Methods for specifying the target difference in a randomised controlled trial : the Difference ELicitation in TriAls (DELTA) systematic review

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Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

<div><p>Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (<i>Tursiops truncatus</i>) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 μg/mL) and concanavalin A (1 μg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using <i>Tursiops truncatus</i>-specific primers (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.</p></div

Development and testing of species-specific ELISA assays to measure IFN-γ and TNF-α in bottlenose dolphins (<i>Tursiops truncatus</i>)

<div><p>Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Though a number of immunological assays are readily available for humans and mice, specific assays for many veterinary species, including cetaceans such as bottlenose dolphins (<i>Tursiops truncatus)</i>, are more limited. Herein, we describe the development of IFN-gamma (IFN-γ) and TNF-alpha (TNF-α) enzyme-linked immunosorbent assays (ELISAs) specific to bottlenose dolphins. Utilizing these assays, we monitored the immune status of bottlenose dolphins from a managed population over a period of eleven months. The ELISA assays developed for bottlenose dolphins were used to measure IFN-γ and TNF-α in serum or in culture supernatants from peripheral blood mononuclear cells (PBMCs) stimulated with varying concentrations of mitogens concanavalin A (ConA) or phytohemagglutinin (PHA). Induction of TNF-α in PBMC cultures was consistently highest with 1 μg/mL ConA, while 1 μg/mL PHA induced the highest secretion of IFN-γ. Serum levels of TNF-α and IFN-γ remained relatively constant for each animal over the time period examined. CBC and plasma chemistry variables measured concurrently in the bottlenose dolphins were then examined as independent predictors of cytokine levels. We found these clinical variables were more likely to predict linear changes in serum IFN-γ and TNF-α levels compared to concentrations of these cytokines in mitogen-stimulated PBMC culture supernatants. Cytokine assays developed will be of substantial benefit in monitoring bottlenose dolphin health as an adjunct to currently available diagnostic tests.</p></div

Bottlenose dolphin (<i>Tursiops truncatus</i>) clinical information.

<p>Bottlenose dolphin (<i>Tursiops truncatus</i>) clinical information.</p

Time course of IFN-γ (ng/mL) levels in supernatants from ConA or PHA stimulated PBMCs isolated from bottlenose dophins (<i>Tursiops truncatus</i>).

<p>PBMCs isolated from <i>Tursiops truncatus</i> were stimulated with 1, 5, or 10 μg/mL PHA or 1 μg/mL ConA for 48 hours in culture. Supernatants were collected and run on an ELISA assay specific for bottlenose dolphin IFN-γ as described in Materials and Methods. Samples were collected monthly over an 11-month period from four bottlenose dolphins (Animals A, B, C, and D). Month of the study is denoted on the x-axis and month 1 corresponds to June 2014.</p

Time course of TNF-α and IFN-γ levels (ng/mL) in bottlenose dolphin (<i>Tursiops truncatus</i>) serum samples.

<p>A serum sample from four bottlenose dolphins (Animals A, B, C, and D) was collected monthly over an 11-month period. Samples were run on ELISA assays specific for bottlenose dolphin TNF-α and IFN-γ as described in Materials and Methods. Month of the study is denoted on the x-axis and month 1 corresponds to June 2014.</p

Time course of TNF-α levels (ng/mL) in supernatants from ConA or PHA stimulated PBMCs isolated from bottlenose dolphins (<i>Tursiops truncatus</i>).

<p>PBMCs isolated from <i>Tursiops truncatus</i> were stimulated with 1, 5, or 10 μg/mL PHA or 1 μg/mL ConA for 48 h in culture. Supernatants were collected and run on an ELISA assay specific for bottlenose dolphin TNF-α as described in Materials and Methods. Samples were collected monthly over an 11-month period from four bottlenose dolphins (Animals A, B, C, and D). Month of the study is denoted on the x-axis and month 1 corresponds to June 2014.</p

Summary of blood-based health indices that independently predicted a linear change in IFN-γ and TNF-α levels in bottlenose dolphins (<i>Tursiops truncatus</i>).

<p>Summary of blood-based health indices that independently predicted a linear change in IFN-γ and TNF-α levels in bottlenose dolphins (<i>Tursiops truncatus</i>).</p

Higher IL-6 and IL-12p40 MSCGT in older dolphins with chronic disease.

<p>RQ values for each cytokine and stimulus were pooled into two groups. Three to four samples from dolphin A were pooled with 4 samples from dolphin D. Similarly, 5 samples from dolphin B were pooled with one to two samples from dolphin C. A, C, E, G: RQ values are plotted against sampling date. Shaded area indicates range from 0.5–2 RQ considered no different from mock. B, D, F, H: Bars indicate geometric mean with 95% confidence interval for the same data points as in A, C, E and G. Stars indicate a statistically significant difference between the groups by two-tailed Mann-Whitney test. *: <i>p</i> ≤ 00332, **: <i>p</i> ≤ 0.0021.</p