Relationship Between Selenium, Copper, Zinc and Their Biomarkers in Blood and Skeletal Muscle Tissue in Adult Horses From Southern Chile.

Microminerals are necessary for all bodily functions. In animal species, selenium (Se), copper (Cu) and zinc (Zn) all form part of antioxidant enzymes. Micromineral deficiencies, particularly Se, are well recognized in large animal species in Chile. Glutathion peroxidase (GPx) is a widely used biomarker for Se nutritional status and to diagnose Se deficiency in horses. Superoxide dismutase (SOD) is a Cu and Zn-dependant antioxidant enzyme, although it is not commonly used as a proxy for the nutritional status of these minerals. Ceruloplasmin (CP) is used as a biomarker of Cu nutritional status. This study´s objective was to explore correlation between said minerals and biomarkers in adult horses from southern Chile. Se, Cu, Zn, GPx, SOD, and CP were measured in whole blood of a group of 32 adult horses (5-15 years old). Further, a second group of 14 adult horses (5-15 years old) underwent gluteal muscle biopsies to determine Cu, Zn, GPx, and SOD. Correlations were determined by means of Pearson´s r coefficient. Significant correlations were found for blood GPx and Se (r = 0.79), blood GPx and SOD (r = -0.6), muscular GPx and SOD (r = 0.78), and Cu and CP (r = 0.48). These results confirm previously described strong association between blood GPx and Se in horses, validating the former´s use as a diagnostic proxy of Se deficiency in Chilean horses, and suggest significant interactions between GPx and SOD in blood and muscle tissue.

Evaluation of peripheral blood polymorphonuclear cell functions after an oral carbohydrate overload in obese and insulin dysregulated horses.

Obesity and insulin dysregulation (ID) are increasingly prevalent conditions in equid populations worldwide. Immune impairment is well described in humans with metabolic dysfunction and is reported but still incompletely understood in horses. This study evaluated the effect of acute induced transient hyperglycemia on apoptosis, phagocytosis and oxidative burst activity of peripheral blood polymorphonuclear cells (PMN) of lean and obese adult horses with or without insulin dysregulation. Seventeen adult horses were allocated into three groups based on their body condition score (BCS) and metabolic status: lean-insulin sensitive (lean-IS), obese-insulin sensitive (obese-IS) and obese-insulin dysregulated (obese-ID). ID was determined by insulin tolerance testing (ITT). Blood glucose elevation was induced through an infeed-oral glucose test (in-feed OGT), and all assessments of PMN functions (apoptosis, phagocytosis and oxidative burst) were done in vitro after isolation from peripheral blood before and 120 min after carbohydrate overload. Results were analyzed using a repeated measures linear mixed model with significance defined at P < 0.05. No differences in apoptosis were observed between experimental groups at any time point. Phagocytic capacity was significantly lower at baseline in the obese-ID group but increased in response to glucose administration when compared to the other two groups. Basal reactive oxygen species production in the obese-IS group differed significantly from the lean-IS and obese-ID groups and decreased significantly in response to glucose administration. Results from this study showed that both metabolic status itself, and oral glucose administration, seem to be factors that alter PMN functionality in horses, specifically phagocytosis and oxidative burst.

Role of Cellular Metabolism in the Formation of Neutrophil Extracellular Traps in Airway Diseases.

Neutrophil extracellular traps (NETs) are a recently described mechanism of neutrophils that play an important role in health and disease. NETs are an innate defense mechanism that participate in clearance of pathogens, but they may also cause collateral damage in unrelated host tissues. Neutrophil dysregulation and NETosis occur in multiple lung diseases, such as pathogen-induced acute lung injury, pneumonia, chronic obstructive pulmonary disease (COPD), severe asthma, cystic fibrosis, and recently, the novel coronavirus SARS-CoV-2. More recently, research into immunometabolism has surged due to the possibility of reprogramming metabolism in order to modulate immune functions. The present review analyzes the different metabolic pathways associated with NETs formation, and how these impact on pathologies of the airways.

Pre-conditioning Strategies for Mesenchymal Stromal/Stem Cells in Inflammatory Conditions of Livestock Species.

Mesenchymal stem/stromal cells (MSCs) therapy has been a cornerstone of regenerative medicine in humans and animals since their identification in 1968. MSCs can interact and modulate the activity of practically all cellular components of the immune response, either through cell-cell contact or paracrine secretion of soluble mediators, which makes them an attractive alternative to conventional therapies for the treatment of chronic inflammatory and immune-mediated diseases. Many of the mechanisms described as necessary for MSCs to modulate the immune/inflammatory response appear to be dependent on the animal species and source. Although there is evidence demonstrating an in vitro immunomodulatory effect of MSCs, there are disparate results between the beneficial effect of MSCs in preclinical models and their actual use in clinical diseases. This discordance might be due to cells' limited survival or impaired function in the inflammatory environment after transplantation. This limited efficacy may be due to several factors, including the small amount of MSCs inoculated, MSC administration late in the course of the disease, low MSC survival rates in vivo, cryopreservation and thawing effects, and impaired MSC potency/biological activity. Multiple physical and chemical pre-conditioning strategies can enhance the survival rate and potency of MSCs; this paper focuses on hypoxic conditions, with inflammatory cytokines, or with different pattern recognition receptor ligands. These different pre-conditioning strategies can modify MSCs metabolism, gene expression, proliferation, and survivability after transplantation.

Tamoxifen triggers the in vitro release of neutrophil extracellular traps in healthy horses.

Neutrophils display an array of biological functions including the formation of neutrophil extracellular traps (NETs), web-like structures specialized in trapping, neutralizing, killing and preventing microbial dissemination within the host. However, NETs contribute to a number of inflammatory pathologies, including severe equine asthma. Tamoxifen (TX) is a selective estrogen receptor modulator which belongs to the triphenylethyllenes group of molecules, and which is used as a treatment in all stages of estrogen-positive human breast cancer. Our previous results suggest that tamoxifen can modulate neutrophil functionality and promote resolution of inflammation; this would partly explain the clinical beneficial effect of this drug in horses with airway inflammation. Enhanced NETs production has been reported with tamoxifen use in humans, but minimal data exists regarding the drug's effect on NETs in horses. The aim of this study is to assess the in vitro effect of TX on NETs formation from peripheral blood of healthy horses. Five clinically healthy mixed-breed adult horses were enrolled in the study. For this, cellular free DNA quantification, immunofluorescence for the visualization of NETs, assessment of different types of NETs, and detection of mitochondrial superoxide. TX induced NETs formation at a concentration of 10 uM. Our results show that only two types of NETs were induced by TX: 95% spread NETs (sprNETs) and 5% aggregated NETs (aggNETs). Furthermore, induction of these NETs could be influenced by mitochondrial ROS. Future research should involve an In vivo study of horses with severe asthma and TX treatment, to evaluate BALF neutrophil NET formation. In conclusion, this in vitro study suggests that the resolution of inflammation by TX in horses with airway inflammation is due to inhibition of other neutrophilic functions but not to NET formation.

Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity nanobody.

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with potential therapeutic applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of Nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.

Metabolomics analysis of bronchoalveolar lavage fluid samples in horses with naturally-occurring asthma and experimentally-induced airway inflammation.

The present work characterized the metabolomic profile of bronchoalveolar lavage fluid (BALF) in healthy horses, experimentally-induced airway inflammation by lipopolysaccharide (LPS) nebulization, and naturally-occurring asthma (n = 3 in each group). All animals underwent clinical and upper airway endoscopic examinations, and bronchoalveolar lavage. BALF supernatant samples were subjected to metabolic analysis based on gas chromatography-mass spectrometry (GC-MS). Overall, 67 peaks were obtained from BALF GC-MS analysis, corresponding to 53 metabolites which were categorized according to chemical class, such as organic acids, fatty acids, nucleosides or their derivatives, amino acids, peptides or their derivatives, carbohydrates, and other compounds. Our results showed that the airway inflammation induction model with LPS produced the same pattern of metabolite changes as in horses with naturally occurring asthma. Metabolic pathway analysis was done by means of Fisher's exact test, for detection of metabolites over-represented in asthma affected-horses and LPS-induced airway inflammation as compared with healthy horses. The most significant altered metabolic pathways were fatty acid biosynthesis, galactose metabolism and citrate cycle. These results suggest that the airway inflammation induction model with LPS is a good study model for asthma-affected horses, due to the similarity of the profile of inflammatory cells (specifically neutrophils) and similar metabolic alterations found in BALF that occur during the inflammatory process of the airways. Further research may increase understanding of metabolomics disturbances and their significance in the pathogenesis of equine asthma.

Pre-conditioning of Equine Bone Marrow-Derived Mesenchymal Stromal Cells Increases Their Immunomodulatory Capacity.

Mesenchymal stem/stromal cells (MSCs) are increasingly explored for the treatment of degenerative and inflammatory diseases in human and veterinary medicine. One of the key characteristics of MSCs is that they modulate inflammation mainly through the secretion of soluble mediators. However, despite widespread clinical use, knowledge regarding the effector mechanisms of equine MSCs, and consequently their effectiveness in the treatment of diseases, is still unknown. The objectives of this study were to determine the mechanisms underlying inhibition of lymphocyte proliferation by equine bone marrow-derived MSCs, and to evaluate the effect of pre-conditioning of equine MSCs with different pro-inflammatory cytokines on inhibition of lymphocyte proliferation. We determined that inhibition of lymphocyte proliferation by equine MSCs depends on activity of prostaglandin-endoperoxide synthase 2 and indoleamine 2,3-dioxygenase. Additionally, pre-conditioning of MSCs with TNF-α, IFN-γ or their combination significantly increased the expression of prostaglandin-endoperoxide synthase 2, indoleamine 2,3-dioxygenase, iNOS and IL-6. This upregulation correlated with an increased inhibitory effect of MSCs on lymphocyte proliferation. In conclusion, pre-conditioning of bone marrow-derived MSC increases their inhibitory effect on lymphocyte proliferation in horses.

Assessment of peripheral blood neutrophil respiratory burst, phagocytosis and apoptosis in obese non-insulin dysregulated horses.

Obesity is a highly prevalent condition in horses. Dysfunctional neutrophil activity has been reported in metabolically healthy obese humans, but minimal data exist regarding horses. The present study evaluated the effect of obesity on apoptosis, phagocytosis and oxidative burst activity of peripheral blood neutrophils from lean and obese non-insulin dysregulated horses. Seven lean (BCS, body condition score 4-6/9) and five obese (BCS 8-9) horses were enrolled in the study. All animals underwent two metabolic tests (OGT, oral glucose test; IRT, insulin response test) before their selection to ensure their metabolic status (non-insulin dysregulated). A single blood sample was obtained from each horse, and a discontinuous density gradient was carried out to isolate neutrophils. Phagocytosis, apoptosis and reactive oxygen species (ROS) production assays were performed for each animal. All statistical analyses were performed with unpaired two-tailed t-tests. Results indicate that neutrophils from obese non-insulin dysregulated horses have a significantly increased ROS production (P < .0001), with no changes observed on phagocytosis (P > .05) or apoptosis (P > .05) when compared to the control group. In conclusion, our results demonstrate that obesity per se, in absence of other endocrine disorders, alters neutrophil reactive oxygen species production. More research is needed to understand the role of obesity on the equine immune system of horses, and its role in the development of endocrine disorders.

Tamoxifen and its metabolites induce mitochondrial membrane depolarization and caspase-3 activation in equine neutrophils.

Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its metabolites (N-desmethyltamoxifen and endoxifen) on the mitochondrial membrane potential assay by flow cytometry, and the activation of effector caspase-3 through immunoblotting, in peripheral blood neutrophils obtained from healthy horses (n = 5). Results show that tamoxifen, N-desmethyltamoxifen and endoxifen depolarize the mitochondrial membrane and activate caspase-3 in healthy equine neutrophils in vitro. These findings suggest that tamoxifen and its metabolites may activate the intrinsic apoptotic pathway in equine neutrophils. However, more studies are necessary to further explore the signalling pathways of these drugs in the induction of apoptosis.

Serological and molecular detection of Anaplasma phagocytophilum in Thoroughbred horses from Chilean racecourses.

Anaplasma phagocytophilum is the causative agent of equine granulocytic anaplasmosis (EGA). This study aimed to perform serological and molecular surveys of A. phagocytophilum in thoroughbred horses from racecourses in Chile. Additionally, hematological findings related to A. phagocytophilum molecular positivity were addressed, and phylogenetic analysis of selected positive samples was performed. Complete blood count and msp2 gene real-time PCR were performed in 457 thoroughbred horses from three racecourses located in three different cities of Chile (Santiago, Viña del Mar and Concepción). Sera from horses in two racecourses (Santiago and Vina del Mar) were tested by Indirect fluorescent antibody test (IFAT) to detect IgG antibodies against A. phagocytophilum. The occurrence of A. phagocytophilum by real-time PCR was 13.6 % (62/457, 95 % CI: 10.8-16.3 %), with the highest occurrence observed in Santiago (26.5 %), followed by Concepción (9%), and the lowest in Viña del Mar (5%). The overall frequency of IgG antibodies to A. phagocytophilum was 7.9 % (23/290, 95 % CI: 4.8-12.7 %), with 9.9 % in Santiago and 6.5 % in Viña del Mar. Only three animals from Santiago Racecourse were positive in both real-time PCR and serology. PCR-positive horses from Santiago racecourse presented significantly lower hemoglobin, mean corpuscular value (MCV), and mean corpuscular hemoglobin concentration (CHCM), and higher eosinophil counts. Phylogenetic analysis based on the msp2 gene showed that A. phagocytophilum sequences found in the present study were closely related with A. phagocytophilum sequences from the USA and Europe. Anaplasma phagocytophilum DNA is detected for the first time in Chile.

Equine bone marrow-derived mesenchymal stromal cells inhibit reactive oxygen species production by neutrophils.

BACKGROUND: Polymorphonuclear neutrophils (PMN) are the largest leukocyte population in the blood of most mammals including horses, and play an important defensive role in many infectious diseases. However, the mechanisms that increase PMN as one of the main cellular subsets in the defense against pathogens could also be involved in the pathophysiology of dysregulated inflammatory conditions. Mesenchymal stem/stromal cells (MSCs) are a heterogeneous population with a modulatory potential on the inflammatory response and are known to interact with nearly all cells of the immune system, including PMN. In this study, the in vitro modulation of equine bone marrow-derived MSCs on equine PMN phagocytosis, ROS production, and NETs generation was assessed. RESULTS: In co-culture with MSCs, unstimulated PMN produce less ROS (2.88 % ±â€¯1.43) than PMN in single culture (5.89 % ±â€¯2.63) (p = 0.016). Moreover, PMN co-cultured with MSCs remain conditioned to produce fewer ROS after PMA stimulation in comparison to PMN in single culture (p < 0.05). Additionally, it was found that incubation with MSC supernatant strongly inhibited ROS production (83 % ±â€¯6.35 less than control) without affecting phagocytosis or capacity for NETosis (p < 0.01). CONCLUSIONS: These results suggest a modulatory effect of equine BM-derived MSCs on PMN respiratory burst, without impairing other important microbicidal functions. This supports the potential use of equine MSCs in excessive or persistent inflammatory conditions in which neutrophils are the main effector cells.

Uterine Inertia due to Severe Selenium Deficiency in a Parturient Mare.

A 12-year-old, multiparous, parturient show jumper embryo-recipient mare presented at a veterinary hospital, seven days past her due date and with a dilated cervix, for evaluation of mild colic. Gastrointestinal or metabolic abnormalities and fetal maldispositions were excluded as causes of dystocia, and a diagnosis of uterine inertia was made. There was no uterine response to oxytocin treatment. A live filly was delivered via C-section, and severe selenium deficiency was eventually confirmed in the mare, her offspring, and in the herd of origin. The filly was born with severe white muscle disease and required intensive treatment. This report suggests that selenium deficiency is an underlying cause of equine uterine inertia in the absence of other causes of dystocia.

Tamoxifen in horses: pharmacokinetics and safety study.

BACKGROUND: Tamoxifen (TAM), a selective modulator of estrogen receptors (SERMs) has been recently explored as a therapeutic option for the oral treatment of airway inflammation in the horse. The objective of this work was to establish pharmacokinetic parameters of TAM and its main metabolites in equines, as well as to determine its clinical safety in short-term treatments. RESULTS: We determined TAM and its three main metabolites (4-OH tamoxifen, endoxifen, and N-desmethyl tamoxifen) in plasma after single administration of 0.25 mg/kg in healthy adult horses (n = 12). A maximum concentration of TAM was achieved 3 h after the oral administration (4.65 pg/mL ± 1.69); 4-OH tamoxifen was the metabolite that reached the highest concentration (78 pg/mL ± 70), followed by N-desmethyl tamoxifen (0.43 pg / mL ± 0.48), and finally endoxifen (0.17 pg/mL ± 0.17). All metabolites showed peak concentration 2 h after oral administration of the drug. Oral TAM bioavailability was 13,15% ± 4,18, with a steady state volume of distribution of 7831 ± 2922 (L/kg). Elimination half-life was 15.40 ± 5.80 h, and clearance was 5876 ± 699 (mL/kg/min). Clinical safety of TAM was determined over a 7-day course of treatment (0.25 mg/kg, orally q 24 h, n = 20). No adverse effects were observed through clinical examination, blood hematology, serum biochemistry, ophthalmological and reproductive examinations. Endometrial edema observed in some mares was attributed to normal cyclic activity. CONCLUSIONS: Tamoxifen has moderate oral bioavailability and a large volume of distribution, with three main metabolites in horses. Additionally, oral TAM administration over a 7-day treatment period demonstrated to be clinically safe, without adverse effects on clinical, hematological or serum biochemical parameters. These data could contribute to the continued research into this drug's potential for the treatment of different inflammatory conditions in equine species.

Tamoxifen induces apoptosis and inhibits respiratory burst in equine neutrophils independently of estrogen receptors.

Neutrophils play an important role in the exacerbation and maintenance of severe equine asthma; persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues. Tamoxifen (TX) is a nonsteroidal estrogen receptor modulator with immunomodulatory effects and induces early apoptosis of blood and bronchoalveolar lavage neutrophils from horses with acute lung inflammation. This study investigated if the in vitro effects of tamoxifen are produced by its action on nuclear (α and ß) and membrane (GPR30) estrogen receptors in healthy equine neutrophils. Results showed that TX inhibits neutrophil respiratory burst induced by opsonized zymosan in a dose-dependent manner. Nuclear (17-ß-Estradiol) and GPR30 cell membrane (G1) estrogen receptor agonists and their antagonists (ICI 182,780 and G15, respectively) do not block or reproduce the effect of TX. Therefore, TX does not inhibit respiratory burst through estrogen receptors. TX (8.5 µM) also increased phosphatidylserine translocation, a marker of early apoptosis, which did not occur with any of the estrogen receptor agonists or antagonists. Thus, tamoxifen generates dose-dependent inhibition of respiratory burst and increased early apoptosis in healthy equine neutrophils, independently of nuclear or membrane estrogen receptors. Further studies are necessary to explore the signaling pathways of tamoxifen-induced ROS inhibition and phosphatidylserine translocation.

Tamoxifen inhibits chemokinesis in equine neutrophils.

Neutrophils are terminally differentiated innate effector cells at the first line of host defense. Neutrophil migration within tissues is complex and involves several steps, during which these cells must be able to interpret a variety of chemical and physical signals. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including equine asthma. Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and bronchoalveolar lavage fluid (BALF) neutrophils, reduction of BALF neutrophil content, and improvement in animals' clinical status. Further, TX dampens chemotactic index and respiratory burst production in vitro. The aim of this study was to provide information on the effect of TX on chemokinesis in peripheral blood neutrophils from five healthy horses. Results showed that neutrophils increased migration and travelled distance in response to IL-8; but in the presence of TX, IL-8 did not produce neutrophil migration. This suggests that TX has an inhibitory effect on the kinesis of equine peripheral blood neutrophils stimulated with IL-8. However, further studies are required to fully understand the signaling pathways of TX on neutrophil chemokinesis.

Role of neutrophils in equine asthma.

Neutrophilic bronchiolitis is the primary lesion in asthma-affected horses. Neutrophils are key actors in host defense, migrating toward sites of inflammation and infection, where they act as early responder cells toward external insults. However, neutrophils can also mediate tissue damage in various non-infectious inflammatory processes. Within the airways, these cells likely contribute to bronchoconstriction, mucus hypersecretion, and pulmonary remodeling by releasing pro-inflammatory mediators, including the cytokines interleukin (IL)-8 and IL-17, neutrophil elastase, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs). The mechanisms that regulate neutrophil functions in the tissues are complex and incompletely understood. Therefore, the inflammatory activity of neutrophils must be regulated with exquisite precision and timing, a task achieved through a complex network of mechanisms that regulates neutrophil survival. The discovery and development of compounds that can help regulate ROS, NET formation, cytokine release, and clearance would be highly beneficial in the design of therapies for this disease in horses. In this review, neutrophil functions during inflammation will be discussed followed by a discussion of their contribution to airway tissue injury in equine asthma.

Microglia and astrocyte activation in the spinal cord of lame horses.

OBJECTIVE: To determine the microglial and astrocyte response to painful lameness in horses. STUDY DESIGN: Ionized calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) expression, cell density and morphology were determined through immunofluorescence within the dorsal horn of equine spinal cord. ANIMALS: A total of five adult horses with acute or chronic unilateral lameness, previously scheduled for euthanasia. METHODS: Musculoskeletal lameness was evaluated in five horses through visual evaluation according to clinical guidelines. Spinal cord samples were obtained immediately after euthanasia, and distal limb lesions were confirmed through dissection and radiography. Iba-1 immunostaining was used for detection and characterization of dorsal horn microglia. GFAP was used for immunostaining of dorsal horn astrocytes. Iba-1 and GFAP labeled cells were quantified in the dorsal horn, and intensity of fluorescence was compared between the ipsi- and contralateral dorsal horn to the affected limb, and between dorsal horn segments of all horses. RESULTS: Iba-1 expression was higher in the ipsilateral dorsal horn of the affected limb in contrast to the contralateral side dorsal horn. GFAP markers did not demonstrate increased astrocytic activity on the dorsal horn ipsilateral side to the distal limb lesion of affected horses. Horses with acute lameness predominantly had a spherical shape microglial phenotype, while cells from chronic lameness cases had variable morphology. Astrocytes evidenced small somas and large processes in both acute and chronic lameness, with higher GFAP localization in the main branches. As in the case of rodents, the localization of microglia and astrocytes in horses was mainly situated within laminae I, II and III. CONCLUSIONS AND CLINICAL RELEVANCE: Iba-1 and GFAP are functional and morphological markers of spinal microglial cells and astrocytes in horses with lameness.

Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses.

The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary Acidic Protein (GFAP) and a polyclonal rabbit antibody against Connexin 43 (Cx-43). For immunodetection of spinal cord dorsal horn microglia, a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Epifluorescence and confocal images were obtained for the morphological and organizational analysis. Evaluation of shape, area, cell diameter, cell process length and thickness was performed on dorsal horn microglia and astrocyte. Morphologically, an amoeboid spherical shape with a mean cell area of 92.4 + 34 µm2 (in lamina I, II and III) was found in horse microglial cells, located primarily in laminae I, II and III. Astrocyte primary stem branches (and cellular bodies to a much lesser extent) are mainly detected using GFAP. Thus, double GFAP/Cx-43 immunolabeling was needed in order to accurately characterize the morphology, dimension and cell density of astrocytes in horses. Horse and rodent astrocytes seem to have similar dimensions and localization. Horse astrocyte cells have an average diameter of 56 + 14 µm, with a main process length of 28 + 8 µm, and thickness of 1.4 + 0.3 µm, mainly situated in laminae I, II and III. Additionally, a close association between end-point astrocyte processes and microglial cell bodies was found. These results are the first characterization of cell morphology and organizational aspects of horse spinal glia. Iba-1 and GFAP/Cx-43 can successfully immune-label microglia and astrocytes respectively in horse spinal cords, and thus reveal cell morphology and corresponding distribution within the dorsal horn laminae of healthy horses. The conventional hyper-ramified shape that is normally visible in resting microglial cells was not found in horses. Instead, horse microglial cells had an amoeboid spherical shape. Horse protoplasmic astroglia is significantly smaller and structurally less complex than human astrocytes, with fewer main GFAP processes. Instead, horse astrocytes tend to be similar to those found in rodent's model, with small somas and large cell processes. Microglia and astrocytes were found in the more superficial regions of the dorsal horn, similarly to that previously observed in humans and rodents. Further studies are needed to demonstrate the molecular mechanisms involved in the neuron-glia interaction in horses.

Modulatory role of regulatory T cells in a murine model of severe equine asthma.

BACKGROUND: It is accepted that T regulatory cells (Treg) control different types of immune responses. In connection with this role, we have recently described an important increase in CD4+, CD25high, Foxp3+ lymphocytes in the airway system of horses coursing with an exacerbation of severe equine asthma (EA). To explore the potential role of this population in the resolution of EA inflammation, we used a murine experimental model in which airway neutrophilic inflammation, which is similar to that observed in EA, is induced in mice by continual exposure to Aspergillus fumigatus contaminated hay. This model has the advantage that in mice we may induce a reduction of the Treg population using low doses of cyclophosphamide (Cy). RESULTS: The results indicated that the percentage of Treg cells increased with allergen exposure, as in horses; and animals partially depleted of Treg cells by treatment with Cy showed increased airway inflammation, demonstrated by an increased percentage of neutrophils and specific immunoglobulins in bronchoalveolar lavage fluid (BALF). Furthermore, a histopathologic study of animals that were pretreated with Cy before antigenic challenge showed higher cellular infiltration in the lung and deeper remodeling changes in the bronchi, including epithelial and goblet cell hyperplasia as well as airway smooth muscle hypertrophy. CONCLUSION: In this murine model of EA, the reduced number and function of Treg induced by low doses of Cy, which directly correlates with increased airway inflammation and lung infiltration, indicates that Treg may play a major role in the regulation and resolution of EA.