Broader Geographic Sharing of Pediatric Donor Lungs Improves Pediatric Access to Transplant.

US pediatric transplant candidates have limited access to lung transplant due to the small number of donors within current geographic boundaries, leading to assertions that the current lung allocation system does not adequately serve pediatric patients. We hypothesized that broader geographic sharing of pediatric (adolescent, 12-17 years; child, <12 years) donor lungs would increase pediatric candidate access to transplant. We used the thoracic simulated allocation model to simulate broader geographic sharing. Simulation 1 used current allocation rules. Simulation 2 offered adolescent donor lungs across a wider geographic area to adolescents. Simulation 3 offered child donor lungs across a wider geographic area to adolescents. Simulation 4 combined simulations 2 and 3. Simulation 5 prioritized adolescent donor lungs to children across a wider geographic area. Simulation 4 resulted in 461 adolescent transplants per 100 patient-years on the waiting list (range 417-542), compared with 206 (range 180-228) under current rules. Simulation 5 resulted in 388 adolescent transplants per 100 patient-years on the waiting list (range 348-418) and likely increased transplant rates for children. Adult transplant rates, waitlist mortality, and 1-year posttransplant mortality were not adversely affected. Broader geographic sharing of pediatric donor lungs may increase pediatric candidate access to lung transplant.

The purinergic system in allotransplantation.

The purine nucleotide adenosine triphosphate (ATP) is a universal source of energy for any intracellular reaction. Under specific physiological or pathological conditions, ATP can be released into extracellular spaces, where it binds and activates the purinergic receptors system (i.e. P2X, P2Y and P1 receptors). Extracellular ATP (eATP) binds to P2X or P2Y receptors in immune cells, where it mediates proliferation, chemotaxis, cytokine release, antigen presentation and cytotoxicity. eATP is then hydrolyzed by ectonucleotidases into adenosine diphosphate (ADP), which activates P2Y receptors. Ectonucleotidases also hydrolyze ADP to adenosine monophosphate and adenosine, which binds P1 receptors. In contrast to P2X and P2Y receptors, P1 receptors exert mainly an inhibitory effect on the immune response. In transplantation, a prominent role has been demonstrated for the eATP/P2X7R axis; the targeting of this pathway in fact is associated with long-term graft function and reduced graft versus host disease severity in murine models. Novel P2X receptor inhibitors are available for clinical use and are under assessment as immunomodulatory agents. In this review, we will focus on the relevance of the purinergic system and on the potential benefits of targeting this system in allograft rejection and tolerance.

Lung transplantation in children following bone marrow transplantation: a multi-center experience.

Allogenic BMT has been successfully performed as a treatment for hematologic diseases with an expected long-term survival. This survival is truncated by respiratory complications including airway obstruction especially BO. Chronic GVHD has been reported to precede almost all cases reported. LTx has become a therapeutic life-saving option for patients with end-stage lung disease that maybe offered for the treatment of GVHD. We report a multi-center experience of pediatric LTx following BMT in 11 patients age- and gender-matched with 11 controls who received LTx for end-stage lung disease secondary to CF. Overall death was 36.4% over a follow-up period of 19 months (range 3-36 months) for the cases and 27.3% for the control group followed for 17 months (range 8-32 months). Median FEV1 one yr post-transplant for the cases was 78% predicted compared with 67.3% predicted for the controls. The median for episodes of infection was comparable at a median of one episode per patient through the entire follow-up period among both groups. Acute rejection episodes were significantly higher in the control group with a median of one episode per patient in the control group compared to none within the cases. Our data suggest that LTx may be a valuable therapeutic option for children with end-stage lung disease post-BMT with comparable survival outcome to that after LTx in children for other indications such as CF. Hospital stay was significantly longer in our case group. Infection rate was comparable between groups albeit type of infection varied. Significantly and of interest is that acute rejection episodes were non-existent in these cases.

Neonatal respiratory failure due to a novel mutation in the surfactant protein C gene.

A full-term infant developed respiratory distress immediately after birth, requiring a prolonged course of extra-corporeal membrane oxygenation, followed by high-frequency ventilation. She was unable to wean off mechanical ventilation, required tracheostomy, and ultimately lung transplantation. A novel mutation in the surfactant C protein gene was identified as the cause of her lung disease.

Respiratory viral infections within one year after pediatric lung transplant.

To characterize epidemiology and risk factors for respiratory viral infections (RVI) in pediatric lung transplant recipients within the first post-transplant year, a retrospective multicenter study of pediatric lung transplant recipients from 1988 to 2005 was conducted at 14 centers in the United States and Europe. Data were recorded for 1 year post transplant. Associations between RVI and continuous and categorical risk factors were assessed using Wilcoxon's rank-sum and chi(2) tests, respectively. Associations between time to RVI and risk factors or survival were assessed by multivariable Cox proportional hazards models. Of 576 subjects, 79 subjects (14%) had 101 RVI in the first year post transplant. Subjects with RVI were younger than those without RVI (median ages 9.7, 13; P<0.01). Viruses detected included adenovirus (n=25), influenza (n=9), respiratory syncytial virus (n=21), parainfluenza virus (n=19), enterovirus (n=4), and rhinovirus (n=22). In a multivariable model for time to first RVI, etiology other than cystic fibrosis (CF), younger age, and no induction therapy were independently associated with risk of RVI. Cytomegalovirus serostatus and acute rejection were not associated with RVI. RVI was independently associated with decreased 12-month survival (hazard ratio 2.6, 95% confidence interval 1.6-4.4). RVI commonly occurs after pediatric lung transplantation with risk factors including younger age and non-CF diagnosis. RVI is associated with decreased 1-year survival.

Nonviral gene delivery with indoleamine 2,3-dioxygenase targeting pulmonary endothelium protects against ischemia-reperfusion injury.

Pulmonary endothelial dysfunction induced by inflammation and inflammation-associated reactive oxygen species is a central component in the pathophysiology of lung transplant ischemia-reperfusion (IR) injury. Indoleamine-2,3-dioxygenase (IDO) is a unique cytosolic enzyme possessing both immune modulating and antioxidant properties. This study investigated whether enhanced pulmonary endothelial IDO activity by a targeted nonviral gene transfer approach ameliorates lung IR injury. Orthotopic syngeneic lung transplants were performed in Lewis rats. A human IDO (hIDO)-expressing plasmid driven by an endothelial cell-specific endothelin-1 promoter was generated and intravenously delivered to donor lung using cationic polymer polyethylenimine. This nonviral gene transfer approach augmented hIDO expression specifically in endothelial cells within lung grafts. Importantly, enhanced IDO activity induced by the hIDO transgene prevented endothelial cell apoptosis, reduced vascular permeability and leukocyte extravasation, and consequently improved graft function and histologic appearance. Furthermore, our in vitro studies showed that increased IDO activity in endothelial cells protected its mitochondrial function and ultrastructure from oxidative stress through stabilization of intracellular redox status. The approach used in these experiments has properties that could eliminate the inherent side effects associated with viral vectors and/or antibody-directed targeted therapy, and thus may represent a potential therapeutic strategy against lung IR injury in patients.

American Society of Transplantation executive summary on pediatric lung transplantation.

Lung transplantation in children poses distinctly different challenges from those seen in the adult population. This consensus statement reviews the experience in the field of pediatric lung transplantation and highlights areas that deserve further investigation.

Analysis of early deaths after isolated lung transplantation.

STUDY OBJECTIVES: To determine the causes of death in patients dying within 30 days after lung transplantation at the University of Florida, to assess the importance of several diagnostic modalities for determining the causes of their decline, and to construct an algorithm for the evaluation of patients with severe respiratory compromise occurring early after lung transplantation. DESIGN: Retrospective review of medical records and pathology slides from all patients dying within 30 days after lung transplantation, and biopsy specimen diagnoses from all lung allograft recipients at the University of Florida. PATIENTS: Nine deaths occurred during the first 30 days after transplantation among 117 patients undergoing 123 isolated lung transplantation operations. RESULTS: Infections accounted for the greatest number of deaths (bacterial pneumonia, four patients; catheter-related bacteremia, one patient). Persistent pneumonia confirmed by biopsy specimen was usually accompanied by histologic manifestations of acute cellular rejection and was associated with poor patient outcome (ie, death or subsequent development of bronchiolitis obliterans syndrome). In two patients, antibody-mediated rejection either was the immediate cause of death (hyperacute rejection, one patient) or preceded a fatal case of pneumonia (accelerated antibody-mediated rejection, one patient). Other causes of death included hypoxic-ischemic encephalopathy secondary to an intraoperative cardiac arrest (one patient), pulmonary venous thrombosis with bacterial colonization of the thrombotic material (one patient), and ischemic reperfusion injury (one patient). In most patients, more than one type of diagnostic technique was needed to ascertain the cause of the catastrophic decline. CONCLUSIONS: The causes of early posttransplant death in our patient group included infections, antibody-mediated rejection, hypoxic-ischemic encephalopathy secondary to cardiac arrest, pulmonary venous thrombosis, and ischemic reperfusion injury. Because these processes often demonstrate overlapping clinical and morphologic features requiring multiple diagnostic techniques for resolution, a systematic multimodality approach to diagnosis is advantageous for determining the causes of decline in individual patients and for estimating the incidences of the different causes of early graft and patient loss in the lung transplant population.

Smad7-dependent regulation of heme oxygenase-1 by transforming growth factor-beta in human renal epithelial cells.

Heme oxygenase-1 (HO-1), a 32-kDa microsomal enzyme, is induced as a beneficial and adaptive response in cells/tissues exposed to oxidative stress. Transforming growth factor-beta1 (TGF-beta1) is a regulatory cytokine that has been implicated in a variety of renal diseases where it promotes extracellular matrix deposition and proinflammatory events. We hypothesize that the release of TGF-beta1 via autocrine and/or paracrine pathways may induce HO-1 and serve as a protective response in renal injury. To understand the molecular mechanism of HO-1 induction by TGF-beta1, we exposed confluent human renal proximal tubule cells to TGF-beta1 and observed a significant induction of HO-1 mRNA at 4 h with a maximal induction at 8 h. This induction was accompanied by increased expression of HO-1 protein. TGF-beta1 treatment in conjunction with actinomycin D or cycloheximide demonstrated that induction of HO-1 mRNA requires de novo transcription and, in part, protein synthesis. Exposure to TGF-beta1 resulted in marked induction of Smad7 mRNA with no effect on Smad6 expression. Overexpression of Smad7, but not Smad6, inhibited TGF-beta1-mediated induction of endogenous HO-1 gene expression. We speculate that the induction of HO-1 in the kidney is an adaptive response to the inflammatory effects of TGF-beta1 and manipulations of the Smad pathway to alter HO-1 expression may serve as a potential therapeutic target.

Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis.

Heme oxygenase-1 (HO-1) is a 32-kDa microsomal enzyme that catalyzes the conversion of heme to biliverdin, releasing iron and carbon monoxide. Induction of HO-1 occurs as a protective response in cells/tissues exposed to a wide variety of oxidant stimuli. The chemotherapeutic effects of cis-diamminedichloroplatinum(II) (cisplatin), a commonly used anticancer drug, are limited by significant nephrotoxicity, which is characterized by varying degrees of renal tubular apoptosis and necrosis. The purpose of this study was to evaluate the functional significance of HO-1 expression in cisplatin-induced renal injury. Our studies demonstrate that transgenic mice deficient in HO-1 (-/-), develop more severe renal failure and have significantly greater renal injury compared with wild-type (+/+) mice treated with cisplatin. In vitro studies in human renal proximal tubule cells demonstrate that hemin, an inducer of HO-1, significantly attenuated cisplatin-induced apoptosis and necrosis, whereas inhibition of HO-1 enzyme activity reversed the cytoprotective effect. Overexpression of HO-1 resulted in a significant reduction in cisplatin-induced cytotoxicity. These studies provide a basis for future studies using targeted gene expression of HO-1 as a therapeutic and preventive modality in high-risk settings of acute renal failure.

Transcriptional regulation and structural organization of the human cytosolic phospholipase A(2) gene.

Cytokines are established regulators of the arachidonic acid cascade in lung cells. The levels of various arachidonic metabolites distinguish the normal and pathogenic states of the human lung. Arachidonyl-selective cytosolic phospholipase A(2) (cPLA(2)) is ubiquitously present in human lung and is most likely the rate-limiting step in eicosanoid generation. We therefore studied the regulation of this pivotal gene in human lung fibroblasts and epithelial cells by proinflammatory cytokines. We demonstrate a dose- and time-dependent induction of human cPLA(2) mRNA by interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma as well as the abrogation of this induction by glucocorticoids. Nuclear runoff studies demonstrate that de novo transcription of the cPLA(2) gene is required for cytokine induction. We have characterized the human cPLA(2) gene, which is encoded by 18 exons and spans in excess of 137 kb. Deletion analysis of a 3.4-kb fragment of the human promoter identified two regions responsible for basal expression of the cPLA(2) gene. Conversely, a CA-dinucleotide repeat in the proximal promoter appears to repress overall promoter activity. Understanding the molecular mechanisms associated with cytokine-dependent expression of the cPLA(2) gene should provide further insight into regulating the level of proinflammatory mediators in pulmonary diseases.

Iron regulates hyperoxia-dependent human heme oxygenase 1 gene expression in pulmonary endothelial cells.

The endothelium of the lung is sensitive to the toxic effects of oxygen, and early evidence of toxicity is characterized by protein leak and extravasation of red blood cells. The overproduction of oxygen free radicals plays a critical role in the pathophysiology of a hyperoxic lung injury. Recently, heme oxygenase 1 (HO-1), the rate-limiting enzyme in the metabolism of heme, has been found to have a protective role in oxidant injury. Our laboratory and others have identified HO-1 as a hyperoxia-inducible protein. In this study, we characterized HO-1 expression and evaluated its regulation in human pulmonary endothelial cells. Hyperoxia results in a relatively small increase in HO-1 expression; however, this induction is potentiated by heme and dramatically potentiated in the presence of free iron. This is probably more reflective of the in vivo situation in which there is extravasation of heme and iron products. We also found that HO-1 expression depended on chelatable iron. The iron chelator desferrioxamine not only inhibited the iron- dependent potentiation of HO-1 in response to hyperoxia but also inhibited both hyperoxia and basal expression. On the basis of inhibitor studies and nuclear run-on assays, we demonstrated that this induction is transcriptionally dependent. We also evaluated 4.5 kb of the human HO-1 promoter region and demonstrated that this region has promoter activity to the stimulus heme; however, there was no evidence of promoter activity to either iron or hyperoxia. This diversity of promoter activity to heme, heavy metals, and hyperoxia is unique to the human HO-1 gene.

Linoleyl hydroperoxide transcriptionally upregulates heme oxygenase-1 gene expression in human renal epithelial and aortic endothelial cells.

Atherogenic lipoproteins such as oxidized LDL are implicated in the pathogenesis of atherosclerosis and renal disease. Fatty acid hydroperoxides and phospholipids such as linoleyl hydroperoxide (LAox or 13-HPODE) and lysophosphatidylcholine (lyso-PC), abundant components of oxidized LDL, mediate the effects of atherogenic lipids. Oxidized LDL has been shown to induce heme oxygenase-1 (HO-1), a microsomal enzyme that is involved in heme detoxification and is a major endogenous source of carbon monoxide. HO-1 is also induced by many other stimuli that shift cellular redox. To identify the constituents and molecular mechanisms of oxidized LDL-mediated HO-1 induction, human renal epithelial cells and aortic endothelial cells were exposed to LAox and lyso-PC. Exposure to LAox (25 microM) showed an approximately 16-fold induction of HO-1 mRNA, whereas exposure to lyso-PC (25 microM) showed only an approximate 2.6-fold increase. Treatment with actinomycin-D (4 microM), a transcriptional inhibitor, as well as nuclear run-on assays, demonstrated that LAox-mediated HO-1 gene induction is dependent on de novo transcription. Cycloheximide did not affect LAox-mediated HO-1 mRNA induction, suggesting that new protein synthesis is not required for transcriptional induction. Transfection of a human HO-1 promoter-reporter gene construct showed that LAox upregulation of HO-1 occurs via mechanisms different from those of known inducers, heme and cadmium. These studies are the first demonstration that LAox induces HO-1 by transcriptional mechanisms and may have implications in the pathogenesis of cell injury in atherosclerosis and progressive renal disease.

Thrombin regulation of endothelin-1 gene in isolated human pulmonary endothelial cells.

Endothelin (ET)-1 is a potent vasoconstrictor elicited from endothelial cells in response to a variety of stimuli and an important mediator for a variety of vascular diseases including pulmonary hypertension. In this paper, we describe the molecular regulation of the ET-1 gene in response to a vasoactive mediator, thrombin, in human pulmonary endothelial cells. Thrombin induces preproET-1 mRNA through a transcriptionally dependent mechanism, with a peak induction after 1 h of exposure. Analysis of chromatin structure identified several DNase I-hypersensitive regions under both basal and thrombin-stimulated conditions that reside in the 5'-promoter region, indicating that the ET-1 promoter is a constitutive promoter. Deletion analysis was employed as a functional assay to identify regions of the ET-1 promoter that are important in transcriptional regulation. We found that sites between -141 and -378 bp are essential for basal activity and that those between -378 and -484 bp are essential for thrombin-stimulated activity. However, full expression under both conditions required an element(s) within -952 bp.

Identification of urokinase as a hyperoxia-inducible gene.

Hyperoxia has deleterious effects on lung form and function; however, the molecular events initiated by oxygen exposure remain unclear. We hypothesized that macrophages function as important intermediaries in the protective response of lung tissues after exposure to hyperoxia. This hypothesis was tested by exposing cultured macrophages (RAW 264.7 cells) to hyperoxia for 24 h and then applying the conditioned medium from these cells to cultured pulmonary epithelial cells or to pulmonary microvascular endothelial cells. We observed that the expression of manganese superoxide dismutase mRNA increased in both target cell lines. Therefore, we next hypothesized that exposure of these macrophages to hyperoxia results in a change in gene expression which could be detected by differential display PCR (ddPCR). This hypothesis was tested by exposing RAW 264.7 cells to > or = 95% oxygen (or normoxia) for 24 h, harvesting RNA, and performing ddPCR. A cDNA fragment upregulated by hyperoxia was identified and reamplified. Verification of differential expression of mRNA was done by Northern analysis. A mRNA which was reproducibly upregulated by hyperoxia, as well as by lipopolysaccharide and interferon gamma, was identified. The differentially expressed PCR product was cloned and sequenced, revealing a product with 99% identity to mouse urokinase mRNA. We speculate that one function of pulmonary macrophages following a hyperoxic exposure is to secrete urokinase.

Hyperoxia-responsive proteins in rat pulmonary microvascular endothelial cells.

Exposure to high partial pressures of oxygen are toxic to the lung, and much of the damage observed is related to injury of the pulmonary microvasculature. In this study, we evaluated the response of the pulmonary microvascular endothelial cell to high oxygen concentrations, using two-dimensional protein gel electrophoresis as a direct molecular assay of differences between cells exposed to room air or hyperoxia. We observed a differential expression of five specific proteins within 24 h of a hyperoxic insult that we termed hyperoxia-responsive proteins. After 4 h of hyperoxia there was a decrease in two of the proteins. From 8 to 24 h we observed a repression of a third and an induction of the other two proteins. One of the induced proteins was also increased by heat shock and hydrogen peroxide and has characteristics similar to heat shock protein (HSP) 32 (heme oxygenase 1). Western analysis using an antibody specific to rat heme oxygenase 1 verified that this oxygen-responsive protein is heme oxygenase 1. The response of the other four hyperoxia-responsive proteins appears to be specific to oxygen and not a general stress response, since they were not changed in response to heat shock or hydrogen peroxide. Based on RNA inhibitor and pulse chase experiments, these changes may result from transcriptional/posttranscriptional mechanisms or hyperoxia-dependent protein turnover.

Effects of vasoactive and inflammatory mediators on endothelin-1 expression in pulmonary endothelial cells.

Endothelin-1 (ET-1) is known to be involved in a variety of pathophysiologic conditions, especially of the pulmonary vasculature. The aim of this study was to investigate physiologic mediators potentially involved in the pathogenesis of pulmonary hypertension, for their effects on ET-1 gene expression at both the transcriptional and translational level. Rat microvascular and pulmonary artery endothelial cells grown in culture were exposed to vasoactive mediators (thrombin or an anoxic gas mixture) and inflammatory mediators (lipopolysaccharide, interleukin 1 alpha, interleukin 1 beta, or tumor necrosis factor alpha) for various time periods. The change in prepro-ET-1 (ppET-1) mRNA levels in these cells in response to stimuli was a time-dependent phenomenon. The inflammatory mediators caused an acute rise in ppET-1 mRNA levels whereby peak induction occurred after 1 h with a rapid decline to control levels by 4 h. The vasoactive mediators elicited a more sustained response whereby a significant elevation in ppET-1 mRNA expression occurred quickly and remained elevated through 4 h. The pattern of induction was more rapid for thrombin than for anoxic gas exposure. Radioimmunoassay analysis demonstrated a similar response for thrombin and the inflammatory mediators in ET-1 mature peptide release, whereas the effect of anoxic gas exposure was divergent. Significant elevations were noted after 6 h for thrombin as well as each of the inflammatory mediators except IL-1 alpha. In response to the anoxic gas exposure, however, a significant rise in ET-1 peptide release was not evident until after 24 h. To determine the level at which ppET-1 mRNA induction is regulated, cells were cotreated with each of the stimuli and actinomycin D or cycloheximide. Results indicate that the induction of ppET-1 mRNA levels is likely due to de novo transcription, as well as mRNA stabilization. In summary, inflammatory and vasoactive agents are important regulators of ET-1 gene expression in rat pulmonary endothelial cells; most important, we observed a differential response at the mRNA or peptide level depending on the mediator involved.

Endothelin-1 expression in myenteric neurons cultured from rat small intestine.

Endothelin is a potent vasoactive peptide. More recently, endothelin-1 (ET-1) has been found in neural tissues such as spinal cord, brain and peripheral ganglion cells. Inagaki (Gastroenterology 101 (1991) 47) reported evidence of ET-1-like immunoreactivity in enteric neurons, but there are no reports of ET-1 peptide or mRNA expression specifically in myenteric neurons. Using a primary culture of myenteric neurons, we set out to evaluate ET-1 peptide and mRNA expression. Myenteric neurons were cultured using a dissection and enzyme dispersion technique. ET-1 reactivity was localized to neurons and ET-1 levels from cells and media were assayed by radioimmunoassay under a variety of media conditions or with depolarizing buffer or veratridine (75 microM). Prepro ET-1 mRNA expression was determined by Northern analysis of total RNA utilizing a rat ET-1 cDNA. ET-1 immunoreactivity was observed almost exclusively in myenteric neurons. Cells contained 0.78 pg/micrograms protein and did not vary with variations in media conditions. Basal release/secretion into media occurred but was not enhanced by depolarizing media or veratridine. High levels of ET-1 mRNA expression were identified. These results of high level constitutive expression of ET-1 linked with previous reports of ET-1 modulation of cholinergic intestinal smooth muscle contraction suggest a neuromodulatory role.