The cell cycle

1) Labile cells (GI tract, blood cells)
- Described as parenchymal cells that are normally found in the G0 phase that can be stimulated to enter the G1
- Undergo continuous replication, and the interval between two consecutive mitoses is designated as the cell cycle
- After division, the cells enter a gap phase (G1), in which they pursue their own specialized activities
•    If they continue in the cycle, after passing the restriction point (R), they are committed to a new round of division
•    The G1 phase is followed by a period of nuclear DNA synthesis (S) in which all chromosomes are replicated
•    The S phase is followed by a short gap phase (G2) and then by mitosis
•    After each cycle, one daughter cell will become committed to differentiation, and the other will continue cycling

2) Stable cells (Hepatocytes, Kidney)

- After mitosis, the cells take up their specialized functions (G0). 
- They do not re-enter the cycle unless stimulated by the loss of other cells

3) Permanent cells (neurons)

- Become terminally differentiated after mitosis and cannot re-enter the cell cycle
- Which cells do not have the ability to differentiate ->  Cardiac myocytes

PHAGOCYTOSIS AND INTRACELLULAR KILLING

A. Phagocytic cells

1. Neutrophiles/Polymorphonuclear cells

PMNs are motile phagocytic cells that have lobed nuclei. They can be identified by their characteristic nucleus or by an antigen present on the cell surface called CD66. They contain two kinds of granules the contents of which are involved in the antimicrobial properties of these cells. 

The second type of granule found in more mature PMNs is the secondary or specific granule. These contain lysozyme, NADPH oxidase components, which are involved in the generation of toxic oxygen products, and characteristically lactoferrin, an iron chelating protein and B12-binding protein.

2. Monocytes/Macrophages

 Macrophages are phagocytic cells . They can be identified morphologically or by the presence of the CD14 cell surface marker. 

B. Response of phagocytes to infection 

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.

 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.

C. Initiation of Phagocytosis 

Phagocytic cells have a variety of receptors on their cell membranes through which infectious agents bind to the cells. These include:

1. Fc receptors – Bacteria with IgG antibody on their surface have the Fc region exposed and this part of the Ig molecule can bind to the receptor on phagocytes. Binding to the Fc receptor requires prior interaction of the antibody with an antigen. Binding of IgG-coated bacteria to Fc receptors results in enhanced phagocytosis and activation of the metabolic activity of phagocytes (respiratory burst).

2. Complement receptors – Phagocytic cells have a receptor for the 3rd component of complement, C3b. Binding of C3b-coated bacteria to this receptor also results in enhanced phagocytosis and stimulation of the respiratory burst. 

3. Scavenger receptors – Scavenger receptors bind a wide variety of polyanions on bacterial surfaces resulting in phagocytosis of bacteria.

4. Toll-like receptors – Phagocytes have a variety of Toll-like receptors (Pattern Recognition Receptors or PRRs) which recognize broad molecular patterns called PAMPs (pathogen associated molecular patterns) on infectious agents. Binding of infectious agents via Toll-like receptors results in phagocytosis and the release of inflammatory cytokines (IL-1, TNF-alpha and IL-6) by the phagocytes.

D. Phagocytosis 

The pseudopods eventually surround the bacterium and engulf it, and the bacterium is enclosed in a phagosome. During phagocytosis the granules or lysosomes of the phagocyte fuse with the phagosome and empty their contents. The result is a bacterium engulfed in a phagolysosome which contains the contents of the granules or lysosomes.

E. Respiratory burst and intracellular killing

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.

1. Oxygen-dependent myeloperoxidase-independent intracellular killing

During phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is formed. Cytochrome B which was part of the specific 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•).

2. Oxygen-dependent myeloperoxidase-dependent intracellular killing 

As the azurophilic granules fuse with the phagosome, myeloperoxidase is released into the phagolysosome. Myeloperoxidase 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).

3. Detoxification reactions 

PMNs and macrophages have means to protect themselves from the toxic oxygen intermediates. These reactions involve the dismutation of superoxide anion to hydrogen peroxide by superoxide dismutase and the conversion of hydrogen peroxide to water by catalase. 

4. Oxygen-independent intracellular 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.

CHEMICAL AGENTS

Chlorine and iodine are most useful disinfectant Iodine as a skin disinfectant and chlorine as a water disinfectant have given consistently magnificent results. Their activity is almost exclusively bactericidal and they are effective against sporulating organisms also. 
Mixtures of various surface acting agents with iodine are known as iodophores and these are used for the sterilization of dairy products.

Apart from chlorine, hypochlorite, inorganic chioramines are all good disinfectants but they act by liberating chlorine. 

Hydrogen peroxide in a 3% solution is a harmless but very weak disinfectant whose primary use is in the cleansing of the wound.
 
Potassium permanganate is another oxidising agent which is used in the treatment of urethntzs. 

Formaldehyde — is one of the least selective agent acting on proteins. It is a gas that is usually employed as its 37% solution, formalin. 

When used in sufficiently high concentration it destroys the bacteria and their spores.

Classification of chemical sterilizing agents

Chemical disinfectant

Interfere with membrane functions

•    Surface acting agents : Quaternary ammonium, Compounds, Soaps and fatty acids

•    Phenols : Phenol, cresol, Hexylresorcinol

•    Organic solvent : Chloroform, Alcohol

Denatures proteins

•    Acids and alkalies : Organic acids, Hydrochloric acid , Sulphuric acid

Destroy functional groups of proteins

•    Heavy metals :  Copper, silver , Mercury

•    Oxidizing agents: Iodine, chlorine, Hydrogen peroxide

•    Dyes : Acridine orange, Acriflavine

•    Alkylating agents : Formaldehyde, Ethylene oxide

Applications and in-use dilution of chemical disinfectants

Alcohols : Skin antiseptic Surface disinfectant, Dilution used 70%

Mercurials : Skin antiseptic Surface disinfectant Dilution Used 0.1 %

Silver nitrate : Antiseptic (eyes and burns)  Dilution Used 1 %

Phenolic compound : Antiseptic skin washes  Dilution Used .5 -5 %

Iodine : Disinfects inanimate object, Skin antiseptic Dilution used  2%

Chlorine compounds  : Water treatment Disinfect inanimate objects , Dillution used 5 %

Quaternary ammonium Compounds : Skin antiseptic , Disinfects inanimate object, Dilution Used < 1 %

Glutaraldehyde: Heat sensitve instruments, Dilution used 1-2 %

Cold sterilization can be achieved by dipping the precleaned instrument in 2% solution of gluteraldehyde for 15-20 minutes. This time is sufficient to kill the vegetative form as well as spores ofthe organisms that are commonly encountered in the dentistry.

Ethylene oxide is an a agent extensively used in gaseous sterilization. It is active against all kinds of bacteria and their spores. but its greatest utility is in sterilizing those objects which are damaged by heat (e.g. heart lung machine). It is also used to sterlise fragile, heat sensitive equipment, powders as well as components of space crafts.

Evaluation of Disinfectants

Two methods which are widely employed are:

 Phenol coefficient test, Kelsey -Sykes test
 
These tests determine the capacity of disinfectant as well as their ability to retain their activity.
 

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.

Cell Functions:
-> Autolysis

- degradative reactions in cells caused by indigenous intracellular enzymes – usually occurs after cell death
- Irreversible (along with Coagulative necrosis or infarcts) – reversible: fatty degeneration, & hydropic degeneration

-> Autolysin:
•    Ab causing cellular lysis in the presence of complement
•    Autolytic enzymes produced by the organism degrade the cell’s own cell wall structures

-> In the presence of cephalosporins & penicillins, growing bacterial cells lyse
•    W/o functional cell wall structures, the bacterial cell bursts

-> Heterolysis: cellular degradation by enzymes derived from sources extrinsic to the cell (e.g., bacteria)

-> Necrosis: sum of intracellular degradative reactions occurring after individual cell death w/in a living organism

MICROBIAL VIRULENCE FACTORS 

Microbial virulence factors are gene products required for a microbial pathogen to establish itself in the host. These gene products are located on the bacterial chromosome, or on mobile genetic elements, such as plasmids or transposons.

Primary pathogens express virulence factors that allow them to cause disease in the normal  host.

Opportunistic pathogens are environmental organisms or normal flora that lack the means to overcome normal host defense mechanisms. They cause disease only when the normal host defenses are breached or deficient. 

Virulence factors can be divided into several categories.

Skin - Propionibacterium acnes, Staphlococcus epidermis , diptheroids; transient colonization by Staphlococcus
aureus

Oral cavity - Viridans Streptococci, Branhamella species, Prevotella melaninogenicus, Actinomyces species, Peptostreptococcus species, other anaerobes

Nasopharynx Oral organisms; transient colonization by S. pneumoniae, Haemophilus species, N. meningitidis  

Stomach Rapidly becomes sterile 

Small intestine Scant

Colon - Bacteroides species, Clostridium species, Fusobacterium species, E. coli, Proteus species, Pseudomonas aeruginosa, Enterococcus species, other bacteria and yeasts 

Vagina - Childbearing years:Lactobacillus species, yeasts, Streptococcus species 

Prepuberty / Postmenopause: colonic and skin flora 

A. Enzyme production can be of several types depending on the needs of the organism, its requirements for survival, and the local environment.
 
1. Hyaluronidase breaks down hyaluronic acid to aid in the digestion of tissue. 
2. Protease digests proteins to enhance the spread of infections. 
3. Coagulase allows coagulation of fibrinogen to clot plasma. 
4. Collagenase breaks down collagen (connective tissues). 

B. Toxins 

1. Exotoxins are heat-labile proteins with specific enzymatic activities produced by many Gram-positive and Gram-negative organisms. Exotoxins are released extracellularly and are often the sole cause of disease. 
a. Some toxins have several domains with discrete biological functions that confer maximal toxicity. An example is A-B exotoxin, where the B subunit binds to host tissue cell glycoproteins and the A subunit enzymatically attacks a susceptible target.
b. Many toxins are ADP-ribosylating toxins

2. Endotoxin is the heat-stable lipopolysaccharide moiety found in the outer membrane of Gram-negative organisms. when released by cell lysls, the lipid A portion of lipopolysaccharide can induce septic shock characterized by fever, acidosis, hypotension, complement consumption, and disseminated intravascular coagulation (DIC).  

C. Surface components 

may protect the organism from immune responses such as phagocytosis or aid in tissue invasion. For example, the polysaccharide capsules of H. influenzae type b and the acidic polysaccharide capsule of Streptococcus pneumoniae interfere with phagocytosis. Other surface proteins, such as adhesins or filamentous appendages (fimbriae, pili), are involved in adherence of invading microorganisms to cells of the host.