Concurrent abnormal non-acid reflux is associated with additional chronic rejection risk in lung transplant patients with increased acid exposure.

Acid reflux has been associated with allograft injury and rejection in lung transplant patients; however, the pathogenic role of non-acid reflux remains debated. We aimed to evaluate the impact of concurrent abnormal non-acid reflux with acid reflux on chronic rejection in lung transplant patients with acid reflux. This was a retrospective cohort study of lung transplant recipients who underwent pre-transplant combined impedance-pH study off acid suppression. Only subjects with acid exposure >4% were included. Non-acid reflux (pH > 4) episodes >27 was considered abnormal per prior normative studies. Chronic rejection was defined as chronic lung allograft dysfunction (CLAD) per International Society for Heart and Lung Transplantation criteria. Time-to-event analyses were performed using Cox proportional hazards and Kaplan-Maier methods, with censoring at death, anti-reflux surgery, or last follow-up. In total, 68 subjects (28 abnormal/40 normal non-acid reflux) met inclusion criteria for the study. Baseline demographic/clinical characteristics were similar between groups. Among this cohort of patients with increased acid exposure, subjects with concurrent abnormal non-acid reflux had significantly higher risk of CLAD than those without on Kaplan-Meier analysis (log-ranked P = 0.0269). On Cox multivariable regression analysis controlling for body mass index, age at transplantation, and proton pump inhibitor use, concurrent abnormal non-acid reflux remained independently predictive of increased CLAD risk (hazard ratio 2.31, confidence interval: 1.03-5.19, P = 0.04). Presence of concurrent abnormal non-acid reflux in lung transplant subjects with increased acid exposure is associated with additional risk of chronic rejection. Non-acid reflux may also contribute to pathogenicity in lung allograft injury/rejection, supporting a potential role for impedance-based testing in this population.

Limited Effect of Prevention Strategies on Incidence of Clinically Detectable Venous Thromboembolism After Lung Transplantation.

BACKGROUND: Thromboembolic complications are common post-lung transplant, leading to significant morbidity. We instituted multiple interventions because of an observed 36.8% incidence of venous thromboembolism (VTE) (Incidence rate (IR) 5.74/1000 pt days) in our recipients. METHODS: Our initiative commenced January 2015 with enoxaparin initiation within 6-8 hours of intensive care unit arrival and continuation for 4-6 weeks. We evaluated the IR of VTE in lung transplant recipients within 90 days of transplant. In 2017, the protocol was modified to extend the time to initiation of prophylaxis to within 72 hours of ICU arrival. In 2019, we further amended our intraoperative vascular access strategy. RESULTS: Eighteen of 26 lung transplant recipients (LTR) met inclusion criteria in the 2015 cohort. Six of 18 (33.3%) developed VTE, 50% of which were upper extremity (UE), line associated. Fifty two of 75 LTR were eligible for enoxaparin prophylaxis in the 2017 cohort. Fifteen of 52 subjects (28.8%) developed VTE, 77.8% of which were UE and line associated. Despite improved adherence in 2017, there was little change in VTE IR (3.90/1000 pt days compared with 3.85/1000 pt days). Twenty six of 43 LTR met protocol inclusion criteria in the 2019 cohort. Ten subjects (38.5%) developed VTE, 67% of which were UE and line associated (IR 5.18/1000 pt days). CONCLUSION: Our prospective study found that LTR remain at high risk for VTE despite aggressive prophylaxis with 4-6 weeks of enoxaparin and adjustment of vascular access approach. Alternative interventions should be investigated to minimize VTE development in this vulnerable population.

Hemibiotrophic Phytophthora infestans Modulates the Expression of SWEET Genes in Potato (Solanum tuberosum L.).

Sugar Efflux transporters (SWEET) are involved in diverse biological processes of plants. Pathogens have exploited them for nutritional gain and subsequently promote disease progression. Recent studies have implied the involvement of potato SWEET genes in the most devastating late blight disease caused by Phytophthora infestans. Here, we identified and designated 37 putative SWEET genes as StSWEET in potato. We performed detailed in silico analysis, including gene structure, conserved domains, and phylogenetic relationship. Publicly available RNA-seq data was harnessed to retrieve the expression profiles of SWEET genes. The late blight-responsive SWEET genes were identified from the RNA-seq data and then validated using quantitative real-time PCR. The SWEET gene expression was studied along with the biotrophic (SNE1) and necrotrophic (PiNPP1) marker genes of P. infestans. Furthermore, we explored the co-localization of P. infestans resistance loci and SWEET genes. The results indicated that nine transporter genes were responsive to the P. infestans in potato. Among these, six transporters, namely StSWEET10, 12, 18, 27, 29, and 31, showed increased expression after P. infestans inoculation. Interestingly, the observed expression levels aligned with the life cycle of P. infestans, wherein expression of these genes remained upregulated during the biotrophic phase and decreased later on. In contrast, StSWEET13, 14, and 32 didn't show upregulation in inoculated samples suggesting non-targeting by pathogens. This study underscores these transporters as prime P. infestans targets in potato late blight, pivotal in disease progression, and potential candidates for engineering blight-resistant potato genotypes.

Esophageal Function and Reflux Evaluations in Lung Transplantation: A Nationwide Survey of UNOS-Accredited Transplant Centers in the United States.

INTRODUCTION: Gastroesophageal reflux disease has been associated with worse lung transplant outcomes. We aimed to assess local practices for esophageal function testing (EFT) across transplant centers. METHODS: This was a survey study of all United Network for Organ Sharing-accredited adult lung transplant centers regarding local EFT practice. RESULTS: Among 39/63 (60%) responded centers, 38.5% required any EFT (35.9% esophageal manometry, 15.4% pH monitoring, and 28.2% pH impedance), while another 28.2% may consider EFT based on symptoms. Five-year transplant volume was higher among centers requiring EFT (253 vs 159, P = 0.04). DISCUSSION: Only a minority of lung transplant centers routinely obtained EFT, supporting the need for guidelines for standardized reflux/esophageal assessment.

Ineffective esophageal motility is associated with acute rejection after lung transplantation independent of gastroesophageal reflux.

BACKGROUND: Gastroesophageal reflux is associated with poorer outcomes after lung transplant, likely through recurrent aspiration and allograft injury. Although prior studies have demonstrated a relationship between impedance-pH results and transplant outcomes, the role of esophageal manometry in the assessment of lung transplant patients remains debated, and the impact of esophageal dysmotility on transplant outcomes is unclear. Of particular interest is ineffective esophageal motility (IEM) and its associated impact on esophageal clearance. AIM: To assess the relationship between pre-transplant IEM diagnosis and acute rejection after lung transplantation. METHODS: This was a retrospective cohort study of lung transplant recipients at a tertiary care center between 2007 and 2018. Patients with pre-transplant anti-reflux surgery were excluded. Manometric and reflux diagnoses were recorded from pre-transplant esophageal function testing. Time-to-event analysis using Cox proportional hazards model was applied to evaluate outcome of first episode of acute cellular rejection, defined histologically per International Society of Heart and Lung Transplantation guidelines. Subjects not meeting this endpoint were censored at time of post-transplant anti-reflux surgery, last clinic visit, or death. Fisher's exact test for binary variables and student's t-test for continuous variables were performed to assess for differences between groups. RESULTS: Of 184 subjects (54% men, mean age: 58, follow-up: 443 person-years) met criteria for inclusion. Interstitial pulmonary fibrosis represented the predominant pulmonary diagnosis (41%). During the follow-up period, 60 subjects (33.5%) developed acute rejection. The all-cause mortality was 16.3%. Time-to-event univariate analyses demonstrated significant association between IEM and acute rejection [hazard ratio (HR): 1.984, 95%CI: 1.03-3.30, P = 0.04], confirmed on Kaplan-Meier curve. On multivariable analysis, IEM remained independently associated with acute rejection, even after controlling for potential confounders such as the presence of acid and nonacid reflux (HR: 2.20, 95%CI: 1.18-4.11, P = 0.01). Nonacid reflux was also independently associated with acute rejection on both univariate (HR: 2.16, 95%CI: 1.26-3.72, P = 0.005) and multivariable analyses (HR: 2.10, 95%CI: 1.21-3.64, P = 0.009), adjusting for the presence of IEM. CONCLUSION: Pre-transplant IEM was associated with acute rejection after transplantation, even after controlling for acid and nonacid reflux. Esophageal motility testing may be considered in lung transplant to predict outcomes.

Pre-Lung transplant reflux testing demonstrates high prevalence of gastroesophageal reflux in cystic fibrosis and reduces chronic rejection risk.

BACKGROUND: Gastroesophageal reflux (GER) has been associated with poor outcomes after lung transplantation for chronic lung disease, including increased risk of chronic rejection. GER is common in cystic fibrosis (CF), but factors influencing the likelihood of pre-transplant pH testing, and the impact of testing on clinical management and transplant outcomes in patients with CF are unknown. AIM: To evaluate the role of pre-transplant reflux testing in the evaluation of lung transplant candidates with CF. METHODS: This was a retrospective study from 2007-2019 at a tertiary medical center that included all patients with CF undergoing lung transplant. Patients with pre-transplant anti-reflux surgery were excluded. Baseline characteristics (age at transplantation, gender, race, body mass index), self-reported GER symptoms prior to transplantation, and pre-transplant cardiopulmonary testing results, were recorded. Reflux testing consisted of either 24-h pH- or combined multichannel intraluminal impedance and pH monitoring. Post-transplant care included a standard immunosuppressive regimen, and regular surveillance bronchoscopy and pulmonary spirometry in accordance with institutional practice as well as in symptomatic patients. The primary outcome of chronic lung allograft dysfunction (CLAD) was defined clinically and histologically per International Society of Heart and Lung Transplantation criteria. Statistical analysis was performed with Fisher's exact test to assess differences between cohorts, and time-to-event Cox proportional hazards modeling. RESULTS: After applying inclusion and exclusion criteria, a total of 60 patients were included in the study. Among all CF patients, 41 (68.3%) completed reflux monitoring as part of pre-lung transplant evaluation. Objective evidence of pathologic reflux, defined as acid exposure time > 4%, was found in 24 subjects, representing 58% of the tested group. CF patients with pre-transplant reflux testing were older (35.8 vs 30.1 years, P = 0.01) and more commonly reported typical esophageal reflux symptoms (53.7% vs 26.3%, P = 0.06) compared to those without reflux testing. Other patient demographics and baseline cardiopulmonary function did not significantly differ between CF subjects with and without pre-transplant reflux testing. Patients with CF were less likely to undergo pre-transplant reflux testing compared to other pulmonary diagnoses (68% vs 85%, P = 0.003). There was a decreased risk of CLAD in patients with CF who underwent reflux testing compared to those who did not, after controlling for confounders (Cox Hazard Ratio 0.26; 95%CI: 0.08-0.92). CONCLUSION: Pre-transplant reflux testing revealed high prevalence of pathologic reflux in CF patients and was associated with decreased risk of CLAD. Systematic reflux testing may enhance outcomes in this patient population.

Interferon-dependent signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections, including COVID-19-related acute respiratory distress syndrome, although its contribution to disease pathogenesis remains poorly understood. Blood and airway immune cells from 52 patients with severe COVID-19 were phenotyped by flow cytometry. Samples and clinical data were collected at 2 separate time points to assess changes during ICU stay. Blockade of type I interferon and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) signaling was performed in vitro to determine their contribution to viral clearance in A2 neutrophils. We identified 2 neutrophil subpopulations (A1 and A2) in the airway compartment, where loss of the A2 subset correlated with increased viral burden and reduced 30-day survival. A2 neutrophils exhibited a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation, with consequent reduced viral catabolism, providing the first discrete mechanism to our knowledge of type I interferon signaling in neutrophils. The identification of this neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Adverse Outcomes Associated With Atrial Arrhythmias After Veno-Venous Extracorporeal Membrane Oxygenation.

Veno-venous extracorporeal membrane oxygenation (VV ECMO) is used as a treatment modality in those who fail to respond to conventional care. Hypoxia and medications used in the intensive care unit may increase risk for atrial arrhythmias (AA). This study aims to evaluate the impact of AA on post-VV ECMO outcome. A retrospective review of patients who were placed on VV ECMO between October 2016 and October 2021. One hundred forty-five patients were divided into two groups, AA and no AA. Baseline characteristic and potential risk factors were assessed. Uni- and multivariate analysis using logistic regression models were constructed to evaluate the predictors of mortality between groups. Survival between groups was estimated by the Kaplan-Meier method using the log-rank test. Advanced age with history of coronary artery disease and hypertension were associated with increased risk to develop AA post-VV ECMO placement ( p value < 0.05). Length on ECMO, time intubated, hospital length of stay, and sepsis were significantly increased in patients in the AA group ( p value < 0.05). There was no difference in the overall mortality between the two groups. AAs were associated with worse hospital course and complications but no difference in overall mortality rate. Age and cardiovascular disease seem to be predisposing risk factors for this. Further studies are needed to investigate potential strategies to prevent AAs development in this population.

IL-33 mediates Pseudomonas induced airway fibrogenesis and is associated with CLAD.

BACKGROUND: Long term outcomes of lung transplantation are impacted by the occurrence of chronic lung allograft dysfunction (CLAD). Recent evidence suggests a role for the lung microbiome in the occurrence of CLAD, but the exact mechanisms are not well defined. We hypothesize that the lung microbiome inhibits epithelial autophagic clearance of pro-fibrotic proteins in an IL-33 dependent manner, thereby augmenting fibrogenesis and risk for CLAD. METHODS: Autopsy derived CLAD and non-CLAD lungs were collected. IL-33, P62 and LC3 immunofluorescence was performed and assessed using confocal microscopy. Pseudomonas aeruginosa (PsA), Streptococcus Pneumoniae (SP), Prevotella Melaninogenica (PM), recombinant IL-33 or PsA-lipopolysaccharide was co-cultured with primary human bronchial epithelial cells (PBEC) and lung fibroblasts in the presence or absence of IL-33 blockade. Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate IL-33 expression, autophagy, cytokines and fibroblast differentiation markers. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of Beclin-1. RESULTS: Human CLAD lungs demonstrated markedly increased expression of IL-33 and reduced basal autophagy compared to non-CLAD lungs. Exposure of co-cultured PBECs to PsA, SP induced IL-33, and inhibited PBEC autophagy, while PM elicited no significant response. Further, PsA exposure increased myofibroblast differentiation and collagen formation. IL-33 blockade in these co-cultures recovered Beclin-1, cellular autophagy and attenuated myofibroblast activation in a Beclin-1 dependent manner. CONCLUSION: CLAD is associated with increased airway IL-33 expression and reduced basal autophagy. PsA induces a fibrogenic response by inhibiting airway epithelial autophagy in an IL-33 dependent manner.

Type I interferon-dependent IFIT3 signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections including COVID-19-related ARDS, although its contribution to disease pathogenesis remains poorly understood. Here, we identified two neutrophil subpopulations (A1 and A2) in the airway compartment of 52 severe COVID-19 subjects, where loss of the A2 subset correlated with increased viral burden and reduced 30-days survival. A2 neutrophils showcased a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation with consequent reduced viral catabolism, providing the first discrete mechanism of type I interferon signaling in neutrophils. The identification of this novel neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Evidence of Advanced Pulmonary Vascular Remodeling in Obstructive Hypertrophic Cardiomyopathy With Pulmonary Hypertension.

BACKGROUND: Elevated mean pulmonary artery pressure (mPAP) is common in patients with hypertrophic cardiomyopathy (HCM) and heart failure symptoms. However, dynamic left ventricular (LV) outflow tract obstruction may confound interpretation of pulmonary hypertension (PH) pathophysiologic features in HCM when relying on resting invasive hemodynamic data alone. RESEARCH QUESTION: Do structural changes to the lung vasculature clarify PH pathophysiologic features in patients with HCM with progressive heart failure? STUDY DESIGN AND METHODS: Clinical data and ultrarare lung autopsy specimens were acquired retrospectively from the National Institutes of Health (1975-1992). Patients were included based on the availability of lung tissue and recorded mPAP. Discarded tissue from rejected lung donors served as control specimens. Histomorphology was performed on pulmonary arterioles and veins. Comparisons were calculated using the Student t test and Mann-Whitney U test; Pearson correlation was used to assess association between morphometric measurements and HCM cardiac and hemodynamic measurements. RESULTS: The HCM cohort (n = 7; mean ± SD age, 43 ± 18 years; 71% men) showed maximum mean ± SD LV wall thickness of 25 ± 2.8 mm, mean ± SD outflow tract gradient of 90 ± 30 mm Hg, median mPAP of 25 mm Hg (interquartile range [IQR], 6 mm Hg), median pulmonary artery wedge pressure (PAWP) of 16 mm Hg (IQR, 4 mm Hg), and median pulmonary vascular resistance of 1.8 Wood units (WU; IQR, 2.4 WU). Compared with control samples (n = 5), patients with HCM showed greater indexed pulmonary arterial hypertrophy (20.7 ± 7.2% vs 49.7 ± 12%; P < .001) and arterial wall fibrosis (11.5 ± 3.4 mm vs 21.0 ± 4.7 mm; P < .0001), which correlated with mPAP (r = 0.84; P = .018), PAWP (r = 0.74; P = .05), and LV outflow tract gradient (r = 0.78; P = .035). Compared with control samples, pulmonary vein thickness was increased by 2.9-fold (P = .008) in the HCM group, which correlated with mPAP (r = 0.81; P = .03) and LV outflow tract gradient (r = 0.83; P = .02). INTERPRETATION: To the best of our knowledge, these data demonstrate for the first time that in patients with obstructive HCM, heart failure is associated with pathogenic pulmonary vascular remodeling even when mPAP is elevated only mildly. These observations clarify PH pathophysiologic features in HCM, with future implications for clinical strategies that mitigate outflow tract obstruction.

Routine Reflux Testing Guides Timely Antireflux Treatment to Reduce Acute and Chronic Rejection After Lung Transplantation.

INTRODUCTION: Gastroesophageal reflux has been associated with poorer lung transplantation outcomes, although no standard approach to evaluation/management has been adopted. We aimed to evaluate the effect of timely antireflux treatment as guided by routine reflux testing on postlung transplant rejection outcomes. METHODS: This was a retrospective cohort study of lung transplant recipients at a tertiary center. All patients underwent pretransplant ambulatory pH monitoring. Timely antireflux treatment was defined as proton pump inhibitor initiation or antireflux surgery within 6 months of transplantation. Patients were separated into 3 groups: normal pH monitoring (-pH), increased reflux (+pH) with timely treatment, and +pH with delayed treatment. Rejection outcomes included acute rejection, bronchiolitis obliterans syndrome, and chronic lung allograft dysfunction per International Society for Heart and Lung Transplantation criteria. Time-to-event analyses using Cox proportional hazard models were applied. Patients not meeting outcomes were censored at death or last clinic visit. RESULTS: One hundred seventy-five patients (59% men/mean 56.3 yr/follow-up: 496 person-years) were included. On multivariable analyses, +pH/delayed treatment patients had higher risks of acute rejection (adjust hazard ratio [aHR]:3.81 [95% confidence interval [CI]: 1.90-7.64], P = 0.0002), bronchiolitis obliterans syndrome (aHR: 2.22 [95% CI: 1.07-4.58], P = 0.03), and chronic lung allograft dysfunction (aHR: 2.97 [95% CI: 1.40-6.32], P = 0.005) than +pH/timely treatment patients. Similarly, rejection risks were increased among +pH/delayed treatment patients vs -pH patients (all P < 0.05). No significant differences in rejection risks were noted between +pH/timely treatment patients and -pH patients. Failure/complications of antireflux treatment were rare and similar among groups. DISCUSSION: Timely antireflux treatment, as directed by pretransplant reflux testing, was associated with reduced allograft rejection risks and demonstrated noninferiority to patients without reflux. A standardized peri-transplant test-and-treat algorithm may guide timely reflux management to improve lung transplant outcomes.

Single-cell transcriptome mapping identifies a local, innate B cell population driving chronic rejection after lung transplantation.

Bronchiolitis obliterans syndrome (BOS) is the main reason for poor outcomes after lung transplantation (LTx). We and others have recently identified B cells as major contributors to BOS after LTx. The extent of B cell heterogeneity and the relative contributions of B cell subpopulations to BOS, however, remain unclear. Here, we provide a comprehensive analysis of cell population changes and their gene expression patterns during chronic rejection after orthotopic LTx in mice. Of 11 major cell types, Mzb1-expressing plasma cells (PCs) were the most prominently increased population in BOS lungs. These findings were validated in 2 different cohorts of human BOS after LTx. A Bhlhe41, Cxcr3, and Itgb1 triple-positive B cell subset, also expressing classical markers of the innate-like B-1 B cell population, served as the progenitor pool for Mzb1+ PCs. This subset accounted for the increase in IgG2c production within BOS lung grafts. A genetic lack of Igs decreased BOS severity after LTx. In summary, we provide a detailed analysis of cell population changes during BOS. IgG+ PCs and their progenitors - an innate B cell subpopulation - are the major source of local Ab production and a significant contributor to BOS after LTx.

Aberrant Expression of ACO1 in Vasculatures Parallels Progression of Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is characterized by mitochondrial dysfunction. However, details about the non-mitochondrial enzymes that sustain the proliferative nature of IPF are unclear. Aconitases are a family of enzymes that sustain metabolism inside and outside mitochondria. It is hypothesized that aconitase 1 (ACO1) plays an important role in the pathogenesis of IPF given that ACO1 represents an important metabolic hub in the cytoplasm. Objectives: To determine if ACO1 expression in IPF lungs shows specific patterns that may be important in the pathogenesis of IPF. To determine the similarities and differences in ACO1 expression in IPF, bleomycin-treated, and aging lungs. Methods: ACO1 expression in IPF lungs were characterized and compared to non-IPF controls by western blotting, immunostaining, and enzymatic activity assay. ACO1-expressing cell types were identified by multicolor immunostaining. Using similar methods, the expression profiles of ACO1 in IPF lungs versus bleomycin-treated and aged mice were investigated. Measurements and main results: Lower lobes of IPF lungs, unlike non-IPF controls, exhibit significantly high levels of ACO1. Most of the signals colocalize with von Willebrand factor (vWF), a lineage marker for vascular endothelial cells. Bleomycin-treated lungs also show high ACO1 expressions. However, most of the signals colocalize with E-cadherin and/or prosurfactant protein C, representative epithelial cell markers, in remodeled areas. Conclusions: A characteristic ACO1 expression profile observed in IPF vasculatures may be a promising diagnostic target. It also may give clues as to how de novo angiogenesis contributes to the irreversible nature of IPF.

Primary Graft Dysfunction: The Role of Aging in Lung Ischemia-Reperfusion Injury.

Transplant centers around the world have been using extended criteria donors to remedy the ongoing demand for lung transplantation. With a rapidly aging population, older donors are increasingly considered. Donor age, at the same time has been linked to higher rates of lung ischemia reperfusion injury (IRI). This process of acute, sterile inflammation occurring upon reperfusion is a key driver of primary graft dysfunction (PGD) leading to inferior short- and long-term survival. Understanding and improving the condition of older lungs is thus critical to optimize outcomes. Notably, ex vivo lung perfusion (EVLP) seems to have the potential of reconditioning ischemic lungs through ex-vivo perfusing and ventilation. Here, we aim to delineate mechanisms driving lung IRI and review both experimental and clinical data on the effects of aging in augmenting the consequences of IRI and PGD in lung transplantation.