IMMUNOLOGY Usep Abdullah Husin.

IMMUNOLOGY Usep Abdullah Husin

Topic Introduction Element of Immunity Immunogenetic Immune Response
Antigen & Immunogen & Vaccine Immunoglobulin Complement System Cytokines

IX. Antigen-Antibody Reaction
X. Immunology in Infection diseases XI. Immunoprophylaxis XII. Hypersensitivity Reaction XIII. Autoimmune Diseases XIV. Immunodeficiency

I. Introduction Immunology “Imunis” All of physiology mechanism =>
foreign agent => - neutralize with or eliminate => without - metabolism tissue damage

History X1st century => China XV1st => variolasi
1798 : E. Jenner : Cowpox => Smallpox 1880 : Vaccine (L. Pasteur) 1908 : => cellular (Metchnikof) => humeral (Ehrlich) >>> 1970 : molecular biology

Imunity changing factor
Genetic Age Metabolism Environment & nutrition Anatomy Microbe Physiology

Function of Immunity Defense Homeostasis Surveillance

Innate (natural) Immunity Acquired (adaptive) Natural : all of creature (+) Such as : 1. Physical hindered Cellular hindered 3. Chemical hindered

With functional characteristic : 1. Specificity Heterogeneity
Adaptive With functional characteristic : 1. Specificity Heterogeneity 2. Differentiate : “SELF” & “NOT SELF” 3. Memory

II. Elements of Immunity
Operator of Immunity : Limphoreticular system Phagocyte cells : MPS, Neutrophyl, Eosinophyl Lymphoid cells : B cell & T cell Mediator cells : Basophile, Mastocyt Which come from : “Hematopoetic Stem Cell” (0,001 % Bone marrow)

NK cell

Lymphoid Tissues Lymphoid Cells Consists of : Lymphoid cells
Lymphoid cell Immunocyt Specific cellular product (Ig & CMI) BM Primary Lymph glands Secondary Lymph gland Immunogen (Prolif. & Dif)

Primary Lymphoid Organ
Thymus gland Function : Maturity T cells BM Thymus gland Circulation Cortex Medulla CD4+ CD4- CD CD4+CD Circulation CD 8+

Bursa Fabricius Only can be found in bird family B cell
BM Secondary lymphoid organ Stem cell B cell Secondary lymphoid organ Consists of : lien, lymph node, Payer Patch, Tonsil as antigen filter Bursa Fabricius Bone Marrow

Secondary lymphoid organs
Bone Marrow Myeloerythroid cells IL-2 SCF IL-7 SCF SCF Hematopoietic sterm cell Lymphoid stem cell ? B cell precursor Virgin B lymphocyte Secondary lymphoid organs IL-3 SCF Thymus IL-2 IL-7 SCF Thymic factors T cell precursor Virgin T lymphocyte

Lymphocyt Circulation
Blood Circulation Tissues Afferent lymph duct Spleen Lymph node Efferent Lymph duct Ductus Thoracicus

ANTIGEN Screening of Antigen PULMO SKIN GIT Resp. Tract. Circulation
Peribronchial Lymphoid Tissues Tonsil PP Spleen Lymph node Regional

Structure Immunology of Spleen
Periarterioler sheet Trabecular artery Red pulp Central arteriole Center germinal

Immunologist Structure of Lymph node
Germinal Center Paracortex Cortex Medulla Efferent Lymph canal Artery Vena

III. Immunogenetic All of immune response processes with genetic basic. “All factors which regulate Immune Response to foreign agents => hereditary” Very widely of scope : HLA & Blood Group Clinical aspects : Blood grouping, tissue/organ transplantation. Autoimmune disease, producing of vaccine, etc.

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’ C C T A A A T T C C ’

HLA Complex Class II Class III Class I C4B 21A Endo 21B BF DP DQ DR
TNF BC centromere    C4A C2 TNF kilobases 500 1000 1500 2000 2500 3000 3500 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.

CLASS I HLA In all nucleus’s cells Such: A, B, C => L M A
 chain Extracellular region CHO CLASS I HLA 101 86 1 S 2 S In all nucleus’s cells 164 NH2 2-microglobulin NH2 Such: A, B, C => L M A S S 203 S 3 Extracellular region S 259 Functions: Immune aware Tissue rejected COOH 282 membrane 306 PO4 cytoplasm 338 COOH 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.

-helix 2 Peptide binding groove 8-strand -pleated sheet 1 N N C C 2m 3 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.

-pleated sheet forming floor of antigen-binding groove
-helix -pleated sheet forming floor of antigen-binding groove N -helix N 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
 chain  chain Class II HLA NH2 NH2 CHO 15 19 S 1 1 S 78 CHO 79 Extracellular region 117 At B cell => macrophage 107 118 CHO S S 2 2 S S Extracellular region 163 173 Functions: T cell aware Tissue rejected 193 200 membrane 214 221 cytoplasm 229 237 COOH COOH Figure 5-5. Schematic representation of a class II HLA molecule. The molecule consists of an MW polypeptide ( chain) noncovalently associated with an MW polypeptide ( chain).

-pleated sheet forming floor of antigen-binding groove
1 NH2 -helix COOH -pleated sheet forming floor of antigen-binding groove COOH NH2 1 -helix 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.

Cell surface Surface Ag- Class I MHC complex Peptide transporter
To cell surfaces 2m Class I MHC Endogenous peptides Exogenous antigen Processing Peptides nucleus RER To cell surfaces Class II MHC Surface Ag- Class II MHC complex Figure 5.7. The pathway of assembly and transport for antigen-MHC complexes containing 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.

Blood Grouping Erythrocyte antigen: A B O Rh ABO Group
Before 20th : transfusion ? 1900 Landsteiner

Sera Ery 1 2 3 4 5 6 Group - + C A B

ABO Genetic ABO single gene ABO with 3 allele A,B,O A,B codominant
KH binding + H substrate Genotype Phenotype Er-Ag Allo ab A anti B anti O/O O O - - A,B anti AO,AA A A + - B anti BO,BB B B - - A anti AB AB AB + + -

Rhesus Factor 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 Rhesus > 30 Ag. Rhesus type Fisher & 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”.

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

6 6 Imunogen 2 2 1 1 1 1 3 1 5 3 5 4 4

TCR endosome MHC II IMMUNOGEEN TH cell lissome MACROPHAGE Antigen-presenting cell (AFC) CD 4 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.

T cell IL-2 Autoactivation MHC II molecules IL-1 CD4 T cell IL-2R proliferation APC TH cell Activation TH cell Release of cytokines and other growth and differentiation factors Costimulation Processed antigens 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.

Helper factors Memory B cell B cell CD4 TCR Ag B cell Progeny MHC II
Proliferation B cell Progeny MHC II differentiation Plasma cell IL-2R B cell Ig Ag receptors 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.

TCR (already triggered) IL-2R TCR target cell (cell death) Ag IL-2 TH cell Tc cell Auto- activation MHC I IL-2R CD4 MHC I CD8 Toxins 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.

V. ANTIGEN and IMMUNOGENICITY
Definition : 1. Immunogen 2. Antigen 3. Immunogenicity 4. Antigenicity Classification : 1. Exogen antigen 2. Endogen antigen : - Xenogeny Ag (Heterolog) - Autolog Ag. - Alogenic Ag.

Immunogenicity Commonly is a macromolecule protein.
1. Molecule antigenisity 2. Molecule size 3. Complexity of Chemistry structure 4. Genetic constitution 5. Method of entry 6. Dosage 7. Digestibility

Determinant Antigenic

Hapten I K Hapten carrier Immunogen I.K agent Hapten

Thymus dependent Ag and Thymus dependent antigen
Thymus independent Ag Receptor Imunogenik B T B Macrophage cell B HLA DR Thymus dependent antigen Plasma cell

Cross Reaction

Adjuvant of Immunogen >>> Immunogenicity antigen pathway
>>> Retention >>> Molecule size Local stimulation

VI. IMMUNOGLOBULIN Definition : The function of Ig :
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 virus Be able to enter respiratory tract Not be broken by enzyme Be able to joint with leukocyte

History 1940 : Tiselius & Kabat  Globulin - AB
1950 : Porter gave papain fragments 1960 : Edelman : Multiple chains Porter : 4 chains 1969 : Edelman, AA chain.from BJ Prot > 1970 : Leder genetic

Three-dimensional structure of an immunoglobulin molecule

H3N+ Vk Fab H3N+ Hinge region Ck Lchain VH Fc H chain CH1 H chain COO-
Pepsin Hchain Papain Cleavage sites H3N+ Lchain Fab H3N+ 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

CDR I N 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. CDR 3 CDR 2 Variable domain Constant domain c

Immunoglobulin Syntnesis Theory
Side Chain Theory Abundant receptors as antibody Instructive Theory Selective Theory Ag DA DNA  globulin DA spontaneous DNA

Clonal selection Ag

Selection of hybridcells In HAT medium
Immunization Myeloma cell culture HPRT -Ig- Myeloma cells (2x107) HPRT+Ig+ Spleen cells (10)8 Selection of hybridcells In HAT medium Assay for antibody Clone antibody-production (positive) hybrids Tumor Induction Freeze hybridoma for future use Mass culture growth Monoclonal antibody Monoclonal Antibody

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 splenocytes From an immunized mouse with polyethylene glycol. The hybrid cells are selected in hypoxanthine-aminopterin- Thymidine (HAT) medium. Unfused myeloma cells are killed By HAT, and unfused splenocytes die out. The hybridomas are cloned, and antibody is produced in tissue Culture 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

VII. Complement Systems
46 + cell Lysis V.ch N V.ch health I V.ch S1 Lysis (+) V.ch 560C 30’ Lysis (-) S1 S1/SN V.ch Lysis

Complement Form & Shape
>>> ß Globulin : > 20 type type : C1Q,R,S, C C9 Widely : C1 C2 C3 (5,6,7,8,9) comp. Biologic function C4 Complement : CoE

The Complement Cascade
Classic Pathway Alternative Pathway Ag:Ab complex C4 C3 C1 C1 C2 C14b C14b(2b)2a C3bBbP C3 C4a H2O Factor D Properdin C3a C3b Factor B C3(H2O) C14b(2b)2a3b or (C3b)BbP C6 C5 C5b C7 C5a C5b67 C8 C9 Terminal Components C5b678(9)n

Diagram of the complement cascade. A: The classic complement pathway
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 C2 C4b C2 C4b C4b C4b C2b C3a C2a C2b C2a C2a C3 C2a C3b C4b C4b C4b C4b C5a C7 C5 C5 C2a C3b C4b C4 C5b C5b C6 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 C9 C9 C9 C8 C7 C8 C6 C6 C9 C5b C7 C5b C9 C9 C3 C3a B Ba C3a B Bb Bb C3 Bb C3b C3b C3b C3b C3b C5

Mechanism of complement regulation
Spontaneous destruction Enzymatic inactivation Specific bind with certain proteins

Complement Biologic Activity
Substance Biologic activity C3a 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

VIII. Cytokines 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 close Mechanism of action: autocrine & paracrine The most important: IL-1,-2,-3,-6,-7 TNF, IFN Synthetic 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

IL-5 TH2 cells, mast cell 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

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

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 IL-13 IL-15 TH2 cells Epithelial cells & Monocyte, non lymphocytic cell IL-4 like effects Mimics IL-2 T-cell effects Mast cell NK activation

TNF  Lymphotoxin Activated macrophages, other somatic cells 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

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

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

IX. Antigen-antibody reaction
Noncovalent binding: Electrostatic force: - NH+ - -OOC - Hydrogen binding force: - OH – H2N Hydrophobic force: Van der Waals force

Antibody affinity AG + AB AGAB K1 > K Affinity K1 K1

AG – AB Reaction Primary Reaction Secondary Reaction Tertiary Reaction
To look  labeling: FARR Immunofluorocent RIA ELISA

Agglutinating reaction Floccules reaction Neutralisms reaction R I C
Secondary reaction Precipitate reaction Agglutinating reaction Floccules reaction Neutralisms reaction R I C Tertiary reaction Such AG – AB reaction in vivo Can be: - advantages - diseases

Schematic figure of antigen-antibody frame work performed

Schematic figure of quantitative precipitation curve
No free Ab & Ag Free Ag Supernatant Free Ab Precipitate Antigen increase Preci pi t ated ant ibody Antigen Antibody Ab-remainder Equivalent Ag-remainder Schematic figure of quantitative precipitation curve

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 hours

Antigen concentration
Log C = D-Do K C = Antigen concentration Do = Intercept with ordinate D = ring diameter K = Slope of line Antigen concentration (n.g/mL) 9 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

Partial identity reaction
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. Identity reaction Nonidentity reaction Partial identity reaction A = A antigen a-A = A anti B = B antigen a-B = B anti A1 = A antigen plus a-A1 = A1 anti more determinant

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 antigen S is unknown specimens can be calculated and compared with standard in the timed interval (Fahey method)

Reaction of partial identity Reaction of nonidentity
Reaction of identity R R R S R R1 R S R R 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

ANTIGEN X QUANTITATION ANTIBODY X QUANTITATION
AgX X AgX/32 AgX/2 X/32 X/2 Antibody x AgX AgX/4 X/4 AgX/16 X/16 AgX/8 X/8 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.

Technique of immunoelectrophoresis
Semisolid agar poured onto glass slide and antigen well and antiserum trough cut out of agar Antigen well filled with human serum Serum separated by electrophoresis

Technique of immunoelectrophoresis
Antiserum trough filled with antiserum to whole human serum Serum and antiserum diffuse into 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 test + + +
Sheep red cell coated with anti sheep red cell antibody Complement reacts with anti sheep red cell antibody and lyses cell Complement Positive test + + + Antigen Antibody to antigen Complement No lysis of antibody coated red cells as complement used up Antibody reacts with antigen and complement combines 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.

Specificity Test DIRECT METHOD + + + INDIRECT METHOD + + + +

Direct method Indirect method + + + + + + Legend Substrate Antigen
Fluorescent heterologous antibody Fluorescent antibody Fluorescent antiglobulin Immune complex Unlabeled antibody 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)
+ + NEUTRALIZING 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.

X. Immunology in Infection Disease
Infection and Infection disease Infection = 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.

Immunocompromised Host Table
Predisposing factors Immune system effects Infection types Immunosupression: X ray, cancer th/, alograft res. Viral Infection: Rubella, EBV Herpes, HIV Hepatitis Tumor 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, UTI Measles, TB, Respiratory Tract Inf., GIT inf. COPD Allergic response. Staph. Inf., TB, Respiratory Tract Inf., bacterium.

Evasi mechanism pattern:
S. aureus : A proteine,coagulase Streptococcus : polisach. caps., streptolysin. Gonococcus & : protease to IG A Meningococcus Intracellular org. : intracellular defense Herves Vi. & EBV : complement inhibit factor.

Immunity and infection
 Multiple defenses  Characteristic : Natural immunity Adaptive immunity

Skin as first line defense
Pathogen bacteria PMN come from blood vascular Tissue macrophage 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

Natural Immunity  Preventing of entry Intact skin Mucous membrane
normal flora  Defense for attacking Humoral mechanism Cellular mechanism as phagocytes, killing microorganism with : Oxidize intra cellular ADCC Cytokine

Specific Immunity The last mechanism is very various depend on the etiology Consist of : Immunity to bacterial infection : toxin extra cell intracellular Immunity to viral infection Immunity to fungal infection Immunity to bacterial toxin ec : C. tetani, V. Cholera, C. Diphtheria The most responsible is IgG

Activating lymphocyte
Plasma cell Activating lymphocyte Organism Toxin Antitoxin Ab-Ag complex Toxin degradation Macrophage PMN 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

Immunity to Extra cellular Infection
Through specific immunoglobulin : IgG & IgM : Opsonisasi IgA for bacterial inside the lumen IgM and Ig (1,2,4) through C  lyses IgG & IgM : agglutination  phagocytes Inhibit Fe uptake by bacteria IgE at mastocyt cell  histamine Bacterial motility <<<

Immunity to Intracellular infection
Phagocytes & humoral immunity is not effective  CMI  with APC pathway  CD4  Cytokine  Activation of CD8 & CD4 as cytolysis cell Another cytolytic cell is NK cell

Immunity to Viral Infection
Vi is non cellular-organism, always intrasel with way : Immune system Infection Various of membrane Antigen Moving antigen

Effector Mechanism Interferon AMI CMI

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

Working Mechanism of Interferon
Virus Host Cell dsRNA Gene activation (A) NK activation, macrophage activation, increase expression of MHC molecules mRNA IFN SYNTHESIS IFN Interferon receptor IFN IFN (B) mRNA Protein synthesis Protein kinase 2,5 A Synthesize dsRNA 2,5 A Phosfhorylated eiF2 Activated endonuclease FIGURE Inhibit protein synthesis

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.

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 : (endonuclease–inactive) endonuclease Active
(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

Humoral Immunity Specific Antibody : Delayed mix with receptor Making immune complex Stimulating viral coagulation

CMI 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 /cutaneus Subcutaneous mycosis Systemic 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

Knowledge of the immune system Defense mechanism like AMI & CMI
XI. Immunoprophylactic Basic of Immunoprophylactic Knowledge of the immune system Immune Response Defense mechanism like AMI & CMI R.I have response of memory. Process : Immunization Active immunization Passive immunization

Active Immunization Requirement : The immunogen : vaccine consist of :
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.”

Vaccine Preparation : Genetic device vaccine Ex. : Hepatitis B
Bacteria cell : Pertusis, Typhoid, BCG Toxoid : Tetanus, Difteri Virus : Poliomyelitis, Morbilli, Rubella,Mumps Polysaccharide capsule : Pneumococcus, Meningococcus, H. Influenza type B

The successful of immunization depend on :
Kind of Vaccine Booster Infection before How 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 protection The disperse specific antigen rate is highest. The biggest risk.

Immunization Program in Indonesia
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 Measles 9 – 15 Table

Suggestion Immunization
Variety of vaccine Immunization Count Time Interval (Weeks) Age (months) Booster : DPT 1 x - 1,5 – 2 Polio DT 4 – 6 Td 12 – 14 (every 10 yrs) Suggestion Immunization MMR >1 years Hepatitis B 3 x Anytime (every 5 yrs)

Immunization Program at Posyandu/ Puskesmas
Variety of Immunization Age 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

Passive Immunization Direct giving AB which needed : Specific Antibody
Homolog AB Heterolog AB Autolog AB Specific Antibody Maternal Gamma Globulin Heterolog Antibody

XII. Hypersensitivity Reaction
Immunity Ag Tolerance Hypersensitive Allergy Allergen

Coombs & Gell : I, II, III : A.M. Hypersensitivity
IV : DTH + C.M.Hypersensitivity V : Stimulatory

Bronchus autonomy muscle Capiler activation   
Type I = Anaphylactic Ag Ag IgE Vasoactive amin Degranulation Bronchus autonomy muscle Capiler activation   

Amine vasoactive substances :
histamines slow-reacting substance of anaphylaxis (SRS-A) ECF-A serotonine

Substances effects to arachidonic acid metabolism :
leukotriens (LTC4 & LTD4) prostaglandin tramboxan People with possibility to hypersensitive reaction : Atopi

Type II reaction : cytotoxic
Binding between Ig G & Ig M (FAB) with cell antigen : phagocytes cytotoxic lysis e.c. : isoimmune reaction autoimmune reaction drugs reaction

Type III reaction : Immune Complex
Antigen-antibody complex abundant  elimination not perfect precipitate in tissue and vascular blood Antigen can produce from ; Pathogen persistent infection Inhalant antigen Autoimmune disease Anomaly because immune complex depends on : Absolute rate antigen-antibody complex Antigen-antibody proportion Antibody >>>  local Antigen >>>  systemic

Type IV reaction : CMI : DTH
cell Tissue damage e.c. : Contact dermatitis Tuberculin test Tissue rejected

Type V reaction Antibody CTC induce self tissue, e.c. thyroid tissue
Secretion 

XIII.Autoimmunity and autoimmune disease
Body complement “Self” Immune system Antibody (Autoantibody) “Not Self” Autoimmunity Autoimmune disease

Pathogenesis Forbidden-clone theory Sequestered-antigen theory
Immunodeficiency Go away from T cell tolerance <<< T suppressor cell function

Forbidden-Clone theory
Positive mutant as antigen Normal lymphocyte Destroyed by normal lymphocyte

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 change Or unknown antigen release + Antibody Cellular injury (Type IV) Or Injury mediated by antigen + Complement Injury immune complex (Type III)

EMBRYONIC LYMPHOID CELL
Foreign Antigen Theory THYROID EMBRYONIC EMBRYONIC LYMPHOID CELL Antigen surfaces Unknown antigen

Unknown antigen that affected by before Sensitized lymphocyte
ADULT THYROID

Activated autoimmune process

Spectrum of Autoimmune Disease
Variety of Diseases Antigen HLA Link Relative Risk Hashimoto’s Thyroid Tyro globulin DR 5 3,2 Primary Miksedema Surface cell DR 3 5,7 Grave’s disease TSH receptor 3,7 Diabetic autoimmune Islet cells DR 5, DR 4, DR 3/4 5,0 - 6,0 - 14,3

Variety of Diseases Antigen HLA Link Relative Risk
Goodpasture Syndrome Glomerulus Basal membrane of lung DR 2 13,1 Primary Cirrhosis Billiary Mitochondria - Colitis ulcerative Colon lypopolisacharida Rheumatoid arthritis Ig G DR 4 4,2 S.L.E. nucleoprotein DR 3 5,8

XVI. Immunodeficiency Pioneer : Bruton found the 8 yrs child who has
hypogamaglobulinemia. As clinical squelae S.I. Disturbance.

ETIOLOGY : Genetic Metabolic & Biochemistry deficiency
Vitamins & mineral deficiency Disturbance Embryogenesis Autoimmune diseases Acquired immunodeficiency.

Classification B Cell Immunodeficiency T Cell Immunodeficiency
B Cell & T Cell Immunodeficiency (combined) Phagocytic Dysfunction

Thank You Dank U well Syukron

FINISH

IMMUNOLOGY Usep Abdullah Husin.

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