Pharmacokinetics of carprofen and firocoxib for medication control in racing greyhounds.

Animals used in sport should be treated as required to ensure animal welfare but any such use of medication should also be controlled to ensure integrity. Pharmacokinetic studies on groups of six greyhounds were performed to measure plasma and urine levels of carprofen and firocoxib to inform medication control advice. Using the standard methodology for medication control the Irrelevant Plasma Concentration was determined as 20 and 2 ng/mL for carprofen and firocoxib, respectively. The Irrelevant Urine Concentration was also determined as 0.3 and 2 ng/mL for carprofen and firocoxib, respectively. These Irrelevant Plasma and Urine Concentrations will allow laboratory Screening Limits, Detection Times and Withdrawal Time advice to be determined and publicised by regulators of greyhound racing. The Screening Limits will also inform Recommended Limits of Detection if meat-containing residues of these medications are fed to greyhounds.

The large-scale capture of eastern grey kangaroos (Macropus giganteus) and red kangaroos (Osphranter rufus) and its application to a population management project.

OBJECTIVE: A large-scale capture method was developed to enable sterilisation of a macropod population in western Sydney from 2005 to 2018. METHODS: Until March 2007, free ranging eastern grey kangaroos and red kangaroos were herded into purpose-built 15 m diameter capture yards (CYs) for darting with a projectile syringe. From March 2007 onwards, animals were free-range darted in large areas without herding. Kangaroos were darted with 1.33-5.10 mg/kg tiletamine/zolazepam and 0.01-0.02 mg/kg medetomidine, ± 0.03 mg/kg acepromazine. Deaths were monitored. Population counts were performed annually. RESULTS: There were 5825 capture events involving 3963 kangaroos. Over 85% of all captures occurred from 2005 to 2008. Of all reported deaths (n = 523), 135 were attributed to ill health. Musculoskeletal injuries incurred during capture were the main project-related cause of death (n = 116). Post capture myopathy was uncommonly diagnosed following capture (n = 19). CONCLUSION: The herding and capture method enabled a large number of kangaroos to be mobilised and captured with low mortality rates, and the use of CYs resulted in fewer capture-related injuries and deaths than free-range capture. The drug doses and combinations used for darting were safe and effective, and the capture technique was successfully applied to a population management project.

Anthelmintic efficacy of single active and combination products against commonly occurring parasites in foals.

Parasite control in foals is complicated by the concurrent presence of biologically diverse parasites with differing levels of anthelmintic resistance. Several combination anthelmintic products are available for use in horses, but information on their efficacies against important equine parasites is scarce. Two trials were performed in New Zealand during 2008 and 2011 on four different farms with substantially different anthelmintic treatment histories. The first trial evaluated the efficacy of an ivermectin/praziquantel/oxibendazole combination, a single active oxibendazole, and a single-active macrocyclic lactone (ML) in 49 foals located on three farms. The second trial evaluated two combination anthelmintic products and three single-active ML products and enrolled a total of 110 foals on three farms. Foals in the second trial were allocated to one of six anthelmintic treatment groups; oxfendazole/pyrantel embonate, pyrantel embonate/ivermectin/praziquantel, ivermectin/praziquantel, abamectin/praziquantel, moxidectin/praziquantel, and a placebo-treated control. In both trials, foals were monitored monthly prior to treatment, and fecal egg counts (FECs) of Parascaris spp., strongylid, and Strongyloides westeri were determined. A "rolling enrolment" process was implemented whereby foals were systematically allocated to a treatment group and treated with the corresponding anthelmintic following the first appearance of Parascaris spp. eggs in the faeces. A generalised linear model was used to evaluate the effect of farm and treatment on Day14 FEC (ln) for each parasite. Three different FECR calculation methods were employed as follows; i) FECR(T) pre and post treatment ii) FECR (C) in the treated group compared with control, and iii) FECR (P) pre- and post- treatment in the treated and control groups. Across both trials, treatment with ML single active products failed to achieve >95% reduction in Parascaris spp. FEC on two of three farms. The pyrantel embonate/oxfendazole and ivermectin/ praziquantel/oxibendazole combinations demonstrated full efficacy against Parascaris spp. This is in contrast to the anti-strongylid efficacies determined, where the pyrantel embonate/oxfendazole combination and single active oxibendazole had reduced efficacy on one farm, while the macrocyclic lactones generally had good efficacy. Strongyloides egg counts were sporadic in both trials, and allowed limited insight into anthelmintic efficacy. The study illustrated the importance of keeping an untreated or placebo-treated control group in studies evaluating anti-Parascaris efficacy and it demonstrated the utility of a rolling enrolment procedure, where foals are enrolled over the course of a defined period of time. Furthermore, the study demonstrated the value of a farm specific FECR monitoring programme and the complexity of parasite control in foals, where combination anthelmintic products can be employed to target multiple species of parasites.

Laparoscopic ovariectomy in eastern grey kangaroos (Macropus giganteus) and red kangaroos (Macropus rufus).

OBJECTIVE: To develop a technique for permanent sterilisation of female eastern grey kangaroos (Macropus giganteus) and red kangaroos (M. rufus) as part of a large-scale macropod management program on an enclosed 1545-ha site in western Sydney. METHODS: Free-ranging female kangaroos (n = 1409: 1285 eastern grey kangaroos, 124 red kangaroos) were anaesthetised via remote anaesthetic drug delivery of tiletamine/zolazepam, medetomidine and acepromazine prior to inhalational anaesthesia using isoflurane-oxygen. A laparoscopic ovariectomy technique was developed using standard laparoscopic equipment to effect permanent sterilisation of the kangaroos. The technique described was also adapted for use on immature animals weighing as little as 1 kg. No direct post-surgical care was provided once the animals had recovered from the anaesthetic. RESULTS: The procedure was simple to perform and had a very high success rate, with an overall project mortality rate of 2.13% (n = 30). Seven kangaroos (0.05% of all operated kangaroos) were euthanased as a direct result of the surgical procedure. Surgical complications were rare but included inadvertent gastrointestinal tract puncture with the trocar, intraoperative haemorrhage and subcutaneous emphysema leading to pouch eversion following surgery. CONCLUSION: The procedure described is a rapid and effective method of permanent fertility control in macropods and carries a low mortality rate.

Bacteroidales recruit IL-6-producing intraepithelial lymphocytes in the colon to promote barrier integrity.

Interactions between the microbiota and distal gut are important for the maintenance of a healthy intestinal barrier; dysbiosis of intestinal microbial communities has emerged as a likely contributor to diseases that arise at the level of the mucosa. Intraepithelial lymphocytes (IELs) are positioned within the epithelial barrier, and in the small intestine they function to maintain epithelial homeostasis. We hypothesized that colon IELs promote epithelial barrier function through the expression of cytokines in response to interactions with commensal bacteria. Profiling of bacterial 16S ribosomal RNA revealed that candidate bacteria in the order Bacteroidales are sufficient to promote IEL presence in the colon that in turn produce interleukin-6 (IL-6) in a MyD88 (myeloid differentiation primary response 88)-dependent manner. IEL-derived IL-6 is functionally important in the maintenance of the epithelial barrier as IL-6-/- mice were noted to have increased paracellular permeability, decreased claudin-1 expression, and a thinner mucus gel layer, all of which were reversed by transfer of IL-6+/+ IELs, leading to protection of mice in response to Citrobacter rodentium infection. Therefore, we conclude that microbiota provide a homeostatic role for epithelial barrier function through regulation of IEL-derived IL-6.

TGF-ß1 alters esophageal epithelial barrier function by attenuation of claudin-7 in eosinophilic esophagitis.

Barrier dysfunction has been implicated in the pathophysiology of eosinophilic esophagitis (EoE). Transforming growth factor-ß1 (TGF-ß1), a potent pleiotropic molecule, is increased in EoE; however, no study has evaluated its influence on esophageal epithelial barrier. We hypothesized that TGF-ß1 regulates barrier dysfunction in EoE. We aimed to determine the role of TGF-ß1 in the epithelial barrier in models of EoE. To examine the impact of TGF-ß1 on esophageal barrier, immortalized human esophageal epithelial (EPC2-hTERT) cells were exposed to TGF-ß1 during the three-dimensional air-liquid interface (3D-ALI) model in vitro. TGF-ß1 exposure diminished EPC2-hTERT barrier function as measured by transepithelial electrical resistance (TEER) and 3 kDa Fluorescein isothiocyanate dextran paracellular flux (FITC Flux), and hematoxylin and eosin (H&E) assessment revealed prominent cellular separation. In analysis of epithelial barrier molecules, TGF-ß1 led to the specific reduction in expression of the tight-junction molecule, claudin-7 (CLDN7), and this was prevented by TGF-ß-receptor I inhibitor. Short hairpin ribonucleic acid (shRNA)-mediated CLDN7 knockdown diminished epithelial barrier function, whereas CLDN7 overexpression resulted in protection from TGF-ß1-mediated barrier dysfunction. In pediatric EoE biopsies CLDN7 expression was decreased and altered localization was observed with immunofluorescence analysis, and the TGF-ß1 downstream transcription factor, phosphorylated SMAD2/3 (pSMAD2/3), was increased. Our data suggest that TGF-ß1 participates in esophageal epithelial barrier dysfunction through CLDN7 dysregulation.

Tryptophan metabolite activation of the aryl hydrocarbon receptor regulates IL-10 receptor expression on intestinal epithelia.

IL-10 is a potent anti-inflammatory cytokine that inhibits the production of proinflammatory mediators. Signaling by IL-10 occurs through the IL-10 receptor (IL-10R), which is expressed in numerous cell types, including intestinal epithelial cells (IECs), where it is associated with development and maintenance of barrier function. Guided by an unbiased metabolomics screen, we identified tryptophan (Trp) metabolism as a major modifying pathway in interferon-γ (IFNγ)-dominant murine colitis. In parallel, we demonstrated that IFNγ induction of indoleamine 2,3-dioxygenase 1, an enzyme that catalyzes the conversion of Trp to kynurenine (Kyn), induces IL-10R1 expression. Based on these findings, we hypothesized that IL-10R1 expression on IEC is regulated by Trp metabolites. Analysis of the promoter region of IL-10R1 revealed a functional aryl hydrocarbon response element, which is induced by Kyn in luciferase-based IL-10R1 promoter assays. Additionally, this analysis confirmed that IL-10R1 protein levels were increased in response to Kyn in IEC in vitro. Studies using in vitro wounding assays revealed that Kyn accelerates IL-10-dependent wound closure. Finally, reduction of murine dextran sodium sulfate colitis through Kyn administration correlates with colonic IL-10R1 expression. Taken together, these results provide evidence on the importance of IL-10 signaling in intestinal epithelia and implicate AHR in the regulation of IL-10R1 expression in the colon.

Studies of the pharmacokinetic profile, in vivo efficacy and safety of injectable altrenogest for the suppression of oestrus in mares.

OBJECTIVE: To investigate the efficacy and safety of the long-acting altrenogest injection (NV Readyserve® injection) for horses. DESIGN: A single-dose pharmacokinetic (PK) study was conducted. The in vivo efficacy study was a blinded, repeated measures design evaluating behaviour scores. The safety study was a non-blinded, controlled, parallel-group, randomised-block design as per the VICH protocol. METHODS: In the PK study, serial blood samples were obtained for analysis of plasma altrenogest for 150 h following the injection and a non-compartmental PK analysis was performed. For the efficacy study, 12 mares in oestrus were treated; they were monitored daily for 10 days for signs of oestrus during teasing and given a behaviour score that was compared with pretreatment scores. A standard safety study was conducted at 1-, 3- and 5-fold the recommended dosage for 84 days. Physical, haematological and biochemical examinations were performed. RESULTS: Mean plasma altrenogest concentrations were greater than ≈0.5 ng/mL for 148 h following administration. Oestrous behaviour was suppressed in all mares within 24 h of administration. Two mares returned to oestrus by day 6 and the rest on days 7-10. In the safety study there were no significant differences in the physical and haematological examinations, but minor biochemical changes in muscle enzymes. There was a low incidence of injection site reactions following the 3- and 5-fold dose, predominantly for pectoral injections. CONCLUSION: These studies support the efficacy and safety of a single dose of Readyserve® injection for the suppression of the signs of oestrus in mares for 5-7 days.

Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis.

Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b(-/-) mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2b(fl/fl)VeCadCre(+)) or intestinal epithelia (Adora2b(fl/fl)VillinCre(+)) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses.

Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis.

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)-1ß, 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.

Fundamental role for HIF-1α in constitutive expression of human ß defensin-1.

Antimicrobial peptides are secreted by the intestinal epithelium to defend from microbial threats. The role of human ß defensin-1 (hBD-1) is notable because its gene (beta-defensin 1 (DEFB1)) is constitutively expressed and its antimicrobial activity is potentiated in the low-oxygen environment that characterizes the intestinal mucosa. Hypoxia-inducible factor (HIF) is stabilized even in healthy intestinal mucosa, and we identified that epithelial HIF-1α maintains expression of murine defensins. Extension to a human model revealed that basal HIF-1α is critical for the constitutive expression of hBD-1. Chromatin immunoprecipitation identified HIF-1α binding to a hypoxia response element in the DEFB1 promoter whose importance was confirmed by site-directed mutagenesis. We used 94 human intestinal samples to identify a strong expression correlation between DEFB1 and the canonical HIF-1α target GLUT1. These findings indicate that basal HIF-1α is critical for constitutive expression of enteric DEFB1 and support targeting epithelial HIF for restoration and maintenance of intestinal integrity.

Detrimental role of the airway mucin Muc5ac during ventilator-induced lung injury.

Acute lung injury (ALI) is associated with high morbidity and mortality in critically ill patients. At present, the functional contribution of airway mucins to ALI is unknown. We hypothesized that excessive mucus production could be detrimental during lung injury. Initial transcriptional profiling of airway mucins revealed a selective and robust induction of MUC5AC upon cyclic mechanical stretch exposure of pulmonary epithelia (Calu-3). Additional studies confirmed time- and stretch-dose-dependent induction of MUC5AC transcript or protein during cyclic mechanical stretch exposure in vitro or during ventilator-induced lung injury in vivo. Patients suffering from ALI showed a 58-fold increase in MUC5AC protein in their bronchoalveolar lavage. Studies of the MUC5AC promoter implicated nuclear factor κB in Muc5ac induction during ALI. Moreover, mice with gene-targeted deletion of Muc5ac⁻/⁻ experience attenuated lung inflammation and pulmonary edema during injurious ventilation. We observed that neutrophil trafficking into the lungs of Muc5ac⁻/⁻ mice was selectively attenuated. This implicates that endogenous Muc5ac production enhances pulmonary neutrophil trafficking during lung injury. Together, these studies reveal a detrimental role for endogenous Muc5ac production during ALI and suggest pharmacological strategies to dampen mucin production in the treatment of lung injury.

Implications of hypoxia on mucosal barrier function.

Epithelial cells which line mucosal surfaces (e.g. lung, intestine) critically function as a semi-permeable barrier to the outside world. Mucosal organs are highly vascular with extensive metabolic demands, and for this reason, are particularly susceptible to diminished blood flow and resultant tissue hypoxia. Recent work from a number of groups have defined the critical molecular and cellular determinants of barrier function in hypoxic/ischemic tissues. Here, we will briefly highlight some of these studies from both a basic and clinical viewpoint and provide a perspective on future work related to tigh tjunction function in mucosal hypoxia.

Expression of BPI (bactericidal/permeability-increasing protein) in human mucosal epithelia.

Among the antimicrobial proteins and peptides of humans is the cationic 55 kDa bactericidal/permeability-increasing protein (BPI), which possesses antibacterial, endotoxin-neutralizing and opsonic activity against Gram-negative bacteria. Although identified originally as an abundant constituent of neutrophil granules, we have recently identified functional expression of BPI by human mucosal epithelia. BPI expression was markedly up-regulated by exposure of epithelia to lipoxins, endogenous anti-inflammatory eicosanoids that are generated in vivo in the context of aspirin treatment (aspirin-triggered lipoxins). Epithelial BPI was found to be surface expressed and fully functional, as measured by antibacterial activity against Salmonella typhimurium as well as lipopolysaccharide (LPS; endotoxin)-neutralizing activity. These results suggest a role for BPI as an effector of epithelial antibacterial activity and as a modulator of epithelial responses to LPS. Both BPI and the lipoxins are currently the subject of intensive biopharmaceutical development, raising the possibility that therapeutic use of BPI or modulation of epithelial BPI expression may be a useful adjunctive therapy for conditions in which epithelial inflammation is associated with Gram-negative infections and/or endotoxin.

Lipid mediators in epithelial cell-cell interactions.

Epithelial cells which line mucosal surfaces (e.g. lung, intestine) play a central role in the coordination of the inflammatory response. In both the healthy and diseased mucosa, epithelia lie anatomically positioned in close proximity to a number of other cell types, including leukocytes, fibroblasts, smooth muscle cells and vascular endothelia. This complex architecture supports a unique microenvironment for biochemical cell-cell crosstalk. Our previous studies and work by others have elucidated lipid mediator signaling networks emanating from epithelial cell-cell interactive pathways, and have defined a number of targets for development of effective therapeutics. This short review will focus on recently defined pathways of lipid mediator function in the mucosa, particularly with regard to the role of the epithelium.

Quantitative exposure assessment for the combustion of meat and bone meal derived from specified risk material in the context of BSE in Ireland.

The probability and severity of an adverse event can be analyzed by quantitative exposure assessment (QEA). This methodology was applied to model the human health risks associated with the combustion of specified risk material (SRM) derived meat and bone meal (MBM) in a combustion facility. The identification of MBM and SRM as significant factors in the spread of bovine spongiform encephalopathy (BSE) has resulted in restrictions on their use and movement, and this has led to a requirement for alternative end-uses for these products. A stochastic (Latin Hypercube sampling) simulation model was developed to assess the exposure and hence the risks associated with the use of SRM-derived MBM in a combustion facility. The model simulates the potential infectivity pathways that SRM-derived MBM follows, including its production from animals potentially infected with sub-clinical BSE and subsequent processing of the material with segregation and heat treatments. A failure probability was included to take account of sub-optimal operating conditions. Two scenarios, reflecting the infectivity risk in different animal tissues as defined by the European Commission's scientific steering committee (SSC), were performed with 100,000 iterations of the model. Model results showed that the societal exposure to humans resulting from the combustion of SRM-derived MBM is extremely small (mean values ranging from 7.57 x 10(-6) ID50/year to 8.38 x 10(-5) ID50/year). The resulting societal risks are significantly less than the background societal risk of approximately 2.5 cases of sporadic CJD in Ireland each year. A sensitivity analysis revealed that the species barrier had a large impact on exposure calculations and hence should be the focus of further scientific investigation to reduce our uncertainty about this parameter. The model predicts that material spillage into untreated effluent represents the biggest risk to humans, indicating that efforts for risk mitigation should be focused on reducing the potential for spillage.

The multiple roles of major histocompatibility complex class-I-like molecules in mucosal immune function.

The human major histocompatibility complex (MHC) on chromosome 6 encodes three classical class-I genes: human leukocyte antigens (HLA) A, B, and C. These polymorphic genes encode a 43- to 45-kDa cell surface glycoprotein that, in association with the 12-kDa beta2-microglobulin molecule, functions in the presentation of nine amino acid peptides to the T-cell receptor of CD8-bearing T lymphocytes and killer inhibitory receptors on natural killer cells. In addition to these ubiquitously expressed, polymorphic proteins, the human genome also encodes several nonclassical MHC class-I-like, or class Ib, genes that, in general, encode nonpolymorphic molecules involved in various specific immunological functions. Many of these genes, including CD1, the neonatal Fc receptor for IgG, HLA-G, HLA-E, the MHC class-I chain-related gene A, and Hfe, are prominently displayed on epithelial cells, suggesting an important role in epithelial cell biology.