Gunnar Mårtensson

BMPR2 Haploinsufficiency as the Inherited Molecular Mechanism for Primary Pulmonary Hypertension

Primary pulmonary hypertension (PPH) is a potentially lethal disorder, because the elevation of the pulmonary arterial pressure may result in right-heart failure. Histologically, the disorder is characterized by proliferation of pulmonary-artery smooth muscle and endothelial cells, by intimal hyperplasia, and by in situ thrombus formation. Heterozygous mutations within the bone morphogenetic protein type II receptor (BMPR-II) gene (BMPR2), of the transforming growth factor beta (TGF-beta) cell-signaling superfamily, have been identified in familial and sporadic cases of PPH. We report the molecular spectrum of BMPR2 mutations in 47 additional families with PPH and in three patients with sporadic PPH. Among the cohort of patients, we have identified 22 novel mutations, including 4 partial deletions, distributed throughout the BMPR2 gene. The majority (58%) of mutations are predicted to lead to a premature termination codon. We have also investigated the functional impact and genotype-phenotype relationships, to elucidate the mechanisms contributing to pathogenesis of this important vascular disease. In vitro expression analysis demonstrated loss of BMPR-II function for a number of the identified mutations. These data support the suggestion that haploinsufficiency represents the common molecular mechanism in PPH. Marked variability of the age at onset of disease was observed both within and between families. Taken together, these studies illustrate the considerable heterogeneity of BMPR2 mutations that cause PPH, and they strongly suggest that additional factors, genetic and/or environmental, may be required for the development of the clinical phenotype.

Persistent high BAL fluid granulocyte activation marker levels as early indicators of bronchiolitis obliterans after lung transplant

The major cause of mortality in the long-term in lung transplant recipients is chronic rejection. This is a fibroproliferative process in the small airways leading to obliterative bronchiolitis and progressive loss of lung function, both constituting the clinical entity bronchiolitis obliterans syndrome (BOS). Granulocyte activation has been implicated as one factor behind BOS. Granulocyte markers in bronchoalveolar lavage (BAL) fluid were prospectively and longitudinally studied in order to identify possible association with BOS. BAL fluid from 266 bronchoscopy procedures performed in twelve single lung, eight bilateral lung and five heart/lung transplant recipients were analysed. The majority (19 of 25) were studied for a period of 2 yrs after surgery. Myeloperoxidase (MPO), eosinophil cationic protein (ECP) and interleukin-8 (IL-8) levels were used as indirect markers of activation and attraction of granulocytes. Five patients developed BOS. Ninety-eight episodes of acute rejection, nine of bacterial infection, 19 of cytomegalovirus pneumonitis, nine of Pneumocystis carinii infection, two of aspergillus infection and two of respiratory syncytial virus infection were diagnosed. BOS patients had significantly higher mean levels of MPO, ECP and IL-8 compared to patients without BOS, irrespective of acute rejection status. Over time, the five patients with BOS had significantly elevated BAL fluid levels of MPO and ECP as well as neutrophil percentages, and in four patients this increase preceded the clinical diagnosis of BOS by several months. Elevated bronchoalveolar lavage fluid neutrophil percentage as well as levels of the granulocyte activation markers myeloperoxidase and eosinophil cationic protein appear to be early signs of development of BOS in lung transplant recipients.

Inflammatory cells and activation markers in BAL during acute rejection and infection in lung transplant recipients: a prospective, longitudinal study

Acute rejection of the transplanted lung is a clinical problem, since it decreases graft survival and predisposes the patient to chronic rejection and obliterative bronchiolitis (OB). In an earlier study, we had indications that eosinophil cationic protein (ECP) from activated eosinophils and hyaluronan (HYA) from fibroblasts were associated with acute pulmonary rejection. This prospective longitudinal study was designed to investigate whether molecules from activated inflammatory cells in bronchoalveolar lavage (BAL) fluid could serve as clinically useful diagnostic markers for acute rejection. BAL fluid from 138 bronchoscopies performed in 10 single lung, four bilateral lung and five heart-lung transplant recipients were analysed. Nine patients were studied for a period of more than 1 yr (mean 13.4 months) after surgery. Differential cell counts were made from the BAL fluid. ECP, myeloperoxidase (MPO), HYA and interleukin-8 (IL-8) were used as indirect markers for activation and attraction of eosinophils, neutrophils and fibroblasts, respectively. Fifty four episodes of acute rejection were diagnosed. Two patients developed OB. Nine episodes of bacterial infection, 13 episodes of cytomegalovirus (CMV) pneumonitis, three of Pneumocystis carinii infection and one of respiratory syncytial virus (RSV) infection were diagnosed. The mean levels of ECP, MPO, HYA and IL-8 were all higher during rejection episodes, but differences were not statistically significant compared to no rejection, when the confounding factors of time, concomitant infection, and repeated measures in the same individual had been accounted for. We could not confirm that measurements of eosinophil cationic protein, myeloperoxidase, hyaluronan and interleukin-8 in bronchoalveolar lavage fluid can be used as diagnostic markers for acute rejection in the postoperative follow-up of lung transplant recipients.

Immunohistochemical differences between hyaluronan- and non-hyaluronan-producing malignant mesothelioma

In many but not all cases, malignant mesothelioma is associated with an elevated content of hyaluronan in pleural fluid. The hyaluronan-producing mesothelioma has not yet been immunohistochemically characterized; therefore, the purpose of this study was to compare the immunohistochemical reactivity patterns in relation to the ability of this tumour to produce hyaluronan. Pleural fluid samples from 33 patients with malignant mesothelioma were analysed for content hyaluronan using a quantitative high performance liquid chromatographic method. Biopsy specimens from the patients were studied immunohistochemically, using monoclonal antibodies against carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), a low molecular weight cytokeratin antigen (CAM 5.2) and vimentin. An elevated hyaluronan content, i.e. > 100 mg.L-1, was noted in 23 patients (70%). There was no reactivity to the monoclonal antibody raised against CEA in any case. There was a significantly higher reactivity to EMA (p = 0.026), a higher reactivity to CAM 5.2 (p = 0.053) and a lower reactivity to vimentin (p = 0.057) in the hyaluronan-producing mesotheliomas as compared to those with normal levels of hyaluronan. Mesotheliomas that produced hyaluronan differed immunohistochemically from those that did not. The connection between the ability to produce different antigens and hyaluronan may relate to the degree of differentiation of the tumour. Both of these characteristics (immunophenotype and ability to produce hyaluronan) may, therefore, be of importance in studies concerning the prognosis and treatment of the malignant mesothelioma.

Ifosfamide in malignant mesothelioma: a phase II study.

Malignant mesothelioma is a rare malignancy with a median survival, ranging from 4 to 18 months in untreated patients. In a phase II study of patients with mesothelioma, the efficacy and toxicity of ifosfamide and mesna was evaluated. Twenty-nine previously untreated patients, with histologically proven and unresectable mesothelioma, entered the study. Three patients were later excluded from the study due to revision of the diagnoses. The patients had to have bidimensionally measurable disease by CT scans and a WHO performance status < or = 3. Eligible patients received ifosfamide 3000 mg/m2 per day for 3 days as a 1-h infusion and mesna 1800 mg/m2 per day for 3 days every third week. Dose modifications were made according to the degree of hematologic, neurologic and renal toxicity. Response to treatment was evaluated in accordance with WHO criteria. The median age of patients was 59 years (range 39-68), 18 patients (69%) had a history of asbestos exposure and the median of treatment cycles was four (range 1-10). No complete responses were observed. One patient obtained a partial response after five cycles with a duration of response of 25 months. Nine patients (35%) had stable disease, while 13 (54%) progressed. The median survival for all patients was 10 months. The toxicity of the treatment was considerable. Thirteen patients (50%) had grade 4 leucopenia, ten patients (38%) had grade 3 or 4 reversible neurotoxicity and ten patients (38%) had grade 3 or 4 nausea and vomiting. Eleven patients (42%) went off the study due to the toxicity of the treatment. In conclusion, ifosfamide did not show any substantial activity of relevance in malignant mesothelioma at the dose level investigated, in spite of considerable toxicity.

Pulmonary hemodynamics as predictors of mortality in patients awaiting lung transplantation

Lung transplantation (LTx) is a therapeutic option for patients with end‐stage lung disease. However, the mortality rate of patients on the waiting list is high. The purpose of this study was to examine the prognostic value of cardio‐pulmonary hemodynamics for death in patients awaiting LTx. Retrospectively, 177 patients with advanced lung disease accepted for LTx at Sahlgrenska University Hospital from January 1990 through December 2003 were studied. Patient demographics, pulmonary function tests, gas exchange and hemodynamic variables were included in the analysis. Death while awaiting LTx was the primary endpoint for all analyses. Mean age was 49 ± 9 years. Main diagnoses were alpha 1 antitrypsin deficiency (n = 56), chronic obstructive pulmonary disease (n = 61), cystic fibrosis (n = 14) and interstitial lung disease (n = 46). Thirty patients died (17%). LTx was performed in 143 cases. By univariate analyses, forced vital capacity (FVC) % of predicted, pulmonary vascular resistance (PVR) and diagnosis were associated with risk for death. In multivariate analysis PVR (HR, 1.22; 95% CI, 1.06–1.41; P = 0.006) and FVC% of predicted (HR, 0.97; 95% CI, 0.94–0.99; P = 0.01) were independently associated with death. Patients with increased PVR and a lower FVC % of predicted awaiting LTx should be considered for a higher organ allocation priority. Assessment of pulmonary hemodynamics needs to be considered during evaluation for LTx.

328: Lung function tests for prediction of BOS

primary graft dysfunction (PGD) following lung transplantion. Methods and Materials: Our institution initiated ARDS-type ventilator management in all lung transplant recipients beginning October 1st, 2004. We reviewed 97 patients (59 single lung, 38 bilateral) after this date and compared their measures of early gas exchange to the previous 97 transplant-type matched controls. Absolute values and gradients in PaO2/FIO2 ratios at 12 and 24 hours posteroperatively as well as the percentage of patients extubated within 48 hours were compared. Results: There were no significant differences in the PaO2/FIO2 ratios or ISHLT PGD grades measured at ICU admission, 12 hours, or 24 hours postoperatively between conventional ventilation and ARDS-type strategies. Neither were significant differences in the 1st 12-hour or 1st 24-hour PaO2/FIO2 gradients seen. Although reintubation rates were reduced using the ARDS ventilation strategy, the percentage of patients intubated beyond 48 hours was increased in this group, although the difference did not reach statistical significance. Conclusions: We were unable to show improvement in early gas exchange in lung transplant patients using an ARDS-type ventilator strategy. Although appealing as a technique to reduce barotrauma that might exacerbate PGD, more carefully controlled randomized trials in larger numbers of patients will be necessary to demonstrate the benefit should one exist.

Yield of surveillance and diagnostic transbronchial lung biopsies (TBB) performed beyond one year after lung transplantation

Abstract Aim: To analyze the outcome of transbronchial lung biopsy (TBB) according to a standard follow-up protocol (protocol TBB), or, on clinical indications (diagnostic TBB) performed at least one year after lung transplantation. Methods: A retrospective review of all protocol and diagnostic TBBs beyond the first year after trans-plantation was conducted in patients transplanted between 1994 – 1999 at Sahlgrenska University Hospital (46 single lung, 26 double lung and 8 heart-lung tx). Results: A total of 357 TBBs were assessed in 80 patients during a follow-up between 1 - 6 years post transplant, 257 were protocol TBBs and the remaining 100 diagnostic TBBs. Protocol TBBs revealed histopathological findings in 26% of the procedures at 1-year follow-up, 17% at 2-year, 18% at 3-year and 8% at 4-year, whereas no findings appeared at 5 and 6-year follow up. Of the histopathological findings 77 % were acute rejection grade 1–2. Diagnostic TBBs were most common between the first and second year post transplant (n=66). Of all diagnostic TBBs, 63% revealed histopathology, they were mainly acute rejections grade 1–3 (31%), nonspecific inflammation (27%) and infections (19%). Conclusion: This study supports the usage of protocol TBB examinations during the first 3 years after lung transplantation. However, beyond this time interval, TBB may only be performed on clinical indications.