In a growing number of laboratories the technique of liquid chromatography-tandem mass spectrometry is used for the quantification of cyclosporin A in whole blood, employing cyclosporin D as the internal standard. Cyclosporin A is extensively metabolized in vivo; in liquid chromatography-tandem mass spectrometry respective metabolites can give rise to both parent and product ions that are isobaric with ions commonly used for the detection of cyclosporin A and cyclosporin D, respectively. In this article it is demonstrated that limited chromatography with co-elution of such metabolites together with cyclosporin A and cyclosporin D can lead to incorrect results.

A convenient liquid chromatography-tandem mass spectrometry method for the quantification of the triazole antifungal agent voriconazole in plasma samples is described. Fenbuconazole is used as an internal standard. After protein precipitation, automated solid-phase extraction is applied. Electrospray ionization in the positive mode is used and the following mass transitions are recorded: voriconazole, 350 -> 127; and fenbuconazol, 337 -> 125. The analytical run time is 4 min. The response was linear from 78 to 5000 mu g/L. The total coefficient of variation (n=16) was 12.6% for a low-concentration pool (143 mu g/L), 4.7% for a medium-concentration pool (419 mu g/L), and 5.0% for a high-concentration pool (4304 mu g/L). The method is proposed for future investigations that should be performed to test the hypothesis that therapeutic drug monitoring of voriconazole is clinically useful.

The Scientific Committee of Molecular Biology Techniques (C-MbT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs), for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories.. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwide.

Aim: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. Results: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 mu g/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. Conclusions: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.

The sixth edition of Samuel Hahnemann's Organon of Medicine is an obligatory work of

reference for homeopathy. Nevertheless, its philosophy can be questioned with

constructivistic and historistic objections. Three Ieveis of content may be distinguished:

(1) practical directions and maxims,

(2) theoretical explanations and hypotheses,

(3) conceptual foundations and premises.

ldeally, these Ieveis should be considered, studied, and taught separately and

gradually. My new German edition of the Organon, published in 2003, tries to meet

these demands. lt contains:

(A) a complete version of Hahnemann's original text, in the original order of

paragraphs, but in modern German with section headings, summary boxes, etc added,

(B) another full version of its content, organized in three Ieveis, as indicated above,

(C) a glossary of about 400 problematic terms.

Background: Lung function and exacerbations of chronic obstructive pulmonary disease (COPD) have been associated with short-term exposure to air pollution. However, the effect of long-term exposure to particulate matter from industry and traffic on COPD as defined by lung function has not been evaluated so far. Our study was designed to investigate the influence of long-term exposure to air pollution on respiratory symptoms and pulmonary function in 55-year-old women. We especially focused on COPD as defined by GOLD criteria and additionally compared the effects of air pollution on respiratory symptoms by questionnaire data and by lung function measurements. Methods: In consecutive cross sectional studies conducted between 1985-1994, we investigated 4757 women living in the Rhine-Ruhr Basin of Germany. NO2 and PM10 exposure was assessed by measurements done in an 8 km grid, and traffic exposure by distance from the residential address to the nearest major road using Geographic Information System data. Lung function was determined and COPD was defined by using the GOLD criteria. Chronic respiratory symptoms and possible confounders were defined by questionnaire data. Linear and logistic regressions, including random effects were used to account for confounding and clustering on city level. Results: The prevalence of COPD (GOLD stages 1-4) was 4.5%. COPD and pulmonary function were strongest affected by PM10 and traffic related exposure. A 7 mu g/m(3) increase in five year means of PM10 (interquartile range) was associated with a 5.1% (95% CI 2.5%-7.7%) decrease in FEV1, a 3.7% (95% CI 1.8%-5.5%) decrease in FVC and an odds ratio (OR) of 1.33 (95% CI 1.03-1.72) for COPD. Women living less than 100 m from a busy road also had a significantly decreased lung function and COPD was 1.79 times more likely (95% CI 1.06-3.02) than for those living farther away. Chronic symptoms as based on questionnaire information showed effects in the same direction, but less pronounced. Conclusion: Chronic exposure to PM10, NO2 and living near a major road might increase the risk of developing COPD and can have a detrimental effect on lung function.

Background: Pulmonary alveolar proteinosis ( PAP) is a rare disease, characterized by excessive intra-alveolar accumulation of surfactant lipids and proteins. Therapeutic whole lung lavages are currently the principle therapeutic option in adults. Not much is known on the kinetics of the wash out process, especially in children. Methods: In 4 pediatric and 6 adult PAP patients 45 therapeutic half lung lavages were investigated retrospectively. Total protein, protein concentration and, in one child with a surfactant protein C mutation, aberrant pro-SP-C protein, were determined during wash out. Results: The removal of protein from the lungs followed an exponential decline and averaged for adult patients 2 - 20 g and < 0.5 to 6 g for pediatric patients. The average protein concentration of consecutive portions was the same in all patient groups, however was elevated in pediatric patients when expressed per body weight. The amount of an aberrant pro-SP-C protein, which was present in one patient with a SP-C mutation, constantly decreased with ongoing lavage. Measuring the optical density of the lavage fluid obtained allowed to monitor the wash out process during the lavages at the bedside and to determine the termination of the lavage procedure at normal protein concentration. Conclusion: Following therapeutic half lung lavages by biochemical variables may help to estimate the degree of alveolar filling with proteinaceous material and to improve the efficiency of the wash out, especially in children.

Background: In a cross-sectional analysis of cystic fibrosis (CF) patients with mild lung disease, reduced surfactant activity was correlated to increased neutrophilic airway inflammation, but not to lung function. So far, longitudinal measurements of surfactant function in CF patients are lacking and it remains unclear how these alterations relate to the progression of airway inflammation as well as decline in pulmonary function over time. Methods: As part of the BEAT trial, a longitudinal study to assess the course of airway inflammation in CF, we studied lung function, surfactant function and endobronchial inflammation using bronchoalveolar lavage fluid from 20 CF patients with normal pulmonary function ( median FEV1 94% of predicted) at three times over a three year period. Results: There was a progressive loss of surfactant function, assessed as minimal surface tension. The decline in surfactant function was negatively correlated to an increase in neutrophilic inflammation and a decrease in lung function, assessed by FEV1, MEF75/25%VC, and MEF25%VC. The concentrations of the surfactant specific proteins A, C and D did not change, whereas SP-B increased during this time period. Conclusion: Our findings suggest a link between loss of surfactant function driven by progressive airway inflammation and loss of small airway function in CF patients with limited lung disease.

Background: The identification of beta-barrel membrane proteins out of a genomic/proteomic background is one of the rapidly developing fields in bioinformatics. Our main goal is the prediction of such proteins in genome/proteome wide analyses. Results: For the prediction of beta-barrel membrane proteins within prokaryotic proteomes a set of parameters was developed. We have focused on a procedure with a low false positive rate beside a procedure with lowest false prediction rate to obtain a high certainty for the predicted sequences. We demonstrate that the discrimination between beta-barrel membrane proteins and other proteins is improved by analyzing a length limited region. The developed set of parameters is applied to the proteome of E. coli and the results are compared to four other described procedures. Conclusion: Analyzing the beta-barrel membrane proteins revealed the presence of a defined membrane inserted beta-barrel region. This information can now be used to refine other prediction programs as well. So far, all tested programs fail to predict outer membrane proteins in the proteome of the prokaryote E. coli with high reliability. However, the reliability of the prediction is improved significantly by a combinatory approach of several programs. The consequences and usability of the developed scores are discussed.