NON-SPECIFIC KILLER CELLS

Several different cells including NK and LAK cells, K cells, activated macrophages and eosinophils are capable of killing foreign and altered self target cells in a non-specific manner. These cells play an important role in the innate immune system.

A. NK and LAK cells

Natural killer (NK) cells are also known as large granular lymphocytes (LGL) because they resemble lymphocytes in their morphology, except that they are slightly larger and have numerous granules.

NK cells can be identified by the presence of CD56 and CD16 and a lack of CD3 cell surface markers.

NK cells are capable of killing virus-infected and malignant target cells but they are relatively inefficient in doing so.

However, upon exposure to IL-2 and IFN-gamma, NK cells become lymphokine-activated killer (LAK) cells, which are capable of killing malignant cells.

Continued exposure to IL-2 and IFN-gamma enables the LAK cells to kill transformed as well as malignant cells. LAK cell therapy is one approach for the treatment of malignancies.

NK and LAK cells have two kinds of receptors on their surface – a killer activating receptor (KAR) and a killer inhibiting receptor (KIR). 

When the KAR encounters its ligand, a killer activating ligand (KAL) on the target cell the NK or LAK cells are capable of killing the target. However, if the KIR also binds to its ligand then killing is inhibited even if KAR binds to KAL. 

The ligands for KIR are MHC-class I molecules. Thus, if a target cell expresses class I MHC molecules it will not be killed by NK or LAK cells even if the target also has a KAL which could bind to KAR. 

Normal cells constitutively express MHC class I molecules on their surface, however, virus infected and malignant cells down regulate expression of class I MHC. Thus, NK and LAK cells selectively kill virus-infected and malignant cells while sparing normal cells.

B. K cells 

Killer (K) cells are not a morphologically distinct type of cell. Rather a K cell is any cell that mediates antibody-dependent cellular cytotoxicity (ADCC). 

In ADCC antibody acts as a link to bring the K cell and the target cell together to allow killing to occur. K cells have on their surface an Fc receptor for antibody and thus they can recognize, bind and kill target cells coated with antibody. 

Killer cells which have Fc receptors include NK, LAK, and macrophages which have an Fc receptor for IgG antibodies and eosinophils which have an Fc receptor for IgE antibodies.

Immunology:

The branch of life science which deals with immune reaction is known as immunology.

Components of Immune System:

The immune system consists of a network of diverse organs and tissue which vary structurally as well as functionally from each other. These organs remain spreaded throughout the body. Basically, immune system is a complex network of lymphoid organs, tissues and cells.

These lym­phoid organs can be categorized under three types depending upon their functional aspects:

i.  Primary lymphoid organ.

ii. Secondary lymphoid organ.

iii.Tertiary lymphoid organ.

White blood cells or leukocytes are the basic cell types which help to give rise to different types of cells which participate in the development of immune response . WBC are classified into granulocytes and agranulocytes depending on the presence or absence of granules in the cyto­plasm.

Agranular leukocytes are of two types, viz., lymphocytes and monocytes. Lymphocytes play pivotal role in producing defensive molecules of immune system. Out of all leukocytes, only lymphocytes possess the quality of diversity, specificity, memory and self-non self recognition as various important aspects of immune response.

Other cell types remain as accessory one; help to activate lymphocytes, to generate various immune effector cells, to increase the rate of anti­gen clearance 

All cells of the immune system have their origin in the bone marrow 

myeloid (neutrophils, basophils, eosinpophils, macrophages and dendritic cells) 

lymphoid (B lymphocyte, T lymphocyte and Natural Killer) cells .

The myeloid progenitor (stem) cell in the bone marrow gives rise to erythrocytes, platelets, neutrophils, monocytes/macrophages and dendritic cells whereas the lymphoid progenitor (stem) cell gives rise to the NK, T cells and B cells. 

For T cell development the precursor T cells must migrate to the thymus where they undergo differentiation into two distinct types of T cells, the CD4+ T helper cell and the CD8+ pre-cytotoxic T cell. 

Two types of T helper cells are produced in the thymus the TH1 cells, which help the CD8+ pre-cytotoxic cells to differentiate into cytotoxic T cells, and TH2 cells, which help B cells, differentiate into plasma cells, which secrete antibodies. 

Function of the immune system is self/non-self discrimination. 

This ability to distinguish between self and non-self is necessary to protect the organism from invading pathogens and to eliminate modified or altered cells (e.g. malignant cells). 

Since pathogens may replicate intracellularly (viruses and some bacteria and parasites) or extracellularly (most bacteria, fungi and parasites), different components of the immune system have evolved to protect against these different types of pathogens.

Bacteria

A bacterial cell has a nuclear apparatus which is a loose arrangement of DNA This is surrounded cytoplasm which contains ribosomes, mesosomes and inclusion granules. The cytoplasm is enclosed within a cytoplasmic membrane. Bacterium has a rigid cell wall  Fimbriae and flagella are the surface adherents. Some bacteria may have a capsule (or loose slime) around the cell wall.

Shape and Size of Bacteria

The bacteria can be spheroidal (coccus), rod or cylindrical (bacillus) and spirillar (spirochaete). Very short bacilli are called as coccobacilli  Some of the bacilli may be curved or comma shaped (Vibrio cholerae).

Arrangement of Bacterial Cells

Streptococci are present in chains; staphylococci in grape-like clusters Cocci in pairs (diplococci) are suggestive of pneumococci, gonococci or menigococci.
Bacilli do not exhibit typical arrangement pattern except the Chinese letter arrangement shown by Corynebacterium diphtheriae

Surface Adherents and Appendages

CAPSULE The gels formed by the capsule adhere to the cell Capsule can be detected by negative staining ,with specific antiserum and observing the capsular swelling phenomenon called as Quellung reaction
Usually weakly antigenic Capsule production is better in vivo as compared to in vitro environment.
Eg. Capsules seen in Pneumococci,  Klebsiella, Escherichia coli, Haemophilus influenzae

Flagella : provide motility to the bacterium. 
Motile organisms: vibrios, pseudomonas, Esch.coli, salmonellae, spirochaetes and spirilla. 
Pathogenic cocci are nomotile.
Flagella measure in length from 3 to 20 µm and in diameter from 0.01 to 0.0 13 µm.
 
Arrangement

Bacteria with one polar flagellum are known as monotrichous; 
Tuft of several polar flagellae is known as lophotrichous
Presence of  Flagellae at both the ends of organism is amphitrichous 
Flagellae distributed all over the surface of the bacterium, it is called peritrichous.
•    Filament is composed of a protein-flagellin. The flagellar antigen is called as H (Hauch) antigen in contrast to somatic antigen which is called as O (Ohne haunch)

PILI (fimbriae) : hair like structures help in attachment also called sex pilli, transfers genetic material through conjugation , Present in Certain Gram negative bacteria. Only Composed of protein pilin  
Gram positive bacterium that has pili is Cornebacterium renale

The Cell Wall

The cell wall of  bacteria is multilayered structure. The external surface of cell wall is smooth in Gram positive bacteria  Gram negative bacteria have convoluted cell surfaces. The average thickness of cell wall is 0.15 to 0.50 .µm. Chemically composed of mucopeptide scaffolding formed by N acetyl glucosamine and N acetyl muramic acid
The cell wall is a three layered structure in Gram negative bacteria: outer membrane middle layer and plasma membrane. The outer membrane consists of lipoprotein and 1ipoppolysaccaride component

Functions of bacterial cell wall

 Provides shape , Gives rigidity , Protection, Surface has receptor sites for phages, Site of  antibody action,  Provides attachment to complement, Contains components toxic to host
 
Cytoplasmic Structures

The Plasma Membrane: This delicate membrane separates rigid cell wall from cytoplasm. It accounts for 30% of total cell weight. Chemically, it is 60% protein, 20-30% lipids and remaining carbohydrates.

 Mesosomes: 
 
 Principal sites of respiratory enzyme , Seen well in Gram positive bacteria as compared to Gram negative batcteria. Attachement of mesosomes to both DNA chromatin and membrane have been noticed thus help in cell division
 
Ribosomes: 

sites of protein synthesis. These are composed of RNA and proteins and constitute upto 4 of total cell protein and 90% of total cellular RNA.
Cytoplasmic Granules: Glycogen  :  Enteric bacteria
Poly-beta & hydroxy Butyrate : Bacillus & Pseudomonas
Babes-Ernst  :Corynebacterium & Yersinia pestis

Nuclear Apparatus

Bacterial DNA represents 2-3% of the cell weight and 10% of the volume of bacterium. Nucleous can be demonstrated by staining it with DNA specific Fuelgen stain .Consists of a single molecule of  double stranded DNA arranged in a circular form. Bacterial chromosome is haploid and replicates by binary fission, the bacteria may have  plasmid an extrachromosomal genetic material.
 

COMPLEMENT

The complement system primarily serves to fight bacterial infections. 

The complement system can be activated by at least three separate pathways. 
1) alternative pathway -
- The alternative pathway of complement activation starts with the spontaneous hydroysis of an internal thioester bond in the plasma complement component C3 to result in C3(H2O).

- The smaller cleavage products C3a, C4a, C5a, sometimes called "anaphylatoxins", act as phagocytes, they cause mast cell degranulation and enhance vessel permeability, thereby facilitating access of plasma proteins and leukocytes to the site of infection

- 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.

2) Lecithin Pathway 

The lectin pathway of complement activation exploits the fact that many bacterial surfaces contain mannose sugar molecules in a characteristic spacing. The oligomeric plasma protein mannan-binding lectin (MBL; lectins are proteins binding sugars) binds to such a pattern of mannose moieties, activating proteases MASP-1 and MASP-2 (MASP=MBL activated serine protease, similar in structure to C1r and C1s). These, by cleaving C4 and C2, generate a second type of C3 convertase consisting of C4b and C2b, with ensuing events identical to those of the alternative pathway.

3) classical pathway

The classical pathway usually starts with antigen-bound antibodies recruiting the C1q component, followed by binding and sequential activation of C1r and C1s serine proteases. C1s cleaves C4 and C2, with C4b and C2b forming the C3 convertase of the classical pathway. Yet, this pathway can also be activated in the absence of antibodies by the plasma protein CRP (C-reactive protein), which binds to bacterial surfaces and is able to activate C1q.

Pharmacology cross reference: humanized monoclonal antibody Eculizumab binds to complement component C5, inhibiting its cleavage and preventing activation of the lytic pathway. This is desirable when unwanted complement activation causes hemolysis, as in paroxysmal nocturnal hemoglobinuria or in some forms of hemolytic uremic syndrome. For the lytic pathway's importance in fighting meningococcal infections, Eculizumab treatment increases the risk of these infections, which may be prevented by previous vaccination.

 BIOLOGICALLY 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 10). 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.

Complement Fixation Test (CFT)

This test is based upon two properties of the complement viz:

a. Complent combines with all antigen-antibody complexes whether or not it is required for that reaction
b. Complement is needed in immunolytic reaction.

Test system

It contains an antigen and a serum suspected to be having antibody to that antigen. The serum is heat treated prior to the test to destroy its complement. Complement Is added in measured quantity to this system. This complement is the form of guinea pig serum which is considered a rich source of complement. The test system is incubated.

Indicator system

To test system, after incubation, is added the indicator system which consists of sheep
RBCs and antibody to sheep RBCs (haemolysin) and another incubation is allowed.
If there is specific antibody in the test system, it will bind to antigen and to this complex the complement will also get fixed. Hence, no complement will be available to combine with indicator system which though contains RBCs and their specific antibody, cannot undergo haemolysis unless complement gets attached. Absence of haemolysis shall indicated positive test or presence of specific antibody in the serum which has been added in the test system. Erythrocytes lysis is obtained in negative test.

GENETIC VARIATION

Two methods are known for genetic variation in bacteria: mutation and gene transfer.

Mutation : Any change in the sequence of bases of DNA, irrespective of detectable changes in the cell phenotype. Mutations may be spontaneous or induced by various agents which are known as mutagens. 

Spontaneous Mutations: Arise from enzymatic imperfections during DNA replications or with transient insertions of transposable elements.

Induced Mutations: Mutation by physical and chemical mutagens.

Physical mutagens  ultraviolet rays and high-energy ionizing radiations. The primary effect of UV rays on DNA is the production of pyrmidine dimers whereas ionizing radiations cause single_stranded breaks the DNA molecules.

Chemical mutagens :Affecting nucleotide sequence

(i) Agents which cause error in base pairing (e.g. nitrous acid and alkylating agents).
(ii) Agents which cause errors in DNA replication (e.g. acridine dyes such as acridine orange and profiavine).
(iii) Base analogs which are incorporated into DNA and cause replication errors (e.g. 5-bromouracil)

Gene Transfer

Transformation: Uptake of naked DNA

Transduction    : Infection by a nonlethal bacteriophage

Conjugation    : Mating between cells in contact

Protoplast fusion

Transformation: Gene transfer by soluble DNA is called as transformation. it requires that DNA be absorbed by the cell, gain entrance to the cytoplasm and undergo recombination with the host genome. 

Artificial Transformation(transfection) :Some of the bacteria (such as Escherichia coli) resist transformation until they are subjected to some special treatment such as CaCl2 to make the bacterium more permeable to DNA. Such modified cells can also take up intact double stranded DNA extracted from viruses or in the shape of plasmids. Though the process is same as transformation, it is 9 as transfection because it results in infection by an abnormal route

Transduction :The type of gene transfer in which the DNA of one bacterial cell is introduced into another bacterial cell by viral infection is known as transduction. This introduces only a small fragment of DNA. Because the DNA is protected from damage by the surrounding phage coat, transduction is an easier to perform and more reproducible process than transduction. ,

Two types of transduction are known.

- Generalized transduction When a bacteriophage picks up fragments of host DNA at random and can transfer any genes

-  Specialised transduction: phage DNA that has been integrated into the host chromosome is excised along with a few adjacent genes, which the phage can then transfer.

After entry into the host cell, the phage DNA gets incorporated into the host chromosome in such a way that the two genomes are linearly contiguous (lysogeny). The phage genome in this stage is known as prophage, The host cell acquires a significant new property as a consequence of lysogeny because it becomes immune to infection by homologous phage. This is hence called as lysogenic conversion and endow toxigenicity to Corynebacterium diphtheriae

Abortive Transduction :phage DNA fails to integrated into the host chromosome, the process is called as abortive transduction The phage DNA does not replicate and along with binary fission Of the host it goes into one of the daughter cells.

Conjugation :This is defined as the transfer of DNA directly from on bacterial. .cell to another by a mechanism that requires cell-to-cell contact. 

The capacity to donate DNA depends upon the possession of the fertility (F) factor. The F pili  also retard male-male union. Concomitant with effective male-female pair formation, the circular DNA bearing the F factor is converted to a linear form that is transferred to the female cell in a sequential manner. DNA replication occurs in the male cell and the newly synthesized, semiconserved DNA molecule remains in the male. This ensures postmating characters of the male.

Conjugation in Different Bacteria: Unusual form of plasmid transfer, called phase mediated conjugation has  been reported to occur with some strains of Staphylococcus aureus.

Protoplast Fusion: Also called as genetic transfusion. Under osmotically buffered Conditions protoplast fusion takes place by joining of cell membrane and generation of cytoplasmic bridges through which genetic material can be exchanged.

Transposons: Transposons  Tn  are  DNA sequences which are incapable of autonomous existence and which transpose blocks of genetic material back and forth between cell Chromosome and smaller replicons such as plasmids. insertion sequences (IS ) are another similar group of nucleotides which can move from one chromosome to another

Genetic material. IS and  Tn are collectively also known as transposable elements or Jumping genes. These are now recognised to play an important role in bringing about vanous types of mutations.