| |
|
| W | 8/25 | Innate Immunity | 1 |
| F | 8/27 | Innate Immunity | 1 |
| M | 8/30 | Cells of the Immune System | 2 |
| W | 9/1 | Cells of the Immune System | 2 |
| F | 9/3 | Cell-Mediated Immunity Overview | 1 |
| M | 9/6 | Labor Day (No classes) | 1 |
| W | 9/8 | Humoral System Overview | 1 |
| F | 9/10 | QUIZ 1, Organs of the Immune System | 2 |
| M | 9/13 | Organs of the Immune System | 2 |
| W | 9/15 | Organs of the Immune System | 2 |
| F | 9/17 | Exam I | |
| M | 9/20 | Leukocyte Migration (Extravasation) | 15 |
| W | 9/22 | Leukocyte Migration (Extravasation) | 15 |
| F | 9/24 | Antigens | 3 |
| M | 9/27 | Antigens | 3 |
| W | 10/29 | Ig Structure and Function | 4 |
| F | 10/1 | Quiz 2, Ig Structure and Function | 4 |
| M | 10/4 | Ig Structure and Function | 4 |
| W | 10/6 | Monoclonal Abs | 4 |
| F | 10/8 | Exam II | |
| M | 10/11 | Ag/Ab Interactions | 6 |
| W | 10/13 | Ag/Ab Interactions | 6 |
| F | 10/15 | Hypersensitivity | 16 |
| FALL BREAK..Oct. 18-22 | |||
| M | 10/25 | Hypersensitivity | 16 |
| W | 10/27 | Hypersensitivity | 16 |
| F | 10/29 | Quiz 3, Genetic Basis of Igs | 5 |
| M | 11/1 | Genetic Basis of Igs | 5 |
| W | 11/3 | Genetic Basis of Igs | 5 |
| F | 11/5 | Genetic Basis of Igs | 5 |
| M | 11/8 | Major Histocompatibility Complex | 7 |
| W | 11/10 | EXAM III | |
| F | 11/12 | Major Histocompatibility Complex | 7 |
| M | 11/15 | Ag Processing and Presentation | 8 |
| W | 11/17 | T-Cell Receptor | 9 |
| F | 11/19 | T-Cell Receptor | 9 |
| M | 11/22 | Development of T-cells | 10 |
| W | 11/24 | Autoimmunity | 20 |
| F | 11/26 | Thanksgiving Break | |
| M | 11/29 | Autoimmunity | 20 |
| W | 12/1 | Clinical Trials for Drug Approval and New Drugs for M.S. | |
| F | 12/3 | Structure and Origin of HIV | 19 |
| M | 12/6 | EXAM IV | |
| W | 12/8 | Infection and Replication | 19 |
| F | 12/10 | HIV, Clinical manifestations | 19 |
| Final Exams.Dec. 13 - 17 | |||
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___________________________________________________________________________
Lecture Outline
Chapter 1
I. Overview
A. Function of Immune System
1. Recognise non-self as foreign
2. Distinguish self from non-self
B. Potential Problems
1. No recognition of non-self as foreign
-Examples?
2. Recognise self as foreign
-Examples?
3. Graft elimination
4. Too actively recognise non-self as foreign
-Examples?
*B. Terms
1. Antigen
2. Antibody
3. Immunization
4. Immunogen
C. Characteristics of Imm Response
1. Specificty
2. Adaptiveness
3. Discrimination
4. Memory
II. Innate Immunity (non-specific)
-Be able to define
A. Anatomic barriers
1. Skin
a) First line of immunologic defense
b) Fatty acids in skin
2. Mucus membranes
B. Physiologic barriers
1. Lysozyme
2. Gastric juices
3. Interferons alpha & beta
a) induced in infected cell by ds RNA of Virus
b) Bind receptor on other cells:
i. Activ. of signalling pathway¦ Gene activation
ii. 2-5 (A) synthase made
-activates RNAse L = degrades viral RNA
iii. Inhibition of viral replication:
-dsRNA-dependent protein kinase inactivates protein synthesis
4. Complement
a) Serum proteins (proteases)
b) Cause cell lysis of more often Gram (-) bacteria
5. Fever
-Why would this be considered a form of protection?
C. Phagocytic and Endocytic barriers
1. Endocytosis
a) Pinocytosis
b) Receptor-mediated endocytosis
2. Phagocytosis
a) Ingestion stimulated by binding of particle to membrane
b) Phagosome + 1o Lysosome c) 2o Lysosome
d) Digestion by acid hydro9lases
e) Release of enzymes, etc. by exocytosis
D. Inflammatory barriers
1. Characteristic:
a) Edema --- Cause?
b) Erythema ---- Cause?
c) Heat ---- Cause?
d) Pain ---- Cause?
2. Three major events:
a) Vasodilation
b) Increased capill. permeability
c) Influx of Phago. cells:
i. Chemotaxis
ii. Margination
iii. Diapedesis
iv. Chemotaxis
III. Acquired Immunity --- How is this different from Innate?
A. Two types of Acquired Immunity
1. Active = make own Ab
a) Artificial -- example?
b) Natural -- example?
2. Passive -- receive immune cells and proteins from another source
a) Artificial -- example?
b) Natural -- example?
B. History of vaccination
1. Edward Jenner (1798)
a) Smallpox vaccine development
b) Cowpox virus
c) Inoculation of boy lead to protection from intentional exposure
2. Louis Pasteur (late 1800's)
a) Chicken cholera
i. Used attenuated bacterium
ii. Why is this different from Jenner's vaccine?
b) Coined term vaccine from "vacca"-Why?
c) Developed rabies vaccine
C. Clonal Selection Theory
1. Niels Jerne and F. MacFarlane Burnet
-Nobel Prize winners
2. Three major points:
a) Lymphocytes have memb. receptors specific for Ag
b) Lymphocyte specificity exists before contact with Ag
c) Binding of Ag to lympho. activates cell
- Get clones of same specificty
II. Immune System Activation
A. Cellular Branch (Cell Mediated Immunity)
1. Macrophage = Expression of foreign peptide on MHC II
2. T-helper + macrophage
a) TCR on T-cell
b) TCR + MHC/pep.
c) macrophage releases IL-1
-binds TH cell
d) Co-stimulatory signal
-B7 + CD28
3. Activation of TH
a) T-cell clones develop
i. Memory T-cells
ii. Effector T-cells
-Live one to two wks.
-Ab or cytokine production
-Activation or inhib. of other immune cells
4. T-cytotoxic cell
a) Tcy binds MHC I /peptide
i. virus-infected cells
ii. tumor cells
iii. Graft cells
b) Tcy binds IL-2 from TH cell
c) Co-stimulatory signal
d) Activation:
i. Tcy clones
ii. Release of perforins --> cell lysis
B. Humoral Branch
1. B-cell Ig receptors bind Ag
2. Binding of IL-4, IL-6, etc. to B-cell
4. B-cell activation:
a) Plasma cell clones develop
i.No Ig receptors
ii. Make soluble Ab
iii. Same specificity as Ig receptors
b) B-Memory cells develop
-May live for years
5. Ag/ Ab complexes form
6. Destruction or Neutralization:
a) macrophage eliminates Ag/Ab complex
-Ab acts as opsonin
b) Ab-dependent cell lysis
c) Ab coats Ag and prevents binding to host cell
Chapter 2
I. Cells of the Immune system
A.. Monocytes/Macrophages (Agranulocytes)
1. Pluripotent stem cells ---> Myeloid cells ---> ---> monocytes
2. Monocytes enter tissues after ~ 8 hrs and become macrophage (mf)
3. Difference between two?
a) mf larger
b) mf has more lysosomes
c) Increased phagocytosis
4. Activation due to:
a) Digestion of Ag
b) IFNg (gamma interferon)
5. Response by mf
a) Increased phago. activity
b) Increased secretion of cytokines
c) Increased expression of MHC II
d) Secretion of inflammatory mediators
-Example?
6. Amoeboid movement thru tissues
7 Fixed mf's
a) Kupffer cells
b) Alveolar mf
c) Microglial cells
B. Granulocytic Cells
1. Pluripotent stem cells ---> Myeloid cells ---> ---> granulocytes
2. Neutrophils
a) 50 - 70% of WBCs
b) Phagocytic
c) Staining properties?
3. Eosinophils
a) Phagocytic
b) Associated with parasitic infection
c) 1 -3 % of WBCs
d) Staining properties?
4. Basophils
a) <1% of WBCs
b) Release of histamine ------> allergic response
c) Staining?
C. Mast cells
1. Pluripotent stem cells ---> Myeloid cells ---> ---> mast cells
2. Found in what tissues mostly?
3. Receptors for Ab which bind allergen
4. Release of histamine -----> allergy
D. Dendritic Cells
1. Ag presenting cells (APC)
-Long dendrite-like processes
2. Bind Ag in tissues then migrate to lymphoid tissues
3. Retain Ag for months or years
4. Follicular Dendritic
a) In lymph nodes
b) High level of Fc receptor for Ab
5. Interdigitating Dendritic
a) Spleen, lymph nodes, thymus
b) Activate T-cells
-High levels of MHC II
6. Langerhans cells in skin
-differentiate to interdig. dendritic cells in lymph nodes
7. Lineage unclear for all dendritic cells
E. Lymphocytes
1. Hematopoiesis
a) Derived from pluripotent stem cells
-Found where?
b) Differentiate into lymphoid precursors
2. B = Ig receptor
3. T-Helper = CD4
4. T-cytotoxic = CD8
a) Don't express IL-2 R until activated
b) Signal sent to nucleus for granule synthesis
c) Two mechanisms of apoptosis:
i. Granzymes = proteases
-enter thru perforin complexes
-see 200 bp DNA fragments quickly (also vial DNA)
-Activate caspase cascade = cysteine proteases
ii. Fas/Fas Ligand
-Fas on traget assoc. with FADD
-Assoc. with procaspase
-Binds Fas Lingand on CTL
-Activation of death signal in target
5. Natural killer Cells (NK)
a) 5 - 10% of lympho. in circulation
b) Defense vs.
i. Tumor cells ii. Virus infected cells iii. Intracellular bacteria
c) How different from T-cells?
d) Activated by alpha and beta INF
-Why considered first line of defense vs. viruses?
e) Kill via Perforins and granzymes
f) Opposing signals Model
i. Inhibitory receptors bind MHC I
_Have veto power. Why?
ii. Activating receptors may bind abnormal glycosylation on cell
-C-type lectins?
II. Organs of Immune System
A. Site of Lympho. Origin = Bone marrow
B. Primary Lymphoid Tissue
- Sites of maturation
1. Cells develop ability to bind Ag
2. B-cells ---> Bone marrow
a) Bursa-equivalent
i. Bursa of Fabricius in chickens
ii. Glick experiment
b) Stromal cell interaction leads to maturation
c) Death of self-recognising cells
3. T-cells ------>Thymus
a) Size decreases with age
i. Be able to reproduce graph drawn in class
ii. Immune Surveillance Theory?
b) 2 lobes with lobules
c) Cortex
i. Nurse cells
ii. Thymic epithelial cells
iii. Immature thymocytes
iv. Death of thymocytes not recognising self-MHC
-Positive selection = Those recognising MHC get life-saving signal
d) Cortical/Medullary Junction
- Interdigitating dendritic cells
e) Medulla
i. Medullary epithelial cells interact with thymocytes
ii. Death of thymocytes recognising self-MHC/peptide
- Negative selection = Those recognising MHC/pep. receive signal to die (apoptosis)
f) Evidence of importance of thymus
i. Thymectomize mice and transplant thymus
ii. DiGeorge's Syndrome
iii. Nude mice
C. Secondary Lymphoid Tissue
- Sites of Ag presentation and activation
1. Lymphocyte receptors bind Ag
2. Lymphatics
a) Carry lymph
i. Derived from plasma
ii. Enters open lymphatic capillaries
-Thinner walls than blood capill.
-One-way valves
b) How are lymphatics like veins?
3. Lymph re-enters blood via:
a) Thoracic duct
b) Right lymphatic duct
4. Types of 2o lymphoid tissues:
a) Lymph nodes
b) Spleen
c) MALT (Mucosal Assoc. Lymphoid Tissue)
d) Lymphoid follicles
D. Lymphoid follicles
a) Aggregates of immune cells surrounded by lymph capillaries
b) What is the difference between a 1o and 2o follicle?
c) Most often found in :
I. Lamina propria of intestines
ii. Upper airways and bronchi
iii. Urogenital tract
E. Lymph Nodes
1. Lymphatics
a) Several afferent lymphatics enter lymph node
b) Single efferent exits
i.Many more lympho. in eff. than aff.
ii. Due to extravasation during Ag challenge
2. Cortex
a) Mostly B-cells
b)1o lymphoid follicles
3. Paracortex
a) T-cells
b) Dendritic cells
4. Medulla
a) Plasma cells
b) Much Ab made
5. Activation:
a) Lymph with Ag percolates through node
b) TH activated 1st
-Migrate to follicles
c) Plasma cells from follicles migrate to medulla
6. Why does a lymph node swell?
F. Spleen
1. Red pulp
a) RBCs
b) mf
2. White pulp
3. Marginal zone
4. Filters blood from splenic artery rather than lymph
5. No lymphatic vessels
-Then why so many lymphocytes?
6. Consequences of splenectomy?
G. MALT
1. In submucosa
2. Common sites?
3. Types
i. Arranged in follicles
-Many plasma cells
-Examples?
ii. Diffuse with little organization
4. M-cells
I. Endocytosis of Ag
ii. Activation of cells in pocket
iii. Activation at inductive sites
iv. Plasma cells migrate
v. IgA produced
-secreted to lumen
III. Tertiary Lymphoid Tissue
A. Cutananeous Assoc. Lymphoid Tissue
1. Keratinocytes in epidermis
2. Langerhans in epidermis
3. Scattered lympho. and mf in dermis
B. Other diffuse regions
Chapter 15
I. Lymphocyte Recirculation
1. Lympho. move from blood to lymph and back
2. Experiment with labeled lympho.
II. Extravasation
A. High Endo thelial Venules (HEV)
1. Where found?
2. Maintained by cytokines due to Ag capture
B. Cell Surface Molecules
1. Cellular Adhesion Mol. (CAMs)
a) On HEV
b) Function:
i. WBC adhesion to endothelium
ii. Strengthen immune cell-cell contacts
2. Integrins
a) CAM receptors
b) On lympho.
c) Leukocyte Adhesion Deficiency
i.Abnormal b subunit synthesis
ii. Consequence?
3. Selectins
a) Lectin-like domain
b) L-Selectins on Lympho.
c) CAM receptors
C. Homing
1. Targeting of lympho. to 2o lymphoid tissue
2. Vascular adressins on HEV
a) Gly CAM -1
b) Binds L-Selectins on naive T-cell
3. Where do memory cells home?
D. Chemokines
1. Inflammatory mediators
2. Lymphocytes bind on surface of HEV
3. Functions:
a) Chemoattractants
b) Activation of integrins on lympho.
4. MIP-1b = B-cells and naive T attracted
5. MCP-1 = T-memory attracted
E. Adhesion ---> Extravasation
1. Gly CAM-1 + L-selectin
2. Rolling lympho.
3. Chemokine binding sends signal internally
a) Integrin activation--¦ ICAM-1 + LFA-1
b) Cytoskeletal changes-¦ . Transendothelial migration
-Know signalling pathway
Chapter 4
I. Terms
A. Haptens
B. Epitope
C. Paratope
D. Immunogen vs. Antigen
II. Immunogenicity
A. Requirements
1. Foreigness
a) Determined during embryonic development
-Self cells sequestered from imm system can look foreign if exposed
b) Foreigness increases with phylogenetic distance
-Exception = Conserved proteins
2. Complexity
a) Heterogeneity
-Heteropolymers vs. Homopolymers
b) Size and heterogeneity
3. Molecular size
a) Best = >100,000 Daltons
b) Poor = 5,000 - 10,000 Daltons
c) < 5000 ---> rarely immunogenic
d) What evidence pts. to size affecting immunogenicity?
4. Charge
a) Polar sites better than non-polar
b) What types of bonds are necessary for Ag/Ab binding?
5. Degradability
a) Lysosomal enzymes must recognise Ag
b) Lg. insoluble molecules better than sm., soluble ones
c) Proteins with D-amino acids not immunogenic
B. Immunogenicity of macromolecules
1. Proteins
a) Tertiary and quaternary structure
b) Charged
c) Broken down by acid hydrolases
2. Carbohydrates
a) Poor to good
b) Can activate T-independent response
-Repeating polymers
c) Glycoproteins are best
-ABO blood group Ags
3. Nucleic Acids
a) Poor
b) Mostly H'phobic
c) Anti-nuclear Ab (lupus)
4. Lipids
a) Poor
b) H'phobic
c) Not presented by MHC
C. B-Cell Epitopes
1. Size and shape determined by paratope
a) Small Ags fit into pocket
b) Lg. Globular Ags bind to planar surface
2. Epitopes H'philic and accessible
3. Due to Sequential or non-sequential AA on protein surface
-What potential effect does denaturing a protein have?
4. Immunodominant epitopes
a) activate immune cells more often
b) Probably charged and/or protruding
D. T-Cell Epitopes
1. Oligomeric peptides
a) MHC binding site determines size
b)Best sizes for MHC I and MHC II?
2. Trimolecular interactions
a) Agretope binds MHC
i.H'phobic
ii. MHC is "promiscuous"
b) Epitope binds TCR
3. Sequential amino acids
a) Processed inside phagocytic cell
b) No tertiary structure remains
4. Amphipathtic peptides
a) Used to predict immunodom.
b) 18 of 23 immunodominant peptides were amphipathic
5. MHC determines immunodominant peptides:
a) L-cell (fibroblast experiment)
b) MHC II genes transfected into L-cells
-Add peptides
c) Look for activation of T-cells
6. Superatigens
a) Potent T-cell mitogens
b) Cross-link TCR and MHC without specificity
c) How can they make us ill?
Chapter 5
Ig Structure and Function
I. Kinetics of immune response
1. Priming Event
2. Latent Phase
a) 1st Ab to appear = IgM
b) 2nd Ab = IgG
3. Exponential Phase
4. Steady State
5. Declining Phase
6. Be familiar with graph
II. Immunoglobulin Structure
A. Light and Heavy Chains
1. 2 Light chains
a) LC has 2 globular domains
b) Both l and k LC in all indiv.
-Not made in same plasma cell
2. Two Heavy Chains
a) Has 4 globular domains
b) CH determines Ab class
-Isotypes
c) Ab's of the same class have essentially identical C-regions
d) Biological activities assoc. with CH
i.Opsonin
ii. c' activation
iii. Mast cell binding
e) ~110 amino acids per globular domain
-much similarity between domains............Why?
2. Variable (V) region
a) Differs between B-cell lines
b) V-region on each chain
c) Three hypervariable regions/chain
-CDRs separated by framework regions
3. Constant (C) region
a) Not involved in Ag binding
b) Subclasses determined here
i. IgG and IgA
ii. 90 - 95% homology
c) Carbo. chains asssoc. with CH2 domains
-important for c' binding
4. Hinge Region
a) Proline-rich
b) Provides flexibility between Fc and Fab
c) Susceptibility to proteases
B. Proteolytic Cleavage
1. Papain
a) Cleaves above disulfides
b) Production of 2 Fab
c) Production of 1 Fc
2. Pepsin
a) Production of divalent F(ab')2
-and Fc fragments
b) Cuts below disulfides
C. Ab Markers
1. Allotypes = Allelic forms of same Ig genes
a) 1-4 AA differences in C- region
b) Three Km allotypes
c) 25 Gm allotypes
d) How would an Ab be raised to distinguish allotypes?
2. Idiotypes = Epitopes from hypervariable regions
a) Anti-idiotypic Abs generated (Network Theory)
i. May play regulatory role for immune system
ii. Limits itself as decreasng levels of Ab made
iii. Can mimic ligand and activate cells
Anti-id may have internal image of ligand
b) How would an Ab be raised to distinguish idiotypes?
3. Isotypes (Ab Classes)
-How would an Ab be raised to distinguish?
III. Isotype Structure and Function
A. Ig G
1. Mol. Wt. 150,000
2. Divalent
3. 1/2 life = 23 days
4. Biological Properties
a) Opsonin
-IgG only one
b) Transfer across placenta
i. First observe at 4 mo. gestation
ii. Maternal Ab gone by 3-4 mo. in neonate
iii. Fc required
c) Agglutination
-involves cells or particulate Ag
d) Precipitation
e) Complement activation
-Two IgG necessary
B. IgM
1. Mol. Wt. 900,000
2. J-chain "joining"
a) For polymerization
b) Necessary for secretion
3. Valence of 10 possible
- Binding of < 10 due to steric hindrance
4. Half life = ~ 5 days
5. Biological Properties
a) B-cell receptor
-H'phobic region near C-term.
b) Earliest Ab made
i.In maturing B-cell
ii. In response to Ag
c) Agglutinatiring on/Precipitation
-Especially effective
d). Complement activation
-Only one IgM needed
6. Four HC constant domains
C. IgA
1. Secretory IgA (s IgA)
a) secretory component
-S-component
b) J-chain
c) Tetravalent
d) 415,000 Daltons
e) Found in body secretions
-Examples?
2. Bio. Properties of secretory IgA
a) Secretion
i. J-chain binds poly Ig receptor
ii. Receptor-mediated endocytosis
iii. Transcytosis and Exocytosis
iv. Proteolysis of poly Ig receptor
-Becomes S-component
b) Defense of mucosal surfaces
i. Vibrio causes cholera via choleragon
ii. IgA binds Vibrio
-inhibits its activity
c) Agglutination/Precipitation
-quite good
d) Huge amts. produced/day
D. IgD
1. Primary Ig receptor on B-cells
2. 180,000 Dalton monomer
3. Membrane spanning region near C-term.
-Hydrophobic
E. IgE
1. Monomer of mol. wt. 200,000
2. Attachment to mast cells or basophil via Fc
a) During sensitization by allergen
b) Allergen cross-links IgE and histamine is released
3. Shortest 1/2 life (~2days)
4. Increases during parasitic infections
5. 4 HC constant domains
IV. Polyclonal Abs
1. What are they?
2. How different from Mabs?
V. Mouse Monoclonal Abs (very specific)
1. Fuse myeloma cell (immortal) + B lymphocyte (from spleen)
2. Select for hybridomas with HAT
a) Lymphocytes die in culture
b) Myelomas die due to HAT
c) How does this work?
3. Test for presence of Mabs
4. Inject Hybridomas into mouse peritoneal cavity
- Amplify Ab production (How?)
READ in Book Clinical Uses for MAbs
VI. Human Mabs
1. Why are human Mabs more desirable than mouse?
2. What have been some problems assoc. with raising human Mabs?
3. Problems overcome by:
a) Lympho. from 2 individuals cultured together
b) Transform cells with Epstein-Barr virus
Chapter 6
Ag/Ab Interactions
I. Cross-reactivity
1. Toxin vs. toxoid
2. Smallpox vs. cowpox
3. ABO blood Ags vs. bacterial components
a. Group A cross-reactivity with Pneymococcal capsular poly sacch.
b. Group B cross-reactivity with some E. coli strains
c) IgM Natural isohemagglutinin
II. Redundancy
A. Epitope Redundancy = Ab binds 2 or more distinct epitopes
B. Paratope Redundancy = Two Abs bind same epitope
III. Ag + Ab Binding
A. Bio. functions needing divalency
1. Agglutination
2. Precipitation
3. C' activation
B. Requirements for Cross-linking
-See handout
IV. Ag/Ab Interaction
A. Know types of Bonds b/ Ag & Ab
1. Relatively low affinity
2. Dissociation by:
a) Low pH....Why?
b) High salt....Why?
B. Affinity vs. Avidity
1. Affinity = Binding energy b/ 1 Paratope and one epitope
2. Avidity
a) Refers to any Ab with more than one paratope
b) Usually not the sum of each K for individual paratope........Why?
c) This is why a multivalent Ab can overcome the problem of low affintiy
II. Immunology Techniques
A. Direct Agglutination
1. Ag natural constituent of cell
2. Ex) Blood typing, hemagglutination assays
B. Passive Agglutination
1. Attach Ag to insoluble matrix
2. Ex) Latex agglutination test (IgG on beads)
C. SDS-PAGE
1. Porous polyacrylamide matrix
2. Boil protein in SDS
-Eliminates what differences between the proteins?
3. Electric current pushes proteins thr polyacrylamide
4 . Separate protein by mol. wt.
-Larger proteins migrate least, found at top of gel
D. Western Blot
1. Blot protein onto nitriocellulose from SDS gel
-Use electric current
2. Incubate with 1st Ab
3. Incubate with 2nd Ab/Peroxidase
4. Add H2O2 as substrate
5. Add color indicator
Ex) a napthol = turns purple in presence of O2
6. Used for HIV Ab detection in serum
E. Solid Phase Assay
1. Ag bound topoloystyrene plate or nitrocellulose
2. Incubate with 1st Ab and then 2nd Ab
-in mobile phase
3. Detect with
a) Autoradiography = radio. label 1st or 2nd Ab
b) ELISA
i. Use enzyme-conjugated 2nd Ab
ii. Use substrate and color indicator
iii. Qualitative = is Ab present?
iv. Quantitative = Do graph of increasing Ab concentrations
-Use linear part of curve
Chapter 16 Hypersensitivity
I. Types
A. Immediate Hypersensitivity
1. Type I
a) IgE-mediated
b) Symptoms 2 - 30 min.
2. Type II (Ab-mediated Cytotoxic)
a) Ab to cell surface Ag triggers c' lysis
b) Erythroblastosis fetalis
Rh- mom and Rh+ baby
c) Transfusion rxns
i.Ab to ABO Ags lyse RBCs
ii. Hb release due to Ab + c' lysis
-Bilirubin toxic
-Fever, chills, nausea
iv. Protein collects in kidney
-glomerulonephritis, hemoglobinuria
-How treated?
c) Symptoms 5-8 hrs.
3. Type III (Immune Complex)
a) Symptoms 2 - 8 hrs.
b) Ag/Ab complexes in tissues
c) Serum sickness (generalized)
i.Horse Ab to snake venom
ii. Activation of c' components
-Anaphylotoxins ---> mast cell release
- Opsonins ----> Phagocyte release of lytic enzymes
iii. Arthritis, glomerulonephritis, rashes with edema
iv. Severity depends on?
d) Localized = Arthus Rxn.
i. Subcutaneous injection
ii. Mild swelling & redness tissue necrosis
e) Autoimmune
i.Lupus = anti-DNA Ab
-Kidney, heart damage
ii. R.A. = damage to synovial membrane
B. Type IV = Delayed Hypersensitivity
1. Cell Mediated Immunity
2. SYMPTOMS IN 24 - 72 HRS.
a) Sensitization of subpopulation of TH.
-TDTH
b) Influx and activ. of macrophages
i.Release of lytic enzymes
ii. Erythema and induration
5. Variety of cytokines released
a) macrophage chemotactic factor
b) g-interferon
c) IL-2
d) Migration Inhibition factor
e) Tumor necrosis factor
6. Examples:
a) TB
i. Mycobacterium tuberculosis
-ingested by alveolar mf
- How does it survive?
ii. Tubercle forms in lung
iii. Calcification
-Ghon complex on x-ray
iv. Intracellular microbes may be reason for delayed hypersensitivity
b) Graft rejection
c) Contact dermatitis
i. Poison oak
ii. Pentadecacatechol (hydrophob.)
- attaches to skin protein
iii. Phago. by Langerhans cells
iv. Presentation by MHC II to TDTH
- Cytokine release
- Redness and pustules
II. IgE-Mediated Hypersensitivity
A. Sensitization Phase
1. Allergen activates Thelper
- Release of IL-4
2. Allergen and IL-4 bind B-cell
- Plasma cells make IgE
3. IgE binds mast cell or basophil
4. Passive transfer of sensitization possible
5. FceRI (Member of Ig Superfamily)
a) IgE receptor
b) Alpha chain interacts with CH3 - CH3 and CH4 - CH4 of IgE
c) Beta chain
i. ITAM
ii. Noncovalenly assoc. with LYN kinase
d) Two gamma
i. ITAM on cytosolic domain of both g
ii. Phosphorylation leads to assoc. with SYK kinase
B. Activation Phase (see handout)
1. Cross-linking of FceRI by allergen
a) Aggregation of receptors
b) Activ. of TPKs due to transauto phosphorylation
i. Assoc. with ITAMs
ii. Activ. of PLC
iii. Formation of IP3 form PIP2
-Calcium release form ER stores
c) Methylation of phospholipids in plasma membrane
i. Formation of phophatidylcholine
ii.Increased membrane fluidity
iii. Opening of calcium channels
d) Calcium increases lead to
i.Microtubule assembly
ii. Granule movement to membrane
e) Activation of Adenylate cyclase
i.Increase in cAMP
ii. cAMP dependent protein kinases activated
iii. Phosphorylation of granule membrane proteins
iv. Increased permeability to water
-Granule swells
v. Fusion of histamine granules with membrane
f) Subsequent decrease in cAMP required for degranulation
-Epinephrine increases cAMP
2. Affinity of IgE for allergen important
a) If low affinity, aggregate will not persist long enough
b) High affinity means aggregate forms long enough to complete cascade
C. Effector Phase
1. Histamine release (primary mediator)
a) Increased vascular permeability
-edema
b) Contraction of smooth muscles
c) Nasal and lacrimal secretions
-mast cells in mucus membrane and conjunctiva
d) Release of mucus from goblet cells
2. Anti-histamines
a) Compete with histamine for histamine receptors
b) Ethylamine group
3. Leukotrienes and Prostaglandins
a) Secondary mediators
b) Cause:
i. smooth muscle contraction
-more powerful than histamine
ii. Increased vasc. permeability
c) Effects longer lasting
D. Allergic Reactions
1. Allergic Rhinitis
a) Hay fever
b) Upper respiratory problems
c) Genetic
2. Asthma
a) Contraction of smooth muscle in airways
b) Mast cells in lower respiratory tract degranulate
-much mucus released
c) Leukotrienes may bear greatest responsibility
d) Why seeing an increase?
3. Systemic Anaphylaxis (Anaphylactic Shock)
a) Explosive release of many mast cells
b) Symptoms:
i. Fluid in respiratory tract
ii. Smooth muscle contraction of bronchioles
iii. Shock due to fluid loss into tissues
c) Intervention with Epinephrine
i. Increases cAMP levels in mast cells
ii. Relaxes smooth muscles in bronchioles
iii. Decreased vascular permeability
4. Food allergies
a) GI distress
i.Vomiting and diarrhea
ii.histamine release of mast cells in GI tract lining
b) Atopic urticaria
- Hives = wheal and flare
E. Diagnosis/Treatment of Allergies
1. Skin sensitivity testing
a) Scratching or intradermal injection of allergen
b) Look for wheal (swelling due to serum) and flare
-What does size correlate with?
2. Hyposensitization
a) Gradually increase dose of allergen
b) Possibly effective due to:
i. Release of blocking Ab
-IgG
ii. Increase in T-cell suppression of IgE production
Not responsible for:
F. Reasons for Allergic Susceptibility
1. MHC presents allergen
2. TCR recognises MHC/allergen
3. Differential response of T-cells to allergen
4. Defect in IgE suppression
5. Increased permeability of respiratory and G.I. tracts to allergen
6. Increased sensitivity to histamine
7. Increased IgE receptors on mast cells
Chapter 5
Organization of Ig Genes
I. Overview of Ig Gene Expression
A. Important Terms
1. Transcription/Translation
2. Exons
3. Introns
4. Processing of heterologous nuclear RNA
5. Leader sequence/signal sequence
B. Ab Synthesis ----> Secretion
1. DNA rearrangements in developing B-cell
2. Transcription of HnRNA in nucleus
- Processing of HnRNA in nucleus
3. mRNA translated on RER
a) Signal sequence synthesized 1st
b) Glycosylation in lumen of RER
c) Heavy and Light chain associate in RER
4. Movement to Golgi
a) Transition vesicles fuse with proximal Golgi
b) Further glycosylation
5. Formation of Secretory Vesicles
-bud from distal Golgi
6. Exocytosis
a) Secretion of soluble Ig
b) Expression of membrane-bounded Ig
i.IgM and IgD
ii. N-terminus extends out from cell
II. Kappa Light Chain Rearrangement
A. Organization of Human Germ line DNA
1. 100 V-genes
2. 5 J genes
3. One CK gene
B. Rearrange in Immature B-Cell
1. In bone marrow
2. V & J join first
a)Recombination Signal sequences complementary
i. Downstream from selected V = 7bp - 12bp - 9bp
ii. Upstream from selected J = 7bp - 23bp - 9bp
b) Loop forms with DNA between V and J
3. Recombinases cut-out loop
a) RAG 1 and RAG 2
b) SCIDS
I. Traced in some humans to mutant RAG genes
ii. Knockout mice lacking RAG genes---> SCIDS
4. Ligation of V gene to J gene by recombinases
C. Transcription of HnRNA
1. Starts at selected Vgene with its leader
2. Stops at end of CKgene
D. Processing to mRNA
-Cut out intervening sequences
E. Translation of mRNA on ribosome
IV. Lamda LC similar to Kappa
1. Similar idea
2. Difference in # of V and J
V. Heavy Chain
A. DNA Rearrangements in immature B-cell
1. D + J 1st
2. V + DJ
3. V encodes CDR1 and CDR2
4. D and J encode CDR3
B. Transcription
1. Starts with selected V
2. Stops at end of last C gene
C. Differential Processing
1. Either Cm or Cd selected
2. m chainprocessing
a) Cut out IVS
b) If membrane-bound, M1 amd M2 selected
-mRNA encodes 40 AA H'phobic C-terminus
d) If secreted, S selected
-mRNA encodes20 AA H'philic C-terminus
D. Translation on ribosome
E. Class Switching
1. In mature B-cell
2.Switch sites = DNA seq. upstream to each C gene
- Multiple repeating nucleotides
3. Assoc. of 5' switch of Cm with 5' switch of appropriate C gene
4. Intervening DNA clipped out by recombinases
5. Induction by cytokines
- Activation of class specific recombinases?
VI. Means of Antibody Diversity
A. HC genes x LC genes
- Due to rearrangements
B. Junctional Flexibility
1. Coding Joint deletions
a) At DJ, VDJ and VJ
b) Produce different amino acids encoded by joint regions
2. Non-productive alignments
-Absent or defective polypeptide
3. Productive alignments
-Functional polypeptide
Not Responsible for:
C. N-Region Nucleotide Addition
1. Only in HCs
2. Nucleotide addition
a) at V-D and D-J
b) Not encoded by the gene
c) Enzymatic addition by terminal deoxynucleotidyl transferase
3. Get random AA additions in HC polypeptide
a) Some productive
b) Some nonproductive
D. Somatic Mutation
1. Mutations in matureB-cell
a) During DNA replication
-after exposure to Ag
b) Not just at joints
2. May increase or decrease in Ig affinity
3. Exp. with phosphorylcholine
a) Sequenced germ line T-15 gene (HC gene)
b) Sequenced Mabs to phosphorylcholine
c) Some reflected Somatic mutation
-Maybe class switching involved
4. Correlated with 2o & 3o responses
5. Mutation rate 106 x greater than in other genes
E. Allelic Exclusion
1. Prevents multispecificity within a single B-cell
a) Only one HC allele and one LC allele expressed
b) Yancopolos and Alt Model
i.1st m successfully made turns off other m
ii. 1st m successfully made turns on kappa LC genes
iii. 1st kappa successfully made turns off the other kappa
iv. l made only if kappa not productive
2. Experiment with transgenic mice carrying rearranged m gene
-How did this demonstrate allelic exclusion?
F. Productive vs. Non-productive rearragements
1. 33.3% of rearrangements successful
2. Only ~3.7% of all immature B-cells develop to maturity
-How was this calculated?
Chapter 7 MHC
I. MHC I
A. MHC I Genes
1. Classical Genes = A,B,C
-Encode single, transmembrane polypeptide
B. MHC I Proteins
1. One polypeptide
a) Ig superfamily
b) Three domains
2. Assoc. noncovalently w/ _2 microglobulin
a) Required for expression of MHC I
b) Ig superfamily
c) Invariant
3. Found on all nucleated cells
4. Co-dominant expression
5. Peptide bound to MHC I
a) Endogenous peptide
b) 9mer best
c) Anchor residues (Pt. of most contact with MHC)
i. Conserved at AA 2 & 3
ii. Carboxy terminal anchor tends to be H'phobic
6. Cleft closed on all sides
a) Limits size
b) Bulging of peptide in middle
i.Accomodates different size peptides
ii. Contact with TCR
7. Promiscuous
II. MHC Class III
A. Encode Soluble Proteins:
1. Complement proteins
2. Heat-shock proteins
3. TNF _ and _
B. Positioned between Class II and Class I genes
III. MHC Clss II
A. MHC II Genes
1. Classical
a) DR, DP, DQ
b) Encode two transmembrane polypeptides
2. Nonclassical
a)LMP 2 and 7
i. Subunits for "Low mol. mass polypeptide"
ii. Found in proteasome
-mediates proteolysis of endogenous proteins
b) TAP 1 and 2
i. Encode RER transmembrane polypeptides
ii. "Transporter of Antigenic Peptides"
iii. Transport endogenous peptides into RER lumen
B._MHC II Proteins
1. _ and _ chains
2. Member of Ig Superfamily
3. Dimer of Heterodimers
a) Two MHC II co-isolated in vitro
b) May need to bind 2 TCR to get appropriate signal
4. Peptide bound to MHC II:
a) 13 -18 AA
b) Cleft open at both ends
5. Co-dominant expression
6. Cell types expressing MHC II
a) Constitutive:
i. B-cells
ii. Dendritic
iii. Thymic epithelial
b) Inducible
I. Activ. T-cells
ii. Macrophage
C. Polymorphism
1. Multiple alleles among members of a population
-probably at least 100 alleles for each MHC
2. MHC highly polymorphic
3. _1, _2 of MHC I most polymorph.
-Regions of TCR contact
4. 5-10% sequence divergence at a single locus
5. _1, _1 most polymorph. of MHCII
-Regions of TCR contact
D. MHC and Susceptibility to Disease
1. MHC as binding site for pathogen
2. Major epitopes on pathogen mimic MHC
-Ignore pathogen
3. Reduction in MHC polymorphism in population
a) Cheetah = much inbreeding
-more susceptible to viral infection
b) Dutch immigrants to So. America
-Population with MHC alleles different from
Chapter 8 Ag Processing and Presentation
A. MHC Restriction
1. Self T-cells only bind self MHC
-Due to positive selection
2. CD4+ cells bind MHC II only
3. CD8+ cells bind MHC I only
B. Cytosolic Processing pathway
1. MHC I + Endogenous polypeptide
2. Ubiquitin binds and targets protein
a) Proteasome digestion
b) LMP subunits may target production of specific peptides
-LMP induced by gamma-interferon
3. In ER, MHC I binds molecular chaperone
a) Calnexin, for example
b) Involved in folding of MHC I
-to a partially folded state
c) Also assoc. with b2 microglobulin
4. TAP transport of peptides into ER
a) ATP-dependent process
b) Association of MHC I/calnexin
c) MHC I binds peptide
5. Dissociation of calnexin
6. Transport to Golgi .. Secretory vesicle plasma membrane
C. Endocytic Processing Pathway
1. MHC II
2. Internalization of protein (exogenous)
a) Receptor-mediated endocytosis of foreign protein........ or
b) Phagocytosis by macrophage (mf)
3. MHC II binds "invariant chain" in ER
a) Involved in folding of alpha and beta
b) Involved in routing of MHC II through endocytic pathway
4. Fusion of Golgi vesicle with lysosome
a) Invariant protein degraded
b) CLIP remains bound to cleft
-prevents premature Ag binding
5. Fusion of endosome/phagosome with lysosome
6. Low pH makes MHC II floppy
-Exchange of CLIP for foreign peptide
7. Exocytosis and stabilization of MHC/peptide
Chapter 9 T-Cell Receptor
A. Structure of alph:beta TCR
1. Var. and constant regions
2. CDR1 & CDR2 encoded by V-genes
3. CDR3 = V-J or V-D-J
4. Member of Ig superfamily
5. alpha:beta genes
a) alpha has V and J genes
b) beta has V, D and J genes
B. Allelic Exclusion = very similar to B-cell
C. TCR vs. Ig Receptor Diversity
1. Lgr. # V and D genes in B-cell
2. No somatic mutations in T-cells
3. N-nucleotide addition in all TCR genes
-Only in Ig HC
4. Alternative joining of D-genes in T-cells
5. Junctional flexibility the same in T & B
6. T-cell has more diversity than B-cell
D. MHC/pep.interaction with TCR
1. CDR1, CDR2 of apha1 and beta1 interact with what parts of MHC?
2. CDR3 of alpha and beta interact with peptide
E. CD3 + alpha:beta = TCR Complex
1. 5 invariant polypeptides in CD3
-form 3 dimers
2. Required for expression of alpha:beta
3. Signal transducer
4. Interaction of Asp. with TCR (Arg) in transmembrane region
5. ITAM in each subunit interacts with Zap 70
6. Several of these polypeptides are members of Ig superfamily
F. Accessory Molecules to TCR
1. CD4 on TH
a) Single polypeptide
- Binds MHC II at beta 2 domain
b) Co-aggregation of CD4 with alpha:beta and CD3
c) Considered a co-receptor
i. Binds MHC
ii. Signal transducer
- Interaction with Lck (Tyr. Protein Kinase)
d) Increases binding affinity by 100x
e) Binding to MHC induces CD4 conformational change
-Binding to other CD4
f) Possible aggregate formation
i.MHC II + TCR
ii. CD4 + MHCII
iii. CD4 + CD4
g) Possible lattice formation
i.Due to MHC II dimer of heterodimers
ii. Due to CD4 tetramers
2. CD8 on Tcy
a) Binds MHC I at a3 domain
b) 2 polypeptides
c) Considered a co-receptor
i. Binds MHC
ii. Signal transducer
- Interaction with Lck (Serine Kinase)
d) Increases binding affinity by 100x
3. Other Accessory Molecules
a) CD2 + LFA-3
b) LFA-1 + ICAM-1
c) CD28 + B7 = co-stimulatory signal
d) Function of accessory Molecules:
i.Prolong time of TCR + MHC/pep. interaction
ii. Strengthens association
iii. Allows more time for cytokine release
Chapter 10 T-Cell Maturation
I. Maturation
A. Progenitor T in bone marrow
1. Migrates to thymus
-1st occurs during embryonic life
2. Chemotactic factor attracts thymocytes
-from thymic epithelial cells
B. Double Negative
-No CD4 or CD8
C. Double Positives
1. Rearrangement of beta gene
2. CD4 and CD8 expressed
3.Rearrangement of alpha gene
4. CD3 expressed
D. Thymic selection
1. Positive selection
a) Thymus cortex
b) Cells not interacting with self-MHC die by apoptosis
2. Negative Selection
a) Thymus medulla
b) Interaction wth bone-marrow derived cells
c) Elimination of cells binding with high affinity to self Ag/MHC
-Apoptosis
E. Single Positives Appear
F. 99% of T-cells die before maturity
-Why?
II. T-cell Signalling
A. TCR + MHC/peptide aggregation
1. Transducers
a) CD3, CD4 or CD8
b) CD45 activates kinases by dephosporylation
2. Kinases activate PLC
3. PIP2 substrate for PLC
a) DAG and IP3 products
b) 2nd messangers
c) Synergistic
4. DAG = remains in membrane
a) Activation of PKC
b) Phosphorylation of inhibitor of NF-kB
c) NF-kB enters nucleus and dimerizes
i. Dimer binds enhancer sequence
ii. Assoc. with promoter proteins on TATA box by Dna folding
d) Activation of IL-2 gene
5. IP3 enters cytosol
a) Release of calcium from ER
b) Activation of calmodulin by binding of calcium
c) Activ. of calcineurin
-Calmodulin-dep. phosphatase
d) NF-ATc activation by removal of phosphate
-T-cell specific
e) NF-ATc enters nucleus and binds AP-1
-Dimer binds enhancer sequence
-Assoc. with promoter proteins on TATA box
f) Activation of IL-2 gene
B. Immunosuppression by Inhibiting Cascade
1. Cyclosporin A and FK506
2. Both bind immunophilins in cytososl
3. Complex blocks calcineurin activity
4. Used to prevent graft rejection
C. Co-Stimulatory Signals
1. Cytokines
2. B7 and CD28 = stronger than cytokine signal
a) Both members of Ig superfamily
b) B7 = monomer
-on APC
c) CD28 = homodimer
-on T-cell
3. Synergistic with signal from TCR
a) Activation of protein Tyrosine kinases
b) May lead to activ. of AP-1
c) May increase half-life of IL-2 mRNA
4. Result:
a) Increased IL-2 secreted from cell
b) Increased IL-2 receptor
Chapter 20: Autoimmunity
I. Ab-mediated
A. Myasthenia gravis
1. Ab vs. Acetylcholine receptor
-blocking Ab
2. Lose muscle activity since no response to nerve signals
3. Induce experimentally
a) Inject animal with Ach recptors from electric eel
b) Cross-reactivity of Ab to electric eel proteins, with Ach R.
B. Graves Disease
1. Ab vs. TSH receptor mimics TSH
-Activating Ab
2. Bypass negative feedback control
a) Normally:
i.Elevated T4 inhibits TSH and TRF release
ii. Decreased T4 allows release of TSH and TRF
b) Ab causes T4 release regardless of T4 levels
3. Hyperthyroidism
-Symptoms?
II. Immune complex-mediated
Systemic Lupus Erythematosus
1. Immune rxns vs many different tissues
2. Skin
a) Butterfly rash
b) Extreme sun sensitivity
i.Release of DNA due to skin cells damage
ii. May be why sun exacerbates symptoms
3. Ab often present to DNA, nucleoproteins, histones, nucleolar RNA
-Fluor. anti-nuclear Ab test
4. Often complexes cause glomerulonephritis
a) Hematuria
b) Proteinuria
c) Complement and phagocyte activation
5. Is there an environmental trigger?
a) Immunize animals with Klebsiella
b) Get Ab to capsular polysacch. of Kleb.
c) Ab cross-reacts with human DNA
III. Ab & T-cell mediated Immunity
Multiple sclerosis:
A. Immune problems:
1. Immune response to myelin sheath
2. Immune cells cross blood-brain barrier
a) Complement activation
b) Lytic enzymes released from phagocytic cells
c) Phagocytosis of Ag/Ab complexes
3. Progressive disability
a) Impaired motor function
b) Impaired sensory function
c) Impaired memory is most common cognitive problem
B. Causes
1. Environmental?
a) Virus?
i.Herpes simplex
-Genome found in some patients
ii. Rubella has homology to myelin basic protein
-Molecular mimicry
b) Farve Island
-Inhabitants did not have M.S. until soldiers came to island
c) More common in No. Hemisphere
d) Clusters in areas such as Key West
2. Genetics
a) Not Mendelian inheritance
b) DR2 allele = 5x greater chance
C. Treatments
1. Immunosuppressants
-Steroids = Stabilize phagocytic membranes
2. Copaxone injections
a) Synthetic peptide of MBP
b) Induce tolerance
3. beta-interferon
a) Tested because anti-viral
b) Appears to change course of disease:
i.decrease exacerbations
ii. decrease lesion size
iii. decrease disability
c) gamma-interferon trials had to be stopped
IV. Causes of Autoimmunity
A. Polyclonal activator
1. T- independent response
2. Polysacch. trigger for B-cells
B. Molecular Mimicry
1. Rheumatic fever
2. Heat shock proteins
a). People with RA have Abs to HSP 65
b). Insulin-dep. Diabetes ? Ab to Hsp 65
cross reacts with enzyme in pancreatic beta-cells
C.. Abnormal Expression of MHC II
1. On cells formerly w/o MHCII
2. Due to gamma-interferon
3. Insulin-dep. Diabetes =high levels of MHC I & II on pancreatic beta-cells
4. Active Lupus = high levels of gamma interferon
Chapter 19 Human Immunodeficiency Virus
I. Two Strains
A. HIV-1
1. Nucleocapsid (p24, p17) surrounds
a) 2 strands RNA
b) 2 molecules of reverse transcriptase
c) Integrase, Protease, Ribonuclease H
2. Viral envelope
a) Lipid bilayer
b) gp 120 & gp 41
c) Also human MHC
B.HIV-2
1. Spreads more slowly than HIV-1
-Why?
2. Mostly found in west Africa
3. 75% homology w/ SIV
-may have 1st arisen as monkey virus
4. Evidence of cross-species transmission in lab technicians
5. Salk vaccine theory
II. Binding and Entry
A. Viral Entry
1. gp120 binds CD4 on T-cell
-T-cell also binds chemokine receptor
2. Fusion of membranes
a) gp 41 (Fusogenic domain)
b) Fusin = co-factor on T-cell
B. Modification of Nucleocapsid by Proteases
C. Replication
1. DNA made via reverse transcriptase
a) RNA used as template-"retrovirus"
c) AZT inhibits
d) High mistake rate
- 1 in 2,000 nucleotides mutated
2. Ribonuclease H chops out RNA
3. 2nd DNA strand made
4. DNA enters nucleus
a) Integration into host genome -via viral integrase
b) Becomes provirus
III. Viral Genome
A. Long Terminal Repeats
1. 5' LTR
a) Enhancer and Promoter sequences
b) Binding site for NF kB
2. 3' LTR
-Polyadenylation site
B. Regulatory Genes
1. Overlapping
2. Different proteins expressed via:
a) Differential processing
b) Reading frame differences
3. Proteins increase rate of transcription or translation of gag, pol and env
4. Rev, Tat, Nef = regulatory proteins
C. Structural Genes
1. Each encodes large polypeptide which is further cleaved
2. gag = encodes capsid proteins
3. pol = encodes:
a) Reverse transcriptase
b) Protease
c) integrase
4. env = encodes gp120 and gp41
IV. Transcription and Assembly
A. RNA Polymerase II
1. Recognition of long terminal repeats
2. NF-kB proteins bound here
a) T-cell activation by other foreigners leads to synthesis of HIV proteins
b) via NF kB activation
B. Three Transcripts made
1. 9kB = nucleic acid core
2. 4 kB = one IVS cut out
-Encodes structural proteins
3. 2kB = two or more IVS cut out
a) Split genes spliced during processing
a) Encodes regulatory proteins
b) Translated first.Why?
c) Rev enters nucleus and binds 4kB and 9kB
-translocation to cytoplasm
C. Translation of Structural Proteins
D. Assembly of Virions
1. Aggregation of 9kB RNA and viral proteins in cytoplasm
2. Budding from cell
3. Protease modifies viral proteins
-Protease inhibitors are a recent treatment
IV. Clinical Aspects
A. Category A
1. >500 CD4/ml
2. Asymptomatic or
3. Flu-like illness
4. Persistent generalized lymphadenopathy
a) Lymph nodes being destroyed since huge viral burden
b) Dendritic cells especially harbor virus
5. Burst of HIV replication
6. Immune response fights but slowly loses
B. Category B
1. CD4 count = 499 - 200
2. Candidiasis
3. Pelvic inflammatory disease
4. Herpes zoster
5. Fever, diarrhea
6. Idiopathic thrombocytopenia
C. Category C
1. <200 CD4/ml
2. Toxoplasmosis
3. Kaposi's sarcoma
4. Pneumocystis carinii
V. Why So Much T-cell Destruction?
1. Complexes of gp120/gp41 may bind T-cell
2. Apoptosis when stimulated with foreign Ag
VI. Vaccines
A. None in Phase III trials
-Lg.-scale tests designed to evaluate effectiveness in humans
B. gp 160 vaccine
1. Abs raised which neutralized lab HIV infection
2. Abs did not neutralize patient HIV isolates
3. Why not effective?
C. Current Vaccine strategies
1. Whole killed viruses
-Require rigorous inactivation
2. Pseudoviruses
a) Empty lipid shells
b) Carry HIV genes
c) Difficult to manufacture in stable form
3. Recruiting Tcy
a) Insert HIV genes into virus which is not harmful
-Ex) Canarypox virus
b) Cells will make HIV proteins and express via MHC I
c) Attract Tcy
4. Combination strategies
a) Canarypox virus 1st
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