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Shear thresholding is a biophysical phenomenon that has only recently been elucidated and may have important ramifications for leukocyte trafficking, platelet adhesion, and targeted drug delivery and contrast agent design. The effect has been observed with in vitro flow assays: as shear increases, the numbers of neutrophils interacting with a ligand-coated surface increases until a peak is observed and then decreases.[1] This unusual behavior of the rolling cellular flux depends on both a biphasic dependence of an effective cellular adhesion rate for unbound cells as well as a biphasic effect of shear on the rolling velocity of cells that are already interacting.[2] Simulations have predicted that as receptor-ligand pairs form encounter complexes on nearby surfaces, the probability of bond formation depends on both the encounter rate and encounter duration, leading to an optimal level of shear to maximize bond formation probability.[3] Once attached, cellular deformability and the influence of forces on individual bonds also contribute.
I have been developing micropatterned surfaces using microfluidic chambers to pattern proteins for cell adhesion assays: the geometrically confined presentation of ligands enables the measurement of kinetic and biophysical interactions that are otherwise difficult to assess accurately. Cells or microbeads with attached ligands accumulate on a patterned surface at low shear. Cell-free buffer is then introduced at varying shear rates to minimize the occurrence of secondary interactions. Microparticles can be observed detaching from an upstream “catch strip” and may or may not form a tether with the downstream feature; a correction for the total cellular flux variation with shear is therefore built into the experiment. An effective cellular kon can be measured from the probability of tether formation on bars of varying widths. I am interested in combining this approach with engineered ligands to evaluate the impact of molecular flexibility, length, and binding pocket characteristics on cellular behavior.
I am interested in computational approaches in biology and have begun several projects that will yield a better mechanistic understanding of inflammatory and other immune responses. I have developed a biophysical model of microbead tethering and rolling. We are collaborating with Dr. Kenneth Breuer’s group at Brown University to deduce nano-scale changes in microparticle separation distances prior to tethering interactions. By coupling independent measurements of nano-scale model inputs with a micro-scale validation of model behavior using micropattern capture analysis, it should be possible to develop a model robust enough to interpret which biophysical and biomolecular characteristics are the most important for leukocyte capture in vivo. The predictive capabilities of the model can be tested by implementation as a design tool for targeted drug delivery and contrast agents.
I am also interested in modeling the signaling events involved in integrin activation. Tethered leukocytes that wish to transition from rolling to arrest and undergo diapedeses face special challenges not encountered by other model cell types, such as migrating fibroblasts. They must slow from traveling at higher velocities while experiencing greater stresses. The small G-protein, Rap-1, has been implicated in leukocyte polarization and motility, but still relatively little is known about the biomolecular pathway through which this molecule transduces the signal from chemokine receptors into changes in integrin affinity and avidity.[4] Our group has begun developing a computational model of the process to better elucidate the time scale of the processes involved and understand which intracellular signaling specializations might be specific to leukocytes.
I am also interested in how cellular interactions influence the outcome of disease. Immune responses tend to polarize towards a TH1 or TH2-directed response, and the decision is critical for the clinical outcome of many illnesses. For example, Leishmania species that elicit a TH2 response, such as donavani, can lead to a case of visceral leishmaniasis and are fatal. The body is able to combat the disease and recover with species that tend to elicit a TH1 response. The type of response the immune system mounts is a complex decision that takes many factors into account. Aside from the nature of the antigen, co-stimulatory molecules expressed on the cell presenting the antigen can also influence the response. Recent studies employing two-photon microscopy have enabled direct visualization of these interactions whithin the lymph node as the immune resonse matures.[5] We have begun incorporating this data into an agent-based model to see how interactions at the cellular level in the lymph node influence polarization.
1. Lawrence, M.B., G.S. Kansas, E.J. Kunkel, and K. Ley, Threshold levels of fluid shear promote leukocyte adhesion through selectins (CD62L,P,E). J Cell Biol, 1997. 136(3): p. 717-27.
2. Kadash, K.E., M.B. Lawrence, and S.L. Diamond, Neutrophil string formation: hydrodynamic thresholding and cellular deformation during cell collisions. Biophys J, 2004. 86(6): p. 4030-9.
3. Chang, K.C. and D.A. Hammer, The forward rate of binding of surface-tethered reactants: effect of relative motion between two surfaces. Biophys J, 1999. 76(3): p. 1280-92.
4. Shimonaka, M., K. Katagiri, T. Nakayama, N. Fujita, T. Tsuruo, O. Yoshie, and T. Kinashi, Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow. J Cell Biol, 2003. 161(2): p. 417-27.
5. Miller, M.J., O. Safrina, I. Parker, and M.D. Cahalan, Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes. J Exp Med, 2004. 200(7): p. 847-56.
Special thanks lab graduate Dr. Chris Paschall for getting me started with HTML!
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