Detection and clinical impact of human leukocyte antigen antibodies in lung transplantation: A systematic review and meta-analysis.

There is significant variability in lung transplant centers' approach to HLA antibodies, creating heterogeneity regarding their clinical significance. Some institutions use beads coated with multiple HLA to screen candidate sera and then use single antigen bead (SAB) to determine antibody identity if the pre-screen is positive. Other centers do not pre-screen, using SAB alone, which may detect low-level antibodies of unknown significance. The primary objective of this study was to review the current literature to identify sources of heterogeneity in the identification of pre- and post-lung transplant HLA antibodies, particularly regarding antibody-detection methods. A random effects model meta-analysis was used to evaluate the relationship between pre-transplant HLA antibodies and the development of de novo donor-specific antibodies (dnDSA) and dnDSA and chronic lung allograft dysfunction (CLAD). Each outcome was stratified by the method of antibody detection (pre-screen followed by SAB vs SAB alone). We identified 13 cohort studies with a total of 3039 patients. The use of pre-screening followed by SAB testing and the use of induction immunosuppression were associated with lower prevalence of dnDSA. Patients with pre-transplant HLA antibodies were more likely to develop dnDSA (hazard ratio [HR] = 1.49, 95% confidence interval [CI]: 1.19-1.86, P < .001). dnDSA was associated with CLAD (HR = 2.02, 95% CI = 1.37-2.97, P < .001). When considering studies using SAB alone, however, pre-transplant antibody status was no longer associated with dnDSA and dnDSA was no longer associated with CLAD. Based on the current literature, SAB-alone testing may detect less clinically relevant antibodies than pre-screening followed by SAB.

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.

Viral metagenomics reveal blooms of anelloviruses in the respiratory tract of lung transplant recipients.

Few studies have examined the lung virome in health and disease. Outcomes of lung transplantation are known to be influenced by several recognized respiratory viruses, but global understanding of the virome of the transplanted lung is incomplete. To define the DNA virome within the respiratory tract following lung transplantation we carried out metagenomic analysis of allograft bronchoalveolar lavage (BAL), and compared with healthy and HIV+ subjects. Viral concentrates were purified from BAL and analyzed by shotgun DNA sequencing. All of the BAL samples contained reads mapping to anelloviruses, with high proportions in lung transplant samples. Anellovirus populations in transplant recipients were complex, with multiple concurrent variants. Quantitative polymerase chain reaction quantification revealed that anellovirus sequences were 56-fold more abundant in BAL from lung transplant recipients compared with healthy controls or HIV+ subjects (p < 0.0001). Anellovirus sequences were also more abundant in upper respiratory tract specimens from lung transplant recipients than controls (p = 0.006). Comparison to metagenomic data on bacterial populations showed that high anellovirus loads correlated with dysbiotic bacterial communities in allograft BAL (p = 0.008). Thus the respiratory tracts of lung transplant recipients contain high levels and complex populations of anelloviruses, warranting studies of anellovirus lung infection and transplant outcome.

Should we reconsider lung transplantation through uncontrolled donation after circulatory death?

Lung transplantation through controlled donation after circulatory death (cDCD) has slowly gained universal acceptance with reports of equivalent outcomes to those through donation after brain death. In contrast, uncontrolled DCD (uDCD) lung use is controversial and requires ethical, legal and medical complexities to be addressed in a limited time. Consequently, uDCD lung use has not previously been reported in the United States. Despite these potential barriers, we present a case of a patient with multiple gunshot wounds to the head and the body who was unsuccessfully resuscitated and ultimately became an uDCD donor. A cytomegalovirus positive recipient who had previously consented for CDC high-risk, DCD and participation in the NOVEL trial was transplanted from this uDCD donor, following 3 h of ex vivo lung perfusion. The postoperative course was uneventful, and the recipient was discharged home on day 9. While this case represents a "best-case scenario," it illustrates a method for potential expansion of the lung allograft pool through uDCD after unsuccessful resuscitation in hospitalized patients.

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.

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.

Early plasma soluble receptor for advanced glycation end-product levels are associated with bronchiolitis obliterans syndrome.

Early epithelial injury after lung transplantation may contribute to development of bronchiolitis obliterans syndrome (BOS). We evaluated the relationship between early postoperative soluble receptor for advanced glycation end-product (sRAGE) levels, a marker of type I alveolar cell injury and BOS. We performed a cohort study of 106 lung transplant recipients between 2002 and 2006 at the University of Pennsylvania with follow-up through 2010. Plasma sRAGE was measured 6 and 24 h after transplantation. Cox proportional hazards models were used to evaluate the association between sRAGE and time to BOS, defined according to ISHLT guidelines. Sixty (57%) subjects developed BOS. The average time to BOS was 3.4 years. sRAGE levels measured at 6 h (HR per SD of sRAGE: 1.69, 95% CI: 1.11, 2.57, p = 0.02) and 24 h (HR per SD of sRAGE: 1.74, 95% CI: 1.14, 2.65, p = 0.01) were associated with an increased hazard of BOS. Multivariable Cox regression indicated this relationship was independent of potential confounders. Elevated plasma sRAGE levels measured in the immediate postoperative period are associated with the development of BOS. Early epithelial injury after transplantation may contribute to the development of fibrosis in BOS.

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.

Delayed disaccharidase development in a rabbit model of intrauterine growth retardation.

Intrauterine growth retardation (IUGR) affects almost 10% of infants born in the United States. It may be responsible for delayed gastrointestinal function and is an important cause of perinatal morbidity and mortality. The New Zealand White rabbit provides an optimal model for the study of naturally occurring IUGR. At term, birth weight is determined by fetal position within the bicornuate uterus. The small intestinal disaccharidase enzymes are indicators of bowel maturity and function. To examine potential differences in disaccharidase development between normal and IUGR fetuses, this rabbit model was investigated. Jejunum was harvested at multiple stages in rabbit development including the third trimester fetus, neonate, and adult. Lactase, maltase, and sucrase enzyme activity, as well as total protein content, was determined. Results were analyzed by the 2-tailed t test and ANOVA. Lactase activity appeared in the mid-third trimester, peaked in the early neonatal period, then declined to adult levels. Maltase activity appeared in the early third trimester and gradually rose to adult levels. Sucrase remained at trace levels until the mid-neonatal period, reaching adult levels by weaning. Both lactase and maltase activity were depressed in IUGR fetuses compared with their normal littermates. This pattern of disaccharidase depression continued into the neonatal period until catch-up growth occurred at 2 wk when levels equalized. This report describes differential small intestinal disaccharidase development between normal and growth-retarded rabbit fetuses in a naturally occurring model of IUGR.

Thermodynamics of three-way multibranch loops in RNA.

RNA multibranch loops (junctions) are loops from which three or more helices exit. They are nearly ubiquitous in RNA secondary structures determined by comparative sequence analysis. In this study, systems in which two strands combine to form three-way junctions were used to measure the stabilities of RNA multibranch loops by UV optical melting and isothermal titration calorimetry (ITC). These data were used to calculate the free energy increment for initiation of a three-way junction on the basis of a nearest neighbor model for secondary structure stability. Imino proton NMR spectra were also measured for two systems and are consistent with the hypothesized helical structures. Incorporation of the experimental data into the mfold and RNA structure computer programs has contributed to an improvement in prediction of RNA secondary structure from sequence.

Identifying sources of stress to native aquatic fauna using a watershed ecological risk assessment framework.

The free-flowing Clinch and Powell River Basin, located in southwestern Virginia, United States, historically had one of the richest assemblages of native fish and freshwater mussels in the world. Nearly half of the species once residing here are now extinct, threatened, or endangered. The United States Environmental Protection Agency's framework for conducting an ecological risk assessment was used to structure a watershed-scale analysis of human land use, in-stream habitat quality, and their relationship to native fish and mussel populations in order to develop future management strategies and prioritize areas in need of enhanced protection. Our analyses indicate that agricultural and urban land uses as well as proximity to mining activities and transportation corridors are inversely related to fish index of biotic integrity (IBI) and mussel species diversity. Forward stepwise multiple regression analyses indicated that coal mining had the most impact on fish IBI followed by percent cropland and urban area in the riparian corridor (R2 = 0.55, p = 0.02); however, these analyses suggest that other site-specific factors are important. Habitat quality measures accounted for as much as approximately half of the variability in fish IBI values if the analysis was limited to sites within a relatively narrow elevation range. These results, in addition to other data collected in this watershed, suggest that nonhabitat-related stressors (e.g., accidental chemical spills) also have significant effects on biota in this basin. The number of co-occurring human land uses was inversely related to fish IBI (r = -0.49, p < 0.01). Sites with > or = 2 co-occurring land uses had >90% probability of having <2 mussel species present. Our findings predict that many mussel concentration sites are vulnerable to future extirpation. In addition, our results suggest that protection and enhancement of naturally vegetated riparian corridors, better controls of mine effluents and urban runoff, and increased safeguards against accidental chemical spills, as well as reintroduction or augmentation of threatened and endangered species, may help sustain native fish and mussel populations in this watershed.

Evolution of regulatory responses to feeding in snakes.

Do animal species that normally consume large meals at long intervals evolve to down-regulate their metabolic physiology while fasting and to up-regulate it steeply on feeding? To test this hypothesis, we compared postfeeding regulatory responses in eight snake species: four frequent feeders on small meals and four infrequent feeders on large meals. For each species, we measured factorial changes in metabolic rate, in activities and capacities of five small intestinal brush border nutrient transporters, and in masses of eight organs that function in nutrient processing after consumption of a rodent meal equivalent to 25% of the snake's body mass. It turned out that, compared with frequent feeders, infrequent feeders digest that meal more slowly; have lower metabolic rates, organ masses, and nutrient uptake rates and capacities while fasting; have higher energy expenditure during digestion; and have higher postfeeding factorial increases in metabolic rate, organ masses, and nutrient uptake rates and capacities. These conclusions, which conform to the hypothesis mentioned above, remain after phylogeny has been taken into account. The small organ masses and low nutrient transporter activities during fasting contribute to the low fasting metabolism of infrequent feeders. Quantitative calculations of partial energy budgets suggest that energy savings drive the evolution of low mass and activities of organs during fasting and of large postfeeding regulatory responses in infrequent feeders. We propose further tests of this hypothesis among other snake species and among other ectotherms.