WikiVet English - User contributions [en]

Innate Immune System

2008-09-15T11:38:42Z

Eayton: /* Phagocytosis */

{{toplink
|backcolour = FFE4E1
|linkpage =WikiBlood
|linktext =WIKIBLOOD
|thispagemap= Innate Immune System (Concept Map) - WikiBlood
|sublink1 =Immunology - WikiBlood
|subtext1 =IMMUNOLOGY
|pagetype =Blood
}}

=Introduction=

The Innate immune system is the body's first barrier of defense to infection. It relies on an older, more generic, and faster acting set of tools than the [[Adaptive Immune System - WikiBlood|adaptive]] system. While the adaptive system is essential for a specific response to infection, it is ultimately the innate system that conquers foreign attackers through means of phagocytosis.

* Non-specific protective mechanisms include such innate factors as:
** '''Physical barriers'''
*** Skin
*** Ciliated mucous membranes
*** Commensal organisms
** '''Humoral factors'''
*** Lysozyme
*** [[Complement - WikiBlood|Complement]]
*** Interferons
** '''Cellular mechanisms'''
*** Phagocytosis
** Factors which regulate '''species specificity'''
*** Membrane receptors for pathogens
*** Nutritional requirements
*** Temperature
*** pH
* Mechanisms of innate immunity are always present and generally unchanging
* Adaptive immunity is acquired only on contact with the infectious agent (antigen) and therefore does not function before first contact with the antigen

=Actions of the Innate Immune System=
==Recognition of Microorganisms==
[[Image:PRRs.jpg|thumb|right|150px|Pattern Recognition Receptors - B. Catchpole, RVC 2008]]
* The innate immune system recognises components of pathogens which are intrinsically foreign (i.e. not present on normal mammalian cells), such as:
**Lipopolysaccharides of gram-negative bacteria
**Peptidoglycans of gram-positive bacteria
**Mannose sugars
**D-isoform amino acids
*These are given away as foreign by expressing '''pathogen-associated molecular patterns''' (PAMPs)
* PAMPs are recognised by '''pattern recognition receptors''' (PRRs) expressed on mammalian cells
** Pattern recognition receptors are expressed on many different cell types, not just on phagocytes
** Not all are expressed by all cells: different cell types express a different range of PRRs
** PRRs are either intracellular, membrane-associated or soluble:
*** Recognition of pathogens via the cellular PRRs results in phagocytosis and inflammation
*** Recognition of pathogens via the humoral PRRs results in various killing mechanisms
* Engagement of PRRs by PAMPs triggers:
** '''Phagocytosis'''
** The expression of '''cytokines''', which brings about [[Inflammation - WikiBlood|inflammation]] and other immune responses

<big><center>'''''Examples of Pattern Recognition Receptors'''''</center></big>

{| style="width:60%; height:200px" border="1" align=center

!'''Receptor'''
!'''Location'''
!'''Ligands'''
|-
| TLR2 (''Toll-like receptor'')
| Cell Membrane
| Peptidoglycan of gram +ve bacteria
|-
| TLR3
| Cell Membrane
| dsRNA of RNA viruses (e.g. avian influenza)
|-
| TLR4
| Cell Membrane
| Lipoplysaccharide from gram-negative bacteria (e.g. E. coli, Salmonella)
|-
| TLR5
| Cell Membrane
| Bacterial flagellin
|-
| TLR9
| Cell Membrane
| Bacterial DNA (CpG DNA)
|-
| C-type lectins
| Soluble
| Carbohydrates, all bacteria, dead cells
|-
| fmlf
| Soluble
| Formyl peptides (i.e. all bacteria)
|-
| Complement receptors
| Soluble
| Fixed complement components (e.g. iC3b)
|-
| NOD2
| Cytoplasm
| Peptidoglycan of gram +ve bacteria
|-
| dsRNA-dependent Protein Kinase Receptor
| Cytoplasm
| ds RNA of RNA viruses
|}

==Phagocytosis==
* Phagocytosis is a very primitive system of defence against infection
** Even exists in invertebrates
* Phagocytosis is a form of endocytosis (cell eating), it is the method of removal of bacteria and dead cells by vesicular internalisation
** The internalised vesicle is referred to as the "phagosome"
** '''Lysosomes''', which contain a large range of enzymes, fuse with the phagosome, killing the microbes in an energy-dependent way
*** Oxygen-dependent degradation utilizes Oxygen and chlorine free-radicals, Hydrogen peroxide, and Nitric oxide
*** Oxygen-independed degradation depends on granules containing proteolytic enzymes such as Defensins, Lysozyme, and cationic proteins
**** In addition, these granules contain antimicrobial elements such as lactoferrin
** Microbes are then digested by a number of different catabolic enzymes
*** Glycosidases: Digest carbohydrates
*** Lipases: Digest lipids
*** Proteases: Digest protein
** Waste products of phagocytosis are either exocytosed or further degraded by the phagocyte
* '''Neutrophils''' and '''macrophages''' are phagocytic
* '''Opsonins''' promote and accelerate phagocytosis
* Phagocytic cells target pathogens by using cell membrane receptors (PRRs) that recognize intrinsically foreign components of microorganisms (pathogen-associated molecular patterns; PAMPs)
Video of phagocytosis of ''Candida albicans'': [http://www.cellsalive.com/qtmovs/mac_mov.htm]

=Tools of Innate Immunity=

==Barriers==
===Physical Barriers===
[[Image:Epithelial barriers.jpg|thumb|right|150px|Epithelial Barriers - B. Catchpole, RVC 2008]]

'''''Skin'''''

The simplest way to avoid infection is to prevent microorganisms gaining access to the body. The skin has an external coating of dead cells (cuticle) that, when intact, is impermeable to most infectious agents.
* Very few pathogens are capable of penetrating the thick stratified squamous epithelium of the skin (and lower urinary tract).
** Infection becomes a problem when there is:
*** Skin loss: e.g. burns
*** A break in the skin: e.g. wounds

'''''Mucus Membranes'''''
* Thin epithelial surfaces are necessary for the normal physiological functions of the bodies mucus membranes (ie absorption and gas exchange).They are therefore more susceptible to infection
** The body uses alternative protective mechanisms in these areas:
*** The '''mucociliary escalator''' of the respiratory tract (assisted by coughing and sneezing)
*** '''Peristalsis, vomiting & diarrhoea''' when necessary removes microorganisms from the GIT

===Biochemical Barriers===
* '''Lactic and fatty acids''' in sweat and sebaceous secretions are directly bacteriocidal
* '''Enzymes''' e.g. lysozyme in saliva, sweat & tears and Gastric acid denature microorganisms
* Mucus itself is acidic, indigestible and traps microorganisms

==='''Commensal Organisms'''===
* Out-compete pathogens at mucosal and epithelial surfaces and produce natural antibiotics
* When commensals are disturbed, infection with opportunistic organisms is increased
** E.g. [[Yeast-like fungi|''Candida'']] (thrush) or [[Clostridium species|''Clostridium difficile'']] (infectious diarrhoea)

==Humoral Factors==
===Lysozyme===
* Lysozyme is one of the major bactericidal agents in secretions
* Helps to protect vulnerable sites such as the eyes and nasal passages
* Exerts bactericidal effects by digesting bacterial cell walls
** Gram-positive bacteria are more sensitive to lysozyme action than gram-negative bacteria
** The outer membrane of gram-negative bacteria helps to protect them

===[[Complement - WikiBlood|Complement]]===
* The Complement system is a group of about 30 proteins within the body fluids of all vertebrates and some invertebrates
* Complement promotes '''phagocytosis''' or causes lysis of an invading organism
* Complement acts as a cascade, like the blood clotting system
** The early enzymes in the cascade are bound to invading bacteria and fungi
*** They have an affinity for components of microbial cell membranes
** This binding initiates a cascade so that the binding of one molecule will eventually lead to the fixation of millions of later molecules
* The early components act as targets for phagocytes
* The later components punch holes in bacteria, causing their lysis

===Interferons===
* Lysozyme and complement have only marginal effects on virus infections because these are intracellular
** The body has evolved non-specific mechanisms to protect against viruses
*** The most notable of these is the interferons
* Interferons are small polypeptides produced mainly by virus-infected cells
** Interact with uninfected cells and render them resistant to infection
*** This resistance is mainly due to the production of enzymes that digest viral nucleic acids

==Cellular responses==
[[Image:LH Macrophage Histology.jpg|thumb|right|125px|'''Macrophage'''© Nottingham Uni]]
* If pathogens breach the barriers formed by the skin and mucus membranes, they must be detected and destroyed by cellular and humoral means
* The cells involved with innate protection are:
** Blood granulocytes, or Polymorphonuclear Cells
*** Notable for their multi-lobed nuclei
*** '''Neutrophils''': phagocytose bacteria
*** '''Eosinophils''': kill parasites by the release of granules
*** '''Basophils/ mast cells''': kill parasites by the release of granules
** Blood '''monocytes''': phagocytose bacteria
** Tissue mast cells and '''macrophages''': phagocytose bacteria
*Effectively, innate cellular response seeks to hold off the infection until the [[Adaptive Immune System - WikiBlood|adaptive]] response can back it up with a more specific attack

===[[Macrophages - WikiBlood|Macrophages]]===
[[Image:Monocytes.jpg|thumb|right|150px|Monocytes - J. Bredl, RVC 2008]]
*The role of macrophages in Innate Immunity is to act as primary '''phagocytes'''
* Macrophages are present within tissues and take the form of distinct, tissue-specific populations:
** Alveolar macrophages
** Tissue histiocytes
** Glomerular macrophages
** Hepatic Küpffer cells
** CNS microglia
** Sinus-lining macrophages of the lymph nodes and spleen
* [[Monocytes - WikiBlood|'''Monocytes''']] (immature macrophages) are circulating phagocytes
** Circulate for 6-8 hours
** Can function as phagocytes within the blood and as newly migrated cells in tissues
** Chiefly function to replace the various tissue macrophage populations

===[[Neutrophils - WikiBlood|Neutrophils]]===
[[Image:Neutrophil 2.jpg|thumb|right|150px|Neutrophils - J. Bredl, RVC 2008]]
* Neutrophils are the principal, highly active '''phagocytes''' in the blood
** Comprise 30-70% of white blood cells depending on species
** Kill and digest microbes in a similar way as macrophages
* Neutrophils can also cause extracellular bacterial killing by disrupting bacterial membranes
** Secrete small antibacterial peptides
*** E.g. defensins and bactenecins
* Neutrophils produce vasoactive peptides
** E.g. histamine and bradykinin
** Cause a great increase in extravasation of blood granulocytes and monocytes and plasma proteins at the site of infection
* Neutrophils are the archetypal cell associated with [[Inflammation - WikiBlood|acute inflammation]]
** Are attracted to sites of inflammation by:
*** Complement activation
*** Cytokine production
*** Changes to vascular endothelium
** Neutrophil activation in an inflammatory lesion results in the release of '''prostaglandins'''
*** Responsible for vasoactive changes and for pain
* The accumulation of dead and dying neutrophils at the site of infection is called '''pus'''
** Their removal from the site after the removal of infection is an important step in the resolution of the lesion

===[[Eosinophils - WikiBlood|Eosinophils]]===
[[Image:Eosinophil.jpg|thumb|right|150px|Eosinophil - J. Bredl, RVC 2008]]
* Eosinophils are less common than neutrophils, and they are not phagocytic
** Make up <5% of the leukocytes in normal blood
* Eosinophil numbers are increased:
** Slightly during the resolution phase of inflammation
** Many-fold in parasite-infected animals
*** The presence of a large proportion of eosinophils in a blood smear is highly indicative of parasitaemia
* Mainly function by targeting the surface of parasites by means of specific antibody or complement
** Release a large range of toxic molecules that break down the parasite integument
* Prominent in [[Allergy - WikiBlood|allergic]] (anaphylactic) reactions

===[[Basophils - WikiBlood|Basophils]] / [[Mast Cells - WikiBlood|Mast Cells]]===
[[Image:Basophil and Lymphocyte.jpg|thumb|right|150px|Basophil - J. Bredl, RVC 2008]]
* Basophils/mast cells are principally localized at epithelial surfaces
** Very small numbers are present in blood
*** less than 0.5% circulating leukocytes
* They have two principal functions:
*# Induction of [[Inflammation - WikiBlood|acute inflammation]]
*#* Trauma and/ or bacterial infection causes the production of '''cytokines''' by the mast cells that induce a classical acute inflammatory response
*# Response to parasite infection
*#* Specific [[Immunoglobulins - WikiBlood|IgE]] binds cells
*#* Subsequent contact with antigen causes the mast cells to degranulate
*#* Release enzymes and vasoactive substances that can result in a high level of mucus secretion and smooth muscle contraction
* Also produce factors that influence local host cell physiology
** Various mediators increase the ratio of phagocyte to microbe

=Innate Immunity to Viruses=
[[Image:Innate viral response.jpg|thumb|right|150px|Innate response to dsRNA - B. Catchpole, RVC 2008]]
Because viruses invade host cells to take over a host's cellular machinery, the innate system has a more difficult time detecting viruses as foreign agents. However, there is a give-away element of the viral attack that the innate system can recognize: the '''double-stranded RNA''' (dsRNA) produced by a virus in its replication phase. Because mammalian cells only ever produce single-stranded RNA, the presence of dsRNA signals a forein intruder. dsRNA can be detected by TLR-3R on the cell surface or intracellularly by the presence of dsRNA-dependent protein kinase.

The innate resonse to viral attack also depends on the presence of '''Type-1 Interferons''', which are produced by all cells on recognition of a viral attack. Interferons serve to increase degradation of mRNA, inhibit protein synthesis, and increase the effectiveness of the adaptive response by increasing antigen presentation to antibody.

Lastly, the final line of defense for the innate response to viruses lies in the actions of [[Natural Killer Cells - WikiBlood|'''Natural Killer (NK) cells''']]. These warriors monitor the production of [[MHC - WikiBlood|MHC]] (Major Histocompatibility Complex) on the surface of cells, which is produced as part of the adaptive response. A cell whose cellular machinery is compromised by viral infection will experience a drop in the amount of MHC it produces. When a cell's MHC production drops, NK cells are triggered to phagocytose these cells. As such, this is a non-specific targeting based simply on the ability of a cell to function normally, which also lends them to playing a role in targeting malignant cells. NK cells are incapable of directly targeting viral infection.

=Innate Immunity to Bacteria=
[[Image:Bacterial innate response.jpg|thumb|right|150px|Bacterial responses - B. Catchpole, RVC 2008]]
The innate response to bacterial infection lies in its first-response role of detection of a foreign organism. By using the above described tools of Pattern-Recognition Receptors (PRRs), the innate response flags up problems while the [[Adaptive Immune System - WikiBlood|adaptive]] response gets itself organized. Once a foreign organism is detected, the innate system responds by engaging in cell warfare via phagocytosis and engaging the [[Inflammation - WikiBlood|inflammatory]] response. The release of inflammatory [[Cytokines - WikiBlood|cytokines]] will cause an increase in vasodilation, vascular permeability and an influx of white blood cells. Neutrophils take on their primary role as phagocytes in this phase. In addition, systemic effects of inflammatory cytokines will sustain a rise in core temperature (fever), the release of acute phase proteins from the [[Liver - Anatomy & Physiology|liver]], and bone marrow mobilization as the need for white blood cells production is increased. Acute phase proteins will bind to bacterial cell walls, enhancing neutrophil, macrophage, and [[Complement - WikiBlood|complement]]-initiated phagocytosis.

=[[Interplay of Innate and Adaptive Immunity - WikiBlood|Interplay of Innate and Adaptive Immunity]]=

=[[Innate Immunity Flashcards - WikiBlood|Innate Immunity Flashcards]]=

=Links=
'''Websites'''
*http://www.cellsalive.com

=References=

=Creators=
[[Rebecca Pocock]]

[[Asher Allison]]

[[Natalie Brown]] (flashcards)

<big><center>[[Immunology - WikiBlood|'''BACK TO IMMUNOLOGY''']]</center></big>

Useful Websites for Immunology

2008-09-15T11:35:49Z

Eayton:

{{toplink
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|linkpage =WikiBlood
|linktext =WIKIBLOOD
|sublink1 =Immunology - WikiBlood
|subtext1 =IMMUNOLOGY
|pagetype =Blood
}}

'''Websites'''

*http://www.blink.biz/immunoanimations/index1.html

*http://www.whfreeman.com/kuby/

*http://www.roitt.com/default.asp

*http://www.cellsalive.com

Immunological Testing Flashcards

2008-09-15T11:31:07Z

Eayton:

{{toplink
|backcolour = f5fffa
|linkpage =Immunological testing - WikiBlood
|linktext =IMMUNOLOGICAL TESTING
|sublink1 =Flash Cards - WikiBlood
|subtext1 =WIKIBLOOD FLASHCARDS
|pagetype =Blood
}}
 
==ELISA testing==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''What does the acronym ELISA stand for?'''
|| <big>
*'''''Enzyme Linked ImmunoSorbent Assay'''''
||[[ELISA testing|Link to Answer Article]]
|-
|<big>'''What type of ELISA requires all the reagents to be added at once?'''
|| <big>
*'''''Homogenous'''''
||[[ELISA testing|Link to Answer Article]]
|-
|<big>'''What are the five basic steps of an ELISA test?'''
|| <big>
*'''''Adsorption of antigen/antibody onto solid phase'''''
*'''''Addition of chosen sample and reagents'''''
*'''''Incubation and washing'''''
*'''''Addition of enzyme-labelled antigen/antibody'''''
*'''''Addition of specific substrate'''''
||[[ELISA testing|Link to Answer Article]]

|}
==Immunofluorescence==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''The adsorption of light by a molecule is known as what?'''
|| <big>
*'''''Excitation'''''
||[[Immunofluorescence|Link to Answer Article]]
|-
|<big>'''What are the fluorescent dyes commonly known as?'''
|| <big>
*'''''Fluorochromes'''''
||[[Immunofluorescence|Link to Answer Article]]
|-
|<big>'''What are the advantages of indirect staining?'''
|| <big>
*'''''Brighter fluorescence'''''
*'''''One preparation can be used to test many serum samples'''''
*'''''Relative expressions of different antigens in the same cell can be tested'''''
*'''''Loss of antibody during conjugation is limited'''''
||[[Immunofluorescence|Link to Answer Article]]
|-
|<big>'''What technique is used to provide a quantitative measure of immunofluorescence?'''
|| <big>
*'''''Flow cytometry'''''
||[[Immunofluorescence|Link to Answer Article]]
|}

==Radioimmunoassay==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''What are radioimmunoassays commonly used to detect?'''
|| <big>
*'''''Hormone levels in blood and tissue fluids'''''
*'''''Serum proteins'''''
*'''''Drugs'''''
*'''''Vitamins'''''
||[[Radioimmunoassay|Link to Answer Article]]
|-
|<big>'''What is the most common isotope used for radioimmunoassay?'''
|| <big>
*'''''Iodine-125'''''
||[[Radioimmunoassay|Link to Answer Article]]
|-
|<big>'''Why is it important to measure the amount of antibody that binds to 50% of the labelled antigen?'''
|| <big>
*'''''Ensures the number of epitopes present exceeds number of antibody-binding sites'''''
||[[Radioimmunoassay|Link to Answer Article]]
|-
|<big>'''What are the disadvantages of the radioimmunoassay?'''
|| <big>
*'''''Substances used are radioactive'''''
*'''''Gamma radiation requires special counting equipment'''''
*'''''Iodine naturally accumulates in the thyroid gland'''''
||[[Radioimmunoassay|Link to Answer Article]]

|}
==Agglutination==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''What are the antibodies that produce agglutination known as?'''
|| <big>
*'''''Agglutinins'''''
||[[Agglutination|Link to Answer Article]]
|-
|<big>'''What constitutes the end of the agglutination test?'''
|| <big>
*'''''The last tube showing distinct agglutination'''''
||[[Agglutination|Link to Answer Article]]
|-
|<big>'''What does the titre represent?'''
|| <big>
*'''''The number of antibodies per unit volume of serum'''''
||[[Agglutination|Link to Answer Article]]
|-
|<big>'''What is the prozone effect?'''
|| <big>
*'''''An inhibition of agglutination caused by an excess of antibodies'''''
||[[Agglutination|Link to Answer Article]]
|-
|<big>'''What agglutination test is often used to test for rhesus disease?'''
|| <big>
*'''''Coombs test'''''
||[[Agglutination|Link to Answer Article]]

Immunological Testing Flashcards

2008-09-15T11:04:09Z

Eayton: /* Immunofluorescence */

Immunofluorescence

2008-09-15T11:02:41Z

Eayton: /* Flow cytometry */

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|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
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==Introduction==
[[Image:763px-Cryptosporidium parvum 01.jpg|thumb|right|150px|Immunofluorescence of ''Cryptosporidium parvum'' spores]]
Immunofluorescence is a technique used to detect cell or tissue-associated antigens using antibodies labelled with fluorescent tags. The stained tissues are then detected by immunofluorescence microscopy (qualitative) or flow cytometry (quantitative). Antibodies bind stably and specifically to their corresponding antigen and the technique makes use of the fact that they can be coupled to fluorescent dyes, such as fluorescein and rhodamine, with no effect on specificity. These conjugates bind to antigens present in a sample and can then be visualised under a microscope with a suitable light source, such as UV light.

==Fluorescent dyes==

If a molecule has the property of fluorescence, it can absorb light of one wavelength (excititation) and emit light of another (emission). Antibodies tagged with these dyes (known as '''fluorochromes''') form immune complexes with specific antigens which can then be indirectly visualised when excited by light of the appropriate wavelength.

===Commonly used fluorochromes===
*'''Fluorescein:''' organic (carbon-based) dye, most widely used. Absorbs blue (490nm) and emits yellow green fluorescence (517nm)
*'''Rhodamine:''' organic dye, absorbs yellow-green light and emits deep red fluorescence (546nm).
**As fluorescein and rhodamine fluorescences are easy to distinguish from one another, it is possible to conjugate them to different antibodies and simultaneously visualise two different antigens on the same cell or tissue.
*'''Phycoerythrin:''' can absorb light from the blue-green (495nm) and the yellow wavelengths, emits bright red fluorescence

==Techniques==
'''Direct staining'''
*An antibody directed against a specific antigen is directly conjugated with the fluorescent dye and applied to the sample.
'''Indirect staining'''
*Utilizes a double layer technique- a primary, unlabelled antibody is applied to the sample, followed by a secondary antibody, an anti-immunoglobulin that has been conjugated to a fluorochrome.
**Indirect staining has several advantages:
***Several secondary antibodies bind to each primary antibody, so the resulting fluorescence is brighter than that of the direct staining.
***One preparation of secondary antibody can be used to test many sera
***By using a mixture of primary antibodies, it is possible to detect the relative expressions of different antigens in the same cell
***Quite often loss of antibody is sustained during the conjugation- in the indirect method the primary antibodies do not need to be conjugated, so this limiting factor is reduced.

==Applications==
*Immunofluorescence is often used to identify populations of cells, and was successfully employed in the identification of the CD4+ and CD8+ subpopulations of T cells
*Identifying bacterial and viral species
*Detecting antigen-antibody complexes in autoimmune diseases
*Detecting complement components in tissues
*Localising hormones, antigens and other cellular products in tissue samples and subcellular compartments
*Mapping the molecular architecture of tissues in relation to gross anatomy

==Developments==
===Confocal microscopy===
*The scanning confocal fluorescent microscope is a recent development that uses computer-aided techniques to produce a high-resolution thin optical section of a sample without the need for further, complex sample preparation. Fluorescent images were previously hard to resolve as the picture was subject to glare from planes above and below that in focus, resulting in a blurred image.
*To obtain an image, a laser light is focused on a fine plane within the sample. The resulting fluorescent emission is collected in a photomultiplier tube (PMT) with a confocal aperture. The fluorescence from planes above and below the optical section fails to reach this aperture, resulting in a sharper image.
*The resolution can be further increased by using low-level illumination, meaning the fluorescent dye can only be excited by two photons. When a pulsed laser is used, it only reaches sufficient intensity to excite fluorescence when the beam is focused onto the focal plane of the microscope. This minimises fluorescence to the desired optical section itself.

===Time-lapse video microscopy===
*Sensitive digital video cameras can be used to record the movement of fluorescently tagged molecules, for example to track the movement of such molecules in cell membranes or during interaction between cells.

===Flow cytometry===
*The flow cytometer is an instrument that directs cells in single file through a narrow chamber illuminated by a laser beam. As fluorescently-tagged cells pass through the laser beam, the fluorochrome is excited and emits light that is detected by a PMT-like detector
*This detector is capable of measuring the strength of emission, so it is possible to sort cells that are negative, slightly positive and highly positive for a specific antigen
*Flow cytometry provides a rapid quantitative technique for antigen detection

Immunological Testing Flashcards

2008-09-15T10:59:55Z

Eayton:

Immunological Testing Flashcards

2008-09-15T10:23:51Z

Eayton:

ELISA testing

2008-09-12T14:00:59Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
}}

==Introduction==
[[Image:ELISA.jpg|thumb|right|150px|Double Antibody Sandwich ELISA]]
The Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay commonly used to detect the presence of an antigen or antibody in a sample. It is a powerful tool in clinical immunology and can be used to determine whether an individual has been exposed to a specified pathogen. Utilizing the principle of antigen-antibody interaction, the test allows easy visualisation of results and, since its introduction in 1971, has quickly replaced radioimmunoassays for diagnostic purposes.

*There are two basic types of ELISA:
**'''Homogenous'''- completed in one step, all reagents added simultaneously. Primarily used to detect small molecules such as digoxin and gentamicin
**'''Heterogenous'''- various reagents added sequentially, primarily used to detect microbial antigens and antibodies

This article describes the '''heterogenous''' type

==Applications==
*Detection and identification of disease agents, e.g. type and subtype
*Identification of specific antibodies, e.g. used in serodiagnosis for epidemiological studies
*Quantification of specific antibody isotypes, e.g. IgM/IgG ratio

==Techniques==

There are many methods of ELISA, but each assay involves these basic steps:
*The adsorption of antibody/antigen to solid phase (the medium)
*The addition of the chosen sample and reagents
*Incubation and washing
*The addition of enzyme-labeled antigen/antibody
*The addition of a specific substrate

'''Competitive VS Non-competitive'''
*As their name implies, competitive assays measure the competition between a pre-titrated (fixed amount) of labeled antigen and an unknown quantity of sample antigen in their affinity to an antibody. The process can be reversed to measure the competition between labeled and unlabeled antibody.
*Competitive techniques:
**Easier to quantify
**Less likely to be influenced by contaminants
**However they are more demanding with regard to the accuracy of the reagents and the purity of the labeled ligand
*Non-competitive assays:
**Errors in dispensing reagents have little effect on the result
**Therefore they are easier to control and yield accurate results
**However they are easily influenced by cross reactions and non-specific binding

'''Solid VS Fluid-phase'''
[[Image:800px-Microtiter plate.jpg|thumb|right|150px|96-well microtiter plate, Copyright Jeffrey M. Vinocur 2006]]

An important consideration is the medium in which the assay is carried out, largely dependent on what is being tested:
*Fluid-phase (''in solution'')
**Main advantage is that the behaviour of molecules in solution is easier to predict
*Solid-phase (''surface of protein-binding material, e.g. plastic'')
**Easier to perform and more sensitive
**The most widely used solid-phase is the 96-well microtiter plater, manufactured as PVC flexible plates or polystyrene rigid plates

----

===Non-competitive ELISA===
[[Image:Sandwich elisa.png|thumb|right|100px|The sandwich ELISA - Copyright J M Vincour]]
'''Double Antibody Sandwich''' (for antigen detection)
# Antibody is adsorbed onto solid phase
# Wash
# Sample serum is added- specific antigen binds to antibody
# Wash
# Enzyme-labeled specific antibody is added- attaches to bound antigen
# Wash
# Enzyme substrate is added (with dye for visualisation)
''visualised product = amount of antigen in the serum''

'''Antibody Class Capture Assay''' (for antibody detection)
# Class-specific antiglobulin is adsorbed onto solid phase
# Wash
# Sample serum is added- class-specific antibody in the serum binds to antiglobulin
# Wash
# Antigen is added- attaches to specific antibody
# Wash
# Enzyme-labeled antibody is added
# Wash
# Enzyme substrate is added
''visualised product = amount of specific antibody in serum''

'''Indirect Method''' (for antibody detection)
# Specific antigen (i.e. not from sample) is adsorbed onto solid phase
# Wash
# Serum is added- any specific antibody present in sample binds to antigen
# Wash
# Enzyme-labeled antiglobulin is added- attaches to antibody
# Wash
# Enzyme substrate is added
''visualised product = amount of antibody in the serum''

===Competitive ELISA===
'''Direct Antibody Competition''' (for antibody detection)
# Antigen is adsorbed onto solid phase
# Wash
# Enzyme-labeled antibody (pre-titrated- optimal colour development) and serum are added
# Wash
# Enzyme substrate is added
''visualised product = amount of antigen in serum sample''

'''Direct Antigen Competition''' (for antigen detection)
# Antigen is adsorbed onto solid phase
# Wash
# Antigen in serum is incubated with enzyme-labelled antibody (again pre-titrated): this is directed against the antigen on the solid phase
# Wash
# Enzyme substrate is added
''visualised product = amount of enzyme-labelled antigen''

Immunological Testing Flashcards

2008-09-12T13:57:32Z

Eayton:

Immunological Testing Flashcards

2008-09-12T13:56:33Z

Eayton: New page: {{toplink |backcolour = f5fffa |linkpage =Immunological testing - WikiBlood |linktext =IMMUNOLOGICAL TESTING |sublink1 =Flash Cards - WikiBlood |subtext1 =WIKIBLOOD FLASHCARDS |pagetype =B...

Cytokines Flashcards

2008-09-12T13:47:03Z

Eayton: New page: {{toplink |backcolour = f5fffa |linkpage =Cytokines - WikiBlood |linktext =CYTOKINES |sublink1 =Flash Cards - WikiBlood |subtext1 =WIKIBLOOD FLASHCARDS |pagetype =Blood }} ==<font col...

{{toplink
|backcolour = f5fffa
|linkpage =Cytokines - WikiBlood
|linktext =CYTOKINES
|sublink1 =Flash Cards - WikiBlood
|subtext1 =WIKIBLOOD FLASHCARDS
|pagetype =Blood
}}
 
==Cytokines==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''Cytokines produced by myeloid cells are commonly known as what?'''
|| <big>
*'''''Monokines'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''Cytokines responsible for directing cell migration are commonly known as what?'''
|| <big>
*'''''Chemokines'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''What type of cytokines are produced by cells in response to viral infection?'''
|| <big>
*'''''Interferons'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''IL-12, produced primarily by macrophages, stimulates what subtype of helper T cells?'''
|| <big>
*'''''Th1'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''What cytokine stimulates local inflammation and endothelial activation?'''
|| <big>
*'''''TNF-α'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''What cytokine activates macrophages and increases MHC expression?'''
|| <big>
*'''''IFN-γ'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''What cytokine, produced primarily by Th2 cells, activates B cells and the IgE switch?'''
|| <big>
*'''''IL-4'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''What two groups can the chemokines be classed as, based on the cysteine residue position?'''
|| <big>
*'''''C-C'''''
*'''''C-X-C'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''What are the main actions of the chemokines?'''
|| <big>
*'''''Lymphoid trafficking'''''
*'''''Wound healing'''''
*'''''Th1/Th2 development'''''
*'''''Angiogenesis'''''
*'''''Lymphoid organ development'''''
*'''''Inflammation'''''
*'''''Cell recruitment'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''What is the main cause of bacterial septic shock?'''
|| <big>
*'''''Bacterial cell wall endotoxins'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''What is the main cause of bacterial toxic shock?'''
|| <big>
*'''''Bacterial superantigens'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|-
|<big>'''The symptoms of bacterial septic and toxic shock are caused mainly by the release of which cytokines?'''
|| <big>
*'''''TNF'''''
*'''''IL-1'''''
||[[Cytokines - WikiBlood|Link to Answer Article]]
|}

 
 
<big><center>[[Cytokines - WikiBlood|'''BACK TO CYTOKINES''']]</center></big>
<big><center>[[Flash Cards - WikiBlood|'''BACK TO WIKIBLOOD FLASHCARDS''']]</center></big>

Cytokines

2008-09-12T13:35:02Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =WikiBlood
|linktext =WIKIBLOOD
|thispagemap = Cytokines (Concept Map) - WikiBlood
|sublink1 =Immunology - WikiBlood
|subtext1 =IMMUNOLOGY
|pagetype =Blood
}}

==Introduction==
The term cytokine is a generic name for the soluble molecules that mediate reactions between cells, acting via specific receptors on those cells. They are particularly important during effector stages of the immune system and the development of haematopoietic cells

==Nomenclature and classification==
Nomenclature of the cytokines was first based on their activity both ''in vivo'' and ''in vitro'', with the name being abbreviated to acronyms. For example, when a factor isolated from antigen-activated lymphocytes was added to non-immune peritoneal macrophages, their migration from capillary tubes was inhibited. This led to the factor being named migration inhibition factor, or MIF for short. It was then discovered that many biological functions were being produced by the same chemicals, which led to the classification of cytokines based on either the cell populations that secrete them or their function:
*'''Monokines'''- produced by myeloid cells (macrophages, monocytes)
*'''Lymphokines'''- produced primarily by lymphocytes
*'''Interleukins'''- produced by leukocytes in general
*'''Chemokines'''- directing cell migration, activating cells in response to infectious agents/tissue damage
*'''Interferons'''- produced by many different cells in response to viral infection

Common cytokines and nomenclature
{| border="1" cellpadding="2"
!width="50"|Name
!width="225"|Abbreviation
!width="225"|Examples
|-
|interleukins || IL || IL-1, IL-2
|-
|interferons || IFN || IFN-alpha
|-
|tumour necrosis factors || TNF || TNF-alpha
|-
|growth factors || GF || NGF, EGF
|-
|colony stimulating factors || CSF || M-CSF, G-CSF
|-
|chemokines || - || RANTES, MCP-1
|}
==Functions of cytokines==
'''Mediating and regulating innate immunity''': bacterial and viral products, such as LPS, stimulate macrophages and natural killer cells to secrete cytokines that primarily act on endothelial cells and leukocytes. They stimulate the early stages of the inflammatory reaction to microbes.

'''Mediating and regulating adaptive immunity''': in response to specific recognition by T lymphocytes, cytokines are produced that have a wide range of functions, including:
*regulating the growth and differentiation of some lymphocyte populations
*recruitment, activation and regulation of specialised effector cells, e.g. mononuclear phagocytes, neutrophils, eosinophils
'''Stimulating haematopoiesis''': produced primarily by bone marrow stromal cells and leukocytes, these cytokines stimulate the growth and differentiation of immature leukocytes
By binding onto specific receptors on cell membranes, cytokines are able to exert their actions by triggering signal-cascade mechanisms that ultimately result in altered gene expression. Cytokines have an incredibly high affnity for their receptors, and are therefore able to act at picomolar concentrations. Cytokines can exert antagonistic effects on each other.

==Important cytokines==
Cytokines primarily produced by '''macrophages''':
*GM-CSF (granulocyte macrophage colony stimulating factor)- stimulates growth and differentiation of granulocytes, macrophages, neutrophils and eosinophils
*IL-1- stimulates Th2 cells and acute phase response
*IL-6- stimulates growth and differentiation of B and T cells and acute phase response
*IL-12- stimulates Th1 cells
*IL-18- stimulates IFN-gamma production by T cells and NK cells, favours Th1 response
*TNF-α- stimulates local inflammation and endothelial activation
Cytokines primarily produced by '''Th1 cells''':
*IL-2- stimulates proliferation and differentiation of T cells, activates NK cells and macrophages
*IFN-γ- activates macrophages, increases expression of MHC I and II molecules, increases antigen presentation
*TNF-β- stimlulates killing mechanisms in T and B cells and endothelial activation
Cytokines primarily produced by '''Th2 cells''':
*IL-4- activates B cells and IgE switch, supresses Th1 cells
*IL-5- stimulates eosinophil growth and differentiation
*IL-10- suppresses macrophage functions
Although neutrophils produce a lower amount of cytokines per cell than other immune cell types, they are often the first and most common cell type present at sites of infection. This makes them a physiologically important source of cytokines, such as IL-12.
==Chemokines==
The chemokines are a superfamily of cytokines, all related in terms of sequence and gene structure. The family is also known as the 'small cytokine' family (scy) or the intercrines. All have a relatively small molecular weight of ~5-10kDa and can be divided in one of two groups based on the position of the cystein residues (important for the tertiary structure):
*C-C subgroup- cysteine residues are adjacent to each other. Important members include:
**Interleukin 8
**MGSA- melanoma growth stimulatory activity
**PF4- platelet factor 4
**βTG- β-thromboglobulin
*C-X-C subgroup- residues are separated by another amino acid. Important members include:
**MCAF- macrophage chemotactic and activating factor
**RANTES
**LD-8
**ACT-2
Chemokines are released by many cell types, and are present in the earliest phase of infection, with actions including the following:
*Lymphoid trafficking
*Wound healing
*Th1/Th2 development
*Angiogenesis/angiostasis
*Lymphoid organ development
*Inflammation
*Cell recruitment
==Cytokines in pathology==
===Bacterial septic shock===
This is the overproduction of cytokines developing a few hours after infection by certain Gram-negative bacteria, including:
*''E. coli''
*''K. pneumoniae''
*''P. aeruginosa''
*''E. aerogenes''
Bacterial cell wall endotoxins are the cause of septic shock, stimulating macrophages to release IL-1 and TNF-α at excessive levels. The condition is often fatal and symptoms include a sudden drop in blood pressure, fever, diarrhoea and blood-clotting in multiple organs.
===Bacterial toxic shock===
This condition is caused by bacterial toxins known as superantigens (antigens that bind simultaneously to MHC II and the beta-V domain of the T cell receptor) that activate large numbers of T cells despite specificity. A number of bacteria have been implicated in the production of superantigens, including:
*''S. aureus''- produces enterotoxins and toxic-shock syndrome toxin
*''M. arthritidis''
The large number of T cells activated by such toxins (between 5-25% of all T cells, compared to less than 0.01% activated towards conventional antigens) means an excessive amount of cytokines produced, such as IL-1 and TNF. These elevated amounts cause the same systemic reactions as seen in bacterial septic shock.
===Lymphoid and myeloid cancers===
The excessive production of cytokines has been linked to some types of cancer, e.g. IL-6 has been shown to be secreted by myeloma cells, plasmacytoma cells and cervical and bladder cancer cells. IL-6 is known to act in an autocrine manner to stimulate cell proliferation.

==[[Cytokines flashcards- Wikiblood|Cytokines Flashcards]]==

==Creators==

[[Edward Ayton]]

Cytokines

2008-09-12T13:17:59Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =WikiBlood
|linktext =WIKIBLOOD
|thispagemap = Cytokines (Concept Map) - WikiBlood
|sublink1 =Immunology - WikiBlood
|subtext1 =IMMUNOLOGY
|pagetype =Blood
}}

==Introduction==
The term cytokine is a generic name for the soluble molecules that mediate reactions between cells, acting via specific receptors on those cells. They are particularly important during effector stages of the immune system and the development of haematopoietic cells

==Nomenclature and classification==
Nomenclature of the cytokines was first based on their activity both ''in vivo'' and ''in vitro'', with the name being abbreviated to acronyms. For example, when a factor isolated from antigen-activated lymphocytes was added to non-immune peritoneal macrophages, their migration from capillary tubes was inhibited. This led to the factor being named migration inhibition factor, or MIF for short. It was then discovered that many biological functions were being produced by the same chemicals, which led to the classification of cytokines based on either the cell populations that secrete them or their function:
*'''Monokines'''- produced by myeloid cells (macrophages, monocytes)
*'''Lymphokines'''- produced primarily by lymphocytes
*'''Interleukins'''- produced by leukocytes in general
*'''Chemokines'''- directing cell migration, activating cells in response to infectious agents/tissue damage
*'''Interferons'''- produced by many different cells in response to viral infection

Common cytokines and nomenclature
{| border="1" cellpadding="2"
!width="50"|Name
!width="225"|Abbreviation
!width="225"|Examples
|-
|interleukins || IL || IL-1, IL-2
|-
|interferons || IFN || IFN-alpha
|-
|tumour necrosis factors || TNF || TNF-alpha
|-
|growth factors || GF || NGF, EGF
|-
|colony stimulating factors || CSF || M-CSF, G-CSF
|-
|chemokines || - || RANTES, MCP-1
|}
==Functions of cytokines==
'''Mediating and regulating innate immunity''': bacterial and viral products, such as LPS, stimulate macrophages and natural killer cells to secrete cytokines that primarily act on endothelial cells and leukocytes. They stimulate the early stages of the inflammatory reaction to microbes.

'''Mediating and regulating adaptive immunity''': in response to specific recognition by T lymphocytes, cytokines are produced that have a wide range of functions, including:
*regulating the growth and differentiation of some lymphocyte populations
*recruitment, activation and regulation of specialised effector cells, e.g. mononuclear phagocytes, neutrophils, eosinophils
'''Stimulating haematopoiesis''': produced primarily by bone marrow stromal cells and leukocytes, these cytokines stimulate the growth and differentiation of immature leukocytes
By binding onto specific receptors on cell membranes, cytokines are able to exert their actions by triggering signal-cascade mechanisms that ultimately result in altered gene expression. Cytokines have an incredibly high affnity for their receptors, and are therefore able to act at picomolar concentrations
Cytokines can exert antagonistic effects on each other

==Important cytokines==
Cytokines primarily produced by '''macrophages''':
*GM-CSF (granulocyte macrophage colony stimulating factor)- stimulates growth and differentiation of granulocytes, macrophages, neutrophils and eosinophils
*IL-1- stimulates Th2 cells and acute phase response
*IL-6- stimulates growth and differentiation of B and T cells and acute phase response
*IL-12- stimulates Th1 cells
*IL-18- stimulates IFN-gamma production by T cells and NK cells, favours Th1 response
*TNF-α- stimulates local inflammation and endothelial activation
Cytokines primarily produced by '''Th1 cells''':
*IL-2- stimulates proliferation and differentiation of T cells, activates NK cells and macrophages
*IFN-γ- activates macrophages, increases expression of MHC I and II molecules, increases antigen presentation
*TNF-β- stimlulates killing mechanisms in T and B cells and endothelial activation
Cytokines primarily produced by '''Th2 cells''':
*IL-4- activates B cells and IgE switch, supresses Th1 cells
*IL-5- stimulates eosinophil growth and differentiation
*IL-10- suppresses macrophage functions
Although neutrophils produce a lower amount of cytokines per cell than other immune cell types, they are often the first and most common cell type present at sites of infection. This makes them a physiologically important source of cytokines, such as IL-12.
==Chemokines==
The chemokines are a superfamily of cytokines, all related in terms of sequence and gene structure. The family is also known as the 'small cytokine' family (scy) or the intercrines. All have a relatively small molecular weight of ~5-10kDa and can be divided in one of two groups based on the position of the cystein residues (important for the tertiary structure):
*C-C subgroup- cysteine residues are adjacent to each other. Important members include:
**Interleukin 8
**MGSA- melanoma growth stimulatory activity
**PF4- platelet factor 4
**βTG- β-thromboglobulin
*C-X-C subgroup- residues are separated by another amino acid. Important members include:
**MCAF- macrophage chemotactic and activating factor
**RANTES
**LD-8
**ACT-2
Chemokines are released by many cell types, and are present in the earliest phase of infection, with actions including the following:
*Lymphoid trafficking
*Wound healing
*Th1/Th2 development
*Angiogenesis/angiostasis
*Lymphoid organ development
*Inflammation
*Cell recruitment
==Cytokines in pathology==
===Bacterial septic shock===
This is the overproduction of cytokines developing a few hours after infection by certain Gram-negative bacteria, including:
*''E. coli''
*''K. pneumoniae''
*''P. aeruginosa''
*''E. aerogenes''
Bacterial cell wall endotoxins are the cause of septic shock, stimulating macrophages to release IL-1 and TNF-α at excessive levels. The condition is often fatal and symptoms include a sudden drop in blood pressure, fever, diarrhoea and blood-clotting in multiple organs.
===Bacterial toxic shock===
This condition is caused by bacterial toxins known as superantigens (antigens that bind simultaneously to MHC II and the beta-V domain of the T cell receptor) that activate large numbers of T cells despite specificity. A number of bacteria have been implicated in the production of superantigens, including:
*''S. aureus''- produces enterotoxins and toxic-shock syndrome toxin
*''M. arthritidis''
The large number of T cells activated by such toxins (between 5-25% of all T cells, compared to less than 0.01% activated towards conventional antigens) means an excessive amount of cytokines produced, such as IL-1 and TNF. These elevated amounts cause the same systemic reactions as seen in bacterial septic shock.
===Lymphoid and myeloid cancers===
The excessive production of cytokines has been linked to some types of cancer, e.g. IL-6 has been shown to be secreted by myeloma cells, plasmacytoma cells and cervical and bladder cancer cells. IL-6 is known to act in an autocrine manner to stimulate cell proliferation.

==[[Cytokines flashcards- Wikiblood|Cytokines Flashcards]]==

==Creators==

[[Edward Ayton]]

Cytokines

2008-09-12T13:17:24Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =WikiBlood
|linktext =WIKIBLOOD
|thispagemap = Cytokines (Concept Map) - WikiBlood
|sublink1 =Immunology - WikiBlood
|subtext1 =IMMUNOLOGY
|pagetype =Blood
}}

==Introduction==
The term cytokine is a generic name for the soluble molecules that mediate reactions between cells, acting via specific receptors on those cells. They are particularly important during effector stages of the immune system and the development of haematopoietic cells

==Nomenclature and classification==
Nomenclature of the cytokines was first based on their activity both ''in vivo'' and ''in vitro'', with the name being abbreviated to acronyms. For example, when a factor isolated from antigen-activated lymphocytes was added to non-immune peritoneal macrophages, their migration from capillary tubes was inhibited. This led to the factor being named migration inhibition factor, or MIF for short. It was then discovered that many biological functions were being produced by the same chemicals, which led to the classification of cytokines based on either the cell populations that secrete them or their function:
*'''Monokines'''- produced by myeloid cells (macrophages, monocytes)
*'''Lymphokines'''- produced primarily by lymphocytes
*'''Interleukins'''- produced by leukocytes in general
*'''Chemokines'''- directing cell migration, activating cells in response to infectious agents/tissue damage
*'''Interferons'''- produced by many different cells in response to viral infection

Common cytokines and nomenclature
{| border="1" cellpadding="2"
!width="50"|Name
!width="225"|Abbreviation
!width="225"|Examples
|-
|interleukins || IL || IL-1, IL-2
|-
|interferons || IFN || IFN-alpha
|-
|tumour necrosis factors || TNF || TNF-alpha
|-
|growth factors || GF || NGF, EGF
|-
|colony stimulating factors || CSF || M-CSF, G-CSF
|-
|chemokines || - || RANTES, MCP-1
|}
==Functions of cytokines==
'''Mediating and regulating innate immunity''': bacterial and viral products, such as LPS, stimulate macrophages and natural killer cells to secrete cytokines that primarily act on endothelial cells and leukocytes. They stimulate the early stages of the inflammatory reaction to microbes.

'''Mediating and regulating adaptive immunity''': in response to specific recognition by T lymphocytes, cytokines are produced that have a wide range of functions, including:
*regulating the growth and differentiation of some lymphocyte populations
*recruitment, activation and regulation of specialised effector cells, e.g. mononuclear phagocytes, neutrophils, eosinophils
'''Stimulating haematopoiesis''': produced primarily by bone marrow stromal cells and leukocytes, these cytokines stimulate the growth and differentiation of immature leukocytes
By binding onto specific receptors on cell membranes, cytokines are able to exert their actions by triggering signal-cascade mechanisms that ultimately result in altered gene expression. Cytokines have an incredibly high affnity for their receptors, and are therefore able to act at picomolar concentrations
Cytokines can exert antagonistic effects on each other

==Important cytokines==
Cytokines primarily produced by '''macrophages''':
*GM-CSF (granulocyte macrophage colony stimulating factor)- stimulates growth and differentiation of granulocytes, macrophages, neutrophils and eosinophils
*IL-1- stimulates Th2 cells and acute phase response
*IL-6- stimulates growth and differentiation of B and T cells and acute phase response
*IL-12- stimulates Th1 cells
*IL-18- stimulates IFN-gamma production by T cells and NK cells, favours Th1 response
*TNF-α- stimulates local inflammation and endothelial activation
Cytokines primarily produced by '''Th1 cells''':
*IL-2- stimulates proliferation and differentiation of T cells, activates NK cells and macrophages
*IFN-γ- activates macrophages, increases expression of MHC I and II molecules, increases antigen presentation
*TNF-β- stimlulates killing mechanisms in T and B cells and endothelial activation
Cytokines primarily produced by '''Th2 cells''':
*IL-4- activates B cells and IgE switch, supresses Th1 cells
*IL-5- stimulates eosinophil growth and differentiation
*IL-10- suppresses macrophage functions
Although neutrophils produce a lower amount of cytokines per cell than other immune cell types, they are often the first and most common cell type present at sites of infection. This makes them a physiologically important source of cytokines, such as IL-12.
==Chemokines==
The chemokines are a superfamily of cytokines, all related in terms of sequence and gene structure. The family is also known as the 'small cytokine' family (scy) or the intercrines. All have a relatively small molecular weight of ~5-10kDa and can be divided in one of two groups based on the position of the cystein residues (important for the tertiary structure):
*C-C subgroup- cysteine residues are adjacent to each other. Important members include:
**Interleukin 8
**MGSA- melanoma growth stimulatory activity
**PF4- platelet factor 4
**βTG- β-thromboglobulin
*C-X-C subgroup- residues are separated by another amino acid. Important members include:
**MCAF- macrophage chemotactic and activating factor
**RANTES
**LD-8
**ACT-2
Chemokines are released by many cell types, and are present in the earliest phase of infection, with actions including the following:
*Lymphoid trafficking
*Wound healing
*Th1/Th2 development
*Angiogenesis/angiostasis
*Lymphoid organ development
*Inflammation
*Cell recruitment
==Cytokines in pathology==
===Bacterial septic shock===
This is the overproduction of cytokines developing a few hours after infection by certain Gram-negative bacteria, including:
*''E. coli''
*''K. pneumoniae''
*''P. aeruginosa''
*''E. aerogenes''
Bacterial cell wall endotoxins are the cause of septic shock, stimulating macrophages to release IL-1 and TNF-α at excessive levels. The condition is often fatal and symptoms include a sudden drop in blood pressure, fever, diarrhoea and blood-clotting in multiple organs.
===Bacterial toxic shock===
This condition is caused by bacterial toxins known as superantigens (antigens that bind simultaneously to MHC II and the beta-V domain of the T cell receptor) that activate large numbers of T cells despite specificity. A number of bacteria have been implicated in the production of superantigens, including:
*''S. aureus''- produces enterotoxins and toxic-shock syndrome toxin
*''M. arthritidis''
The large number of T cells activated by such toxins (between 5-25% of all T cells, compared to less than 0.01% activated towards conventional antigens) means an excessive amount of cytokines produced, such as IL-1 and TNF. These elevated amounts cause the same systemic reactions as seen in bacterial septic shock.
===Lymphoid and myeloid cancers===
The excessive production of cytokines has been linked to some types of cancer, e.g. IL-6 has been shown to be secreted by myeloma cells, plasmacytoma cells and cervical and bladder cancer cells. IL-6 is known to act in an autocrine manner to stimulate cell proliferation.

==[[Host invasion by microorganisms flashcards- Wikiblood|Host Invasion Flashcards]]==

==Creators==

[[Edward Ayton]]

Western blot

2008-09-12T13:15:57Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
}}

Also known as immunoblotting, the Western blot technique is used to identify specific proteins or antibodies in complex mixtures and has largely overtaken the use immunoelectrophoresis in research and diagnosis.

===Principle===
In the Western blot technique, a protein mixture is electrophoretically separated onto a denaturing gel, separating the proteins according to molecular weight. The bands can then be identified by applying enzyme-/radio-labeled antibodies to the mixture and the resulting complexes visualised either by ELISA technique or autoradiography.

===Method===
# Protein mixture is treated with sodium dodecyl sulfate (SDS), a strong dissociating agent
# Mixture is separated by electrophoresis- takes place in SDS polyacrylamide gel (SDS-PAGE)
# The gel is removed and applied to a protein-binding sheet of nitrocellulose/nylon and an electric current passed through it
# The antigens of interest are detected using enzyme-linked antibodies
# An ELISA reaction is used to detect the position of the antibodies

===Applications===
*'''HIV testing'''- this is the most widely used application of this test, used to determine whether a patient is producing antibodies specific to viral proteins present during an HIV infection

Western blot

2008-09-12T13:02:54Z

Eayton: New page: Also known as immunoblotting, the Western blot technique is used to identify specific proteins or antibodies in complex mixtures and has largely overtaken the use immunoelectrophoresis in ...

Also known as immunoblotting, the Western blot technique is used to identify specific proteins or antibodies in complex mixtures and has largely overtaken the use immunoelectrophoresis in research and diagnosis.

===Principle===
In the Western blot technique, a protein mixture is electrophoretically separated onto a denaturing gel, separating the proteins according to molecular weight. The bands can then be identified by applying enzyme-/radio-labeled antibodies to the mixture and the resulting complexes visualised either by ELISA technique or autoradiography.

===Method===
# Protein mixture is treated with sodium dodecyl sulfate (SDS), a strong dissociating agent
# Mixture is separated by electrophoresis- takes place in SDS polyacrylamide gel (SDS-PAGE)
# The gel is removed and applied to a protein-binding sheet of nitrocellulose/nylon and an electric current passed through it
# The antigens of interest are detected using enzyme-linked antibodies
# An ELISA reaction is used to detect the position of the antibodies

===Applications===
*'''HIV testing'''- this is the most widely used application of this test, used to determine whether a patient is producing antibodies specific to viral proteins present during an HIV infection

Complement Fixation

2008-09-12T12:40:41Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
}}

Complement fixation is a test that exploits the fact that antibody-antigen complexes are able
to activate the complement system, using the mechanism to show the presence of a specific antibody in a serum sample.

===Method===
A mixture of antibody, complement and antigen is incubated and an indicator system (normally antibody-coated sheep red blood cells) is added.
*As the complement reaction with the immune complex produces no visible result, sheep red blood cells are coated with anti-sheep red blood cell antibody. The complement in the mixture will react with this antibody and lyse the cells.
An indication of a positive test is the absence of lysis of the red blood cells, as the complement has already been used up by the antigen-antibody system. Controls are included to ensure that none of the reagents have taken up the complement non-specifically (e.g. contaminated serum)

===Applications===
*'''Wasserman reaction'''- used in the diagnosis of syphilis, the test consists of a mixture of Wasserman antigen, dilutions of the patients serum and complement (normally sourced from guinea pigs).
*Virus detection- tissue samples are innoculated with blood or tissue samples from a patient and tested using complement fixation

Immunodiffusion

2008-09-12T12:40:22Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
}}

Immunodiffusion reactions are diagnostic tests that use the diffusion of particles through a medium (such as agar) to measure:
*relative concentrations of antibodies/antigens
*relative purity of an antigen preparation

There are two types of immunodiffusion reaction, both being carried out on a semisolid medium:
*'''radial immunodiffusion''': also known as the Mancini method. The medium is prepared with a suitable dilution of antiserum and the antigen sample placed on top and allowed to diffuse. As diffusion takes place, large insoluble complexes form creating a ring of precipitation (precipitin). By comparing the area of the ring with a standard curve, the concentration of the antigen sample can be determined.
*'''double immunodiffusion''': also known as the Ouchterlony method. Antigen and antibody preparations are placed on the medium and both allowed to diffuse radially from the wells towards each other. This establishes a concentration gradient, forming an area of equivalence and line of precipitin.

Agglutination

2008-09-12T12:40:07Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
}}

Agglutination is the clumping together of particles, a reaction that can be used as an immunological test by exploiting the interaction between antibody and a particulate antigen. The antibodies that produce such a reaction are known as agglutinins.

===Method===
The agglutination test is often carried out in round-bottomed test tubes, with doubling dilutions of the antiserum (i.e. 1:2, 1:4, 1:8...). The particulate antigen is then added and the mixture incubated at 37 degrees C. The last tube showing clear agglutination constitutes the end of the test. The dilution at which the test ends is known as the '''titre'''- the number of antibodies per unit volume of the serum.
*An excess of antibody can inhibit agglutination reactions, again in much the same way as a precipitation reaction- this is known as the '''prozone effect'''. At high antibody concentrations, the number of epitopes is outnumbered by antibody binding sites. This means the antibodies bind to the antigen univalently, so are unable to cross-link antigen. This often happens in the first few tubes of the test, so the agglutination often only happens in those containing more dilute antiserum.

===Applications===
[[Image:Coombs test schematic.png|thumb|right|100px|Coombs test- copyright A Rad 2006]]
*'''Widal test''': used to test for antibodies to certain bacteria in patient serum, e.g. salmonella
*'''Blood typing''': also known as '''haemagglutination''', the reaction can be used to determine blood type, i.e. an anti-A serum will agglutinate with type A red blood cells but not with B or O.
*'''Virus diagnosis''': some viruses, such as the myxoviruses, cause agglutination of the red blood cells. Antibodies inhibit this process and can be detected in a patient's serum for diagnosis.
*'''Coombs test''': agglutination of red blood cells is an indication that certain antibodies are present on the cell surface, e.g. testing for Rh disease in newborn babies.
*Hormone assay by agglutination inhibition- red blood cells coated with a specific hormone produce agglutination with a hormone-specific antibody. The addition of free hormone (the test sample) will block the antigen-binding sites, preventing agglutination. By comparing the activity of the test sample against that of a known standard hormone, a quantitative result can be obtained.

Complement Fixation

2008-09-12T12:39:05Z

Eayton: New page: Complement fixation is a test that exploits the fact that antibody-antigen complexes are able to activate the complement system, using the mechanism to show the presence of a specific anti...

Complement fixation is a test that exploits the fact that antibody-antigen complexes are able to activate the complement system, using the mechanism to show the presence of a specific antibody in a serum sample.

===Method===
A mixture of antibody, complement and antigen is incubated and an indicator system (normally antibody-coated sheep red blood cells) is added.
*As the complement reaction with the immune complex produces no visible result, sheep red blood cells are coated with anti-sheep red blood cell antibody. The complement in the mixture will react with this antibody and lyse the cells.
An indication of a positive test is the absence of lysis of the red blood cells, as the complement has already been used up by the antigen-antibody system. Controls are included to ensure that none of the reagents have taken up the complement non-specifically (e.g. contaminated serum)

===Applications===
*'''Wasserman reaction'''- used in the diagnosis of syphilis, the test consists of a mixture of Wasserman antigen, dilutions of the patients serum and complement (normally sourced from guinea pigs).
*Virus detection- tissue samples are innoculated with blood or tissue samples from a patient and tested using complement fixation

Agglutination

2008-09-12T12:06:49Z

Eayton:

Agglutination is the clumping together of particles, a reaction that can be used as an immunological test by exploiting the interaction between antibody and a particulate antigen. The antibodies that produce such a reaction are known as agglutinins.

===Method===
The agglutination test is often carried out in round-bottomed test tubes, with doubling dilutions of the antiserum (i.e. 1:2, 1:4, 1:8...). The particulate antigen is then added and the mixture incubated at 37 degrees C. The last tube showing clear agglutination constitutes the end of the test. The dilution at which the test ends is known as the '''titre'''- the number of antibodies per unit volume of the serum.
*An excess of antibody can inhibit agglutination reactions, again in much the same way as a precipitation reaction- this is known as the '''prozone effect'''. At high antibody concentrations, the number of epitopes is outnumbered by antibody binding sites. This means the antibodies bind to the antigen univalently, so are unable to cross-link antigen. This often happens in the first few tubes of the test, so the agglutination often only happens in those containing more dilute antiserum.

===Applications===
[[Image:Coombs test schematic.png|thumb|right|100px|Coombs test- copyright A Rad 2006]]
*'''Widal test''': used to test for antibodies to certain bacteria in patient serum, e.g. salmonella
*'''Blood typing''': also known as '''haemagglutination''', the reaction can be used to determine blood type, i.e. an anti-A serum will agglutinate with type A red blood cells but not with B or O.
*'''Virus diagnosis''': some viruses, such as the myxoviruses, cause agglutination of the red blood cells. Antibodies inhibit this process and can be detected in a patient's serum for diagnosis.
*'''Coombs test''': agglutination of red blood cells is an indication that certain antibodies are present on the cell surface, e.g. testing for Rh disease in newborn babies.
*Hormone assay by agglutination inhibition- red blood cells coated with a specific hormone produce agglutination with a hormone-specific antibody. The addition of free hormone (the test sample) will block the antigen-binding sites, preventing agglutination. By comparing the activity of the test sample against that of a known standard hormone, a quantitative result can be obtained.

Immunodiffusion

2008-09-11T12:50:22Z

Eayton:

Immunodiffusion reactions are diagnostic tests that use the diffusion of particles through a medium (such as agar) to measure:
*relative concentrations of antibodies/antigens
*relative purity of an antigen preparation

There are two types of immunodiffusion reaction, both being carried out on a semisolid medium:
*'''radial immunodiffusion''': also known as the Mancini method. The medium is prepared with a suitable dilution of antiserum and the antigen sample placed on top and allowed to diffuse. As diffusion takes place, large insoluble complexes form creating a ring of precipitation (precipitin). By comparing the area of the ring with a standard curve, the concentration of the antigen sample can be determined.
*'''double immunodiffusion''': also known as the Ouchterlony method. Antigen and antibody preparations are placed on the medium and both allowed to diffuse radially from the wells towards each other. This establishes a concentration gradient, forming an area of equivalence and line of precipitin.

Immunodiffusion

2008-09-11T12:50:10Z

Eayton: New page: Immunodiffusion reactions are diagnostic tests that use the diffusion of particles through a medium (such as agar) to measure: *relative concentrations of antibodies/antigens *relative pur...

Immunodiffusion reactions are diagnostic tests that use the diffusion of particles through a medium (such as agar) to measure:
*relative concentrations of antibodies/antigens
*relative purity of an antigen preparation

There are two types of immunodiffusion reaction, both being carried out on a semisolid medium:
*'''radial immunodiffusion''': also known as the Mancini method. The medium is prepared with a suitable dilution of antiserum and the antigen sample placed on top and allowed to diffuse. As diffusion takes place, large insoluble complexes form creating a ring of precipitation (precipitin). By comparing the area of the ring with a standard curve, the concentration of the antigen sample can be determined.
*'''double immunodiffusion'''also known as the Ouchterlony method. Antigen and antibody preparations are placed on the medium and both allowed to diffuse radially from the wells towards each other. This establishes a concentration gradient, forming an area of equivalence and line of precipitin.

Agglutination

2008-09-11T11:54:28Z

Eayton: New page: Agglutination is the clumping together of particles, a reaction that can be used as an immunological test by exploiting the interaction between antibody and a particulate antigen. The anti...

File:Coombs test schematic.png

2008-09-11T11:52:35Z

Eayton: Copyright A Rad 2006

ELISA testing

2008-09-11T10:03:47Z

Eayton: /* Techniques */

{{toplink
|backcolour = FFE4E1
|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
}}

==Introduction==
[[Image:ELISA.jpg|thumb|right|150px|Double Antibody Sandwich ELISA]]
The Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay commonly used to detect the presence of an antigen or antibody in a sample. It is a powerful tool in clinical immunology and can be used to determine whether an individual has been exposed to a specified pathogen. Utilizing the principle of antigen-antibody interaction, the test allows easy visualisation of results and, since its introduction in 1971, has quickly replaced radioimmunoassays for diagnostic purposes.

*There are two basic types of ELISA:
**'''Homogenous'''- completed in one step, all reagents added simultaneously. Primarily used to detect small molecules such as digoxin and gentamicin
**'''Heterogenous'''- various reagents added sequentially, primarily used to detect microbial antigens and antibodies

This article describes the '''heterogenous''' type

==Applications==
*Detection and identification of disease agents, e.g. type and subtype
*Identification of specific antibodies, e.g. used in serodiagnosis for epidemiological studies
*Quantification of specific antibody isotypes, e.g. IgM/IgG ratio

==Techniques==

There are many methods of ELISA, but each assay involves these basic steps:
*The adsorption of antibody/antigen to solid phase (the medium)
*The addition of the chosen sample and reagents
*Incubation and washing
*The addition of enzyme-labeled antigen/antibody
*The addition of a specific substrate

'''Competitive VS Non-competitive'''
*As their name implies, competitive assays measure the competition between a pre-titrated (fixed amount) of labeled antigen and an unknown quantity of sample antigen in their affinity to an antibody. The process can be reversed to measure the competition between labeled and unlabeled antibody.
*Competitive techniques:
**Easier to quantify
**Less likely to be influenced by contaminants
**However they are more demanding with regard to the accuracy of the reagents and the purity of the labeled ligand
*Non-competitive assays:
**Errors in dispensing reagents have little effect on the result
**Therefore they are easier to control and yield accurate results
**However they are easily influenced by cross reactions and non-specific binding

'''Solid VS Fluid-phase'''
[[Image:800px-Microtiter plate.jpg|thumb|right|150px|96-well microtiter plate, Copyright Jeffrey M. Vinocur 2006]]

An important consideration is the medium in which the assay is carried out, largely dependent on what is being tested:
*Fluid-phase (''in solution'')
**Main advantage is that the behaviour of molecules in solution is easier to predict
*Solid-phase (''surface of protein-binding material, e.g. plastic'')
**Easier to perform and more sensitive
**The most widely used solid-phase is the 96-well microtiter plater, manufactured as PVC flexible plates or polystyrene rigid plates

----

===Non-competitive ELISA===[[Image:Sandwich elisa.png|thumb|right|100px|The sandwich ELISA - Copyright J M Vincour]]
'''Double Antibody Sandwich''' (for antigen detection)
# Antibody is adsorbed onto solid phase
# Wash
# Sample serum is added- specific antigen binds to antibody
# Wash
# Enzyme-labeled specific antibody is added- attaches to bound antigen
# Wash
# Enzyme substrate is added (with dye for visualisation)
''visualised product = amount of antigen in the serum''

'''Antibody Class Capture Assay''' (for antibody detection)
# Class-specific antiglobulin is adsorbed onto solid phase
# Wash
# Sample serum is added- class-specific antibody in the serum binds to antiglobulin
# Wash
# Antigen is added- attaches to specific antibody
# Wash
# Enzyme-labeled antibody is added
# Wash
# Enzyme substrate is added
''visualised product = amount of specific antibody in serum''

'''Indirect Method''' (for antibody detection)
# Specific antigen (i.e. not from sample) is adsorbed onto solid phase
# Wash
# Serum is added- any specific antibody present in sample binds to antigen
# Wash
# Enzyme-labeled antiglobulin is added- attaches to antibody
# Wash
# Enzyme substrate is added
''visualised product = amount of antibody in the serum''

===Competitive ELISA===
'''Direct Antibody Competition''' (for antibody detection)
# Antigen is adsorbed onto solid phase
# Wash
# Enzyme-labeled antibody (pre-titrated- optimal colour development) and serum are added
# Wash
# Enzyme substrate is added
''visualised product = amount of antigen in serum sample''

'''Direct Antigen Competition''' (for antigen detection)
# Antigen is adsorbed onto solid phase
# Wash
# Antigen in serum is incubated with enzyme-labelled antibody (again pre-titrated): this is directed against the antigen on the solid phase
# Wash
# Enzyme substrate is added
''visualised product = amount of enzyme-labelled antigen''

ELISA testing

2008-09-11T10:01:02Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
}}

==Introduction==
[[Image:ELISA.jpg|thumb|right|150px|Double Antibody Sandwich ELISA]]
The Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay commonly used to detect the presence of an antigen or antibody in a sample. It is a powerful tool in clinical immunology and can be used to determine whether an individual has been exposed to a specified pathogen. Utilizing the principle of antigen-antibody interaction, the test allows easy visualisation of results and, since its introduction in 1971, has quickly replaced radioimmunoassays for diagnostic purposes.

*There are two basic types of ELISA:
**'''Homogenous'''- completed in one step, all reagents added simultaneously. Primarily used to detect small molecules such as digoxin and gentamicin
**'''Heterogenous'''- various reagents added sequentially, primarily used to detect microbial antigens and antibodies

This article describes the '''heterogenous''' type

==Applications==
*Detection and identification of disease agents, e.g. type and subtype
*Identification of specific antibodies, e.g. used in serodiagnosis for epidemiological studies
*Quantification of specific antibody isotypes, e.g. IgM/IgG ratio

==Techniques==

There are many methods of ELISA, but each assay involves these basic steps:
*The adsorption of antibody/antigen to solid phase (the medium)
*The addition of the chosen sample and reagents
*Incubation and washing
*The addition of enzyme-labeled antigen/antibody
*The addition of a specific substrate

'''Competitive VS Non-competitive'''
*As their name implies, competitive assays measure the competition between a pre-titrated (fixed amount) of labeled antigen and an unknown quantity of sample antigen in their affinity to an antibody. The process can be reversed to measure the competition between labeled and unlabeled antibody.
*Competitive techniques:
**Easier to quantify
**Less likely to be influenced by contaminants
**However they are more demanding with regard to the accuracy of the reagents and the purity of the labeled ligand
*Non-competitive assays:
**Errors in dispensing reagents have little effect on the result
**Therefore they are easier to control and yield accurate results
**However they are easily influenced by cross reactions and non-specific binding

'''Solid VS Fluid-phase'''
[[Image:800px-Microtiter plate.jpg|thumb|right|150px|96-well microtiter plate, Copyright Jeffrey M. Vinocur 2006]]

An important consideration is the medium in which the assay is carried out, largely dependent on what is being tested:
*Fluid-phase (''in solution'')
**Main advantage is that the behaviour of molecules in solution is easier to predict
*Solid-phase (''surface of protein-binding material, e.g. plastic'')
**Easier to perform and more sensitive
**The most widely used solid-phase is the 96-well microtiter plater, manufactured as PVC flexible plates or polystyrene rigid plates

----

===Non-competitive ELISA===[[Image:Sandwich elisa.png|thumb|right|100px|The sandwich ELISA - Copyright J M Vincour]]
'''Double Antibody Sandwich''' (for antigen detection)
# Antibody is adsorbed onto solid phase
# Wash
# Sample serum is added- specific antigen binds to antibody
# Wash
# Enzyme-labeled specific antibody is added- attaches to bound antigen
# Wash
# Enzyme substrate is added
''visualised product = amount of antigen in the serum''

'''Antibody Class Capture Assay''' (for antibody detection)
# Class-specific antiglobulin is adsorbed onto solid phase
# Wash
# Sample serum is added- class-specific antibody in the serum binds to antiglobulin
# Wash
# Antigen is added- attaches to specific antibody
# Wash
# Enzyme-labeled antibody is added
# Wash
# Enzyme substrate is added
''visualised product = amount of specific antibody in serum''

'''Indirect Method''' (for antibody detection)
# Antigen is adsorbed onto solid phase
# Wash
# Serum is added- any specific antibody present in sample binds to antigen
# Wash
# Enzyme-labeled antiglobulin is added- attaches to antibody
# Wash
# Enzyme substrate is added
''visualised product = amount of antibody in the serum''

===Competitive ELISA===
'''Direct Antibody Competition''' (for antibody detection)
# Antigen is adsorbed onto solid phase
# Wash
# Enzyme-labeled antibody (pre-titrated- optimal colour development) and serum are added
# Wash
# Enzyme substrate is added
''visualised product = amount of enzyme-labelled antibody''

'''Direct Antigen Competition''' (for antigen detection)
# Antigen is adsorbed onto solid phase
# Wash
# Antigen in serum is incubated with enzyme-labelled antibody (again pre-titrated): this is directed against the antigen on the solid phase
# Wash
# Enzyme substrate is added
''visualised product = amount of enzyme-labelled antigen''

ELISA testing

2008-09-11T09:59:17Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
}}

==Introduction==
[[Image:ELISA.jpg|thumb|right|150px|Double Antibody Sandwich ELISA]]
The Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay commonly used to detect the presence of an antigen or antibody in a sample. It is a powerful tool in clinical immunology and can be used to determine whether an individual has been exposed to a specified pathogen. Utilizing the principle of antigen-antibody interaction, the test allows easy visualisation of results and, since its introduction in 1971, has quickly replaced radioimmunoassays for diagnostic purposes.

*There are two basic types of ELISA:
**'''Homogenous'''- completed in one step, all reagents added simultaneously. Primarily used to detect small molecules such as digoxin and gentamicin
**'''Heterogenous'''- various reagents added sequentially, primarily used to detect microbial antigens and antibodies

This article describes the '''heterogenous''' type

==Applications==
*Detection and identification of disease agents, e.g. type and subtype
*Identification of specific antibodies, e.g. used in serodiagnosis for epidemiological studies
*Quantification of specific antibody isotypes, e.g. IgM/IgG ratio

==Techniques==

There are many methods of ELISA, but each assay involves these basic steps:
*The adsorption of antibody/antigen to solid phase (the medium)
*The addition of the chosen sample and reagents
*Incubation and washing
*The addition of enzyme-labeled antigen/antibody
*The addition of a specific substrate

'''Competitive VS Non-competitive'''
*As their name implies, competitive assays measure the competition between a pre-titrated (fixed amount) of labeled antigen and an unknown quantity of sample antigen in their affinity to an antibody. The process can be reversed to measure the competition between labeled and unlabeled antibody.
*Competitive techniques:
**Easier to quantify
**Less likely to be influenced by contaminants
**However they are more demanding with regard to the accuracy of the reagents and the purity of the labeled ligand
*Non-competitive assays:
**Errors in dispensing reagents have little effect on the result
**Therefore they are easier to control and yield accurate results
**However they are easily influenced by cross reactions and non-specific binding

'''Solid VS Fluid-phase'''
[[Image:800px-Microtiter plate.jpg|thumb|right|150px|96-well microtiter plate, Copyright Jeffrey M. Vinocur 2006]]

An important consideration is the medium in which the assay is carried out, largely dependent on what is being tested:
*Fluid-phase (''in solution'')
**Main advantage is that the behaviour of molecules in solution is easier to predict
*Solid-phase (''surface of protein-binding material, e.g. plastic'')
**Easier to perform and more sensitive
**The most widely used solid-phase is the 96-well microtiter plater, manufactured as PVC flexible plates or polystyrene rigid plates

----

===Non-competitive ELISA===[[Image:Sandwich elisa.png|thumb|right|100px|The sandwich ELISA - Copyright J M Vincour]]
'''Double Antibody Sandwich''' (for antigen detection)
# Antibody is adsorbed onto solid phase
# Wash
# Sample is added- specific antigen binds to antibody
# Wash
# Enzyme-labeled specific antibody is added- attaches to bound antigen
# Wash
# Enzyme substrate is added
''visualised product = amount of antigen in the sample''

'''Antibody Class Capture Assay''' (for antibody detection)
# Class-specific antiglobulin is adsorbed onto solid phase
# Wash
# Sample is added- class-specific antibody in the sample binds to antiglobulin
# Wash
# Antigen is added- attaches to specific antibody
# Wash
# Enzyme-labeled antibody is added
# Wash
# Enzyme substrate is added
''visualised product = amount of specific antibody in sample''

'''Indirect Method''' (for antibody detection)
# Antigen is adsorbed onto solid phase
# Wash
# Sample is added- any specific antibody present in sample binds to antigen
# Wash
# Enzyme-labeled antiglobulin is added- attaches to antibody
# Wash
# Enzyme substrate is added
''visualised product = amount of antibody in the sample''

===Competitive ELISA===
'''Direct Antibody Competition''' (for antibody detection)
# Antigen is adsorbed onto solid phase
# Wash
# Enzyme-labeled antibody (pre-titrated- optimal colour development) and sample are added
# Wash
# Enzyme substrate is added
''visualised product = amount of enzyme-labelled antibody''

'''Direct Antigen Competition''' (for antigen detection)
# Antigen is adsorbed onto solid phase
# Wash
# Antigen in sample is incubated with enzyme-labelled antibody (again pre-titrated): this is directed against the antigen on the solid phase
# Wash
# Enzyme substrate is added
''visualised product = amount of enzyme-labelled antigen''

ELISA testing

2008-09-11T09:57:49Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =Immunology - WikiBlood
|linktext =IMMUNOLOGY
|sublink1 =Immunological testing - WikiBlood
|subtext1 =IMMUNOLOGICAL TESTING
|pagetype =Blood
}}

==Introduction==
[[Image:ELISA.jpg|thumb|right|150px|Double Antibody Sandwich ELISA]]
The Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay commonly used to detect the presence of an antigen or antibody in a sample. It is a powerful tool in clinical immunology and can be used to determine whether an individual has been exposed to a specified pathogen. Utilizing the principle of antigen-antibody interaction, the test allows easy visualisation of results and, since its introduction in 1971, has quickly replaced radioimmunoassays for diagnostic purposes.

*There are two basic types of ELISA:
**'''Homogenous'''- completed in one step, all reagents added simultaneously. Primarily used to detect small molecules such as digoxin and gentamicin
**'''Heterogenous'''- various reagents added sequentially, primarily used to detect microbial antigens and antibodies

This article describes the '''heterogenous''' type

==Applications==
*Detection and identification of disease agents, e.g. type and subtype
*Identification of specific antibodies, e.g. used in serodiagnosis for epidemiological studies
*Quantification of specific antibody isotypes, e.g. IgM/IgG ratio

==Techniques==

There are many methods of ELISA, but each assay involves these basic steps:
*The adsorption of antibody/antigen to solid phase (the medium)
*The addition of the chosen sample and reagents
*Incubation and washing
*The addition of enzyme-labeled antigen/antibody
*The addition of a specific substrate

'''Competitive VS Non-competitive'''
*As their name implies, competitive assays measure the competition between a pre-titrated (fixed amount) of labeled antigen and an unknown quantity of sample antigen in their affinity to an antibody. The process can be reversed to measure the competition between labeled and unlabeled antibody.
*Competitive techniques:
**Easier to quantify
**Less likely to be influenced by contaminants
**However they are more demanding with regard to the accuracy of the reagents and the purity of the labeled ligand
*Non-competitive assays:
**Errors in dispensing reagents have little effect on the result
**Therefore they are easier to control and yield accurate results
**However they are easily influenced by cross reactions and non-specific binding

'''Solid VS Fluid-phase'''
[[Image:800px-Microtiter plate.jpg|thumb|right|150px|96-well microtiter plate, Copyright Jeffrey M. Vinocur 2006]]

An important consideration is the medium in which the assay is carried out, largely dependent on what is being tested:
*Fluid-phase (''in solution'')
**Main advantage is that the behaviour of molecules in solution is easier to predict
*Solid-phase (''surface of protein-binding material, e.g. plastic'')
**Easier to perform and more sensitive
**The most widely used solid-phase is the 96-well microtiter plater, manufactured as PVC flexible plates or polystyrene rigid plates

----

===Non-competitive ELISA===
'''Double Antibody Sandwich''' (for antigen detection)
# Antibody is adsorbed onto solid phase
# Wash
# Sample is added- specific antigen binds to antibody
# Wash
# Enzyme-labeled specific antibody is added- attaches to bound antigen
# Wash
# Enzyme substrate is added
''visualised product = amount of antigen in the sample''

'''Antibody Class Capture Assay''' (for antibody detection)
# Class-specific antiglobulin is adsorbed onto solid phase
# Wash
# Sample is added- class-specific antibody in the sample binds to antiglobulin
# Wash
# Antigen is added- attaches to specific antibody
# Wash
# Enzyme-labeled antibody is added
# Wash
# Enzyme substrate is added
''visualised product = amount of specific antibody in sample''

'''Indirect Method''' (for antibody detection)
# Antigen is adsorbed onto solid phase
# Wash
# Sample is added- any specific antibody present in sample binds to antigen
# Wash
# Enzyme-labeled antiglobulin is added- attaches to antibody
# Wash
# Enzyme substrate is added
''visualised product = amount of antibody in the sample''

===Competitive ELISA===
'''Direct Antibody Competition''' (for antibody detection)
# Antigen is adsorbed onto solid phase
# Wash
# Enzyme-labeled antibody (pre-titrated- optimal colour development) and sample are added
# Wash
# Enzyme substrate is added
''visualised product = amount of enzyme-labelled antibody''

'''Direct Antigen Competition''' (for antigen detection)
# Antigen is adsorbed onto solid phase
# Wash
# Antigen in sample is incubated with enzyme-labelled antibody (again pre-titrated): this is directed against the antigen on the solid phase
# Wash
# Enzyme substrate is added
''visualised product = amount of enzyme-labelled antigen''

File:Sandwich elisa.png

2008-09-11T09:55:41Z

Eayton: (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)

(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. Copyright J M Vincour

Immune Tolerance Flashcards

2008-09-11T09:47:21Z

Eayton: /* Immune tolerance */

==Immune tolerance==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''What is the definition of tolerance?'''
|| <big>
*'''''A state of immune unresponsiveness towards antigens'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''Where does central tolerance develop?'''
|| <big>
*'''''Thymus'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''What are the two processes of selection known as?'''
|| <big>
*'''''Positive selection'''''
*'''''Negative selection'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''What are the four ways peripheral tolerance can be achieved in T cells?'''
|| <big>
*'''''Ignorance'''''
*'''''Anergy'''''
*'''''Cell death'''''
*'''''Immune deviation/suppression'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''What is the process in which one T cell response is selectively induced over another?'''
|| <big>
*'''''Immune deviation'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''What is the definition of tolerance?'''
|| <big>
*'''''A state of immune unresponsiveness towards antigens'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''What subtype are regulatory T cells?'''
|| <big>
*'''''CD4+'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''What part of the body do Treg cells develop?'''
|| <big>
*'''''Thymus'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''What typical inhibitory cytokines do Treg cells secrete?'''
|| <big>
*'''''IL-4'''''
*'''''IL-10'''''
*'''''TGF-β'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''The mechanism of suppression of Treg cells depends on the binding of which receptors?'''
|| <big>
*'''''Treg CTLA-4 to target cell B7'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|-
|<big>'''What is the transcription repressor unique to Treg cells?'''
|| <big>
*'''''FoxP3'''''
||[[Immune Tolerance - WikiBlood|Link to Answer Article]]
|}

Equipment Sterilisation

2008-09-11T09:22:22Z

Eayton:

{{toplink
|linkpage =WikiClinical
|linktext =WikiClinical
|maplink = WikiClinical Content Map
|pagetype = Clinical
}}
 
==Introduction==
Sterilisation is the use of physical or chemical means to destroy all organisms, including microbial spores and viruses. There are several techniques that can be used;
*Heat
*Irradiation
*Filtration
*Chemicals
Other techniques include freezing, lysis, dessicationa and ultrasonification, but these are not often employed due to lower efficiency

==Heat==
Due to cost-effectiveness, efficiency and ease-of-use, heat is the preferred method of sterilisation.

'''Dry heat'''
Hot air can be used to sterilise items such as glassware, although due to poor diffusion and penetration this method is not as efficient as moist heat.
*Incineration- useful for items such as bandages and paper dishes
*Oven baking- useful for items not containing water and not readily permeable by steam, e.g. wood, glass syringes.
*Sterilisation in this case normally means processing at 171 degrees for over an hour

'''Moist heat'''
The most effective form of heat sterilisation is saturated steam under pressure
*Achieved using an autoclave
*Pressure aids the penetration of the steam into materials
*Used to sterilise heat resistant materials and pharmaceuticals

'''Immersion in boiling water'''
*Kills vegetative bacteria, but not all bacterial spores
*Addition of 2% sodium bicarbonate improves efficiency

'''Pasteurisation'''
*Used to reduce bacteria in fluids

==Irradiation==
Gamma radiation is the method of choice for sterilising large batches of items such as needles and syringes
*Although the cost is high, the process is 100% effective
*Mechanism involves production of free radicals- destroys DNA
Ultraviolet radiation is not normally used as its efficiency as a sterilant is low
*Requires direct exposure
*It is used however in hospitals to stop the growth of bacteria in water, e.g. air hoods in virology laboratories

==Filtration==
Used to produce particle and pyrogen-free fluids
*Work by electrostatic attraction and physical pore size

==Chemical agents==
Although the use of chemical agents has been overtaken by the development of gamma radiation techniques, two alkylating gases are still used:
*'''Ethylene oxide'''- used to sterilise single-use medical items such as heart valves
**Not often used due to high toxicity
*'''Formaldehyde'''- used to decontaminate rooms, e.g. isolation rooms
**Irritates mucuos membranes
**Relatively high humidity required for effectiveness

Immune Tolerance Flashcards

2008-09-10T15:13:12Z

Eayton: New page: ==Immune tolerance== {| border="3" cellpadding="8" !width="400"|'''Question''' !width="400"|'''Answer''' !width="150"|'''Article''' |- |<big>'''What is the...

Immune Tolerance

2008-09-10T14:39:42Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =WikiBlood
|linktext =WIKIBLOOD
|thispagemap= Immune Tolerance(Concept Map) - WikiBlood
|sublink1 =Immunology - WikiBlood
|subtext1 =IMMUNOLOGY
|pagetype =Blood
}}

=Immune Tolerance=
Immunological tolerance is the state of unresponsiveness towards particular antigens, whereby immune responses are prevented or suppressed. Tolerance is required to prevent:
* Potentially harmful inflammatory responses towards innocuous substances, such as air-borne or food antigens
* To prevent an immune attack against host tissue- described as '''self-tolerance'''

=T Cell Tolerance=

During T cell development within the thymus, genes encoding the T cell receptor are rearranged, resulting in adult cells that are able to recognise antigen fragments displayed by the host MHC molecule. Some receptors however will be self-reactive, i.e. they bind too strongly to antigens expressed by the host's own tissues ('''autoantigens'''). As these cells will induce immune reactions that could be damaging to the host ([[Autoimmune Diseases - WikiBlood|Autoimmunity]]), they must be deleted or suppressed.

==Central Tolerance==
[[Image:Thymic selection of t cells.jpg|thumb|right|150px|Negative selection of t cells- copyright Brian Catchpole]]
*Takes place within the thymus
*Involves the processes '''positive''' and '''negative''' selection
** During positive selection, cells passing through the thymic cortex encounter cortical epithelial cells expressing MHC molecules. Those with a suitable level of binding affinity for the MHC recieve 'survival' signals and apoptosis is prevented (at the same time cells lose either their CD4 or CD8 co-receptor)
**During negative selection, cells passing through the corticomedullary junction and thymic medulla once again encounter MHC molecules, on epithelial cells, dendritic cells and macrophages, this time bound to self-peptide. Cells bearing receptors that bind too strongly to this complex are deleted through mechanisms that induce apoptosis.

==Peripheral Tolerance==
It is inevitable that some self-reactive T cells will get through the thymic selection process and into the periphery, as:
*Some self-antigens are not expressed in the thymus
*Some antigens will not show sufficient affinity to MHC to form the MHC:self-peptide complex required for negative selection in the thymus
*Conversely some T cell receptors will not have enough affinity for their respective self-antigen to induce apoptosis
Peripheral tolerance is the process in which unresponsiveness towards self-antigen is developed outside the primary lymphoid organs. There are four ways this may be achieved in T cells:
* Ignorance
* Anergy
* Cell death
* Immune deviation/suppression

===Ignorance===
Can occur if;
* Self-reactive T cells cannot penetrate an endothelial barrier
* Self-antigen is present in very low amount
* Self-antigen is present on cells that do not express/express low amount of MHC
* T cells are not present in sufficient numbers to mount effective response
* Self-antigen is presented without co-stimulation- can lead to ignorance or anergy, depending on type of antigen and affinity to TCR

===Anergy===
* Defined as a state where the T cell is still alive, but fails to respond to stimulation from its specific T cell receptor and other receptors required for activation
* Easily induced in T cells ''in vivo'' by activation of T cell receptor without co-stimulation
* Induced ''in vitro'' by injection of potent superantigens (antigens that stimulate T cells with different receptor types, using the same T cell receptor V gene)
* Can be caused by downregulation of T cell receptors as a result of chronic stimulation
** Anergy induced without co-stimulation can be reversed by IL-2

===Cell death===
Peripheral deletion of T cells requires the engagement of:
* Fas by Fas ligand
** Deficiencies in Fas ligand lead to lymphoproliferative disorders
** After activation by an antigen, T cells upregulate expression of Fas ligand
** Some tissues, such as the testis and retina, constitutively express Fas ligand to protect themselves from activated T cells
* TNF via TNF receptor
* CTLA-4 recently implicated
* Subsequent signalling cascade activates proteases, such as IL-1beta Converting Enzyme (ICE), that leads to apoptosis

===Immune deviation===
* Th2-derived cytokines, such as IL-10, typically support antibody production, but also down-regulate macrophage effector functions, such as antigen presentation, thereby suppressing inflammatory responses
* Likewise, Th1-derived gamma-IFN can prevent Th0-Th2 differentiation
** This process is described as '''immune deviation''', i.e. one response being selectively induced over the other
* In the case of self-antigens, autoimmune diseases such as diabetes are caused by Th1 cells and can be prevented by 'antigen-primed' Th2 cells.

=Mucosal tolerance=
Mucosal tolerance is the systemic unresponsiveness towards antigens administered across the mucosal surfaces
*As the highest antigenic load of the body surfaces occurs in the GI tract, it is also known as oral tolerance
*When oral tolerance towards food antigens breaks down, inflammatory autoimmune responses are induced
*Gut associated lymphoid tissue is important for developing oral tolerance:
**Animals that lack Peyer’s patches and mesenteric lymph nodes do not develop oral tolerance
**It is thought the liver and spleen may also play a role

==Mechanisms==
*High doses of antigen can cause anergy or cell death
*Low doses can induce a T cell response:
**The antigen is taken up and presented, inducing a Th2-like cell response
**This cell response produces cytokines that suppress the Th1 inflammatory response, such as IL-10 and TGF-beta
***Although the cellular response is antigen-specific, the cytokines released are not. TGF-beta is known to inhibit the proliferation and function of B-cells, cytotoxic T cells and NK cells. This means tolerance induction to one antigen suppress an immune response to a second associated antigen- this mechanism has been used to suppress some autoimmune diseases by feeding with an antigen isolated from the affected tissue. This is known as ‘’bystander suppression’’.

==Other mucosal surfaces==
*Nasal deposition of some peptides can be used to induce tolerance, controlling both humoral and cellular responses
*Administration of antigen in aerosol form to the lung has been used to control both allergic and autoimmune responses

=Regulatory T Cells=
A number of cell populations identified during studies on autoimmunity and organ transplantation have shown the capacity to suppress responses to self-antigen and regulate rejection. Although once considered a tentative theory, this form of tolerance is now considered a major mechanism in the protection of host tissue from immune attack.
[[Image:T reg cells.JPG|thumb|right|150px|Regulatory T cells- copyright Brian Catchpole]]
* Known as '''regulatory T cells''', these CD4+ cells are antigen-specific
* Currently thought to develop in the thymus
* They usually release inhibitory cytokines, e.g. IL-4, IL-10 and TGF-beta
* When their TCRs bind to an antigen, they do not proliferate themselves but suppress the proliferation of other ''naive'' T cells responding to that antigen
* Mechanism of suppression is dependent on:
** CTLA-4 on the regulatory T cell binding with B7 on the target T cell
* Both cells binding the same antigen
* Regulatory T cells are unique in their use of a transcription repressor known as FoxP3
** Encoded by a gene on the X chromosome, rare deficiencies in FoxP3 are characterised by autoimmunity, primarily towards gut tissue, the thyroid, pancreative beta-cells and the skin. Sufferers are unable to produce regulatory T cells and the only known treatment is a bone marrow transplant from a MHC-identical sibling.

==[[Immune tolerance flashcards - Wikiblood|Immune tolerance flashcards]]==

Immune Tolerance

2008-09-10T14:38:39Z

Eayton:

{{toplink
|backcolour = FFE4E1
|linkpage =WikiBlood
|linktext =WIKIBLOOD
|thispagemap= Immune Tolerance(Concept Map) - WikiBlood
|sublink1 =Immunology - WikiBlood
|subtext1 =IMMUNOLOGY
|pagetype =Blood
}}

=Immune Tolerance=
Immunological tolerance is the state of unresponsiveness towards particular antigens, whereby immune responses are prevented or suppressed. Tolerance is required to prevent:
* Potentially harmful inflammatory responses towards innocuous substances, such as air-borne or food antigens
* To prevent an immune attack against host tissue- described as '''self-tolerance'''

=T Cell Tolerance=

During T cell development within the thymus, genes encoding the T cell receptor are rearranged, resulting in adult cells that are able to recognise antigen fragments displayed by the host MHC molecule. Some receptors however will be self-reactive, i.e. they bind too strongly to antigens expressed by the host's own tissues ('''autoantigens'''). As these cells will induce immune reactions that could be damaging to the host ([[Autoimmune Diseases - WikiBlood|Autoimmunity]]), they must be deleted or suppressed.

==Central Tolerance==
[[Image:Thymic selection of t cells.jpg|thumb|right|150px|Negative selection of t cells- copyright Brian Catchpole]]
*Takes place within the thymus
*Involves the processes '''positive''' and '''negative''' selection
** During positive selection, cells passing through the thymic cortex encounter cortical epithelial cells expressing MHC molecules. Those with a suitable level of binding affinity for the MHC recieve 'survival' signals and apoptosis is prevented (at the same time cells lose either their CD4 or CD8 co-receptor)
**During negative selection, cells passing through the corticomedullary junction and thymic medulla once again encounter MHC molecules, on epithelial cells, dendritic cells and macrophages, this time bound to self-peptide. Cells bearing receptors that bind too strongly to this complex are deleted through mechanisms that induce apoptosis.

==Peripheral Tolerance==
It is inevitable that some self-reactive T cells will get through the thymic selection process and into the periphery, as:
*Some self-antigens are not expressed in the thymus
*Some antigens will not show sufficient affinity to MHC to form the MHC:self-peptide complex required for negative selection in the thymus
*Conversely some T cell receptors will not have enough affinity for their respective self-antigen to induce apoptosis
Peripheral tolerance is the process in which unresponsiveness towards self-antigen is developed outside the primary lymphoid organs. There are four ways this may be achieved in T cells:
* Ignorance
* Anergy
* Cell death
* Immune deviation/suppression

===Ignorance===
Can occur if;
* Self-reactive T cells cannot penetrate an endothelial barrier
* Self-antigen is present in very low amount
* Self-antigen is present on cells that do not express/express low amount of MHC
* T cells are not present in sufficient numbers to mount effective response
* Self-antigen is presented without co-stimulation- can lead to ignorance or anergy, depending on type of antigen and affinity to TCR

===Anergy===
* Defined as a state where the T cell is still alive, but fails to respond to stimulation from its specific T cell receptor and other receptors required for activation
* Easily induced in T cells ''in vivo'' by activation of T cell receptor without co-stimulation
* Induced ''in vitro'' by injection of potent superantigens (antigens that stimulate T cells with different receptor types, using the same T cell receptor V gene)
* Can be caused by downregulation of T cell receptors as a result of chronic stimulation
** Anergy induced without co-stimulation can be reversed by IL-2

===Cell death===
Peripheral deletion of T cells requires the engagement of:
* Fas by Fas ligand
** Deficiencies in Fas ligand lead to lymphoproliferative disorders
** After activation by an antigen, T cells upregulate expression of Fas ligand
** Some tissues, such as the testis and retina, constitutively express Fas ligand to protect themselves from activated T cells
* TNF via TNF receptor
* CTLA-4 recently implicated
* Subsequent signalling cascade activates proteases, such as IL-1beta Converting Enzyme (ICE), that leads to apoptosis

===Immune deviation===
* Th2-derived cytokines, such as IL-10, typically support antibody production, but also down-regulate macrophage effector functions, such as antigen presentation, thereby suppressing inflammatory responses
* Likewise, Th1-derived gamma-IFN can prevent Th0-Th2 differentiation
** This process is described as '''immune deviation''', i.e. one response being selectively induced over the other
* In the case of self-antigens, autoimmune diseases such as diabetes are caused by Th1 cells and can be prevented by 'antigen-primed' Th2 cells.

=Mucosal tolerance=
Mucosal tolerance is the systemic unresponsiveness towards antigens administered across the mucosal surfaces
*As the highest antigenic load of the body surfaces occurs in the GI tract, it is also known as oral tolerance
*When oral tolerance towards food antigens breaks down, inflammatory autoimmune responses are induced
*Gut associated lymphoid tissue is important for developing oral tolerance:
**Animals that lack Peyer’s patches and mesenteric lymph nodes do not develop oral tolerance
**It is thought the liver and spleen may also play a role

==Mechanisms==
*High doses of antigen can cause anergy or cell death
*Low doses can induce a T cell response:
**The antigen is taken up and presented, inducing a Th2-like cell response
**This cell response produces cytokines that suppress the Th1 inflammatory response, such as IL-10 and TGF-beta
***Although the cellular response is antigen-specific, the cytokines released are not. TGF-beta is known to inhibit the proliferation and function of B-cells, cytotoxic T cells and NK cells. This means tolerance induction to one antigen suppress an immune response to a second associated antigen- this mechanism has been used to suppress some autoimmune diseases by feeding with an antigen isolated from the affected tissue. This is known as ‘’bystander suppression’’.

==Other mucosal surfaces==
*Nasal deposition of some peptides can be used to induce tolerance, controlling both humoral and cellular responses
*Administration of antigen in aerosol form to the lung has been used to control both allergic and autoimmune responses

=Regulatory T Cells=
A number of cell populations identified during studies on autoimmunity and organ transplantation have shown the capacity to suppress responses to self-antigen and regulate rejection. Although once considered a tentative theory, this form of tolerance is now considered a major mechanism in the protection of host tissue from immune attack.
[[Image:T reg cells.JPG|thumb|right|150px|Regulatory T cells- copyright Brian Catchpole]]
* Known as '''regulatory T cells''', these CD4+ cells are antigen-specific
* Currently thought to develop in the thymus
* They usually release inhibitory cytokines, e.g. IL-4, IL-10 and TGF-beta
* When their TCRs bind to an antigen, they do not proliferate themselves but suppress the proliferation of other ''naive'' T cells responding to that antigen
* Mechanism of suppression is dependent on:
** CTLA-4 on the regulatory T cell binding with B7 on the target T cell
* Both cells binding the same antigen
* Regulatory T cells are unique in their use of a transcription repressor known as FoxP3
** Encoded by a gene on the X chromosome, rare deficiencies in FoxP3 are characterised by autoimmunity, primarily towards gut tissue, the thyroid, pancreative beta-cells and the skin. Sufferers are unable to produce regulatory T cells and the only known treatment is a bone marrow transplant from a MHC-identical sibling.

==[[Host invasion by microorganisms flashcards- Wikiblood|Host invasion flashcards]]==

Host Invasion by Microorganisms Flashcards

2008-09-10T14:31:34Z

Eayton:

==Host invasion==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''By what three routes can pathogens enter the body?'''
|| <big>
*'''''Contact'''''
*'''''Aerosol'''''
*'''''Orofecal'''''
||[[Host invasion by microorganisms - WikiBlood|Link to Answer Article]]
|}

==Viruses==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''Naked viruses contain only...?'''
|| <big>
*'''''RNA/DNA and protein coat'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''Name three functions of viral structural proteins'''
|| <big>
*'''''Protection of the genome'''''
*'''''Attachment to host cell'''''
*'''''Fusion of envelope to host cell membrane'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''Name the two main innate defences against viral infection'''
|| <big>
*'''''Interferon'''''
*'''''Natural killer cells
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''Upon viral infection, which antibody isotype is increased at mucosal surfaces?'''
|| <big>
*'''''IgA'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''What do antibodies stop free virus particles from doing?'''
|| <big>
*'''''Binding to host cell, entering and uncoating'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''By what mechanism is complement able to damage the virion envelope?'''
|| <big>
*'''''Virolysis'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''When coupled with antibodies, what mechanisms does complement aid in immune defence?'''
|| <big>
*'''''Blocking the virus receptor'''''
*'''''Lysis of infected cells'''''
*'''''Opsonisation of free particles or infected cells'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''What are the main cytokines CD4+ T cells release to recruit macrophages?'''
|| <big>
*'''''IFN-γ'''''
*'''''TNF'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|}

==Bacteria==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''Describe three ways prokaryotic DNA differs from eukaryotic DNA'''
|| <big>
*'''''Few repeated sequences'''''
*'''''Most of the DNA is transcibed'''''
*'''''No intervening sequences within structural genes'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''These surface components allow bacteria to move around'''
|| <big>
*'''''Flagella'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''Bacteria with an outer layer of lipopolysaccharide are classed as...'''
|| <big>
*'''''Gram-negative'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''Bacteria with a round shape are classed as...'''
|| <big>
*'''''Cocci'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''Bacteria with a long shape are classed as...'''
|| <big>
*'''''Rods or bacilli'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''What are the four rules summarising the cause and effect relationship between bacteria and infection?'''
|| <big>
*'''''Koch's postulates'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''What is the condition, characterised by excessive cytokine production, that is caused by bacterial components such as LPS?'''
|| <big>
*'''''Endotoxin shock'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''By what mechanism do most bacteria replicate?'''
|| <big>
*'''''Binary fission'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''By what three mechanisms can bacteria transfer DNA?'''
|| <big>
*'''''Transformation'''''
*'''''Transduction'''''
*'''''Conjugation'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|}

==Parasites==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''Do protozoa have a eukaryotic or prokaryotic cell structure'''
|| <big>
*'''''Eukaryotic'''''
||[[Parasites - WikiBlood|Link to Answer Article]]
|-
|<big>'''What are the two main groups of helminth parasite?'''
|| <big>
*'''''Nematodes'''''
*'''''Platyhelminthes'''''
||[[Parasites - WikiBlood|Link to Answer Article]]
|-
|<big>'''What is the main cell type that mediates the destruction of larger parasites?'''
|| <big>
*'''''Eosinophils'''''
||[[Parasites - WikiBlood|Link to Answer Article]]
|-
|<big>'''What T cell subtype is required to fight intracellular protozoa'''
|| <big>
*'''''Th1'''''
||[[Parasites - WikiBlood|Link to Answer Article]]
|-
|<big>'''What T cell subtype is required to fight intestinal worms?'''
|| <big>
*'''''Th2'''''
||[[Parasites - WikiBlood|Link to Answer Article]]
|-
|<big>'''How do antibodies defend against parasitic infection?'''
|| <big>
*'''''Directly damaging protozoa'''''
*'''''Activating complement'''''
*'''''Blocking attachment to host cells'''''
*'''''Enhancing macrophage phagocytosis'''''
*'''''Antibody-dependent cell-mediated cytotoxicity'''''
||[[Parasites - WikiBlood|Link to Answer Article]]

Host Invasion by Microorganisms Flashcards

2008-09-10T14:29:02Z

Eayton:

Host Invasion by Microorganisms Flashcards

2008-09-10T14:22:59Z

Eayton:

Host Invasion by Microorganisms Flashcards

2008-09-10T13:59:36Z

Eayton:

Host Invasion by Microorganisms Flashcards

2008-09-10T13:58:45Z

Eayton:

{{toplink
|backcolour = f5fffa
|sublink1 =Flash Cards - WikiBlood
|subtext1 =WIKIBLOOD FLASHCARDS
|pagetype =Blood
}}
 
==Host invasion==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''By what three routes can pathogens enter the body?'''
|| <big>
*'''''Contact'''''
*'''''Aerosol'''''
*'''''Orofecal'''''
||[[Host invasion by microorganisms - WikiBlood|Link to Answer Article]]
|}

==Viruses==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''Naked viruses contain only...?'''
|| <big>
*'''''RNA/DNA and protein coat'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''Name three functions of viral structural proteins'''
|| <big>
*'''''Protection of the genome'''''
*'''''Attachment to host cell'''''
*'''''Fusion of envelope to host cell membrane'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''Name the two main innate defences against viral infection'''
|| <big>
*'''''Interferon'''''
*'''''Natural killer cells
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''Upon viral infection, which antibody isotype is increased at mucosal surfaces?'''
|| <big>
*'''''IgA'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''What do antibodies stop free virus particles from doing?'''
|| <big>
*'''''Binding to host cell, entering and uncoating'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''By what mechanism is complement able to damage the virion envelope?'''
|| <big>
*'''''Virolysis'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''When coupled with antibodies, what mechanisms does complement aid in immune defence?'''
|| <big>
*'''''Blocking the virus receptor'''''
*'''''Lysis of infected cells'''''
*'''''Opsonisation of free particles or infected cells'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''What are the main cytokines CD4+ T cells release to recruit macrophages?'''
|| <big>
*'''''IFN-γ'''''
*'''''TNF'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|}

==Bacteria==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''Describe three ways prokaryotic DNA differs from eukaryotic DNA'''
|| <big>
*'''''Few repeated sequences'''''
*'''''Most of the DNA is transcibed'''''
*'''''No intervening sequences within structural genes'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''These surface components allow bacteria to move around'''
|| <big>
*'''''Flagella'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''Bacteria with an outer layer of lipopolysaccharide are classed as...'''
|| <big>
*'''''Gram-negative'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''Bacteria with a round shape are classed as...'''
|| <big>
*'''''Cocci'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''Bacteria with a long shape are classed as...'''
|| <big>
*'''''Rods or bacilli'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''What are the four rules summarising the cause and effect relationship between bacteria and infection?'''
|| <big>
*'''''Koch's postulates'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''What is the condition, characterised by excessive cytokine production, that is caused by bacterial components such as LPS?'''
|| <big>
*'''''Endotoxin shock'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''By what mechanism do most bacteria replicate?'''
|| <big>
*'''''Binary fission'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|-
|<big>'''By what three mechanisms can bacteria transfer DNA?'''
|| <big>
*'''''Transformation'''''
*'''''Transduction'''''
*'''''Conjugation'''''
||[[Bacteria - WikiBlood|Link to Answer Article]]
|}

Host Invasion by Microorganisms Flashcards

2008-09-10T13:26:01Z

Eayton:

Host Invasion by Microorganisms Flashcards

2008-09-10T13:25:39Z

Eayton:

Host Invasion by Microorganisms Flashcards

2008-09-10T13:02:08Z

Eayton: New page: {{toplink |backcolour = f5fffa |linktext =Host invasion by microorganisms |sublink1 =Flash Cards - WikiBlood |subtext1 =WIKIBLOOD FLASHCARDS |pagetype =Blood }} ==<font color="purple"...

{{toplink
|backcolour = f5fffa
|linktext =Host invasion by microorganisms
|sublink1 =Flash Cards - WikiBlood
|subtext1 =WIKIBLOOD FLASHCARDS
|pagetype =Blood
}}
 
==Host invasion==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''By what three routes can pathogens enter the body?'''
|| <big>
*'''''Contact'''''
*'''''Aerosol'''''
*'''''Orofecal'''''
||[[Host invasion by microorganisms - Wikiblood|Link to Answer Article]]
|}

==Viruses==
{| border="3" cellpadding="8"
!width="400"|'''Question'''
!width="400"|'''Answer'''
!width="150"|'''Article'''
|-
|<big>'''Naked viruses contain only...?'''
|| <big>
*'''''RNA/DNA and protein coat'''''
||[[Viruses - WikiBlood|Link to Answer Article]]
|-
|<big>'''Name three functions of viral structural proteins'''
|| <big>
*'''''Protection of the genome'''''
*'''''Attachment to host cell'''''
*'''''Fusion of envelope to host cell membrane'''''
||[[Viruses - Immunology#Viral proteins|Link to Answer Article]]
|-
|<big>'''Name the two main innate defences against viral infection'''
|| <big>
*'''''Interferon'''''
*'''''Natural killer cells
||[[Viruses - Immunology#Immunity to viruses|Link to Answer Article]]
|-
|<big>'''Upon viral infection, which antibody isotype is increased at mucosal surfaces?'''
|| <big>
*'''''IgA'''''
||[[Viruses - Immunology#Adaptive immunity|Link to Answer Article]]
|-
|<big>'''What do antibodies stop free virus particles from doing?'''
|| <big>
*'''''Binding to host cell, entering and uncoating'''''
||[[Viruses - Immunology#Adaptive immunity|Link to Answer Article]]
|-
|}

Host invasion by microorganisms

2008-09-10T11:45:11Z

Eayton:

{{toplink
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|linkpage =WikiBlood
|linktext =WIKIBLOOD
|thispagemap = Host invasion by microorganisms (Concept Map) - WikiBlood
|sublink1 =Immunology - WikiBlood
|subtext1 =IMMUNOLOGY
|pagetype =Blood
}}

===Introduction===

One of the main causes of disease is infection by microorganisms, with four types so far identified:
*Viruses
*Bacteria
*Fungi
*Parasites- comprising protozoa and helminths

All exist as either:
*Primary pathogens ('''obligate'''): presence always corresponds with disease
*Secondary ('''opportunistic'''): prerequisites must be fulfilled before disease occurs

Microorganisms can enter the body by three routes:
*'''Contact''': typically seen in dermatological infections
*'''Aerosol''': typically respiratory infections
*'''Orofecal''':typically enteric infections

===[[Viruses - WikiBlood|Viruses]]===

===[[Bacteria - WikiBlood|Bacteria]]===

===[[Parasites - WikiBlood|Parasites]]===

===[[Fungi - Wikiblood|Fungi]]===
===References===
*<div id="Janeway">{{citation|initiallast = Murphy|initialfirst = K|2last = Travers|2first = P|finallast = Walport|finalfirst = M|year = 2008|title = Janeway's Immunobiology|ed = 7th |city = New York|pub = Garland Science Publishing}}</div>

==[[Host invasion by microorganisms flashcards- Wikiblood|Host invasion flashcards]]==

File:Conjugative plasmids.png

2008-09-09T14:41:01Z

Eayton: Description : This image is a line drawing of bacterial conjugation. The image shows, going from the top to the bottom, two bacteria before, during, and after conjugation. On the top then are two bacterium, before conjugation, each with their own chromoso

Description : This image is a line drawing of bacterial conjugation. The image shows, going from the top to the bottom, two bacteria before, during, and after conjugation. On the top then are two bacterium, before conjugation, each with their own chromosomal DNA. Only one bacterium shows a plasmid. In the middle, are the same two bacterium during conjugation. A pilius (connection) forms between the two bacteria and a linear copy of the plasmid is transported through the pilius to the other bacterium. On the bottom, are the same two bacterium after conjugation. The pilius is now gone and each bacterium has a plasmid.
copyright Magnus Manske

File:Binary fission.png

2008-09-09T14:37:17Z

Eayton: copyright J W Schmidt

File:S aureus.jpg

2008-09-09T14:21:52Z

Eayton:

File:PapillomavirusCapsid.jpg

2008-09-09T14:09:36Z

Eayton:

Cytokines

2008-09-09T13:35:06Z

Eayton:

==Introduction==
The term cytokine is a generic name for the soluble molecules that mediate reactions between cells, acting via specific receptors on those cells. They are particularly important during effector stages of the immune system and the development of haematopoietic cells

==Nomenclature and classification==
Nomenclature of the cytokines was first based on their activity both ''in vivo'' and ''in vitro'', with the name being abbreviated to acronyms. For example, when a factor isolated from antigen-activated lymphocytes was added to non-immune peritoneal macrophages, their migration from capillary tubes was inhibited. This led to the factor being named migration inhibition factor, or MIF for short. It was then discovered that many biological functions were being produced by the same chemicals, which led to the classification of cytokines based on either the cell populations that secrete them or their function:
*'''Monokines'''- produced by myeloid cells (macrophages, monocytes)
*'''Lymphokines'''- produced primarily by lymphocytes
*'''Interleukins'''- produced by leukocytes in general
*'''Chemokines'''- directing cell migration, activating cells in response to infectious agents/tissue damage
*'''Interferons'''- produced by many different cells in response to viral infection

Common cytokines and nomenclature
{| border="1" cellpadding="2"
!width="50"|Name
!width="225"|Abbreviation
!width="225"|Examples
|-
|interleukins || IL || IL-1, IL-2
|-
|interferons || IFN || IFN-alpha
|-
|tumour necrosis factors || TNF || TNF-alpha
|-
|growth factors || GF || NGF, EGF
|-
|colony stimulating factors || CSF || M-CSF, G-CSF
|-
|chemokines || - || RANTES, MCP-1
|}
==Functions of cytokines==
'''Mediating and regulating innate immunity''': bacterial and viral products, such as LPS, stimulate macrophages and natural killer cells to secrete cytokines that primarily act on endothelial cells and leukocytes. They stimulate the early stages of the inflammatory reaction to microbes.

'''Mediating and regulating adaptive immunity''': in response to specific recognition by T lymphocytes, cytokines are produced that have a wide range of functions, including:
*regulating the growth and differentiation of some lymphocyte populations
*recruitment, activation and regulation of specialised effector cells, e.g. mononuclear phagocytes, neutrophils, eosinophils
'''Stimulating haematopoiesis''': produced primarily by bone marrow stromal cells and leukocytes, these cytokines stimulate the growth and differentiation of immature leukocytes
By binding onto specific receptors on cell membranes, cytokines are able to exert their actions by triggering signal-cascade mechanisms that ultimately result in altered gene expression. Cytokines have an incredibly high affnity for their receptors, and are therefore able to act at picomolar concentrations
Cytokines can exert antagonistic effects on each other

==Important cytokines==
Cytokines primarily produced by '''macrophages''':
*GM-CSF (granulocyte macrophage colony stimulating factor)- stimulates growth and differentiation of granulocytes, macrophages, neutrophils and eosinophils
*IL-1- stimulates Th2 cells and acute phase response
*IL-6- stimulates growth and differentiation of B and T cells and acute phase response
*IL-12- stimulates Th1 cells
*IL-18- stimulates IFN-gamma production by T cells and NK cells, favours Th1 response
*TNF-α- stimulates local inflammation and endothelial activation
Cytokines primarily produced by '''Th1 cells''':
*IL-2- stimulates proliferation and differentiation of T cells, activates NK cells and macrophages
*IFN-γ- activates macrophages, increases expression of MHC I and II molecules, increases antigen presentation
*TNF-β- stimlulates killing mechanisms in T and B cells and endothelial activation
Cytokines primarily produced by '''Th2 cells''':
*IL-4- activates B cells and IgE switch, supresses Th1 cells
*IL-5- stimulates eosinophil growth and differentiation
*IL-10- suppresses macrophage functions
Although neutrophils produce a lower amount of cytokines per cell than other immune cell types, they are often the first and most common cell type present at sites of infection. This makes them a physiologically important source of cytokines, such as IL-12.
==Chemokines==
The chemokines are a superfamily of cytokines, all related in terms of sequence and gene structure. The family is also known as the 'small cytokine' family (scy) or the intercrines. All have a relatively small molecular weight of ~5-10kDa and can be divided in one of two groups based on the position of the cystein residues (important for the tertiary structure):
*C-C subgroup- cysteine residues are adjacent to each other. Important members include:
**Interleukin 8
**MGSA- melanoma growth stimulatory activity
**PF4- platelet factor 4
**βTG- β-thromboglobulin
*C-X-C subgroup- residues are separated by another amino acid. Important members include:
**MCAF- macrophage chemotactic and activating factor
**RANTES
**LD-8
**ACT-2
Chemokines are released by many cell types, and are present in the earliest phase of infection, with actions including the following:
*Lymphoid trafficking
*Wound healing
*Th1/Th2 development
*Angiogenesis/angiostasis
*Lymphoid organ development
*Inflammation
*Cell recruitment
==Cytokines in pathology==
===Bacterial septic shock===
This is the overproduction of cytokines developing a few hours after infection by certain Gram-negative bacteria, including:
*''E. coli''
*''K. pneumoniae''
*''P. aeruginosa''
*''E. aerogenes''
Bacterial cell wall endotoxins are the cause of septic shock, stimulating macrophages to release IL-1 and TNF-α at excessive levels. The condition is often fatal and symptoms include a sudden drop in blood pressure, fever, diarrhoea and blood-clotting in multiple organs.
===Bacterial toxic shock===
This condition is caused by bacterial toxins known as superantigens (antigens that bind simultaneously to MHC II and the beta-V domain of the T cell receptor) that activate large numbers of T cells despite specificity. A number of bacteria have been implicated in the production of superantigens, including:
*''S. aureus''- produces enterotoxins and toxic-shock syndrome toxin
*''M. arthritidis''
The large number of T cells activated by such toxins (between 5-25% of all T cells, compared to less than 0.01% activated towards conventional antigens) means an excessive amount of cytokines produced, such as IL-1 and TNF. These elevated amounts cause the same systemic reactions as seen in bacterial septic shock.
===Lymphoid and myeloid cancers===
The excessive production of cytokines has been linked to some types of cancer, e.g. IL-6 has been shown to be secreted by myeloma cells, plasmacytoma cells and cervical and bladder cancer cells. IL-6 is known to act in an autocrine manner to stimulate cell proliferation.

Cytokines

2008-09-09T11:09:49Z

Eayton: