Michael Ebsen

Hypoxia-induced intrauterine growth retardation: effects on pulmonary development and surfactant protein transcription.

Background and Objectives: Preterm infants with intrauterine growth retardation (IUGR) reveal an increased risk for the development of acute and chronic pulmonary disorders, i.e. bronchopulmonary dysplasia (BPD). In order to investigate the effect of IUGR on pulmonary development, an easily reproducible animal model for fetal growth restriction has been established using hypoxia as a sole intervention in the last third of pregnancy. Methods: Date-mated mice were randomly assigned to either being kept at a fraction of inspired oxygen (FiO2) of 0.10 (hypoxic group) starting at day 14 or under normoxic conditions until day 17.5 of gestation (control group). Variables of somatic growth were assessed and standardized histomorphometric analyses of pulmonary tissue were performed. Expression of surfactant proteins (SP)-A, -B, -C and -D was determined by quantitative rt-PCR as biochemical indicators for lung development and maturation. Results: Fetuses were delivered preterm at 0.87 of gestation. Those grown under hypoxic conditions revealed significantly lower birth weights (median: 0.69 vs. 0.97 g in controls; p < 0.001), body lengths (median: 17.5 vs. 20.2 mm in controls; p < 0.001) and fronto-occipital diameters (median: 9.4 vs. 10.1 mm in controls; p < 0.001) compared to controls. Histomorphometric analyses were found to be without significant differences between both groups. On the transcriptional level, however, mRNA expression of SP-A, -B and -C but not SP-D could be shown to be significantly reduced in hypoxic fetuses compared to normoxic controls. Conclusions: In conclusion, hypoxic conditions from day 14 to 17.5 led to IUGR in preterm mice and to significant alterations of the developing surfactant system. We speculate restricted development of SP gene expression to be a causal factor for the increased risk of acute and chronic pulmonary disorders in preterm infants with IUGR.

Infection of Murine Precision Cut Lung Slices (PCLS) with Respiratory Syncytial Virus (RSV) and Chlamydophila pneumoniae Using the Krumdieck Technique

The Krumdieck technique allows the investigation of the so-called precision cut lung slices (PCLS) with a special microtome. It is thus possible to evaluate morphologic changes over a longer period of time using only a small group of animals. Chlamydophila pneumoniae (Cp) and respiratory syncytial virus (RSV) proved to be important causes of pneumonia, rhinitis and exacerbations of asthma bronchiale, as well as of lower respiratory tract infections in young children. PCLS should be tested for their suitability as an in vitro model for these infections. The PCLS were infected with Cp and RSV over different periods of time. Investigations were carried out by light and transmission electron microscopy (TEM). Furthermore, immunofluorescence (IF) studies with antibodies against bacterial or viral proteins and cell-specific markers were done using confocal laser scanning microscopy (CLSM). Non-infected and infected PCLS showed a well-preserved morphology up to 72 hours. After short infection intervals, typical inclusions of Cp or RSV were detected in vacuoles of different cell types. Infection and cell types could be verified using IF. Cytopathic effects were not prominent. Ciliary beat was detectable up to 96 hours after infection. This in vitro technique offers the possibility of studying mechanisms and effects of bacterial and viral infections on viable tissue complexes.

Synthetic and natural surfactant differentially modulate inflammation after meconium aspiration.

ObjectiveMeconium aspiration syndrome remains a relevant cause of neonatal respiratory failure and is associated with severe pulmonary changes including surfactant inactivation and pronounced inflammatory changes. The present study investigated the effect of two different surfactant preparations—recombinant surfactant protein C surfactant (rSP-C Surf) and natural bovine surfactant—on pulmonary gas exchange and inflammatory response.Design and subjectsTwenty-three newborn piglets were intubated, mechanically ventilated, received 5 ml/kg 20% sterile meconium for induction of lung injury, and were randomized thereafter for controls (n=7), rSP-C Surf (n=8), or natural surfactant (n=8). Surfactants were given as an intratracheal bolus (75 mg/kg) and animals were further ventilated.Measurements and resultsLung function variables, arterial blood gas samples and lung tissues were obtained. Histological evaluation was performed in right lung tissue using an established score. Cytokine mRNA expression (left lung tissue) was quantified using TaqMan real-time PCR (ΔΔCT method, normalized to controls). In addition to significant improvement in gas exchange and lung function, histological evaluation showed significantly lower sum scores in the rSP-C Surf group than in controls). Cytokine mRNA expression of IL-1β in whole lung tissue was significantly lower after administration of rSP-C Surf than in natural surfactant and controls whereas IL-10 mRNA expression was significantly induced in both surfactant groups.ConclusionsSurfactant administration improved both gas exchange and pulmonary inflammatory cytokine transcription. Mechanisms underlying the differential inflammatory response in both surfactant preparations need to be further addressed.

Ultrastructural pathology of primary ciliary dyskinesia: report about 125 cases in Germany

BackgroundPrimary ciliary dyskinesia (PCD) is a rare genetically induced disorder of cilia inducing mainly respiratory diseases. Transmission electron microscopy (TEM) analysis of ciliary ultrastructure is classically used for diagnosis. We report our experience of TEM investigations in a large series of patients.MethodsTEM analysis performed of 742 biopsies from patients with suspected PCD was reviewed retrospectively. Ultrastructural defects were analysized further in 125 cases with changes typical for PCD.ResultsIn 18.1% of patients diagnosis of PCD was made because of morphological alterations, in 68.2% secondary changes were seen. In 13.7% material was not feasible for analysis. Mostly defects of dynein arms were detected in PCD (96.8%). In particular defects of the inner arms (51.2%) and combined dynein defects (37.6%) were found. Total loss of dynein arms was dominant. Only in 3.2% deficiencies of central structures were found alone. Associated situs inversus or dextracardia was reported clinically in 21.4%.ConclusionsTEM analysis is possible in most patients and a useful tool for diagnosis of PCD. Functional and genetic analysis should be done additionally. Registers should be installed to collect all available informations and push further research.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1629267757580889.

Diagnostik der primären ziliären Dyskinesie

ZusammenfassungCharakteristikaDie primäre ziliäre Dyskinesie (PCD) ist eine seltene angeborene Erkrankung der Zilien, die sich zumeist im respiratorischen System manifestiert.DiagnostikBei klinischem Verdacht auf das Vorliegen einer PCD und/oder bei positivem Screening (erniedrigte nasale NO-Werte) sollten Patienten zeitnah weitere diagnostische Maßnahmen durchlaufen. In Zentren, in denen eine Hochfrequenzvideomikroskopieanalyse (HVMA) des Zilienschlags zur Verfügung steht, ist eine initiale nasale NO-Messung zum Screening nicht zwingend erforderlich. Als erste diagnostische Maßnahme zur Sicherung oder zum Ausschluss einer PCD sollte eine HVMA erfolgen. Bei auffälligem Befund sind eine transmissionselektronenmikroskopische Analyse (TEM) der Ultrastruktur und eine hochauflösende immunfluoreszenzmikroskopische Analyse (IF) der Zilien anzuschließen. Obligat für die Diagnosestellung sind mindestens 2 kongruente pathologische Befunde aus HVMA, TEM oder IF. Wenn eine PCD-Variante ohne Hinweis auf einen ultrastrukturellen Defekt vorliegt, muss ein identischer pathologischer Zilienschlag mittels HVMA an insgesamt 3 unabhängigen Terminen belegt werden. Danach sollte auf Basis der erhobenen Befunde für HVMA, TEM und IF eine gerichtete genetische Abklärung angestrebt werden. Ein eindeutiger genetischer Befund kann die Diagnose ebenfalls sichern. VorgehenBei Verdacht auf eine PCD soll Kontakt mit einem Diagnosezentrum aufgenommen werden. Ein Referenzzentrum für PCD-Diagnostik evaluiert unklare Befunde.AbstractCharacteristicsPrimary ciliary dyskinesia (PCD) is a rare congenital disease of the cilia which is mostly manifested in the respiratory system.DiagnosticsWhen there is a clinical suspicion of the presence of PCD and/or a positive screening result with reduced nasal nitrogen oxide (NO) values, further diagnostic measures should be initiated as soon as possible. In centers where high-frequency video microscopy analyses (HVMA) of beating of cilia are available, an initial nasal NO measurement for screening must not necessarily be carried out. As the first diagnostic measure for confirmation or exclusion of PCD, HVMA should be carried out. If the findings are conspicuous transmission electron microscopic analysis (TEM) of the ciliary structure and high-resolution immunofluorescence (IF) microscopic analysis of the cilia should follow. Mandatory for diagnosis are at least two congruent pathological findings from HVMA, TEM or IF. When a PCD variant with no evidence of ultrastructural defects is present, an identical pathological beating of cilia must be demonstrated with HVMA on three independent occasions. Following that a targeted genetic clarification should be attempted based on the findings for HVMA, TEM and IF. A clear genetic result can also confirm the diagnosis.ApproachWhen PCD is suspected contact with a diagnostic center should be made. A reference center for PCD diagnostics will evaluate uncertain findings.

Surfactant replacement and open lung concept--comparison of two treatment strategies in an experimental model of neonatal ARDS.

BackgroundSeveral concepts of treatment in neonatal ARDS have been proposed in the last years. The present study compared the effects of open lung concept positive pressure ventilation (PPVOLC) with a conventional ventilation strategy combined with administration of two different surfactant preparations on lung function and surfactant homoeostasis.MethodsAfter repeated whole-lung saline lavage, 16 newborn piglets were assigned to either PPVOLC (n = 5) or surfactant treatment under conventional PPV using a natural bovine (n = 5) or a monomeric protein B based surfactant (n = 6).ResultsComprehensive monitoring showed each treatment strategy to improve gas exchange and lung function, although the effect on PaO2 and pulmonary compliance declined over the study period in the surfactant groups. The overall improvement of the ventilation efficiency index (VEI) was significantly greater in the PPVOLC group. Phospholipid and protein analyses of the bronchoalveolar lavage fluid showed significant alterations to surfactant homoeostasis in the PPVOLC group, whereas IL-10 and SP-C mRNA expression was tendentially increased in the surfactant groups.ConclusionThe different treatment strategies applied could be shown to improve gas exchange and lung function in neonatal ARDS. To which extent differences in maintenance of lung function and surfactant homeostasis may lead to long-term consequences needs to be studied further.

Surfactant Protein A Detection in Primary Pulmonary Adenocarcinoma without Bronchioloalveolar Pattern

Background: Immunohistochemical studies in human lung carcinoma reported positive staining of tumor cells for surfactant protein A (SP-A), especially in peripheral airway cell carcinoma, which include bronchioloalveolar carcinoma and in some reports also papillary subtypes. Objective: The purpose of this study was to determine the SP-A expression in tumor cells of lung adenocarcinoma without a bronchioloalveolar pattern, classified according to the WHO. Methods: In total, 169 primary adenocarcinomas of the lung (109 acinar, 32 solid with mucin, 24 papillary and 4 mucinous) were examined by immunohistochemistry for SP-A expression. Results: Twenty-five percent of acinar, 38% of papillary and 3% of solid adenocarcinoma with mucin showed a positive intracytoplasmic SP-A reaction of the tumor cells. None of the mucinous adenocarcinomas stained for SP-A. This study included the largest number of acinar adenocarcinomas and solid adenocarcinomas with mucin studied for SP-A. We clearly demonstrated that also primary lung adenocarcinoma without a bronchioloalveolar pattern can express SP-A. A positive staining of hyperplastic type II cells surrounding the tumors or entrapped in the tumor could clearly be differentiated from the SP-A-positive stain of tumor cells. Conclusion: These results support the theory that SP-A-producing cells may generate not only bronchioloalveolar and papillary carcinoma, but also other subtypes of lung adenocarcinoma.

Positive End-Expiratory Pressure Modifies Response to Recombinant and Natural Exogenous Surfactant in Ventilated Immature Newborn Rabbits

Background and Objectives: Different types of surfactant preparations were shown not to exert uniform response in preterm infants suffering from respiratory distress syndrome (RDS). Therefore, the effects of a recombinant surfactant protein C (rSP-C) based preparation and a natural surfactant were compared applying different levels of positive end-expiratory pressure (PEEP) in experimental RDS. Methods: Preterm rabbits (n = 7–14 per group; 27 days gestation; term 30 days) were randomized for receiving either 100 mg/kg rSP-C or natural bovine surfactant and were compared with saline treated controls. Animals were ventilated for 30 min with either 0.3 or 0 kPa PEEP at standardized tidal volumes and lung mechanics were measured as well as lung histology and mRNA expression of surfactant associated proteins B and C by real-time PCR. Results: The PEEP level applied (0.3 vs. 0 kPa) largely influenced dynamic compliance after administration of rSP-C surfactant (4.45 vs. 2.58 ml/kg), whereas natural surfactant improved compliance regardless of the PEEP applied (4.86 vs. 4.24 ml/kg) compared to controls (2.41 vs. 1.55 ml/kg). Accordingly, administration of PEEP significantly increased alveolar count in all groups as well as SP-C mRNA expression, whereas SP-B expression and protein content both remained unchanged. Conclusion: Response to rSP-C surfactant depends on the PEEP level applied in our model of neonatal RDS. These findings should be considered for the conception of clinical trials regarding treatment strategies in neonatal RDS.

Reactive alveolar epithelium in chondroid hamartoma of the lung.

OBJECTIVE To determine the morphologic characteristics of the nonciliated epithelium found in chondroid hamartoma of the lung. STUDY DESIGN The morphologic characteristics and immunohistochemical reaction for surfactant protein A of the nonciliated epithelium in chondroid hamartoma of the lung was studied by immunohistochemistry. Alveolar epithelium in normal lung tissue and lung tissue surrounding primary lung cancer or metastatic lung lesions was used as a control. RESULTS In all cases, the nonciliated epithelium in chondroid hamartoma showed the morphologic criteria of hyperplastic alveolar type II cells and a very strong positive surfactant protein A reaction in the cytoplasm when compared with alveolar epithelium of the normal lung. Similar hyperplastic type II cells were also found in the alveolar lung around metastatic or primary lung tumors. CONCLUSION These findings may indicate that the nonciliated cells found in chondroid hamartoma of the lung are hyperplastic type II cells. This suggests that the alveolar epithelium found in chondroid hamartoma of the lung is a secondary reaction around the hamartoma and not a primary component of the lesion.