ASP2408 and ASP2409, novel CTLA4-Ig variants with CD86-selective ligand binding activity and improved immunosuppressive potency, created by directed evolution.

The CTLA4-Ig therapeutics abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) co-stimulatory ligands. Both compounds preferentially bind CD80, yet CD86 has been implicated as the dominant co-stimulatory ligand. Using directed evolution methods, novel CTLA4-Ig variants were created with selective CD86 binding affinity, a property that confers increased immunosuppressive potency and potentially improved efficacy and safety profiles. Relative to abatacept (wild-type CTLA4-Ig), ASP2408 and ASP2409 have 83-fold and 220-fold enhanced binding affinity to CD86 while retaining 1.5-fold and 5.6-fold enhanced binding affinity to CD80, respectively. Improvements in CD86 binding affinity correlates with increased immunosuppressive potencyin vitroandin vivo Our results highlight the power of directed evolution methods to obtain non-intuitive protein engineering solutions and represent the first examples of CD86-selective CTLA4-Ig compounds that have entered clinical trials.

Applications of flexible ultrasonic transducer array for defect detection at 150 °C.

In this study, the feasibility of using a one dimensional 16-element flexible ultrasonic transducer (FUT) array for nondestructive testing at 150 °C is demonstrated. The FUT arrays were made by a sol-gel sprayed piezoelectric film technology; a PZT composite film was sprayed on a titanium foil of 75 µm thickness. Since the FUT array is flexible, it was attached to a steel pipe with an outer diameter of 89 mm and a wall thickness of 6.5 mm at 150 °C. Using the ultrasonic pulse-echo mode, pipe thickness measurements could be performed. Moreover, using the ultrasonic pulse-echo and pitch-catch modes of each element of FUT array, the defect detection was performed on an Al alloy block of 30 mm thickness with a side-drilled hole (SDH) of f3 mm at 150 °C. In addition, a post-processing algorithm based on the total focusing method was used to process the full matrix of these A-scan signals of each single transmitter and multi-receivers, and then the phase-array image was obtained to indicate this defect- SDH. Both results show the capability of FUT array being operated at 150 °C for the corrosion and defect detections.

Design and fabrication of an integrated dual-mode ultrasonic probe.

In this paper, we present an integrated dual-mode ultrasonic probe that is capable of transmitting and receiving longitudinal and shear waves (LW and SW, respectively) simultaneously. The probe consists of a thick piezoelectric film LW ultrasonic transducer and a substrate with a specific shape. An SW is generated inside the substrate by mode conversion. In the probe design, two factors were theoretically investigated: impedance matching between the substrate and the material being measured to improve measurement sensitivity and mode conversion efficiency at the slanted face of the substrate. The developed probe was tested with a selected silicone liquid sample to demonstrate its capability to measure the properties of a viscous liquid using both LW and SW.

Ultrasonic probes having three orthogonal polarizations.

Ultrasonic probes which can simultaneously generate and receive 2 orthogonal polarized shear (S(perpendicular) and S(//)) and longitudinal (L) waves in screw shape have been presented. Thick piezoelectric films are directly fabricated onto the heads of such probes as L wave integrated ultrasonic transducers. S(perpendicular) and S(//) waves are obtained using mode conversion method. Potential applications of such probes are discussed.

Integrated high-temperature piezoelectric plate acoustic wave transducers using mode conversion.

Piezoelectric thick (>66 microm) films have been directly coated onto aluminum (Al) substrates using a sol-gel spray technique. With top electrode, these films serve as integrated ultrasonic transducers (IUT), which normally operate as thickness longitudinal wave transducers. When such IUT are located at the edges of the metallic plates, they can excite and detect symmetrical, antisymmetric and shear horizontal types of plate acoustic waves (PAW) using mode conversion methods. In 2 mm thick Al plates, 2 line defects of 1 mm width and 1 mm depth were clearly detected at temperatures up to 150 degrees C in pulse-echo mode. Results indicated that, for 2 mm thick aluminum plates, shear horizontal PAW were the best for the line defect detection. Also, the experimental results agree well with those obtained by a finite-difference-based method.

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase.

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.

Effects of EGF and IGF-1 on proliferation of cultured human proximal tubule cells after oxidant stress.

BACKGROUND: Both insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF) stimulate proliferation of various renal tubule epithelial cells in culture including proximal tubule cells. In some epithelial cells, the effects of EGF and IGF-1 are additive or synergistic. The effects of EGF and IGF-1 in cultured tubule epithelial cells following injury are limited. METHODS: Immortalized human proximal tubules cultured in serum-free defined medium were exposed to 0.3-1.5 mM peroxide for 1 h then washed and growth factors were added. ATP was measured by chemiluminescence, proliferation by [3H]thymidine uptake, and receptor expression by flow cytometry. RESULTS: Immediately after 1.5 mM peroxide exposure, ATP levels were depressed to as low as approximately 15% of normal but had recovered to near normal levels by 4 h. Proliferation was depressed in a dose-dependent manner by peroxide. At the lowest doses of peroxide both EGF (20 ng/mL) and IGF-1 (390 ng/mL) stimulated proliferation. As the concentration of peroxide increased, EGF lost its ability to stimulate proliferation and in fact antagonized IGF-1 which when added alone remained effective at stimulating proliferation even at the highest levels of peroxide exposure. EGF and peroxide depressed EGF receptor expression but there were no changes in IGF-1 receptor expression with any maneuver. CONCLUSION: The effects of EGF to antagonize IGF-1 are distal to IGF-1 receptor expression. The effects of these growth factors under control conditions do no translate to effects after injury.