PATHOGENESIS

Virulence is a quantitative measure of pathogenicity and is measured by the number of organism required to cause disease. The 50% lethal dose (LD50) is the number of organisms needed to kill half the host and 50% infectious dose is the number of organisms needed to cause infection in half the hosts. The infectious dose varies greatly among the pathogenic bacteria. For example, Shigella and Salmonella both cause diarrhea by infecting the gastrointestinal tract but the infectious dose of Shigella is less than 100 organisms whereas the infectious dose of Salmonella is on the order of 100,000 organisms.

The infectious dose of bacteria depends primarily on their virulence factors: whether pills allow them to adhere well to mucous membrane, whether they produce exotoxins or endotoxins, whether they possess a capsule to protect them from phagocytosis, and whether they can survive various non-specific host defences such as acid in stomach.

A micro-organism is a pathogen if is capable of causing disease;

1. Some organisms are frequently pathogens, whereas other cause disease rarely. Some bacterial pathogens are obligate intracellular parasites, e.g. Chlamydia and Rickettsia, because they can grow only within host cell. Many bacteria are facultative parasites because they can grow within cells, outside cells, or on bacteriologic media.

2. Members of normal flora cause some bacterial infections. Members of the normal flora are permanent residents of the body and vary according to anatomic site.

Opportunistic pathogens cause rarely diseases in immunocompetent people but can cause serious infections in immunocompromised patients. These opportunists are frequently members of body normal flora and cause disease if is the opportunity for infection, like reduced host defences such as acid in stomach.

TYPES OF BACTERIAL INFECTIONS

Bacteria cause diseases by 2 major mechanisms: 1. Toxin production and

2. Invasion and inflamation.

Colonisation refers to the presence of a new organism that is neither a member of normal flora or pathogen bacteria.

Stages of bacterial pathogenesis

Transmission from an external source into the portal of entry

Evasion of primary host defences such as skin or stomach acid

Adherence to mucous membrane, usually by bacterial pills.

Colonisation by growth of bacteria at the site of adherence

Diseases symptoms caused by toxin production or invasion accompanied by inflammation

Host responses, both non-specific and specific (immunity)

Progression or resolution of the disease

Adherence to mucous membrane (cell surfaces)

Some bacteria have specialised structures: adhesins e.g. pills, or produce substances, e.g. capsule or glycocalyx that allow them to adhere to the surface of human cells, thereby enhancing their ability to cause disease. These adherence mechanisms are essential for organisms that attach to mucous membrane. For example, the pills of Neisseria gonorrhoea and Escherichia coli mediate the attachment of the organism to the urinary tract epithelium and the glycocalyx of Staphylococcus epidermidis and certain viridans streptococci allows the organisms to adhere strongly to the endothelium of heart valves. Foreign bodies such as artificial heart valves and artificial joints, predispose to infections, because bacteria can adhere to these surfaces.

Invasiveness of the tissue

Several enzymes secreted by invasive bacteria play a role in pathogenesis. They are invasins. The most prominent are:

Collagenase and hyaluronidase, which degrade collagen and hyaluronic acid, allowing the bacteria to spread through subcutaneous tissue. They are especially important in cellulite caused by Streptococcus pyogenes

Coagulase, which is produced by Stapylococcus aureus accelerates the formation of a fibrin clot from its precursor, fibrinogen. This clot may protect the bacteria from phagocytosis by walling off the infected area and by coating the organisms with a layer of fibrin.

Immunoglobulin A (Ig A) protease, which degrade Ig A, allowing the organism to adhere to mucous membranes. Is produced by Neisseria gonorrhoea, Haemophilus influenzae and Streptococcus pneumoniae.

Leukocidins, which can destroy both leukocytes and macrophages.

Several factors contribute to invasiveness by limiting the ability of the host defence mechanisms, especially phagocytosis, to operate effectively. The most important of these factors is the capsule external to the cell wall of several important pathogens such as S. pneumoniae and Neisseria meningitidis. The polysaccharide capsule prevents the phagocyte from adhering to the bacteria. Anticapsular antibodies allow more effective phagocytosis to occur a process called opsonisation. The vaccine against S. pneumoniae, H. influenzae and N. meningitidis contain capsular polysaccharides that induce protective anticapsular antibodies.

A second group of antiphagocytic factors are the cell wall proteins of the gram - positive cocci, such as M protein and the group A streptococci (S. pyogenes) and protein A of Stapylococcus aureus.

The virulence factors of different bacteria are:

Gram positive cocci

Streptococcus pneumoniae: polysaccharides capsule

Streptococcus pyogenes: M protein

Staphylococcus aureus: protein A

Gram negative cocci

Neisseria meningitidis: polysaccharide capsule

Gram positive rods

Bacillus anthracis: polypeptide capsule

Gram negative rods

Haemophylus influenzae: polysaccharide capsule

Klebsiella pneumoniae: polysaccharide capsule

Escherichia coli: protein pills

Salmonella typhi: polysaccharide capsule

Yersinia pestis: V and W proteins

Bacteria cause two types of inflammation: pyogenic and granulomatous. In pyogenic (pus producing) inflammation, neutrophils are the predominant cells. Some of the most important pyogenic bacteria are Gram positive cocii.

In granulomatous inflammation, macrophages and T cells predominate. The most important organism in this category is Mycobacterium tuberculosis. The bacterial antigens stimulate the cell- mediated immune system, resulting in sensitised T lymphocyte and macrophage activity. Phagocitosis by macrophages kills most of bacteria, but some survive and grow within the macrophages in the granuloma. Several bacteria and fungal pathogens typically invade, survive and grow within reticuloendothelial cells. They are intracellular pathogens and commonly cause granulomatous lesions. These bacteria may be Mycobacterium, Legionella, Brucella, Listeria and the fungus Histoplasma. These organisms are not obligate intracellular parasites, like Chlamydia and Rickettsia. They can be cultured on microbiologic media in laboratory but prefer an intracellular location within the body. The precise mechanism by which these bacteria survive into cells is unclear, but some of them, e.g. Legionela, inhibit the fusion of lysosomes with phagosomes, thereby avoiding the degradative enzymes in the lysosomes.

The invasion of cells by bacteria is dependent of the interaction of specifically bacterial surface proteins called invasins and specific cellular receptors belonging to the integrin family of transmembrane adhesion proteins. The movement of bacteria into the cell is a function of actin microfilaments. Once inside the cell, these bacteria typically reside within cell vacuoles such as phagosomes. Some remain there, other migrates into the cytoplasm, and some move from the cytoplasm into adjacent cells through tunnels formed from actin. Infection of the surrounding cells in this manner allows the bacteria to evade host defences.

Toxin production

The second major mechanism by which bacteria cause disease is the production of toxins. A comparison of the main features of exotoxins is shown in Table 7-3.

Exotoxins

Exotoxins are produced by several gram - positive and gram - negative bacteria, in contrast to endotoxins, which are present only in gram-negative bacteria.

The essential characteristic of exotoxins is that, the bacteria secrete them, whereas endotoxin is a component of the cell wall. Exotoxins are polypeptides whose genes are frequently located on plasmids or lysogenic bacterial viruses (bacteriophages). In general, these polypeptides consist of 2 domains or subunits, one responsible for the binding to the cell membrane and entry into the cell and the other possessing the toxic activity.

Exotoxins are among the most toxic substances known. For example, the fatal dose of tetanus toxin for a human is estimated to be less than 1 mg. Because some purified exotoxins can reproduce all aspects of the disease, we can conclude that certain bacteria play no other role in pathogenesis than to synthesise the exotoxin. Exotoxin polypeptides are good antigens and induce the synthesis of productive antibodies called antitoxins, some of which are useful in prevention or treatment of diseases such as botulism and tetanus. When treated with formaldehyde (or acid or heat), the exotoxin polypeptides are converted into toxoids, which are used in protective vaccines because they retain their antigenicity but have lost their toxicity.

Mechanism of action of the important exotoxins produced by toxigenic bacteria differs significantly as described below and summarised.

A. Gram - positive bacteria

Diphtheria toxin is produced by Corynebacterium diphteriae. The tox gene, which codes for exotoxin, is carried by a temperate bacteriophage. Only lysogenised strains by this phage cause diphteria. Nonlysogenized C. diphtheria can be found in the throats of some healthy people.

Diphtheria toxin inhibits protein synthesis by ADP - ribosylation of elongation factor 2 (EF-2). The exotoxin activity depends on 2 functions mediated by different domains of the molecule: A and B. the toxin is synthesised as a single polypeptide (MW 62000) that is non-toxic because the active site of the enzyme is masked. Intact extracellular toxin binds to an eukariotic cell by its B bond. A single proteolyctic nick plus reaction of the sulfhydryl bonds yields 2 active polypeptides. After proteolytic cleavage and reduction of the disulphide bond, A region is activated.

Fragment A, a 22000-molecular weight peptide at the amid-terminal end of the exotoxin is an enzyme that catalyses the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to EF-2, thereby inactivating it. The ADP - ribosylation of EF-2 freezes the translocation complex and protein synthesis stops. The reaction is as follow:

EF-2 + NAD EF -2 - ADP-ribose + Nicotinamide

Fragment B, a 40000-molecular weight peptide at the carboxyl-terminal end, binds to receptors on the outer membrane of eukaryotic cells and mediates transport of fragment A into the cells.

To summarise, the exotoxin binds to cell membrane via a region near its carboxyl end. The toxin is transported across the membrane, and the proteolytic nick and reduction of the disulphide bonds occur. This releases the active fragment A, which inactivates EF-2. The enzymatic activity is specific for EF-2; no other protein is ADP-ribosylated.

The specificity is due to the presence in EF-2 of a unique amino acid, a modified histidine, called diphthamide. The reaction occurs in all eukaryotic cells; there is no tissue or organ specificity. Prokaryotic and mitochondrial protein synthesis is not affected, because a different, nonsusceptible elongation factor is involved. The enzyme activity is remarkably potent; a single molecule of fragment A will kill within a few hours.

Pseudomonas aeruginosa exotoxin has the same mode of action.

Other organisms whose exotoxins act by ADP-ribosylation are E coli, Vibrio cholerae and Bordetella Pertussis.

Tetanus toxin, produced by Clostridium tetani is a neurotoxin that prevents release of the inhibitory neurotransmitter glycine. This causes muscle spasms. Tetanus toxin tetanospasmine is composed of 2 polypeptide subunits encoded by plasmid DNA. The heavy chain of the polypeptide binds to gangliosides in the membrane of the neurone; the light chain exerts the toxic activity. The toxin released at the side of the peripheral wound may travel either by retrograde axon transport or in bloodstream to the anterior horn and interstitial neurones of the spinal cord. Blockage of release of the inhibitory transmitter leads to convulsive contraction of the voluntary muscles best exemplified by spasm of the jaw and neck muscles (lockjaw).

Botulism toxin, produced by Clostridium botulinicum, is a neurotoxin that blocks the release of acetylcholine at the synapse, producing paralysis. Genes of a temperate bacteriophage encode the toxin. 1 g of toxin is lethal for humans; it is the most toxic compounds known. The toxin is composed of 2 polypeptide subunits held together by disulphide bonds. One of the subunits binds to a receptor on the neurone; the mechanism by which the other subunit inhibits acetylcholine release is unknown.

Clostridum difficile produces two exotoxins, both of which are involved in the pathogenesis of pseudomembranous colitis. Exotoxin A is an enterotoxin that may cause watery diarrhea. Exotoxin B is a cytotoxin that damages the colonic mucosa and auses pseudomembranes to form. Exotoxin B can disaggregate actin filaments in the cytoskeleton.

Clostridium perfingens and other species of clostridia that cause gas gangrene produce multiple toxins: 7 lethal factors and 5 enzymes. Alpha toxin is the best known. Is a phospholipase that hydrolyzes lecithin in the cell membrane, resulting in the widespread cell death. The other 4 enzymes are collagenase, protease, hyaluronidase and deoxyribonuclease (DNase). The 7 lethal toxins are a heterogenous group with hemolytic and necrotizing activity.

Bacillus anthracis, the agent of antrax, produce 3 exotoxins: edema factor, protective antigen and lethal factor. Edema factor is an adenylate cyclase that requires protective antigen for its entry into human cell. The bacterial adenylate cyclase raises the c AMP concentration within the cell, resulting in loss of chloride ions and water and consequent edema formation in the tissue. The mode of action of lethal factor is unknown.

Superantigens: certain proteins act as superantigens. In contrast to the usual antigen, which activates one helper T cells, superantigens (TSST) binds directly to class II major histocompatibility (MHC) proteins, withou intracellular processing. This complex interacts with the variable portion of the beta () chain of the T cell receptor of many helper T cells. This activates the T cells. This cause the release of large amounts of interleukins, especially interleukin 1 and interleukin 2. They produce many of the signs and simptomes of toxic shock. Toxic shock syndrome toxin (TSST) is produced by certain strains of Staphylococcus aureus. The staphylococcal enterotoxins that cause food poisoning have a similar mode of action. Certain viral proteins, eg, those of mouse mammary tumor virus (a retrovirus), also posses superantigen activity.

Erythrogenic toxin, produced by Streptococcus pyogenes, causes the rash characteristic of scarlat fever. The mechanism of action the toxin is unnknown. The ADN that codes the toxin resides on a temperate bacteriophage. Notlysogenic bacteria do not cause scarlat fever, although they can cause pharyngitis.

Gram negative bacteria

The heat-labile enterotoxin produced by E. coli causes watery, nonbloody diarrhea by stimulating adenilate cyclase activity in cells in the small intestine. The resulting ingrease of the concentration of cyclic adenoside monophosphate (c AMP) causes excretion of the chloride ion, inhibition of sodium iod absorbtion, and signifiant fluid and electrolyte loss into the lumen of the gut. The heat labile toxin which is inactivated at 65⁰C for 30 minutes, is composed of 2 subunits, a B subunit, which binds to a ganglioside receptor in the cell membrane, and a A subunit, which enters the cell and mediates the transfer of ADP ribose from NAD to a stimulatory coupling protein (Gs protein). This locks the Gs protein in the on position, thereby continually stimulating adenylate cyclase to synthesise cAMP. The genes for heat labile toxin ant for heat stable toxin are carried on plasmids.

The heat stable toxin produced by E. coli is a polypeptide that is not inactivated by boiling for 30 minutes. The heat-stable toxin affects cGMP rather than cAMP. It stimulates guanylate cyclase and thus increases the concentration of cGMP, wich inhibits the reasorbtion of sodium ions and causes diarrhea.

Verotoxin is an exotoxin produced by strains of E. coli with the O157:H7 serotype. These strains cause bloody diarrhea and are the cause of outbreaks associated with eating undercooked hamburger in fast food restaurants. The toxin is named for its cytotoxic effect on Vero monkey cells in culture. The toxin inactivates protein synthesis by removing adenine from 28 S r ARN in the large subunit of the human ribosome. The enterotoxin produced by Shigella and the toxin ricin, which is produced by the Ricinus plant, are the same mode of action as does verotoxin. Ricin coupled to monoclonal antibody to human tumor antigens has been used experimentally to kill human cancer cells.

The enterotoxin produced by V cholerae, the agent of cholera, and Bacillus cereus, a cause of diarrhea, act in a similar manner similar to the heat labile toxin of E.coli. causes watery, nonbloody diarrhea by stimulating adenilate cyclase activity in cells in the small intestine. The resulting ingrease of the concentration of cyclic adenoside monophosphate (c AMP) causes excretion of the chloride ion, inhibition of sodium iod absorbtion, and signifiant fluid and electrolyte loss into the lumen of the gut.

Bordetella pertussis and Bacillus anthracis also produce toxins that increase the c AMP concentration, but these toxins act by different mechanisms.

The exotoxin of Bordetella pertussis, the cause of whooping cough, is an exotoxin that enhances adenylate cyclase. Inactivation of thie inhibitory regulator results in the stimulation of adenylate cyclase activity.

ENDOTOXINS

Endotoxins are integral part of the cell wall of both gram-negative rods and cocci, in contrast to exotoxins, which are released from the cell. Endotoxins are lipopolysaccarides LPS), whereas exotoxins are polypeptides; The enzymes that produce the lipopolysaccaride are encodes by genes on bacterial chromosome. The toxicity of endotoxins is low in comparrison with that of exotoxins. Endotoxins are weakly antigenic. They induce protective antibodoes so poorly that multiple episodes of toxicity can occur. No toxoid have been produced from endotoxins and endotoxin are not used as antigens in any avaible vaccine.

The structure of the lipopolysaccharide: the toxic portion of the molecule is lipid A, which is composed of disaccrides with several fatty acid attached. Hydroximiristic acid is always one of the fatty acid and is found only in lipid A. The other fatty acids differ according to species. The polysaccharide core is in middle of the molecule and has the same chemical composition within members of the genus. The repeat unit of sugars on the exterior differs in each species and frecvently differs between strains of a single species. It is O or somatic antigen and is composed of 3,4,5 sugars repeated up 25 times.

Mode of action of endotoxin

Activating macrophages to produce IL1 (fever), TNF (fever and hypotension) nitric oxid (hypotension).

Activating of the alternative pathway of the complement: C3a produce hypotension and edema and C5a neutrophil chemotaxis.

Activating of the coagulation system through Hageman factor (factor XII)

The biologic efects of endotoxin

The biologic efects of endotoxin include:

Fever due to release of macrophages of endogenous pyrogen (interleukin 1), which acts on hhypotathalamic temperature regulatory center.

Hypotenion, shock and impaired perfusion of essential organs owing to bradykinin induced vasodilatation, increased vascular permeability, and decreased peripheral resistance. Nitric acid a potent vasodilatator also causes hypotension.

Disseminated intravascular coagulation due to activation of the coagulation system through Hageman factor (factor XII), resultind in thrombosis, a petechial or purpuric rash, and tissue ischemia;

Activation of the alternative pathway of the complement cascade (C3a, C5a), resulting in inflammation and tissue damage. C3a produce hypotension and edema and C5a neutrophil chemotaxis.

Activation of macrophages to produce IL1 (fever), TNF (fever and hypotension) nitric oxid (hypotension), increasing their phagocytic ability.

Activation of many clones of B lymphocytes, increasing antibody production. Endotoxin is a polyclonal activator of B cell.

The evidence that endotoxin produce these effects comes from the following 2 findings: 1. Purified lipopolysaccharide, free of the organism, reproduces the effects; 2. Antiserum against the core glycolipid of endotoxin can block these effects.

Endotoxin can not cause these effects directly. They elicid the production of host factors such interleukin-1 and tumor necrosis factor (TNF - cachetin) from macrophages. TNF is the central mediator because purified recombinant TNF reproduces the effects of endotoxin and antiserum against TNF bloocks the effects of the endotoxin.

Endotoxin are the most important cause of septic shock, which is characterized by fever, hypotension, and disseminated intravascular coagulation (CID).

All endotoxins produce the same generalized effects of fever and shock, although the endotoxin of some organism are more effective that those of others. The findings of fever and hypotension are salient features of septic shock.

The endotoxins of gram negative bacteria are the best established cause of septic shock. The features of toxic shock are:

In septic shock the bacteria are in bloodstream, and culture blood is usually positive. In toxic shock the toxin is circulating in blood and blood culture are usually negative.

Septic shock can cause of death of the patient even though antibiotics have killed the bacteria in the patients blood and blood culture is negative. This occurs because septic shock is mediated by cytokines: tumor necrosis factor and interleukin-1, that continue to act even though the bacteria that induce the cytokines are no longer present.

HOST DEFENCES MECHANISMS

INTRODUCTION

The main function of the immune system is to prevent or limit infections by microorganisms such as bacteria, viruses, fungi and parasites. Protection is provided by the antibody - mediated arms of immune system (humoral immunity) and cell mediated immunity. Two other additional components of the immune system are complement and phagocytosis.

Host defences may be natural (innate) or acquired (adaptive)

NATURAL (INNATE) RESISTTANCE is innate; is not acquired through contact with antigen. It is non-specific and includes host defences such:

barriers to infections agents (skin and mucous membranes - keratin layer of intact skin: mechanical barrier); fatty acids of the skin (inhibit growth of bacteria and other microorganisms); respiratory cilia (elevate mucus containing trapped organisms);

normal flora of throat, colon, and vagina (inhibits colonisation by pathogens)

low pH of vagina and stomach (inhibits or kills certain pathogens)

certain cells (phagocytes, eg, neutrophils, macrophage - ingest and kill bacteria),

certain plasma proteins: complement (eg,C3 is an oppsonin), transferrin and lactoferrin (sequester iron required for bacterial growth); interferon's (inhibit virus replication), C - reactive protein, mannose - binding proteins and other acute phase plasma proteins, which are synthesised by the liver, binds to the surface of bacteria and activate complement), and

Other processes such phagocytosis and inflammation (limit spread of microrganisms).

IMMUNITY occurs after exposure to an agent, is specific, and mediated by lymphocytes: B - lymphocytes, T lymphocytes namely helper T and citotoxic lymphocytes, natural killer lymphocytes (cells). Immunity may be active and passive.

Active immunity is induced after contact with foreign antigens, e.g. microorganisms This contact consist of clinical or subclinical infection or exposure to microbial products: toxins (natural immunity), immunisation with live or killed infectious agents or their antigens and toxoids (artificial immunity). In all these instances, the host produces an immune response, consisting of antibodies and activated helper and cytotoxic lymphocytes. The immune response may be primary or secondary.

Passive immunity is based on antibodies performed in other host. Administration of antibody against diphtheria, tetanus, botulism makes large amounts of antitoxin immediately available to neutralise toxins. Likewise, performed antibodies to certain viruses (rabies and hepatitis A and B viruses, can be injected during the incubation period to limit viral multiplication. The disadvantages of passive immunisation are the short life - span of this antibody and possible hypersensibility reactions if globulin's of another species are used.

Antibodies cross the placenta and protect new-borns. It is passive immunity of the new-borns. 

The antibody mediated immunity - humoral immunity:

efence against extracellular, encapsulated, pyogenic bacteria,

opsonise bacteria, making them easier to phagocytes

eutralise toxins and viruses,

is accomplished by lymphocytes B and plasma cells

The cell mediated immunity

efence against intracellular bacteria, fungi, parasites

kills virus infected cells and tumour cell

Is accomplished by lymphocytes T: helper T cells and citotoxic cells and macrophages.

Both the cell mediated and antibody responses are characterised by three important features:

they exhibit remarkable diversity (they can respond to millions of different antigens;

they have a long memory (they can respond many years after the initial exposure because memory T cells and memory B cells are produced;

They exhibit exquisite specific (their action is specifically directed against the antigen that initiated the response.

The antibody mediated immunity (humoral immunity)

Antibody synthesis typically involves the co-operation of 3 cells: macrophages, helper T lymphocytes and B - lymphocytes. After processing by a macrophage, fragments of antigens appear on the surface of the macrophage in association with class II MHC proteins. This molecules bind to specific receptors on the surface of helper T lymphocytes, which then produces lymphokines such as interleukin 2, 4, 5. These factors activate the antigen specific B -lymphocytes. The activated B - lymphocytes proliferates and differentiates to form many plasma cells that secrete large amounts of antibodies - immunoglobulins.

Certain antigens can activate B - lymphocytes directly, without the help of helper T lymphocytes (e.g. bacterial polysaccharides) and are called T cell independent antigens.

ANTIGENS

Antigens are molecules that induce an immune response and react with antibodies or B and T antigen cell receptor. An antigen is immunogenic. In most cases, antigens are immunogens.

A hapten is a molecule that is not immunogenic by itself but can react with specific antibody. Haptens are usually small molecules. Many drugs, e.g. penicillin's and simple chemical groups like - COOH, SO3H are haptens. Haptens cannot stimulate a primary or secondary response by themselves. They can do so when covalently bound to a carrier protein. Hapten - carrier conjugate induce antibody against the hapten. The carrier protein is immunogenic. A hapten covalently bound to a carrier protein can induce antibody to the hapten.

In this process the hapten interacts with an Ig M receptor on the B cell and hapten - carrier protein is internalised. A peptide of carrier protein is presented in association with class II MHC protein to the helper T lymphocytes. The activated helper T lymphocytes then produce interleukins, which stimulate the B - lymphocytes to produce antibody to the hapten. A hapten alone cannot induce antibody, because the helper T lymphocytes are not activated by the hapten.

NATURAL ANTIGENS

The majority of antigenic substances are species - specific, and some are even organ specific within an animal species. Human proteins can easily be distinguished from the proteins of other animals by antigen - antibody reactions and will cross react only with protein of closely related species.

Whiting a single species, kidney protein may be distinguished from lung protein, etc. Exceptions of this species - specificity are certain antigens that are widely distributed among animals, particularly protein of the lens and the eye and so - called heterophil antigen, which is present in the organs of mice, dogs, cats, horses and chickens as well as in red cells of sheep and in some bacteria.

MICROORGANISMS, BACTERIAL ANTIGENS

Most microorganisms contain not just one, but many antigens to each of which antibodies may develop in the course of infection. Among these antigens may be capsular polysaccharides, protein pills, somatic protein or lipoprotein - carbohydrate complexes, protein exotoxins and enzymes produced by the organism.

Many hormones also are antigenic.

ANTIGENIC SPECIFICITY

The interaction of antigen and antibody is highly specific and means that an antigen will react only with antibodies elicited by its own kind or by a closely related kind of antigen. This characteristic is frequently used in the diagnostic laboratory to identify microorganisms.

Antibody usually can distinguish between the homologous antigen, which stimulated their formation, and heterologous, related antigen.

The specificity of an antibody population depends on its ability to discriminate between antigens and related structures by combining with them to a different extend.

Antigenic determinants (Epitopes): Epitopes are small chemical groups on the antigen molecules that can elicit and can react with antibody.

In general, a determinant is roughly 5 amino acids or 3 - 5 monosacharides (sugars) in size. The overall 3 - dimensional structure is the main criterion of antigenic specificity.

An antigen can have one or more determinants. Most antigens have many determinants, i.e. they are multivalent, but there are also antigens with only one determinant - this antigen is monovalent. An antigen may have only one determinant - may be a small group that is an essential part of the molecule and may repeat itself, (the determinant is the same and the antigen is monodeterminant) or may have different determinants - antigen polideterminant.

A protein antigen is polideterminant and monovalent.

A polysaccaridic antigen is monodeterminant and polyvalent.

Epitopes interacts with an Ig. M, D receptor on the B cell and with specific antibody, with antibody - combining site, named paratope.

Antigen and antibody bind by weak forces such as hydrogen bonds and van deer Walls forces rather than by covalent bonds. The strength of the binding (the affinity) is proportionate to the fit of the antigen and its antibody - combining site, i.e. its ability to form more of these bonds (depends on closeness of fit between the configuration of the antigenic determinant site and the combining site of antibody. The affinity of antibodies increases with successive exposure to the specific antigens. The antibody with the best fit and the strongest binding are said to have high affinity for the antigen. They have little tendency to dissociate from antigen after binding it (i.e. they have high avidity); early in the process of immunisation, antibody may have relatively low affinity; as immunisation proceeds, antibody of increasing higher affinity is made.

In spite of the very great antigenic specificity, cross - reactions occur between antigenic determinants of closely related structure and their antibodies. The sharing of similar antigenic determinants by molecules of different origin leads to unexpected and unpredictable cross-reactions, e.g. between human group red cells and type 14 pneumococci. Some microorganisms share antigens (e.g. Rickettsia and Proteus OX19, OX2).

With gentle formaldehyde treatment of toxins, the original antigenity may be preserved, whereas the toxicity of the molecule thus converted to a toxoid, which is immunogenic but non-toxic.

IMMUNOGENICITY

The features of molecules that determine immunogenicity are as follows.

Foreignness: in general, molecules recognised, as self are not immunogenic. The immune system is tolerant to self - molecules. To be immunogenic, molecules must be recognised as nonself or foreign.

In addition, the genetic constitution of host (HLA genes) determines whether a molecule is immunogenic. Different strains of same species of animal may respond differently to the same antigen.

Molecular size: the most potent immunogens are proteins with high molecular weights, i.e. above 100,000. Generally, molecules with molecular weights below 10,000 Dal. Are weakly immunogenic and very small ones, e.g. amino acid are nonimmunogenic. Certain small molecules, e.g. haptens, become immunogenic only when linked to a carrier protein.

Chemical - Structural Complexity: a certain amount of chemical complexity is reguired; e.g. amino acid homopolymers are less immunogenic that heteropolimers containing 2 or 3 different amino acid.

Dosage, Route, and Timing of Administration: these also affect immunogenicity. Every individual - child or adult should be adequately immunised against infectious diseases. The schedule of administration, dose and method of administration (intramuscularly, oral, etc) vary with each product.

Rate of absorption and elimination of antigen: one of feature that determines the effectiveness of an antigen as a stimulus for antibody production is its rate of absorption and elimination from the site of administration. Antigens differ greatly in their rate of excretion, but the major portion of injected antigen is eliminated from the host within hours or days. In general, the antibody response will be higher and more sustained if the antigen is absorbed slowly from its depot at site of injection. For this reason, many immunising preparations employ physical methods to delay absorption. Toxoids are often absorbed onto aluminium hydroxide (DTP - toxoids of diphtheria and tetanus, aluminium hydroxide absorbed). Bacterial or viral suspensions are sometimes prepared with adjutants, which delay absorption and promote tissue reaction to fix the antigen at its site of injection.

Adjutants: enhance the immune response to an immunogen. They are chemical unrelated to the immunogen and may act non-specific stimulating the immunoreactive cells or by release of immunogen slowly. Some human vaccine contain adjutants as aluminium hydroxide or lipids e.g. diphtheria and tetanus vaccine contain adjutants as aluminium hydroxide).

Age: immunity is less than optimal at both end of life, i.e. in the new-borne and the elderly.

The reason for the relatively poor immune response in new-borne is unclear, but they appear to have inadequate T cell function. In new-borne, antibodies are provided primarily by the transfer of maternal Ig. G across the placenta. Maternal antibodies decay and so that little remains by 3-6 months of age and the risk of infection in the child are high. Colostrum also contains antibodies, especially secretors Ig. A, which can protect new-bore against various infections.

The new-borne response to certain protein antigens is good; hence poliovirus immunisation can begin at 2 months of age. However young children respond poorly to certain polysaccharide antigens; therefore, vaccines for protection from certain pathogens, e.g. Streptococcus pneumoniae, should not be given until 18-24 months of age. 

ANTIBODIES

Antibodies are globulin protein (immunoglobulins) that react specificaly with the antigen that stimulated their production. They make up 20 % of the protein in blood plasma. Blood contain three types of globulins: alpha, beta an gamma, based on their electrophoretic migration rate. Antibodies are gamma globulins. There are five classes of antibodies: Ig G, Ig M, Ig D, Ig E.

IMMUNOGLOBULIN STRUCTURE

All immunoglogulins have similar structural patterns but great diversity of antigenic properties and amino acid sequence.

Immunoglobulins are glicoproteins make up of light (L) and heavy (H) polypeptide chains. The term light and heavy refer to molecular weight; L chains have a molecular weight of about 25, 000, whereas heavy (H) chains have a molecular weight of 50,000 - 70,000. The simplest antibody molecules has a Y sharpe and consist of 4 polypeptide chain: 2 H chains and 2 L chains. The 4 chains are linked by disulfide bonds. An individual antibody molecule always consists of identical H chains and identical L chains. This is primarly the result of 2 phenomena: allelic exclusion and regulation whitin the B cell, wich ensure the synthesis of either kappa or lambda L chains but not both.

L and H chains are subdivided into variable (V), amino terminal and constant (C) carboxi terminal regions. The regions are composed of 3 - dimensionally folded, repeating segments called domains. Each domain is approximatly 110 amino acid long.

An L chain consists of one variable (VL) and one constant (CL) domain. Most H chains consist of one variable (VH) and constant (CH) domains. Ig G, Ig A have 3 CH domains (CH1, CH2, CH3), whereas Ig M and E have 4 domains (CH1, CH2, CH3, CH4).

The variable regions are responsible for antigen binding, whereas constant regions are responsible for various biologic functions of Ig, eg, comlement activation and binding to cell surface receptors.

The variable regions of both L and H chains have 3 extremely variable (hypervariable) amino acid sequences at the amino - terminal end and form the antigen binding site. Only 5 -10 amino acid in each hypervariable region form the antigen - binding site. Antigen - antibody binding involves electrostatic and van der Waals forces and hydrogen and hydrophobic bondes rather than covalent bonds. The remarkable specificity of antibodies is due to these hypervariable regions.

L chains belong to one of 2 types: kappa (k) or lambda (l), on the basis of amino acid differences in their constant region. Both types occur in all classes of immunoglobulins, but any one immunoglobulin molecule contains only one type of L chain. In humans, the ratio of immunoglobulins containing kappa chains to those containing lambda chains is approximately 2:1. The amino - terminal portion of each L chain participates in the antigen binding site.

H chains, belong, on the basis of amino acid differences in their constant region, to one of 5 types and are designated: gamma (g), alpha (a), miu (m), epsilon (e), delta (d). H chain are distinct for each of 5 immunoglobulins classes (classes of antibodies): Ig G, Ig M, Ig D, Ig E. The amino terminal portion of each H chain participates in the antigen - binding site. The carboxy terminal portion form the Fc fragment.

If an antibody molecule is treated with a proteolytic enzime such as papain, peptide bonds in the hinge region is broken, producing 2 identical Fab fragments, wich carry the antigen - binding sites (combining site), and one Fc fragment, which has the biologic activities.

Each Fab - fragment is univalent , containing a single antigen binding site and is formed by the variable regions of both the light and heavy chains. The antigen binding site is located on the amino terminal end of antibody molecule and is composed of certain hipervariabile folded segments whitin the variable region. It has been estimated that of 650 amino acid residues of an L and H chain pair, between 15 and 30 amino acid residues may be involved in each antibody combining. The specificity of the antigen binding site is a function of the amino acid sequence of the hypervariable region and its 3- dimensional configuration.

The CH1 contains carbohydrate; CH2 domain contains the comlement binding site and the CH3 domains is the site of attacment of Ig G to receptors on neutrophils and macrophages.

IZOTYPES are defined by antigenic (amino acid) differences in their constant regions. Although different antigenically, all isotypes are found in all normal humans. For example, Ig G and IgA are different isotype; the constant region of their H chains gamma (g), alpha (a), is different antigenically. The 5 immunoglobulins classes: Ig G, Ig A, Ig E, Ig M are different isotype; their H chains are antigenically different. The Ig G isotype is subdivided into four subtypes (subclasses): Ig G1, Ig G2, Ig G3, Ig G4, based on antigenic differences of their heavy chains. Similarly, Ig A1 and Ig A2 are different isotype (the antigenity of the constant region of their H chain is different). L chains belong to one of 2 types: kappa (k) or lambda (l), on the basis of amino acid differences in their constant region. Both types occur in all classes of immunoglobulins, but any one immunoglobulin molecule contains only one type of L chain. Kappa (k) and lambda (l) L chains are different isotypes (their constant regions also differ antigenically).

ALLOTYPES: on the other hand, are additional antigenic features of immunoglobulins that vary among individuals. They vary because the genes that code for L and H chains are polymorphic and individuals can have different alleles. For example , the gamma chain contains an allotype called Gm, which is due to a one or more amino acid difference that provides a different antigenicity to the molecule. Each individual inherits different allelic genes that code for one or aother amino acid at the Gm site.

IDIOTYPE: are the antigenic determinants formed by the specific amino acid in the hipervariable region. Each idiotype is unique for the immunoglobulin produced by a specific clone of antibody producing cells. Anti - idiotype antibody reacts only with the hypervariable region of the specific immunoglogulin molecule that induce it.

BIOLOGIC ACTIVITIES OF ANTIBODIES

placentar transfer, complement fixation, attachment site for various cells: attachment to phagocitic cells, degranulation of mast cells, skin fixation and other biologic activities. 

HUMAN IMMUNITY

Humoral (antibody-mediated) immunity is directed primarily against (1) toxin-induced diseases, (2) infections in which virulence is related to polysaccharide capsules (e.g., pneumococci, meningococci, Hemophilus influenzae), and (3) certain viral infections. In this chapter the kinetics of antibody synthesis,i.e., the primary and secondary responses, are described. The functions of the various immunoglobulins are summarized in this chapter.

THE PRIMARY RESPONSE

When an antigen is first encountered, antibodies are detectable in the serum after a longer lag period than occurs in the secondary response. The lag period is typically 7-10 days but can be longer depending on the nature and dose of the antigen and the route of administration (e.g., parenteral or oral). A small clone of B cells and plasma cells specific for the antigen is formed. The serum antibody concentrations continues to rise for several weeks, then declines and may drop to very low levels. The first antibodies to appear are IgM followed by IgG or IgA. IgM levels decline earlier than IgG levels.

THE SECONDARY RESPONSE

When there is a second encounter with the same antigen or a closely related (or cross-reacting) one, months or years after the primary response, there is a rapid antibody response (the lag period is typically only 3-5 days ) to higher levels than the primary response. This is attributed to the persistence of antigen-specific memory cells after the first contact. These memory cells proliferate to form a large clone of specific B cells and plasma cells, which mediate the secondary antibody response.

During the secondary response, the amount of IgM produced is similar to that after the first contact with antigen. However, a much larger amount of IgG antibody is produced and the levels tend to persist much longer than in the primary response.

With each succeeding exposure to the antigen, the antibodies tend to bind antigen more firmly. Antibody binding improves because mutations occur in the DNA that encodes the antigen-binding site. Some mutations result in the insertion of different amino acids in the hypervariable region that result in a better fit and cause the antigen to be bound more strongly. The subset of plasma cells with these improved hypervariable regions are more strongly (and frequently) selected by antigen and therefore constitute an increasingly larger part of the population of antibody-producing cells. This process is called affinity maturation.

RESPONSE TO MULTIPLE ANTIGENS ADMINISTERED SIMULTANEOUSLY

When 2 or more antigens are administered at the same time, the host reacts by producing antibodies to all of them. Competition of antigens for antibody-producing mechanisms occurs experimentally but appears to be of little significance in medicine. Combined immunization is widely used,eg, the diphteria, pertussis, tetanus (DPT) vaccine or the measles, mumps, rubella (MMR) vaccine.

FUNCTION OF ANTIBODIES

The primary function of antibodies is to protect against infectious agents or their products. Antibodies provide resistance because they can (1) neutralize toxins and virus and (2) opsonize microorganisms. Opsonization is the process by which antibodies make microorganisms more easily ingested by phagocytic cells. This occurs by either of 2 reactions: (1) The Fc portion of IgG interacts with its receptors on the phagocyte surface to facilitate ingestion; or (2) IgG or IgM activates complement to yield C3b, which interacts with its receptors on the surface of the phagocyte.

Antibodies can be induced actively in the host or acquired passively and are thus immediately available for defense. In medicine, passive immunity is used in the neutralization of the toxins of diphteria, tetanus, and botulism by anatoxins and in the inhibition of such virus as rabies and hepatitis A and B virus early in the incubation period.

ANTIBODIES IN THE FETUS

IgM is the antibody made in greatest amounts by the fetus. Small amounts of fetal IgG and IgA are made also. Note, however, that the fetus has more total IgG than IgM because maternal IgG passes the placenta in large amounts.

TESTS FOR EVALUATION OF HUMORAL IMMUNITY

Evaluation of humoral immunity consists primarily of measuring the amount of each of the 3 important immunoglobulins,i.e., IgG, IgM, and IgA, in the patient s serum. This is usually done by radial immunodiffusion. Immunoelectrophoresis can also provide valuable information.

IMMUNOGLOBULIN CLASSES

Ig G

Each Ig G molecule consists of 2 L chains and 2 H chains linked by disulfide bonds (molecular formula H2L2, monomeric molecule) . Because it has 2 identical antigens - binding sites, it is said to be divalent. There are 4 subclasses, IgG1 - IgG4, based on antigenic differences in the H chains and on the number and location of disulfide bonds. IgG1 makes up most (65%) of the total Ig G. IgG2 antibody is directed against polysaccharide antigens and is important in host defense against encapsulated bacteria.

Ig G is the predominant antibody in the secondary response and constitues an important defenses against bacteria and viruses. Ig G is the only antibody to cross the placenta; only its Fc portion binds to receptors on the surface of placental cells. It is the most important immunoglobulins in newborne.

Ig G is one of two immunoglobulins that can activate complement, Ig M is the other.

Ig G is the immunoglobulins that opsonizes. It can opsonize, ie, enhance phagocytosis, because there are receptors for the gamma (g) H chain on the surface of phagocytes. Ig M does not opsonise directly because there are not receptors on the phagocyte surface for the miu (m) chain. However, Ig M activates complement, and the resulting C3b can opsonize because there are binding sites for C3b both on Ig G and the surface oFf phagocytes.

Ig A

Ig A is the main immunoglobulin in secretion such as colostrum, saliva, tears, and respiratory, intestinal, and genital tract secretions. It prevent attachment of microorganisms, eg bacteria and viruses, to mucous membranes. Each secretory Ig A molecule (MW 400,000) consists of 2 H2L2 units (dimers) plus one molecule each of J (joining) chain and secretory component. Only Ig A and Ig M have J chains. Only these immunoglogulins exist as multimers (dimers and pentamers, respectively). The J chain initites the polimerization process, and the multimers are held together by disulfide bonds between their Fc regions.

The secretory component is a polypeptide synthesized by epithelial cells that provides for Ig A passage to mucosal surface. It also protects Ig A being degraded in the intestinal tract. In serum, some Ig A exists as monomeric H2L2 (MW 170,000)

Ig M

Ig M is the main immunoglogulins produced early in the primary responses. It is present as a monomer on the surface of virtually all B cells, where it function as an antigen - binding receptor The surface monomer Ig M has a differen heavy chain from that of serum Ig M.

In serum, it is a pentamer composed of 5 monomers (5 H2L2 units) plus one molecule of J (joining) chain. The pentamer (MW 900,000) has a total of 10 antigen binding sites and a valence of 10. It is produced in the primary response to an antigen. Fixes complement. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody reactions against bacteria and virusses. It can be produced by fetus in certain infections, but does not cross the placenta. It has the highest avidity of the immunoglobulins; its interaction with antigen can involve all 10 of its binding site.

Ig E

The Fc region of Ig E binds to the surface of mast cells and basophils. Bound IgE serves as a receptor for antigen (allergen), and this antigen - antibody complex triggers allergic responses of the immediate (anphylactic) type. Although Ig Eis present in trace amounts in normal serum (0.004%), person with allergic reactivity have greatly increase amounts and Ig e may appear in external secretions. Ig E does not fix complement and does not cross the placenta. The serum Ig E is ussualy increased in helminth infections. Because worms are too large to be ingested by phagocytes, they are killed by eosinophils that release Worm - destroying enzymes. Ige specific to worm proteins binds to receptors on eosinophils, triggering the antibody - dependent cellular cytotoxicity (ADDC) response.

Mediates immediate hypersensitivity (anphylactic by causing release of mediators from mast cells and basophils upon exposure to antigen (allergogen). Defends against worm infection by causing release of enzymes from eosinophils. Does not fix complement. Most host defense against helminth infections.

Ig D

This immunoglobulin has no known antibody function. Found on the surface of many B cells as well in serum, and may function as an antigen receptor. It is present in small amounts in serum.

IMMUNOGLOBULIN GENES

GENE REARRANGEMENT

To produce the very large number of different immunoglobulin molecules (106-109) without requiring excessive numbers of genes, special genetic mechanisms,e.g., DNA rearrangement and RNA splicing, are used.

Each immunoglobulin chain consists of a distinct variable (V) and constant (C) regions. For each type oc immunoglobulin chain, i.e., kappa light chain (kL), lambda light chain (l L), and the 5 heavy chains ( g H, a H, m H, e H and d H) there is a separate pool of gene segments located on different chromosomes. Each pool contains a set of different V gene segments widely separated from the D (diversity, seen only in H chains), J (joining), and C gene segments.

In the synthesis of an H chain, for example, a particular V region is translocated to lie close to a D segment, several J segments, and a C region.These genes-VDJ transcribed into mRNA.

During B cell differentiation the first translocation brings a VH gene near a Cm gene, leading to the formation of Ig M as the first antibody produces in a primary response.

The V region of each L chain is encoded by 2 gene segments (V+J). The V region of each H chain is encoded by 3 gene segments (V + D + J). These various segments are united into one functional V gene by DNA rearrangement. Each of these assembled V genes is then transcribed with the appropriate C genes and spliced to produce an mRNA that codes for the complete peptide chain.

L and H chains are synthesised separately on polysomes and then assembled in the cytoplasm by means of disulfide bonds to form H2L2 units. Finally, an oligosaccharide is added to the constant region of the heavy chain and the immunoglobulin molecule is released from the cell.

The diversity of the B cell antigen receptor:The gene organisation mechanism outlined above permits the assembly of a very large number of different molecules. Antibody diversity depends on (1) multiple gene segments. (2) their rearrangement into different sequences, (3) the combining of different L and H chains in the assembly of immunoglobulin molecules,and (4) mutations. A fifth mechanism called junctional diversity applies primarily to the antibody heavy chain. Junctional diversity occurs by the addition of new nucleotides at the splice junctions between the V-D and D-J gene segments.

The diversity of the T cell antigen receptor is also dependent on the joining of V, D, and J gene segments and the combining of different alpha and beta polypeptide chains. However, unlike antibodies, mutations do not play a significant role in the diversity of the T cell receptor.

IMMUNOGLOBULIN SWITCHING (ISOTYPE SWITCHING)

Initially, all B cells carry Ig M specific for an antigen and produce Ig M antibody in response to exposure to that antigen. Later, gene rearrangement permits the elaboration of antibodies of the same antigenic specificity but of different immunoglobulin classes. Note that the antigenic specificity remains the same for the lifetime of the B cell and plasma cell because the specificity is determined by the variable regions genes (V, D, and J genes on the heavy chain and V and J genes on the light chain) no matter which heavy chain constant region is being utilised.

In class switching, the same assembled VH gene can sequentially associate with different CH genes so that the immunoglobulins produces later (IgG, IgA, or IgE) are specific for the same antigen as the original IgM but have different biologic characteristics. A different molecular mechanism is involved in the switching from IgM to IgD. In this case, a single mRNA consisting of VDJ Cm Cd is initially transcribed and is then spliced into separate VDJ Cd and VDJ Cm mRNAs. Mature B cells can, in this manner, express both IgM and IgD (alternative RNA splicing). Note that once a B cell has class switched past a certain H-chain gene, it can no longer make that class of H chain because the intervening DNA is excised and discarded. Class switching occurs only with heavy chains; light chains do not undergo class switching.

The control of class switching is dependent on at least two factors. One is the concentration of various interleukins. For example, IL - 4 enhances the production of IgE whereas IL - 5 increases IgA. The other is the interaction of the CD 40 protein on the B cell with CD 40 ligand protein on the helper T cell.

In hyper-IgM syndrome, the failure to interact properly result in an inability of the B cell to switch to the production of either IgG, IgA, or IgE. Therefore, only IgM is made.

A single B cell expresses only one L-chain (either k or l) and one H-chain allele; either the paternal or maternal set is expressed but not both. This is called allelic exclusion. Each individual contains a mixture of B cells, some expressing the paternal genes and others the maternal ones. The mechanism of this exclusion is unknown.

MONOCLONAL ANTIBODIES

Antibodies that arise in an animal in response to typical antigens are heterogeneous, because they are formed by several different clones of plasma cells: i.e., they are polyclonal. Antibodies that arise from a single clone of cells, e.g., in a plasma cell tumor ( myeloma), are homogeneous; i.e. they are monoclonal. Monoclonal antibodies also can be made in the laboratory by fusing a myeloma cell with an antibody-producing cell. Such hybridomas produce virtually unlimited quantities of monoclonal antibodies that are useful in diagnostic tests and in research.

One of the most important scientific advances of this century is the hybridoma cell, which has the remarkable ability to produce large quantities of a single molecular species of immunoglobuline. These immunoglobulins, which are known as monoclonal antibodies, are called monoclonal because they are made by a clone of cells that arose from a single cell. Note, however, that this single cell is, in fact, formed by the fusion of 2 different cells: i.e. it is a hybrid, hence the term hybridoma.

Hybridoma cells are made in the following manner: (1) An animal, e.g. a mouse, is immunised with the antigen of interest. (2) Spleen cells from this animal are grown in a culture dish in the presence of mouse myeloma cells. The strain of myeloma cells chosen has 2 important attributes: it grows indefinitely in culture, and it does not produce immunoglobulines. (3) Fusion of the cells is encouraged by adding certain chemicals, e.g. polyethylene glycol. (4) The cells are grown in a special culture medium (HAT medium) that supports the growth of the fused, hybrid cells but not of the parental cells.(5) The resulting clones of cells are screened for the production of antibody to the antigen of interest.

Chimeric monoclonal antibodies consisting of mouse variable regions and human constant regions are being made for use in treating human diseases such as leukemia. The advantages of the human constant chain are that human complement is activated (whereas it is not if the constant regions is mouse-derived) ant that antibodies against the monoclonal antibody are not formed (whereas antibodies are formed if the constant region is mouse-derived. The advantage of the mouse variable regions is that it is much easier to obtain monoclonal antibodies against, for example, a human tumor antigen by inoculating a mouse with the tumor cells. Chimeric antibodies can kill tumor cells either by complement-mediated cytotoxicity or by delivering toxins, e.g., diphtheria toxin, specifically to the tumor cell.

B CELLS

B cells perform 2 important functions:(1) They differentiate into plasma cells and produce antibodies, and (2) they are antigen presenting cells (APCs).

Origin

During embryogenensis, B cell precursors are recognized first in the fetal liver. From there they migrate to the bone marrow, which is their main location during adult life. Unlike T cells, tey do not require the tymus for maturation. Pre-B cells lack surface immunoglobulins and light chain but do have chains in the cytoplasm. The maturation of B cells has two phass: the antigen - independent phase consist of stem cells,pre-B cells and B cells, whereas the antigen - dependent phase consists of the cells that arise subsequent to the interaction of antigen with the B cells, e.g., activated B cell and plasma cells.B cells display surface IgM, which serves as a receptor for antigens. This surface IgM is a monomer, in contrast to circulating IgM, which is a pentamer. Surface IgD on some B cells may also be an antigen receptor.

B cells constitute about 30% of the recirculating pool of small lymphocytes, and their life span is short, i.e. days or weeks. Approximately 10⁹ B cells are produces each day. Within lymph nodes, they are located in germinal centers; within the spleen, they are found in the white pulp. They are also found in the gut-associated lymphoid tissue (Peyers patches).

Clonal selection

The antigen select a B cell endowed with the preexisting capacity to make the antibody.

It is clonal selelction, accounts for antibody formation. Each individual has a large pool of B lymphocytes (about 10⁷). Each immunologically responsive B cell bears a surface receptor (either IgM or IgD) that can react with one antigen (or closely realted group of antigens); i.e. there are about 107 different specificities.

An antigen interacts with the B lymphocyte that shows the best fit with its immunoglobulin surface receptor. After the antigen binds, B cell is stimulated to proliferate and form a clone of cells. These selected B cells soon become plasma cells and secrete antibody specific for the antigen. Plasma cells synthesize the immunoglobulins with the same antigenic specificity (i.e. they have the same heavy chain and the same light chain) as those carried by the selected B cell. Antigenic specificity does not change when heavy-chain class switching occurs.

Activation of B cells

In the following example, the B cell is the APC. Multivalent antigen binds to surface IgM (or IgD) and cross-links adjacent immunoglobulin molecules. The immunoglobulins aggregate to form patches and eventually migrate to one pole of the cell to form a cap. Endocytosis of the capped material follows, the antigen is processed, and epitopes appear on the surface in conjunction with class II MHC proteins. This complex is recognized by helper T cell with a receptor for the antigen on its surface TCR.The T cell now produces various lymphokines (IL-2, IL- 4 and IL-5) that stimulate the growth and differentiation of the B cell. Many plasma cells that produce large amounts of immunoglobulins specific for the epitope are the end result. Plasma cells secrete thousands of antibody molecules per second for a few days and then die. Some activated B cells form memory cells, which can remain quiescent for long periods but are capable of being activated rapidly upon reexposure to antigen. Most memory B cells have surface IgG that serves as antigen receptor, but some have IgM. Memory T cells secrete interleukins that enhance antibody production by the memory B cells. The presence of these cells explains the rapid appearance of antibody in the secondary response.

T CELL

T cell perform several important functions, which can be divided into to main categories, namely regulatory and effector. The regulatory functions are mediated primarly by helper (CD4-positive) T cells, which produce interleukins. For example, helper T cells make (1) IL-4 and IL-5, which help B cell produce antibodies; (2) IL-2 which activates CD4 and CD8 cells: and (3) gamma interferon, which activates macrophages, the main mediators of delayed hypersensitivity against intracellular organisms such as Mycobacterium tuberculosis. The effector functions are carries out primarly by cytotoxic (CD8 positive) T cells, which kill virus-infected cells, tumor cells, and allografts.

THE ANTIGEN SPECIFIC T CELL RECEPTOR

The T cell receptor is a transmembrane heterodimeric protein composed of two disulfide - linked chains. There are two different classes ot T cell receptorr. In one, the two chains are known as a and b, and in other as g and d g and d - expressing T cell are relatively infrequent in humans and seem to be predisposed toward recognition of bacterial antigens, eg, the highly conserved heat shock proteins of certain mycobacteria. a and b cells make up of the predominant T cell phenotype and are subdivided by their expression of other cell surface markers, the proteins known as CD4 and CD8, into helper and cytotoxic functional classes respectively.

The T cell receptor proteins have variable and constant regions, similar to antibodies. The variable regions are located at amino terminals of the polypeptide chain farthest away from the cell membrane. Both chains contribute to the variable domain that has been shown to interact with antigen.

Generation of diversity in the T cell receptor is accomplished in a fashion largely analogous to that decribed earlier for immunoglobulins. Thus, there are multiple variable region exons contributing a repertoire of different antigen specificieties; multiple V, D, J segments that can combine in different ways just as for antibodies; and random combination of a large number of alfa and beta chains. There are two differences from the situation described earlier for antibodies: (1) no evidence for somatic mutation in T cell receptors has been obtain, and (2) the potential for increasing the repertoire of potential antigen specificities by junctional diversity is much greater for T cell receptors than for antibodies. In essence however, the encoding of T cell receptors is very much like that described for immunoglobulins. For example, the variable regions of the alfa and gamma chains of the T cell receptor are like the variable regions of immunoglobulin light-chains in having V and J segments, whereas the beta and delta chains are like immunoglobulin heavy chains in being encoded by V, D, and J segments.

In all functional antigen-specific T cell, the T cell receptor chains are noncovalently associated with the five proteins of the CD3 complex. The CD3 complex is responsible for transducing the signal received by T cell receptor on recognition of antigen to the inside of the cell. All five proteins of the CD3 complex are transmembrane proteins and interact with tyrosine kinase on the inside of the membrane. It is this interaction that begins the biochemical events of signal transducing leading to gene transcription, cell activation, and initiation of the functional activities of T cell. 

The process of activation T cells does not function as a simply on-off switch. The binding of an epitope to the T cell receptor can result in full activation, partial activation in which only certain lymphokines are made, or no activation, depending on which of the signal transduction pathways is stimulated by that particular epitope. This important observation may have profound implications for our understanding of how helper T cells shape our response to infectious agents.

MAJOR HISTOCOMPATIBILITY COMPLEX & TRANSPLANTATION

The success of tissue and organ transplants depends on the donors and recipients human leukocyte antigens (HLA) encoded by the HLA genes. These proteins are alloantigens:i.e.,they differ among members ot the same species. If the HLA proteins on the donors cells differe from those on the recipients cells, an immune response occurs in the recipient. The genes for the HLA proteins are clusterd in the major histocompatibility complex (MHC), located on the short arm of chromosome 6.

There are 3 genes at the class I locus (A, B, and C) and 3 genes at the class II locus (DP, DQ, and DR). We inherit one set of class I and one set of class II genes from each parent. Therefore, our cells can express as many as 6 different class I and 6 different class II proteins. Furthermore, there are multiple alleles at each gene locus. Each of these MHC proteins can present peptides with a different amino acid sequence. This explains, in part, our ability to respond to many different antigens.

Three of these genes (HLA-A, HLA-B, and HLA-C) code for the class I MHC proteins. Several HLA loci determine the class II MHC proteins, i.e., DP, DQ and DR.

Each person has 2 haplotypes, i.e., 2 sets of these genes, one on the paternal and the other on the maternal chromosome 6. These genes are very diverse (polymorphic) (i.e., there are many alleles of the class I and class II genes). For example, there are at least 40 HLA-A genes, 80 HLA-B genes, and 10 HLA-C genes, but any individual inherits only an single allele at each locus from each parent and thus can make no more than 2 class I and II proteins at each gene locus. Expression of these genes is codominant; i.e., the proteins encoded by both the paternal and maternal genes are produces. Each person can make as many as 12 HLA proteins: 3 at class I loci and 3 at class II loci, from both chromosomes.

In addition to the major antigns encoded by the HLA genens, there are an unknown number of minor antigens encoded by genes at sites other then the HLA locus. These minor antigens can induce a weak immune response that can result in slow rejection of a graft. The cumulative effect of several minor antigens can lead to a more rapid rejection response. There are no laboratory tests for minor antiges.

Between the class I and class II loci is a third locus, sometimes called class III. This locus contains several immunologically important genes, namely 2 cytokines (tumor necrosis factor and lymphotoxin) and two complement components (C2 and C4).

MHC PROTEINS

Class I MHC Proteins

These are glycoproteins found on the surface of virtually all nucleated cells. There are approximately 20 different proteins encoded by the allelic genes at the A locus , 40 at the B locus and 8 at the C locus. The complete class I protein is composed of a 45,000-molecular-weight heavy chain noncovalently bound to a b 2 - microglobulin (b 2 - microglobulin is encoded on chromosome 15). The heavy chain is highly polymorphic and is similar to an immunoglobulin molecule; it has hypervariable regions in its N-terminal region. The polymorphism of these molecules is important in the recognition of self and nonself. Stated abother way, if these molecules were more similar, our ability to acept foreign grafts would be correspondingly improved. The heavy chain also has a constant region where the CD8 protein of the cytotoxic T cell binds.

Class I proteins are detected in the laboratory by reacting lymphocytes (as antigen) with a battery of specific antibodies plus complement. If lymphocyte and antibody match, the cell is lysed. This test along with the test for class II proteins, is used to identify the haplotypes of donors being considered for transplant surgery.

Class II MHC Proteins

These are glycoproteins found on the surface of certain cells: APC including macrophages, B cells, dendritic cells of the spleen, and Langerhans cells of the skin. They are highly polymorphic glycoproteins composed of 2 polypeptides (MW 33,000 and 28,000) that are noncovalently bound. Like class I proteins, they have hypervariable regions that provide much of the polymorphism. Unlike class I proteins, which have only one chain encodend by the MHC locus ( b 2 - microglobulin is encoded on chromosome 15), both chains of the class II proteins are encoded by the MHC locus. The 2 peptides also have a constant region where the CD4 proteins of the helper T cells bind.

BIOLOGIC IMPORTANCE OF MHC

The ability of T cells to recognize antigen is dependent on association of the antigen with either class I or class II proteins.

This requirement to recognize antigen in association with a self MHC protein is called MHC restriction.

There are many different alleles within the class I and class II MHC genes; hence, there are many different MHC proteins. These various MHC proteins bind to different peptide fragments. The polymorphism of The MHC genes and the proteins they encode are a means of presenting many different antigens to the T cell receptor. Note that class I and class II MHC proteins can only present peptides; other types of molecules do not bind and therefore can not be presented.

Cytotoxic T cells respond to antigen in association with class I MHC proteins. Thus, a cytotoxic T cell that kills a virus-infected cell will not kill a cell infected with the same virus if the cell does not also express the appropriate class I proteins. This finding was determined, using inbred animals, by mixing virus-infected cells and cytotoxic T cells bearing different class I proteins and observing that no killing of the virus-infected cells occurred.

Helper T cells recognize class II proteins. Helper-cell activity depends in general on both the recognition of the antigen on the antigen-presenting cells and the presence on these cells of self class II MHC proteins.

T cells recognize antigens only when the antigens are presented on the surface of cells (in association with either class I or II MHC proteins), whereas B cells do not have that requirement and can recognize soluble antigens in plasma with their surface monomer IgM acting as the antigen receptor.

MHC genes and proteins are also important in 2 other medical contexts. One is that many autoimmune diseases occur in people who carry certain MHC gnes and the other is that the succes of organ transplants is, in large part, determined by the compatibility of the MHC genes of the donor and recipient.

TRANSPLANTATION

An autograft (transfer of an individuals own tissue) is always accepted.

A syngenic graft is a transfer of tissue between genetically identical individuals, i.e., identical twins, and usually takes permanently.

A xenograft, a transfer of tissue between different species, is always rejected by an immunocompetent recipient.

An allograft is a graft between genetically different members of the same species, e.g., from one human to another. Allografts are usually rejected unless the recipient is given immunosuppresive drugs. The severity and rapidity of the rejection will vary depending on the degree of the differences between the donor and the recipient at the MHC loci.

Allograft Rejection

Unless immunosuppresive measures are taken, allografts are rejected by a process called the allograft reaction.

The acceptance or rejection of a transplant is determined, in large part, by the class I and class II MHC proteins on the donor cells,with claII playing the major role. The protein encoded by the DR locus are especially important. These alloantigens activate T cells, both helper and cytotoxic, which bear T cells receptors specific for the alloantigens. The activated T cells proliferate and then react agains the alloantigens on the donor cells. CD 8-positive T cells activated (cytotoxic T cells) do most of the killing of the allograft cells.

IMPORTANT CYTOKINES

Mediators Affecting Lymphocytes

(1) IL-1 is a protein produced mainly by macrophages. It activates a wide variety of target cells, eg, T and B lymphocytes, neutrophils, epithelial cells, and fibroblasts,to grow, differentiate, or synthesize specific products. For example, it stimulates helper T cells to differentiate and produce IL-2. In addition, it is endogenous pyrogen which acts on the hypothalamus to cause the fever associated with infections and other inflammatory reactions.

(2) IL-2 is a protein produced mainly by helper T cells that stimulates both helper and cytotoxic T cells to grow. Resting T cells are stimulated by antigen (or other stimulators) both to produce IL-2 and to form IL-2 receptors on their surface, thereby acquiring the capacity to respond to IL-2. Interaction of IL-2 with its receptor stimulates DNA synthesis. IL-2 acts synergistically with IL-4 to stimulate the growth of B cells.

(3) IL-4 and IL-5 are proteins produced by helper T cells; they promote the growth and differentiation of B cells, respectively. IL-4 enhances humoral immunity by increasing the number of Th-2 cells, the subset of helper T cells that produces IL-4 and IL-5. IL-4 is required for isotype switching, ie, the switching from one class of antibody to another, within the antibody-producing cell. It also enhances the synthesis of IgE and hence may predispose to type I hypersensitivity. IL-5 enhances the synthesis of IgA and stimulates the production and activation of eosinophils. Eosinophils are an important host defense against many helminths (eg, Strongyloides) and are increased in immediate hypersensitivity (allergic) reaction.

(4) Other interleukins (IL- 6 through IL-12) have been described. IL-6 is produced by helper T cells and stimulates B cells to differentiate. It is also produced by macrophages and is an important endogenous pyrogen (induces fever).

IL-10 and IL-12 regulate the production of Th-1 cells, the cells that mediate delayed hypersensitivity. IL-12 is produced by macrophages and promotes the development of Th-1 cells by stimulating gamma interferon production whereas IL-10 is produced by Th-2 cells and inhibits the development of Th-1 cells by limiting gamma interferon production. The relative amounts of IL-4, IL-10, and IL-12 drive the differentiation of Th-1 and Th-2 cells and therefore enhance either cell-mediated or humoral mediated immunity, respectively. This is likely to have important medical consequences because the main host defense against certain infections is either cell-mediated or humoral immunity. For example, Leishmania infections in mice ae lethal if a humoral response predominates but is controlled if a vigorous cell-mediated response ocurs. The other lymphokines are of lesser medical importance than those discussed above.

(5) The main function of transforming growth factor-b (TGF-beta) is to inhibit the growth and activities of T cells. It is viewed as an anti-cytokine because, in addition to its action on T cells, it can inhibit many functions of macrophages, B cells, neutrophils, and NK cells by counteracting the action of other activationg factors. Although it is a negative regulator of the immune response, it stimulates wound healing by enhancing the synthesis of collagen. It is produced by many types of cells, including T cells, B cells, and macrophages. In summary, the role of TGF-b is to supress the immune response when it is no longer needed after an infection and to promote the healing process.

Mediators Affecting Macrophages & Monocytes

1.Chemotactic factor attracts monocytes, which then become macrophages.

2. Migration inhibitory factor inhibits the migration of normal macrophages in vitro and may act to retain macrophages at the site of a delayed hypersensitivity reaction in vivo.

3. Macrophage-activating factors, like migration inhibitory factor, are produced by lymphocytes and can activate macrophages to phagocytize certain organisms, eg. M tuberculosis, Gamma interferon (see below) is one of the most important macrophage-activating factors.

Mediators Affecting Polymorphonuclear Leukocytes

1.Tumor necrosis factor activates the phagocytic and killing activities of neutrophils and increases the synthesis of adhesion molecules by endothelial cells. The adhesion molecules mediate the attachment of neutrophils at the site of infection.

2.Leukocyte-inhibitory factor inhibits migration of neutrophils, analogous to migration-inhibitory factor (above).

3.Chemotactic factors for neutrophils, basophils and eosinophils selectively attract each cell type.

Mediators Affecting Stem Cells

IL-3 is made by activated helper T cells and supports the growth and differentiation of bone marrow stem cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim) is made by T lymphocytes and macrophages. It stimulates the growth of granulocytes and macrophages and enhances the antimicrobial activity of macrophages. It is used clinically to improve regeneration of these cells after bone marrow transplantation. Granulocyte colony-stimulating factor (G-CSF, filgrastim) is made by various cells, eg. macrophages, fibroblasts and endothelial cells. It enhances the development of neutrophils from stem cells and is used clinically to prevent infections in patients who have received cancer chemotherapy. The stimulation of neutrophil production by G-CSF and GM-CSF results in the increased number of these cells in the peripheral blood after infection.

Mediators With Other Effects

1. Interferons are glycoproteins that block virus replication and exert many immunomodulating functions. Alpha interferon (from leukocytes) and beta interferon (from fibroblasts) are induced by viruses (or double-stranded RNA) and have antiviral activity. Gamma interferon is a limphokine produced primarily by the Th-1 subset of helper T cells. It is one of the most potent activators of the phagocytic activity of macrophages, NK cells and neutrophils, thereby enhancing their ability to kill microorganisms and tumor cells. For example, it greatly increases the killing of intracellular bacteria, such as M tubercullosis, by macrophages. It also increases the synthesis of class I and II MHC proteins in a variety of cell types. This enhances antigen presentantion by these cells.

2. Tumor necrosis factor (TNF-a is an inflammatory mediator released primarily by macrophages. It has many important effects that differ depending on the concentration. At low concentration, it increases the synthesis of adhesion molecules by endothelial cells, which allows neutrophils to adhere to blood vessel walls at the site of infection. It also activates the respiratory burst within neutrophils, thereby enhancing the killing power of these phagocytes. It increases lymphokine synthesis by helper T cells and stimulates the growth of B cells. At high concentrations, it is an important mediator of endotoxin-induced septic shock; antibody to TNF-a prevents the action of endotoxin. TNF-a is also known as cachectin because it inhibits lipoprotein lipase in adipose tissue, thereby reducing the utilisation of fatty acids. This results in cachexia. TNF-a, as its name implies, causes the death and necrosis of certain tumors in experimental animals. It may do this by promoting intravascular coagulation that causes infection of the tumor tissue. The role of TNF-a in the death of human tumor cells is unclear.

3. Lymphotoxin (also known as TNF-b) is made by activated T lymphocytes and causes effects similar to those of TNF-a. It binds to the same receptor as TNF-a and hence has the same effects as TNF-a

4. Nitric oxide (NO) is an important mediator made by macrophages in response to the presence of endotoxin, a lipopolysaccharide found in the cell wall of gram-negative bacteria. NO causes vasodilatation and therefore may be an important mediator of the hypotension seen in septic shock. Inhibitors of NO synthase, the enzyme that catalyzes the synthesis of NO from arginine, can prevent the hypotension asociated with septic shock.

FEATURES OF T CELLS

T cells constitute 65-80% of the recirculating pool of small lymphocytes. Within lymph nodes, they are located in the inner, subcortical region, not in the germinal centers. (B cells constitutes most of the remainder of the pool of small lymphocytes and are found primarily in the germinal centers of lymph nodes.) The life span of T cells is long: months or years. They can be stimulated to divide when exposed to certain mitogens, e.g., phytohemagglutinin or concanavalin A ( endotoxin, a lipopolysaccharide found on the surface of gram-negative bacteria, is a mitogen for B cells but not T cells). Most human T cells have receptors for sheep erythrocytes on their surface and can form rosettes with them; this finding serves as a means of identifying T cells in a mixed population of cells.

CD4 & CD8 Types of T cells

Within the thymus, perhaps within the outer epithelial cells, T cell progenitors differentiate under the influence of thymic hormones (thymosins and thymopoetins) into T cells subpopulations. These cells are characterized by certain surface glycoproteins (CD3, CD4 and CD3). All T cells have CD3 proteins on their surface in association with T cell antigen receptors. The CD3 complex of 5 transmembrane proteins is involved with tranmitting, from the outside of the cell to the inside, the information that the antigen receptor is occupied.. One of the CD3 transmembrane proteins, the zeta chain, is linked to a tyrosine kinase called fyn, whinch is involved with signal transduction. The signal is transmitted via several second messengers, which are described in the section on activation, below. CD4 is a single transmembrane polypwptide whereas CD8 consists of 2 transmembrane polypeptides. They may signal via tyrosine kinase (the lck kinase) also.

T cells are subdivided into 2 major categories on the basis of whether they have CD4 or CD8 proteins on their surface. Mature T cells have either CD4 or CD8 proteins but not both.

CD4 lymphocytes perform the following halper functions:

(1) they help B cells develop into antibody-producing plasma cells;

(2) they help CD8 T cells to become activated cytotoxic T cells

(3) they help macrophages effect delayed hypersensitivity:

These functions are performed by 2 subpopulations of CD4 cells: Th-1 cells help the delayed hypersensitivity response by producing primarly IL-2 and gamma interferon, whereas Th-2 cells perform the B cell helper function by producing primarly IL-4 and IL-5. One important regulator of the balance between Th-1cells and Th-2 cells is interleukin-12 (IL-12), which is produced by macrophages. IL-12 increases the number of Th-1 cells, thereby enhancing host defences against organisms that are controlled by a delayed hypersensitivity response. Another important regulator is gamma interferon which inhibits the production of Th-2 cells. CD4 cells make up to 65 % of peripheral T cells and predominate in the thymic medulla, tonsils, and blood.

CD8 lymphocites perform cytotoxic functions: that is, they kill virus-infected cells, tumor and allograft cells. They kill by either of two mechanisms, namely, the release of porfirins, which destroy cell membranes, or the induction of programmed cell death (apoptosis). CD8 cells predominate in human bone marrow and gut lymphoid tissue and consitute about 35% of peripheral T cells.

Activation of T Cells

The activation of helper T cells requires that they recognize a complex on the surface of antigen-presenting cells (APCs), eg. macrophages consisting of both the antigen and a class II MHC protein. Within the cytoplasm of the macrophage, the foreign protein is cleaved into small peptides that associate with the class II MHC proteins. The complex is transported to the surface of the macrophage, where the antigen, in association with a class II MHC protein, is presented to the receptor on the CD4-positive helper cell. Similar events occur within a virus infected cell, except that the cleaved viral peptide associate with a class I rather than a class II MHC protein. The complex is transported to the surface, where the viral antigen is presented to the receptor on a CD8-positive cytotoxic cell.

The MHC genes and the proteins they encode are a means of presenting many different antigens to the T cell receptor. Class I and class II MHC proteins can only present peptides; other types of molecules do not bind and therefore can not be presented.

The first step in the activation process is the interaction of the antigen with the T cell receptor specific for that antigen. IL-1 produced by the macrophages is also necessary for efficient helper T cell activation. When the T cell receptor interacts with the antigen-MHC protein complex, the CD4 protein on the surface of the helper T cell also interacts with the the class II MHC protein. In addition to the binding of the CD4 protein with the MHC class II protein, other proteins interact to stabilize the contact between the T cell and the APCs eg, (1) LFA-1 protein on T cells (both CD4 or CD8) binds to ICAM-1 protein on the APCs, and (2) CD28 protein on T cells binds to B7 protein on the APCs. The binding of CD28 to B7 is required for full activation of T cells.

T cells recognize only polypeptide antigens. Furthermore, they recognize those polypeptides only when they are presented in association with MHC proteins. Helper T cells recognize antigen in association with class II MHC proteins, whereas cytotoxic cells recognize antigen in association with class I MHC proteins. This is called MHC restriction: ie, the 2 types of T cells (CD4 helper and CD8 cytotoxic) are restricted because they are able to recognize antigen only when the antigen is presented with the proper class of MHC protein. This restriction is mediated by specific binding sites primarly on the T cell receptor, but also on the CD4 and CD8 proteins that bind to specific regions on the class II and class I MHC proteins, respectively.

Generally speaking, class I MHC proteins present endogenously synthesized antigens, eg, viral proteins, whereas class Ii MHC proteins present the antigen of extracellular microorganisms that have been phagocytized, eg, bacterial proteins. One important consequence of these observations is that killed vaccines do not activate the cytotoxic (CD8-positive) T cells, because the virus does not replicate within cells therefore viral epitopes are not presented in association with class I MHC proteins.

B cells, on the other hand, can interact directly with antigens via their surface immunoglobulins (IgM and IgD). Antigens do not have to be presented to b cells in association with class II MHC proteins, unlike T cells. Note that B cells can present the antigen, after internalization and processing, to helper T cells in association with class II MHC proteins located on the surface of the B cells. Unlike the antigen receptor on T cells, which recognize only peptides, the antigen receptors on B cells recognize many different types of molecules, such as peptides, polysaccharides, nucleic acids, and small chemicals,eg, penicillin.

These differences between T cells and B cells explain the hapten-carrier relationship. To stimulate hapten-specific antibody, the hapten must covalently bound to the carrier protein. A B cell specific for the hapten internalizes the hapten-carrier conjugate, processes the carrier protein, and presents a peptide to a helper t cell bearing a receptor for that peptide. The helper T cell then secretes lymphokines that activate the B cell to produce antibodies to the hapten.

When the antigen-MHC protein complex on the APC interact with the T cell receptor, a signal is transmitted by the CD3 protein complex through several pathways that eventually lead to a larga influx of calcium into the cell. Calcium activates calcineurin, a serine phosphatase. Calcineurin moves to the nucleus and is involved in the activation of the genes for IL-2 and the IL-2 receptor.

The end result of this series of events is the activation of the helper T cell to produce various lymphokines (IL-2, as well as the IL-2 receptor). IL-2 , also known as T cell growth factor, stimulates the helper T cell to multiply into a clone of antigen-specific helper T cells. Most cells of this clone perform effector and regulatory functions, but some become memory cells, which are capable of being rapidly activated upon exposure to antigen at a later time. (Cytotoxic T cells and B cells also form memory cells) Note that IL-2 stimulates CD8 cytotoxic cells as well as CD4 helper T cells. Activated CD-4 positive T cells also produce another lymphokine called gamma interferon, which increases the expression of class II MHC proteins on APCs. This enhances the ability of APCs to present antigen to T cells and upregulates the immune response.

NATURAL KILLER CELLS

Natural killer (NK) cells play an important role in the innate host defenses. They specialize in killing virus-infected cells and tumor cells by secreting cytotoxins (perforins) similar to those of cytotoxic T lymphocytes. They are called natural killer cells because they are active without prior exposure to the virus, are not enhanced by exposure, and are not specific for any virus. They can kill without antibody, but antibody enhances their effectiveness, a process called antibody-dependent cellular cytotoxicity (ADCC). From 5 to 10 % of peripheral lymphocytes are NK cells.

NK cells are lymphocytes with some T cell markers, but they do not have to pass through the thymus in order to mature. They have no immunologic memory and, unlike cytotoxic T cells, have no T cell receptor; also, killing does not require recognition of MHC proteins. IL-2 activated NK cells (LAK cells) are being used for the treatment of certain cancers.

EFFECTOR FUNCTIONS OF T CELLS

There are 2 important components of host defenses mediated by T cells: delayed hypersensitivity and cytotoxicity.

A. Delayed Hypersensitivity reactions are produced particularly against antigens of intracellular microorganisms including certain fungi, e.g., Histoplasma and Coccidioides, and certain intracellular bacteria, e.g., mycobacteria. Delayed hypersensitivity is mediated by macrophages and CD4 cells, in particular by the Th-1 subset of CD4 cells. Important lymphikines for these reactions include gamma interferon, macrophage activation factor, and macrophage migration inhibition factor. CD4 cells produce the interleukins, and macrophages are the ultimate effectors of delayed hypersensitivity. A deficiency of cell-mediated immunity manifests itself as a marked susceptibility to infection by such microorganisms.

B. Cytotoxicity: The cytotoxic response is concerned primarily with graft rejection and with destroying virus-infected cells and tumor cells. In response to virus-infected cells, the CD8 lymphocytes must recognize both viral antigen and class I molecules on the surface of infected cells. Helper (CD4) lymphocytes recognize viral antigens bound to class Ii molecules on an APC, e.g., a macrophage. The helper T cells secrete IL-2, which stimulates the virus-specific cytotoxic T cell to form a clone of cells. These cytotoxic T cells kill the virus-infected cells by inserting perforins through the membrane, the cell contents are lost; and the cell dies. After killing the virus-infected cell, the cytotoxic T cell itself is not damaged and can continue to kill other cells infected with the same virus. Cytotoxic T cells have no effect on free virus, only on virus-infected cells.

In addition to direct killing by cytotoxic T cells, virus-infected cells can be destroyed by a combination of IgG and phagocytic cells. In this process, called antibody-dependent cellular cytotoxicity (ADCC), antibody bound to the surface of the infected cell is recognized by IgG receptors on the surface of phagocytic cells,eg, macrophages or NK cells, and the infected cell is killed. The ADCC process can also kill helminths (worms). In this case, IgE is the antibody involved and eosinophils displays receptors for the epsilon heavy chain. The major basic protein located in the granules of the eosinophils is released and damages the surface of the worm.

Many tumor cells develop new antigen on their surface. These antigens bound to class I proteins are recognized by cytotoxic T cells, which are stimulated to proliferate by IL-2. The resultant clone of cytotoxic T cells can kill the tumor cells, a phenomenon called immune surveillance.

In response to allografts, cytotoxic (CD8) cells recognize the class I MHC molecules on the surface of the foreign cells. Helper (CD4) cells recognize the foreign class II molecules on certain cells in the graft, e.g., macrophages and lymphocytes. The activated helper cells secrete IL-2, which stimulates the cytotoxic cell to form a clone of cells. These cytotoxic cells kill the cells in the allograft.

REGULATORY FUNCTIONS OF T CELLS

T cells play a central role in regulating both the humoral (antibody) and cell-mediated arms of the immune system.

A. Antibody Production: Antibody production by B cells usually requires the participation of helper T cells (T cell-dependent response), but antibodies to some antigens, e.g., polymerized (multivalent) macromolecules such as bacterial capsular polysaccharide, are T cell-independent. In the following example illustrating the T cell-dependent response, B cells are used as the APC, although macrophages commonly perform this function. In this instance, antigen binds to surface IgM or IgD, is internalized within B cell, and is fragmented. Some of the fragments return to the surface in association with class II MHC molecules. These interact with the receptor on the helper T cell, which is stimulated to produce lymphokines, e.g., IL-2, B cell growth factor (IL-4), and B cell differentiation factor (IL-5). These factors stimulate the B cell do divide and differentiate into many antibody-producing plasma cells. However, interleukins alone are not sufficient to activate B cells. A membrane protein on activated helper T cells must interact with a protein called CD40 on the surface of the resting B cells to stimulate the differentiation of B cells serve to strengthen the interaction between the helper T cell and the antigen-presenting B cell; e.g., CD28 on the T cell interacts with B7 on the B cell and LFA-a on the T cell interacts with ICAM-1 on the B cell. (There are also ICAM proteins on the T cell that interact with LFA proteins on the B cell).

In the T cell-dependent response, all classes of antibody are made (IgG, IgM, IgA, etc.), whereas in the T cell-independent response, primarily IgM is made. This indicates the lymphokines produced by the t cell are needed for class switching. The T cell-dependent response generates memory B cells whereas the T cell-independent response does not, so a secondary antibody response does not occur in the latter. The T cell-independent response is the main response to bacterial capsular polysaccharides, because these molecules are not effectively processed and presented by APCs and hence do not activate helper T cells. The most likely reason for this is that polysaccharides do not bind to class II MHC proteins whereas peptide antigens do.

B. Cell-Mediated Immunity: In cell-mediated response, the initial events are similar to those described above for antibody production. The antigen is processed by macrophages, is fragmented, and is presented in conjunction with class II MHC molecules on the surface. These interact with the receptor on the helper T cell, which is then stimulated to produce lymphokines such as IL-2, which stimulates the specific helper and cytotoxic T cells to grow.

C. Suppression of Certain immune Responses: Certain T cells can supress antibody production. Failure of such regulation may result in unrestrained antibody production to self antigens, which can cause autoimmune diseases. There may not be a specific population of T cells that mediates suppression. there is evidence that in some situations CD8 cells can suppress, but inhibitory lymphokines produced by CD4 cells also can play this role.

When there is an imbalance in numbers or activity between CD4 and CD8 cells, cellular immune mechanisms are greatly impaired. For example, in lepromatous leprosy there is unrestricted multiplication of Mycobacterium leprae, a lack of delayed hypersensitivity to M. leprae antigens, a lack of cellular immunity to that organism, and an excess of CD8 cells in lesions. removal of some CD8 cells can restore cellular immunity in such patients and limit M.leprae multiplication. In acquired immunodeficiency syndrome (AIDS), the normal ratio of CD4: CD8 cells (> 1.5) is greatly reduced. Many CD4 cells are destroyed by the human immunodeficiency virus (HIV), and the number of CD8 cells increases. This imbalance, i.e., a loss of helper activity and an increase in suppressor activity, results in a susceptibility to opportunistic infections and certain tumors.

One important part of the host response to infection is the increased expression of class I and class II MHC proteins induced by various cytokines, especially interferons such as gamma interferon. The increased amount of MHC proteins leads to increased antigen presentation and a more vigorous immune response. However, certain viruses can suppress the increase in MHC protein expression, thereby enhancing their survival. For example, hepatitis B virus, adenovirus, and cytomegalovirus can prevent an increase in class I MHC protein expression, thereby reducing the cytotoxic T cell response against cells infected by these viruses.

EFFECT OF SUPERANTIGENS ON T CELLS

Certain proteins, particularly staphylococcal enterotoxins and toxic shock syndrome toxin, act as superantigens. In contrast to the usual antigen, which activates one (or a few) helper T cells, superantigens activate a large number of helper T cells. For example, toxic shock syndrome toxin binds directly to class II MHC proteins without internal processing of the toxin. This complex interacts with the variable portion of the beta chain (Vb) of the T cell receptor of many T cells. This activates the T cells, causing the release of IL-2 from the T cells and IL-1 from macrophages. The se interleukins account for many of the findings seen in toxin-mediated staphylococcal diseases. Certain viral proteins,e.g., those of mouse mammary tumor virus (a retrovirus), also possess superantigen antivity.

COMPLEMENT

The complement system consists of approximately 20 proteins that are present in normal human (and other animal) serum. The term complement refers o the ability of these proteins to complement (augment) the effects of other components of the immune system, eg.antibody. Complement is an important component of our innate host defenses.

There are 3 main effects of complement: (1) lysis of cell such as bacteria, allografts, and tumor cells; (2) generation of mediators that participate in inflammation and attract phagocytes; and (3) opsonization, ie. enhancement of phagocytosis. Complement proteins are synthesized mainly by the liver. Complement is heat-labile (it is inactivated by heating serum at 56 C for 30 minutes). Immunoglobulins are not inactivated at this temperature.

ACTIVATION

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

Sequential activation of complement components occurs via one of 2 pathways: the classic pathway and the alternative pathway. Of the two pathways, the alternative one is more important the first time we are infected by a microorganism, since the antibody required to trigger the classic pathway is not present. Both pathways lead to the production of C3b, the central molecule of the complement cascade. C3b has 2 important functions: (1) It combines with other complement components to generate C5 convertase, the enzyme that leads to the production of the membrane attack complex, and (2) it opsonizes bacteria because phagocytes have receptors for C3b on their surface.

(1) The classic pathway

The complement-binding site on the heavy chain of IgM and IgG is unavailable to the C1 component of complement if antigen is not bound to these antibodies. This means that complement is not activated by IgM and IgG despite being present in the blood at all times. However, when antigen binds to its specific antibody, a conformational shift occurs and the C1 component can bind and initiate the cascade. Antigen-antibody complexes activates C1 to form a protease, which cleaves C2 and C4 to form C4b2b complex.

The latter is C3 convertase, which cleaves C3 molecules into 2 fragments, C3a and C3b. C3a is an anaphylatoxin. C3b forms a complex with C4b2b, producing a new enzyme, C5 convertase (C4b2b3b), which cleaves C5 to form C5a and C5b. C5a is an anaphylatoxin and a chemotactic factor. C5b binds to C6 and C7 to form a complex that interacts with C8 and C9 to produce the membrane attack complex (C5b6789), which causes cytolysis. Note that the b fragment continues in the main pathway whereas the a fragment is split off and has other activities.

(2) In the alternative pathway, many unrelated cell surface substances, eg. bacterial lipopolysaccharides (endotoxin), fungal cell walls, and viral envelopes, can initiate the process by binding C3 and factor B. This complex is cleaved by a protease, factor D, to produce C3bBb. This acts as a C3 convertase to generate more C3b. The alternative pathway can proceed if is sufficient C3b. Another component that enhances activation of the alternative pathway is properdin, which protects C3b and stabilizes the C3 convertase. C3bBbP(C3b)[DV1] n acts as a C5 convertase.

REGULATION OF THE COMPLEMENT SYSTEM

The first regulatory step in the classic pathway is the level of the antibody itself. Several serum proteins regulate the complement system at different stages.

(1) C1 inhibitor is an important regulator of the classic pathway. It inactivates the protease activity of C1. Activation of the classic pathway proceeds past this point by generating sufficient C1 to overwhelm the inhibitor.

(2) Regulation of the alternative pathway is mediated by the binding of factor H to C3b and cleavage of this complex by factor I, a protease. This reduces the amount of C5 convertase available. The alternative pathway can proceed past this regulatory point if sufficient C3b attaches to cell membranes. Attachment of C3b to cell membranes protects it from degradation by factors H and I.

(3) Protection of human cells from lysis by the membrane attack complex of complement is mediated by decay-accelerating factor (DAF), a glycoprotein located on the surface of human cells. DAF acts by destabilizing C3 convertase and C5 convertase. This prevents the formation of the membrane attack complex.

BIOLOGIC EFFECTS OF THE COMPLEMENT SYSTEM

Opsonization

Cells, antigen-antibody complexes, and viruses are phagocytized much better in the presence of C3b. This is due to the presence of C3b receptors on the surface of many phagocytes.

Chemotaxis

C5a and the C567 complex attract neutrophils. They migrate especially well toward C5a. C5a also enhances the adhesiveness of neutrophils to the enodthelium.

Anaphylatoxin

C3a, C4a, and C5a cause degranulation of mast cells with release of mediators, eg. histamine, leading to increased vascular permeability and smooth muscle contraction, especially contraction of the bronchioles leading to bronchospasm. C5a is, by far, the most potent of the anaphylatoxins. Anaphylaxis caused by these complement components is less common that anaphylaxis caused by type I (IgE mediated) hypersensitivity.

Cytolysis

Insertion of the C5b6789 complex into the cell membrane leads to killing or lysis of many types of cells including erythrocytes, bacteria, and tumor cells. Cytolysis is not an enzymatic process; rather, it appears that insertion of the complex results in disruption of the membrane and the entry of water and electrolytes into the cell.

Inherited (or acquired) deficiency of some complement components, especially C5-C8, greatly enhances susceptibility to Neisseria bacteremia and other infections. A deficiency of C3 leads to severe, reccurent pyogenic sinus and respiratory tract infections.

Inherited deficiency of C1 esterase inhibitor results in angioedema. When the inhibitor is reduced, an overproduction of esterase occurs. This leads to an increase in anaphylatoxins, which cause capillary permeability and edema.

Acquired deficiency of decay-accelerating factor on the surface of cells result in an increase in complement-mediated hemolysis. Clinically, this appears as the disorder paroxysmal nocturnal hemoglobinuria.

In transfusion mismatches, (e.g. when type A blood is given by mistake to a person who is type B ), antibody to the A antigen in the recipient binds to A antigen on the donor red cells, complement is activated, and large amounts of anaphylatoxins and membrane attack complexes (C5b6789) are generated. The anaphylatoxins cause shock, and the membrane complexes cause red cell hemolysis.

Immune complexes bind complement, and thus complement levels are low in immune complex diseases (acute glomerulonephritis and systemic lupus erythematous).

Binding (activating) complement attracts polymorphonucleas leukocytes, which release enzymes that damage tissue.

PHAGOCYTOSIS

As a part of the inflammatory response, bacteria are engulfed (phagocytized) by polymorphonuclear neutrophils (PMNs) and macrophages. PMNs make up approximately 60% of the leukocytes in the blood, and their number increase significantly during infection (leukocytosis). It should be noted, however, that in certain bacterial infections such as typhoid fever, a decrease in the number of leukocytes (leukopenia) is found. The increase in PMNs is caused by the production of granulocyte-stimulating factors (G-CSF and GM-CSF) by macrophages soon after infection.

The process of phagocytosis can be divided into 3 steps : migration, ingestion and killing. Migration of PMNs to the site of the organisms is due to the chemotactic factors, such as complement component C5a and kallikrein, which - in addition to being chemotactic - is the enzyme that catalyzes the formation of bradykinin. Adhesion of PMNs to the endothelium at the site of infection is mediated first by the interactin of the PMNs with selectin proteins on the endothelium and then by the interaction of integrin proteins called LFA proteins, located on the PMN surface, with ICAM proteins on the endothelial cell surface. ICAM proteins on the endothelium are increased by inflammatory mediators, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), which are produced by macrophages in response to the presence of bacteria. The increase in the level of ICAM proteins ensures that PMNs selectively adhere to the site of infection. Increased permeability of capillary as a result of histamine, kinnis and prostaglandins allows PMNs to migrate through the capillary wall to reach the bacteria. This migration is called diapedesis and takes several minutes to occur.

The bacteria are ingested by the invagination of the PMN cell membrane around the bacteria to form a vacuole (phagosome). This engulfment in enhancd by the binding of IgG antibodies (opsonins) to the surface of the bacteria, a process called opsonizaton. The C3b component of complement enhances organization. (The outer cell membranes of both PMNs and macrophages have receptors both for the Fc portion of IgG and for C3b.) Even in the absence of antibody, the C3b component of complement, which can be generated by the alternative pathway, can opsonize. This is particularly important for bacterial and fungal organisms whose polysaccharides activate the alternative pathway.

At the time of engulfment, a new metabolic pathway, known as the respiratory burst, is triggered; this results in the production of 2 microbicidal agents, the superoxide radical and hydrogen peroxide. These highly reactive compounds are synthesized by the following reactins :

O2 + 1e- O2-

2O2- + 2H+ H2O2 +O2

In the first reaction, molecular oxygen is reduced by an electron to form the superoxide radical, which is weakly bactericidal. In the next step, the enzyme superoxide dismutase catalyzes the formation of hydrogen peroxide from 2 superoxide radicals. Hydrogen paroxide is more toxic than superoxide but is not effective against catalase-producing organisms such as staphylococci.

The killing of the organism within the phagosome is a 2-step process that consists of degranulation followed by production of hypochlorite ions (see below), which are probably the most important microbicidal agents.

In degranulation, the 2 types of granules in the cytoplasm of the neutrophil fuse with the phagosome, emptying their contents in the process. These granules are lysosomes that contain a variety of enzymes essential to the killing and degradation that accur within the phagolysosome.

1. The larger lysosomal granules, which constitute about 15% of the total, contain the important enzyme myeloperoxidase, as well as lysozyme and several other degradative enzymes. Myeloperoxidase, which is green, makes a major contribution to the color of pus.

2. The smaller granules, which make up the remaining 85%, contain lactoferrin and additional degradative enzymes such as proteases, nucleases and lipases. Lysosomal granules can empty into the extracellular space as well as into the phagosome. Outside the cell, the degradative enzymes can attack structures too large to be phagocytized, such as fungal mycelia, as well as extracellular bacteria.

The actual killing of microorganisms occurs by a variety of mechanisms, which fall into 2 categories: oxygen-dependent and oxygen-independent. The most important oxygen-dependent mechanism is the production of the highly reactive hypochlorite ion by myeloperoxidase according to the following reaction :

Cl- + H2O2  ClO- +H2O

In this reaction, chloride ion plus H2O2, which was produced by the respiratory burst, yields hypochloride ion in the presence of myeloperoxidase. Hypochloride by itself cell walls but can also react with H2O2 to produce singlet oxygen, which damages cells by reacting with double bonds in the fatty acids of membrane lipids.

Rare individuals are genetically deficient in myeloperoxidase, yet their defense systems can kill bacteria, albeit more slowly. In these persons, the respiratory burst that produces H2O2 and superoxide ion seems to be sufficient, but with 2 caveats: if an organism produces catalase, H2O2 will be ineffective, and if an organism produces superoxide dismutase, superoxide ion will be oneffective.

The oxygen-independent mechanisms are important under anaerobic conditions. These mechanisms involve lactofferin, which chelates iron from the bacteria; lysozyme, which degrades peptidoglycan in the bacterial cell wall; cationic proteins, which damage bacterial membranes; and low pH.

Macrophages also migrate, engulf, and kill bacteria by using essentially the same processes as PMNs do, but there are several differences.

1. Macrophages do not possess myeloperoxidase and so cannot make hypochlorite ion; however, they do produce H2O2 and superoxide by respiratory burst.

2. Certain organisms such as the agents of tuberculosis, brucellosis and toxoplasmosis are preferentially ingested by macrophages rather than PMNs and may remain viable and multiply within these cells; granulomas formed during these infections contain many of these macrophages.

3. Macrophages secrete plasminogen activator, an enzyme that converts the proenzyme plasminogen to active plasmin, which dissolves the fibrin clot.

In childrea with genetic defects in phagocytic process, repeated infections occur and chronic granulomatous diseases. The phagocyte cannot kill the ingested bacteria owing to a defect in NADPH oxidase and a resultant failure to generate H2O2 or in other infections lysosomal granules cannot fuse with the phagosome, so that even though bacteria is ingested, they survive.

INFLAMMATORY RESPONSE

The presence of foreign bodies such as bacteria within the body provokes a protective inflammatory response. This response is characterized by the clinical findings of redness, swelling, warmyh and pain at the site of infection. These signs are due to increased blood flow, increased capillary permeability and the escape of fluid and cells into the tissue spaces. The increased permeability is due to several chemical mediators, of which histamine, prostaglandins and leukotrienes are the most important. Bradykinin is an important ediator of pain. Of the cells that appear at the site, neutrophils and macrophages, both of which perform phagocytic functions, arrive early. Neutrophils predominate in acute pyogenic infections, whereas macrophages are more prevalent in chronic or granulomatous infections. Certain proteins, known collectively as the acute-phase response, are also produced early in inflamations. The best known of these is C-reactive protein, which is synthesized by the liver and is thought to play a role in activating the alternative pathway of complement by binding to the surface of bacteria. The importance of the inflammatory response in limiting infection is emphasized by the ability of anti-inflammatory agents such as corticosteroids to lower resistance to infection.

The inflammation is the results of: mast cells degranulation; complement activation; antigen antibody interaction and production of the cytokines.

Mediators Affecting Lymphocytes

(1) IL-1 is a protein produced mainly by macrophages. It activates a wide variety of target cells, eg, T and B lymphocytes, neutrophils, epithelial cells, and fibroblasts, to grow, differentiate or synthesize specific products. For example, it stimulates helper T cells to differentiate and produce IL-2. In addition, it is endogenous pyrogen which acts on the hypothalamus to cause the fever associated with infections and other inflammatory reactions.

IL-5 enhances the synthesis of IgA and stimulates the production and activation of eosinophils. Eosinophils are an important host defense against many helminths (eg, Strongyloides) and are increased in immediate hypersensitivity (allergic) reaction.

IL-6 is produced by helper T cells and stimulates B cells to differentiate. It is also produced by macrophages and is an important endogenous pyrogen (induces fever).

Mediators Affecting Macrophages & Monocytes

1.Chemotactic factor attracts monocytes, which then become macrophages.

Mediators Affecting Polymorphonuclear Leukocytes

1.Tumor necrosis factor activates the phagocytic and killing activities of neutrophils and increases the synthesis of adhesion molecules by endothelial cells. The adhesion molecules mediate the attachment of neutrophils at the site of infection.

2.Leukocyte-inhibitory factor inhibits migration of neutrophils, analogous to migration-inhibitory factor (above).

3.Chemotactic factors for neutrophils, basophils and eosinophils selectively attract each cell type.

Mediators Affecting Stem Cells

IL-3 is made by activated helper T cells and supports the growth and differentiation of bone marrow stem cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim) is made by T lymphocytes and macrophages. It stimulates the growth of granulocytes and macrophages and enhances the antimicrobial activity of macrophages. It is used clinically to improve regeneration of these cells after bone marrow transplantation. Granulocyte colony-stimulating factor (G-CSF, filgrastim) is made by various cells, eg. macrophages, fibroblasts and endothelial cells. It enhances the development of neutrophils from stem cells and is used clinically to prevent infections in patients who have received cancer chemotherapy. The stimulation of neutrophil production by G-CSF and GM-CSF results in the increased number of these cells in the peripheral blood after infection.

Tumor necrosis factor (TNF-a is an inflammatory mediator released primarily by macrophages. It has many important effects that differ depending on the concentration. At low concentration, it increases the synthesis of adhesion molecules by endothelial cells, which allows neutrophils to adhere to blood vessel walls at the site of infection. It also activates the respiratory burst within neutrophils, thereby enhancing the killing power of these phagocytes. It increases lymphokine synthesis by helper T cells and stimulates the growth of B cells.

2. The inflammation ocurs when antigen binds to IgE on the surface of mast cells with the consequent release of several mediators.

Some important mediators and their effects are as follows:

(1) Histamine occurs in granules of tissue mast cells and basophils in a preformed state. Its release cause vasodilation, increased capillary permeability, and smooth-muscle contraction. Clinically, disorders such as allergic rhinitis (hay fever), urticaria, and angioedema can occur. The bronchospasm so prominent in acute anaphylaxis is due, in part, to histamine release. Antihistamine drugs block histamine receptors sites and can be relatively effective in allergic rhinitis but not in asthma.

(2) Slow-reacting substance of anaphylaxis (SRS-A) consists of several leukotrienes, which do not exist in a preformed state but are produced during anaphylactic reactions. This occounts for the slow onset of the effect of SRS-A. Leukotriens are formed from arrachidonic acid by the lipoxygenase pathway and cause increased vascular permeability and smooth-muscle contraction. They are the principal mediators in the bronchoconstriction of asthma and are not influenced by antihistamines.

(3)Eosinophil chemotactic factor of anaphylaxis (ECF-A) is a tetrapeptide that exists preformed in mast cell granules. When released during anaphylaxis, it attracts eosinophils that are prominent in immediate allergic reactions. The role of eosinophils in type I hypersensitivity reactions is uncertain, but they do release histaminase and arylsulfatase, which degrade 2 important mediators, histamine and SRS-A, respectively. Eosinophils may therefore reduce the severity of the type I response.

(4) Serotonin (Hydroxytryptamine) is preformed in mast cells and blood platelets. When released during anaphylaxis, it causes capillary dilation, increased vascular permeability, and smooth-muscle contraction but is of minor importance in human anaphylaxis.

(5) Prostaglandins and thromboxanes are released to leukotrienes. They are derived from arachidonic acid via the cyclooxygenase pathway. Prostaglandins cause dilation and increased permeability of capillaries and bronchoconstriction. Thromboxanes aggregate platelets.

The above-mentioned mediators are active only for a few minutes after release; they are enzymatically inactivated and resynthesized slowly. Manifestations of anaphylaxis vary among species because mediators are released at different rates in different amounts and tissues vary in their sensitivity to them. For example, the respiratory tract is a principal shock organ in humans, but the liver plays that role in dogs.

C3a, C4a, and C5a cause degranulation of mast cells with release of mediators, eg. histamine, leading to increased vascular permeability and smooth muscle contraction, especially contraction of the bronchioles leading to bronchospasm. C5a is, by far, the most potent of the anaphylatoxins. Anaphylaxis caused by these complement components is less common that anaphylaxis caused by type I (IgE mediated) hypersensitivity.

HYPERSENSITIVITY ( ALLERGY)

When an immune response results in exaggerated or inappropriate reactions harmful to the host, the term hypersensitivity or allergy is used. The clinical manifestations of these reactions are typical in a given individual and occur on contact with the specific antigen to which the individual is hypersensitive. The first contact of the individual with the antigen sensitizes, ie, induces the antibody, and then the subsequent contacts licit tje allergic response.

Hypersensitivity reactions can be subdivided into 4 main types. Tupes I, II and III are antibody-mediated, whereas type IV is cell-mediated. Type I reactions are mediated by IgE, whereas types II and III are mediated by IgG.

TYPE I : IMMEDIATE ( ANAPHYLACTIC) HYPERSENSITIVITY

An immediate hypesensitivity reaction ocurs when antigen binds to IgE on the surface of mast cells with the consequent release of several mediators. The process begins when an antigen induces formation of IgE antibody, which binds firmly by its Fc portion to basophils and mast cells. Reexposure to the same antigen results in cross-linking of the cell-bound IgE and release of pharmacologically active mediators within minutes (immediate reaction). Cyclic nucleotides and calcium play essential roles in release of the mediators.

The clinical manifestations of type I hypersensitivity can appear in various forms, eg, urticaria (also known as hives), eczema, rhinitis and conjunctivitis (also known as hay fever), and asthma. The most severe form is systemic anaphylaxis, in which severe bronchoconstriction and hypotension (shock) can be life-threatening. No single mediator accounts for all the manifestations of type I hypersensitivity reactions. Some important mediators and their effects are as follows:

(1) Histamine occurs in granules of tissue mast cells and basophils in a preformed state. Its release cause vasodilation, increased capillary permeability, and smooth-muscle contraction. Clinically, disorders such as allergic rhinitis (hay fever), urticaria, and angioedema can occur. The bronchospasm so prominent in acute anaphylaxis is due, in part, to histamine release. Antihistamine drugs block histamine receptors sites and can be relatively effective in allergic rhinitis but not in asthma.

(2) Slow-reacting substance of anaphylaxis (SRS-A) consists of several leukotrienes, which do not exist in a preformed state but are produced during anaphylactic reactions. This occounts for the slow onset of the effect of SRS-A. Leukotriens are formed from arrachidonic acid by the lipoxygenase pathway and cause increased vascular permeability and smooth-muscle contraction. They are the principal mediators in the bronchoconstriction of asthma and are not influenced by antihistamines.

(3)Eosinophil chemotactic factor of anaphylaxis (ECF-A) is a tetrapeptide that exists preformed in mast cell granules. When released during anaphylaxis, it attracts eosinophils that are prominent in immediate allergic reactions. The role of eosinophils in type I hypersensitivity reactions is uncertain, but they do release histaminase and arylsulfatase, which degrade 2 important mediators, histamine and SRS-A, respectively. Eosinophils may therefore reduce the severity of the type I response.

(4) Serotonin (Hydroxytryptamine) is preformed in mast cells and blood platelets. When released during anaphylaxis, it causes capillary dilation, increased vascular permeability, and smooth-muscle contraction but is of minor importance in human anaphylaxis.

(5) Prostaglandins and thromboxanes are released to leukotrienes. They are derived from arachidonic acid via the cyclooxygenase pathway. Prostaglandins cause dilation and increased permeability of capillaries and bronchoconstriction. Thromboxanes aggregate platelets.

The above-mentioned mediators are active only for a few minutes after release; they are anzymatically inactivated and resynthesized slowly. Manifestations of anaphylaxis vary among species because mediators are released at different rates in different amounts and tissues vary in their sensitivity to them. For example, the respiratory tract is a principal shock organ in humans, but the liver plays that role in dogs.

In contrast to anaphylactic reactions, which are IgE - mediated, anaphylactoid reactions, which appear clinically similar to anaphylactic ones, are not IgE-mediated. In anaphylactoid reactions, the inciting agents, usually drugs or iodinated contrast media, directly induce the mast cells and basophils to release their mediators without the involvement of IgE.

ATOPY

Atopic disoders are immediate-hypersensitivity reactions that exhibit a strong familial predisposition and are associated with elevated IgE levels. The predisposition to atopy is genetic, and symptoms are induced by exposure to the specific allergens. these antigens are typically found in the environment (pollens and house dust) or in foods (shelfish and nuts). Exposure of nonatopic individuals to these substances does not elicit an allergic reaction. Common clinical manifestations include hay fever, asthma, eczema, and urticaria. Many sufferers give immediate-type reactions to skin tests (injection, patch, or scratch) containing the offending antigen.

Atopic hypersensitivity is transferable by serum (it is antibody-mediated), not by lymphoid cells. In the past, this observation was used for diagnosis in the passive cutaneous anaphylaxis (Prausnitz-Kustner) reaction, which consists of taking serum from the patient and injecting it into the skin of normal person. Some hours later the test antigen, injected into the sensitized site, will yield an immediate wheal--and-flare reaction. Thist test is now impractical because of the danger of transmitting certain viral infections. Radioallergosorbant test (RAST) permit the identification of specific IgE against potentially offending allergens if suitable specific antigns for in vitro tests are available.

The cause of atopy is uncertain. Reduced numbers of suppressor T cells and a predisposition to an abnormally high IgE response have been proposed.

Drug Hypersensitivity

Drugs, particularly antimicrobial agents such as penicilin, are now among the most common causes of hypersensitivity reactions. Usually it is not the intact drug that induces antibody formation. Rather, a metabolit product of the drug, which acts as a hapten and binds to a body protein, does so. The resulting antibody can react with the hapten or the intact drug to give rise to type I hypersensitivity. When reexposed to the drug, the person may exibit rashes, fever, or local or systemic anaphylaxis of varying severity. reactions to very small amounts of the drug can occur,eg, in a skin test with the hapten. A clinically useful example is the skin te st using penicilloyl-polylysine to reveal an allergy to penicillin.

Treatment & Prevention of Anaphylactic Reactions

Treatment includes drugs to counteract the action of mediators, maintenance of an airway, and support of respiratory and cardiac function. Epinephrine, antihistamines, corticosteroids, or cromolyn sodium, either single or in combination. should be given. Cromolyn sodium prevents release of mediators, eg, histamine, from mast cell granules. Prevention relies on identification of the allergen by a skin test and avoidance of that allergen.

TYPE II : CYTOTOXIC HYPERSENSITIVITY

Cytotoxic hypersensitivity occurs when antibody directed at antigen of the cell membrane activates complement. This generates a membrane-attack complex, which damages the cell membrane. The antibody (IgG or IgM) attaches to the antigen via the Fab region and acts as a bridge to complement via Fc region. As a result, there is complement-mediated lysis as a hemolytic anemias, ABO transfusion reactions, or Rh hemolytic disease. In addition to causing lysis, complement activation attracts phagocytes to the site, with consequent release of enzymes that damage cell membranes.

Drugs (eg, penicillins, phenacetin, quinidine) can attach to surface proteins on red blood cells and initiate antibody formation. Such autoiommune antibodies (IgG) then interact with the cell surface and result in hemolysis. The direct antiglobulin (Coombs) test is typically positive. Other drugs (eg, quinine) can attach to platelets and induce autoantibodies that lyse them to produce thrombocytopenia with bleeding tendency. Others (eg, hydralazine) may modify host tissue and favor the production of autoantibodies directed at cell DNA, with results resembling those of systemic lupus erythematosus. Certain infections, eg, Mycoplasma pneumoniae infection, can induce antibodies that cross-react with red cell antigens, resulting in hemolytic anemia. In rheumatic fever antibodies against the group A streptococci cross-react with cardia tissue. In Goodpastures syndrome, antibody to basement membranes of kidneys and lungs form, resulting in severe damage to the membranes through activity of complement-attracted leukocytes.

TYPE III : IMMUNE- COMPLEX HYPERSENSITIVITY

Immune-complex hypersensitivity occurs when antigen-antibody complexs induce an inflammatory response in tissues. Normally, immune complexes are promptly removed by the reticuloendothelial system, but occasionally they persist and are deposited in tissues, resulting in several disorders. In persistent microbial or viral infections, immune complexes may be deposited in organs, eg, the kidneys, resulting in damage. In autoiimune disorders, self antigens may elicit antibodies that bind to organ antigens or deposit in organs as complexes, especially in joints (arthritis), kidneys (nephritis), or blood vessels (vasculitis).

Whereveer immune complexes are deposited, they activate the complement system. Polymorphonuclear cells are attracted to the site, and inflammation and tissue injury occur. Two typical type III hypersensitivity reactions are the Arthus reaction and serum sickness.

Arthus Reaction

If animals are given an antigen repeatedly until they have high levels of IgG antibody and that antigen is then injected subcutaneously or intradermally, intense edema and hemorrhage develop,,reaching a peak in 3-6 hours. A clinical manifestation of the Arthus reaction hypersensitivity pneumonitis (allergic alveolitis) associated with the inhalation of thermophilic actinomycetes (farmers lung).

Serum sickness

Following the injection of foreign serum (or certain drugs), the antigen is excreted slowly. During this time, antibody production starts. Typical serum sickness results in fever, urticaria, arthralgia, lymphadenopathy, splenomegaly, and eosinophilia a few days to 2 weeks after injection of the foreign serum or drug.

Immune-complex Diseases

Many clinical disorders associated with immune complexes have been described, although the antigen that initiates the disease is often in doubt. Several representative examples are: glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus.

TYPE IV: DELAYED ( CELL-MEDIATED) HYPERSENSITIVITY

Delayed hypersensitivity is a function of helper (CD4) T lymphocytes, not antibody. It can be transferred by immunologically committed (sensitized) T cells, not by serum. The response is delayed (it starts hours or days after contact with the antigen) and often lasts for days. It consists mainly of mononuclear cell infiltration (macrophages and helper CD4 T cells) and tissue induration, as typified by the tuberculin skin test.

A. Contact Hypersensitivity: This manifestation of cell-mediated hypersensitivity occurs after sensitization with simple chemicals (nickel, formaldehyde), some cosmetics, soap and other substances.

B. Tuberculin-Type Hypersensitivity. The mechanism is the following: The macrophage ingests the antigen, processes it, and presents an epitope on its surface in association with class II MHC protein. The helper T (Th-1) cell is activated and produces gamma interferon, which activates macrophages. These two types of cells mediate delayed hypersensitivity. They produce monokines (lymphokines): mediators affecting macrophages, monocytes and lymphocytes.

1.Chemotactic factor attracts monocytes, which then become macrophages.

2. Migration inhibitory factor inhibits the migration of normal macrophages in vitro and may act to retain macrophages at the site of a delayed hypersensitivity reaction in vivo.

3. Macrophage-activating factors, like migration inhibitory factor, are produced by lymphocytes and can activate macrophages to phagocytize certain organisms, eg. M tuberculosis, Gamma interferon (see below) is one of the most important macrophage-activating factors.

Delayed hypersensitivity to antigen of microorganisms occurs in many infectious diseases and has been used as an aid in diagnosis. It is typified by the tuberculin reaction. When a patient previously exposed to Mycobacterium tuberculosis is injected with a small amount of tuberculin (PPD) intradermally, there is little reaction in the first few hours. Gradually, however, induration and redness develop and reach a peack in 48-72 hours. A positive skin test indicates that the person has been infected with the agent, but it does not confirm the presence of current disease.

Cell-mediated hypersensitivity develops in many viral infections: however serologic tests are more specific than skin tests both for diagnosis and for assessment of immunity. In protozoan and helminthic infections, skin tests may be positive, but they are generally not as useful as specific serologic tests.