E.A. van de Graaf

Polymorphisms in innate immunity genes associated with development of bronchiolitis obliterans after lung transplantation

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.

Diabetes before and after lung transplantation in patients with cystic fibrosis and other lung diseases.

Diabet. Med. 29, e159–e162 (2012)

Effects of nutritional status and dietetic interventions on survival in Cystic Fibrosis patients before and after lung transplantation

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.

Pharmacokinetics and Toxicity of Tacrolimus Early After Heart and Lung Transplantation

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.

A Promoter Polymorphism in the CD59 Complement Regulatory Protein Gene in Donor Lungs Correlates With a Higher Risk for Chronic Rejection After Lung Transplantation

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.

Serum thymus and activation regulated chemokine levels post‐lung transplantation as a predictor for the bronchiolitis obliterans syndrome

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.

Anti-ETAR and anti-AT1R autoantibodies are elevated in patients with endstage cystic fibrosis

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.

Humoral immunity and complement effector mechanisms after lung transplantation

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.

Mannose-binding lectin deficiency linked to cytomegalovirus (CMV) reactivation and survival in lung transplantation.

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.

Measuring plasma exudation in nasal lavage fluid and in induced sputum as a tool for studying respiratory tract inflammation.

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.