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B CELLS

T CELLS

ANTIBODIES


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Immunogens or Antigens

Immunogen or antigen:

* Antigens are molecules that react with antibodies. In

most cases, antigens are immunogens

* However, in the case of haptens, an antigen may not beimmunogenic by itself, but can react with specific

antibodies


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Haptens:

- Low molecular weight substances

- These substances not immunogenic by itself

- If couple to a larger carrier molecule (albumin, globulins), they

become immunogenic

- Examples :

simple chemicals and drugs:

penicillin, sulphonamid, aspirin, cosmetic, tranquillizers, neomycin

skin ointment


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Epitopes or Antigenic determinants:

* Sites on or within antigen with whichantibodies react

* Antibodies are specific for epitopes


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Types of Antigens

Exogenous Antigens1- Bacterial antigens:

a- Antigens related to bacterial cells

- Somatic antigen (O): part of cell wall gm ve bacter.

- Capsular antigen: usually polysaccharide

- Flagellar Ag (H) : a protein made of flagellin

- Fimbrial Ag: surface antigens in fimbriated bacilli

b- Antigen secreted by bacteria:- Exotoxins- Enzymes

2- Viral antigens:a- protein coat viral antigens

b- Soluble antigens (soluble nucleoproteins as in influenza)


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Types Of Antigens

Endogenous antigens

Human tissue antigens:

a- Blood group antigens:

A, B and Rh antigens

b- Histocompatibility antigens:Glycoprotein molecules on all nucleotide cells:

- Major histocompatibility complex antigens (MHC)

- Human leukocyte antigen (HLA)-important for organtransplantation success (alloantigens)


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The MHC proteins act as "signposts" that

serve to alert the immune system if foreign

material is present inside a cell.

They achieve this by displaying fragmented

pieces ofantigens on the host cell's surface.

These antigens may be selfor nonself.


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Name Function Expression

MHC class IEncodes heterodimeric peptide-bindingproteins, as well as antigen-processing

molecules

All nucleated cells. MHC class I proteins

contain an chain & 2-micro-globulin.They present antigen fragments to

cytotoxic T-cells via the CD8 receptor on

the cytotoxic T-cells and also bind

inhibitory receptors on NK cells.

MHC class II

Encodes heterodimeric peptide-binding

proteins and proteins that modulate

antigen loading onto MHC class II

proteins in the lysosomal compartment

On most immune system cells,

specifically on antigen-presenting cells.

MHC class II proteins contain &

chains and they present antigen

fragments to T-helper cells by binding to

the CD4 receptor on the T-helper cells.

MHC class III region

Encodes for other immune components,

such as complement components (e.g.,

C2, C4, factor B) and some that encode

cytokines (e.g., TNF-) and also hsp.

Variable


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Major Histocompatibility Complex Antigens

(MHC)

* MHC has an important function in presentation of antigens to T-

cells

* Helper T-cells recognize foreign antigens on surface ofAPCs(antigen-presenting cells), only when these antigens are

presented in the groove ofMHC II molecule

* Cytotoxic T-cells will only recognize antigens, on the surfaces ofvirus infected cells or tumor cells only when these antigens are

presented in the groove ofClass I molecule (MHC restriction)


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Superantigens (SAgs)

* They activate multiple clones ofT-lymphocytes

* Bacterial toxins:

Staph.aureus toxic shock syndrome toxin (TSST) and enterotoxins

Strpt.pyogenes pyrogenic toxin A

* They have the ability to bind both class II MHC molecules and TCR

chain

* They act as a clamp between the two, providing a signal for T-cell

activation


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Superantigens (SAgs)

* They are active at very low concentration causing release oflarge

amounts ofcytokines

* The massive T-cell activation and release oflarge amounts ofcytokines cause systemic toxicity

* This method ofstimulation is not specific for the pathogen

* It does not lead to acquired immunity i.e no memory


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Antigen Binding And Recognition Molecules

Antigens are recognized by and bind to:1) B-cell receptors (BCR) :

- These are membrane-bound immunoglobulins

(IgM and IgD) on B-cells

- BCRs can be secreted in plasma as antibodies

2) T-cell receptors (TCR)

- and chains anchored to T-cells

- There is a groove which binds small peptides

presented by MHC on surface of APCs

3) MHC molecules

They are essential for presentation of peptides so that they can be

recognized and bind to TCRs


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Factors influencing Immunogenicity

1-Foreigness :Foreign substances are immunogenic

2- Molecular size:

High molecular weight increase immunogenicty

3- Chemical structure complexity:

High complexity increase immunogenicty

4- Route of administration:

Parenteral routes are more immunogenic to oral route


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Factors influencing Immunogenicity

5- Method of administration:

a- Antigen dose:

Appropriate dose optimum antigenicity

Low dose low- zone tolerance

High dose high-zone tolerance

b- Adjuvant:Substance when injected with an antigen

enhance immunogenicty


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Cells of Immune System

Stem cells of bone marrowdifferentiate into

cytokines (IL-&, IL-3)

colony stimulating factor

Lymphoid series Myeloid series

B-lymphocytes T-lymphocytes NK

monocytes-macrophages dendritic cells eosinophils mast cells


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The Life Of The B Cell

B lymphocytes are formed within the bone marrow andundergo their development there

They have the following functions:

To interact with antigenic epitopes, using theirimmunoglobulin receptors

To subsequently develop into plasma cells, secreting largeamounts of specific antibody, or

To circulate as memory cells

To present antigenic peptides to T cells, consequent uponinteriorization and processing of the original antigen


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* B cells become

plasma cells, which

produce antibodies

when a foreign antigen

triggers the immune

response


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B-lymphocytes

in bone marrow

* The lymphoid stem cells differentiate into B cells

* B-cell precursors mature and differentiate into immunocompetentB-cells with a single antigen specificity

* Immature B-cells that express high affinity receptors for selfantigens, die or fail to mature

i.e negative selection or clonal deletion

* This process induces central self tolerance and reduces autoimmunediseases


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B-lympocytes* Immature B cells express IgM receptors on the surface

* Mature B cells express IgM, IgD molecules on surfaces

* IgM and IgD molecules serve as receptors for antigens

* Memory B-cells express IgG or IgA or IgE on the surface

* B-cells bear receptors for Fc portion of IgG and a receptor for C3 component of the

complement

* They express an array of molecules on their surfaces that are important in B-cellsinteractions with other cells


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Mechanism of Humoral immunity

* Antibodies induce resistance through:

1) Antitoxin neutralize bacterial toxins(diphtheria,tetanus)

Antitoxin are developed actively as a result of:

a- Previous infection

b- Artificial immunization

c- Transferred passively as antiserum

* Neutralization of toxin with antitoxin prevents a combination with

tissue cells


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Mechanism of Humoral immunity

2) Antibodies attach to the surface of bacteria and

a- act as opsonins and enhance phagocytosis

b- prevent the adherence of microorganisms totheir target cells, e.g. IgA in the gut

c- Activate the complement and lead to bacterial lysis

d- Clump bacteria (agglutination) leading to

phagocytosis


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Activation of B cells to make antibody


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T-Lymphocytes

T-lymphocytes migrate from bone marrow to enter thymus

1) In the outer cortex of thymus:

- T-lymphocytes acquire specific receptors (TCRs)

- This receptor commits lymphocytes to a single antigen

specificity

- Responding by proliferation and production of a

clone of cells (clonal selection)

- They differentiate to express CD3, both CD4 and

CD8 coreceptors (double positive cells)


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* T lymphocytes become

CD4+ (helper T cells)

or* CD8+ cells (which in turn

can become killer T cells)

also called cytotoxic T cells


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T-Lymphocytes

2) In the medulla of thymus:- TCRs recognize MHC molecules, loaded with normal

self-peptides

- TCRs capable of binding with low affinity to MHCwill receive positive selection signals to divide and

establish clones

- TCRs that bind too strongly to MHC undergo

negative selection

- This selection process will eliminate the potentially

most harmful self reactive T-cells (central self

tolerance)


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T-Lymphocytes

3)Immature T-cells express both CD4 and CD8 (DP)

As they mature

* T-cell with TCRs that have affinity to bind to MHCclass II will become helper T-cells with CD4

molecule only

* T-cell with TCRs that have affinity to bind with MHC

class I will become cytotoxic T-cells with CD8

molecule only


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T-Lymphocytes

4) Mature positively selected T-cells are MHC restricted

* CD4 T-cells are MHC II restricted and only recognize

specific foreign peptide only when they are presentedin association with specific MHC II molecules

* CD8 T-cells are MHC I restricted and recognize

specific foreign peptidees only when they are

presented in association with specific MHC I

molecules


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T-cell surface markers

These are molecules that by which we can identify

T-cells and divide them to subsets

They are required to for interactions between T-cells and APC andfor antigen recognition


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T-cell subpopulation

1) CD4 T helper lymphocytes (TH)

- TH lymphocytes recognize antigen on the surface of APC in

association with class II MHC molecules

- They are activated and secrete several cytokines

- There are two main subsets of TH cells (THI and TH2)

- The two subsets are differentiated on basis of the cytokines they

produce


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1) CD4 T helper lymphocytes Subsets

Th1 produce mainly :

- Cytokines of CMI and inflammation

e.g. IFN-, TNF- , IL-3 and IL-2

TH2 produce mainly:

- Cytokines that stimulate B-cells

- Suppressor cytokines

e.g. Il-4, IL-5, IL-6 and IL-10


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2) CD8 Cytotoxic T-lymphocytes (CTLs)

* They constitute 35% of peripheral T-cells

* CTLs recognize antigen on suurface of target cells (infected APC or

other infected nucleotid cell) in association with MHC-I

* They are activated and kill the virus infected cell or tumor cell


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Professional APCs

Dendritic cells, macrophages, and B-lymphocytes

Dendritic cells:

- They are the most efficient APCs

- They are the main inducers of primary immune response

- Presenting antigen to and activating native T-cells in the recognition

phase

- They express class I and class II MHC molecules

- Dendritic cells are primarly located under skin and mucosa of most

organs

- They capture foreign antigens and transport them to local lymph nods

- They present antigen to native helper T-cells


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Macrophages

* Derived from myeloid stem cells in bon marrow

* They exist as free cells in blood e.g. monocytes and fixed cells in

tissues e.g. Kupffer cells of liver

* They are important link between innate and aquired immune

responses

* They are activated and attracted to the site of foreign material by

action of different cytokines

e.g IFN- , C5a


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Functions of Macrophages1) Phagocytosis

2) Opsonization

3) APCs: they ingest foreign material, process it, and fragments ofantigen are presented on its surface

(in association with MHC molecules) for interaction with T-cells

4) Macrophages may kill antibody coated infected cells or tumour cellsthrough release of lytic enzymes

5) They produce IL-1, IL-6, IL-12, IL-15, TNF-alpha

6) They secret prostaglandins and synthesize complement components


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Natural killer (NK) Cells

* Large granular lymphocytes which lack most surface markers of B and

T-cells

* They comprise 5-10% of the peripheral lymphocytes

* They function mainly in innate immunity

* They have spontaneous non-specific cytotoxic activity on virusinfected cells, tumour cells and graft cells

* They are not MHC restricted and MHC I inhibits their killing functions

* The mechanism of NK mediated cytolysis is as that of CTLs


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NK cells differ from CTLs in

1)They are non-specific

2)They act spontaneously without prior recognition or activation

3)They do not require antigen presentation by MHC

4)They destroy cells coated with antibodies,

a mechanism called antibody dependant cellular cytotoxicity(ADDCC)


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Antibodies produced by B-cells of the immune system

recognize foreign antigens and mark them for destruction


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Activation of helper T cells


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Activation of cytotoxic T cells


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Immunoglobulin (Ig)

Immunoglobulins are glycoprotein molecules

that are produced by plasma cells in response

to an immunogen and which function as

antibodies.


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FUNCTIONS OF Ig

Antigen binding

- Primary function

- Highly specific Effector function

- Complement fixation resulting in cell lysis

- Binding to phagocytes, lymphocytes, andother cells


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Plasma immunoglobulins play a major role in

the bodys defense mechanisms

B cells are responsible for their synthesis

They are circulating, humoral antibodies

Plasma immunoglobulins are synthesized

mainly by plasma cells in response to antigen

exposure


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IMMUNOGLOBULINS

Contain a minimum of two identicallight (L) chains (23 kDa) and two identical

heavy (H) chains (5375 kDa), held together as

a tetramer (L2H2) by disulfide bonds The half of the light (L) chain toward the

carboxyl terminal is referred to as the constant

region (CL), while the amino terminal half is

the variable region of the light chain (VL).


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STRUCTURE OF IMMUNOGLOBULIN G

(IgG)

Each light chain

consists of a variable

(VL) and a constant

(CL) region

Hinge region confers

flexibility in binding

to antigenic sites


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(IgG)

Digestion of an

immunoglobulin by the

enzyme papain produces

two antigen-bindingfragments (Fab) and one

crystallizable

fragment (Fc), which is

responsible for functionsof Ig


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(IgG)

Each heavy chain

consists of a variable

region (VH) and a

constant region that

is divided into three

domains (CH1, CH2,

and CH3).


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(IgG)

The CH2 domaincontains thecomplement-binding

site CH3 domain contains

a site that attaches to

receptors onneutrophils andmacrophages


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(IgG)

The antigen-binding

site is formed by the

hypervariable

regions, also called

complementarity-

determining regions

(CDRs)


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VARIABLE REGIONS

No two variable regions from different

humans have been found to have identical

amino acid sequences

The interactions between antibodies and

antigens involve noncovalent forces and

bonds (electrostatic and van der Waals forces

and hydrogen and hydrophobic bonds)


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CONSTANT REGIONS

Responsible for the class-specific effector

functions of the different immunoglobulin

molecules

Some immunoglobulins such as immune IgG

exist only in the basic tetrameric structure,

while others such as IgA and IgM can exist as

higher order polymers of two, three (IgA), orfive (IgM) tetrameric units


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Both IgA and

IgM have a J

chain, but only

secretory IgA

has a secretory

component


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THE LIGHT CHAIN

There are two general types of light chains,kappa () and lambda (), which can bedistinguished on the basis of structuraldifferences in their CL regions

A given immunoglobulin molecule alwayscontains two or two light chainsnever amixture of and

Inhumans, the chains are more frequentthan chains in immunoglobulin molecules


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THE HEAVY (H) CHAIN

Five classes of H chain have been found in humans,

distinguished by differences in their CH regions. They

are designated , , , and

The and chains each have four CH domains rather

than the usual three.

The type of H chain determines the class of

immunoglobulin and thus its effector function.

There are five immunoglobulin classes: IgG, IgA, IgM,

IgD, and IgE.


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The L chains and H chains are synthesized as

separate molecules and are subsequently

assembled within the B cell or plasma cell into

mature immunoglobulin molecules, all of

which are glycoproteins

Both light & heavy chains are products of

multiple genes


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Antibody diversity depends on gene

rearrangements

Each person is capable of generating

antibodies directed against perhaps 1 million

different antigens


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Both overproduction and underproduction of

Immunoglobulins may result in disease states

A severe reduction in synthesis of an

immunoglobulin class due to a genetic

abnormality can result in a serious

immunodeficiency disease


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Monoclonal antibodies (mAb or moAb) are

antibodies that are identical because they are

produced by one type of immune cell that are

all clones of a single parent cell.


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In the 1970s the B-cell cancer myeloma was

known, and it was understood that these

cancerous B-cells all produce a single type of

antibody


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A process of producing monoclonal antibodies

involving human-mouse hybrid cells was

described by Jerrold Schwaber in 1973 and

remains widely cited among those using

human-derived hybridomas


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Monoclonal antibodies are typically made by

fusing the spleen cells from a mouse or rabbit

that has been immunized with the desired

antigen with myeloma cells.


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Polyethylene glycol is used to fuse adjacent

plasma membranes, but the success rate is

low so a selective medium is used in which

only fused cells can grow.

This is because myeloma cells have lost the

ability to synthesize hypoxanthine-guanine-

phosphoribosyl transferase (HGPRT).


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Immortal myeloma

cells are fused with

HGPRT-containing Bcells from rabbit spleen

Resulting hybridoma

cells are immortalizedand produce

antibodies


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The selective culture medium is called HAT medium

because it contains:

Hypoxanthine

Aminopterin Thymidine

This medium is selective for fused, (hybridoma) cells

because unfused myeloma cells cannot growbecause they lack HGPRT.


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Unfused normal spleen cells cannot grow

indefinitely because of their limited life span.

However, hybridoma cells are able to grow

indefinitely because the spleen cell partner

supplies HGPRT and the myeloma partner is

immortal because it is a cancer cell.


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The Complement System

The complement system is an important

soluble component of the innate immune

system

It is a series of plasma enzymes, regulatory

proteins, and proteins that are activated in acascading fashion, resulting in cell lysis.

The complement system comprises about

20 Plasma Proteins

The Complement System


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The Complement System There are four pathways of the complement system:

1. the classic activation pathway activated by antigen/antibodyimmune complexes

2. the MBL activation pathway activated by microbes withterminal mannose groups

3. the alternative activation pathway activated by microbes or

tumor cells4. the terminal pathway that is common to the first three

pathways and leads to the membrane attack complex thatlyses cells

The series of enzymes of the complement system are serine

proteases


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Clinical assessment of immunity requires

investigation of the four major components of the

immune system that participate in host defense andin the pathogenesis of autoimmune diseases:

(1) humoral immunity (B cells)

(2) cell-mediated immunity (T cells, monocytes)

(3) phagocytic cells of the reticuloendothelial system

(macrophages), as well as polymorphonuclear

leukocytes

(4) complement


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Clinical problems that require an evaluation of

immunity include chronic infections, recurrent

infections, unusual infecting agents, and

certain autoimmune syndromes


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Defects in cellular immunity generally result in

viral, mycobacterial, and fungal infections

An extreme example of deficiency in cellular

immunity is AIDS

Antibody deficiencies result in recurrent

bacterial infections, frequently with organismssuch as S.pneumoniae and Haemophilus

influenzae


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Disorders of phagocyte function are

frequently manifested by recurrent skin

infections, often due to Staphylococcusaureus

Finally, deficiencies of early and late

complement components are associated with

autoimmune phenomena and recurrent

Neisseria infections


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IgG Increases in:

a) Chronic granulomatous infectionsb) Infections of all typesc) Hyperimmunizationd) Liver disease

e) Malnutrition (severe)f) Dysproteinemiag) Disease associated with hypersensitivitygranulomas, dermatologic disorders, and IgGmyelomah) Rheumatoid arthritis


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IgG Decreases in:

a) Agammaglobulinemia

b) Lymphoid aplasia

c) Selective IgG, IgA deficiency

d) IgA myeloma

e) Bence Jones proteinemia

f) Chronic lymphoblastic leukemia

I M I (i d l ) i


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IgM Increases (in adults) in:

a) Waldenstrm's macroglobulinemia

b) Trypanosomiasisc) Actinomycosisd) Carrin's disease (bartonellosis)e) Malaria

f) Infectious mononucleosisg) Lupus erythematosush) Rheumatoid arthritisI) Dysgammaglobulinemia (certain cases)


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In the newborn, a level of IgM above 20 ng./dl

is an indication ofinutero stimulation of the

immune system and stimulation by the rubella

virus, the cytomegalovirus, syphilis, ortoxoplasmosis.

I M D i


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IgM Decreases in:

a) Agammaglobulinemia

b) Lymphoproliferative disorders (certain

cases)

c) Lymphoid aplasiad) IgG and IgA myeloma

e) Dysgammaglobulinemia

f) Chronic lymphoblastic leukemia

I A I i


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IgA Increases in:

a) Wiskott-Aldrich syndrome

b) Cirrhosis of the liver (most cases)

c) Certain stages of collagen and other

autoimmune disorders such as rheumatoidarthritis and lupus erythematosus

d) Chronic infections not based on

immunologic deficienciese) IgA myeloma

IgA Decreases in


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IgA Decreases in:

a) Hereditary ataxia telangiectasia

b) Immunologic deficiency states (e.g.,dysgammaglobulinemia, congenital andacquired agammaglobulinemia, andhypogammaglobulinemia)c) Malabsorption syndromesd) Lymphoid aplasiae) IgG myeloma

f) Acute lymphoblastic leukemiag) Chronic lymphoblastic leukemia

IgD


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IgD

1. Increases in:

a) Chronic infectionsb) IgD myelomas

IgE

1. Increases in:

a) Atopic skin diseases such as eczema

b) Hay fever

c) Asthmad) Anaphylactic shock

e) IgE-myeloma


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IgE Decreases in:

a) Congenital agammaglobulinemia

b) Hypogammaglobulinemia due to faulty

metabolism or synthesis of immunoglobulins

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