SBC 252: BASIC IMMUNOLOGY

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Course Outline

• Introduction• Components of the immune system- cells and

tissues• Types of immunity- innate and adaptive• The complement system of innate immunity• Basics of immunoglobulins• Autoimmune diseases• Tumour immunology• Congenital immunological disorders• Evolution and development of the immune system

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References

• Medical immunology, revised and expanded, 5th edition. Virella G, 2001

• Immunobiology: The immune system in health and disease, 6th edition. Janeway , Travels, Walport and Ahlomochik, 2005

• Cell and Molecular biology: concepts and experiments, 3rd Edition, Karp G, 2003

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Objectives

• Student should :

-Explain the response of a body to infections-using both innate and adaptive mechanisms.

-Explain the generation of diversity by theimmunoglobulin that facilitates its specificityin function.

-Explain how tumor cells arise in the body andthe future prospects .

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-Explain the evolution of immune system

-Observe and name different types of immunecells in human blood.

Course delivery

-Lectures, assignments and presentations,practical

Assessment

-one written CATs, Assignment (30%), Universityfinal exam (70%)

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• Immunology -Branch of biomedical scienceconcerned with the response of the organismto immunogenic (antigenic) challenge, therecognition of self from nonself, and all thebiological ( in vivo ), serological ( in vitro ),physical, and chemical aspects of immunephenomena.

Introduction-Definations and History of immunology

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• immunity -A state of acquired or innateresistance or protection from a pathogenicmicroorganism or its products or from the effectof toxic substances such as snake or insectvenom.

• Or

-is the ability to resist infection.

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• innate immunity -It is the immunity present inall individuals at all times and doesn’t increasewith repeated exposure to a given pathogen, anddiscriminates between groups of similarpathogens / Natural or native immunity that ispresent from birth and is designed to protect thehost from injury or infection without previouscontact with the infectious agent.

e.g. protection by the skin, mucous membranes,lysozyme in tears, stomach acid.

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• adaptive immunity-immunity that results inprotection from an infectious disease agent asa consequence of clinical or subclinicalinfection with that agent or by deliberateimmunization against that agent withproducts from it.

-Its mediated by B and T lymphocytes followingexposure to specific antigen.

-It is characterized by specificity, immunologicalmemory, and self/nonself recognition.

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- immune response is the response made by thehost to defend itself against a pathogen.

- The primary immune response is the adaptiveimmune response to an initial exposure toantigen.

-Adaptive immune response -a specific immuneresponse that is characterised by theproduction of antibodies against a particularpathogen.

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• The immune system is the name used todescribe the tissues, cells, and moleculesinvolved in adaptive immunity, or innateimmunity sometimes the totality of hostdefense mechanisms.

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Historical development

-The fundamental observation that led to thedevelopment of immunology as a scientificdiscipline was that an individual mightbecome resistant for life to a certain diseaseafter having contracted it only once.

-The term immunity, derived from the Latinimmunis (exempt), was adopted to designatethis naturally acquired protection againstdiseases such as measles or smallpox.

-The emergence of immunology as a disciplinewas closely tied to the development ofmicrobiology.

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-1796 – Edward Jenners discovered that cowpox(viccinia) induced protection against humansmall pox. 1979 WHO announced that smallpox had been eradicated.

-19th Century microbiologists and Robert Kochproved that infectious diseases are caused bymicro-organisms, which are responsible for aparticular disease / pathology. Four broadcategories of disease causing microorganisms(pathogens) are: viruses, bacteria, pathogenicfungi and parasites (complex eukaryoticorganisms).

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-In 1880s’ Louis pasteur devised a vaccineagainst cholera in chickens and a rabiesvaccine.

- Early 1890’s Emil Von Bohring and ShibasaburoKitasaso discovered that serum of animals’immune to diphtheria or tetatus contained aspecific antitoxic activity, the antibody thatconferred a short l ived immunity onunimmunized individuals.

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-Infectious diseases could therefore beprevented by exposure to killed or attenuatedorganisms or to compounds extracted fromthe infectious agents.

-The impact of immunization against infectiousdiseases such as tetanus, measles, mumps,poliomyelitis, and smallpox, can be graspedwhen we reflect on the fact that thesediseases, which were at one time significantcauses of mortality and morbidity, are noweither extinct or very rarely seen.

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-Both- innate and adaptive immune responsesprovide the defense system of the body,ensuring we become ill only relatively rarely.

- Immunology now transcends its earlyboundaries and has become a more generalbiomedical discipline. Today, the study ofimmunological defense mechanisms is still ani m p o r t a n t a r e a o f r e s e a r c h , b u timmunologists are involved in a much widera r r a y o f p r o b l e m s e . g s e l f - n o n s e l fdiscrimination, control of cell and tissuedifferentiation, transplantation, cancerimmunotherapy, etc.

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• The focus of interest has shifted toward thebasic understanding of how the immunesystem works in the hope that this insight willallow novel approaches to its manipulation.

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THE COMPONENTS OF THE IMMUNE SYSTEM

Definations to remember

-Immune system- Name used to describe thetissues, cells and molecules involved in innateand adaptive immunity or sometimes totality ofhost defence mechanisms.

-Immunity- Ability to resists infections.

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-Antigen- any molecule that can bind specificallyto an antibody. The name arise from theirability to generate antibodies. Some antigensdo not by themselves elicit antibodyproduction.

-Antigens that can induce antibody productionare called immunogens.

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A: CELLS OF THE IMMUNE SYSTEM

-Cells of the immune system originate in thebone marrow.

- After they mature, they then migrate to guardthe peripheral tissues, circulating in the bloodand in the lymphatic system.

-All the cellular elements of blood: the red bloodcells that transport oxygen, the platelets thattrigger blood clotting in damaged tissues, andthe white blood cells of the immune system,derive from the same progenitor or precursorcells- the hematopoietic stem cells in thebone marrow.

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-Since these stem cells can give rise to all of the different types of blood cells, they are often known as pluripotent hematopoietic stem cells.

-Initially, they give rise to stem cells of more limited potential, which are the immediate progenitors of red blood cells, platelets, and the two main categories of white blood cells.

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Types of stem cells:

A) Common myeloid progenitor

- gives rise to different types of leukocytes(white blood cells), erythrocytes, and themegakaryocytes that produce platelets.

-The precursor of the granulocyte, macrophages,dendritic cells and the mast cells of theimmune system.

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i. Macrophages

-Are one of the three types of phagocyte in theimmune system and are distributed widely inthe body tissues, where they play a criticalpart in innate immunity.

-They are the mature form of monocytes, whichcirculate in the blood and differentiatecontinuously into macrophages uponmigration into the tissues.

-Their activation results in: phagocytosis andactivation of bactericidal mechanisms andantigen presentation.

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ii. Dendritic cells

-Are specialized to take up antigen and display itfor recognition by T lymphocytes.

- Immature dendritic cells migrate from theblood to reside in the tissues and are bothphagocytic and macropinocytic (ingest largeamounts of the surrounding extracellularfluid).

-They continously migrate from the tissuebearing self antigens and induce tolerance asthey lack the necessary co-stimulatingmolecules.

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-Upon encountering a pathogen, they rapidlymature, express co-stimulatory molecules andmigrate to lymph nodes.

iii. Mast cells

-Their blood-borne precursors are not welldefined.

- They differentiate in the tissues.

-They mainly reside near small blood vessels andwhen activated, they release substances thataffect vascular permeability.

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-They play a role in orchestrating allergicresponses.

-They play a part in protecting mucosal surfacesagainst pathogens.

-Their activation results in release of granulescontaining histamine and other active agents.

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iv. Granulocytes / polymorphonuclear (PMN) leukocytes

-Are called granulocytes because they havedensely staining granules in their cytoplasm.

-Are called polymorphonuclear leukocytesbecause of their oddly shaped nuclei. Thereare three types of granulocyte:

- all are relatively short lived

- are produced in increased numbers duringimmune responses, when they leave theblood to migrate to sites of infection orinflammation.

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a) Neutrophils- Are phagocytic cell of theimmune system.

- are the most numerous and most importantcellular component of the innate immuneresponse: hereditary defic iencies inneutrophil function lead to overwhelmingbacterial infection, which is fatal if untreated.

- Their activation results in phagocytosis andactivation of bectericidal mehanisms.

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b)Eosinophils -are important chiefly in defenseagainst parasitic infections, because their

numbers increase during a parasitic infection.-Their activation results in killing of antibody-coated parasites.

c)Basophils- play a role in allergic inflammation.

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Granulocytes (polymorphonuclear leukocytes)

B. Common lymphoid progenitor

-Give rise to the T and B lymphocytesresponsible for adaptive immunity and thenatural killer (NK) cells of innate immunity. Alsogive rise to dendritic cells that appearindistinguishable from those derived from thecommon nyeloid progenitor but majority ofdendritic cells in the body arise from commonmyeloid progenitor.

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Lyphocytes

- Most lymphocytes are small, featureless cellswith few cytoplasmic organelles and much of thenuclear chromatin inactive.

-The small lymphocytes have no functional activityuntil they encounter antigen and co-stimulatorymolecules (molecules induced by innateimmunity), which provide signals necessary totrigger their proliferation and the differentiationof their specialized functional characteristics.

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-There are two major types of lymphocyte:

a) B lymphocytes or B cells- when activateddifferentiate into plasma cells that secreteantibodies

b)T lymphocytes or T cells- there are two mainclasses.

- One class differentiates on activation intocytotoxic T cells which kill cells infected withviruses.

- The second class differentiates into cells thatactivate other cells such as B cells andmacrophages

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-T and B lymphocytes are distinguished by:

a) their sites of differentiation: T cells in thethymus and B cells in the bone marrow

b)by their antigen receptors- Lymphocytesmount a specific immune response againstvirtually any foreign antigen.

This is possible because each individuallymphocyte matures bearing a unique variant(differing slightly/ changeable) of a prototype(full size functional model) antigen receptor.

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Thus, a population of T and B lymphocytescollectively bear a huge repertoire ofreceptors that are highly diverse in theirantigen-binding sites.

-The B-cell antigen receptor (BCR) is amembrane-bound form of the antibody thatthe B cell will secrete after activation anddifferentiation to plasma cells. Antibodymolecu les a s a c la ss are known asimmunoglobulins (Ig) therefore, the antigenreceptor of B lymphocytes is known asmembrane immunoglobulin (mIg).

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-The T-cell antigen receptor (TCR) is adapted todetect antigens derived from foreign proteinsor pathogens that have entered into host cells.

-Mature T and B lymphocytes circulate betweenthe blood and peripheral lymphoid tissues.After encounter with antigen, B cellsdifferentiate into antibody secreting plasmacells, whereas T cells differentiate intoeffector T cells with a variety of functions.

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Natural killer cells

-They lack antigenspecific receptors and are part ofthe innate immune system.

-They circulate in the blood as large lymphocyteswith distinctive cytotoxic granules.

- They are able to recognize and kill someabnormal cells e.g.some tumor cells and virus-infected cells e.g. herpes viruses.

- Are important in the innate immune defenseagainst intracellular pathogens.

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B) Lymphoid organs

-Lymphoid organs are organized tissuescontaining large numbers of lymphocytes in aframework of nonlymphoid cells.

-In these organs, the interactions lymphocytesmake with nonlymphoid cells are importanteither to lymphocyte development, to theinitiation of adaptive immune responses, or tothe sustenance of lymphocytes.

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-Lymphoid organs-can be divided broadly intocentral or primary lymphoid organs- wherelymphocytes are generated,

and peripheral or secondary lymphoid organs-where adaptive immune responses areinit iated and where lymphocytes aremaintained.

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Central lympoid organs- Are the bone marrow and the thymus ( a large

organ in the upper chest).

-Both B and T lymphocytes originate in the bonemarrow but only B lymphocytes mature there.

- T lymphocytes migrate to the thymus tou n d e r g o t h e i r m a t u r a t i o n . ( T h u s Blymphocytes are so-called because they arebone marrow derived, and T lymphocytesbecause they are thymus derived).

-Lymphocytes recirculate between theperipheral lymphoid organs and the blooduntil they encounter their specific antigens.

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-Once they have completed their maturation,both types of lymphocyte enter thebloodstream, from which they migrate to theperipheral lymphoid organs, where adaptiveimmune responses are initiated and thelymphocytes are maintained.

- T cells receive survival signals from dendriticcells in the periphery. B cells receive survivalsignals from lymphoid follicles.

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Distribution of organs in the body

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-Lymphocytes arise from stem cells in bonemarrow, and differentiate in the centrallymphoid organs (yellow), B cells in bonemarrow and T cells in the thymus.

-They migrate from these tissues and are carriedin the bloodstream to the peripheral orsecondary lymphoid organs (blue), the lymphnodes, the spleen, and lymphoid tissuesassociated with mucosa, like the gut-associated tonsils, Peyer's patches, andappendix.

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-The peripheral lymphoid organs are the sites oflymphocyte activation by antigen, andlymphocytes recirculate between the bloodand these organs until they encounter antigen.Lymphatics drain extracellular fluid from theperipheral tissues, through the lymph nodesand into the thoracic duct, which empties intothe left subclavian vein.

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-This fluid, known as lymph, carries antigen tot h e l y m p h n o d e s a n d r e c i r c u l a t i n glymphocytes from the lymph nodes back intothe blood.

-Lymphoid tissue is also associated with othermucosa such as the bronchial linings.

-Pathogens can enter the body by many routesand set up an infection anywhere, but antigenand lymphocytes will eventually encountereach other in the peripheral lymphoid organsthe lymph nodes, the spleen, and the mucosallymphoid tissues.

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- Lymphocytes are continually recirculatingthrough these tissues, to which antigen is alsocarried from sites of infection, primarily withinmacrophages and dendritic cells.

-Within the lymphoid organs, specialized cellssuch as mature dendritic cells display theantigen to lymphocytes.

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The peripheral lymphoid organs

include the lymph nodes, the spleen, andlymphoid tissues associated with mucosa (e.g.the gut-associated tonsils, Peyer's patches,and appendix).

-The organs :

-Are specialized to trap antigen

-They allow the initiation of adaptive immuneresponses

-They provide signals that sustain recirculatinglymphocytes.

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a) The lymph nodes

The organization of lymph node. 2016/9/13 49

- A lymph node consists of an outermost cortexand an inner medulla.

- The cortex is composed of an outer cortex ofB cells organized into lymphoid follicles, anddeep or paracortical areas made up mainly ofT cells and dendritic cells.

-Lymph nodes are highly organized lymphoids t ructures located at the po ints ofconvergence of vessels of the lymphaticsystem.

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-Lymphatic system is an extensive system ofvessels (lymphatic vessels or lymphatics) thatcollects extracellular fluid from the tissues andreturns it to the blood.

-This extracellular fluid is produced continuouslyby filtration from the blood, and is calledlymph.

Lymph draining from the extracellular spaces ofthe body carries antigens in phagocyticdendritic cells and macrophages from thetissues to the lymph node via the afferentlymphatics, where they are trapped.

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- Lymph leaves by the efferent lymphatic in themedulla.

- The medulla consists of strings of macrophagesand antibody-secreting plasma cells known asthe medullary cords.

- Naive lymphocytes enter the node from thebloodstream through specialized postcapillaryvenules and leave with the lymph through theefferent lymphatic.

-In the lymph nodes, B lymphocytes are localizedin follicles. The T cells are more diffuselydistributed in surrounding paracortical areas,also referred to as T-cell zones.2016/9/13 52

- Some of the B-cell follicles include germinalcenters, where B cells undergo intenseproliferation after encountering their specificantigen and their cooperating T cells.

- This segregation of B and T lymphocytespromotes the crucial interactions that occurbetween B and T cells upon encounteringantigen (this is the case in other peripherallymphoid tissues).

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-It is a fist-sized organ just behind the stomachthat collects antigen from the blood.

- It also collects and disposes of senescent red blood cells (aging or approaching an advanced age). The bulk of the spleen is composed of red pulp, which is the site of red blood cell disposal / destruction interspersed with lymphoid white pulp.

-The lymphocytes surround the arterioles entering the organ, forming areas of white pulp.

b. The spleen

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-Lymphocytes and antigen- loaded dendriticcells come together in the periarteriolarlymphoid sheath.

- In each area of white pulp, blood carryinglymphocytes and antigen flows from atrabecular artery into a central arteriole. Cellsand antigen then pass into a marginal sinusand drain into a trabecular vein.

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-The marginal sinus is surrounded by a marginalzone of lymphocytes.

-Within the marginal sinus and surrounding thecentral arteriole is the periarteriolar lymphoidsheath (PALS), made up of T cells.

-The follicles consist mainly of B cells; insecondary follicles a germinal center issurrounded by a B-cell corona.

-NB//Although the organization of the spleen issimilar to that of a lymph node, antigen entersthe spleen from the blood rather than fromthe lymph.

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c)The gut-associated lymphoid tissues (GALT)

- The bulk of the tissue is B cells, organized in alarge and highly active domed follicle. T cellsoccupy the areas between follicles. Theantigen enters across a specialized epitheliummade up of multi-fenestrated ( M) cells.

-GALT Include the tonsils, adenoids, andappendix, and specialized structures calledPeyer's patches in the small intestine.

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-They collect antigen from the epithelialsurfaces of the gastrointestinal tract.

-The Peyer's patches are the most importantand highly organized of these tissues.

-The antigen is collected by specialized epithelialcells called multi-fenestrated or M cells.

-The lymphocytes form a follicle consisting of alarge central dome of B lymphocytessurrounded by smaller numbers of Tlymphocytes.

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- Bronchial-associated lymphoid tissue (BALT)are more diffuse aggregates of lymphocytesthat protect the respiratory epithelium.

-Those that protect other mucosa are known asmucosal associated lymphoid tissue (MALT).Collectively, the mucosal immune system isestimated to contain as many lymphocytes asall the rest of the body, and they form aspecialized set of cells obeying somewhatdifferent rules.

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Organization of typical gut-associated lymphoid tissue2016/9/13 62

NB// Although very different in appearance, thelymph nodes, spleen, and mucosal-associatedlymphoid tissues all share the same basicarchitecture.

-Each of these tissues operates on the sameprinciple, trapping antigen from sites ofinfection and presenting it to migratory smalllymphocytes, thus inducing adaptive immuneresponses.

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-The peripheral lymphoid tissues also providesustaining signals to the lymphocytes that donot encounter their specific antigen, so thatthey continue to survive and recirculate untilthey encounter their specific antigen.

- This is important in maintaining the correctnumbers of circulating T and B lymphocytes,and ensures that only those lymphocytes withthe potential to respond to foreign antigenare sustained.

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-Small B and T lymphocytes that have maturedin the bone marrow and thymus but have notyet encountered antigen are referred to asnaive lymphocytes

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TYPES OF IMMUNITY

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A. Innate immunity

- serves as a first line of defense-.

- Form early barriers to the establishment ofthe infection in the host.

- T h e in n a te i m m u n e m ech a n i s m s a c timmediately, and are followed by earlyinduced responses, which can be activated byinfection but do not generate lastingprotective immunity.

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-Only if an infectious organism can breach theseearly lines of defense will an adaptive immuneresponse ensue, with the generation ofantigen-specific effector cells that specificallytarget the pathogen, and memory cells thatcan prevent reinfection with the samemicroorganism.

-Innate immunity depends upon germline-encoded receptors to recognize features thatare common to many pathogens.

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i. Innate immunity is the front line of host defense.

-Microorganisms that cause pathology in humansand animals enter the body at different sites andproduce disease by a variety of mechanisms.Many different infectious agents can causepathology, and those that do are referred to aspathogenic microorganisms or pathogens.

-Invasions by microorganisms are initiallycountered, in all vertebrates, by innate defensemechanisms that preexist in all individuals andact within minutes of infection.

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-Only when the innate host defenses arebypassed, evaded, or overwhelmed is aninduced or adaptive immune responserequired.

-Our bodies are constantly exposed tomicroorganisms present in the environment,including infectious agents that have beenshed from infected individuals.

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-Contact with these microorganisms may occurthrough external or internal epithelial surfaces:the respiratory tract mucosa provides a routeof entry for airborne microorganisms, thegastrointestinal mucosa for microorganisms infood and water; insect bites and woundsallow micro-organisms to penetrate the skin;and direct contact between individuals offersopportunities for infection of the skin andreproductive mucosa.

Examples- Table 1

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b. Stages of resonse to an infection / infectiuos agent by the innate system

i). The epithelial surface barrier

-The epithelial surfaces of the body serve as an effective barrier against most microorganisms, and are rapidly repaired if wounded.

-provide a physical barrier between the internal milieu and the external world that contains pathogens. Epithelial cells are held together by tight junctions, which effectively form a seal against the external environment.

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-Epithelia comprise the skin and the linings of thebody's tubular structures -the gastrointestinal,respiratory, and urinogenital tracts. Infectionsoccur only when the pathogen colonize or crossthrough these barriers.

-The dry, protective layers of the skin present amore formidable barrier and pathogen entrymost often occurs through the internal epithelialsurfaces, when the barrier is breached e.g. inwounds and burns.

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-In the absence of wounding or disruption,pathogens normally cross epithelial barriersby binding to molecules on internal epithelialsurfaces, or establish an infection by adheringto and colonizing these surfaces.

This specific attachment allows the pathogen toinfect the epithelial cell, or to damage it sothat the epithelium can be crossed, or, in thecase of colonizing pathogens, to avoid beingdislodged by the flow of air or fluid across theepithelial surface.

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-The internal epithelia ( are known as mucosalepithelia) secrete a viscous fluid called mucus,which contains many glycoproteins called mucins.E.g. the respiratory tract.

-Microorganisms coated in mucus are expelled inthe flow of mucus driven by the beating ofepithelial cilia. People with defective mucussecretion or inhibition of ciliary movementfrequently develop lung infections caused bybacteria that colonize the epithelial surface.

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- In the gut, peristalsis mechanism keep bothfood and infectious agents moving through.Failure of peristalsis is accompanied byovergrowth of bacteria within the intestinallumen.

-The surface epithelia produce chemicalsubstances that are microbicidal or inhibitmicrobial growth e.g. the antibacterialenzyme lysozyme and phospholipase A aresecreted in tears and saliva. The salivacontains several histidine-histidine richpeptides with antimicrobial properties.

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-The acid pH of the stomach and the digestiveenzymes, bile salts, fatty acids and lysolipidsfound in the upper gastrointestinal tractcreate a substantial chemical barrier toinfection.

-Antibacterial and antifungal peptides called cryptidins or α-defensins are made by Paneth cells, which are resident in the base of the crypts in the small intestine beneath the epithelial stem cells.

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- Antimicrobial peptides, the β-defensins, aremade by other epithelia, primarily in the skinand respiratory tract.

- Antimicrobial peptides play a role in theimmune defense of many organisms,including insects. They are cationic peptidesthat are thought to kill bacteria by damagingthe bacterial cell membrane.

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-The antimicrobial protein secreted into thefluid that bathes the epithelial surfaces of thelung contains two proteins surfactant proteinsA and D that bind to and coat the surfaces ofpathogens so that they are more easilyphagocytosed by macrophages that have leftthe subepithelial tissues to enter the alveoli ofthe lung.

Coating of a particle with proteins that facilitateits phagocytosis is known as opsonization.

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-Most epithelial surfaces are associated with anormal flora of nonpathogenic bacteria thatcompete with pathogenic microorganisms fornutrients and for attachment sites on cells.

- The normal flora can also produce antimicrobialsubstances e.g. colicins (anti-bacterial proteinsmade by Escherichia coli) and lactic acid

- Colicins prevent colonization by other bacteria.When the nonpathogenic bacteria are killed byantibiotic treatment, pathogenic microorganismsfrequently replace them and cause disease.

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b) recognition, ingestion and killing ofpathogens by phagocytes (macrophages andn e u t r o p h i l s )-If a microorganism crosses an epithelial barrier

and begins to replicate in the tissues of thehost, it is immediately recognized by themononuclear phagocytes, or macrophages,that reside in tissues.

-Macrophages mature continuously fromcirculating monocytes that leave thecirculation to migrate into tissues throughoutthe body.

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-They are found in especially large numbers inconnective tissue, in association with thegastrointestinal tract, in the lung (where theyare found in both the interstitium and thealveoli), along certain blood vessels in the liver(Kupffer cells), and throughout the spleen,where they remove senescent blood cells.

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- The second major family of phagocytes then e u t r o p h i l s , o r p o l y m o r p h o n u c l e a rneutrophilic leukocytes (PMNs or polys) areshort-lived cells that are abundant in theblood but are not present in normal, healthytissues.

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-Macrophages are the first to encounterpathogens in the tissues but they are soon re-inforced by the recruitment of large numbersof neutrophils to sites of infection.

-These two phagocytic cells play a key role ininnate immunity by recognizing, ingesting,and destroying many pathogens without theaid of an adaptive immune response.

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-Macrophages and neutrophils recognizepathogens by means of cell-surface receptorsthat discriminate between the surfacemolecules displayed by pathogens and thoseof the host e.g. the macrophage mannosereceptor (found on macrophages but not onmonocytes or neutrophils), scavengerreceptors ( bind many charged ligands), andC D 1 4 ( a r e c e p t o r f o r b a c t e r i a llipopolysaccharide (LPS) found predominantlyon monocytes and macro-phages ).

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- Pathogens can also interact with macrophagesand neutrophils through receptors forcomplement borne on these cells.

-Ligation of many of the cell-surface receptorst h a t r e c o g n i z e p a t h o g e n s l e a d s t ophagocytosis of the pathogen, followed by itsdeath inside the phagocyte.

-Phagocytosis is an active process, in which thebound pathogen is first surrounded by thephagocyte membrane and then internalized ina membrane-bounded vesicle known as aphagosome, which becomes acidified.

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- Macrophages and neutrophils have granulescalled lysosomes, that contain enzymes,proteins, and peptides that can mediate anintracellular antimicrobial response.

-The phagosome fuses with one or morelysosomes to generate a phagolysosome inwhich the lysosomal contents are released todestroy the pathogen.

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-Upon phagocytosis, macrophages andneutrophils also produce a variety of othertoxic products that help kill the engulfedmicroorganism.

-These include: hydrogen peroxide (H2O2), thesuperoxide anion (O2 ) , and nitric oxide (NO),which are directly toxic to bacteria.

- They are generated by lysosomal NADPHoxidases and other enzymes in a processknown as the respiratory burst, as it isaccompanied by a transient increase inoxygen consumption.

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-NO is produced by a high-output form of nitricoxide synthase (iNOS2).

- Superoxide is generated by a multicomponentmembrane associated NADPH oxidase in aprocess called repiratory burst and isconverted by enzyme superoxide mutase intohydrogen peroxide.

-Further chemical and enzymatic reactionsproduce a range of toxic chemicals fromhydrogen peroxide including hydroxyl radical(OH•) , hypochlorite (OCl-) , hypobromite (OBr-)ions which can kill the microorganism

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-Neutrophils are short-lived cells and die soonafter accomplishing a round of phagocytosis.Dead and dying neutrophils are a majorcomponent of the pus that forms in someinfections. bacteria that give rise to suchinfections are thus known as pyogenicbacteria.

-Macrophages are long-lived and continue togenerate new lysosomes.

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-Patients with chronic granulomatous diseasehave a genetic deficiency of NADPH oxidasei.e. their phagocytes do not produce toxicoxygen derivatives and are less able to killingested microorganisms and clear theinfection.

-People with this defect are unusuallysusceptible to bacterial and fungal infections,especially in infancy.

-Sea star rely entirely on innate immunity(macrophages) for their defense againstinfection.

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-A key feature that distinguishes pathogenicfrom nonpathogenic micro-organisms is theirability to overcome innate immune defenses.Pathogens have developed a variety ofstrategies to avoid being immediatelydestroyed by macrophages.

Examples:

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1. Many extracellular pathogenic bacteria coatthemselves with a thick polysaccharidecapsule that is not recognized by anyphagocyte receptor.

2.Other pathogens e.g. mycobacteria, haveevolved ways to grow inside macrophagephagosomes by inhibiting fusion with alysosome.

3. Microoranisms enter the body in sufficientnumbers to overwhelm the immediate innatehost defenses and establish a focus ofinfection.

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c) Initiation of an inflammatory response

• Inflammation-A defense reaction of livingtissue to injury.

-Inflammation results from the interactionbetween pathogens and tissue macrophageswhere macrophages are activated to releasecytokines and other mediators that set up astate of inflammation in the tissue and bringsneutrophils and plasma proteins to the site ofinfection.

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-An inflammatory response is usually initiatedwithin minutes of infection or wounding.

- Inflammation plays three essential roles incombating infection:

a) To deliver additional effector molecules andcells to sites of infection to augment thekilling of invading microorganisms by thefront-line macrophages.

b) To provide a physical barrier preventing thespread of infection.

c) To promote the repair of injured tissue (thisis a non immunological role).

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• Inflammatory responses are characterized bypain, redness, heat, and swelling at the site ofan infection, reflecting three types of changein the local blood vessels:

1.The first is an increase in vascular diameter,leading to increased local blood flow hencethe heat and redness and a reduction in thevelocity of blood flow especially along thesurfaces of small blood vessels.

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2. The endothelial cells lining the blood vessel areactivated to express adhesion molecules thatpromote the binding of circulating leukocytes.

-The combination of slowed blood flow andinduced adhesion molecules allows leukocytes toattach to the endothelium and migrate into thetissues, a process known as extravasation.

-These changes are initiated by the cytokinesproduced by activated macrophages.

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-Once inflammation has begun, the first cellsattracted to the site of infection areneutrophils.

-They are followed by monocytes, whichdifferentiate into more tissue macrophages. Inthe later stages of inflammation, otherleukoc ytes s uch a s eos i n op hi l s an dlymphocytes also enter the infected site.

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3. An increase in vascular permeability.

Instead of being tightly joined together, theendothelial cells lining the blood vessel wallsbecome separated, leading to exit of fluid andproteins from the blood and their localaccumulation in the tissue.

This accounts for the swelling, or edema, andpain as well as the accumulation of plasmaproteins that aid in host defense.

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4. Clotting in microvessels in the site of infection.This contributes to host defense by preventingspread of the pathogen via the blood (not animmunological response).

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• These changes are induced by a variety ofinf lammatory mediators re leased as aconsequence of the recognition of pathogens.

-These include the lipid mediators of inflammationprostaglandins, leukotrienes, and platelet-activating factor (PAF) which are rapidlyproduced by macrophages through enzymaticpathways that degrade membrane phospholipids.

-Their actions are followed by those of thecytokines and chemokines (chemoattractantcytokines) that are synthesized and secreted bymacrophages in response to pathogens.

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- E.g. The cytokine tumor necrosis factor-α (TNF-α), is a potent activator of endothelial cells.

- Pathogen recognition can rapidly triggers aninflammatory response through activation of thecomplement cascade. The cleavage productspeptide C5a, of the complement reaction is apotent mediator of inflammation, with severaldifferent activities:

-It increases vascular permeability and induces theexpression of some adhesion molecules

- it acts as a powerful chemoattractant forneutrophils and monocytes

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-it activates phagocytes and local mast cellswhich are in turn stimulated to releasegranules containing the inflammatorymolecule histamine and TNF-α.

• If wounding has occurred, the injury to bloodvessels immediately triggers two otherprotective enzyme cascades:

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a) The kinin system- an enzymatic cascade ofplasma proteins that is triggered by tissuedamage to produce several inflammatorymediators, including the vasoactive peptidebradykinin. This causes:

- an increase in vascular permeability thatpromotes the influx of plasma proteins to thesite of tissue injury.

- pain, which, draws attention to the problemand leads to immobilization of the affectedpart of the body, which helps to limit thespread of any infectious agents.

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b) The coagulation system

-is an enzymatic cascade of plasma enzymes(proenzymes or zymogens) that is triggeredfollowing damage to blood vessels.

This leads to the formation of a fibrin clot, whichprevents any microorganisms from enteringthe bloodstream (normal role of fibrin is toprevent blood loss).

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NB// Both these cascades have an importantrole in the inflammatory response topathogens even if wounding or gross tissueinjury has not occurred, as they are alsotriggered by endothelial cell activation.

Thus, within minutes of the penetration oftissues by a pathogen, the inflammatoryresponse causes an influx of proteins and cellsthat will control the infection.

It also forms a physical barrier to limit thespread of infection and makes the host fullyaware of the local infection.

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Receptors of the innate immune system.

-Although the innate immune system lacks thespecificity of adaptive immunity, it candistinguish nonself from self.

-receptors that activate the innate immuneresponse are those that recognize pathogensdirectly and those that signal for a cellularresponse.

-Proteins that recognize features common tomany pathogens occur as secreted moleculesand as receptors on cells of the innateimmune system.

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-The binding of pathogens by these receptorsgives rise to very rapid responses.

• Receptors with specificity for pathogensurfaces (recognize patterns of repeatingstructural motifs).

-The surfaces of microorganisms bear repeatingpatterns of molecular structure.

The innate immune system recognizes suchpathogens by means of receptors that bindfeatures of these regular patterns; thesereceptors are sometimes known as pattern-recognition molecules.

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a) The mannan-binding lectin (MBL) / mannosebinding lectin

- It initiates the MB-lectin pathway ofcomplement activation. pathogen recognitionand discrimination from self is due torecognition of a particular orientation ofcertain sugar residues and their spacing,which is found only on pathogenic microbesand not on host cells.

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-MBL is a member of the collectin (e.g. SP-A and SP-D)family of proteins, composed of between two to sixclusters of carbohydrate-binding lectin domainsthat interact with each other via a collagen-likedomain.

• Collectin: A family of structurally related calcium-dependent proteins or sections with a collagen-likedomain that bind sugars, such as mannose-bindingprotein.

-Collectins may bind to C1q, thereby activating thecomplement system, and participate in innateimmunity through their action as microbialrecognition receptors.

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-Within each cluster are three separate bindingsites that have a fixed orientation relative toeach other; all three sites can therefore onlybind when their ligands mannose and fucoseresidues in bacterial cell-wall polysaccharideshave the appropriate spacing.

- The collectin C1q is able to bind directly topathogen surfaces and initiate complementactivation through the classical pathway.

- Other collectins are made in the liver as partof the acute-phase response.

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- All collectins have multiple carbohydrate-recognition domains attached to a collagenhelix and are thought to bind pathogensurfaces in a similar way to mannan-bindinglectin.

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-The interaction of these soluble receptors withp a t h o g e n s l e a d s t o b i n d i n g o f t h ereceptor:pathogen complex by phagocytes,either through direct interaction with thepathogen-binding receptor, or throughreceptors for complement, thus promotingphagocytosis and killing of the boundpathogen.

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b) Macrophage mannose receptor

-it is a multipronged molecule with severalcarbohydrate-recognition domains.

-It is a transmembrane cell-surface receptor andthus it can function directly as a phagocyticreceptor.

-Found on the cell surface of the Phagocytes andthey recognize pathogen surfaces directly. Thisreceptor is a cell-bound C-type lectin that bindscertain sugar molecules found on the surface ofmany bacteria and some viruses, including thehuman immunodeficiency virus (HIV).2016/9/13 119

c) Scavenger receptors- a phagocytic receptorsthat recognize certain anionic polymers andalso acetylated low-density lipoproteins.

T h e s e r e c e p t o r s a r e a s t r u c t u r a l l yheterogeneous set of molecules, existing in atleast six distinct molecular forms.

Some scavenger receptors recognize structuresthat are shielded by sialic acid on normal hostcells. These receptors are involved in theremoval of old red blood cells that have lostsialic acid, as well as in the recognition andremoval of pathogens.

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• Receptors on phagocytes that signal thepresence of pathogens.

-Binding of pathogens by macrophages cantrigger the induced responses of innateimmunity, and responses that eventually leadto the induction of adaptive immunity.

a) .Toll receptors- a family of evolutionarilyconserved transmembrane receptors.

- function exclusively as signaling receptors.Were first described in the fruit-fly. They donot to recognize and bind pathogens directly,

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but are involved in signaling the appropriateresponse to different classes of pathogen.

-In the fruit-fly, the Toll receptor itself triggers theproduction of antifungal peptides in response tofungal infections.

- In mammals, a Toll-family protein, called Toll-likereceptor 4, or TLR-4, signals the presence of LPSby associating with CD14, the macrophagereceptor for LPS.

- TLR-4 is also involved in the immune response torespiratory syncytial virus.

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-Mammalian Toll-like receptor, TLR-2, signalsthe presence microbial constituents includingthe proteoglycans of gram-positive bacteria,although how it recognizes these is not known.

There are at least nine distinct Toll proteinsfamily in mammals.

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The complement system and innate immunity

-Complement is a system of plasma proteinsthat interacts with pathogens to mark themfor destruction by phagocytes.

-Although first discovered as an effector arm ofthe antibody response, complement is alsoactivated early in infection in the absence ofantibodies.

-Complement first evolved as part of the innateimmune system, where it still plays animportant role.

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-The complement system is made up of a largenumber of distinct plasma proteins that reactwith one another to opsonize pathogens andinduce a series of inflammatory responses thathelp to fight infection.

-A number of complement proteins are proteasesthat are themselves activated by proteolyticcleavage ( these enzymes are referrred aszymogens-e.g. The digestive enzyme pepsin is storedinside cells and is secreted as an inactive precursorenzyme, pepsinogen, which is only cleaved to pepsin inthe acid environment of the stomach. The advantage isthat the host is not autodigested).

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- The precursor zymogens are widelydistributed throughout body fluids and tissueswithout adverse effect.

- At sites of infection, they are activated locallyand trigger a series of potent inflammatoryevents.

- The complement system activates through atriggered-enzyme cascade. In such a cascade,an active complement enzyme generated bycleavage of its zymogen precursor thencleaves its substrate, another complementzymogen, to its active enzymatic form.

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-This in turn cleaves and activates the nextzymogen in the complement pathway. In thisway, the activation of a small number ofcomplement proteins at the start of thepathway is hugely amplif ied by eachsuccessive enzymatic reaction, resulting in therapid generation of a disproportionately largecomplement response. E.g. The bloodcoagulation system -is a triggered-enzymecascade where, a small injury to a blood vesselwall can lead to the development of a largethrombus.

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• Three ways in which the complement systemprotects against infection:

a).it generates large numbers of activatedcomplement proteins that bind covalently topathogens, opsonizing them for engulfment byphagocytes bearing receptors for complement.

b).the Small fragments of some complementproteins act as chemoattractants to recruit morephagocytes to the site of complement activation,and also to activate these phagocytes.

c). the terminal complement components damagecertain bacteria by creating pores in the bacterialmembrane.

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• three distinct pathways through whichcomplement can be activated on pathogensurfaces.

-These pathways depend on different moleculesfor their initiation, but they converge togenerate the same set of effector molecules.

Pathways:

• Classical pathway

• MB- lectin pathway

• Alternative pathway

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• The nomenclature of complement proteins

-All components of the classical complementpathway and the membrane-attack complexare designated by the letter C followed by anumber.

The native components have a simple numberdesignation e.g. C1 and C2, but unfortunately,the components were numbered in the orderof their discovery rather than the sequence ofreactions, which is C1, C4, C2, C3, C5, C6, C7,C8, and C9.

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-The products of the cleavage reactions aredesignated by added lower-case letters, thelarger fragment being designated b and thesmaller a e.g. C4 is cleaved to C4b, the largefragment of C4 that binds covalently to thesurface of the pathogen, and C4a, a smallfragment with weak pro-inflammatoryproperties.

-The components of the alternative pathway are designated by different capital letters e.g. factor B and factor D.

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-Their cleavage products are designated by theaddition of lower-case a and b: thus, the largefragment of B is called Bb and the small fragmentBa.

- In the mannose-binding lectin pathway, the firstenzymes to be activated are known as themannan-binding lectin-associated serineproteases MASP-1 and MASP-2, after which thepathway is essentially the same as the classicalpathway.

- Activated complement components are oftendesignated by a horizontal line. The large activefragment of C2 was originally designated C2a.

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-The formation of C3 convertase activity ispivotal in complement activation, leading tothe production of the principal effectormolecules, and initiating the late events.

-In the classical and MB-lectin pathways, the C3convertase is formed from membrane-boundC4b complexed with C2b.

- In the alternative pathway, a homologous C3convertase is formed from membrane-boundC3b complexed with Bb. The alternativepathway can act as an amplification loop for allthree pathways, as it is initiated by the bindingof C3b.

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The pathways

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A. The classical pathway

- is triggered by antibody or by direct binding ofcomplement component C1q to the pathogensurface. The activation of C1 complexactivates the pathway.

- plays a role in both innate and adaptiveimmunity.

- C1q, links the adaptive humoral immuneresponse to the complement system bybinding to antibodies complexed withantigens.

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C1q can, also bind directly to the surface ofcertain pathogens and trigger complementactivation in the absence of antibody.

-C1q is part of the C1 complex, which comprisesa single C1q molecule bound to two moleculeseach of the zymogens C1r and C1s.

-C1q is a calcium-dependent sugar bindingprotein, a lectin, belonging to the collectinfamily of proteins, which contains bothcollagen-like and lectin domains hence thename collectin.

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-It has six globular heads, linked together by acollagen-like tail, which surround the(C1r:C1s)2 complex

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The C1 complex- a complex of C1q, C1r, and C1s.

-Binding of more than one of the C1q heads to apathogen surface causes a conformationalchange in the (C1r:C1s)2 complex, which leadsto activation of an autocatalytic enzymaticactivity in C1r; the active form of C1r thencleaves its associated C1s to generate anactive serine protease.

-Once activated, the C1s enzyme cleaves C4 andthen C2 to generate two large fragments, C4band C2b, which together form the C3convertase of the classical pathway.

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-In the first step, C1s cleaves C4 to produce C4b,which binds covalently to the surface of thepathogen. The covalently attached C4b thenbinds one molecule of C2, making itsusceptible to cleavage by C1s. C1s cleaves C2to produce the large fragment C2b, which isitself a serine protease.

- The complex of C4b with the active serineprotease C2b remains on the surface of thepathogen as the C3 convertase of the classicalpathway.

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-Its most important activity is to cleave largenumbers of C3 molecules to produce C3bmolecules that coat the pathogen surface. Thecleavage product, C3a, initiates a localinflammatory response.

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-b. The mannan-binding lectin pathway(homologous to the classical pathway)

- is triggered by mannan-binding lectin, a normalseru m cons t i tuen t t ha t b i n ds so meencapsulated bacteria

-The mannan-binding lectin (MBL) protein , is acollectin, like C1q. MBL binds specifically tomannose residues and to certain other sugarswhich are accessible and arranged in a patternthat allows binding on many pathogens. Onvertebrate cells, these are covered by othersugar groups, especially sialic acid.

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-MBL initiates complement activation by bindingto pathogen surfaces.

It is present at low concentrations in normalplasma of most individuals but its productionby the liver is increased during the acute-phase reaction of the innate immuneresponse.

-Mannan-binding lectin, is a six-headedmolecule that forms a complex with twoprotease zymogens- MASP-1 and MASP-2.

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2016/9/13 146Mannan-binding lectin

-MASP-1 and MASP-2 are closely homologous toC1r and C1s ( all the 4 enzymes may havefrom gene dupl icat ion of a commonprecursor).

-When the MBL complex binds to a pathogensurface, MASP-1 and MASP-2 are activated tocleave C4 and C2, forming a C3 convertasefrom C2b bound to C4b.

-People deficient in mannan-binding lectinexperience a substantial increase in infectionsduring early childhood.

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c. The alternative pathway of complement- It is initiated through the spontaneous hydrolysis of

C3 .- This pathway can proceed on many microbial

surfaces in the absence of specific antibody, and itleads to the generation of a distinct C3 convertasedesignated C3b,Bb.

- C3 is abundant in plasma, and C3b is produced at asignificant rate by spontaneous cleavage (alsoknown as 'tickover'). This occurs through thespontaneous hydrolysis of the thioester bond in C3to form C3(H2O) which has analtered conformation,allowing binding of the plasma protein factor B.

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-The binding of B by C3(H2O) then allows a plasmaprotease, factor D to cleave factor B to Ba and Bb,the latter remaining associated with C3(H2O) toform the C3(H2O)Bb complex.

This complex is a fluid-phase C3 convertase. It isonly formed in small amounts and can cleavemany molecules of C3 to C3a and C3b.

-Much of this C3b is inactivated by hydrolysis, butsome attaches covalently, through its reactivethioester group to the surfaces of host cells or topathogens.

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-C3b bound in this way is able to bind factor B,allowing its cleavage by factor D to yield thesmall fragment Ba and the active protease Bb.This results in formation of the alternativepathway C3 convertase, C3b,Bb.

• WHY COMPLEMENT SYSTEM

-The main effect of complement activation is todeposit large quantities of C3b on the surfaceof the infecting pathogen, where it forms acovalently bonded coat that signal theultimate destruction of the pathogen byphagocytes.

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-In the classical and the MB-lectin pathways, aC5 convertase is formed by the binding of C3bto C4b,2b to yield C4b,2b,3b. By the sametoken, the C5 convertase of the alternativepathway is formed by the binding of C3b tothe C3 convertase to form C3b2,Bb.

-C5 is captured by these C5 convertasecomplexes through binding to an acceptor siteon C3b, and is then rendered susceptible tocleavage by the serine protease activity of C2bor Bb.

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-This reaction, which generates C5b and C5a, ismuch more limited than cleavage of C3, as C5can be cleaved only when it binds to C3b thatis part of the C5 convertase complex.

Thus, complement activation by both thealternative, MB-lectin and classical pathwaysleads to the binding of large numbers of C3bmolecules on the surface of the pathogen, thegeneration of a more limited number of C5bmolecules, and the release of C3a and C5a.

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-The terminal complement proteins (C5 (C5a, C5b);C6; C7; C8; C9)

- The terminal components of complementassembles to form a membrane-attack complex.

- The end result is a pore in the lipid bilayermembrane that destroys membrane integrity.This kills the pathogen by destroying the protongradient across the pathogen cell membrane.

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Regulation of all three pathways of complement activation

- The cells are safeguarded from uncontrolledactivation by:

1. generation of the effector molecules ofcomplement through the sequent ia lactivation of zymogens.

2. The activation of zymogens usually occurs ona pathogen surface, and the activatedcomplement fragments produced in theensuing cascade of reactions usually bindnearby or are rapidly inactivated by hydrolysis

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3. All complement components are activatedspontaneously at a low rate in plasma. Activatedcomplement components will sometimes bindproteins on host cells. The potentially damagingconsequences are prevented by a series ofcomplement control proteins which regulate thecomplement cascade at different points i.e.

• Proteins that regulate the complement system

• Control proteins of the classical and alternative pathways

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Name and symbol Role in regulation

C1 inhibitor (CIINH) Binds to activated Cir, CIS removing it form C1q

C4-binding protein (C4Bp) Binds C4b displacing C2b, cofactor for C4b cleavage by I

Complement receptor 1 (CR1) Binds C4b or C3b displacing Bb cofactor 1

Factor H (H) Binds C3b displacing Bb cofactor 1

Factor I (i) Serine protease that cleaves C3b and C4b aided by H, MCP, C4BP or

CR1

Decay-accelerating factor (DAF) Membrane protein that displaces Bb from C3b and C2b from C4b

Membrane cofactor protein (MCP) Membrane protein that promotes C3b and C4b inactivation by I

CD59 (protectin) Prevents formation of membrane attack complex on autologous or

allogenic cells. Widely expressed on membranes.

Adaptive immune response

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-The lymphocytes of the adaptive immunesystem have evolved to provide a moreversatile means of defense which, in addition,provides increased protection againstsubsequent reinfection with the samepathogen.

-The cells of the innate immune system play acrucial part in the initiation and subsequentdirection of adaptive immune responses, aswell as participating in the removal ofpathogens that have been targeted by anadaptive immune response.

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-There is normally a delay of 4 -7 days beforethe initial adaptive immune response takeseffect, the innate immune response has acritical role in controlling infections during thisperiod.

- The innate immune response makes a crucialcontribution to the activation of adaptiveimmunity:

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1.The inflammatory response increases the flow oflymph containing antigen and antigen-bearing cellsinto lymphoid tissue, while complement fragmentson microbial surfaces and induced changes in cellsthat have taken up microorganisms provide signalsthat synergize in activating lymphocytes whosereceptors bind to specific microbial antigens.

2.Macrophages that have phagocytosed bacteria andbecome activated can also activate T lymphocytes.

3. dendritic cells are specialize in presenting antigento T lymphocytes and initiating adaptive immunity.

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- The induction of an adaptive immune responsebegins when a pathogen is ingested by animmature dendritic cell in the infected tissue. - -immature dendritic cell are resident in mosttissues and are relatively long-lived, turning overat a slow rate.

- Dendritic cells derive from the bone marrowprecursor as macrophages, and migrate to theirperipheral stations, where they survey the localenvironment for pathogens.

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-All tissue-resident dendritic cells migratethrough the lymph to the regional lymphnodes where they interact with recirculatingnaive lymphocytes.

-If the dendritic cells fail to be activated, theyinduce tolerance to the antigens of self thatthey bear.

-The immature dendritic cell carries receptorson its surface that recognize common featuresof many pathogens e.g. bacterial cell wallproteoglycans.

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-binding of a bacterium to these receptorsstimulates the dendritic cell to engulf thepathogen and degrade it intracellularly.

- Immature dendritic cells also continually takeup extracellular material, including any virusparticles or bacteria that may be present, bythe receptor-independent mechanism ofmacropinocytosis nasd these are presented to

T lymphocytes.

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- When a dendritic cell takes up a pathogen ininfected tissue, it becomes activated andtravels to a nearby lymph node. The dendriticcell matures into a highly effective antigen-presenting cell (APC) and undergoes changesthat enable it to activate pathogen-specificlymphocytes that it encounters in the lymphnode.

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- Recogn i t ion mechan ism u sed by thelymphocytes of the adaptive immuneresponse enables recognition of an almostinfinite diversity of antigens, with eachdifferent pathogen being targeted specifically.-The specificity of these receptors isdetermined by a unique genetic mechanismt h a t o p e r a t e s d u r i n g l y m p h o c y t edevelopment in the bone marrow and thymusto generate millions of different variants ofthe genes encoding the receptor molecules.

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-Although an individual lymphocyte carriesreceptors of only one specificity, thespecificity of each lymphocyte is different.

This ensures that the millions of lymphocytes inthe body collectively carry millions of differentantigen receptor specificities, the lymphocytereceptor repertoire of the individual.

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-During a person's lifetime these lymphocytesundergo a process akin to natural selection;only those lymphocytes that encounter anantigen to which their receptor binds will beactivated to proliferate and differentiate intoeffector cells.

-This selective mechanism was first proposed inthe 1950s by Macfarlane Burnet to explainwhy antibodies, which can be induced inresponse to virtually any antigen, are producedin each individual only to those antigens towhich he or she is exposed. Burnet called thisthe clonal selection theory.

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Clonal selection theory-Each lymphocyte progenitor gives rise to many

lymphocytes, each bearing a distinct antigenreceptor.

-Lymphocytes with receptors that bind ubiquitousself antigens are eliminated before they becomefully mature, ensuring tolerance to such selfantigens.

-When antigen interacts with the receptor on amature naive lymphocyte, that cell is activatedand starts to divide, giving rise to a clone ofidentical progeny, all of whose receptors bindthe same antigen.

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- Antigen specificity is thus maintained as theprogeny proliferate and differentiate intoeffector cells.

-Once antigen has been eliminated by theseeffector cells, the immune response ceasesalthough some lympphocytes are retained tomediate immunological memory.

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• Basic principles of clonal selection hypothesis

1.Each lymphocyte bears a single type ofreceptor with a unique specificity

2. Interaction between a foreign molecule and alyphocyte receptor capable of binding thatmolecule with high aff in ity leads tolymphocyte activation.

3. The differentiated effector cells derived froman activated lymphocyte will bear receptors ofidentical specificity to those of the parentalcell from which that lymphocyte was derived.

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4. Lymphocytes bearing receptors specific forubiquitous self molecules are deleted at anearly stage in lymphoid cell development andare therefore absent from the repertoire ofmature lymphocytes.

The structure of the antibody molecule

-Antibodies are glycoprotein substancesproduced by B lymphoid cells in response tostimulation with an immunogen.

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-They possess the ability to react in vitro and invivo specifically and selectively with the antigenicdeterminants or epitopes eliciting theirproduction or with an antigenic determinantclosely related to the homologous antigen.

-Are molecules are immunoglobulins found in theblood and body fluids formed in response toimmunogens.

-Antibodies may be produced by hybridomatechnology in which antibody-secreting cells arefused by polyethylene glycol (PEG) treatmentwith a mutant myeloma cell line.

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-Monoclonal antibodies are widely used inresearch and diagnostic medicine and havepotential in therapy.

-Immunoglobulins are not restricted to theplasma but may be found in other body fluidsor tissues, such as urine, spinal fluid, lymphnodes, spleen, etc. Immunoglobulins do notinclude the components of the complementsystem.

-Immunoglobulins (antibodies) constituteapproximately 1 to 2% of the total serumproteins in health.

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-Abs are produced in very large quantities inresponse to antigen, they can be studied bytraditional biochemical techniques.

- Antibody molecules are roughly Y-shapedmolecules consisting of three equal-sizedportions, loosely connected by a flexibletether (determined by X-ray crystallography).

-The two arms of the Y end in regions that varybetween different antibody molecules, the Vregions. These are involved in antigen binding.

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- The stem of the Y, or the C region, is far lessvariable and is the part that interacts witheffector cells and molecules.

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-All antibodies are constructed in the same wayfrom paired heavy and light polypeptidechains.

- More subtle differences confined to the Vregion account for the specificity of antigenbinding.

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General structural features of immunoglobulins (Ig G)

-Are large molecules, having a molecularweight of approximately 150 kDa,composed of two different

kinds of polypeptide chain of approximately 50kDa, termed the heavy or H chain, and theother, of 25 kDa, termed the light or L chain .

-Each IgG molecule consists of two heavy chainsand two light chains.

-The two heavy chains are linked to each otherby disulfide bonds and each heavy chain islinked to a light chain by a disulfide bond.

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- In any given immunoglobulin molecule, thetwo heavy chains and the two light chains areidentical, giving an antibody molecule twoidentical antigen-binding sites and thus theability to bind simultaneously to two identicalstructures.

- Two types of light chain, termed lambda (λ)and kappa (κ), are found in antibodies.

- A given immunoglobulin either has κ chains orλ chains, never one of each.

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- No functional difference has been foundbetween antibodies having λ or κ light chains,and either type of light chain may be found inantibodies of any of the five major classes.

- The ratio of the two types of light chain variesfrom species to species e.g. In mice, theaverage κ to λ ratio is 20:1; in humans it is 2:1and in cattle it is 1:20. The reason for thisvariation is unknown.

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- Distortions of this ratio can sometimes be used todetect the abnormal proliferation of a clone of Bcells. These would all express the identical lightchain, and thus an excess of λ light chains in aperson might indicate the presence of a B-celltumor producing λ chains.

-The class and the effector function of an antibody isdefined by the structure of its heavy chain.

-There are five main heavy-chain classes or isotypes,some of which have several subtypes, and thesedetermine the functional activity of an antibodymolecule.

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-The five major classes of immunoglobulin areimmunoglobulin M (IgM), immunoglobulin D (IgD),immunoglobulin G (IgG), immunoglobulin A (IgA),and immunoglobulin E (IgE).

-Their heavy chains are denoted by the correspondinglower-case Greek letter (μ, δ, γ, α, and ,respectively).

-IgG is by far the most abundant immunoglobulin andhas several subclasses (IgG1, 2, 3, and 4 in humans).Their distinctive functional properties are conferredby the carboxy-terminal part of the heavy chain,where it is not associated with the light chain.

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Lymphocyte recognition of Ag-The antigen-recognition molecules of B cells are

the Ig. These proteins are produced by B cellsin a vast range of antigen specificities, each Bcell producing immunoglobulin of a singlespecificity.

-Membrane-bound immunoglobulin on the B-cell surface serves as the cell's receptor forantigen, and is known as the B-cell receptor(BCR).

- Ig of the same antigen specificity is secreted asantibody by terminally differentiated B cells/the plasma cells.

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- The secretion of antibodies, which bindpathogens or their toxic products in theextracellular spaces of the body, is the maineffector function of B cells in adaptive immunity.

- Functions of Ab are:

- to bind specifically to molecules from thepathogen that elicited the immune response

- to recruit other cells and molecules to destroythe pathogen once the antibody is bound to ite.g. binding by antibody neutralizes viruses andmarks pathogens for destruction by phagocytesand complement.

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-These functions are structurally separated inthe antibody molecule, one part of whichspecifically recognizes and binds to thepathogen or antigen whereas the otherengages different effector mechanisms.

-The antigen-binding region varies extensivelybetween antibody molecules and is thusknown as the variable region or V region. Thisvariability of antibody molecules allows eachantibody to bind a different specific antigen,and the total repertoire of antibodies made bya single individual is large enough to ensurethat virtually any structure can be recognized.

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-The region of the antibody molecule that engagesthe effector functions of the immune systemdoes not vary and is thus known as the constantregion or C region.

- It comes in five main forms, which are eachspecialized for activating different effectormechanisms.

- The membrane-bound B-cell receptor does nothave these effector functions, as the C regionremains inserted in the membrane of the B cell.

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-Its function is as a receptor that recognizes andbinds antigen by the V regions exposed on thesurface of the cell and transmits a signal thatcauses B-cell activation leading to clonalexpansion and specific antibody production.

-The antigen-recognition molecules of T cells aremade solely as membrane-bound proteins andonly function to signal T cells for activation.

-These T-cell receptors (TCRs) are related toimmunoglobulins both in their protein structurehaving both V and C regions and in the geneticmechanism that produces their great variability.

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- T-cell receptor does not recognize and bindantigen directly, but instead recognizes shortpeptide fragments of pathogen proteinantigens which are bound to MHC moleculeson the surfaces of other cells.

-The MHC molecules are glycoproteins encodedin the large cluster of genes known as themajor histocompatibility complex (MHC).

-Their most striking structural feature is a cleftrunning across their outermost surface, inwhich a variety of peptides can be bound.

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-MHC molecules show great genetic variation inthe population, and each individual carries upto 12 of the possible variants, which increasesthe range of pathogen-derived peptides thatcan be bound.

-T-cell receptors recognize features both of thepeptide antigen and of the MHC molecule towhich it is bound, a phenomenon called MHCrestriction (i.e any given T-cell receptor isspecific not for a foreign peptide antigen, butfor a unique combination of a peptide and aparticular MHC molecule).

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How are antigen receptors with an almostinfinite range of specificities encoded by afinite number of genes?

-The genes for immunoglobulin variable regionsare inherited as sets of gene segments, eachencoding a part of the variable region of oneof the immunoglobulin polypeptide chains .

- During B-cell development in the bonemarrow, these gene segments are irreversiblyjoined by DNA recombination to form astretch of DNA encoding a complete variableregion.

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- Because there are many different genesegments in each set, and different genesegments are joined together in different cells,each cell generates unique genes for thevariable regions of the heavy and light chainsof the immunoglobulin molecule.

- Once a functional receptor is produced,further rearrangement is prohibited. Thuseach lymphocyte expresses only one receptorspecificity.

- This mechanism has three importantconsequences:

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• It enables a limited number of gene segments togenerate a vast number of different proteins.

• It enables each cell to express a unique receptorspecificity, because each cell assembles adifferent set of gene segments

• All the progeny of that cell will inherit genesencoding the same receptor specificity becausegene rearrangement involves an irreversiblechange in a cell's DNA.

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The potential diversity of lymphocyte receptorsgenerated in this way is enormous. Just a fewhundred different gene segments cancombine in different ways to generatethousands of different receptor chains.

- The diversity of lymphocyte receptors isfurther amplified by junctional diversity,created by adding or subtracting nucleotidesin the process of joining the gene segments,and by the fact that each receptor is made bypairing two different variable chains, eachencoded in distinct sets of gene segments.

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- A thousand different chains of each typecould thus generate 106 distinct antigenreceptors through this combinatorial diversity.

- Only a subset of these randomly generatedreceptor specificities survive the selectiveprocesses that shape the peripherallymphocyte repertoire.

- There are lymphocytes of at least 108different specificities in an individual at anyone time. These provide the raw material onwhich clonal selection acts.

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• The main distinctions between B and T-l y m p h o c y t e r e c e p t o r s a r e t h a t t h eimmunoglobulin that serves as the B-cellantigen receptor has two identical antigen-recognition sites and can also be secreted,whereas the T-cell antigen receptor has asingle antigen recognition site and is always acell-surface molecule.

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Lymphocyte development and survival

- Lymphocyte maturation and survival areregulated by signals received through theirantigen receptors.

- Strong signals received through the antigenreceptor by an immature lymphocyte cause it todie or undergo further receptor rearrangement,and in this way self-reactive receptor specificitiesare deleted from the repertoire.

- A complete absence of signals from the antigenreceptor can also lead to cell death.

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- In order to survive, lymphocytes mustperiodically receive certain signals from theirenvironment via their antigen receptors.Survival signals are delivered by other cells inthe lymphoid organs and must derive, at leastin part, from the body's own molecules, theself antigens, as altering the self environmentalters the life-span of lymphocytes in thatenvironment.

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-Developing B cells in the bone marrow interactwith stromal cells.Their final maturation andcontinued recirculation depend on survivalsignals received from the B cell follicles ofperipheral lymphoid tissue.

-T lymphocytes receive survival signals from selfmolecules on specialized epithelial cells in thethymus during development, and from thesame molecules expressed by dendritic cells inthe lymphoid tissues in the periphery.

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-The self ligands that interact with the T-cellreceptor to deliver these signals are composed ofknown cell-surface molecules complexed withundefined peptides from other self proteins inthe cell. These same cell-surface moleculesfunction to present foreign intracellular antigensto T cells.

-Lymphocytes that fail to receive survival signals,and those that are clonally deleted because theyare self-reactive, undergo a form of cell suicidecalled apoptosis or programmed cell death.Dying cells are finally phagocytosed byspecialized macrophages in the liver and spleen.

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Lymphocytes proliferate

-Occurs in response to antigen in peripherallymphoid organs.

-Lymphocyte activation and proliferation isinitiated in the draining lymphoid tissues,where naive lymphocytes and activatedantigen-presenting cells can come together.On recognizing its specific antigen, a smalllymphocyte stops migrating and enlarges. Thechromatin in its nucleus becomes less dense,nucleoli appear, the volume of both thenucleus and the cytoplasm increases, and newRNAs and proteins are synthesized.

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-This happens within a few hours producing anew from of lymphocyte known as alymphoblast.

-The lymphoblasts begin to divide, normallyduplicating themselves 2-4 times every 24hours for 3 to 5 days, so that one naivelymphocyte gives rise to a clone of around1000 daughter cells of identical specificity.These then differentiate into effector cells.

-B cells differentiated effector cells are theplasma cells that secrete antibody.

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-T cells effector cells are able to destroy infectedcells or activate other cells of the immunesystem. These changes also affect therecirculation of antigen-specific lymphocytes.Changes in the cell-adhesion molecules theyexpress on their surface allow effectorlymphocytes to migrate into sites of infectionor stay in the lymphoid organs to activate Bcells.

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-After a naive lymphocyte has been activated, ittakes 4 to 5 days before clonal expansion isc o m p l e t e a n d t h e l y m p h o c y t e s h a v edifferentiated into effector cells. That is whyadaptive immune responses occur only after adelay of several days.

-Effector cells have only a limited life-span and,once antigen is removed, most of the antigen-specific cells generated by the clonal expansionof small lymphocytes undergo apoptosis.

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-Some persist after the antigen has beeneliminated forming the memory cells andform the basis of immunological memory,which ensures a more rapid and effectiveresponse on a second encounter with apathogen and thereby provides lastingprotective immunity.

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• Comparing the antibody response of anindividual to a first or primary immunizationwith the response elicited in the sameindividual by a secondary or boosterimmunization with the same antigen.

- The secondary antibody response occurs aftera shorter lag phase, achieves a markedlyhigher level, and produces antibodies ofhigher affinity, or strength of binding, for theantigen.

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When the animal is later challenged with a mixtureof antigens A and B, a very rapid and intensesecondary response to A occurs. This illustratesimmunological memory- the ability of theimmune system to make a second response tothe same antigen more efficiently and effectively,providing the host with a specific defense againstinfection.

-This is the main reason for giving boosterinjections after an initial vaccination. Note thatthe response to B resembles the initial orprimary response to A, as this is the firstencounter of the animal with antigen B.

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-Immunological memory enables successfulvaccination and prevents reinfection withpa thog ens th at hav e been repe l l edsuccessfully by an adaptive immune response.

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-First encounter with an antigen produces aprimary response.

Antigen A introduced at time zero encounterslittle specific antibody in the serum. After alag phase, antibody against antigen A (blue)appears; its concentration rises to a plateau,and then declines. When the serum is testedfor antibody against another antigen, B(yellow), there is none present, demonstratingthe specificity of the antibody response.

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TUMOUR IMMUNOLOGY

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Immune responses

-subdivided into humoral (antibody-mediated)and cel lu lar (cel l -mediated) immuneresponses.

• Humoral (B-cell) immune responses functionspredominantly in the elimination of solubleantigens and the destruction of extracellularmicroorganisms

• Cell-mediated (T-cell) immunity is moreimportant for the elimination of intracellularorganisms (e.g. viruses).

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-One of the important functions of cellularimmunity is to execute surveillance againstmalignant cells.

• Qn: Do tumour cells show differences fromtheir normal cellular counterparts that theimmune system can recognize?

• Are tumour associated antigens specific forthe particular cancer involved?

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Surface Antigens on Tumour Cells

-Tumour antigens fall into 2 broad categories:

• Tumour specific antigens (TSA)- are unique,found only on tumour cells and are eminentlypositioned as targets for immunologic attack.

• tumour-associated antigens (TAA)- are foundan tumour cells and some normal cells as well.

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Unique Tumour Specific Antigens

-are found only on tumour cells and not onother cells of the host. E.g

• (a) Virally Induced Antigens e.g those inducedby viruses. In mammals, several RNA virsuses(eg: retroviruses) and DNA viruses (eg: herpesviruses) cause malignant transformation.

e.g.Marek’s disease in chickens, caused by aherpes virus. The virus infects the lymphocyteand transforms it resulting in a unique proteinon the membrane of the transformedlymphocyte.

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-All tumours due to this virus carry this surfaceantigen and antibodies to the protein aredetectable in host sera.

-Animals are prophylactically protected byimmunization against Marek’s disease, usingeither the whole virus or the TSA.

-Tumour cells induced by RNA viruses expressantigens coded for by the viral proteininaddition to their own cell surface antigense.g. human T cell leukaemia virus (HTLV) inman.

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(b) Chemically or Physically Induced TumourAntigens

Tumours induced by chemical carcinogens suchas methyl cholanthrene (MCA) express uniqueand individually distinct tumour antigens.Lead to alterations in both major and minorhistocompatibility antigens. The samemechanism operates in tumours due toultraviolet light or radiation.

(c) Antigens of Spontaneous Tumours

Spontaneous tumours have no known inducingagent.

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Tumour Associated Antigens

- are, to a large extent, specific to the tumoursthat display them, some normal cells may alsoexpress such antigens at particular stages ofdifferentiation.

(a) Oncofoetal antigens

Oncogenes are found in cancer-causing viruses.

-Most oncogenes are present in the hostcell,where they function in regulated cell growth.

The host cell gene is called a proto-oncogene.

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-Proteins encoded by proto-oncogenes areexpressed by normal cells, and over-expressedon tumour cells qualify them as tumour-associated antigens.

-Tumour cells can sometimes “switch on” geneswhich are associated with growth anddevelopment of the foetus.

-Antigens which are associated normally withembryogenesis and are not detectable in theadult begin to appear during tumour growth.

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-E.g.The prototype oncofoetal antigen carcinoembryonic antigen (CEA), which is animproperly glycosylated glycoprotein foundon foetal gut and human colon cancer cells,but not on normal adult colonic cells.

• Alpha fetoprotein is a secreted tumourantigen and is the foetal equivalent ofalbumin.

-It is found in the serum of patients withhepatomas and teratomas and can be used asa marker for the presence of such cancers.

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(b) Differentiation antigens

Are unique to the histogenetic type of tumour .

There are antigens common to melanomas (aform of skin cancer that arises frommelanocytes - the cells that produce pigment)or neuroblastomas (A malignant tumorcomposed of neuroblasts, originating in theautonomic nervous system or the adrenal)and they reflect the stage of differentiation atwhich particular tumour cells are arrested.

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• Tissue specific tumour antigens representdifferentiation antigens of the stages of celllineage from which tumours arise. Hence,they are not entirely specific to tumour cellsand populations of normal cells of the samelineage will carry similar antigens. An immuneresponse against such antigens carries thedanger of attack against normal cells as well.

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