Amanda Smith, Ed.M. Union City High School, Union City, NJ

1. Lecture Slides for Curriculum “Macrophage Morphological Changes Due to iNOS Activation by LPS and Its Implication in Chronic Asthma” Amanda Smith, Ed.M. Union City High School, Union City, NJ AAI Summer Research Program for Teachers

2. Introduction to the Immune System Lesson 1

3. Do Now • Think back to the Online Stem Cell Lab, health class, and past science classes. • How many different cell types can you name that are related to the immune system? • List them with their corresponding functions in your notebook.

4. Innate vs. Adaptive Immunity Innate Adaptive Slow response (a few days) Specific response to molecules that caused it Repeated exposure to molecules will cause increased responses • Immediate, rapid response • Generalized response, nonspecific • No “memory”

5. Response Question 1 • You get the flu vaccine. A few weeks later, your sibling gets the flu and exposes you to the virus. Which part of your immune system will be prepared to fight off the flu virus? • Innate Immune System • Adaptive Immune System

6. Hematopoietic Stem Cells • Found in bone marrow • Give rise to blood and immune cells • Form two lineages: myeloid and lymphoid

7. Myeloid Lineage • Gives rise to eight types of cells found in body tissues, focusing on three • Mast Cells • Degranulate hormonal mediators when activated, involved with inflammation • Activated by IgE antibodies, physical/chemical injury, or complement proteins • Morphology: typical round cellular shape • Defining Organelles: secretory granules and lipid bodies • A Visual Interpretation Image at: http://www.mastcellaware.com/about.html

8. Myeloid Lineage • Macrophages • Matured monocytes that migrate into tissues from the circulatory system • Role is to phagocytize dead cells and pathogens • Produce NO, O2-, and H2O2 to break down material. • Specific names for specific locations, ex: Kupffer cells in liver • Morphology: monocyte is a spherical shape, but macrophage shape depends on what tissue it goes and matures. • Defining Organelles: extensive Rough ER, lysosomes/phagosomes, pseudopods

9. Myeloid Lineage • Dendritic Cells • Main role is to process antigen material and present it to other cells on its surface. • AKA antigen presenting cell • Morphology: form branch like projections (dendrites) during stage of development • Defining Organelles: Golgi, ER, (proteasome – protein complex that degrades proteins) Image at: https://www.boundless.com/microbiology/immunology--2/antigen-presenting-cells/dendritic-cells/

10. Response Question 2 • A cut on your arm went untreated, and now, it is infected by bacteria. Which of the myeloid lineage cells are involved with fighting off the infection? • Mast Cells • Macrophages • Dendritic Cells • A & B • A, B & C

11. Lymphoid Lineage • Gives rise to three major types of cells • Natural Killer Cells (NK) • Role: limits the spread of tumors and microbial infections by inducing apoptosis in cells, limiting tissue damage • Morphology: large granular lymphocyte, spherical • Defining Organelles: cytoplasmic lytic granules, secretory lysosomes, golgi, vesicles, microtubules Image at: http://imagebank.hematology.org/AssetDetail.aspx?AssetID=4105&AssetType=Asset

12. Lymphoid Lineage • B Cells • Role: produce antibodies, become antigen-presenting cells, become memory B cells • Memory B cells “remember” specific antigens and can launch fast immune response if antigen is encountered again. • Live for about 10 years. • Morphology: spherical, with very large nucleus and scant cytoplasm around it, has B-cell receptor on cell surface • Defining Organelles: large ER and Golgi, lysosomes Image at: http://commons.wikimedia.org/wiki/File:Lymphocyte2.jpg

13. Lymphoid Lineage • T Cells • Several types including T Helper and cytotoxic T cells • Role: secrete cytokines after activation by APC to help aid active immune responses, and destroy virally infected and tumor cells by binding to antigens on their surface, respectively • Morphology: Spherical with T-cell receptor on cell surface, covered with short microvilli • Defining Organelles: ER, Golgi Image at: http://www.iayork.com/MysteryRays/2008/03/30/whats-in-a-name-are-cytotoxic-t-lymphocytes-cytotoxic/

14. Response Question 3 • What type of cell is also derived from hematopoietic stem cells that we have not gone over? • Chondroblasts (cartilage cells) • Erythrocytes (red blood cells) • Endothelium (cells that line blood vessels) • Hepatocytes (liver cells)

15. Interactions between cells • Dendritic Cells present antigen to activate immature T cells to become CD4 or CD8 cells. • TH2 cells and costimulatory molecules released by TH1 cells activate B cells to produce antibodies.

16. Discussion • What are the two major lineages that arise from hematopoietic stem cells? • What types of cells can naïve T cells differentiate into? • What is the relationship between dendritic cells and naïve T cells? • How are T cells related to B cells? • What is the difference between an antigen and an antibody?

17. Review Activity • Graphic Organizer • Show the major lineages and what cells are formed. • Connect lineages with lines and descriptor words. • Start with hematopoietic stem cells.

18. TEACHER USE ONLY! View diagram showing all cell types derived from hematopoietic stem cells at http://www.allthingsstemcell.com/category/hematopoietic-stem-cells/ Have students make their own/use worksheet.

19. Nitric Oxide, Inflammation, and the Immune Response Lesson 2

20. Do Now • What do you think are some possible effects of the immune system over-responding (responding too much)? • Would the effects be positive, negative, or neutral? Explain your reasoning.

21. Inflammation • Part of the immune response to pathogens • Caused by vasodilation for the movement of plasma and immune cells into the injured tissues. • Can be acute or chronic. • Chronic inflammation occurs if the stimulus for the inflammation persists. • Mediated by macrophages and released chemicals such as IL-1 and TNF-alpha

22. Nitric Oxide & Inflammation • Nitric Oxide (NO) is released by macrophages to destroy the source of inflammation. • NO is produced by the protein inducible nitric oxide synthase (iNOS). • iNOS is formed in after injury and inflammatory mediators are released. • An increase in NO production can lead to tissue injury. • Occurs when there’s an increase in inflammatory cytokines being released

23. Chronic Asthma • Can be caused by allergies, environmental pollutants, microbial infections, or genetics • NO normally helps to regulate airway vascular tone and pulmonary surface tension. • Over expression disrupts innate lung defenses that modulate lung inflammation, leading to chronic inflammation.

24. Article Review • Read over questions for article. • Read “The Inflammatory Response in the Pathogenesis of Asthma.” • Highlight important information to help you answer questions as you read. • Complete related worksheet & Inflammatory Response Flow Chart.

25. Intro to Aseptic Technique & Lab Procedures Lesson 3

26. Do now • What does it mean when somebody is septic or goes into septic shock? • What does Aseptic mean? • How can you practice being aseptic in a lab setting? List as many possible ways as you can.

27. Aseptic Technique • Origin: septikos – rotten; a – without • Purpose: to keep the sterile cell culture free of microorganisms from the environment • Four basic ways to achieve this • Sterile work area • Good personal hygiene • Sterile reagents & media • Sterile handling

28. 1. Sterile Work Area • Use a cell culture hood. • Maintain a clear work surface. • Disinfect work surface before and after use with 70% ethanol. Image at: http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/cell-culture-equipment/laminar-flow-hood.html

29. 2. Good personal hygiene • Wash your hands before and after working with cell cultures. • Tie your hair back. • Wear personal protective equipment. • Gloves • Lab coat Image at: http://web.princeton.edu/sites/ehs/labsafetymanual/sec6c.htm

30. 3. Sterile Reagents & Media • Commercial materials (bought from a company) are already sterilized. • Materials prepared in the lab should be prepared with sterile instruments using aseptic technique.

31. 4. Sterile Handling • Wipe outside of containers with 70% ethanol . • Use sterile glass or disposable plastic materials. • Do not cross contaminate. • Do not unwrap materials until the moment you are going to use them. • Cap or seal with tape bottles, well plates, etc. to prevent contamination. • Perform experiments rapidly (but safely!) to minimize exposure to contamination.

32. Video http://media.invitrogen.com.edgesuite.net/Cell-Culture/videos/SterileTechnique.html?CID=ccbvid2

33. The Micropipette • Used to measure small volumes accurately • Uses disposable tips to prevent cross contamination • Can be set at a fixed volume or variable – you specify within a range Image at: http://faculty.ksu.edu.sa/moncef/Pages/OperatingtheMicropipette.aspx

34. Setting the Volume • Turn the volumeter adjustment on top. • Stay within the micropipette’s range. • Decimal appears only for smaller range pipettes – make sure to turn the adjustment to the correct volume. Image at: http://www.medicine.mcgill.ca/physio/vlab/bloodlab/eryfrag2_n.htm Table image at: http://www.scienceteacherprogram.org/biology/JBodenmiller10.html

35. Drawing liquid Image at: http://www.karantinapertaniansby.com/en/?hal=detil_artikel&id=2 1. Push pipette plunger down to FIRST stop while OUT of liquid. 2. Place tip into liquid, release plunger to draw liquid up. 3. Push back down on plunger to first stop to release liquid. 4. Push plunger further to second stop to expel any remaining liquid from tip. 5. Release plunger to go back to starting position.

36. Practice! • Learn how to use a micropipette to make a serial dilution. • Virtual Lab Activity

37. Hemocytometer • Originally used to count blood cells, now used for various cell types. • Cells first stained with trypan blue to determine viable from non-viable cells. • Used to calculate amount of cells in a solution from a 10 microliter sample. Image at: http://www.sigmaaldrich.com/catalog/product/sigma/z359629?lang=en&region=US

38. Practice! • Hemocytometer virtual lab