Evaluating insulindysregulation in horses: A two-step insulin-tolerance test using porcine zinc insulin.

In insulin dysregulation, hyperinsulinemia (HI) can be accompanied by peripheral insulin resistance (IR) in horses, which can be diagnosed with an insulin-tolerance test (ITT). The administration of 0.1 IU/kg body weight of recombinant regular human insulin (RHI) should elicit a 50 % reduction of the initial blood glucose concentration at 30 min after insulin administration in insulin sensitive horses. Compared to RHI, porcine zinc insulin (PZI) is veterinary-approved and therefore easier accessible for many practitioners. The aim of this study was to compare the insulin and glucose dynamics during a standard ITT with RHI to an ITT performed with PZI. Twelve Icelandic horses were subjected to an ITT with RHI (ITT-RHI) and with PZI (ITT-PZI) at same dosages in a randomised crossover design. The insulin and glucose dynamics that resulted from these tests were compared, and the consistency of classification into insulin-sensitive and IR categories was evaluated. No complications were observed with the use of either RHI or PZI in ITT. A good correlation of the test results was observed (r = 0.88; P < 0.001). The blood glucose concentrations and the percentage reduction in glucose concentration did not differ significantly between the two tests (P = 0.053), but four out of twelve horses were classified as IR in the ITT-RHI whereas with the ITT-PZI seven out of twelve horses were classified as IR with the 50 % glucose reduction from baseline. Based on the Youden index, when using the ITT-PZI, an adjusted cut-off value for blood glucose reduction of 40 % at 30 min resulted in better test performance. With consideration for the seemingly weaker effect of PZI and the adjusted cut-off value, PZI can be an appropriate substitute to RHI in an ITT.

Comparison of a modified 2-step insulin response test performed with porcine zinc insulin and an oral glucose test to detect hyperinsulinemic Icelandic horses.

Both, oral and intravenous (IV) testing protocols, are recommended and still used to detect insulin dysregulation (ID) in equids. However, IV tests mainly focus on peripheral insulin resistance (IR), while oral tests assess hyperinsulinemia (HI), which are different aspects of ID. The objective of this study was to describe if horses with HI also demonstrate IR and consequently can be detected by a modified 2-step insulin response test (2-step IRT) performed with a veterinary approved porcine zinc insulin (PZI). Twelve Icelandic horses were subjected to an OGT and 2-step IRT in a crossover study. Serum insulin concentrations during the OGT revealed that six horses were hyperinsulinemic (HI) while six were not (NON-HI). To describe the glucose response to IV injected PZI, the decline of plasma glucose concentration within the first 30 min was analyzed. Glucose reduction was similar in horses with and without HI during the 2-step IRT over time. Additionally, none of the horses reached a glucose reduction of ≥ 50% at 30 min. The results of the present study indicated that a comparable insulin mediated glucose uptake may be observed in horses with and without HI during a modified 2-step IRT. While six out of twelve horses were identified as HI by the OGT, all twelve horses were identified as IR by the modified 2-step IRT performed with PZI underlining the importance, but difficulty in choosing the right diagnostic tool in clinical settings to assess ID.

[Fulminant sepsis after Capnocytophaga canimorsus infection]. / Fulminante Sepsis nach Capnocytophaga-canimorsus-Infektion.

Capnocytophaga canimorsus is a Gram-negative bacterium present in the oral cavities of cats and dogs. It is known for its capability to cause life-threatening sepsis following a dog bite, especially in immunocompromised patients. We report the case of a 67-year-old man who suffered from septic shock and multiorgan failure presumably following contamination of a pre-existing minor skin lesion by canine saliva. The purpose of this report is to raise awareness of Capnocytophaga canimorsus among physicians when faced with a patient presenting with fulminant septic shock and Gram-negative bacteremia, who has been exposed to dogs.

Trans- and paracellular calcium transport along the small and large intestine in horses.

Intestinal calcium absorption plays a key role in the maintenance of calcium homeostasis and may either occur by paracellular or transcellular mechanisms. The horse has some unique peculiarities in calcium homeostasis compared to other species including a high absorptive capacity for calcium in the intestine, high plasma calcium concentrations, high renal excretion, and low plasma concentrations of vitamin D metabolites. So far, knowledge about the underlying mechanisms and the regulation of intestinal calcium absorption is still limited concerning this species. Several studies have documented that intestinal calcium transport in horses is not as dependent on vitamin D as in other species. However, published data on other potential regulatory mechanisms are still lacking. In the present study, paracellular and transcellular transport mechanisms for intestinal calcium transport along the intestinal axis were identified in horses using the Ussing chamber technique. Furthermore, the expression of respective transport proteins including transient receptor potential vanilloid member 6, calbindin-D9k and calcium ATPase type 1 in line with the determined calcium flux rates was documented. In respect to regulation of transepithelial calcium transport, novel regulatory proteins for maintaining calcium homeostasis such as B-box and SPRY-domain containing protein and calmodulin were investigated for the first time in equine intestinal tissues in this study. This provides the basis for a new approach for a better understanding of equine calcium homeostasis regulation.

Comparison of three different methods for the quantification of equine insulin.

BACKGROUND: Exact analysis of equine insulin in blood samples is the key element for assessing insulin resistance or insulin dysregulation in horses. However, previous studies indicated marked differences in insulin concentrations obtained from sample analyses with different immunoassays. Most assays used in veterinary medicine are originally designed for use in human diagnostics and are based on antibodies directed against human insulin, although amino acid sequences between equine and human insulin differ. Species-specific assays are being used more frequently and seem to provide advantages compared to human-specific assays. The aim of this study was to compare three immunoassays, one porcine-specific insulin enzyme-linked immunosorbent assay (ELISA), advertised to be specific for equine insulin, one porcine-specific insulin radioimmunoassay (RIA) and one human-specific insulin chemiluminescence immunoassay (CLIA), all three widely used in veterinary laboratories for the analysis of equine insulin. Furthermore, we tested their clinical applicability in assessing insulin resistance and dysregulation by analysis of basal blood and blood samples obtained during a dynamic diagnostic stimulation test (OGT) with elevated insulin concentrations. RESULTS: Insulin values obtained from the ELISA, RIA and CLIA, investigated for analyses of basal blood samples differed significantly between all three assays. Analyses of samples obtained during dynamic diagnostic stimulation testing with consecutively higher insulin concentrations revealed significantly (p < 0.001) lower insulin concentrations supplied by the CLIA compared to the ELISA. However, values measured by ELISA were intermediate and not different to those measured by RIA. Calculated recovery upon dilution, as a marker for assay accuracy in diluted samples, was 98 ± 4 % for ELISA, 160 ± 41 % for RIA and 101 ± 11 % for CLIA. CONCLUSIONS: Our results indicate that insulin concentrations of one sample measured by different methods vary greatly and should be interpreted carefully. Consideration of the immunoassay method and reliable assay-specific reference ranges are of particular importance especially in clinical cases where small changes in insulin levels can cause false classification in terms of insulin sensitivity of horses and ponies.

In vitro anticancer activity of Betulinic acid and derivatives thereof on equine melanoma cell lines from grey horses and in vivo safety assessment of the compound NVX-207 in two horses.

Betulinic acid, a pentacyclic triterpene, and its derivatives are promising compounds for cancer treatment in humans. Melanoma is not only a problem for humans but also for grey horses as they have a high potential of developing melanoma lesions coupled to the mutation causing their phenotype. Current chemotherapeutic treatment carries the risk of adverse health effects for the horse owner or the treating veterinarian by exposure to antineoplastic compounds. Most treatments have low prospects for systemic tumor regression. Thus, a new therapy is needed. In this in vitro study, Betulinic acid and its two derivatives B10 and NVX-207, both with an improved water solubility compared to Betulinic acid, were tested on two equine melanoma cell lines (MelDuWi and MellJess/HoMelZh) and human melanoma (A375) cell line. We could demonstrate that all three compounds especially NVX-207 show high cytotoxicity on both equine melanoma cell lines. The treatment with these compounds lead to externalization of phosphatidylserines on the cell membrane (AnnexinV-staining), DNA-fragmentation (cell cycle analysis) and activation of initiator and effector caspases (Caspase assays). Our results indicate that the apoptosis is induced in the equine melanoma cells by all three compounds. Furthermore, we succeed in encapsulating the most active compound NVX-207 in 2-Hydroxyprolyl-ß-cyclodextrine without a loss of its activity. This formulation can be used as a promising antitumor agent for treating grey horse melanoma. In a first tolerability evaluation in vivo the formulation was administered every one week for 19 consecutive weeks and well tolerated in two adult melanoma affected horses.

Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent.

BACKGROUND: Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. RESULTS: In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis. CONCLUSION: Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.

Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release.

Leukocytes and their functional capacities are used extensively as biomarkers in immunological research. Commonly employed indicators concerning leukocytes are as follows: number, composition in blood, response to discrete stimuli, cytokine release, and morphometric characteristics. In order to employ leukocytes as biomarkers for disease and therapeutic monitoring, physiological variations and influencing factors on the parameters measured have to be considered. The aim of this report was to describe the ranges of selected leukocyte parameters in a sample of healthy horses and to analyse whether age, sex, breed, and sampling time point (time of day) influence peripheral blood leukocyte composition, cell morphology and release of cytokines ex vivo. Flow cytometric comparative characterisation of cell size and complexity in 24 healthy horses revealed significant variance. Similarly, basal release of selected cytokines by blood mononuclear cells also showed high variability [TNFα (65-16,624pg/ml), IFNγ (4-80U/ml), IL-4 (0-5069pg/ml), IL-10 (49-1862pg/ml), and IL-17 (4-1244U/ml)]. Each animal's age influenced leukocyte composition, cell morphology and cytokine release (TNFα, IL-4, IL-10) ex vivo. Geldings showed smaller monocytes and higher spontaneous production of IL-10 when compared to the mares included. The stimulation to spontaneous release ratios of TNFα, IL-4 and IL-17 differed in Warmblood and Thoroughbred types. Sampling time influenced leukocyte composition and cell morphology. In summary, many animal factors - age being the dominant one - should be considered for studies involving the analysis of equine leukocytes. In addition, high inter-individual variances argue for individual baseline measurements.

Impact of tetrodotoxin application and lidocaine supplementation on equine jejunal smooth muscle contractility and activity of the enteric nervous system in vitro.

By blocking the enteric nervous system (ENS) using tetrodotoxin (TTX), previous studies have documented the contractility-enhancing (CE) effects of lidocaine in equine intestinal smooth muscle (SM) at the level of SM cells and/or interstitial cells of Cajal (ICC). The present study examined the impact of ENS deactivation on CE lidocaine effects, and investigated the effects of lidocaine on ENS activity. TTX application did not affect the CE effects of lidocaine, indicating that these were not mediated by TTX-sensitive sodium channels. Application of TTX or ≥100 mg/L lidocaine reduced ENS activity. Although such concentrations of lidocaine exceed therapeutic blood concentrations, tissue concentrations may be higher with the potential to reduce ENS activity and impair intestinal motility in vivo. Improved understanding of underlying mechanisms is relevant for therapeutic use of lidocaine in horses with postoperative ileus.

Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines.

Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1ß, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with their corresponding equine cytokines. Dot Blot (DB) and Western Blot (WB) analyses were performed using recombinant equine cytokines as positive controls. Immunohistochemistry (IHC) was carried out on equine tissue and flow cytometry on equine PBMC as positive controls. As expected, three equine IL-1ß antibodies detected equine IL-1ß in DB, WB and IHC. For these, reactivity in IHC has not been described before. One of them was also found to be suitable for intracellular staining of equine PBMC and flow cytometric analysis. Two antibodies raised against ovine GM-CSF cross-reacted with equine GM-CSF in DB, WB and IHC. For these anti-GM-CSF mAbs this is the first experimental description of cross-reactivity with equine GM-CSF (one mAb was predicted to be cross-reactive in WB in the respective data sheet). The other clone additionally proved to be appropriate in flow cytometric analysis. Two mAbs targeting porcine IL-18 cross-reacted in IHC, but did not show specificity in the other applications. No reactivity was shown for the remaining five antibodies in DB, although cross-reactivity of two of the antibodies was described previously. The results obtained in this study can provide beneficial information for choosing of antibodies for immunological tests on equine cytokines.

In vitro studies on intestinal peptide transport in horses.

Published data on the physiology of nutrient transport across the equine intestine are limited, and the existence and relevance of peptide transporters are still unknown in the horse. In the present study, the equine intestinal peptide transport was investigated by Ussing chamber experiments using the radioisotope tracer technique and by uptake studies into brush border membrane vesicles (BBMV). Jejunal mucosae of 16 healthy adult horses were used. Tissue samples were mounted in Ussing chambers, and electrophysiological parameters as well as unidirectional flux rates of the radiolabelled dipeptide glycylglutamine (Gly-Gln) were determined. The short-circuit current (Isc) response to the luminal addition of Gly-Gln was significantly greater compared to the Isc response to glycylsarcosine (Gly-Sar) addition (P<0.01). Positive net flux rates were determined indicating absorption of the dipeptide. The addition of Gly-Sar reduced the flux rates significantly (P<0.01), suggesting that both peptides compete for the same transport system. The flux rates were not affected by changes in luminal pH value. Uptake studies into BBMV demonstrated an uphill transport in both the absence and the presence of an inwardly directed H+-gradient with the H+-mediated uphill transport being significantly greater than the transport under equilibrium conditions (P<0.001). A Na+-gradient did not cause an uphill transport. The Gly-Gln uptakes displayed Michaelis-Menten kinetics with the Km value for the H+-dependent Gly-Gln uptake being significantly different from the Km value for the Gly-Gln uptake under equilibrium conditions (P<0.05). In conclusion, the study demonstrated for the first time that dipeptides are transcellularly transported across the equine small intestine. The results indicate the presence of at least 2 transport systems for peptide absorption in the horse: 1 secondary active H+-mediated cotransport and 1 that is capable of an uphill transport energized by a mechanism other than a H+ or a Na+-gradient.

[Examination of horses with acute colic - clinical pathology and diagnostic imaging]. / Untersuchung des akut kolikkranken Pferdes -labordiagnostische und bildgebende Verfahren.

The article summarizes the relevant clinical pathological assessment of horses with acute colic. A minimal laboratory evaluation should include the patient's haematocrit (or packed cell volume), total protein, and lactate concentration in the blood. Haematocrit and total protein provide an indication of the severity of dehydration (haematocrit < 0.45 l/l is evidence of no to mild dehydration whereas > 0.5 l/l points to a severe dehydration). The degree of dehydration is often associated with the severity of the colic. Additionally, the blood lactate concentration rises with increasing intestinal compromise with a concentration of > 4 mmol/l indicating a guarded prognosis. However, it is crucial to assess laboratory values only in the context of the clinical findings. If an abdominocentesis is performed, the leukocyte count and the protein and lactate concentrations offer valuable information regarding the type of colic, the severity of the lesion, further therapy, and prognosis of the colic. Reddish discolouration of peritoneal fluid may be a sign of a strangulating obstruction. Transcutaneous abdominal ultrasonography may provide a crucial insight into the colic cause and severity in a relatively short time, even for inexperienced examiners. In regards to small intestinal lesions, dilated small intestinal loops can often be imaged ultrasonographically before they can be palpated transrectally. The occurrence of free peritoneal fluid and dilated small intestine as well as the evaluation of the intestinal wall and the extent of the gastric wall, allow a better management of the acute colic patient. In ponies and foals, radiography as a further diagnostic imaging modality of the abdomen is of great value. It can help to visualise sand impactions, meconium impactions, or gastrointestinal atresia in the neonate.

Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti-human CD14 mAb and automated magnetic activated cell sorting.

REASONS FOR PERFORMING STUDY: CD14 positive (CD14+) cells are the precursor cells of monocyte-derived dendritic cells (DCs). In horses their potent antigen-presenting capacity and ability to induce an effective immune response classify these cells suitable for several therapeutic approaches such as for equine sarcoid. However, in horses, the generation efficiency of DCs from adherent peripheral blood mononuclear cells (PBMCs) is currently still poor. OBJECTIVES: Establishment of a simple short protocol to enhance DC generation in horses by using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell sorting (MACS) system. METHODS: Peripheral blood mononuclear cells were isolated from fresh heparinised blood samples of 3 horses and primarily stained for flow cytometric analysis (FACS) with a mAb against human CD14 as well as a secondary phycoerythrin (PE) conjugated antibody to determine the initial percentage of CD14 cells in the sample. Peripheral blood mononuclear cells were used for automated MACS using the same primary and secondary antibodies and analysed by FACS. CD14+ selected cells were cultured for 4 days adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to the culture media. Dendritic cell generation was assessed analysing cell morphology and surface marker expression (hCD83, hCD86, eqMHCII). RESULTS: Prior to selection, the mean percentage of CD14+ cells in the total cell population was 5.5%, further gaiting of this cell population resulted in 78.46% CD14+ monocytes. After our positive selection the mean percentage of CD14+ cells in the population was 98% without affecting viability. After culture, DC yield was 2-fold higher than in previous published outcomes. CONCLUSIONS: The additional CD14 cell separation step after PBMC isolation significantly amplified the number of CD14+ cells, increasing the number of generated DCs. POTENTIAL RELEVANCE: The number of DCs available is critical for further use of these cells and the herein described protocol will therefore help to improved DC generation for therapeutic approaches in horses.

[Examination of horses with acute colic: clinical and legal aspects]. / Untersuchung des akut kolikkranken Pferdes - klinische und forensische Aspekte.

The article provides hands-on advice and scientific background information regarding the management of the equine acute colic patient. It summarizes essential information on the patient's history, clinical examination, and legal aspects. The scope of the history is based on the clinical appearance of the patient. In horses with violent and barely controllable signs of pain, it is limited to colic-associated information such as onset, course and severity of colic signs, and the last time of defecation, as well as prior therapeutic intervention. In these patients, the clinical examination is focussed primarily on the assessment of cardiovascular parameters, rectal temperature, and the patient's behaviour. The nasogastric intubation is an essential part of the examination of a horse with colic to prevent a gastric rupture due to gastric distension. Transrectal palpation is equally important and should always be performed unless there are important reasons to object to this procedure. In most cases, a thorough patient history and clinical examination of an acute colic patient allows the examining veterinarian to make a tentative diagnosis and a prognostic evaluation. This helps with the decision to start a conservative or a surgical therapy. Due to the existing obligatory documentation requirement and the duty to inform patient owners, it is of utmost importance to promptly document one's findings, therapeutic measures, consultations, and information of the patient owner in written form. This supports further therapy and it can also be of relevance in a potentially resulting lawsuit.

In vivo induction of interferon gamma expression in grey horses with metastatic melanoma resulting from direct injection of plasmid DNA coding for equine interleukin 12.

Whole blood pharmacokinetics of intratumourally injected naked plasmid DNA coding for equine Interleukin 12 (IL-12) was assessed as a means of in vivo gene transfer in the treatment of melanoma in grey horses. The expression of induced interferon gamma (IFN-g) was evaluated in order to determine the pharmacodynamic properties of in vivo gene transduction. Seven grey horses bearing melanoma were injected intratumourally with 250 µg naked plasmid DNA coding for IL-12. Peripheral blood and biopsies from the injection site were taken at 13 time points until day 14 post injection (p.i.). Samples were analysed using quantitative real-time PCR. Plasmid DNA was quantified in blood samples and mRNA expression for IFN-g in tissue samples. Plasmid DNA showed fast elimination kinetics with more than 99 % of the plasmid disappearing within 36 hours. IFN-g expression increased quickly after IL-12 plasmid injection, but varied between individual horses. Intratumoural injection of plasmid DNA is a feasible method for inducing transgene expression in vivo. Biological activity of the transgene IL-12 was confirmed by measuring expression of IFN-g.

In vitro effects of lidocaine on the contractility of equine jejunal smooth muscle challenged by ischaemia-reperfusion injury.

REASONS FOR PERFORMING STUDY: Post operative ileus (POI) in horses is a severe complication after colic surgery. A commonly used prokinetic drug is lidocaine, which has been shown to have stimulatory effects on intestinal motility. The cellular mechanisms through which lidocaine affects smooth muscle activity are not yet known. OBJECTIVES: To examine the effects of lidocaine on smooth muscle in vitro and identify mechanisms by which it may affect the contractility of intestinal smooth muscle. HYPOTHESIS: Ischaemia and reperfusion associated with intestinal strangulation can cause smooth muscle injury. Consequently, muscle cell functionality and contractile performance is decreased. Lidocaine can improve basic cell functions and thereby muscle cell contractility especially in ischaemia-reperfusion-challenged smooth muscle. METHODS: To examine the effects of lidocaine on smooth muscle function directly, isometric force performance was measured in vitro in noninjured and in vivo ischaemia-reperfusion injured smooth muscle tissues. Dose-dependent response of lidocaine was measured in both samples. To assess membrane permeability as a marker of basic cell function, release of creatine kinase (CK) was measured by in vitro incubations. RESULTS: Lidocaine-stimulated contractility of ischaemia-reperfusion injured smooth muscle was more pronounced than that of noninjured smooth muscle. A 3-phasic dose-dependency was observed with an initial recovery of contractility especially in ischaemia-reperfusion injured smooth muscle followed by a plateau phase where contractility was maintained over a broad concentration range. CK release was decreased by lidocaine. CONCLUSION: Lidocaine may improve smooth muscle contractility and basic cell function by cellular repair mechanisms which are still unknown. Improving contractility of smooth muscle after ischaemia-reperfusion injury is essential in recovery of propulsive intestinal motility. POTENTIAL RELEVANCE: Characterisation of the cellular mechanisms of effects of lidocaine, especially on ischaemia-reperfusion injured smooth muscle, may lead to improved treatment strategies for horses with POI.

Electrophysiological characterization of electrolyte and nutrient transport across the small intestine in horses.

The aim of this study was to characterize the transport mechanisms of electrolytes and nutrients across the jejunum of nine healthy horses electrophysiologically. The stripped mucosa was mounted in Ussing chambers and tissue conductances (G(t)) and short circuit currents (I(sc)) were continuously monitored. After blocking the sodium and potassium channels with amiloride, tetraethylammonium chloride (TEA) and barium, chloride secretion was stimulated by carbachol and forskolin. Subsequently, chloride channels were inhibited by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, CFTR(inh)-172, N-(2-naphtalenyl)-(3.5-dibromo-2.4-dihydroxyphenyl)methylene glycine hydrazide (GlyH-101) and glibenclamide and their dose-response effect was investigated. The response to glucose, l-alanine and glycyl-l-glutamine was determined at two different mucosal pH values (pH 7.4 and 5.4 respectively). Mean basal I(sc) was -0.47 +/- 0.31 microEq/cm(2)h and mean G(t) was 22.17 +/- 1.78 mS/cm(2). Amiloride and TEA did not alter the baseline I(sc). Barium, carbachol and forskolin significantly increased I(sc). Irrespective of the dose, none of the chloride inhibitors changed I(sc). All nutrients induced a significant increase in I(sc) with the increase being significantly higher at pH 7.4 than at pH 5.4. In conclusion, there is evidence that chloride secretion in horses may be different from respective transport mechanisms in other species. The glucose absorption is suggestive of a sodium-dependent glucose cotransporter 1. However, a decrease in luminal pH did not stimulate current response to peptides as shown for other mammals.

Necrosis of the nasal conchae in 12 horses.

Twelve horses with a history of a unilateral malodorous nasal discharge were examined clinically, endoscopically and radiographically. The head of one of the horses was examined by computed tomography. Tissue samples were taken for microbiology from four of the horses and for histology from three. The conchal necrosis was unilateral in 11 of the horses and bilateral in the other, and no particular concha was affected more than the others. The affected tissue was removed transendoscopically with a polypectomy snare while the horses were sedated and standing. Mild disinfectants were used locally in six of the horses. After the removal of all the necrotic tissue, the affected concha healed without any complications in all 12 horses.