Bernd Schmidt

Impact of pulmonary hypertension on gas exchange and exercise capacity in patients with pulmonary fibrosis

Pulmonary hypertension is a relevant interceding morbidity in patients with pulmonary fibrosis that has significant impact on exercise tolerance and outcome. The aim of this study was to further characterize the exercise intolerance, dyspnoea and ventilatory inefficiency of patients with pulmonary fibrosis in the presence or absence of pulmonary hypertension via cardiopulmonary exercise testing. Thirty-four patients underwent pulmonary function testing, symptom-limited exercise testing on a bicycle and dyspnoea evaluation according to the BORG scale. Pulmonary hypertension was assessed by echocardiography and in a subset of patients right heart catheterization. Sixteen of 34 patients with pulmonary fibrosis revealed pulmonary hypertension. While all study patients did not differ in lung functions and demographic characteristics, patients suffering from pulmonary hypertension showed a significantly impaired exercise tolerance and worsened ventilatory inefficiency. The extent of pulmonary artery pressure elevation impacted significantly on ventilatory inefficiency. In addition, the increased ventilatory requirements significantly influenced the extent of dyspnoea in patients with pulmonary hypertension. We conclude that pulmonary hypertension has a significant impact on exercise capacity and dyspnoea in patients with interstitial lung disease (ILD). The further impairment of exercise capacity as well as the extent of dyspnoea in patients with interceding PHT is attributable to a significantly impaired ventilatory inefficiency.

An Improved Method for the Isolation of Free‐Circulating Plasma DNA and Cell‐Free DNA from Other Body Fluids

Cell‐free DNA is of increasing interest in different fields, such as cancer research, prenatal diagnosis, and the diagnosis of nonmalignant diseases. The quantity of free‐circulating DNA in plasma, serum, and other body fluids is usually low and its isolation is still a challenge. Here we evaluate the application of the new commercially available NucleoSpin Plasma XS Kit for the isolation of cell‐free DNA from plasma, serum, and cell‐free bronchial lavage samples. The NucleoSpin columns proved superior to the QIAamp system in terms of DNA yield, purity, and retrieval of small DNA fragments. The procedure is fast and easy to standardize and might be valuable for laboratories working in the field of CNAPS.

Cell-free DNA resuscitated for tumor testing

Extracellular DNA floating around in blood plasma provides an accessible template for detecting mutations associated with tumors. A new technique is able to quantify such mutated DNA and predict relapse in individuals with colorectal cancer. The technique complements other approaches, such as the analysis of tumor cells in the plasma (pages 985–990).

INCREASED SPONTANEOUS INTERLEUKIN-10 RELEASE FROM ALVEOLAR MACROPHAGES IN ACTIVE PULMONARY SARCOIDOSIS

The study was designed to determine whether alveolar macrophages (AM) in acute pulmonary sarcoidosis release in vitro the anti-inflammatory cytokine interleukin (IL)-10. To learn more about the coherence between IL-10 and proinflammatory cytokines in active sarcoidosis, the release of interferon (IFN)- γ, macrophage inhibitory protein (MIP)-1 α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied and additionally compared to normal controls and patients with pneumonia and interstitial lung fibrosis. AM were obtained by bronchoalveolar lavage from 13 patients with active sarcoidosis, 8 patients with interstitial lung fibrosis, 10 patients with bacterial pneumonia, and 14 normal controls. The spontaneous and stimulated (tumor necrosis factor [TNF]- α, IL-1 β) cytokine release was measured in the supernatant of cultured AM by enzyme-linked immunosorbent assay (ELISA). Unstimulated AM from sarcoidosis patients released more IL-10, IFN- γ, MIP-1 α, and GM-CSF than normal controls and patients with pneumonia and interstitial lung disease. Stimulation with TNF- α or IL-1 β increased the MIP-1 α and GM-CSF release from AM of normal controls and patients with pneumonia and interstitial lung disease: however, no further enhancement of MIP-1 α and GM-CSF production was observed in AM from sarcoidosis patients. Exogenous IL-10 reduced the spontaneous and stimulated MIP-1 α and GM-CSF release in sarcoidosis to a lesser extent than in controls and patients with fibrosis and pneumonia. The up-regulated IL-10 in active pulmonary sarcoidosis may be a compensatory response to the enhanced expression of proinflammatory cytokines in order to down-regulate the inflammatory process. The results suggest an involvement of the anti-inflammatory cytokine IL-10 in the immunopathogenesis of sarcoidosis.

Therapeutic implication of BAL in patients with neutropenia

Bronchoalveolar lavage (BAL) is a practicable procedure establishing the etiology of pneumonia. In patients with neutropenia, empirical antimicrobial treatment is mandatory immediately after diagnosis of infection, usually before results of BAL are available. We evaluated the impact of BAL on treatment and outcome of pneumonia in immunocompromised patients with a special regard to neutropenia. Bronchoscopy with BAL was performed in 58 episodes of clinical documented pneumonia in patients with hematological malignancies (88%) or solid tumors (12%), in 30 cases patients had neutropenia, in 28 cases patients had no neutropenia. In 93% of cases, BAL was performed under empirical antimicrobial treatment. BAL fluid was cultivated for bacteria, fungi, and tested for Pneumocystis jirovecii and cytomegalovirus (CMV). BAL revealed positive bacterial results in 67% of cases. Gram-positive microorganisms were detected in 95% of positive BAL results, gram-negative microorganisms in 23%, mixed bacterial cultures occurred in 41%. Positive fungi cultures were found in 59%. P. jirovecii was detected in 5% of cases tested and CMV in 8%. There was no significant difference between neutropenic and non-neutropenic patients. BAL results directed a change of therapy in only six of 58 episodes (5%). Overall mortality related to pneumonia was 16%. In this patient setting, the yield of BAL rarely has a significant influence on treatment and outcome of pneumonia. The early beginning of antimicrobial treatment reduces the diagnostic yield of BAL. In patients with pneumonia during neutropenia, its use should be well considered.

Detection of cell-free DNA in bronchial lavage fluid supernatants of patients with lung cancer.

Abstract: Recently, it was shown that it is possible to isolate free circulating DNA from plasma/serum of patients with benign and malignant diseases. In addition, several groups were able to detect tumor‐associated alterations in these nucleic acids. We wondered whether any nucleic acids are detectable in cell‐free bronchial lavage supernatants, which until now have been discarded after cell harvest. Additionally, we wanted to find out if it is possible to detect tumor‐associated alterations in these DNA molecules. DNA was isolated from cell‐free lavage supernatants from 30 lung cancer patients, and the DNA was examined for microsatellite alterations. Intact DNA could be isolated from all cell‐free bronchial lavage supernatants. Microsatellite alterations were found in lavage supernatants of 12 of 30 patients and in lavage cells of 6 of 30 patients. Altogether, alterations were found in 14 of 30 patients. Thus, we could demonstrate for the first time that it is possible to isolate intact DNA from cell‐free bronchial lavage supernatants. Their quantity and quality are sufficient for further amplification via polymerase chain reaction. Altogether, tumor‐associated changes were detected in the DNA of 47% of the patients that were analyzed.

Detection of tumor-specific mRNA in cell-free bronchial lavage supernatant in patients with lung cancer.

Abstract: Bronchoscopy is a standard procedure in the workup of patients with suspicious pulmonary lesions. We wondered whether it is possible to isolate malignancy‐associated mRNA from cell‐free lavage supernatant. Extracellular mRNA from cell‐free lavage supernatant of 25 patients with lung cancer (23 with non‐small cell lung cancer, 2 with small cell lung cancer) was isolated, reverse‐transcribed, and amplified by reverse transcriptase polymerase chain reaction. The quantity and quality of the isolated RNA were checked after cDNA synthesis by amplification with β‐actin‐specific primers. Afterwards, a panel of eight genes known to be expressed in lung tumors was used for the detection of tumor‐associated mRNA expression in lavage supernatant and serum. mRNA coding for β‐actin could be isolated from lavage supernatant of all 25 patients. In addition, the expression of at least one tumor‐associated gene was detectable in all patients. These results show that intact mRNA can be isolated from cell‐free lavage supernatant and that its quantity and quality are sufficient for the detection of tumor‐associated gene expression alterations. This may open new possibilities for the diagnosis of lung cancer.

Mature mediastinal teratoma with subtotal unidirectional pancreatic differentiation

Tumor-like lesions of the mediastinum consisting exclusively of mature pancreatic tissue are rare. Most authors regard these lesions as developmental abnormalities. We report the case of a 17-year-old male who presented with progressive dyspnea due to a large mediastinal tumor with accompanying pericardial effusion. Percutaneous core needle biopsies showed differentiated pancreatic tissue with chronic inflammation and cystic transformation. On this basis, heterotopic pancreatic tissue of the mediastinum, a lesion occasionally reported in the literature, was suspected and the lesion was excised. The histopathological workup of the surgically excised lesion, in the first place, supported the primary diagnosis. However, after extensive sampling of the lesion, the diagnosis had to be changed to mature teratoma with subtotal unidirectional pancreatic differentiation. We hypothesize that at least some of those cases reported in the literature as heterotopic pancreatic tissue of the mediastinum may be in fact unidirectionally differentiated teratomas and should therefore be regarded and treated as true neoplasms rather than developmental abnormalities.

Comparison of Nucleosomes and Quantitative PCR Using Diverse DNA Isolation Methods

Reliable standardization for the isolation and detection methods prior to quantification of cell-free DNA are still lacking. QIAamp DNA Blood Midi Kit (Q), the NucleoSpin-Kit (MN), and the MagNA-Pure isolation system (RD) were compared on the amount of DNA isolated versus the immunological quantification of circulating nucleosomes using the Cell Death Detection ELISA plus (RD). Forty-four plasma samples were used with each system in parallel at laboratories in Berlin and Munich. Subsequently, DNA was quantified by real-time PCR on a LightCycler 480 (RD) in Berlin using targets for s-globin. Correlations of plasma nucleosomes and cell-free DNA measured by real-time PCR ranged between R = 0.32 and R = 0.64 and were comparable between the two laboratories. Large differences in the amounts of cell-free DNA were observed between the diverse isolation methods: the RD isolation system gave the highest DNA yield followed by MN and Q systems. Most comparable results with nucleosome ELISA were achieved using the Roche system.