Lymphocytic Bronchiolitis Research Articles

Diagnostic alignment to optimize inter-rater reliability among lung transplant pathologists

BackgroundPoor agreement among lung transplant pathologists has been reported in the assessment of rejection. In addition to acute rejection (AR) and lymphocytic bronchiolitis (LB), acute lung injury (ALI) and organizing pneumonia (OP) were recently identified as histopathologic risk factors for chronic lung allograft dysfunction (CLAD). Therefore, maximizing inter-rater reliability (IRR) for identifying these histopathologic risk factors is important to guide individual patient care and to support incorporating them in inclusion criteria for clinical trials in lung transplantation. MethodsNine pathologists across eight North American lung transplant centers were surveyed for practices in the assessment of lung transplant transbronchial biopsies. We conducted seven diagnostic alignment sessions with pathologists discussing histomorphologic features of CLAD high-risk histopathology. Then, each pathologist blindly scored 75 digitized slides. Fleiss’ kappa, accounting for agreement across numerous observers, was used to determine IRR across all raters for presence of any high-risk finding and each individual entity. ResultsIRR (95% confidence intervals) and % agreement for any high-risk finding (AR, LB, ALI and/or OP) and each individual finding is as follows: Any Finding, k = 0.578 (0.487, 0.668), 78.9%; AR, k = 0.582 (0.481, 0.651), 79.1%; LB, k = 0.683 (0.585, 0.764), 83.5%; ALI, k = 0.418 (0.312, 0.494), 70.9%; OP, k = 0.621 (0.560, 0.714), 81.0%. ConclusionsAfter pre-study diagnostic alignment sessions, a multi-center group of lung transplant pathologists seeking to identify histopathology high-risk for CLAD achieved good IRR.

Disulfiram, an Anti-alcoholic Drug, Targets Macrophages and Attenuates Acute Rejection in Rat Lung Allografts.

Macrophages contribute to post-transplant lung rejection. Disulfiram (DSF), an anti-alcoholic drug, has an anti-inflammatory effect and regulates macrophage chemotactic activity. Here, we investigated DSF efficacy in suppressing acute rejection post-lung transplantation. Male Lewis rats (280-300g) received orthotopic left lung transplants from Fisher 344 rats (minor histocompatibility antigen-mismatched transplantation). DSF (0.75mg/h) monotherapy or co-solvent only (50% hydroxypropyl-β-cyclodextrin) as control was subcutaneously administered for 7 days (n = 10/group). No post-transplant immunosuppressant was administered. Grades of acute rejection, infiltration of immune cells positive for CD68, CD3, or CD79a, and gene expression of monocyte chemoattractant protein and pro-inflammatory cytokines in the grafts were assessed 7 days post-transplantation. The DSF-treated group had significantly milder lymphocytic bronchiolitis than the control group. The infiltration levels of CD68+ or CD3+ cells to the peribronchial area were significantly lower in the DSF than in the control groups. The normalized expression of chemokine ligand 2 and interleukin-6 mRNA in allografts was lower in the DSF than in the control groups. Validation assay revealed interleukin-6 expression to be significantly lower in the DSF than in the control groups. DSF can alleviate acute rejection post-lung transplantation by reducing macrophage accumulation around peripheral bronchi and suppressing pro-inflammatory cytokine expression.

Effect of β-Nicotinamide Adenine Dinucleotide on Acute Allograft Rejection After Rat Lung Transplantation.

Rat single-lung transplantation was performed in 2 groups (n = 8 per group), using Brown-Norway donors and major histocompatibility complex-mismatched Lewis recipients. Recipients of the NAD+ group received 1000 mg/kg NAD+ intraperitoneally before transplantation and daily thereafter until euthanasia, whereas the control group received saline solution. At autopsy on day 5, blood samples were analyzed and the lung allograft was assessed by bronchioalveolar lavage, histology, and immunochemistry. The NAD+ group maintained an intact compliant lung tissue, a strong trend of lower acute cellular rejection (A3 versus A3-A4) and significantly less lymphocytic bronchiolitis (B0-B2R versus B1R-Bx). In addition, a trend of fewer alveolar CD68+ macrophages and significantly fewer interstitial CD163+ macrophages was observed. Bronchoalveolar lavage in the NAD+ group showed significantly fewer proinflammatory cytokines interleukin (IL)-6, IL-13, TNFα, and a protective IL-6/IL-10-ratio. In blood samples, we observed significantly fewer neutrophils, and proinflammatory GRO/KC in the NAD+ group. NAD+ might be a promising substance in prevention of acute allograft rejection in lung transplantation.

Pulmonary Histopathologic Findings in Pediatric Patients After Hematopoietic Stem Cell Transplantation: An Autopsy Study.

Pathologic characterization of pulmonary complications following hematopoietic stem cell transplantation (HSCT) is limited. We describe lung findings in pediatric patients who died following HSCT and attempt to identify potential clinical associations. Pathology databases at Texas Children's Hospital and the Children's Hospital of Philadelphia were queried (2013-2018 CHOP and 2017-2018 TCH). Electronic medical records and slides were reviewed. Among 29 patients, 19 received HSCT for hematologic malignancy, 8 for non-malignant hematologic disorders, and 2 for metastatic solid tumors. Twenty-five patients (86%) showed 1 or more patterns of acute and organizing lung injury. Sixty-two percent had microvascular sclerosis, with venous involvement noted in most cases and not correlating with clinical history of pulmonary hypertension, clinical transplant-associated thrombotic microangiopathy, irradiation, or graft-versus-host disease. Features suggestive of graft-versus-host-disease were uncommon: 6 patients had lymphocytic bronchiolitis, and only 2 patients had evidence of bronchiolitis obliterans (both clinically unexpected), both with a mismatched unrelated donor transplant. Acute and subacute alveolar injury (diffuse alveolar damage or organizing pneumonia) is common in pediatric patients who died following HSCT and is difficult to assign to a specific etiology. Microvascular sclerosis was frequent and did not correlate with a single distinct clinical feature.

СТРУКТУРНО-ФУНКЦИОНАЛЬНАЯ ХАРАКТЕРИСТИКА БРОНХИОЛИТОВ, ВЫЗВАННЫХ БЫСТРОРАСТУЩИМИ ШТАММАМИ НЕТУБЕРКУЛЕЗНЫХ МИКОБАКТЕРИЙ

The relevance of the research is associated with the need to study changes in the lungs caused by rapidly growing strains of nontuberculous mycobacteria (NTM). Objective: to analyze results obtained by the complex study of the lungs for evaluation of the frequency and nature of terminal bronchiole lesions in patients with nontuberculous mycobacterial (NTM)infection caused by rapidly growing strains. Materials and methods. We carried out clinical, radiological, morphological, and spirometric studies of the lungs of 35 patients with rapidly growing NTM strains isolated by real-time PCR. All the patients underwent multislice CT (MSCT) of the chest using the Somatom Emotion 16 scanner (Siemens, Germany) based on the high resolution computed tomography (HRCT) algorithm with 0.8 mm slice thickness and 1.5 mm slice increment. Lung ventilation was studied using the standard spirometry. Morphological studies were performed using resection samples from 11 patients with pulmonary TB comorbidity and 7 patients with only TB (the comparison group). Results. Rapidly growing strains of NTM cause exudative lymphocytic bronchiolitis, cell infiltration of the peribronchial tissue, which is not typical for TB infection. Later, lymphocytic bronchiolitis of terminal bronchioles develops into chronic constrictive bronchiolitis accompanied by the development of bronchiolectasis, atelectasis, and emphysema confirmed by HRCT results. They show prevailing bronchogenic lesions of small airways, accompanied by obstructive-type pulmonary ventilation disorders in 80% of cases (FEV1 is more than 70% of the predicted values). The results of the complex study should be considered in diagnosis and treatment of patients with NTM infection of the lungs.

USE OF INHALED CORTICOSTERIODS/LONG-ACTING BETA AGONIST (ICS-LABA) IN PATIENTS WITH BRONCHIOLITIS CAUSED BY SJÖGREN SYNDROME DECREASES NEED FOR ORAL STEROIDS

USE OF INHALED CORTICOSTERIODS/LONG-ACTING BETA AGONIST (ICS-LABA) IN PATIENTS WITH BRONCHIOLITIS CAUSED BY SJÖGREN SYNDROME DECREASES NEED FOR ORAL STEROIDS

Performance of Donor-Derived Cell-Free DNA (dd-cfDNA) for Discriminating Acute Lung Allograft Injury in a Multicenter Cohort

Purpose Histological patterns of acute allograft injury including perivascular acute rejection (AR), lymphocytic bronchiolitis (LB), organizing pneumonia (OP) and acute lung injury (ALI) are frequent after lung transplant and have been associated with worse outcomes. Circulating dd-cfDNA has been proposed as a biomarker to discriminate acute allograft injury in lung recipients. We sought to determine the performance characteristics of three plasma dd-cfDNA thresholds for distinguishing acute lung allograft injury in a prospective, multicenter cohort. Methods The cohort was drawn from the Clinical Trials in Organ Transplantation-20 study and comprised 126 first adult lung recipients with 320 plasma samples that paired with a lung biopsy performed early within the first posttransplant year. Total cfDNA was isolated from ∼1mL of plasma using automated nucleic acid purification and the dd-cfDNA fraction determined at CareDx using next generation sequencing and validated bioinformatics. Biopsies were binned into typical sampling timeframes of 1, 3, and 6 months posttransplant. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for discriminating between a biopsy with vs. without acute allograft injury (defined as any of AR, LB, OP or ALI) was estimated at each timeframe for each of the following dd-cfDNA thresholds; 0.5%, 0.85%, and 1.0%. Results The Table reports the performance characteristics of each dd-cfDNA threshold at each early posttransplant timeframe. At 1month posttransplant the NPV, indicating the probability of a biopsy without acute allograft injury having a concurrent plasma dd-cfDNA value below the threshold, was poor for all evaluated thresholds. However, by 6months posttransplant the NPV improved, in particular for the 0.5% threshold. Conclusion In this prospective real-world cohort, a dd-cfDNA threshold of 0.5% achieved a high NPV for acute histological allograft injury at 6 months posttransplant. Histological patterns of acute allograft injury including perivascular acute rejection (AR), lymphocytic bronchiolitis (LB), organizing pneumonia (OP) and acute lung injury (ALI) are frequent after lung transplant and have been associated with worse outcomes. Circulating dd-cfDNA has been proposed as a biomarker to discriminate acute allograft injury in lung recipients. We sought to determine the performance characteristics of three plasma dd-cfDNA thresholds for distinguishing acute lung allograft injury in a prospective, multicenter cohort. The cohort was drawn from the Clinical Trials in Organ Transplantation-20 study and comprised 126 first adult lung recipients with 320 plasma samples that paired with a lung biopsy performed early within the first posttransplant year. Total cfDNA was isolated from ∼1mL of plasma using automated nucleic acid purification and the dd-cfDNA fraction determined at CareDx using next generation sequencing and validated bioinformatics. Biopsies were binned into typical sampling timeframes of 1, 3, and 6 months posttransplant. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for discriminating between a biopsy with vs. without acute allograft injury (defined as any of AR, LB, OP or ALI) was estimated at each timeframe for each of the following dd-cfDNA thresholds; 0.5%, 0.85%, and 1.0%. The Table reports the performance characteristics of each dd-cfDNA threshold at each early posttransplant timeframe. At 1month posttransplant the NPV, indicating the probability of a biopsy without acute allograft injury having a concurrent plasma dd-cfDNA value below the threshold, was poor for all evaluated thresholds. However, by 6months posttransplant the NPV improved, in particular for the 0.5% threshold. In this prospective real-world cohort, a dd-cfDNA threshold of 0.5% achieved a high NPV for acute histological allograft injury at 6 months posttransplant.

Prolonged Cold Ischemia and Indirect Alloimmunity Are Fundamental for the Development of Chronic Lung Allograft Dysfunction

<h3>Purpose</h3> Lung transplantation has the worst long-term survival in solid organ transplantation due to chronic lung allograft dysfunction (CLAD). Lymphatic vessels play central roles in alloimmunity by bridging innate and adaptive immune response. The purpose of this project is to study the role of lymphatic endothelium in CLAD development and to analyse the impact of prolonged donor cold ischemia on CLAD development in the experimental lung transplantation in mouse. <h3>Methods</h3> Single mismatch orthotopic left lung transplantation (left lungs from HLA-A2 knock-in mice on a C57BL/6J background or C57BL/6J as donor mice were orthotopically transplanted to C57BL/6J recipient mice) was used as a CLAD model. Donor lungs were induced to 4 hours cold ischemia at 4°C. CLAD development was analysed by Masson's trichrome staining. Lymphatic vessels were stained by anti-podoplanin and anti-LYVE-1 antibodies. Pro-inflammatory macrophages were stained by anti-iNOS antibody, anti-inflammatory macrophages were stained by anti-CD206 antibody. VEGFC source in macrophages were identified by dual immunofluorescence. <h3>Results</h3> Short cold allograft ischemia (1h, 4°C) resulted in the development of lymphocytic bronchiolitis (LB) 8 weeks after the surgery. Prolonged cold allograft ischemia resulted in the development of lymphocytic bronchiolitis (LB) already one week after the surgery and led to the late stage CLAD 8 weeks after reperfusion. All histological features of bronchiolitis obliterate syndrome and restrictive allograft syndrome (RAS) were observed in 8-weeks allogeneic lungs and were similar to human findings. The lungs from syngeneic recipients showed no pathological changes. In the allogeneic recipients, the lymphatic density increased during the CLAD development. VEGFC expression was observed in iNOS and CD206 positive macrophages. <h3>Conclusion</h3> Prolonged cold lung ischemia seems to facilitate the development of experimental late stage CLAD. Indirect alloimmunity and deteriorated ischemia-reperfusion injury are fundamental for the late stage CLAD development. Experimental allogeneic lung transplantation with prolonged cold ischemia leads to a lymphatic phenotype similar to human RAS. Proinflammatory and anti-inflammatory macrophages are likely to drive lymphangiogenesis by increased VEGFC expression.

Clinical Validation of a Plasma Donor-derived Cell-free DNA Assay to Detect Allograft Rejection and Injury in Lung Transplant.

Background.Lung transplant patients are vulnerable to various forms of allograft injury, whether from acute rejection (AR) (encompassing acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), chronic lung allograft dysfunction (CLAD), or infection (INFXN). Previous research indicates that donor-derived cell-free DNA (dd-cfDNA) is a promising noninvasive biomarker for the detection of AR and allograft injury. Our aim was to validate a clinical plasma dd-cfDNA assay for detection of AR and other allograft injury and to confirm and expand on dd-cfDNA and allograft injury associations observed in previous studies.Methods.We measured dd-cfDNA fraction using a novel single-nucleotide polymorphism-based assay in prospectively collected plasma samples paired with clinical-pathologic diagnoses. dd-cfDNA fraction was compared across clinical-pathologic cohorts: stable, ACR, AMR, isolated lymphocytic bronchiolitis, CLAD/neutrophilic-responsive allograft dysfunction (NRAD), and INFXN. Performance characteristics were calculated for AR and combined allograft injury (AR + CLAD/NRAD + INFXN) versus the stable cohort.Results.The study included 195 samples from 103 patients. Median dd-cfDNA fraction was significantly higher for ACR (1.43%, interquartile range [IQR]: 0.67%–2.32%, P = 5 × 10−6), AMR (2.50%, IQR: 2.06%–3.79%, P = 2 × 10−5), INFXN (0.74%, IQR: 0.46%–1.38%, P = 0.02), and CLAD/NRAD (1.60%, IQR: 0.57%–2.60%, P = 1.4 × 10−4) versus the stable cohort. Area under the receiver operator characteristic curve for AR versus stable was 0.91 (95% confidence interval [CI]: 0.83-0.98). Using a ≥1% dd-cfDNA fraction threshold, sensitivity for AR was 89.1% (95% CI: 76.2%-100.0%), specificity 82.9% (95% CI: 73.3%-92.4%), positive predictive value, 51.9% (95% CI: 37.5%-66.3%), and negative predictive value, 97.3% (95% CI: 94.3%-100%). For combined allograft injury area under the receiver operator characteristic curve was 0.76 (95% CI: 0.66-0.85), sensitivity 59.9% (95% CI: 46.0%-73.9%), specificity 83.9% (95% CI: 74.1%-93.7%), positive predictive value, 43.6% (95% CI: 27.6%-59.6%), and negative predictive value, 91.0% (95% CI: 87.9%-94.0%).Conclusions.These results indicate that our dd-cfDNA assay detects AR and other allograft injury. dd-cfDNA monitoring, accompanied by standard clinical assessments, represents a valuable precision tool to support lung transplant health and is appropriate for further assessment in a prospective randomized-controlled study.

Acute rejection and post lung transplant surveillance.

PurposeThe purpose of this review is to summarize the current evidence on the evaluation and treatment of acute rejection after lung transplantation.ResultsDespite significant progress in the field of transplant immunology, acute rejection remains a frequent complication after transplantation. Almost 30% of lung transplant recipients experience at least one episode of acute cellular rejection (ACR) during the first year after transplant. Acute cellular rejection, lymphocytic bronchiolitis, and antibody-mediated rejection (AMR) are all risk factors for the subsequent development of chronic lung allograft dysfunction (CLAD). Acute cellular rejection and lymphocytic bronchiolitis have well-defined histopathologic diagnostic criteria and grading. The diagnosis of antibody-mediated rejection after lung transplantation requires a multidisciplinary approach. Antibody-mediated rejection may cause acute allograft failure.ConclusionsAcute rejection is a risk factor for development of chronic rejection. Further investigations are required to better define risk factors, surveillance strategies, and optimal management strategies for acute allograft rejection.

Severe Acute Cellular Rejection With High-Grade Lymphocytic Bronchiolitis Following Transition from Tacrolimus to Belatacept in a Lung Transplantation Recipient: A Case Report

This case report describes a lung transplantation recipient who developed severe acute cellular rejection with high-grade lymphocytic bronchiolitis after transition to a calcineurin-free regimen using belatacept. A 53-year-old man who had undergone lung transplantation 3 years prior developed progressive chronic kidney disease related to tacrolimus. He was transitioned off tacrolimus to belatacept to prevent the need for dialysis. He was admitted 2 months later with acute hypoxemic respiratory failure. Video-assisted thoracic surgery biopsy showed acute fibrinous and organizing pneumonia and A4B2 rejection. He subsequently developed chronic lung allograft dysfunction. This case illustrates the potential increased risk of acute rejection associated with belatacept maintenance immunosuppression.

Variability in azithromycin practices among lung transplant providers in the International Society for Heart and Lung Transplantation Community

Chronic lung allograft dysfunction (CLAD) is the most important long-term complication after lung transplant (LTx), and clinical experience suggests significant variability in its management. We sought to capture azithromycin practices among LTx providers internationally. A survey was distributed via the International Society for Heart and Lung Transplantation and completed by 103 respondents (15 countries). Azithromycin indications, timing, and dosing varied significantly, and 37 (36%) reported inconsistency even within their center. Thirty (29%) reported initiating azithromycin prophylactically (during initial transplant hospitalization). Of 73 others, only 10 (14%) reported waiting until CLAD diagnosis (with persistent ≥20% pulmonary function decline). Most initiated azithromycin after a CLAD risk-factor and/or event, including 59 (81%) for a persistent ≥10% decrement in FEV1, 32 (44%) for lymphocytic bronchiolitis, and 27 (37%) for bronchoalveolar lavage neutrophilia. Azithromycin prescribing patterns appear to vary significantly, and further study is needed to elucidate the optimal timing and indications for its initiation after LTx.

Cathepsin B promotes collagen biosynthesis, which drives bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is a major complication after lung transplantation (LTx). BOS is characterised by massive peribronchial fibrosis, leading to air trapping-induced pulmonary dysfunction. Cathepsin B, a lysosomal cysteine protease, has been shown to enforce fibrotic pathways in several diseases. However, the relevance of cathepsin B in BOS progression has not yet been addressed. The aim of the study was to elucidate the function of cathepsin B in BOS pathogenesis.We determined cathepsin B levels in bronchoalveolar lavage fluid (BALF) and lung tissue from healthy donors (HD) and BOS LTx patients. Cathepsin B activity was assessed via a fluorescence resonance energy transfer-based assay and protein expression was determined using Western blotting, ELISA and immunostaining. To investigate the impact of cathepsin B in the pathophysiology of BOS, we used an in vivo orthotopic left LTx mouse model. Mechanistic studies were performed in vitro using macrophage and fibroblast cell lines.We found a significant increase of cathepsin B activity in BALF and lung tissue from BOS patients, as well as in our murine model of lymphocytic bronchiolitis. Moreover, cathepsin B activity was associated with increased biosynthesis of collagen and had a negative effect on lung function. We observed that cathepsin B was mainly expressed in macrophages that infiltrated areas characterised by a massive accumulation of collagen deposition. Mechanistically, macrophage-derived cathepsin B contributed to transforming growth factor-β1-dependent activation of fibroblasts, and its inhibition reversed the phenotype.Infiltrating macrophages release active cathepsin B, thereby promoting fibroblast activation and subsequent collagen deposition, which drive BOS. Cathepsin B represents a promising therapeutic target to prevent the progression of BOS.

Free Airway C4d after Lung Transplantation - A Quantitative Analysis of Bronchoalveolar Lavage Fluid

In recent years, the utility of vascular complement factor 4d (C4d) deposition as diagnostic tool for antibody mediated rejection (AMR) after lung transplantation, has become a controversial issue. We aimed to pinpoint the problematic nature of C4d as biomarker with a simple experiment.We quantified C4d in broncho-alveolar lavage (BAL) of lung transplant patients with diverse post-transplant complications in 3 different settings of clinically clear cases of: 1/ chronic lung allograft dysfunction (CLAD); 2/ acute complications acute rejection (AR), lymphocytic bronchiolitis (LB), antibody-mediated rejection (AMR) and respiratory infection (INF); 3/ patients with parallel C4d immunostaining and Anti-HLA. All groups were compared to BAL of stable patients. C4d was measured via standard ELISA.C4d was increased in CLAD, predominantly in RAS (p = 0.0026) but not in BOS (p = 0.89). C4d was increased in all acute events, AR (p = 0.0025), LB (p < 0.0001), AMR (p = 0.0034), infections (p < 0.0001). In patients with parallel C4d immunostaining and serum HLA antibodies, C4d was increased in C4d-/HLA- (p = 0.0011); C4d-/HLA+ (p = 0.013); HLA+/C4d + (p = 0.0081). A correlation of systemic C-reactive protein (CRP) with C4d was found in all patients (r = 0.49; p < 0.0001). We hypothesize that free C4d in BAL may only be representative of a general immune response in the transplanted lung.

Risk Factors for Acute Rejection in the First Year after Lung Transplant. A Multicenter Study.

Rationale: Acute rejection, manifesting as lymphocytic inflammation in a perivascular (acute perivascular rejection [AR]) or peribronchiolar (lymphocytic bronchiolitis [LB]) distribution, is common in lung transplant recipients and increases the risk for chronic graft dysfunction.Objectives: To evaluate clinical factors associated with biopsy-proven acute rejection during the first post-transplant year in a present-day, five-center lung transplant cohort.Methods: We analyzed prospective diagnoses of AR and LB from over 2,000 lung biopsies in 400 newly transplanted adult lung recipients. Because LB without simultaneous AR was rare, our analyses focused on risk factors for AR. Multivariable Cox proportional hazards models were used to assess donor and recipient factors associated with the time to the first AR occurrence.Measurements and Main Results: During the first post-transplant year, 53.3% of patients experienced at least one AR episode. Multivariable proportional hazards analyses accounting for enrolling center effects identified four or more HLA mismatches (hazard ratio [HR], 2.06; P ≤ 0.01) as associated with increased AR hazards, whereas bilateral transplantation (HR, 0.57; P ≤ 0.01) was associated with protection from AR. In addition, Wilcoxon rank-sum analyses demonstrated bilateral (vs. single) lung recipients, and those with fewer than four (vs. more than four) HLA mismatches demonstrated reduced AR frequency and/or severity during the first post-transplant year.Conclusions: We found a high incidence of AR in a contemporary multicenter lung transplant cohort undergoing consistent biopsy sampling. Although not previously recognized, the finding of reduced AR in bilateral lung recipients is intriguing, warranting replication and mechanistic exploration.

Evidence for Environmental-Human Microbiota Transfer at a Manufacturing Facility with Novel Work-related Respiratory Disease.

Rationale: Workers' exposure to metalworking fluid (MWF) has been associated with respiratory disease.Objectives: As part of a public health investigation of a manufacturing facility, we performed a cross-sectional study using paired environmental and human sampling to evaluate the cross-pollination of microbes between the environment and the host and possible effects on lung pathology present among workers.Methods: Workplace environmental microbiota were evaluated in air and MWF samples. Human microbiota were evaluated in lung tissue samples from workers with respiratory symptoms found to have lymphocytic bronchiolitis and alveolar ductitis with B-cell follicles and emphysema, in lung tissue samples from control subjects, and in skin, nasal, and oral samples from 302 workers from different areas of the facility. In vitro effects of MWF exposure on murine B cells were assessed.Measurements and Main Results: An increased similarity of microbial composition was found between MWF samples and lung tissue samples of case workers compared with control subjects. Among workers in different locations within the facility, those that worked in the machine shop area had skin, nasal, and oral microbiota more closely related to the microbiota present in the MWF samples. Lung samples from four index cases and skin and nasal samples from workers in the machine shop area were enriched with Pseudomonas, the dominant taxa in MWF. Exposure to used MWF stimulated murine B-cell proliferation in vitro, a hallmark cell subtype found in the pathology of index cases.Conclusions: Evaluation of a manufacturing facility with a cluster of workers with respiratory disease supports cross-pollination of microbes from MWF to humans and suggests the potential for exposure to these microbes to be a health hazard.

Recent advances in the understanding of bronchiolitis in adults.

Bronchiolitis is injury to the bronchioles (small airways with a diameter of 2 mm or less) resulting in inflammation and/or fibrosis. Bronchioles can be involved in pathologic processes that involve predominantly the lung parenchyma or large airways, but, in some diseases, bronchioles are the main site of injury (“primary bronchiolitis”). Acute bronchiolitis caused by viruses is responsible for most cases of bronchiolitis in infants and children. In adults, however, there is a wide spectrum of bronchiolar disorders and most are chronic. Many forms of bronchiolitis have been described in the literature, and the terminology in this regard remains confusing. In clinical practice, a classification scheme based on the underlying histopathologic pattern (correlates with presenting radiologic abnormalities) facilitates the recognition of bronchiolitis and the search for the inciting cause of the lung injury. Respiratory bronchiolitis is the most common form of bronchiolitis in adults and is usually related to cigarette smoking. Currently, the diagnosis of respiratory bronchiolitis is generally achieved based on the clinical context (smoking history) and chest CT findings. Constrictive (obliterative) bronchiolitis is associated with airflow obstruction and is seen in various clinical contexts including environmental/occupational inhalation exposures, transplant recipients (bronchiolitis obliterans syndrome), and many others. Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) is increasingly recognized and can be associated with progressive airflow obstruction related to constrictive bronchiolitis (“DIPNECH syndrome”). Diffuse aspiration bronchiolitis is a form of aspiration-related lung disease that is often unsuspected and confused for interstitial lung disease. Novel forms of bronchiolitis have been described, including lymphocytic bronchiolitis and alveolar ductitis with emphysema recently described in employees at a manufacturing facility for industrial machines. Bronchiolitis is also a component of vaping-related lung injury encountered in the recent outbreak.

Work-related adverse respiratory health outcomes at a machine manufacturing facility with a cluster of bronchiolitis, alveolar ductitis and emphysema (BADE)

ObjectivesFour machine manufacturing facility workers had a novel occupational lung disease of uncertain aetiology characterised by lymphocytic bronchiolitis, alveolar ductitis and emphysema (BADE). We aimed to evaluate current workers’ respiratory...

Single Cell Profiling Identifies Novel B Cell Populations That Drive Chronic Rejection after Lung Transplantation

Despite immunosuppressive T cell-targeting therapies, the survival rate after LTx remains as low as 50% after 5 years, mainly due to the development of BOS. The T cell centric paradigm is being increasingly challenged in solid organ transplantation. We recently demonstrated in a new mouse model of lymphocytic bronchiolitis (LB) after orthotopic LTx that B cells played a central role in the development of BOS. Alarming experiences from the past strongly suggest that a systemic B cell depletion must be considered with caution if at all. We hypothesized that a subclassification of B cells based on their individual expression profiles in rejecting versus non-rejecting murine lung grafts can provide insights into which B cell subset should be specifically targeted in priority to prevent lung graft rejection. C57BL/6J mice received C57BL/6J (non-rejecting group) or HLA-A2 knock-in (LB group) lung grafts by an orthotopic LTx surgery. One month later, single cell suspensions of lung grafts were submitted to single cell RNA-sequencing. An unsupervised clustering generated an atlas of 15 cell clusters. While the general distribution of cells amongst the different clusters was homogenous between the two groups, major quantitative shifts were observed. As such, classical monocyte, leukocyte and T cell numbers were increased respectively by 3.93-, 2.78- and 2.27-fold in the LB grafts. B cells, identified based on the expression of Cd79a, Cd79b and Ig genes, were further subdivided into 7 clusters. Clusters 0 and 2 were respectively enriched by 4.12- and 2.92-fold in the grafts that developed LB versus syngeneic lung grafts, while clusters 3 and 4 were overrepresented by 5.21- and 6.78-fold in the absence of LB. Markers for cluster 0 appeared redundant with other clusters, making it difficult to consider for further investigations. Cluster 2 displayed the expression of genes involved either in B cell activation, or differentiation into plasma cells, or regulatory/inhibitory roles, or a combination of the functions above depending on the context. Since the set of markers defining cluster 2 does not clearly relate to any described B cell subtype, we plan to functionally investigate these cells by classical immunology approaches (mixed lymphocyte reaction and ex vivo antibody production).

A Multi-Center Study of BAL CXCR3 Chemokines during Allograft Injury after Lung Transplantation

The occurrence of acute allograft injuries remains one of the most important factors responsible for the development of early chronic lung allograft dysfunction. We and others have previously demonstrated a consistent association between acute lung injury (ALI), acute perivascular rejection (AR) and lymphocytic bronchiolitis (LB) and the subsequent development of CLAD. However, the pathogenesis of these allograft injuries remains poorly understood. CXCL9, CXCL10, and CXCL11 are CXC chemokines induced by interferon-γ and act as potent chemoattractants of mononuclear cells (e.g., activated T-cells and NK cells). We hypothesized that episodes of ALI, AR and LB would be associated with increased activity of these CXCR3 chemokines, as measured in the BAL at the time of the injury. The study cohort consisted of the first 200 lung transplant recipients enrolled in Clinical Trials in Organ Transplantation-20 (NCT02631720), a prospective multi-center observational study collecting serial clinical data and biologic samples from 5 North American transplant centers. Recipients received a median (Q1,Q3) of 5 (3, 6) bronchoscopies during the first-year post-transplant. Transbronchial biopsies (TBBXs) with no histopathologic evidence of allograft injury were classified as "healthy". BALF CXCL9, CXCL10 and CXCL11 mean fluorescence intensities (MFIs) were measured using luminex bead assays. To evaluate differences in CXCR3 chemokine levels between allograft injuries and healthy biopsies, multivariable linear mixed effects models were constructed taking into account repeated measurements from recipients. There were 844 TBBXs with 249(30%) episodes of allograft injury observed. There were 173(20%) episodes of AR, 30(4%) episodes of LB and 42(5%) episodes of ALI. Biopsies with AR, LB or ALI were associated with significant elevations in all three chemokines compared with healthy biopsies (p<0.01). For CXCL9 and CXCL10, ALI had the highest MFIs, followed by AR and LB. For CXCL11, AR had the highest MFIs followed by ALI and LB. We demonstrate for the first time in a multicenter study, CXCR3 chemokine elevations during episodes of acute allograft injury. These findings support the association of CXCR3 chemokines during episodes of AR, LB and ALI, and may potentially offer novel therapeutic targets to prevent and treat these deleterious events.