Canadian Guidelines for Controlled Pediatric Donation After Circulatory Determination of Death-Summary Report

OBJECTIVES: Create trustworthy, rigorous, national clinical practice guidelines for the practice of pediatric donation after circulatory determination of death in Canada. METHODS: We followed a process of clinical practice guideline development based on World Health Organization and Canadian Medical Association methods. This included application of Grading of Recommendations Assessment, Development, and Evaluation methodology. Questions requiring recommendations were generated based on 1) 2006 Canadian donation after circulatory determination of death guidelines (not pediatric specific), 2) a multidisciplinary symposium of national and international pediatric donation after circulatory determination of death leaders, and 3) a scoping review of the pediatric donation after circulatory determination of death literature. Input from these sources drove drafting of actionable questions and Good Practice Statements, as defined by the Grading of Recommendations Assessment, Development, and Evaluation group. We performed additional literature reviews for all actionable questions. Evidence was assessed for quality using Grading of Recommendations Assessment, Development, and Evaluation and then formulated into evidence profiles that informed recommendations through the evidence-to-decision framework. Recommendations were revised through consensus among members of seven topic-specific working groups and finalized during meetings of working group leads and the planning committee. External review was provided by pediatric, critical care, and critical care nursing professional societies and patient partners. RESULTS: We generated 63 Good Practice Statements and seven Grading of Recommendations Assessment, Development, and Evaluation recommendations covering 1) ethics, consent, and withdrawal of life-sustaining therapy, 2) eligibility, 3) withdrawal of life-sustaining therapy practices, 4) ante and postmortem interventions, 5) death determination, 6) neonatal pediatric donation after circulatory determination of death, 7) cardiac and innovative pediatric donation after circulatory determination of death, and 8) implementation. For brevity, 48 Good Practice Statement and truncated justification are included in this summary report. The remaining recommendations, detailed methodology, full Grading of Recommendations Assessment, Development, and Evaluation tables, and expanded justifications are available in the full text report. CONCLUSIONS: This process showed that rigorous, transparent clinical practice guideline development is possible in the domain of pediatric deceased donation. Application of these recommendations will increase access to pediatric donation after circulatory determination of death across Canada and may serve as a model for future clinical practice guideline development in deceased donation

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.

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.

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.

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.

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.

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.

Autophagy and heart disease: implications for cardiac ischemia-reperfusion damage.

Survival of myocytes and mesenchymal cells in the heart is tightly regulated by a number of adaptive processes that are invoked with the changes that occur within the parenchyma and stroma. Autophagy is implicated in cellular housekeeping duties and maintenance of the integrity of the intracellular milieu by removal of protein aggregates and damaged organelles, whereas under pathophysiological conditions, the chronic up-regulation of autophagy may lead to significant disturbance of homeostatic conditions. Nonetheless, the role of autophagy in heart disease in the context of cardiac ischemia-reperfusion injury is currently unclear. This review will focus upon the role of autophagy as it pertains to ischemia reperfusion damage in the heart.

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.

Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts.

3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are cholesterol-lowering drugs that exert other cellular effects and underlie their beneficial health effects, including those associated with myocardial remodeling. We recently demonstrated that statins induces apoptosis and autophagy in human lung mesenchymal cells. Here, we extend our knowledge showing that statins simultaneously induces activation of the apoptosis, autophagy and the unfolded protein response (UPR) in primary human atrial fibroblasts (hATF). Thus we tested the degree to which coordination exists between signaling from mitochondria, endoplasmic reticulum and lysosomes during response to simvastatin exposure. Pharmacologic blockade of the activation of ER-dependent cysteine-dependent aspartate-directed protease (caspase)-4 and lysosomal cathepsin-B and -L significantly decreased simvastatin-induced cell death. Simvastatin altered total abundance and the mitochondrial fraction of proapoptotic and antiapoptotic proteins, while c-Jun N-terminal kinase/stress-activated protein kinase mediated effects on B-cell lymphoma 2 expression. Chemical inhibition of autophagy flux with bafilomycin-A1 augmented simvastatin-induced caspase activation, UPR and cell death. In mouse embryonic fibroblasts that are deficient in autophagy protein 5 and refractory to autophagy induction, caspase-7 and UPR were hyper-induced upon treatment with simvastatin. These data demonstrate that mevalonate cascade inhibition-induced death of hATF manifests from a complex mechanism involving co-regulation of apoptosis, autophagy and UPR. Furthermore, autophagy has a crucial role in determining the extent of ER stress, UPR and permissiveness of hATF to cell death induced by statins.

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.

K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts.

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.

The equivalence between off-resonance and on-resonance pulse sequences and its application to steady-state free precession with diffusion in inhomogeneous fields.

We show that the spin dynamics of any pulse sequence with off-resonant pulses is identical to that of a modified sequence with on-resonant pulses, including relaxation and diffusion effects. This equivalence applies to pulse sequences with arbitrary offset frequency deltaomega(0) which may exceed the RF field strength omega(1). Using this approach, we examine steady-state free precession (SSFP) in grossly inhomogeneous fields. We show explicitly that the magnitude of the magnetization for each mode at an offset frequency deltaomega(0) is equal to that for SSFP with on-resonance pulses of rescaled amplitude, with the same dependence on relaxation times and diffusion coefficient. The rescaling depends on offset frequency and RF field strength. The theoretical results have been tested experimentally and excellent agreement is found.

Coexistence of Alzheimer disease neuropathology with herpes simplex encephalitis.

Several unusual features were observed during routine histopathological confirmation of a clinical diagnosis of Alzheimer's disease (AD) in an 85-year-old, right-handed, married male. The patient presented with a 12-year history of slowly progressive cognitive impairment, which increased in severity just prior to death. Detailed postmortem examination of the frontal lobes revealed a significant number of neuritic plaques and neurofibrillary tangles. Multifocal spongiform encephalopathic changes, mononuclear perivascular infiltrates, subcortical demyelination and gliosis were also found. Of particular interest were well-defined neuronal and astrocytic intranuclear inclusion bodies (Cowdry type I and I), suggestive of viral disease. Electron microscopy, immunohistochemical and immunohistofluorescent studies confirmed a Herpes simplex type I encephalitis (HSV-I). These histological results and the clinical history of progression suggest that reactivation of a latent viral infection may have contributed to the rapid progression of dementia prior to death. The present analysis underscores the fact that multiple etiologic factors may act simultaneously to produce dementia. While one such process may be identified or diagnosed (in the present case AD), it is necessary to be open to the possibility that another mechanism may come into play during the time course of that illness. A differential diagnosis may be difficult when the symptoms of the two disease processes are very similar. Such may be the case if there is reactivation of a previously undiagnosed herpes virus infection. With the development of PCR and in situ hybridization diagnosis will be simplified and more definitive.

Surgical management of isolated hemangioblastomas of the spinal cord.

OBJECTIVE: Intramedullary hemangioblastomas are rare tumors, accounting for just 3% of all intraspinal neoplasms. The purpose of this study is to define the occurrence of isolated intramedullary hemangioblastomas and to analyze the role of the radiological studies and surgery for these lesions. METHODS: The charts of 19 consecutive patients operated on for isolated spinal intramedullary hemangioblastoma were reviewed. Preoperatively, all patients underwent magnetic resonance imaging and nine underwent spinal angiography. For all patients, the surgical approach was via posterior laminectomy. RESULTS: Our study sample comprised 6 women and 13 men, with an average age of 31.5 years (range, 16-75 yr). The mean prodrome was 20.8 months. Pain was the most common complaint. In all cases, the neoplasms were associated with a syrinx or edema. Gross total resection was achieved in all patients. At last follow-up examination (mean, 50.1 mo), 13 patients (68%) had improved and 6 patients (32%) had stabilized as compared with their preoperative clinical status. CONCLUSION: Isolated intramedullary hemangioblastomas typically have an indolent clinical course. These tumors have characteristic imaging properties on magnetic resonance imaging and angiography. Surgical removal of these lesions results in excellent long-term functional outcome.

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.

Relevance of torsional effects to the conformational equilibria of 1,5-diaza-cis-decalins: a theoretical and experimental study.

1,5-Diaza-cis-decalin populates two conformations in which the nitrogen atoms are either gauche (N-in) or anti (N-out) to one another. The equilibrium mixture of the two conformers depends on the substituents at the nitrogen atom, as well as the reaction conditions. Ab initio (HF/6-31G, B3LYP/6-31+G) and molecular mechanics (Amber) calculations have been performed to examine the possible role of stereoelectronics and steric effects in controlling the equilibrium of substituted 1,5-diaza-cis-decalins. In the present study, N,N'-diethyl- and N,N'-bistrifluoroethyl-1,5-diaza-cis-decalins have been synthesized, and the equilibrium mixtures have been measured using 1H and 13C NMR experiments. Steric effects appear to control the equilibria between the two conformational isomers of 1,5-diaza-cis-decalin while torsional effects appear to dominate the equilibria for the N,N'-dialkyl derivatives.

Evaluation of cytotoxic T-lymphocyte responses in human and nonhuman primate subjects infected with human immunodeficiency virus type 1 or simian/human immunodeficiency virus.

Cytotoxic T-lymphocyte (CTL) responses have been implicated as playing an important role in control of human immunodeficiency virus (HIV) infection. However, it is technically difficult to demonstrate CTL responses consistently in nonhuman primate and human subjects using traditional cytotoxicity assay methods. In this study, we systematically evaluated culture conditions that may affect the proliferation and expansion of CTL effector cells and presented a sensitive method for detection of cytotoxicity responses with bulk CTL cultures. We confirmed the sensitivity and specificity of this method by demonstration of vigorous CTL responses in a simian-HIV (SHIV)-infected rhesus macaque. The expansion of epitope-specific CTL effector cells was also measured quantitatively by CTL epitope-major histocompatibility complex tetramer complex staining. In addition, two new T-cell determinants in the SIV gag region are identified. Last, we showed the utility of this method for studying CTL responses in chimpanzee and human subjects.