Equine Lung Research Articles

In search of epigenetic hallmarks of different tissues: an integrative omics study of horse liver, lung, and heart.

DNA methylation and microRNA (miRNA) expression are epigenetic mechanisms essential for regulating tissue-specific gene expression and metabolic processes. However, high-resolution transcriptome, methylome, or miRNAome data is only available for a few model organisms and selected tissues. Up to date, only a few studies have reported on gene expression, DNA methylation, or miRNA expression in adult equine tissues at the genome-wide level. In the present study, we used RNA-Seq, miRNA-seq, and reduced representation bisulfite sequencing (RRBS) data from the heart, lung, and liver tissues of healthy cold-blooded horses to identify differentially expressed genes (DEGs), differentially expressed miRNA (DE miRNA) and differentially methylated sites (DMSs) between three types of horse tissues. Additionally, based on integrative omics analysis, we described the observed interactions of epigenetic mechanisms with tissue-specific gene expression alterations. The obtained data allowed identification from 4067 to 6143 DMSs, 9733 to 11,263 mRNAs, and 155 to 185 microRNAs, differentially expressed between various tissues. We pointed out specific genes whose expression level displayed a negative correlation with the level of CpG methylation and miRNA expression and revealed biological processes that they enrich. Furthermore, we confirmed and validated the accuracy of the Next-Generation Sequencing (NGS) results with bisulfite sequencing PCR (BSP) and quantitative PCR (qPCR). This comprehensive analysis forms a strong foundation for exploring the epigenetic mechanisms involved in tissue differentiation, especially the growth and development of the equine heart, lungs, and liver.

Validation of three-dimensional thoracic electrical impedance tomography of horses during normal and increased tidal volumes

Objective. Data from two-plane electrical impedance tomography (EIT) can be reconstructed into various slices of functional lung images, allowing for more complete visualisation and assessment of lung physiology in health and disease. The aim of this study was to confirm the ability of 3D EIT to visualise normal lung anatomy and physiology at rest and during increased ventilation (represented by rebreathing). Approach. Two-plane EIT data, using two electrode planes 20 cm apart, were collected in 20 standing sedate horses at baseline (resting) conditions, and during rebreathing. EIT data were reconstructed into 3D EIT whereby tidal impedance variation (TIV), ventilated area, and right-left and ventral-dorsal centres of ventilation (CoVRL and CoVVD, respectively) were calculated in cranial, middle and caudal slices of lung, from data collected using the two planes of electrodes. Main results. There was a significant interaction of time and slice for TIV (p < 0.0001) with TIV increasing during rebreathing in both caudal and middle slices. The ratio of right to left ventilated area was higher in the cranial slice, in comparison to the caudal slice (p = 0.0002). There were significant effects of time and slice on CoVVD whereby the cranial slice was more ventrally distributed than the caudal slice (p < 0.0009 for the interaction). Significance. The distribution of ventilation in the three slices corresponds with topographical anatomy of the equine lung. This study confirms that 3D EIT can accurately represent lung anatomy and changes in ventilation distribution during rebreathing in standing sedate horses.

9 Respiratory microbiota in horses are individual-specific rather than biogeographically variable

Respiratory disease in horses is associated with transportation, aspiration of oral contents, and primary infectious diseases, and has significant implications for both horse welfare and economic stability of equine industry. Regional conditions within the respiratory tract vary widely in temperature, nutrient availability, pH, and oxygen tension, creating different niches for microbial flora, some of which may not be identified by bacteriologic culture. Proliferation of commensal microbes likely contributes to initiation and progression of clinical disease. Understanding normal microbial populations is essential to investigating dysbiosis in the development of infectious respiratory disease. However, microbial populations within the equine lung have not been investigated with next generation sequencing technologies. Therefore, the study objective was to describe microbial population variability between respiratory tract locations in healthy horses. Samples were collected from 4 healthy adult horses, 2 mares and 2 geldings, aged 10–25 years, Thoroughbred, Standardbred, and Warmblood (2) breeds. Additional inclusion criteria were no history of respiratory or systemic disease within 2 mo of study, and no transportation within 1 week of sample collection at the University of Illinois Veterinary Teaching Hospital. Samples were collected by nasopharyngeal lavage (NPL) and transtracheal aspirate (TTA) premortem, and bybronchoalveolar lavage (BAL) of 6 distinct regions within the lung immediately post-mortem. Microbial DNA was extracted and full-length 16S ribosomal DNA sequencing was performed, followed by microbial profiling analysis with TADA Nextflow and R. There was a large amount of diversity, with over 1800 taxa identified, reduced to 160 taxa after prevalence filtering. The number of taxa and diversity were highly variable across sample locations. Actinobacteria, proteobacteria, and firmicutes were the predominant phyla. There was no significant difference in α or β diversity between the 3 sampling locations (NPL, TTA, BAL). When NPL and TTA were combined, there was a significant difference in α diversity compared with lung for Chao1, ACE and PD indices. There was significant difference in β diversity between horses. Overall, microbiota in respiratory samples of heathy horses were highly variable. Taxa were more similar between regions within the same horse, than between the same region of different horses, suggesting that respiratory microbiota are unique to individuals. These findings do not support presence of a core respiratory microbiome across individuals.

Prevalence of Equine Lung Worm and Its Associated Risk Factors in Kersa district Jimma Zone, South West Ethiopia.

Dictyocaulus arnfieldi parasites directly affect the health and production of working equines, which contributes to the reduction in their work output and ultimately in the income of the owner and the community. A cross-sectional study was conducted from November 2019 to march2020 in and around kersa distinct south west Ethiopia. With the objectives of determining the prevalence and assessing the possible risk factors of lungworm infection in equine. A total of 384 faecal samples from equine species (124 donkeys, 200 horses, 60 mules) were collected and examined for the presence of eggs of parasites using modified Bearmann technique. Out of these, 384(53.1%) equines were found positive for lung worm. The prevalence of lung worm in donkeys, horses, and mules was64.5 %, 49.0%, and43.3% respectively with statistical significant variation (x2 =10.14, P = 0.006). Age of equines was found to have a significant association with the prevalence of Dictyocaulus arnfieldi infection (P&lt;0.05), the prevalence to being in age of greater in young animal. Assessment of the two body condition scores with their prevalence revealed a significant variation, the prevalence was very high in poor body condition groups (x2 =299.99, P = 0.000). However, there was no statistically significant (p&gt;0.05) between the occurrence of equine lungworm and the factors sex. It is concluded that prevalence of equine lungworm in the study area associated with young and emaciated equines were more affected by the lung worm infection. Therefore, Due attention needs to be given to equine health services by district veterinary services office so that equines are handled well in order to earn their maximum potential benefits and grazing management and regular strategic deworming of the whole herd with anthelminthic rather than treating infested individuals is recommended.

Equine alveolar macrophages and monocyte-derived macrophages respond differently to an inflammatory stimulus.

Alveolar macrophages (AMs) are the predominant innate immune cell in the distal respiratory tract. During inflammatory responses, AMs may be supplemented by blood monocytes, which differentiate into monocyte-derived macrophages (MDMs). Macrophages play important roles in a variety of common equine lower airway diseases, including severe equine asthma (SEA). In an experimental model, an inhaled mixture of Aspergillus fumigatus spores, lipopolysaccharide, and silica microspheres (FLS), induced SEA exacerbation in susceptible horses. However, whether equine AMs and MDMs have differing immunophenotypes and cytokine responses to FLS stimulation is unknown. To address these questions, alveolar macrophages/monocytes (AMMs) were isolated from bronchoalveolar lavage fluid and MDMs derived from blood of six healthy horses. Separately, AMMs and MDMs were cultured with and without FLS for six hours after which cell surface marker expression and cytokine production were analyzed by flow cytometry and a bead-based multiplex assay, respectively. Results showed that regardless of exposure conditions, AMMs had significantly higher surface expression of CD163 and CD206 than MDMs. Incubation with FLS induced secretion of IL-1β, IL-8, TNF-α and IFN-γ in AMMs, and IL-8, IL-10 and TNF-α in MDMs. These results suggest that AMMs have a greater proinflammatory response to in vitro FLS stimulation than MDMs, inferring differing roles in equine lung inflammation. Variability in recruitment and function of monocyte-macrophage populations warrant more detailed in vivo investigation in both homeostatic and diseased states.

Retrospective study of pneumonia in non-racing horses in California.

Pneumonia is a significant disease of horses. Although pneumonia has traditionally been studied in racehorses, little information is available for non-racing horses. Non-racing horses that died with pulmonary lesions (n = 156) were available from cases submitted for autopsy from January 2015 to June 2020. Bronchopneumonia (35%), interstitial pneumonia (29%), embolic pneumonia (21%), granulomatous pneumonia (13%), and pleuritis (2%) were observed in the examined horses. Seventy-four horses died or were euthanized because of pulmonary diseases, and 82 horses died or were euthanized because of non-pulmonary causes but had lung lesions. Of the horses that died from pulmonary causes, the most common finding was bronchopneumonia, with abscesses and/or necrosis in the cranioventral aspect of the lung. Bacteria isolated from cases of bronchopneumonia were Streptococcus equi subsp. zooepidemicus (48.5%), Klebsiella pneumoniae (12.1%), and Actinobacillus equuli subsp. haemolyticus (9.1%). The most common extrapulmonary lesions responsible for death in horses that also had lesions in the lung were mainly in the gastrointestinal system (30%), multiple systems (septicemia and/or toxemia; 27%), and musculoskeletal system (12%). The main postmortem findings in cases of bronchopneumonia of non-racing horses were similar to those reported previously in racehorses. However, some non-racing horses also had interstitial and granulomatous pneumonia, patterns not described previously in racehorses in California, likely as a result of the inclusion of extended age categories for non-racing horses. We also found that the equine lung was frequently affected in cases of sepsis and gastrointestinal problems of infectious origin.

Giant Multinucleated Cells Are Associated with Mastocytic Inflammatory Signature Equine Asthma.

Simple SummaryLung inflammation is commonly assessed in asthmatic horses by bronchoalveolar lavage fluid (BALF) cytology. Among the cell types commonly found in equine BALF samples, macrophages are the most abundant. The clinical significance of the abnormal cytological appearance of macrophages in samples from diseased horses is largely disregarded. The present work focuses on cytological alterations observed in macrophages during a chronic inflammatory disease such as equine asthma. Our data, although limited in number, support macrophage fusion (resulting in multinucleated giant cells) as a mechanism of interest in the classification of equine asthma phenotypes, as it was significantly associated with the inflammatory signature and chronicity of the disease.Equine asthma is currently diagnosed by the presence of increased neutrophil (>5%), mast cell (>2%), and/or eosinophil (>1%) differential cell count. Macrophages are normal resident cells within the alveoli. Their presence in BALF is considered normal, but the clinical implication of the presence of activated or fused macrophages (giant multinucleated cells, GMC) is currently overlooked. We aimed to assess the prevalence, cytological determinants, and clinical significance of increased GMC counts in BALF of 34 asthmatic horses compared to 10 controls. Counts were performed on 15 randomly selected high magnification fields per cytospin slide (40×), and expressed as GMC:single macrophage (GMC:M) ratio. Regression models were used for statistical analysis. GMC was frequently observed in both asthmatic and control horses, with an increased prevalence of equine asthma (p = 0.01). GMC:M ratio was significantly higher in severe vs. mild to moderate equine asthmatic and control horses. In asthmatic horses, an increased GMC:M ratio was significantly associated with BALF mastocytosis (p = 0.01), once adjusting for age and the presence and severity of clinical signs of the horses. Tachypnea was the only clinical sign that tended to be positively associated with GMC:M ratio after adjustment (p = 0.08). In conclusion, our data suggest that a relationship might exist between molecular mechanisms regulating GMC formation and mast cell recruitment in the equine lung. The same mechanisms could lead to tachypnea even in the absence of respiratory effort at rest. We suggest including GMC count in the basic cytological assessment of BALF samples to gain more insights into their role in equine asthma.

MORPHOLOGICAL FEATURES AND MORPHOMETRIC PARAMETERS OF THE LUNGS OF SEXUALLY MATURE HORSES (EQUUS FERUS CABALLUS L., 1758)

The lungs, which form part of the respiratory apparatus, provide gas exchange between the environment and the tissues of the human and animal body. Therefore, lung diseases are one of the most pressing issues for modern humane and veterinary medicine. This problem is caused by the progressive growth of diseases in mammals such as bronchial asthma, bronchitis, bronchopneumonia, pneumonia, coronavirus infections, etc. Therefore, to effectively solve this issue in terms of prevention, effective treatment, and prompt differential diagnosis of respiratory diseases, an essential area in morphology is the study of the respiratory apparatus, namely the lungs, in clinically healthy animals, to develop marker test criteria that will serve as indicators of the norm in the differential diagnosis of these diseases. The purpose of this study was to investigate the macro- and microscopic structure of the lungs, conduct a morphometric assessment of their morphological structures in domestic sexually mature horses (class Mammals, species – domestic horse (Equus ferus caballus L., 1758). The object of this study was the lungs of clinically healthy sexually mature horses (n=5). Fresh lungs of the animals under study were subjected to anatomical preparation. For histological studies, generally accepted methods of fixing pieces of material and making histological sections were used, which were then stained with haematoxylin and eosin and according to Van Gieson’s method. The basis of the lungs in horses are pyramidal or cone-shaped lobules. Part of the structure of the lobes are acini covered with a thin layer of connective tissue. The microscopic structure of acini is formed by alveolar ducts, alveolar sacs, and alveoli. According to the results of research, the alveolar tree in horses is shortened and wide and has a bubble shape. The results of morphometric studies showed that the average volume of pulmonary alveoli in clinically healthy horses is 699.80±106.42 thous. μm3. The respiratory part of the lungs in horses occupies 54.8±7.4% of the total area of the lung parenchyma, the connective tissue base – 45.2±7.4%. Such studies of morphological features and morphometric parameters of equine lungs are of practical importance in veterinary medicine since they are markers and criteria for pathomorphological diagnosis of diseases associated with the respiratory system

Nebulisation of dexamethasone sodium phosphate for the treatment of severe asthmatic horses.

Inhaled corticosteroids are effective in the treatment of equine asthma. A recent study reported that nebulisation of injectable dexamethasone had low systemic bioavailability in healthy horses and could represent a cost-effective therapy for equine inflammatory lung diseases. To determine the effects of dexamethasone nebulisation on lung function in severe asthmatic horses. It was hypothesised that dexamethasone administered by nebulisation would be more effective than the same dose administered orally. Randomised blinded experimental study in severe asthmatic horses. Twelve severe asthmatic horses in clinical exacerbation were randomly assigned to treatment with 5mg of dexamethasone sodium phosphate by nebulisation (n=6) or by oral administration (n=6), once daily for 7days. Lung function was evaluated at baseline, after four treatment days (D4) and 24h after the last dose (D8). The presence of residual bronchospasm was assessed on D8 with N-butylscopolammonium bromide administration (0.3mg/kg i.v.). A respiratory clinical score was performed daily. Serum cortisol concentration was measured at baseline, D4 and D8. The pulmonary elastance was unchanged in both groups while pulmonary resistance was significantly improved in the oral group on D8 (mean reduction in 1cmH2 O/L/s [CI: 0.34-1.65, P=0.003]). All horses had residual bronchospasm at the end of the study. There was a group difference in the respiratory clinical score as it was significantly reduced in the oral group, from D5 to D8. Serum cortisol concentration decreased in all subjects. Low number of horses and lack of placebo group. Considering the lack of improvement of lung function and the hypothalamic-pituitary-adrenal axis suppression, the results of this study do not support aerosolisation of an injectable form of dexamethasone for the treatment of severe equine asthma at the dose and with the nebuliser evaluated.

Glucocorticosteroids administration is associated with increased regulatory T cells in equine asthmatic lungs

Recurrent inflammation in severe equine asthma causes a remodeling of the airways leading to incompletely reversible airway obstruction. Despite the improvement of clinical signs and lung function with glucocorticoids (GC), inflammation, translated by an increased percentage of neutrophils, persists in the airways. Regulatory T cells (Treg) have been shown to have anti-inflammatory properties and play an important role in balancing the immune response by suppressing effector lymphocyte activity. However, interactions between Treg, neutrophils and glucocorticosteroids in vivo are unclear, particularly in asthma. Furthermore, the effects of GC on Treg in the airway of asthmatic horses have not been investigated. We hypothesized that horses with severe asthma display a decreased population of pulmonary Treg when compared to heathy controls, and that treatment with GC lead to an increased pulmonary Treg cell population only in affected horses. Using lung function measurements and flow cytometry with surface antigens CD4 and FoxP3, we investigated Treg in airway luminal cells obtained by bronchoalveolar lavage fluid (BALF) from 6 asthmatic horses in exacerbation of the disease and 6 aged-match controls, kept in the same environment, before and following a 2-week treatment with dexamethasone. Results showed that the number of Treg increases only in the lungs of asthmatic horses following GC therapy, despite continued presence of increased numbers of neutrophils. Our results support the complexity of the interaction between Treg, neutrophils and GC.

Phylogenetic Analysis and Characterization of a Sporadic Isolate of Equine Influenza A H3N8 from an Unvaccinated Horse in 2015.

Equine influenza, caused by the H3N8 subtype, is a highly contagious respiratory disease affecting equid populations worldwide and has led to serious epidemics and transboundary pandemics. This study describes the phylogenetic characterization and replication kinetics of recently-isolated H3N8 virus from a nasal swab obtained from a sporadic case of natural infection in an unvaccinated horse from Montana, USA. The nasal swab tested positive for equine influenza by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). Further, the whole genome sequencing of the virus confirmed that it was the H3N8 subtype and was designated as A/equine/Montana/9564-1/2015 (H3N8). A BLASTn search revealed that the polymerase basic protein 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), and matrix (M) segments of this H3N8 isolate shared the highest percentage identity to A/equine/Tennessee/29A/2014 (H3N8) and the polymerase basic protein 2 (PB2), neuraminidase (NA), and non-structural protein (NS) segments to A/equine/Malaysia/M201/2015 (H3N8). Phylogenetic characterization of individual gene segments, using currently available H3N8 viral genomes, of both equine and canine origin, further established that A/equine/Montana/9564-1/2015 belonged to the Florida Clade 1 viruses. Interestingly, replication kinetics of this H3N8 virus, using airway derived primary cells from multiple species, such as equine, swine, bovine, and human lung epithelial cells, demonstrated appreciable titers, when compared to Madin–Darby canine kidney epithelial cells. These findings indicate the broad host spectrum of this virus isolate and suggest the potential for cross-species transmissibility.

Maintenance of equine anaesthesia over the last 50years: Controlled inhalation of volatile anaesthetics and pulmonary ventilation.

In the first edition of this journal, Barbara Weaver wrote a review titled 'Equine Anaesthesia', stating that, at that time, it was quickly becoming accepted practice that many horses were being anaesthetised 'by essentially similar procedures, i.e. premedication, induction and then maintenance by controlled inhalation'. To celebrate the 50th anniversary of the first edition of this journal, this review covers the development of understanding and practice of inhalational anaesthesia and controlled ventilation in horses over the last 50years. We review how the perceived benefits of halothane led to its widespread use, but subsequently better understanding of halothane's effects led to changes in equine anaesthetic practice and the utilisation of different inhalation agents (e.g. isoflurane and sevoflurane). We discuss how more recently, better understanding of the effects of the 'newer' inhalation agents' effects has led to yet more changes in equine anaesthetic practice, and while, further new inhalation agents are unlikely to appear in the near future, further enhancements to anaesthetic practice may still lead to improved outcomes. We review advances in our understanding of the anatomy and pathophysiology of the equine lung as well of the effects of anaesthesia on lung function and how these predispose to some of the common problems of gas exchange and ventilation during anaesthesia. We identify the aims of optimal mechanical ventilation for anaesthetic management and whether the various methods of ventilatory support during equine anaesthesia achieve them. We also highlight that further developments in equipment and optimal ventilator modes are likely in the near future.

Equine lung decellularization: a potential approach for in vitro modeling the role of the extracellular matrix in asthma

Contrary to conventional research animals, horses naturally develop asthma, a disease in which the extracellular matrix of the lung plays a significant role. Hence, the horse lung extracellular matrix appears to be an ideal candidate model for in vitro studying the mechanisms and potential treatments for asthma. However, so far, such model to study cell–extracellular matrix interactions in asthma has not been developed. The aim of this study was to establish a protocol for equine lung decellularization that maintains the architecture of the extracellular matrix and could be used in the future as an in vitro model for therapeutic treatment in asthma. For this the equine lungs were decellularized by sodium dodecyl sulfate detergent perfusion at constant gravitational pressure of 30 cmH2O. Lung scaffolds were assessed by immunohistochemistry (collagen I, III, IV, laminin, and fibronectin), scanning electron microscopy, and DNA quantification. Their mechanical property was assessed by measuring lung compliance using the super-syringe technique. The optimized protocol of lung equine decellularization was effective to remove cells (19.8 ng/mg) and to preserve collagen I, III, IV, laminin, and fibronectin. Moreover, scanning electron microscopy analysis demonstrated maintained microscopic lung structures. The decellularized lungs presented lower compliance compared to native lung. In conclusion we described a reproducible decellularization protocol that can produce an acellular equine lung feasible for the future development of novel treatment strategies in asthma.

Characterization of respiratory dendritic cells from equine lung tissues

BackgroundDendritic cells (DCs) are professional antigen-presenting cells that have multiple subpopulations with different phenotypes and immune functions. Previous research demonstrated that DCs have strong potential for anti-viral defense in the host. However, viruses including alphaherpesvirinae have developed strategies to interfere with the function or maturation of DCs, causing immune dysfunction and avoidance of pathogen elimination. The goal of the present study was to isolate and characterize equine lung-derived DCs (L-DCs) for use in studies of respiratory viruses and compare their features with equine blood-derived DCs (B-DCs), which are currently used for these types of studies.ResultsWe found that L-DCs were morphologically similar to B-DCs. Overall, B-DCs demonstrated higher expression of CD86 and CD172α than L-DCs, but both cell types expressed high levels of MHC class II and CD44, as well as moderate amounts of CD163, CD204, and Bla36. In contrast, the endocytic activity of L-DCs was elevated compared to that of B-DCs. Finally, mononuclear cells isolated from lung (L-MCs), which are used as precursors for L-DCs, expressed more antigen-presenting cell-associated markers such as MHC class II and CD172α compared to their counterparts from blood.ConclusionsOur results indicate that L-DCs may be in an earlier differentiation stage compared to B-DCs. Concurrent with this observation, L-MCs possessed significantly more antigen-uptake capacity compared to their counterparts from blood. It is likely that L-DCs play an important role in antigen uptake and processing of respiratory pathogens and are major contributors to respiratory tract immunity and may be ideal tools for future in vitro or ex vivo studies.

Expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinase-2 and -9 in horses with chronic airway inflammation.

OBJECTIVE To examine whether expression of extracellular matrix metalloproteinase inducer (EMMPRIN) can be detected in equine lungs and whether it correlates with matrix metalloproteinase (MMP)-2 and -9 expression in bronchoalveolar lavage fluid (BALF) of horses with chronic inflammation of the lungs (ie, lower airway inflammation [LAI]). ANIMALS 29 horses with signs of chronic respiratory tract disease, which were classified as the LAI (n = 17) and LAI with respiratory distress (RDLAI [12]) groups, and 15 control horses. PROCEDURES BALF, tracheal aspirate, and blood samples were obtained, and EMMPRIN expression was determined from BALF cells and RBCs by use of western blotting. Activities of MMP-2 and -9 were determined with zymography. RESULTS Expression of EMMPRIN protein was identified in BALF cells of all horses. Expression of EMMPRIN protein was highest for the RDLAI group and was correlated with MMP-2 and -9 protein expression, MMP-9 gelatinolytic activity, and airway neutrophilia. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that EMMPRIN was involved in the pathophysiologic processes of asthma in horses. However, additional studies of horses and other species are warranted to elucidate the regulation of EMMPRIN expression in asthmatic lungs.

Expression and Localization of Equine Tissue-Specific Divalent Ion-Transporting Channel Proteins

Divalent ions such as calcium, iron, and copper have roles in physiological functions such as maintaining ionic homeostasis. Ion channels, present in the cell membrane, control the flow of ions across the cell membrane. Hyperkalemic periodic paralysis, iron-deficiency anemia, and horse colic are related to ion channel malfunctions. Equine ion channels have not been studied as much as those in other species. In the present study, we focused on expression and localization of calcium, iron, and copper ion channels in equine duodenum, heart, kidney, liver, lung, ovary, uterus, and testis tissues. Gene and protein expressions were quantified by using real-time polymerase chain reaction and western blotting, respectively. Localization of each channel was identified via immunohistochemical analysis. Gene (mRNA) and protein levels of Nckx3 and Trpv2 were high in uterus tissue. Ferroportin (Ireg1) mRNA levels varied, but its protein level was notably high in liver. Hephaestin (Heph) mRNA level was high in duodenum, whereas its protein level was high in liver. Nckx3 was highly expressed in ovary, uterus, and testis. Trpv2 was expressed in duodenum, liver, lung, and kidney. Ferroportin was expressed in duodenum, liver, ovary, and uterus. Hephaestin was expressed in duodenum, ovary, and testis. Copper transporter 1 (Ctr1) was expressed in all tissues, whereas Atp7a was expressed in duodenum, kidney, and testis. Each protein was expressed in their respective structures, including gland, epithelium, and myocyte. Taken together, the regulation of each ion channel is important to secretion, absorption, and contraction in the various organ. The results of this study could form a basis for elucidating Nckx3-, Trpv2-, Ireg1-, Heph-, Ctr1-, and Atp7a-related diseases in horse.

The equine pasture asthma-restricted lung transcriptome: A tool to decipher the pathophysiology of airway hyper-responsiveness in horses

The equine pasture asthma-restricted lung transcriptome: A tool to decipher the pathophysiology of airway hyper-responsiveness in horses

Evaluation of contractile phenotype in airway smooth muscle cells isolated from endobronchial biopsy and tissue specimens from horses.

OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture. SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4). PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens from freshly harvested equine lungs and from healthy and heaves-affected horses. Immunologic techniques were used to evaluate the contractile phenotype of ASM cells in culture. RESULTS Characteristic ASM cells were successfully cultured from endobronchial tissue or biopsy specimens from both healthy and heaves-affected horses, and their contractile phenotype was maintained for up to 7 passages. Moreover, the capacity of cells at the seventh passage to contract in a collagen gel in response to methacholine was maintained. CONCLUSIONS AND CLINICAL RELEVANCE ASM cells isolated from equine endobronchial tissue and biopsy specimens were able to maintain a contractile phenotype in long-term cell cultures, suggesting they could be used for tissue engineering and in vitro studies of equine ASM cells.

Pharmacological indices and pulmonary distribution of rifampicin after repeated oral administration in healthy foals.

The treatment of equine lung infections by Rhodococcus equi with rifampicin is empirically based because pharmacokinetic/pharmacodynamic (PK/PD) indices and pivotal clinical outcome data are not available. To evaluate the pharmacokinetics and pulmonary distribution of rifampicin into epithelial lining fluid (ELF) and bronchoalveolar lavage cells (BALC) to predict antimicrobial activity in the lung using PK/PD indices. Controlled, randomised, two-period, crossover, repeated-dose study with an initial arm to measure disposition after i.v. administration of rifampicin. Pharmacokinetics and lung distribution were evaluated in six healthy foals treated with 10 mg/kg bwt rifampicin i.v. (initial arm) and with repeated oral doses of rifampicin at 10 mg/kg bwt and 20 mg/kg bwt once per day for 10 days (crossover arms). ELF and BALC were sampled by bronchoalveolar lavage 24 h after the last oral dosing. Rifampicin and 25-O-desacetyl rifampicin were quantified using liquid chromatography tandem-mass spectrometry. Enzyme induction by rifampicin was confirmed by evaluation of plasma 4β-OH-cholesterol:cholesterol ratios. The distribution volume of rifampicin administered i.v. was ~0.85 L/kg. Terminal elimination half-life was ~11 h. Orally given rifampicin was slowly absorbed (Tmax , range: 2.5-8.0 h) and eliminated with apparent half-lives of ~6-8 h. Trough concentrations in ELF and BALC were 1.01 ± 0.20 μg/mL and 1.25 ± 0.29 μg/mL, respectively, after 10 mg/kg bwt rifampicin and 2.71 ± 1.25 μg/mL and 3.09 ± 1.63 μg/mL, respectively, after 20 mg/kg bwt rifampicin. The average ratios of area under the plasma concentration time curve during an administration interval of 24 h (AUC0-24 h ) to minimum inhibitory concentration (MIC) were 145 and 322 h, respectively, for less susceptible strains of R. equi (MIC90 : 0.5 μg/mL). The clearance and bioavailability of rifampicin after repeated oral dosing were not evaluated. Treatment with rifampicin at 10 mg/kg bwt administered once per day is suitable to generate drug concentrations above the MIC90 in the ELF and BALC of foals. Future clinical studies with rifampicin in combination with macrolide antibiotics with low drug interaction potential are required to translate the PK/PD indices into protocols for the treatment of R. equi lung infections.

Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation.

BackgroundData obtained in both animal models and clinical trials suggest that cell-based therapies represent a potential therapeutic strategy for lung repair and remodeling. Recently, new therapeutic approaches based on the use of stem cell derivatives (e.g., conditioned medium (CM) and microvesicles (MVs)) to regenerate tissues and improve their functions were proposed. The aim of this study was to investigate the immunomodulatory effects of equine amniotic mesenchymal cell derivatives on lipopolysaccharide (LPS)-induced cytokine production in equine alveolar macrophages, which may be beneficial in lung inflammatory disorders such as recurrent airway obstruction (RAO) in horses. RAO shares many features with human asthma, including an increased number of cells expressing mRNA for interleukin (IL)-4 and IL-5 and a decreased expression of IFN-γ in bronchoalveolar lavage fluid (BALF) of affected horses.MethodsThe release of TNF-α, IL-6, and TGF-β1 at different time points (1, 24, 48, and 72 h) was measured in equine alveolar macrophages stimulated or not with LPS (10 and 100 ng/mL) in the presence or absence of 10 % CM or 50 × 106 MVs/mL.Cytokines were measured using commercially available ELISA kits. For multiple comparisons, analysis of variance was used with Tukey post-hoc test. Differences were considered significant at p ≤ 0.05.ResultsSignificant modulatory effects of CM on LPS-induced TNF-α release at 24 h, and of both CM and MVs on TNF-α release at 48 h were observed. A trend toward a modulatory effect of both CM and MVs on the release of TGF-β and possibly IL-6 was visible over time.ConclusionsResults support the potential use of CM and MVs in lung regenerative medicine, especially in situations in which TGF-β may be detrimental, such as respiratory allergy. Further studies should evaluate the potential clinical applications of CM and MVs in equine lung diseases, such as RAO and other inflammatory disorders.