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FcGAMMA
RECEPTOR MEDIATED CELLULAR FUNCTION
Fcgamma receptors (FcgammaR) are a
group of partially polymorphic cell surface receptors on a variety of
immuno competent cells. After contact with complexed (or monomeric) IgG
the FcgammaR induce cellular functions such as phagocytosis, antibody
mediated cellular cytotoxicity (ADCC), generation of reactive oxygen
species etc. Experimentally the receptors may be detected by blotting
methods or functionally by phagocytosis of specific target cells. The
allogenic FcgammaR forms are demonstrated by serology (indirect
immunofluorescence, granulocyte aggregation) or by allele specific PCR.
Immunophagocytosis as one example of FcgammaR mediated cellular
functions is inhibited in
vitro by antibodies of different specificities, e.g. antibody against
FcgammaRI, II and III and HLA antigens. Thus it is possible to determine
which of the different FcgammaR is involved in phagocytosis of specific
immunologic targets. On the other hand inhibition of monocyte immune
phagocytosis enables the detection of antibodies specific for HLA class
I and II antibodies which play an important role in graft rejection. The
underlying mechanism is the simultaneous binding of the Fab’ portion
of an HLA antibody to its corresponding antigen on the monocyte and
binding of the Fc portion of the same antibody molecule to a FcgammaR on
the same cell. Subsequently, the blocked FcgammaR is unable to induce
phagocytosis of immunoglobulin coated red blood cells which act as an
indicator for the process (Prof. Dr. med. J. Neppert, Dr. rer. Nat. B.
Flesch, ref. 1-3, 7-12). POLYMERASE
CHAIN REACTION (PCR)
The rapid production of large quantities of specific DNA sequence
took a leap forward with the development of the polymerase chain
reaction (PCR). The PCR requires two nucleotide oligomers that hybridize
to the complementary DNA strands in a region of interest. The oligomers
serve as primers for a DNA polymerase that extends each strand. Repeated
cycling of the PCR yields large amounts of each DNA molecule of interest
in a matter of hours as opposed to days and weeks associated with
cloning techniques.
The PCR amplification of a specific DNA sequence can be
accomplished with a purified DNA sample or a small region within a
complex mixture of DNA. The nucleotide sequence of the DNA to be
amplified must be known or it must be cloned in a vector where the
sequence of the flanking DNA has been established. The product of PCR is
a double-stranded DNA molecule and the reaction is completed in each
cycle when all of the template molecules have been copied. In order to
initiate a new round of replication the sample is heated to melt the
double-stranded DNA and, in the presence of excess oligonucleotide
primers, cooled to permit hybridization of the single-stranded template
with free oligomers. A new cycle of DNA replication will initiate in the
presence of DNA polymerase and all four dNTPs. Heating to about 95°C
as required for melting DNA inactivates most polymerases, but a heat
stable polymerase, termed Taq DNA polymerase isolated from Thermus
aquaticus, is now employed, obviating the need for fresh polymerase
after each cycle. This has permitted the automation of PCR with each DNA
molecule capable of being amplified one million-fold.
When the DNA to be amplified is present in very low
concentrations relative to the total DNA in the sample, it is possible
to amplify the DNA region of interest along with other spurious
sequences. In this situation the specificity of the amplification
reaction can be enhanced by nested PCR. After conducting the first PCR
with one set of primers for 10-20 cycles, a small aliquot is removed for
the second PCR. However, the second PCR is conducted with a new set of
primers that the complementary to the template DNA just downstream of
the first set of primers, or nested between the original set of primers.
This process amplifies the DNA region of interest twice with greatly
enhanced specificity.
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