Genetically modified natural killer cells specifically recognizing the tumor-associated antigens ErbB2/HER2 and EpCAM

The continuously growing natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origin without affecting normal human cells. Based on this selectivity, the potential of NK-92 cells for adoptive therapy is currently being investigated in phase I clinical studies. To further enhance the antitumoral activity of NK-92 cells and expand the range of tumor entities suitable for NK-92-based therapies, here by transduction with retroviral vectors we have generated genetically modified NK-92 cells expressing chimeric antigen receptors specific either for the tumor-associated ErbB2 (HER2/neu) antigen or the human Epithelial Cell Adhesion Molecule (Ep-CAM). Both antigens are overexpressed by many tumors of epithelial origin. The chimeric antigen receptors consist of either the ErbB2 specific scFv(FRP5) antibody fragment or the Ep-CAM specific scFv(MOC31), a flexible hinge region derived from CD8, and transmembrane and intracellular regions of the CD3 zeta chain. Transduced NK-92-scFv(FRP5)-zeta or NK-92-scFv(MOC31)-zeta cells express high levels of the fusion proteins on the cell surface as determined by FACS analysis. In europium release assays no difference in cytotoxic activity of NK-92 and transduced NK-92 cells towards ErbB2 or Ep-CAM negative targets was found. However, even at low effector to target ratios transduced NK-92 cells specifically and efficiently lysed established ErbB2 or Ep-CAM expressing tumor cells that were completely resistant to cytolytic activity of parental NK-92 cells. Similarly, ErbB2-positive primary breast cancer cells isolated from pleural effusions of patients with recurrent disease were selectively killed by NK-92-scFv(FRP5)-zeta. In an in vivo model in immunodeficient mice treatment with retargeted NK-92-scFv(FRP5)-zeta, but not parental NK-92 cells resulted in markedly delayed growth of ErbB2 transformed cancer cells. These results demonstrate that efficient retargeting of NK-92 cytotoxicity can be achieved, and might allow the generation of potent cell-based therapeutics for the treatment of ErbB2 and Ep-CAM expressing malignancies. This therapeutic approach might be applicable for a large variety of different cancers where suitable cell surface antigens have been identified

eKompetenz in den Regionalen Arbeitsvermittlungszentren des Kantons Bern

Inhaltsverzeichnis: 1 Einleitung – das Wichtigste im Überblick; 2 Gesellschaftliche und politische Rahmenbedingungen; 2.1 Begriffsdefinitionen; 2.2 Von der Industrie- zur Informationsgesellschaft; 2.3 Strategie des Bundes für eine Informationsgesellschaft Schweiz; 2.4 Forschungsstand zur Medien- und IKT-Nutzung in der Schweiz; 2.5 Regionale Arbeitsvermittlungszentren und ihre Kunden; 3 Empirische Studie I: Befragung der Klientel und Mitarbeitenden; 3.1 Ziel und Fragestellung; 3.2 Datenerhebung und Stichprobe; 3.3 Ergebnisse; 3.4 Fazit; 4 Empirische Studie II: Design Forschung; 4.1 Ausgangslage; 4.2 Fragebogen; 4.3 Flankierende Design Forschung; 4.4 Ausblick; 5 Weiterbildungskonzept; 5.1 Ausgangslage; 5.2 Arbeitsmarktliche Massnahmen BECO; 5.3 Die Resultate der BFH-Studie unter dem Blickwinkel der Förderung der Internet-Kompetenz; 5.4 Informationsgesellschaft Schweiz und Fördermassnahmen; 5.5 Kritik an Fördermassnahmen zur Überwindung der digitalen Spaltung; 5.6 Förderung der Internet-Kompetenz durch Schulung und Medienräume; 5.7 Fazit und Erwägungen; 6 Prototyp; 6.1 Einleitung Geschäftsprozess/Webapplikation; 6.2 Internetseite www.treffpunkt-arbeit.ch versus SSI-Terminal; 6.3 Ziel und Ablauf der Prototyperstellung; 6.4 Geschäftsprozess "Stellensuche von Arbeitslosen"; 6.5 SSI-Terminal; 6.6 Anforderungen für Prototyp; 6.7 Umsetzung Prototyp; 7 Literaturverzeichnis ; Anhan

Das Stigma von Suchterkrankungen verstehen und überwinden = Understanding and overcoming the stigma of substance use disorders

Understanding and overcoming the stigma of substance use disorders Abstract. Stigma does harm to individuals with substance use disorders (SUD), and it increases the burden of SUDs. It presents a barrier to help seeking, results in lower treatment quality and increases social and health related consequences of SUDs. This applies to both the individual, societal and economic consequences of substance use. Moreover, stigmatizing persons with addictions is an ethical problem, since it discriminates against a certain group and infringes on their human dignity. Dealing with substance use disorders without stigma is possible. Eliminating the stigma of SUDs means finding better ways to deal with SUDs and to make these ways available to everyone. Instead of devaluing, marginalizing and disciplining persons with SUD, empowerment and appreciation need to be at the core of dealing with SUD in prevention, treatment and every day life

Preclinical Evaluation of a Bone-Marrow Autograft Culture Procedure for Generating Lymphokine-Activated Killer Cells in Vitro

Despite the use of high dose chemoradiotherapy for the treatment of acute leukemia. relapse continues to be a major cause of death in patients given an autologous bone marrow transplant. Further augmentation of pretransplant chemotherapy causes life threatening toxicity to nonhematopoietic tissues and the effectiveness of currently available ex vivo purging methods in reducing the relapse rate is unclear. Recently, data from experimental models have suggested that bone marrow-derived lymphokine (IL-2)-activated killer (BM-LAK) cells might be used to eliminate residual leukemic cells both in vivo and in vitro. To evaluate this possibility clinically, a procedure was developed for culturing whole marrow harvests with IL-2 prior to use as autografts, and a number of variables examined that might affect either the generation of BM-LAK cells or the recovery of the primitive hematopoietic cells. The use of Dexter long term culture (LTC) conditions, which expose the cells to horse serum and hydrocortisone. supported LAK cell generation as effectively as fetal calf serum (FCS) -containing medium in seven-day cultures. Maintenance of BM-LAK cell activity after a further seven days of culture in the presence of IL-2 was also tested. As in the clinical setting. patients would receive IL-2 in vivo for an additional week immediately following infusion of the cultured marrow autograft. Generation ofBM-LAK activity was dependent on the presence of IL-2 and could be sustained by further incubation in medium containing IL-2. Primitive hematopoietic cells were quantitated by measuring the number of in vitro colony-forming progenitors produced after five weeks in secondary Dexter-type LTC. Maintenance of these 'LTC-initiating cells' was unaffected by lL-2 in the culture medium. These results suggest that LAK cells can be generated efficien tly in seven-day marrow autograft cultures containing IL-2 under conditions that allow the most primitive human hematopoietic cells currently detectable to be maintained

Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting

Background: On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors. Methods: Here, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92. Results: Unmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals. Conclusions: These observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance