1a. Objectives (from AD-416):
The objective of this cooperative research project is to develop cytokine assays for Atlantic bottlenose dolphins.
1b. Approach (from AD-416):
To begin with, proinflammatory molecules (IL-1beta, IL-8, TNF-alpha, IL-12), and type I (IFN-gamma), as well as type II (e.g., IL-10, IL-13) cytokine reagents will be proposed for development. The reasons for this selected set is as follows. As the name implies, pro-inflammatory cytokines play a critical role in mediating inflammatory processes. Type I cytokines are critical for cell-mediated immune responses to intracellular pathogens. Type II responses facilitate the production of specific classes of antibody. These cytokines would be expressed as recombinant proteins as follows: Mature proteins would be expressed using an expression vector with its own signal sequence and transfected into yeast (Pichia pastoris). Expressed proteins will be purified by high performance liquid chromatography (HPLC). A collaborator has expertise with this system. Bioassays will be used for testing the bioactivity of recombinant cytokines. Several of these assays have been successfully used for measuring bioactivity of cytokines from a wide variety of species (e.g., cattle, swine, poultry, and fish). Once bioactivity of each cytokine has been confirmed, polyclonal antibodies (pAb) to select recombinant cytokines will be produced in rabbits. Monoclonal antibodies (mAb) to specific cytokines (e.g., IFN-gamma and IL-13) would be produced in mice. Development of ELISA assays. ELISAs will be developed using two pAb, two mAb, or a mAb and a pAb along with the appropriate recombinant cytokine. Standard curves will be used to quantify the levels of cytokines in blood or in cultures of cetacean leukocytes. Development of ELISAs will allow for the evaluation of functional changes in the T. truncatus immune system in response to environmental insults, infectious diseases, and/or vaccination. This will be of substantial benefit to monitoring bottlenose dolphin health.
3. Progress Report:
We prepared a cDNA library from bottlenose dolphin splenic tissue total RNA. The dolphin spleen tissue sample was obtained from personnel with the US Navy Marine Mammal Program. The cDNA library was sent to Kingfisher Biotechnology, Inc. Target cytokine genes were then amplified from the cDNA library using the appropriate PCR primer sets. The amplified PCR products were cloned into an expression vector for the yeast Pichia pastoris and sequenced. Using this Pichia pastoris expression system, the cytokine proteins were purified. To date, we have purified bottlenose dolphin interlekin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, IL-8, IL-1beta, and interferon (IFN)-gamma. We immunized rabbits with recombinant dolphin IL-8 and produced a rabbit anti-IL-8 polyclonal antibody. Recombinant cytokines were used for western blot analysis. The antibody we produced reacted with dolphin IL-8, but not bovine IL-4. Immunohistochemical (IHC) assays have been performed on bottlenose dolphin frozen tissue sections using samples of dolphin lymph node, spleen or liver. We have found that anti-bovine IFN-gamma and IL-10, anti-swine IL-6, and anti-human TGF-beta antibodies cross-react with dolphin tissues and can be used to detect these cytokines in dolphin tissue or cells. In addition, the anti-dolphin IL-8 works for IHC staining of dolphin spleen. An enzyme-linked immunosorbent assay (ELISA) is in development using recombinant dolphin IL-8 as a standard and using the rabbit anti-IL-8 polyclonal Ab as capture and biotinylated anti-dolphin IL-8 as the detecting Ab.