Effect of nutritional antioxidant supplementation on systemic and pulmonary antioxidant status,airway inflammation and lung function in heaves-affected horses

An oxidant/antioxidant imbalance in favour of oxidants has been identified as playing a decisive role in the pathogenesis of chronic inflammatory airway diseases. Nutritional antioxidant supplementation might reduce oxidative damage by enhancement of the antioxidant defence, thereby modulating inflammatory processes. In a placebo-controlled, blind study, it was tested whether a dietary antioxidant supplement administered for 4 weeks would improve lung function and reduce airway inflammation in heaves-affected horses. Eight horses in clinical remission of heaves were investigated at rest and after a standardised exercise test before and after treatment with an antioxidant supplement (consisting of a mixture of natural antioxidants including vitamins E and C and selenium from a variety of sources) or placebo (oatfeed pellets without additive). Pulmonary function and exercise tolerance were monitored; systemic and pulmonary lining fluid uric acid, glutathione and 8-epi-PGF(2alpha) were analysed, and bronchoalveolar lavage (BAL) cytology and inflammatory scoring of the airways were performed. The antioxidant treatment significantly improved exercise tolerance and significantly reduced endoscopic inflammatory score. Plasma uric acid concentrations were significantly reduced, suggesting downregulation of the xanthine-dehydrogenase and xanthine-oxydase pathway. Haemolysate glutathione showed a nonsignificant trend to increase, while plasma 8-epi-PGF(2alpha) remained unchanged. Pulmonary markers and BAL cytology were not significantly affected by antioxidant supplementation. The present study suggests that the antioxidant supplement tested modulated oxidant/antioxidant balance and airway inflammation of heaves-affected horses.     

Cytokine expression by peripheral blood neutrophils from heaves-affected horses before and after allergen challenge

Heaves, also known as recurrent airway obstruction, is a common condition of horses characterised by pulmonary neutrophilia and reversible airway obstruction. This study evaluated the role of neutrophils in producing cytokines and chemokines that might be involved in the recruitment and activation of inflammatory cells in horses with heaves. Peripheral neutrophils were isolated from heaves-affected (n = 9) and control (n = 4) horses before and after 5 h of natural inhalation challenge. Expression of mRNA of two pro-inflammatory cytokines, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta, along with two chemokines, IL-8 and macrophage inflammatory protein (MIP)-2, was evaluated. After exposure to mouldy hay, horses with heaves had significant airway obstruction and increased numbers of neutrophils in bronchoalveolar lavage samples, compared to control horses. However, there were no differences in the expression of mRNAs of TNF-alpha, IL-1beta, IL-8 and MIP-2 between the two groups, suggesting that the release of cytokines and chemokines by peripheral blood neutrophils is not necessary for the development of heaves.     

Comparison of prednisolone and dexamethasone effects in the presence of environmental control in heaves-affected horses

This study was designed to compare the efficacy of oral prednisolone and intramuscular (IM) dexamethasone in heaves-affected horses with environmental control. A total of 16 horses, aged 8-20years, with heaves were included in the study. Complete examinations were performed on Day 0 (before treatment), Day 13 (after treatment) and Day 30. Clinical variables, arterial blood gases, mucus scoring and carina evaluation (during endoscopy), and bronchoalveolar lavage (BAL) cytological analysis were all assessed. The horses were randomly assigned to receive either oral prednisolone (1mg/kg) or IM dexamethasone (0.1mg/kg). The animals were clinically scored and mucus accumulation evaluated. Results were analysed by repeated measures ANOVA with time (days of treatment) and treatment as the main effects. When combined with environmental control, prednisolone and dexamethasone treatments had similar effects on heaves score, blood gases and endoscopic scores. However, dexamethasone had a more beneficial effect on BAL cytology.     

Effect of chronic airway inflammation and exercise on pulmonary and systemic antioxidant status of healthy and heaves-affected horses

In heaves-affected horses the relation between oxidant status, airway inflammation (AI) and pulmonary function (PF) is unknown. The oxidant status of blood and pulmonary epithelial lining fluid (PELF) of healthy (H, n = 6) and heaves-affected horses in clinical remission (REM, n = 6) and in crisis (CR, n = 7) was assessed at rest, during and after standardised exercise test by measurement of reduced and oxidised glutathione, glutathione redox ratio [GRR%]; uric acid and 8-epi-PGF2alpha. Oxidant status was related to PF parameters (mechanics of breathing and arterial blood gas tension) and Al parameters (bronchoalveolar lavage [BAL] neutrophil % and AI score). Haemolysate glutathione was significantly different between groups and was correlated with PF and AI parameters; GRR in PELF was increased during CR and was correlated with PF and AI parameters. Exercise induced an increase of plasma uric acid that was significantly higher both in REM and CR. PELF 8-epi-PGF2alpha was significantly increased in CR and correlated with PF and AI parameters. These results suggest that oxidative stress occurring in heaves is correlated with PF and AI and may be locally assessed by PELF glutathione status, uric acid and 8-epi-PGF2alpha. Systemic repercussions are reflected by assay of GSH in resting horses and by uric acid in exercising horses.     

Comparison of inulin with urea as dilutional markers of bronchoalveolar lavage in healthy and heaves-affected horses

Solute analysis in bronchoalveolar lavage fluid involves the use of dilutional markers to correct for variable recovery of pulmonary epithelial lining fluid (PELF). Urea is the best characterised endogenous marker, whereas inulin appears to meet the requirements of an exogenous marker. In horses, the use of inulin has never been investigated and the impact of lower airway diseases such as heaves, on PELF recovery is unknown. In this study, five healthy and five heaves-affected horses underwent airway endoscopy and bronchoalveolar lavage. PELF recovery from bronchoalveolar lavage was calculated by the inulin and the urea method. The inulin method was compared to the urea method and differences between healthy and heaves-affected horses were analysed. From a technical and analytical point of view, inulin fulfilled the requirements of a marker of dilution as well as urea. When both healthy and heaves-affected horses groups were pooled together, PELF recovery calculated by the inulin method was significantly higher than by the urea method (6.43+/-4.08% versus 0.789+/-0.299%, P < 0.005). No significant differences were observed between healthy and heaves-affected horses, neither by the inulin nor by the urea method. Inulin did not present major advantages over urea, but the combined use of both markers can improve the standardisation of studies comparing PELF compounds, by providing upper limits (inulin dilution) and lower limits (urea dilution) of PELF recovery.     

Effects of the bronchoalveolar lavage procedure on lung function in horses with clinical exacerbation of recurrent airway obstruction

OBJECTIVE: To evaluate whether bronchoalveolar lavage (BAL) alters respiratory mechanics of horses with recurrent airway obstruction (ie, heaves) over a 48-hour period. ANIMALS: 6 horses affected with heaves. PROCEDURES: Horses were subjected to a complete BAL procedure, which included sedation with xylazine and butorphanol, intratracheal administration of lidocaine, and instillation and aspiration of two 250-mL boluses of saline (0.9% NaCl) solution through an endoscope (study 1). To evaluate the effects of saline solution, horses were subjected to the same procedure without saline solution instillation and aspiration (study 2). Lastly, the endoscope was similarly introduced into the lower airways, without sedation or saline instillation and aspiration (study 3). Respiratory mechanics were performed at baseline (time 0) and at 3, 6, 12, 24, and 48 hours after each procedure. RESULTS: In study 1, BAL induced a significant decrease in pulmonary resistance lasting up to 6 hours. This may have resulted from clearance of mucus in large airways. We also observed a significant increase in lung elastance and transpulmonary pressure at 12 hours after BAL in all 3 studies, which may be attributed to a circadian effect. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that the temporal effects of BAL procedures on lung mechanics should be taken into account when designing research protocols involving horses with heaves. Future studies should address the immediate effects of BAL on lung function.     

Cytokine induction in pulmonary airways of horses with heaves and effect of therapy with inhaled fluticasone propionate

Work in humans and laboratory animals has identified a central role for cytokines and chemokines in development and persistence of lower airway inflammation. The objectives of this study were to determine interleukin (IL)-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha induction in bronchoalveolar lavage (BAL) of control horses and horses with heaves both during remission and exacerbation of the disease, and to determine the effect of therapy with inhaled fluticasone propionate on the cytokine profile of horses with heaves. IL-1 beta and TNF-alpha mRNA expression was significantly higher in horses with heaves after exposure to moldy hay compared to either values obtained during clinical remission or to healthy controls. IL-8 mRNA expression and protein concentrations were significantly higher in horses with heaves than in controls. Both IL-4 and IFN-gamma mRNA expression was increased at various times in heaves-susceptible horses compared to controls. IL-2, IL-5 and IL-10 mRNA expression was not detected in BAL cells of either group. Therapy with inhaled fluticasone propionate after induction of a severe heaves exacerbation resulted in complete resolution of clinical signs, normalization of pulmonary function tests, and significant decrease in BAL neutrophilia. This was associated with a significant decrease in IL-4 mRNA expression and increase in IFN-gamma/IL-4 ratio in horses with heaves. These results demonstrate the clinical efficacy of inhaled fluticasone propionate for the treatment of heaves and suggest a role for cytokines in the development of lower airway inflammation in heaves-susceptible 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.     

Chronic exacerbation of equine heaves is associated with an increased expression of interleukin-17 mRNA in bronchoalveolar lavage cells

Recent finding suggests that T-cells may be involved in the pathogenesis of heaves in horses. However, little is known concerning their possible contribution to pulmonary neutrophilia, a characteristic finding in heaves. Interleukin (IL)-17 is a cytokine secreted by activated T-cells that indirectly promotes the maturation, chemotaxis and activation of neutrophils. We therefore hypothesized that IL-17 may be involved in the recruitment of neutrophils into the airways and that its mRNA expression would be increased in bronchoalveolar lavage (BAL) cells of horses with heaves. Heaves susceptible horses (n=4) and control horses (n=4) when in pasture (clinical remission) and after 35 days of continuous exposure to moldy hay were studied. BAL and respiratory mechanics measurements were performed at both time periods. The mRNA expression of IL-17 in BAL was studied using real-time polymerase chain reaction (PCR) and CD3-zeta was used as a marker of T-cell numbers. There was no significant difference in IL-17 mRNA expression between groups of horses while in pasture. However, stabling resulted in an increased expression of IL-17 in all horses with heaves but in none of the control horses. These preliminary results suggest that IL-17 may contribute in the pathogenesis of horses with heaves following chronic antigen challenge.     

The effects of a single acupuncture treatment in horses with severe recurrent airway obstruction

REASONS FOR PERFORMING STUDY: Acupuncture may be recommended for horses with 'heaves' because it is being increasingly applied to treat human asthma. Therefore, its efficacy was investigated in horses with this asthma-like disease. OBJECTIVE: To evaluate the efficacy of a single acupuncture treatment for the relief of airway obstruction in heaves-affected horses. METHODS: The efficacy of a single acupuncture treatment was tested in 10 heaves-affected horses, and the effect of removal from the dusty stall environment in 5 heaves-affected horses. Before treatment, horses were stabled to induce airway obstruction and, apart from trips to the laboratory for pulmonary function measurements, they remained stabled for the duration of each treatment. The severity of airway obstruction was quantified by measurement of lung function before treatment (baseline), and at 20, 60, 120 and 240 mins and 24 h after the following treatments administered in random order: halter restraint and patting, a single acupuncture treatment by an experienced acupuncturist, and a single acupuncture treatment using predetermined points (recipe) by a veterinarian with no acupuncture training. In a second study, horses were untreated and remained either in their stall or in a paddock for all measurements of lung function, after baseline readings were made. RESULTS: In the first study, after all treatments, there was a temporal improvement in maximal change in pleural pressure, pulmonary resistance, dynamic compliance, respiratory rate, and tidal volume that lasted less than 24 h. There was no specific effect of acupuncture treatment. In the second study, removal from the dusty environment did not produce an improvement in lung function in the first 6 h. We conclude that most of the improvements in lung function observed in the study were due to handling. CONCLUSIONS: Assessed objectively, a single acupuncture treatment during an attack of heaves causes no more improvement in lung function than does handling the horse. POTENTIAL RELEVANCE: Acupuncture should not replace conventional medical treatments for heaves.     

Airway mucus in recurrent airway obstruction--short-term response to environmental challenge

Mucus accumulation and neutrophilic inflammation in the airways are hallmarks of heaves. Endoscopically visible mucus accumulations, however, have not been studied during exposure to dusty hay and allergens (ie, environmental challenge). We hypothesized that (1) heaves-affected horses have increased mucus accumulation compared with controls, (2) mucus accumulations increase in heaves-affected horses during environmental challenge, and (3) environmental challenge also induces neutrophilic inflammation and mucus accumulation in control horses. Mucus accumulation was graded endoscopically (mucus grades [MGs] 1-5), and airway inflammation was evaluated by bronchoalveolar lavage fluid (BALF) cytology before (0 hours) and during (6, 24, 48 hours) environmental challenge. Large amounts of mucus (MG 4-5) were specific for heaves-affected horses in this study. Variation among controls was considerable, however, and intermediate grades (MG 2-3) were nonspecific, showing complete overlap between the 2 groups. Median mucus accumulations (25th, 75th percentiles) increased in heaves-affected horses from MG 2.5 (1.5, 3.5) at baseline to MG 3.5 (2.0, 4.0), 4.0 (3.0, 4.0), and 4.0 (4.0, 4.0) at 6, 24, and 48 hours, respectively. MG values did not increase in controls--overall MG 1.0 (1.0, 2.0)--even though controls also showed a moderate increase of BALF neutrophils. Mucus accumulations before and especially after exposure to dust and allergens are increased in heaves-affected horses compared with controls. Healthy controls show considerable variability in mucus accumulation but, despite an influx of neutrophils into the airways, no increase of mucus accumulation after exposure to hay dust.     

Lack of clinical efficacy of a phosphodiesterase-4 inhibitor for treatment of heaves in horses

Phosphodiesterase-4 (PDE 4) enzyme inhibitors have been shown to have anti-inflammatory properties in various animal disease processes and therefore could be effective drugs for the treatment of equine airway diseases. The purpose of this study was to evaluate the efficacy and adverse effects of the PDE 4 inhibitor L-826,141 in horses with heaves. In a blinded parallel design, horses with heaves exposed daily to moldy hay were given a placebo for 14 days and then administered either L-826,141 (n = 6; loading dose of 1 mg/kg IV followed by 0.5 mg/kg IV q48h) or dexamethasone (n = 6; 0.04 mg/kg IV q24h) from days 15 to 29 (study 1). Pulmonary function and bronchoalveolar (BAL) cytology were evaluated weekly from baseline (day 0) to 29 days. In study 2, horses were treated with L-826,141 (1.0 mg/kg IV q24h) for 8 days. Although ex vivo lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha and LTB4 production by fresh blood were inhibited up to 90% after repeated administrations of L-826,141, this treatment failed to improve lung function. In contrast, dexamethasone (positive control) treatment resulted in significant improvement in lung mechanics and airway function in all horses. Neither drug had a significant effect on BAL total cell counts and differential cytology. Administration of the PDE 4 inhibitor L-826,141 for up to 14 days to horses with heaves was not associated with an improvement in airway function or inflammation. These findings suggest that the PDE 4 enzyme is not a key mediator of lung inflammation in heaves.     

Effects of 8-epi-PGF2α on isolated bronchial smooth muscle of healthy and heaves-affected horses

8-Epi-PGF2alpha, a prostaglandin-like compound generated by oxidative stress, has been shown to be an in vitro bronchoconstrictor in airways from healthy laboratory animals and healthy humans, but it has never been studied in diseased airways. Here, the bronchoconstrictive capacity of 8-epi-PGF2alpha on isolated bronchial rings (BR) of healthy and heaves-affected horses was evaluated by comparing the maximal effect and the potency of 8-epi-PGF2alpha to those of (1) acetylcholine (ACh), (2) its stereoisomer PGF2alpha and (3) its synthetic receptor agonist, U46619. Furthermore, the potential capacity of 8-epi-PGF2alpha to enhance the cholinergic (ACh) responsiveness of bronchial smooth muscle was investigated. 8-Epi-PGF2alpha contracted BR with a rank order of efficacy of Ach > U44619 > PGF2alpha > 8-epi-PGF2alpha in both healthy and heaves-affected horses. The contractile maximal response elicited by 8-epi-PGF2alpha was significantly smaller than that elicited by the other drugs, but was significantly higher in BR from heaves-affected horses than in those sampled in healthy horses, whilst pD2 values were similar. A subthreshold concentration of 8-epi-PGF2alpha (10-7 M) did not induce in vitro cholinergic hyper-responsiveness in BR of either healthy or heaves-affected horses. In conclusion, it has been demonstrated that 8-epi-PGF2alpha is an in vitro bronchoconstrictor of minor importance in healthy horses, but whose efficacy is significantly increased in heaves-affected horses.     

Respiratory and metabolic effects of massive administration of isotonic saline solution in heaves-affected and control horses

REASONS FOR PERFORMING STUDY: It has been suggested that rapid administration of isotonic saline solution relieves airway obstruction in heaves-affected horses, but the efficacy and tolerability of this treatment is poorly documented. OBJECTIVES: To determine the respiratory and metabolic effects of a rapid administration of isotonic saline solution in control horses and those with heaves. METHODS: Thirty litres isotonic saline solution were administered i.v. to 5 control (Group C) and 9 heaves-affected horses (Group H). Pulmonary function testing and arterial blood gas analysis were performed prior to saline infusion, at 90 and 180 mins after starting the infusion, and 1, 3, 6 and 10 days later. PCV, plasma total solids, plasma electrolytes (Na+, Cl- and K+) and venous blood gas analysis were performed prior, during and after saline infusion. RESULTS: Rapid saline administration was shown to have no beneficial effects on lung function during infusion in Group H. On the contrary, they had significantly increased pulmonary resistance (RL) at 180 mins following the initiation of saline administration compared to baseline. In Group C, saline administration resulted in a significant increase in RL at 180 mins and a significant decrease in PaO2 at 90 mins. These results suggest a transient decrement in lung function caused by rapid saline administration. Group H had a metabolic alkalosis with hypochloraemia at baseline compared to control horses. This resolved following i.v. saline administration and returned to baseline within 6 h after treatment had been discontinued. CONCLUSION: The administration of saline to Group H did not improve airway function significantly, but resulted in mild and transient side effects. POTENTIAL RELEVANCE: Rapid administration of isotonic saline alone is not an effective therapy for heaves in horses.     

Clinical signs, evaluation of bronchoalveolar lavage fluid, and assessment of pulmonary function in horses with inflammatory respiratory disease

OBJECTIVE: To evaluate the association among clinical signs, results of cytologic evaluation of bronchoalveolar lavage (BAL) fluid, and measures of pulmonary function in horses with inflammatory respiratory disease. ANIMALS: 9 healthy horses, 5 horses with inflammatory airway disease (IAD), and 9 horses with chronic obstructive pulmonary disease (COPD). PROCEDURES: Clinical examination, lung function tests, and BAL were performed on each horse. RESULTS: Standard lung mechanics of horses with exacerbated COPD differed significantly from those of healthy horses; however, there were few differences among horses with IAD, horses with COPD during remission, and healthy horses. Most variables for forced expiration (FE) in horses with COPD or IAD differed significantly from those for healthy horses. Results of clinical examination had low to moderate sensitivity and predictive values for a diagnosis of COPD (range, 67 to 80%). Results of FE tests had high sensitivity, specificity, and predictive values for a diagnosis of COPD (79 to 100%), and results of standard lung mechanics tests had low sensitivity and predictive values (22 to 69%). Percentage of neutrophils in BAL fluid was highly sensitive (100%) but moderately specific (64%) for a diagnosis of COPD. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical examination is moderately accurate for establishing a diagnosis of COPD. Forced expiration tests can specifically detect early signs of airway obstruction in horses with COPD and IAD that may otherwise be inapparent. Cytologic evaluation of BAL fluid allows early detection of inflammatory respiratory disease, but it is not specific for COPD.     

Inhalation of organic dusts and lipopolysaccharide increases gelatinolytic matrix metalloproteinases (MMPs) in the lungs of heaves horses

We report the effects of mouldy hay/straw exposure, inhaled hay dust suspension (HDS) and inhaled lipopolysaccharide (LPS) on bronchoalveolar lavage fluid (BALF) gelatinolytic matrix metalloproteinase (MMP) levels and degree of activation in healthy (n = 6) and heaves- (previously termed chronic obstructive pulmonary disease) affected (n = 6 or 7) horses. Gelatinolytic MMPs in BALF were quantified by zymography, and gelatinases were shown by Western immunoblotting to be MMP-2 and MMP-9. Hay/straw and HDS challenges increased BALF total gelatinolytic activity only in heaves horses, with the majority of gelatinolytic activity comprising pro- and active MMP-9. The 5 h duration hay/straw challenge increased BALF gelatinolytic MMP activity in heaves horses at 5 and 24 h after the start of this challenge, with activity returning to baseline by Day 4. In contrast to hay/straw and HDS challenges, LPS inhalation increased BALF gelatinolytic MMP activity in both groups. For all challenges, absolute BALF neutrophil counts were highly significantly correlated (P<0.0001) with levels of proMMP-9 and active MMP-9, but not with levels of MMP-2 (P>0.05). As gelatinolytic MMPs are pro-inflammatory agents, they may contribute to lung dysfunction and tissue destruction in heaves horses exposed to airborne organic stable dusts.     

Temporal regulation of cytokine mRNA expression in equine recurrent airway obstruction

Acute and chronic inflammation of the airway remains an important health problem for equids. "Heaves" or recurrent airway obstruction (RAO) remains one of the most commonly diagnosed conditions affecting the lung of older horses in Europe and the United States. The typical clinical signs of RAO include non-productive coughing, serous nasal discharge, labored expiratory effort, and flaring of the nostrils. Auscultation of the lungs of the affected horse often reveals abnormal respiratory sounds, described as crackles and wheezes, throughout the area of the lung field. These clinical signs occur secondary to an inflammatory response that results in bronchospasm, excessive mucus production and airway obstruction. This inflammatory response is characterized by the presence of excessive mucus and inflammatory cells, primarily neutrophils, in the small airways. Most evidence suggests that RAO is the result of a pulmonary hypersensitivity to inhaled antigens. Exposure of affected horses to hay dust, pollens, and mold spores leads to neutrophil accumulation in the lung and bronchospasm. The identification of allergen-specific IgE in bronchoalveolar lavage (BAL) fluid and sera of affected horses supports the involvement of a late phase, IgE-mediated, hypersensitivity reaction in the pathogenesis of equine RAO. The production of IgE antibodies is regulated by the cytokines IL-4 and IL-13. Using a quantitative PCR method we have reported that horses with RAO exhibit a modified Type 2 cytokine response characterized by the production of IL-4 and IL-13 mRNA, but not IL-5 mRNA in BAL cells. Interferon-gamma mRNA was also elevated, suggesting a mixed response. While these results are consistent with equine RAO being the result of an aberrant Type 2 cytokine response to inhaled allergens, others have failed to find any evidence of elevated Type 2 cytokine mRNA in BAL from horses with "heaves". It is likely that these disparate results could be the result of differences in the clinical stage of the affected animals or the timing of sample collection. Here, we report a diverse pattern of cytokine gene expression when sampling a group of affected horses over a period of time.     

蒙古马母体与胎儿胃肠道NF-κB信号通路相关基因mRNA相对表达水平研究

[目的] 研究蒙古马母体与胎儿胃肠道不同区段NF-κB信号通路相关基因的mRNA相对表达水平。[方法] 选取3匹体况良好的妊娠蒙古马进行屠宰,屠宰过程中取出胎儿,分别采集母体及胎儿的胃、十二指肠、空肠、回肠、盲肠、大结肠、小结肠和直肠组织;采用实时荧光定量PCR方法（qPCR）,对NF-κB信号通路上6个相关基因（NF-κB p50、NF-κB p65、NFKBIA、IL-1β、TNF-α和IL-8）的mRNA相对表达量进行分析,比较母体和胎儿胃肠道不同区段组织中各基因的表达差异。[结果] NF-κB信号通路上6个基因在蒙古马母体和胎儿胃肠道不同区段8个组织中均有表达;总体来看,NF-κB p50、NF-κB p65和NFKBIA基因的mRNA相对表达量在母体和胎儿上均较高,IL-1β、TNF-α和IL-8基因的mRNA相对表达量均较低。在胃肠道区段8个组织中,6个基因的mRNA相对表达量基本呈现母体高于胎儿的特征;在回肠和盲肠组织中,母体NF-κB p50基因的mRNA相对表达量显著（P<0.05）高于胎儿;在大结肠组织中,母体NF-κB p65基因和NFKBIA基因的mRNA相对表达量显著（P<0.05）高于胎儿。[结论] NF-κB信号通路上6个基因在蒙古马母体和胎儿胃肠道8个组织中均有表达,且存在一定差异;母体部分肠道区段组织中的NF-κB p50、NF-κB p65、NFKBIA基因mRNA相对表达量显著高于胎儿。     

Partial divergence of cytokine mRNA expression in bronchial tissues compared to bronchoalveolar lavage cells in horses with recurrent airway obstruction

The aim of this study was to investigate mRNA levels of cytokines in bronchial epithelium in horses with recurrent airway obstruction (RAO) during acute crisis and remission. Additionally, cytokine mRNA levels in endobronchial biopsies and bronchoalveolar lavage (BAL) cells were compared. Seven RAO horses were examined while in respiratory crisis following provocation and again while in remission after 2 months on pasture, during which time six healthy horses on pasture were also examined. Quantitative real-time PCR (RT-PCR) was used to assess mRNA expression for cytokines IL-5, IL-6, IL-8, IL-10, IL-17 and transforming growth factor beta1 (TGF-beta1) in endobronchial biopsies and bronchoalveolar lavage. Expression of IL-8 mRNA was significantly upregulated during crisis in both endobronchial biopsies and BAL cells (p=0.036), while there was a similar trend for upregulation of IL-10 mRNA only in BAL cells that approached significance (p=0.059). Moreover, during crisis the expression of IL-8 mRNA in BAL cells was positively correlated to relative IL-6 mRNA expression (r(s)=0.971, p=0.001) and bronchial epithelial expression of IL-10 and TGF-beta1 mRNA were positively correlated (r(s)=0.943, p=0.005). In comparing the relationship of mRNA expression in BAL to biopsy in individual RAO horses, there was a positive correlation with IL-6 to IL-8 mRNA expression in BAL during respiratory crisis (r(s)=0.971, p=0.001) that also correlated positively with IL-8 expression in biopsies on pasture (r(s)=0.986, p<0.0001 for both). Regarding RAO horses at pasture versus controls neither the cytokine mRNA levels in endobronchial biopsy nor in BAL cells differed significantly. These results further support previous findings that IL-8 mRNA in both BAL cells and bronchial epithelium is upregulated in RAO horses during crisis. However, apart from IL-8, it appears that expression of other cytokines, including IL-5, IL-6, IL-10, IL-17 and TGF-beta1 in bronchial epithelium does not necessarily mirror cytokine expression in BAL cells in individual horses with RAO. Accordingly, examination of markers of inflammation in endobronchial tissue provides complementary but not necessarily identical information to that obtained in BAL cells. Given the potential for repeated sampling over time bronchial biopsy can serve as an invaluable additional tool for investigation of time-dependent changes in inflammatory process in this animal model of asthma.