Avoidance of Profound Hypothermia During Initial Reperfusion Improves the Functional Recovery of Hearts Donated After Circulatory Death.

The resuscitation of hearts donated after circulatory death (DCD) is gaining widespread interest; however, the method of initial reperfusion (IR) that optimizes functional recovery has not been elucidated. We sought to determine the impact of IR temperature on the recovery of myocardial function during ex vivo heart perfusion (EVHP). Eighteen pigs were anesthetized, mechanical ventilation was discontinued, and cardiac arrest ensued. A 15-min standoff period was observed and then hearts were reperfused for 3 min at three different temperatures (5°C; N = 6, 25°C; N = 5, and 35°C; N = 7) with a normokalemic adenosine-lidocaine crystalloid cardioplegia. Hearts then underwent normothermic EVHP for 6 h during which time myocardial function was assessed in a working mode. We found that IR coronary blood flow differed among treatment groups (5°C = 483 ± 53, 25°C = 722 ± 60, 35°C = 906 ± 36 mL/min, p < 0.01). During subsequent EVHP, less myocardial injury (troponin I: 5°C = 91 ± 6, 25°C = 64 ± 16, 35°C = 57 ± 7 pg/mL/g, p = 0.04) and greater preservation of endothelial cell integrity (electron microscopy injury score: 5°C = 3.2 ± 0.5, 25°C = 1.8 ± 0.2, 35°C = 1.7 ± 0.3, p = 0.01) were evident in hearts initially reperfused at warmer temperatures. IR under profoundly hypothermic conditions impaired the recovery of myocardial function (cardiac index: 5°C = 3.9 ± 0.8, 25°C = 6.2 ± 0.4, 35°C = 6.5 ± 0.6 mL/minute/g, p = 0.03) during EVHP. We conclude that the avoidance of profound hypothermia during IR minimizes injury and improves the functional recovery of DCD hearts.

Physiologic Changes in the Heart Following Cessation of Mechanical Ventilation in a Porcine Model of Donation After Circulatory Death: Implications for Cardiac Transplantation.

Hearts donated following circulatory death (DCD) may represent an additional source of organs for transplantation; however, the impact of donor extubation on the DCD heart has not been well characterized. We sought to describe the physiologic changes that occur following withdrawal of life-sustaining therapy (WLST) in a porcine model of DCD. Physiologic changes were monitored continuously for 20 min following WLST. Ventricular pressure, volume, and function were recorded using a conductance catheter placed into the right (N = 8) and left (N = 8) ventricles, and using magnetic resonance imaging (MRI, N = 3). Hypoxic pulmonary vasoconstriction occurred following WLST, and was associated with distension of the right ventricle (RV) and reduced cardiac output. A 120-fold increase in epinephrine was subsequently observed that produced a transient hyperdynamic phase; however, progressive RV distension developed during this time. Circulatory arrest occurred 7.6±0.3 min following WLST, at which time MRI demonstrated an 18±7% increase in RV volume and a 12±9% decrease in left ventricular volume compared to baseline. We conclude that hypoxic pulmonary vasoconstriction and a profound catecholamine surge occur following WLST that result in distension of the RV. These changes have important implications on the resuscitation, preservation, and evaluation of DCD hearts prior to transplantation.

The varying effects of obesity and morbid obesity on outcomes following cardiac transplantation.

The purpose of this study was to compare the outcomes of patients undergoing cardiac transplantation stratified by body mass index (BMI, kg m(-)(2)). The Alberta Provincial Project for Outcome Assessment in Coronary Heart Disease registry captured 220 cardiac transplantations in Alberta, Canada from January 2004 to April 2013. All recipients were stratified by BMI into five groups (BMI: <20, 20-24.9, 25-29.9, 30-<34.9 and ⩾35). Patient characteristics were analyzed by analysis of variance and χ(2) analyses. Kaplan-Meier was used to examine survival differences. Preoperative characteristics demonstrated significant increases in metabolic syndrome, prior myocardial infarction and prior coronary artery bypass graft in patients with morbid obesity. Intra-operatively, there was an increase in cardiopulmonary bypass time in patients with morbid obesity (P<0.01). Postoperative analysis revealed increased rates of early complications (<30 days), associated with a BMI >35. Long-term survival was also significantly decreased in patients with morbid obesity. Of interest, obesity (BMI, 30-34.9) was not associated with decreased survival. These findings suggest that, post-cardiac transplantation, patients who have a BMI ⩾35 have lower long-term survival compared with all other BMI groups. However, patients with BMI 30-34.9 did not have significantly worse outcomes and should not be excluded for heart transplantation based on BMI.

Ex vivo perfusion of the swine heart as a method for pre-transplant assessment.

We describe a cost-effective, reproducible circuit in a porcine, ex vivo, continuous warm-blood, bi-ventricular, working heart model that has future possibilities for pre-transplant assessment of marginal hearts donated from brain stem dead donors and hearts donated after circulatory determination of death (DCDD). In five consecutive experiments over five days, pressure volume loops were performed. During working mode, the left ventricular end systolic pressure volume relationship (LV ESPVR) was 23.1±11.1 mmHg/ml and the LV preload recruitable stroke work (PRSW) was 67.8±7.2. (Standard PVAN analysis software) (Millar Instruments, Houston, TX, USA) All five hearts were perfused for 219±64 minutes and regained normal cardiac function on the perfusion system.They displayed a significant upward and leftward shift of the end systolic pressure volume relationship, a significant increase in preload recruitable stroke work and minimal stiffness. These hearts could potentially be considered for transplantation. The circuit was effective during reperfusion and working modes whilst proving to be successful in maintaining cardiac function in excess of four hours. Using an autologous prime of approximately 20% haematocrit (Hct), electrolytes and blood gases were easy to control within this period using standard perfusion techniques.

Hearts from DCD donors display acceptable biventricular function after heart transplantation in pigs.

Cardiac transplantation is in decline, in contrast to other solid organs where the number of solid organ transplants from donors after circulatory death (DCD) is increasing. Hearts from DCD donors are not currently utilized due to concerns that they may suffer irreversible cardiac injury with resultant poor graft function. Using a large animal model, we tested the hypothesis that hearts from DCD donors would be suitable for transplantation. Donor pigs were subjected to hypoxic cardiac arrest (DCD) followed by 15 min of warm ischemia and resuscitation on cardiopulmonary bypass, or brainstem death (BSD) via intracerebral balloon inflation. Cardiac function was assessed through load-independent measures and magnetic resonance imaging and spectroscopy. After resuscitation, DCD hearts had near normal contractility, although stroke volume was reduced, comparable to BSD hearts. DCD hearts had a significant decline in phosphocreatine and increase in inorganic phosphate during the hypoxic period, with a return to baseline levels after reperfusion. After transplantation, cardiac function was comparable between BSD and DCD groups. Therefore, in a large animal model, the DCD heart maintains viability and recovers function similar to that of the BSD heart and may be suitable for clinical transplantation. Further study is warranted on optimal reperfusion strategies.

T cell recognition of major histocompatibility complex class II complexes with invariant chain processing intermediates.

Peptides from the lumenal portion of invariant chain (Ii) spanning residues 80-106 (class II-associated Ii peptide [CLIP]) are found in association with several mouse and human major histocompatibility complex (MHC) class II allelic variants in wild-type and presentation-deficient mutant cells. The ready detection of these complexes suggests that such an intermediate is essential to the MHC class II processing pathway. In this study, we demonstrate that T cells recognize CLIP/MHC class II complexes on the surface of normal and mutant cells in a manner indistinguishable from that of nominal antigenic peptides. Surprisingly, T cell hybrids specific for human CLIP bound to murine MHC class II molecule I-Ab and a new monoclonal antibody 30-2 with the same specificity, recognize two independent epitopes expressed on this peptide/class II complex. T cell recognition is dependent on a Gln residue (position 100) in CLIP, whereas the 30-2 antibody recognizes a Lys residue-at position 90. These two residues flank the 91-99 sequence that is conserved among human, mouse, and rat Ii, potentially representing an MHC class II-binding site. Our results suggest that the COOH-terminal portion of CLIP that includes TCR contact residue Gln 100 binds in the groove of I-Ab molecule. Moreover, both T cells and the antibody recognize I-Ab complexed with larger Ii processing intermediates such as the approximately 12-kD small leupeptin-induced protein (SLIP) fragments. Thus, SLIP fragments contain a CLIP region bound to MHC class II molecule in a conformation identical to that of a free CLIP peptide. Finally, our data suggest that SLIP/MHC class II complexes are precursors of CLIP/MHC class II complexes.

Coordinate defects in human histocompatibility leukocyte antigen class II expression and antigen presentation in bare lymphocyte syndrome.

The human immunodeficiency, type II bare lymphocyte syndrome (BLS), has been attributed to a defect in the transcription of class II histocompatibility genes. Immunocompetence, as assessed by functional exogenous antigen presentation, was not restored in immortalized B cells, derived from a BLS patient, after transfection with HLA-DR class II structural genes. Incubation of protein antigens, as well as infectious virus, with DR-transfected BLS cells failed to induce activation of antigen-specific helper T lymphocytes. Peptide antigens were presented by class II molecules displayed on BLS cells, although the conformation of these class II proteins was altered as indicated by epitope mapping. This defect in antigen presentation was independent of the specific class II DR allele transfected into BLS cells. Genetic complementation analysis has been used with BLS cells to demonstrate that the defect in class II gene transcription is linked to the absence of a trans-acting factor. Similarly, functional class II dimers were restored after in vitro fusion of cells derived from two distinct BLS complementation groups, implying that specific transcriptional control elements are shared by a gene critical for antigen presentation and genes encoding HLA class II antigens. Thus, two important functionally linked pathways of class II molecules, structural gene expression and antigen presentation, share a common regulatory pathway defective in BLS.

Synthesis of ammonium cyanate and urea by reduction of nitric oxide on platinum, rhodium, and ruthenium catalysts.

Nearly quantitative conversion of nitric oxide (NO) into N(2)H(4)CO has been obtained in the reduction of NO with carbon monoxide and hydrogen over platinum and rhodium catalysts. Depending on the temperature of collection, N(2)H(4)CO is isolated as ammonium cyanate or its isomer, urea. The process is an effective way of recovering fixed nitrogen from dilute industrial streams containing NO. Ammonium cyanate may play a role in the reduction of NO in automotive exhaust control.

Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.

Naturally processed T cell epitopes from human glutamic acid decarboxylase identified using mice transgenic for the type 1 diabetes-associated human MHC class II allele, DRB1*0401.

The identification of class II binding peptide epitopes from autoimmune disease-related antigens is an essential step in the development of antigen-specific immune modulation therapy. In the case of type 1 diabetes, T cell and B cell reactivity to the autoantigen glutamic acid decarboxylase 65 (GAD65) is associated with disease development in humans and in nonobese diabetic (NOD) mice. In this study, we identify two DRB1*0401-restricted T cell epitopes from human GAD65, 274-286, and 115-127. Both peptides are immunogenic in transgenic mice expressing functional DRB1*0401 MHC class II molecules but not in nontransgenic littermates. Processing of GAD65 by antigen presenting cells (APC) resulted in the formation of DRB1*0401 complexes loaded with either the 274-286 or 115-127 epitopes, suggesting that these naturally derived epitopes may be displayed on APC recruited into pancreatic islets. The presentation of these two T cell epitopes in the islets of DRB1*0401 individuals who are at risk for type 1 diabetes may allow for antigen-specific recruitment of regulatory cells to the islets following peptide immunization.

Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.

A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.

Impact of Obesity on Heart and Lung Transplantation: Does Pre-Transplant Obesity Affect Outcomes?

Increasing prevalence of obesity has led to a rise in the number of prospective obese heart and lung transplant recipients. The optimal management strategy of obese patients with end-stage heart and lung failure remains controversial. This review article discusses and provides a summary of the literature surrounding the impact of obesity on outcomes in heart and lung transplantation. Studies on transplant obesity demonstrate controversy in terms of morbidity and mortality outcomes and obesity pre-transplantation. However, the impact of obesity on outcomes seems to be more consistently demonstrated in lung rather than heart transplantation. The ultimate goal in heart and lung transplantation in the obese patient is to identify those at highest risk of complication that may warrant therapies to mitigate risk by addressing comorbid conditions.

Feasibility of Lung Transplantation From Donation After Circulatory Death Donors Following Portable Ex Vivo Lung Perfusion: A Pilot Study.

BACKGROUND: Donation after circulatory death (DCD) has the potential to significantly alleviate the shortage of transplantable lungs. We report our initial experience with the use of portable ex vivo lung perfusion (EVLP) with the Organ Care System Lung device for evaluation of DCD lungs. METHODS: We performed a retrospective review of the DCD lung transplantation (LTx) experience at a single institution through the use of a prospective database. RESULTS: From 2011 to 2015, 208 LTx were performed at the University of Alberta, of which 11 were DCD LTx with 7 (64%) that underwent portable EVLP. DCD lungs preserved with portable EVLP had a significantly shorter cold ischemic time (161 ± 44 vs 234 ± 60 minutes, P = .045), lower grade of primary graft dysfunction at 72 hours after LTx (0.4 ± 0.5 vs 2.1 ± 0.7, P = .003), similar mechanical ventilation time (55 ± 44 vs 103 ± 97 hours, P = .281), and hospital length of stay (29 ± 11 vs 33 ± 10 days, P = .610). All patients were alive at 1-year follow-up after LTx with improved functional outcomes and acceptable quality of life compared with before LTx, although there were no intergroup differences. CONCLUSIONS: In our pilot cohort, portable EVLP was a feasible modality to increase confidence in the use of DCD lungs with validated objective evidence of lung function during EVLP that translates to acceptable clinical outcomes and quality of life after LTx. Further studies are needed to validate these initial findings in a larger cohort.

Canada's longest experience with extracorporeal membrane oxygenation as a bridge to lung transplantation: a case report.

INTRODUCTION: Extracorporeal membrane oxygenation (ECMO) is a method of enabling gas exchange through an external membrane used to treat respiratory failure in critically ill patients. ECMO as a bridge to lung transplantation has been investigated as a potential method of reducing lung transplantation waitlist mortality. Herein we describe a case of ECMO as a bridge-to-lung transplantation for the duration of 35 days, which is the longest documented length of ECMO support before successful transplantation in Canada. CASE DESCRIPTION: The prospective recipient was a 28-year-old female suffering from stage 4 pulmonary sarcoidosis. Given an acute exacerbation of her chronic respiratory failure, ECMO had to be initiated. She remained on ECMO for 35 days until a suitable set of donor lungs became available. The recipient had a prolonged course in hospital but was successfully discharged home where she continues to have good lung function. She remains alive and well at home 5 months post-transplantation and continues to improve and gain strength. CONCLUSION: Our case provides hope that in the future we may be able to expand the population of recipients who may be candidates for lung transplantation. This case adds to the growing literature on the role of ECMO as a bridge-to-lung transplantation with the potential to reduce patient deaths while wait-listed for lung transplantation as well as increase the number of transplantations being performed.

2-Methoxyestradiol: A Hormonal Metabolite Modulates Stimulated T-Cells Function and proliferation.

BACKGROUND: 2-Methoxyestradiol (2ME2) is an endogenous metabolite of estrogen that is nonestrogenic and has been studied in cancer as an antimitotic agent that is beneficial by its selectivity for cancer cells without toxicity to nonmalignant cells. Because the effect of 2ME2 in a transplant rejection setting remains unknown, we hypothesized that 2ME2 can inhibit stimulated T-cell function. METHODS: Human peripheral blood mononuclear cells (PBMCs) were cultured and pretreated with 2ME2 before stimulation. The cultured medium was collected for enzyme-linked immunosorbent assays, and whole-cell lysates were collected for Western immunoblotting. Proliferation and apoptosis assays were performed and analyzed by means of flow cytometry. RESULTS: Tumor necrosis factor -α and interferon-γ cytokine production in 2ME2-treated stimulated PBMCs were modestly reduced relative to control samples. T-cell proliferation was blunted by treatment with 2ME2, and a decrease in apoptosis correlated with a decrease in caspase-9 activity. Additionally, 2ME2 was able to block stress-induced senescence caused by stimulation of T-cells. CONCLUSIONS: 2ME2 is a hormone-based therapy that blunts stimulated T-cell proliferation and does not induce apoptosis or stress-induced senescence. Stimulated T-cells treated with 2ME2 are still able to produce normal levels of cytokines. Therefore, 2ME2 may lead to an oral immunomodulatory adjunct therapy with a low side effect profile for individuals undergoing transplantation.

Autophagy is a regulator of TGF-ß1-induced fibrogenesis in primary human atrial myofibroblasts.

Transforming growth factor-ß(1) (TGF-ß(1)) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-ß(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-ß(1)-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-ß(1) to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-ß(1) promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-ß(1) in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3ß indicated the localization of punctate LC3ß with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-ß(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

The middle latency auditory evoked potential may be abnormal in dementia.

The P1 potential (50 msec) of the middle latency auditory evoked potential was lacking in 12 of 31 (39%) patients with probable Alzheimer's disease and seven of 12 (58%) demented patients with Parkinson's disease. Component P1 was not present in one normal control subject and one nondemented Parkinson's disease patient. Clinical and experimental evidence suggests that abnormalities of P1 in dementia may be due to cholinergic dysfunction.

The P1 component of the middle latency auditory evoked potential predicts a practice effect during clinical trials in Alzheimer's disease.

Thirty-five patients with probable Alzheimer's disease who were enrolled in an experimental drug trial of linopirdine underwent repeated testing that included recording the middle latency auditory evoked potential (MLAEP), the Mini-Mental State Examination (MMSE), and the Alzheimer's Disease Assessment Scale Cognitive Subscale (ADASCOG). Patients lacking the P1 component of the MLAEP exhibited a significantly greater decline in cognitive function as measured by the ADASCOG over 56 weeks. This decline appeared to be due to a less robust practice effect, which was maximal in all patients at 16 weeks. At the end of 56 weeks the entire group of patients was near baseline with respect to the ADASCOG. This lack of the annualized decline expected from other longitudinal studies may be explained by practice and placebo effects. The MMSE did not exhibit a practice effect and showed the expected decline in scores.

The agraphia of Alzheimer's disease.

Hypothesizing that agraphia in Alzheimer's disease (AD) reflects disturbances in multiple cognitive domains, we evaluated writing samples from 33 patients meeting strict criteria for probable AD. We found agraphia to be common on a standard narrative writing task. When compared with 41 education- and age-matched normal control subjects, AD patients had significantly lower writing scores, wrote significantly fewer words, mentioned significantly fewer categories of information, and were significantly more likely to make writing errors. On stepwise regression procedures, neuropsychological measures of visuoperceptual impairment and disease severity were the strongest predictors of agraphia, but other analyses indicated that measures of language, praxis, and attention could also contribute significantly to agraphia. On two writing tasks, we failed to confirm the previous contention that agraphia is a marker for familial AD. However, there was a highly significant interaction between family history, oral naming, and writing: patients with nonfamilial AD, but not those with a family history of dementia, showed a strong correlation between naming and writing performance. We conclude that agraphia in AD can be variously determined and that agraphia is not a reliable marker for familial disease.

Forgetting in H.M.: a second look.

Forgetting was assessed in the amnesic patient H.M. using forced-choice and yes-no picture recognition at four delay intervals: 10 min, 24 hr, 72 hr, and 1 week after learning. In order to make H.M.'s initial recognition performance comparable to that of control subjects who viewed each slide for 1 sec, H.M. viewed each slide for 20 sec. H.M. displayed normal forgetting in forced-choice and yes-no recognition, although he was impaired in yes-no recognition at the 24-hr delay interval. These data contradict Huppert and Piercy's hypothesis that medical temporal-lobe pathology is associated with rapid forgetting.