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Rationale: Primary graft dysfunction (PGD) is a severe form of acute lung injury, leading to increased early morbidity and mortality after lung transplant. Obesity is a major health problem, and recipient obesity is one of the most significant risk factors for developing PGD. Objectives: We hypothesized that T-regulatory cells (Tregs) are able to dampen early ischemia-reperfusion events and thereby decrease the risk of PGD, whereas that action is impaired in obese recipients. Methods: We evaluated Tregs, T cells, and inflammatory markers, plus clinical data, in 79 lung transplant recipients and 41 liver or kidney transplant recipients and studied two groups of mice on a high-fat diet (HFD), which did ("inflammatory" HFD) or did not ("healthy" HFD) develop low-grade inflammation with decreased Treg function. Measurements and Main Results: We identified increased levels of IL-18 as a previously unrecognized mechanism that impairs Tregs' suppressive function in obese individuals. IL-18 decreases levels of FOXP3, the key Treg transcription factor, decreases FOXP3 di- and oligomerization, and increases the ubiquitination and proteasomal degradation of FOXP3. IL-18-treated Tregs or Tregs from obese mice fail to control PGD, whereas IL-18 inhibition ameliorates lung inflammation. The IL-18-driven impairment in Tregs' suppressive function before transplant was associated with an increased risk and severity of PGD in clinical lung transplant recipients. Conclusions: Obesity-related IL-18 induces Treg dysfunction that may contribute to the pathogenesis of PGD. Evaluation of Tregs' suppressive function together with evaluation of IL-18 levels may serve as a screening tool to identify obese individuals with an increased risk of PGD before transplant.

Primary graft dysfunction (PGD) is a syndrome encompassing a spectrum of mild to severe lung injury that occurs within the first 72 hours after lung transplantation. PGD is characterized by pulmonary edema with diffuse alveolar damage that manifests clinically as progressive hypoxemia with radiographic pulmonary infiltrates. In recent years, new knowledge has been generated on risks and mechanisms of PGD. Following ischemia and reperfusion, inflammatory and immunological injury-repair responses appear to be key controlling mechanisms. In addition, PGD has a significant impact on short- and long-term outcomes; therefore, the choice of donor organ is impacted by this potential adverse consequence. Improved methods of reducing PGD risk and efforts to safely expand the pool are being developed. Ex vivo lung perfusion is a strategy that may improve risk assessment and become a promising platform to implement treatment interventions to prevent PGD. This review details recent updates in the epidemiology, pathophysiology, molecular and genetic biomarkers, and state-of-the-art technical developments affecting PGD., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: The role of genetics in the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) from direct or indirect lung injury has not been specifically investigated. The aim of this study was to identify genetic variants contributing to ALI/ARDS from pulmonary or extrapulmonary causes., Methods: We conducted a multistage genetic association study. We first performed a large-scale genotyping (50K ITMAT-Broad&#95;CARe Chip) in 1717 critically ill Caucasian patients with either pulmonary or extrapulmonary injury, to identify single nucleotide polymorphisms (SNPs) associated with the development of ARDS from direct or indirect insults to the lung. Identified SNPs (p≤0.0005) were validated in two separated populations (Stage II), with trauma (Population I; n=765) and pneumonia/pulmonary sepsis (Population II; n=838), as causes for ALI/ARDS. Genetic variants replicating their association with trauma related-ALI in Stage II were validated in a second trauma-associated ALI population (n=224, Stage III)., Results: In Stage I, non-overlapping SNPs were significantly associated with ARDS from direct/indirect lung injury, respectively. The association between rs1190286 (POPDC3) and reduced risk of ARDS from pulmonary injury was validated in Stage II (p<0.003). SNP rs324420 (FAAH) was consistently associated with increased risk of ARDS from extrapulmonary causes in two independent ALI-trauma populations (p<0.006, Stage II; p<0.05, Stage III). Meta-analysis confirmed these associations., Conclusions: Different genetic variants may influence ARDS susceptibility depending on direct versus indirect insults. Functional SNPs in POPDC3 and FAAH genes may be driving the association with direct and indirect ALI/ARDS, respectively.

Background: Packed red blood cell (PRBC) transfusion is associated with acute lung injury (ALI) development after trauma, but this risk may not be constant through time after trauma. We hypothesized that the relationship between PRBC delivery and ALI risk varies through time after injury., Methods: Data were collected prospectively from 1999 to 2006. Inclusion criteria include the following: older than 13 years, surgical intensive care unit admission, and Injury Severity Score of 16 or greater. Exclusion criteria included discharge/death within 24 hours of admission. Patients were followed up prospectively for ALI development for 5 days after trauma. Discrete time models were fit to test the association of timing of PRBC delivery with the development of ALI while controlling for patient demographics, resuscitation variables, Injury Severity Score, and Acute Physiology and Chronic Health Evaluation III scores., Results: At total of 602 patients were included. Median age was 33 years, 77% were male, and 50% were African American. Using a discrete time-survival model, the relation between transfusion and ALI development was found to vary by transfusion time window (p < 0.0001). The major effect of PRBC delivery on ALI risk occurred in the first 24 hours after trauma; this finding persisted in multivariable modeling (adjusted odds ratio, 1.07 per unit; 95% confidence interval, 1.02-1.11, p < 0.001). Cumulative incidence of ALI approached 50% in patients receiving 6 U of PRBC or more in the first 24 hours., Conclusion: The association between PRBC transfusion and ALI development in patients with trauma is time dependent, with PRBC delivery in the first 24 hours after injury driving the overall relation. Each PRBC unit during this period increases odds of subsequent ALI development by 7%., Level of Evidence: Prognostic/epidemiologic study, level II.

Purpose: Endocan is a proteoglycan expressed by endothelial cells in the lung that may inhibit leukocyte recruitment and thus prevent the development of acute lung injury (ALI). We tested the association of serum endocan levels with subsequent development of ALI after major trauma., Materials and Methods: This was a single-center nested case-control study within a prospective cohort study of major trauma patients. Using an enzyme-linked immunosorbent assay test, we measured endocan levels from admission serum in 24 controls (no ALI) and 24 cases (ALI within 5 days of trauma). Multivariable logistic regression was used to test the association of admission serum endocan levels with subsequent ALI., Results: Patients who developed ALI had lower levels of endocan on admission (mean, 3.5 ± 1.4 ng/mL vs 4.9 ± 2.6 ng/mL in controls; P = .02). For each 1-unit increase in serum endocan level, the odds ratio for ALI development decreased (0.69; 95% confidence interval, 0.49-0.97; P = .03). Lower endocan levels remained associated with a higher incidence of ALI after adjustment for age and illness severity., Conclusions: Lower levels of serum endocan on admission are associated with subsequent development of ALI in trauma patients. These observations may be explained by endocan-mediated blockade of leukocyte recruitment in the lung., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: We used a gene - based replication strategy to test the reproducibility of prior acute lung injury (ALI) candidate gene associations., Methods: We phenotyped 474 patients from a prospective severe trauma cohort study for ALI. Genomic DNA from subjects' blood was genotyped using the IBC chip, a multiplex single nucleotide polymorphism (SNP) array. Results were filtered for 25 candidate genes selected using prespecified literature search criteria and present on the IBC platform. For each gene, we grouped SNPs according to haplotype blocks and tested the joint effect of all SNPs on susceptibility to ALI using the SNP-set kernel association test. Results were compared to single SNP analysis of the candidate SNPs. Analyses were separate for genetically determined ancestry (African or European)., Results: We identified 4 genes in African ancestry and 2 in European ancestry trauma subjects which replicated their associations with ALI. Ours is the first replication of IL6, IL10, IRAK3, and VEGFA associations in non-European populations with ALI. Only one gene - VEGFA - demonstrated association with ALI in both ancestries, with distinct haplotype blocks in each ancestry driving the association. We also report the association between trauma-associated ALI and NFKBIA in European ancestry subjects., Conclusions: Prior ALI genetic associations are reproducible and replicate in a trauma cohort. Kernel - based SNP-set analysis is a more powerful method to detect ALI association than single SNP analysis, and thus may be more useful for replication testing. Further, gene-based replication can extend candidate gene associations to diverse ethnicities.

Rationale: Cognitive and psychiatric morbidity is common and potentially modifiable after acute lung injury (ALI). However, practical measures of neuropsychological function for use in multicenter trials are lacking., Objectives: To determine whether a validated telephone-based neuropsychological test battery is feasible in a multicenter trial. To determine the frequency and risk factors for long-term neuropsychological impairment., Methods: As an adjunct study to the Acute Respiratory Distress Syndrome Clinical Trials Network Fluid and Catheter Treatment Trial, we assessed neuropsychological function at 2 and 12 months post-hospital discharge., Measurements and Main Results: Of 406 eligible survivors, we approached 261 to participate and 213 consented. We tested 122 subjects at least once, including 102 subjects at 12 months. Memory, verbal fluency, and executive function were impaired in 13% (12 of 92), 16% (15 of 96), and 49% (37 of 76) of long-term survivors. Long-term cognitive impairment was present in 41 of the 75 (55%) survivors who completed cognitive testing. Depression, post-traumatic stress disorder, or anxiety was present in 36% (37 of 102), 39% (40 of 102), and 62% (63 of 102) of long-term survivors. Enrollment in a conservative fluid-management strategy (P = 0.005) was associated with cognitive impairment and lower partial pressure of arterial oxygen during the trial was associated with cognitive (P = 0.02) and psychiatric impairment (P = 0.02)., Conclusions: Neuropsychological function can be assessed by telephone in a multicenter trial. Long-term neuropsychological impairment is common in survivors of ALI. Hypoxemia is a risk factor for long-term neuropsychological impairment. Fluid management strategy is a potential risk factor for long-term cognitive impairment; however, given the select population studied and an unclear mechanism, this finding requires confirmation.

Rationale: Despite advances in clinical management, there are currently no reliable diagnostic and therapeutic targets for acute respiratory distress syndrome (ARDS). The inflammasome/caspase-1 pathway regulates the maturation and secretion of proinflammatory cytokines (e.g., IL-18). IL-18 is associated with injury in animal models of systemic inflammation., Objectives: We sought to determine the contribution of the inflammasome pathway in experimental acute lung injury and human ARDS., Methods: We performed comprehensive gene expression profiling on peripheral blood from patients with critical illness. Gene expression changes were assessed using real-time polymerase chain reaction, and IL-18 levels were measured in the plasma of the critically ill patients. Wild-type mice or mice genetically deficient in IL-18 or caspase-1 were mechanically ventilated using moderate tidal volume (12 ml/kg). Lung injury parameters were assessed in lung tissue, serum, and bronchoalveolar lavage fluid., Measurements and Main Results: In mice, mechanical ventilation enhanced IL-18 levels in the lung, serum, and bronchoalveolar lavage fluid. IL-18-neutralizing antibody treatment, or genetic deletion of IL-18 or caspase-1, reduced lung injury in response to mechanical ventilation. In human patients with ARDS, inflammasome-related mRNA transcripts (CASP1, IL1B, and IL18) were increased in peripheral blood. In samples from four clinical centers, IL-18 was elevated in the plasma of patients with ARDS (sepsis or trauma-induced ARDS) and served as a novel biomarker of intensive care unit morbidity and mortality., Conclusions: The inflammasome pathway and its downstream cytokines play critical roles in ARDS development.

Acute Lung Injury (ALI) is a syndrome with high associated mortality characterized by severe hypoxemia and pulmonary infiltrates in patients with critical illness. We conducted the first investigation to use the genome wide association (GWA) approach to identify putative risk variants for ALI. Genome wide genotyping was performed using the Illumina Human Quad 610 BeadChip. We performed a two-stage GWA study followed by a third stage of functional characterization. In the discovery phase (Phase 1), we compared 600 European American trauma-associated ALI cases with 2266 European American population-based controls. We carried forward the top 1% of single nucleotide polymorphisms (SNPs) at p<0.01 to a replication phase (Phase 2) comprised of a nested case-control design sample of 212 trauma-associated ALI cases and 283 at-risk trauma non-ALI controls from ongoing cohort studies. SNPs that replicated at the 0.05 level in Phase 2 were subject to functional validation (Phase 3) using expression quantitative trait loci (eQTL) analyses in stimulated B-lymphoblastoid cell lines (B-LCL) in family trios. 159 SNPs from the discovery phase replicated in Phase 2, including loci with prior evidence for a role in ALI pathogenesis. Functional evaluation of these replicated SNPs revealed rs471931 on 11q13.3 to exert a cis-regulatory effect on mRNA expression in the PPFIA1 gene (p = 0.0021). PPFIA1 encodes liprin alpha, a protein involved in cell adhesion, integrin expression, and cell-matrix interactions. This study supports the feasibility of future multi-center GWA investigations of ALI risk, and identifies PPFIA1 as a potential functional candidate ALI risk gene for future research.

Background: Peroxiredoxin 6 (PRDX6) is involved in redox regulation of the cell and is thought to be protective against oxidant injury. Little is known about genetic variation within the PRDX6 gene and its association with acute lung injury (ALI). In this study we sequenced the PRDX6 gene to uncover common variants, and tested association with ALI following major trauma., Methods: To examine the extent of variation in the PRDX6 gene, we performed direct sequencing of the 5' UTR, exons, introns and the 3' UTR in 25 African American cases and controls and 23 European American cases and controls (selected from a cohort study of major trauma), which uncovered 80 SNPs. In silico modeling was performed using Patrocles and Transcriptional Element Search System (TESS). Thirty seven novel and tagging SNPs were tested for association with ALI compared with ICU at-risk controls who did not develop ALI in a cohort study of 259 African American and 254 European American subjects that had been admitted to the ICU with major trauma., Results: Resequencing of critically ill subjects demonstrated 43 novel SNPs not previously reported. Coding regions demonstrated no detectable variation, indicating conservation of the protein. Block haplotype analyses reveal that recombination rates within the gene seem low in both Caucasians and African Americans. Several novel SNPs appeared to have the potential for functional consequence using in silico modeling. Chi2 analysis of ALI incidence and genotype showed no significant association between the SNPs in this study and ALI. Haplotype analysis did not reveal any association beyond single SNP analyses., Conclusions: This study revealed novel SNPs within the PRDX6 gene and its 5' and 3' flanking regions via direct sequencing. There was no association found between these SNPs and ALI, possibly due to a low sample size, which was limited to detection of relative risks of 1.93 and above. Future studies may focus on the role of PRDX6 genetic variation in other diseases, where oxidative stress is suspected.

Rationale: Acute lung injury (ALI) acts as a complex genetic trait, yet its genetic risk factors remain incompletely understood. Large-scale genotyping has not previously been reported for ALI., Objectives: To identify ALI risk variants after major trauma using a large-scale candidate gene approach., Methods: We performed a two-stage genetic association study. We derived findings in an African American cohort (n = 222) using a cardiopulmonary disease-centric 50K single nucleotide polymorphism (SNP) array. Genotype and haplotype distributions were compared between subjects with ALI and without ALI, with adjustment for clinical factors. Top performing SNPs (P < 10(-4)) were tested in a multicenter European American trauma-associated ALI case-control population (n = 600 ALI; n = 2,266 population-based control subjects) for replication. The ALI-associated genomic region was sequenced, analyzed for in silico prediction of function, and plasma was assayed by ELISA and immunoblot., Measurements and Main Results: Five SNPs demonstrated a significant association with ALI after adjustment for covariates in Stage I. Two SNPs in ANGPT2 (rs1868554 and rs2442598) replicated their significant association with ALI in Stage II. rs1868554 was robust to multiple comparison correction: odds ratio 1.22 (1.06-1.40), P = 0.0047. Resequencing identified predicted novel splice sites in linkage disequilibrium with rs1868554, and immunoblots showed higher proportion of variant angiopoietin-2 (ANG2) isoform associated with rs1868554T (0.81 vs. 0.48; P = 0.038)., Conclusions: An ANGPT2 region is associated with both ALI and variation in plasma angiopoietin-2 isoforms. Characterization of the variant isoform and its genetic regulation may yield important insights about ALI pathogenesis and susceptibility.

Background: In observational studies using acute lung injury (ALI) as an outcome, a spectrum of lung injury and difficult-to-interpret chest radiographs (CXRs) may hamper efforts to uncover risk factor associations. We assessed the impact of excluding patients with difficult-to-classify or equivocal ALI diagnosis on clinical and genetic risk factor associations for ALI after trauma., Methods: This study was of a prospective cohort of 280 critically ill trauma patients. The primary outcome was the development of ALI. Patients were classified into one of three groups: (1) definite ALI (patients who fulfilled the American-European Consensus Conference [AECC] criteria for ALI), (2)equivocal ALI (patients who had difficult-to-interpret CXRs), and (3) definite non-ALI. We compared clinical and genetic ALI risk factor associations between two classification schemes: AECC classification (definite ALI vs rest) and alternative classification (definite ALI vs definite non-ALI, excluding equivocal ALI)., Results: Ninety-three (35%) patients were classified as definite ALI, 67 (25%) as equivocal, and 104 (39%) as definite non-ALI. Estimates of clinical and genetic ALI risk factor associations were farther from the null using the alternative classification. In a multivariable risk factor model, the C statistic of the alternative classification was significantly higher than that derived from the AECC classification (0.82 vs 0.74; P < .01)., Conclusions: The ability to detect ALI risk factors may be improved by excluding patients with equivocal or difficult-to-classify ALI. Such analyses may provide improved ability to detect clinical and genetic risk factor associations in future epidemiologic studies of ALI.

Acute lung injury (ALI) is a syndrome with significant morbidity and mortality, but its genetic susceptibility is not clearly understood. In the present study, we characterized functional promoter single nucleotide polymorphisms (SNPs) in the phase II antioxidant gene NQO1 (NAD(P)H:quinone oxidoreductase1) to evaluate its role in susceptibility to ALI. Three previously uncharacterized SNPs in the NQO1 promoter were selected for investigation. Luciferase assays were performed using constructs of each promoter polymorphism to evaluate function. Functional SNPs were genotyped in a prospective cohort of major trauma patients (N = 264) and assessed for association with development of ALI. The A/C SNP at -1221 decreased in vitro transcription of NQO1 at baseline and after exposure to hyperoxia and other oxidant stressors. Patients heterozygous for the -1221 C allele were at significantly lesser risk of ALI after major trauma compared with patients with wild-type alleles, even after adjustment for APACHE III score, and mechanism of trauma [OR, 0.46 (95% CI 0.23, 0.90); P = 0.024]. This study demonstrated that the AC genotype at position -1221 in the NQO1 gene caused decreased transcription and was associated with a lower incidence of ALI following major trauma. These novel findings may have important implications in diseases with oxidant stress aetiologies.

OBJECTIVE The authors designed an automated electronic system that incorporates data from multiple hospital information systems to screen for acute lung injury (ALI) in mechanically ventilated patients. The authors evaluated the accuracy of this system in diagnosing ALI in a cohort of patients with major trauma, but excluding patients with congestive heart failure (CHF). DESIGN Single-center validation study. Arterial blood gas (ABG) data and chest radiograph (CXR) reports for a cohort of intensive care unit (ICU) patients with major trauma but excluding patients with CHF were screened prospectively for ALI requiring intubation by an automated electronic system. The system was compared to a reference standard established through consensus of two blinded physician reviewers who independently screened the same population for ALI using all available ABG data and CXR images. The system's performance was evaluated (1) by measuring the sensitivity and overall accuracy, and (2) by measuring concordance with respect to the date of ALI identification (vs. reference standard). MEASUREMENTS One hundred ninety-nine trauma patients admitted to our level 1 trauma center with an initial injury severity score (ISS) >/= 16 were evaluated for development of ALI in the first five days in an ICU after trauma. Main RESULTS The system demonstrated 87% sensitivity (95% confidence interval [CI] 82.3-91.7) and 89% specificity (95% CI 84.7-93.4). It identified ALI before or within the 24-hour period during which ALI was identified by the two reviewers in 87% of cases. CONCLUSIONS An automated electronic system that screens intubated ICU trauma patients, excluding patients with CHF, for ALI based on CXR reports and results of ABGs is sufficiently accurate to identify many early cases of ALI.

Objective: We sought to develop a simple point score that would accurately capture the risk of hospital death for patients with acute lung injury (ALI)., Design: This is a secondary analysis of data from two randomized trials. Baseline clinical variables collected within 24 hours of enrollment were modeled as predictors of hospital mortality using logistic regression and bootstrap resampling to arrive at a parsimonious model. We constructed a point score based on regression coefficients., Setting: Medical centers participating in the Acute Respiratory Distress Syndrome Clinical Trials Network (ARDSnet)., Patients: Model development: 414 patients with nontraumatic ALI participating in the low tidal volume arm of the ARDSnet Acute Respiratory Management in ARDS study. Model validation: 459 patients participating in the ARDSnet Assessment of Low tidal Volume and elevated End-expiratory volume to Obviate Lung Injury study. Model Validation: 459 patients participating in the ARDSnet Assessment of Low tidal Volume and elevated End-expiratory volume to Obviate Lung Injury trial., Interventions: None., Measurements and Main Results: Variables comprising the prognostic model were hematocrit <26% (1 point), bilirubin >or=2 mg/dL (1 point), fluid balance >2.5 L positive (1 point), and age (1 point for age 40-64 years, 2 points for age >or=65 years). Predicted mortality (95% confidence interval) for 0, 1, 2, 3, and 4+ point totals was 8% (5% to 14%), 17% (12% to 23%), 31% (26% to 37%), 51% (43% to 58%), and 70% (58% to 80%), respectively. There was an excellent agreement between predicted and observed mortality in the validation cohort. Observed mortality for 0, 1, 2, 3, and 4+ point totals in the validation cohort was 12%, 16%, 28%, 47%, and 67%, respectively. Compared with the Acute Physiology Assessment and Chronic Health Evaluation III score, areas under the receiver operating characteristic curve for the point score were greater in the development cohort (0.72 vs. 0.67, p = 0.09) and lower in the validation cohort (0.68 vs. 0.75, p = 0.03)., Conclusions: Mortality in patients with ALI can be predicted using an index of four readily available clinical variables with good calibration. This index may help inform prognostic discussions, but validation in nonclinical trial populations is necessary before widespread use.

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