FIS: Immunology Christopher F. Cuff, Ph.D. Rm. 2070 293-4622 CCUFF@HSC.WVU.EDU

1. FIS: Immunology Christopher F. Cuff, Ph.D. Rm. 2070 293-4622 CCUFF@HSC.WVU.EDU

2. Goals • Enable biomedical scientists to understand the role of microbes in health and disease at a level sufficient to integrate new knowledge in the field as it applies to his/her discipline. • Enable students to understand the nature and use of research tools developed from the study of microbiology and immunology (such as genetic manipulation and antibodies)

3. Microbiology and Immunology A study of infectious organisms to understand the mechanisms of pathogenesis A study of the host’s immune system to understand the mechanisms involved in the clearance of infection

4. Microbes as Infectious Agents Multicellular - parasites, fungi Unicellular - bacteria, fungi, protozoan parasites Subcellular - viruses, prions Infectious Agents: -bind to the host, replicate, in some cases produce toxins, -cause damage to the hosts’ tissues, -elicit an immune response from the host. Distinguish from ‘normal flora’ that contributes to health of the host.

5. Red Blood Cell Bacteria Viruses

6. Bacteria staphylococci streptococci spirochetes bacilli

7. Viruses HSV-1 Hepatitis B HIV Ebola

8. Fungi unicellular multi-cellular

9. Parasites (unicellular and multi-cellular) unicellular multi-cellular

10. ‘Koch’sPostulates’ The same organism must be found in all cases of a given disease. The organism must be isolated and grown in pure culture. The organisms from pure culture must reproduce the disease when inoculated into a healthy, susceptible animal. The organism must then be again isolated from the experimentally-infected animal. Robert Koch (1843-1910) First to demonstrate that microorganisms cause a human disease (anthrax)

11. The Immune System: System to maintain the integrity of the hosts’ internal environment during insult from pathogens and toxins in the external environment.

12. Immunity is Mediated by: Innate Factors (non-specific) & Adaptive Factors (specific)

13. Innate Immunity Anatomic Barriers Intact epithelium (keratinized and non-keratinized) Mucous Cilia Smooth muscle contraction

14. Innate Immunity Anatomic Barriers Physiologic (Chemical) Barriers temperature pH enzymes (i.e. lysozyme, & peroxidase) complement and acute phase reactants Endocytic/Phagocytic Barriers

15. Phagocytic cells - internalize particles Granulocytes (neutrophils, eosinophils)

16. Macrophages (monocytes)

17. Endocytosis: internalization of macromolecules (phagocytosis: specialized internalization of particles Such as whole bacteria) Receptor Mediated Endocytosis: Endocytosis following binding of macromolecules to surface receptors Pinocytosis: Non-specific membrane invagination

18. Macromolecules binding to receptors on cell surface Extracellular Intracellular Primary Lysozomes from Golgi complex

19. Primary Lysozomes from Golgi complex Macromolecules binding to receptors on cell surface Extracellular Intracellular

20. Endosome Lysosome Fusion Macromolecules binding to receptors on cell surface Extracellular Intracellular Primary Lysozomes from Golgi complex

21. Degradation of Macromolecules Macromolecules binding to receptors on cell surface Extracellular Primary Lysozomes from Golgi complex

22. reactive oxygen intermediates e.g. superoxide anions (O2-) and hydrogen peroxide (H202) reactive nitrogen intermediates e.g. nitric oxide (NO) antibacterial proteins lysozyme and defensins proteolytic enzymes elastase and cathepsin B, cathepsin L, and cathepsin G Antimicrobial and cytotoxic activities of phagocytes (in lysozomes) Oxygen-dependent Oxygen-independent

23. Inflammation: Non-specific reaction that occurs as a result of local tissue damage and the presence of foreign substances.

24. 1. Pain 2. Heat 3. Redness 4. Swelling 5. Loss of Function Five Cardinal Signs of Inflammation

25. Neutrophils Rolling Along Endothelium

26. Release of Vasoactive and Chemotactic Substances SITE OF INFLAMMATION

27. Margination: Neutrophil stops rolling and sticks to endothelium Diapedesis: Neutrophil squeezes out of blood vessel between endothelial cells SITE OF INFLAMMATION

28. Chemotaxis: Neutrophil migrates toward site of tissue damage along the concentration gradient of chemotactic substances.

29. Margination: Neutrophil stops rolling and sticks to endothelium. Diapedesis: Neutrophil squeezes out of blood vessel between endothelial cells. Chemotaxis: Neutrophil migrates toward site of tissue damage along the concentration gradient of chemotactic substances. Interactions between endothelial cells and neutrophils are mediated by molecules expressed on the surface of both the endothelial cells and the neutrophils.

30. The Complement System Involved in inflammation and resistance to bacterial (and to a lesser extent fungal and viral) infections. Complement deficiencies put patients in grave danger.

31. The Complement System A system of about 20 proteins that are produced by the liver and found in blood serum. Produced in an inactive form and are sequentially activated to produce biologically active complexes. Function of these complexes is 2-fold: direct killing of pathogens through formation of the ‘membrane attack complex’ (MAC) enhance the inflammatory response by opsonizing bacteria and increasing phagocyte chemotaxis

32. Complement components are sequentially activated in a process often referred to as the ‘complement cascade’ The complement cascade can be activated through 3 distinct pathways: 1) the classical pathway 2) the alternative pathway 3) lectin activation pathway

33. Complement components polymerize to form a Membrane Attack Complex (MAC) that inserts into the membrane and forms holes in the pathogen

34. Complement enhances the activity of phagocytic cells by opsonizing bacteria C3b

35. Chemotaxis: Neutrophil migrates toward site of tissue damage along the concentration gradient of chemotactic substances. C5a

36. Alternative Pathway and Lectin Mediated Pathway • of Complement Activation: • Collection of serum proteins that are sequentially activated • to enhance the inflammatory response. • Initiated by the spontaneous cleavage of C3 and recognition • of bacterial mannose-containing surface molecules (mannans) • 3) C5-C9 forms the MAC,C3b opsonizes the pathogen, • thereby promoting phagocytosis, and C5a promotes • chemotaxis of phagocytes to the site of inflammation.

37. Pathogen Anatomic Barriers Inflammatory Response (chemical and phagocytic barriers) -local release of vasoactive and chemotactic substances -neutrophil infiltration (margination, diapedesis, chemotaxis) -complement activation resulting in MAC, opsonization, and increased neutrophil migration

38. Immune Cell PAMP The Innate Arm of the Immune System Express Receptors for Components of Microbial Pathogens PAMPs - ‘pathogen-associated molecular patterns’ PRRs - ‘pattern recognition receptors’ PRR

39. PRRs are Soluble or Expressed on the Cell Surface Soluble PRRs Mannan Binding Lectin (MBL) C-reactive Protein (CRP) Serum Amyloid Protein (SAP) LPS Binding Protein (LBP) Cell Bound PRRs Toll-Like Receptors (TLRs) TLR2, TLR3, TLR4, TLR5, TLR9 CD14 Bind to various bacterial cell wall components and enhance killing

40. PAMPs Bacterial lipopolysaccharide (LPS) – the outer coating of certain bacteria (Gram negative) Bacterial cell wall peptidoglycan – the rigid outer layer of bacteria Bacterial flagellin – the protein that makes flagella, which are used for motility Mannan from yeast or bacteria Unmethylated CpG DNA – nucleic acid bases Double stranded RNA (dsRNA) from viruses

41. Immune Cell PAMP Binding of PAMPs by PRRs can stimulate cells of the innate arm of the immune system to become: -more phagocytic, -more effective and initiating adaptive immune responses. Danger, Danger !!!!

42. Innate Immunity Anatomic/Physiologic barriers Phagocytosis/Endocytosis by granulocytes and macrophages When Innate Immunity Fails to Eliminate the Pathogen, Specific Immune Responses are Initiated

43. Properties of Specific Adaptive Immunity 1. Specificity (exquisite) 2. Memory

44. 2o anti-A Immune Response Pathogen A Pathogen A 1o anti-B = 0 14 0 6 14 Time (Days) Specificity and Memory 1o anti-A + Pathogen B

45. Cells of the Immune System Specific Immunity: Directed against foreign substances (termed antigens) following the failure of innate immunity B-lymphocytes (Bursa-derived) T-lymphocytes (Thymus-derived) Antigen Presenting Cells (APC)

46. B-cells B-cells produce and secrete proteins that bind to, and neutralize antigens (antibodies) . B-cells that have contacted antigens further differentiate into either memory cells or plasma cells. B-cells do not require APCs to become activated.

47. T-Lymphocytes T-helper cells provide signals for the growth (proliferation) and differentiation of other immunologic effector cells. (Th) T-cytotoxic cells can kill abnormal cells in the body - for example infected cells or tumor cells. (Tc) T-regulatory cells can inhibit inflammatory responses. (Treg or Ts)

48. B-cells and T-cells are Antigen-Specific (Clonal) Each B-cell recognizes a unique antigen, but does not require an APC. Each T-cell recognizes a unique antigen on the surface of an APC. Clonal Selection: Antigen Stimulates Specific Clones to Generate Antigen-Specific Responses

49. Expression of Differentiation Markers is used to Distinguish T-cells and B-cells TCR TCR sIg CD4 CD8 T-cytotoxic B-cell T-helper

50. Antigen-Presenting Cells (APC) -dendritic cells, macrophages, and in some cases, B-cells. -process antigens by endocytosis (or phagocytosis), degrading them into simple molecular fragments, and re-expressing these fragments on their cell surface.