1. The Immunohistochemistry Studies of Tumor Necrosis Factor Αlpha and Interferon Gamma Expression in Pseudocapillaria tomentosa Infected and Uninfected Zebrafish Oregon State University Lalee Lo Dr. Jan Marie Spitsbergen Dr. Susan Tornquist

2. What is immunohistochemistry? Immuno- The immune system relies on antibodies and antigen. Histo- is the study of cells and tissue Immunohistochemistry exploits the characteristics of antibodies and antigens. -Primary antibody attaches to Antigen -Secondary antibody with tag attaches to Primary Antibody.

3. What is immunohistochemistry?

4. Tumor Necrosis Factor Alpha -apoptotic cell death -cellular proliferation and differentiation -inflammation -regulation of immune cells

5. Interferon Gamma -immune proteins produced in response to challenges by: -viruses -bacteria -parasites -inflammation

6. Pseudocapillaria tomentosa A common fish nematode known to cause: -Inflammation of the gut and intestine of zebrafish. -Aggressive neoplasm (abnormal growth of tissue) of the intestine.

7. Why Zebra Fish? -Low maintenance and feed cost -Ability to reproduce year round -Zebra fish embryos develop rapidly, progressing from eggs to larvae in under three days and contain duplicate genes.

8. Current Research -TNF α and IFN γ function in homeostasis. -TNF α is produced by practically every cell. -Higher levels of TNF α and IFN γ have been observed in inflamed tissue.

9. Purpose of Research To determine whether the levels of TNF α and IFN γ elevate in response to parasitic infection.

10. Goals and Objectives Hypothesis: Pseudocapillaria tomentosa infected zebrafish would have stronger staining of tissues with chromogen in immunohischemistry studies indicating elevated tissue levels of TNF α and IFN γ

11. Experiment Set #1 Date of Birth Date of Carcinogen Exposure Carcinogen Treatment Date of Parasite Treatment Parasite Treatment Lot #Sampling Date # of Fish 9/10/2006None 5/10/200 7 Pseudoc apillaria ZRN 14-9 8/10/20 0720 9/10/2006None 5/10/200 7 Pseudoc apillaria ZRN 14-10 8/10/20 0720 9/10/2006None 5/10/200 7None ZRN 14-11 8/10/20 0720 9/10/2006None 5/10/200 7None ZRN 14-12 8/10/20 0720

12. Experiment Set #2 Date of Birth Date of Carcinogen Exposure (age: 30 days) Carcinogen Treatment Date of Parasite Treatment Parasite Treatment Lot #Sampling Date# of Fish 1/5/20062/8/06DMSO Control 3/20/200 6 NoneZRN 14-5 5 fish at 3 wk and 5 wk, 20 fish at 17 wk, 30-50 fish at 29 wk post-parasite 74 1/5/20062/8/06DMSO Control 3/20/200 6 Pseudocap illaria ZRN 14- 5b See Above.67 1/5/20062/8/06DMBA 1 ppm 3/20/200 6 NoneZRN 14-6 See Above.74 1/5/20062/8/06DMBA 1 ppm 3/20/200 6 Pseudocap illaria ZRN 14- 6b See Above.90

13. Experiment Tank Feed Setup Zebrafish were raised in flowing well water in 30 gallon tanks at 27 Celsius +/- 2 -fed ad libitum with Aquatox (Zieger, Gardners PA) flake fish feed and brine shrimp. -Necropies occurred at 11 months of age. 12 weeks post infection.

14. Methods of Analysis: Paraffin Sectioning Paraffin wax, is widely used on fruits, vegetables, and candy to retard moisture loss and spoilage. The zebra fish tissue set in paraffin blocks, this allows the tissue to be cut thin.

15. Methods of Analysis: Prep Immuno-histochemistry Protocol (IHC): -Xylene and ethanol. Dissolves the paraffin. -Blocking solution Binds to any sites that are not antibody bound. -Peroxidase Block Block endogenous peroxidase.

16. Methods of Analysis: Protocol Optimizing the IHC protocol: -Finding the optimal TNF α and IFN γ antibody concentration. Antigen Retrieval Methods -Enzyme vs Steam Antigen Retrieval Steam Antigen Retrieval worked better.

17. Methods of Analysis: Protocol Compared various buffers for the steam heat treatment -pH 6 citrate buffer -Tris buffered saline (TBS) at pH 7.2 -Dako antigen retrieval buffer For consistency reasons, we decided to use the Dako antigen retrieval buffer.

18. Methods of Analysis: Antibody ABCam TNF α stain -Mouse derived antibody ABCam IFN γ stain -Rabbit derived antibody DAKO Envision Plus Immunohistochemistry polymer system

19. Methods of Analysis: Antibody

20. Methods of Analysis: Protocol Primary Antibody -Abcam TNF α (dilution 1:10,000) -Abcam IFN γ (dilution 1:1,000) Secondary Antibody Chromogen (chemical compound that changes color when bound) Hematoxylin and then add mounting media.

21. Methods of Analysis: MPO MPO (Myeloperoxidase) specifically stains the cytoplasm of neutrophils of zebrafish. Neutrophils produce reactive oxygen. DNA damage likely plays a role in the neoplasia associated with chronic inflammation.

22. Results Myeloperoxidase MPO expression found that the intestine of zebrafish chronically infected with Pseudocapillaria (3 months post infection in experiment 2) showed increased numbers of neutrophils compared to intestine of uninfected fish.

23. Myeloperoxidase in the Intestine MPO is negative in uninfected intestine MPO is positive in infected intestine

24. Myeloperoxidase in Neutrophils in Hematopoietic Tissue in the Kidney MPO in neutrophils in impression smear from kidney MPO in neutrophils in hematopoietic tissue of anterior kidney

25. Results TNF α Experiment Set #1 No variation between infected and uninfected group. TNF α staining was strongest in: -Head skeletal muscle -Intestine

26. Experiment Set #1 TNF Alpha: Tissue StainedNegative Control

27. Experiment Set #1 TNF Alpha: Eye StainedNegative Control

28. Experiment Set #1 Tumor Necrosis Factor Alpha Tissues Staining Strongly Tissues Staining Moderately Tissues Staining Lightly Skeletal muscle of head and at bases of fins (derived from neural crest) Neurosensory epithelium of nose Bile duct mucosal surface Intestine mucosal surface (entire intestine) Taste BudsExocrine pancreas acinar cell Ultimobranchial glandChloride cell of gill filament and lamellae Outer plexiform layer of retina of eye Heart ventricular myocardium Heart atrial myocardium

29. Results IFN γ Experiment Set #1 No variation between infected and uninfected group. IFN γ staining was strongest in: -heart -inflamed skeletal muscle and connective tissue

30. Experiment Set #1 IFN Gamma: Heart StainedNegative Control

31. Experiment Set #1 IFN Gamma: Ventricle StainedNegative Control

32. Experiment Set #1 Interferon Gamma Tissues Staining Strongly Tissues Staining Moderately Tissues Staining Lightly Endocardium of heartNeurosensory epithelium of nose Pseudobranch epithelium Macrophages in inflammation in skeletal muscle Taste BudsAmeloblastic epithelium of tooth Inflammatory cells in connective tissue Hemopoietic tissue in kidney (multifocal) Meninges and ventricles of brain Chloride cells of gill filament Ganglia of cranial nerves Developing oocytes (perinucleolar and vitellogenic) Tooth pulp

33. Experiment Set #2 TNFα Elevation Infected Intestine Uninfected intestine. Red chromogen indicates TNFα expression. Infected intestine. Red chromogen indicates TNFα expression.

34. Experiment Set #2 TNFα Elevation Base of Dorsal Fin Lymphocytes and macrophages in inflamed skeletal muscle (long arrow). Normal high expression of TNFα in skeletal muscle at base of fin (short arrow). High TNFα expression in capillaries in inflamed skeletal muscle (arrowhead).

35. Experiment Set #2 IFNγ Inflammation M: Macrophages in inflamed skeletal muscle Arrow: Heart M M M M

36. Experiment Set #2 IFNγ Inflammation Arrow points to macrophages expressing IFNγ in inflamed skeletal muscle of trunk Arrow points to macrophages expressing IFNγ in skeletal muscle near optic nerve ( * )

37. Discussion Experiment Set #1 Comparison of the infected vs uninfected showed no difference in TNF α and IFN γ at the 12 week post infection sampling.

38. Discussion Experiment Set #2 5 weeks post infection showed there is a difference in TNF α and IFN γ levels. Experiment set #1 did not catch this difference as the levels most likely dropped after the peak earlier on.

39. Goals and Objectives Hypothesis: Pseudocapillaria tomentosa infected zebrafish would have stronger staining of tissues with chromogen in immunohischemistry studies indicating elevated tissue levels of TNF α and IFN γ

40. Conclusion -TNF α and IFN γ is present in zebrafish cells and function in cell homeostasis. -No difference in TNF α and IFN γ levels between infected and uninfected at the 12 weeks post infection. -Higher levels appeared at 5 weeks post infection.

41. Special Thanks to: Mentor Dr. Jan Marie Spitsbergen

42. Additional Thanks -Dr. Kate Fields -Wanda Crannell -Dr. Susan Tornquist

43. Additional Thanks -The researchers at the Center for Fish Disease Research. -NIH (NIEHS) -John Fryer Salmon Disease Lab -Marine and Freshwater Biomedical Science Center -Environmental Health Science Center at Oregon State University

44. Questions?