Microbiology Exam 1 Review

Lectures 1 & 2: Introduction Lymphatic System: o Lymphatics drain all tissues and transport lymphocytes and antigens from the tissues to organized lymph where the lymphocytes can interact with the antigens. o As blood circulates, the plasma seeps into surrounding tissues via capillariesmost returns to the blood, but some (now called lymph) flows from the connective tissue into lymphatic vessels and eventually to the left Subclavian vein. o One-way valvesalso, lymph is not pumped, it moves via muscle/body movement. o Primary Lymphoid Organs: lymphocyte development and maturation: Bone Marrow: Major site of hematopoeisis B Cells develop and mature here o Requires stromal cell contact, stromal cell-secreted cytokines, and selection Thymus: Bi-lobed organ above the heart T cell development and selection occur here DiGeorges Syndrome: congenital birth defect where the thymus fails to develop. Therefore, there is an absence of circulating T cells, and an increased number of infections. Thymic Atrophy: the thymus atrophies with age, and is at its largest at puberty. o Secondary Lymphoid Organs: environment for lymphocyte-antigen interaction Lymph Nodes: Encapsulated bean-shaped cells Contain networks of lymphocytes, macrophages, and Dendritic cells FIRST lymph tissue to encounter an antigen As lymph travels through lymph nodes, antigens are trapped by Phagocytic and Dendritic cells Spleen: Large and ovoid organ in upper left quadrant of abdomen Contains red and white pulp Adapted to filtering blood and trapping blood-born antigens MALT: Mucosal Associated Lymphoid Tissue Loose clusters of lymphoid tissue Appendix, Peyers patches, tonsils

CALT: Cutaneous Associated Lymphoid Tissue Loose clusters of lymphoid tissue

Hematopoiesis: o Formation and development of red and white blood cells from stem cells. o Begins in the yolk sack, then the fetal liver and spleen o Bone marrow is the final, major site. o This process initiates in the bone marrow from pluripotent hematopoietic stem cells (PHSC). These cells are self-renewing and can develop into any type of blood cell. These cells represent only 1-2% of total bone marrow.

Clinical Importance of stem cells: BM transplants: must carefully match the MHC to prevent graft rejection Graft vs. Host Disease (GVHD) Gene Therapy: SCID (bubble boy) ADA deficiency (replace the gene in HSCs) CD Markers o Proteins present on the surface of cells that help us identify the type of cell

o o

Common CD Markers: CD4: T Helper Cells CD8: Cytotoxic T Cells CD3: All T Cells CD 14: Macrophages CD 16, CD56: Natural Killer Cells CD19-CD21: B Cells CD34: Stem Cells Lymphoid Cells: continuously circulating, non-phagocytic, considered nave until they come into contact with their first antigenthen they grow and differentiate into memory and effector cells. o B Lymphocytes Develop in the bone marrow They have membrane-bound ligands that serve as receptors for antigens They can develop into plasma and memory cells All of the clonal progeny from a given B cell secrete antibodies with the same antigen binding specificity o T Lymphocytes Develop in the Thymus Have TCR receptors for antigensTCR, however, only recognizes antigen in association with MHC 2 Major subsets: T Helper Cells (CD4) o Recognize exogenous antigens associated with Class II MHC o Become activated after they recognize an antigen-class II MHC on an antigen-presenting cell (APC) o Once activated, these cells divide and give rise to clones which secrete various cytokines, and also play a major role in B and Cytotoxic T Cell activation o T Helper Cell 1: activates Cytotoxic T cells and macrophages o T Helper Cell 2: activates B cells Cytotoxic T Cells (CD8) o Recognizes endogenous antigen associated with Class I MHC o Become activated after they recognize an antigen-class I MHC complex o Secrete few cytokines o Hitman: directly recognizes and kills target cells (virus-infected and intracellular bacteria o Natural Killer Cells No B or T Cell markers, no specific antigen receptors. Perform Cytotoxic activity against a wide array of tumors

Receptors for antibody-ADCC o Chediak-Higashi: autosomal recessive diseaseno NK cells. There is therefore an increased incidence of lymphomas in the patient. Monocyte/Macrophages o Monocytes Exist in the blood Develop in the bone marrow and then enter the blood to further differentiate into Monocytes. Circulate in the blood for 8 hours, then migrate to tissue and differentiate into specific tissue macrophages o Macrophages Main function is phagocytosis, this activity can be enhanced by T Helper Cell cytokines Activated macrophages are more efficient at eliminating pathogens than resting macrophages because: Increased phagocytic activity Increased ability to activate T Helper Cells Higher level of Class II MHC on the cell surface o Tissue Macrophages Alveolar macrophages: lung Histiocytes: connective tissues Kupfer Cells: liver Mesangial Cells: kidney Microglial Cells: brain Neutrophils o Granulated cytoplasm, stains with acidic or basic dyes o Multi-lobed nucleus o Form in the bone marrow, then are released into the blood, migrate for 7-10 hours, then plant in tissue and live for 3 days o Bone marrow releases Neutrophils in response to infection Leukocytosis: Excess release of leukocytes (in this scenario, Neutrophils). Body is fighting disease and begins to release immature leukocytes because the bone marrow has run out of mature ones. Good way to test for disease. o First at the site of inflammation Eosinophils o Granulated cytoplasm stains with acidic dye o Bilobed nucleus o Phagocytic, major role against parasites o The secretion of eosinophilic granules results in damage to the parasite membrane Basophils

o Granulated cytoplasm stains with basic dye o Lobed nucleus o Non-phagocytic o Deals with the release of pharmacologically active substances contained within granules o Allergies Mast Cells o Precursors are formed in the bone marrow and released into the blood. They do not differentiate until they leave the blood and enter the tissue. o Allergies Dendritic Cells o Express very high levels of Class II MHC, therefore, are better APCs than macrophages and B Cells o After capturing antigen in the tissue, these cells migrate to the blood or lymph and circulate to various lymphoid organs in order to present antigen to T Cells Tissue Dendritic Cells o Langerhans Cells: epidermis and mucous membranes o Interstitial: most organs o Interdigitating: T cell zones of secondary lymph tissue and thymic medulla o Circulating: blood o Follicular: exclusive to lymph nodes (B Cell Rich) Do not express Class II MHC Do not function as APCs for TH cell activation Bind Ag-Ab complexes and retain them for weeks-years Role in formation of memory B cells

Lecture 3: Complement What is complement? o A number of blood proteins produced by the liver, spleen and macrophages primarily. Originally discovered because of their ability to lyse antibody-coated RBCs when activated. o Components of complement consist of proteins C1-9, factors B, D and P, and numerous inhibitors of the pathway. o Some factors are cleaved into proteolytic fragments. For example, C3 cleaved into C3a and C3b. The b fragment is usually the larger and has the biological activity. 3 Pathways o Classical, Alternative, Lectin o When activated, all 3 pathways: Induce inflammation Lyse certain infectious agents

Stimulate B cell production Opsonize infectious agents Clear immune complexes

o Complement Activation: o Lectin: Mannan binding lectin binds to CHO on bacteria o Classical: Antibody and antigen (immune complexes) IgM and IgG o Alternate: spontaneous lysis of C3, if it binds to bacteria Complement Effectors: o Opsonization: Bacteria and other cells are delivered to phagocytes for destructionthe molecules that aid in this process are called opsonins. Opsonins: IgG, C3b, iC3b, C4b Occurs via complement receptors

Clearance of Immune Complexes Immune Complexes (Ag and Ab complexes) are insoluble lattices They trigger inflammation and type III hypersensitivity reactions High levels of C3b disrupts immune complexes, making them soluble C3b on immune complexes can bind to the C1 receptor on rbcs and be taken to the liver and spleen where they can be removed by the professional phagocytes

Anaphylatoxins C3a and C5a split products diffuse away from the site of complement activation Can cause degranulation of mast cells and basophils without IgE Result: MAJOR Role in inflammatory response C5a is a major chemotactic protein for inflammation Increase vascular permeability, stimulate phagocytosis

 o Pore forming molecules C5-C9 Lyses the cell Critical for protection against Neisseria infections. So a deficiency in C5-C9 would result in an increase in Neisseria infections. What goes up must come down o After the threat, Complement inhibitors turn off the response. o C1INH dissociates C1r or C1q Deficiencies of Complement and its Regulatory Proteins MAC

C1q, C1r, C1s, C4, C2 immune complex diseases, infections with pyogenic bacteria Alternative pathway Factor B, properdin Neisserial infections Classical Pathway Both pathways C3 Recurrent bacterial infections, immune complex disease Recurrent meningococcal and gonococcal infections Overuse of C1, 4, 2 Edema at mucosal surfaces

C5, C6, C7 or C8 Deficiencies in complement regulatory proteins C1-INH (hereditary angioedema)

Lecture 4: Adaptive (Acquired) Immune Response Lag time for the immune response to begin is about 2 weeks after exposure. Then, there is an immune response from IgM. The next exposure to that same antigen has no lag time, and a much more robust response from IgG. Characteristics of Adaptive Immunity: o Diversity: Immune system has the diversity to deal with the unlimited array of microbes we deal with on a daily basis o Specificity: Given the amount of diversity our immune system deals with daily, it MUST have specific ways to deal with each different entity o Memory: The first contact with an antigen imprints some information about that antigen and imparts memory This is why the second response is better, faster and more robust. Memory is the basis for immunization. o Self Vs. Non-Self Recognition The immune system can recognize one antigen and distinguish it from another. During ontogeny, the immune system must learn the difference between self and non-self entities The result of not being able to determine self from non-self is autoimmunity Cellular Components of Acquired Immune Response o B Cells Humoral Immunity. This basically means antibodies. Protection in the blood that can protect you at all bodily surfaces. Mainly focuses on extracellular bacteria. o T Cells Cell Mediated Immunity. These cells secrete cytokines and are involved in killing things. Also help mediate intracellular dilemmas. B Cells o Develop in the bone marrow o Terminally differentiate in secondary lymph organs (lymph nodes, spleen, MALT, CALT) to plasma cells. o Plasma cells secrete large amounts of antibody that bind antigen and help clear foreign substance from the body. o Once a B Cell binds an antigen at a receptor, it has the option to become a plasma cell and secrete antibodies, rather than remain with membrane bound antibodies. Antibody o Definition: an antigen-binding protein that is secreted from plasma cells Aids in: o Opsonization o Complement o ADCC (antibody dependent cellular cytotoxicity) o Links Innate to acquired immune system

T Cells o Made in the bone marrow, but complete differentiation in the Thymus. They recognize antigens just like B cells and antibodies can. o T Cells must also recognize antigen on the surface of other cells that is in association with MHC. o T Helper Cells (CD4) Key role is in adaptive immune response They bind antigens with class II MHC They also produce cytokines that direct the immune system Interferon-y is a cytokine that directs macrophages to kill intracellular bacteria IL-4 and IL-5 are cytokines that help B cells to make antibodies o Cytotoxic T Cells (CD8) Directly lyse and kill viral infected cells They bind to antigens with Class I MHC Can kill cells via direct lyse, or apoptosis Apoptosis: Tc cell binds to Fas molecule on the surface of the viral infected cell. This sends a signal to the cell to kill itself using apoptosis. A calcium dependent endonuclease is then activated and chops up the cellular DNA

Lectures 5 & 6: Humoral Immunity (B Cells & Antibodies) Another name for antibody is immunoglobulin Antibody Structure: o 2 identical heavy and 2 identical light chains o Both chains have variable and constant regions o Antigens will bind to the variable region. The constant region is more responsible for biological activity. o Isotypes are the IgM, IgG, etc. o The hinge region gives the antibody flexibility to reach out and grab antigen

o Antibody Gene Rearrangement

Heavy Chain: 5 classes: ,,,, and (determines the isotype of the antibody) V, D and J segments Are located on the same chromosome Pick one V, one D, and one L to make up the variable region of the heavy chain. Chromosome 14 o Light Chain: 2 classes: and V and J segments Located on different chromosomes Pick one V and one J to make up the variable region of the light chain Chromosome 22 is light chain Chromosome 2 is light chain Ig Gene Rearrangement o The heavy chain rearranges first, then the light chain rearranges. o Recombination Activating Genes (RAG) Recombination is catalyzed by a VDJ recombinase, which is a product of RAG-1 and RAG-2 genes. Recombinase is responsible for the rearranging, and only exists in B and T cells. o Heavy Chain Rearrangement: Recombinase picks a D and a J segment, then picks a V segment Next, still in the nucleus, transcription and splicing occur to form mRNA In the cytoplasm, translation occurs for the final protein product: specific Ig heavy chain The heavy chain then waits in the cytoplasm for the light chain because it cannot function without it. o Light Chain Rearrangement: Recombinase picks a V and a J for the chain Next, still in the nucleus, transcription and splicing occur to form mRNA In the cytoplasm the protein is then translated The light chain then finds and joins the heavy chain in the cytoplasm o DEFECTS in gene rearrangement: SCID: Severe Combined Immuno Deficiency Disease Omenn Syndrome (recombinase disorder) Deletion of RAG-1 or RAG-2 Missense Mutation: RAGs only have partial activity N-Nucleotide Addition o Occurs via the enzyme TdT

TdT is a very random type of enzyme (as opposed to DNA polymerase). After gene rearrangement, there is a hairpin structure that exists. In order to join new segments, TdT cuts randomly in order to seal things together. Adds another level of diversity. o TdT is very important in looking at cancers and B and T Cell lineages. TdT is present very early in the development of these cells, therefore treatment for cancer depends on the state and development the cells are in (which can be seen from presence of TdT). Allelic Exclusion o B Cells are diploid, however a given B cell will only expressed rearranged Heavy Chain genes from 1 chromosome, and Light Chain genes from 1 chromosome. This ensures antigenic specificity. o When a B cell has two specificities, there is a major problem because in selection in bone marrow, only one receptor is tested. So technically, autoreactive receptors could be passed into the body because they are not tested due to the presence of other receptors that were tested on the cell. Immunoglobulin Classes/Isotypes o IgG: Gamma heavy chain o IgM: Mu heavy chain o IgA: Alpha heavy chain o IgE: Epsilon heavy chain o IgD: Delta heavy chain IgG o Predominant antibody in serum, HIGH AFFINITY antibody that predominates during the second immune response o Only antibody that can Opsonize, adcc, and cross the placenta o Also activates complement o 4 isotypes, 1-4, named in decreasing concentration in serum. IgM o ALWAYS MADE FIRST, FIRST ANTIBODY MADE o Secreted as a pentomer when differentiated o Activates Classical Complement o HIGH AVIDITY, LOW AFFINITY o Presence suggests first exposure IgA o Mucosal o Dimer, isotypes 1 and 2 o Secretory piece is the polyIg receptor stolen from the cell membrane that sterically hinders other enzymes in the mucosa from breaking down IgA IgE o Mast and basophils o Parasites and allergies o Type I Hypersensitivity

IgD o Membrane bound in mature B Cells o Antigen receptor on B cell B Cell Development and Activation o Antigen Independent In the bone marrow B cells develop from hematopoietic stem cells though a series of defined intermediates o Antigen Dependent In the periphery Development of mature B cells to memory and plasma cells B Cell Development o Pro-B stage: Stromal Cells and TdT o Pro-B Pre-B Stage: IL-7 and RAG-1/2 o IgM is present at immature to mature B o IgD is present at mature B o B Cell Markers: CD 19, 20, 21 o Anergy: if a potential autoreactive cell is released it will be turned off B Cell/Pre B Cell Receptors o Pre B cell receptors wait for signals, test functionality, and then receive a signal to turn into immature B cells Antigen Dependent B Cell Development o Nave B cells are activated when they come into contact with antigen, and can either differentiate into Memory B Cells or Plasma Cells that secrete antibody o Occurs in the periphery o Includes responses to T Cell Dependent and Independent antigens T Cell Dependent/Independent Antigens o Dependent: require direct contact of a B Cell with a Helper T Cell in order to activate the B Cell (CD40-CD40L interaction). Can also develop IgG. B Cell binds antigen, engulfs and chews it, then presents it to the T Helper Cell via CD40-CD40L, then is able to make IgG, IgA, IgE. o Independent: does not require T Cells, and is much weaker, and can ONLY make the antibody IgM. This is because of the lack of class switching that is dependent on T Helper Cell cytokines. Also, no memory response. Transduction of activating signals: o When antigens bind to the B cell receptor, there is a crosslinking of B cell receptors. Most importantly: Ig alpha and beta are required for this. o SYK is B Cell specific, if it is turned on all the timeCANCER B Cell Co-Receptors o Lower the threshold of antigens needed to initiate B Cell response o CD-21 is an EBV receptor

o 3 protein complex: CD19, CR2, TAPA-1 Activation Signals o Nave B Cells are in G0 o Competence Signal: G0-G1 o Progression Signal: G1-Scell division and differentiation o The T Cell now cranks out cytokines that tell the B Cell it is alright to switch classes and proliferate. B Cell Activation o TD Ags require 2 competence signals because the first signal generated by binding Ag is weak. o The second signal is the binding of CD40 on the B cell with CD40L on an activated TH cell. o After both competence signals, the B cell will begin to express cytokine receptors. o After binding to an activated TH cell, the TH cell will secrete cytokines that bind to the B cell and give it the progression signal Relevance of B Cell Activation o Goal is to push the cell through the cell cycle o Proteins such as TK play an important role in cell cycle regulation o If B Cells were constitutively activated, there would be cancer. T Helper Cell Role in Humoral Response o Formation of the B/T conjugate (1st competence signal) o Contact dependent help mediated by CD40-CD40L (second competence signal) o Progression signal induced by T Helper Cell Cytokines (IL 2, 4, 5) Ag Dependent B Cell Development

o Affinity Maturation o Higher affinity antibodies are positively selected, lower affinity antibodies are signaled to die via apoptosis. o Mutations will occur in the already rearranged variable regions The new antibodies produced can increase or decrease affinity

X Linked Hyper IgM Syndrome: T Helper Cells do not express CD40L o (IL4IgE and IgG1) o (IL5IgA) Before antigen contact, only Mu and Delta can be used, after, all can be used. Plasma and Memory B Cells o To differentiate into plasma cells, IL-1 and CD23, produced by FDCs, deliver the signal o To differentiate into a memory B cell, the activated B cell must bind to an antigen complex bound to or secreted from the FDC membrane

Lecture 7: Major Histocompatibility Complex (HLA) Chromosome 6 o Class I: A B and C alleles Presents antigens to Cytotoxic T cells Responsible for viral, endogenous. Alpha1 and 2 make the antigen binding cleft Beta2 microglobulin is required for proper folding of alpha chain o Class II: DP, DQ, DR Presents antigens to T Helper Cells Responsible for bacterial, exogenous. APCs including macrophages, B Cells, and Dendritic Cells Alpha1 and Beta1 form the antigen binding cleft Antigen Processing o Process by which antigens get loaded into the HLA and presented to Cytotoxic T cells or Helper T cells o Peptides come in 2 types: Endogenous: intracellular pathogens, viruses, bind to Class I Exogenous: extracellular pathogens & bacteria, bind to Class II Endogenous Class I MHC Antigen Processing o Proteosomes cut up the antigens and prepare them for MHC attachment o TAP1 and TAP2 provide a tunnel from the cytoplasm to the ER for the cut up antigen o Antigen is presented to the surface for Cytotoxic T Cells via golgi vesicle transport Exogenous Class II MHC Antigen Processing o APCs can express class I and II o They prevent endogenous antigens from binding to Class II with Invariant chains o Antigen is taken into the cell via phagocytosis, brought to the Class II in the ER, and then brought to the surface via exocytosis Cytokine and Viral Regulation of HLA Expression o IFN-y and TNF can increase the expression of Class II MHC. o Certain viruses also increase MHC expression (but it can also be decreased) o CMV proteins can bind to Beta2 microglobulin to prevent the proper assembly of Class I MHC molecules

Lecture 8: Cytokines Cytokine Secreted by: Target cell or tissue TH cells Hepatocytes hypothalamus Activity

IL-1

Monocytes macrophages, B cells dendritic cells, endothelial cells TH1 cells

Costimulatory Acute phase protein induction Fever

IL-2 (SKID)

Activated TH cells and CTLs Hematopoietic cells B cells

Proliferation, enhanced killing Growth factor

IL-3

TH cells

IL-4

TH2 cells

Activation, class switching to IgE + IgG Activation, class switching to IgA Differentiation into plasma cells and Ab production Acute phase protein induction Fever Differentiation

IL-5

TH2 cells

B cells

IL-6

Macrophages, TH2 cells

B cells Hepatocytes hypothalamus

IL-7 Bone Marrow /thymic stromal cells Macrophages B and T cell precursors

IL-8

Neutrophils

Chemotaxis

IL-10

Macrophages

Macrophages, TH cells

Down-regulation of proinflammatory response Promotes differentiation of TH cells into TH1 cells Inhibits viral replication Activation Induces cytokine secretion, cachexia Granulocyte development Granulocyte and macrophage development

IL-12

Macrophage

TH cells

IFN- and IFN- IFN- TNF-

Leukocytes and fibroblasts TH1 Macrophages

Infected cells

Macrophages Inflammatory cells

G-CSF

Macrophages and TH cells Macrophages and TH cells

Bone marrow precursors Bone marrow precursors

GM-CSF

Interferons o Alpha and beta are anti-viral o IFN alpha is used to treat HBV, HCV, and HHV-8 o IFN beta is used to treat multiple sclerosis o IFN gamma is used to activate macrophages to stimulate intracellular killing and inhibits the T Helper 2 response Used to treat chronic granulomatous disease TNF o Can kill tumor cells o Alpha: made by macrophages, T Cells and fibroblasts o Beta: made by activated T and B Cells

Lectures 9 & 10: Cell Mediated Immunity T Cell Receptor (TCR) o Is NEVER secreted o Must recognize antigen with MHC, not free antigen CD3 o Signaling molecule for T Cells (like Igalpha and Igbeta in B cells) o Non-covalently attached to the T Cell Receptor o Responsible for T Cell activation and differentiation TCR Gene Rearrangement o Occurs in a similar manner to Ig gene in B Cells o Beta Chain is like the heavy chain o Alpha chain is like the light chain T Cell Development o Development and maturation occurs in the thymus o Pro-T Cells are released from the bone marrow and migrate to the thymus and undergo either positive or negative selection Thymus o Cortex: has immature lymphocytes, where selection takes place, and has Nurse cells o Medulla: has cells that survive selection, and more mature thymocytes T Cell Development o When Pro-T cells leave the bone marrow, they cannot recognize antigen. They undergo differentiation in the thymus o Intimate association with thymic stromal cells is required for development. o Ontology CD4 stabilizes MHC II/TCR interactionT Helper Cells CD8 stabilizes MHC I/TCR interactionCytotoxic T Cells Thymic Selection o Positive: If you bind self MHC you go on to negative selection, if not, you die. o Negative: If you bind self MHC or self ag+mhc TIGHTLY, you die, if you bind and move on, youre good.

o T Cell Activation o T Cells that have passed selection and matured in the Thymus, leave and enter the periphery in search of antigens. They are nave until they actually encounter antigen. o Initial contact with antigen is in secondary lymphoid tissue (spleen, lymph nodes, etc) o The interaction of T Cell Receptors with antigen+MHC initiates activation, but other signals are required also APCs o Must present antigen to T Helper Cells with co-stimulatory molecules. o B7, present on Dendritic Cells, the ONLY CELLS that can activate nave T Cells Macrophages can activate nave T cells, but only when activate by IFN-y B Cells can activate nave T Cells after contact with antigen. T Helper Cell Activation o T Helper Cell and APC exchange mutual activation signals o Cytokines IL4, IFN-y from the T Helper Cell activate B cells and macrophages Macrophages secrete IL1, IL6, and TNF alpha o New cell surface markers T Helper cell expressed IL2R and secrete IL2 Co-stimulatory molecules CD28(Th) and B7 (B) Adhesion molecules Signaling in T Cells

o Superantigens o Bind to regions of TCR and MHC outside of the peptide binding cleft. This means less specificity and activation of numerous T cells o Endogenous o Exogenous Cell Mediated Immunity o Extracellular infectious agents are cleared by antibodies, complement and phagocytes. o Intracellular infectious agents require cell mediated immunity o The goal is to destroy the intracellular agent by destroying the host cell that harbors it o Viruses ALWAYS require CMI for effective clearance o Intracellular bacteria and parasites induce delayed type hypersensitivity via Th1 and macrophage activation

T Cell Subsets and Interleukins

TH subsets
divided into 3 functional subsets based on the cytokines they secrete
TH0
secrete IL-3, GM-CSF, IFN-, IL-2, IL-4, and IL-5 can differentiate into TH1 or TH2

TH1 (Cell Mediated Immunity)

delayed type hypersensitivity helping the development of CD8+ TC cells produces IFN-, IL-2, TNF-, IL-3 and GM-CSF down regulates TH2 response via IFN-

TH2 (Humoral Immunity)

help produce antibody class switching proliferates eosinophil and mast cell precursors produces IL-4, IL-5, and IL-10 downregulates TH1 responses via IL-10

Delayed Hypersensitivity Reaction o Macrophages are often targets for infection with intracellular pathogens

Like humoral immunity, CMI requires TH cell help (TH1-IFN- and IL-2) Effector cells CTL (cytotoxic T lymphocyte), CD8+

CMI

class I MHC restricted and endogenous peptides clonal expansion of CTLs and full effector function requires IL-2 from an activated TH1 cell perforin
membrane punching, pore forming molecule

granzymes
enzymes that damage the target cell

cytokines
IFN- and TNF- are secreted by CTLs induce metabolic changes in target cells that lead to apoptosis

Fas and FasL

CTLs express FasL and target cells express Fas interaction between Fas and FasL causes apoptosis

Effector Cells in Cell-Mediated Immunity

Effector Cell CTL CD Marker TCR CD3 CD8 CD2 CD16 CD56 CD2 Ag Recognition Specific, TCR MHC Recognition Required for Killing Yes, class I Effector Molecules Perforin, granzymes, TNF-, IFN- As above

NK cell

ADCC: specific by IgG; otherwise recognizes lectins

No, MHC I recognition inhibits

Effector Cells in Cell-Mediated Immunity

Effector Cell Macrophage CD Marker CD14 Ag Recognition Nonspecific MHC Recognition Required for Killing No Effector Molecules TNF-, Enzymes, NO, oxygen radicals

Lecture 11: Ontogeny Primary Vs. Secondary Lymphoid Tissue o Primary: B ad T Cells are derived here.thymus and bone marrow

o Secondary: B and T Cells respond to antigen herespleen, LN, MALT, BALT, GALT Dendritic Cells o Interdigitating (IDC): only capable of activating nave T cells o Follicular (FDC): Do not express class II MHC, have long dendrites with C and AB receptors, retain immune complexes and help form memory and plasma cells. Secondary Lymphoid Tissues o When antigen enters the body, it goes to Lymph nodes: clear antigen in the tissue Antigens can come in whenever they want, but can never leave There are separate compartments for B and T cells Cortex o Resting B Cells and follicular Dendritic cells o Once contacted by antigen, FDCs and dividing B cells create germinal center Para Cortex o T Cells and IDC (increased Class II) Medulla o Plasma cells that secrete Ab Germ Center: very importantclass switching, affinity maturation, and differentiation into Memory/Plasma cells occur here. Spleen: clears antigen in the blood Antigens are carried by the Splenic artery, deals with blood borne antigens. Red and white pulp, we are concerned with white. B Cells are in the marginal zone T Cells surround the artery in the PALS MALT: clears antigen in GI or Respiratory Response to Antigen o Innate immune system binds to antigen and delivers it to the draining lymph node. (antigens can also enter the lymph node by themselves) o Activation begins in the lymph node. Presentation of antigens to T cells (primary activation)looking for activation so we can get the effector cells (for example, effector B cell would be plasma cell that makes antibodies). o Effector cells leave via lymphatics and enter circulation. They are traveling to the site of infection, and they know where to go because of innate inflammation. Lymphocyte Trafficking o Flow of B and C Cells though secondary lymphoid tissue is directed by CAMs and specialized endothelial cells called HEVs. o Nave lymphocytes constantly move throughout the body because they need to find antigen or else they will die.

Memory cell does not have to do this because it remembers and knows where the antigen will be. More restricted pattern of circulation. o Lymphocytes change their circulation pattern by expressing different proteins on their surfaces. (CAMs which recognize HEVs in lymph nodes) o CAMs on the lymphocytes recognize CAMs on the endothelial cells Transmigration o Rollingattachmentmigration.

o o

Leukocyte Adhesion Deficiency (LAD) Recurrent bacterial infections, impaired wound healing Leukocytes cannot leave the blood vessels and enter the tissue because of CAM deficiency/malfunction Really high white cell counts during infection (leukemia high).bone marrow is still getting the signal that it needs to make more cells, but the problem is that they cannot get to where they need to be. One of the first signs of LAD is Umbilytis. Really infected umbilical cord remnant. Swelling, no puss (no white cells) Collection of lymphocytes, plasma cells and phagocytes Lung and intestine Plasma cells secrete large amounts of IgA M Cell: capable of taking up bacteria and pathogens, but does not kill like a macrophage would.it just samples things. It basically shuttles cells to a cluster of immune cells such as dendritic cells, B and T Cells After activation, lymphocytes enter the lymph, go through MLN and thoracic duct, then they pass from the blood back into the lamina propria and become IgA secreting plasma cells

MALT o o o o

************Lecture 12: Immunodeficiency Diseases************* Autosomal Recessive are rare, X-Linked recessive are more common (affects mostly men) Development of Humoral Immunity in Infants o Most immunodeficiencies do not get diagnosed until after 6 months because of maternal IgG still in the infants system. o Vaccines are not given until after 12 months.

o Phagocytic Deficiencies o Phagocytosis is a primary means for removing extracellular bacteria from the body, therefore these defects reveal an inability to clear infection. o Could be from a reduction in the number of phagocytes, or decrease in their function o Hallmark: Recurrent bacterial or fungal infections, anywhere from mild skin ro life threatening systsemic Frequent infections with: S. aureus, S. pneumonia, E.coli, Pseudomonas, Candida, Aspergillus o Chronic Granulomatous Disease Deficiency: NADPH oxidase, failure to generate O2 radicals Symptoms: Recurrent infection with catalase, bacteria, fungi o Leukocyte Adhesion Deficiency Deficiency: CD18 Symptoms: Recurrent infections, failure to form pus, omphalitis o Chediak-Higashi Syndrome Deficiency: Granule Structural Defect Symptoms: Albinism, no NK cells, recurrent infection o G6PD Deficiency Deficiency: enzyme in hexose monophosphate Symptoms: same as CGD o MPO Deficiency Deficiency: Granule enzyme Symptoms: Mild to Non o Job Syndrome Deficiency: T Helper 1 Cells cannot make IFN-y, PMNs dont respond to chemotactic stimuli

Symptoms: Coarse facies, cold abscess, retained primary teeth, increase IgE, eczema Complement Deficiencies o Similar manifest to phagocytic and antibody deficiencies o Hallmarks: defects in Opsonization, lytic activity, clearing of immune complexes o Classical Pathway Deficiencies Deficiency: C1q, r or s, C2, C4 Symptoms: Increased immune complex diseases, increased infection with pyogenic bacteria o Alternative Pathway Deficiencies Deficiency: Factor B, properdin Symptoms: Increased Neisserial infections o Both Pathway Deficiencies Deficiency: C3EARLY IN LIFE Symptoms: recurrent bacterial infections, immune complex disease Deficiency: C5-C8 (MAC)LATER IN LIFE Symptoms: recurrent meningococcal & gonococcal infections o Regulatory Proteins Deficiency: C1-INH Symptoms: Overuse of C1, 2 and 4edema at mucosal surfaces B Cell Deficiencies (Defects in Humoral Immunity) o Recurrent bacterial infection with normal immunity to virus and fungal parasites o Staph and Strep are most common o Bruton X-linked hypo-y globulinemia Deficiency: tyrosine kinase, blocked B Cell maturation Symptoms: Decrease in all classes of Ig, NO CIRCULATING B CELLS, pre-B cells in bone marrow are normal, CMI normal o X-linked Hyper IgM syndrome Deficiency: CD40L on activated T cells Symptoms: increased IgM, no other isotypes, otherwise normal o Selective IgA Deficiency (MOST COMMON) Deficiency: IgA Symptoms: repeated sinupulmonary and GI infections o Common Variable hypo-y-globulinemia Deficiency: unknown Symptoms: Late teen onset, Ig levels decrease over time, autoimmunity increases over time o Transient hypo-y-globulinemia of infancy Deficiency: delayed onset of normal IgG synthesis Symptoms: Detected 5th-6th month, resolves by 16-30th month, pylogenic bacterial infections

T Cell Deficiencies o Can affect both the humoral and cell-mediated response o Very severe, increased viral and fungal pathogens o Selective T Cell Deficiencies: DiGeorge Syndrome: Deficiency: thymic aplasia Symptoms: lots of malformations, decreased T cell number, absent T cell response MHC Class I Deficiency: Deficiency: TAP1 Symptoms: decrease in CD8 T Cells, recurrent viral infection, other things normal MHC Class II Deficiency (Bare Lymphocyte Syndrome): Deficiency: transctiption factor defect, Class II MHC does not express Symptoms: Decrease in CD4 cells, no GVHD, decreased Igs, observed as SCID Combined Immunodeficiency Diseases o Patients susceptible to any type of pathogen o COMPLETE B and T cell deficiencies (SCID) Deficiency: defect in common y of the IL2 receptor (which is present in IL4, 7, 9 and 15), X-linked Symptoms: Chronic diarrhea, skin mouth and throat lesions, opportunistic infections, cells unresponsive to mitogens Deficiency: Adenosine Deaminase Deficiency Symptoms: same Deficiency: Mutations in RAG1 or RAG2 Symptoms: total absence of B and T Cells o Partial B and T Cell Deficiencies Wiskott-Aldrich Syndrome Deficiency: cytoskeletal glycoprotein Symptoms: thrombocytopenia, eczema, immunodeficiency Ataxia Telangiectasia Deficiency: kinase involved in the cell cycle Symptoms: ataxia, telangiextasia, deficiency of IgA and IgE

Lecture 13: Vaccinations Modes of acquisition of specific immunity o Natural Passive: placental transfer Active: recovery from disease o Artificial

Passive: antiserum Active: immunization Passive acquisition o Never activates an immune response. o For example, giving someone antibodies or IgG and fetal immunity, or IgA and breast milk o Used to provide immediate protection, there is no memory response Active acquisition o always activates an immune response o Natural is exposure to a disease and an immune response o Artificial is getting vaccinated and having an immune response o Boosters Whole Organism Vaccines o Attenuated: measles, mumps, rubella, oral polio, VZV, Yellow fever, influenza, vaccinia, ROTAVIRUS, TB, oral typhoid Organism loses capacity to cause disease, but still grows in the host and mimics natural infection. Organisms are grown under abnormal conditions and select mutants for this. Ex: BCG is grown in bile for more than 13 years Advantages: Mimic natural infection 1 dose Humoral and Cell-Mediated immunity Disadvantages: Reversion to virulent form Contaminating viruses Complications similar to the natural disease Measles--encephalitis o Inactivated: cholera, pertussis, plague, typhoid, influenza, polio (IPV), rabies, hepatitis A Kill the pathogen by heat or chemical means so it can no longer replicate in the host. Requires repeated boosters Produces humoral response Purified Macromolecules o Capsular Polysaccharide Vaccines Only IgM response, repeat doses do not booster Pneumococcal vaccine-23 stereotypes Meningococcal Stereotypes A, C, Y and W135, not B o Conjugate Polysaccharide Vaccines

Polysaccharide conjugated to protein antigenchanges the immune response from T Cell independent to T Cell dependent Positive booster response, increased immunogenicity in infants Hib, MCV4, pediatric pneumococcal disease o Toxoid Vaccines Anti-toxoid antibodies will bind to toxin and neutralize its effects Difficult to make in high quantities o Recombinant Antigen Vaccines Any gene for an immunologic protein can be isolated and cloned in bacteria, yeast or mammalian cells HBV, HPV Induces humoral immunity Recombinant Vector Vaccines o Introduce genes encoding major antigen of virulent pathogens into attenuated viruses o Vaccinia, HSV, HIV, o CMI and Abs Timing and Spacing of Vaccines o If received live vaccine Wait 2 weeks before giving antibody o If received antibody Wait at least 3 months before giving vaccine o Inactivated vaccines are generally not affected by circulating antibody to antigen Live vs. Inactivated o Live Injected Vaccines First dose usually provides coverage Second insures seroconversion o Inactivated Vaccines First dose does not provide protection May not develop until 2nd or 3rd dose Antibody titer wanes over the years o Comparisons Tetanus Ab levels decline Requires boosters Hib Rare in children under 2 Does not require boosters Herd Immunity: you are less likely to get disease if those around you are immune. Adverse Reactions o Local: common with inactivated vaccines, mild and self limiting, within a few hours of injection

o Systemic: common with whole cell or live attenuated vaccines o Allergic: due to a vaccine or vaccine component. Rare and should be reported to CDC Immunosuppressed Persons o Should NEVER receive live attenuated vaccines o Inactivated vaccines can be given, but response may be minimal o Cancer patients can receive live vaccines 3 months after chemo is finished o HIV+ MMR and varicella can be given to the asymptomatic Symptomatic or full blown aids patients should NEVER receive MMR

Summary of Bacterial Vaccines

Organism C. diphtheriae B. pertussis C. tetani H. influenzae Vaccine DTP DTP, DTaP DTP Hib Vaccine Type Toxoid Killed, toxoid Toxoid Capsular Polysaccharide and protein 7 capsular serotypes 23 capsular serotypes Available in U.S. Yes Yes Yes Yes Recommended Vaccine in U.S. Yes Yes Yes Yes

S. pneumoniae

PCV
Pediatric

Yes Yes

Yes Yes

PPSV Adult

Summary of Bacterial Vaccines

Organism N. meningitidis S. typhi Vaccine MCV/ MPV Ty21a Vaccine Type 4 capsular serotypes Live, attenuated Killed Available in U.S. Yes Yes No Recommended Vaccine in U.S. Yes For travelers No longer available in U.S. For travelers No

ViCPS Y. pestis

Capsular polysaccharide Killed

Yes No

B. anthracis

AVA

Toxoid

Yes

For high-risk groups

Summary of Bacterial Vaccines

Organism

Vaccine

Vaccine Type

Available in U.S.

Recommended Vaccine in U.S.

M. tuberculosis
B. burgdorferi Vibrio cholerae (O1)

BCG

Live, attenuated
Osp A flagellar protein Oral: subunit toxoid, attenuated bacterium

No
No No

No
No No

Summary of Viral Vaccines

Virus Rabies Vaccine
Rabavert

Vaccine type Available in US Killed Yes

Recommended

in US For exposed

Influenza Fluvirin Flumist Polio IPOL (salk)

Hepatitis A HBV HPV

Killed live killed

killed

Yes Yes Yes

Yes

Yes Yes* Yes

For travelers Yes Not yet

Havrix
Recombivax HB

recombinant Yes recombinant Yes

Gardasil

Summary of Viral Vaccines

Virus Measles Mumps Rubella Varicella Zoster Vaccine MMR MMR MMR Varivax (Proquad w/MMR) Vaccine type Available in US Live, attenuated Yes Yes Yes Yes
Recommended

in US Yes Yes Yes Yes

Adenovirus
Polio (sabin) Small pox Yellow fever Vaccinia, dryvax YF-vax

Yes (military) Military

No Limited Yes No High risk For travelers

Lecture 14: Diagnostic Principles Brightfield Microscopy: shows mostly bacteria, not viruses o Used for parasites and eggs, Gram-stain, and blood smears

Darkfield Microscopy: used for thin bacteria, spirochetes, Treponema pallidum, Borrelia, Leptospira Phase Contrast Microscopy: Creates 3D image, shows internal details of microbes Fluorescent Microscopy: brightly illuminated against background Electron Microscopy: very small organisms, viral particles Molecular Analysis o DNA Probe DNA-specific probes tagged with fluorescent or radioactive compounds Used to diagnose STDs, CMV, HCV, HIV, HPV, Cancer, type HLA o PCR (RFLP) Amplification of DNA Real time for HIV Mainly used for viruses HIV, HSV, HPV, Hantavirus Also antibiotic resistance MRSA RFLP: DNA is cleaved by restriction enzymes Distinguish HSV-1 from HSV-2, identify the spread of pathogens between points. Salmonella o Western Blotting Used to identify specific proteins THE TEST TO CONFIRM HIV o Microarrays Can test or screen for a large number of organisms DNA, RNA or antibody probes are spotted onto chip Chemiluminesence Serologic Techniques o Precipitation Reactions Ab and soluble Ag interaction forms a precipitate o Agglutination reactions Ab and Ag interaction causes clumping Hemagglutination: blood type groups Bacterial agglutination Passive agglutination: spread pattern of rbcs o RIA and ELISA RIA: competitive binding of radiolabeled Ag and unlabeled AG to high affinity Ab Greater radioactivity means fewer antigens in the sample. Used to detect HBV surface antigen ELISA: similar to RIA but uses enzyme instead of radioactive label o Complement Fixation: Rbc + complement = indicator

+ equals no hemolysis of RBCs (white) Complement inactivated by Ab-Ag complex - equals hemolysis of RBCs (red) Flow Cyometry Study individual cell populations, uses fluorochrome labeled Abs HLA, cancer detections, immune disorder screening Presented as histogram or dot plot Differential analysis: WBC expression of CD4 and CD8

Lecture 15: Hypersensitivity What is Hypersensitivity? o Failure of the immune system o Reactions that are unwarranted and harmful to the host o The immune response itself is responsible for the induction of disease o Requires sensitization and is antigen specific o Classified by Gell and Coombs

Gell and Coombs Classification of Hypersensitivity

Type I
II III IV

Description Immediate
Cytotoxic Immune complexes Delayed

Effectors IgE
IgG and IgM IgG and IgM T cells (48 hrs)

Basic Characteristics o First contact with the allergen does not produce symptoms, but the individual becomes sensitized o Re-exposure to the same antigen elicits a reaction which is highly specific o The severity of the reaction can increase and decrease Type I Hypersensitivity

Induced by certain types of antigens and has all of the hallmarks as a regular immune responsewhat distinguishes it from a normal immune response? Secretion of IgE from plasma cells IgE binds to a high affinity FcR on the surface of tissue mast cells and blood basophils IgE coated mast cells and basophils are considered to be sensitized Future exposure crosslinks the mIgE on the sensitized cells and causes the degranulation of these cells resulting in the release of pharmacologically active mediators o Atopic persons have a genetic defect affecting the regulation of IgE response. This allows non-parasitic antigens to stimulate inappropriate IgE production leading to HS I reactions. o Most allergic IgE responses occur in the mucous . Common Allergens o Small proteins, usually present in dried up particles and then rehydrate upon inhalation and get presented to T Helper cells by APCs o Leads to the development of T Helper 2 cells in response to the increase in IgE IgE o Normal range is 0.1-0.4 micrograms per millimeter o Severely allergic is greater than 1 o The half life of IgE is about 2 to 3 days ,however when bound to an FcR on a mast cell or basophil it can be stable for weeks to months Mast Cells and Basophils o Mucosa and epithelial surfaces, all vascularizing tissues except for the CNS and the retina o Contain granules with pre-formed (primary, immediate) pharmacologically active mediators o Once the FcR has been crosslinked, degranulation occurs in seconds Primary Mediators (immediate) o Heparin, TNF-alpha, proteases, degradative enzymes, inflammatory mediators ECF (late stage asthma) and NCF o Histamine Binds to histamine receptors H1, H2, and H3 Binds to H1 on nearby smooth muscle cells and endothelial cells lining blood vessels. This induces vascular permeability and eventually causes inflammation Smooth muscle contraction: constricts the airways and increases the secretion of mucous Secondary Mediators (late) o IL-4 o PAF: chemotactic for leukocytesactivates Neutrophils, Eosinophils and platelets o Prostaglandins and leukotrienes

Effects are felt after the primary, but are more potent and long lasting

Mast Cell Mediators

Mediators Stored and Released Histamine Effect

Heparin
Eosinophil chemotactic factor A (multiple chemokines)

Smooth muscle contraction, increased vascular permeability Anticoagulant

Chemotactic

Mast Cell Mediators

Mediators Newly Effect Synthesized from Arachidonic Acid Prostaglandin D2, E2, Increased smooth F2 muscle contraction & vascular permeability Leukotriene C4, D4, Increased smooth E4 (lipoxygenase muscle contraction & pathway) permeability Leukotriene B4 Chemotactic for neutrophils
Systemic vs. Organ Specific Anaphylaxis o Systemic: occurs when allergens enter the blood Widespread activation of mast cells, increased vascular permeability, widespread constriction of smooth muscle.

Fluid leaving the blood leads to a rapid drop in blood pressure and anaphylactic shockmany organ systems are damaged because they are not functionally impaired. Mortality is usually associated with asphyxiation due to airway constriction

Systemic Anaphylaxis Most common allergen is penicillinalso, insect stings and peanuts and brazil nuts Treatment: Rapid administration of epinephrine o This stimulates reformation of tight junctions, reduces permeability, prevents fluid loss from blood, decreases swelling, increases BP. o Organ Specific: occurs when allergens effect target organs (most commonly respiratory, GI and connective) Urticaria/Hives o Activate mast cells in skin. Can get hives from food allergies if the allergen gets carried through the blood o Most common cause is insect bites o Atopic dermatitis (eczema): chronic itching with skin rash, history of increased IgE and allergies Food Allergies o Allergens in the gut that pass across the epithelial wall and bind IgE on mast cells o The increase in vascular permeability allows allergen to enter the blood and can cause hives if they settle in the skin

Also increase the fluid from blood in the lumen combined with contraction of smooth muscle in the stomach diarrhea, vomiting and cramping

Type II Hypersensitivity o Antibody mediated hypersensitivity against our own cells or receptors or membranes o Mediated by IgG or IgM, antibodies against tissue antigens o Causes Local complement activation Influx of leukocytes Tissue destruction via ADCC, degranulation and oxygen radicals o Cytotoxic Type II HS Direct lysis of rbcs Drugs, transfusion reactions, Erythroblastosis fetalis o Second pregnancy, T cell dependent IgG response to Rh Graft rejection o Non-Cytotoxic Type II HS Cell or tissue is altered in function Graves Disease o IgG autoantibodies are formed against the TSH receptor, the antibodies bind the receptor and stimulate unregulated production of thyroid hormones Myasthenia Gravis Type III Hypersensitivity o Caused by high levels of circulating immune complexes IgG or IgM

o o

Systemic damage because the circulating immune complexes overwhelm the immune systems ability to remove them These complexes deposit in various tissues and activate complementneutrophils try to remove the complexes and this results in degranulation and tissue damage

o Serum Sickness o 7-10 days following antiserum treatment, the patient would get chills, fever, vasculitis and occasional glomerulonephritis o Today: anti-venom, streptokinase for MI, and large doses of penicillin Arthus Reaction o if antigen is injected beneath the skin and it forms immune complexes with antibodies that have diffused from the blood into connective tissue o Complement is activated and inflammatory response ensuesappears as localized areas of redness and swelling Type IV Hypersensitivity o