Immunology and Immuno-technology

Desalegn Amenu (M.Sc.) Wollega University, 2014Desalegn Amenu (M.Sc.) Wollega University, 2014Desalegn Amenu (M.Sc.) Wollega University, 2014Desalegn Amenu (M.Sc.) Wollega University, 2014 Page i

WU, Nekemte

May, 2014

Immunology and Immuno-technology 2014

Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega University, 2014University, 2014University, 2014University, 2014 Page ii

Table of contents

Table of contents ............................................................................................................................... ii

List of tables ...................................................................................................................................... v

Preface .............................................................................................................................................. ix

1. Historical Background of Immunology ......................................................................................... 1

1. Innate (Nonspecific) Immune Response .................................................................................... 2

1.1. Overview of the immune system ........................................................................................ 2

1.2. Innate host defenses against infection: Anatomical barriers ............................................... 4

1.3. Innate host defenses against infection: Humoral barriers ................................................... 5

1.4. Innate host defenses against infection: Cellular barriers .................................................... 6

1.5. Phagocyte response to infection ......................................................................................... 6

1.6. Non-specific killer cells ..................................................................................................... 9

1.7. Determinants recognized by the innate immune response .................................................. 9

2. Complement ............................................................................................................................. 10

2.1. Complement Functions .................................................................................................... 10

2.2. Pathways of Complement Activation ............................................................................... 11

2.2.1. Classical Pathway ..................................................................................................... 12

2.2.2. Lectin Pathway ......................................................................................................... 15

2.2.3. Alternative Pathway ................................................................................................. 16

2.3. Membrane Attack (Lytic) Pathway .................................................................................. 20

2.4. Biologically Active Products of Complement Activation ................................................ 21

3. Antigens ................................................................................................................................... 22

3.1. Factors influencing immunogenicity ................................................................................ 22

3.2. Chemical nature of immunogens ...................................................................................... 23

3.3. Types of antigens ............................................................................................................. 23

3.4. Antigenic determinants recognized by B cells and Ab ..................................................... 24

3.5. Determinants recognized by T cells ................................................................................. 25

3.6. Superantigens ................................................................................................................... 25


Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega University, 2014University, 2014University, 2014University, 2014 Page iii

4. Immunoglobulins: Structure and Function ............................................................................... 27

4.1. General Functions of Immunoglobulins ........................................................................... 27

4.2. Basic Structure of Immunoglobulins ................................................................................ 28

5.5.5.5.5.5.5.5. Structure of the Variable Region ...................................................................................... 29

5.6.5.6.5.6.5.6. Immunoglobulin Fragments: Structure/Function Relationships ....................................... 30

5.7. Human Immunoglobulin Classes, Subclasses, Types and Subtypes ..................................... 32

5.8.5.8.5.8.5.8. Structure and Some Properties of Ig Classes and Subclasses ........................................... 33

6. Immunoglobulins: Isotypes, Allotypes and Idiotypes .................................................................. 37

7. Immunoglobulins: Genetics ........................................................................................................ 41

8. Antibody Formation .................................................................................................................... 50

8.1. General Characteristics of the Antibody Response ................................................................... 50

8.2. Antibody Formation.............................................................................................................. 51

8.3. Kinetics of antibody responses to T-dependent Ag .............................................................. 52

8.4. Specificity of 1o and 2o responses ....................................................................................... 53

8.5. Qualitative changes in Ab during 1o and 2o responses........................................................ 54

8.6. Cellular events during 1o and 2o responses to T-dependent Ag .......................................... 56

9. Immunization ............................................................................................................................... 59

10. Cells of the Immune System and Antigen Recognition ............................................................. 64

11. Major Histocompatibility Complex and T Cell Receptors ........................................................ 71

12. Antigen Process and Presentation .............................................................................................. 79

13. Cell-Cell Interactions in Immune Responses ............................................................................. 86

14. Cytokines ................................................................................................................................... 96

15. Immunoregulation ................................................................................................................... 105

16. MHC: genetics and role in transplantation .............................................................................. 108

17. Tolerance and Autoimmunity................................................................................................... 124

17.1. Tolerance Introduction: ..................................................................................................... 124

17.2. Autoimmunity ................................................................................................................... 127


Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega University, 2014University, 2014University, 2014University, 2014 Page iv

18. Tumor Immunology ................................................................................................................. 130

19. Immunodeficiency ................................................................................................................... 136

20. Antibody and Antigen Reaction .............................................................................................. 140

21. Serological Techniques ............................................................................................................ 148

22. Human Immunodeficiency Virus (HIV) .................................................................................. 169


Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega University, 2014University, 2014University, 2014University, 2014 Page v

List of tables

Table 1: Complete protein ............................................................................................................... 11

Table 2: Biological Activity of classical pathway products. ............................................................ 15

Table 3: Regulation of the Classical Pathway ................................................................................ 15

Table 4: CD Marker cell ................................................................................................................. 67

Table 5: types of cytokines .............................................................................................................. 91

Table 6: Immunoglobulin regulation ............................................................................................. 105

Table 7: Factors which determine induction of immune response or tolerance following challenge

with antigen. .................................................................................................................................. 127


Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega University, 2014University, 2014University, 2014University, 2014 Page vi

List of figures

Figure 1: overview of immune system .............................................................................................. 3

Figure 2: Cells of Immune system ..................................................................................................... 4

Figure 3: phagocytic cells .................................................................................................................. 7

Figure 4: Phagocytosis ...................................................................................................................... 7

Figure 5: Nitric oxide-dependent killing ........................................................................................... 9

Figure 6: nitric oxide toxic ................................................................................................................ 9

Figure 7: PAMP and PRR ............................................................................................................... 10

Figure 8: Path way of complement activation ................................................................................. 12

Figure 9: Generation of C3 convertase in the classical pathway ..................................................... 13

Figure 10: Generation of C5convertase in the classical pathway .................................................... 14

Figure 11: Generation of C3 convertase in the lectin pathway ........................................................ 16

Figure 12: Generation of C5 convertase in the lectin pathway ........................................................ 17

Figure 13: Spontaneous activation of C3 ......................................................................................... 18

Figure 14: Regulation of activated C3 by DAF ............................................................................... 19

Figure 15: Regulation by CR1, Factor H and Factor I ..................................................................... 20

Figure 16: Stabilized C3 convertase of the alternative pathway ...................................................... 21

Figure 17: antigenic determinants.................................................................................................... 25

Figure 18: T –cell Receptor ............................................................................................................. 26

Figure 19: Immunoglobulin ............................................................................................................. 27

Figure 20: Basic structure of immunoglobulin (Heavy and Light chain) ....................................... 28

Figure 21: variable index of Ig ........................................................................................................ 29

Figure 22: Immunoglobulin fragments ............................................................................................ 30

Figure 23: Antigen Binding and FC receptor .................................................................................. 31

Figure 24: F(ab’) 2 fragments ......................................................................................................... 31

Figure 25: Immunoglobulin classes ................................................................................................. 32

Figure 26: Immunoglobulin J Chain and Cµ4 ................................................................................ 34

Figure 27: Immunoglobulin Tail piece ........................................................................................... 35

Figure 28: Immunoglobulin structure and αβ ................................................................................ 35

Figure 29: Immunoglobulin secretory piece .................................................................................... 36

Figure 30: IgE Tail piece ................................................................................................................ 36

Figure 31: IgE C€4 ......................................................................................................................... 36

Figure 32: Ig Isotopes Kappa .......................................................................................................... 37

Figure 33: Ig Allotypes .................................................................................................................... 38

Figure 34: Ig Idiotypes location....................................................................................................... 41


Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega University, 2014University, 2014University, 2014University, 2014 Page vii

Figure 35: Light chain gene families ............................................................................................... 42

Figure 36: Gene rearrangement and Expression ............................................................................... 43

Figure 37: Heavy chain gene family ................................................................................................ 44

Figure 38: VDJ arrangement ........................................................................................................... 45

Figure 39: VDJ Transcription .......................................................................................................... 45

Figure 40: Mechanism of DNA rearrangements .............................................................................. 46

Figure 41: Ig genes are expressed in a B cell (Heavy chain) ........................................................... 47

Figure 42: Ig genes are expressed in a B cell (light chain) .............................................................. 48

Figure 43: antibody formation ......................................................................................................... 51

Figure 44: antibody formation after immunization (10 and (20) ....................................................... 53

Figure 46 : antibody affinity ............................................................................................................ 54

Figure 47: affinity maturation (10) ................................................................................................... 55

Figure 48: affinity maturation (20) ................................................................................................... 55

Figure 49: Cellular events during 1o ............................................................................................... 56

Figure 50: Cellular events during 2o responses .............................................................................. 56

Figure 51: Cellelular events during secondary response .................................................................. 57

Figure 52.class switching ................................................................................................................ 58

Figure 53: polyadenylation sites ...................................................................................................... 59

Figure 54: types of immunity .......................................................................................................... 60

Figure 55: Newer adjuvant formulations ......................................................................................... 63

Figure 56: stem cell of immune system ........................................................................................... 67

Figure 57: B cell and T cell ............................................................................................................ 68

Figure 58: B and T cell origination ................................................................................................. 70

Figure 59: lymphatic lymphocytes .................................................................................................. 71

Figure 60: B and T cell differentiation ............................................................................................ 71

Figure 61: Structure of MHC class I ............................................................................................... 73

Figure 62: Structure of MHC class II .............................................................................................. 74

Figure 63: Role of TCR in the immune response ............................................................................ 75

Figure 64: TCR a heterodimer ......................................................................................................... 76

Figure 65: TCR β chains ............................................................................................................... 77

Figure 66: Antigen presenting cell .................................................................................................. 77

Figure 67: TCR and CD3 Recognition ............................................................................................ 77

Figure 68: MHC class I pathway ..................................................................................................... 81

Figure 69: MHC class II pathway.................................................................................................... 82

Figure 70: random VDJ recombination ........................................................................................... 85


Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega University, 2014University, 2014University, 2014University, 2014 Page viii

Figure 71: TCP selection ................................................................................................................. 86

Figure 72: Central role of Th cells in immune response .................................................................. 87

Figure 73: Cytokine production ....................................................................................................... 88

Figure 74: B cell proliferation ......................................................................................................... 90

Figure 75: Cell-cell interactions in primary Ab response ................................................................ 91

Figure 76: Process of Antigen presentation ..................................................................................... 93

Figure 77: Fas- and TNF-mediated killing ..................................................................................... 94

Figure 78: Granule-mediated killing ............................................................................................... 94

Figure 79: cytokine production, bactericidal and tumoricidal activities ......................................... 95

Figure 80: Receptors for cytokines are heterodimers (A) ................................................................ 97

Figure 81: Receptors for cytokines are heterodimers (B) ................................................................ 98

Figure 82: Interferon ..................................................................................................................... 100

Figure 83: activate NK cells and monocytes, proliferative phase................................................. 101

Figure 85: Cytokine networks ....................................................................................................... 104

Figure 86: Immunoregulation by antibody ................................................................................... 106

Figure 87: The human MHC gene complex ................................................................................... 109

Figure 88: The mouse MHC complex ........................................................................................... 111

Figure 89: Co-dominant expression of MHC antigens ................................................................... 112

Figure 90: Activation of CTL and Mechanism of Allograft Destruction ....................................... 114

Figure 92: types of hypersensitivity reaction ................................................................................. 121

Figure 93: Mechanism of damage in type-III hypersensitivity ...................................................... 122

Figure 94: Mechanisms of damage in delayed hypersensitivity .................................................... 123

Figure 95: Latex agglutination reaction ........................................................................................ 143


Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega Desalegn Amenu (M.Sc.) Wollega University, 2014University, 2014University, 2014University, 2014 Page ix

Preface

Immunology and immunotechnology is an advanced science dealing with how the human immune

system organized, function and the different types of serological techniques are applied. It is a very

vast subject covering a wide area of technology. This teaching material is prepared based on the

existing curriculum of immunology and immunotechnology and consists of different chapters with

different subtopics. Therefore, the material is designed to present clear and concise understanding

about Immunology and immunotechnology; and it is primarily suitable for students following

Bachelor and Master programme in Biology, Medical and any Microbiology. Finally, it is quite

obvious that it had demanded a lot of effort in preparing this material. However, it should be noted

that even then, there could be constructive comments which are helpful in improving this lecture

note. Thus, it will be well accepted and acknowledged for the contribution.


Desalegn Amenu (M.Sc, Microbiology) Desalegn Amenu (M.Sc, Microbiology) Desalegn Amenu (M.Sc, Microbiology) Desalegn Amenu (M.Sc, Microbiology) Page 1

1. . . . Historical Background of ImmunologyHistorical Background of ImmunologyHistorical Background of ImmunologyHistorical Background of Immunology

Immunology is defined as the study of the molecules, cells, organs, and systems responsible for the

recognition and disposal of foreign material. Immunology began as a branch of microbiology. The

study of infectious disease and the body’s response to them has a major role for the development of

immunology. Moreover, the concept of germ theory of disease has contributed to the field of

immunology.

It was Edward Jenner who first studied the response of the body to foreign substances. He

observed that dairy maids who had naturally contracted a mild infection called cowpox seemed to

be protected against smallpox, a horribly disfiguring disease and a major killer. In 1796, Jenner

inoculated an eight year-old boy with fluid from cowpox blisters on the hand of a dairymaid. The

boy contracted cowpox. Then two month later Jenner inoculated him with fluid from a small pox

blister, the boy only developed a small sore at the site of inoculation. His exposure to the mild

disease cowpox had made him immune to the small pox infection. These were some of the vital

events occurred in the history of immunology following Jenner’s achievement. In 1879, the first

human pathogen, gonococcus, was isolated by Neisser. In 1883, Klebs and Loeffler isolated

diphtheria bacilli which led to the production of the first defined antigen, diphtheria toxin, by Roux

and Yersin in 1888. In the same year the first antibodies, serum bactericidins, were reported by

Nuttal and Pasteur.

In 1890, von Behring and Kitasato discovered antitoxins that led to the development of toxoids for

diphtheria and tetanus. In 1900, Land Steiner discovered the blood group antigens and their

corresponding antibodies. This led to the ability to give blood transfusion without provoking

reactions. It was in 1916 that the first journal of immunology began publication in which many of

new findings published on it. In general, immunology has always depended on and stimulated the

application of technology, such as the use of microscopy, electrophoresis,

immunoelectrofluorescence, etc. Thus Immunology and Serology immunology has not become an

inborn discipline but has maintained close associations with many other fields of medical sciences.

Immunology is the study of our protection from foreign macromolecules or invading organisms

and our responses to them. These invaders include viruses, bacteria, protozoa or even larger

parasites. In addition, we develop immune responses against our own proteins (and other

molecules) in autoimmunity and against our own aberrant cells in tumor immunity.



Our first line of defense against foreign organisms is barrier tissues such as the skin that stop the

entry of organism into our bodies. If, however, these barrier layers are penetrated, the body

contains cells that respond rapidly to the presence of the invader. These cells include macrophages

and neutrophils that engulf foreign organisms and kill them without the need for antibodies.

Immediate challenge also comes from soluble molecules that deprive the invading organism of

essential nutrients (such as iron) and from certain molecules that are found on the surfaces of

epithelia, in secretions (such as tears and saliva) and in the blood stream. This form of immunity is

the innate or non-specific immune system that is continually ready to respond to invasion.

A second line of defense is the specific or adaptive immune system which may take days to

respond to a primary invasion (that is infection by an organism that has not hitherto been seen). In

the specific immune system, we see the production of antibodies (soluble proteins that bind to

foreign antigens) and cell-mediated responses in which specific cells recognize foreign pathogens

and destroy them. In the case of viruses or tumors, this response is also vital to the recognition and

destruction of virally-infected or tumorigenic cells. The response to a second round of infection is

often more rapid than to the primary infection because of the activation of memory B and T cells.

We shall see how cells of the immune system interact with one another by a variety of signal

molecules so that a coordinated response may be mounted. These signals may be proteins such as

lymphokines which are produced by cells of the lymphoid system, cytokines and chemokines that

are produced by other cells in an immune response, and which stimulate cells of the immune

system.

1.1.1.1. Innate (NonspInnate (NonspInnate (NonspInnate (Nonspececececific) Immune Resific) Immune Resific) Immune Resific) Immune Respppponseonseonseonse

1.1.1.1.1.1.1.1. OverviewOverviewOverviewOverview ofofofof thethethethe immuneimmuneimmuneimmune ssssyyyystemstemstemstem

We are constantly being exposed to infectious agents and yet, in most cases, we are able to

resist these infections. It is our immune system that enables us to resist infections. The

immune system is composed of two major subdivisions, the innate or non-specific immune system

and the adaptive or specific immune system (Figure 1). The innate immune system is our first

line of defense against invading organisms while the adaptive immune system acts as a second

line of defense and also affords protection against re- exposure to the same pathogen. Each of

the major subdivisions of the immune system has both cellular and humoral components by

which they carry out their protective function (Figure 1). In addition, the innate immune system

also has anatomical features that function as barriers to infection. Although these two arms of



the immune system have distinct functions, there is interplay between these systems (i.e.,

components of the innate immune system influence the adaptive immune system and vice versa).

There are two phases to the immune response: pathogen recognition and pathogen

removal. Although the innate and adaptive immune systems both function to protect against

invading organisms, they differ in a number of ways. The adaptive immune system requires

some time to react to an invading organism, whereas the innate immune system includes defenses

that, for the most part, are constitutively present and ready to be mobilized upon infection.

Second, the adaptive immune system is antigen specific and reacts only with the organism that

induced the response. In contrast, the innate system is not antigen specific and reacts equally

well to a variety of organisms. Finally, the adaptive immune system demonstrates

immunological memory. It “remembers” that it has encountered an invading organism and

reacts more rapidly on subsequent exposure to the same organism. In contrast, the innate immune

system does not demonstrate immunological memory.

Figure 1: overview of immune system

All cells of the immune system have their origin in the bone marrow. They include myeloid

(neutrophils, basophils, eosinophils, macrophages, and dendritic cells) and lymphoid cells (B

lymphocytes, T lymphocytes, and natural killer cells) (Figure 2).

Immunology and Immuno

Desalegn Amenu (M.Sc, Microbiology) Desalegn Amenu (M.Sc, Microbiology) Desalegn Amenu (M.Sc, Microbiology) Desalegn Amenu (M.Sc, Microbiology)

Figure 2: Cells of Immune system

The main function of the

distinguish between self and

pathogens and to eliminate modified or altered cells (

replicate intracellular (virus

fungi and parasites), different co

these different types of pathogens.

does not necessarily mean

eliminate the infection before

high, when the virulence of

Although the immune syste

effects as well. During inflammation,

local discomfort and collateral da

by the immune response.

toward self tissues resulting in autoimmune disease.

1.2.1.2.1.2.1.2. Innate host defenses against iInnate host defenses against iInnate host defenses against iInnate host defenses against i

1) Mechanical factors:Mechanical factors:Mechanical factors:Mechanical factors: epithelial s

infectious agents. Thus, the

desquamation of skin epitheli

adhered to the epithelial surf

and the gastrointestinal tract

Immunology and Immuno-technology


Cells of Immune system

the immune system is self/non-self discrimination.

self and non-self is necessary to protect the organism

eliminate modified or altered cells (e.g. malignant cells). Since pathogens

ses and some bacteria and parasites) or extracellular

fungi and parasites), different components of the immune system have evolved

pathogens. It is important to remember that infecti

diseases, since in most cases the immune system will

ore disease occurs. Disease occurs only when the

of the invading organism is great or when imm

immune system, for the most part, has beneficial effects, there can be detri

inflammation, which is the response to an invading organis

fort and collateral damage to healthy tissue as a result of the toxic products produced

In addition, in some cases the immune response

resulting in autoimmune disease.

Innate host defenses against iInnate host defenses against iInnate host defenses against iInnate host defenses against innnnfection: Anatofection: Anatofection: Anatofection: Anatommmmical barriersical barriersical barriersical barriers

ithelial surfaces form a physical barrier that is very i

the skin acts as our first line of defense against invading organis

epithelium also helps remove bacteria and other infectious

faces. Movement due to cilia or peristalsis helps

t free from microorganisms. The flushing action

technology 2014

Page 4

nation. This ability to

organism from invading

). Since pathogens may

extracellular (most bacteria,

evolved to protect against

ection with an organism

system will be able to

the bolus of infection is

munity is compromised.

s, there can be detrimental

organism, there may be

hy tissue as a result of the toxic products produced

response can be directed

that is very impermeable to most

invading organisms. The

ectious agents that have

helps to keep air passages

action of tears and saliva



helps prevent infection of the eyes and mouth. The trapping affect of mucus that lines the

respiratory and gastrointestinal tract helps protect the lungs and digestive systems from infection.

2) CheCheCheChemmmmicalicalicalical factorfactorfactorfactorssss:::: fatty acids in sweat inhibit the growth of bacteria. Lysozyme and

phospholipase found in tears, saliva and nasal secretions can breakdown the cell wall of bacteria

and destabilize bacterial membranes. The low pH of sweat and gastric secretions prevents growth

of bacteria. Defensins (low molecular weight proteins) found in the lung and gastrointestinal

tract have antimicrobial activity. Surfactants in the lung act as opsonins (substances that promote

phagocytosis of particles by phagocytic cells).

3) BiologicalBiologicalBiologicalBiological factors:factors:factors:factors: the normal flora of the skin and in the gastrointestinal tract can

prevent the colonization of pathogenic bacteria by secreting toxic substances or by competing with

pathogenic bacteria for nutrients or attachment to cell surfaces.

1.3.1.3.1.3.1.3. Innate host defenses againstInnate host defenses againstInnate host defenses againstInnate host defenses against ininininffffeeeecccctitititioooon: Hun: Hun: Hun: Hummmmoral barriersoral barriersoral barriersoral barriers

i. AnatoAnatoAnatoAnatommmmicalicalicalical barriersbarriersbarriersbarriers are very effective in preventing colonization of tissues by microorganisms.

However, when there is damage to tissues the anatomical barriers are breached and infection may

occur. Once infectious agents have penetrated tissues, another innate defense mechanism

comes into play, namely acute inflammation. Humoral factors play an important role in

inflammation, which is characterized by edema and the recruitment of phagocytic cells. These

humoral factors are found in serum or they are formed at the site of infection.

ii. The The The The complcomplcomplcompleeeement systment systment systment systeeeemmmm is the major humoral non-specific defense mechanism. Once activated

complement can lead to increased vascular permeability, recruitment of phagocytic cells, and lysis

and opsonization of bacteria.

iii. DependingDependingDependingDepending onononon thethethethe severityseverityseverityseverity of the tissue injury, the coagulation system may or may not be

activated. Some products of the coagulation system can contribute to the non-specific defenses

because of their ability to increase vascular permeability and act as chemotactic agents for

phagocytic cells. In addition, some of the products of the coagulation system are directly

antimicrobial. For example, beta-lysin, a protein produced by platelets during coagulation

can lyse many Gram positive bacteria by acting as a cationic detergent.

iv. ByByByBy bindingbindingbindingbinding ironironironiron, an essential nutrient for bacteria, lactoferrin and transferrin limit bacterial

growth.

v. LysLysLysLysoooozzzzyme yme yme yme breaksbreaksbreaksbreaks down the cell wall of bacteria.

vi. CytoCytoCytoCytokikikikinesnesnesnes have various effects depending on the balance. Interferons are proteins that can

limit virus replication in cells. Some interleukins induce fever and the production of acute phase



proteins, some of which are antimicrobial because they can opsonize bacteria.

1.4.1.4.1.4.1.4. Innate host defenses againstInnate host defenses againstInnate host defenses againstInnate host defenses against ininininffffeeeecccctitititioooon: Celln: Celln: Celln: Celluuuular lar lar lar bbbbarriersarriersarriersarriers

1. Part of the inflammatory response is the recruitment of polymorphonuclear eosinophils and

macrophages to sites of infection. These cells are the main line of defense in the non-specific

immune system.

2. Neutrophils, Polymorphonuclear cells (PMNs), are recruited to the site of infection where

they phagocytose invading organisms and kill them intracellularly. In addition, PMNs

contribute to collateral tissue damage that occurs during inflammation.

3. Tissue macrophages and newly recruited monocytes, which differentiate into macrophages, also

function in phagocytosis and intracellular killing of microorganisms. In addition, macrophages

are capable of extracellular killing of infected or altered self target cells. Furthermore,

macrophages contribute to tissue repair and act as antigen- presenting cells, which are required

for the induction of specific immune responses.

4. Natural killer (NK) and lymphokine activated killer (LAK) cells can nonspecifically kill virus

infected and tumor cells. These cells are not part of the inflammatory response but they are

important in nonspecific immunity to viral infections and tumor surveillance.

5.5.5.5. EosinophilsEosinophilsEosinophilsEosinophils have proteins in granules that arehave proteins in granules that arehave proteins in granules that arehave proteins in granules that are eeeeffffffffective in ective in ective in ective in kkkkiiiilling clling clling clling ceeeertrtrtrtaaaaiiiin parasites.n parasites.n parasites.n parasites.

1.5. Phagocyte response to infectionPhagocyte response to infectionPhagocyte response to infectionPhagocyte response to infection

i. Circulating PMNs and monocytes respond to danger (SOS) signals generated at the site of an

infection. SOS signals include N-formyl-methionine containing peptides released by bacteria,

clotting system peptides, complement products and cytokines released from tissue macrophages

that have encountered bacteria in tissue. Some of the SOS signals stimulate endothelial cells

near the site of the infection to express cell adhesion molecules such as ICAM-1 and selectins

which bind to components on the surface of phagocytic cells and cause the phagocytes to adhere

to the endothelium. Vasodilators produced at the site of infection cause the junctions between

endothelial cells to loosen and the phagocytes then cross the endothelial barrier by “squeezing”

between the endothelial cells in a process called diapedesis (Figure 3). Once in the tissue spaces

some of the SOS signals attract phagocytes to the infection site by chemotaxis (movement toward

an increasing chemical gradient). The SOS signals also activate the phagocytes, which results in

increased phagocytosis and intracellular killing of the invading organisms.



Figure 3: phagocytic cells

ii. Phagocytosis:Phagocytosis:Phagocytosis:Phagocytosis: Phagocytic cells have a variety of receptors on their cell membranes through

which infectious agents bind to the cells (Figure 4A). These include Fc receptors,

complement receptors, scavenger receptors, and toll-like receptors. After attachment of a

bacterium, the phagocyte begins to extend pseudopods around the bacterium (Figure 4B). The

pseudopods eventually surround the bacterium and engulf it, and the bacterium is enclosed in a

phagosome (Figure 4C). During phagocytosis the granules or lysosomes of the phagocyte

fuse with the phagosome and empty their contents (Figure 4D). The result is a bacterium

engulfed in a phagolysosome which contains the contents of the granules or lysosomes.

Figure 4: Phagocytosis

iii. RespiratoryRespiratoryRespiratoryRespiratory burstburstburstburst. During phagocytosis there is an increase in glucose and oxygen consumption

which is referred to as the respiratory burst. The consequence of the respiratory burst is that a

number of oxygen-containing compounds are produced which kill the bacteria being

phagocytosed. This is referred to as oxygen-dependent intracellular killing. In addition,

bacteria can be killed by pre-formed substances released from granules or lysosomes when they

fuse with the phagosome. This is referred to as oxygen-independent intracellular killing.



a) OxygenOxygenOxygenOxygen----dependentdependentdependentdependent mmmmyeloperoxidayeloperoxidayeloperoxidayeloperoxidasssseeee (MPO)-independent intracellular killing. During

phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is

formed. Cytochrome B which was part of the granule combines with the plasma membrane

NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the

NADPH. The result is the production of superoxide anion. Some of the superoxide anion is

converted to H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion

can react with H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The

result of all of these reactions is the production of the toxic oxygen compounds superoxide

anion (O2-), H2O2, singlet oxygen (1O2) and hydroxyl radical (OH•).

b) OxygenOxygenOxygenOxygen----dependentdependentdependentdependent MMMMPPPPOOOO----dependentdependentdependentdependent intracellintracellintracellintracelluuuularlarlarlar killing. As the azurophilic granules fuse with

the phagosome, myeloperoxidase is released into the phagolysosome. MPO utilizes H2O2 and

halide ions (usually Cl-) to produce hypochlorite, a highly toxic substance. Some of the

hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions

is the production of toxic hypochlorite (OCl-) and singlet oxygen (1O2).

c) Detoxification Detoxification Detoxification Detoxification reactionsreactionsreactionsreactions. PMNs and macrophages have means to protect themselves from the toxic

oxygen intermediates. These reactions involve the disputation of superoxide anion to hydrogen

peroxide by superoxide dismutase and the conversion of hydrogen peroxide to water by catalase.

d) OxygenOxygenOxygenOxygen----ininininddddependent ependent ependent ependent intracellularintracellularintracellularintracellular killing. In addition to the oxygen-dependent mechanisms of

killing there are also oxygen–independent killing mechanisms in phagocytes: cationic proteins

(cathepsin) released into the phagolysosome can damage bacterial membranes; lysozyme

breaks down bacterial cell walls; lactoferrin chelates iron, which deprives bacteria of this required

nutrient; hydrolytic enzymes break down bacterial proteins. Thus, even patients who have defects

in the oxygen- dependent killing pathways are able to kill bacteria. However, since the oxygen-

dependent mechanisms are much more efficient in killing, patients with defects in these pathways

are more susceptible and get more serious infections.

e) Nitric oxideNitric oxideNitric oxideNitric oxide----dependent killingdependent killingdependent killingdependent killing. Binding of bacteria to macrophages, particularly binding via Toll-

like receptors, results in the production of TNF-alpha, which acts in an autocrine manner to induce

the expression of the inducible nitric oxide synthetase gene (i-nos ) resulting in the production of

nitric oxide (NO). If the cell is also exposed to interferon gamma (IFN-gamma) additional nitric

oxide will be produced. Nitric oxide released by the cell is toxic and can kill microorganism in

the vicinity of the macrophage (Figure 6).



Figure 5: Nitric oxide-dependent killing

1.6.1.6.1.6.1.6. NonNonNonNon----specific killer cellsspecific killer cellsspecific killer cellsspecific killer cells

a) Several different cells including

capable of killing foreign and

important role in the innate im

b) Innate response to virus infection

receptors on their surface, NK

encounters its ligand on a ta

receptor also binds its ligand

constitutively express MHC

cells down regulate expression of MHC class I. Thus, NK

transformed cells while sparing nor

c) InnateInnateInnateInnate responseresponseresponseresponse totototo extracellextracellextracellextracelluuuu

cells with the ability to engage and d

Activated eosinophils relea

eosinophil peroxidase (a

ribonuclease that is an eosinophil

1.7.1.7.1.7.1.7. DeterDeterDeterDetermmmminants recognized by the innate iminants recognized by the innate iminants recognized by the innate iminants recognized by the innate im

a) Determinants recognized by components of the innate (

those recognized by the adaptive (specific)

receptors recognize discrete d

the adaptive immune system to recognize and

components of the innate

pathogens but not in the host.

immune system. The broad



dependent killing

Figure 6: nitric oxide toxic

Several different cells including NK cells, activated macrophages, eosinophils,

and altered self target cells in a non-specific m

nate immune system.

ection and altered self (transformed cells): NK cells

NK receptor and inhibitory receptor (Figure 7).

arget cell, the NK cell is signaled to kill. However,

ligand (MHC class I) then the killing signal is rep

C class I on their surface, however virus infected and tra

xpression of MHC class I. Thus, NK cells selectively kill

ring normal cells.

uuuularlarlarlar mmmmiiiicroorganiscroorganiscroorganiscroorganismmmmssss (parasites): eosinophils are a specialized group of

cells with the ability to engage and damage large extracellular parasites, such as schistoso

release their granule components including

(a cationic hemoprotein), and eosinophil

eosinophil-specific toxin that is very potent at killing

inants recognized by the innate iminants recognized by the innate iminants recognized by the innate iminants recognized by the innate immmmmune rune rune rune reeeesponsesponsesponsesponse

inants recognized by components of the innate (nonspecific) immune system

adaptive (specific) immune system. Antibodies

te determinants and demonstrate a high degree of specificity, enabling

the adaptive immune system to recognize and react to a particular pathogen.

innate immune system recognize broad molecular

host. Thus, they lack a high degree of specificity seen in the adaptive

The broad molecular patterns recognized by the innate

technology 2014

Page 9

: nitric oxide toxic

eosinophils, and mast cells are

manner. These play an

K cells have two kinds of

When the NK receptor

e NK cell is signaled to kill. However, if the inhibitory

pressed. Normal cells

e, however virus infected and transformed

ells selectively kill virus-infected and

(parasites): eosinophils are a specialized group of

mage large extracellular parasites, such as schistosomes.

major basic protein,

cationic protein (a

killing many parasites).

) immune system differ from

Antibodies and the B and T cell

onstrate a high degree of specificity, enabling

react to a particular pathogen. In contrast,

molecular patterns found in

degree of specificity seen in the adaptive

innate immune system have



been called PAMPS (pathogen

called PRRs (pattern recognition rece

that may be present on a nu

variety of different pathogens. Exa

Figure 7: PAMP and PRR

2.2.2.2. ComplementComplementComplementComplement

2.1.2.1.2.1.2.1. Complement FunctionsComplement FunctionsComplement FunctionsComplement Functions

Historically, the term complement (C) was used to refer to a heat

able to lyse bacteria (activity is destroyed (inactivated) by heating serum at 56 degrees C for 30

minutes). However, complement is now known to contribute to

well. Complement can opsonize bacteria for enhanced phagocytosis; it can recruit and activate

various cells including polymorphonuclear cells (PMNs) and macrophages; it can participate in

regulation of antibody responses and

apoptotic cells. Complement can also have detrimental effects for the host; it contributes to

inflammation and tissue damage and it can trigger anaphylaxis.

Complement comprises over 20 different serum

variety of cells including, hepatocytes, macrophages and gut epithelial cells. Some complement

proteins bind to immunoglobulins or to membrane components of cells. Others are proenzymes



(pathogen associated molecular patterns) and the receptors for PAMPS are

called PRRs (pattern recognition receptors). A particular PRR can recognize

a number of different pathogens enabling the receptor to recognize a

variety of different pathogens. Examples of some PAMPs and PRRs are illustr

: PAMP and PRR

Historically, the term complement (C) was used to refer to a heat-labile serum component that was


minutes). However, complement is now known to contribute to host defenses in other ways as



regulation of antibody responses and it can aid in the clearance of immune complexes and


inflammation and tissue damage and it can trigger anaphylaxis.

Complement comprises over 20 different serum proteins (see Table 1) that are produced by a



technology 2014

Page 10

receptors for PAMPS are

A particular PRR can recognize a molecular pattern

ber of different pathogens enabling the receptor to recognize a

nd PRRs are illustrated in Figure 8.

labile serum component that was


host defenses in other ways as



it can aid in the clearance of immune complexes and


proteins (see Table 1) that are produced by a





that, when activated, cleave one or more other complement proteins. Upon cleavage some of the

complement proteins yield fragments that activate cells, increase vascular permeability or opsonize

bacteria.

Table 1: Complete protein

C1(qrs), C2, C3, C4, C5. C6, C7, C8 and C9

Factors B, D, H, I and Properdin (P)

Mannose binding lectin (MBL), MBL-associated serine proteases (MASP-1 and MASP-2)

C1 inhibitor (C1-INH, serpin), C4-binding protein (C4-BP), decay accelerating factor (DAF)

Complement receptor 1 (CR1) protein S (vitronectin)

2.2.2.2.2.2.2.2. Pathways of Complement ActivationPathways of Complement ActivationPathways of Complement ActivationPathways of Complement Activation

Complement activation can be divided into four pathways (figure 1): the classical pathway, the

lectin pathway, the alternative pathway and the membrane attack (or lytic) pathway. Both classical

and alternative pathways lead to the activation of C5 convertase and result in the production of

C5b which is essential for the activation of the membrane attack pathway.



Figure 8: Path way of complement activation

2.2.1.2.2.1.2.2.1.2.2.1. Classical PathwayClassical PathwayClassical PathwayClassical Pathway

C1 activation

C1, a multi-subunit protein containing three different proteins (C1q, C1r and C1s), binds to the Fc

region of IgG and IgM antibody molecules that have interacted with antigen. C1 binding does not

occur to antibodies that have not completed with antigen and binding requires calcium and

magnesium ions. (N.B. In some cases C1 can bind to aggregated immunoglobulin [e.g. aggregated

IgG] or to certain pathogen surfaces in the absence of antibody). The binding of C1 to antibody is

via C1q and C1q must cross link at least two antibody molecules before it is firmly fixed. The

binding of C1q results in the activation of C1r which in turn activates C1s. The result is the

formation of an activated “C1qrs”, which is an enzyme that cleaves C4 into two fragments C4a

and C4b.



Figure 9: Generation of C3 convertase in the classical pathway

C4 and C2 activation (generation of C3 convertase).

The C4b fragment binds to the membrane and the C4a fragment is released into the

microenvironment. Activated “C1qrs” also cleaves C2 into C2a and C2b. C2a binds to the

membrane in association with C4b, and C2b is released into the microenvironment. The resulting

C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and C3b.



Figure 10: Generation of C5convertase in the classical pathway

C3 activation (generation of C5 convertase)

C3b binds to the membrane in association with C4b and C2a, and C3a is released into the

microenvironment. The resulting C4bC2aC3b is a C5 convertase. The generation of C5 convertase

is the end of the classical pathway. Several of the products of the classical pathway have potent

biological activities that contribute to host defenses. Some of these products may also have

detrimental effects if produced in an unregulated manner. Table 2 summarizes the biological

activities of classical pathway components. If the classical pathway were not regulated there would

be continued production of C2b, C3a, and C4a. Thus, there must be some way to regulate the

activity of the classical pathway. Table 3 summarizes the ways in which the classical pathway is

regulated. The importance of C1-INH in regulating the classical pathway is demonstrated by the

result of a deficiency in this inhibitor. C1-INH deficiencies are associated with the development of

hereditary angioedema.



Table 2: Biological Activity of classical pathway products.

Table 3: Regulation of the Classical Pathway

Compone

nt

Regulation

All C1-INH; dissociates C1r and C1s from C1q

C3a C3a inactivator (C3a-INA;Carboxypeptidase B); inactivates C3a

C3b Factors H and I; Factor H facilitates the degradation of C3b by Factor I

C4a C3-INA

C4b C4 binding protein(C4-BP) and Factor I; C4-BP facilitates

degradation of C4b by Factor I; C4-BP also prevents association of C2a with C4b thus

blocking the formation of C3 convertase

2.2.2.2.2.2.2.2.2.2.2.2. Lectin Pathway Lectin Pathway Lectin Pathway Lectin Pathway

The lectin pathway (Figure 4) is very similar to the classical pathway. It is initiated by the binding

of mannose-binding lectin (MBL) to bacterial surfaces with mannose-containing polysaccharides

(mannans). Binding of MBL to a pathogen results in the association of two serine proteases,

MASP-1 and MASP-2 (MBL-associated serine proteases). MASP-1 and MASP-2 are similar to

C1r and C1s, respectively and MBL is similar to C1q. Formation of the MBL/MASP-1/MASP-2

tri-molecular complex results in the activation of the MASPs and subsequent cleavage of C4 into

C4a and C4b. The C4b fragment binds to the membrane and the C4a fragment is released into the

Component Biological Activity

C2b Prokinin; cleaved by plasmin to yield kinin, which results in edema C3a Anaphylotoxin; can activate basophils and mast cells to degranulate resulting

in increased vascular permeability and contraction of smooth muscle cells,

which may lead to anaphylaxis C3b Opsonin; promotes phagocytosis by binding to complement receptors

Activation of phagocytic cells C4a Anaphylotoxin (weaker than C3a) C4b Opsonin; promotes phagocytosis by binding to complement receptors



microenvironment. Activated MASPs also cleave C2 into C2a and C2b. C2a binds to the

membrane in association with C4b and C2b is released into the microenvironment. The resulting

C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and C3b. C3b binds to the

membrane in association with C4b and C2a and C3a is released into the microenvironment. The

resulting C4bC2aC3b is a C5 convertase. The generation of C5 convertase is the end of the lectin

pathway. The biological activities and the regulatory proteins of the lectin pathway are the same as

those of the classical pathway.

Figure 11: Generation of C3 convertase in the lectin pathway

2.2.3.2.2.3.2.2.3.2.2.3. Alternative Pathway Alternative Pathway Alternative Pathway Alternative Pathway

The alternative pathway begins with the activation of C3 and requires Factors B and D and Mg++

cation, all present in normal serum.

1.1.1.1. Amplification loop of C3b formation

In serum there is low level spontaneous hydrolysis of C3 to produce C3i. Factor B binds to C3i and

becomes susceptible to Factor D, which cleaves Factor B into Bb. The C3iBb complex acts as a C3

convertase and cleaves C3 into C3a and C3b. Once C3b is formed, Factor B will bind to it and

becomes susceptible to cleavage by Factor D. The resulting C3bBb complex is a C3 convertase

that will continue to generate more C3b, thus amplifying C3b production. If this process continues

unchecked, the result would be the consumption of all C3 in the serum. Thus, the spontaneous

production of C3b is tightly controlled.



Figure 12: Generation of C5 convertase in the lectin pathway

2.2.2.2. Control of the amplification loopControl of the amplification loopControl of the amplification loopControl of the amplification loop

As spontaneously produced C3b binds to autologous host membranes, it interacts with DAF (decay

accelerating factor), which blocks the association of Factor B with C3b thereby preventing the

formation of additional C3 convertase. In addition, DAF accelerates the dissociation of Bb from

C3b in C3 convertase that has already formed, thereby stopping the production of additional C3b.

Some cells possess complement receptor 1 (CR1). Binding of C3b to CR1 facilitates the enzymatic

degradation of C3b by Factor I. In addition, binding of C3 convertase (C3bBb) to CR1 also

dissociates Bb from the complex. Thus, in cells possessing complement receptors, CR1 also plays a

role in controlling the amplification loop. Finally, Factor H can bind to C3b bound to a cell or in

the in the fluid phase and facilitate the enzymatic degradation of C3b by Factor I. Thus, the

amplification loop is controlled by either blocking the formation of C3 convertase, dissociating C3

convertase, or by enzymatically digesting C3b. The importance of controlling this amplification

loop is illustrated in patients with genetic deficiencies of Factor H or I. These patients have a C3

deficiency and increased susceptibility to certain infections.



Figure 13: Spontaneous activation of C3

3. Stabilization of C convertase by . Stabilization of C convertase by . Stabilization of C convertase by . Stabilization of C convertase by activator (protector) surfacesactivator (protector) surfacesactivator (protector) surfacesactivator (protector) surfaces

When bound to an appropriate activator of the alternative pathway, C3b will bind Factor B, which

is enzymatically cleaved by Factor D to produce C3 convertase (C3bBb). However, C3b is

resistant to degradation by Factor I and the C3 convertase is not rapidly degraded, since it is

stabilized by the activator surface. The complex is further stabilized by properdin binding to

C3bBb. Activators of the alternate pathway are components on the surface of pathogens and

include: LPS of Gram-negative bacteria and the cell walls of some bacteria and yeasts. Thus, when

C3b binds to an activator surface, the C3 convertase formed will be stable and continue to generate

additional C3a and C3b by cleavage of C3.



Figure 14: Regulation of activated C3 by DAF

4. Generation of C5 convertaseGeneration of C5 convertaseGeneration of C5 convertaseGeneration of C5 convertase

Some of the C3b generated by the stabilized C3 convertase on the activator surface associates with

the C3bBb complex to form a C3bBbC3b complex. This is the C5 convertase of the alternative

pathway. The generation of C5 convertase is the end of the alternative pathway. The alternative

pathway can be activated by many Gram-negative (most significantly, Neisseria meningitidis and

N. gonorrhoea), some Gram-positive bacteria and certain viruses and parasites, and results in the

lysis of these organisms. Thus, the alternative pathway of C activation provides another means of

protection against certain pathogens before an antibody response is mounted. A deficiency of C3

results in an increased susceptibility to these organisms. The alternate pathway may be the more

primitive pathway and the classical and lectin pathways probably developed from it.

Remember that the alternative pathway provides a means of non-specific resistance against

infection without the participation of antibodies and hence provides a first line of defense against a

number of infectious agents.

Many gram negative and some gram positive bacteria, certain viruses, parasites, heterologous red

cells, aggregated immunoglobulins (particularly, IgA) and some other proteins (e.g. proteases,

clotting pathway products) can activate the alternative pathway. One protein, cobra venom factor

(CVF), has been extensively studied for its ability to activate this pathway.



Figure 15: Regulation by CR1, Factor H and Factor I

2.3.2.3.2.3.2.3. Membrane Attack (Lytic) Pathway Membrane Attack (Lytic) Pathway Membrane Attack (Lytic) Pathway Membrane Attack (Lytic) Pathway

C5 convertase from the classical (C4b2a3b), lectin (C4b2a3b) or alternative (C3bBb3b) pathway

cleaves C5 into C5a and C5b. C5a remains in the fluid phase and the C5b rapidly associates with

C6 and C7 and inserts into the membrane. Subsequently C8 binds, followed by several molecules

of C9. The C9 molecules form a pore in the membrane through which the cellular contents leak

and lysis occurs. Lysis is not an enzymatic process; it is thought to be due to physical damage to

the membrane. The complex consisting of C5bC6C7C8C9 is referred to as the membrane attack

complex (MAC).

C5a generated in the lytic pathway has several potent biological activities. It is the most potent

anaphylotoxin. In addition, it is a chemotactic factor for neutrophils and stimulates the respiratory

burst in them and it stimulates inflammatory cytokine production by macrophages. Its activities are

controlled by inactivation by carboxypeptidase B (C3-INA). Some of the C5b67 complex formed

can dissociate from the membrane and enter the fluid phase. If this were to occur it could then bind

to other nearby cells and lead to their lysis. The damage to bystander cells is prevented by Protein

S (vitronectin). Protein S binds to soluble C5b67 and prevents its binding to other cells.



Figure 16: Stabilized C3 convertase of the alternative pathway

2.4.2.4.2.4.2.4. Biologically Active Products of Complement ActivationBiologically Active Products of Complement ActivationBiologically Active Products of Complement ActivationBiologically Active Products of Complement Activation

Activation of complement results in the production of several biologically active molecules which

contribute to resistance, anaphylaxis and inflammation.

Kinin production.

C2b generated during the classical pathway of C activation is a prokinin which becomes

biologically active following enzymatic alteration by plasmin. Excess C2b production is prevented

by limiting C2 activation by C1 inhibitor (C1-INH) also known as serpin which displaces C1rs

from the C1qrs complex (Figure 16). A genetic deficiency of C1-INH results in an overproduction

of C2b and is the cause of hereditary angioneurotic edema. This condition can be treated with

Danazol which promotes C1-INH production or with ε-amino caproic acid which decreases

plasmin activity.

Anaphylotoxins

C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which cause

basophil/mast cell degranulation and smooth muscle contraction. Undesirable effects of these

peptides are controlled by carboxypeptidase B (C3a-INA).

Chemotactic Factors



C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of neutrophils,

basophils and macrophages and causes induction of adhesion molecules on vascular endothelial

cells.

Opsonins

C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and

promote phagocytosis. Other Biologically active products of C activation Degradation products of

C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors and modulate their

functions.

In summary, the complement system takes part in both specific and non-specific resistance and

generates a number of products of biological and pathophysiological significance (Table 4). There

are known genetic deficiencies of most individual C complement components, but C3 deficiency is

most serious and fatal. Complement deficiencies also occur in immune complex diseases (e.g.,

SLE) and acute and chronic bacterial, viral and parasitic infections.

3. Antigens

1) Definitions

2) Immunogen - a substance that induces a specific immune response.

3) Antigen (Ag) - a substance that reacts with the products of a specific immune response.

4) Hapten - a substance that is nonimmunogenic but which can react with the products of a specific

immune response. Haptens are small molecules which could never induce an immune response

when administered by themselves but which can when coupled to a carrier molecule. Free haptens,

however, can react with products of the immune response after such products have been elicited.

Haptens have the property o Antigenicity but not immunogenicity.

5) Epitope or Antigenic Determinant - the portion of an antigen that combines with the products

of a specific immune response. Antibody (Ab) - a specific protein which is produced in response

to an immunogen and which reacts with an antigen.

3.1.3.1.3.1.3.1. FactorsFactorsFactorsFactors influencinginfluencinginfluencinginfluencing imimimimmmmmuuuunnnnogeniciogeniciogeniciogenicittttyyyy

a) Contribution of the immunogen: The immune system normally discriminates between self and

non-self such that only foreign molecules are immunogenic. There is not absolute size above

which a substance will be immunogenic. However, in general, the larger the molecule the more

immunogenic it is likely to be. In general, the more complex the substance is chemically the

more immunogenic it will be. The antigenic determinants are created by the primary sequence of

residues in the polymer and/or by the secondary, tertiary or quaternary structure of the molecule.

The physical form, such as whether it is particulate or soluble can affect immunogenicity. In



general, particulate antigens are more immunogenic than soluble ones and denatured antigens

more immunogenic than the native form. Antigens that are easily degradable and phagocytosed

are generally more immunogenic. This is because for most antigens (T-dependant antigens, see

below) the development of an immune response requires that the antigen be phagocytosed,

processed and presented to helper T cells by an antigen presenting cell (APC)

b) Contribution of the Biological System: Genetic factors of the host can determine immunogenicity

of an antigen. Some substances are immunogenic in one species but not in another. Similarly, some

substances are immunogenic in one individual but not in others (i.e. responders and non-

responders). The species or individuals may lack or have altered genes that code for the receptors

for antigen on B cells and T cells or they may not have the appropriate genes needed for the APC

to present antigen to the helper T cells. Age can also influence immunogenicity. Usually the very

young and the very old have a diminished ability to mount an immune response in response to an

immunogen.

c) Method of Administration: The dose of administration of an immunogen can influence its

immunogenicity. There is a dose of antigen above or below which the immune response will not be

optimal. For example, high doses of antigen can often be tolerogenic and blunt the immune

response. Generally the subcutaneous route is for effective for inducing an immune response than

the intravenous or intragastric routes. The route of antigen administration can also alter the nature

of the response. Substances that can enhance the immune response to an immunogen are called

adjuvants. The use of adjuvants, however, is often hampered by undesirable side effects such as

fever and inflammation.

3.2.3.2.3.2.3.2. Chemical nature of immunogensChemical nature of immunogensChemical nature of immunogensChemical nature of immunogens

a) The vast majority of immunogens are proteins. These may be pure proteins or they may be

glycoproteins or lipoproteins. In general, proteins are usually very good immunogens. Pure

polysaccharides and lipopolysaccharides are good immunogens. Nucleic acids are usually poorly

immunogenic. However they may become immunogenic when single stranded or when complexed

with proteins. In general lipids are non-immunogenic, although they may be haptens. Some

glycolipids and phospholipids can stimulate T cells and produce a cell-mediated immune response

3.3.3.3.3.3.3.3. Types of antigensTypes of antigensTypes of antigensTypes of antigens

a) T-independent antigens are antigens which can directly stimulate the B cells to produce antibody

without the requirement for T cell help. In general, polysaccharides are T- independent antigens.

The responses to these antigens differ from the responses to other antigens. These antigens are

characterized by the same antigenic determinant repeated many times. Many of these antigens



can activate B cell clones specific for other antigens (polyclonal activation). T-independent

antigens can be subdivided into Type 1 and Type 2 based on their ability to polyclonally activate

B cells. Type 1 T-independent antigens are polyclonal activators while Type 2 are not. T-

independent antigens are generally more resistant to degradation and thus they persist for longer

periods of time and continue to stimulate the immune system.

b) T-dependent antigens are those that do not directly stimulate the production of antibody without

the help of T cells. Proteins are T-dependent antigens. Structurally these antigens are

characterized by a few copies of many different antigenic determinants.

c) Hapten-carrier conjugates are immunogenic molecules to which haptens have been covalently

attached. The immunogenic molecule is called the carrier. Structurally these conjugates are

characterized by having native antigenic determinants of the carrier as well as new

determinants created by the hapten (haptenic determinants). The actual determinant created by

the hapten consists of the hapten and a few of the adjacent residues, although the antibody

produced to the determinant will also react with free hapten. In such conjugates the type of

carrier determines whether the response will be T-independent or T-dependent.

3.4.3.4.3.4.3.4. Antigenic determinants recognized by B cells and AbAntigenic determinants recognized by B cells and AbAntigenic determinants recognized by B cells and AbAntigenic determinants recognized by B cells and Ab

Antigenic determinants recognized by B cells and the antibodies secreted by B cells are created by

the primary sequence of residues in the polymer (linear or sequence determinants) and/or by the

secondary, tertiary or quaternary structure of the molecule (conformational determinants). In

general antigenic determinants are small and are limited to 4-8 residues. Although, in theory, each

4-8 residues can constitute a separate antigenic determinant, in limited to approximately 4-8

residues. (Amino acids and or sugars). The combining site of f an antibody will accommodate an

antigenic determinant of the number of antigenic determinants per antigen is much lower than what

would theoretically be possible. Usually the antigenic determinants are limited to those portions of

the antigen that are accessible to antibodies as illustrated in the Figure 17 (antigenic determinants

are indicated in black).



Figure 17: antigenic determinants

3.5.3.5.3.5.3.5. DeterDeterDeterDetermmmminants recognized by T cellsinants recognized by T cellsinants recognized by T cellsinants recognized by T cells

a) Antigenic determinants

amino acids in proteins. T cells

why polysaccharides are generally

antigens. The determinants

recognition of the determinant

deg r aded into smal l e r pep t i d e s

the peptides associate with

(MHC) and it is the complex

cells can recognize lipids in

antigenic determinants are sm

theory, each 8-15 residues can

of antigenic determinants per

The antigenic determinants

molecules. This is why there

3.6.3.6.3.6.3.6. SuperantigensSuperantigensSuperantigensSuperantigens

a) When the immune system encounters a conv

(1 in 104 -105 ) of the

activated (monoclonal/oligoclonal response).

polyclonally activate a large

superantigens (Figure 18). Ex



antigenic determinants

inants recognized by T cellsinants recognized by T cellsinants recognized by T cellsinants recognized by T cells

inants recognized by T cells are created by the

ino acids in proteins. T cells do not recognize polysaccharide or nucleic acid

generally T-independent antigens and proteins are generally T

inants need not be located on the exposed surface

inant by T cells requires that the antigen

pep t i d e s . Free pep t i d e s a r e not recognized

with molecules coded for by the major Histoco

mplex of MHC molecules + peptide that is recognized

in conjunction with a MHC-like molecule ca

mall and are limited to approximately 8-15 amino

15 residues can constitute a separate antigenic determinant, in

per antigen is much less than what would theoretically

are limited to those portions of the antigen

This is why there can be differences in the responses of different individuals.

When the immune system encounters a conventional T-dependent antigen, only a s

T cell population is able to recognize the

onoclonal/oligoclonal response). However, there are so

rge fraction of the T cells (up to 25%). These

Examples of superantigens include: Staphylococcal enterotoxins

technology 2014

Page 25

the primary sequence of

do not recognize polysaccharide or nucleic acid antigens. This is

are generally T-dependent

t be located on the exposed surface of the antigen since

the antigen b e p r o t eo ly t i ca l l y

not recognized by T cells, rather

Histocompatibility complex

recognized by T cells. Some T

alled CD1. In general

ino acids. Although, in

ant, in practice, the number

theoretically be possible.

that can bind to MHC

ses of different individuals.

dependent antigen, only a small fraction

e antigen and become

However, there are some antigens which

These antigens are called

Staphylococcal enterotoxins (food



poisoning), Staphylococcal toxic

toxins (scalded skin syndro

bacterial superantigens are

other microorganisms as well. The

part, due to hyper activation of the immune

cytokines by activated T cells.

Figure 18: T –cell Receptor



Staphylococcal toxic shock toxin (toxic shock syndrome), Staphylococcal

syndrome) and Streptococcal pyrogenic exotoxins (shock).

the best studied there are superantigens associated

well. The diseases associated with exposure to superantigens are, in

part, due to hyper activation of the immune system and subsequent release

T cells.

cell Receptor

technology 2014

Page 26

e), Staphylococcal exfoliating

(shock). Although the

associated with viruses and

diseases associated with exposure to superantigens are, in

release of biologically active



4.4.4.4. Immunoglobulins: Immunoglobulins: Immunoglobulins: Immunoglobulins: Structure and FunctionStructure and FunctionStructure and FunctionStructure and Function

1. Definition

Immunoglobulins (Ig) Glycoprotein molecules which are produced by plasma cells in response to

an immunogen and which function as antibodies. The immunoglobulins derive their name from

the finding that when antibody-containing serum is placing in an electrical fields the

antibodies, which were responsible for immunity, migrated with the globular proteins (Figure 19).

Figure 19: Immunoglobulin

4.1.4.1.4.1.4.1. GeneralGeneralGeneralGeneral FunctionsFunctionsFunctionsFunctions ofofofof ImmunoglobulinsImmunoglobulinsImmunoglobulinsImmunoglobulins

Ag binding - Immunoglobulins bind specifically to one or a few closely related antigens. Each

immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies

is the primary function of antibodies and can result in protection of the host.

Valency. The valence of antibody refers to the number of antigenic determinants that an individual

antibody molecule can bind. The valence of all antibodies is at least two and in some instances

more.

Effector Functions - Often the binding of an antibody to an antigen has no direct biological effect.

Rather, the significant biological effects are a consequence of secondary "effector functions" of

antibodies. The immunoglobulins mediate a variety of these effector functions. Usually the

ability to carry out a particular effector function requires that the antibody bind to its antigen. Not

every immunoglobulin will mediate all effector functions. Fixation of complement - lysis of cells,

release of biologically active molecules Binding to various cell types - phagocytic cells,

lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins and

the binding can activate the cells to perform some function. Some immunoglobulins also bind to



receptors on placental trophoblasts. The binding results in transfer of the immunoglobulin across

the placenta and the transferred maternal antibodies provide immunity to the fetus and newborn

4.2.4.2.4.2.4.2. BasicBasicBasicBasic StructureStructureStructureStructure of Immunoglobulinsof Immunoglobulinsof Immunoglobulinsof Immunoglobulins

The basic structure of the immunoglobulins is illustrated in the Figure 2. Although different

immunoglobulins can differ structurally they all are built from the same basic unit.

A. Heavy and Light Chains - All immunoglobulins have a four chain structure as their basic unit. They

are composed of two identical light chains (23Kd) and two identical heavy chains (50-70Kd).

Figure 20: Basic structure of immunoglobulin (Heavy and Light chain)

B. DisulfideDisulfideDisulfideDisulfide bondsbondsbondsbonds

1. Inter-chain - The heavy and light chains and the two heavy chains are held together by inter-

chain disulfide bonds and by non-covalent interactions. The number of interchain disulfide bonds

varies among different immunoglobulin molecules.

Intra-chain - Within each of the polypeptide chains there are also intra-chain disulfide bonds.

C. Variable (V) and Constant (C) Regions - After the amino acid sequences of many different

heavy chains and light chains were compared, it became clear that both the heavy and light chain

could be divided into two regions based on variability in the amino acid sequences.

1. Light Chain - VL (110 aa) and CL (110 aa)

2. Heavy Chain - VH (110 aa) and CH (330-440 aa)

D. Hinge Region - The region at which the arms of the antibody molecule forms a Y is called the

hinge region because there is some flexibility in the molecule at this point.



E. Domains - 3D images of the immunoglobulin molecule shows that it is not straight as depicted

in Figure 2. Rather, it is folded into globular regions each of which contains an intra-chain

disulfide bond. These regions are called domains.

1. Light Chain Domains - VL and CL

2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)

F. Oligosaccharides - Carbohydrates are attached to the CH2 domain in most immunoglobulins.

However, in some cases carbohydrates may also be attached at other locations.

Figure 21: variable index of Ig

5.5.5.5.5.5.5.5. StructureStructureStructureStructure ofofofof thethethethe VariableVariableVariableVariable RegionRegionRegionRegion

A. Hypervariable (HVR) or complementarity determining regions (CDR)

Comparisons of the amino acid sequences of the variable regions of Ig's show that most of the

variability resides in three regions called the hypervariable regions or the complementarity

determining regions as illustrated in Figure 3. Antibodies with different specificities (i.e. different

combining sites) have different CDR's while antibodies of the exact same specificity have

identical CDR's (i.e. CDR --> Ab Combing site). CDR's are found in both the H and the L chains.

B. Framework regions

The regions between the CDR's in the variable region are called the framework regions (FR)

(Figure 22). Based on similarities and differences in the framework regions the immunoglobulin

heavy and light chain variable regions can be divided into groups and subgroups. These represent

the products of different variable region genes.



5.6.5.6.5.6.5.6. ImmunoglobulinImmunoglobulinImmunoglobulinImmunoglobulin Fragments: Structure/Function Relationships Fragments: Structure/Function Relationships Fragments: Structure/Function Relationships Fragments: Structure/Function Relationships

Immunoglobulin fragments produced by proteolytic digestion have proven very useful in

elucidating structure/function relationships in immunoglobulins.

A. Fab - Digestion with papain breaks the immunoglobulin molecule in the hinge region before

the H-H inter-chain disulfide bond Figure 4. This results in the formation of two identical

fragments that contain the light chain and the VH and CH1 domains of the heavy chain.

1. Antigen binding - These fragments were called the Fab fragments because they contained the

antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original

molecule was divalent. The combining site of the antibody is created by both VH and VL. An

antibody is able to bind a particular antigenic determinant because it has a particular combination

of VH and VL. Different combinations of a VH and VL result in antibodies that can bind

different antigenic determinants.

Figure 22: Immunoglobulin fragments

B.Fc - Digestion with papain also produces a fragment that contains the remainder of the two

heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it

was easily crystallized. Effector functions - The effector functions of immunoglobulins are

mediated by this part of the molecule. Different functions are mediated by the different domains

in this fragment (See Figure 5). Normally the ability of an antibody to carry out an effector

function requires the prior binding of an antigen. However, there are exceptions to this rule.



Figure 23: Antigen Binding and FC receptor

Figure 24: F(ab’) 2 fragments

C. F(ab')2 - Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after

the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites

(Figure 23). This fragment was called F (ab') 2 because it was divalent. The Fc region of the

molecule is digested into small peptides by pepsin. The F(ab')2 binds antigen but it does not



mediate the effector functions of antibodies.

5.7. . . . HumanHumanHumanHuman ImmunoglobulinImmunoglobulinImmunoglobulinImmunoglobulin Classes, Subclasses,Classes, Subclasses,Classes, Subclasses,Classes, Subclasses, TypesTypesTypesTypes andandandand SubtypesSubtypesSubtypesSubtypes

A.Immunoglobulin classes - The immunoglobulins can be divided into 5 different classes based on

differences in the amino acid sequences in the constant region of the heavy chains. All

immunoglobulins within a given class will have very similar heavy chain constant regions. These

differences can be detected by sequence studies or more commonly by serological means (i.e. by

the use of antibodies directed to these differences).

1. IgG - Gamma (γ) heavy chains

2. IgM - Mu (µ) heavy chains

3. IgA - Alpha (α) heavy chains

4. IgD - Delta (δ) heavy chains

5. IgE - Epsilon (ε) heavy chains

Figure 25: Immunoglobulin classes

B.Immunoglobulin Subclasses - The classes of immunoglobulins can be divided into subclasses

based on small differences in the amino acid sequences in the constant region of the heavy chains.

All immunoglobulins within a subclass will have very similar heavy chain constant region amino

acid sequences. Again these differences are most commonly detected by serological means.

1. IgG Subclasses

a) IgG1 - Gamma 1 (γ1) heavy chains b) IgG2 - Gamma 2 (γ2) heavy chains c) IgG3 -

Gamma 3 (γ3) heavy chains d) IgG4 - Gamma 4 (γ4) heavy chains

2. IgA Subclasses

a) IgA1 - Alpha 1 (α1) Heavy chains b) IgA2 - Alpha 2 (α2) heavy chains

C. Immunoglobulin Types - Immunoglobulins can also be classified by the type of light chain that

they have. Light chain types are based on differences in the amino acid sequence in the

constant region of the light chain. These differences are detected by serological means.



1. Kappa light chains (κ)

2. Lambda light chains (λ)

D.Immunoglobulin Subtypes - The light chains can also be divided into subtypes based on differences

in the amino acid sequences in the constant region of the light chain.

1. Lambda subtypes a)

Lambda 1

(λ1)

b) Lambda 2 (λ2)

c) Lambda 3 (λ3)

d) Lambda 4 (λ4)

E. Nomenclature - Immunoglobulins are named based on the class, or subclass of the heavy chain

and type or subtype of light chain. Unless it is stated precisely you are to assume that all subclass,

types and subtypes are present. IgG means that all subclasses and types are present.

F. Heterogeneity - Immunoglobulins considered as a population of molecules are normally very

heterogeneous because they are composed of different classes and subclasses each of which has

different types and subtypes of light chains. In addition, different immunoglobulin molecules

can have different antigen binding properties because of different VH and VL regions.

5.8.5.8.5.8.5.8. StructureStructureStructureStructure andandandand SomeSomeSomeSome PropertiesPropertiesPropertiesProperties ofofofof IgIgIgIg ClassesClassesClassesClasses andandandand SubclassesSubclassesSubclassesSubclasses

A IgG

1. Structure - The structures of the IgG subclasses are presented in Figure 7. All IgG's are monomers

(7S immunoglobulin). The subclasses differ in the number of disulfide bonds and length of the

hinge region.

2. Properties - Most versatile immunoglobulin because it is capable of carrying out all of the

functions of immunoglobulin molecules.

1) IgG is the major Ig in serum - 75% of serum Ig is IgG

2) IgG is the major Ig in extra vascular spaces

3) Placental transfer - IgG is the only class of Ig that crosses the placenta. Transfer is mediated by

receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally; IgG2 do not

cross well.

4) Fixes complement - Not all subclasses fix equally well; IgG4 does not fix complement

5) Binding to cells - Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for



the Fc region of IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc

receptors. A consequence of binding to the Fc receptors on PMN's, monocytes and macrophages is

that the cell can now internalize the antigen better. The antibody has prepared the antigen for

eating by the phagocytic cells. The term opsonin is used to describe substances that enhance

phagocytosis. IgG is a good opsonin. Binding of IgG to Fc receptors on other types of cells results

in the activation of other functions.

B. IgM

1. Structure - The structure of IgM is presented in Figure 8. IgM normally exists as a pentamer (19S

immunoglobulin) but it can also exist as a monomer. In the pentameric form all heavy chains are

identical and all light chains are identical. Thus, the valence is theoretically 10. IgM has an extra

domain on the µ chain (CH4) and it has another protein covalently bound via a S-S bond called

the J chain. This chain functions in polymerization of the molecule into a pentamer.

Figure 26: Immunoglobulin J Chain and Cµ4

Properties

a) IgM is the 3rd most common serum Ig.

b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when

it is stimulated by antigen.

c) As a consequence of its pentameric structure, IgM is a good complement fixing Ig. Thus, IgM

antibodies are very efficient in leading to the lysis of microorganisms.

d) As a consequence of its structure, IgM is also a good agglutinating Ig. Thus, IgM antibodies

are very good in clumping microorganisms for eventual elimination from the body.



e) IgM binds to some cells via Fc receptors.

f) B cell surface Ig - Surface IgM exists as a monomer and lacks J chain but it has extra 20

amino acids at the C-terminal end to anchor it into the membrane (Figure 27). Cell surface IgM

functions as a receptor for antigen on B cells. Surface IgM is noncovalently associated with two

additional proteins in the membrane of the B cell called Ig-α and Ig-β as indicated in Figure 10.

These additional proteins act as signal transducing molecules since the cytoplasmic tail of the Ig

molecule itself is too short to transduce a signal. Contact between surface immunoglobulin and an

antigen is required before a signal can be transuded by the Ig-α and Ig-β chains. In the case of T-

independent antigens, contact between the antigen and surface immunoglobulin is sufficient to

activate B cells to differentiate into antibody secreting plasma cells. However, for T-dependent

antigens, a second signal provided by helper T cells is required before B cells are activated.

Figure 27: Immunoglobulin Tail piece

Figure 28: Immunoglobulin structure and αβ

C IgA

Structure - Serum IgA is a monomer but IgA found in secretions is a dimer as presented in Figure

27. When IgA exits as a dimer, a J chain is associated with it. When IgA is found in secretions is

also has another protein associated with it called the secretory piece or T piece; sIgA is sometimes

referred to as 11S immunoglobulin. Unlike the remainder of the IgA which is made in the plasma

cell, the secretory piece is made in epithelial cells and is added to the IgA as it passes into the

secretions (Figure 28). The secretory piece helps IgA to be transported across mucosa and also

protects it from degradation in the secretions.



Figure 29: Immunoglobulin secretory piece

Properties

1) IgA is the 2nd most common serum Ig.

2) IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. Since it is found in

secretions secretory IgA is important in local (mucosal) immunity.

3) Normally IgA does not fix complement, unless aggregated.

4) IgA can bind to some cells - PMN's and some lymphocytes.

5) IgD

3. Structure - The structure of IgD is presented in the Figure 13. IgD exists only as a monomer.

2. Properties

IgD is found in low levels in serum; its role in serum uncertain.

IgD is primarily found on B cell surfaces where it functions as a receptor for antigen. IgD on the

surface of B cells has extra amino acids at C-terminal end for anchoring to the membrane. It also

associates with the Ig-α and Ig-β chains.

IgD does not bind complement.

IgE

Structure - The structure of IgE is presented in Figure 31. IgE exists as a monomer and has an

extra domain in the constant region.

Properties

IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast

cells even before interacting with antigen.

Figure 30: IgE Tail piece

Figure 31: IgE C€4



Involved in allergic reactions - As a consequence of its binding to basophils an mast cells, IgE

is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the

release of various pharmacological mediators that result in allergic symptoms. IgE also plays a

role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring

IgE levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE

and binding of eosinophils to IgE-coated helminths results in killing of the parasite.

IgE does not fix complement.

6. . . . Immunoglobulins: Isotypes, Allotypes and Idiotypes Immunoglobulins: Isotypes, Allotypes and Idiotypes Immunoglobulins: Isotypes, Allotypes and Idiotypes Immunoglobulins: Isotypes, Allotypes and Idiotypes

IIII. . . . IsotypesIsotypesIsotypesIsotypes

Definition - Isotypes are antigenic determinants that characterize classes and subclasses of heavy

chains and types and subtypes of light chains. If human IgM is injected into a rabbit the rabbit will

recognize antigenic determinants on the heavy chain and light chain and make antibodies to them.

If that antiserum is absorbed with human IgG the antibodies to the light chain determinants and

any determinants in common between human IgM and IgG will be removed and the resulting

antiserum will be react only with human IgM. Indeed, the antibodies will only react with the

constant region of the µ chain. Antibodies to the variable region are rare perhaps because only a

few copies of each different variable region are represented in the IgM and thus effective

immunization does not occur. The determinants that are recognized by such antibodies are called

isotypic determinants and the antibodies to those determinants are called anti-isotypic antibodies.

Each class, subclass, type and subtype of immunoglobulin has its unique set of isotypic

determinants.

Location - Heavy chain isotypes are found on the Fc portion of the constant region of the molecule

while light chain isotypes are found in the constant region. The location of isotypic determinants is

illustrated in Figure 32.

Figure 32: Ig Isotopes Kappa

Occurrence - Isotypes are found in ALL NORMAL individuals in the species. The prefix Iso



means same in all members of the species. Some individuals with immunodeficiencies may lack

one or more isotypes but normal individuals have all isotypes.

Importance - Antibodies to isotypes are used for the quantitating Ig classes and subclasses in

various diseases, in the characterization of B cell leukemia and in the diagnosis of various

immunodeficiency diseases.

II. Allotypes

A. Definition - Allotypes are antigenic determinants specified by allelic forms of the Ig genes.

B. Allotypes represent slight differences in the amino acid sequences in the heavy or light chains of

different individuals. Even a single amino acid difference can give rise to an allotypic

determinant, although in many cases the several amino acid substitutions have occurred.

C. Allotypic differences are detected by using antibodies directed against allotypic determinants.

These antibodies can be prepared by injecting the Ig from one person into another. In practice

however we obtain anti-allotype antisera from women who have had multiple pregnancies or

from people who have received blood transfusions or from some patients with rheumatoid

arthritis.

B. Location - In man the allotypic differences are localized to the constant region of the heavy and

light chains as illustrated in the Figure 33.

Figure 33: Ig Allotypes

C. Occurrence - Individual allotypes are found in individual members of a species. All allotypes are

not found in all members of the species. The prefix Allo means different in individuals of a

species

D. Human Ig Allotypes

Nomenclature - Human Ig allotypes are named on the basis of the heavy or light chain on which



it is located. Thus, an allotype on a Gamma 1 heavy chain is given the name: G1m(3). An

allotype on a Kappa light chain is given the name: Km(1). Table 1 lists some human allotypes.

Genetics

1. Codominant autosomal genes - Allotypes that represent amino acid substitutions at the same

position in a heavy or light chain (eg. G1m(3) and G1m(17) or Km(1) and Km(3) are inherited as

codominant autosomal genes.

eg. Km(1)/Km(3) X Km(1)/Km(1)

⇓

Km(1)/Km(1) and Km(1)/Km(3)

2. Allelic Exclusion - Although in a heterozygote both alleles are expressed, any individual Ig

molecule will only have one allotype. This is because an individual B cell can only expresses one

allele. This is called allelic exclusion. Allotypes that represent amino acid substitutions at

different locations in a molecule (eg. G1m(1) and G1m(17)) can be found on the same molecule.

eg. In a G1m(1,17) individual both allotypes can be on the same heavy chain

G1m(17) Gm1(1)

| |

214 355-358

F. Importance

1. Monitoring bone marrow grafts - Bone marrow grafts that produce a different allotype from the

recipient can be used to monitor the graft.

2. Forensic medicine - Km and Gm allotypes are detectable in blood stains and semen and are useful

in forensic medicine.

3. Paternity testing - The immunoglobulin allotypes are one of the characteristics used in legal cases

involving paternity.

III. Idiotypes (Id)

A. Definition - Unique antigenic determinants present on individual antibody molecules or on

molecules of identical specificity.

Identical specificity means that all antibodies molecules have the exact same hypervariable



regions.

To understand what idiotypes are, it is helpful to understand how they are detected.

DNP-BSA ⇒ Strain A ⇒ anti-DNP Ab

⇓

?? ⇐ Strain A

⇐ purified anti-DNP Ab

Antigenic determinants created by the combining site of an antibody are called idiotypes and

the antibodies elicited to the idiotypes are called anti-Id antibodies. Idiotypes are the

antigenic determinants created by the hypervariable regions of an antibody and the anti-

idiotypic antibodies are those directed against the hypervariable regions of an antibody.

B. Location - Idiotypes are localized on the Fab fragment of the Ig molecules as illustrated in

Figure 3. Specifically, they are localized at or near the hypervariable regions of the heavy and

light chains. In many instances the actual antigenic determinant (i.e. idiotype) may include some

of the framework residues near the hypervariable region. Idiotypes are usually determinants created

by both heavy and light chain HVR's although sometimes isolated heavy and light chains will

express the idiotype.

C. Importance

1. V region marker - Id's are a useful marker for a particular variable region.

2. Regulation of immune responses - there is evidence that immune responses may be regulated

by anti-Id antibodies directed against our own Id's.

3. Vaccines - In some cases anti-idiotypic antibodies actually stimulate B cells to make antibody and

thus they can be used as a vaccine. This approach is being tried to immunize against highly

dangerous pathogens that cannot be safely used as a vaccine.

4. Treatment of B cell tumors - Anti-idiotypic antibodies directed against an idiotype on malignant

B cells can be used to kill the cells. Killing occurs because of complement fixation or because

toxic molecules is attached to the antibodies.



Figure 34: Ig Idiotypes location

7. Immunoglobulins: Immunoglobulins: Immunoglobulins: Immunoglobulins: GeneticsGeneticsGeneticsGenetics

I. History

Amino acid sequencing data revealed that a single C region could be associated with many

different V regions. Also, it was shown that a single Idiotypes could be associated with different C

regions (eg. IgM and IgG). To explain these data it was suggested that perhaps the two regions of the

Ig molecule were coded for by separate genes and that the V and C region genes were somehow

joined before an Ig molecule was made (i.e. there were two genes for one polypeptide). This was a

revolutionary concept but with the advent of recombinant DNA technology, it has been shown to be

the correct. The Ig heavy and light chains are coded for by three separate gene families each one on

a separate chromosome - one for the heavy chain and one for each of the light chain types. Each

of these gene families has several V region genes and one or more C region genes. The V and C

regions genes are not however immediately adjacent to each other.

II. Light chain gene families

1.Germ line gene organization - The organization of the κ and λ light chain genes in the germ line or

undifferentiated cells is depicted in Figure 1.



Figure 35: Light chain gene

a. Lambda light chains - The λ

of λ chain, and approximately

exons, one (L) that codes for

region. Upstream of each of

J and C exons are separated by introns

b. Kappa light chains - The κ

is only one type of κ light

each of which has a leader exon and a V exon.

located between the V and C

2.Gene rearrangement and Expression

As a cell differentiates into a

various genes (exons) and th

commits to become a B cell making

level such that one of the V

recombination event which

which V gene is used is not tot

nearest to the J regions. However

V genes and J regions can be generated.



gene families

λ gene family is composed of 4 C region genes, one for each

approximately 30 V region genes. Each of the V region genes

for a leader region and the other (V) that codes for

of the C genes there is and additional exon called

J and C exons are separated by introns (intervening non-coding sequences).

light chain gene family contains only one C region gene, since

light chain. There are many V region genes (approximately

a leader exon and a V exon. In the κ gene family there

C genes. All of the exons are separated by introns.

Expression

mature B cell that will make a light chain, there i

the gene begins to be expressed as depicted in

cell making a light chain, there is a rearrangement of

V genes is brought next to one of the J regions. This

removes the intron between the V and J regions.

which V gene is used is not totally random; there is some preference for the

However, with time all V genes can be used so that all combinations of

V genes and J regions can be generated.

technology 2014

Page 42

gene family is composed of 4 C region genes, one for each subtype

region genes is composed of two

for most of the variable

called J (joining). The L, V,

contains only one C region gene, since there

genes (approximately 250)

there are several J exons

introns.

is a rearrangement of the

in Figure 2. As a cell

of the genes at the DNA

regions. This occurs by a

intron between the V and J regions. The selection of

for the use of V genes

time all V genes can be used so that all combinations of



Figure

A consequence of this DNA

a promoter (P), which is assoc

located in the intron between the J

pre-mRNA is made which contains sequences

for the introns between L and

(spliced) in the nucleus and the

and C exons contiguous.

The mRNA is translated in the cytoplasm and

into the lumen of the endoplasmic

endoplasmic reticulum and the

V region of the mature light

region by sequences in the C gene.

III. Heavy chain gene family

1.Germ line gene organization

In the heavy chain gene family

Each of the C genes is actually composed of several exons, one for each



Figure 36: Gene rearrangement and Expression

rearrangement is that the gene becomes transcriptionally ac

associated with the V gene, is brought close to an enhancer (E), which is

located in the intron between the J and C regions. As transcription initiates

mRNA is made which contains sequences from the L, V J and C regions

and V and between J and C (See Figure 36). This pre

the remaining introns are removed. The resulting

The mRNA is translated in the cytoplasm and the leader is removed as the

endoplasmic reticulum. The light chain is assembled with

the Ig is secreted via the normal route of secretory

chain is coded for by sequences in the V gene

region by sequences in the C gene.

family

organization - The organization of the heavy chain genes

family there are many C genes, one for each class and subclass

actually composed of several exons, one for each

technology 2014

Page 43

transcriptionally active because

an enhancer (E), which is

As transcription initiates from the promoter a

regions as well as sequences

pre-mRNA is processed

resulting mRNA has the L, V J

the protein is transported

assembled with a heavy chain in the

secretory proteins. The region

and J region and the C

genes is depicted in Figure 3.

e many C genes, one for each class and subclass of Ig.

domain and another for



the hinge region. In the heavy chain gene family there are many V region genes, each composed

of a leader and V exon. In addition to several J exons, the heavy chain gene family also contains

several additional exons called the D (diversity) exons. All of the exons are separated by introns as

depicted in Figure 37.

Figure 37: Heavy chain gene family

2. GeneGeneGeneGene rearrangementsrearrangementsrearrangementsrearrangements andandandand expressionexpressionexpressionexpression

As a cell differentiates into a mature B cell that will make a heavy chain, there is a rearrangement

of the various genes segments (exons) and the gene begins to be expressed as depicted in Figures

4 and 5. As a cell commits to become a B cell making a heavy chain, there are two

rearrangements at the DNA level. First, one of the D regions is brought next to one of the J regions

and then one of the V genes is brought next to the rearranged DJ region. This occurs by two

recombination events which remove the introns between the V, D and J regions. As with the light

chains the selection of the heavy chain V gene is not totally random but eventually all of the V

genes can be used.

A consequence of these DNA rearrangements is that the gene becomes transcriptionally active

because a promoter (P), which is associated with the V gene, is brought close to an enhancer (E),

which is located in the intron between the J and Cµ regions. As transcription initiates from the

promoter a pre-mRNA is made which contains sequences from the L, V, D, J Cµ and Cδ regions

as well as sequences for the introns between L and V, between J and Cµ , and between Cµ and Cδ



(Figure 38).

Figure 38: VDJ arrangement

Figure 39: VDJ Transcription



The pre-mRNA is processed (spliced) in the nucleus and the remaining introns, including

those between the exons in the C genes, are removed See Figure 5). The pre-mRNA can be

processed in two ways, one to bring the VDJ next to the Cµ gene and the other to bring the VDJ

next to the Cδ gene. The resulting mRNAs have the L, V, D, J and Cµ or Cδ exons

contiguous and will code for a µ and a δ chain, respectively.

The mRNAs are translated in the cytoplasm and the leader is removed as the protein is transported

into the lumen of the endoplasmic reticulum. The heavy chain is assembled with a light chain in

the endoplasmic reticulum and the Ig is secreted via the normal route of secretory proteins. The

region V region of the mature heavy chain is coded for by sequences in the V gene, D region and J

region and the C region by sequences in the C gene.

Mechanism of DNA rearrangements

Flanking the V, J and D exons there are unique sequences referred to as recombination signal

sequences (RSS), which

function in recombination. Each

RSS consists of a conserved

nonamer and a conserved

heptamer that are separated by

either 12 or 23 base pairs as

illustrated in Figure 6. The 12bp

and 23 bp spaces correspond to

one or two turns of the DNA

helix.

Figure 40: Mechanism of DNA rearrangements



Recombination only occurs between a 1 turn and a 2 turn signal. In the case of the λ light chains

there is a 1 turn signal upstream of the J exon and a 2 turn signal downstream of Vλ. In the case of

the κ light chains there is a 1 turn signal downstream of the Vκ gene and a 2 turn signal

upstream of the J exon.. In the case of the heavy chains there are 1 turn signals on each side of the

D exon and a 2 turn signal downstream of the V gene and a 2 turn signal upstream of the J

exon. Thus, this ensures that the correct recombination events will occur.

The recombination event results in the removal of the introns between V and J in the case of the

light chains or between the V, D, and J in the case of the heavy chains. The recombination

event is catalyzed by two proteins, Rag-1 and Rag-2. Mutations in the genes for these proteins

results in a severe combined immunodeficiency disease (both T and B cells are deficient), since

these proteins and the RSS are involved in generating both the B and T cell receptors for antigen.

Order of gene expression in Ig gene families

An individual B cell only produces one type of light chain and one class of heavy chain. (N.B.

The one exception is that a mature B cell can produce both µ and δ heavy chains but the antibody

specificity is the same since the same VDJ region is found on the µ and δ chains). Since any B

cell has both maternal and paternal chromosomes which code for the Ig genes there must be some

orderly way in which a cell expresses its Ig genes so as to ensure that only one type of light chain

and one class of heavy chain is produced? The order in which the Ig genes are expressed in a B

cell is depicted in Figure 41.

Figure 41: Ig genes are expressed in a B cell (Heavy chain)



Heavy chain (Figure 41) - A cell first attempts to rearrange one of its heavy chain genes; in some

cells the maternal chromosome is selected and in others the paternal chromosome is selected. If the

rearrangement is successful so that a heavy chain is made, then no further rearrangements occur in

the heavy chain genes. If, on the other hand, the first attempt to rearrange the heavy chain genes is

unsuccessful (i.e. no heavy chain is made), then the cell attempts to rearrange the heavy chain genes

on its other chromosome. If the cell is unsuccessful in rearranging the heavy chain genes the second

time, it is destined to be eliminated.

Figure 42: Ig genes are expressed in a B cell (light chain)

Kappa light chain (Figure 42) - When a cell successfully rearranges a heavy chain gene, it then

begins to rearrange one of its κ light chain genes. It is a random event whether the

maternal or paternal κ light chain genes are selected. If the rearrangement is unsuccessful (i.e. it

does not produce a functional κ light chain), then it attempts to rearrange the κ genes on the

other chromosome. If a cell successfully rearranges a κ light chain gene, it will be a B cell that

makes an Ig with a κ light chain.

Lambda light chain (Figure 42) - If a cell is unsuccessful in rearranging both of its κ light chain genes,

it then attempts to make a λ light chain. It is a random event whether the maternal or paternal λ

light chain genes are selected. If the rearrangement is unsuccessful (i.e. it does not produce a

functional λ light chain), then it attempts to rearrange the λ genes on the other chromosome. If a

cell successfully rearranges a λ light chain gene, it will be a B cell that makes an Ig with a λ light

chain.



The orderly sequence of rearrangements in the Ig gene families explains:

1)Why an individual B cell can only produce one kind of immunoglobulin with one kind of heavy

and one kind of light chain.

2) Why a individual B cell can only make antibodies of one specificity.

3) Why there is allelic exclusion in Ig allotypes at the level of an individual Ig molecule but co-

dominant expression of allotypes in the organism as a whole.

Origin of Antibody Diversity

Background - Antibody diversity refers to the sum total of all the possible Ab specificities

that an organism can make. It is estimated that we can make 107 - 108 different Ab molecules.

One of the major questions in immunology has been how can we make so many different antibody

molecules. Theories which have attempted to explain the origin of antibody diversity fall into two

major categories.

Germ line theory - This theory states that we different V region gene for each possible antibody

we can make.

Somatic mutation theory - This theory state that we have only one or a few V region genes

and the diversity is generated by somatic mutations which occur in these genes.

Current Concepts - Our current thinking is that both the germ line and somatic mutation

theories have some merit. It is thought that antibody diversity is generated by the following

mechanisms.

1. Large number of V genes

a) 30 lambda V genes b) 300 kappa V genes c) 1000 heavy chain V genes

2.V-J and V-D-J joining - The region where the light chain V gene and J region or the heavy chain V

gene and D and J regions come together is in the 3rd hyper variable region. Since it is random which

V and which J or D regions come together, there is a lot of diversity that can be generated by V-J

and V-D-J joining.

3.Junctional diversity (Inaccuracies in V-J and V-D and D-J recombination) - (Figure 9).

Recombination between V-J and V-D-J is not always perfect and additional diversity can arise

by errors that occur in the recombination event that brings the V region next to the J or D regions

or the D region next to the J region. It is estimated that these inaccuracies can triple the diversity

generated by V-J and V-D-J joining. The diversity generated by this mechanisms is occurring in

the 3rd hypervariable region and thus, is directly affecting the combining site of the Ab.

4.N region insertion - At the junction between D and J segments there is often an insertion



of a series of nucleotides which is catalyzed by the enzyme terminal transferase. (Terminal

transferase catalyzes the radon polymerization of nucleotides into DNA without the need for a

template. This leads to further diversity in the 3rd hypervariable region.

5.Somatic Mutation - There is evidence that somatic mutations are occurring in the V gene,

particularly in the place that codes for the 2nd hypervariable region. Thus, somatic mutation

probably contributes to Ab diversity to some extent.

6.Combinatorial Association - Any individual B cell has the potential to make any one of the possible

heavy chains and any one of the possible light chains. Thus, different combinations of heavy and

light chains within an individual B cell adds further diversity.

7.Multispecificity - Due to cross reactions between antigenic determinants of similar structure an

antibody can often react with more than one antigenic determinant. This is termed

multispecificity. Multispecificity also contributes to Ab diversity.

The process of gene rearrangement of the heavy and light chains and the combinatorial association

of these chains occurs during B cell development and is independent of antigen. Clones of B cells

expressing all of the possible antibody specificities are produced during development and antigen

simply selects those clones which have the appropriate receptor.The selected clones are then

activated, proliferate and differentiate into antibody secreting plasma cells.

T Cell Receptor For Antigen

T cells also have a receptor for antigen on their surfaces. This receptor is not an

immunoglobulin molecule but it is composed of two different polypeptide chains which have

constant and variable regions analogous to the immunoglobulins. Diversity in the T cell receptor

is also generated in the same way as described for antibody diversity (e.g. by VJ and VDJ joining

of gene segments and combinatorial association). However, no somatic mutation has been

observed in T cells.

8. 8. 8. 8. AntibodyAntibodyAntibodyAntibody FormationFormationFormationFormation

8.1. 8.1. 8.1. 8.1. GeneralGeneralGeneralGeneral CharacteristicsCharacteristicsCharacteristicsCharacteristics ooooffff thethethethe AntibodyAntibodyAntibodyAntibody ResponseResponseResponseResponse

A.A.A.A. Self/non-self discrimination - One characteristic feature of the specific immune system is that



it normally distinguishes between self and non-self and only reacts against non-self.

B.B.B.B. Memory - A second feature of the specific immune response is that it demonstrates memory. The

immune system "remembers" if it has seen an antigen before and it reacts to secondary exposures

to an antigen in a manner different than after a primary exposure. Generally only an exposure to the

same antigen will illicit this memory response.

C.C.C.C. Specificity - A third characteristic feature of the specific immune system is that there is a high degree

of specificity in its reactions. A response to a particular antigen is specific for that antigen or a

few closely related antigens.

8.2. . . . AntibodyAntibodyAntibodyAntibody FormationFormationFormationFormation

A. Fate of the immunogen

1. Clearance after primary injection - The kinetics of Ag clearance from the body after a primary

administration is depicted in Figure 1.

A.A.A.A. Equilibrium phase - The first phase is called the equilibrium or equilibration phase. During this time

the Ag equilibrates between the vascular and extra vascular compartments by diffusion. This is

normally a rapid process. Since particulate antigens don't diffuse, they do not show this phase.

B.B.B.B. Catabolic decay phase - In this phase the host's cells and enzymes metabolize the antigen. Most

of the antigen is taken up by macrophages and other phagocytic cells. The duration will

depend upon the immunogen and the host.

Figure 43: antibody formation



c) Immune elimination phase - In this phase newly synthesized antibody combines with the

antigen producing antigen/antibody complexes which are phagocytosed and degraded. Antibody

appears in the serum only after the immune elimination phase is over.

2. Clearance after secondary injection - If there is circulating antibody in the serum injection of

the antigen for a second time results in a rapid immune elimination. If the is no circulating antibody

then injection of the antigen for a second time results in all three phases but the onset of the immune

elimination phase is accelerated.

8.3. .3. .3. .3. KineticsKineticsKineticsKinetics ofofofof antibodyantibodyantibodyantibody responsesresponsesresponsesresponses totototo TTTT----dependentdependentdependentdependent AgAgAgAg

1. Primary (1o) Ab response - The kinetics of a primary antibody response to an antigen is

illustrated in Figure 43.

a) Inductive, latent or lag phase - In this phase the Ag is recognized as foreign and the cells begin to

proliferate and differentiate in response to the antigen. The duration of this phase will vary

depending on the antigen but it is usually 5-7 days.

b) Log or Exponential Phase - In this phase the Ab concentration increases exponentially as the

B cells that were stimulated by the antigen differentiate into plasma cells which secrete antibody.

c) Plateau or steady-state phase - In this phase Ab synthesis is balanced by Ab decay so that there in

no net increase in Ab concentration.

d) Decline or decay phase - In this phase the rate of Ab degradation exceeds that of Ab synthesis and

the level of Ab falls. Eventually the level of Ab may reach base line levels.



Figure 44: antibody formation after immunization (10 and (20)

2. Secondary (2o), memory or anamnestic response

a).Lag phase - In a secondary response there is a lag phase by it is normally shorter than that

observed in a primary response.

b) Log phase - The log phase in a secondary response is more rapid and higher Ab levels are

achieved.

c) Steady state phase

d) Decline phase - The decline phase is not as rapid and Ab may persist for months,

years or even a lifetime.

8.4. Specificity of 1o and 2o responses

Ab elicited in response to an antigen is specific for that antigen although it may also cross react

with other antigens which are structurally similar to the eliciting antigen. In general secondary

responses are only elicited by the same antigen used in the primary response. However, in some

instances a closely related antigen may produce a secondary response, but this is a rare

exception.



8.5. QualitativeQualitativeQualitativeQualitative changeschangeschangeschanges inininin AbAbAbAb duringduringduringduring 1111oooo andandandand 2222oooo responsesresponsesresponsesresponses

1. Ig class variation - In the primary response the major class of Ab produced is IgM whereas in

the secondary response it is IgG (or IgA or IgE) (Figure 4). The antibodies that persist in

the secondary response are the IgG antibodies.

2. Affinity - The affinity of the IgG Ab produced increases progressively during the response,

particularly after low doses of antigen (Figure 5). This is referred to as affinity maturation.

Affinity maturation is most pronounced after secondary challenge with antigen.

Figure 45 : antibody affinity

One explanation for affinity maturation is clonal selection as illustrated in Figure 47. A second

explanation for affinity maturation is that, after a class switch has occurred in the immune response,

somatic mutations occur which fine tunes the antibodies to be of higher affinity. There is

experimental evidence for this mechanism, although it is not known how the somatic mutation



mechanism is activated after exposure to antigen.

Figure 46: affinity maturation (10)

Figure 47: affinity maturation (20)

Avidity - As a consequence of increased affinity, the avidity of the antibodies increases during the

response.

Cross-reactivity - As a result of the higher affinity later in the response there is also an increase in

detectible cross reactivity. An explanation for why increasing affinity results in an increase in

detectible cross reactivity is illustrated by the following example. If a minimum affinity of 10-6 is

needed to detect a reaction, early in an immune response the reaction of a cross reacting antigen

with an affinity of 10-3 will not be detected. However, late in a response when the affinities

increase 1000 fold, the reaction with both the immunizing and cross reacting antigens will be

detected.



8.6. CellularCellularCellularCellular eventseventseventsevents duringduringduringduring 1111oooo andandandand 2222oooo responsesresponsesresponsesresponses totototo TTTT----dependentdependentdependentdependent AgAgAgAg

1. Primary response (Figure 49)

a) Lag phase - Clones of T and B cells with the appropriate antigen receptors bind antigen,

become activated and begin to proliferate. The expanded clones of B cells differentiate into

plasma cells which begin to secrete antibody.

b) Log phase - The plasma cells initially secrete IgM antibody since the Cµ heavy chain gene is

closest to the rearranged VDJ gene. Eventually some B cells switch from making IgM to

IgG, IgA or IgE. As more B cells proliferate and differentiate into antibody secreting cells the

antibody concentration increases exponentially

Figure 48: Cellular events during 1o

Figure 49: Cellular events during 2o responses

c) Stationary phase - As antigen is depleted, T and B cells are no longer activated. In addition,

mechanisms which down regulate the immune response come into play. Furthermore, plasma cells

begin to die. When the rate of antibody synthesis equals t h e r a t e o f a n t i b o d y decay

the stationary phase is reached.

d) Decline phase - When no new antibody is produced because the antigen is no longer present to



activate T and B cells and the residual antibody slowly is degraded, the decay phase is reached.

2. Secondary response (Figure 50)

Not all of the T and B cells that are stimulated by antigen during primary challenge with antigen

die. Some of them are long lived cells and constitute what is referring to as the memory cell

pool. Both memory T cells and memory B cells are produced and memory T cells survive longer

than memory B cells. Upon secondary challenge with antigen not only are virgin T and B cells

activated, the memory cells are also activated and thus there is a shorter lag time in the secondary

response. Since there is an expanded clone of cells being stimulated the rate of antibody

production is also increased during the log phase of antibody production and higher levels are

achieved. Also, since many if not all of the memory B cells will have switched to IgG (IgA or IgE)

production, IgG is produced earlier in a secondary response. Furthermore since there is an expanded

clone of memory T cells which can help B cells to switch to IgG (IgA or IgE) production, the

predominant class of Ig produced after secondary challenge is IgG (IgA or IgE).

Figure 50: Cellelular events during secondary response

Ab response to T-independent Ag

Responses to T-independent Ag are characterized by the production of almost exclusively IgM Ab

and no secondary response. Secondary exposure to the Ag results in another primary response to the

Ag as illustrated in Figure 52.

G. Class switching



During an antibody response to a T- dependent antigen a switch occurs in the class of Ig produced

from IgM to some other class (except IgD). Our understanding of the structure of the

immunoglobulin genes helps explain how class switching occurs (Figure 52).

Figure 51.class switching

During class switching another DNA rearrangement occurs between a switch site (Sµ) in the intron

between the rearranged VDJ regions and the Cµ gene and another switch site before one of the

other heavy chain constant region genes. The result of this recombination event is to bring the

VDJ region close to one of the other constant region genes, thereby allowing expression of a new

class of heavy chain. Since the same VDJ gene is brought near to a different C gene and since the

antibody specificity is determined by the hyper variable regions within the V region, the antibody

produced after the switch occurs will have the same specificity as before. Cytokines secreted by T

helper cells can cause the switch to certain isotypes.

MembraneMembraneMembraneMembrane andandandand secretedsecretedsecretedsecreted immunoglobulinimmunoglobulinimmunoglobulinimmunoglobulin

The specificity of membrane immunoglobulin on a B cell and the Ig secreted by the plasma cell

progeny of a B cell is the same. An understanding of how the specificity of membrane and secreted

Ig from an individual B cell can be the same comes from an understanding of immunoglobulin



genes (Figure 11). There are two potential polyA sites in the immunoglobulin gene. One after the

exon for the last heavy chain domain and the other after the exons that code for the trans-

membrane domains. If the first polyA site is used, the pre-mRNA is processed t o p r o d u c e a

s e c r e t e d protein. If the second polyA site is used, the pre-mRNA is processed to produce a

membrane form of the immunoglobulin. However, i n a l l cases the same VDJ region is used

and thus the specificity of the antibody remains the same. All C regions genes have these

additional membrane p i e c e s a s s o c i a t e d w i t h them and thus after class switching other

classes of immunoglobulins can be s e c r e t ed o r e xp r es s ed on t he surface of B cells.

Figure 52: polyadenylation sites

9. 9. 9. 9. ImmuniImmuniImmuniImmunizzzzationationationation

Immunization is the means of providing specific protection against most common and damaging

pathogens. The mechanism of immunity depends on the site of the pathogen and also the

mechanism of it pathogenesis. Thus, if the mechanism of pathogenesis involves exotoxin, the

only immune mechanism effective against it would be neutralizing antibodies that would prevent

its binding to the appropriate receptor and promoting its clearance and degradation by phagocytes.

Alternatively, if the pathogen produces disease by other means, the antibody will have to react

with the organism and eliminate by complement-mediated lysis or phagocytosis and intracellular

killing. However, if the organism is localized intracellular, it will not be accessible to antibodies



while it remains inside and the cell harboring it will have to be destroyed and, only then antibody

can have any effect. Most viral infections and intracellular bacteria and protozoa are examples of

such pathogens. In this case, the harboring cells can be destroyed by elements of cell mediated

immunity or if they cause the infected cell to express unique antigens recognizable by antibody,

antibody-dependent and complement mediated killing can expose the organism to elements of

humoral immunity. Alternatively, cells harboring intracellular pathogen themselves can be

activated to kill the organism. Such is the case with pathogens that have the capability of

surviving within phagocytic cells.

Figure 53: types of immunity

Passive Immunity:

Immunity can be gained, without the immune system being challenged with an antigen, by

transfer of serum or gamma globulins from an immune donor to a non-immune individual.

Alternatively, immune cells from an immunized individual may be used to transfer immunity.

Passive immunity may be acquired naturally or artificially.

Naturally acquired passive immunity: Immunity is transferred from mother to fetus through

placental transfer of IgG or colostral transfer of IgA.

Artificially acquired passive immunity: Immunity is often artificially transferred by injection

with gamma globulin from other individuals or from an immune animal. Passive transfer of



immunity with immune globulin or gamma globulin is practiced in numerous acute infections

(diphtheria, tetanus, measles, rabies, etc.), poisoning (insect-, reptile-bites, botulism), and as a

prophylactic measure (hypogammaglobulinemia). In these situations, gamma globulin of human

origin is preferable although specific antibodies raised in other species (usually horse) are

effective and used in some cases (e.g., poisoning, diphtheria, tetanus, gas gangrene, botulism,

etc.). While this form of immunization has the advantage of providing immediate protection, it

is effective for a short duration only and often results in pathological complications, such as

serum sickness characterized by rash, fever, arthralgia, vasculitis, nephritis, etc., and

anaphylaxis. Homologous immunoglobulin may carry the risk of transmitting hepatitis and HIV

and other blood borne diseases.

Passive transfer of cell-mediated immunity (immunity that is transferred by cells and not by

antibody) can also be accomplished in certain diseases (cancer, immunodeficiency). However, it

is difficult to find histocompatible (matched) donors and there is severe risk of graft versus host

disease.

Active Immunity:

This refers to immunity produced by the body following exposure to antigens.

Naturally acquired active immunity: Exposure to different pathogens leads to sub clinical or

clinical infections, which normally result in a protective immune response against these

pathogens.

Artificially acquired active immunity: Immunization may be achieved by administering live or

dead pathogens or their components. Vaccines used for active immunization consist of live

(attenuated: capable of producing very mild or no symptoms) organism, killed whole organism,

microbial components or secreted, detoxified toxins (toxoid).

Live vaccines: Live organisms are used for immunization against a number of viral infections.

Live vaccines for measles, mumps, rubella and chicken pox (varicella) are used routinely. A live

bacterial vaccine consisting of a strain of Mycobacterium bovis, Bacillus Calmet Geurin (BCG) is

used against tuberculosis in many African, European and Asian countries but not many others.

Whereas many studies have shown the efficacy of BCG vaccine, a number of studies also cast

doubt on its benefits.



Live vaccines normally produce self-limiting non-clinical infections and lead to subsequent

immunity, both humoral and cell-mediated, the latter being essential for intracellular pathogens.

However, they carry a serious risk of causing overt disease in immunocompromised individuals.

Furthermore, since live vaccines are often attenuated (made less pathogenic) by passage in animal

or thermal mutation, they can revert to their pathogenic form and cause serious illness. It is for

this reason, polio live (Sabin) vaccine, which was used for many years, has been replaced in many

countries by the inactivated (Salk) vaccine.

Killed vaccines: These consist of whole organisms inactivated by heat, chemicals or UV irradiation

treatment. Many killed viral and bacterial vaccines are available. Some of these are used to

immunize people at risks (e.g. influenza, hepatitis A, etc.) while others are used to immunize

travelers to different countries (e.g. cholera, typhoid etc.). Pertussis (whooping cough) whole

bacterial vaccine was used routinely until a few years ago, but due to its serious side effect, it has

been replaced by a formulation of acellular components.

Sub-unit vaccines: Some vaccines consist of subcomponents of the pathogenic organisms, usually

proteins or polysaccharides. Since polysaccharides are relatively weak T-independent antigens,

and produce only IgM responses without immunologic memory, they are made more

immunogenic and T-dependent by conjugation with proteins (e.g., haemophilus, meningococcus,

pneumococcus, etc.). Hepatitis-B, rabies vaccines consist of antigenic proteins cloned into a

suitable vector (e.g., yeast). These subunit vaccines are designed to reduce the problems of

toxicity and risk of infection. When the pathogenic mechanism of an agent involves a toxin, a

modified form of the toxin (toxoid) is used as vaccine (e.g., diphtheria, tetanus, etc.). Toxoids,

although lose their toxicity, they remains immunogenic.

Other novel vaccines: A number of novel approaches to active immunization are in the

investigative stage and are used only experimentally. These include anti-idiotype antibodies,

DNA vaccines and immunodominant peptides (recognized by the MHC molecules) and may be

available in the future. Anti-idiotype antibodies against polysaccharide antibody produce long

lasting immune responses with immunologic memory. Viral peptide genes cloned into vectors,

when injected transfect host cells and consequently produce a response similar to that produced

against live-attenuated viruses (both cell-mediated and humoral). Immunodominant peptides are

simple and easy to prepare and, when incorporated into MHC polymers, can provoke both



humoral and cell mediated responses.

Adjuvants: Weaker antigens may be rendered more immunogenic by the addition of other

chemicals. Such chemicals are known as adjuvants. There are many biological and chemical

substances that have been used in experimental conditions (Table 1). However, only Aluminum

salts (alum) are approved for human use and it is incorporated in DTP vaccine. Furthermore,

pertussis itself has adjuvant effects. Adjuvants used experimentally include mixtures of oil and

detergents, with (Freund’s complete adjuvant) or without certain bacteria (Freund’s incomplete

adjuvant). Bacteria most often used in an adjuvant are Mycobacteria (BCG) and Nocardia. In

some instance sub-cellular fractions of these bacteria can also be used effectively as adjuvants.

Newer adjuvant formulations include synthetic polymers and oligonucleotides. Most adjuvants

recognize TOLL-like receptors thus activating mononuclear phagocytes and inducing selective

cytokines that can enhance Th1 or Th2 responses, depending on the nature of the adjuvant.

Figure 54: Newer adjuvant formulations

The protective immunity conferred by a vaccine may be life-long (measles, mumps, rubella,

small pox, tuberculosis, yellow fever, etc.) or may last as little as a few months (cholera). The

primary immunization may be given at the age of 2-3 months (diphtheria, pertussis, tetanus,

recommended age range polio), or 13-15 months (mumps, measles, rubella). The currently

recommended schedule of routine immunization in the USA (recommended by CDC and AIP)



is summarized in Figure 55. This schedule is revised on yearly basis or as need by the CDC

Advisory Committee on Immunization Practice (AICP).

Prophylactic versus therapeutic immunization: Most vaccines are given prophylactic ally, i.e.,

prior to exposure to the pathogen. However, some vaccines can be administered therapeutically,

i.e., post exposure (e.g., rabies virus). The effectiveness of this mode of immunization depends on

the rate of replication of the pathogen, incubation period and pathogenic mechanism. For this

reason, only a booster shot with tetanus is sufficient if the exposure to the pathogen is within less

than 10 years and if the exposure is minimal (wounds are relative superficial). In a situation

where pathogen has a short incubation period, the pathogenic mechanism is such that only a small

amount of pathogenic molecules could be fatal (e.g., tetanus and diphtheria) and/or bolus of

infection is relatively large, both passive and active post exposure immunization are essential.

Passive prophylactic immunization is also normal in cases of defects in the immune system, such

as hypogammaglobulinemias.

Adverse effects of immunization: Active immunization may cause fever, malaise and

discomfort. Some vaccine may also cause joint pains or arthritis (rubella), convulsions,

sometimes fatal (pertussis), or neurological disorders (influenza). Allergies to egg may develop

as a consequence of viral vaccines produced in egg (measles, mumps, influenza, yellow fever).

Booster shots result in more pronounced inflammatory effects than the primary immunization.

The noticeable and serious side effects documented have been those following the DTP vaccine

(Table 2). Most of these were attributable to the whole pertussis component of the vaccine and

have been eliminated since the use of the acellular pertussis preparation.

10. Ce10. Ce10. Ce10. Cellllls ofls ofls ofls of the Immune the Immune the Immune the Immune SSSSyyyyststststem and Antigen Recognitionem and Antigen Recognitionem and Antigen Recognitionem and Antigen Recognition

1) OverviewOverviewOverviewOverview

a) The immune system has developed to protect the host from pathogens and other foreign

substances. Self/non-self discrimination is one of the hallmarks of the immune system. There are

two mains sites where pathogens may reside: extracellularly in tissue spaces or intracellularly

within a host cell; and the immune system has different ways of dealing with pathogens at these



sites. Although immune responses are tailored to the pathogen and to where the pathogen

resides, most pathogens can elicit both an antibody and a cell- mediated response, both of which

may contribute to ridding the host of the pathogen. However, for any particular pathogen an

antibody or a cell-mediated response may be more important for defense against the pathogen

b) Extracellular pathogens: antibodies are the primary defense against extracellular pathogens

and they function in three major ways:

i) Neutralization - by binding to the pathogen or foreign substance antibodies can block the

association of the pathogen with their targets. For example, antibodies to bacterial toxins

can prevent the binding of the toxin to host cells thereby rendering the toxin ineffective.

Similarly, antibody binding to a virus or bacterial pathogen can block the attachment of the

pathogen to its target cell thereby preventing infection or colonization.

ii) Opsonization - Antibody binding to a pathogen or foreign substance can opsonize the material and

facilitate its uptake and destruction by phagocytic cells. The Fc region of the antibody interacts

with Fc receptors on phagocytic cells rendering the pathogen more readily phagocytosed.

iii) Complement activation - Activation of the complement cascade by antibody can result in

lysis of certain bacteria and viruses. In addition, some components of the complement cascade

(e.g. C3b) opsonize pathogens and facilitate their uptake via complement receptors on phagocytic

cells.

c) Intracellular pathogens: Because antibodies do not get into host cells, they are ineffective against

intracellular pathogens. The immune system uses a different approach to deal with these kinds

of pathogens. Cell-mediated responses are the primary defense against intracellular pathogens and

the approach is different depending upon where the pathogen resides in the host cell (i.e., in the

cytosol or within vesicles). For example, most viruses and some bacteria reside in the cytoplasm

of the host cell, however, some bacteria and parasites actually live within endosomes in the

infected host cell. The primary defense against pathogens in the cytosol is the cytotoxic T

lymphocyte (Tc or CTL). In contrast, the primary defense against a pathogen within vesicles

is a subset of helper T lymphocytes (Th1).

i) Cytotoxic T cells (CTL) - CTLs are a subset of T lymphocytes that express a unique antigen on



their surface called CD8. These cells recognize antigens from the pathogen that are

displayed on the surface of the infected cell and kill the cell thereby preventing the spread of the

infection to neighboring cells. CTLs kill by inducing apoptosis in the infected cell.

ii) Th1 helper T cells - Th cells are a subset of T cells that express a unique antigen on their surface

called CD4. A subpopulation of Th cells, Th1 cells, is the primary defense against intracellular

pathogens that live within vesicles. Th1 cells recognize antigen from the pathogen that are

expressed on the surface of infected cells and release cytokines that activate the infected cell.

Once activated, the infected cell can then kill the pathogen. For example, Mycobacterium

tuberculosis, the causative agent of tuberculosis, infects macrophages but is not killed because it

blocks the fusion of lysosomes with the endosomes in which it resides. Th1 cells that recognize

M. tuberculosis antigens on the surface of an infected macrophage can secrete cytokines that

activate macrophages. Once activated the lysosomes fuse with endosomes and the M.

tuberculosis bacteria are killed.

2) Cells of the immune systemCells of the immune systemCells of the immune systemCells of the immune system

a) All cells of the immune system originate from a hematopoietic stem cell in the bone marrow,

which gives rise to two major lineages, a myeloid progenitor cell and a lymphoid

progenitor cell (Figure 1). These two progenitors give rise to the myeloid cells (monocytes,

macrophages, dendritic cells, mast cells, and granulocytes) and lymphoid cells (T cells, B

cells and NK cells), respectively. These cells make up the cellular components of the innate

(non-specific) and adaptive (specific) immune systems.



Figure 55: stem cell of immune system

b) Cells of the innate immune system – Cells of the innate immune system include

phagocytic cells (monocyte/macrophages and PMNs), NK cells, basophils, mast cells, eosinophils

and platelets. The roles of these cells have been discussed previously (see nonspecific immunity,

lecture 1). The receptors of these cells are pattern recognition receptors (PRRs) that recognize

broad molecular patterns found on pathogens (pathogen associated molecular patterns, PAMPS).

c) Cells that link the innate and adaptive immune systems – A specialized subset of cells called

antigen presenting cells (APCs) are a heterogeneous population of leukocytes that play an

important role in innate immunity and also act as a link to the adaptive immune system by

participating in the activation of helper T cells (Th cells). These cells include dendritic cells and

macrophages. A characteristic feature of APCs is the expression of a cell surface molecule

encoded by genes in the major histocompatibility complex, referred to as class II MHC molecules.

B lymphocytes also express class II MHC molecules and they also function as APCs, although

they are not considered as part of the innate immune system. In addition, certain other cells

(e.g., thymic epithelial cells) can express class II MHC molecules and can function as APCs.

d) Cells of the adaptive immune system – Cells that make up the adaptive (specific) immune system

include the B and T lymphocytes. After exposure to antigen, B cells differentiate into plasma

cells whose primary function is the production of antibodies. Similarly, T cells can differentiate

into either cytotoxic (CTL) or T helper (Th) cells of which there are two types Th1 and Th2

cells. There are a number of cell surface markers that are used in clinical laboratories to

distinguish B cells, T cells and their subpopulations. These are summarized in Table 4.

Table 4: CD Marker cell

Marker B cell CTL T‐helper

Antigen R BCR (surface Ig) TCR TCR

CD3 ‐‐ + +

CD4 ‐‐ ‐‐ +

CD8 ‐‐ + ‐‐

CD19/ CD20 + ‐‐ ‐‐

CD40 + ‐‐ ‐‐



3) Specificity of the adaptive immune response

a) Specificity of the adaptive immune response resides in the Ag receptors on T and B cells, the

TCR and BCR, respectively. The TCR and BCR are similar in that each receptor is specific for

one antigenic determinant but they differ in that BCRs are divalent while TCRs are monovalent

(Figure 2).

Figure 56: B cell and T cell

b) Each B and T cell has a receptor that is unique for a particular antigenic determinant and there are

a vast array of different antigen receptors on both B and T cells (discussed in more detail in

lecture 11). The question of how these receptors are generated was the major focus of

immunologists for many years. Two basic hypotheses were proposed to explain the generation of

the receptors: the instructionist (template) hypothesis and the clonal selection hypothesis.

i) Instructionist hypothesis – The instructionist hypothesis states that there is only one common

receptor encoded in the germline and that different receptors are generated using the Ag as a

template. Each Ag would cause the one common receptor to be folded to fit the Ag. While this

hypothesis was simple and very appealing, it was not consistent with what was known about

protein folding (i.e. protein folding is dictated by the sequence of amino acids in the protein). In

addition this hypothesis did not account for self/non-self discrimination in the immune system. It

could not explain why the one common receptor did not fold around self Ag.



ii) Clonal selection hypothesis – The clonal selection hypothesis states that the germline encodes

many different Ag receptors - one for each antigenic determinant to which an individual will be

capable of mounting an immune response. Ag selects those clones of cells that have the

appropriate receptor. The four basic principles of the clonal selection hypothesis are:

(1) Each lymphocyte has a SINGLE type of Ag receptor with a unique specificity.

(2) Interaction between the foreign molecule and Ag receptor capable of binding that molecule with

a high affinity leads to lymphocyte activation.

(3) The differentiated effector cell derived from an activated lymphocyte will have the same Ag

receptor as the parental lymphocyte; thus they are clones.

(4) Lymphocytes bearing Ag receptors for self molecules are deleted early in lymphoid

development and are absent from the repertoire of mature lymphocytes.

c) The clonal selection hypothesis is now generally accepted as the correct hypothesis to explain how

the adaptive immune system operates. It explains many of the features of the immune

response: 1) the specificity of the response; 2) the signal required for activation of the response

(i.e. Ag); 3) the lag in the adaptive immune response (time is required to activate cells and to

expand the clones of cells); and 4) self/non-self discrimination.

4) DevelopDevelopDevelopDevelopmmmment of the immune systement of the immune systement of the immune systement of the immune system

a) All immune cells arise from the hematopoietic stem cell. PMNs pass from the circulation into the

tissues. Mast cells are identifiable and thought to be “resident” in most tissues. B cells mature

in the fetal liver and bone marrow. T cells mature in the thymus. NK cells likely originate

in the bone marrow. Lymphocytes recirculate through secondary lymphoid tissues such as the

spleen where cells such as dendritic cells act as APCs.



Figure 57: B and T cell origination

5) LyLyLyLymmmmphocyte recirculationphocyte recirculationphocyte recirculationphocyte recirculation

a) There are relatively few T or B lymphocytes with a receptor for any particular antigen (1/10,000 –

1/100,000), the chances for a successful encounter between an antigen and the appropriate

lymphocyte are slim. However, the chances for a successful encounter are greatly enhanced by

the recirculation of lymphocytes through the secondary lymphoid organs. Lymphocytes in

the blood enter the lymph nodes and percolate through the lymph nodes (Figure 4). If they

do not encounter an antigen in the lymph node, they leave via the lymphatics and return to the

blood via the thoracic duct. It is estimated that 1-2% of lymphocytes recirculate every hour. If

the lymphocytes in the lymph nodes encounter an antigen, which has been transported to the

lymph node via the lymphatics, the cells become activated, divide and differentiate to become a

plasma cell, Th or CTL cell. After several days the effector cells can leave the lymph nodes via

the lymphatics and return to the blood via the thoracic duct and then make their way to the

infected tissue site.



Figure 58: lymphatic lymphocytes

b) Naïve (virgin) lymphocytes enter the lymph nodes from the blood via High Endothelial Venules

(HEVs). Homing receptors on the lymphocytes direct the cells to the HEVs. In the lymph nodes,

lymphocytes with the appropriate Ag receptor encounter Ag, which has been transported to the

lymph nodes by dendritic cells or macrophages. After activation the lymphocytes express new

receptors that allow the cells to leave the lymph node and reenter the circulation. Receptors on

the activated lymphocytes recognize cell adhesion molecules expressed on endothelial cells near

the site of an infection and chemokines produced at the infection site help attract the activated

cells (Figure 59).

Figure 59: B and T cell differentiation

11. Major Histocompatibility Complex and T Cell Receptors

1) Role of MHC in the iRole of MHC in the iRole of MHC in the iRole of MHC in the immmmmune responsemune responsemune responsemune response

a) Cell-cell interactions of the adaptive immune response are critically important in protection

from pathogens. These interactions are orchestrated by the immunological synapse whose



primary components are the T cell Ag receptor (TCR) and the Major histocompatibility complex

(MHC) molecule. The major function of the TCR is to recognize Ag in the correct context of

MHC and to transmit an excitatory signal to the interior of the cell. Since binding of peptide

within the MHC is not covalent, there are many factors while help stabilize the immunological

synapse.

b) There are two types of MHC (class I and class II) which are recognized by different subsets of T

cells. The cytotoxic T cell (CTL) recognizes Ag peptide in the context of MHC class I. The T

helper cell (Th) recognizes Ag presented in MHC class II.

2) StructStructStructStructuuuure of MHC class Ire of MHC class Ire of MHC class Ire of MHC class I

a) The molecule: Class I MHC molecules are composed of two polypeptide chains, a long α chain

and a short β chain called β2-microglobulin (60). The α chain has four regions. First, a

cytoplasmic region, containing sites for phosphorylation and binding to cytoskeletal elements.

Second, a transmembrane region containing hydrophobic amino acids by which the molecule is

anchored in the cell membrane. Third, a highly conserved α3 immunoglobulin (Ig)-like domain to

which CD8 binds. Fourth, a highly polymorphic peptide binding region formed from the α1 and

α2 domains. The β2- microglobulin associates with the chain and helps maintain the proper

conformation of the molecule.

A B



Figure 60: Structure of MHC class I

b) The Ag-binding groove: An analysis of which part of the class I MHC molecules is most variable

demonstrates that variability is most pronounced in the α1 and α2 domains, which comprise

the peptide binding region (Figure 61B). The structure of the peptide binding groove, revealed by

X-ray crystallography, shows that the groove is composed of two α helices forming a wall on each

side and eight β-pleated sheets forming a floor. The peptide is bound in the groove and the

residues that line the groove make contact with the peptide. These are the residues that are

the most polymorphic. The groove will accommodate peptides of approximately 8-10 amino

acids long. Whether a particular peptide will bind to the groove will depend on the amino acids

that line the groove. Because class I molecules are polymorphic, different class I molecules will

bind many different peptides. Each class I molecule will bind only certain peptides and will have

a set of criteria that a peptide must have in order to bind to the groove. For every class I

molecule, there are certain amino acids that must be a particular location in the peptide before it

will bind to the MHC molecule. Interactions at the N and C-terminus of the peptide are critical

and “lock” the peptide within the grove. These sites in the peptide are referred to as the “anchor

sites”. The ends of the peptide are buried within the closed ends of the class I binding

groove while the center bulges out for presentation to the TCR.

3) ) ) ) StructStructStructStructuuuure of MHC class IIre of MHC class IIre of MHC class IIre of MHC class II

a) The molecule: Class II MHC molecules are composed of two polypeptide chains, an α and a β

chain of approximately equal length (Figure 62). Both chains have four regions: first, a

cytoplasmic region containing sites for phosphorylation and binding to cytoskeletal

elements; second, a transmembrane region containing hydrophobic amino acids by which the

molecule is anchored in the cell membrane, third, a highly conserved α2 domain and a highly

conserved β2 domain to which CD4 binds and fourth, a highly polymorphic peptide binding

region formed from the α1 and β1 domains.



Figure 61: Structure of MHC class II

b) The Ag-binding groove: As with Class I MHC molecules, an analysis of which part of the class II

MHC molecule is most variable demonstrates that variability is most pronounced in the α1 and β1

domains, which comprise the peptide binding region (Figure 62 A). The structure of the peptide

binding groove, revealed by X-ray crystallography, shows that, like class I MHC molecules, the

groove is composed of two α helices forming a wall on each side and eight β-pleated sheets

forming a floor. Both the α1 and β1 chain contribute to the peptide binding groove. The peptide

is bound in the groove and the residues that line the groove make contact with the peptide. These

are the residues that are the most polymorphic. The groove of Class II molecules is open at one

end so that the groove can accommodate longer peptides of approximately 13-25 amino acids long

with some of the amino acids located outside of the groove. Whether a particular peptide will

bind to the groove will depend on the amino acids that line the groove. Because class II

molecules are polymorphic, different class II molecules will bind different peptides. Like

class I molecules, each class II molecule will bind only certain peptides and will have a set of

criteria that a peptide must have in order to bind to the groove (i.e. “anchor sites”).

4) IIIImmmmportant aspects of MHCportant aspects of MHCportant aspects of MHCportant aspects of MHC

a) Although there is a high degree of polymorphism for a species, an individual has maximum of six

different class I MHC products and only slightly more class II MHC products (considering only

the major loci). Each MHC molecule has only one binding site. The different peptides a given

MHC molecule can bind all bind to the same site, but only one at a time. Because each MHC

molecule can bind many different peptides, binding is termed degenerate. MHC polymorphism is

determined only in the germline. There are no recombinatorial mechanisms for generating

diversity. MHC molecules are membrane-bound; recognition by T cells requires cell-cell contact.

Alleles for MHC genes are co-dominant. Each MHC gene product is expressed on the cell

surface of an individual nucleated cell. A peptide must associate with a given MHC of that

particular individual otherwise no immune response can occur. Mature T cells must have a T cell



receptor that recognizes the peptide associated with MHC. Cytokines (especially interferon-γ)

increase level of expression of MHC. Polymorphism in MHC is important for survival of the

species.

b) How do peptides get into the MHC groove (Figure 62)? Peptides from the cytosol associate

with class I MHC and are recognized by CTL cells. The peptides enter the endoplasmic

reticulum and bind in the MHC class I groove. This complex is then exported to the cell

surface through the golgi. MHC class II molecules are formed with an invariant (Ii) chain as a

place holder while in the ER and Golgi. The Ii chain is cleaved and removed once the complex

is in a vesicle. Peptides from within the vesicle associate with class II MHC and are then

exported to the cell surface where they are recognized by Th cells.

5) Role of TCR in the immune response

a) The TCR is a surface molecule found on T cells that recognizes Ag presented in the correct

MHC context. The TCR is similar to immunoglobulin (Ig) and is part of the Ig superfamily.

There are two types of TCRs, the predominant αβ which is commonly found in lymphoid

tissues, and the γδ which is found at mucosal surfaces.

6) Structure of the TCR (αβ)

Figure 62: Role of TCR in the immune response

The TCR is a heterodimer composed of one α and one β chain of approximately equal length

(Figure 64). Each chain has a short cytoplasmic tail but it is too small to be able to transduce an

activation signal to the cell. Both chains have a transmembrane region comprised of hydrophobic

amino acids by which the molecule is anchored in the cell membrane.



Figure 63: TCR a heterodimer

Both chains have a constant region and a variable region similar to the immunoglobulin chains.

The variable region of both chains contains hyper variable regions that determine the specificity

for antigen.

6)6)6)6) IIIImmmmportant aspects of the TCRportant aspects of the TCRportant aspects of the TCRportant aspects of the TCR

a) Each T cell bears a TCR of only one specificity (i.e. there is allelic exclusion). The αβ TCR

recognizes Ag only in the context of cell-cell interaction and in the correct MHC. The γδ TCR

recognizes Ag in an MHC-independent manner in response to certain viral and bacterial Ag.

8) ) ) ) Genetic basis for receGenetic basis for receGenetic basis for receGenetic basis for receppppttttoooor generationr generationr generationr generation

a) The genetic basis for the generation of the vast array of antigen receptors on B cells has been

discussed previously (see lecture on Ig genetics). The generation of a vast array of TCRs is

accomplished by similar mechanism. The germline genes for the TCR β genes are composed of

V, D and J gene segments that rearrange during T cell development to produce many different

TCR β chains (Figure 64). The germline genes for the TCR α gene are composed of V and

J gene segments which rearrange to produce α chains. The specificity of the TCR is determined

by the combination of α and β chains.



Figure 64: TCR β chains

9) TCR and CD3 complex

a) The TCR is closely associated with a group of 5 proteins collectively called the CD3 complex

(Figure 65). The CD3 complex is composed of one γ, one δ, two ε and 2 ξ chains.

All of the proteins of the CD3 complex are invariant and they do not contribute to the Ag

specificity in any way. The CD3 complex is necessary for cell surface expression of the TCR

during T cell development as it stabilizes the receptor. In addition, the CD3 complex

transduces activation signals to the cell following antigen interaction with the TCR.

Figure 65: Antigen presenting cell

Figure 66: TCR and CD3 Recognition

10)10)10)10) The The The The ““““imimimimmmmmunological synapseunological synapseunological synapseunological synapse””””



a) The interaction between the TCR and MHC molecules are not very strong. Accessory molecules

are necessary to help stabilize the interaction (Figure 66 and 67). These include: 1) CD4

binding to Class II MCH, which ensures that Th cells only interact with APCs; 2) CD8 binding to

Class I MHC, which ensures that CTL cells can interact with target cells; 3) CD2 binding to

LFA-3; and 4) LFA-1 binding to ICAM-1. The accessory molecules are invariant and do not

contribute to the specificity of the interaction, which is solely determined by the TCR. The

expression of accessory molecules can be increased in response to cytokine, which is one way

that cytokines can modulate immune responses.

b) In addition to accessory molecules which help stabilize the interaction between the TCR and

antigen in association with MHC molecules, other molecules are also needed for T cell

activation. Two signals are required for T cell activation – one is the engagement of the TCR

with Ag/MHC and the other signal comes from the engagement of co- stimulatory molecules with

their ligands. One of the most important (but not the only) co-stimulatory molecule is CD28

on T cells which must interact with B7-1 (CD80) or B7-2 (CD86) on APCs. Like accessory

molecules the co-stimulatory molecules are invariant and do not contribute to the specificity of the

interaction. The multiple interactions of TCR with Ag/MHC and the accessory and co-

stimulatory molecules with their ligands have been termed the “immunological synapse.”

c) Not only is co-stimulation necessary for T cell activation, a lack of co-stimulation may result in

energy (i.e., inability to respond to antigen) or down-regulation of the response. There are a

number of possible outcomes of a T cell receiving one or both of the signals necessary for

activation. Engagement of the TCR with Ag/MHC but no co-simulation results in energy.

Engagement of only the co-stimulatory molecule has no effect. Engagement of TCR with

Ag/MHC and co-stimulatory molecules with their ligand results in activation. Engagement

of the TCR with Ag/MHC and engagement of B7 ligand with CTLA-4, molecules similar to

CD28, results in down-regulation of the response. CTLA-4/B7 interaction sends an inhibitory

signal to the T cell rather than an activating signal. This is one of the ways that immune

responses are regulated. CTLA-4 is expressed on T cells later in an immune response and

this helps to turn off the response.

11)11)11)11) Key steps inKey steps inKey steps inKey steps in T cell T cell T cell T cell aaaactictictictivvvvationationationation



a) The APC must process and present peptides to T cells. T cells must receive a co- stimulatory

signal, usually from CD28/CD80 or CD86 interaction. Accessory adhesion molecules must help

to stabilize the binding of T cells and the APC. Signals from the cell surface must be

transmitted to the nucleus via second messengers. Cytokines, produced by the activated cell, help

to drive cell proliferation.

12. 12. 12. 12. Antigen Process and Antigen Process and Antigen Process and Antigen Process and PPPPrrrreeeessssenenenenttttaaaationtiontiontion

1) 1) 1) 1) CCCCoooommmmparison of BCR andparison of BCR andparison of BCR andparison of BCR and TCRTCRTCRTCR

a) B cells and T cells recognize different substances as antigens and in a different form. The B cell

uses cell surface-bound immunoglobulin as a receptor and the specificity of that receptor is the

same as the immunoglobulin that it is able to secrete after activation. B cells recognize the

following antigens in soluble form: 1) proteins (both conformational determinants and

determinants exposed by denaturation or proteolysis); 2) nucleic acids; 3) polysaccharides; 4)

some lipids; 5) small chemicals (haptens).

b) In contrast, the overwhelming majority of antigens for T cells are proteins, and these must be

fragmented and recognized in association with MHC products expressed on the surface of

nucleated cells, not in soluble form. T cells are grouped functionally according to the class of

MHC molecules that associate with the peptide fragments of protein: helper T cells recognize

only those peptides associated with class II MHC molecules, and cytotoxic T cells recognize only

those peptides associated with class I MHC molecules.

2) 2) 2) 2) Ag processing and presentationAg processing and presentationAg processing and presentationAg processing and presentation

a) Antigen processing and presentation are processes that occur within a cell that result in

fragmentation (proteolysis) of proteins, association of the fragments with MHC molecules,

and expression of the peptide-MHC molecules at the cell surface where they can be recognized by

the T cell receptor on a T cell. However, the path leading to the association of protein fragments

with MHC molecules differs for class I and class II MHC. MHC class I molecules present

degradation products derived from intracellular (endogenous) proteins in the cytosol. MHC class



II molecules present fragments derived from extracellular (exogenous) proteins that are located in

an intracellular compartment.

b) MHC class I pathway - All nucleated cells express class I MHC. As shown in Figure 1, proteins

are fragmented in the cytosol by proteosomes (a complex of proteins having proteolytic activity)

or by other proteases. The fragments are then transported across the membrane of the endoplasmic

reticulum by transporter proteins. (The transporter proteins and some components of the

proteosome have their genes in the MHC complex). Synthesis and assembly of class I

heavy chain and beta2 microglobulin occurs in the endoplasmic reticulum. Within the

endoplasmic reticulum, the MHC class I heavy chain, beta2microglobulin and peptide form a

stable complex that is transported to the cell surface.



Figure 67: MHC class I pathway

c) MHC class II pathway - Whereas all nucleated cells express class I MHC, only a limited group of

cells express class II MHC, which includes the antigen presenting cells (APC). The principal APC

are macrophages, dendritic cells (Langerhans cells), and B cells, and the expression of class II

MHC molecules is either constitutive or inducible, especially by interferon-gamma in the case of

macrophages. As shown in Figure 69, exogenous proteins taken in by endocytosis are fragmented

by proteases in an endosome. The alpha and beta chains of MHC class II, along with an invariant

chain, are synthesized, assembled in the endoplasmic reticulum, and transported through the Golgi

and trans-Golgi apparatus to reach the endosome, where the invariant chain is digested, and

the peptide fragments from the exogenous protein are able to associate with the class II MHC

molecules, which are finally transported to the cell surface.



Figure 68: MHC class II pathway

d) Important aspects of Ag processing - One way of rationalizing the development of two different

pathways is that each ultimately stimulates the population of T cells that is most effective in

eliminating that type of antigen. Viruses replicate within nucleated cells in the cytosol and

produce endogenous antigens that can associate with MHC class I. By killing these infected cells,

CTL cells help to control the spread of the virus. Bacteria mainly reside and replicate

extracellularly. By being taken up and fragmented inside cells as exogenous antigens that can

associate with MHC class II molecules, helper Th2 T cells can be activated to assist B cells to

make antibody against bacteria, which limits the growth of these organisms. Some bacteria grow

intracellularly inside the vesicles of cells like macrophages. Inflammatory Th1 T cells help to

activate macrophages to kill the intracellular bacteria. Fragments of self, as well as non-

self, proteins associate with MHC molecules of both classes and are expressed at the cell

surface. Which protein fragments bind is a function of the chemical nature of the groove for that

specific MHC molecule.

3) Self MHC restriction

a) In order for a T cell to recognize and respond to a foreign protein antigen, it must

recognize the MHC on the presenting cell as self MHC. This is termed self MHC restriction.

Helper T cells recognize antigen in context of class II self MHC. CTL cells recognize antigen in

context of class I self MHC. The process whereby T cells become restricted to recognizing self

MHC molecules occurs in the thymus.

4) Ag presenting cells (APCs)

a) The three main types of antigen presenting cells are dendritic cells, macrophages and B cells,

although other cells, that express class II MHC molecules, (e.g., thymic epithelial cells) can act



as antigen presenting cells in some cases. Dendritic cells, which are found in skin and other

tissues, ingest antigens by pinocytosis and transport antigens to the lymph nodes and spleen. In

the lymph nodes and spleen they are found predominantly in the T cells areas. Dendritic cells are

the most effective antigen presenting cells and can present antigens to naïve (virgin) T cells.

Furthermore, they can present internalized antigens in association with either class I or class II

MHC molecules (cross presentation), although the predominant pathway for internalized antigen is

the class II pathway. The second type of antigen presenting cell is the macrophage. These cells

ingest antigen by phagocytosis of pinocytosis. Macrophages are not as effective in presenting

antigen to naïve T cells but they are very good in activating memory T cells. The third type of

antigen presenting cell is the B cell. These cells bind antigen via their surface Ig and ingest

antigens by pinocytosis. Like macrophages these cells are not as effective as dendrite cells in

presenting antigen to naïve T cells. B cells are very effective in presenting antigen to

memory T cells, especially when the antigen concentration is low because surface Ig on the B

cells binds antigen with a high affinity.

5) Presentation of superAgPresentation of superAgPresentation of superAgPresentation of superAg

a) Superantigens are antigens that can polyclonally activate T cells (see lecture on antigens) to

produce large quantities of cytokines that can have pathological effects. These antigens

must be presented to T cells in association with class II MHC molecules but the antigen does not

need to be processed. In the case of a superantigen the intact protein binds to class II MHC

molecules and to one or more Vβ regions of the TCR. The antigen is not bound to the

peptide binding groove of the MHC molecule or to the antigen binding site of the TCR.

Thus, any T cell that uses a particular Vβ in its TCR will be activated by a superantigen,

resulting in the activation of a large numbers of T cells. Each superantigen will bind to a

different set of Vβ regions.

6) ThyThyThyThymmmmic educationic educationic educationic education

a) Both Th and CTL cells are self-MHC restricted. In addition, T cells do not normally

recognize self antigens. How are self MHC restricted T cells generated and why are self reacting

T cells not produced? Random VDJ rearrangements in T cells would be expected to

generate some T cells that can recognize non-self MHC and some T cells that can recognize self



antigens. It is the role of the

self-MHC restricted and unable

Functional T cells in the periph

because APC or target cells prese

individual does not need function

associated with foreign MHC.

periphery that can recognize

damage of healthy, normal tissues.

b) As a result of random VDJ reco

TCRs of all specificities

specificities are retained. There are two sequential steps shown in Figure 3. First, T cells

the ability to bind to self M

retained. This is known as positi

cells having a TCR that recognizes

self MHC molecules associated

cells and macrophages are killed.

are retained. As a result of

foreign antigen survive. Each

and is released into the periphery retai



the thymus to ensure that the only T cells that get

unable to react with self antigen.

periphery have to recognize foreign antigens associ

cells present foreign antigen associated with self

functional T cells in the periphery that recognize antigen

MHC. An individual especially does not want functional

recognize self antigens associated with self MHC because they could lead to

al tissues.

VDJ recombination events occurring in immature T cells within the thy

are produced. Processes in the thymus deter

specificities are retained. There are two sequential steps shown in Figure 3. First, T cells

self MHC molecules expressed by cortical thy

positive selection. Those that do not bind, undergo

recognizes self MHC survive. Next, T cells with the

ociated with self molecules expressed by thymic epithelial cells,

killed. This is known as negative selection. Those

of these two steps, T cells having a TCR that recogn

survive. Each T cell that survives positive and negative selection

e periphery retains its specific T cell receptor (TCR).

technology 2014

Page 84

that get to the periphery are

ssociated with self MHC,

self MHC. However, an

nize antigen (self or foreign)

functional T cells in the

ens associated with self MHC because they could lead to

ing in immature T cells within the thymus,

determine which TCR

specificities are retained. There are two sequential steps shown in Figure 3. First, T cells with

ymic epithelial cells are

bind, undergo apoptosis. Thus, T

with the ability to bind to

epithelial cells, dendritic

Those that do not bind

recognizes self MHC and

selection in the thymus



Figure 69: random VDJ reco

c) While positive and negative

expressing CD4 or CD8 antige

is CD4-CD8-. In the thym

precedes a cell becomes eith

or CD8+ cells depends on

cell is presented with a class I

a cell is presented with a class

cell.



VDJ recombination

negative selection is occurring in the thymus the imm

antigens on their surface. Initially the pre-T cell

mus it becomes CD4+CD8+ and as positive

her a CD4+ or CD8+ cell. The commitment to beco

which class of MHC molecule the cell encounters.

ted with a class I molecule it will down regulate CD4 and beco

class II MHC molecule it will down regulate CD8

technology 2014

Page 85

mature T cells are also

that enters the thymus

and negative selection

become either a CD4+

the cell encounters. If a CD4+CD8+

become a CD8+ cell. If

gulate CD8 and become a CD4+



Figure 70: TCP selection

7) Negative selection in the Negative selection in the Negative selection in the Negative selection in the peripheryperipheryperipheryperiphery

a) Positive and negative selection in the thymus is not a 100% efficient process. In addition, not all

self antigens may be expressed in the thymus. Thus some self reactive T cells may get to the

periphery. Thus, there are additional mechanisms that are designed to eliminate self reactive T

cells in the periphery. These will be discussed in the tolerance lecture.

8) ) ) ) B cell sB cell sB cell sB cell seeeelelelelectctctctionionionion

a) Since B cells are not MHC-restricted there is no need for positive selection of B cells.

However, negative selection (i.e., elimination of self-reactive clones) of B cells is required.

This occurs during B cell development in the bone marrow. However, negative selection of B

cells is not a critical as for T cells since, in most instances, B cells require T cell help in order to

become activated. Thus, if a self reactive B cell does get to the periphery it will not be activated

due to lack of T cell help

13. CeCeCeCellllllll----CeCeCeCelllll Intl Intl Intl Inteeeeractions ractions ractions ractions inininin Immune ReImmune ReImmune ReImmune Ressssponsesponsesponsesponses

1) 1) 1) 1) Central role of Th Central role of Th Central role of Th Central role of Th cells in immune responsecells in immune responsecells in immune responsecells in immune response

a) As depicted in Figure 1, after Th cells recognize specific antigen presented by an APC, they can

initiate several key immune processes. These include: 1) selection of appropriate effector

mechanisms (e.g., B cell activation or CTL generation); 2) induction of proliferation of

appropriate effector cells and 3) enhancement of the functional activities of other cells (e.g.,



granulocytes, macrophages, NK cells).

Figure 71: Central role of Th cells in immune response

b) There are four subpopulations of Th cells, Th0, Th1, Th2, and Th17 cells. When naïve Th0

cells encounters Ag in secondary lymphoid tissues, they are capable of differentiating

into inflammatory Th1 cells, helper Th2 cells, or pathogenic Th17 cells, which are distinguished by

the cytokines they produce (Figure 73). Whether a Th0 cells becomes a Th1, Th2, or Th17 cell

depends upon the cytokines in the environment, which is influenced by Ag. For example some

antigens stimulate IL-4 production which favors the generation of Th2 cells while other antigens

stimulate IL-12 production, which favors the generation of Th1 cells. Th1, Th2, and Th17 cells

affect different cells and influence the type of immune response, as shown in Figure 3 for Th1 and

Th2.

i) Cytokines produced by Th1 cells activate macrophages and participate in the generation

of CTL cells, resulting in a cell-mediated immune response.

ii) In contrast cytokines produced by Th2 cells help to activate B cells, resulting in antibody

production. In addition, Th2 cytokines also activate granulocytes.

iii) A relatively recent discovery, Th17 cells (designated as such by their production of IL-17)

differentiate (in humans) in response to IL-1, IL-6, and IL-23 (TGF-β is important for Th17

differentiation in mice, although not in humans). IL-17 enhances the severity of some

autoimmune diseases including MS, inflammatory bowel disease, and rheumatoid arthritis.



Figure 72: Cytokine production

d) Equally important, each subpopulation

produced by Th1 cells inhibits

produced by Th2 cells inhibits

shown, IL-4 inhibits production

response is directed to the type

– cell-mediated responses for intrac

pathogens.



: Cytokine production

subpopulation can exert inhibitory influences on

inhibits proliferation of Th2 and differentiation of

inhibits production of IFN-γ by Th1 cells. In

production of Th1 and differentiation of Th17 cells.

type of response that is required to deal with the

sponses for intracellular pathogens or antibody responses for extracellular

technology 2014

Page 88

on the other. IFN- γ

of Th17 cells and IL-10

addition, although not

cells. Thus, the immune

with the pathogen encountered

llular pathogens or antibody responses for extracellular



2) Cell-cell interactions in Ab responses to exogenous T

Hapten-carrier model:

i) Historically one of the major

required for antibody production

understanding of this process ca

Studies with hapten-carrier conjugates established that: 1) Th2 cells recognized the carrier

determinants and B cells recognized haptenic deter

specific B cells and carrier-specific

both in antigen recognition and in antigen presentation.

ii) B cells occupy a unique positi

and class II MHC molecules on t

antibody having the same specificity as that expressed

addition they can function as

conjugate model, the mechani

Ig receptor, the hapten-carrier

carrier protein are presented

production of cytokines that enable

soluble anti-hapten antibodies (Fig

A.



responses to exogenous T-dependent Ag a)

ajor findings in immunology was that both T

production to a complex protein. A major contribution to our

process came from studies on the formation of

carrier conjugates established that: 1) Th2 cells recognized the carrier

ants and B cells recognized haptenic determinants; 2) interactions between

specific Th cells was self MHC restricted; and 3) B cells

both in antigen recognition and in antigen presentation.

position in immune responses because they express immunoglobulin

cules on their cell surface. They therefore are

e specificity as that expressed by their immunoglobulin recept

as an antigen presenting cell. In terms

echanism is thought to be the following: the hapten

carrier is brought into the B cell, processed, and peptide

ented to a helper T cell (Figure 4A). Activation of the

that enable the hapten-specific B cell to become acti

ies (Figure 74 A).

technology 2014

Page 89

cells and B cells were

ajor contribution to our

anti-hapten antibodies.

carrier conjugates established that: 1) Th2 cells recognized the carrier

tions between hapten-

Th cells was self MHC restricted; and 3) B cells can function

express immunoglobulin (Ig)

They therefore are capable of producing

immunoglobulin receptor; in

of the hapten-carrier

hapten is recognized by the

peptide fragments of the

of the T cell results in the

become activated to produce



B.

Figure 73: B cell proliferation

iii) Note that there are multiple signals delivered to the B cells in this model of Th2 cell- B cell

interaction. As was the case for activation of T cells where the signal derived from the TCR

recognition of a peptide-MHC molecule was by itself insufficient for T cell activation, so too for

the B cell. Binding of an antigen to the immunoglobulin receptor delivers one signal to the B

cell, but that is insufficient. Second signals delivered by co-stimulatory molecules are required;

the most important of these is CD40L on the T cell that binds to CD40 on the B cell to initiate

delivery of a second signal.

b) Cell-cell interactions in primary Ab response

i) B cells are not the best antigen presenting cell in a primary antibody response; dendritic

cells or macrophages are more efficient. Nevertheless, with some minor modifications the hapten-

carrier model of cell-cell interactions described above also applies to interactions in a primary

antibody response (Figure 75). In a primary response the Th2 cell first encounters antigen

presented by dendritic cells or macrophages. The “primed” Th2 cell can then interact with B

cells that have encountered antigen and are presenting antigenic peptides in association with class

II MHC molecules. The B cells still requires two signals for activation – one signal is the

binding of antigen to the surface Ig and the second signal comes from CD40/CD40 ligand

engagement during Th2/B cell-cell interaction. In addition, cytokines produced by the Th2 cells

help B cells proliferate and differentiate into antibody secreting plasma cells.



Figure 74: Cell-cell interactions in

c) Cell-cell interactions in secondary

i) As a consequence of

Memory B cells have a high affinity Ig r

bind and present antigen at

dendritic cells. In addition,

Thus, B/Th cell interactions

necessary (although it can

dendritic cells or macrophages.

ii) Cytokines and class switching:

proliferation and differentiation

Different cytokines influence

(Table 5). In this way the antibody

antibodies for parasitic worm

Cytokine IgG1

IL-4 ↑

IL-5

IFN-γ ↓

TGF-β



cell interactions in primary Ab response

cell interactions in secondary Ab response

of a primary response, many memory T and

ory B cells have a high affinity Ig receptor (due to affinity maturation), which allows

at much lower concentrations than that required

addition, memory T cells are more easily activated

interactions are sufficient to generate secondary antibody

occur) to “prime” memory Th cells with antigen

acrophages.

switching: cytokines produced by activated Th2 cells

differentiation of B cells, they also help regulate the

influence the switch to different classes of Ab with different

antibody response is tailored to suit the pathogen encountered (e.g. IgE

antibodies for parasitic worm infections).

Table 5: types of cytokines

IgG1 IgG2a IgG2b IgG3

↑ ↓ ↓

↓ ↑ ↑

↑ ↓

technology 2014

Page 91

B cells are produced.

tion), which allows them to

that required for macrophages or

ated than naïve T cells.

antibody responses. It is not

ith antigen presented by

cells not only stimulate

class of Ab produced.

different effort functions

tailored to suit the pathogen encountered (e.g. IgE

IgA IgE IgM

↑ ↓

↑

↓ ↓

↑ ↓



3) Cell-cell interactions in Ab responses to exogenous T-independent Ag

a) Antibody responses to T-independent antigens do NOT require cell-cell interactions. The

polymeric nature of these antigens allows for cross-linking of antigen receptors on B cells

resulting in activation. No secondary responses, affinity maturation or class switching occurs.

Responses to T-independent antigens are due to the activation of a subpopulation of B cells

called CD5+ B cells (also called B1 cells), which distinguishes them from conventional B cells

that are CD5- (also called B2 cells).

b) CD5+ cells are the first B cells to appear in ontogeny. They express surface IgM but little

or no IgD and they produce primarily IgM antibodies from minimally somatically mutated germ

line genes. Antibodies produced by these cells are of low affinity and are often polyreactive

(bind multiple antigens). Most of the IgM in serum is derived from CD5+ B cells. CD5+ B

cells do not give rise to memory cells. An important characteristic of these cells is that

they are self-renewing, unlike conventional B cells which must be replaced from the bone

marrow. CD5+ B cells are found in peripheral tissues and are the predominant B cell in the

peritoneal cavity. B1 cells are a major defense against many bacterial pathogens that

characteristically have polysaccharides in their cell walls. The importance of these cells in

immunity is illustrated by the fact that many individuals with T cell defects are still able to resist

many bacterial pathogens

4) Cell-cell interactions in cell-mediated immune response: generation of CTL in response to

exogenous Ag in cytosol



Figure 75: Process of Antigen presentation

a) Cytotoxic T lymphocytes are not fully mature when they exit the thymus. They have a functional

TCR that recognizes antigen, but they cannot lyse a target cell. They must differentiate into fully

functional effector CTL cells. Cytotoxic cells differentiate from a "pre-CTL" in response to two

signals: specific Ag in the context of MHC class I on a stimulator cell, and cytokines produced

by Th1 cells (especially IL-2 and IFN-γ) (Fig 75).

b) Features of CTL-mediated lysis- CTL killing is Ag-specific. To be killed by a CTL, the target

cell must bear the same class I MHC-associated Ag that triggered pre-CTL differentiation. CTL

killing requires cell contact. CTL are triggered to kill when they recognize the target Ag

associated with a cell surface MHC molecule. Adjacent cells lacking the appropriate target

MHC-Ag are not affected. CTLs are not injured when they lyse target cells; therefore, each CTL

is capable of killing sequentially numerous target cells.

c) Mechanisms of CTL killing - CTLs utilize several mechanisms to kill target cells, some of

which require direct cell-cell contact and others that result from the production of certain

cytokines. In all cases death of the target cells is a result of apoptosis.

i) Fas- and TNF-mediated killing (Figure 76): Once generated CTLs express Fas ligand on their

surface, which binds to Fas receptors on target cells. In addition, TNF-α secreted by CTLs can

bind to TNF receptors on target cells. The Fas and TNF receptors are a closely related family of

receptors, which when they encounter their ligands, form trimers of the receptors. These receptors

also contain death domains in the cytoplasmic portion of the receptor, which after

trimerization can activate caspases that induce apoptosis in the target cell.



Figure 76: Fas- and TNF-mediated killing

ii) Granule-mediated killing (Figure 77): Fully differentiated CTLs have numerous granules

that contain perforin and granzymes. Upon contact with target cells, perforin is released

and it polymerizes to form channels in the target cell membrane. Granzymes, which are serine

proteases, enter the target cell through the channels and activate caspases and nucleases in the

target cell resulting in apoptosis.

Figure 77: Granule-mediated killing



5) Cell-cell interact ions in cel l-mediated immune response: activation of macrophages in

response to endogenous Ag in vesicles

a) Macrophages play a central role in the immune system. They are involved in 1) the initial

defense against pathogens as part of the innate immune system, 2) Ag presentation to Th cells,

and 3) various effector functions (e.g., cytokine production, bactericidal and tumoricidal

activities) (Figure 78). Indeed macrophages play an important role not only in immunity but also

in reorganization of tissues. However, because of their potent activities, macrophage can also

do damage to tissues.

Figure 78: cytokine production, bactericidal and tumoricidal activities

b) Many of these macrophage functions can only be performed by activated macrophages.

Macrophage activation can be defined as quantitative alterations in the expression of various gene

products that enable the activated macrophage to perform some function that cannot be

performed by the resting macrophage.



c) Macrophage activation is an important function of Th1 cells. When Th1 cells get activated by

an APC such as a macrophage, they release IFN-γ, which is one of two signals required to

activate a macrophage. Lipopolysaccharide (LPS) from bacteria or TNF-α produced by

macrophages exposed to bacterial products deliver the second signal.

d) As discussed in the lecture on the innate immune response (lecture 1), effector

mechanisms employed by macrophages include production of 1) TNF-α, which can

induce apoptosis, 2) nitric oxide and other reactive nitrogen intermediates, 3) reactive oxygen

intermediates, and 4) cationic proteins and hydrolytic enzymes.

e) Macrophage activation by Th1 cells is very important in protection against many different

pathogens. For example, Pneumocystis carinii, an extracellular pathogen, is controlled in

normal individuals by activated macrophages; it is, however, a common cause of death in AIDS

patients because they are deficient in Th1 cells. Similarly, Mycobacterium tuberculosis, an

intracellular pathogen that resides in vesicles, is not efficiently killed by macrophages unless

they are activated; hence this infection is a problem in AIDS patients.

14. Cytokines

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

They were initially identified as products of immune cells that act as mediators and

regulators of immune processes but many cytokines are now known to be produced by cells other

than immune cells and they can have effects on non-immune cells as well. Cytokines are

currently being used clinically as biological response modifiers for the treatment of various

disorders. The term cytokine is a general term used to describe a large group of proteins but

there are other terms that are commonly used to describe particular kinds of cytokines. These

include: monokines (cytokines produced by mononuclear phagocytic cells), lymphokines

(cytokines produced by activated lymphocytes, especially Th cells), interleukins

(cytokines that acts as mediators between leukocytes), and chemokines (small cytokines primarily

responsible for leukocyte migration).

2) Cytokines function as part of a larger inter-related system of proteins and signaling cascades, the

cytokine network. These are complex interactions in which different cells can respond differently



to the same cytokine depending upon other signals received by the cell. Cytokine signaling is

very flexible and can induce both protective and damaging responses. One cytokine often

influences the synthesis of other cytokines. They can produce cascades, or enhance or

suppress production of other cytokines. In addition, they can often influence the action of other

cytokines. The effects can be: antagonistic, additive, or synergistic.

3) Cytokines are not typically stored as preformed proteins. Rather their synthesis is initiated by

gene transcription and their mRNAs are short lived. They are produced as needed in immune

responses. Genes encoding cytokines can produce variants through alternative splicing to

yield proteins with slightly different but biologically significant bioactivities.

4) Many individual cytokines are produced by many cell types involved in both the innate and

adaptive immune response. Individual cytokines also act on many cell types (i.e., they are

pleotropic) and in many cases cytokines have similar actions (i.e., they are redundant).

Redundancy is due to the nature of the cytokine receptors.

5) Receptors for cytokines are heterodimers (sometimes heterotrimers) many of which can be

grouped into families based on common structural features; one subunit is common to all

members of a given family. Some examples are shown in Figure 78 (type 1) and Figure 79 (type

2).

Figure 79: Receptors for cytokines are heterodimers (A)



Figure 80: Receptors for cytokines are heterodimers (B)

a) Type 1 cytokine receptors (IL-2R family) are the largest family of cytokine receptors.

This family is divided into three subsets based on common components: IL2Rγ, common β,

and gp130 (Figure 1A). These receptors lack intrinsic protein tyrosine kinase activity. Ligand

(cytokine) binding leads to receptor dimerization and initiation of intracellular signaling.

b) Type 2 cytokine receptors (IFNR family) is denoted by conserved cysteines in the

extracellular domains of the subunits. The extracellular domains also have tandem Ig- like

domains characteristic of this cytokine receptor family. These receptor subunits also have

intrinsic tyrosine kinase activity (denoted by the * in Figure 1B).

c) Chemokine receptors all have seven transmembrane segments linked to GTP-binding proteins.

They are selectively expressed on particular lymphocyte populations and are named based on

the family of chemokines to which they bind; CCR (the CC receptor) binds CC chemokines as its

ligand while the CXCR binds CXC chemokines as its ligand (chemokines naming convention will

be discussed below).

6) Since the subunit common to all members of the family functions in binding cytokine and in

signal transduction, a receptor for one cytokine can often respond to another cytokine in the same

family. Thus, an individual lacking IL-2, for example, is not adversely affected because

other cytokines (IL-15, IL-7, IL-9, etc.) assume its function. Similarly, a mutation in a cytokine

receptor subunit other than the one in common often has little effect. On the other hand, a

mutation in the common subunit has profound effects. For example, a mutation in the gene for

the IL-2Rγ subunit causes human X-linked severe combined immunodeficiency (XSCID)



characterized by a complete or nearly complete T and B cell defects.

7) Cytokines bind to specific receptors on target cells with high affinity and the cells that

respond to a cytokine are either: 1) the same cell that secreted cytokine (autocrine); 2) a nearby

cell (paracrine) or 3) a distant cell reached through the circulation (endocrine). Cellular

responses to cytokines are generally slow (hours) because they require new mRNA and protein

synthesis.

8) Categories of cytokines: Cytokines can be grouped into different categories based on their

functions or their source but it is important to remember that because they can be produced by

many different cells and act on many different cells, any attempt to categorize them will be

subject to limitations.

a) Mediators of the innate immune response: Cytokines that play a major role in the innate immune

system include: TNF-α, IL-1, IL-10, IL-12, type I interferons (IFN-α and IFN- β), IFN-γ,

and chemokines.

i) TNF-α: Tumor necrosis factor alpha is produced by activated macrophages is response

to microbes, especially the lipopolysaccharide (LPS) of Gram negative bacteria. It is an

important mediator of acute inflammation. It mediates the recruitment of neutrophils and

macrophages to sites of infection by stimulating endothelial cells to produce adhesion molecules

and by producing chemokines which are chemotactic cytokines. TNF- α also acts on the

hypothalamus to produce fever and it promotes the production of acute phase proteins.

ii) IL-1: Interleukin 1 is another inflammatory cytokine produced by activated macrophages. Its

effects are similar to that of TNF-α and it also helps to activate T cells.

iii) IL-10: Interleukin 10 is produced by activated macrophages and Th2 cells. It is predominantly

an inhibitory cytokine. It inhibits production of IFN-γ by Th1 cells, which shifts immune

responses toward a Th2 type. It also inhibits cytokine production by activated macrophages

and the expression of class II MHC and co- stimulatory molecules on macrophages,

resulting in a dampening of immune responses.

iv) IL-12: Interleukin 12 is produced by activated macrophages and dendritic cells. It stimulates the



production of IFN-γ and induces the differentiation of Th cells to become Th1 cells. In addition,

it enhances the cytolytic functions of Tc and NK cells.

v) Type I interferons: Type I interferons (IFN-α and IFN-β) are produced by many cell types and

they function to inhibit viral replication in cells. They also increase expression of class I MHC

molecules on cells making them more susceptible to killing by CTLs. Type I interferons also

activate NK cells.

vi) INF-γ: Interferon gamma is an important cytokine produced by primarily by Th1 cells,

although it can also be produced by Tc and NK cells to a lesser extent. It has numerous functions

in both the innate and adaptive immune systems as depicted in Figure 2.

Figure 81: Interferon

vii) Chemokines: Chemokines are chemotactic cytokines produced by many kinds of leukocytes

and other cell types. They represent a large family of molecules that function to recruit leukocytes

to sites of infection and play a role in lymphocyte trafficking by determining which cells will

cross the epithelium and where they are directed to go. There are four families of chemokines

based on spacing of conserved cysteine. Two examples are the α-chemokines which

have a CXC structure (two cysteines with a different amino acid in between) and the β-

chemokines which have a CC structure (two neighboring cysteines). Individual chemokines



(within the same family) often bind more than one receptor.

b) Mediators of the adaptive immune response: Cytokines that play a major role in the

adaptive immune system include: IL-2, IL-4, IL-5, TGF-β, IL-10 and IFN-γ.

i) IL-2: Interleukin 2 is produced by Th cells, although it can also be produced by Tc cells to a

lesser extent. It is the major growth factor for T cells. It also promotes the growth of B cells and

can activate NK cells and monocytes as depicted in Figure 82. IL-2 acts on T cells in an

autocrine fashion. Activation of T cells results in expression of IL-2R and the production of IL-2.

The IL-2 binds to the IL-R and promotes cell division. When the T cells are no longer being

stimulated by antigen, the IL-2R will eventually decay and the proliferative phase ends Figure 83.

Figure 82: activate NK cells and monocytes, proliferative phase

ii) IL-4: Interleukin 4 is produced by macrophages and Th2 cells. It stimulates the development

of Th2 cells from naïve Th cells and it promotes the growth of differentiated Th2 cells resulting

in the production of an antibody response. It also stimulates Ig class switching to the IgE isotype.

iii) IL-5: Interleukin 5 is produced by Th2 cells and it functions to promote the growth and

differentiation of B cells and eosinophils. It also activates mature eosinophils.



iv) TGF-β: Transforming growth factor beta is produced by T cells and many other cell types. It is

primarily an inhibitory cytokine. It inhibits the proliferation of T cells and the activation of

macrophages. It also acts on PMNs and endothelial cells to block the effects of pro-

inflammatory cytokines.

c) Stimulators of hematopoesis: Some cytokines stimulate the differentiation of

hematopoietic cells. These include GM-CSF which promotes the differentiation of bone marrow

progenitors, M-CSF, which promotes growth and differentiation of progenitors into monocytes

and macrophages and G-CSF (also known as pluripoietin), which promotes production of PMNs.

d) Interleukin 17: IL-17 is proinflammatory cytokine approximately 150 amino acids long. The

IL-17 family includes sex members which share sequence homology but differential tissue

expression. IL-17 is produced by Th17 cells and its overexpression has been associated with

autoimmune disease including multiple sclerosis, rheumatoid arthritis, and inflammatory bowel

disease.

9) Cytokine networks: Although the focus of most research and this paper has been on the

production and action of cytokines on cells of the immune system, it is important to

remember that many of them have effects on other cells and organ systems. A schematic diagram

showing some of the interactions in the cytokine network is presented in Figure 5. In fact, the

cytokine network is quite complex and represents a series of overlapping and inter-related

connections amongst cytokines. Within this network, one cytokine may induce or suppress its

own synthesis, induce or suppress the synthesis of other cytokines, induce or suppress synthesis of

cytokine receptors (both its own and other cytokine Rs), and antagonize or synergize with other

cytokines.





Figure 83: Cytokine networks



15. Immunoregulation

1) The magnitude of an immune response is det

activation of lymphocytes and negative regulato

response. Regulatory mechanis

immune response.

2) Regulation in response to Ag has been discussed previously.

a) Recognition of antigen in the abse

b) Recognition of antigen with CTLA

activation,

c) Cytokines with stimulatory or inh

d) Idiotype/anti-idiotype interac

e) Dose and route of Ag exposure can induce diff

can protect and in another can

Table 6: Immunoglobulin regulation



agnitude of an immune response is determined by the balance between antigen

phocytes and negative regulatory influences that prevent or da

egulatory mechanisms can act at the recognition, activation or effector phases of an

Regulation in response to Ag has been discussed previously.

antigen in the absence of co-stimulation resulting in ener

antigen with CTLA-4 engagement of B7 resulting in do

ulatory or inhibitory activities on immune cells,

ctions leading to stimulation or inhibition of immune responses.

and route of Ag exposure can induce differential Th responses(Table

can protect and in another can tolerate.

: Immunoglobulin regulation

technology 2014

Page 105

ined by the balance between antigen-driven

y influences that prevent or dampen the

recognition, activation or effector phases of an

energy,

own regulation of T cell

inhibition of immune responses.

6) which in one case



3) Regulation by antibody

a) Soluble antibody can compete

activation (Figure 2A). In this case the regulation is

b) In addition antigen antibody co

inhibitory signal to B cells (Figure 2B). H

c) Antibody can also regulate activation (enhance)

this case, Ab binds Ag form

system. Complement activ

(Figure 2C).

Figure 84: Immunoregulation by antibody



Soluble antibody can compete with antigen receptors on B cells and block or prevent B cell

In this case the regulation is occurring at the recognition level.

addition antigen antibody complexes can bind to Fc receptors on B cells, sending an

inhibitory signal to B cells (Figure 2B). Here regulation occurs at the activation level.

Antibody can also regulate activation (enhance) by maintaining a source of antigen for APC. In

ming an immune complex which binds and activated the co

ment activation allows for ligation to the complement rece

: Immunoregulation by antibody

technology 2014

Page 106

lls and block or prevent B cell

rring at the recognition level.

xes can bind to Fc receptors on B cells, sending an

re regulation occurs at the activation level.

a source of antigen for APC. In

plex which binds and activated the complement

nt receptor on the APC



4) Regulation by cytokines

a) Cytokines are positive or negative regulators. They act at many stages of the immune response,

but their activity is dependent upon the other cytokines present in the microenvironment as well as

receptor expression on effector cells. Cytokines regulate the type and extent of the immune

response generated.

5) Regulation by regulatory T cells (Tregs): Regulatory T cells (Tregs) are recently described

populations of cells that can regulate immune responses. They do not prevent initial T cell

activation; rather, they inhibit a sustained response and prevent chronic and potentially damaging

responses. They do not have characteristics of Th1, Th2, or Th17 cells but they can suppress both

Th1 and Th2 responses.

a) Naturally occurring Tregs – The thymus gives rise to CD4+/CD25+/Foxp3+ cells that function as

Tregs. These Tregs suppress immune responses in a cell contact-dependent manner but the

mechanism of suppression has not been established.

b) Induced Tregs – In the periphery some T cells are induced to become Tregs by antigen and either

IL-10 or TGF-β. Tregs induced by IL-10 are CD4+/CD25+/Foxp3- and are referred to as Tr1

cells. These cells suppress immune responses by secretion of IL10. Tregs induced by TGF-β

are CD4+/CD25+/Foxp3+ and are referred to as induced Tregs. These cells suppress by secretion

of TGF-β.

c) CD8+ Tregs – Some CD8+ cells can also be induced by antigen and IL-10 to become a Treg cell.

These cells are CD8+/Foxp3+ and they suppress by a cell contact-dependent mechanism or by

secretion of cytokines. These cells have been demonstrated in vitro but it is not known whether

they exist in vivo.

6) Genetic factors influencing immunoregulation

a) MHC-linked genes help control response to infection. Certain HLA haplotypes are associated



with individuals who are responders or nonresponder, those who are susceptible or resistant.

b) Non-MHC genes are also involved in immunoregulation. An example is a gene related to

macrophage activity encoding a transporter protein involved in transport of nitrite (NO2-) into the

phagolysosome, natural resistance-associated macrophage protein-1 (Nramp1). Polymorphisms in

this gene could change the activity of macrophages.

c) Cytokine, chemokine, and their receptors are involved in immunoregulation as discussed

previously. Polymorphisms in the genes encoding these, in particular the receptors, have been

shown to correlate to susceptibility to infection or generation of autoimmune disease.

16. MHC: genetics and role in transplantation

Histocompatibility (transplantation) antigens: Antigens, on tissues and cells, which determine their

rejection when grafted between two genetically different individuals; Major histocompatibility

(MHC) antigens: Histocompatibility antigens which cause a very strong immune response and are

most important in rejection of organs and tissues; MHC complex: Group of genes on a single

chromosome encoding the MHC antigens; HLA (human leukocyte antigens): MHC antigens of

man (first detected on leukocytes); H-2 antigens: MHC antigens of mouse; Xenograft: Grafts

between members of different species (also known as heterologous or xenogeneic grafts or hetero-

grafts); Allograft: Grafts between two members of the same species (also known as allogeneic

grafts or homo-grafts); Isograft/syngeneic : Grafts between members of the same species with

identical genetic makeup (identical twins or inbred animals); Haplotype: a group of genes on a

single chromosome.

Principles of Transplantation:

An immunocompetent host recognizes the foreign antigens on grafted tissues (cells), and mounts

an immune response, which results in rejection (host -vs- graft reaction). On the other hand if an

immunocompromised host is grafted with foreign immunocompetent lymphoid cells, the

immunoreactive T-cells present in the graft recognize the foreign antigens on the host tissue and

cause their damage (graft -vs- host reaction).



Host-versus-graft-reaction:

The duration of graft survival follows the order, xeno- < allo- < iso- = auto- graft. The time of

rejection also depends on the antigenic disparity between the donors and recipient. While the

MHC antigens are the major contributors in rejection, the minor histocompatibility antigens

also play a significant role. Rejection due to disparity in several minor histocompatibility

antigens may be as quick or quicker than rejection mediated by an MHC antigen. Like in other

immune responses, there is immunological memory and secondary response in graft rejection.

Thus, once a graft is rejected by a recipient, a second graft from the same donor, or a donor

with the same histocompatibility antigens, will be rejected in a much shorter time.

Graft-versus-host (GVH) reaction:

Histoincompatible lymphoid cells when injected into an immunosuppressed host are readily

accepted. However, the immunocompetent T lymphocytes found in the grafted cells recognize

the alloantigens found on the host and proliferate and progressively cause damage to the host

tissues and cells. This condition is known as graft-versus-host (GVH) disease and is often fatal.

Common manifestations of GVH reaction are diarrhea, erythema, weight loss, malaise, fever,

joint pains, etc. and ultimately death.

Figure 85: The human MHC gene complex



The MHC Gene Complex:

The MHC complex contains a number of genes, which control several antigens,most of which

influence allograft rejection. These antigens (and their genes) can be divided into three major

classes: class I, class II and class III. The class I and class II antigens are expressed on cells and

tissues whereas as class III antigens are associated with proteins in serum and other body-fluids

(e.g.C4, C2, factor B, TNF). While antigens from class I and class II gene products play a critical

role in transplantation, those from class III gene products have no direct role in immune

responses that determine graft survival.

Human MHC:

The human MHC is located on chromosome 6.

Class I MHC:

The class I gene complex contains three major loci of highest significance, B, C and A and some

undefined loci of less significance (Figure 1). Each these loci codes for a polypeptide, α-chain

that contains antigenic determinants that are polymorphic (has many alleles). Each α-chain

associates with a β-2 microglobulin molecule (β-chain), encoded by a gene outside the MHC

complex. The α-β-chain complex is expressed on the cell surface as the class-I MHC antigen.

Without a functional β-2 microglobulin chain, the class I antigen will not be expressed on the

cells surface. Individuals with defective a β-2 microglobulin gene do not express any class I

antigen and hence they have a deficiency of cytotoxic T cells.

Class II MHC:

The class II gene complex also contains at least three loci, DP, DQ and DR; each of these loci

codes for one α- and a variable number of ß-chain polypeptides which associate together to form

the class II antigens. Like the class I antigens, the class II antigens are also polymorphic. The

DR locus contains more than one, possibly 4, functional β-chain genes.

MHC Polymorphism: MHC complex is the most polymorphic in the genome. This means that

there is an astonishing allelic diversity found within MHC. In humans, the most conspicuously-

diverse loci, HLA-A, HLA-B, and HLA-DRB1, have roughly 250, 500, and 300 known alleles

respectively. This helps protect the species from extinction that could result from infections and



other diseases. However, it is for this very reason, it is extremely difficult to match the donor and

the recipient.

Mouse MHC:

The mouse MHC is located on chromosome 17.

Class I MHC:

It consists of two major loci, K and D. Unlike the human MHC, the mouse class I gene complex

loci are not together but they are separated by class II and class III genes (Figure 87).

Figure 86: The mouse MHC complex

Class II MHC:

The class II gene complex of mouse contains two loci, A and E each of which code for one α-

and one ß- chain polypeptide, which form one class II molecule. The mouse class II gene

complex is also known as the I-region and the genes in this complex are referred to as Ir

(immune response) genes since they determine the magnitude of immune responsiveness of

different mouse strains to certain antigens. Products of A and E loci are also termed IA and IE

antigens, collectively known as Ia antigens.

MHC

ANTIGENS:

Nomenclature:

HLA specificities are identified by a letter for locus and a number (A1, B5, etc.), and the

haplotypes are identified by individual specificities (e.g., A1, B7, Cw4, DP5, DQ10 DR8).

Specificities which are defined by genomic analysis (PCR), are named with a letter for the locus

and a four digit number (e.g. A0101, B0701, C0401, etc.) Specificities of Mouse MHC (H-2) are

identified by a number.Each strain is homozygous and has a unique haplotype. The MHC



haplotype in these strains is designated by a ‘small’ letter (a, b, d, k, q, s, etc.). For example,

the MHC haplotype of Balb/c, an inbred strain of mouse, is H2d.

Inheritance:

MHC genes are inherited as a group (haplotype), one from each parent. Thus, a heterozygous

human inherits one paternal and one maternal haplotype, each containing three class-I (B, C and

A) and three class II (DP, DQ and DR) loci. A heterozygous individual will therefore inherit a

maximum of 6 class I specificities (Figure 3: top). Similarly, the individual will also inherit DP

and DQ genes and express both parental antigens. Since the class II MHC molecule consists of

two chains (α and β), with some antigenic determinants (specificities) on each chain, and DR

α- and β-chains can associate in cis (both from the same parent) or trans (one from each parent)

combination, an individual can have additional DR specificities (Figure 3: bottom). Also, there

are more than one functional DR β-chain genes (not shown in the figure). Hence, many DR

specificities can be found in any one individual.

Figure 87: Co-dominant expression of MHC antigens

Crossover:

Haplotypes, normally, are inherited intact and hence antigens encoded by different loci are



inherited together (e.g., A2; B27; Cw2; DPw6; DQw9; DRw2). However, on occasions, there is

crossing over between two parental chromosomes, thereby resulting in new recombinant

haplotypes. Thus any specificity encoded by one locus, may combine with specificities from

other loci. This results in a vast heterogeneity in the MHC make-up in a given population.

MHC antigen expression on cells:

MHC antigens are expressed on the cell surface in a co-dominant manner: products of both

parental genes are found on the same cells. However, not all cells express both class I and class

II antigens. While class I antigens are expressed on all nucleated cells and platelets (and red

blood cells in the mouse), the expression of class II antigens is more selective. It is expressed

on B lymphocytes, a proportion of macrophages and monocytes, skin associated (Langerhans)

cells, dendritic cells and occasionally on other cells.

MHC detection by serological test:

The MHC class I antigens are detected by serological assays (Ab and C). Tissue typing sera for

the HLA were obtained, in the past, from multiparous women who were exposed to the child=s

paternal antigens during the parturition and subsequently developed antibodies to these antigens.

More recently they are being produced by the monoclonal antibody technology. With most

laboratories switching to PCR for tissue typing, the use of serology is rapidly diminishing.



Figure 88: Activation of CTL and Mechanism of Allograft Destruction

MHC detection by mixed leukocyte reaction (MLR):

It has been observed that lymphocytes from one donor, when cultured with lymphocytes from an

unrelated donor, are stimulated to proliferate. It has been established that this proliferation is

primarily due to a disparity in the class II MHC (DR) antigens and T cells of one individual

interact with allogeneic class-II MHC antigen bearing cells (B cells, dendritic cells, langerhans

cells, etc.). This reactivity was termed mixed leukocyte reaction (MLR) and has been used for

studying the degree of histocompatibility. In this test, the test lymphocytes (responder cells)are

mixed with irradiated or mitomycin-C treated leukocytes from the recipient, containing B-

lymphocytes and monocytes (stimulator cells).

The cells are culturedfor 4-6 days. The responder T-cells will recognize the foreign class II

antigens found on the donor and undergo transformation (DNA synthesis and enlargement:

blastogenesis) and proliferation (mitogenesis). The T cells that respond to foreign class II

antigens are typically CD4+ TH-1 type cells. These changes are recorded by the addition of

radioactive (tritiated, 3H) thymidine into the culture and monitoring its incorporation into DNA.

Generation of cytotoxic T lymphocytes

Another consequence of the MHC antigen and T cell interaction is the induction of cytotoxic T-

lymphocytes. When T-lymphocytes are cultured in the presence of allogeneic lymphocytes, in

addition to undergoing mitosis (MLR), they also become cytotoxic to cells of the type that

stimulated MLR (Figure 4). Thus, T-lymphocytes of 'x' haplotype cultured over 5-7 days with B

lymphocytes of 'y' haplotype will undergo mitosis and the surviving T-lymphocytes become

cytotoxic to cells of the 'y' haplotype. The cytotoxic T-lymphocytes (CTL) primarily recognize

class-I antigens and are CD8+.

Allograft Rejection



The clinical significance of the MHC is realized in organ transplantation. Cells and tissues are

routinely transplanted as a treatment for a number of diseases. However, reaction of the host

against alloantigens of the graft (HVG) results in its rejection and is the major obstacle in organ

transplantation. The rejection time of a graft may vary with the antigenic nature of the graft and

the immune status of the host and is determined by the immune mechanisms involved (Figure 5).

Hyper-acute rejection:

This occurs in instances when the recipient has preformed high titer antibodies. A graft may

show signs of rejection within minutes to hours due to immediate reaction of antibodies and

complement.

Accelerated (2nd set; secondary) rejection:

Transplantation of a second graft, which shares a significant number of antigenic determinants

with the first one, results in a rapid (2-5 days) rejection. This is due to presence of T-

lymphocytes sensitized during the 1st graft rejection. Accelerated rejection is mediated by

immediate production of lymphokines, activation of monocytes and macrophages and induction

of cytotoxic lymphocytes.

Acute (1st set; primary) rejection:

The normal reaction, which follows the first grafting of a foreign transplant, takes 1-3 weeks. This

is known as acute rejection and is mediated by T lymphocytes sensitized to class-I and class-II

antigens of the allograft, lymphokines, activated of monocytes and macrophages.

Chronic rejection:

Some grafts may survive for months or even years, but suddenly exhibit symptoms of rejection.

This is referred to as chronic rejection, the mechanism of which is not entirely clear. The

hypotheses are that this may be due infection, causes which led to failure of the first organ, loss of

tolerance induced by the graft, etc.

Fetus as an Allograft: The fetus in an out-bred mammalian species bears antigens derived from

both the father and the mother. Thus, truly, the fetus is an allograft and the mother should



normally recognize the fetus as foreign and reject the fetus. Nonetheless, such rejections seldom

occur. Thus, mammals have adapted in a way that allows implantation of their embryos in the

mother's womb and their subsequent survival. There are multiple mechanisms that play a role, of

which the most important being the unique structure and function of placenta.

Immunologically privileged sites and tissues: There are certain locations in the body in which

allografts are not readily rejected. These include the brain, anterior chamber of the eye, testis,

renal tubule, uterus, etc. This stems from the fact that such sites may lack of good lymphatic

drainage. Also, such tissues may express molecules such as Fas ligand that kills any immune cell

that may come in contact with these tissues. Additionally, such tissues, may have other immune

suppressor mechanisms. Similarly, there are some tissues that can be transplanted without

matching and without being rejected. Such tissues are called immunologically privileged tissues.

Corneal graft is an excellent example that enjoys the highest success rate of any form of organ

transplantation. The low incidence of graft rejection is impressive despite the fact that HLA

antigen matching of donor and recipient is not normally performed. There are many explanations

as to why such grafts are accepted. The avascularity of the graft bed prevents corneal

alloantigens from reaching the regional lymphoid tissues. Also, the corneal antigens may be

masked. Together, such mechanisms fail to activate the immune system of the recipient.

Procedures to Enhance Graft Survival

In clinical practice, the most successful transplantation programs have been with kidneys and

corneas. However, other organs are being transplanted with increasing frequency and success.

The success in these programs has been due to a better understanding of immunological

mechanisms, definition of MHC antigens and development of more effective

immunosuppressive agents.

Donor selection:

Based on extensive experiences with renal transplants certain guidelines can be followed in

donor selection and recipient preparation for most organ transplants. The most important in

donor selection is the MHC identity with the recipient; an identical twin is the ideal donor.

Grafts from HLA-matched sibling have 95-100% chance of success. One haplotype-identical

parent or sibling must match at HLA D region. A two-haplotype-distinct donor with reasonable



match for D-region antigen can also be used. Organ from two or one DR matched cadaver has

been used also with some success. In every case, an ABO-compatibility is essential.

Recipient preparation:

The recipient must be screened for donor-specific anti-HLA antibodies and be negative, must be

infection free and must not be hypertensive. One to five transfusions of 100-200 ml whole blood

from the donor at 1-2 week intervals improves the graft survival and is practiced when possible.

Immunosuppression:

Immunosuppressive therapy is most essential part of allo-transplantation. The most recent and

effective family of agents is cyclosporine, tacrolimus (formerly, FK-506; Prograft®) and

rapamycin (Rapmune®). Cyclosporine and tacrolimus inhibits IL-2 synthesis following Ag-

receptor binding whereas rapamycin interferes with signal transduction following IL2-IL2R

interaction. Thus, all these three agents block T cell proliferation in response to antigen. Other

chemical agents used to prevent graft rejection and their modes of action have been listed in Table

2. Whole body irradiation is used in leukemia patients before bone marrow transplantation.

Antisera against T cells (anti-thymocyte globulin: ATG) or their surface antigens (CD3, CD4,

CD45on activated T-cells, CD25: IL-2 receptors) are being used also to achieve

immunosuppression.

Strategies for bone marrow transplantation:

In bone marrow transplantation, the most crucial factor in donor selection is class II MHC

compatibility. Once again, an identical twin is the ideal donor. From poorly matched grafts, T

lymphocytes can be removed using monoclonal antibodies. The recipient must be

immunosuppressed. Malignant cells must be eliminated from the recipient blood (in case of

blood-borne malignancies). Methotrexate, cyclosporin and prednisone are often used to control

GVH disease.

Other grafts:



Corneal grafts do not contain D region antigens and consequently survival is frequent. Small

grafts are better and their survival is improved by corticosteroid use.

Skin allograft have very poor success rate and immunosuppressive therapy is relatively

ineffective. Nevertheless, they are often used to provide a temporary covering to promote healing

in severe skin damage. Indeed, there will be no rejection, if the host and donor are perfectly

matched (identical twins) or the recipient is tolerant to the donor MHC antigens (bone marrow

chimeras).

17. 17. 17. 17. HypersensitivityHypersensitivityHypersensitivityHypersensitivity reactireactireactireactioooonsnsnsns

Hypersensitivity refers to excessive undesirable (damaging, discomfort producing and sometimes

fatal) reactions produced by the normal immune system. Hypersensitivity reactions require a pre-

sensitized (immune) state of the host. Hypersensitivity reactions can be divided into four types:

type I, type II, type III and type IV, based on the mechanisms involved and time taken for the

reaction. Frequently, a particular clinical condition (disease) may involve more than one type of

reaction.

Type I HypersensitiType I HypersensitiType I HypersensitiType I Hypersensitivvvviiiitytytyty

It is also known as immediate or anaphylactic hypersensitivity. The reaction may involve skin

(urticaria and eczema), eyes (conjunctivitis), nasopharynx (rhinorrhea, rhinitis), bronchopulmonary

tissues (asthma) and gastrointestinal tract (gastroenteritis). The reaction may cause from minor

inconvenience to death.

Type I hypersensitivity is mediated by IgE. The primary cellular component in this

hypersensitivity is mast cell or basophil. The reaction is amplified and/or modified by platelets,

neutrophils and eosinophils. A biopsy of the reaction site demonstrates mainly mast cells and

eosinophils. The mechanism of reaction involves preferential production of IgE, in response to

certain antigens, often called allergens (Figure 1). The precise mechanism as to why some

individuals are more prone to type-I hypersensitivity is not clear. However, it has been shown

that such individuals preferentially produce more of TH2 cells that secrete IL-4, IL-5 and IL-13

which in turn favor IgE class switch. IgE has very high affinity for its receptor (Fcε; CD23) on

mast cells and basophils. A subsequent exposure to the same allergen cross links the cell-

bound IgE and triggers the release of various pharmacologically active substances (Figure 1).



Cross-linking of IgE Fc-receptor is important in mast cell triggering. Mast cell degranulation is

preceded by increased Ca++ influx, which is a crucial process; ionophores which increase

cytoplasmic Ca++ also promote degranulation of mast cells, whereas, agents which deplete

cytoplasmic Ca++ suppress degranulation.

The agents released from mast cells and their effects are listed in Table 1. Mast cells may

be triggered by other stimuli such as exercise, emotional stress, chemicals (e.g., photographic

developing medium, calcium ionophores, codeine, etc.), anaphylotoxins (e.g., C4a, C3a, C5a,

etc.). These reactions mediated by agents without IgE-allergen interaction are not typical

hypersensitivity reactions, although they produce the same symptoms.

The reaction is amplified by PAF (platelet activation factor) that causes platelet aggregation and

release of histamine, heparin and vasoactive amines. Eosinophil chemotactic factor of anaphylaxis

(ECF-A) and neutrophil chemotactic factors attract eosinophils and neutrophils, respectively,

which release various hydrolytic enzymes that cause necrosis. Eosinophil may also control the

local reaction by releasing arylsulphatase, histaminase, phospholipase-D and prostaglandin-E,

although this role of eosinophils is now in question.

Cyclic nucleotides appear to play a significant role in the modulation of immediate

hypersensitivity reaction, although their exact function is ill understood. Substances which alter

cAMP and cGMP levels significantly alter the allergic symptoms. Thus, substances that increase

intracellular cAMP seem to relieve allergic symptoms, particularly broncho-pulmonary ones, and

are used therapeutically. Conversely, agents that decrease cAMP or stimulate cGMP aggravate

these allergic conditions.

Diagnostic tests for immediate hypersensitivity include skin (prick and intradermal) tests resulting

in wheal and flare reaction, measurement of total IgE and specific IgE antibodies against the

suspected allergens. Total IgE and specific IgE antibodies are measured by a modification of

enzyme immunoassay (ELISA). Increased IgE levels are indicative of atopic condition, although

IgE may be elevated in some non atopic diseases (e.g., myelomas, helminthic infection, etc.).



There appears to be a genetic predisposition for atopic diseases and there is evidence for

HLA (A2) association.

Symptomatic treatment is achieved with antihistamines that block histamine receptors.

Chromolyn sodium inhibits mast cell degranulation, probably, by inhibiting Ca++ influx. Late

onset allergic symptoms, particularly bronchoconstriction which is mediated by leukotrienes are

treated with leukotriene receptor blockers (Singulair, Accolate, Lukast) or inhibitors of

cyclooxygenase pathway (Zileutoin). Symptomatic, although short-term, relief from

bronchoconstriction is provided by bronchodilators (inhalants) such as isoproterenol derivatives

(Terbutaline, Albuterol). Thophylline elevates cAMP by inhibiting cAMP-phosphodiesterase and

inhibits intracellular Ca++ release is also used to relieve bronchopulmonary symptoms.

IgG antibodies against the Fc portions of IgE that binds to mast cells has been approved for

treatment of certain allergies, as it can block mast cell sensitization. Hyposensitization

(immunotherapy or desensitization) is another treatment modality which is successful in a

number of allergies, particularly to pollen. The mechanism is not clear, but there is a correlation

between appearance of IgG (blocking) antibodies and relief from symptoms. Suppressor T cells

that specifically inhibit IgE antibodies may play a role.

Type II Hypersensitivity

It is also known as cytotoxic hypersensitivity and may affect a variety of organs and tissues.

The antigens are normally endogenous, although exogenous chemicals (haptens) that can attach to

cell membranes can also lead to type II hypersensitivity. Drug-induced hemolytic anemia,

granulocytopenia and thrombocytopenia are such examples. The reaction time is minutes to

hours. It is mediated, primarily, by antibodies of IgM or IgG class and complement (Figure 92).

Phagocytes a n d K ( killer) cells m ay a l s o play a role.



Figure Figure Figure Figure 89898989: types of hypersensitivity reaction : types of hypersensitivity reaction : types of hypersensitivity reaction : types of hypersensitivity reaction

The lesion contains antibody, complement and neutrophils. Diagnostic tests include detection of

circulating antibody against tissues involved and the presence of antibody and complement in the

lesion (biopsy) by immunofluorescence. The staining pattern is normally smooth and linear, such

as that seen in Goodpasture’s nephritis (renal and lung basement membrane) and pemphigus

(skin intercellular protein, desmosome).

Type III HType III HType III HType III Hyyyypersensitivitypersensitivitypersensitivitypersensitivity

It is also known as immune complex hypersensitivity. The reaction may be general (e.g., serum

sickness) or may involve individual organs including skin (e.g., systemic lupus erythematosus,

Arthus reaction), kidneys (e.g., lupus nephritis), lungs (e.g., aspergillosis), blood vessels (e.g.,

polyarteritis), joints (e.g., rheumatoid arthritis) or other organs. This reaction may be the

pathogenic mechanism of diseases caused by many microorganisms.

The reaction may take 3-10 hours after exposure to the antigen (as in Arthus reaction). It is

mediated by soluble immune complexes. They are mostly of IgG class, although IgM may also be

involved. The antigen may be exogenous (chronic bacterial, viral or parasitic infections), or

endogenous (non-organ specific autoimmunity: e.g., systemic lupus eythematosus-SLE). The

antigen is soluble and not attached to the organ involved. Primary components are soluble

immune complexes and complement (C3a, 4a and 5a). The damage is caused by platelets and

neutrophils (Figure 3).



The lesion contains primarily neutrophils and deposits of immune complexes and

complement. Macrophages infiltrating in later stages may be involved in the healing process.

Figure 90: Mechanism of damage in type-III hypersensitivity

The affinity of antibody and size of immune complexes are important in production of disease and

determining the tissue involved. Diagnosis involves examination of tissue biopsies for deposits of

Ig and complement by immunofluorescence. The immunofluorescent staining in type III

hypersensitivity is granular (as opposed to linear in type II: Goodpasture). Presence of immune

complexes in serum and depletion in complement level are also diagnostic. Treatment includes

anti-inflammatory agents.

Type IV HypersensitivityType IV HypersensitivityType IV HypersensitivityType IV Hypersensitivity

It is also known as cell mediated or delayed type hypersensitivity. The classical example of this

hypersensitivity is tuberculin (Montoux) reaction that peaks 48 hours after the injection of

antigen (PPD or old tuberculin). The lesion is characterized by induration and erythema.



Figure 91: Mechanisms of damage in delayed hypersensitivity

Type IV hypersensitivity is involved in the pathogenesis of many autoimmune and infectious

diseases (tuberculosis, leprosy, blastomycosis, histoplasmosis, toxoplasmosis, leishmaniasis, etc.)

and granulomas due to infections and foreign antigens. Another form of delayed

hypersensitivity is contact dermatitis (poison ivy, chemicals, heavy metals, etc.) in which the

lesions are more papular. Type IV hypersensitivity can be classified into three categories

depending on the time of onset and clinical and histological presentation (Table 7).

Mechanisms of damage in delayed hypersensitivity include T lymphocytes and monocytes and/or

macrophages. The pathogenesis is triggered primarily by helper T (TH1) cells that secrete

cytokines that activate and recruit macrophages, which cause the bulk of the damage (Figure 3).

The delayed hypersensitivity lesions mainly contain monocytes and T cells.

Major lymphokines involved in delayed hypersensitivity reaction include monocyte chemotactic

factor, interleukin-2, interferon-γ, TNF α, etc. Diagnostic tests in vivo include delayed

cutaneous reaction (e.g. Montoux test) and patch test (for contact dermatitis). In vitro tests

for delayed hypersensitivity include mitogenic response, lympho-cytotoxicity and IL-2

production.



17. Tolerance and Autoimmunity

17.1. Tolerance Introduction:

Tolerance refers to the specific immunological non-reactivity to an antigen resulting from

a previous exposure to the same antigen. While the most important form of tolerance is non-

reactivity to self antigens, it is possible to induce tolerance to non-self (foreign) antigens. When

an antigen induces tolerance, it is termed tolerogen.

Tolerance to self antigens: We normally do not mount a strong immune response against our

own (self) antigens, a phenomenon called self-tolerance. When the immune system

recognizes a self antigen and mounts a strong response against it, autoimmune disease develops.

Nonetheless, the immune system has to recognize self-MHC to mount a response against a

foreign antigen. Thus, the immune system is constantly challenged to discriminate self vs non-

self and mediate the right response.

Induction of tolerance to non-self : Tolerance can also be induced to non-self (foreign) antigens

by modifying the antigen, by injecting the antigen through specific routes such as oral,

administering the antigen when the immune system is developing, etc. Certain bacteria and

viruses have devised clever ways to induce tolerance so that the host does not kill these microbes.

Ex: Patients with lepromatous type of leprosy do not mount an immune response against

Mycobacterium leprae.

Tolerance to tissues and cells:

Tolerance to tissue and cell antigens can be induced by injection of hemopoietic (stem) cells in

neonatal or severely immunocompromised (by lethal irradiation or drug treatment) animals. Also,

grafting of allogeneic bone marrow (or thymus) in early life results in tolerance to the donor type

cells and tissues. Such animals are known as chimeras. These findings are of significant

importance in bone marrow grafting.

Immunologic features of tolerance:



Tolerance is different from non-specific immunosuppression, and immunodeficiency. It is an

active antigen dependent process in response to the antigen. Like immune response, tolerance is

specific and like immunological memory, it can exist in T-cell, B cells or both and like

immunological memory, tolerance at the T cell level is longer lasting than tolerance at the B cell

level.

Induction of tolerance in T cells is easier and requires relatively smaller amounts of tolerogen

than tolerance in B cells. Maintenance of immunological tolerance requires persistence of antigen.

Tolerance can be broken naturally (as in autoimmune diseases) or artificially (as shown in

experimental animals, by x-irradiation, certain drug treatments and by exposure to cross reactive

antigens).

Tolerance may be induced to all epitopes or only some epitopes on an antigen and tolerance

to a single antigen may exist at B cell level or T cells level or at both levels.

Mechanisms of tolerance induction:

The exact mechanism of induction and maintenance of tolerance is not fully understood.

Experimental data, however, point to several possibilities.

Clonal deletion: T and B lymphocytes during development come across self antigens and such

cells undergo clonal deletion through a process known as apoptosis or programmed cell death.

For example, T cells that develop in the thymus first express neither CD4 nor CD8. Such cells

next acquire both CD4 and CD8 called double-positive cells and express low levels of αβ

TCR. Such cells undergo positive selection after interacting with class I or class II MHC

molecules expressed on cortical epithelium. During this process, cells with low affinity for

MHC are positively selected. Unselected cells die by apoptosis, a process called “death by

neglect”. Next, the cells loose either CD4 or CD8. Such T cells then encounter self-

peptides presented by self MHC molecules expressed on dendritic cells. Those T cells with high

affinity receptors for MHC + self-peptide undergo clonal deletion also called negative selection

through induction of apoptosis. Any disturbance in this process can lead to escape of auto-

reactive T-cells that can trigger autoimmune disease. Likewise, differentiating early B cells

when they encounter self-antigen, cell associated or soluble, undergo deletion. Thus, clonal

deletion plays a key role in ensuring tolerance to self antigen.



Peripheral tolerance: The clonal deletion is not a fool proof system and often T and B cells fail

to undergo deletion and therefore such cells can potentially cause autoimmune disease once they

reach the peripheral lymphoid organs. Thus, the immune system has devised several additional

check points so that tolerance can be maintained.

Activation-induced cell death: T cells upon activation not only produce cytokines or carryout

their effector functions but also die through programmed cell death or apoptosis. In this process,

the death receptor (Fas) and its ligand (FasL) play a crucial role. Thus, normal T cells express

Fas but not FasL. Upon activation, T cells express FasL which binds to Fas and triggers

apoptosis by activation of caspase-8. The importance of Fas and FasL is clearly demonstrated by

the observation that mice with mutations in Fas (lpr mutation) or FasL (gld mutation) develop

severe lymphoproliferative and autoimmune disease and die within 6 months while normal mice

live up to 2 years. Similar mutations in these apoptotic genes in humans leads to a

lymphoproliferative disease called autoimmune lymphoproliferative syndrome (ALPS).

Clonal anergy: Auto-reactive T cells when exposed to antigenic peptides on antigen presenting

cells (APC) that do not possess the co-stimulatory molecules CD80 (B7-1) or CD86 (B7-2)

become anergic (nonresponsive) to the antigen. Also, while activation of T cells through CD28

triggers IL-2 production, activation of CTLA4 leads to inhibition of IL-2 production and anergy.

Also, B cells when exposed to large amounts of soluble antigen down-regulate their surface IgM

and become anergic. These cells also up-regulate the Fas molecules on their surface. An

interaction of these B cells with Fas-ligand bearing T cells results in their death via apoptosis.

Clonal ignorance: T cells reactive to self-antigen not represented in the thymus will mature and

migrate to the periphery, but they may never encounter the appropriate antigen because it is

sequestered in inaccessible tissues. Such cells may die out for lack of stimulus. Auto-reactive B

cells, that escape deletion, may not find the antigen or the specific T-cell help and thus not be

activated and die out.

Anti-idiotype antibody: These are antibodies that are produced against the specific idiotypes of

other antibodies. Anti-idiotypic antibodies are produced during the process of

tolerization and have been demonstrated in tolerant animals. These antibodies may prevent the



B cell receptor from interacting with the antigen.

Regulatory T cells (Formerly called suppressor cells): Recently, a distinct population of T cells

has been discovered called regulatory T cells. Regulatory T cells come in many flavors,

but the most well characterized include those that express CD4+ and CD25+. Because

activated normal CD4 T cells also express CD25, it was difficult to distinguish regulatory T

cells and activated T cells. The latest research suggests that regulatory T cells are defined by

expression of the forkhead family transcription factor Foxp3. Expression of Foxp3 is required for

regulatory T cell development and function. The precise mechanism/s through which regulatory T

cells suppress other T cell function is not clear. One of the mechanisms include the production

of immunosuppressive cytokines such as TGF-β and IL-10. Genetic mutations in Foxp3 in

humans leads to development of a severe and rapidly fatal autoimmune disorder known as

Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX)syndrome. This

disease provides the most striking evidence that regulatory T cells play a critical role in preventing

autoimmune disease.

Table 7: Factors which determine induction of immune response or tolerance following challenge

with antigen.

determinant favor immune response favor tolerance

physical form of antigen

large, aggregated, complex

molecules;

soluble, aggregate-free, relatively

smaller, less complex molecules, Ag

not processed by APC or processed

route of Ag administration sub-cutaneous or intramuscular oral or sometimes intravenous

dose of antigen

optimal dose very large (or sometime very small)

dose

age of responding animal

older and immunologically

Newborn (mice), immunologically

differentiation state of

cells

fully differentiated cells;

memory T and memory B cells

relatively undifferentiated: B cells

with only IgM (no IgD), T cells (e.g.

cells in thymic cortex)

17.2. Autoimmunity



Definition:

Autoimmunity can be defined as breakdown of mechanisms responsible for self-tolerance and

induction of an immune response against components of the self. Such an immune response may

not always be harmful (e.g., anti-idiotype antibodies or recognition of self-MHC molecules).

However, in numerous (autoimmune) diseases it is well recognized that products of the immune

system cause severe damage to the self.

Effector mechanisms in autoimmune diseases:

Both antibodies and effector T cells and their products can be involved in the damage in

autoimmune diseases.

General classification:

Autoimmune diseases are generally classified on the basis of the organ or tissue involved. These

diseases may fall in an organ-specific category in which the immune response is directed against

antigen(s) associated with the target organ being damaged or a non-organ-specific (also sometimes

referred to as systemic) in which the antibody is directed against an antigen or many antigens not

associated with the target organ and the disease is seen throughout the body (Table 1). The

antigen involved, in most autoimmune diseases is evident from the name of the disease (Table 1).

GenetGenetGenetGenetiiiicccc prepreprepreddddispositionispositionispositionisposition forforforfor autoimmautoimmautoimmautoimmuuuunity:nity:nity:nity:

Studies in mice and observations in humans suggest a genetic predisposition for autoimmune

diseases. Association between certain HLA types and autoimmune diseases has been noted

(HLA: B8, B27, DR2, DR3, DR4, DR5 etc.).

EtiologyEtiologyEtiologyEtiology ofofofof aaaautoimmunityutoimmunityutoimmunityutoimmunity disease:disease:disease:disease:

The exact etiology of autoimmune diseases is not known. However, various theories have been

offered. These include sequestered antigen, escape of auto-reactive clones, loss of Regulatory T

cells, cross-reactive antigens including exogenous antigens (pathogens) and altered self antigens

(chemical and viral infections).

Sequestered antigen: Lymphoid cells may not be exposed to some self-antigens during their

differentiation, because they may be late-developing antigens or may be confined to specialized

organs (e.g., testes, brain, eye, etc.). A release of antigens from these organs, resulting from

accidental traumatic injury or surgery, can result in the stimulation of an immune response and



6

initiation of an autoimmune disease.

Escape of auto-reactive clones: The negative selection in the thymus may not be fully

functional to eliminate self reactive cells. Not all self antigens may be represented in the thymus

or certain antigens may not be properly processed and presented.

Lack of regulatory T cells: There are fewer regulatory T-cells in many autoimmune diseases.

Cross reactive antigens: Antigens on certain pathogens may have determinants which cross react

with self antigens and an immune response against these determinants may lead to effector cells

or antibodies against tissue antigens. Post streptococcal nephritis and carditis, anticardiolipin

antibodies during syphilis and association between Klebsiella and ankylosing spondylitis are

examples of such cross reactivity.

Diagnosis:Diagnosis:Diagnosis:Diagnosis:

Diagnosis of autoimmune diseases is based on symptoms and detection of antibodies (and/or very

rarely T cells) reactive against antigens of tissues and cells involved. Antibodies against

cell/tissue-associated antigens are detected by immunofluorescence. Antibodies against soluble

antigens are normally detected by ELISA or radioimmunoassay (see table above). In some

cases, a biological /biochemical assay may be used (e.g., Graves diseases, pernicious anemia).

Treatment:

The goals of treatment of autoimmune disorders are to reduce symptoms and control the

autoimmune response while maintaining the body's ability to fight infections. Treatments vary

widely and depend on the specific disease and symptoms: Anti-inflammatory (corticosteroid)

and immunosuppressive drug therapy (such as cyclophosphamide, azathioprine, cyclosporine )

is the present method of treating autoimmune diseases. Extensive research is being carried out

to develop innovative treatments which include: anti-TNF alpha therapy against arthritis, feeding

antigen orally to trigger tolerance, anti-idiotype antibodies, antigen peptides, anti-IL2 receptor



antibodies, anti-CD4 antibodies, anti-TCR antibodies, etc.

Models of autoimmune diseases:

There are a number of experimental and natural animal models for the study of autoimmune

diseases. These experimental models include experimental allergic encephalomyelitis(EAE)

which mimics Multiple Sclerosis, experimental thyroiditis, adjuvant induced arthritis, etc.

Naturally occurring models of autoimmune diseases include hemolytic anemia in NZB mice,

systemic lupus erythematosus in NZB/NZW (BW), BXSB and MRL lpr/lpr mice and diabetes in

NOD (non-obese diabetic) mice.

18. Tumor Immunology

Malignant Transformation:

The proliferation of normal cells is carefully regulated. However, such cells when exposed to

chemical carcinogens, irradiation and certain viruses may undergo mutations leading to their

transformation into cells that are capable of uncontrolled growth, producing a tumor or neoplasm.

A tumor may be

1) Benign, if it is not capable of indefinite growth and the host survives.

2) Malignant, if the tumor continues to grow indefinitely and spreads (metastasizes), eventually

killing the host.

This uncontrolled growth may be due to upregulation of oncogenes (cancer-inducing genes)

and/or downregulation of tumor suppressor genes (that normally inhibit tumor growth often by

inducing cell death).

Evidence for existence of an immune response against tumors

The following criteria serve as evidence that tumors can elicit an immune response.

1. Certain tumors regress spontaneously (e.g., melanomas, neuroblastomas).suggesting an

immunological response.

2. Tumors that have severe mononuclear cell infiltration have a better prognosis than those that

lack it.

3. Some tumor metastases regress after removal of primary tumor which reduces the tumor



load, thereby inducing the immune system to kill the residual tumor..

4. Although chemotherapy leads to rejection of a large number of tumor cells, the few tumor cells

that evade the action of the drugs can outgrow and kill the host. However, the immune system

may be able to mount an attack against the few tumor cells that are spared by the

chemotherapeutic agent.

5. There is an increased incidence of malignancies in immunodeficient patients such as AIDS

patients who are susceptible to Kaposi sarcoma and transplant patients who are susceptible to

Epstein Barr virus (EBV)- induced lymphoma.

6. Tumor-specific antibodies and T lymphocytes (detected in cytotoxicity and proliferative

response assays) have been observed in patients with tumors.

7. The young and the old population have an increased incidence of tumors. These members of the

population often have an immune system that is compromised.

8. Hosts can be specifically immunized against various types of tumors demonstrating tumor Ags

can elicit an immune response.

Tumor antigens

Tumorigenesis may lead to expression of new antigens or alteration in existing antigens

that are found on normal cells. These antigens may include membrane receptors,

regulators of cell cycle and apoptosis, or molecules involved in signal transduction

pathways. There are 2 main types of tumor antigens.

1. Tumor-specific transplantation antigens (TSTA) which are unique to tumor cells and not

expressed on normal cells. They are responsible for rejection of the tumor.

2. Tumor associated transplantation antigens (TATA) that are expressed by tumor cells and

normal cells.

Although chemical- , UV- or virus-induced tumors express neo-antigens, majority of the tumors are

often weakly immunogenic or non-immunogenic. In most cases, tumor-specific transplantation

Ags cannot be identified easily. Also, some of these antigens may be secreted while others include

membrane-associated molecules.

Tumor associated transplantation antigens (TATA)

The majority of tumor Ags are the tumor associated transplantation antigens (TATA). They may

be expressed at higher levels on tumor cells when compared to normal cells. Alternatively, they



may be expressed only during development of cells and lost during adult life but re-expressed in

tumors. These include the tumor-associated developmental Ags (TADA) and tumor-associated

viral Ags (TAVA).

Tumor-associated developmental Ags (TADA) or Onco-fetal antigens

These include alpha-fetoprotein (AFP) and carcino-embryonic antigen (CEA) found secreted in

the serum. AFP is found in patients with hepatocellular carcinoma whereas CEA is found in

colon cancer. These are important in diagnosis.

Virus-induced tumors:

Viruses that cause tumors include

DNA viruses:

1. Papova (papilloma, polyoma) viruses. Ex. Papilloma virus causes cervical cancer.

2. Hepatitis virus: Hepatitis B virus causes hepatocellular cancer.

3. Adenoviruses

RNA viruses:

Retroviruses: Human T-lymphotropic viruses (HTLV-I and HTLV-II) causes Adult T cell

leukemia.

Virus-induced tumors express tumor-associated viral Ags (TAVA). These are cell surface

antigens that are distinct from antigens on the virion itself. However, these transplantation-

associated viral Ags are shared by all tumors induced by the same virus, regardless of tissue

origin of the tumor or animal in which the tumor exists.

Chemically-induced tumors

Chemically-induced tumors are different from virally-induced tumors in that they are extremely

heterogeneous in their antigenic characteristics. Thus, any two tumors induced by the same

chemical, even in the same animal, rarely share common tumor specific antigens. These unique

antigens on chemically-induced tumors are referred to as tumor- specific transplantation antigens

(TSTA).

Syngeneic, Allogeneic and Xenogeneic Tumors:

A tumor that grows in an animal strain will also grow in another animal belonging to the same

inbred strain obtained by repeated brother-sister matings. These animals express the same MHC

molecules and are referred to as syngeneic. However, most normal animal populations are

allogeneic and have various MHC haplotypes. Thus, a tumor transferred from one animal to

another animal belonging to an outbred strain is rejected because of the allo-MHC rather than the



TSTA. A tumor transferred from an animal belonging to one species to another animal

belonging to a different species is rapidly rejected because the animals are xenogeneic.

Immune rImmune rImmune rImmune reeeesponse to tsponse to tsponse to tsponse to tuuuumorsmorsmorsmors::::

Evidence for immunity against malignancy comes mostly from experimental studies, wherein mice

were immunized by administering irradiated tumor cells or following removal of a primary tumor

challenged with the same live tumor. These animals were found to be resistant to rechallenge with

the same live tumor. While Abs may develop against few cancers, cell-mediated immunity plays a

critical role in tumor rejection. Thus, immunity can be transferred, in most cases, from an animal,

in which a tumor has regressed, to a naïve syngeneic recipient by administration of T

lymphocytes. The T helper (Th) cells recognize the tumor Ags that may be shed from tumors and

internalized, processed and presented in association with class II MHC on antigen presenting cells.

These Th cells when activated will produce cytokines. Thus, the Th cells provide help to B cells

in Ab production. The cytokines such as IFN-γ may also activate macrophages to become

tumoricidal. Furthermore, the Th cells also provide help to tumor-specific cytotoxic T cells (CTL)

by inducing their proliferation and differentiation. The CTL recognize tumor Ags in the context

of class I MHC and mediate tumor cell lysis. In tumors that exhibit decreased MHC Ags, natural

killer (NK) cells are important in mediating tumor rejection.

HowHowHowHow tumors evade immune systtumors evade immune systtumors evade immune systtumors evade immune systeeeem:m:m:m:

According to the Immune Surveillance Theory, cancer cells that arise in the body are eliminated

by the immune system. However, due to impaired immune reactivity, the cancer cells escape

destruction.

Tumors evade immune recognition by several mechanisms. Some tumors may evade the

immune system by secreting immunosuppressive molecules such as interleukin-10 (IL-10) or

transforming growth factor-beta (TGF-β) and others may induce regulatory cells particularly

the CD4+CD25+ FoxP3+ T regulatory cells or myeloid derived suppressor cells (MDSC)

which have both granulocyte and macrophage markers (Gr- 1+CD11b+). Also, some tumors

may shed their antigens which in turn may interact and block antibodies and T cells from

reacting with the tumor cells. Tumors may not express neo-antigens that are immunogenic or

they may fail to express co-stimulatory molecules required for the activation of T cells. In

addition, certain tumors are known to lack or be poor expressers of MHC antigen. Such



tumors are however, susceptible to NK cell cytotoxicity. Another reason for failure of

immune surveillance may be the fact that in the early development of a tumor, the amount of

antigen may be too small to stimulate the immune system (low dose tolerance) or due to the

rapid proliferation of malignant cells (high dose tolerance), the immune system is quickly

overwhelmed. Tumor cells may express the death inducing ligand, FasL (CD95L) whereas the

T cells express the death receptor, Fas (CD95), thereby leading to killing of the T cells.

However, CTL have been shown to express FasL and some tumors may express Fas.

ImmunothImmunothImmunothImmunotheeeerapyrapyrapyrapy

Immunotherapy has been used as a novel mode to treat cancer. Both active and passive means of

stimulating the non-specific and specific immune system have been employed, in some cases with

significant success.

1) Active Immunotherapy: Wherein the host actively participates in mounting an

immune response a) Specific activation using vaccines:

i) Hepatitis B vaccine useful against development of hepatocellular cancer.

ii) Human Papilloma virus (HPV) vaccine (Gardasil) has been successfully used to prevent

cervical cancers

b) Nonspecific activation which results in stimulation of generalized immune response is

achieved by immunization with:

i) Bacillus Calmette-Guerin (BCG) mycobacteria. ii) Corynebacterium parvum

These microbes lead to activation of macrophages which are tumoricidal.

2) Passive Immunotherapy: This involves transfer of preformed Abs, immune cells and other

factors into the hosts.

a) Specific: Preformed Abs or CTL directed against tumor Ags are used in the treatment of

tumors i) Antibodies against tumor Ags (e.g. Abs against Her2/Neu for treatment of

breast cancer)

ii) Abs against interleukin-2 receptor (IL-2R) are used in the treatment of Human T lymphotropic

virus (HTLV-1)-induced adult T cell leukemia as this virus infects T cells and leads to production

of IL-2 that binds to IL-2R and induces the T cell proliferation.

iii) Abs against CD20 expressed on all B cells are used in the treatment of non Hodgkin’s

B cell lymphoma.

These Abs bind to tumor Ags on the cell surface and activate complement (C’) to

mediate tumor cell lysis. In addition, Fc receptor bearing cells such as NK cells,

macrophages and granulocytes may bind to the Ag-Ab complexes on tumor cell surface



and mediate tumor cell killing through Ab-dependent cell-mediated cytotoxicity.

iv) Abs conjugated to toxins, radioisotopes and anti-cancer drugs have also been used. These enter

the cells and inhibit protein synthesis because of the toxin. e.g. anti-CD20 conjugated to

Pseudomonas toxin or ricin toxin has been used in the treatment of B cell tumors.

There are several problems with the use of Abs

(1) Abs are not efficient because the tumor Ags are associated with class I MHC Ags.

(2) The tumors may shed Ag or Ag-Ab complexes. Thus, immune cells cannot mediate tumor

destruction.

(3) Some Abs may not be cytotoxic.

(4) Abs may bind nonspecifically to immune cells expressing the Fc receptors which include NK

cells, B cells, macrophages and granulocytes without binding to tumor cells.

v) Dendritic cells pulsed with tumor Ags may be administered which can present tumor Ags in the

context of class II MHC to tumor-specific Th cells. As tumor Ags are usually not known,

tumor lysates are used. The Th cells may in turn produce cytokines which lead to development

of CTL activity. On the other hand, the dendritic cells may be transfected with the gene for

tumor Ags, in which case, the Ags will associate with the Class I MHC and elicit a CTL

response.

b) Nonspecific:

i) Adoptive Transfer of lymphocytes:

(1) Lymphokine-activated killer (LAK) cells which are IL-2 activated T cells and NK cells can be

used in the treatment of melanoma and renal cell carcinoma

(2) Tumor-infiltrating lymphocytes (TIL) include T cells and NK cells. While the infiltrating NK

cells will kill tumors nonspecifically, the CTL will be able to kill specific tumor targets.

ii) Cytokines

(1) Interleukin-2 (IL-2): Activates T cells/NK cells which express IL-2 receptors and leads to their

proliferation. Used in the treatment of renal cell carcinoma and melanoma,

(2) Interferon-alfa (IFNα): Activates NK cell activity against tumors and also used in the treatment

of Kaposi sarcoma, renal cell carcinoma and melanomas.

(3) IFN-γ: Ιncreases class II MHC expression; used in the treatment of ovarian

cancers. (4) Tumor necrosis factor (TNF)-α: Kills tumor cells.

(5) Granulocyte-macrophage colony stimulating factor (GM-CSF): Useful in overcoming

neutropenia due to chemo- or radiation therapy

iii) Cytokine gene transfected tumor cells may also be used which can activate T or LAK cells that



can mediate anti-tumor immunity.

19. Immunodeficiency

Immunodeficiency is the failure of the immune system to protect against disease or malignancy.

Primary Immunodeficiency is caused by genetic or developmental defect in the immune system.

These defects are present at birth but may show up later on in life. Secondary or Acquired

Immunodeficiency is the loss of immune function as a result of exposure to disease agents,

environmental factors, immunosuppression, or aging.

Types of Primary Immunodeficiency Disorders

Defect in the hematopoietic stem cells results in reticular dysgenesis that leads to generalized

immune defects and subsequent susceptibility to infections. This condition is fatal if left untreated,

but can be successfully treated with bone marrow transplantation.

Myeloid Lineage deficiency: This deficiency involves myeloid progenitor cells and affects innate

immunity. Inasmuch as, phagocytosis is affected, the patients are susceptible to bacterial infections.

Congenital Agranulomatosis:

Patients have a decrease in the neutrophil count. It is due to a defect in the myeloid progenitor cell

differentiation into neutrophils. These patients are treated with granulocyte-macrophage colony

stimulating factor (GM-CSF) or G-CSF.

Chronic Granulomatous Disease (CGD):

This is characterized by defective reactive oxygen species (ROS) production which normally kills

phagocytosed bacteria. However, they exhibit inflammatory reaction with neutrophils,

macrophages and T cells resulting in the formation of granulomas. The disease is detected by a

negative reaction in the nitroblue tetrazolium test which in normal individual turns blue due to

reduction by superoxide anions. This is an autosomal recessive or X-linked trait. Treatment is

with interferon-γ (IFN-γ).

Leukocyte adhesion Deficiency (LAD):

Lack of CD18 (β chain) on T cells and macrophages impairs adhesion of these cells to

endothelium thereby preventing inflammation. Treatment is with bone marrow (devoid of T cells

and MHC-matched) transplantation or gene therapy.

Lymphoid lineage immunodeficiency:

If the lymphoid progenitor cells are defective, then both the T and B cell lineages are affected and



result in the severe combined immunodeficiency (SCID). They are less common but are very

severe. Such infants suffer from recurrent infections especially by opportunistic microorganisms.

These include the following disorders.

Patients having both T and B cell deficiency lack recombinase activating genes (RAG1 and 2)

that are responsible for the T cell receptor and Ig gene rearrangements. These patients are

athymic and are diagnosed by examining the T cell receptor (TCR) gene rearrangement. They

also lack B cells although they do have Abs in early infant life because of passive transfer from

mother. NK cells are normal in these patients. This is an autosomal recessive trait.

Interleukin-2 Receptor (IL-2R) may be lacking in patients thereby preventing signaling by IL-2

and other cytokines which act as growth factors. This would lead to defect in the proliferation

of T cells, B cells and NK cells. This is an autosomal recessive trait.

Adenosine deaminase (ADA) is an enzyme responsible for converting adenosine to inosine.

ADA deficiency leads to accumulation of adenosine which results in production of toxic

metabolites that interfere with DNA synthesis. The patients have defects in T, B and NK cells.

These SCID are autosomal recessive traits. Treatment is by gene therapy or stem cell

transplantation.

T cell deficiency affects both cell-mediated and humoral immunity. The patients are susceptible to

viral, protozoal and fungal infections. Infection with viruses such as cytomegalovirus or attenuated

measles vaccine can be life-threatening in these patients.

DiGeorge Syndrome or congenital thymic aplasia patients lack a thymus. This deficiency results

from deletion of a region on chromosome 22 during development of 3rd and 4th pharyngeal

pouch. Because of this, this deficiency is also responsible for facial and heart defects. This is an

autosomal dominant trait. Treatment is with a thymic graft.

B cell deficiency may result from the absence of B cells, plasma cells, total Igs or selective

Igs. These patients have recurring infection with extracellular bacteria, but are capable of

eliciting an immune response against intracellular bacteria as well as viruses and fungi.



X-linked Agammaglobulinemia (X-LA): In such patients, mature B cells are absent as they

exist in the pre B cell stage with H chains rearranged but not L chains. They also have no Igs

and suffer from recurrent bacterial infections.

X-linked hyper-IgM Syndrome: Such patients exhibit deficiency in IgG, IgA and IgE but

elevated IgM levels. This is due to a defect in the differentiation of IgM producing cells to cells

producing other Igs.

Selective Deficiency of Ig classes: IgA deficiency results from a defect in the class switching.

Such patients suffer from respiratory and genitourinary tract infections.

Common Variable Immunodeficiency: Also known as Late Onset hypogammaglobulinemia.

B cells fail to differentiate into plasma cells. Treatment is with Igs.

Complement defects:

Defects in C components results in immunodeficiency. The patients are susceptible to bacterial

infections.

Acquired or Secondary Immunodeficiencies:

All acquired immunodeficiencies have been outdone by the AIDS that is caused by Human

Immunodeficiency Virus (HIV)-1. It was first discovered in 1981 and the patients exhibited fungal

infections with opportunistic organisms such as Pneumocystis carinii and in other cases, with a skin

tumor known as Kaposi sarcoma. HIV-1 and 2 have been discovered with the strain frequently

found in N. America being HIV-1. HIV is spread through homosexual and promiscuous

heterosexual intercourse, infected blood and body fluids as well as during delivery from mother to

offspring. HIV was discovered by Luc Montagnier in Paris and Robert Gallo in Bethesda in 1983.

It is a retrovirus with RNA that is reverse transcribed to DNA by reverse transciptase (RT)

following entry into the cell. The DNA is integrated into the cell genome as a provirus that is

replicated along with the cell. HIV-1 does not replicate in most other animals but infects

chimpanzees although it does not induce AIDS in them. Severe combined immunodeficient mice

(SCID) reconstituted with human lymphocytes can be infected with HIV-1.

HIV-1 virion has an envelope made up of the outer lipid bilayer of the host cell in which are

embedded glycoproteins composed of the transmembrane gp41 along with the associated gp120.



The gp120 binds the CD4 expressed on host cells. Within the viral envelope is the viral core or

nucleocapsid consisting of a layer of matrix protein composed of p17 and an inner capsid made up

of p24. The viral genome consists of 2 ssRNA associated with 2 reverse transcriptase (RT)

molecules as well as other enzymes including a protease and an integrase.

Replication cycle and targets of therapy:

The virus attaches to the CD4 molecule on Th cells, monocytes and dendritic cells through the

gp120 of HIV. For HIV infection, a coreceptor is required. The coreceptor is a chemokine

receptor such as CXCR4 or CCR5. CCR5 expressed predominantly on macrophages and CXCR4

on CD4+ T cells serve as coreceptors for HIV infection. After the fusion of HIV envelope and the

host membrane, the nucleocapsid enters the cell. The RT synthesizes viral DNA which is

transported to the nucleus where it integrates with the cell DNA in the form of a provirus. The

provirus can remain latent till the cell is activated when the provirus also undergoes transcription.

The virions consisting of the transcribed viral RNA and proteins are produced. These are budded

out of the host cell membrane from where they acquire the envelope. Thus, therapeutic agents have

been developed that target viral entry and fusion, as well as serve as RT, protease and integrase

inhibitors. Highly active anti-retroviral therapy is a cocktail of 3 or more such agents.

ImmunologicalImmunologicalImmunologicalImmunological Changes:Changes:Changes:Changes:

The virus replicates rapidly and within ~2 weeks the patient develops fever. The viral load in the

blood increases significantly and peaks in 2 months after which there is a sudden decline because

of the latent virus found in germinal centers of the lymph nodes. CTL develop very early whereas

antibodies can be detected between 3-8weeks. The CTL killing of Th cells around 4-8 weeks

leads to decrease in CD4+ T cells. When the CD4+ T cell count decreases below 200/mm3, full

blown AIDS develops.

ImmunotheImmunotheImmunotheImmunotherrrrapapapapyyyy::::

There are several barriers to development of an effective HIV vaccine.

1) Attenuated vaccine itself may induce the disease

2) Heat-killed virus is not antigenic

3) CD4+ T cells may be destroyed by the vaccine

4) Antigenic variation of HIV due to induction of mutations results in escape from CTL.

5) Low immunogenicity of the virus by downregulation of MHC molecules

6) Lack of animal models in which HIV grows

7) Lack of in vitro tests to study HIV infection and growth.

The following immunological agents have been considered in developing vaccines



1) The use of soluble CD4 which could compete for binding with gp120.

2) Anti-gp120 Abs may bind to CD4 and block entry of the HIV

3) Chemokines that compete for the co-receptors may inhibit binding and entry of HIV.

4) Immunization with deletion mutants to reduce pathogenicity.

5) Vaccination with some recombinant proteins

6) Gene encoding proteins introduced into virus vectors may be used for vaccination

7) IL-2 to boost the Th cells.

20. 20. 20. 20. Antibody and Antigen ReactionAntibody and Antigen ReactionAntibody and Antigen ReactionAntibody and Antigen Reaction

In Vitro Antigen Antibody Reactions and Their Role in the Diagnosis of Disease

There are different types of antigen antibody interaction these include

1. Precipitation reaction

2. Agglutination reaction

3. Complement fixation reaction

4. Enzyme Immuno Assay (EIA)

5. Radio Immuno Assay (RIA)

Precipitation Reaction

Precipitins can be produced against most proteins and some carbohydrates and carbohydrates- lipid

complexes. Various system are available in which precipitation tests are performed in semisolid

media such as agar or agarose, or nongel support medium such as cellulose acetate. Agar has been

found to interfere with the migration of charged particles and has been largely replaced as an

immunodiffusion medium by agarose. Agarose is a transparent, colorless, neutral gel. In the

clinical laboratory several applications of the precipitation reaction are used. These methods

include:

Immunodiffusion

Electroimmunodiffusion

Immunodiffusion: These are of two types: single and double immuodiffusion.

I. Double diffusion

This technique also referred to as the Ouchterlony method, may be used to determine the

relationship between antigen and antibodies.

Principle: Antibody dilutions and specific soluble antigens are placed in adjacent wells. If the well

size and shape, distance between wells, temperature, and incubation time are optimal, these



solutions diffuse out, bind to each other, cross-link, and form a visible precipitate at the point of

equivalence perpendicular to the axis line between the wells the precipitation bands will be

compared with a standard antigen. The precise location of the band depends on the concentration

and rate of diffusion of antigen and antibody. In a condition of antibody excess, the band will be

located nearer the antigen well. If two antigens are present in the solution that can be recognized

by the antibody, two precipitin bands form independently. Antibodies associated with autoimmune

disorders such as rheumatoid arthritis and systemic lupus erythematosus can be identified by

double diffusion.

Identity

An identity reaction is indicated when the precipitin band forms a single smooth area. This

precipitin is formed between the antibody and the two test antigens fuses, indicating that the

antibody is precipitating identical antigen specificities in each preparation. This does not mean that

the antigens are necessarily identical; they are only identical insofar as the antibody can distinguish

the difference.

Nonidentity

A non-identity pattern (Fig 6-1B) is expressed when the precipitation line cross each other. They

intersect or cross because the sample contains no antigenic determinants in common.

Partial Identity

In a partial identity pattern, the precipitation lines merge with spur formation. This merger

indicated that the antigen are non identical but possess common determinants.

Single Radial ImmunodiffusionSingle Radial ImmunodiffusionSingle Radial ImmunodiffusionSingle Radial Immunodiffusion

This is a simple and specific method for identification and quantization of a number of proteins

found in human serum and other body fluids.

Principle: Radial immunodiffusion is based on a technique using a precipitin reaction in which

specific antibody is added to a buffered agarose medium, serum containing the test antigen is

placed in a well centered in the agarose. The diameter of the resulting precipitin zone is related to

the concentration of antigen placed in a well.

Eletroimmuno diffusion (EIDEletroimmuno diffusion (EIDEletroimmuno diffusion (EIDEletroimmuno diffusion (EID)



EID is a variation of the double immunodiffusion reaction in a support medium such as cellulose

acetate or agarose through the use of an electric current that enhances the mobility of reactants and

increase their movement towards each other. Antibody is placed in the well favoring its migration

in the direction of the cathode; antigens that tend to be more negatively charged and placed in the

well that favors migration of the anode. Precipitin bands form at a point of equivalence in a shorter

periods of time. Electro immunodiffusion method, like immunodiffusion procedures, are classified

into one-or-two-dimensional, singles, or double diffusion when a voltage is applied across the gels

to move the antigens and antibodies together, immuno-double diffusion becomes counter current

immuno electrophoresis (CIE) radial immunodiffusion (RID) becomes electro immunoassay (EIA).

Counter Current immunoelectrophoresis (CIE) is a variation of the classic precipitin procedure; it

merely adds an electrical current to help antigens and antibodies move towards each other more

quickly than in simple diffusion. The procedure takes advantage of the net electric charge of the

antigens and antibodies being tested in a particular test buffer. Variables such as types of gel,

amount of current, a concentration of antigen and antibody must be carefully controlled for

maximum reactivity. The sensitivity of CIE is 10 to 20 times greater than in immuno-double

diffusion, however, it is more expensive than other techniques such as immunodiffusion.

Electro immunoassayElectro immunoassayElectro immunoassayElectro immunoassay

Antigens may be quantitiated by electrophoresis than in an antibody-containing gel electro

immunoassay. This technique combines the speed of electrophoresis with the accuracy and

sensitivity of radioimmunoassay.

Agglutination ReactionAgglutination ReactionAgglutination ReactionAgglutination Reaction

Precipitation and agglutination are the visible expression of the aggregation of antigens and

antibodies through the formation of a framework in which antigen particles or molecules alternate

with antibody molecules. Agglutination of particles to which soluble antigen has been absorbed

produces a serum method of demonstrating precipitins. Example of artificial carriers includes latex

particles and colloidal charcoal. Cells unrelated to the antigen, such as erythrocytes coated with

antigen in a constant amount can be used as a biologic carriers. Whole bacterial cells can contain

an antigen that will bind with antibodies produced in response to that antigen when it was

introduced into the host. Agglutination tests are easy to perform and in some cases are the most



sensitive tests currently available. These tests have a wide range of applications in the clinical

diagnosis of noninfectious immune disorders and infectious diseases.

Latex agglutinationLatex agglutinationLatex agglutinationLatex agglutination

In latex agglutination procedures, antibody molecules can be bound to the surface of latex beads.

Many antibody molecules can be bound to each latex particle, increasing the potential number of

exposed antigen-binding sites. If an antigen is present in a test specimen, the antigen will bind to

the combining sites of the antibody exposed on the surface of the latex heads, forming visible

cross-linked aggregates of latex beads and antigen. In some test systems, latex particles can be

coated with antigen. In the presence of serum antibodies, these particles agglutinate in to large

visible clumps. Examples of tests based on latex agglutination reaction include C-reactive protein,

IgG rheumatoid factors, and IgM rheumatoid factors.

Figure Figure Figure Figure 92929292: : : : Latex agglutination reaction

Direct bacterial agglutinationDirect bacterial agglutinationDirect bacterial agglutinationDirect bacterial agglutination



Direct whole pathogens can be used to detect antibodies directed against pathogens. The most basic

tests are those that measure the antibody produced by the host to determinants on the surface of a

bacterial agent in response to infection with that bacterium. In a thick suspension of the bacteria,

the binding of specific antibodies to surface antigens of the bacteria causes the bacteria to clump

together in visible aggregates. This type agglutination is called bacterial agglutination. Because

tube testing allows more time for antigen-antibody reaction, it is considered to be more sensitive

than slide testing.

Indirect or passive hemagglutination

Hemagglutination is agglutination of red blood cells, and tests for antibody detection. In the

indirect or passive hemagglutination technique, erythrocytes are coated with substances such as

extracts of bacterial cells, protozoa or purified polysaccharides or proteins. Erythrocyte of animals

such as sheep or rabbits, or from group “O” humans, function as carrier for detecting and

titrating the corresponding antibodies by agglutination. This technique is called indirect or passive

hemagglutination testing because it is not the antigen of the erythrocytes themselves but the

passively attached antigens that are bound by antibody .For example, in rubella antibody test;

erythrocytes are coated with rubella antigen. In the presence of antibody, agglutination occurs.

Hemagglutination Inhibition techniqueHemagglutination Inhibition techniqueHemagglutination Inhibition techniqueHemagglutination Inhibition technique

Hemagglutination inhibition test is used to detect some viral antibodies, for example, rubella. A

known quantity of rubella viral antigen is mixed with dilutions of the patient’s serum, to which

red blood cells are added. If the serum lucks antibody, the virus will spontaneously attach to the

red cells, link together, and agglutinate. If antibody to the virus is present, all of the virus particles

will be bound by antibody, which prevents or inhibits hemagglutination. The serum is therefore

positive for the antibodies. The highest dilution of serum that totally inhibits agglutination of red

cells determines the antibody titer of the serum. Disadvantage of this technique include: time

consuming, & subjective bias in the interpretation for results. Negative results do not always

indicate the absence of antibody. In some case false negative results can occur from a low titer of

antibody. Nonspecific inhibitors can cause false positive results.

Complement Fixation ReactionComplement Fixation ReactionComplement Fixation ReactionComplement Fixation Reaction

Complement fixation is a classic method for demonstrating the presence of antibody in serum. This

method consists of two components. The first component is an indicator system consisting of a

combination of sheep red cells; complement-fixing antibody produced against the sheep red cells in



another animal, and an exogenous source of complement, usually guinea pig serum. When these

three components are combined in an optimal concentration, the anti-sheep cell antibody,

hemolysin, can bind to the surface of the red cells. Complement can subsequently bind to this

antigen antibody complex and cause cell lysis. The second component consists of a known antigen

and patient serum, which are added to a suspension of sheep erythrocytes, hemolysin, and a

complement. The two components of the complement fixation procedure are tested in sequence.

Patient serum is first added to the known antigen, and complement is added to the solution. If the

serum contains antibody to the antigen, the resulting antigen antibody complexes will bind all of

the complement. Sheep red cells and hemolysin are then added. If complement has not been bound

by an antigen antibody complex formed from the patient serum and known antigen, it is available

to bind to the indicator system of indicates both a lack of antibody and a negative complement

fixation test. If the patient’s serum does contain a complement fixing antibody appositive result

will be demonstrated by the lack of haemolysis.

Immunofluorescent Test (IFT)Immunofluorescent Test (IFT)Immunofluorescent Test (IFT)Immunofluorescent Test (IFT)

The fluorescent techniques are extremely specific and sensitive. This technique consists of labeling

antibody with fluorescein isothiocyantate, a fluorescent compound with an affinity for proteins to

form a complex, conjugate. The fluorescent assay includes: direct immunofluoresent assay and

indirect immunofluoresent assay.

Direct Immunofluorescent assayDirect Immunofluorescent assayDirect Immunofluorescent assayDirect Immunofluorescent assay

In this technique, Fluorescein- conjugated antibody is used to detect antigen- antibody reactions.

This method can be applied to the detection of hepatitis B virus & chlamydia. A fluorescent

microscope is required to observe the production of color; fluorescein gives a yellow- green light.

Indirect ImmunoflIndirect ImmunoflIndirect ImmunoflIndirect Immunofluorescent Assay (IFA)uorescent Assay (IFA)uorescent Assay (IFA)uorescent Assay (IFA)

This method is based on the fact that antibodies not only react with homologous antigens but can

act as antigens and react with antibody. In the indirect immunofluorescent assay, the antigen source

to the specific antibody being tested is fixed to the surface of a microscopic slide. The patent’s

serum is diluted and placed on the slide to cover the antigen source. If antibody is present in the

serum, it will bind to its specific antigen unbound antibody is then removed by washing the slide,

finally antihuman globulin conjugated to a fluorescent substance that will fluoresce when exposed

to a fluorescent substance that will fluoresce when exposed to ultraviolet light is placed on the

slide. This conjugated marker of human antibody will bind to the antibody already bound to the



antigen on the slide and will serve as a marker for the antibody when viewed under a fluorescent

microscope.

Enzyme Immuno Assay (EIA)Enzyme Immuno Assay (EIA)Enzyme Immuno Assay (EIA)Enzyme Immuno Assay (EIA)

An enzyme labeled antibody or enzyme labeled antigen conjugate is used in immunologic assays

for detection of antigens or antibodies, in a patient’s serum e.g. HIV antibody, HIV antigen,

hepatitis A antibody. Various enzymes are employed in enzyme immunoassay. The most

commonly used enzymes are peroxidase and alkaline phosphatase. In EIA, a plastic bead or plastic

plate is coated with antigen. The antigen reacts with antibody in the patient serum. The bead or

plate is then incubated with an enzyme-labeled antibody conjugate, if antibody is present on the

bead or plate. The enzyme activity is measured spectrophotometrically after the addition of the

specific chromogneic substrate. Test result is calculated by comparing the spectrophotometer

reading of patient serum to that of a control serum.

Radio Immunoassay (RIA)Radio Immunoassay (RIA)Radio Immunoassay (RIA)Radio Immunoassay (RIA)

In radioimmunoassay, radioisotopes can be used to measure the concentration of antigen or

antibody in serum sample. If antibody concentration is being measured, radioactive labeled

antibody competes with patient unlabeled antibody for binding sites on a known amount of antigen.

The main advantage of the radioimmunoassay method is the extreme sensitivity and ability to

detect trace amounts. In addition, a large number of tests can be performed in a relatively short

time period. The disadvantage is the hazards and instability of isotopes.

Factors Affecting Antigen Antibody ReactionsFactors Affecting Antigen Antibody ReactionsFactors Affecting Antigen Antibody ReactionsFactors Affecting Antigen Antibody Reactions

Many factors affect the interaction between antigen and antibody; these include specificity, cross

reactivity, temperature PH, ionic strength, concentration, and intermolecular specificity.

Specificity: The ability of a particular antibody to combine with one antigen instead of another is

referred to as specificity. This property resides in the portion of the antigen binding fragment of an

immunoglobulin molecule. Antigen-antibody reactions can show a high level of specificity.

Specificity exists when the binging sites of antibodies directed against determinants of one antigen.

Cross reactivity: When some of the determinants of an antigen are shared by similar antigenic

determinants on the surface apparently unrelated molecules, a proportion of the antibodies directed

against one kind of antigen will also react with the other kind of antigen. This is called cross

reactivity. Antibodies directed against a protein in one species may also react in a detectable



manner with the homologous protein in another species, which is another example of cross

reactivity.

Temperature: The optimum temperature needed to reach equilibrium in an antibody-antigen

reaction differs for different antibodies. IgM antibodies are cold reacting with thermal-range 4-

220C, and IgG antibodies are warm reacting, with an optimum temperature of reaction at 370C.

pH: Although the optimum pH for all reactions has not been determined, a pH of 7.0 is used for

routine laboratory testing.

Ionic strength: The concentration of salt in the reaction medium has an effect on antibody uptake

by the membrane bound erythrocyte antigens. Sodium and chloride ions in solution have inhibition

effect. These ions cluster around and partially neutralize the opposite charges on antigen and

antibody molecules, which hinders the association of antibody with antigen. Reducing or lowering

the ionic strength of a reaction medium such as low-ionic strength salt can enhance antibody

uptake.

Concentration: Under normal condition the concentration of antigen and antibody should be

optimal but sometime this thing fail to be happen in which excess antibody or antigen

concentration will result in false reaction, sometimes known as zonal reaction. When the

concentration of antigen is excess it is known as post zone reaction; excess antibody is referred as

prozone reaction. This phenomenon can by overcome by serial dilution until optimum amount of

antigen and antibody will present.

Bond strength and inter molecular attractive force Bonding of an antigen to an antibody takes place

because of the formation of multiple, reversible, intermolecular attraction between an antigen and

amino acids of the binding site. The bonding of antigen to antibody is exclusively non covalent.

The attractive force of non-covalent bonds is weak when compared to covalent bonds, but the

formation of multiple non-covalent bonds produces considerable total- binding energy. The

strength of a single antigen- antibody bond is termed antibody affinity.

The strongest bonding develops when antigens and antibodies are close to each other and when the

shapes of both the antigenic determinate and the antigen-binding site conform to each other. This

complementary matching is referred to as goodness of fit.



21. 21. 21. 21.

Serological TechniquesSerological TechniquesSerological TechniquesSerological Techniques

Materials Necessary for Basic Serologic TestsMaterials Necessary for Basic Serologic TestsMaterials Necessary for Basic Serologic TestsMaterials Necessary for Basic Serologic Tests

As discussed in the previous chapter, wide verities of serologic techniques are available to detect

either an antibody or antigen. For the detection of this unknown substance from patient’s

specimen, the specimen should be collected and prepared appropriately. In addition the equipment

that is used for testing should be free from any contaminants so as to get true result. The following

are some of the equipment used in routine serology.

Glasswares

Dirty glass-wares easily affect serological test. After using all the glass wares (test tube, beaker,

pipette, etc.) they should be socked in detergent for several hours and rinsed several times in tap

water. Finally allow drying by placing in a dry oven or dust free place. Test tubes and pipettes

should not be scratched or broken, which will interfere with the reading of a test.

Types of glassware include:

Test tube

Glass slides

Serologic pipette with a size of l0ml, 5ml, 2ml&1ml.

Constant temperature device

Incubator and water bath are usually used in serologic tests. These materials are electrically

operated and have thermostat that hold the temperature within the required limits. These devices

should be checked prior to use by installing a thermometer

Rotating machines

Rotating machines are required to facilitate antigen antibody reactions. Such machine has a flat

plate, which rotate at a prescribed rate of speed. A knob located on the front part of the machine

controls the number of revolution per minute.

Collection Preparation and Preservation of Specimen for Serologic Tests

Specimens that are used for serologic test include: serum, plasma & cerebrospinal fluid. Serum or

plasma sample could be obtained from venous blood, which can be performed by the laboratory

personnel however. Cerebrospinal fluid should be collected by a physician or a trained nurse. For

serum or plasma sample, first 2-3 ml of venous blood is collected using sterile syringe and needle



from a patient. If serum is required, allow the whole blood to clot at room temperature for at least

one hour and centrifuge the clotted blood for 10 minutes at 2000 rpm. Then transfer the serum to a

labeled tube with a pasture pipette and rubber bulb.

Plasma sample is obtained by treating fresh blood with an anticoagulant, centrifuge and separate

the supernatant. The specimen should be free from hemolysin blood. Finally, seal the specimen

containing tube; the tube should be labeled with full patient’s identification (Age, Sex, code no,

etc). The test should be performed within hours after sample collection, if this could not be done

preserve it at- 200c.

Shipment of Serological SpecimenShipment of Serological SpecimenShipment of Serological SpecimenShipment of Serological Specimen

Most health center and clinic laboratories often are limited in the diagnostic procedures that can be

carried out and have to ship serologic specimens to other laboratories. Before shipment the

following things should be considered.

Don’t ship whole blood unless the tests to be performed require whole blood.

Do not inactivate serum or plasma before mailing.

Keep the specimen and packing container in the refrigerator until time of shipment but if shipment

requires several days, freeze the specimen.

Then ship the specimen by the fastest route.

Complement Inactivation

Complement inactivation is important because it is known to interfere with different tests. In

activation of complement can be achieved by heating the serum or plasma at 560C for 30 minutes.

If more than four hours has elapsed since inactivation, a specimen should be re-inactivated with

same temperature 10 minutes.

Serial DilutionSerial DilutionSerial DilutionSerial Dilution

Dilution is the act of making a weaker solution from a stronger one. This is usually done by adding

a water or saline, which contains none of the material being diluted. Dilution is usually expressed

as one unit of the original solution to the total number of units of final solution. Serial dilution

means decreasing the volume of serum progressively by maintaining a constant volume of fluid

most commonly, serial dilutions are twofold, that is, each dilution is half as concentrated as the

preceding one. The total volume in each tube is the same.



Determination of Endpoint and TiterDetermination of Endpoint and TiterDetermination of Endpoint and TiterDetermination of Endpoint and Titer

If we take the above example again, after serially diluting the patient’s serum, equal amount of

an antigen is added in each dilution to observe immunolgic reaction. The last tube that shows

visible immunologic reaction is known as end point of the test, the dilution of the antiserum at the

end point is known as the titer. The reciprocal of the greatest reacting dilution of the serum is

considered as the measure of titer or the concentration of the antibody. For example, it the highest

dilution of the serum that shows a visible reaction is at 1:32 dilution, the titer of the test is

expressed as 32.

TreponematosesTreponematosesTreponematosesTreponematoses

It is a chronic inflammatory disease, primarily it affects the skin and mucous membrane, and

during latent period other organs and tissues may be affected. Medically important species is

mainly T. palladium but there are other pathogenic treponemes like T. pertenue, T. endemicum &

T. carateum. Each of these organisms are obligate parasite of humans, morphologically identical,

cannot be cultured in vitro and have similar laboratory diagnosis and treatment.

These diseases differ in there:

Geographical distribution,

pathogenicity and

Degree of virulence.

T. pertenue

Cause a disease known as yaws. Its geographical distribution is West Africa, central Africa south

East Asia. T.pertenue is transmitted through exposed skin. Hand, face, legs and feet are parts of the

body most affected, it produce raised granular papilloma on the skin. In the later stage,

disfigurement of the infected area will be resulted.

T. endemicumT. endemicumT. endemicumT. endemicum

Cause endemic syphilis. It is widely distributed in sub-Sahara Africa and transmitted through

exposed skin and oral mucosa.

T.carateum

Cause a disease pinta, has geographical distribution of central and South America, transmitted

through exposed skin. The organisms produce itchy red papules on the uncovered part of the body.

In the later stage when infected area healed loss of the normal pigment will be resulted.



Syphilis

Syphilis is caused by a spirochete bacterium, Treponema pallidum.

Morphology and Metabolism of T. PallidumMorphology and Metabolism of T. PallidumMorphology and Metabolism of T. PallidumMorphology and Metabolism of T. Pallidum

Microscopically T. pallidum appears as fine, spiral (8 to 24 coils) organism, approximately 6-15

μm long. They are not cultivatable with any consistency in artificial laboratory media out side the

host. T. pallidum are extremely susceptible to a variety of physical and chemical agents. However,

they may remain viable for up to 5 days in tissue specimens removed from diseased animals and

from frozen specimens. Syphilis is a venereal disease. It can be acquired by kissing a person with

active oral lesion. There are very few cases of transfusion-acquired syphilis. In addition, syphilis

may be transmitted transplacentally to the fetus. Spirochetes can be transmitted to the fetus during

the last trimester of pregnancy.

Stages of SyphilisStages of SyphilisStages of SyphilisStages of Syphilis

Untreated syphilis is a chronic disease with sub acute symptomatic periods separated by

asymptomatic intervals, during which the diagnosis can be made serologically. The progression of

untreated syphilis is generally divided in to stages. Initially, T.pallidum penetrates intact mucous

membranes or enters the body through tiny defects in the epithelium. Upon entrance, the

microorganism is carried by the circulatory system to every organ of the body. Spirochetemia

occurs very early in infection, even before the first lesions have appeared or blood tests become

reactive. Before chemical or serologic manifestations develop patients are said to be ‘incubating

syphilis”. The incubation period usually lasts about 3 weeks but can range from 10-90 days.

Primary syphilisPrimary syphilisPrimary syphilisPrimary syphilis

At the end of the incubation period, a patient develops a characteristics primary inflammatory

lesion called a chance at the point of initial inoculation and multiplication of the spirochetes. The

chancre begins as a papule and erodes to form a gradually enlarging ulcer with a clean base and

indurate edge. Generally it is relatively painless and commonly located around the genitalia, but in

about 10% of cases lesions may appear almost any where else on the body e.g. Throat, lip, hands.

Most of the patients with primary syphilis will develop swelling of inguinal lymph nods. The

primary chancre will persist for 1 to 5 weeks and will heal completely within about 4 to 6 weeks.

Primary syphilis is diagnosed by its characteristics chancre with positive serological test and

detection of T.pallidum by dark field examination from the lesion.



Secondary syphilisSecondary syphilisSecondary syphilisSecondary syphilis

Within 2 to 8 weeks after the appearance of the primary chancre, a patient may develop the sign

and symptoms of secondary syphilis when organisms gain access to the circulation from the

infected site. The secondary stage is characterized by a generalized illness that usually begins with

symptoms suggesting a viral infection headache, sore throat, low-grade fever and occasionally

nasal discharges. Blood tests reveal a moderate increase in leukocytes with a relative increase in

lymphocytes. The disease progresses with the development of lymhadenopathy and lesions of the

skin and mucous membranes corresponding to the spread of the organism in the body by way of

the circulating blood. The lesions contain a large number of spirochetes, and when located on

exposed surfaces, are highly contagious. Macular lesions are common, and a rash invariably

involves the genitalia and often is prominent on the palms and soles. Secondary syphilis usually

resolves itself within 2 to 6 weeks, even in the absence of therapy. It may be diagnosed by typical

skin rash and positive syphilis serology test.

Latent syphilisLatent syphilisLatent syphilisLatent syphilis

After resolution of untreated secondary syphilis, the patient enters a latent non-infectious state in

which diagnosis can be made only by serologic method. During the first 2-4 years of infection, one

fourth of patients will show relapses of manifestation of secondary syphilis. During these relapses,

patients are infectious, and the underlying spirochetemia may be passed translucently to the fetus.

Relapses are extremely rare after four years of latency. About one third of patients entering latency

are eventually spontaneously cured of the disease, one third will never develop further clinical

manifestation of the disease and the remaining one third will eventually develop late syphilis.

Late (Tertiary) syphilisLate (Tertiary) syphilisLate (Tertiary) syphilisLate (Tertiary) syphilis

The first manifestations of late syphilis are usually seen from 3-10 years after primary infection.

About 15% of untreated syphilitic individuals eventually develop late benign syphilis characterized

by the presence of destructive granulomas. These granulomas, or gummas, may produce lesions

resembling segments of circles that often heal with superficial scarring. Treponems are rarely

found in the lesions, which are referred to as benign gummas. Of untreated patients 10% develop

cardiovascular manifestations. T. pallidum may damage large blood vessels such as aorta and

coronary arthritis. This condition is usually fatal. In about 8% of untreated patients, late syphilis

involves the CNS. Initially CNS disease is asymptomatic and can be detected only by examination

of cerebrospinal fluid. In symptomatic neurosyphilis, spirochetes may also involve the brain tissue

and cause destructions of the brain parenchyma (paresis), dorsal root of the spinal cord (tabes).



Congenital syphilisCongenital syphilisCongenital syphilisCongenital syphilis

Congenital syphilis is caused by maternal spirochetemia and transplacental transmission of the

microorganism usually after 18 weeks of gestation. Congenital syphilis is diagnosed in three

fourths of the cases in patients over 10 years of age. About half of damage to the fetus depends on

the stage of the disease and the number of treponemes circulating in pregnant women at the time of

transmission. Early congenital syphilis appears either at birth or up to two years of age, the

manifestation includes cutanuous lesion, mucous membrane lesion like thick nasal discharge

containing T.pallidum. Late congenital syphilis may be characterized by fissuring around the

mouth, anus, skeletal lesions, and perforation of the palate and the collapse of nasal bones to

produce a saddle-nose deformity.

Antibodies in SyphilisAntibodies in SyphilisAntibodies in SyphilisAntibodies in Syphilis

In the treponemes, two classes of antigen have been recognized those restricted to one or a few

species and those shared by many different spirochetes. Infection with T.pallidum, T.pertenue,

T.carateum T.endemicum produces similar antibody response. Specific and non-specific antibodies

are produced in the immunocompetent host.

Specific antibodySpecific antibodySpecific antibodySpecific antibody

Antibodies in early or untreated early latent syphilis are predominantly IgM antibodies. The early

immune response to infection is rapidly followed by the appearance of IgG antibodies. The greatest

elevation in IgG concentration is seen in secondary syphilis.

Non – specific (nontreponemal or reagin) antibodies

Are produced by infected patients against components of their own or other mammalian cells.

Reagin is widespread in nature and can be isolated from any mammalian tissues as well as from

treponemes. Although patients with syphilis almost always produce these antibodies, patients with

other infectious disease, like measles, hepatitis, leprosy, Brucelosis, malaria, rickettsia, also

produce them. Patients can alsoexhibit reagin with non-infectious conditions such as drug addition,

old age, pregnancy and recent immunization.

Collection and Handling of Syphilitic Specimen from LesionCollection and Handling of Syphilitic Specimen from LesionCollection and Handling of Syphilitic Specimen from LesionCollection and Handling of Syphilitic Specimen from Lesion

The treponemal lesion is infectious. As a result wearing a rubber glove is vital to protect one self

from infection. The following procedure should be followed to get a representative sample.



Cleanse the area of chancre with swab moistened with physiological saline. Apply gentle pressure

on the area to squeeze the sample from the depth of the lesion. Collect the sample of serous

exudates on a cover glass and invert it on a slide. Deliver immediately the preparation to the

laboratory for examination by dark-field microscopy.

Tests for SyphilisTests for SyphilisTests for SyphilisTests for Syphilis

Either demonstration of microorganism in a lesion or serologic testing confirms the clinical

diagnosis of syphilis in the laboratory. The serologic methods for syphilis measure the presence of

two types of antibodies: treponemal and non treponemal.

Serologic procedures for syphilis include the following.

1. Nontreponemal method e.g. Venereal Disease research laboratory (VDRL) and the rapid plasma

regain (RPR) procedures.

2. Treponemal methods e.g. Fluorescent Treponema pallidum antibody absorption (FTA-ABS) and

microhemagglutination Treponema pallidum MHA-TP).

Nontreponemal methods

The VDRL and RPR are the two most widely used nontreponemal serologic procedures. Each is a

flocculation or agglutination test in which soluble antigen particles coalesce to form larger particles

visible as clumps when they are aggregated by antibodies. The VDRL procedure is recommended

when a patient suspected of having syphilis has a negative dark field microscopy result or when

atypical lesions are present. It is further recommended that a quantitative VDRL assessment be

made quarterly for 1 year after treatment for syphilis, or that the adequacy of treatment in both

early and latent syphilis be monitored. The VDRL procedure can be performed on cerebrospinal

fluid for the detection of neurosyphilis. The RPR test can be performed on unheated serum or

plasma using a modified VDRL antigen suspension of choline chloride with EDTA. The RPR test

card test antigen also contains charcoal for macroscopic reading. It is about as

specific as, and possibly more sensitive than, the VDRL slide test.

Treponemal methods

The FTA – ABS and MHA represent treponemal methods. Reactive (Positive) regain test can be

confirmed with these two specific treponemal antigen tests. These procedures, however, should not

be used as primary screening methods. Procedures such as the FTA-ABS and MHA can be used to

confirm that a positive non-treponemal test result has been caused by syphilis rather than one of

the other biologic conditions that can produce positive VDRL, or they can determine quantitative

titer of antibody, which is useful in following response to therapy. The FTA-Abs uses a killed



suspension of T. pallidum spirochetes as the antigen. The micro hemagglutination assay for T.

pallidum is based on agglutination by specific antibodies in the patient’s serum with sheep

erythrocytes sensitized to T. pallidum antigen. The Treponema pallidum immobilization test (TPI)

method is obsolete.

Sensitivity of commonly used serologic tests for syphilis

Detection of syphilis by serologic methods is related both to the stage of the disease and to the test

method. In the primary stage, about 30% of cases become serologically active after one week and

90% of patients demonstrate reactivity after three weeks. Reagin titers increase rapidly during the

first four weeks of infection and then remain stationary for approximately six months. Patients in

the secondary stage of syphilis are serologically positive. During latent syphilis there is a gradual

return of non-reactive serologic manifestations with non-treponemal method. About one third of

patients in the latent stage will remain seroreactive and presumably infectious. In late syphilis,

treponemal tests are generally reactive, non-treponemal methods are non reactive.

Venereal Disease Research Laboratory (VDRL) Qualitative Slide TestVenereal Disease Research Laboratory (VDRL) Qualitative Slide TestVenereal Disease Research Laboratory (VDRL) Qualitative Slide TestVenereal Disease Research Laboratory (VDRL) Qualitative Slide Test

Principle: heat inactivated serum is mixed with a buffered saline suspension of cardiolipin–

lecithin–cholesterol antigen. This serum-antigen mixture is microscopically examined for

flocculation.

Specimen collection and preparation

The specimen should include all identification, it must include the patient’s full name, the date

the specimen is collected and the patient’s hospital identification number. Blood should be drawn

by an aseptic technique. The required specimen is a minimum of 2 ml of clotted blood. The

specimen should be promptly centrifuged and an aliquot of the serum removed. Severely lipemic or

hemolyzed serum is unsuitable for testing. Before testing, the serum must be heat in activated at

56C0 for 30 minutes. In activated serum should be reheated at 56C0 for 10 minutes if tested more

than 4hours after the original in activation. Cerebrospinal fluid is also an appropriate fluid for

testing.

Reagents requiredReagents requiredReagents requiredReagents required

VDRL antigen – a colorless, alcoholic solution containing 0.03% cardiolipin, 0.9% cholesterol, and

sufficient purified lecithin to produce standard reactivity. Each lot must be serologically



standardized by comparison with an antigen of know reactivity. Ampules should be stored in the

dark at either at 6C0 to 10 C0or at room temperature antigen that contains precipitate should be

discarded. VDRL- buffered saline Contains 1% sodium chloride, PH 6.0 + 0.1, it should be stored

in screw capped or glass stopper bottles.

Equipment requiredEquipment requiredEquipment requiredEquipment required

� VDRL test slide with paraffin of ceramic ring.

� 18 gauges hypodermic needle without bevel (it will deliver 60 drops) ml of reagents.

� 30 ml flat-bottomed glass with stopper, narrow mouth bottle

� Syringe (1-2ml)

� Rotator

� Serological graduated pipette

� Water bath 56C0

Preparation of working antigen suspension

1. Dispense 0.4 ml of buffered saline to the bottom of the 30ml, round, glass-stopper bottle.

2. Rapidly add 0.5ml of antigen drop by drop directly by rotating the bottle in a circular motion on

a flat surface. The pipette tip should remain in upper third of the bottle. Take care to avoid

splashing saline on the pipette. Blow the last drop of antigen from the pipette without touching the

pipette to the saline.

3. Continue to rotate the bottle for 10 seconds.

4. Add 4.1 ml of buffered saline with a 5 ml pipette

5. Place the stopper on the bottle and shake up and down approximately 30 times in 10 seconds.

The antigen suspension is ready for use, but it must be gently mixed at the time of use. Do not

force back and forth through the needle & syringe as this may lead to break down of antigen

particles and loss of their activity.

Note: The working antigen suspension can be stabilized by adding 50 μl of benzoic acid to 5ml of

the diluted working solution instead of discarding within 24 hours. The temperature of the buffer

saline and antigen should be in the range of 230 to 29C0. The antigen suspension must be used on

the day of preparation.

Quality controlQuality controlQuality controlQuality control

Include positive control sera of graded reactivity each time serologic testing is performed. The

antigen suspension to be used each day is first examined with these control sera. Store control sera

frozen at -200C or liquid form for 7 to 10 days. Thaw, mix thoroughly, and heat in activate at



560C before use. Check antigen – dispensing needle at the time of use to be sure that it accurately

deliver 60-drops/ml reagents. Clean needles and syringes by rinsing with water, alcohol and

acetone. Remove needle from syringe after cleaning.

Procedure

1. Pipette 0.5 ml of inactivated patient serum in to one of the rings of the ceramic-ringed slide.

Pipette additional specimen and controls in to additional rings.

2. Add one drop of antigen suspension to each serum with a calibrated 18-gauge needle and

syringe held in a vertical position.

3. Rotate the slide on a mechanical rotator for 4 minutes. In externally dry climate, cover the slide

with a lid containing moistened filter paper to prevent evaporation during rotation.

4. Examine each specimen microscopically with the low (10x) objective.

Note: The test should be performed at a temperature range of 230-290C.

Reporting Results

Non reactive: No clumping or very slight roughness weakly reactive: Small clumps

Reactive: medium and large clumps

Weakly reactive: small clumps

Note: All reactive and weakly reactive specimens (sera) should be tested quantitatively to estimate

the antibody titer.

False negative reactions it can occur in a variety of situations like:

� Technical error (e.g. unsatisfactory antigen preparation or techniques.

� The presence of inhibitors in the patient’s serum

� Low antibody titer patients may have syphilis, but the reagin concentration is too low to produce a

reactive test result.

It may be caused by several factors: an infection that is too recent to have produced antibodies, the

effect of treatment, latent or inactive disease, or patients who have not produced protective

antibodies because of immunological tolerance. These seronegative patients may demonstrate a

positive reaction with more sensitive treponemal tests such as the FTA-ABS.

Inappropriate temperature

Prozone reaction

Weakly reactive results can be caused by

Very early infection

Lessening of the activity of the disease after treatment.

Improper technique or questionable reagents



False – positive reactions can also be observed. Of all positive serologic tests for syphilis, 10% to

30% may be false biologic positive reactions. Non-syphilitic positive VDRL reactions have been

reported with cardiolipin type of antigen in rheumatic fever, pneumococcal pneumonia, infectious

hepatitis, leprosy, malaria, pregnancy, aging individuals, and rheumatoid arthritis. Contaminated or

hemolyzed specimens can also produce false positive results.

VDRL quantitative test

Principle: Retest quantitatively to an end-point titer all sera that produce reactive, weakly reactive

or questionably nonreactive results in the qualitative VDRL slide test.


Same as VDRL Qualitative test for undiluted serum

Preparation of serial dilution.

A. Pipette 0.05 ml of 0.9% saline in to ring number 2,3 &4 on ceramic slide do not spread the saline.

B. Pipette 0.05 ml of serum to ring numbers 1 and 2. Draw the serum and saline mixture up and down

in the pipette tip in ring number 2 to mix. Aspirate 0.05 ml of diluted serum and spread the

remaining dilution over the entire area of the circle with the pipette tip.

C. Transfer 0.05ml of the diluted (1:2) serum in ring number 2 to ring number 3. Draw the serum and

saline mixture. Aspirate 0.05 ml of diluted serum and spread the remaining dilution over the entire

area of the circle with the pipette tip.

D. Transfer 0.05 ml of the diluted (1:4) serum in ring number 3 to ring number 4 Draw the serum and

saline mixture up and down in the pipette tip in ring number 3 to mix. Aspirate 0.05 ml of diluted

serum and spread the remaining dilution over the entire area of the circle with the pipette tip.

Discard 0.05 ml of the diluted (1:8) serum from ring number 4 unless greater dilutions are needed

for strongly relative serum, and spread the remaining dilution over the entire area of the circle with

the pipette tip.

Reagent, Supplies, and equipment

In addition to the VDRL qualitative test the following reagent and piece of equipment are needed.

0.9% saline

Preparation – weigh 0.9gm of sodium chloride to a leit volumetric flask. Dilute to the calibration

mark with distilled water

Safety pipette (50ml or 0.05ml)

Procedure:

1. Add one drop of antigen suspension to each diluted serum with a calibrated 18-gauge needle and

syringe held in a vertical position.

2. Rotate the slide on a mechanical rotator for 4 minutes.



3. Examine each specimen microscopically (10x objective)

Note: the test should be performed at a temperature range of 230 to 290C.

Reporting results

Report the titer in terms of the highest dilution that produces a reactive (weakly reactive) result.

Rapid Plasma Reagin (RPR) Card Test

Principle

A cardiolopin lecithin-cholesterol antigen coated with carbon particle is mixed with patient’s

serum. Then flocculation reaction is observed macroscopically in the presence of cardiolipin

antigen.


No special preparation is required before specimen collection. The specimen should be labeled

with all patient’s identification fresh serum or plasma sample can be used. It is not important to

heat inactivate the specimen before testing.

Reagents & Equipment

Note: Except for the antigen, all other components should be stored at room temperature in a dry

place in the original kit packaging.

Provided in the test kit

RPR card test antigen

It contains cardiolipin, lecithin, cholesterol, EDTA, charcoal, chorine chloride, and distilled water.

Store the antigen suspension in ampules or in plastic dispensing bottle at 20 to 80C. Unopened

ampules have a shelf life of 12 months from the date of manufacture.

Opened antigen ampules has stability for 3 months

Needle, 18-gauge, without bevel. The needle should deliver 60+2 drops of antigen suspension per

milliliter when held in a vertical position.

Specially prepared, plastic-coated cards

Serological pipette

Dispenser, 0.05 ml/drop

Stirrer

Other material

Rotator

Humidifier cover containing a moistened sponge

0.9% saline

Fluorescent Treponema Pallidum AntibodyFluorescent Treponema Pallidum AntibodyFluorescent Treponema Pallidum AntibodyFluorescent Treponema Pallidum Antibody



Absorption Test

Principle: Patients serum is added on the slide coated with T. pallidum antigen followed by

addition of fluorescent-tagged anti-human globulin and examine under fluorescent microscope.

It’s the most sensitive confirmatory test. Specimen collection and preparation. The serum should

be heat inactivated. The specimen should be labeled with all the identification.

Reagent supplied

Treponema pallidum antigen: Extracted from rabbit testicular tissue

Store at 60

c -100C in lyophilized form

Conjugate (fluorescent labeled anti-human globulin)

Positive control

Equipment

. Test tubes, pipette

. Incubator

. Fluorescent microscope

Procedure

1. Coat the slide with T.pallidum antigen by adding a drop of suspension of T.pallidum on a clean

slide and keep in oven at low temperature

2. Take out and wash by rinsing with tap water to remove excess unbounded T.pallidum

3. Add the patient’s serum to the coated antigen and incubate, rinse by tap water to remove

excess antibody, if the pt serum has an antibody, and to remove the whole serum if it doesn’t

contain antibody.

4. Add conjugate (fluorescent tagged antihuman globulin) and rinse with tap water, to remove

excess conjugate, if serum contain antibody and to remove the whole conjugate, if it doesn’t

contain antibody.

5. Examine under fluorescent microscope.

The fluorochromes usually used are

Fluorescein isothyocyanate – yellow green

Rudamin - Red color

Reporting results

Fluorescence indicates the presence of specific antibody to T.pallidum. Non – fluorescence indicate

the absence of specific antibody to T.pallidum.

Preparation of Control Sera

1. Collect all reactive sera and store in freezer



2. Collect all non reactive sera and store in freezer

3. After collecting a large amount thaw at room temperature

4. Filter to remove the particles

5. Add mertiolet (preservative) 1 mg/ml serum.

6. Make a serial dilution of reactive with non reactive sera

7. Perform several tests each day. Select the dilution, which always gives reactive, weakly reactive

and non-reactive result.

8. Prepare a large quantity of those dilutions

9. Distribute in a small container (alginate) and store in a freezer.

10. Control sera of graded reactivity should be included each time when serologic procedures are

performed.

NB: If the control sera fail to give the desired (known) result (do not produce the established

relativity) pattern the result of the specimen is unacceptable so to have acceptable result the

following should be done:

- Prepare another antigen suspension

- Test temperature must be adjusted at room temperature (23-290C)

- Adjust equipment

- Use commercially prepared controls

- Strictly follow the manufactures procedure.

Agglutination Test for Febrile DiseaseAgglutination Test for Febrile DiseaseAgglutination Test for Febrile DiseaseAgglutination Test for Febrile Disease

When any pathogenic microorganism invades the human body, the natural response is the

production of antibodies. The host and microbial factors influence the rate of antibody formation,

the type and amount of antibodies produced, and the persistence of antibody in the circulation.

Among the antibodies produced in response to certain pathogenic microorganism are febrile

agglutinins. The microorganism that elicits the production of febrile agglutinin is characterized by

presence of persistent fever & frequently difficult to grow in laboratory cultures. Some of the

causative agents of febrile diseases are salmonella species, rickettsial and brucella abortus.

Typhoid and Paratyphoid FeverTyphoid and Paratyphoid FeverTyphoid and Paratyphoid FeverTyphoid and Paratyphoid Fever

The etiological agent is Salmonella species; it occurs in human only. Sometimes it is termed as

enteric fever since they colonize the intestine. Salmonella of medically important species are

S.typhi (typhoid fever), S.paratyphi A and B (paratyphoid fever). Typhoid and paratyphoid fever is



transmitted through ingestion of contaminated food or water. They contaminate usually by carriers

like rodents, hens, cows, etc. Typhoid or enteric fever is a clinical syndrome characterized by

fever, headache, splenomegaly, leucopenia & cough. Its incubation period ranges from 7 to 14

days. In 5% to10% of untreated patients relapse may occur the symptoms in relapse are milder than

the initial illness and begin about two weeks after discontinuation of antimicrobial therapy. The

carrier state is assymptomatic and in 1 - 3% of carriers there is continuous excretion of S.typhi for

a minimum of one year. The gall bladder is the site of persistent intestinal infection.

Identification of salmonellaIdentification of salmonellaIdentification of salmonellaIdentification of salmonella

Salmonella species can be identified based on their antigenic structure they possess. They have

three different antigenic structures.

O- antigen (somatic antigen)

It is lipopolysaccharide of the outer membrane, which is heat and alcohol stable antigen.

Salmonella is divided in to five distinct serogroups (A-E) on the basis of somatic antigen.

H-antigen (flagellar antigen)

H-antigen is protein, which makes the perithrchous flagella. It is heat and alcohol labile.

Salmonella is further subdivided in to more than 1200 serotypes on the basis of flagellar antigens.

Vi- Antigen:

This is the antigen that determines the virulence, the ability to cause disease, of the organism.

Preparation of antigen suspension

Salmonella antigen suspension is available commercially and it’s also possible to prepare in the

laboratory.

A. Preparation of H antigen (flagellar antigen)

Procedure

1. Inoculate bacteria from a single colony in to a broth and incubate for 6hrs.

2. View a drop of the culture in a wet film to confirm that most of the bacteria are motile and

therefore sufficiently flagellated for the tests.

3. Kill the culture by adding formaldehyde to a concentration of 0.2% and incubate for several

hours at 370C

B. Preparation of O antigen

Procedure

1. Suspend the bacteria from an agar culture in saline and heat for 30 minute at 1000C to remove

the flagella.



2. Centrifuge to separate the bacteria from the detached flagella.

3. Resuspend the bacteria in saline.

Alternatively

1. Remove the flagella by mixing a dense saline suspension of the bacteria with an equal volume of

absolute ethanol

2. Incubate for 20 hr at 370C

3. Dilute the suspension with saline.

Widal test

Widal test is a serological test, which is commonly used to diagnose typhoid and paratyphoid

fever. The patient’s serum is tested for O and H antibodies

Rapid slide (Screening) test

1. Clean the glass slides supplied in the kit well and wipe it free of water.

2. Place one drop of undiluted test serum in each of the first circle (1to4) and one drop of positive

control serum in each of the last two circles.

3. Place one drop of antigen O, H, A (H) and B (H) in circle 1,2,3, &4 respectively and O antigen

in circle five and H antigen in circle 6

4. Mix the contents of each circle with separate applicator stick and spread to fill the whole area of

the individual circle.

5. Rotate the slide for one minute and observe for agglutination.

If agglutination is visible, quantitative estimation of the titer of the appropriate antibodies should

be done

Tube agglutination method

Procedure

1. Take a set of 8 clean dry test tubes for each serum to be tested.

2. Place 1.9ml of saline in tube 1 and 1 ml of saline in other tuber (2-8)

3. Transfer 0.1 ml of undiluted serum to tube 1. Mix thoroughly. The resultant dilution of serum is

1:20.

4. Further dilutions are done in the following

a) Transfer 1ml of the diluted serum from tube 1 and place in tube 2 this leads to 1:40 dilutions in

tube 2

b) Repeat the transfer process for tube 7 after mixing.

c) Leave 1 ml of saline in tube 8 at the ‘saline control’

Note. Tube 1 has a serum dilution of 1:20, 1:40 (2), 1:80 (3), and 1:160 (4), 1:320 (5), 1:640 (6) &

1:1280 (7).



5. Add one drop of appropriate antigen in each (use only that antigen suspension which has given a

positive reaction in the screening test).

Note: Each antigen (O, H, AH, BH) will require a series of 8 tubes for determine the titer of their

corresponding antibodies.

6. Mix well and incubate overnight (16-18 hrs) at 370C.

7. Examine agglutination macroscopically.

8. Two tubes for positive control O and it antigen should be included.

InterpretationInterpretationInterpretationInterpretation

1. Only a titer above 1:80 should be considered as significant.

2. A rise in titer (done each week) is considered to be definite evidence of infection. A single test

result is considered of diagnostic value only when it is usually high (above 160).

3. Antibiotic treatment in typhoid fever often prevents a rise in titer,

4. A negative test does not rule out the possibility of infection because of the tine when the blood

sample was taken in relation to the stage of the disease.

5. Positive results should always be interpreted with reference to clinical findings.

Rickettsial DiseaseRickettsial DiseaseRickettsial DiseaseRickettsial Disease

Rickettsiae resemble viruses in that they are obligate intercellular parasite and unable to survive as

free-living organisms. They are about the size of the large viruses and can just be seen with the

light microscope. Unlike viruses rickettsiae contain both RNA and DNA multiply by binary

fission, they have cell wall that contains muramic acid and enzyme. Based on their antigenic

structure, the genus rickettsia has been divided into three main groups: typhus group (R.

prowazeki, R. typhi), scrub typhus group (R. tsutsugamushi), and spotted fever group (R.conori,

R.siberica, and R.rickettsi R.conoripijperi). Man is an accidental host of rickettsia species except R.

prowazeki; they live in intestinal tract of louse, fleas, ticks and mites. Reservoirs host include,

dogs, rats, mice, rodents Rickettsial disease can be acquired by inhaling of dried infected vector

faces, through damaged skin bite of an infected vector ticks, mite, etc. The infection is

characterized by high continuous fever, severe head ach and body pains, marked weakness,

enlarged spleen

Laboratory diagnosis

Embryonated egg inoculation technique used for culturing viruses can also be used for isolating

rickettsiae however it require costly materials and performed in reference laboratory.



SerologySerologySerologySerology

In rickettsial infection, specific IgM antibodies are produced followed by IgG response in the later

stages. The most reliable and useful serological techniques to diagnose rickettsial infections are

immunofluorescent assay and complement fixation test; however, this test is not available in

district laboratory due to its cost.

WeilWeilWeilWeil----felix reactionfelix reactionfelix reactionfelix reaction

A Weil Felix test is a type of agglutination test most commonly used serologic test. The reaction is

based on similarity of particular antigenic determinant, which occur in most species of pathogenic

rickettsia and in the OX-19, OX -2 strains of Proteus vulgaris and OX-K strains of Proteus

mirabilis. In other word, Proteus antigen is used to detect rickettsial antibody. This could be an

example of hetrophile antigen antibody reaction. Weil Felix test has similar principle and

procedure with Widal test.

Note: False negative reactions are common in scrub typhus. False positive reactions may occur in

Proteus infections, relapsing fever, brucellosis and other acute febrile illnesses. A rise in titer in

two consecutive specimen collected in interval is significant than a rise in titer in single specimen

and a rise in titer in single specimen should not be taken as a positive sample.

Brucella AbortusBrucella AbortusBrucella AbortusBrucella Abortus

Brucella aborts, gram-negative bacilli, is the causative microorganism of brucellosis. If is a

zoonoses that infects humans by accident. The agents of brucellosis are normal flora of the genital

and urinary tract of many animals including pigs, cows, and dogs. Most human acquire brucellosis

because of the ingestion of contaminated food products or through occupational exposure. Farmers,

veterinarians, and slaughterhouse workers are particularly prone to infection.


Because of the difficulty of isolating this organism by the culturing technique, many cases of

brucellosis are diagnosed serologically by identifying the presence of antibodies. Antibodies

usually appear within 2 to 3 weeks after infection. An antibody titer of 1:80 to 1:60 strongly

suggests infection.

Human Chorionic Gonadotropin Hormon (HCG)Human Chorionic Gonadotropin Hormon (HCG)Human Chorionic Gonadotropin Hormon (HCG)Human Chorionic Gonadotropin Hormon (HCG)

HCG and PregnancyHCG and PregnancyHCG and PregnancyHCG and Pregnancy

Human chorionic gonadotropin (HCG) is a hormone secreted by placenta during pregnancy. Its

production stimulates secretion of progesterone by the ovary. Adequate levels of progesterone are

necessary for successful implantation and prevent any further release of egg from the ovary.



Human chorionic gonadotrophin is a glycoprotein, has alpha and beta sub units. The alpha subunit

usually cross reacts with the alpha subunit of leutenizing hormone; however, the beta subunit is

specific for HCG. It appears in urine, blood and amniotic fluid. The serum and urine level rise

rapidly during gestation, reaching a peak at six to eight weeks, after which there is a steady

decline.

Pregnancy TestsPregnancy TestsPregnancy TestsPregnancy Tests

Laboratory tests for pregnancy are based on the detection of human chorionic gonadotrophin

hormone in serum or urine; mainly there are two types of test.

I. Biologic animal Bioassay (A-Z test)

This test is performed in laboratory animal (female mouse). i.e. Patient’s urine is injected in to a

female mouse after certain period, the mouse will be killed and the ovary will be examined for sign

of pregnancy. However, this test cannot be used for early diagnosis. Moreover it is time consuming

and requires steady supply of laboratory animals.

II. Immunologic test. Immunologic test. Immunologic test. Immunologic test

Immunologic test could be qualitative and quantitative. Qualitative estimation of HCG in urine is

used for early detection and confirmation of pregnancy. Quantitative estimation of HCG in serum

has of value in case of preeclamptic toxemia, hydatidiform mole and choriocarcinoma. Compared

to biologic animal assay, immunologic test is less expensive and quicker test.

10.3 Specimen Collection

An early morning urine specimen is preferable because this is the most concentrated and contains

the highest level of HCG. However, specimen collected at any time may be used with a specific

gravity at least 1.010. Urine must be collected in a clean detergent free container. If it cannot be

tested immediately, it should be refrigerated at 40C for not longer than 48 hours. Specimen

preserved with boric acid is also suitable for testing. When tested, the urine and test reagents

should be at room temperature. If the urine is cloudy it should be filtered or centrifuged and the

supernatant fluid used. Specimens that are heavily contaminated or contains large amount of

proteins or blood, are not usually suitable for testing.

There are two types of immunologic test commonly available and provided in a form of kit.

Rapid latex slide test: have two types

I. Indirect latex slide test

II. Direct latex slide test

Tube test (haemagglutination inhibition technique)



A. Rapid latex slide test

I. Indirect latex slide test

Principle

Urine specimen is first treated with anti-HCG and then reacted with the latex suspension. If the

urine contains HCG, the anti HCG will be neutralized and then the latter will not be available to

the HCG coated latex particles for bringing about agglutination.

Reagents and materials

Antiserum that contain HCG antibody

Latex reagent coated with HCG

Positive and negative controls

Mixing sticks and slides

Procedure

Note: In all case it is better to refer manufacturer manual

1. Place one drop of urine sample on the ring of the slide provided by the manufacture. 2. Add one

drop of anti-HCG reagent to the urine specimen placed on the slide. Mix the two fluids well with

applicator stick.

Note. In order to maintain the same volume, always hold the dropper in the same vertical position

and use the same vertical position and use the same kind of dropper for both urine specimen and

the antiserum.

3. Rock the slide gently for about 30 seconds

4. Gently shake the vial with latex antigen and then add one drop.

5. Mix again with applicator stick and observe the appearance of agglutination at 2 minutes under a

bright light.

Reporting

Latex particle agglutinated _______ Negative (non-reactive)

Homogenous suspension of latex particles without any sign of agglutination _______Positive

(Reactive)

II. Direct latex slide testII. Direct latex slide testII. Direct latex slide testII. Direct latex slide test

Principle

The reaction is based on the reaction between HCG in urine and the latex particles coated with anti

HCG. In positive result agglutination will be observed.

B. Haemagglutination inhibition test (tube test)

It is more sensitive than slide test



Principle: similar with latex slide test except the HCG is coated on red cells, not on polystyrene

particles.

Procedure

1. Add a drop of urine and drop of anti-HCG antiserum in a small tube.

2. Add red cell coated with HCG

3. Mix the contents of the tube and leave at room temperature (20-280C) for 1-2 hrs to allow time

for the red cells to settle.

4. If the urine contains HCG it will combine with the antibody. This will leave no antibody to react

with the HCG on the red cells.

5. If the urine contains no HCG, the anti HCG antibody will react with the HCG on the red cells

and cause their agglutination.

Reporting

Reactive __________Non-agglutinated cells settle in the bottom of the tube.

Non-reactive _______Agglutinated cells settle and cover the bottom of the tube.

Factors Affecting Pregnancy Tests

Different factors influence the result of pregnancy test these are False Negative may occur in

conditions like:

Error in reading – inappropriate interpretation of procedure

Test is performed too early-The concentration of HCG is below the sensitivity of the test, which is

capable of detecting reliably. The sensitivity of a test, the recommended time of testing will be

included in the information supplied by the manufacture.

Urine too diluted -falsely low levels of HCG may be due to a diluted urine (low specific gravity)

Ectopic pregnancy implantation of the ovum outside the uterine cavity

False positive may occur in conditions like

Error in reading- inappropriate interpretation of test procedure

Luteinizing hormone cross-reaction

Test performed at time of ovulation or in menopausal women

Proteinuria and hematuria

Recent pregnancy -test performed less than 10 days after abortion of full-term delivery.

Detergents on glassware and slide used in the test, it must be well rinsed to remove trace of

detergent even the smallest trace of detergents may affect the performance of the test.

Drug interference- aldomet, marijuana, aspirin in large doses, etc.

HCG treatment for infertility



Trophoblastic disease e.g. molar pregnancy or choriocarcinoma

HCG secreted by malignant tumor (ovary, breast, lung, kidney)

Testicular tumor (in male)

Use of pregnancy test

Situations in which pregnancy testing is indicated include pregnancy test usually ordered to

investigate some conditions like ectopic pregnancy, threatened abortion, hydatiform mole, and

choriocarcinoma. It also used for checking a woman of childbearing age is pregnant before

carrying out medical or surgical investigation, x-ray or drug therapy that could be harmful to an

embryo.

22. 22. 22. 22. Human Immunodeficiency Virus (HIV)Human Immunodeficiency Virus (HIV)Human Immunodeficiency Virus (HIV)Human Immunodeficiency Virus (HIV)

Disease Characteristics and Clinical Manifestation

Infection with HIV produces a chronic infection with symptoms that range from a symptomatic to

the end stage complications of AIDS. Typically, patients in the early stages of HIV infection are

either completely asymptomatic or may show mild, chronic swelling of lymph nodes. The early

phase may last from many months to many years after viral exposure. During the early period after

primary infection, widespread dissemination of virus occurs and a sharp decrease in the number of

CD4 T- Cells in peripheral blood is manifested. The early burst of virus in the blood is often

accompanied by flulike symptom. This phase is followed by a prolonged period of clinical latency

range 7 to 11 years. During this period the patient is usually a symptomatic. Due to different

factors, there is a variation in the duration of clinical latency. The quantity of CD4 lymphocytes

continues to diminish as the disease progresses and when the number of cells reaches a critically

low level the risk of opportunistic infection increases. Clinical symptoms of the later phase of the

disease include extreme weight loss, fever and multiple secondary infections. The end stage of

AIDS is characterized by the occurrence of opportunistic infection like M. tuberculosis,

Salmonella, P. carinii, etc.

LLLLaboratory Diagnosisaboratory Diagnosisaboratory Diagnosisaboratory Diagnosis

A ‘window’ period of seronegativity exists from the time of initial infection to 6 or 12 weeks or

longer. Serological screening tests designed to detect HIV antibodies are usually enzyme linked

immunosorbent assay and dot blot assay; western blot assay is commonly used confirmatory test.

Enzyme Linked ImmunoSorbent Assay (ELISA)Enzyme Linked ImmunoSorbent Assay (ELISA)Enzyme Linked ImmunoSorbent Assay (ELISA)Enzyme Linked ImmunoSorbent Assay (ELISA)



The indirect ELISA is the most commonly utilized test that is supplied in the form of kit. In this

type of assay, an antigen coated on a solid phase combine with the patient’s serum containing

antibody, the antigen antibody complex will interact with conjugate (enzyme Labeled with anti

human immunoglobulin) then a color change is observed up on addition of a substrate. The

intensity of the color gives an indication of the amount of bound antibody.

Dot blot assay (HIV spot test)Dot blot assay (HIV spot test)Dot blot assay (HIV spot test)Dot blot assay (HIV spot test)

Dot blot assay are rapid and easy to perform. In this type of assay antigens are coated on micro

particles that are trapped within a membrane. These antigen bind with HIV antibody on the

patient’s sample than a color production is observed when the conjugate is added on antigen

antibody complex.

Western blot assay

Western blot assay is the most widely accepted confirmatory assay for the detection of HIV;

however, it is time consuming and expensive test. In the western blot procedure, purified HIV-1

viral antigens are electrophoresed on sulfate polyacryamide gel (SDS gels) and the separated

polypeptides are then transferred on to sheets of nitrocellulose paper incubated with the serum

specimen. Any antibody that binds to the separated peptides present on the nitrocellulose paper is

detected by a secondary antihuman antibody, conjugated to enzyme substrate. Antibody

specificities against known viral components are considered true positive results, whereas

antibodies specific against non-viral cellular contaminants are nonspecific, false-positive results.

Viral hepatitis is the most common liver disease worldwide. The viral agents of acute hepatitis can

be divided in to twomajor groups

1. 10 hepatitis viruses: A, B, C, D & E

2. 20 hepatitis viruses: Epstein- Barr virus, cytomegalovirus, herpes virus, etc.

Primary hepatitis viruses attack primarily the liver and have little direct effect on other organ

system. Secondary viruses involve the liver secondary in the curse of systemic infection of another

body system.

Hepatitis A virusHepatitis A virusHepatitis A virusHepatitis A virus (Infectious or short(Infectious or short(Infectious or short(Infectious or short---- incubation hepatitis)incubation hepatitis)incubation hepatitis)incubation hepatitis)

Hepatitis A virus is a small, single stranded RNA virus when seen by electron microscope.

Infection can be acquired by ingestion of virus in contaminated food or water from hands or other

objects contaminated with infected feces (fecal routes), after exposure within 2-6 weeks clinical

symptoms will develop. In acute phase of infection, the highest titers of HAV can be detected in

stool sample. Shortly after the onset of fecal shedding, an IgM antibody is detectable in serum,



followed within a few days by the appearance of an IgG antibody. IgM anti-HA is almost always

detectable in patients with acute HA. IgG anti-HA, a manifestation of immunity, peaks after the

acute illness and remains detectable indefinitely, perhaps lifelong. The finding of IgM anti- HA in

a patient with acute viral hepatitis is highly diagnostic of acute HA. Demonstration of IgG anti-HA

indicates previous infection. The presence of IgG anti-HA protects against subsequent infection

with HA virus, but it is not protective against HBV or other viruses.

Laboratory diagnosisLaboratory diagnosisLaboratory diagnosisLaboratory diagnosis

Testing methods for hepatitis A virus include the following:

1. Total antibody by enzyme immunoassay (EIA)

2. IgM antibody by RIA

3. HA antigen by radioimmunoassayt (RIA)

Hepatitis B virus

(Long term or serum hepatitis)

Hepatitis B virus is a double stranded, DNA virus. It is the classic example of a virus acquired

through blood transfusion. It has various antigens hepatitis B surface antigen, an outer coat,

hepatitis B antigen that is an inner core component and hepatitis B core antigen. The major routes

of transmission of hepatitis B virus included blood transfusion, sexual inter course, transplacental

and sharing of contaminated needle. The incubation period of hepatitis B virus may range from 6-

26 weeks. Infected patients manifest hepatitis B virus in all body fluids including blood, feces,

urine, saliva, semen, tear and milk.


Serum that is collected in acute stage of illness can be tested by:

Counterimmunoelectrophoresis

Enzyme Linked immuno sorbent assay

Reverse passive Hemagglutination test

Reverse passive hemagglutination is the commonly employed test since it is less expensive and

sensitive test.

Hepatitis C virusHepatitis C virusHepatitis C virusHepatitis C virus

Viral hepatitis caused by hepatitis c virus, the identification of this virus is not clear, sometime it

known as non-A/non-B hepatitis. This virus is commonly acquired by contaminated blood and

blood products.


Hepatitis C virus antibody can be detected from serum usually by radioimmunoassay.



Hepatitis D virus (Delta virus)Hepatitis D virus (Delta virus)Hepatitis D virus (Delta virus)Hepatitis D virus (Delta virus)

It is defective or incomplete RNA virus that is unable by itself to cause infection, i.e., transmitted

through blood products. HBV is required as a helper to initiate delta infection only persons with

acute or chronic HBV infection can be infected with delta agent.

Laboratory diagnosisLaboratory diagnosisLaboratory diagnosisLaboratory diagnosis---- Radio immuno assayRadio immuno assayRadio immuno assayRadio immuno assay

Hepatitis E virusHepatitis E virusHepatitis E virusHepatitis E virus

This is responsible for large water borne out breaks incubation period 6 weeks.

Laboratory diagnosis: Specific test for IgM & IgG antihepatitis E virus.

C - reactive protein

The main biologic sign of inflammation is an increase in the erythrocyte sedimentation rate (ESR).

In addition an increase in plasma concentrations of a group of proteins known as acute-phase

proteins is a good indicator of local inflammatory activities and tissue damage. The acute phase

proteins include C-reactive protein (CRP), inflammatory mediators (e.g. complement components

c3 and c4, fibrinogen, etc. CRP is prominent among the acute-phase proteins because it provides

fast and adequate information of the actual clinical situation; as a result CRP is a direct and

quantitative measure of the acute-phase reactions.

Measures of CRP add to the diagnostic procedure in selected cases (e-g. in the differentiation

between a bacterial and a viral infection). An extremely elevated CRP is suggestive of a possible

bacterial infection. The CRP level may be useful also for monitoring the effect of treatment and for

early detection of postoperative complications or intercurrent infections. The CRP is a parameter

for inflammatory activity. CRP is a method of choice for screening for inflammatory and malignant

diseases and monitoring therapy in inflammatory disease. Elevations of CRP occur in nearly to

diseases states, including bacterial infection, viral infections, and myocardial infarction specificity

rules out CRP as a definitive diagnostic tool.

The CRP test has been widely used to detect infection in circumstances where microbial diagnosis

is difficult. These conditions include septicemia and meningitis in neonates, infections in

immunosuppressed patients, serious post operative infections etc. CRP levels rise following the

tissue injury or surgery. In uncomplicated cases the level of CRP peaks about 2 days

postoperatively and gradually returns to normal levels within 7 to 10 days. CRP is synthesized

more rapidly than other acute phase proteins; assays of CRP are the measurement of choice in

suspected inflammatory conditions.



Tests for CRPTests for CRPTests for CRPTests for CRP

Rapid latex agglutination testRapid latex agglutination testRapid latex agglutination testRapid latex agglutination test

Principle: The test is based on the reaction between patient serum containing CRP as the antigen &

the corresponding antibody coated to the treated surface of latex particle. The coated particles

enhance the detection of an agglutinate reaction when antigen is present in the serum being tested.

Specimen- Serum

Reagent & materials required

CRP latex reagent

Glycine – saline buffer

Capillary pipette

Applicator sticks

Glass slide

Serologic pipettes & rubber bulb

Quality control

Include positive & negative control serum.

Procedure

1. Deliver one drop of undiluted serum on a slide by using capillary Pipette.

2. Deliver one drop of positive and negative control on separate (other) circle of the slide

3. Add one drop of CRP latex reagent to each serum specimen & to each control

4. Mix the suspension using separate applicator sticks.

5. Tilt the slide back & forth slowly for two minutes observe for agglutination macroscopically.

Reporting

Positive reaction – agglutination

Negative reaction– absence of agglutination

Streptolysin O

Streptolysin O is a hemolytic factor produced by most strains of GroupA beta- hemolytic

streptococci (S. pyogenes). Streptococci are gram-positive cocci in chain, non-motile, facultative

anaerobes. It produce toxin like streptolysin O & streptolysin S and enzymes like DNAase,

streptokinase. It is oxygen & heat labile immunogenic enzyme with molecular weight range from

50,000-75,000 Dalton, which cause lysis of red cells under reduced condition. It can severely

damage or destroy PMN leukocytes, also able to destroy adjacent cells and tissues and thus

contribute to the spread of organism from local sites.

Antistreptolysin O (ASO)Antistreptolysin O (ASO)Antistreptolysin O (ASO)Antistreptolysin O (ASO)



Is specific neutralizing antibody produced after infection with these organisms & it appears in

serum from 1 week-1month after the onset of a streptococcal infection. It combines and neutralizes

the heamolytic activity of streptolysin O.

Streptolysin S

Oxygen stable non-antigenic toxic enzyme with molecular weight of 20,000 Dalton. It hemolyze

red cells and phagocytic cells by direct cell to cell contact also it is responsible for the surface

heterolysis observed around colonies of group A streptococci grown on blood agar plate.

SeroSeroSeroSerological testlogical testlogical testlogical test

Antistreptolysin O test is used to diagnose conditions post streptococcal resulting from a

streptococcal infection especially in diagnosis of rheumatic fever and glomerulonephritis when

it’s not possible to isolate Group A streptococci in culture (most complication develop at a stage

when it is not possible to isolate group A streptococcus in culture). The principle of the test

depends on the following factors. Antistretolysin O can react specifically with SLO and inhibits the

heamolytic activity. The amount of ASO can be estimated by dilution of patient’s serum in the

presence of constant amount of SLO to the point where there is still complete prevention of

haemolysis. The occurrence of ASO depends on the production of SLO by streptococci in the

infected host.

Commercially available test are:

Antistreptolysin O latex slide test- used for screening a significant raise in ASO titer

Antistreptolysin O titration test –used to determine the titer of ASO antibody.

Rapid Antistreptolysin O latex agglutination test

Principle: In the presence of ASO antibody a visible agglutination reaction will be exhibited when

a serum specimen combine with latex particle coated with streptolysin

O antigen.

Specimen

Clear, haemolysis free serum

Reagent & equipment required

Latex particle coated with streptolycin

0.9% NaCl solution

Glass slide with six cells

Applicator sticks (stirrer)



Control reagent

Other material required

Timer

Test tubes

Pasture pipettes and rubber bulb

Serologic pipette and safety bulb

Quality control

Positive control-a prediluted serum containing at least 200 lu/ml of ASO. This control should

exhibit visible agglutination at the end of the 3-minute test period. Negative control serum a

prediluted serum containing less than 100 Iu/ml of ASO. This control should exhibit a smooth or

slightly granular appearance at the end of the 3-minute test period. A positive and negative control

should be tested and read concurrently with each group of patient sera.

Procedure

1. Dilute the serum by saline in 1:2

2. Label the slide for positive control dilution negative control and patient sera

3. Pipette 50μl of the controls and patient sera onto the appropriately labeled cell (well)

4. Add one drop of latex reagent to each well

5. Mix the specimen with separate applicator stick spread the mixture evenly over the well.

Rotate the slide for exactly 3 minutes. Examine with a bright light

Reporting

Positive – agglutination

Negative- No agglutination

Agglutination demonstrates 200 lu/ml or more ASO. Positive results should be retested

quantitatively.

A titer of 200Iu/ml or greater may be associated with rheumatic fever or glomerulonephritis.

Antistreptolysin O titration kit

In the titration test, a constant amount of streptolysin O antigen reagent is added to a series of

dilutions of the patient’s serum. Following a period of incubation, Group O washed human or

rabbit red cells are added. The tubes are then examined for lysis of the red cells. Haemolysis

occurs in those tuber in which there is insufficient antibody to neutralize the antigen. The highest

dilution of serum showing no haemolysis is the ASO titer; the titer of ASO antibody in the serum

is directly proportional to the reciprocal of the serum dilution.

Bibliography



1. Cheesbrough Monica. Medical Laboratory Manual for Tropical Countries; vol ll

2000.Cambridge Butter worth.Heinemann.Ltd.

2. P.Stities Daniel. Basic and Clinical Immunology; 8thed, 1994,USA 4.Fischbach Frances,

Manual of Laboratory and Diagnostic tests; 4ed 1992,Lippincott.

3. 5. Turgeon L.M, Immunology and Serology in Laboratory Medicine,2nded, 1996,Mosby.

4. Male, Brostoff, Roth and Roitt: Immunology. 6th ed.

5. Male, Brostoff, Roth and Roitt: Immunology. 7th ed.

Documents

Immunology and Immuno-technology