Robust segregation of donor and recipient cells from single-cell RNA-sequencing of transplant samples

BackgroundSingle-cell RNA-sequencing (scRNA-seq) technology has revealed novel cell populations in organs, uncovered regulatory relationships between genes, and allowed for tracking of cell lineage trajectory during development. It demonstrates promise as a method to better understand transplant biology; however, fundamental bioinformatic tools for its use in the context of transplantation have not been developed. One major need has been a robust method to identify cells as being either donor or recipient genotype origin, and ideally without the need to separately sequence the donor and recipient.MethodsWe implemented a novel two-stage genotype discovery method (scTx) optimized for transplant samples by being robust to disparities in cell number and cell type. Using both in silico and real-world scRNA-seq transplant data, we benchmarked our method against existing demultiplexing methods to profile their limitations in terms of sequencing depth, donor and recipient cell imbalance, and single nucleotide variant input selection.ResultsUsing in silico data, scTx could more accurately separate donor from recipient cells and at much lower genotype ratios than existing methods. This was further validated using solid-organ scRNA-seq data where scTx could more reliably identify when a second genotype was present and at lower numbers of cells from a second genotype.ConclusionscTx introduces the capability to accurately segregate donor and recipient gene expression at the single-cell level from scRNA-seq data without the need to separately genotype the donor and recipient. This will facilitate the use of scRNA-seq in the context of transplantation

A method for translational rat ex vivo lung perfusion experimentation

The application of ex vivo lung perfusion (EVLP) has significantly increased the successful clinical use of marginal donor lungs. While large animal EVLP models exist to test new strategies to improve organ repair, there is currently no rat EVLP model capable of maintaining long-term lung viability. Here, we describe a new rat EVLP model that addresses this need, while enabling the study of lung injury due to cold ischemic time (CIT). The technique involves perfusing and ventilating male Lewis rat donor lungs for 4 h before transplanting the left lung into a recipient rat and then evaluating lung function 2 h after reperfusion. To test injury within this model, lungs were divided into groups and exposed to different CITs (i.e., 20 min, 6 h, 12 h, 18 h and 24 h). Experiments involving the 24-h-CIT group were prematurely terminated due to the development of severe edema. For the other groups, no differences in the ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO₂/FIO₂) were observed during EVLP; however, lung compliance decreased over time in the 18-h group (P = 0.012) and the PaO₂/FIO₂ of the blood from the left pulmonary vein 2 h after transplantation was lower compared with 20-min-CIT group (P = 0.0062). This new model maintained stable lung function during 4-h EVLP and after transplantation when exposed to up to 12 h of CIT

Effect of ventilator-induced lung injury on the development of reperfusion injury in a rat lung transplant model

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Cricotracheal resection for adult subglottic stenosis : Factors predicting treatment failure

Objectives/Hypothesis Identify predictors of decannulation failure after cricotracheal resection (CTR) and thyrotracheal anastomosis (TTA) in patients with subglottic stenosis (SGS). Study Design Retrospective cohort study. Methods Charts of patients undergoing CTR and TTA for SGS at the University Health Network, Toronto, Ontario, Canada between 1988 and 2017 were reviewed. Patient, pathology, treatment, and outcome data were collected. The end points for statistical analysis were development of restenosis and permanent tracheostomy. Results One hundred fourteen patients (n = 114) were eligible for inclusion in this review. The mean age at primary resection was 46.9 years, 95 (83%) were females, and 19 (17%) were males. The rate of restenosis and permanent tracheostomy was 13% and 5%, respectively. Sixty-two patients (54%) underwent a CTR and TTA, and 52 patients (46%) underwent a CTR, laryngofissure, and TTA. Traumatic stenosis (odds ratio [OR] = 10.3, P = .017), longer T-tube duration (OR = 1.2, P = .011), combined glottic/subglottic stenosis (OR = 10.47, P = .010), start of the stenosis at the vocal cords (OR = 6.6, P = .029), postoperative minor complications (OR = 13.6, P = .028), and need for repeat surgery (OR = 44.1, P <.001) were associated with an increased risk of requiring permanent tracheostomy. Conclusions CTR and TTA are excellent surgical approaches for adult patients with subglottic stenosis. In this study, 5% of patients required permanent tracheostomy. Factors predicting treatment failure include traumatic stenosis, longer T-tube duration, combined glottic/subglottic stenosis, start of stenosis at the level of vocal cords, postoperative minor complications, and need for repeat surgery. Level of Evidence 4 Laryngoscope, 2019Peer reviewe

Improved results of induction chemoradiation before surgical intervention for selected patients with stage IIIA-N2 non–small cell lung cancer

ObjectiveOptimal management of stage IIIA-N2 non–small cell lung cancer remains controversial. The surgical arm of the North American Intergroup 0139 trial was adopted as the standard treatment for patients with resectable N2 disease at the University Health Network. Results after 7 years of experience are reported.MethodsThis is a retrospective study of consecutive patients with biopsy-proved T1-3 N2 M0 lung cancer who underwent induction chemoradiation before surgical intervention from January 1997 through August 2004. Induction chemotherapy consisted of cisplatin, 50 mg/m2, on days 1 and 8; etoposide, 50 mg/m2, on days 1 to 5, weeks 1 and 5; and concurrent daily external beam radiotherapy to 45 Gy. Lung resection was performed within 6 weeks of completion of chemoradiation, followed by 2 further cycles of consolidation chemotherapy.ResultsBetween January 1997 and August 2004, 40 patients were treated according to this protocol (25% T1, 62.5% T2, 7.5% T3, and 5% T4). Overall and disease-free median survivals were 40 and 37.1 months, respectively, whereas overall and disease-free 3-year survivals were 51.7% and 52.3%, respectively. R0 resection was achieved in 92.5%. The overall operative mortality rate was 7.5% (0% for lobectomy and 27% for pneumonectomy). Notably, all mortalities occurred within the first 2 years of our experience with this regimen.ConclusionChemoradiation before pulmonary resection in carefully selected patients with surgically resectable stage IIIA (N2) non–small cell lung cancer can lead to improved overall and disease-free survival

Implications of High Sensitivity Troponin Levels After Lung Transplantation

Trends in high-sensitivity cardiac troponin I (hs-cTnI) after lung transplant (LT) and its clinical value are not well stablished. This study aimed to determine kinetics of hs-cTnI after LT, factors impacting hs-cTnI and clinical outcomes. LT recipients from 2015 to 2017 at Toronto General Hospital were included. Hs-cTnI levels were collected at 0–24 h, 24–48 h and 48–72 h after LT. The primary outcome was invasive mechanical ventilation (IMV) &gt;3 days. 206 patients received a LT (median age 58, 35.4% women; 79.6% double LT). All patients but one fulfilled the criteria for postoperative myocardial infarction (median peak hs-cTnI = 4,820 ng/mL). Peak hs-cTnI correlated with right ventricular dysfunction, &gt;1 red blood cell transfusions, bilateral LT, use of EVLP, kidney function at admission and time on CPB or VA-ECMO. IMV&gt;3 days occurred in 91 (44.2%) patients, and peak hs-cTnI was higher in these patients (3,823 vs. 6,429 ng/mL, p &lt; 0.001 after adjustment). Peak hs-cTnI was higher among patients with had atrial arrhythmias or died during admission. No patients underwent revascularization. In summary, peak hs-TnI is determined by recipient comorbidities and perioperative factors, and not by coronary artery disease. Hs-cTnI captures patients at higher risk for prolonged IMV, atrial arrhythmias and in-hospital death

Survival After Lung Transplantation for Chronic Hypersensitivity Pneumonitis: Results From a Large International Cohort Study

Repeated exposure to antigens via inhalation is the primary cause of hypersensitivity pneumonitis, a form of interstitial pneumonia. The chronic form of hypersensitivity pneumonitis leads to progressive loss of respiratory function; lung transplantation is the only therapeutic option for chronically ill patients. The ESTS Lung Transplantation Working Group conducted a retrospective multicentred cohort study to increase the body of knowledge available on this rare indication for lung transplantation. Data were collected for every patient who underwent lung transplant for hypersensitivity pneumonitis in participating centres between December 1996 and October 2019. Primary outcome was overall survival; secondary outcome was freedom from chronic lung allograft dysfunction. A total of 114 patients were enrolled from 9 centres. Almost 90% of patients were diagnosed with hypersensitivity pneumonitis before transplantation, yet the antigen responsible for the infection was identified in only 25% of cases. Eighty per cent of the recipients received induction therapy. Survival at 1, 3, and 5 years was 85%, 75%, and 70%, respectively. 85% of the patients who survived 90 days after transplantation were free from chronic lung allograft dysfunction after 3 years. The given study presents a large cohort of HP patients who underwent lung transplants. Overall survival rate is higher in transplanted hypersensitivity pneumonitis patients than in those suffering from any other interstitial lung diseases. Hypersensitivity pneumonitis patients are good candidates for lung transplantation

Surgical therapy of thymic tumours with pleural involvement: an ESTS Thymic Working Group Project

OBJECTIVES Surgery for thymic epithelial tumours (TETs) with pleural involvement is infrequently performed. Thus, the value of surgical therapy for primary or recurrent TETs with pleural involvement is not sufficiently defined yet. METHODS Twelve institutions contributed retrospective data on 152 patients undergoing surgery (1977-2014) on behalf of the ESTS Thymic Working group. Outcome measures included overall (OS), cause-specific (CSS) and disease-free (DFS) survival as well as freedom from recurrence (FFR). RESULTS In 70.4% of cases, pleural involvement was present at the time of primary intervention, whereas 29.6% had surgery for recurrent disease involving the pleura. Pleural involvement resulted from thymomas (88.8%) and thymic carcinomas (11.2%). Forty extrapleural pneumonectomies (EPPs), 23 total pleurectomies (TPs), and 88 local pleurectomies (LPs) were performed (completeness of resection in 76.8%). OS for the entire patient population at 1, 3, 5 and 10 years was 96.4%, 91.0%, 87.2% and 62.7%, respectively. There was no statistically significant difference regarding FFR and OS for patients with local or advanced disease undergoing EPP, TP or LP. Thymic carcinomas in comparison with thymomas had a negative impact on OS [hazard ratio 6.506, P  = 0.002], CSS and FFR. Incomplete resections predicted worse OS [hazard ratio 6.696, P  = 0.003]. CONCLUSIONS Complete resection remains the mainstay of treatment for TETs with pleural involvement. Study populations treated with EPP, TP and LP had similar survival that may be factual as observed, but in the presence of selection bias, we can further conclude from the results that EPP, TP and LP are equally effective procedures. Procedural choice depends upon the extent of tumour distribution. EPPs, TPs and LPs performed within a multimodality setting seem to be efficient procedures for local control of disease, as they yield excellent results regarding OS, DFS, CSS and FFR

Pentraxin 3 deficiency enhances features of chronic rejection in a mouse orthotopic lung transplantation model

Chronic lung allograft dysfunction (CLAD) is a serious complication after lung transplantation and thought to represent chronic rejection. Increased expression of Pentraxin 3 (PTX3), an acute phase protein, was associated with worse outcome in lung transplant patients. To determine the role of recipient PTX3 in development of chronic rejection, we used a minor alloantigen-mismatched murine orthotopic single lung transplant model. Male C57BL/10 mice were used as donors. Male PTX3 knockout (KO) mice and their wild type (WT) littermates on 129/SvEv/C57BL6/J background were used as recipients. In KO recipients, 7/13 grafted lungs were consolidated without volume recovery on CT scan, while only 2/9 WT mice showed similar graft consolidation. For grafts where lung volume could be reliably analyzed by CT scan, the lung volume recovery was significantly reduced in KO mice compared to WT. Interstitial inflammation, parenchymal fibrosis and bronchiolitis obliterans scores were significantly higher in KO mice. Presence of myofibroblasts and lymphoid aggregation was significantly enhanced in the grafts of PTX3 KO recipients. Recipient PTX3 deficiency enhanced chronic rejection-like lesions by promoting a fibrotic process in the airways and lung parenchyma. The underlying mechanisms and potential protective role of exogenous PTX3 as a therapy should be further explored