[German translation and validation of the Stress Appraisal Measure (SAM)]. / Deutsche Übersetzung und Validierung des Stress Appraisal Measure (SAM).

OBJECTIVE: In the present study, the German-language version of the Stress Appraisal Measure (SAM) by Peacock and Wong was validated in a student population. SAM is a relatively short questionnaire (28 items) that evaluates a current, stress-triggering event. The theoretical background is provided by the stress model of Lazarus and Folkman. METHOD: 85 students (age: 23; 59 female, 26 male) were exposed to two stress scenarios in order to test whether they were suited to provoke stress. A factor analysis was performed and the internal consistency of the seven SAM scales was determined. In addition, the convergent validity of SAM with State and Trait Anxiety Inventory (STAI), Coping Inventory for Stressful Situations (CISS) and specific emotion scales was investigated via Pearson's product-moment correlation. RESULTS: The two stress scenarios were suited to evoke stress. The factor structure and the internal consistency of the individual scales, as well as the convergent validity of SAM were replicated with minor limitations in the present German version. Some items (especially from the fifth factor) were only replicated partially. CONCLUSION: SAM can also be employed in the German language version.

An ultra scale-down analysis of the recovery by dead-end centrifugation of human cells for therapy.

An ultra scale-down method is described to determine the response of cells to recovery by dead-end (batch) centrifugation under commercially defined manufacturing conditions. The key variables studied are the cell suspension hold time prior to centrifugation, the relative centrifugal force (RCF), time of centrifugation, cell pellet resuspension velocities, and number of resuspension passes. The cell critical quality attributes studied are the cell membrane integrity and the presence of selected surface markers. Greater hold times and higher RCF values for longer spin times all led to the increased loss of cell membrane integrity. However, this loss was found to occur during intense cell resuspension rather than the preceding centrifugation stage. Controlled resuspension at low stress conditions below a possible critical stress point led to essentially complete cell recovery even at conditions of extreme centrifugation (e.g., RCF of 10000 g for 30 mins) and long (~2 h) holding times before centrifugation. The susceptibility to cell loss during resuspension under conditions of high stress depended on cell type and the age of cells before centrifugation and the level of matrix crosslinking within the cell pellet as determined by the presence of detachment enzymes or possibly the nature of the resuspension medium. Changes in cell surface markers were significant in some cases but to a lower extent than loss of cell membrane integrity.

Potent and multiple regulatory actions of microglial glucocorticoid receptors during CNS inflammation.

In CNS, glucocorticoids (GCs) activate both GC receptor (GR) and mineralocorticoid receptor (MR), whereas GR is widely expressed, the expression of MR is restricted. However, both are present in the microglia, the resident macrophages of the brain and their activation can lead to pro- or anti-inflammatory effects. We have therefore addressed the specific functions of GR in microglia. In mice lacking GR in macrophages/microglia and in the absence of modifications in MR expression, intraparenchymal injection of lipopolysaccharide (LPS) activating Toll-like receptor 4 signaling pathway resulted in exacerbated cellular lesion, neuronal and axonal damage. Global inhibition of GR by RU486 pre-treatment revealed that microglial GR is the principal mediator preventing neuronal degeneration triggered by lipopolysaccharide (LPS) and contributes with GRs of other cell types to the protection of non-neuronal cells. In vivo and in vitro data show GR functions in microglial differentiation, proliferation and motility. Interestingly, microglial GR also abolishes the LPS-induced delayed outward rectifier currents by downregulating Kv1.3 expression known to control microglia proliferation and oxygen radical production. Analysis of GR transcriptional function revealed its powerful negative control of pro-inflammatory effectors as well as upstream inflammatory activators. Finally, we analyzed the role of GR in chronic unpredictable mild stress and aging, both known to prime or sensitize microglia in vivo. We found that microglial GR suppresses rather than mediates the deleterious effects of stress or aging on neuronal survival. Overall, the results show that microglial GR acts on several key processes limiting pro-inflammatory actions of activated microglia.

The high post-test probability of a cytological examination renders further investigations to establish a diagnosis of epithelial malignant pleural mesothelioma redundant.

The aim of the study was to establish in a prospective and blinded manner the diagnostic yield of morphology, immunocytochemistry (ICH) and electron microscopy (EM) in the cytological analysis of malignant pleural mesothelioma (MPM). Pleural fluid from consecutive patients, 14 with a histologically proven MPM, 12 with a malignant pleuritis due to adenocarcinoma (AC), and 13 with a reactive pleural effusion (RM), was separately analyzed. Smears were incubated with monoclonal antibodies (Tag72, Ber-Ep4, anti-CEA, EMA). These were considered suggestive for MPM when only EMA stained positive, for AC when three out of four markers stained positive, and for RM when no marker stained positive. The post-test probability of the morphological, ICH, and EM analysis were 92, 100, 92% or MPM, 91, 100, 86% for AC, and 88, 88, 90% for RM, respectively. We concluded that the high post-test probability of a combined morphological and ICH diagnosis of MPM warrants to cease further diagnostic procedures in these patients. Electron microscopy did not add to accuracy of diagnosis.

Synthesis and SAR of 3- and 4-substituted quinolin-2-ones: discovery of mixed 5-HT(1B)/5-HT(2A) receptor antagonists.

Quinolin-2-ones bearing a heteroaryl-piperazine linked by a two carbon chain at the 3- or 4-position were synthesised and evaluated as mixed 5-HT(1B)/5-HT(2A) receptor antagonists. Potent mixed antagonists were obtained with thieno[3,2-c]pyridine derivatives. In this series, compound 2.1 (SL 65.0472) proved to be functional antagonist at both the 5-HT(2A) receptor (rat in vivo 5-HT-induced hypertension model) and the 5-HT(1B) receptor (dog in vitro saphenous vein assay).

Antiplatelet and antithrombotic activity of SL65.0472, a mixed 5-HT1B/5-HT2A receptor antagonist.

The antiplatelet and antithrombotic activity of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-di-hydroquinoline-acetamide), a mixed 5-HT1B/5-HT2A receptor antagonist was investigated on 5HT-induced human platelet activation in vitro, and in rat, rabbit and canine platelet dependent thrombosis models. SL65.0472 inhibited 5-HT-induced platelet shape change in the presence of EDTA (IC50 values = 35, 69 and 225 nM in rabbit, rat and human platelet rich plasma (PRP)), and also inhibited aggregation induced in human PRP by 3-5 microM 5-HT + threshold concentrations of ADP (0.5-1 microM) or collagen (0.3 microg/ml) with mean IC50 values of 49 +/- 13 and 48 +/- 6 nM respectively. SL65.0472 inhibited thrombus formation when given both intravenously 5 min and orally 2 h prior to assembly of an arterio-venous (A-V) shunt in rats as from 0.1 and 0.3 mg/kg respectively. It was active in a rabbit A-V shunt model with significant decreases in thrombus weight as from 0.1 mg/kg i. v. and at 10 mg/kg p.o. The delay to occlusion in an electric current-induced rabbit femoral artery thrombosis model was increased by 251% (p <0.05) after 20 mg/kg p.o. SL65.0472 (30 microg/kg i.v.) virtually abolished coronary cyclic flow variations (7.2 +/- 1.0/h to 0.6 +/- 0.6/h, p <0.05) and increased minimum coronary blood flow (1.2 +/- 0.8 ml/min to 31.8 +/- 8.4 ml/min, p <0.05) in a coronary artery thrombosis model in the anaesthetised dog. Finally, SL65.0472 significantly increased the amount of blood lost after rat tail transection at 3 mg/kg p.o. Thus the anti-5-HT2A component of SL65.0472 is reflected by its ability to inhibit 5-HT-induced platelet activation, and platelet-rich thrombus formation.

[Trends in the prevalence of caries in Dutch children]. / Trends in de prevalentie van tandcariës bij de Nederlandse jeugd.

Have there been changes in the trend of caries experience among Dutch children in the late 1990s? To answer this question a meta-analysis was carried out on epidemiological data collected in 5-, 6-, 11- and 12-year-old children living in the Netherlands between 1980 and 1999. From results of the present analysis it appears that after the mid 1980s a halt in the decline of caries experience among 5- and 6-year-olds occurred whereas among 11- and 12-year-olds, the earlier decrease in mean DMFS-scores stopped in the mid nineties. No significant trend in the caries experience among children of Turkish or Moroccan origin was found.

Effects of SL 65.0472, a novel 5-HT receptor antagonist, on 5-HT receptor mediated vascular contraction.

5-hydroxytryptamine (5-HT) contracts vascular smooth muscle and pharmacological and molecular biological data suggest that these effects are mediated primarily by stimulation of 5-HT(1B) and 5-HT(2A) receptor subtypes. We have studied the properties of 7-fluoro-2-oxo-4-[2-[4-(thieno[3,2-c] pyridin-4-yl) piperazin-1-yl] ethyl]-1,2-dihydroquinoline-1-acetamide (SL 65.0472 ), a novel 5-HT receptor antagonist, in isolated vascular preparations contracted by 5-HT or sumatriptan. In canine isolated saphenous vein strips (putatively 5-HT(1B)-mediated contraction), SL 65.0472 antagonised sumatriptan-induced contractions in a competitive manner (pA(2) 8. 17+/-0.36). 5-HT contracts rabbit aorta by stimulation of 5-HT(2A) receptors. SL 65.0472 displaced the 5-HT concentration response curve in rabbit aorta rightwards with a significant reduction in maximum. The apparent pK(B) value was 8.58+/-0.18. 5-HT-induced contractions of human coronary arteries are mediated by a mixed population of 5-HT(1B) and 5-HT(2A) receptors. SL 65.0472 produced rightward parallel shifts of the 5-HT concentration response curves in all tissues studied (pA(2) 8.8+/-0.14, n=7). In conclusion, SL 65. 0472 is a potent antagonist of vascular smooth muscle contraction in vitro mediated by 5-HT receptor stimulation.

Microbial hydroxylation of natural drimenic lactones.

Incubation of confertifolin and isodrimenin with Mucor plumbeus, Aspergillus niger or Rhizopus arrhizus gave in good yields the corresponding 3 beta-hydroxy derivatives. From isodrimenin, the known natural 7 alpha-hydroxy derivative (futronolide) was also obtained and its structure was definitely established by X-ray crystallographic study of its acetate derivative.

Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions.

Cytological slides of serous fluids of 41 malignant mesotheliomas, 88 metastatic adenocarcinomas, and 25 reactive effusions were immunostained with the antibodies anti-CEA, MOC-31, Leu-M1, Ber-EP4, and B72.3. Most mesotheliomas and all reactive fluids failed to stain with these antibodies. The sensitivity of the five markers to detect carcinoma cells differed remarkably. Especially MOC-31, Ber-EP4, and B72.3 stained with a high number of carcinoma cases and the complemetary value of Ber-EP4 and B72.3 to immunostain carcinoma cells was impressive: 94% of the metastatic adenocarcinoma cases reacted with Ber-EP4 or B72.3 in contrast to 1 of 41 malignant mesotheliomas.

Expression of platelet-derived growth factor (PDGF) and PDGF receptors in human malignant mesothelioma in vitro and in vivo.

The expression of platelet-derived growth factor (PDGF) and PDGF receptors was studied in human normal and malignant mesothelial cells in vitro and in vivo. Staining with anti-cytokeratin and ME1 antibodies and ultrastructural analysis confirmed the mesothelial nature of the cell lines used to study PDGF and PDGF receptor expression in vitro. Using antibodies, mesothelioma cell lines were found to express PDGF and both the PDGF alpha- and the PDGF beta-receptor, whereas cultured normal mesothelial cells expressed PDGF and PDGF alpha-receptor. This PDGF and PDGF receptor staining pattern largely reflects the earlier described mRNA expression in these cell lines. The only exception was the immunocytochemical detection of PDGF alpha-receptors in the mesothelioma cell lines, which is different from the inability to detect alpha-receptor transcripts on Northern blots. Expression was also investigated in mesothelial cells in vivo. Expression of PDGF was observed in malignant mesothelioma cells on frozen tissue sections. In pleural effusions, a double immunofluorescence staining procedure for PDGF and epithelial membrane antigen (EMA) revealed PDGF expression by EMA-positive malignant mesothelioma cells. PDGF beta-receptors and occasionally PDGF alpha-receptors were detected in frozen tissue sections of malignant mesotheliomas, whereas mesothelioma cells in effusions showed faint expression of only the PDGF beta-receptor. In contrast, in effusions containing non-malignant mesothelial cells, only a very low level of PDGF alpha-receptor could be detected. Taken together, these results indicate that the pattern of PDGF and PDGF receptor expression in mesothelial cells in vivo largely corresponds to expression of PDGF and its receptors in vitro. Malignant mesothelioma cell lines thus constitute a good model system for studies on the role of PDGF in this malignancy. Furthermore, the data reported in this paper are consistent with the idea that an autocrine growth stimulatory effect of PDGF via PDGF receptors may play a role in the pathogenesis of malignant mesothelioma.

In vivo and in vitro interaction of the novel selective histamine H1 receptor antagonist mizolastine with H1 receptors in the rodent.

The interaction of mizolastine (CAS 108612-45-9, SL 85.0324) with histamine H1 receptors has been evaluated in the rodent. Mizolastine inhibited with high affinity (IC50 = 47 nmol/l) the binding of [3H]pyrilamine to histamine H1 receptors in guinea pig cerebellar membranes and sections. The order of potency of mizolastine and various H1 antagonists in this binding assay was the following: cyproheptadine > pyrilamine > mequitazine > mizolastine > astemizole > terfenadine > cetirizine > loratadine. Mizolastine also potently antagonized the contractile effects of histamine in the guinea pig ileum (pA2 = 8.5) and histamine-induced stimulation of phosphoinositide turnover in rat cortical slices (IC50 = 0.35 mumol/l). In contrast, this compound displayed very low affinity for serotonergic, noradrenergic and muscarinic cholinergic receptors as evidenced in both binding assays and functional tests. In guinea pig cerebellar membranes, [3H]mizolastine labelled in a saturable and reversible manner a single population of binding sites with Kd and Bmax values of 1.1 nmol/l and 635 fmol/mg protein, respectively. [3H]Mizolastine binding in guinea pig cerebellar membranes was inhibited by histamine (IC50 = 30 mumol/l) and by drugs that possess affinity for the H1 receptor such as pyrilamine (IC50 = 1 nmol/1), DL-chlorphenyramine (IC50 = 6.4 nmol/l) terfenadine (IC50 = 6 nmol/l) and loratadine (IC50 = 50 nmol/l). At concentrations lower than 10 mumol/l, the H2 receptor ligands dimaprit and cimetidine and the H3 receptor ligands burimamide and 4-methyl-histamine failed to displace [3H]mizolastine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemistry of malignant mesothelioma: OV632 as a marker of malignant mesothelioma.

In pleural or ascitic effusions the cytomorphological distinction of adenocarcinoma cells, reactive mesothelial cells, and malignant mesothelioma cells often causes a diagnostic dilemma. The value of immunocytochemistry was investigated on cytological smears of 24 well-established cases of malignant mesothelioma, a selected series of 31 metastatic adenocarcinomas, and 20 smears of patients without known malignancy. In these smears we scored the immunoreactivity with a panel of four monoclonal antibodies. In addition to antibodies for epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA), the monoclonal antibody MOC31 and the ovarian carcinoma specific antibody OV632 were incorporated in the panel. With none of these four antibodies was immunostaining of reactive mesothelial cells found. CEA- and MOC31-positive tumour cells were frequent in metastatic adenocarcinomas, but occurred rarely in malignant mesotheliomas. EMA-positive tumour cells were found in all metastatic adenocarcinomas (100 per cent) and in most malignant mesotheliomas (83 per cent). In addition to the expected reactivity of OV632 with ovarian carcinomas, 22 of 24 malignant mesotheliomas contained immunopositive tumour cells, while only a small proportion of non-ovarian adenocarcinomas reacted with this antibody. This selective staining of malignant mesothelioma cells, but not reactive mesothelial cells, with OV632 now permits the positive identification of malignant mesothelioma cells in male patients.

Cytopathology of malignant mesothelioma. Reappraisal of the diagnostic value of collagen cores.

The identification of malignant mesothelial cells in cytological smears prepared from serous effusions is still hampered by the lack of features specific for mesothelial differentiation. We examined the diagnostic value of collagen cores within clusters of tumour cells in cytological smears prepared from effusions from 43 patients with malignant mesothelioma and of 62 cases of metastatic adenocarcinoma. In Giemsa-stained smears collagen cores were detected in 51% of the cases of malignant mesothelioma and in none of the smears with metastatic adenocarcinoma. Using the Azan stain, collagen cores were detected in 64% of the malignant mesotheliomas and 4% of the adenocarcinomas. Immunoelectron microscopy demonstrated that the collagen cores are largely composed of collagen type III fibrils and some elastin embedded in a homogenous extracellular matrix. It can be concluded that the presence of collagen cores within clusters of tumour cells is highly suggestive of mesothelial differentiation and a common finding in malignant mesothelioma.

Characterization of three human malignant mesothelioma cell lines.

Three human malignant mesothelioma cell lines, designated Mero-14, Mero-25, and Mero-41, have been isolated from effusions and from autopsy material of confirmed cases of malignant mesothelioma. Light and electron microscopy, cytogenetics, growth requirements, and intermediate filament expression of these cell lines were studied and, where possible, compared with the original tumor material of the patient. Cytologic and ultrastructural morphology was consistent with the mesothelial nature of the cells. All cell lines displayed a hyperdiploid karyotype similar to that of the tumor cells obtained directly from the patient. All three malignant mesothelioma cell lines had marker chromosomes 1, 3, 9, and 22, as well as other markers that were occasionally present in these cell lines and in other malignant mesotheliomas studied. Growth kinetic studies in medium supplemented with epidermal growth factor (EGF) showed increased proliferation and a decreased proliferation in medium supplemented with hydrocortisone (HC) or EGF plus HC. The three malignant mesothelioma cell lines were positive for the cytokeratins 7, 8, 18, and 19 based on immunofluorescence and immunoblotting tests with chain-specific monoclonal antibodies. The characteristics of these cell lines support the assumption that Mero-14, Mero-25 and Mero-41 are derived from malignant mesotheliomas and have retained their original character.