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Biomedical subjects

D Roos

Publications and source records attributed to D Roos.

At least 271 records · Page 15Linked to original sources
PubMed1985

Immunologic studies with LFA-1- and Mo1-deficient lymphocytes from a patient with recurrent bacterial infections.

A patient and his parents, deficient for lymphocyte function associated antigen-1 (LFA-1) and Mo1 (OKM1), were studied with respect to leukocyte surface marker expression and functional properties. The patient had a history of severe recurrent bacterial infections. Two siblings had already died of bacterial infections. The patient's granulocytes, monocytes, and lymphocytes expressed low but detectable amounts (less than or equal to 10%) of LFA-1 and Mo1. Intracellularly, LFA-1 and Mo1 (OKM1) were detectable and LFA-1 expression was enhanced on patient T cells stimulated with phytohemagglutinin. Granulocytes and monocytes of both the patient's parents expressed markedly decreased amounts of LFA-1 and Mo1. Lymphocytes of the mother expressed 40 to 60% of the amount of LFA-1 expressed on control lymphocytes, but his father's lymphocytes showed a normal LFA-1 expression. Granulocytes of the patient and of his deceased sister showed normal phagocytosis, but they had a dysfunction in the activation of the oxidative metabolism. Functional activities mediated by patient T cells were all normal. Moreover, all lymphocyte functions, including killer (K), natural killer (NK), cytotoxic T cell activity, helper activity for in vitro immunoglobulin (Ig) production by normal B cells, and PHA-induced proliferation were inhibitable by anti-LFA-1 monoclonal antibodies. K and NK activity mediated by patient leukocytes was 100-fold more sensitive to the inhibiting effect of anti-LFA-1 antibody than K and NK activity of normal donor leukocytes. Thus, although the amount of LFA-1 expressed was strongly reduced, it was still sufficient and required for the functional activity exhibited by patient T cells. The major functional defect observed with leukocytes of the patient and his father was an apparent B cell defect. B cells of the father and of the patient failed to produce Ig in the pokeweed mitogen (PWM)-driven system. The B cells of patient and of his father only produced Ig when cultured with T cells of the father, and not with normal donor T cells or T cells of the mother, in the presence of exogenous interleukin 2 (IL 2). In addition, the father's B cells produced Ig when cocultivated with patient T cells in the IL 2-driven system. This restriction of helper T cell activity is noteworthy because PWM- and IL 2-driven Ig synthesis by normal lymphocytes show no histocompatibility requirements between cooperating T and non-T cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Surface↗
PubMed1985

Characterization of the interaction of human eosinophils and neutrophils with opsonized particles.

The interaction of human eosinophils with opsonized particles was compared with that of human neutrophils. When eosinophils are stimulated with serum-opsonized zymosan particles, the lag time in H2O2 production is twice as long as found with neutrophils. Moreover, the concentration of these IgG + C3-coated particles required for optimal stimulation is about four times as high for eosinophils as for neutrophils. Under these conditions, the two cell types generate similar amounts of H2O2. However, eosinophils produce twice as much H2O2 as do neutrophils when stimulated with the soluble agent phorbol myristate acetate. Thus, although the oxidase capacity of eosinophils is larger than that of neutrophils, opsonized zymosan is a weak trigger for this activity in eosinophils. This phenomenon may be due to differences between the two cell types in the plasma membrane receptors or in the receptor oxidase transducing signal. The following are indications for the first possibility. i) IgG interacts poorly with the Fc gamma receptors on the eosinophil surface compared with those on neutrophils. This was shown by the inability of IgG-coated zymosan or IgG-coated latex to trigger any substantial H2O2 production by eosinophils unless brought into close contact with these cells by centrifugation. In contrast, neutrophils are stimulated by these particles both in suspension and in a pellet. The dissimilarity of the Fc gamma receptors on eosinophils and neutrophils was also shown with respect to antigenicity, determined by the monoclonal antibodies 3G8 and CLB-FcR-1. ii) Eosinophils contain about half as many receptors for C3b and C3bi on their surface as do neutrophils, also detected with monoclonal antibodies. The interaction of IgG subclasses with functional Fc gamma receptors on eosinophils and neutrophils showed that eosinophils release twice as much H2O2 as do neutrophils upon interaction with IgG1-, IgG2-, or IgG3-coated Sepharose beads, but this difference becomes fivefold with IgG4-coated Sepharose. This might be of relevance to the situation of chronic antigenic stimulation, e.g., in chronic schistosomiasis, in which eosinophil numbers and IgG4 antibody levels are elevated.

Antibodies, Monoclonal↗
PubMed1984

Cytochrome b, flavins, and ubiquinone-50 in enucleated human neutrophils (polymorphonuclear leukocyte cytoplasts).

Neutrophilic granulocytes contain an oxidase system in their plasma membrane that can be activated to generate superoxide radicals and hydrogen peroxide. Cytochrome b, flavoprotein, and ubiquinone-50 have been proposed as components of this oxidase system. These components have been quantitated, but the results are obscured by different isolation procedures for plasma membranes from resting and activated neutrophils. This problem has now been avoided by the use of enucleated neutrophils (polymorphonuclear leukocyte cytoplasts), which are almost completely devoid of intracellular structures but contain an intact, activatable oxidase system (Roos, D., Voetman, A.A., and Meerhof, L.J. (1983) J. Cell Biol. 97, 368-377). Membranes of resting and phorbol myristate acetate-stimulated cytoplasts contain equal amounts of cytochrome b (4 pmol/milliunit of alkaline phosphatase) and also equal amounts of noncovalently bound FAD (2 pmol/milliunit of alkaline phosphatase). These findings refute the hypothesis that incorporation of cytochrome b and/or a flavoprotein into the plasma membrane constitutes the mechanism of activation of the oxidase system. Ubiquinone-50 is present neither in intact neutrophils nor in cytoplasts, excluding a role for this compound in the generation of bactericidal oxygen species by neutrophils.

Cell Fractionation↗
PubMed1984

Excretion of superoxide by phagocytes measured with cytochrome c entrapped in resealed erythrocyte ghosts.

Resealed erythrocyte membranes (ghosts) filled with (Fe3+)cytochrome c were used as an assay system to measure the release of superoxide (O-2) from human phagocytes into the incubation medium. Neutrophils, activated by either opsonized zymosan particles or the soluble stimulus phorbol myristate acetate, released O-2, which subsequently entered the ghosts and reduced (Fe3+)cytochrome c. This reaction was dependent on the time of incubation, the concentration of neutrophils, the concentration of stimulus, and the concentration of ghosts. The reaction was completely inhibited by superoxide dismutase and by 4,4'-diisothiocyano-2,2'-disulfonic acid, a specific blocker of anion channels in membranes. The reduction of (Fe3+)cytochrome c free in solution was about four times as fast as the reduction of (Fe3+)cytochrome c in the ghosts. Human eosinophils stimulated by phorbol myristate acetate reacted similarly to human neutrophils; the rate of O-2 production/cell was about twice as high for eosinophils as for neutrophils. In contrast, eosinophils stimulated with opsonized zymosan particles only reduced (Fe3+)cytochrome c free in solution, but not (Fe3+)cytochrome c in ghosts. This lack of reaction was not due to production of an inhibitor or below threshold generation of O-2 for the ghost assay. These results indicate: 1) activated human neutrophils and eosinophils can release O-2 or a similar product into the incubation medium; and 2) reduction of (Fe3+)cytochrome c free in solution is no proof for O-2 excretion by phagocytes.

Animals↗
PubMed1984

Pyruvate kinase and the "high ATP syndrome".

The erythrocytes of a patient with the so-called "high ATP syndrome" were characterized by a high ATP content and low 2,3-diphosphoglycerate level. The pyruvate kinase activity was specifically increased (about twice the normal level). After separation of the erythrocytes according to age by discontinuous Percoll density centrifugation, the pyruvate kinase activity was found to be increased in all Percoll fractions. Pyruvate kinase of the patient's cells was characterized by a decreased K0.5 for the substrate phosphoenolpyruvate and no inhibition by ATP. The Michaelis constant (Km) value for ADP, the nucleotide specificity, the thermostability, pH optimum, and immunological specific activity were normal. It is concluded that the high pyruvate kinase activity is due to a shift in the R(elaxed) in equilibrium T(ight) equilibrium to the R(elaxed) form.

2,3-Diphosphoglycerate↗
PubMed1984

Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli.

A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D-galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta-galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta-galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.

Blood Bactericidal Activity↗
PubMed1984

Organelle-depleted human neutrophil cytoplasts used to study fmet-leu-phe receptor modulation and cell function.

Organelle-depleted human neutrophil cytoplasts were used to study N-formylmethionylleucylphenylalanine (fmet-leu-phe) receptor regulation and adaptation, aggregation, chemotaxis, and chemoattractant-elicited changes in shape, volume, and surface charge. Cytoplasts aggregated in response to the chemoattractant fmet-leu-phe, but the magnitude of the response was less than that in neutrophils. Unlike neutrophils, cytoplasts did not exhibit a second wave of aggregation with the addition of cytochalasin B and also failed to disaggregate. In chemotactic assays, cytoplasts migrated poorly into cellulose nitrate filters, and compared with intact neutrophils required a 100-fold greater concentration of fmet-leu-phe to elicit shape change. In contrast to neutrophils, cytoplasts did not decrease their surface charge in response to fmet-leu-phe and did not exhibit an increase in the binding of fmet-leu-[3H]phe after stimulation with phorbol myristate acetate. However, receptor adaptation and induced changes in membrane potential, as measured by using the membrane probe di-O-C5(3), were similar in cytoplasts and in neutrophils. The data suggest that the presence of functional intracellular organelles is required for normal aggregation and disaggregation, chemotaxis, and shape change induced by fmet-leu-phe, and also peptide receptor upregulation in response to secretagogues. The data show that peptide receptor adaptation occurs in the absence of secretory granules and is independent of receptor upregulation .

Carbocyanines↗
PubMed1984

Cryopreservation of enucleated human neutrophils (PMN cytoplasts).

Previously, we have shown that enucleated human neutrophils (PMN cytoplasts), when activated by particulate or fluid stimuli, generate superoxide and hydrogen peroxide at rates comparable (per unit area of plasma membrane) to those observed with intact neutrophils. Moreover, PMN cytoplasts also ingest and, to a certain extent, kill bacteria. We now report that PMN cytoplasts can be cryopreserved with maintenance of their functional activity. The PMN cytoplasts were frozen in a medium with 10% (v/v) fetal calf serum and 10% (v/v) dimethyl sulfoxide, and stored at -70 degrees C. After thawing and washing, the recovery was 75%. The content of alkaline phosphatase and lactate dehydrogenase, the consumption of oxygen and generation of hydrogen peroxide, and the rate of phagocytosis of Staphylococcus aureus bacteria was the same for fresh and cryopreserved PMN cytoplasts. Identical values were obtained after preservation in liquid nitrogen. These results open possibilities to store neutrophil material, allowing longitudinal follow-up of patients, comparative studies between different patients, exchange of material between laboratories, and storage of reference material for experiments in series.

Blood Bactericidal Activity↗
PubMed1983

Continuous therapy with sulfamethoxazole-trimethoprim in patients with chronic granulomatous disease.

In a retrospective study, the effect of long-term treatment with sulfamethoxazole-trimethoprim was evaluated in nine male patients with chronic granulomatous disease. During this treatment, a marked reduction was observed in the number of infectious episodes, the number of causative agents, and the number of surgical interventions. Furthermore, a significant reduction in days of hospitalization per year was found. The mean observation period was six years before and four years during treatment. Transient alopecia was observed in one patient during therapy. We conclude that prophylactic treatment with sulfamethoxazole-trimethoprim is beneficial in patients with chronic granulomatous disease.

Adolescent↗
PubMed1983

Granulocytes without degranulation: neutrophil function in granule-depleted cytoplasts.

Neutrophils respond to a variety of stimuli by generating superoxide anion, degranulating, and aggregating. Because it has been suggested that fusion of granules with the plasmalemma (degranulation) is necessary for aggregation and superoxide anion generation, we have tested whether these responses can be demonstrated in "neutrophilic cytoplasts" (granule-free vesicles of cytoplasm enclosed by plasmalemma). When examined by electron microscopy, cytoplasts were found to be approximately 4 microns in diameter and essentially granule free. Cytoplasts exposed to fMet-Leu-Phe (0.1 microM) generated superoxide anion after a lag of 16 sec but released no detectable beta-glucuronidase, lysozyme, or elastase. Aggregation of cytoplasts, as measured by changes in light transmission, was also activated by fMet-Leu-Phe; no lag period was observed. Electron microscopy of the aggregates demonstrated clusters of cytoplasts with a scalloped appearance. Superoxide anion generation and aggregation of cytoplasts were also activated by phorbol 12-myristate 13-acetate, concanavalin A, and leukotriene B4. Exposure of cytoplasts to the dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)] led to dye uptake and enhancement of fluorescence, implying that the vesicles were sealed and maintained a membrane potential across the plasmalemma. Exposure of DiOC6(3)-loaded cytoplasts to fMet-Leu-Phe and PMA caused a rapid loss of dye fluorescence that was not inhibited by CN-, compatible with their lack of mitochondria. Exposure of dye-loaded cytoplasts to concanavalin A or leukotriene B4 caused an increase in fluorescence--i.e., a hyperpolarization. These results demonstrate that degranulation is not a prerequisite for aggregation or superoxide anion generation. The retention of ionic gradients and changes in membrane potential, as measured by DiOC6(3) fluorescence changes, suggest a fundamental role for ionic movements in activating superoxide anion generation and aggregation.

Cytochrome c Group↗
PubMed1983

Functional activity of enucleated human polymorphonuclear leukocytes.

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.

Blood Bactericidal Activity↗
PubMed1983

Granulocytes without degranulation: neutrophil function in granule-depleted neutroplasts.

Neutroplasts, which are vesicles consisting of cytoplasm enclosed by plasmalemma, have been prepared and found to be incapable of degranulation in response to f-Met-Leu-Phe. However, neutroplasts generate superoxide anion in response to f-Met-Leu-Phe and PMA, and therefore, degranulation is not essential for superoxide anion generation. In addition, neutroplasts aggregate and show shape changes in response to f-Met-Leu-Phe and PMA, and thus degranulation is not essential for aggregation. Neutroplasts take up the carbocyanine dye DiOC6(3), thereby providing evidence for ionic gradients. Dye-loaded neutroplasts show fluorescence changes in response to a variety of stimuli. This response is not CN--inhibitable; therefore, activation of neutroplasts is associated with changes in membrane potential at the plasmalemma, suggesting a role for ion fluxes in the activation sequence for aggregation and superoxide anion generation.

Anions↗
PubMed1983

Low beta-adrenergic receptor concentration on human thymocytes.

Several agents are known that can elevate cyclic AMP levels in lymphoid cells, e.g. isoproterenol, PGE1 and adenosine. We have studied the cyclic AMP increasing effect of these agents on thymocytes from mouse and man and on human peripheral T lymphocytes. In contrast to mouse thymocytes and human peripheral T lymphocytes, human thymocytes appeared to be insensitive to isoproterenol, but did respond to PGE1 and adenosine. Furthermore, the density of beta-adrenergic receptors on the cells was determined by measuring the specific binding of 3H-dihydroalprenolol. A correlation was found between the receptor density on the cells and the rise in intracellular cyclic AMP induced by isoproterenol: human thymocytes appeared to have very few beta-adrenergic receptors, in contrast to thymocytes from mouse or to T lymphocytes from human blood. We conclude that the development of beta-adrenergic receptors in T cell ontogeny is different for mice and human beings. Comparison of animal models with the situation in man should be made with caution.

Adenosine↗
PubMed1983

Enzyme analysis of lymphoproliferative diseases: a useful addition to cell surface phenotyping.

Lymphocytes from patients with acute and chronic T-cell malignancy or chronic T gamma lymphocytosis were characterized by studying the activity of three enzymes involved in purine metabolism and by determining the isoenzyme pattern of lactate dehydrogenase (LDH) in addition to analysis of surface marker expression with monoclonal antibodies. Four clinically different types of disease were distinguished on the basis of the enzyme parameters. Lymphocytes from patients with acute lymphocytic leukemia (T-ALL) showed an enzyme profile similar to that of normal thymocytes, i.e., an elevated level of adenosine deaminase (ADA) activity as compared with normal T lymphocytes, reduced activities of purine 5'nucleotidase (5'NT) and purine nucleoside phosphorylase (PNP), and a binomial distribution of the LDH isoenzyme pattern. Cells from "null"-ALL patients had an ADA/PNP ratio that was intermediate between that of normal T cells and that of T-ALL cells or thymocytes, but their 5'NT activity and LDH isoenzyme pattern were thymocyte-like. Patients with chronic T-cell proliferation were subdivided into those with chronic T gamma lymphocytosis and those with proven chronic T malignancy. The lymphocytes from these patients had ADA and PNP activities within the ranges of those of normal T lymphocytes. However, the ADA activity and/or the ADA/PNP ratio were consistently higher in the cells from the patients with chronic T gamma lymphocytosis than in those with chronic T malignancy. The enzyme profile of the cells from the T gamma patients was similar to that of T gamma cells of normal individuals. The cells from patients with chronic T malignancies showed a heterogeneous enzyme pattern as compared with that of normal T lymphocytes. Analysis with monoclonal antibodies enabled us to distinguish null-ALL patients from the other leukemias studied, but a distinction between chronic and acute T-cell proliferation disease, for instance, was not possible with monoclonal antibodies alone. Our data demonstrate that the enzyme profiles studied provide supplementary information for classification and diagnosis of lymphoproliferative diseases to that obtained with cell surface markers alone.

5'-Nucleotidase↗
PubMed1982

Oxidative damage to lysosomal enzymes in human phagocytosing neutrophils.

During phagocytosis neutrophils from 8 patients with chronic granulomatous disease released 2-3 times more activity of lysozyme and beta-glucuronidase than did normal neutrophils. This difference was caused by the partial inactivation of these enzymes by normal neutrophils. The inactivation of granule enzymes depends on oxidative products and takes place mainly in the phagolysosomes. Myeloperoxidase is involved in this phenomenon.

Granulomatous Disease, Chronic↗
PubMed1982

Discrepancies in the oxygen balance of whole human neutrophils and neutrophil homogenates.

The oxygen consumption, superoxide production and hydrogen peroxide generation was studied in human neutrophils phagocytosing zymosan particles. Application of sodium azide, as an inhibitor of catalase, and/or 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), as an inhibitor of glutathione reductase, led to the conclusion that neutrophils convert about half of the oxygen consumed in the respiratory burst to hydrogen peroxide; the other half is used for formation of organic peroxides, disulfide bridges, etc. These products are rapidly degraded to water by catalase and/or the glutathione redox cycle. Reduction of exogenous cytochrome C accounted for only about 15% of the consumed oxygen. Neutrophil homogenates contain a badly damaged oxidase system, because oxygen consumption and hydrogen peroxide formation were only about one-tenth of that observed with whole cells. In contrast, cytochrome-C reduction was about three times as high as that found with intact cells. Probably, cytochrome C partly reconstitutes damaged oxidase systems, thus artificially increasing the oxidase activity. We conclude that cytochrome-C reduction is not a good parameter to characterize cell-free oxidase preparations.

Cell-Free System↗