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PHAR 753PHARMACOLOGY AND MEDICINAL CHEMISTRY SERIES

Immunology AndInflammation

Winter 2007

Reading Materials:1. Goodman&Gilman’s The Pharmacological Basis of Therapeutics, Tenth Ed.

Chapter 53, pp. 1463-14802. Wilson and Gisvold’s Textbook of Organic Medicinal and Pharmaceutical

Chemistry, 10th Ed.3. Wei-Chiang Shen and Stan G Louie’s Immunology for Pharmacy Students

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PHAR 753

LEARNING OBJECTIVES

Be able to differentiate between innate and acquired immune responses

Be able to classify the cells of the immune system into their main groupings and theirsubgroups. Know their characteristics and functions in the immune response.

Know which organs contribute to the immune response and how they do it.

Be able to define antigen and antigen presenting cell

Be able to differentiate the five main groups of immunoglobulins: their structures, theirfunctions, their main location in tissues, the genetics of their formation

Be able to define major histocompatibility complex and know they roles in the immuneresponse

Be able to define complement molecules and know their synthetic pathways and theyroles in the immune response

Know how immune system responds to infection and how the pathogens escape theimmune response

Know examples of immunodeficiency diseases and their causes

Be able to explain the process of cell migration and inflammation

Be able to differentiate types of hypersensitivity reactions and their biologicalconsequences

Be able to differentiate types of organ transplants and their potential rejection reactions

Know methods of suppressing the immune system and be able to explain themechanism of action and other aspects of immunosuppressive drugs covered in class

Know types and names of preparations that target or affect the immune system, theirmechanism of actions, their specific indications, their side effects, and other aspectscovered in class

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INNATE AND ACQUIRED (ADAPTIVE) IMMUNITIES

The immune system involves a vast network of cells and molecules with a commongoal: to distinguish between self and nonself and destroy the nonself.

INNATE IMMUNITY-Fast in response-Nonspecific-Lacking in memory of the challenge

ACQUIRED (ADAPTIVE) IMMUNITY-Slow in response-Highly specific-Has memory of previous infections

Innate Immunity

Physical defenses Cellular defensesChemical and biochemical defenses

skin

mucous membranes

tears

urine

sneezing

granulocytes

monocytes

macrophages

spermine in sperm

acid in stomach

lysozyme in most secretions

cilia lining trachea

sebaceous gland secretions

commensal organisms in gut and vagina

Examples of the physical, chemical, and biochemical defensesIntact skin is impenetrable to most bacteria, and fatty acids produced by the skin aretoxic to many organisms.Epithelial surface of trachea is cleansed by ciliary actionEpithelial surface of urinary track is cleansed by flushing action of urinepH changes in the stomach and vagina (both are acidic) can destroy many bacteria.Commensal bacteria in the vagina can metabolize glycogen secreted by the epitheliumto produce lactic acid.Commensal bacteria occupy a particular ecological niche, and prevent pathogens fromgaining access to it.

ACQUIRED (ADAPTIVE) IMMUNITY-Antibody-mediated immunity-Cell-mediated immunity

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CELLS OF THE IMMUNE SYSTEM

Myeloid Lineage (producing phagocytes and other cells)

Lymphoid Lineage (producing lymphocytes)

Pluripotent Stem Cell

Myeloid

Lymphoid

New Stem Cell

Monocytes Macrophages

Granulocytes

Neutrophils

Eosinophils

Basophils

erythrocytes and platelets

B-lymphocytes

T-lymphocytes

Natural Killer Cells

Natural Killer T-Cells

Mast cells

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Myeloid Lineage

Polymorphonuclear Granulocytes (neutrophils, eosinophils, basophils)They are called polymorphonuclear granulocytes because they contain polymorphicmultilobed nucleus and many cytoplasmic granules. They are produced in the bonemarrow at a rate of 80 million cells per minute. The granules are usually stored inlysosomal pouches and consist of hydrolytic enzymes, oxidative enzymes, ironchelating agents, histamine, leukotrienes, and other chemicals. Granulocytes are fullydifferentiated, short-lived, and end stage cells. So, they are mature cells that do notreproduce.

Phagocytes (neutrophils, eosinophils, monocytes/macrophages)They are called phagocytes because they engulf foreign particles and form intracellularvacuole bodies called phagosomes. Phagosomes fuse with lysosomes to formphagolysosomes and chemoenzymatically kill or destroy the engulfed pathogens.

Monocytes- have phagocytic capabilities- when activated release cytokines- half-life of monocytes in bloodstream is about 10 h- migrate into tissues and differentiated into macrophages- regulate and program the immune response

Macrophages- terminally differentiated monocytes- have phagocytic capabilities- when activated release cytokines (IL-1 and TNF)- kill more slowly than neutrophils but have a much broader spectrum- act as antigen presenting cells (APCs)- develop in the tissues to have specialized functions (Examples: Kupffer’s

cells in liver, alveolar cells in lung, spleen cells, peritoneal cells, microglial cells inCNS, osteoclast cells in bone)

Neutrophils- primary innate defense against pathogenic bacteria- make up 50-75% of the leukocytes- have multilobed nuclei- respond to chemical motility factors, such as complement mediators released

from infected or inflamed tissues- phagocytose invading microbes- contain lysosomes and phagosomes

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Eosinophils- can function as phagocytes- make up 2-4% of blood leukocytes- have bilobed nuclei- contain histamine and leukotrienes- associated with the allergic response- have diagnostic role in parasitic infections and in allergies- unlike neutrophils, need not phagocytose a parasite to kill it- release oxidative substances

Basophils (categorized as auxiliary cells)- basophils present at only 0.2% of the leukocytes (basophils are circulating cells)- have multilobed nuclei- release the inflammatory mediators associated with allergy- have high-affinity immunoglobulin E (IgE) receptors

Mast Cells (also auxiliary cells)- release the inflammatory mediators associated with allergy- prevalent in the skin, lungs, and nasal mucosa- have high-affinity immunoglobulin E (IgE) receptors- mast cells lie close to the blood vessels in all tissues

Platelets (auxiliary cell)- derived from megakaryocytes in the bone marrow- contain granules- involved in blood clotting and the immune response (especially in inflammation)- express class I MHC molecules; receptor for IgG and low affinity receptor for IgE

Innate immune response distinguishes self and non-self through pattern-basedrecognition of evolutionarily conserved structures of foreign entities.Pattern recognition molecules that are expressed by many different cell types, e.g.,macrophages, neutrophils, and NK cells, and present as surface receptors, are calledpattern recognition receptors (PRRs). The structures recognized by PRRs includepathogen cell wall components, bacterial and viral DNA, viral dsRNA, bacterial proteins,microbial lipoproteins, etc.PRRs include CD14 co-receptor, Toll-like receptors (TLRs), and scavenger receptors,etc.

Analysis of the various TLR signaling paths showed that they all use theMyD88/IRAK/IκB/NK-κB pathway to induce new gene transcription.

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Lymphoid Lineage

B CellsB lymphocytes are produced and mature in the bone marrow. They represent about 5-15% of the circulating lymphoid pool. They carry a number of markers including somesurface immunoglobulin markers (mostly IgM and IgD).

Bursa of Fabricius: A lymphoepithelial organ found at the junction of the hindgut andcloaca in birds, which is the site of B cell maturation.

Plasma B cellFully differenciated and nonreproducing B cells. It is highly specialized and programmedto make only one clone of antibody. Its function is to synthesize, store and releaseenormous amounts of immunoglobulins.

Memory B cellAlso called Resting or Primed B cell. It responds rapidly to the same antigen for which itwas originally programmed, to produce a clone of antibody secreting B cells.

B cell clonal selection

B cells 1 2 n

2 2

2 2 2 2

2 2

plasma cells memory cells

2 2 2 2 2 2

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T CellsT lymphocytes are produced in the bone marrow but mature in the thymus. Contain adefinitive marker: T-cell antigen receptor (TCR). There are two defined types of TCR:TCRαβ is a heterodimer of two disulphide-linked polypeptides (α and β ); TCRγδconsists of γ and δ polypeptides. Both receptors interact with a set of five polypeptides,the CD3 complex, to form the T-cell receptor complex (TCR-CD3 complex). About 85-95% of the T-cells express TCRαβ and about 5-15% express TCRγδ.

*CD (Cluster Determinant): cell surface molecules of lymphocytes or myeloid cells that aredistinguishable with monoclonal antibodies and may be used to differentiate different cellpopulation. T cells have three main types of cluster determinants; CD3, CD4 and CD8. The CD3complexes with T cell receptor (TCR) as part of the transducer process for transferring a signalinto the T cell after antigen binding to the T cell receptor.

TCRγδ T cells are mostly found in mucosal epithelia representing the majority of intra-epithelial lymphocytes. TCRγδ T cells that reside in the epithelia express the CD8marker, but those in the circulation do not (CD4- CD8-).They may play an important role in protecting the mucosal surfaces from bacterialinfections (cytotoxic).

TCRαβ T cells usually express either CD4 or CD8 markers, which determines whetherthey recognize antigen in association with major histocompatibility complex (MHC)*class I or class II molecules. But, a small subset double negative (CD4- CD8-) TCRαβ Tcells has also been described.

TCRγδ

CD4-CD8-

CD8+

TCRαβ

CD4+ CD8+

cytotoxic cells

MHC class IIrestricted

(mostly TH cells)

MHC class Irestricted

(mostly Tc cells)

Major T cells subsets

<10% humanblood T cells>90% human blood T cells

TCRγδ TCRγδTCRαβ TCRαβ

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The subset that carries CD4 is mainly responsible for ‘helping’ the induction of immuneresponse, and known as T-Helper (TH). They recognize their specific antigens inassociation with MHC class II molecules.

*Major Histocompatibility Complex (MHC) is referred to a cluster of genes (genetic loci) thatdetermines the histocompatibility antigens of cell membranes. The products, called MHCmolecules, are presented on the surface of cells. Foreign or non-self cells or tissue or aninfected cell express different MHC antigens, which allows the body’s immune system todifferentiate foreign antigens from the body’s own cells.

Functionally, T-helper has been divided into two groups:TH1 cells, secrete IL-2, IL-12 and IFNγ, mediate several functions associated withcytotoxicity and local inflammatory reactions, particularly play a significant role incombating intracellular pathogens (virus, bacteria, parasites).

TH2 cells, secrete IL-4, IL-5, and IL-6, which stimulate B cells to proliferate and produceantibodies (humoral immunity).

The subset that carries CD8 is mainly cytotoxic, known as cytotoxic T cells (Tc). Theyrecognize their antigens in association with MHC class I molecules.

APC

CD4+

T cellCD8+

T cell

virus-infected cell

TCRCD4 CD8TCR

MHC class IIwith processed

antigen

MHC class Iwith processed

antigen

T suppressor (Ts) cellsThese cells dampen the immune response of the B and T cells after the antigen isremoved.

T memory cellsT cells that contain the information that recognizes specific antigen.

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Natural Killer (NK) cellsNK cells are produced and mature in the bone marrow. They represent about 15% ofblood lymphocytes and are known as lymphocytes having no conventional surfaceantigen receptors (TCR or Immunoglobulin). The presence of CD56 and/or CD16,and the absent of CD3 is currently used as a definitive marker for NK cells in human.Since they have an IL-2 receptor, they can be activated by IL-2. NK cells kill certaintumor cells, virus-infected cells, as well as IgG coated objects.

Natural Killer T-CellsPhenotypically similar to NK cells. They combine several features of both T lymphocytesand NK cells. NKT cells carry a TCR, but unlike T cells, the TCR repertoire is much lessdiverse. Upon activation, NKT cells quickly secrete cytokines that can help theactivation and differentiation of B and T cells. Like NK cells, they may also carry outcytolysis. The quick response and the limited range of antigen recognition of NKT cellsmark them as cells contributing primarily to the innate immune response.

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oligosaccharide conjugate

triggering receptor

+

self MHC class I

inhibitoryreceptor

–

self peptide

oligosaccharide conjugate

triggering receptor

+

foreign MHC class I with foreign peptide

NO SIGNAL

oligosaccharide conjugate

triggering receptor

+

no MCH class Imolecule

NO SIGNAL

oligosaccharide conjugate

triggering receptor

+

self MHC class I

viral peptide

NO SIGNAL

normal cell

foreign cell

mutant cell

virus-infectedcell

NKcell

NKcell

NKcell

NKcell

not killed

killed

killed

killed

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The Lymphoid System

Primary Lymphoid Tissues-Bone Marrow (site of maturation of B cells)

-Thymus (site of maturation of T cells)Located in thorax, consists of two lateral lobes (bilobed). Each lateral lobe is composedof many lobules. The primary lobules are made of a number of small follicles or nodules.Within each follicle the lymphoid cells (thymocytes) are arranged into a medulla (inner)and a cortex (outer).

Cortex:-mostly contains lymphocytes (immature T-cells)-supported by a network of branched cortical epithelial cells (Thymic origin)-positive selections and a limited degree of negative selections occur in cortex-T cell receptor gene rearrangements-dense of lymphocytes (thymocytes).

Medulla:-less dense than cortex-negative selections (to remove autoimmune T cells) occur in medulla-T-cell maturation-The gene AIRE (Autoimmune Regulator) is expressed in medulla

Thymus involutes with age. Atrophy begins at puberty and continues through life.Cortical thymocytes are sensitive to corticosteroids. Acute increase in steroids(pregnancy, stress, drugs) promotes thymic atrophy.

Secondary Lymphoid Organs or Tissuesa) Spleen (responds predominantly to blood-borne antigens)b) Lymph nodes (respond to antigens circulating in the lymph)c) Tonsilsd) Adenoidse) Peyer’s patches (in the intestine)f) Lamina propriag) Bronchus-associated lymphoid tissueh) Urogenital lymphoid tissuei) Bone marrow (also contained mature T-cells and other plasma cells)

c – h respond to antigens which have penetrated the mucosal barriers.

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ANTIGENS

Antigen: any molecule that can be specifically recognized by B cells or T cells or bothresulted in the production of antibodies or the disruption of cells.

Each antibody recognizes and binds to a particular part of the antigen called anantigenic determinant or epitope. A single antigen can have several different epitopes orrepeated epitopes.

T-cells also recognize antigens, but only antigens that originated from within cells (smallpolypeptide fragments which are presented on the surface of other host cells).

ANTIGEN PRESENTING CELLS (APCs): A variety of cell types that carry antigen in aform that can stimulate lymphocytes. Interaction between the APCs and T cells is thefirst cellular interaction to occur following antigen challenge, and its outcome dictatesthe subsequent events.

APCs are a heterogenous population of leukocytes with immunostimulatory capacity.Some induce TH cells, some communicate with other leukocytes. Cells other thanleukocytes, such as endothelial or epithelial cells, which normally do not express class IIMHC molecules, can also present antigen when stimulated by cytokines (IFNγ andTNF), and the class II MHC is induced.

Dendritic Cells are irregular shaped cells that can arise from myeloid or lymphoidprecursors in either in the thymus or bone marrow, or from more differentiated cell typesin the blood or tissue. Dendritic cells express MHC class II, therefore, they are importantAPCs. Dendritic cells in nonlymphoid organs called interstitial DCs. Those in areas oflymphoid organs populated primarily by T cells are called interdigitating cells.

Langerhans’ cells (dendritic cells - specialized APCs) in the skin can migrate(carrying antigen from the skin) via the afferent lymphatics into the lymph nodeparacortex, where they ‘interdigitate’ with many TH cells. They express class II MHCmolecules.

Another specialized APCs, the follicular dendritic cells (found in the secondary folliclesof the B cell areas of the lymph nodes and spleen), can present antigen to B cells. Theylack class II MHC molecules but have the complement receptor CR1 (CD35) forinteraction with immune complexes.

APCs found in thymus are rich in self-antigens and class II MHC molecules. They play arole in deleting T cells that react against self-antigen, which is referred to as ‘negativeselection’.Activated B cells are also rich in class II MHC molecules. So, they can process andpresent antigens.

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ANTIGEN RECEPTOR MOLECULES

A. ImmunoglobulinsB. T-cell Antigen ReceptorC. Major Histocompatibility Complex

A. IMMUNOGLOBULINS (Antibodies)

Humoral immunity involves a number of soluble proteins called antibodies(immunoglobulins), which are a group of glycoproteins present in the serum of allmammals. They are produced in large quantities by plasma cells, which are developedfrom B cells.

Specific antibodies are synthesized following exposure to an antigen. There were twotheories of how specific antibodies are formed rapidly:

1. Instructive theory: the antigen acts as a template that directs the folding of thenascent antibody chain.

2. Selective theory: the combining site of an antibody molecule is completelydetermined before it ever encounters antigen.

The structure of antibodies (immunoglobulins)

The general structure of the immunoglobulins looks like a letter Y, with the antigen-binding regions at the bifurcated end.IgG is the most abundant Ig in the blood serum and it has a smallest mass weight(150kD). It can be cleaved into three active 50 kD fragments by the proteolytic action ofpapain. Two of these fragments bind antigen, therefore, they are called Fab. The otherone is called Fc (Fragment crystallizable).Fc:- mediates protective responses called effector functions, such as complement

fixation or binds to various cell of immune system and some phagocytic cells.- recognized by membrane transport systems

The Fc and the two Fab units of the intact antibody are joined by a hinge that allowsegmental flexibility to enhance the formation of antibody-antigen complexes byenabling both combining sites on an antibody to bind a multivalent antigen.

Immunoglobulin G consists of two kind of polypeptide chains, a 25 kD light (L) chain anda 50 kD heavy (H) chain. Each subunit of IgG is constructed from two L and 2 H chains,which are bonded by disulfide bonds.

The light chain exists in two distinct forms called kappa type and lambda type. Either ofthe light chain types may combine with any of the heavy chains.

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variable region

hinge region

antigen binding sites

light chain(212 residues)

Heavy chain(450 residues)

VH

CH1

CH2

CH3

VL

CL

IgG1

NH2

COOH

Fab

Fccarbohydrate unit

The L chains contain 212 amino acid residues, which consist of a variable region(residues 1 to 108) and a constant region (residues 109 to 212).The H chains contain 450 amino acid residues, which consist of a variable region(residues 1 to 108) and a constant region (residues 109 to 450).Three segments in the variable regions of both the L chain and the H chain have farmore variability than do the rest of the regions. These hypervariable regions are calledcomplementarity-determining regions (CDRs), because they determine antibodyspecificity.

Antibody Types and Characteristics

Five distinct classes of immunoglobulin molecules are known in human: IgG, IgM, IgA,IgD, IgE.

The class and subclass of an immunoglobulin molecule are determined by its heavychain type. For example, the four human IgG subclasses (IgG1, IgG2, IgG3, and IgG4)have heavy chain called γ1, γ2, γ3, and γ4, which differ slightly, although all are stillrecognized as γ heavy chain.

Immunoglobulin G (IgG) – (originally called γ-globulin)- the major human Ig (70%).- MW = 150 kD- Provides maternal protection/can be transferred across placenta- Fc = γ

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- 12 - 15 mg/mL- Half life = 3 weeks- Monomer- Chain structure = κ2γ2 or λ2γ2

- Four subclasses: IgG1, IgG2, IgG3, and IgG4

Immunoglobulin M (IgM)- the first Ig formed during immunization- MW = 950 kD- 1 mg/mL- Half life = < 1 week- responsible for the A, B, and O blood types- Fc = µ- Pentamer with one J fragment.- Chain structure = (κ2µ2)5 or (λ2µ2)5

J-chain

VL

CL

VH

Cµ1

Cµ2

Cµ3

Cµ4

IgM

carbohydrate units

Immunoglobulin A (IgA)- found in exocrine gland secretions (external secretions, such as saliva, tears,

milk, bronchial mucus, intestinal mucus).- First line of defense against bacterial and viral antigens.- consist of two IgG-like units linked together at the Fc region by a J fragment.- MW = 180-500 kD- Fc = α- 1 – 3 mg/mL- dimer- Chain structure = (κ2α2)n or (λ2α2)n

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- Two subclasses: IgA1 an IgA2.- IgA contain a secretory component (70 kDa), which is synthesized by the

epithelial cell and attached to the IgA during it transfer through epithelial celllayers. Bound secretory component facilitates the transport of sIgA intosecretions, as well as protecting it from proteolytic attack. IgA1 is thepredominant subclass in serum, whereas IgA2 predominates in secretions.

Immunoglobulin D (IgD)- present on the surface of B cells- the benefits are not yet known- MW = 175 kD- half life is only 3 days- Fc = δ- Antigen receptor that activate Ig production- < 0.1 mg/mL- Chain structure = κ2δ2 or λ2δ2

Immunoglobulin E (IgE)- MW = 200 kD- 0.001 mg/mL- responsible for hypersensitivity or allergy reactions- found on mast cells and basophils- Fc portion is part of mast cells- IgE-antigen complexes cross-link receptors on the surfaces of mast cells to

trigger the release of granules, such as histamine- Fc = ε- Chain structure = κ2ε2 or λ2ε2

Why vertebrates can produce a huge number of different specific antibodies?

1. variable and constant regions are encoded by separate genes2. several hundred genes encode the variable regions of L and H chains3. 4-6 J (joining) segment genes4. 12 D (diversity) genes5. Total number of combination is more than 100 millions possible antibodies.

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Light Chain gene recombination

V1 1 2 3 4 5V2 V3 Vn CK5' 3'

VK J1 - J5

V1 V2 4 5 CK 3'

V2 4 5 CK 3'5'

5'

V2 4 CK

CKVK

germ line DNA

B cell rearrangedDNA

primary RNA transcript

mature mRNA

Kappa chain polypeptide

rearrangement

transcription

splicing

translation

Kappa chain (light chain) synthesis

CK

NH2 COOH

Heavy chain gene recombination

V1 V2 µVn5' 3'

V1 - V200 J1 - J6

germ line DNA

B cell rearranged

DNA

primary RNA transcript

maturemRNA

heavy chain polypeptide

rearrangement

transcription

translation

1 2 12 1 2 6 δ γ3 γ1 α1 γ2 γ4 ε α2

D1 - D12 C Region Genes

V2 2 5 6 µ 3'δ γ3 γ1 α1 γ2 γ4 ε α25'

transcription

V2 2 5 6 µ 3'δ5'

alternative splicing

V2 2 5 µ δV2 2 5

translation

V CµNH2 COOH V CδNH2 COOH

Heavy chains biosynthesis

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Antigen-Antibody Reactions

There are a number of ways antibody can inactivate antigen.

1. Neutralization: Toxin and bacteria can be neutralized by antibody. Once theybind to antibody they are no longer toxic as their active site structures areblocked and they cannot bind to their target any more.

2. Precipitation: Some soluble antigens react with antibody to form insolubleprecipitates.

3. Agglutination: antibodies can bind to bacterial cells, mask the negative ioniccharges on the cell surface and immobilize the bacteria by aggregation.

4. Bacteriolysis: a complement-mediated reaction. The activated complementsdisrupt the cell membrane of bacteria and eventually kill the bacteria.

B. T CELL ANTIGEN RECEPTOR

Antigen recognition by T lymphocytes is mediated by T cell receptor (TCR). There aretwo forms of TCR, namely αβ and γδ TCRs. To be active, they need to be physicallyassociated with a series of polypeptides called CD3. CD3 complex is required for signaltransduction, following antigen recognition by the TCR heterodimer. CD3 comprises of 5invariants polypeptides: γ, δ, ε, ζ and η.During T cell activation (TCR binds with antigen), cytoplasmic portions of severalcomponents of CD3 become phosphorylated, catalyzed by protein kinase C, tyrosinekinases. Tyrosine phosphorylation on CD3 ζ is likely involved in the signal transductionpathway that communicates signals from the surface to the nucleus.

C. MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)

The MHC is referred to a cluster of genes (genetic loci) that determines thehistocompatibility antigens of cell membranes. The products, which are called MHCmolecules or MHC antigens (small polypeptide) are presented on the surface of cells.Foreign or non-self cells or tissue or an infected cell express different MHC antigens,which allows the body’s immune system to differentiate foreign antigens from the body’sown cells. For example, the cells of the allograft (in organ transplant) will expressalloantigens, which will be recognized as foreign by the recipient T cells. The T cells willthen kill the transplanted cell. This is also the case with an infected cell.

Human MHC is known as Human Lymphocyte Antigen System (HLA). There arethree classes of MHC molecules that are recognized so far:

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I. Class I MHC moleculesThis class of MHC molecules is found on the membranes of all nucleated cells andtissues. They can be detected using antibody or serological screening. The structurecomprised of a glycosylated heavy chain (45 kDa) consisting three globular domainscalled α1, α2, and α3, which are closely associated with a non-MHC-encoded peptide,β2-microglobulin (12 kDa).Examples of class I HLA:HLA-AHLA-BHLA-CHLA-E - J

II. Class II MHC moleculesThis class of MHC molecules is found on B lymphocytes, macrophages, and otherantigen presenting cells. They can be detected using T lymphocytes. The structure is aheterodimer of heavy (α) and light (β) glycoprotein chains. Both seem to have the sameoverall structures consisting two domains each: α1 and α2 or β1 and β2. Both the α- andβ-chains are coded by genes located in the MHC.Class II MHC molecules in human:HLA-DRHLA-DQHLA-DPEtc.

III. Class III MHC moleculesThe genetic loci is located in between class I and class II loci. The function of theproducts is not clearly understood.

MHC PolymorphismOne of the characteristics of the MHC is the extreme degree of polymorphism encodedwithin it. Polymorphism is not evenly spread throughout the MHC. The amino acidsequence variability in class I antigens is clustered in three main regions of the α1 andα2 domains, where as the α3 domain appears to be more conserved. For human class IIMHC, most polymorphism occurs in DR and DQβ chains. However, DPβ chain is slightlyless polymorphic.

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THE COMPLEMENT SYSTEM

Complement is a system of more than 35 different proteins and cofactors thatcontinuously circulate in the bloodstream or as cell-bound proteins acting as asecondary defense system. They help to kill the intruders that are missed by theneutrophils and the macrophages. The proteins normally circulate as inactiveproenzymes but in the presence of antigen the pathway will be initiated in a tightlyregulated cascade fashion to convert the inactive proenzymes to the active enzymes.

Complement proteins are produced mainly in the liver. The most abundant complementprotein is called C3.

The complement system is also one of the major effector pathways of the process ofinflammation. Deficiencies of complement proteins increase susceptibility to infectionsby pyogenic (abscess forming) bacteria and illnesses associate with the production ofauto-antibodies and immune complexes. So, complements may play a role in defenseagainst bacteria and the disposal of immune complexes.

NomenclatureThe proteins of the classical pathway are each assigned a number and react in theorder: C1q, C1r, C1s, C4, C2, C3, C5, C6, C7, C8, C9.The enzymatically active form identified by a bar drawn above its notation.The cleavage products of complement proteins are identified by suffix letters, a, b, etc.

Activation of and self/non-self distinction by complement proteins

The Classical Pathway – adaptive responseThis pathway requires a trigger in the form of an antigen-Ab complex. Therefore it isinvolved in the acquired (adaptive) immunity. IgG and IgM are the only antibodiesinvolved.

The Alternative Pathway – innate, non-specific reactionThe alternative pathway involved in the innate immunity and requires no Ab or antigento be active. Usually it is triggered by direct environmental influence of the microbialsurface. In this case, C3b is the initiating factor.

The Lectin Activation PathwayThe central player in this pathway is mannose-binding lectin, a protein produced mainlyin the liver but also present in the blood and tissues. Mannose-binding lectin binds toyeast (Candida albicans), viruses (HIV, influenza A), bacteria (Salmonella,Streptococci), parasites (Leismania). But, mannose-binding lectin does not bind to thecarbohydrates found in healthy human cells and tissues.

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CLASSICAL PATHWAY

ALTERNATIVE PATHWAY

C4

C3

C4b

C4a

C3a

C3b

C2 C2b

C4b2a

C3bBb

B Ba

C3

(C3 convertase)

(C3 convertase)

C3a

C3a

C3b

C4b2a3b

C3bBb3b

(C5 convertase)

(C5 convertase)

C5

C5a

C5b

Factor D

antigen-antibody

C1qr2s2 C1qr2s2

C1s

C1s

C6

C7

C8

C9

C5b6789n(MAC)

C1 is a pentamolecular Ca2+-dependent complex consisting of a single C1q molecule,two C1r and two C1s molecules. Multiple binding of the globular domains of C1q to IgGin an immune complex, for example, leads to comformational change in the C1complex. This makes one of the C1r molecules activates itself by autocatalysis andcleaves the other C1r to yield active enzyme. These two enzymes then cleave the twoC1s molecules to give active serine proteases. C1s cleaves the thioester-containingprotein C4 into C4a and the unstable intermediate C4b, some of which is quicklyattacked by the immediate vicinity amino or hydroxyl groups (proteins or carbohydratesfrom cell surface molecules) to form covalent amide or ester bonds. The rest ishydrolyzed by water. Surface bound C4b now acts as a binding site for the zymogenC2. When combined with C4b, C2 becomes a substrate for C1s and is cleaved to C2band C2a. The C2a segment remains bound, giving C4b2a.Both classical and alternative pathways generate a C3 convertase. In the classicalpathway, c1 is activated by complexed antibody and splits both C4 and C2. The smallfragments C4a and C2b, are lost and the major components form C4b2a. In thealternative pathway, C3b binds factor B, which is split releasing a small fragment, Ba.The major fragment Bb remains bound to form C3bBb. This converts more C3.The C3 convertases may bind C3b to yield C5 convertase enzymes. These activate thenext component of the complement system, C5 (the first component of the lyticpathway). C5b binds C6 and C7 to give C5b67, which is hydrophobic and has amembrane binding site allowing the complex close to the reaction site. C8 binds to asite on C5b and penetrate the membrane, where it can polymerize a number of C9molecules to generate the membrane attack complex (MAC).

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In the lectin activation pathway:In the blood, mannose binding lectin (MBL) complexes with another protein calledMASP (MBL-associate Serine Protein). When the mannose-binding lectin binds itstarget, the MASP protein functions like a convertase C1r and C1s that can cut C4 andC2. The resulting C4b2a can clip C3 complement protein to make C3b. Because C3 isso abundant in the blood, this happens very efficiently. The C3b fragments can thenbind to the surface of the bacterium, and the complement chain reaction is initiated.

Activation of the complement system will generate proteins, which have the followingeffects:

1) Opsonization: coat microorganisms for uptake by phagocytes. (opsonicfragments: C3b and iC3b bind to the membranes of target cells)

2) Chemotaxis: attract phagocytes to site of infection. Fragments of complementcan bind specific receptors on polymorphs and macrophages, and causechemotaxis. (C3a and C5a)

3) Lysis: damage the plasma membranes of cells, gram-negative bacteria,enveloped viruses, etc. Insertion of a hydrophobic ‘plug’ into lipid membranebilayers, leading to osmotic disruption of the target cell. (C5b6789n)

Complement can distinguish self and non-self by the covalent binding of C3b(deposition) to particles such as microorganisms and immune complexes, while self-cellsurfaces carry molecules that effectively limit C3b and other components deposition.

Biological Effects of C3a and C5a: stimulate chemotaxis, activate leukocytes, triggerdegranulation of basophils and mast cells (therefore they are called anaphylatoxins)

Biological effects of C3b and C4b: binding of bacteria, viruses and immunecomplexes to neutrophils, monocytes, and macrophages; endocytosis, phagocytosisand respiratory burst by complement receptor activation; phagocytosis and cytotoxicityvia IgA (ADCC and NK activation); erythrocyte CR1 binding to clear immune complexes;localization of immune complexes and APCs; solubilization of immune complexes bydisruption of lattices.

24

IMMUNE RESPONSE TO INFECTION

1. AGAINST BACTERIA INFECTION

Immune response to bacteria infection largely depends on the type of bacterial cell wall,and the mechanism of their pathogenicity.There are four main types of bacterial cell wall: 1) gram-positive bacterial cell wall; 2)gram-negative cell wall; 3) mycobacterial cell wall; and 4) spirochaetes cell wall.The mechanisms of pathogenicity of bacteria range between the following two extremepatterns: 1) toxicity without invasiveness; 2) invasiveness without toxicity. Mostpathogenic bacteria are intermediate between these extremes.

Events in immune response against bacteria not involving B or T cells

1. Recognition of common bacterial components by the innate immunesystem. This event does not involve the antigen-specific receptors of B- or T-cells.

2. Activation of complement via the alternative pathway, which may result inkilling the susceptible bacteria using the lytic complex (C5-C9), or releasing thechemotactic products, C3a and C5a, which cause smooth muscle contraction,mast cell degranulation, attracting and activating neutrophils. Derivatives of C3can bind to the bacteria and induce interactions with phagocytes.

3. Chemotaxis: more phagocytes are attracted to the site of infection due tocomplement activation and chemotactic effects of bacterial products.

4. Cytokine release: macrophages release cytokines (e.g. TNF and IL-1), whichactivate other phagocyte cells, increase their adhesion to endothelium andfacilitate passage into infected tissue.

5. Lymphokine release: TNF and microbial components can activate NK cells torelease lymphokines (e.g. IFNγ), which in turn can activate macrophages.

6. Adjuvant effects: bacterial components are recognized by the immune systemas a non-specific signal that boosts immune activity.

25

histamine

natural killer

macrophage

mast cell

bacteria

activate complementsC3a and C5a

produce formylpeptides

produce muramylpeptides, peptidoglycan

produce polyanions, lipoproteins, lipoteichoic acid, lipoarabinomannan, lipopolysaccharide

TNF

cytokines

IFNγ

increase vacular

permeability

chemotaxis

activation of phagocytes

activation of endothelium

adhesion ofphagocytes

activation of phagocytes

Phagocytosis

Most bacteria are killed by phagocytes. Following attachment of the phagocyte to thebacteria, long pseudopodia surround the bacteria. Fusion of pseudopodia brings thebacteria into the cell, forming a phagosome. Fusion of the phagosome with a lysosomeproduces a phagolysosome and results in release of the lysosome contents, whichdigest the bacteria. Some of the digested materials are presented on the cell surface tohelper T lymphocytes via class II MHC molecules.

26

phagosome

phagolysosome

non-antigenicproducts

antigenic product

lyso-some

MHC class IIwith processed antigen

Antigen (microorganisms, etc)

pseudopodia

Phagocytosis and processing of exogenous antigen by macrophages

The killing mechanisms of phagocytic cells are via:

a. oxygen-dependent mechanism; an enzyme in the phagosome membranereduces oxygen to superoxide anion (•O2

-), a reactive oxygen intermediate.b. Nitric oxide (NO) formation. Activation by IFNγ is needed to express nitric oxide

synthetase (NOS), which is then optimally triggered by TNF. NOS reacts oxygenwith the guanidine nitrogen of L-arginine to give NO and citrulline.Tetrahydrobiopterin is needed as a cofactor.

c. oxygen-independent killing (the mechanism is not clear).d. cationic proteins with antibiotic-like properties (e.g., defensins, which are cystein-

and arginine-rich cationic peptides of 30-33 aa, found in human neutrophilpolymorphs). They form ion-permeable channels in lipid bilayers.

e. other antimicrobial mechanisms (e.g., lysozyme, lactoferrin). Lactoferrin – foundin neutrophil polymorphs – can bind iron.

Natural Killer Cells and Tc cells are also speculated to be involved in bacterial killing bydirect contact.

2. AGAINST VIRAL INFECTION

Different viruses infect different cell types, for example, HIV infects CD4+ cells andEpstein-Barr virus infects B cell.

Immune response to viral infection depends on the species of the virus and the phase ofthe infection. There are a number of ways the immune system response to viralinfection.

27

Interferons (IFNs) are a group of several unrelated class of proteins, some of which areglycosylated, released from many different cell types in response to viral infection,double-stranded RNA, lipopolysaccharide, etc. Interferons can protect the host cells byinducing a state of resistance in the host cells to virus infection.

IFNα is the largest group of IFN, consists of more than 20 variants, made by leukocytesin response to viruses or nucleic acids

IFNβ is made by fibroblast in response to viruses and nucleic acids

IFNα and IFNβ can inhibit cell growth, which is clinically useful in certain rare cancers(renal cell cancer and hairy cell leukaemia).

IFNγ is produced by activated T cells and natural killer cells in reponse to immunestimuli. IFNγ can acts as a positive feedback (activate T cells further), activatemacrophages and enhance their capacity to act as APCs.

Antibody is only effective against extracellular viruses, but not against those that havebeen in the cell. IgG and IgM active in the plasma and tissue fluid. Secretory IgA onlyactives on epithelial surfaces. Antibody in association with complements (C1 – C9) canlyse viral infected cells, or directly damage enveloped viruses.

Tc cells recognize viral-infected cells from the presence of viral antigens in themembrane and kill the infected cells.

Natural Killer (NK) cells may be involved in immune response to cytomegalovirus(CMV).

3. AGAINST FUNGI (little is known: will not be discussed)

28

4. AGAINST PROTOZOA (Plasmodium, Leishmania, Trypanosoma)

Table: General immune responses in protozoal infections

Antibody-independent Antibody-dependentprotozoa

mechanism importance mechanism importance

Trypanosomabruceifree in blood

-

Lysis withcomplementwhich also

opsonizes forphagocytosis

++++

Plasmodiuminsideerythrocyte

Cytokines

Macrophageactivation

+++ at liverstage

+++ at bloodstage

Blockinvasion,

opsonizes forphagocytosis

+++

Trypanosomacruziinsidemacrophage

Macrophageactivation by

cytokines andkilling by NO

and ROIs

+++in cronicphase

Limits spreadin acuteinfection,

sensitizes forADCC

++

Leishmaniainsidemacrophage

Macrophageactivation by

cytokines andkilling by NO

and ROIs

++++ Limits spread +

29

5. AGAINST WORMS (Helminths)

neutrophileosinophilmacrophage

mast cellTB

IgGIgE

platelet

cytokines

TNFTNF

NONO

ROIROI

complement

parasite antigen

ECF

ECF-P

ESP

IgE

effector moleculesAPC

solubleantigen

Antigens released by the parasite stimulate macrophages and T cells, which theninteract with B cells to produce specific antibody. Specific IgE antibody sensitizes localmast cells, which degranulate when come into contact with antigen and release effectormolecules. The mast cells also release eosinophil chemotactic factors (ECF), whichtogether with parasite-derived chemotactic factors (ECF-P) attract eosinophils towardthe worm. Eosinophils are also attracted toward the worm and stimulated to proliferateby eosinophil stimulation promoter (ESP) derived from antigen-stimulated T cells. Theydamage the worm tegument mainly by releasing major basic (cytotoxic) proteins (foundin the granules). Antibodies and complements at high levels damage the worm.However, at lower levels antibody sensitizes neutrophils, macrophages, eosinophils,and platelets for antibody-dependent cell-mediated cytotoxicity. Neutrophils andmacrophages may act by releasing toxic nitric oxide (NO) and reactive oxygenintermediate (ROI).

30

ESCAPING THE IMMUNE SYSTEM

“THE TRICKS OF THE PATHOGENS”

Antigen masking

Parasites acquire a coating of host antigens that cover and mask their own antigenicmaterial to avoid host immune response. For example, the blood fluke Schistosoma, livefree in the venous circulation and therefore exposed to many immune components,become coated with host-derived molecules, such as blood glycolipids, MHC molecules,etc. The worm is completely camouflaged and appears as ‘self’.

Some viruses incorporate host cell genes that code for cell surface proteins into theirgenomes, which permits the virus to synthesize and incorporate the proteins into theircapsids, which will be ignored by the host immune system.

Antigenic variation

Parasites rapidly change their antigenic pattern. The patient’s immune system doesproduce antibodies, which kill most of the parasites, but some of the parasites survivewith new antigenic characteristics. This event is repeated over and over until thepatient’s immune system exhausted.

Viruses under go drift (less significant change in antigenic properties) and shift(significant change). The immune system will not recognize virus that has undergone ashift. It will recognize a drift, but the fit between the antibody and the viral antigen willnot be as tight.

Bacteria can have multiple different serotypes.Streptococcus pneumoniae has more than 90 different serotypes.Salmonella typhimurium, a common cause of food poisoning, can alternate expressionof two antigenically distinct flagellins, proteins of the bacterial flagella.Neisseria gonorrhea has several variable antigens.

Overwhelm immune system

Parasites can release large amounts of antigen that antibody supply is exhausted.

HIV virus overwhelms the immune system by rapid reproduction of the virus. The virusreproduces faster than the immune system can eliminate it.

Enzymatic destruction of antibodies

Some bacteria and parasites produce proteases that hydrolyze the antibodies thatrecognize either the bacteria or their toxins.

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Hiding from the Immune response

Herpes simplex virus, the cause of cold sores, first infects epithelial cells and thenspreads to sensory neurons serving the area of infection. Neurons are a favored site forlatent viruses to lurk because they express very small number of MHC class Imolecules.

Attack the cells of the immune system

HIV and HTLV viruses infect T cells of the immune system

Anthrax toxin may reduce dendritic cell function by blocking kinase signaling.

Hide inside cells

Mycobacterium tuberculosis can reside in a patient’s macrophages for years. It inhibitsphagosome-lysosome fusion.

Listeria is engulfed by macrophages and inserted into a phagosome where it should bedestroyed. However, Listeria can produce a pore-forming protein that dissolved thephagosome membrane. Listeria reproduces inside the macrophage and then spreads toadditional cells.

Yersinia pestis also survive inside macrophage. It produces a toxin that is a cysteineprotease that cleaves proteins the macrophage would use to communicate with othermacrophages.

Other mechanisms:Block glucocorticoid signaling (Anthrax toxin), interfere with the generation and transportof MHC molecules (CMV, EBV, adenovirus, herpes virus, Chlamydia, Yersinia,Leishmania, TB), inhibiting the caspase enzymes and other mechanisms leading toapoptosis, inhibition of humoral immunity (Herpes simplex, CMV, vaccinia), inhibition ofinflammatory response (CMV, vaccinia, EBV), immunosuppression of host by producingIL-1 homolog that inhibits TH1 cells and reduces IFN-γ production (EBV).

32

IMMUNODEFICIENCY

Immunodeficiency diseases cause increased susceptibility to infection in patients

Primary immunodeficiency diseases – due to intrinsic defects in cells of the immunesystem – genetic defects

Secondary immunodeficiency diseases – due to extrinsic factors, e.g., irradiation,drugs, malnutrition, or infection.Examples: Acquired Immunodeficiency Syndrome (AIDS)

Specific immunodeficiency diseases – abnormalities of T or B cells

-B cell deficienciesX-linked agammaglobulinemia (X-LA) – patients have no B-cell and most ofimmunoglobulins. (lost of Btk [Bruton’s tyrosine kinase – only in X chromosome]. Btkcontributes to intracellular signaling from the B-cell receptor and is necessary for thegrowth and differentiation of pre-B cells)

-T cell deficienciesSevere combined immunodeficiency (SCID) – very few lymphocytes (T and/or Bcells) in the blood, no lymphoid cells in the thymus. (specific abnormality: Adenosinedeaminase deficiency, purine nucleotide phosphorylase deficiency, etc)

MHC class II deficiency – patients do not have CD4+ T cells leading to a deficiency inantibodies. Affected infants have recurrent infections.

Non-specific immunodeficiency diseases – abnormalities of phagocytes andcomplementsDeficiencies of the classical pathway components C1, C2, C4 cause immune-complexdiseases such as systemic lupus erythematosus.Deficiencies of the alternative pathway components C3, factor H, factor I increasesusceptibility to pyogenic infections.Deficiencies of the lytic pathway increase susceptibility to Neisseria gonorrheae and Nmeningitidis.Hereditary Angioneurotic Oedema (HLA) – deficiencies of the C1 inhibitor (compoundthat is responsible for dissociation of activated C1).

Chronic granulomatous disease (CGD)Incapable of forming oxygen radicals and hydrogen peroxide in their phagocytes.

Leukocyte adhesion deficiency (LAD)Deficiencies of complement receptor 3 (CR3 – on the β chain) on phagocytes. So, theycannot bind to iC3b on opsonized microorganisms. Lymphocyte function associatedantigen (LFA-1) shares the same β chain. Therefore, leukocyte-endothelial celladhesion is also affected.

33

CELL MIGRATION AND INFLAMMATION

As a defense system, the cells of immune system need to migrate from and to varioustissues in the body via the blood lymphatics. To make this possible they have topenetrate the vascular endothelium, which is varies throughout the body.

In general, leukocyte migration depends on:1. the surface charge of the interacting cells2. the haemodynamic shear force in the vascular3. the expression of complementary sets of adhesion molecules on the endothelial

cells and on the leukocytes.

There are four steps involved in leukocyte migration:- Induction/Activation- Rolling- Adhesion (pavementing)- Migration (diapedesis)

haemodynamic shear force and surface charge prevent adhesion

rolling

site of inflammationtissue damage

immune reactions

penetration(diapedesis)

IL-1

chemotaxis

TNF-α

adhesion(pavementing)

activation

blood vessel

endotheliumC5a and f-met

During bacterial infection, the macrophages release IL-1 and TNF-α that induceendothelial cells that line nearby blood vessels to express a protein on their surfacescalled selectin. Selectin interacts with selectin-ligand on the neutrophils as they pass by.This interaction between selectin and its ligand is only strong enough to causeneutrophils to slow down and roll along the inner surface of the blood vessel. Whenthere are other chemoattractant molecules, such as C5a, LPS, f-met (formylmethionine), present, the neutrophil rushes a preformed protein called integrin to itssurface. Integrin can interact with another adhesion molecule on the surface of the

34

endothelial cell called ICAM. This interaction is very strong, and it causes the neutrophilto stop rolling. Once the neutrophil has stopped, it can be influenced bychemoattractants to penetrate out the endothelial cells and migrate to the site ofinflammation.

Actually, the patterns of cell migration are complex. For examples, neutrophils are thefirst cells accumulated at the site of inflammation caused by infection; large numbers ofCD4+ T cells and mononuclear phagocytes are found during chronic autoimmune;eosinophils are commonly accumulated in the sites of parasitic infections, etc.These complex patterns of cell migration depend on:

1. the type and state of activation of the cells2. the type of adhesion molecules expressed by the vascular endothelial cells3. the presence of chemotactic molecules, which may selectively attract particular

population of leukocytes.

Intercellular Adhesion Molecules: are membrane-bound proteins, which allow onecell to interact with another.

Families of adhesion molecules

The Selectins (or lectin cellular adhesion molecules): transmembrane moleculeswhere the extracellular region contains a N-terminal domain, which has lectin-likeproperties (it binds to carbohydrate residues), hence, they are called selectins.Their ligands usually carry carbohydrate moieties.Examples of the selectins: ELAM-1, GMP-140, and MEL-14.

The immunoglobulin super family: Expressed or inducible on vascular endothelialcells.Example: ICAM-1, ICAM-2, VCAM.

Table: The properties of some adhesion molecules

Adhesionmolecules onendothelium

Ligands onleukocytes

Type ofleukocyte Stimulants

Time ofexpression

ELAM Asialo-Lewis X neutrophils TNFα or IL1 4 – 12 h

ICAM-1 LFA-1monocytes,lymphocytes

TNF, IL-1, IFNγ 24 – 96 h

ICAM-1 CR3 phagocytes TNF, IL-1, IFNγ 24 – 96 hICAM-2 LFA-1 lymphocytes constitutiveVCAM VLA-4 lymphocytes TNF, IL-1 24 – 96 h

Time of expression of ELAM may explain why neutrophils appear early at the sites ofacute inflammation.

35

ICAM-1 and VCAM are important incontrolling lymphocytes migration.ICAM-1 and VCAM are both inducedby cytokines with a similar timecourse. The different is that IFNγinduces ICAM-1 but not VCAM. It issuggested that different blend ofinducers at different sites could affectthe composition of adhesionmolecules present on those places.

Integrins: consist of two non-covalently bound polypeptides (α and β).The β1 integrins are involved in binding of the cells to extracellular matrixThe β2 integrins are involved in leukocyte adhesion to endothelium or to other immunecellsThe β3 integrins (cytoadhesins) are involved in the interactions of platelets andneutrophils at inflammatory sites.

Integrintype ligands expression

VLA-1 collagen T cell, fibroblastsVLA-2 collagen Activated T cells, plateletsVLA-3 Laminin, collagen, fibronectin Kidney, thyroidVLA-4 VCAM, fibronectin Most haemopoietic cellsVLA-5 fibronectin Some leukocytes, plateletsVLA-6 laminin Widely distributedLFA-1 ICAM-1, ICAM-2 Most leukocytesCR3 C3b, C4b, ICAM-1, +endothelium Mononuclear phagocytes, neutrophilsCR4 C3b, C4b, +endothelium macrophages

Vascular addressins: expressed on high endothelial venules (HEV) at different sitesaround the body, e.g. in the lymph nodes and in mucosal lymphoid tissue. Thesemolecules modulate lymphocyte traffic into secondary lymphoid tissues (control normallymphocyte traffic) and inflammatory sites.

0 1 2 3 4 5

ICAM-2

ICAM-1 or VCAM

ELAM

days

TNFα stimulation

GMP-140

36

INFLAMMATION

Inflammation is the body’s response to infection, antigen challenge (venom), physicaldamage, etc.

The cells of immune systems are widely distributed throughout the body. But, if infectionoccurs, it is necessary to concentrate them and their products at the site of infection.

Three major events occur during inflammation:1. An increase in blood supply to the site of injury2. An increase in capillary permeability in the area – to allows antibody,

complement, and molecule of other plasma enzyme systems to reach the site ofinjury

3. Migration of leukocytes from blood vessel to the site

The process of inflammationInflammation reaction is mediated by cytokines (TNF, IL) and vasoactive mediators(histamine, serotonin, PAF, prostaglandins, leukotrienes) released by auxiliary cells(mast cells, basophils and platelets).First, fast-acting mediators or preformed mediators (histamine, serotonin,bradykinin, TNF, IL) modulate the immune response. Once, leukocytes have arrived atthe site of infection or inflammation, they release newly synthesize mediators(leukotrienes, prostaglandins, thromboxanes, platelet-activating factor,monohydroxyeicosatetraenoic acids (HETE), and hydroperoxyeicosatetraenoic acid(HPETE), which control the later accumulation and activation of other cells.

37

Mediators of inflammation

mediator source Actions

histamineMast cells,basophils

Increased vascular permeability, smoothmuscle contraction, chemokinesis

5-hydroxytryptamine(5HT)=serotonin

Platelets, mastcells (rodent)

Increased vascular permeability, smoothmuscle contraction

Platelet activatingfactor (PAF)

Basophils,neutrophils,

macrophages

Mediator release from platelets, increasedvascular permeability, smooth muscle

contraction, neutrophil activationNeutrophil

chemotactic factor(NCF)

Mast cells Neutrophil chemotaxis

IL-8 lymphocytes Monocyte localization

C3a Complement C3Mast cell degranulation, smooth muscle

contraction

C5a Complement C5

Mast cell degranulation, smooth musclecontraction, neutrophil and macrophage

chemotaxis, neutrophil activation,increased capillary permeability

bradykininKinin system(kininogen)

Vasodilatation, smooth muscle contraction,increased capillary permeability

Fibrinopeptides andfibrin breakdown

productsClotting system

Increased vascular permeability, neutrophiland macrophage chemotaxis

Prostaglandin E2

(PGE2)Cyclooxygenase

pathway

Vasodilatation, increased vascularpermeability produced by histamine and

bradykininLeukotriene B4

(LTB4)Lipoxygenase

pathwayNeutrophil chemotaxis, synergizes with

PGE2 in increasing vascular permeabilityLeukotriene D4

(LTD4)Lipoxygenase

pathwaySmooth muscle contraction, increasing

vascular permeability

38

HYPERSENSITIVITY REACTIONS

Type of Hypersensitivity

Type I: immediate hypersensitivity, occurs when an IgE response is directed againstinnocuous environmental antigens, such as pollen, house-dust mites or animal dander,etc.

Type II: antibody-dependent cytotoxic hypersensitivity, occurs when antibody binds toeither self antigen or foreign antigen on cells, and leads to phagocytosis, killer cellactivity or complement-mediated lysis.

Type III: occurs when immune-complexes are formed in large quantities, or cannot becleared adequately by the reticulo-endothelial system (RES), leading to serum-sicknesstype reactions.

Type IV: delayed type hypersensitivity (DTH), occurs when antigens are trapped in amacrophage and cannot be cleared. T-cells are then stimulated to elaboratelymphokines, which mediate a range of inflammatory responses.

Type I: Antibody-mediated anaphylactic reactions or immediate hypersensitivity

Manifested as commonly known allergy (changed reactivity) reactions, such as hayfever, asthma, foods or drugs allergy, eczema, urticaria, bee venom allergy, etc.

1. Mediated by IgE attached to mast cells and basophils2. Require pre-exposure before reaction can occur3. Reactions occur soon after subsequent exposure to the antigen4. Antigens (allergens) include pollen, house-dust mites, mold spores, animal

dander and saliva proteins.

Many drugs can cause allergy-like reactions possibly due to their effects on thedegranulation in mast cells.E.g., narcotic drugs (morphine and codeine), polymyxin B sulfate, d-tubocurarine iodide,iodinated organic compounds, etc.Drugs, such as penicillin, may also conjugate to serum proteins and act as haptens toelicit immunological response.

Steps in type I hypersensitivity reactions:

Activation phaseThe antigens binds to at least two IgE attached to mast cell or basophils leading togranulation

39

Effector phasepreformed mediators

-histamine: binds to both the H1 and H2 receptors.-neutrophil chemotactic factors-eosinophil chemotactic factors-heparin

Newly synthesized mediators (slow reacting substances)-leukotrienes: prolong constriction of smooth muscle leading to antihistamineresistant asthma-tromboxanes and prostaglandins: vasoactive, bronchoconstrictive, chemotacticfor neutrophils, eosinophils, basophils, and monocytes (macrophages).-Platelet-Activating Factor (PAF): induces platelets to aggregate and releasehistamine and arachidonic acid metabolites leading to bronchoconstriction andvasodilatation.

The inflammatory response in asthmatic bronchi

IgE

chemotacticfactors

cytokinesLTB4

PAF

spasmogens

histamineLTPGPAF

immediate responsebronchoconstriction

E

N

B

M

P

cytotoxic proteins

lysosomal enzymes

complementprostaglandins

microtrombi histamine

bronchialhyperreactive

ASTHMA

hypersecretion of mucussmooth muscle hypertropy

cellular infiltration

bronchus

40

Factors involved in the development of allergy

a. Genetic factors: allergic parents tended to have a higher proportion of allergicchildren than parents who were not allergic.

b. T-cell deficiency: There are reduced numbers of CD3+ T cells and CD8+ Tscells in patients with severe atopic eczema. It is not clear in patients with rhinitisor asthma. It seems that this T cells deficiency is a cause rather than aconsequence of atopic disease.

c. Mediator feedback: It was found that mast cell mediators such as histamine,prostaglandins and leukotrienes have an anti-inflammatory role. In contrast to theinflammatory effects of histamine mediated by H1 receptors (increase bloodvessel permeability, induce urticaria, induce asthma, induce gut permeability),the anti-inflammatory effects are mediated by H2 receptors, via the secondarymessenger cAMP (suppressor cell activation, reduce chemotaxis release, reducehistamine release, reduce C2 production).

d. Environmental factors: SO2, NO, diesel-exhaust particulates, fly ash, smoking.e. Quantity of Exposuref. Nutritional Statusg. Presence of chronic underlying infections or acute viral illnesses.

Type I reactions can possibly be reduced by desensitizing the patient to the antigen byadministering small doses of the antigen over long periods of time. This is also called‘HYPOSENSITIZATION’.

Proposed mechanisms include:a. increase synthesis of IgG which bind the antigen before it reaches the IgE bound

to mast cells and basophilsb. Induction of tolerance to the antigen.c. Induction of T suppressor cells

Type II: Cytolytic or Cytotoxic Reactions

Occurs when IgM and IgG bind to antigen on the surface of the patient’s cells andactivate the complement cascade leading to destruction of cells by the complementproteins and attraction of granulocytes and macrophages to the area. Also, whenantibody binds to a certain receptor and blocks its physiological functions.

-mismatched blood transfusion (mainly IgM)-Rh incompatibility-drug allergies-some autoimmune diseases (myasthenia gravis: caused by immunoglobulins binding toAch receptors on motor end plates of muscles; Lambert-Eaton Syndrome: caused bydefective release of acetylcholine from the neuron due to IgG mediated disruption of ionchannel on the neuron itself and not on the endplate; Graves’s Disease: caused byimmunoglobulins binding to the thyroid-stimulating hormone (TSH) receptor of thyroidcells, which activates the thyroid cell leading to hyperthyroidism).

41

Type III: Immune Complex-mediated Reactions

Occurs when complexes of antigen and IgM or IgG accumulate in the circulation or intissue and activate the complement cascade. The chemotactic complement proteins(C3a and C5a) attract granulocytes to the area, which then degranulate releasing lyticenzymes and other destructive agents.-the initial stage is the formation of soluble antibody-antigen complexes which are toosmall to be cleared by the reticuloendothelial system.-detrimental to the kidney because immune complex are easily trapped in the renalglomeruli – cause glomerulonephritis.-The condition of immune complex-associated symptoms is known as serum sickness.

Type IV: Cell-mediated Reactions or Delayed-Type Hypersensitivity (DTH)

Mediated by T-cells rather than immunoglobulins. The activated T-cells releaselymphokines (cytokines) that attract and activate macrophages. The latter cause thelocal damage.Also known as delayed-type hypersensitivity because it requires 1 – 2 days afterexposure to the antigen before the reaction is seen.Exp. -skin tuberculin test

-rejection of a transplant organ-contact sensitivity (poison oak, poison ivy)-identification of an intracellular parasite (viruses, bacteria, fungi)

42

Table: Immune Constituents of Hypersensitivity Reactions

HypersensitivityReactions

Ig Involved Complms.Involved

Major Immunecells involved

Principalmediators

Type IIntermediate reactions-early-phase reaction

-late-phase reactions

IgE

None

No

No

Mast cells

Eosinophils,PMNs,

lymphocytes

Histamine,leukotrienes

Multiplecytokines

Type IICytotoxic reactions IgG, IgM

IgGYesNo

NonePMNs, NK cells,

macrophages

None

Type IIIImmune complex-mediated reactions

IgG, IgM Yes PMNs Lysosomalenzymes

Type IVCell-mediated

reactionsNone No T cells,

macrophagesMultiple

cytokines

PMNs: polymorphonuclear leukocytes; NK: natural killer.

43

ORGAN TRANSPLANTION AND REJECTION REACTIONS

Organ transplantation is a common clinical practice, which is intended to make good afunctional deficit. However, unless the donor and recipient are genetically identical, thegraft antigens will promote an immunological rejection response.

Examples of Clinical Transplantation:- kidney : in the case of end-stage renal failure- heart: in the case of terminal cardiac failure- lung or heart/lung: in the cases of pulmonary hypertension, cystic fibrosis- liver: in the cases of cirrhosis, cancer, biliary atresia- cornea: dystrophy, keratitis- pancreas or islets: in the case of diabetes- bone marrow: in the cases of immunodeficiency, leukemia- small bowel: in the case of cancer- skin: in the case of burns

There are four different types of organ transplants:

1. Autograft: from one part of the body to another. Do not elicit rejection.

2. Isograft: between genetically identical twins. Do not elicit rejection.

3. Allograft: between members of the same species. Common clinical transplant.The cells of the donor organ will express alloantigens, which can be recognizedas foreign by the recipient.

4. Xenograft: between members of different species (e.g., monkey to human). Ingeneral, the donor organ is rapidly rejected.

Two components of the immune system that play a main role in rejection reactions are:a. Major histocompatibility complex (MHC) antigensb. T cells (especially TH cells).

However, some other cells or molecules are also involved, for example, antibody causegraft damage, macrophages may involved in inflammatory reaction in grafted tissue.

Processes in rejection reaction:

The T cells involved in rejection recognize the MHC antigens expressed on the graft,which positively select T cell production and maturation in the thymus. T helper cells areactivated by antigen presenting cells to release lymphokines. The most importantlymphokines in cellular rejection are IL-2 (activates Tc cells and cause cell-mediatedcytotoxicity) and IFNγ (induces MHC-antigen expression, boost APC activity, activatelarge granular lymphocytes and macrophages). Lymphokines also activate B cells toproduce anti-graft antibodies, which trigger antibody-dependent cell mediated

44

cytotoxicity). These antibodies together with complements also cause damage to thevascular endothelial resulting in hemorrhage, platelet aggregation, graft thrombosis, etc.

TREATMENT OF REJECTION: See Below

45

METHODS OF SUPPRESSING THE IMMUNE SYSTEM

“HUMAN INTERVENTION”

1. Radiation TherapySuppresses the stem cells in the bone marrow

2. Drug Therapy

Glucocorticoids

Calcineurin InhibitorsCyclosporine (Sandimmune™; Neoral™)FK-506 (Tacrolimus, Prograf™; topical form: Protopic™)

Antiproliferative/antimetabolic agentsRapamycin (Sirolimus, Rapamune™)Azathioprine (Imuran™)Mycophenolate (CellCept™)

Non-steroidal anti-inflammatory drugs (NSAIDS)

3. Specific Immune suppressionAntibodies

Drug Therapy

APC

macrophage

THTc

Tc

Tc

Tc

IL-2IL-2R

IFNγcell division

STEROIDSCYCLOSPORINE

FK506AZATHIOPRINE

RAPAMYCIN

graft cells

MYCOPHENOLATE

46

Cyclosporine (Sandimmune; Neoral)

HN C

HC N C

HC N C

HC N C

HC N CH2

C

N

CHCNHCCN

HCCN

HCCN

HC

O O O O

OOO

CN

CH

CO

O

CH3

H3C H3CH3C CH3

H3C

H3C

HOCH3

H

CH3 CH3

H

CH3H3C H CH3

H

CH3H3C

CH3

CH3O CH3

H3C

H3C

O

MeLeu MeVal

MeGly

CH3MeLeu

ValCH3H3CMeLeu

AlaD-Ala

MeLeu

Cyclosporine

A cyclic peptide of 11 amino acids, produced by the fungus species Beauveria nivea.Cyclosporine is lipophilic or highly hydrophobic.

Mechanism of action: Inhibits antigen-triggered signal transduction in T cells,preventing expression of many lymphokines, e.g. IL-2. Cyclosporine forms a complexwith a cytoplasmic receptor protein, cyclophilin (a member of immunophilin). Thecomplex binds to calcineurin resulted in the inhibition of Ca2+-stimulateddephosphorylation of the cytosolic component of NFAT. Therefore, the cytoplasmiccomponent of NFAT cannot translocate to the nucleus, where it complexes with nuclearcomponents required for complete T-cell activation, including transactivation of IL-2 andother lymphokine genes.Cyclosporine is also reported to increase expression of transforming growth factor β(TGF-β), a potent inhibitor of IL-2-stimulated T-cell proliferation and generation ofcytotoxic T cells.

47

FK-506 (Tacrolimus)

H3C

O OH

O

CH3O

O

CH3

CH3

OCH3

O

N

O

CH2

OCH3

H3C

HO

H3CO

FK506

H

A macrolide antibiotic produced by Streptomyces tsukubaensis.

Mechanism of action: Tacrolimus (FK506) binds to FK506-binding protein-12 (FKBP-12), which is structurally related to cyclophilin. The complex then binds to cancineurin +calcium + calmodulin resulted in the inhibition the calcineurin phosphatase activity. So,although the intracellular receptor of FK506 is different from that of cyclosporine, theyshare a common pathway for immunosuppression.

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Rapamycin (Sirolimus, Rapamune)

NO

H3COH

O

H3COO

H3CO

O

H3C

CH3

CH3

CH3

HOH3C

H

H

OH

OCH3H

O

CH3

Rapamycin

A macrocyclic lactone produced by Streptomyces hygroscopicus.

Mechanism of action: Rapamycin inhibits T-cell activation and proliferation downstreamof IL-2R and other T-cell growth factor receptors. The action of rapamycin is alsoinitiated by a binding to the immunophilin, FKBP-12, but the complex does not bind tocalcineurin. Instead, it binds and inhibits the mammalian kinase, target of rapamycin(mTOR), which is important in T cell proliferation.

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TCR IL-2R

Protein Tyrosine Kinases (Src,

Ick, fyn, Zap70)

PIP2DG

PLC γ1

PO4IP3PKC

X

X-PO4

RAS

Raf-1

Mek

MAP Kinase

Vav orGRB2/sos

increaseCa2+ conc.

calcineurin

NFATc NFATcPO4

increase immediateearly genes fos/jun

NFATn NFATc

IL-2 geneNUCLEUS

translocationIL-2 mRNA

IL-2

Cdk 2

proliferation

cyclophilin or FKBP

cyclosporin or FK506

rapamycin

FKBP

mTOR

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Azathioprine (Imuran)

N

N NH

N

S

N

N

NO2

H3CN

N NH

N

SH

Azathioprine 6-Mercaptopurine

Azathioprine is an imidazolyl derivative of 6-mercaptopurine, a purine anti-metabolite.

Mechanism of action: In the presence of nucleophiles, such as glutathione,azathioprine is cleaved to 6-mercaptopurine. The latter compound can be converted tothe 6-mercaptopurine nucleotides that inhibit DNA biosynthesis. Also, it can beconverted to 6-thioinosine 5’-phosphate (T-IMP). The latter can inhibit de-novo pathwayof purine biosynthesis.

Mycophenolate (CELLCEPT – Roche-Syntex)

O

O OH

CH3

OCH3

CH3

O

ON

O

Mycophenolate mofetil

Mycophenolate is commercially prepared as the ester prodrug to increase absorption ofthe drug. The parent acid is isolated from fermentation broths of several Penicilliumspecies.

Mechanism of action: selectively inhibit the proliferation of lymphocytes by inhibition ofIMPDH (inosine monophosphate dehydrogenase), which is the rate-limiting enzyme inGMP biosynthesis. GMP is a precursor of GTP and deoxy-GTP nucleotides necessaryfor RNA and DNA synthesis. This inhibition is more pronounced in lymphocytesbecause they do not use the purine salvage reactions, which recycle purine bases fromnucleic acids and nucleotides.

51

PREPARATIONS THAT TARGET OR AFFECT THE IMMUNE SYSTEM

1. Vaccines

2. Antibodies

Immunoglobulins

Monoclonal Antibodies

3. Cytokines

Interferons

Interleukins

Interleukin receptor antagonist

4. Growth Factors

Epoetin alfa

Darbopoetin alfa

Granulocyte-Colony Stimulating Factor

Granulocyte-Macrophage-Colony Stimulating Factor

5. Synthetic Drugs

6. Immunomodulators whose main indication is RA

7. Herbal Products

VACCINES

Vaccine is a suspension of attenuated or killed microorganisms (viruses, bacteria, orrickettsiae), administered for prevention, amelioration, or treatment of infectiousdiseases. Vaccines have been very effective in preventing people, especially infantsand children, from getting sick caused by infectious diseases. However, it has beensuggested that the increased use of vaccines in infants and children increases the riskof allergic problems, such as asthma, among children. The logic behind this is thatvaccination mainly stimulates TH2 cell production, which leads to B cell developmentand activation of the immune response for inflammation. TH1 cells usually activatemacrophages and killer cells. In the normal exposure to antigen mixtures, both type of Thelper cells are produced, which provide check and balance system between the two.However, vaccination provides protection so that the antigen is intercepted before TH1

cells can develop, which leads to an excess of TH2 cells relative to TH1 cells.

Methods of vaccine production

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The objective of vaccine development is to alter a pathogen or its toxins in such a waythat they become innocuous without losing their antigenicity, because antibodies and Tcells recognize only particular parts of antigens, the epitopes, and not the wholeorganism or toxin.

- Killed (inactivated) pathogensTreating the pathogen with denaturing disinfectant to kill the pathogen but recover thesurface antigens.

- Live/attenuated pathogensPassing the pathogen through many generations of host animals to yield low virulentstrain.

- Live/attenuated related strainFor example, cowpox virus used in place of smallpox virus

- Cellular antigen from a pathogenIsolate the surface antigen from the pathogen, purify it and reconstitute into a vaccinepreparation.

- Genetically engineered pathogensClone a piece of DNA encoding the surface antigen from the pathogen and overproduce the antigen in E. coli or other hosts.

Types of Vaccinesa. simple vaccine: contains only one kind of antigen or strain

b. multivalent vaccine: contains two or more kinds of antigens or strains thatcause the same disease (Important for full protection!)

c. polyvalent vaccine: contains two or more kinds of antigens or strains that causedifferent diseases. (Just for convenience)

Types of dosinga. single-dose vaccine: needed only once during life time

b. multiple-dosing regimen: needed several doses to get maximum protection

c. booster dose: needed to bolster or reinforce protection after a certain period oftime

d. co-administered vaccine: only possible if they don’t interfere each other.

Form of vaccinesa. fluid vaccine (in saline buffer)

53

b. adsorbed vaccine (on a matrix of aluminum or calcium phosphate)

Types of Vaccines based on pharmacological usesa. Viral Vaccines- Smallpox vaccine- Influenza vaccine- Polio vaccine- Rubella vaccine- Measles vaccine- Mumps vaccine- Chickenpox vaccine- Hepatitis vaccines- Rotavirus vaccine

b. Bacterial Vaccines- Pertussis vaccine- Haemophilus influenzae type B conjugate vaccine- Tuberculosis vaccine- Cholera vaccine- Meningococcal polysaccharide vaccine- Pneumococcal vaccine

c. Toxoids- Tetanus toxoid- Diphtheria toxoid- DPT

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54

ANTIBODIES

There are two major uses of antibodies for the treatment of human diseases:1. As drugs to neutralize and eliminate toxic molecules2. As drugs to eliminate target cells

a. Antibody-mediated cell growth controlb. Antibody-coated target cells can be engulfed by phagocytes

(reticuloendothelial clearance).c. Complement-mediated cytotoxicity (CMC)d. Antibody-dependent cell-mediated cytotoxicity (ADCC).

IMMUNOGLOBULINS

-Human Immunoglobulins

-Animal Immunoglobulins

Digoxin Immune Fab (Digibind®)It is the Fab fragment of γ-globulin isolated from sheep (ovine) immunized againstdigoxin. Therefore, it is used in severe digoxin over dosing. It tightly binds digoxinshifting the equilibrium away from drug receptor complex formation. Digoxin is clearedfrom the blood in about 30 minutes.

Digoxin ReceptorDigoxin-Receptor

Complex

Digibind®Digoxin-Digibind®

Complex

++

-Antithymocyte globulin (THYMOGLOBULIN)

Is a purified gamma globulin from the serum of rabbit immunized with humanthymocytes. It contains antibodies that bind to CD2, CD3, CD4, CD8, CD11a, CD18,CD25, CD44, CD45, and HLA class I and II molecules present on the surface of humanT lymphocytes.

Mechanism of action: Deplete circulating T-lymphocytes by direct killing, and blocklymphocyte function by binding to cell surface molecules involved in the regulation ofcell function.

Atgam (Upjohn)Anti-thymocyte equine immunoglobulin (horse IgG).

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Indication: rejection of transplanted kidney, aplastic anemia in patients who are notsuitable for a bone marrow transplant

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56

MONOCLONAL ANTIBODIES

Monoclonal antibody (Mab) is an antibody synthesized by a single clone of B-lymphocytes or plasma cells.

Polyclonal antibodies are multiple immunoglobulins responding to different epitopes onan antigen molecule.

constant region(Fc)

antigen binding region (Fab)

mouse antibody chimeric antibody(66% human)

humanized antibody(>90% human)

fully human antibody

antigen-binding region from

mouse

specific antigen-binding sites from mouse

Nomenclature

The World Health Organization and the US Adopted Name Council (USAN) have thefollowing rules for naming monoclonal antibodies, Fab fragments and their conjugatedand/or radiolabeled products.

1) suffix –mab is to be used for ‘monoclonal antibodies’ and fragments

2) the source of monoclonal antibodies is identified as follows:

‘a’ for rat‘e’ for hamster‘i’ for primate‘o’ for mouse‘u’ for human‘xi’ for chimera

For examples: -umab (human)

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-omab (mouse)-ximab (chimera)

Identification of animal sources will aid healthcare providers in determining if the patientmay have a hypersensitivity reaction to the product.

3) The general disease state subclass is coded into the name:viral (-vir)bacterial (-bac-)immune or immunomodulator (-lim-)Tumors: colon (-col-)

melanoma (-mel-)mammary (-mar-)prostate (-pr(o)-)gonad –testis (-got-)

-ovary (-gov-)miscellaneous (-tum-)

cardiovascular (-cir-)

4) The sequence of stems is: target disease state – animal source - mab

1. MONOCLONAL ANTIBODIES (Mab) THAT ARE SPECIFIC FOR A PARTICULARRECEPTOR

1.1. Mab that attach to a T cell receptor; used to stop rejection of transplantedorgans.

-Muromonab-CD3 (Orthoclone OKT3 Sterile Solution)Monoclonal antibody from mice that interacts with CD3 marker of human T cells. It candecrease T cell numbers in the blood within minutes. It is used daily for 10-14 days, butcannot be used again due to patient’s antibodies production against the murineantibodies.

Indication: rejection of transplanted kidney

Side effects: fluid retention, difficulty in breathing, fever.

-Daclizumab (Zenapax – Roche)A humanized monoclonal IgG1κ produced by rDNA that targets human T cells (Anti-IL-2receptor). It binds the IL-2 receptor α-chain of activated T cells.

Indication: the prevention of rejection of renal allografts. It has also been used in otherorgan transplants. Normally, it is used in combination with cyclosporine or tacrolimusand corticosteroids and is not indicated for acute rejection episodes.

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Recently, it has been experimentally used for the treatment of multiple sclerosis (AnnNeurol. 2004 Dec;56(6):864-7). From 19 patients under investigation, sustained clinicalimprovement (10 patients) or stabilization (9 patients) was observed.

-Basiliximab (Simulect® - Novartis)A chimeric monoclonal IgG1κ. Anti-IL-2 receptor similar to daclizumab.

1.2. Mab that being used for the treatment of malignancies

Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrugtherapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complementdependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment.Modified from Carter P: Improving the efficacy of antibody-based cancer therapies.Nat Rev Cancer 2001;1:118-129.

For Non-Hodgkins Lymphomas-Rituximab (Rituxan® and MabThera® - Genentech)The first antibody approved (in the US: 1997) for the treatment of cancer. A chimeric(Chinese hamster/human) monoclonal IgG1κ that binds CD20 Human B-lymphocyte-restricted differentiation antigen, which is distributed on more than 90% of B cell non-Hodgkin’s lymphomas. But, it is also found in normal B cells.

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-Tositumomab I-131 (Bexxar® – Corica Corp.; GlaxoSmithKline) – approved by FDA inJune 2003.

Tositumomab is murine IgG2aλ monoclonal antibody that is linked covalently withiodine-131. It is an anti-CD20 radioimmunoconjugate approved for the treatment of non-Hodgkin’s lymphomas. When administered as a single dose, it showed impressivetumor response rates with an acceptable toxicity profile.

Mechanism of action: In addition to induce normal immunological reactions, such asinducing apoptosis, mediating antibody-dependent cellular cytotoxicity, andcomplement-dependent cellular cytotoxicity, radioimmunoconjugates deliver cytotoxicionization radiation. The high-energy beta particles emitted by I-131 are cytotoxic overdistances of about 1-2 mm, therefore also kill cells that are inaccessible to the antibody.

At the present time, the use of I-131 tositumomab is limited to patients with diseaserefractory to rituximab therapy.

-Y-90-labeled ibritumomab tiuxetan (Zevalin® - Biogen Idec) – approved by FDA inFebruary 2002.A murine IgGκ, anti CD20; radiolabeled (Yttrium 90).

Anti- HER2 antibodies (HER2 is an oncogene in breast cancer)-Trastuzumab (Herceptin)A humanized monoclonal IgG1κ indicated for the treatment of metastatic breastcarcinomas that over-express the HER2 gene. (The HER2 protein stimulates celldivision).

Mechanism of action: Anti-HER2 antibodies inhibit HER2-mediated signaling in cancercells, ultimately upregulating the levels and activity of p27(Kip1) protein, which lead tocell cycle G(1) arrest and growth inhibition. It is a complex mechanism of action: at leastsix signaling targets and pathways are modulated by trastuzumab.

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Bind to epidermal growth factor receptor-Cetuximab (Erbitux)A chimeric monoclonal antibody (IgG1κ) that bind to epidermal growth factor receptor(EGFR). The EGRF is frequently over-expressed and/or abnormally activated in tumors,therefore, monoclonal antibodies directed at the EGRF can prevent ligand binding whichin turn inhibit the growth of cancer cells.

Indication: metastatic colorectal cancer

Recent studies revealed that a combination of cetuximab with the tyrosine kinaseinhibitor gefitinib resulted in a synergistic effect on cell proliferation and in superiorinhibition of EGFR-dependent signaling and induction of apoptosis.

Antiangiogenic Monoclonal Antibodies-Bevacizumab (Avastin® – Genentech)A recombinant humanized monoclonal antibody (IgG1). Bevacizumab specificallyinhibits vascular endothelial growth factor (VEGF) and has activity in multiple cancer celllines. Bevacizumab has a long half-life, allowing intravenous administration once everytwo-three weeks.

Indication: metastatic colorectal cancer

Recent Warning: increased risk of serious arterial thromboembolic events includingcerebrovascular accidents (stroke), myocardial infarctions, transient ischemic attacks,and angina.Other potential side effects: hypertension, GI perforation, bleeding, proteinuria.

Mab that carry highly toxic antibiotics

-Gemtuzumab ozogamicin (Mylotarg - Wyeth)Is a humanized IgG4κ anti-CD33 monoclonal antibody covalently linked via a suitablelinker to calicheamicin, a cytotoxic natural product isolated from Micromonosporaechinospora. CD33 is expressed on the malignant blast cells in most cases of acutemyeloid leukemia (AML), and immature normal cells of myelomonocytic lineage, but noton normal hematopoietic pluripotent stem cells. Engagement of Mylotarg with CD33results in internalization of the immunoconjugate and hydrolytic release of the toxiccalicheamicin moiety. Calicheamicin binds DNA in the minor groove and causes double-strand DNA breaks, leading to cell death.

Indication: elderly patient with relapsed acute myeloid leukemia (AML).

1.3. Mab that being used as antiviral-Palivizumab (MEDI-493; Synagis)Palivizumab is a humanized monoclonal antibody (IgG1κ). Anti Respiratory SyncytialVirus (RSV). The viral F glycoprotein involved in the fusion of the viral envelope with the

61

host cell membrane during the early stage of infection. So, binding of palivizumab to theF glycoprotein prevents RSV infection.

Indication: preventive treatment of RSV in infants.

1.4. Mab for Crohn’s Disease and Rheumatoid Arthritis

-Infliximab (cA2; Remicade® – Centocor)A chimeric IgGκ that binds to both free and membrane bound TNF-α.Indicated for the treatment of rheumatoid arthritis and Crohn’s Disease and, mostrecently, ankylosing spondylitis.

Recent study also showed that a combination of infliximab and ciprofloxacin tended tobe more effective that infliximab alone to treat perianal Crohn’s disease.

Side effects: Risk of infections. There are reports that large number of cases oftuberculosis in patients receiving infliximab therapy.Recent Drug Warning: severe hepatic reactions including acute liver failure,jaundice/cholestatis, and hepatitis, including autoimmune hepatitis, have been rarelyreported in patients receiving REMICADE. But, a causal relationship between remicadeand the side effects has not been established.

-Adalimumab (HUMIRA - Abbott)A humanized monoclonal antibody (IgG1κ) against TNF-α.Indication: rheumatoid arthritis.Warning: Risk of infections.

1.5. Mab for Multiple Sclerosis and Crohn’s Disease

-Natalizumab (Tysabri - Biogen)A humanized IgG4k. Anti-α4-integrin. A voluntary suspension of natalizumab marketingwas announced on Feb 28, 2005.

1.6. Mab for cardiovascular applications

-Abciximab (ReoPro®)Abciximab is a Fab fragment of the chimeric human-murine monoclonal antibody (IgG1)that has high affinity to and a slow rate of dissociation from glycoprotein IIb/IIIa. The GPIIb/IIIa complex (a platelet surface integrin) is one of the most important receptorsinvolved in platelet aggregation. So, abciximab inhibits platelet aggregation and used forthrombotic arterial diseases including myocardial infarction, unstable angina, peripheralartery disease, and stroke.

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1.7. Mab for Asthma and Allergic Rhinitis

-Omalizumab (Xolair® - developed by Tanox Inc., and licensed by both Genentech andNovartis for US marketing).Omalizumab is a recombinant humanized monoclonal antibody (IgG1κ) selectivelybinds to circulating IgE, therefore, prevents binding of IgE to mast cells and othereffector cells. Without surface-bound IgE, these cells are unable to recognizedallergens, thus preventing cellular activation by antigens and the subsequent allergicreactions. It is used in the treatment of adolescent and adult patients with moderate tosevere allergic asthma inadequately controlled with inhaled corticosteroids.

1.8. Mab for Psoriasis

-Efalizumab (Raptiva® - Genentech)Efalizumab is a recombinant humanized monoclonal IgG1κ antibody that binds CD11a,a cell surface protein that plays a key role in the T cell-mediated steps leading to thepathogenesis of psoriasis. Therefore, efalizumab inhibits the interaction of CD11a (LFA-1) with various ICAM molecules. Because ICAM-1 (CD54) is expressed on activatedendothelial cells and antigen presenting cells (APCs), the antibody inhibits both the APC- T cell interaction and the T cell adhesion to endothelial cells, preventingtransendothelial migration.

2. MONOCLONAL ANTIBODIES THAT CARRY DIAGNOSTIC AGENTS TO TARGETORGANS (ENHANCING SELECTIVITY)

Diagnosis of ovarian and colorectal cancer-OncoScint OV103 and OncoScint CR103 – CytogenUsed for determining the location and extent of ovarian and colorectal carcinoma.Consists of Indium-111, which bound to a murine monoclonal antibody that targetstumor cells.

-CEA-ScanA murine monoclonal antibody fragment linked to 99mTc. CEA-Scan is reactive withcarcinoembryonic antigen, a tumor marker for cancer of the colon and rectum. It isindicated with other standard diagnostic modalities for the detection of recurrent and/ormetastatic colorectal cancer.

Cardiac Imaging-MyoScintMyoScint is a cardiac imaging agent indicated for detecting the presence and location ofmyocardial injury in patients with suspected myocardial infarction. It is a murinemonoclonal antibody Fab fragment linked to 111In. it binds with high affinity andspecificity to human cardiac myosin, which is exposed following a loss of integrity of themyocyte cell membrane. Myoscint is used when electrocardiography and cardiacenzymes are nondiagnostic.

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Prostate Cancer-ProstaScintProstaScint is a monoclonal antibody imaging agent linked to 111In specific to prostatecancer and its metastates. ProstaScint images can help in identifying when the cancerhas spread outside the prostatic bed to regional lymph nodes or to distant soft tissuesites.

Small-cell lung cancer-VerlumaVerluma is a monoclonal antibody Fab fragment linked to 99mTc. It identifies advanced-stage disease in patients with small-cell lung cancer (SCLC). In the clinical trial involving89 patients with confirmed SCLC, Tc-labeled Verluma accurately determined whetherthe disease was extensive or limited 82 percent of the time. If the test indicatedextensive disease, the result was true in 94 percent of the patients. However, if the testindicated limited disease, it was less valuable as a diagnositc aid, failing to imagetumors in some body organs in approximately 23 percent of the patients. Because ofthese false negative readings, additional standard diagnostic tests, such as a bone orCT scan or a bone marrow biopsy should be performed when limited disease is found.

64

ONCOANTIGEN TEST KITS

Carcinoembryonic Antigen (CEA)Carcinoembryonic antigen is a test that measures the amount of carcinoembryonicantigen (CEA) in blood.CEA is a protein that normally occurs in fetal gut tissue. After birth, detectable serumlevels essentially disappear. However, CEA may increase in the presence of variousdisorders such as colon cancer. This test may also be used to determine theresponsiveness of cancer patients to treatment (to determine if cancer is spreading orgoing into remission).The normal range is 0 to 2.5 mcg/L (less than 3 ng/mL). Normal value ranges may varyslightly among different laboratories.

Prostate Specific Antigen (PSA)The PSA test measures the amount of PSA (prostate-specific antigen) in the blood.PSA is a glycoprotein found in prostatic epithelial cells. It can be detected at a low levelin the blood of all adult men.

The PSA level is elevated in men with BPH (benign prostatic hypertrophy) and prostatecancer. Other inflammatory diseases, such as prostatitis, may also increase the PSAlevel.

PSA test kit contains anti-PSA antibody

CA-125CA-125 is a protein that is more prevalent in ovarian cancer cells than in other cells.This protein enters the blood stream and can be measured by a blood test. The test ismost appropriately used to follow women who have already been diagnosed withovarian cancer. In these cases, the CA-125 is a very good indicator of whether a patientis responding to treatment for her cancer, and whether a patient remains in remissionafter treatment. In general, the CA-125 is not a good test to screen healthy women forovarian cancer.

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65

CYTOKINES AS DRUGS

INTERFERONS

α-Interferons (INTRON A – Schering-Plough; ROFERIN-A – Roche; ACTIMMUNE –Genentech)Produced by recombinant DNA in E. coli

Indication: Hairy cell leukemiaMalignant melanomaFollicular lymphomaKaposi’s SarcomaChronic hepatitis BRoferin-A has an indication for Chronic Granulomatous Disease, which is an inheriteddeficiency in phagocytic oxidation metabolism.

Non-naturally occurring rDNA α-interferon (Infergen™; interferon alfacon-1)

β-interferonsInterferon Beta-1a (Avonex-Biogen; Rebif-Serono)Produced by rDNA in Chinese Hamster ovary cells

Indication:Neurological exacerbations in relapsing-remitting multiple sclerosis

Interferon Beta-1b (Betaseron-Chiron/Berlex)Produced by rDNA in E. coliIndication: Neurological exacerbations in relapsing-remitting multiple sclerosis

γ-InterferonsInterferon gamma-1b (Actimmune)Produced by rDNA in E. coliCauses activation of phagocytes

Indication:Reduce the frequency and severity of infections associated with chronic granulomatousdisease.Severe and malignant osteopetrosis (a hereditary disease marked by abnormally densebone)

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66

INTERLEUKINS

Interleukin-2 (IL-2, aldesleukin, PROLEUKIN™ – Chiron/Cetus)

Initially known as T-cell Growth Factor.Indication: cancer, especially metastatic renal cell carcinoma

IL-2 is not directly cytotoxic, but induces a T-cell response cytolytic for tumor cells.

The half-life of IL-2 is short, therefore, it is administered via continuous infusion ormultiple intermittent dosing.

Maximum tolerated doses: 600,000 U/kg

Interleukin-11 (IL-11; NEUMEGA; Oprelvekin™)

IL-11 is known as a platelet growth factor.Commercially produced by rDNA in E. coli and is one amino acid shorter than thehuman IL-11.

Indication: to increase platelets and decrease the need for platelet transfusionsfollowing myelosuppresive chemotherapy in patients with nonmyeloid malignancies.

It is administered by subcutaneous injection into the abdomen, thigh, hip and upperarm.

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INTERLEUKIN RECEPTOR ANTAGONISTS

-Anakinra (KINERET – Amgen, Inc.)A recombinant human IL-1 receptor antagonist for the treatment of rheumatoid arthritis.IL-1 receptor antagonist blocks the binding of IL-1 (a cytokine present in excess inrheumatoid arthritis patients as part of the inflammatory responses). When given aloneor in combination with methotrexate, it improves clinical sign and symptoms, decreasesradiographic progression, improves patient function, decreases pain and fatigue, andslows bone erosion. Anakinra is generally well tolerated. Side effects include increasethe possibility of serious infection, such as tuberculosis and other opportunisticinfections. Anakinra should not be given in combination with anti-TNF agents.

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GROWTH FACTORS

GRANULOCYTE-COLONY STIMULATING FACTOR (G-CSF)

Filgrastim (NEUPOGEN™ – Amgen)Produced by rDNA in E. coliIndication: neutropenia due to non-myeloid cancer chemotherapy or severe cronic

neutropeniato reduce the duration of neutropenia after bone marrow transplant

*G-CSF could promote growth of malignant myeloid cells

Pegfilgrastim (NEULASTA – Amgen)Produced by rDNA in E. coli.Indication is the same as filgrastim (myelosuppressive regiments). Stimulate bonemarrow to produce more white blood cells.

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GRANULOCYTE-MACROPHAGE-COLONY STIMULATING FACTOR (GM-CSF)Sargramostim (LEUKINE – Immunex; PROKINE – Hoechst-Roussel)

Produced by rDNA in yeastIndication: Hasten myeloid reconstitution after autologous bone marrow replacement.

ERYTHROPOIETIN

EPOETIN ALFA (EPOGEN™ – Amgen; PROCRIT™ – Ortho Biotech; EPREX™ –Johnson&Johnson)

Indication: Kidney dialysisChemotherapy in non-myeloid malignancies

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DARBEPOETIN ALFA (ARANESP – Amgen)Differs from epoetin alfa by having two extra carbohydrate chains, which gives it greatermetabolic stability resulting in a three-fold longer half-life.Indication: Anemia associated with chronic renal failure and cancer chemotherapy.

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SYNTHETIC DRUGS

LEUKOTRIENE D4 RECEPTOR INHIBITORSZafirleukast (Accolate)Montelukast Sodium (Singulair)

5-LIPOOXYGENASE INHIBITORSZileuton (Zyflo)

Fatty acidin diet esterified fatty acids

Triggering of an immune response

mash cells, macrophages,neutrophils, lymphocytesinitiate a reaction

activation of phospholipase A2or other acylhydrolases

(e.g. phospholipids)

COOH

arachidonic acid cyclooxygenase-2or

cyclooxygenase-1

cytokinesgrowth factorsendotoxin

dexamethasone

COOH

false substrates

5,8,11,14-eicosatetraynoic acid

lipoxygenases (LP)

aspirinindomethacin COOHO

OOOH

PGG2

COOHO

OOH

PGH2COOH

OH

thromboxane synthase

dazoxiben

TXA2OO

other thromboxanes

PGD2PGE2PGF2

etc.

prostacyclinsynthase

PGI2

urinary metabolites

COOH

5-HPETE

OOH12-HPETE

12-HETE

5-LP12-LPzileuton

COOH

C5H11

O

LTA4

LT synthase

COOH

C5H11LTC4

LTB4

OH

SCys-Gly

H

γ-Glu

LTD4LTE4 Chemotaxis, spasmogens, inflammation, mucus production, etc.

zafirlukastmontelukast Na(LTD4 receptorantagonist)

chemotaxis

rofecoxib (Vioxx)celecoxib (Celebrex)valdecoxib (Bextra)

(NSAIDS)

Zafirleukast (Accolate)

Zafirleukast is a reversible and competitive inhibitor of the cysteinyl-leukotriene receptor(leukotriene D4 (LTD4) receptor antagonist).

Indications: Asthma (for both early and late responses). It is intended for chronic userather than for acute exacerbations.Dosing: 20 mg bid, 1 h before or 2 h after meals.

Metabolism: half-life is about 10 h (98% bound to serum albumin). Metabolized by theCYP450 3A4 and CYP2C9.

69

Zafirleukast but not montelukast may interact with warfarin and increase prothrombin-times.

N

HN O

O

H3C

OCH3

NH

S

O

O

CH3

O

Zafirlukast

Montelukast Sodium (Singulair)

NCl

S

HO

O-

O

Na+

Montelukast Sodium

The mechanism of action of montelukast sodium is similar with that of zafirleukast.

Indications: Chronic asthma. Approved for pediatric use. Recently approved for allergicrhinitis.

Contraindications: acute asthma attacks including status asthmatis; monotherapy forexercise-induced bronchospasm.

Dosing: Available as 5 mg chewable tablet (for kids) and 10 mg tablets. Can beadministered once daily. It is recommended that children take it in the evening, but thereis no evidence whether morning or evening dosing is better.

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Zileuton (ZYFLO)

S N

CH3

HO NH2

O

Zileuton

Zileuton inhibits the 5-lipoxygenase enzyme responsible for the conversion ofarachidonic acid to 5-HPETE, which prevents the formation of subsequent leukotrienes.

Indication: Asthma

Metabolism: half-life is about 2.5 h. Drug-drug interactions are reported but not muchabout their clinical significance.

Adverse effects: Zileuton decreases the steady-state clearance of theophillin andwarfarin. 4%-5% of patients taking zileuton have an elevation of liver enzymes.

Dosing: 600 mg qid.

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MAST CELL STABILIZERS

Ca2+influx

allergen, anti-IgE, lectins

complements(C3a and C5a)

drugs, e.g.,codeine, morphine, synthetic ACTH

calcium ionophoremellitincompound 48/80

preformedmediators

newly generatedmediatorscromolyn sodium

nedocromil sodiumlodoxamide tromethamine

olopatadine HCl

-Cromolyn Sodium (INTAL)

O

O OCH2CHCH2O

NaOOC O

O

COONa

OH

cromolyn sodium

Cromolyn is one of the first-line agents in the treatment of mild and moderate asthma.

Mechanism of action: In asthma, it is not clearly known, but a variety of activities thatmay related to its therapeutic effect in asthma were reported:1) inhibiting mediator release from bronchial mast cells2) ability to reverse increased functional activation in leukocytes obtained from the bloodof asthmatic patient3) suppression of the activating effects of chemotactic peptides on human neutrophils,eosinophils, and monocytes4) inhibition of parasympathetic and cough reflexes5) inhibition of leukocytes trafficking in asthmatic airways

For asthma, cromolyn is given by inhalation (usually by aerosol spray or nebulizer).It is ineffective in treating on-going broncho-constriction.Approved for all ages

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-Nedocromil Sodium (ALCOCRIL)

ON

O

COONa

O

CH2CH2CH3

NaOOC

H3CH2C

nedocromil sodium

In many aspects nedocromil sodium is similar to cromolyn. It is also used in thetreatment of mild and moderate asthma. It is more effective than cromolyn. Approved forused in asthmatic patients 12 years old and older.

-Lodoxamide Tromethamine (ALOMIDE - Alcon)

NH

NH

Cl

CN

-O

O

O OO-

O

CH2OHH3N

CH2OHCH2OH

2

Lodoxamide tromethamine

Lodoxamide is an anti allergic medication for eyes (0.1% ophthalmic solution).

Mechanism of action: prevent the antigen-stimulated release of histamine and otherinflammatory mediators or it may also prevent the calcium influx into mast cells.Lodoxamide shows no vasoconstrictor, antihistaminic, cyclooxygenase inhibition, orother anti-inflammatory activity.

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-Olopatadine HCl (PATANOL – Alcon)

O

NHCH3

CH3

O

OH

Cl-

Olopatadine HCl

Olopatadine is also an anti allergic medication for eyes.

Mechanism of action: inhibits the release of histamine from mast cells.

Patanol is 0.1% solution of olopatadine.

-Amlexanox Oral Paste (APHTHASOL; Aptheal -5% amlexanox paste-)

O N

O

NH2

COOOH

Amlexanox

Amlexanox inhibits the formation and/or release of inflammatory mediators such ashistamine and leukotrienes. Used for aphthous ulcers in patients with functioningimmune systems. The drug is applied directly to each ulcer in the mouth at their earliestsign or symptoms. Repeat four times a day after thoroughly cleaning the mouth aftereach meal.

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TNF-α INHIBITORS

-Thalidomide (THALOMID)

N

O

ONH

O

OThalidomide

Thalidomide is best known for the severe, life-threatening birth defects it has causedwhen administrated to pregnant women.

Indication: erythema nodosum leprosum (ENL).

Mechanism of action: unclear, but it has been reported to decrease circulating TNF-α inpatients with ENL but to increase it in patients with HIV-seropositive.

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IMMUNOMODULATORS WHOSE MAIN INDICATION IS RA

-Etanercept (ENBREL)

A dimeric fusion protein consists of two extracellular ligand-binding protein portions ofthe human 75 kDa (p75) tumor necrosis factor (TNF) receptor linked to the Fc portion ofhuman IgG1. Total 934 aa.

Indication: moderate to severe rheumatoid arthritis, psoriasis, anklyosing spondylitis.

Mechanism of action: binds TNF which in turn slowing down or stopping jointdestruction. Clinical responses usually seen within 1 – 2 weeks.

Administered subcutaneously (60% bioavailability)

Side effect: increase the susceptibility to infectious disease

-Infliximab (cA2; REMICADE – Centocor)A chimeric IgGκ that binds to both free and membrane bound TNF.

-Adalimumab (HUMIRA - Abbott)A humanized monoclonal antibody (IgG1κ) against TNF-α.Indication: rheumatoid arthritis.Warning: Risk of infections.

-Anakinra (KINERET – Amgen, Inc.)A IL-1 receptor antagonist.

-Leflunomide (ARAVA)

NO CH3

NHO

CF3

NHO CH3

NHO

CF3Leflunomide A771726

Leflunomide is the prodrug of the active anti-rheumatic metabolite A77 1726.A77 1726 suppresses lymphocyte proliferation and also inhibits inflammation cytokineproduction.It has been reported that leflunomide is superior to methotrexate in improving arthritisand joint function in established adjuvant-induced arthritis.

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Mechanism of action: A77 1726 inhibits dihydroorotate dehydrogenase required forpyrimidine biosynthesis. It also inhibits tyrosine kinase activity instituted by cytokinesand growth factors, suggesting that the drug interferes with T- and B-cell proliferation.

Side effects: liver injuries and liver toxicity.

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IMMUNORESTORATIVE AGENTS (IMMUNOSTIMULANTS)

-Levamisole HCl (ERGAMISOL)

N

SNHHCl

Levamisole HCl

Restore depressed immune function of B cells, T cells, monocytes, and macrophages,which is due to chemotherapy in cancer patient. Therefore, its clinically indication is asan adjuvant treatment with fluorouracil after surgical resection in patients with colorectalcarcinoma.

-Imiquimod (ALDARA – 3M Pharmaceutical)

N

N

N

NH2

CH3

CH3Imiquimod

Indication: Treatment of external genital and perianal warts caused by humanpapillomavirus, as well as actinic keratosis (=precancerous skin condition that developin sun-exposed skin).

Mechanism of action: Unclear. It is proposed that imiquimod can stimulate the immunesystem to produce interferon and similar molecules that attack the wart virus.

Available as 5% and 1% cream. Apply it to the effected area three times a week beforegoing to bed and left it on the skin for 6-10 h.