Search PubMed⌕ Search

Biomedical subjects

D Roos

Publications and source records attributed to D Roos.

At least 217 records · Page 12Linked to original sources
PubMed1990

[Hepatitis C virus (HCV) antibodies in German blood donors--a pilot study].

A new EIA-test to detect Hepatitis C antibody (ORTHO Diagnostic Systems) has been evaluated in four blood transfusion services in FRG (Hamburg, Hannover, Springe, Frankfurt). Among 3,123 specimens, 18 (0.58%) reacted initially positive, 13 (0.42%) remained positive after repeat testing. A detailed analysis revealed remarkable differences: blood donors from the northern part of Germany were less frequently positive than donors from the southern region (0.24% vs. 0.79%), repeat donors had a higher HCV-antibody incidence than first-time donors (0.48% vs. 0.29%). Similar differences were observed between remunerated and non-remunerated donors (0.24% vs. 0.53%), as well as between donors from rural and urban areas (0.34% vs. 0.46%). In contrast to other investigators, only a weak correlation between elevated ALT-levels (greater than 50 IU/ml) and anti-HCV seropositivity rate has been found.

Antigens, Viral↗
PubMed1989

Platelet-activating factor (PAF) acts as an intercellular messenger in the changes of cytosolic free Ca2+ in human neutrophils induced by opsonized particles.

Addition of opsonized particles to human neutrophils in suspension leads to a biphasic elevation in the cytosolic free Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i during the second phase (greater than 3 min) is pronounced (about 400 nM), in contrast to the rise during the first phase, which is relatively small (less than 100 nM). The second and large rise in [Ca2+]i is brought about by messenger(s) released from the cell after addition of opsonized particles. This second rise in [Ca2+]i is not observed in the presence of the platelet-activating factor (PAF) antagonist WEB 2086, indicating that PAF can act as an intercellular messenger affecting Ca2+ homeostasis in human neutrophils.

Azepines↗
PubMed1989

A rapid turbidimetric assay of phagocytosis and serum opsonizing capacity.

An in vitro assay has been developed to measure the opsonizing capacity of serum and the extent of bacterial uptake by phagocytes. Various micro-organisms were preopsonized for 10 min with a serum concentration previously determined to be optimal for the respective types of micro-organism. Subsequently, neutrophils from a healthy donor were added to the preopsonized bacteria in a cuvette of a spectrophotometer. The decrease in turbidity at 400 nm, resulting from the uptake of the micro-organisms by the neutrophils, was measured for 20-30 min and the area under the curves was taken as a measure of the opsonizing capacity of the serum or the phagocytic capacity of the neutrophils. The results correlated well with standard opsonophagocytic assays. By excluding Ca2+ from the buffer of the assay, phagocytosis was distinguished from the combined response of phagocytosis and aggregation. In the presence of Ca2+ ions, both phagocytosis and aggregation contributed to the decrease in turbidity. In the absence of Ca2+, phagocytosis was normal, but aggregation was completely inhibited. Phagocytosis in the absence of Ca2+ was also observed using microscopic and radiometric methods of evaluation. Neutrophils from a patient with a deficiency of leukocyte adhesion molecules, ingested as many bacteria as did normal neutrophils without Ca2+. Experiments with NaF, to inhibit phagocytosis, indicated that the change in turbidity measured in the absence of Ca2+ was mainly caused by phagocytosis, not by attachment of bacteria to the neutrophils. The opsonizing capacity of sera, as determined in our assay, depended both on antibodies and on an intact complement system and the inter-assay variance was less than 5%. We found a close correlation between turbidity changes measured in the presence or absence of Ca2+, suggesting that both phagocytosis and aggregation are opsonin-dependent. This assay is applicable to a variety of opsonizing fluids and micro-organisms, and can be used for assessing the phagocytic capacity of patients' neutrophils as well as for assessing the opsonizing capacity of patients' sera.

Blood Bactericidal Activity↗
PubMed1989

Pyrimidine 5'-nucleotidase deficiency: improved detection of carriers.

The activities of pyrimidine 5'-nucleotidase (P5N) and the nucleotide pools in the erythrocytes from 19 members of a Dutch family with P5N deficiency were measured. In the erythrocytes of 5 (out of 6) apparent heterozygotes (based on P5N activities), an increased amount of UDP-N-acetylhexosamines was found. This increase was also found in the erythrocytes of 2 (out of 3) questionable heterozygotes (P5N activity below normal range, but not below normal mean--2 X SD) and not in the erythrocytes of family members with a normal P5N activity nor in erythrocytes from healthy donors. We conclude that analysis of the ribonucleotide patterns, in combination with determination of P5N activity, allows a more accurate diagnosis of heterozygosity for P5N.

5'-Nucleotidase↗
PubMed1989

Studies on the incorporation of precursors into purine and pyrimidine nucleotides via 'de novo' and 'salvage' pathways in normal lymphocytes and lymphoblastic cell-line cells.

The incorporation of radioactive precursors into purine and pyrimidine nucleotides via 'de novo' and 'salvage' pathways was measured in normal lymphocytes, resting as well as proliferating, and lymphoblastic cell-line cells (MOLT-3). Lymphocytes stimulated with anti-CD3 were taken as actively proliferating lymphocytes (35% in the S-phase, 40 h after stimulation). The incorporation of the precursors in the purine and pyrimidine ribonucleotides was measured by a combination of anion-exchange high-performance liquid chromatography (HPLC) and on-line radioactivity measurement. The actively proliferating normal lymphocytes and MOLT-3 cells incorporated 30-500 times more of the various precursors in the ribonucleotides compared to normal resting lymphocytes. The imbalance in the nucleotide pool found in proliferating normal and lymphoblastic cells was reflected in the incorporation pattern of the various precursors. The activities of the branch-point enzymes IMP dehydrogenase and CTP synthetase most likely determine the differences in the composition of the nucleotide pools between resting and proliferating cells.

Adenosine Deaminase↗
PubMed1989

Comparison between the chromate inhibition test and a cytochemical method for the determination of glucose-6-phosphate dehydrogenase deficiency in erythrocytes.

The sensitivity and specificity of the chromate inhibition test for the determination of glucose-6-phosphate dehydrogenase (G6PD) deficiency in erythrocytes were compared with a cytochemical staining method. Fifty blood samples were used in a double blind study. The samples were selected from 600 blood samples on the basis of two biochemical criteria, viz. either G6PD activity less than 4.8 IU/g Hb as analysed spectrophotometrically and/or G6PD activity less than glutathione reductase (GSSG-R) activity. The cytochemical assay was taken as reference because it has been proved to be sensitive and specific for the detection of heterozygous and homo/hemizygous forms of deficiency. Cytochemically, one hemizygously deficient patient, 19 heterozygotes and 30 normals were detected. When applying the chromate inhibition test a somewhat different result was obtained with the same samples: one of the 30 normals was classified as heterozygously deficient (3% false positives) and 5 of the 19 heterozygously deficient patients were classified as normal (26% false negatives). It is concluded that the chromate inhibition test is a more sensitive biochemical test than the fluorescence spot test or spectrophotometric assays. However, it is less reliable than the cytochemical test for the detection of heterozygously G6PD deficient patients.

Chromates↗
PubMed1989

Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils.

Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low-mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high-mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X-linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.

Amino Acid Sequence↗
PubMed1989

1-O-hexadecyl-2-Q-methylglycerol, a novel inhibitor of protein kinase C, inhibits the respiratory burst in human neutrophils.

To assess the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the activation of the human neutrophil respiratory burst, we have utilized an ether lipid of the type 1-O-alkyl-2-O-methylglycerol (AMG), recently shown to be an inhibitor of this kinase. AMG-C16 (with an hexadecyl chain at the sn-1 position) was found to inhibit the respiratory burst induced by sub-optimal concentrations of phorbol 12,13-dibutyrate. Respiratory burst activity was recovered by subsequent addition of a supraoptimal dose of phorbol 12-myristate 13-acetate, indicating that in the presence of the inhibitor only the activation of the NADPH:O2 oxidoreductase via protein kinase C is inhibited, but not the oxidoreductase itself. The respiratory burst induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) was also inhibited in the presence of AMG-C16, the extent of inhibition being dependent on the concentration of fMLP. At the concentrations applied in these studies, AMG-C16 had no effect on cell viability, did not affect the formation of inositol phosphates induced by fMLP, and did not affect the characteristics of the Ca2+ fluxes induced by the same stimulus. In a cell-free assay system, AMG-C16 had no effect on the activity of cAMP-dependent or Ca2+/calmodulin-dependent protein kinase but inhibited protein kinase C in a dose-dependent fashion. To characterize the inhibitory action of AMG-C16 on the respiratory burst activity in more detail, we studied protein phosphorylation in relation to respiratory burst activity in neutrophil cytoplasts. We focused on the phosphorylation of the 47-kDa protein, because this protein is functionally associated with the NADPH:O2 oxidoreductase. At suboptimal concentrations of phorbol 12,13-dibutyrate, AMG-C16 inhibited phosphorylation of proteins, including that of the 47-kDa protein. Recovery of protein phosphorylation in parallel to recovery of respiratory burst activity was obtained by addition of increasing doses of phorbol 12,13-dibutyrate. Recovery of respiratory burst activity at intermediate concentrations of fMLP did not result in a proportional increase in 47-kDa protein phosphorylation; phosphorylation of the 47-kDa protein was recovered only at high concentrations of fMLP. From these data we conclude that protein kinase C is involved in the activation of the respiratory burst by phorbol esters and fMLP. However, with fMLP as a stimulus, a second signal seems to be triggered, which is insensitive to AMG-C16.

Calcium↗
PubMed1989

The 40-kDa Fc gamma receptor (FcRII) on human neutrophils is essential for the IgG-induced respiratory burst and IgG-induced phagocytosis.

Neutrophils express two types of receptor for the Fc region of IgG, FcRII and FcRIII. Per neutrophil, 10,000 to 20,000 molecules of FcRII (40 kDa) and 100,000 to 200,000 molecules of FcRIII (50 to 80 kDa) are expressed. Via these receptors, neutrophils bind IgG complexes that contain more than one IgG molecule. This binding activates functional processes, such as the respiratory burst and phagocytosis. We studied the contribution of FcRII and FcRIII in the activation of these processes, using well-defined complexes (both large and small) in combination with mAb against FcRII and FcRIII. Small (dimeric) IgG complexes appeared to bind via FcRIII. However, binding to FcRIII alone, when FcRII is blocked by an anti-FcRII mAb, did not induce a respiratory burst. Induction of the respiratory burst by a large immune complex, such as Staphylococcus aureus Wood opsonized with IgG antibodies, was mediated by binding to FcRII, because it was blocked by an anti-FcRII mAb but not by an anti-FcRIII mAb. This indicates that these IgG-opsonized bacteria can cross-link FcRII and activate the cells without the need to adhere to the FcRIII. The respiratory burst induced by IgG-latex was not inhibited by an anti-FcRII mAb, because the avidity for FcRII of IgG-latex, a particle of the same size as a Staphylococcus but with a two to three times higher IgG content, is increased by its simultaneous binding to FcRIII. This enhanced avidity results in removal of anti-FcRII mAb from the FcRII by IgG-latex. This increased avidity of large complexes for FcRII, created by concurrent binding to FcRIII, is not necessary for activation of human neutrophils, because neutrophils from patients with paroxysmal nocturnal hemoglobinuria, with about 10% of the normal FcRIII expression, showed a normal metabolic response upon addition of IgG-latex. Phagocytosis of IgG-opsonized 14C-labeled S. aureus Wood was inhibited equally well by anti-FcRII mAb and by anti-FcRII in combination with anti-FcRIII mAb. Thus, FcRII is not only essential for the IgG-induced activation of the NADPH oxidase system, but also for the IgG-induced phagocytosis.

Antigens, Differentiation↗
PubMed1989

Binding characteristics of dimeric IgG subclass complexes to human neutrophils.

Immune complexes were prepared by incubation of human IgG paraproteins with F(ab')2 fragments of the mAb K35 against the kappa-L chain of human IgG. The composition of these complexes was analyzed by centrifugation over sucrose gradients, by gel filtration, by RIA with either IgG Sepharose or K35 Sepharose and by double-labeling studies. The results indicated that the complexes consist of saturated tetramers composed of two IgG molecules cross-linked by two F(ab')2 fragments of the mAb. These complexes were used to study the binding of the different IgG subclasses to human neutrophils at 4 degrees C. Human neutrophils bound IgG3 complexes approximately three times faster than IgG1 complexes. Binding of IgG2 or IgG4 dimers to the neutrophils was undetectable. The same number of IgG1 complexes and IgG3 complexes bound to the neutrophils, but considerable inter-donor variation was found (mean number of Fc gamma R per neutrophil: 190,000, range 120,000 to 400,000). The Ka for the binding of IgG1 complexes to neutrophils (median 11 x 10(7) M-1) was lower than the Ka for the binding of IgG3 complexes (median 47 x 10(7) M-1). Competition studies between labeled IgG1 complexes or IgG3 complexes and unlabeled complexes showed that the Fc gamma R of human neutrophils do not display an IgG subclass specificity. Incubation of neutrophils with a mAb against the FcRIII completely blocked the binding of IgG1 complexes and IgG3 complexes. Incubation with a mAb against the FcRII reduced the affinity of the complexes for the neutrophils but had no effect on the maximum number of complexes bound. This indicates that one complex may bind simultaneously to one FcRIII and to one FcRII.

Animals↗
PubMed1989

1,2-Diacylglycerol accumulation in human neutrophils does not correlate with respiratory burst activation.

Measurements of the level of 1,2-diacylglycerol (1,2-DG) during activation of the respiratory burst of human neutrophils by formyl-methionyl-leucyl-phenylalanine (fMLP) in the presence of platelet-activating factor (PAF) or by opsonized particles show that a correlation between accumulation of 1,2-DG and O2 consumption does not exist. Inhibition of protein kinase C activity with staurosporine before addition of opsonized particles demonstrates that the first phase of the respiratory burst is not inhibited, whereas the second phase, which is accompanied by a rise in the content of 1,2-DG, is strongly inhibited. This study indicates that accumulation of 1,2-DG cannot be the sole signal for the initiation of the respiratory burst in human neutrophils.

Alkaloids↗
PubMed1989

Dual mechanisms in priming of the chemoattractant-induced respiratory burst in human granulocytes. A Ca2+-dependent and a Ca2+-independent route.

After interaction with so-called priming agents, the respiratory burst in human granulocytes does not become activated, but is enhanced upon subsequent stimulation with the chemoattractant FMLP. Investigating the mechanism of the priming reaction, we found that a transient rise in the cytosolic free calcium concentration [( Ca2+]i) suffices to irreversibly prime human granulocytes. Thus, platelet-activating factor (PAF) induced a transient increase in [Ca2+]i and primed the cells to an enhanced respiratory burst upon subsequent interaction with FMLP. Artificially, the transient rise in [Ca2+]i was mimicked by addition and subsequent removal of the Ca2+ ionophore ionomycin; this treatment too, primed the respiratory burst of the granulocytes. The priming induced by ionomycin was completely abolished when [Ca2+]i changes were buffered during exposure of the cells to the ionophore. The priming induced by PAF was only partially inhibited under [Ca2+]i-buffering conditions during priming, indicating that multiple pathways exist in the priming of granulocytes by PAF.

Buffers↗
PubMed1989

Deficiency of pyrimidine 5'-nucleotidase in human leukocytes.

Both erythrocytes and leukocytes from a patient with erythrocyte pyrimidine 5'-nucleotidase (P5N) deficiency were shown to contain increased amounts of pyrimidine nucleotides. These findings suggested that the leukocytes were also deficient for P5N. Measurement of the P5N activity in lysates from lymphocytes or granulocytes, in the presence of inhibitors for non-specific 5'-nucleotidase or alkaline phosphatase, indeed showed a deficiency for P5N in lymphocytes and granulocytes of the patient with erythrocyte P5N deficiency. However, the P5N deficiency in the leukocytes did not cause clinical disturbances in addition to the weak haemolytic anaemia.

5'-Nucleotidase↗
PubMed1989

Monitoring of platelet morphology during storage of platelet concentrates.

During storage, human platelet concentrates progressively lose the capacity to survive and function in vivo after transfusion. A shape transformation from disc to sphere is the most reliable in vitro determinant for the loss of the in vivo survival of platelets. To find an objective measurement for platelet morphology, we studied the effect of anticoagulant, temperature, and storage on the apparent median platelet volume (MPV) as determined by a particle counter and on changes in platelet shape as measured and by light microscopy. Changes in MPV, light transmission, and morphology score by light microscopy were observed within 1 minute after collection of blood in CPDA. As compared to blood immediately fixed on withdrawal, in CPDA blood, the MPV increased from 4.1 to 5.7 fl, and light transmission difference decreased from 22 to 7 percent. A partial restoration of these determinants was found when the whole blood was incubated for 30 minutes at 37 degrees C, before preparation of platelet-rich plasma. In the first 5 days of platelet storage, the MPV increased from 4.6 to 5.0 fl; thereafter, it started to decrease. An increase in fragmented platelets after 5 days was observed on light microscopy. The light transmission difference showed a slow disc-to-sphere transformation during storage. This transformation accelerated from Day 5 to Day 7; after 11 days, only spheres were detected. After 7 days the swirling pattern scores were still in accordance with the presence of discs, whereas the other structure-associated determinants showed already spheric and even fragmented platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1989

A phosphoprotein of Mr 47,000, defective in autosomal chronic granulomatous disease, copurifies with one of two soluble components required for NADPH:O2 oxidoreductase activity in human neutrophils.

The NADPH:O2 oxidoreductase (NADPH oxidase) of human neutrophils is converted from a dormant to an active state upon stimulation of the cells. We have studied the soluble fraction that is required for NADPH oxidase activation in a cell-free system. Human neutrophils were separated in a membrane-containing and a soluble fraction. The soluble fraction was separated on carboxymethyl (CM) Sepharose in 10 mM 4-morpholino-ethanesulfonic acid buffer of pH 6.8. Reconstitution of the NADPH oxidase activity, measured as O2 consumption, was only found when the membrane fraction was combined with the flowthrough of the CM Sepharose column as well as with a fraction that eluted at 125 mM NaCl. This result indicates that at least two soluble components are necessary for reconstitution of the NADPH oxidase activity: one that does not bind to CM Sepharose and one that does bind. These components were designated soluble oxidase component (SOC) I and SOC II, respectively. Boiling destroyed the activity in both fractions. In the soluble fraction of human lymphocytes and thrombocytes neither SOC I nor SOC II activity was found. SOC II copurified with a 47-kD phosphoprotein, previously found defective in patients with the autosomal form of chronic granulomatous disease (CGD). Inactive soluble fractions of cells from autosomal CGD patients were reconstituted with a SOC II fraction from control cells. The result of this experiment indicates that autosomal CGD patients are normal in SOC I but defective in SOC II.

Blood Platelets↗