Postwachstum und Raumentwicklung : Denkanstöße für Wissenschaft und Praxis

Dieses von Mitgliedern des Arbeitskreises "Postwachstumsökonomien" (2016 - 2020) der ARL verfasste Positionspapier ordnet die internationale Postwachstumsdebatte ein und diskutiert ihre Relevanz für die Raumentwicklung und die raumwissenschaftliche Forschung. Neben sektoralen Zugängen und Befunden werden konkrete Vorschläge zur Postwachstumsorientierung in Planung, Forschung und Lehre gemacht. Dabei wird zwischen kurzfristig erreichbaren Veränderungen und mittel- bis langfristig auszulegenden Maßnahmen unterschieden

Induction of neutralizing antibodies specific for the envelope proteins of the koala retrovirus by immunization with recombinant proteins or with DNA

Background: The koala retrovirus (KoRV) is the result of a transspecies transmission of a gammaretrovirus with fatal consequences for the new host. Like many retroviruses, KoRV induces lymphoma, leukemia and an immunodeficiency that is associated with opportunistic infections in the virus-infected animals. We recently reported the induction of neutralizing antibodies by immunization with the recombinant ectodomain of the transmembrane envelope protein p15E of KoRV. Since the neutralization titers of the p15E-specific sera were only moderate, we investigated the use of the surface envelope protein gp70 to induce neutralizing antibodies. Findings: We immunized rats and goats with the recombinant gp70 protein of the KoRV, an unglycosylated protein of 52kD (rgp70/p52) or with the corresponding DNA. In parallel we immunized with recombinant rp15E or with a combination of rp15E and rgp70/p52. In all cases binding and neutralizing antibodies were induced. The gp70-specific sera had titers of neutralizing antibodies that were 15-fold higher than the p15E-specific sera. Combining rp15E and rgp70/p52 did not significantly increase neutralizing titers compared to rgp70/p52 alone. High titers of neutralizing antibodies specific for gp70 were also induced by immunization with DNA. Since KoRV and PERV are closely related, we investigated cross-neutralization of the antisera. The antisera against p15E and gp70 of PERV and KoRV inhibited infection by both viruses. Conclusion: The envelope proteins of the KoRV may therefore form the basis of an effective preventive vaccine to protect uninfected koalas from infection and possibly an immunotherapeutic treatment for those already infected

Xenotransplantation und porzine endogene Retroviren (PERV): Evaluierung des Risikos und Prävention der Virusübertragung

Die Xenotransplantation porziner Zellen, Gewebe und Organe bietet eine Möglichkeit, den immensen Mangel an geeigneten allogenen Transplantaten zu überbrücken. Die Grundvoraussetzung ist hierbei neben der Lösung immunologischer und physiologischer Probleme hinsichtlich Akzeptanz und Funktionalität des Xenotransplantats im Rezipienten, die mikrobiologische Sicherheit. Den zu den γ-Retroviren, von welchen viele in der Lage sind, Leukämien und Immundefizienzen auszulösen, gehörenden porzinen endogenen Retroviren wird dabei eine besondere Bedeutung beigemessen, da sie im Genom aller Schweine integriert sind und nicht wie die meisten anderen Pathogene durch SPF-Zucht eliminiert werden können. Auch die Entfernung der Viren durch knock-out-Verfahren wird als äußert schwierig angesehen, da PERVs mit mehr als 50 Kopien an verschiedenen Loci des porzinen Genoms integriert sind. Obwohl PERVs humane Zellen in vitro infizieren können, wurde weder in Infektionsstudien noch in ersten experimentellen Xenotransplantationen eine PERV-Übertragung beobachtet. In Anbetracht der besonderen Situation bei einer Xenotransplantation, der Exposition porziner Zellen und Organe im immunsupprimierten Organismus über mehrere Jahre, ist das Risiko einer PERV-Übertragung auf den Rezipienten nicht auszuschließen. Vor diesem Hintergrund war das Ziel der vorliegenden Arbeit, einen Beitrag zur Evaluierung der Virussicherheit sowie zur Prävention der Virusübertragung bei der Xenotransplantation zu leisten. Nachdem im Rahmen dieser Arbeit gezeigt werden konnte, dass die PERV-Expression in unterschiedlichen Schweinerassen, innerhalb eines Wurfes derselben Rasse sowie gewebsspezifisch variiert und die Selektion geeigneter Schweine mit einer stabilen und geringen PERV-Expression somit schwierig ist, wurden weitere Strategien zur Prävention der PERV-Übertragung entwickelt. Basierend auf dem Mechanismus der RNA-Interferenz wurden shRNA-transgene Schweine mit um 95% verminderter PERV-Expression generiert, die in Zukunft zu mikrobiologisch sicheren Xenotransplantaten beitragen könnten. Weiterhin erfolgte im Rahmen dieser Arbeit die Entwicklung von Impfstoffstrategien zur Prävention der PERV-Übertragung. Auf Basis des viralen Hüllproteins konnten in zwei Spezies (Ratten und Ziegen) neutralisierende Antikörper gegen PERV sowie das phylogenetisch eng verwandte Koala Retrovirus (KoRV) induziert werden, die in der Lage waren eine PERV- bzw. KoRV-Infektion in vitro zu 100% zu neutralisieren. Ein solcher Impfstoff könnte nach Klärung eventueller negativer Auswirkungen auf die Akzeptanz des Transplantats zur Impfung der Rezipienten eingesetzt werden. Weiterhin konnten im Rahmen dieser Arbeit erste Hinweise auf eine erhöhte PERVExpression in porzinen Melanomen gewonnen werden. Diese Beobachtung demonstriert die große Bedeutung der bisher unternommenen Anstrengungen geeignete Strategien zur Prävention der PERV-Übertragung zu entwickeln.Xenotransplantation of porcine cells, tissues and organs may offer a solution to the severe shortage of suitable allografts. In addition to overcoming immunological rejection and problems with the physiological functionality of the xenograft, a basic requirement for xenotransplantation is to ensure microbiological safety. Porcine endogenous retroviruses that belong to the family of γ-retroviruses of which many members are able to induce leukemias and immunodeficiencies are of particular importance. They are an integrated part of the porcine genome and cannot be eliminated by selective breeding under SPF conditions, as is the case for most other pathogens. Eliminating these viruses may prove difficult due to the fact that over 50 PERV copies are integrated into the porcine genome at different loci. Although PERVs are able to infect human cells in vitro, PERV transmission has not been observed during infectivity studies or following the first experimental xenotransplantations. However, given the special situation during xenotransplantation, i.e. the presence of porcine cells and organs within an immunosuppressed host over a period of several years, the risk of a PERV transmission cannot be excluded. Against this background, the aim of this dissertation was to contribute to the evaluation of safety as well as to the prevention of virus transmission during xenotransplantation. The demonstration of variable PERV expression in different breeds of pig, in litters of the same race and even in different tissues of individual animals, indicates that selection of pigs with a low and stable level of PERV expression may prove difficult. Additional strategies for preventing PERV transmission were therefore developed. Based on the mechanism of RNA interference, shRNA transgenic pigs demonstrating a drastically (95%) reduction of PERV expression were generated. Such animals could provide a source of virologically safe xenografts in the future. In addition, as part of this work, vaccine strategies to prevent PERV transmission were addressed. Antibodies able to fully neutralize both PERV and the phylogenetically related koala retrovirus (KoRV) in vitro were induced in two species using an immunogen based of the viral envelope protein. After resolving the negative consequences on xenograft acceptance that potentially might occur, such a vaccine could be used to pre-vaccinate xenograft recipients. Finally, this work provides the first evidence of elevated PERV expression in porcine melanomas, again demonstrating the importance of developing appropriate measures to prevent PERV transmission to humans

Xenotransplantation and porcine endogenous retroviruses (PERV): Evaluation of the risk and prevention of virus transmission

0\. Titelblatt und Inhaltsverzeichnis 1\. Einleitung 1 2\. Material 20 3\. Methoden 27 4\. Ergebnisse 53 5\. Diskussion 98 6\. Zusammenfassung 125 7\. Literaturverzeichnis I 8\. Anhang aDie Xenotransplantation porziner Zellen, Gewebe und Organe bietet eine Möglichkeit, den immensen Mangel an geeigneten allogenen Transplantaten zu überbrücken. Die Grundvoraussetzung ist hierbei neben der Lösung immunologischer und physiologischer Probleme hinsichtlich Akzeptanz und Funktionalität des Xenotransplantats im Rezipienten, die mikrobiologische Sicherheit. Den zu den ?-Retroviren, von welchen viele in der Lage sind, Leukämien und Immundefizienzen auszulösen, gehörenden porzinen endogenen Retroviren wird dabei eine besondere Bedeutung beigemessen, da sie im Genom aller Schweine integriert sind und nicht wie die meisten anderen Pathogene durch SPF-Zucht eliminiert werden können. Auch die Entfernung der Viren durch knock-out-Verfahren wird als äußert schwierig angesehen, da PERVs mit mehr als 50 Kopien an verschiedenen Loci des porzinen Genoms integriert sind. Obwohl PERVs humane Zellen in vitro infizieren können, wurde weder in Infektionsstudien noch in ersten experimentellen Xenotransplantationen eine PERV-Übertragung beobachtet. In Anbetracht der besonderen Situation bei einer Xenotransplantation, der Exposition porziner Zellen und Organe im immunsupprimierten Organismus über mehrere Jahre, ist das Risiko einer PERV- Übertragung auf den Rezipienten nicht auszuschließen. Vor diesem Hintergrund war das Ziel der vorliegenden Arbeit, einen Beitrag zur Evaluierung der Virussicherheit sowie zur Prävention der Virusübertragung bei der Xenotransplantation zu leisten. Nachdem im Rahmen dieser Arbeit gezeigt werden konnte, dass die PERV-Expression in unterschiedlichen Schweinerassen, innerhalb eines Wurfes derselben Rasse sowie gewebsspezifisch variiert und die Selektion geeigneter Schweine mit einer stabilen und geringen PERV-Expression somit schwierig ist, wurden weitere Strategien zur Prävention der PERV- Übertragung entwickelt. Basierend auf dem Mechanismus der RNA-Interferenz wurden shRNA-transgene Schweine mit um 95% verminderter PERV-Expression generiert, die in Zukunft zu mikrobiologisch sicheren Xenotransplantaten beitragen könnten. Weiterhin erfolgte im Rahmen dieser Arbeit die Entwicklung von Impfstoffstrategien zur Prävention der PERV-Übertragung. Auf Basis des viralen Hüllproteins konnten in zwei Spezies (Ratten und Ziegen) neutralisierende Antikörper gegen PERV sowie das phylogenetisch eng verwandte Koala Retrovirus (KoRV) induziert werden, die in der Lage waren eine PERV- bzw. KoRV-Infektion in vitro zu 100% zu neutralisieren. Ein solcher Impfstoff könnte nach Klärung eventueller negativer Auswirkungen auf die Akzeptanz des Transplantats zur Impfung der Rezipienten eingesetzt werden. Weiterhin konnten im Rahmen dieser Arbeit erste Hinweise auf eine erhöhte PERVExpression in porzinen Melanomen gewonnen werden. Diese Beobachtung demonstriert die große Bedeutung der bisher unternommenen Anstrengungen geeignete Strategien zur Prävention der PERV-Übertragung zu entwickeln.Xenotransplantation of porcine cells, tissues and organs may offer a solution to the severe shortage of suitable allografts. In addition to overcoming immunological rejection and problems with the physiological functionality of the xenograft, a basic requirement for xenotransplantation is to ensure microbiological safety. Porcine endogenous retroviruses that belong to the family of ?-retroviruses of which many members are able to induce leukemias and immunodeficiencies are of particular importance. They are an integrated part of the porcine genome and cannot be eliminated by selective breeding under SPF conditions, as is the case for most other pathogens. Eliminating these viruses may prove difficult due to the fact that over 50 PERV copies are integrated into the porcine genome at different loci. Although PERVs are able to infect human cells in vitro, PERV transmission has not been observed during infectivity studies or following the first experimental xenotransplantations. However, given the special situation during xenotransplantation, i.e. the presence of porcine cells and organs within an immunosuppressed host over a period of several years, the risk of a PERV transmission cannot be excluded. Against this background, the aim of this dissertation was to contribute to the evaluation of safety as well as to the prevention of virus transmission during xenotransplantation. The demonstration of variable PERV expression in different breeds of pig, in litters of the same race and even in different tissues of individual animals, indicates that selection of pigs with a low and stable level of PERV expression may prove difficult. Additional strategies for preventing PERV transmission were therefore developed. Based on the mechanism of RNA interference, shRNA transgenic pigs demonstrating a drastically (95%) reduction of PERV expression were generated. Such animals could provide a source of virologically safe xenografts in the future. In addition, as part of this work, vaccine strategies to prevent PERV transmission were addressed. Antibodies able to fully neutralize both PERV and the phylogenetically related koala retrovirus (KoRV) in vitro were induced in two species using an immunogen based of the viral envelope protein. After resolving the negative consequences on xenograft acceptance that potentially might occur, such a vaccine could be used to pre-vaccinate xenograft recipients. Finally, this work provides the first evidence of elevated PERV expression in porcine melanomas, again demonstrating the importance of developing appropriate measures to prevent PERV transmission to humans

Induction of neutralizing antibodies specific for the envelope proteins of the koala retrovirus by immunization with recombinant proteins or with DNA

Background: The koala retrovirus (KoRV) is the result of a transspecies transmission of a gammaretrovirus with fatal consequences for the new host. Like many retroviruses, KoRV induces lymphoma, leukemia and an immunodeficiency that is associated with opportunistic infections in the virus-infected animals. We recently reported the induction of neutralizing antibodies by immunization with the recombinant ectodomain of the transmembrane envelope protein p15E of KoRV. Since the neutralization titers of the p15E-specific sera were only moderate, we investigated the use of the surface envelope protein gp70 to induce neutralizing antibodies. Findings: We immunized rats and goats with the recombinant gp70 protein of the KoRV, an unglycosylated protein of 52kD (rgp70/p52) or with the corresponding DNA. In parallel we immunized with recombinant rp15E or with a combination of rp15E and rgp70/p52. In all cases binding and neutralizing antibodies were induced. The gp70-specific sera had titers of neutralizing antibodies that were 15-fold higher than the p15E-specific sera. Combining rp15E and rgp70/p52 did not significantly increase neutralizing titers compared to rgp70/p52 alone. High titers of neutralizing antibodies specific for gp70 were also induced by immunization with DNA. Since KoRV and PERV are closely related, we investigated cross-neutralization of the antisera. The antisera against p15E and gp70 of PERV and KoRV inhibited infection by both viruses. Conclusion: The envelope proteins of the KoRV may therefore form the basis of an effective preventive vaccine to protect uninfected koalas from infection and possibly an immunotherapeutic treatment for those already infected

Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs

Background: Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission. Methods: We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV-infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in-solution hybridization analysis and real-time RT PCR, respectively. Results: All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild-type control animals. Conclusion: This strategy may lead to animals compatible with PERV safe xenotransplantation

Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation

Background: Multi-transgenic pigs produced for use in xenotransplantation have to be screened for the presence and expression of porcine endogenous retroviruses (PERV) to select animals with low PERV load. The production of transgenic pigs may also be associated with the integration of the transgene adjacent to or into the locus of a PERV provirus, potentially leading to an enhanced virus expression. Methods: Non-transgenic animals, single-transgenic, and multi-transgenic pigs were screened for the presence of PERV-A, -B, and -C and recombinant PERV-A/C using polymerase chain reaction (PCR). PERV expression was determined by real time reverse transcriptase-PCR. An assay based on the activation of PERV in peripheral blood mononuclear cells by mitogens was used to discriminate between low and high PERV producer animals. Results: All animals carried PERV-A and -B. A total of 176 from 181 (97.2%) animals carried PERV-C in the germ line and 18 from 64 animals carried PERV-A/C in the genome of lymphoid cells but not in the germ line. The expression of PERV was very low in all animals and not different between transgenic pigs and non-transgenic animals. PERV expression differed between various pig lines. The highest expression was found in mini-pigs and crossing other pig lines with mini-pigs resulted in increased PERV expression in the progeny. However, expression of viral proteins and particle release were not observed in all transgenic animals. Conclusions: No evidence for elevated PERV expression in (multi-) transgenic pigs was observed. Differences in PERV expression correlated with the genetic background of the animals, not with the specific transgene. Mini-pigs consistently had the highest level of PERV expression and animals with a mini-pig background had a higher level of expression compared with animals without mini-pig background

Post-growth and spatial development

This position paper, drawn up by members of the ARL’s Working Group on Post-Growth Economies (2016 – 2020), situates the international post-growth debate and discusses its relevance for spatial development and spatial science research. In addition to sectoral approaches and findings, the authors make concrete proposals for post-growth focuses in planning, research and teaching, distinguishing between changes that can be achieved in the short term and measures designed for the medium to long term