Multicentre evaluation of performance of C-reactive protein analysis over a one-year period.

This communication deals with a longitudinal evaluation of C-reactive protein (CRP) analysis during a one-year period using a single lot of liquid control sera (3 levels) (BIOREF-CRP levels 1, 2 and 3) in different laboratories. A total of 652 sets of data were returned from 20 participating laboratories using 13 different reagent-measuring device combinations. The use of the control materials was defined in a standard operating procedure. Data was returned to the organizers on a monthly basis and questions could be asked or problems presented during the evaluation period. Although the performance of different reagents varied, the control materials were shown to be stable over the whole of the evaluation period when stored at 4-7 degrees C in a refrigerator/cold room. Typical problems were encountered, examples of which are presented here in graphical and tabular form.

Expression and characterization of the substance P (NK1) receptor in the rat pituitary and AtT20 mouse pituitary tumor cells.

Although substance P is known to take part in the regulation of the anterior pituitary, no conclusive evidence for the expression of the tachykinin NK1 receptor has been found yet in the pituitary or pituitary derived cells. With the reverse transcription-polymerase chain reaction (RT-PCR) method we could detect the low abundant transcripts of the NK1 receptor in the rat pituitary and in the AtT20 cell line (clone D16v). Furthermore, the functional expression of the NK1 receptor in AtT20 cells was confirmed by activation of the phosphatidylinositol-calcium second messenger system when the cells were treated with substance P. In addition, binding studies also indicated the functional expression of this receptor in AtT20 cells. Thus we provide the first evidence that the NK1 receptor is expressed in AtT20 cells and the rat pituitary.

Synaptosomal uptake and release of dopamine in rat striatum after hypoxia.

Hypoxia induces alterations of central monoaminergic transmission and of behavior. We studied the effect of hypoxia on adult and newborn rats to obtain more information about long-lasting changes of dopamine (DA) transmission caused by neonatal hypoxia. One single exposure of adult rats to hypoxia leads to short-term alterations of DA uptake: decreased affinity of the uptake carrier to DA (Km, 269.5% versus control) and a sharp increase of Vmax up to 301.4% resulting in an increase of total uptake of DA into the striatum synaptosomes. The K+-evoked DA release decreased to 69.5%. After 1 week of recovery all parameters are normalized. Chronic postnatal hypoxia (postnatal day 2-11) caused long-lasting changes of DA release and uptake opposite to those observed in adult rats. Three months after hypoxia, the K+-stimulated DA release was enhanced (132% of control), and the uptake was reduced due to decreased affinity of the uptake carrier system for the substrate (Km, 187% of control value). In conclusion, the alterations observed after chronic postnatal hypoxia reflect special adaptive processes that are related to the high plasticity of the immature neonatal brain and contribute to an increased DA function in the nigrostriatal system.

In vitro generated mast cells investigated by monoclonal antibodies.

In the present study we have investigated the development of mast cells under in vitro conditions after depletion of mature mast cells of male Wistar rats by intraperitoneal application of distilled water. The studies were carried out both by monoclonal antibodies (MAb) against rat peritoneal mast cells and by cytochemical techniques. During cultivation the amount of MAb-positive cells increased from less than 5% to 64% after 3 weeks, which was accompanied by increased histamine levels and cell size. After 2 weeks the first Alcian blue stained mast cells were detectable, while safranin-positive granules appeared only after 3 weeks. These findings suggest that there are mast cell precursors in the peritoneum which may differentiate into mature mast cells. The antigen on the cell surface recognized by the MAb seems to be expressed during mast cell maturation.

Investigation of mast cell differentiation in vivo by use of monoclonal antibodies.

In the present study mast cell differentiation/maturation was studied in vivo after depletion of mature mast cells from the peritoneal cavity by injection of distilled water. The reconstituting cell population was characterized by use of different staining methods. Additionally, the monoclonal antibody (MAb) IWF F2, which recognizes a membrane antigen of rat mast cells, was used to follow up mast cell differentiation/maturation in the course of the experiment. The antigen expression was studied both by immunofluorescence of antigen-bearing cells and by MAb inhibition of compound 48/80-stimulated histamine release from mast cells. In the course of the experiment the amount of antigen-positive cells increased continuously from less than 5% to control level (1st and 22nd days, respectively). The expression of the membrane antigen detectable by the MAb precedes the appearance of cytochemically identifiable mast cells for several days. The mast cells mature morphologically and functionally as indicated by increasing size, histamine content and MAb inhibition of compound 48/80-stimulated histamine release. The results obtained suggest the MAb IWF F2 to be a useful methodical tool for additional characterization of mast cell differentiation/maturation processes.

Monoclonal antibodies against rat mast cells.

Four hybridomas have been isolated which secrete monoclonal antibodies against surface determinants of rat peritoneal mast cells. This could be demonstrated by an indirect immunofluorescence assay. The monoclonal antibodies did also react with a portion of mononuclear phagocytes from the peritoneal cavity but not with other lymphatic cells.

Influence of postnatal hypoxia on 32P-labeling of polyphosphoinositides and phosphatidic acid in striatum synaptosomes from rat brain.

Striatum synaptosomes prepared from adult rats which had been exposed to postnatal hypoxia incorporate 32P-phosphate into phosphatidylinositol-4,5-trisphosphate (PI-4,5-P2) with decreased rate. 32P-incorporation amounted to 57% of the control for PI-4,5-P2 labeling and was slightly diminished for phosphatidic acid and PI-4-P. Exposure to hypoxia of adult rats did not affect inositol phospholipid labeling. The inhibitory effect of dopamine on 32P-phosphate incorporation was reduced only after postnatal hypoxia. 32P-incorporation rates and the dopamine inhibitory effect were not influenced by external calcium. A working hypothesis is suggested for the dopamine action on specific receptors which may be linked to the polyphosphoinositide metabolism and membrane calcium release. The long lasting effects of an early postnatal hypoxia on 32P-incorporation rates into polyphosphoinositides and phosphatidic acid could reflect the role of the proposed dopamine receptor interaction.

The relations between erythrocytic and CNS parameters following postnatal hypobaric hypoxia of the rat.

In the present paper the relations between selected erythrocytic criteria and alterations of biochemical brain parameters following hypoxia were investigated in order to find out the relevance of erythrocytic criteria for the diagnosis of hypoxia-induced alterations of the CNS during development. Newborn Wistar rats (n = 80) with their mother were exposed to hypobaric hypoxia, pO2 = 11.3 kPa, from day 2-10 of life, daily for 11 h. On the 11th day of life the animals had a lower body (38%), liver (33%) and brain (17%) weight, an increased number of reticulocytes, a lower median density of peripheral red blood cells (rbc) and an increased packed cell volume (PCV) indicating stimulation of erythropoiesis. Hypoxia caused a decrease of protein concentration, of AChE activity and of the amount of DNA in the brain tissue. In addition to these parameters the cholesterol level and the protein/DNA ratio are diminished and the carboanhydrase activity is increased in the cortex. In order to quantify the changes of various brain parameters in each animal in the sense of altered development a score of these results was calculated. It can be concluded that the hypoxia-induced changes of erythrocytic parameters correlate with those of the neurochemical indices.

Monoclonal antibodies against rat mast cells differentiate between subtypes.

Four monoclonal mouse anti rat mast cell antibodies were selected which detect an antigenic determinant occurring on connective tissue mast cells of the rat. A strong antigen density was found on peritoneal mast cells whereas pleural and mesenteric mast cells exhibit considerably smaller amounts of the antigen. It does not occur on lung mast cells and basophils, thus permitting a mast cell subtype differentiation according to the expression of a surface antigen. The monoclonal antibodies do not react with IgE or IgE Fc-receptor determinants and do not interfere with the histamine secretion from peritoneal mast cells.

Influence of a perinatal hypoxia on the carbonic anhydrase activity in different brain regions of the rat.

Homogenates of striatum, hippocampus and cerebral cortex of the rat brain were investigated for carbonic anhydrase activities. During ontogeny the enzyme activities increase with proceeding gliogenesis. At the 5th postnatal day (PD) prenatal hypoxia (17th day of gestation (GD)--birth) is followed by a 4-fold increase of carbonic anhydrase (CA) activity in the hippocampus. After postnatal hypoxia (2nd-10th day of life) CA activity in all investigated brain regions rise. At the 11th PD the values were 190 (cerebral cortex), 242 (striatum) and 176 (hippocampus) per cent of control. Noradrenaline (10(-6)M) enhanced CA activity in cerebral cortex and hippocampus about 2-fold. Dopamine (10(-6)M) caused an increase in the striatum up to 210% of control. The increased CA activity after hypoxia is assumed to be a functional response to activation of the glia by catecholamines.

Effect of early and late postnatal hypoxia on subcellular synaptosomal fractions from cerebral cortex of rats. I. An electron-microscopical and biochemical study.

Synaptosomal fractions of brain cortex of rats exposed to moderate intermittent hypobaric hypoxic conditions during the first or second decade of the postnatal life were investigated electron microscopically one day or up to one week after the hypoxia experiment. Mitochondria and synaptosomes with impaired morphology were observed to occur more frequently in fractions obtained from hypoxic animals. Synaptosomes showed lesions of the outer membrane and loss of the synaptoplasmatic content, or condensation of the synaptosomal content with increasing electron density, deformation of the synaptosomes in size and shape, disappearance of structural details, degeneration of intrasynaptosomal mitochondria in form of strong condensation and, finally, formation of intrasynaptosomal vacuoles with dense core inclusions. The mitochondrial reaction was characterized by two antagonistic configurations: condensation of the matrix with secondary enlargement of the intracistal spaces and of the outer compartment and, finally, the decay of the matrix condensate in several crumbs. On the other hand, swelling of the matrix with strong enlargement of the whole mitochondrium, later defects in the outer membrane and the degeneration into empty mitochondrial ghosts, appearing in the upper fractions. Some characteristic marker enzymes were tested in the fractions. The results hint at an elevated vulnerability of synaptosomes obtained from animals exposed to postnatal hypoxia, as indicated by the lowered quotient of LDH activities after and before addition of Triton X-100. Furthermore, there are clues to a decrease in the number of synaptic connections within the cortex of hypoxia exposed animals, as is concluded from the diminished AChE activity in the subcellular fractions of these animals. However, no different effect of early or late postnatal hypoxia exposition was evident in the biochemical parameters.

Effect of early and late postnatal hypoxia on subcellular synaptosomal fractions from cerebral cortex of rats. II. A quantitative ultrastructural study.

Synaptosomal subfractions were isolated by discontinuous sucrose gradient centrifugation from the P2-fraction of cerebral cortices of juvenile rats exposed to moderate intermittent hypobaric hypoxia from day 2-11 (early postnatal period) or from day 12-21 (late postnatal period) after birth. Ultrastructural investigations were carried out on fractions A and E of the preparation. Quantitative parameters of synaptosomes and mitochondria (volume density, numerical density, profile size frequency distribution) were estimated in a stereological analysis. Early postnatal hypoxia exposition strengthened the irreversible swelling of mitochondria. The effect on synaptosomes was less pronounced, very large synaptosomes showed significant condensation. Late postnatal hypoxia exposition resulted in strong condensation of synaptosomes in all size classes and similar reaction in mitochondria. Both hypoxia experiments produced an enhanced vulnerability of the synaptosomal membranes against the fractionation procedure and the appearance of monstrous large condensed synaptosomes. The results are discussed in view of hypothetical biochemical mechanisms underlying these changes.