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Cytotoxicity

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Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are a chemical substance, an immune cell or some types of venom e.g. from the puff adder or brown recluse spider.

[edit] Cell Physiology

Treating cells with a cytotoxic compound can result in a variety of cell fates. The cells may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of cell lysis. The cells can stop actively growing and dividing (a decrease in cell viability), or the cells can activate a genetic program of controlled cell death (apoptosis).

Cells undergoing necrosis typically exhibit rapid swelling, lose membrane integrity, shut down metabolism and release their contents into the environment. Cells that undergo rapid necrosis in vitro do not have sufficient time or energy to activate apoptotic machinery and will not express apoptotic markers.[1] Apoptosis is characterized by well defined cytological and molecular events including a change in the refractive index of the cell, cytoplasmic shrinkage, nuclear condensation and cleavage of DNA into regularly sized fragments.[2] Cells in culture that are undergoing apoptosis eventually undergo secondary necrosis. They will shut down metabolism, lose membrane integrity and lyse.[2][3]

[edit] Measuring Cytotoxicity

Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds, if they are interested in developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical.

Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components.[3] Alternatively, membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the outside. One commonly measured molecule is lactate dehydrogenase (LDH).[4] Protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. The live-cell protease is only active in cells that have a healthy cell membrane, and loses activity once the cell is compromised and the protease is exposed to the external environment. The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity.[5]

Cytotoxicity can also be monitored using the MTT or MTS assay. This assay measures the reducing potential of the cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored formazan product. A similar redox-based assay has also been developed using the fluorescent dye, resazurin. In addition to using dyes to indicate the redox potential of cells in order to monitor their viability, researchers have developed assays that use ATP content as a marker of viability.[3] Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction.[6]

Cytotoxicity can also be measured by the Sulforhodamine B (SRB) assay, WST assay and clonogenic assay.

A label-free approach to follow the cytotoxic response of adherent animal cells in real-time is based on electric impedance measurements when the cells are grown on gold-film electrodes. This technology is referred to as Electric cell-substrate impedance sensing or short ECIS. Label-free real-time techniques provide the kinetics of the cytotoxic response rather than just a snapshot like in many colorimetric endpoint assays.

[edit] Immune System Cytotoxicity

Antibody-dependent cell-mediated cytotoxicity (ADCC) describes the cell-killing ability of certain lymphocytes, which requires the target cell being marked by an antibody. Lymphocyte-mediated cytotoxicity, on the other hand, does not have to be mediated by antibodies; nor does complement-dependent cytotoxicity (CDC), which is mediated by the complement system.

Three groups of cytotoxic lymphocytes are distinguished:

[edit] References

  1. ^ Promega Corporation (2006) Protocols and Applications Guide. Cell Viability.
  2. ^ a b Promega Corporation (2007) Protocols and Applications Guide. Apoptosis.
  3. ^ a b c Riss TL, Moravec RA (February 2004). "Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays". Assay Drug Dev Technol 2 (1): 51–62. doi:10.1089/154065804322966315. PMID 15090210
  4. ^ Decker T, Lohmann-Matthes ML (November 1988). "A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity". J. Immunol. Methods 115 (1): 61–9. PMID 3192948
  5. ^ Niles AL, Moravec RA, Eric Hesselberth P, Scurria MA, Daily WJ, Riss TL (July 2007). "A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers". Anal. Biochem. 366 (2): 197–206. doi:10.1016/j.ab.2007.04.007. PMID 17512890
  6. ^ Fan F, Wood KV (February 2007). "Bioluminescent assays for high-throughput screening". Assay Drug Dev Technol 5 (1): 127–36. doi:10.1089/adt.2006.053. PMID 17355205

[edit] External links

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http://en.wikipedia.org/wiki/Cytotoxicity

 

 

Cytotoxic T cell

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Antigen presentation stimulates T cells to become either "cytotoxic" CD8+ cells or "helper" CD4+ cells.

A cytotoxic T cell (also known as TC, CTL, T-Killer cell, cytolytic T cell, CD8+ T-cells or killer T cell) belongs to a sub-group of T lymphocytes (a type of white blood cell) that are capable of inducing the death of infected somatic or tumor cells; they kill cells that are infected with viruses (or other pathogens), or are otherwise damaged or dysfunctional. Most cytotoxic T cells express T-cell receptors (TCRs) that can recognize a specific antigenic peptide bound to Class I MHC molecules, present on all nucleated cells, and a glycoprotein called CD8, which is attracted to non-variable portions of the Class I MHC molecule. The affinity between CD8 and the MHC molecule keeps the TC cell and the target cell bound closely together during antigen-specific activation. CD8+ T cells are recognized as TC cells once they become activated and are generally classified as having a pre-defined cytotoxic role within the immune system.

[edit] Cytotoxic T cell development

Development of single positive T cells in the thymus

Hematopoietic stem cells in the bone marrow migrate into the thymus, where they undergo VDJ recombination of their beta-chain TCR DNA to form a developmental form of the TCR protein, known as pre-TCR. If that rearrangement is successful, the cells then rearrange their alpha-chain TCR DNA to create a functional alpha-beta TCR complex. This highly-variable genetic rearrangement product in the TCR genes helps create millions of different T cells with different TCRs, helping the body's immune system respond to virtually any protein of an invader. The vast majority of T cells express alpha-beta TCRs (αβ T cells), but some T cells in epithelial tissues (like the gut) express gamma-delta TCRs (γδ T cells), which recognize non-protein antigens.

T cells with functionally stable TCRs express both the CD4 and CD8 co-receptors and are therefore termed "double-positive" (DP) T cells (CD4+CD8+). The double-positive T cells are exposed to a wide variety of self-antigens in the thymus and undergo two selection criteria:

  • (1) positive selection, in which those double-positive T cells that bind too weakly to MHC-presented self antigens undergo apoptosis because of their inability to recognize MHC-protein complexes.
  • (2) negative selection, in which those double-positive T cells that bind too strongly to MHC-presented self antigens undergo apoptosis because their propensity (an often intense natural inclination or preference) to become autoreactive could lead to autoimmunity.

Only those T cells that bind to the MHC-self-antigen complexes weakly are positively selected. Those cells that survive positive and negative selection differentiate into single-positive T cells (either CD4+ or CD8+), depending on whether their TCR recognizes an MHC class I-presented antigen (CD8) or an MHC class II-presented antigen (CD4). It is the CD8+ T-cells that will mature and go on to become cytotoxic T cells following their activation with a class I-restricted antigen.

[edit] Cytotoxic T cell activation

With an exception of some cell types, such as non-nucleated cells (including erythrocytes), Class I MHC is expressed by all host cells. When these cells are infected with a virus (or another intracellular pathogen), the cells degrade foreign proteins via antigen processing. These result in peptide fragments, some of which are presented by MHC Class I to the T cell antigen receptor (TCR) on CD8+ T cells.

The activation of cytotoxic T cells is dependent on several simultaneous interactions between molecules expressed on the surface of the T cell and molecules on the surface of the antigen-presenting cell (APC). For instance, consider the two signal model for TC cell activation.

Signal

T cell

APC

Description

first signal

TCR

peptide-bound MHC class I molecule

There is a second interaction between the CD8 coreceptor and the class I MHC molecule to stabilize this signal.

second signal

CD28 molecule on the T cell

either CD80 or CD86 (also called B7-1 and B7-2)

CD80 and CD86 are known as costimulators for T cell activation. This second signal can be assisted (or replaced) by stimulating the TC cell with cytokines released from helper T cells.

Once activated, the TC cell undergoes clonal expansion with the help of a cytokine called Interleukin-2 (IL-2) that is a growth and differentiation factor for T cells. This increases the number of cells specific for the target antigen that can then travel throughout the body in search of antigen-positive somatic cells.

[edit] Cytotoxic T cell effector functions

When exposed to infected/dysfunctional somatic cells, TC cells release the cytotoxins perforin and granulysin. Perforin forms pores in the target cell's plasma membrane allowing granzymes, types of serine proteases, to enter the target cell, which then activate a series of enzymes, the caspase cascade, that eventually lead to apoptosis (programed cell death). A second way to induce apoptosis is via cell-surface interactions between the TC and the infected cell. When a TC is activated it starts to express the surface protein FAS ligand (FasL), which can bind to Fas molecules expressed on the target cell. However, this Fas-Fas ligand interaction is thought to be more important to the disposal of unwanted T lymphocytes during their development or to the lytic activity of certain TH cells than it is to the cytolytic activity of TC effector cells.

[edit] Cytotoxic T cell role in disease pathogenesis

During HBV infection cytotoxic T cells play an important pathogenetic role. They contribute to nearly all of the liver injury associated with HBV infection and, by killing infected cells and by producing antiviral cytokines capable of purging HBV from viable hepatocytes, cytotoxic T cells also eliminate the virus.[1] Recently platelets have been shown to facilitate the accumulation of virus-specific cytotoxic T cells into the infected liver.[2]

[edit] References

  1. ^ Iannacone M. et al (2006). "Pathogenetic and antiviral immune responses against hepatitis B virus". Future Virology 1 (2): 189-196. doi:10.2217/17460794.1.2.189 Future Virology]
  2. ^ Iannacone M. et al (2005). "Platelets mediate cytotoxic T lymphocyte-induced liver damage". Nat Med 11: 1167–1169. 

http://en.wikipedia.org/wiki/Cytotoxic_T_cell

 

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