Patient and parent perspectives of adolescent Laparoscopic Adjustable Gastric Banding (LAGB)

Introduction Adolescent obesity is a significant global health challenge and severely obese adolescents commonly experience serious medical and psychosocial challenges. Consequently, severe adolescent obesity is increasingly being treated surgically. The limited available research examining the effectiveness of adolescent bariatric surgery focuses primarily on bio-medical outcomes. There is a need for a more comprehensive understanding of the behavioural, emotional and social factors which affect adolescents’ and parents’ experience of weight-loss surgery. Materials/Methods Patient and parents’ perspectives of adolescent LAGB were examined using a qualitative research methodology. Individual, semi-structured interviews were conducted with eight adolescent patients and five parents. Thematic analysis was used to identify key themes in the qualitative data. Results Patients and parents generally considered adolescent LAGB to be a life-changing experience, resulting in physical and mental health benefits. Factors considered to facilitate weight-loss following surgery included parental support and adherence to treatment guidelines. Many adolescents reported experiencing surgical weight-loss stigma and challenging interpersonal outcomes after weight-loss for which they felt unprepared. Conclusion Patients and parents perceived LAGB positively. There are opportunities to improve both the experience and outcomes of adolescent LAGB through parental education and enhancements to surgical aftercare programs

Targeting gene expression to endothelium in transgenic animals: A comparison of the human ICAM-2, PECAM-1 and endoglin promoters

It is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs.This work was supported in part by grants from Ministerio de Ciencia y Tecnologia and Comunidad de Madrid to CB.Peer Reviewe

Transgenic perspectives in xenotransplantation, 2001

Mark B. Nottle; Anthony J. F. D'Apice; Peter J. Cowan; Andrew C. Boquest; Sharon J. Harrison; Christopher G. Grupe

Targeting gene expression to endothelium in transgenic animals: a comparison of the human ICAM-2, PECAM-1 and endoglin promoters

The definitive version is available from www.blackwell-synergy.comIt is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs.Peter J. Cowan, Trixie A. Shinkel, Nella Fisicaro, James W. Godwin, Carmelo Bernabéu, Nuria Almendro, Carlos Rius, Andrew J. Lonie, Mark B. Nottle, Peter L. Wigley, Kathy Paizis, Martin J. Pearse and Anthony J. F. d'Apic

Rhesus monkeys and baboons develop clotting factor VIII inhibitors in response to porcine endothelial cells or islets

BackgroundXenotransplantation of porcine organs holds promise of solving the human organ donor shortage. The use of α-1,3-galactosyltransferase knockout (GTKO) pig donors mitigates hyperacute rejection, while delayed rejection is currently precipitated by potent immune and hemostatic complications. Previous analysis by our laboratory suggests that clotting factor VIII (FVIII) inhibitors might be elicited by the structurally restricted xenoantibody response which occurs after transplantation of either pig GTKO/hCD55/hCD59/hHT transgenic neonatal islet cell clusters or GTKO endothelial cells.MethodsA recombinant xenoantibody was generated using sequences from baboons demonstrating an active xenoantibody response at day 28 after GTKO/hCD55/hCD59/hHT transgenic pig neonatal islet cell cluster transplantation. Rhesus monkeys were immunized with GTKO pig endothelial cells to stimulate an anti-non-Gal xenoantibody response. Serum was collected at days 0 and 7 after immunization. A two-stage chromogenic assay was used to measure FVIII cofactor activity and identify antibodies which inhibit FVIII function. Molecular modeling and molecular dynamics simulations were used to predict antibody structure and the residues which contribute to antibody-FVIII interactions. Competition ELISA was used to verify predictions at the domain structural level.ResultsAntibodies that inhibit recombinant human FVIII function are elicited after non-human primates are transplanted with either GTKO pig neonatal islet cell clusters or endothelial cells. There is an apparent increase in inhibitor titer by 15 Bethesda units (Bu) after transplant, where an increase greater than 5 Bu can indicate pathology in humans. Furthermore, competition ELISA verifies the computer modeled prediction that the recombinant xenoantibody, H66K12, binds the C1 domain of FVIII.ConclusionsThe development of FVIII inhibitors is a novel illustration of the potential impact the humoral immune response can have on coagulative dysfunction in xenotransplantation. However, the contribution of these antibodies to rejection pathology requires further evaluation because "normal" coagulation parameters after successful xenotransplantation are not fully understood

Sustained function of genetically modified porcine lungs in an ex vivo model of pulmonary xenotransplantation

BACKGROUND Xenotransplantation could provide a solution to the donor shortage that is currently the major barrier to solid-organ transplantation. The ability to breed pigs with multiple genetic modifications provides a unique opportunity to explore the immunologic challenges of pulmonary xenotransplantation. METHODS Explanted lungs from wild-type and 3 groups of genetically modified pigs were studied: (i) α1,3-galactosyltransferase gene knockout (GTKO); (ii) GTKO pigs expressing the human complementary regulatory proteins CD55 and CD59 (GTKO/CD55-59); and (iii) GTKO pigs expressing both CD55-59 and CD39 (GTKO/CD55-59/CD39). The physiologic, immunologic and histologic properties of porcine lungs were evaluated on an ex vivo rig after perfusion with human blood. Results Lungs from genetically modified pigs demonstrated stable pulmonary vascular resistance and better oxygenation of the perfusate, and survived longer than wild-type lungs. Physiologic function was inversely correlated with the degree of platelet sequestration into the xenograft. Despite superior physiologic profiles, lungs from genetically modified pigs still showed evidence of intravascular thrombosis and coagulopathy after perfusion with human blood. CONCLUSIONS The ability to breed pigs with multiple genetic modifications, and to evaluate lung physiology and histology in real-time on an ex vivo rig, represent significant advances toward better understanding the challenges inherent to pulmonary xenotransplantation.Glen P. Westall, Browyn J. Levvey, Evelyn Salvaris, Julian Gooi, Sylvana Marasco, Frank Rosenfeldt, Chris Egan, Robin McEgan, Mark Mennen, Prue Russell, Simon C. Robson, Mark B. Nottle, Karen M. Dwyer, Greg I. Snell, Peter J. Cowa

Life supporting pig-to-primate xenotransplantation model using transgenic GalT KO pigs: The Padua experience

Background: Survival of pig organs transplanted into primates is limited due to the early occurrence of acute humoral xenograft rejection (AHXR). Generation of a1,3-galactosyltransferase gene-knockout (GTKO) and/or human complement regulatory protein (hCRP) transgenic pigs avoided occurrence of hyperacute rejection in pig-to-primate xenotransplantation. The aim of this study was to investigate the added value deriving from further genetic modifications, including the addition of coagulation regulating molecules, in a life supporting pig to primate renal xenotransplantation model. Methods: Twelve bilaterally nephrectomized cynomolgus monkeys received a kidney from GTKO pigs. Animals in Group 1 (n = 4) received a kidney from GTKO, CD55 donor pigs. Animals in Group 2 (n = 6) were transplanted with a kidney from GTKO pigs transgenic for human CD39, CD55, CD59 and fucosyltransferase (TF) proteins. Animals in Group 3 (n = 2) received a kidney from GTKO pigs transgenic for human CD55, endothelial protein C receptor (EPCR) and thrombomodulin (TM). All recipients received cyclophosphamide (up to 4 doses perioperatively), cyclosporin A (35 mg/kg twice a day), mycophenolate sodium (40\u201360 mg/kg daily) and steroids. Complete hematological and biochemical analyses were routinely performed in all xenotransplanted animals. Pre- and post-transplantation sera were tested for binding of IgM and IgG to donor porcine aortic endothelial cells (PAEC) by flow cytometry. Tissue sections from all explanted kidneys were stained with hematoxylin eosin. The xenografts were also examined for the presence of IgM, IgG, C5b-9 and fibrin by immunohystochemistry. In addition, cellular infiltrates were characterized. Results: Median survival of the animals in the study was 8, 16 and 29 days for Group 1, 2 and 3, respectively. Except for one animal euthanized due to abdominal bleeding, all primates were euthanized in the presence of kidney failure. In all cases a humoral immune response against donor PAEC was observed. Indeed, anti PAEC antibodies IgM and IgG increased as early as on day 4 and 7 days, respectively. Antibodies persisted throughout the post-operative period. Higher antibody titers were observed in Group 2 animals. At euthanasia all xenografted kidneys presented grade II and III AHXR with considerable deposition of IgM, IgG, C5b9 and fibrin. Interestingly, failure in animals from Group 3 occurred in the presence of a more pronounced deposition of IgG. Conclusion: Preliminary data suggest that, in our model, the use of Gal deficient donor kidneys multitransgenic pig with endothelial specific human EPCR and TM proteins extend recipient's survival. This in vivo data confirms that control of coagulation derangements is beneficial in solid organ xenotransplantation