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Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Phenotypic and Functional Impacts of Genetic Modification on Natural Killer Cells to Express A Chimeric Antigen Receptor (CAR) or High Affinity CD16 Receptor with Specificity to the Tumor-associated Antigen ErbB2
No full textAccording to the National Institute of Health in 2018, new cases of cancer exceeded 18 million with 9.6 million deaths caused by cancer worldwide. It is well known that cancer is a leading cause of death worldwide, yet conventional medical treatments such as chemotherapy are unable to fully treat the disease which has encouraged the development of new therapies. Initially, cell therapies involving T cells were explored and paved the way for further enhancements in genetic modification that could be key to closing the current gap in treatment; however, different forms of genetic modification can impact a treatment in a multitude of ways. This is what inspired the evaluation of the NK-92 cell line efficiency, expandability, and safety amongst genetically modified variants expressing a CAR or high affinity CD16 receptor with endogenous IL-2 production. In this study, I have characterized features of a second generation ErbB2-specific CAR-engineered NK-92 with NK-92 expressing a recombinant Fc receptor to the parental NK-92 cell line with respect to cell surface receptor expression using flow cytometry, growth characteristics, cytotoxicity against ErbB2/HER2 positive and negative breast cancer cell lines, cytokine release, and outgrowth profiles post-irradiation. Cell surface receptors were comparable with a few exceptions. CD2, CD11a, CD52, CD54, CD56, and CD337 (NKp30) expression was maintained amongst all cell lines while CD3 and CD158a expression remained negative. FcγRIII/IL-2 NK-92 gained expression of CD314 (NKG2D) when compared to the other NK-92 cell lines, making it preactivated for target cells expressing ligands of NKG2D. Although WT NK-92 expressed CD336 (NKp44), expression of this receptor was not present on NK-92/5.28.z and FcγRIII/IL-2 NK-92 cell lines. Expansion of all NK-92 cell lines was comparable: WT NK-92 and NK-92/5.28.z were able to expand up to 1.0 x 106 cells/mL while FcγRIII/IL-2 NK-92 was able to expand up to 1.2 x 106 cells/mL. All expansion halted after two to three days with the addition of radiation, especially a dose 7.5 Gy or more. NK-92/5.28.z and FcγRIII/IL-2 NK-92 cell lines were able to lyse almost 100% of ErbB2(+) target cells without irradiation. However, even with the addition of 10 Gy irradiation, NK-92/5.28.z cells were able to maintain 100% lysis of target cells while FcγRIII/IL-2 NK-92 cells dropped down to 75% lysis. Cytokine profiles were similar for NK-92 WT and FcγRIII/IL-2 NK-92 in the presence of ErbB2(+) target cells: both NK-92 cell lines secreted more granzyme B than IFN-γ whereas NK-92/5.28.z secreted more IFN-γ in the presence of ErbB2(+) target cells than granzyme B. The addition of radiation did not affect the trend in cytokine profiles for any NK-92 cell lines. The data found in the experiments included in this study can be used for selecting which NK-92 variant should enter clinical trials as well as for screening other NK-92 variants for similar purposes. The goal of this study was to address some of the issues revolving around NK cell use in cellular therapy by characterizing and comparing a preclinical NK-92 cell expressing high affinity Fc-γ receptor combined with intracellular IL-2 against an NK-92 cell modified to express a second-generation CAR consisting of an intracellular CD3 ζ domain and CD28 costimulatory molecule. The efficiency of each NK-92 cell line seen in the various experiments can be compared to other cell therapies in order to further determine whether another treatment is optimal or if combinatorial treatments are an option.:Abstract IV
List of Figures VII
List of Tables VIII
Abbreviations IX
Symbols and Units XII
1. Introduction 1
1.1 The Natural Killer Cell 2
1.1.1 Maturation 2
1.1.2 Cells of the Adaptive Immune System 4
1.1.3 Function 6
1.1.4 NK Cell Activation Receptors 6
1.1.5 NK Cell Inhibition Receptors 7
1.1.6 Targeted Natural Cytotoxicity 8
1.1.7 Antibody-dependent Cellular Cytotoxicity 9
1.1.8 Death-receptor-mediated Apoptosis 10
1.1.9 IFN-γ 11
1.1.10 Intracellular Signaling Cascades in ADCC-mediated VS Natural Cytotoxicity 12
1.2 Establishment of a Natural Killer Cell Line 14
1.2.1 NK-92 Cells 15
1.2.2 NK-92/5.28.z 15
1.2.3 FcγRIII/IL-2-expressing Preclinical Clone 18
1.3 Therapeutic Monoclonal Antibodies (mAbs) 18
1.4 The Tumor Cell 19
1.5 Objectives/Motivation 19
2. Materials and Methods 21
2.1 Cell Lines and Culture Conditions 22
2.2 Fluorescence-activated Cell Sorting (FACS) 22
2.2.1 Acquisition of Fluorescence-labeled Cells 22
2.2.2 Verification of ErbB2 Expression on Breast Cancer Cell Line 22
2.2.3 Analysis of Expression of the ErbB2 Chimeric scFv Receptor on NK-92/5.28.z Cells 23
2.2.4 Phenotypic Characterization of NK-92 cells 23
2.3 Growth Characteristics 26
2.4 Dye Exclusion for Cell Viability Determination 26
2.5 Radiation of NK-92 cells 26
2.6 Statistical Analysis 26
2.7 Potency Assay 27
2.8 Cytokine Profile in Steady State and Post-activation 28
2.9 Impact of γ-radiation on Cell Growth Kinetics 29
2.10 Impact of Radiation on Potency 29
2.11 Impact of Radiation on Cytokine Profile 29
2.12 Impact of Radiation on Outgrowth of NK-92 30
3. Results 31
3.1 Transgene Expression (Fc Receptor and CAR) on NK-92 32
3.1.1 ErbB2-CAR Expression 32
3.1.2 Fc Receptor Expression 33
3.1.3 Cell Surface Receptor Expression on NK-92 Variants 34
3.2 Impact on Growth Characteristics 35
3.3 Functional Cytotoxicity Against ErbB2-positive and Negative Cell Lines 37
3.4 Cytokine Profile in Steady State and Post-activation 38
3.5 Impact of γ-radiation on Cell Growth Kinetics 41
3.6 Impact of Radiation on Potency 43
3.7 Cytokine Profile Post-radiation 44
3.8 Impact of Radiation on Outgrowth 47
4. Discussion 49
5. Summary 62
5.1 Summary 63
5.2 Zusammenfassung 65
6. Appendix 67
6.1 Declaration of Compliance with Legal Requirements 68
6.2 Supplementary Data 69
References 76
Acknowledgements 87
Publications 88
Declaration of Authorship 89Laut dem National Institute of Health überstiegen im Jahr 2018 neue Krebsfälle 18 Millionen mit 9,6 Millionen krebsbedingten Todesfällen weltweit. Es ist bekannt, dass Krebs weltweit eine der häufigsten Todesursachen ist, jedoch können konventionelle medizinische Behandlungen, wie eine Chemotherapie, nicht vollständig behandeln, was die Entwicklung neuer Therapien gefördert hat. Zunächst wurden Zelltherapien mit T-Zellen erprobt, welche den Weg für weitere genetische Verbesserungen geebnet haben, die in der Lage sein könnten die derzeitige Behandlungslücke zu schließen. Verschiedene Formen einer genetischen Veränderung können eine Behandlung auf vielfältige Weise beeinflussen. Dies war der Grund für die Bewertung der Effizienz, Erweiterbarkeit und Sicherheit der NK-92-Zelllinie und ihrer genetisch veränderten Varianten, die jeweils einen chimären Antigenrezeptor (CAR) oder einen hochaffinen CD16-Rezeptor zusammen mit einem endogen exprimierten IL-2 exprimieren. In dieser Arbeit habe ich Eigenschaften einer mit einem ErbB2- spezifischen CAR der 2. Generation ausgestatteten NK-92 Zelllinie mit einer NK-92 Zelllinie mit rekombinanter Fc Rezeptor Expression und mit parentalen NK-92 Zellen in Bezug auf die Oberfächenrezeptorexpression mittels Durchflusszytometrie, Wachstumseigenschaften, Zyototoxizität gegen ErbB2/HER2-positive und negative Brustkrebszelllinien, Zytokinfreisetzung und Auswachsen nach einer Bestrahlung charakterisiert. Zelloberflächenrezeptoren waren mit wenigen Ausnahmen vergleichbar. Die Expression von CD2, CD11a, CD52, CD54, CD56 und CD337 (NKp30) wurde unter allen Zelllinien aufrechterhalten, während die Expression von CD3 und CD158a negativ blieb. FcγRIII/IL-2 NK-92 regulierte, im Vergleich zu den anderen NK-92-Zelllinien, die Expression von CD314 (NKG2D) hoch, wodurch sie für Zeilzellen preaktiviert ist, welche NKG2D Liganden exprimieren. Obwohl Wildtyp NK-92 Zellen eine Expression von CD336 (NKp44) aufzeigt, war die Expression dieses Rezeptors auf NK-92/5.28.z- und FcγRIII / IL-2-NK-92 nicht vorhanden. Die Expansion aller NK-92-Zelllinien war vergleichbar: NK-92 WT und NK-92 / 5.28.z konnten zu einer Dichte von 1,0 x 10 6 Zellen/ml expandieren, während sich FcγRIII /IL-2 NK-92 auf bis zu 1,2 x 10 6 Zellen wachsen können. Eine Bestrahlung der NK-92 Zellen führte in allen Fällen zu einer Inhibition des Zellwachstums innerhalb von zwei bis drei Tagen, insbesondere bei einer Dosis von 7,5 Gy oder mehr. Ohne Bestrahlung konnten NK-92/5.28.z- und FcγRIII/IL-2-NK-92-Zelllinien fast 100% der ErbB2 (+) - Zielzellen lysieren. Selbst nach einer Bestrahlung mit 10 Gy-konnten NK-92/5.28.z-Zellen eine 100% ige Lyse der Zielzellen aufrechterhalten, während die Zyototoxizität von FcγRIII/IL-2 NK-92 auf 75% Lyse abfiel. Die Zytokinprofile waren in Gegenwart von ErbB2 (+) - Zielzellen für NK-92 WT und FcγRIII / IL-2 NK-92 ähnlich: Beide NK-92-Zelllinien sekretierten mehr Granzym B als IFN-γ, während NK-92/5.28.z in Gegenwart von ErbB2 (+) – Zielzellen mehr IFN-γ als Granzym B sekretierte. Eine Bestrahlung hatte keinen Einfluss auf die Zytokinprofile irgendeiner NK-92-Zelllinie. Die in dieser Arbeit gewonnenen experimentellen Daten können zur Auswahl einer geeigneten NK-92-Variante für klinische Studien, sowie zum Screening anderer NK-92-Varianten für ähnliche Zwecke verwendet werden. Das Ziel dieser Arbeit war es, einige der Fragestellungen zu adressieren, die sich im Hinblick auf eine therapeutische Anwendung NK-Zell basierter Zelltherapie ergeben. Hierzu wurde eine präklinische NK-92-Zelllinie, die einen hochaffinen Fc-γ-Rezeptor in Kombination mit intrazellulärem IL-2 exprimiert, mit einer NK-92-Zelle verglichen, die einen 2. Generation CAR exprimiert, der aus einer intrazellulären CD3-ζ-Domäne und einem kostimulatorischen CD28-Molekül besteht. Die Effizienz jeder NK-92-Zelllinie, die in den verschiedenen Experimenten beobachtet wurde, kann mit anderen Zelltherapien verglichen werden, um weiter zu bestimmen, ob eine andere Behandlung optimal ist oder ob kombinatorische Behandlungen eine Option sind.:Abstract IV
List of Figures VII
List of Tables VIII
Abbreviations IX
Symbols and Units XII
1. Introduction 1
1.1 The Natural Killer Cell 2
1.1.1 Maturation 2
1.1.2 Cells of the Adaptive Immune System 4
1.1.3 Function 6
1.1.4 NK Cell Activation Receptors 6
1.1.5 NK Cell Inhibition Receptors 7
1.1.6 Targeted Natural Cytotoxicity 8
1.1.7 Antibody-dependent Cellular Cytotoxicity 9
1.1.8 Death-receptor-mediated Apoptosis 10
1.1.9 IFN-γ 11
1.1.10 Intracellular Signaling Cascades in ADCC-mediated VS Natural Cytotoxicity 12
1.2 Establishment of a Natural Killer Cell Line 14
1.2.1 NK-92 Cells 15
1.2.2 NK-92/5.28.z 15
1.2.3 FcγRIII/IL-2-expressing Preclinical Clone 18
1.3 Therapeutic Monoclonal Antibodies (mAbs) 18
1.4 The Tumor Cell 19
1.5 Objectives/Motivation 19
2. Materials and Methods 21
2.1 Cell Lines and Culture Conditions 22
2.2 Fluorescence-activated Cell Sorting (FACS) 22
2.2.1 Acquisition of Fluorescence-labeled Cells 22
2.2.2 Verification of ErbB2 Expression on Breast Cancer Cell Line 22
2.2.3 Analysis of Expression of the ErbB2 Chimeric scFv Receptor on NK-92/5.28.z Cells 23
2.2.4 Phenotypic Characterization of NK-92 cells 23
2.3 Growth Characteristics 26
2.4 Dye Exclusion for Cell Viability Determination 26
2.5 Radiation of NK-92 cells 26
2.6 Statistical Analysis 26
2.7 Potency Assay 27
2.8 Cytokine Profile in Steady State and Post-activation 28
2.9 Impact of γ-radiation on Cell Growth Kinetics 29
2.10 Impact of Radiation on Potency 29
2.11 Impact of Radiation on Cytokine Profile 29
2.12 Impact of Radiation on Outgrowth of NK-92 30
3. Results 31
3.1 Transgene Expression (Fc Receptor and CAR) on NK-92 32
3.1.1 ErbB2-CAR Expression 32
3.1.2 Fc Receptor Expression 33
3.1.3 Cell Surface Receptor Expression on NK-92 Variants 34
3.2 Impact on Growth Characteristics 35
3.3 Functional Cytotoxicity Against ErbB2-positive and Negative Cell Lines 37
3.4 Cytokine Profile in Steady State and Post-activation 38
3.5 Impact of γ-radiation on Cell Growth Kinetics 41
3.6 Impact of Radiation on Potency 43
3.7 Cytokine Profile Post-radiation 44
3.8 Impact of Radiation on Outgrowth 47
4. Discussion 49
5. Summary 62
5.1 Summary 63
5.2 Zusammenfassung 65
6. Appendix 67
6.1 Declaration of Compliance with Legal Requirements 68
6.2 Supplementary Data 69
References 76
Acknowledgements 87
Publications 88
Declaration of Authorship 8
Phenotypic and Functional Impacts of Genetic Modification on Natural Killer Cells to Express A Chimeric Antigen Receptor (CAR) or High Affinity CD16 Receptor with Specificity to the Tumor-associated Antigen ErbB2
No full textAccording to the National Institute of Health in 2018, new cases of cancer exceeded 18 million with 9.6 million deaths caused by cancer worldwide. It is well known that cancer is a leading cause of death worldwide, yet conventional medical treatments such as chemotherapy are unable to fully treat the disease which has encouraged the development of new therapies. Initially, cell therapies involving T cells were explored and paved the way for further enhancements in genetic modification that could be key to closing the current gap in treatment; however, different forms of genetic modification can impact a treatment in a multitude of ways. This is what inspired the evaluation of the NK-92 cell line efficiency, expandability, and safety amongst genetically modified variants expressing a CAR or high affinity CD16 receptor with endogenous IL-2 production. In this study, I have characterized features of a second generation ErbB2-specific CAR-engineered NK-92 with NK-92 expressing a recombinant Fc receptor to the parental NK-92 cell line with respect to cell surface receptor expression using flow cytometry, growth characteristics, cytotoxicity against ErbB2/HER2 positive and negative breast cancer cell lines, cytokine release, and outgrowth profiles post-irradiation. Cell surface receptors were comparable with a few exceptions. CD2, CD11a, CD52, CD54, CD56, and CD337 (NKp30) expression was maintained amongst all cell lines while CD3 and CD158a expression remained negative. FcγRIII/IL-2 NK-92 gained expression of CD314 (NKG2D) when compared to the other NK-92 cell lines, making it preactivated for target cells expressing ligands of NKG2D. Although WT NK-92 expressed CD336 (NKp44), expression of this receptor was not present on NK-92/5.28.z and FcγRIII/IL-2 NK-92 cell lines. Expansion of all NK-92 cell lines was comparable: WT NK-92 and NK-92/5.28.z were able to expand up to 1.0 x 106 cells/mL while FcγRIII/IL-2 NK-92 was able to expand up to 1.2 x 106 cells/mL. All expansion halted after two to three days with the addition of radiation, especially a dose 7.5 Gy or more. NK-92/5.28.z and FcγRIII/IL-2 NK-92 cell lines were able to lyse almost 100% of ErbB2(+) target cells without irradiation. However, even with the addition of 10 Gy irradiation, NK-92/5.28.z cells were able to maintain 100% lysis of target cells while FcγRIII/IL-2 NK-92 cells dropped down to 75% lysis. Cytokine profiles were similar for NK-92 WT and FcγRIII/IL-2 NK-92 in the presence of ErbB2(+) target cells: both NK-92 cell lines secreted more granzyme B than IFN-γ whereas NK-92/5.28.z secreted more IFN-γ in the presence of ErbB2(+) target cells than granzyme B. The addition of radiation did not affect the trend in cytokine profiles for any NK-92 cell lines. The data found in the experiments included in this study can be used for selecting which NK-92 variant should enter clinical trials as well as for screening other NK-92 variants for similar purposes. The goal of this study was to address some of the issues revolving around NK cell use in cellular therapy by characterizing and comparing a preclinical NK-92 cell expressing high affinity Fc-γ receptor combined with intracellular IL-2 against an NK-92 cell modified to express a second-generation CAR consisting of an intracellular CD3 ζ domain and CD28 costimulatory molecule. The efficiency of each NK-92 cell line seen in the various experiments can be compared to other cell therapies in order to further determine whether another treatment is optimal or if combinatorial treatments are an option.:Abstract IV
List of Figures VII
List of Tables VIII
Abbreviations IX
Symbols and Units XII
1. Introduction 1
1.1 The Natural Killer Cell 2
1.1.1 Maturation 2
1.1.2 Cells of the Adaptive Immune System 4
1.1.3 Function 6
1.1.4 NK Cell Activation Receptors 6
1.1.5 NK Cell Inhibition Receptors 7
1.1.6 Targeted Natural Cytotoxicity 8
1.1.7 Antibody-dependent Cellular Cytotoxicity 9
1.1.8 Death-receptor-mediated Apoptosis 10
1.1.9 IFN-γ 11
1.1.10 Intracellular Signaling Cascades in ADCC-mediated VS Natural Cytotoxicity 12
1.2 Establishment of a Natural Killer Cell Line 14
1.2.1 NK-92 Cells 15
1.2.2 NK-92/5.28.z 15
1.2.3 FcγRIII/IL-2-expressing Preclinical Clone 18
1.3 Therapeutic Monoclonal Antibodies (mAbs) 18
1.4 The Tumor Cell 19
1.5 Objectives/Motivation 19
2. Materials and Methods 21
2.1 Cell Lines and Culture Conditions 22
2.2 Fluorescence-activated Cell Sorting (FACS) 22
2.2.1 Acquisition of Fluorescence-labeled Cells 22
2.2.2 Verification of ErbB2 Expression on Breast Cancer Cell Line 22
2.2.3 Analysis of Expression of the ErbB2 Chimeric scFv Receptor on NK-92/5.28.z Cells 23
2.2.4 Phenotypic Characterization of NK-92 cells 23
2.3 Growth Characteristics 26
2.4 Dye Exclusion for Cell Viability Determination 26
2.5 Radiation of NK-92 cells 26
2.6 Statistical Analysis 26
2.7 Potency Assay 27
2.8 Cytokine Profile in Steady State and Post-activation 28
2.9 Impact of γ-radiation on Cell Growth Kinetics 29
2.10 Impact of Radiation on Potency 29
2.11 Impact of Radiation on Cytokine Profile 29
2.12 Impact of Radiation on Outgrowth of NK-92 30
3. Results 31
3.1 Transgene Expression (Fc Receptor and CAR) on NK-92 32
3.1.1 ErbB2-CAR Expression 32
3.1.2 Fc Receptor Expression 33
3.1.3 Cell Surface Receptor Expression on NK-92 Variants 34
3.2 Impact on Growth Characteristics 35
3.3 Functional Cytotoxicity Against ErbB2-positive and Negative Cell Lines 37
3.4 Cytokine Profile in Steady State and Post-activation 38
3.5 Impact of γ-radiation on Cell Growth Kinetics 41
3.6 Impact of Radiation on Potency 43
3.7 Cytokine Profile Post-radiation 44
3.8 Impact of Radiation on Outgrowth 47
4. Discussion 49
5. Summary 62
5.1 Summary 63
5.2 Zusammenfassung 65
6. Appendix 67
6.1 Declaration of Compliance with Legal Requirements 68
6.2 Supplementary Data 69
References 76
Acknowledgements 87
Publications 88
Declaration of Authorship 89Laut dem National Institute of Health überstiegen im Jahr 2018 neue Krebsfälle 18 Millionen mit 9,6 Millionen krebsbedingten Todesfällen weltweit. Es ist bekannt, dass Krebs weltweit eine der häufigsten Todesursachen ist, jedoch können konventionelle medizinische Behandlungen, wie eine Chemotherapie, nicht vollständig behandeln, was die Entwicklung neuer Therapien gefördert hat. Zunächst wurden Zelltherapien mit T-Zellen erprobt, welche den Weg für weitere genetische Verbesserungen geebnet haben, die in der Lage sein könnten die derzeitige Behandlungslücke zu schließen. Verschiedene Formen einer genetischen Veränderung können eine Behandlung auf vielfältige Weise beeinflussen. Dies war der Grund für die Bewertung der Effizienz, Erweiterbarkeit und Sicherheit der NK-92-Zelllinie und ihrer genetisch veränderten Varianten, die jeweils einen chimären Antigenrezeptor (CAR) oder einen hochaffinen CD16-Rezeptor zusammen mit einem endogen exprimierten IL-2 exprimieren. In dieser Arbeit habe ich Eigenschaften einer mit einem ErbB2- spezifischen CAR der 2. Generation ausgestatteten NK-92 Zelllinie mit einer NK-92 Zelllinie mit rekombinanter Fc Rezeptor Expression und mit parentalen NK-92 Zellen in Bezug auf die Oberfächenrezeptorexpression mittels Durchflusszytometrie, Wachstumseigenschaften, Zyototoxizität gegen ErbB2/HER2-positive und negative Brustkrebszelllinien, Zytokinfreisetzung und Auswachsen nach einer Bestrahlung charakterisiert. Zelloberflächenrezeptoren waren mit wenigen Ausnahmen vergleichbar. Die Expression von CD2, CD11a, CD52, CD54, CD56 und CD337 (NKp30) wurde unter allen Zelllinien aufrechterhalten, während die Expression von CD3 und CD158a negativ blieb. FcγRIII/IL-2 NK-92 regulierte, im Vergleich zu den anderen NK-92-Zelllinien, die Expression von CD314 (NKG2D) hoch, wodurch sie für Zeilzellen preaktiviert ist, welche NKG2D Liganden exprimieren. Obwohl Wildtyp NK-92 Zellen eine Expression von CD336 (NKp44) aufzeigt, war die Expression dieses Rezeptors auf NK-92/5.28.z- und FcγRIII / IL-2-NK-92 nicht vorhanden. Die Expansion aller NK-92-Zelllinien war vergleichbar: NK-92 WT und NK-92 / 5.28.z konnten zu einer Dichte von 1,0 x 10 6 Zellen/ml expandieren, während sich FcγRIII /IL-2 NK-92 auf bis zu 1,2 x 10 6 Zellen wachsen können. Eine Bestrahlung der NK-92 Zellen führte in allen Fällen zu einer Inhibition des Zellwachstums innerhalb von zwei bis drei Tagen, insbesondere bei einer Dosis von 7,5 Gy oder mehr. Ohne Bestrahlung konnten NK-92/5.28.z- und FcγRIII/IL-2-NK-92-Zelllinien fast 100% der ErbB2 (+) - Zielzellen lysieren. Selbst nach einer Bestrahlung mit 10 Gy-konnten NK-92/5.28.z-Zellen eine 100% ige Lyse der Zielzellen aufrechterhalten, während die Zyototoxizität von FcγRIII/IL-2 NK-92 auf 75% Lyse abfiel. Die Zytokinprofile waren in Gegenwart von ErbB2 (+) - Zielzellen für NK-92 WT und FcγRIII / IL-2 NK-92 ähnlich: Beide NK-92-Zelllinien sekretierten mehr Granzym B als IFN-γ, während NK-92/5.28.z in Gegenwart von ErbB2 (+) – Zielzellen mehr IFN-γ als Granzym B sekretierte. Eine Bestrahlung hatte keinen Einfluss auf die Zytokinprofile irgendeiner NK-92-Zelllinie. Die in dieser Arbeit gewonnenen experimentellen Daten können zur Auswahl einer geeigneten NK-92-Variante für klinische Studien, sowie zum Screening anderer NK-92-Varianten für ähnliche Zwecke verwendet werden. Das Ziel dieser Arbeit war es, einige der Fragestellungen zu adressieren, die sich im Hinblick auf eine therapeutische Anwendung NK-Zell basierter Zelltherapie ergeben. Hierzu wurde eine präklinische NK-92-Zelllinie, die einen hochaffinen Fc-γ-Rezeptor in Kombination mit intrazellulärem IL-2 exprimiert, mit einer NK-92-Zelle verglichen, die einen 2. Generation CAR exprimiert, der aus einer intrazellulären CD3-ζ-Domäne und einem kostimulatorischen CD28-Molekül besteht. Die Effizienz jeder NK-92-Zelllinie, die in den verschiedenen Experimenten beobachtet wurde, kann mit anderen Zelltherapien verglichen werden, um weiter zu bestimmen, ob eine andere Behandlung optimal ist oder ob kombinatorische Behandlungen eine Option sind.:Abstract IV
List of Figures VII
List of Tables VIII
Abbreviations IX
Symbols and Units XII
1. Introduction 1
1.1 The Natural Killer Cell 2
1.1.1 Maturation 2
1.1.2 Cells of the Adaptive Immune System 4
1.1.3 Function 6
1.1.4 NK Cell Activation Receptors 6
1.1.5 NK Cell Inhibition Receptors 7
1.1.6 Targeted Natural Cytotoxicity 8
1.1.7 Antibody-dependent Cellular Cytotoxicity 9
1.1.8 Death-receptor-mediated Apoptosis 10
1.1.9 IFN-γ 11
1.1.10 Intracellular Signaling Cascades in ADCC-mediated VS Natural Cytotoxicity 12
1.2 Establishment of a Natural Killer Cell Line 14
1.2.1 NK-92 Cells 15
1.2.2 NK-92/5.28.z 15
1.2.3 FcγRIII/IL-2-expressing Preclinical Clone 18
1.3 Therapeutic Monoclonal Antibodies (mAbs) 18
1.4 The Tumor Cell 19
1.5 Objectives/Motivation 19
2. Materials and Methods 21
2.1 Cell Lines and Culture Conditions 22
2.2 Fluorescence-activated Cell Sorting (FACS) 22
2.2.1 Acquisition of Fluorescence-labeled Cells 22
2.2.2 Verification of ErbB2 Expression on Breast Cancer Cell Line 22
2.2.3 Analysis of Expression of the ErbB2 Chimeric scFv Receptor on NK-92/5.28.z Cells 23
2.2.4 Phenotypic Characterization of NK-92 cells 23
2.3 Growth Characteristics 26
2.4 Dye Exclusion for Cell Viability Determination 26
2.5 Radiation of NK-92 cells 26
2.6 Statistical Analysis 26
2.7 Potency Assay 27
2.8 Cytokine Profile in Steady State and Post-activation 28
2.9 Impact of γ-radiation on Cell Growth Kinetics 29
2.10 Impact of Radiation on Potency 29
2.11 Impact of Radiation on Cytokine Profile 29
2.12 Impact of Radiation on Outgrowth of NK-92 30
3. Results 31
3.1 Transgene Expression (Fc Receptor and CAR) on NK-92 32
3.1.1 ErbB2-CAR Expression 32
3.1.2 Fc Receptor Expression 33
3.1.3 Cell Surface Receptor Expression on NK-92 Variants 34
3.2 Impact on Growth Characteristics 35
3.3 Functional Cytotoxicity Against ErbB2-positive and Negative Cell Lines 37
3.4 Cytokine Profile in Steady State and Post-activation 38
3.5 Impact of γ-radiation on Cell Growth Kinetics 41
3.6 Impact of Radiation on Potency 43
3.7 Cytokine Profile Post-radiation 44
3.8 Impact of Radiation on Outgrowth 47
4. Discussion 49
5. Summary 62
5.1 Summary 63
5.2 Zusammenfassung 65
6. Appendix 67
6.1 Declaration of Compliance with Legal Requirements 68
6.2 Supplementary Data 69
References 76
Acknowledgements 87
Publications 88
Declaration of Authorship 8
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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