Evaluation of SNAP cPL, Spec cPL, VetScan cPL Rapid Test, and Precision PSL Assays for the Diagnosis of Clinical Pancreatitis in Dogs.

BACKGROUND: The sensitivity, specificity, and agreement of 4 diagnostic assays (SNAP canine pancreatic lipase (cPL), specific cPL (Spec cPL), VetScan cPL Rapid Test, and Precision PSL) for pancreatitis in dogs have not been directly compared. HYPOTHESIS/OBJECTIVES: To determine the level of agreement among each of the 4 assays and a clinical suspicion score, level of agreement among the assays, and sensitivity and specificity of each assay in a clinically relevant patient group. ANIMALS: Fifty client-owned dogs with clinical signs of gastrointestinal disease. METHODS: Prospective study. History, physical examination, complete blood count, serum biochemistry, abdominal ultrasound examination, and the 4 diagnostic assays for pancreatitis were performed. Intraclass correlation coefficients (ICC) were used to determine the level of agreement between each assay and a clinical suspicion score determined by a panel of 5 board-certified veterinary internists. RESULTS: The ICC between the clinical suspicion score and the 4 assays were SNAP cPL, 0.61; Spec cPL, 0.68; VetScan cPL Rapid Test, 0.68; and Precision PSL, 0.60. The sensitivities of the assays ranged from 73.9 to 100.0%, whereas the specificities were SNAP cPL, 71.1-77.8%; Spec cPL, 74.1-81.1%; VetScan cPL Rapid Test, 76.9-83.8%; and Precision PSL, 64.0-74.3%. CONCLUSIONS AND CLINICAL IMPORTANCE: A good to excellent level of agreement was demonstrated among the 4 assays. The previously unreported sensitivity and specificity of the VetScan cPL Rapid Test were 73.9-83.3% and 76.9-83.8%, respectively. Results of any of the 4 diagnostic assays alone, in the absence of supporting clinical findings, are insufficient to establish a diagnosis of clinical pancreatitis in dogs.

Platelets Inhibit Migration of Canine Osteosarcoma Cells.

The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition.

Anti-platelet therapy in small animal medicine.

Thromboembolism is a significant complication in many commonly encountered diseases, and can be a devastating sequel to otherwise treatable conditions. Platelets play an essential role in the hemostatic process and, consequently, are associated with thrombus formation. Platelets adhere to denuded vascular subendothelium, recruit additional platelets and cells, aggregate, and provide the catalytic surface for thrombin production and fibrin formation. Therapy to prevent unwanted thrombus formation and thromboembolic crises is essential in the management of hypercoagulable patients. Unfortunately, many of the medications used in veterinary medicine that inhibit or modulate coagulation factors, such as the heparins, are cost prohibitive, only effective when administered by injection or require frequent drug monitoring, and are therefore poor choices for long term at home therapy. While the role of the platelet in pathologic thrombus formation is not fully understood, veterinarians often resort to anti-platelet therapy in the management of patients at risk for thromboembolic complications, because many anti-platelet medications are inexpensive, require minimal drug monitoring, and can be given orally. The aim of this review is to discuss the anti-platelet therapies that are currently being used or being considered for use to inhibit platelet function and reduce thromboembolic complications in hypercoagulable dogs and cats.

Effects of oral cyclosporine on canine T-cell expression of IL-2 and IFN-gamma across a 12-h dosing interval.

The duration of immunosuppressive effects following oral cyclosporine in dogs is unknown. This study used flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to evaluate the effects of high-dose oral cyclosporine across a 12-h dosing interval. Expression of interleukin-2 (IL-2) and interferon-gamma (IFN-γ) was compared before and after 8 days of cyclosporine at 10 mg/kg every 12 h in six healthy dogs. Samples were collected at 0, 2, 4, and 8 h postdosing for analysis of unactivated and activated T-cell and whole blood cytokine expression using flow cytometry and qRT-PCR, respectively, and at 0, 2, 4, 6, 8, and 10 h postdosing for measurement of cyclosporine concentrations. Flow cytometry and qRT-PCR both demonstrated significant marked reductions in IL-2 and IFN-γ levels at 0, 2, 4, and 8 h after dosing compared to pretreatment levels (P < 0.05) for activated samples, with less consistent effects observed for unactivated samples. Both flow cytometry and qRT-PCR are viable techniques for measuring cyclosporine pharmacodynamics in dogs, yielding comparable results with activated samples. Two hours postdrug administration is the preferred time for concurrent assessment of peak drug concentration and cytokine expression, and T-cell activation is needed for optimal results.

Pharmacokinetic evaluation of oral dantrolene in the dog.

The pharmacokinetics of dantrolene and its active metabolite, 5-hydroxydantrolene, after a single oral dose of either 5 or 10 mg/kg of dantrolene was determined. The effects of exposure to dantrolene and 5-hydroxydantrolene on activated whole-blood gene expression of the cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) were also investigated. When dantrolene was administered at a 5 mg/kg dose, peak plasma concentration (Cmax ) was 0.43 µg/mL, terminal half-life (t1/2 ) was 1.26 h, and area under the time-concentration curve (AUC) was 3.87 µg·h/mL. For the 10 mg/kg dose, Cmax was 0.65 µg/mL, t1/2 was 1.21 h, and AUC was 5.94 µg·h/mL. For all calculated parameters, however, there were large standard deviations and wide ranges noted between and within individual dogs: t1/2 , for example, ranged from 0.43 to 6.93 h, Cmax ratios ranged from 1.05 to 3.39, and relative bioavailability (rF) values ranged from 0.02 to 1.56. While activated whole-blood expression of IL-2 and IFN-γ as measured by qRT-PCR was markedly suppressed following exposure to very high concentrations (30 and 50 µg/mL, respectively) of both dantrolene and 5-hydroxydantrolene, biologically and therapeutically relevant suppression of cytokine expression did not occur at the much lower drug concentrations achieved with oral dantrolene dosing.

Oral cyclosporine treatment in dogs: a review of the literature.

Cyclosporine is an immunomodulatory drug used to treat an increasing spectrum of diseases in dogs. Cyclosporine is a calcineurin inhibitor, ultimately exerting its inhibitory effects on T-lymphocytes by decreasing production of cytokines, such as interleukin-2. Although, in the United States, oral cyclosporine is approved in dogs only for treatment of atopic dermatitis, there are many other indications for its use. Cyclosporine is available in 2 oral formulations: the original oil-based formulation and the more commonly used ultramicronized emulsion that facilitates oral absorption. Ultramicronized cyclosporine is available as an approved animal product, and human proprietary and generic preparations are also available. Bioavailability of the different formulations in dogs is likely to vary among the preparations. Cyclosporine is associated with a large number of drug interactions that can also influence blood cyclosporine concentrations. Therapeutic drug monitoring (TDM) can be used to assist in attaining consistent plasma cyclosporine concentrations despite the effects of varying bioavailability and drug interactions. TDM can facilitate therapeutic success by guiding dose adjustments on an individualized basis, and is recommended in cases that do not respond to initial oral dosing, or during treatment of severe, life-threatening diseases for which a trial-and-error approach to dose adjustment is too risky. Pharmacodynamic assays that evaluate individual patient immune responses to cyclosporine can be used to augment information provided by TDM.

Expression of the thrombopoietin gene in tissues from healthy dogs.

Thrombopoietin (THPO) is the major cytokine that regulates megakaryopoiesis and platelet production. Several human and murine studies have demonstrated that THPO is primarily synthesized in the liver, but the kidney, spleen and bone marrow are also sites of expression. The aim of this study was to determine THPO mRNA levels in a range of canine tissues by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Samples of bone marrow (n = 5), liver (n = 10), lung (n = 10), renal cortex (n = 10), renal medulla (n = 5) and spleen (n = 10) were obtained from 10 healthy, hound-cross dogs aged 6-8 months. The highest THPO mRNA levels were found in the liver, followed by the bone marrow, spleen, lung and kidney. There was a 13-fold difference in expression between liver and kidney. The bone marrow showed high levels of THPO mRNA in the absence of disease. The liver and bone marrow are likely to be the major sites of THPO production in the dog.

Cyclooxygenase expression and platelet function in healthy dogs receiving low-dose aspirin.

BACKGROUND: Low-dose aspirin is used to prevent thromboembolic complications in dogs, but some animals are nonresponsive to the antiplatelet effects of aspirin ("aspirin resistance"). HYPOTHESIS/OBJECTIVES: That low-dose aspirin would inhibit platelet function, decrease thromboxane synthesis, and alter platelet cyclooxygenase (COX) expression. ANIMALS: Twenty-four healthy dogs. METHODS: A repeated measures study. Platelet function (PFA-100 closure time, collagen/epinephrine), platelet COX-1 and COX-2 expression, and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) were evaluated before and during aspirin administration (1 mg/kg Q24 hours PO, 10 days). Based on prolongation of closure times after aspirin administration, dogs were divided into categories according to aspirin responsiveness: responders, nonresponders, and inconsistent responders. RESULTS: Low-dose aspirin increased closure times significantly (62% by Day 10, P < .001), with an equal distribution among aspirin responsiveness categories, 8 dogs per group. Platelet COX-1 mean fluorescent intensity (MFI) increased significantly during treatment, 13% on Day 3 (range, -29.7-136.1%) (P = .047) and 72% on Day 10 (range, -0.37-210%) (P < .001). Platelet COX-2 MFI increased significantly by 34% (range, -29.2-270%) on Day 3 (P = .003) and 74% (range, -19.7-226%) on Day 10 (P < .001). Urinary 11-dTXB(2) concentrations significantly (P = .005, P < .001) decreased at both time points. There was no difference between aspirin responsiveness and either platelet COX expression or thromboxane production. CONCLUSIONS AND CLINICAL IMPORTANCE: Low-dose aspirin consistently inhibits platelet function in approximately one-third of healthy dogs, despite decreased thromboxane synthesis and increased platelet COX expression in most dogs. COX isoform expression before treatment did not predict aspirin resistance.

The effects of cyclosporine on platelet function and cyclooxygenase expression in normal dogs.

BACKGROUND: Cyclosporine has been shown to alter platelet plasma membranes and have a hypercoagulable effect in humans, leading to thromboembolic complications. HYPOTHESIS/OBJECTIVES: Our hypothesis was that by modulating platelet reactivity, cyclosporine increases the risk of thromboembolic complications. The objective was to determine the effects of cyclosporine on primary hemostasis in normal dogs. ANIMALS: Eight healthy, intact female dogs. METHODS: A repeated-measures design utilized flow cytometry to evaluate platelet expression of platelet reactivity markers (P-selectin and phosphatidylserine) and COX-1 and COX-2 during the administration of 2 cyclosporine dosages (19 mg/kg q12h [immunosuppressive dosage] and 5 mg/kg q24h [atopy dosage]). Urine 11-dehydro-thromboxane-B(2) (11-dTXB(2) ) concentration was normalized to urine creatinine concentration, and platelet function was analyzed by PFA-100. RESULTS: After a week of the immunosuppressive dosage, all platelet reactivity markers showed a significant decrease in mean fluorescent intensity (MFI). After the atopy dosage, only P-selectin and COX-2 MFI demonstrated a change from baseline, decreasing by 29% (P = .013) and 31% (P = .003), respectively. Urinary 11-dTXB(2) -to-creatinine ratio significantly increased at all time points during the immunosuppressive dosage, but no significant change occurred during administration of the atopy dosage. PFA-100 closure times using collagen/ADP cartridges increased by 62% (P = .008) with the immunosuppressive dosage and decreased by 45% with the atopy dosage (P = .035). No significant changes in closure times occurred with collagen/epinephrine cartridges. CONCLUSIONS AND CLINICAL IMPORTANCE: Our study suggests that, similar to what is observed in humans, cyclosporine alters the platelet plasma membrane and increases thromboxane production in dogs, especially at immunosuppressive dosages.

Evidence of selective packaging and different α-granule subtypes in canine platelets.

Platelets are circulating megakaryocytic cytoplasmic fragments that are required to maintain vascular integrity and prevent haemorrhage. During activation, platelets release hundreds of bioactive proteins, mainly by secretion of α-granule content. These proteins include von Willebrand factor (vWF) and fibrinogen, major adhesive proteins involved in haemostasis. Human and mouse platelet α-granules are packaged selectively to contain different molecules and platelets can differentially release these proteins on specific receptor activation. Confocal laser scanning microscopy was used to analyse vWF and fibrinogen localization within canine platelet α-granules. In resting canine platelets, the majority of platelet vWF and fibrinogen is located in separate α-granule populations. These findings provide evidence that canine platelets contain different α-granule subtypes, suggesting that selective protein packaging occurs during canine platelet ontology.

Pharmacodynamic monitoring of canine T-cell cytokine responses to oral cyclosporine.

BACKGROUND: Pharmacodynamic assays measure the immunosuppressive effects of cyclosporine on T-cells and offer an alternative assessment of efficacy in individual patients. OBJECTIVE: To assess the immunosuppressive effects of high and low dosage cyclosporine on canine T-cells and to develop a novel testing system for individualized dose adjustment. ANIMALS: Seven healthy female Walker hounds. METHODS: Experimental study using a paired comparison design. Flow cytometry was used to measure T-cell expression of IL-2, IL-4, and IFN-γ. Cytokine expression 8 days after oral administration of high and low dosages of cyclosporine was compared to baseline and washout values, respectively. The high dosage was initially 10 mg/kg q12h and was then adjusted to attain established immunosuppressive trough blood drug concentrations (>600 ng/mL). The low dosage was 5 mg/kg q24h. RESULTS: High dosage cyclosporine resulted in significant decreases in IL-2 and IFN-γ expression (P = .0156, P = .0156), but not IL-4 expression (P = .2188). Low dosage cyclosporine was associated with a significant decrease in IFN-γ expression (P = .0156), while IL-2 expression was not affected (P = .1094). CONCLUSIONS AND CLINICAL IMPORTANCE: T-cell function is suppressed at trough blood drug concentrations exceeding 600 ng/mL, and is at least partially suppressed in some dogs at low dosages. Direct evaluation of T-cell function could be an effective, more sensitive alternative to measuring blood drug concentrations for monitoring immunosuppressive therapy.

Platelet cyclooxygenase expression in normal dogs.

BACKGROUND: Human platelets express both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Variation in COX-2 expression could be a mechanism for variable response to aspirin. HYPOTHESIS/OBJECTIVES: The hypotheses were that circulating canine platelets express COX-1 and COX-2, and that aspirin alters COX expression. The objective was to identify changes in platelet COX expression and in platelet function caused by aspirin administration to dogs. ANIMALS: Eight female, intact hounds. METHODS: A single population, repeated measures design was used to evaluate platelet COX-1 and COX-2 expression by flow cytometry before and after aspirin (10 mg/kg Q12h for 10 days). Platelet function was analyzed via PFA-100(®) (collagen/epinephrine), and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) was measured and normalized to urinary creatinine. Differences in COX expression, PFA-100(®) closure times, and urine 11-dTXB(2 ): creatinine ratio were analyzed before and after aspirin administration. RESULTS: Both COX-1 and COX-2 were expressed in canine platelets. COX-1 mean fluorescent intensity (MFI) increased in all dogs, by 250% (range 63-476%), while COX-2 expression did not change significantly (P = 0.124) after aspirin exposure, with large interindividual variation. PFA-100(®) closure times were prolonged and urine 11-dTXB(2) concentration decreased in all dogs after aspirin administration. CONCLUSIONS AND CLINICAL IMPORTANCE: Canine platelets express both COX isoforms. After aspirin exposure, COX-1 expression increased despite impairment of platelet function, while COX-2 expression varied markedly among dogs. Variability in platelet COX-2 expression should be explored as a potential mechanism for, or marker of, variable aspirin responsiveness.

Cyclosporine A affects the in vitro expression of T cell activation-related molecules and cytokines in dogs.

Cyclosporine is a powerful immunosuppressive drug that is being used with increasing frequency to treat a wide range of immune-mediated diseases in the dog. To date, ideal dosing protocols that will achieve immunosuppression with cyclosporine in dogs remain unclear, and standard methods that can measure effectiveness of immunosuppression have not been established. The aim of our study was to evaluate the effects of in vitro cyclosporine exposure on a panel of molecules expressed by activated T cells to ascertain their potential as biomarkers of immunosuppression in dogs. Blood was drawn from six healthy dogs, and peripheral blood mononuclear cells (PBMC) were isolated and activated. Half of the cells were incubated with 200 ng/mL cyclosporine prior to activation, and the other half were not exposed to cyclosporine. Samples were analyzed using flow cytometry, and the expression of intracellular cytokines IL-2, IL-4, and IFN-γ was evaluated after 6, 12, and 24h of drug exposure. Each cytokine exhibited a time-dependent suppression profile, and all but two samples activated in the presence of cyclosporine showed lower cytokine expression than untreated controls. We also evaluated the expression of the surface T cell activation molecules CD25 and CD95 by flow cytometry after 36 h of drug exposure. Expression of these surface molecules decreased significantly when activated in the presence of cyclosporine. Our results suggest that suppressed expression of the markers related to T cell activation could potentially be utilized as an indicator of the efficacy of cyclosporine therapy in dogs.

Alloreactive (CD4-Independent) CD8+ T cells jeopardize long-term survival of intrahepatic islet allografts.

Despite success of early islet allograft engraftment and survival in humans, late islet allograft loss has emerged as an important clinical problem. CD8+ T cells that are independent of CD4+ T cell help can damage allograft tissues and are resistant to conventional immunosuppressive therapies. Previous work demonstrates that islet allografts do not primarily initiate rejection by the (CD4-independent) CD8-dependent pathway. This study was performed to determine if activation of alloreactive CD4-independent, CD8+ T cells, by exogenous stimuli, can precipitate late loss of islet allografts. Recipients were induced to accept intrahepatic islet allografts (islet 'acceptors') by short-term immunotherapy with donor-specific transfusion (DST) and anti-CD154 mAb. Following the establishment of stable long-term islet allograft function for 60-90 days, recipients were challenged with donor-matched hepatocellular allografts, which are known to activate (CD4-independent) CD8+ T cells. Allogeneic islets engrafted long-term were vulnerable to damage when challenged locally with donor-matched hepatocytes. Islet allograft loss was due to allospecific immune damage, which was CD8- but not CD4-dependent. Selection of specific immunotherapy to suppress both CD4- and CD8-dependent immune pathways at the time of transplant protects islet allografts from both early and late immune damage.

Activation and maturation of alloreactive CD4-independent, CD8 cytolytic T cells.

The goal of this study was to determine the in vivo conditions that promote activation of the (CD4-independent) CD8+ T cell-mediated rejection pathway. We have previously noted that hepatocellular but not islet allografts readily activate this rejection pathway. In the current study, we utilized these two cell transplant models to investigate whether differences in host cell recruitment to the graft site, expression of T-cell activation markers by CD8+ graft infiltrating cells (GICs), and/or development of delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte cell-mediated effector functions could account for the differential transplant outcomes. The collective results demonstrate that recruitment of CD8+ T cells to the site of transplant, CD103 or CD69 expression on CD8+ GICs, and activation of alloreactive DTH responses are insufficient to initiate CD4-independent, CD8-dependent transplant rejection. Instead, rejection by alloreactive (CD4-independent) CD8+ T cells correlated with expression of CD25, CD154 and CD43 by CD8+ GICs, in vitro alloproliferation by recipient CD8+ T cells, and the development of in vivo allospecific cytolytic effector function. These results suggest that tissue-derived factors influence the activation and maturation of (CD4-independent) CD8+ T cells into cytolytic effectors, which correlates with transplant rejection.

Laparoscopic Nissen fundoplication in childhood.

An obese, 10-year-old boy presented with symptoms of pain and apnea due to gastroesophageal reflux that was unresponsive to medication. A laparoscopic Nissen fundoplication was performed, and the child was discharged from the hospital within 36 hours of the operation. The rationale and technique of a laparoscopic Nissen fundoplication for symptomatic gastroesophageal reflux in childhood is described.