Biology Reference
In-Depth Information
T A B L E
6.2. Techniques for Measuring Cytokines
Lymphokine secretion:
1. Extracellular
i. ELISA
ii. Radioimmunoassay
iii. Sensitive cell lines
iv. ELISPOT
2. Intracellular
i. Flow cytometry
DNA fragmentation:
1. JAM test
2. DNA quanti®cation
Membrane stability:
1.
51
Chromium release
2. CARE-LASS (calcein-release-assay)
3. Time-resolved (BATDA)
4. Intracellular protein
Lymphokine Secretion Studies
HIV infection has been shown to lead to a variety of disturbances in the regu-
lation of cytokine expression. These disturbances include a general increase in
the expression of proin¯ammatory cytokines (TNF-a, IL-1, and IL-6) and anti-
viral IFN-a and -b, an increase in type 2 T-helper cytokines ( IL-4, IL-10, and
TGF-b), and a decrease in type 1 T-helper cytokines ( IL-2, IL-12, IL-15, and
IFN-g) (Fan et al., 1993). These changes may play a role in the pathogenesis
and dynamics of HIV infection ( Bailer et al., 1999; Valdez and Lederman,
1997). TGF-b has been shown to be immunosuppressive and is up-regulated by
the Tat protein of HIV ( Reinhold et al., 1999). TGF-b up-regulation has been
postulated to lead to immunosuppression of both B- and T-lymphocyte func-
tion, including inhibition of other cytokines such as IFN-a and -g (Sharma et
al., 1995). Our own studies suggest that HIV-speci®c TGF-b may be one cause
of nonspeci®c immunosuppression in HIV infection.
Many studies of cytokines have used IFN-g as a surrogate marker for CTL
function. IFN-g is produced by NK cells and T lymphocytes. IFN-g induces the
synthesis of host proteins important in defense against intracellular pathogens.
These include MHC class I and II molecules, TNF-a, adhesion molecules,
NADPH oxidase, and nitric oxide. IFN-g also down-regulates Th2 responses in
addition to having a modest direct antiviral activity ( Valdez and Lederman,
1997).
Measurement of IFN-g typically involves in vitro bulk studies that use
restimulation of T cells with HIV-speci®c peptides, proteins, or recombinant
viruses. IFN-g is measured in a variety of ways. These include techniques for
measuring secreted
IFN-g that
range
from
enzyme-linked immunosorbant
