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 capillaries…most

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 valves…also, 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 DiGeorge’s 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, Peyer’s 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 spleeno 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.

o

o 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 HSC’s)

CD Markers

o Proteins present on the surface of cells that help us identify the type of cello 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 “naïve” until they come into contact with their first antigen…then 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 specificityo T Lymphocytes

Develop in the Thymus Have TCR receptors for antigens…TCR, however, only recognizes antigen in

association with MHC 2 Major subsets:

T Helper Cells (CD4)o Recognize exogenous antigens associated with Class II MHCo 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 macrophageso T Helper Cell 2: activates B cells

Cytotoxic T Cells (CD8)o Recognizes endogenous antigen associated with Class I MHCo Become activated after they recognize an antigen-class I MHC

complexo Secrete few cytokineso Hitman: directly recognizes and kills target cells (virus-infected

and intracellular bacteriao 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/Macrophageso 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 macrophageso 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

Neutrophilso Granulated cytoplasm, stains with acidic or basic dyeso Multi-lobed nucleuso Form in the bone marrow, then are released into the blood, migrate for 7-10 hours,

then plant in tissue and live for 3 dayso 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 dyeo Bilobed nucleuso Phagocytic, major role against parasiteso The secretion of eosinophilic granules results in damage to the parasite membrane

Basophilso Granulated cytoplasm stains with basic dyeo Lobed nucleuso Non-phagocytico Deals with the release of pharmacologically active substances contained within granuleso Allergies

Mast Cellso 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 Cellso Express very high levels of Class II MHC, therefore, are better APC’s than macrophages

and B Cellso 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 membraneso Interstitial: most organso Interdigitating: T cell zones of secondary lymph tissue and thymic medullao Circulating: bloodo Follicular: exclusive to lymph nodes (B Cell Rich)

Do not express Class II MHC Do not function as APC’s 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 RBC’s 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 Pathwayso Classical, Alternative, Lectino 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 bacteriao Classical: Antibody and antigen (immune complexes)

IgM and IgGo Alternate: spontaneous lysis of C3, if it binds to bacteria

Complement Effectors:o Opsonization:

Bacteria and other cells are delivered to phagocytes for destruction…the molecules that aid in this process are called opsonins.

Opsonins: IgG, C3b, iC3b, C4b Occurs via complement receptors

o 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 rbc’s and be

taken to the liver and spleen where they can be removed by the professional phagocytes

o 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 MAC

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

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 basiso Specificity: Given the amount of diversity our immune system deals with daily, it MUST

have specific ways to deal with each different entityo 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 Responseo 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 marrowo 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 Opsonizationo Complemento 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 chainso Both chains have variable and constant regionso 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 Rearrangemento Heavy Chain:

5 classes: μ,δ,γ,α, and ε (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: κ and λ 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 λ light chain Chromosome 2 is κ light chain

Ig Gene Rearrangemento 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 Additiono Occurs via the enzyme TdT

o 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 Exclusiono 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/Isotypeso IgG: Gamma heavy chaino IgM: Mu heavy chaino IgA: Alpha heavy chaino IgE: Epsilon heavy chaino IgD: Delta heavy chain

IgGo Predominant antibody in serum, HIGH AFFINITY antibody that predominates during the

second immune responseo Only antibody that can Opsonize, adcc, and cross the placentao Also activates complemento 4 isotypes, 1-4, named in decreasing concentration in serum.

IgMo ALWAYS MADE FIRST, FIRST ANTIBODY MADEo Secreted as a pentomer when differentiatedo Activates Classical Complemento HIGH AVIDITY, LOW AFFINITYo Presence suggests first exposure

IgAo Mucosalo Dimer, isotypes 1 and 2o 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 basophilso Parasites and allergieso Type I Hypersensitivity

IgDo Membrane bound in mature B Cellso Antigen receptor on B cell

B Cell Development and Activationo Antigen Independent

In the bone marrow B cells develop from hematopoietic stem cells though a series of defined

intermediateso Antigen Dependent

In the periphery Development of mature B cells to memory and plasma cells

B Cell Developmento Pro-B stage: Stromal Cells and TdTo Pro-B Pre-B Stage: IL-7 and RAG-1/2o IgM is present at immature to mature Bo IgD is present at mature Bo B Cell Markers: CD 19, 20, 21o Anergy: if a potential autoreactive cell is released it will be turned off

B Cell/Pre B Cell Receptorso 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 Naïve 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 peripheryo Includes responses to T Cell Dependent and Independent antigens

T Cell Dependent/Independent Antigenso 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-Receptorso Lower the threshold of antigens needed to initiate B Cell responseo CD-21 is an EBV receptor

o 3 protein complex: CD19, CR2, TAPA-1 Activation Signals

o Naïve B Cells are in G0o Competence Signal: G0-G1o Progression Signal: G1-S…cell division and differentiationo 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 Ag’s 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 cycleo Proteins such as TK play an important role in cell cycle regulationo If B Cells were constitutively activated, there would be cancer.

T Helper Cell Role in Humoral Responseo 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 Maturationo 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 FDC’s, deliver the signalo 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 6o 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. APC’s including macrophages, B Cells, and Dendritic Cells Alpha1 and Beta1 form the antigen binding cleft

Antigen Processingo Process by which antigens get loaded into the HLA and presented to Cytotoxic T cells or

Helper T cellso 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 Processingo Proteosomes cut up the antigens and prepare them for MHC attachmento TAP1 and TAP2 provide a tunnel from the cytoplasm to the ER for the cut up antigeno Antigen is presented to the surface for Cytotoxic T Cells via golgi vesicle transport

Exogenous Class II MHC Antigen Processingo APC’s can express class I and IIo They prevent endogenous antigens from binding to Class II with Invariant chainso 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

Activity

IL-1 Monocytes macrophages, B cells dendritic cells, endothelial cells

TH cells Hepatocytes hypothalamus

Costimulatory Acute phase

protein induction

Fever

IL-2 (SKID) TH1 cells Activated TH cells and CTL’s

Proliferation, enhanced killing

IL-3 TH cells Hematopoietic cells

Growth factor

IL-4 TH2 cells B cells Activation, class switching to IgE + IgG

IL-5 TH2 cells B cells Activation, class switching to IgA

IL-6 Macrophages, TH2 cells

B cells Hepatocytes hypothalamus

Differentiation into plasma cells and Ab production

Acute phase protein induction

Fever

IL-7 Bone Marrow /thymic stromal

B and T cell precursors

Differentiation

cells

IL-8 Macrophages Neutrophils Chemotaxis

IL-10 Macrophages Macrophages, TH

cells Down-regulation

of pro-inflammatory response

IL-12 Macrophage TH cells Promotes differentiation of TH cells into TH1 cells

IFN-α and IFN-β

Leukocytes and fibroblasts

Infected cells Inhibits viral replication

IFN-γ TH1 Macrophages Activation

TNF-α Macrophages Inflammatory cells

Induces cytokine secretion, cachexia

G-CSF Macrophages and TH cells

Bone marrow precursors

Granulocyte development

GM-CSF Macrophages and TH cells

Bone marrow precursors

Granulocyte and macrophage development

Interferons

o Alpha and beta are anti-viralo IFN alpha is used to treat HBV, HCV, and HHV-8o IFN beta is used to treat multiple sclerosiso IFN gamma is used to activate macrophages to stimulate intracellular killing and inhibits

the T Helper 2 response Used to treat chronic granulomatous disease

TNFo Can kill tumor cellso Alpha: made by macrophages, T Cells and fibroblastso Beta: made by activated T and B Cells

Lectures 9 & 10: Cell Mediated Immunity

T Cell Receptor (TCR)o Is NEVER secretedo Must recognize antigen with MHC, not free antigen

CD3o Signaling molecule for T Cells (like Igalpha and Igbeta in B cells)o Non-covalently attached to the T Cell Receptoro Responsible for T Cell activation and differentiation

TCR Gene Rearrangemento Occurs in a similar manner to Ig gene in B Cellso Beta Chain is like the heavy chaino Alpha chain is like the light chain

T Cell Developmento Development and maturation occurs in the thymuso 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 cellso Medulla: has cells that survive selection, and more mature thymocytes

T Cell Developmento When Pro-T cells leave the bone marrow, they cannot recognize antigen. They undergo

differentiation in the thymuso Intimate association with thymic stromal cells is required for development.o Ontology

CD4 stabilizes MHC II/TCR interaction…T Helper Cells CD8 stabilizes MHC I/TCR interaction…Cytotoxic T Cells

Thymic Selectiono 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, you’re good.

o

T Cell Activationo T Cells that have passed selection and matured in the Thymus, leave and enter the

periphery in search of antigens. They are naïve 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… APC’s

o Must present antigen to T Helper Cells with co-stimulatory molecules.o B7, present on Dendritic Cells, the ONLY CELLS that can activate naïve T Cells

Macrophages can activate naïve T cells, but only when activate by IFN-y B Cells can activate naïve T Cells after contact with antigen.

T Helper Cell Activationo T Helper Cell and APC exchange mutual activation signalso 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

Superantigenso Bind to regions of TCR and MHC outside of the peptide binding cleft. This means less

specificity and activation of numerous T cellso Endogenouso Exogenous

Cell Mediated Immunityo Extracellular infectious agents are cleared by antibodies, complement and phagocytes.o Intracellular infectious agents require cell mediated immunityo The goal is to destroy the intracellular agent by destroying the host cell that harbors ito Viruses ALWAYS require CMI for effective clearanceo Intracellular bacteria and parasites induce delayed type hypersensitivity via Th1 and

macrophage activation

Delayed Hypersensitivity Reaction

o Macrophages are often targets for infection with intracellular pathogens

Lecture 11: Ontogeny

Primary Vs. Secondary Lymphoid Tissueo Primary: B ad T Cells are derived here….thymus and bone marrow

o Secondary: B and T Cells respond to antigen here…spleen, LN, MALT, BALT, GALT Dendritic Cells

o Interdigitating (IDC): only capable of activating naïve T cellso 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 cellso Once contacted by antigen, FDC’s 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 important…class 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 Traffickingo Flow of B and C Cells though secondary lymphoid tissue is directed by CAM’s and

specialized endothelial cells called HEV’s.o Naïve lymphocytes constantly move throughout the body because they need to find

antigen or else they will die.

o 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. (CAM’s which recognize HEV’s in lymph nodes)

o CAM’s on the lymphocytes recognize CAM’s on the endothelial cells Transmigration

o Rolling—attachment—migration.

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)

MALTo Collection of lymphocytes, plasma cells and phagocyteso Lung and intestineo Plasma cells secrete large amounts of IgAo 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

o 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

************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 Deficiencieso 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 functiono 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, Aspergilluso 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 don’t respond to

chemotactic stimuli

Symptoms: Coarse facies, cold abscess, retained primary teeth, increase IgE, eczema

Complement Deficiencieso Similar manifest to phagocytic and antibody deficiencieso Hallmarks: defects in Opsonization, lytic activity, clearing of immune complexeso Classical Pathway Deficiencies

Deficiency: C1q, r or s, C2, C4 Symptoms: Increased immune complex diseases, increased infection with

pyogenic bacteriao Alternative Pathway Deficiencies

Deficiency: Factor B, properdin Symptoms: Increased Neisserial infections

o Both Pathway Deficiencies Deficiency: C3…EARLY IN LIFE

Symptoms: recurrent bacterial infections, immune complex disease Deficiency: C5-C8 (MAC)…LATER IN LIFE

Symptoms: recurrent meningococcal & gonococcal infectionso Regulatory Proteins

Deficiency: C1-INH Symptoms: Overuse of C1, 2 and 4…edema at mucosal surfaces

B Cell Deficiencies (Defects in Humoral Immunity)o Recurrent bacterial infection with normal immunity to virus and fungal parasiteso Staph and Strep are most commono 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 normalo 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 timeo 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 Deficiencieso Can affect both the humoral and cell-mediated responseo Very severe, increased viral and fungal pathogenso 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 Ig’s, observed as

SCID Combined Immunodeficiency Diseases

o Patients susceptible to any type of pathogeno 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 immunityo 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

milko Used to provide immediate protection, there is no memory response

Active acquisition o always activates an immune responseo Natural is exposure to a disease and an immune responseo Artificial is getting vaccinated and having an immune responseo Boosters

Whole Organism Vaccineso 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 Macromoleculeso Capsular Polysaccharide Vaccines

Only IgM response, repeat doses do not booster Pneumococcal vaccine-23 stereotypes Meningococcal

Stereotypes A, C, Y and W135, not Bo Conjugate Polysaccharide Vaccines

Polysaccharide conjugated to protein antigen…changes 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 Vaccineso Introduce genes encoding major antigen of virulent pathogens into attenuated viruseso Vaccinia, HSV, HIV, o CMI and Abs

Timing and Spacing of Vaccineso If received live vaccine

Wait 2 weeks before giving antibodyo If received antibody

Wait at least 3 months before giving vaccineo Inactivated vaccines are generally not affected by circulating antibody to antigen

Live vs. Inactivatedo 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 vaccineso Allergic: due to a vaccine or vaccine component. Rare and should be reported to CDC

Immunosuppressed Personso Should NEVER receive live attenuated vaccineso Inactivated vaccines can be given, but response may be minimalo Cancer patients can receive live vaccines 3 months after chemo is finishedo HIV+

MMR and varicella can be given to the asymptomatic Symptomatic or full blown aids patients should NEVER receive MMR

Lecture 14: Diagnostic Principles

Brightfield Microscopy: shows mostly bacteria, not viruseso 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.

Salmonellao 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 Techniqueso Precipitation Reactions

Ab and soluble Ag interaction forms a precipitateo Agglutination reactions

Ab and Ag interaction causes clumping Hemagglutination: blood type groups Bacterial agglutination Passive agglutination: spread pattern of rbc’s

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 labelo Complement Fixation:

Rbc + complement = indicator

+ equals no hemolysis of RBCs (white) Complement inactivated by Ab-Ag complex

- equals hemolysis of RBCs (red)o 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 systemo Reactions that are unwarranted and harmful to the hosto The immune response itself is responsible for the induction of diseaseo Requires sensitization and is antigen specifico Classified by Gell and Coombs

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 specifico The severity of the reaction can increase and decrease

Type I Hypersensitivityo Induced by certain types of antigens and has all of the hallmarks as a regular immune

response…what 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 APC’s

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 millimetero Severely allergic is greater than 1o 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-4o PAF: chemotactic for leukocytes…activates Neutrophils, Eosinophils and platelets

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

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 shock…many 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 penicillin…also, 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/Hiveso Activate mast cells in skin. Can get hives from food allergies if the allergen gets carried

through the bloodo Most common cause is insect biteso 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 cellso The increase in vascular permeability allows allergen to enter the blood and can cause

hives if they settle in the skin

o 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 membraneso Mediated by IgG or IgM, antibodies against tissue antigenso 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

Grave’s Diseaseo 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 IgMo Systemic damage because the circulating immune complexes overwhelm the immune

system’s ability to remove themo These complexes deposit in various tissues and activate complement…neutrophils try to

remove the complexes and this results in degranulation and tissue damage

o

Serum Sicknesso 7-10 days following antiserum treatment, the patient would get chills, fever, vasculitis

and occasional glomerulonephritiso Today: anti-venom, streptokinase for MI, and large doses of penicillin

Arthus Reactiono if antigen is injected beneath the skin and it forms immune complexes with antibodies

that have diffused from the blood into connective tissueo Complement is activated and inflammatory response ensues…appears as localized areas

of redness and swelling Type IV Hypersensitivity

o