Immunoglobulin (Ig)

Immunoglobulins are glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that they migrate with globular proteins when antibody-containing serum is placed in an electrical field

FUNCTION
1. 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.

2. The significant biological effects are a consequence of secondary "effector functions" of antibodies.Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn.

STRUCTURE OF IMMUNOGLOBULINS

The basic structure of the immunoglobulins is illustrated in figure 2. Although different immunoglobulins can differ structurally, they all are built from the same basic units.

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)

B. Disulfide bonds

1. Inter-chain disulfide bonds - 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 inter-chain disulfide bonds varies among different immunoglobulin molecules.

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

C. Variable (V) and Constant (C) Regions

When 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. These are the:

1. Light Chain - VL (110 amino acids) and CL (110 amino acids)

2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)\(x = {-b \pm \sqrt{b^2-4ac} \over 2a}\)h the arms of the antibody molecule forms a Y. It is called the hinge region because there is some flexibility in the molecule at this point.

E. Domains

Three dimensional images of the immunoglobulin molecule show that it is not straight as depicted in figure 2A. Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond (figure 2B-D). 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. 

IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS

Immunoglobulin fragments produced by proteolytic digestion –

A.  Fab 
Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 6. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.

Antigen binding – These fragments are  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. 

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 . 

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 . This fragment is called F(ab’)2because it is divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab’)2binds antigen but it does not mediate the effector functions of antibodies.
 

INNATE (NON-SPECIFIC) IMMUNITY

The elements of the innate (non-specific) immune system include anatomical barriers, secretory molecules and cellular components. 

Among the mechanical anatomical barriers are the skin and internal epithelial layers, the movement of the intestines and the oscillation of broncho-pulmonary cilia. 

Associated with these protective surfaces are chemical and biological agents.

A. Anatomical barriers to infections

1. Mechanical factors

The epithelial surfaces form a physical barrier that is very impermeable to most infectious agents. Thus, the skin acts as our first line of defense against invading organisms. The desquamation of skin epithelium also helps remove bacteria and other infectious agents that have adhered to the epithelial surfaces. 

2. Chemical factors

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. Biological 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.

B. Humoral barriers to infection

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.

1. Complement system – The complement system is the major humoral non-specific defense mechanism (see complement chapter). Once activated complement can lead to increased vascular permeability, recruitment of phagocytic cells, and lysis and opsonization of bacteria. 

2. Coagulation system – Depending on the severity 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.

3. Lactoferrin and transferrin – By binding iron, an essential nutrient for bacteria, these proteins limit bacterial growth.

4. Interferons – Interferons are proteins that can limit virus replication in cells.

5. Lysozyme – Lysozyme breaks down the cell wall of bacteria. 

6. Interleukin -1 – Il-1 induces fever and the production of acute phase proteins, some of which are antimicrobial because they can opsonize bacteria.

C. Cellular barriers to infection

Part of the inflammatory response is the recruitment of polymorphonuclear eosinophiles and macrophages to sites of infection. These cells are the main line of defense in the non-specific immune system.

1. Neutrophils – Polymorphonuclear cells  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.

2. Macrophages – 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.

3. Natural killer (NK) and lymphokine activated killer (LAK) cells – NK and 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. 

4. Eosinophils – Eosinophils  have proteins in granules that are effective in killing certain parasites.

THE PLASMIDS

The extrachromosomal genetic elements, called as plasmids are autonomously replicating , cyclic ,double stranded DNA molecules which are distinct from the cellular chromosome 

Classification

Plasmids can be broadly classified as conjugative and nonconjugative. 

Conjugative plasmids are large and self-transmissible i.e. they have an apparatus through which they can mediate their own transfer to another cell after coming in contact with the same. Example:  RF and certain bacteriocinogen plasmids.

Nonconjugative plasmids are small in size and can be mobilised for transfer into another cell only through the help of a conjugative plasmid. To this group belong some ‘r’ determinants and few bacteriocinogenic plasmids. Plasmids can also be transferred without cell contact by the process of transfection.

Properties of plasmids

Double stranded DNA , Autonomously replicate in host cell, Plasmd specific, Free DNA is transferred b transfection

Significance of Plasmids :The spread of resistance to antibiotics is one such well known example. These also play an important  role in the geochemical  cycle by spreading genes for the degradation of complex organic compounds.
 

CROSS INFECTION AND STERLIZATION IN DENTISTRY

Cross infection is defined as the transmission of infectious agents amongst patients and staff with in hospital environment.

Routes of Infection 
Two routes are important : transdermal  and respiratory. 

 In transdermal route microorganisms enter the tissues of the recipient by means of injection through intact skin or mucosa (usually due to an accident involving a sharp instrument) or via defects in the skin e.g. recent cuts and abrasions.
 
Microorganisms causing cross infection in dentistry

Transmitted through skin 

Bacteria : Treponema pallidum, Staphylococcus aureus

Viruses :Hepatitis virus, HIV ,Herpes simplex virus, Mumps, Measles , Epstein-Barr virus

Fungi: Dermatomycoses, Candidiasis, 

Transmitted through aerosols

Bordetella pertussis, Myco.tuberculosis, Streptococcus pyogenes, Influenza virus
Rhinovirus,  Rubella 
 

PHYSICAL AGENTS

Heat occupies the most important place as a physical agent.

Moist Heat : This is heating in the presence of water and can be employed in the following ways:

Temperature below 100°C: This includes holder method of Pasteurization where 60°C for 30 minutes is employed for sterilization and in its flash modification where in objects are subjected to a temperature of 71.1°C for 15 seconds. This method does not destroy spores.

Temperatures Around 100°C : Tyndallization is an example of this methodology in which steaming of the object is done for 30 minutes on each of three consecutive days. Spores which survive the heating process would germinate before the next thermal exposure and would then be killed.

Temperatures Above 100°C : Dry saturated steam acts as an excellent agent for sterilization. Autoclaves have been designed on the principles of moist heat.

Time-temperature relationship in heat sterilization
Moist heat   (autoclaving)

121°C       15 minutes
126°C         10 minutes
134 C          3 minutes

Dry heat

>160°C    >120 minutes
>170°C    >60minutes
>180°C    >30 minutes

Mechanism of microbial inactivation 

The autoclaving is in use for the sterilization of many ophthalmic and parentral products. surgical dressings, rubber gloves, bacteriological media as well a of lab and hospital reusable goods.

Dry Heat: Less efficient,  bacterial spores are most resistant. Spores may require a temperature of 140° C for three hours to get killed.
Dry heat sterilization is usually carried out by flaming as is done in microbiology laboratory to sterilize the inoculating loop and in hot air ovens in which a number of time-temperature combinations can be used. It is essential that hot air should circulate between the objects to be sterilized. Microbial inactivation by dry heat is primarily an oxidation process.

Dry heat is employed for sterilization of glassware glass syringes, oils and oily injections as well as metal instruments.    -

Indicators of Sterilization:  
These determine the efficacy of heat sterilization and can be in the form of spores of Bacillus stearothermophilus (killed at 121C in 12 minutes) or in the form of chemical indicators, autoclave tapes and thermocouples.

Ionizing Radiations

Ionizing radiations include X-rays, gamma rays and beta rays, and these induce defects in the microbial DNA synthesis is inhibited resulting in cell death. Spores are more resistant to ionizing radiations than nonsporulating bacteria.

The ionizing radiations are used for the sterilization of single use disposable medical items.

Mechanism of microbial inactivation by moist heat

Bacterial spores

•    Denaturation of  spore_epzymes
•    Impairment of germination
•    Damage to cell membrane
•    Increased sensitivity to inhibitory agents
•    Structural damage
•    Damage to chromosome

Nonsporulating bacteria

•    Damage to cytoplasmic membrane
•    Breakdown of RNA
•    Coagulation  of proteins
•    Damage to bacterial chromosome

Ultraviolet Radiations : 
wave length 240-280 nm have been found to be most efficient in sterilizing. Bacterial spores are more resistant to U.V. rays than the vegetative forms. Even viruses are sometimes more resistant than vegetative bacteria.

Mechanism of Action :

Exposure to UV rays results in the formation of purine and pyrimidine diamers between adjacent molecules in the same strand of DNA. This results into noncoding lesions in DNA and bacterial death.
Used to disinfect drinking water, obtaining pyrogen free water, air disinfection (especially in safety laboratories, hospitals, operation theatres) and in places where dangerous microorganisms are being handled.

Filteration

Type of Filters

Various types of filters that are available are    /
Unglazed ceramic filter (Chamberland and Doulton filters)
Asbestos filters (Seitz, Carlson and Sterimat filters)
Sintered glass filters

Membrane filters

Membrane filters are widely used now a days. Made up of cellulose ester and are most suitable for preparing_sterile solutions. The range of pore size in which these are available is 0.05-12 µm whereas the required pore size for sterlization is in range of 0.2-0.22 p.m.

Radioimmunoassays (RIA)

It is an extremely sensitive technique in which antibody or antigen is labelled with a radioactive material. The amount of radioactive material in the antigen-antibody complex can be measured with which concentration of antigen or antibody can be assayed. After the reaction ‘free’ and ‘bound’ fractions of antigen are separated and their radioactivity-measured.