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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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Immunogens or Antigens
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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Immunogens or Antigens
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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STRUCTURE OF IMMUNOGLOBULIN G
(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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STRUCTURE OF IMMUNOGLOBULIN G
(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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STRUCTURE OF IMMUNOGLOBULIN G
(IgG)
The CH2 domaincontains thecomplement-binding
site CH3 domain contains
a site that attaches to
receptors onneutrophils andmacrophages
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STRUCTURE OF IMMUNOGLOBULIN G
(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