Peripheral Blood Gene Expression Changes Associated With Primary Graft Dysfunction After Lung Transplantation.

Recipient responses to primary graft dysfunction (PGD) after lung transplantation may have important implications to the fate of the allograft. We therefore evaluated longitudinal differences in peripheral blood gene expression in subjects with PGD. RNA expression was measured throughout the first transplant year in 106 subjects enrolled in the Clinical Trials in Organ Transplantation-03 study using a panel of 100 hypothesis-driven genes. PGD was defined as grade 3 in the first 72 posttransplant hours. Eighteen genes were differentially expressed over the first year based on PGD development, with significant representation from innate and adaptive immunity genes, with most differences identified very early after transplant. Sixteen genes were overexpressed in the blood of patients with PGD compared to those without PGD within 7 days of allograft reperfusion, with most transcripts encoding innate immune/inflammasome-related proteins, including genes previously associated with PGD. Thirteen genes were underexpressed in patients with PGD compared to those without PGD within 7 days of transplant, highlighted by T cell and adaptive immune regulation genes. Differences in gene expression present within 2 h of reperfusion and persist for days after transplant. Future investigation will focus on the long-term implications of these gene expression differences on the outcome of the allograft.

The Perioperative Lung Transplant Virome: Torque Teno Viruses Are Elevated in Donor Lungs and Show Divergent Dynamics in Primary Graft Dysfunction.

Primary graft dysfunction (PGD) is a principal cause of early morbidity and mortality after lung transplantation, but its pathogenic mechanisms are not fully clarified. To date, studies using standard clinical assays have not linked microbial factors to PGD. We previously used comprehensive metagenomic methods to characterize viruses in lung allografts >1 mo after transplant and found that levels of Anellovirus, mainly torque teno viruses (TTVs), were significantly higher than in nontransplanted healthy controls. We used quantitative polymerase chain reaction to analyze TTV and shotgun metagenomics to characterize full viral communities in acellular bronchoalveolar lavage from donor organs and postreperfusion allografts in PGD and non-PGD lung transplant recipient pairs. Unexpectedly, TTV DNA levels were elevated 100-fold in donor lungs compared with healthy adults (p = 0.0026). Although absolute TTV levels did not differ by PGD status, PGD cases showed a smaller increase in TTV levels from before to after transplant than did control recipients (p = 0.041). Metagenomic sequencing revealed mainly TTV and bacteriophages of respiratory tract bacteria, but no viral taxa distinguished PGD cases from controls. These findings suggest that conditions associated with brain death promote TTV replication and that greater immune activation or tissue injury associated with PGD may restrict TTV abundance in the lung.

Protein Quantitative Trait Loci Analysis Identifies Genetic Variation in the Innate Immune Regulator TOLLIP in Post-Lung Transplant Primary Graft Dysfunction Risk.

The authors previously identified plasma plasminogen activator inhibitor-1 (PAI-1) level as a quantitative lung injury biomarker in primary graft dysfunction (PGD). They hypothesized that plasma levels of PAI-1 used as a quantitative trait could facilitate discovery of genetic loci important in PGD pathogenesis. A two-stage cohort study was performed. In stage 1, they tested associations of loci with PAI-1 plasma level using linear modeling. Genotyping was performed using the Illumina CVD Bead Chip v2. Loci meeting a p < 5 × 10(-4) cutoff were carried forward and tested in stage 2 for association with PGD. Two hundred ninety-seven enrollees were evaluated in stage 1. Six loci, associated with PAI-1, were carried forward to stage 2 and evaluated in 728 patients. rs3168046 (Toll interacting protein [TOLLIP]) was significantly associated with PGD (p = 0.006). The increased risk of PGD for carrying at least one copy of this variant was 11.7% (95% confidence interval 4.9-18.5%). The false-positive rate for individuals with this genotype who did not have PGD was 6.1%. Variants in the TOLLIP gene are associated with higher circulating PAI-1 plasma levels and validate for association with clinical PGD. A protein quantitative trait analysis for PGD risk prioritizes genetic variations in TOLLIP and supports a role for Toll-like receptors in PGD pathogenesis.

Objective Estimates Improve Risk Stratification for Primary Graft Dysfunction after Lung Transplantation.

Primary graft dysfunction (PGD) is a major cause of early mortality after lung transplant. We aimed to define objective estimates of PGD risk based on readily available clinical variables, using a prospective study of 11 centers in the Lung Transplant Outcomes Group (LTOG). Derivation included 1255 subjects from 2002 to 2010; with separate validation in 382 subjects accrued from 2011 to 2012. We used logistic regression to identify predictors of grade 3 PGD at 48/72 h, and decision curve methods to assess impact on clinical decisions. 211/1255 subjects in the derivation and 56/382 subjects in the validation developed PGD. We developed three prediction models, where low-risk recipients had a normal BMI (18.5-25 kg/m(2) ), chronic obstructive pulmonary disease/cystic fibrosis, and absent or mild pulmonary hypertension (mPAP<40 mmHg). All others were considered higher-risk. Low-risk recipients had a predicted PGD risk of 4-7%, and high-risk a predicted PGD risk of 15-18%. Adding a donor-smoking lung to a higher-risk recipient significantly increased PGD risk, although risk did not change in low-risk recipients. Validation demonstrated that probability estimates were generally accurate and that models worked best at baseline PGD incidences between 5% and 25%. We conclude that valid estimates of PGD risk can be produced using readily available clinical variables.

Preoperative plasma club (clara) cell secretory protein levels are associated with primary graft dysfunction after lung transplantation.

Inherent recipient factors, including pretransplant diagnosis, obesity and elevated pulmonary pressures, are established primary graft dysfunction (PGD) risks. We evaluated the relationship between preoperative lung injury biomarkers and PGD to gain further mechanistic insight in recipients. We performed a prospective cohort study of recipients in the Lung Transplant Outcomes Group enrolled between 2002 and 2010. Our primary outcome was Grade 3 PGD on Day 2 or 3. We measured preoperative plasma levels of five biomarkers (CC-16, sRAGE, ICAM-1, IL-8 and Protein C) that were previously associated with PGD when measured at the postoperative time point. We used multivariable logistic regression to adjust for potential confounders. Of 714 subjects, 130 (18%) developed PGD. Median CC-16 levels were elevated in subjects with PGD (10.1 vs. 6.0, p<0.001). CC-16 was associated with PGD in nonidiopathic pulmonary fibrosis (non-IPF) subjects (OR for highest quartile of CC-16: 2.87, 95% CI: 1.37, 6.00, p=0.005) but not in subjects with IPF (OR 1.38, 95% CI: 0.43, 4.45, p=0.59). After adjustment, preoperative CC-16 levels remained associated with PGD (OR: 3.03, 95% CI: 1.26, 7.30, p=0.013) in non-IPF subjects. Our study suggests the importance of preexisting airway epithelial injury in PGD. Markers of airway epithelial injury may be helpful in pretransplant risk stratification in specific recipients.

Donor age and early graft failure after lung transplantation: a cohort study.

Lungs from older adult organ donors are often unused because of concerns for increased mortality. We examined associations between donor age and transplant outcomes among 8860 adult lung transplant recipients using Organ Procurement and Transplantation Network and Lung Transplant Outcomes Group data. We used stratified Cox proportional hazard models and generalized linear mixed models to examine associations between donor age and both 1-year graft failure and primary graft dysfunction (PGD). The rate of 1-year graft failure was similar among recipients of lungs from donors age 18-64 years, but severely ill recipients (Lung Allocation Score [LAS] >47.7 or use of mechanical ventilation) of lungs from donors age 56-64 years had increased rates of 1-year graft failure (p-values for interaction = 0.04 and 0.02, respectively). Recipients of lungs from donors <18 and ≥65 years had increased rates of 1-year graft failure (adjusted hazard ratio [HR] 1.23, 95% CI 1.01-1.50 and adjusted HR 2.15, 95% CI 1.47-3.15, respectively). Donor age was not associated with the risk of PGD. In summary, the use of lungs from donors age 56 to 64 years may be safe for adult candidates without a high LAS and the use of lungs from pediatric donors is associated with a small increase in early graft failure.

Donor surfactant protein D (SP-D) polymorphisms are associated with lung transplant outcome.

Chronic lung allograft dysfunction (CLAD) is the major factor limiting long-term success of lung transplantation. Polymorphisms of surfactant protein D (SP-D), an important molecule within lung innate immunity, have been associated with various lung diseases. We investigated the association between donor lung SP-D polymorphisms and posttransplant CLAD and survival in 191 lung transplant recipients consecutively transplanted. Recipients were prospectively followed with routine pulmonary function tests. Donor DNA was assayed by pyrosequencing for SP-D polymorphisms of two single-nucleotide variations altering amino acids in the mature protein N-terminal domain codon 11 (Met(11) Thr), and in codon 160 (Ala(160) Thr) of the C-terminal domain. CLAD was diagnosed in 88/191 patients, and 60/191 patients have died. Recipients of allografts that expressed the homozygous Met(11) Met variant of aa11 had significantly greater freedom from CLAD development and better survival compared to those with the homozygous Thr(11) Th variant of aa11. No significant association was noted for SP-D variants of aa160. Lung allografts with the SP-D polymorphic variant Thr(11) Th of aa11 are associated with development of CLAD and reduced survival. The observed genetic differences of the donor lung, potentially with their effects on innate immunity, may influence the clinical outcomes after lung transplantation.

Gene set enrichment analysis identifies key innate immune pathways in primary graft dysfunction after lung transplantation.

We hypothesized alterations in gene expression could identify important pathways involved in transplant lung injury. Broncho alveolar lavage fluid (BALF) was sampled from donors prior to procurement and in recipients within an hour of reperfusion as part of the NIAID Clinical Trials in Organ Transplantation Study. Twenty-three patients with Grade 3 primary graft dysfunction (PGD) were frequency matched with controls based on donor age and recipient diagnosis. RNA was analyzed using the Human Gene 1.0 ST array. Normalized mRNA expression was transformed and differences between donor and postreperfusion values were ranked then tested using Gene Set Enrichment Analysis. Three-hundred sixty-two gene sets were upregulated, with eight meeting significance (familywise-error rate, FWER p-value <0.05), including the NOD-like receptor inflammasome (NLR; p < 0.001), toll-like receptors (TLR; p < 0.001), IL-1 receptor (p = 0.001), myeloid differentiation primary response gene 88 (p = 0.001), NFkB activation by nontypeable Haemophilus influenzae (p = 0.001), TLR4 (p = 0.008) and TLR 9 (p = 0.018). The top five ranked individual transcripts from these pathways based on rank metric score are predominantly present in the NLR and TLR pathways, including IL1ß (1.162), NLRP3 (1.135), IL1α (0.952), IL6 (0.931) and CCL4 (0.842). Gene set enrichment analyses implicate inflammasome-mediated and innate immune signaling pathways as key mediators of the development of PGD in lung transplant patients.

Hypoalbuminemia and early mortality after lung transplantation: a cohort study.

Hypoalbuminemia predicts disability and mortality in patients with various illnesses and in the elderly. The association between serum albumin concentration at the time of listing for lung transplantation and the rate of death after lung transplantation is unknown. We examined 6808 adults who underwent lung transplantation in the United States between 2000 and 2008. We used Cox proportional hazard models and generalized additive models to examine multivariable-adjusted associations between serum albumin and the rate of death after transplantation. The median follow-up time was 2.7 years. Those with severe (0.5-2.9 g/dL) and mild hypoalbuminemia (3.0-3.6 g/dL) had posttransplant adjusted mortality rate ratios of 1.35 (95% CI: 1.12-1.62) and 1.15 (95% CI: 1.04-1.27), respectively. For each 0.5 g/dL decrease in serum albumin concentration the 1-year and overall mortality rate ratios were 1.48 (95% CI: 1.21-1.81) and 1.26 (95% CI: 1.11-1.43), respectively. The association between hypoalbuminemia and posttransplant mortality was strongest in recipients with cystic fibrosis and interstitial lung disease. Hypoalbuminemia is an independent risk factor for death after lung transplantation.

Titrated oxygen requirement and prognostication in idiopathic pulmonary fibrosis.

The supplemental oxygen flow rate is a common bedside measure of gas exchange impairment. We aimed to determine whether a titrated oxygen requirement (TOR) predicted mortality in idiopathic pulmonary fibrosis (IPF). We examined 104 adults with IPF enrolled in a prospective cohort study and a validation cohort of 151 adults with a variety of interstitial lung diseases (ILDs). The TOR was defined as the lowest oxygen flow rate required to maintain an oxyhaemoglobin saturation of 96% while standing. Cox proportional hazards models and time-dependent receiver operating characteristic curves were used to examine survival time. A higher TOR was associated with a greater mortality rate independent of forced vital capacity and 6-min walk test results in IPF (adjusted hazard ratio (per 1 L·min(-1)) 1.16, 95% CI 1.06-1.27). The TOR was at least as accurate as pulmonary function and 6-min walk testing at predicting 1-yr mortality. Findings were similar in other ILDs. The TOR is a simple, inexpensive bedside measurement that aids prognostication in IPF.

Elevated plasma long pentraxin-3 levels and primary graft dysfunction after lung transplantation for idiopathic pulmonary fibrosis.

Primary graft dysfunction (PGD) after lung transplantation may result from ischemia reperfusion injury (IRI). The innate immune response to IRI may be mediated by Toll-like receptor and IL-1-induced long pentraxin-3 (PTX3) release. We hypothesized that elevated PTX3 levels were associated with PGD. We performed a nested case control study of lung transplant recipients with idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD) from the Lung Transplant Outcomes Group cohort. PTX3 levels were measured pretransplant, and 6 and 24 h postreperfusion. Cases were subjects with grade 3 PGD within 72 h of transplantation and controls were those without grade 3 PGD. Generalized estimating equations and multivariable logistic regression were used for analysis. We selected 40 PGD cases and 79 non-PGD controls. Plasma PTX3 level was associated with PGD in IPF but not COPD recipients (p for interaction < 0.03). Among patients with IPF, PTX3 levels at 6 and 24 h were associated with PGD (OR = 1.6, p = 0.02 at 6 h; OR = 1.4, p = 0.008 at 24 h). Elevated PTX3 levels were associated with the development of PGD after lung transplantation in IPF patients. Future studies evaluating the role of innate immune activation in IPF and PGD are warranted.

Elevated plasma clara cell secretory protein concentration is associated with high-grade primary graft dysfunction.

Primary graft dysfunction (PGD) is the leading cause of early posttransplant morbidity and mortality after lung transplantation. Clara cell secretory protein (CC16) is produced by the nonciliated lung epithelium and may serve as a plasma marker of epithelial cell injury. We hypothesized that elevated levels of CC16 would be associated with increased odds of PGD. We performed a prospective cohort study of 104 lung transplant recipients. Median plasma CC16 levels were determined at three time points: pretransplant and 6 and 24 h posttransplant. The primary outcome was the development of grade 3 PGD within the first 72 h after transplantation. Multivariable logistic regression was performed to evaluate for confounding by donor and recipient demographics and surgical characteristics. Twenty-nine patients (28%) developed grade 3 PGD within the first 72 h. The median CC16 level 6 h after transplant was significantly higher in patients with PGD [13.8 ng/mL (IQR 7.9, 30.4 ng/mL)] than in patients without PGD [8.2 ng/mL (IQR 4.5, 19.1 ng/mL)], p = 0.02. Elevated CC16 levels were associated with increased odds of PGD after lung transplantation. Damage to airway epithelium or altered alveolar permeability as a result of lung ischemia and reperfusion may explain this association.

Circulating KL-6, a biomarker of lung injury, in obstructive sleep apnoea.

In obstructive sleep apnoea (OSA), oxidative stress contributes to endothelial dysfunction in the peripheral circulation. In the lung, oxidative stress can lead to alveolar injury. The present authors hypothesised that patients with OSA would have biomarker evidence of increased alveolar wall permeability. Sleep characteristics, brachial artery flow-mediated dilation and plasma KL-6 levels were observed in 11 otherwise healthy patients with OSA and 10 controls. Median (interquartile range) plasma KL-6 levels were higher in patients with OSA compared with controls: 317 (232-506) U.mL(-1) versus 226 (179-257) U.mL(-1), respectively. Higher plasma KL-6 levels were associated with greater time spent asleep with an oxyhaemoglobin saturation <90%, lower nadir saturation, more frequent desaturation of >4% during sleep and lower brachial artery flow-mediated dilation. Adjustment for nadir saturation or flow-mediated dilation attenuated the association between plasma KL-6 levels and OSA. Circulating KL-6 levels are elevated in some patients with obstructive sleep apnoea, possibly reflecting increased alveolar wall permeability.

Racial and ethnic disparities in idiopathic pulmonary fibrosis: A UNOS/OPTN database analysis.

We previously reported poorer survival among non-Hispanic blacks and Hispanics with idiopathic pulmonary fibrosis (IPF) compared to non-Hispanic whites at our center. In the current study, we hypothesized that these disparities would exist in a nationwide cohort of wait-listed patients with IPF. We performed a retrospective cohort study of 2635 patients with IPF listed for lung transplantation between 1995 and 2003 at 94 transplant centers in the United States. The age-adjusted mortality rate was higher among non-Hispanic blacks [hazard ratio (HR) = 1.24, 95% confidence interval (CI) 1.06-1.45, p = 0.009] and Hispanics (HR = 1.29, 95% CI 1.06-1.56, p = 0.01) compared to non-Hispanic whites. These findings persisted after adjustment for transplantation, medical comorbidities and socioeconomic status. Worse lung function at the time of listing appeared to explain some of these differences (HR for non-Hispanic blacks after adjustment for forced vital capacity percent predicted = 1.16, 95% CI 0.98-1.36, p = 0.09; HR for Hispanics = 1.21, 95% CI 0.99-1.48, p = 0.056). In summary, black and Hispanic patients with IPF have worse survival than whites after listing for lung transplant.

Racial and ethnic disparities in survival in lung transplant candidates with idiopathic pulmonary fibrosis.

Minority patients have worse outcomes than nonminority patients in a variety of pulmonary diseases. We aimed to compare the survival of Black and Hispanic patients to that of others with idiopathic pulmonary fibrosis (IPF). We performed a retrospective cohort study of patients with IPF who were evaluated for lung transplantation at our center. Kaplan-Meier survival curves and Cox proportional hazards models were used to compare survival between groups. Black and Hispanic patients had spirometry, lung volumes and diffusion capacity that were similar to others, but had worse exercise capacity. Minority patients had a significantly increased risk of death compared to others independent of transplantation status (hazard ratio = 3.3, 95% CI 1.2-8.9, p = 0.02). Differences in exercise capacity, pulmonary hemodynamics and socioeconomic factors appeared to account for some of the differences in survival. Black and Hispanic patients with IPF had an increased risk of death following referral for lung transplantation. This finding may be due to differences in disease progression and/or differences in access to medical care among minority patients. Future studies should confirm our findings in a larger cohort. The elimination of racial and ethnic disparities in outcome should be a priority for clinicians and researchers in this field.