Cytokine Immunoassays: New Methods to Evaluate Steller Sea Lion Immune Health Mary Bozza, Research Associate Alaska SeaLife Center

Steller Sea Lion Immune Health Do Steller sea lions bioaccumulate contaminants to toxic levels? How can we measure toxicity? Toxins can cause immune system disruption Marine contaminants linked to immunosuppression include: Organochlorines, PCB’s, Cadmium

Steller Sea Lion Immune Health Alaska SeaLife Center Contaminants Project: Endocrinology, Immunology and Toxicology S. Aktkinson, B. Middlebrooks, Q. Li Individual Health Assessment of Steller Sea Lions in Alaska Burek, Beckmen, Rea, and Gellat

Project Goals: I.Develop assays to measure Steller sea lion cytokines in serum and from white blood cells in vitro II.Evaluate immunocompetence: Establish baseline cytokine profiles to assess immune health of individual Steller sea lions Correlate cytokine profiles with immune and overall health parameters body burdens of contaminants to determine toxicity

What are Cytokines? Proteins secreted by immune cells in response to inflammation or other stress Called “interleukins,” they act as chemical messengers to regulate inflammation Routinely used to study other mammalian immune systems including human & mouse Commercially available reagents may be available to measure Steller sea lion cytokines if appropriate assays are developed

An immunoassay uses an antibody to recognize the protein of interest; the concentration of the antibody- protein complex is measured Challenge: Antibodies to Steller sea lion cytokines do not exist! Proteins are not available to generate or validate antibodies Solution: Make Steller sea lion protein using molecular cloning techniques Use the protein to validate existing cross-reactive antibodies to develop an assay

Project Goals: I.Develop assays to measure Steller sea lion cytokines in serum and from white blood cells in vitro II. Evaluate immunocompetence : Establish baseline cytokine profiles to assess immune health of individual Steller sea lions Correlate cytokine profiles with immune and overall health parameters body burdens of contaminants to determine toxicity

6. Validate assay using cross- reactive antibodies 1. SSL immune cells cultured in vitro with a bacterial extract (LPS) 2. Isolate mRNA 4. Clone and sequence full-length cytokine genes by PCR 3. Construct a cDNA library 5. Protein Expression Goal I: Develop Assays

Step 1. LPS Stimulation of Mononuclear Cells Collect whole blood Place cells in culture dish, add LPS (lipopolysaccharide) to stimulate immune response Centrifuge blood over a Ficoll- Hypaque gradient to isolate mononuclear cells Cell Signaling Transcription Step 2. Isolate mRNA TranslationSecretion

Step 2: Isolate mRNA Size fractionation by electrophoresis (1% agarose gel) 1.Total RNA from HS LPS stimulated mononuclear cells 2.PolyA+ Control RNA (human placenta) 1 2

Step 3. Construct a cDNA Library Isolate mRNA transcripts from tissues or cells. Convert transcripts into double-stranded DNA fragments called cDNA. ChromosomeGenomic DNAmRNAcDNA

Insert cDNA into plasmid vectors that can replicate in bacteria. Introduce vectors into bacteria. Amplify library. cDNA cDNA inserted into plasmid plasmid in bacteria amplified library

Advantages of a cDNA Library Contains only protein-coding DNA (genes) Represents the entire collection of genes expressed in cell or tissue type Vector with inserted gene can be used to produce protein Can amplify and store the library for long term use Clone many other genes from stimulated white blood cell cDNA library New libraries from Steller sea lion cells or cell lines New libraries from other species cells or cell lines

6. Validate assay using cross- reactive antibodies 1. SSL mononuclear cells cultured in vitro with a bacterial extract (LPS) 2. Isolate mRNA 4. Clone and sequence full-length cytokine genes by PCR 3. Construct a cDNA library 5. Protein Expression

Step 7. Assay Development ELISA (enzyme linked immunosorbent assay) or RIA (radioimmunoassay) Test commercially available antibodies for binding to protein Validate assay using purified protein Validate assay using serum

Ways to Use New Assays Collect blood samples Cytokine ELISAs (Inflammation, Infection or Disease) Stimulate WBC in vitro (Immuno competence) Cytokine ELISAs White blood cellsSerum

Ways to Use New Assays Collect blood samples Cytokine Profiles by RT-PCR (Inflammation, Infection or Disease) Cytokine ELISAs (Inflammation, Infection or Disease) Stimulate WBC in vitro (Immuno competence) Cytokine Profiles by RT-PCR Cytokine ELISAs White blood cellsSerum

Steller Sea Lion Immune Health Do Steller sea lions bioaccumulate contaminants to toxic levels? How can we measure toxicity? Toxins can cause immune system disruption Marine contaminants linked to immunosuppression include: Organochlorines, PCB’s, Cadmium

What’s next? LPS stimulation done cDNA library is being constructed Next step: PCR Cloning Collaboration Vectors, methods for protein production Which genes to clone, proteins to assay? Additional libraries?

Acknowledgements Dr. Shannon Atkinson Don Calkins Dr. Jo-Ann Mellish ASLC Mammal Dept. ASLC Veterinary Services Dept. Carol Stephens Blood Donors: Woody, Kiska, Sugar, Tina and Pender Jon Moreland Lizabeth Moundalexis Annette D’Alessandro & Angie Steeves