Medical Immunology: Block 2

Ex) Mycobacterium, Listeria, Legionella

Bacteria can survive intracellularly, including in macrophages/neutrophils

Generalized Response: CD4 Th1 cell-mediated immunity is best response

Neutrophils/Macrophages cannot kill resistant bacteria.
Infected phagocytes express IL-12 and NK cell-activating molecules.
Activated NK cells, produce IFN-γ (less specific than Th1) to activate macrophages

CD4 activated by Antigen-MHC II complex presentation, costimulatory w/ IL-12 from dendritic cell.

At site of infection , CD4 Th1 cell recognizes antigen-MHC complex and CD40L(Th1)-CD40(APC) interactions and secretes IFN-γ (More Specific than NK)
Macrophages activated by IFN-γ to heightened ROS/RNS species, also secrete TNF-α, IL-1, chemokines for inflammation and IL-12 to activate T Cells

Activated by antigen-MHC I complex to activate CTL
CD8 recognized peptide-MHC I complex on infected macrophage and responds by killing macrophage (Perforins, Granzymes Fas-FasL)

IFN-γ from Th1 cell induced class switching to IgG.
IgG activates classical complement, C3b/IgG opsonize bacteria to help macrophage effector function

Antibody INEFFECTIVE when bacteria is in host cell.

Inhibit lysosome fusion to phagosomes
Escape from phagolysosome into cytoplasm
Inactivate oxidative and lysosomal products

Granulomas - persistance of bacteria causes chronic stimulation of T Cell/ Macrophages
Differences in individual resistance determine dominate type of immune response generate

Viruses: Obligate Intracellular microbes. Use host cell surface molecules to enter cell. Use host cellular machinery to replicate. Located in CYTOPLASM

Antibody contain spread of virus, CTL eliminate virus

Virally Infected Cells produce Type I Interferons (IFN-α/IFN-β)
Type I Interferons: Inhibit viral replication, increase expression of MHC I, and activate NK

NK Cells: recognize infected cells: They lack MHC I but may have NK activating receptors, both of which activated NK Cells

Nk Cells kill virally-infected cell by release cytotoxic molecules (granzymes/perforins_ and cytokines (IFN-γ)

CD8 cells activate by cross presentation of antigen-MHC I on dendrtitic cells ( CD4 Costimulus by IL-12)
CD8 Effector T cell (CTL) recognize antigen-MHC I complex and release perforins and granzymes to induce apoptosis
Interaction of FAS(Target)-FAS ligand(CTL) also causes apoptosis
CTL can move to the next cell

Only effective against Extracellular stage of infections: Early in infection before entry to host cells, or when released from infected cells.
Functions: Neutralizing antibodys (Mostly IgA). Binds to virus, and prevents viral entry into cells. Can be IgG or IgA

Continually change of surface antigens
Interfere with antigen presentation, processing or expression of MHC I
Produce immunosuppressive cytokines

Viral-antibody immune complexes can be deposited in blood vessels to systemic vasculitis
Viral proteins have AA sequences present in self-antigens. Can cause Cross-reaction immune response directed to self-antigens

Sites: Circulation, Epithelial Surfaces, Connective Tissue, Tissue Spaces.
Bacteria cause induction of inflammation, and production of toxins
Immunity Principally mediated by antibody

Phagocytosis/Killing by macrophages, monocytes, and neutrophils
Activation of complement by alternative and lectin pathway resulting in Opsonization (C3b), Cell Lysis (MAC), Recruitment of phagocytosis (C5a/C3a) by anaphylatoxins.
Production of cytokines for local inflammation and ehnance adaptive immuen response

B-Cells activated by antigen-MHC II complex w/ costimulation by CD4 Th2(prduced by IL-4) cells. Affinity Maturation + Isotype Switching. Plasma Cells Secreate Antibodies

Effector Functions:
Neutralization or microbes/toxins by IgG or IgA (mucosal) by prevention of attachment of microbe, preventing spread of microbes, inhibiting binding and/or effects of toxins
Opsonization: IgG coats microbes: Fcγ Binds phagocytes
Complement: Activation of classic complement by IgG and IgA
ADCC: Cytotoxicity, IgG coating causes release of mediators from Innate Immune Cells by attachment of Fcγ

Contribute to response by releasing cytokines
IFN-Extracellular Bacteria causes class switching to IgG and enhancement of macrophage activation
TNF: Inflammation w/ recruitment

Genetic Variation of Surface Antigens
Bacterial Capsules prevent phagocytosis and complement activation
Secrete molecules to degrade complement components or antibodies
Interfere with complement activation sequence
Impaired host integrity of epithelial or mucosal surfaces by toxins

LPS: causes Inflammation and septic Shock
Toxic Shock: toxins act as superantigens to activate large amount of CD4 T Cells.
Generation of Disease-producing Antibodies: Rheumatic Fever M-protein of β-strep cross react with myocardial proteins and myosin
Glomerulonephritis: antigen-antibody complexes deposit in kidneys

Hoest defense dependant on location of parasite
Most parasites cause chronic infections because of weak innate immunity and ability to evade adaptive immunity
Protozoa: CD8 cell lysis & CD4 Th1 Activaiton of Macrophages
Helminth: Activated Eosinophils produce Major Basic Protein (MBP)

Phagocytosis
NK cells through ADCC

Protozoa can survive within Macrophages. CD4 Th1 cells secrete IFN-γ to cause Macrophage activation.
Immunity to other host cells mediated by CD8 CTL.

Protozoa found in extracellular sites eliminated by antibody by complement-mediated lysis, neutralization or opsonization.

Major: Th2 cell activation causing production of IgE and activation of eosinophils and mast cells. IL-4 causes class switching to IgE, IL-5 activated Eosinophils, IL-13 induces epithelial cell turnover and mucus production from goblet cell to expel worms
IgE: arm mast cells and eosinophils to degranulate. Eosinophils produce Major Basic Protein to kill parasites.

Th1: IFN-γ isotype switching to IgG to activate complement and ADCC

Masking, shedding, or varying surface antigens.
Disguising surface with host antigens
Interfering with host's immune response
Sequester within host

Enlargment og liver/spleen due to increase in macrophages
Formation of Immunocomplexes and deposition in kidney
Autoantibodies against RBC, lymphocytes, DNA or crossreaction
Non-Specific Immunosupression

Fungi live either intra or extracellularly so immune response similar to extra or intracellular bacteria
Innate: Mostly phagocytosis by neutrophils/macrophages
Adaptive: mechanisms involving both CD4 and CD8 T Cells, mainly ADCC

Formation of a capsule to inhibit phagocytosis: Crytococcus

Evasion of Macrophage killing: Histoplasma

Formation of granulomas cause injury

Immune Cells - Found in Sruface Epithelium and Lamina Propria
Also contains Mesenteric Lymph Nodes along lining of cavity which are organized similar to other 2ndary lymphoid tissues.
The presence of these lymph nodes also allows for adaptive immune responses to mucosal infection to develop locally

- Goblet Cells
- Paneth Cells
- Intraepithelial Lymphocytes
- Lamina Propria
- Peyer's Patches : M Cells, Dome Area, Efferent Lymphatics
- Isolated Lymphoid Follicles

Secrete mucus which contains components such as lysozyme, lactoferrin, peroxidase and secretory IgA.
Functions to protect against attachement and colonization of pathogenic microbes

Specialized cells found at thebase of the crypts.
Produce antimicrobial proteins

Contain CD4 & CD8 T cells, Plasma Cells, Mast Cells, Dendritic Cells, and Macrophages

Also contain lymphatics that drains to the mesenteric lymph nodes

Integrated in Epithelium and contain B & T Cell Areas
Lumen separated from Peyer's Patches by M-Cells which lack microvilli
"Dome" Area immediately under epithelium w/ B cells similar to Marginal zone of B-1 B cells.
Antigens enter through M-Cells
NO Afferent but Efferent lymphs which drain to mesenteric lymph nodes

Single follicles consisting of Mostly B Cells overlayed by M-Cells

Specialized for Uptake of Antigens and transport to basal side
Have Deep invaginations on basolateral side forming pockets which contain lymphocytes, dendritic cells and macrophages.
Dendritic cells take up antigen fro T Cell presentation

Besides M Cells found in Lamina Propria and can take up antigen independently by extension through epithelial cells.
Migrate to T Cell areas of Peyer's patches or draining lymphatics to T Cell Area of Mesenteric Lymph Nodes for presentation

Naive B/T Cells enter Peyer's Patches or Mesenteric Lymph Nodes through HEVs.
B/T Cells are activated, proliferate, and differentiate into effector/memory cells.
Effector Cells from Peyer's Patches leave through efferent lymphatics, through mesenteric lymph nodes to the blood, migrate back to musocal tissue.
Effector cells from Mesenteric Lymph Nodes leave and travel to mucosal tissue of provoking antigen

Controlled by adhesion molecules and chemokines
Dendritic Cells induce correct homing molecules for type of mucosa in which cells were activated

Effector Lymphocytes:Express Integrin α4:β7 which binds MAdCAM-1

Effector Lymphocytes: Express CCR9 which binds CCL25

IEL: Express Integrin αE:β7 which binds to E-cadherins expressed by epithelial cells and allows movement within epithelium

Effector T Cells: produce cytokines or killing infected host cells

Effector B Cells: differentiate into plasma cells produce dimeric IgA induced by TGF-β

Basis for Immunoprecipitation with soluble antigen.
At optimal concentrations of antigen (ag) and antibody(Ab) extensve cross linking occurs.
Precipitation occurs of growing Ag-Ab aggregate crosslinking into 3D lattice

Excess Antibody: No cross linking, little precipitate

Equivalence: Optimal precipitation, Crosslinking occurs

Excess Antigen: No Cross-linking, very little precipitate. Precipitate dissociation

Formation of immune complexes measured in photoelectic cell as an optical density.

Direct Relationship between complex concentration and optical density.

Quantitative Assay Determined concentration of serum components: Complement, Ig

Either Antigen OR Antibody incorporated into agar.

Other component placed in well cut into agar and diffuses out until equivalence point.

Visible line of precipitate on agar. Determines relative concentrations of Ag or Ab.

Usually compared to STANDARD CURVE of known concentrations

Quantitative Assay: Blood Proteins (Ig)

Primary assay for Blood Group Detection (ABO + Rh(D) +/-)

Blood added to commercially prepared antibody to different blood group antigens.

If Agglutiation (clumping) occurs, Antigen is present.

Person develops antibodies to antigens not present on self blood cells:
IgM for ABO
IgG for Rh(D) Factor

Antigen or Specific Antibody attached to color plastic beads

When added to other component area of color develops (Pregnancy Test has antibodies of hCG)

Replaced by more sensitive methods

Special kind of Agglutination.

Involves spontaneous agglutination of non-human RBC by certain virus.

Reaction blocked if if anti-viral antibodies present in serum to binds to virus.

Serial dilutions of serum can be used to determine concentration ( TITER) of anti-viral antibodies present

Antigen/Specific Antibody attached to solid support and immoblized.

Other component added to and will attach if specific

Ligand (usually 2nd antibody) that has enzyme attached added to attached to antibody/antigen from 2nd step.

Enzyme-ligand conjugate added to colorless substrate that will produce colored product which is determined by Spectrophotometer.

Quantitative Assay Used to TITER blood samples.

Titer is usually reciprical of greatest dilution that is able to show positive ELISA reaction with antigen.

Determine acute vs convalescent stage of disease.

Must be 4 fold increase in titer.

Same as ELISA except reagents are labeled with radioisotope.

Quantitative Assay

Separates proteins in electric field on gel.
Proteins are than transfered to "membrane".
Membrane treated with antibody for specific antigen.
Either ELISA or RIA antibody used to detect antigen-specific antibody.

***Useful in characterizing antibody reactivities in patients with certain pathogens (HIV)***

Proteins placed in well on support media and separated based on charge. Support media stained than scanned by densitometry to provide analytical pattern of protein

Combines electrophoretic separation of serum protein followed by immunologic detection using specific antibodies.
Interpretation made by comparing to standard reference.
Qualitative but can be used to determine under production of certain isotypes (immunodeficeny) or overpruction of proteins

qualitatively detects presence of antigens or antibody on cells or tissue
Tissue or cells attached to glass slide. Flourescent or enzyme labeled antibody applied to material.

Used in the Detection of Ag-AB complexes or complement complexes associated with autoimmune diseases
Localizing presence of specific Ags or hormones in tissue
Detecting treponemal-specific antibody from a patient with syphilis

Studies individual cell for reactivity with specific fluorescent markers.
Stream of single cells pass through laser beam(s) which causing light scattering and fluorescence.
Can also sort cells of interest, used in leukemias, and measurement of T Cell subpopulations as prognosis indicator for AIDS
Forward Scatter: Size
Side Scatter: Granularity
Colored Lasers: Detection of fluorescence

Plotted FCS v SSC

OUTDATED
Step 1: Mix antigen with serum of suspected specific antibody. ** If antigen-antibody complexes for they will fix complement***

Step 2: Add Antibody coated Sheep RBCs as indicator system. If complement depleted in Step 1, RBC will remain intact

Uses patient serum as source of complement to lyse antibody-coated Sheep RBC.

Measurement of hemolytic activity of serum is taken at the 50% hemolysis level:
Classic: CH50
Alternative: AH50

Determines Activity of complement associated with certain diseases.

*****Assays using specific antibodies for various complement and nephelometry or ELISA determine complement concentrations