Edward Cantu

Role of flow cytometry to define unacceptable HLA antigens in lung transplant recipients with HLA-specific antibodies

Background. Antidonor HLA-specific antibodies have been associated with hyperacute rejection and primary graft failure in lung transplant recipients. Thus, transplant candidates with HLA-specific antibodies generally undergo prospective crossmatching to exclude donors with unacceptable HLA antigens. However, the need to perform a prospective crossmatch limits the donor pool and is associated with increased waiting list times and mortality. A virtual crossmatch strategy using flow cytometry, which enables precise determination of HLA-specific antibody specificity, was compared to prospective crossmatching in sensitized lung transplant candidates. Methods. In all, 341 lung transplant recipients were analyzed retrospectively (April 1992 to July 2003). Sixteen patients with HLA-specific antibodies underwent transplantation based on flow cytometric determination of antibody specificity and 10 underwent prospective crossmatching. Results. Freedom from bronchiolitis obliterans syndrome (BOS) at three years was similar in those undergoing a virtual crossmatch, those undergoing prospective crossmatching, and those without HLA-specific antibodies (80.4%±13.4, 85.7%±13.2, and 73.8%±2.8, respectively, P=0.88). Three-year survival was also comparable (87.5%±8.3, 70.0%±14.5, and 78.5%±2.4, respectively, P=0.31). Elimination of prospective crossmatching for sensitized patients was associated with a significant decrease in time on the waiting list (P<0.01) and in waiting list mortality (P<0.05). All 16 patients undergoing a virtual crossmatch had negative retrospective crossmatches. Conclusions. By carefully determining the specificity of HLA-specific antibodies, flow cytometry methodologies enable the prediction of negative crossmatch results with up to 100% accuracy, enabling the determination of appropriateness of donors. Using this virtual crossmatch strategy, crossmatching can be safely omitted prior to lung transplantation, thereby decreasing waiting list time and mortality rates for candidates with HLA-specific antibodies.

Arginase 1 is an innate lymphoid-cell-intrinsic metabolic checkpoint controlling type 2 inflammation

Group 2 innate lymphoid cells (ILC2s) regulate tissue inflammation and repair after activation by cell-extrinsic factors such as host-derived cytokines. However, the cell-intrinsic metabolic pathways that control ILC2 function are undefined. Here we demonstrate that expression of the enzyme arginase-1 (Arg1) during acute or chronic lung inflammation is a conserved trait of mouse and human ILC2s. Deletion of mouse ILC-intrinsic Arg1 abrogated type 2 lung inflammation by restraining ILC2 proliferation and dampening cytokine production. Mechanistically, inhibition of Arg1 enzymatic activity disrupted multiple components of ILC2 metabolic programming by altering arginine catabolism, impairing polyamine biosynthesis and reducing aerobic glycolysis. These data identify Arg1 as a key regulator of ILC2 bioenergetics that controls proliferative capacity and proinflammatory functions promoting type 2 inflammation.

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.

Primary graft dysfunction.

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.

Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing

BackgroundFungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources.ResultsHere, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which are routinely acquired during DNA library preparation, to annotate output sequence data, and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure.ConclusionsWe find increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in subjects with increasingly severe pulmonary and immunologic deficits. We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci, a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.

Latent Class Analysis Identifies Distinct Phenotypes of Primary Graft Dysfunction After Lung Transplantation

BACKGROUND There is significant heterogeneity within the primary graft dysfunction (PGD) syndrome. We aimed to identify distinct grade 3 PGD phenotypes based on severity of lung dysfunction and patterns of resolution. METHODS Subjects from the Lung Transplant Outcomes Group (LTOG) cohort study with grade 3 PGD within 72 h after transplantation were included. Latent class analysis (LCA) was used to statistically identify classes based on changes in PGD International Society for Heart & Lung Transplantation grade over time. Construct validity of the classes was assessed by testing for divergence of recipient, donor, and operative characteristics between classes. Predictive validity was assessed using time to death. RESULTS Of 1,255 subjects, 361 had grade 3 PGD within the first 72 h after transplantation. LCA identified three distinct phenotypes: (1) severe persistent dysfunction (class 1), (2) complete resolution of dysfunction within 72 h (class 2), and (3) attenuation, without complete resolution within 72 h (class 3). Increased use of cardiopulmonary bypass, greater RBC transfusion, and higher mean pulmonary artery pressure were associated with persistent PGD (class 1). Subjects in class 1 also had the greatest risk of death (hazard ratio, 2.39; 95% CI, 1.57-3.63; P < .001). CONCLUSIONS There are distinct phenotypes of resolution of dysfunction within the severe PGD syndrome. Subjects with early resolution may represent a different mechanism of lung pathology, such as resolving pulmonary edema, whereas those with persistent PGD may represent a more severe phenotype. Future studies aimed at PGD mechanism or treatment may focus on phenotypes based on resolution of graft dysfunction.

Cognitive Function, Mental Health, and Health-related Quality of Life after Lung Transplantation

RATIONALE Cognitive and psychiatric impairments are threats to functional independence, general health, and quality of life. Evidence regarding these outcomes after lung transplantation is limited. OBJECTIVES Determine the frequency of cognitive and psychiatric impairment after lung transplantation and identify potential factors associated with cognitive impairment after lung transplantation. METHODS In a retrospective cohort study, we assessed cognitive function, mental health, and health-related quality of life using a validated battery of standardized tests in 42 subjects post-transplantation. The battery assessed cognition, depression, anxiety, resilience, and post-traumatic stress disorder (PTSD). Cognitive function was assessed using the Montreal Cognitive Assessment, a validated screening test with a range of 0 to 30. We hypothesized that cognitive function post-transplantation would be associated with type of transplant, cardiopulmonary bypass, primary graft dysfunction, allograft ischemic time, and physical therapy post-transplantation. We used multivariable linear regression to examine the relationship between candidate risk factors and cognitive function post-transplantation. MEASUREMENTS AND MAIN RESULTS Mild cognitive impairment (score, 18-25) was observed in 67% of post-transplant subjects (95% confidence interval [CI]: 50-80%) and moderate cognitive impairment (score, 10-17) was observed in 5% (95% CI, 1-16%) of post-transplant subjects. Symptoms of moderate to severe anxiety and depression were observed in 21 and 3% of post-transplant subjects, respectively. No transplant recipients reported symptoms of PTSD. Higher resilience correlated with less psychological distress in the domains of depression (P < 0.001) and PTSD (P = 0.02). Prolonged graft ischemic time was independently associated with worse cognitive performance after lung transplantation (P = 0.001). The functional gain in 6-minute-walk distance achieved at the end of post-transplant physical rehabilitation (P = 0.04) was independently associated with improved cognitive performance post-transplantation. CONCLUSIONS Mild cognitive impairment was present in the majority of patients after lung transplantation. Prolonged allograft ischemic time may be associated with cognitive impairment. Poor physical performance and cognitive impairment are linked, and physical rehabilitation post-transplant and psychological resilience may be protective against the development of long-term impairment. Further study is warranted to confirm these potential associations and to examine the trajectory of cognitive function after lung transplantation.

Oxidant stress regulatory genetic variation in recipients and donors contributes to risk of primary graft dysfunction after lung transplantation

OBJECTIVE Oxidant stress pathway activation during ischemia reperfusion injury may contribute to the development of primary graft dysfunction (PGD) after lung transplantation. We hypothesized that oxidant stress gene variation in recipients and donors is associated with PGD. METHODS Donors and recipients from the Lung Transplant Outcomes Group (LTOG) cohort were genotyped using the Illumina IBC chip filtered for oxidant stress pathway genes. Single nucleotide polymorphisms (SNPs) grouped into SNP sets based on haplotype blocks within 49 oxidant stress genes selected from gene ontology pathways and literature review were tested for PGD association using a sequencing kernel association test. Analyses were adjusted for clinical confounding variables and population stratification. RESULTS Three hundred ninety-two donors and 1038 recipients met genetic quality control standards. Thirty percent of patients developed grade 3 PGD within 72 hours. Donor NADPH oxidase 3 (NOX3) was associated with PGD (P = .01) with 5 individual significant loci (P values between .006 and .03). In recipients, variation in glutathione peroxidase (GPX1) and NRF-2 (NFE2L2) was significantly associated with PGD (P = .01 for both). The GPX1 association included 3 individual loci (P values between .006 and .049) and the NFE2L2 association included 2 loci (P = .03 and .05). Significant epistatic effects influencing PGD susceptibility were evident between 3 different donor blocks of NOX3 and recipient NFE2L2 (P = .026, P = .017, and P = .031). CONCLUSIONS Our study has prioritized GPX1, NOX3, and NFE2L2 genes for future research in PGD pathogenesis, and highlights a donor-recipient interaction of NOX3 and NFE2L2 that increases the risk of PGD.

Depletion of Pulmonary Intravascular Macrophages Prevents Hyperacute Pulmonary Xenograft Dysfunction

Background. Recent years have brought dramatic progress in the field of xenotransplantation, with the development of transgenic swine and various other means of overcoming the rejection mediated by xenoreactive antibodies. Although progress has been rapid with kidney and heart xenografts, progress with pulmonary xenografts has lagged behind. Recent findings have suggested that donor pulmonary intravascular macrophages may play a critical role in the hyperacute dysfunction of pulmonary xenografts. Methods. The function of pulmonary xenografts from pigs depleted of pulmonary intravascular macrophages was compared with the function of xenografts from normal pigs. Results. Pulmonary xenografts from pigs from which pulmonary intravascular macrophages were depleted survived (23.5±0.9 hours) about five times longer than normal (macrophage sufficient) xenografts (4.4±1.41 hours) (P< 0.0001). At 21 hours postreperfusion, the left pulmonary arterial flow was 225.0±34 ml/min in lungs depleted of pulmonary intravascular macrophages, whereas all normal xenografts had failed. Conclusions. These findings indicate that donor macrophages play a critical role in pulmonary xenograft dysfunction. This finding has broad implications for xenotransplantation, suggesting that porcine macrophages might pose a barrier to the engraftment and function of a variety of porcine organ xenografts.

Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor

Functional tissue regeneration is required for the restoration of normal organ homeostasis after severe injury. Some organs, such as the intestine, harbour active stem cells throughout homeostasis and regeneration; more quiescent organs, such as the lung, often contain facultative progenitor cells that are recruited after injury to participate in regeneration. Here we show that a Wnt-responsive alveolar epithelial progenitor (AEP) lineage within the alveolar type 2 cell population acts as a major facultative progenitor cell in the distal lung. AEPs are a stable lineage during alveolar homeostasis but expand rapidly to regenerate a large proportion of the alveolar epithelium after acute lung injury. AEPs exhibit a distinct transcriptome, epigenome and functional phenotype and respond specifically to Wnt and Fgf signalling. In contrast to other proposed lung progenitor cells, human AEPs can be directly isolated by expression of the conserved cell surface marker TM4SF1, and act as functional human alveolar epithelial progenitor cells in 3D organoids. Our results identify the AEP lineage as an evolutionarily conserved alveolar progenitor that represents a new target for human lung regeneration strategies.