Comparison of a microsphere-based platform with a multiplex flow cytometric assay for determination of circulating cytokines in the mouse.

BACKGROUND: Measuring expression profiles of inflammatory biomarkers is important in monitoring the polarization of immune responses; therefore, results should be independent of quantitation methods if they are to be accepted as validated clinical pathology biomarkers. To evaluate effects of differing quantitation methods, the seven major circulating Th1/Th2/Th17 cytokines interleukin 2 (IL-2), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-4, IL-6, IL-10 and IL-17A were quantified in plasma of lipopolysaccharide (LPS)-treated mice with two different multiplex platforms. METHODS: Female C57BL6 mice were treated orally with vehicle or dexamethasone, followed by LPS intravenously. Plasma samples were analyzed 0.5, 1, 2, 4, and 6 h post-LPS challenge with assays at Myriad-RBM and compared to assays performed on a BD Accuri C6 flow cytometer. RESULTS: IL-17A response to LPS was limited but sustained, and the response for the remaining cytokines were early and transient; dexamethasone reduced expression of all 7 cytokines. TNF-α and IL-6 levels were similar across both assays, and IL-4 levels were generally very low. Plasma levels of remaining cytokines were variably lower with BD assays than Myriad-RBM assays. CONCLUSIONS: The present findings demonstrate that quantitation of circulating biomarkers of inflammation can be achieved using multiplexed flow cytometry, but careful consideration must be taken for assay validation when cross-referencing with another multiplexed assay.

Development of a Functional Observational Battery in the Minipig for Regulatory Neurotoxicity Assessments.

A functional observational battery (FOB) is recommended as the first-tier neurotoxicity screening in the preclinical safety pharmacology testing guidelines. Minipigs have increasingly been used in regulatory toxicology studies; however, no current FOB protocol is available for neurotoxicity testing in these species. Hence, a minipig FOB instrument was developed. A complete crossover study with Sinclair minipigs was performed to evaluate physiologic, neurologic, and behavioral effects of amphetamine, ketamine, and diazepam. The treated minipigs were first observed in their home cage, were video-recorded for 10 minutes in an open field, and then went through a complete neurologic examination. Both ketamine and diazepam were shown to reduce the freezing and behavior shifts of treated minipigs, while increasing their exploratory behaviors. Both drugs also caused muscular and gait impairment. The effects of ketamine and diazepam were consistent with their roles as central nervous system (CNS) suppressants. Unique effects were also observed with ketamine and diazepam treatments, which may reflect their unique mechanisms of action. Consistent with its role as a CNS stimulant, amphetamine caused the treated minipigs to be hyperactive and to display increased freezing and behavior shifts and reduced exploring activities. These effects of amphetamine were opposite to those observed with ketamine and diazepam. Amphetamine also increased locomotion in the treated minipigs. The present effects of amphetamine, ketamine, and diazepam are in agreement with observations by others. In conclusion, the minipig is a suitable species for FOB evaluation of pharmaceuticals in preclinical safety pharmacology testing.

The importance of minipigs in dermal safety assessment: an overview.

The use of miniature swine as a non-rodent species in safety assessment has continued to expand for over a decade and their use has become routine, particularly in pharmacology as a model for human integumentary diseases. Translational preclinical swine study data are now favorably compared and contrasted to human data, and miniature swine models provide important information in dermal safety assessment and skin pharmacology. For example, the miniature swine model has been well-accepted for cutaneous absorption and toxicity studies due to swine integument being morphologically and functionally similar to human skin. Subsequently, this model is important to dermal drug development programs, and it is the animal model of choice for assessment of dermal absorption, local tolerance and systemic toxicity following dermal exposures. In conclusion, the miniature swine model has an important role to play in the safety assessment of pharmaceutical products and in multiple aspects of human dermal drug development.

The Miniature Swine as a Model in Experimental and Translational Medicine.

The use of the miniature swine as a nonrodent species in research has continued to expand for over a decade, and they are becoming routinely used both in experimental pharmacology and as a therapeutic model for human diseases. Miniature swine models are regularly used for studies designed to assess efficacy and safety of new therapeutic compounds given through different routes of exposure and are used as an alternative model to rodents, canines, or nonhuman primates. Translational preclinical swine study data presented here support the current understanding that miniature swine are the animal model of choice for the assessment of drugs targeting endocrine, dermal, and ocular disorders. Because research investigators need to be familiar with some of the important features of the models developed in the miniature swine in order to place clinical and experimental findings in their proper perspective, relevant references and data from these models will be presented, compared, and partially illustrated.

Miniature Swine Breeds in Toxicology and Drug Safety Assessments: What to Expect during Clinical and Pathology Evaluations.

The use of miniature swine as a nonrodent species in safety assessment has continued to expand for over a decade, and they are becoming routinely used in toxicology and in pharmacology as well as a model for human diseases. Miniature swine models are regularly used for regulatory toxicity studies designed to assess safety of new therapeutic compounds given through different routes of exposure and are used as an alternative model to the canine or the nonhuman primate. Translational preclinical swine study data presented support the current finding that miniature swine are the animal model of choice for assessment of drug absorption, tolerance, and systemic toxicity following systemic exposures. Because research investigators need to be familiar with important anatomic and histopathologic features of the miniature swine in order to place toxicopathologic findings in their proper perspective, clinical and anatomic pathology data from a large number of Sinclair, Hanford, Yucatan, and Göttingen breeds from control groups from a wide variety of studies performed between 2004 and 2014 will be presented, compared, and partially illustrated.

Miniature swine model of phototoxicity testing.

BACKGROUND/PURPOSE: This study determined the threshold doses for 'solar erythema' and for phototoxic responses to 8-methoxypsoralen (8-MOP) in white skin Hanford and grey skin Yucatan miniature swine. METHODS: For threshold erythema determinations, the UVR exposures included both UVA (315-400 nm) and UVB (290-315 nm) radiation by positioning one fluorescent 'sunlamp' among 10 'PUVA' lamps. With this configuration the UVR exposures ranged from 0.5-2.8 times the 'instrumental MED' (MEDi) for Hanford and from 1.0-5.6 times the MEDi for Yucatan. For phototoxicity determinations (i.e., with and without topically-applied graduated concentrations of 8-MOP), the UVB component was minimized by extinguishing the sunlamp, thus permitting higher UVA exposures. RESULTS: The Hanford had the lower UV erythema dose threshold (1.0-1.4 times the MEDi) and the erythema that developed was readily observable. The exposure doses for the phototoxicity test were 5 J/cm(2) of UVA in 35 minutes or 10 J/cm(2) in 70 minutes. The phototoxic (vascular) response to 8-MOP was observed in the two highest concentrations (0.01% and 0.1%) in Hanford pigs, in a dose-related manner. Microscopic evidence of a dose-related response was also observed as the concentration of 8-MOP increased. CONCLUSION: This verified that the Hanford miniature swine is the preferable strain for phototoxic effects. In contrast, UVR exposure of the Yucatan pig skin produced tanning rather than erythema, confirming that the Yucatan is the more appropriate strain for studying the melanization response. Thus, Hanford and Yucatan miniature swine have cutaneous photobiological responses that reflect their respective strain differences.