Major Histocompatibility complex OR

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Major Histocompatibility complexOR

• MHC

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MHC

• The principal function of T cells are
• Defense against intracellular microbes
• Activation of other cells(B cells ,Macrophages )
• peptide recognition by T and B cells is
  different.
• B cells recognize soluble as well as cell
  associated Ags
• In contrast ,T cells recognize peptide which is
  displaying only by APC in associated with MHC
  proteins.

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MHC

• MHC genes are the most polymorphic genes
• MHC genes are codominantly expressed in each
  individual
• There are two main type of MHCclass I II
• The physiologic function of MHC molecules is
  presentation of peptides to T cells ,control of
  immune responsiveness to all proteins and graft
  rejection
• MHC-I present cytosolic peptides to T cytotoxic
  cells and MHC-II present endocytosed peptides to
  Th cells

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MHC genes
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• HLA-G ( a role in Ag recog.by NK cells)
• HLA-H (involved in iron metabolism
• HLA-DM (peptide binding to class II)

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• Class III
• C4,B,C2
• TNF,LT
• joined to class II
• Proteasome genes,TAP,DM

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MHC I structure
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MHC-II structure
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Conformational structure
MHC-II
MHC-I
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Peptide location
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Characteristics of peptide-MHC interaction

• Each class I or II has a single peptide binding
  cleft that are accommodate many different
  peptides
• Slow on-rate and very slow off-rate
• The MHC molecules of an individual dont
  discriminate between self and non self

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T cell receptor and MHC interaction
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Polymorphism of class II

• HLA-DPA 12
• HLA-DPB 88
• HLA-DQA 17
• HLA-DQB 42
• HLA-DR gt400

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Polymorphism of class I

• HLA-A gt280
• HLA-B gt500
• HLA-C gt130

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MHC genes and graft rejection
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HLA expression
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Peptide presentation by MHC-II
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Peptide presentation by MHC-I
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MHC I and peptide presentation
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Features of peptide binding to MHC
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HLA and diseases
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• Testing of DNA sequences permits detection of
  many more subtypes or "splits" of HLA antigens or
  alleles.
• In serological typing, some antigens are
  difficult to identify and may even mask the
  presence of others. DNA typing can routinely
  define antigens at the allele level, assuring no
  ambiguity in interpretations.
• DNA typing does not require live blood cells from
  the patient, permitting more flexible sample
  requirements. Thus, LabCorp can perform DNA-based
  HLA typing on

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More interesting facts

• Erythrocytes will adsorb some Class I antigens
  viz. Bg blood group system (B7,A28, B57.)
• HLA B most polymorphic system and studies have
  shown is most significant followed by A and then
  C
• 45Kd glycoprotein comprising three heavy chain
  domains, non-covalently associated

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TYPING METHODS

• SEROLOGY used to be the gold standard. Now
  being superceded by molecular techniques as they
  become more robust and time efficient
• CELLULAR rarely used now. Orginally used for
  Class II typing
• MOLECULAR fast becoming the method of choice.
  Many laboratories test of choice.

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SEROLOGY

• Complement Dependent Cytotoxicity (CDC)
• Viable peripheral blood lymphocytes are obtained
  by discontinous density gradient centrifugation
  using Ficoll / Tryosil or Ficoll / Sodium
  Metrizoate at a density of 1.077 at 19º - 22ºC.
• Microlymphocytotoxic test 3 stages

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Microlymphocyototoxic test

• 1.Viable lymphocytes are incubated with HLA
  specific antibodies. If the specific antigen is
  present on the cell the antibody is bound.
• 2.Rabbit serum as a source of complement is
  added, incubate. If antibody is bound to the HLA
  antigen on the cell surface it activates the
  complement which damages the cell membrane making
  it permeable to vital stains.

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Microlymphocyototoxic test 2

• 3.Results are visualised by adding dye usually a
  fluorochrome eg Ethidium Bromide although both
  Trypan Blue and Eosin have been used in the past.
• If the reaction has taken place the EB enters the
  cell and binds to the DNA.
• For ease double staining is normally used. We use
  a cocktail of Ethidium Bromide and Acridine
  Orange, quenched using Bovine Haemoglobin to
  allow simultaneous visualisation of both living
  and dead cells.

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Microlymphocytotoxicity test 3

• Test is left for 10 minutes and then read using
  an inverted fluorescient microscope.
• A mixture of T and B lymphocytes can be used for
  HLA Class I typing.
• B lymphocytes are required for HLA Class II
  typing by serology. (Normal population 85-90 T
  and 10-15 B cells)
• This can be achieved using a number of methods.

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• Easily performed does not require expensive
  equipment.
• Takes around three hours to perform
• Low level resolution, with good antisera reliable
  results
• Requires large volumes of blood
• Requires viable lymphocytes
• Difficult to find good antisera for rarer
  antigens in different populations

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molecular

• DNA extraction from the nucleated cells following
  cell lysis and protein digestion.
• polymerase chain reaction (PCR)

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Molecular Methods 4

• Electrophoresis is used following amplification.
  PCR product is run out on an agarose gel
  containing ethidium bromide. Each product moves
  according to its size and is compared to a
  molecular weight marker.
• Interpretation every tube should produce an
  identical sized product as internal control and
  either a specific band or not dependent on
  whether the allele(s) is/are present or not.
• Results are visualised using 312nm UV
  transillumination and recorded either by video
  imaging or polaroid photograghy.