Emanuele Cozzi | Università degli Studi di Padova (original) (raw)

Papers by Emanuele Cozzi

Veterinary Research Communications, 2007

Xenotransplantation is one of the possible avenues currently being explored to address the shorta... more Xenotransplantation is one of the possible avenues currently being explored to address the shortage problem of human organs. With this in mind, this article will briefly review the current situation with respect to the immunological, physiological and biosafety aspects related to the transplantation of pig organs into primates. Acute humoral xenograft rejection (AHXR) currently remains the central immunological obstacle and the development of strategies for both a better control of the elicited anti-pig humoral immune response or the prevention of the onset of coagulation disorders that accompany AHXR are the two primary focuses of research. To date, porcine xenografts have been shown to sustain the life of nonhuman primates for several months. Such preclinical studies have also demonstrated the absence of insurmountable physiological incompatibilities between pig and primate. In addition, reassuring findings regarding biosafety aspects have been generated and pro-active research aimed at the identification of an organ source with a higher safety profile is also underway. These advancements, in conjunction with ongoing research in pig genetic engineering, immunosuppression and tolerance are expected to further extend the survival of porcine xenografts transplanted into primates. However, until further physiological, efficacy and safety data are generated in relevant primate models, clinical xenotransplantation should not be considered.

Transplantation Journal, 2004

Xenotransplantation, 2009

The microcirculation was assessed in the livers of human decay accelerating factors (hDAF) and wi... more The microcirculation was assessed in the livers of human decay accelerating factors (hDAF) and wild-type transgenic rats by fluorescent intravital microscopy, histology and histomorphology to determine the benefits of hDAF expression for the microcirculation of a rat liver xenograft perfused with human blood. Male hDAF transgenic rats (group A; n = 20) and wild-type Sprague-Dawley rats (group B; n = 20) were xenoperfused with human blood, while other male wild-type Sprague-Dawley rats (group C; n = 10) were perfused with allogeneic blood. Following plasma and leukocyte staining with fluorescein sodium, and platelet staining with rhodamine, the right lobe of the liver was assessed by intravital microscopy, counting the numbers of perfused sinusoids and leukocytes adhering to the endothelium per mm(2), and calculating the acinar perfusion index (Pi). The liver underwent histological assessment at the end of each experiment. Mean +/- SEM values were calculated and the Mann-Whitney U-test was used for statistical analysis. The number of perfused sinusoids was higher in the group of hDAF rat livers (group A) and controls (group C) than in the group of non-transgenic rat livers perfused with human blood (group B) (P < 0.05), although only group C still had a significantly more perfused sinusoids than the other groups after 90 min of perfusion (P < 0.05). The acinar perfusion index was higher in groups A and C than in group B (P < 0.05); here again, only group C still had a significantly higher Pi than group B after 90 min of perfusion (P < 0.05). There was a massive accumulation of leukocytes that peaked after 5 min and persisted throughout the perfusion in all three groups. Histology showed portal and subendothelial hepatic vein hemorrhage, necrosis and inflammatory reaction, which were particularly evident in group B. In our study, rat livers transgenic for hDAF were better protected against early tissue damage by perfusion with human blood, but this did not result in a longer xenograft survival.

Xenotransplantation, 2006

Young people, and students in particular, generally have a positive attitude to xenotransplantati... more Young people, and students in particular, generally have a positive attitude to xenotransplantation. We reported previously that university students attending their first year approved of the idea of transplanting animal organs into humans. The aim of this study was to evaluate the impact of a 3-yr course at various faculties on the university students' understanding of and attitude to xenotransplantation. University students in their fourth year at five different faculties of Padua University (Italy), who had previously been surveyed in exactly the same way 3 yr earlier, were re-administered an anonymous 19-item questionnaire on their attitude to xenotransplantation. A total of 453 students completed the fourth year questionnaire (84 males, 369 females; mean age 24.6 yr, range 21-36 yr). Students were attending courses in Medicine (22.1%), Veterinary Science (16.5%), Agriculture (10.4%), Educational Sciences (22.1%) and Psychology (28.9%). In their fourth year, 85% of the students knew that animal organs could, at some stage, be transplanted into humans, 81.5% approved of this idea and 84% would accept an animal organ to save their life if necessary (these percentages were 88%, 78% and 76%, respectively 3 yr earlier). A significantly larger proportion of the students who approved of xenotransplantation were attending science courses rather than art courses, while no differences emerged as regards gender. Those who refused xenotransplantation justified their position mainly on ethical-moral (41.7%) and immunological (35.7%) grounds. As in their first year, so too in their fourth, University students were well informed about the feasibility of transplanting animal organs into human beings and those attending science courses were more likely to accept this idea than art students. Approval of xenotransplantation was much the same among fourth year males and females, whereas in the first year, male students had approved more than female students.

Xenotransplantation, 2007

In July 2005, in the context of the fourth and last Call for Proposals of the European Union (EU)... more In July 2005, in the context of the fourth and last Call for Proposals of the European Union (EU) 6th Framework Programme (FP6), a research topic entitled ‘‘Application of post-genomics to xenotransplantation research’’ was included in the thematic priority area 1: ‘‘Life sciences, genomics and biotechnology for health.’’ This thematic priority is considered strategically important for Europe since life sciences and biotechnology are viewed as critical research areas that may contribute significantly to the Lisbon Objective of the EU (i.e. for Europe to become the most competitive knowledge-based economy in the world by 2010). From a scientific standpoint, the overall objective of this thematic priority is to apply the knowledge generated in sequencing the human genome (and the genomes of many other species) with the aim of improving human health and to stimulate industrial and economic growth. Indeed, the major emphasis of the proposed research must be to develop basic scientific knowledge through to its application (‘‘translational’’ approach). The funding instrument selected by the EU to promote a xenotransplantation research programme was that of an Integrated Project. Typically, these EU funded projects involve 10–15 partners who are expected to work together in a collaborative program with support spread over up to 5 years. In addition to the groups selected for their expertise on several scientific levels, the EU stipulates that partners who are experts in ethical, social, and regulatory issues must be included in the project. Many first-class investigators from European research centers, with excellent track records in xenotransplantation research, could have taken part in this research initiative. However, collaborators were necessarily limited by stringent EU requirements. The structure of such EU research projects is bound by specific definitions and with clear funding limits. (Indeed, the funding provided must be considered as a contribution to the research, and does not cover all costs.) Amongst the requirements with which each successful application must comply, the following are of critical importance: • Commitment to promote integration of research capacities (public and private) across Europe to increase coherence and achieve critical mass. • Allocation of at least 15% of the budget to (‘‘high-tech’’) small and medium enterprises (SMEs). • Involvement of all key stakeholders (including appropriate industry, healthcare providers and physicians, policy makers, regulatory authorities, patient associations, and experts on ethical matters, etc.) in implementing the theme. The proposed transdisciplinary collaboration between all stakeholders must ensure that due account is taken of the ethical and societal concerns, our obligations towards future generations and the rest of the world. • Ensuring that new knowledge is disseminated and translated into new therapies and clinical practice. • A very high ethical profile of the research proposed for funding.

Xenotransplantation, 2007

Cyclophosphamide (CYP) and methotrexate (MTX) have been used as immunosuppressants in induction o... more Cyclophosphamide (CYP) and methotrexate (MTX) have been used as immunosuppressants in induction or maintenance protocols in a large variety of xenotransplantation models. Combining the use of transgenic porcine organs expressing human decay-accelerating factor (hDAF) with immunosuppressive therapy that included the use of CYP or MTX, survival of primate recipients of life-supporting renal xenografts has been prolonged. However, both drugs can cause significant systemic toxicity and, in particular, gastrointestinal (GI) toxicity. To date only limited data have been reported on the histopathological features deriving from the use of such agents in non-human primates. Cyclophosphamide or MTX was used as part of the immunosuppressive regimen in 15 bilaterally nephrectomized non-human primate (Macaca fascicularis) recipients of a life-supporting hDAF porcine kidney. At post-mortem, a detailed analysis of the GI tract in animals receiving either CYP or MTX was performed. Paraffin-embedded sections of each portion of the GI tract were prepared and stained with hematoxylin and eosin (H&E). In some animals, additional investigations by immunohistochemistry (CD3, CD5, CD20, CD79 alpha cy, lambda, and kappa light chains) and by in situ hybridization for EBV encoded RNA (EBER) were undertaken. The xenografted animals from the CYP group had a mean survival of 31 days (range: 0 to 90 days); animals from the MTX group survived a median of 14 days (range: 0 to 39 days). GI complications were the most frequent cause of euthanasia after renal failure. In CYP-treated animals GI-tract lesions were primarily characterized by diffuse, severe lymphoplasmocytic mucosal inflammatory infiltrate. Variable degrees of villi atrophy and fusion, gut-associated lymphoid tissue (GALT) and goblet cell hyperplasia were also observed. In MTX-treated primates, findings were consistent with severe villi atrophy associated with mild-to-moderate disseminated lymphoplasmocytic infiltration. In conclusion, GI tract lesions are an early and consistent finding when CYP or MTX are used as induction agents in this model. The two compounds induce different types of GI tract damage, however, in agreement with their different mechanisms of action. Whilst CYP primarily determines inflammatory lesions, MTX leads to a degenerative type of damage. This study indicates that immunosuppressive drugs can cause severe GI tract damage in primate recipients of renal xenografts and may be responsible for life-threatening lesions.

Xenotransplantation, 2003

Methotrexate (MTX) has been used successfully as an immunosuppressant in rodent xenotransplantati... more Methotrexate (MTX) has been used successfully as an immunosuppressant in rodent xenotransplantation models, but the data generated so far with MTX in pig-to-baboon cardiac transplantation studies have been disappointing. The potential of this agent was consequently explored in a life-supporting pig-to-primate renal model using the cynomolgus monkey as the recipient species. Introductory in vitro and in vivo pharmacokinetic and pharmacodynamic studies with MTX were conducted in three cynomolgus monkeys. Subsequently, 10 cynomolgus monkey recipients of a life-supporting kidney from human decay-accelerating factor transgenic pigs were administered MTX intravenously according to three different regimens. All the animals also received cyclosporine A and steroids. In addition, mycophenolate sodium (MPS) was administered post-operatively in two of the three groups of transplanted animals. At clinically relevant concentrations, MTX is able in vitro to inhibit the mixed lymphocyte reactions (MLR) in cynomolgus monkeys. After intravenous administration, moreover, exposure of cynomolgus monkeys to MTX appeared to be higher than had been previously reported in baboons. Graft function was observed in the transplanted animals, which survived from 0 to 41 days. All but two animals revealed acute humoral rejection in the explanted graft and developed diarrhea. Diarrhea was the cause of euthanasia in five cases. It was unrelated to the administration of MPS and associated with severe histopathological signs of enteritis. This study demonstrates that the pharmacokinetic and pharmacodynamic profiles of MTX vary substantially between non-human primate species. In vitro, MTX has immunosuppressive properties in the cynomolgus monkey at clinically relevant concentrations. In vivo, MTX has a very narrow therapeutic window in cynomolgus monkeys, however, as it does in baboons. We conclude that MTX is scarcely effective as an immunosuppressant, be it for induction or maintenance, in pig-to-cynomolgus monkey renal xenotransplantation.

Xenotransplantation, 2008

Despite being still at the experimental level, xenotransplantation may become an effective strate... more Despite being still at the experimental level, xenotransplantation may become an effective strategy to overcome the scarcity of human organs. However, at the present time there is considerable resistance to this kind of biomedical technology. The aim of the present study was to identify novel strategies to reduce patients' negative affective reactions towards xenotransplantation helping them to understand the advantages of xenotransplantation in a more analytical fashion and increase their acceptance for this approach. The study was conducted in a group of patients with liver cirrhosis waiting for liver transplantation. They were presented with hypothetical scenarios and asked to choose among either two or three alternative types of donor defined by their species (e.g., livers from humans vs. other species) and availability (low for human donors and high for livers from non-human species). Patients were unwilling to accept xenotransplantation if they were presented with livers from humans (chosen by 97.5% of participants) vs. livers from genetically modified pigs (2.5%). On the other hand, a different group of patients was significantly more willing to accept xenotransplantation if they were presented with three different types of donors: respectively, human beings (74.4%), genetically modified pigs (25.6%) and genetically modified dogs. In addition, human livers were judged significantly more attractive than genetically modified livers from pigs, monkeys, dogs, or sheep and pig livers were rated as significantly more attractive than livers from monkeys, dogs, or sheep (for all comparisons P < 0.01). These results demonstrate that paradigms from other fields, like decision-making, might help to communicate more effectively the potential of xenotransplantation, modulating patients' affective reactions and allowing them to understand the potential strengths of this biomedical technology.

Xenotransplantation, 2009

Xenotransplantation, 2007

Xenotransplantation, 2013

Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs whe... more Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs when pig organs are transplanted into primates. The coagulopathy reported in this model is a very complex process that involves simultaneously coagulation factors, platelets and phospholipid-bearing cells (i.e., leukocytes, red blood cells, and endothelial cells). Choosing whole blood for coagulation analysis theoretically appears more favorable compared with plasma. Whole blood rotation thromboelastometry (ROTEM(®) ) is a point-of-care global coagulation analyzer able to evaluate the characteristics of clot formation and lysis by dynamic monitoring. The aim of this study was to record thromboelastographic profiles, performed by ROTEM(®) , in a series of immunosuppressed nephrectomized primates that received a life-supporting kidney. Of the eight primates, n = 4 received a pig kidney transgenic for human decay-accelerating factor (hDAF/Gal+); n = 2, an α 1,3-galactosyltransferase gene-knockout (GT-KO) pig kidney transgenic for human CD39, CD55, CD59 and fucosyltransferase (HTF); and n = 2, a GT-KO pig kidney transgenic for hDAF. Blood samples were collected before and at least once per week after transplantation till euthanasia. Intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM) were evaluated. Thromboelastographic parameters considered were clotting time (CT, seconds) and clot formation time (CFT, seconds) in INTEM and EXTEM and maximum clot firmness (MCF, mm) in FIBTEM. The correlations between CT in INTEM and activated partial thromboplastin time (aPTT), CT in EXTEM and PT, CFT in INTEM and EXTEM, and platelet counts and MCF in FIBTEM and fibrinogen plasma levels were also considered. In all animals, thromboelastographic profiles showed progressive prolongation of CT (activation of coagulative cascade) in INTEM. A close correspondence was observed between (i) the prolongation of the CFT values (propagation of clot formation), both in INTEM and EXTEM, and the decrease in platelet counts; (ii) the reduction in MCF values (clot firmness) ​​in FIBTEM and the decrease in fibrinogen plasma levels. No concordance between CT in INTEM and aPTT and between CT in EXTEM and PT was observed. Our study demonstrated that ROTEM(®) analyzer could be a useful and complementary tool to study the consumptive coagulopathy, either "compensated" or "non-compensated," that takes place when transgenic pig kidneys are transplanted into primates. Larger and prospective studies are needed to confirm our results and to evaluate the role of ROTEM(®) to guide the management of consumptive coagulopathy in order to prolong the survival of the transplanted organ.

Transplantation Proceedings, 2000

Transplantation, 2005

Fibrin deposition is central to the acute humoral rejection process occurring in the presence of ... more Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates. To assess whether strategies aimed at preventing fibrin formation may extend xenograft survival, we administered high daily doses of recombinant human antithrombin (rhAT) (500 U/kg twice daily) to obtain both anticoagulant and anti-inflammatory effects in immunosuppressed primate recipients of porcine kidneys. Some degree of consumptive coagulopathy developed in both rhAT-treated (n=3) and untreated (n=3) primates. No major differences in the coagulation parameters analyzed were observed between the 2 groups. Similarly, no difference in survival was seen between rhAT-treated (20.6+/-4 days; range: 15-23 days) and untreated animals (17.3+/-11.6 days; range: 7-30 days), although the rhAT-treated primates had a higher bleeding tendency. Despite the high daily dose of rhAT, considerable fibrin deposition was observed in the graft as early as 2 weeks after transplantation. These results suggest that a high daily dose of rhAT fails to influence survival or prevent fibrin formation and deposition in the graft in our pig-to-primate model. However, the potential role of rhAT administered in combination with heparins or other clotting inhibitor concentrates in this model remains to be determined.

Transplantation, 2000

It is not known whether the pig liver is capable of functioning efficiently when transplanted int... more It is not known whether the pig liver is capable of functioning efficiently when transplanted into a primate, neither is there experience in transplanting a liver from a transgenic pigs expressing the human complement regulator human complement regulator decay accelerating factor (h-DAF) into a baboon. The objective of this study was to determine whether the porcine liver would support the metabolic functions of non-human primates and to establish the effect of hDAF expression in the prevention of hyperacute rejection of porcine livers transplanted into primates. Five orthotopic liver xenotransplants from pig to baboon were carried out: three from unmodified pigs and two using livers from h-DAF transgenic pigs. The three control animals transplanted with livers from unmodified pigs survived for less than 12 hr. Baboons transplanted with livers from h-DAF transgenic pigs survived for 4 and 8 days. Hyperacute rejection was not detected in the baboons transplanted with hDAF transgenic pig livers; however, it was demonstrated in the three transplants from unmodified pigs. Baboons transplanted with livers from h-DAF transgenic pigs were extubated at postoperative day 1 and were awake and able to eat and drink. In the recipients of hDAF transgenic pig livers the clotting parameters reached nearly normal levels at day 2 after transplantation and remained normal up to the end of the experiments. In these hDAF liver recipients, porcine fibrinogen was first detected in the baboon plasma 2 hr postreperfusion, and was present up to the end of the experiments. One animal was euthanized at day 8 after development of sepsis and coagulopathy, the other animal arrested at day 4, after an episode of vomiting and aspiration. The postmortem examination of the hDAF transgenic liver xenografts did not demonstrate rejection. The livers from h-DAF transgenic pigs did not undergo hyperacute rejection after orthotopic xenotransplantation in baboons. When HAR is abrogated, the porcine liver maintains sufficient coagulation and protein levels in the baboon up to 8 days after OLT.

Veterinary Research Communications, 2007

Xenotransplantation is one of the possible avenues currently being explored to address the shorta... more Xenotransplantation is one of the possible avenues currently being explored to address the shortage problem of human organs. With this in mind, this article will briefly review the current situation with respect to the immunological, physiological and biosafety aspects related to the transplantation of pig organs into primates. Acute humoral xenograft rejection (AHXR) currently remains the central immunological obstacle and the development of strategies for both a better control of the elicited anti-pig humoral immune response or the prevention of the onset of coagulation disorders that accompany AHXR are the two primary focuses of research. To date, porcine xenografts have been shown to sustain the life of nonhuman primates for several months. Such preclinical studies have also demonstrated the absence of insurmountable physiological incompatibilities between pig and primate. In addition, reassuring findings regarding biosafety aspects have been generated and pro-active research aimed at the identification of an organ source with a higher safety profile is also underway. These advancements, in conjunction with ongoing research in pig genetic engineering, immunosuppression and tolerance are expected to further extend the survival of porcine xenografts transplanted into primates. However, until further physiological, efficacy and safety data are generated in relevant primate models, clinical xenotransplantation should not be considered.

Transplantation Journal, 2004

Xenotransplantation, 2009

The microcirculation was assessed in the livers of human decay accelerating factors (hDAF) and wi... more The microcirculation was assessed in the livers of human decay accelerating factors (hDAF) and wild-type transgenic rats by fluorescent intravital microscopy, histology and histomorphology to determine the benefits of hDAF expression for the microcirculation of a rat liver xenograft perfused with human blood. Male hDAF transgenic rats (group A; n = 20) and wild-type Sprague-Dawley rats (group B; n = 20) were xenoperfused with human blood, while other male wild-type Sprague-Dawley rats (group C; n = 10) were perfused with allogeneic blood. Following plasma and leukocyte staining with fluorescein sodium, and platelet staining with rhodamine, the right lobe of the liver was assessed by intravital microscopy, counting the numbers of perfused sinusoids and leukocytes adhering to the endothelium per mm(2), and calculating the acinar perfusion index (Pi). The liver underwent histological assessment at the end of each experiment. Mean +/- SEM values were calculated and the Mann-Whitney U-test was used for statistical analysis. The number of perfused sinusoids was higher in the group of hDAF rat livers (group A) and controls (group C) than in the group of non-transgenic rat livers perfused with human blood (group B) (P < 0.05), although only group C still had a significantly more perfused sinusoids than the other groups after 90 min of perfusion (P < 0.05). The acinar perfusion index was higher in groups A and C than in group B (P < 0.05); here again, only group C still had a significantly higher Pi than group B after 90 min of perfusion (P < 0.05). There was a massive accumulation of leukocytes that peaked after 5 min and persisted throughout the perfusion in all three groups. Histology showed portal and subendothelial hepatic vein hemorrhage, necrosis and inflammatory reaction, which were particularly evident in group B. In our study, rat livers transgenic for hDAF were better protected against early tissue damage by perfusion with human blood, but this did not result in a longer xenograft survival.

Xenotransplantation, 2006

Young people, and students in particular, generally have a positive attitude to xenotransplantati... more Young people, and students in particular, generally have a positive attitude to xenotransplantation. We reported previously that university students attending their first year approved of the idea of transplanting animal organs into humans. The aim of this study was to evaluate the impact of a 3-yr course at various faculties on the university students' understanding of and attitude to xenotransplantation. University students in their fourth year at five different faculties of Padua University (Italy), who had previously been surveyed in exactly the same way 3 yr earlier, were re-administered an anonymous 19-item questionnaire on their attitude to xenotransplantation. A total of 453 students completed the fourth year questionnaire (84 males, 369 females; mean age 24.6 yr, range 21-36 yr). Students were attending courses in Medicine (22.1%), Veterinary Science (16.5%), Agriculture (10.4%), Educational Sciences (22.1%) and Psychology (28.9%). In their fourth year, 85% of the students knew that animal organs could, at some stage, be transplanted into humans, 81.5% approved of this idea and 84% would accept an animal organ to save their life if necessary (these percentages were 88%, 78% and 76%, respectively 3 yr earlier). A significantly larger proportion of the students who approved of xenotransplantation were attending science courses rather than art courses, while no differences emerged as regards gender. Those who refused xenotransplantation justified their position mainly on ethical-moral (41.7%) and immunological (35.7%) grounds. As in their first year, so too in their fourth, University students were well informed about the feasibility of transplanting animal organs into human beings and those attending science courses were more likely to accept this idea than art students. Approval of xenotransplantation was much the same among fourth year males and females, whereas in the first year, male students had approved more than female students.

Xenotransplantation, 2007

In July 2005, in the context of the fourth and last Call for Proposals of the European Union (EU)... more In July 2005, in the context of the fourth and last Call for Proposals of the European Union (EU) 6th Framework Programme (FP6), a research topic entitled ‘‘Application of post-genomics to xenotransplantation research’’ was included in the thematic priority area 1: ‘‘Life sciences, genomics and biotechnology for health.’’ This thematic priority is considered strategically important for Europe since life sciences and biotechnology are viewed as critical research areas that may contribute significantly to the Lisbon Objective of the EU (i.e. for Europe to become the most competitive knowledge-based economy in the world by 2010). From a scientific standpoint, the overall objective of this thematic priority is to apply the knowledge generated in sequencing the human genome (and the genomes of many other species) with the aim of improving human health and to stimulate industrial and economic growth. Indeed, the major emphasis of the proposed research must be to develop basic scientific knowledge through to its application (‘‘translational’’ approach). The funding instrument selected by the EU to promote a xenotransplantation research programme was that of an Integrated Project. Typically, these EU funded projects involve 10–15 partners who are expected to work together in a collaborative program with support spread over up to 5 years. In addition to the groups selected for their expertise on several scientific levels, the EU stipulates that partners who are experts in ethical, social, and regulatory issues must be included in the project. Many first-class investigators from European research centers, with excellent track records in xenotransplantation research, could have taken part in this research initiative. However, collaborators were necessarily limited by stringent EU requirements. The structure of such EU research projects is bound by specific definitions and with clear funding limits. (Indeed, the funding provided must be considered as a contribution to the research, and does not cover all costs.) Amongst the requirements with which each successful application must comply, the following are of critical importance: • Commitment to promote integration of research capacities (public and private) across Europe to increase coherence and achieve critical mass. • Allocation of at least 15% of the budget to (‘‘high-tech’’) small and medium enterprises (SMEs). • Involvement of all key stakeholders (including appropriate industry, healthcare providers and physicians, policy makers, regulatory authorities, patient associations, and experts on ethical matters, etc.) in implementing the theme. The proposed transdisciplinary collaboration between all stakeholders must ensure that due account is taken of the ethical and societal concerns, our obligations towards future generations and the rest of the world. • Ensuring that new knowledge is disseminated and translated into new therapies and clinical practice. • A very high ethical profile of the research proposed for funding.

Xenotransplantation, 2007

Cyclophosphamide (CYP) and methotrexate (MTX) have been used as immunosuppressants in induction o... more Cyclophosphamide (CYP) and methotrexate (MTX) have been used as immunosuppressants in induction or maintenance protocols in a large variety of xenotransplantation models. Combining the use of transgenic porcine organs expressing human decay-accelerating factor (hDAF) with immunosuppressive therapy that included the use of CYP or MTX, survival of primate recipients of life-supporting renal xenografts has been prolonged. However, both drugs can cause significant systemic toxicity and, in particular, gastrointestinal (GI) toxicity. To date only limited data have been reported on the histopathological features deriving from the use of such agents in non-human primates. Cyclophosphamide or MTX was used as part of the immunosuppressive regimen in 15 bilaterally nephrectomized non-human primate (Macaca fascicularis) recipients of a life-supporting hDAF porcine kidney. At post-mortem, a detailed analysis of the GI tract in animals receiving either CYP or MTX was performed. Paraffin-embedded sections of each portion of the GI tract were prepared and stained with hematoxylin and eosin (H&E). In some animals, additional investigations by immunohistochemistry (CD3, CD5, CD20, CD79 alpha cy, lambda, and kappa light chains) and by in situ hybridization for EBV encoded RNA (EBER) were undertaken. The xenografted animals from the CYP group had a mean survival of 31 days (range: 0 to 90 days); animals from the MTX group survived a median of 14 days (range: 0 to 39 days). GI complications were the most frequent cause of euthanasia after renal failure. In CYP-treated animals GI-tract lesions were primarily characterized by diffuse, severe lymphoplasmocytic mucosal inflammatory infiltrate. Variable degrees of villi atrophy and fusion, gut-associated lymphoid tissue (GALT) and goblet cell hyperplasia were also observed. In MTX-treated primates, findings were consistent with severe villi atrophy associated with mild-to-moderate disseminated lymphoplasmocytic infiltration. In conclusion, GI tract lesions are an early and consistent finding when CYP or MTX are used as induction agents in this model. The two compounds induce different types of GI tract damage, however, in agreement with their different mechanisms of action. Whilst CYP primarily determines inflammatory lesions, MTX leads to a degenerative type of damage. This study indicates that immunosuppressive drugs can cause severe GI tract damage in primate recipients of renal xenografts and may be responsible for life-threatening lesions.

Xenotransplantation, 2003

Methotrexate (MTX) has been used successfully as an immunosuppressant in rodent xenotransplantati... more Methotrexate (MTX) has been used successfully as an immunosuppressant in rodent xenotransplantation models, but the data generated so far with MTX in pig-to-baboon cardiac transplantation studies have been disappointing. The potential of this agent was consequently explored in a life-supporting pig-to-primate renal model using the cynomolgus monkey as the recipient species. Introductory in vitro and in vivo pharmacokinetic and pharmacodynamic studies with MTX were conducted in three cynomolgus monkeys. Subsequently, 10 cynomolgus monkey recipients of a life-supporting kidney from human decay-accelerating factor transgenic pigs were administered MTX intravenously according to three different regimens. All the animals also received cyclosporine A and steroids. In addition, mycophenolate sodium (MPS) was administered post-operatively in two of the three groups of transplanted animals. At clinically relevant concentrations, MTX is able in vitro to inhibit the mixed lymphocyte reactions (MLR) in cynomolgus monkeys. After intravenous administration, moreover, exposure of cynomolgus monkeys to MTX appeared to be higher than had been previously reported in baboons. Graft function was observed in the transplanted animals, which survived from 0 to 41 days. All but two animals revealed acute humoral rejection in the explanted graft and developed diarrhea. Diarrhea was the cause of euthanasia in five cases. It was unrelated to the administration of MPS and associated with severe histopathological signs of enteritis. This study demonstrates that the pharmacokinetic and pharmacodynamic profiles of MTX vary substantially between non-human primate species. In vitro, MTX has immunosuppressive properties in the cynomolgus monkey at clinically relevant concentrations. In vivo, MTX has a very narrow therapeutic window in cynomolgus monkeys, however, as it does in baboons. We conclude that MTX is scarcely effective as an immunosuppressant, be it for induction or maintenance, in pig-to-cynomolgus monkey renal xenotransplantation.

Xenotransplantation, 2008

Despite being still at the experimental level, xenotransplantation may become an effective strate... more Despite being still at the experimental level, xenotransplantation may become an effective strategy to overcome the scarcity of human organs. However, at the present time there is considerable resistance to this kind of biomedical technology. The aim of the present study was to identify novel strategies to reduce patients' negative affective reactions towards xenotransplantation helping them to understand the advantages of xenotransplantation in a more analytical fashion and increase their acceptance for this approach. The study was conducted in a group of patients with liver cirrhosis waiting for liver transplantation. They were presented with hypothetical scenarios and asked to choose among either two or three alternative types of donor defined by their species (e.g., livers from humans vs. other species) and availability (low for human donors and high for livers from non-human species). Patients were unwilling to accept xenotransplantation if they were presented with livers from humans (chosen by 97.5% of participants) vs. livers from genetically modified pigs (2.5%). On the other hand, a different group of patients was significantly more willing to accept xenotransplantation if they were presented with three different types of donors: respectively, human beings (74.4%), genetically modified pigs (25.6%) and genetically modified dogs. In addition, human livers were judged significantly more attractive than genetically modified livers from pigs, monkeys, dogs, or sheep and pig livers were rated as significantly more attractive than livers from monkeys, dogs, or sheep (for all comparisons P < 0.01). These results demonstrate that paradigms from other fields, like decision-making, might help to communicate more effectively the potential of xenotransplantation, modulating patients' affective reactions and allowing them to understand the potential strengths of this biomedical technology.

Xenotransplantation, 2009

Xenotransplantation, 2007

Xenotransplantation, 2013

Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs whe... more Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs when pig organs are transplanted into primates. The coagulopathy reported in this model is a very complex process that involves simultaneously coagulation factors, platelets and phospholipid-bearing cells (i.e., leukocytes, red blood cells, and endothelial cells). Choosing whole blood for coagulation analysis theoretically appears more favorable compared with plasma. Whole blood rotation thromboelastometry (ROTEM(®) ) is a point-of-care global coagulation analyzer able to evaluate the characteristics of clot formation and lysis by dynamic monitoring. The aim of this study was to record thromboelastographic profiles, performed by ROTEM(®) , in a series of immunosuppressed nephrectomized primates that received a life-supporting kidney. Of the eight primates, n = 4 received a pig kidney transgenic for human decay-accelerating factor (hDAF/Gal+); n = 2, an α 1,3-galactosyltransferase gene-knockout (GT-KO) pig kidney transgenic for human CD39, CD55, CD59 and fucosyltransferase (HTF); and n = 2, a GT-KO pig kidney transgenic for hDAF. Blood samples were collected before and at least once per week after transplantation till euthanasia. Intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM) were evaluated. Thromboelastographic parameters considered were clotting time (CT, seconds) and clot formation time (CFT, seconds) in INTEM and EXTEM and maximum clot firmness (MCF, mm) in FIBTEM. The correlations between CT in INTEM and activated partial thromboplastin time (aPTT), CT in EXTEM and PT, CFT in INTEM and EXTEM, and platelet counts and MCF in FIBTEM and fibrinogen plasma levels were also considered. In all animals, thromboelastographic profiles showed progressive prolongation of CT (activation of coagulative cascade) in INTEM. A close correspondence was observed between (i) the prolongation of the CFT values (propagation of clot formation), both in INTEM and EXTEM, and the decrease in platelet counts; (ii) the reduction in MCF values (clot firmness) ​​in FIBTEM and the decrease in fibrinogen plasma levels. No concordance between CT in INTEM and aPTT and between CT in EXTEM and PT was observed. Our study demonstrated that ROTEM(®) analyzer could be a useful and complementary tool to study the consumptive coagulopathy, either "compensated" or "non-compensated," that takes place when transgenic pig kidneys are transplanted into primates. Larger and prospective studies are needed to confirm our results and to evaluate the role of ROTEM(®) to guide the management of consumptive coagulopathy in order to prolong the survival of the transplanted organ.

Transplantation Proceedings, 2000

Transplantation, 2005

Fibrin deposition is central to the acute humoral rejection process occurring in the presence of ... more Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates. To assess whether strategies aimed at preventing fibrin formation may extend xenograft survival, we administered high daily doses of recombinant human antithrombin (rhAT) (500 U/kg twice daily) to obtain both anticoagulant and anti-inflammatory effects in immunosuppressed primate recipients of porcine kidneys. Some degree of consumptive coagulopathy developed in both rhAT-treated (n=3) and untreated (n=3) primates. No major differences in the coagulation parameters analyzed were observed between the 2 groups. Similarly, no difference in survival was seen between rhAT-treated (20.6+/-4 days; range: 15-23 days) and untreated animals (17.3+/-11.6 days; range: 7-30 days), although the rhAT-treated primates had a higher bleeding tendency. Despite the high daily dose of rhAT, considerable fibrin deposition was observed in the graft as early as 2 weeks after transplantation. These results suggest that a high daily dose of rhAT fails to influence survival or prevent fibrin formation and deposition in the graft in our pig-to-primate model. However, the potential role of rhAT administered in combination with heparins or other clotting inhibitor concentrates in this model remains to be determined.

Transplantation, 2000

It is not known whether the pig liver is capable of functioning efficiently when transplanted int... more It is not known whether the pig liver is capable of functioning efficiently when transplanted into a primate, neither is there experience in transplanting a liver from a transgenic pigs expressing the human complement regulator human complement regulator decay accelerating factor (h-DAF) into a baboon. The objective of this study was to determine whether the porcine liver would support the metabolic functions of non-human primates and to establish the effect of hDAF expression in the prevention of hyperacute rejection of porcine livers transplanted into primates. Five orthotopic liver xenotransplants from pig to baboon were carried out: three from unmodified pigs and two using livers from h-DAF transgenic pigs. The three control animals transplanted with livers from unmodified pigs survived for less than 12 hr. Baboons transplanted with livers from h-DAF transgenic pigs survived for 4 and 8 days. Hyperacute rejection was not detected in the baboons transplanted with hDAF transgenic pig livers; however, it was demonstrated in the three transplants from unmodified pigs. Baboons transplanted with livers from h-DAF transgenic pigs were extubated at postoperative day 1 and were awake and able to eat and drink. In the recipients of hDAF transgenic pig livers the clotting parameters reached nearly normal levels at day 2 after transplantation and remained normal up to the end of the experiments. In these hDAF liver recipients, porcine fibrinogen was first detected in the baboon plasma 2 hr postreperfusion, and was present up to the end of the experiments. One animal was euthanized at day 8 after development of sepsis and coagulopathy, the other animal arrested at day 4, after an episode of vomiting and aspiration. The postmortem examination of the hDAF transgenic liver xenografts did not demonstrate rejection. The livers from h-DAF transgenic pigs did not undergo hyperacute rejection after orthotopic xenotransplantation in baboons. When HAR is abrogated, the porcine liver maintains sufficient coagulation and protein levels in the baboon up to 8 days after OLT.