ACVIM consensus statement on the diagnosis of immune thrombocytopenia in dogs and cats.

Immune thrombocytopenia (ITP) is the most common acquired primary hemostatic disorder in dogs. Immune thrombocytopenia less commonly affects cats but is an important cause of mortality and treatment-associated morbidity in both species. Immune thrombocytopenia remains a diagnosis of exclusion for which diagnostic guidelines are lacking. Primary, or non-associative, ITP refers to autoimmune platelet destruction. Secondary, or associative, ITP arises in response to an underlying disease trigger. However, evidence for which comorbidities serve as ITP triggers has not been systematically evaluated. To identify key diagnostic steps for ITP and important comorbidities associated with secondary ITP, we developed 12 Population Evaluation/Exposure Comparison Outcome (PECO) format questions. These questions were addressed by evidence evaluators utilizing a literature pool of 287 articles identified by the panelists using a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations that then were integrated by diagnosis and comorbidity domain chairs. The revised PECO responses underwent a Delphi survey process to reach consensus on final guidelines. A combination of panel expertise and PECO responses were employed to develop algorithms for diagnosis of ITP in dogs and cats, which also underwent 4 iterations of Delphi review. Comorbidity evidence evaluators employed an integrated measure of evidence (IME) tool to determine evidence quality for each comorbidity; IME values combined with evidence summaries for each comorbidity were integrated to develop ITP screening recommendations, which also were subjected to Delphi review. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The final consensus statement provides clinical guidelines for the diagnosis of, and underlying disease screening for, ITP in dogs and cats. The systematic consensus process identified numerous knowledge gaps that should guide future studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Treatment of Immune Thrombocytopenia.

The Characteristics and Function of Small Extracellular Vesicles Derived from Human Bone Marrow and Umbilical Cord Mesenchymal Stromal Cells Are Influenced by Cell Culture Conditions.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC-EVs) have been proposed as a novel therapeutic tool with numerous clinically related advantages. However, their characteristics and functionality are dependent on the source of MSCs and their cell culture conditions. Fetal bovine serum (FBS) provides a source of nutrients and growth factors to the cultured cells. However, certain pitfalls are associated with its supplementation to the culture media, including introduction of exogenous FBS-derived EVs to the cultured cells. Thus, recent practices recommend utilization of serum-free (SF) media or EV-depleted FBS. On the contrary, evidence suggests that the immunomodulatory ability of MSC-EVs can be improved by exposing MSCs to an inflammatory (IF) environment. The objective of this study was to (1) compare EVs isolated from two tissue sources of MSCs that were exposed to various cell culture conditions and (2) to evaluate their anti-inflammatory effects. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) and umbilical cord-derived mesenchymal stromal cells (UC-MSCs) were exposed to either a SF media environment, an IF environment, or media supplemented with 5% EV-depleted FBS. Following isolation of MSC-EVs, the isolates were quantified and evaluated for particle size, phenotypic changes, and their immunomodulatory potential. A statistically significant difference was not identified on the yield and protein concentration of different isolates of EVs from BM-MSCs and UC-MSCs, and all isolates had a circular appearance as evaluated via electron microscopy. A significant difference was identified on the phenotype of different EVs isolates; however, all isolates expressed classical markers such as CD9, CD63, and CD81. The addition of BM-derived MSC-EVs from FBS environment or UC-derived MSC-EVs from IF environment resulted in statistically significant downregulation of IL-6 messenger RNA (mRNA) in stimulated leukocytes. This study confirms that EVs produced by different MSC sources and cell culture conditions affect their phenotype and their immunomodulatory capacities.

Heterologous prime-boost H1N1 vaccination exacerbates disease following challenge with a mismatched H1N2 influenza virus in the swine model.

Influenza A viruses (IAVs) pose a significant threat to both human and animal health. Developing IAV vaccine strategies able to elicit broad heterologous protection against antigenically diverse IAV strains is pivotal in effectively controlling the disease. The goal of this study was to examine the immunogenicity and protective efficacy of diverse H1N1 influenza vaccine strategies including monovalent, bivalent, and heterologous prime-boost vaccination regimens, against a mismatched H1N2 swine influenza virus. Five groups were homologous prime-boost vaccinated with either an oil-adjuvanted whole-inactivated virus (WIV) monovalent A/swine/Georgia/27480/2019 (GA19) H1N2 vaccine, a WIV monovalent A/sw/Minnesota/A02636116/2021 (MN21) H1N1 vaccine, a WIV monovalent A/California/07/2009 (CA09) H1N1, a WIV bivalent vaccine composed of CA09 and MN21, or adjuvant only (mock-vaccinated group). A sixth group was prime-vaccinated with CA09 WIV and boosted with MN21 WIV (heterologous prime-boost group). Four weeks post-boost pigs were intranasally and intratracheally challenged with A/swine/Georgia/27480/2019, an H1N2 swine IAV field isolate. Vaccine-induced protection was evaluated based on five critical parameters: (i) hemagglutination inhibiting (HAI) antibody responses, (ii) clinical scores, (iii) virus titers in nasal swabs and respiratory tissue homogenates, (iv) BALf cytology, and (v) pulmonary pathology. While all vaccination regimens induced seroprotective titers against homologous viruses, heterologous prime-boost vaccination failed to enhance HAI responses against the homologous vaccine strains compared to monovalent vaccine regimens and did not expand the scope of cross-reactive antibody responses against antigenically distinct swine and human IAVs. Mismatched vaccination regimens not only failed to confer clinical and virological protection post-challenge but also exacerbated disease and pathology. In particular, heterologous-boosted pigs showed prolonged clinical disease and increased pulmonary pathology compared to mock-vaccinated pigs. Our results demonstrated that H1-specific heterologous prime-boost vaccination, rather than enhancing cross-protection, worsened the clinical outcome and pathology after challenge with the antigenically distant A/swine/Georgia/27480/2019 strain.

Rabies: who should care?

Rabies is the deadliest viral infection known, with no reliable treatment, and although it is entirely preventable, rabies continues to kill more than 60,000 people every year, mostly children in countries where dog rabies is endemic. America is only 1 generation away from the time when rabies killed more than 10,000 animals and 50 Americans every year, but 3 to 5 Americans continue to die annually from rabies. Distressingly, > 50,000 Americans undergo rabies prevention therapy every year after exposure to potentially rabid animals. While enormous progress has been made, more must be done to defeat this ancient but persistent, fatal zoonosis. In the US, lack of public awareness and ambivalence are the greatest dangers imposed by rabies, resulting in unnecessary exposures, anxiety, and risk. Veterinarians have a special role in informing and reassuring the public about prevention and protection from rabies. This summary of current facts and future advances about rabies will assist veterinarians in informing their clients about the disease.

A Novel Neuraminidase Virus-Like Particle Vaccine Offers Protection Against Heterologous H3N2 Influenza Virus Infection in the Porcine Model.

Influenza A viruses (IAVs) pose a global health threat, contributing to hundreds of thousands of deaths and millions of hospitalizations annually. The two major surface glycoproteins of IAVs, hemagglutinin (HA) and neuraminidase (NA), are important antigens in eliciting neutralizing antibodies and protection against disease. However, NA is generally ignored in the formulation and development of influenza vaccines. In this study, we evaluate the immunogenicity and efficacy against challenge of a novel NA virus-like particles (VLPs) vaccine in the porcine model. We developed an NA2 VLP vaccine containing the NA protein from A/Perth/16/2009 (H3N2) and the matrix 1 (M1) protein from A/MI/73/2015, formulated with a water-in-oil-in-water adjuvant. Responses to NA2 VLPs were compared to a commercial adjuvanted quadrivalent whole inactivated virus (QWIV) swine IAV vaccine. Animals were prime boost vaccinated 21 days apart and challenged four weeks later with an H3N2 swine IAV field isolate, A/swine/NC/KH1552516/2016. Pigs vaccinated with the commercial QWIV vaccine demonstrated high hemagglutination inhibition (HAI) titers but very weak anti-NA antibody titers and subsequently undetectable NA inhibition (NAI) titers. Conversely, NA2 VLP vaccinated pigs demonstrated undetectable HAI titers but high anti-NA antibody titers and NAI titers. Post-challenge, NA2 VLPs and the commercial QWIV vaccine showed similar reductions in virus replication, pulmonary neutrophilic infiltration, and lung inflammation compared to unvaccinated controls. These data suggest that anti-NA immunity following NA2 VLP vaccination offers comparable protection to QWIV swine IAV vaccines inducing primarily anti-HA responses.

Equine Platelet Lysate Gel: A Matrix for Mesenchymal Stem Cell Delivery.

A variety of bioscaffolds have been developed as carriers for the delivery of mesenchymal stem cells (MSCs), however, many of them are unable to provide direct cell nourishment, a critical factor for survival and retention of MSCs at the site of delivery. Platelet lysate is a plasma-derived product rich in growth factors that can be turned into a gel matrix following the addition of calcium chloride. Our objective was to characterize growth factor and cytokine release of equine platelet lysate gel (ePL gel) encapsulated with MSCs over time and to measure the viability and proliferation of ePL gel-encapsulated MSCs for up to 14 days. The release of interleukin-1ß (IL-1ß), interleukin-10 (IL-10), transforming growth factor beta (TGF-ß), vascular endothelial growth factor (VEGF), and platelet-derived growth factor BB (PDGF-BB), as well as fibrinogen degradation, were measured from ePL gel with and without equine bone marrow-derived MSCs and compared with MSCs in monolayer. MSC proliferation and viability within the gel were assessed up to 14 days. Compared with monolayer MSC cultures, significantly higher concentrations of IL-1ß, IL-10, and TGF-ß were measured from supernatants collected from ePL gel containing MSCs at various time points. Significantly lower concentrations of PDGF-BB were measured in the supernatant when MSCs were incorporated in ePL gel while VEGF tended to be increased compared with MSCs in monolayer. Incorporation in ePL gel for up to 14 days did not appear to affect viability and proliferation rates of MSCs as these were found to be similar to those measured in monolayer cell culture. ePL gel may have the potential to serve as bioscaffold for MSC delivery since it appears to support the proliferation and viability of MSCs for up to 14 days.

Investigation of platelet measurands in dogs with hematologic neoplasia.

INTRODUCTION: Thrombocytopenia has been associated with some neoplastic processes, including hematologic neoplasia. There is no information regarding specific changes in platelet measurands in dogs with hematologic neoplasia compared with healthy dogs. The objectives of our study were to establish RIs, evaluate platelet measurands in dogs with hematologic neoplasia, and compare these measurands in patients with hematologic malignancies with or without thrombocytopenia. METHODS: This was a retrospective study. Platelet measurands were determined using the ADVIA 120 Hematology analyzer when a CBC was performed and included the platelet count, MPV, platelet distribution width (PDW), plateletcrit (PCT), mean platelet component (MPC), platelet component distribution width (PCDW), mean platelet mass (MPM), platelet mass distribution width (PMDW), and number of large platelets. Reference intervals were determined retrospectively using data from 129 healthy dogs. Patients with hematologic neoplasia (n = 50) were identified through retrospective evaluation of medical records from the Auburn University Veterinary Teaching Hospital and separated into thrombocytopenic (n = 20) and nonthrombocytopenic groups (n = 30). RESULTS: Platelet count and PCT were significantly higher in older healthy dogs compared with younger dogs. Significant differences were identified when comparing healthy dogs with those with hematologic neoplasia without thrombocytopenia for PDW, PCDW, PMDW, and the number of large platelets, indicating the presence of more heterogeneous platelets. Thrombocytopenic dogs with hematologic neoplasia had significantly decreased MPCs and increased MPVs, MPMs, and PCDWs compared with nonthrombocytopenic dogs with neoplasia. CONCLUSIONS: Dogs with hematologic neoplasia had more heterogeneous platelets, whereas thrombocytopenic patients with neoplasia had more activated platelets.

Cancer of unknown primary in a mare: case report and comparative pathology review.

A 25-y-old Percheron mare was admitted to the teaching hospital because of lethargy and intractable dyspnea. Thoracoabdominal ultrasound examination identified severe peritoneal effusion, mild bilateral pleural effusion, and a diffuse pulmonary nodular pattern. Cytology of peritoneal fluid revealed a hypercellular sample with clusters of neoplastic polygonal cells and admixed macrophages. Euthanasia was followed by postmortem examination; marked bi-cavitary effusion was present, and innumerable up to 4-cm diameter, round-to-floriform nodules were diffusely evident throughout serosal surfaces as well as the pulmonary and hepatic parenchyma. Disseminated adenocarcinoma, predominantly affecting lung and liver with widespread serosal implantation, was confirmed on light microscopy. Neoplastic cells had strong immunolabeling for pancytokeratin and lacked immunoreactivity to vimentin, napsin A, and Pax8. Cytokeratin 7 and thyroid transcription factor-1 were non-contributory given absent and inconsistent internal control reactivity, respectively. Such results, combined with the lack of a major mass that would indicate a primary site, were supportive of carcinoma of unknown primary site, which remains a conundrum in human oncology, and is poorly explored in veterinary medicine, mainly as a result of clinical and diagnostic limitations.

Fibrinogen-Depleted Equine Platelet Lysate Affects the Characteristics and Functionality of Mesenchymal Stem Cells.

Fetal bovine serum (FBS) is widely used to culture mesenchymal stem cells (MSCs) in the laboratory; however, FBS has been linked to adverse immune-mediated reactions prompting the search for alternative cell culture medium. Platelet lysate (PL) as an FBS substitute has been shown to promote MSCs growth without compromising their functionality. Fibrinogen contained in PL has been shown to negatively impact the immune modulating properties of MSCs; therefore, we sought to deplete fibrinogen from PL and compare proliferation, viability, and immunomodulatory capacities of MSCs in FBS or PL without fibrinogen. We depleted fibrinogen from equine platelet lysate (ePL) and measured platelet-derived growth factor-beta (PDGF-ß), transforming growth factor-beta (TGF-ß) and tumor necrosis factor-alpha (TNF-α) through ELISA. First, we determined the ability of 10% ePL or fibrinogen-depleted lysate (fdePL) compared with 10% FBS to suppress monocyte activation by measuring TNF-α from culture supernatants. We then evaluated proliferation, viability, and immunomodulatory characteristics of bone marrow-derived MSCs (BM-MSCs) cultured in FBS or ePL with or without fibrinogen. Growth factor concentrations decreased in ePL after fibrinogen depletion. Lipopolysaccharide (LPS)-stimulated monocytes exposed to ePL and fdePL produced less TNF-α than LPS-stimulated monocytes in 10% FBS. BM-MSCs cultured in fdePL exhibited lower proliferation rates, but similar viability compared with BM-MSCs in ePL. BM-MSCs in fdePL did not effectively suppress TNF-α expression from LPS-stimulated monocytes compared with BM-MSCs in FBS. Depleting fibrinogen results in a lysate that suppresses TNF-α expression from LPS-stimulated monocytes, but that does not support proliferation and immune-modulatory capacity of BM-MSCs as effectively as nondepleted lysate.

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. METHODS: Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. RESULTS: Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS. CONCLUSION: ePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS.

Innate immune responses of equine monocytes cultured in equine platelet lysate.

Platelet lysate (PL) has been extensively used for the laboratory expansion of human mesenchymal stem cells (MSC) in order to avoid fetal bovine serum (FBS) which has been associated with immune-mediated host reactions and transmission of bovine-origin microbial contaminants. Before suggesting the routine use of PL for MSC culture, we wanted to further investigate whether PL alone might trigger inflammatory responses when exposed to reactive white blood cells such as monocytes. Our objectives were to evaluate the inflammatory profile of equine monocytes cultured with equine PL (ePL) and to determine if ePL can modulate the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated monocytes. In a first experiment, equine monocytes were isolated and incubated with donor horse serum (DHS), FBS, six individual donors ePL or pooled ePL from all horses. In a second experiment, monocytes were stimulated with E. coli LPS in the presence of 1, 5 or 10% DHS and/or pooled ePL. After 6h of incubation, cell culture supernatants were assayed via ELISA for production of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin 1ß (IL-1ß) as well as for the anti-inflammatory Interleukin 10 (IL-10). Equine monocytes incubated with pooled ePL produced significantly less TNF-α and significantly more IL-10 than monocytes incubated in FBS. A statistically significant difference was not identified for the production of IL-1ß. The second experiment showed that pooled ePL added to LPS-stimulated equine monocytes resulted in a significant reduction in TNF-α and IL-1ß production. IL-10 production was not significantly upregulated by the addition of ePL to LPS-stimulated monocytes. Finally, the addition of ePL to LPS-stimulated monocytes in the presence of various concentrations of DHS resulted to statistically significant decrease of TNF-α and IL-1ß compared to the control groups. This is the first study to demonstrate that ePL suppresses the release of pro-inflammatory cytokines from stimulated equine monocytes. These results encourage further exploration of PL as a homologous media substitute for FBS but also opens the possibility of investigating its use as means to suppress cell-mediated inflammation.

Platelet lysate obtained via plateletpheresis performed in standing and awake equine donors.

BACKGROUND: Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. STUDY DESIGN AND METHODS: Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and coagulation panels and then again at 8, 16, 24, and 48 hours after the procedure. To produce platelet lysate, the platelet concentrate underwent two freeze-thaw cycles followed by centrifugation and filtration processing. The platelet lysate was then analyzed for cellular debris, fibrinogen, and growth factors. RESULTS: The collected platelet concentration contained a mean platelet yield of 390 × 103 /µL. Donor platelet count decreased from a mean of 193 × 103 /µL to 138 × 103 /µL after plateletpheresis, but no individual was at risk for hemorrhage. Pooled platelet lysate had minimal cellular residue and contained growth factor concentrations at 6.1 ng/mL for transforming growth factor-ß1, at 3.5 ng/mL for platelet-derived growth factor-BB, and at 13.8 ng/mL for vascular endothelial growth factor-A. CONCLUSION: Plateletpheresis using commercially available apheresis equipment is a feasible option for collecting platelet concentrate from equine donors. The lysate generated from the apheresis product contains growth factors and has potential to be used as a fetal bovine serum substitute for cell culture.