Collaborative academic medical product development: An 8-year review of commercialization outcomes at the Institute of Translational Health Sciences.

INTRODUCTION: The Institute of Translational Health Sciences (ITHS), a Clinical and Translational Science Award (CTSA)-funded program at the University of Washington (UW), established the Drug and Device Advisory Committee (DDAC) to provide product-specific scientific and regulatory mentoring to investigators seeking to translate their discoveries into medical products. An 8-year retrospective analysis was undertaken to evaluate the impact of the DDAC programs on commercialization metrics. METHODS: Tracked metrics included the number of teams who consulted with the DDAC, initiated a clinical trial, formed a startup, or were successful obtaining federal small business innovation awards or venture capital. The review includes historical comparisons of the startup rates for the UW School of Medicine and the Fred Hutchinson Cancer Research Center, two ITHS-affiliated institutions that have had different DDAC utilization rates. RESULTS: Between 2008 and 2016, the DDAC supported 161 unique project teams, 28% of which went on to form a startup. The commercialization rates for the UW School of Medicine increased significantly following integration of the DDAC into the commercialization programs offered by the UW technology transfer office. CONCLUSIONS: A formalized partnership between preclinical consulting and the technology transfer programs provides an efficient use of limited development funds and a more in-depth vetting of the business opportunity and regulatory path to development.

Deformability limits of Plasmodium falciparum-infected red blood cells.

Splenic filtration of infected red blood cells (RBCs) may contribute to innate immunity and variable outcomes of malaria infections. We show that filterability of individual RBCs is well predicted by the minimum cylindrical diameter (MCD) which is calculated from a RBC's surface area and volume. The MCD describes the smallest diameter tube or smallest pore that a cell may fit through without increasing its surface area. A microfluidic device was developed to measure the MCD from thousands of individual infected RBCs (IRBCs) and uninfected RBCs (URBCs). Average MCD changes during the blood-stage cycle of Plasmodium falciparum were tracked for the cytoadherent strain ITG and the knobless strain Dd2. The MCD values for IRBCs and URBCs raise several new intriguing insights into how the spleen may remove IRBCs: some early-stage ring-IRBCs, and not just late-stage schizont-IRBCs, may be highly susceptible to filtration. In addition, knobby parasites may limit surface area expansions and thus confer high MCDs on IRBCs. Finally, URBCs, in culture with IRBCs, show higher surface area loss which makes them more susceptible to filtration than naive URBCs. These findings raise important basic questions about the variable pathology of malaria infections and metabolic process that affect volume and surface area of IRBCs.

Fibronectin in aging extracellular matrix fibrils is progressively unfolded by cells and elicits an enhanced rigidity response.

While the mechanical properties of a substrate or engineered scaffold can govern numerous aspects of cell behavior, cells quickly start to assemble their own matrix and will ultimately respond to their self-made extracellular matrix (ECM) microenvironments. Using fluorescence resonance energy transfer (FRET), we detected major changes in the conformation of a constituent ECM protein, fibronectin (Fn), as cells fabricated a thick three-dimensional (3D) matrix over the course of three days. These data provide the first evidence that matrix maturation occurs and that aging is associated with increased stretching of fibronectin fibrils, which leads to at least partial unfolding of the secondary structure of individual protein modules. A comparison of the conformations of Fn in these 3D matrices with those constructed by cells on rigid and flexible polyacrylamide surfaces suggests that cells in maturing matrices experience a microenviroment of gradually increasing rigidity. In addition, further matrix stiffening is caused by active Fn fiber alignment parallel to the contractile axis of the elongated fibroblasts, a cell-driven effect previously described for other fibrillar matrices. The fibroblasts, therefore, not only cause matrix unfolding, but reciprocally respond to the altered Fn matrix properties by up-regulating their own rigidity response. Consequently, our data demonstrate for the first time that a matured and aged matrix has distinctly different physical and biochemical properties compared to a newly assembled matrix. This might allow cells to specifically recognise the age of a matrix.

Microfluidic approaches to malaria pathogenesis.

Malaria is a major poverty-related human infectious disease of the world. Over a billion individuals are under threat and several million die from malaria every year. The nature of disease, especially fatal disease, has been the subject of many studies. The consensus is that parasite-induced cytoadherance of red blood cells precipitates capillary blockage and inflammatory responses in affected organs. Reduced deformability of infected erythrocytes may also contribute to disease. What is not very clear is why people with significant parasite burdens display large variations in disease outcomes. Technologies which allow a detailed description of the cytoadherance properties of infected erythrocytes in individual patients, and which allow a complete description of the flow capabilities of red blood cell populations in that patient, would be very useful. Here we review the recent introduction of microfluidic technology to study malaria pathogenesis, including the fabrication processes. The devices are cheap, versatile, portable and require very small patient samples. With greater use in research laboratories and field sites, we eventually expect microfluidic methods to play important roles in malaria diagnosis, as well as prognosis.

Microfluidic modeling of cell-cell interactions in malaria pathogenesis.

The clinical outcomes of human infections by Plasmodium falciparum remain highly unpredictable. A complete understanding of the complex interactions between host cells and the parasite will require in vitro experimental models that simultaneously capture diverse host-parasite interactions relevant to pathogenesis. Here we show that advanced microfluidic devices concurrently model (a) adhesion of infected red blood cells to host cell ligands, (b) rheological responses to changing dimensions of capillaries with shapes and sizes similar to small blood vessels, and (c) phagocytosis of infected erythrocytes by macrophages. All of this is accomplished under physiologically relevant flow conditions for up to 20 h. Using select examples, we demonstrate how this enabling technology can be applied in novel, integrated ways to dissect interactions between host cell ligands and parasitized erythrocytes in synthetic capillaries. The devices are cheap and portable and require small sample volumes; thus, they have the potential to be widely used in research laboratories and at field sites with access to fresh patient samples.

Single molecule fluorescence studies of surface-adsorbed fibronectin.

The conformation of the extracellular matrix protein fibronectin plays a critical role in regulating cell function, including cell adhesion and migration. While average conformations of large ensembles of adhesion proteins have been previously measured, cells may sensitively respond to conformational outliers. We therefore applied both single molecule imaging and spectroscopy techniques to map a range of conformational states of individual fibronectin molecules adsorbed to glass, as well as to measure their conformational fluctuations in time. Single-step photobleaching experiments confirmed single molecule sensitivity. Single molecule spectra showed fluctuations in the peak wavelength, both over a spatial ensemble of molecules and in a single molecule over time, most likely indicating the different conformational states fibronectin can assume upon surface adsorption. Single-pair fluorescence resonance energy transfer (FRET) revealed that a fraction of fibronectin molecules existed in conformations that allowed for energy transfer between the labeled cysteine residues of the two dimeric arms folded upon each other, and that fluctuations occurred in the FRET efficiency. Fluorescence polarization experiments identified two possible sources of FRET fluctuations: changes in fluorophore orientation and conformational fluctuations of fibronectin over a time scale of seconds.

Fibronectin conformational changes induced by adsorption to liposomes.

One of the major drawbacks of drug delivery techniques that utilize liposomes as carriers is that they are often cleared from the body before they can deliver their therapeutic cargo. It is well known that serum proteins can adsorb to these drug delivery vehicles and influence their uptake by phagocytic cells. For this reason, protein adsorption to liposomes has been extensively quantified, and strategies have been developed to minimize protein adsorption to improve drug delivery. However, the conformation of proteins on surfaces can play an even greater role in controlling cell behavior than the quantity of adsorbed protein. We have therefore used fluorescence resonance energy transfer (FRET) to measure changes in the structure of fibronectin (Fn)--a key serum protein involved in phagocytosis--upon interaction with phosphatidylcholine (PC) liposomes. Our experiments reveal that fibronectin opens up from its inactive, compact conformation upon interaction with gel phase PC liposomes. We also used FRET to estimate a physiologically relevant dissociation constant, KD=1.1 nM, for the interaction. Conformational changes in serum proteins may result in the exposure of otherwise concealed recognition sites and therefore influence the interaction of liposomes with phagocytic cells.

Liquid-crystalline collapse of pulmonary surfactant monolayers.

During exhalation, the surfactant film of lipids and proteins that coats the alveoli in the lung is compressed to high surface pressures, and can remain metastable for prolonged periods at pressures approaching 70 mN/m. Monolayers of calf lung surfactant extract (CLSE), however, collapse in vitro, during an initial compression at approximately 45 mN/m. To gain information on the source of this discrepancy, we investigated how monolayers of CLSE collapse from the interface. Observations with fluorescence, Brewster angle, and light scattering microscopies show that monolayers containing CLSE, CLSE-cholesterol (20%), or binary mixtures of dipalmitoyl phosphatidylcholine(DPPC)-dihydrocholesterol all form bilayer disks that reside above the monolayer. Upon compression and expansion, lipids flow continuously from the monolayer into the disks, and vice versa. In several respects, the mode of collapse resembles the behavior of other amphiphiles that form smectic liquid-crystal phases. These findings suggest that components of surfactent films must collapse collectively rather than being squeezed out individually.