Targeting glycoprotein VI to disrupt platelet-mediated tumor cell extravasation.

Activated platelets coat circulating tumor cells, protecting them from shear stress in the blood stream and promoting their evasion from immune surveillance. Platelets promote tumor cell dissemination to distant organs by releasing transforming growth factor-ß1 (TGF-ß1) into the tumor microenvironment, which induces phenotypic changes to the epithelial-mesenchymal transition. This process facilitates tumor cell transendothelial extravasation and formation of early metastatic niches. Development of antiplatelet agents that interrupt the platelet-tumor cell axis but do not interfere with physiological hemostatic mechanisms is critical. The glycoprotein VI (GPVI), a member of the immunoreceptor family that is co-expressed with the fragment crystallizable (Fc) receptor γ-chain, is exclusively expressed in platelets and megakaryocytes, and blocking the receptor or genetic deficiency has minimal impact on bleeding. Tumor cell-expressed galectin-3, which contains a collagen-like peptide domain, binds to platelet GPVI-dimers, and the receptor-ligand activates platelets to form a protective heteroaggregate coat around tumor cells. This review highlights the potential of targeting the GPVI/FcR γ-chain complex to inhibit platelet activation by galectin-3 expressing tumor cells, disrupting the platelet-tumor cell amplification loop while maintaining the function of platelets in hemostasis.

Research: Comparing ASTM F1671 with a Modified Dot-Blot Method to Evaluate Personal Protective Materials.

Effective personal protective equipment (PPE) is critical in preventing the spread of infectious diseases. Appropriate test systems and test soils are needed to adequately evaluate PPE. ASTM test method F903, which specifies the test method setup also used in ASTM F1670 and F1671, has been used for decades to test liquid (ASTM F1670) or viral (ASTM F1671) penetration resistance of PPE fabrics. However, an alteration of the bacteriophage propagation method detailed in the standard was necessary to obtain consistent titers of virus. In this study, modification of the nutrient broth provided consistently higher titers of virus and the use of the top agar in smaller increments prevented premature solidification. This study then compared the standard ASTM F1671 (using bacteriophage ϕχ174) with a modified dot-blot method to assess viral penetration of PPE materials. The results indicated that ASTM F1671 and the dot-blot apparatus methods were equivalent. The dot-blot method described here is less labor intensive and faster than the ASTM F1671 method. However, using the dot-blot system, which uses antibodies to detect the bacteriophage and signal amplification, does not indicate if virus viability or infectivity is retained, whereas the ASTM F1671 method indicates both. Nonetheless, the method presented in this investigation is a substantial improvement of a standard method for viral challenge testing of PPE materials.

Research: Using Clinically Relevant Test Soils to Evaluate Personal Protective Materials.

Effective personal protective equipment (PPE) is critically important to preventing the spread of infectious diseases. Appropriate test systems and test soils are needed to adequately evaluate PPE. ASTM test method F903, which specifies the test method setup also used in ASTM F1670/F1670M-17a and ASTM F1671/F1671M-13, has been used for decades to test liquid penetration resistance of fabrics. All three standards require at least 60 mL of challenge liquid, such as synthetic blood solution (F1670) or bacteriophage in nutrient broth (F1671). The three ASTM test methods also are labor intensive and prone to exhibiting problems with leakage around the gaskets. Previous work comparing the F903 test apparatus with a modified dot-blot apparatus to evaluate the visual penetration of a blood test soil in series of commercially available gowns and drapes demonstrated that the methods are comparable and revealed that penetration through PPE material may depend on the test solution. The study described here evaluated a series of clinically relevant test soils (blood, vomit, urine, and feces) in penetration of PPE garments using the modified dot-blot apparatus. The results indicated that a vomit test soil penetrates PPE material more often than blood, urine, or fecal test soils and that the blood test soil has the least number of PPE failures. Incorporating clinically relevant, chemically defined test soils to evaluate PPE material should be considered to protect healthcare workers and reduce the spread of infectious material.

Evaluation of Apparatus Used to Test Liquid through Protective Materials: Comparison of a Modified Dot-Blot Apparatus to the ASTM Penetration Cell.

Personal protective equipment (PPE), such as gowns used in the latest Ebola outbreak in Western Africa, are critical in preventing the spread of deadly diseases. Appropriate test systems and test soils are needed to adequately evaluate PPE. ASTM F903, Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Liquid, has been used for decades to test fabrics' resistance to liquid penetration. However, this test apparatus requires at least 60 mL of test solutions, is labor intensive, and has problems with leakage around the gaskets. We compared the F903 test apparatus to a modified dot-blot apparatus to evaluate the visual penetration of a blood test soil. A series of commercially available gowns and drapes were tested in each apparatus. Using blood test soil at 2 psi, there was no statistically significant difference between the two methods except for in one gown. By comparing this gown in the ASTM test apparatus with and without a screen, the particular screen selected did not account for the difference between the dot-blot and F903 apparatuses; however, it is conceivable that a particular screen/fabric combination could account for this difference. The modified dot-blot apparatus was evaluated using three different test solutions: blood, vomit, and a labeled protein (goat anti-rabbit immunoglobulin G-horseradish peroxidase [GaR IgG-HRP]) in a blood test soil solution. This testing revealed significant difference in penetration for some of the PPE garments. The modified dot-blot had several large advantages over the ASTM apparatus-over six times less specimen volume and no edge or gasket leakage. In addition, nitrocellulose can be easily incorporated into the modified dot-blot apparatus, enabling the trapping of viruses and proteins that penetrate PPE-thus permitting the use of antibodies to quickly and sensitively detect penetration.

Performance characteristics of the AmpliSeq Cancer Hotspot panel v2 in combination with the Ion Torrent Next Generation Sequencing Personal Genome Machine.

Next-Generation Sequencing is a rapidly advancing technology that has research and clinical applications. For many cancers, it is important to know the precise mutation(s) present, as specific mutations could indicate or contra-indicate certain treatments as well as be indicative of prognosis. Using the Ion Torrent Personal Genome Machine and the AmpliSeq Cancer Hotspot panel v2, we sequenced two pancreatic cancer cell lines, BxPC-3 and HPAF-II, alone or in mixtures, to determine the error rate, sensitivity, and reproducibility of this system. The system resulted in coverage averaging 2000× across the various amplicons and was able to reliably and reproducibly identify mutations present at a rate of 5%. Identification of mutations present at a lower rate was possible by altering the parameters by which calls were made, but with an increase in erroneous, low-level calls. The panel was able to identify known mutations in these cell lines that are present in the COSMIC database. In addition, other, novel mutations were also identified that may prove clinically useful. The system was assessed for systematic errors such as homopolymer effects, end of amplicon effects and patterns in NO CALL sequence. Overall, the system is adequate at identifying the known, targeted mutations in the panel.

In silico prediction of Ebola Zaire GP(1,2) immuno-dominant epitopes for the Balb/c mouse.

BACKGROUND: Ebola is a Filovirus (FV) that induces a highly communicable and deadly hemorrhagic fever. Currently, there are no approved vaccines to treat FV infections. Protection from FV infection requires cell mediated and humoral immunity. Glycoprotein GP(1,2) Fc Zaire, a recombinant FV human Fc fusion protein, has been shown to confer protection against mouse adapted Zaire Ebola virus. The present studies are focused upon identifying immunodominant epitopes using in silico methods and developing tetramers as a diagnostic reagent to detect cell mediated immune responses to GP(1,2) Fc. METHODS: The GP(1,2) Ebola Zaire sequence from the 1976 outbreak was analyzed by both BIMAS and SYFPEITHI algorithms to identify potential immuno-dominant epitopes. Several peptides were synthesized and screened in flow-based MHC stability studies. Three candidate peptides, P8, P9 and P10, were identified and, following immunization in Balb/c mice, all three peptides induced IFN-γ as detected by ELISpot and intracellular staining. RESULTS: Significantly, P8, P9 and P10 generated robust cytotoxic T-cell responses (CTL) as determined by a flow cytometry-based Caspase assay. Antigen specific cells were also detected, using tetramers. Both P9 and P10 have sequence homology with highly conserved regions of several strains of FV. CONCLUSIONS: In sum, three immunodominant sequences of the Ebola GP(1,2) have been identified using in silico methods that may confer protection against mouse adapted Ebola Zaire. The development of tetramer reagents will provide unique insight into the potency and durability of medical countermeasure vaccines for known bioterrorism threat agents in preclinical models.

Toll-like receptor 4 signaling pathway mediates proinflammatory immune response to cobalt-alloy particles.

Metal orthopedic implant debris-induced osteolysis of hip bone is a major problem in patients with prosthetic-hips. Although macrophages are the principal targets for implant-wear debris, the receptor(s) and mechanisms underlying these responses are not fully elucidated. We examined whether the TLR4 pathway mediates immune response to metal-on-metal (MoM) implant-generated wear particles. Human monocytes (THP-1) were exposed to Co-alloy particles at increasing particle:cell ratio for 24 h. Challenge with particles caused up-regulation of IL-1ß, TNF-α and IL-8, and mediated degradation of cytosolic I-κB and nuclear translocation of NF-κB. Blocking antibodies against TLR4 or gene silencing of MyD88 and IRAK-1 prevented particle-induced I-κB/NF-κB activation response and markedly inhibited IL-8 release. Particle-mediated IL-8 response was not observed in TLR4-negative HEK293T cells; whereas transfection-based TLR4-overexpression in HEK293T enabled particle-sensitivity, as observed by I-κB degradation and IL-8 expression in response to particles. Results demonstrate that Co-alloy particles trigger immune response via the TLR4-MyD88-dependent signaling pathway.

Effects of ultrasound and ultrasound contrast agent on vascular tissue.

BACKGROUND: Ultrasound (US) imaging can be enhanced using gas-filled microbubble contrast agents. Strong echo signals are induced at the tissue-gas interface following microbubble collapse. Applications include assessment of ventricular function and virtual histology. AIM: While ultrasound and US contrast agents are widely used, their impact on the physiological response of vascular tissue to vasoactive agents has not been investigated in detail. METHODS AND RESULTS: In the present study, rat dorsal aortas were treated with US via a clinical imaging transducer in the presence or absence of the US contrast agent, Optison. Aortas treated with both US and Optison were unable to contract in response to phenylephrine or to relax in the presence of acetylcholine. Histology of the arteries was unremarkable. When the treated aortas were stained for endothelial markers, a distinct loss of endothelium was observed. Importantly, terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling (TUNEL) staining of treated aortas demonstrated incipient apoptosis in the endothelium. CONCLUSIONS: Taken together, these ex vivo results suggest that the combination of US and Optison may alter arterial integrity and promote vascular injury; however, the in vivo interaction of Optison and ultrasound remains an open question.

Examining the role of nickel and NiTi nanoparticles promoting inflammation and angiogenesis.

Nickel titanium (NiTi, or Nitinol) alloy is used in several biomedical applications, including cardiac, peripheral vascular, and fallopian tube stents. There are significant biocompatibility issues of metallic implants to nickel ions and nano-/micro-sized alloy particles. Our laboratories have recently shown that microscale CoCr wear particles from metal-on-metal hips crosslink with the innate immune signaling Toll-like receptor 4 (TLR4), prompting downstream signaling that results in interleukin (IL)-1ß and IL-8 gene expression. In vivo, NiTi alloy can also generate wear particles on the nanoscale (NP) that have thus far not been studied for their potential to induce inflammation and angiogenesis that can, in turn, contribute to implant (e.g. stent) failure. Earlier studies by others demonstrated that nickel could induce contact hypersensitivity by crosslinking the human, but not the mouse, TLR4. In the present work, it is demonstrated that NiCl2 ions and NiTi nanoparticles induce pro-inflammatory and pro-angiogenic cytokine/chemokine expression in human endothelial and monocyte cell lines in vitro. These observations prompt concerns about potential mechanisms for stent failure. The data here showed a direct correlation between intracellular uptake of Ni2+ and generation of reactive oxygen species. To determine a role for nickel and NiTi nanoparticles in inducing angiogenesis in vivo, 1-cm silicone angioreactors were implanted subcutaneously into athymic (T-cell-deficient) nude mice. The angioreactors contained Matrigel (a gelatinous protein mixture that resembles extracellular matrix) in addition to one of the following: PBS (negative control), VEGF/FGF-2 (positive control), NiCl2, or NiTi NP. The implantation of angioreactors represents a potential tool for quantification of angiogenic potentials of medical device-derived particles and ions in vivo. By this approach, NiTi NP were found to be markedly angiogenic, while Ni2+ was less-so. The angioreactors may provide a powerful tool to examine if debris shed from medical devices may promote untoward biological effects.

Induction of nicotinamide-adenine dinucleotide phosphate oxidase and apoptosis by biodegradable polymers in macrophages: implications for stents.

The drug-eluting stent platform has a limited surface area, and a polymer carrier matrix is coated to enable sufficient loading of drugs. The development of a suitable polymer has been challenging because it must exhibit biocompatibility with the intravascular milieu. The use of biodegradable polymers seems to be attractive because it enables drug release as it degrades and is eventually eliminated from the body leaving the permanent metallic stent polymer-free. The aim of this study was to investigate the biocompatibility of biodegradable polymers using the human monocyte cell line. Cultured monocytes differentiated into functional macrophages (THP-1) were incubated with various polymers including poly-L-lactide (PLA), polycaprolactone (PCL), or poly-D, L-lactide-co-glycolide (PLGA) for up to 5 days. Exposure of cells to the polymers resulted in macrophage-polymer adhesion and induced marked pro-oxidant species as measured by calcein AM uptake assay and flow cytometric analysis of 2',7'-dichlorofluorescin fluorescence, respectively. Real-time reverse-transcription polymerase chain reaction and Western blot analysis of expression of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases revealed enhanced expression of NADPH oxidase subunits in response to PLA and PLGA compared with that of PCL. Flow cytometric analysis of fluorescein isothiocyanate-Annexin V and propium iodide-stained PLA and PGLA polymer-exposed THP-1 cells showed early and late apoptotic changes. Similarly, exposure to the PLA and PGLA polymers, but not to the PCL polymer, resulted in enhanced staining for cleaved poly(ADP-ribose) polymerase-1, a protein fragment produced by caspase cleavage. These results indicate that biodegradable polymers are associated with cell adhesion, NADPH oxidase-induced generation of reactive oxygen species and excess apoptosis.

Paclitaxel potentiates inflammatory cytokine-induced prothrombotic molecules in endothelial cells.

To overcome the limitations of balloon expandible metal stent-induced neointimal smooth muscle cell proliferation, drug-coated stent devices have been developed. Drug eluting stents release high concentrations of antiproliferative agents, such as paclitaxel, to reduce neointimal hyperplasia. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), is known to cause severe endothelial dysfunction and accelerate atherosclerotic lesion progression. The interaction of TNF-alpha and paclitaxel on the release of prothrombotic molecules was examined in endothelial cells. Treatment of endothelial cells with paclitaxel had no direct effect on tissue factor (TF) expression, but TNF-alpha increased TF. Cotreatment of paclitaxel with TNF-alpha markedly augmented the release of TF. TNF-alpha induced release of plasminogen activator inhibitor but no synergism occurred with paclitaxel. Treatment of endothelial cells with paclitaxel and TNF-alpha reduced expression of thrombomodulin and protein C receptor. Tissue factor pathway inhibitor expression was reduced by prolonged treatment with either paclitaxel or TNF-alpha. The adhesion molecule, CD62 E, was induced by TNF-alpha; however, CD31, CD62 P, and CD106 were not affected by paclitaxel and TNF-alpha. Apoptosis was not observed with cotreatment of endothelial cells with paclitaxel and TNF-alpha. CD59-positive microparticles were released in response to TNF-alpha, but the release was not augmented by paclitaxel. Paclitaxel and TNF-alpha increased the nitrotyrosination of proteins. These findings indicate that paclitaxel enhances TNF-alpha-induced release of TF, and downregulated thrombomodulin, increased protein nitration, which may subsequently favor prothrombotic intimal surface.

Evaluation of one-way valves used in medical devices for prevention of cross-contamination.

BACKGROUND: One-way valves used in day use devices (used on multiple patients throughout a day without reprocessing between patients) are intended to reduce the potential for cross-contamination between patients resulting from the backflow of patient fluids. One-way valves are typically designed to withstand high levels of back pressure before failure; however, they may not be explicitly designed as a means of infection control as used in medical device applications. METHODS: Five different medical grade one-way valves were placed in low pressure configurations. After flushing in the intended direction of flow, bacteriophage, bacteria, or dye was placed patient side for 24 hours. The upstream device side of the valve was then evaluated for microbial growth or presence of visible dye. RESULTS: Leakage (ie, backflow) of the microorganisms occurred with a variety of one-way valve designs across a range of fluid properties tested. CONCLUSIONS: This study describes testing of the one-way valves (component-level testing) for the potential of cross-contamination. Although day use medical device systems may use numerous other factors to prevent patient cross-contamination, this work demonstrates that one-way valves themselves may not prevent leakage of contaminated fluid if the fluid is able to reach the upstream side of the one-way valve.

Inhibition of mammalian target of rapamycin modulates expression of adhesion molecules in endothelial cells.

Neointimal hyperplasia often follows angioplasty-induced arterial injury or stenting and results in restenosis. Previous reports have suggested that arterial injury activates complement which amplifies inflammatory responses that may initiate and sustain neointimal hyperplasia. The effects of rapamycin on complement-induced expression of intracellular adhesion molecules (ICAMs) were examined in porcine arterial endothelial cell (PAEC) line that was transformed with large T antigen. Porcine complement was activated by treating sera with zymosan (PO ZYM) to generate C5b-9. C5b-9 binds to PAEC in a concentration- and time-dependent manner. PO ZYM-induced expression of ICAMs was maximally induced by 18h. Rapamycin reduced the expression of vascular cell adhesion molecule (VCAM) and P-selectin in a concentration-dependent manner. Adhesion of monocytes was reduced by rapamycin and the inhibition was prevented by antibodies to vascular cell adhesion molecule, P-selectin and endothelial-leukocyte adhesion molecule (ELAM). In summary, inhibition of the mammalian target of rapamycin down regulates complement-induced ICAMs expression which may modulate inflammatory responses that follow stent implant-induced restenosis during percutanous coronary interventions.

Wear particles derived from metal hip implants induce the generation of multinucleated giant cells in a 3-dimensional peripheral tissue-equivalent model.

Multinucleate giant cells (MGCs) are formed by the fusion of 5 to 15 monocytes or macrophages. MGCs can be generated by hip implants at the site where the metal surface of the device is in close contact with tissue. MGCs play a critical role in the inflammatory processes associated with adverse events such as aseptic loosening of the prosthetic joints and bone degeneration process called osteolysis. Upon interaction with metal wear particles, endothelial cells upregulate pro-inflammatory cytokines and other factors that enhance a localized immune response. However, the role of endothelial cells in the generation of MGCs has not been completely investigated. We developed a three-dimensional peripheral tissue-equivalent model (PTE) consisting of collagen gel, supporting a monolayer of endothelial cells and human peripheral blood mononuclear cells (PBMCs) on top, which mimics peripheral tissue under normal physiological conditions. The cultures were incubated for 14 days with Cobalt chromium alloy (CoCr ASTM F75, 1-5 micron) wear particles. PBMC were allowed to transit the endothelium and harvested cells were analyzed for MGC generation via flow cytometry. An increase in forward scatter (cell size) and in the propidium iodide (PI) uptake (DNA intercalating dye) was used to identify MGCs. Our results show that endothelial cells induce the generation of MGCs to a level 4 fold higher in 3-dimentional PTE system as compared to traditional 2-dimensional culture plates. Further characterization of MGCs showed upregulated expression of tartrate resistant alkaline phosphatase (TRAP) and dendritic cell specific transmembrane protein, (DC-STAMP), which are markers of bone degrading cells called osteoclasts. In sum, we have established a robust and relevant model to examine MGC and osteoclast formation in a tissue like environment using flow cytometry and RT-PCR. With endothelial cells help, we observed a consistent generation of metal wear particle- induced MGCs, which heralds metal on metal hip failures.

Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice.

Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use.