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.

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.

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.

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.

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.

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.

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.

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.

Functional consequences of ultrastructural geometry in "backwards" fluid-transporting epithelia.

Many fluid-transporting epithelia possess dead-end, long, and narrow channels opening in the direction to which fluid is being transported (basal infoldings, lateral intercellular spaces, etc.). These channels have been thought to possess geometrical significance as standing-gradient flow systems, in which active solute transport into the channel makes the channel contents hypertonic and permits water-to-solute coupling. However, some secretory epithelia (choroid plexus, Malpighian tubule, rectal gland, etc.) have "backwards" channels opening in the direction from which fluid is being transported. It is shown that these backwards channels can function as standing-gradient flow systems in which solute transport out of the channel makes the channel contents hypotonic and results in coupled water flow into the channel mouth. The dependence of the transported osmolarity (isotonic or hypertonic) on channel radius, length, and other parameters is calculated for backwards channels for values of these parameters in the physiological range. In addition to backwards channels' being hypotonic rather than hypertonic, they are predicted to differ from "forwards" channels in that some restrictions are imposed by the problem of solute exhaustion, and in the presence of a sweeping-in effect on other solutes which limits the solutes that may be transported.

Rediscovery of the yellow-fronted gardener bowerbird.

The long-lost bowerbird Amblyornis flavifrons was found in the Foja Mountains of west New Guinea, and its bower and display were discovered. The bower is a stick tower on a rimmed moss platform, adorned with separate piles of fruit of three different colors. The displaying male extends toward the female a blue fruit set against his golden crest. These observations support a derivation of bower ornamentation from ritual courtship feeding and a transfer of ornamentation from the male's plumage to the bower.

Zoological classification system of a primitive people.

The Fore people of the New Guinea Highlands classify all animals in one of nine higher categories ("tábe aké"), and these are further subdivided into lower categories ("ámana aké"). There are 182 lower categories for vertebrates alone. The nearly one-to-one correspondence between Fore amana ake and species as recognized by European taxonomists reflects the objective reality of the gaps separating sympatric species. In 90 percent of the cases, when a species of animal unknown to the Fore is presented for naming, it is called by the name of the Fore species considered its closest relative by zoologists. The origin of Fore classification is probably utilitarian.

Colonization of exploded volcanic islands by birds: the supertramp strategy.

After volcanic explosions or tidal waves had defaunated several islands near New Guinea, bird species number rapidly returned to equilibrium on coral islets and rapidly returned to quasi-steady-state values limited by regrowth of vegetation in lowland forest of larger islands. However, reequilibration in montane forest has been limited by slow dispersal of the birds. Colonists have been drawn disproportionately from r-selected "supertramrip" species, which maintain much higher population densities than do K-selected faunas, perhaps due to selection for resource overexploitation by the latter.

Distributional Ecology of New Guinea Birds: Recent ecological and biogeographical theories can be tested on the bird communities of New Guinea.

The concepts by which MacArthur and Wilson have transformed the science of ecology in the past decade, and the results of ecological studies such as mine on New Guinea bird communities, have implications for conservation policies. For example, primary tropical rain forest, the most species-rich and ecologically complex habitat on earth, has for millions of years served as the ultimate evolutionary source of the world's dominant plant and animal groups. Throughout the tropics today, the rain forests are being destroyed at a rate such that little will be left in a few decades. When the rain forests have been reduced to isolated tracts separated by open country, the distribution of obligate rain forest species will come to resemble bird distributions on New Guinea land-bridge islands after severing of the land bridges. The smaller the tract, the more rapidly will forest species tend to disappear and be replaced by the widespread second-growth species that least need protection (13). This ominous process is illustrated by Barro Colorado Island, a former hill in Panama that became an island when construction of the Panama Canal flooded surrounding valleys to create Gatun Lake. In the succeeding 60 years several forest bird species have already disappeared from Barro Colorado and been unable to recolonize across the short intervening water gap from the forest on the nearby shore of Gatun Lake. The consequences of the species-area relation (Fig. 1) should be taken into consideration during the planning of tropical rain forest parks (13). In a geographical area that is relatively homogeneous with regard to the fauna, one large park would be preferable to an equivalent area in the form of several smaller parks. Continuous nonforest strips through the park (for example, wide highway swaths) would convert one rain forest "island" into two half-size islands and should be avoided. If other considerations require that an area be divided into several small parks, connecting them by forest corridors might significantly improve their conservation function at little further cost in land withdrawn from development. Modern ecological studies may also be relevant to the understanding of human populations. For instance, during a long period of human evolution there appear to have been not one but two coexistent hominid lines in Africa, the Australopithecus robustus-A. boisei ("Zinjanthropus") line, which became extinct, and the Australopithecus africanus-A. habilis line, which led to Homo sapiens (27). The need to maintain niche differences between these lines must have provided one of the most important selective pressures on the ancestors of modern man in the late Pliocene and early Pleistocene. Thus, any attempt to understand human evolution must confront the problem of what these ecological segregating mechanisms were. To what extent were contemporaneous species of the two lines separated by habitat, by diet, by size difference, or by foraging technique, and were their local spatial distributions broadly overlapping or else sharpened by behavioral interactions as in the case of the Crateroscelis warblers of Fig. 6? To take another example, there are striking parallels between the present distributions of human populations and of bird populations on the islands of Vitiaz and Dampier straits between New Guinea and New Britain. Some of these islands were sterilized by cataclysmic volcanic explosions within the last several centuries. The birds that recolonized these islands have been characterized as coastal and small-island specialists of high reproductive potential, high dispersal powers, and low competitive ability, unlike the geographically closer, competitively superior, slowly dispersing, and breeding birds of mainland New Guinea (10, 11, 13). It remains to be seen whether the people of the Vitiaz-Dampier islands, the Polynesians, and other human populations that colonize insular or unstable habitats also have distinctive population ecologies.

Changes in the cell membranes of the bullfrog gastric mucosa with Acid secretion.

The effective area, resistance, and configuration of the apical and basolateral cell membranes of the bullfrog gastric mucosa were studied as a function of acid secretion rate, by alternating-current impedance methods. The drop in transepithelial resistance with acid secretion is attributed to the great increase in apical membrane area (hence conductance) associated with tubulovesicles. There is no evidence of a change in basolateral membrane resistance or of apical membrane premeability per unit area.

Extinctions and introductions in the new zealand avifauna: cause and effect?

New Zealand, like many other islands, has suffered extinctions of native species and successful introductions of exotic species. It has been uncertain whether the introductions caused the extinctions or whether the extinctions permitted the introductions. On New Zealand's Hauraki Gulf islands, which are unusual in their near lack of introduced mammalian predators and complete lack of mammalian browsers, exotic bird species abundant in mainland New Zealand forest and reaching these islands are virtually absent from unmodified forest. Exotic bird species disappeared from Cuvier Island's forest after elimination of mammalian predators and browsers. Hence extinctions of native species were not due to competition from introduced species but to other factors (such as mammalian predators and habitat alteration). Only after decimation of native species and forest alteration by browsing mammals could exotic birds invade forest.