Outline the oxygen dependent mechanism of microbial killing.

Oxygen dependent microbial killing is caused by Reactive oxygen species (ROS)

Reactive Oxygen Species. ROS are produced by the rapid assembly and activation of a multicomponent enzyme, phagocyte oxidase which oxidizes NADPH and, in the process, reduces oxygen to the superoxide anion. In neutrophils, this oxidative reaction is tightly linked to phagocytosis,
and is called the respiratory burst. Phagocyte oxidase is an enzyme complex consisting of at least seven proteins. In resting neutrophils, different components of the enzyme are located in the plasma membrane and the cytoplasm. In response to activating stimuli, the cytosolic protein components
translocate to the phagosomal membrane, where they assemble and form the functional enzyme complex. Thus, the ROS are produced within the phagolysosome, where they can act on ingested particles without damaging the host cell. 

The azurophilic granules of neutrophils contain the enzyme myeloperoxidase (MPO), which, in the presence of a halide such as Cl−, converts H2O2 to hypochlorite, the active ingredient in household bleach. The latter is a potent anti-microbial agent that destroys microbes by halogenation or by oxidation of proteins and lipids. 

The H2O2-MPO-halide system is the most efficient bactericidal system of neutrophils. Nevertheless, inherited deficiency of MPO only causes a modest increase in susceptibility to infection, emphasizing the redundancy of microbicidal mechanisms in leukocytes. H2O2 also is converted to hydroxyl radical (OH•), another powerful destructive agent

Give pathogenesis of Apoptosis.

Apoptosis result from the activation of enzymes called caspases. Like many proteases, casepases exist as inactive proenzymes or zymogens and must undergo enzymatic cleavage to become active.
The process of apoptosis may be divided into an Initiation phase during which some caspases become catalytically active and an execution phase during which other caspases trigger the degradation of critical cellular components. The activation of caspases depends on a finely tuned balance between production of pro-apoptotic and anti-apoptotic proteins.
Two distinct pathways converge on caspase activation:
A. The mitochondrial pathway
B. The death receptor pathway

The mitochondrial pathway or the intrinsic pathway is the major mechanism of apoptosis in all mammalian cells. It results from increased permeability of the mitochondrial outer membrane with consequent release of death inducing.

What do you mean by Apoptosis? What are the causes of Apoptosis?

Apoptosis:

Apoptosis is a pathway of cell death that is induced by a tightly regulated suicide program in which cells
destined to die activate intrinsic enzymes that degrade the cells’ own nuclear DNA and nuclear and cytoplasmic proteins.

Causes of Apoptosis:

Apoptosis occurs normally both during development and throughout adulthood, and serves to remove unwanted, aged, or potentially harmful cells. It is also a pathologic event when diseased cells become damaged beyond repair and are eliminated.

Apoptosis in Physiologic Situations:

• The destruction of cells during embryogenesis, including implantation, organogenesis, developmental involution, and metamorphosis.
• Involution of hormone-dependent tissues upon hormone withdrawal, such as endometrial cell breakdown during the menstrual cycle, ovarian follicular atresia in menopause, the regression of the lactating breast after weaning, and prostatic atrophy after castration.
• Cell loss in proliferating cell populations, such as immature lymphocytes in the bone marrow and thymus and B lymphocytes in germinal centers that fail to express useful antigen receptors.
• Elimination of potentially harmful self-reactive lymphocytes, either before or after they have completed their maturation, so as to prevent reactions against one’s own tissues.
• Death of host cells that have served their useful purpose, such as neutrophils in an acute inflammatory response, and lymphocytes at the end of an immune response.

Apoptosis in Pathologic Conditions:

• DNA damage. Radiation, cytotoxic anticancer drugs, and hypoxia can damage DNA, either directly or via production of free radicals.
• Accumulation of misfolded proteins.
• Cell death in certain infections, particularly viral infections, in which loss of infected cells is largely due to apoptosis that may be induced by the virus (as in adenovirus and HIV infections) or by the host immune response (as in viral hepatitis).
• Pathologic atrophy in parenchymal organs after duct obstruction, such as occurs in the pancreas, parotid gland, and kidney.

Write down the mechanism of irreversible cell injury.

Mechanisms of cell injury are as follows:

• Depletion of ATP: Reduction in ATP levels is fundamental cause of necrotic cell death. ATP depletion and decreased ATP synthesis are frequently associated with both hypoxic and chemical injury. The  major causes of ATP depletion are reduced supply of oxygen and nutrients, mitochondrial damage, and the actions of some toxins.
• Mitochondrial Damage: Mitochondria are critical players in cell injury and cell death by all pathways. Mitochondria can be damaged by increases of cytosolic Ca2+, reactive oxygen species, oxygen deprivation and mutations in mitochondrial genes in some inherited diseases.
• Influx of Calcium and Loss of Calcium Homeostasis: Calcium ions are important mediators of cell injury.
• Accumulation of Oxygen-Derived Free Radicals: Cell injury induced by free radicals, particularly reactive oxygen species, is an important mechanism of cell damage in many pathologic conditions, such as chemical and radiation injury, ischemia-reperfusion injury, cellular aging, and microbial killing by phagocytes.
• Defects in Membrane Permeability: Early loss of selective membrane permeability, leading ultimately to overt membrane damage, is a consistent feature of most forms of cell injury. Membrane damage may affect the functions and integrity of all cellular membranes.
• Damage to DNA and Proteins.

What are the morphologic changes in cell injury.

Morphologic changes in reversible cell injury:

Light microscopy:

Cellular swelling is the first manifestation of almost all forms of injury to cells. It causes some pallor, increased turgor, and increase in weight of the organ. On microscopic examination, small clear vacuoles may be seen within the cytoplasm; these represent distended and pinched-off segments of the ER. This pattern of nonlethal injury is sometimes called hydropic change or vacuolar degeneration. Cells may also show increased eosinophilic staining, which becomes much more pronounced with progression to necrosis. 

The ultrastructural changes of reversible cell injury include: 

1.Plasma membrane alterations, such as blebbing, blunting, and loss of microvilli 

2.Mitochondrial changes, including swelling and the appearance of small amorphous densities 

3.Dilation of the ER, with detachment of polysomes; intracytoplasmic myelin  figures may be present 

4.Nuclear alterations, with disaggregation of granular and fibrillar elements.

Consequences of Hypoxic injury

Consequences of hypoxic injury:

• As the oxygen tension within the cell falls, there is loss of oxidative phosphorylation and decreased generation of ATP. The depletion of ATP results in failure of the sodium pump, leading to efflux of potassium, influx of sodium and water, and cell swelling. 
• There is also influx of Ca2+, with its many deleterious effects. 
• There is progressive loss of glycogen and decreased protein synthesis. The functional consequences may be severe at this stage.
• The cytoskeleton disperses, resulting in the loss of ultrastructural features such as microvilli and the formation of blebs at the cell surface. 
• Myelin figures derived from degenerating cellular membranes, may be seen within the cytoplasm or extracellularly. They are thought to result from unmasking of phosphatide groups, promoting the uptake and intercalation of water between the lamellar stacks of membranes. 
• At this time the mitochondria are usually swollen, as a result of loss of volume control in these organelles; the ER remains dilated; and the entire cell is markedly swollen, with increased concentrations of water, sodium, and chloride and a decreased concentration of potassium. 
• If ischemia persists, irreversible injury and necrosis ensue.

Describe briefly the sources and possible consequences of increased cytosolic calcium in cell injury.

Sources of calcium:

1. Intracellular source: Mitochondria, Endoplasmic reticulum.

2. Extracellular source.

Consequences of increased cytosolic calcium:

• The accumulation of Ca2+ in mitochondria results in opening of the mitochondrial permeability transition pore and failure of ATP generation. 
• Increased cytosolic Ca2+ activates a number of enzymes with potentially deleterious effects on cells. These enzymes include phospholipases (which cause membrane damage), proteases (which break down both  membrane and cytoskeletal proteins), endonucleases (which are responsible for DNA and chromatin  fragmentation), and ATPases (thereby hastening ATP depletion).
• Increased intracellular Ca2+ levels also result in the induction of apoptosis, by direct activation of caspases and by increasing mitochondrial permeability.

What are the morhological types of irreversible cell injury?

Morphological pattern of irreversible cell injury are as follows: 

A. Necrosis

• Coagulative necrosis
• Liquefactive necrosis
• Gangrenous necrosis
• Caseous necrosis
• Fat necrosis
• Fibrinoid necrosis

B. Apoptosis.

What are the causes of cell injury?

Causes of Cell Injury: 

The causes of cell injury range from the external gross physical violence of an automobile accident to subtle internal abnormalities, such as a genetic mutation causing lack of a vital enzyme that impairs normal metabolic function. Most injurious stimuli can be grouped into the following broad categories.

1. Oxygen Deprivation. Causes of hypoxia include reduced blood flow (celled ischemia), inadequate oxygenation of the blood due to cardiorespiratory failure, and decreased oxygen-carrying capacity of the blood, as in anemia or carbon monoxide poisoning (producing a stable carbon monoxyhemoglobin that blocks oxygen carriage) or after severe blood loss.

2. Physical Agents. Physical agents capable of causing cell injury include mechanical trauma, extremes of temperature (burns and deep cold), sudden changes in atmospheric pressure, radiation, and electric shock.

3. Chemical Agents and Drugs. Oxygen at high concentrations is toxic, Arsenic, Cyanide, Mercuric salts.

4. Infectious Agents. Rickettsiae, Bacteria, Fungi, and higher forms of parasites.

5. Immunologic Reactions. Injurious reactions to endogenous self-antigens are responsible for several autoimmune diseases.

6. Genetic Derangements.

7. Nutritional imbalance. Protein-calorie deficienciency, Deficiencies of specific vitamins, Atherosclerosis, Obesity.

What is cell injury? What are the types of cell injury?

Cell injury:

When cells are stressed so severely that they are no longer able to adapt or when cells are exposed to inherently damaging agents or suffer from intrinsic abnormalities. Injury may progress through a reversible stage and culminate in cell death.

Types of cell injury:

1. Reversible cell injury.

2. Irreversible cell injury or cell death.

Classification of free radicals.

Free radicals can be classified as:

• Reactive oxygen species
• Superoxide anion
• Hydrogen Peroxide (H2O2)
• Hydroxyl radical (OH)
• Hypochlorous acid (HOCl)
•  Reactive nitrogen Species:
• Nitric oxide (NO)
• Peroxynitrite anion (ONOO)

Role of free radicals in cell injury OR mechanism of cell injury by free radicals.

The effects of reactive oxygen species are wide ranging, but three reactions are particularly relevant to cell injury:

1. Lipid peroxidation of membrane: Free radicals in the presence of oxygen may cause peroxidation of lipids within plasma and organellar membrane
2. Oxidative Modification of protein: Oxidative modification enhances degradation of critical proteins by the multicatalytic proteosome complex, raising havoc throughout cell.
3. Lesions in DNA: Reactions with thymine in nuclear and mitochondrial DNA produce single stranded breaks in DNA.   

Mechanism of production of free radicals.

The following mechanisms are involved in production of free radicals.

1. Absorption of radiant energy
2. Enzymatic metabolism of exogenous chemicals or drugs
3. The reduction-oxidation reactions that occur during normal metabolic process
4. Re-perfusion injury
5. Oxygen Toxicity  

What are free radicals?

Free radicals are chemical species that have a single unpaired electron in an outer orbit.

Reduced reactive oxygen forms are produced as an unavoidable byproduct of mitochondrial respiration. Some of these forms are free radicals

What are the common causes of cell injury?

The common causes of cell injury are as follows:

• Oxygen deprivation

• Hypoxia

• Physical agents
• Mechanical Trauma
• Extremes of temperature
• Sudden changes in atmospheric pressure
• Radiation
• Electric shock

• Chemical agents and drugs:
• Oxygen in high concentration
• Arsenic
• Cyanide
• Mercuric salt
• Insecticides
• Carbon-mono-oxide
• Asbestos

• Infectious agents:
• Virus, Bacteria, Fungi, Helminths, Rickettsiae

• Immunological reactions
• Genetic derangements
• Nutritional imbalance

Describe The Biochemical Mechanism Leading To Cell Membrane Damage

The following biochemical mechanism contribute to cell membrane damage:
A. Mitochondrial dysfunction- Defective mitochondrial function results in decreased phospholipid synthesis, which affects all cellular membrane. At the same time increase cytosolic Ca++ activate phospholipases and leading to breakdown of phospholipids. The net result is a depletion of phospholipids from the mitochondria and other dellular membranes and accumulation of free fatty acids. In the mitochondria this changes causes permeability defects.

B. Loss of membrane phospholipids: This is because activation of endogenous phospholipases by increased levels of cytosolic calcium, phospholipid loss can also occur secondary to decreased ATP-dependant reacylation or diminished de novo synthesis of phospholipids.

C. Cytoskeletal abnormalities: Activation of proteases by increased cytosolic Calcium may cause damage to elements of the cytoskeleton.

D. Reactive oxygen species: Partially reduced oxygen free radicals cause injury to cell membranes and to other cell constituents.

E. Lipid breakdown products: These includes unestirified free acids, acyl carnitine and lysophospholipids catabolic products that are known to accumulate in injured cells as a result of phospholipid degradation. They have detergent effect on membrane.