Serum neopterin for early assessment of severity of severe acute respiratory syndrome

Neopterin and C-reactive protein (CRP) concentrations were determined in serum samples from 129 severe acute respiratory syndrome (SARS) patients and 156 healthy blood donors. In the patients with confirmed SARS, an early neopterin elevation was detected already at the day of onset of symptoms and rose to a maximum level of 45.0 nmol/L 3 days after the onset. All SARS patients had elevated neopterin concentrations (>10 nmol/L) within 9 days after the onset. The mean neopterin concentrations were 34.2 nmol/L in acute sera of SARS patients, 5.1 nmol/L in convalescent sera, and 6.7 nmol/L in healthy controls. In contrast, the mean CRP concentrations in both acute and convalescent sera of SARS patients were in the normal range (<10 mg/L). Serum neopterin level in SARS patients was associated with fever period and thus the clinical progression of the disease, while there was no significant correlation between the CRP level and the fever period. Serum neopterin may allow early assessment of the severity of SARS. The decrease of neopterin level was found after steroid treatment, which indicates that blood samples should be collected before steroid treatment for the neopterin measurement.     

Genetic variability of the extraneuronal monoamine transporter EMT (SLC22A3)

The extraneuronal monoamine transporter EMT (HGNC Nomenclature SLC22A3) is the molecular correlate of the classical uptake₂ system responsible for the non-neuronal inactivation of circulating and centrally released catecholamines. Because of its functional profile and expression pattern, EMT is regarded as a candidate gene for diseases related to the sympathetic nervous system and neuropsychiatric disorders. We describe the first investigation of the genetic variability of the EMT gene in human. Six single-nucleotide substitutions and one deletion were detected within the assumed core promoter, the exonic and flanking intronic sequences and the 3'-untranslated region in 100 Caucasian individuals. No amino acid changes were found and Tajima's D was positive (D=2.91; P<0.01). However, the synonymous nucleotide substitution 1233G→A might serve as a cryptic splice acceptor site. Analysis of linkage disequilibrium between polymorphisms yielded 12 possible haplotypes accounting for more than 90% of all haplotypes. Knowledge of the sequence variation and frequency of the underlying polymorphisms in this member of the amphiphilic solute facilitator family of transporters provides the basis for subsequent association studies and candidate gene approaches. Electronic Publication     

The Kallikrein-Kinin system and muscle metabolism—Clinical aspects

The influence of synthetic bradykinin (BK) on disturbed protein and carbohydrate metabolism was studied in chemical and manifest maturity-onset diabetics, in surgical patients and in alloxan diabetic rats. BK,mixed with insulin and injected subcutaneously twice daily in alloxan diabetic rats lowered the morning blood glucose concentration in a dose-dependent way, whereas in a control group treated with insulin only no decrease was seen. Accelerated local blood flow or enhanced vascular permeability as a cause of increased glucose uptake could be ruled out by control experiments using papaverine and eledoisin. Better metabolic control in the BK/insulin-treated group was also indicated by lower arterial levels of free fatty acids and of -hydroxybutyrate, normalized hepatic glycogen content and better growth of body weight. In healthy man an intravenous infusion of BK (80 g/h) did not influence normal fasting blood glucose concentrations, whereas elevated glucose levels in maturity-onset diabetics were continuously reduced within 100 min by 12.2±1.4%. A comparable diabetic group receiving saline alone showed no spontaneous drop of blood glucose concentration. An improvement of pathological carbohydrate metabolism by infusion of BK i.v. could also be demonstrated using the intravenous glucose tolerance test in chemical and manifest maturity-onset diabetics and in surgical patients: in all groupsk values of the glucose tolerance test were significantly increased by BK. This effect was neither due to stimulated insulin release nor to changed glucose pool or to increased renal glucose loss, which was even reduced by BK. Interestingly, normalk values in healthy volunteers were not further improved by BK. A stimulated protein breakdown, which occurs after surgery due to peripheral insulin resistance, can also be restricted by intravenous infusion of BK: in surgical patients urinary nitrogen excretion was reduced by 50% during infusion of BK and was accelerated again after cessation of the infusion. These results indicate that BK can improve the efficacy of exogenous insulin in insulin-deficient animals and depressed insulin sensitivity in maturity-onset diabetics and surgical patients.     

Active detachment involves inhibition of cell-matrix contacts of malignant melanoma cells by secretion of melanoma inhibitory activity

Melanoma inhibitory activity (MIA) has been identified as a small protein secreted from malignant melanoma cells. Recent results revealed a direct interaction of MIA and epitopes within extracellular matrix proteins including fibronectin. The aim of this study was to analyze functional consequences mediated by this interaction. Here we show that MIA interferes specifically with attachment of melanoma cells to fibronectin, a phenomenon we refer to as active detachment. Antibodies inhibiting binding of alpha4beta1 and alpha5beta1 integrins to fibronectin cross-react specifically with MIA, suggesting that MIA shares significant structural homology with the binding pockets of these integrins and thereby masks the respective epitopes on extracellular matrix molecules. Several peptides derived from fibronectin and from a phage display screening were tested with respect to a potential MIA-inhibitory effect. In vitro tests identified two peptides affecting MIA function; both inhibited growth of melanoma metastases in vivo. In summary, we conclude that MIA may play a role in tumor progression and spread of malignant melanomas via mediating active detachment of cells from extracellular matrix molecules within their local milieu. Further, our results suggest that inhibiting MIA functions in vivo may provide a novel therapeutic strategy for metastatic melanoma disease.     

Heterogeneity in the complement-dependent bacteriolysis within the species of Borrelia burgdorferi

Sixteen Borrelia burgdorferi strains, including all three species, were compared in a colorimetric bactericidal assay for their ability to escape the complement-dependent bacteriolysis on incubation in normal human serum free of specific antibodies (NHS). The species B. afzelii was found to be serum resistant (EB1, EB3, FEM1, FEM2, Pko), whereas strains of the species B. garinii were found to be serum sensitive (1/B29, G1, G2, PSth, PBr, PTrob). Six strains, mainly B. burgdorferi sensu stricto, were only partially sensitive (Z25, 297, B31, PKa-I, PBi). All strains activated the complement cascade in NHS, whereas only four strains (G1, G2, PBr, PSth) could activate complement in the presence of EGTA-Mg. After complement activation, covalently bound C3 fragments (C3b, iC3b) were detected on serum-sensitive as well as serum-resistant borrelial strains. Heterogeneity, however, was observed between serum-resistant and serum-sensitive strains with respect to deposition of C6 and C9. Whereas serum-sensitive strains were strongly positive for C6 and C9 and were, therefore, killed by the terminal complement complex (TCC), serum-resistant strains were devoid of C6 and C9 on their cell surface. The serum resistance may, therefore, be due to an absent or only transient formation of TCC on the bacterial surface. Received: 17 September 1996     

Changes in cerebral endothelial barrier antigen, without alteration of permeability for intravenously injected leptin in diet-induced obesity in rats

Leptin, a potent anorectic, 16-kDa, adipose tissue-derived protein, predominantly acts in hypothalamic nuclei, signaling obesity and modulating ingestive behavior. To reach this brain area, leptin, probably has to cross the blood-brain barrier (BBB). In some cases of obesity, enhanced leptin levels in the blood do not result in anorectic effects, probably due to an altered leptin transport across the BBB. Therefore, we investigated the BBB in lean and diet-induced obese Lewis rats. To obtain information about the presence of microvessels with barrier dysfunction we examined three brain areas (hypothalamus, cortex, hippocampus) using a monoclonal antibody which detects intact microvessels of the BBB (anti-endothelial barrier antigen, anti-EBA). The results showed a significantly reduced EBA staining in the brain sections of the obese animals, except the hippocampus, compared to the control group. In a second step we injected I125-labeled leptin intravenously (i.v.) in permanent i.v.-cannulated, unrestrained Lewis rats (lean and obese). We measured the radioactivity in the cerebrospinal fluid after puncture of the cisterna magna, in the blood and brain tissue 90 min after injection. The leptin content in the cerebrospinal fluid and brain was not reduced in obese compared to lean rats, thus showing a similar transport capacity of the BBB in both experimental groups. Therefore, the results of the in vivo investigations do not indicate an impairment of the BBB in diet-induced obesity, despite the immunohistological findings. Further functional and morphological studies are necessary to evaluate the specific role of other organs and distinct forms of leptin (free and protein-bound) in the pathogenesis of diet-induced obesity.     

Tissue array technology for testing interlaboratory and interobserver reproducibility of immunohistochemical estrogen receptor analysis in a large multicenter trial

Semiquantitative immunohistochemical assessment of estrogen receptor (ER) is used to predict the likelihood of response to antiestrogen therapy in breast carcinoma. If semiquantitative immunohistochemical analysis leads to therapeutic decisions, the importance of standardization and quality control increases. ER assessment reproducibility was studied among 172 laboratories using tissue microarray slides with 20 tissue spots negative and 10 tissue spots expressing ER at low, medium, or high levels. More than 80% of the laboratories demonstrated ER positivity in the medium- and high-expressing tissue spots, but only about 43% succeeded with tissue spots with low expression. Poor interlaboratory agreement was based on insufficient retrieval efficacy as shown by additional tests using autoclave pretreatment. The immunohistochemical scores used to quantify therapeutic target molecules remain inconclusive as long as progress toward standardized immunohistochemical procedures and evaluation is not achieved. Tissue microarray technology has proved its suitability for large-scale immunohistochemical trials, giving rise to new dimensions in control assessment.     

Behavioral phenotyping of mice in pharmacological and toxicological research

The evaluation of behavioral effects is an important component for the in vivo screening of drugs or potentially toxic compounds in mice. Ideally, such screening should be composed of monitoring general health, sensory functions, and motor abilities, right before specific behavioral domains are tested. A rational strategy in the design and procedure of testing as well as an effective composition of different well-established and reproducible behavioral tests can minimize the risk of false positive and false negative results in drug screening. In the present review we describe such basic considerations in planning experiments, selecting strains of mice, and propose groups of behavioral tasks suitable for a reliable detection of differences in specific behavioral domains in mice. Screening of general health and neurophysiologic functions (reflexes, sensory abilities) and motor function (pole test, wire hang test, beam walking, rotarod, accelerod, and footprint) as well as specific hypothesis-guided testing in the behavioral domains of learning and memory (water maze, radial maze, conditioned fear, and avoidance tasks), emotionality (open field, hole board, elevated plus maze, and object exploration), nociception (tail flick, hot plate), psychiatric-like conditions (porsolt swim test, acoustic startle response, and prepulse inhibition), and aggression (isolation-induced aggression, spontaneous aggression, and territorial aggression) are described in further detail. This review is designed to describe a general approach, which increases reliability of behavioral screening. Furthermore, it provides an overview on a selection of specific procedures suitable for but not limited to behavioral screening in pharmacology and toxicology.     

Genetic ablation of FLRT3 reveals a novel morphogenetic function for the anterior visceral endoderm in suppressing mesoderm differentiation

During early mouse development, the anterior visceral endoderm (AVE) secretes inhibitor and activator signals that are essential for establishing the anterior–posterior (AP) axis of the embryo and for restricting mesoderm formation to the posterior epiblast in the primitive streak (PS) region. Here we show that AVE cells have an additional morphogenetic function. These cells express the transmembrane protein FLRT3. Genetic ablation of FLRT3 did not affect the signaling functions of the AVE according to the normal expression pattern of Nodal and Wnt and the establishment of a proper AP patterning in the epiblast. However, FLRT3^−/− embryos showed a highly disorganized basement membrane (BM) in the AVE region. Subsequently, adjacent anterior epiblast cells displayed an epithelial-to-mesenchymal transition (EMT)-like process characterized by the loss of cell polarity, cell ingression, and the up-regulation of the EMT and the mesodermal marker genes Eomes, Brachyury/T, and FGF8. These results suggest that the AVE acts as a morphogenetic boundary to prevent EMT and mesoderm induction in the anterior epiblast by maintaining the integrity of the BM. We propose that this novel function cooperates with the signaling activities of the AVE to restrict EMT and mesoderm induction to the posterior epiblast.     

Genetic and biochemical characterization of Malassezia pachydermatis with particular attention to pigment-producing subgroups.

The aim of the study was the characterization of Malassezia pachydermatis and its pigment-producing subgroup using biochemical tests and RAPD. It was of interest to determine whether particular RAPD patterns could be used to indicate pigment production, as well as a close genetic relatedness to Malassezia furfur. Therefore, 210 strains of M. pachydermatis were examined for morphology, catalase and ss-glucosidase activity, lipid and carbohydrate assimilation and the tryptophan-dependent synthesis of pigments. Of these, 114 strains were subjected to RAPD analyses. A multivariate logistic regression model was applied to classify M. pachydermatis isolates regarding their pigment production by using genetic and biological parameters. Biological and RAPD findings showed a high biological and genetic diversity within the species M. pachydermatis and within pigment producers. RAPD analysis revealed 28 genotypes within 114 strains tested. Pigment producing strains could not be assigned to a common RAPD profile, but a genetic relatedness of pigment-producing M. pachydermatis with M. furfur can be assumed. A particular RAPD pattern allowed statistically significant probability of pigment production (P<0.001) and might be used as a tool to rapidly detect pigment producing M. pachydermatis, e.g. in Malassezia-associated pityriasis versicolor. The reported method is useful for identification of pigment producing M. pachydermatis isolates and has advantages over established tests.