There is a growing experience demonstrating benefit of mesenchymal stromal cell (MSC)-based cell therapies in preclinical models of asthma. In the current study, conditioned media (CM) and, in particular, the extracellular vesicle fraction obtained from the CM were as potent as the MSCs themselves in mitigating Th2/Th17-mediated allergic airway inflammation in a mouse model of severe refractory clinical asthma. Moreover, human MSC CM and extracellular vesicles were effective in this immunocompetent mouse model. These data add to a growing scientific basis for initiating clinical trials of MSCs or extracellular vesicles derived from MSCs in severe refractory asthma and provide further insight into the mechanisms by which the MSCs may ameliorate the asthma.

Myxomatous mitral valve disease (MMVD) is functionally and histologically identical to mitral valve prolapse (MVP) in humans. Currently, there are no medical treatments that can delay the progression of this valvular disease or associated cardiac remodelling. Therefore, there is a need to understand the molecular pathology associated with MMVD and MVP better, and thus identify potential therapeutic targets. Circulating exosomes contain small RNA, including miRNA, which reflect cell physiology and pathology. This study explored the association between circulating exosomal miRNA (ex-miRNA) content and MMVD, heart failure due to MMVD (MMVD-CHF) and ageing, which is strongly associated with MMVD. Ex-miRNA was isolated from old normal/healthy dogs (n = 6), young normal dogs (n = 7), dogs with MMVD (n = 7) and dogs with MMVD-CHF (n = 7). Separately, total plasma miRNA was isolated from normal dogs (n = 8), dogs with MMVD (n = 8) and dogs with MMVD-CHF (n = 11). Using reverse transcription quantitative polymerase chain reaction, exosomal miR-181c (p = 0.003) and miR-495 (p = 0.0001) significantly increased in dogs with MMVD-CHF compared to the other three groups. Exosomal miR-9 (p = 0.002) increased in dogs with MMVD and MMVD-CHF compared to age-matched (old) normal dogs. Exosomal miR-599 (p = 0.002) decreased in dogs with MMVD compared to old normal dogs. In total plasma, 58 miRNA were deemed significantly different (p < 0.04) between normal dogs, dogs with MMVD and dogs with MMVD-CHF. However, in contrast to ex-miRNA, none of the miRNA in total plasma remained statistically significant if the false discovery rate was <15%. Changes in ex-miRNA are observed in dogs as they age (miR-9, miR-495 and miR-599), develop MMVD (miR-9 and miR-599) and progress from MMVD to CHF (miR-181c and miR-495). Ex-miRNA expression-level changes appear to be more specific to disease states than total plasma miRNA.RESPONSIBLE EDITOR Elena Aikawa, Harvard Medical School, USA

Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve EV characterization as a precursor for myriad downstream investigations. Identifying expression of EV surface epitopes can aid in determining EV enrichment and allow for comparisons of sample phenotypes. This study was designed to test a rigorous method of indirect fluorescent immunolabeling of single EV with subsequent evaluation using nanoparticle tracking analysis (NTA) to simultaneously determine EV concentration, particle size distribution, and surface immunophenotype. In this study, EV were isolated from canine and human cell cultures for immunolabeling and characterized using NTA, transmission electron microscopy, and Western blotting. Indirect fluorescent immunolabeling utilizing quantum dots (Qd) resulted in reproducible detection of individual fluorescently labeled EV using NTA. Methods were proposed to evaluate the success of immunolabeling based on paired particle detection in NTA light scatter and fluorescent modes. Bead-assisted depletion and size-exclusion chromatography improved specificity of Qd labeling. The described method for indirect immunolabeling of EV and single vesicle detection using NTA offers an improved method for estimating the fraction of EV that express a specific epitope, while approximating population size distribution and concentration.

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Background Diagnosis of pituitary pars intermedia dysfunction (PPID) using the thyrotropin‐releasing hormone (TRH) stimulation test requires blood collection 10 minutes after TRH injection; it is unknown if small differences in timing affect test results. Objective To determine whether early or late sampling results in a significant (≥10%) difference in plasma adrenocorticotropic hormone (ACTH) concentration compared to standard 10‐minute sampling. Animals Twenty‐four healthy adult horses with unknown PPID status. Methods In this prospective study, subjects underwent a single TRH stimulation test, with blood collected exactly 9 minutes (early), 10 minutes (standard), and 11 minutes (late) after injection. ACTH was measured by chemiluminescent immunoassay. Two aliquots of the 10‐minute plasma sample were analyzed separately to assess intra‐assay variability. Data were reported descriptively and bias was calculated using Bland‐Altman plots. Significance was set at P = .05. Results Minor variability was observed between the paired 10‐minute sample aliquots (range, 0%‐6%; median 3%). Overall variability of early or late samples compared to the corresponding paired (average) 10‐minute standard concentration ranged from 0% to 92% (median 10%). Seventy‐five percent of horses (18/24) tested had at least 1 early or late reading that differed by ≥10% from its corresponding 10‐minute standard concentration, and 21% of horses (5/24) would have a different interpretation of testing result with either early or late sampling. Incidence of ≥10% variability was independent of PPID status (P = .59). Conclusions and Clinical Importance Precise timing of sample collection is critical to ensure accurate assessment of PPID status given the observation of significant variability associated with minor alterations in timing of sample collection.

TEG measurements of MA and G are more reproducible than assessment of K, within samples and between operators. The highest test variability was thus observed within the early phase of clot formation.

A 15-year-old, 558-kg Quarter Horse gelding was examined because of peripheral edema and bicavitary (pleural and abdominal) effusion, identified via field ultrasonography before referral. Two years earlier, the horse was evaluated for a similar clinical presentation, including peripheral edema, bicavitary effusion, and fever. The gelding was diagnosed with Anaplasma phagocytophilum infection by polymerase chain reaction (PCR) a on peripheral blood. He was treated with oxytetracycline b (approximately 10 mg/kg IV q12h for 5 days) and made a full clinical recovery.Initial evaluation during the most recent episode revealed a normal body temperature, tachycardia (60 BPM; reference range (RR): 26-50 1 ), and a normal respiratory rate and effort. Thoracic auscultation identified muffled left-sided heart sounds and bilateral absence of bronchovesicular sounds ventrally. The horse had marked pitting ventral, preputial, and pectoral edema. There were prominent jugular pulses, extending one-half to one-third up the neck.Arterial oxygen tension c was within reference range. A subsequent serum chemistry analysis revealed low total protein levels (3.9 g/dL; RR: 5.6-7.0) characterized by panhypoproteinemia, but was otherwise unremarkable.Complete blood count (CBC) d analysis revealed mild anemia (hematocrit [HCT] 30%; RR: 32-50), leukopenia (4.8 9 10 3 /lL; RR: 5.9-11.2), and lymphopenia (0.96 9 10 3 /lL; RR: 1.6-5.2), consistent with inflammation. The serum fibrinogen (375 mg/dL; RR: 100-400) and platelet count (159 9 10 3 /lL; RR: 100-400) were within normal limits. No granulocytic inclusions were noted.Abdominal and thoracic ultrasound examination confirmed the presence of moderate, hypoechoic bicavitary effusion, and identified mild dilatation of the liver sinusoids. Bilateral thoracentesis yielded moderate volumes (4 L from the left hemithorax, 3 L from the right hemithorax) of a neutrophilic transudate. As the horse's clinical signs could be attributable to heart failure, an echocardiogram was subsequently performed. The examination revealed a structurally normal heart with normal cardiac measurements, but mild pulmonic valve insufficiency and mild tricuspid valve regurgitation. Moderate pericardial effusion was present, resulting in irregular motion of the right cardiac chambers, consistent with mild tamponade. Regional epicardial thickening with fibrin accumulation was also noted. An electrocardiogram identified a second-degree atrioventricular block. Subsequent pericardiocentesis yielded 5 L of serosanguinous fluid, which was characterized as a mixed inflammatory exudate with a total protein of 3.8 g/dL (RR: <2), HCT of 2%, red blood cell count of 0.334 M/lL, and a total nucleated cell count of 6.02 9 10 3 /lL (RR: <5). Cytologic evaluation revealed 52% nondegenerate neutrophils, 39% lymphocytes, and 9% monocytes. While peripheral blood contamination could not be entirely ruled out, the lack of visible platelets in the sample, as well as the relative increase in inflammatory cells compared to peripheral blood made a ...