B cell activation and antibody production

• One of the first scientists to explain the antibodies

and it’s work is the german scientist karl landsteiner
• In 1900, he was awarded the nobel prize in medicine
because he discovered the ABO blood system which
is a very important sytem to understand immunology.
• As you know there are 4 blood groups, which are
A,B,AB and O, and they reflect the antigens that are
present on the surface of the RBC’s and in the
endothelium (in blood vessels).
• Look at the figure to know the antigens and
antibodies of each blood group
Note: the O blood group has an antigen called H antigen
• The ABO blood group antigens are carbohydate antigens .
• The A and B antigens are dominant over the H antigen (O blood group) and co-dominant with
each other.
• The A and B genes code for proteins (enzymes) which add carbohydrate antigens to the H
antigen
• These enzymes add galactose in the case of B antigen and N-acetyl-galactoseamine in the
case of A antigen.
• Carbohydrate antigens stimulate the production of IgM antibodies

Phases of humoral immune response

• When we are born we will have almost 1011 naïve (not experienced ) B cells each with specific
antigen receptor (BCR).
• Humoral immune responses are initiated by specific B cell recognition of antigen in secondary
lymphoid organs, so antigens are the main stimulant for the activation of these cells.
• Sometimes during the activation we might need the help of T-cells.
• After activation, there will be proliferation
(clonal expansion).
• As seen in the figure, B cells use IgM and IgD
mainly as their surface receptors.
• In early stages of their development before they
acquire their receptors (Ig's) they are called Pro-B
cells then Pre-B cells and then B cells which have
the receptors , and finally when they do their
function and secrete these Ig's they are called
plasma cells.
Note: The main receptors in B cells are IgM and IgD,
but in late stages, they might acquire other Ig's such
as IgG.
• IgD only acts as a surface receptor, which cannot be secreted, whereas IgM can be
secreted when needed.
• The main stimulus that triggers B cells and the humoral immune response is the presence of an
antigen, when the cells recognize the antigen they will be activated which leads to proliferation,
they divide to produce many cells of the same type that recognized the antigen.
• This activation will lead to different responses as seen in the figure above:
1- Antibody secretion: cells secrete IgM as a response when they are exposed to the antigen for the
first time.
2- Isotype switching: occurs when there is need for another type of Ig such as IgG, IgE and IgA.
3- Affinity maturation: in late stages of antigen exposure, we will see that the affinity between the
paratope of the antibody and the epitope of the antigen is increased due to somatic mutations in the
variable region which lead to better attachment and fit of the antibody to the antigen.
4- Memory B cells: which are long living cells that circulate for a longer time, and when the second
exposure of the antigen happens, these cells will respond faster and more aggressively than the
Naive (virgin) cells.
• When the antigen is eliminated ,the immune response will be inhibited.

Primary and secondary immune responses

• The production of memory cells is the reason for
the presence of a very important phenomena that
distinguishes the adaptive response from the
innate response which is the difference between
the primary and secondary immune response.
• The cells that respond in the primary response
are Naive B cells , this response usually takes
about a week ( delayed) , and its main Ig is IgM
(sometimes in late stages the cells will produce
IgG but in low amounts) ,and it has a low peak
(the number of Ig's produced is low).
• In the second exposure, the responding cells will
be memory cells which are faster (2 -3 days
usually) and more diverse, there will be a higher
peak and different Ig's (not only IgM).
• Another thing that distinguishes the secondary
from the primary response is the type of the
immunogen, in the first response cells will
recognize carbohydrates mainly which are less
dangerous from proteins, and then the cells will produce IgM, while in the secondary response it
will be proteins (such as Rh system antigens) and there will be involvement of T cells and different
Ig's.
Note: if the secondary response was also by a carbohydrate antigen, there will be NO isotype
switching, only IgM will be produced (similar to the primary response).
Note: all non-protein antigens induce primary immune response only no matter how many time they
infect the body.

• In the figure (you can see that there are different responses , in the spleen and other lymphoid
organs like tonsils, there will be 2 pathways of B cell development depending on the antigen :
1- The response for a protein antigen and the help of
T cells (T dependent) , meeting will happen in the
germinal centers and the affinity of antibodies will
increase by little modifications in the variable region.
Also by help of T cells, there will also be isotype
switching in which other Ig's are formed such as IgG,
IgA and IgE . The B cells that undergo this pathway
are called follicular B cells.
2- Response to other antigens like polysaccharides
and lipids by Marginal B cells, this response is T
independent and is mainly done through the
production of IgM only .
3-The third response in nearly similar to the second one but involves B-1 cells ,and happens in
mucosal tissues and peritoneal cavity.

• As you see, the figure represents how the

antigen is brought to follicular B cells in
the lymph nodes (which are involved in T-
dependent pathway as we said before).
• Normally the meeting between the B cell
and the antigen happens in the secondary
lymphoid organs.
• The PROTEIN antigen will be brought
either directly (small antigens usually)
through conduits or by presenting cells for
larger antigens (macrophages in the
subscapular sinus and dendritic cells in
the medulla).
• Macrophages are present in lymph nodes/
and normal tissues where they engulf the
antigen and then go to the regional lymph node and present it to different cells (B and T cells)
each type has a specific location in the lymph node (cortex and paracortex respectively).
• B-cell receptor recognizes the antigens by its hypervariable regions, this leads to the initiation of
signals that would lead to the activation of the B-cell, but B-cell receptor is not enough for the
activation, another signals (co-stimulation) should enhance this pathway of B Cell Activation, for
example, we have:
- The alfa and beta chain that are Associated
with the BCR. they have long cytoplasmic tails
which play a role in this process,
- There are two mechanisms with two different
receptors that will help in the activation of B
cells :
1. through the interaction between CR2/CD21
with the complement coated microbe, it will
enhance the BCR signaling which in the End will
lead to the proliferation and differentiation of the B
cell, the complement receptor 2 will recognize the
antigens that are coated by complement (C3d).
2. (TLR) The tool like receptors also play a role in
the activation by recognizing pathogen associated
molecular patterns (PAMP) that are present only on
pathogens .
• Some viruses are very clever, they use The receptors that are present on certain immune cells in
order to infect and destroy them, for example, *the HIV virus enters the T helper cell through its
CD4 receptor on its surface, another important virus that affects the B CELL is called Epstein-
Barr virus EBV, it enters the B-cell through the CD21/CR2, this virus causes long-standing flu-like
symptoms that may extend to a month or a year or more, the clinical disease that it causes is
called infectious mononucleosis.

• Naïve B cell binding to antigen will increase:

1. Increased expression of cytokine receptor,
which will increase the responsiveness of these
cells to cytokines.
2. Increased expression of CCR7 (Chemokine
receptor) : for migration of B cell to T cell zones
where the interaction between them happens.
3. Increased expression of B7 (B works as APC) to
aid in the presentation of Ag’s.
4. expression of proteins that promote survival and
proliferation.
sequence of events in humoral immune responses to T-cell dependent protein
antigens
• Immune responses are initiated by the recognition of antigens by B cells and helper T cells.
• The activated lymphocytes migrate toward one
another and interact, resulting in B cell proliferation
and differentiation.
• Restimulation of B cells by helper T cells in
extrafollicular sites leads to early isotype switching
and short-lived plasma cell generation.
• The late events occur in germinal centers and
include somatic mutation and the selection of high-
affinity cells (affinity maturation), additional isotype
switching, memory B cell generation, and the generation of long-lived plasma cells.
• Some diseases are caused by the failure of T and B-cell interactions.

Germinal centers in secondary lymphoid organs

• immunoflourescence technique (Right picture), the left one is seen by light microscope.
A, Histology of a secondary follicle with a germinal center in a lymph node. The germinal center is
contained within the follicle and includes a
basal dark zone and an adjacent light zone.
The mantle zone is the parent follicle within
which the germinal center has formed.
B, Cellular components of the germinal center.
A secondary follicle has been stained with an
anti-CD23 antibody (green), which brightly
stains follicular dendritic cells in the light zone
and dimly stains naive B cells in the mantle zone. Anti-Ki67 (red), which detects cycling cells, stains
mitotically active B cell blasts in the dark zone.

Immunologic synapse in which different

immunologic cells meet together such as B
cells, T cells and monocytes , and this is
important for coordination between these cells
to happen.
• The B cell can also be activated by a helper T cell , this is done by tow
mechanisms after the T cell recognize the antigen expressed B cells ,
these tow mechanisms include:
1. The release of cytokines from T cells that will bind to their receptors on
B cells.
2. By other costimulatory pathways through the CD40 ligand on T cells
that has its receptor on B cells (CD40).
• there is a disease called hyper IgM syndrome, it's a genetic disease that
affects the gene that codes for the CD40 ligand (CD156) on helper T
cells, the absence of this ligand will lead to the hyper IgM secretion ,
where the patient synthesizes huge amounts of IgM and only little
amounts of IgA , igG.

• we have 5 classes of Ig’s, and 9 different subclasses

• different cytokines released from the helper T-cell
after it interacts with the B cell , causes the B cell to
differentiate to plasma cell and do isotype switching ,
from IgM to IgG, IgA, IgE, for example
1. When the T-cell secrets IFN-gamma, the B-cell will
switch from IgM to IgG that has a very important role in
opsonization, phagocytosis, complement activation, and
neonatal immunity especially in the first 9 months to one
year.
2. When the T cell secrets interleukin 4 the B cell will
switch to IgE, which is important in the immunity
against helminths and hypersensitivity reactions.
3. When the T cell secrets other cytokines like TGF-beta
the B cell will switch to IgA which is important in mucosal
immunity.

• First, you have to know the order of the gene locus of the
constant heavy chain for different immunoglobulins types
on the DNA, the order is IgM, IgD, IgG, IgE, IgA .
• As you can see the igA is located at the end of the DNA
locals so it has the highest chance to be missing.
• In this picture it is explained how the B-cell do isotype
switching when it transforms to a plasma cell that secret
immunoglobulins , as we said this is controlled by the
type of a cytokine that the T helper cell secrets let's take
for example when the helper T-cell secrets interleukin 4
this cytokine will affect enzymes that are present in the
 nucleus these enzymes will do recombination on the DNA and this recombination will delete a
segment on the DNA that contains the locus for the heavy chains
that the cell don't want to secret, and keep the locus for the IgE
antibody this will lead to the shortening of the DNA, and this is a
very special process that doesn't happen in any cell in the body.

• There's a process that happens in the germline center of different

lymphoid organs that will lead to Stronger binding of immunoglobulin
secreted by the plasma cell, this process is called affinity maturation
(somatic mutations)
• After the B-cell has been activated it will proliferate and as it
proliferates it will undergo this process some of the B cells will
succeed at making their immunoglobulins with high affinity , the
cells that failed at this would undergo apoptosis.
• This process is done by making point mutations on
the variable chains locals on the DNA, these mutations
are specifically done on the complementary
determining regions (the hypervariable regions).
• You should also know that the Kd number which is the
dissociation constant, decreases as the Affinity gets
stronger(As the cell accumulates mutation on its
hypervariable regions).

• This image shows how alternative processing of

the primary RNA transcript will result in switching
the immunoglobulin from a transmembrane form
into a secreted form,
• If the polyadenylation happened after the
cytoplasmic and transmembrane segment, the
immunoglobulin translated would be a
transmembrane immunoglobulin (membrane
bound).
• And if the polyadenylation happened Before the
cytoplasmic and transmembrane segment, it
would be as in a secreted form , and this
depends on the state of the B cell.
• The important thing to know is that it happens at the level of the RNA not the DNA, which means
that the whole gene Gets transcribed, then the processing happens.
Note: the difference between membrane and secreted form of the antibody is the transmembrane
and the cytoplasmic segments.
Helpful videos
https://www.youtube.com/watch?v=C7pmDXsF7CA

https://www.youtube.com/watch?v=JHgUzLCWqvw

questions:
1- IFN gamma stimulates the production of:
a) Monomeric IgA
b) IgG class 1 and 3
c) Dimeric IgA
d) IgE

2- What is the exact anatomical site for affinity maturation of B cells for antigen?
a) Spleen
b) Germinal centers
c) Lymph nodes
d) Thymus
e) Bone marrow
3- First step after binding of an Ag on BCR:
a) Isotype switching
b) Affinity maturation
c) Proliferation and expansion
d) Production of plasma cells
e) Non of the above
4- The effect of CCR7 is:
a) Proliferation
b) Migration
c) Activation
d) Maturation

5- All true about T-independent immune response except:

a) Production of IgM mainly at first exposure
b) Against non-protein carbohydrate Ag
c) Production of Short lived plasma cells
d) Caused by the follicular B cells
e) No isotype switching

6- EBV receptor is:

a) CD21
b) CD8
c) CD4
d) CD64
e) CD16

Answers: 1-b 2-b 3-c 4-b 5- d 6-a

RH blood group system
• This system was dicovered by coombs and
landestiener in 1930.
• The antigens of this sytsem are protein
antigens, so the antibody that will form is IgG
which can cross the placenta..
• The locus for RH group is present on
chromosome 1.
• Rh positive: has the Rh (D) antigen, no anti-
Rh Ab, .
• Rh negative: no Rh (d) antigen, has anti-Rh
Ab.
• Negative can donate and positive and
receive but not vice versa
• 90% of people are positive Rh, while 10% percent are negative Rh, Rn is a protein antigen on the
surface of RBC’s, Rh system have 3 antigens which are C, D and E, D is the most important one.
• If a pregnant lady with a negative Rh, and her husband is Rh positive (remember that Rh positive
is dominant over Rh negative). So their baby will have a positive Rh blood.
• The mother’s blood circulation is separated from the baby’s circulation, during the delivery, the
mother’s blood and the baby’s blood are exposed to each other.
• So during labor, the mother will form antibodies against the Rh antigens, but this is not dangerous
now because the fetus is already delivered, and only low amount of antibody is produced, also
IgM antibody can’t cross the placenta.
• If the second or third pregnancy results in Rh positive
baby, this will be dangerous because the fetus is still
in the uterus, high amount of antibody, IgG antibody
is produced which can cross the placenta, this will
result in destruction of the fetal blood and jaundice
and the baby will be lost in the middle of the second
or third pregnancy, and the mother may become
infertile (secondary infertility) this process is called
hydrops fetalis
• What we do to prevent this from happening is that we
test the blood type for the mother before or in the first
months of pregnancy if it’s negative of not, if it’s
negative we test the father also, if he is negative everything will be fine, I he is positive we have to
solve this problem.
• To solve this problem, during delivery we test the baby if he has a positive or negative Rh, if the
baby is positive we give the mother an injection within 2-3 days of delivery called Rhogam (anti-D
Abs).
• The mechanism of action of this injection: it destroys Rh Ag on the surface of neonatal RBC’s
before their recognition by maternal immunity and blocks production of anti-Rh Abs by binding to
Fc gamma 2B (FCyRIIb) receptors on B cells and this will lead to feedback inhibition (inhibitory
receptor).
Some notes about the previous lecture:
• No matter how many times we are exposed to ABO system, we will always IgM antibodies and not
IgG because they are carbohydrate antigens.
• During the first year of our life, we get our antibodies from the mother, then we will start making
our antibodies
• some bacteria and food has the same Ags of ABO system, so we have antibodies even if we are
not exposed to blood products.

Effector mechanisms of humoral immunity

• immune response has three stages, the first is recognition then activation then killing mechanism
or effector mechanisms of humoral immune response.
• We can stimulate the effector mechanisms of Humoral immunity through “vaccines”.
• The first vaccine was against small pox that is a very dangerous disease and it’s very easy
transmitted and communicable through droplets, this dangerous disease will lead to death of 50%
in affected patients (very high mortality) and if they are treated they will have deafness or
blindness and skin rash on his face and hands in 90% or 100% of the patients who survived.
• Note: the small pox was irradiated from the world in 1980.
• A good vaccine has to have these features: it should give a life-long immunity against the infection
(agent), without side effects or problems, easy to manufacture and give and cheap.
• Tetanus, diphtheria and other bacteria that release toxins which are neutralized by toxiods
vaccine. Tetanus toxoids release Ab that inhibit the toxin from certain bacteria and its
neutralization is by IgG system, while polio neutralization is IgA system. Also the pneumococcal
pneumonia is neutralized by opsonization and phagocytosis.
• Note: polio vaccine have oral attenuated and injection forms.

Effector functions of antibodies

• Effector mechanisms of Ab’s are either direct by Ab itself or through the complement system
(complement the action).
• The direct functions are:
1. neutralization of microbes and toxins
The importance of neutralization of toxins and microbes: blocks penetration of MO through barriers,
blocks binding of organisms and infection of cells, blocks binding of toxin to cellular receptors.
 2. Opsonization and phagocytosis of microbes
3. antibody dependent cellular cytotoxicity.
The remaining functions through complement activation:
1. Inflammation
2. Lysis of microbes
3. Phagocytosis of microbes opsonized with
complement fragments (C3b).

Classes and functions of Antibodies

There are 5 classes and 9 subclasses of antibodies:
- IgD it appears with IgM on the surface of B cell.
- IgE is monomer, has the lowest level in serum, it’s
receptor is present at the surface of mast cell and
basophils in case of allergic reactions, aids in actions
against helminthic and parasitic infections, isotype
switching by IL-4.
- IgG has four subclasses, monomer, has the highest level in
serum, has a long half-life, function in neonatal immunity
(can cross placenta), also it is has many functions and in
relation with ADCC by NK cells, isotype switching by:
interferon gamma.
- IgA in mucosal immunity, secreted mainly as a dimer,
isotype switching by IL-5, selective igA deficiency is the
most common deficiency in adults.
- IgM (natural antibody) is the first antigen to be secreted,
pentamer in the secreted form (10 fabs), monomer in the
receptor form, can’t cross placenta, used for CHO ags
mainly, best in complement fixation, short half-life (5-7)
days, antigen receptor of naïve B cell.

neutralization of microbes and toxins

- The Ab prevents infection and it works against
toxins by preventing toxins from binding with its
receptors on cell surface.
- Without Ab’s there will be cell necrosis for example,
but blocking Ab will inhibit this .
- The first function can be directly with Ab on a toxin
or pathogens by blocking them and prevent
infection, damage, toxicity and in many applications
Ab block antigens and prevent diseases.
- that means Ab inhibits toxicity from certain bacteria
or toxic molecule.
Note: this mechanism is done through the variable
region of the antibody, not the FC region.
Fc receptors:
1- Fc alpha (CD 89): it binds IgA, it is found on neutrophils , eosinophils and monocytes also their
function is unknown.
2- Fc epsilon (CD23): is found on B cell, eosinophils, basophils and Langerhans cells and it has low
affinity receptor (FcER2) for IgE and it’s function is unknown. The high affinity (FCER1) is in basophils
and mast cells (it makes mast cell degranulation in allergic (anaphylaxis) reactions).
3- IgG has 3 receptors
• Fc gamma receptor 1 has high affinity
for immunoglobulins.
• Fc gamma receptor 2 has intermediate
affinity for immunoglobulins.
• Fc gamma receptor 3 is low affinity for
immunoglobulins (it has 3a and 3b that
have relation to ADCC).

➢ Fc gamma 1 (CD64) is one chain and

used in phagocytosis + opsonization +
activation of phagocytes.

➢ Fc gamma 2a and 2c (CD32) used in

phagocytosis + opsonization + activation of phagocytes.
But 2b is used in feed-back inhibition (for example RhoGAM drug in RH-incompatibility) it is present
of B cells, macrophages and DC.

➢ Fc gamma 3 (CD16) has a and b chains that work in ADCC like NK cell.
ADCC (antibody dependent cellular cytotoxicity):
• MO is coated with igG.
• NK has an Fc receptor FCyRIII ( CD16), which is
marker to detect NK.
• CD16 binds the segment of igG.
• NK cell kills the target MO.

-The activation of Complement through the classical pathway is thought to be a part of adaptive
humoral immunity (specific or acquired immune response) because it starts with the antibody.
- Complement system which is a group of proteins (40-50), is part of the innate immunity (natural
immunity or non-specific immunity) by the two other pathways:
1. Alternative pathway.
2. Lectin pathway.

Classical pathway
• It is called classical Because it’s the first pathway to describe the
Antibody antigen immune complex formation.
• When we have either two molecules of IgG molecules or one
molecule of IgM attached to its antigen; the C1 molecule attaches.
• If the antibody is alone (not attached to antigen)," no immune
complex", the complement (C1) will not attach and there will be no
action. But when the antibody recognizes the antigen then the site for
complement C1q attachment to the immune complex or to the antibody becomes available (FC
portion).
• when we have IgG, we need two adjacent molecules of it.
• IgM is a pentamer (5 immunoglobulin molecules) and one molecule is
enough.
• So IgM is more potent complement activator than IgG in the classical
pathway, BUT both IgG & IgM lead to complement classical pathway
activation.
• C1 is the first molecule of complement system (50 proteins) and it
consists of C1q, C1r & C1s (two copies for each).
• In the classical pathway, we start with C1 molecule when it becomes
activated (and C1 becomes an enzyme "CD protease" that digests
the other complements (converts C4 to C4b (stay) & C4a, C4a (lost
and called anaphylatoxin which will go to another site and leads to
inflammation), and C2 → C2a (stay, big) & C2b (lost, small)
(Exception).
• Complement activation : is an enzymatic activity (serine protease)
of the complement molecules, the small part of this molecule will be
lost while the large part will stay to form the next action and it is
 usually enzymatic action (proteolysis). When it continues to the end it causes the complement
functions.
• So classical pathway starts with C1 then the cleavage of C4 converting it to C4b, and the
conversion of C2 to c2a Thus, forming C3 convertase (C4b2a), and then the common pathway of
complement activation starts.
• C3 convertase is an enzyme that converts C3 to it’s active form.
Note: if there’s no antibodies, the complement system can kill and recognize microbes through:
alternative pathway & Lectin pathway.

alternative pathway
• it Is a spontaneous and slow process.
• C3 → C3a (LOST) & C3b, becomes C3
convertase.
• On normal cells of our body there are
inhibitors that inhibits this pathway, while on
the surface of microbes these inhibitors are
absent.
• In Alternative pathway, factor D plays a role
in the cleavage of Factor B.
• properdin and factor D are proteins involved
in the alternative pathway.

Lectin pathway (or binding Lectin pathway or mannose binding Lectin pathway)
• Certain receptors or certain ligands like mannose.
• Mannose is located on cell surface of bacteria not in our normal cells.
• Mannose binding Lectin is a molecule similar to C1 and has other molecules and it shares certain
similarities with the classical pathway.
• Mannose-binding Lectin is a type of collectins (a protein family).
• Mannose activation specific proteins (MASP) 1 & 2 recognize mannose on the surface of
microbes ,then it works on C4 & C2 < similar to classical pathway but classical pathway needs
antibodies (specificity of antibody to antigen),► Formation of C3 convertase of the Lectin pathway
(recognition mechanism).
In conclusion, Activation of the complement through three recognition pathways: Classical pathway
(start with immune complex), alternative pathway through C3 convertase or C3 take over and
spontaneous activation of the C3, and the Lectin pathway C3 convertase by a mechanism for
recognition. All pathways lead to the C3 activation, when C3 becomes activated (means C3 → C3a &
C3b), then it is released and does certain action. C3 conversion will lead to activation or formation of
C5 convertase. C5 is converted into C5b (stays) and C5a. (C5a & C3a &C4a are called
anaphylatoxins which will cause inflammation). After that from (C5-C9) there are no enzymatic action.
Notes: C5 convertase is (C4b2ac3b), C3b works as
an opsonin.
• In Steps from C1 to C5, there are digestion and
conversion of the factor (complement factor),
complement factor B will stay and complement
factor A will be lost and cause problem.
• When we reach C5, there is addition without an
enzymatic action (C5 not enzyme), after that
there will be insertion (or attachment) of C6 and
Then C7, then C8 then there is a multiple copies
of C9 which form membrane attack complex
(MAC) which will form a hole or (attach itself and
penetrate to cell wall), water will go through these
halls which will cause the cell to lyse.
• In complement activation, the enzymatic action
will end at C5 and then (C6, C7, C8, C9) all of
them will make what we call ** lytic ** it will cause
a damage to the cell by making a hole in the cell
wall (plasma membrane) which is called **
membrane attack complex ** (MAC) this is a
killing mechanism of how the Bacteria or a virus
will die, or even human cell or animal cell.
• So, if the complement activation reaches to C9 it
will kill the cell and make a pore in the cell wall
so cell will die.

Actions of complement
1. Complement mediated cytolysis (MAC) leads to
osmotic lysis of any cell (virus or bacteria) and it
will kill them.
2. (C4a, C5a, C3a) are called anaphylatoxins (When someone is injured there will be an
inflammation and the area will become red, swollen and warm Because these anaphylatoxins that
are released in this area will cause recruitment of the phagocytic cells to that area. This will cause
vasodilatations and increased permeability of blood vessels (more blood) and the cells
(leukocytes) become activated by anaphylatoxins, this will lead to destruction of the microbes by
leukocytes.
3. The microbes with the complement on it will be recognized by phagocytes (similar to antibodies,
they can recognize the presence of the complement on the surface of the other cells and then
phagocytes will engulf it (opsonization and phagocytosis) and this is done by complement
receptors.
Receptors for fragments of C3:
1. Type 1 complement receptors (CR1) (CD35) mainly in Red blood cells → it’s function: clearance of
immune complex to prevent autoimmune disease like SLE.
• when there is a toxin in the blood and the
antibody recognizes it, we don’t want it to
stay in the blood, we aim to remove it
from the circulation, How? by
complement receptor type 1 (CD35)
mainly on Red blood cells (this receptor
will carry the RBC and the immune
complex and every 120 days CD35 will
remove aged red blood cells from
circulation through spleen and get rid of
immune complexes.
2. Type 2 Complement receptors (CR2) (CD21) → leads to activation of the B lymphocytes. (it’s a
receptor for EBV: Epstein–Barr virus which causes infectious mononucleosis).
• Complement receptors 3&4 lead to phagocytosis.
• All the complement receptors in general mainly for phagocytosis.
• as a quick summary: if the complement continues till the end it will form MAC and it will kill the cell
by (lysis). Or if it stops there will be another action through complement receptors.

Complement system is a very potent system, so if it was left activated with no control it will lead to
many problems and disease.

Regulators of complement are two types:

1. In the serum (soluble)*plasma proteins: (C1 esterase inhibitor).
2. In the cell surface: Decay- accelerating factor, CD59, CD55 (in human cells and animal cells that
can stop the complement activation).
Two diseases caused by deficiency of these regulators:
Since the regulation of complement system in our bodies is very efficient, these diseases are rare
actually, but still they are true and could happen sometimes.

1. The decay- accelerating factor deficiency: there is a disease called Paroxysmal nocturnal
hemoglobinuria. This disease may be found in Japan, leads to break in Red blood cells and platelet,
hemoglobin will get out from red blood cells so we will find it in serum and urine.
2. C1 esterase inhibitor deficiency leads to hereditary angioneurotic edema.
Helpful videos:
https://www.youtube.com/watch?v=dTb0iEUS1oA

Questions:
1- Paroxysmal nocturnal hemoglobinuria is associated with:

a) C1 inhibitor
b) Factor i
c) Factor H
d) DAF
e) CD59

2- CR1 is found on:

a) Basophils
b) RBC’s
c) Eosinophils
d) Mast cells
e) None of the above

3- EBV receptor:

a) CD8
b) CD4
c) CR2
d) CD16
e) CD64

4- Hereditary angioneurotic edema is associated with:

a) CR2
b) CR3
c) C1 inhibitor
d) CR4
e) DAF

5-Which of the following has receptor for IgE:

a) TH1 cells
b) Mast cells
c) TH2 cells
d) CTLs

6- ADCC of NK cells is mediated by which Ig:

a) IgA
b) IgG
c) IgE
d) IgM
e) IgD

7- Which molecule is not involved in B cell activation:

a) CD21
b) CD16
c) CD19
d) Surface igM

Answers: 1- d 2-b 3-c 4-c 5-b 6- b 7-b

 Regional Immunity
• Although the same basic principles underlie immune functions all over the body, the immune
system still displays various characteristics in different body regions according to their specific
buildup and function. Such body regions include:
1. Those whose physiological roles (their functions) require direct interaction with the outside
environment, so they are covered by epithelial barrier tissues and are in direct interaction with
their respective normal flora systems. These areas display immune response peculiarities different
from elsewhere in the body, thus giving rise to such immune system arms as the:
→ Mucosal Immune System: specialized Immune System in body systems lined with mucosa,
mainly immunity in the Gastrointestinal tract, and other mucosal tissues like Genitourinary tract
and Respiratory tract.
→Cutaneous Immune System: Immune System interacting with the skin, thus protects it.
2. Those areas which are particularly susceptible to inadvertent injury by inflammatory and immune
responses, and therefore experience immune privilege.
These regions are: Brain, eyes, testis and fetus of the pregnant woman.

Immunity in areas exposed to the environment:

• The basic organization of those immune systems is:
1. Integration of innate and adaptive immune mechanisms (incorporate & work together to achieve
the strongest possible Immune Response).
2. Outer epithelial layers to prevent microbial invasion (prevent microbial entry to the body).
3. Underlying connective tissues (e.g. lamina propria in mucosal areas, dermis in skin) which contain
scattered immune cells of various types (e.g. lymphocytes, dendritic cells, macrophages), some of
which may be peculiar to that specific region.

→In mucosal areas, these immune cells might take also the form of local specified arrangements
called mucosa-associated lymphoid tissues (i.e. MALTs).

4. Draining local lymph nodes (lymph drained from those areas goes to local Lymph Node where the
Immune Response might take place.
5. Preferential homing of lymphocytes to the specific region in which they were initially activated.
6. Importance of regulation to prevent unnecessary responses to nonharmful substances.

Mucosal Immune System:

The main features of the mucosal immune system are:
1. Relatively impermeable epithelial barriers.
2. Secretion of mucin and defensins.
3. Localized subepithelial collections of lymphoid tissue (i.e. MALTs).
4. Constant sampling of antigens beyond the barrier by immune cells from within.
5. Integration of proinflammatory and regulatory signals generated by microbial products binding to
immune receptors on epithelial and dendritic cells (to differentiate between harmful & harmless
objects).
6. Reliance on secretory IgA-mediated humoral responses.
7. Stimulation of particular types of effector and regulatory T cell responses.
Immunity in the Gastrointestinal Tract:
• The gastrointestinal tract (GIT) is characterized by its large surface area and abundance of normal
flora, which creates challenges in identifying potentially harmful microbes and telling them apart
from the much more numerous normal flora microbes.
• Both the innate and adaptive immune
responses play an important part towards
achieving that goal.
• Various GI epithelial cell types play different
roles related to immunity:
1) Goblet cells: located at the top of the intestinal
villi and secrete mucus.

2) Paneth cells: located at the bottom of the crypts

and secrete antibacterial peptides.
3) Microfold (M) cells: found in the dome
structures overlying lymphoid tissues and act in
antigen sampling.

→ 1&2 play roles in innate immunity, while 3 plays a role in adaptive immunity.
→ Ag sampling is taking the Ag from the lumen to the underling tissue, specifically to the immune
cells, to determine whether it’s harmful or not.

Innate immunity in the GIT:

1) Mucins: they are secreted and cell surface glycoproteins produced by mucosal goblet cells and
submucosal glands, the composition and rate of production of mucin is affected by various stimuli
(e.g. cytokines, TNF, neutrophil products).

These mucins would then form:

→ Secreted mucus: a single layer in the small intestine, and two layers in the colon, with the
bacteria trapped away from the epithelial surface
→ Glycocalyx(membrane bound/cell surface mucin): which is formed by the combination of
membrane-bound mucins with various glycolipids,
Mucins would provide a barrier against microbes , decoy function, and matrix for display of
antimicrobial peptides.
2) Antibacterial peptides: produced by the intestinal epithelium cells, affect the integrity of microbial
structure.
→Defensins: disrupt the integrity of microbial outer membranes (in Gram negative bacteria)
- -defensins: (e.g. HD5, HD6): produced by the small intestinal Paneth cells as well as by
neutrophils.
- -defensins: produced by colonic crypt cells.
→C-type lectins (e.g. REGIII): bactericidal substances which bind to peptidoglycan (in Gram +ve
bacteria).
3) Pattern recognition receptors
→ Membrane-bound: Toll-like receptors (TLRs)
→ Cytoplasmic: nucleotide oilgomerization domain (NOD)-like receptors (NLRs)
- They recognize pathogen-associated molecular patterns (PAMPs) (e.g. lipopolysaccharide,
peptidoglycans), thus promoting responses
against pathogens whilst suppressing those
against commensals.
TLR and NLR activation might lead to:
1. Tightening of interepithelial junctions (Increase
integrity, decrease permeability)
2. Promoting intestinal motility and epithelial
proliferation (Increase integrity)
3. Stimulating the secretion of antimicrobial peptides
and IgA.
- Compartmentalization may help in guiding the
nature of the immune response upon TLR
activation.

4) Innate lymphoid cells (ILCs): derived from the lymphoid lineage but lack specific antigen
receptors. They are activated by cytokines (some of which are called alarmins), after which they
exhibit helper functions analogous to TH subsets.
ILC types include:
→ ILC2s: activated by IL-25 + IL-33 to secrete IL-5 (which activates eosinophils) and IL-13 (which
increases mucus production). Both those functions help in fighting off parasitic infestations. Exhibit
TH2
→ ILC3s: activated by the alarmin IL-1β + IL-23 to produce IL-17 + IL-22, thus promoting
inflammation, defensin production, and tight junction function. Exhibit TH17

Adaptive Immunity in the GIT:

• The major features of adaptive immunity in the GIT are:
1. Relative importance of humoral immunity in the form of secretory IgA.
2. Cell-mediated immune responses are primarily mediated by TH17 cells.
3. Importance of Treg cells in controlling immune responses towards tolerance of antigens from food
and commensal microbes.
‫ال‬
→Gut-associated lymphoid tissue (GALT) takes the form of such structures as:
• Peyer’s patches(at the beginning of the digestive tract in the intestine)
• Tonsils (whether lingual, palatine or nasopharyngeal)
• As well as smaller aggregates of lymphoid follicles
- Those follicles are primarily made of B cells, TH cells, follicular dendritic cells and
macrophages.
- The dome areas lie between the follicles and the overlying epithelium, richer in B cells, while
parafollicular areas are particularly richer in T cells compared to other GALT areas.
→ M- Cells: M cells move particulate matter from the lumen first by endocytosis, then by moving the
endosomes across the cytoplasm to the basolateral membrane.
- There, they would be delivered through
exocytosis to B cells and dendritic cells
whether within GALT or scattered in the
lamina propria.
- Unlike macrophages or dendritic cells, M
cells do not process the matter which it
transports and can’t present them to T
cells.
- The antigens delivered to the lamina
propria would then initiate immune
responses whether locally or in draining
lymph nodes.
- During TH cell activation, dendritic cells secrete retinoic acid to imprint a gut-homing phenotype on
B cells and effector T cells through integrin- and chemokine receptor-dependent mechanisms.

Humoral immunity in the GIT:

→ Secretory IgA (whether produced locally or, in the case of breastfeeding, secreted in colostrum or
breast milk) is the main antibody isotype in the GIT, and it mainly acts through neutralization( direct
binding with the Ag and stop it’s pathogenicity) or reducing microbial motility( i.e by binding to
bacterial flagella).
→ Chemicals which mediate IgA isotype switching include the cytokines TGF-β and APRIL, retinoic
acid, and nitric oxide.
→ IgA-producing plasma cells in the lamina propria secrete IgA in the form of a dimer of 4-chain basic
units where a J chain is bound to the Fc
regions of the chains.
The dimer would then bind to the poly-Ig
receptor, move across the cytosol
through transcytosis, then released from
the apical membrane through cleavage
of the poly-Ig receptor (on the basal
area of the epithelium, can transport
both IgA and IgM that’s why it’s called
poly), thus giving rise to the secretory
component).
Other Aspects of Adaptive Immunity ( T cells):
• Most of the intraepithelial T cells are CD8+ with a significant T
cell population, while elsewhere in the gut the CD4+ T cells
predominate.
• As antigen-presenting cells, dendritic cells and macrophages
sample antigens which have breached the epithelial barrier,
and sometimes even send projections across the epithelium to
take samples from the lumen.

• The TH17 pattern predominates in the gut, with less occurrence

of the TH2 and TH1.
• Treg cells are also relatively abundant in the gut, as their
formation is usually induced by TGF and retinoic acid, and
they usually exert their action through producing IL-10.

Immunity in Other Mucosal Areas:

Immunity in the respiratory tract→ it shares many features with that of the GIT.
• It produces mucus and defensins + cathelicidins (which have antimicrobial properties)
• It also produces surfactant proteins (which are involved in viral neutralization, microbial
clearance and inflammatory suppression)
Note: The mucociliary escalator is an active mechanism to keep the tract free from any foreign debris

Immunity in the genitourinary tract→ shows less prominence of MALTs compared to other
mucosal areas(mostly scattered), and it is peculiar in the fact that most of the antibodies in genital
secretions are of the IgG isotype (Remember: In the GIT IgA predominates)

Cutaneous Immune System

• The other Immune System that is exposed to the
environment is Cutaneous Immune System.
(Innate)→ Besides providing a physical barrier function,
the epidermal keratinocytes produce antimicrobial
peptides (e.g. defensins, cathelicidins) and various
cytokines.
→ If this barrier is breached, dermal macrophages, mast
cells and ILCs (Innate lymphoid cells) initiate innate
immune responses. i.e: ILCs 1, which exhibit TH1 cells,
and they are activated by IL-18 and produce IFN
→ PAMPs and damage-associated molecular patterns
(DAMPs) activate pattern recognition receptors (e.g.
TLRs)
(Adaptive)→ Antigen processing and presentation in the skin is carried out by the epidermal
Langerhans cells and the dermal langerin-expressing dendritic cells .These APC interact with the
cutaneous T cell population, 95% of which have a memory phenotype (98% of which reside in the
dermis and only 2% in the epidermis)
→ Upon T cell activation, vitamin D appears to play a major role in imprinting homing pattern on
them. (Remember: In GIT mucosa retinoic acid plays this role).

Immune-Privileged Areas
Areas that are overly sensitive to the adverse effects of immune reactions, therefore the immune
response is tweaked away towards a more suppressed one. These areas include:
1) Brain. 2) Eyes. 3) Testis. 4) Fetus of the pregnant mother

• Mechanisms which suppress immune and inflammatory responses in the Brain:

1. The tight junctions among the local microvascular endothelium (i.e. blood-brain barrier)
2. The action of neuropeptides
3. Scarcity of dendritic cells (relatively low amount of them)
4. The relatively high threshold for microglial activation
→ Even though, a basic level of immune response still takes place there.
The evidence: The occurrence of opportunistic infections in the brain and the presence of local
lymphatics.
• Features which contribute to immune privilege of the Eye:
1. Corneal avascularity
2. The blood-eye barrier near the anterior chamber (bacause of the tight junctions)
3. Absence of lymphatics there
4. Presence of soluble factors with immunosuppressive and antiinflammatory properties in the
aqueous humor.

• Features which contribute to immune privilege of the testis:

1. A blood-tissue barrier which limits cellular access into testicular tissue
2. The antiinflammatory effects of androgens (i.e Testosterone)
3. immunosuppressive effects of TGFβ

• Fetus of the Pregnant Mother: the fetus represents a naturally-occurring allograft, so various
mechanisms are needed to prevent the maternal Immune System from attacking it and
terminating the pregnancy. These mechanisms are:
1. Trophoblastic expression of HLA-G : a nonpolymorphic class I MHC molecule which inhibits NK
cell function and don’t express cytotoxic T cells
2. Lack of costimular molecules in the trophoblast cells
3. functional inhibition of immune responses in the decidua(part of placenta)
4. Treg cells’ role in maternal tolerance of the fetus.
Helful videos:
https://www.youtube.com/watch?v=H-0S7R0cn6U

questions:
1- the J chain is produced by:
a) Helper T cells
b) Dendritic cells
c) Mucosal epithelial cells
d) Macrophages
e) Plasma cells
2- Which of the following substances is a C-type lectin:
a) Mucin
b) Retinoic acid
c) HD5
d) REGIII (alpha)
e) TNF

3- All of the following are immune privileged sites, except:

a) Testes
b) Eyes
c) Brain
d) Breast
e) Fetus

4- All of the following are chemicals that mediate IgA isotype switching except:
a) TGF-β
b) APRIL
c) retinoic acid
d) nitrous oxide.
e) All of them are correct.
5- An example on alarmin is:
a) IL-23
b) IL-17
c) IL-22
d) IL-13
e) IL-1 (Beta)
6- All of the following are related to immunity in the GIT, except:
a) Mucins
b) Defensins
c) M cells
d) TLR and NLR
e) IgM
Answers: 1-e 2-d 3-d 4-d 5-e 6-e
 Immunological Tolerance and Autoimmunity
Introduction
• In the context of immunological memory, immunological tolerance means the unresponsiveness
of the immune system to an antigen it has previously encountered (i.e. tolerogen).
• This runs in contrast to immune responses, where
repeated exposure to an immunogen would sensitize the
immune system and strengthen its reactivity.
• This phenomenon is particularly important when dealing
with self antigens, where self-tolerance would protect
against autoimmunity and the diseases that might ensue
(i.e. autoimmune diseases).
• The basic principle behind self-tolerance is the recognition
and inactivation of lymphocyte clones which express
receptors that recognize self antigens with high affinity.
• As the recombination of antigen receptor genes is a
random process, the products of their expression may be
self-reactive from the start.
• Therefore, central tolerance refers to the mechanisms
which take place in the primary lymphoid organs to ensure
the inactivation of self-reactive immature lymphocyte
clones.
• In contrast, peripheral tolerance inactivates the self-reactive mature lymphocytes which have
managed to reach the periphery.
• The antigens presented to immature lymphocytes during their development are dealt with as self
antigens, and so their recognition would lead to negative selection of clones with receptors
complementary to such antigens.
• Those antigens may be either expressed locally in the primary lymphoid organs or imported after
their capture from the periphery.
• If the self antigen is not presented in the primary lymphoid organ or is expressed only in adult life,
peripheral tolerance would deal with lymphocyte clones reactive against it through inducing
apoptosis, anergy, or active suppression through regulatory T (Treg) cells.
• Also, sequestration of self antigens in immune-privileged areas away from the immune system
would prevent potential immune responses from taking place.

T Cell Tolerance
• During T cell development in the thymus, negative
selection through clonal deletion may occur to DP T cells
in the cortex or SP T cells in the medulla.
• If the thymocyte’s TCR recognizes a self antigen-HLA
complex on the APC, the ensuing strong signal would
cause clonal deletion.
• In contrast, Treg cell formation might result from :
1. a slightly weaker signal
2. specific APC type involved
3. local cytokine influences.
• These Treg populations would then be released to the
periphery where they would play an important part in
mediating peripheral tolerance.

• Functional unresponsiveness (i.e. anergy) of the mature

CD4+ T cell usually happens when the TCR recognizes
the HLA-antigen complex but without the costimuli
originating from innate immunity.
• Such costimulation mostly arises from CD28 on the T cell
binding to B7-1 (CD80) and B7-2 (CD86) on the APC.
• Anergy takes the form of either:
- Blockage of TCR-induced signal transduction.
- Attentuation of immune receptor signaling through either:
▪ Ubiquitination.
▪ Activation of inhibitor receptors: These usually
belong to the CD28 family (e.g. CTLA-4, PD-1).

• CTLA-4 inhibits costimulation through competing with CD28 in

order to cause endocytosis of the corresponding B7 molecule.
• This would cause anergy in the activated T cell (i.e. cell-
intrinsic mechanism of action of CTLA-4).
• As the activation of immune-related receptors is mostly related
to adding phosphate groups to them, the phosphatase
activity of PD-1 would inhibit them.
• PD-1 activation follows its binding to its ligands, PD-L1 and
PD-L2, which are found on APCs and other tissue cells.

• Treg cells are CD4+ T cells which suppress immune

responses and which usually express high levels of:
- IL-2 receptor α chain (CD25).
- FoxP3 transcription factor.
• Treg cells may be divided according to their mode of formation into:
- Thymic Treg (tTreg) cells (i.e. natural Tregs): which form in the thymus upon thymocyte
exposure to self antigens.
- Peripheral Treg (pTreg) cells (i.e. adaptive or inducible Tregs): which form in peripheral
tissues upon naïve mature CD4+ T cell exposure to antigens (self or foreign) in the absence of
strong innate immune responses.
• Factors which enhance Treg cell formation include IL-2, transforming growth factor-β (TGF-β) and
retinoic acid.
• Treg cells, in turn, would have the following
effects:
- Cell-extrinsic mechanism of action of
CTLA-4: where the Treg cells reduce the
amount of B7 on APCs and thus decrease
their responsiveness to T cells.
- IL-2 consumption: thus depriving other
immune cells of it.
• Production of immunosuppressive cytokines:
such as:
- TGF-β: which inhibits macrophage
activation and promotes Treg and IgA
formation as well as tissue repair.
- IL-10: which acts on dendritic cells and macrophages to inhibit the formation of IL-12 (and thus
IFNγ), MHC class II molecules as well as costimulators.
B Cell Tolerance
• As for T cells, B cells encounter events whether centrally or
peripherally to ensure unresponsiveness against self
antigens.
• In the bone marrow, if the immature B cell is highly self-
reactive, the strong signal would cause the cell to undergo
receptor editing.
• This means that the cell would change its light chain
structure to modify its reactivity, but if these editing attempts
fail, the clone would get deleted by apoptosis.
• In contrast, a weak signal would prompt the cell to undergo
anergy instead.

• Peripherally, the absence of self-reactive TH cells as well as

innate immune responses (e.g. complement system) would
deprive B cells from the additional stimuli it needs to get
activated.
• This, in combination with persistent BCR stimulation, would
prompt B cells to undergo anergy or deletion.
• In some cases, B cell binding to self antigens might activate
inhibitory receptors, meaning that B cell activation would need
additional signals from the BCR coreceptors, innate immune
receptors as well as TH cell help.

Autoimmunity
• Breakdown of the aforementioned mechanisms of self-tolerance is proposed to be the main
reason behind autoimmunity and the ensuing tissue injury and disease.
• Such diseases may be systemic (e.g. systemic lupus erythematosus) or organ-specific (e.g.
myasthenia gravis, type 1 diabetes, multiple sclerosis), but they all tend to be chronic, progressive
and self-perpetuating.
• In general, autoimmune diseases are proposed to take place in a genetically-susceptible
individual exposed to an environmental trigger (e.g. infection, local tissue injury).

Role of Genetics in Autoimmunity

• Genetic susceptibility which increases the likelihood of autoimmunity may be due to:
- Multiple genetic polymorphisms: which interplay with one another and with the environment to
give rise to complex polygenic traits. Genes implicated in such combinations include:
▪ MHC genes: presumably because certain MHC molecules are more likely to bind a self
antigen and present it to T cells.
▪ Other genes: where some alleles are associated with an increased risk of autoimmunity, as
with:
 -
Complement proteins: where their deficiency may reduce immune complex clearance, thus
giving rise to lupus-like autoimmune diseases.
- CD25: where some variants may reduce Treg function, thus giving rise to multiple sclerosis and
type 1 diabetes.
- FcγRIIB: where a variant might lead to reduced B cell inhibition, thus giving rise to lupus-like
autoimmune diseases.
- Insulin: where some polymorphisms may cause reduced thymic expression, thus causing
impaired central tolerance and subsequent diabetes.
• Single gene (i.e. Mendelian) defects: which, though rarer than polygenic ones, have a stronger
association with autoimmunity, clearer pathogenic mechanisms and higher penetrance. Examples
include:
- AIRE protein: which is involved in the expression of
many peripheral self antigens in the thymus, thus
defects in its expression would lead to the escape of
many self-reactive lymphocytes to the periphery.
These would then attack such endocrine organs as
the adrenals, parathyroids and pancreatic islets,
thus giving rise to autoimmune polyendocrine
syndrome type 1 (APS1). Moreover, autoantibodies
against IL-17 would cause susceptibility to fungal
infections in APS1 patients.
-
- FoxP3 protein: which is important for Treg function,
thus defects in it production would lead to immune
dysregulation, polyendocrinopathy, enteropathy,
X-linked (IPEX) syndrome.

Role of Environment in Autoimmunity

• Environmental triggers for autoimmunity in
genetically-susceptible individuals include:
- Microbial agents: which might dysregulate the
immune system through:
▪ Bystander activation: where the innate
immune responses secondary to infection
would lead the APCs to express costimulators
and secrete T cell-activating cytokines thus
overcoming the tolerance mechanisms in
place.
▪ Molecular mimicry: where immune
responses initiated by microbial antigens
would eventually attack self antigens due to
structural similarity between both antigen
types (e.g. rheumatic fever).
• Injury of immune-privileged areas (e.g. due to
trauma, ischemia, inflammation): where antigens normally sequestered and shielded from the
immune system would get released and exposed to it, thus causing its sensitization and prompting
it to attack the tissues of origin (e.g. posttraumatic uveitis or orchitis).
Helpful videos
https://www.youtube.com/watch?v=vDwNpDT-8L0

https://www.youtube.com/watch?v=rHx30H3dUKQ

questions:
1- which protein’s defective production is associated with IPEX syndrome:
a) CTLA-4
b) FoxP3
c) CD25
d) AIRE protein
e) Complement protein
2- regarding autoimmune polyendocrine syndrome 1 (APS1), which physiological function would not be
affected:
a) Parathyroid function
b) Defense against fungal infections
c) Adrenal function
d) Regulation of blood glucose concentration
e) Thyroid function
3- which disease mechanism is most closely related to the pathogenesis of rheumatic fever:
a) Mendelian defects
b) Bystander activation
c) Multiple genetic polymorphisms
d) Breakdown of central tolerance
e) Molecular mimicry

4- when compared to multiple genetic polymorphisms, single gene defects______

a) Have unclear pathogenic mechanisms
b) Have less interplay with the environment to cause disease
c) Have lower penetrance
d) Are commoner
e) Have weaker association with autoimmunity
5-regarding autoimmunity, which disease is associated with variants in the FC(gamma)RIIB gene?
a) Multiple sclerosis
b) Lupus-like disease
c) Parathyroid disease
d) Adrenal disease
e) Diabetes mellitus
Answers: 1-b 2-e 3-e 4-b 5-b
 Transplantation
• Transplantation is an advanced and major method of treatment. It helps patients with organs
disorders, like heart failure and kidney diseases, to have a more healthy and comfortable life.
• Transplantation: taking an organ or living tissue and implanting it in another part of the body or in
another body.
• When an organ in the human body is damaged, or a significant amount of blood is lost, a
transplantation process could be done.
Note: the first successful transplantation was blood transfusion.
• Transplantation can be classified depending on the organ that is transplanted and depending on
the relationship between the donor and the recipient.
• Depending on the organ that is transplanted, there are a lot of types of transplantation such as
blood transfusion, cornea transplantation, bone marrow transplantation, solid organs
transplantation, etc.
• Blood transfusion is the simplest and easiest form of transplantation. When we do a blood
transfusion, only RBCs are transferred from the donor to the recipient, WBC and platelets must
not be transferred so that the recipient doesn’t develop autoimmune issues. The donor’s unit is to
be tested, centrifuged and divided to portions to ensure that only RBCs are transferred to the
recipient and to ensure that no transfusion-transmitted infections, such as AIDS, malaria, and
Hepatitis B, are transmitted to the recipient.
Notes:
1. Transfusion-transmitted infections are infections that are transmitted from a person to another
through blood.
2. RBCs are short lived, enucleated and don’t have MHC molecules. So, rejection of blood occurs
only when rejected because of the ABO or the Rh systems.
3. In 1900s Karl Landsteiner created the ABO system, then he worked with Comb and described the
Rh blood group in 1930.

• Cornea transplantation is also one of the easiest forms of transplantation and the easiest in
terms of immunity. The cornea has neither blood vessels and they get O2 from the air. That’s why
its transplantation doesn’t require the ABO checking. So, an immune response is not a factor that
causes rejection of the cornea transplant. One of the best countries in cornea transplantation is
sirilanka.
• Bone marrow transplantation is the most difficult transplantation immunologically; because
rejection can happen in both ways (the host might reject the graft and the graft might reject the
host).
• Solid organs transplantation is any transplantation of an internal organ that has a firm tissue
consistency and is neither hollow (such as the organs of the gastrointestinal tract) nor liquid (such
as blood). Such organs include the heart, kidney, liver, lungs, and pancreas. The most widespread
solid organ transplantation is the kidney transplantation.
→The most important limiting factor is the availability of organs (neglecting the religious and cultural
factors).
Notes:
1. The first successful kidney transplant was between twins.
2. A person can live with half a kidney, and we have two kidneys so it is common to do
transplantation of kidneys.
3. liver transplantation is more difficult than kidney.

Types of grafts transplantation (5 types) depending on the relationship:

1) Autogenic or autologous (Autograft): The recipient and the donor are the same individual, it is
always accepted.
• Examples:
- skin, such as in burn victims.
- Bone marrow transplantation: in chemotherapy of cancer patients, the bone marrow of the
patient is damaged. The transplantation process causes a lot of pain, so under general
anesthesia, we get some bone marrow from the patient, most likely from the iliac crest or the
sternum, and it is stored like a blood unit in some place. Afterwards, a higher dose of
chemotherapy is given to the patient. After 2 to 3 days, when the bone marrow is highly damaged
or is very weak, the stored unit of bone marrow is given to him.
- When removing the thyroid gland, the parathyroid gland is associated with it. The parathyroid is
important for the control of calcium metabolism, so, it’s taken and injected under the skin (of the
arm for example), so maybe cells live and do their function there, in controlling calcium
metabolism.
2) Syngeneic (isograft or syngraft): between two completely genetically identical members of a
specie, (same strain), identical twins, same mice strains.
- The first successful kidney transplantation was between identical twins.
3) Allogeneic (Allograft): between two individuals of the same species, but are not identical (always
rejected).
4) Xenogeneic (xenograft): between two different species.
- Pig to human; aortic valve replacement from the pig (because of the genetic similarity between
those two). Size of the valve and anatomical shape are very similar also.
- Trials of HIV suggested transplantation of bone marrow and immune cells from monkeys to humans
to aid their immunity.
5) Mechanical (metallic): transplantation of metal: metallic heart, heart valve, screw, etc

After WWII, peter Medawar did a lot of experiments on inbred strains of mice for skin transplantation
(Inbred means that all the strains offspring are identical twins and homozygous for all traits)
1. Strain A skin graft was given to strain B, after 1-2 weeks→ graft rejection occurs (first set rejection)
2. If we give the same mouse the same graft from the same donor, rejection is faster, after 3-7 days
(because of memory and amplification). This confirms that the mediators for the transplantation
process are immune cells. (second set rejection)
3. if you take lymphocytes from the host (B with skin from A) and give them to an identical twin
(strain B), then give him the same skin graft (from A) rejection occurs.
 (1) (2) (3)

Medawar stated the laws of transplantation as the following:

A. transplantation between members of the same strain is always accepted.
B. transplantation between members of different strains is always rejected.
C. If we cross mate between members of both strains (A with B), transplantation from parents to sons
is always accepted.
D. If we cross mate between members of both strains (A with B), transplantation from sons to parents
is always rejected.
(an immune response determines the acceptance in a transplantation)
Note : medawar expected that there are almost 50 genes responsible for the rejection process.
Types of graft rejection:

1) Hyperacute rejection (occurs immediately

within 2-3 min).
- The recipient has performed Abs against
donor Ag, organ, ABO, etc.
- Happens when: when the antigens are
completely mismatched, giving the wrong type
of blood.
- Consequences: complement
activation/inflammation/thrombosis/ endothelial
damage/loss of the organ (The organ must be
removed immediately to save patient’s life).

2) Acute rejection (takes 1-2 weeks, so we

can give the patient a drug to reduce the
rejection and achieve successful
transplantation).
- Some of the organ’s antigens differ from the
recipient antigens, which develop specific
immune response (mainly cellular but might be
humoral too).
- this rejection leads to parenchymal cell
damage, interstitial inflammation and
endothelialitis,

3) Chronic rejection (takes place years after

transplantation)
-The major cause of failure of vascularized
organ allograft.
- Chronic rejection can cause accelerated
atherosclerosis.
- Consequences: chronic inflammation/intimal smooth muscle proliferation, thus vessel
occlusion (no solution so far).

4) Graft vs Host Disease (GVHD), some of the donor cells are immune cells which causes these
cells to attack the host (the recipient), and the patient will have two diseases instead of one, it is seen
mainly in BM and stem cell transplantation.
Consequences: skin rash/ fever/ joints pain/ kidney and liver failures.
Alloantigen recognition
1) Direct alloantigen recognition
• Happens in organ transplantation
in the case of allogeneic
transplantation which is the most
frequent one, APCs present MHC
molecules of the donor graft which
are going to be recognized by T
cells of the recipient (alloreactive
T-cells), a mismatch in MHC
causes non-specific immune
stimulation.
• Specific T cells won’t attack the
mismatched MHC molecules as
antigens, but the mismatch is recognized, so T
cells recognize unprocessed allogeneic MHC
molecules on the graft’s APCs, so the T cells are
from the recipient and the APCs are from the
graft. and it involves both CD8+ and CD4+
cells.
2) Indirect alloantigen recognition
• Sometimes, APCs of the recipient (macrophages,
etc.) go inside the graft of the donner and pick up
some antigens and present them to their own T
cells.
• So here, the antigen is from the graft and the
APCs and T-Cells are from the recipient (Indirect
Alloantigen Recognition), and it involves only
CD4+ cells.
Important note: mainly the rejection in transplantation is mainly through cell mediated immunity
(mainly CD8+ cytotoxic T-lymphocytes) , not humoral immunity.

Compatibility testing:
1) ABO system
• The most important test because the ABO antigens are not only present on RBC’s but also in
the endothelium of every blood vessel of every organ.
• the ABO antigens are carbohydrates linked to cell surface proteins and lipids.
• Individuals who express a particular ABO antigen are tolerant to that antigen, but individuals who
do not express that antigen produce natural antibodies that recognize the antigen.
• When doing a blood transfusion, we should consider that blood antigens of the donor must not
meet with the antibodies that have specificity for them (To avoid hyperacute rejection; clumps
formation).
• All blood groups have H antigen on the surface.
• There are enzymes that the A or B genes code for that adds a carbohydrates to the H antigen; the
A gene enzymes add N-acetyl-galactosamine, the B gene enzymes add galactose, to the H
antigen carbohydrates. (Look at the picture).
• The most important thing to
consider when we are doing
transplantation to any organ
is blood group compatibility.
Note: AB blood group is an
example of a co-dominant
inheritance,
• When we have a human with a blood group A for example, his RBC’s will have A antigen so any
cell of the immune system
that recognize this antigen
will be killed, the same
principle applies to all blood
groups.
• When we are born, we will
have the antigens of the
blood groups but we will not
have the antibodies to the
other blood groups. But when we reach 1 year of age, we will be exposed to A and B antigens
from the surface of bacteria so we will make the antibodies against them.
• As we know, O- is the universal donor and AB+ is the universal acceptor.
• IgM is the natural antibody synthesized without overt exposure.
2) HLA (MHC) typing.
• In kidney transplantation, the larger the number of MHC (HLA) alleles that are matched between
the donor and recipient, the better the graft survival (it can reach to 80%).
• HLA matching had a more profound influence on graft survival before modern immunosuppressive
drugs were routinely used, but current data still show significantly greater survival of grafts when
donor and recipient have fewer HLA allele mismatches.
• Of all the class I and class II loci, matching at HLA-A, HLA-B, and HLA-DR is most important for
predicting survival of kidney allograft. (Although current typing protocols in many centers include
HLA-C, most of the available data in predicting graft outcome refer only to HLA-A, HLA-B, and
HLA-DR mismatches).

3) Cross matching test.

• a test tube experiment for incompatibility in which serum from the recipient and RBC’s from the
donor are mixed together and observed.
• If there was no reaction (no lysis, no performed Ab’s or Ag-Ab reaction) between the 2 specimens,
then they are compatible.
• The less (cell lysis, performed Ab’s or Ag-Ab reaction) the more compatible is the graft.
• This test is important for minor and less common blood groups.
• We also do Ab cross matching in cases of solid organs transplantations.
4) Mixed lymphocyte reaction (MLR)
• in this test we mix lymphocytes (mononuclear
cells) from both the donor and the recipient, this
step is called primary mixed lymphocyte reaction.
• If there is recognition from one group of cells to
the other, there will be proliferation of these cells
and eventually there will be lysis of cells and
cytokine secretion.
• If this reaction happens, this indicates that we
can’t do transplantation between them.

Examples of drugs that can suppress the immune attack, they prevent acute
rejections:
➢ Cyclosporine (FK506): Inhibitor for the
calcineurin/calmodulin pathway (improved the
transplantation significantly, the best drug).
Calcineurin is an activator of T-cells, so by inhibiting this
pathway, we suppress the immune system.
➢ Corticosteroids: widespread cytokine production inhibitor
(IL-2, IL-4, IL-5)
➢ Anti-IL-2-receptor-antibody: Inhibition of IL-2 binding .
➢ Azathioprine (Imuran) and mycophenolate: inhibits the
proliferation of immune cells.
➢ Anti-TCR (OKT3, thymoglobulin)
➢ CTLA4-Ig
➢ Monoclonal antibodies.

Helpful videos:
https://www.youtube.com/watch?v=GWWBlBS9hwM
questions:
1- which immune suppressive work on inhibiting TCR-MHC/antigen interaction?
a) Cyclosporine
b) OKT3 thymoglobulin
c) Mycophenolate
d) FK506
e) Azathioprine
2- transplanting tissues and organs between 2 individuals not of the same species is called:
a) Allogenic
b) Autologous
c) Xenogeniec
d) Metallic
e) Syngeniec
3- which rejection type is due to complement activation and endothelial damage?
a) Acute humoral rejection
b) Hyperacute rejection
c) Chronic rejection
d) Graft versus host disease
e) Acute cellular rejection
4- which tissue is the easiest immunologically to transplant ?
a) Cornea
b) Liver
c) Heart
d) Kidney
e) BM
5- the most important matching step is:
a) MHC class 1
b) MHC class 2
c) Cross match
d) ABO blood group
e) MLR
6- Graft versus host disease is seen in which of the following transplants:
a) Cartilage
b) Heart
c) Kidney
d) Bone marrow
e) Pancreas

Answers: 1-b 2-c 3-b 4-a 5-d 6-d

 Immunity to tumors
• As we are said earlier that one of the functions of the immune system is to fight cancer.
- The evidence is that immunodeficiency patients (like AIDS patients) have a higher tendency to
have cancers more than usual, like: primary CNS lymphoma, Non-Hodgkin lymphoma, Kaposi
sarcoma and it indicates that there is failure of the immune system.
- so if someone has an immune deficiency disease , he\she will be more susceptible to have cancer
• Immune system fights and recognizes tumor cells.
• Malignancy (Cancer): abnormal proliferation of cells due to dysregulation in cell cycle control,
usually it is due to increased division of the cells (miosis) “more than what is needed “leading to
accumulation of tumor cells OR the cells do not die leading to abnormal steady state of number of
cells .also, they lose the cell adhesion inhibition which leads to uncontrolled division of cells
Note: DNA mutations or abnormalities is usually responsible for this dysregulation in cell cycle
control.
• Steady state of number of cells: this means that you need to have equal numbers of dividing
cells and dying cells.
• cells die in the process of apoptosis so, if we have a decrease in this process it will lead to an
accumulation of tumor cells.
Note: Almost all the cells in the body except the first part of the dedendum can become cancerous .
So we say skin cancer, blood cancer, bone cancer… to identify from where this cancer is coming
from.
• even if you have a biopsy of two different
cancers (medullary breast carcinoma (A) &
malignant melanoma in skin (B)), we will
see malignant cells and lymphocytic
infiltration that is fighting the cancer, the
lymphocytic cells indicate that the immune
system can recognize this cancer and start to
fight it; hopefully these cancers will have a
better prognosis.
• Red arrows indicate malignant cells. Yellow
arrows indicate lymphocyte -rich
inflammatory infiltrates that are fighting the
cancer.
• Immunohistochemical staining of resected tumors can be used to enumerate different types of T
cells associated with the tumor, such as an infiltrate of CD8+ T cells in a colonic carcinoma.

One proof of tumor immunity is an experiment on mouse:

1. Bring a mouse and rub its skin with a carcinogen material (MCA) until a cancer develops.
- the carcinogen is MCA which is a chemical substance that causes skin cancer.
2. After that, you resect the tumor from the mouse.
• If you try to re-transplant it to another site in the same mouse, it will not be accepted (rejected). So
you will see that there is no tumor cell
growth because this mouse was exposed to
this cancer or antigen before and rejected it,
so this mouse developed immunity against
the tumor and it will be resistant.
• If you transplanted the tumor cells into
another similar mouse (twins) “two inbred
strains of mice individuals: exact genetic
makeup” you will see tumor growth, but if
you before this isolate CD8+ T cells and
inject them into this mouse, there will be
transfusion of immunity by T cells leading
to the eradication of the tumor cells .
- The value of this experiment is to show tumor
immunity.

Tumor antigens
• Antigen is a molecule that the Immune System recognizes it but not necessarily fight it.
Why?
- because some of these antigens, after recognition, are tolerated and do not activate the Immune
system.
• If they activate the immune system, they are known as immunogens.
- In the pathogenesis of certain cancers there is a mutation in certain genes which control the cell
cycle, and this leads to the overexpression of certain genes called oncogenes.
- tumor antigens are of different types according to the different types of tumor.
- The oncogene: RAS mutation, Philadelphia chromosome (Bcr/Abl rearrangements) in
Chronic Myelogenous Leukemia, the product of this is p210.
Note: the change is either a change to the gene or an over expression of the gene such as in the
case of breast cancer where HER2 gene is overexpressed.
Ex 1: Products of oncogenic viruses: One of the positive things is that in regions that have an
increase in Cervical Carcinoma in female, it was proven to be effective to have vaccination against
papilloma virus because it is protective against this cancer.
- Papillomavirus’s E6-E7 proteins are recognized antigens in Cervical Carcinoma, because part of
the pathogenesis of this cancer is this viral infection. So, Antigens of this virus are considered as
tumor antigens.
Ex 2: EBNA-1 protein of EBV cause EBV-associated lymphomas, nasopharyngeal carcinoma.
Ex 3: Human herpesvirus 8 (HHV-8) this is a virus that causes Kaposi sarcoma.
Ex 4: alpha-phetoprotein is normally expressed in embryonic period. After we are born we will stop
expressing this protein unless we have certain types of cancer, like colon , GI or liver cancer.
- We can use it as marker in hepatocellular carcinoma which is
associated with alpha-phetoprotein expression.
Ex 5: If a patient has prostatitis or some problems like prostate
hypertrophy, the PSA [prostate specific antigen] will increase
but in cancer it will be much increased.
Ex 6: We use CD20 as a marker for B cell lymphoma.
These antigen are useful in:
1. To demonstrate that there is immunity.
2. Sometimes we use them for diagnosis, like if someone have
liver or GI mass we can check for alpha-phetoprotein, this
helps us to know the type of cancer and the way of
treatment.
3. When we eradicate and treat the cancer, the concentration
of the antigen will decrease. But if the concentration
increased again this means that the cancer has come back
to the patient.

• Part of the virulence factors of cancers is that they develop or

they become resistant to the immune responses.
• They expect that the malignant cells normally express tumor
antigens that can be presented by MHC1 molecules on cell
surface.
• MHC1 recognized by T-cells specific for that antigen -it will be
CD8+ [CTLs]. {This is what is expected and what we hope}
• We may use other mechanisms like ADCC by NK cells.
• Anti-tumor immunity is mainly by T-cells, CD8+ recognition of
tumor antigens leading to T-cell activation and anti-cancer
function.
• If the cancer is small, we will see a lot of immune cells and anti-
tumor cells will attack but sometimes these mechanisms fail,
how? 1-3
- All of these mechanism will lead to failure of T-cell recognition
(CTL recognition of tumor antigen) and these are evasion
mechanisms:
1. failure to produce tumor antigen so this leads to antigen
loss and we will have variant of tumor cell which lead to
lack T-cell recognition of tumor.
2. Mutation in MHC genes or genes needed for antigen
processing will lead to MHC deficient tumor.
- MHC1 and MHC2 are not present on cell surface until they
have antigen on it.
3. secretion of immunosuppressive proteins like TGF-B & IL-
10 and expression of inhibitory cell surface proteins like
CTLA-4 and PDL-1 or not expressing co-stimulatory molecules to activate T-cells.
 Tumor immune-editing
- RAG: one of the enzymes or one of the gene products that are needed for T-cell and B-cell
development << another name for it is recombination activation gene >>
- RAG KO mouse → doesn’t have this gene (knockout) → immune deficiency mouse.

First experiment
1. Tumor cells are transplanted into a
mouse with functional immune system
(normal mouse), the cancer cells grow
with time.
2. The cells are isolated and transplanted
into another normal mouse,
- Result: the cells are also able to live in a
new mouse.
- Conclusion: These cancerous cells,
growing under the selection pressure -
drive natural selection pressure- of a
normal immune system are being edited
to yield stronger and more evading cells.
On the Other Hand (second experiment):
1. Tumor cells are transplanted into RAG KO mouse (immune-deficient).
2. Tumor cells are isolated and transferred to a normal mouse.
Result: Tumor that is originally transplanted in immune-deficient mouse is rejected when
transplanted into a normal mouse.
Conclusion: Tumors removed from RAG KO without adaptive immunity will not experience the
same selection pressure that will allow the evading cells to prevail, so it will remain
immunogenic and will be rejected after transfer into a normal mouse.

Tumor vaccines
• Vaccines: substances which are capable of inducing permanent immunity against a dangerous
substance without causing the disease.
• The only usable and successful tumor vaccine right now is for papilloma virus which causes
cervical cancer.
• it is possible to use the immunity to kill cancers.
• Tumor Vaccines are not accepted and not standard to the way of treatment just like
immunotherapy & not widely used.
Three characteristics of a good vaccine:
1- Protective
2- No side effects
3- long term of protection by T & B- cells.

There are 3 examples of successful immunotherapy:

Ex 1: Stimulation of the immune system by vaccine or antigens as in the case of urinary bladder
cancer (transitional cell carcinoma), BCG vaccine is given (used against Tuberculosis) Around
the cancer. This attracts monocytes and lymphocytes,
allowing them to infiltrate around it and kill cancer cells
causing the shrinkage of it.
Ex 2: is hyper-nephroma or renal cell carcinoma: T
cells specific for tumor antigens can be harvested from a
patient’s tumor tissue or blood, expanded and activated in
vitro, and re-infused into cancer patients. Lymphocytes are
taken from blood, put in culture, and IL-2 is added for
activation. when injected in humans, it cures
Hypernephroma and renal cell carcinoma. LAKC
(Lymphokine Activated Killer Cells) is the name of the cells
produced by this procedure.
Ex 3: Anti-Tumor Monoclonal Abs
• Tumors can be used as antigens so, that we can get monoclonal Abs from Immune System. It is
Alternative to chemotherapy which kills cells in non-specific way which may lead to killing normal
cells, so we can use monoclonal Abs which are specific for cancer cells.
• We prefer monoclonal Abs because it is more acceptable from the body and have low side effects.
Ex: Rituximab (Mabthera): an anti CD-20 used in Non-Hodgkin Lymphoma, Acute and Chronic
Leukemia.
Note: These drugs are expensive.
Cancers of the immune system and blood
• leukemia and lymphoma have the same type of cells, but the difference between leukemia
and lymphoma is:
- Leukemia: cancer has reached blood.
- Lymphoma: cancer is only in lymph nodes or tissues, hasn’t reached the blood yet.

1- Myeloproliferative disorder: is a disease of the bone marrow, it can develop into acute myeloid
leukemia. (AML)
2- Polycythemia vera: disease of the bone marrow that results in increased production of RBC’s and
results in hemoglobin level of greater than 18.5 g.

- Hematopoietic tissues can transform to cancer:

1- Early hematopoietic tissue-early B cells (such as pro then pre-B cell) can transform into cancer
forming acute lymphoblastic leukemia
- More common in children and more aggressive than chronic lymphocytic leukemia.
- the lymphocytes that become cancer are usually B cells with chances of 95-90%, while T cells only
by 10%.
2- Late/mature hematopoietic tissue – late B cells (monocytes or myeloid series) forming chronic
lymphocytic leukemia /lymphoma .
- Which is More common in adults and it is associated with retro-viruses such as Adult B cell
leukemia / lymphoma because of HTLV1 virus. (Human T-lymphotropic Virus 1) . Cancer can also
affect RBC or platelets in this case.

● Multiple myeloma: where plasma cells (B cells) are affected by cancer, and this results in high
levels of immunoglobulins ‫الاااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااااا‬

Helpful video:
https://www.youtube.com/watch?v=K09xzIQ8zsg
questions:
1- if an early B or T lymphocyte becomes cancerous, it will become which type of cancer:
a) Acute myeloid leukemia
b) Acute lymphoblastic leukemia
c) Chronic lymphocytic leukemia
d) Multiple myeloma
e) Essential thrombocytosis
2- all are true about tumor vaccines, except:
a) Use overexpressed but unmutated cellular proteins
b) Use of products of mutated genes to stimulate immunity
c) They are well established and widely used
d) Use of HSP-associated antigens
e) Use of whole inactivated cells or cell lysate
3- with which malignancy the use of local BCG injections is successful?
a) Skin cancer
b) Renal cell carcinoma
c) Brain cancer
d) Breast cancer
e) Urinary bladder cancer
4- if plasma cell becomes cancerous it will become which type of cancer?
a) Chronic lymphocytic leukemia
b) Essential thrombocytosis
c) Acute lymphoblastic leukemia
d) Multiple myeloma
e) Acute myeloid leukemia
5- monoclonal antibodies against CD52 or CD20 is approved against which cancer:
a) Breast
b) B cell lymphoma
c) Ovarian
d) Gastrointestinal
e) Melanoma
6- which viral vaccine has been successful in preventing a type of cancer that it is recommended to
use?
a) Polio vaccine for nervous system cancers
b) Tuberculosis vaccine for lung cancer
c) HIV virus vaccine for non-Hodgkin lymphoma
d) Human papilloma virus to prevent cervical cancer
e) Smallpox vaccine for skin cancer
Answers: 1-b 2-c 3-e 4-d 5-b 6-d
 Immunity against Microbes
Introduction:
• Defense against microbes is the main function of the immune system, where different microbial
targets are fought by its various arms.
• In order to appreciate the multitude of roles played by different immune system components in
this task, one might study them according to microbial nature in terms of relative structural
complexity:
- Acellular (viruses which can’t reproduce outside living cells) and Unicellular Microbes
(prokaryotic: bacteria, eukaryotic: protozoa, yeasts).
- Multicellular Microbes (helminths .
• In addition to the nature of the target, the immune response mechanisms themselves also show
immense variation.
• They can be innate (quick response/ no memory) or adaptive (long response/ memory/ specific),
and the immune cell role may be frontline (contacts the microbe) or supportive (support the
frontlines).
• Knowledge of immune response against infection may help in identifying the evasive mechanisms
microbes adopt whilst causing disease as well as designing strategies for artificial induction of
immunity through vaccination.

Immunity Against Acellular and Unicellular Microbes:

• Immunity against acellular and unicellular microbes may be studied according to the nature of
their initial encounter with immune cells:
- Extracellular Microbes: where free microbes located outside host cells are attacked by
immune cells.
- Intracellular Microbes: where immune cells start attacking body cells hosting microbes inside
their cytoplasm.
• Note that the above classification scheme means that the immune system may attack the same
type of microbe differently according to its location (e.g. free virions vs. viruses within the host
cell). Also, note that ‘intracellularity’ here does not apply to the microbes still within immune cell
phagosomes.

Immunity Against Extracellular Microbes:

• Professional phagocytes (i.e. neutrophils and monocytes-macrophages) identify microbial
products, swallow them, then kill them intracellularly.
• Pattern recognition receptors (PRRs) (e.g. TLRs (toll like receptors), mannose receptors,
scavenger receptors, complement) as well as antibodies aid in this process through opsonization
and stimulation of killing activity.
• Receptors on phagocytes which help mediate opsonization are:
- Complement receptors CR1 and CR3: which identify various C3 derivatives.
- immunoglobulin receptors FcγRI and FcγRIIa: which identify IgG1 and IgG3.
• T-independent antigens are non-protein molecules (e.g. polysaccharides, lipids) which induce
antibody production without TH cell help.
• Such antibodies are usually produced in the spleen (people who had undergone splenectomy are
more susceptible to these pathogens), and they are important in opsonizing polysaccharide-rich
 encapsulated bacteria (e.g. Streptococcus pneumoniae, Neisseria species.) also they will not
produce memory like protein molecules.
• The body can’t recognize non-protein the same way as protein molecules (protein molecule are
capable to activate T helper cells while non-protein can’t).
• Besides bacteria, phagocytosis and intracellular killing is important in facing different types of
unicellular microbes outside host cells, including fungi (e.g. Candida species) as well as protozoa
(e.g. Plasmodium species., Toxoplasma species.).
• Through activating the complement system, formation of the membrane attack complex (MAC)
is important for killing bacteria with thin cell walls (e.g. Neisseria species) as well as enveloped
extracellular virions.
• The complement system may be activated through the classical pathway (through IgG1, IgG2,
IgG3 or IgM action), alternative pathway, or lectin pathway.
• The mere binding of the antibody to microbial molecules necessary for pathogenesis (e.g. surface
structures, toxins) would protect against them through steric hindrance (i.e. antibody-mediated
neutralization). This is mostly done through IgG, IgM, or IgA (especially in mucosal surfaces).
• The inflammatory response happens due to complex interaction between inflammatory cells (i.e.
immune cells (leukocytes or dendritic cells), platelets and endothelium) and inflammatory
mediators.
• The degree and type of contribution of each component depends on the initial tissue injury, thus
determining the eventual character of this response and its outcome.
• Among the responses mediated by cells of the innate immune system are:
• Chemokine secretion: by macrophages, which helps in chemotaxis.
• Secretion of IL-1, IL-6 and IL-23: by macrophages and dendritic cells, thus stimulating the
development of ILC3s and TH17 cells. This may be in response to PRRs that identify β-
glucans on fungal surfaces (e.g. TLRs, dectins).
• Secretion of IL-12: by macrophages and dendritic cells, thus stimulating the development of
ILC1s and TH1 cells.

Innate immunity Adaptive immunity

Frontline Phagocytosis Antibody-Mediated Neutralization

Complement-Dependent Lysis (neutralize microbial target (ex:
virus/bacterial toxin))

Supportive Supportive Functions of: TH Cell Function

• Neutrophils & Monocytes Macrophages.
• Mast Cells, Basophils & Eosinophils
• Innate Lymphoid Cells (ILCs)

Note: Neutrophils & Monocytes-Macrophages can be supportive AND frontline through phagocytosis.
• Various TH cell subsets also play different roles in terms of the immune system mechanisms they
favor when facing extracellular microbes:
- TH1 cells (similar to ILC1): develop in response to IL-12 and they mainly produce IFNγ and
express CD40L. They are potent activators of macrophage function, especially when they
 encounter microbes that are resistant to intracellular killing (e.g. Mycobacterium tuberculosis,
Mycobacterium leprae, Histoplasma species). This might lead to granulomatous inflammation
with destructive effects on nearby host tissues.
- TH2 cells (similar to ILC2): which secrete IL-4 and IL-5, thus inhibiting macrophage function
whilst switching antibody isotype to IgE and IgG4. Though that may be beneficial against
helminths, a poor TH1 over TH2 response may prove detrimental to the host whilst dealing with
some protozoa (e.g. Leishmania species).
- TH17 cells (similar to ILC3): develop in response to IL-1, IL-6 and IL-23 and secrete IL-17, thus
recruiting neutrophils and stimulating inflammation and tight junction function. This
response is important for fighting off bacteria as well as Candida spp...) Especially in mucosal
tissues.

Immunity against Intracellular Microbes:

• If the microbe happens to be found within the cell’s cytoplasm, the aforementioned mechanisms
become useless to fight it.
• This applies not only to virus-infected cells, but in the case of some other microbes as well (e.g.
Listeria monocytogenes, Cryptococcus neoformans (yeast), Plasmodium species, Toxoplasma
species. (protozoa)).
• In these cases, some immune cells are prompted to form pores in the plasma membrane of the
infected cell, which will in turn cause:
• Osmotic lysis.
• Induction of apoptosis: either through the action of granzymes (entering through the pores)
or membrane bound FasL and TNF.
Both of those mechanisms may be performed by:
- Innate immune system: through the action of NK cells and monocytes-macrophages. They
would perform antibody-dependent cellular cytotoxicity (ADCC) through IgG1 and IgG3 binding
to the FcgRIII molecules on their surface through their Fab region.
- Adaptive Immune system: through TC cells.
• The innate and adaptive arms complement each other in fighting intracellular microbes; while
the adaptive arm through cytotoxic T cells attacks the infected cells which express class I HLA
(MHC) molecules, but if the microbes shut down their expression, the innate arm targets those
cells.
• The main cytokines which mediate and support immunity against intracellular microbes are the
interferons (IFNs).
• They are divided into different types (e.g. type I, type II) with different modes of action (e.g.
direct antiviral intracellular action, stimulation of HLA class I expression), but they all share
functional relation with the TH1 cell subset and related cells (e.g. ILC1s, NK cells, TC cells).

The doctor said about the peak incidence of innate

and adaptive and the innate is faster response and
the cells involved. in (a)

←the virus here is extracellular microbe

←the virus here is intracellular microbe

in the first box we conclude that the cell successfully
phagocyte the microbe so the phagocytosis as a
mechanism of killing the extracellular microbe was
successful process but if the microbe escape the
phagosome the mechanism will change from
extracellular to intracellular and because of that the body
will activate the CD8+ cytotoxic T lymphocyte to kill the
infected cell.
So the same microbe could be intracellular or
extracellular according to the place of it, and according to
that the immune response will change.
Immunity against Multicellular Microbes:
• The main effector immune cells which attack helminths are the eosinophils.
• They are supported by the TH2 cell subset and mast cells, which produce:
• IL-5: which stimulates eosinophil production from the bone marrow.
• IL-4: which stimulates the production of IgE, which would then bind to both the parasitic
surface and the eosinophils’ FcεR. This would cause the eosinophils to degranulate onto the
parasite, thus attacking its tegument (parasite ‫ )الغشاء الخاجي لل‬and nervous system.

Microbial Evasion of the Immune System:

• In response to the aforementioned attack options of the immune system, microbes have adopted
myriad mechanisms to evade the immune system and avoid destruction by it:
• ‘Hit and Run’ Raid: where infection, replication and shedding all take place quickly before
adaptive immunity defenses come into play (e.g. common cold, rotavirus).
• Inaccessibility: where infection takes place in an immune-privileged area which prevent the
immune system to get there (e.g. rabies).
• ‘Silent Infection’: where microbial antigens are not displayed on the host cell’s surface (HLA).
This happens with herpes simplex virus (HSV) and cytomegalovirus (CMV).
• Molecular Mimicry’: where microbial antigens mimic host antigens, thus leading to poor
immune response (e.g. Mycoplasma pneumoniae).
• ‘Antigenic Variation’: where the change in the microbial antigenic makeup limits if not
altogether cancels the value of immunological memory in mounting increasingly stronger
immune responses. Examples include HIV, influenza viruses (i.e. antigenic shift and drift with
these mechanisms the influenza could change its genetic material and so their proteins),
rhinoviruses, Trypanosoma brucei, Plasmodium species, Escherichia coli, and Neisseria
gonorrhoeae.
• ‘Antigenic Shedding’ (e.g. Entamoeba histolytica which get rids of its antigens).
• Nonspecific, Nonproductive ‘Switch-On’ of Lymphocytes as with polyclonal activation of T
cells (by superantigens) or of B cells due to staphylococci.
• Presence of Fc Receptors on Microbe or Induced on Host Cell: would cause nonspecific
binding of IgG antibodies in an ‘upside-down’ direction (e.g. some streptococci).
• Antibody Destruction (e.g. Neisseria gonorrhoeae, Haemophilus influenzae).
• TH Cell Destruction (e.g. HIV).
• ‘Exhaustion’ of TC Cell Response (long-standing activation of these cells which leads to
upregulation of inhibitory receptors or inhibition of their function) (e.g. HIV, viral
hepatitis).
• Encouraging T-reg Cell Formation (e.g. Leishmania spp.). It will suppress the immune
system.
• Cytokine Inhibition: as with poxviruses and Epstein-Barr virus (EBV).
Vaccination:
• The main idea behind vaccination is allowing the immune system to develop immunological
memory against a specific pathogen whilst avoiding contracting disease because of it.
• The nature of the pathogen would determine the chances of success of developing a vaccine, as
such factors as latency, antigenic variation and animal reservoirs would make those chances
significantly lower.
• From an immunological point of view, vaccines may be divided into:
- Live attenuated vaccines: where the germ is administered in a weakened state without killing
it. This method has a number of advantages:
o Microbial replication ensures sustained antigen delivery to the body.
o Enhancement of antigen binding to MHC class I molecules: due to intracellular microbial
replication, thus maximizing Tc cell response.
o Preferential antigen concentration in the target tissues.
However, the use of live attenuated vaccines poses two main problems:
o Immunodeficient patients may develop disease even due to weak microbes.
o Possible reversion of the microbe to its pathogenic, wild-type form which can the disease with
its full pathogenic potential.
Examples include: the BCG, measles, mumps, rubella, oral polio and varicella vaccines.

- Inactivated Vaccines: where the microbe is killed before administration. This eliminates the
risks of live attenuated vaccines but reduces its beneficial effects (e.g. Injectable flu and polio
vaccines, rabies).

- Subunit Vaccines: where the vaccine is prepared from a certain microbial portion which would
induce the formation of protective antibodies against the whole microbe (e.g. HBsAg (hepatitis B
surface antigen), human papilloma virus (HPV) which can cause cervical cancer in women).

- Conjugate Vaccines: If the microbial antigen is a weak immunogen, it may be bound to an

adjuvant to stimulate the immune response (e.g. Haemophilus influenzae type b, pneumococci,
meningococci).

- Toxoid Vaccines: where the microbial toxin is weakened before administration to the host (e.g.
diphtheria, pertussis, tetanus).

- Experimental Vaccines: have been designed for effective delivery of genes coding for microbial
antigens to the inside of the host cells, thus hopefully eliciting a better cell-mediated immune
response. These genes may be delivered using either lipid micelles or viral vectors.
Helpful videos:
https://www.youtube.com/watch?v=fHzs9FcnkdE
https://www.youtube.com/watch?v=kuaClgGHyZ4
questions:
1- which of the following evasive mechanisms is used by poxvirus?
a) (hit and run) raid
b) Cytokine inhibition
c) Inaccessibility
d) Encouraging T-reg cell formation
e) Exhaustion of cytotoxic T cell response
2-to which vaccine type does the rabies vaccine belong?
a) Live attenuated vaccine
b) Subunit vaccine
c) Toxoid vaccine
d) Conjugate vaccine
e) Inactivated vaccine
3- which of the following antibody isotypes cannot activate the classical complement pathway?
a) IgG3
b) IgM
c) IgG4
d) IgG1
e) IgG2
4- which microbe uses antigen shedding to evade the immune system?
a) Plasmodium
b) Mycoplasma pneumoniae
c) Trypanosoma brucei
d) Entamoeba histolytica
e) Leishmania
5-to which vaccine type does the human papilloma virus (HPV) belong?
a) Live attenuated vaccine
b) Subunit vaccine
c) Toxoid vaccine
d) Inactivated vaccine
e) Conjugate vaccine

Answers: 1-b 2-e 3-c 4-d 5-b

 Congenital and Acquired Immunodeficiencies
Classifications of Immunodeficiencies
• Immunodeficiency diseases: are defects in one or more components of the immune system
which can lead to serious and often fatal disorders.
• These diseases are broadly classified into two groups according to their causes:
- Congenital (primary) immunodeficiencies: are genetic defects that result in an increased
susceptibility to infection that is frequently manifested early in infancy and childhood
(embryonic intrauterine life)
o They are very rare (a doctor may never see them through his whole professional life).
o the highest interest in them are for research purposes because they explain how the
immune system works.
- Acquired (secondary) immunodeficiencies: are not inherited diseases but develop as a
consequence of malnutrition, disseminated cancer, treatment with immunosuppressive drugs,
or infection of cells of the immune system (such as HIV)
o Much commoner.
o Less interests for research purposes
Note: Adaptive immune deficiencies are more common than innate immune deficiencies.
When we say that there is immune deficiency, we say that it is unable to mount an immune response
in general, while immune tolerance is an antigen specific unresponsiveness.

General features of immunodeficiency diseases

• The nature of the infection in a particular patient depends largely on the component of the immune
system that is defective.
• Immunodeficiencies are associated with the arms of the immune system:
- Cellular innate immunity (Phagocytes, monocytes, etc...)
- Humoral innate immunity (complement, interferons)
- Cellular Adaptive immunity (T-cells)
- Humoral Adaptive immunity (B-cells and antibodies)
• Each type of the immune system has its own characteristic defects.

CONGENITAL IMMUNODEFEICIENCIES
Congenital defects in the cellular innate immunity
• These defects are the rarest among the congenital immunodeficiencies.
Chronic Granulomatous Disease:
- Incidence of 4 in 1 million in Jordan.
- Incidence of 1 in 1 million in the US.
- It involves a defect in phagocytes.
- When phagocytes engulf a pathogen, they kill it by a specific toxic substance (Reactive oxygen
species) in a process known as respiratory burst.
- This is accomplished by an enzyme called NADPH oxidase. This enzyme of composed of
multiple subunits. In chronic granulomatous disease one of these subunits genes is defective,
thus no enzymes will be synthesized, no respiratory burst, no killing.
 - Two thirds of cases are X-linked recessive (more in males than in females)
- One third of cases are autosomal recessive
- Why there is cases with x-linked and some with autosomal?? Because not the same subunit is
always defective and the distributions of there genes on chromosomes are not the same.
- The disease is characterized by recurrent infections with intracellular bacteria and fungi and
the formation of granulomas.
- We do nitro blue
tetrazolium (NTB)
test or burst test to
detect this disease.
Note: this is a functional defect not a numerical defect of phagocytes.

Congenital defects in the humoral innate immunity

Complement system deficiencies:
• All components reported deficient.
• Deficiencies in the complement system can be classified as deficiencies in the early or late
components of the complements system:
- Early components (C1, C2,C3,C4): characterized by a recurrent pyogenic (pus forming)
bacterial infections which can be streptococcus, staphylococcus or Hemophilus strains and
autoimmune disorders (Such as SLE) because of less clearance of immune complexes.
- Late components (C5, C6, C7,C8,C9): characterized by recurrent neisserial infections.

Congenital defects in the adaptive (acquired) immunity (humoral and cellular)

• This type describes deficiencies involving T(cellular) and B (humoral) lymphocytes
Defects in T-cells(cellular):
- T-cells are mainly involved in killing intracellular bacteria, viruses (mainly herpes strains), certain
parasites (pneumocystis jiroveci which is also called pneumocystis carinii previously) and many
types of fungi.
- A defect in T-cells will cause a recurrent infection in one of these mentioned microbes
- At diagnosis we observe a normal level of immunoglobulins and a reduced delayed type
hypersensitivity reaction (this type of hypersensitivity involves T-cell presence)
Defects in B-cells(humoral):
- B-cells are involved in immunoglobulins secretion, and extracellular killing.
- A defect in B-cells causes recurrent infections by pyogenic bacteria, enteric bacteria, some
viruses and parasites. Why pyogenic infections? Because a defect in B-cell causes a defect in
immunoglobulins like IgM and IgG which are involved in early complement components activation
in the classical pathway thus no early components, no killing of pyogenic bacteria.
- At diagnosis we observe a reduced level of immunoglobulins
and a normal level in delayed type hypersensitivity reactions
(B-cell have no role in this type of hypersensitivity)
Note: Please don’t be confused between the acquired immunity
(third line of defense) and the acquired type of
immunodeficiencies(secondary).

Severe combined immunodeficiencies (Congenital)

• As the name states, this type is characterized by a combination of both T and B-cells
immunodeficiencies.
• When a diagnosis shows a decrease in immunoglobulins and a decrease in delayed type
hypersensitivity reactions (negative test) as the same time, this shows that both B and T-cells and
defective.
• So, what happens at the microscopic level? When we take a biopsy from the spleen or the lymph
nodes, we can see abnormalities in the T and B cells regions (no germinal centers or T or B cells
zones).
• As the name state all these diseases are severe.
• The problem starts by the marriage of relatives. When they birth to their first child, some
symptoms appear on the child and the child dies within 1 to 2 years of birth. This child has shown
a severe combined immunodeficiency.
• So why didn’t his parents treat him soon? Because parents usually don’t consider it as a severe
problem at the beginning and when they try to treat him, it will be too late.
• When they give birth to the second child and notice that he has the same symptoms they consider
an early treatment this time. So, we must also consider that these deficiencies develop early in
life.
• These diseases are a reflect of multiple genetic mutation in B and T cells like mutations in their
enzymes, pathways, development or receptors.

Congenital Antibody Deficiencies (Humoral adaptive defects)

- Though these defects are congenital, but the disease doesn’t appear at the first year of birth.
This is because antibodies defects are compensated by antibodies from the mother (the
mother supplies the child with antibodies either by breast feeding or colostrum or by igG
through the placenta.
X-linked Agammaglobulinemia (Bruton’s agammaglobulinemia):
- As the name states, it is an agammaglobulinemia which means that there is no
immunoglobulins in the blood at all.
- Caused by mutations in the gene encoding an enzyme called Bruton tyrosine kinase (Btk) that
results in a failure of B cells maturation. This results in a deficiency in both B-cells and isotypes
of immunoglobulins
Selective IgA deficiency:
- It is under the category of hypoglobulinemia, which means that there is a reduction in the
immunoglobulins level in serum but not a complete depletion. A reduction means that there is
still some B-cells available but in a reduced quantity. Note that the reduction here is selective
only for IgA.
- Most common type of congenital immunodeficiencies in adults
- Occurs in 1 in every 400 adults
- Occurs in 1 in every 700 adults (from book)
Common Variable Immunodeficiency:
- Under the category of hypoglobulinemia as well.
- Patients are born normal then they acquire mutations.
- Also characterized in a reduction in serum immunoglobulins as well as B-cells.

Defects in T Lymphocyte Activation and Function (Adaptive cellular defects)

- The only disease to be discussed is the DiGeorge syndrome.
- DiGeorge syndrome: is a T-cell deficiency that usually affects children, characterized by a
mutation in a gene located on chromosome 22 locus 11 which codes for T-box 1 (TBX 1)
transcription factor. This mutation causes a defect in the 3rd and 4th pharyngeal pouches of the
 embryonic period. These pouches will later give rise to the thymus and the parathyroid glands
thus in this disease no thymus or parathyroid glands will be developed, no T-cells maturation(T-
cell maturation takes part in the thymus gland), hypocalcemia and parathormone hormone (PTH)
deficiency (parathyroid gland is involved in calcium and PTH secretion). These patients will suffer
from tetani as a reflect of PTH deficiency. Also results in some congenital heart diseases.
- There are dysmorphic features in these patients like low set ears and elongated philtrum.
- Please note that this type of defects begin at the beginning of the birth (the mother can’t
compensate T-cell defects like in B-cell defects)

Acquired (secondary) Immunodeficiencies

- As said previously they are much more commoner.
- Also there are less interesting for researchers (only
HIV is used in research purposes)
Protein-Calorie Malnutrition:
- The most common worldwide secondary
immunodeficiency is protein calorie malnutrition
- 10% of people have a form or another of malnutrition
(very common)
- Of course malnutrition is common between the poor,
refugees and in Africa for example
- Deficient intake of protein, fat, vitamins, and minerals,
will adversely affect maturation and function of the
cells of the immune system
- Mumps and measles are usually simple diseases that are easily stopped by our immune system
but in patients with malnutrition there will be decrease in the functions of the immune system
(secondary immunodeficiency) thus these two diseases can become major killers.
Diabetes Mellitus (Type 2):
Patients usually suffer from inflammations, diabetic foot (gangrene in limbs in general),
emphysematous cholecystitis (infection of the gallbladder by gas producing microbes),
emphysematous cystitis (infection of the urinary bladder by gas producing microbes)

HIV(AIDS):
- Viruses can cause immunodeficiencies as well. A small
example is the herpes simplex 1 virus (HSV- 1)
which causes cold sore (inflammation on the lips). After
it causes cold sore, the immune system suppresses it
and this virus becomes latent in the body. When the
patient is infected by a pneumococcus (a bacteria)
which causes pneumococcal pneumonia, the bacteria
will suppress the immune system thus helping the virus
to become active again.
- HIV is classified under the family of retroviruses which are RNA viruses.
- These group of viruses are characterized by having the reverse transcriptase enzyme which helps
in the conversion of RNA to proviral DNA.
- As shown in the figure, this virus is composed of two identical strands of RNA and some enzymes
(reverse transcriptase, integrase, and protease) which are both packaged in a cone-shaped core
composed of p24 capsid protein with a surrounding p17 protein matrix.
- All of the previous structures are further coated by a phospholipid membrane derived from the
host cells.
- Also there are some membrane proteins as gp41 and gp120 (gp=glycoprotein).
- HIV targets T-helper cells (CD4+). When this virus
enters the body it uses a tooth tip recognition
mechanism to find T-helper cell.
- When it finds the helper T-cell it binds with it at the
CD4 co-receptor of the T-helper cell by the virus
gp120 membrane protein (CD4 co-receptor with
gp120 interaction). This accomplishes the first signal
but a second signal is needed for the virus to enter the
T- helper cell.
- The second signal is accomplished by the binding of
the gp41 membrane protein of the virus with the
chemokine receptor of the T-helper cell.
- Now we know that we need two signals so that HIV
can infect a T- helper cell. HIV enters the cell by fusing
its membrane with the plasma membrane of the cells
thus releasing its genetic material inside to T-helper
cell.
- When the genetic material is inside it fuses with the
genome of the host and controls it. How can an RNA
fuse with a DNA? It doesn’t do that directly as an
RNA actually. The RNA need first to be converted to
a DNA and this is the function of the reverse
transcriptase enzyme.
- As the proviral DNA integrates with and controls the
host genome through the integrase enzyme , it
shifts the machinery toward the production of new
viral RNA and proteins. These products assemble to
thousands of new viruses.
- These viruses secrets the protease enzyme thus killing the cell and getting out to infect more
cells.
- Please note that HIV doesn’t directly kill patients but it causes a secondary immunodeficiency to
the patient thus now he becomes susceptible to other infections like pneumocystis jiroveci (easily
taken down by a functional immune system) which causes pneumocystis pneumonia.
- As a summary HIV causes an immunodeficiency thus helping other types of bacteria to infect the
patient and cause serious diseases.
- There are two types of HIV (HIV-1 and HIV-2). In 1980 this virus caused an epidemic infection by
the jiroveci bacteria in the USA thus people where dying by this weak bacteria.
HIV Pathogenesis
- When we say pathogenesis, we mean the way how a disease develops and
progresses in the absence of treatment. In other words, the stages from
which the virus enters the body until it causes the disease (AIDS is our
concentration now) or death
- The pathogenesis of HIV is considered from a clinical point of view. Which is
given the term CLINICAL COURSE.
HIV can be transmitted through several ways:
o By blood: either by the transfusion of virally contaminated blood from a
person to another or by intravenous injections used by multiple drug
abusers or through needle stick (doctors can stick themselves by
mistake sometimes during procedures)
o Vertical transmission: which mean the transmission from a mother to
its child during pregnancy (a pregnant mother shares its blood circulation
with its child)
o Sexual transmission: through sexual courses.
HIV pathogenesis can be divided into four main stages:
The occupational stage (primary infection): is the stage from which the
virus enters the body until the symptoms of the disease starts to appear
phenotypically
Acute HIV stage (2 weeks to 3-6 months): as the symptoms appear this takes us to this phase.
The symptoms are manifestoed as fever, headaches, sore throat with pharyngitis, generalized
lymphadenopathy and rashes. At the beginning of this stage the immune system sharply decreases
while the virus sharply increases. But at the end of this stage the immune system activates and
increases T- lymphocytes and immunoglobulins levels in an attempt to fight the virus. So the virus
levels starts to decrease. Note that the disease can be contagious during this period.
Clinical latency period (5-10 years): here the virus regains
his position and wins again by infecting the T-cells. This causes
the immune system to slowly decrease and starts to become
deficient. Please know that in this period the patient feel well at
the beginning of this period and the symptoms are gone (that’s
why its called latent). At the end of this period constitutional
symptoms starts to appear as exhaustion, loss of wight, fever,
loss of appetite.
The AIDS stage: please don’t be confused. AIDS means
acquired immune deficiency syndrome. So, it is a syndrome,
not a symptom. When we say that a patient is suffering from
AIDS, we don’t mean that he is suffering from a rash,
headache, pharyngitis or fever but we mean that he’s suffering
from an immunodeficiency syndrome. So at this stage the patient becomes completely
immunodeficient. Also, at this stage opportunistic infections start to take place, like our previously
discussed jiroveci example because the patient is IMMUNODEFICIENT). These secondary
opportunistic infections can lead to death.
Note: Normal levels of T-helper cells are 1000 per cubic square which can decrease to less than 200
during HIV infection.
Note: The good understanding of this virus has helped us in synthesizing drugs as anti- reverse
transcriptase and anti-protease to change this disease from a major killer to be just a chronic disease
like diabetes for example but this is accomplished when there is an early diagnoses of this virus.

A, Plasma viremia, blood CD4+ T cell counts,

and clinical stages of disease. About 12 weeks
after infection, blood-borne virus (plasma
viremia) is reduced to very low levels
(detectable only by sensitive reverse
transcriptase–polymerase chain reaction
assays) and stays this way for many years.
Nonetheless, CD4+ T cell counts steadily
decline during this clinical latency period
because of active viral replication and T cell
infection in lymph nodes. When CD4+ T cell
counts drop below a critical level (about
200/mm3), the risk for infection and other clinical features of AIDS is high.
B, Immune response to HIV infection. A CTL response to HIV is detectable by 2 to 3 weeks after the
initial infection and peaks by 9 to 12 weeks. Marked expansion of virus-specific CD8+ T cells occurs
during this time, and up to 10% of a patient’s CTLs may be HIV specific at 12 weeks. The humoral
immune response to HIV peaks at about 12 weeks.

Helpful videos:
https://www.youtube.com/watch?v=GoEXNXYWNsA

https://www.youtube.com/watch?v=HJnlQinO8V8

questions:
1- which HIV antigen binds to CD4 co-receptor?
a) gp41
b) gp120
c) HLA-DR
d) HLA-DP
e) Reverse transcriptase
2- which immunodeficiency causes problems with neisserial organisms?
a) B cell defect
b) Early complement component defect
c) Late complement component defect
d) Phagocyte defect
e) T cell defect
3- which of the following primary immunodeficiencies has the highest incidence in adults?
a) DiGeorge syndrome
b) TAP deficiency
c) Chronic granulomatous disease
d) Selective IgA deficiency
e) Wickott-aldrich syndrome
4- which of the following infections indicate the most severe T cell defect?
a) Reactivation of herpes simplex
b) Tuberculosis
c) Reactivation of herpes zoster
d) Low virulence mycobacteria
e) Candidiasis
5- which type of the following groups of diseases is genetically based but is rare?
a) Autoimmune diseases
b) Secondary immunodeficiency
c) Allergy
d) Atopy
e) Primary immunodeficiency
6- which phase of HIV infection occurs after the sero-conversion?
a) Progressive Generalized lymphadenopathy stage
b) Progressive stage
c) Asymptomatic stage
d) Death
e) AIDS stage

Answers: 1-b 2-c 3-d 4-d 5-e 6-c

 Introduction:
• Hypersensitivity reactions (HS): Excessive
immune responses that cause damage to
tissue.)overreaction of our Immune system).
- In the right of the picture we have gell and coombs
classification of the four different types of
hypersensitivity. (I, II, III, IV)
- In the left we have the antigens that could induce
and initiate hypersensitivity.
• They can occur in response to three different types
of antigen:

1. Infectious agents 2. Environmental substances 3. Self antigens

→The immune system sometimes
overreacts to infections and causes
disease, when an overzealous immune
response contributes to the symptoms
of infection, the resultant disease is a
type of hypersensitivity.
Examples:
- Viruses : influenza & HPV
- Bacteria :streptococcus pyogenes
- Parasites: schistosoma
→ Hypersensitivity can occur in → Normal host molecules can trigger
response to innocuous (harmless) immune responses, referred to as
environmental antigens, one example autoimmunity; when these cause
of which is allergy (type1). hypersensitivity, autoimmune disease
For example, in hay fever‫حمى القش‬, is the result, such as insulin dependent
grass pollens ‫ حبوب اللقاح‬themselves are diabetes mallets (IDDM),
incapable of causing damage; the hashimoto's thyroiditis, brain
immune response to the pollen is what diseases, rheumatoid arthritis (RA)
causes the harm. ,Systemic lupus erythematous(
SLE),Multiple sclerosis (MS) and many
other diseases.
.
Examples :
- Dust
- Feces of house dust mite/ insect
venoms
- Drugs: beta lactams (penicillin
and cephalosporin...). (Remember
that penicillin is a hapten.)
- Foods /Nickel/ Pollens (pollens of
olives tree)/ fungal spores.

1. Hypersensitivity to Infectious Agents →(Not all infections are capable of causing hypersensitivity
reactions).
• For example, although the common cold (rhinoviruses) elicits a strong immune response, it
never appears to cause harm.
• Other respiratory viruses such as influenza can cause hypersensitivity.
• Influenza virus damages epithelial cells in the respiratory tract but can sometimes elicit an
exaggerated immune response, which is far more damaging than the virus itself.
- Influenza can trigger high levels of cytokine secretion, sometimes referred to as a cytokine
storm. The cytokines attract leukocytes to the lungs and trigger vascular changes that lead to
hypotension and coagulation.
- In severe influenza, inflammatory cytokines also spill out into the systemic circulation, causing ill
effects in remote parts of the body, such as the brain.( Remote sites: any part of our body could
be infected.)
- This is analogous to the cytokine response seen in septic shock, which also leads to a type of
cytokine storm.
- Infections that are capable of eliciting hypersensitivity do not do so in every case.

• Hepatitis B virus (HBV) infection can result in chronic hepatitis in some individuals who make an
overzealous response.
• Another very different example of an infection causing hypersensitivity →immune complex
disease caused by streptococci.
The response depends on the infecting dose of virus and the immune response genes of the individual.
Infectious dose (how many MOs enter our body) or genetically (whether we are going to be
susceptible to develop hypersensitivity reactions).

2. Hypersensitivity to Environmental Substances

• For environmental substances to trigger hypersensitivity reactions, they must gain access to the
immune system.
1. Dust triggers a range of responses because it is able to enter the lower extremities of the
respiratory tract, an area rich in adaptive immune response cells. Dust can mimic parasites and may
stimulate an antibody response.
→ If the dominant antibody is immunoglobulin E (IgE), it may subsequently trigger immediate
hypersensitivity, which manifests as allergy symptoms such as asthma or rhinitis.)type1)
→If the dust stimulates IgG antibodies (type 2 or 3), it may trigger a different kind of hypersensitivity,
such as farmer’s lung. (type3)

2. Smaller molecules sometimes diffuse into the skin and may act as haptens, triggering a delayed
hypersensitivity reaction. This is the basis of contact dermatitis (type 4) caused by nickel.
- Hapten-: small molecule which induces hypersensitivity when it binds with protein carrier.

3. Drugs administered orally, by injection, or onto the surface of the body can elicit hypersensitivity
reactions mediated by IgE (type 1) or IgG (type 2 or 3) antibodies or by T-cells(type 4).
Immunologically mediated hypersensitivity reactions to drugs are quite common, and even very tiny
doses of drug can trigger life-threatening reactions. These are all classified as idiosyncratic adverse
drug reactions.

3. Hypersensitivity to Self Antigens

• A degree of immune response to self antigens is normal and present in most people. When these
become exaggerated or when tolerance to other antigens breaks down, hypersensitivity reactions
can occur. This is autoimmune disease.
- Is hypersensitivity good or bad?? Hypersensitivity is not always bad, True, it could lead to death
(anaphylaxis)and tissue damage in sever effects, but minor Hypersensitivity could means that you have
excessive immune response and better defense against infections and cancer cells.
-Sometimes little bite hypersensitivity could be good for our bodies.
-Allergic people are more immune to many infectious diseases as well as to malignancy.

TYPES OF HYPERSENSITIVITY REACTION)4 types)

• The hypersensitivity classification system used here was first described by Gell and Coombs.
• The system classifies the different types of hypersensitivity reactions by the types of immune
responses involved.
• Each type of hypersensitivity reaction produces characteristic clinical disease whether the trigger
is an environmental, infectious, or self antigen.
• For example, in type III hypersensitivity, the clinical result is similar whether the antigen is
Streptococcus, a drug, or an auto antigen such as DNA. (Immune complexes diseases)
• Hypersensitivity reactions are reliant on the adaptive immune system (specific immune response).
• Previous exposure to antigen is required to prime the adaptive immune response to produce
IgE (type I), IgG (types II and III), or T cells (type IV).
• Because previous exposure is required, hypersensitivity reactions do not take place when an
individual is first exposed to antigen.
• Adaptive and innate are involved →in each type of hypersensitivity reaction; the damage is
caused by different aspects of the adaptive and innate systems.

❖ Type I=allergy=immediate hypersensitivity (mast cells +basophiles+ eosinophils+ IgE+

(seconds-minutes))
- Type I hypersensitivity is mediated through the degranulation of mast cells and eosinophils. The
effects are felt within minutes of exposure. (This type is the fastest and the most serious type).

❖ Type II (IgG/IgM+ immune hemolysis diseases+ macrophage)

- Type II hypersensitivity is caused by IgG reacting with antigen present on the surface of cells.
- The bound Ig then interacts with complement or with Fc receptor on macrophages.
- These innate mechanisms then damage the target cells using processes that may take several
hours, as in the case of drug-induced hemolysis.

❖ Type III
- IgG is also responsible for type III hypersensitivity.
- In this case, immune complexes of antigen and antibody form and either cause damage at the
site of production or circulate and cause damage elsewhere.
- Immune complexes take some time to form and to initiate tissue damage. Poststreptococcal
glomerulonephritis is a good example of immune complex disease.

❖ Type IV
- The slowest form of hypersensitivity is that mediated by T cells, so-called type IV
hypersensitivity.
- This can take 2 to 3 days to develop and is referred to as delayed hypersensitivity.

DIAGNOSIS AND TREATMENT OF HYPERSENSITIVITY

- There are major differences in how the types of hypersensitivity reaction are diagnosed and
treated.
- For example, although skin tests are used to diagnose both type I and type IV hypersensitivity, the
exact type of testing depends on the type of disease suspected.
- Treatment for each type of hypersensitivity is also different.
- Because many diseases are caused by an overlap of different types of hypersensitivity, one
criticism of the Gell and Coombs classification system is that it is simplistic.
- However, some knowledge of this system makes it easier to understand how the different
disorders come about and how they can be effectively diagnosed and treated.
- Prick test: for type 1 (minutes). Patch test: for type 4 (2-3 days).
--
Immediate Hypersensitivity = (Type I) = Allergy = anaphylactic type

• Atopy: is an immediate hypersensitivity reaction to

environmental antigens mediated by IgE.
• Such reactions tend to run in families; these families are
said to have inherited the atopy trait. )Genetic
susceptibility to develop type one hypersensitivity
reaction)
• There are 40% of the population in general that have the
genes to develop HS type I reaction.)They have history of
atopy) .
• Allergy: was originally defined as any altered reactivity to
exogenous antigens .It is now often used synonymously
with atopy.
• Any person with a history of atopy has the susceptibility to develop type I of HS reaction which is
IgE mediated type .
• Allergy is defined as immediate hypersensitivity mediated by IgE.
• In normal conditions: IgE is the least common Ig found in our circulation
• (Has the lowest concentration in the serum), but in type I of HS, the concentration of IgE will be
very high.

Laypeople, and many clinicians, refer to any hypersensitivity reaction to exogenous substances as
“allergy,” a term that originally meant any altered reaction to external substances.
Most texts define allergy as an immediate hypersensitivity mediated by IgE antibodies. This more
restrictive definition is used because it helps to explain the specific diagnosis and treatment of
hypersensitivities mediated by IgE.

• Allergic diseases include anaphylaxis, angioedema, urticaria, rhinitis, asthma, and some types of
dermatitis )Atopic dermatitis)or eczema.

1. The systemic type of allergy is called anaphylactic shock/ anaphylaxis: which is characterized
by low blood pressure, the patient usually dies due to low BP.
• THUS, we have to give adrenaline (epinephrine) and noradrenalin (nor epinephrine) in order
to save his/her life.
2. Bronchial asthma: this condition could be life-threatening situation due to the smooth muscles
contraction, so it is characterized by what is called "wheezing" & if it lasts for longer it will be called
status asthmaticus, in this condition, the patient could die.
3. Rhinitis: occurs seasonally, when the plants produce their pollens. (At spring time and might also
occur at summer time)
4. Urticaria: the skin lesions that occur in this condition are called wheal-and-flare reaction which is
edema + erythema (redness) + itching.

- In urticaria, the mast cells degranulation will take place and these mast cells have an enzyme
called tryptase.
- SO, if you measure the level of tryptase and the result is high, this means mast cell degranulation
or basophil degranulation occurred. {Type I HS}
- When we do the skin-prick test we induce urticarial lesions as well.
- Exposure to cold temperature causes cold urticaria, while exercising induces an urticaria called
cholinergic urticaria.

**Mast cells can be degranulated without IgE. **

• However, mast cells, eosinophils and basophils can also be activated by other stimuli. For
example, activated complement generated by infections can activate mast cells, as can signals
transmitted by the nervous system—for example, in response to changes in temperature. It is
important to remember that the symptoms can be caused by other conditions in addition to allergy.
• What are the causes of Urticaria)mast cells degranulation)?
1. Mast cell degranulation is responsible for many cases of acute urticaria.
2. skin-prick test.
3. Cold temperature
4. Exercise.
5. Morphine

5. Atopic eczema: skin lesions and chronic itching feeling (due to histamine that is produced by mast
cells), accompanied with edema and erythema.

• The distinction between true allergy and other reactions is important because some of the
treatments for allergy would be inappropriate for other types of reactions.
The difference between allergy and other conditions. This concept is so important because if a patient is diagnosed
wrong, this could lead to his death. One example of this is one of the symptoms of allergy which is the redness of the
eye. This symptom can be from allergy, but it can be as well from herpes virus infecting the eye and causing what is
called an eye injection. If the doctor diagnosis a patient wrongly, as in says that they have allergy because of their
red eyes, when in fact they have the herpes virus, a wrong drug could be prescribed such as steroids. The virus will
cause an over infection leading to liver failure due to hepatitis. A simple mistake can cause someone their life.

• Allergies can occur in isolation in an individual. For example, an individual may have allergy to
penicillin and no other allergies at all.
• More often, individuals develop different types of allergy at different times in their lives. Typically:
1. Atopic eczema develops in →an infant.
2. Then food allergies develop as→ a toddler.
3. Finally allergic rhinitis and asthma develop in →elementary school.

- This phenomenon is referred to as the allergic march.

- About half of toddlers with atopic eczema develop asthma, and two-thirds develop rhinitis.
• It is common for allergies to improve spontaneously over time. The best example of this is allergic
eczema due to eggs, which disappears in the majority of young children.
• Allergies are usually very rapid reactions mediated by IgE; the symptoms develop within
minutes of exposure to antigen.
• However, some allergic reactions continue for a long time, such as when the environmental
antigen cannot be easily avoided, and they develop into a late-phase reaction characterized by T-
cell infiltrates.

• ALLERGEN: Antigens that trigger allergic reactions are referred to as allergens, which must be
able to gain entry to the body.
- Some allergens are present in the environment as small particles or low molecular-weight
substances that penetrate the body after being inhaled, eaten, or administered as drugs.
- Inhaled antigens include pollens, fungal spores, and the feces of the house dust mite )Many
allergens, including house dust mite feces, are enzymes)
- This characteristic may allow them to partially digest innate immune system barriers.
- Some insect venoms, which are injected directly into the skin, are allergens.

• An important part of the treatment of allergy is identification and avoidance of allergens which
is facilitated by:
1. Careful history taking.
- For example, a patient with a runny nose (rhinitis) is likely to be sensitive to aeroallergens
(airborne allergens).
- If symptoms occur predominantly in the summer, grass pollen is the likely culprit.
- If symptoms occur year-round and mainly indoors, sensitivity to house dust mite feces is likely.
Note: House dust mite allergy occurs in areas where a cold climate dictates the need for central
heating, heavy bedding, and thick carpets—the habitat of this mite.

2.knowledge of cross-reacting allergens.

Allergists use detailed knowledge about allergens to provide patients with avoidance strategies.
Here are some examples:

• Peanut allergy is the most common

allergic cause of severe reactions
and death.
- Many people with peanut allergy are
allergic to the peanut protein Ara h2.
This protein is very stable and is not
destroyed by cooking or by gastric
acid.
- In people who are allergic to Ara h2,
minute quantities of peanut in food
can cause severe systemic reactions
even in cooked foods.
- Other individuals are allergic to a
different peanut protein, Ara h8,
which cross-reacts with proteins
found in other foods (hazelnut,
carrot, and apple) and with birch
pollen.
- People with Ara h8 allergy can have nasal symptoms when birch trees are in blossom.
- Importantly, Ara h8 is an unstable protein that is destroyed by heating and by gastric acid.
- This means that cooked foods containing peanut are somewhat less risky for people with allergy
to Ara h8.
- Additionally, because the Ara h8 protein is destroyed by gastric acid, it does not have systemic
effects and tends to cause symptoms mainly in the mouth and lips.
- The coexistence of springtime nasal symptoms and allergy to this food group is referred to as the
oral allergy syndrome.
• Another example of cross-reactivity is between latex and some foods (banana +avocado+
kiwifruit).

(Skin-prick testing is not possible when patients have taken antihistamines)

• In penicillin allergy, the allergen is the β-lactam core of the penicillin molecule.
• Patients experience symptoms with different members of the penicillin family, such as amoxicillin
and flucloxacillin.
• Patients with penicillin allergy can react to other families of antibiotics, such as cephalosporins,
which also contain β-lactam ring structures.
❖ As shown by these examples, people with allergies often need to be referred to specialists to have
their allergies investigated in detail. This can be done by skin-prick or blood testing.
DEGRANULATING CELLS→ These cells release the mediators that cause the symptoms of allergy.

The major cells involved in allergy:

1. Mast cells→ Mast cells are resident in a wide number of tissues, rather like macrophages. Mast cells
express receptors for IgE and FcεRI (high-affinity IgE receptor); their degranulation is caused by IgE and
other activating agents (temperatures changes or exercise).

2. Eosinophils→ Eosinophils migrate into tissues where type I hypersensitivity is taking place, rather like
neutrophils attracted to sites of inflammation.
Eotaxin attracts eosinophils to the site of inflammation→ Eosinophils will be attracted to site of infection by
eotaxin(eosinophils chemotaxin), IL5, IL-13→IL-13 plays a major role in type I HS & recruitment of
eosinophils as IL-5.
3. Third type of degranulating cell is the basophil. These have a similar appearance to mast cells and they
have FcεRI as well, but they stay in circulation. It is unknown whether they have a special function.

1. All CD4+ T cells arise from developing T cells, which have the capacity to differentiate into either a
TH1, TH17, or a TH2 direction.
2. Antigen-presenting cells (APCs) appear to make decisions about whether precursor T cells will
develop in a TH1, TH17, or TH2 direction (TH1 or TH2 cytokine profile), leading to expression of
either IL-4(TH2) or IFN-γ (TH1).
3. APC through TLR on their surfaces determines which cytokines must be produced in order to
make the polarization step.
4. T cells can 'polarize' into TH1 or TH2 effector cells types in response to distinct cytokines.
5. In circumstances that are not well understood, an APC favors the production of TH2 cells. This
may be more likely to happen if the antigen is present at a mucosal surface or is associated with
molecules that stimulate certain pattern recognition molecules on the APC.
6. Stimulation of Toll-like receptor (TLR) 2 appears to eventually favor TH2 responses. Hence,
GATA3 is induced, and the stimulated T cell produces small quantities of IL-4. With each
successive round of T-cell proliferation, epigenetic changes become more established, and the
daughter T cells can become more and more polarized toward a TH2 phenotype. This can
occur only if Tregs do not inhibit the polarization. Once a TH2 response is established, high-
level IL-4 secretion will stimulate production of IgE by B cells.
7. During most responses, a mixture of TH1 and TH2 cells are produced, although one type of
response will tend to dominate, depending on the triggering infection. TH1 and TH2 cells are able
to produce positive feedback for their own type of cells, which could lead to extreme
polarization of the immune response.

• TLR2 & GATA3: activation of TH2 & suppression of TH1 (polarization to TH2)→ Cytokines that
are produced by TH2 will suppress TH1 and vice versa. If the Ag binds to a specific type of TLR,
like TLR2, it this will activate TH2 and produce transcription factor GATA3, leading to the
production of IL-4 and IL-13 causing TH1 suppression.

- Antigens(allergens) bind to →TLR2 (APC)→ T helper produce TF GATA3 →activation of TH2

→production of IL13,IL5 and IL4
- IL4 →suppression TH1+positive feedback (TH2) +induce B cells to produce IgE
- IgE→binds to Fc receptor on the surface of mast cells → in the second exposure to the same
antigen → antigen will bind with the fab portion of the IgE on the surface of mast cell (from the first
exposure)→ lead to degranulation
8. B cells class switch to IgE production when they are co-stimulated by interleukin 4 (IL-4) secreted
by T-helper 2 (TH2) cells (IgE is called reaginic antibody). (The Stimulation of TH2 will lead to type
I HS)
9. Once IgE is produced, it binds to the high-affinity receptor FcεRI, expressed on resting mast cells
resident in tissues and eosinophils that have been activated and that have migrated into tissues.
- Mast cells are responsible for initiating the symptoms of allergic reactions after allergen and
IgE have interacted.

10. If antigen cross-links the IgE bound to the FcεRI, mast cells release IL-4. IL-4 also inhibits the
production of IFN-γ by TH1 T cells. (in the second exposure )
- Thus once a T-cell response to an antigen has deviated toward production of TH2 cytokines,
positive feedback sustains and enhances the response.
- In addition, TH2 T cells secrete other cytokines (IL-5, IL-13, eotaxin), which help perpetuate the
TH2 response by stimulating the maturation and migration of eosinophils and by switching off TH1
cells.
- IgE binds to the FcεRI with such high affinity that although IgE is found at a thousand fold lower
concentration than IgG in serum, mast cells are constantly coated with IgE against different
antigens.
- Very high levels of IgE are seen in patients infected with parasites, such as in schistosomiasis.
- Total levels of IgE are also high in people who have inherited the atopy trait.
- Levels of IgE specific for given allergens can be measured using skin-prick testing or
enzyme-linked immunosorbent assay (ELISA) when investigating the cause of allergic
symptoms.
- IgE class switching will be stimulated by IL-4, TH2, skin prick test, and ELISA which is used to
detect the specific IgE that is involved in the reaction.

• Which direction this differentiation takes is dictated by whether TBX21, RORγt, or GATA3 becomes the
dominant transcription factor.
- TH1-polarized cells are characterized by expressing the transcription factor TBX21, which promotes the
secretion of IFN-γ.
- IL-12(which is secreted by macrophages): activation of TH1 and IFN -γ production (polarization to
TH1)→The same thing regarding TH1 but the transcription factor here is T-bet that leads to the production
of IL-12 which will affect TH-1 to produce IFN- γ . IFN- γ will suppress TH2.
- TH17 cells are regulated by the transcription factor RORγt.
- TH2 cells express the transcription factor GATA3, which promotes the secretion of IL-4 and associated
cytokines IL-5 and IL-13.
T-reg
• A fourth population of CD4+ T cells is the regulatory T cells (Tregs).
• Tregs play an important role in peripheral tolerance and inhibit both TH1 and TH2 cells in an
antigen dependent fashion.
• Tregs inhibit other T cells by secreting cytokines, such as transforming growth factor beta (TGF-β)
and IL-10.
- (In most people, Tregs prevent T-helper responses from becoming over polarized toward
extremes of either TH1 or TH2 cytokine production)
- (Extreme polarization is normally prevented by Tregs. In the absence of Tregs, an immune
response may become over polarized toward TH2, and allergy may develop)
- (Tregs may also have a role during infection; for example, they may prevent overzealous
responses that would otherwise lead to hypersensitivity)
This is the basis of allergen immunotherapy.
- For example, in nonallergic individuals, the Tregs are the dominant T-cell type specific for
environmental allergens.
- In other words, in nonallergic individuals, a polarized response to environmental allergens cannot
develop because Tregs would inhibit it. Tregs can also be induced in an individual who has
become allergic.

• Immediate type of Hypersensitivity occurs within seconds or minutes after the exposure to the
allergens.
• Certain mediators are going to be produced. When allergen cross-links IgE bound to cells by
FcεRI, cells release the mediators of the early-phase reaction. (Early phase) (Mast cells
mediators).
• Then, 1 or 2 hours after the exposure and under the
influence of cytokines that are produced, eosinophils
can be recruited into the area of infection by IL-5 which
also stimulates the production of eosinophils. SO, IL-5
stimulates maturation & production of eosinophils. (Late
phase) (Eosinophils mediators).

• Most adaptive immune system responses in humans

produce a mixture of antibodies and cytotoxic T
lymphocytes (CTLs).
• The precise balance of which type of response
dominates depends on the type of pathogen the immune
system is responding to:
- The immune system responds to chronic intracellular infection, such as tuberculosis (TB) → with
the production of a mainly T-helper 1 (TH1) response, with resulting activation of CTLs and
macrophages by interferon gamma (IFN-γ). High levels of IgG are produced in TH1 responses.
- TH17 cells respond to extracellular infections by activating neutrophils and stimulating epithelial
cells to secrete antimicrobial peptides.
- In TH2 responses, production of IgG and CTLs is inhibited.
- TB and allergy represent opposite poles of the adaptive immune response. Most responses are
much less extreme and involve a mix of TH1, TH17, and TH2 cells.
PREDISPOSITION TO ALLERGY
There are three important facts to consider in the epidemiology of allergy:
• Allergy is very common and affects up to 40% of the population in the developed world.
• Allergy runs in families and appears to have a genetic basis.
• Twin studies have shown that 80% of the risk of allergy is genetic. Affected family members may
have different allergies (such as asthma, hay fever, or eczema( in response to different allergens.
It is the risk of allergy that is inherited, not the specific allergy.
• The prevalence of allergy has increased in the developed world and is beginning to increase in
many parts of the developing world.
• Understanding the biologic basis of these epidemiologic facts would help us prevent and treat
allergies(

1.Genetics of Allergy )filaggrin gene + CD14 gene)

• Although allergy clearly has a hereditary component, it has not been possible to find mutations or
polymorphisms that occur in everyone affected by allergy.
• Polymorphisms in several different genes have been implicated, and these probably work together
to affect the risk of allergy.
Polymorphisms in the gene for filaggrin are a well-established cause of allergy.

- Filaggrin is a protein expressed in the keratinocytes of

the skin. It has a role in forming the cytoskeleton of
these cells, particularly in and around the tight junction
between keratinocytes, maintaining the epithelial barrier.
- In addition, filaggrin is broken down to smaller peptides,
which are hygroscopic and act as a natural
moisturizing factor (NMF).
- Finally, NMF helps keep the pH of the skin low, which is
thought to help prevent invasion by pathogens.
- There is considerable variation in the gene for filaggrin.
These variants are regarded as polymorphisms because
they occur in more than 1% of the population.
- These variants are less effective at maintaining the epithelial barrier or moisturizing the skin.
- Filaggrin variants are associated with all types of allergy and, for example, are implicated in 50%
of cases of severe atopic eczema.

- Environmental factors place children who have inherited filaggrin variants at extra risk of
developing allergy. For example, exposure of children with filaggrin variants to cats in early
childhood increases the risk of developing hay fever (allergic rhinitis caused by grass pollen) in
later life.
- This implies that the filaggrin variants cause systemic changes in immunity so that other organ
systems are affected and sensitization to other allergens is increased.
- Importantly, exposure to cats later in life does not have the same effect; thus the timing of the
exposure to the environmental factor is also critical.
Summary : There are genes called filaggrins which are responsible of making the skin moisturized
changes/variation any and in them will cause the dryness of the skin (the tight junctions will become
loose and the patient becomes more susceptible to type I HS reaction.) (Causes positive stimulation
of TH2 and immediate type of HS).
▪ Variants in other immune system genes have also been associated with allergy. For example,
lipopolysaccharide (LPS) is released from a wide range
of organisms.
▪ Variants in the CD14 gene (the receptor for LPS) and LPS
levels interact to positively or negatively affect the risk of
allergies.
This Figure shows a hypothetical timeline that illustrates
how genetic and environmental factors can interact at
critical time points to become manifest as allergy.

- People who work with live-stock are exposed to

mycobacterium Ag leading to TH1 activation & TH2
suppression. (-)
- Some people are sensitive to cat dander; sometimes it
causes stimulation or suppression of TH2 (But usually
stimulation) (+/-)
- Some people that are infected by rhinovirus or common
cold virus suffer from massive edema & manifestations of type I HS (TH2 activation) (+)

2. Environmental Factors and Allergy

• The increasing incidence of allergies in the developed world is suggestive of the effects of a
changing environmental factor, in particular, increasing urbanization.
• Studies have shown, for example, that growing up on farms and being exposed to livestock
decreases the risk for allergies.
→ This led to the idea that exposure to animals and their excreta and to bacteria in general steered
the immune system away from a TH2 pattern and the development of allergies. ( they will be
more exposed to environmental pathogens, which will activate the production of TH1, which will
suppress TH2 production
• This thinking was referred to as the hygiene hypothesis, which posits that the increase in
allergies in the developed world is caused by reduced exposure to microorganisms in early life.
• For people who live in an extra clean environment, TH2 production will be activated rather than
TH1.Which means that they will be more susceptible to type I hypersensitivity reactions, more
allergic consequences.
• The hygiene hypothesis inspired clinical trials that exposed children to harmless microorganisms
that have been investigated for their impact on allergy.
• These have included studies on so called “probiotic” bacteria in milk and live vaccines.
Neither of these approaches showed clear benefits in preventing allergy.
• However, we now know that the hygiene hypothesis is an oversimplification. For example,
infections with some worms, such as schistosomes, decrease the risk of allergies even though
these worms provoke a TH2 response.
• Allergy is also becoming more common in parts of the world where exposure to infectious
microorganisms is still very common, such as in some parts of South America. Current thinking is
that infections can both increase or decrease the subsequent risk of allergy development,
depending on the exact timing of exposure and the individual’s genotype.
• In addition, other environmental changes may have contributed to the rise of allergy, such as
some types of air pollution and the overabundance of food, causing obesity. Both obesity and
airborne pollution are known to trigger inflammation.
• As knowledge of epigenetics improves, it is becoming clearer that environmental factors can act in
utero or even by exposing the mother (or even the grandmother) before conception.
• Research into these environmental factors will hopefully lead to the discovery of interventions that
can prevent allergy.
• Mycobacteria are common environmental organisms, most of which do not cause disease in
healthy humans.
There are two overlapping ways in which mycobacteria
may prevent over- polarized TH2 responses from
developing in early life:
1- Mycobacteria can survive inside macrophages and
provoke a strong TH1 response.(Mycobacterium tuberculosis
has evolved evasion mechanisms that enable it to cause
disease in healthy humans -it overcomes the TH1 response-
).According to the hygiene hypothesis, exposure to
nonvirulent mycobacteria in rural environments—for example,
in animal feces—stimulates T cells to secrete IFN-γ, which
inhibits TH2 responses.

2- The second explanation is that nonvirulent mycobacteria

may induce conditions that favor the production of Tregs.
Instead of producing TH2 cells, individuals may then produce
Tregs that are specific for environmental allergens.

o Vaccination with Mycobacterium vaccae.

o Drugs: CpG and TLR9 stimulate IL-12.

The hygiene hypothesis may be translated into clinical benefits in the future:
• Vaccination with Mycobacterium vaccae, a nonvirulent mycobacterium, has been shown to
reduce the severity of eczema in allergic children.
• Drugs can also be used to mimic microorganisms and steer the immune response away from TH2
polarization. For example, CpG is a nucleotide motif found in viruses that binds to Toll- like
receptor 9 and stimulates IL-12 secretion by APCs. Synthetic CpG administered to animals can
switch off allergic TH2 responses.

MEDIATORS OF EARLY PHASE OF ALLERGY

• The early phase of allergy is caused by mediators released by mast
cells when IgE bound to FcεRI is cross-linked by allergen.
• Anaphylaxis is the most serious type of allergy and can occur
when allergen enters the body from any route.
• During anaphylaxis, mast cells rapidly synthesize prostaglandins and
leukotrienes through the cyclooxygenase and lipoxygenase pathways.
• These mediators cause vasodilation )low BP) and an increase in
vascular permeability.
• In addition, fluid shifts from the vascular to the extravascular space,
and a fall in vascular tone occurs.
• The result of widespread mast cell activation is a dramatic fall in blood
pressure, which is characteristic of anaphylaxis.
• Mast cells in the skin, but not the airway, release histamine, which
contributes to swelling and fluid shift.
• In other forms of allergy, more localized changes in blood vessels
occur that are restricted to the site of allergen entry. For example, in
allergic rhinitis, inhaled allergens stimulate mast cells in the nasal
 mucosa. Subsequent vasodilation and edema in the nose
causes nasal stuffiness and sneezing. Leukotrienes increase
mucus secretion, which causes the discharge characteristic of
allergic rhinitis. Increased mucus secretion in the bronchi also
occurs in asthma and contributes to the airflow obstruction.
• However, in the lungs, leukotrienes cause smooth muscle
contraction, which has the most dramatic effects on airflow
reduction.
• Degranulating mast cells also release enzymes. These activate
messenger molecules such as complement and kinins.
• Mast cell tryptase has a special role in clinical medicine in the diagnosis of anaphylaxis.
• All of these effects can take place within minutes of exposure to allergen, and symptoms persist
while exposure to allergen continues. Even if the person is able to avoid the allergen, late phase
response may occur.
MEDIATORS OF LATE PHASE OF ALLERGY
• Type I hypersensitivity reactions are generally characterized by immediate symptoms after
exposure to allergens. For example, a patient with asthma who is allergic to cats will develop
airway obstruction characterized by wheezing seconds after exposure.
• The symptoms improve after an hour or so as the immediate response dies down.
• Several hours after the acute episode, the airflow in the bronchi may deteriorate again, reflecting
the migration of leukocytes—particularly eosinophils—into the bronchi in response to
chemokines.
• The late phase may last several hours.
• In some individuals, this process becomes self-perpetuating as TH2 cells in the bronchial wall
secrete cytokines such as IL-4 and attractant chemokines. The result is chronic allergic
inflammation in the airways.
• Mediators released by eosinophils include peroxidase, eosinophil major basic protein, and cationic
protein, which all cause direct damage to bronchial tissue.
• As a result of the chronic allergic inflammation, the bronchial smooth muscle is hypertrophic, and
mucus secretion is increased; airflow becomes persistently, rather than intermittently, reduced.

TREATMENT
• Allergies produce a spectrum of symptoms that range from mild, such as nasal blockage in
rhinitis, to life-threatening, as in the case of severe asthma or anaphylaxis.
• The treatment for allergy is tailored to the individual patient’s circumstances and symptoms.
• General measures in the treatment of allergy include identifying and avoiding possible allergens.
• This is not always possible when the allergen is widespread in the environment, such as with
grass pollen. Other treatments involve the use of drugs or desensitization.
Drug Treatments
• Some drugs block the end effects of mediator release; for example, β2-adrenergic agonists,
such as salbutamol, mimic the effects of the sympathetic nervous system and work mainly by
preventing smooth bronchial muscle contraction in asthma.
• Epinephrine (adrenaline) is an important drug and can be lifesaving in anaphylaxis. In
anaphylaxis, the blood pressure falls dramatically because fluid shifts out of blood vessels and
into the tissues when vessel permeability increases. Epinephrine stimulates both α- and β-
adrenergic receptors, decreases vascular permeability, increases blood pressure, and reverses
airway obstruction.
• Antihistamines block specific histamine receptors and have an important role in allergies that
affect the skin, nose, and mucus membranes.
• Antihistamines are much slower acting than epinephrine in the treatment
of anaphylaxis and are not very useful in asthma because histamine is
not an important allergic mediator released by mast cells in the lung.
• Specific receptor antagonists block the effects of leukotrienes.
Montelukast, for example, reduces the amount of airway inflammation in
asthma.
• Corticosteroids are widely used in the prevention of symptoms in
patients with allergy. Corticosteroids can prevent the immediate
hypersensitivity reaction, the late phase, and chronic allergic
inflammation.
• To avoid side effects, corticosteroids are often given topically in
allergies; for example, inhaled steroids are used in asthma.
• Sodium cromoglycate has some effects in preventing allergy attacks. It is thought to work by
stabilizing mast cells and reducing degranulation.
• Other drugs in development aim to block the TH2 cytokine pathway or prevent IgE binding to the
FcεR. Interesting approaches have also been taken to reduce the allergenicity of environmental
allergens. For example, one biotechnology company has produced genetically modified cats that
do not produce the cat allergen FelD1. These cats do not provoke allergic symptoms in sensitized
patients, but at over $1000 per animal, they are not likely to be a popular solution.
Allergen Immunotherapy
• Allergen immunotherapy, or desensitization (allergy shots), is a well-established technique that
aims to improve allergy symptoms caused by specific allergens.
• It is most useful when single allergens are involved in the symptoms, and it is often used to
prevent anaphylaxis resulting from insect stings or to reduce symptoms from hay fever due to
grass pollen allergy.
• Although treatment starts with very small doses of allergen given by injection, there is a risk for
precipitating a full-blown anaphylactic attack. Therefore trained staff must perform the
desensitization with access to resuscitation equipment.
• Over time, the patient is given injections with increasing quantities of allergen until sufficient
allergen is being given to dampen the allergic response.
• Another type of treatment, sublingual immunotherapy, is used to treat hay fever. Patients are
given small doses of purified grass pollen under the tongue for several months, again dampening
allergy symptoms.
• Large doses of bee venom allergen can induce regulatory cells in people who are stung
frequently.
• Immunotherapy injections work in the same way. As the dose builds up, regulatory T cells are
induced. These secrete IL-10, which reduces TH2 cell activity and thus reduces IgE secretion. At
the same time, specific IgG secretion increases. These regulatory effects are achieved only
when a high dose of allergen is delivered.
• Sublingual immunotherapy induces regulatory T cells in a slightly different way. The same dose of
pollen placed in the nose of someone with pollen allergy would cause severe symptoms. But
because the mouth contains few mast cells, doses of grass pollen cannot cause any allergy
symptoms when administered orally. The APCs in the mouth and associated lymph nodes tend to
induce T-regulatory cells (Tregs) rather than TH2 cells. These Tregs secrete IL-10, which leads to
reduced IgE production; and so sublingual exposure to grass pollen eventually leads to reduced
allergy symptoms, even when pollen arrives in the nose.
• These two types of treatment illustrate how changing the dose (in the case of injection
desensitization) or route of delivery (in the case of sublingual desensitization) affects immunologic
outcomes.
Type (2) hypersensitivity

• Type II hypersensitivity reactions are a consequence of

immunoglobulin G (IgG) or IgM binding to the surface
of cells.
1. Antibody binding frequently damages red blood cells,
either through activation of complement or because the
antibodies opsonize the target erythrocytes. This is
referred to as immune-mediated hemolysis.
2. Antibody binding may also damage solid tissues, where the antigen may be cellular or part of the
extracellular matrix (eg, basement membrane).
3. Less often, antibodies may modify the function of cells by binding to receptors for hormones,
which is illustrated in autoimmune thyroid disease. Hyperacute graft rejection is also a version of
type II hypersensitivity.
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IMMUNE-MEDIATED HEMOLYSIS

Red Blood Cell Antigens

- Red blood cells express a variety of antigen systems. (ABO system+ Rh system)
- The rhesus (Rh) and ABO antigen systems contain alleles inherited in a mendelian fashion.
- The genetics and immunology of the clinically important rhesus and ABO systems differ.
- The rhesus blood group system consists of three loci )C, D, and E ( of which D is the most
important.
- Most individuals express a D locus antigen and are therefore rhesus positive.
- About one in six people are homozygous for a null D allele; they express no D antigen and are
therefore rhesus negative.
- D is a conventional protein antigen, and rhesus-negative individuals make IgG anti-D after
exposure to the antigen.

• The A and B blood group antigens are oligosaccharide

molecules produced on the surface of red cells. These
sugars are inherited in a co-dominant fashion:
1. An individual can inherit the A antigen (blood group A).
2. The B antigen (blood group B).
3. Both A and B (blood group AB).
4. A null allele (blood group O).
• The A and B antigens are similar to oligosaccharides
expressed on bacteria.
- Natural antibodies produced by B1 cells recognize A and B.
- Thus anti-A and anti-B are IgM natural antibodies, physiologically produced as a defense against
bacteria and capable of cross-reacting with A or B red cell antigens.
- Hence, individuals who are blood group O and have inherited neither A nor B antigen will produce
IgM against A and B from birth, regardless of whether they have been exposed to these antigens.
- Individuals who have inherited A antigen but not B antigen produce anti-B only, and so forth.

• Other red cell antigens are nonallelic; the same molecule is expressed by everyone. For example,
the I antigen is expressed by adults on the surface of red cells. I behaves as a regular self
antigen and should not normally elicit anti-I antibodies.
• Antigens of the ABO and rhesus systems are alloantigens—they differ from person to person.
• The antibodies produced against these
antigens can cause type II hypersensitivity
when cells are transferred from one individual
to another, such as in blood transfusion or
during pregnancy.
• Antigens of the rhesus and I systems can also
act as autoantigens. They can cause type II
hypersensitivity when they become targets of
autoimmunity.

Anti–Red Cell Antibodies

→IgM antibodies against red cell antigens are produced as natural antibodies against A and B or as
autoantibodies against I in some types of autoimmune hemolytic anemia (AIHA).
- IgM antibodies are highly effective at activating complement and rapidly cause damage through
activation of the membrane attack complex.
→ IgG antibodies are produced against rhesus antigens, either as a response to allogeneic
stimulation or in some types of AIHA.
- IgG is not very effective in activating complement and does not cause hemolysis in the circulation.
Instead, IgG-coated red cells are recognized by Fc receptors on resident macrophages in the liver
and spleen. The IgG-coated red cells are then destroyed by phagocytosis.

Types of Immune-Mediated Hemolysis

1. Alloimmune Hemolysis
- The rhesus antigens behave like conventional antigen—exposure is required to produce IgG
antibody.
- This most frequently occurs in pregnancy when IgG antibodies against rhesus antigens cross the
placenta and cause hemolytic disease of the newborn.
- These IgG antibodies cause fairly gradual destruction of red cells. This is because the IgG-coated
red cells are only slowly recognized by macrophages in the spleen, which have Fc receptors for
IgG.
Incompatibility in the ABO system is the most common cause of serious blood transfusion reactions.
For example, an A-positive individual who requires a transfusion possesses natural antibodies
against B-positive cells. If B-positive cells are inadvertently transfused, they will be rapidly hemolyzed
in the circulation. The hypersensitivity reaction can take place within seconds of the donor cells
entering the recipient. IgM reactions are very fast because pentameric IgM is able to efficiently
activate complement. This is because each IgM molecule is able to aggregate antigen more
effectively and because of the greater numbers of Fc components, which activate the early classic
complement cascade
• Units of blood for transfusion contain mainly red cells and very little antibody-containing plasma.
• The recipient’s antibodies and the donor’s red cell antigens must be checked for compatibility prior
to transfusion.
• In most routine clinical situations, considerable precautions are taken to ensure patients receive
the correct type of blood. Mismatched transfusions should be 100%preventable, but every year, a
handful of deaths are caused by transfusion errors.
• In emergencies, when the laboratory does not have time to determine the recipient’s blood group,
O cells—which have neither A nor B antigens—can be used for transfusion into any type of
recipient.
2. Autoimmune Hemolysis
• Autoimmune hemolytic anemia can be triggered by infections or drugs, or it can be part of
generalized autoimmune diseases such as systemic lupus erythematosus (SLE).
• Autoantibodies can also be produced by malignant clones of B cells in diseases such as chronic
lymphocytic leukemia or lymphoma. However, most cases of AIHA are not explained.
• Red cell antigens can become targets for IgG and IgM autoantibodies.
• The most common type of AIHA is caused by IgG autoantibodies against rhesus antigens. The
antibody-coated red cells are only slowly removed by the spleen, and the onset of anemia is
gradual.
• The I antigen is generally the target when IgM antibodies cause AIHA. Much more rapid and
dangerous intravascular hemolysis occurs as a result of complement activation.
• Another feature of IgM antibodies is that they often bind red cells best at temperatures below
37°C.
• These cold hemagglutinins can cause red cells to aggregate in vessels in the hands and feet,
which may cause ischemic damage. Similar alloimmune and autoimmune processes can affect
platelets and neutrophils.

Type II Autoimmune Hypersensitivity against Solid Tissue

• Autoantibodies can also attack and damage components of solid tissues. For example, in
Goodpasture syndrome, IgG autoantibodies bind a glycoprotein in the basement membrane of the
lung and glomeruli. Anti–basement membrane antibody activates complement, which can trigger
an inflammatory response.
• Goodpasture syndrome can be diagnosed by finding antibodies to glomerular basement
membrane in patient’s serum on indirect immunofluorescence.
• In a variety of other comparable conditions, IgG antibodies bind to other cells or to tissue
components. For example, in the blistering skin condition pemphigus, antibodies bind on the
intercellular cement protein desmoglein.
• In myasthenia gravis, IgG binds to the acetylcholine receptor in skeletal muscle, causing
widespread weakness.
• A characteristic that this group of diseases shares with AIHA is that the diagnosis can be made by
detecting the autoantibody in blood samples. Treatment is aimed at removing or blocking the
autoantibody.

TYPE II HYPERSENSITIVITY AND ANTIBODIES THAT AFFECT CELL FUNCTION

• In other situations, antibodies bind to cells and affect their function.
• These antibodies can simply stimulate the target organ function without causing much target
organ damage, such as in Graves disease.

Graves Disease
• Graves disease is the most common cause of hyperthyroidism, often affecting young women
with a family history.
• Graves disease is linked to the human leukocyte antigen (HLA) allele DR3.
• In Graves disease the thyroid is stimulated by an autoantibody that
binds onto the thyroid-stimulating hormone (TSH) receptor.
• The anti-TSH receptor antibody mimics the effects of the hormone.
• Graves disease is thus a special kind of type II hypersensitivity.
• In pregnant women with Graves disease, IgG thyroid-stimulating
antibody can cross the placenta and cause transient neonatal
hyperthyroidism.
• Graves disease is associated with exophthalmos (protruding eyes)
resulting from T cells infiltrating the orbit of the eye.
• Exophthalmos is thought to be caused by an orbital antigen that
cross-reacts with a thyroid antigen.

Type (3) hypersensitivity

• The antigens can be derived from infection, innocuous
environmental substances, or autoantigen.
• Immune complexes (Ab-Ag complex) are usually cleared
by the complement system, but when this does not
happen, immune complex diseases may arise.
• Immune complexes are lattices of antigen and
antibody; they may be localized to the site of antigen
production or may circulate in the blood.
• They are produced as part of the normal immune
response and are usually cleared by mechanisms that
involve complement. Immune complexes cause disease in a number of situations.
- Farmer’s lung and the Arthur reaction→local immune complex disease.
- Poststreptococcal glomerulonephritis →circulating immune complex disease.
- Systemic lupus erythematosus (SLE) →Systemic immune complex disease.

ANTIGENS IN IMMUNE COMPLEXES

• Antigens that can form immune complexes must be polyvalent; that is, each antigen molecule
must be able to bind more than one antibody molecule.
• For immune complexes to develop, antigen must be present long enough to elicit an antibody
response.
• Immune complexes usually form when antigen is in slight excess of antibody.
• Immune complexes may form when antigen is produced from one of three sources:
1- Infectious antigens. 2 -Innocuous environmental antigens. 3-autoantigens

1- Infectious Antigens
• Most infections are short lived and are controlled by the immune response. Even in such rapidly
controlled infections, immune complexes may cause hypersensitivity, such as after streptococcal
infection.
• Infections such as hepatitis B are not always controlled and can cause sustained high levels of
antigen in blood (antigenemia), resulting in more chronic immune complex disease.

2- Innocuous Environmental Antigens

• Harmless environmental antigens can elicit an immunoglobulin G (IgG) response if they are small
enough to enter the tissues.
• A good example is fungal spores, which cause the localized immune complex disease known as
farmer’s lung.
• Drugs are also environmental antigens and sometimes cause localized immune complexes, such
as the Arthus reaction.
• Drugs can also cause circulating immune complexes. This leads to a disorder referred to as
serum sickness.
• Serum sickness: The name was coined in the period before antibiotics were available, when
patients with infections were given immune horse serum. Nowadays, serum sickness most often
happens as a result of the use of mouse monoclonal antibodies to treat cancer or autoimmune
disease. Repeated exposure leads to production of antimouse antibodies and circulating immune
complexes. Serum sickness then causes fever, rash, and joint pain. This problem can be
overcomed by genetically manipulating the mouse antibodies to humanize them.

3-Autoantigens
• Autoantigens can only cause immune complex disease in the presence of autoantibodies. DNA is
an antigen in SLE.
• SLE is the most prevalent immune complex disease. DNA is released into the circulation when
cells die, especially if innate immune system mechanisms usually responsible for clearing DNA
are defective. DNA that is not rapidly cleared can elicit an antibody response.

ANTIBODIES IN IMMUNE COMPLEXES

• Immune complexes will form only when the ratio between antigen and antibody is exactly right.
- At low levels of antibody, each antigen molecule binds several immunoglobulin molecules.
- When antibody and antigen levels are approximately equal, or antibody levels are slightly in
excess, large complexes can form.
- When antibody exceeds
antigen, small complexes form.
• During an infection, this means
that immune complexes will be
produced very transiently at the
point where antibody levels are
increasing. Immune complexes
rarely become persistent during
infections and do so only when
the infection cannot be cleared by
antibodies (e.g., hepatitis virus
infections).
CLEARANCE OF IMMUNE COMPLEXES
• Immune complexes can form in normal individuals when antibodies are produced during
infections.
• Two mechanisms involving complement can clear immune complexes:

Complement Breaks Down Large Soluble Complexes

- Immune complexes of antigen and immunoglobulin contain high numbers of immunoglobulin Fc in
close proximity, which activates complement through the classical pathway .
- Small molecular components, especially activated C3, are produced through activation of the
complement pathway. These molecules insert themselves into and break up the lattice of the
immune complex.
Complement Receptor 1 Transfers Complexes to Phagocytes
- Red blood cells transfer circulating immune complexes from tissues and blood to the phagocytes
of the liver and spleen.
- Red cells express complement receptor 1 (CR1), the receptor for activated C3.
- Immune complexes bind to the complement receptor CR1 on red cells, which then circulate
through the liver and spleen.
In the liver and spleen,
receptors take up the
immune complexes and in
doing so stimulate the
macrophages to
phagocytose them.
- This mechanism is highly
efficient and can entirely
remove immune complexes from the circulation in a few minutes. Furthermore, because the
spleen is home to a large population of B cells, antigens originally present in the periphery are
rapidly presented to B cells to boost antibody production.
Failure of Clearance
- These immune complex clearance mechanisms can be saturated in situations where there is
excessive, ongoing production of immune complexes—for example, in antigenemia resulting from
chronic infection.
- Some individuals lack complement and, because the mechanisms described cannot function, are
predisposed to immune complex disease.

MECHANISMS OF INFLAMMATION IN IMMUNE COMPLEX DISEASE

• Immune complexes that are not cleared rapidly cause damage by activating components of the
innate immune system.
• Immune complexes activate complement→Although this process helps to clear complexes, low-
molecular-weight anaphylatoxins are produced, which increase permeability of blood vessels
and are chemotactic for leukocytes.
• Complexes bind to and activate cells such as neutrophils, mast cells, and platelets.
• Neutrophils and mast cells release proteolytic enzymes, which damage blood vessels and initiate
inflammation.
• Activated platelets bind to the endothelium and form thrombi.
• If antigen is present predominantly at one site, immune complexes cause localized damage—for
example, the Arthus reaction and farmer’s lung.
• Small complexes produced when antigen is in excess enter the circulation and form circulating
immune complexes.
• Circulating complexes cause damage to blood vessels that range from inflammation of the vessel
walls to occlusion of the vessel and ischemic damage. Immune complex disease is one cause of
vessel inflammation (vasculitis).
• Circulating immune complexes cause damage at specific sites, especially the joints, skin, and
kidney. Immune complexes can become deposited in joints and cause synovitis, which causes
pain and swelling.

IMMUNE COMPLEX DISEASE IN THE KIDNEY

• Involvement of the kidney in type III hypersensitivity is a common cause of renal failure.
• The kidney is often affected because blood pressure in the glomerulus is four times higher than
that in the systemic circulation. High blood pressure increases immune complex deposition in
vessel walls.
• Glomerular cells also express the complement receptor CR1, which may predispose to immune
complex deposition at this site.
• Synovial cells also express CR1, which may explain why joints are also often involved in
circulating immune complex disease.
• Immune complex disease in the kidney can result in two clinically defined syndromes:
1. Nephrotic syndrome, in which protein leaks into the urine and there is gradual-onset renal failure.
2. Nephritis, in which there is rapid-onset renal failure, blood and protein in the urine, and
hypertension.
→Both types of disease are produced by inflammation in the glomeruli (glomerulonephritis).
• In the nephrotic syndrome, immune complexes are deposited in the glomerular basement
membrane where they activate complement. This usually causes subtle damage to the basement
membrane, which allows proteins to leak into the urine.
• In nephritis, by comparison, there is a cellular infiltrate in addition to complement activation.
Neutrophils are attracted into the glomeruli, and the resulting inflammation causes blood and
protein to leak into the urine, impairing the ability of the kidney to excrete toxic metabolites.
• Which type of glomerular lesion is produced depends on several factors, including the size of
immune complexes, the rate at which they are produced, and the duration of immune
complex production.

• In poststreptococcal glomerulonephritis, the renal disease is dramatic but short lived because
infection is brought under control by the immune response.
• When drugs cause immune complex–mediated kidney disease, stopping the drug improves
kidney function.
• In SLE, the immune complexes contain autoantigens, and therefore the renal disease has gradual
onset but is not self-limiting.
• Immune complexes are not the only immunologic cause of glomerulonephritis. Renal damage
can also occur in Goodpasture syndrome and when immunoglobulin light chains damage
the kidney in multiple myeloma.
• Laboratory tests are crucial in the investigation of nephritis and the nephrotic syndrome. Indirect
immunofluorescence is used to find antibodies implicated in immune complex disease (e.g.,
anti-DNA antibodies) or other types of autoantibody (antiglomerular basement membrane
antibody). Sometimes it is necessary to perform direct immunofluorescence on a renal biopsy
specimen to determine what type of process is causing damage.

TREATMENT OF IMMUNE COMPLEX DISEASE

• Antigen avoidance is possible in some cases of type III hypersensitivity—for example, farmer’s
lung or some drugs and vaccines. In the case of autoantigens, however (e.g., DNA), avoidance is
clearly not possible.
• In autoimmune causes of immune complex disease, corticosteroids block some of the damage
caused by effector cells, such as neutrophils.
• Cyclophosphamide is an alkylating agent that impairs DNA synthesis and prevents rapid
proliferation of cells such as lymphocytes. Although cyclophosphamide has some effects on T
cells, its main benefit is in reducing B-cell proliferation and hence autoantibody levels.
Cyclophosphamide is often used in severe SLE.
Type (4) hypersensitivity(Delayed Hypersensitivity):
• Delayed hypersensitivity reactions mediated by T cell.
• Multiple sclerosis (MS) and rheumatoid arthritis
(RA) are two important autoimmune diseases
mediated by delayed hypersensitivity reactions.
• Delayed hypersensitivity was originally defined as
reactions that take place 2 to 3 days after
exposure to antigen, such as tuberculin skin
testing.
• Delayed hypersensitivity is characterized by T-
helper 1 (TH1) cells driving inflammatory responses mediated by macrophages.
• TH17 T cells appear to have a more variable role in delayed hypersensitivity.
• Delayed hypersensitivity can be a physiologic reaction to pathogens that are hard to clear,
such as hepatitis B virus (HBV) and Mycobacterium tuberculosis (TB).
• Mycobacterial infections trigger the most extreme delayed hypersensitivity reactions, which are
characterized by granuloma formation, extensive cell death, and the appearance of caseous
necrosis.
• Delayed hypersensitivity can also occur in response to innocuous environmental antigens,
such as nickel in some cases of contact dermatitis.
• These antigens must have a low molecular weight in order to enter the body. The very small size
of these substances means that they must act as haptens to become antigenic. Contact
dermatitis can occur as a result of exposure to a wide range of other chemicals, including
cosmetics and plant extracts such as poison ivy.
• Delayed hypersensitivity reactions also take place against autoantigens. For example, in type 1
diabetes mellitus (T1DM), T cells respond to pancreatic islet cell antigens, damaging the islets and
eventually preventing insulin secretion.
• The T cells responsible for causing hypersensitivity in the pancreatic islets appear to be mainly
TH1 cells. TH2 and TH17 cells have little, if any, role in causing damage in this setting.
------------------------------------------------------------------------------------------------------------------------------------------------------------------
DELAYED HYPERSENSITIVITY REACTIONS ARE DRIVEN BY T-HELPER 1 CELLS
1. Delayed hypersensitivity reactions are initiated when
tissue macrophages recognize the presence of danger
signals and initiate an inflammatory response.
2. Dendritic cells loaded with antigen migrate to local lymph
nodes, where they present antigen to T cells.
3. Specific T-cell clones proliferate in response to antigens,
which migrate to the site of inflammation.
4. T cells and macrophages stimulate one another through
the cytokine network. (IFN-Y&IL-12)
5. Tumor necrosis factor (TNF) is secreted by both
macrophages and T cells and stimulates much of the damage in delayed hypersensitivity.
Because of the need for antigen presentation by T cells, delayed hypersensitivity reactions are often
associated with specific human leukocyte antigen (HLA) alleles, as in T1DM and celiac disease.

TYPE IV HYPERSENSITIVITY DISEASE

• There are two common autoimmune diseases caused by delayed hypersensitivity: rheumatoid
arthritis and multiple sclerosis.

Rheumatoid Arthritis (RA)

• RA is a chronic disabling condition.
• RA affects up to 1% of the population.
• RA tends to present in the third to sixth decades of life.
• In RA, the synovial membrane that lines the joints and tendon sheaths is swollen up to 100
times its normal size.
• Although most disabling symptoms arise in the joints and tendons, RA also affects the skin,
lungs, and eyes and is a systemic connective tissue disease.
• RA has many features of delayed hypersensitivity with persistent TH1 and TH17 reactions and
TNF secretion.
• However, patients with RA also develop unusual auto-antibodies, which play an important role in
diagnosis.
• The antigens that drive RA appear to be citrullinated proteins.
Note: Citrullination is the conversion of the amino acid arginine to the amino acid citrulline.

• Many proteins, such as fibrin, do not normally contain citrulline unless they have been citrullinated.
• Citrullination of proteins such as fibrin may take place as a result of endogenous enzymes
activated by inflammation.
• Evidence suggests that this may be triggered by smoking, for example, in which citrullination
takes place in the lungs. Bacteria that cause mouth infections, such as Porphyromonas, may
also secrete enzymes that cause citrullination.
• Autoreactive T cells and B cells can recognize citrullinated proteins.
• The result is production of antibodies against citrullinated protein. These are referred to as anti–
cyclic citrullinated peptide (CCP) antibodies.
• Anti-CCP antibodies are produced in some healthy people who will never develop RA. However,
anti-CCP antibodies are present in most people with RA and in people who will eventually develop
RA; these antibodies can be present at least 10 years before the onset of symptoms. The T and B
cells that respond to CCP are not present in the synovium at this stage.
• It is not yet known why the synovium becomes the focus of inflammation in RA. It may be that
immune complexes deliver citrullinated proteins and antibodies to the synovium.
• Alternatively, proteins in the synovium itself, such as specific types of collagen, could become
citrullinated.
• Once RA has become established, the synovium becomes infiltrated by chronic inflammatory
cells, including T cells and macrophages.
• The T cells are a mixture of TH1 and TH17 cells. Cytokines secreted by these cells, notably TNF
and interleukin 17 (IL-17), attract and activate neutrophils that cause damage to the synovium.
• Osteoclasts are activated and destroy bone at the joint margins, creating erosions.
• Persistent IL-6 secretion triggers an acute-phase response, although in this case the acute phase
may last several years. Features of this include fatigue, weight loss, and elevated levels of C-
reactive protein (CRP).
• People with RA frequently have a family history of the disease. RA is associated with HLA-DR4.
• Associations between disease and specific HLA alleles provide evidence for the role of T cells in
the development of disease. RA is also more common in smokers and those infected with
Porphyromonas, which gives some support to the idea that protein citrullination is a key step in
the development of this disease.
• Although there are still gaps in the understanding of how RA develops, treatment has improved
considerably. RA can now be treated with monoclonal antibodies against the cytokines TNF, IL-
1, and IL-6 and against B cells. All of these are effective, but they carry some special risks.

Multiple Sclerosis
• MS is a chronic, disabling neurologic disease, and half of those diagnosed are disabled within 15
years of onset.
• Early in MS, bouts of inflammation recur and produce demyelinating plaques in different parts of
the central nervous system (CNS).
• Later in MS, chronically progressive disease leads to extensive axonal loss.
• MS affects about 1 in 1000 people in Northern Europe and central North America. Closer to the
equator, the prevalence is usually much lower.
• Individuals who move from lower- to higher-risk areas acquire an increased risk for developing
MS, suggesting that environmental factors are more important than genes for the geographic
variation in prevalence.
• One theory is that people who live closer to the equator are protected from MS because higher
amounts of ultraviolet light enable them to synthesize more vitamin D.
→On the other hand, several strands of evidence support infections as being an environmental
trigger for MS:
- Demyelination that superficially resembles MS is occasionally seen after documented infections,
such as measles.
- Infections can precipitate relapses in patients with MS.
- The risk of developing MS is greater in people who have had Epstein-Barr virus infection.

→Genes are involved to a lesser extent in MS; the concordance rate among identical twins is only
30%. Genes that affect the synthesis of vitamin D have been linked to MS; higher vitamin D levels
appear to be protective. This may be because, apart from its role in regulating calcium metabolism,
vitamin D acts as a hormone that promotes macrophage maturation. The involvement of vitamin D
in immunologic disease is thus plausible.
Therefore MS most likely results from the interplay between susceptibility genes and the environment.

- Initially in MS, acute attacks occur during which inflammatory lesions consisting of TH1 and TH17
cells and macrophages develop in the affected nervous tissue. The inflammatory lesions cause
the reversible, relapsing disability typical of early MS.
- Although active inflammation is present in the vicinity, myelin loss impairs the ability of neurons to
conduct impulses, resulting in neurologic symptoms.
- Once the inflammation settles, the disability improves. Between attacks, there is usually good
recovery of function, at least early in the disease.
- The chronic disability that usually occurs later in MS is the result of another process, axonal loss.
Although demyelinated nerve cells can remyelinate to some extent, axon loss from the nerve cell
is irreversible.
- Although T cells are thought to initiate much of the damage in MS, there are also B cells in the
CNS that secrete antibodies against a wide variety of brain components, including myelin basic
protein. These antibodies may participate in inflammation and also provide a marker of antibody
production in the CNS, which can sometimes help make a diagnosis of MS.

Treatment of Delayed Hypersensitivity Reactions

• In delayed hypersensitivity, it is sometimes possible to avoid the relevant environmental antigens.
For example, some types of contact dermatitis are improved by avoiding exposure to nickel.
• In celiac disease, avoiding dietary gluten improves symptoms and reduces levels of
antiendomysium antibodies.
• In these examples, an exogenous antigen is driving an autoimmune disease.
• Where the cause is an endogenous antigen, treatment is more complex. Options currently
used are anti-inflammatory drugs— which mainly have effects on effectors of delayed
hypersensitivity, especially macrophages—and immunosuppressive drugs, which have effects on
T cells.
Antiinflammatory Drugs
• Antiinflammatory drugs work by cutting down the mediators released during inflammation, usually
by cells of the innate system.
• For example, nonsteroidal antiinflammatory drugs (NSAIDs) — such as aspirin, ibuprofen, and
indomethacin—inhibit arachidonic acid metabolism.
• Endogenous corticosteroids suppress the immune response during physiologic stress.
Corticosteroids are often used as immunosuppressive drugs during the treatment of
autoimmunity and allergy and after transplantation. Their effects are mediated by affecting gene
transcription when used at low to moderate doses.
• Corticosteroids bind specific receptors that transport them to the nucleus and are responsible for
binding to regulatory gene sequences. At higher doses they affect cell signaling directly.
• Although corticosteroids are thought to affect the transcription of 1% of all genes in a wide range
of cells, their dominant therapeutic effects are on phagocytes. Effects on lymphocytes may be
largely a result of poor antigen processing and costimulation provided by phagocytes.
• The side effects of corticosteroids are well known. From the immunologic point of view,
immunosuppression is a particular concern, and it may result in reactivation of infections
normally controlled by macrophages, such as tuberculosis.
• Corticosteroids have some effects in MS, but only at high intravenous doses.
• Corticosteroids probably work in MS by reducing the actions of macrophages. Corticosteroids are
of some value in RA but are too toxic for long-term use.
• Newer approaches introduced in the past 20 years exploit the increasing knowledge of cells and
molecules in inflammation. For example, drugs that block TNF, IL-6, the costimulatory
molecule CD80, and antibodies against B cells are all now routinely used in RA.
• These biologic drugs have been very effective, and for the first time RA patients can go into
lasting remission and avoid irreversible disability.
• Recombinant interferon (IFN)-β has been used in some patients with MS; it delays the
development of acute attacks of nervous system inflammation. There is some evidence to suggest
that IFN-β has long-term benefits and can prevent the chronic disability associated with
demyelination.
• Type I interferons (IFN-α and IFN-β) have potent antiviral effects and weaker immunostimulatory
effects, increasing antigen presentation and activating natural killer cells. It is therefore surprising
that IFN-β is effective in MS, in which antiinflammatory effects would be expected to be more
beneficial.
• One possibility is that IFN-β reduces the migration of T cells across the blood-brain barrier. This
illustrates the pleomorphism of cytokines; that is, that any one cytokine can have multiple effects
on many different cell types. More recently, other potent antiinflammatory and immunosuppressive
drugs have been found to be more effective in MS.

Immunosuppressive Drugs
• Immunosuppressive drugs inhibit the specific immune response that drives delayed
hypersensitivity and are most relevant in autoimmune delayed hypersensitivity, when antigen
cannot be avoided.
• Immunosuppressive drug are most often used in transplant recipients.
• The benefits of immunosuppressive drugs must be balanced against their dangerous side effects,
particularly the increased the risk for infection.
• For example, in T1DM, pancreatic islet cell function can be maintained while patients receive
immunosuppressive drugs. However, the drugs would have to be given for life, and the side
effects are unacceptable; insulin replacement is a safer option. Immunosuppressive drugs have
not been widely tested in MS.
Immunologically Mediated Drug Reactions
• Drug reactions are common and affect up to 15% of hospital patients.
• The majority of reactions are predictable and are directly related to the pharmacologic effects of
the drug. For example, a patient given an incorrectly high dose of a sedative drug will sleep longer
than expected. Other side effects are less predictable and are described as being idiosyncratic.
• Some of these reactions may occur when a patient lacks an enzyme that is responsible for
metabolizing a drug. For example, a patient who has low levels of the appropriate metabolizing
enzyme will experience excessive sleepiness, even after the correct dose of sedative.
• Idiosyncratic drug reactions also commonly
have an immunologic basis. Some of these
effects are mediated by the innate immune
system. For example, morphine can
stimulate mast cell degranulation, leading to
histamine release and the development of the
itchy rash urticaria. Reactions can also
involve the adaptive system, and they can
cause any type of hypersensitivity.
• These reactions occur only after a patient has
previously been exposed to a drug so that
antibodies or reactive T cells can develop.
• It is important to diagnose the cause of these reactions because repeat exposure can lead to life-
threatening reactions.
• Laboratory tests can provide indirect evidence of immunologic hypersensitivity. For example,
elevated blood mast cell tryptase levels suggest mast cell involvement through innate
mechanisms or type I hypersensitivity. An allergist will try to confirm the presence of specific IgE
against the drugs, either by skin testing or blood testing.
Review of Hypersensitivity Reactions
• The Gell and Coombs classification of hypersensitivity is an oversimplification, and many diseases
overlap the different types. For example:
- Asthma is generally classified as an allergic disorder causing immediate symptoms mediated
by IgE. However, the late phases of the type I reactions in asthma and atopic dermatitis are
characterized by T-cell infiltrates more typical of type IV reactions.
- Although celiac disease and RA are both type IV reactions, autoantibodies (anti–tissue
transglutaminase and anti-CCPs) play an important role.
Nonetheless, understanding the different types of hypersensitivity guides the diagnosis and treatment
of these important conditions. For example, all allergies are best diagnosed by identifying specific IgE
by blood or skin tests. These tests would not be used for any other type of hypersensitivity.

Helpful videos:
https://www.youtube.com/watch?v=ic6qpoolfDA

https://www.youtube.com/watch?v=74gkBLnJz3A
Questions (for the past 2 lectures)
1- sulfonamide hemolytic anemia is a classical example of which type of a hypersensitivity reaction?
a) Type IV
b) Type III
c) Type I
d) Does not induce any hypersensitivity reaction
e) Type II
2-binding of an antigen to TLR2 stimulates the production of which of the following cells?
a) CTL cell
b) GATA3 precursor of TH2
c) NK cell precursor
d) Tbet precursor of TH1
e) Dendritic cell precursor
3- prick test is the best of choice in order to make a diagnosis of which of the following diseases?
a) SLE
b) Contact dermatitis
c) Farmers lung disease
d) Bronchial asthma
e) Grave’s disease
4- patch test is the test of choice in order to make diagnosis of which of the following diseases?
a) Bronchial asthma
b) Peanuts allergy
c) Eczema
d) SLE
e) Nickle ring allergy
5- which of the following antibodies is indirectly a strong indicator of the presence of the celiac
disease?
a) Anti-mitochondrial antibodies
b) Anti-double stranded antibody
c) Anti-endomysial antibody
d) Anti-TSH antibody
e) Anti-single stranded DNA antibody
6- hepatitis B virus and post streptococcus glomerulonephritis induce which type of hypersensitivity?
a) Type 1
b) Type 2
c) Type 3
d) Type 4
e) Doesn’t induce hypersensitivity
7- which type of vaccines is used for treatment of type 1 hypersensitivity?
a) DPT
b) BCG
c) Vibrio cholera enterotoxin
d) Streptococcus polysaccharide capsular vaccine
e) Mycobacterium vaccae
8- what is the immunoglobulin that is produced when frequently administering and antigen in the
treatment of hypersensitivity?
a) IgM
b) IgG
c) IgA
d) IgE
e) IgD
9- rheumatoid arthritis is an example of which hypersensitivity?
a) Type 1
b) Type 2
c) Type 3
d) Type 4
e) Non of the above
10- hemolytic disease of the newborn does not occur when:
a) An RH positive mother conceives and RH positive child.
b) An RH negative mother conceives and RH negative child.
c) An RH positive mother conceives and RH negative child.
d) All of the above
e) Non of the above
11- which one takes the longest time to develop after exposure to the antigen?
a) Contact dermatitis
b) Hay fever
c) Post-streptococcal glomerunephritis
d) Drug induced hemolysis
e) Peanut allergy
12- celiac disease is associated with:
a) HLA-DR3
b) HLA-DRB1
c) HLA-DQ2
d) HLA-DR2
e) HLA-B27
Answers: 1-e 2-b 3-d 4-e 5-c 6-c 7-e 8-b 9-d 10-d 11- a 12-c
 Blood Banking & Immunohematology
• The first lifesaving transfusion was performed almost 200 years ago by James Blundell in 1818.
• The safety of blood transfusion has steadily improved since the first US blood bank was founded
in the 1940s. Tests were developed and implemented to detect the infectious diseases recognized
as transmitted in blood products.
• New molecular diagnostic techniques are now being investigated to improve the sensitivity of
the tests used for donor blood analysis.
• Nevertheless, transfusion continues to require the removal of blood from one human being for
infusion into another.
• This “living transplant” carries with it the complexities of its human source and thereby brings
with it the potential of undesirable side effects in the recipient.
• Some risks of transfusion are now known, and others have yet to be described. Consequently, the
need for transfusion must be judged carefully in light of these risks.

BLOOD GROUPS
• The first blood group system was described at the turn of the 20th century by Karl Landsteiner.
He observed that erythrocytes from some individuals clumped when mixed with the serum of
others but not with their own.
• Using this agglutination technique, he classified an individual's erythrocytes into four types: A, B,
AB, and O.
• It is now recognized that A and B represent carbohydrate antigens on the erythrocyte.
• Group O individuals have neither of these antigens on their erythrocytes, whereas erythrocytes
from AB individuals have both A and B antigens.
• The ABO system is the most important blood group system for transfusion purposes.
• Knowledge about blood groups has expanded to include a diverse and numerous array of
antigenic determinants on erythrocytes. Approximately 600 erythrocyte antigens are known, of
which 207 belong to 23 recognized blood group systems.
• Each blood group system has members, each
of which may be composed of one or more
different antigens. Each antigen is controlled by
one gene.
• The antigenic determinants of a blood group
are produced either directly (for proteins) or
indirectly (for carbohydrates) by alleles at a
single gene locus or at other gene loci so
closely linked that crossing over is
extremely rare.
• For any antigen of a blood group, a single allele
is present at that locus and other alleles are
therefore excluded.
• A specific antigen on the erythrocyte surface is
usually detected in the blood bank laboratory by
reacting erythrocytes with sera known to
contain antibodies reactive with that antigen. This test defines a phenotype.
ERYTHROCYTE ANTIGENS
❖ H & ABO
• Antigenic determinants of the H and ABO systems are carbohydrate moieties whose specificity
resides in the terminal sugars of an oligosaccharide.
• On erythrocyte and endothelial surfaces, most of the antigens
are bound to glycosphingolipids.
• Genetic control is via the production of transferase enzymes
that conjugate terminal sugars to a stem carbohydrate.
• The H and ABO systems have separate gene loci and are
independent of each another (Figure 17-1).
• The H gene codes for a fucosyl transferase enzyme that adds
fucose to precursor chains and completes the stem chain. The
H gene is rarely absent; this phenotype (hh) is called Oh, or
Bombay, type.
• In the absence of a complete stem chain, additional sugars cannot be added despite the presence
of A or B transferase, and high-titer anti-H is produced.
• The ABO blood groups are determined by allelic genes A, B, and O (Table 17-1).

• The A-group transferase adds N-acetylglucosamine to the completed stem chain.

• The B-group transferase adds a terminal D-galactose. The O gene produces no transferase to
modify the blood group substance (see Figure 17-1).
• Both groups A and B can be divided into subgroups.
• Many subgroups of A have been described, but most are rare. The most important are A1 and
A2. Differences between subtypes of group A appear to be quantitative, that is, in the number of
antigenic sites per erythrocyte surface.
• AB blood can also be divided into A1B and A2B types.
• Although less frequently detected, subgroups of group B can also be distinguished. Subgroups of
group B, like those of group A, demonstrate a continuum in the number of antigenic sites per
erythrocyte.
• The naturally occurring antibodies to groups A and B are thought to be stimulated by very
common substances. Intestinal bacteria are known to have substances chemically similar to
and therefore antigenically cross-reactive with A and B.
• Antibodies to A or B antigens (or both) are first detected in children at 3–6 months of age, peaking
at 5–10 years of age and falling with age and in some immunodeficiency states.
• Two other systems directly interact with the ABO and H systems: Lewis and secretor. Secretion
of ABH substances in body fluids (saliva, sweat, milk, etc) is controlled by the allelic genes Se
and se.
• These genes are independent of ABO and are inherited in a mendelian dominant manner.
• Eighty percent of people are Se; they secrete Lewis antigens in addition to ABH substances.
Typing of body fluids for these antigens has been useful in forensic investigations.

❖ Rh (Rhesus)
• The Rhesus blood group system is second in importance only to the ABO system.
• Anti-Rh antibodies are the leading cause of hemolytic disease of the newborn (HDN) and may
also cause delayed hemolytic transfusion reactions.
• Recent investigations have elucidated the genetic basis of the primary Rh antigens: D, C, c, E, e.
• The Rh locus on chromosome 1 consists of two adjacent structural genes designated RHD and
RHCE.
• The RHD gene encodes the D polypeptide present on the erythrocyte in Rh-positive individuals.
• The RHD gene is completely absent in the genome of Rh-
negative individuals, which explains why no D antigen
counterpart (d) has ever been found in Rh-negative people.
• The RHCE gene encodes for both C/c and E/e proteins via
alternative splicing events.
• Previous theories explaining the genetic basis of the Rh system
gave rise to different nomenclatures.
• In the Wiener nomenclature, multiple Rh alleles were
designated as either R or r with one of many superscripts. R
alleles produced the antigen Rho(D) in a particular phenotype
in addition to two other antigens; r alleles denote the absence
of Rho.
• In the Fisher and Race system (Table 17-2), three allelic gene
pairs were thought to commonly produce five antigens (the
remaining antigens are rare variants). Each antigen (D, C, c, E,
and e) has a corresponding designation in the Wiener system
(ie, D = Rho, C = rh′ , etc). C and c, as well as E and e, function as alleles. No d antigen was
known, so d describes the absence of D.
• The Rh antigens were believed to be inherited as two sets of three, one from each parent.
• Clinically, Rh-positive (Rh+) means the presence of D (Rho) and Rh-negative (Rh–) indicates the
absence of D (Rho). D is the most immunogenic of the Rh antigens.
• Slightly less than half of Rh+ people are homozygous for D. Because there are no antisera to
detect the absence of D, determination of zygosity depends on family studies or gene
amplification techniques.
• Roughly 15% of whites are Rh–. Rh-negativity is less common in other races.
• Erythrocytes with less than the normal number of D antigen sites are described and designated
weak D (previously termed Du). A weak D can appear as D negative (Rh–) in testing if blood is
typed only with routine anti-D antisera but is detected if the indirect anti-globulin test is used.
• Blood-banking standards require all donor blood to be tested using methods that detect weak D
antigen. If weak D is detected, the blood unit is labeled Rh-positive.
Other Erythrocyte Antigens
• Many of the remaining 20 blood group systems are rarely implicated in transfusion reactions.
Antibodies to the Kidd, Duffy, Kell, and MNS systems, however, are known for their ability to
cause hemolysis if antigen-positive blood is transfused into a sensitized recipient.
• In general, hemolytic antibodies are IgG and react at 37°C (body temperature). IgM antibodies
rarely cause hemolysis.
• Antibodies to Kidd antigens are a frequent cause of delayed hemolytic transfusion reaction and
can cause HDN. These antibodies are often difficult to identify in test systems because of poor
reactivity. Four antigenic phenotypes have been described: Jk (a+ b- ), Jk (a- b+, Jk (a+ b+), and
Jk (a- b-)
• The antigens of the Duffy system (Fya and Fyb) are controlled by codominant alleles. Antibodies
to Fya are more commonly associated with delayed hemolytic transfusion reactions than are those
to Fyb. Many blacks have a third allele, which produces the Fy (a- b-) phenotype.
• Duffy antigens on erythrocytes serve as receptors for the entry of Plasmodium vivax into the
erythrocytes. Fy (a- b-) individuals who lack Duffy antigens are resistant to P.vivax infection.
• The Kell system, as first described, included the allelic pair K and k, k antigen being the more
frequent. The system now includes two additional allelic pairs and several variants. The K antigen
is highly immunogenic, with one of 20 individuals transfused with K+ cells developing antibody.
• Antibodies to Kell antigen cause HDN, hemolytic transfusion reactions, and, occasionally,
autoimmune hemolytic anemia.
• Individuals of the McLeod phenotype lack Kx antigen, which is a precursor in the synthesis of Kell
antigens. These individuals have erythrocyte and neuromuscular system abnormalities. The
McLeod phenotype is also associated with some cases of chronic granulomatous disease.

METHODS FOR DETECTION OF ANTIGEN & ANTIBODIES TO ERYTHROCYTES

1-Antiglobulin Tests
• Antibody or complement adsorbed onto erythrocytes is detected by using antibodies to human
serum globulins (AHG).
• AHG reagents are produced either in animals or in tissue culture by using monoclonal antibody
techniques. These reagents may be polyspecific (a mixture of antibodies to IgG, complement,
and heavy and light chains) or monospecific (antibodies to specific immunoglobulin or
components of complement).
• The direct antiglobulin test (DAT) detects antibody or complement coating the surface of
erythrocytes, whereas the indirect antiglobulin test (IAT) identifies antibody in serum or plasma.
• To perform the DAT (Figure 17-2) erythrocytes are washed with saline to remove unbound
antibody or complement and then AHG is added. If antibody is present on the erythrocytes, the
Fab portion of AHG attaches to the Fc portion of the erythrocyte-bound antibody.
• Bridging of AHG Fab molecules between erythrocytes results in visually detectable agglutination.
• The DAT is used in the investigation of autoimmune or drug-induced hemolytic anemia, HDN, and
suspected hemolytic transfusion reactions.
• The IAT detects serum or plasma antibodies, which can attach in vitro to erythrocytes (see Figure
17-2). This test differs from the DAT in that before an IAT is performed, the serum or plasma to be
tested is incubated with erythrocytes so that antibody, if present, binds to erythrocyte antigen.
• The erythrocytes are then washed to remove any
unbound globulin, and AHG is added.
• If agglutination is observed, antibodies to erythrocyte
antigens are present.
• The IAT is used by blood banks in three ways:
- First, to identify the presence and specificity of
recipient plasma antibody, plasma is tested using
panels of reagent erythrocytes with known
antigens on their surface.
- Second, to select donor blood that is free of
specific erythrocyte antigens, commercial
reagents, containing known erythrocyte antibodies,
are used to test donor blood for the absence of the
antigen.
- Third, to confirm the absence of an antigen– antibody reaction, recipient plasma is tested
against donor blood cells (crossmatch).

2-Pretransfusion Testing
• Blood is tested prior to transfusion to prevent clinically significant destruction of the transfused
erythrocytes.
• Clinically significant antibodies are those known to have caused unacceptably shortened
erythrocyte survival in vivo or frank hemolysis.
• Generally, these antibodies react at 37°C (body temperature) and in the indirect antiglobulin test.
• Prior to transfusion, the recipient's erythrocytes and plasma are tested for ABO and Rho (D) types
and for antibodies to erythrocyte antigens, often called the “type and screen.” Additionally, the
recipient's plasma is tested for compatibility with the erythrocytes from the intended donor
(crossmatch).

3-Type & Screen

• ABO and Rho (D) types are determined by mixing the recipient's erythrocytes with anti-A, anti-B,
and anti-D antisera.
• The ABO group is then confirmed by testing the recipient's plasma against commercial reagent A
and B cells to detect isoagglutinins.
• The recipient's plasma is screened for alloantibodies that may not be demonstrated in the
crossmatch.
• In antibody screens, suspensions of reagent O erythrocytes that contain known erythrocyte
antigens on their surface are incubated at 37°C with the recipient's plasma.
• If antigen–antibody complexes are formed, hemolysis or agglutination of erythrocytes is
observed.
• The screen is completed by the IAT and again observed for agglutination. In the crossmatch,
compatibility between donor and recipient is determined.
• Donor cells are combined with recipient plasma, centrifuged, and observed for hemolysis or
agglutination (called the “immediate spin” crossmatch).
• If the recipient either has a history of previous erythrocyte antibody or has had antibody detected
during the antibody-screening procedure, the IAT using recipient plasma and donor red cells must
be performed before a crossmatch may be considered compatible.
• A variety of methods to increase the sensitivity of the IAT has been developed. These methods
add albumin, low-ionic-strength solution (LISS), polybrene, or polyethylene glycol (PEG) to the
test system.
• Reagent erythrocytes can also be treated with proteolytic enzymes to enhance the reactivity of
some erythrocyte antigens (Rh and Kidd) and to abolish the reactivity of others (M, N, Fya , and
Fyb).

TRANSFUSION REACTIONS
• Blood transfusion has become increasingly safe, but a variety of adverse reactions, only some of
which are preventable, continues to occur (Table 17-3).
• Patients who are transfused must be monitored during infusion for immediate reactions and over
time to detect delayed reactions.

1-Hemolytic Reactions
• The transfusion of incompatible blood may cause immediate hemolysis.
• Immediate hemolytic transfusion reactions, which are fatal in approximately 10–40% of cases,
generally occur when ABO-incompatible blood is transfused.
• The cause is most often managerial or clerical error, such as transfusing patients with units
intended for other recipients.
• Two thirds of these errors occur in areas other than the hospital blood bank. Incompatible
transfusions involving other blood groups are usually less severe, but deaths have been reported.
• The most common presentation of a hemolytic transfusion reaction is fever or fever with chills.
• Other signs or symptoms are chest pain, hypotension, nausea, flushing, dyspnea, and
hemoglobinuria.
• The hemolytic transfusion reaction may progress to shock, disseminated intravascular coagulation
(DIC), and renal failure.
• Delayed hemolytic transfusion reactions occur 3–10 days after transfusion and may be clinically
undetected.
• This reaction occurs from an anamnestic immune response to transfused erythrocytes in a
previously sensitized person with undetectable antibody in pretransfusion testing.
• Presenting symptoms are fever, anemia, and jaundice. The patient's transfused erythrocytes are
coated with antibody demonstrated by a positive DAT.
• The antibody specificity is identified by removing it from the surface of the coated transfused
erythrocytes by a procedure called elution. The eluted antibody is then tested against a panel of
reagent erythrocytes by the IAT. The frequency of delayed hemolytic transfusion reactions is 1 per
4000 units of blood transfused. Mortality from delayed hemolytic transfusion reactions is
uncommon.

2-Febrile Reactions
• In the past, febrile nonhemolytic transfusion reactions (FNHTR) were thought to be caused by
cytotoxic or agglutinating antibodies in the recipient, directed against donor leukocyte antigens.
• Leukocyte reduction filters used at the time of red cell or platelet transfusion decreased the
amount of leukocytes transfused and should have eradicated FNHTRs.
• When this anticipated effect was not observed, researchers looked for other causes to explain the
fever, chills, and rare rigors that describe a FNHTR.
• It was observed that during storage, cytokines (IL-1β, IL-6, TNFα) are released from leukocytes
present in red cell and platelet components.
• These cytokines are known to have pyrogenic activity and thus may be the cause of this adverse
reaction.
• FNHTR must be distinguished from fever associated with hemolytic transfusion reactions and from
the high fever (>40°C) and rigors associated with bacterial contamination of blood components.
• Only one in eight patients with a febrile reaction has another reaction on subsequent transfusion.
Recurrent febrile reactions are often controlled with antipyretics, leukocyte-reduced
components, or recently collected components.

3-Transfusion-Related Acute Lung Injury

• High-titer leukocyte antibodies in donor plasma can cause pulmonary edema.
• Donor antibodies bound to recipient granulocytes (or infrequently, recipient antibodies bound to
donor granulocytes) activate complement.
• Complement activation leads to the sequestration of antibody– granulocyte complexes in the lung
microvasculature.
• The presence of activated complement fragments and leukocyte enzymes or free radicals are
thought to cause lung injury with resultant pulmonary edema. The sequelae are fever, dyspnea,
and marked hypoxemia.
• The acute respiratory distress occurs within 1–6 hours of a transfusion and often requires
aggressive respiratory support.
• Although some deaths have been reported, most patients with transfusion-related acute lung
injury (TRALI) improve within 48–96 hours if promptly treated. The risk of TRALI is approximately
1 per 5000 units transfused.
4-Allergic Reactions
• Allergic reactions to transfusion are characterized by itching, hives, and local erythema. Rarely
they are accompanied by cardiopulmonary instability.
• They are thought to be caused by infused plasma proteins and occur in 1–2% of transfusions.
• Patients with a history of allergy more frequently have allergic reactions to blood.
• Mild reactions can be treated with antihistamines and the transfusion continued.
• Pretreatment with antihistamines often prevents recurrent allergic reactions.
• If the allergic reaction is severe, washed erythrocytes may be indicated.
• After transfusion of as little as 10–15 mL of a blood component, some IgA-deficient recipients with
antiIgA experience anaphylactic reactions. Fortunately, these reactions are rare.
• The reaction is due to the IgA present in transfused plasma and is prevented by transfusing
plasma-free or IgA-deficient components.
• Other transfusion reactions include those caused by bacterial contamination of blood components,
congestive heart failure due to intravascular volume overload, and donor erythrocyte destruction
prior to infusion.
• Erythrocytes may be destroyed by inadvertent overheating, improper freezing technique, or mixing
with nonisotonic solutions.

Transfusion-Transmitted Infections
• Transfusion may be complicated by a variety of infectious
microorganisms, only some of which can be detected by
current donor-screening methods (Table 17-4).
• The most frequently reported posttransfusion infections in
developed countries are various bacterial contaminants,
hepatitis, cytomegalovirus (CMV), human immunodeficiency
virus-1 (HIV-1), and human T-cell lymphotrophic virus I/II
(HTLV-I/II).
• Elimination of potentially infected blood depends on successful
donor screening by medical history, aseptic blood collection,
and adequate laboratory testing of the donated blood.
• The presence of hepatitis B surface antigen (HBsAg),
antibody to hepatitis B core antigen (anti-HBc), antibody to
hepatitis C virus (anti-HCV), anti-HIV 1/2, HIV-1 antigen
(p24), anti-HTLV I/II, and syphilis (STS) is currently tested in
all US blood donors.
• The prevalence of posttransfusion hepatitis (PTH) is estimated
to be <1%. PTH is caused by hepatitis B virus in 5% of cases
and by hepatitis C virus in 95% of cases.
• Of transfusion recipients who develop posttransfusion
hepatitis, 50% develop chronic hepatitis; 10% of these develop
cirrhosis. All blood components can potentially transmit
hepatitis, except those that can be pasteurized, such as albumin and other plasma proteins.
• CMV is transmitted to CMV-seronegative transfusion recipients by leukocytes contaminating
erythrocyte and platelet components.
• Roughly 50% of blood donors are infected with CMV, which limits availability of CMV-negative
blood.
• CMV disease causes significant morbidity and mortality in severely immunocompromised patients.
• When possible, CMV seronegative blood should be given to low-birthweight infants (<1250 g),
CMV-seronegative pregnant women, and CMV-seronegative recipients of CMV-seronegative
bone marrow or organ transplants.
• HIV-1 infection due to transfusion is rare since implementation of donor HIV-1 antibody testing
(March 1985). HIV-1 can be transmitted by erythrocytes, platelets, cryoprecipitate, fresh-frozen
plasma, and possibly other blood components.
• The risk of infection by transfusion is now estimated to be about 1 in 675,000 per unit transfused.
The virus can be transmitted by blood collected from donors who have been recently infected but
don't yet have detectable levels of HIV antigen or antibodies (called the “window period”).
• Even though HIV-2 infection is rare in the United States, isolated cases are reported in parts of
Europe and West Africa. Consequently, all US blood donations are screened for antibodies to
both HIV-1 and HIV-2 as well as to HIV p24 antigen.
• To date 3 US blood donors were found to have been infected with HIV-2 since HIV-2 testing was
implemented in 1992.
• Human T-lymphotrophic viruses type I and type II (HTLV-I and HTLV-II) are also retroviruses
known to be transmitted by transfused blood products. Donor screening histories and serologic
testing for evidence of HTLV-I/II infection has reduced the risk of transfusion-transmitted HTLV-I/II
infection to 1:641,000.
• Both viruses are associated with a slowly progressive spinal cord disorder known as tropical
spastic paraparesis/HTLV-associated myelopathy (TSP-HAM).
• Blood donors found to be infected with HTLV-I or HTLV-II by serologic testing have been shown to
have an increased incidence of infections (bladder–kidney infections with HTLV-I and bladder–
kidney infections, bronchitis, and oral herpes with HTLV-II) when compared with seronegative
donor controls. In addition, HTLV-I can cause adult T-cell leukemia.

Other Diseases Transmitted by Transfusion

• Bacterial contamination of blood products is an important cause of morbidity and mortality.
• The source of blood product contamination is either silent bacteremia in the donor or skin
contaminants at the venipuncture site.
• Storage of products at standard refrigerator temperatures (4°C) retards the growth of most
bacteria so the risk of transfusion-transmitted bacteria in red blood cells is about 1:500,000 units
transfused.
• In stark contrast, platelet products carry a much higher bacterial contamination risk of 1:12,000
units transfused due to their storage at room temperature.
• Gram-negative organisms are more often found in refrigerated products, whereas in platelets
stored at ambient temperature the organisms are gram-positive.
• The mortality rate from transfused bacterially contaminated blood products has been estimated to
be as high as 25%.
• Epstein–Barr virus (EBV) may be transmitted by transfusion. In most cases it results in
asymptomatic seroconversion, but it can cause a mononucleosis syndrome.
• Posttransfusion syphilis is now rare. There is a low prevalence of syphilitic infection in blood
donors, and all donors are screened for antibody. Since the organism does not survive cold
storage for more than 96 hours, it can be transmitted only by fresh blood or platelets.
• Malaria remains a disease of major worldwide importance. The parasite can be present in
erythrocytes of carriers for years after infection. No available laboratory tests are simple and
sensitive enough to screen the blood donor population; therefore, blood banks in the United
States rely on histories taken at the time of donation. Donors who have traveled to areas where
malaria is endemic are deferred for 12 months.
• Other parasites are transmitted by transfusion. In the United States, Babesia microti, the
causative agent of babesiosis, is the second most common parasitical infection transmitted by
blood products.
• Trypanosoma cruzi, which causes Chagas' disease, is a very rare cause of transfusion-
transmitted parasitic infection in the United States; however, in the endemic countries of Central
and South America, blood transfusion is a common source for this infection.
• Microfilariasis is a transfusion risk in the tropical areas of the world where Wuchereria bancrofti,
Loa loa, and other filarial parasites are found. Transfusion-transmitted leishmaniasis is also
reported.

Immunologic Mechanisms of Transfusion Reactions

• Hemolytic transfusion reactions are caused by antigen–antibody complexes on the erythrocyte
membrane.
• These complexes activate Hageman factor (factor XIIa) and complement and induce the
production of several cytokines. Hageman factor activates the kinin system.
• Bradykinins thus generated increase capillary permeability and dilate arterioles, causing
hypotension.
• Complement is activated and leads to intravascular hemolysis as well as to histamine release
from mast cells.
• Hageman factor and free incompatible erythrocyte stroma activate the intrinsic clotting cascade,
with consequent DIC.
• Systemic hypotension with renal vasoconstriction and the formation of intravascular thrombi lead
to renal failure.
• When complement activation is incomplete, the reaction is less severe.
• Erythrocytes coated with C3b are cleared from the circulation by phagocytes, resulting in
extravascular hemolysis.
• The mechanism of graft-versus-host disease (GVHD) depends on the engraftment and clonal
expansion of donor lymphocytes in the recipient. Donor lymphocytes recognize recipient tissue
antigens as “foreign” and cause a clinical syndrome characterized by fever, skin rash, hepatitis,
and diarrhea.
• In transfusion-associated GVHD (TA-GVHD), bone marrow is also a target of donor
lymphocytes, and a significant aplasia results.
• Most cases of TA-GVHD are poorly responsive to treatment and result in death. Gamma
irradiation of lymphocyte-containing blood components to preclude lymphocyte activation and
expansion prevents TA-GVHD.
• Patients at risk for TA-GVHD are fetuses receiving intrauterine transfusions, patients transfused
with HLA-matched platelets, newborns undergoing exchange transfusion, patients with T-cell
immunodeficiencies, and patients severely immunosuppressed by intensive irradiation and
chemotherapy.
• There are rare reports of graft-versus-host disease following transfusion of blood from a
haploidentical donor into an immunocompetent recipient. Consequently, designated blood
donations collected from blood relatives are now irradiated before transfusion.

RH ISOIMMUNIZATION
• The D antigen is a common, strongly immunogenic antigen, 50 times more immunogenic than the
other Rh antigens.
• The prevalence of antibody formation to Rh+ blood depends on the dose of Rh+ cells: 1 mL of
cells sensitizes 15% of individuals exposed; 250 mL sensitizes 60–70%.
• After the initial exposure to Rh+ cells, weak IgM antibody can be detected as early as 4 weeks.
This is followed by a rapid conversion to IgG antibody.
• A second exposure to as little as 0.03 mL of Rh+ erythrocytes may result in the rapid formation of
IgG antibodies.
• The majority of potential transfusion reactions to Rh can be prevented by transfusing Rh–
individuals with Rh- blood.
• Immunization and antibody formation to D antigen still occur owing to occasional Rh sensitization
during pregnancy or to transfusion errors, particularly during emergencies.
• Immunization to other Rh antigens may occur because donor blood is typed routinely for D but not
for other Rh antigens.
• Hemolytic disease of the newborn occurs with the passage of Rh+ cells from the fetus to the
circulation of the Rh- mother. Once anti-D antibody is formed in the mother, IgG but not IgM anti-D
antibodies cross the placenta, causing hemolysis of fetal erythrocytes. Rh- mothers become
sensitized during pregnancy or at the time of delivery as a result of transplacental fetal
hemorrhage. Following delivery, 75% of women will have had transplacental fetal hemorrhage.
• Some obstetric complications increase the risk of transplacental fetal hemorrhage:
antepartum hemorrhage, toxemia of pregnancy, cesarean section, external version, and manual
removal of the placenta.
• Transplacental fetal hemorrhage can also occur following spontaneous or therapeutic abortion,
amniocentesis, chorionic villus sampling (CVS), or percutaneous umbilical cord sampling (PUBS).
• Overall Rh immunization occurs in 8–9% of Rh- women following the delivery of the first Rh+
ABO-compatible baby and in 1.5–2.0% of Rh- women who deliver Rh+ ABO-incompatible babies.

Rh Prophylaxis
• Rh immunization can now be suppressed almost entirely in antepartum or postpartum Rh- women
if high-titer anti-Rh immunoglobulin (RhIG) is administered within 72 hours after the potentially
sensitizing dose of Rh+ cells. The protective mechanism of RhIG administration is not clear.
• RhIG does not effectively block Rh antigen from immunosuppressive cells by competitive
inhibition, since effective doses of RhIG do not cover all D antigen sites. Intravascular hemolysis
and rapid clearance of RhIG-coated erythrocytes is also unlikely.
• Although this mechanism appears to explain the 90% protective effect of ABO incompatibility
between mother and fetus, RhIG-induced erythrocyte hemolysis is extravascular. Rh+ fetal cells
are removed primarily by highly phagocytic cells in the spleen and liver.
• The most likely mechanism is a negative modulation of the primary immune response, which
thereby depresses antibody formation.
• Antigen–antibody complexes are bound to cells bearing Fc receptors in the lymph nodes and
spleen. These cells presumably stimulate suppressor T-cell responses, which prevent antigen-
induced B cell proliferation and antibody formation.
• A prophylactic dose of 300 µg of RhIG intramuscularly prevents Rh immunization following
exposure to up to 15 mL of Rh+ erythrocytes, which corresponds to 30 mL of fetal whole blood.
• Initial recommendations were that 300 µg of RhIG be given to nonimmunized Rh- mothers within
72 hours after delivery of an Rh+ infant.
• The postpartum dose of RhIG decreased the incidence of anti-D development to 1% in Rh-
women giving birth to Rh+ infants.
• To further decrease the chances of developing anti-D in this population of women, antepartum
RhIG is also now administered at 28 weeks' gestation.
• A dose of RhIG is also indicated for an Rh- woman after any terminated pregnancy,
amniocentesis, CVS, PUBS, and fetal surgery or manipulation. Additional doses may have to be
given in cases of massive transplacental fetal hemorrhage.
• Large doses of RhIG can effectively suppress immunization following inadvertent transfusion of
Rh+ blood into Rh- patients if given within 72 hours of transfusion. Once Rh immunization is
demonstrated by the IAT, administration of RhIG is ineffective.

BLOOD COMPONENT THERAPY

• Improvements in the medical care of previously fatal illnesses has placed increasing demands on
the blood supply.
• As the need for blood products has expanded, the pool of eligible blood donors has decreased
due to more intensive screening and testing.
• The separation of a whole-blood donation into its component parts (fresh-frozen plasma, platelets,
and erythrocytes) has helped stretch a limited blood supply.

Erythrocytes
• During acute blood loss, 1 hour or more is required for equilibration of intravascular and
extravascular fluids and an accurate assessment of the fall in the hemoglobin level.
• Generally, a loss of 20% of blood volume can be corrected with crystalloid (electrolyte)
solution alone, which can then be supplemented with colloid (protein) solution. Whole blood is
indicated if blood loss exceeds one third of blood volume.
• Operative blood loss of 1000–1200 mL rarely requires transfusion in an otherwise healthy adult. If
increased oxygen-carrying capacity is required, erythrocyte transfusion is indicated (Table 17-5).
• A decreased hemoglobin level is tolerated better in a patient with chronic anemia than in a patient
with acute blood loss. Patients with a slow decline in their hemoglobin level compensate for the
decreased oxygen-carrying capacity by increasing their cardiac output.
• 2,3-Diphosphoglycerate is also increased in patients with chronic anemia, shifting the
oxyhemoglobin dissociation curve to the right. This rightward shift enhances oxygen release to
the tissues.
Platelets
• Platelets function to control bleeding by acting as hemostatic plugs on vascular endothelium.
• Platelet abnormalities that require platelet transfusion may be either quantitative or qualitative.
• The vast majority of platelet transfusions are given to supplement decreased numbers of
circulating platelets due to suppressed production, pooling, or dilution.
• Platelets are available as either platelet concentrates (recovered from a whole-blood donation)
or as plateletpheresis (collected by using a cytopheresis instrument).
• The transfusion of one platelet concentrate is expected to increase the platelet count of a 70-kg
adult by 5000–10,000/µL.
• A plateletpheresis is equivalent to four to eight platelet concentrates because both have the
same number of platelets.
• The survival of transfused platelets decreases in patients who are actively bleeding; who have
splenomegaly, fever, infection, or DIC; or who are sensitized to platelet antigens.
• The transfusion of ABO-incompatible platelets may be associated with slightly decreased platelet
survival.
• Much discussion ensues whenever the subject of indications for the appropriate use of platelet
transfusions arises. Little good clinical evidence addresses the indications for platelet therapy.
• General guidelines suggest that stable, afebrile thrombocytopenic adults and older children are
not at high risk of serious bleeding unless their platelet counts fall below 5,000–10,000 µL.
• Indications for transfusion of unstable patients are more problematic. Bleeding patients should be
more aggressively transfused, and many experts suggest transfusion when platelet counts fall
below 30,000–50,000 µL.
• Thrombocytopenic patients undergoing invasive procedures do not generally experience
increased complications unless their platelet counts are less than 50000 µL however, the patient's
clinical situation and the site of the procedure or surgery should influence the decision to
transfuse.
• Patients undergoing surgery on the eye, brain, spinal cord, or airway are at higher risk of serious
sequelae due to bleeding and may require higher platelet counts for safety.
Plasma Products
• Fresh-frozen plasma (FFP), stored plasma, and cryoprecipitate are valuable sources of
coagulation factors.
• Stored plasma and FFP may often be used interchangeably. Levels of factors V and VIII in stored
plasma are half those in FFP, but levels of other factors are equivalent.
• Cryoprecipitate was initially produced to provide therapeutic doses of factor VIII and von
Willebrand's factor. This use has been greatly supplanted by the development of recombinant
or treated factor VIII, which have lower infectious risks to recipients.
• Cryoprecipitate is now most often used to treat bleeding in patients with fibrinogen less than 100
mg/dL.
• FFP is used for treating isolated congenital factor deficiencies, for which a safer factor concentrate
product is not available. It is also used to correct warfarin overdoses in patients with
significant bleeding. FFP is also used to treat thrombotic thrombocytopenic purpura and C1
esterase inhibitor deficiency.
• Massively transfused patients with a prothrombin time or partial thromboplastin time greater than
1.5 times normal and platelet counts above 50,000/µL may benefit from FFP treatment.
• FFP or plasma should never be used for volume expansion because colloid solutions without
infectious risk are available (ie, albumin).

Helpful videos:
https://www.youtube.com/watch?v=9DnlP6AgQdQ

https://www.youtube.com/watch?v=g-61CDnGXrY

questions:
1- which of the following blood group antigens act as the receptor for P.flaciparum species of
malaria?
a) Blood group O
b) Blood group B
c) Blood group A
d) Kell
e) Duffy blood group FYa FYb type
2- which of the following enzymes is missing in Bombay blood group (hh)?
a) Lactose transferase
b) N-acetyl glucose amine transferase
c) Fucosyl transferase
d) N-acetyl galactose amine transferase
e) Galactose transferase
3- all of the following blood group genotypes are classified as Rh- except:
a) Cde
b) CDE
c) Cde
 d) CdE
e) cdE
4- a blood group that contains anti-A, anti-B and anti-H antibodies:
a) A
b) B
c) AB
d) Bombay
e) O
5- fresh frozen plasma is indicated in:
a) Massive blood transfusion for thalassemia
b) Open heart surgery
c) Hemophilia A and hemophilia B
d) As a plasma expander
e) For total parenteral nuitrition
6- not transmitted by refrigerated blood:
a) HIV
b) HBV
c) HCV
d) Syphilis
e) CMV
Answers: 1-e 2-c 3-b 4-d 5-a 6-d
 Tests for Antigen-Antibody Reactions
Introduction:
• The combination of Antibody with its specific Antigen plays important role in clinical immunology
laboratory (CIL) tests.
• Immunoassays tests: detect either Antigen or Antibody & vary from easily performed manual to
highly complex automated ones.

Nature of Antigen-Antibody Reactions:

(1) Key & Lock Concept:
• Key (Antigen) fits into lock (Antibody).
• X-Ray crystallography shows that antigenic determinant (epitope) nestles in cleft formed by
antibody combining site which is located in Fab region of immunoglobulin (Ig) & is constructed
from hypervariable regions (HVR) of Heavy & Light chains.
(2) Non-covalent Bonds:
• All tight multiple bonds between Antigen and Antibody are non-covalent in nature & it includes:
- hydrogen bond.
- electrostatic bonds
- Van der Waals forces
- hydrophobic bonds.
(3) Reversibility:
• As Antigen-Antibody reactions occur via non-covalent bonds, they are by their nature reversible.

Antibody Affinity & Avidity

1- Affinity
• strength of binding between single epitope & single
(individual) antibody combining site.
• sum of attractive & repulsive forces operating
between epitope & Antibody combining site.

2- Avidity
• It is the overall strength of binding between
multivalent Antigen (with many epitopes) &
multivalent Antibodies (with many Antibody
combining sites).
• influenced by both: valency of Antibody & valency of Antigen.
Antibody Specificity & Cross Reactivity
Specificity
• ability of individual Antibody combining site to bind to only one epitope, or ability of population of
Antibody molecules to bind to only one Antigen.
• In general, there is high degree of specificity in Antigen-Antibody reactions.
• Antibodies can distinguish the differences in:
- 1° (Primary) structure of Antigen.
- Isomeric forms of Antigen.
- 2° (secondary) & 3° (tertiary) structure of Antigen.
Cross reactivity
• Ability of individual Antibody combining site to
bind to more than 1 epitope, or ability of
population of Antibody molecules to bind to
more than 1 Antigen.
• Cross reactions result from cross reacting
Antigen which has either:
(1) Shared epitope in common with immunizing
Antigen
(2) Structurally Similar epitope to the one on
immunizing Antigen (multi-specificity).

Factors affecting measurement of Antigen-Antibody reactions:

• Detection (direct or indirect) of Antigen-Antibody complexes depends on:
(1) Affinity: higher affinity of Antibody for Antigen →more stable interaction→ easier detection.

(2) Avidity: Reactions between multivalent Antigen & multivalent Antibodies are more stable→
easier detection.

(3) Antigen to Antibody ratio:

- As size of formed complexes is related to concentration of
Antigen and Antibody.
- The concentration of antigen to antibody in this reaction, they
are in equivalence leading to lattice formation or clumping so it
will be detected in the lab→→

(4) Physical form of Antigen:

- Particulate →agglutination (clumping) of Antigen by Antibody.
(Antibody= agglutinin).
- Soluble→ precipitation of Antigen by production of large insoluble Antigen-Antibody complexes.
(Ab= precipitin).
 Agglutination Tests (easy to perform vs semi-quantitative ONLY)
• Theoretically, ALL Antibodies can agglutinate particulate Antigens but IgM (due to its high
valency) is particularly good agglutinin → it may be concluded that Antibody may be of IgM class if
it is a good agglutinating Antibody.
• Direct: Antigens normally found on surfaces of particles as bacteria (ex: a patient who has
meningitis, so you suspect that there is bacteria in CSF so I will bring foreign anti-serum to
S.coccus pneumonia then take a sample from CSF, if the antigen is found there will be
agglutination which means that it is positive)
• Passive: Antigens NOT normally found on surfaces of inert carrier particles including RBCs
(Hemagglutination), latex (most frequently solid phase used), gelatin, & silicates.
• Reverse passive: Antibody, rather than Antigen, coating inert carrier particles.
• Agglutination inhibition
• Co-agglutination: Ab coating Bacteria as inert carrier particles, S.aureus most frequently used,
because of protein A on its outer surface which naturally adsorbs Fc of Antibody molecules.

Qualitative agglutination test

• To detect Antigen or Antibody.
• Use: ABO blood group (A, B, & O) typing.
(1) RBCs mixed with IgM Antibodies to blood group A & B Antigens to
detect Antigens on RBCs & determine blood groups.
(2) Serum mixed with RBCs of known blood groups to detect Antibodies
to that blood group Antigen in serum & determine blood group.

Quantitative agglutination test

• To measure Antibodies to particulate Antigens.
• Principle: fixed number of RBCs, bacteria or other particulate Antigens is added to serial dilutions
of serum with Antibody → titer (maximum dilution with visible agglutination) → result reported as
reciprocal of titer.
• Use: Diagnosis of bacterial Infections (fourfold rise in Antibody titer in paired dilutions of serum
samples is significant) as Widal test for typhoid fever and Brucellosis .

Quantitative hemagglutination test:

• Row (6) (Prozone effect) it started with negative cause it has
High [Antibody] in lower dilutions → NO visible agglutination.
• Further dilutions → Low [Antibody] in higher dilutions →
visible agglutination.
• Why? Antibody excess → very small complexes which do not
clump to form visible agglutination.
Passive hemagglutination
• Principle: RBCs coated with soluble Antigen (e.g. viral
Antigen, polysaccharide, or hapten) → agglutination of RBCs
by specific Antibody.
• Procedure: just like agglutination test.
• Applications: detection of Antibodies to soluble Antigens, &
viral Antigens.

Coomb's (Antiglobulin/ Antiglobulin mediated agglutination) Test

• Use: detection of Incomplete Antibodies (NO structural difference, rather functional definition
only) = non-agglutinating Antibodies which can bind to but can NOT agglutinate RBCs (i.e. can
NOT cause hemagglutination).
• Why? →due to Antigen/Antibody ratio being in Antigen excess or Antibody excess, or in some
cases electrical charges on RBCs preventing effective cross linking of RBCs required for
visible agglutination.
• Principle: addition of second anti-Ig directed against incomplete Antibodies →cross link RBCs
→ visible agglutination

Applications
• Detection of anti-rhesus factor (Rh) incomplete Antibodies:
(1) Direct Coombs test to detect anti-Rh Antibodies on newborn’s RBCs.
(2) Indirect Coombs test to detect anti-Rh Antibodies in mother’s serum.

Hemagglutination Inhibition
• Principle: modified agglutination test to measure ability of soluble antigen to inhibit
hemagglutination of Antigen-coated RBCs by Antibodies.
• How: fixed amount of Antibodies is mixed with fixed amount of RBCs coated with Antigen, &
different amounts of sample tested with Antigen.
• Quantitative: serial dilution of sample → titer of soluble
Antigen in unknown sample.
• Results:
- positive test: -ve/ NO hemagglutination (soluble
Antigen in sample competes with Antigen coated on
RBCs for binding to Antibodies).
- negative test: +ve hemagglutination (NO soluble Ag in
sample).
Precipitation Reaction
• Soluble Antibody + soluble Antigen interacting in aqueous solution → insoluble visible lattice
precipitate of Antigen-Antibody complexes depending on valency of both Antibody (must be
bivalent Fab fragments) & Antigen (must be bivalent/ polyvalent; no precipitate if Antigen contains
only single copy of each epitope).

Precipitation Tests
• Measurement of precipitation by light scattering:
- Turbidimetry: measure of turbidity or cloudiness of solution.
- Nephelometry: measures scattered light as an index of [solution].
• Uses: Quantification of Igs, kappa & lambda light chains, & other serum proteins (as complement
components, C reactive protein, & several clotting factors).
• Passive immunodiffusion Techniques:
- Single Radial Immunodiffusion.
- Double Immunodiffusion.
• Electrophoretic Techniques:
- Immunoelectrophoresis (IEA).
- Countercurrent IEA (CIE).
- Rocket IEA.
- Immunoblotting.

Passive (NO electric current) Immunodiffusion

(a) Single Radial (Mancini method)
- Antibody incorporated into agar gel during its pouring, & different dilutions of Antigen placed in
wells cut into agar.
 - ONLY (Single) Antigen diffuses passively out in all directions, reacts with Antibody
→equivalence point → precipitin ring.
- Amount of Antibody is constant →diameter of precipitin ring is proportional to log of [Antigen]
→running different [standard Antigen] → generation of standard curve from which amount of
Antigen in unknown sample could be quantitated.
- Quantitative test.
- Common use: assaying of Ig levels in patient samples.
- Mixture of Antigens or Antibodies → >1 Antigen-Antibody reaction → >1 ring in test.

(b) Double (Ouchterlony method)

• Multi-specific Antibody in central well, & different Antigens in surrounding wells → BOTH
(Double) Antigen and Antibody diffuse passively independently from wells, react with each other
→ equivalence point →precipitin bands:
(1) position of bands between wells allows for Antigens to be compared with one another.
(2) density (thickness) of bands reflect amounts of immune complexes formed.

Diffusion patterns:

Immunoelectrophoresis (IEA) (= Electrophoresis + Double)

• Electrophoresis of complex mixture of Antigens placed in well cut into
agar gel, → Antigens are separated according to their electric charge.
• Antibodies added in trough cut in agar gel parallel to line of Antigens
separation.
• Antibodies diffuse into agar → precipitin lines produced in equivalence
zone where Antigen-Antibody reaction occurs.
• Use: qualitative analysis of complex mixtures of Antigens, & crude
quantitative measure (thickness of line).
• Commonly used for analysis of components in patients serum. Serum
is placed in well & Antibody to whole serum in trough. By
comparisons to normal serum, deficiencies of ≥1 serum components
or overabundance of some serum components (thickness of line) can
be determined.
• can also be used to evaluate purity of isolated serum proteins.

Countercurrent immunoelectrophoresis (CIE)

• Antigen and Antibody are placed in wells cut into agar gel & are electrophoresed into each other
where they form precipitin line.
• Advantage: speed.
• Limitation: only if Antigen and Antibody have opposite
electric charges.
• Primarily qualitative, but quantity might be measured from
thickness of band.
Rocket IEA (Laurell Technique; one-dimension IEA)
Detection of:
- Major classes of Igs.
- Myelomas.
- Malignant lymphomas.
- Others lymphoproliferative disorders
Rocket IEA
- Standards in wells 1, 2 & 3.
- Patients samples in wells 4, 5 &
6.
- Well 4 contains NO Antigen
because NO ring is formed.
- Well 5 contains low [Antigen].
- Well 6 contains high [Antigen].

Western blot (protein blot) for detection of solubilized specific target protein in sample:
(confirmatory test for HIV, Bovine spongiform encephalopathy (BSE), Lyme disease, & HBV)
(1) Separation of proteins from known infected case by Gel electrophoresis →cathode - (longer
molecules) to anode + (shorter molecules).
(2) Electro blotting for uniform & effective transfer of separated proteins from within the gel retaining
same pattern of original gel separation onto sheet of special blotting paper/ membrane
(nitrocellulose or polyvinylidene difluoride) to make them accessible for staining with specific
Antibodies → process checked with staining membrane with Coomassie Brilliant Blue or Ponceau
S dyes.

Radioimmunoassay (RIA)/ Enzyme-Linked Immunosorbent Assay (ELISA)

RIA
- based on measurement of radioactivity associated with immune complexes.
- Radiolabel may be on either antigen or antibody.
ELISA
- based on measurement of enzymatic reaction associated with immune complexes.
- Enzyme may be linked to either antigen or antibody.

I. Competitive RIA/ELISA
• Principle: detection & measurement of Antigen in unknown
sample.
• How: using known amounts of standard unlabeled Antigen →
generation of standard curve relating radioactivity (cpm; counts
per minute)/ Enzyme bound versus amount of Antigen.
• Use: to quantitate serum proteins, hormones & drugs
metabolites.
• The key to assay is easy separation of immune complexes from
other components includes free Antigen → 3 different ways:
(1) Farr Technique (ammonium sulphate): Addition of
ammonium sulphate (33-50% [final]) → precipitation
of Immunoglobuins but NOT many Antigens.
(2) Anti-Ig Antibody: Addition of second anti-Ig Antibody
directed against first Antibody→ precipitation of
immune complexes.
(3) Solid phase RIA or ELISA (most common method
used today): Antibody immobilization onto surface of plastic bead or coating onto surface of
plastic micro titer plate → simple washing.

II. Non-competitive RIA/ELISA for measurement of antigen or antibody

(1) Bead is coated with Antigen & is used to measure Antibody in
unknown sample. Amount of labeled second Antibody which binds is
proportional to amount of first Antibody in sample which bound to
Antigen.
o Commonly used in Radio allegro sorbent test (RAST) which
specifically measures igE Antibodies directed against particular
allergens in allergic patients. A known allergen is used as Ag &
covalently coupled to paper disc which is then tied with patient’s
serum. The amount of specific IgE bound is determined using
labeled anti-IgE Antibodies.

(2) Bead is coated with antibody & is used to measure Antigen in

unknown sample.
o Amount of labeled second Antibody which binds is proportional to
amount of Antigen in sample which bound to first Antibody.
Tests for Cell Associated Antigens Immunofluorescence (IF)
• Principle: detection of Antigen in or on cell or tissue by fluorescence
emitted by bound Antibody labeled with fluorescent dye/ fluorochrome
molecule (fluorescein, rhodamine, or one of many others).
(1) Direct IF:
Antibody specific to Ag is directly tagged with fluorochrome.

(2) Indirect IF:

• Antibody specific for Antigen is unlabeled, & second anti-Ig Antibody
directed against first Antibody is tagged with fluorochrome.
• More sensitive than direct IF since there is amplification of signal.

(3) Flow Cytometry:

• Commonly used to Identify & enumerate cells bearing particular

Antigen.
• Cells in suspension are labeled with fluorescent tag by either direct or
indirect IF, then analyzed on flow cytometer.

Complement Fixation (CF) Tests

• Principle: measurement of antigen-antibody complexes which rely on their ability for fixation
(consumption) of complement (as free antigens or antibodies do not).
• Limitation: only work with complement fixing Antibodies (IgG & IgM are best).
• Use:
- to quantitate antigen-antibody reactions.
- most commonly to assay for Antibody in test sample but they can be modified to measure Ag.
How?
o Antigen + Antibody? in test serum →
antigen-antibody complexes.
o + complement → CF.
o + Indicator system (Antibody-coated
RBCs):
(1) Positive test result: antigen-
antibody complexes → CF (fixation
of some complement) → -ve/ NOT
all RBCs lyse.
(2) negative test result: No antigen-antibody complexes →NO CF → all complement available →
+ve/ lysis of all RBCs.
• Measuring amount of RBCs lysis by measuring release of hemoglobin into medium → indirect
quantitation of Antigen-Antibody complexes in tube.
Helpful videos:
https://www.youtube.com/watch?v=YJ0-qQslqqQ

https://www.youtube.com/watch?v=zUGikX9ZB9U

questions:
1- all of the following immunoassays depend upon lattice formation, except:

a) Immunoelectrophoresis
b) Indirect coombs test
c) Direct coombs test
d) Complement fixation
e) Hemagglutination inhibition

2- indirect coomb’s test looks for antibodies in:

a) Mother’s RBC’s
b) Mother’s serum
c) Child’s RBC’s
d) Child’s serum
e) Non of the above

3- a test to identify and enumerate cells bearing particular antigen:

a) Direct immunofluorescence
b) inDirect immunofluorescence
c) flow cytometry
d) rocket immunoelectrophoresis
e) counteract immunoelectrophoresis

4- which of the following uses protein antigens separated by molecular weight:

a) ELISA
b) Western blotting
c) Agglutination test
d) Eastern blotting
e) Complement fixation

5- agglutination test (direct) is used to confirm the diagnosis of:

a) Lyme disease
b) HIB
c) BSE
d) Salmonella
e) HBV

6- widal test is:

a) Direct agglutination test

b) Indirect agglutination test
c) Passive agglutination
d) Co-agglutination test
e) Reverse agglutination test Answers: 1-d 2-b 3-c 4-b 5-d 6- a