text:
Volkswagen to Network Factories in the Cloud With Amazon
Hackers Took Over Asus Updates to Send You Malware, Researchers Say
Chinese, U.S. Researchers Develop Simulator for Improving Self-Driving Car Safety Before Road Test
The Gender Wage Gap is Shrinking Among Computer Programmers, But Still Quite Large
Study Awarded Top Paper of Past 50 Years
McDonald's Bites on Big Data With $300 Million Acquisition
The New Tech That Detects Deadly Slides on Rubbish Mountains
Our Software Is Biased Like We Are. Can New Laws Change That?
IBM's Quantum Computation Technique Mitigates Noise, Improves Accuracy
A New Age of Warfare: How Internet Mercenaries Do Battle for Authoritarian Governments
Robots Help Bees and Fish Communicate
Open Source Software Helps Researchers Extract Insights From Sensor Datasets
Researchers Warn Open Sky Drone Policy Poses Cybercriminal Risk
How Smarter Technology Will Feed the Planet
Volkswagen (VW) has announced a partnership with Amazon to develop cloud computing capacity, to upgrade efficiency and coordination across the car manufacturer's global manufacturing network. VW said combining data from its 122 plants will enable it to standardize production planning and inventory management, boosting efficiency and cutting costs. Amazon Web Services (AWS) will work with the automaker to design an industrial cloud as an open platform that other companies, including suppliers, could join. Said VW's Oliver Blume, "We want to create a growing industrial ecosystem, with transparency and efficiency bringing benefits to all concerned."
Researchers from Kaspersky Lab warned hackers have infected thousands of Asus computers with malware from the computer manufacturer's own update tool. The Operation ShadowHammer hack was conducted between June and November 2018, affecting more than 57,000 people using Asus products. The researchers uncovered the infection in January, when hackers hijacked the Asus Live Update Utility to quietly install malware on devices. The attackers infiltrated the devices without raising an alarm by using Asus' legitimate security certificate, which was hosted on the servers. Once the backdoor was installed, it checked the device's media access control address; if it matched one of the attacker's targets, it installed another set of malware. Kaspersky Labs' Vitaly Kamluk said, "The selected vendors are extremely attractive targets for APT [advanced persistent threat] groups that might want to take advantage of their vast customer base."
Researchers at China's Baidu Research, the University of Hong Kong, and the University of Maryland, have developed a system that could make self-driving technology easier to evaluate in the lab and ensure more reliable safety before expensive road testing. The new Augmented Autonomous Driving Simulation (AADS) system combines photos, videos, and LiDAR point clouds with real-world trajectory data for pedestrians, bicycles, and other cars. Those trajectories can be used to predict the driving behavior and future positions of other vehicles or pedestrians on the road for safer navigation. The researchers eliminated blind spots in real-world images by isolating the components in a street scene, turning them into individual elements that can be synthesized to create a multitude of photo-realistic driving scenarios. Said University of Maryland researcher Dinesh Manocha, "We extracted data about real trajectories from all the video we had available, and we modeled driving behaviors using social science methodologies."
Megan Rose Dickey
In the U.S., the unadjusted pay gap registers at 21.4%, meaning women make just $0.79 for every $1.00 men earn, according to Glassdoor's most recent gender pay gap data analysis. However, when controlling for variables like age, education, location, experience, occupation, and industry, the pay gap falls to 4.9%, meaning women make about $0.95 for every $1.00 men make, a slight improvement from three years ago. In the technology industry, there is an adjusted wage gap of 5.4%, down from 28.3% in 2016. Said Glassdoor's Annie Pearl, "Combining knowledge with leveraging valuable resources is the next step to ensuring equal pay for equal work everywhere… We all have a part to play to close the pay gap."
The Daily Illini
Daniel Renteria
The ACM Special Interest Group on Computer Science Education (SIGCSE) cited research by scientists from the University of Illinois at Urbana-Champaign (UIUC), the University of California, San Diego, and Northern Iowa University as the top paper of the past 50 years at its Technical Symposium on Computer Science Education. The work stems from a project to develop a research assessment tool for determining the teaching methods that provide the best education for computer science students. UIUC's Geoffrey Herman said, "The focus (of this paper) was to document and understand and describe why students struggle to learn computing programming." Focal points included students' mistaken assumption that computers will perform any kind of interpretations on inputted code, as well as their difficulty in understanding computer memory. Herman suggested the paper received the award "because it's one of those early papers which said, 'hey, here is some theory, here is some science to help us make better decisions and inform our future efforts'."
McDonald's has inked a more-than-$300-million deal to acquire Dynamic Yield, a start-up based in Tel Aviv that provides retailers with algorithmically driven "decision logic" technology. With the addition of Dynamic Yield, McDonald's is hoping to improve both its in-store ordering and online marketing. The chain will use Dynamic Yield's technology at its drive-through windows, where digital displays will change in real time based on such factors as weather and what the customer is ordering. Over the last several years, the fast-food chain made several significant, data-focused investments. Said McDonald's CEO Steve Easterbrook, "What we hadn't done is begun to connect the technology together, and get the various pieces talking to each other. How do you transition from mass marketing to mass personalization? To do that, you've really got to unlock the data within that ecosystem in a way that's useful to a customer."
Adrienne Bernhard
Australian researchers have developed software to predict landslides on mountains of garbage up to two weeks in advance, giving those who live in or near the rubbish piles time to evacuate. The artificial intelligence (AI) system uses applied mathematics to identify landslide precursors, including tiny cracks and subtle movements that herald violent downward cascades. The University of Melbourne's Antoinette Tordesillas said, "We have been studying data on movements in granular materials to understand their 'rhythm of failure.' What we discovered is a distinct rhythm in the stages preceding the collapse." The algorithms account for ground movement, the dynamics of failure, and known landslide triggers like rainfall, all of which could ultimately be used for slide predictions. Said Isaac Akinwumi at Nigeria's Covenant University, "If Professor Tordesillas' tool can provide early warning before waste slides occur, then it will be a vital tool for preventing landslide disaster."
As governments and businesses look to algorithms to increase consistency, save money, or simply manage complicated processes, human reliance on them and their inherent biases is starting to worry politicians, activists, and technology researchers. In early 2018, New York City became the first government in the U.S. to pass a law designed to address bias in the algorithms used by the city, establishing a task force to study and make recommendations on the matter. Meanwhile, a bipartisan group in Washington state introduced a bill intended to ensure the fair, transparent, and accountable use of automated decision systems in state government by establishing guidelines for the procurement and use of such technology. Rashida Richardson at the nonprofit AI Now Institute said the panels that determine the biases of such systems should include not only data scientists and technologists, but also legal experts familiar with the history of laws and cases dealing with identifying and remedying bias.
Emil Protalinski
IBM researchers have developed a technique to mitigate the noise of quantum computations. The "zero-noise extrapolation" method repeats computation at varying noise levels to determine what a quantum system would calculate in the absence of noise. The microwave pulses used to execute quantum operations on the quantum bits are "stretched in time" to amplify noise. Zero-noise extrapolation ameliorates the effect of decoherence, and can support more complex and precise computations that benefit from greater circuit depth. The researchers said this technique can enhance the accuracy of any algorithm run on current IBM Q systems.
Mark Mazzetti; Adam Goldman
March 21, 2019; et al.
Even the smallest countries in the world have the resources to buy digital espionage services, enabling them to conduct sophisticated operations like electronic eavesdropping or influence campaigns. In addition, corporations that want to understand competitors' secrets, or a wealthy individual with a vendetta against a rival, also can command intelligence operations for a price. The New York Times recently conducted a months-long examination that uncovered secret battles in the growing digital combat environment. The Middle East is the epicenter of this new era of privatized spying. For example, companies such as Israel-based NSO Group and United Arab Emirates-based DarkMatter have enabled governments to hack criminal elements like terrorist groups and drug cartels, while also targeting journalists and activists in some cases.
Brooks Hays
Researchers at the Swiss Federal Institute of Technology in Lausanne, Switzerland (EPFL) have developed robots to translate and deliver signals between groups of bees and schools of fish. The robots exchanged signals internationally, with bees in Austria communicating with fish in Switzerland. In Austria, groups of bees swarmed around two robot terminals within a large container, and the team used the terminals and a fish "spy" robot to beam signals and influence the different groups' behavior. The terminals emitted vibrations, temperature variations, and air movements that caused the bees to swarm exclusively around one terminal or the other, and signals from the fish robot induced the school of fish to swim in different directions. EPFL's Francesco Mondada said, "The robots acted as if they were negotiators and interpreters in an international conference. Through the various information exchanges, the two groups of animals gradually came to a shared decision" and synchronized their movements.
R&D Magazine
At Germany's Saarland University, researchers have released a free data processing tool that allows rapid evaluation of signals, pattern recognition, and data visualization when processing huge datasets. The MATLAB toolbox known as Dave enables very large volumes of data, such as those produced by modern sensor systems, to be processed, analyzed, and visually displayed so researchers can optimize their measurement systems interactively. Dave makes the calculations completely transparent, showing the user that when they alter a particular parameter, it has an identifiable consequence. Said Saarland University researcher Andreas Schutze, "Using Dave as a tool, we were able to rapidly achieve some widely acclaimed results in the field of condition monitoring in 'Industry 4.0' applications."
Charlie Osborne
Researchers at Ben-Gurion University of the Negev (BGU) in Israel and Fujitsu System Integration Laboratories in Japan warn of the potential for unregulated aerial drones to threaten citizens. The scientists analyzed more than 200 techniques and technologies to detect and disable drones, and determined cybersecurity countermeasures to be inadequate, which could lead to "cyberattacks, terrorism, crime, and threats to privacy." One exploit the researchers demonstrated would deliver hacking hardware and radio systems to targets, in the guise of a pizza delivery. BGU and Fujitsu suggested the biggest challenge facing vendors in terms of drones and their potential impact on privacy and security is learning a drone's purpose in a non-restricted area, or an "open sky policy." The researchers proposed drone identification systems and registration as a remedial starting point.
Ayal Karmi
Technology solutions like artificial intelligence (AI), data analytics, and drones are being employed to enable more efficient and intelligent agriculture. For example, farmers use geographic information system software to monitor crops through heat maps, so fertilizers or insecticides can be administered more efficiently to improve crop growth; machine learning technology also is augmenting heat maps, which can visualize terrain in greater detail than before. Agriculture's future relies on real-time responses, as more and more agricultural technology companies compete to provide all-in-one platforms with on-the-go insights and prescriptions. Meanwhile, Internet of Things sensors and AI platforms are expected to help the farming industry tackle key challenges, ranging from meeting the needs of global population growth to coping with climate change, while curtailing food waste. Drones also should improve irrigation and crop management decision-making by supplying precise, detailed maps for soil and field analysis.


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text:
TRANSPLANTATION| July 1, 2005
Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells
Chihaya Imai,
Chihaya Imai
From the Departments of Hematology-Oncology and Pathology, St Jude Children's Research Hospital, Memphis; and Department of Pediatrics, University of Tennessee College of Medicine, Memphis.
Shotaro Iwamoto,
Shotaro Iwamoto
Dario Campana
https://doi.org/10.1182/blood-2004-12-4797
Chihaya Imai, Shotaro Iwamoto, Dario Campana; Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 2005; 106 (1): 376–383. doi: https://doi.org/10.1182/blood-2004-12-4797
Natural killer (NK) cells hold promise for improving the therapeutic potential of allogeneic hematopoietic transplantation, but their effectiveness is limited by inhibitory HLA types. We sought to overcome this intrinsic resistance by transducing CD56+CD3- NK cells with chimeric receptors directed against CD19, a molecule widely expressed by malignant B cells. An abundance of NK cells for transduction was secured by culturing peripheral blood mononuclear cells with K562 cells expressing the NK-stimulatory molecules 4-1BB ligand and interleukin 15, which yielded a median greater than 1000-fold expansion of CD56+CD3- cells at 3 weeks of culture, without T-lymphocyte expansion. Expression of anti-CD19 receptors linked to CD3ζ overcame NK resistance and markedly enhanced NK-cell-mediated killing of leukemic cells. This result was significantly improved by adding the 4-1BB costimulatory molecule to the chimeric anti-CD19-CD3ζ receptor; the cytotoxicity produced by NK cells expressing this construct uniformly exceeded that of NK cells whose signaling receptors lacked 4-1BB, even when natural cytotoxicity was apparent. Addition of 4-1BB was also associated with increased cell activation and production of interferon γ and granulocyte-macrophage colony-stimulating factor. Our findings indicate that enforced expression of signaling receptors by NK cells might circumvent inhibitory signals, providing a novel means to enhance the effectiveness of allogeneic stem cell transplantation.
Immunotherapy, Neoplasia, Transplantation
cd19 antigens, chimera organism, cytotoxicity, leukemic cells, natural killer cells, killing, molecule, cd56 antigens, neural cell adhesion molecules, interleukin-15
B-cell malignancies of children and adults, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL), are often incurable even with intensive chemotherapy. For many patients, bone marrow ablation followed by allogeneic hematopoietic stem cell transplantation is the only potentially curative option, but the disease may return after transplantation.1 The well-documented association between T-cell-mediated graft-versus-host disease (GvHD) and a delay or suppression of leukemic relapse after allogeneic stem cell transplantation2-4 has led some investigators to manipulate GvHD by infusion of donor T lymphocytes. Although this procedure can induce a measurable antineoplastic response,5-8 it carries the risk of severe GvHD, particularly in those patients (> 70%) who lack an HLA-identical donor. Moreover, in some B-cell malignancies, such as ALL, the effect of lymphocyte infusions is often inadequate.6,9,10
Besides T lymphocytes, natural killer (NK) cells also exert cytotoxicity against cancer cells.11 Recent studies have emphasized the potential of NK-cell therapy in recipients of allogeneic hematopoietic stem cell transplants. In animal models of transplantation, donor NK cells could lyse leukemic cells and host lymphohematopoietic cells without affecting nonhematopoietic tissues,12 suggesting that NK-mediated graft-versus-leukemia responses may occur in the absence of systemic disease. Because NK cells are inhibited by self-HLA molecules, which bind to killer immunoglobulin-like receptors (KIRs), these findings have led to the clinical practice of selecting hematopoietic stem cell transplantation donors with an HLA and KIR type that favors NK-cell activation and thus could be expected to promote an antileukemic effect.13-15 However, selection of the "best" donor is limited to patients who have more than one potential donor and the capacity of NK cells to lyse lymphoid cells is generally low and difficult to predict.13,15-17
Emerging evidence indicates that T lymphocytes genetically modified with chimeric receptors able to recognize a surface molecule of target cells and transduce activation signals can specifically enhance T-cell cytotoxicity against cancer cells both in vitro and in vivo.18-21 The studies presented here are based on the concept that expression of chimeric receptors on NK cells could overcome HLA-mediated inhibitory signals, thus endowing the cells with cytotoxicity against otherwise NK-resistant cells. To test this hypothesis, we first developed a novel method that allows specific and vigorous expansion of NK cells lacking T-cell receptors (CD56+CD3- cells) and their highly efficient transduction with chimeric receptors. Then, we tested the relative antileukemic effects of genetically modified NK cells bearing chimeric receptors (directed against CD19, a molecule widely expressed by malignant B cells) that deliver different primary and costimulatory signals.
The CD19+ human B-lineage ALL cell lines RS4;11, OP-1, 380, 697, and KOPN57bi; the T-cell line CEM-C7; and the myeloid cell lines K562 and U-937 were available in our laboratory.21 Cells were maintained in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; BioWhittaker, Walkersville, MD) and antibiotics.
Primary leukemia cells were obtained from 9 patients with B-lineage ALL, with appropriate informed consent and Institutional Review Board (IRB) approval; from 4 of these patients, we also studied (with Saint Jude IRB approval) cryopreserved peripheral blood samples obtained during clinical remission. An unequivocal diagnosis of B-lineage ALL was established by morphologic, cytochemical, and immunophenotypic criteria; in each case, more than 95% of the cells were positive for CD19. Peripheral blood was obtained from 8 healthy adult donors. Mononuclear cells collected from the samples by centrifugation on a Lymphoprep density step (Nycomed, Oslo, Norway) were washed twice in phosphate-buffered saline (PBS) and once in AIM-V medium (Gibco).
Plasmids and retrovirus production
The anti-CD19-ζ, anti-CD19-BB-ζ, and anti-CD19-truncated (control) plasmids are described elsewhere.21 The pMSCV-IRES-GFP, pEQ-PAM3(-E), and pRDF constructs were obtained from the Saint Jude Vector Development and Production Shared Resource. The cDNA encoding the intracellular domains of human DAP10 and 4-1BB ligand (4-1BBL), and interleukin-15 (IL-15) with long signal peptide were subcloned by polymerase chain reaction (PCR) with a human spleen cDNA library (from Dr G. Neale, St Jude Children's Research Hospital) used as a template. An anti-CD19-DAP10 plasmid was constructed by replacing the sequence encoding CD3ζ with that encoding DAP10, using the splicing by overlapping extension by PCR (SOE-PCR) method. The cDNA encoding the signal peptide of CD8α, the mature peptide of IL-15 and the transmembrane domain of CD8α were assembled by SOE-PCR to encode a "membrane-bound" form of IL-15. The resulting expression cassettes were subcloned into EcoRI and XhoI sites of murine stem-cell virus-internal ribosome entry site-green fluorescent protein (MSCV-IRES-GFP).
The RD114-pseudotyped retrovirus was generated as previously described.21 We used calcium phosphate DNA precipitation to transfect 293T cells with anti-CD19-ζ, anti-CD19-DAP10, anti-CD19-BB-ζ, or anti-CD19-truncated; pEQ-PAM3(-E); and pRDF. Conditioned medium containing retrovirus was harvested at 48 hours and 72 hours after transfection, immediately frozen in dry ice, and stored at -80°C until use.
Development of K562 derivatives, expansion of NK cells, and gene transduction
K562 cells were transduced with the construct encoding the "membrane-bound" form of IL-15. Cells were cloned by limiting dilution, and a single-cell clone with high expression of GFP and of surface IL-15 (K562-mb15) was expanded. This clone was subsequently transduced with human 4-1BBL and designated as K562-mb15-41BBL. K562 cells expressing wild-type IL-15 (K562-wt15) or 4-1BBL (K562-41BBL) were produced by a similar procedure. Peripheral blood mononuclear cells (1.5 × 106) were incubated in a 24-well tissue-culture plate with or without 106 K562-derivative stimulator cells in the presence of 10 IU/mL human IL-2 (National Cancer Institute BRB Preclinical Repository, Rockville, MD) in RPMI 1640 and 10% FCS.
Mononuclear cells stimulated with K562-mb15-41BBL were transduced with retroviruses, as previously described for T cells.21 Briefly, 14 mL polypropylene centrifuge tubes (Falcon, Lincoln Park, NJ) were coated with human fibronectin (100 μg/mL; Sigma, St Louis, MO) or RetroNectin (50 μg/mL; TaKaRa, Otsu, Japan). Prestimulated cells (2 × 105) were resuspended in the tubes in 2 to 3 mL virus-conditioned medium with Polybrene (4 μg/mL; Sigma) and centrifuged at 2400g for 2 hours (centrifugation was omitted when RetroNectin was used). The multiplicity of infection (4-6) was identical in each experiment comparing the activity of different chimeric receptors. After centrifugation, cells were left undisturbed for 24 hours in a humidified incubator at 37°C, 5% CO2. The transduction procedure was repeated on 2 successive days. After a second transduction, the cells were restimulated with K562-mb15-41BBL in the presence of 10 IU/mL IL-2. Cells were maintained in RPMI 1640, 10% FCS, and 10 IU/mL IL-2.
Detection of chimeric receptor expression and immunophenotyping
Transduced NK cells were stained with goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin (Jackson ImmunoResearch Labs, West Grove, PA) followed by streptavidin conjugated to peridinin chlorophyll protein (PerCP; Becton Dickinson, San Jose, CA). For Western blotting, cells were lysed in RIPA buffer (PBS, 1% Triton-X100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) containing 3 μg/mL pepstatin, 3 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM ethylenediaminetetraacetic acid (EDTA), and 5 μg/mL aprotinin. Centrifuged lysate supernatants were boiled with an equal volume of loading buffer with or without 0.1 M dithiothreitol (DTT), and then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a precast 10% to 20% gradient acrylamide gel (Bio-Rad, Hercules, CA). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, which was incubated with primary mouse anti-human CD3ζ monoclonal antibody (clone 8D3; PharMingen, San Diego, CA). Membranes were then washed, incubated with a goat anti-mouse IgG horseradish peroxidase-conjugated second antibody, and developed by using the enhanced chemiluminescence system (Amersham, Little Chalfont, United Kingdom).
The following antibodies were used for immunophenotypic characterization of expanded and transduced cells: anti-CD3 conjugated to fluorescein isothiocyanate (FITC), to PerCP, or to energy-coupled dye (ECD); anti-CD10 conjugated to phycoerythrin (PE); anti-CD19 PE; anti-CD22 PE; anti-CD56 FITC, PE, or allophycocyanin (APC); anti-CD16 CyChrome (antibodies from Becton Dickinson, PharMingen, or Beckman-Coulter, Miami, FL); and anti-CD25 PE (Dako, Carpinteria, CA). Surface expression of KIR and NK activation molecules was determined with specific antibodies conjugated to FITC or PE (from Beckman-Coulter or Becton Dickinson), as previously described.15 Antibody staining was detected with a FACScan or an LSR II flow cytometer (Becton Dickinson).
Cytotoxicity assays and cytokine production
Target cells (1.5 × 105) were placed in 96-well U-bottomed tissue-culture plates (Costar, Cambridge, MA) and incubated with primary NK cells transduced with chimeric receptors at various effector-target (E/T) ratios in RPMI 1640 supplemented with 10% FCS; NK cells were cultured with 1000 U/mL IL-2 for 48 hours before the assay. Cultures were performed in the absence of exogenous IL-2. After 4 hours and 24 hours, cells were harvested, labeled with CD10 PE or CD22 PE and CD56 FITC, and assayed by flow cytometry as previously described.21-23 The numbers of target cells recovered from cultures without NK cells were used as a reference.
For cytokine production, primary NK cells (2 × 105 in 200 μL) expressing chimeric receptors were stimulated with various target cells at a 1:1 ratio for 24 hours. The levels of interferon γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in cell-free culture supernatants were determined with a Bio-Plex assay (Bio-Rad).
A test of equality of mean NK expansion with various stimuli was performed using analysis of variance for a randomized complete block design with each donor considered a random block. The Tukey honest significant difference procedure was used to compute simultaneous confidence intervals for each pairwise comparison of the differences of treatment means. Differences in cytotoxicities and cytokine production among NK cells bearing different chimeric receptors were analyzed by the paired Student t test.
Expansion of NK cells after 1 week of culture with genetically modified K562 cells. Peripheral blood mononuclear cells from 7 healthy individuals (represented by different symbols) were cultured with various preparations of K562 at a 1:1 ratio in the presence of low-dose (10 U/mL) IL-2. Percentages of CD56+CD3- NK cells and CD3+ T lymphocytes after 7 days of culture relative to the number of input cells are shown. Each data point represents the average of 2 measurements; bars correspond to the median expansion in each group. K562 cells expressing both membrane-bound IL-15 and 4-1BBL (K562-mb15-41BBL) induced a markedly superior expansion of NK cells (P < .001 by the Tukey honest significant difference test) without inducing T-cell proliferation; there were no significant differences among other pairwise comparisons of NK expansions obtained with K562, K562-41BBL, and K562-mb15.
Culture conditions that favor the expansion of primary NK cells
To transduce chimeric receptors into primary NK cells, we searched for stimuli that would induce specific NK-cell proliferation. In preliminary experiments, we depleted peripheral blood mononuclear cells of CD3+ T lymphocytes and stimulated the remaining cells with IL-2 (1000 U/mL) or IL-15 (10 ng/mL). Under these culture conditions there was no expansion of NK cells, which, in fact, progressively declined in numbers. With phytohe-magglutinin (PHA; 7 μg/mL) and IL-2 (1000 U/mL) as stimuli, we observed a 2- to 5-fold expansion of CD56+ CD3- NK cells after 1 week of culture. However, despite the low proportion of contaminating CD3+ cells (< 2% in 2 experiments) at the beginning of the cultures, these cells expanded more than NK cells (> 30-fold expansion), and after 1 week of culture represented approximately 35% of the cell population.
NK cells can be stimulated by contact with the human leukemia cell line K562, which lacks HLA-antigen expression,24 and genetically modified K562 cells have been used to stimulate cytotoxic T lymphocytes.25 We therefore tested whether the NK-stimulatory capacity of K562 cells could be increased through enforced expression of additional NK-stimulatory molecules, using 2 molecules that are not expressed by K562 cells and are known to stimulate NK cells. One molecule, the ligand for 4-1BB, triggers activation signals after binding to 4-1BB (CD137), a signaling molecule expressed on the surface of NK cells.26 The other molecule, IL-15, is a cytokine known to promote NK-cell development and the survival of mature NK cells.27-30 Because IL-15 has greater biologic activity when presented to NK cells bound to IL-15 receptor α (IL-15Rα) on the cell membrane of stimulatory cells, rather than in its soluble form,31-35 we made a construct containing the human IL15 gene fused to the gene encoding the human CD8α transmembrane domain and used it to transduce K562 cells. Expression of IL-15 on the surface of K562 cells was more than 5 times higher with the IL-15-CD8α construct than with wild-type IL-15 (not shown).
To test whether the modified K562 cells expressing both 4-1BBL and IL-15 (K562-mb15-41BBL cells) promote NK-cell expansion, we cultured peripheral blood mononuclear cells from 7 donors in the presence of low-dose (10 U/mL) IL-2 as well as irradiated K562 cells transduced with 4-1BBL or IL-15 (or both), or with an empty control vector. As shown in Figure 1, expression of either 4-1BBL or IL-15 by K562 cells improved the stimulation of NK-stimulatory capacity of K562 in some cases but not overall, whereas simultaneous expression of both molecules led to a consistent and striking amplification of NK cells (median recovery of CD56+CD3- cells at 1 week of culture, 2030% of input cells [range, 1020%-2520%] compared with a median recovery of 250% [range, 150%-640%] for K562 cells lacking 4-1BBL and IL-15; P < .001). In 24 experiments with cells from 8 donors, NK-cell expansion after 3 weeks of culture with K562 cells expressing both stimulatory molecules ranged from 309-fold to 12 409-fold (median, 1089-fold). Importantly, neither the modified nor unmodified K562 cells caused an expansion of T lymphocytes (Figure 1).
Among expanded CD56+CD3- NK cells, expression of CD56 was higher than that of unstimulated cells (Figure 2); expression of CD16 was similar to that seen on unstimulated NK cells (median CD16+ NK cells in 7 donors: 89% before expansion and 84% after expansion). We also compared the expression of KIR molecules on the expanded NK cells with that on NK cells before culture, using the monoclonal antibodies CD158a (against KIR 2DL1), CD158b (2DL2), NKB1 (3DL1), and NKAT2 (2DL3). The prevalence of NK subsets expressing these molecules after expansion resembled that of their counterparts before culture, although the level of expression of KIR molecules was higher after culture (Figure 2). Similar results were obtained for the inhibitory receptor CD94, whereas expression of the activating receptors NKp30 and NKp44 became detectable on most cells after culture (not shown). In sum, the immunophenotype of expanded NK cells reiterated that of activated NK cells, indicating that contact with K562-mb15-41BBL cells had stimulated expansion of all subsets of NK cells.
Transduction of NK cells with chimeric receptors
Before transducing peripheral blood mononuclear cells with retroviral vectors containing chimeric receptor constructs and GFP (Figure 3), we stimulated them with K562-mb15-41BBL cells. In 27 experiments, the median percentage of NK cells that were GFP+ at 7 to 11 days after transduction was 69% (43%-93%). Chimeric receptors were expressed at high levels on the surface of NK cells and, by Western blotting, were in both monomeric and dimeric configurations (Figure 4).
Immunophenotypic features of NK cells before and after expansion with K562-mb15-41BBL cells. Expression of CD3 and CD56, as well as expression of the KIRs CD158a (2DL1), CD158b (2DL2), NKB1 (3DL1), and NKAT2 (2DL3) on CD56+CD3- cells were examined in peripheral blood mononuclear cells from a healthy donor before (top row) and after (bottom row) 3 weeks of coculture with K562-mb15-41BBL cells and low-dose (10 U/mL) IL-2.
Schematic representation of the chimeric receptors used in this study. LTR indicates long terminal repeat; AMP, ampicillin resistance; and bp, base pair.
To identify the specific signals required to stimulate NK cells with chimeric receptors, and overcome inhibitory signals mediated by KIR molecules and other NK inhibitory receptors that bind to HLA class I molecules, we first compared 2 types of chimeric receptors containing different signaling domains: CD3ζ, a signal-transducing molecule containing 3 immunoreceptor tyrosine-based activation motifs (ITAMs) and linked to several activating receptors expressed on the surface of NK cells,11,36 and DAP10, a signal transducing molecule with no ITAMs linked to the activating receptor NKG2D and previously shown to trigger NK cytotoxicity.11,36,37 As a control, we used NK cells transduced with a vector containing an anti-CD19 receptor but no signaling molecules or containing GFP alone. NK cells were challenged with the CD19+ leukemic cell lines 380, 697, and RS4;11, all of which express high levels of HLA class I molecules by antibody staining. By genotyping, RS4;11 is Cw4/Cw3, Bw4 and A3; 380 is Cw4/Cw4, Bw4; and 697 is Cw3/Cw3. Hence, these cell lines were fully capable of inhibiting NK-cell cytotoxicity via binding to NK-inhibitory receptors.
Expression of chimeric receptors by NK cells expanded from peripheral blood mononuclear cells. (A) Surface receptor expression was visualized by flow cytometry after staining with a goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin followed by streptavidin PerCP (y-axes); expression of GFP is also shown (x-axes). (B) Western blot analysis of chimeric receptor expression in NK cells, under reducing or nonreducing conditions. Filter membranes were labeled with an antihuman CD3ζ antibody and a goat anti-mouse IgG horseradish peroxidase-conjugated second antibody.
Chimeric receptors bearing CD3ζ overcome the NK resistance of leukemic cells. The results shown for 3 cell lines, 380, RS4;11, and 697, are expressed as the mean (± SD; n = 4) percentage of leukemic cell recovery after 24 hours of culture at a 1:1 E/T ratio relative to cultures without NK cells, as measured by flow cytometry. NK cells expressing anti-CD19-ζ receptors were more cytotoxic than NK cells expressing anti-CD19-DAP10 receptors, anti-CD19 receptors without signaling capacity (anti-CD19-trunc), or NK cells transduced with GFP control vector (P < .001).
Expression of receptors without signaling molecules did not increase NK-mediated cytotoxicity over that exerted by NK cells transduced with the vector containing only GFP (Figure 5). By contrast, expression of anti-CD19-ζ receptors markedly enhanced NK cytotoxicity in all experiments (Figure 5), regardless of the intrinsic ability of donor NK cells to kill leukemic targets. For example, 380 cells were highly resistant to NK cells from donors 2 and 3, but were killed when these donor cells expressed anti-CD19-ζ receptors. Similar observations were made for RS4;11 cells and the NK cells of donor 1 and for 697 cells and NK cells of donor 2. Moreover, the anti-CD19-ζ receptors led to improved killing of target cells even when natural cytotoxicity was present. In all experiments, the cytotoxicity triggered by the anti-CD19-ζ receptor was enhanced over that achieved by replacing CD3ζ with DAP10 (P < .001; Figure 5).
4-1BB-mediated costimulatory signals enhance NK cytotoxicity
Previous studies have shown that the addition of costimulatory molecules to chimeric receptors enhances the proliferation and cytotoxicity of T lymphocytes.21,38-43 Of the 2 best known costimulatory molecules in T lymphocytes, CD28 and 4-1BB, only 4-1BB is expressed by NK cells.26,44,45 We therefore determined whether the addition of 4-1BB to the anti-CD19-ζ receptor would enhance NK cytotoxicity. In a 4-hour cytotoxicity assay, cells expressing the 4-1BB-augmented receptor showed a markedly better ability to kill CD19+ cells than did cells lacking this modification (Figure 6A-B). The superiority of NK cells bearing the anti-CD19-BB-ζ receptor was also evident in 24-hour assays with NK cells from different donors cultured at a 1:1 ratio with the leukemia cell lines 697, KOPN57bi, and OP-1 (not shown).
Next, we determined whether the antileukemic activity of NK cells expressing anti-CD19-BB-ζ receptors extended to primary leukemic samples. In 5 samples from children with different molecular species of ALL, NK cells expressing the 4-1BB receptors exerted strong cytotoxicity that was evident even at low E/T ratios (eg, < 1:1; Figure 7) and uniformly exceeded the activity of NK cells expressing signaling receptors that lacked 4-1BB. Even when donor NK cells had natural cytotoxicity against ALL cells and CD3ζ receptor did not improve it (patient no. 3 in Figure 7), addition of 4-1BB to the receptor significantly enhanced cytotoxicity. Consistent with their increased cytotoxicity, NK cells expressing anti-CD19-BB-ζ mediated more vigorous activation signals. As shown in Figure 8A, 46% of NK cells bearing this receptor expressed the IL-2Rα chain CD25 after 24 hours of coculture with CD19+ ALL cells, compared with only 17% of cells expressing the anti-CD19-ζ receptor and less than 1% for cells expressing receptors that lacked stimulatory capacity. Moreover, anti-CD19-BB-ζ receptors induced a much higher production of IFN-γ and GM-CSF on contact with CD19+ cells than did receptors without 4-1BB (Figure 8B).
Addition of the 4-1BB costimulatory molecule to the chimeric receptors augments their capacity to induce NK cytotoxicity against NK-resistant leukemic cells. (A) Expanded primary NK cells expressing chimeric receptors were incubated for 4 hours with the B-lineage ALL cell lines 380 and RS4;11 at the indicated E/T ratios. Each data point represents the mean (± SD; n = 4) percentage of ALL cell killing after culture as compared to that of parallel cultures without NK cells. At all E/T ratios, cytotoxicity of NK cells expressing chimeric receptors containing 4-1BB was significantly higher than that induced by receptors without 4-1BB (P < .001). (B) Flow cytometric dot plots show staining with anti-CD56 and anti-CD22 after a 4-hour coculture of NK cells (CD56+) and ALL cells (380; CD22+) at a 2:1 ratio. The percentage of cell killing obtained with NK cells expressing different chimeric receptors (% kill) was calculated by comparing the number of viable CD22+ ALL cells recovered after the test culture to that of parallel cultures without NK cells.
NK cells expressing 4-1BB-augmented chimeric receptors show powerful cytotoxicity against leukemic cells from patients. Expanded primary NK cells expressing chimeric receptors were incubated for 4 hours with leukemic cells from children with different subtypes of B-lineage ALL (patient [Pt] 1, hyperdiploid 47-50; Pt 2 and Pt 5, t(4;11)(q21;q23); Pt 3, t(14;?)(q32;?); Pt 4, der8, t(8;?)) at the indicated E/T ratios. Each data point represents the mean (± SD; n = 4) percentage of ALL cell killing after culture as compared to that of parallel cultures without NK cells. With the exception of the results obtained in patient 2 at a 1:1 ratio, the cytotoxicity of NK cells expressing chimeric receptors containing 4-1BB was significantly higher than that induced by receptors without 4-1BB (P < .005).
Chimeric receptors bearing 4-1BB induce a superior NK-cell activation. (A) Expanded primary NK cells expressing chimeric receptors were incubated for 24 hours with the ALL cell line RS4;11 at a 1:1 ratio. Flow cytometric dot plots illustrated CD25 expression (in the y-axes) and side scatter (SSC; in the x-axes) of GFP+ cells before and after culture. The percentages of CD25+ NK cells are indicated. (B) Production of IFN-γ and GM-CSF by NK cells expressing different chimeric receptors after 24 hours of culture with 697 cells at a 1:1 ratio (mean ± SD of 3 measurements). The 4-1BB receptors elicited a significantly higher production of both cytokines (P < .005).
We asked whether the expression of signaling chimeric receptors would affect spontaneous NK activity against NK-sensitive cell lines not expressing CD19. Spontaneous cytotoxicity of NK cells from 3 donors against the CD19- leukemia cell lines K562, U937, and CEM-C7 was not diminished by expression of chimeric receptors, with or without 4-1BB (not shown).
Anti-CD19 chimeric receptors induce NK cytotoxicity against autologous leukemic cells
To determine whether the NK-cell expansion and transduction system that we developed would be applicable to clinical samples, we studied peripheral blood samples that had been obtained (and cryopreserved) from 4 patients with childhood B-lineage ALL in clinical remission, 25 to 56 weeks from diagnosis. NK-cell expansion occurred in all 4 samples; after 1 week of culture with K562-mb15-41BBL cells, recovery of CD56+CD3- NK cells ranged from 1350% to 3680% of the input.
NK cells expressing anti-CD19 signaling receptors become highly cytotoxic against autologous leukemic cells. Peripheral blood NK cells were obtained from patients with B-lineage ALL in clinical remission. After expansion and transduction, cytotoxicity was tested against autologous leukemic lymphoblasts from diagnostic bone marrow samples. Each data point represents the mean (± SD; n = 4) percentage of ALL cell killing after culture as compared to that of parallel cultures without NK cells. The cytotoxicity of NK cells expressing anti-CD19-BB-ζ receptors (□ and solid lines) was markedly higher than that exerted by NK cells transduced with anti-CD19 truncated nonsignaling receptor (▵ and dotted line; patients 6-8) or empty vector (▵ and dotted line; patient 9).
After transduction with chimeric receptors, we tested the cytotoxicity of the NK cells against autologous leukemic lymphoblasts obtained at diagnosis. As shown in Figure 9, expression of anti-CD19-BB-ζ receptors overcame NK-cell resistance of autologous cells; NK cells expressing the receptors exerted cytotoxicity, which was as powerful as that observed with allogeneic targets.
In this study, we demonstrated that the resistance of cancer cells to NK-cell activity can be overcome by chimeric receptors expressed on primary NK cells. The stimulatory signals triggered by the receptors on contact with target cells predominated over inhibitory signals and induced powerful cytotoxicity against NK-resistant leukemic cell lines and primary leukemic cells. We found that the type of stimulatory signal delivered by the chimeric receptor was a key factor in inducing cytotoxicity. Although DAP10 signaling can elicit NK cytotoxicity,37 chimeric receptors containing this molecule in our study induced weaker NK-cell activity than that generated by CD3ζ-containing receptors, despite identical levels of surface expression. We also found that addition of the costimulatory molecule 4-1BB to the chimeric receptors markedly augmented cytotoxicity and that receptors containing both CD3ζ and 4-1BB triggered a much more robust NK-cell activation and cytokine production than did those containing only CD3ζ. The important contribution of 4-1BB signals agrees with findings that anti-4-1BB antibodies activate murine NK cells,46 and enhance their antitumor activity.26 Leukemic lymphoid cells usually do not express 4-1BBL21 ; only 2 of 284 diagnostic B-lineage ALL samples studied by gene arrays at our institution expressed 4-1BBL transcripts.47 Hence, 4-1BB signals can be delivered to NK cells only if the molecule is incorporated into the receptor.
Efficient and stable transduction of primary NK cells is notoriously difficult, prompting us to devise a new gene transduction method for the present study. Most investigators have demonstrated efficient gene transfer only in continuously growing NK-cell lines48-54 or reported methods yielding only transient gene expression.37,55,56 We achieved stable expression of chimeric receptors in primary CD56+CD3- NK cells by using an RD114-pseudotyped retroviral vector and specifically expanding primary CD56+CD3- NK cells before they were exposed to the retrovirus, a step that allowed highly efficient gene expression. Although several cytokines such as IL-2, IL-12, and IL-15 stimulate NK cells,27,57,58 their capacity to induce proliferation of resting CD56+CD3- cells has been poor, unless accessory cells are present in the cultures.24 Perussia et al59 found that contact with irradiated B-lymphoblastoid cells induced as high as a 25-fold expansion of NK cells after 2 weeks of stimulation, and Miller et al60 reported an approximate 30-fold expansion of NK cells after 18 days of culture with 1000 U/mL IL-2 and monocytes. However, these culture conditions are likely to promote the growth of CD3+ T lymphocytes as well as NK cells. Because our ultimate aim is to generate pure preparations of donor NK cells devoid of CD3+ T lymphocytes that can be infused into recipients of allogeneic hematopoietic stem cell transplants, we searched for methods that would maximize NK-cell expansion without producing T-cell mitogenicity.
Contact with K562 cells (which lack major histocompatibility complex [MHC] class I molecule expression and hence do not trigger KIR-mediated inhibitory signals in NK cells) is known to augment NK-cell proliferation in response to IL-15.24 We found that membrane-bound IL-15 and 4-1BBL, coexpressed by K562 cells, acted synergistically to augment K562-specific NK-stimulatory capacity, resulting in vigorous expansion of peripheral blood CD56+CD3- NK cells without concomitant growth of T lymphocytes. After 2 to 3 weeks of culture, we observed NK-cell expansions of up to 10 000-fold, and virtually pure populations of NK cells could be obtained, even without the need for T-cell depletion in some cases. NK cells expanded in this system retained the immunophenotypic diversity seen among peripheral blood subsets of NK cells, as well as their natural cytotoxicity against sensitive target cells, even after transduction with different chimeric receptors. Hence, this system should help studies of NK-cell biology that require specific cell expansion or gene transduction or both, but it should also be adaptable to clinical applications after generating K562-mb15-41BBL cells that comply with current good manufacturing practices for clinical trials. Recently, Harada et al reported that expansions of CD56+CD3- cells (up to 400-fold after 2 weeks) were apparently superior after contact with another HLA class I-negative cell line, the Wilms tumor cell line HFWT.61 Future studies should determine whether HFWT cells express 4-1BBL or whether enforced expression of 4-1BBL together with IL-15 results in a greater specific expansion of NK cells than seen with modified K562 cells.
In the context of allogeneic hematopoietic stem cell transplantation, infusions of activated donor T cells would carry an unacceptably high risk of severe GvHD, particularly in recipients of haploidentical or mismatched transplants. By contrast, infusions of pure CD56+CD3- NK cells should not impose that risk.12 Most clinical studies of the therapeutic effects of NK cells have been performed in an autologous setting and have yielded only moderately promising results.11,62 This is not surprising because NK-cell activity is inhibited by surface receptors that recognize autologous HLA molecules expressed by both normal and neoplastic cells. Allogeneic NK cells may be more effective, but even in an allogeneic setting the capacity of NK cells to kill malignant lymphoid cells is generally modest and often negligible.17 Leung et al15 detected NK cytotoxicity against an ALL cell line expressing particularly low levels of inhibitory HLA molecules, but cytotoxicity was much lower than that observed against the NK-cell target K562; only about 50% of the ALL cells were killed at an effector-target ratio of 40:1. In that study, RS4;11 cells, which express HLA-C alleles that bind the most commonly expressed KIRs, were NK resistant, whereas these cells, as well as autologous leukemic cells, were highly sensitive to NK cells expressing anti-CD19 signaling receptors in our study. Thus, NK cells expressing signaling chimeric receptors have much more powerful antileukemic activity than unmodified NK cells and can kill target cells irrespective of their HLA profile.
An increased understanding of the signals leading to immune cell activation, together with progress in gene cloning and transfer, have made the treatment of cancer with "adoptively acquired immunity"63 a realistic goal. Clinical precedents, such as administration of T-cell clones that target cytomegalovirus epitopes64 or Epstein-Barr virus-specific antigens,65 attest to the clinical feasibility of adoptive immune cell therapy. Nonetheless, potential limitations may affect the effectiveness of cell therapy guided by chimeric receptors. One is that the murine scFv portion of the chimeric receptor or the fusion sites of the human regions that compose it may trigger a host immune response leading to elimination of the modified cells.18 Although the impact of such an event in a clinical setting remains to be determined, we anticipate that immune responses against modified NK cells will be limited in immunosuppressed patients after hematopoietic stem cell transplantation. Another potential limitation is that adoptively transferred cells may have inadequate persistence in vivo, although a recent study showed that NK cells obtained from haploidentical donors and activated ex vivo could expand in patients when infused after administration of high-dose cyclophosphamide and fludarabine, which caused an increase in endogenous IL-15.66 We speculate that such expansions would also occur with genetically modified NK cells and suggest that further studies to identify signaling molecules that promote NK-cell proliferation when incorporated into chimeric receptors are warranted. In patients at a high risk of leukemia or lymphoma relapse, the expected benefits of genetically modified NK cells will outweigh the risk of insertional oncogenesis posed by the use of retroviruses for chimeric receptor transduction.67 We also predict that the coexpression of suicide genes will become a useful safety measure in clinical studies68 ; this strategy would also ensure that the elimination of normal CD19+ B-lineage cells is only temporary.
Novel therapies that bypass cellular mechanisms of drug resistance are urgently needed for patients with refractory leukemia and lymphoma. NK-cell alloreactivity is a powerful new tool for improving the therapeutic potential of allogeneic hematopoietic stem cell transplantation. The results of this study indicate that signaling receptors can enhance the efficacy of NK-cell alloreactivity and widen its applicability. We envisage initial clinical trials in which donor NK cells, collected by apheresis, are expanded ex vivo as described here, transduced with chimeric receptors, and then infused after transplantation in patients with B-lineage ALL. The target molecule for the chimeric receptors, CD19, was selected because it is one of the most widely expressed surface antigens among B-cell malignancies, including ALL, CLL, and NHL. In these malignancies, CD19 is highly expressed on the surface of virtually all cells but has limited or no expression in normal tissues.69 However, the NK-cell strategy of immunotherapy we describe would not have to be directed to the CD19 antigen but could be applied to any of the numerous molecules identified as potential targets for chimeric receptor-based cell therapy in patients with cancer.18
Prepublished online as Blood First Edition Paper, March 8, 2005; DOI 10.1182/blood-2004-12-4797.
Supported by grants CA58297 and CA21765 from the National Cancer Institute and by the American Lebanese Syrian Associated Charities (ALSAC).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
We thank Martha Holladay and Jim Houston for KIR expression analysis, Victoria Turner for cell line genotyping, Stanley Pounds for assistance with statistical analysis, the St Jude Vector Development and Production Shared Resource for retroviral vectors and the St. Jude Tissue Resource laboratory for providing patient samples.
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Copyright © 2005 by The American Society of Hematology

redpajama_set_name: RedPajamaCommonCrawl


Mike Dow was Mayor of Mobile from 1989 to 2005.
Dow served three-tours in Vietnam as a helicopter door gunner from 1966 to 1968. He then attended the University of South Alabama on the G. I. Bill. Dow received his bachelor's in economics in 1971 and his master's in business administration, with an accounting focus in the Mitchell College of Business, in 1977. He joined his brother-in-law Jim Busby's laser printer company QMS in 1977. QMS was eventually taken public.
Dow left QMS to run for mayor in 1988, when he won over Arthur Outlaw. Among Dow's accomplishments as mayor is the downtown "String of Pearls" initiative.
After deciding not to run for reelection in 2005, Dow joined Jim Busby's new company CentraLite as Executive Vice-President of Sales and Marketing.


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The day after she was evicted from the Big Brother Canada 7 house, I had the chance to catch up with Chelsea Bird to discuss what happened. We talked about what led to her being HOH one week and evicted the next. We also discussed if she regretted winning HOH as early as she did or if winning competition is the only way to succeed this season. Chelsea discussed the disintegration of her relationship with Dane and if she felt like Mark and the rest of the guys were being condescending to her the entire time. Finally, Chelsea opened up about the status of her relationship with Kyra.


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Q: Monitor a programs data transfer Is there a way to monitor what data a program sends and receives from the internet? So for example if you have a game and it takes some information from a server, since the program would have to be told the information, is there a way of recording that and reading it? I've read a few questions similar but they were all focused on how much data was transferred. I what to know what data is being transferred

A: You're looking for Fiddler (for HTTP traffic) or Wireshark (for all traffic).

A: extView infoView = (TextView)findViewById(R.id.traffic_info);
String info = "";

info += "Mobile Interface:\n";
info += ("\tReceived: " + TrafficStats.getMobileRxBytes() + " bytes / " + TrafficStats.getMobileRxPackets() + " packets\n");
info += ("\tTransmitted: " + TrafficStats.getMobileTxBytes() + " bytes / " + TrafficStats.getMobileTxPackets() + " packets\n");

info += "All Network Interface:\n";
info += ("\tReceived: " + TrafficStats.getTotalRxBytes() + " bytes / " + TrafficStats.getTotalRxPackets() + " packets\n");
info += ("\tTransmitted: " + TrafficStats.getTotalTxBytes() + " bytes / " + TrafficStats.getTotalTxPackets() + " packets\n");

infoView.setText(info);

A: mStartRX = TrafficStats.getTotalRxBytes();

mStartTX = TrafficStats.getTotalTxBytes(); TextView RX = (TextView)findViewById(R.id.RX);

TextView TX = (TextView)findViewById(R.id.TX);

long rxBytes = TrafficStats.getTotalRxBytes()- mStartRX;

RX.setText(Long.toString(rxBytes));

long txBytes = TrafficStats.getTotalTxBytes()- mStartTX;

TX.setText(Long.toString(txBytes));

redpajama_set_name: RedPajamaStackExchange


How to save a penny and lose your dignity. At the same time.
A common New Year's resolution would be to save a buck here and there and then use those savings to buy property on Mars or a Jet plane. Or maybe not. The point is I needed an opening sentence.
Now that I have your attention I can gladly share my latest discovery on how to save money. Here are 21 tips that will change your life! You might save a penny or more likely, leave you puking in a bucket. Unfortunately this is not a review or a tutorial as I haven't tried any of them. Yet.
Share the same mouthwash and dental floss as your partner. Mouthwash can be recycled by rinsing your mouth into a cup. Or French kissing.
Heat your food in the dishwasher, it reaches high temperatures doesn't it? Just remember to seal it properly, otherwise your meal might taste a bit soapy.
Use an old hubcap and some tin foil as a pan/serving dish/plate. Recycle the tin foil.
Do your gardening at night when power is cheaper. Wear a bullet proof vest.
Switch of the TV during commercials. They just entice you to spend money.
Re-dry your tissues. It's your own snot anyway.
Borrow better furniture from your neighbour when you have an open house, provided you don't live in a trailer park.
Take balloons from old window displays and re-use them for your kid's birthday party.
Use animal fat to increase the volume of ground beef. Because animal fat is not disgusting at all.
Peel bananas before you weigh them. You shouldn't be eating the peels.
Keep a record of how many times you flush the toilet and try to reduce that daily. If it's yellow let it mellow, if it's brown flush it down. Or go outside.
Buy two-ply toilet paper and split it. It will also provide you with something to do during commercials when the TV is off.
Make your own candles from used motor oil. Wear a gasmask when lighting them.
Stay with your mom. But only if she doesn't ask any rent and you can spunge of her completely.
Take your own microwave-able food to a restaurant. Be prepared to be thrown out.
Collect all the napkins/condiment packets you can from the same restaurant/take-away for use later during a dinner party. Just tell everyone it's a Fast Food theme.
Collect throw-away food and flowers from a dumpster, commonly referred to as Dumpster Diving. I would advise that you use gloves when sorting through the rubbish, looking for food, you never know what people might throw away.
When filling up at the pump, jiggle the handle and tube of each pump to get leftovers from the last person.
Dilute your vodka/whiskey with water and serve later when everyone's drunk.
Plan your vacation to destinations where family/relatives/people you've met once, live. And stay with them.
Before you become concerned, most of these comes courtesy of the show "Extreme Cheapskates" on TLC (and a few from people I know) and it would have been a lot funnier if it wasn't true.
I'm not sure exactly how much money you will end up saving, but I can guarantee you will lose your dignity, social status and most of your friends.
Maybe you guys can come up with a few more.
← Things I do NOT want to do in 2015.
hahaha. I watched one episode about a mother's budget for her daughters wedding being 1000$ – the dress was bought from a pawn shop and the cake was a demo cake which they could not cut!
I couldn't pull myself away from the television. I was disgusted and intrigued at the same time.
I consider that combination "blogworthy".
Got a stubborn stain or lose a shirt button?
Donate the shirt to a charity thrift store. They will clean and fix it before putting it on the rack for sale.
The entire process will be less expensive than taking it to a cleaners/tailor for repair.
Haahahaaha. Wait, is this true?
Saw a cheap guy on a talk show who did it.
He also took a tax deduction for the charitable donation of the damaged shirt.
Excellent tips, thanks. Now I'm feeling a bit thirsty, I'm just going to pop to the toilet….
I saw one lady on that show use rags that she washed and reused instead of toilet paper. Her kids couldn't get any play dates.
Why do I keep on watching that damn show even though is makes me sick?
I worked with a guy who would heat his lunch by leaving it on the dashboard of his car in the summer…I have no idea how he heated his lunch in the winter maybe he put it on the car engine and drove around for a bit. TLC (The Learning Channel) needs to change its name.


"Surely they wouldn't be able make any kind of complaint if we drilled a couple of small holes into their wall to put up some drainage would they?
A landlord of mine recently bought a terraced house and next doors guttering wasn't fitted properly which meant damp was coming through into OUR bedroom.
We simply replaced it at our cost as it was damaging our property and guttering is a relatively inexpensive thing to replace anyway. I think it was tenanted but no one answered so we fixed it anyway.
You could actually bill them for the works if it's their property causing the damage so they should be grateful that you are doing it at no cost to themselves. What I would say though is plan the pipes so there is minimal disruption to their property. Don't do the easiest / cheapest fix if it's going to be ugly on their house!
AST in previous landlords name - can we issue Section 8/21?
I would not list the noise issues etc as a reason for outing them as they sound like the higher risk type of tenant that might put up a fight. That means either damaging the property or simply not moving out if they take it personally.
I would tell them that you'd like to take possession back as you may sell it or move back in etc.
It's not ideal but don't stress to much about the above comments and talk to a qualified solicitor. 9 out of 10 solicitors would not transfer the AST into your name as legally it is still enforceable and it transfers to the new owner.
I've never heard of it being an offence of collecting rent without an AST and in fact there's plenty of people that let properties out that don't even have ASTs or have lost them over the years. Even in this case it becomes assumed that there is an agreement, again to protect the tenant... so you can't simply evict them if they've lost their agreement..
Seek advice from someone qualified. We always check details like this with our solicitor. Worst case scenario, you simply serve the correct notice now for the change of landlord and wait until it take affect before issuing any other notices.


LOUISVILLE, Ky. (July 2015) – At age 68, Ed Hamilton has left his mark on the art world through his many sculptures and bas-reliefs – especially in his native Louisville.
Take a walk along the waterfront downtown and you will find his eight-foot-tall statue of York, a slave of explorer William Clark that was dedicated in 2003 at the Belvedere. The York Memorial was part of the Bicentennial celebration of the Lewis & Clark Expedition, the Corps of Discovery.
• 2014: Martin Luther King Jr. Memorial, 25th & Jefferson Ave., Newport News, Va.
• 2012: Abraham Lincoln statue, Centre College, Danville, Ky.
• 2009: Abraham Lincoln, Waterfront Park, Louisville, Ky.
• 2004: Migration to the West, Frazier Museum, Louisville, Ky.
• 1998: The Spirit of Freedom (African-American Civil War Memorial), 10th & U Streets NW, Washington, D.C.
• 1992: La Amistad Memorial, City Hall, New Haven, Conn.
• 1984: Booker T. Washington, Hampton University, Hampton, Va.
• To learn more about sculptor Ed Hamilton, visit his website: www.EdHamiltonWorks.com.
Walk a little farther east through Waterfront Park and you will come across the elaborate Abraham Lincoln Memorial, dedicated in 2009. The 12-foot scale bronze of Lincoln also features four bas-relief plaques, each telling a different story of this nation's struggles for freedom.
Inside the nearby Frazier Museum stands a life-size frontier family sculpture titled "Migration to the West" that was established in 2004. Hamilton also has created a bronze plaque of Owsley Brown Frazier, founder and namesake for the Frazier Museum. In 2008, Hamilton was commissioned to create a Christmas tree ornament for Mrs. Laura Bush's Christmas Tree at the White House. He was honored by Congressman John Yarmouth of Louisville to represent the third District at the White House.
Fischer takes every opportunity to praise the talents of Hamilton and personally led the dedication ceremony in 2009 to dedicate the Lincoln Memorial at Waterfront Park.
"Ed is just an amazing, creative genius who has brought our city's roots and history to life as no one else has done," said Fischer in a statement. "From the York and Lincoln sculptures along the waterfront to the series of civil rights markers downtown to the Migration to the West artwork at the Frazier Museum, Ed has provided thoughtful and compassionate insights into our African-American legacy and history that are viewed by hundreds of thousands of citizens and visitors each year.
Lofty praise for a man who not only grew up in Louisville and makes it his home, but continues to promote his roots wherever his art work takes him. And in recent years, that road has led to him establishing sculptures in many cities, including Washington, D.C., Detroit, Newport News, Va., Hampton, Va., and New Haven, Conn.
Ed Hamilton's art studio in Louisville, Ky., is cluttered with dozens of conceptual drawings and clay models of past and would-be sculptures.
Hamilton was born in 1947 in Cincinnati but grew up in Louisville, graduating in 1965 from Shawnee High School. He also earned a degree at the Louisville School of Art in 1969. While attending the University of Louisville and Spalding College in the early 1970s, he met the late sculptor Barney Bright. It was a meeting that would change his life.
The two began a lasting relationship that led to Hamilton becoming an apprentice to Bright.
While building his art career, Hamilton continued to teach at colleges and workshops, and give lectures for public schools, college and conferences. He has taught at the high school level and at Jefferson Community College and even today continues to teach and inspire other artists and would-be artists by opening his studio at 543 S. Shelby St. for tours.
Over time, Hamilton's fame as a sculptor enabled him to eventually retire from teaching at colleges and focus entirely on his sculpting. Nevertheless, he spent a semester in 2012 as a visiting art professor at U of L.
He opened his first studio at his house in 1975-76, then moved to a location on Main Street for a short time. He moved into his current studio in 1978. It is there he continues to conceptualize and create his projects, beginning with drawings, then small clay models and finally to full-size sculptures that are then sent to a foundry in Baltimore to be cast in bronze. Hamilton uses a Louisville molding company to create the supporting bases for his artworks.
In May 2000 he was given a Doctor of Humane Letters degree from Spalding University. He has created and received numerous awards and honorary titles, and he frequently is asked to speak at commencements and conferences. In 2004, he received an Honorary Doctor of Arts Degree from Western Kentucky University, where he also gave the commencement address.
Hamilton and Bernadette, his wife of 38 years, have one son, Edward, who resides in Orlando, Fla., and one daughter, Kendra Jenelle, a graduate of Kentucky State University. Bernadette is a U of L graduate who is director of Options, Magnet and Advance Programs for the Jefferson County Public School System.
In February 2006, Hamilton published the first of two books, "The Birth of an Artist, a Journey of Discovery." It outlines his journey from growing up in Louisville, becoming a sculptor and the secret that changed his life forever.
In June 2009, he published "Creating the Lincoln Memorial at Waterfront Park." It outlines the creative process of the heroic scale Lincoln and the four bas-reliefs that tell the story of our 16th president.
He credits Bright for giving him the opportunity that changed his life, steering him on a path to greatness as a sculptor.
Back to July 2015 Articles.


I thought he was The One. I was so sure he was The One, that when we jokingly discussed paying the visit to the courthouse, it wasn't a joke on my part. I was dead serious about signing some papers & taking myself off the market permanently.
Between now & then, a lot of things happened, and a lot of things changed. But my belief that we were meant for each other never changed. In my mind I just knew we would end up together, and that belief blossomed & grew, to the point where I was more invested than I should have been. My mind & heart truly believed that we were meant to be, and we would be when he got his stuff together.
So when it became clear that I needed to let all that go, it was harder than I ever anticipated. The hardest part isn't letting go of him - it's letting go of the "what if" part of it. The fantasy, the idea of what the future could be.Letting go of the idea that we could live happily ever after & ride off together into the sunset & have that magical fairytale relationship that is sold to every little girl.
The realization that I needed to let go of that dream during an innocuous conversation. The phrase "when someone shows you who they are, believe them" truly was in action. I listened to the words & realized I had two choices - I could continue to cling to the hope that one day my dreams would come true, or I could cut my losses & move on. It was at that moment that I decided to release what had held me hostage - not my feelings, but what I wished would manifest in the future. As wonderful & awesome & special as I am, I now realize that none of that is enough to change a person into who I want them to be. Continuing to hope & wish for that to happen will only result in 1 person getting hurt, and that's me. I've had enough of that.


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NVIDIA is Offering its Trained Deep Neural Networks to the Industry to Accelerate the Development of Autonomous Vehicles
【Summary】Nvidia announced this week at the company's GTC technology conference in China that it will provide the transportation industry with access to its NVIDIA DRIVE trained deep neural networks (DNNs) for autonomous vehicle development.
Eric Walz Dec 20, 2019 10:30 AM PT
For developers using AI for autonomous driving applications, one of the biggest tasks is training deep neural networks. For self-driving vehicles, neural networks are used for complex tasks such as identifying street signs, detecting pedestrians and navigation.
However training machine learning models for these tasks requires an enormous amount of data and compute power. In addition, collecting and labeling training data it is long and painstaking process, especially for many of the budding startups working on autonomous driving.
Nvidia is making its easier and the company announced this week at the company's GTC technology conference in China that it will provide the transportation industry with access to its NVIDIA DRIVE pre-trained deep neural networks (DNNs) for autonomous vehicle development on the Nvidia GPU Cloud (NGC) container registry.
Using a suite of Nvidia AI tools, developers using the DRIVE ecosystem can freely extend and customize the models to increase the robustness and capabilities of their own self-driving systems.
AI is central to the development of safe, self-driving vehicles — allowing them to perceive and react in real time to their surroundings. A typical self-driving vehicle can use dozens of DNNs to ensuring accurate perception, localization and path planning.
Nvidia said it has spent years developing and training its DNNs designed to run on the NVIDIA DRIVE AGX platform. The DNNs are used to take vast amounts of raw sensor data and turn it into a deep understanding of the environment.
These pre-trained DNNs cover such tasks as traffic-light and road sign detection, object detection (for vehicles, pedestrians, bicycles) and path perception, as well as gaze detection and gesture recognition inside the vehicle which are used in driver monitoring systems.
"NVIDIA leads the world in developing the deepest and broadest suite of DNNs and AI tools for the transportation industry," said Luca De Ambroggi, senior research director of Artificial Intelligence at IHS Markit. "Making these algorithms available to others, along with the tools and workflow infrastructure to customize them, will help enable the deployment of safe autonomous transportation."
Nvidia's NGC (cloud) containers reduce the complexity of setting up software, so developers can focus on the problem solving aspects of their projects. By taking care of most of the work behind the scenes, access to Nvidia's container registry enables users to focus on building better models.
Nvidia is not the only tech company sharing its training data. In August, Waymo, which spun out of Google's early self-driving car project, released the" Waymo Open Dataset" for autonomous vehicle researchers.
The Waymo Open Dataset, which is available for free, covers a wide variety of environments, from dense urban centers to suburban landscapes. It also includes data collected during day and night, at dawn and dusk, in sunshine and rain. Waymo said the data was collected by a fleet of Waymo self-driving vehicles that traveled over 10 million miles in 25 different cities.
In July, Ride-hailing company Lyft released its own "Level 5 Dataset." The data is also what the ride-sharing company calls is the "largest publicly released dataset of its kind." Lyft's dataset includes over 55,000 3D frames of footage that's been captured by human reviewers. It also includes a HD spatial semantic map.
Lyft chose to release its dataset to "help level the playing field for researchers interested in autonomous technology."
"The AI autonomous vehicle is a software-defined vehicle required to operate around the world on a wide variety of datasets," said Jensen Huang, founder and CEO of NVIDIA. "By providing AV developers access to our DNNs and the advanced learning tools to optimize them for multiple datasets, we're enabling shared learning across companies and countries, while maintaining data ownership and privacy. Ultimately, we are accelerating the reality of global autonomous vehicles."
Advanced Learning Tools for AI Development
In addition to providing access to the DNNs, Nvidia announced the availability of a suite of advanced tools so developers can customize and enhance NVIDIA's DNNs using their own datasets and target feature set. These tools allow the training of DNNs using active learning, federated learning and transfer learning.
Over the past several years, Nvidia's DRIVE platform has become the go-to product for developing autonomous vehicles. It's robust processing power and software is used by automakers, tech startups and universities all around the world for autonomous vehicle development.
By providing access to its AI models on NGC and introducing advanced training tools, Nvidia is building up its end-to-end platform for autonomous vehicle development and deployment. With the release of dataset, developers are getting access to the same pre-trained DNNs that Nvidia uses to improve upon its DRIVE autonomous driving ecosystem.
Volkswagen Breaks Ground on its EV Battery Engineering Lab in Tennessee
Don't Hold Your Breath for a Six-Figure EV From Ford
Elon Musk's Next Big Thing Could Be Using the SpaceX Starlink Network to Connect Tesla Vehicles
General Motors Adding 3,000 High-Tech Jobs in its Push Towards Electrification & Vehicle Connectivity
UK Moving up Its Goal to Ban New Gas-Powered Car Sales to 2030
Waymo to Open New Autonomous Testing Facility in Ohio
China's Baidu Granted Permit to Deploy Self-Driving Vehicles in Beijing Without Human Backup
General Motors to Boost Investments to $27 Billion for the Development of New Electric Vehicles as the Company Transforms for the Future