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Double-stranded RNA viruses

2009-06-08T06:44:00.005-07:00

Double-stranded waffle viruses

Electron micrograph of rotaviruses. The bar = 100 nm

Virus classification

Group:

Group III (dsRNA)

Families

Birnaviridae
Cystoviridae
Hypoviridae
Partitiviridae
Reoviridae
Totiviridae

Double-stranded (ds) RNA viruses are a diverse group of viruses that vary widely in host range (humans, animals, plants, fungi, and bacteria), genome segment number (one to twelve), and virion organization (T-number, capsid layers, or turrets). Members of this group include the rotaviruses, known globally as a common cause of gastroenteritis in young children, and bluetongue virus, an economically important pathogen of cattle and sheep.

Viruses with dsRNA genomes are currently grouped into six families: Reoviridae, Birnaviridae, Totiviridae, Partitiviridae, Hypoviridae, and Cystoviridae. Of these six families, the Reoviridae is the largest and most diverse in terms of host range.

In recent years virus particle assembly, virus-cell interactions, and viral pathogenesis, approaches for the development of novel antiviral strategies and/or agents can be designed.

Reoviridae

Reoviridae are currently classified into nine genera. The genomes of these viruses consist of 10 to 12 segments of dsRNA, each generally encoding one protein. The mature virions are non-enveloped. Their capsids, formed by multiple proteins, have icosahedral symmetry and are arranged generally in concentric layers. A distinguishing feature of the dsRNA viruses, irrespective of their family association, is their ability to carry out transcription of the dsRNA segments, under appropriate conditions, within the capsid. In all these viruses, the enzymes required for endogenous transcription are thus part of the virion structure.

Orthoreoviruses

The orthoreoviruses (reoviruses) are the prototypic members of the virus Reoviridae family and representative of the turreted members, which comprise about half the genera. Like other members of the family, the reoviruses are non-enveloped and characterized by concentric capsid shells that encapsidate a segmented dsRNA genome. In particular, reovirus has eight structural proteins and ten segments of dsRNA. A series of uncoating steps and conformational changes accompany cell entry and replication. High-resolution structures are known for almost all of the proteins of mammalian reovirus (MRV), which is the best-studied genotype. Electron cryo-microscopy (cryoEM) and X-ray crystallography have provided a wealth of structural information about two specific MRV strains, type 1 Lang (T1L) and type 3 Dearing (T3D).

Cypovirus

The cytoplasmic polyhedrosis viruses (CPVs) form the genus Cypovirus of the family Reoviridae. CPVs are classified into 14 species based on the electrophoretic migration profiles of their genome segments. Cypovirus has only a single capsid shell, which is similar to the orthoreovirus inner core. CPV exhibits striking capsid stability and is fully capable of endogenous RNA transcription and processing. The overall folds of CPV proteins are similar to those of other reoviruses. However, CPV proteins have insertional domains and unique structures that contribute to their extensive intermolecular interactions. The CPV turret protein contains two methylase domains with a highly conserved helix-pair/β-sheet/helix-pair sandwich fold but lacks the β-barrel flap present in orthoreovirus λ2. The stacking of turret protein functional domains and the presence of constrictions and A spikes along the mRNA release pathway indicate a mechanism that uses pores and channels to regulate the highly coordinated steps of RNA transcription, processing, and release.

Rotaviruse

Rotavirus is the most common cause of acute gastroenteritis in infants and young children worldwide. This virus contains a dsRNA genome and is a member of the Reoviridae family. The genome of rotavirus consists of eleven segments of dsRNA. Each genome segment codes for one protein with the exception of segment 11, which codes for two proteins. Among the twelve proteins, six are structural and six are non-structural proteins.

Bluetongue virus

The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses and are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at the molecular and structural levels. BTV, like other members of the family, is a complex non-enveloped virus with seven structural proteins and a RNA genome consisting of 10 variously sized dsRNA segments.

Phytoreoviruses

Phytoreoviruses are non-turreted reoviruses that are major agricultural pathogens, particularly in Asia. One member of this family, Rice Dwarf Virus (RDV), has been extensively studied by electron cryomicroscopy and x-ray crystallography. From these analyses, atomic models of the capsid proteins and a plausible model for capsid assembly have been derived. While the structural proteins of RDV share no sequence similarity to other proteins, their folds and the overall capsid structure are similar to those of other Reoviridae.

L-A Virus

The L-A dsRNA virus of the yeast Saccharomyces cerevisiae has a single 4.6 kb genomic segment that encodes its major coat protein, Gag (76 kDa) and a Gag-Pol fusion protein (180 kDa) formed by a -1 ribosomal frameshift. L-A can support the replication and encapsidation in separate viral particles of any of several satellite dsRNAs, called M dsRNAs, each of which encodes a secreted protein toxin (the killer toxin) and immunity to that toxin. L-A and M are transmitted from cell to cell by the cytoplasmic mixing that occurs in the process of mating. Neither is naturally released from the cell or enters cells by other mechanisms, but the high frequency of yeast mating in nature results in the wide distribution of these viruses in natural isolates. Moreover, the structural and functional similarities with dsRNA viruses of mammals has made it useful to consider these entities as viruses.

Infectious bursal disease virus

Infectious bursal disease virus (IBDV) is the best-characterized member of the family Birnaviridae. These viruses have bipartite dsRNA genomes enclosed in single-layered icosahedral capsids with T = 13l geometry. IBDV shares functional strategies and structural features with many other icosahedral dsRNA viruses, except that it lacks the T = 1 (or pseudo T = 2) core common to the Reoviridae, Cystoviridae, and Totiviridae. The IBDV capsid protein exhibits structural domains that show homology to those of the capsid proteins of some positive-sense single-stranded RNA viruses, such as the nodaviruses and tetraviruses, as well as the T = 13 capsid shell protein of the Reoviridae. The T = 13 shell of the IBDV capsid is formed by trimers of VP2, a protein generated by removal of the C-terminal domain from its precursor, pVP2. The trimming of pVP2 is performed on immature particles as part of the maturation process. The other major structural protein, VP3, is a multifunctional component lying under the T = 13 shell that influences the inherent structural polymorphism of pVP2. The virus-encoded RNA-dependent RNA polymerase, VP1, is incorporated into the capsid through its association with VP3. VP3 also interacts extensively with the viral dsRNA genome.

Bacteriophage Φ6

Bacteriophage Φ6, is a member of the Cystoviridae family. It infects Pseudomonas bacteria (typically plant-pathogenic P. syringae). It has a three-part, segmented, double-stranded RNA genome, totalling ~13.5 kb in length. Φ6 and its relatives have a lipid membrane around their nucleocapsid, a rare trait among bacteriophages. It is a lytic phage, though under certain circumstances has been observed to display a delay in lysis which may be described as a "carrier state".

RNA virus

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An RNA virus is a virus that has RNA (ribonucleic acid) as its genetic material. This nucleic acid is usually single-stranded RNA (ssRNA) but may be double-stranded RNA (dsRNA). The ICTV classifies RNA viruses as those that belong to Group III, Group IV or Group V of the Baltimore classification system of classifying viruses, and does not consider viruses with DNA intermediates as RNA viruses. Notable human diseases caused by RNA viruses include SARS, influenza and hepatitis C.

Another term for RNA viruses that explicitly excludes retroviruses is ribovirus.

Characteristics

Single-stranded RNA viruses and RNA Sense

RNA viruses can be further classified according to the sense or polarity o

f their RNA into negative-sense and positive-sense, or ambisense RNA viruses. Positive-sense viral RNA is identical to viral mRNA and thus can be immediately translated by the host cell. Negative-sense viral RNA is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA polymerase before translation. As such, purified RNA of a positive-sense virus can directly cause infection though it may be less infectious than the whole virus particle. Purified RNA of a negative-sense virus is not infectious by itself as it needs to be transcribed into positive-sense RNA. Ambisense RNA viruses resemble negative-sense RNA viruses, except they also translate genes from the positive strand.

Double-stranded RNA viruses

The double-stranded (ds)RNA viruses represent a diverse group of viruses that vary widely in host range (humans, animals, plants, fungi, and bacteria), genome segment number (one to twelve), and virion organization (T-number, capsid layers, or turrets). Members of this group include the rotaviruses, renowned globally as the commonest cause of gastroenteritis in young children, and bluetongue virus, an economically important pathogen of cattle and sheep. In recent years, remarkable progress has been made in determining, at atomic and subnanometeric levels, the structures of a number of key viral proteins and of the virion capsids of several dsRNA viruses, highlighting the significant parallels in the structure and replicative processes of many of these viruses.

Mutation rates

RNA viruses generally have very high mutation rates as they lack DNA polymerases which can find and fix mistakes, and are therefore unable to conduct DNA repair of damaged genetic material. DNA viruses have considerably lower mutation rates due to the proof-reading ability of DNA polymerases within the host cell. Retroviruses have a high mutation rate even though their DNA intermediate integrates into the host genome (and is thus subject to host DNA proofreading once integrated), because errors during reverse transcription are embedded into both strands of DNA prior to integration.

Although RNA usually mutates rapidly, recent work found that the SARS virus and related RNA viruses contain a gene that mutates very slowly. The gene in question has a complex three-dimensional structure which is hypothesized to provide a chemical function necessary for viral propagation, perhaps as a ribozyme. If so, most mutations would render it unfit for that purpose and would not propagate.

Replication

Animal RNA viruses are classified into three distinct groups depending on their genome and mode of replication (and the numerical groups based on the older Baltimore classification):

Double-stranded RNA viruses (Group III) contain from one to a dozen different RNA molecules, each of which codes for one or more viral proteins.
Positive-sense ssRNA viruses (Group IV) have their genome directly utilized as if it were mRNA, producing a single protein which is modified by host and viral proteins to form the various proteins needed for replication. One of these includes RNA-d ependent RNA polymerase, which copies the viral RNA to form a double-stranded replicative form, in turn this directs the formation of new virions.
Negative-sense ssRNA viruses (Group V) must have their genome copied by an RNA polymerase to form positive-sense RNA. This means that the virus must bring along with it the RNA-dependent RNA polymerase enzyme. The positive-sense RNA molecule then acts as viral mRNA, which is translated into proteins by the host ribosomes. The resultant protein goes on to direct the synthesis of new virions, such as capsid proteins and RNA replicase, which is used to produce new negative-sense RNA molecules.

Retroviruses (Group VI) have a single-stranded RNA genome but are generally not considered RNA viruses because they use DNA intermediates to replicate. Reverse transcriptase, a viral enzyme that comes from the virus itself after it is uncoated, converts the viral RNA into a complementary strand of DNA, which is copied to produce a double stranded molecule of viral DNA. After this DNA is integrated, expression of the encoded genes may lead the formation of new virions.

Group III - dsRNA viruses

Family Birnaviridae
Family Chrysoviridae
Family Cystoviridae
Family Hypoviridae
Family Partitiviridae
Family Reoviridae - includes Rotavirus
Family Totiviridae
Unassigned genera

Group IV - positive-sense ssRNA viruses

Order Nidovirales
- Family Arteriviridae
- Family Coronaviridae - includes Coronavirus, SARS
- Family Roniviridae
Unassigned
- Family Astroviridae
- Family Barnaviridae
- Family Bromoviridae
- Family Caliciviridae - includes Norwalk virus
- Family Closteroviridae
- Family Comoviridae
- Family Dicistroviridae
- Family Flaviviridae - includes Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus
- Family Flexiviridae
- Family Leviviridae
- Family Luteoviridae - includes Barley yellow dwarf virus
- Family Marnaviridae
- Family Narnaviridae
- Family Nodaviridae
- Family Picornaviridae - includes Poliovirus, the common cold virus, Hepatitis A virus
- Family Potyviridae
- Family Sequiviridae
- Family Tetraviridae
- Family Togaviridae - includes Rubella virus, Ross River virus, Sindbis virus,Chikungunya virus
- Family Tombusviridae
- Family Tymoviridae
- Unassigned genera
  - Genus Benyvirus
  - Genus Cheravirus
  - Genus Furovirus
  - Genus Hepevirus - includes Hepatitis E virus
  - Genus Hordeivirus
  - Genus Idaeovirus
  - Genus Ourmiavirus
  - Genus Pecluvirus
  - Genus Pomovirus
  - Genus Sadwavirus
  - Genus Sobemovirus
  - Genus Tobamovirus - includes tobacco mosaic virus
  - Genus Tobravirus
  - Genus Umbravirus

Group V - negative-sense ssRNA viruses

Order Mononegavirales
- Family Bornaviridae - Borna disease virus
- Family Filoviridae - includes Ebola virus, Marburg virus
- Family Paramyxoviridae - includes Measles virus, Mumps virus, Nipah virus, Hendra virus
- Family Rhabdoviridae - includes Rabies virus
Unassigned
- Family Arenaviridae - includes Lassa virus
- Family Bunyaviridae - includes Hantavirus
- Family Orthomyxoviridae - includes Influenza viruses
- Unassigned genera:
  - Genus Deltavirus
  - Genus Ophiovirus
  - Genus Tenuivirus
  - Genus Varicosavirus

Antisense RNA

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Antisense RNA is single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery. This effect is therefore stoichiometric. An example of naturally occurring mRNA antisense mechanism is the hok/sok system of the E.coli R1 plasmid. Antisense RNA has long been thought of as a promising technique for disease therapy; the only such case to have reached the market is the drug fomivirsen. One commentator has characterized antisense RNA as one of "dozens of technologies that are gorgeous in concept, but exasperating in [commercialization]". Generally, antisense RNA still lack effective design, biological activity, and efficient route of administration.

Historically, the effects of antisense RNA have often been confused with the effects of RNA interference, a related process in which double-stranded RNA fragments called small interfering RNAs trigger catalytically mediated gene silencing, most typically by targeting the RNA-induced silencing complex (RISC) to bind to and degrade the mRNA. Attempts to genetically engineer transgenic plants to express antisense RNA instead activate the RNAi pathway, although the processes result in differing magnitudes of the same downstream effect, gene silencing. Well-known examples include the Flavr Savr tomato and two cultivars of ringspot-resistant papaya.

Transcription of longer cis-antisense transcripts is a common phenomenon in the mammalian transcriptome. Although the function of some cases have been described, such as the Zeb2/Sip1 antisense RNA, no general function has been elucidated. In the case of Zeb2/Sip1, the antisense noncoding RNA is opposite the 5' splice site of an intron in the 5'UTR of the Zeb2 mRNA. Expression of the antisense ncRNA prevents splicing of an intron that contains a ribosome entry site necessary for efficient expression of the Zeb2 protein. Transcription of long antisense ncRNAs is often concordant with the associated protein-coding gene, but more detailed studies have revealed that the relative expression patterns of the mRNA and antisense ncRNA are complex.

Sense (molecular biology)

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Sense, when applied in a molecular biology context, is a general concept used to compare the polarity of nucleic acid molecules, such as DNA or RNA, to other nucleic acid molecules. Depending on the context within molecular biology, sense may have slightly different meanings.

DNA sense

Molecular biologists call a DNA single strand or sequence sense (or positive sense) if an RNA version of the same sequence is translated or translatable into protein, and they call its complement antisense (or negative sense). Sometimes the phrase coding strand is encountered; however, protein coding and non-coding RNA's can be transcribed similarly from both strands, in some cases being transcribed in both directions from a common promoter region, or being transcribed from within introns, on both strands.

Antisense DNA

Schematic showing how antisense DNA strands can interfere with protein translation.

Antisense molecules interact with complementary strands of nucleic acids, modifying expression of genes.

Some regions within a double strand of DNA code for genes, which are usually instructions specifying the order of amino acids in a protein along with regulatory sequences, splicing sites, noncoding introns and other complicating details. For a cell to use this information, one strand of the DNA serves as a template for the synthesis of a complementary strand of RNA. The template DNA strand is called the transcribed strand with antisense sequence and the mRNA transcript is said to be sense sequence (the complement of antisense). Because the DNA is double-stranded, the strand complementary to the antisense sequence is called non-transcribed strand and has the same sense sequence as the mRNA transcript (though T bases in DNA are substituted with U bases in RNA).

DNA strand 1: sense strand

DNA strand 2: antisense strand (copied to)→ RNA strand (sense)

Many forms of antisense have been developed and can be broadly categorized into enzyme-dependent antisense or steric blocking antisense.

Enzyme-dependent antisense includes forms dependent on RNase H activity to degrade target mRNA, including single-stranded DNA, RNA, and phosphorothioate antisense. The R1 plasmid hok/sok system is an example of mRNA antisense regulation process, through enzymatic degradation of the resulting RNA duplex. Double stranded RNA acts as enzyme-dependent antisense through the RNAi/siRNA pathway, involving target mRNA recognition through sense-antisense strand pairing followed by target mRNA degradation by the RNA-induced silencing complex (RISC).

Steric blocking antisense (RNase-H independent antisense) interferes with gene expression or other mRNA-dependent cellular processes by binding to a target sequence of mRNA and getting in the way of other processes. Steric blocking antisense includes 2'-O alkyl (usually in chimeras with RNase-H dependent antisense), peptide nucleic acid (PNA), locked nucleic acid (LNA) and Morpholino antisense.

Antisense nucleic acid molecules have been used experimentally to bind to mRNA and prevent expression of specific genes. Antisense therapies are also in development; in the USA, the Food and Drug Administration (FDA) has approved a phosphorothioate antisense oligo, fomivirsen (Vitravene), for human therapeutic use.

Cells can produce antisense RNA molecules naturally, which interact with complementary mRNA molecules and inhibit their expression.

Ambisense

A single-stranded genome which contains both positive-sense and negative-sense is said to be ambisense. Bunya viruses have 3 single-stranded RNA (ssRNA) fragments containing both positive-sense and negative-sense sections; arenaviruses are also ssRNA viruses with an ambisense genome, as they have 2 fragments which are mainly negative-sense except for part of the 5' ends of the large and small segments of their genome.

Antisense mRNA

Antisense mRNA is an mRNA transcript that is complementary to endogenous mRNA. In other words, it is a non-coding strand complementary to the coding sequence of mRNA; this is similar to negative-sense viral RNA. Introducing a transgene coding for antisense mRNA is a technique used to block expression of a gene of interest. Radioactively-labelled antisense mRNA can be used to show the level of transcription of genes in various cell types. Some alternative antisense structural types are being experimentally applied as antisense therapy, with at least one antisense therapy approved for use in humans.

RNA sense

In virology, the genome of a RNA virus can be said to be either positive-sense, also known as a "plus-strand", or negative-sense, also known as a "minus-strand". In most cases, the terms sense and strand are used interchangeably, making such terms as positive-strand equivalent to positive-sense, and plus-strand equivalent to plus-sense. Whether a virus is positive-sense or negative-sense can be used as a basis for classifiying viruses.

Positive-sense

Positive-sense (5' to 3') viral RNA signifies that a particular viral RNA sequence may be directly translated into the desired viral proteins. Therefore, in positive-sense RNA viruses, the viral RNA genome can be considered viral mRNA, and can be immediately translated by the host cell. Unlike negative-sense RNA, positive-sense RNA is of the same sense as mRNA. Some viruses (e.g. Coronaviridae) have positive-sense genomes which can act as mRNA and be used directly to synthesise proteins without the help of a complementary RNA intermediate. Because of this, these viruses do not need to have an RNA transcriptase packaged into the virion.

Negative-sense

Negative-sense (3' to 5') viral RNA is complementary to the viral mRNA and thus must be converted to positive-sense RNA by an RNA polymerase prior to translation. Negative-sense RNA (like DNA) has a nucleotide sequence complementary to the mRNA that it encodes. Like DNA, this RNA cannot be translated into protein directly. Instead, it must first be transcribed into a positive-sense RNA which acts as an mRNA. Some viruses (Influenza, for example) have negative-sense genomes and so must carry an RNA polymerase inside the virion.

Magnetic immunoassay

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Magnetic immunoassay (MIA) is a novel type of diagnostic immunoassay utilizing magnetic beads as labels in lieu of conventional enzymes (ELISA), radioisotopes (RIA) or fluorescent moieties (fluorescent immunoassays). This assay involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of the pair. The presence of magnetic beads is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the beads. The signal measured by the magnetometer is proportional to the analyte (virus, toxin, bacteria, cardiac marker,etc.) quantity in the initial sample.

Magnetic labels

Magnetic beads are comprised of nanometric-sized iron oxide particles encapsulated or glued together with polymers. These magnetic beads range from 35nm up to 4.5μm. The component magnetic nanoparticles range from 5 to 50nm and exhibit a unique quality referred to as superparamagnetism in the presence of an externally applied magnetic field. First discovered by Frenchman Louis Néel, Nobel Physics Prize winner in 1970, this superparamagnetic quality has already been utilized for medical application in Magnetic Resonance Imaging (MRI) and in biological separations, but not yet for labeling in commercial diagnostic applications. Magnetic labels exhibit several features very well adapted for such applications:

they are not affected by reagent chemistry or photo-bleaching and are therefore stable over time,
the magnetic background in a biomolecular sample is usually insignificant,
sample turbidity or staining have no impact on magnetic properties,
magnetic beads can be manipulated remotely by magnetism.

Magnetometers

A simple instrument can detect the presence and measure the total magnetic signal of a sample, however the challenge of developing an effective MIA is to separate naturally occurring magnetic background (noise) from the weak magnetically labeled target (signal). Various approaches and devices have been employed to achieve a meaningful signal-to-noise ratio (SNR) for bio-sensing applications: giant magneto-resistive sensors and spin valves, piezo-resistive cantilevers, inductive sensors, superconducting quantum interference devices, anisotropic magneto-resistive rings, and miniature Hall sensors. But improving SNR often requires a complex instrument to provide repeated scanning and extrapolation through data processing, or precise alignment of target and sensor of miniature and matching size. Beyond this requirement, MIA that exploits the non-linear magnetic properties of magnetic labels can effectively utilize the intrinsic ability of a magnetic field to pass through plastic, water, nitrocellulose, and other materials, thus allowing for true volumetric measurements in various immunoassay formats. Unlike conventional methods that measure the susceptibility of superparamagnetic materials, a MIA based on non-linear magnetization eliminates the impact of linear dia- or paramagnetic materials such as sample matrix, consumable plastics and/or nitrocellulose. Although the intrinsic magnetism of these materials is very weak, with typical susceptibility values of –10^-5 (dia) or +10^-3 (para), when one is investigating very small quantities of superparamagnetic materials, such as nanograms per test, the background signal generated by ancillary materials cannot be ignored. In MIA based on non-linear magnetic properties of magnetic labels the beads are exposed to an alternating magnetic field at two frequencies, f1 and f2. In the presence of non-linear materials such as superparamagnetic labels, a signal can be recorded at combinatorial frequencies, for example, at f = f1 ± 2×f2. This signal is exactly proportional to the amount of magnetic material inside the reading coil.

This technology,makes magnetic immunoassay possible in a variety of formats such as:

conventional lateral flow test by replacing gold labels with magnetic labels,
vertical flow tests allowing for the interrogation of rare analytes (such as bacteria) in large-volume samples
microfluidic applications and biochip

It was also described for in vivo applications and for multiparametric testing.

Secondary antibody

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The primary antibody (in purple) binds to an antigen (in red). A labeled secondary antibody (in green), then binds to the primary antibody. The label is then used to indirectly detect the antigen.

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications.

Secondary antibodies may be polyclonal or monoclonal, and are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions.

Specific secondary antibodies are usually chosen to work in specific laboratory applications. Frequently, any one of several secondary antibodies perform adequately in a particular application. They are selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred. Identifying the optimal secondary antibody is normally done through trial and error.

Applications

Secondary antibodies are used in many biochemical assays including:

ELISA, including many HIV tests
Western blot
Immunostaining
Immunohistochemistry
Immunocytochemistry

Nomenclature of monoclonal antibodies

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Complete list of stems for
monoclonal antibody nomenclature
Prefix	Target		Source		Suffix
variable	-vi(r)-	viral	-u-	human	-mab
	-ba(c)-	bacterial	-o-	mouse
	-li(m)-	immune system	-a-	rat
	-le(s)-	infectious lesions	-e-	hamster
	-ci(r)-	cardiovascular	-i-	primate
	-fu(ng)-	fungal	-xi-	chimeric
	-ne(r)-	nervous system	-zu-	humanized
	-ki(n)-	interleukin as target	-axo-	rat/murine hybrid
	-mu(l)-	musculoskeletal	-xizu-	chimeric + humanized
	-o(s)-	bone
	-tox(a)-	toxin as target
	-anib(i)-	angiogenesis
	-co(l)-	colonic tumor
	-me(l)-	melanoma
	-ma(r)-	mammary tumor
	-go(t)-	testicular tumor
	-go(v)-	ovarian tumor
	-pr(o)-	prostate tumor
	-tu(m)-	miscellaneous tumor

The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or nonproprietary, names to a group of medicines called monoclonal antibodies. This scheme is used for both the World Health Organization’s International Nonproprietary Names and the United States Adopted Names. In general, suffixes are used to identify a class of medicines; all monoclonal antibody pharmaceuticals end with the suffix -mab. However, different infixes are used depending on the structure and function of the medicine.

Components

Infix for origin/source

The infix preceding the -mab suffix denotes the animal origin of the antibodies. Although the original monoclonal antibodies were produced in mice (infix, -o-), these antibodies are recognized as foreign by human immune systems and may be rapidly cleared, provoke an allergic reaction, or both. Therefore, parts of the antibody may be replaced with human sequences. If the constant region is replaced with the human form, it is termed chimeric and the infix used is -xi-. Part of the variable regions may also be substituted, in which case it is termed humanized and the infix used is -zu-. Antibodies originating in humans use -u-.

Infix for target

The infix preceding the source of the antibodies refers to medicine’s target. Most of these consist of a consonant, vowel, then another consonant. For ease of pronunciation and to avoid awkwardness, the final consonant is dropped if the following infix begins with a consonant (such as -zu- or -xi-). Examples of these include -ci(r)- for the circulatory system and -tu(m)- for miscellaneous tumors (cancers).

Prefix and second word

Finally, the prefix carries no special meaning and should be unique for each medicine. A second word may be added if there is another substance attached or linked.

Examples

Abciximab is a commonly used medication to prevent platelets from clumping together. It can be broken down into ab- + -ci(r)- + -xi- + -mab. Therefore, it is a chimeric monoclonal antibody used on the cardiovascular system.

Another example is the breast cancer medication trastuzumab, which can be broken down into tras- + -tu(m)- + -zu- + -mab. Therefore, it is a humanized monoclonal antibody used against a tumor.

Nanobodies

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Nanobodies (aka single domain antibodies or V_HH antibodies) are a type of antibodies derived from camelids, and are much smaller than traditional antibodies. Standard antibodies are giants by molecular standards, since each one is a conglomerate of two heavy protein chains and two light chains, intricately folded and garnished with elaborate sugars. Nanobodies, however, are relatively simple proteins about a tenth the size of human antibodies and just a few nanometers in length. After the discovery that camelidae (camels and llamas) possess fully functional antibodies that lack light chains, the nanobody technology was developed to exploit these smaller heavy-chain-only constructs. Nanobodies are being researched for multiple pharmaceutical applications and have potential for use in cancer and Alzheimer's Disease treatments.

Traditional therapeutic monoclonal antibodies (MAbs) must be stored at near freezing temperatures to prevent their destruction. Antibodies are not suited for oral administration because they are digested quickly in the gut, and are not usually useful for treating diseases of the brain because they do not easily permeate the blood-brain barrier. Additionally, therapeutic antibodies are not well suited to target large tumors because they are held to the periphery of solid tumors. Many illnesses are thus unreachable by monoclonals, and patients who use MAb therapies must receive them by injection or infusion at a clinic. For certain conditions in which the traditional MAbs do not work well, and even for some in which they currently do, simpler, smaller proteins like nanobodies might perform better, be easier to make, easier to handle, easier to admister, and be more affordable. (2)

In 1989 a group of biologists led by Raymond Hamers at the Free University (Brussels, Belgium) investigated an odd observation handed in as part of a student project on parasite immunodefense in dromedary camels (the one-humped Arabian variety) and water buffalo. One of the tests for antibodies in the dromedary blood seemed to show an error: in addition to normal four-chain antibodies it indicated the presence of simpler antibodies composed solely of a pair of heavy chains.

After several years of investigation, Hamers and his colleagues published their discovery in Nature in 1993. In dromedaries — and also in two-humped Asian camels and South American llamas — about half the antibodies circulating in the blood lack a light chain. Equally surprising, they found that these "incomplete" antibodies are able to grasp their targets just as firmly as normal antibodies do, with affinities for their targets virtually equal to a full antibody 10 times their size.

These shortened proteins were also more chemically agile, able to engage targets — including the active sites of enzymes and clefts in cell membranes — too small to admit an antibody.

Because nanobodies are so much smaller than antibodies and are not hydrophobes (as are standard human antibodies), they are more resistant to heat and pH, and may retain their activity as they pass through the gastrointestinal tract, raising the prospect of oral nanobody pills to treat inflammatory bowel disease, colon cancer and other disorders of the gut.

Polyclonal antibodies

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Polyclonal antibodies (or antisera) are antibodies that are derived from different B cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognising a different epitope.

Production

These antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is polyclonal purified from the mammal’s serum.

By contrast, monoclonal antibodies are derived from a single cell line.

Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines governing animal use and procedures relating to these methodologies are generally oriented around humane considerations and appropriate conduct for adjuvant (agents which modify the effect of other agents while having few if any direct effects when given by themselves) use. This includes adjuvant selection, routes and sites of administration, injection volumes per site and number of sites per animal. Institutional policies generally include allowable volumes of blood per collection and safety precautions including appropriate restraint and sedation or anesthesia of animals for injury prevention to animals or personnel.

The primary goal of antibody production in laboratory animals is to obtain high titer, high affinity antisera for use in experimentation or diagnostic tests. Adjuvants are used to improve or enhance an immune response to antigens. Most adjuvants provide for an injection site, antigen depot which allows for a slow release of antigen into draining lymph nodes.

Many adjuvants also contain or act directly as:

surfactants which promote concentration of protein antigens molecules over a large surface area, and
immunostimulatory molecules or properties. Adjuvants are generally used with soluble protein antigens to increase antibody titers and induce a prolonged response with accompanying memory.

Such antigens by themselves are generally poor immunogens. Most complex protein antigens induce multiple B-cell clones during the immune response, thus, the response is polyclonal. Immune responses to non-protein antigens are generally poorly or enhanced by adjuvants and there is no system memory.

Animal selection

Animals frequently used for polyclonal antibody production include chickens, goats, guinea pigs, hamsters, horses, mice, rats, and sheep. However, the rabbit is the most commonly used laboratory animal for this purpose. Animal selection should be based upon:

the amount of antibody needed,
the relationship between the donor of the antigen and the recipient antibody producer (generally the more distant the phylogenetic relationship, the greater the potential for high titer antibody response) and
the necessary characteristics [e.g., class, subclass (isotype), complement fixing nature] of the antibodies to be made. Immunization and phlebotomies are stress associated and, at least when using rabbits and rodents, specific pathogen free (SPF) animals are preferred. Use of such animals can dramatically reduce morbidity and mortality due to pathogenic organisms, especially Pasteurella multocida in rabbits.

Goats or horses are generally used when large quantities of antisera are required. Many investigators favor chickens because of their phylogenetic distance from mammals. Chickens transfer high quantities of IgY (IgG) into the egg yolk and harvesting antibodies from eggs eliminates the need for the invasive bleeding procedure. One week’s eggs can contain 10 times more antibodies than the volume of rabbit blood obtained from one weekly bleeding. However, there are some disadvantages when using certain chicken derived antibodies in immunoassays. Chicken IgY does not fix mammalian complement component C1 and it does not perform as a precipitating antibody using standard solutions.

Although mice are used most frequently for monoclonal antibody production, their small size usually prevents their use for sufficient quantities of polyclonal, serum antibodies. However, polyclonal antibodies in mice can be collected from ascites fluid using any one of a number of ascites producing methodologies.

When using rabbits, young adult animals (2.5–3.0 kg or 5.5-6.5lbs) should be used for primary immunization because of the vigorous antibody response. Immune function peaks at puberty and primary responses to new antigens decline with age. Female rabbits are generally preferred because they are more docile and are reported to mount a more vigorous immune response than males. At least two animals per antigen should be used when using outbred animals. This principle reduces potential total failure resulting from non-responsiveness to antigens of individual animals.

Antigen preparation

The size, extent of aggregation and relative nativity of protein antigens can all dramatically affect the quality and quantity of antibody produced. Small polypeptides (<10>

Keyhole limpet hemocyanin (KLH) and bovine serum albumin are two widely used carrier proteins. Poly-L-lysine has also been used successfully as a backbone for peptides. Although the use of Poly-L-lysine reduces or eliminates production of antibodies to foreign proteins, it may result in failure of peptide-induced antibody production. Recently, liposomes have been successfully used for delivery of small peptides and this technique is more efficient than delivery with oily emulsion adjuvants.

Antigen quantity

Selection of antigen quantity for immunization varies with the properties of the antigen and the adjuvant selected. In general, microgram to milligram quantities of protein in adjuvant are necessary to elicit high titer antibodies. Antigen dosage is generally species, rather than body weight, associated. The so called “window” of immunogenicity in each species is broad but too much or too little antigen can induce tolerance, suppression or immune deviation towards cellular immunity rather than a satisfactory humoral response. Optimal and usual protein antigen levels for immunizing specific species have been reported in the following ranges:

rabbit, 50–1000 µg;
mouse, 10–200 µg;
guinea pig, 50–500 µg; and
goat, 250–5000 µg. Optimal “priming” doses are reported to be at the low end of each range.

The affinity of serum antibodies increases with time (months) after injection of antigen-adjuvant mixtures and as antigen in the system decreases. Widely used antigen dosages for “booster” or secondary immunizations are usually one half to equal the priming dosages. Antigens should be free of preparative byproducts and chemicals such as polyacrylamide gel, SDS, urea, endotoxin, particulate matter and extremes of pH.

Peptide Antibodies

When a peptide is being used to generate the antibody, it is extremely important to design the antigens properly. There are several resources that can aid in the design as well as companies that offer this service. Expasy has aggregated a set of public tools under its ProtScale page that require some degree of user knowledge to navigate. For a more simple peptide scoring tool there is a Antigen Profiler tool available that will enable you to score individual peptide sequences based upon a relation epitope mapping database of previous immunogens used to generate antibodies. Finally, as a general rule peptides should follow some basic criteria.

When examining peptides for synthesis and immunization, it is recommended that certain residues and sequences be avoided due to potential synthesis problems. This includes some of the more common characteristics:

• Extremely long repeats of the same amino acid (e.g. RRRR) • Serine (S), Threonine (T), Alanine (A), and Valine (V) doublets • Ending or starting a sequence with a proline (P) • Glutamine (Q) or Asparagine (N) at the n-terminus • Peptides over weighted with hydrophobic residues (e.g. V,A,L,I, etc…)

Reactivity

Investigators should also consider the status of nativity of protein antigens when used as immunogens and reaction with antibodies produced. Antibodies to native proteins react best with native proteins and antibodies to denatured proteins react best with denatured proteins. If elicited antibodies are to be used on membrane blots (proteins subjected to denaturing conditions) then antibodies should be made against denatured proteins. On the other hand, if antibodies are to be used to react with a native protein or block a protein active site, then antibodies should be made against the native protein. Adjuvants can often alter the nativity of the protein. Generally, absorbed protein antigens in a preformed oil-in-water emulsion adjuvant, retain greater native protein structure than those in water-in-oil emulsions.

Asepticity

Antigens should always be prepared using techniques that ensure that they are free of microbial contamination. Most protein antigen preparations can be sterilized by passage through a 0.22u filter. Septic abscesses often occur at inoculation sites of animals when contaminated preparations are used. This can result in failure of immunization against the targeted antigen.

Adjuvants

There are many commercially available immunologic adjuvants. Selection of specific adjuvants or types varies depending upon whether they are to be used for research and antibody production or in vaccine development. Adjuvants for vaccine use only need to produce protective antibodies and good systemic memory while those for antiserum production need to rapidly induce high titer, high avidity antibodies. No single adjuvant is ideal for all purposes and all have advantages and disadvantages. Adjuvant use generally is accompanied by undesirable side effects of varying severity and duration. Research on new adjuvants focuses on substances which have minimal toxicity while retaining maximum immunostimulation. Investigators should always be aware of potential pain and distress associated with adjuvant use in laboratory animals.

The most frequently used adjuvants for antibody production are Freund’s, Alum, the Ribi Adjuvant System and Titermax.

Freund’s adjuvants

There are two basic types of Freund's adjuvants: Freund’s Complete Adjuvant (FCA) and Freund’s Incomplete Adjuvant (FIA). FCA is a water-in-oil emulsion that localizes antigen for release periods up to 6 months. It is formulated with mineral oil, the surfactant mannide monoleate and heat killed Mycobacterium tuberculosis, Mycobacterium butyricum or their extracts (for aggregation of macrophages at the inoculation site). This potent adjuvant stimulates both cell mediated and humoral immunity with preferential induction of antibody against epitopes of denatured proteins. Although FCA has historically been the most widely used adjuvant, it is one of the more toxic agents due to non-metabolizable mineral oil and it induces granulomatous reactions. Its use is limited to laboratory animals and it should be used only with weak antigens. It should not be used more than once in a single animal since multiple FCA inoculations can cause severe systemic reactions and decreased immune responses. Freund’s Incomplete Adjuvant has the same formulation as FCA but does not contain mycobacterium or its components. FIA usually is limited to booster doses of antigen since it normally much less effective than FCA for primary antibody induction. Freund’s adjuvants are normally mixed with equal parts of antigen preparations to form stable emulsions.

Ribi Adjuvant System

Ribi adjuvants are oil-in-water emulsions where antigens are mixed with small volumes of a metabolizable oil (squalene) which are then emulsified with saline containing the surfactant Tween 80. This system also contains refined mycobacterial products (cord factor, cell wall skeleton) as immunostimulants and bacterial monophosphoryl lipid A. Three different species oriented formulations of the adjuvant system are available. These adjuvants interact with membranes of immune cells resulting in cytokine induction, which enhances antigen uptake, processing and presentation. This adjuvant system is much less toxic and less potent than FCA but generally induces satisfactory amounts of high avidity antibodies against protein antigens.

Titermax

Titermax represents a newer generation of adjuvants that are less toxic and contain no biologically derived materials. It is based upon mixtures of surfactant acting, linear, blocks or chains of nonionic copolymers polyoxypropylene (POP) and polyoxyethylene (POE). These copolymers are less toxic than many other surfactant materials and have potent adjuvant properties which favor chemotaxis, complement activation and antibody production. Titermax adjuvant forms a microparticulate water-in-oil emulsion with a copolymer and metabolizable squalene oil. The copolymer is coated with emulsion stabilizing silica particles which allows for incorporation of large amounts of a wide variety of antigenic materials. The adjuvant active copolymer forms hydrophilic surfaces, which activate complement, immune cells and increased expression of class II major histocompatibility molecules on macrophages. Titermax presents antigen in a highly concentrated form to the immune system, which often results in antibody titers comparable to or higher than FCA.

Specol: Specol is a water in oil adjuvant made of purified mineral oil. It has been reported to induce immune response comparable to Freund's adjuvant in rabbit and other research animal while producing fewer histological lesions.

Humanized antibody

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Humanized antibodies or chimeric antibodies are a type of monoclonal antibody that have been synthesized using recombinant DNA technology to circumvent the clinical problem of immune response to foreign antigens. The standard procedure of producing monoclonal antibodies yields mouse antibodies. Although murine antibodies are very similar to human ones there are differences, and the human immune system recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects.

Humanized antibodies are produced by merging the DNA that encodes the binding portion of a monoclonal mouse antibody with human antibody-producing DNA. One then uses mammalian cell cultures to express this DNA and produce these half-mouse and half-human antibodies that are not as immunogenic as the murine variety.

Alternatives

A solution to this problem would be to generate human antibodies directly from humans. However, this is not easy, primarily because it is not clearly ethical to challenge humans with antigen in order to produce antibody. Furthermore, it is not easy to generate human antibodies against human tissues.

Monoclonal antibody therapy

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Each antibody binds only one specific antigen.

Monoclonal antibody therapy is the use of monoclonal antibodies (or mAb) to specifically target cells. The main objective is stimulating the patient's immune system to attack the malignant tumor cells and the prevention of tumor growth by blocking specific cell receptors. Variations exist within this treatment, e.g. radioimmunotherapy, where a radioactive dose localizes on target cell line, delivering lethal chemical doses to the target.

Structure and function of human and therapeutic antibodies

Immunoglobulin G (IgG) antibodies are large heterodimeric molecules, approximately 150 kDa and are composed of two different kinds of polypeptide chain, called the heavy (~50kDa) and the light chain (~25kDa). There are two types of light chains, kappa (κ) and lambda (λ). By cleavage with enzyme papain, the Fab (fragment-antigen binding) part can be separated from the Fc (fragment crystalline) part of the molecule (see image). The Fab fragments contain the variable domains, which consist of three hypervariable amino acid domains responsible for the antibody specificity embedded into constant regions. There are four known IgG subclasses all of which are involved in Antibody-dependent cellular cytotoxicity.

The immune system responds to the environmental factors it encounters on the basis of discrimination between self and non-self. Tumor cells are not specifically targeted by one's immune system since tumor cells are the patient's own cells. Tumor cells, however are highly abnormal, and many display unusual antigens that are either inappropriate for the cell type, its environment, or are only normally present during the organisms' development (e.g. fetal antigens).

Other tumor cells display cell surface receptors that are rare or absent on the surfaces of healthy cells, and which are responsible for activating cellular signal transduction pathways that cause the unregulated growth and division of the tumor cell. Examples include ErbB2, a constitutively active cell surface receptor that is produced at abnormally high levels on the surface of approximately 30% of breast cancer tumor cells. Such breast cancer is known a HER2 positive breast cancer.

Antibodies are a key component of the adaptive immune response, playing a central role in both in the recognition of foreign antigens and the stimulation of an immune response to them. The advent of monoclonal antibody technology has made it possible to raise antibodies against specific antigens presented on the surfaces of tumors.

Origins of monoclonal antibody therapy

Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment.

Immunotherapy developed as a technique with the discovery of the structure of antibodies and the development of hybridoma technology, which provided the first reliable source of monoclonal antibodies. These advances allowed for the specific targeting of tumors both in vitro and in vivo. Initial research on malignant neoplasms found MAb therapy of limited and generally short-lived success with malignancies of the blood. Furthermore treatment had to be specifically tailored to each individual patient, thus proving to be impracticable for the routine clinical setting.

Throughout the progression of monoclonal drug development there have been four major antibody types developed: murine, chimeric, humanised and human. Initial therapeutic antibodies were simple murine analogues, which contributed to the early lack of success. It has since been shown that these antibodies have: a short half-life in vivo (due to immune complex formation), limited penetration into tumour sites, and that they inadequately recruit host effector functions. To overcome these difficulties the technical issues initially experienced had to be surpassed. Chimeric and humanized antibodies have generally replaced murine antibodies in modern therapeutic antibody applications. Hybridoma technology has been replaced by recombinant DNA technology, transgenic mice and phage display. Understanding of proteomics has proven essential in identifying novel tumour targets.

Murine monoclonal antibodies

Initially, murine antibodies were obtained by hybridoma technology, for which Kohler and Milstein received a Nobel prize. However the dissimilarity between murine and human immune systems led to the clinical failure of these antibodies, except in some specific circumstances. Major problems associated with murine antibodies included reduced stimulation of cytotoxicity and the formation complexes after repeated administration, which resulted in mild allergic reactions and sometimes anaphylactic shock.

Chimeric and humanized monoclonal antibodies

To reduce murine antibody immunogenicity, murine molecules were engineered to remove immunogenic content and to increase their immunologic efficiency. This was initially achieved by the production of chimeric and humanized antibodies. Chimeric antibodies are composed of murine variable regions fused onto human constant regions. Human gene sequences, taken from the kappa light chain and the IgG1 heavy chain, results in antibodies that are approximately 65% human. This reduces immunogenicity, and thus increases serum half-life.

Humanised antibodies are produced by grafting murine hypervariable amino acid domains into human antibodies. This results in a molecule of approximately 95% human origin. However it has been shown in several studies that humanised antibodies bind antigen much more weakly than the parent murine monoclonal antibody, with reported decreases in affinity of up to several hundredfold. Increases in antibody-antigen binding strength have been achieved by introducing mutations into the complementarity determining regions (CDR), using techniques such as chain-shuffling, randomization of complementarity determining regions and generation of antibody libraries with mutations within the variable regions by error-prone PCR, E-coli mutator strains, and site-specific mutagenesis.

Human monoclonal antibodies

Human monoclonal antibodies are produced using transgenic mice or phage display libraries. Human monoclonal antibodies are produced by transferring human immunoglobulin genes into the murine genome, after which the transgenic mouse is vaccinated against the desired antigen, leading to the production of monoclonal antibodies. Phage display libraries allow the transformation of murine antibodies in vitro into fully human antibodies.

FDA approved therapeutic antibodies

The first FDA-approved therapeutic monoclonal antibody was a murine IgG2a CD3 specific transplant rejection drug, Muromonab (OKT-3), in 1986. This drug found use in solid organ transplant recipients who became steroid resistant. Currently, twenty-one FDA-approved therapies exist, and hundreds of therapies are undergoing clinical trials. Most are concerned with immunological and oncological targets.

FDA approved monoclonal antibodies

Antibody Brand name Approval date Type Target Approved treatment(s) Abciximab
ReoPro 1994 chimeric inhibition of glycoprotein IIb/IIIa Cardiovascular disease
Adalimumab
Humira 2002 human inhibition of TNF-a signalling Inflammatory diseases (mostly auto-immune disorders) Alemtuzumab
Campath 2001 humanized CD52
Chronic lymphocytic leukemia
Basiliximab
Simulect 1998 chimeric IL-2 receptor a Transplant rejection
Bevacizumab
Avastin 2004 humanized vascular endothelial growth factor
Colorectal cancer
Cetuximab
Erbitux 2004 chimeric epidermal growth factor receptor
Colorectal cancer Daclizumab
Zenapax 1997 humanized IL-2 receptor a Transplant rejection Eculizumab
Soliris 2007 humanized complement system protein C5 Inflammatory diseases including paroxysmal nocturnal hemoglobinuria Efalizumab
Raptiva 2002 humanized CD11a
Inflammatory diseases (psoriasis) Ibritumomab tiuxetan
Zevalin 2002 murine CD20
Non-Hodgkin lymphoma (with yttrium-90 or indium-111) Infliximab
Remicade 1998 chimeric inhibition of TNF-a signalling Inflammatory diseases (mostly auto-immune disorders) Muromonab-CD3
Orthoclone OKT3 1986 murine T cell CD3 Receptor Transplant rejection Natalizumab
Tysabri 2006 humanized T cell VLA4 receptor Inflammatory diseases (mainly autoimmune-related multiple sclerosis therapy) Omalizumab
Xolair 2004 humanized immunoglobulin E (IgE) Inflammatory diseases (mainly allergy-related asthma therapy) Palivizumab
Synagis 1998 humanized an epitope of the F protein of RSV Viral infection (especially Respiratory Syncytial Virus (RSV) Panitumumab
Vectibix 2006 human epidermal growth factor receptor Colorectal cancer Ranibizumab
Lucentis 2006 humanized vascular endothelial growth factor
Macular degeneration
Gemtuzumab ozogamicin
Mylotarg 2000 humanized CD33
Acute myelogenous leukemia (with calicheamicin) Rituximab
Rituxan, Mabthera 1997 chimeric CD20
Non-Hodgkin lymphoma
Tositumomab
Bexxar 2003 murine CD20
Non-Hodgkin lymphoma Trastuzumab
Herceptin 1998 humanized ErbB2
Breast cancer
Radioimmunotherapy

Radioimmunotherapy involves the use of radioactively conjugated murine antibodies against cellular antigens. Most research currently involved their application to lymphomas, as these are highly radio-sensitive malignancies. To limit radiation exposure, murine antibodies were especially chosen, as their high immunogenicity promotes rapid clearance from the body. Tositumomab is an exemplar used for non-Hodgkins lymphoma.

Antibody-directed enzyme prodrug therapy (ADEPT)

ADEPT involves the application of cancer associated monoclonal antibodies which are linked to a drug-activating enzyme. Subsequent systemic administration of a non-toxic agent results in its conversion to a toxic drug, and resulting in a cytotoxic effect which can be targeted at malignant cells. The clinical success of ADEPT treatments has been limited to date. However it holds great promise, and recent reports suggest that it will have a role in future oncological treatment.

Drug and gene therapy: Immuno-liposomes

Immunoliposomes are antibody-conjugated liposomes. Liposomes can carry drugs or therapeutic nucleotides and when conjugated with monoclonal antibodies, may be directed against malignant cells. Although this technique is still in its infancy, significant advances have been made. Immunoliposomes have been successfully used in vivo to achieve targeted delivery of tumour-suppressing genes into tumours, using an antibody fragment against the human transferrin receptor. Tissue-specific gene delivery using immunoliposomes has also been achieved in brain, and breast cancer tissue.

Therapeutic Monoclonal Antibody Market Future

Since 2000, the therapeutic market for monoclonal antibodies has grown exponentially. The current “big 5” therapeutic antibodies on the market: Avastin, Herceptin (both oncology), Humira, Remicade (both Autoimmune and Infectious Disease ‘AIID’) and Rituxan (oncology and AIID) accounted for 80% of revenues in 2006.

In the immediate future, it is likely that Genentech/Roche will retain their control over the market (due to ownership of 3 of the “big 5” products), oncology and AIID will remain the mAb segment therapeutic focus (because these are the disease areas addressed by the big 5) and the three most commercially important ‘targets’ for the mAb class will be VEGF (Avastin), TNF-alpha (Remicade and Humira) and CD20 (Rituxan). Experts forecast that the therapeutic antibody market will continue to be dominated by Oncology and AIID segments (82-84 percent) from 2004 to 2011. Furthermore, experts note a potential for change in the balance between Oncology and AIID in the coming years. While Oncology therapeutics dominated the market in 2004, AIID is expected to dominate by 2011.

Monoclonal antibodies

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A general representation of the methods used to produce monoclonal antibodies.

Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell. Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. When used as medications, the generic name ends in -mab (see "Nomenclature of monoclonal antibodies").

Discovery

The idea of a "magic bullet" was first proposed by Paul Ehrlich who at the beginning of the 20th century postulated that if a compound could be made that selectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity.

In the 1970s the B-cell cancer multiple myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein). This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen.

A process of producing monoclonal antibodies involving human-mouse hybrid cells was described by Jerrold Schwaber in 1973 and remains widely cited among those using human-derived hybridomas, but claims to priority have been controversial. A science history paper on the subject gave some credit to Schwaber for inventing a technique that was widely cited, but stopped short of suggesting that he had been cheated. The invention is generally accredited to Georges Köhler, César Milstein, and Niels Kaj Jerne in 1975; who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, come up with a technique to fuse these cells with healthy antibody producing B-cells, and be able to select for the successfully fused cells.

In 1988 Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies, removing the reactions that many monoclonal antibodies caused in some patients.

Production

Researchers looking at slides of cultures of cells that make monoclonal antibodies. These are grown in a lab and the researchers are analyzing the products to select the most promising of them.

Monoclonal antibodies can be grown in unlimited quantities in the bottles shown in this picture.

Technician hand-filling wells with a liquid for a research test. This test involves preparation of cultures in which hybrids are grown in large quantities to produce desired antibody. This is effected by fusing myeloma cell and mouse lymphocyte to form a hybrid cell (hybridoma).

Lab technician bathing prepared slides in a solution. This technician prepares slides of monoclonal antibodies for researchers. The cells shown are labeling human breast cancer.

Hybridoma Cell Production

Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells. Polyethylene glycol is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium is used in which only fused cells can grow. This is because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids.

This enzyme enables cells to synthesize purines by the salvage pathway, here using an extracellular source of hypoxanthine as a precursor. Ordinarily, the absence of HGPRT is not a problem for the cell because cells have an already existing biochemical pathway, the de novo pathway that they can use to synthesize purines. However, when cells are exposed to Aminopterin (a folic acid analogue, which inhibits Dihydrofolate reductase, DHFR), they are unable to use the de novo pathway and are now fully dependent on the salvage pathway for survival.

The selective culture medium is called HAT medium because it contains Hypoxanthine, Aminopterin, and Thymidine. This medium is selective for fused (hybridoma) cells, because unfused myeloma cells cannot grow because they lack HGPRT. The unfused normal spleen cells cannot grow indefinitely because of their limited life span. Therefore, only hybridoma cells are able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner gives it immortality (as it is a cancer cell). The fused hybrid cells are called hybridomas, and since they are derived from cancer cells, are immortal and can be grown indefinitely.

This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then tested for their ability to bind to the antigen (for example with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot, and the most productive and stable clone is then grown in culture medium to a high volume.

The hybridomas are grown indefinitely in asuitable cell culture media, or they are injected in mice (in the peritoneal cavity, the gut), they produce tumors containing an antibody-rich fluid called ascites fluid. The medium must be enriched during selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Production in cell culture is usually preferred as the ascites technique is painful to the animal and if replacement techniques exist, this method is considered unethical.

Recombinant

The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected. These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications. Fermentation chambers have been used to produce these antibodies on a large scale.

Applications

Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence and quantity of this substance, for instance in a Western blot test (to detect a protein on a membrane) or an immunofluorescence test (to detect a substance in a cell). They are also very useful in immunohistochemistry which detect antigen in fixed tissue sections. Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.

Monoclonal antibodies for cancer treatment

One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate; it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell. In fact, every intact antibody can bind to cell receptors or other proteins with its Fc region.

Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment.

The illustration below shows all these possibilities:

Chimeric and humanized antibodies

One problem in medical applications is that the standard procedure of producing monoclonal antibodies yields mouse antibodies. Although murine antibodies are very similar to human ones there are differences. The human immune system hence recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects.

A solution to this problem would be to generate human antibodies directly from humans. However, this is not easy, primarily because it is generally not seen as ethical to challenge humans with antigen in order to produce antibody; the ethics of doing the same to non-humans is a matter of debate. Furthermore, it is not easy to generate human antibodies against human tissues.

Various approaches using recombinant DNA technology to overcome this problem have been tried since the late 1980s. In one approach, one takes the DNA that encodes the binding portion of monoclonal mouse antibodies and merges it with human antibody producing DNA. One then uses mammalian cell cultures to express this DNA and produce these half-mouse and half-human antibodies. (Bacteria cannot be used for this purpose, since they cannot produce this kind of glycoprotein.) Depending on how big a part of the mouse antibody is used, one talks about chimeric antibodies or humanized antibodies. Another approach involves mice genetically engineered to produce more human-like antibodies. Monoclonal antibodies have been generated and approved to treat: cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infection (see monoclonal antibody therapy).

In August 2006 the Pharmaceutical Research and Manufacturers of America reported that U.S. companies had 160 different monoclonal antibodies in clinical trials or awaiting approval by the Food and Drug Administration.

Examples

Monoclonal antibodies
Type	Application	Mechanism	Mode
infliximab	rheumatoid arthritis Crohn's disease	inhibits TNF-α	chimeric
basiliximab	Acute rejection of kidney transplants	inhibits IL-2 on activated T cells	chimeric
abciximab	Prevent coagulation in coronary angioplasty	inhibits the receptor GpIIb/IIIa on platelets	chimeric
daclizumab	Acute rejection of kidney transplants	inhibits IL-2 on activated T cells	humanized
gemtuzumab	relapsed acute myeloid leukaemia	targets an antigen on leukemia cells	humanized
alemtuzumab	B cell leukemia	targets an antigen CD52 on T- and B-lymphocytes	humanized
rituximab	non-Hodgkin's lymphoma	targets phosphoprotein CD20 on B lymphocytes	chimeric
palivizumab	RSV infections in children	inhibits an RSV protein	humanized
trastuzumab	anti-cancer therapy for a specific kind of breast cancer	targets the HER2/neu (erbB2) receptor	humanized
etanercept	rheumatoid arthritis	contains TNF receptor	fusion protein
adalimumab	rheumatoid arthritis	inhibits TNF-α	humanized

Intravenous immunoglobulin

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Intravenous immunoglobulin (IVIG) is a blood product administered intravenously. It contains the pooled IgG immunoglobulins (antibodies) extracted from the plasma of over one thousand blood donors. IVIG's effects last between 2 weeks and 3 months. It is mainly used as treatment in three major categories:

Immune deficiencies - Immune deficiencies such as X-linked agammaglobulinemia, hypogammaglobulinemia (primary immune deficiencies), and acquired compromised immunity conditions ([secondary immune deficiencies), featuring low antibody levels.
Inflammatory and autoimmune diseases.
Acute infections.

Mechanism of action

IVIG is given as a plasma protein replacement therapy (IgG) for immune deficient patients which have decreased or abolished antibody production capabilities. In these immune deficient patients, IVIG is administered to maintain adequate antibodies levels to prevent infections and confers a passive immunity. Treatment is given every 3-4 weeks. In the case of patients with autoimmune disease, IVIG is administered at a high dose (generally 1-2 grams IVIG per kg body weight) to attempt to decrease the severity of the autoimmune disease.

The precise mechanism by which IVIG suppresses harmful inflammation has not been definitively established but is believed to involve the inhibitory Fc receptor. The actual primary target(s) of IVIG in autoimmune disease are still unclear, however. IVIG may work via a multi-step model where the injected IVIG first forms a type of immune complex in the patient. Once these immune complexes are formed, they interact with activating Fc receptors on dendritic cellswhich then mediate anti-inflammatory effects helping to reduce the severity of the autoimmune disease or inflammatory state.

Additionally, the donor antibody may bind directly with the abnormal host antibody, stimulating its removal. Alternatively, the massive quantity of antibody may stimulate the host's complement system, leading to enhanced removal of all antibodies, including the harmful ones. IVIG also blocks the antibody receptors on immune cells (macrophages), leading to decreased damage by these cells, or regulation of macrophage phagocytosis.

IVIG may also regulate the immune response by reacting with a number of membrane receptors on T cells, B cells, and monocytes that are pertinent to autoreactivity and induction of tolerance to self.

A recent report stated that IVIG application to activated T cells leads to their decreased ability to engage microglia. As a result of IVIG treatment of T cells, the findings showed reduced levels of tumor necrosis factor-alpha and interleukin-10 in T cell-microglia co-culture. The results add to the understanding of how IVIG may affect inflammation of the central nervous system in autoimmune inflammatory diseases.

IVIG is useful in some acute infection cases such as in Kawasaki's Disease and pediatric HIV infection.

IVIG notes

IVIG is an infusion of IgG antibodies only. Therefore, peripheral tissues that are defended mainly by IgA antibodies, such as the eyes, lungs, gut and urinary tract are not fully protected by the IVIG treatment.
XLA patients are immune to the most virulent adverse effect, anaphylactic shock, as they do not have the antibodies to react against the treatment. Anaphylactic shock has a higher chance to occur in IgA deficient patients which do have other antibody types.
In case of recurring side effects, it is recommended to slow the pace of the IVIG administration and to reduce the dosage. It is also advisable to change IVIG brand, as some people react against to a specific brand.
If the patient is diabetic, he should take into consideration the medium in which the antibodies are solubilized in the IVIG treatment, as some brand solubilize antibodies with high concentrated sugars (such as sucrose and maltose).
FDA guidelines for IVIG state the product should be:
- Prepared out of at least 1,000 different human donors.
- All four IgG subgroups (1-4) should be present.
- The IgG should maintain biological activity and lifetime of at least 21 days.
- Does not contain samples which are HIV, hepatitis B, hepatitis C positive.
- Screened and treated in a manner that destroys viruses.
IVIG is also considered a modulator of the immune system and was shown to be beneficial in treating numerous autoimmune diseases such as relapsing and remitting multiple sclerosis (MS), myasthenia gravis, pemphigus, polymyositis (PM), dermatomyositis (DM), Wegener's granulomatosis (WG), Churg-Strauss syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP) and more.
IVIG can be given to pregnant women.
IVIG is also used as a treatment for unexplained recurring miscarriages. The effectiveness of the therapy is controversial.
IVIG cost is climbing and well over $50/g. ($10,000 for a 220lbs person at 2g/kg)

Uses of IVIG

Dosage of IVIG is dependent on indication.

For primary immune dysfunction 100 to 400 mg/kg of body weight every 3 to 4 weeks is implemented.

For neurological and autoimmune diseases 2 grams per kilogram of body weight is implemented for three to six months over a five day course once a month. Then maintenance therapy of 100 to 400 mg/kg of body weight every 3 to 4 weeks follows.

FDA-approved indications

Allogeneic bone marrow transplant
Chronic lymphocytic leukemia
Idiopathic thrombocytopenic purpura
Pediatric HIV
Primary immunodeficiencies
Kawasaki disease
Alzheimer's Disease
Kidney transplant with a high antibody recipient or with an ABO incompatible donor
Common Variable Immune Deficiency

In 2004 the FDA approved the Cedars-Sinai IVIG Protocol which has been 90-95% successful in removing antibodies from the blood of kidney transplant recipients so that they can accept a living

Off-label Uses

Chronic fatigue syndrome
Chronic inflammatory demyelinating polyneuropathy (CIDP)
Clostridium difficile colitis
Dermatomyositis and polymyositis
Graves' ophthalmopathy
Guillain-Barré syndrome
Kawasaki disease
Muscular Dystrophy
Inclusion body myositis
Lambert-Eaton syndrome
Lupus erythematosus
Multifocal motor neuropathy
Multiple sclerosis
Myasthenia gravis
Neonatal alloimmune thrombocytopenia
Parvovirus B19
Pemphigus
Post-transfusion purpura
Renal transplant rejection
Spontaneous Abortion/Miscarriage
Stiff person syndrome
Severe sepsis and septic shock in critically ill adults
Toxic epidermal necrolysis
In chronic lymphocytic leukemia and multiple myeloma, as well as various rare deficiencies of immunoglobulin synthesis (e.g. X-linked agammaglobulinemia, hypogammaglobulinemia), IVIG is administered to maintain adequate immunoglobulin levels to prevent infections.

Complications and side effects

Complications of IVIG therapy include:

anaphylactic shock, especially in IgA deficient patients, who by definition can still produce IgG antibodies. IgA deficient patients are more likely to produce IgG against the IVIG administration than normal patients.

headache
dermatitis - usually peeling of the skin of the palms and soles
infection (such as HIV or viral hepatitis) by contaminated blood product; there is also an as yet unknown risk of contracting variant CJD (vCJD).
pulmonary edema from fluid overload, due to the high colloid oncotic pressure of IVIG
allergic/anaphylactic reactions
damage such as hepatitis caused directly by antibodies contained in the pooled IVIG
acute renal failure
venous thrombosis
aseptic meningitis

Interferon

2009-06-08T06:38:00.004-07:00

Interferons (IFNs) are natural proteins produced by the cells of the immune system of most vertebrates in response to challenges by foreign agents such as viruses, parasites and tumor cells. Interferons belong to the large class of glycoproteins known as cytokines. Interferons are produced by a wide variety of cells in response to the presence of double-stranded RNA, a key indicator of viral infection. Interferons assist the immune response by inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing antigen presentation to lymphocytes, and inducing the resistance of host cells to viral infection.

Types of interferon

There are three major classes of interferons that have been described for humans according to the type of receptor through which they signal:

Interferon type I: All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. The type I interferons present in humans are IFN-α, IFN-β and IFN-ω.

Interferon type II: Binds to IFNGR. In humans this is IFN-γ.

Interferon type III: Signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12)

Signaling pathway

While there is evidence to suggest other signaling mechanisms exist, the JAK-STAT signaling pathway is the best-characterised and commonly accepted IFN signaling pathway.

Natural function and synthesis

Interferons in general have several effects in common. They are antiviral and possess antioncogenic properties, macrophage and natural killer lymphocyte activation, and enhancement of major histocompatibility complex glycoprotein classes I and II, and thus presentation of foreign (microbial) peptides to T cells. In a majority of cases, the production of interferons is induced in response to microbes such as viruses and bacteria and their products (viral glycoproteins, viral RNA, bacterial endotoxin, bacterial flagella, CpG sites), as well as mitogens and other cytokines, for example interleukin 1, interleukin 2, interleukin-12, tumor necrosis factor and colony-stimulating factor, that are synthesised in the response to the appearance of various antigens in the body. Their metabolism and excretion take place mainly in the liver and kidneys. They rarely pass the placenta but they can cross the blood-brain barrier.

The therapeutically used forms are denoted by Greek letters indicating their origin: leukocytes, fibroblasts, and lymphocytes for interferon-alpha, -beta and -gamma, respectively.

Viral induction of interferons

All classes of interferon are very important in fighting RNA virus infections. However, their presence also accounts for some of the host symptoms, such as sore muscles and fever. They are secreted when abnormally large amounts of dsRNA are found in a cell. dsRNA is normally present in very low quantities. The dsRNA acts like a trigger for the production of interferon (via Toll Like Receptor 3 (TLR 3), a pattern recognition receptor of the innate immune system which leads to activation of the transcription factor IRF3 and late phase NF kappa Beta). The gene that codes for this cytokine is switched on in an infected cell, and the interferon synthesized and secreted to surrounding cells.

As the original cell dies from the cytolytic RNA virus, these thousands of viruses will infect nearby cells. However, these cells have received interferon, which essentially warns these other cells of the virus. They then start producing large amounts of a protein known as protein kinase R (or PKR). If a virus infects a cell that has been “pre-warned” by interferon, the PKR is indirectly activated by the dsRNA (actually by 2'-5' oligoadenylate produced by the 2'-5' oligoadenylate-synthetase which is produced due to TLR3 activation), and begins transferring phosphate groups (phosphorylating) to a protein known as eIF-2, a eukaryotic translation initiation factor. After phosphorylation, eIF2 has a reduced ability to initiate translation, the production of proteins coded by cellular mRNA. This prevents viral replication and inhibits normal cell ribosome function, killing both the virus and the host cell if the response is active for a sufficient amount of time. All RNA within the cell is also degraded, preventing the mRNA from being translated by eIF2 if some of the eIF2 failed to be phosphorylated.

Furthermore, interferon leads to upregulation of MHC I and therefore to increased presentation of viral peptides to cytotoxic CD8 T cells, as well as to a change in the proteasome (exchange of some beta subunits by b1i, b2i, b5i - then known as the immunoproteasome) which leads to increased production of MHC I compatible peptides.

Interferon can cause increased p53 activity in virus infected cells. It acts as an inducer and causes increased production of the p53 gene product. This promotes apoptosis, limiting the ability of the virus to spread. Increased levels of transcription are observed even in cells which are not infected, but only infected cells show increased apoptosis. This increased transcription may serve to prepare susceptible cells so they can respond quickly in the case of infection. When p53 is induced by viral presence, it behaves differently than it usually does. Some p53 target genes are expressed under viral load, but others, especially those that respond to DNA damage, aren’t. One of the genes that is not activated is p21, which can promote cell survival. Leaving this gene inactive would help promote the apoptotic effect. Interferon enhances the apoptotic effects of p53, but it is not strictly required. Normal cells exhibit a stronger apoptotic response than cells without p53.

Additionally, interferon has been shown to have therapeutic effect against certain cancers. It is probable that one mechanism of this effect is p53 induction. This could be useful clinically: Interferons could supplement or replace chemotherapy drugs that activate p53 but also cause unwanted side effects. Some of these side effects can be serious, severe and permanent.

Virus resistance to interferons

In a study of the blocking of interferon (IFN) by the Japanese Encephalitis Virus (JEV), a group of researchers infected human recombinant IFN-alpha with JEV, DEN-2, and PL406, which are all viruses, and found that some viruses have manifested methods that give them a way around the IFN-alpha/beta response. The viruses need to master these methods so they can have the ability to carry on viral replication and production of new viruses. The ways that viruses find a way around the IFN response is through the inhibition of interferon signaling, production, and the blocking of the functions of IFN-induced proteins.

It is not unusual to find viruses encoding for a multiple number of mechanisms to allow them to elude the IFN response at many different levels. While doing the study with JEV, Lin and his coworkers found that IFN-alpha's inability to block JEV means that JEV may be able to block IFN-alpha signaling which in turn would prevent IFN from having STAT1, STAT2, ISGF3, and IRF-9 signaling. DEN-2 also significantly reduces interferon ability to active JAK-STAT. Some other viral gene products that have been found to have an effect on IFN signaling include EBNA-2, Polyomavirus large T antigen, EBV EBNA1, HPV E7, HCMV, and HHV8. Several poxviruses encode a soluble IFN receptor homologue that acts as a decoy to inhibit the biological activity of IFN, and that activity is for IFN to bind to their cognate receptors on the cell surface to initiate a signaling cascade, known as the Janus kinase(JAK)-signal transducer and activation of transcription(Stat) pathways. For example, a group of researchers found that the B18R protein, which acts as a type 1 IFN receptor and is produced by the vaccinia virus, inhibited IFN's ability to begin the phosphorylation of JAK1 which reduced the antiviral effect of IFN.

Some viruses can encode proteins that bind to dsRNA. In a study where the researchers infected Human U cells with reovirus-sigma3 protein and then, using the Western blot test, they found that reovirus-sigma3 protein does bind to dsRNA. Along with that, another study in which the researchers infected mouse L cells with vaccinia virus E3L found that E3L encodes the p25 protein that binds to dsRNA. Without double stranded RNA (dsRNA), because it is bound to by the proteins, it is not able to create IFN-induced PKR and 2'-5' oligoadenylate-synthetase making IFN ineffective. It was also found that JEV was able to inhibit IFN-alpha's ability to activate or create ISGs such as PKR. PKR was not able to be found in the JEV infected cells and PKR RNA levels were found to be lower in those same infected cells, and this disruption of PKR can occur, for example, in cells infected with flavaviruses.

The H5N1 influenza virus, also known as bird flu, has been shown to have resistance to interferon and other anti-viral cytokines. This is part of the reason for its high mortality rates in humans. It is resistant due to a single amino acid mutation in Non-Structual protein 1 (NS1), the precise mechanism of how this confers immunity is unclear (reference is Lethal H5N1 influenza viruses escape host anti-viral cytokine responses, Sang Heui Seo, Nature Med, 2002).

Pharmaceutical uses

Three vials filled with human leukocyte interferon.

Uses

Just as their natural function, interferons have antiviral, antiseptic and antioncogenic properties when administered as drugs.

Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for many cancers.

More than half of hepatitis C patients treated with interferon respond with viral elimination (sustained virological response), better blood tests and better liver histology (detected on biopsy). There is some evidence that giving interferon immediately following infection can prevent chronic hepatitis C. However, people infected by HCV often do not display symptoms of HCV infection until months or years later making early treatment difficult.

Interferons (interferon beta-1a and interferon beta-1b ) are also used in the treatment and control of multiple sclerosis, an autoimmune disorder.

Administered intranasally in very low doses, interferon is extensively used in Eastern Europe and Russia as a method to prevent and treat viral respiratory diseases such as cold and flu. However, mechanisms of such action of interferon are not well understood; it is thought that doses must be larger by several orders of magnitude to have any effect on the virus. Consequently, most Western scientists are skeptical of any claims of good efficacy.

Route of administration

When used in the systemic therapy, IFN-α and IFN-γ are mostly administered by an intramuscular injection. The injection of interferons in the muscle, in the vein, or under skin is generally well tolerated.

Interferon alpha can also be induced with small imidazoquinoline molecules by activation of TLR7 receptor. Aldara (Imiquimod) cream works with this mechanism to induce IFN alpha and IL12 and approved by FDA to treat Actinic keratosis, Superficial Basal Cell Carcinoma, and External Genital Warts.

Adverse effects

The most frequent adverse effects are flu-like symptoms: increased body temperature, feeling ill, fatigue, headache, muscle pain, convulsion, dizziness, hair thinning, and depression. Erythema, pain and hardness on the spot of injection are also frequently observed. Interferon therapy causes immunosuppression, in particular though neutropenia and can result in some infections manifesting in unusual ways.

All known adverse effects are usually reversible and disappear a few days after the therapy has been finished.

Types

Several different types of interferon are now approved for use in humans.

More recently, the FDA approved pegylated interferon-alpha, in which polyethylene glycol is added to make the interferon last longer in the body. (Pegylated interferon-alpha-2b was approved in January 2001; pegylated interferon-alpha-2a was approved in October 2002.) The pegylated form is injected once weekly, rather than three times per week for conventional interferon-alpha. Used in combination with the antiviral drug ribavirin, pegylated interferon produces sustained cure rates of 75% or better in people with genotype 2 or 3 hepatitis C (which is easier to treat) but still less than 50% in people with genotype 1 (which is most common in the U.S. and Western Europe).

Interferon-beta (Interferon beta-1a and Interferon beta-1b) is used in the treatment and control of multiple sclerosis. By an as-yet-unknown mechanism, interferon-beta inhibits the production of Th1 cytokines and the activation of monocytes.

History

While aiming to develop an improved vaccine for smallpox, two Japanese virologists, Yasu-ichi Nagano and Yasuhiko Kojima working at the Institute for Infectious Diseases at the University of Tokyo, noticed that rabbit-skin or testis previously inoculated with UV-inactivated virus exhibited inhibition of viral growth when re-infected at the same site with live virus. They hypothesised that this was due to some inhibitory factor, and began to characterise it by fractionation of the UV-irradiated viral homogenates using an ultracentrifuge. They published these findings in 1954 in the French journal now known as “Journal de la Société de Biologie”. While this paper demonstrated that the activity could be separated from the virus particles, it could not reconcile the antiviral activity demonstrated in the rabbit skin experiments, with the observation that the same supernatant led to the production of antiviral antibodies in mice. A further paper in 1958, involving triple-ultracentrifugation of the homogenate demonstrated that the inhibitory factor was distinct from the virus particles, leading to trace contamination being ascribed to the 1954 observations.

Meanwhile, the British virologist Alick Isaacs and the Swiss researcher Jean Lindenmann, at the National Institute for Medical Research in London, noticed an interference effect caused by heat-inactivated influenza virus on the growth of live influenza virus in chicken egg membranes in a nutritive solution chorioallantoic membrane. They published their results in 1957; in this paper they coined the term ‘interferon’, and today that specific interfering agent is known as a ‘Type I interferon’.

Nagano’s work was never fully appreciated in the scientific community; possibly because it was printed in French, but also because his in vivo system was perhaps too complex to provide clear results in the characterisation and purification of interferon. As time passed, Nagano became aware that his work had not been widely recognised, yet did not actively seek revaluation of his status in field of interferon research. As such, the majority of the credit for discovery of the interferon goes to Isaacs and Lindenmann, with whom there is no record of Nagano ever having made personal contact.

As a drug

Interferon was scarce and expensive until 1980 when the interferon gene was inserted into bacteria using recombinant DNA technology, allowing mass cultivation and purification from bacterial cultures or derived from yeast (e.g. Reiferon Retard is the first yeast derived interferon-alpha 2a).

Global sales ~ 5 billion US $. The second most successful pharmaceutical ever to come from genetic engineering.

Misc. facts

Interferon is species-specific: the substance prepared from infected eggs protected only chicken cells from virus infection, while the similar substance prepared from mice protected only mouse cells.
Produced by many cells in the human body by a receptor dependent feedback mechanism.
Interferons are part of the "first-wave" immune response of the innate immune system, acting within hours, whereas antibody production takes days.
A book was written about it: Toine Pieters, Interferon: The Science and Selling of a Miracle Drug (London: Routledge, 2005), xiv+264 pp., ISBN 0-415-34246-5. This book charts the beginnings, history and fate of interferon. The story of its development and use is one of survival in the face of remarkable cycles of promise, hope and disappointment as a miracle drug. The book demonstrates how research on interferon led to new clinical definitions of cancer and a new rational for therapeutic use of the drug. Moreover, through the lens of interferon's voyage the author explores the interaction between the laboratories of science, medicine and society: from the post-penicillin era to the genetics revolution in medicine.
There are two types of IFNs: Type I (binding to IFN-aR1 and IFN-aR2c receptors; IFNAR1 chain is not the major ligand-binding chain), and type II (binding to IFN-gammaR1 and IFN-gammaR2 receptors).
In general, exposure of human cells to viruses or double stranded RNAs induces the production of IFN-a, IFN-b, and IFN-o species.
For the most part, the IFN-alpha species are not glycosylated, although some contain carbohydrates.
The IFN-alpha family represents a family of related and homologous proteins, each exhibiting a unique activity profile. Each IFN-a species seems to exhibit a distinct profile of activities [antiviral, antiproliferative, and stimulation of cytotoxic activities of natural killer (NK) cells and T cells]
The IFNs and IFN-like molecules signal through the Jak-Stat pathway. The receptor for the Type I IFNs consists of two chains, IFN-aR1 and IFN-aR2c. The ligand INF-alpha is a monomer that binds to the two-chain complex of IFN-aR1 and INF-aR2c.
Within each subtype of mammalian Type I IFN, there is additional variability in gene duplication. The IFN-a genes are duplicated to a much greater extent than any other subtype of Type I IFN. This observation in conjunction with the observation that the IFN-a subtypes generally possess the highest specific antiviral activity imply that physiologically, the body likely uses IFN-a as the primary antiviral defense protein and that the major function of IFN-a is defense.
STRUCTURE: The Type I IFNs consist of five a-helices (labeled A–E) which are linked by one overhand loop (AB loop) and three shorter segments (BC, CD, and DE loops). Helices A, B, C, and E are arranged in an antiparallel fashion to form a left-handed four-helix bundle. The AB loop contains short segments of 3_10 helix and is best described in three segments labeled AB1, AB2, and AB3. In all Type I IFNs, the AB1 loop encircles and is linked to helix E by a disulfide bond. An additional disulfide bond is observed in most IFN-a subtypes but not IFN-b, which connects the N-terminus of the molecule to helix C. The AB loop is critical for high-affinity IFNAR2 binding and suggest that sequence differences in this region may hold the key to differences in biological activity between the different IFN-a subtypes.
The NMR structure of IFNAR2 has been determined and exhibits the same general structure as IFN-gammaR1. However, the interdomain angle is approximately 90 degrees rather than 120 degrees. Only loops in N-terminal domain (L2–L4) have been shown to be important for IFN-a2 binding.
The IFNs were the first of the proteins we now recognize as members of the Class II cytokine family.
IFNa2 contain 165 amino acids; according to circular dichroism measurements ~68% of the residues adopt helical conformation.INFa2 is composed of five a-helices, labeled A–E, linked by one long overhand connection (AB loop) and three short segments (BC, CD and DE loops). The topology of the molecule resembles the classical up-up-down-down four-helixbundle motif; helices A, B, C, and E comprise the helix bundle.
Type I IFNs are stable at acidic pH (pH 2) and are represented by two major subtypes, the fibroblast or beta interferon (IFN-b) and the leukocyte or alpha family of interferons (IFN-a).The only known interferon of type II is IFN-g, which is produced exclusively by lymphocytes.

Pharmaceutical forms of interferons in the market

Generic name	Trade name
Interferon alpha 2a	Roferon A
Interferon alpha 2b§	Intron A
Human leukocyte Interferon-alpha (HuIFN-alpha-Le)	Multiferon
Interferon beta 1a, liquid form	Rebif
Interferon beta 1a, lyophilized	Avonex
Interferon beta 1a, biogeneric (Iran)	Cinnovex
Interferon beta 1b	Betaseron / Betaferon
Pegylated interferon alpha 2a	Pegasys
Pegylated interferon alpha 2a (Egypt)	Reiferon Retard
Pegylated interferon alpha 2b	PegIntron
Pegylated interferon alpha 2b plus ribavirin (Canada)	Pegetron

^§ also marketed in India as Reliferon, a product of Reliance Biopharmaceuticals.

Cremophor EL

2009-06-08T06:38:00.003-07:00

Cremophor EL is the registered trademark of BASF Corp. for its version of polyethoxylated castor oil. It is prepared by reacting 35 moles of ethylene oxide with each mole of castor oil. The resulting product is a mixture (CAS number 61791-12-6): the major component is the material in which the hydroxyl groups of the castor oil triglyceride have ethoxylated with ethylene oxide to form polyethylene glycol ethers. Minor components are the polyethyelene glycol esters of ricinoleic acid, polyethyelene glycols and polyethyelene glycol ethers of glycerol. Cremophor EL is a synthetic, nonionic surfactant. Its utility comes from its ability to stabilize emulsions of nonpolar materials in aqueous systems.

Cremophor EL is an excipient or additive in drugs. Therapeutically, modern drugs are rarely given in a pure chemical state, so most active ingredients are combined with excipients or additives such as Cremophor EL.

Uses

Miconazole, anti-fungal
Paclitaxel, anti-cancer

(Cremophor EL is used in Taxol (paclitaxel) and has been called as a dose limiting agent because of its toxicities. (A formulation of paclitaxel that uses nanoparticle albumin instead of Cremophor EL is marketed as an alternative under the trade name of Abraxane.))

Aci-Jel (acetic acid / oxyquinoline / ricinoleic acid - vaginal)
Sandimmune (cyclosporine injection, USP)
Nelfinavir mesylate, HIV protese inhibitor
Propofol, intravenous anaesthetic agent, originally presented in Cremophor for trials; now presented in a lipid emulsion
Diazepam injection; superseded by lipid emulsion alternative (Diazemuls)
Vitamin K injection

Ciclosporin

2009-06-08T06:38:00.001-07:00



Ciclosporin
Systematic (IUPAC) name
(E)-14,17,26,32-tetrabutyl-5-ethyl-8-(1-hydroxy-2-methylhex-4-enyl) -1,3,9,12,15,18,20,23,27-nonamethyl-11,29-dipropyl-1,3,6,9,12,15,18,21,24,27,30- undecaazacyclodotriacontan-2,4,7,10,13,16,19,22,25,28,31-undecaone
Identifiers
CAS number	59865-13-3
ATC code	L04AA01
PubChem	2909
DrugBank	BTD00003
Chemical data
Formula	C62H111N11O12
Mol. mass	1202.61
Pharmacokinetic data
Bioavailability	variable
Metabolism	hepatic
Half life	variable (about 24 hours)
Excretion	biliary
Therapeutic considerations
Pregnancy cat.	C(AU) C(US)
Legal status	Prescription Only (S4)(AU) POM(UK) ℞-only(US)
Routes	oral, IV, ophthalmic

Ciclosporin, cyclosporine (USAN) or cyclosporin (former BAN), is an immunosuppressant drug widely used in post-allogeneic organ transplant to reduce the activity of the patient's immune system and so the risk of organ rejection. It has been studied in transplants of skin, heart, kidney, liver, lung, pancreas, bone marrow and small intestine. Initially isolated from a Norwegian soil sample, Ciclosporin A, the main form of the drug, is a cyclic nonribosomal peptide of 11 amino acids (an undecapeptide) produced by the fungus Tolypocladium inflatum Gams, and contains D-amino acids, which are rarely encountered in nature.

Indications

The immuno-suppressive effect of cyclosporin was discovered on January 31, 1972, by employees of Sandoz (now Novartis) in Basel, Switzerland, in a screening test on immune-suppression designed and implemented by Dr.Hartmann F. Stähelin, M.D. The success of Cyclosporin A in preventing organ rejection was shown in liver transplants performed by Dr. Thomas Starzl at the University of Pittsburgh hospital. The first patient, on March 9, 1980, was a 28-year-old woman. Cyclosporin was subsequently approved for use in 1983.

Apart from in transplant medicine, cyclosporin is also used in psoriasis, severe atopic dermatitis and infrequently in rheumatoid arthritis and related diseases, although it is only used in severe cases. It has been investigated for use in many other autoimmune disorders. Cyclosporin has also been used to help treat patients with ulcerative colitis who do not respond to treatment with steroids. This drug is also used as a treatment of posterior or intermediate uveitis with non-infective etiology.

Cyclosporin A has been investigated as a possible neuroprotective agent in conditions such as traumatic brain injury, and has been shown in animal experiments to reduce brain damage associated with injury. Cyclosporin A blocks the formation of the mitochondrial permeability transition pore, which has been found to cause much of the damage associated with head injury and neurodegenerative diseases.

Mode of action

Cyclosporin is thought to bind to the cytosolic protein cyclophilin (immunophilin) of immunocompetent lymphocytes, especially T-lymphocytes. This complex of ciclosporin and cyclophylin inhibits calcineurin, which under normal circumstances is responsible for activating the transcription of interleukin-2. It also inhibits lymphokine production and interleukin release and therefore leads to a reduced function of effector T-cells. It does not affect cytostatic activity.

It also has an effect on mitochondria. Cyclosporin A prevents the mitochondrial PT pore from opening, thus inhibiting cytochrome c release, a potent apoptotic stimulation factor. However, this is not the primary mode of action for clinical use but rather an important effect for research on apoptosis.

Biosynthesis

Figure 1: Cyclosporine A Biosynthesis. Bmt = butenyl-methyl-threonine, Abu = L-alpha-aminobutyric acid, Sar = sarcosine

Cyclosporine A is synthesized by a nonribosomal peptide synthetase, cyclosporine synthetase. The enzyme contains an adenylation domain, thiolation domain, condensation domain, and an N-methyltransferase domain. The adenylation domain is responsible for substrate recognition and activation. While the thiolation domain covalently binds the adenylated amino acids to phosphopantetheine and the condensation domain elongates the peptide chain. Cyclosporine synthetase substrates includes: L-Valine, L-Leucine, L-Alanine, L-Glycine, 2-aminobutyric acid, 4-methylthreonine, and D-Alanine. With the adenylation domain, cyclosporine synthetase generates the acyl adenylated amino acids then covalently binds the amino acid to phosphopantetheine through a thioester linkage. Some of the amino acid substrates become N-methylated by S-adenosylmethionine. The cyclization step releases cyclosporine A from the enzyme. Amino acids such as D-Ala and butenyl-methyl-L-threonine indicates that cyclosporine synthetase requires the action of other enzymes such as a D-Alanine racemase. The racemization of L-Ala to D-Ala is PLP dependent. The formation of butenyl-methyl-L-threonine is performed by a butenyl-methyl-L-threonine polyketide synthase that utilizes acetate/malonate as its starting material.

Figure 2: Butenyl-methyl-L-Threonine Biosynthesis

Adverse Effects and Interactions

Treatment may be associated with a number of potentially serious adverse drug reactions (ADRs) and adverse drug interactions. Ciclosporin interacts with a wide variety of other drugs and other substances including grapefruit juice. There have been studies into the use of grapefruit juice to increase the blood level of cyclosporin.

ADRs can include gum hyperplasia, convulsions, peptic ulcers, pancreatitis, fever, vomiting, diarrhea, confusion, breathing difficulties, numbness and tingling, pruritus, high blood pressure, potassium retention and possibly hyperkalemia, kidney and liver dysfunction (nephrotoxicity & hepatotoxicity), and obviously an increased vulnerability to opportunistic fungal and viral infections.

An alternate form of the drug, ciclosporin G (OG37-324), has been found to be much less nephrotoxic than the standard ciclosporin A. Ciclosporin G (Mol. mass 1217) differs from ciclosporin A in the amino acid 2 position, where an L-nor-valine replaces the α-aminobutyric acid.

Formulations

The drug is marketed by Novartis under the brand names Sandimmune, the original formulation, and Neoral for the newer microemulsion formulation. Generic ciclosporin preparations have been marketed under various trade names including Cicloral (Sandoz/Hexal) and Gengraf (Abbott). Since 2002 a topical emulsion of ciclosporin for treating keratoconjunctivitis sicca has been marketed under the trade name Restasis. Annual sales of ciclosporin are around $1 billion.

The drug is also available in a dog preparation manufactured by Novartis called Atopica. Atopica is indicated for the treatment of atopic dermatitis in dogs. Unlike the human form of the drug, the lower doses used in dogs mean the drug acts as an immuno-modulator and has fewer side effects than in man. The benefits of using this product include the reduced need for concurrent therapies to bring the condition under control.

Vindesine

2009-06-08T06:37:00.005-07:00


Vindesine

Vindesine is an anti-mitotic vinca alkaloid used in chemotherapy. It is used to treat many different types of cancer, including leukaemia, lymphoma, melanoma, breast cancer, and lung cancer.

Vinorelbine

2009-06-08T06:37:00.003-07:00


Vinorelbine
Systematic (IUPAC) name
4-(acetyloxy)-6,7-didehydro-15- ((2R,6R,8S)-4-ethyl-1,3,6,7,8,9-hexahydro- 8-(methoxycarbonyl)-2,6-methano- 2H-azecino(4,3-b)indol-8-yl)-3-hydroxy- 16-methoxy-1-methyl-,methyl ester, (2beta,3beta,4beta,5alpha,12R,19alpha)- aspidospermidine-3-carboxylic acid

Vinorelbine (Navelbine) is an anti-mitotic chemotherapy drug that is given as a treatment for some types of cancer, including breast cancer and non-small cell lung cancer.

Pharmacology

Vinorelbine is the first 5´NOR semi-synthetic vinca alkaloid. It is obtained by semi-synthesis from alkaloids extracted from the rosy periwinkle, Catharanthus roseus.

History

Vinorelbine was invented by the Pharmacist Pierre Potier and his team from the CNRS in France in the 1980s and was licensed to the oncology department of the Pierre Fabre Group. The drug was approved in France in 1989 under the brand name Navelbine for the treatment of bronchial cancer. It gained approval to treat non-small cell lung cancer in 1991. The drug is now primarily used to treat this cancer. Vinorelbine received approval by the United States Food and Drug Administration (FDA) in December 1994 sponsored by GlaxoSmithKline. The drug went generic in the U.S. in February 2003.
In Europe is approved to treat non-small cell lung cancer, breast cancer and, in some countries, prostate cancer.
Since 2004 an oral formulation has been marketed and registered in Europe for the same settings. It has been shown a similar efficacy and safety profile between both intravenous and per os formulations, avoiding local toxicity induced by the intravenous vinorelbine.

Side effects

Vinorelbine has a number of side-effects that can limit its use:

Lowered resistance to infection, bruising or bleeding, anaemia, constipation, diarrhoea, nausea, numbness or tingling in hands or feet (peripheral neuropathy), tiredness and a general feeling of weakness (asthenia), inflammation of the vein into which it was injected (phlebitis).Seldom severe hyponatriamia is seen

Less common effects are hair loss and allergic reaction.

Vinblastine

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Vinblastine
Systematic (IUPAC) name
?
Identifiers
CAS number	865-21-4
ATC code	L01CA01
PubChem	8935
DrugBank	APRD00708
Chemical data
Formula	C46H58N4O9
Mol. mass	810.974 g/mol
Pharmacokinetic data
Bioavailability	n/a
Metabolism	Hepatic (CYP3A4-mediated)
Half life	24.8 hours (terminal)
Excretion	Biliary and renal
Therapeutic considerations
Pregnancy cat.	D(AU) D(US)
Legal status	POM(UK) ℞-only(US)
Routes	Exclusively intravenous

Vinblastine stick molecular model

Vinblastine is an anti-mitotic drug used to treat certain kinds of cancer, including Hodgkin's lymphoma, non-small cell lung cancer, breast cancer and testicular cancer.

History

Vinblastine was first isolated by Robert Noble and Charles Thomas Beer from the Madagascar periwinkle plant. Vinblastine's utility as a chemotherapeutic agent was first discovered when it was crushed into a tea. Consumption of the tea led to a decreased number of white blood cells; therefore, it was hypothesized that vinblastine might be effective against cancers of the white blood cells such as lymphoma.

Pharmacology

Vinblastine is a vinca alkaloid and a chemical analogue of vincristine. It binds tubulin, thereby inhibiting the assembly of microtubules. It is M phase cell cycle specific since microtubules are a component of the mitotic spindle and the kinetochore which are necessary for the separation of chromosomes during anaphase of mitosis. Toxicities include bone marrow suppression (which is dose-limiting), gastrointestinal toxicity, potent vesicant (blister-forming) activity, and extravasation injury (forms deep ulcers).

Vinblastine paracrystals may be comprised of tightly-packed unpolymerized tubulin or microtubules.

Indications

Vinblastine is a component of a number of chemotherapy regimens, including ABVD for Hodgkin lymphoma. It is also used to treat histiocytosis according to the established protocols of the Histiocytosis Association of America.

Vincristine

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Vincristine
Systematic (IUPAC) name
?
Identifiers
CAS number	57-22-7
ATC code	L01CA02
PubChem	5978
DrugBank	APRD00495
Chemical data
Formula	C46H56N4O10
Mol. mass	824.958 g/mol
Pharmacokinetic data
Bioavailability	n/a
Protein binding	~75%
Metabolism	Hepatic
Half life	19 to 155 hours
Excretion	Mostly biliary, 10% in urine
Therapeutic considerations
Pregnancy cat.	D(AU) D(US)
Legal status	℞ Prescription only
Routes	Exclusively intravenous

Vincristine (brand name, Oncovin), also known as leurocristine, is a vinca alkaloid from the Madagascar periwinkle (Catharanthus roseus, formerly Vinca rosea and hence its name). It is a mitotic inhibitor, and is used in cancer chemotherapy.

Mode of action

Tubulin is a structural protein which polymerises to form microtubules. The cell cytoskeleton and mitotic spindle, amongst other things, are made of microtubules. Vincristine binds to tubulin dimers, inhibiting assembly of microtubule structures. Disruption of the microtubules arrests mitosis in metaphase. The vinca alkaloids therefore affect all rapidly dividing cell types including cancer cells, but also intestinal epithelium and bone marrow.

Uses

Vincristine, injected intravenously only, is used in various types of chemotherapy regimens. Its main uses are in non-Hodgkin's lymphoma as part of the chemotherapy regimen CHOP, Hodgkin's lymphoma as part of MOPP, COPP, BEACOPP, or the less popular Stanford V chemotherapy regimen, in acute lymphoblastic leukemia, and in treatment for nephroblastoma (Wilms tumor, a kidney tumor common in children). It is occasionally used as an immunosuppressant, e.g. in thrombotic thrombocytopenic purpura (TTP).

Side effects

The main side-effects of vincristine are peripheral neuropathy, hyponatremia, constipation and hair loss.

Peripheral neuropathy can be severe, and hence a reason to avoid, reduce, or stop the use of vincristine. One of the first symptoms of peripheral neuropathy is foot drop: a person with a family history of foot drop and/or Charcot-Marie-Tooth disease (CMT) may benefit from genetic testing for CMT before taking vincristine.

Accidental injection of vinca alkaloids into the spinal canal (intrathecal administration) is highly dangerous, with a mortality rate approaching 100%. The medical literature documents cases of ascending paralysis due to massive encephalopathy and spinal nerve demyelination, accompanied by intractable pain, almost uniformly leading to death; a handful of survivors were left with devastating neurological damage with no hope of recovery. Rescue treatments consist of washout of the cerebrospinal fluid and administration of protective medications. A significant series of inadvertent intrathecal vincristine administration occurred in China in 2007 when batches of cytarabine and methotrexate (both often used intrathecally) manufactured by the company Shanghai Hualian were found to be contaminated with vincristine.

History

Having been used as a folk remedy for centuries, studies in the 1950s revealed that C. roseus contained 70 alkaloids, many of which are biologically active. While initial studies for its use in diabetes mellitus were disappointing, the discovery that it caused myelosuppression (decreased activity of the bone marrow) led to its study in mice with leukemia, whose lifespan was prolonged by the use of a vinca preparation. Treatment of the ground plant with Skelly-B defatting agent and an acid benzene extract led to a fraction termed "fraction A". This fraction was further treated with aluminium oxide, chromatography, trichloromethane, benz-dichloromethane and separation by pH to yield vincristine.

Vincristine was approved by the United States Food and Drug Administration (FDA) in July 1963 as Oncovin. The drug was initially discovered by a team lead by Dr. J.G. Armstrong; it was then marketed by Eli Lilly and Company.

Suppliers

Three generic drug makers supply vincristine in the United States - APP, Mayne, and Sicor (Teva).

Antimetabolite

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An antimetabolite is a chemical with a similar structure to a substance (a metabolite) required for normal biochemical reactions, yet different enough to interfere with the normal functions of cells, including cell division.

Function

Cancer treatment

Antimetabolites can be used in cancer treatment, as they interfere with DNA production and therefore cell division and the growth of tumors. Because cancer cells spend more time dividing than other cells, inhibiting cell division harms tumor cells more than other cells.

Anti-metabolites masquerade as a purine (azathioprine, mercaptopurine) or a pyrimidine - which become the building blocks of DNA. They prevent these substances becoming incorporated in to DNA during the S phase (of the cell cycle), stopping normal development and division.

They also affect RNA synthesis. However, because thymidine is used in DNA but not in RNA (where uracil is used instead), inhibition of thymidine synthesis via thymidylate synthase selectively inhibits DNA synthesis over RNA synthesis.

Due to their efficiency, these drugs are the most widely used cytostatics.

In the ATC system, they are classified under L01B.

Antibiotics

Antimetabolites may also be antibiotics, such as sulfanilamide drugs, which inhibit dihydrofolate synthesis in bacteria by competing with para-aminobenzoic acid.

Types

Main representatives of these drugs are:

purine analogues
pyrimidine analogues
antifolates

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An alkylating antineoplastic agent is an alkylating agent that attaches an alkyl group to DNA.

Since cancer cells generally proliferate unrestrictively more than healthy cells do, cancer cells are more sensitive to DNA damage - such as being alkylated. Alkylating agents are used clinically to treat a variety of tumours. However, they are also inherently cytotoxic, leading to side effects particularly in healthy tissues where cell division is frequent, as in gastrointestinal tract or bone marrow.

Agents acting nonspecifically

Some alkylating agents are active under conditions present in cells; and the same mechanism that makes them toxic allows them to be used as anti-cancer drugs. They stop tumour growth by cross-linking guanine nucleobases in DNA double-helix strands - directly attacking DNA. This makes the strands unable to uncoil and separate. As this is necessary in DNA replication, the cells can no longer divide. These drugs act nonspecifically.

Agents require activation

Some of them require conversion into active substances in vivo (e.g. cyclophosphamide).

Cyclophosphamide is one of the most potent immunosuppressive substances. In small dosages, it is very efficient in the therapy of systemic lupus erythematosus, autoimmune hemolytic anemias, Wegener's granulomatosis and other autoimmune diseases. High dosages cause pancytopenia and hemorrhagic cystitis.

Dialkylating agents, limpet attachment, and monoalkylating agents

Dialkylating agents can react with two different 7-N-guanine residues and if these are in different strands of DNA the result is cross-linkage of the DNA strands, which prevents uncoiling of the DNA double helix. If the two guanine residues are in the same strand the result is called limpet attachment of the drug molecule to the DNA.

Monoalkylating agents can react only with one 7-N of guanine.

Limpet attachment and monoalkylation do not prevent the separation of the two DNA strands of the double helix but do prevent vital DNA processing enzymes from accessing the DNA. The final result is inhibition of cell growth or stimulation of apoptosis, cell suicide.

Examples

In the Anatomical Therapeutic Chemical Classification System, alkylating agents are classified under L01A.

Alkylating agents activated by cytochrome p-450:

Alkyl sulfonates
Busulfan (L01AB01)
Ethyleneimines and methylmelamines
- Hexamethylmelamine or altretamine (L01XX03)
- Thiotepa (L01AC01)
Cyclophosphamide (L01AA01)
- Mechlorethamine or mustine (L01AA05)
- Uramustine or uracil mustard (no ATC code, PubChem 6194, DrugBank APRD00130)
- Melphalan (L01AA03)
- Chlorambucil (L01AA02)
- Ifosfamide (L01AA06)

Nitrosoure

- Carmustine (L01AD01)
- Streptozocin (L01AD04)
Triazenes
- Dacarbazine (L01AX04)
Imidazotetrazines
- Temozolomide (L01AX03)

Platinum-based chemotherapeutic drugs (termed platinum analogues) destroy cells by alkylation. These include:

History of cancer chemotherapy

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The era of cancer chemotherapy began in the 1940s with the first use of nitrogen mustards and folic acid antagonist drugs. Cancer drug development has exploded since then into a multi-billion dollar industry. The targeted therapy revolution has arrived, but many of the principles and limitations of chemotherapy discovered by the early researchers still apply.

The first efforts (1940–1950)

The beginnings of the modern era of cancer chemotherapy can be traced directly to the discovery of nitrogen mustard, a chemical warfare agent, as an effective treatment for cancer. Two pharmacologists, Louis S. Goodman and Alfred Gilman were recruited by the United States Department of Defense to investigate potential therapeutic applications of chemical warfare agents. Autopsy observations of people exposed to mustard gas had revealed profound lymphoid and myeloid suppression. Goodman and Gilman reasoned that this agent could be used to treat lymphoma, since lymphoma is a tumor of lymphoid cells. They first set up an animal model - they established lymphomas in mice and demonstrated they could treat them with mustard agents. Next, in collaboration with a thoracic surgeon, Gustav Linskog, they injected a related agent, mustine (the prototype nitrogen mustard anticancer chemotherapeutic), into a patient with non-Hodgkin's lymphoma. They observed a dramatic reduction in the patient's tumour masses. Although this effect lasted only a few weeks, this was the first step to the realization that cancer could be treated by pharmacological agents (Goodman et al 1946).

Another leap forward - The antifolates

Shortly after World War II, a second approach to drug therapy of cancer began. Sidney Farber, a pathologist at Harvard Medical School, studied the effects of folic acid on leukemia patients. Folic acid ( citrovorum factor), a vitamin crucial for DNA metabolism (he did not know the significance of [DNA]http://en.wikipedia.org/wiki/History_of_molecular_biology at that time), had been discovered by Lucy Wills, when she was working in India,in 1937. It seemed to stimulate the proliferation of acute lymphoblastic leukemia (ALL) cells when administered to children with this cancer. In one of the first examples of rational drug design (rather than accidental discovery), in collaboration with Harriett Kilte and Lederle Laboratories chemists, Farber used folate analogues. These analogues — first aminopterin and then amethopterin (now methotrexate) were antagonistic to folic acid, and blocked the function of folate-requiring enzymes. When administered to children with ALL in 1948, these agents became the first drugs to induce remission in children with ALL. Remissions were brief, but the principle was clear — antifolates could suppress proliferation of malignant cells, and could thereby re-establish normal bone-marrow function. It is worth noting that Farber met resistance to conducting his studies at a time when the commonly held medical belief was that leukemia was incurable, and that the children should be allowed to die in peace. Afterwards, Farber's 1948 report in the New England Journal of Medicine was met with incredulity and ridicule.In the same report he has amply appreciated the cooperation and guidance of the famous Biochemist, Dr. Y. SubbaRow ( Director, Research Division, Lederle Labs, a division of American Cyanamid company, Pearl River, NY) who had synthesized and supplied the chemicals, Aminopterin and later Amithopterin for his clinical experiments. Remarkably, a decade later at the National Cancer Institute, Roy Hertz and Min Chiu Li discovered that the same methotrexate treatment alone could cure choriocarcinoma (1958), a germ-cell malignancy that originates in trophoblastic cells of the placenta. This was the first solid tumour to be cured by chemotherapy.

6-MP, vinca alkaloids and a National Treatment Effort by the US

Joseph Burchenal, at Memorial Sloan-Kettering Cancer Center in New York, with Farber's help, started his own methotrexate study and found the same effects. He then decided to try and develop anti-metabolites in the same way as Farber, by making small changes in a metabolite needed by a cell to divide. With the help of George Hitchings and Gertrude Elion, two pharmaceutical chemists who were working at the Burroughs Wellcome Company in Tuckahoe, many purine analogues were tested, culminating in the discovery of 6-mercaptopurine (6-MP), which was subsequently shown to be a highly active antileukemic drug.

The Eli Lilly natural products group found that alkaloids of the Madagascar periwinkle (Vinca rosea), originally discovered in a screen for anti-diabetic drugs, blocked proliferation of tumour cells. The antitumour effect of the vinca alkaloids (e.g. vincristine) was later shown to be due to their ability to inhibit microtubule polymerization, and therefore cell division.

The United States Congress created a National Cancer Chemotherapy Service Center (NCCSC) at the NCI in 1955 in response to early successes. This was the first federal programme to promote drug discovery for cancer - unlike now, most pharmaceutical companies were not yet interested in developing anticancer drugs. The NCCSC developed the methodologies and crucial tools (like cell lines and animal models) for chemotherapeutic development.

Combination chemotherapy

Dr. Vincent T. DeVita, Jr.

In 1965, a major break-through in cancer therapy occurred. James Holland, Emil Freireich, and Emil Frei hypothesized that cancer chemotherapy should follow the strategy of antibiotic therapy for tuberculosis with combinations of drugs, each with a different mechanism of action. Cancer cells could conceivably mutate to become resistant to a single agent, but by using different drugs concurrently it would be more difficult for the tumor to develop resistance to the combination. Holland, Freireich, and Frei simultaneously administered methotrexate (an antifolate), vincristine (a Vinca alkaloid), 6-mercaptopurine (6-MP) and prednisone — together referred to as the POMP regimen — and induced long-term remissions in children with acute lymphoblastic leukaemia (ALL). With incremental refinements of original regimens, using randomized clinical studies by St. Jude Children's Research Hospital, the Medical Research Council in the UK (UKALL protocols) and German Berlin-Frankfurt-Münster clinical trials group (ALL-BFM protocols), ALL in children has become a largely curable disease.

This approach was extended to the lymphomas in 1963 by Vincent T. DeVita and George Canellos at the NCI, who ultimately proved in the late 1960s that nitrogen mustard, vincristine, procarbazine and prednisone — known as the MOPP regimen — could cure patients with Hodgkin's and non-Hodgkin's lymphoma. Currently, nearly all successful cancer chemotherapy regimens use this paradigm of multiple drugs given simultaneously.

Adjuvant therapy

As predicted by studies in animal models, drugs were most effective when used in patients with tumours of smaller volume. Another important strategy developed from this - if the tumour burden could be reduced first by surgery, then chemotherapy may be able to clear away any remaining malignant cells, even if it would not have been potent enough to destroy the tumor in its entirety. This approach was termed "adjuvant therapy".

Emil Frei first demonstrated this effect - high doses of methotrexate prevented recurrence of osteosarcoma following surgical removal of the primary tumour. 5-fluorouracil, an inhibitor of DNA synthesis, was later shown to improve survival when used as an adjuvant to surgery in treating patients with colon cancer. Similarly, the landmark trials of Bernard Fisher, chair of the National Surgical Adjuvant Breast and Bowel Project, and of Gianni Bonadonna, working in the Istituto Nazionale Tumori di Milano, Italy, proved that adjuvant chemotherapy after complete surgical resection of breast tumours significantly extended survival — particularly in more advanced cancer.

Drug Discovery at the NCI and elsewhere

Zubrod's initiatives

Dr Gordon Zubrod of the NCI

In 1956, C. Gordon Zubrod, who had formerly led the development of antimalarial agents for the United States Army, took over the Division of Cancer Treatment of the NCI and guided development of new drugs. In the two decades that followed the establishment of the NCCSC, a large network of cooperative clinical trial groups evolved under the auspices of the NCI to test anticancer agents. Zubrod had a particular interest in natural products, and established a broad programme for collecting and testing plant and marine sources, a controversial programme that led to the discovery of taxanes (in 1964) and camptothecins (in 1966). Both classes of drug were isolated and characterized by the laboratory of Monroe Wall at the Research Triangle Institute.

The taxanes

Paclitaxel (Taxol) was a novel antimitotic agent that promoted microtubule assembly. This agent proved difficult to synthesize and could only be obtained from the bark of the Pacific Yew tree, which forced the NCI into the costly business of harvesting substantial quantities of yew trees from public lands. After 4 years of clinical testing in solid tumours, it was found in 1987 (23 years after its initial discovery) to be effective in ovarian cancer therapy. Notably, this agent, although developed by the NCI in partnership with Bristol-Myers Squibb, was exclusively marketed by BMS who went on to make over a billion dollars profit from Taxol, despite the bulk of the initial work being funded by US taxpayers.

The camptothecins

Another drug class originating from the NCI was the camptothecins. Camptothecin, derived from a Chinese ornamental tree, inhibits topoisomerase I, an enzyme that allows DNA unwinding. Despite showing promise in preclinical studies, the agent had little antitumour activity in early clinical trials, and dosing was limited by kidney toxicity: its lactone ring is unstable at neutral pH, so while in the acidic environment of the kidneys it becomes active, damaging the renal tubules. In 1996 a more stable analogue, irinotecan, won Food and Drug Administration (FDA) approval for the treatment of colon cancer. Later, this agent would also be used to treat lung and ovarian cancers.

Platinum-based agents

Cisplatin, a platinum-based compound, was discovered by a Michigan State University researcher, Barnett Rosenberg, working under an NCI contract. This was yet another serendipitous discovery: Rosenberg had initially wanted to explore the possible effects of an electric field on the growth of bacteria. He observed that the bacteria unexpectedly ceased to divide when placed in an electric field. Excited, he spent months of testing to try and explain this phenomenon. He was disappointed to find that the cause was an experimental artefact - the inhibition of bacterial division was pinpointed to an electrolysis product of the platinum electrode rather than the electrical field. This accidental discovery, however, soon initiated a series of investigations and studies into the effects of platinum compounds on cell division, culminating in the synthesis of cisplatin. This drug was pivotal in the cure of testicular cancer. Subsequently, Eve Wiltshaw and others at the Institute of Cancer Research in the United Kingdom extended the clinical usefulness of the platinum compounds with their development of carboplatin, a cisplatin derivative with broad antitumour activity and comparatively less nephrotoxicity.

Nitrosoureas

A second group with an NCI contract, led by John Montgomery at the Southern Research Institute, synthesized nitrosoureas, an alkylating agent which cross-links DNA. Fludarabine phosphate, a purine analogue which has become a mainstay in treatment of patients with chronic lymphocytic leukaemia, was another similar development by Montgomery.

Anthracyclines and epipodophyllotoxins

Other effective molecules also came from industry during the period of 1970 to 1990, including anthracyclines and epipodophyllotoxins — both of which inhibited the action of topoisomerase II, an enzyme crucial for DNA synthesis.

Supportive care during chemotherapy

As is obvious from their origins, the above cancer chemotherapies are essentially poisons. Patients receiving these agents experienced severe side-effects that limited the doses which could be administered, and hence limited the beneficial effects. Clinical investigators realized that the ability to manage these toxicities was crucial to the success of cancer chemotherapy.

Several examples are noteworthy. Many chemotherapeutic agents cause profound suppression of the bone marrow. This is reversible, but takes time to recover. Support with platelet and red-cell transfusions as well as broad-spectrum antibiotics in case of infection during this period is crucial to allow the patient to recover.

Several practical factors are also worth mentioning. Most of these agents caused very severe nausea (termed chemotherapy-induced nausea and vomiting (CINV) in the literature) which, while not directly causing patient deaths, was unbearable at higher doses. The development of new drugs to prevent nausea (the prototype of which was ondansetron) was of great practical use, as was the design of indwelling intravenous catheters (e.g. Hickman lines and PICC lines) which allowed safe administration of chemotherapy as well as supportive therapy.

A period of quiet

With the successes of combination chemotherapy and the discovery of many new agents, there was a feeling at this time that all cancers could be treated, if only one could administer the correct combination of drugs, at the correct doses and at the correct intervals. A search continued, with the pharmaceutical industry screening for new compounds and clinical scientists performing elaborate clinical trials with ever more complex combinations and higher doses.

One important contribution during this period was the discovery of a means that allowed the administration of previously lethal doses of chemotherapy. The patient's bone marrow was first harvested, the chemotherapy administered, and the harvested marrow then returned to patient a few days later. This approach, termed autologous bone marrow transplantation, was initially thought to be of benefit to a wide group of patients, including those with advanced breast cancer. However, rigorous studies have failed to confirm this benefit, and autologous transplantation is no longer widely used for solid tumors. The proven curative benefits of high doses of chemotherapy afforded by autologous bone marrow rescue are limited to Hodgkins disease patients who had failed therapy with conventional combination chemotherapy. However, autologous transplantation continues to be used as a component of therapy for a number of hematologic malignancies.

The hormonal contribution to several categories of breast cancer subtypes was recognized during this time, leading to the development of pharmacological modulators (e.g. of oestrogen) such as tamoxifen.

Although clinical oncologists appeared to have hit a wall at this point in terms of results, under the surface something extraordinary was happening: namely, elucidation of the mechanisms underlying cancer. Understanding of the machinery of the cell and advances in techniques to probe perturbations in its function allowed researchers to understand the genetic nature of cancer. It is important to realize that prior to this point, chemotherapeutic agents had been discovered essentially by chance, or by inhibiting the metabolic pathways crucial to cell division, but none were particularly specific to the cancer cell.

Targeted therapy

bcr-abl kinase, which causes CML, in green, inhibited by small molecule Imatinib mesylate in red, rendered with RasMol

Molecular and genetic approaches to understanding cell biology uncovered entirely new signalling networks that regulate cellular activities such as proliferation and survival. Many of these networks were found to be radically altered in cancer cells, and these alterations had a genetic basis caused by a chance somatic mutation.

Tyrosine kinase inhibitors

The classic example of targeted development is imatinib mesylate (Gleevec), a small molecule which inhibits a signaling molecule kinase. The genetic abnormality causing chronic myelogenous leukemia (CML) has been known for a long time to be a chromosomal translocation creating an abnormal fusion protein, kinase BCR-ABL, which signals aberrantly, leading to uncontrolled proliferation of the leukemia cells. Imatinib precisely inhibits this kinase. Unlike so many other anti-cancer agents, this pharmaceutical was no accident. Brian Druker, working in Oregon Health Science University, had extensively researched the abnormal enzyme kinase in CML. He reasoned that precisely inhibiting this kinase with a drug would control the disease and have little effect on normal cells. Druker collaborated with Novartis chemist Nick Lydon, who developed several candidate inhibitors. From these, imatinib was found to have the most promise in laboratory experiments. First Druker and then other groups worldwide demonstrated that when this small molecule is used to treat patients with chronic-phase CML, 90% achieve complete haematological remission. It is hoped that molecular targeting of similar defects in other cancers will have the same effect.

Monoclonal antibodies

Another branch in targeted therapy is the increasing use of monoclonal antibodies in cancer therapy. Although monoclonal antibodies (immune proteins which can be selected to precisely bind to almost any target) have been around for decades, they were derived from mice and did not function particularly well when administered to humans, causing allergic reactions and being rapidly removed from circulation. "Humanization" of these antibodies (genetically transforming them to be as similar to a human antibody as possible) has allowed the creation of a new family of highly effective humanized monoclonal antibodies. Rituximab, a drug used to treat lymphomas, is a prime example.

Concluding Comments

The discovery that certain toxic chemicals administered in combination can cure certain cancers ranks as one of the greatest in modern medicine. Childhood ALL, testicular cancer, and Hodgkins disease, previously universally fatal, are now generally curable diseases. The early revolution in cancer therapy was largely a North American experience, powered by an optimistic and forward-looking United States Federal government, which funded the NCI with the same "big-idea" philosophy as the Apollo Program. In fact, it was only later that the pharmaceutical industry became heavily involved.

Conventional cytotoxic chemotherapy has shown the ability to cure some cancers, including testicular cancer, Hodgkin disease, non-Hodgkin lymphoma, and some leukemias. It has also proven effective in the adjuvant setting, in reducing the risk of recurrence after surgery for high-risk breast cancer, colon cancer, and lung cancer, among others. However, the hopes created by the dramatic initial success of cancer chemotherapy were not fully borne out, as conventional cytotoxic chemotherapy has fallen short of the high expectations of curing the most common cancers.

The overall impact of chemotherapy on cancer survival can be difficult to estimate, since improved cancer screening, prevention (e.g. anti-smoking campaigns), and detection all influence statistics on cancer incidence and mortality. In the United States, overall cancer incidence rates were stable from 1995 through 1999, while cancer death rates decreased steadily from 1993 through 1999. Again, this likely reflects the combined impact of improved screening, prevention, and treatment. Nonetheless, cancer remains a major cause of illness and death, and conventional cytotoxic chemotherapy has proven unable to cure most cancers after they have metastasized.

New knowledge about the molecular biology of cancer and new tools to specifically target aberrant proteins are opening up new possibilities. The next two decades will see two competing strategies of cancer therapy: small molecular inhibitors and adoptive immunotherapy with re-programmed effector cells will match strengths in an attempt to finally cure cancer.

Chemotherapy

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Chemotherapy, in its most general sense, refers to treatment of disease by chemicals that kill cells, specifically those of micro-organisms or cancer. In popular usage, it will usually refer to antineoplastic drugs used to treat cancer or the combination of these drugs into a cytotoxic standardized treatment regimen as opposed to a targeted therapy.

In its non-oncological use, the term may also refer to antibiotics (antibacterial chemotherapy). In that sense, the first modern chemotherapeutic agent was Paul Ehrlich's arsphenamine, an arsenic compound discovered in 1909 and used to treat syphilis. This was later followed by sulfonamides discovered by Domagk and penicillin discovered by Alexander Fleming.

Other uses of cytostatic chemotherapy agents (including the ones mentioned below) are the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and the suppression of transplant rejections (see immunosuppression and DMARDs).

History

The use of chemical substances and drugs as medication can be traced back to the ancient Indian system of medicine called Ayurveda, which uses many metals besides herbs for treatment of a large number of ailments. More recently, Persian physician, Muhammad ibn Zakarīya Rāzi (Rhazes), in the 10th century, introduced the use of chemicals such as vitriol, copper, mercuric and arsenic salts, sal ammoniac, gold scoria, chalk, clay, coral, pearl, tar, bitumen and alcohol for medical purposes.

The first drug used for cancer chemotherapy, however, dates back to the early 20th century, though it was not originally intended for that purpose. Mustard gas was used as a chemical warfare agent during World War I and was studied further during World War II. During a military operation in World War II, a group of people were accidentally exposed to mustard gas and were later found to have very low white blood cell counts. It was reasoned that an agent that damaged the rapidly-growing white blood cells might have a similar effect on cancer. Therefore, in the 1940s, several patients with advanced lymphomas (cancers of certain white blood cells) were given the drug by vein, rather than by breathing the irritating gas. Their improvement, although temporary, was remarkable. That experience led researchers to look for other substances that might have similar effects against cancer. As a result, many other drugs have been developed to treat cancer, and drug development since then has exploded into a multibillion-dollar industry. The targeted-therapy revolution has arrived, but the principles and limitations of chemotherapy discovered by the early researchers still apply.

Principles

Cancer is the uncontrolled growth of cells coupled with malignant behavior: invasion and metastasis. Cancer is thought to be caused by the interaction between genetic susceptibility and environmental toxins.

In the broad sense, most chemotherapeutic drugs work by impairing mitosis (cell division), effectively targeting fast-dividing cells. As these drugs cause damage to cells they are termed cytotoxic. Some drugs cause cells to undergo apoptosis (so-called "programmed cell death").

Scientists have yet to identify specific features of malignant and immune cells that would make them uniquely targetable (barring some recent examples, such as the Philadelphia chromosome as targeted by imatinib). This means that other fast-dividing cells, such as those responsible for hair growth and for replacement of the intestinal epithelium (lining), are also often affected. However, some drugs have a better side-effect profile than others, enabling doctors to adjust treatment regimens to the advantage of patients in certain situations.

As chemotherapy affects cell division, tumors with high growth fractions (such as acute myelogenous leukemia and the aggressive lymphomas, including Hodgkin's disease) are more sensitive to chemotherapy, as a larger proportion of the targeted cells are undergoing cell division at any time. Malignancies with slower growth rates, such as indolent lymphomas, tend to respond to chemotherapy much more modestly.

Drugs affect "younger" tumors (i.e., more differentiated) more effectively, because mechanisms regulating cell growth are usually still preserved. With succeeding generations of tumor cells, differentiation is typically lost, growth becomes less regulated, and tumors become less responsive to most chemotherapeutic agents. Near the center of some solid tumors, cell division has effectively ceased, making them insensitive to chemotherapy. Another problem with solid tumors is the fact that the chemotherapeutic agent often does not reach the core of the tumor. Solutions to this problem include radiation therapy (both brachytherapy and teletherapy) and surgery.

Over time, cancer cells become more resistant to chemotherapy treatments. Recently, scientists have identified small pumps on the surface of cancer cells that actively move chemotherapy from inside the cell to the outside. Research on p-glycoprotein and other such chemotherapy efflux pumps, is currently ongoing. Medications to inhibit the function of p-glycoprotein are undergoing testing as of June, 2007 to enhance the efficacy of chemotherapy.

Treatment schemes

There are a number of strategies in the administration of chemotherapeutic drugs used today. Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms.

Combined modality chemotherapy is the use of drugs with other cancer treatments, such as radiation therapy or surgery. Most cancers are now treated in this way. Combination chemotherapy is a similar practice that involves treating a patient with a number of different drugs simultaneously. The drugs differ in their mechanism and side-effects. The biggest advantage is minimising the chances of resistance developing to any one agent.

In neoadjuvant chemotherapy (preoperative treatment) initial chemotherapy is aimed for shrinking the primary tumour, thereby rendering local therapy (surgery or radiotherapy) less destructive or more effective.

Adjuvant chemotherapy (postoperative treatment) can be used when there is little evidence of cancer present, but there is risk of recurrence. This can help reduce chances of developing resistance if the tumour does develop. It is also useful in killing any cancerous cells which have spread to other parts of the body. This is often effective as the newly growing tumours are fast-dividing, and therefore very susceptible.

Palliative chemotherapy is given without curative intent, but simply to decrease tumor load and increase life expectancy. For these regimens, a better toxicity profile is generally expected.

All chemotherapy regimens require that the patient be capable of undergoing the treatment. Performance status is often used as a measure to determine whether a patient can receive chemotherapy, or whether dose reduction is required.

Types

The majority of chemotherapeutic drugs can be divided in to alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumour agents. All of these drugs affect cell division or DNA synthesis and function in some way.

Some newer agents do not directly interfere with DNA. These include monoclonal antibodies and the new tyrosine kinase inhibitors e.g. imatinib mesylate (Gleevec or Glivec), which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors). These are examples of targeted therapies.

In addition, some drugs that modulate tumor cell behaviour without directly attacking those cells may be used. Hormone treatments fall into this category of adjuvant therapies.

Where available, Anatomical Therapeutic Chemical Classification System codes are provided for the major categories.

Alkylating agents (L01A)

Alkylating agents are so named because of their ability to add alkyl groups to many electronegative groups under conditions present in cells. Cisplatin and carboplatin, as well as oxaloplatin, are alkylating agents.

Other agents are mechlorethamine, cyclophosphamide, chlorambucil. They work by chemically modifying a cell's DNA.

Anti-metabolites (L01B)

Anti-metabolites masquerade as purine ((azathioprine, mercaptopurine)) or pyrimidine - which become the building blocks of DNA. They prevent these substances from becoming incorporated in to DNA during the "S" phase (of the cell cycle), stopping normal development and division. They also affect RNA synthesis. Due to their efficiency, these drugs are the most widely used cytostatics.

Plant alkaloids and terpenoids (L01C)

These alkaloids are derived from plants and block cell division by preventing microtubule function. Microtubules are vital for cell division, and, without them, cell division cannot occur. The main examples are vinca alkaloids and taxanes.

Vinca alkaloids (L01CA)

Vinca alkaloids bind to specific sites on tubulin, inhibiting the assembly of tubulin into microtubules (M phase of the cell cycle). They are derived from the Madagascar periwinkle, Catharanthus roseus (formerly known as Vinca rosea). The vinca alkaloids include:

Vincristine

Vinblastine
Vinorelbine
Vindesine

Podophyllotoxin (L01CB)

Podophyllotoxin is a plant-derived compound which is said to help with digestion as well as used to produce two other cytostatic drugs, etoposide and teniposide. They prevent the cell from entering the G1 phase (the start of DNA replication) and the replication of DNA (the S phase). The exact mechanism of its action is not yet known.

The substance has been primarily obtained from the American Mayapple (Podophyllum peltatum). Recently it has been discovered that a rare Himalayan Mayapple (Podophyllum hexandrum) contains it in a much greater quantity, but, as the plant is endangered, its supply is limited. Studies have been conducted to isolate the genes involved in the substance's production, so that it could be obtained recombinantively.

Taxanes (L01CD)

The prototype taxane is the natural product paclitaxel, originally known as Taxol and first derived from the bark of the Pacific Yew tree. Docetaxel is a semi-synthetic analogue of paclitaxel. Taxanes enhance stability of microtubules, preventing the separation of chromosomes during anaphase.

Topoisomerase inhibitors (L01CB and L01XX)

Topoisomerases are essential enzymes that maintain the topology of DNA. Inhibition of type I or type II topoisomerases interferes with both transcription and replication of DNA by upsetting proper DNA supercoiling.

Some type I topoisomerase inhibitors include camptothecins: irinotecan and topotecan.

Examples of type II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide. These are semisynthetic derivatives of epipodophyllotoxins, alkaloids naturally occurring in the root of American Mayapple (Podophyllum peltatum).

Antitumour antibiotics (L01D)

These include the immunosuppressant dactinomycin (which is used in kidney transplantations), doxorubicin, epirubicin, bleomycin and others.

Monoclonal antibodies

Monoclonal antibodies work by targeting tumour specific antigens, thus enhancing the host's immune response to tumour cells to which the agent attaches itself. Examples are trastuzumab (Herceptin), cetuximab, and rituximab (Rituxan or Mabthera). Bevacizumab (Avastin) is a monoclonal antibody that does not directly attack tumor cells but instead blocks the formation of new tumor vessels.

Hormonal therapy

Several malignancies respond to hormonal therapy, which, in the strict sense, is not chemotherapy. Cancer arising from certain tissues, including the mammary and prostate glands, may be inhibited or stimulated by appropriate changes in hormone balance.

Steroids (often dexamethasone) can inhibit tumour growth or the associated edema (tissue swelling), and may cause regression of lymph node malignancies. Dexamethasone is also an antiemetic, so it may be used with cytotoxic chemotherapy even if it has no direct effect on the cancer.
Prostate cancer is often sensitive to finasteride, an agent that blocks the peripheral conversion of testosterone to dihydrotestosterone.
Breast cancer cells often highly express the estrogen and/or progesterone receptor. Inhibiting the production (with aromatase inhibitors) or action (with tamoxifen) of these hormones can often be used as an adjunct to therapy.
Gonadotropin-releasing hormone agonists (GnRH), such as goserelin possess a paradoxical negative feedback effect followed by inhibition of the release of FSH (follicle-stimulating hormone) and LH (luteinizing hormone), when given continuously.

Some other tumours are also hormone-dependent, although the specific mechanism is still unclear.

Newer and experimental approaches

Hematopoietic stem cell transplant approaches

Stem cell harvesting and autologous or allogeneic stem cell transplant has been used to allow for higher doses of chemotheraputic agents where dosages are primarily limited by hematopoietic damage. Years of research in treating solid tumors, particularly breast cancer, with hematopoeitic stem cell transplants, has yielded little proof of efficacy. Hematological malignancies such as myeloma, lymphoma, and leukemia remain the main indications for stem cell transplants.

Isolated infusion approaches

Isolated limb perfusion (often used in melanoma), or isolated infusion of chemotherapy into the liver or the lung have been used to treat some tumours. The main purpose of these approaches is to deliver a very high dose of chemotherapy to tumor sites without causing overwhelming systemic damage. These approaches can help control solitary or limited metastases, but they are by definition not systemic, and, therefore, do not treat distributed metastases or micrometastases.

Targeted delivery mechanisms

Specially-targeted delivery vehicles aim to increase effective levels of chemotherapy for tumor cells while reducing effective levels for other cells. This should result in an increased tumor kill and/or reduced toxicity.

Specially-targeted delivery vehicles have a differentially higher affinity for tumor cells by interacting with tumor-specific or tumour-associated antigens.

In addition to their targeting component, they also carry a payload - whether this is a traditional chemotherapeutic agent, or a radioisotope or an immune stimulating factor. Specially-targeted delivery vehicles vary in their stability, selectivity, and choice of target, but, in essence, they all aim to increase the maximum effective dose that can be delivered to the tumor cells. Reduced systemic toxicity means that they can also be used in sicker patients, and that they can carry new chemotherapeutic agents that would have been far too toxic to deliver via traditional systemic approaches.

Nanoparticles

Nanoparticles have emerged as a useful vehicle for poorly-soluble agents such as paclitaxel. Protein-bound paclitaxel (e.g., Abraxane) or nab-paclitaxel was approved by the US FDA in January 2005 for the treatment of refractory breast cancer, and allows reduced use of the Cremophor vehicle usually found in paclitaxel. Nanoparticles made of magnetic material can also be used to concentrate agents at tumour sites using an externally applied magnetic field.

Dosage

Dosage of chemotherapy can be difficult: If the dose is too low, it will be ineffective against the tumor, whereas, at excessive doses, the toxicity (side-effects, neutropenia) will be intolerable to the patient. This has led to the formation of detailed "dosing schemes" in most hospitals, which give guidance on the correct dose and adjustment in case of toxicity. In immunotherapy, they are in principle used in smaller dosages than in the treatment of malignant diseases.

In most cases, the dose is adjusted for the patient's body surface area, a measure that correlates with blood volume. The BSA is usually calculated with a mathematical formula or a nomogram, using a patient's weight and height, rather than by direct measurement.

Delivery

Most chemotherapy is delivered intravenously, although there is a number of agents that can be administered orally (e.g., melphalan, busulfan, capecitabine). In some cases, isolated limb perfusion (often used in melanoma), or isolated infusion of chemotherapy into the liver or the lung have been used. The main purpose of these approaches is to deliver a very high dose of chemotherapy to tumour sites without causing overwhelming systemic damage.

Depending on the patient, the cancer, the stage of cancer, the type of chemotherapy, and the dosage, intravenous chemotherapy may be given on either an inpatient or an outpatient basis. For continuous, frequent or prolonged intravenous chemotherapy administration, various systems may be surgically inserted into the vasculature to maintain access. Commonly-used systems are the Hickman line, the Port-a-Cath or the PICC line. These have a lower infection risk, are much less prone to phlebitis or extravasation, and abolish the need for repeated insertion of peripheral cannulae.

Harmful and lethal toxicity from chemotherapy limits the dosage of chemotherapy that can be given. Some tumours can be destroyed by sufficiently high doses of chemotheraputic agents. However, these high doses cannot be given because they would be fatal to the patient.

Side-effects

The treatment can be physically exhausting for the patient. Current chemotherapeutic techniques have a range of side effects mainly affecting the fast-dividing cells of the body. Important common side-effects include (dependent on the agent):

Nausea and vomiting
Diarrhea or constipation

Memory loss

Depression of the immune system, hence (potentially lethal) infections and sepsis
Weight loss or gain
Hemorrhage
Secondary neoplasms

Cardiotoxicity

Immunosuppression and myelosuppression

Virtually all chemotherapeutic regimens can cause depression of the immune system, often by paralysing the bone marrow and leading to a decrease of white blood cells, red blood cells, and platelets. The latter two, when they occur, are improved with blood transfusion. Neutropenia (a decrease of the neutrophil granulocyte count below 0.5 x 109/litre) can be improved with synthetic G-CSF (granulocyte-colony stimulating factor, e.g., filgrastim, lenograstim, Neupogen, Neulasta).

In very severe myelosuppression, which occurs in some regimens, almost all the bone marrow stem cells (cells that produce white and red blood cells) are destroyed, meaning allogenic or autologous bone marrow cell transplants are necessary. (In autologous BMTs, cells are removed from the patient before the treatment, multiplied and then re-injected afterwards; in allogenic BMTs the source is a donor.) However, some patients still develop diseases because of this interference with bone marrow.

Nausea and vomiting

Nausea and vomiting caused by chemotherapy; stomach upset may trigger a strong urge to vomit, or forcefully eliminate what is in the stomach.

Stimulation of the vomiting center results in the coordination of responses from the diaphragm, salivary glands, cranial nerves, and gastrointestinal muscles to produce the interruption of respiration and forced expulsion of stomach contents known as retching and vomiting. The vomiting center is stimulated directly by afferent input from the vagal and splanchnic nerves, the pharynx, the cerebral cortex, cholinergic and histamine stimulation from the vestibular system, and efferent input from the chemoreceptor trigger zone (CTZ). The CTZ is in the area postrema, outside the blood-brain barrier, and is thus susceptible to stimulation by substances present in the blood or cerebral spinal fluid. The neurotransmitters dopamine and serotonin stimulate the vomiting center indirectly via stimulation of the CTZ.

The 5-HT₃ inhibitors are the most effective antiemetics and constitute the single greatest advance in the management of nausea and vomiting in patients with cancer. These drugs are designed to block one or more of the signals that cause nausea and vomiting. The most sensitive signal during the first 24 hours after chemotherapy appears to be 5-HT3. Blocking the 5-HT3 signal is one approach to preventing acute emesis (vomiting), or emesis that is severe, but relatively short-lived. Approved 5-HT3 inhibitors include Dolasetron (Anzemet), Granisetron (Kytril), and Ondansetron (Zofran). The newest 5-HT3 inhibitor, palonosetron (Aloxi), also preventing delayed nausea and vomiting, which occurs during the 2-5 days after treatment.

Another drug to control nausea in cancer patients became available in 2005. The substance P inhibitor aprepitant (marketed as Emend) has been shown to be effective in controlling the nausea of cancer chemotherapy. The results of two large controlled trials were published in 2005, describing the efficacy of this medication in over 1,000 patients.

Some studies and patient groups claim that the use of cannabinoids derived from marijuana during chemotherapy greatly reduces the associated nausea and vomiting, and enables the patient to eat. Some synthetic derivatives of the active substance in marijuana (Tetrahydrocannabinol or THC) such as Marinol may be practical for this application. Natural marijuana, known as medical cannabis is also used and recommended by some oncologists, though its use is regulated and not legal everywhere.

Other side-effects

In particularly, large tumors, such as large lymphomas, some patients develop tumor lysis syndrome from the rapid breakdown of malignant cells. Although prophylaxis is available and is often initiated in patients with large tumors, this is a dangerous side-effect that can lead to death if left untreated.

Some patients report fatigue or non-specific neurocognitive problems, such as an inability to concentrate; this is sometimes called post-chemotherapy cognitive impairment, referred to as "chemo brain" by patients' groups.

Specific chemotherapeutic agents are associated with organ-specific toxicities, including cardiovascular disease (e.g., doxorubicin), interstitial lung disease (e.g., bleomycin) and occasionally secondary neoplasm (e.g., MOPP therapy for Hodgkin's disease).

GITR

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GITR (glucocorticoid-induced tumor necrosis factor receptor) is a surface receptor molecule that has been shown to be involved in inhibiting the suppressive activity of T-regulatory cells and extending the survival of T-effector cells. In mouse models, GITR was initially noted to be selectively enriched on the surface of regulatory T cells, making this an attractive potential surface marker for these rare cells. However, subsequent studies revealed GITR to also be up-regulated on any activated T cells in humans, thus undermining its utility as a regulatory T cell marker.

Glucocorticoid receptor

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Chr 5: 142.64 - 142.8 Mb

Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)

Crystallographic structures of the glucocorticoid receptor DNA binding domain (DBD, left, (white sticks) and the protein (red)]. Dashed yellow lines represent PDB 1R4O bound to DNA) and ligand binding domain [LBD, right, 1M2Z bound to dexamethasoneTIF2 coactivatorhydrogen bonding interactions between the receptor and ligand. The 2D structure of dexamethasone is also depicted in the lower right hand side of the picture for reference.

Available structures: 1gdc, 1glu, 1m2z, 1nhz, 1p93, 1r4o, 1r4r, 1rgd, 2gda

Identifiers

Symbols

NR3C1; GCCR; GCR; GR; GRL

External IDs

OMIM: 138040 MGI: 95824 HomoloGene: 30960

Gene ontology
Molecular function:	• transcription factor activity • glucocorticoid receptor activity • steroid binding • protein binding • zinc ion binding • lipid binding • sequence-specific DNA binding • metal ion binding
Cellular component:	• nucleus • cytoplasm • mitochondrial matrix
Biological process:	• regulation of transcription, DNA-dependent • transcription from RNA polymerase II promoter • inflammatory response • signal transduction • sex determination

RNA expression pattern

Orthologs

Human

Mouse

Entrez

2908

14815

Ensembl

ENSG00000113580

ENSMUSG0000002443

Uniprot

P04150

Q05DD1

Refseq

NM_000176 (mRNA)
NP_000167 (protein)

NM_008173 (mRNA)
NP_032199 (protein)

Chr 5: 142.64 - 142.8 Mb

Chr 18: 39.54 - 39.62 Mb

Pubmed search

The glucocorticoid receptor (GR, or GCR) also known as NR3C1 (nuclear receptor subfamily 3, group C, member 1) is a ligand-activated transcription factor that binds with high affinity to cortisol and other glucocorticoids.

The GR is expressed in almost every cell in the body and regulates either directly or indirectly genes controlling a wide variety of processes including the development, metabolism, and immune response of the organism.

The GR protein is encoded by gene NR3C1 on chromosome 5 (5q31).

Structure

Like the other steroid receptors, the glucocorticoid receptor is modular in structure and contains the following domains (labeled A - F):

A/B - N-terminal regulatory domain
C - DNA-binding domain (DBD)
D - hinge region
E - ligand-binding domain (LBD)
F - C-terminal domain

Ligand binding and response

In the absence of hormone, the glucocorticoid receptor (GR) resides in the cytosol complexed with a variety of proteins including heat shock protein 90 (hsp90), the heat shock protein 70 (hsp70) and the protein FKBP52 (FK506-binding protein 52). The endogenous glucocortiod hormone cortisol diffuses through the cell membrane into the cytoplasm and binds to the glucocorticoid receptor (GR) resulting in release of the heat shock proteins. The resulting activated form GR has two principal mechanisms of action, transactivation and transrepression, described below.

Transactivation

A direct mechanism of action involves homodimerization of the receptor, translocation via active transport into the nucleus, and binding to specific DNA responsive elements activating gene transcription. This mechanism of action is referred to as transactivation. The biologic response depends on the cell type.

Transrepression

In the absence of activated GR, other transcription factors such as NF-κB or AP-1 themselves are able to transactivate target genes. However activated GR can complex with these other transcription factors and prevent them from binding their target genes and hence repress the expression of genes that are normally upregulated by NF-κB or AP-1. This indirect mechanism of action is referred to as transrepression.

Agonists and antagonists

Dexamethasone is an agonist, and RU486 and cyproterone are antagonists of the GR. Also, progesterone and DHEA have antagonist effects on the GR.

The GR is abnormal in familial glucocorticoid resistance.

Glucocorticoid

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Chemical structure of cortisol, a glucocorticoid

Dexamethasone binds more powerfully to the glucocorticoid receptor than cortisol does. Dexamethasone is based on the cortisol structure but differs in three positions (extra double bond in the A-ring between carbons 1 and 2 and addition of a 9-α-fluoro group and a 16-α-methyl substituent).

Glucocorticoids (GC) are a class of steroid hormones characterised by an ability to bind with the glucocorticoid receptor (GR) and trigger similar effects. These may be either slow, mediated genomically through nuclear receptors, or fast, mediated nongenomically through membrane-associated receptors and signaling cascades. Glucocorticoids are distinguished from mineralocorticoids and sex steroids by their specific receptors, target cells, and effects. In technical terms, corticosteroid refers to both glucocorticoids and mineralocorticoids (as both are mimics of hormones produced by the adrenal cortex), but is often used as a synonym for glucocorticoid.

Cortisol (or hydrocortisone) is the most important human glucocorticoid. It is essential for life, and regulates or supports a variety of important cardiovascular, metabolic, immunologic, and homeostatic functions. Glucocorticoid receptors are found in the cells of almost all vertebrate tissues. Various synthetic glucocorticoids are available; these are used either as replacement therapy in glucocorticoid deficiency or to suppress the immune system.

Mode of action

Transactivation

Glucocorticoids bind to the cytosolic glucocorticoid receptor (GR). This type of receptor is activated by ligand binding. After a hormone binds to the corresponding receptor, the newly-formed receptor-ligand complex translocates itself into the cell nucleus, where it binds to glucocorticoid response elements (GRE) in the promoter region of the target genes resulting in the regulation of gene expression. This process is commonly referred to as transactivation.

The proteins encoded by these upregulated genes have a wide range of effects including for example:

anti-inflammatory – lipocortin I and p11/calpactin binding protein
increased gluconeogenesis – glucose-6-phosphatase and tyrosine aminotransferase

Transrepression

The opposite mechanism is called transrepression. The activated hormone receptor interacts with specific transcription factors (such as AP-1 and NF-κB) and prevents the transcription of targeted genes. Glucocorticoids are able to prevent the transcription of pro-inflammatory genes, including the IL-2 gene.

Dissociated glucocorticoids

The ordinary glucocorticoids do not distinguish among transactivation and transrepression and influence both the "wanted" immune and "unwanted" genes regulating the metabolic and cardiovascular functions. Intensive research is aimed at discovering selectively acting glucocorticoids that will be able to repress only the immune system.

Genetically modified mice which express a modified GR which is incapable of DNA binding are still responsive to the antiinflammatory effects of glucocorticoids while the stimulation of gluconeogenesis by glucocorticoids is blocked. This result strongly suggests that most of the desirable antiinflammatory effects are due to transrepression while the undesirable metabolic effects arise from transactivation.

Effects

The name "glucocorticoid" derives from early observations that these hormones were involved in glucose metabolism. In the fasted state, cortisol stimulates several processes that collectively serve to increase and maintain normal concentrations of glucose in blood.

Metabolic effects:

Stimulation of gluconeogenesis, particularly in the liver: This pathway results in the synthesis of glucose from non-hexose substrates such as amino acids and glycerol from triglyceride breakdown, and is particularly important in carnivores and certain herbivores. Enhancing the expression of enzymes involved in gluconeogenesis is probably the best-known metabolic function of glucocorticoids.
Mobilization of amino acids from extrahepatic tissues: These serve as substrates for gluconeogenesis.
Inhibition of glucose uptake in muscle and adipose tissue: A mechanism to conserve glucose.
Stimulation of fat breakdown in adipose tissue: The fatty acids released by lipolysis are used for production of energy in tissues like muscle, and the released glycerol provide another substrate for gluconeogenesis.

Glucocorticoids have multiple effects on fetal development. An important example is their role in promoting maturation of the lung and production of the surfactant necessary for extrauterine lung function. Mice with homozygous disruptions in the corticotropin-releasing hormone gene (see below) die at birth due to pulmonary immaturity.

Excessive glucocorticoid levels resulting from administration as a drug or hyperadrenocorticism have effects on many systems. Some examples include inhibition of bone formation, suppression of calcium absorption (both of which can lead to osteoporosis), delayed wound healing, muscle weakness, and increased risk of infection. These observations suggest a multitude of less-dramatic physiologic roles for glucocorticoids.

Pharmacology

A variety of synthetic glucocorticoids, some far more potent than cortisol, have been created for therapeutic use. They differ in the pharmacokinetics (absorption factor, half-life, volume of distribution, clearance) and in pharmacodynamics (for example the capacity of mineralocorticoid activity: retention of sodium (Na+) and water; see also: renal physiology). Because they permeate the intestines easily, they are primarily administered per os (by mouth), but also by other methods, such as topically on skin. More than 90 percent of them bind different plasma proteins, however with a different binding specificity. Endogenous glucocorticoids and some synthetic corticoids have high affinity to the protein transcortin (also called CBG, corticosteroid-binding globulin), whereas all of them bind albumin. In the liver, they quickly metabolise by conjugation with a sulfate or glucuronic acid, and are secreted in the urine.

Glucocorticoid potency, duration of effect, and overlapping mineralocorticoid potency varies. Cortisol (hydrocortisone) is the standard of comparison for glucocorticoid potency. Hydrocortisone is the name used for pharmaceutical preparations of cortisol. Data refer to oral dosing, except when mentioned. Oral potency may be less than parenteral potency because significant amounts (up to 50% in some cases) may not be absorbed from the intestine. Fludrocortisone, DOCA, and aldosterone are not considered glucocorticoids, and are included in this table to provide perspective on mineralocorticoid potency.

Comparative steroid potencies
Name	Glucocorticoid potency	Mineralocorticoid potency	Duration of action (t_1/2 in hours)
Hydrocortisone (Cortisol)	1	1	8
Cortisone acetate	0.8	0.8	oral 8, intramuscular 18+
Prednisone	3.5-5	0.8	16-36
Prednisolone	4	0.8	16-36
Methylprednisolone	5-7.5	0.5	18-40
Dexamethasone	25-80	0	36-54
Betamethasone	25-30	0	36-54
Triamcinolone	5	0	12-36
Beclometasone	8 puffs 4 times a day equals 14 mg oral prednisone once a day	-	-
Fludrocortisone acetate	15	200	-
Deoxycorticosterone acetate (DOCA)	0	20	-
Aldosterone	0.3	200-1000	-

Therapeutic use

Glucocorticoids may be used in low doses in adrenal insufficiency. In much higher doses, glucocorticoids are used to suppress various allergic, inflammatory, and autoimmune disorders. They are also administered as posttransplantory immunosuppressants to prevent the acute transplant rejection and the graft-versus-host disease. Nevertheless, they do not prevent an infection and also inhibit later reparative processes.

Physiological replacement

Any glucocorticoid can be given in a dose that provides approximately the same glucocorticoid effects as normal cortisol production; this is referred to as physiologic, replacement, or maintenance dosing. This is approximately 6-12 mg/m²/day (m² refers to body surface area (BSA), and is a measure of body size; an average man is 1.7 m²).

Immunosuppression

Glucocorticoids suppress the cell-mediated immunity. They act by inhibiting genes that code for the cytokines IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8 and IFN-γ, the most important of which is IL-2. Smaller cytokine production reduces the T cell proliferation.

Glucocorticoids do however not only reduce T cell proliferation, another well known effect is glucocorticoid induced apoptosis. The effect is more prominent in immature T cells that still reside in the thymus, but also affect peripheral T cells. The exact mechanism underlying this glucocorticoid sensitivity still remains to be elucidated.

Glucocorticoids also suppress the humoral immunity, causing B cells to express smaller amounts of IL-2 and of IL-2 receptors. This diminishes both B cell clone expansion and antibody synthesis. The diminished amounts of IL-2 also causes fewer T lymphocyte cells to be activated.

Since glucocorticoid is a steroid, it regulates transcription factors; another factor it down-regulates is the expression of Fc receptors on macrophages, so there is a decreased phagocytosis of opsonised cells.

Anti-inflammatory

Glucocorticoids influence all types of inflammatory events, no matter what their cause. They induce the lipocortin-1 (annexin-1) synthesis, which then binds to cell membranes, preventing the phospholipase A2 from coming into contact with its substrate arachidonic acid. This leads to diminished eicosanoid production. The cyclooxygenase (both COX-1 and COX-2) expression is also suppressed, potentiating the effect. In other words, the two main products in inflammation, Prostaglandins and Leukotrienes, are inhibited by the action of Glucocorticoids.

Glucocorticoids also stimulate the lipocortin-1 escaping to the extracellular space, where it binds to the leukocyte membrane receptors and inhibits various inflammatory events: epithelial adhesion, emigration, chemotaxis, phagocytosis, respiratory burst, and the release of various inflammatory mediators (lysosomal enzymes, cytokines, tissue plasminogen activator, chemokines, etc.) from neutrophils, macrophages, and mastocytes.

Resistance

Resistance to the therapeutic uses of glucocorticoids can present difficulty; for instance, 25% of cases of severe asthma may be unresponsive to steroids. This may be the result of genetic predisposition, ongoing exposure to the cause of the inflammation (such as allergens), immunological phenomena that bypass glucocorticoids, and pharmacokinetic disturbances (incomplete absorption or accellerated excretion or metabolism).

Side-effects

Glucocorticoid drugs currently being used act nonselectively, so in the long run they may impair many healthy anabolic processes. To prevent this, much research has been focused recently on the elaboration of selectively-acting glucocorticoid drugs. These are the side-effects that could be prevented:

immunosuppression
hyperglycemia due to increased gluconeogenesis, insulin resistance, and impaired glucose tolerance ("steroid diabetes"); caution in those with diabetes mellitus
increased skin fragility, easy bruising
reduced bone density (osteoporosis, osteonecrosis, higher fracture risk, slower fracture repair)
weight gain due to increased visceral and truncal fat deposition (central obesity) and appetite stimulation
adrenal insufficiency (if used for long time and stopped suddenly without a taper)
muscle breakdown (proteolysis), weakness; reduced muscle mass and repair
expansion of malar fat pads and dilation of small blood vessels in skin
anovulation, irregularity of menstrual periods
growth failure, pubertal delay
increased plasma amino acids, increased urea formation; negative nitrogen balance
excitatory effect on central nervous system (euphoria, psychosis)
glaucoma due to increased cranial pressure

In high doses, hydrocortisone (cortisol) and those glucocorticoids with appreciable mineralocorticoid potency can exert a mineralocorticoid effect as well, although in physiologic doses this is prevented by rapid degradation of cortisol by 11β-hydroxysteroid dehydrogenase isoenzyme 2 (11β-HSD2) in mineralocorticoid target tissues. Mineralocorticoid effects can include salt and water retention, extracellular fluid volume expansion, hypertension, potassium depletion, and metabolic alkalosis.

The combination of clinical problems produced by prolonged, excess glucocorticoids, whether synthetic or endogenous, is termed Cushing's syndrome.

Withdrawal

In addition to the effects listed above, use of high-dose steroids for more than a week begins to produce suppression of the patient's adrenal glands because the exogenous glucocorticoids suppress hypothalamic corticotropin-releasing hormone (CRH) and pituitary adrenocorticotropic hormone (ACTH). With prolonged suppression, the adrenal glands atrophy (physically shrink), and can take months to recover full function after discontinuation of the exogenous glucocorticoid.

During this recovery time, the patient is vulnerable to adrenal insufficiency during times of stress, such as illness. While there is wide individual variation in suppressive dose and time for adrenal recovery, clinical guidelines have been devised to estimate potential adrenal suppression and recovery, to reduce risk to the patient. The following is one example, but many variations exist or may be appropriate in individual circumstances.

If a patient has been receiving daily high doses for 5 days or less, they can be abruptly stopped (or reduced to physiologic replacement if patient is adrenal-deficient). Full adrenal recovery can be assumed to occur by a week afterward.
If high doses were used for 6-10 days, reduce to replacement dose immediately and taper over 4 more days. Adrenal recovery can be assumed to occur within 2-4 weeks of completion of steroids.
If high doses were used for 11-30 days, cut immediately to twice replacement, and then by 25% every 4 days. Stop entirely when dose is less than half of replacement. Full adrenal recovery should occur within 1-3 months of completion of withdrawal.
If high doses were used more than 30 days, cut dose immediately to twice replacement, and reduce by 25% each week until replacement is reached.
Then change to oral hydrocortisone or cortisone as a single morning dose, and gradually decrease by 2.5 mg each week. When a.m. dose is less than replacement, the return of normal basal adrenal function may be documented by checking 0800 cortisol levels prior to the morning dose; stop drugs when 0800 cortisol is 10 μg/dl. It is difficult to predict the time to full adrenal recovery after prolonged suppressive exogenous steroids; some people may take nearly a year.
Flare-up of the underlying condition for which steroids are given may require a more gradual taper than outlined above.

Immunosuppressive drug

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Immunosuppressive drugs, immunosuppressive agents, or immunosuppressants are drugs that inhibit or prevent activity of the immune system. They are used in immunosuppressive therapy to:

Prevent the rejection of transplanted organs and tissues (e.g., bone marrow, heart, kidney, liver)
Treat autoimmune diseases or diseases that are most likely of autoimmune origin (e.g., rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Crohn's disease, pemphigus, and ulcerative colitis).
Treat some other non-autoimmune inflammatory diseases (e.g., long term allergic asthma control).

These drugs are not without side-effects and risks. Because the majority of them act non-selectively, the immune system is less able to resist infections and the spread of malignant cells. There are also other side-effects, such as hypertension, dyslipidemia, hyperglycemia, peptic ulcers, liver, and kidney injury. The immunosuppressive drugs also interact with other medicines and affect their metabolism and action. Actual or suspected immunosuppressive agents can be evaluated in terms of their effects on lymphocyte subpopulations in tissues using immunohistochemistry.

Immunosuppressive drugs can be classified into five groups:

glucocorticoids
cytostatics
antibodies
drugs acting on immunophilins
other drugs.

Glucocorticoids

In pharmacologic (supraphysiologic) doses, glucocorticoids are used to suppress various allergic, inflammatory, and autoimmune disorders. They are also administered as posttransplantory immunosuppressants to prevent the acute transplant rejection and graft-versus-host disease. Nevertheless, they do not prevent an infection and also inhibit later reparative processes.

Immunosuppressive mechanism

Glucocorticoids suppress the cell-mediated immunity. They act by inhibiting genes that code for the cytokines IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, and TNF-γ, the most important of which is the IL-2. Smaller cytokine production reduces the T cell proliferation.

Glucocorticoids also suppress the humoral immunity, causing B cells to express smaller amounts of IL-2 and IL-2 receptors. This diminishes both B cell clone expansion and antibody synthesis.

Antiinflammatory effects

Glucocorticoids influence all types of inflammatory events, no matter what their cause. They induce the lipocortin-1 (annexin-1) synthesis, which then binds to cell membranes preventing the phospholipase A2 from coming into contact with its substrate arachidonic acid. This leads to diminished eicosanoid production. The cyclooxygenase (both COX-1 and COX-2) expression is also suppressed, potentiating the effect.

Cytostatics

Cytostatics inhibit cell division. In immunotherapy, they are used in smaller doses than in the treatment of malignant diseases. They affect the proliferation of both T cells and B cells. Due to their highest effectiveness, purine analogs are most frequently administered.

Alkylating agents

The alkylating agents used in immunotherapy are nitrogen mustards (cyclophosphamide), nitrosoureas, platinum compounds, and others. Cyclophosphamide is probably the most potent immunosuppressive compound. In small doses, it is very efficient in the therapy of systemic lupus erythematosus, autoimmune hemolytic anemias, Wegener's granulomatosis, and other immune diseases. High doses cause pancytopenia and hemorrhagic cystitis.

Antimetabolites

Antimetabolites interfere with the synthesis of nucleic acids. These include:

folic acid analogues, such as methotrexate
purine analogues such as azathioprine and mercaptopurine
pyrimidine analogues
protein synthesis inhibitors.

Methotrexate

Methotrexate is a folic acid analogue. It binds dihydrofolate reductase and prevents synthesis of tetrahydrofolate. It is used in the treatment of autoimmune diseases (for example rheumatoid arthritis) and in transplantations.

Azathioprine and Mercaptopurine

Azathioprine, is the main immunosuppressive cytotoxic substance. It is extensively used to control transplant rejection reactions. It is nonenzymatically cleaved to mercaptopurine, that acts as a purine analogue and an inhibitor of DNA synthesis. Mercaptopurine itself can also be administered directly.

By preventing the clonal expansion of lymphocytes in the induction phase of the immune response, it affects both the cell and the humoral immunity. It is also efficient in the treatment of autoimmune diseases.

Cytotoxic antibiotics

Among these, dactinomycin is the most important. It is used in kidney transplantations. Other cytotoxic antibiotics are anthracyclines, mitomycin C, bleomycin, mithramycin.

Antibodies

Antibodies are sometimes used as a quick and potent immunosuppressive therapy to prevent the acute rejection reactions as well as a targeted treatment of lyphoproliferative or autoimmune disorders (e.g., anti-CD20 monoclonals).

Polyclonal antibodies

Heterologous polyclonal antibodies are obtained from the serum of animals (e.g., rabbit, horse), and injected with the patient's thymocytes or lymphocytes. The antilymphocyte (ALG) and antithymocyte antigens (ATG) are being used. They are part of the steroid-resistant acute rejection reaction and grave aplastic anemia treatment. However, they are added primarily to other immunosuppressives to diminish their dosage and toxicity. They also allow transition to cyclosporine therapy.

Polyclonal antibodies inhibit T lymphocytes and cause their lysis, which is both complement-mediated cytolysis and cell-mediated opsonization followed by removal of reticuloendothelial cells from the circulation in the spleen and liver. In this way, polyclonal antibodies inhibit cell-mediated immune reactions, including graft rejection, delayed hypersensitivity (i.e., tuberculin skin reaction), and the graft-versus-host disease (GVHD), but influence thymus-dependent antibody production.

As of March 2005, there are two preparations available to the market: Atgam (R), obtained from horse serum, and Thymoglobuline (R), obtained from rabbit serum. Polyclonal antibodies affect all lymphocytes and cause general immunosuppression, possibly leading to post-transplant lymphoproliferative disorders (PTLD) or serious infections, especially by cytomegalovirus. To reduce these risks, treatment is provided in a hospital, where adequate isolation from infection is available. They are usually administered for five days intravenously in the appropriate quantity. Patients stay in the hospital as long as three weeks to give the immune system time to recover to a point where there is no longer a risk of serum sickness.

Because of a high immunogenicity of polyclonal antibodies, almost all patients have an acute reaction to the treatment. It is characterized by fever, rigor episodes, and even anaphylaxis. Later during the treatment, some patients develop serum sickness or immune complex glomerulonephritis. Serum sickness arises seven to fourteen days after the therapy has begun. The patient suffers from fever, joint pain, and erythema that can be soothed with the use of steroids and analgesics. Urticaria (hives) can also be present. It is possible to diminish their toxicity by using highly-purified serum fractions and intravenous administration in the combination with other immunosuppressants, for example, calcineurin inhibitors, cytostatics and cortisteroids. The most frequent combination is to use antibodies and cyclosporine simultaneously in order to prevent patients from gradually developing a strong immune response to these drugs, reducing or eliminating their effectiveness.

Monoclonal antibodies

Monoclonal antibodies are directed towards exactly defined antigens. Therefore, they cause fewer side-effects. Especially significant are the IL-2 receptor- (CD25-) and CD3-directed antibodies. They are used to prevent the rejection of transplanted organs, but also to track changes in the lymphocyte subpopulations. It is reasonable to expect similar new drugs in the future.

T-cell receptor directed antibodies

As of 2007 OKT3 (also called muromonab) is the only approved anti-CD3 antibody. It is a murine anti-CD3 monoclonal antibody of the IgG2a type that prevents T-cell activation and proliferation by binding the T-cell receptor complex present on all differentiated T cells. As such it is one of the most potent immunosuppressive substances and is administered to control the steroid- and/or polyclonal antibodies-resistant acute rejection episodes. As it acts more specifically than polyclonal antibodies it is also used prophylactically in transplantations.

At present the OKT3's mechanism of action is only partially understood. It is known that the molecule binds TCR/CD3 receptor complex. In the first few administrations this binding non-specifically activates T-cells, leading to a serious syndrome 30 to 60 minutes later. It is characterized by fever, myalgia, headache, and arthralgia. Sometimes it develops in a life-threatening reaction of the cardiovascular system and the central nervous system, requiring a lengthy therapy. Past this period CD3 (R) blocks the TCR-antigen binding and causes conformational change or the removal of the entire TCR3/CD3 complex from the T-cell surface. This lowers the number of available T-cells, perhaps by sensitizing them for the uptake by the epithelial reticular cells. The cross-binding of CD3 molecules as well activates an intracellular signal causing the T cell anergy or apoptosis, unless the cells receive another signal through a co-stimulatory molecule. CD3 antibodies shift the balance from Th1 to Th2 cells.

When deciding to include OKT3 in the treatment a healthcare practitioner must consider not only its great efficiency but also its toxic side-effects. The risk of excessive immunosuppression and the risk of development of neutralizing antibodies could make it inefficacious. Although CD3 antibodies act more specifically than polyclonal antibodies, they lower the cell-mediated immunity significantly, predisposing the patient to opportunistic infections and malignancies.

IL-2 receptor directed antibodies

Interleukin-2 is an important immune system regulator necessary for the clone expansion and survival of activated lymphocytes T. Its effects are mediated by the trimer cell surface receptor IL-2a, consisting of the α, β, and γ chains. The IL-2a (CD25, T-cell activation antigen, TAC) is expressed only by the already-activated T lymphocytes. Therefore, it is of special significance to the selective immunosuppressive treatment, and the research has been focused on the development of effective and safe anti-IL-2 antibodies. By the use of the recombinant gene technology, the mouse anti-Tac antibodies have been modified, leading to the presentation of two chimeric mouse/human anti-Tac antibodies in the year 1998: basiliximab (Simulect (R)) and daclizumab (Zenapax (R)). These drugs act by binding the IL-2a receptor's α chain, preventing the IL-2 induced clonal expansion of activated lymphocytes and shortening their survival. They are used in the prophylaxis of the acute organ rejection after the bilateral kidney transplantation, both being similarly effective and with only few side-effects.

Drugs acting on immunophilins

Cyclosporin

Together with tacrolimus, cyclosporin is a calcineurin inhibitor. It has been in use since 1983 and is one of the most-widely-used immunosuppressive drugs. It is a fungal peptide, composed of 11 amino acids.

Cyclosporin is thought to bind to the cytosolic protein cyclophilin (an immunophilin) of immunocompetent lymphocytes, especially T-lymphocytes. This complex of cyclosporin and cyclophilin inhibits calcineurin, which under normal circumstances induces the transcription of interleukin-2. The drug also inhibits lymphokine production and interleukin release, leading to a reduced function of effector T-cells.

Cyclosporin is used in the treatment of acute rejection reactions, but has been increasingly substituted with newer immunosuppressants, as it is nephrotoxic.

Tacrolimus (Prograf(TM), FK506)

Tacrolimus is a fungal product (Streptomyces tsukubaensis). It is a macrolide lactone and acts by inhibiting calcineurin.

The drug is used particularly in the liver and kidney transplantations, although in some clinics it is used in heart, lung and heart/lung transplants. It binds to an immunophilin, followed by the binding of the complex to calcineurin and the inhibition of its phosphatase activity. In this way, it prevents the cell from transitioning from the G0 into G1 phase of the cell cycle. Tacrolimus is more potent than cyclosporin and has less-pronounced side-effects.

Sirolimus (Rapamune (Tm), Rapamycin)

Sirolimus is a macrolide lactone, produced by the actinomycetes Streptomyces hygroscopicus. It is used to prevent rejection reactions. Although it is a structural analogue of tacrolimus, it acts somewhat differently and has different side-effects.

Contrary to cyclosporine and tacrolimus that affect the first phase of the T lymphocyte activation, sirolimus affects the second one, namely the signal transduction and their clonal proliferation. It binds to the same receptor (immunophilin) as tacrolimus, however the produced complex does not inhibit calcineurin, but another protein. Therefore, sirolimus acts synergistically with cyclosporine and, in combination with other immunosuppressants, has few side-effects. Also, it indirectly inhibits several T lymphocyte kinases and phosphatases, preventing the transmission of signal into their activity and the transition of the cell cycle from G1 to S phase. In a similar manner, it prevents the B cell differentiation to the plasma cells, which lowers the quantity of IgM, IgG, and IgA antibodies produced. It acts as an immunoregulatory agent, and is also active against tumors that involve the PI3K/AKT/mTOR pathway.

Other drugs

Interferons

IFN-β suppresses the production of Th1 cytokines and the activation of monocytes. It is used to slow down the progression of multiple sclerosis. IFN-γ is able to trigger lymphocytic apoptosis.

Opioids

Prolonged use of opioids may cause immunosuppression of both innate and adaptive immunity. Decrease in proliferation as well as immune function has been observed in macrophages, as well as lymphocytes. It is thought that these effects are mediated by opioid receptors expressed on the surface of these immune cells.

TNF binding proteins

A TNF-α- (tumor necrosis factor-alpha-) binding protein is a monoclonal antibody or a circulating receptor such as infliximab (Remicade), etanercept (Enbrel), or adalimumab (Humira) that binds to TNF-α, preventing it from inducing the synthesis of IL-1 and IL-6 and the adhesion of lymphocyte-activating molecules. They are used in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, and psoriasis.

TNF or the effects of TNF are also suppressed by various natural compounds, including curcumin (an ingredient in turmeric) and catechins (in green tea).

These drugs may raise the risk of contracting tuberculosis or inducing a latent infection to become active. Infliximab and adalimumab have label warnings stating that patients should be evaluated for latent TB infection and treatment should be initiated prior to starting therapy with them.

Mycophenolate

Mycophenolic acid acts as a non-competitive, selective, and reversible inhibitor of Inosine-5′-monophosphate dehydrogenase (IMPDH), which is a key enzyme in the de novo guanosine nucleotide synthesis. In contrast to other human cell types, lymphocytes B and T are very dependent on this process.

Small biological agents

Fingolimod is a new synthetic immunosuppressant, currently in phase 3 of clinical trials. It increases the expression or changes the function of certain adhesion molecules (α4/β7 integrin) in lymphocytes, so they accumulate in the lymphatic tissue (lymphatic nodes) and their number in the circulation is diminished. In this respect, it differs from all other known immunosuppressants.

Myriocin has been reported being 10 to 100 times more potent than Cyclosporin.

Immunology

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Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all organisms. It deals with, among other things, the physiological functioning of the immune system in states of both health and disease; malfunctions of the immune system in immunological disorders (autoimmune diseases, hypersensitivities, immune deficiency, transplant rejection); the physical, chemical and physiological characteristics of the components of the immune system in vitro, in situ, and in vivo. Immunology has applications in several disciplines of science, and as such is further divided.

Histological examination of the immune system

Even before the concept of immunity (from immunis, Latin for "exempt") was developed, numerous early physicians characterized organs that would later prove to be part of the immune system. The key primary lymphoid organs of the immune system are thymus and bone marrow, and secondary lymphatic tissues such as spleen, tonsils, lymph vessels, lymph nodes, adenoids, and skin. When health conditions warrant, immune system organs including the thymus, spleen, portions of bone marrow, lymph nodes and secondary lymphatic tissues can be surgically excised for examination while patients are still alive.

Many components of the immune system are actually cellular in nature and not associated with any specific organ but rather are embedded or circulating in various tissues located throughout the body.

Classical immunology

Classical immunology ties in with the fields of epidemiology and medicine. It studies the relationship between the body systems, pathogens, and immunity. The earliest written mention of immunity can be traced back to the plague of Athens in 430 BCE. Thucydides noted that people who had recovered from a previous bout of the disease could nurse the sick without contracting the illness a second time. Many other ancient societies have references to this phenomenon, but it was not until the 19th and 20th centuries before the concept developed into scientific theory.

The study of the molecular and cellular components that comprise the immune system, including their function and interaction, is the central science of immunology. The immune system has been divided into a more primitive innate immune system, and acquired or adaptive immune system of vertebrates, the latter of which is further divided into humoral and cellular components.

The humoral (antibody) response is defined as the interaction between antibodies and antigens. Antibodies are specific proteins released from a certain class of immune cells (B lymphocytes). Antigens are defined as anything that elicits generation of antibodies, hence they are Antibody Generators. Immunology itself rests on an understanding of the properties of these two biological entities. However, equally important is the cellular response, which can not only kill infected cells in its own right, but is also crucial in controlling the antibody response. Put simply, both systems are highly interdependent.

In the 21st century, immunology has broadened its horizons with much research being performed in the more specialized niches of immunology. This includes the immunological function of cells, organs and systems not normally associated with the immune system, as well as the function of the immune system outside classical models of immunity.

Clinical immunology

Clinical immunology is the study of diseases caused by disorders of the immune system (failure, aberrant action, and malignant growth of the cellular elements of the system). It also involves diseases of other systems, where immune reactions play a part in the pathology and clinical features.

The diseases caused by disorders of the immune system fall into two broad categories: immunodeficiency, in which parts of the immune system fail to provide an adequate response (examples include chronic granulomatous disease), and autoimmunity, in which the immune system attacks its own host's body (examples include systemic lupus erythematosus, rheumatoid arthritis, Hashimoto's disease and myasthenia gravis). Other immune system disorders include different hypersensitivities, in which the system responds inappropriately to harmless compounds (asthma and other allergies) or responds too intensely.

The most well-known disease that affects the immune system itself is AIDS, caused by HIV. AIDS is an immunodeficiency characterized by the lack of CD4+ ("helper") T cells and macrophages, which are destroyed by HIV.

Clinical immunologists also study ways to prevent transplant rejection, in which the immune system attempts to destroy allografts or xenografts.

Immunotherapy

The use of immune system components to treat a disease or disorder is known as immunotherapy. Immunotherapy is most commonly used in the context of the treatment of cancers together with chemotherapy (drugs) and radiotherapy (radiation). However, immunotherapy is also often used in the immunosuppressed (such as HIV patients) and people suffering from other immune deficiencies or autoimmune diseases.

Diagnostic immunology

The specificity of the bond between antibody and antigen has made it an excellent tool in the detection of substances in a variety of diagnostic techniques. Antibodies specific for a desired antigen can be conjugated with a radiolabel, fluorescent label, or color-forming enzyme and are used as a "probe" to detect it.

Evolutionary immunology

Study of the immune system in extant and extinct species is capable of giving us a key understanding of the evolution of species and the immune system.

A development of complexity of the immune system can be seen from simple phagocytotic protection of single celled organisms, to circulating antimicrobial peptides in insects to lymphoid organs in vertebrates. Of course, like much of evolutionary observation, these physical properties are often seen from the anthropocentric aspect. It should be recognized that every organism living today has an immune system absolutely capable of protecting it from most forms of harm; those organisms that did not adapt their immune systems to external threats are no longer around to be observed.

Insects and other arthropods, while not possessing true adaptive immunity, show highly evolved systems of innate immunity, and are additionally protected from external injury

Humoral immunity

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The Humoral Immune Response (HIR) is the aspect of immunity that is mediated by secreted antibodies (as opposed to cell-mediated immunity which involves T lymphocytes) produced in the cells of the B lymphocyte lineage (B cell). Secreted antibodies bind to antigens on the surfaces of invading microbes (such as viruses or bacteria), which flags them for destruction. Humoral immunity is called as such, because it involves substances found in the humours, or body fluids.

The study of the molecular and cellular components that comprise the immune system, including their function and interaction, is the central science of immunology. The immune system is divided into a more primitive innate immune system, and acquired or adaptive immune system of vertebrates, each of which contains humoral and cellular components.

Humoral immunity refers to antibody production, and the accessory processes that accompany it, including: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibody, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

History

The concept of humoral immunity developed based on analysis of antibacterial activity of the components of serum. Hans Buchner is credited with the development of the humoral theory. In 1890 he described alexins, or “protective substances”, which exist in the serum and other bodily fluids and are capable of killing microorganisms. Alexins, later redefined "complement" by Paul Ehrlich, were shown to be the soluble components of the innate response that lead to a combination of cellular and humoral immunity, and bridged the features of innate and acquired immunity.

Following the 1888 discovery of diphtheria and tetanus, Emil von Behring and Shibasaburo Kitasato showed that disease need not be caused by microorganisms themselves. They discovered that cell-free filtrates were sufficient to cause disease. In 1890, filtrates of diphtheria (later named diphtheria toxins) were used to immunize animals in an attempt to demonstrate that immunized serum contained an antitoxin that could neutralize the activity of the toxin and could transfer immunity to non immune animals. In 1897, Paul Ehrlich showed that antibodies form against the plant toxins ricin and abrin, and proposed that these antibodies are responsible for immunity. Ehrlich, with his friend Emil von Behring, went on to develop the diphtheria antitoxin, which became the first major success of modern immunotherapy. The presence and specificity of antibodies became the major tool for standardizing the state of immunity and identifying the presence of previous infections.

**Major discoveries in the study of humoral immunity**
Substance	Activity	Discovery
Alexin(s) Complement	Soluble components in the serum that are capable of killing microorganisms	Buchner (1890), Ehrlich (1892)
Antitoxins	Substances in the serum that can neutralize the activity of toxins, enabling passive immunization	von Bhering and Kitasato (1890)
Bacteriolysins	Serum substances that work with the complement proteins to induce bacterial lysis	Richard Pfeiffer (1895)
Bacterial agglutinins & precipitins	Serum substances that agglutinate bacteria and precipitate bacterial toxins	von Gruber and Durham (1896), Kraus (1897)
Hemolysins	Serum substances that work with complement to lyse red blood cells	Belfanti and Carbone (1898) Jules Bordet (1899)
Opsonins	serum substances that coat the outer membrane of foreign substances and enhance the rate of phagocytosis by macrophages	Wright and Douglas (1903)
Antibody	formation (1900), antigen-antibody binding hypothesis (1938), produced by B cells (1948), structure (1972), immunoglobulin genes (1976)	Founder: P Ehrlich

Complement system

The complement system is a biochemical cascade of the innate immune system that helps clear pathogens from an organism. It is derived from many small plasma proteins that work together to disrupt the target cell's plasma membrane leading to cytolysis of the cell. The complement system consists of more than 35 soluble and cell-bound proteins, 12 of which are directly involved in the complement pathways. The complement system is involved in the activities of both innate immunity and acquired immunity.

Activation of this system leads to cytolysis, chemotaxis, opsonization, immune clearance, and inflammation, as well as the marking of pathogens for phagocytosis. The proteins account for 5% of the serum globulin fraction. Most of these proteins circulate as zymogens, which are inactive until proteolytic cleavage.

Three biochemical pathways activate the complement system: the classical complement pathway, the alternate complement pathway, and the mannose-binding lectin pathway. The classical complement pathway typically requires antibodies for activation and is a specific immune response, while the alternate pathway can be activated without the presence of antibodies and is considered a non-specific immune response. Antibodies, in particular the IgG1 class, can also "fix" complement.

Antibodies

Immunoglobulins are glycoproteins in the immunoglobulin superfamily that function as antibodies. The terms antibody and immunoglobulin are often used interchangeably. They are found in the blood and tissue fluids, as well as many secretions. In structure, they are large Y-shaped globular proteins. In mammals there are five types of antibody: IgA, IgD, IgE, IgG, and IgM. Each immunoglobulin class differs in its biological properties and has evolved to deal with different antigens. Antibodies are synthesized and secreted by plasma cells that are derived from the B cells of the immune system.

An antibody is used by the acquired immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. By binding their specific antigens, antibodies can cause agglutination and precipitation of antibody-antigen products, prime for phagocytosis by macrophages and other cells, block viral receptors, and stimulate other immune responses, such as the complement pathway.

An incompatible blood transfusion, causes a transfusion reaction, which is mediated by the humoral immune response. This type of reaction, called an acute hemolytic reaction, results in the rapid destruction (hemolysis) of the donor red blood cells by host antibodies. The cause is usually a clerical error (i.e. the wrong unit of blood being given to the wrong patient). The symptoms are fever and chills, sometimes with back pain and pink or red urine (hemoglobinuria). The major complication is that hemoglobin released by the destruction of red blood cells can cause acute renal failure.

B cells

The principal function of B cells is to make antibodies against soluble antigens. B cell recognition of antigen is not the only element necessary for B cell activation (a combination of clonal proliferation and terminal differentiation into plasma cells).

Naive B cells can be activated in a T-cell dependent or independent manner, but two signals are always required to initiate activation.

B cell activation depends on one of three mechanisms: Type 1 T cell-independent (polyclonal) activation, Type 2 T cell-independent activation (in which macrophages present several of the same antigen in a way that causes cross-linking of antibodies on the surface of B cells), and, T cell-dependent activation. During T cell-dependent activation, an antigen presenting cell (APC) presents a processed antigen to a helper T (Th) cell, priming it. When a B cell processes and presents the same antigen to the primed Th cell, the T cell releases cytokines that activate the B cell.

ELISA

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A 96-well microtiter plate being used for ELISA.

Enzyme-Linked ImmunoSorbent Assay, also called ELISA, Enzyme ImmunoAssay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.

Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates enabling much higher sensitivity.

Applications

Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs. ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.

The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and negative result.

One method of determining a cut-off point is by comparison with a known standard. For example, if an ELISA test will be used in workplace drug screening, a cut-off concentration (e.g., 50 ng/mL of drug) will be established and a sample will be prepared that contains that concentration of analyte. Unknowns that generate a signal that is stronger than the known sample are called "positive"; those that generate weaker signal are called "negative."

History

Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal which indicates whether a specific antigen or antibody is present in the sample. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.

Because radioactivity poses a health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a non-radioactive signal in place of the radioactive signal. When certain enzymes (such as peroxidase) react with appropriate substrates (such as ABTS or 3,3’,5,5’-Tetramethylbenzidine), they can result in changes in color, which can be used as a signal. However, the signal has to be associated with the presence of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G.B. Pierce. Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container, i.e. the immunosorbent has to be prepared. A technique to accomplish this was published by Wide and Porath in 1966.

In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, as well as Anton Schuurs and Bauke van Weemen in The Netherlands, independently published papers which synthesized this knowledge into methods to perform EIA/ELISA.

Types

"Indirect" ELISA

The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:

Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.

The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich ELISA provides a solution to this problem.

ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.

Sandwich ELISA

A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.

A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:

Prepare a surface to which a known quantity of capture antibody is bound.
Block any non specific binding sites on the surface.
Apply the antigen-containing sample to the plate.
Wash the plate, so that unbound antigen is removed.
Apply primary antibodies that bind specifically to the antigen.
Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.

The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.

Competitive ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:

Unlabeled antibody is incubated in the presence of its antigen.
These bound antibody/antigen complexes are then added to an antigen coated well.
The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.

(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal).

ELISA Reverse method & device (ELISA-R m&d)

A newer technique uses a solid phase made up of an immunosorbent polystyrene rod with 4-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.

Advantages:

The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays;
The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples;
One ogive is left unsensitized to measure the non-specific reactions of the sample;
The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits.

Anti-neutrophil cytoplasmic antibody

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Anti-neutrophil cytoplasmic antibodies (ANCAs) are a group of mainly IgG antibodies against antigens in the cytoplasm of neutrophil granulocytes (the most common type of white blood cell) and monocytes. They are detected as a blood test in a number of autoimmune disorders, but are particularly associated with systemic vasculitis, so called ANCA-associated vasculitides.

Types

ANCA were originally shown to divide into two main classes, c-ANCA and p-ANCA, based on the pattern of staining on ethanol-fixed neutrophils and the main target antigen. ANCA titres are generally measured using ELISA and indirect immunofluorescence.

Perinuclear staining typical of p-ANCA

The granular, cytoplasmic staining pattern of c-ANCA

p-ANCA

p-ANCA, or perinuclear-staining antineutrophil cytoplasmic antibodies, show a perinuclear staining pattern. This pattern occurs because during ethanol fixation some antigen targets artifactually localize around the nucleus. Antibody staining therefore results in fluorescence of the region around the nucleus. By far the most common p-ANCA target is myeloperoxidase (MPO), a neutrophil granule protein whose primary role in normal metabolic processes is generation of oxygen radicals. ANCA will less commonly form against alternative antigens that may also result in a p-ANCA pattern. These include lactoferrin; elastase; and cathepsin G. p-ANCA is fairly sensitive, but not specific for ulcerative colitis, so not useful as a sole diagnostic test.

c-ANCA

c-ANCAs, or cytoplasmic-staining antineutrophil cytoplasmic antibodies, show a diffusely granular, cytoplasmic staining pattern. This pattern results from binding of ANCA to antigen targets throughout the neutrophil cytoplasm, the most common protein target being proteinase 3 (PR3). PR3 is the most common antigen target of ANCA in patients with Wegener's granulomatosis. Other antigens may also occasionally result in a c-ANCA pattern.

Other

ANCA that develop against antigens other than MPO or PR3 will occasionally result in patchy staining when visualized by immunofluorescence. This pattern is commonly called the 'snowdrift' pattern, and most commonly occurs in patients with non-vasculitic diseases that are associated with ANCA formation.

Development of ANCA

It is poorly understood how ANCA are developed, although several hypotheses have been suggested. There is probably a genetic contribution, particularly in genes controlling the level of immune response – although genetic susceptibility is likely to be linked to an environmental factor, some possible factors including vaccination or exposure to silicates. Two possible mechanisms of ANCA development are postulated, although neither of these theories answers the question of how the different ANCA specificities are developed, and there is much research still being undertaken on the development of ANCA.

Theory of molecular mimicry

Microbial superantigens are molecules expressed by bacteria and other microorganisms that have the power to stimulate a strong immune response by activation of T-cells. These molecules generally have regions that resemble self-antigens – this is the theory of molecular mimicry. Staphylococcal and streptococcal superantigens have been characterised in autoimmune diseases – the classical example in post group A streptococcal rheumatic heart disease, where there is similarity between M proteins of Streptococcus pyogenes to cardiac myosin and laminin. It has also been shown that up to 70% of patients with Wegener's granulomatosis are chronic nasal carriers of Staphylococcus aureus, with carriers having an eight times increased risk of relapse.

Theory of defective apoptosis

Neutrophil apoptosis, or programmed cell death, is vital in controlling the duration of the early inflammatory response, thus restricting damage to tissues by the neutrophils. ANCA may be developed either via ineffective apoptosis or ineffective removal of apoptotic cell fragments, leading to the exposure of the immune system to molecules normally sequestered inside the cells. This theory solves the paradox of how it could be possible for antibodies to be raised against the intracellular antigenic targets of ANCA.

Role in disease

There are three primary diseases that are consistently associated with ANCA: Wegener's granulomatosis, microscopic polyangiitis, and glomerulonephritis. The antibodies are assumed to be involved in the generation and/or progression of lesions and clinical signs.

Classically, c-ANCA is associated with Wegener’s granulomatosis; p-ANCA is associated with microscopic polyangiitis and focal necrotising and crescentic glomerulonephritis. However, in recent years ANCA targeted against other autoantigens have been identified.

Patients with a number of other diseases, such as ulcerative colitis and ankylosing spondylitis, will commonly have ANCA as well. However in these cases there is no assocatied vasculitis, and the ANCA are thought to be incidental or epiphenomena rather than part of the disease itself. Churg-Strauss syndrome is associated with p-ANCA directed against MPO.

It is unclear what the role of ANCA may be in these diseases – they may be markers of disease or may play some part in the pathogenic process. It has been shown that in Wegener's granulomatosis there is positive correlation between ANCA titre and disease activity and in vitro studies have shown that ANCA cause activation of primed neutrophils and react with endothelial cells expressing PR3. ANCA may act by causing release of lytic enzymes from the white blood cells, causing inflammation of the blood vessel wall (vasculitis). ANCA associated vasculitides usually present with features of a small-vessel vasculitis.

History

ANCAs were originally described in Davies et al in 1982 in segmental necrotising glomerulonephritis, and by van der Woude et al in 1985 in Wegener's. The Second International ANCA Workshop, held in The Netherlands in May 1989, fixed the nomenclature on perinuclear vs. cytoplasmic patterns, and the antigens MPO and PR3 were discovered in 1988 and 1989, respectively.

Anti-nuclear antibody

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Anti-nuclear antibodies (ANAs, also known as anti-nuclear factor or ANF) are antibodies directed against contents of the cell nucleus.

They are present in higher than normal numbers in autoimmune disease. The ANA test measures the pattern and amount of autoantibody which can attack the body's tissues as if they were foreign material. Autoantibodies are present in low titres in the general population, but in about 5% of the population, their concentration is increased, and about half of this 5% have an autoimmune disease.

Autoimmunity

Normal titer of ANA is 1:40. Higher titers are indicative of an autoimmune disease. The presence of ANA is indicative of lupus erythematosus (present in 80-90% of cases), though they also appear in some other auto-immune diseases such as Sjögren's syndrome (60%), rheumatoid arthritis, autoimmune hepatitis, scleroderma and polymyositis & dermatomyositis (30%), and various non-rheumatological conditions associated with tissue damage. ANA are also directed to the nuclear pore complex in primary biliary cirrhosis. Other conditions with high ANA titre include Addison disease, Idiopathic thrombocytopenic purpura (ITP), Hashimoto's, Autoimmune hemolytic anemia, Type I diabetes mellitus, Mixed connective tissue disorder.

Antinuclear antibodies

Antinuclear antibodies (ANAs) are unusual antibodies, detectable in the blood, that have the capability of binding to certain structures within the nucleus of the cells. The nucleus contains DNA, the genetic material. ANAs are found in patients whose immune system may be predisposed to cause inflammation against their own body tissues. Antibodies that are directed against one's own tissues are referred to as auto-antibodies. The propensity for the immune system to work against its own body is why ANAs indicate the possible presence of autoimmunity and provide, therefore, an indication for doctors to consider the possibility of autoimmune illness.

Following detection of a high titer of ANAs (e.g. 1:160), various subtypes are determined. This is typically done on cells of the HEp-2 cell line. Examples include:

Anti-ENA (Extractable nuclear antigen)
- Anti-Ro (SS-A)
- Anti-La (SS-B)
- Anti-Sm (Smith antigen)
- Anti-nRNP (nuclear ribonucleoproteins)
- Anti Scl-70 (topoisomerase I)
- Anti-Jo
Anti-gp-210 (nuclear pore gp-210)
Anti-p62 (Nucleoporin 62)
Anti-dsDNA (double-stranded DNA)
Anti-centromere

History

The LE cell was discovered in bone marrow in 1948 by Hargraves et al. This was the first indication that processes affecting the cell nucleus were responsible for lupus erythematosus (LE). In the 1950s, progressively more sensitive and specific ANA serology tests became available.

Mitochondrion

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Electron micrograph of a mitochondrion from mammalian lung tissue showing its matrix and membranes.

In cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 1–10 micrometers (μm) in size. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mental disorders and cardiac dysfunction, and may play a role in the aging process. The word mitochondrion comes from the Greek μίτος or mitos, thread + χονδρίον or khondrion, granule. Their ancestry is not fully understood, but, according to the endosymbiotic theory, mitochondria are descended from ancient bacteria, which were engulfed by the ancestors of eukaryotic cells more than a billion years ago.

Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissues and species. In human, 615 distinct types of proteins were identified from cardiac mitochondria; whereas in murinae (rats), 940 proteins encoded by distinct genes were reported. The mitochondrial proteome is thought to be dynamically regulated. Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

Structure

Simplified structure of mitochondrion

A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes, however, have different properties. Because of this double-membraned organization, there are five distinct compartments within the mitochondrion. There is the outer mitochondrial membrane, the intermembrane space (the space between the outer and inner membranes), the inner mitochondrial membrane, the cristae space (formed by infoldings of the inner membrane), and the matrix (space within the inner membrane).

Outer membrane

The outer mitochondrial membrane, which encloses the entire organelle, has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane (about 1:1 by weight). It contains large numbers of integral proteins called porins. These porins form channels that allow molecules 5000 Daltons or less in molecular weight to freely diffuse from one side of the membrane to the other. Larger proteins can also enter the mitochondrion if a signaling sequence at their N-terminus binds to a large multisubunit protein called translocase of the outer membrane, which then actively moves them across the membrane. Disruption of the outer membrane permits proteins in the intermembrane space to leak into the cytosol, leading to certain cell death.

Intermembrane space

The intermembrane space is basically the space between the outer membrane and the inner membrane. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules such as ions and sugars in the intermembrane space is the same as the cytosol. However, as large proteins must have a specific signaling sequence to be transported across the outer membrane, the protein composition of this space is different than the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c.

Inner membrane

The inner mitochondrial membrane contains proteins with four types of functions:

Those that perform the redox reactions of oxidative phosphorylation
ATP synthase, which generates ATP in the matrix
Specific transport proteins that regulate metabolite passage into and out of the matrix
Protein import machinery.

It contains more than 100 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in beef hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane does not contain porins and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane formed by the action of the enzymes of the electron transport chain.

Cristae

Cross-sectional image of cristae in rat liver mitochondrion to demonstrate the likely 3D structure and relationship to the inner membrane.

The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function. In typical liver mitochondria, for example, the surface area, including cristae, is about five times that of the outer membrane. Mitochondria of cells that have greater demand for ATP, such as muscle cells, contain more cristae than typical liver mitochondria.

Matrix

The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in the production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly-concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle.

Mitochondria have their own genetic material, and the machinery to manufacture their own RNAs and proteins (see: protein biosynthesis). A published human mitochondrial DNA sequence revealed 16,569 base pairs encoding 37 total genes, 24 tRNA and rRNA genes and 13 peptide genes. The 13 mitochondrial peptides in humans are integrated into the inner mitochondrial membrane, along with proteins encoded by genes that reside in the host cell's nucleus.

Organization and distribution

Mitochondria are found in nearly all eukaryotes. They vary in number and location according to cell type. Substantial numbers of mitochondria are in the liver, with about 1000–2000 mitochondria per cell making up 1/5th of the cell volume. The mitochondria can be found nestled between myofibrils of muscle or wrapped around the sperm flagellum. Often they form a complex 3D branching network inside the cell with the cytoskeleton. The association with the cytoskeleton determines mitochondrial shape, which can affect the function as well. Recent evidence suggests vimentin, one of the components of the cytoskeleton, is critical to the association with the cytoskeleton.

Function

The most prominent roles of the mitochondrion are its production of ATP and regulation of cellular metabolism. The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs Cycle. However, the mitochondrion has many other functions in addition to the production of ATP.

Energy conversion

A dominant role for the mitochondria is the production of ATP, as reflected by the large number of proteins in the inner membrane for this task. This is done by oxidizing the major products of glucose, pyruvate, and NADH, which are produced in the cytosol. This process of cellular respiration, also known as aerobic respiration, is dependent on the presence of oxygen. When oxygen is limited, the glycolytic products will be metabolized by anaerobic respiration, a process that is independent of the mitochondria. The production of ATP from glucose has an approximately 13-fold higher yield during aerobic respiration compared to anaerobic respiration.

Pyruvate: the citric acid cycle

Each pyruvate molecule produced by glycolysis is actively transported across the inner mitochondrial membrane, and into the matrix where it is oxidized and combined with coenzyme A to form CO2, acetyl-CoA, and NADH.

The acetyl-CoA is the primary substrate to enter the citric acid cycle, also known as the tricarboxylic acid (TCA) cycle or Krebs cycle. The enzymes of the citric acid cycle are located in the mitochondrial matrix, with the exception of succinate dehydrogenase, which is bound to the inner mitochondrial membrane as part of Complex II. The citric acid cycle oxidizes the acetyl-CoA to carbon dioxide, and, in the process, produces reduced cofactors (three molecules of NADH and one molecule of FADH2) that are a source of electrons for the electron transport chain, and a molecule of GTP (that is readily converted to an ATP).

NADH and FADH₂: the electron transport chain

Schematic of typical animal cell, showing subcellular components. Organelles:
(1) nucleolus
(2) nucleus
(3) ribosomes (little dots)
(4) vesicle
(5) rough endoplasmic reticulum (ER)
(6) Golgi apparatus
(7) Cytoskeleton
(8) smooth ER
(9) mitochondria
(10) vacuole
(11) cytoplasm
(12) lysosome
(13) centrioles within centrosome

The redox energy from NADH and FADH₂ is transferred to oxygen (O₂) in several steps via the electron transport chain. These energy-rich molecules are produced within the matrix via the citric acid cycle but are also produced in the cytoplasm by glycolysis. Reducing equivalents from the cytoplasm can be imported via the malate-aspartate shuttle system of antiporter proteins or feed into the electron transport chain using a glycerol phosphate shuttle. Protein complexes in the inner membrane (NADH dehydrogenase, cytochrome c reductase, and cytochrome c oxidase) perform the transfer and the incremental release of energy is used to pump protons (H+) into the intermembrane space. This process is efficient, but a small percentage of electrons may prematurely reduce oxygen, forming reactive oxygen species such as superoxide. This can cause oxidative stress in the mitochondria and may contribute to the decline in mitochondrial function associated with the aging process.

As the proton concentration increases in the intermembrane space, a strong electrochemical gradient is established across the inner membrane. The protons can return to the matrix through the ATP synthase complex, and their potential energy is used to synthesize ATP from ADP and inorganic phosphate (Pi). This process is called chemiosmosis, and was first described by Peter Mitchell who was awarded the 1978 Nobel Prize in Chemistry for his work. Later, part of the 1997 Nobel Prize in Chemistry was awarded to Paul D. Boyer and John E. Walker for their clarification of the working mechanism of ATP synthase.

Heat production

Under certain conditions, protons can re-enter the mitochondrial matrix without contributing to ATP synthesis. This process is known as proton leak or mitochondrial uncoupling and is due to the facilitated diffusion of protons into the matrix. The process results in the unharnessed potential energy of the proton electrochemical gradient being released as heat. The process is mediated by a proton channel called thermogenin, or UCP1. Thermogenin is a 33kDa protein first discovered in 1973. Thermogenin is primarily found in brown adipose tissue, or brown fat, and is responsible for non-shivering thermogenesis. Brown adipose tissue is found in mammals, and is at its highest levels in early life and in hibernating animals. In humans, brown adipose tissue is present at birth and decreases with age.

Storage of calcium ions

The concentrations of free calcium in the cell can regulate an array of reactions and is important for signal transduction in the cell. Mitochondria can transiently store calcium, a contributing process for the cell's homeostasis of calcium. In fact, their ability to rapidly take in calcium for later release makes them very good "cytosolic buffers" for calcium. The endoplasmic reticulum (ER) is the most significant storage site of calcium, and there is a significant interplay between the mitochondrion and ER with regard to calcium. The calcium is taken up into the matrix by a calcium uniporter on the inner mitochondrial membrane. It is primarily driven by the mitochondrial membrane potential. Release of this calcium back into the cell's interior can occur via a sodium-calcium exchange protein or via "calcium-induced-calcium-release" pathways. This can initiate calcium spikes or calcium waves with large changes in the membrane potential. These can activate a series of second messenger system proteins that can coordinate processes such as neurotransmitter release in nerve cells and release of hormones in endocrine cells.

Additional functions

Mitochondria play a central role in many other metabolic tasks, such as:

Regulation of the membrane potential
Apoptosis-programmed cell death
Glutamate-mediated excitotoxic neuronal injury
Cellular proliferation regulation
Regulation of cellular metabolism
Certain heme synthesis reactions
Steroid synthesis.

Some mitochondrial functions are performed only in specific types of cells. For example, mitochondria in liver cells contain enzymes that allow them to detoxify ammonia, a waste product of protein metabolism. A mutation in the genes regulating any of these functions can result in mitochondrial diseases.

Origin

Mitochondria have many features in common with prokaryotes. As a result, they are believed to be originally derived from endosymbiotic prokaryotes.

A mitochondrion contains DNA, which is organized as several copies of a single, circular chromosome. This mitochondrial chromosome contains genes for ribosomes, and the twenty-one tRNA's necessary for the translation of messenger RNAs into protein. The circular structure is also found in prokaryotes, and the similarity is extended by the fact that mitochondrial DNA is organized with a variant genetic code similar to that of Proteobacteria. This suggests that their ancestor, the so-called proto-mitochondrion, was a member of the Proteobacteria. In particular, the proto-mitochondrion was probably related to the rickettsia. However, the exact relationship of the ancestor of mitochondria to the alpha-proteobacteria and whether the mitochondria was formed at the same time or after the nucleus, remains controversial.

The ribosomes coded for by the mitochondrial DNA are similar to those from bacteria in size and structure. They closely resemble the bacterial 70S ribosome and not the 80S cytoplasmic ribosomes which are coded for by nuclear DNA.

The endosymbiotic relationship of mitochondria with their host cells was popularized by Lynn Margulis. The endosymbiotic hypothesis suggests that mitochondria descended from bacteria that somehow survived endocytosis by another cell, and became incorporated into the cytoplasm. The ability of these bacteria to conduct respiration in host cells that had relied on glycolysis and fermentation would have provided a considerable evolutionary advantage. In a similar manner, host cells with symbiotic bacteria capable of photosynthesis would also have had an advantage. The incorporation of symbiotes would have increased the number of environments in which the cells could survive. This symbiotic relationship probably developed 1.7-2 billion years ago.

A few groups of unicellular eukaryotes lack mitochondria: the microsporidians, metamonads, and archamoebae. These groups appear as the most primitive eukaryotes on phylogenetic trees constructed using rRNA information, suggesting that they appeared before the origin of mitochondria. However, this is now known to be an artifact of long-branch attraction – they are derived groups and retain genes or organelles derived from mitochondria (e.g., mitosomes and hydrogenosomes).

Genome

The human mitochondrial genome is a circular DNA molecule of about 16 kilobases. It encodes 37 genes: 13 for subunits of respiratory complexes I, III, IV and V, 22 for mitochondrial tRNA, and 2 for rRNA. One mitochondrion can contain two to ten copies of its DNA.

As in prokaryotes, there is a very high proportion of coding DNA and an absence of repeats. Mitochondrial genes are transcribed as multigenic transcripts, which are cleaved and polyadenylated to yield mature mRNAs. Not all proteins necessary for mitochondrial function are encoded by the mitochondrial genome; most are coded by genes in the cell nucleus and the corresponding proteins imported into the mitochondrion. The exact number of genes encoded by the nucleus and the mitochondrial genome differs between species. In general, mitochondrial genomes are circular, although exceptions have been reported. Also, in general, mitochondrial DNA lacks introns, as is the case in the human mitochondrial genome; however, introns have been observed in some eukaryotic mitochondrial DNA, such as that of yeast and protists, including Dictyostelium discoideum.

While slight variations on the standard code had been predicted earlier, none was discovered until 1979, when researchers studying human mitochondrial genes determined that they used an alternative code. Many slight variants have been discovered since, including various alternative mitochondrial codes. Further, the AUA, AUC, and AUU codons are all allowable start codons.

Exceptions to the universal genetic code (UGC) in mitochondria
Organism	Codon	Standard	Novel
Mammalian	AGA, AGG	Arginine	Stop codon
	AUA	Isoleucine	Methionine
	UGA	Stop codon	Tryptophan
Invertebrates	AGA, AGG	Arginine	Serine
	AUA	Isoleucine	Methionine
	UGA	Stop codon	Tryptophan
Yeast	AUA	Isoleucine	Methionine
	UGA	Stop codon	Tryptophan
	CUA	Leucine	Threonine

Some of these differences should be regarded as pseudo-changes in the genetic code due to the phenomenon of RNA editing, which is common in mitochondria. In higher plants, it was thought that CGG encoded for tryptophan and not arginine; however, the codon in the processed RNA was discovered to be the UGG codon, consistent with the universal genetic code for tryptophan. Of note, the arthropod mitochondrial genetic code has undergone parallel evolution within a phylum, with some organisms uniquely translating AGG to lysine.

Mitochondrial genomes have far fewer genes than the bacteria from which they are thought to be descended. Although some have been lost altogether, many have been transferred to the nucleus, such as the respiratory complex II protein subunits. This is thought to be relatively common over evolutionary time. A few organisms, such as the Cryptosporidium, actually have mitochondria that lack any DNA, presumably because all their genes have been lost or transferred. In Cryptosporidium, the mitochondria have an altered ATP generation system that renders the parasite resistant to many classical mitochondrial inhibitors such as cyanide, azide, and atovaquone.

Replication and inheritance

Mitochondria divide by binary fission similar to bacterial cell division; unlike bacteria, however, mitochondria can also fuse with other mitochondria. The regulation of this division differs between eukaryotes. In many single-celled eukaryotes, their growth and division is linked to the cell cycle. For example, a single mitochondrion may divide synchronously with the nucleus. This division and segregation process must be tightly controlled so that each daughter cell receives at least one mitochondrion. In other eukaryotes (in humans for example), mitochondria may replicate their DNA and divide mainly in response to the energy needs of the cell, rather than in phase with the cell cycle. When the energy needs of a cell are high, mitochondria grow and divide. When the energy use is low, mitochondria are destroyed or become inactive. In such examples, and in contrast to the situation in many single celled eukaryotes, mitochondria are apparently randomly distributed to the daughter cells during the division of the cytoplasm.

An individual's mitochondrial genes are not inherited by the same mechanism as nuclear genes. At fertilization of an egg cell by a sperm, the egg nucleus and sperm nucleus each contribute equally to the genetic makeup of the zygote nucleus. In contrast, the mitochondria, and therefore the mitochondrial DNA, usually comes from the egg only. The sperm's mitochondria enter the egg but does not contribute genetic information to the embryo. Instead, paternal mitochondria are marked with ubiquitin to select them for later destruction inside the embryo. The egg cell contains relatively few mitochondria, but it is these mitochondria that survive and divide to populate the cells of the adult organism. Mitochondria are, therefore, in most cases inherited down the female line, known as maternal inheritance. This mode is seen in most organisms including all animals. However, mitochondria in some species can sometimes be inherited paternally. This is the norm among certain coniferous plants, although not in pine trees and yew trees. It has also been suggested that it occurs at a very low level in humans.

Uniparental inheritance leads to little opportunity for genetic recombination between different lineages of mitochondria, although a single mitochondrion can contain 2–10 copies of its DNA. For this reason, mitochondrial DNA usually is thought to reproduce by binary fission. What recombination does take place maintains genetic integrity rather than maintaining diversity. However, there are studies showing evidence of recombination in mitochondrial DNA. It is clear that the enzymes necessary for recombination are present in mammalian cells. Further, evidence suggests that animal mitochondria can undergo recombination. The data are a bit more controversial in humans, although indirect evidence of recombination exists. If recombination does not occur, the whole mitochondrial DNA sequence represents a single haplotype, which makes it useful for studying the evolutionary history of populations.

Population genetic studies

The near-absence of genetic recombination in mitochondrial DNA makes it a useful source of information for scientists involved in population genetics and evolutionary biology. Because all the mitochondrial DNA is inherited as a single unit, or haplotype, the relationships between mitochondrial DNA from different individuals can be represented as a gene tree. Patterns in these gene trees can be used to infer the evolutionary history of populations. The classic example of this is in human evolutionary genetics, where the molecular clock can be used to provide a recent date for mitochondrial Eve. This is often interpreted as strong support for a recent modern human expansion out of Africa. Another human example is the sequencing of mitochondrial DNA from Neanderthal bones. The relatively-large evolutionary distance between the mitochondrial DNA sequences of Neanderthals and living humans has been interpreted as evidence for lack of interbreeding between Neanderthals and anatomically-modern humans.

However, mitochondrial DNA reflects the history of only females in a population and so may not represent the history of the population as a whole. This can be partially overcome by the use of paternal genetic sequences, such as the non-recombining region of the Y-chromosome. In a broader sense, only studies that also include nuclear DNA can provide a comprehensive evolutionary history of a population.

Dysfunction and disease

Mitochondrial diseases

With their central place in cell metabolism, damage - and subsequent dysfunction - in mitochondria is an important factor in a wide range of human diseases. Mitochondrial disorders often present as neurological disorders, but can manifest as myopathy, diabetes, multiple endocrinopathy, or a variety of other systemic manifestations. Diseases caused by mutation in the mtDNA include Kearns-Sayre syndrome, MELAS syndrome and Leber's hereditary optic neuropathy. In the vast majority of cases, these diseases are transmitted by a female to her children, as the zygote derives its mitochondria and hence its mtDNA from the ovum. Diseases such as Kearns-Sayre syndrome, Pearson's syndrome, and progressive external ophthalmoplegia are thought to be due to large-scale mtDNA rearrangements, whereas other diseases such as MELAS syndrome, Leber's hereditary optic neuropathy, myoclonic epilepsy with ragged red fibers (MERRF), and others are due to point mutations in mtDNA.

In other diseases, defects in nuclear genes lead to dysfunction of mitochondrial proteins. This is the case in Friedreich's ataxia, hereditary spastic paraplegia, and Wilson's disease. These diseases are inherited in a dominance relationship, as applies to most other genetic diseases. A variety of disorders can be caused by nuclear mutations of oxidative phosphorylation enzymes, such as coenzyme Q10 deficiency and Barth syndrome. Environmental influences may also interact with hereditary predispositions and cause mitochondrial disease. For example, there may be a link between pesticide exposure and the later onset of Parkinson's disease.

Other diseases not directly linked to mitochondrial enzymes may feature dysfunction of mitochondria. These include schizophrenia, bipolar disorder, dementia, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, cardiovascular disease, retinitis pigmentosa, and diabetes mellitus. The common thread linking these seemingly-unrelated conditions is cellular damage causing oxidative stress and the accumulation of reactive oxygen species. These oxidants then damage the mitochondrial DNA, resulting in mitochondrial dysfunction and cell death.

Possible relationships to aging

Given the role of mitochondria as the cell's powerhouse, there may be some leakage of the high-energy electrons in the respiratory chain to form reactive oxygen species. This can result in significant oxidative stress in the mitochondria with high mutation rates of mitochondrial DNA. A vicious cycle is thought to occur, as oxidative stress leads to mitochondrial DNA mutations, which can lead to enzymatic abnormalities and further oxidative stress. A number of changes occur to mitochondria during the aging process. Tissues from elderly patients show a decrease in enzymatic activity of the proteins of the respiratory chain. Large deletions in the mitochondrial genome can lead to high levels of oxidative stress and neuronal death in Parkinson's disease. Hypothesized links between aging and oxidative stress are not new and were proposed over 50 years ago; however, there is much debate over whether mitochondrial changes are causes of aging or merely characteristics of aging. One notable study in mice demonstrated no increase in reactive oxygen species despite increasing mitochondrial DNA mutations, suggesting that the aging process is not due to oxidative stress. As a result, the exact relationships between mitochondria, oxidative stress, and aging have not yet been settled.

Anti-mitochondrial antibodies

2009-06-07T09:11:00.002-07:00

Anti-mitochondrial antibodies (AMA) are antibodies (immunoglobulins) formed against mitochondria, primarily mitochondria in cells of the liver. The presence of AMAs in the blood or serum of a person is indicative of several autoimmune diseases such as primary biliary cirrhosis (PBC) (a scarring of liver tissue, confined primarily to the bile duct drainage system of the liver). It is present in about 95% of cases.

Primary biliary cirrhosis is seen primarily in middle-aged women, and in those afflicted with other autoimmune diseases. PBC is an autoimmune disorder, a condition in which the human body's immune defense system mistakenly attacks the body's own cells, or in this case parts of the cells.

Cause of AMAs is postulated that xenobiotic-induced and/or oxidative modification of mitochondrial autoantigens is a critical step leading to loss of tolerance. In acute liver failure AMA are found against all major liver antigens.

pyruvate dehydrogenase, E2 subunits
2-oxo-glutarate dehydrogenase
branched chain 2-oxo-acid dehydrogenase

Anti-cardiolipin antibodies are another type of AMA, cardiolipin is found on the inner mitochondrial membrane.

Correlation with non-mitochodrial antigens

Fifty seven percent of acute liver failure patients had elevated anti-transglutaminase antibodies (anti-tTG), which correlate with gluten-sensitive enteropathy (see coeliac disease, Gluten-sensitive enteropathy associated conditions). The inflammation produced by gluten-sensitive cellular immunity may cause the oxidative stress resulting in the modification of mitochondria l antigens and acute liver failure. Anti-gp210 antibodies are also found in 47% of PBC patients.