The Resurgence of Xenotransplantation.

There has been an upsurge of interest in xenotransplantation in recent years. This resurgence can attributed to a combination of factors. First, there has been a dramatic improvement in efficacy in several preclinical models, with maximum xenograft survival times increasing to 950 days for islets, 945 days for hearts, and 310 days for kidneys. Second, the rapid development of genome editing technology (particularly the advent of clustered regularly interspaced short palindromic repeats/Cas9) has revolutionized the capacity to generate new donor pigs with multiple protective genetic modifications; what once took many years to achieve can now be performed in months, with much greater precision and scope. Third, the specter of porcine endogenous retrovirus (PERV) has receded significantly. There has been no evidence of PERV transmission in clinical trials and preclinical models, and improved screening methods and new options for the treatment or even elimination of PERV are now available. Balancing these positive developments are several remaining challenges, notably the heavy and often clinically inapplicable immunosuppression required to prevent xenograft rejection. Nonetheless, the potential for xenotransplantation as a solution to the shortage of human organs and tissues for transplantation continues to grow.

CD39 overexpression does not attenuate renal fibrosis in the unilateral ureteric obstructive model of chronic kidney disease.

Chronic kidney disease has multiple etiologies, but its single, hallmark lesion is renal fibrosis. CD39 is a key purinergic enzyme in the hydrolysis of ATP and increased CD39 activity on regulatory T cells (Treg) is protective in adriamycin-induced renal fibrosis. We examined the effect of overexpression of human CD39 on the development of renal fibrosis in the unilateral ureteric obstructive (UUO) model, a model widely used to study the molecular and cellular factors involved in renal fibrosis. Mice overexpressing human CD39 (CD39Tg) and their wild-type (WT) littermates were subjected to UUO; renal histology and messenger RNA (mRNA) levels of adenosine receptors and markers of renal fibrosis were examined up to 14 days after UUO. There were no differences between CD39Tg mice and WT mice in the development of renal fibrosis at days 3, 7, and 14 of UUO. Relative mRNA expression of the adenosine A2A receptor and endothelin-1 were higher in CD39Tg than WT mice at day 7 post UUO, but there were no differences in markers of fibrosis. We conclude that human CD39 overexpression does not attenuate the development of renal fibrosis in the UUO model. The lack of protection by CD39 overexpression in the UUO model is multifactorial due to the different effects of adenosinergic receptors on the development of renal fibrosis.

Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

Altered glycosylation in donor mice causes rejection of strain-matched skin and heart grafts.

Differential protein glycosylation in the donor and recipient can have profound consequences for transplanted organs, as evident in ABO-incompatible transplantation and xenotransplantation. In this study, we investigated the impact of altered fucosylation on graft acceptance by using donor mice overexpressing human α1,2-fucosyltransferase (HTF). Skin and heart grafts from HTF transgenic mice were rapidly rejected by otherwise completely matched recipients (median survival times 16 and 14 days, respectively). HTF skin transplanted onto mice lacking T and B cells induced an natural killer cell-mediated innate rejection crisis that affected 50-95% of the graft at 10-20 days. However, in the absence of adaptive immunity, the residual graft recovered and survived long-term (>100 days). Experiments using "parked" grafts or MHC class II-deficient recipients suggested that indirect rather than direct antigen presentation plays a role in HTF skin graft rejection, although the putative antigen(s) was not identified. We conclude that altered glycosylation patterns on donor tissue can trigger a powerful rejection response comprising both innate and adaptive components. This has potential implications for allotransplantation, in light of increasing recognition of the variability of the human glycome, and for xenotransplantation, where carbohydrate remodeling has been a lynchpin of donor genetic modification.

Control of IBMIR in neonatal porcine islet xenotransplantation in baboons.

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.

Pre-transplant predictors of one yr weight gain after kidney transplantation.

Clinically useful predictors of weight gain could be used to reduce the epidemic of post-kidney transplant obesity and resulting co-morbidities. The purpose of this study was to identify predictors of weight gain at 12 months following kidney transplant in a cohort of 96 recipients. Demographic, clinical, and environmental data were obtained at transplant and 12 months. Descriptive, correlational, and Bayesian network analysis were used to identify predictors. For the 52 (55.9%) recipients who gained weight, the average amount gained was 9.18 ± 6.59 kg. From the 15 baseline factors that met inclusion criteria, Bayesian network modeling identified four baseline predictors for weight gain: younger age, higher carbohydrate consumption, higher trunk fat percentage, and higher perception of mental health quality of life. Three are modifiable through either pre- or immediate post-transplant clinical intervention programs.

Protective effects of transgenic human endothelial protein C receptor expression in murine models of transplantation.

Thrombosis and inflammation are major obstacles to successful pig-to-human solid organ xenotransplantation. A potential solution is genetic modification of the donor pig to overexpress molecules such as the endothelial protein C receptor (EPCR), which has anticoagulant, anti-inflammatory and cytoprotective signaling properties. Transgenic mice expressing human EPCR (hEPCR) were generated and characterized to test this approach. hEPCR was expressed widely and its compatibility with the mouse protein C pathway was evident from the anticoagulant phenotype of the transgenic mice, which exhibited a prolonged tail bleeding time and resistance to collagen-induced thrombosis. hEPCR mice were protected in a model of warm renal ischemia reperfusion injury compared to wild type (WT) littermates (mean serum creatinine 39.0 ± 2.3 µmol/L vs. 78.5 ± 10.0 µmol/L, p < 0.05; mean injury score 31 ± 7% vs. 56 ± 5%, p < 0.05). Heterotopic cardiac xenografts from hEPCR mice showed a small but significant prolongation of survival in C6-deficient PVG rat recipients compared to WT grafts (median graft survival 6 vs. 5 days, p < 0.05), with less hemorrhage and edema in rejected transgenic grafts. These data indicate that it is possible to overexpress EPCR at a sufficient level to provide protection against transplant-related thrombotic and inflammatory injury, without detrimental effects in the donor animal.

Antioxidant network expression abrogates oxidative posttranslational modifications in mice.

Antioxidant enzymatic pathways form a critical network that detoxifies ROS in response to myocardial stress or injury. Genetic alteration of the expression levels of individual enzymes has yielded mixed results with regard to attenuating in vivo myocardial ischemia-reperfusion injury, an extreme oxidative stress. We hypothesized that overexpression of an antioxidant network (AON) composed of SOD1, SOD3, and glutathione peroxidase (GSHPx)-1 would reduce myocardial ischemia-reperfusion injury by limiting ROS-mediated lipid peroxidation and oxidative posttranslational modification (OPTM) of proteins. Both ex vivo and in vivo myocardial ischemia models were used to evaluate the effect of AON expression. After ischemia-reperfusion injury, infarct size was significantly reduced both ex vivo and in vivo, ROS formation, measured by dihydroethidium staining, was markedly decreased, ROS-mediated lipid peroxidation, measured by malondialdehyde production, was significantly limited, and OPTM of total myocardial proteins, including fatty acid-binding protein and sarco(endo)plasmic reticulum Ca(²+)-ATPase (SERCA)2a, was markedly reduced in AON mice, which overexpress SOD1, SOD3, and GSHPx-1, compared with wild-type mice. These data demonstrate that concomitant SOD1, SOD3, and GSHPX-1 expression confers marked protection against myocardial ischemia-reperfusion injury, reducing ROS, ROS-mediated lipid peroxidation, and OPTM of critical cardiac proteins, including cardiac fatty acid-binding protein and SERCA2a.

Transgenic overexpression of CD39 protects against renal ischemia-reperfusion and transplant vascular injury.

The vascular ectonucleotidases CD39[ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1), EC 3.6.1.5] and CD73[EC 3.1.3.5] generate adenosine from extracellular nucleotides. CD39 activity is critical in determining the response to ischemia-reperfusion injury (IRI), and CD39 null mice exhibit heightened sensitivity to renal IRI. Adenosine has multiple mechanisms of action in the vasculature including direct endothelial protection, antiinflammatory and antithrombotic effects and is protective in several models of IRI. Mice transgenic for human CD39 (hCD39) have increased capacity to generate adenosine. We therefore hypothesized that hCD39 transgenic mice would be protected from renal IRI. The overexpression of hCD39 conferred protection in a model of warm renal IRI, with reduced histological injury, less apoptosis and preserved serum creatinine and urea levels. Benefit was abrogated by pretreatment with an adenosine A2A receptor antagonist. Adoptive transfer experiments showed that expression of hCD39 on either the vasculature or circulating cells mitigated IRI. Furthermore, hCD39 transgenic kidneys transplanted into syngeneic recipients after prolonged cold storage performed significantly better and exhibited less histological injury than wild-type control grafts. Thus, systemic or local strategies to promote adenosine generation and signaling may have beneficial effects on warm and cold renal IRI, with implications for therapeutic application in clinical renal transplantation.

Anti-inflammatory and anticoagulant effects of transgenic expression of human thrombomodulin in mice.

Thrombomodulin (TBM) is an important vascular anticoagulant that has species specific effects. When expressed as a transgene in pigs, human (h)TBM might abrogate thrombotic manifestations of acute vascular rejection (AVR) that occur when GalT-KO and/or complement regulator transgenic pig organs are transplanted to primates. hTBM transgenic mice were generated and characterized to determine whether this approach might show benefit without the development of deleterious hemorrhagic phenotypes. hTBM mice are viable and are not subject to spontaneous hemorrhage, although they have a prolonged bleeding time. They are resistant to intravenous collagen-induced pulmonary thromboembolism, stasis-induced venous thrombosis and pulmonary embolism. Cardiac grafts from hTBM mice to rats treated with cyclosporine in a model of AVR have prolonged survival compared to controls. hTBM reduced the inflammatory reaction in the vein wall in the stasis-induced thrombosis and mouse-to-rat xenograft models and reduced HMGB1 levels in LPS-treated mice. These results indicate that transgenic expression of hTBM has anticoagulant and antiinflammatory effects that are graft-protective in murine models.

The effect of core stability training on balance and mobility in ambulant individuals with multiple sclerosis: a multi-centre series of single case studies.

BACKGROUND: Core stability training is popular in the management of people with multiple sclerosis (MS); however, scientific evidence to support its effectiveness is scarce. OBJECTIVE: To explore the effectiveness of core stability training on balance and mobility. METHOD: A multi-centre series of eight single case studies was undertaken. Eight ambulant individuals with stable MS participated in 16 face-to-face core stability training sessions, delivered by a neurophysiotherapist, plus a daily home exercise programme. A range of outcomes were measured: 10-m timed walk, 12-item MS walking scale, timed get up and go, functional reach tests, timed single leg stance, visual analogue scales of two activities, and the Activities-specific Balance Confidence Scale. RESULTS: Visual analysis of trend, level and slope demonstrated improvement in five subjects (62%) in seven measures. This was confirmed by the two standard deviation band method of analysis for six measures. Analysis of group data (repeated measures within subjects analysis of variance) indicated significant improvement between baseline and intervention phases for timed walk (p = 0.019), MSWS-12 Scale (p = 0.041), forward (p = 0.015) and lateral reach (p = 0.012). In general, no further improvements were made following withdrawal of the intervention. CONCLUSIONS: This study provides preliminary evidence of the effectiveness of an 8-week core stability training programme in improving balance and mobility in ambulant people with MS. Variations in response to intervention are evident. Assessor-blinded randomized controlled studies are required to confirm these findings and determine patient characteristics which identify those who benefit most from this intervention.

Pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein C and TAFI.

Incompatibility between pig thrombomodulin (TM) and primate thrombin is thought to be an important factor in the development of microvascular thrombosis in rejecting pig-to-primate xenografts. To examine this interaction at the molecular level, we cloned pig TM and measured its ability to bind human thrombin and act as a cofactor for the activation of human protein C and TAFI. The 579-residue pig TM protein showed approximately 69% sequence identity to human TM. Within the EGF domains necessary for binding of thrombin (EGF56), protein C (EGF4) and TAFI (EGF3), all of the amino acids previously identified as critical for the function of human TM, with the exception of Glu-408 in EGF5, were conserved in pig TM. Comparison of transfected cells expressing pig or human TM demonstrated that both proteins bound human thrombin and inhibited its procoagulant activity. However, pig TM was a poor cofactor for the activation of human protein C and TAFI, with domain swapping showing that EGF5 was the most important determinant of compatibility. Thus, while pig TM may be capable of binding thrombin generated in the vicinity of xenograft endothelium, its failure to promote the activation of human protein C remains a significant problem.

Postgraduate training options in dentistry--the RCSI roadmap.

This paper presents the RCSI roadmap for postgraduate training in dentistry. The importance of an approved training element is stressed in all aspects of this pathway to ensure that the trainee is provided with the best teaching and will emerge with the highest standards. It is critical in today's world that all healthcare professionals enter into lifelong learning in order to maintain the best level of care for our patients.

Infection of naïve, free-living brushtail possums (Trichosurus vulpecula) with the nematode parasite Parastrongyloides trichosuri and its subsequent spread.

Despite the importance of spatial processes in host-parasite interactions, parasite dispersal has been the subject of few experimental studies. Introduced marsupial common brushtail possums (Trichosurus vulpecula) are a major environmental and agricultural pest in New Zealand. Parastrongyloides trichosuri, an intestinal rhabdiasoid nematode parasite specific to possums, is being evaluated as a self-disseminating delivery system for engineered fertility control vaccines. This study addressed whether an artificial infection could be established in a naïve, free-living possum population, by measuring the post-release dynamics of possum-parasite interactions at the release site, and by following the spread of the parasite into surrounding possum populations. Infection was established efficiently by applying infective larvae to the skin of possums on a single occasion. All experimentally infected possums recaptured 3 weeks after infection had parasite eggs in their faeces. Over the subsequent 2.5 years, infection spread steadily over an area of about 6000 ha. Infection persisted at the original release site for the 3.5 years of the study and at a nearby site infected by natural spread for more than 3 years. Seasonal changes in faecal egg counts were similar at the two sites. The rapid establishment of the parasite and its spread provide additional support for its ongoing development as a vaccine delivery system.

Human Endothelial Protein C Receptor Overexpression Protects Intraportal Islet Grafts in Mice.

Islet transplantation can potentially cure type 1 diabetes mellitus, but it is limited by a shortage of human donors as well as by islet graft destruction by inflammatory and thrombotic mechanisms. A possible solution to these problems is to use genetically modified pig islets. Endothelial protein C receptor (EPCR) enhances protein C activation and regulates coagulation, inflammation, and apoptosis. We hypothesized that human EPCR (hEPCR) expression on donor islets would improve graft survival and function. Islets from an hEPCR transgenic mouse line strongly expressed the transgene, and hEPCR expression was maintained after islet isolation. Islets were transplanted from hEPCR mice and wild-type (WT) littermates into diabetic mice in a marginal-dose syngeneic intraportal islet transplantation model. The blood glucose level normalized within 5 days in 5 of 7 recipients of hEPCR islets, compared with only 2 of 7 recipients of WT islets (P < .05). Transplanted hEPCR islets had better preserved morphology and more intense insulin staining than WT grafts, and they retained transgene expression. The improved engraftment compared with WT islets suggests that inflammation and coagulation associated with the transplant process can be reduced by hEPCR expression on donor tissue.

Litigation.

The law in relation to clinical practice is specialised and complex. Most practitioners have a rudimentary understanding of the basic concepts. This chapter explores the principles of duty of care, breach and causation, and how-using case law-they are applied to neonatal care. It considers the roles and responsibilities of the practitioner and the organisation in providing standards of care that are supportable in the current clinical governance and medicolegal climate. The problems facing practitioners who find themselves involved in a case many years after the actual clinical events are also discussed, and some risk management suggestions are made. Detailed and good-quality published data on the extent and nature of neonatal cases is lacking in the UK but should be improved by the changes that have occurred following the establishment of the National Health Service Litigation Authority. Finally, consideration is given to the difficulties parents, practitioners and experts can face in dealing with information and expectations in adverse clinical outcomes when litigation is inevitable.

Anti-xenograft immune responses in alpha 1,3-galactosyltransferase knock-out mice.

Although originally generated to test the effect of eliminating the alpha-Gal epitope on HAR, it is becoming increasingly clear that GalT KO mice offer a convenient and inexpensive model to investigate many aspects of the anti-xenorgraft immune response. Clearly, not all aspects of anti-xenograft rejection responses are identical in mice and primates, which should be kept in mind when interpreting results of GalT KO mouse studies. However, with this and other mouse models it is possible to test a large number of variables, which is impractical for both logistical and financial reasons with primates. Furthermore the short gestation time and large litter size of mice means that genetic strategies targeting different aspects of the anti-xenograft immune response can be combined and subsequently tested to identify the optimal combination of genetic and therapeutic approaches to achieve long term xenograft survival. In this regard the GalT KO mouse has been and will continue to be a valuable small animal model for the study of all facets of xenograft rejection involving anti-Gal antibodies.

Prevention of hyperacute rejection in a model of orthotopic liver xenotransplantation from pig to baboon using polytransgenic pig livers (CD55, CD59, and H-transferase).

INTRODUCTION: The search for alternative sources for transplant organs leads us to the search for animals as an inexhaustible source of organs. The objective of this study was to analyze whether livers from polytransgenic pigs expressing the human complement regulatory proteins CD55 (hDAF), CD59, and alfa alpha1,2-fucosyltransferase (H-transferase), protected against hyperacute rejection after orthotopic liver xenotransplantation to a baboon and also to study pig liver function in a nonhuman primate. MATERIALS AND METHODS: Nine liver transplants from pig to baboon were divided into two groups: a control group (n = 4) of genetically unmodified pigs and an experimental group (n = 5) of pigs transgenic for CD55, CD59, and H-transferase as donors. All the donating piglets obtained through hysterectomy were maintained in specific pathogen-free conditions. The selection of transgenic pig donors followed demonstration of transgene expression using monoclonal antibodies (antiCD55, antiCD59) and immunohistological studies on liver biopsies. RESULTS: All animals in the control group developed hyperacute rejection with survival rates less than 16 hours without function of transplanted livers. In the experimental group none of the animals suffered hyperacute rejection. Survival in this group was between 13 and 24 hours. The livers were functional, producing bile and maintaining above 35% prothrombin activity. Only in one case was there primary dysfunction of the xenograft. CONCLUSION: Polytransgenic livers for complement regulatory proteins prevent hyperacute rejection when xenotransplanted into a baboon.

The launch of MCBEND 10.

MCBEND 10 is the latest release of the general radiation transport Monte Carlo code from the ANSWERS Software Service of Serco Assurance. MCBEND is developed within a Nuclear Code Development (NCD) partnership between Serco Assurance and BNFL. The ANSWERS vision is 'to provide easy-to-use software that meets the current and emerging needs of the user community'. In the case of MCBEND, this vision focuses on the key areas of accuracy, understanding of uncertainties, efficiency and user-friendliness. MCBEND 10 is a major launch of the code with many new and enhanced features. New developments in MCBEND 10 include automatic splitting mesh generation, point energy adjoint for neutrons, calculation of uncertainty in the results due to material cross section uncertainties and a unified source facility. Enhanced features include improved temperature treatment, extended scoring of sensitivity to geometry perturbations, geometry improvements, extensions to formulae and improved user guide image. The user-friendliness of the MCBEND code has been further enhanced by recent developments to the visualisation tools, VISAGE and VISTA-RAY. Developments have been made to the three-dimensional visualisation tool, VISTA-RAY, to simplify the detailed checking of a model, with the option to use a mouse-pointer to select regions of interest for further detail and to visually highlight incorrectly defined areas. A further development to VISTA-RAY is the inclusion of the capability to overlay a representation of a user-designated set of results from a MCBEND analysis on the model. Improvements have also been made to the graphical user interface LaunchPad for submitting and controlling calculation submission, with a common user-image across all the systems. Recent enhancements to LaunchPad include a job-scheduler to simplify processing multiple tasks. A selection of the new developments in MCBEND 10, VISTA-RAY and LaunchPad will be described in this paper.