Patents, Ethics and Stem Cell Research : The Case of hESC Innovation in Australia, Europe and the United States

Embryonic stem cells offer potentially a ground-breaking insight into health and diseases and are said to offer hope in discovering cures for many ailments unimaginable few years ago. Human embryonic stem cells are undifferentiated, immature cells that possess an amazing ability to develop into almost any body cell such as heart muscle, bone, nerve and blood cells and possibly even organs in due course. This remarkable feature, enabling embryonic stem cells to proliferate indefinitely in vitro (in a test tube), has branded them as a so-called miracle cure . Their potential use in clinical applications provides hope to many sufferers of debilitating and fatal medical conditions. However, the emergence of stem cell research has resulted in intense debates about its promises and dangers. On the one hand, advocates hail its potential, ranging from alleviating and even curing fatal and debilitating diseases such as Parkinson s, diabetes, heart ailments and so forth. On the other hand, opponents decry its dangers, drawing attention to the inherent risks of human embryo destruction, cloning for research purposes and reproductive cloning eventually. Lately, however, the policy battles surrounding human embryonic stem cell innovation have shifted from being a controversial research to scuffles within intellectual property rights. In fact, the ability to obtain patents represents a pivotal factor in the economic success or failure of this new biotechnology. Although, stem cell patents tend to more or less satisfy the standard patentability requirements, they also raise serious ethical and moral questions about the meaning of the exclusions on ethical or moral grounds as found in European and to an extent American and Australian patent laws. At present there is a sort of a calamity over human embryonic stem cell patents in Europe and to an extent in Australia and the United States. This in turn has created a sense of urgency to engage all relevant parties in the discourse on how best to approach patenting of this new form of scientific innovation. In essence, this should become a highly favoured patenting priority. To the contrary, stem cell innovation and its reliance on patent protection risk turmoil, uncertainty, confusion and even a halt on not only stem cell research but also further emerging biotechnology research and development. The patent system is premised upon the fundamental principle of balance which ought to ensure that the temporary monopoly awarded to the inventor equals that of the social benefit provided by the disclosure of the invention. Ensuring and maintaining this balance within the patent system when patenting human embryonic stem cells is of crucial contemporary relevance. Yet, the patenting of human embryonic stem cells raises some fundamental moral, social and legal questions. Overall, the present approach of patenting human embryonic stem cell related inventions is unsatisfactory and ineffective. This draws attention to a specific question which provides for a conceptual framework for this work. That question is the following: how can the investigated patent offices successfully deal with patentability of human embryonic stem cells? This in turn points at the thorny issue of application of the morality clause in this field. In particular, the interpretation of the exclusions on ethical or moral grounds as found in Australian, American and European legislative and judicial precedents. The Thesis seeks to compare laws and legal practices surrounding patentability of human embryonic stem cells in Australia and the United States with that of Europe. By using Europe as the primary case study for lessons and guidance, the central goal of the Thesis then becomes the determination of the type of solutions available to Europe with prospects to apply such to Australia and the United States. The Dissertation purports to define the ethical implications that arise with patenting human embryonic stem cells and intends to offer resolutions to the key ethical dilemmas surrounding patentability of human embryonic stem cells and other morally controversial biotechnology inventions. In particular, the Thesis goal is to propose a functional framework that may be used as a benchmark for an informed discussion on the solution to resolving ethical and legal tensions that come with patentability of human embryonic stem cells in Australian, American and European patent worlds. Key research questions that arise from these objectives and which continuously thread throughout the monograph are: 1. How do common law countries such as Australia and the United States approach and deal with patentability of human embryonic stem cells in their jurisdictions? These practices are then compared to the situation in Europe as represented by the United Kingdom (first two chapters), the Court of Justice of the European Union and the European Patent Office decisions (Chapter 3 onwards) in order to obtain a full picture of the present patenting procedures on the European soil. 2. How are ethical and moral considerations taken into account at patent offices investigated when assessing patentability of human embryonic stem cell related inventions? In order to assess this part, the Thesis evaluates how ethical issues that arise with patent applications are dealt with by: a) Legislative history of the modern patent system from its inception in 15th Century England to present day patent laws. b) Australian, American and European patent offices presently and in the past, including other relevant legal precedents on the subject matter. c) Normative ethical theories. d) The notion of human dignity used as the lowest common denominator for the interpretation of the European morality clause. 3. Given the existence of the morality clause in form of Article 6(1) of the Directive 98/44/EC of the European Parliament and of the Council of 6 July 1998 on the legal protection of biotechnological inventions which corresponds to Article 53(a) European Patent Convention, a special emphasis is put on Europe as a guiding principle for Australia and the United States. Any room for improvement of the European morality clause and Europe s current manner of evaluating ethical tensions surrounding human embryonic stem cell inventions is examined. 4. A summary of options (as represented by Australia, the United States and Europe) available as a basis for the optimal examination procedure of human embryonic stem cell inventions is depicted, whereas the best of such alternatives is deduced in order to create a benchmark framework. This framework is then utilised on and promoted as a tool to assist Europe (as represented by the European Patent Office) in examining human embryonic stem cell patent applications. This method suggests a possibility of implementing an institution solution. 5. Ultimately, a question of whether such reformed European patent system can be used as a founding stone for a potential patent reform in Australia and the United States when examining human embryonic stem cells or other morally controversial inventions is surveyed. The author wishes to emphasise that the guiding thought while carrying out this work is to convey the significance of identifying, analysing and clarifying the ethical tensions surrounding patenting human embryonic stem cells and ultimately present a solution that adequately assesses patentability of human embryonic stem cell inventions and related biotechnologies. In answering the key questions above, the Thesis strives to contribute to the broader stem cell debate about how and to which extent ethical and social positions should be integrated into the patenting procedure in pluralistic and morally divided democracies of Europe and subsequently Australia and the United States.

A Hybrid Multilevel Inverter Topology for an Open-End Winding Induction-Motor Drive Using Two-Level Inverters in Series With a Capacitor-Fed H-Bridge Cell

In this paper, a new five-level inverter topology for open-end winding induction-motor (IM) drive is proposed. The open-end winding IM is fed from one end with a two-level inverter in series with a capacitor-fed H-bridge cell, while the other end is connected to a conventional two-level inverter. The combined inverter system produces voltage space-vector locations identical to that of a conventional five-level inverter. A total of 2744 space-vector combinations are distributed over 61 space-vector locations in the proposed scheme. With such a high number of switching state redundancies, it is possible to balance the H-bridge capacitor voltages under all operating conditions including overmodulation region. In addition to that, the proposed topology eliminates 18 clamping diodes having different voltage ratings compared with the neutral point clamped inverter. On the other hand, it requires only one capacitor bank per phase, whereas the flying-capacitor scheme for a five-level topology requires more than one capacitor bank per phase. The proposed inverter topology can be operated as a three-level inverter for full modulation range, in case of any switch failure in the capacitor-fed H-bridge cell. This will increase the reliability of the system. The proposed scheme is experimentally verified on a four-pole 5-hp IM drive.

In vitro protection of umbilical cord blood-derived primitive hematopoietic stem progenitor cell pool by mannose-specific lectins via antioxidant mechanisms.

BACKGROUND: Earlier we reported that an oral administration of two mannose-specific dietary lectins, banana lectin (BL) and garlic lectin (GL), led to an enhancement of hematopoietic stem and progenitor cell (HSPC) pool in mice. STUDY DESIGN AND METHODS: Cord blood derived CD34+ HSPCs were incubated with BL, GL, Dolichos lectin (DL), or artocarpin lectin (AL) for various time periods in a serum- and growth factor free medium and were subjected to various functional assays. Reactive oxygen species (ROS) levels were detected by using DCHFDA method. Cell fractionation was carried out using lectin-coupled paramagnetic beads. RESULTS: CD34+ cells incubated with the lectins for 10 days gave rise to a significantly higher number of colonies compared to the controls, indicating that all four lectins possessed the capacity to protect HSPCs in vitro. Comparative analyses showed that the protective ability of BL and GL was better than AL and DL and, therefore, further experiments were carried out with them. The output of long-term culture-initiating cell (LTC-IC) and extended LTC-IC assays indicated that both BL and GL protected primitive stem cells up to 30 days. The cells incubated with BL or GL showed a substantial reduction in the ROS levels, indicating that these lectins protect the HSPCs via antioxidant mechanisms. The mononuclear cell fraction isolated by lectin-coupled beads got enriched for primitive HSPCs, as reflected in the output of phenotypic and functional assays.CONCLUSION: The data show that both BL and GL protect the primitive HSPCs in vitro and may also serve as cost-effective HSPC enrichment tools.

Visiting the cell biology of Salmonella infection

Salmonella, a Gram-negative facultative intracellular pathogen is capable of infecting vast array of hosts. The striking ability of Salmonella to overcome every hurdle encountered in the host proves that they are true survivors. In the host, Salmonella infects various cell types and needs to survive and replicate by countering the defense mechanism of the specific cell. In this review, we will summarize the recent insights into the cell biology of Salmonella infection. Here, we will focus on the findings that deal with the specific mechanism of various cell types to control Salmonella infection. Further, the survival strategies of the pathogen in response to the host immunity will also be discussed in detail. Better understanding of the mechanisms by which Salmonella evade the host defense system and establish pathogenesis will be critical in disease management. (C) 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Terminal solid solubilities at 900–1000°C in the magnesium oxide-zinc oxide system measured using a magnesium fluoride solid-electrolyte galvanic cell

The terminal solid solubilities of the periclase (MgO-rich) and zincite (ZnO-rich) solid solutions in the MgO---ZnO system have been determined by measuring the activity of MgO using a solid-state galvanic cell of the type 02(g), Pt/MgO, MgF2//MgF2//{χMgO+(1-χ)ZnO}(s, sln), MgF2/Pt, O2(g) in the temperature range 900–1050°C. The ZnO activity was calculated by graphical Gibbs-Duhem integration. The activity-composition plots of both components exhibit a strong positive deviation from ideality and are characterised by a miscibility gap. The terminal solid solubilities of the periclase and zincite solid solutions obtained from the activity-composition plots are found to be in reasonable agreement with those reported in the literature.

Proteomic Identification of Haptoglobin alpha 2 as a Glioblastoma Serum Biomarker: Implications in Cancer Cell Migration and Tumor Growth

Glioblastoma (GBM; grade IV astrocytoma) is the most malignant and common primary brain tumor in adults. Using combination of 2-DE and MALDI-TOF MS, we analyzed 14 GBM and 6 normal control sera and identified haptoglobin alpha 2 chain as an up-regulated serum protein in GBM patients. GBM-specific up-regulation was confirmed by ELISA based quantitation of haptoglobin (Hp) in the serum of 99 GBM patients as against lower grades (49 grade III/AA; 26 grade II/DA) and 26 normal individuals (p = 0.0001). Further validation using RT-qPCR on an independent set (n = 78) of tumor and normal brain (n = 4) samples and immunohistochemcial staining on a subset (n = 42) of above samples showed increasing levels of transcript and protein with tumor grade and were highest in GBM (p = < 0.0001 and < 0.0001, respectively). Overexpression of Hp either by stable integration of Hp cDNA or exogenous addition of purified Hp to immortalized astrocytes resulted in increased cell migration. RNAi-mediated silencing of Hp in glioma cells decreased cell migration. Further, we demonstrate that both human glioma and mouse melanoma cells overexpressing Hp showed increased tumor growth. Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration.

Gonadal cell surface receptor for plasma retinol-binding protein A method for its radioassay and studies on its level during spermatogenesis

A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps: direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme.The binding of retinol-binding protein to the receptor is saturable and reverible. The interaction shows a Kd value of 2.1 · 10−10 M. The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testoterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifically induced by testoterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome.

Catalytic hydrogen combustion for treatment of combustible gases from fuel cell processors

Catalytic combustion of H-2 was carried out over combustion synthesized noble metal (Pd or Pt) ion-substituted CeO2 based catalysts using a feed stream that simulated exhaust gases from a fuel cell processor The catalysts showed a high activity for H-2-combustion and complete conversion was achieved below 200 C over all the catalysts when O-2 was used in a stoichiometric amount With higher amounts of O-2 the reaction rates Increased and complete conversions were possible below 100 C The reaction was also carried out over Pd-impregnated CeO2 The conversions of H-2 with stoichiometric amount of O-2 were found to be higher over Pd-substituted compound The mechanism of the reaction over noble metal-substituted compounds was proposed on the basis of X-ray photoelectron spectroscopy studies The redox couples between Ce and metal ions were established and a dual site redox mechanism was pi posed for the reaction (C) 2010 Elsevier B V All rights reserved

Signaling different pathways of cell death: Abrin induced programmed necrosis in U266B1 cells

Abrin is a type II ribosome-inactivating protein comprising of two subunits, A and B. Of the two, the A-subunit harbours the RNA-N-glycosidase activity and the B subunit is a galactose specific lectin that enables the entry of the protein inside the cell. Abrin inhibits protein synthesis and has been reported to induce apoptosis in several cell types. Based on these observations abrin is considered to have potential for the construction of immunotoxin in cell targeted therapy. Preliminary data from our laboratory however showed that although abrin inhibited the protein synthesis in all cell types, the mode of cell death varied. The aim of the present study was therefore to understand different death pathways induced by abrin in different cells. We used the human B cell line, U266B1 and compared it with the earlier studied T cell line Jurkat, for abrin-mediated inhibition of protein translation as well as cell death. While abrin triggered programmed apoptosis in Jurkat cells in a caspase-dependent manner, it induced programmed necrosis in U266B1 cells in a caspase-independent manner, even when there was reactive oxygen species production and loss of mitochondrial membrane potential. The data revealed that abrin-mediated necrosis involves lysosomal membrane permeabilization and release of cathepsins from the lysosomes. Importantly, the choice of abrin-mediated death pathway in the cells appears to depend on which of the two events occurs first: lysosomal membrane permeabilization or loss of mitochondrial membrane potential that decides cell death by necrosis or apoptosis. (C) 2010 Elsevier Ltd. All rights reserved.

Rapid burst of H2O2 by plant growth regulators increases intracellular Ca2+ amounts and modulates CD4(+) T cell activation

The identification of small molecules that affect T cell activation is an important area of research. Three molecules that regulate plant growth and differentiation, but not their structurally similar analogs, were identified to enhance primary mouse CD4(+) T cell activation in conjunction with soluble anti-CD3 stimulation: Indoleacetic acid (natural plant auxin), 1-Napthaleneacetic acid (synthetic plant auxin) and 2,4-Dichlorophenoxyacetic acid (synthetic plant auxin and herbicide). These effects are distinct in comparison to Curcumin, the well known phenolic immunomodulator, which lowers T cell activation. An investigation into the mechanisms of action of the three plant growth regulators revealed a rapid induction of reactive oxygen species (ROS), mainly comprising H2O2 . In addition, these three molecules synergize with soluble anti-CD3 signaling to enhance intracellular Ca2+ concentrations Ca2+](i), leading to greater T cell activation, e.g. induction of CD25 and IL-2. Enhanced production of TNF alpha and IFN gamma by CD4+ T cells is also observed upon plant growth regulator treatment with soluble anti-CD3. Interestingly, maximal IL-2 production and CD4(+) T cell cycle progression are observed upon activation with soluble anti-CD3 and phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Additionally, stimulation with PMA and Ionomcyin (a Ca2+ ionophore), which activates T cells by circumventing the TCR, and plant growth regulators also demonstrated the role of the strength of signal (SOS): T cell cycle progression is enhanced with gentle activation conditions but decreased with strong activation conditions. This study demonstrates the direct effects of three plant growth regulators on CD4(+) T cell activation and cycling. (C) 2010 Elsevier B.V. All rights reserved.

Cell-dynamical simulation of magnetic hysteresis in the two-dimensional Ising system

We present results from numerical simulations using a ‘‘cell-dynamical system’’ to obtain solutions to the time-dependent Ginzburg-Landau equation for a scalar, two-dimensional (2D), (Φ2)2 model in the presence of a sinusoidal external magnetic field. Our results confirm a recent scaling law proposed by Rao, Krishnamurthy, and Pandit [Phys. Rev. B 42, 856 (1990)], and are also in excellent agreement with recent Monte Carlo simulations of hysteretic behavior of 2D Ising spins by Lo and Pelcovits [Phys. Rev. A 42, 7471 (1990)].

NK-and T-cell function in chronic myeloid leukemia patients treated with interferon-? monotherapy

Chronic myeloid leukemia (CML) is one of the most studied human malignancies. It is caused by an autonomously active tyrosine kinase BCR-ABL, which is a result from a translocation between chromosomes 9 and 22 in the hematopoietic stem cell. As an outcome, a Philadelphia (Ph) chromosome is formed. BCR-ABL causes disturbed cell proliferation among other things. Although targeted tyrosine kinase inhibitor therapy has been developed in the beginning of the millenium and the survival rate has increased significantly, it is still not known why some patients benefit more from the treatment than others. Furthermore, the therapy is not considered to be curative. Before the era of tyrosine kinase inhibitors, the first-line treatment for CML was interferon-? (IFN-?). However, only a small proportion of patients benefitted from the treatment. Of these patients, a few were able to discontinue the treatment without renewal of the disease. The mechanism of IFN-? is not completely understood, but it is believed that differences in the immune system can be one of the reasons why some patients have better therapy response. Kreutzman, Rohon et al. have recently discovered that patients who have been able to stop IFN-? treatment have an increased number of NK- and T-cells. They also have a unique clonal T-cell population and more cytotoxic CD8+ T-cells and less CD4+ T-cells. The aim of this master’s thesis was to study the function of T- and NK-cells in IFN-? treated patients. Although it was shown earlier that IFN-? treated patients have increased NK-cell count, the function of these cells was unknown. Therefore, we have now investigated the killing potential of patients’ NK-cells, their activation status and cell surface antigen expression. In addition, we have also studied the activation status of patients’ T-cells and their cytotoxic properties. We observed that NK-cells from patients treated with IFN-? are unable to kill leukemic cells (K562) than NK-cells from healthy controls. In addition, patients on IFN-? treatment have more active T-cells and their NK-cells have an undifferentiated immunoregulatory phenotype. Patients that have been able to stop the treatment have anergic T-and NK-cells. As a conclusion our results suggest that IFN-? therapy induces increased NK-cell count, NK-cell immunoregulatory functions and more active T-cells. After stopping IFN-? therapy, NK- and T-cells from CML patients restore anergy typical for CML.

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide-and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.