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Biomedical subjects

E Vollmer

Publications and source records attributed to E Vollmer.

At least 19 recordsLinked to original sources
PubMed2006

Characterisation of guinea pig precision-cut lung slices: comparison with human tissues.

Precision-cut lung slices (PCLS) allow comparison of the airway responses of different species under identical experimental conditions. The aim of this study was to establish and characterise PCLS from guinea pigs (GPs) and to compare them with human PCLS. GP PCLS were prepared according to previously published procedures with the exception that the agarose solution and the initial incubation medium contained isoproterenol to avoid post mortem airway contraction. The median effective concentrations (EC50, expressed as nM) for agonist-induced bronchoconstriction in GP and human PCLS, respectively, were: leukotriene D4 (1.8, 5.0); thromboxane (16, 1.3); serotonin (69, unresponsive); histamine (217, 2,170); and methacholine (231, 234). Allergen-induced bronchoconstriction of passively sensitised PCLS was attenuated by histamine or thromboxane-prostanoid receptor antagonists and was almost completely prevented by their combination with leukotriene receptor antagonists. Airways pre-contracted with methacholine were relaxed by the beta-agonist salbutamol or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Simultaneous studies of airways and vessels are possible with, for example, EC50 values for endothelin-1 of 37 nM (pulmonary arteries), 10 nM (pulmonary veins) and 9.6 nM (airway). When compared with previous findings in rat and mouse, these data show that guinea pig lungs are a more appropriate model for human airway pharmacology than lungs from rats or mice.

Animals↗
PubMed2006

Real-time PCR assay for improved detection of Mycobacterium tuberculosis complex in paraffin-embedded tissues.

OBJECTIVE: To test the usefulness of a commercially available real-time polymerase chain reaction (PCR) kit for the detection of Mycobacterium tuberculosis complex (MTBC) in formalin-fixed, paraffin-embedded tissues. RESULTS: The examination of 24 specimens of patients with a final diagnosis of TB shows that the real-time PCR assay exhibits a higher sensitivity (66.7%) for the detection of MTBC DNA than an alternative in-house IS6110 PCR (33.3%), whereas staining detected acid-fast bacilli in only two cases (8.3%). CONCLUSION: The real-time PCR assay provides a highly sensitive and specific means for the detection of MTBC DNA in histopathological specimens.

Biopsy↗
PubMed2006

The HOPE technique opens up a multitude of new possibilities in pathology.

Fixation of tissues with formalin results in well-preserved morphology but to a high degree leads to degradation of nucleic acids, which substantially constricts the spectrum of applicable molecular techniques. The novel HOPE-fixative with subsequent paraffin embedding, as an alternative to formalin, has been shown to result in a morphological preservation comparable to formalin-fixed, paraffin-embedded specimens. Due to a similar workflow like in formalin-fixation and paraffin embedding, the HOPE technique can be successfully established within any pathological institute. We have shown that DNA, RNA and proteins are protected in HOPE-fixed, paraffin-embedded tissues for at least eight years. Moreover, we described procedures which permit successful application of all common molecular techniques such as in situ hybridization targeting either DNA or RNA, immunohistochemistry without antigen retrieval and for formalin-refractory antigens, PCR, RT-PCR, Western blot, Northern blot, and transcription microarrays to HOPE-fixed, paraffin-embedded tissues. Furthermore, HOPE-fixed tissues can be used for the construction of tissue microarrays for enhanced high-throughput analyses on the molecular level. Using the HOPE technique as its crucial methodological base, ex vivo model systems could be established, e.g. for the simulation of early events in human infections and detection of chemotherapy resistances in human cancer. In addition to tissues, cell-culture preparations have been prepared utilizing the HOPE technique, which were then successfully applied to in situ hybridization targeting mRNA or immunocytochemistry with excellent preservation of morphological details. Taken together, the HOPE technique to date represents an alternative fixation that is, in contrary to other procedures, scientifically broadly analyzed. Therefore new possibilities are opened up especially within the rapidly growing field of molecular pathology.

Fixatives↗
PubMed2005

Fatal outcome of a hyperinfection syndrome despite successful eradication of Strongyloides with subcutaneous ivermectin.

We report the case of a 77-year-old man who developed a Strongyloides hyperinfection syndrome following immunosuppressive therapy more than 60 years after he moved away from an area endemic for Strongyloides stercoralis. Successful eradication of the nematode was achieved with an off label subcutaneous formulation of ivermectin. However, the patient subsequently died from acute respiratory distress syndrome (ARDS). Despite a high wormload in the stool and sputum of the patient and delayed infection control measures in the hospital, testing of the medical staff revealed a very low risk of Strongyloides transmission among healthcare workers.

Aged↗
PubMed2005

Improved detection of mycobacterial DNA by PCR in formalin-fixed, paraffin-embedded tissues using thin sections.

PCR is a unique methodology allowing for the sensitive detection of mycobacterial DNA-sequences in cases in which no fresh material can be obtained for classic analyses. Despite the limitations of this technique, for example the less satisfactory quality of DNA from paraffin-embedded specimens and the high effort necessary to control contamination, PCR still represents a useful additional tool for routine diagnostic examinations of mycobacterial infections. Fragmentation of the DNA extracted from formalin-fixed, paraffin-embedded samples on the one hand and the rigid cell wall of mycobacteria on the other hand are obstacles to detecting the DNA of these microorganisms by PCR. Here, we describe a simple mechanical procedure that allows us to improve the detection of mycobacterial DNA with the use of thin (1 microm) sections instead of thicker sections. This could be explained by a gentle, mechanical opening of the acid fast mycobacterial cell wall. Thus, even the application of heat/cold shock treatments is not necessary. This inexpensive fast procedure can also be used for the detection of other infectious agents.

DNA, Bacterial↗
PubMed2005

Phenotype and genotype associations of lung carcinoma with atypical adenomatoid hyperplasia, squamous cell dysplasia, and chromosome alterations in non-neoplastic bronchial mucosa.

The frequency of preneoplastic lesions of the lung and bronchial mucosa as well as potential genotype alterations in spatial relationship to pulmonary malignancies still need intensive investigations in order to understand the occurrence and manifestation of lung cancer in detail. To investigate the contemporary manifestation of lung cancer precursor lesions, peripheral (non-neoplastic) lung parenchyma and bronchial mucosa of operated lung carcinomas were analyzed at distinct distances (1, 2, 3, and 4 cm) from the tumor boundary for pre-neoplastic lesions--atypical adenomatoid hyperplasia (AAH) and squamous cell dysplasia (SCD), in 150 surgical specimens. Short-term tissue cultures of additional 55 primary and secondary lung tumors and their surrounding non-neoplastic bronchial mucosa were performed at the same distances in order to search for chromosome alterations, i.e. genotype aberrations. In phenotype observations, atypical adenomatoid hyperplasia was noted in 19/150 (13%) cases, and squamous cell dysplasia in 46/150 (31%) cases. The degree of cellular atypia decreased with increasing distance from the tumor boundary in both AAH and SCM. AAH was observed more frequently in adenocarcinomas, SCQ more frequently in squamous cell carcinomas. In genotype observations, the average number of abnormal metaphases measured 4.5/10 high power fields (HPF) in primary lung carcinomas, and only 2/10 in metastases. Data indicate that the so-called preneoplastic lesions in the lung are not completely tumor-precursor lesions, but, in addition, induced by the tumor itself.

Bronchi↗
PubMed2004

HOPE technique enables Western blot analysis from paraffin-embedded tissues.

In contrast to the spectrum of biochemical analyses of fresh material, that of archived specimens is widely restricted. Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. In this study, we investigated whether HOPE-fixed tissue can be analyzed by Western blotting. Furthermore, a comparison with conventionally fixed and frozen material was made. The specimens used were tumor-free and obtained from lobectomies for lung cancer. All four antibodies tested, i.e., antibodies specific for focal adhesion kinase, surfactant protein A, PI-3-kinase, and IKKalpha, worked well if used for immunoblotting of HOPE-fixed and frozen tissue. By contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Our findings show that HOPE fixation maintains the antigenicity of proteins better than formalin fixation. The possibility for performing Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues.

Blotting, Western↗
PubMed2004

[Transcriptional activity of surfactant-apoproteins A1 and A2 in non small cell lung carcinomas and tumor-free lung tissues].

UNLABELLED: Objective of this study was to investigate the balance of surfactant-apoprotein A1 and A2 (SP-A1 and SP-A2) at transcriptional level in tumor and tumor-free lung-tissues of patients with non-small-cell lung-carcinomas. MATERIALS AND METHODS: Nonfixed snap-frozen tumor and tumor-free sections of lungs, excised from 21 patients with adenocarcinomas of the lung and 6 patients with squamous cell carcinomas (control group) were analyzed. This was accomplished by RT-PCR using a SP-A1/SP-A2 consensus sequence, followed by enzymatic restriction. RESULTS: Tumor-containing tissues of adenocarcinomas showed higher SP-A1/SP-A2 ratios than the tumor free tissues. There was a significant difference in expression of SP-A1/SP-A2-mRNA in intra-individual tumor and tumor-free lung-tissues of adenocarcinoma patients (p = 0,05). In cases of squamous cell carcinomas this was not observed. CONCLUSIONS: A novel method to investigate the transcriptional activity of surfactant-apoproteins A1 and A2 in snap-frozen lung tissues was established. It was shown, that adenocarcinoma-tissues display higher SP-A1/SP-A2 than the corresponding tumor-free tissues and that the variation of SP-A-mRNA expression rises in cases of higher tumor-grading.

Adenocarcinoma↗
PubMed2004

[Bronchological bioptic diagnosis of lung cancer -- cytology and/or histology?].

INTRODUCTION: Bronchial carcinomas are commonly diagnosed through histological analysis of forceps biopsies. The use of flexible bronchoscopic biopsy techniques has led to an increase in the number of cytological investigations. The present study has the aim to assess the diagnostic efficiency of histology, cytology and their combined use. METHOD: In a retrospective analysis, 3630 cytological and/or histological samples that had been obtained bronchoscopically between 1/97 and 12/03 prior to surgery in 1436 patients, who were operated due to bronchial carcinoma, were compared to corresponding histological findings in the resected material. RESULTS: In 1888 preoperatively analysed areas the resected material ultimately allowed the detection of malignant lesions. Among these cases, histologic analysis of forceps biopsies yielded 399, cytological analysis 728, and their combined use 801 correct diagnoses prior to surgery. The combination of both procedures led to an increase of sensitivity by 16 % and 13.0 % compared to histology and cytology, respectively, as separate procedures. Histologically there were 149 cases, cytologically 1061 cases, and in combination 1040 cases that were falsely diagnosed as negative prior to surgery. The use of histology and/or cytology allowed to correctly detect 1250 areas of the resected material as free of malignant lesions. Specificity was 99.8 % for both methods. CONCLUSION: Currently bronchial carcinomas are often diagnosed preoperatively by minimally invasive cytological procedures. The present results demonstrate cytology and histology to be procedures that are complementary to each other. Their combined use offered the greatest diagnostic yield and therefore should be considered standard in the diagnosis of bronchial carcinoma.

Biopsy↗
PubMed2003

G-CSF application in patients with severe bacterial pneumonia increases IL-10 expression in neutrophils.

In severe pneumonia, the application of granulocyte-colony stimulating factor (G-CSF) was associated with reduced complications possibly by an induction of anti-inflammatory cytokines. It is not clear, whether G-CSF induces interleukin-10 (IL-10) synthesis in neutrophils. In a randomized study, 15 patients with severe community acquired pneumonia were treated either by a single dose of G-CSF and antibiotic therapy (n=8) or antibiotics alone (n=7). Messenger ribonucleic acid (mRNA) expression of IL-10 and tumor necrosis factor alpha of peripheral blood leukocytes was measured using in-situ hybridization (ISH) and reverse-transcription-polymerase-chain-reaction (RT-PCR). In addition, the cytokine release of lipopolysaccharide (LPS)-stimulated whole blood was measured by ELISA. We detected increased IL-10 mRNA by ISH (140 +/- 8% vs. -11 +/- 5%, P<0.01) and RT-PCR (126 +/- 16% vs. -28 +/- 3%, P<0.01) in the G-CSF-treated group only. In contrast, LPS-stimulated whole blood cells in vitro released significantly less IL-10 compared to the control group (-38.2 +/- 97 vs. -14.8 +/- 6 pg/ml, P<0.02). There was no significant effect on IL-10 serum protein levels and the TNF-alpha release and expression. IL-10 mRNA was detected predominantly in cluster designation 66b (CD66b) positive nucleated blood cells indicating that polymorphonuclear leukocytes are the main source of IL-10 expression after G-CSF stimulation. G-CSF induces transcription of IL-10 mRNA in neutrophils without increased release. This may be due to posttranscriptional effects.

Analysis of Variance↗
PubMed2003

HOPE-fixation enables improved PCR-based detection and differentiation of Mycobacterium tuberculosis complex in paraffin-embedded tissues.

Standard PCR-based detection of mycobacterial DNA in paraffin-embedded specimens may lack sufficient sensitivity because of the degradation of nucleic acids caused by routinely used formalin fixation. Therefore, we set up an approach that aimed at improving the results by applying the novel HOPE-fixative in PCR-detection of mycobacteria in paraffin-embedded tissues. Comparison of PCR-results using DNA extracted from either HOPE- or formalin-fixed specimens in BCG-infected SCID-mice revealed a more than 100fold enhanced sensitivity for the HOPE-fixed material. Owing to the preservation of DNA from degradation in HOPE-fixed tissues, even differentiation within the M. tuberculosis complex was possible by spoligotyping. We therefore conclude that the HOPE-fixative is a useful tool for molecular pathology that enhances the sensitivity of PCR-based methods for the detection of pathogens in paraffin-embedded tissues compared to formalin-fixation. Owing to the better preserved DNA, improved differentiation of mycobacteria from archived materials is possible. These results promise new and a substantially wider range of possibilities in the field of molecular pathology.

Animals↗
PubMed2003

The early allergic response in small airways of human precision-cut lung slices.

To study the role of small airways in the early allergic response (EAR), the method of human precision-cut lung slices (PCLS) was developed and used to examine the bronchoconstriction elicited by passive sensitisation and allergen provocation. Viable human PCLS of 250-microm thickness containing airways <1.5 mm in outer diameter were prepared from lung lobes obtained from lung resection and taken into culture. According to the low release of lactate dehydrogenase and the constant ciliary beat frequency, human PCLS were viable for at least 3 days. Following overnight passive sensitisation with serum from allergic individuals, administration of grass-pollen extract or activating immunoglobulin E antibody resulted in immediate airway contraction that was quantified by videomicroscopy. The extent of the EAR increased with decreasing airway size (outer airway diameter), with the strongest response occurring in the terminal bronchioles. Histamine receptor antagonism was ineffective, and leukotriene or thromboxane receptor antagonism attenuated the early allergic response only in some cases. However, simultaneous blockade of leukotriene and thromboxane receptors almost completely prevented the early allergic response in the precision-cut lung slices from all individuals, suggesting such a dual treatment as a potential future asthma therapy.

Acetates↗
PubMed2003

Alveolar macrophages are the main source for tumour necrosis factor-alpha in patients with sarcoidosis.

Tumour necrosis factor (TNF)-alpha is known to play a major role in the formation of noncaseating granuloma, a hallmark of sarcoidosis. The main cellular source in situ is still ambiguous. Serial sections of transbronchial biopsies from 14 patients with and 12 without sarcoidosis were studied, using immunohistochemistry (IHC), for TNF-alpha, T-cells (CD3), macrophages (CD68), and epithelial cells (MNF116). TNF-alpha spontaneously released (sr-TNF-alpha) by freshly isolated bronchoalveolar lavage cells, isolated from the same patients and cultured without any stimulus over a 24-h period was measured using an enzyme-linked immunosorbent assay. IHC revealed colocalisation of TNF-alpha with CD68 cells only. Cases with TNF-alpha tissue immunoreactivity exhibited higher sr-TNF-alpha (1,667 +/- 504 pg x mL(-1)) than cases without tissue immunoreactivity (211 +/- 60 pg x mL(-1)). In an explorative approach, a subgroup of patients could be identified and characterised by the presence of alveolar macrophage aggregates. It was found that sr-TNF-alpha was highest in this subgroup (2,700 +/- 769 pg x mL(-1)) compared with patients with normal histology (221 +/- 61 pg x mL(-1)) or with prominent granuloma (460 +/- 137 pg x mL(-1)), whereas in most clinical parameters this subgroup was intermediate. The findings from this study strongly corroborate the view that alveolar macrophages are the main cellular source for tumour necrosis factor-alpha in the initial phase of sarcoidosis. The authors suggest that in these patients, aggregates of alveolar macrophages may represent at least predecessors to granulomas if not granulomas in statu nascendi.

Adult↗
PubMed2002

[Small bowel obturation ileus related to ingestion of a medical device].

We report the case of a 50-year-old woman who was admitted to the hospital with an acute abdomen and underwent exploratory laparotomy. Intraoperatively, we suspected Crohn's disease of the ileocecal valve and decided to treat conservatively. However, in the further clinical course the patient became severely sick and developed a necrotizing ileocecal inflammation in combination with multiple perforations, 4-quadrants peritonitis and several other complications. Histopathology revealed an unspecific inflammation of the ileocecal valve. Furthermore, numerous little cellulose sponges were found in the resected intestinal segment. They were obstructing the lumen and were identical with the contents of an appetite suppressing medical device. A connection between the ingestion of this medical device and the penetrating inflammation of the ileocecal valve has to be suspected.

Administration, Oral↗
PubMed2002

Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues.

We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.

Cells, Cultured↗
PubMed2001

Cost-effective gel documentation using a web-cam.

In search for a cost effective gel documentation system applicable for different fields of molecular biology, we analyzed the capabilities of a cheap CCD-camera originally designed to capture images for transmission through the internet (web-cam) with regard to gel documentation. The camera was connected to a personal computer by universal serial bus (USB) and used for the documentation of DNA separated on agarose gels and stained by ethidium-bromide using the software provided with the camera. The web-cam provided digital images of sufficient quality for routine documentation and combined the low set-up costs of a Polaroid system with the low running costs of video capture systems, hence is ideal as a start-up system and as augmentation to existing equipment.

Computers↗