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IMMUNOLOGY QUESTIONS
1. Components of the non-specific immune system

The skin and mucous membranes act as a barrier to infectious agents. Antimicrobial substances(cationic
peptides(defensins)), lysozyme, lactoferrin and secretory(IgA) are found in secretions at mucosal surfaces,
e.g. tears, mucus and saliva and are part of the non-specific(innate) immune system. Different defensins
can disrupt bacterial, viral and fungal membranes. Lysozyme induces lysis of bacteria by cleaving the
polysaccharide backbone of the peptidoglycan of Gram positive bacteria. Lactoferrin, an iron-binding
protein, deprives microbes of the free iron they need for growth.

Lactoperoxidase may be inhibitory to many microorganisms and is found in milk and saliva.

Beta- lysin is effective against many gram-positive bacteria and is found in thromobocytes and normal
serum.

The acidic environment of the stomach, bladder and kidneys and the bile of the intestines inactivates many
viruses and bacteria.

Body temperature, and especially fever, limits or prevents the growth of many microbes. Additionally, the
immune system is more efficient at elevated temperatures.

Bacterial components are excellent activators of the innate antigen-nonspecific protective and
inflammatory responses. The bacterial cell walls(teichoic acid) and peptidoglycan fragments of gram
positive bacteria and especially the lipopolysaccharide(LPS) of gram negative bacterial cell walls can be
recognized by pathogen-associated molecular pattern recognistion(PAMP) receptors, including the cell
surface Toll-like receptors(TLR’s) and the cytoplasmic peptidoglycan receptors NOD1, NOD2 and cryopyrin.
Binding of these PAMP’s to receptors on macrophages, Langerhans cells and dendritic cells activate kinase
cascades that promote cytokine production(IL-1, IL-6 and TNF), protective responses and maturation of
dendritic cells. NK cells, NKT and gamma/delta T cells residing in tissue also respond, produce cytokines
and re-inforce cellular responses.

The alternative complement pathway is activated by bacterial cell surfaces and their components in the
absence of antibody and, with mannose-binding proteins, can activate the classic complement pathway to
produce:-

1. Chemotactic factors(C5a) to attract neutophils and macrophages to the site of infection

2. Anaphylotoxins(C3a and C5a) to stimulate mast cell release of histamine and thereby increase vascular
permeability, allowing access to the infection site
3. Opsonins(C3b) which bind to bacteria and promote their phagocytosis
4. A B-cell activator(C3d)

Part of the inflammatory response is the recruitment of polymorphonuclear eosinophils and macrophages to
the site of infection. These cells are the main line of defense in the non-specific immune system.
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2. Phagocytes (types) and phagocytosis

Phagocytes are divided into mononuclear and polymorphonuclear group. In mononuclear group are monocyte
which spend only short period of time in blood-after that they migate to tissues and become macrophages.
Macrophages live for months and are able to phygocyte and digest more than 100 bacetrias. Tissuse
machrophages are histocytes in skin, alveolar machrophages, Kupfer cells in liver,microglia in CNS and
osteoclasts. Monocytes and macrophages present first line of body s defense.

Polymorphonuclear cells are neutrophils which posses microphagic properties meaning only ingestion but not
digestion of 10 bacteria, basophils releasing some factors in allergic reaction and eosinophils which have the
most important function of killing parasites. Polymorphonuclear cells maturate in bone marrow, migrate to
blood and have quite a short life span.

Phagocytosis is a process composed of chemotaxis, recognition, ingestion and as last digestion. Chemotaxis is
a process of attraction of phagocytes to places where needed, enabled by certain chemotaxic substances such
as cytokines, complement products etc. Recognition by neutrophils and macrophages is done by surface
receptors specific for attacking microorganisms. Ingestion is process in which microorganisms are transported
into the phagocyte by first joining membranes and retraction. Digestion starts when the microorganism is
ingested, the phagosome is joined with a lysosome which is full enzymes such as proteases ,oxsidases etc.,
forming a phagolysosome.

3.Humural factors contributing to non specific imunnity

Those factors can be found in plasma and extracellular fluid and they have a role in stimulating phagocyte
action. Those substances are: Complement-system proteins, Beta lysine-protein which cause apoptosis of
Gram positive bacterias and are released from thrombocytes, Lysozyme-enzyme released from phagocytes
which degrade the cell wall, Interferon-(types gamma,alfa,beta,omega) proteins which act as antiviral,
antiproliferative and immunoregulatory. Alfa interferon is secreted by leukocytes and it causes activation of
NK cells. Beta interferon is secreted by fibroblasts whit antiviral function. Gamma interferon is secreted by T
cells with immunoregulatory action. C-reactive protein is a beta globulin which speeds up activation of
complement and phagocytosis. Normal antibodies found in the serum are against antigens on red blood cells
of different species.

4.Lymphocytic cells contrubuting to nonspecific, immunity NK , K cells:

Although humoral (antibody-mediated specific immunity) is an important host defense against many bacterial
and viral diseases, in many other bacterial infections and viral infections, it is primarily the cell-mediated arm
that imparts resistance and aids in recovery. Furthermore, cell-mediated immunity is important in defense
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against fungi, parasites, and tumors and in the rejection of organ transplants. Its suppression results in
overwhelming infections or tumors.

The constituents of the non-specific immune system includes several cell types:

(1) macrophages, which present the antigen to T cells;

(2) helper T cells, which participate in antigen recognition and in regulation (helper and suppressor) functions;

(3) natural killer (NK) cells, which can inactivate pathogens; and

(4) cytotoxic T cells, which can kill virus-infected cells with or without antibody. Macrophages and helper T
cells produce cytokines that activate helper and cytotoxic T cells, leading to the killing of the pathogen or tumor
cell.

Infection with some viruses, namely, measles virus and cytomegalovirus, can suppress cell-mediated immunity
against other microorganisms.

The terms primary and secondary response are associated primarily with antibody formation, but the timing of
the T-cell response also follows the same pattern. After the initial exposure to the antigen, the specific T cell
proliferates to form a small clone of cells, i.e., a primary response occurs. Then, on subsequent exposure to the
antigen, the small clone expands and many more specific T cells are formed. These cells constitute the
secondary response.

Although the interactions between various cells and various cytokines are complex, the result is relatively
simple: In the person with competent cellular immunity, opportunistic pathogens rarely or never cause disease,
and the spread of other agents—e.g., certain viruses (e.g., herpesviruses) or tumors (e.g., Kaposi's sarcoma)—is
limited. The assessment of the competence of cell-mediated immunity is therefore important.

5. Antigens, Haptens, Epitopes.

Almost all of the proteins and carbohydrates associated with an infectious agent, whether a bacterium,
fungus, virus, or parasite, are considered foreign to the human host and have the potential to induce
an immune response. A protein or carbohydrate that challenges the immune system and can initiate an
immune response is called an immunogen.
Immunogens may contain more than one antigen (e.g., bacteria).

An antigen is a molecule that is recognized by specific antibody or T cells.

An epitope (antigenic determinant) is the actual molecular structure that interacts with a single
antibody molecule or T-cell receptor.
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Within a protein, an epitope may be formed by a specific sequence (linear epitope) or a three-
dimensional structure (conformational epitope).
Antigens and immunogens usually contain several epitopes, each capable of binding to a different
antibody molecule. A monoclonal antibody recognizes a single epitope.

Not all molecules are immunogens. In general, proteins are the best immunogens, carbohydrates are weaker
immunogens, and lipids and nucleic acids are poor immunogens.

Haptens (incomplete immunogens) are often too small to immunize (i.e., initiate a response) an individual but
can be recognized by antibody.

Haptens can be made immunogenic by attachment to a carrier molecule, such as a protein. For example,
dinitrophenol conjugated to bovine serum albumin is an immunogen for the dinitrophenol hapten.

6. Properties of immunogenicity foreigness special type of antigen-cross reacting ones, sequestered

antigens: tissue specific antigens

Unlike antibodies that can recognize conformational epitopes, T cell antigenic peptides must be linear
epitopes. A T-cell antigen must be a peptide of 8 to 12 amino acids with a hydrophobic backbone that
binds to the molecular cleft of the class I or class II MHC molecule and exposes a T-cell epitope to the
TCR(T cell receptor). Owing to these constraints, there may be only one T-cell antigenic peptide in a
protein.

The Endogenous route of antigen presentation is adopted by all nucleated cells, which proteolytically
process a set of intracellular proteins and display the peptides to the CD8 T cells to distinguish “self,” “non-
self,” and the absence of intracellular infections.

The Exogenous route of antigen presentation occurs with APC’s(Antigen presenting cells), which process
and present phagocytosed proteins to CD4 T cells.

Dendritic cells can cross these routes exhibiting the phenomenon of Cross-presentation whereby they
present exogenous antigen to CD8 T cells to initiate antiviral and antitumour responses.

During a viral infection, large quantities of viral proteins are produced and degraded into peptides and
become the predominant source of peptides, occupying the class I MHC molecules to be presented to CD8
T cells.

Transplanted cells(grafts) express proteins on their MHC molecules, which differ from those of the host
and therefore may be recognized as foreign. Host tissue possesses tissue specific antigens.

Tumour cells often express peptides derived from abnormal or embryonic proteins, which may elicit responses
in the host because the host was not tolerised to these proteins. Expression of these “foreign” peptides on
MHC I at the cell surface allows the T cell to “see” what is going on within the cell.
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7.Immunoglobulin structure

Immunoglobulins are composed of at least two heavy chains and two light chains, a dimer of dimers. They are
subdivided into classes and subclasses based on the structure and antigenic distinction of their heavy chains.
IgG, IgM, and IgA are the major antibody forms, whereas IgD and IgE make up less than 1% of the total
immunoglobulins. The IgA and IgG classes of immunoglobulin are divided further into subclasses based on
differences in the Fc portion. There are four subclasses of IgG, designated as IgG1 through IgG4, and two IgA
subclasses (IgA1 and IgA2)

Antibody molecules are Y-shaped molecules with two major structural regions that mediate the two major
functions of the molecule. The variable-region/antigen-combining site must be able to identify and specifically
interact with an epitope on an antigen. A large number of different antibody molecules, each with a different
variable region, are produced in every individual to recognize the seemingly infinite number of different
antigens in nature.

The Fc portion (stem of the antibody Y) interacts with host systems and cells to promote clearance of antigen
and activation of subsequent immune responses. The Fc portion is responsible for fixation of complement and
binding of the molecule to cell surface immunoglobulin receptors (FcR) on macrophages, natural killer cells,
and T cells.

For IgG and IgA, the Fc portion interacts with other proteins to promote transfer across the placenta and the
mucosa, respectively. In addition, each of the different types of antibody can be synthesized with a
membrane-spanning portion to make it a cell surface antigen receptor.

IgG and IgA have a flexible hinge region rich in proline and susceptible to cleavage by proteolytic enzymes.
Digestion of IgG molecules with papain yields two Fab fragments and one Fc fragment. Each Fab fragment has
one antigen-binding site. Pepsin cleaves the molecule, producing an F(ab')2 fragment with two antigen-
binding sites and a pFc' fragment.

The different types and parts of immunoglobulin can also be distinguished using antibodies directed against
different portions of the molecule. Isotypes (IgM, IgD, IgG, IgA, IgE) are determined by antibodies directed
against the Fc portion of the molecule (iso meaning the same for all people.) Allotypic differences occur for
antibody molecules with the same isotype but contain protein sequences that differ from one person to
another (in addition to the antigen-binding region). (Every one ["allo"] of them cannot have the same IgG.) The
idiotype refers to the protein sequences in the variable region that generate the large number of antigen-
binding regions. (There are many different idiots in the world.)

On a molecular basis, IgM and IgA consist of multimers of the two heavy and two light chain basic structure.
The heavy and light chains of immunoglobulin are fastened together by interchain disulfide bonds. Two types
of light chains-κ and λ-are present in all five immunoglobulin classes, although only one type is present in an
individual molecule. Approximately 60% of human immunoglobulin molecules have κ light chains, and 40%
have λ light chains. There are five types of heavy chains, one for each isotype of antibody (IgM, μ; IgG, γ; IgD,
δ; IgA, α; and IgE, Ε). Intrachain disulfide bonds define molecular domains within each chain. Light chains have
a variable and a constant domain. The heavy chains have a variable and three (IgG, IgA) or four (IgM, IgE)
constant domains. The variable domains on the heavy and light chains interact to form the antigen-binding
site. The constant domains from each chain make up the Fc portion, provide the molecular structure to the
immunoglobulin and define the interaction of the antibody molecule with host systems, hence its ultimate
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function. The heavy chain of the different antibody molecules can also be synthesized with a membrane-
spanning region to make the antibody an antigen-specific cell surface receptor for the B cell.

8.Biological and chemical properties of Immunoglobulins

General. All immunoglobulins appear to be glycoproteins but the carbohydrate content ranges from 2-3% for
IgG to 12-14% for IgM, IgD and IgE. Each class possesses a characteristic type of heavy chain. Thus IgG
possesses chains; IgM, chains; IgA, chains, IgD, chains and IgE, chains. Variation in heavy chain
structure within a class gives rise to immunoglobulin subclasses. For example the human IgG pool consists of
four subclasses reflecting four distinct types of heavy chain. The physicochemical properties of the
immunoglobulins vary between the different classes. Note that IgA occurs in a dimeric form in association with
a protein chain termed the secretory piece. The diversity of structure of the different classes suggests that they
perform different functions, in addition to their primary function of antigen binding. In spite of this diversity all
antibodies have a common basic structure.

Main Immunoglobulins and its characteristics:

IgG. IgG comprises approximately 85% of the immunoglobulins in adults. Production of IgG requires T-cell
help. IgG, as a class of antibody molecules, has the longest half-life (23 days) of the five immunoglobulin
classes, crosses the placenta, and is the principal antibody in the anamnestic or booster response. IgG shows
high avidity (binding capacity) for antigens, fixes complement, stimulates chemotaxis, and acts as an opsonin to
facilitate phagocytosis.

IgM. IgM is the first antibody produced in response to antigenic challenge and can be produced in a T-cell-
independent manner. IgM makes up 5% to 10% of the total immunoglobulins in adults and has a half-life of 5
days. IgM is the most efficient immunoglobulin for fixing (binding) complement. A single IgM pentamer can
activate the classical complement pathway. Monomeric IgM is found with IgD on the B-cell surface, where it
serves as the receptor for antigen. Because IgM is relatively large, it remains in the blood and spreads
inefficiently from the blood into tissue. IgM is particularly important for immunity against polysaccharide
antigens on the exterior of pathogenic microorganisms. It also promotes phagocytosis and promotes
bacteriolysis by activating complement through its Fc portion. IgM is also a major component of rheumatoid
factors (autoantibodies).

IgA IgA comprises 5% to 15% of the serum immunoglobulins and has a half-life of 6 days. In addition to serum
IgA, a secretory IgA appears in body secretions and provides localized immunity. IgA production requires
specialized T-cell help and mucosal stimulation. Adjuvants, such as cholera toxin and attenuated Salmonella
bacteria, can promote an IgA response. IgA binds to a poly-Ig receptor on epithelial cells for transport across
the cell. The poly-Ig receptor remains bound to IgA and is then cleaved to become the secretory component
when secretory IgA is secreted from the cell. An adult secretes approximately 2 gm of IgA per day. Secretory
IgA appears in colostrum, intestinal and respiratory secretions, saliva, tears, and other secretions. IgA-deficient
individuals have an increased incidence of respiratory tract infections.

IgD. IgD accounts for less than 1% of serum immunoglobulins. IgD exists primarily as membrane IgD, which
serves with IgM as an antigen receptor on early B-cell membranes to help initiate antibody responses by
activating B cell growth. IgD and IgM are the only isotypes that can be expressed together by the same cell.
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IgE. IgE accounts for less than 1% of the total immunoglobulins and has a half-life of approximately 2.5 days.
Most IgE is bound to Fc receptors on mast cells, on which it serves as a receptor for allergens and parasite
antigens. When sufficient antigen binds to the IgE on the mast cell, the mast cell releases histamine,
prostaglandin, platelet-activating factor, and cytokines. IgE is important for protection against parasitic
infection and is responsible for anaphylactic hypersensitivity (Type 1) (rapid allergic reactions).

9. Complement system

The classical complement cascade is initiated by binding to the Fc portion of antibody that is bound to cell
surface antigens, or in an immune complex with soluble antigens. Aggregation of antibody (IgG or IgM, not
IgA or IgE) changes the structure of the heavy chain to allow binding to complement.

The first complement component, designated C1, consists of a complex of three separate proteins designated
C1q, C1r, and C1s. One molecule each of C1q and C1s with two molecules of C1r constitutes the C1 complex
or recognition unit.

C1q facilitates binding of the recognition unit to cell surface antigen-antibody complexes.

Activation of the classical complement cascade requires linkage of C1q to two IgG antibodies through their Fc
regions. In contrast, one pentameric IgM molecule attached to a cell surface may interact with C1q to initiate
the classical pathway.

Binding of C1q activates C1r (referred to now as C1r*) and in turn C1s (C1s*).

C1s* then cleaves C4 to C4a and C4b, and C2 to C2a and C2b. The convention is that the b fragment is bigger
and bound to something. The ability of a single recognition unit to split numerous C2 and C4 molecules
represents an amplification mechanism in the complement cascade.

The union of C4b and C2b produces C4b2b, which is known as C3 convertase.

This complex binds to the cell membrane and cleaves C3 into C3a and C3b fragments.

The C3b protein has a unique thioester bond that will covalently attach C3b to a cell surface or be hydrolyzed.

The C3 convertase amplifies the response by splitting many C3 molecules. The interaction of C3b with C4b2a
bound to the cell membrane produces C4b3b2b, which is termed C5 convertase. This activation unit splits C5
into C5a and C5b fragments and represents yet another amplification step.

Various factors produced by the cascade have functions:-

1. Chemotactic factors(C5a) to attract neutophils and macrophages to the site of infection

2. Anaphylotoxins(C3a and C5a) to stimulate mast cell release of histamine and thereby increase vascular
permeability, allowing access to the infection site
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3. Opsonins(C3b) which bind to bacteria and promote their phagocytosis

4. A B-cell activator(C3d)

The terminal stage of the classical pathway involves creation of the membrane attack complex, which is also
called the lytic unit.The five terminal complement proteins (C5 through C9) associate into a membrane attack
complex on target cell membranes to mediate injury.

10.Pathways of complement activation:

Activation of Complement

Several complement components are proenzymes, which must be cleaved to form active enzymes. Activation of
the complement system can be initiated either by antigen–antibody complexes or by a variety of
nonimmunologic molecules

Sequential activation of complement components occurs via one of three pathways: the classic pathway, the
lectin pathway, and the alternative pathway (see below). Of these pathways, the lectin and the alternative
pathways are more important the first time we are infected by a microorganism because the antibody
required to trigger the classic pathway is not present. The lectin pathway and the alternative pathway are,
therefore, participants in the innate arm of the immune system.

All three pathways lead to the production of C3b, the central molecule of the complement cascade. The
presence of C3b on the surface of a microbe marks it as foreign and targets it for destruction. C3b has two
important functions: (1) It combines with other complement components to generate C5 convertase, the enzyme
that leads to the production of the membrane attack complex. (2) It opsonizes bacteria because phagocytes have
receptors for C3b on their surface.

1. In the classic pathway, antigen–antibody complexes1 activate C12 to form a protease, which cleaves C2
and C4 to form a C4b,2b complex. The latter is C3 convertase, which cleaves C3 molecules into two
fragments, C3a and C3b. C3a, an anaphylatoxin, is discussed below. C3b forms a complex with
C4b,2b, producing a new enzyme, C5 convertase (C4b,2b,3b), which cleaves C5 to form C5a and C5b.
C5a is an anaphylatoxin and a chemotactic factor (see below). C5b binds to C6 and C7 to form a
complex that interacts with C8 and C9 to produce the membrane attack complex (C5b,6,7,8,9), which
causes cytolysis. Note that the "b" fragment continues in the main pathway, whereas the "a" fragment is
split off and has other activities.

2. In the lectin pathway, mannan-binding lectin (MBL) (also known as mannose-binding protein) binds to
the surface of microbes bearing mannan (a polymer of the sugar, mannose). This activates proteases
associated with MBL that cleave C2 and C4 components of complement and activate the classic
pathway. Note that this process bypasses the antibody-requiring step and so is protective early in
infection before antibody is formed
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In the alternative pathway, many unrelated cell surface substances, e.g., bacterial lipopolysaccharides
(endotoxin), fungal cell walls, and viral envelopes, can initiate the process by binding C3(H2O) and factor B.
This complex is cleaved by a protease, factor D, to produce C3b,Bb. This acts as a C3 convertase to generate
more C3b.

11. T lymphocytes(development of the T cells). The T cell receptor for antigen

T cell precursors develop into T cells in the thymus. Contact with the thymic epithelium and hormones
such as thymosin, thymulin and thymopoietin II in the thymus promote extensive proliferation and
differentiation of the individual’s T cell population during fetal development. While T cell precursors are in
the thymus, genetic events similar to those for immunoglobulin generate numerous TCR’s, each expressed
on a different T cell clone. T cells that can not interact with MHC molecules do not grow, and those that
react with the host(self-reactive) are forced into committing suicide(apoptosis). The remaining T cells
differentiate into the subpopulations of T cells.

The TCR(T cell receptor) complex is a combination of the antigen recognition structure(TCR) cell-activation
machinery(CD3). The specificity of the TCR determines the antigenic response of the T cell. Each TCR
molecule is made up of 2 polypeptide chains, consisting of constant and variable regions. TCR’s are
estimated to be able to recognize 1015 separate epitopes.

The CD3 complex is found on all T cells and consists of gamma, delta and epsilon polypeptide chains. The
CD3 complex is the signal transduction unit for the TCR. Tyrosine protein kinases associate with the CD3
complex when antigen is bound to the TCR complex, promoting a cascade of protein phosphorylations,
activation of phospolipase C(PLC) and other events.

CD4 proteins associate with Class II MHC and CD8 with Class I MHC.

There are various accessory molecules on the T cell, which include several protein receptors on the cell
surface that interact with proteins on APC’s and target cells, leading to activation of the T cell, promoting
tighter interactions between the cells, or facilitating the killing of the target cell. Accessory molecules
include:-

CD45RA(naïve T cell) or CD45RO(memory T cell) that orchestrate transmembrane protein tyrosine

phosphatase(PTP)

CD28 which interacts with B7(co-stimulatory molecules)

CD40L which interacts with CD40 on DC(dendritic cells), macrophages and B cells(i.e. APC’s)

FasL which intiates apoptosis in a target cell that expresses Fas on its cell surface.

Adhesion molecules tighten the interaction of the T cell with the APC or target cell and may also promote
activation. Adhesion molecules include LFA-1, which interacts with the intercellular adhesion molecules
ICAM-1, ICAM-2 and ICAM-3 on the target cell. CD2 binds to LFA-3 on the target cell and promotes cell-to-
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cell adhesions and T cell activation. Very late antigens(VLA-4 and VLA-5) are expressed on activated cells to
enhance the interaction.

12. Functions of T lymphocyte subsets

The T helper cells(CD4) activate and control immune and inflammatory responses by specific cell-cell
interactions and by releasing cytokines(soluble messengers). Helper T cells interact with peptide antigens
presented on class II MHC molecules expressed on APC’s(dendritic cells, macrophages and B cells). The
array of cytokines secreted by a specific CD4 T cell in response to antigenic challenge defines the type of
CD4 T cell.

All 3 type of T cell, namely TH0, TH1 and TH3, produce GM-CSF, IL-3, INF-alpha and some chemokines. THO
cells specifically produce IL-2, IFN-gamma and IL-4. TH0 cells respond to antigen and can be converted to
either TH1 or TH2 cells, depending on the cytokines produced by the antigen-presenting cells.

TH1 cells promote inflammatory responses, which are especially important for controlling
intracellular(mycobacterial and viral) and fungal infections and promoting certain subtypes of IgG antibody
production.

TH2 cells promote antibody responses.

TH3 subtypes help promote production of IgA.

TH17 cells secrete IL-17 and IL-23 in response to bacterial infection to promote inflammation.

T-regulatory cells(Treg) express CD4 and CD25, prevent spurious activation of T cells and control the
immune response.

TH1 and TH2 responses are antagonistic and TH3 responses suppress TH1 and TH2 responses.

CD8 T cells are categorized as cytolytic and suppressor T cells, but can make cytokines similar to CD4 cells.
Activated CD8 T cells “patrol” the body for virus-infected or tumour cells, which are identified by antigenic
peptides presented by class I MHC molecules. Class I MHC molecules are found on all cells.

13. B lymphocytes(development and B cell surface markers)

B cells are derived from pluripotent stem cells. The stem cells reside primarily in the bone marrow, but can
also be isolated from the fetal blood in umbilical cords and as rare cells in adult blood. Specific cell surface
interactions with the marrow and specific cytokines lead to the appropriate differentiation. The “bursal
equivalent” of the thymus in the Peyer’s patches promotes the development of B lymphocytes. The bone
marrow, like the thymus, is considered a primary lymphoid organ. Peyer’s patches is part of the secondary
lymphoid tissue. The cells of the primary and secondary lymphoid organs express cell surface adhesion
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molecules(Adhesins) that inteact with cell surface adhesion molecules expressed on B cells. Differentiation
also occurs in the fetal liver and spleen.

A number of B cell surface markers exist. CR1 and CR2 markers allow B cells to be identified by the
presence of immuoglobulins, class II MHC molecules and receptors for the C3b and C3d products of the
complement cascade.

Activated B cells develop into either memory cells which express the CD45RO cell surface marker and
circulate until activated by specific antigen and plasma cells.

B cells naturally have IgD and IgM immunoglobulins on their cell surface which allow the classical complement
pathway to occur.

14. Collaboration between B and T lymphocytes

T-independent antigens cross-link sufficient numbers of surface antibody to stimulate growth of the
antigen-specific B cells. In contrast, production of antibody to T-dependent antigens requires receptor
interaction of the B cell, CD40L(T cell) and the action of cytokines.

Different combinations of cytokines produced by helper T cells induce class switching.

TH1-helper responses(Interferon-gamma) promote the production of IgG.

TH2-helper responses(IL4, IL-5, IL-6) promote production of IgG, IgE and IgA.

IgA production is especially promoted by IL-5 and transforming growth factor- beta(TGF-beta)(TH3).

Memory cells are developed with T-cell help. Terminal differentiation produces the ultimate antibody
factory, the plasma cell.

15. Antigen processing and presentation. Antigen presenting cell(APC)

Antigen presenting cells include dendritic cells, macrophages and B cells).

Activation of an antigen-specific T cell response requires a combination of cytokine and cell-cell receptor
interactions. Unlike cell surface immunoglobulin on the B cell, which senses the presence of soluble foreign
molecules floating past the cell, the TCR on the T cell must be presented with the relevant epitope, which
is cleared from the protein and cradled in a molecular holder on the surface of an APC, allowing the T cell
to “touch” and respond to it.
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Class I and II MHC molecules provide the molecular cradle for the peptide.

The CD8 molecule on cytolytic/suppressor T cells binds to and promotes the interaction with class I MHC
molecules on T cells.

The CD4 molecule on T Helper cells binds to and promotes interactions with class II MHC molecules on
APC’s.

The MHC molecules are encoded within the major histocompatibility complex(MHC) gene locus. The MHC
gene locus contains a cluster of genes important to the immune response.

Class I MHC molecules are found on all nucleated cells and are the major determinant of “self.” HLA
consists of 2 chains, a variable heavy chain and a light chain. Differences in the heavy chain of the HLA
molecule between individuals(allotypic differences) elicit the T cell response prevents graft(tissue)
transplantation. There are 3 major HLA genes: HLA-A, HLA-B and HLA-C.

The class II MHC molecules are encoded by the DP, DQ and DR loci. Class II MHC molecules present
antigens exogenously by APC’s to CD4-expressing T cells, whereas class I MHC molecules present antigen
endogenously to CD8 T cells.

Cross-presentation can occur with dendritic cells, whereby exogenous antigen is presented to CD8 T cells to
initiate anti-viral and anti-tumour responses.

16. Cytokines

Cytokines are a diverse group of non-antibody proteins that act as mediators between cells.

Cytokines can be grouped as:-

1. Monokines, which are produced by mononuclear phagocytic cells

2. Lymphokines, which are produced by activated lymphocytes, especially TH cells
3. Interleukins, which are mediators between lymphocytes
4. Chemokines, which are small cytokines responsible for lymphocytic migration

Cytokines can be grouped into different categories based on their functions or their origin.

Mediators of innate immunity include TNF-alpha, IL-1, IL-10, IL-12, Type I interferons, IFN-gamma and
chemokines.

TNF-alpha(tumour necrosis factor alpha) is produced by activated macrophages in response to microbes,

especially the LPS of gram-negative bacteria. It is an important mediator of inflammation by recruiting
neutrophils and macrophages to the site of infection. It also acts on the hypothalamus to produce fever
and it promotes the production of acute phase proteins.
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IL-1 is also an inflammatory cytokine produced by activated macrophages. It has similar effects to TNF-
alpha and it helps to activate T cells.

IL-10 is produced by activated macrophages and TH2 cells. It is predominantly an inhibitory cytokine.

IL-12 is produced by activated macrophages and dendritic cells. It stimulates the production of INF-gamma
and induces the differentiation of TH cells to become TH1 cells. It also enhances the cytolytic functions of
Tc and NK cells.

Type I interferons(IFN-alpha and IFN-beta) are produced by many cell types and they function to inhibit
viral replication in cells. They also enhance expression of type I MHC molecules making them more
susceptible to killing by CD8 T cells.

IFN-gamma is produced mainly by TH1 cells. It has numerous functions in both non-specific and cell-
mediated immunity.

Chemokines are chemotactic cytokines produced by many kinds of leukocytes and other cell types. Their
function is primarily to recruit lymphocytes to sites of infection.

Mediators of adaptive immunity include IL-2, IL-4, IL-5, TGF-beta, IL-10 and IFN-gamma.

IL-2 is produced by TH cells, although it can be produced by Tc cells to a lesser extent. It is the major
growth factor for T cells. It also promotes B cell growth and can activate NK cells and monocytes.

IL-4 is produced by macrophages and TH2 cells. It stimulates the growth of TH2 cells from naïve TH cells
and promotes the growth of differentiated TH2 cells resulting in the productions of an antibody response.
It also stimulates Ig class switching to IgE.

IL-5 is produced by TH2 cells and it functions to promote the growth and differentiation of B cells and
eosinophils.

TGF-beta is produced by T cells and many other cell types. It is primarily an inhibitory cytokine. For
example, it inhibits the proliferation of T cells and the activation of macrophages.

Interleukin 17 is a proinflammatory cytokine. Its over-expression has been noted in autoimmune diseases such
as rheumatoid arthritis.

17. Cell-mediated immune reactions

On ingestion of bacteria and stimulation of Toll-like receptors(TLR’s) by bacterial components, the

immature dendritic cell(iDC) matures to a dendritic cell(DC), ceases to phagocytose and moves to the
lymph nodes to process and deliver their internalized antigen for presentation to T cells. Antigenic
peptides(>11 amino acids) produced from phagocytosed proteins(exogenous route) are bound to class II
MHC molecules and presented by APC’s to naïve CD4 T cells.
 Page 14 of 25

The CD4 T cells are activated by a combination of:-

1. Antigenic peptide in the MHC II complex with the T cell receptor and CD4
2. Co-stimulatory signals provided by the interaction of CD28 molecule on the T cell with the B7 molecule
on the dendritic cell(DC)
3. IL-1, IL-12 and other cytokines produced by the DC cells.

In addition, IL-6 produced by the DC inhibits the production of suppressive cytokines(TGF-beta and IL-10)
by CD4+CD25+ regulatory T cells(Treg) tgo allow the activation of naïve T cells.

The TH0 cells produce IL-2, INF-gamma and IL-4.

Bacterial cells interact with B cells to produce IgM.

Complement also activates B cells to produce IgM.

Swollen lymph nodes are an indication of lymphocyte activation in response to antigenic challenge.

The conversion of TH0 cells to TH1 cells is promoted by IL-12 and re-inforced by IFN-gamma.

CD4 TH1 T cells:-

1. Promote and re-inforce inflammatory responses, e.g. IFN-gamma activation of macrophages and
growth of T and B cells(IL-2) to expand the immune response.
2. Promote B cells to produce complement-binding antibodies(IgM, IgG upon class-switching).

CD8 T cells are not especially important for anti-bacterial immunity.

CD4 TH2 cells responses occur in the absence of IL-12 at more distant lymph nodes. These responses are
also initiated by dendritic cells and later by B-cell presentation of antigen. Binding of antigen to the cell
surface antibody on B cells activates the B cells and also promotes uptake, processing of the antigen and
presentation of antigenic peptides on class II MHC molecules to the CD4 TH2 cell.

The TH2 cell produces IL-4, IL-5, IL-6, IL-10 and IL-13 which enhance IgG production and, depending on
other factors, the production of IgE or IgA.

The TH2 response also promotes terminal differentiation of B cells to plasma-cell factories. CD4+CD25+
regulatory T cells(Treg) curtail both TH1 and TH2 responses and promote the development of some of the
antigen-specific cells into memory T cells.

18. Immunotolerance(autotolerance, acquired immune tolerance.

Tolerance is specific immunologic unresponsiveness, i.e., an immune response to a certain antigen (or
epitope) does not occur, although the immune system is otherwise functioning normally. In general, antigens
that are present during embryonic life are considered "self" and do not stimulate an immunologic response, i.e.,
we are tolerant to those antigens. The lack of an immune response in the fetus is caused by the deletion of self-
 Page 15 of 25

reactive T-cell precursors in the thymus. On the other hand, antigens that are not present during the process of
maturation, i.e., that are encountered first when the body is immunologically mature, are considered "nonself"
and usually elicit an immunologic response. Although both B cells and T cells participate in tolerance, it is T-
cell tolerance that plays the primary role.

The main process by which T lymphocytes acquire the ability to distinguish self from nonself occurs in the fetal
thymus. This process, called clonal deletion involves the killing of T cells ("negative selection") that react
against antigens (primarily self MHC proteins) present in the fetus at that time. (Note that exogenous substances
injected into the fetus early in development are treated as self.) The self-reactive cells die by a process of
programmed cell death called apoptosis. Tolerance to self acquired within the thymus is called central
tolerance, whereas tolerance acquired outside the thymus is called peripheral tolerance.

For negative selection and clonal deletion to be efficient, the thymic epithelial cells must display a vast
repertoire of "self" proteins. A transcriptional regulator called the "autoimmune regulator" (AIRE) enhances the
synthesis of this array of self proteins. Mutations in the gene encoding the AIRE protein result in the
development of an autoimmune disease called autoimmune polyendocrinopathy. The AIRE transcription factor
also functions in the peripheral lymphoid organs such as the spleen and lymph nodes where it contributes to
peripheral tolerance.

Peripheral tolerance is necessary because some antigens are not expressed in the thymus and therefore some
self-reactive T cells are not killed in the thymus. There are several mechanisms involved in peripheral tolerance:
some self-reactive T cells are killed, some are not activated, and others are suppressed by regulatory T cells
producing inhibitory cytokines. Clonal anergy is the term used to describe self-reactive T cells that are not
activated because proper costimulation does not occur. Clonal ignorance refers to self-reactive T cells that
ignore self antigens. These self-reactive T cells are either kept ignorant by physical separation from the target
antigens, e.g., the blood–brain barrier, or ignore self antigens because the antigens are present in such small
amounts.

B cells also become tolerant to self by two mechanisms: (1) clonal deletion, probably while the B-cell
precursors are in the bone marrow and (2) clonal anergy of B cells in the periphery. However, tolerance in B
cells is less complete than in T cells, an observation supported by the finding that most autoimmune diseases are
mediated by antibodies.

Whether an antigen will induce tolerance rather than an immunologic response is largely determined by the
following:

1. The immunologic maturity of the host; e.g., neonatal animals are immunologically immature and do not
respond well to foreign antigens (for instance, neonates will accept allografts that would be rejected by
mature animals).

2. The structure and dose of the antigen, e.g., a very simple molecule induces tolerance more readily than
a complex one, and very high or very low doses of antigen may result in tolerance instead of an immune
response. Purified polysaccharides or amino acid copolymers injected in very large doses result in
"immune paralysis"—a lack of response.
 Page 16 of 25

Other aspects of the induction or maintenance of tolerance are as follows:

1. T cells become tolerant more readily and remain tolerant longer than B cells.

2. Administration of a cross-reacting antigen tends to terminate tolerance.

3. Administration of immunosuppressive drugs enhances tolerance, e.g., in patients who have received
organ transplants.

4. Tolerance is maintained best if the antigen to which the immune system is tolerant continues to be
present.

The adult host usually exhibits tolerance to tissue antigens present during fetal life that are recognized as
"self." However, in certain circumstances tolerance may be lost and immune reactions to host antigens
may develop, resulting in autoimmune diseases. The most important step in the production of
autoimmune disease is the activation of self-reactive helper (CD4) T cells. These self-reactive Th-1 or
Th-2 cells can induce either cell-mediated or antibody-mediated autoimmune reactions, respectively.
Most autoimmune diseases are antibody-mediated.

Examples of autoimmune diseases include Myasthenia gravis and Graves’ disease in which antibodies are
produced against receptors. Rheumatoid arthritis represents an autoimmune disease in which antibodies are
produced against cell components other than receptors, namely IgG in joints.

19. Type I – immediate hypersensitivity reactions

Type I is early type of hypersensitivity which happens after antigen reacts with IgE present on surface of
basophils and mast cells which release anaphylactic mediators. We know 2 types of this reaction, namely
anaphylactic shock and atopy.

Anaphylactic shock is a generalized hypersensitivity reaction which can happen to all people and it is the most
serious condition in medicine. Agents that can cause it include medicines, serum proteins, insect stings etc. At
first, introduction of antigen into the body starts the process of sensibilization meaning production of IgE,
which binds to mast cells and basophils. When the same antigen is introduced again after a while, it binds to
the Fab segment of IgE, which is bound to mast cells and basophils. Afterwards mast cells and basophils
undergo a degranulation process causing a release of histamine, which acts as a vasodilator and
bronchoconstrictor. There are also some other mediators.

Symptoms appear after 20-30 minutes resulting in a blood pressure drop, oedema, smooth muscle
constrictions caused by bradykinin and compromised coagulation due to heparin release, vomiting, diarrhea
and collapse.

Treatment is applying anti-histamine drugs, adrenaline and calcium.

 Page 17 of 25

Atopy is a non-systemic anaphylactic reaction mediated as well by IL-5 and IL-4, but the mechanism is the
same as in generalized anaphylactic shock. It appears usually on nasal mucosa, respiratory tract, skin, or the
gastrointestinal tract. It can be also caused by high levels of IgE due to T suppressor cell deficiency. Allergens
are usually pollen, grass, dust, some types of food or animal hair.

20. Type II – cytotoxic reactions

Mediators in these reactions are IgG and IgM on the cell surface as integral parts. There are 3 ways to achieve
cytotoxic effect. The first way is by opsonization and phagocytosis with macrophages, meaning that the target
cell possesses antigen which binds to antibodies being produced against this antigen. The fab fragment of
antibodies binds to antigen and the Fc fragment is free for binding to phagocytes. The second way is by
activation of the complement system meaning that antibodies IgG and IgM activate the complemeny system,
which results in the production of C5b6789 main phospolipase which lyses the target cell. The third method is
destruction of the target cell with NK cells, contact mediated by antibodies. NK cell possesses FCR receptor for
IgG which is bound to antigen, therefore NK cells cannot recognize each specific antigen, but they can react
with Fc fragments of antibody, or C3 of the complement system bound to antigen.

Manifestations of cytotoxic reactions are foetal erythroblastosis, myasthenia gravis caused by production of
antibodies against acetylcholine receptors, graves disease with antibodies against TSH and different anaemia
with antibodies against red blood cells.

21. Type III – immune-complex mediated reactions

These reactions are a consequence of antigen-antibody complexes, which precipitate and cause damage to
tissues, where there is high blood pressure, or slow blood flow like in the glomerulus and choroidal plexus.
This is also an early type of reaction. There are 2 manifestations. The first one is Arthus reaction and the
second reaction is known as serum disease.

Arthus reaction is caused by antigen-antibody complex precipitants where antibodies are in higher
concentration. Precipitation happens in blood vessels causing complement activation, vasoactive amines
release and infiltration of polymorphonuclear lymphocytes which bind to the Fc fragment of the antibody and
the C3 complex of complement. Now phagocytosis occurs. Phagocytosis is accompanied with different tissue
active substance releases from phagocytes and the number of eosinophiles and mononuclear cells increases at
the site.

Serum disease is also caused by antigen-antibody complex, but here antigen is in greater concentration.
Usually, it happens after heterologic horse serum application(vaccination against clostridium tetani) followed
by IgG and IgM production. IgG and IgM form precipitants with heterologic serum if it is not excreted quickly
enough precipitants being trapped in blood vessels. These complexes are phagocytosed and cause activation
of the classical complement pathway.

Manifestations starts from 7-14 days after heterologic serum application with increase of temperature, joint
pain, proteinuria, lymph node enlargement and general kidney damage. The scale of damage depends on the
dosage of antigen.
 Page 18 of 25

22.Typle IV- Cell mediated-delayed type of reactions

It is based on cellular mechanism mediated by by T-lymphocytes. It is normal immune response mediated by

T-lymphocytes but in this case Ag is not harmfull. It starts by processing Ag in APC and its presentation to T
cells which secrete cytokines activating macrophages releasing imflamatory mediatiors. This type of reaction
appears only when same Ag is appllyed to organism,manifesting after 24h whit inflammation at application
site. Delayed type of reaction can be passively transfered from one organism to other whit sensibilised T cells
but not whit serum containing Ab. In case of congenital aplasia of tymus and followed deffiency of T cells this
reaction cannot occur.

Examples of this reactions are tuberculine reaction where PPD(purified protein derivate) is dermally applied
and 72h later local inflammation appears due to infiltrations of mononuclear WBC, contac dermatitis caused
by some chemicals diffusing trought skin and causing sensibilisiation of T cells.

23. Tumour associated antigens. Immune response to tumour antigens

 In order for the immune system to react against a tumor, the latter must have antigens that are
recognized as foreign.

 Alterations in gene expression occur in tumourigenesis.

 Tumourigenesis may lead to expression of new antigens(Neoantigens) or alterations in existing

antigens found on normal cells.

 Antigens - membrane receptors, regulators of cell cycle and apoptosis + molecules involved in signal
transduction pathways.

 2 main types of tumour antigens:-

 1. Tumour-specific transplantation antigens(TSTA) – Unique to tumour cells & not expressed on

normal cells. Responsible for rejection of tumour

 2. Tumour associated transplantation antigens(TATA) – Expressed by both tumour and normal cells

 The majority of tumor antigens are also present on normal cells and are referred to as tumor
associated transplantation antigens.

 May be expressed at higher levels on tumour cells compared to normal cells.

 May be expressed only during development of cells and lost during adult life but re-expressed in
tumors.
 Page 19 of 25

 Tumour-associated developmental antigens or onco-fetal antigens include Alpha-fetoprotein(AFP) and

Carcino-embryonic antigen(CEA)

 Both found secreted in the serum

 AFP is found in patients with hepatocellular carcinoma

 CEA is found in colon cancer

 Both are important for diagnosis

 AFP is produced only as a secreted protein whereas CEA is found both on cell membranes and in
secreted fluids.

 The normal range of AFP concentrations in humans is 0-20 ng/ml. This level rises considerably in
patients with hepatomas and non-seminal testicular carcinoma. A 5-fold or higher rise in this protein is
used for monitoring hepatomas and testicular cancers. AFP level may also be raised in some non-
malignant conditions, such as cirrhosis, in hepatitis and other forms of liver damage.

CEA levels in normal people range up to 2.5 ng/ml, but they increase significantly in certain malignancies,
particularly colo-rectal cancers. They may also rise in some non-malignant conditions (such as chronic
cirrhosis, pulmonary emphysema and heavy smoking). Levels that are 4 to 5 times normal have been used to
predict recurrence of colo-rectal tumors.

24. Immunological factors favouring tumour growth. Mechanisms of tumour rejection

 According to immune surveillance theory, cancer cells that arise in the body are eliminated by the
immune system.

 Due to impaired immune reactivity, tumour cells may escape destruction.

 Tumour cells evade immune recognition by several mechanisms:-

 1. Tumors may not express neo-antigens that are immunogenic or they may fail to express co-
stimulatory molecules required for the activation of T cells.

 2. Certain tumours are known to lack or be poor expressers of MHC antigen.

 3. In the early development of a tumor, the amount of antigen may be too small to stimulate the
immune system (low dose tolerance) or, due to the rapid proliferation of malignant cells (high dose
tolerance), the immune system is quickly overwhelmed.

 4. Some tumors may evade the immune system by secreting immunosuppressive molecules and others
may induce regulatory cells particularly the CD4+CD25+ FoxP3+ T regulatory cells.

 5. Some tumors may shed their antigens which in turn may interact and block antibodies and T cells
from reacting with the tumor cells.
 Page 20 of 25

 Tumour rejection is initiated by cell-mediated reactions which attack these "nonself" tumor cells and
limit their proliferation. Such immune responses probably act as a surveillance system to detect and
eliminate newly arising clones of neoplastic cells. In general, the immune response against tumor cells is
weak and can be overcome experimentally by a large dose of tumor cells. Some tumor cells can escape
surveillance by "modulation," i.e., internalizing the surface antigen so that it no longer presents a target
for immune attack.

 The cell-mediated immune responses that affect tumor cells in vitro include natural killer (NK) cells,
which act without antibody, killer (K) cells, which mediate antibody-dependent cytolysis (antibody-
dependent cellular cytotoxicity), cytotoxic T cells, and activated macrophages. Whether these immune
responses function to prevent or control tumors in vivo is unknown.

 Tumor antigens can stimulate the development of specific antibodies as well. Some of these antibodies
are cytotoxic, but others, called blocking antibodies, enhance tumor growth, perhaps by blocking
recognition of tumor antigens by the host. Spontaneously arising human tumors may have new cell
surface antigens against which the host develops both cytotoxic antibodies and cell-mediated immune
responses.

25.Agglutination test

This test uses the agglutination reaction meaning Ag-Ab reaction where Ag’s are particles bound together
whit Ab’s, forming precipitins. Reaction becomes visible only when optimal proportion between Ab s and Ag s
is achieved and visible precipitin can be seen. Agglutination test is used for confirming presence of Ag or Ab.
There are direct and indirect agglutination tests.

Direct agglutination is preformed when epitope is integral part of test Ag, namely when we use corpuscular Ag
testing for Ab presence or when we test sample for Ag presence adding Ab. Agglutination occurs as
consequence of reaction and binding Ab whit Ag .Ig Ab s have different abilities to cause agglutination,
depending on valency number whit IgM as the stronges due to possession of 10 valent spots. Test Ag used for
reaction are most often suspensions of MO,RBC and synthetic latex particles artificially covered whit Ag.
Rection is preformed in tube, glass and plastic plates for tipisation of blood groups, MO identification etc.

Indirect or passive agglutination is when on inert particle like latex naturally without epitope soluble Ag is
bound and artificial particle achieves state of corpuscular Ag capable of reacting. Ag bound in that case can be
Ig s which react whit Anti Ig Ab causing agglutination. When ladding RBC whit purified Ab we can prove specific
Ag in test sample. Ladding techniques include passive adsorption or chemical binding. Indirect test whit RBC is
used to detect Ab against ricketias and latex test for reaumathoid factor, etc.

26.Complement fixation test

The complement fixation test is an immunological medical test that can be used to detect the presence of
either specific antibody or specific antigen in a patient's serum. It was widely used to diagnose infections,
particularly with microbes that are not easily detected by culture methods, and in rheumatic diseases.
 Page 21 of 25

The complement fixation test is based on complement fixation reaction which is a system of serum proteins
that react with Ag-Ab complexes. If this reaction occurs on a cell surface, it will result in the formation of trans-
membrane pores and therefore destruction of the cell. The basic steps of a complement fixation test are as
follows:[1]

1. Serum is isolated from the patient.

2. Patients naturally have different levels of complement proteins in their serum. To negate any effects
this might have on the test, the complement proteins in the patient's serum must be destroyed and
replaced by a known amount of standardized complement proteins.
1. The serum is heated in such a way that all of the complement proteins—but none of the
antibodies—within it are destroyed. (This is possible because complement proteins are much
more susceptible to destruction by heat than antibodies.)
2. A known amount of standard complement proteins are added to the serum. (These proteins are
frequently obtained from guinea pig serum.)
3. The antigen of interest is added to the serum.
4. Sheep red blood cells (sRBCs) [2] which have been pre-bound to anti-sRBC antibodies are added to the
serum. The test is considered negative if the solution turns pink at this point and positive otherwise.

If the patient's serum contains antibodies against the antigen of interest, they will bind to the antigen in step 3
to form antigen-antibody complexes. The complement proteins will react with these complexes and be
depleted. Thus when the sRBC-antibody complexes are added in step 4, there will be no complement left in
the serum. However, if no antibodies against the antigen of interest are present, the complement will not be
depleted and it will react with the sRBC-antibody complexes added in step 4, lysing the sRBCs and spilling their
contents into the solution, thereby turning the solution pink.

27.ELISA and RIA

ELISA (enzyme linked immunoabsorbant assay) is a method relying on enzymatic action changing substrate.
Reaction has 2 stages, first immunological meaning Ag binding to Ab which is not visible and second chemical
stage where enzyme acts on substrate resulting in color change determined by spectroscopy. Proving Ag s
whit ELISA is preformed by direct, inhibitional or competitive ELISA.

In direct method known Ab is boud to plate on which tested sample is later applied and in the case of Ag
presence it binds on Ab on the plate. Than we put again Ab on which enzyme is present and they bind to Ag s
on the plate, substrate is added and change of color means positive result.

Proving presence of Ab is done by direct competitive ELISA method in which known Ag s are bound to plate,
tested serum is added whit suspected Ab presence which Ab s bind to Ag on the plate. Than suspected Ab s
on which enzyme is bound are added again and in case of Ab presence in tested serum no free places on plate
are left free for binding whit Ab carrying enzyme, plate is washed and supstrate added. Absence of color
means positive result.
 Page 22 of 25

Disadvantage of ELISA is sometimes appearing false positive result. Enzymes most often used are peroxidase
and alkaline phosphatase.

RIA or radioimmunoassay s a very sensitive technique used to measure concentrations of Ag like hormone
levels in the blood etc., by use of Ab. To perform a radioimmunoassay, a known quantity of an Ag is made
radioactive, frequently by labeling it with gamma-radioactive isotopes. This radiolabeled Ag is then mixed with
a known amount of Ab for that Ag, and as a result, the two chemically bind to one another. Then, a sample of
serum from a patient containing an unknown quantity of that same Ag is added. This causes the unlabeled
-"cold"Ag from the serum to compete with the radiolabeled Ag-"hot" for Ab binding sites. As the
concentration of "cold" Ag is increased, more of it binds to the Ab, displacing the radiolabeled variant, and
reducing the ratio of Ab-bound radiolabeled Ag to free radiolabeled Ag. The bound Ag are then separated
from the unbound ones, and the radioactivity of the free Ag remaining in the supernatant is measured using a
gamma counter, allowing us to determent exact concentration of Ag in patients.

28. Transplantation immunity

We have auto-transplantation, which is when the tissue is being transplanted from one place of an organism
to another place on the same organism. This is an autograft. There are no immunological problems.

Iso-transplantation is when the transplantation occurs from one place of an organism to another organism
that is mono-zygotic twins. There are also no immunological problems faced with this type of transplantation.

Allogenic transplantation is the transplantation between two different organisms from the same species, but
they are not genetically identical.

Xeno-transplantations is the transplantation between different species. Evidence that transplantation reaction
is immunological reaction:-

1. It has property of immunological memory

2. It is generalized
3. It is depends on the state of the immunological system and immunological competency of the
organism
4. It depends on the gene product of the HLA gene

Rejection reaction can not occur in auto-transplantation. Molecules that are recognized by the host of the
graft as foreign molecules leads to rejection reaction are called tissue specific antigens, which are coded on
the genes in different places of the genome of organisms. Meanwhile, major histocompatibility complex is one
of which products cause the most powerful rejection of grafts for unrelated organisms.

This complex of a human being is called HLA(Human leukocyte antigen).

2 classes of these HLA genes have relevant products for the immune response, named HLA-1 and HLA-2.
 Page 23 of 25

The major function of MHC product is to present antigenic peptides to T lymphocytes. Products of this highly
polymorphic complex express on graft cells are recognized by allo-reactive lymphocytes.

The process of transplantation rejection begins when CD4 T lymphocytes of a host recognize allogenic
products of HLA-2 class on antigen presenting cells(APC’s) on a graft, which is called direct presentation.

The other way is when foreign antigens are captured and presented by APC’s of a host, which is called indirect
presentation.

The most important APC’s causing rejection reaction are dendritic cells found in grafts, or in organ.

When talking about people, even endothelial cells of a graft can activate allo-reactive CD4+ T lymphocytes.
Activated CD4+ stimulate proliferation and differentiation of allo-reactive CD8+ lymphocytes.

Effector mechanisms which are used by immune system for graft rejection are:-

1. Allo-reactive T lymphocytes produce cytokines attracting macrophages which damage transplantated

tissue
2. Allo-reactive cytotoxic lymphocytes directly kill endothelial cells of the graft
3. Allo-antibodies which specifically react with antigens of endothelial cells of graft causing activation of
complement

29. Host-versus-graft responses cause transplant rejection

Allogenic transplantation is the transplantation between two different organisms from the same species, but
they are not genetically identical.

Rejection reaction can not occur in auto-transplantation. Molecules that are recognized by the host of the
graft as foreign molecules leads to rejection reaction are called tissue specific antigens, which are coded on
the genes in different places of the genome of organisms. Meanwhile, major histocompatibility complex is one
of which products cause the most powerful rejection of grafts for unrelated organisms.

This complex of a human being is called HLA(Human leukocyte antigen).

2 classes of these HLA genes have relevant products for the immune response, named HLA-1 and HLA-2.

The major function of MHC product is to present antigenic peptides to T lymphocytes. Products of this highly
polymorphic complex express on graft cells are recognized by allo-reactive lymphocytes.

The process of transplantation rejection begins when CD4 T lymphocytes of a host recognize allogenic
products of HLA-2 class on antigen presenting cells(APC’s) on a graft, which is called direct presentation.

The other way is when foreign antigens are captured and presented by APC’s of a host, which is called indirect
presentation.
 Page 24 of 25

The most important APC’s causing rejection reaction are dendritic cells found in grafts, or in organ.

When talking about people, even endothelial cells of a graft can activate allo-reactive CD4+ T lymphocytes.
Activated CD4+ stimulate proliferation and differentiation of allo-reactive CD8+ lymphocytes.

Effector mechanisms which are used by immune system for graft rejection are:-

1. Allo-reactive T lymphocytes produce cytokines attracting macrophages which damage transplantated

tissue
2. Allo-reactive cytotoxic lymphocytes directly kill endothelial cells of the graft

Allo-antibodies which specifically react with antigens of endothelial cells of graft causing activation of
complement

30. The tempo of transplantation rejection(hyper-acute rejection, acute rejection, chronic rejection)

Clinical manifestations and mechanisms of transplant rejection are a consequence of activation of Tc CD8+ and
TH CD4+ cells which both activate complement, macrophages and B lymphocytes, which leads to transplant
rejection.

Rejection can be:-

1. Hyper-acute, which is characterized by an extreme fast rejection in minutes or hours caused by

occlusion of blood vessels, which causes necrosis because of lack of blood. This type of rejection is
caused mostly because of free sensibilised host and incompatibility of blood groups(ABO or HR
system).
2. Acute rejection is manifested in 2 forms. (a) Vascular form being very similar to hyper-acute, but it has
lower intensity and speed and it is a consequence of antibody production against HLA molecules of
graft endothelial cells which then activate complement leading to necrosis. (b) Cellular type when Tc
and TH lymphocytes are activated.

Chronic rejection, which is caused as a result of acute rejection when fibrosis of tissue occurs.

31.Prevention of transplantation reaction

There are 2 approaches used in clinics, first one being suppression acceptor s immune response and second
being lowering graft s immunogenicity. First approach is achieved by immunsupresive therapy which
nonspecifically inhibits unwanted immune system using medicines like cyclosporine A blocking activation of T
 Page 25 of 25

cells. Second approach namely lowering graf s immunogenicity meaning basically finding out
histocompatibility Ag of a donor and acceptor by checking compatibility blood groups and MHC molecules.

Even beside that all protective measures tissues of 2 different organism are never 100% compatible.

Another approach is the adoption of Monoclonal antibodies in immunosuppressive regimens, both to prevent
rejection and to treat rejection episodes. Muromonab (OKT3) is a monoclonal antibody against CD3, and
basiliximab and daclizumab are monoclonal antibodies against the IL-2 receptor.