Effect of strenuous exercise stress on chemiluminescence response of equine alveolar macrophages

Bronchoalveolar lavage samples were collected using a fibreoptic endoscope from horses at specified times before and after single bouts of exercise. Lucigenin-dependent phagocytic chemiluminescence was used to assess the effect of exercise on the alveolar macrophage metabolic activity in response to stimulation by opsonised zymosan. A profound suppressive effect on the chemiluminescence production was present throughout the first three days after exercise. However, the cellular composition of lavage fluids was not altered by the exercise. It is suggested that strenuous exercise may jeopardize the antimicrobial function of alveolar macrophages which may lead to an increase in susceptibility to infection.     

The effect of strenuous exercise on mRNA concentrations of interleukin-12, interferon-gamma and interleukin-4 in equine pulmonary and peripheral blood mononuclear cells

The effect of strenuous exercise on the mRNA concentrations of interleukin-12p35 subunit (IL-12p35), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in equine pulmonary and peripheral blood mononuclear cells (PBMCs) was investigated. We hypothesized that strenuous exercise would suppress the expression of IL-12p35, IFN-gamma and augment the expression of IL-4. Eleven horses were randomly divided into two groups, a stall-confined control group (n=5) and an exercise-conditioned treatment group (n=6). Bronchoalveolar and PBMCs were obtained from horses in the treatment group prior to the commencement of a 9-week conditioning program and 24h after the completion of a maximum exercise test conducted in week 12. Samples were obtained simultaneously from control horses. Differential counts were performed on the bronchoalveolar lavage cells. Real-time PCR was performed on the pulmonary and PBMCs to quantitate cytokine expression using equine-specific primers and Taqman probes. Target gene expression was normalized to 18s rRNA expression. With the exception of IL-4 in the BALF cells, mRNA for the three cytokines was detected in the mononuclear cells from all horses at both sampling times. There were no significant differences in the cytokine mRNA concentrations between the two groups of horses at either of the sampling times. These findings demonstrate that strenuous treadmill exercise does not exert a deleterious effect on gene expression for IL-12p35, IFN-gamma or IL-4 when assessed in horses 24h following the intense physical activity.     

Effect of lipopolysaccharide infusion on gene expression of inflammatory cytokines in normal horses in vivo

Horses are exquisitely sensitive to bacterial endotoxin and endotoxaemia is common in colic cases. In this study, gene expression of inflammatory cytokines was characterised in the blood of healthy horses following i.v. administration of lipopolysaccharide (LPS). Six horses received an LPS infusion and 6 controls received an equivalent volume of saline. Gene expression of genes encoding interleukin (IL)‐1α, IL‐1β, IL‐6, IL‐8, and tumour necrosis factor‐α (TNF‐α) was quantified by real‐time PCR. Gene expression of all inflammatory cytokines was upregulated following administration of LPS. Interleukin‐1α, IL‐1β, IL‐8 and TNF‐α gene expression peaked at 60 min, while IL‐6 expression peaked at 90 min post LPS infusion. Interleukin‐1β and IL‐6 messenger RNA expression levels were above the baseline values 3 h post LPS infusion, whereas IL‐1α, IL‐8 and TNF‐α expression levels returned to baseline values by 3 h after LPS infusion. It was concluded that LPS infusion upregulated gene expression of inflammatory cytokines in the blood of healthy horses.     

Functional characterization of equine dendritic cells propagated ex vivo using recombinant human GM-CSF and recombinant equine IL-4

Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone marrow ex vivo with various cytokines. However, all other model systems, including equine, still require the laborious isolation procedures to obtain DC. In this study, we have adapted the methods developed for the human system to generate large numbers of equine DC from PBMC precursors using recombinant human GM-CSF and recombinant equine IL-4. Our report is the first documentation of ex vivo generated DC from PBMC in a domesticated animal model system. Equine DC derived from PBMC were rigorously characterized by analyzing morphological, phenotypic, and functional properties and were determined to have similar attributes as DC generated from human PBMC. Equine DC appeared stellate with large projectiles and veils and had cell surface antigens at similar levels as those defined on human and murine DC. Furthermore, functional attributes of the DC included rapidly capturing antigens by pinocytosis, receptor-mediated endocytosis, and phagocytosis, activating naive T cells in a mixed leukocyte reaction to a much greater extent than macrophage or lymphoblasts, presenting soluble and particulate antigen 10-100 fold more effectively to T cells on a per cell basis than macrophage or lymphoblasts, and presenting soluble and particulate antigen to both CD4+ and CD8+ T cells. Taken together, our study provides a framework by which equine DC can now be readily produced from PBMC precursors and presents an impetus for and model by which DC can be simply generated in other animal model systems.     

Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis

Reasons for performing study: There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis‐related laminitis than the black walnut extract (BWE) model. Objectives: To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. Methods: Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature >38.9°C (DEV group, n = 8) or at onset of Obel grade 1 lameness (OG1 group, n = 8). Control horses (CON group, n = 8) were anaesthetised 24 h after NG administration of 6 l of deionised water. Laminar tissue was collected from horses while under anaesthesia, followed by humane euthanasia. Real time‐quantitative PCR was used to assess laminar mRNA concentrations of genes involved in inflammatory signalling. Results: Increased mRNA concentrations (P<0.05) for IL‐1β, IL‐6, IL‐12p35, COX‐2, E‐selectin and ICAM‐1 were present in laminae from horses with OG1 lameness but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL‐2, IL‐4, IL‐10, TNF‐α, IFN‐γ or COX‐1 at either the DEV or OG1 time points. Conclusions: There was a notable difference in the temporal pattern of inflammatory events between the BWE and CHO models, with the majority of laminar inflammatory events appearing to occur at or near the onset of lameness in the CHO model, whereas many of these events peak earlier in the developmental stages in the BWE model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leucocyte emigration and proinflammatory cytokine expression. Potential relevance: The similar (although somewhat delayed) inflammatory response in the CHO model of laminitis indicates that inflammatory signalling is a consistent entity in the pathophysiology of laminitis.     

Cloning and expression of recombinant equine interleukin-3 and its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes

Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL).Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culicoides nubeculosus extract with or without IL-3. Sulfidoleukotriene (sLT) production was measured in supernatants by ELISA and mRNA expression of IL-4, IL-13 and thymic stromal lymphopoietin (TSLP) assessed in cell lysate by quantitative real-time PCR.Recombinant equine IL-3 (req-IL-3) had a dose dependent effect on sLT production by stimulated equine PBL and significantly increased IL-4, IL-13 and TSLP expression compared to non-primed cells.IL-3 priming significantly increased Culicoides-induced sLT production in IBH-affected but not in non-affected horses and was particularly effective in young IBH-affected horses (≤3 years).A functionally active recombinant equine IL-3 has been produced which will be useful for future immunological studies in horses. It will also allow improving the sensitivity of cellular in vitro tests for allergy diagnosis in horses.     

In vitro and ex vivo effects of the phosphodiesterase 4 inhibitor,rolipram, on thromboxane production in equine blood

Phosphodiesterase 4 (PDE4) inhibitors have been shown to inhibit equine neutrophil function in vitro and may be of benefit in recurrent airway obstruction (RAO), an allergy-based respiratory disease characterized by inflammatory cell recruitment and activation within the lungs following exposure of susceptible horses to allergens in mouldy hay. The aim of this study was to evaluate the inhibitory effects of the PDE4 inhibitor, rolipram, in an in vitro assay of thromboxane (Tx) production. The assay was then used to monitor the activity of this compound in vivo in normal and RAO-affected horses. Rolipram and the structurally distinct PDE4 inhibitor, denbufylline, attenuated both lipopolysaccharide (LPS)-induced and unstimulated Tx production in blood from normal horses. Thromboxane production appeared to involve a calcium-dependent interaction between leucocytes and platelets (LPS-induced Tx production = 2.3 +/- 0.4, 4.5 +/- 1.1 and 20.8 +/- 3.6 ng/mL for platelets, leucocytes and blood, respectively) and rolipram-inhibited Tx production via an effect on leucocytes. Inhibition of ex vivo LPS induced Tx production was detected after intravenous administration of rolipram (5 microg/kg) to normal ponies. This dose did not significantly affect either lung function or neutrophil accumulation when administered to three horses with clinical signs of RAO. This study suggests that inhibition of Tx production in equine blood can be used to measure PDE4 activity. However, PDE4 inhibitors with improved therapeutic profiles are required for evaluation in RAO.     

喜树碱对NIH/3T3细胞TLR3表达及活化的影响

为研究喜树碱(CPT)对小鼠成纤维细胞NIH/3T3中Toll样受体3(TLR3)的表达及活化的影响,本实验利用CCK-8试剂盒测定了CPT在不同浓度下、不同作用时间对NIH/3T3细胞的毒性作用,并确定CPT对NIH/3T3细胞的基本无毒浓度为1μg/mL。通过流式细胞术检测CPT作用NIH/3T3细胞表明,TLR3平均荧光强度与对照组相比显著增强,表明TLR3表达增加。此外,ELISA试剂盒检测结果显示CPT作用后IFN-β表达量增加,并高于阳性对照组,差异极显著(p<0.01)。本研究表明CPT对NIH/3T3细胞的TLR3具有显著的激动作用。     

脂多糖对小鼠子宫内膜Toll样受体4介导的炎性信号通路的影响

革兰阴性菌感染可引起子宫内膜炎,机体的Toll样受体4(TLR4)可接受革兰阴性菌的细胞壁主要成分脂多糖(LPS)的刺激引发一系列炎症反应。为了研究LPS对TLR4介导的炎性信号通路的影响,本试验以小鼠为模型,分别用不同浓度的LPS对小鼠进行在体子宫灌注和处理体外培养的子宫内膜上皮细胞系。组织学观察显示,灌注不同浓度LPS后子宫内膜组织中炎性细胞增多。通过RT-PCR对各组中TLR4和核转录因子κB(NF-κB)、IL-6的mRNA表达水平进行检测,发现LPS刺激能够增强TLR4和NF-κB、IL-6 mRNA表达,影响TLR4介导的炎性信号通路。     

Cloning, expression and bioinformatics analysis of the duck TLR 4 gene

1. Toll-like receptors (TLRs) are type I transmembrane proteins that play an essential role in the innate immune system. Studies on the structure and function of TLRs can be applied to the development of new approaches to control diseases of humans and animals.

2. A 3432-bp cDNA encoding duck toll-like receptor 4 (duTLR4) was cloned from duck splenic lymphocytes using RT-PCR and rapid amplification of cDNA ends.

3. The encoded protein, which was predicted to contain 843 amino acids, had a molecular weight of 96·01?kDa and included an archetypal toll/interleukin-1 receptor domain, a transmembrane domain, and a distinctive arrangement of extracellular leucine-rich repeat regions similar to chicken TLR4, human TLR4, and mouse TLR4. The duTLR4 showed 82·1% amino acid sequence identity with previously described chicken TLR4, and 43·2–45·2% sequence identity with mammalian homologs.

4. RT-qPCR analysis indicated that the duTLR4 gene was strongly expressed in the liver, kidney, spleen, intestine, and brain.     

Differential gene expression of CYP3A isoforms in equine liver and intestines

Tydén, E., Löfgren, M., Pegolo, S., Capolongo, F., Tjälve, H., Larsson, P. Differential gene expression of CYP3A isoforms in equine liver and intestines. J. vet. Pharmacol. Therap.  35 , 588–595. Recently, seven CYP3A isoforms – CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 – have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin‐isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K_m for the LIPA substrate in the liver and the intestine, while the maximum velocity (V_max) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses.     

IL-4, IL-5 and IFN-gamma mRNA expression in pulmonary lymphocytes in equine heaves

Heaves is a common condition of horses of cold climate that is characterized by small airway inflammation and obstruction following exposure of susceptible horses to moldy hay and straw. It has been shown that helper T lymphocytes (Th) orchestrate the inflammatory response in asthma and in various animal models of allergic lung diseases by the release of Th2-type cytokines. Results of previous studies indicate that a predominant expression of Th2-type response by airway cells may also be present in heaves. To evaluate the temporal mRNA expression of Th1 (IFN-gamma) and Th2 (IL-4, IL-5) type cytokines in heaves and their relationship to clinical disease, we studied the pulmonary mechanics and cytokine mRNA expression (IL-4, IL-5 and IFN-gamma) in bronchoalveolar lavage lymphocytes of horses with heaves (n=6) and control (n=6) before and after 24h and 9 days of continuous natural inhalation challenge. Starting 24h after challenge horses with heaves, but not control horses, had a significant increase in pulmonary elastance and the number of lymphocytes expressing mRNA for IL-4 and IL-5. These changes were further increased at 9 days, at which time the number of cells positive for IFN-gamma mRNA was decreased. In this study we have shown that BAL lymphocytes of horses with heaves during clinical exacerbation have a predominant Th2-type cytokine response and that this response coincides in time with the presence of airway obstruction.     

In vitro and ex vivo pharmacodynamics of selected non-steroidal anti-inflammatory drugs in equine whole blood

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX), and the inhibition of COX-2 rather than COX-1 can limit the onset of NSAID-related adverse effects. The pharmacodynamic properties of eltenac, naproxen, tepoxalin, SC-560 and NS 398 in healthy horses were investigated using an in vitro whole blood assay. To predict COX selectivity in clinical use, eltenac and naproxen were also studied ex vivo after intravenous administration. SC-560 acted as a selective COX-1 inhibitor, tepoxalin as a dual inhibitor with potent activity against COX-1, and NS 398 as a preferential COX-2 inhibitor. Eltenac was a preferential COX-2 inhibitor in vitro but un-selective in the ex vivo study. Naproxen maintained its non-selectivity both in vitro and ex vivo. These findings have demonstrated that in vitro studies may not accurately predict in vivo NSAID selectivity for COX and should be confirmed using an ex vivo whole blood assay.     

Effect of exercise on the partitioning of equine respiratory resistance

Pleural, tracheal, pharyngeal and mask pressures as well as airflow and tidal volume of five ponies on a treadmill (incline 8.3 degrees) were recorded simultaneously while resting, walking, trotting slowly, trotting fast, after standing for 30 secs and 5 mins after the end of the exercise. The curves obtained were used to calculate total pulmonary resistance (RL), lower airway resistance (RLA) and upper airway resistance (RUA). The latter was also divided into nasal resistance (Rnas) and laryngeal plus extrathoracic tracheal resistance (Retr + lar). Furthermore, the inspiratory and expiratory components of each of these R values were estimated. Levels of RL, RLA and RUA were increased significantly during exercise but, during the recovery period, the values were significantly lower than those pre-exercise. RUA represented 82 per cent of RL at rest and this percentage did not change significantly during and after exercise. The nasal resistance to RUA ratio was always higher than 0.5. The fact that RL increased with exercise intensity was due to the increase of RUA during inspiration, and mainly a result of the increase of RLA during expiration. At fast trot, RLA represented 5 and 50 per cent of RL during inspiration and expiration respectively. It was concluded that heavy exercise induces in ponies an increase of RL, one reason for which could be the partial collapse of the extrathoracic and intrathoracic airways during inspiration and expiration respectively.