o Pyrogen = something that causes fever Physical barriers Skin most microbes cannot penetrate unbroken keratinized skin

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1 DAY 1 2/03/2015 9:53 AM BARRIERS General Barriers Nutrition malnourished are more susceptible Acute phase reactants reactants whose concentrations o Iron sequestration o C reactive protein (complement activation, phagocytosis) Fever exogenous pyrogen (microbial components endotoxins in Gram (-) bacteria) stimulate endogenous pyrogen (IL-1) which stimulates fever o Pyrogen = something that causes fever Physical barriers Skin most microbes cannot penetrate unbroken keratinized skin o Dry, mildly acidic (ph 4-6), sebum oily film Mucosa lines the respiratory, digestive, and genitourinary systems; traps some microbes; iron binding proteins (lactoferrin) Respiratory tract mucociliary elevator (coughing and sneezing) brings microbes away from lungs Gastrointestinal tract peristalsis Genitourinary tract flushing action of urine o Short female urethra predisposes to more UTI s Eye conjunctiva (mucus secreting epithelial membrane) tears Chemical barriers Interferons (alpha and beta) low MW glycoprotein called cytokines that are stimulated by viral infection and act nonspecifically against any viral infection o Host species specific (human interferon works best in humans) o Once stimulate interferon acts against any virus Gastric acid ph 1 Lysozyme degrades peptidoglycan o Found in tears, mucosa, some secretions, phagocytes o Effective especially against Gram (+) Complement System A complex of 20 plasma proteins (C ) Some act as an enzyme cascade (product of one reaction catalyzes the next) Must be cleaved into biologically active fragments Complement System Activation

2 o Classical pathway: Antigen-Antibody Complexes o Alternative pathway: plasma protein, properdin and pathogen surfaces (usually repetitive structures) o Lectin (mannose binding lectin) pathway: lectin binds to mannose on pathogen surfaces Fragments (smaller a fragments stay in plasma larger b fragments bind to cell surfaces) o C5a inflammatory mediator acts directly on vascular endothelial cells vasodilation/permeability o C3a another inflammatory mediator (works with C5a) o C3b most abundant complement component involved with opsonization (C3b coats microbial cell phagocytes can better identify and engulf cell) Membrane attack complex (MAC) composed of C5b6789n o Many copies of C9 to create rest of pore o Gram (-) bacterial cells are lysed; Gram (+) are mainly insensitive Normal Flora (microbiota) Many are commensal, but some are mutualistic (helpful) Sterile sites in the body where NO microorganisms should be found: deep tissue, blood, cerebrospinal (CSF), pleural, peritoneal fluids Important to know which sites are colonized so that they can be minimized when specimens are collected!! (for every 1 facultative colony! have 1000 anaerobe colonies present that might not show up on plate) o Large intestine (colon): many anaerobes (Clostridium Basteroides); family Enterobacteriaceae (Escherichia coli); enterococci o Skin: coryneform bacteria, staphylococci (staphylococcus epidermidis); Propionibacterium o Urinary tract (UT): many skin flora o Vagina: the UT flora; Lactobacillus species and other anaerobes; viridans streptococci o Sputum: coryneform bacteria, staphylococci, streptococci Pathogens and Pathogenesis Virulence (degree of pathology caused by organism) quantitates pathogenicity (microbe s ability to cause disease) - (doesn t imply lethality)

3 Frank Pathogen (Primary Pathogen): causes infectious disease under virtually any circumstance (Shigella species) Opportunistic Pathogen: causes infectious disease only under certain circumstances (when given the opportunity) (Pseudomonas aeruginosa) can be super severe CELLS OF THE IMMUNE SYSTEM INNATE & ADAPTIVE IMMUNITY Lymphoid cells (non-phagocytic leukocytes) don t engulf things Lymphoid progenitors B lymphocytes precursors of antibody-producing cells (plasma cells) and memory cells (adaptive immunity) T lymphocytes helper and cytotoxic leukocytes (adaptive immunity) Natural killer (null cells) (NK) lymphocyte (mainly innate but also adaptive) o Large leukocyte o Non-specifically kill nucleated cells that are lacking a normal marker MHC1 " Because!! All nucleated cells supposed to have MHC1 o Virally infected; malignant cells that lack MHC1 are seen as abnormal and are killed by the NK cell o NK inserts a pore forming protein (perforin) into cell membrane, and inserts toxic granzymes Mononuclear cells (phagocytic leukocytes)! Myeloid progenitors Monocytes and fixed macrophages in lung, liver, bone marrow, and lymph nodes are part of the mononuclear macrophage system (formerly the reticuloendothelial system RES) Monocytes mature into macrophages and dendritic cells o Macrophage (M ) phagocytic leukocytes (both innate and adaptive responses); leave the bloodstream to migrate into the infected tissues; are also involved with Ag presentation to B and T cells and can be activated to enhance killing of internal pathogens o Dendritic cells circulating cells that can differentiate between foreign and self molecules (innate); migrate to blood or lymph and present processed antigens (Ag) to T cells (adaptive) Granulocytes (polymorphonuclear leukocytes) mainly innate o Basophils stain with basic dyes; non-phagocytic; release mediators (vasoactive agents); role in allergic responses (hypersensitivity); mainly stay in blood

4 o o Eosinophils stain with acidic dyes; non-phagocytic; can leave blood and enter tissue; defend (extracellularly) against protozoans and helminthes; some role in allergy Neutrophils (polymorphonuclear neutrophils PMN) stain only under neutral conditions; major phagocytic cell in acute inflammation (innate immunity major defense) ORGANS OF THE IMMUNE SYSTEM INNATE & ADAPTIVE IMMUNITY Primary lymphoid organs where lymphocytes mature (learn self from non-self and become antigenically committed ) Thymus primary lymphoid organ; lymphocytes processed here to become T cells Bone marrow primary lymphoid organ; lymphocytes processed here become B cells Secondary lymphoid organs capture Ag and allow lymphocytes and other cells to interact Spleen filters blood and traps microorganisms and Ag; macrophages and dendritic cells then process Ag to present to lymphocytes Lymph nodes located throughout the body, also filter and trap microorganisms and Ag from lymph (from regional tissue); lymphocytes (macrophages), dendritic, B and T cells also found here Mucosal associated lymphoid tissue (MALT) found in tonsils, adenoids, Peyers patches in gut; many pathogens try to gain entry here, but many are stopped by macrophages and lymphocytes. Also GALT gut associated lymphoid tissue (cells and antigens interacting basically) Inflammation (Innate Immunity) Acute: PMN s main phagocyte; adscess in walling off structure. Staphylococcus aureus, meningococcus microbial exs. o Complement components C3a and C5a (inflammatory mediators) trigger mast cell to contribute additional inflammatory (vascular permeability) mediators o Chemotactic factors elicit phagocytic movement in capillaries along endothelial cells o PMN s pass thu gaps in dilated capillary endothelial cells ad enter infected tissue diapedesis

5 DAY 2 2/03/2015 9:53 AM PHAGOCYTOSIS ENGULFING AND DESTROYING INVADERS BY PROFESSIONAL CELLS Mechanism of Phagocytosis Chemotaxis of phagocytes (follow chemical attractant gradient) and diapedesis into infected tissue Adherence of invader to phagocyte surface if C3b and/or specific Ab can bind to invader, this is called opsonization, and phagocytosis is greatly facilitated because phagocytes have Ab and C3b receptors o Nothing (+/-) o Ab (+) o C3b (++) o Ab and complement C3b (++++) Pseudopodia extend around invader (false feet) Phagocyte receptors sequentially attach to microbe surface, and plasma membrane ultimately encloses it (zipper or Velcro mechanism) The engulfed microbe is now inside phagocytic vacuole called phagosome Granules (lysosomes) fuse with phagosome creating a phagolysosome where killing components work on microbe. Often fusion occurs simultaneously with engulfment o Lysozymes are in lysosomes PMN first phagocyte to act in acute inflammation, and the primary phagocyte; has two types of lysosomal granules that contain chemicals and enzymes to kill microbes Primary Azurophilic granule (take up blue dye when stain) o Myeloperoxidase (oxygen dependent killing); other oxygen dependent killing (production of hydrogen peroxide and other toxic oxygen products) o Lysozyme (degrade peptidoglycan) independent of oxygen; cationic antimicrobial proteins Secondary Specific o Lactoferrin (iron chelator) pulls iron away from microbes o Lysozyme obviously everywhere (ubiquitous) o Acid hydrolyses for killing or degrading microbes o **Note all this normally occurs in phagolysosome so doesn t affect human Macrophage enters later in acute inflammation; macrophage phagocytosis and killing are similar to that of the PMN

6 Macrophage also able to engulf and degrade damaged host tissue sanitary engineers scooping up waste Most resident macrophages are down-regulated ; surface receptors are not mobile; have low level of engulfment During (mainly chronic) inflammation macrophages become larger; have more enzymes; mobile Ab and C3b receptors; increased phagocytic function After presents processed Ag to T cells, they produce cytokines with feedback to activate macrophages fully Fully activated angry killer macrophages is more able to kill engulfed microbes Unlike PMN s, macrophages can become activated with greater killing power, especially for intracellular microbes; macrophages can present processed Ag to T helper cells (adaptive immunity) **SIDE NOTE another way besides interaction with Ab and C3b = TLR receptors on macrophages and dendritic cells can also recognize lots of things ADAPTIVE/SPECIFIC IMMUNITY Cellular and Humoral (Ab) response to specific Ags achieve cell and AB SPECIFICITY (one cell for one antigen). Responses to virtually ALL Ag results in cell and Ab DIVERSITY (all the different Ab can make in response to different Ag) Ag presenting cells (APC) present processed Ag to specific T cells (via specific T cell receptor (TCR) to elicit adaptive (humoral or cellular) immunity Much of the interaction between APC and T cells takes place in the secondary lymphoid organs: spleen, lymph nodes, MALT, GALT Two types of presentation: exogenous and endogenous o Exogenous (from outside): macrophage, dendritic, and B cells have MHC2 (major histocompatibility component) on their surfaces. MCH2 is folded to form a groove to present (non-specific in presentation) exogenous processed (mainly bacterial or fungal) Ag (from engulfed pathogens) to specific T(h) cell via TCR o Endogenous: all host nucleated cells have MHC1 (formed in endoplasmic reticulum) which is the major determinant of SELF. Virally infected (or tumor) cell present processed (endogenous) Ag via MCH1 to the cytotoxic T lymphocyte (CTL) via TCR. This signals the CTL that the cell is foreign,

7 non-self ; CTL then kills the virally infected (or tumor) cell. T(h) cells also required. ADAPTIVE/SPECIFIC IMMUNITY T CELLS There are two major types of T cells T HELPER T(h) CD4 cells make up 65% of the peripheral (circulating) T cells. After interacting with foreign Ag, they become activated, make cytokines; help activate B cells and T(c) CYTOTOXIC T T(c) CD8 cells comprise about 35% of peripheral T cells. T(c) will become an activated cytotoxic T lymphocyte (CTL); these kill (like NK cells) virally infected or tumor cells. They recognize foreign Ag presented via MHC1. Kill by similar mechanisms as NK cells o Perforin polyperforin polymerized inserted in cell membrane, allowing water to enter the cell to lyse it osmotically o Granzymes T(c) enzymes enter infected or tumor cell (often after polyperforin inserted) and activate other enzymes which ultimately induce the virally infected cell to undergo apoptosis (programmed cell death) o Tumor necrosis factor (TNF) make by macrophages and INF alpha and beta also help destroy virally infected or tumor cells There is LITTLE or NO experimental evidence for Suppressor T cells; down-regulation of the immune response is most likely due to cytokines produced by T(h) or T(c) There are surface glycoproteins on T(h) and T(c) that allow cell/cell interaction with APCs and transmission of activation signals CD4 found on T(f) associates with MHC2 on APC, and strengthens interaction between MHC2 and TCR (specific T cell receptor for specific Antigen) CD8 found on T(c) associates with MHC1 molecule on infected cell and strengthens interaction between MHC1 and TCR Specific TCR will bind to MHC1 or MHC2, receive presented Ag, and become an activated T cell (specific) TCR have a constant and a variable region: variable region ensures SPECIFICITY for specific Ag Diversity all the host s TCR can recognize 10^15 presented antigenic epitopes. This happen PRIOR to Ag encounter; by somatic mutation, DNA splicing rearrangement (recombination), mrna splicing

8 In each case, a SPECIFIC set of T cells is activated by the SPECIFIC Ag (CLONAL SELECTION) one that is needed will multiply Self-reacting cells are eliminated during development. This is not perfect, as autoimmune conditions occur Super Ags NONSPECIFIC, uncontrolled activation of many T cell clones damaging to host via cytokine storm There is an abnormal nonspecific activation of T cells by presence of a SUPER Ag (staphylococcal TSST-1 toxin [toxic shock syndrome]; AIDS virus) Super Ag bind Ag presenting MCH2 to TCR of ALLT cells regardless of their specificity binds to outside AND NOT in Ag-presenting groove ADAPTIVE/SPECIFIC HUMORAL IMMUNITY B CELLS B cells have three major functions Present Ag Differentiate into Ab producing plasma cells (humoral response) Differentiate into memory B cells effect memory for humoral response Elaboration B cells present processed exogenous Ag via its MHC2 to the specific T(h) TCR B cells will effect Ab production (humoral response) o Ig is the surface structure on B cells for Ag recognition-start of humoral response " Each B cells has about 10^5 Ig (Immunoglobulin) molecules with identical SPECIFICITY " Both IgM and IgD Ig classes on B cell " Each B cell is specific for one Ag epitope " Because the Ig B cell receptors are bivalent (two Ag binding sites per molecule), the Ig can be cross-linked, and T independent Ag can effect the B cell directly o T-independent Ag (weaker, not so long lasting) ONLY B cell is needed to Ab formation " These Ags are usually polymers (capsular polysaccharide) present large number of identical Ag epitopes to specific B cell " Activates the specific B cell (CLONAL SELECTION) to divide and differentiate into an Ab producing plasma cell " Only IgM produced (no switching to IgG)

9 DAY 3 2/03/2015 9:53 AM ADAPTIVE/SPECIFIC HUMORAL IMMUNITY Ab Antigens (Ab) Antibody generators Epitope: antigenic determinant site; many Ag have several different epitopes (not all epitopes stimulate Ab production) Immunogen stimulates Ab production Hapten not immunogenic, but can bind with Ab Self vs. Nonself making Ab against self is damaging to host (immunopathologic) or autoimmune many of these cross reacting Ab/cells are eliminated as B and T cells mature (in bone marrow and thymus prior to Ag exposure) Basic types of specific humoral immunity (where Ab comes from) Natural active Ab formed in body following infection with the etiological agent (e.g. measles) Artificial active Ab formed in body following immunization with a vaccine (e.g. MMR vaccine) Natural passive preformed Ab passed from mother to fetus or neonate (e.g. fetus gets immune mom s anti-measles Ab)! in order for fetus to get Pertussis Ab, immunize mom in 3 rd trimester Artificial passive Ab produce in another animal and given to individual (e.g. immune globulin given after exposure to Hepatitis A)! only given when needed after exposure (horses or pools of human serum) Ab Production Polyclonal heterogeneous (Abs with different specificity) b/c formed by DIFFERENT plasma cell clones (animal immunization to complex Ag, e.g. typhoid vaccine) Monoclonal homogenous (identical specificity) b/c formed from SAME plasma cell clone ( Hybridoma technology single epitope Ag yields single Ab from plasma cell clone) Ab structure Ab are Immunoglobulins (Ig) Immunoglobulins (Ig) large globular glycoproteins found in serum (gamma globulin fraction) and tissue fluids; 5 basic classes: IgA, IgG, IgM, IgD, IgE (most is known about IgG) Heavy (H) and Light (L) chains each containing constant and VARIABLE regions define the two functional parts of Ig molecule