Annette M. Müller

HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues.

We have developed a novel method for tissue fixation, including subsequent paraffin-embedding and sectioning, that allows the complete pathological analysis of all types of human soft tissues. Furthermore, it maintains additional positive features relevant to immunohistochemistry and molecular pathology. The so-called HOPE-technique (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) comprises a protection-solution with an organic buffer, acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature. Although the exact mechanism of protection has still to be elucidated, it seems rather unlikely that chemical bindings occur during the whole process of fixation, which is described and compared with the standard formalin-paraffin technique. Essentially, HOPE-fixed sections show formalin-like morphology. However, the sections are somewhat difficult to handle because of their fragility. This is due to the absence of any type of protein cross-linking and the dynamic processes of immersion and outflow of the HOPE protection solution. HOPE-fixed sections provide an excellent preservation of proteins and antigenic structures for differential analysis by immunohistochemical and/or enzyme histochemical techniques. However, their most remarkable feature is the extremely low degradation of nucleic acids (DNA and RNA) combined with good results obtained by in situ hybridization techniques. In conclusion, HOPE fixation may become a valuable additional tool in modern pathology.

Expression of BLIMP1/PRMT5 and concurrent histone H2A/H4 arginine 3 dimethylation in fetal germ cells, CIS/IGCNU and germ cell tumors

BackgroundMost testicular germ cell tumors arise from intratubular germ cell neoplasia unclassified (IGCNU, also referred to as carcinoma in situ), which is thought to originate from a transformed primordial germ cell (PGC)/gonocyte, the fetal germ cell. Analyses of the molecular profile of IGCNU and seminoma show similarities to the expression profile of fetal germ cells/gonocytes. In murine PGCs, expression and interaction of Blimp1 and Prmt5 results in arginine 3 dimethylation of histone H2A and H4. This imposes epigenetic modifications leading to transcriptional repression in mouse PGCs enabling them to escape the somatic differentiation program during migration, while expressing markers of pluripotency.ResultsIn the present study, we show that BLIMP1 and PRMT5 were expressed and arginine dimethylation of histones H2A and H4 was detected in human male gonocytes at weeks 12–19 of gestation, indicating a role of this mechanism in human fetal germ cell development as well. Moreover, BLIMP1/PRMT5 and histone H2A and H4 arginine 3 dimethylation was present in IGCNU and most seminomas, while downregulated in embryonal carcinoma (EC) and other nonseminomatous tumors.ConclusionThese data reveal similarities in marker expression and histone modification between murine and human PGCs. Moreover, we speculate that the histone H2A and H4 arginine 3 dimethylation might be the mechanism by which IGCNU and seminoma maintain the undifferentiated state while loss of these histone modifications leads to somatic differentiation observed in nonseminomatous tumors.

Expression of von Willebrand factor by human pulmonary endothelial cells in vivo

Background and Objectives: Whether von Willebrand factor (vWf) is variably expressed by endothelial cells (EC) of the human vascular tree or not is not known. Studies on animals showed that the varying degrees of vWf expression in pulmonary vessels was a reflection of EC heterogeneity. Neither the influence of age or sex nor that of pathophysiological factors such as pulmonary hypertension (PH) on vWf expression has been systematically analysed up to now. However, such information is essential for the design and delivery of site-selective drugs and genes as well as for the analysis of EC culture systems. Methods: The variable degrees of vWf expression in lung tissue specimens from 64 patients (age: 6 weeks to 86 years) with and without PH were studied immunohistochemically and analysed statistically. Results: CD31-specific antibody was used as a control stain for EC. It produced equally strong staining reactions in all pulmonary EC. In contrast, vWf-specific antibody yielded negative or weakly positive staining reactions in capillary EC. The staining intensities increased hand in hand with vessel calibres. Besides, they increased statistically significantly with age and PH. Sex had no influence on vWf expression. Conclusion: These findings show that (1) vWf expression by pulmonary EC is heterogeneous and specific for individual types of vessels, (2) age and PH enhance vWf expression, (3) vWf expression is indicative of altered EC activation but not of EC defects, and (4) elevated plasma levels of vWf correlate positively with raised coagulatory activities in older patients and in patients with PH.

Correlation of age with in vivo expression of endothelial markers

The importance of endothelial senescence as a pathogenetic factor in age-related vascular alterations has almost exclusively been studied in vitro. However, the in vitro-findings have rarely been compared with histomorphological changes in aging human tissue or in age-related degenerative diseases. Therefore, we compared the expression of the endothelial marker CD34 and the procoagulant protein von Willebrand factor (vWf) in lung endothelium by conventional immunohistochemistry and confocal laser scan microscopy (CLSM), taking the age of the patients into consideration. The staining reactions were statistically analysed by covariance analysis. With age the endothelial staining intensity of CD34 increased in arteries and veins, but decreased in arterioles, capillaries and venules. For vWf, on the contrary, the endothelial staining intensity increased with age in all types of vessels. CLSM confirmed a mosaic staining pattern. This study demonstrates age-associated phenotypical alterations of CD34 and vWf. Whether the down-regulation of CD34 correlates with an age-associated reduction of the angiogenetic properties of EC or an age-related over-expression of vWf as a relevant cofactor for the raised coagulatory activity and the increase in thrombotic diseases resp coronary heart disease in older patients, remains subject to debate.

Fetal transesophageal echocardiography: clinical introduction as a monitoring tool during cardiac intervention in a human fetus.

Because of insufficient imaging by maternal transabdominal fetal echocardiography (TAE) in a human fetus with aortic atresia, imperforate atrial septum and progressive cardiac failure, we assessed the feasibility of fetal transesophageal echocardiography (TEE) as a monitoring tool during fetal cardiac intervention at 24 + 6 weeks of gestation. Percutaneous fetoscopic intraesophageal deployment of the ultrasound catheter was achieved and did not result in any maternal or fetal complications. Fetal TEE permitted substantially clearer definition of fetal cardiac anatomy and intracardiac device manipulations than conventional maternal TAE. Despite the employment of various devices, no sufficiently large opening could be achieved within the atrial septum. Although the fetus tolerated the procedure remarkably well and satisfactory fetoplacental flow could be documented at the end of the procedure, the fetus died from progressive cardiac failure 3 days after the intervention. Fetoscopic TEE is feasible in the human fetus and permits substantially clearer definition of fetal cardiac anatomy and intracardiac manipulations than conventional maternal TAE. Based on the observation of spontaneous closure of multiple iatrogenic perforations of the atrial septum, specialized devices are required in order to improve the technical success rate of septoplasty methods and hence the survival odds of these high‐risk patients. Copyright

Endothelial VE-cadherin expression in human lungs

Cell adhesion molecule vascular endothelial cadherin (VE-cadherin) is the major component of endothelial adherence junctions, maintaining endothelial cell integrity. Studies dealing with constitutive VE-cadherin expression patterns in different pulmonary vessel types (arteries, arterioles, capillaries, venules, veins) or with the influence of physiological factors such as age or sex on VE-cadherin expression have not been published yet. Knowledge of constitutive resp. varying expression patterns not only fundamentally contribute to understanding the role of VE-cadherin in the pathogenesis of pulmonary diseases but also help to develop therapies based on immunotargeting. Hence, endothelial VE-cadherin expression was studied in regular lung tissue. Fifty-eight specimens of regular lung tissue (30 females, 28 males between 1 month and 75 years old) were immunohistochemically stained with an antibody against VE-cadherin. There was strong endothelial expression of VE-cadherin in arteries, arterioles, and capillaries but almost no expression in veins and venules. Neither age nor sex had any influence on the expression pattern or staining intensity. There is a vessel type-specific expression pattern for VE-cadherin in regular human lung tissue, which is not influenced by age or sex. Further studies will have to prove whether this is influenced by pathological conditions, e.g., ARDS.

Prenatal Diagnosis and Evaluation of Sonographic Predictors for Intervention and Adverse Outcome in Congenital Pulmonary Airway Malformation

Objective To describe antenatal findings and evaluate prenatal risk parameters for adverse outcome or need for intervention in fetuses with congenital pulmonary airway malformation (CPAM). Methods In our retrospective study all fetuses with a prenatal diagnosis of CPAM detected in our tertiary referral center between 2002 and 2013 were analyzed. Sonographic findings were noted and measurements of mass-to-thorax-ratio (MTR), congenital pulmonary airway malformation volume-ratio (CVR) and observed to expected lung-to head-ratio (o/e LHR) were conducted and correlated to fetal or neonatal morbidity and mortality and/or need for prenatal intervention. Results 67 fetuses with CPAM were included in the study. Hydropic fetuses were observed in 16.4% (11/67) of cases, prenatal intervention was undertaken in 9 cases; 7 pregnancies were terminated. The survival rate of non-hydropic fetuses with conservatively managed CPAM was 98.0% (50/51), the survival rate for hydropic fetuses with intention to treat was 42.9% (3/7). 10 (18.2%) children needed respiratory assistance. Fetuses with a CVR of <0.91 were significantly less likely to experience adverse outcome or need for prenatal intervention with sensitivity, specificity and positive/negative predictive value of 0.89, 0.71, 0.62 and 0.93, respectively. A MTR (mass-to-thorax-ratio) of < 0.51 had a positive predictive value of 0.54 and a negative predictive value of 0.96 of adverse events with a sensitivity of 0.95 and a specificity of 0.63. The negative predictive value for o/e LHR of 45% was 0.84 with sensitivity, specificity and positive predictive value of 0.73, 0.68 and 0.52, respectively. Conclusions The majority of cases with CPAM have a favorable outcome. MTR and CVR are able to identify fetuses at risk, the o/e LHR is less sensitive.

Bronchopulmonary sequestration with massive pleural effusion: pleuroamniotic shunting vs intrafetal vascular laser ablation

To assess the incidence of complications among a relatively large cohort of fetuses with bronchopulmonary sequestration (BPS) and the success of two different intrauterine treatment modalities.

E-cadherin, E-selectin and vascular cell adhesion molecule: immunohistochemical markers for differentiation between mesothelioma and metastatic pulmonary adenocarcinoma?

Abstract. Pleural mesotheliomas, especially pure epithelioid mesotheliomas, may histologically be easily confused with peripheral pulmonary adenocarcinomas or pleural carcinosis. As there is no specific antibody for mesotheliomas, today a panel of immunohistochemical markers is used for the differential diagnosis of these two tumour entities. In search of further significant antibodies for application onto formalin-fixed, paraffin-embedded tissue, we immunohistochemically investigated the expression pattern of three adhesion molecules: vascular cell adhesion molecule (VCAM), E-selectin and E-cadherin. A comparatively large number of 44 mesotheliomas (15 epithelioid, 15 biphasic, 14 sarcomatoid) and 18 peripheral pulmonary adenocarcinomas were analysed. While for these two tumour entities there were no significant differences of the staining patterns for VCAM and E-selectin, there were significant differences in the expression of E-cadherin: while nearly all adenocarcinomas stained positively, there was almost no staining reaction of the mesotheliomas. Therefore, E-cadherin – in contrast to E-selectin and VCAM – appears to be a further relevant immunohistochemical marker for the distinction between adenocarcinomas and mesotheliomas.

Expression of CD34 in Pulmonary Endothelial Cells in vivo1

Objectives: The morphological phenotype of endothelial cells (EC) is specific for individual types of vessels. There are, however, no studies on the phenotypical diversity of EC in human lung tissue. The influences exerted by physiological factors (e.g. age, sex) or pathological factors (e.g. pulmonary hypertension) on EC have not been ascertained up to now. Methods: In order to determine the influence of pulmonary hypertension (PH), age and sex on EC, we localized the expression of the endothelial marker CD34 immunohistologically by light microscopy and laser scanning microscopy in lung tissue specimens from children and adults. Results: Capillary EC showed a stronger staining reaction than EC in arteries, veins, arterioles or venules. The staining intensity increased with age in veins and arteries and decreased with age in venules, arterioles and capillaries. Sex exerted no statistically significant influence. CD34 was generally more strongly expressed in specimens with PH than in those without. Conclusion: The study demonstrates for the first time that (1) CD34 is heterogeneously expressed by human pulmonary EC, and (2) the physiological/pathophysiological factors age/PH influence CD34 expression. Hence a correlation between CD34 expression and its role as adhesion molecule and a link between CD34 expression and maturation are subject of discussion.