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.

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.

Characterizing flow pathways in a sandstone aquifer: Tectonic vs sedimentary heterogeneities.

Sandstone aquifers are commonly assumed to represent porous media characterized by a permeable matrix. However, such aquifers may be heavy fractured when rock properties and timing of deformation favour brittle failure and crack opening. In many aquifer types, fractures associated with faults, bedding planes and stratabound joints represent preferential pathways for fluids and contaminants. In this paper, well test and outcrop-scale studies reveal how strongly lithified siliciclastic rocks may be entirely dominated by fracture flow at shallow depths (≤180m), similar to limestone and crystalline aquifers. However, sedimentary heterogeneities can primarily control fluid flow where fracture apertures are reduced by overburden pressures or mineral infills at greater depths. The Triassic St Bees Sandstone Formation (UK) of the East Irish Sea Basin represents an optimum example for study of the influence of both sedimentary and tectonic aquifer heterogeneities in a strongly lithified sandstone aquifer-type. This fluvial sedimentary succession accumulated in rapidly subsiding basins, which typically favours preservation of complete depositional cycles including fine grained layers (mudstone and silty sandstone) interbedded in sandstone fluvial channels. Additionally, vertical joints in the St Bees Sandstone Formation form a pervasive stratabound system whereby joints terminate at bedding discontinuities. Additionally, normal faults are present through the succession showing particular development of open-fractures. Here, the shallow aquifer (depth≤180m) was characterized using hydro-geophysics. Fluid temperature, conductivity and flow-velocity logs record inflows and outflows from normal faults, as well as from pervasive bed-parallel fractures. Quantitative flow logging analyses in boreholes that cut fault planes indicate that zones of fault-related open fractures characterize ~50% of water flow. The remaining flow component is dominated by bed-parallel fractures. However, such sub-horizontal fissures become the principal flow conduits in wells that penetrate the exterior parts of fault damage zones, as well as in non-faulted areas. The findings of this study have been compared with those of an earlier investigation of the deeper St Bees Sandstone aquifer (180 to 400m subsurface depth) undertaken as part of an investigation for a proposed nuclear waste repository. The deeper aquifer is characterized by significantly lower transmissivities. High overburden pressure and the presence of mineral infillings, have reduced the relative impact of tectonic heterogeneities on transmissivity here, thereby allowing matrix flow in the deeper part of the aquifer. The St Bees Sandstone aquifer contrasts the hydraulic behaviour of low-mechanically resistant sandstone rock-types. In fact, the UK Triassic Sandstone of the Cheshire Basin is low-mechanically resistant and flow is supported both by matrix and fracture. Additionally, faults in such weak-rocks are dominated by granulation seams representing flow-barriers which strongly compartmentalize the UK Triassic Sandstone in the Cheshire Basin.

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.

Fractional flow in fractured chalk; a flow and tracer test revisited.

A multi-borehole pumping and tracer test in fractured chalk is revisited and reinterpreted in the light of fractional flow. Pumping test data analyzed using a fractional flow model gives sub-spherical flow dimensions of 2.2-2.4 which are interpreted as due to the partially penetrating nature of the pumped borehole. The fractional flow model offers greater versatility than classical methods for interpreting pumping tests in fractured aquifers but its use has been hampered because the hydraulic parameters derived are hard to interpret. A method is developed to convert apparent transmissivity and storativity (L(4-n)/T and S(2-n)) to conventional transmissivity and storativity (L2/T and dimensionless) for the case where flow dimension, 2

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.

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.

A multi-directional tracer test in the fractured Chalk aquifer of E. Yorkshire, UK.

A multi-borehole radial tracer test has been conducted in the confined Chalk aquifer of E. Yorkshire, UK. Three different tracer dyes were injected into three injection boreholes and a central borehole, 25 m from the injection boreholes, was pumped at 330 m(3)/d for 8 days. The breakthrough curves show that initial breakthrough and peak times were fairly similar for all dyes but that recoveries varied markedly from 9 to 57%. The breakthrough curves show a steep rise to a peak and long tail, typical of dual porosity aquifers. The breakthrough curves were simulated using a 1D dual porosity model. Model input parameters were constrained to acceptable ranges determined from estimations of matrix porosity and diffusion coefficient, fracture spacing, initial breakthrough times and bulk transmissivity of the aquifer. The model gave equivalent hydraulic apertures for fractures in the range 363-384 microm, dispersivities of 1 to 5 m and matrix block sizes of 6 to 9 cm. Modelling suggests that matrix block size is the primary controlling parameter for solute transport in the aquifer, particularly for recovery. The observed breakthrough curves suggest results from single injection-borehole tracer tests in the Chalk may give initial breakthrough and peak times reasonably representative of the aquifer but that recovery is highly variable and sensitive to injection and abstraction borehole location. Consideration of aquifer heterogeneity suggests that high recoveries may be indicative of a high flow pathway adjacent, but not necessarily connected, to the injection and abstraction boreholes whereas low recoveries may indicate more distributed flow through many fractures of similar aperture.

American society of transplantation symposium on B cells in transplantation: harnessing humoral immunity from rodent models to clinical practice.

There is growing awareness that B cells and alloantibodies are important mediators of both acute and chronic allograft injury. Unfortunately, few therapies are clinically available to mitigate the function of B cells or the effects of established alloantibody. As a result, many sensitized people await transplantation without a suitable donor, and several rejection syndromes are emerging that appear to involve B cells either as antibody producers or as antigen-presenting cells. In recognition of this unmet need in transplantation, the American Society of Transplantation organized a Symposium on B cells in Organ Transplantation to foster interest in this topic amongst basic researchers attending the annual meeting of the American Association of Immunologists. This manuscript will give an overview of the presentations from this symposium including the current risks of allosensitization, adaptive accommodation, approaches toward B-cell tolerance for allo- and xenoantigens and clinical application of these concepts in ABO incompatible neonatal cardiac transplantation.

A novel tool for B-cell tolerance research: characterization of mouse alloantibody development using a simple and reliable cellular ELISA technique.

In animal-based transplantation research, the measurement of anti-donor antibodies in transplant recipients is limited by lack of an appropriate technique. We have developed a novel immunoassay capable of quantifying antibody bound to cell-surface major histo- compatability complex (MHC) and non-MHC antigens, using splenocytes from wild-type and MHC-deficient mice as antigen-bearing target cells. We utilized our "cellular ELISA" (CELISA) technique to study the development of tolerance versus immunity in the B-cell compartment in response to neonatal exposure to allogeneic fetal liver cells (FLC). This neonatal tolerance protocol typically induces permanent acceptance of donor-type and third-party cardiac allografts, but rejection of both donor-type and third-party skin grafts occurs. C3H/He (C3H; H-2(k)) mice were injected as neonates with BALB/c (BALB; H-2(d)) FLC and transplanted as adults with C57BL/6 (B6; H-2(b)) cardiac grafts. Despite long-term acceptance of third-party B6 cardiac grafts, serum contained increased anti-B6 IgG and IgM levels as measured by CELISA; IgM production was elevated by 2 weeks posttransplant and remained stable, while IgG production increased rapidly between 2 and 5 weeks posttransplant. In another experimental setting, CELISA assays were able to detect that neonatal injection of C3H mice with FLC from wild-type B6 mice or from MHC class II-deficient or class I/II-deficient (B6 background) mice (CI(+)CII(+), CI(+)CII(-), CI(-)CII(-), respectively) prevented sensitization to B6 antigens by subsequent skin transplants but did not induce graft acceptance, whereas FLC from MHC class I-deficient-only (CI(-)CII(+)) did not prevent B6 sensitization. The CELISA technique is a simple and sensitive means for quantifying alloantibodies in mice and will assist in further delineating the role of the B-cell compartment in neonatally induced cardiac allograft acceptance.

Induction of human histo-blood group A antigen expression in mouse cells by gene therapy using lentiviral vectors harbouring human ABH-related glycosyltransferase genes.

We recently discovered that ABO incompatibility, which evokes a rapid humoral immune response in adult heart transplantation, is not a barrier in infant heart transplantation, and that infant recipients of ABO-incompatible hearts develop specific B-cell tolerance to donor A/B antigens. An animal model of ABO-incompatible heart transplantation would allow detailed investigation of the mechanism(s) of acceptance of ABO-incompatible grafts, using experimental methods that would not be possible in humans. To determine the feasibility of such a model, the human alpha-1,2-fucosyltransferase (H-transferase; for H antigen expression) gene was cloned into a lentiviral vector, and the human alpha-1,3-N-acetylgalactosaminyltransferase (A-transferase; for A antigen expression) gene was cloned into a bicistronic lentiviral vector also containing the green fluorescent protein (GFP) gene; these replication-deficient vectors were denoted H-trs and A-GFP, respectively. Synthesis of the human histo-blood group A antigen in humans is dependent on expression of these two glycosyltransferases. HeLa cells, a human cell line known to be of blood group O origin, expressed cell surface A antigen as measured by cellular ELISA when transfected with A-GFP alone, and the level of A antigen expression was enhanced by transfection with H-trs in addition to A-GFP. Cell surface H antigen expression was observed on both mouse fibroblast and mouse endothelial cells only when infected with H-trs lentiviral particles. Expression of A antigen was dependent on infection with both H-trs and A-GFP lentiviral particles, approaching levels on human group A cells. These collective results indicate that expression of human histo-blood group A antigen at the cell surface can be induced in mouse cells by infection with H-trs and A-GFP, and that such A antigen expression is dependent on H-trs expression.

Crossing the ABO barrier in infant heart transplantation at the Hospital for Sick Children.

Heart transplantation in infancy is generally associated with excellent clinical results. Unfortunately, heart transplantation cannot be offered to many infants who would benefit from this therapy due to a shortage of organ donors of suitable size for infants, and traditional limitations that constrain donor availability still further, such as the requirement for ABO-compatibility. At the Hospital for Sick Children, we questioned the need to apply this requirement to infants, based on available scientific evidence regarding the immaturity of certain immune responses. Our experience with 23 ABO-incompatible infant heart transplants demonstrates that this procedure can be performed safely in infants. Moreover, we have shown that donor-specific B-cell tolerance develops following ABO-incompatible transplantation. The consistency of the clinical and laboratory outcomes of ABO-incompatible infant heart transplantation to date would suggest that there is no scientific rationale to require ABO-compatibility between donors and recipients for infant transplantation.

Quality of life and function following cardiac transplantation in adolescents.

Quality of life and functional status are important outcome measures following heart transplantation. The present study evaluated the quality of life and function of 10 adolescent heart transplant recipients at the Hospital for Sick Children. Subjects were surveyed using a visual analog quality of life scale, the Children's Depression Inventory, The Pediatric Quality of Life Scale 4.0, and the Functional Status IIR. Results demonstrated excellent perceived quality of life and psychologic well-being, comparable to healthy norms. Subscale results for physical, social, and emotional function provide evidence for positive responses to transplantation. As well, results on factors such as self-esteem, school, interpersonal function, and mood demonstrate gender differences that may influence outcomes. Studies are currently underway to further delineate these important quality of life, function, and psychosocial issues to ensure optimal outcomes are achieved in our patients.

Defining critical windows in the development of the human immune system.

Identifying critical windows in immune system development is crucial for determination of either safety or vulnerability to exposure to specific agents during rapidly changing phases of ontogeny. These phases in the human range from postconception early gestation through adolescence. A detailed understanding of these windows will facilitate avoidance of environmental toxins as well as allow improved planning for unavoidable exposures. Critical windows of immune development will be influenced by concomitant development, maturation and growth of other organ systems, thus the influence of potentially toxic exposures must be determined within a co-ordinated multisystem and multidisciplinary approach.