Basophils and mast cells Basophils and mast cells

and their importance in and their importance in

immune responsesimmune responses

Mast cellsMast cells

Mucosal mast cellsMucosal mast cells - in the mucous - in the mucous membranes of respiratory and gastrointestinal membranes of respiratory and gastrointestinal tract, produce histamine, serotonin, heparin, tract, produce histamine, serotonin, heparin, tryptase, leukotriene C4 ..., participate tryptase, leukotriene C4 ..., participate in parasitosis and allergy in parasitosis and allergy

CConnective tissue mast cellsonnective tissue mast cells - the connective - the connective tissue, producing tryptase, chymase, tissue, producing tryptase, chymase, prostaglandinD2 ..., are multiplicated in fibrosis, prostaglandinD2 ..., are multiplicated in fibrosis, in parasitosis and allergy are not participating in parasitosis and allergy are not participating

Mast cell functionsMast cell functions

Defense against parasitic infectionsDefense against parasitic infections

In pathological circumstances, responsible In pathological circumstances, responsible for the early type of hypersensitivity for the early type of hypersensitivity (immunopathological reaction typeI)(immunopathological reaction typeI)

Apply during inflammation, in angiogenesis, Apply during inflammation, in angiogenesis, in tissue remodeling in tissue remodeling

Mast cell activationMast cell activation

Mast cells can be stimulated to Mast cells can be stimulated to degranulate by:degranulate by:

cross-linking of IgE Fc receptorscross-linking of IgE Fc receptors

by anafylatoxins (C3a, C4a, C5a)by anafylatoxins (C3a, C4a, C5a)

direct injury, alcohol, some antibioticsdirect injury, alcohol, some antibiotics

Mast cell activation by cross-linking Mast cell activation by cross-linking of IgE Fc receptors of IgE Fc receptors

Establishing of multivalent antigen (multicellular parasite) Establishing of multivalent antigen (multicellular parasite) to IgE on highaffinnity Fc receptor for IgE (Fcto IgE on highaffinnity Fc receptor for IgE (FcRI) RI)

Aggregation of several molecules FcAggregation of several molecules FcRI RI

Initiate mast cell degranulation (cytoplasmic granules Initiate mast cell degranulation (cytoplasmic granules mergers with the surface membrane and release their mergers with the surface membrane and release their contents) contents)

Activation of arachidonic acid metabolism (leukotriene C4, Activation of arachidonic acid metabolism (leukotriene C4, prostaglandin D2) prostaglandin D2)

Start of production of cytokines (TNF, TGFStart of production of cytokines (TNF, TGF, IL-4, 5,6 ...) , IL-4, 5,6 ...)

Mast cell activation schemeMast cell activation scheme

Secretory products of mast cellsSecretory products of mast cells

Cytoplasmatic granulesCytoplasmatic granules: hydrolytic enzymes, : hydrolytic enzymes, proteoglycans (heparin, chondroitin sulphate), biogenic proteoglycans (heparin, chondroitin sulphate), biogenic amines (histamine, serotonin) amines (histamine, serotonin)

HistamineHistamine causes vasodilation, increased vascular causes vasodilation, increased vascular permeability, erythema, edema, itching, contraction of permeability, erythema, edema, itching, contraction of bronchial smooth muscle, increases intestinal peristalsis, bronchial smooth muscle, increases intestinal peristalsis, increased mucus secretion of mucosal glands in the increased mucus secretion of mucosal glands in the respiratory tract and GIT (helps eliminate the parasite) respiratory tract and GIT (helps eliminate the parasite)

Arachidonic acid metabolitesArachidonic acid metabolites (leukotriene C4, (leukotriene C4, prostaglandin D2) prostaglandin D2)

CytokinesCytokines (TNF, TGF (TNF, TGF , IL-4, 5,6 ...), IL-4, 5,6 ...)

The role of mast cells in development of The role of mast cells in development of allergyallergy

BasophilsBasophils Differentiate from myeloid precursor Differentiate from myeloid precursor

They are considered to be the circulating form of They are considered to be the circulating form of mast mast

Receptor equipment, containing granules, the Receptor equipment, containing granules, the mechanisms of stimulation and functions are very mechanisms of stimulation and functions are very similar to mast cellssimilar to mast cells

They are responsible for the emergence of They are responsible for the emergence of anaphylactic shock anaphylactic shock

basophil activation markers: CD 63, (CD 203) basophil activation markers: CD 63, (CD 203)

Immune mechanisms Immune mechanisms

of inflammation of inflammation

(Local and systemic (Local and systemic

reactions)reactions)

InflammationInflammation

* Is a summary of physiological responses to breach the Is a summary of physiological responses to breach the

integrity of the organism, leading to protection against integrity of the organism, leading to protection against infection of damaged sites, localization of damage and infection of damaged sites, localization of damage and healing. healing.

* * The first signals to the development of inflammatory The first signals to the development of inflammatory

responses come from mast cells, phagocytes, and the responses come from mast cells, phagocytes, and the substances released from damaged cells and substances released from damaged cells and extracellular components of matter. extracellular components of matter.

Local body's response to Local body's response to inflammationinflammation

ManifestationsManifestations - pain (dolor), fever (calor), redness (rubor), pain (dolor), fever (calor), redness (rubor), swelling (tumor) and loss of function (funkcio laesa) swelling (tumor) and loss of function (funkcio laesa)

- increased permeability of blood vessels (vasoactive increased permeability of blood vessels (vasoactive amines, complement components C3a, C5a, leukotrienes amines, complement components C3a, C5a, leukotrienes ..., swelling at site of inflammation) ..., swelling at site of inflammation)

- increased expression of adhesion molecules on endothelia - increased expression of adhesion molecules on endothelia

- activation of coagulation, fibrinolytic, kinin and complement - activation of coagulation, fibrinolytic, kinin and complement system system - influence of local nerve endings (prostaglandins, pain) - influence of local nerve endings (prostaglandins, pain)

- changes in temperature (IL-1, IL-6, TNF, prostaglandins) - changes in temperature (IL-1, IL-6, TNF, prostaglandins)

Systemic response to inflammationSystemic response to inflammation

- depends on the extent of damage and duration of local - depends on the extent of damage and duration of local inflammation inflammation

- - fever fever (proinflammatory cytokines TNF, IL-1, IFN (proinflammatory cytokines TNF, IL-1, IFN stimulate hypothalamic center of thermoregulation) stimulate hypothalamic center of thermoregulation)

- mobilization of tissue metabolism - mobilization of tissue metabolism

- induction of expression of Hsp (heat-shock-proteins; - induction of expression of Hsp (heat-shock-proteins; function as chaperones) function as chaperones)

- production of - production of acute phase proteinsacute phase proteins (CRP, SAP, C3, C4; (CRP, SAP, C3, C4; opsonization and complement activation) by liver after opsonization and complement activation) by liver after stimulation with cytokines (TNF-α, IL-1, IL-6)stimulation with cytokines (TNF-α, IL-1, IL-6)

- increased hepatic synthesis of certain serum transport proteins increased hepatic synthesis of certain serum transport proteins (ceruloplasmin, transferrin) (ceruloplasmin, transferrin)

- increased synthesis of protease inhibitors (- increased synthesis of protease inhibitors ( macroglobulin) macroglobulin)

- - leukocytosisleukocytosis

Septic shockSeptic shock - - the massive penetration of microorganisms the massive penetration of microorganisms into the bloodstream into the bloodstream (TNF) (TNF)

Anaphylactic shockAnaphylactic shock - basophil degranulation and complement - basophil degranulation and complement activation with allergen (histamine) activation with allergen (histamine)

Repair of damaged tissueRepair of damaged tissue

- elimination of damaged cells with phagocytes - elimination of damaged cells with phagocytes

- activation of fibroplastic mechanisms - activation of fibroplastic mechanisms

- activation of angiogenesis - activation of angiogenesis

- regeneration and tissue remodeling - regeneration and tissue remodeling

Physiological Physiological

mechanisms of mechanisms of

regulation of the regulation of the

immune systemimmune system

Regulation by antigenRegulation by antigen

Induce immune responses and extinctionInduce immune responses and extinction

Affinity maturation of B lymphocytes Affinity maturation of B lymphocytes

Maintaining immunological memoryMaintaining immunological memory

Antigenic competition Antigenic competition

Threshold density of the complex MHC II-gp Ag on Threshold density of the complex MHC II-gp Ag on APC APC

Regulation by antagonistic peptidesRegulation by antagonistic peptides

AgonistAgonist - antigenic peptide, which induce full T - antigenic peptide, which induce full T cell response (proliferation, differentiation, Tcell response (proliferation, differentiation, THH or or TTCC and stimulation of effector functions) and stimulation of effector functions)

AntagonistAntagonist - (partial agonist) peptide structurally - (partial agonist) peptide structurally similar to antigen-peptide, which induce similar to antigen-peptide, which induce qualitatively different response of T lymphocytes qualitatively different response of T lymphocytes (production of only some cytokines, anergy, ...) (production of only some cytokines, anergy, ...)

Negative signals induced by antagonist may Negative signals induced by antagonist may overcome positive signals induced by agonist overcome positive signals induced by agonist (which is in the body in excess), it is used by (which is in the body in excess), it is used by some microorganisms some microorganisms

Regulation by antibodiesRegulation by antibodies

Antibodies competes with the BCR for antigen Antibodies competes with the BCR for antigen (negative regulator of B lymphocyte stimulating) (negative regulator of B lymphocyte stimulating)

IgG immune complexes bind to the BCR and FcIgG immune complexes bind to the BCR and FcR R on B cells, resulting in blocking activation of B on B cells, resulting in blocking activation of B lymphocytes lymphocytes

Regulation via idiotypic network Regulation via idiotypic network

Regulation by cytokines and cellular Regulation by cytokines and cellular contactcontact

Interaction APC - T lymphocyte Interaction APC - T lymphocyte

Interaction TInteraction THH1 – macrophages1 – macrophages

Interaction TInteraction THH2 - B lymphocytes 2 - B lymphocytes

Mutual regulation of activity TMutual regulation of activity THH1 versus T1 versus THH2 2

Development of leukocyte subpopulations Development of leukocyte subpopulations

Negative regulation of effector cells:Negative regulation of effector cells:

CTLA-4 - T cell inhibitory receptor, binds ligands CD80 and CTLA-4 - T cell inhibitory receptor, binds ligands CD80 and

CD86CD86

Inhibitory receptors of NK cellsInhibitory receptors of NK cells

Self-destruction interaction of the apoptotic receptor Fas with Self-destruction interaction of the apoptotic receptor Fas with

ligand FasL on the surface of activated T lymphocytes ligand FasL on the surface of activated T lymphocytes

Interaction of APC with T lymphocyteInteraction of APC with T lymphocyte

T cell:T cell:

TCR - antigen-specific receptor (signal 1)TCR - antigen-specific receptor (signal 1)

CD4 or CD8 - coreceptor (MHCgp binding)CD4 or CD8 - coreceptor (MHCgp binding)

CD 28 - costimulatory receptor (signal 2, binds CD 80, CD 86)CD 28 - costimulatory receptor (signal 2, binds CD 80, CD 86)

CTLA-4 - inhibitory receptor (binds CD 80, CD 86)CTLA-4 - inhibitory receptor (binds CD 80, CD 86)

CD-40LCD-40L

APC:APC:

MHC gp I + antigenic peptideMHC gp I + antigenic peptide

MHC gp II + antigenic peptideMHC gp II + antigenic peptide

CD 80, CD 86 - costimulatory ligandsCD 80, CD 86 - costimulatory ligands

CD 40 CD 40

Suppression mediated by T Suppression mediated by T lymphocyteslymphocytes

Mutual negative interaction TMutual negative interaction THH1 and T1 and THH2 cytokine-mediated 2 cytokine-mediated

(T(THH2 lymphocytes produce IL-4 and IL-10 that suppress the 2 lymphocytes produce IL-4 and IL-10 that suppress the

immune response based on TH1 cells)immune response based on TH1 cells)

CD 8CD 8++ T TSS - suppressor T cells has not yet been isolated - suppressor T cells has not yet been isolated

as a separate subset (partly identical with T as a separate subset (partly identical with TCC) )

              - negatively regulate the activation of other T cells              - negatively regulate the activation of other T cells

Soluble suppressor factors - some CD 8Soluble suppressor factors - some CD 8++ T lymphocytes T lymphocytes produce a soluble form of TCR produce a soluble form of TCR

Clonal elimination or anergy of T lymphocytes after contact Clonal elimination or anergy of T lymphocytes after contact with antigen on the surface of other cells than APC (lacking with antigen on the surface of other cells than APC (lacking costimulating signals)costimulating signals)

Regulatory T cells (TRegulatory T cells (Trr1 CD 41 CD 4++) help to maintain tolerance to ) help to maintain tolerance to

autoantigensautoantigens

Factors influencing the outcome of the Factors influencing the outcome of the immune responseimmune response

The same antigen can induce an active immune The same antigen can induce an active immune response or an active state of tolerance, the result response or an active state of tolerance, the result of response depends on many factors:of response depends on many factors:

State of the immune systemState of the immune system

Properties of antigen Properties of antigen

Dose of antigenDose of antigen

Route of antigen administration Route of antigen administration

CytokinesCytokines

(Tissue hormones) (Tissue hormones)

CytokinesCytokines

Regulatory proteins and glycoproteins produced by Regulatory proteins and glycoproteins produced by leukocytes and other cells leukocytes and other cells

Essential regulators of the immune systemEssential regulators of the immune system

Apply also outside the immune system Apply also outside the immune system (angiogenesis, tissue regeneration, carcinogenesis, (angiogenesis, tissue regeneration, carcinogenesis, treatment of many brain functions, embryonic treatment of many brain functions, embryonic development ...) development ...)

Cytokines - secreted Cytokines - secreted      - membrane (CD 80, CD86, CD40L,      - membrane (CD 80, CD86, CD40L, FasL ..) FasL ..)

Pleiotropic effectPleiotropic effect

Operates in a cascadeOperates in a cascade

Cytokine Network Cytokine Network

Cytokine system is redundant Cytokine system is redundant

Effects of cytokinesEffects of cytokines - autocrine - autocrine - paracrine - paracrine

- endocrine - endocrine

Are known as interleukins (exception: TNF, Are known as interleukins (exception: TNF, lymphotoxin, TGF, interferons, CSF and growth lymphotoxin, TGF, interferons, CSF and growth factors) factors)

B cells communicate via cytokines with other

inflammatory cells, such as T cells and macrophages

Distribution of cytokines by functionDistribution of cytokines by function

Proinflammatory cytokines (IL-1, IL-6,IL- 8,IL- 12,IL- 18, TNF)Proinflammatory cytokines (IL-1, IL-6,IL- 8,IL- 12,IL- 18, TNF)

Antiinflammatory cytokines (IL-1Ra, IL-4, IL-10, TGFAntiinflammatory cytokines (IL-1Ra, IL-4, IL-10, TGF))

Cytokines with the activity of hematopoietic cells growth factorCytokines with the activity of hematopoietic cells growth factor (IL-2, 3, 4, 5, 6, 7, 9, 11, 14, 15, CSF, SCF, LIF, EPO) (IL-2, 3, 4, 5, 6, 7, 9, 11, 14, 15, CSF, SCF, LIF, EPO)

Cytokines applying in TCytokines applying in THH2 humoral immunity (IL-4, 5, 9, 13) 2 humoral immunity (IL-4, 5, 9, 13)

Cytokines applying in the cell-mediated immunity TCytokines applying in the cell-mediated immunity THH1 1 (IL-2, 12, IFN (IL-2, 12, IFN, GM-CSF, lymphotoxin) , GM-CSF, lymphotoxin)

Cytokines with anti-virus effect (IFN-Cytokines with anti-virus effect (IFN-, IFN-, IFN-, IFN- , IFN- ))

Cytokine receptorsCytokine receptors Consisting of 2 or 3 subunits Consisting of 2 or 3 subunits

One subunit binds cytokine, other are associated One subunit binds cytokine, other are associated with cytoplasmic signaling molecules (protein with cytoplasmic signaling molecules (protein kinases) kinases)

Signaling subunit is shared by several different Signaling subunit is shared by several different cytokine receptors - called receptor family cytokine receptors - called receptor family

Signaling through these receptors may lead to Signaling through these receptors may lead to proliferation, differentiation, activation of effector proliferation, differentiation, activation of effector mechanisms or blocking the cell cycle and mechanisms or blocking the cell cycle and induction of apoptosis induction of apoptosis

HLA systemHLA system

(MHC glycoproteins) (MHC glycoproteins)

MHC glycoproteins class I MHC glycoproteins class I (Major histocompatibility complex)(Major histocompatibility complex)

The function of MHCgpI is presentation of peptide The function of MHCgpI is presentation of peptide fragments from inside the cell (which are produced fragments from inside the cell (which are produced by cell, including viral peptides if are present) on by cell, including viral peptides if are present) on the cell surface so to T lymphocytes (cytotoxic the cell surface so to T lymphocytes (cytotoxic CD8+) CD8+)

Present on all nuclear cells of the organism Present on all nuclear cells of the organism

3 isotype classical human MHC gp.3 isotype classical human MHC gp. (HLA - A,-B,-C)(HLA - A,-B,-C)

3 isotype-classical MHC gp.3 isotype-classical MHC gp. (HLA - E,-F,-G; (HLA - E,-F,-G; molecule CD1) molecule CD1)

Structure of MHC gp IStructure of MHC gp I

MHC gp class I consists of transmembrane MHC gp class I consists of transmembrane chain chain and non-covalently associated and non-covalently associated 22mikroglobulin mikroglobulin

chain has 3 domains, 2 N-terminal (chain has 3 domains, 2 N-terminal (1, 1, 2 - 2 - binding site for peptides) and 1 C-terminal binding site for peptides) and 1 C-terminal domain (domain (3 - anchored in the cytoplasmic 3 - anchored in the cytoplasmic membrane, a structure similar to imunoglobulin membrane, a structure similar to imunoglobulin domain)domain)

Binding of peptide is necessary for a stable Binding of peptide is necessary for a stable conformation of MHCgp and thus ensure its long conformation of MHCgp and thus ensure its long presentation on the cell surface presentation on the cell surface

Peptides binding to MHCgpIPeptides binding to MHCgpI MHC gp I bind peptides with a length of 8 to 10 MHC gp I bind peptides with a length of 8 to 10

aminoacidesaminoacides

Certain MHC gp molecule binds peptides sharing Certain MHC gp molecule binds peptides sharing common structural features - common structural features - coupling motifcoupling motif (critical are (critical are aminoacides near the end of peptide) aminoacides near the end of peptide)

The binding of endogenous peptides occurs in the The binding of endogenous peptides occurs in the endoplasmic reticulum during biosynthesis of MHC gp endoplasmic reticulum during biosynthesis of MHC gp

After a string After a string and and 22mikroglobulin create in the ER, mikroglobulin create in the ER, folding into the correct conformation and the mutual folding into the correct conformation and the mutual association and the association of an appropriate association and the association of an appropriate peptide, the complex is further processed in the Golgi peptide, the complex is further processed in the Golgi apparatus and then is presented on the cell surface apparatus and then is presented on the cell surface

Linked peptides derived from proteins degraded Linked peptides derived from proteins degraded proteasome, which cleaves cytoplasmic proteins for proteasome, which cleaves cytoplasmic proteins for destruction (labeled with ubiquitin), peptide fragments destruction (labeled with ubiquitin), peptide fragments are transported into the ER by specific membrane are transported into the ER by specific membrane pump pump

Peptides binding to MHC gp IPeptides binding to MHC gp I

Peptides binding to MHC gp IPeptides binding to MHC gp I

Non-classical MHC gpNon-classical MHC gp II

HLA - E,-F,-G; CD1 moleculesHLA - E,-F,-G; CD1 molecules

Structurally similar to classical MHC gp Structurally similar to classical MHC gp

Are less polymorphic Are less polymorphic

There are only on some cells There are only on some cells

They specialize in binding of specific ligands They specialize in binding of specific ligands

HLA-E and HLA-G - occurs on the trophoblast cells HLA-E and HLA-G - occurs on the trophoblast cells

Complexes of HLA-E and HLA-G with peptides are Complexes of HLA-E and HLA-G with peptides are recognized by inhibiting receptors of NK cells and recognized by inhibiting receptors of NK cells and contribute to the tolerance of the fetus in utero contribute to the tolerance of the fetus in utero

CD1 molecules - bind glycolipids (recognized by CD1 molecules - bind glycolipids (recognized by NK-T lymphocytes) NK-T lymphocytes)

MHC glycoproteins class IIMHC glycoproteins class II

The function of MHC gpII is the presentation of The function of MHC gpII is the presentation of peptide fragments from protein whitch were peptide fragments from protein whitch were engulfed by antigen presenting cell on the cell engulfed by antigen presenting cell on the cell surface to T lymphocytes (auxiliary CD4) surface to T lymphocytes (auxiliary CD4)

Occur on the APC (dendritic cells, monocytes, Occur on the APC (dendritic cells, monocytes, macrophages, B lymphocytes) macrophages, B lymphocytes)

3 isotypes of MHC gpII (DR, DQ, DP) 3 isotypes of MHC gpII (DR, DQ, DP)

Structure of MHC gp IIStructure of MHC gp II MHC gp II consist of 2 non-covalently associated MHC gp II consist of 2 non-covalently associated

transmembrane subunits transmembrane subunits and and

The peptide binding site consists of N-terminal The peptide binding site consists of N-terminal domains domains 1 and 1 and 1 1

Binding of peptide is necessary for a stable MHC Binding of peptide is necessary for a stable MHC gp conformation and thus ensure its long gp conformation and thus ensure its long

presentation on the cell surfacepresentation on the cell surface

Binding of peptides to MHC gp IIBinding of peptides to MHC gp II

MHC gpII bind peptides with a length of 15 to 35 MHC gpII bind peptides with a length of 15 to 35 aminoacides (but possibly longer - because the peptide aminoacides (but possibly longer - because the peptide binding site is open at both ends) binding site is open at both ends)

Certain MHC gp molecule binds peptides sharing common Certain MHC gp molecule binds peptides sharing common structural features - structural features - coupling motif coupling motif

After a string After a string and and are created in ER, fold into the correct are created in ER, fold into the correct conformation and the mutual associated are connected with conformation and the mutual associated are connected with another transmembrane chain called invariant chain, which another transmembrane chain called invariant chain, which blocks the binding site for the peptide, this complex is blocks the binding site for the peptide, this complex is further processed in the Golgi apparatus, secretory vesicles further processed in the Golgi apparatus, secretory vesicles isolated from GA merge with endosomes, then split the isolated from GA merge with endosomes, then split the invariant chain and peptide fragments from cell absorbed invariant chain and peptide fragments from cell absorbed proteins bind into binding site of MHC gp and the complex is proteins bind into binding site of MHC gp and the complex is then presented on cell surfacethen presented on cell surface

Peptides binding to MHC gp IIPeptides binding to MHC gp II

Polymorphism of MHC glycoproteinsPolymorphism of MHC glycoproteins HLA complex is located on chromosome 6 HLA complex is located on chromosome 6

For MHC gp is typical high polymorphism (except For MHC gp is typical high polymorphism (except the non-classical MHC gp, and DR the non-classical MHC gp, and DR chain) chain)

Codominant inheritance of alelic forms Codominant inheritance of alelic forms (Individual has 3 cell surface isotypes (Individual has 3 cell surface isotypes of HLA molecules (HLA-A,-B,-C) mostly of HLA molecules (HLA-A,-B,-C) mostly in 2 different alelic forms) in 2 different alelic forms)

Polymorphism has a protective significance at Polymorphism has a protective significance at individual and population level individual and population level

Ppolymorphism MHC gp causes complications in Ppolymorphism MHC gp causes complications in transplantation transplantation

HLA typing = determmination of HLA HLA typing = determmination of HLA antigens on the surface of lymphocytesantigens on the surface of lymphocytes

Carry out during the testing before transplantation Carry out during the testing before transplantation and in determination of paternityand in determination of paternity 1) Serotyping - 1) Serotyping - Microlymfocytotoxic test Microlymfocytotoxic test Allospecific serumsAllospecific serums (obtained from multiple natal to 6 weeks after (obtained from multiple natal to 6 weeks after

birth, obtained by vaccination of volunteers, or commercially birth, obtained by vaccination of volunteers, or commercially prepared sets of typing serums (monoclonal antibodies)) prepared sets of typing serums (monoclonal antibodies))

PrinciplePrinciple - the incubation of lymphocytes with typing serums in - the incubation of lymphocytes with typing serums in the the presence of rabbit complement, then is added the presence of rabbit complement, then is added the vital vital dye which stained dead cells dye which stained dead cells

- cells carrying a specific HLA are killed by cytotoxic Ab - cells carrying a specific HLA are killed by cytotoxic Ab against the Ag, the percentage of dead cells is a against the Ag, the percentage of dead cells is a measure measure of serum toxicity (forces and antileukocyte antibody of serum toxicity (forces and antileukocyte antibody titre)titre)

Positive reaction is considered more than 10% dead cells Positive reaction is considered more than 10% dead cells

(serological typing can be done also by flow cytometry (serological typing can be done also by flow cytometry

2) Molecular genetic methods2) Molecular genetic methods For typing are used hypervariable sections in the II. exon For typing are used hypervariable sections in the II. exon

genes coding for HLA class II; to determine HLA class I is genes coding for HLA class II; to determine HLA class I is used polymorphism in II. and III. exon coding genesused polymorphism in II. and III. exon coding genes

2a) PCR-SSP2a) PCR-SSP = Polymerase chain reaction with sequential specific = Polymerase chain reaction with sequential specific primers primers

Extracted DNA is used as a substrate in a set of PCR Extracted DNA is used as a substrate in a set of PCR reactionsreactions

Each PCR reaction contains primers pair specific for a Each PCR reaction contains primers pair specific for a certain allele (or group of alleles) certain allele (or group of alleles)

Positive and negative reactions are evaluated by Positive and negative reactions are evaluated by electrophoresis, each combination of alleles has a electrophoresis, each combination of alleles has a specific electrophoretic painting specific electrophoretic painting

2b) PCR-SSO2b) PCR-SSO

PCR reaction with sequence-specific oligonucleotides PCR reaction with sequence-specific oligonucleotides Multiplication of hypervariable sections of genes Multiplication of hypervariable sections of genes coding HLA coding HLA

Hybridization with enzyme or radiolabeled DNA probes Hybridization with enzyme or radiolabeled DNA probes specific for individual alleles specific for individual alleles

2c) PCR-SBT2c) PCR-SBT

Sequencing based typingSequencing based typing

The most accurate method of HLA typing.The most accurate method of HLA typing.

We get the exact sequence of nucleotides, which We get the exact sequence of nucleotides, which compares with a database of known sequences of HLA compares with a database of known sequences of HLA alleles alleles

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