Liver transplantation in children: Experience with the development of an Australian pilot programme. The Queensland Liver Transplantation Programme

Children with end-stage liver disease now form a major sub-group of patients considered suitable for liver transplantation (ltp), and enjoy better survival statistics after transplantation than do adults. Since June 1984, a paediatric ltp programme has been developed in Brisbane with an initial working relationship and ongoing close links with two USA centres (Pittsburgh, and the UCLA Medical Center). Fourteen children with end-stage liver disease have been referred to the Queensland Liver Transplantation Programme for formal assessment. Following frank, informed discussion with their parents, 10 of these children were offered the option of ltp. During the transition stage, two infants with biliary atresia were referred to UCLA at their parents' request and, subsequently, eight children aged from 9 months to 6 years have been placed on a transplant candidacy list in Brisbane. A donor procurement team with access to a Queensland Government jet has been available to cover all mainland States except Western Australia. Six of the children have now had orthotopic ltp (two children at the UCLA Medical Center; four children at the Royal Children's Hospital, Brisbane). One UCLA patient died with a non-functioning graft, and one Brisbane patient died 5 weeks post-transplant with rejection, hepatic artery thrombosis and sepsis. The other four children are alive and well, three with normal liver function and one with unexplained intrahepatic cholestasis, during the 1-20 month follow-up to date. Three further children have died of their liver disease without a donor of an appropriate blood group and size being found, and one patient still awaits a suitable donor. The experience of these authors suggests that ltp is a major advance in the treatment of paediatric liver disease, and that the procedure can be carried out successfully in Australia with initial results comparable with leading overseas centres. The procedure requires the full array of services of a major paediatric tertiary care facility, an intensive team effort with awareness of the special needs of children, and a widespread procurement capability. A major problem for Australia is the procurement of sufficient numbers of optimal paediatric donor livers.

Biomaterial templates for the culture and transplantation of retinal pigment epithelial cells: A critical review

The idea of retinal cell transplantation as a potential treatment for age-related retinal degeneration, a leading cause of blindness in the Western world, has been around for a number of decades. To date, however, it has not been entirely successful; one of the main reasons for this is the lack of an ideal substratum for the retinal cells, specifically for the growth of retinal pigment epithelial cells prior to transplantation. This chapter reviews the reasoning behind this potential treatment, the development of animal transplantation models for human trials, the prerequisites of an ideal substratum, the past and current research on substratum materials, and the potential for future developments in this area.

Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice

Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.

Washout hole for bi-ventricular assist device (BVAD) centrifugal blood pump

The shortage of donor hearts for patients with end stage heart failure has accelerated the development of ventricular assist devices (VAD) that act as a replacement heart. Mechanical devices involving pulsatile, axial and centrifugal devices have been proposed. Recent clinical developments indicate that centrifugal devices are not only beneficial for bridge to transplantation applications, but may also aid myocardial recovery. The results of a recent study have shown that patients who received a VAD have extended lives and improved quality of life compared to recipients of drug therapy. Unfortunately 25% of these patients develop right heart failure syndrome, sepsis and multi-organ failure. It was reported that 17% of patients initially receiving an LVAD later required a right ventricular assist device (RVAD). Hence, current research focus is in the development of a bi-ventricular assist device (BVAD). Current BVAD technology is either too bulky or necessitates having to implant two pumps working independently. The latter requires two different controllers for each pump leading to the potential complication of uneven flow dynamics and the requirements for a large amount of body space. This paper illustrates the combination of the LVAD and RVAD as one complete device to augment the function of both the left and right cardiac chambers with double impellers. The proposed device has two impellers rotating in counter directions, hence eliminating the necessity of the body muscles and tubing/heart connection to restrain the pump. The device will also have two separate chambers with independent rotating impeller for the left and right chambers. A problem with centrifugal impellers is the fluid stagnation underneath the impeller. This leads to thrombosis and blood clots.This paper presents the design, construction and location of washout hole to prevent thrombus for a Bi-VAD centrifugal pump. Results using CFD will be used to illustrate the superiority of our design concept in terms of preventing thrombus formation and hemolysis.

The legality of tissue transplants for the benefit of family members in the UK and Australia : implications for saviour siblings

The ethics of creating ‘saviour siblings’ for the benefit of another has received much attention, but little consideration has been given to the legal position of those saviours born who may be asked to provide tissue for transplantation to another during childhood. This article examines the ethical issues surrounding minor donation as well as the existing legal framework in the UK and Australia that regulates minors providing tissue for the benefit of another. Against this background the position of minor saviours, who are called upon to donate bone marrow or peripheral blood stem cells, is examined. This analysis suggests that the law does not provide sufficient protection for minor saviours who are called upon to donate to another. It is argued that specific ethical obligations are owed to saviours—that ought to be reflected in the law—in order to protect them from exploitation while they remain minors.

Establishment of a preclinical ovine model for tibial segmental bone defect repair by applying bone tissue engineering strategies

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. The field of tissue engineering has emerged as an important approach to bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. The subsequent gap between research and clinical translation, hence commercialization, is referred to as the ‘Valley of Death’ and describes a large number of projects and/or ventures that are ceased due to a lack of funding during the transition from product/technology development to regulatory approval and subsequently commercialization. One of the greatest difficulties in bridging the Valley of Death is to develop good manufacturing processes (GMP) and scalable designs and to apply these in pre-clinical studies. In this article, we describe part of the rationale and road map of how our multidisciplinary research team has approached the first steps to translate orthopaedic bone engineering from bench to bedside byestablishing a pre-clinical ovine critical-sized tibial segmental bone defect model and discuss our preliminary data relating to this decisive step.

Simulation and development of a mock circulation loop with variable compliance

Heart disease is attributed as the highest cause of death in the world. Although this could be alleviated by heart transplantation, there is a chronic shortage of donor hearts and so mechanical solutions are being considered. Currently, many Ventricular Assist Devices (VADs) are being developed worldwide in an effort to increase life expectancy and quality of life for end stage heart failure patients. Current pre-clinical testing methods for VADs involve laboratory testing using Mock Circulation Loops (MCLs), and in vivo testing in animal models. The research and development of highly accurate MCLs is vital to the continuous improvement of VAD performance. The first objective of this study was to develop and validate a mathematical model of a MCL. This model could then be used in the design and construction of a variable compliance chamber to improve the performance of an existing MCL as well as form the basis for a new miniaturised MCL. An extensive review of literature was carried out on MCLs and mathematical modelling of their function. A mathematical model of a MCL was then created in the MATLAB/SIMULINK environment. This model included variable features such as resistance, fluid inertia and volumes (resulting from the pipe lengths and diameters); compliance of Windkessel chambers, atria and ventricles; density of both fluid and compressed air applied to the system; gravitational effects on vertical columns of fluid; and accurately modelled actuators controlling the ventricle contraction. This model was then validated using the physical properties and pressure and flow traces produced from a previously developed MCL. A variable compliance chamber was designed to reproduce parameters determined by the mathematical model. The function of the variability was achieved by controlling the transmural pressure across a diaphragm to alter the compliance of the system. An initial prototype was tested in a previously developed MCL, and a variable level of arterial compliance was successfully produced; however, the complete range of compliance values required for accurate physiological representation was not able to be produced with this initial design. The mathematical model was then used to design a smaller physical mock circulation loop, with the tubing sizes adjusted to produce accurate pressure and flow traces whilst having an appropriate frequency response characteristic. The development of the mathematical model greatly assisted the general design of an in vitro cardiovascular device test rig, while the variable compliance chamber allowed simple and real-time manipulation of MCL compliance to allow accurate transition between a variety of physiological conditions. The newly developed MCL produced an accurate design of a mechanical representation of the human circulatory system for in vitro cardiovascular device testing and education purposes. The continued improvement of VAD test rigs is essential if VAD design is to improve, and hence improve quality of life and life expectancy for heart failure patients.

Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

Low sodium haemodialysis reduces interdialytic fluid consumption but paradoxically increases post-dialysis thirst

Background Interdialytic weight gain (IDWG) can be reduced by lowering the dialysate sodium concentration ([Na]) in haemodialysis patients. It has been assumed that this is because thirst is reduced, although this has been difficult to prove. We compared thirst patterns in stable haemodialysis patients with high and low IDWG using a novel technique and compared the effect of low sodium dialysis (LSD) with normal sodium dialysis (NSD). Methods Eight patients with initial high IDWG and seven with low IDWG completed hourly visual analogue ratings of thirst using a modified palmtop computer during the dialysis day and the interdialytic day. The dialysate [Na] was progressively reduced by up to 5 mmol/l over five treatments. Dialysis continued at the lowest attained [Na] for 2 weeks and the measurements were repeated. The dialysate [Na] then returned to baseline and the process was repeated. Results Baseline interdialytic day mean thirst was higher than the dialysis day mean for the high IDWG group (49.9±14.0 vs 36.2±16.6) and higher than the low weight gain group (49.9±14.0 vs 34.1±14.6). This trend persisted on LSD, but there was a pronounced increase in post-dialysis thirst scores for both groups (high IDWG: 46±13 vs 30±21; low IDWG: 48±24 vs 33±18). The high IDWG group demonstrated lower IDWG during LSD than NSD (2.23±0.98 vs 2.86±0.38 kg; P<0.05). Conclusions Our results indicate that patients with high IDWG experience more intense feelings of thirst on the interdialytic day. LSD reduces their IDWG, but paradoxically increases thirst in the immediate post-dialysis period.

Polydipsia : a feature of peritoneal dialysis

Background Some dialysis patients fail to comply with their fluid restriction causing problems due to volume overload. These patients sometimes blame excessive thirst. There has been little work in this area and no work documenting polydipsia among peritoneal dialysis (PD) patients. Methods We measured motivation to drink and fluid consumption in 46 haemodialysis patients (HD), 39 PD patients and 42 healthy controls (HC) using a modified palmtop computer to collect visual analogue scores at hourly intervals. Results Mean thirst scores were markedly depressed on the dialysis day (day 1) for HD (P<0.0001). The profile for day 2 was similar to that of HC. PD generated consistently higher scores than HD day 1 and HC (P = 0.01 vs. HC and P<0.0001 vs HD day 1). Reported mean daily water consumption was similar for HD and PD with both significantly less than HC (P<0.001 for both). However, measured fluid losses were similar for PD and HC whilst HD were lower (P<0.001 for both) suggesting that the PD group may have underestimated their fluid intake. Conclusion Our results indicate that HD causes a protracted period of reduced thirst but that the population's thirst perception is similar to HC on the interdialytic day despite a reduced fluid intake. In contrast, the PD group recorded high thirst scores throughout the day and were apparently less compliant with their fluid restriction. This is potentially important because the volume status of PD patients influences their survival.

Cholecystokinin and leptin: their influence upon the eating behaviour and nutrient intake of dialysis patients

Background We have used serial visual analogue scores to demonstrate disturbances of the appetite profile in dialysis patients. This is potentially important as dialysis patients are prone to malnutrition yet have a lower nutrient intake than controls. Appetite disturbance may be influenced by accumulation of appetite inhibitors such as leptin and cholecystokinin (CCK) in dialysis patients. Methods Fasting blood samples were drawn from 43 controls, 50 haemodialysis (HD) and 39 peritoneal dialysis (PD) patients to measure leptin and CCK. Hunger and fullness scores were derived from profiles compiled using hourly visual analogue scores. Nutrient intake was derived from 3 day dietary records. Results Fasting CCK was elevated for PD (6.73 ± 4.42 ng/l vs control 4.99 ± 2.23 ng/l, P < 0.05; vs HD 4.43 ± 2.15 ng/l, P < 0.01). Fasting CCK correlated with the variability of the hunger (r = 0.426, P = 0.01) and fullness (r = 0.52, P = 0.002) scores for PD. There was a notable relationship with the increase in fullness after lunch for PD (r = 0.455, P = 0.006). When well nourished PD patients were compared with their malnourished counterparts, CCK was higher in the malnourished group (P = 0.004). Leptin levels were higher for the dialysis patients than controls (HD and PD, P < 0.001) with pronounced hyperleptinaemia evident in some PD patients. Control leptin levels demonstrated correlation with fullness scores (e.g. peak fullness, r = 0.45, P = 0.007) but the dialysis patients did not. PD nutrient intake (energy and protein intake, r = -0.56, P < 0.0001) demonstrated significant negative correlation with leptin. Conclusion Increased CCK levels appear to influence fullness and hunger perception in PD patients and thus may contribute to malnutrition. Leptin does not appear to affect perceived appetite in dialysis patients but it may influence nutrient intake in PD patients via central feeding centres.