Extracellular Microvesicles (MV's) Isolated from 5-Azacytidine-and-Resveratrol-Treated Cells Improve Viability and Ameliorate Endoplasmic Reticulum Stress in Metabolic Syndrome Derived Mesenchymal Stem Cells.

Extracellular vesicles (EVs), a spherical membrane fragments including exosomes, are released from several cell types, including mesenchymal stromal cells (MSCs), constitutively or under stimulation. As MVs cargo include DNA, RNA, miRNA, lipids and proteins their have gain special attention in the field of regenerative medicine. Depending on the type of transferred molecules, MVs may exert wide range of biological effects in recipient cells including pro-inflammatory and anti-apoptotic action. In presented paper, we isolated MVs form adipose derived mesenchymal stem cells (ASC) which underwent stimulation with 5-azacytydine and resveratrol (AZA/RES) in order to improve their therapeutic potential. Then, isolated MVs were applied to ASC with impaired cytophysiological properties, isolated from equine metabolic syndrome diagnosed animals. Using RT-PCR, immunofluorescence, ELISA, confocal microscopy and western blot, we have evaluated the effects of MVs on recipient cells. We have found, that MVs derived from AZA/RES treated ASC ameliorates apoptosis, senescence and endoplasmic reticulum (ER) stress in deteriorated cells, restoring their proper functions. The work indicates, that cells treated with AZA/RES through their paracrine action can rejuvenate recipient cells. However, further research needs to be performed in order to fully understand the molecular mechanisms of these bioactive factors action. Graphical Abstract Graphical abstract of presented study.

Effects of advanced age and pituitary pars intermedia dysfunction on components of the acute phase reaction in horses.

Age, neurodegenerative disorders, and dysfunction of insulin secretion may be correlated with increased systemic concentrations of acute phase markers. Thus, the study aimed to determine the effect of age, pituitary pars intermedia dysfunction (PPID), and insulin dysregulation (ID) associated with PPID, on markers of the acute phase reaction. Twenty-nine mix-breed horses of both sexes were classified into groups: (1) healthy adult controls, (2) healthy non-PPID geriatric horses, (3) PPID ID+ horses, and (4) PPID ID- horses. Whole blood proinflammatory cytokine gene expression and serum concentrations of pro- and anti-inflammatory cytokines and acute phase proteins were measured. The data were analyzed using the Mann-Whitney U-test, and correlations between groups of data were assessed using Spearman's correlation coefficient. The tests were statistically significant if P < 0.05. No differences in the whole blood cytokine gene expression, serum cytokine concentrations, or acute phase proteins were noted between the groups. In the PPID ID group, there was a strong correlation between the ACTH concentration after the administration of thyrotropin-releasing hormone and the expression of IL-8 (r = 0.941; P = 0.0321). In the PPID ID+ group, there was a strong correlation between basal insulin concentrations and serum amyloid A (SAA; r = 0.936; P = 0.0083) as well as between postprandial insulin concentrations and SAA (r = 0.965; P = 0.001). These data suggest that neurodegeneration in horses moderately affects circulating markers of inflammation and that ID in horses with PPID influences acute phase inflammatory markers.

Effects of equine metabolic syndrome on inflammation and acute-phase markers in horses.

Obesity and metabolic disorders are associated with systemic low-grade chronic inflammation, both in humans and animals. The aim of the study is to assess the effects of obesity and hyperinsulinemia on individual components of the acute-phase reaction in equine metabolic syndrome (EMS) horses. Eight mixed-breed EMS and six control, age-matched horses of both sexes were included in the study. Animals were classified as EMS or control based on the assessment of BCS, cresty neck score, and basal insulin >50 µU/mL and/or insulin responses to the oral sugar test (OST) >60 µU/mL. Peripheral venous blood was collected. The expression of proinflammatory cytokines, the concentration of circulating cytokines, and acute-phase proteins (serum amyloid A, C-reactive protein, haptoglobin, activin A, and procalcitonin) were measured. The data were analyzed using the Mann-Whitney test, whereas correlations were examined using Spearman's correlation coefficient. The tests were statistically significant if P ≤ 0.05. There were no differences in cytokine gene expression, circulating cytokine concentrations, or concentrations of acute-phase proteins between the EMS and the control groups. There was a strong correlation between the basal concentration of insulin and the serum concentrations of IL-6 (r = 0.71, P < 0.05). Activin A was positively correlated with post-OST insulin concentrations (r = 0.707, P = 0.05), indicating that this marker of inflammation could warrant further investigation in horses with EMS.

Prevalence and severity of ultrasonographic pulmonary findings in horses with asthma - a preliminary study.

Asthma is one of the most common non-infectious respiratory diseases in horses. Ultrasound examination is a widely available non-invasive additional diagnostic tool. To date, there are no studies focusing on ultrasonographic findings in horses with asthma. The aim of this study was to analyse the prevalence and severity of ultrasound lesions in lung tissue in horses with asthma. Lung ultrasonography was carried out on six healthy horses (controls) and 12 horses with asthma (six with mild and six with severe asthma). The sonographic changes in three lung sections were assessed using a scoring system. The most common changes present in all the animals were comet- tail artefacts. More advanced lesions were present in horses with severe asthma. Statistically significant differences in the overall average intensity of the ultrasound changes were seen between the controls and the study group and between the horses with mild and severe asthma. The lesions were usually located in the caudal lung regions, but they were also present in other areas as the disease progressed. Ultrasonography is a useful additional diagnostic tool enabling an assessment of the stage of the asthma progression. It is a very sensitive technique that visualizes minor lesions in the lung tissue even in clinically healthy animals. Due to its low specificity, it cannot replace endoscopy and the bronchoalveolar lavage in horses with asthma.

Assessment of sand accumulation in the gastrointestinal tract and its excretion with stool in silesian foals.

The aim of this study was to assess sand accumulation in the gastrointestinal tract and fecal sand excretion in Silesian foals using three diagnostic methods and taking into account the sex and age of the animals. Another aim of the study was to compare the three diagnostic methods. The study was carried out on 21 clinically healthy Silesian foals (10 females and 11 males) from 9-28 weeks old grazed on permanent pasture. The sand intake was assessed using a sedimentation test, abdominal ultrasonography and a quantitative evaluation of sand per 100 g of stool. In the sedimentation test, the sand was palpable in the stool of 57.1% of the horses, and clearly visible in 42.9% of the animals. The ultrasound examination revealed the presence of sand in the gastrointestinal tract in 66.7% of the horses. It was limited to a single location in 60% of the horses, while it was present in several regions in 40% of the horses. The mean amount of sand was 0.14 ± 0.33 g per 100 g of stool. It did not exceed 0.1g in 71.4% foals, while it ranged from 0.1-0.5 g in 23.8% foals. In 4.8% of the animals, it amounted to 1.6 g per 100 g of stool. There was no correlation between age and gender and the results. There was a positive correlation between the ultrasound examination and the sedimentation test. Sand may be accumulated in the gastrointestinal tract of foals without any clinical signs. The amount of sand excreted in the stool is not an indicator of the amount of sand accumulated in the gastrointestinal tract. An abdominal ultrasound examination should be combined with a sedimentation test for more specific results.

Conjunctival aerobic bacterial flora in healthy Silesian foals and adult horses in Poland.

BACKGROUND: Commensal bacterial and fungal flora of the conjunctival sac has been described in horses and other animals. The identification of commensal flora of the conjunctival sac may aid in the diagnosis of ocular inflammatory diseases, such as conjunctivitis or more severe ulcerative keratitis, common in horses. Moreover, damage of ocular protective barriers may lead to an opportunistic infection. The study was carried out in Silesian horses kept at a single breeding center in South-western Poland, in order to limit any breed-dependant and climate-dependant variables affecting the results. Following an ophthalmic examination that revealed no abnormalities, sterile swabs were collected from conjunctival sac in 26 adult horses and 11 foals. The obtained swabs were subjected to bacterial culture testing. In case of Staphylococcus spp. isolation, susceptibility to methicillin was evaluated. RESULTS: Forty- three bacterial isolates, representing eleven genera of bacteria were cultured from 30 (81%) horses. Gram-positive bacteria were the dominant isolates (72%) (p < 0.001). The most commonly isolated Gram-positive bacteria were Staphylococcus spp., while Moraxella spp. were the most frequently isolated Gram-negative bacteria. There was no significant influence of sex and age on the frequency and type of microbial isolates. CONCLUSIONS: Commensal flora is present in the conjunctival sac of healthy horses in Poland. Age does not affect the abundance and type of microbial isolates.

The detection of capsaicin and dihydrocapsaicin in horse serum following long-term local administration.

BACKGROUND: Capsaicin and dihydrocapsaicin are alkaloids with analgesic effects in humans and animals. When used locally, both of them minimalise pain sensation by defunctionalising nerve endings. According to the Federation Equestrian International Prohibited Substances List, these are substance banned in horse competitions. The aim of the study was to determine the detection time of capsaicin in both plasma and serum after long-term use of a gel recommended for commercial use and applied as intended. The objective of the study was to select the best material for the detection of capsaicin as a doping substance in horses. METHODS: Nine healthy mature horses were administered 0.1% capsaicin topically in the form of a commercial analgesic gel (15 g of the gel per limb) to the front limbs every 24 hours for five days with a polar fleece bandage. Blood serum and plasma were collected prior to gel application and in the 12th, 18th, 24th, 36th, 42nd, 48th, 60th, 84th, 108th, 132nd, 156th hour after the gel application. Qualitative and quantitative analysis was performed using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). RESULTS: The concentration of capsaicin in the serum samples did not exceed the lower limit of quantification. Capsaicin was not detected in the plasma samples during the entire study period. Dihydrocapsaicin was not detected in blood serum or plasma. CONCLUSION: The presented results suggest that capsaicin is not detected in horse serum in the 24-hour-periodfollowing its last application according to the dosage regimen used by owners and veterinarians for therapy rather than doping, based on a five day gel application and a polar bandage.

Morphology and elemental analysis of free range and stabled Polish Konik horses hair using Energy-dispersive X-ray spectroscopy (EDS).

Hair is a more biologically stable material than other tissues and contains a relatively constant level of minerals, drugs or toxins. The content of essential elements in hair depends on dietary and non-dietary factors. The aim of this study was to assess and compare the effect of diet on the elemental composition and morphological properties of the hair of healthy Polish Konik horses. Mane hair was collected from 19 horses living in The National Park in Popielno in Poland. Six of these horses were free-ranged all year with permanent access to pasture, while 13 horses were kept in the stable and fed oats, hay and straw. The samples were analyzed using a Scanning Electron Microscope combined with Energy-dispersive X-ray spectroscopy (SEM-EDS). Each hair was analyzed for its microscopic appearance and elemental Mg, Si, S, Ca, Mn, Co, Zn and Se content. No significant changes in the morphological properties and elemental composition were observed. There was inter- individual variability in element content between horses in both groups, but this did not affect their health. The comparison of the present results with other studies shows that the level of elements in the hair of Polish Konik horses is significantly lower than in healthy horses of other breeds. The traditional feeding plan containing pasture, oats and hay does not contain a sufficient amount of main mineral elements. Further investigation is needed to explain the effect of diet and maintenance on the mineral balance of horses.

Searching for ivermectin resistance in a Strongylidae population of horses stabled in Poland.

BACKGROUND: There are no available studies describing the possible resistance of strongyles to ivermectin in horses in Poland. One hundred seventy three horses from nine stud farms from South-Western Poland were studied. The effectiveness of ivermectin was studied on the 14th day after ivermectin administration using the fecal egg count reduction test, and a long-term observation of the egg reappearance period was carried out. The fecal study was carried out using a modified McMaster method, which typically detects 20 eggs per gram of stool. The results were subjected to statistical analysis that enabled quantification of the eggs in the stool samples. RESULTS: The study revealed high efficacy of ivermectin on the 14th day after administration without a shortening of the egg reappearance period. CONCLUSION: The results indicate that strongyles resistance to ivermectin in Poland is not a serious problem.