Cytokine Immunoassays: New Methods to Evaluate Steller Sea Lion Immune Health

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

2. 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

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

4. Project Goals: • Develop assays to measure Steller sea lion cytokines in serum and from white blood cells in vitro • 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

5. 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

7. 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 lioncytokines 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

8. Project Goals: • 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

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

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

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

12. Chromosome Genomic DNA mRNA cDNA Step 3. Construct a cDNA Library • Isolate mRNA transcripts from tissues or cells. • Convert transcripts into double-stranded DNA fragments called cDNA.

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

14. 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

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

16. 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

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

18. Ways to Use New Assays Collect blood samples White blood cells Serum 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

19. 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

20. 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?

21. 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