III. CONTROL OF MICROORGANISMS IN THE ENVIRONMENT AND IN THE BODY

Because of their roles in decay and disease, control of microorganisms both in the environment and in our bodies has been a major goal of science. Much of the improvement in health and nutrition which have occurred in this century can be directly or indirectly correlated with increased knowledge of microbial control. In this unit the various methods which have been discovered to control the growth of microorganisms both in the environment and in our bodies will be reviewed.

A. Definitions

l. Disinfection - Any process which kills or removes infectious microorganisms.

2. Sterilization - Any process which kills or removes all organisms.

a. Commercial sterilization - This is used for canned foods. Only enough heat is applied to destroy the endospores of C. botulinum. It does not destroy the spores of some thermophiles, but they are of no practical importance because they will not grow at room temperatures.

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3. Bacteriostasis - Inhibition of bacterial growth. Note that organisms are not necessarily killed, they are simply prevented from reproducing.

4. Antisepsis - Any process which prevents the growth of microbes in living tissue (sepsis) by either killing or inhibiting their growth.

5. Asepsis - The absence of pathogens from an area or object. As in aseptic surgery.



6. Sanitization - This is essentially disinfection combined with making inanimate objects aesthetically clean.

B. Classification of control methods

l. Inhibition - These methods prevent growth and reproduction. They include the following.

a. Low temperatures

b. Air drying

c. Freeze drying (lyophilization)

d. High osmotic pressure

e. Inhibitory chemicals and drugs

2. Removal - This constitutes the physical removal of the organisms. This is accomplished by basically two methods.

a. Filtration

b. Centrifugation

3. Destruction - These methods kill or destroy the organisms. They include the following.

a. Incineration

b. High temperatures - ovens and autoclaves

c. Chemicals

d. Radiation

e. Mechanical methods - ultrasonic vibration

C. Factors which govern the effectiveness of controls

l. Characteristics of the organisms - This is the resistance of an organism to the control method employed. Several factors are found here.

a. Numbers of organisms - High numbers of organisms leads to

of clumps. Organisms inside of the clump are shielded from the control method.

b. Waxy cell walls or gelatinous sheaths will confer a high degree of resistance to water based liquid disinfectants.

c. Formation of spores - Spores are much more resistant to

destruction than are vegetative cells.

2. Characteristic of the control agent - A good control agent should have certain important characteristics.

a. It should not be highly toxic to humans or tissue corrosive.

b. It should be relatively cheap.

c. It should be easy to apply.

d. It should control the target organisms.

e. For liquid control agents, the surface tension is very

important. Surface tension may be thought of as the degree of wetness of the agent. The lower the surface tension, the more closely the active agent will come in contact with the organism. Most liquid disinfectants have a surface tension reducing agent added. Frequently this is a soap or detergent which has the effect of making the water wetter.

3. Interaction of agent and organism - The interaction of an organism with a control agent is complex and frequently involves a number of factors.



a. Temperature - Warm liquid disinfectants are more effective than colder ones. This is because increasing the temperature decreases surface tension and at the same time increases the rate of chemical reactions.

b. Organic matter - Extra organic matter can decrease the

effectiveness of control agents in two ways.

(1) It may shield organisms from the control agent.

(2) It may react with the control agent thereby reducing its concentration.

c. Time - Every agent and concentration requires a minimum amount of time to act. Less than this minimum will result in loss of control.

d. pH - Many liquid control agents have an optimum pH at which they work best. Deviation from this optimum value results in loss of activity.

D. Sterilization

l. Dry heat

a. Incineration - This is sterilization by burning. Soiled bandages, tissues, mouth wipes, sputum cups, and other disposable items are sterilized in this manner. It has the advantage of being cheap, fast, and effective. Items contaminated with radioactive materials must never be incinerated. They return as radioactive fallout!

b. Direct flaming - This is used principally to sterilize inoculating loops in the laboratory or clinic. The loop is placed in the flame until it grows red.



c. Ovens - In order to insure sterility using dry ovens, a temperature of l65 degrees C. must be maintained for two hours. Items often sterilized by this method include.

(l) Glassware

(2) Syringes

(3) Powders

(4) Gauze dressings

(5) Oily substances

2. Moist heat

a. Boiling water - This is not suitable for sterilization as the temperature of boiling water (or live steam) never exceeds l00 C. It is however an excellent disinfectant. One somewhat tedious method known as Tyndalization utilizes boiling water to sterilize. It requires that the items to be sterilized be exposed to boiling water for 30 minutes three successive days with incubation for 24 hours between each boiling. This permits any spores that were not destroyed by the water to germinated into vegetative

cells which can be destroyed by boiling.



b. Compressed steam - When steam is placed under pressure the temperature rises. This is the principle of the pressure cooker and the autoclave which is the main instrument for sterilization work in microbiology. In autoclaves the pressure of the steam is increased to about l5 to l8 pounds per square inch this pressure yields a temperature of l2l C. Which will insure sterilization in 15 to 30 minutes.

3. Sterilization without heat

a. Radiation - All living things and their spores can be destroyed by adequate levels of high energy radiation. There are several types.

(l) Ultraviolet (UV) light - This is satisfactory for air and smooth surfaces but for little else as UV has no penetrating power.

(2) Gamma radiation - This is a high energy ionizing radiation produced by many radioactive substances. A major source for sterilization is the isotope known as cobalt 60. This type of radiation has good penetrating power and therefore can be used to sterilize prepackaged materials such as petri dishes, bandages, and other similar type materials.



(a) Food irradiation - This is a technique for preserving and making food safe. While it is possible to sterilize food completely with this technique, more often it is used to reduce microbial numbers thereby increasing shelf life or for destruction of potential pathogens. While irradiation does not make food radioactive, it may, in some cases, change flavor. See page 795 of your text for more information. The following web address also contains useful information. http://www.foodsafety.org

b. Chemicals - Certain gaseous agents have been used to sterilize materials that cannot be sterilized by any other way. These include heat and water sensitive instruments as well as machinery that is launched to other planets. There are two principle agents.

(l) Ethylene oxide - This gas is usually applied in special autoclaves. It is more expensive that steam autoclaving and takes longer. It has the advantage in that it does not harm most surfaces.

(2) Beta-Propiolactone - This is a liquid at 20 C but vaporizes at 25 C. It is even more effective at killing microorganisms and their spores than is ethylene oxide. It is more expensive, more corrosive to human tissues, and may be carcinogenic. These drawbacks have limited its use.

c. Filtration - Filters for air and liquids can be designed that will remove all microorganisms.

(l) Membrane filters - These are synthetic polymer filters that have holes so small that all microorganisms will be removed.



(2) High-efficiency particulate air (HEPA) filters - These filters not only clean the air but remove all microbes thereby rendering the air sterile. These filters are used for air supplies to operating rooms, isolation rooms, and "clean" rooms where sensitive electronic equipment is assembled.

E. Disinfection - Although disinfection may be done with heat as in

Pasteurization (exposure to 63 C. for 30 minutes, or 72 C for 15 seconds) it is generally considered to be the application of a chemical substance to an inanimate object. The principal disinfecting agents in use today include the following substances.

l. Halogens - This is a family of elements that readily form salts. In terms of disinfection, only two are of significance, chlorine and iodine. Both seem to work by forming salts with protein thereby inactivating them, and also by oxidation.

a. Chlorine - This is one of the most widely used disinfectants. It is utilized in several different forms.

(l) Chlorine gas - Extremely effective, but highly toxic to humans. It requires special equipment for handling and is used for large scale disinfection such as in municipal swimming pools and in city water supplies.

(2) Calcium hypochlorite (CaOCl) - This is readily storable in powdered form and is used in l to 5% solutions. It releases chlorine gas when dissolved in water. It is widely used to disinfect home swimming pools.

(3) Sodium hypochlorite (NaOCl) - This is commercially available 5.25% solutions as ordinary laundry bleach. It is one of the best general purpose disinfectants around and certainly among the cheapest.

(4) Organic forms - There are several different organic compounds of chlorine which are good general purpose disinfectants. They are frequently substituted for calcium hypochlorite in home pools. Chloramines are one example that are often added to water supplies to control taste problems when chlorine reacts with other ammonia forms.



(5) Chlorine dioxide (ClO2) is a gas that is used to disinfect rooms, especially those contaminated with anthrax spores.

b. Iodine - This is most commonly used in alcoholic solution known as tinctures of iodine. It is a common skin disinfectant used for cuts and abrasions as well as a skin preparation in surgery.

(l) Organic forms - Like chlorine, organic compounds containing iodine are good disinfectants and frequently less harsh to the skin than tinctures preparations. These compounds are known as iodophors. Betadine is a common example.

c. Fluorine - This is the most reactive of the halogens. It is not used in disinfectants but is frequently added to water supplies in the ionic form known as fluoride. It is extremely effective in preventing dental diseases.

2. Heavy metals - It has long been known that heavy metal compounds are toxic to microorganisms as well as human beings. While lead and arsenic compounds were once used, today these have been discontinued due to their extreme toxicity to humans. Heavy metals seem to function by denaturing proteins.

a. Mercuric chloride - This was once used extensively in dilute solutions as a general disinfectant. Today it has been replaced in most cases by improved disinfectants which are not as harsh.

b. Organic compounds - These include Mercurochrome, Merthiolate and Metaphen. They are not as irritating as inorganic forms of mercury and are mainly bacteriostatic.

c. Silver nitrate - This was most commonly used in a l% solution to wash out the eyes of newborns to prevent gonorrhea eye infections, but has been largely replaced by antibiotics. It is also used in styptic pencils to stop bleeding of minor cuts. Recently silver compounds have enjoyed a resurgence as impregnations for surgical dressings where antibiotic resistance bacteria have become a problem.

d. Zinc chloride - This is an antimicrobial used in mouth washes. Zinc oxide is used as an anti-mildew agent in paints.

e. Copper sulfate - This is used as an algicide in swimming pools and other bodies of water.

3. Alcohols - There are two principal ones in use. The effectiveness of both is enhanced by the addition of small amounts of iodine. They seem to function by protein denaturization although they can also disrupt membranes and dissolve many lipids.

a. Ethyl alcohol - Widely used as a skin disinfectant, instrument disinfectant, and thermometer disinfectant. It is most effective as a 70% solution.

b. Isopropyl alcohol - This is a 70% solution sold as rubbing alcohol. It is as effective as ethyl and is cheaper and easier to obtain.

4. Phenol compounds

a. Phenol is famous as the disinfectant first used by Joseph Lister in the l880's in antiseptic surgery. It is however very corrosive to human tissues and today is usually the starting point for the synthesis of derivatives. Phenol and all of its derivatives seem to work by protein denaturization and injury of cell membranes.

b. Phenol derivatives (phenolics) - There are many of these available. Three of the more common ones are listed below.

(l) Cresols - More effective than phenol but still

corrosive. They are used as general purpose disinfectants. One of these, O-phenylphenol is the main ingredient in Lysol.

(2) Bis-phenols - These are frequently combined with surface tension reducers such as soap. Lysol falls into this category as does hexachlorophene which is the active ingredient in phisohex. Because of the possibility of neural damage in infants, hexachlorophene compounds are no longer as widely used as they once were. Triclosan is another compouind which is used in antibacterial soaps and other areas.



c. Biguanides - The princiapal compound is chlorohexidine. Although this is not a phenol compound it is similar in structure and function to hexachlorophene and has no reported toxic effects. It is often used as an alternative in soaps, and on skin and mucous membranes.

5. Oxidizing agents - In diluted form, strong oxidizing agents usually make good antiseptics. These agents kill by oxidizing organic molecules.



a. Hydrogen peroxide - This is used in 3% solutions for irrigating wounds. In this capacity it is not a good general antiseptic because the catalase from the cells rapidly break it down into water and oxygen. It is useful for anaerobes in wounds because of the oxygen released. On inanimate objects it works much better against all classes of organisms and is even sporicidal.. Contact lens disinfection and the disinfection of food packaging are two examples of current use.



b. Ozone (O3) - This is an extremely reactive form of oxygen that is generated by high energy electrical sparks and UV light. Currently there are generators being used to disinfect swimming pools.



c. Benzoyl peroxide - This is used for anaerobic wounds and to treat acne caused by anaerobic bacteria.



6. Aldehydes - These are extremely reactive substances that alter the shape of proteins by forming covalent cross links within and between chains. There are two major ones in use, formaldehyde and glutaraldehyde. Both are highly effective but are also extremely noxious to humans. Formaldehyde in solution (formalin) is a sterilizing agent and is frequently used to inactivate viruses for vaccine preparation. Glutaraldehyde is used in mortuaries for embalming and also for sterilization of various pieces of clinical equipment.

7. Surface active agents - Most soaps and detergents are surface tension reducing compounds (surfactants). Based upon chemical structure, these can be divided into three classes.

a. Anionic - The surfactant activity resides in negative charges on the molecule. Most soaps are found here.

b. Nonionic - These lack electrical charges and rely on emulsifying action exclusively.

c. Cationic - The surfactant activity resides in positive charges on the molecule. These also have disinfectant activity. The quaternary ammonium compounds (quats) are found here. The quats include such trade name items as Zephiran and Cepacol. The quats have low toxicity for humans, are effective against many microbes, and are stable in high dilutions.

8. Dyes - Certain dyes have good antibacterial activity. Crystal violet and malachite green inhibit Gram negative organisms. Acridine dyes (acriflavine and tryptoflavine) are effective against Gram positive organisms. Crystal violet is also effective against fungi.



9. Organic acids - These are frequently used in food, shampoos, and cosmetics to inhibit growth of molds. Sorbic acid and benzoic acid are examples.

10. Strength of disinfectants - At one time the principal method of evaluating disinfectants was by comparing them to a standard solution of phenol. This has been largely replaced by use-dilution tests. The procedure is as follows.



a. Three bacteria are usually used in the test. They are Salmonella choleraesuis, Staphylococcus aureus, and Pseudomonas aeruginosa.



b. Metal carrier rings are dipped into broth culture and allowed to dry at 37 degrees C.for a short period of time.



c. The carrier rings are then placed into the test disinfectant at a dilution recommended by the manufacturer, and left for 10 minutes at 20 degrees C.



d. The treated rings are transferred to a microbial growth medium. The effectiveness is gauged by how many cultures grow.



e. This method can be used to test for effectiveness against fungi, viruses, endospore formers, and Mycobacterium.



F. Antimicrobial chemotherapy - In the previous discussion were concerned with controlling microorganisms in the external environment and on the skin. In this section we will examine the control of these organisms once they have enter the body. Antimicrobial chemotherapy refers to the treatment of infectious disease by chemicals which ideally (but rarely) destroy the microbe without harming the infected person.

l. Characteristics of antimicrobial drugs

a. Definitions

(l) Antimicrobial - Any substance which can be used to kill or inhibit the growth of a microorganism in the body.

(2) Antibiotic - Antimicrobial agents obtained from organisms.

(3) Semisynthetic - Drugs which are made by modifying an antibiotic.

(4) Synthetic - Drugs which are completely laboratory made.

(5) Broad spectrum - A drug which is effective against a wide range of different organisms.

(6) Narrow spectrum - A drug which is effective against only one type or group of organisms, for example, useful only against Gram + organisms.

b. Therapeutic range - The dosage range between the minimum dosage necessary for clinical control (therapeutic dose) and the dosage which is toxic to human cells (toxic dose). The wider the therapeutic range, the more useful the drug.

c. Static versus cidal - Some drugs inhibit growth and are termed static while others kill and are termed cidal. For cidal drugs there are frequently two different doses.

(l) MIC (Minimum Inhibitory Concentration) - Lowest concentration which inhibits microbial growth.

(2) MBC (Minimum Bactericidal Concentration) - Lowest concentration which kills (cidal).

2. Modes of action

a. Inhibition of cell wall synthesis - Certain drugs block peptidoglycan synthesis and therefore inhibit procaryotes but not eucaryotes.

b. Inhibition of protein synthesis - Procaryotes utilize different sized ribosomes. Drugs which effect those ribosomes generally do not affect eucaryotes.

c. Disruption of membranes - Many groups of organisms have unique molecules in their membranes. Blockage of these molecules disrupts membranes. Most antifungals work this way.

d. Inhibition of nucleic acid synthesis - This is usually a blockage of RNA as DNA blockers cannot differentiate between microbe and human.

e. Antimetabolites (Enzyme inhibition) - These are drugs which block synthesis of some key enzyme or intermediate compound. The best example is the blockage of folic acid synthesis in bacteria by the sulfa drugs.

3. Antibacterial drugs

a. Penicillins - Natural forms are effective against G+ organisms and a few G-. Semisynthetic forms exist which are broad spectrum. Penicillins function by blocking cell wall synthesis and are bacteriostatic. They have a very wide therapeutic range.



b. Carbapenems - These represent a new class of antibiotics Which are considered to be semisynthetic penicillin. Primaxin is a representative trade name. These drugs work by inhibition of cell wall synthesis and have a very wide range of activity.



c. Cephalosporins - Similar to penicillins. They are derived from a fungus and also block cell wall synthesis. They are effective against G+ organisms. In high concentrations they are bacteriocidal. They are often used as substitutes for penicillins.



d. Monobactams - A new class of drug, similar to penicillin, but not affected by penicillinase, the enzyme produced by many penicillin resistant bacteria. Affects only a selected few gram negative organisms.

e. Macrolides - The best known antibiotic in this group is erythromycin. It is a broad spectrum antibiotic effective against G+, G-, and Chlamydia It is a protein synthesis blocking drug. It is frequently used as a penicillin alternative. It is the drug of choice in treating Legionnaire's disease. New semisynthetic forms known as ketolides have been developed to deal with antibiotic resistance problems. Ketex is an example.



f. Streptogramins - These are related to the macrolides and block protein synthesis. The principal one is Synercid, effective against a broad range of gram positive bacteria that are resistant to other antibiotics.



g. Oxazolidiones - This group was approved in 2001, the first new class of antibiotic in 25 years! They are synthetic protein synthesis blocerks effective against Gram positive bacteria. One of them, Zyvox is used against MRSA (multiple resistant S. aureus).

h. Aminoglycosides - These are primarily effective against G- organisms. They inhibit protein synthesis and disrupt cell membranes. They are all bacteriocidal. They are produced by various species of the genus Streptomyces They include streptomycin, neomycin, kanamycin, paromycin, gentamicin, and tobramicin.

i. Tetracyclines - Broad spectrum antibiotic which inhibit protein synthesis. They are bacteriostatic. They include oxytetracycline (trade name - Terramycin), chlortetracycline (trade name -Aureomycin), and tetracycline.

j. Chloramphenicol - A broad spectrum antibiotic which is bacteriostatic. It functions by blocking protein synthesis. It has very dangerous side effects including aplastic anemia and a narrow therapeutic range. It should therefore not be used for trivial conditions.

k. Polypeptides -These are polypeptide chains isolated from the genus of bacteria known as Bacillus. They function by destroying membranes. They have many toxic side effects (renal, neural, and respiratory) and are used primarily in topical preparations. Polymyxin B and Bacitracin are two examples. Vancomysin, a polypeptide obtain from a species of Streptomyces found in the jungles of Borneo, is a cell wall inhibitor, and has served as the drug of last resort to treat MRSA. Recently resistant forms to vancomysin has been discovered.



l. Sulfonamides - These are folic acid blockers. Folic acid is a key component of an essential coenzyme in many bacteria. The most widely used sulfa today is a combination of trimethoprim and sulfamethoxazole (TMP-SMZ).



m. Rifamycins - The drugs are RNA blockers. The most important is rifampin which is effective against the mycobacteria that cause TB.



n. Isoniazid (INH) - This is a synthetic which is bacteriostatic and effect almost exclusively against the mycobacteria. It seems to block the synthesis of mycolic acid. It is used with rifampin and streptomycin to treat TB.



o. Ethambutol - This drug is effective only against mycobacteria where it seems to inhibit the incorporation of mycolic acid into the cell wall. It is a weak drug used secondarily to avoid resistance problems.



p. Nitrofurans - These are very wide spectrum synthetics and affect fungi and protozoa as well as bacteria. They have limited internal use with the exception of urinary tract infections.



q. Fluroquinolones - These interferes with DNA synthesis. The most widely used are norfloxacin and ciprofloxacin (Cipro). These are broad spectrum antibiotics. Cipro is used against anthrax.



4. Antifungal drugs - Fungi, being eucaryotic, are more difficult to treat than are bacteria. The following are the principal antifungal drugs.

a. Griseofulvin - Applied topically to treat superficial mycoses. This is produced by a species of Penicillium

b. Polyenes - These are derived from Streptomyces. There are two principal ones.



(1) Nystatin - Used in cream form for yeast infections.

(2) Amphotericin B - Used to treat systemic mycoses. Many toxic side effects.

c. Azoles - One group is the imidazoles, which contain miconazole, clotrimazole, and ketoconazole. Clotrimazole and miconazole are largely topical, but ketoconazole can be taken internally as an alternative to amphotericin B. A new group, the triazoles, include fluconazole and itraconazole. These are rapidly replacing ketoconazole.



d. Allylamines - New class that are used with fungi that are resistant to the azoles.



e. Echinocandins - The first new class of antifungals developed in 40 years. They block cell wall synthesis. Cancidas is an example.



f. Flucytosine - Blocks RNA synthesis, but is narrow spectrum and highly toxic.



g. Pentamidine isethionate - Used for treatment of pneymocystis pneumonia, a frequent problem in AIDS.



d. Tolnaflate - This is frequently used as an alternative for miconazole in treating athlete's foot which is a fungal disease.



5. Antiviral drugs - These are far and few between, this is especially distressing when one considers that probably 60% of all infectious disease in developed countries are viral in origin, with bacteria accounting only for about 15%. There are no drugs available which have the effectiveness that antibiotics have on bacteria The following antiviral drugs seem to have at least some effect.

a. Ribavirin - Used against some RNA viruses.



b. Lamivudine - Used against heptatitis B.

c. Acyclovir - DNA viruses, especially genital herpes.

d. Ganciclovir and famiciclovir - Herpes viruses and the cytomegalovirus.



e. Amantadine hydrochloride - Certain RNA viruses, influenza viruses.



f. Interferon - This is an antiviral protein produced by our body. Genetic engineering has made large quantities available for chemotherapy, and it is currently used to treat hepatitis.



f. Azidothymidine (AZT) - This is one of the first drugs used against AIDS. Many more are currently available. See page 577 of your textbook for a listing of the current drugs and their modes of action.

6. Antiprotozoan drugs - There are several of these, many which are quite toxic. The most famous is chloroquine used in treatment of malaria. Metronidazole is probably the most widely used antiprotozan drug

7. Effects of improper antimicrobial drug use - Uncontrolled use of antimicrobial drugs have many dangerous side effects. These include the following.

a. Toxic effects on human tissue.

b. Evolution of resistant strains of pathogens.

c. Development of drug allergies.

d. Disturbance of the natural body flora - This often leads to superinfections. The natural ecology of the organisms that live on and in us is disrupted. This permits normal flora organisms to invade and become pathogenic.

8. Sensitivity testing - It is important to know what organisms are sensitive to various antibiotics. This is determined by several different kinds of testing. The simplest of these is the Kirby-Bauer or disk method. Paper disks are saturated with antimicrobial drugs and laid upon a petri dish which has been seeded with the test organism. After incubation the plate is observed for zones of inhibition around the various disks. The wider the zone, the more effective is the antibiotic against the test organism.



9. Generic versus trade names - Generic names are the scientific names for a drug, for example, oxytetracycline. Trade names are company names for a given drug. Terramyacin is a trade name for oxytetracycline. When a new drug is discovered by a company they are granted a 20 year patent on that drug. They will normally give it a trade name and no one else can produce that drug during this time without a license from the patent owner. After 20 years, the drug goes into the public domain and anybody can manufacture it, but they cannot use the trade name. Rather they must use its generic name. Generic drugs are of the same standards as the trade name equivalent, but usually are much less expensive as the manufacturer does not have to pay for the research and development costs.























IV. BODY DEFENSES: RESISTANCE TO INFECTION

During the course of evolution the body has developed a series of defenses against microbial invasion. Indeed, many of the drugs discussed in the last section only inhibit the organism's growth, allowing our defense systems time to catch up. In this section the

various mechanisms of defense will be examined.

A. Cells and fluids of the body which are involved in defense - Before it is possible to discuss the various defense mechanisms it is necessary to review the tissues involved in these processes.

l. Blood - This is a major line of defense in the body as the blood contains numerous substances involved in the defense process. The composition of whole blood consists of two major parts or fractions.

a. Plasma - This is the liquid portion of the blood. It contains a number of proteins which are involved in defense. Among the most important are the gamma globulins or antibodies, and the proteins known as complement.

b. Formed elements - These are the blood cells. There are three major groups, erythrocytes or red cells, leukocytes or white cells, and thrombocytes or platelets. All are derived from the bone marrow. In terms of defense against infection, only the white cells play a direct role, and they will be considered further.

(l) Leukocytes - The white blood cells are the major infection fighters. All of them are amoeboid and can move about on their own. They are divided into two major classes.

(a) Granular - These cells possess heavy granulation in their cytoplasm and have a lobed or multilobed nucleus.

/l/ Neutrophils - These are also termed polymorphonuclear (PMN) cells. They are the most abundant white cell constituting about 65% of the total white cell population. They are very active and highly phagocytic. They can leave the blood and enter the tissues.

/2/ Eosinophils - These make up about 2 to 4% of the total white cells. They are phagocytic and are usually elevated in people suffering allergies and parasitic infections.

/3/ Basophils - These make up about l% of the total white cell count. They release vasoactive substances such as heparin and histamine which can redirect blood flow.

(b) Agranular - The cytoplasm of these cells is clear and lacks granulation. The nucleus is usually round and entire or indented like a horse shoe.

/l/ Lymphocytes - The constitute about 25% of the total white cell count. They exist in two physiological forms, T-cells and B-cells. Lymphocytes play a key role in specific immunity.

/2/ Monocytes - These constitute about 5% of the total white cell population. These cells leave the circulation soon after formation and enter the tissues where they become large phagocytic cells known as macrophages.

(c) Origin of leukocytes - In the embryo all white cells have their origin in the bone marrow. After birth the granulocytes still continue to originate there, but the agranulocytes can proliferate in other parts of the body, especially in the lymphoid tissue.

2. Macrophages - These are large phagocytic cells found throughout the body. They may wander about the tissues in which case they are wandering macrophages, or they may be fixed into place, and termed histiocytes. Many fixed macrophages line certain of the blood and lymph channels where they survey the fluids that pass by.

a. Mononuclear phagocytic system (reticuloendothelial system) - This is the name given to the macrophage system. This system of macrophages cleans and purifies the body fluids of both living and non-living foreign material. In addition, damaged body tissue is also phagocytized by this system. The macrophages of the MPS (RE system) also play an important role in inducing immunity as will be discussed later.

3. Mast cells - These are cells that resemble basophils in terms of their contents, vasoactive substances. They are found distributed throughout the connective tissues of the body. They play a role in many defense processes and also in mediating allergic responses.

B. Mechanisms of defense - The body has evolved two major types of defense against infection. The first group are termed non-specific because they react to any type of invasion. The second group is highly specific in that the invading object is specifically targeted. This is known as the immune response. Most infections usually involve both non-specific and specific responses.

l. Non-specific defenses - It is convenient to think of the defenses of the body as a series of defensive lines which an infectious organism must breach if it is to be successful. The first two lines are non-specific while the third and final line is specific.

a. First line of defense - prevention of entry - The intact skin and mucous membranes that guard the body openings constitute a physical barrier to most microbes. In addition, the skin has other antimicrobial weapons.

(l) Natural flora - These are the organisms that normally live on the skin. They normally will out compete any pathogen which is not adapted to the skin. In addition, their secretions make the skin an inhospitable place for pathogens.

(2) Acidity - The pH of the skin is normally somewhat acid, and most pathogens do not like acidity. This acidity is due to organic acids produced by the normal flora as well organic acids produced by the sebaceous glands of the skin.

(3) Lysozyme - This is an enzyme produced by the skin and also found in body secretions such as tears. It hydrolyses the walls of many bacterial species.

b. Second line of defense - If the first line is breached, then a series of non-specific mechanisms are triggered. These collectively represent the second line mechanisms.

(l) Phagocytosis - This is the ingestion of material by the phagocytic cells of the RE system as well as the granulocytes. In most invasions, the phagocytic cells destroy the invaders before they can establish a foot hold. Regardless of the defensive response, it is ultimately the phagocytes that clean up the mess.

(2) Properdin system - This is part of the plasma protein system known as complement. The complement system can be thought of as a bomb that when triggered, explodes in contact with a foreign cell. The classical pathway requires specific antibody to light the complement fuse and thus is a part of specific immunity. Properdin is known as the alternative pathway because the properdin protein will light the fuse without antibody. Properdin is in turn triggered by various components of microorganisms including endotoxin. As a large number of different microbes can initiate the properdin system, it is non-specific. Details of the complement system will be considered under specific immunity.

(3) Interferon - This is a protein produced by cells when invaded by viruses. It does not protect the invaded cell, but is absorbed by adjacent cells where it stimulates the synthesis of an antiviral protein which protects those cells.

(a) There are several kinds of inteferons produced by different cells. Gamma interferon is produced by lymphocytes and has roles other than virus protection.

(b) Interferon also has anti-cancer properties. During recent years interferon genes have been cloned into bacteria so that large quantities are now available. It has not shown activity against the more common cancers such as breast, lung, and colon, but does have good affect on rarer forms.

(c) There is also hope that interferon may prove to be a good antiviral drug. The results are mixed, and many side effects have been found. Extensive research still continues as it has been discovered that there are many different subclasses of interferon, and one or more of these may prove to be useful in both cancer and viral chemotherapy.

(4) Inflammation - This is the response of the body to any irritating agent. The overall function is to remove the source of irritation, and repair any damage which may have been done. The components of the inflammatory response are as follows.

(a) Hyperemia - This is increased blood flow to the point of irritation. It is brought about by the dilation of the blood vessels supplying the irritated area. This dilation is brought about by chemical substances released by damaged tissues, Mast cells, and Basophils. If the inflammatory response is at the surface of the body the hyperemia will result in redness (rubor) and warmth (calor), two of the cardinal signs of inflammation. Increased blood flow to the area permits the mobilization of white cells and other defensive agents carried by the blood.

(b) Swelling (tumor) - Swelling of inflamed areas is due to accumulation of tissue fluid. This increased tissue fluid is due to increase permeability of the capillary blood vessels. This increase in permeability is brought about by the substances released by damaged cells, Mast cells, and basophils. Increased fluid in the area brings additional nutrients and other vital substances. Swelling causes compression of the nerve endings in the area. This in turn causes pain (dolor), the fourth cardinal sign of inflammation.

(c) Pus formation - Injured cells release substances that attract phagocyte cells, especially neutrophils. These cells congregate at the site of infection where they ingest microorganisms, dead and dying cells, and other debris. The combination of tissue fluid, white cells, and cellular debris constitutes pus.

(d) Defensive fibrin - The excess fluid from the circulation brought with it the clotting protein fibrinogen. It now forms a fibrin meshwork around the infected area. This along with fiber forming cells forms a capsule which walls off the infected area preventing the spread of the organisms which may be there. A closed fibrous sac such as this is termed an abscess.

(e) Repair - Eventually all of the microbes are destroyed and the dead tissue phagocytized. Abscesses located at the surface of the body rupture and drain to the outside. Abscesses deep in the body must be reabsorbed by the body. Eventually the cavity fills up with scar tissue and the process is complete.

(5) Natural Killer (NK) cells - These are large granular lymphocytes. They are non-specific and kill both tumor cells and virus invaded cells. NK contact the target cells and destroy their membranes. These may be the primary defense against cancer.

(6) Fever - This is a general increase in body temperature. It is brought about by a resetting of the temperature control center located in the hypothalamus of the brain. The center is reset by certain substances known as pyrogens. Some of these are chemical agents released by microorganisms. Others, such as Interleukin 1, are released by phagocytic cells of the body in response to microbial invasion. Increased body temperature is thought to be defensive in two ways.



(a) Many microorganisms will not grow well at the increased body temperatures. One reason is sensitivity to heat, but another is that at higher temperature the liver spleen sequesters iron and zinc, to elements that bacteria require in high concentrations for proper growth.



(b) The antimicrobial chemical reactions of the body defense systems are speeded up and intensified by elevated body temperatures.



2. Specific resistance - The third line of defense - This is immunity, and as the name implies, it is specifically targeted. It relies upon molecules and cells which are specifically targeted against an offending agent.

3. Types of immunity - There are two basic kinds of immunity.

a. Humoral (Antibody) mediated immunity (HMI) - This depends upon complex protein molecules termed antibodies to attack and neutralize invading organisms.

b. Cell mediated immunity (CMI) - Here cells are the effectors of the immune response, attacking and destroying microorganisms and other cells.

4. Antigens - Antigens by definition are substances which can be recognized by the immune system. If they can provoke an immune response, either CMI or HMI, they are termed immunogens. Chemically, antigens and immunogens are usually either proteins or polysaccharides. In order to be antigenic, a molecule must exhibit a large degree of complexity.

5. Cellular basis of immunity - There are three principal cell types which play a role in specific immunity. They are as follows.

a. Antigen presenting cells - These cells encounter the antigen for the first time. They process the antigen and display components of the antigen on their own cell membrane so that the other key cells in the immune response system can recognize it and evoke a response, if appropriate. The major antigen presenting cell is the macrophage, but certain skin cells, the Langerhans dendritic cells, as well as B-lymphocytes can also function as APCs.

b. T - lymphocytes (T-cells) - These are lymphocytes that have been processed in the thymus gland. There is a hormone (or a series of hormones) termed thymosin which is produced by the thymus gland. This hormone induces the maturation of immature lymphocytes into T-cells. There are three major categories of T-cells. They all appear identical, but can be differentiated from one another by the receptors (molecules) found on the surface of their membranes. Fully 80% of circulating lymphocytes are T-cells.

(l) Helper T cells (T4 or TH) - This is the primary regulatory cell of the immune system. It is ultimately responsible for recognizing the antigen displayed by the macrophage and then signaling for either HMI, CMI, or both. It may be thought of as the commander and chief of the immune system. This is the cell that the AIDS virus destroys.



(2) T suppressor cells (Ts) - These cells also have a regulatory role but it is directed toward suppression of an immune response. These cells play a role in

terminating immune responses that have run their course. They also may play a role in self tolerance.

(3) T killer or cytotoxic T cells (T8) or TC) - These cells have the ability to make contact with other cells and destroy them. They seem to be especially effective against virus infected cells and cancer cells.



(4) T delayed hypersensitivity cells (TDH) - These delayed hypersensitivity cells. They exhibit both the CD4 and CD8 markers and play a role in certain allergic responses.

c. B-lymphocytes (B-cells) - These are cells that produce antibody. They are recruited by T-helper cells. They have receptors on their membrane that interacts with antigen. When stimulated by antigen and helper T-cells, they undergo rapid mitosis to form a clone of large lymphocytes termed plasma cells. These are active antibody producing cells. They produce antibody which is specific for the antigen which stimulated the B-cell. B-cells appear identical to T-cells and can only be separated based upon their membrane receptors.

d. Memory cells - Both T and B lymphocytes produce memory cells that exist for long periods of time and remember the antigen. If the antigen makes a subsequent appearance, these memory cells immediately initiate an immune response. Consequently an immune response can be mounted immediately, destroying the antigen carrying organisms before they can establish an infection. This is why we are usually immune to a disease that we have had previously.

e. Summary - The following is a summary of the cellular events and interactions that occur during the immune response.

(l) Antigen reacts with receptors found on the surface of B lymphocytes and/or cytotoxic T lymphocytes. The antigen must also be processed by an APC (macrophage) and presented to a T-helper cell.

(2) The now activated T-helper cell divides to form a colony of cells which are sensitive to the particular antigen. The helper cells now communicate with B-cells, cytotoxic T-cells, or both, stimulating an immune response against the antigen. The activated helper T cells only interact with B-cells and cytotoxic T-cells which have been activated by the same antigen.

(3) The B-cells which have been activated produce antibody which binds with the antigen thereby altering it in some fashion. If the antigen is attached to cells, or inside of cells, cytotoxic T cells which attach to these cells and destroy them.

(4) Once the immune response has run its course and the antigen has been cleared, T-suppressor cells will bring a halt to the various immune reactions and thereby prevent an over reaction which could harm the body.

(5) A population of memory cells, both B and T, will continue to circulate. When an antigen is seen for the first time, there may be only four or five lymphocytes which have receptors that will fit it and become activated. These cells must proliferate to reach sufficient numbers to be effective. For this reason, initially an immune response may take 5 to 10 days to become powerful enough to clear the antigen. However, following antigen clearing, thousands of memory cells for that antigen will remain. If they ever meet the antigen again they will be present in sufficient numbers to provoke an immediate response thereby destroying the antigen before disease can result.

(6) The cellular interactions of the immune response are

diagramed on the following page.





6. Intercellular recognition and communication - The

cellular interactions described previously rely on precise recognition and communication between the various cells of the immune response system. The interactions can be broken down into two phases: recognition and communication.

a. Recognition - The cells of the immune response system must be able to recognize each other as well as all other body cells. The basis of this recognition is a set of antigens found on the surface of the cells. These are referred to as the MHC (major histocompatibility complex) antigens or the HLA (human leukocyte antigens). These surface antigens are all genetically controlled and serve as identification markers for antigen presentation and recognition. Only identical twins will have absolutely identical HLAs. There are two major classes.

(l) Class I - These antigens are found on all

nucleated cells in the body.

(2) Class II - These are found only on antigen presenting cells.

Both classes are involved in antigen presentation. It appears that antigen must be combined with these MHC antigens in order to be recognized. The origin of the antigen determines which class of MHC will be involved.



(1) Exogenous (outside) antigens - These must be combined with class II cell antigens. The APC processes the exogenous antigen and then combines parts of it with its own MHC II antigens which are then read by the helper T cell. This type of presentation can turn on both HMI and CMI.



(2) Endogenous (inside) antigen - This antigen comes from inside the body cell, perhaps from a virus infection, another intracellular parasite, or a cancer. This can be combined with MHC I proteins found on the surface of any nucleated cell and presented to cytotoxic T cells. This type of presentation can effect only CMI. It is primarily a defense against our own body cells that have become diseased and potentially threaten the other cells. Therefore, any cell can call in the defense of cytotoxic T cells.

Note that for both exogenous and endogenous antigen, a dual recognition is necessary in order to stimulate the immune system. Both the antigen and MHC protein, class I or class II, are necessary.

b. Communication - Cytokines (lymphokines and monokines) - The cells of the immune system have receptors on their surfaces that respond to antigen and other receptors that respond to chemical signals. These signals are proteins released by various white cells and are termed cytokines. Lymphocyte cytokines are termed lymphokines and those released by monocytes are termed monokines. At this point over 30 have been discovered. Some of the major known cytokines and their functions are listed below.

Cytokine Origin Target Action
Interleukin-l (Il-1) monocytes and macrophages TH cell activation and proliferation. Promotes fever and inflammation.
Interleukin-2 (Il-2) TH cells TH and TC cells and B cells. growth and proliferation. activates natural killer cells.
Interleukin-3 (Il-3) TH cells hematopoietic stem cells and mast cells growth and proliferation
Interleukin-4 (Il-4) TH cells B cells Costimulates activated B cells. stimulates secretions of IgE
Interleukin-5 (Il-5) TH cells B cells Costimulates activated B cells. IgA secretion
Interleukin-6 (Il-6) TH B cells Converts B cells into plasma cells.
Gamma interferon TH cells many many
Colony stimulating factor (CSF) TH cells and macrophages hemopoietic tissue proliferation of granulocytes
Tumor necrosis factor (TNF) monocytes and macrophages tumor cells destruction
Macrophage chemotactic factor (MCF) TH cells macrophages attracts to site of CMI reactions
Macrophage inhibiting factor (MIF) TH cells macrophages inhibits movement from area of CMI reactions
Macrophage activating factor (MAF) TH cells macrophages activates macrophages during CMI responses






An example of cytokine communication in an infection would be as follows.

a. A macrophage engulfs an invading organism and couples with a helper T-cell. It releases IL-l which activates the helper T- cell. IL-l also stimulates fever which helps fight infection.



b. The activated helper T-cell produces IL-2 which stimulates other helper and/or cytotoxic T-cells to grow and divide. The helper T-cells release Il-4 and Il-5 which causes B-cells to proliferate.

c. As the number of B-cells increases, the helper T-cells release Il-6 which instructs the B-cells to become plasma cells.



d. Helper T-cells produce gamma interferon which has many effects.



(l) Like IL-2, it also activates killer T-cells.

(2) It increases the ability of B-cells to produce antibody.

(3) It attracts and activates macrophages at the site of

infection.



(4) It activates NK cells and also has antiviral effects.

e. The cytokine cascade amplifies the immune response until the invading organism is overwhelmed by sheer strength of numbers.



7. Antigen specificity - clonal selection - It was pointed out earlier that each lymphocyte had a unique antigen receptor. As noted above the antigen receptor for B cells are IgD and IgM antibodies attached the surface of the cell membrane. How is it possible for each lymphocyte to have a uniquely shaped antigen receptor and be able to produce an antibody that is exactly the same shape?



(1) During development of a lymphocyte there are about 200 or so gene segments that code for antibody and antigen receptors. During the maturation of the lymphocyte, the variable regions are assembled randomly by "mixing and matching" various gene segments. With several hundred fragments to choose from, right away it becomes obvious that millions of possible amino acid sequences in the final antibody are possible.



(2) The different variable regions for both light chains and heavy chains are not attached randomly to the different constant regions which also increases variability.



(3) The genes for B-cell receptors and antibodies show hypermutability. They mutate very rapidly during development increasing the possible receptor types even further.



(4) Once an antigen finds a receptor it fits, the lymphocyte which bears that receptor has been "selected." With appropriate costimulation it will rapidly divide forming a clone of identical cells which are also sensitive to the same antigen. In the case of B-cells this clone becomes a clone of antibody secreting cells all of which produce antibody specific for the antigen which did the selecting.8.Humoral mediated immunity - This is immunity which is mediated by antibodies produced by B-cells. A more recent term used for antibodies is immunoglobulin.

a. Immunoglobulins - These are large complex protein molecules that belong to that class of plasma proteins known as gamma globulins. Gamma globulin, antibody, and immunoglobulin all mean the same thing. Immunoglobulins are synthesized upon stimulation of a B-cell by an appropriate antigen, and is specific for that antigen.

b. Structure of immunoglobulins - Each immunoglobulin is composed of 4 polypeptide chains.

(l) Two of the chains are large and are termed heavy (H) chains.

(2) Two of the chains are small and are termed light (L) chains.

(3) Both the heavy and light chains have two regions: a constant region and a variable region. The constant regions are the same for all heavy chains within a given immunoglobulin class and the same is true for the light chains. The variable regions are unique for each immunoglobulin and give them their specificity



c. Classification of immunoglobulins - Immunoglobulins are divided into 5 classes based upon the constant region of the heavy chain. Within each class, the constant region of every heavy chain is identical. The classes are presented below.

(l) IgG (Immunoglobulin G) - The most abundant in the body, accounting for about 80% of all serum Ig, circulates in the plasma and can cross the placenta.

(2) IgM - Present in much lesser quantities than IgG (5 to 10%) , but still a circulating molecule. It is the first immunoglobulin that a plasma cell will make following antigen exposure. The cells will eventually switch to IgG production. IgM also serves as an antigen receptor on the surface of B cells.

(3) IgA - Secreted on to the surfaces of the body that open to the outside. Found in tears and other surface secretions.

(4) IgD - Found attached to the surface of B-cells where it serves as the receptor for antigen along with IgM.

(5) IgE - Does not circulate. Attaches to Mast cells in the tissues and mediates allergic responses.

d. Functions of immunoglobulins - Ig usually does not destroy antigen directly. Usually they mark antigen for destruction by other agents of the defense system such as phagocytic cells or complement. The principal functions that immunoglobulins are involved in are as follows.

(l) Precipitation - Soluble molecules (mostly proteins) such as bacterial toxins are precipitated out of solution into masses which can then be phagocytized. Such molecules are sometimes called anti-toxins and preciptins. IgG

(2) Agglutinins - These react with antigens on the

surface of the cells causing the cells to clump together (agglutinate). IgG, IgA, and IgM (the best). Agglutinated cells are easily phagocytized.

(4) Opsonins - These react with surface antigens to form

a coat on cells. This facilitates phagocytosis.

IgG.

(5) Neutralization - These react with viruses and bacterial exotoxins by destroying their ability to infect cells. IgG and IgA.

(6) Lysins - These immunoglobulins cause the breakup

of cells. They do this by targeting the cells with complement proteins. The complement proteins then react on the surface of the cell causing holes to appear in the membrane. The complement proteins may be thought of as a bomb. The bomb is placed on the target cell and then activated by immunoglobulin.

IgG and IgM.



e. Complement - Complement is a major mediator of HMI. Immunoglobulin react with antigens and in doing so often cause complement undergo a series of reactions, the so called fixation of complement. Complement gets its name from the fact that it is often a required "complementary" factor in immunoglobulin mediated responses. Complement consists of ll different plasma proteins. Once the fixation process begins, it is much like knocking over dominoes, one protein reacts and in doing so causes the next to react all the way down the line. Complement fixation that requires immunoglobulin is termed the classical pathway while complement fixation that is non-specific, does not require immunoglobulin is termed the alternative pathway or the properdin system.

(l) Designation of complement components - All of the proteins in the complement system are designated by the letter C with a number following. Thus we find Cl - C9. The reason there are not ll proteins is that the first three complement proteins combine to form Cl. Subfragments are designated with an addition letter, for example, C5a or C3a. The additional components of the properdin system are designated

P, B, and D.

(2) Fixation of complement - classical pathway - This requires the formation of an antigen-immunoglobulin complex. The fixation process may, for simplicity, be divided into three phases.

(a) Recognition - The first complement component (Cl) is bound to the antigen-immunoglobulin complex. The binding converts Cl into an active enzyme that begins to work on the other complement proteins and leads to the second phase.

(b) Activation - Active Cl now activates C4, C2, and C3, in that order.

(c) Formation of the membrane attack complex - The now active enzymes formed during the activation phase now react with the remaining complement components, C5 - C9, to form a chemical complex which destroys the cell membrane. This results in lysis of the cell.

(3) Fixation of complement - the alternative pathway - In this method the process begins at C3 when factor B activates it. Cl, C2, and C4 are not involved. Immunoglobulin is not required. The triggers for the alternate pathway include components found in the cell walls of G- bacteria, bacterial capsules, fungal cell walls, and other chemical substances.

(4) Other defense roles of complement - Complement plays a number of other defensive roles in addition to cell lysis.

(a) Endotoxin inactivation.

(b) Virus neutralization.

(c) Initiation of inflammation.

/l/ Dilation of blood vessels.

/2/ Chemotaxis of neutrophils, monocytes, and

eosinophils.

(d) Immune adherence (opsonization) - Attachment of antigen to macrophages.

(e) Increased induction of antibody formation.

(f) Stimulation of B-cell lymphokine production.

9. Cell Mediated Immunity - In CMI, not only is the effector always a cell, but the target is also a cell. It may be one of the following.

a. A host cell that contains an intracellular parasite (TB is an example).

b. A transformed cell (one with new antigens) due either to a virus infection or the cell has become cancerous.

c. A foreign tissue graft.

The target cell is always killed by either contact with a T-cell or a macrophage. The sequence of events in a CMI response is as follows.

a. The macrophage identifies the foreign cell and communicates antigen information to a helper T-cell.Conversely, any transformed body cell can present endogenous antigen combined with its class I MHC antigen directly to a cytotoxic T cell, without intervention of an APC.



b. The helper T-cell population then releases a series of lymphokines that have the following effects.

(l) Macrophages are attracted to the antigen bearing cells, inhibited from leaving the area, and activated so that they become much more phagocytically active.

(2) Cytotoxic T-cells are stimulated to proliferate (by IL-2). These then also attach to the antigen bearing target cells.

c. Because the entire CMI response is mediated by white cells, all CMI responses have an inflammatory appearance about them. It must be remembered that the CMI response is directed against specific antigen on the surfaces of the target cell.

10. Tolerance - The immune system normally recognizes self and does not mount an attack against our own cells. This is known as tolerance. Tolerance is due to the fact that during the development of the immune system, any lymphocyte which has receptors that will react with self antigens is destroyed. Therefore, none of our immune cells ordinarily will react with self antigens. Suppressor T cells may also play a role in inducing tolerance.

a. Besides normal self tolerance, there is a special case whereby the body tolerates foreign antigens. This is the case of the mother's immune system accepting the fetus which bears some of the father's antigens.

G. Active immunity against disease - Immunity against disease causing organisms is obtained by exposure to these organisms, or at least some of their antigens. Once an immune response has been mounted against the antigens of a particular organism, memory cells will remain, and if that antigen is ever encountered again, an immediate and overwhelming immune response will be launched, preventing the organism from establishing an infection. It is the memory response which makes us immune.

l. Natural immunity - This is immunity which is developed due to exposure to organisms by natural means. Sometimes an actual disease resulting infection occurs, but immunity can develop during low level exposure that does not result in obvious disease.

2. Artificial immunity - This is immunity which is developed by the deliberate exposure of a non-immune organism to antigen from a disease causing organism. This is the basis of vaccination. There are several ways in which this is accomplished.

a. Injection of live organisms - Here live but attenuated organisms are injected into a non-immune person. Even though the organism has no disease causing ability (virulence) it still has surface antigens which will provoke an immune response.

b. Injection of dead organisms - Here the organism is first killed or inactivated before injection. Again, the surface antigens remain intact so that immunity is stimulated.

c. Injection of toxoids - These are toxins from organisms that have been converted to an inactive form. These are especially useful in the prevention of diseases which are mediated primarily by a toxin released by the disease causing organism. Tetanus and diphtheria are cases in point.

d. Injection of antigens - Here antigens are removed from the organism and injected. The antigens of course provoke an immune response that will work against the antigens in the future when they enter the body attached to the organism. These antigens may be obtained in two ways.

(l) Processing disease causing organisms - Here pure cultures are treated in such a manner that antigens may be stripped off and utilized.

(2) Genetic cloning - In this technique the DNA gene which codes for an antigen is isolated, removed, and cloned into another microorganism which will then produce large quantities of pure antigen. This technique is new and holds great promise for vaccines in the future, especially against organisms that are difficult to produce vaccines against by more traditional methods. When vaccines are developed against herpes and AIDS, they will probably be obtained by this method.



e. Nucleic acid vaccines - These are still experimental but have great promise. Here a nucleic acid (DNA or RNA) that codes for a protein is inserted into cells. The protein is produced and then serves as the antigen for an immune response.



f. Plant produced vaccines - Plants can be genetically engineered to produce virtually any type of product. These "antigens" would be eaten with the plant itself. The heavy walls of plant cells protects the antigen from breakdown by stomach acids and allows it to enter the intestinal tract where it can invoke an immune response. Several are currently under test. A highly desirable target is bananas because the are eaten raw, esteemed by children, and grow in the tropics where many diseases occur.

3. Primary and secondary immune responses

a. Primary response - This is the sequence of events that occurs the first time an antigen is encountered. The first time that an antigen is seen, there may be only a few cells that are capable of reacting with it. These must form clones in order to produce a sufficiently aggressive immune response. This takes anywhere from 5 to l0 days to develop a good immune attack, and the build up of weapons is gradual.

b. Secondary response - This is the sequence of events that occurs during a subsequent encounter with the antigen. There will now be hundreds or even thousands of memory cells that are capable of reacting with the antigen. Consequently, the reaction occurs much faster and is always much more intense. A good response can occur within hours of being exposed in HMI and about 24 hours for a CMI response. The secondary response is known as the memory response, and it is the basis of immunity.

c. Boosters - After a period of time, if there has not been exposure to a given antigen, the immune response may decline as immunoglobulin is metabolized and memory cells die out. Consequently it may be necessary to provoke another response by administering a small amount of antigen. This is termed a booster and serves to

recharge the immune response.

H. Passive immunity - This is immunity that is given to a person by

providing them with Immunoglobulin or immune cells from an immune

person or animal. It is frequently used with toxin diseases where it is necessary to establish a high antitoxin level immediately. It is also used to prevent the development of an infection when there is a high probability of exposure to an antigen. This is the gamma globulin shot. This is only a transitory type of immunity.

l. Natural passive immunity - There is one example of this, the immunity transferred from the mother to her child. IgG can cross the placenta and provide a measure of immunity for the newborn while its immune system is developing.

I. Allergic responses - This is the over reaction of the immune response system to some antigen. There are two types of allergic responses based upon the two types of immunity.

l. Immediate hypersensitivity - This is allergy mediated by immunoglobulin. The sequence of events is as follows.

a. Exposure to an antigen results in the synthesis of IgE.

b. IgE does not circulate but rather attaches to the membranes of Mast cells and basophils.

c. A subsequent exposure to the antigen results in an antigen-antibody response occurring on the surface of Mast cells and basophils. This causes them to release chemical substances such as histamine and heparin which affect the circulation and also the excitable tissues.

d. The manifestations of the allergic response depend upon which part of the body the reactions occur.

(l) Anaphylaxis - This is a highly acute and generalized allergic response that affects most tissues of the body. There is a massive vasodilation and loss of fluid from the circulation. The edema that occurs as a result of this can block the respiratory passages causing rapid death. If this does not occur, cardiac shock can result which can also result in death. Anaphylaxis is always life threatening and must be treated immediately. It is usually countered by giving vasoconstrictive agents such as epinephrine which also opens respiratory passages.

2. Delayed hypersensitivity - This is an allergic manifestation of CMI. Unlike the immediate response that can occur in minutes to hours, delayed response may require several days. The process involves an intense attack by the T-cells and macrophages on any cells that bear the antigen. Because of the nature of CMI, delayed hypersensitivity reactions appear inflammatory. Basically, DH reactions are really CMI type responses. The tuberculin skin test and the poison ivy reaction are two good examples.



J. Diagnostic immunology - Besides vaccination, immune responses can serve as diagnostic tests. For example, the detection of antibodies against a particular disease causing organism indicates that the person is either currently infected, or has been infected in the past. The extreme specificity of immune reactions makes these types of test extremely reliable. A wide variety of techniques have been developed to take advantage of this specificity. These include precipitation reactions, agglutination reactions, complement fixation tests, fluorescent antibody tests, and Enzyme-linked immunosorbent assay (ELISA). Chapter 18 of your text describes these tests in more detail.