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CD59 is a complement regulatory protein that inhibits membrane attack complex formation. A soluble form of CD59 (sCD59) is present in various body fluids and is associated with cellular damage after acute myocardial infarction. Lung transplantation (LTx) is the final treatment for end-stage lung diseases, however overall survival is hampered by chronic lung allograft dysfunction development, which presents itself obstructively as the bronchiolitis obliterans syndrome (BOS). We hypothesized that, due to cellular damage and activation during chronic inflammation, sCD59 serum levels can be used as biomarker preceding BOS development. We analyzed sCD59 serum concentrations in 90 LTx patients, of whom 20 developed BOS. We observed that BOS patients exhibited higher sCD59 serum concentrations at the time of diagnosis compared to clinically matched non-BOS patients (p = 0.018). Furthermore, sCD59 titers were elevated at 6 months post-LTx (p = 0.0020), when patients had no BOS-related symptoms. Survival-analysis showed that LTx patients with sCD59 titers ≥400 pg/ml 6 months post-LTx have a significant (p < 0.0001) lower chance of BOS-free survival than patients with titers ≤400 pg/ml, 32% vs. 80% respectively, which was confirmed by multivariate analysis (hazard ratio 6.2, p < 0.0001). We propose that circulating sCD59 levels constitute a novel biomarker to identify patients at risk for BOS following LTx.

Complement activation leads primarily to membrane attack complex formation and subsequent target cell lysis. Protection against self-damage is regulated by complement regulatory proteins, including CD46, CD55, and CD59. Within their promoter regions, single-nucleotide polymorphisms (SNPs) are present that could influence transcription. We analyzed these SNPs and investigated their influence on protein expression levels. A single SNP configuration in the promoter region of CD59 was found correlating with lower CD59 expression on lung endothelial cells (p = 0.016) and monocytes (p = 0.013). Lung endothelial cells with this SNP configuration secreted more profibrotic cytokine IL-6 (p = 0.047) and fibroblast growth factor β (p = 0.036) on exposure to sublytic complement activation than cells with the opposing configuration, whereas monocytes were more susceptible to antibody-mediated complement lysis (p < 0.0001). Analysis of 137 lung transplant donors indicated that this CD59 SNP configuration correlates with impaired long-term survival (p = 0.094) and a significantly higher incidence of bronchiolitis obliterans syndrome (p = 0.046) in the recipient. These findings support a role for complement in the pathogenesis of this posttransplant complication and are the first to show a deleterious association of a donor CD59 promoter polymorphism in lung transplantation., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Objectives: Peri- and postoperative complications diminish the outcome of lung transplantation (LTx) in patients with cystic fibrosis (CF). We hypothesized that the degree of pathological findings on pre-LTx high resolution computed tomography (HRCT) is associated with higher morbidity and mortality in CF., Methods: All our CF patients undergoing LTx between 2001 and 2011 were included. HRCT examinations were evaluated according to a scoring system for pulmonary disease in CF patients, the Severe Advanced Lung Disease (SALD) score and for pleural involvement., Results: Fifty-three patients were included. Dominant infectious/inflammatory disease according to the SALD score was observed in 10 patients (19%). Five (50%) of those patients died within one week after LTx, compared to 2 (5%) patients without dominant infectious/inflammatory disease (p<0.001). This difference in survival percentage remained also significant in multivariate analysis. Patients with infectious/inflammatory disease received more packed red blood cells; 26 versus 8 in the first week (p<0.001). Pleural thickening was associated with higher requirement (10 units) for blood transfusion during LTx, compared to patients with normal pleura (4 units)., Conclusions: The analysis of HRCT in CF patients according to the SALD score showed that dominant infectious/inflammatory disease is associated with a higher mortality after LTx. If confirmed in other studies, HRCT might aid estimation of surgical risk in some adult CF patients.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal fibrosing lung disease with a median survival of approximately 3 years after diagnosis. The only medical option to improve survival in IPF is lung transplantation (LTX). The purpose of this study was to evaluate trajectory data of IPF patients listed for LTX and to investigate the survival after LTX., Methods and Results: Data were retrospectively collected from September 1989 until July 2011 of all IPF patients registered for LTX in the Netherlands. Patients were included after revision of the diagnosis based on the criteria set by the ATS/ERS/JRS/ALAT. Trajectory data, clinical data at time of screening, and donor data were collected. In total, 98 IPF patients were listed for LTX. During the waiting list period, 30 % of the patients died. Mean pulmonary artery pressure, 6-min walking distance, and the use of supplemental oxygen were significant predictors of mortality on the waiting list. Fifty-two patients received LTX with a median overall survival after transplantation of 10 years., Conclusions: This study demonstrated a 10-year survival time after LTX in IPF. Furthermore, our study demonstrated a significantly better survival after bilateral LTX in IPF compared to single LTX although bilateral LTX patients were significantly younger.

Annually, about 8000 heart and lung transplantations are successfully performed worldwide. However, morbidity and mortality still pose a major concern. Renal failure in heart and lung transplant recipients is an essential adverse cause of morbidity and mortality, often originating in the early postoperative phase. At this time of clinical instability, the kidneys are exposed to numerous nephrotoxic stimuli. Among these, tacrolimus toxicity plays an important role, and its pharmacokinetics may be significantly altered in this critical phase by fluctuating drug absorption, changed protein metabolism, anemia and (multi-) organ failure. Limited understanding of tacrolimus pharmacokinetics in these circumstances is hampering daily practice. Tacrolimus dose adjustments are generally based on whole blood trough levels, which widely vary early after transplantation. Moreover, whole blood trough levels are difficult to predict and are poorly related to the area under the concentration-time curve. Even within the therapeutic range, toxicity may occur. These shortcomings of tacrolimus monitoring may not hold for the unbound tacrolimus plasma concentrations, which may better reflect tacrolimus toxicity. This review focuses on posttransplant tacrolimus pharmacokinetics, discusses relevant factors influencing the unbound tacrolimus concentrations and tacrolimus (nephro-) toxicity in heart and lung transplantation patients., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Lung disease in cystic fibrosis (CF) involves excessive inflammation, repetitive infections and development of bronchiectasis. Recently, literature on emphysema in CF has emerged, which might become an increasingly important disease component due to the increased life expectancy. The purpose of this study was to assess the presence and extent of emphysema in endstage CF lungs., Methods: In explanted lungs of 20 CF patients emphysema was semi-quantitatively assessed on histology specimens. Also, emphysema was automatically quantified on pre-transplantation computed tomography (CT) using the percentage of voxels below -950 Houndfield Units and was visually scored on CT. The relation between emphysema extent, pre-transplantation lung function and age was determined., Results: All CF patients showed emphysema on histological examination: 3/20 (15%) showed mild, 15/20 (75%) moderate and 2/20 (10%) severe emphysema, defined as 0-20% emphysema, 20-50% emphysema and >50% emphysema in residual lung tissue, respectively. Visually upper lobe bullous emphysema was identified in 13/20 and more diffuse non-bullous emphysema in 18/20. Histology showed a significant correlation to quantified CT emphysema (p = 0.03) and visual emphysema score (p = 0.001). CT and visual emphysema extent were positively correlated with age (p = 0.045 and p = 0.04, respectively)., Conclusions: In conclusion, this study both pathologically and radiologically confirms that emphysema is common in end-stage CF lungs, and is age related. Emphysema might become an increasingly important disease component in the aging CF population.

After lung transplantation (LTx), circulating mononuclear cell composition and their subsets may be predictive for the bronchiolitis obliterans syndrome (BOS). We investigated the cellular composition in patients developing BOS, or not, by analyzing peripheral blood taken at multiple time points after transplantation. PBMCs of 11 BOS and 39 non-BOS patients were analyzed by FACS for monocytes, dendritic cells, NK-, NKT-, B- and T cells as well as B- and T cell subsets. Analysis of blood samples taken monthly during the first year post-LTx showed that circulating NK, NKT and dendritic cell percentages were not indicative of BOS development, whereas increases in T cells, monocytes and lowered fractions of B cells were related to BOS development. B- and T cell subset analysis at month 5 post-LTx indicated that IgM+IgD- memory B cells and central memory CD8+ T cells were decreased, whereas NKT cells were increased in BOS patients compared to non-BOS patients. Prior to BOS diagnosis, the composition of specific mononuclear cells on a group level differs from patients remaining BOS free. However, given the overlap in percentages of cellular frequencies between the patient groups investigated, this analysis does not allow prediction or risk stratification for development of BOS in individual patients., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Autoantibodies against endothelin-1 type A receptor (ETAR) are present in systemic sclerosis complicated by lung fibrosis and pulmonary hypertension. As increased serum levels and local overproduction of endothelin-1 in the airways are reported in cystic fibrosis (CF) patients, we reasoned that anti-ETAR antibodies could be prevalent in endstage CF patients prior to lung transplantation (LTx). Also, ETAR autoantibodies are frequently associated with autoantibodies against the angiotensin II type 1 receptor (AT1R). We analyzed the presence of anti-ETAR and anti-AT1R autoantibodies in 43 LTx patients (chronic obstructive pulmonary disease (COPD), n=20; CF, n=13; interstitial lung disease (ILD), n=1). We observed overall higher anti-ETAR and anti-AT1R autoantibody titers in sera taken prior to LTx in the CF patient group as compared to COPD. No difference was found in autoantibody levels between patients with CF versus ILD. In sera taken post-LTx we found the same difference in anti-ETAR and anti-AT1R autoantibody titers between patients with CF versus COPD. No difference was found in antibody titers between sera taken prior to or 6 months after LTx. There was no association between autoantibody levels and other relevant demographic parameters, and we found no association between autoantibody titers and the development of the bronchiolitis obliterans syndrome. Both autoantibody titers were strongly correlated. We hypothesize that due to prolonged exposure to bacterial infection, increased levels of AT1R and ETAR result in a deregulated immune response causing autoantibody formation. Further research is expedient to elucidate the occurrence of autoantibodies against ETAR and AT1R and their role in disease progression., (Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Lung transplantation (LTx) is the final treatment option for patients with endstage lung diseases including chronic obstructive pulmonary disease, cystic fibrosis, and interstitial lung disease. Survival after LTx is severely hampered by the development of the bronchiolitis obliterans syndrome (BOS) which is hallmarked by excessive fibrosis and scar tissue formation leading to small airway obliteration and eventually organ failure. The pathophysiology of BOS is incompletely understood. During the past years both anti-HLA and non-HLA antibodies have been identified that correlate with transplantation outcome. Also, the involvement of autoimmunity on BOS progression has been demonstrated, including autoantigens Type V collagen and K-alpha tubulin. Both allo- and autoantibodies binding to its respective antigen trigger the binding of C1q and sequential complement activation which can lead to either cell damage or activation, both processes which fit into the current model of BOS pathogenesis. In this review we will discuss both HLA, non-HLA and autoantibodies associated with disease progression, but also elaborate on the subsequent complement effector mechanisms, complement regulation, and the potential influence of regulatory mechanisms on graft survival., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Bactericidal/permeability increasing protein fold containing family A (BPIFA) 1, is a secreted protein of the upper airways that shares structural homology with BPI and exhibits comparable antimicrobial capacities. We hypothesized that CF patients have circulating IgG or IgA anti-BPIFA1 autoantibodies, similarly as reported for BPI autoantibodies., Methods: We analyzed pre- and post-transplantation sera from 67 endstage lung disease patients who underwent lung transplantation (LTx) because of COPD (n=27), CF (n=25), and ILD (n=15)., Results: Anti-BPIFA1 (48%) and anti-BPI (92%) were elevated in CF patients compared to healthy controls, with anti-BPIFA1 IgG isotype being most prevalent, whereas anti-BPI is of the IgA isotype. Levels of anti-BPI autoantibodies significantly declined post-LTx, whereas anti-BPIFA1 did not. No relation was found between autoantibodies against BPIFA1 and BPI., Conclusion: Our results indicate that BPIFA1 is a novel target for autoantibodies in CF. The function of these autoantibodies needed to be investigated in future studies., (© 2013.)

Background: This study retrospectively investigated nutritional status, dietetic intervention and intake in Cystic Fibrosis (CF) patients before and after lung transplantation (LTX)., Methods: Body Mass Index (BMI), Fat Free Mass Index (FFMI) and nutritional intake were retrieved from 75 out-patients aged 15-53 years. Patients were seen every 3-4 months during the waiting list time (range 0-81 months) and up to 116 months after LTX. Survival was measured in months., Results: The median BMI at baseline was 19.2 kg/m(2) (range: 15.3 to 28.4 kg/m(2)) with 29 patients (39%) below ≤18.5 kg/m(2). FFMI (measured in 65 patients) had a median of 15.2 kg/m(2) (range: 11.1 to 22.4 kg/m(2)) with 39 patients (60%) ≤16.7 kg/m(2) (men) or ≤14.6 kg/m(2) (women). Median energy intake was 2800 kcal, 239 kcal higher than the estimated energy requirement. However, 8 patients consumed ≥500 kcal less than recommended. Protein intake was 104 (range 60-187) g or 1.9 g/kg per day. Despite dietetic intervention with oral nutritional supplements (ONS) (36 patients), tube feeding (12 patients), or both (13 patients), BMI and FFMI hardly improved pre-LTX. LTX was performed in 51 patients (68%); 10 patients died during follow-up, median survival time was 41 months. A BMI ≤18.5 kg/m(2) was more prevalent in patients who died before LTX (6/9) or who died after LTX (4/10) than in patients who were still alive on the waiting list (5/15) or who survived LTX (14/41). Results for FFMI were comparable. From 6-12 months post-LTX, BMI and FFMI markedly improved, especially in underweight patients., Conclusion: A BMI ≤18.5 kg/m(2) and an FFMI ≤16.7 kg/m(2) (men) or ≤14.6 kg/m(2) (women) appears to impair survival in LTX candidates with CF. Patients maintained a low body weight before LTX. After LTX weight gain is achieved., (© 2013. Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. All rights reserved.)

Objective: Bilateral transverse thoracosternotomy (clamshell incision) is a widely used approach in bilateral sequential lung transplantation, but the closure technique is associated with sternal dehiscence. This study compares the incidence of sternal dehiscence between the crossed and uncrossed closure techniques., Methods: In 129 patients who underwent transplantation through a clamshell incision, the sternum was closed using either the crossed or the uncrossed method based on the surgeon's preference. The position of the sternal parts was evaluated on lateral chest radiographs and scored as normal, override, or separation., Results: We observed sternal override in 38 patients and separations in 18 patients. The sternum was closed using the uncrossed method in 79 patients and the crossed method in 50 patients. There were significantly fewer overrides (n = 6, 12.0%) and separations (n = 6, 12.0%) of the sternal parts using the crossed closure technique compared with the uncrossed technique (32 overrides, 41.0%; and 12 separations, 15.1%; P < .001). Reconstructive surgery was only performed in patients with separation of the sternal parts (n = 10)., Conclusions: Using the crossed closure technique for the sternum after bilateral sequential lung transplantation reduces the incidence of sternal dehiscence compared with the uncrossed closure technique and, therefore, reduces the necessity of reconstructive surgery., (Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Background: This study investigated the prevalence of pulmonary hypertension (PH) in cystic fibrosis (CF) patients awaiting lung transplantation (LTx) and its influence on survival. We also explored the feasibility of using echocardiography as a first assessment for diagnosing PH., Methods: The study included 93 CF patients (46 women [50%]) evaluated for LTx between 2001 and 2010. Median age was 29 years. PH was defined as a mean pulmonary artery pressure (mPAP) measured by right heart catheterization (mPAP(cath)) of ≥ 25 mm Hg with a wedge pressure of ≤ 15 mm Hg. Echocardiographic results were divided into 3 categories based on current guidelines as "unlikely," "possible," or "likely" to have PH., Results: In 23 patients (25%) the mPAP(cath) was between 25 and 35 mm Hg, and 1 (1%) had severe PH (mPAP(cath) of ≥ 35 mm Hg). PH did not influence survival after enlistment (p = 0.7) and after LTx (p = 0.8). For 62 patients (67%), the sPAP(echo) could be measured, and PH was unlikely in 24 (39%). In another 19 patients (20%), PH was unlikely based on the absence of tricuspid regurgitation. The negative-predictive value (NPV) of measuring PH by echocardiography was 88% in whom PH was estimated to be unlikely (n = 43); whereas in 24 patients with a measurable low sPAP(echo), the NPV was 96%., Conclusions: PH exists in 26% of end-stage CF patients and has no effect on survival on the waiting list for LTx or after LTx. Echocardiography might be used as the first tool to rule out PH, showing a NPV of 88%., (Copyright © 2013 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background and Objective: The development of bronchiolitis obliterans syndrome (BOS) after lung transplantation is characterized by inflammation, remodeling and fibrosis. Both YKL-40 and matrix metalloproteinase (MMP)-9 have shown to be involved in these processes. We measured serial YKL-40 and MMP-9 serum levels in lung transplant recipients and assessed their usefulness as biomarker for BOS. Furthermore, we investigate the relationship between these two potential biomarkers of BOS and MMP-7., Design: Ten patients with BOS (BOS(pos)) and 10 matched patients without BOS (BOS(neg)) were included. Serial serum samples were collected after lung transplantation and prior to BOS. YKL-40, MMP-9 and MMP-7 serum levels were determined by ELISA., Results: The median concentrations of YKL-40 did not differ between BOS(pos) and BOS(neg) patients (p > 0.05). The median concentration of MMP-9 in BOS(pos) patients was significantly higher than in BOS(neg) patients (p < 0.0001). For MMP-9 as possible risk factor for BOS, a cut off value of 145 ng/ml has a sensitivity of 90% and a negative predictive value of 83%. Longitudinal analysis of YKL-40 and MMP-9 serum levels from the early post-transplant period onwards did not reveal a significant trend in time in both serum levels preceding BOS. In BOS(neg) patients MMP-9 showed an inverse relationship with MMP-7, that was absent in BOS(pos) patients., Conclusions: From the moment of transplantation onwards, patients who eventually developed BOS had significantly increased MMP-9 serum levels in comparison with patients who did not develop BOS. Therefore, increased MMP-9 serum levels might be useful as risk factor for BOS.

Aims: The aims of the study are to investigate the prevalence of diabetes in patients with cystic fibrosis compared with patients without cystic fibrosis, and its impact on the outcome after lung transplantation., Methods: Data were reviewed from 77 lung transplantation recipients in our centre between 2001 and 2010; 43 patients had cystic fibrosis and 34 patients had other lung diseases (no cystic fibrosis). To define diabetes, we used the American Diabetes Association definition., Results: Before lung transplantation, diabetes was diagnosed in 63% of patients with cystic fibrosis and 6% of patients without cystic fibrosis (P<0.001). In both groups, approximately 60% of the patients at risk developed new-onset diabetes after transplantation. The mortality in patients with cystic fibrosis was higher in patients with diabetes diagnosed before lung transplantation compared with those without (44 vs. 6%, P=0.04). Diabetes remained an independent factor in multivariate analyses., Conclusions: Diabetes diagnosed before lung transplantation has a negative effect on survival after lung transplantation in patients with cystic fibrosis. Pre-existing diabetes is common in patients with cystic fibrosis, in contrast to patients without cystic fibrosis. Development of new-onset diabetes after transplantation is similar in both groups., (© 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.)

Objective: To describe patients diagnosed with idiopathic pulmonary fibrosis (IPF) registered for lung transplantation and to evaluate the current referral guidelines for lung transplantation in the Netherlands., Design: Retrospective study., Method: All patients diagnosed with interstitial lung disease and registered for lung transplantation from September 1989-June 2010 were included in this study. Patients who had been diagnosed with IPF according to the American Thoracic Society-European Respiratory Society criteria were included. Clinical data of these patients at the time of screening for lung transplantation and survival data were collected., Results: In total, 289 patients with IPF were registered for lung transplantation. After a first waiting list. During the waiting period, 30 patients (33%) died, 7 were taken off the list due to newly developed comorbidity and excessive physical deterioration, 51 underwent transplantation and 2 were still on the waiting list at the time of study closure. At the time of screening, the mean FVC% predicted of these patients was 51% (SD: 19.0) and the mean diffusing capacity was 27% of predicted (SD: 9.3)., Conclusion: One-third of the IPF patients on the waiting list died before donor lungs became available. The mean diffusing capacity of 27% of predicted at the time of screening was considerably lower than advised in the international guidelines for placement on the waiting list. This study, therefore, shows that the timing of screening IPF patients for lung transplantation can be improved in the Netherlands.

Background: Caveolin 1 (Cav-1) is the primary structural component of cell membrane invaginations called 'caveolae'. Expression of Cav-1 is implicated in the pathogenesis of pulmonary fibrosis. Genetic polymorphisms in the CAV1 gene influence the function of Cav-1 in malignancies and associate with renal allograft fibrosis. Chronic allograft rejection after lung transplantation, called 'bronchiolitis obliterans syndrome' (BOS), is also characterised by the development of fibrosis.In this study, we investigated whether CAV1 genotypes associate with BOS and whether Cav-1 serum levels are influenced by the CAV1 genotype and can be used as a biomarker to predict the development of BOS., Methods: Twenty lung transplant recipients with BOS (BOSpos), ninety without BOS (BOSneg) and four hundred twenty-two healthy individuals donated DNA samples. Four SNPs in CAV1 were genotyped. Serial Cav-1 serum levels were measured in a matched cohort of 10 BOSpos patients and 10 BOSneg patients. Furthermore, single-time point Cav-1 serum levels were measured in 33 unmatched BOSneg patients and 60 healthy controls., Results: Homozygosity of the minor allele of rs3807989 was associated with an increased risk for BOS (odds ratio: 6.13; P = 0.0013). The median Cav-1 serum level was significantly higher in the BOSpos patients than in the matched BOSneg patients (P = 0.026). Longitudinal analysis did not show changes in Cav-1 serum levels over time in both groups. The median Cav-1 serum level in the group of 43 BOSneg patients was lower than that in the healthy control group (P = 0.046).In lung transplant recipients, homozygosity of the minor allele of rs3807989 and rs3807994 was associated with increased Cav-1 serum levels., Conclusion: In lung transplant recipients, the CAV1 SNP rs3807989 was associated with the development of BOS and Cav-1 serum levels were influenced by the CAV1 genotype.

Alloreactive T cells that infiltrate the graft after lung transplantation (LTx) play a role in chronic rejection. Chemokines such as thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and monocyte chemotactic protein-1 (MCP-1) are produced locally in the lung and attract T cells via chemokine receptor 4 (CCR4). In a TARC gradient, cells expressing CCR4(++) migrate more efficiently than CCR4(+) -expressing cells. In this study, we compared the CCR4 expression of T cells in blood from 20 lung transplant recipients to healthy controls. We then examined whether CCR4 expression is associated with the occurrence of chronic rejection. The CCR4(++) expression was decreased on CD4 T cells from LTx patients (P < 0·0001) when compared to healthy controls. The analysis of CD4 T cell subsets showed that this decrease was present on central memory, effector memory and terminally differentiated T cells (P = 0·0007, P < 0·0001 and P = 0·05, respectively), while a trend was found for naive CD4 T cells (P = 0·06). Also, the expression of CCR4(+) on regulatory T cells (T(regs) ) was decreased in LTx patients when compared to healthy controls (P = 0·02). Interestingly, the CCR4(++) expression on CD4 effector memory T cells was decreased in patients developing chronic rejection sometimes more than a year before the clinical diagnosis when compared to patients who did not (P = 0·04). The analysis of CD8 T cell subsets only showed the CCR4(+) expression to be increased significantly on effector memory and terminally differentiated CD8 T cells (P = 0·02, P = 0·03, respectively) in LTx patients, but no relation was found in chronic rejection. In conclusion, the expression of CCR4 on T cell subsets was altered after LTx and appears to be related to chronic rejection., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

Despite the use of immunosuppressives mainly influencing T and B cell responses, the prevalence of the bronchiolitis obliterans syndrome (BOS) after lung transplantation is high. Mannose-binding lectin (MBL) is a pattern recognition molecule of complement and an important component of the innate immunity. MBL is associated with rejection, infection and survival in other solid organ transplantations. In this study the relation between functional MBL levels and cytomegalovirus (CMV) reactivations and the development of BOS and survival after lung transplantation was investigated. MBL levels were measured in 85 patients before and in 57 of these patients after lung transplantation. The relation of MBL on survival, CMV reactivation and the development of BOS were investigated with Kaplan-Meier (log-rank) survival analysis. MBL levels decreased on average by 20% (P < 0·001) after transplantation and eventually returned to pretransplant levels. Fourteen of the 85 patients had deficient pretransplant MBL levels and these patients had a tendency towards a better survival compared to those with normal MBL levels (P = 0·08). Although no correlation was found between MBL deficiency and the development of BOS, more CMV reactivations occurred in recipients with deficient versus normal levels of MBL (P = 0·03). Our results suggest that MBL deficiency is associated with CMV reactivations and a longer overall survival, but not with the development of BOS., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

Venous thromboembolism (VTE), which includes pulmonary embolism (PE) and deep vein thrombosis (DVT), is a common occurrence in patients undergoing surgery and is a potentially fatal complication. Especially after lung transplantation, vascular complications can compromise the function of the allograft and limit survival. Typically, the risk of pulmonary infarction after PE in lung transplant recipients is high because the absence or poor development of the collateral bronchial circulation may predispose lung transplant recipients to pulmonary infarction. This article reports 2 cases of PE with associated pulmonary infarction after lung transplantation with significant morbidity.

Background: Passenger leukocytes of donor origin are transferred to the patient resulting in circulatory microchimerism after lung transplantation (LTx). This chimeric state has been shown to occur in the total leukocyte fraction as well as unseparated peripheral blood mononuclear cells (PBMCs). In this study we determined the microchimerism levels of B cells, monocytes, natural killer (NK) and T cells and dendritic cell (DC) subsets (mDC1, mDC2 and pDC) during the first year after lung transplantation., Methods: To identify circulating donor cells, 11 donor-patient combinations were selected, which were mismatched for HLA-B8. Analysis consisted of flow cytometry on a minimum of 1 million PBMCs taken monthly up to 1 year after LTx., Results: Levels of microchimerism were found to be stable after LTx for all cell types investigated, although for NK+T cells an above-baseline chimerism of donor cells from the donor lung was observed in the first month after transplantation. Circulating PBMCs consisted of, on average, 0.002%, 1.7%, 0.03% and 0.001% of B cells, monocytes, NK+T cells and DCs, respectively, indicating that overall levels of microchimerism differed between the cell types investigated. In 2 patients no B-cell chimerism and in 1 patient no DC chimerism could be detected. Cell types and DC subsets of recipient origin were normally distributed. Conversely, monocytes, B cells and DCs of donor origin were increased and donor NK+T cells were decreased in number, compared with the recipient ratios. Analysis of circulating recipient DCs showed a normal distribution of mDC1s (70%), mDC2s (5%) and pDCs (25%). However, circulating donor DCs consisted of 80%, 20% and <1% of DC subsets mDC1, MDC2 and pDC, indicating that donor plasmacytoid dendritic cells were not detectable in the circulation., Conclusions: In the first year after lung transplantation a stable microchimerism was detected for all cell types investigated. However, donor pDCs were consistently absent in all samples investigated, which may be linked with graft rejection often observed after LTx., (Copyright © 2011 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

The production of IgG HLA antibodies after lung transplantation (LTx) is considered to be a major risk factor for the development of chronic rejection, represented by the bronchiolitis obliterans syndrome (BOS). It has recently been observed that elevated levels of IgM HLA antibodies also correlates with the development of chronic rejection in heart and kidney transplantation. This study investigates the relationship between IgM and IgG antibodies against HLA and MICA after lung transplantation. Serum was collected from 49 patients once prior to transplantation and monthly for up to 1 year after lung transplantation was analyzed by Luminex to detect IgM and IgG antibodies against HLA and MICA. The presence of either IgM or IgG HLA and/or MICA antibodies prior to or after transplantation was not related to survival, gender, primary disease, or the development of BOS. Additionally, the production of IgG alloantibodies was not preceded by an increase in levels of IgM, and IgM levels were not followed by an increase in IgG. Under current immune suppressive regimen, although the presence of IgM antibodies does not correlate with BOS after LTx, IgM( high) IgG( low) HLA class I antibody titers were observed more in patients with BOS compared to patients without BOS.

Background: The mechanisms that lead to the fibrotic obliteration in bronchiolitis obliterans syndrome (BOS) may involve the interactions between T-helper (Th)1 and Th2 cytokines. The aim of this study is to determine the Th1 and Th2 cytokine and chemokine profiles in serum and exhaled breath condensate (EBC) in lung transplant recipients and to assess their usefulness as biomarkers to predict the development of BOS., Methods: Serum and EBC from 10 patients with BOS (BOS(pos)) and 10 patients without BOS (BOS(neg)), matched for clinical and demographic variables, were analyzed with a multiplex immunoassay to measure a panel of 27 cytokines and chemokines., Results: The pro-inflammatory cytokines in serum were elevated in lung transplant recipients compared with controls. BOS(pos) patients had significantly lower concentrations of interleukin (IL)-4, IL-13, and vascular endothelial growth factor (VEGF) compared with BOS(neg) patients. The concentration of IL-5, however, was significantly higher in BOS(pos) patients. Levels of IL-4 and IL-5 were hardly detectable in EBC. IL-13 and VEGF, both decreased in serum in BOS(pos) patients, were also decreased in EBC in BOS(pos) patients compared with BOS(neg) patients. Longitudinal analysis of cytokines and chemokines in serum and EBC from the time of lung transplantation onwards did not reveal significant trends in cytokines and chemokines that preceded the diagnosis of BOS., Conclusions: Levels of pro-inflammatory cytokines were increased in lung transplant recipients compared with controls. From the moment of transplantation onwards, there is a different pattern of Th2 cytokines in serum in BOS(pos) patients than in BOS(neg) patients., (Copyright 2010 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Pulmonary epithelium is the primary target of injury in the development of bronchiolitis obliterans syndrome (BOS) after lung transplantation. Matrix metalloproteinases (MMP)-8 and -9 already have been implicated in the pathogenesis of BOS. MMP-7, which is involved in the repair of the lung epithelium, has not been studied in this respect. We hypothesized that genetic polymorphisms in MMP7 influence its expression and correlate with serum MMP-7 levels and the development of BOS., Methods: DNA was collected from 110 lung transplant recipients, including 21 patients with BOS. We genotyped 7 single nucleotide polymorphisms in MMP7 and measured serum MMP-7 levels. The control group comprised 422 healthy individuals., Results: BOS(pos) patients had lower levels of MMP-7 than BOS(neg) patients (7.87 vs 10.18 ng/ml). Significant differences in genotype and haplotype distribution between the BOS(pos) and BOS(neg) patients and controls were found. An increased risk for BOS development was found in patients homozygous for the major alleles of rs17098318, rs11568818, and rs12285347, and for the minor allele rs10502001 (odds ratio, 3.88-5.30). Haplotypes constructed with 3 or 4 risk alleles correlated with lower MMP-7 levels., Conclusions: Genetic polymorphisms of MMP7 predispose to the development of BOS. Patients carrying these risk alleles express lower levels of MMP-7, which may contribute to aberrant tissue repair and culminate in the development of BOS., (Copyright 2010 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Activation of the immune system is suggested to prevent transplant tolerance and to promote the development of bronchiolitis obliterans syndrome (BOS). The innate immune system is activated by the interaction of pathogen-associated molecular patterns of microorganisms with Toll-like receptors (TLRs). Activation of innate immunity via TLRs was shown to be a barrier to the induction of transplantation tolerance after lung transplantation. We hypothesized that polymorphisms in 10 genes coding for TLR1 to TLR10 might contribute to an altered immune response and the subsequent development of BOS., Methods: DNA was collected from 110 lung transplant recipients. Twenty patients developed BOS. The control group comprised 422 individuals. Sixty-four single-nucleotide polymorphisms (SNPs) in 10 genes coding for TLR1 to TLR10 were genotyped., Results: The genotype distribution of TLR2 (rs1898830 and rs7656411), TLR4 (rs1927911) and TLR9 (rs352162 and rs187084) was significantly different between BOS(pos) patients and BOS(neg) patients and controls. The BOS(pos) group had significantly more patients with 3 or 4 of these risk alleles compared with the BOS(neg) and control groups., Conclusions: Polymorphisms in TLR2, TLR4 and TLR9 that recognize bacterial and viral pathogens are associated with BOS after lung transplantation., (Copyright 2010 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Objective: To describe the clinical presentation, diagnosis, and prognosis of a cohort of Dutch patients with idiopathic pulmonary fibrosis (IPF), a serious and rapidly progressive lung disease belonging to the idiopathic interstitial pneumonias., Design: Retrospective study of patient records., Method: The data from the clinical presentation, diagnosis, treatment and survival of all patients with IPF, diagnosed in the St. Antonius Hospital in Nieuwegein and University Medical Center Utrecht (UMCU), both in the Netherlands, during the period 1998-2007 were investigated. For the diagnosis, the criteria of the American Thoracic Society and the European Respiratory Society from 2002 were adhered to., Results: The records of 113 patients satisfied the inclusion criteria. Mean age at the time of presentation was 61.9 (SD: 12.7) years and a strong male predominance was observed (90 men vs. 23 women). The most common complaints and symptoms at presentation were dyspnoea, cough, basal crepitations and clubbing of the nails. Lung function tests revealed restrictive lung function impairment and a reduced diffusing capacity. In 72% of cases the diagnosis IPF was histologically confirmed by open lung biopsy, which revealed a pattern of usual interstitial pneumonia (UIP). In 17% of patients it concerned a familial form of the disease with diagnosis at a younger age (average 52 years; SD: 14.8). The medical treatment mostly consisted of corticosteroids, which for half of the patients were administered in combination with an immunosuppressant such as azathioprine or cyclophosphamide. After screening, 28 patients were eligible for lung transplantation. Of these, 12 patients underwent a lung transplantation in the study period, 9 died and 7 are still on the waiting list. The median survival period was 3.9 years., Conclusion: In the cohort studied, IPF presented as a rapidly progressive disease with only a marginal response to medical treatment and a poor prognosis. It is important to differentiate IPF from other fibrotic interstitial lung diseases and to refer to a specialist centre, especially in the case of patients who could be eligible for a lung transplant or for participation in trials with new drugs.

The main reason for mortality after lung transplantation is the bronchiolitis obliterans syndrome (BOS), which represents chronic rejection. As soluble CD30, which is produced mainly by activated T helper 2 (Th2) cells, was shown to be related to development of BOS, we aimed to investigate the relation between development of BOS and Th2 chemoattractant thymus and activation regulated chemokine (TARC/CCL17). In 54 patients we measured serum TARC levels prior to transplantation by enzyme-linked immunosorbent assay, and in 44 of these patients sera were analysed at months 1, 2 and 3 after lung transplantation. In addition, longitudinal measurements were performed in sera from eight healthy controls and 14 patients, the latter taken over a period of 2 years post-transplantation from seven patients developing BOS plus seven clinically matched BOS-free patients. Median serum TARC levels post-transplantation of patients who developed BOS were significantly lower than those of the matched BOS-free patients (P = 0.05). A receiver operating characteristics analysis (area under the curve 0.77), together with a Kaplan-Meyer analysis, showed that serum TARC levels below 325 pg/ml in the first month post-transplantation can predict development of BOS post-transplantation (P = 0.001). In contrast, pretransplant serum TARC levels were not significantly different between patients developing BOS, BOS-free patients or healthy controls. In conclusion, pretransplantation serum TARC levels do not predict the development of BOS post-transplantation, but measurement of the serum TARC levels in the first month directly after transplantation can provide us with a tool to identify the group at risk of developing BOS.

Background: The purpose of this study was to determine the utility of post-transplant serum soluble CD30 levels as a biomarker for the development of the bronchiolitis obliterans syndrome (BOS) after lung transplantation during a tacrolimus/mycophenolate mofetil-based regimen., Methods: Soluble CD30 (sCD30) concentrations were measured prior to transplantation and in 175 samples taken after transplantation in 7 patients developing BOS and 7 non-BOS patients closely matched for age, underlying diseases, follow-up and gender., Results: High pre-transplant sCD30 levels dropped significantly after lung transplantation, but in the post-transplant samples no differences could be detected between patients developing BOS or not, and no changes were found prior to or during the development of BOS., Conclusions: After transplantation, sCD30 levels are consistently suppressed, but BOS is not prevented, indicating that sCD30 cannot be used as a biomarker to predict BOS after transplantation in the regimen employed.

Background: The development of bronchiolitis obliterans syndrome (BOS) after lung transplantation is associated with viral infections. Natural killer (NK) cells are involved in the lysis of viral infected cells, and their activation is largely controlled by activating and inhibitory killer immunoglobulin-like receptors (KIRs). We hypothesized that KIR ligand incompatibility and recipients' individual KIRs could influence the development of BOS and the incidence of cytomegalovirus reactivation after lung transplantation., Methods: The KIR gene contents were determined in 48 patients who received a lung transplant, and human leukocyte antigen (HLA)-Cw and HLA-Bw4 typing was performed on their respective donors., Results: BOS developed in 7 patients and cytomegalovirus reactivation occurred in 16. BOS developed in 5 of 19 patients homozygous for KIR haplotype A compared with 2 of 27 patients with KIR haplotype AB and B (homozygous; p = 0.03; log-rank test). In none of the patients with BOS was the activating KIR2DS5 gene detected, whereas it was present in 35% of patients without BOS (p = 0.04; log-rank test). No correlation was found between KIR gene content and cytomegalovirus reactivation., Conclusion: Our results suggest that the lack of activating KIRs may play an important role in the development of BOS but not in the control of cytomegalovirus reactivation after lung transplantation.

Background: It has become evident that, besides cellular allogeneic immune responses against airway epithelial cells (AEC), humoral responses also contribute significantly to the pathogenesis of bronchiolitis obliterans syndrome (BOS) after lung transplantation (LTx). Antibody responses against transplanted lungs are directed against HLA and non-HLA antigens, but the identity of the latter antigens is presently unknown., Methods: The main purpose of this study is to identify non-HLA target antigens on donor lungs recognized by patients' antibodies after LTx. Serum samples were taken before and 6 months after lung transplantation from 11 patients (4 men and 6 women, median age 44 years, range 18 to 63 years). Protein expression libraries were made from the luminal side containing AEC of discarded the donor bronchus, which was snap frozen in liquid N(2) during the organ harvesting procedure. Subsequently, all sera were analyzed for reactivity against library-encoded antigens by serologic analysis of recombinant cDNA expression libraries (SEREX). Recognized gene products were sequenced and analyzed by the NCBI/BLAST server., Results: From a total of +/-3 x 10(4) gene products analyzed, six different non-HLA antigens were recognized by individual patient sera. Gene analysis indicated that they consisted of both polymorphic (PSMC4, F3, LOC284058, PLUNC, ZNF33A) and non-polymorphic (XP&#95;931864) antigens. Cross-sectional analysis indicated that some antigens were recognized by 4 of 10 patient sera tested., Conclusions: Antibodies directed against non-HLA antigens are present after LTx, and can be identified using the SEREX technique. Identification of target antigens recognized after LTx will improve our understanding of the pathogenesis of BOS. Monitoring of the antibody response may be used to predict BOS.

Background: The purpose of this study was to determine the clinical relevance of soluble CD30 (sCD30) concentrations in sera from lung transplantation (LTx) candidates., Methods: Soluble CD30 concentrations were determined by enzyme-linked immunoassay in pre-transplantation sera of 38 LTx candidates and in serial samples taken after LTx from 10 patients., Results: LTx candidates did not have increased serum sCD30 concentrations when compared with healthy controls (mean +/- SE: 22.3 +/- 2.7 vs 26.2 +/- 3.4 U/ml). No relation could be found between age and serum sCD30 concentrations, neither in the pre-transplant patients nor in the healthy controls. In addition, no differences in sCD30 values could be detected between patients sorted by their original type of lung disease. Measurement of sCD30 in pre-transplant sera of LTx patients showed that the median concentration was 20 U/ml and that LTx patients with low sCD30 (< or = 20 U/ml) had a statistically significant (p = 0.039) longer period of freedom from bronchiolitis obliterans syndrome (BOS) compared to those with a high sCD30 concentration (> 20 U/ml). Furthermore, sCD30 concentrations in sera taken at several timepoints after LTx remained stable, although a peak could be observed before the clinical manifestation of acute rejection (AR)., Conclusions: Soluble CD30 before lung transplantation is significantly associated with an increased risk of BOS after transplantation.

Background: The inflammatory process in chronic obstructive pulmonary disease (COPD) is characterised by the presence of neutrophils in the lung that are able to synthesise de novo several inflammatory mediators. The local chronic persistent inflammatory response is accompanied by systemic effects such as cytokine induced priming of peripheral leucocytes and muscle wasting. The preactivation or priming of peripheral blood neutrophils was used to gain more insight into the mechanisms of this systemic inflammatory response., Methods: Gene arrays were performed on peripheral blood neutrophils obtained from healthy donors after stimulation in vitro with tumour necrosis factor (TNF)-alpha, granulocyte-macrophage colony stimulating factor (GM-CSF), or both. The expression of many inflammatory genes was regulated in these cells following stimulation. The expression of inflammatory genes in peripheral blood neutrophils in healthy subjects and those with COPD was measured by real time RT-PCR after stimulation with TNFalpha, GM-CSF, interleukin (IL)-8, fMLP, TNFalpha + GM-CSF, and lipopolysaccharide (LPS)., Results: The genes regulated in the gene array with TNFalpha/GM-CSF stimulated neutrophils included cytokines (such as IL-1beta), chemokines (such as IL-8), and adhesion molecules (such as ICAM-1). Disease severity as measured by forced expiratory volume in 1 second (FEV(1)) in COPD patients correlated with expression of several of these genes including IL-1beta (r = -0.540; p = 0.008), MIP-1beta (r = -0.583; p = 0.003), CD83 (r = -0.514; p = 0.012), IL-1 receptor 2 (r = -0.546; p = 0.007), and IL-1 receptor antagonist (r = -0.612; p = 0.002)., Conclusions: These data are consistent with the hypothesis that progression of COPD is associated with the activation of neutrophils in the systemic compartment. De novo expression of inflammatory mediators by peripheral blood neutrophils suggests a pro-inflammatory role for these cells in the pathogenesis of COPD.

During the late-phase asthmatic response, eosinophils migrate to the bronchial tissue and cause severe damage. In this study we compared in vivo primed eosinophils from patients with allergic asthma with eosinophils from healthy control subjects in their adhesion behavior to tumor necrosis factor-alpha-activated endothelium under flow conditions (0.8 dyn/cm2). More eosinophils from patients with asthma adhered to activated endothelium, compared with cells from healthy control subjects (1,237 +/- 126 versus 887 +/- 94 cells/mm2, respectively). In the presence of blocking antibodies directed against very late antigen-4 and E-selectin, the residual binding of the cells of individuals with allergic asthma was significantly higher than that of the healthy control subjects (353 +/- 64 versus 123 +/- 31 cells/mm2, respectively, P < 0.02). In addition, secondary tethering or formation of clusters of the eosinophils of patients with allergic asthma was significantly increased compared with the healthy control subjects (cluster indices 1.8 +/- 0.3 versus 0.8 +/- 0.2, respectively, P < 0.05). Because patient cells showed an enhanced interaction with platelets during the perfusions, the role of P-selectin on platelets was investigated. Blocking antibodies directed against P-selectin reduced the enhanced binding and clustering of eosinophils of patients with allergic asthma. We conclude that P-selectin-bearing platelets contribute to secondary tethering processes of eosinophils to activated endothelium. Therefore, platelets might play an important role in the chronic inflammatory processes of these patients.

We performed nasal lavage (NAL) combined with induced sputum to determine exudative inflammation in the upper and lower airways in patients with chronic sinusitis and in controls. To monitor plasma exudation into the respiratory lumen and loss of size-selectivity of the mucosa, we determined the sample-to-serum ratio of albumin and alpha-2-macroglobulin, Qa1b and Qa2m, and the dilution independent Relative Coefficient of Excretion, RCE=Qa2m/Qa1b. To detect low protein levels in NAL and induced sputum we adapted an ELISA system for alpha-2-macroglobulin described by Out et al. [Clin. Chim. Acta, 165 (1987) 277-288], and modified this into a sensitive ELISA for albumin. Dithiothreitol, added to increase sputum solubility, did not interfere with the analysis, nor did N-ethylmaleimide, added to block dithiothreitol. In this study plasma exudation in induced sputum is significantly increased in patients with chronic sinusitis, compared to controls. Plasma exudation in NAL is also increased in patients, although not significant. The RCE in NAL and sputum is well-correlated in one of the three study visits. There is much variation in sample protein-levels partly due to differences in dilution and the heterogeneity of the studied population. Determination of plasma exudation together with RCE in NAL and induced sputum is a good, non-invasive way to quantify inflammation of airway mucosa.

We have investigated whether IL-8 is present in airway secretions from patients with asthma and chronic obstructive pulmonary disease (COPD) to obtain information on its possible role in airway inflammation in obstructive airways disease. In the bronchoalveolar lavage fluid (BALF) from 11 clinically stable patients with asthma the levels of IL-8 were increased compared to 10 healthy subjects (median: controls 21.5 pg/ml, asthma 244 pg/ml: p < 0.005). In the patients with asthma the levels of IL-8 correlated with the percentage neutrophils in the BALF (r = 0.81; p < 0.001) and with a parameter of the permeability of the respiratory membrane, the quotient (alpha 2-macroglobulin in BALF)/(alpha 2-macroglobulin in serum) (r = 0.66; p < 0.025). In the sputum sol phase of 9 patients with symptomatic asthma the levels of IL-8 were lower than in 9 patients with COPD (asthma: 6.4 ng/ml; COPD: 16.3 ng/ml; p < 0.02) and significantly correlated with those of neutrophilic myeloperoxidase (MPO; r = 0.85; p < 0.005). The increased levels of IL-8 in the airway secretions from both patients with asthma and COPD may be markers of an ongoing inflammatory process, which is more pronounced in patients with COPD. In patients with asthma the strong correlation between the levels of IL-8 and the percentage neutrophils and/or the levels of MPO points to a role of IL-8 in the recruitment and activation of neutrophils in the airway lumen.

We measured surfactant protein A and phosphatidylcholine in the bronchoalveolar lavage fluid from healthy volunteers and several groups of patients with lung diseases to obtain information on surfactant in the lung. We developed three types of enzyme-linked immunosorbent assays that used combinations of polyclonal antiserum and monoclonal antibodies. Phosphatidylcholine was assessed by enzymatic measurement. The median amounts of surfactant protein A in bronchoalveolar lavage fluid determined by the enzyme-linked immunosorbent assay with monoclonal antibodies were as follows: control subjects (n = 10), 2.82 mg/L (range, 0.92 to 5.17 mg/L); patients with asthma (n = 13), 1.89 mg/L (range, 0.45 to 2.95 mg/L); and patients with pulmonary sarcoidosis (n = 20), 2.98 mg/L (range, 0.68 to 7.02 mg/L). The median phosphatidylcholine concentrations in the bronchoalveolar lavage fluid were as follows: control subjects (n = 10), 20 mumol/L (range, 3 to 37 mumol/L); patients with asthma (n = 12), 24 mumol/L (range, 3 to 55 mumol/L); and patients with pulmonary sarcoidosis (n = 20), 26 mumol/L (range, 4 to 76 mumol/L). As a group, the patients with asthma had less surfactant protein A in bronchoalveolar lavage fluid than did the control subjects (Mann-Whitney U test, p less than 0.05). The surfactant protein A levels measured by the enzyme-linked immunosorbent assay with polyclonal antiserum and by a competitive enzyme-linked immunosorbent assay were also lower in bronchoalveolar lavage fluid from patients with asthma than in that from control subjects. The phosphatidylcholine concentrations in all groups were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated complement factors within the lung may induce several local biological effects. In order to investigate local complement activation we have developed non-competitive two-site ELISAs of C3a and total C3 in bronchoalveolar lavage fluid (BALF). For the assay of C3a, both C3 and C3(H2O) were removed from the samples by precipitation with polyethylene glycol. It was necessary to add carrier proteins to BALF to remove C3 and C3(H2O) fully. The ELISA of C3a has the lowest limit of detection reported thus far, namely 0.045 nM (= 0.405 ng/ml). In BALF from healthy persons (n = 9) the C3a concentration was 0.20 nM (0.12-0.31 nM) (median, range). C3a was higher in BALF from patients with asthma or with sarcoidosis; asthma (n = 10), 0.45 nM (0.20-5.79 nM); sarcoidosis (n = 19), 1.31 nM (0.095-5.65 nM) (Mann-Whitney U test, p less than 0.005). In BALF from patients with Pneumocystis carinii pneumonitis (n = 10) the C3a concentration was 0.18 nM (0.07-0.57 nM). C3a concentrations in BALF may reflect local complement activation in the lung and/or diffusion into the lumen. This was studied by normalizing C3a concentrations in BALF into values for epithelial lining fluid (ELF), and calculating serum-to-ELF quotients of C3a, and C3a/total C3 quotients.

We have measured Immunoglobulin G (IgG) subclasses in serum and bronchoalveolar lavage fluid (BALF) from 12 non-smoking patients with stable asthma and 9 non-smoking healthy volunteers to obtain information on their possible role in local immunological reactions. The quotients (concentration of IgG subclass in BALF)/(concentration of IgG subclass in serum) were calculated. In controls QIgG3 were lower than QIgG1, QIgG2 and QIgG4. The IgG subclasses in BALF and epithelial lining fluid (ELF) from patients with asthma were significantly higher than in controls, mainly due to increased leakage from the blood. Again QIgG3 were lower than Q of other subclasses. In the analysis of local production of IgG, albumin or ceruloplasmin was used as reference protein. Several patients showed a local production or a preferential accumulation of one or more IgG subclasses. We conclude that in healthy persons the IgG subclasses in ELF originate from the systemic circulation by passive permeation. In patients with asthma, the permeability of the respiratory membrane may be increased resulting in increased concentrations of subclasses in lung-lining fluid. In some patients with asthma, an additional local production of IgG subclasses occurs.

Immunoglobulins in the epithelial lining fluid of the respiratory tract either originate from the systemic circulation or they are locally produced by plasma-cells in the mucosa. For polymeric immunoglobulins (dimer IgA and IgM) specific receptors on the surface of epithelial cells allow facilitated transport of these immunoglobulins into the lining fluid. Local inflammatory reactions in the lung will damage the respiratory membrane, which results in an increased leakage of proteins into the lining fluid. This increased leakage complicates the differentiation between local production of immunoglobulins and passive permeation. In asthma patients inflammation of the respiratory epithelium is found. This inflammation may be the result of non-immunologically triggered processes as well as of immunological reactions. In the experimental part of this paper we analyse possible local production of immunoglobulins and other proteins within the lungs of patients with asthma. This is performed with the use of steady-state protein concentrations in serum and bronchoalveolar lavage fluid. Information on the permeability of the respiratory membrane can be obtained by the measurement of proteins that are not produced within the lungs. Information on local production can be obtained by comparing the epithelial lining fluid to serum concentration ratios of proteins with identical molecular masses.

In patients with stable asthma, we assayed plasma proteins in the bronchoalveolar lavage fluid to obtain information on plasma exudation into the airways. Fourteen nonsmoking patients with asthma who were in a stable period of their disease and eight nonsmoking healthy volunteers were studied. The ratios of the concentrations of albumin, ceruloplasmin (CP), and alpha-2-macroglobulin (A2M) between blood and epithelial lining fluid were calculated (cQalb, cQCP, and cQA2M). The cQalb was increased in the patients (Mann-Whitney U test, p less than 0.05). In 10 patients the bronchial hyperreactivity was assessed with histamine provocation tests. Significant relationships between the cQalb, cQCP, and cQA2M on the one hand and PC15 on the other hand were found (Spearman's rank correlation: r = -0.62, p less than 0.05; r = -0.61, p less than 0.05; r = -0.79, p less than 0.01, respectively). Fourteen patients were treated with two inhalations of 200 micrograms glucocorticosteroids per day in a 3-month prospective study. Three of them were excluded from further study because of an intercurrent exacerbation of asthmatic symptoms during therapy. In the 11 patients with stable asthma, the cQalb and cQA2M decreased after treatment with inhaled steroids (Wilcoxon's matched pairs signed rank test, p less than 0.03). Our results show that in patients with stable asthma, there is an increased plasma exudation into the airways, most likely caused by an increased respiratory membrane permeability. The plasma exudation correlated with the bronchial hyperreactivity to histamine, and it decreased after corticosteroid therapy.

In 6 healthy controls and 23 patients with pulmonary diseases the influx of urea during bronchoalveolar lavage was measured by comparing the concentrations of albumin and urea in the sequential samples recovered. It varied between -28 and 151 mumol/l. In the bronchoalveolar lavage fluid and serum we measured alpha-2-macroglobulin (A2M) and ceruloplasmin (CP). The bronchoalveolar lavage fluid to serum ratios were calculated (QCP and QA2M). QA2M/QCP was taken as a measure of the respiratory membrane permeability; it varied between 0.05 and 0.53. Influx of urea during lavage was higher according as the QA2M/QCP ratio was higher. We conclude that concentrations of substances in the epithelial lining fluid calculated with the urea correction method have to be corrected for the influx of urea.

We have measured lactoferrin and secretory IgA (sIgA) in the unconcentrated bronchoalveolar lavage fluid (BALF) from nonsmoking healthy volunteers (n = 10) and nonsmoking patients with stable asthma (n = 14). The median concentrations and the ranges of lactoferrin were controls, 0.13 mg/L (0.01-0.43 mg/L); asthma, 0.41 mg/L (0.07-7.51 mg/L). For sIgA the results were controls, 0.48 mg/L (0.12-1.47 mg/L); asthma, 1.29 mg/L (0.65-14.6 mg/L). The concentrations in the epithelial lining fluid (ELF) were calculated on the basis of urea in BALF and serum. SIgA and lactoferrin levels in the BALF and ELF from the patients with asthma were higher than in controls (Mann-Whitney U-test, p less than 0.03). Our results indicate that in patients with stable asthma the airway epithelial cells are activated, resulting in an enhanced secretion of lactoferrin and enhanced secretory transport of sIgA into the airway lumen.

In order to obtain information on the role of IgM in the pathogenesis of asthma, we assayed IgM in the broncho-alveolar lavage fluid (BALF) from 14 non-smoking patients with asthma and 9 non-smoking healthy persons, using an ELISA. The concentrations of IgM in the epithelial lining fluid (ELF) were calculated on the basis of urea in serum and BALF. The concentrations of IgM in ELF after correction for serum IgM, cQIgM, from the patients as a group were higher than those from controls (Mann-Whitney U test, p less than 0.05). The IgM in BALF and serum, analysed by sucrose density gradient centrifugation was pentameric. The transudation of IgM from blood into ELF was compared with that of alpha 2 macroglobulin (A2M), a protein with a similar molecular mass. In 6 patients the cQIgM/cQA2M value was above the upper value of the range for controls suggesting abnormal local production of IgM. The presence of the secretory component (SC) linked to IgM was determined by ELISA. The percentage of SC-IgM in BALF was increased in 5 out of 7 patients tested. The increased local IgM concentrations and its increased local production may suggest a role for local IgM-mediated reactions in the inflammatory events associated with asthma.