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Ekkehard Vollmer

Publications and source records attributed to Ekkehard Vollmer.

At least 19 recordsLinked to original sources
PubMed2006

Enhanced molecular analyses by combination of the HOPE-technique and laser microdissection.

As part of an investigation aimed at illuminating the possibilities and limits of the HOPE-fixation and paraffin-embedding technique we here describe a novel procedure which was developed in order to combine the benefits of the HOPE-technique with the capabilities of laser microdissection. The presented procedure avoids the need for amplification of template-RNA thus facilitating reliable and reproducible results. The excellent preservation of nucleic acids, proteins, and morphology in HOPE-fixed, paraffin-embedded tissues enhances the molecular applications available to date with materials acquired by laser microdissection when compared to formalin fixed, paraffin-embedded tissues, thus substantially extending the methodological panel in tissue based research.

Journal Article↗
PubMed2006

High-throughput DNA methylation profiling using universal bead arrays.

We have developed a high-throughput method for analyzing the methylation status of hundreds of preselected genes simultaneously and have applied it to the discovery of methylation signatures that distinguish normal from cancer tissue samples. Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases.

Base Sequence↗
PubMed2006

A vicious circle of alveolar macrophages and fibroblasts perpetuates pulmonary fibrosis via CCL18.

RATIONALE: Recently, models of macrophage activation have been revised. Macrophages stimulated with Th2 cytokines have been classified as alternatively activated. OBJECTIVES: This article examines the expression and regulation of CC chemokine ligand 18 (CCL18), a marker of alternative activation, by human alveolar macrophages (AMs). METHODS: AM were obtained from bronchoalveolar lavage (BAL) fluid of patients with idiopathic pulmonary fibrosis, sarcoidosis, or hypersensitivity pneumonitis (n = 69) and healthy volunteers (n = 22). Expression of CCL18 was determined by quantitative reverse transcriptase-polymerase chain reaction, in situ hybridization, flow cytometry, and immunohistochemistry, respectively. MEASUREMENTS AND MAIN RESULTS: Spontaneous CCL18 production by BAL-derived cells was markedly increased in patients with pulmonary fibrosis and correlated negatively with pulmonary function test parameters. CCL18 gene expression and protein production were up-regulated in normal AMs after Th2 cytokine stimulation and/or coculture with human lung fibroblasts. Native collagen significantly up-regulated CCL18 expression in normal AMs activated with Th2 cytokines via a mechanism mediated by beta2-integrin/ scavenger receptor(s). Culture supernatants of AMs from patients with idiopathic pulmonary fibrosis increased collagen production by normal lung fibroblasts partly mediated via CCL18. CONCLUSIONS: Our findings suggest that AMs from patients with pulmonary fibrosis disclose a phenotype of alternative activation and might be a part of a positive feedback loop with lung fibroblasts perpetuating fibrotic processes.

Biopsy↗
PubMed2006

Pulmonary responses to overventilation in late multiple organ failure.

BACKGROUND: Patients with multiple organ failure (MOF) require mechanical ventilation for several days. The enormous significance of the ventilation strategy for the outcome of these patients is well appreciated. However, most studies have focused on the onset and the early phase of MOF. It was the aim of the current study to investigate the effect of ventilation in the course of MOF. METHODS: Using a model where mice develop MOF 7-14 days after intraperitoneal injection of zymosan, the authors analyzed lung functions, signaling pathways, and mediator release in response to protective ventilation (end-expiratory pressure -3 cm H2O; end-inspiratory pressure -10 cm H2O) and overventilation (-22.5 cm H2O) in isolated lungs ex vivo. RESULTS: On day 7, pulmonary compliance, pulmonary resistance, and tidal volume were normal, but vascular resistance was elevated compared with untreated animals. During ex vivo ventilation, these lungs showed enhanced nuclear factor-kappaB activation, Akt kinase phosphorylation, and cytokine release, and this was further aggravated by overventilation. After 14 days, zymosan-treated animals were characterized by pulmonary hypertension, reduced tidal volume, elevated pulmonary resistance, and increased mediator production. However, in these lungs, neither nuclear factor-kappaB activation nor cytokine production where enhanced by overventilation. CONCLUSIONS: The zymosan model is characterized by pulmonary inflammation, diminished lung functions, and chronic hypertension. Mechanical ventilation with high distending pressures further augmented cytokine production in this chronic model of MOF only if it significantly augmented tidal volume. The authors speculate that these findings may be explained on the basis of different degrees of lung stretch.

Animals↗
PubMed2006

Interleukin-18 expression by alveolar epithelial cells type II in tuberculosis and sarcoidosis.

The aims of the present study were to characterize the localization of interleukin-18 (IL-18) expression in lung tissue specimens from patients with pulmonary tuberculosis, sarcoidosis and controls, and to determine whether human alveolar epithelial cells type II (AEC-II) are able to generate IL-18 in primary culture. IL-18 was determined using semiquantitative reverse-transcription-PCR and localized in lungs using in situ hybridization. IL-18 protein levels were determined using the enzyme-linked immunosorbent assay and western blot analysis. Alveolar epithelial cells type II (AEC-II) were stimulated in vitro by proinflammatory cytokines, lipopolysaccharides, and whole cell lysate from Mycobacterium tuberculosis. IL-18 mRNA expression was significantly increased in the lungs affected by tuberculosis and sarcoidosis. In situ hybridization revealed different sites of expression in tuberculosis and sarcoidosis lungs, with AEC-II as one major source of IL-18 in the alveolar compartment. Basal IL-18 expression could be detected in normal AEC-II. Whole cell lysate from M. tuberculosis, but not lipopolysaccharide, led to a strong increase of IL-18 mRNA accumulation in AEC-II. Resting AEC-II secreted only small amounts of IL-18, but intracellular IL-18 protein levels increased in a time-dependent manner during culture. Proinflammatory cytokines tumour necrosis factor-alpha, IL-1beta, and interferon-gamma altered IL-18 mRNA expression and mature protein secretion of human AEC-II. These findings indicate a possible role for AEC-II and AEC-II-derived IL-18 in pathomechanisms of granulomatous lung diseases.

Cytokines↗
PubMed2005

CCR2 and CXCR3 agonistic chemokines are differently expressed and regulated in human alveolar epithelial cells type II.

The attraction of leukocytes from circulation to inflamed lungs depends on the activation of both the leukocytes and the resident cells within the lung. In this study we determined gene expression and secretion patterns for monocyte chemoattractant protein-1 (MCP-1/CCL2) and T-cell specific CXCR3 agonistic chemokines (Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11) in TNF-alpha-, IFN-gamma-, and IL-1beta-stimulated human alveolar epithelial cells type II (AEC-II). AEC-II constitutively expressed high level of CCL2 mRNA in vitro and in situ , and released CCL2 protein in vitro . Treatment of AEC-II with proinflammatory cytokines up-regulated both CCL2 mRNA expression and release of immunoreactive CCL2, whereas IFN-gamma had no effect on CCL2 release. In contrast, CXCR3 agonistic chemokines were not detected in freshly isolated AEC-II or in non-stimulated epithelial like cell line A549. IFN-gamma, alone or in combination with IL-1beta and TNF-alpha resulted in an increase in CXCL10, CXCL11, and CXCL9 mRNA expression and generation of CXCL10 protein by AEC-II or A549 cells. CXCL10 gene expression and secretion were induced in dose-dependent manner after cytokine-stimulation of AEC-II with an order of potency IFN-gamma > > IL-1beta > or = TNF-alpha. Additionally, we localized the CCL2 and CXCL10 mRNAs in human lung tissue explants by in situ hybridization, and demonstrated the selective effects of cytokines and dexamethasone on CCL2 and CXCL10 expression. These data suggest that the regulation of the CCL2 and CXCL10 expression exhibit significant differences in their mechanisms, and also demonstrate that the alveolar epithelium contributes to the cytokine milieu of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response.

Cells, Cultured↗
PubMed2005

Inter-laboratory validation of PCR-based detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues.

The present study is based on the initiative for quality assurance in pathology of the German Society of Pathology and the Professional Association of German Pathologists. Four panel laboratories with experience and expertise in polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis were selected to establish the prerequisites for continuous external laboratory trials, in particular, by providing pre-tested specimens and evaluation criteria for participating institutes. In the first step, the four panel laboratories performed an internal trial to test their own reliability and reproducibility. Paraffin sections and DNA preparations from 34 tissues (25 clinical specimens and 9 controls) totalling to 66 samples were evaluated by each panel institute according to their own protocols. The methodologies differed and are described in detail. Despite these differences, a high degree of inter-laboratory reliability was achieved. In this report, we summarise our results including the correlation with the histology and provide recommendations for applying PCR-based methodology for the detection of mycobacterial DNA in surgical specimens. Supplementary data are available online at http://www.charite.de/ch/patho (rubric "Forschung"). Pre-tested specimens are now available for the external trial and can be ordered from the steering institute via Oligene (http://www.oligene.com/). All molecular pathology laboratories are invited to participate in this quality assurance initiative.

Animals↗
PubMed2005

Human lung cancer cells express functionally active Toll-like receptor 9.

BACKGROUND: CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology. METHODS: The expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system. RESULTS: We found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis. CONCLUSIONS: Here we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology.

Biomarkers, Tumor↗
PubMed2005

Lung carcinoma-associated atypical adenomatoid hyperplasia, squamous cell dysplasia, and chromosome alterations in non-neoplastic bronchial mucosa.

This article analyzes phenotype and genotype alterations of the lung in association with lung cancer. The frequency of phenotype preneoplastic lesions (atypical adenomatoid hyperplasia (AAH) and squamous cell dysplasia (SCD)) was analyzed at distinct distances from the tumor boundary in 150 lung carcinomas. AAH was noted in 19/150 (13%) cases and more frequently seen in adeno carcinomas, squamous cell dysplasia was noted in 46/150 (31%) cases and more frequently seen in squamous cell carcinomas. The degree of cellular atypia decreased with increasing distance from tumor boundary in both AAH and SCD. At similar distances, genotype (chromosome) alterations of surrounding bronchial mucosa were studied in additional 55 primary and secondary lung tumors by karyotype analysis. Numerical chromosome aberrations occur frequently in primary lung carcinomas and adjacent bronchial mucosa, and affect at average 4.5/10 metaphases in primary lung cancer and 2/10 metaphases in metastases. Most abnormal metaphases were induced by chromosome losses, only few by additional copies, i.e. trisomy, etc. Losses of y chromosome were seen in both malignancy and adjacent bronchial mucosa, and interpreted as "tumor related", losses of chromosome 21 in adjacent bronchial mucosa were non-tumor related in adenocarcinoma and metastases, losses of chromosome 19 in adjacent bronchial mucosa occurred independently in squamous cell and large cell carcinomas. The data suggest the hypothesis that preneoplastic lesions in the lung might be partly induced by the tumor itself.

Aged↗
PubMed2005

Tissue microarrays from HOPE-fixed specimens allow for enhanced high throughput molecular analyses in paraffin-embedded material.

In this study, we describe a technique that allows for the production and the use of tissue microarrays (TMA) from HOPE-fixed, paraffin-embedded specimens. The combination of these two technologies unites the advantages of the high throughput aspects in TMA, with good preservation of nucleic acids, proteins, and morphology of HOPE-fixed tissues, thus substantially widening the capabilities presently available for molecular studies in paraffin-embedded tissues.

Biomarkers↗
PubMed2004

Alveolar epithelial cells type II are major target cells for C. pneumoniae in chronic but not in acute respiratory infection.

Pulmonary presence of Chlamydia pneumoniae is associated with acute and chronic infections. We show that unapparent chlamydial infection in four out of 31 chronic obstructive pulmonary disease (COPD) patients (12.9%) is characterized by a significant increase in infected alveolar epithelial cells type II (18.2 +/- 3.5% vs. 2.3 +/- 0.9; IHC/ISH) compared to a newly established model of acute chlamydial infection (ACIM) in vital lung specimens from pulmonary lobectomy. Expression of cHSP60 demonstrated pathogen viability and virulence in the ACIM. We conclude that target cells differ in acute and chronic chlamydial infection and suggest the ACIM as a novel tool to analyze the host-pathogen-interactions in acute respiratory infections.

Acute Disease↗
PubMed2004

The HOPE-technique permits Northern blot and microarray analyses in paraffin-embedded tissues.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

Blotting, Northern↗
PubMed2003

Phosphoinositide 3-OH kinase inhibition prevents ventilation-induced lung cell activation.

In acute respiratory distress syndrome patients, protective ventilation strategies reduce mortality and proinflammatory mediator levels. It has been suggested that some of the side effects of mechanical ventilation are caused by the excessive release of mediators capable of causing pulmonary inflammation and tissue destruction (biotrauma). Selective inhibition of this process might be used to minimize the side effects of artificial mechanical ventilation. This study was designed to identify the cell types and specific signaling mechanisms that are activated by ventilation with increased pressure/volume (overventilation). In isolated perfused mouse lungs, overventilation caused nuclear translocation of nuclear factor-kappaB (NF-kappaB) and enhanced expression of interleukin-6 mRNA in alveolar macrophages and alveolar epithelial type II cells. The phosphoinositide 3-OH kinase inhibitor Ly294002 prevented nuclear translocation of NF-kappaB and the subsequent release of interleukin-6 and macrophage inflammatory protein-2alpha in overventilated but not in endotoxic lungs. Similar results were obtained in rats in vivo, where Ly294002 prevented NF-kappaB activation by overventilation but not by endotoxin. These findings show that alveolar macrophages and alveolar epithelial type II cells contribute to the ventilation-induced release of proinflammatory mediators and that selective inhibition of this process is possible without inhibiting the activation of NF-kappaB by endotoxin.

Animals↗
PubMed2003

Stretch activates nitric oxide production in pulmonary vascular endothelial cells in situ.

Whereas endothelial responses to shear stress have been studied extensively, the responses to circumferential vascular stretch are yet poorly defined. Circumferential stretch in pulmonary microvessels is largely determined by the transmural pressure gradient, hence by both vascular perfusion and alveolar ventilation pressures. Here, we have studied the production of nitric oxide (NO) by the endothelial nitric oxide synthase (eNOS) in two different models of vascular stretch in the intact lung: In isolated-perfused rat lungs, vascular stretch was induced by elevation of vascular pressure. In situ digital fluorescence microscopy revealed stretch-dependent NO production, which was localized to capillary endothelial cells and inhibited by NOS blockers. In isolated-perfused mouse lungs, vascular stretch was generated by ventilation with elevated negative pressure. Stretch-induced phosphorylation of Akt and eNOS in lung endothelial cells was demonstrated by immunohistochemistry and increased NO production by in situ fluorescence microscopy. Stretch-induced endothelial responses in both models were abrogated by pretreatment with phosphatidylinositol-3-OH kinase inhibitors. These findings demonstrate that circumferential stretch activates NO production in pulmonary endothelial cells by a signaling cascade involving phosphatidylinositol-3-OH kinase, Akt, and eNOS and that this response is independent from the mechanical factors causing vascular distension.

Animals↗
PubMed2003

Toll-like receptor 2 is expressed by alveolar epithelial cells type II and macrophages in the human lung.

The ability of the host to recognize pulmonary invasion by pathogenic organisms and establish an appropriate host response to infection requires innate immune defense mechanisms. Early bacterial clearance in the lung is mediated by alveolar macrophages (AM) and polymorphonuclear neutrophils. Additionally alveolar epithelial cells type II (AEC-II) may act as immunoregulatory cells. The toll-like receptors (TLR) are part of this innate immune defense, recognizing conserved patterns on microorganisms. Toll-like receptor 2 (TLR2) is crucial in detecting components of gram-positive bacteria and mycobacteria. Signals initiated by the interaction of TLR2 with bacterial components direct the subsequent inflammatory response. The detection of TLR2 mRNA in human lung tissue prompted us to localize the expression of mRNA and protein at the cellular level using a novel method for tissue fixation. We utilized HOPE-fixed lung specimen sections for targeting mRNA by in situ hybridization and protein by immunohistochemistry using the monoclonal antibody TL2.1. In normal lung areas the expression of TLR2 mRNA and protein was found to be located in cells resembling AEC-II and AM. Expression of mRNA was verified by RT-PCR and DNA sequencing. These results indicate a potential mechanism of increased immunosurveillance at the alveolar level controlling the localized infection.

Epithelium↗
PubMed2003

Atypical adenomatous hyperplasia of lung: its incidence and analysis of clinical, glycohistochemical and structural features including newly defined growth regulators and vascularization.

BACKGROUND: Adenomatous hyperplasia of the peripheral lung has been suggested to be a preneoplastic lesion leading to peripherally localized lung carcinomas. The paucity of data about cellular and vascular characteristics of this lesion in comparison to normal lung prompted this investigation. MATERIAL AND METHODS: We describe results of two investigations comprising 75 cases and 70 cases, respectively, with atypical adenomatous hyperplasia (AAH) of the lung, respectively: (a) a prospective study part with thorough analysis of surgical lung specimens (lobes and lungs) for light microscopical detection of the lesion; and (b) a retrospective study part with immuno- and lectin histochemical analysis of AAH and non-neoplastic lung parenchyma monitoring expression of growth-related markers and changes in vascularization patterns. Sections of the individual cases were examined by an image-analyzing system including automated measurement of staining intensities and structure analysis. RESULTS: The prospective study part revealed an incidence of AAH in 2/31 cases with squamous cell carcinoma and in 5/32 cases with adenocarcinomas. No relation to pT- or pN stages was detectable, high grade AAHs were seen to be close to the tumor lesions (<2 cm distance) and those with low grade at greater distances. Statistically significantly increased levels of expression of anti-apoptotic bcl-2, macrophage migration inhibitory factor (MIF) capable to suppress p53 activities, heparin-binding lectin, interleukin-2, galectin-1 and of binding sites for the endogenous lectins galectins-1, -3 and -7 were determined. In addition, alveolar-lining cells, which express these markers, formed spatial clusters, which harbor different levels of structural entropy. AAH displayed an increased level of vascularization characterized by regular size and increased number of newly formed vessels. INTERPRETATION: The prospective and retrospective study parts point to a close association of AAH with peripherally localized adenocarcinoma of the lung. AAH is characterized by pronounced alteration of expression of several growth-related markers and probably non-reversible changes in vascularization.

Adenocarcinoma↗
PubMed2003

HOPE fixation of cytospin preparations of human cells for in situ hybridization and immunocytochemistry.

In primary or cultured cells, in situ hybridization (ISH) or immunocytochemistry (ICC) is often performed on tissue that has been fixed by paraformaldehyde or Carnoy's. Recently we reported an optimized HOPE (HEPES-glutamic acid buffer-mediated organic solvent protection effect) fixation protocol for ISH targeting mRNA in lung tissues. We have now examined whether HOPE fixation could also be used on in vitro cultured cells for targeting mRNA by ISH or proteins by ICC on cytospin preparations. Using the myeloid stem cell line KG-1a as a model system, we showed that HOPE fixation can be applied for ISH and ICC on cultured cells. HOPE can be used with cells and tissues and with a broad spectrum of immunohistocytochemical and molecular techniques.

Buffers↗