I. Introduction II. Element of Immunity III. Immunogenetic IV. Immune Response V. Antigen &...

I. IntroductionII. Element of ImmunityIII. ImmunogeneticIV. Immune ResponseV. Antigen & Immunogen & VaccineVI. ImmunoglobulinVII. Complement SystemVIII. Cytokines

IX. Antigen-Antibody Reaction X. Immunology in Infection

diseasesXI. ImmunoprophylaxisXII. Hypersensitivity ReactionXIII. Autoimmune DiseasesXIV. Immunodeficiency

Immunology “Imunis”All of physiology mechanism => foreign agent => - neutralize with

or - eliminate => without

- metabolism tissue

damage

X1st century => ChinaXV1st => variolasi1798 : E. Jenner : Cowpox => Smallpox1880 : Vaccine (L. Pasteur)1908 : => cellular (Metchnikof) => humeral (Ehrlich)>>> 1970 : molecular biology

GeneticAgeMetabolismEnvironment & nutritionAnatomyMicrobePhysiology

1. Defense2. Homeostasis3. Surveillance

Innate (natural)Immunity Acquired (adaptive)

Natural : all of creature (+) Such as : 1. Physical hindered

2. Cellular hindered

3. Chemical hindered

Adaptive

With functional characteristic :1. Specificity Heterogeneity

2. Differentiate : “SELF” & “NOT SELF”

3. Memory

Operator of Immunity : Limphoreticular system

Phagocyte cells : MPS, Neutrophyl, EosinophylLymphoid cells : B cell & T cellMediator cells : Basophile, Mastocyt

Which come from : “Hematopoetic Stem Cell”

(0,001 % Bone marrow)

NK cell

Consists of : Lymphoid cells Lymphoid tissues

Lymphoid Cells

Lymphoid cell Immunocyt

Specific cellular product (Ig & CMI)

BM Primary Lymph glands Secondary Lymph

Immunogen

(Prolif. & Dif)

Thymus glandFunction : Maturity T cells

BM Thymus gland Circulation

Cortex Medulla

CD4- CD8- CD4+CD8+ Circulation

Only can be found in bird family B cell

BM Secondary lymphoid organ

Stem cell B cell

Secondary lymphoid organConsists of : lien, lymph node, Payer Patch, Tonsilas antigen filter

Bursa FabriciusBone Marrow

Hematopoietic sterm cell

Myeloerythroid cells

Lymphoid stem cell

?Secondary lymphoid organs

IL-2SCF

IL-7SCF

B cell precursor

Virgin B lymphocyte

Bone Marrow

Thymus

IL-3SCF

T cellprecursor

VirginT lymphocyte

SCFThymicfactors

IL-2IL-7

Blood Circulation

Tissues

Afferent lymph duct

Lymph node

Efferent Lymph duct

Ductus Thoracicus

Spleen

PULMO SKIN GIT Resp. Tract. Circulation

Peribronchial Lymphoid Tissues

Tonsil

Lymph node Regional Spleen

ANTIGEN

Periarterioler sheet

Centralarteriole

Red pulp

Center germinal

Trabecular artery

Structure Immunology of Spleen

Medulla

Cortex

Paracortex

Germinal Center

Artery

Efferent Lymph canal

Immunologist Structure of Lymph

All of immune response processes with genetic basic. “All factors which regulate Immune Response to foreign agents => hereditary”

Very widely of scope : HLA & Blood GroupClinical aspects : Blood grouping, tissue/organ transplantation.Autoimmune disease, producing of vaccine,

MHC = HLA (man)Genetic: position: short arm of

Chromosome 6 length: 3,5 x 106 bps

5’ C C A T T T A A C C - - - 3’3’ C C T A A A T T C C - - - 5’

Class II

Figure 5-1. Organization of the HLA complex on the short arm of human chromosome 6. Regions encoding the 3 classes of MHC proteins are indicated by

braces. Endo denotes a cluster of genes within the class II region that encode protease components and peptide transport proteins required for processing endogenous antigens (see text). Class III proteins are unrelated to class I and II and are not

involved in antigen presentation. Among proteins encoded in the class III region are tumor necrosis factors and , and complement factors C2, C4, B and F.

Figure 5-1. Organization of the HLA complex on the short arm of human chromosome 6. Regions encoding the 3 classes of MHC proteins are indicated by

braces. Endo denotes a cluster of genes within the class II region that encode protease components and peptide transport proteins required for processing endogenous antigens (see text). Class III proteins are unrelated to class I and II and are not

involved in antigen presentation. Among proteins encoded in the class III region are tumor necrosis factors and , and complement factors C2, C4, B and F.

0 500 1000 1500 2000 2500 3000 3500kilobases

centromere

Class III Class I

21BC4B 21A

BFTNF BCDRDQ

EndoDP

TNFC4A C2

CLASS I HLA

In all nucleus’s cells

Such: A, B, C=> L M A

Functions:- Immune aware- Tissue rejected

Figure 5-2. Schematic representation of a class I HLA protein. The molecule consists of an MW 44,000 polymorph transmembrane polypeptide ( chain) non covalently

associated with an MW 12,000 non polymorph polypeptide (2-microglobulin). The 3 extracellular domains of the chain are designated 1, 2, and 3. The binding site for immunogenic peptides (T cell determinants, is formed by the cleft between the 1

and 2 domains.

cytoplasm

membrane

COOH338PO4

306282

COOH259

2-microglobulin

Extracellularregion

CHO chain

Extracellularregion

Figure 5.3. Diagrammatic structure of a class I HLA molecule (side view). In this ribbon diagram of the polypeptide backbone, the polypeptides are oriented as in Fig 3-2, but only the extracellular region is depicted. The peptide binding site as a cleft (or groove) formed by 8 strands of –pleated sheet and a pair of -helices from the 1 and 2

domains. The –sheet structure forms the floor and the type helices the walls of the cleft. –strands are depicted is broad arrows and –helices as narrow coils.

-helixPeptidebindinggroove

28-strand-pleated

sheet1

-helix

-pleated sheet forming floor of antigen-binding

groove

-helixN

Figure 5-4. Peptide-binding site of a class I HLA molecule, viewed along an axis

perpendicular to the cell surface. Eight strands of -pleated sheet contributed by the 1

and 2 domains forms the floor of the site, and 2 -helices, one from each of the 2 domains, form the walls. The groove accommodates peptides 8-9 amino acid residues

long, leaving them partially accessible for interaction with the T cell antigen receptors.

Figure 5-4. Peptide-binding site of a class I HLA molecule, viewed along an axis

perpendicular to the cell surface. Eight strands of -pleated sheet contributed by the 1

and 2 domains forms the floor of the site, and 2 -helices, one from each of the 2 domains, form the walls. The groove accommodates peptides 8-9 amino acid residues

long, leaving them partially accessible for interaction with the T cell antigen receptors.

Class II HLA

At B cell => macrophage

Functions:- T cell aware- Tissue rejected

Extracellularregion

membrane

cytoplasm

COOH COOH

Extracellularregion

chain chain

NH2 NH2

229 237

214193

221200

173163

118107 117

Figure 5-5. Schematic representation of a class II HLA molecule. The molecule consists of an MW 34.000 polypeptide ( chain) noncovalently associated with an MW

29.000 polypeptide ( chain).

Figure 5-6. Structure of the peptide binding site of a class II HLA molecule. The binding site is similar to that of class I molecules, except that it is formed by the 1 and 1 domains of the class II molecule and is relatively open at both ends to

accommodate longer peptides.

-helix

-pleated sheet forming floor of antigen-binding

groove

Figure 5.7. The pathway of assembly and transport for antigen-MHC complexescontaining class I (top) and class II (bottom) HLA molecules. MHC polypeptide of initially expressed in the rough endoplasmic reticculum (RER). Class I proteins sequentially bind endogenous peptides and 2-microglobulin (2m) in the RER lumen and are than transported to the cell surface. Class II proteins associate with invariant chain (li) in the RER and so are prevented from binding endogenous peptides, they are translocated instead to an endosomal compartment, where li dissociates and is replaced by exogenous peptides.

Endogenouspeptides

nucleus

Peptides Processing Exogenous antigen

Class IIMHC

To cellsurfaces Surface Ag-

Class II MHCcomplex

To cellsurfaces

Surface Ag-Class I MHC

complexClass IMHC

Peptide transporter

Cell surface

Erythrocyte antigen: A B O Rh

ABO GroupBefore 20th : transfusion ?1900 Landsteiner

Sera Ery

1 2 3 4 5 6 Group

1 - + + + + - C

2 - - + + - - A

3 - + - - + - B

4 - + - - + - B

5 - - + + - - A

6 - + + + + - C

ABO single gene ABO with 3 allele A,B,OA,B codominant KH binding + H substrate

Genotype

AB AB AB

Phenotype Er-Ag

O O A,B anti

A anti B anti

A antiB anti

Allo ab

Levine & Stetson (1939) => Ag + Asera from post partum mother Ag + S.I rabbit by Rhesus of

erythrocyte => “Rh factor” => Rh. Ag.Natural antibody (-), except by

“immunization”

Genetic of RhesusGenetic of Rhesus

> 30 Ag. Rhesus typeFisher & Race

3 gene with allele partner’s => 5 determinant antigen D, C, E, E, C.

Wiener 1 gene locus => “multiple complex allele”DA Rho, rh’, rh”, hr’, hr”.

Definition:“Self” & “not self”“Virgin” lymphocyte (109/day), with IG & TCR => 108 antigen type“Clonal restriction”“Clonal selection”Each others cells communication.

Imunogen

IMMUNOGEENendosome

lissome

MHC IITH cell

MACROPHAGEAntigen-presenting

cell (AFC)

Figure 3-3. Capture, processing, and presentation of antigen by an APC. The immunogen is captured by phagocytosis, receptor-mediated endocytosis, or pinocytosis and is broken down into fragments. Some fragments (antigens) become associated with class II MHC proteins and are transported to the cell surface, where they can be recognized by CD4 T cells. TCR, T cell receptor.

MHC IImolecules

Processedantigens

Costimulation

APCTH cell

Activation

Autoactivation

proliferation T cell

T cell

TH cell

Release of cytokines and other growth and differentia-tion factors

Figure 3-4. The cell activation. The APC presents an antigen in the context of class II MHC to the TH cell and also provides a costimulatory signal. The 2 signals lead to

activation of the TH cell. The APC also releases IL-1, which acts on both the APC and the TH cell to promote activation. Activation leads to IL-2 receptor expression and IL-2

secretion by the TH cell, resulting in autocrine growth stimulation.

Figure 3-4. The cell activation. The APC presents an antigen in the context of class II MHC to the TH cell and also provides a costimulatory signal. The 2 signals lead to

activation of the TH cell. The APC also releases IL-1, which acts on both the APC and the TH cell to promote activation. Activation leads to IL-2 receptor expression and IL-2

secretion by the TH cell, resulting in autocrine growth stimulation.

IL-2RMHC II

Ig Ag receptors

B cell

Helper factorsB cell

B cell

Proliferation Progeny

differentiation Plasma cell

MemoryB cell

Antibody

Figure 3-5. B cell activation. Antigen binding to the surface immunoglobulins, coupled with soluble or contact-mediated helper factors from an activated TH cell, lead to

proliferation and differentiation. Cytokines involved in TH cell help include IL-2, IL-4 and IL-6.

Figure 3-5. B cell activation. Antigen binding to the surface immunoglobulins, coupled with soluble or contact-mediated helper factors from an activated TH cell, lead to

proliferation and differentiation. Cytokines involved in TH cell help include IL-2, IL-4 and IL-6.

Figure 3-6. To cell activation requires contact with specific antigen in the context of a class I MHC molecule on the surface of a target cell. It also requires IL-2 from a nearby activated TH cell. The activated Tc cell kills the target cell either by secreting cytotoxins

(as shown) or by inducing it to commit suicide.

Figure 3-6. To cell activation requires contact with specific antigen in the context of a class I MHC molecule on the surface of a target cell. It also requires IL-2 from a nearby activated TH cell. The activated Tc cell kills the target cell either by secreting cytotoxins

(as shown) or by inducing it to commit suicide.

Auto-activation

TCR(already

triggered)

TH cell Tc cell

Ag target cell(cell death)

MHC IToxins

Definition :

1. Immunogen2. Antigen3. Immunogenicity4. Antigenicity

Classification :1. Exogen antigen2. Endogen antigen : - Xenogeny Ag. (Heterolog) - Autolog Ag. - Alogenic Ag.

Commonly is a macromolecule protein.1. Molecule antigenisity2. Molecule size3. Complexity of Chemistry structure4. Genetic constitution5. Method of entry 6. Dosage7. Digestibility

Determinant Antigenic

Hapten

HaptenHapten carriercarrier ImmunogenImmunogen

I KI K

I.K agentI.K agent HaptenHapten

Thymus dependent Ag andThymus independent Ag

Macrophage cell

Plasma cell

Imunogenik

HLA DR

Receptor

Cross Reaction

>>> Immunogenicity antigen pathway

>>> Retention>>> Molecule size

Local stimulation

>>> Immunogenicity antigen pathway

>>> Retention>>> Molecule size

Local stimulation

Definition : Protein as humoral immunity effectors

molecule

The function of Ig : Binding Ag Biological activity Thus as complex molecule

Example Antibody to Viral It has particular part which

could :Binding virusBe able to enter respiratory tractNot be broken by enzymeBe able to joint with leukocyte

1940 : Tiselius & Kabat Globulin - AB

1950 : Porter gave papain 3 fragments

1960 : Edelman : Multiple chains Porter : 4 chains

1969 : Edelman, AA chain.from BJ Prot> 1970 : Leder genetic

Three-dimensional structure of an immunoglobulin moleculeThree-dimensional structure of an immunoglobulin molecule

Vk Fab

LchainH chain

Hinge region

CH2 CH3

H chain

PepsinPapain Cleavage sites

Hchain

LchainH3N+

Figure 6-1. Schematic model of an IgG1 (x) human antibody Molecule showing the basic 4-chain structure and domains. Sites of enzymatic cleavage by pepsin and papain are shown

Constant domain

Variable domain

NCDR I

Figure 6-5. Three dimensional structure of a light chain, in this ribbon

diagram tracing the polypeptide backbone, -strands are shown as wide ribbons, other region as

narrow string. Each of the 2 globular domains consists of a barrel-shaped assembly of

7-9 antiparallel -strands. The three hypervariable regions

(CDR1, CDR2, & CDR3) are flexible loop that project outward from the amino-terminal end of the VL

domain.

Side Chain Theory Abundant receptors as antibody

Instructive Theory

Selective Theory

DNA globulin

spontaneous

Clonal selectionAg

Immunization

HPRT+Ig+

Spleen cells(10)8

Myeloma cell culture

HPRT -Ig-

Myeloma cells(2x107)

Selection of hybridcellsIn HAT medium

Assay for antibodyClone antibody-production (positive) hybrids

TumorInduction

Monoclonal AntibodyMonoclonal antibody

Mass culture growthFreeze hybridomafor future use

Figure 12-40. Formation of hybridomas between mouse cells and myeloma cells. Mouse myeloma cells that do not produce their own immunoglobulins and lack hypoxanthine and phosphoribosyl transferase (HPRT) are fused to splenocytesFrom an immunized mouse with polyethylene glycol.The hybrid cells are selected in hypoxanthine-aminopterin-Thymidine (HAT) medium. Unfused myeloma cells are killedBy HAT, and unfused splenocytes die out.The hybridomas are cloned, and antibody is produced in tissueCulture or by ascites formation. (Reproduced, with permission,From Diamond BA, Yelton DE, Scharff MD: Monoclonal Antibodies: A new technique for producing serologic reagents. N Engl J Med 1981; 304: 1344

46 + cell Lysis

health

S1 Lysis (+)V.ch

560C 30’Lysis (-)

S1/SN V.chLysis

Complement Form & Shape

>>> ß Globulin : > 20 type 11 type : C1Q,R,S, C2 C9

Widely : C1C2C3 (5,6,7,8,9) comp. Biologic

function

C4Complement : CoE

Classic Pathway

The Complement CascadeThe Complement Cascade

Alternative Pathway

Ag:Ab complex

C1 C1C4

C14bC2

C14b(2b)2a

C3bBbP

C14b(2b)2a3b (C3b)BbPor

C5b67 C8

C5b678(9)n

Factor D ProperdinFactor B

C3(H2O)

Terminal Components

Diagram of the complement cascade. A: The classic complement pathway. A doublet of IgG antibody molecules on a surface can bind and activate C1, a 3-part molecule composed of C1q, C1r & C1s. C1q has a core & 6 radiating arms, each of which ends in a pod. The pod recognizes & binds to the Fc fragment of the IgG. On activation the C1 binds & cleaves C4. The small fragment, C4a, is release. The large fragment, C4b, binds to the target to continue the cascade. In the presence of magnesium ion, C2 recognizes and binds to C4b. B: Once C2 is bound to C4b, it can be cleaved by C1. A small fragment C2b, is release, and the large fragment, C2a, remains bound to the C4b. This newly formed complex of 2 protein fragment can now bind and cleave C3. This molecule is, in turn, cleaved into 2 fragments, C3a & C3b. The small fragment, C3a, is release, & the large fragment, C3b, can bind covalently to a suitable acceptor, C3b molecules that bind directly to the C4b continue the cascade. C: The complex formed of C2a, C4b & C3b can bind and cleave C5. A small fragment of C5, C5a is released. The large fragment, C5b, does not bind covalently. It is stabilized by binding to C6. When C7 binds, the complex of C5b, C6 & C7 becomes hydro phonic. It is partially lipid-soluble and can insert into the lipid of the cell membrane bilayer.

C4a C4a

C4b C4bC4b

C2aC2b

C2aC3b

C2a C3b

C5b C5bC6 C7

D: When the C5b67 binds C8, a small channel is formed in the cell membrane. Multiple molecules of C9 can bind and markedly enlarge the channel. The channel has a hydrophobic outer surface and hydrophilic central channel that allows passage of water and ions. E: The alternative complement pathway. In the presence of magnesium ions, C3b on a surface can bind factor B, just as C4b can bind C3, factor D, a fluid-phase factor, can cleave bound factor B into 2 fragments, Ba & Bb. Ba is released. The C3bBb complex can now bind an additional molecule of C3 and cleave it, just as C4b2a can bind & cleave C3. C3a is release, & the new complex of C3bBbC3b, usually written (C3b)2Bb, can bind C5 to continue the cascade

C3bC3bC3b C3b

Mechanism of complement regulation

1. Spontaneous destruction2. Enzymatic inactivation3. Specific bind with certain proteins

Complement Biologic Activity

Substance Biologic activityC3a Smooth muscle control,

capillary permeability, mastocyt degranulation

C3b Ossification

C3c PMN mobilization

C4a Smooth muscle control, capillary permeability

C52 = C3a

C5a-des-arg Chemotaxis, release En Hidrol from neutrophyl

BB Migration & induction inhibition, monocyt & macrophage spread

Definition:protein (peptide/glycoprotein) as product of a cell group => mediator/communicator between cells for immune system regulation.

Today >>> 100 types, contain of: - lymphokine - monokine>>> local effect & very closeMechanism of action: autocrine & paracrineThe most important: IL-1,-2,-3,-6,-7 TNF, IFNSynthetic cytokine: Recombinant DNA

Actions of IL-1 and TNF on hematopoietic & lymphoid tissue (A) and nonlymphoid cells & tissue (B). Activities of the two individual cytokines differ in some respects

Major properties of human interleukins and other immunoregulatory cytokines

Earlier Terms

Principal Cell

Source

Principal Effects

Interleukins

IL-1 and Lymphocyte-activating factor, B cell activating factor, hematopoietin

Macrophages, other APCs, other somatic cells

•Costimulation of APCs and T cells•B cell proliferation & Ig production•Acute-phase response of liver•Phagocyte activation•Inflammation & fever•hematopoiesis

Earlier Terms

Principal Cell Source

Principal Effects

IL-2 T cell growth factor

Activated TH1

cells, TC cells,

NK cells

•Proliferation of activated T cells•Nk and TC cell functions•B cell proliferation & Ig G2 expression

IL-3 Multi-colony-stimulating factor

T lymphocyte Growth of early hematopoietic progenitors

IL-4 B cell growth factor I, B cell stimulatory factor I

TH2 cells, mast cells

•B cell proliferation, Ig E expression & class II MCH expression•TH2 & Tc- cell proliferation & function•Eosinophil & mast cell growth & function•Inhibition of monokine production

Earlier Terms

Principal Effects

IL-5 TH2 cells, mast

Eosinophil growth & function

IL-6 IFN-2, hepatocyte-stimulating factor, hybridoma growt factor

Activated TH2 cells, APCs, other somatic cells

•Synergistic effects with IL-1 or TNF to costimulator T cell•Acute-phase response of liver•B-cell proliferation & Ig production•Thrombopoiesis

IL-7 Thymic & marrow stromal cells

•T & B lymphopoiesis•Tc cell function

IL-8 Macrophages, other somatic cells

Chemoattractant for neutrophils & T cells

Earlier Terms

Principal Effects

IL-9 Cultured T cell Some hematopoietic & thymopoietic effects

IL-10 Cytokine synthesis inhibitory factor

Activated TH2, CD8 T, & B lymphocytes, macrophages

•Inhibition of cytokine production by TH1 cells, NK cells & APCs•Promotion of B cell proliferation & antibody responses•Suppression of cellular immunity•Mast cell growth

IL-11 Stromal cells •Synergistic effects on hematopoiesis & thrombopoiesis

Earlier Terms

Principal Effects

IL-12 Cytotoxic lymphocyte maturation factor, NK cell stimulatiory factor

B cells,

macrophages

•Proliferation & function of activated Tc & NK cells•IFN production•TH1 cell induction, supresses TH2 cell functions•Promotion of cell-mediated immune responses

TH2 cells

Epithelial cells &Monocyte, non lymphocytic cell

IL-4 like effects

Mimics IL-2 T-cell effectsMast cell NK activation

Earlier Terms

Principal Effects

TNF Lymphotoxin

Activated

macrophages,

other somatic

•IL-1 like effect•Vascular thrombosis & tumor necrosis

INF dan

Leukocyte interferons, type I interferons

Macrophages ; neutrophils, other somatic cells

•Antiviral effect•Induction of class I MHC on all somatic cells•Activation of macrophages & NK cells

Earlier Terms

Principal Cell

Source

Principal Effects

INF Immune interferon, type II interferon

Activated TH1 & NK cells

•Induction of class I MHC on all somatic cells•Induction of class II MHC on APCs & somatic cells•Activation of macrophages, neutrophils & NK cells•Promotion of cell-mediated immunity•Induction of high endothelial venules•Antiviral effect

Earlier Terms

Principal Cell

Source

Principal Effects

TGF Activated T lymphocytes, platelets, macrophages, other somatic cells

•Anti-inflammatory (supression of cytokine production & class II MHC expression•Anti-proliferative for macrophages & lymphocyte•Promotion of B-cell expression of Ig A•Promotion of fibroblast proliferation & wound healing

Noncovalent binding:1. Electrostatic force: - NH+ - -OOC -2. Hydrogen binding force: - OH –

3. Hydrophobic force: 4. Van der Waals force

Antibody affinityAntibody affinity

AG + AB AGAB

K1 > K2 Affinity

AG – AB ReactionAG – AB Reaction

Primary ReactionSecondary ReactionTertiary Reaction

Primary ReactionTo look labeling:

FARRImmunofluorocentRIA ELISA

Secondary reactionPrecipitate reaction Agglutinating reactionFloccules reactionNeutralisms reactionR I C

Tertiary reaction

Such AG – AB reaction in vivo

Can be: - advantages

- diseases

Antigen Antibody

Schematic figure of antigen-antibody frame work performed

Schematic figure of quantitative precipitation curve

Ab-remainder Equivalent Ag-remainder

Antigen Antibody

Precipitated antibody

Supernatant

Precipitate

Free Ab

No free Ab & Ag

Free Ag

Antigen increase

Single radial diffusion in agar

(radial immunodiffusion)Petri dished is filled with semisolid agar solution containing antibody to antigen S. After agar hardens, the center well is filled with a precisely measured amount of material containing antigen S

Antigen S is allowed to diffuse radially from the center well for 24-48 hours

Standard curve for single radial diffusion. Relationship between ring diameter and

antigen concentration is described by the line constructed from known amounts of antigen. Equation and curve for timed interval (Fahey)

method

Log C = D-Do KC = Antigen concentrationDo = Intercept with ordinateD = ring diameterK = Slope of lineA

Identity reactionIdentity reaction

Nonidentity reaction

Partial identity reaction

A = A antigen a-A = A antiB = B antigen a-B = B antiA1 = A antigen plus a-A1 = A1 anti more determinant

Schematic figure of 3 type Ouchterlony double diffuse reaction. B, Ouchterlony double diffusion bowl shows identity reaction between 1 & 2 fraction, partially identity reaction between all of Rabbit gammaglobuline (RGG) and 2 & 3 fraction and nonidentity reaction between 1 & 3 fraction.

Single radial diffusion in agar

(radial immunodiffusion)

Where antigen S meet corresponding antibody to S in the agar, precipitation results. After reaction proceeds to completion or at a timed interval, a sharp border or a ring is formed

By serial dilution of a known standard quantity of antigen S-S/1,S/2, S/4,S/8- rings of progressively decreasing size are formed. The amount of antigenS is unknown specimens can be calculated and compared with standard in the timed interval (Fahey method)

Reaction of identity Reaction of nonidentity

Reaction of partial identity

Reaction patterns in angular double imunodiffusion (Ouchterlony). R = antigen R, S = antigen S, R1 = antigen R1, R = antibody to R , S = antibody to S. reaction of identity: Precisely similar precipitin lines have formed in the reaction of R with R . Note that the lines intersect at a point. Reaction of nonidentity: precipitin lines completely cross owing to separate interaction of R with R and S with S when R and S are non cross reacting antigens. Reaction of partial identity: R reacts with both R and R1 but forms lines that do not form a complete cross. Antigenic determinants are partially shared between R and R1

X/32 X/2

Antibodyx

AgX/16

AgX/32

Semiquantitative analysis of antigen and antibody by double immunodiffussion. Antigen X (Ag X) is serially diluted and placed circumferentially in wells surrounding the central well containing antibody against antigen X. Precipitin lines form with decreasing thickness until no longer visible at dilution of 1:32 of antigen X. on the right, a similar pattern is generated but with serial 2-fold dilutions of antibody X (X). Formation of a single precipitin line indicates that a single antigen-antibody reaction has occurred.

ANTIGEN X QUANTITATION

ANTIBODY X QUANTITATION

Technique of immunoelectrophoresis

Semisolid agar poured onto glass slide and antigen well andantiserum trough cut out of agar

Antigen well filled with humanserum

Serum separated by electrophoresis

Technique of immunoelectrophoresis

Antiserum trough filled with antiserum to whole human serum

Serum and antiserum diffuseinto agar

Precipitin lines form for individual serum proteins

Comparison of patterns of zone electrophoresis and

immunoelectrophoresis of normal human serum

albumin

Complement Fixation Test

Indicator system

Positive testComplement Sheep red cell coated with

anti sheep red cell antibodyComplement reacts with anti sheep

red cell antibody and lyses cell

Antigen Antibody to antigen Complement

Antibody reacts with antigen and complement combines

No lysis of antibody coated red cells as

complement used up

Complement-fixation test. The indicator system (sheep red cells coated with antibody to sheep red cells) is normally lysed in the presence of complement (fresh guinea-pig serum) -top. If another antibody-antigen system is first mixed with the complement it will no longer

be available to lyse the indicator system –bottom.

Complement-fixation test. The indicator system (sheep red cells coated with antibody to sheep red cells) is normally lysed in the presence of complement (fresh guinea-pig serum) -top. If another antibody-antigen system is first mixed with the complement it will no longer

be available to lyse the indicator system –bottom.

DIRECT METHOD

INDIRECT METHOD

Specificity Test

Direct method Indirect method

Legend

Substrate AntigenFluorescent antibody

Fluorescent antiglobulin

Immune complex

Unlabeled antibody

Unlabeled antiglobulin

Fluorescent heterologous

antibody

Mechanism of immunofluorescence techniques. Direct method (top): Antigen in substrate detected by direct labeling with fluorescent antibody. (bottom): Antigen-antibody (immune) complex in substrate labeled with fluorescent antiglobulin reagent. Indirect method (top): incubation of antigen in

substrate with unlabeled antibody forms immune complex. Labeling performed with fluorescent antiglobulin reagent. (bottom): Immune complex in substrate reacted with unlabeled antiglobulin reagent and then stained with fluorescent antiglobulin reagent directed at unlabeled antiglobulin.

Mechanism of immunofluorescence techniques. Direct method (top): Antigen in substrate detected by direct labeling with fluorescent antibody. (bottom): Antigen-antibody (immune) complex in substrate labeled with fluorescent antiglobulin reagent. Indirect method (top): incubation of antigen in

substrate with unlabeled antibody forms immune complex. Labeling performed with fluorescent antiglobulin reagent. (bottom): Immune complex in substrate reacted with unlabeled antiglobulin reagent and then stained with fluorescent antiglobulin reagent directed at unlabeled antiglobulin.

BLOCKING METHOD (Indirect method)BLOCKING METHOD (Indirect method)

NEUTRALIZING METHODNEUTRALIZING METHOD

Specificity test. Direct method (Left): Substrate antigen fails to react with fluorescent antiglobulin reagent. No fluorescence results. (Right): Immune complex –substrate fails to react with fluorescent

antibody directed again unrelated antigen. No fluorescence results. Indirect method (Top): Unlabeled specific antiglobulin is replaced by unrelated antibody. In second step, fluorescent antiglobulin can not

react directly with antigen in substrate that has not bound specific antiglobulin. No fluorescence results. (Bottom): First step performed by reacting specific antibody with substrate antigen. In second

stage, the specific conjugate is replaced by unrelated fluorescent heterologous antibody. No fluorescence results. Blocking method Substrate antigen is incubated with unlabeled specific antibody prior to addition of specific fluorescent antibody . Decreased fluorescence results. Neutralizing method

Substrate antigen is incubated with specific fluorescent antibody after it is absorbed with specific antigen substrate. No fluorescence results.

Infection and Infection diseaseInfection = microorganism invasion local / systemic alteration.

Pathogenic M.O. has evasive mechanism with its photogenic factors.

Balance disturbance defense <<< iatrogenic disease.

Defense mechanism <<< Immune

Compromised Host.

Infection and Infection diseaseInfection = microorganism invasion local / systemic alteration.

Pathogenic M.O. has evasive mechanism with its photogenic factors.

Balance disturbance defense <<< iatrogenic disease.

Defense mechanism <<< Immune

Compromised Host.

Predisposing factors Immune system effects Infection typesImmunosupression: X ray, cancer th/,

alograft res.Viral Infection: Rubella, EBV Herpes, HIV HepatitisTumor

Malnutrition

Smoking, Dust inhalation

Chronic endocrine diseases.

Primary I.D

CMI & humoral immunity decrease.

Viral replication in limfoid cell which cause immune function disturbance.

Immune cells replaced by tumor cells.Lymphoid hypolasia Lymphocyte << Phagocytes <<“Inflam. lung change”Immune Compl. to spore

Fag. Activ. <<

CMI & Hum. <<

Pulmonal inf., bacterium, fungal inf., UTI.

Secondary bacterial inf.

Bacterium, pneumonia, UTIMeasles, TB, Respiratory Tract Inf., GIT inf.

COPDAllergic response.

Staph. Inf., TB, Respiratory Tract Inf., bacterium.

Evasi mechanism pattern:S. aureus : A

proteine,coagulaseStreptococcus : polisach. caps.,

streptolysin. Gonococcus & : protease to IG AMeningococcusIntracellular org. : intracellular defenseHerves Vi. & EBV : complement inhibit.

factor.

Evasi mechanism pattern:S. aureus : A

proteine,coagulaseStreptococcus : polisach. caps.,

streptolysin. Gonococcus & : protease to IG AMeningococcusIntracellular org. : intracellular defenseHerves Vi. & EBV : complement inhibit.

factor.

Multiple defenses

Characteristic : Natural immunity Adaptive immunity

Schematic form of phagocytes by poly morphonuclear leukocyte (PMN) and tissue macrophage after penetrating skin and the

pathogen bacteria entry to the deeper part of the tissue. PMN are more efficient in

phagocyting than macrophage. Attention to PMN which are mobilized to the tissue and vascular in

inflammatory response

Skin as first line defense

Tissue macrophage

Pathogenbacteria

PMN come from blood vascular

Natural Immunity Preventing of entry

Intact skin Mucous membrane normal flora

Defense for attackingHumoral mechanismCellular mechanism as phagocytes, killing microorganism with :

Oxidize intra cellularADCCCytokine

The last mechanism is very various depend on the etiology

Consist of :Immunity to bacterial infection :

toxin extra cell intracellular

Immunity to viral infectionImmunity to fungal infectionImmunity to bacterial toxin

ec : C. tetani, V. Cholera, C. Diphtheria

The most responsible is IgG

Schematic form of immunology mechanism in neutralizing toxin by antibody. Toxin-antitoxin complex, which is neutralized, is showed being

ingested and destroyed in two type of phagocyte cells

Organism Toxin

Activating lymphocyte

Plasma cell

Antitoxin

Ab-Ag complex

Toxin degradation

Macrophage

Immunity to Extra cellular Infection

Through specific immunoglobulin :IgG & IgM : OpsonisasiIgA for bacterial inside the lumenIgM and Ig (1,2,4) through C lysesIgG & IgM : agglutination phagocytesInhibit Fe uptake by bacteriaIgE at mastocyt cell histamineBacterial motility <<<

Immunity to Intracellular

infection

Phagocytes & humoral immunity is not effective CMI with APC pathway CD4 Cytokine Activation of CD8 & CD4 as cytolysis cellAnother cytolytic cell is NK cell

Immunity to Viral Infection

Vi is non cellular-organism, always intrasel with way :

Immune system Infection Various of membrane AntigenMoving antigen

InterferonAMICMI

Function :Delayed viral replication (type I = & )Activation immunity system(Type II = )

Working Mechanism of Interferon

IFN SYNTHESIS

Gene activation

IFNIFN IFN

Protein synthesis

2,5 A Synthesize

Activated endonuclease

Protein kinase

PhosfhorylatedeiF2

Inhibit protein synthesis

Interferonreceptor

NK activation, macrophage activation, increase expression of MHC molecules

Host Cell

FIGURE

Figure :Proposed mechanisms of induction of interferon synthesis and production of resistance to virus infection.Cell (A) is induced to produce interferon

(IFN) by the presence of double stranded RNA (dsRNA).The interferon ( or ß depending on the type of cell) is released and binds to receptors on other cells.

FIGURE

The interferon ( or ß depending on the type of cell) is released and binds to receptors on other cells.This interaction can cause activation of host effectors functions and induce an intiviral state in neighboring cells (B). mRNA = messenger RNA; 2, 5-A = 2’, 5’ –oligoadenylate.

Working Mechanism :(2’ – 5’ oligoadenylate synthetase-inactive) + ds RNA

2’ – 5’ oligoadenylate synthetase – Active

(endonuclease–inactive) endonuclease Active

RNA degradation

Protein Kinase + ds RNA

elF 2 active (elF 2 INACTIVE)

Impaired protein synthesis

Mx protein(and its analogues in other

species)

Specific influenza virus inhibition in mice

Specific Antibody :Delayed mix with receptorMaking immune complexStimulating viral coagulation

AB was not effective for intracellular viral

Could changed membrane cell Antigen Example :

Oncogenic vi., Vaccinia vi., Influenza vi.Paramyxo vi., Toga virus, Papova, Rubella, Rabies.

Form main defense on viral infection.The effectors is Tc (CD 8 & cd 4).

Immunity for Fungal Infection

Manifestation of Fungal Infection :

Superficial mycosis /cutaneusSubcutaneous mycosisSystemic mycosis

Defense Mechanism ?AMI or CMI ?

Cutaneus as DTH Subcutaneous and systemic depend on

activity from neutrophyl, macrophage, lymphocyte, NK cell ?

Although we have immunity to fungal

infection :Patient with neutropenia easy to get infection as:Candidiasis, Aspergilosis, Zigomycosis.Patient with CMI disturbance easy to get infection as : Cryptococcosis, Histoplasmosis, Coccidiomycosis

Basic of ImmunoprophylacticKnowledge of the immune system

Immune Response

Defense mechanism like AMI & CMIR.I have response of memory.Process : Immunization

Active immunization Passive immunization

Active Immunization

Immunity gets actively.

Requirement :Immune System must be normal.Booster

The immunogen : vaccine consist of :Conventional vaccine : Toxoid

“Killed Vaccine” Subunit Vaccine “Attenuated Living

Vac.”

Genetic device vaccineEx. : Hepatitis B

Vaccine Preparation :Bacteria cell : Pertusis, Typhoid, BCGToxoid : Tetanus, DifteriVirus : Poliomyelitis, Morbilli, Rubella,MumpsPolysaccharide capsule :

Pneumococcus, Meningococcus, H. Influenza type B

The successful of immunization depend on :Kind of VaccineBoosterInfection beforeHow to give

Immunization target in Indonesia :Neonatal until child with school age

Kind of Immunization :Obliged :

Diphtheria, Pertusis, Tetanus (DPT)Tuberculosis (BCG)Polio (Sabin)Measles

Immunization Procedure

Each country is different.

Conditions to give immunization :

Less protectionThe disperse specific antigen rate

is highest.

The biggest risk.

Variety of vaccine

Immunization Count

Time Interval(Weeks)

Age(months)

Basic immunization

BCG 1 x - 0 – 11

DPT 3 x 4 – 8 2 – 11

Polio 4 x 6 – 8 2 – 11

Measles 1 x - 9 – 15

Immunization Program

in Indonesia

Variety of vaccine

Immunization Count

Time Interval(Weeks)

Age(months)

Booster :DPT 1 x - 1,5 – 2

Polio 1 x - 1,5 – 2

DT 1 x - 4 – 6

Td 1 x - 12 – 14(every 10 yrs)

Suggestion ImmunizationMMR 1 x >1 years

Hepatitis B 3 x Anytime(every 5 yrs)

Immunization Program at Posyandu/ Puskesmas

Variety of Immunization

BCG, DPT I, Polio I 2 months

HB I, DPT II, Polio II 3 months

HB II, DPT III, Polio III 4 months

HB III, Measles, Polio IV 9 months

Direct giving AB which needed :

Homolog ABHeterolog ABAutolog AB

Specific AntibodyMaternalGamma GlobulinHeterolog Antibody

Allergen Allergy

Immunity

Tolerance

Hypersensitive

Coombs & Gell :I, II, III : A.M. HypersensitivityIV : DTH + C.M.HypersensitivityV : Stimulatory

Type I = AnaphylacticAg

Vasoactive amin

Capiler activation

Bronchus autonomy muscleDegranulation

Amine vasoactive substances :histaminesslow-reacting substance of anaphylaxis (SRS-A)ECF-Aserotonine

Substances effects to arachidonic acid metabolism :

leukotriens (LTC4 & LTD4)prostaglandintramboxan

People with possibility to hypersensitive reaction : Atopi

Binding between Ig G & Ig M (FAB) with cell antigen :phagocytescytotoxiclysise.c. : isoimmune reactionautoimmune reactiondrugs reaction

Type II reaction : cytotoxic

Antigen-antibody complex abundant elimination not perfect precipitate in tissue and vascular blood

Antigen can produce from ;Pathogen persistent infectionInhalant antigenAutoimmune disease

Anomaly because immune complex depends

on :Absolute rate antigen-antibody complex

Antigen-antibody proportion

Antibody >>> localAntigen >>> systemic

Type III reaction : Immune Complex

Type IV reaction : CMI : DTH

Tissue damagee.c. : • Contact dermatitis• Tuberculin test• Tissue rejected

Antibody CTC induce self tissue, e.c. thyroid tissue

Secretion

Type V reaction

XIII.Autoimmunity and autoimmune disease

Immune system

“Self”

“Not Self” Antibody

(Autoantibody)

Autoimmunity

Autoimmune disease

Body complement

Pathogenesis1. Forbidden-clone theory2. Sequestered-antigen theory3. Immunodeficiency4. Go away from T cell tolerance5. <<< T suppressor cell function

Forbidden-Clone theory

Normal lymphocyte

Positive mutant as antigen

Destroyed by normallymphocyte

Normal lymphocyte

Negative mutant as antigenic(Forbidden-Clone)

Survive, become sensitive and attack target tissue

Immune Deficiency Theory

Positive mutant as antigenic

Immunoglobulin deficiency lymphocyte

Positive mutant as antigenic

Position & negative mutant attack tissue target

Microbe pathogen survive

Antigen changeOr unknown antigen release

Cellular injury(Type IV)OrInjury mediated by antigen

+ Antibody

+ Complement

Injury immune complex (Type III)

THYROID EMBRYONIC

Antigen surfaces

Unknown antigen

EMBRYONIC LYMPHOID

ADULT THYROID

Unknown antigen that affected by

before

Sensitized lymphocyte

Activated autoimmune process

Spectrum of Autoimmune Disease

Variety ofDiseases

Antigen HLA Link Relative Risk

Hashimoto’s Thyroid

Tyro globulin

DR 5 3,2

PrimaryMiksedema

Surface cell DR 3 5,7

Grave’s disease

TSHreceptor

DR 5 3,7

Diabetic autoimmune

Islet cells DR 5, DR 4, DR 3/4

5,0 - 6,0 - 14,3

Variety ofDiseases

Antigen HLA Link Relative Risk

Goodpasture Syndrome

GlomerulusBasal

membrane of lung

DR 2 13,1

Primary Cirrhosis Billiary

Mitochondria - -

Colitis ulcerative

Colon lypopolisachari

Rheumatoid arthritis

Ig G DR 4 4,2

S.L.E. nucleoprotein DR 3 5,8

Pioneer :Bruton found the 8 yrs child who hashypogamaglobulinemia.As clinical squelae S.I. Disturbance.

GeneticMetabolic & Biochemistry deficiencyVitamins & mineral deficiencyDisturbance Embryogenesis Autoimmune diseasesAcquired immunodeficiency.

B Cell ImmunodeficiencyT Cell ImmunodeficiencyB Cell & T Cell Immunodeficiency

(combined)Phagocytic Dysfunction

I. Introduction II. Element of Immunity III. Immunogenetic IV. Immune Response V. Antigen &...

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