Stable Lung Transplant Recipients Research Articles

Alterations of lung microbiota in lung transplant recipients with pneumocystis jirovecii pneumonia

BackgroundIncreasing evidence revealed that lung microbiota dysbiosis was associated with pulmonary infection in lung transplant recipients (LTRs). Pneumocystis jirovecii (P. jirovecii) is an opportunistic fungal pathogen that frequently causes lethal pneumonia in LTRs. However, the lung microbiota in LTRs with P. jirovecii pneumonia (PJP) remains unknow.MethodsIn this prospective observational study, we performed metagenomic next-generation sequencing (mNGS) on 72 bronchoalveolar lavage fluid (BALF) samples from 61 LTRs (20 with PJP, 22 with PJC, 19 time-matched stable LTRs, and 11 from LTRs after PJP recovery). We compared the lung microbiota composition of LTRs with and without P. jirovecii, and analyzed the related clinical variables.ResultsBALFs collected at the episode of PJP showed a more discrete distribution with a lower species diversity, and microbiota composition differed significantly compared to P. jirovecii colonization (PJC) and control group. Human gammaherpesvirus 4, Phreatobacter oligotrophus, and Pseudomonas balearica were the differential microbiota species between the PJP and the other two groups. The network analysis revealed that most species had a positive correlation, while P. jirovecii was correlated negatively with 10 species including Acinetobacter venetianus, Pseudomonas guariconensis, Paracandidimonas soli, Acinetobacter colistiniresistens, and Castellaniella defragrans, which were enriched in the control group. The microbiota composition and diversity of BALF after PJP recovery were also different from the PJP and control groups, while the main components of the PJP recovery similar to control group. Clinical variables including age, creatinine, total protein, albumin, IgG, neutrophil, lymphocyte, CD3+CD45+, CD3+CD4+ and CD3+CD8+ T cells were deeply implicated in the alterations of lung microbiota in LTRs.ConclusionsThis study suggests that LTRs with PJP had altered lung microbiota compared to PJC, control, and after recovery groups. Furthermore, lung microbiota is related to age, renal function, nutritional and immune status in LTRs.

Diagnostic accuracy of eNose ‘breathprints’ for therapeutic drug monitoring of Tacrolimus trough levels in lung transplantation

In order to prevent long-term immunity-related complications after lung transplantation, close monitoring of immunosuppressant levels using therapeutic drug monitoring (TDM) is paramount. Novel electronic nose (eNose) technology may be a non-invasive alternative to the current invasive procedures for TDM. We investigated the diagnostic and categorization capacity of eNose breathprints for Tacrolimus trough blood plasma levels (TACtrough) in lung transplant recipients (LTRs). We performed eNose measurements in stable LTR attending the outpatient clinic. We evaluated (1) the correlation between eNose measurements and TACtrough, (2) the diagnostic capacity of eNose technology for TACtrough, and (3) the accuracy of eNose technology for categorization of TACtrough into three clinically relevant categories (low: <7 µg ml−1, medium: 7–10 µg ml−1, and high: >10 µg ml−1 ). A total of 186 measurements from 86 LTR were included. There was a weak but statistically significant correlation (r = 0.21, p = 0.004) between the eNose measurements and TACtrough. The root mean squared error of prediction for the diagnostic capacity was 3.186 in the training and 3.131 in the validation set. The accuracy of categorization ranged between 45%–63% for the training set and 52%–69% in the validation set. There is a weak correlation between eNose breathprints and TACtrough in LTR. However, the diagnostic as well as categorization capacity for TACtrough using eNose breathprints is too inaccurate to be applicable in TDM.

Changes in HCMV immune cell frequency and phenotype are associated with chronic lung allograft dysfunction.

Human cytomegalovirus (HCMV) infection is common and often severe in lung transplant recipients (LTRs), and it is a risk factor associated with chronic lung allograft dysfunction (CLAD). The complex interplay between HCMV and allograft rejection is still unclear. Currently, no treatment is available to reverse CLAD after diagnosis, and the identification of reliable biomarkers that can predict the early development of CLAD is needed. This study investigated the HCMV immunity in LTRs who will develop CLAD. This study quantified and phenotyped conventional (HLA-A2pp65) and HLA-E-restricted (HLA-EUL40) anti-HCMV CD8+ T (CD8 T) cell responses induced by infection in LTRs developing CLAD or maintaining a stable allograft. The homeostasis of immune subsets (B, CD4T, CD8 T, NK, and γδT cells) post-primary infection associated with CLAD was also investigated. At M18 post-transplantation, HLA-EUL40 CD8 T responses were less frequently found in HCMV+ LTRs (21.7%) developing CLAD (CLAD) than in LTRs (55%) keeping a functional graft (STABLE). In contrast, HLA-A2pp65 CD8 T was equally detected in 45% of STABLE and 47.8% of CLAD LTRs. The frequency of HLA-EUL40 and HLA-A2pp65 CD8 T among blood CD8 T cells shows lower median values in CLAD LTRs. Immunophenotype reveals an altered expression profile for HLA-EUL40 CD8 T in CLAD patients with a decreased expression for CD56 and the acquisition of PD-1. In STABLE LTRs, HCMV primary infection causes a decrease in B cells and inflation of CD8 T, CD57+/NKG2C+ NK, and δ2-γδT cells. In CLAD LTRs, the regulation of B, total CD8 T, and δ2+γδT cells is maintained, but total NK, CD57+/NKG2C+ NK, and δ2-γδT subsets are markedly reduced, while CD57 is overexpressed across T lymphocytes. CLAD is associated with significant changes in anti-HCMV immune cell responses. Our findings propose that the presence of dysfunctional HCMV-specific HLA-E-restricted CD8 T cells together with post-infection changes in the immune cell distribution affecting NK and γδT cells defines an early immune signature for CLAD in HCMV+ LTRs. Such a signature may be of interest for the monitoring of LTRs and may allow an early stratification of LTRs at risk of CLAD.

Dd-cfDNA Levels Are Independent of Allograft Longevity in Stable Lung Transplant Recipients

<h3>Purpose</h3> Donor-derived cell free DNA (dd-cfDNA) is a well-studied biomarker of early allograft injury, though kinetics of dd-cfDNA outside the early period is understudied. We measured levels of plasma dd-cfDNA in stable lung transplant recipients (LTRs) >2-years post-transplant to determine if dd-cfDNA varies with allograft longevity. <h3>Methods</h3> We performed a prospective, observational study of LTRs that were > 2-years post-transplant and had stable lung function. Plasma dd-cfDNA (CareDx) was measured at outpatient visits. Patients were considered at baseline if they were hospital-free in the prior 30-days, FEV1 was >90% baseline, and they were sans fever, cough, or dyspnea. Patients treated with augmented immunosuppression for ALAD / CLAD were excluded. LTRs were categorized into three allograft-age cohorts (2-3, 3-5, or > 5 years post-transplant). Statistics were performed using SSPS (IBM); chi-squared testing and ANOVA was used for binary and continuous data, respectively. <h3>Results</h3> Baseline dd-cfDNA was measured in 59 LTRs meeting inclusion criteria. Overall median dd-cfDNA was 0.45% (IQR 0.30-0.65). (Fig. 1). Median plasma dd-cfDNA was 0.51% (IQR 0.34-0.63), 0.35% (IQR 0.12-0.59), and 0.43% (IQR= 0.29-0.67) for LTRs 2-3years, 3-5 years and >5-years post-transplant, respectively (p=0.91). Baseline demographics were similar between cohorts for all variables except transplant type (Table 1). <h3>Conclusion</h3> Plasma dd-cfDNA is <1% in clinically stable LTRs > 2-years post-transplant and does not vary with allograft survival. Future studies will determine whether changes in dd-cfDNA from baseline can predict CLAD onset.

Donor Age and Recipient-Donor Sex and Height Mismatch Are Associated with Abnormal Pulmonary Reactance (X5) in Stable Recipients After Bilateral Lung Transplantation

<h3>Purpose</h3> Respiratory system reactance at 5Hz (X₅) reflects tissue elastance and inertance and is measured using oscillometry, a novel measurement of respiratory mechanics. Abnormal reactance (stiffness) has been observed in baseline and chronic lung allograft dysfunction. This study aims to assess predictors of pulmonary reactance in stable lung transplant (LTx) recipients. <h3>Methods</h3> A cross-sectional study was performed on bilateral lung transplant (LTx) recipients at 2 Australian centres between Jan-20 and Jun-21. Spirometry and oscillometry (TremoFlo C-100) were performed to obtain FEV₁ and FVC and X₅, respectively. Patients with acute and chronic lung allograft dysfunction (LAD) were excluded. Baseline lung allograft dysfunction (BLAD) was defined as failure to achieve FEV₁ or FVC >80%-predicted. Oostveen equations were used to generate predicted X₅ values. Abnormal X₅ was defined as Z-score ≤ -1.64. Logistic regression measured associations between baseline transplant predictors and risk of abnormal X_5. <h3>Results</h3> A total of 326 bilateral lung transplant recipients underwent oscillometry. 173 patients with LAD were excluded and 153 included for final analysis. Patients were at median (IQR) 1.9 (2.7) years after transplant. The median (IQR) concurrent FEV₁ as %-baseline FEV₁ was 96% (7). Abnormal X₅ was detected in 21 (13.7%) and normal X₅ in 132 (86.3%). Increasing donor age (OR 1.03, 95%CI 1.00-1.06, p=0.047), recipient-donor sex mismatch (Male:Female OR 5.15, 95%CI 1.70-15.59, p=0.004), donor-recipient height difference (OR 0.95, 95% CI 0.90-0.99, p=0.027) and spirometric BLAD (OR 7.21, 95%CI 2.59-20.06, p<0.001) were associated with an increased risk of abnormal X_5. <h3>Conclusion</h3> Undersizing of lungs, recipient-donor sex mismatch and increasing donor age are significantly associated with abnormal X₅. Differences in lung volume due to sex and size matching may partly explain this association. Increasing age is associated with decreased lung elastic recoil. Preliminary analysis is limited by the use of recipient anthropometrics to generate predicted values. Longitudinal studies are required to determine if the reactance trajectory may help predict patients at risk of poor outcomes.

Novel role for tumor suppressor gene, liver kinase B1, in epithelial–mesenchymal transition leading to chronic lung allograft dysfunction

Epithelial-mesenchymal transition (EMT) has been implicated to play a role in chronic lung allograft dysfunction (CLAD). Liver kinase B1 (LKB1), a tumor suppressor gene, can regulate EMT. However, its role in CLAD development following lung transplantation remains unknown. Using qRT-PCR, biopsies from lung transplant recipients with bronchiolitis obliterans syndrome (BOS) demonstrated significant downregulation of LKB1 (p=.0001), compared to stable biopsies. To determine the role of LKB1 in EMT development, we analyzed EMT in human bronchial epithelial cell line BEAS-2B. Knockdown of LKB1 by siRNA significantly dysregulated mesenchymal markers expression in BEAS-2B cells. Following incubation of human primary bronchial epithelial cell or BEAS-2B cells with exosomes isolated from BOS or stable lung transplant recipients, LKB1 expression was inhibited when incubated with BOS-exosome. Incubation with BOS-exosomes also decreased LKB1 expression and induced EMT markers in air-liquid interface culture method. Our results provide novel evidence that exosomes released from transplanted lungs undergoing chronic rejection are associated with inactivated tumor suppressor gene LKB1 and this loss induces EMT leading to the pathogenesis of CLAD following human lung transplantation.

Biological Variation of Donor-Derived Cell-Free DNA in Stable Lung Transplant Recipients.

Prior studies demonstrate that donor-derived cell-free DNA (dd-cfDNA) in lung transplant recipients may serve as a marker of allograft injury for detecting allograft rejection and infection. Clinical interpretation of dd-cfDNA requires understanding its biological variation in stable lung transplant patients in order to identify abnormal results suggesting underlying allograft dysfunction. This study establishes the biological variation and reference change values (RCV) of dd-cfDNA in stable lung transplant recipients using an analytically validated assay with an established analytic coefficient of variation (CVA). The AlloSure® assay, a targeted, sequencing-based approach, was used to measure plasma dd-cfDNA in a cohort of lung transplant patients at 4 centers that used dd-cfDNA to monitor for allograft dysfunction in preference to surveillance transbronchial biopsy. Patients with stable allograft function and ≥3 dd-cfDNA samples were included. Intraindividual coefficient of variation (CVI), interindividual CV (CVG), index of individuality (II) and the RCV were calculated. Thirty-five patients with a combined 124 dd-cfDNA samples were included in the final analysis. The median dd-cfDNA was 0.31% (interquartile range 0.18%-0.68%), the 97.5th percentile and 95th percentile were 1.3% and 1.0%, respectively. In 30 stable patients with an average of 3.7 tests, the CVI was 25%, CVG 19%, II 1.33, and RCV 70%. In stable lung transplant patients, fluctuations in dd-cfDNA levels of up to 70% or levels less than 1% are within normal biological variation. With further validation, these thresholds may be incorporated into surveillance monitoring algorithms to identify potentially abnormal results indicating allograft dysfunction.

Bronchoalveolar lavage cytokine-based risk stratification of minimal acute rejection in clinically stable lung transplant recipients

Acute cellular rejection (ACR) remains the most significant risk factor for chronic lung allograft dysfunction (CLAD). While clinically significant or higher-grade (≥A2) ACR is generally treated with augmented immunosuppression (IS), the management of clinically stable grade A1 ACR remains controversial. At our center, patients with clinically stable grade A1 ACR are routinely not treated with augmented IS. While the overall outcomes in this group of patients at our center are equivalent to patients with stable A0 pathology, CLAD and death rates remain overall high. We hypothesized that a distinct cytokine signature at the time of early minimal rejection state would be associated with worse outcomes. Specifically, we aimed to determine whether bronchoalveolar lavage (BAL) biomarkers at the time of first clinically stable grade A1 ACR (CSA1R) are predictive of subsequent CLAD or death. Among all adult, bilateral, first lung transplants, performed 2010-2016, transbronchial biopsies obtained within the first-year post-transplant were categorized as clinically stable or unstable based on the presence or absence of ≥10% concurrent drop in forced expiratory volume in 1 second (FEV1). We assessed BAL samples obtained at the time of CSA1R episodes, which were not preceded by another ACR (i.e., first episodes). Twenty-one proteins previously associated with ACR or CLAD were measured in the BAL using a multiplex bead assay. Association between protein levels and subsequent CLAD or death was assessed using Cox Proportional Hazards models, adjusted for relevant peri-transplant clinical covariates. We identified 75 patients with first CSA1R occurring at a median time of 98 days (range 48.5-197) post-transplant. Median time from transplant to CLAD or death was 1247 (756.5-1921.5) and 1641 days (1024.5-2326.5), respectively. In multivariable models, levels of MCP1/CCL2, S100A8, IL10, TNF-receptor 1, and pentraxin 3 (PTX3) were associated with both CLAD development and death (p < 0.05 for all). PTX3 remained significantly associated with both CLAD and death after adjusting for multiple comparisons. Our data indicate that a focused BAL protein signature, with PTX3 having the strongest association, may be useful in determining a subset of CSA1R patients at increased risk and may benefit from a more aggressive management strategy.

Connective Tissue Growth Factor Is Overexpressed in Explant Lung Tissue and Broncho-Alveolar Lavage in Transplant-Related Pulmonary Fibrosis.

BackgroundConnective tissue growth factor (CTGF) is an important mediator in several fibrotic diseases, including lung fibrosis. We investigated CTGF-expression in chronic lung allograft dysfunction (CLAD) and pulmonary graft-versus-host disease (GVHD).Materials and MethodsCTGF expression was assessed by quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry in end-stage CLAD explant lung tissue (bronchiolitis obliterans syndrome (BOS), n=20; restrictive allograft syndrome (RAS), n=20), pulmonary GHVD (n=9). Unused donor lungs served as control group (n=20). Next, 60 matched lung transplant recipients (BOS, n=20; RAS, n=20; stable lung transplant recipients, n=20) were included for analysis of CTGF protein levels in plasma and broncho-alveolar lavage (BAL) fluid at 3 months post-transplant, 1 year post-transplant, at CLAD diagnosis or 2 years post-transplant in stable patients.ResultsqPCR revealed an overall significant difference in the relative content of CTGF mRNA in BOS, RAS and pulmonary GVHD vs. controls (p=0.014). Immunohistochemistry showed a significant higher percentage and intensity of CTGF-positive respiratory epithelial cells in BOS, RAS and pulmonary GVHD patients vs. controls (p<0.0001). BAL CTGF protein levels were significantly higher at 3 months post-transplant in future RAS vs. stable or BOS (p=0.028). At CLAD diagnosis, BAL protein content was significantly increased in RAS patients vs. stable (p=0.0007) and BOS patients (p=0.042). CTGF plasma values were similar in BOS, RAS, and stable patients (p=0.74).ConclusionsLung CTGF-expression is increased in end-stage CLAD and pulmonary GVHD; and higher CTGF-levels are present in BAL of RAS patients at CLAD diagnosis. Our results suggest a potential role for CTGF in CLAD, especially RAS, and pulmonary GVHD.

Bronchiolitis obliterans syndrome is associated with increased senescent lymphocytes in the small airways

Immunosuppression therapy is ineffective at preventing bronchiolitis obliterans syndrome (BOS), primarily a disease of the small airways (SAs). Our previous reports show increased senescent CD28null T and natural killer T (NKT)-like cells in the peripheral blood of patients with BOS and increased cytotoxic, proinflammatory lymphocytes in the SAs. We hypothesized that the cytotoxic, proinflammatory lymphocytes in the SAs would be steroid-resistant senescent CD28null lymphocytes. Intracellular cytotoxic mediator granzyme B, interferon (IFN)-γ and tumor necrosis factor (TNF)-α proinflammatory cytokines, and CD28 were measured in the blood, bronchoalveolar lavage, large airway, and SA brushing T and NKT-like cells from 10 patients with BOS, 11 stable lung transplant recipients, and 10 healthy age-matched controls. SA brushings were cultured in the presence of ±1 µmol/liter prednisolone, ±5 mg/liter theophylline, and ±2.5 ng/ml cyclosporine A, and IFN-γ and TNF-α proinflammatory cytokines were assessed using flow cytometry. Increased SA CD28null T and NKT-like cells were identified in patients with BOS compared with that in the controls and stable transplant recipients. Loss of CD28 was associated with increased T and NKT-like cells expressing granzyme B, IFN-γ, and TNF-α. Loss of CD28 expression by CD8+ T cells was significantly associated with forced expiratory volume in 1 sec (R = 0.655, p = 0.006) and with time after transplantation (R = -0.552, p = 0.041). Treatment with prednisolone + theophylline + cyclosporin A inhibited IFN-γ and TNF-α production by SA CD28null CD8+ T and NKT-like cells additively. BOS is associated with the loss of CD28 in SA cytotoxic, proinflammatory senescent T and NKT-like lymphocytes. Treatment options that target the proinflammatory nature of these cells in the SAs may improve graft survival.

Molecular phenotyping of rejection-related changes in mucosal biopsies from lung transplants.

Diagnosing lung transplant rejection currently depends on histologic assessment of transbronchial biopsies (TBB) with limited reproducibility and considerable risk of complications. Mucosal biopsies are safer but not histologically interpretable. Microarray-based diagnostic systems for TBBs and other transplants suggest such systems could assess mucosal biopsies as well. We studied 243 mucosal biopsies from the third bronchial bifurcation (3BMBs) collected from seven centers and classified them using unsupervised machine learning algorithms. Using the expression of a set of rejection-associated transcripts annotated in kidneys and validated in hearts and lung transplant TBBs, the algorithms identified and scored major rejection and injury-related phenotypes in 3BMBs without need for labeled training data. No rejection or injury, rejection, late inflammation, and recent injury phenotypes were thus scored in new 3BMBs. The rejection phenotype correlated with IFNG-inducible transcripts, the hallmarks of rejection. Progressive atrophy-related changes reflected by the late inflammation phenotype in 3BMBs suggest widespread time-dependent airway deterioration, which was especially pronounced after two years posttransplant. Thus molecular assessment of 3BMBs can detect rejection in a previously unusable biopsy format with potential utility in patients with severe lung dysfunction where TBB is not possible and provide unique insights into airway deterioration. ClinicalTrials.gov NCT02812290.

Myeloid-Derived Suppressor Cells in Lung Transplantation.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immune cells from the myeloid lineage. MDSCs expand in pathological situations, such as chronic infection, cancer, autoimmunity, and allograft rejection. As chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplantation (LTx), MDSCs may play a role in its pathophysiology. We assessed phenotype and frequency of MDSCs in peripheral blood from lung transplant recipients and its relationship to post-transplant complications and immunosuppression. Granulocytic (G)-MDSC were identified and quantified by flow cytometry of blood from 4 control subjects and 20 lung transplant patients (stable n = 6, infection n = 5; CLAD n = 9). G-MDSC functionality was assessed in vitro by their capability to block CD4 and CD8 T cell proliferation. More G-MDSC could be assessed using EDTA tubes compared to heparin tubes (p = 0.004). G-MDSC were increased in stable lung transplant recipients vs. non-transplant controls (52.1% vs. 9.4%; p = 0.0095). The infection or CLAD groups had lower G-MDSCs vs. stable recipients (28.2%p = 0.041 and 33.0%; p = 0.088, respectively), but were not different among CLAD phenotypes. G-MDSC tended to correlate with cyclosporine A and tacrolimus levels (r2 = 0.18; r2 = 0.17). CD4 and CD8 cells proliferation decreased by 50 and 80% if co-cultured with MDSCs (1:6 and 1:2 MDSC:T-cell ratio, respectively). In conclusion, circulating MDSCs are measurable, functional and have a G-MDSC phenotype in lung transplant patients. Their frequency is increased in stable patients, decreased during post-transplant complications, and related to level of immunosuppression. This study may pave the way for further investigations of MDSC in the context of lung transplantation.

Host miRNA Profile of Aspergillus Colonization Resulting in CLAD in Lung Transplant Recipients

Purpose Chronic lung allograft dysfunction (CLAD) has been associated with Aspergillus colonization after lung transplantation . We sought to identify the miRNA profile that distinguishes Aspergillus colonization with CLAD from Aspergillus colonization without CLAD in bronchoalveolar lavages (BALs) collected from lung transplant recipients (LTRs). Methods We used the Nanostring nCounter Human miRNA v3 panel to measure human miRNAs in BALs collected from LTRs with Aspergillus colonization and CLAD (n=7) against those with Aspergillus colonization without CLAD (n=10) and control stable LTRs without Aspergillus colonization (n=9). The miRNA profile was compared using the permutation test of 100000 trials for each of the comparisons. We used mirDip ( http://ophid.utoronto.ca/mirDIP/index.jsp#r ) to obtain their gene targets. The targets were filtered by their integrated score to be greater or equal to 90%. Subsequently, we used pathDIP ( http://ophid.utoronto.ca/pathDIP/ ) to determine pathway enrichment. Results We found 92 differentially expressed miRNAs in samples from Aspergillus colonization with CLAD compared to colonization without CLAD and controls (p-value = 90% resulted in 8 miRNAs which targeted 320 genes. These miRNAs were differentially expressed by more than two folds, and included hsa-miR-195-5p, hsa-miR-214-3p, hsa-miR-199b-5p, hsa-miR-424-5p, hsa-miR-181c-5p, hsa-miR-145-5p, hsa-miR-32-3p and hsa-miR-128-3p (Figure 1b). Conclusion This pilot study suggests that patients with Aspergillus colonization with CLAD may have a distinctive miRNA profile compared to patients with Aspergillus colonization without CLAD. Further longitudinal studies are required to evaluate whether these miRNAs can help identify Aspergillus colonized patients who may later develop CLAD.

A Pilot Study to Investigate the Balance between Proteases and α1-Antitrypsin in Bronchoalveolar Lavage Fluid of Lung Transplant Recipients.

The neutrophilic component in bronchiolitis obliterans syndrome (BOS, the main form of chronic lung rejection), plays a crucial role in the pathogenesis and maintenance of the disorder. Human Neutrophil Elastase (HNE), a serine protease responsible of elastin degradation whose action is counteracted by α1-antitrypsin (AAT), a serum inhibitor specific for this protease. This work aimed to investigate the relationship between HNE and AAT in bronchoalveolar lavage fluid (BALf) from stable lung transplant recipients and BOS patients to understand whether the imbalance between proteases and inhibitors is relevant to the development of BOS. To reach this goal a multidisciplinary procedure was applied which included: (i) the use of electrophoresis/western blotting coupled with liquid chromatography-mass spectrometric analysis; (ii) the functional evaluation of the residual antiprotease activity, and (iii) a neutrophil count. The results of these experiments demonstrated, for the first time, the presence of the complex between HNE and AAT in a number of BALf samples. The lack of this complex in a few specimens analyzed was investigated in relation to a patient’s lung inflammation. The neutrophil count and the determination of HNE and AAT activities allowed us to speculate that the presence of the complex correlated with the level of lung inflammation.

BOS is associated with decreased HDAC2 from steroid resistant lymphocytes in the small airways.

Immunosuppression therapies including corticosteroids fail to prevent bronchiolitis obliterans syndrome (BOS), primarily a disease of the small airways, following lung transplantation. We reported increases in steroid-resistant proinflammatory lymphocytes and their loss of histone deacetylase 2 (HDAC2), an important mediator of steroid action, in the blood of stable lung transplant recipients. We noted similar increases in the steroid-resistant lymphocytes in both the blood and small airways in BOS compared with the large airways. We hypothesized that these small airway cells would also exhibit a loss of HDAC2, and that these changes could be reversed by treatment with theophylline (HDAC2 activator). Blood, bronchoalveolar lavage and large and small airway brushings were collected from lung transplant patients with BOS (n=12) or stable lung function (n=18) and healthy aged-matched controls (n=13). Intracellular proinflammatory cytokines [interferon (IFN-γ) and tumour necrosis factor (TNF)-α and HDAC2 were measured in CD8+ T, natural killer (NK)T-like and NK cells from cultured small airway brushings±5mg/l theophylline±1µM prednisolone using flow cytometry. Increased small airway CD8 T, NKT-like and NK cells were identified in BOS versus stable transplant and controls. In BOS, these cells exhibited increased IFN-γ/TNF-α and a loss of HDAC2. HDAC2 expression by small airway CD8+ T cells correlated with forced expiratory volume in 1 s (FEV1 ) (R=0·880, P=0·031). Theophylline and prednisolone synergistically up-regulated HDAC2 in CD8+ T cells. BOS is associated with loss of HDAC2 from steroid-resistant proinflammatory CD8+ T, NK T-like and NK cells in the small airways. Therapeutically increasing HDAC2 in these lymphocytes may reduce steroid resistance and improve graft survival.

Role of C1q-binding anti-HLA antibodies as a predictor of lung allograft outcome.

Presence of anti-human leukocyte antigen donor-specific antibodies (DSAs) is associated with poor outcome after lung transplantation. Currently, DSAs are detected using the Luminex technique, which may be overly sensitive. The new C1q assay allows for the exclusive detection of complement (C1q)-binding antibodies, involved in antibody-mediated rejection. We investigated whether early detection of complement-binding DSAs is associated with chronic lung allograft dysfunction (CLAD) and survival.From 2009 to 2012, lung transplant recipients from three transplantation centres were screened for the presence of DSA and their complement-binding capacity during the 6-12 months post-transplantation in a stable condition.The analysis included 168 patients. The 3-year rates of freedom from CLAD and graft survival were lower for patients with complement-binding DSAs (33.6% and 53.7%, respectively), as compared with patients with non-complement-binding DSAs (61.9% and 77.4%, respectively) and patients without DSA (70% and 84.9%, respectively) (p<0.001 and p=0.001, respectively). Detection of complement-binding DSA was associated with a risk of graft loss that was nearly tripled after adjustment for clinical, functional, histological and immunological factors (hazard ratio 2.98, 95% CI 1.33-6.66; p=0.008).Assessment of the C1q-binding capacity of DSA appears to be useful in identifying stable lung transplant recipients at high risk of lung allograft loss.

Conversion from Twice-Daily to Once-Daily Tacrolimus in Stable Lung Transplant Recipients: Short-Term Effects on Renal Function and Diabetic Parameters

Conversion from Twice-Daily to Once-Daily Tacrolimus in Stable Lung Transplant Recipients: Short-Term Effects on Renal Function and Diabetic Parameters

Bronchiolitis obliterans syndrome is associated with increased p-glycoprotein expression and loss of glucocorticoid receptor from steroid-resistant proinflammatory CD8+ T cells.

Immunosuppressive therapy fails to suppress the production of proinflammatory cytokines, particularly by CD8+ T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8+ cells, and we hypothesized that the drug membrane efflux pump, p-glycoprotein-1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and interferon (IFN)-γ/tumour necrosis factor (TNF)-α proinflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged-matched controls (±prednisolone±Pgp inhibitor, cyclosporin A±GCR activator, Compound A) using flow cytometry. Both Pgp+ and Pgp- lymphocyte subsets from all subjects produced IFN-γ/TNF-α proinflammatory cytokines. Pgp expression was increased in CD8+ Pgp+ T cells and correlated with IFN-γ/TNF-α expression and BOS grade. Reduced GCR was observed in CD8+ Pgp- T, natural killer (NK)T-like and NK cells from stable patients compared with controls, and reduced further in CD8+ Pgp- T cells in BOS. The addition of 2·5ng/ml cyclosporin A and 1µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp+ T cells from BOS patients. The addition of 10µM Compound A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp- T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid-resistant proinflammatory CD8+ T cells. Treatments that inhibit Pgp and up-regulate GCR in CD8+ T cells may improve graft survival.

Low Levels of Exhaled Surfactant Protein A Associated With BOS After Lung Transplantation.

BackgroundThere is no clinically available marker for early detection or monitoring of chronic rejection in the form of bronchiolitis obliterans syndrome (BOS), the main long-term complication after lung transplantation. Sampling and analysis of particles in exhaled air is a valid, noninvasive method for monitoring surfactant protein A (SP-A) and albumin in the distal airways.MethodsWe asked whether differences in composition of exhaled particles can be detected when comparing stable lung transplant recipients (LTRs) (n = 26) with LTRs who develop BOS (n = 7). A comparison between LTRs and a matching group of healthy controls (n = 33) was also conducted. Using a system developed in-house, particles were collected from exhaled air by the principal of inertial impaction before chemical analysis by immunoassays.ResultsSurfactant protein A in exhaled particles and the SP-A/albumin ratio were lower (P = 0.002 and P = 0.0001 respectively) in the BOS group compared to the BOS-free group. LTRs exhaled higher amount of particles (P < 0.0001) and had lower albumin content (P < 0.0001) than healthy controls.ConclusionsWe conclude that low levels of SP-A in exhaled particles are associated with increased risk of BOS in LTRs. The possibility that this noninvasive method can be used to predict BOS onset deserves further study with prospective and longitudinal approaches.

Parametric Response Mapping of Bronchiolitis Obliterans Syndrome Progression After Lung Transplantation.

Bronchiolitis obliterans syndrome (BOS) remains a major complication after lung transplantation. Air trapping and mosaic attenuation are typical radiological features of BOS; however, quantitative evaluation remains troublesome. We evaluated parametric response mapping (PRM, voxel-to-voxel comparison of inspiratory and expiratory computed tomography [CT] scans) in lung transplant recipients diagnosed with BOS (n = 20) and time-matched stable lung transplant recipients (n = 20). Serial PRM measurements were performed prediagnosis, at time of BOS diagnosis, and postdiagnosis (Tpre , T0 , and Tpost , respectively), or at a postoperatively matched time in stable patients. PRM results were correlated with pulmonary function and confirmed by microCT analysis of end-stage explanted lung tissue. Using PRM, we observed an increase in functional small airway disease (fSAD), from Tpre to T0 (p = 0.006) and a concurrent decrease in healthy parenchyma (p = 0.02) in the BOS group. This change in PRM continued to Tpost , which was significantly different compared to the stable patients (p = 0.0002). At BOS diagnosis, the increase in fSAD was strongly associated with a decrease in forced expiratory volume in 1 s (p = 0.011). Micro-CT confirmed the presence of airway obliteration in a sample of a BOS patient identified with 67% fSAD by PRM. We demonstrated the use of PRM as an adequate output to monitor BOS progression in lung transplant recipients.