Amikacin disaggregates platelet clumps in EDTA blood samples from cats and dogs when added postcollection.

BACKGROUND: Pseudothrombocytopenia may lead to the erroneous diagnosis of thrombocytopenia, resulting in unnecessary testing and treatment. The addition of exogenous substances to blood samples prior to collection has been shown to mitigate platelet (PLT) clumps in blood samples. Postcollection additives aiming to disaggregate PLT clumps have been largely unexplored. OBJECTIVES: We aimed to determine if the addition of amikacin to blood samples postcollection aids in the disaggregation of PLT clumps in cats and dogs. METHODS: For this prospective study, EDTA-collected blood samples from 28 cats and 17 dogs were obtained from a hospital population at UC Davis Veterinary Medical Teaching Hospital. Samples had PLT clumps detected on blood smears and thrombocytopenia per analyzer count. Amikacin was added to samples postcollection, and an additional CBC was performed. Flow cytometry was performed to assess PLT-fibrinogen binding in amikacin-treated aliquots. RESULTS: PLT-clumped samples treated with amikacin significantly increased PLT numbers by 134% and decreased mean platelet volume (MPV) values by 14% (P ≤ 0.0001) in cats, and increased PLT numbers by 32% (P = 0.04) and increased MPV values by 9% (P = 0.02) in dogs. Mean cell volume (MCV) slightly increased (<4%) for both species. No other CBC parameters were substantially affected by the addition of amikacin. Flow cytometry showed decreased PLT-fibrinogen binding in the majority of cats but was not significant (P > 0.05). CONCLUSIONS: Adding amikacin to PLT-clumped blood samples postcollection may be a convenient solution for pseudothrombocytopenia in cats and dogs. Future studies are needed to elucidate the mechanism of amikacin and its effectiveness under different storage conditions. This is the first reported use of amikacin postcollection to disaggregate PLT clumps in blood samples from animals.

A potential early clinical phenotype of necrotizing meningoencephalitis in genetically at-risk pug dogs.

BACKGROUND: Necrotizing meningoencephalitis (NME) in the pug dogs is a fatal neuroinflammatory disease associated with rapid progression and poor response to conventional immunosuppressive therapy. Diagnosis is typically made after severe neurological abnormalities have manifested. HYPOTHESIS/OBJECTIVE: Pug dogs at genetic risk for NME might manifest neurological abnormalities before developing pathognomonic clinical signs of NME. ANIMALS: Thirty-six pug dogs less than 4 years of age asymptomatic for NME. METHODS: Prospective observational cohort study with germline genome-wide genotyping. Neurological examinations were performed 4 weeks apart to document reproducible findings of central nervous system disease. Magnetic resonance imaging, cerebrospinal fluid analysis, and testing for infectious diseases were performed in all pugs with reproducible abnormalities detected on neurological examination. RESULTS: The overall risk allele frequency in this cohort was 40%; 5 (14%) dogs were high risk, 19 (53%) dogs were medium risk, and 12 (33%) dogs were low genetic risk for NME. Reproducible abnormalities detected on neurological examination were identified in 8/24 (33%) genetically at-risk dogs and 0/12 (0%) low risk dogs. Clinical abnormalities included multifocal spinal pain in 8/8, reduced menace response in 5/8, and lateralizing postural reaction deficits in 5/8 pugs. There was a strong association between genotype risk and the presence of this clinical phenotype (P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings suggest the presence of a novel early clinical phenotype of NME in apparently asymptomatic genetically at-risk pugs which might be used to plan early diagnostic and therapeutic clinical trials.

Lipoprotein profile of pleural and peritoneal transudates in dogs and cats.

BACKGROUND: Current diagnostic evaluation of transudative effusions rarely aids in identifying an underlying etiology. Lipoproteins in the fluid might reflect the site or nature of vessel involvement. OBJECTIVES: Improve the classification and diagnostic utility of pleural and peritoneal transudates in dogs and cats by investigating lipoprotein patterns in effusions. Compare these patterns with other peritonaeal and pleural fluid variables and underlying diseases. ANIMALS: Samples of transudates and serum from 18 cats and 37 dogs with transudative effusion (total nucleated cell count [TNCC] <5000 cells/µL) were analyzed. METHODS: Lipoprotein fractions, triglyceride, and cholesterol (CHO) concentrations were prospectively determined in paired fluid and serum samples. Standard fluid measurements were retrospectively collected. RESULTS: Two distinct fluid lipoprotein patterns were noted. Fluids rich in VLDL+IDL were associated with chronic kidney disease, acquired portosystemic shunts or protein-losing enteropathy (group I). Fluids rich in denser lipoproteins were associated with underlying heart disease, caudal vena cava syndrome or intracavitary neoplasia (group II). Group I and group II also had significant differences between fluid concentrations of CHO (x̄ = 8 vs 110 mg/dL) and TP (x̄ = 0.6 vs 3.8 g/dL), respectively. Five peritoneal transudates were triglyceride-rich (>100 mg/dL) and associated with pancreatitis. CONCLUSIONS AND CLINICAL IMPORTANCE: Protein-poor (TP <1.5 g/dL) and protein-rich (TP >2.5 g/dL) transudates were associated with distinct lipoprotein patterns and specific groups of disease. Effusions secondary to pancreatitis might be transudative and rich in triglycerides.

Multipotent Stromal Cells and Viral Interaction: Current Implications for Therapy.

Multipotent stromal cells (MSCs) are widely utilized in therapy for their immunomodulatory properties, but their usage in infectious viral diseases is less explored. This review aimed to collate the current novel use of MSCs in virus-associated conditions, including MSC's susceptibility to virus infection, antiviral properties of MSCs and their effects on cell-based immune response and implementation of MSC therapy in animal models and human clinical trials of viral diseases. Recent discoveries shed lights on MSC's capability in suppressing viral replication and augmenting clearance through enhancement of antiviral immunity. MSC therapy may maintain a crucial balance between aiding pathogen clearance and suppressing hyperactive immune response.

Leukocyte and cytokine variables in asymptomatic Pugs at genetic risk of necrotizing meningoencephalitis.

BACKGROUND: Necrotizing meningoencephalitis (NME, aka Pug dog encephalitis) is an inflammatory brain condition associated with advanced disease at initial presentation, rapid progression, and poor response to conventional immunomodulatory therapy. HYPOTHESIS/OBJECTIVES: That genetic risk for NME, defined by a common germline DNA haplotype located on chromosome 12, is associated with altered blood cytokine concentrations and leukocyte subsets in asymptomatic Pugs. ANIMALS: Forty Pug dogs asymptomatic for NME from a hospital sample. METHODS: Prospective observational cohort study, including germline genome-wide genotyping, plasma cytokine determination by multiplexed profiling, and leukocyte subset characterization by flow cytometric analysis. RESULTS: Seven (18%) dogs were high risk, 10 (25%) medium risk, and 23 (58%) low risk for NME, giving a risk haplotype frequency of 30%. High and medium risk Pugs had significantly lower proportion of CD4+ T cells (median 22% [range, 7.3%-38%] vs 29% [range, 16%-41%], P = .03) and higher plasma IL-10 concentrations than low-risk Pugs (median 14.11 pg/mL [range, 9.66-344.19 pg/mL] vs 12.21 pg/mL [range, 2.59-18.53 pg/mL], P = .001). No other variables were significantly associated with the NME haplotype-based risk. CONCLUSIONS AND CLINICAL IMPORTANCE: These data suggest an immunological underpinning to NME and a biologic rationale for future clinical trials that investigate novel diagnostic, preventative, and therapeutic strategies for this disease.

Feline adipose-derived mesenchymal stem cells induce effector phenotype and enhance cytolytic function of CD8+ T cells.

BACKGROUND: Feline adipose-derived mesenchymal stem cells (ASCs) engage with a variety of immune cells and have been used in several clinical trials for the treatment of inflammatory and immune-dysregulated diseases in cats, but the impact they exert on the functional characteristics on T cells, particularly CD8+ T cells, remains to be elucidated. METHODS: Modified mixed leukocyte reaction was performed between feline ASCs and PBMCs. Changes of cell cycle stages, phenotype and cellular senescence were determined through flow cytometry and gene expression analysis. Cytotoxicity assay was performed to evaluate CD8+ T cell effector function. RESULTS: Feline ASCs induce cell cycle arrest on CD8+ T cells in a contact-dependent manner, downregulate CD8 surface expression, and shift their phenotype toward terminally differentiated effector cells (CD57+, CD45R+, CD62L-). CD8 T cells interacted with feline ASCs also upregulated granzyme B, IL-2 and KLRG-1 expression and have enhanced cytotoxic potential, evident by the increased percentage of lysis on target cells. CONCLUSIONS: Our findings suggest that feline ASCs (1) alter CD8+ T cells toward terminally differentiated, proinflammatory effector phenotype with limited proliferative capacity, and (2) enhance their cytotoxic potential through granzyme B upregulation. These cytotoxic CD8+ T cells could aid in disease cure in cases caused by an underlying, unresolved viral infection.

Stem cell therapy prior to full-mouth tooth extraction lacks substantial clinical efficacy in cats affected by chronic gingivostomatitis.

OBJECTIVES: The aim of this pilot study was to determine the safety, efficacy and immunomodulatory function of systemically administered adipose-derived mesenchymal stem cells (ASCs) in cats affected by feline chronic gingivostomatitis (FCGS) prior to full-mouth tooth extractions. METHODS: Five client-owned cats affected with FCGS that did not undergo full-mouth tooth extractions for FCGS treatment received two intravenous injections of 20 million fresh, allogeneic or autologous ASCs. An oral examination with photographs, a complete blood count, blood immune cell phenotyping and a biochemical profile were completed at 0 and 6 months after treatment. RESULTS: Four cats completed the study and one cat exited the study 3 months after treatment. While the treatment was determined to be clinically safe, no positive clinical response was observed in three cats and a mild response was noted in two cats. Furthermore, none of the cats exhibited immune modulation, as evidenced by no alteration in circulating CD8+ T cells, normalization of the CD4:CD8 ratio or neutrophil counts. CONCLUSIONS AND RELEVANCE: Unlike the reported efficacy of ASCs in treating cats with non-responsive FCGS after full-mouth tooth extraction, the systemic administration of ASCs prior to full-mouth tooth extraction lacks substantial clinical efficacy and is not recommended at this time.

Do allogeneic bone marrow derived mesenchymal stem cells diminish the inflammatory response to lipopolysaccharide infusion in horses? A pilot study.

Endotoxemia is a leading cause of morbidity and mortality in the equine industry, with colic being the most common cause of endotoxemia in horses. The objective of this study was to evaluate the safety and potential efficacy of a single dose of allogeneic equine bone marrow derived mesenchymal stem cells (BM-MSCs) in horses after the IV administration of lipopolysaccharide (LPS). Six horses were administered an IV infusion of 30 ng/kg LPS (O55:B5 Escherichia coli) in 500 ml saline over 30 min. Immediately after infusion test horses (n = 3) were administered 100 × 106 allogeneic BM-MSCs diluted in saline IV and control horses (n = 3) were administered saline. Clinicopathological data, pro-inflammatory cytokine measurements and sCD14 concentrations were compared between groups. No adverse reactions were observed in horses administered BM-MSCs intravenously. There were no significant differences between test and control horses with regard to clinicopathological values or pro-inflammatory cytokine production. At no time point did concentrations of sCD14 exceed the reference range in any horse. Results suggest that administration of a single IV dose of freshly cultured MSCs is safe and well-tolerated in horses with induced endotoxemia. Further study to evaluate their efficacy as a potential therapeutic in a larger number of horses with clinical disease is required.

Intra-articular Administration of Allogeneic Adipose Derived MSCs Reduces Pain and Lameness in Dogs With Hip Osteoarthritis: A Double Blinded, Randomized, Placebo Controlled Pilot Study.

This study was conducted to investigate the therapeutic effect of allogeneic adipose-derived MSCs on dogs with hip osteoarthritis (OA). Twenty dogs with bilateral osteoarthritis of the coxofemoral (hip) joint, diagnosed by a veterinarian through physical examination and radiographs were randomly allocated into four groups. Group 1 served as a placebo control and were injected with 0.9% sodium chloride (saline) (n = 4). Group 2 were injected with a single dose of 5 million MSCs (n = 5). Group 3 received a single dose of 25 million MSCs (n = 6) and Group 4 received a single dose of 50 million MSCs (n = 5). Intra-articular administration of allogeneic MSCs into multiple joints did not result in any serious adverse events. The average lameness score of the dogs in the placebo control group (-0.31) did not show improvement after 90 days of intra-articular saline administration. However, the average lameness score of the all MSC-treated dogs was improved 2.11 grade at this time point (P < 0.001). Overall, sixty five percent (65%) of the dogs that received various doses of MSCs showed improvement in lameness scores 90 days after intra-articular MSC administration. Our results showed that intra-articular administration of allogeneic adipose derived MSCs was well-tolerated and improved lameness scores and reduced pain in dogs associated with hip OA. All doses of MSCs were effective. Subsequent studies with more animals per group are needed to make a conclusion about the dose response. The improved lameness effect was present up to 90 days post-injection. Serum interleukin 10 was increased in a majority of the dogs that received MSCs and that also had improved lameness.

Placenta-derived multipotent mesenchymal stromal cells: a promising potential cell-based therapy for canine inflammatory brain disease.

BACKGROUND: Canine inflammatory brain disease (IBD) is a severe inflammatory disorder characterized by infiltration of activated immune cell subsets into the brain and spinal cord. Multipotent mesenchymal stromal cells (MSCs) are a promising therapy for IBD, based on their potent pro-angiogenic, neuroprotective, and immunomodulatory properties. The aims of this study were to compare the immunomodulatory attributes of canine adipose-derived MSCs (ASCs) and placenta-derived MSCs (PMSCs) in vitro. These data will serve as potency information to help inform the optimal MSC cell source to treat naturally occurring canine IBD. METHODS: Indoleamine 2,3 dioxygenase (IDO) activity and prostaglandin E2 (PGE2) concentration at baseline and after stimulation with interferon gamma (IFNγ) and/or tumor necrosis factor alpha (TNFα) were measured from canine ASC and PMSC cultures. Leukocyte suppression assays (LSAs) were performed to compare the ability of ASCs and PMSCs to inhibit activated peripheral blood mononuclear cell (PBMC) proliferation. IDO activity and PGE2; interleukin (IL)-2, IL-6, and IL-8; TNFα; and vascular endothelial growth factor (VEGF) concentrations were also measured from co-culture supernatants. Cell cycle analysis was performed to determine how ASCs and PMSCs altered lymphocyte proliferation. RESULTS: Activated canine MSCs from both tissue sources secreted high concentrations of IDO and PGE2, after direct stimulation with IFNγ and TNFα, or indirect stimulation by activated PBMCs. Both ASCs and PMSCs inhibited activated PBMC proliferation in LSA assays; however, PMSCs inhibited PBMC proliferation significantly more than ASCs. Blocking PGE2 and IDO in LSA assays determined that PGE2 is important only for ASC inhibition of PBMC proliferation. Activated ASCs increased IL-6 and VEGF secretion and decreased TNFα secretion, while activated PMSCs increased IL-6, IL-8, and VEGF secretion. ASCs inhibited lymphocyte proliferation via cell cycle arrest in the G0/G1 and PMSCs inhibited lymphocyte proliferation via induction of lymphocyte apoptosis. CONCLUSION: Our results demonstrate that ASCs and PMSCs have substantial in vitro potential as a cell-based therapy for IBD; however, PMSCs more potently inhibited lymphocyte proliferation by inducing apoptosis of activated lymphocytes. These data suggest that the mechanism by which ASCs and PMSCs downregulate PBMC proliferation differs. Additional studies may elucidate additional mechanisms by which canine MSCs modulate neuroinflammatory responses.

Histological, Immunological, and Genetic Analysis of Feline Chronic Gingivostomatitis.

Feline chronic gingivostomatitis (FCGS) is an immune-mediated inflammatory condition affecting the oral mucosa that results in substantial pain and suffering. The goal of this study was to complete an in-depth immunohistochemistry analysis of affected FCGS mucosa, to perform and compare immune cell phenotypes in the blood of FCGS and healthy controls cats, and to determine a transcriptomic profile of the affected and normal oral mucosa of FCGS cats. We hypothesized that cats with FCGS would have circulating activated CD8+ T cells and that tissues would be infiltrated with activated B and T cells with a highly proinflammatory transcriptome. We found that oral mucosal tissues from cats with FCGS have high tissue infiltration of B cells and that T cells include both CD4+ and CD8+ lymphocytes. Cells positive for CD25 (IL2 receptor, indicative of lymphocyte activation) and FOXP3 (indicative of regulatory T cells) were scattered throughout the mucosa. Compared to healthy individuals, cats with FCGS had high circulating CD8+ effector memory cells with a concurrent decrease in central memory cells and evidence of circulating activated CD8+ T cells (CD25+, CD62L-). Gene expression in the affected tissues was enriched for genes associated with T-cell signaling, cell adhesion molecules, leukocyte migration, inflammatory signaling pathways, extracellular matrix-receptor interactions, cytokine-cytokine receptor interactions, and natural killer cell-mediated cytotoxicity, among others. These data are essential to understand disease pathogenesis, to inform mechanism of action studies for future and current therapies, and to help select prognostic biomarkers and potency assays for stem cell treatment of FCGS.

A multicenter experience using adipose-derived mesenchymal stem cell therapy for cats with chronic, non-responsive gingivostomatitis.

BACKGROUND: The ability of mesenchymal stem cells (MSCs) to modulate immune responses inspired a series of clinical trials addressing oral mucosal inflammation. We previously reported on the safety and efficacy of fresh, allogeneic and autologous, adipose-derived mesenchymal stem cells (ASCs) to treat feline gingivostomatitis (FCGS), an oral mucosal inflammatory disease that shares similarities with human oral lichen planus. METHODS: To meet clinical demand and goals for future commercialization, we determined the feasibility of shipping fresh ASCs to distant clinics and extended our pilot studies to expand safety and efficacy data for shipped and non-shipped ASCs in a cohort of 18 FCGS cats enrolled locally and at a few different locations within the USA. RESULTS: We found that ASCs retained their viability, phenotype, and function after shipment. ASCs administered systemically resulted in a 72% positive response rate, identical to that noted in our previous studies. Cats that responded to ASC therapy had a significant decrease in circulating globulin concentration and histological evidence of decreased CD3+ T cells and CD20+ B cells in the oral mucosa. Responder cats also had significantly decreased percentages of CD8lo cells in blood prior to and at 3 months post-ASC therapy. CD8lo cells may serve as a potential "predictor" for response to systemic ASC therapy. CONCLUSION: Fresh feline ASCs can be successfully shipped and administered to cats with FCGS. ASCs modulate the immune response and demonstrate efficacy for chronic oral mucosal inflammatory lesions that are characterized by CD8+ T cell inflammation and T cell activation. FCGS is a potentially useful naturally occurring large animal model of human oral inflammatory diseases.

Horses with equine recurrent uveitis have an activated CD4+ T-cell phenotype that can be modulated by mesenchymal stem cells in vitro.

Equine recurrent uveitis (ERU) is an immune-mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T-cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T-cell phenotype. Adipose-derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4+ T-cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T-cells and B-cells in horses with ERU were similar to normal horses. However, CD4+ T-cells from horses with ERU expressed higher amounts of IFNγ indicating a pro-inflammatory Th1 phenotype. When co-incubated with MSCs, activated CD4+ T-cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell-cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4+ T-cell activation phenotype through a combination of cell-cell contact and prostaglandin signaling.

Influence of Donor's Age on Immunomodulatory Properties of Canine Adipose Tissue-Derived Mesenchymal Stem Cells.

Osteoarthritis challenges traditional therapies and remains a leading cause of lameness in older dogs. Regenerative medicine offers new strategies, typically involving the injection of autologous adipose-derived mesenchymal stem cells (MSCs). Conversely, allogenic MSCs are appealing candidates to palliate patient morbidity and cell preparation time. Regardless of the source of cells, identifying critical donor characteristics, such as age, is essential to obtain the most competent MSCs. The objectives of this study were to determine the influence of donor's age on proliferation, gene expression, and immunomodulatory properties of MSCs in dogs. Canine adipose tissue-derived MSCs (cAD-MSCs) were isolated from the falciform-ligament adipose tissues of nine pairs of gender-matched young (<2 years) or old (>7 years) client-owned dogs undergoing abdominal surgery. Growth kinetics, transcriptome before and after stimulation by tumor necrosis factor alpha and interferon gamma, MSC-induced lymphocyte suppression assay, and secretion of prostaglandin E2 (PGE2) and indoleamine 2,3-dioxygenase (IDO) were compared between cells obtained from young or old dogs. The doubling times at passages 2 and 3 were shorter when MSCs were isolated from young (34.8 ± 1.8 h and 46.3 ± 2.3 h) rather than old dogs (56.5 ± 8.0 h and 123.8 ± 46.7 h, P < 0.05). The MSC transcriptomes from both populations were similar without stimulation, while stimulation resulted in a 3-fold greater expression of osteogenic gene, fibroblast growth factor 10, in cells from old dogs. cAD-MSCs from young dogs suppressed proliferation of activated T cells more strongly (P < 0.05), although secretion of PGE2 and IDO did not differ between groups. In conclusion, donors' age affected proliferation, immunomodulatory properties of cAD-MSCs, and increased expression of osteogenic gene under proinflammatory conditions in our population of dogs. Collectively, our results provide evidence to support further evaluation of allogenic MSC therapies derived from young donors as alternatives to autologous MSC therapy in older dogs.

Mechanisms utilized by feline adipose-derived mesenchymal stem cells to inhibit T lymphocyte proliferation.

BACKGROUND: Feline adipose-derived mesenchymal stem cells (ASCs) have been successfully used in clinical trials for the treatment of immune-mediated diseases with T cell dysregulation. However, the immunomodulatory pathways utilized by feline ASCs to suppress T cell activation have not been fully determined. We investigated the mechanisms used by feline ASCs to inhibit T cell proliferation, including the soluble factors and the cell-cell contact ligands responsible for ASC-T cell interaction. METHODS: The immunomodulatory activity of feline ASCs was evaluated via cell cycle analysis and in vitro mixed leukocyte reaction using specific immunomodulatory inhibitors. Cell-cell interactions were assessed with static adhesion assays, also with inhibitors. RESULTS: Feline ASCs decrease T cell proliferation by causing cell cycle arrest in G0-G1. Blocking prostaglandin (PGE2), but not IDO, partially restored lymphocyte proliferation. Although PDL-1 and CD137L are both expressed on activated feline ASCs, only the interaction of intercellular adhesion molecule 1 (ICAM-1, CD54) with its ligand, lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), was responsible for ASC-T cell adhesion. Blocking this interaction reduced cell-cell adhesion and mediator (IFN-γ) secretion and signaling. CONCLUSIONS: Feline ASCs utilize PGE2 and ICAM-1/LFA-1 ligand interaction to inhibit T cell proliferation with a resultant cell cycle arrest in G0-G1. These data further elucidate the mechanisms by which feline ASCs interact with T cells, help define appropriate T cell-mediated disease targets in cats that may be amenable to ASC therapy, and may also inform potential translational models for human diseases.

ESTABLISHING HEMATOLOGY AND SERUM CHEMISTRY REFERENCE INTERVALS FOR WILD HAWAIIAN MONK SEALS ( NEOMONACHUS SCHAUINSLANDI).

Hematology and serum chemistry reference intervals have been previously established for the endangered Hawaiian monk seal ( Neomonachus schauinslandi) as an imperative measure for health assessments. Monitoring the health of the wild population depends upon reference intervals that are context specific; hence we developed reference intervals from fresh samples, as opposed to frozen, from wild monk seals. This study builds on the number of parameters from previous efforts by using samples collected between 2004 and 2015 from wild monk seals. Blood samples were analyzed by a single veterinary diagnostic laboratory within 24 hr of collection from apparently healthy, wild seals during research activities. Reference intervals were determined based on the analytical steps outlined by the American Society for Veterinary Clinical Pathology. These comprehensive hematology and serum chemistry reference intervals enable more consistent and systematic interpretation of results, which will guide individual and population-level health assessment and decision-making research and recovery activities.

Safety and tracking of intrathecal allogeneic mesenchymal stem cell transplantation in healthy and diseased horses.

BACKGROUND: It is currently unknown if the intrathecal administration of a high dose of allogeneic mesenchymal stem cells (MSCs) is safe, how MSCs migrate throughout the vertebral canal after intrathecal administration, and whether MSCs are able to home to a site of injury. The aims of the study were: 1) to evaluate the safety of intrathecal injection of 100 million allogeneic adipose-derived MSCs (ASCs); 2) to assess the distribution of ASCs after atlanto-occipital (AO) and lumbosacral (LS) injection in healthy horses; and 3) to determine if ASCs homed to the site of injury in neurologically diseased horses. METHODS: Six healthy horses received 100 × 106 allogeneic ASCs via AO (n = 3) or LS injection (n = 3). For two of these horses, ASCs were radiolabeled with technetium and injected AO (n = 1) or LS (n = 1). Neurological examinations were performed daily, and blood and cerebrospinal fluid (CSF) were evaluated prior to and at 30 days after injection. Scintigraphic images were obtained immediately postinjection and at 30 mins, 1 h, 5 h, and 24 h after injection. Three horses with cervical vertebral compressive myelopathy (CVCM) received 100 × 106 allogeneic ASCs labeled with green fluorescent protein (GFP) via AO injection and were euthanized 1-2 weeks after injection for a full nervous system necropsy. CSF parameters were compared using a paired student's t test. RESULTS: There were no significant alterations in blood, CSF, or neurological examinations at any point after either AO or LS ASC injections into healthy horses. The radioactive signal could be identified all the way to the lumbar area after AO ASC injection. After LS injection, the signal extended caudally but only a minimal radioactive signal extended further cranially. GFP-labeled ASCs were not present at the site of disease at either 1 or 2 weeks following intrathecal administration. CONCLUSIONS: The intrathecal injection of allogeneic ASCs was safe and easy to perform in horses. The AO administration of ASCs resulted in better distribution within the entire subarachnoid space in healthy horses. ASCs could not be found after 7 or 15 days of injection at the site of injury in horses with CVCM.

Hounsfield units are a useful predictor of pleural effusion cytological type in dogs but not in cats.

All categories of pleural effusion subjectively display as soft tissue opacity on computed tomography (CT). Quantitative measurement using Hounsfield units (HU) has the potential to bring additional information regarding the nature of the fluid in a noninvasive way. The purposes of this retrospective cross-sectional analytical study were to compare Hounsfield units of different pleural effusion categories in dogs and cats, assess association between specific cytologic parameters and Hounsfield units, and evaluate the effect of dependent vs. nondependent aspect of the effusion pool on Hounsfield unit. A total of 111 patients (74 dogs and 37 cats) with pleural effusion, that underwent thoracic CT and diagnostic thoracocentesis, were included in the study. Effusions were cytologically categorized as exudate, transudate, modified transudate, hemorrhage, or chyle. Significant differences existed in Hounsfield units between categories in dogs (P < 0.0001) but not in cats (P = 0.334). Canine chylous effusion (6.1 ± 4.7 HU (mean ± standard deviation)) and transudate (5.6 ± 2.0) were significantly lower than exudate (20.3 ± 9.5) and hemorrhage (21.4 ± 9.2). No significant differences were found between modified transudate (13.6 ± 10.3) and other categories. Significant, weak linear correlation was identified in dogs between Hounsfield units and total protein (P = 0.018, R2  = 0.089), red blood cells (P = 0.021, R2  = 0.077), and total nucleated cells (P = 0.013, R2  = 0.089). The Hounsfield units of dependent effusion was not significantly higher than the nondependent effusion, except for canine chylous effusion (P = 0.008). Fourteen Hounsfield units was identified as the most clinically useful threshold: <14 HU identified transudate or chylous effusion with a sensitivity of 100% and a specificity of 69%. A threshold >14 HU had a specificity of 100% and a sensitivity of 69% for identifying exudate, modified transudate, or hemorrhage.

A Comparison of Bone Marrow and Cord Blood Mesenchymal Stem Cells for Cartilage Self-Assembly.

Joint injury is a common cause of premature retirement for the human and equine athlete alike. Implantation of engineered cartilage offers the potential to increase the success rate of surgical intervention and hasten recovery times. Mesenchymal stem cells (MSCs) are a particularly attractive cell source for cartilage engineering. While bone marrow-derived MSCs (BM-MSCs) have been most extensively characterized for musculoskeletal tissue engineering, studies suggest that cord blood MSCs (CB-MSCs) may elicit a more robust chondrogenic phenotype. The objective of this study was to determine a superior equine MSC source for cartilage engineering. MSCs derived from bone marrow or cord blood were stimulated to undergo chondrogenesis through aggregate redifferentiation and used to generate cartilage through the self-assembling process. The resulting neocartilage produced from either BM-MSCs or CB-MSCs was compared by measuring mechanical, biochemical, and histological properties. We found that while BM constructs possessed higher tensile properties and collagen content, CB constructs had superior compressive properties comparable to that of native tissue and higher GAG content. Moreover, CB constructs had alkaline phosphatase activity, collagen type X, and collagen type II on par with native tissue suggesting a more hyaline cartilage-like phenotype. In conclusion, while both BM-MSCs and CB-MSCs were able to form neocartilage, CB-MSCs resulted in tissue more closely resembling native equine articular cartilage as determined by a quantitative functionality index. Therefore, CB-MSCs are deemed a superior source for the purpose of articular cartilage self-assembly.