The effect on consent rates for deceased organ donation in Wales after the introduction of an opt-out system.

Organ transplantation saves and transforms lives. Failure to secure consent for organ retrieval is widely regarded as the single most important obstacle to transplantation. A soft opt-out system of consent for deceased organ donation was introduced into Wales in December 2015, whilst England maintained the existing opt-in system. Cumulative data on consent rates in Wales were compared with those in England, using a two-sided sequential procedure that was powered to detect an absolute difference in consent rates between England and Wales of 10%. Supplementary risk-adjusted logistic regression analysis examined whether any difference in consent rates between the two nations could be attributed to variations in factors known to influence UK consent rates. Between 1 January 2016 and 31 December 2018, 8192 families of eligible donors in England and 474 in Wales were approached regarding organ donation, with overall consent rates of 65% and 68%, respectively. There was a steady upward trend in the proportion of families consenting to donation after brain death in Wales as compared with England and after 33 months, this reached statistical significance. No evidence of any change in the donation after circulatory death consent rate was observed. Risk-adjusted logistic regression analysis revealed that by the end of the study period the probability of consent to organ donation in Wales was higher than in England (OR [95%CI] 2.1 [1.26-3.41]). The introduction of a soft opt-out system of consent in Wales significantly increased organ donation consent though the impact was not immediate.

Kidney and liver transplantation in the elderly.

BACKGROUND: Transplant surgery is facing a shortage of deceased donor organs. In response, the criteria for organ donation have been extended, and an increasing number of organs from older donors are being used. For recipients, the benefits of transplantation are great, and the growing ageing population has led to increasing numbers of elderly patients being accepted for transplantation. METHODS: The literature was reviewed to investigate the impact of age of donors and recipients in abdominal organ transplantation, and to highlight aspects of the fine balance in donor and recipient selection and screening, as well as allocation policies fair to young and old alike. RESULTS: Overall, kidney and liver transplantation from older deceased donors have good outcomes, but are not as good as those from younger donors. Careful donor selection based on risk indices, and potentially biomarkers, special allocation schemes to match elderly donors with elderly recipients, and vigorous recipient selection, allows good outcomes with increasing age of both donors and recipients. The results of live kidney donation have been excellent for donor and recipient, and there is a trend towards inclusion of older donors. Future strategies, including personalized immunosuppression for older recipients as well as machine preservation and reconditioning of donor organs, are promising ways to improve the outcome of transplantation between older donors and older recipients. CONCLUSION: Kidney and liver transplantation in the elderly is a clinical reality. Outcomes are good, but can be optimized by using strategies that modify donor risk factors and recipient co-morbidities, and personalized approaches to organ allocation and immunosuppression.

A self-assembling ß-peptide hydrogel for neural tissue engineering.

We report a new class of ß-peptide based hydrogel for neural tissue engineering. Our ß-peptide forms a network of nanofibres in aqueous solution, resulting in a stable hydrogel at physiological conditions. The hydrogel shows excellent compatibility with neural cells and provides a suitable environment for cells to adhere and proliferate.

In situ normothermic regional perfusion for controlled donation after circulatory death--the United Kingdom experience.

Organs recovered from donors after circulatory death (DCD) suffer warm ischemia before cold storage which may prejudice graft survival and result in a greater risk of complications after transplant. A period of normothermic regional perfusion (NRP) in the donor may reverse these effects and improve organ function. Twenty-one NRP retrievals from Maastricht category III DCD donors were performed at three UK centers. NRP was established postasystole via aortic and caval cannulation and maintained for 2 h. Blood gases and biochemistry were monitored to assess organ function. Sixty-three organs were recovered. Forty-nine patients were transplanted. The median time from asystole to NRP was 16 min (range 10-23 min). Thirty-two patients received a kidney transplant. The median cold ischemia time was 12 h 30 min (range 5 h 25 min-18 h 22 min). The median creatinine at 3 and 12 months was 107 µmol/L (range 72-222) and 121 µmol/L (range 63-157), respectively. Thirteen (40%) recipients had delayed graft function and four lost the grafts. Eleven patients received a liver transplant. The first week median peak ALT was 389 IU/L (range 58-3043). One patient had primary nonfunction. Two combined pancreas-kidney transplants, one islet transplant and three double lung transplants were performed with primary function. NRP in DCD donation facilitates organ recovery and may improve short-term outcomes.

In situ miRNA delivery from a hydrogel promotes osteogenesis of encapsulated mesenchymal stromal cells.

Hydrogels are attractive candidates for use in tissue-engineering and the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports demonstrate that mechanosensitive miRNAs, miR-100-5p and miR-143-3p can enhance MSC osteogenesis, using a complex multi-step procedure to transfect, encapsulate and differentiate the cells. In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we show that particle size, charge, and hydrogel mechanical properties all influence the diffusion of embedded transfection agent complexes. By incorporating both MSCs and transfection agents into the hydrogels we demonstrate successful in situ transfection of encapsulated MSCs. Comparing the efficacy of pre- and in situ transfection of miR-100-5p/miR-143-3p on the osteogenic capacity of hydrogel-encapsulated MSCs, our data demonstrates superior mineralisation and osteogenic gene expression following in situ transfections. Overall, we demonstrate a simple, one-pot system for in situ transfection of miRNAs to enhance MSC osteogenic potential and thus demonstrates significant promise to improve the efficiency of MSC differentiation in hydrogels for bone tissue-engineering applications. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are sensitive to cues from their surrounding microenvironment. Osteogenesis is enhanced in MSCs grown on stiffer substrates, but this is limited when using hydrogels for bone tissue-engineering. Modulating pro-osteogenic genes with mechanosensitive microRNAs (miRNAs) represents a potential tool to overcome this challenge. Here we report a hydrogel platform to deliver miRNAs to encapsulated MSCs. We characterise effects of hydrogel composition and transfection agent type on their mobility and transfection efficiency, demonstrating successful in situ transfection of MSCs and showing that miRNAs can significantly enhance osteogenic mineral deposition and marker gene expression. This system was simpler and more effective than conventional 2D transfection prior to encapsulation and therefore holds promise to improve MSC differentiation in bone tissue-engineering.

Review paper: a review of the cellular response on electrospun nanofibers for tissue engineering.

Electrospinning has been employed extensively in tissue engineering to generate nanofibrous scaffolds from either natural or synthetic biodegradable polymers to simulate the cellular microenvironment. Electrospinning rapidly produces fibers of the nanolength scale and the process offers many opportunities to tailor the physical, chemical, and biological properties of a material for specific applications and cellular environments. There is growing evidence that nanofibers amplify certain biological responses such as contact guidance and differentiation, however this has not been fully exploited in tissue engineering. This review addresses the cellular interactions with electrospun scaffolds, with particular focus on neural, bone, cartilage, and vascular tissue regeneration. Some aspects of scaffold design, including architectural properties, surface functionalization and materials selection are also addressed.

Polyethylene glycol-gelatin hydrogels with tuneable stiffness prepared by horseradish peroxidase-activated tetrazine-norbornene ligation.

Tetrazine-norbornene ligation has previously been applied in bioorthognal polymer crosslinking to form hydrogels suitable for 3D cell culture. However, the tetrazine group is prone to reduction by the free thiol in a biological environment, reducing the crosslinking efficiency and shortening the storage of tetrazine containing linkers. Here, we introduce a method to form a tetrazine group in situ by catalytic oxidation of the dihydrogen tetrazine using horse radish peroxidase (HRP). Enzymatic oxidation is highly efficient at a low HRP concentration and does not require hydrogen peroxide, allowing for rapid gelation when HRP was added to an aqueous solution of 4-arm PEG dihydrogentetrazine and gelatin norbornene. The storage modulus of the resultant gels can be varied by changing the concentration of the crosslinker, which is in the range of 1.2-3.8 kPa. Human mesenchymal stem cells encapsulated within these gels, with varying stiffness, display varied interactions and morphologies and can be maintained with prolonged culture periods of at least 32 days of 3D culture. The enzymatic activation of tetrazine-norbornene is therefore an attractive addition to the tetrazine ligation that is highly suitable for cell related studies in tissue engineering.

Polylysine-functionalised thermoresponsive chitosan hydrogel for neural tissue engineering.

Foetal mouse cortical cells were cultured on 2D films and within 3D thermally responsive chitosan/glycerophosphate salt (GP) hydrogels. The biocompatibility of chitosan/GP 2D films was assessed in terms of cell number and neurites per cell. Osmolarity of the hydrogel was a critical factor in promoting cell survival with isotonic GP concentrations providing optimal conditions. To improve cell adhesion and neurite outgrowth, poly-D-lysine (PDL) was immobilised onto chitosan via azidoaniline photocoupling. Increase in PDL concentrations did not alter cell survival in 2D cultures but neurite outgrowth was significantly inhibited. Neurons exhibited a star-like morphology typical of 2D culture systems. The effects of PDL attachment on cell number, cell morphology and neurite outgrowth were more distinct in 3D culture conditions. Neurones exhibited larger cell bodies and sent out single neurites within the macroporous gel. Immobilised PDL improved cell survival up to an optimum concentration of 0.1%, however, further increases resulted in drops in cell number and neurite outgrowth. This was attributed to a higher cell interaction with PDL within a 3D hydrogel compared to the corresponding 2D surface. The results show that thermally responsive chitosan/GP hydrogels provide a suitable 3D scaffolding environment for neural tissue engineering.

Interaction of embryonic cortical neurons on nanofibrous scaffolds for neural tissue engineering.

The interaction of murine embryonic cortical neurons on randomly orientated electrospun scaffolds of poly(L-lactide) (P(L)LA) and poly(lactide-co-glycolide) (PLGA) is investigated in this study. The scaffolds were surface treated with different concentrations of KOH to partially hydrolyze the surface and therefore change the surface tension. Hydrophilicity did not significantly influence the number of primary and secondary branches; however, it had a considerable effect on neurite extension. For scaffolds with surface tensions of 40-47 dyn cm(-1) there was a significantly greater overall neurite length for both the primary and secondary branches compared with more hydrophilic scaffolds. Another major finding of this work was that the interfibre distance influenced how the neurites extended. When the interfibre distance was greater than approximately 15 microm the neurites followed the fibres and avoided regions of very high fibre density. At interfibre distances less than approximately 15 microm, the neurites traversed between the fibres. Therefore, this study provided little evidence that contact guidance was the dominating cue in directing neurite extension, instead inferring that chemical cues, possibly from adjacent neurons had induced directional change.

Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.

Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells.

Uncovering a rare but critical complication following thyroid surgery: an audit across the UK and Ireland.

OBJECTIVE: Serious wound infection after thyroidectomy is uncommon, but actual incidence is not well documented in the literature. In the past a patient in our unit died secondary to fulminant streptococcal sepsis after thyroidectomy for benign disease. This prompted us to audit experience of serious wound infection among British Association of Endocrine Surgery (BAES) members. DESIGN: A questionnaire was posted to BAES members inquiring about experience of major wound infection following cervicotomy, incidence of minor wound infection, and prophylactic and therapeutic antibiotic usage. MAIN OUTCOME: Eight respondents experienced a case of fulminant wound infection after cervicotomy (8% total respondents). Five patients died and, in 6 patients, cases of streptococci were cultured. Then, 9% of respondents used prophylactic antibiotics routinely, 16% sometimes and 75% never. The most commonly used antibiotic was augmentin, and the most common reasons for use among those with a selective policy were re-operative cases (38%) and immunocompromised patients (38%). Also, 40% of respondents experienced major wound infection requiring intravenous antibiotics or surgical drainage. The most common choices of antibiotic used before sensitivities were obtained were augmentin (43%) and flucloxacillin (35%). CONCLUSIONS: Although rare, fulminant streptococcal wound infection after cervicotomy does occasionally occur and carries a high mortality.

Collision cross section predictions using 2-dimensional molecular descriptors.

Traditional methods for deriving computationally-generated collision cross sections for comparisons with ion mobility-mass spectrometry data require 3-dimensional energy-minimized structures and are often time consuming, preventing high throughput implementation. Here, we introduce a method to predict ion mobility collision cross sections of lipids and peptide analogs important in prebiotic chemistry and other fields. Using less than 100 2-D molecular descriptors this approach resulted in prediction errors of less than 2%.

Morphology and gelation of thermosensitive xyloglucan hydrogels.

Galactose modified xyloglucan is a thermally reversible hydrogel that is increasingly used in the biomedical field due to the ease of altering the gelation time and temperature by modifying the galactose removal ratio. However there is little information concerning the morphology and rheological properties of the hydrogel under physiological conditions. Differential scanning microcalorimetry (DSmicroC) showed the thermal gelation process to occur over a broad temperature range (5-50 degrees C). The rheological properties of the hydrogels were investigated as a function of concentration, temperature and ionic strength. The final elastic moduli of the hydrogels increased with increases in concentration. Isothermal rheology suggests that the gelation occurred in two distinct stages, which was influenced by the solution media. Scanning electron microscopy (SEM) was used to characterize the morphology of the xyloglucan which were thermally gelled at 37 degrees C. The resultant morphology was strongly dependent on the concentration of the hydrogel. Strong hydrogels were only obtained at 3 wt.% at 37 degrees C, and the morphology characterized by an open 3-dimensional network, comprised of thin membranes. It is proposed that the first stage of the isothermal gelation is the formation and growth of the thin membranes, followed by the formation of a three dimensional network.

The effect of surface hydrophilicity on the behavior of embryonic cortical neurons.

The aim of this study was to investigate the interaction of mouse embryonic cortical neurons on P(L)LA and PLGA substrates, which were partially hydrolysed using potassium hydroxide (KOH). The chemical and topographical properties of the surfaces were characterized, and it was discovered that there was a decrease in the hydrophilicity for the P(L)LA with increasing concentration of KOH. This was due to chemical modifications to the surfaces of the substrates. Alternatively for the PLGA substrate, only the 0.1 M KOH treated sample had a significantly different hydrophilicity highlighting that surface erosion resulted at higher concentrations. The morphology of the neurons grown on the two substrates were compared to poly(D)lysine (positive control). The neurons formed colonies on all of the substrates, but were dramatically reduced in size in the case of the 0.1 M KOH treated substrates. This finding was attributed to the increases in cell spreading and the size of the cells, as they were larger, more elongated and bipolar like those on the positive control. However, there was a significant decrease in the total number of live cells per unit area. Therefore, on these materials when there was increased cellular spreading there was significantly higher cell death. Furthermore, unlike the 0, 0.2, and 0.4 M KOH treated substrates, there was an absence of large bundles of axons that extended between colonies on the 0.1 M sample, instead exhibiting short axons that grew in free space.

Morphology and gelation of thermosensitive chitosan hydrogels.

The morphology of physical hydrogels is often difficult to examine due to the delicate nature of the system and therefore has not been studied in detail. Chitosan/GP (glycerophosphate salt) is a significant hydrogel in the biomedical and cosmetic fields as it is thermosensitive and contains less than 5% polysaccharide. The morphology of this system was examined with laser scanning confocal microscopy (LSCM) to image the gel morphology. The images indicate that the gel is quite heterogeneous, and power spectra reveal a fractal-like morphology. A study of composition found that increasing chitosan concentration increased the amount of polymer-rich phase present in the gel, and that the smallest aggregates decreased in size.

Prednimustine in refractory non-Hodgkin's lymphoma: a phase II study of the Northern California Oncology Group.

Fifty-six patients with advanced non-Hodgkin's lymphoma (NHL) were entered into a phase II study of prednimustine, an ester of chlorambucil and prednisolone. All patients were refractory to extensive prior combination chemotherapy. Therapy with prednimustine, 100 mg/m2/day orally, was given for three consecutive days every 2 weeks. The overall response rate in 43 evaluable patients was 30% (13/43), with 9% (4/43) achieving complete response (CR) and 21% (9/43) achieving partial response (PR). In the favorable histology subgroup (23 patients), the response rate was 39% (9/23), with 4% (1/23) achieving CR and 35% (8/23) achieving PR. In the unfavorable histology subgroup (20 patients), responses were seen in 20% (4/20) with 15% (3/20) achieving CR, all in heavily pretreated diffuse histiocytic lymphoma. Toxicity of this regimen was mild, with leukopenia below 3,000/mm3 in 22% and thrombocytopenia below 90,000/mm3 in 16% of patients. A positive correlation was observed between response and hematologic toxicity, indicating the potential for a dose-escalation schedule in future trials. These data confirm activity of prednimustine in NHL refractory to standard treatment. In view of its relatively mild toxicity, we conclude that prednimustine is an appropriate agent to test in combination chemotherapy regimens in this group of lymphomas.

Graft function and other risk factors as predictors of cardiovascular disease outcome.

The high incidence of cardiovascular disease after renal transplantation is related to a high prevalence and accumulation of risk factors before and after transplantation. Hypertension, posttransplantation diabetes, and hyperlipidemia are well-recognized risk factors for the development of cardiovascular events after renal transplantation and are strongly associated with immunosuppressive therapy. Hyperhomocysteinemia is a potential risk factor for cardiovascular disease in renal transplant recipients, but although a growing matter of study, a direct association with immunosuppressive agents is not yet proven. In addition to treatment intervention, risk management should also involve tailoring the immunosuppressive regimen to minimize the more indirect cardiovascular risk factors such as renal dysfunction and acute rejection.

Renal allograft rejection. Tubular epithelial cells present alloantigen in the presence of costimulatory CD28 antibody.

Renal tubular epithelial cells can be induced to express potentially immunogenic levels of class II MHC antigens but fail to stimulate the activation of allospecific T lymphocytes. The current series of experiments was performed to determine whether the failure of lymphocyte activation in this system is caused by defective T cell costimulation. It was found that cultured renal epithelial cells expressed class II MHC antigens and the immunoregulatory adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and lymphocyte function-associated antigen-3 after stimulation with IFN-gamma, but that the B7 ligand for CD28 was not expressed. Mixed cell culture experiments were set up in which lymphocytes were mixed with IFN-gamma-treated allogeneic renal cells. Lymphoproliferation and IL-2 production were only observed if bivalent anti-CD28 antibodies were titrated into these cultures. The requirement for antigen stimulation was retained by these lymphocytes, as no proliferation was observed after stimulation by class II MHC antigen nonexpressing, resting renal cells. Further experiments demonstrated that the effectiveness of the anti-CD28 antibody-mediated signal was enhanced by cross-linking with a secondary anti-kappa-chain antibody. These data are consistent with the concept that a costimulatory signal generated by ligation of CD28 is of central importance to the development of an immune response to alloantigen. Furthermore, these results indicate that tubular epithelial cells within a rejecting renal allograft are unlikely to initiate direct activation of infiltrating allospecific lymphocytes.

The value of posttransplant monitoring of interleukin (IL)-2, IL-3, IL-4, IL-6, IL-8, and soluble CD23 in the plasma of renal allograft recipients.

Over the past few years, the central role of cytokines in the amplification of the immune response has been reported and several studies have examined the relationship between the plasma level of individual lymphokines during renal allograft rejection. The aim of the present investigation was to study simultaneously IL-2, IL-3, IL-4, IL-6, IL-8, and soluble CD23. Analysis of results has allowed both the prognostic value and any possible interrelationships between the measured cytokines to be determined. We studied 16 renal transplant recipients for the first 14 days after transplantation. Seven patients showed clinical evidence of acute allograft rejection and 5 showed excellent stable graft function with no signs of rejection. Primary nonfunction was seen in 4 patients. The plasma levels of each cytokine were measured by commercially available ELISA and immunoradiometric assay kits. As reported in previous studies, plasma IL-2 levels, whenever found at detectable levels, were predictive of impending graft rejection. Serial monitoring of IL-4 and IL-6 was more reliable for the differential diagnosis of rejection, particularly toward the end of the first week after transplantation. IL-3, IL-8, and soluble CD23 were not diagnostic or predictive of rejection, due to the occurrence of significantly high levels in transplant patients who showed no evidence of clinical rejection. While the value of cytokine monitoring has been shown in this study, it should be remembered that infection, although not seen in these studies, may have a profound affect on the results obtained.