Stefan Hirt

Matrix metalloproteinase-9 in bronchiolitis obliterans syndrome after lung transplantation

Bronchiolitis obliterans syndrome (BOS) is a severe complication after lung transplantation (LTX). In a retrospective cohort study 12 stable healthy recipients (non-BOS) and eight patients with BOS were enrolled after LTX and matrix metalloproteinases (MMP)-9, TIMP-1 and cell characteristics in bronchoalveolar lavage (BAL) samples (n = 145) were analysed. BALs from patients with BOS were further divided according to whether they were obtained before (pre-BOS) or after manifestation of BOS (BOS group). The MMP-9/TIMP-1 ratio was significantly increased in the BOS group compared with non-BOS or pre-BOS; furthermore, the ratio was negatively correlated with forced expiratory volume in one second. In zymography, the active form of MMP-9 was detected predominantly in the BOS group. In addition, zymography showed the banding pattern of neutrophil-derived MMP-9, indicating that polymorphonuclear neutrophils (PMNs) were the main source of MMP-9. According to that, MMP-9 was significantly correlated with the number of PMN. In immunocytochemistry, MMP-9 was also associated predominantly with PMN. This is the first study to evaluate the expression of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases-1 over time during manifestation of a fibroproliferative lung disease in patients. It demonstrates development of bronchiolitis obliterans syndrome after lung transplantation is associated with an imbalance of matrix metalloproteinases-9/tissue inhibitors of metalloproteinase-1 ratio.

Preserved Frank–Starling mechanism in human end stage heart failure

OBJECTIVE The goal of the present study was to examine the ability of failing myocardium to respond to enhanced preload with an increase in force development. METHODS The effect of various preload conditions (2.5-15 mN) on force development was studied in right ventricular trabeculae carneae from explanted human failing hearts with ischemic cardiomyopathy (ICM, n = 5, 42 preparations) or idiopathic dilated cardiomyopathy (DCM, n = 9, 77 preparations). To determine the severity of cardiac impairment we measured the positive inotropic effect of beta-adrenoceptor stimulation and calcium (ISO/Ca2+ ratio) and the expression of atrial natriuretic peptide (ANP) mRNA in all hearts. RESULTS (1) Force of contraction increased with stepwise augmentation of preload (length at 2.5 mN preload to length of maximal force development) from 3.7 +/- 0.5 (ICM) and 2.7 +/- 0.4 (DCM) to 8.3 +/- 0.9 and 6.5 +/- 0.8 mN/mm2, respectively (p < 0.05). (2) The ISO/Ca2+ ratio was 0.40 +/- 0.04 (ICM) and 0.35 +/- 0.03 (DCM), respectively. (3) ANP mRNA was expressed in all preparations, albeit at greatly varying levels (ICM 22.5 +/- 6.1 and DCM 18.7 +/- 4.7 normalized arbitrary units). (4) Contraction experiments performed in left ventricular tissue (n = 3, 32 preparations) essentially confirmed the results. CONCLUSION The Frank-Starling mechanism is preserved in terminally failing human hearts irrespective of the underlying etiology. We found no relation between the severity of cardiac impairment as assessed by either ANP expression or the ISO/Ca2+ ratio and the ability of failing human myocardium to respond to enhanced preload with an increase in force development.

Surfactant replacement in reperfusion injury after clinical lung transplantation

Background: Reperfusion injury remains a significant risk factor in the immediate postoperative course after lung transplantation. We report on our initial clinical experience of surfactant replacement in reperfusion injury after clinical lung transplantation. Methods and results: In 31 consecutive patients, lung (8 single lung, 16 bilateral lung) or heart-lung (7) transplantation was performed. In 6 patients, severe reperfusion injury developed and was treated with continuously nebulized surfactant. Compliance of the allograft increased 40 ± 25 % within 3 h following treatment with surfactant. Alveolar arterial oxygen gradient decreased by 23 ± 11 % after 3 h and by 35 ± 20 % after 6 h. Normal graft function was reestablished within 1–3 days after transplantation. All treated recipients were extubated until the 6th postoperative day. The 30-day mortality for the 31 recipients was 3.3 %, the 1-year survival 84 %. Conclusions: Surfactant replacement may become a clinical method for treatment of reperfusion injury after lung transplantation.

Detection of CMV Pneumonitis after Lung Transplantation Using PCR of DNA from Bronchoalveolar Lavage Cells

Background: Cytomegalovirus (CMV) is known as a common pathogen causing infections after lung transplantation. Rapid diagnosis of CMV infection is important for the initiation of a specific treatment. Objective: Evaluation of methods for the rapid diagnosis of CMV pneumonitis. Methods: The detection rates of CMV DNA in bronchoalveolar lavage (BAL) and bronchial brushes by polymerase chain reaction (PCR), of viral antigens (p52 and IE1) in BAL and of pp65 antigen in peripheral blood leukocytes were compared to the clinical status after lung transplantation. In 28 patients, 105 BAL, 96 blood samples and 14 brushes were analyzed. Results: In 6 patients, a total of eight episodes of CMV pneumonitis occurred. Five of the 6 with positive CMV antigens in BAL (p52 or IE1) showed signs of CMV pneumonitis. All episodes of CMV pneumonitis were detected by the PCR of BAL cells. Fourteen samples positive for CMV pp65 antigen in blood were negative in BAL PCR. In these cases, no clinical signs of pulmonary CMV infection occurred. Overall sensitivity, specificity, and positive and negative predictive values for the detection of CMV pneumonitis by PCR of BAL cells were 100, 98.9, 88.9 and 100%, respectively. In brush samples, PCR did not provide additional information to the results of the PCR of BAL cells. Conclusions: PCR of DNA from BAL cells is suitable for reliable and rapid detection of CMV pneumonitis.

Eosinophilic alveolitis in BAL after lung transplantation

Abstract Lung transplantation has become a therapeutic option for patients with end stage lung disease. However, outcome after transplantation is complicated by episodes of rejection and infections. Bronchoalveolar lavage is a valuable tool in monitoring patients after transplantation, since it allows the detection of pathogens. A marker specifically indicating rejection from changes in BAL fluid has not been found yet. Especially changes in differential cell count, like lymphocytosis or an increase in polymorphnuclear granulocytes, are unspecific. The role of high eosinophil levels in BAL has not been elucidated yet. We analyzed 25 BAL samples and clinical data of 4 patients who underwent lung transplantation and presented with recurrent episodes of eosinophilic alveolitis in BAL. All patients demonstrated a deterioration of clinical condition, lung function, and blood gas analysis during times of eosinophilia in BAL, compared to previous examinations. In all cases, eosinophilia in BAL was accompanied by rejection. All patients were finally treated with high doses of steroids, resulting in improvement of all parameters. Eosinophilia was not associated with significant changes in the IL-5 concentration in BAL or the pattern of IL-5 expression in BAL cells. In conclusion, eosinophilic alveolitis may indicate acute rejection in patients after lung transplantation, if other causes of eosinophilia are excluded.

Differential coupling of m-cholinoceptors to Gi/Go-proteins in failing human myocardium.

Muscarinic acetylcholine receptors (mAChRs) mediate their main cardiac effects via pertussis toxin-sensitive G-proteins. Physiological effects differ considerably between atrium and ventricle, and it is unknown to which extent these differences derive from selective receptor-G-protein coupling or further downstream events. We have characterized specific coupling between mAChRs and Gi/Go-protein isoforms in atrial and ventricular myocardium by agonist-dependent photoaffinity labeling with [(32)P]azidoanilido GTP (aaGTP) and immunoprecipitation in sarcolemmal membranes from terminally failing human hearts. The total amount of mAChRs, as determined by specific binding of [(3)H]QNB, was significantly higher in right-atrial (RA +/- SEM, 959 +/- 68 fmol/mg, n = 4) than in left-ventricular membranes (LV, 582 +/- 53 fmol/mg, n = 6). Standardized immunoblots revealed that Gialpha-2 was the predominant subtype in both regions. A 40-kDa splice variant of Goalpha (Goalpha-1 and/or Goalpha-3) was almost exclusively detectable in RA. Levels of Gialpha-3 and a 39-kDa splice variant of Goalpha (Goalpha-2) were also higher in RA. Basal aaGTP binding was higher in RA than in LV for all Gialpha/Goalpha subtypes. The carbachol (10 micromol/l)-induced increase in aaGTP binding was significantly higher in RA than in LV for Goalpha-1/3 (336 +/- 95% of LV, n = 4) and for Gialpha-3 (211 +/- 83%), lower for Gialpha-2 (42 +/- 5%), and was similar in both regions for Goalpha-2 (130 +/- 62%). The differential coupling of mAChRs in human RA and LV suggests that the initiation of different physiological responses to mAChR stimulation starts with signal sorting at the receptor-G-protein level.

Adhesion molecules in patients after lung transplantation.

Leukocyte adhesion molecules, such as intercellular adhesion molecule (ICAM)‐1 and its ligands, are involved in inflammatory processes of the lung. For ICAM‐1, differential expression during different kinds of complications after transplantation has been proposed.

Dissociation of the effects of forskolin and dibutyryl cAMP on force of contraction and phospholamban phosphorylation in human heart failure.

Forskolin and dibutyryl cyclic adenosine monophosphate (cAMP) stimulate force of contraction independent of beta-adrenoceptor stimulation. We studied their effects on force of contraction and phosphorylation of regulatory proteins in isolated electrically driven trabeculae carneae from failing human ventricles. The phosphorylation state of the regulatory protein phospholamban was studied because its phosphorylation usually faithfully follows contractility. For comparison, the phosphorylation state of the inhibitory subunit of troponin was studied. The phosphorylation state was inferred from in vitro phosphorylation of homogenates with cAMP-dependent protein kinase in the presence of radioactive gamma[32P]ATP Proteins were separated by electrophoresis, and radioactivity in the proteins of interest was quantified. The maximal positive inotropic effects occurred at 30 microM forskolin and were attenuated in comparison with the maximal effects to dibutyryl cAMP (1 mM). Both forskolin and dibutyryl cAMP enhanced phospholamban phosphorylation. However, phospholamban phosphorylation in intact trabeculae treated with 30 microM forskolin and 1 mM dibutyryl cAMP was comparable. It is suggested that phospholamban phosphorylation can be dissociated from inotropy at least in isolated trabeculae from failing human hearts.

Lung transplantation for acute pulmonary failure

Lung transplantation as well the combined replacement of the heart and both lungs could be established as a routine procedure at many cardiac surgical units during the last decade. Meanwhile, transplantation of the lung represents a realistic therapeutic option in a number of end-stage parenchymal and vascular pulmonary disorders. A prerequisite for lung transplant surgery is, however, the absence of transplant specific contraindications. With respect to the scarcity of suitable organ donors the potential recipient must, in addition, be able to survive a certain waiting time. Comparing with patients suffering from renal failure, who are able to survive endstage organ damage for many years using dialysis techniques, biotechnological bridging was impossible in severe heart or lung failure for many years. Recently, assist devices allow bridging in acute isolated lung failure for a certain period of time for either spontaneous recovery or subsequent lung transplantation. However, with respect to the enormous effort required in intensive care, medical costs and with respect to the donor shortage this therapeutic concept must be restricted to carefully selected cases.

Effects of ischaemia and preservation on the ultrastructure of the bronchiolar epithelium

In ten cases of clinical human single-lung transplantation, the nontransplanted Euro-Collins-preserved contralateral lungs were examined using electron microscopy to determine the effects of ischaemia on the bronchiolar epithelium. Existing structural damage at the time of transplantation was characterized using this approach, and nine nonpreserved canine single lungs were also investigated to identify the impact of ischaemia. The study revealed a significant correlation between the duration of ischaemia and the mitochondrial surface-to-volume ratio, which can serve as a morphometric criterion for mitochondrial damage, in canine lungs. However, this correlation was not found in the human donor lungs. Further examination of human donor lungs showed slight to moderate damage to the endoplasmic reticulum and nuclear chromatin. In addition, various degrees of damage to mitochondrial structure, ranging from inconspicuous to severe, were found. The mitochondrial surface-to-volume ratio can be considered to be a suitable criterion for the quantification of ischaemic damage of the bronchiolar epithelium under experimental conditions. Ultrastructural analysis of human donor lungs revealed intact bronchiolar epithelial cell structures at the time of transplantation, reflecting adequate organ preservation with Euro-Collins solution.