was detected in ViruSAL-treated cells at concentrations that completely abrogated viral infectivity. Moreover, intranasal instillation of ViruSAL to a rat model did not result in any toxicity or pathological changes. Together these findings highlight the potential for ViruSAL as a novel and potent antiviral for use within clinical and prophylactic settings.

A novel proprietary formulation, ViruSAL, has previously been demonstrated to inhibit diverse enveloped viral infections in vitro and in vivo. We evaluated the ability of ViruSAL to inhibit SARS-CoV-2 infectivity, using physiologically relevant models of the human bronchial epithelium, to model early infection of the upper respiratory tract. ViruSAL potently inhibited SARS-CoV-2 infection of human bronchial epithelial cells cultured as an air-liquid interface (ALI) model, in a concentration- and time-dependent manner. Viral infection was completely inhibited when ViruSAL was added to bronchial airway models prior to infection. Importantly, ViruSAL also inhibited viral infection when added to ALI models post-infection. No evidence of in vitro cellular toxicity was detected in ViruSAL treated cells at concentrations that completely abrogated viral infectivity. Moreover, intranasal instillation of ViruSAL to a rat model did not result in any toxicity or pathological changes. Together these findings highlight the potential for ViruSAL as a novel and potent antiviral for use within clinical and prophylactic settings.

Some free fatty acids derived from milk and vegetable oils are known to have potent antiviral and antibacterial properties. However, therapeutic applications of short- to medium-chain fatty acids are limited by physical characteristics such as immiscibility in aqueous solutions. We evaluated a novel proprietary formulation based on an emulsion of short-chain caprylic acid, ViroSAL, for its ability to inhibit a range of viral infections in vitro and in vivo. In vitro, ViroSAL inhibited the enveloped viruses Epstein–Barr, measles, herpes simplex, Zika and orf parapoxvirus, together with Ebola, Lassa, vesicular stomatitis and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) pseudoviruses, in a concentration- and time-dependent manner. Evaluation of the components of ViroSAL revealed that caprylic acid was the main antiviral component; however, the ViroSAL formulation significantly inhibited viral entry compared with caprylic acid alone. In vivo, ViroSAL significantly inhibited Zika and Semliki Forest virus replication in mice following the inoculation of these viruses into mosquito bite sites. In agreement with studies investigating other free fatty acids, ViroSAL had no effect on norovirus, a non-enveloped virus, indicating that its mechanism of action may be surfactant disruption of the viral envelope. We have identified a novel antiviral formulation that is of great interest for the prevention and/or treatment of a broad range of enveloped viruses, particularly those of the skin and mucosal surfaces.

Oral ingestion remains the preferred method of drug administration. Its disadvantages with regard to biologic agents reflect the fact that systemic bioavailability is influenced by the chemical properties of the drug and by the gastrointestinal physiological and pathologic features of the patient. Unfortunately, many candidate biologic agents are labile or cannot readily cross the gastrointestinal epithelium. 6 month embargo - AC

Liver inflammation is a common extraintestinal manifestation in inflammatory bowel disease (IBD), yet, the mechanisms driving gut-liver axis inflammation remain poorly understood. IBD leads to a breakdown in the integrity of the intestinal barrier causing an increase in portal and systemic gut-derived antigens, which challenge the liver. Here, we examined the role of platelet activating factor receptor (PAFR) in colitis-associated liver damage using dextran sulfate sodium (DSS) and anti-CD40-induced colitis models. Both DSS and anti-CD40 models exhibited liver inflammation associated with colitis. Colitis reduced global PAFR protein expression in mouse livers causing an exclusive re-localization of PAFR to the portal triad. The global decrease in liver PAFR was associated with increased sirtuin 1 while relocalized PAFR expression was limited to Kupffer cells (KCs) and co-localized with toll-like receptor 4. DSS activated the NLRP3-inflammasome and increased interleukin (IL)-1β in the liver. Antagonism of PAFR amplified the inflammasome response by increasing NLRP3, caspase-1, and IL-1β protein levels in the liver. LPS also increased NLRP3 response in human hepatocytes, however, overexpression of PAFR restored the levels of NLPR3 and caspase-1 proteins. Interestingly, KCs depletion also increased IL-1β protein in mouse liver after DSS challenge. These data suggest a protective role for PAFR-expressing KCs during colitis and that regulation of PAFR is important for gut-liver axis homeostasis.

Intestinal permeation enhancers (PEs) offer an attractive strategy to enable oral peptide administration. However, optimal presentation of peptide and PE from solid-dosage forms is offset by slow dissolution rates in the small intestine, which reduces the likelihood that the PE can reach the threshold concentration for sufficient permeability enhancement. The purpose of this study was to design a PE-based liquid dispersion that can improve intestinal permeation of macromolecules across Caco-2 monolayers and isolated rat/human intestinal mucosae mounted in Ussing chambers. An enhancer screen in monolayers based on permeability (TEER, Papp [14C]-mannitol) and cytotoxicity (MTT assay) initially identified methyl 10-hydroxydecanoate (10-OHC10CH3) as a candidate. 10-OHC10CH3 (20 mM) increased the Papp of fluorescent dextran of 4 kDa (FD4) (167-fold), 10 kDa (FD10) (429-fold), and 40 kDa (FD40) (520-fold) across monolayers. Blends of 10-OHC10CH3 with low molecular weight PEGs (0.2–1 kDa) formed liquid dispersions in which enhancement capacity across monolayers of 10-OHC10CH3 was increased over 10-OHC10CH3 alone in the order PEG200 < PEG400 < PEG600 < PEG1000. Finally, a 1:5 ratio of 10-OHC10CH3 (10–20 mM)/PEG600 (50–100 mM) increased the Papp of [14C]-mannitol across rat and human intestinal mucosae. This study highlights the potential future role for non-aqueous, PE-based liquid dispersions in oral delivery of macromolecules.

Bacterially derived short chain fatty acids (SCFAs), such as butyrate, are vital in maintaining the symbiotic relationship that exists between humans and their gastrointestinal microbial populations. A key step in this process is the transport of SCFAs across colonic epithelial cells via MCT1 transporters. This study investigated MCT1 protein abundance in various human intestinal tissues. Initial RT-PCR analysis confirmed the expected MCT1 RNA expression pattern of colon > small intestine > stomach. Using surgical resection samples, immunoblot analysis detected higher abundance of a 45 kDa MCT1 protein in colonic tissue compared to ileum tissue (P

The symbiotic relationship between humans and their intestinal microbiomeis supported by urea nitrogen salvaging. Previous studies have shown thatcolonic UT-B urea transporters play a significant role in this important physi-ological process. This current study investigated UT-A and UT-B urea trans-porter expression along the human gastrointestinal tract. Initial end-pointPCR experiments determined that UT-A RNA was predominantly expressedin the small intestine, while UT-B RNA was expressed in stomach, small intes-tine, and colon. Using western blotting experiments, a strong 40–60 kDa UT-B signal was found to be abundant in both ileum and colon. Importantly, thissignal was deglycosylated by PNGaseF enzyme treatment to a core protein of30 kDa in both tissues. Further immunolocalization studies revealed UT-Btransporter proteins were present at the apical membrane of the villi in theileum, but predominantly at the basolateral membrane of the colonic surfaceepithelial cells. Finally, a blind scoring immunolocalization study suggestedthat there was no significant difference in UT-B abundance throughout thecolon (NS, ANOVA,N=5–21). In conclusion, this current study suggestedUT-B to be the main human intestinal urea transporter. Intriguingly, thesedata suggested that the same UT-B isoform was present in all intestinalepithelial cells, but that the precise cellular location varied. Science Foundation Ireland

SUMMARYFasciola hepatica is a parasitic trematode that causes serious losses to livestock producers, and also zoonotic disease. The limitations of chemotherapy for the control of fasciolosis have led to significant interest in the development of vaccines to protect cattle and sheep from infection. However, relatively few studies have concentrated on the mechanisms of invasion of the gut by newly excysted juvenile liver flukes (NEJ) and the host response triggered by this event. The aim of this work was to develop an in vitro model to study invasion by NEJ, while also reducing the requirement for challenge infections of experimental animals. Fasciola hepatica metacercariae were excysted in vitro and placed into compartments containing rat distal jejunal sheets. Variations in incubation medium, chamber size and incubation temperature were used to identify optimal conditions for NEJ migration across the gut. Histological examination showed increased migration until 120 min post-incubation. The use of RPMI, without gassing at 39 °C, as the incubation medium was found to be optimal, with 40·5% of NEJ migrating after 150 min. This study describes a readily-reproducible method for studying the migration of F. hepatica NEJ within the definitive host. It will be useful for identifying potential drug and vaccine targets.

Background: A novel emulsion with efficacy as an agent for eliminating biofilms was selected. The aim of this study was to examine efficacy and effect of a formulation of ML:8 against commensal bacteria harvested from ex vivo human colonic tissues. Methods: Mucosal sheets, obtained at the time of surgery, were exposed for 2 minutes to one of four solutions: Krebs-Hensleit (KH) solution, saline (NaCl; 0.9%), povidone iodine (1%), or ML:8 (2%); n = 4. Lumenal surfaces were swabbed for culture under aerobic or anaerobic conditions. Following treatment, each sheet was mounted in Ussing chambers and voltage clamped. Tissues were challenged with carbachol. Permeability coefficient (Papp) was determined using mannitol fluxes. At the end of each experiment, tissues were examined histologically. Results: Similar colony forming units grew in aerobic and anaerobic conditions in both control and NaCl treated tissues. Iodine reduced and ML:8 virtually abolished viable bacteria. Basal electrophysiological parameters were not different between treatments. Transepithelial electrical resistance values did not differ between groups. All tissues responded to carbachol, although this was attenuated in iodine treated tissue. Papp values were slightly elevated in all treated tissues but this did not reach significance. Histopathological assessment revealed no overt damage to tissues. Conclusion: Brief exposure to ML:8 reduced culturable bacterial burden from human intestinal tissues harvested at the time of surgical resection. Such gnotobiotic tissues retain structural and functional integrity. This is a novel approach to reduce bacterial burden. Gastroenterol Res. 2015;8(5):247-252 doi: http://dx.doi.org/10.14740/gr675w

Colorectal cancer is one of the most common cancers worldwide, with an overall increased incidence annually. Despite improvements in treatment and surveillance, almost 50% develop recurrent and/or distant disease. Unknown cellular processes are the fundamental cause for treatment failure and metastatic disease. The interplay of chronic inflammation and carcinogenesis is well established. Recent work has highlighted the role of nuclear receptors and co-regulators in the inflammation to carcinogenesis process. Orphan nuclear receptors have been shown to be involved in numerous cellular processes, including both at a transcriptional and a non-genomic level. There is a significant emphasis to identify ligands that will interact and modify these nuclear receptors, with the long-term aim of developing novel pharmaceutical therapies. The identification of orphan nuclear receptor ligands will also help increase our current understanding of their role in cellular signaling, by enabling manipulation of these receptors. This review aims to provide a brief overview of some key orphan nuclear receptors which may be involved in colorectal cancer.

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 trinitrobenzene sulfonic acid 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 with 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 interleukin (IL)-1β, IL-6, and tumor necrosis factor (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 that the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis. © 2014 Society for Mucosal Immunology.

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.

The effect of a bovine whey protein concentrate partially digested with lipase to release free fatty acids (WPD) on growth and viability of Streptococcus mutans , and its adherence to hydroxylapatite (HA) in phosphate-buffered saline were examined. The effects of other whey mixtures and individual whey proteins, β-lactoglobulin (BLG), lactoferrin (LF), caseinoglycomacropeptide (CGMP) and α-lactalbumin were also investigated. Bacterial viability was assessed by incubation with whey protein and measuring colony-forming units. S. mutans binding was quantified using Syto-13 nucleic-acid binding dye and Alamar Blue, which measures cellular metabolic activity. WPD was bactericidal, and inhibited S. mutans growth and adhesion to HA. Other wheys inhibited S. mutans adhesion, particularly acid whey, as well as the parent compound for WPD, Carbelac 80, and a whey protein isolate. BLG, CGMP and LF inhibited S. mutans adhesion. A mixture containing 56% BLG and 20% CGMP was as effective an inhibitor of bacterial adhesion as WPD.

Studies investigating the activating and inhibitory actions of bioflavonoids on colonic function have yielded conflicting results. At low concentrations, flavonoids may stimulate chloride secretion while at higher concentrations they may have antisecretory actions in the colon. Naringenin (4',5,7-trihydroxyflavanone), found predominantly in citrus fruits, confers a protective effect against colorectal cancer and is purported to modulate secretory function in colonic cell lines. The aim of this study was to investigate the effects of naringenin on ion transport in rat and human colonic mucosae. Naringenin inhibited basal and stimulated chloride secretion in rat and human colonic mucosae mounted in Ussing chambers (IC(50) 330 μMol/L and 360 μMol/L respectively) and did not alter intracellular cAMP generation. Naringenin inhibited chloride secretion in MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide) loaded crypts stimulated with forskolin. In BCECF (2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein acetoxymethyl ester) loaded crypts, naringenin caused an intracellular acidification (ΔpH/min=0.05 ± 0.004) which was sensitive to the Na-K-Cl co-transporter (NKCC) inhibitor bumetanide. In addition, the antisecretory effect of naringenin was not inhibited by blockade of barium sensitive basolateral K(+) transporters or by inhibition of Na+/H(+) exchange by amiloride. We propose that the antisecretory action of naringenin is due to inhibition of basolateral NKCC1 in rat and human colon.

Background/Aims: Methods: 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. Results: 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 typhimuriuminvasion of epithelia in both E12 monolayers and of isolated rat ileal mucosae. However, neither chloride secretion nor mucus disruption altered numbers of adhering bacteria. Conclusion: 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.

Objective—To measure the expression of cyclooxygenase-2 (COX-2) mRNA in gastric biopsy specimens serially obtained from horses before, during, and after an 8-day intermittent feed-deprivation trial and to investigate the mucosal location of COX-2. Animals—9 mixed-breed horses for retrieval of gastric biopsy specimens and 16 additional horses for immunohistochemical analysis. Procedures—Gastric biopsy specimens were obtained from 6 horses; 3 of these horses and 3 more participated in an intermittent feed-deprivation trial 9 weeks later. A quantitative PCR assay was used to determine the amount of COX-2 mRNA in biopsy specimens from nonulcerated and ulcerated gastric mucosa. Immunohistochemical staining of specimens by use of a polyclonal anti–COX-2 antibody was performed on full-thickness postmortem gastric biopsy specimens. Results—COX-2 mRNA was expressed in all glandular gastric mucosal specimens but was only detectable in nonglandular mucosal specimens when ulceration was present or during ulcer healing. Positive staining for COX-2 was present in 12 of 14 nonulcerated glandular mucosal sections. Although such staining was weak or absent in nonulcerated nonglandular sections, stronger staining was evident in regenerating epithelium at the rims of erosions and ulcers. Conclusions and Clinical Relevance—COX-2 was constitutively present in equine glandular gastric mucosa, although its contribution to mucosal protection remains unclear. Our finding of COX-2 mRNA expression in ulcer margins during healing may support a role for the products of this enzyme in mucosal repair. The potential roles of COX-2 should be considered when COX-2–selective inhibitors are prescribed for horses with gastric ulcers. (Am J Vet Res 2010;71:1312–1320)

The effect of a partially lipase-digested whey protein concentrate (WPD) containing free fatty acids on the adherence of Salmonella enterica serovar Typhimurium to Caco-2 cells was investigated. A short concurrent exposure of the Caco-2 monolayers and WPD caused dose-dependent inhibition of bacterial adhesion. Pre-treatment of S. Typhimurium with WPD resulted in inhibition of adhesion equivalent to that with concurrent administration, whereas treatment with WPD did not significantly inhibit adhesion. WPD was not bactericidal towards S. Typhimurium at up to 50 mg ml −1 for up to 1 h, but was bacteriostatic over 12 h. High concentrations of WPD (>20 mg ml −1 ) were cytotoxic to newly-confluent cell monolayers, depending on the duration of exposure. Lactoferrin (LF), caseinoglycomacropeptide (CGMP), and gamma globulins all significantly inhibited S. Typhimurium adhesion.

Epidemiological studies have correlated consumption of dietary phytoestrogens with beneficial effects on colon, breast and prostate cancers. Genomic and non-genomic mechanisms are responsible for anti-carcinogenic effects but, until now, the effect on human colon was assumed to be passive and remote. No direct effect on human colonic smooth muscle has previously been described. Institutional research board approval was granted. Histologically normal colon was obtained from the proximal resection margin of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended under 1 g of tension in organ baths containing oxygenated Krebs solution at 37 °C. After an equilibration period, tissues were exposed to diarylpropionitrile (DPN) (ER β agonist) and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) (ER α agonist) or to the synthetic phytoestrogen compounds genistein ( n = 8), daidzein ( n = 8), fisetin ( n = 8) and quercetin ( n = 8) in the presence or absence of fulvestrant (oestrogen receptor antagonist). Mechanism of action was investigated by inhibition of downstream pathways. The cholinergic agonist carbachol was used to induce contractile activity. Tension was recorded isometrically. Phytoestrogens inhibit carbachol-induced colonic contractility. In keeping with a non-genomic, rapid onset direct action, the effect was within minutes, reversible and similar to previously described actions of 17 β oestradiol. No effect was seen in the presence of fulvestrant indicating receptor modulation. While the DPN exerted inhibitory effects, PPT did not. The effect appears to be reliant on a p38/mitogen activated protein kinase mediated induction of nitric oxide production in colonic smooth muscle. The present data set provides the first description of a direct effect of genistein, daidzein, fisetin and quercetin on human colonic smooth muscle. The presence of ER in colonic smooth muscle has been functionally proven and the β isoform appears to play a predominant role in exerting non-genomic effects.

Background and purpose: Prostaglandin F2α (PGF2α) is implicated in the pathogenesis of inflammatory bowel disease and colorectal cancer. This study investigates the effects of PGF2α on electrophysiological parameters in isolated human colonic mucosa. Experimental approach: Ion transport was measured as changes in short-circuit current across human colonic epithelia mounted in Ussing chambers. Colonic crypts were isolated by calcium chelation and cyclic adenosine monophosphate (cAMP) was measured by ELISA. Key Results: PGF2α stimulated chloride secretion in a concentration-dependent manner with an EC50 of 130 nM. The PGF2α induced increase in chloride secretion was inhibited by AL8810 (10 µM), a specific PGF2α receptor antagonist. In addition, PGF2α (1 µM) significantly increased levels of cAMP in isolated colonic crypts. Conclusions and implications: PGF2α stimulated chloride secretion in samples of human colon in vitro through a previously unrecognizd cAMP-mediated mechanism. These findings have implications for inflammatory states.

The effects of two absorption promoters, (sodium caprate (C(10)) and melittin), on intestinal permeability and viability were measured in intact rat and human colonic epithelia mounted in Ussing chambers. Apical-side addition of C(10) (10 mM) and melittin (10-50 microM) rapidly reduced the transepithelial electrical resistance (TEER) and increased the apparent permeability coefficient (Papp) of [(14)C]-mannitol and FITC-dextran-4 kDa (FD4) across colonic mucosae from both species. Effects of C(10) on flux were greater than those of melittin at the concentrations selected. C(10) irreversibly decreased TEER, but the effects of melittin were partially reversible. Enhanced permeability of polar sugars (0.18-70 kDa) in colonic mucosae with C(10) was accompanied by significant release of lactate dehydrogenase (LDH) from the luminal surface as well as by inhibition of electrogenic chloride secretion induced by the muscarinic agonist, carbachol (0.1-10 microM). Although melittin did not alter electrogenic chloride secretion in rat or human colonic mucosae, it caused leakage of LDH from rat tissue. Gross histology and electron microscopy of rat and human colonic mucosae demonstrated that each permeation enhancer can induce colonic epithelial damage at concentrations required to increase marker fluxes. C(10) led to more significant mucosal damage than melittin, characterised by sloughing and mucosal erosion. Overall, these results indicate that while C(10) and melittin increase transport of paracellular flux markers across isolated human and rat colonic mucosae in vitro, these effects are associated with some cytotoxicity.

While the concept of a role of estrogen in gastrointestinal (in particular, colonic) malignancy has generated excitement in recent years, no review has examined the role of this potent and omnipresent steroid hormone in physiological states or its contribution to the development of benign pathological processes. Understanding these effects (and mechanisms therein) may provide a platform for a deeper understanding of more complex disease processes.A literature search was conducted using the PubMed database and the search terms were "estrogen," "estrogen AND gastrointestinal tract," "estrogen AND colon," "estrogen AND esophagus," "estrogen AND small intestine," "estrogen AND stomach," "estrogen AND gallbladder," and "estrogen AND motility." Bibliographies of extracted studies were further cross-referenced. In all, 136 full-text articles were selected for review. A logical organ-based approach was taken to enable extraction of data of clinical relevance and meaningful interpretation thereof. Insight is provided into the hypotheses, theories, controversies, and contradictions generated over the last five decades by extensive investigation of estrogen in human, animal, and cell models using techniques as diverse as autoradiographic studies of baboons to human population analysis.Effects from esophagus through to the colon and rectum are summarized in this first concise collection of data pertaining to estrogenic actions in gastrointestinal health and disease. Mechanisms of these actions are discussed where possible. Undoubtedly, this hormone exerts many actions yet to be elucidated, and its potential therapeutic applications remain, as yet, largely unexplored.

The critical metabolic functions of the liver often eclipse any perception of its role as an immune organ. However, the liver as a mediator of systemic and local innate immunity and an important site of immune regulation is now an accepted concept. Complex repertoires of lymphoid and non-lymphoid cells are key to hepatic defense and immunoregulation. Hepatic cells of myeloid lineage include Kupffer cells and dendritic cells. Intrahepatic lymphocytes are distinct both in phenotype and function from their counterparts in any other organ and include both conventional (CD4+ and CD8+ alphabeta T cell receptor (TCR)+ T cells, B cells, natural killer (NK) cells) and nonconventional lymphoid cells (natural killer T (NKT) cells, gamma delta TCR+ T cells, CD4- CD8- T cells). Many hepatic T cells express the TCR at an intermediate level and the great majority of them either coexpress NK cell markers (NKT cells) or they are apoptosing peripheral T cells. The percentage of activated (CD69+) and memory (CD45RB low+) lymphocytes is much higher while naive (CD62L high) and resting T cells as well as B lymphocytes are underrepresented in the liver. The discovery of major populations of lymphoid cells in the liver that differ phenotypically, functionally and even perhaps developmentally from populations in other regions has been key to the evolving perception of the liver as a regulatory lymphoid organ. This chapter will focus on these populations and how they contribute to immune surveillance against malignant, infectious and autoimmune disease of the liver.

Background Classical effects of oestrogen involve activation of target genes after binding nuclear receptors. Oestrogenic effects too rapid for DNA transcription (non-genomic) are known to occur. The effect of oestrogen on colonic motility is unknown despite the prevalence of gastrointestinal symptoms in pregnant and premenopausal women. Methods Histologically normal colon was obtained from proximal resection margins of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended in organ baths under 1 g of tension. After equilibration, they were exposed to 17β-oestradiol (n = 8) or bovine serum albumin (BSA)-conjugated 17β-oestradiol (n = 8). Fulvestrant, an oestrogen receptor antagonist, was added to some baths (n = 8). Other strips were exposed to calphostin C or cycloheximide. Carbachol was added in increasing concentrations and contractile activity was recorded isometrically. Results Oestrogen inhibited colonic contractility (mean difference 19·7 per cent; n = 8, P < 0·001). In keeping with non-genomic, rapid-onset steroid action, the effect was apparent within minutes and reversible. It was observed with both 17β-oestradiol and BSA-conjugated oestrogen, and was not altered by cycloheximide. Effects were inhibited by fulvestrant, suggesting receptor mediation. Conclusion Oestrogen decreases contractility in human colonic smooth muscle by a non-genomic mechanism involving cell membrane coupling.

Background and purpose: Kinins are acknowledged as important regulators of intestinal function during inflammation; however, their effects on human intestinal ion transport have not been reported. Here, we used muscle-stripped human colonic tissue and cultured T84-cell monolayers to study bradykinin (BK) actions on human intestinal ion transport. Experimental approach: Ion transport was measured as changes in short-circuit current (Isc) across colonic epithelia mounted in Ussing chambers. Key results: In intact tissue, there was a distinct polarity to BK-elicited Isc responses. Whereas basolateral BK stimulated sustained responses (EC50=0.5±0.1 μM), those to apical BK were more rapid and transient (EC50=4.1±1.2 nM). In T84 cells, responses to both apical and basolateral BK were similar to those seen upon apical addition to intact tissues. Cross-desensitization between apical and basolateral domains was not observed. BK-induced responses were largely due to Cl− secretion as shown by their sensitivity to bumetanide and removal of Cl− from the bathing solution. Studies using selective agonists and antagonists indicate responses to BK are mediated by B2 receptors. Finally, responses to basolateral BK in intact tissues were inhibited by tetrodotoxin (1 μM), atropine (1 μM), capsaicin (100 μM) and piroxicam (10 μM). BK-stimulated prostaglandin (PG)E2 release from colonic tissue. Conclusions: BK stimulates human colonic Cl− secretion by activation of apical and basolateral B2 receptors. Responses to apical BK reflect a direct action on epithelial cells, whereas those to basolateral BK are amplified by stimulation of enteric nerves and PG synthesis. British Journal of Pharmacology (2008) 155, 558–566; doi:10.1038/bjp.2008.288; published online 7 July 2008

Intestinal lymphoid tissues and Peyer's patches (PP) are innervated sites of immune surveillance in the gastrointestinal tract. Following infection with F. hepatica, neuronal hyperplasia and significantly increased eosinophil and mast cell trafficking to colonic PP sites were evident in rat tissues. Nerve-eosinophil associations were significantly elevated in infected colon and colonic PP, as were colonic tissue levels of the circulatory recruitment factors IL-5 and eotaxin. Increased immunoreactivity for neuronal plasticity markers GAP-43 and neural cell adhesion molecule (NCAM) was also found in infected tissues. Such neuronal alterations in the PP during enteric parasitism may have functional consequences on particular or pathogen uptake.

Sixty twin-bearing ewes were allocated to one of four dietary treatments investigating the effects of supplementary iodine or cobalt during late pregnancy on lamb serum immunoglobulin G (IgG), triiodothyronine (T3), thyroxine (T4) and vitamin E concentrations, and lamb IgG absorption efficiency. Ewes were offered grass silage ad libitum supplemented with 800 g per ewe per day of a 190 g/kg crude protein (CP) concentrate from day 126 of gestation until parturition plus one of the following supplements (n = 15 per treatment); no supplement (C); 26.6 mg iodine per day for final 3 weeks pre partum (I-3); 26.6 mg iodine/day for final week pre partum (I-1); 20 mg cobalt/day for final 3 weeks pre partum (Co-3). Lambs were blood sampled at 24 and 72 h post partum for serum IgG and vitamin E concentrations. Ten lambs from C and I-3 were blood sampled at 1 h post partum for serum IgG, vitamin E, T3 and T4 concentrations. There were no differences in serum IgG, vitamin E or T4 values (P > 0.05) at 1 h post partum between lambs born to the C and I-3 ewes. T3 levels were lower in I-3 compared with C progeny (P < 0.05). Supplemental iodine reduced colostral IgG absorption efficiency (P < 0.001) and lamb serum IgG concentrations at 24 and 72 h post partum (P < 0.001). Serum vitamin E concentration in I-3 and I-1 lambs was lower than in Co-3 lambs at 24 h post partum, while at 72 h post partum I-3, I-1 and Co-3 lambs had significantly lower concentrations than C lambs (P < 0.001). Supplementing the ewe’s diet with 26.6 mg/day of iodine for the final week of pregnancy reduced lamb serum IgG concentration at 24 and 72 h post partum. The lower total and free T3 values in the progeny of I-3-treated ewes suggest interference in the synthesis and metabolism of thyroid hormones when ewes receive excessive dietary iodine for 3 weeks immediately pre partum. Based on these findings, the indications are that the toxicity level for iodine in the diet of the pregnant ewe should be lowered to 20 mg per ewe per day, equivalent to 40% of its current level. The finding that high-level cobalt supplementation during the final 3 weeks of pregnancy will have a negative effect on serum vitamin E concentration at 72 h post partum is a new and significant finding and previously has not been reported in the literature.

To examine whether myosin light chain kinase (MLCK) inhibitors can reduce intestinal epithelial permeability increases in vitro.Isolated rat, mouse and human colonic tissue mucosae and Caco-2 monolayers were exposed to cytochalasin D (cD) and sodium caprate (C10), in the absence and presence of the MLCK inhibitors, ML-9 and D PIK. Transepithelial electrical resistance (TEER) and Papp of [14C]-mannitol or FITC-dextran 4000 (FD-4) were measured. Western blots were used to measure MLC phosphorylation.Increases in Papp of [14C]-mannitol and decreases in TEER were induced by tight junction openers. These changes were attenuated by ML-9. D-PIK offset the FD-4 Papp increase induced by C10 in Caco-2 only, while ML-9 and PIK inhibited MLC directly, cD induced constriction of peri-junctional actin in Caco-2 monolayers, but this was prevented by ML-9. Although mannitol fluxes across colonic mucosae from dextran-sulphate (DSS)-treated mice were higher than control, they were not ameliorated by either ML-9 or PIK in vitro.ML-9 inhibits paracellular permeability increases in several intestinal epithelial models. D-PIK reduced stimulated paracellular fluxes in Caco-2 monolayers, but not in tissue. Pre-established increases were not modified by two MLCK inhibitors in a mouse model of IBD.

The onset of obesity occurs as a result of an imbalance between nutrient consumption/absorption and energy expenditure. Gastrointestinal (GI) motility plays a critical role in the rate of consumption of foods, digestion, and absorption of nutrients. Various segments of the GI tract coordinate in a complex yet precise way, to control the process of food consumption, digestion, and absorption of nutrients. GI motility not only regulates the rates at which nutrients are processed and absorbed in the gut, but also, via mechanical and neurohormonal methods, participates in the control of appetite and satiety. Altered GI motility has frequently been observed in obese patients, the significance of which is incompletely understood. However, these alterations can be considered as potential contributing factors in the development and maintenance of obesity and changed eating behavior. Therapies aimed at regulating or counteracting the observed changes in GI motility are being actively explored and applied clinically in the management of obese patients.

Histamine is a primary mediator of the inflammatory response in mammals. Degranulation of intestinal mast cells results in the release of mast cell mediators such as histamine. Histamine stimulates epithelial ion transport in a range of mammalian tissues via specific histamine receptors. The aim of this study was to assess a potential role of tissue mast cells and of exogenous histamine in the regulation of ion transport in avian mucosa. Broiler chicken ileal histamine release and secretory responses to mast cell degranulation were determined in vitro with the use of ELISA and Ussing chamber techniques. Pharmacological degranulation of mucosal mast cells using compound 48/80 (15 μg/mL) resulted in histamine release and an immediate-onset transient increase in transmural short-circuit current. The response to compound 48/80 was subject to tachyphylaxis and was significantly reduced in the presence of the histamine H1 antagonist mepyramine, but was unaffected by the cyclooxygenase inhibitor piroxicam. Prior incubation with the mast cell stabilizer ketotifen prevented compound 48/80-induced increase in transmural short-circuit current. In conclusion, degranulation of avian intestinal mast cells would appear to result in histamine release that stimulates epithelial ion transport via histamine H1 receptor activation. Although prostaglandin E2 is a potent secretagogue in the avian small intestine epithelium, prostanoid production appears to have little role to play in mast cell-mediated epithelial ion transport.

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFκB activity in control and TNF-α treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative κB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFκB activity. As a surrogate marker of NFκB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-α enhanced NFκB activity in bovine mammary epithelial monolayers. p65 NFκB homodimer was identified in both control and TNF-α treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative κB consensus sequence, which specifically bound TNF-α inducible p50/p65 heterodimer. TNF-α induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-α induced IL-8 release. High dose interleukin-1β (IL-1β) induced IL-8 release, however significantly less potently than TNF-α. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-α or IL-1β but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-α in activation of NFκB and release of IL-8 from bovine mammary epithelial cells.

Equine recurrent airway obstruction (RAO) has been used as a naturally occurring model of human asthma. However, it is unknown whether there is an early-phase response in RAO. The aim of this study was to determine whether exposure to organic dust induces immediate changes in lung function in RAO-affected horses, which could be mediated by airway mast cells. Six RAO-affected horses in remission and six control horses were challenged with hay-straw dust suspension by nebulization. Total respiratory resistance at 1 Hz, measured by forced oscillation, was increased from 0.62 ± 0.09 cmH2O·l−1·s (mean ± SE) to 1.23 ± 0.20 cmH2O·l−1·s 15 min after nebulization in control horses ( P = 0.023) but did not change significantly in the RAO group. Total respiratory reactance at 1 Hz ( P = 0.005) was significantly lower in the control horses (−0.77 ± 0.07 cmH2O·l−1·s) than in the RAO group (−0.49 ± 0.04 cmH2O·l−1·s) 15 min after nebulization. Bronchoalveolar lavage fluid (BALF) histamine concentration was significantly elevated 10 and 20 min postnebulization in control horses but not in RAO horses. Minimum reactance at 1 Hz in the early postnebulization period significantly correlated with both prechallenge BALF mast cell numbers ( r = −0.65, P = 0.02) and peak BALF histamine concentration postnebulization ( r = −0.61, P = 0.04). In conclusion, RAO horses, unlike human asthmatic patients, do not exhibit an early-phase response. However, healthy control horses do demonstrate a mild but significant early (

Electrogenic ion transport in human colon is a surrogate marker for colonic mucosal function, and may be manipulated by a variety of hormonal, neural, immune and paracrine mediators. Polyamines are present in vast quantities in the colonic lumen and appear to be integral to cellular function. This study explores some of the mechanisms of polyamine action on colonic tissue through study of their effects on differential secretory pathways, as well as examining their actions on intracellular cAMP and Ca(2+) accumulation. Human colonic mucosa was mounted in Ussing chambers and treated with polyamines (spermine, spermidine and putrescine) with changes in ion transport recorded. In separate experiments colonic crypts were treated with polyamines and intracellular cAMP levels determined by ELISA and intracellular calcium concentrations were quantified by fluorescent imaging. Polyamines at physiological concentrations (1mM) exert no effects on basal mucosal chloride secretion or transepithelial electrical resistance. Polyamines inhibit electrogenic ion secretion as stimulated by forskolin (cAMP-mediated), but not carbachol (Ach-mediated). All the polyamines used in this study inhibited intracellular cAMP accumulation, according to potency (spermine>spermidine>putrescine). Spermine increased intracellular Ca(2+) in a PKC-dependent manner, likely due to its effects on the extracellular calcium-sensing receptor (CaSR). Polyamines act to prevent cAMP-mediated Cl(-) hypersecretion in the colon, acting through CaSR to inhibit PKC-mediated [Ca(2+)]i release from intracellular stores.

Bacterial drug resistance represents one of the most crucial problems in present day antibacterial chemotherapy. Of particular concern to public health is the continuing worldwide epidemic spread of Salmonella enterica serovar Typhimurium phage type DT104 harbouring a genomic island called Salmonella genomic island I (SGI-1). This island contains an antibiotic gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides and tetracyclines. These resistance genes are assembled in a mosaic pattern, indicative of several independent recombinational events. The mobility of SGI-1 coupled with the ability of various antibiotic resistance genes to be integrated and lost from the chromosomal resistance locus allows for the transfer of stable antibiotic resistance to most of the commonly used antibiotics and adaptation to new antibiotic challenges. This, coupled with the incidence of increasing fluoroquinolone resistance in these strains increases the risk of therapeutic failure in cases of life-threatening salmonellosis. Fluoroquinolone resistance has largely been attributed to mutations occurring in the genes coding for intracellular targets of these drugs. However, efflux by the AcrAB-TolC multi-drug efflux pump has recently been shown to directly contribute to fluoroquinolone resistance. Furthermore, the resistance to chloramphenicol-florfenicol and tetracyclines in DT104 isolates, is due to interaction between specific transporters for these antibiotics encoded by genes mapping to the SGI-1 and the AcrAB-TolC tripartite efflux pump. The potential for the use of efflux pump inhibitors to restore therapeutic efficacy to fluoroquinolones and other antibiotics offers an exciting developmental area for drug discovery.

Rationale for this study – This study has two purposes. The first is to explore an instrument of evaluation of the approaches to study (deep, strategic, and surface) adopted by students in the pre-clinical years of their veterinary degree program. The second is to examine relationships between these approaches and a broad range of further factors deemed relevant to the veterinary medicine context. We envisage that a greater knowledge of how these students learn will aid curriculum reform in a way that will enrich the learning experience of veterinary students. Methodology – A questionnaire consisting of the 52-question Approaches to Study Inventory (ASI) and an additional 49 questions relating mainly to teaching, assessment, and study skills was distributed to 215 veterinary medicine (MVB) students in their pre-clinical years of study. Factor analysis was used to ensure that the ASI section of the questionnaire maintained previously reported structure. The internal reliability of the approaches measured was tested using Cronbach alpha analysis. The approaches were described as frequency distributions. Associations between the parameters (deep, strategic, and surface) and 49 additional context-specific factors were investigated using loglinear analysis. Results – (1) Factor analysis revealed that the integrity and structure of the instrument in this context was generally comparable to previous studies. (2) The impact of a high workload was evident in the surface approach, with fear of failure becoming a strong motivating factor and syllabus boundness a widely used strategy. (3) Associations made between the approaches and 49 context-specific factors showed strong associations between both workload and lack of prior knowledge with the surface approach. (4) Grades were associated positively with both the deep and strategic approaches but negatively with the surface approach. (5) A range of learning and study skills were associated positively with the deep and strategic approaches and negatively with the surface approach. Conclusion – The ASI proved to be a reliable and insightful instrument, highlighting specific surface learning tendencies present in the group as well as a deep learning approach, the pattern of which deviates from previous studies on this subject. This study also confirms the value of some teaching practices as a means of supporting deep learning and perhaps challenging surface learning strategies. The prevalent perception of a high workload is notable, as is its positive association with surface learning.

Intercellular cell adhesion molecule-1 (ICAM-1) is an important member of the immunoglobulin superfamily (IgSF) of proteins that is centrally involved in trafficking of leukocytes to endothelial and epithelial barriers. This review will first outline the traditional role of ICAM-1 in regulating leukocyte trafficking across the endothelium. It will then deal with more novel aspects of epithelial ICAM-1 expression, particularly its induction upon apical epithelial surfaces and the potential functional consequences for both pathogen invasion and host defence. Signaling mechanisms related to structural rearrangements of the F-actin cytoskeleton which may facilitate pathogen adherence or invasion will also be briefly discussed in the context of antigen sampling through intestinal Peyer's patches. Finally, precedence for pathogen targeting of other IgSF members will be addressed, with emphasis on key cell-cell adhesion proteins as potential pathogen docking structures.

Most therapeutic agents are delivered orally. Consequently, the major classes of therapeutically useful chemicals are partially lipophilic, small molecular weight compounds. They have reasonable permeability coefficient values across cell membranes, including those of intestinal epithelia and vascular endothelia. In contrast, large molecular weight biotechnology compounds have limited usefulness by non-injected routes as a consequence of their low membrane permeability and variable solubility. However, a wide range of infectious agents have developed strategies or have hijacked physiological routings in order to enter the host by the oral route. Efforts to address such issues have refreshed interest in mechanisms by which different types of payloads (including particulates and microorganisms) translocate across gut epithelia and then distribute to target tissues. Special attention is given to the potential role of the enteric nervous system and its plasticity.

Adherence to the tooth surface by Streptococcus mutans is an important step in initiation of dental caries. Current in vitro methods used to study bacterial adherence are time-consuming and may involve the use of radiolabels. The aim of this study was to develop a more convenient, high-throughput, microtitre-plate assay of bacterial adherence to hydroxylapatite. S. mutans was labelled with the fluorescent indicator BCECF/AM and fluorescence measured using a spectrofluorometer. Fluorescence microscopy confirmed label uptake. Optimal labelling occurred at 120 min with 50 μM BCECF/AM in DMSO. Viability was similar in control untreated bacterial cells, bacteria treated with DMSO alone or with the label for up to 4 h. Preliminary adherence experiments were performed using four commercially available types of hydroxylapatite. Fluorescence from pre-labelled bacteria was measured for bound cells. The assay was then optimised with respect to time and bacterial concentration using Fluka crude hydroxylapatite. Time course studies demonstrated that adherence reached saturation by 30 min incubation when using 1×10 7 cfu/ml labelled bacteria to 1 mg hydroxylapatite, coated with PBS or saliva. The fluorescence-based adherence assay was highly reproducible in repeated analyses and was useful in demonstrating interference with adherence. In conclusion, this microtitre-plate assay offers a more convenient approach to examine streptococcal adherence and could be used to screen for potential anti-adhesive agents.

Aims Prostaglandin D2 is released by mast cells and is important in allergies. Its role in gastrointestinal function is not clearly defined. This study aimed to determine the effect of exogenous PGD2 on ion transport in ex vivo normal human colonic mucosa. Materials and methods Mucosal sheets were mounted in Ussing chambers and voltage clamped to zero electric potential. Ion transport was quantified as changes in short-circuit current. In separate experiments epithelial monolayers or colonic crypts, isolated by calcium chelation, were treated with PGD2 and cAMP levels determined by ELISA or calcium levels were determined by fluorimetry. Key findings PGD2 caused a sustained, concentration-dependent rise in short-circuit current by increasing chloride secretion (EC50 = 376 nM). This effect of PGD2 is mediated by the DP1 receptor, as the selective DP1 receptor antagonist BW A686C inhibited PGD2-induced but not PGE2-induced rise in short-circuit current. PGD2 also increased intracellular cAMP in isolated colonic crypts with no measurable influence on cytosolic calcium. PGD2 induces chloride secretion in isolated human colonic mucosa in a concentration-dependent manner with concomitant elevation of cytoplasmic cAMP in epithelial cells. Significance The involvement of DP2 receptor subtypes has not previously been considered in regulation of ion transport in human intestine. Since inflammatory stimuli may induce production of eicosanoids, selective regulation of these pathways may be pivotal in determining therapeutic strategies and in understanding disease.

Urea transporters (UTs) encoded by the Slc14a1 (UT-B) and Slc14a2 (UT-A) genes facilitate the movement of urea across plasma membranes. Urea flux into the gastrointestinal tract enhances the growth...

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.