Showing papers by "Simon C. Watkins published in 2011"

Sunitinib facilitates the activation and recruitment of therapeutic anti-tumor immunity in concert with specific vaccination

Abstract: The multikinase inhibitor sunitinib malate (SUT) has been reported to reduce levels of myeloid suppressor cells and Treg cells in cancer patients, hypothetically diminishing intrinsic impediments for active immunization against tumor-associated antigens in such individuals. The goal of this study was to identify longitudinal immune molecular and cellular changes associated with tumor regression and disease-free status after the treatment of established day 7 s.c. MO5 (B16.OVA) melanomas with SUT alone (1 mg/day via oral gavage for 7 days), vaccination using ovalbumin (OVA) peptide-pulsed dendritic cell [vaccine (VAC)] alone, or the combination of SUT and VAC (SUT/VAC). We observed superior anti-tumor efficacy for SUT/VAC combination approaches, particularly when SUT was applied at the time of the initial vaccination or the VAC boost. Treatment effectiveness was associated with the acute loss of (and/or failure to recruit) cells bearing myeloid-derived suppressor cells or Treg phenotypes within the tumor microenvironment (TME) and the corollary, prolonged enhancement of Type-1 anti-OVA CD8(+) T cell responses in the tumor-draining lymph node and the TME. Enhanced Type-1 T cell infiltration of tumors was associated with treatment-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and CXCR3 ligand chemokines in vascular/peri-vascular cells within the TME, with SUT/VAC therapy benefits conditionally negated upon adminsitration of CXCR3 or VCAM-1 blocking antibodies. These data support the ability of a short 7 day course of SUT to (re)condition the TME to become more receptive to the recruitment and prolonged therapeutic action of (VAC-induced) anti-tumor Tc1 cells.

Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane.

Abstract: Cells survive exposure to bacterial pore-forming toxins, such as streptolysin O (SLO), through mechanisms that remain unclear. Previous studies have suggested that these toxins are cleared by endocytosis. However, the experiments reported here failed to reveal any evidence for endocytosis of SLO, nor did they reveal any signs of damage to endosomal membranes predicted from such endocytosis. Instead, we illustrate that SLO induces a characteristic form of plasma membrane blebbing that allows cells to shed SLO by the process known as ectocytosis. Specifically, ‘deep-etch’ electron microscopy of cells exposed to SLO illustrates that the toxin is rapidly sequestered into domains in the plasmalemma greatly enriched in SLO pores, and these domains bleb outwards and bud from the cell surface into the medium. Such ectocytosis is even observed in cells that have been chemically fixed before exposure to SLO, suggesting that it is caused by a direct physical action of the toxin on the cell membrane, rather than by an active cellular reaction. We conclude, therefore, that ectocytosis is an important means for SLO clearance and hypothesize that this is a primary method by which cells defend themselves generally against pore-forming toxins.

α-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion

Abstract: α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic β-cells, α-synuclein might therefore play a similar role by interacting with organelles ...

Functional Characterization of an scFv-Fc Antibody that Immunotherapeutically Targets the Common Cancer Cell Surface Proteoglycan CSPG4

Abstract: Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

Complement Proteins C3 and C4 Bind to Collagen and Elastin in the Vascular Wall: A Potential Role in Vascular Stiffness and Atherosclerosis

Abstract: Circulating infl ammatory mediators including complement activation products participate in the pathogenesis of cardiovascular diseases. As such, previous reports demonstrating the presence of complement proteins within atherosclerotic plaque and on the luminal surface would be anticipated. In contrast, we have recently made the unexpected observation that complement proteins also deposit along the external elastic lamina of mouse aortas in the absence of luminal deposition or plaque development. This suggests that complement activation may play a critical role in the pathogenesis of vascular stiffness and atherosclerosis through a mechanism initiated within the adventitia rather than on the endothelial surface. This hypothesis was tested in the current study by ultrastructural identifi cation of the C3- and C4-binding targets within the adventitia of the mouse aorta. The results demonstrate extensive binding of C3 and C4 to both collagen and elastin fi bers within the adventitia in both ApoE(/) and C57Bl/6J control mice, as well as the presence of C3 and C4 within perivascular adipose tissue. These observations suggest a potential “outside-in” mechanism of vascular stiffness during which perivascular adipose may produce C3 and C4 that bind to collagen and elastin fi bers within the adventitia through covalent thiolester bonds, leading to increased vascular stiffness. Clin Trans Sci 2011; Volume 4: 146‐152

Extracellular superoxide dismutase in macrophages augments bacterial killing by promoting phagocytosis.

Abstract: Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and limits inflammation and injury in response to many pulmonary insults. To test the hypothesis that EC-SOD has an important role in bacterial infections, wild-type and EC-SOD knockout (KO) mice were infected with Escherichia coli to induce pneumonia. Although mice in the EC-SOD KO group demonstrated greater pulmonary inflammation than did wild-type mice, there was less clearance of bacteria from their lungs after infection. Macrophages and neutrophils express EC-SOD; however, its function and subcellular localization in these inflammatory cells is unclear. In the present study, immunogold electron microscopy revealed EC-SOD in membrane-bound vesicles of phagocytes. These findings suggest that inflammatory cell EC-SOD may have a role in antibacterial defense. To test this hypothesis, phagocytes from wild-type and EC-SOD KO mice were evaluated. Although macrophages lacking EC-SOD produced more reactive oxygen species than did cells expressing EC-SOD after stimulation, they demonstrated significantly impaired phagocytosis and killing of bacteria. Overall, this suggests that EC-SOD facilitates clearance of bacteria and limits inflammation in response to infection by promoting bacterial phagocytosis.

A role for zinc in regulating hypoxia-induced contractile events in pulmonary endothelium

Abstract: We previously reported that zinc thiolate signaling contributes to hypoxic contraction of small, nonmuscularized arteries of the lung. The present studies were designed to investigate mechanisms by which hypoxia-released zinc induces contraction in isolated pulmonary endothelial cells and to delineate the signaling pathways involved in zinc-mediated changes in the actin cytoskeleton. We used fluorescence-based imaging to show that hypoxia induced time-dependent increases in actin stress fibers that were reversed by the zinc chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). We further showed that hypoxia-induced phosphorylation of the contractile protein myosin light chain (MLC) and assembly of actin stress fibers were each TPEN sensitive. Hypoxia and zinc-induced inhibition of MLC phosphatase (MLCP) were independent of the regulatory subunit (MYPT1) of MLCP, and therefore hypoxia-released zinc likely inhibits MLCP at its catalytic (PP1) subunit. Inhibition of PKC by Ro-31–8220 and a dominant-negative construct of PKC-e attenuated hypoxia-induced contraction of isolated pulmonary endothelial cells. Furthermore, zinc-induced phosphorylation of MLC (secondary to inhibition of MLCP) was PKC dependent, and hypoxia-released zinc promoted the phosphorylation of the PKC substrate, CPI-17. Collectively, these data suggest a link between hypoxia, elevations in labile zinc, and activation of PKC, which in turn acts through CPI-17 to inhibit MLCP activity and promote MLC phosphorylation, ultimately inducing stress fiber formation and endothelial cell contraction.

Span-wise wind fluctuations in open terrain as applicable to small flying craft

Abstract: Micro air vehicles (MAVs) are typically of low mass and moment of inertia and have flight speeds comparable to birds and the larger insects. Such craft traverse the lower levels of the atmospheric boundary layer (ABL) which is a significantly different environment than that experienced by larger manned aircraft, which spend the majority of their time in relatively clean air and fly at speeds significantly higher than typical wind speeds in the ABL. Here a new series of measurements dedicated to understanding spatial and temporal velocity fields that MAVs experience are presented. Atmospheric wind measurements were taken by sampling four multi-hole dynamic pressure probes spanned perpendicular to the oncoming wind at spans of between 0·014m and up to 0·45m. It was noted that the variation of both longitudinal velocity and flow pitch angle against spacing followed a fractional power law and as such large variations were present even for the smallest inter-probe separations. This effect is thought to explain the increasing piloting difficulties experienced in maintaining good roll control for decreasing scales of craft.

An unexpected role for the clock protein timeless in developmental apoptosis.

Abstract: Background: Programmed cell death is critical not only in adult tissue homeostasis but for embryogenesis as well. One of the earliest steps in development, formation of the proamniotic cavity, involves coordinated apoptosis of embryonic cells. Recent work from our group demonstrated that c-Src protein-tyrosine kinase activity triggers differentiation of mouse embryonic stem (mES) cells to primitive ectoderm-like cells. In this report, we identified Timeless (Tim), the mammalian ortholog of a Drosophila circadian rhythm protein, as a binding partner and substrate for c-Src and probed its role in the differentiation of mES cells. Methodology/Principal Findings: To determine whether Tim is involved in ES cell differentiation, Tim protein levels were stably suppressed using shRNA. Tim-defective ES cell lines were then tested for embryoid body (EB) formation, which models early mammalian development. Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate. Cells retained within the centers of the failed cavities strongly expressed the pluripotency marker Oct4, suggesting that further development is arrested without Tim. Immunoblots revealed reduced basal Caspase activity in the Tim-defective EBs compared to wild-type controls. Furthermore, EBs formed from Tim-knockdown cells demonstrated resistance to staurosporine-induced apoptosis, consistent with a link between Tim and programmed cell death during cavitation. Conclusions/Significance: Our data demonstrate a novel function for the clock protein Tim during a key stage of early development. Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate. As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent. These findings reveal a new function for Tim in the coordination of ES cell differentiation, and raise the intriguing possibility that circadian rhythms and early development may be intimately linked. © 2011 O'Reilly et al.

A review of power generation from wind in australia

Abstract: With recent surge in fossil fuel prices, demands for cleaner renewable energy sources, wind turbines have become a viable technology for power generation. Greenhouse gases such as carbon dioxide emitted into our atmosphere have resulted on what many scientists believe is due to climate change. To tackle climate change, many of the world's developed countries have agreed on a policy to cute down on its carbon emission. Australia has agreed on a renewable energy target of 20 per cent by the year 2020. Twenty per cent of Australia's energy generation must come from renewable energy sources by this period. This paper will examine the importance wind energy as an alternative source of energy to fossil fuel based energy both globally and locally. Wind energy is fastest growing renewable energy in the world. It has the potential to rival the cost competitiveness of conventional energy sources. Wind energy is a reliable and sustainable with a mature technology and a proven track record.

Performance of Adaptive Airfoil Control System Using Shape Memory Alloy Actuators for UAV

Abstract: .Shape memory alloy (SMA) is lightweight, produces high force and large deflection which makes it a suitable candidate for actuator in the adaptive airfoil system design. The deflection of a variable cambered wing is controlled by means of resistive heating of SMA actuator and cooling in the surrounding air. Experiments were conducted under wind tunnel conditions not only to verify the robustness of the adaptive airfoil control system but also to investigate the effects of its application on the aerodynamic behaviour of the wing. The change of forces acting on the wing with and without the adaptive airfoil control system was investigated. The results proved that the use of SMA actuators in the wing model is reliable as significant change in lift to drag ratio was detected when the wing was morphed. The adaptive airfoil control system was able to maintain the same L/Dmax for changing lift coefficient.

The influence of wheel–housing shape on vehicle aerodynamic performance

Abstract: Despite intense research into the flow around a road vehicle, the drag coefficient has plateaued at about 0.3 and further reductions will come from wheel associated areas. The wheel housings have had relatively little attention despite contributing significant disturbances to the airflow. A simple two wheeled body was developed to reproduce wheel effects of a typical passenger car. Benchmarking experiments were performed in a wind tunnel using a double–symmetry testing concept. CFD techniques were used for parametric study. A number of parameters were analysed and the flow structures indicate that wheel–housing shape has a significant influence on the vehicle aerodynamics.

Pressure fluctuations over an airfoil in smooth and turbulent flow at low Reynolds number

Abstract: Tests on a 2-D airfoil are presented relevant to micro air vehicle (MAV) flight. A thin, pressure-tapped, flat plate airfoil was tested at a Reynolds number of 75000 under various levels of turbulence. The turbulence intensity ranged from as low as 1.2% (nominally smooth flow) to as high as 12.6% with length scale of 0.31m. Smoke flow visualization and time varying pressure measurements were taken to identify various properties of the flow over the airfoil when exposed to smooth and turbulent conditions. Statistical analysis of the data revealed that in both conditions, majority of the fluctuations occurred at the lower frequencies while the higher frequency fluctuations possessed very little energy. Laminar Separation Bubbles (LSBs) formed in smooth flow and grew rapidly with increase in Angle of Attack. In turbulence however, the growth of LSBs was severely curtailed due to the shear layer attaining transition much earlier. On earlier attainment of transition, a significantly large portion of the shear layer rolled upon reattachment leading to the formation of strong vortical cores. As these vortical cores formed and advected downstream, they imparted large fluctuations in pressure and velocity. The rate of formation of the vortical cores was found not to be a function of oncoming turbulence, but rather a characteristic of airfoil geometry.