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.

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.

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.

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.

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.