NITRIC OXIDE-DEPENDENT 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 (figure 12). Nitric oxide released by the cell is toxic and can kill microorganism in the vicinity of the macrophage.

STRUCTURE AND SOME PROPERTIES OF IG CLASSES AND SUBCLASSES

A.  IgG

1. Structure

 All IgG’s are monomers (7S immunoglobulin). The subclasses differ in the number of disulfide bonds and length of the hinge region.

2. Properties

IgG is the most versatile immunoglobulin because it is capable of carrying out all of the functions of immunoglobulin molecules.

a) IgG is the major Ig in serum – 75% of serum Ig is IgG

b) IgG is the major Ig in extra vascular spaces

c) Placental transfer – IgG is the only class of Ig that crosses the placenta. Transfer is mediated by a receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally well; IgG2 does not cross well.

d) Fixes complement – Not all subclasses fix equally well; IgG4 does not fix complement

e) Binding to cells – Macrophages, monocytes and neutrophils and some lymphocytes have Fc receptors for the Fc region of IgG.  A consequence of binding to the Fc receptors on such cells  is that the cells can now internalize the antigen better. The antibody prepares the antigen for killing by the phagocytic cells. The term opsonin is used to describe substances that enhance phagocytosis. (Coating of the surface of pathogen by antibody is called opsonization).IgG is a good opsonin. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions.

IgM

1. Structure
 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 mu 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.

2. Properties

a) IgM is the third 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 an extra 20 amino acids at the C-terminus to anchor it into the membrane . Cell surface IgM functions as a receptor for antigen on B cells.

IgA

1. Structure

Serum IgA is a monomer but IgA found in secretions is a dimer as presented in Figure 10. 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 . The secretory piece helps IgA to be transported across mucosa and also protects it from degradation in the secretions.

2. Properties

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

b) 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.

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

d) IgA can binding to some cells – PMN’s and some lymphocytes.

IgD

1. Structure

 IgD exists only as a monomer.

2. Properties

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

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

c) IgD does not bind complement.

E. IgE

1. Structure

IgE exists as a monomer and has an extra domain in the constant region.

2. Properties

a) 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.

b) Involved in allergic reactions – As a consequence of its binding to basophils and 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.

c) 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.

d) IgE does not fix complement.

NORMAL MICROBIAL FLORA 

A. Properties. Normal microbial flora describes the population of microorganisms that usually reside in the body. The microbiological flora can be defined as either 
1) Resident flora - A relatively fixed population that will repopulate if disturbed, 

2) Transient flora - that are derived from the local environment. These microbes usually reside in the body without invasion and can
even prevent infection by more pathogenic organisms, a phenomenon known as bacterial interference. 
The flora have commensal functions such as vitamin K synthesis. However, they may cause invasive disease in immunocompromised hosts or if displaced from their normal area. 

B. Location. Microbial flora differ in composition depending on their anatomical locations and microenvironments. The distribution of normal microbial flora.

Classification:

Neutrophiles (pH = 7.0)
- P. aeruginosaqo
- Clostridium sporogenes
- Proteus species

Acidophiles (pH < 7.0)
- Thiobacillus thiooxidans
- Sulfollobus acidocaldaarius
- Bacillus acidocaldarius

Alkaliphiles (pH > 7.0)
- Nitrobacter species
- Streptococcus pneumoniae

Measurement of Bacterial of Growth

A convenient method is to determine turbidity by photoelectric colorimeter or spectrophotometer. 
The cell number can be counted as total cell number as well as viable count. Viable Count Viable number of bacteria can be counted by inoculating the suspension onto solid growth medium and counting the number of colonies. Since each colony is the end product of one viable bacterium, their count gives the number of viable bacteria in the suspension.
Total number of bacteria can be ascertained in specially designed chambers such as Coulter counter.
 

Test for Antigen - Antibody Reactions

Antigens are those substance that stimulates the production of antibodies which, when enter into the body it reacts specifically in a manner that are clearly visible. 

Some antigens may not induce antibody production, but instead creates immunological tolerance. 
An antigen introduced into the body produces only specific antibodies and will react with only those specific antigens. 
These antibodies appear in the serum and tissue fluids. All antibodies are considered as immunoglobulin. They are mainly of five classes; IgG, IgA, IgM, IgD and IgE. 

Antigen- antibody reactions are known as serological reactions and are used as serological diagnostic tests for the identification of infectious diseases.

The reaction occurs mainly in three stages; 

1. The initial interaction between the antigen and antibody, which produces no visible effects. It is a reversible and rapid reaction.
2. The secondary stage leads to the demonstration proceedings, such as precipitation, agglutination, etc.
3. The tertiary reaction follows the neutralization or destruction of injurious antigens. These results in clinical allergy and other immunological diseases.

There are certain characteristics for antigen-antibody reactions;

1. Reaction is specific.
2. The whole molecules participate in the reaction, and not just a part of it.
3. No denaturation of antigen or antibody occurs during the reaction.
4. The combination usually occurs at the surface.
5. The combination is firm, but reversible
6. Agglutinins formed after agglutination usually are formed by both antigen and antibody together.
7. They can combine in varying proportions.

Measurement of antigen and antibody are made in terms of mass or as units or titre.

Serological reactions include;

1. Precipitation reaction

a soluble antigen combining with the specific antibody in the presence of electrolytes at a suitable temperature and pH forming insoluble precipitins.  Commonly used tests are ring test, slide test, tube test, immunodiffusion, etc.

Radial Immunodiffusion 

In radial immunodiffusion antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed .
This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples.

Immunoelectrophoresis 

In immunoelectrophoresis, a complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs .

This tests is used for the qualitative analysis of complex mixtures of antigens

This test can also be used to evaluate purity of isolated serum proteins.

Countercurrent electrophoresis

In this test the antigen and antibody are placed in wells punched out of an agar gel and the antigen and antibody are electrophoresed into each other where they form a precipitation line. 

2. Agglutination reaction 

when a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. When the antigen is an erythrocyte the term hemagglutination is used.

Applications of agglutination tests

i. Determination of blood types or antibodies to blood group antigens.
ii. To assess bacterial infections
e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.

Passive hemagglutination 

The agglutination test only works with particulate antigens. However, it is possible to coat erythrocytes with a soluble antigen (e.g. viral antigen, a polysaccharide or a hapten) and use the coated red blood cells in an agglutination test for antibody to the soluble antigen . This is called passive hemagglutination. 
The test is performed just like the agglutination test.

Applications include detection of antibodies to soluble antigens and detection of antibodies to viral antigens.

Coomb's Test (Antiglobulin Test)

DIRECT ANTIGLOBULIN TEST (DAT)

The DAT is used to detect IgG or C3 bound to the surface of the red cell.  In patients with hemolysis, the DAT is useful in determining whether there is an immune etiology.  
A positive DAT can occur without hemolysis
Immune causes of hemolysis including autoimmune hemolytic anemias, drug induced hemolysis, and delayed or acute hemolytic transfusion reactions are characterized by a positive DAT.

INDIRECT ANTIGLOBULIN TEST (IAT)

The IAT (antibody screen) is performed by incubating patient serum with reagent screening red cells for approximately 20 minutes and then observing for agglutination.  If the antibody screen is positive, additional testing is required to determine the specificity of the antibody. 

The IAT is used to detect red cell antibodies in patient serum.  Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both.

3. Complement fixation test (CFT)

the ability of antigen antibody complexes to fix complement is made use in this test. Complement is something which takes part in any immunological reaction and absorbed during the combining of antigen with its specific antibody. 

The best example of CFT is the Wassermann reaction done for the detection of Syphilis.

4. Neutralization test

different types of these are available. Virus neutralization, toxin neutralization, etc. are some of its kind.

5. Opsonization

this makes use of the determination of opsonic index, which is the ratio of the phagocytic activity of patient’s blood to the phagocytic activity of the normal patient’s for a given bacterium.

6. Immunfluorescence 

the method of labeling the antibodies with fluorescent dyes and using them for the detection of antigens in tissues.

7. Radioimmunoassay (RIA)

 is a competitive binding radioisotopes and enzymes are used as labels to conjugate with antigens or antibodies.

8. Enzyme Immuno Assay (EIA)

 the assays based on the measurement of enzyme labeled antigen or antibody. The most common example is ELISA used to detect HIV.

9. Immunoelectroblot

 it uses the sensitivity of Enzyme immunoassay with a greater specificity. Example is Western blot done for the serodiagnosis of HIV infection.