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Einleitung: Die im Genom der Schweine integrierten porzinen endogenen Retroviren (PERV) gehören zu den potentiellen humanpathogenen Erregern, die eines der Risiken bei der Xenotransplantation darstellen. Für die Abschätzung des Infektionsrisikos von PERV sind drei Verfahrensweisen von Bedeutung: Erstens die Evaluierung der PERV-Freisetzung aus porzinen Zellen und Geweben; Zweitens die Etablierung eines In vivo-Infektionsmodells und Drittens ein retrospektives Screenen von Patienten. Methoden: Die PERV-Expression in Inselzellen von Schweinen der Deutschen Landrasse wurde in vitro und in vivo evaluiert. Anschließend wurde PERV auf nicht-humanen Primatenzellen passagiert. In einem zweiten Modellversuch wurde murinen Zellen in vitro und SCID-Mäusen in vivo zellfreies PERV appliziert. Schließlich wurden Seren von Patienten analysiert. Ergebnisse: Die untersuchten Inselzellen setzten keine Viruspartikel frei und konnten somit weder humane Zellen noch BALB/c-Mäuse infizieren. Die verwendeten Affenzellen produzierten infektiöses PERV mit geringer Replikation. Weder in den murinen Modellversuchen noch in den untersuchten Patienten wurde eine Übertragung von PERV beobachtet. Schlussfolgerung: Schweine der Deutschen Landrasse könnten als Ausgangsbasis für die Zucht sicherer Schweine für die Xenotransplantation dienen. Da keine Infektion verschiedener muriner Zellen mit PERV beobachtet wurde, muss angenommen werden, dass Publikationen anderer Arbeitsgruppen, die eine PERV-Infektion in SCID-Mäusen diagnostizierten, ein falsch-positives Ergebnis aufgrund von Mikrochimärismus oder aufgrund von Pseudotypisierungen mit murinen endogenen Retroviren wiedergeben. Unsere Befunde werden dadurch erhärtet, dass der Rezeptor für PERV-A auf murinen Zellen nicht exprimiert wird und diese auch in vitro nicht infiziert werden konnten. In Übereinstimmung mit den weltweit etwa 200 behandelten Patienten konnte auch in den beiden neuen Studien keine Übertragung von PERV festgestellt werden. / Objective: Porcine endogenous retroviruses (PERVs) are integrated in the porcine genome and are able to infect human cells in vitro. Therefore, PERVs are one of the possible pathogens which poses a risk for xenotransplantations. In this study three significant methods were used to evaluate the infectious risk of PERV: The release of PERV particles from porcine cells and tissues, the formation of an in vivo infection model and a retrospective screening of patients. Methods: Islet cells from german landrace pigs were co-cultivated with human cells in vitro and were transplanted in BALB/c mice in vivo. Serial passaging experiments were performed with nonhuman primate cells. Murine cells were incubated in vitro and SCID mice in vivo with PERV. Sera of patients who were treated ex vivo with porcine liver cells and who had received islet cells were investigated for antibodies against PERV. Results: No virus release were observed in german landrace islet cells, thus they were neither able to infect human cells nor BALB/c mice. The used nonhuman primate cells released low replicating PERVs. None of the murine cells could be infected by PERV and no provirus integration was observed in different SCID mice organs. PERV-specific antibodies were found in none of the investigated patients. Conclusion: German landrace pigs could be used as a source for breeding safe genetically modified pigs suitable for xenotransplantation. Since there were no detectable PERV infection of different murine cells and SCID mice, it have to be supposed, that previously reported PERV transmissions to SCID mice might be due to microchimerism or to pseudotyping of murine endogenous retroviruses. Our results were confirmed by the fact, that the receptor for PERV-A is not expressed on murine cells and that these cells could not be infected in vitro. The absence of a PERV transmission in the investigated patients, correspond to the results obtained from approximately twohundred treated patients worldwide.

Devido ao consumo predatório e ao risco inerente de extinção de várias espécies de peixes, pesquisadores têm buscado técnicas alternativas de reprodução para viabilizar tanto o cultivo como a conservação ex situ. Os transplantes xenogênicos de gônadas representam uma técnica reprodutiva inovadora e já existem resultados promissores em diversas espécies de mamíferos, aves, insetos e também de peixes. No entanto, a baixa eficiência na geração de gametas alógenos consiste em uma das limitações desta técnica. Um dos possíveis motivos seria o desencadeamento de reações de rejeição por parte do hospedeiro, comum em transplante de órgãos e tecidos humanos. Dentro deste contexto, visamos identificar e avaliar a interferência destes mecanismos durante o processo de enxerto de gônada de truta arco-íris entre linhagens diferentes e salmão do Atlântico, identificando possíveis graus de respostas imunológicas utilizando enxertos autógenos, isógenos, alógenos e xenógenos. Tais avaliações foram feitas pela análise histopatológica dos tecidos transplantados e dos locais dos implantes. Com os resultados obtidos, pudemos avaliar que a resposta imunológica em implantes de tecidos alogênicos e xenogênicos é bem evidente. Já no enxerto de células germinativas dissociadas houve apenas um discreto processo inflamatório em todas as variações dos enxertos, o que pode representar uma adaptação técnica viável e mais eficaz para a obtenção de gametas. / Due the predatory consumption and the inherent risk of extinction of various species of the fish, investigate the alternatives of reproduction to make viable the cultivation as an ex situ. Xenogeneic transplants of gonads represent an innovative reproductive technique and those that can already be found in various species of animals, birds, insects and fish. However, a low efficiency in the range of allogenic gametes is one of the limitation of this technique. A probable cause would be related to an immune response process that would lead to rejection of the transplanted tissue, as observed in organs and tissues transplantation in human. In this context, we aimed to identify and to evaluate the presence of immune rejection mechanisms during the process of gonads tissues transplantation among different rainbow trout lineages and to Atlantic salmon, observing possible response degrees according to the nature of the graft: autogenic, isogenic, allogeneic or xenogeneic transplantations. Histopathological analysis of the transplanted tissues and of the implanted sites were conducted. Results showed a clear evidence of an immune response in allogeneic and xenogeneic implants. Dissociated gonad cells grafts showed a discrete inflammatory process disregarding the graft nature, that may be an indication of a feasible and more efficient technique adaption to obtain viable gametes.

<p>One main hurdle to xenotransplantation, i.e. transplantation between different species, is the immunological barrier that the organ meets in the recipient. The aim of this thesis was to characterise xenogeneic rejection mechanisms by using the concordant mouse-to-rat heart transplantation model.</p><p>Graft-infiltrating immune cells could be isolated from both rejecting and non-rejecting grafts using ex vivo propagation, a technique based on incubation of graft biopsies in culture medium for 48 hours. The numbers of recovered T lymphocytes were considerably higher in grafts undergoing cell-mediated rejection than in grafts undergoing acute vascular rejection (AVR) or in non-rejecting transplants. Thus, ex vivo propagation should be a valuable tool for further studies of cell-mediated rejection.</p><p>Cytokine patterns in the grafts, as measured by a quantitative real-time RT-PCR method, showed that AVR and cell-mediated rejection are associated with an increase of both pro-inflammatory cytokines (IL-1β and TNF-α) and more specific cytokines (IL-2, IL-10, IL-12p40 and IFN-γ). These data differed considerably from the patterns seen in the spleens of the recipients. Cell-mediated xenograft rejection was also found to be associated with a local accumulation of hyaluronan.</p><p>Oral administration of xenogeneic cells stimulated a production of antibodies that could induce hyperacute rejection of cardiac xenografts when passively transferred to graft recipients. This is in contrast to several models for autoimmune diseases and allogeneic transplantation where oral administration of antigens is an effective way to induce unresponsiveness. Hence, future attempts to induce oral tolerance in xenotransplantation should be done with caution.</p>

<p>Xenotransplantation, i.e. transplantation between different species, could be a possible solution to the present shortage of organ donors. The immunological response to a xenograft is strong and difficult to suppress. It is driven both by the humoral and cellular part of the immune system. The aim of this thesis was to characterise and modulate this response in a concordant mouse-to-rat model, using both vascularised and non-vascularised grafts.</p><p>Exposure of mouse cells or tissue to the circulation of a rat, either through transplantation or transfusions, easily evoked an immune response, consisting of IgM antibodies. A response that was aimed both at antigens present on mouse mononuclear cells and on erythrocytes. A non-immunosuppressed rat rejected a mouse heart graft within three days. The combined use of cyclosporine A (CyA) and deoxyspergualin (DSG) as immunosuppression prevented the rejection of vascularised heart transplants as well as of non-vascularised pancreatic islet grafts. This acceptance was sustained for the heart transplant also after the termination of DSG treatment, but not for the pancreatic islet graft. Furthermore, a second heart graft was accepted when transplanted under monotherapy with CyA 56-154 days after the first transplantation. This finding was interpreted as a humoral unresponsiveness, which could not be reproduced when the primary heart was substituted with a cellular graft, consisting of pancreatic islets or heart cells, or by blood transfusions. However, the rejection of a mouse heart after blood transfusions occurred in the absence of antibodies directed against mouse erythrocytes, in contrast to the observations in non-transfused animals. This indicates that a partial humoral tolerance restricted to the response against erythrocytes can be induced. This mechanism may offer a possibility to induce total humoral tolerance against a xenograft if the appropriate antigens are administered in conjunction with CyA and DSG.</p>

Xenotransplantation, i.e. transplantation between different species, could be a possible solution to the present shortage of organ donors. The immunological response to a xenograft is strong and difficult to suppress. It is driven both by the humoral and cellular part of the immune system. The aim of this thesis was to characterise and modulate this response in a concordant mouse-to-rat model, using both vascularised and non-vascularised grafts. Exposure of mouse cells or tissue to the circulation of a rat, either through transplantation or transfusions, easily evoked an immune response, consisting of IgM antibodies. A response that was aimed both at antigens present on mouse mononuclear cells and on erythrocytes. A non-immunosuppressed rat rejected a mouse heart graft within three days. The combined use of cyclosporine A (CyA) and deoxyspergualin (DSG) as immunosuppression prevented the rejection of vascularised heart transplants as well as of non-vascularised pancreatic islet grafts. This acceptance was sustained for the heart transplant also after the termination of DSG treatment, but not for the pancreatic islet graft. Furthermore, a second heart graft was accepted when transplanted under monotherapy with CyA 56-154 days after the first transplantation. This finding was interpreted as a humoral unresponsiveness, which could not be reproduced when the primary heart was substituted with a cellular graft, consisting of pancreatic islets or heart cells, or by blood transfusions. However, the rejection of a mouse heart after blood transfusions occurred in the absence of antibodies directed against mouse erythrocytes, in contrast to the observations in non-transfused animals. This indicates that a partial humoral tolerance restricted to the response against erythrocytes can be induced. This mechanism may offer a possibility to induce total humoral tolerance against a xenograft if the appropriate antigens are administered in conjunction with CyA and DSG.

One main hurdle to xenotransplantation, i.e. transplantation between different species, is the immunological barrier that the organ meets in the recipient. The aim of this thesis was to characterise xenogeneic rejection mechanisms by using the concordant mouse-to-rat heart transplantation model. Graft-infiltrating immune cells could be isolated from both rejecting and non-rejecting grafts using ex vivo propagation, a technique based on incubation of graft biopsies in culture medium for 48 hours. The numbers of recovered T lymphocytes were considerably higher in grafts undergoing cell-mediated rejection than in grafts undergoing acute vascular rejection (AVR) or in non-rejecting transplants. Thus, ex vivo propagation should be a valuable tool for further studies of cell-mediated rejection. Cytokine patterns in the grafts, as measured by a quantitative real-time RT-PCR method, showed that AVR and cell-mediated rejection are associated with an increase of both pro-inflammatory cytokines (IL-1β and TNF-α) and more specific cytokines (IL-2, IL-10, IL-12p40 and IFN-γ). These data differed considerably from the patterns seen in the spleens of the recipients. Cell-mediated xenograft rejection was also found to be associated with a local accumulation of hyaluronan. Oral administration of xenogeneic cells stimulated a production of antibodies that could induce hyperacute rejection of cardiac xenografts when passively transferred to graft recipients. This is in contrast to several models for autoimmune diseases and allogeneic transplantation where oral administration of antigens is an effective way to induce unresponsiveness. Hence, future attempts to induce oral tolerance in xenotransplantation should be done with caution.

This thesis focuses on the evolution and framing of xenotransplantation (XTP) policy debate in New Zealand from 1998 to 2011. Its aim is providing a better understanding of both the science-society interface and the importance of issue framing policy debate in understanding of the scientific debate in New Zealand and its relationship with the public. A qualitative study, this thesis draws upon a variety of public science commentary and debate and poses the research question: How did xenotransplantation’s introduction and explanation to the New Zealand public inform its current status as a Restricted Procedure under New Zealand law; and what ethical implications arise from this public policy debate for public participation in bio-medical research in New Zealand?

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