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Biomarkers are
biological molecules that accurately and reliably indicate physiologic or
disease state. While the term biomarkers has been used more commonly in recent
times, biomarkers have been successfully used to diagnose and characterize
disease states for many decades. Two well-known biomarkers are glucose for
diabetes and cholesterol for heart disease. It is clear that biomarkers are the
key reagents for the development of robust in vitro assays critical to
patient diagnosis and treatment (6,12,18). Validation of biomarkers comes from
correlation of the presence of the biomarker to the in vivo disease
state. We have an opportunity through the use of sophisticated in vitro
microfluidics and in vivo imaging technology to bridge preclinical and
clinical studies and to understand the role of the specific molecules in the
disease process. The development and validation of novel biomarkers is a goal
that can only be realized as we reconcile the inherent differences between
humans and animal models of human disease that are relevant to predicting the
outcome of human clinical therapies. New molecular imaging tools and other
technologies have been brought to bear on creating more sophisticated animal
models of human disease for use in development and validation of novel
biomarkers and the subsequent use of these molecules for both in vitro
and in vivo diagnostic assays (2,11).
Pamela Reilly Contag,
Caliper Life Sciences
Summary
The requirement for accurate
and precise molecular diagnostics to characterize the human disease state to
deliver the appropriate and most effective therapies is driving the field to
develop more and novel biomarkers of disease. Certain classes of molecules lend
themselves to diagnosis of disease state because of their intrinsic regulatory
or messenger role in pathophysiology (2,10). Kinases and other immune system
molecules can be reliable indicators or biomarkers of disease or physiologic
state (3,5,15,17). For example, before there was an accurate diagnostic for HIV
viremia, a drop in the level of CD4 expression was indicative of a reduction in
T-helper cells and a hallmark of AIDS and is still used as prognostic for
disease outcome along with HIV viremia (9,13,14,16). In many cases, the innate
and adoptive immune system is an ideal source of biomarkers. Activation of these
molecules upon infection and inflammation can be readily monitored by in
vitro assays. However, in vitro monitoring is not always indicative
of what is happening in real time in vivo (8). One way to validate these
types of in vitro studies is with the appropriate in vivo animal
model to track the expression and activity of the biomarker of interest as the
disease is also being monitored in real time (2,11).
One approach to the physiologic
context limitations of in vitro assays is to build the in vitro
assay side by side with the in vivo animal model to combine the
multiparameter aspect of in vitro with the
multidimensional aspect of in vivo to create a total portrait of
the disease state. Validation of the in vitro assay against the
predictive animal model can then be performed using more sophisticated in
vivo endpoints. Real-time animal model disease progression data may then
be mirrored in a human clinical trial by using either the in vivo or
likely the in vitro assay from tissues or cells of humans.
click to enlarge
FIGURE 1. IN VITRO IKK2
MICROFLUIDIC PROTEIN KINASE ASSAY. Peaks show ratio of substrate to
phosphorylated product in the presence of a purified protein kinase. The
biphasic curve would be altered upon the action of an inhibitor or activator by
the increase or decrease of phosphorylated product.
We describe here a robust
microfluidic chip assay of IKB phosphorylation by I kappa kinase 2 (IKK2) and
corresponding in vivo imaging assays that model a relationship between
the IKK2 phosphorylation of IKB and the subsequent degradation of IKB by the
proteosome. This potentially represents a platform for the development of
biomarkers that span in vitro activity, predicting therapeutic efficacy
of a drug in whole animals with the ultimate goal of translation to the clinic.
click to enlarge
FIGURE 2. IKK2 is a protein
kinase that is up-regulated in inflammatory responses and cancers. LPS is a
small molecule that activates IKK2, which in turn phosphorylates IKB. The
phosphorylated IKB is directed to the proteosome and degraded and thus releases
NFKB (inhibited by unphosphorylated IKB) for entry into the nucleus. This
nuclear NFKB transcription factor acts on other genes including its own
regulator IKB. Any low level of inflammation that activates tumor necrosis
factor also activates NFKB and apoptosis.
FIGURE 3. IKB promoter
regulating expression of firefly luciferase, then used to create a transgenic
mouse line that emits light concomitantly when the IKB gene is transcribed.
click to enlarge
FIGURE
4. IKB-luc induction with LPS is demonstrated in a transgenic animal model that
has a firefly luciferase gene driven by the IKB mouse promoter and thus, as IKB
is transcribed, light is emitted that can be detected noninvasively by optical
imaging methodology.
Introduction
Across the drug development
continuum, biomarkers are often used in both preclinical and clinical studies as
well as in patient diagnostics. Biomarkers of physiologic state in vitro
and in vivo are used to diagnose disease and to determine disease
progression. In clinical trials, this information may determine patient
selection and diagnose therapeutic outcome. When used broadly in the clinic,
biomarkers may help determine customized therapies. Some of the challenges in
the identification of biomarkers include understanding the role of a specific
biomarker to a clinically relevant problem; developing either an indirect or
direct readout of physiologic state; determining the comparable pathways between
animal models and humans; and finally the conversion of the biomarker into a
robust assay and subsequent validation and approval of the assay in clinical
applications (2,11).
IKK2 is a protein kinase that
is up-regulated in inflammatory responses and cancers and thus can be implicated
in several diseases. We use an animal model of the induction of IKK2 by
lipopolysaccharide (LPS), the endotoxin molecule on the surface of Gram-negative
bacteria that induces the inflammatory response. LPS activates IKK2, which in
turn phosphorylates IKB. In vitro phosphorylation activity can be
quantitated in a robust microfluidic assay depicted in Figure 1. In vivo,
the phosphorylated IKB is directed to the proteosome and degraded and thus
releases NFKB (inhibited by unphosphorylated IKB) for entry into the nucleus.
This nuclear NFKB transcription factor acts on other genes including its own
regulator IKB. Any low level of inflammation that activates tumor necrosis
factor also activates NFKB and apoptosis (1). See Figure 2.
While the Protein Kinase IKK2
microfluidic assay has been shown to be both precise and accurate, subsequent
correlation of this protein kinase activity to a biological event in vivo
may provide validation for the in vitro assay as well as
create an in vivo assay for drug screening.
The creation of transgenic mice
to report activation of different genes that are immunoregulated or
immunomodulatory has been described (19-21). Also reported is the production of
a specific transgenic animal model that has a firefly luciferase gene driven by
the IKB mouse promoter and thus, as IKB is transcribed, light is emitted that
can be detected noninvasively by optical imaging methodology (20) (Figures 3 and
4). A similar image is generated when TNF is used to activate NFKB, which in
turn activates IKB transcription (data not shown).
The IKB-phosphorylated protein
has been monitored in a transgenic mouse developed by Gross and colleagues (4).
The IKB alpha subunit was fused to luciferase so that the IKB protein
degradation could be evaluated by optical imaging. Under normal conditions, LPS
activation phosphorylates IKB, which is then degraded by the proteosome.
Bortezomib is a drug that inhibits proteosome degradation, and it is evident by
the continued emission of light that the IKB is not being degraded by the
proteosome (4).
Discussion
The discovery and validation of
biomarkers of human disease in animal models and in humans is being facilitated
by new technologies such as microfluidics for high-quality in vitro
assays and molecular imaging for in vivo animal studies.
We have demonstrated that the
kinase phosphorylation measured in a IKK2 microfluidic kinase assay can also be
monitored in an in vivo model of inflammation and can be inhibited by
treatment with a known anti-inflammatory drug. Thus, the models and assays
described here can provide a method to discover and utilize biomarkers for
disease diagnosis, prognosis, and compound efficacy determination. The potential
for using several biomarkers in multiple parameter in vivo studies has
also been shown to be useful in evaluating drug treatments (7). These
multiparameter and multidimensional models may also be instrumental in the
discovery of related surrogate biomarkers (e.g., specific proteins) that could
be assayed ex vivo using a simple and rapid microfluidic assay as another
tool for assessment of drug candidates throughout the development process and
into clinical trials. It is thus a likely prediction that novel biomarkers will
be discovered, validated, and turned into robust and accurate in vitro
and in vivo assays through the use of animal model assays with endpoints
that are relevant to human disease.
References
1.
Bubici, C., et al. “The NF-kappaB-mediated control of ROS and JNK
signaling.” Histol Histopathol 21, 69-80; 2006.
2. Feigin, A. “Evidence from
biomarkers and surrogate endpoints.” NeuroRx 1, 323-30; 2004.
3. Griffin , J. W. et al. “Macrophage systems in peripheral nerves. A review.” J
Neuropathol Exp Neurol 52, 553-60; 1993.
4. Gross, S., and D. Piwnica-Worms.
“Real-time imaging of ligand-induced IKK activation in intact cells and in
living mice.” Nat Methods 2, 607-14; 2005.
5. Hacker, H. et al.
“Specificity in Toll-like receptor signalling through distinct effector
functions of TRAF3 and TRAF6.” Nature 439, 204-7; 2006.
6. Herder, C. et al.
“Inflammation and type 2 diabetes: results from KORA Augsburg.” Gesundheitswesen
67(Suppl 1), S115-21; 2005.
7. Kadurugamuwa, J.L., et al.
“Reduction of astrogliosis by early treatment of pneumococcal meningitis
measured by simultaneous imaging, in vivo, of the pathogen and host
response. Infect Immun 73, 7836-43; 2005.
8. Kirimanjeswara, G.S. et al.
“The complex mechanism of antibody-mediated clearance of Bordetella from the
lungs requires TLR4.” J Immunol 175, 7504-11; 2005.
9. Kornfeld, H. et al.
“Lymphocyte activation by HIV-1 envelope glycoprotein.” Nature 335,
445-8; 1988.
10. Pawelec, G. et al.
“Pathways to a robust immune response in the elderly.” Immunol All Clin N
Am 23, 1-13; 2003.
11. Pien, H.H. et al. “Using
imaging biomarkers to accelerate drug development and clinical trials.” Drug
Disc Today 10, 259-66; 2005.
12. Sampietro, T. et al.
“Inflammatory markers and serum lipids in idiopathic dilated cardiomyopathy.”
Am J Cardiol 96, 1718-20; 2005.
13. Sanchez-Ramon, S. et al.
“Low blood CD8+ T-lymphocytes and high circulating monocytes are predictors of
HIV-1-associated progressive encephalopathy in children.” Pediatrics
111, E168-75; 2003.
14. Sandberg, J.K. et al.
“Selective loss of innate CD4(+) V alpha 24 natural killer T cells in human
immunodeficiency virus infection.” J Virol 76, 7528-34; 2002.
15. Sato, S. et al. Essential
function for the kinase TAK1 in innate and adaptive immune responses.” Nat
Immunol 6, 1087-95; 2005.
16. Stuhler, G., and S.F.
Schlossman. “Antigen organization regulates cluster formation and induction of
cytotoxic T lymphocytes by helper T cell subsets.” Proc Natl Acad Sci
USA
94, 622-7; 1997.
17. Takahashi, H. et al.
“Pulmonary surfactant proteins A and D: innate immune functions and biomarkers
for lung diseases.” Curr Pharm Des 12, 589-98; 2006.
18. Turina, M. et al.
“Short-term hyperglycemia in surgical patients and a study of related cellular
mechanisms.” Ann Surg 243, 845-53; 2006.
19. Zhang, N. et al.
“NF-kappaB and not the MAPK signaling pathway regulates GADD45beta expression
during acute inflammation.” J Biol Chem 280, 21400-8; 2005.
20. Zhang, N. et al.
“Regulation of IkappaBalpha expression involves both NF-kappaB and the MAP
kinase signaling pathways.” J Inflam (London ) 2, 10; 2005.
21. Zhang, N. et al. “An
inducible nitric oxide synthase-luciferase reporter system for in vivo testing
of anti-inflammatory compounds in transgenic mice. J Immunol 170,
6307-19, 2003.
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