Preliminary Single-Center Canadian Experience of Human Normothermic Ex Vivo Liver Perfusion: Results of a Clinical Trial.

After extensive experimentation, outcomes of a first clinical normothermic machine perfusion (NMP) liver trial in the United Kingdom demonstrated feasibility and clear safety, with improved liver function compared with standard static cold storage (SCS). We present a preliminary single-center North American experience using identical NMP technology. Ten donor liver grafts were procured, four (40%) from donation after circulatory death (DCD), of which nine were transplanted. One liver did not proceed because of a technical failure with portal cannulation and was discarded. Transplanted NMP grafts were matched 1:3 with transplanted SCS livers. Median NMP was 11.5 h (range 3.3-22.5 h) with one DCD liver perfused for 22.5 h. All transplanted livers functioned, and serum transaminases, bilirubin, international normalized ratio, and lactate levels corrected in NMP recipients similarly to controls. Graft survival at 30 days (primary outcome) was not statistically different between groups on an intent-to-treat basis (p = 0.25). Intensive care and hospital stays were significantly more prolonged in the NMP group. This preliminary experience demonstrates feasibility as well as potential technical risks of NMP in a North American setting and highlights a need for larger, randomized studies.

The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions.

The 13th Banff Conference on Allograft Pathology was held in Vancouver, British Columbia, Canada from October 5 to 10, 2015. The cardiac session was devoted to current diagnostic issues in heart transplantation with a focus on antibody-mediated rejection (AMR) and small vessel arteriopathy. Specific topics included the strengths and limitations of the current rejection grading system, the central role of microvascular injury in AMR and approaches to semiquantitative assessment of histopathologic and immunophenotypic indicators, the role of AMR in the development of cardiac allograft vasculopathy, the important role of serologic antibody detection in the management of transplant recipients, and the potential application of new molecular approaches to the elucidation of the pathophysiology of AMR and potential for improving the current diagnostic system. Herein we summarize the key points from the presentations, the comprehensive, open and wide-ranging multidisciplinary discussion that was generated, and considerations for future endeavors.

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells.

Regulatory T cell (Treg)-based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3(+) Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood.

ABH-Glycan Microarray Characterizes ABO Subtype Antibodies: Fine Specificity of Immune Tolerance After ABO-Incompatible Transplantation.

Organ transplantation from ABO blood group-incompatible (ABOi) donors requires accurate detection, effective removal and subsequent surveillance of antidonor antibodies. Because ABH antigen subtypes are expressed differently in various cells and organs, measurement of antibodies specific for the antigen subtypes in the graft is essential. Erythrocyte agglutination, the century-old assay used clinically, does not discriminate subtype-specific ABO antibodies and provides limited information on antibody isotypes. We designed and created an ABO-glycan microarray and demonstrated the precise assessment of both the presence and, importantly, the absence of donor-specific antibodies in an international study of pediatric heart transplant patients. Specific IgM, IgG, and IgA isotype antibodies to nonself ABH subtypes were detected in control participants and recipients of ABO-compatible transplants. Conversely, in children who received ABOi transplants, antibodies specific for A subtype II and/or B subtype II antigens-the only ABH antigen subtypes expressed in heart tissue-were absent, demonstrating the fine specificity of B cell tolerance to donor/graft blood group antigens. In contrast to the hemagglutination assay, the ABO-glycan microarray allows detailed characterization of donor-specific antibodies necessary for effective transplant management, representing a major step forward in precise ABO antibody detection.

Imaging Tolerance Induction in the Classic Medawar Neonatal Mouse Model: Active Roles of Multiple F1-Donor Cell Types.

The immature immune system is uniquely susceptible to tolerance induction and thus an attractive target for immunomodulation strategies for organ transplantation. Newborn mice injected with adult semi-allogeneic lymphohematopoietic cells accept transplants without immunosuppressive drugs. Early in vivo/in situ events leading to neonatal tolerance remain poorly understood. Here, we show by whole body/organ imaging that injected cells home to lymphoid organs and liver where various F1-donor cell types selectively alter neonatal immunity. In host thymus, F1-donor dendritic cells (DC) interact with developing thymocytes and regulatory T cells suggesting a role in negative selection. In spleen and lymph nodes, F1-donor regulatory T/B cells associate with host alloreactive cells and by themselves prolong cardiac allograft survival. In liver, F1-donor cells give rise to albumin-containing hepatocyte-like cells. The neonatal immune system is lymphopenic, Th-2 immunodeviated and contains immature DC, suggesting susceptibility to regulation by adult F1-donor cells. CD8a T cell inactivation greatly enhances chimerism, suggesting that variable emerging neonatal alloreactivity becomes a barrier to tolerance induction. This comprehensive qualitative imaging study systematically shows contribution of multiple in vivo processes leading simultaneously to robust tolerance. These insights into robust tolerance induction have important implications for development of strategies for clinical application.

Chemical Basis for Qualitative and Quantitative Differences Between ABO Blood Groups and Subgroups: Implications for Organ Transplantation.

Blood group ABH(O) carbohydrate antigens are carried by precursor structures denoted type I-IV chains, creating unique antigen epitopes that may differ in expression between circulating erythrocytes and vascular endothelial cells. Characterization of such differences is invaluable in many clinical settings including transplantation. Monoclonal antibodies were generated and epitope specificities were characterized against chemically synthesized type I-IV ABH and related glycans. Antigen expression was detected on endomyocardial biopsies (n = 50) and spleen (n = 11) by immunohistochemical staining and on erythrocytes by flow cytometry. On vascular endothelial cells of heart and spleen, only type II-based ABH antigens were expressed; type III/IV structures were not detected. Type II-based ABH were expressed on erythrocytes of all blood groups. Group A1 and A2 erythrocytes additionally expressed type III/IV precursors, whereas group B and O erythrocytes did not. Intensity of A/B antigen expression differed among group A1 , A2 , A1 B, A2 B and B erythrocytes. On group A2 erythrocytes, type III H structures were largely un-glycosylated with the terminal "A" sugar α-GalNAc. Together, these studies define qualitative and quantitative differences in ABH antigen expression between erythrocytes and vascular tissues. These expression profiles have important implications that must be considered in clinical settings of ABO-incompatible transplantation when interpreting anti-ABO antibodies measured by hemagglutination assays with reagent erythrocytes.

Accuracy of influenza vaccination rate estimates in United States nursing home residents.

The US Center for Medicare and Medicaid Services (CMS) requires nursing homes and long-term-care facilities to document residents' vaccination status on the Resident Assessment Instrument (RAI). Vaccinating residents can prevent costly hospital admissions and deaths. CMS and public health officials use RAI data to measure vaccination rates in long-term-care residents and assess the quality of care in nursing homes. We assessed the accuracy of RAI data against medical records in 39 nursing homes in Florida, Georgia, and Wisconsin. We randomly sampled residents in each home during the 2010-2011 and 2011-2012 influenza seasons. We collected data on receipt of influenza vaccination from charts and RAI data. Our final sample included 840 medical charts with matched RAI records. The agreement rate was 0·86. Using the chart as a gold standard, the sensitivity of the RAI with respect to influenza vaccination was 85% and the specificity was 77%. Agreement rates varied within facilities from 55% to 100%. Monitoring vaccination rates in the population is important for gauging the impact of programmes and policies to promote adherence to vaccination recommendations. Use of data from RAIs is a reasonable approach for gauging influenza vaccination rates in nursing-home residents.

Developing a More Tailored Approach to Patient and Public Involvement with Children and Families in Pediatric Clinical Research: Lessons Learned.

Listening to, and acting on, the voices of children and families during clinical research and innovation is fundamental to ensuring enhanced pediatric health care, medicines development, and technological advances. While this is often discussed as an important step in ensuring patient-centered care, involving children and families across the life cycle of clinical research is not currently routine. The pediatric research community needs to address how to meaningfully involve children and families if they are to succeed in designing clinical research that suits the needs of pediatric patients and their families. This paper describes how an international community working under the umbrella International Children's Advisory Network (iCAN) and European Young Person's Advisory Group Network (eYPAGnet) has involved children and families in the design and delivery of pediatric clinical research. It offers practical solutions through various case studies assessed against seven patient engagement quality criteria within the Patient Engagement Quality Guidance (PEQG) tool, highlighting some of the lessons learnt from involving and engaging with children and families across different stages of clinical research, including pediatric trials for drug development programs.

Influence of aeration implements, phosphorus fertilizers, and soil taxa on phosphorus losses from grasslands.

Attenuation of rainfall within the solum may help to move contaminants and nutrients into the soil to be better sequestered or utilized by crops. Surface application of phosphorus (P) amendments to grasslands may lead to elevated concentrations of P in surface runoff and eutrophication of surface waters. Aeration of grasslands has been proposed as a treatment to reduce losses of applied P. Here, results from two small-plot aeration studies and two field-scale, paired-watershed studies are supplemented with previously unpublished soil P data and synthesized. The overall objective of these studies was to determine the impact of aeration on soil P, runoff volume, and runoff P losses from mixed tall fescue [Lolium arundinaceum (Schreb.) Darbysh.]-bermudagrass (Cynodon dactylon L.) grasslands fertilized with P. Small-scale rainfall simulations were conducted on two soil taxa using three types of aeration implements: spikes, disks, and cores. The-field scale study was conducted on four soil taxa with slit and knife aeration. Small-plot studies showed that core aeration reduced loads of total P and dissolved reactive P (DRP) in runoff from plots fertilized with broiler litter and that aeration was effective in reducing P export when it increased soil P in the upper 5 cm. In the field-scale study, slit aeration reduced DRP losses by 35% in fields with well-drained soils but not in poorly drained soils. Flow-weighted concentrations of DRP in aerated fields were related to water-soluble P applied in amendments and soil test P in the upper 5 cm. These studies show that the overall effectiveness of mechanical soil aeration on runoff volume and P losses is controlled by the interaction of soil characteristics such as internal drainage and compaction, soil P, type of surface-applied manure, and type of aeration implement.

Equivalent outcomes for pediatric heart transplantation recipients: ABO-blood group incompatible versus ABO-compatible.

ABO-blood group incompatible infant heart transplantation has had excellent short-term outcomes. Uncertainties about long-term outcomes have been a barrier to the adoption of this strategy worldwide. We report a nonrandomized comparison of clinical outcomes over 10 years of the largest cohort of ABO-incompatible recipients. ABO-incompatible (n = 35) and ABO-compatible (n = 45) infant heart transplantation recipients (< or =14 months old, 1996-2006) showed no important differences in pretransplantation characteristics. There was no difference in incidence of and time to moderate acute cellular rejection. Despite either the presence (seven patients) or development (eight patients) of donor-specific antibodies against blood group antigens, in only two ABO-incompatible patients were these antibodies implicated in antibody-mediated rejection (which occurred early posttransplantation, was easily managed and did not recur in follow-up). Occurrence of graft vasculopathy (11%), malignancy (11%) and freedom from severe renal dysfunction were identical in both groups. Survival was identical (74% at 7 years posttransplantation). ABO-blood group incompatible heart transplantation has excellent outcomes that are indistinguishable from those of the ABO-compatible population and there is no clinical justification for withholding this lifesaving strategy from all infants listed for heart transplantation. Further studies into observed differing responses in the development of donor-specific isohemagglutinins and the implications for graft accommodation are warranted.

Absence of donor-specific anti-HLA antibodies after ABO-incompatible heart transplantation in infancy: altered immunity or age?

Specific B-cell tolerance toward donor blood group antigens develops in infants after ABO-incompatible heart transplantation, whereas their immune response toward protein antigens such as HLA has not been investigated. We assessed de novo HLA-antibodies in 122 patients after pediatric thoracic transplantation (28 ABO-incompatible) and 36 controls. Median age at transplantation was 1.7 years (1 day to 17.8 year) and samples were collected at median 3.48 years after transplantation. Antibodies were detected against HLA-class I in 21 patients (17.2%), class II in 18 (14.8%) and against both classes in 10 (8.2%). Using single-antigen beads, donor-specific antibodies (DSAs) were identified in six patients (all class II, one additional class I). Patients with DSAs were significantly older at time of transplantation. In patients who had undergone pretransplant cardiac surgeries, class II antibodies were more frequent, although use of homografts or mechanical heart support had no influence. DSAs were absent in ABO-incompatible recipients and class II antibodies were significantly less frequent than in children with ABO-compatible transplants. This difference was present also when comparing only children transplanted below 2 years of age. Therefore, tolerance toward the donor blood group appears to be associated with an altered response to HLA beyond age-related effects.

HNF4 and HNF1 as well as a panel of hepatic functions are extinguished and reexpressed in parallel in chromosomally reduced rat hepatoma-human fibroblast hybrids.

Rat hepatoma-human fibroblast hybrids of two independent lineages containing only 8-11 human chromosomes show pleiotropic extinction of thirteen out of fifteen hepatic functions examined. Reexpression of the entire group of functions most often occurs in a block, and except for one discordant subclone, correlates with loss of human chromosome 2. The extinguished cells and their reexpressing derivatives have been examined for the expression of seven liver-enriched transcription factors. C/EBP, LAP, DBP, HNF3, and vHNF1 expression are not systematically extinguished in parallel with the hepatic functions. However, HNF1 and HNF4 show a perfect correlation with phenotype: these factors are expressed only in the cells showing pleiotropic reexpression. Since recent evidence indicates that HNF4 controls HNF1 expression, it can be proposed that the HNF4 gene is the primary target of the pleiotropic extinguisher.

Anomeric specificity of glucose-stimulated insulin release: evidence for a glucoreceptor?

The effects on insulin secretion of alpha and beta anomers of D-glucose were studied in the in vitro perfused rat pancreas. Both phases of insulin release showed consistent stereospecificity for alpha-glucose; this specificity indicates an action of glucose independent of intracellular glucose metabolism.

Controlled drinking by alcoholics? New findings and a reevaluation of a major affirmative study.

Controlled drinking has recently become a controversial alternative to abstinence as an appropriate treatment goal for alcoholics. In this study we reexamine the evidence underlying a widely cited report by Sobell and Sobell of successful controlled drinking by a substantial proportion of gamma (physically dependent) alcoholic subjects in a behavior therapy experiment. A review of the evidence, including official records and new interviews, reveals that most subjects trained to do controlled drinking failed from the outset to drink safely. The majority were rehospitalized for alcoholism treatment within a year after their discharge from the research project. A 10-year follow-up (extended through 1981) of the original 20 experimental subjects shows that only one, who apparently had not experienced physical withdrawal symptoms, maintained a pattern of controlled drinking; eight continued to drink excessively--regularly or intermittently--despite repeated damaging consequences; six abandoned their efforts to engage in controlled drinking and became abstinent; four died from alcohol-related causes; and one, certified about a year after discharge from the research project as gravely disabled because of drinking, was missing.

A critical review of hydrological data collection for assessing preservation risk for urban waterlogged archaeology: A case study from the City of York, UK.

Environmental change caused by urban development, possibly augmented by climate change, may result in accelerated decay of in situ archaeological resources. Damage may be related to changes in hydrological processes. Such archaeological resources have to be considered in environmental planning. In this paper we highlight the need for improved hydrological data from urban archaeological sites using the case study of the City of York, UK, arguably one of the most well studied and well preserved urban archaeological environments globally. We suggest that the quality of hydrological data collected during routine surveys and experimental work must be improved and standardised in order for us to produce reliable archaeological risk models for urban sites.

Groundwater flow velocities in a fractured carbonate aquifer-type: Implications for contaminant transport.

Contaminants that are highly soluble in groundwater are rapidly transported via fractures in mechanically resistant sedimentary rock aquifers. Hence, a rigorous methodology is needed to estimate groundwater flow velocities in such fractured aquifers. Here, we propose an approach using borehole hydraulic testing to compute flow velocities in an un-faulted area of a fractured carbonate aquifer by applying the cubic law to a parallel plate model. The Cadeby Formation (Yorkshire, NE England) - a Permian dolostone aquifer present beneath the University of Leeds Farm - is the fractured aquifer selected for this hydraulic experiment. The bedding plane fractures of this dolostone aquifer, which are sub-horizontal, sub-parallel and laterally persistent, largely dominate the flow at shallow (<~40 mBGL) depths. These flowing bedding plane discontinuities are separated by a rock matrix which is relatively impermeable (Kwell-test/Kcore-plug~104) as is common in fractured carbonate aquifers. In the workflow reported here, the number of flowing fractures - mainly bedding plane fractures - intersecting three open monitoring wells are found from temperature/fluid conductivity and acoustic/optical televiewer logging. Following well installation, average fracture hydraulic apertures for screened intervals are found from analysis of slug tests. For the case study aquifer, this workflow predicts hydraulic apertures ranging from 0.10 up to 0.54 mm. However, groundwater flow velocities range within two order of magnitude from 13 up to 242 m/day. Notably, fracture apertures and flow velocities rapidly reduce with increasing depth below the water table; the upper ~10 m shows relatively high values of hydraulic conductivity (0.30-2.85 m/day) and corresponding flow velocity (33-242 m/day). Permeability development around the water table in carbonate aquifer-types is common, and arises where high pCO2 recharge water from the soil zone causes calcite/dolomite dissolution. Hence, agricultural contaminants entering the aquifer with recharge water are laterally transported rapidly within this upper part. Computation of groundwater flow velocities allows determination of the Reynolds number. Values of up ~1, indicating the lower limit of the transition from laminar to turbulent flow, are found at the studied site, which is situated away from major fault traces. Hence, turbulent flow is likely to arise in proximity to tectonic structures, such as normal faults, which localize flow and enhance karstification. The occurrence of turbulent flow in correspondence of such tectonic structures should be represented in regional groundwater flow simulations.

Waiting before birth: outcomes after fetal listing for heart transplantation.

Following fetal diagnosis of a profound heart defect, transplantation (HTx) is an alternative to pregnancy termination or neonatal surgical palliation. Retrospective review of the cardiac and transplant databases of fetal listings for HTx between 1990 and July 2006 was undertaken to describe outcomes after listing. We identified 26 fetal listings (of 269 total listings). Diagnoses included congenital heart disease (n = 24) and cardiomyopathy (n = 2). Seven patients were delisted after birth: in five cases parents opted for surgical palliation, two clinically improved. One patient died wait-listed (stillborn). Time wait-listed as a fetus ranged from 1-41 days (median 19 days). Eighteen patients underwent HTx (median weight 2.8 kg, range 2.1-10.9 kg); median days wait-listed after birth was 22 (4 h-123 days). Two fetuses were surgically delivered at 36 weeks gestation when a donor organ became available; 11 were transplanted as neonates (<30 days). The median age at HTx was 1 month (4 h-2.6 months). Fetal listing for HTx increases the potential window of opportunity for a donor organ to become available; patients had low wait-list mortality and a fair intermediate-term outcome. Well-defined criteria for eligibility for fetal listing and priority allocation to infants over fetuses seem rational approaches for centers that offer fetal listing.

Evaluation of the safety and bioactivity of purified and semi-purified glucoraphanin.

The anti-carcinogenic effects of broccoli have been attributed to sulforaphane, the hydrolysis product of glucoraphanin (GRP). Here we determined if purified GRP, in the absence of the plant-derived hydrolyzing enzyme myrosinase, could affect pulmonary and hepatic ethoxyresorufin O-deethylase (EROD) and/or NAD(P)H-quinone oxidoreductase 1 (NQO1) activity. Male F344 rats were administered semi-synthetic, semi-purified or purified GRP (240 mg/kg: 550 micromol/kg rat daily for 4 days) by gavage. Hepatic and pulmonary NQO1 activity increased ( approximately 20%), but not EROD. Varying doses of semi-purified GRP (30, 60, or 120 mg/kg rat daily for 4 days) again caused no change in EROD activity, although a dose-dependent increase in NQO1 was seen. Urinary excretion of mercapturic acids showed no difference between preparations, and recovery increased with decreasing dose. Histopathologic examination revealed no abnormal tissues other than cecum, where inflammation was dose dependent; mild at 120 mg/kg and severe at 240 mg/kg, a greatly supra-physiological dose. We conclude that GRP 30-60 mg/kg p.o. is safe and effectively enhances NQO1 in all tissues evaluated.

Mitochondrial transcription factor A (TFAM) shapes metabolic and invasion gene signatures in melanoma.

Mitochondria are central key players in cell metabolism, and mitochondrial DNA (mtDNA) instability has been linked to metabolic changes that contribute to tumorigenesis and to increased expression of pro-tumorigenic genes. Here, we use melanoma cell lines and metastatic melanoma tumors to evaluate the effect of mtDNA alterations and the expression of the mtDNA packaging factor, TFAM, on energetic metabolism and pro-tumorigenic nuclear gene expression changes. We report a positive correlation between mtDNA copy number, glucose consumption, and ATP production in melanoma cell lines. Gene expression analysis reveals a down-regulation of glycolytic enzymes in cell lines and an up-regulation of amino acid metabolism enzymes in melanoma tumors, suggesting that TFAM may shift melanoma fuel utilization from glycolysis towards amino acid metabolism, especially glutamine. Indeed, proliferation assays reveal that TFAM-down melanoma cell lines display a growth arrest in glutamine-free media, emphasizing that these cells rely more on glutamine metabolism than glycolysis. Finally, our data indicate that TFAM correlates to VEGF expression and may contribute to tumorigenesis by triggering a more invasive gene expression signature. Our findings contribute to the understanding of how TFAM affects melanoma cell metabolism, and they provide new insight into the mechanisms by which TFAM and mtDNA copy number influence melanoma tumorigenesis.