Tumor necrosis factor activity in the circulation of horses given endotoxin

Serum and plasma from horses injected with endotoxin was examined for cytotoxic activity. Each of the cell lines, L929 and WEHI 164 clone 13, was sensitive to the cytotoxic effects of equine serum; however, a precipitation artifact caused by the use of isopropanol in the WEHI assay limited the use of this assay to samples containing less than 2 mg of protein/ml. In foals treated with a sublethal IV bolus of 5 micrograms of lipopolysaccharide (LPS)/kg and in adult horses given a low-dose continuous infusion of LPS (30 ng/kg/h for 4 hours), cytotoxic activity was detected in all serum or plasma samples taken between 30 minutes and 4 hours after LPS infusion began. In horses given either continuous or bolus LPS infusions, circulating cytotoxic activity peaked at 1 to 2 hours before decreasing sharply. The onset of pyrexia after LPS infusion coincided with the appearance of circulating cytotoxic activity, but the temperature remained high, even after cytotoxic activity disappeared. Treatment of horses with flunixin meglumine (1 mg/kg) appeared to blunt the pyrexic effect of low-dose continuous LPS infusion, but had no significant effect on circulating cytotoxic activity. Incubation of serum samples with an antibody raised against a portion of human tumor necrosis factor (TNF) resulted in the removal of greater than 90% of serum cytotoxicity, suggesting strongly that the cytotoxic activity was attributable to TNF. These findings are consistent with the hypothesis that TNF is an early acting mediator of the effects of endotoxin in the horse.     

Microvascular circulation of the descending colon in horses.

The microvascular circulation of the descending colon was studied in 5 adult horses, using microangiography and light microscopy combined with gross studies and scanning electron microscopy of vascular replicas. After heparinization, horses were euthanatized, and 3 segments of the descending colon and its mesentery containing 1 vascular arcade were removed from each horse. The fecal balls were gently massaged from the lumen, and the blood was flushed free of the circulation with isotonic NaCl. In 5 segments, the vascular system was injected with a modified radiopaque medium and evaluated radiographically. Specimens examined radiographically also were prepared for histologic examination, using standard methods. Ten segments were injected with 1 of 2 types of plastics and studied grossly or by scanning electron microscopy. Arcuate arteries gave rise to a descending colonic rete that surrounded the vein and supplied numerous descending colonic lymph nodes. The rete also supplied the mesocolon and the descending colonic tissue. Short filamentous vessels arising from the rete directly penetrated the mesenteric tenia to supply an intermuscular plexus between the longitudinal and circular muscle layers of the muscularis externa. Larger vessels arising from either side of the rete divided into the long- and short-terminal arteries that supplied an extensive submucosal plexus, which was continuous around the circumference. The submucosal plexus supplied the mucosa, the tunica muscularis, and the serosa. Vessels running centrifugally from the submucosal plexus formed an intermuscular plexus between the longitudinal and circular muscle layers of the muscularis externa. The intermuscular plexus at the mesenteric angle also was supplied by vessels branching from the short-terminal arteries as they penetrated the muscularis externa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of alpha 1-acid glycoprotein from horses, and its evaluation as an acute-phase reactive protein in horses.

Equine alpha 1-acid glycoprotein (alpha 1AG) was isolated from equine serum by successive ammonium precipitation, anion- and cation-exchange chromatographies, and gel filtration. Purified equine alpha 1AG had a molecular weight of 46,000 +/- 1,000, and contained 31.4% carbohydrate. Gel isoelectric focusing revealed an isoelectric point range of 2.8 to 3.7. With immunoelectrophoresis, it was found that alpha 1AG migrated to the alpha 1-globulin region. Single radial immunodiffusion was used for quantitative measurement of alpha 1AG in equine serum. In clinically normal foals, serum alpha 1AG was undetectable (less than or equal to 20 micrograms/ml) in less than or equal to 7-day-old foals, but was detected by 14 days. The alpha 1AG concentration (mean +/- SD) increased to reach mean adult values of 99.23 +/- 26.90 micrograms/ml by 1 year of age. The alpha 1AG concentration in pregnant mares decreased at 2 to 3 months before parturition, then gradually increased until 1 day after parturition, when a brief decrease was observed. The concentration increased again at 2 weeks after foaling, then a decrease was observed, after which the alpha 1AG concentration increased again by 2 to 4 months after parturition. The concentration of serum alpha 1AG quickly rose to peak values 2 to 3 days after castration and jejunojejunostomy in adult horses, returning to baseline values by 14 to 28 days after surgery. The alpha 1AG was concluded to be an acute-phase reactive protein in horses.     

Microvascular circulation of the cecum in horses

The microvascular circulation of the cecum was studied in 15 adult horses, using microangiography and light microscopy combined with gross studies and scanning electron microscopy of vascular replicas. After heparinization, the horses were euthanatized and the cecum was transected at the cecocolic junction. Blood was flushed free of the circulation with isotonic NaCl and the cecal lumen was slightly distended. In 6 horses, the vascular system was injected with a modified radiopaque medium and evaluated radiographically. Sections evaluated radiographically were also prepared for histologic examination by standard methods. Eight horses were injected with 1 of 2 types of plastics and studied grossly or by scanning electron microscopy. In 1 additional horse, the lateral and medial arteries were injected with different colored plastic for gross studies. The lateral and medial cecal arteries appear to supply the respective areas of the cecum with minimal mixing of the 2 circulations. The major vascular supply to the cecal apex appeared to be through the medial cecal artery. Both the lateral and medial cecal arteries gave rise to cecal retia, which formed a mesh-like network around the respective veins. Vessels from these retia supplied the cecal tissue and the cecal lymph nodes. The continuation of the retia was through long terminal arteries that coursed around the circumference in the submucosa, forming an extensive submucosal plexus. This plexus supplied both the mucosa, and the tunica muscularis and serosa. Vessels within the longitudinal and circular muscle layers of the muscularis externa ran parallel to the muscle fibers, and consequently, perpendicular to each other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrocortisone levels in the urine and blood of horses treated with ACTH.

An investigation was undertaken to demonstrate whether therapeutic treatment with ACTH raises hydrocortisone (cortisol) levels in horse urine above the limit (1000 ng/ml) established by the International Conference of Racing Authorities with the aim of controlling the abuse of cortisol and ACTH in equine sports. ACTH (200 iu) was administered i.m. to 3 Thoroughbred horses; urine and blood samples were collected at intervals afterwards and analysed by an immunoenzymatic system (ELISA) and HPLC-MS. To ascertain post exercise cortisol levels in untreated horses, 101 urine and 103 serum samples were taken from horses immediately after racing and analysed by ELISA. The peak urine level of cortisol, detected 8 h after ACTH administration, was around 600 ng/ml using either ELISA or HPLC-MS. The peak serum cortisol concentration was found to be around 250 ng/ml by ELISA, but consistently less by HPLC-MS. Mean cortisol levels in post race horses were 135.1+/-72.1 ng/ml in urine and 90.1+/-41.7 ng/ml in serum. High levels of the metabolite 20beta-dihydrocortisol in urine and the cortisol precursor 11beta-desoxycortisol in serum were found. The latter showed high cross-reactivity with cortisol on ELISA. In our experiment, treatment with ACTH 200 iu i.m. did not raise urinary cortisol levels above the 1000 ng/ml threshold proposed by the ICRA.     

Microvascular circulation of the ascending colon in horses

Microvascular circulation of the ascending colon in healthy horses was studied using microangiography, light microscopy, and scanning electron microscopy. The pelvic flexure with 30 cm of ventral and dorsal colon attached was removed from 14 adult horses immediately after horses were euthanatized. The lumen was flushed with warm water, and this section of the ascending colon was placed in a 37-C bath of isotonic NaCl. In sections from 8 horses, colic vessels were perfused with a radio-opaque medium for microangiography. After angiographic evaluation, tissue sections were prepared for light microscopic observation, using standard histologic methods. In sections from 6 horses, injection replicas were made by perfusing the vessels with 2 types of plastics. The results of microangiography, light microscopy, and scanning electron microscopy of vascular replicas were correlated, providing a comprehensive documentation of the microvasculature of the ascending colon at the pelvic flexure. Arteries branched from mesenteric colic vessels approximately every 2 cm toward the colonic tissue. Immediately after branching, arterial vessels formed an anastomotic plexus, the colonic rete. However, each branch from the colic vessel eventually continued into the colonic tissue. A second set of vessels originated from the colonic tissue. A second set of vessels originated from the colonic rete and supplied the mesenteric lymph nodes. Arterial vessels penetrated the tunica muscularis into the submucosa 3 to 4 cm toward the antimesenteric border forming a submucosal vascular network. From the submucosal arterioles, branching took place at right angles to supply the mucosal capillaries. Capillaries surrounded the colonic glands and anastomosed at the luminal surface, forming a superficial luminal honeycomb-appearing vascular plexus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of infusion of adenosine triphosphate-magnesium chloride on cardiopulmonary and clinicopathologic variables, cytokine activity, and endothelin concentration in horses administered a low dose of endotoxin

OBJECTIVE: To evaluate systemic effects of i.v. infusion of ATP-MgCl2 subsequent to infusion of a low dose of endotoxin in horses. ANIMALS: 12 adult horses. PROCEDURE: Horses were administered endotoxin (lipopolysaccharide [LPS]) or saline (0.9% NaCl) solution i.v., during a 30-minute period. Immediately thereafter, horses in each group were infused i.v. with ATP-MgCl2 or saline solution. Two weeks later, horses were administered the opposite solution (LPS or saline solution), but it was followed by the same infusion as 2 weeks previously (ie, ATP-MgCl2 or saline solution). Cardiopulmonary and clinicopathologic variables, cytokine activity, and endothelin (ET) concentrations were recorded. RESULTS: IV infusion of ATP-MgCl2 after administration of a low dose of endotoxin failed to attenuate the cardiopulmonary, clinicopathologic, and cytokine alterations that develop secondary to endotoxin exposure. The combination of LPS and ATP-MgCl2 potentiated pulmonary hypertension, leukopenia, and neutropenia when compared with the combination of LPS and saline solution. The combination of LPS and ATP-MgCl2 resulted in thrombocytopenia. Endothelin concentration was increased in jugular venous and pulmonary arterial plasma in horses receiving LPS and ATP-MgCl2. Similar increases were not observed with LPS and saline solution. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of ATP-MgCl2 did not protect horses from systemic effects of experimentally induced endotoxemia. Furthermore, the use of ATP-MgCl2 during endotoxemia may worsen the cardiopulmonary and clinicopathologic status of affected horses. Because ATP and other adenine nucleotides are released from cells during shock, their potential role in the development of hemodynamic derangements, leukocyte adherence, and coagulopathies during endotoxemic episodes warrants further investigation.     

Values of urine specific gravity for thoroughbred horses treated with furosemide prior to racing compared with untreated horses.

The distribution of specific gravity values for 2,599 urine samples collected from racing Thoroughbred horses that were known to have received furosemide prior to racing was compared with that for 1,669 urine samples from racing Thoroughbred horses that reportedly had not received furosemide. Values of specific gravity for furosemide-treated horses were significantly lower (P < 0.001) than those for horses that had not received furosemide, and the proportion of horses with urine specific gravity either <1.010 or <1.012 was significantly greater (P < 0.001) among the furosemide-treated horses. These data indicate that evaluation of urine specific gravity would be a useful component of drug testing programs for regulation of furosemide use.     

Characterization of the inflammatory infiltrate and cytokine expression in the skin of horses with recurrent urticaria

Background – Recurrent urticaria (RU) is a common skin disease of horses, but little is known about its pathogenesis. Hypothesis/Objective – The aim of this study was to characterize the inflammatory cell infiltrate and cytokine expression pattern in the skin of horses with RU. Animals – Biopsies of lesional and nonlesional skin of horses with RU (n = 8) and of skin from healthy control horses (n = 8) were evaluated. Methods – The inflammatory cell infiltrate was analysed by routine histology. Immunohistochemistry was used to identify T cells (CD3), B cells (CD79), macrophages (MAC387) and mast cells (tryptase). Expression of T‐helper 2 cytokines (interleukins IL‐4, IL‐5 and IL‐13), a T‐helper 1 cytokine (interferon‐γ), IL‐4 receptor α and thymic stromal lymphopoietin was assessed by quantitative RT‐PCR. Results – In subepidermal lesional skin of RU‐affected horses, increased numbers of eosinophils (P ≤ 0.01), CD79‐positive (P ≤ 0.01), MAC387‐positive (P ≤ 0.01) and tryptase‐positive cells (P ≤ 0.05) were found compared with healthy horses. Subepidermal lesional skin of RU‐affected horses contained more eosinophils (P ≤ 0.05) and tryptase‐positive cells (P ≤ 0.05) compared with nonlesional skin. There was no significant difference in infiltrating cells between nonlesional skin and skin of healthy horses. Expression of IL‐4 (P ≤ 0.01), IL‐13 (P ≤ 0.05), thymic stromal lymphopoietin (P ≤ 0.05) and IL‐4 receptor α (P ≤ 0.05) was increased in lesional skin of RU‐affected horses compared with control horses. Expression of IL‐4 was higher (P ≤ 0.05) in lesional compared with nonlesional RU skin. Conclusions and clinical importance – Analysis of cytokine expression and inflammatory infiltrate suggests that T‐helper 2 cytokines, eosinophils, mast cells and presumptive macrophages play a role in the pathogenesis of equine RU.     

Cyathostome fecal egg count trends in horses treated with moxidectin, ivermectin or fenbendazole

Commercial preparations of fenbendazole (Safe-Guard, Intervet), ivermectin (Eqvalan, Merial) or moxidectin (Quest, Fort Dodge) were administered once to horses scheduled for routine parasiticide treatment. In total, 93 horses from six cooperating farms were used in the study. Computer generated, random allocation of horses to treatment group was conducted at each farm. Fecal egg counts were determined for all horses on trial days 0, 56, 84 and 112, with corresponding calendar dates that were unique to each farm. Only strongyle egg counts from animals which were positive at day 0 were used for analysis of variance and comparisons. Counts for the three treatment groups were similar at day 0, moxidectin相似文献   

Exercise-induced pulmonary haemorrhage in the horses: results of a detailed clinical, post mortem and imaging study. VII. Ventilation/perfusion scintigraphy in horses with EIPH

Detailed post mortem examination of the lungs of horses with exercise-induced pulmonary haemorrhage (EIPH) has demonstrated significant small airway disease and intense bronchial arterial proliferation in the dorsocaudal lungfields. The purpose of this study was to investigate ventilation and perfusion distribution in the lungs of a similar group of horses to compare changes in the live animal with the previously reported post mortem findings. Thoracic radiography and ventilation/perfusion (V/Q) scintigraphy were performed on five racing Thoroughbreds with recent histories of EIPH. Parametric images of V/Q ratios for left and right lungfields were also generated from the scan images. In all horses, ventilation and perfusion deficits were demonstrated in the dorsocaudal areas of the lung corresponding closely to the observed radiographic lesions. In particular, the perfusion images and V/Q ratio displays indicated that, in affected areas of lung, pulmonary arterial perfusion was the more seriously impaired. This finding appears to confirm the post mortem evidence of reduced pulmonary arterial perfusion and bronchial arterial dominance in these areas. Ventilation deficits in the same areas also confirmed the likelihood of partial airway obstruction consistent with the small airway disease noted in previous post mortem observations. These results suggest that the vascular and airway lesions demonstrated in detailed post mortems of horses with EIPH are also functionally important in affected horses, even at rest. As a consequence of the apparent persistent, insidious and progressive nature of the lesions associated with EIPH there are serious long term implications for management of the condition.     

Effects on plasma endotoxin and eicosanoid concentrations and serum cytokine activities in horses competing in a 48-, 83-, or 159-km endurance ride under similar terrain and weather conditions

OBJECTIVE: To determine plasma endotoxin concentration in horses competing in a 48-, 83-, or 159-km endurance race and its importance with regard to physical, hematologic, or serum and plasma biochemical variables. ANIMAL: 3 horses. PROCEDURE: Weight and rectal temperature measurements and blood samples were obtained before, during, and after exercise. Blood samples were analyzed for plasma endotoxin concentration; serum antiendotoxin antibody titers; thromboxane B2 (TxB2) and 6-keto-prostaglandin F1alpha (PGF1alpha) concentrations; tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) activities; WBC, plasma protein, lactate, serum electrolyte, and calcium concentrations; PCV; and creatine kinase activity. RESULTS: Detection of plasma endotoxin increased during exercise for horses competing at all distances but occurred more frequently in the 48- and 83-km groups. Plasma lactate concentration was significantly greater when endotoxin was concurrently detected. Endotoxin in plasma was not significantly associated with success of race completion. Plasma TxB2 and PGF1alpha concentrations and serum IL-6 activity significantly increased with exercise. Horses that had an excellent fitness level (as perceived by their owners) had greater decreases in serum antiendotoxin antibody titers during exercise than did horses perceived as less fit. In horses with better finish times, TxB2 and PGF1alpha concentrations were significantly greater and TNFalpha activity was significantly less than that of slower horses. CONCLUSIONS AND CLINICAL RELEVANCE: Endotoxemia developed during endurance racing, but was significantly correlated with increased plasma lactate concentration and not with other variables indicative of endotoxemia. Plasma TxB2 and PGF1alpha concentrations and serum TNFalpha activity may be associated with performance success.     

Characterisation of lymphocyte subpopulations in the skin and circulation of horses with sweet itch (Culicoides hypersensitivity)

Circulating lymphocyte numbers are elevated in horses with the allergic skin disease sweet itch and skin lesions are typified by an infiltrate of eosinophils and mononuclear cells, the latter of which have not been fully characterised. The aim of the present study was to characterise the lymphocyte subpopulations in the circulation and skin of ponies with sweet itch by flow cytometry and a newly developed modified alkaline phosphatase immunohistochemical technique. Sweet itch ponies were found to have significantly greater numbers of circulating CD5+ and CD4+ T-lymphocytes than normal animals. Increased numbers of CD3+ T-lymphocytes, most of which were CD4+, and eosinophils were present in the skin of these animals following intradermal injection of a Culicoides antigen extract (97 +/- 21 vs. 449 +/- 49 CD3+ T-lymphocytes/mm2 in deep dermis of vehicle vs. antigen injected sites; 83 +/- 8% CD4+ T-lymphocytes at antigen injected site). T-lymphocytes, which are thought to be important in the pathogenesis of human allergic skin disease, may therefore contribute to the development of sweet itch lesions via the release of cytokines which can cause eosinophil accumulation and activation. An understanding of the pathology of this disease may lead to a more rational approach to therapy.     

Intradermal testing in healthy horses and horses with chronic obstructive pulmonary disease, recurrent urticaria, or allergic dermatitis.

OBJECTIVE: To compare responses to a variety of intradermally injected allergens among healthy horses and horses with chronic obstructive pulmonary disease (COPD), recurrent urticaria (RU), and atopic dermatitis-insect hypersensitivity (allergic dermatitis [AD]). DESIGN: Case-control study. ANIMALS: 86 horses. PROCEDURE: Results of intradermal testing for horses with COPD, RU, or AD were compared with results for healthy horses. RESULTS: Compared with healthy horses, horses with COPD, RU, and AD were significantly more likely to have positive (> or = 3+) reactions to intradermal allergens (molds, weeds, trees, grasses-crops, and insects) 30 minutes (immediate reaction), 4 hours (late-phase reactions), and 24 hours (delayed-phase reactions) after exposure. In addition, diseased horses reacted to a significantly higher number of allergens in each allergen group than did healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE: Reactions to individual allergens should not be used to determine that horses have hypersensitivity. Overall patterns of reactivity to intradermal allergens may be helpful in management when used in conjunction with a compatible history and evidence of potential exposure to allergens in horses with conditions associated with hypersensitivity to environmental allergens.     

谷氨酰胺对热应激肉鸡S-IgA、血液内毒素和细胞因子的影响

选用1日龄科宝-500肉鸡240只,随机分为6个处理,每处理4个重复,每重复10只,Ⅰ组饲喂玉米-豆粕型基础日粮,Ⅱ、Ⅲ、Ⅳ、Ⅴ和Ⅵ组分别在基础日粮中添加0.4%、0.8%、1.2%、1.6%和2.0%的谷氨酰胺,试验时间15～42日龄,共28d。试验期间从每天09：00-17：00温度维持在（35±1）℃8h,17：00至次日09：00温度维持在（30±1）℃,鸡舍相对湿度控制在70%～80%。试验测定了热应激条件下21、28、35、42日龄肉鸡小肠黏膜分泌型免疫球蛋白含量A,血浆中血液内毒素含量和白介素-1、肿瘤细胞坏死因子含量。结果表明,日粮中添加谷氨酰胺显著提高了21、28、35、42日龄热应激肉鸡小肠黏膜分泌型免疫球蛋白含量（P〈0.05）,降低血浆中血液内毒素、白介素-1、肿瘤细胞坏死因子的含量（P〈0.05）。因此,在基础日粮中添加一定水平的谷氨酰胺可改善热应激肉鸡的肠道免疫性能。     

Endotoxin-induced production of interleukin 6 by equine peritoneal macrophages in vitro.

A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (IL-6) in vitro in response to endotoxin. Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages. Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5). Culture medium supernatants were collected after 3, 6, 12, and 24 hours' incubation and were frozen at -70 C until assayed for IL-6 activity. Supernatant IL-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B 13.29 clone B.9, which is dependent on IL-6 for survival. Results indicated that equine peritoneal macrophages produce IL-6 in vitro and that supernatant medium IL-6 activity was significantly (P less than 0.05) increased by exposure to endotoxin. Significant (P less than 0.05) time and treatment effects on macrophage IL-6 production were apparent. The IL-6 activity peaked at 6 or 12 hours' incubation, then remained high through 24 hours' incubation, regardless of endotoxin exposure. Medium IL-6 activity during 3 and 6 hours' incubation was significantly (P less than 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak IL-6 activity was similar among all endotoxin concentrations. Endotoxin concentration did not have an effect on medium IL-6 activity from macrophages exposed to endotoxin for 12 or 24 hours.     

Effects of an infusion of dopamine on the cardiopulmonary effects of Escherichia coli endotoxin in anaesthetised horses

Horses with colic may be endotoxaemic and subsequently develop hypotension during anaesthesia for surgical operation. The aim of this study was to evaluate the efficacy of dopamine as a means to improve cardiovascular function in anaesthetised endotoxaemic horses. Nine horses (five in group 1 and four in group 2) were anaesthetised with thiopentone and guaifenesin and anaesthesia was maintained with halothane. After approximately one hour, facial artery pressure, heart rate, pulmonary artery pressure, cardiac output, temperature, pHa, PaCO2, PaO2, base excess, packed cell volume, plasma protein concentration and white cell count were measured (time 0). Escherichia coli endotoxin was infused intravenously over 15 minutes in both groups. Group 2 horses were given an intravenous infusion of dopamine (5 micrograms kg-1 min-1) starting five minutes after the start of the endotoxin infusion and continuing for 60 minutes. Measurements were made at 15 minute intervals for 120 minutes. In group 1, one horse died during the endotoxin infusion and in two other horses mean facial artery pressures decreased to 50 mm Hg. Total pulmonary vascular resistance and packed cell volume were significantly increased. Cardiac output, cardiac index and change in mean arterial pressure were significantly greater in group 2 horses than in group 1 horses. Conversely, diastolic pulmonary artery pressure, total vascular resistance and total pulmonary resistance were significantly less in group 2 than in group 1. PaO2, base excess and white blood cell count were significantly decreased in both groups. It was concluded that dopamine improved cardiovascular function in the presence of endotoxaemia and attenuated the rate of haemoconcentration, but had no effect on the development of decreased PaO2 or metabolic acidosis.