Biomedical Engineering Reference
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types that play a role in the disease and the signaling pathways that are affected. The
former point is one that previously has been difficult to characterize because no
method was available that allowed the different cell types of peripheral blood to be
analyzed individually. Measurements were typically performed on the blood as a
whole, lysing and mixing all the different cellular subsets together. Information
regarding the complexity of the disease was lost and large changes in small cell
subsets were hidden due to lack of change in predominant cell subsets. With phospho
flow, disease complexity can be described, and harnessed, for more effective drug
development.
15.3 UNDERSTANDING DISEASE MECHANISM: AUTOIMMUNITY
AS CASE STUDY
Drug discovery has recently focused much attention on intracellular signaling in the
pursuit of rational drug design. Even as cell line assays reach new heights, there is a
growing realization that cell lines do not perfectly recapitulate the signaling biology
present in human disease, a difficulty compounded by the limitations of existing
pharmacodynamic assays. These factors contribute to the high rate of failure of drug
candidates during clinical trials that had looked promising early in the development
pipeline. But the difficulty transitioning from high-throughput cell line assays
to working with heterogeneous primary tissues requires new tools and a new
perspective.
15.3.1 Dynamic Signaling Profiles in Primary Samples
Flow cytometry is an ideal platform for resolving heterogeneous samples. The
technology has been embraced by immunologists for its ability to resolve all the
major populations of interest in a complex mixture of cells, even cell populations that
make up less than 0.1% of the sample. Only recently, however, has the flow cytometer
been recognized as a uniquely powerful proteomics platform for investigating
signaling biology. Signaling networks in eukaryotic cells make extensive use of
phosphorylation and dephosphorylation to control the activity of signaling kinases
and other classes of molecules used in signaling cascades. From our studies and those
of other research groups, we have learned that when cells undergo profound changes,
such as differentiation, activation, or transformation, there are underlying alterations
to intracellular signaling pathways that allow the cell to adapt its functional responses
to environmental stimuli. The most important modifications do not appear to be
in the basal activity of signaling networks, but rather involve changes in transduc-
tion of incoming signals from the extracellular environment. These changes are
revealed when the cells are probed with extracellular stimuli such as cytokines and
growth factors.
The ability to resolve heterogeneous populations is of critical value as disease
samples often appear to respond normally when analyzed in bulk, for example, in
whole blood lysates. This result is an artifact of averaging, because only a small subset
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