Effects of low altitude exposure on 24-hour blood pressure and adrenergic activity.
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Adolescent↗
Biomedical subjects
G Berton
Publications and source records attributed to G Berton.
Studies on the NADPH oxidase of phagocytes. Production of a monoclonal antibody which blocks the enzymatic activity of pig neutrophil NADPH oxidase.
We describe in this paper a monoclonal antibody to pig NADPH oxidase which inhibits enzymatic activity. This antibody, designated 1H8.2, was selected from a group of monoclonal antibodies produced against active preparations of purified NADPH oxidase and which showed selectivity of binding. 1H8.2 is an IgM restricted in binding to pig NADPH oxidase and showing higher binding to NADPH oxidase purified from phorbol myristate acetate-stimulated than from resting neutrophils. The antibody inhibits by about 90% the oxidase activity at 20-50 micrograms/ml. Inhibition is due to a decrease of the Vmax of the oxidase, and the Km is not affected. Incubation of the NADPH oxidase with 1H8.2 in the presence of concentrations of NADPH up to 25-fold the Km does not prevent the inhibition. Together with the evidence that the antibody does not inhibit the neutrophil superoxide dismutase-insensitive NADPH cytochrome c reductase and the liver NADPH-cytochrome c reductase this observation indicates that the 1H8.2 does not bind to an epitope belonging to the NADPH-binding site. Experiments of immunoprecipitation of iodinated membrane proteins and of immunoaffinity purification showed that 1H8.2 recognizes a heterodimer of apparent molecular mass of 16/18 and 14 kDa. These polypeptides can be involved in the NADPH oxidase activity or represent still unrecognized molecules able to modulate its function.
Animals↗
Generation of superoxide anion by alveolar macrophages in sarcoidosis: evidence for the activation of the oxygen metabolism in patients with high-intensity alveolitis.
We studied superoxide anion (O2) generation by alveolar macrophages (AM) isolated from bronchoalveolar lavages (BAL) of patients with sarcoidosis, and assayed immediately after the isolation or after maintenance in culture for 2 days. In assays of cells freshly isolated from BAL, AM of patients with active sarcoidosis with a high-intensity lymphocytic alveolitis produced more O2- in response to phorbol myristate acetate than AM of patients with inactive sarcoidosis. Also, after 2 days of cultivation sarcoid AM were heterogeneous in their capability to metabolize oxygen, although both AM of active and inactive sarcoid patients produced higher amounts of O2- than AM of healthy subjects. In vitro treatment with recombinant interferon-gamma (rIFN-gamma) caused an enhancement of the capability of AM of inactive sarcoid patients to produce O2- in response to PMA. AM of patients with active sarcoidosis did not respond to rIFN-gamma when they already produced O2- vigorously. However, they became sensitive to the activating effect of rIFN-gamma after the down-modulation of their capability to produce O2-, that occurred upon prolonged cultivation. Monocytes isolated from blood of sarcoid patients and assayed immediately or after different times of cultivation did not produce more O2- than control monocytes and monocyte-derived macrophages, thus indicating that the activation of AM in sarcoidosis is likely a local phenomenon. These studies strengthen the notion that T lymphocyte-macrophage interaction is a critical event in the pathogenesis of sarcoidosis and establish that the enhanced capability to metabolize oxygen to highly reactive intermediates by AM is one of the consequence of this interaction.
Adult↗
[Changes in arterial pressure and adrenergic activity during a holiday at a mountain at low altitude].
To answer the question often asked by hypertensive patients whether it is advisable for them to spend a holiday in the mountains for fear of a rise in blood pressure, 12 untreated mild hypertensive and 12 normotensive subjects underwent 24-hour non-invasive blood pressure monitoring, plasma catecholamines assay (HPLC) and plasma renin activity determination at home (sea level) and in a mountain resort (1210 m). The 2 evaluations (at home and in the mountains) were performed in a cross-over fashion. The ICR Spacelabs Pressuromaster and the Del Mar Avionics Pressurometer IV were used: blood pressure was measured every 8 minutes during daytime and every 15 minutes during sleep. Blood pressure was also recorded during a 1.5 to 3 hour excursion by cable-car to a height of 3006 m. Average 24-hour blood pressure at sea level was 134/86.7 mmHg in the hypertensive subjects and 115.8/73.6 mmHg in the normotensive ones. At 1210 m, it rose to 140.6/90.2 mmHg (n.s.) and to 120.8/74.9 mmHg (n.s.) respectively. Twenty-four-hour profiles showed that the blood pressure difference was present only during waking hours, while during sleep blood pressure levels were similar at sea level and in the mountains.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
The Km of NADH dehydrogenase is decreased in mitochondria of cystic fibrosis cells.
The kinetic properties of the NADH dehydrogenase of the mitochondrial respiratory chain, assayed as NADH-dependent rotenone-sensitive cytochrome c reductase have been studied in mitochondria isolated from mononuclear white blood cells in patients affected by cystic fibrosis. Data reported here show that the apparent Km of the enzyme for NADH is significantly decreased in cystic fibrosis mitochondria. These findings are independent of the age or the clinical state of the disease and have also been obtained with mitochondria isolated from cultured skin fibroblasts. These observations support the notion that cystic fibrosis is possibly accompanied by alterations of intracellular membranes and these are evident also in circulating cells and cultured fibroblasts.
Adolescent↗
Interferon-gamma activates human neutrophil oxygen metabolism and exocytosis.
Here we have investigated the ability of recombinant interferon-gamma (rIFN-gamma) to modulate human neutrophil (PMN) functions. PMN incubated in the presence of rIFN-gamma showed an enhanced hydrogen peroxide production in response to Con A, FMLP, PMA or immune complexes. The effect of rIFN-gamma was dose dependent, being half maximal at 2 U/ml, and required between 90 min and 240 min of incubation to reach optimal response. The enhancing effect of rIFN-gamma on the respiratory response of PMN was not blocked by polymixin B sulphate, but an anti-rIFN-gamma monoclonal antibody and cycloheximide and actinomycin D were effective inhibitors. The enhancement of the response to Con A was not accompanied by an enhanced binding of the lectin. Neither the kinetic properties of the Con A-stimulated NADPH oxidase nor the expression of cytochrome b-245 were altered in rIFN-gamma-treated PMN. rIFN-gamma also enhanced granule secretion in response to Con A, FMLP and PMA. Initial studies on the possible alterations of transmembrane signalling in rIFN-gamma-treated PMN showed that neither inositol phosphates formation nor cytoplasmic calcium transients were altered.
Calcium↗
[Reproducibility of ambulatory blood pressure monitoring].
Short-term blood pressure variability has been studied extensively with monitoring techniques. To assess whether or not and to what extent average 24-hour blood pressure varies when repeatedly recorded, 12 mild to moderate hypertensive subjects underwent 3 non-invasive blood pressure monitorings at monthly intervals. When the average 24-hour blood pressure of the whole group was evaluated no substantial differences were found between the 3 recordings, even though slightly lower blood pressure values were recorded at the first monitoring. However, when the within-period comparison was studied in the single patients, remarkable discrepancies of up to 20.9/15.8 mmHg were detected. Systolic and diastolic blood pressure variations greater than or equal to 5 mmHg were recorded in 58% of the subjects. These differences were unrelated to the number of faulty readings. Casual blood pressure varied even to a greater extent between the first and the subsequent visits; no correlation was found between casual and 24-hour blood pressure variations. The results of the present study indicate that ambulatory blood pressure may vary even to a great extent when repeatedly recorded. This finding entails important clinical implications, as the evaluation of the hypertensive patient is usually made with a single blood pressure monitoring.
Adult↗
Desensitization of macrophage oxygen metabolism on immobilized ligands: different effect of immunoglobulin G and complement.
During adhesion and spreading to immobilized immune complexes, casein-elicited mouse peritoneal macrophages produced superoxide anion. This production was time-dependent, ceased after a couple of hours, and was due to interaction with immunoglobulins G (IgG) because neither immobilized antigen alone nor immunoglobulins M with or without complement-derived fragments were efficient stimuli. Cultivation of macrophages on immobilized IgG for 24 hr caused desensitization of the response to an unrelated stimulus like zymosan. Desensitization was due neither to inhibition of binding and uptake of zymosan nor to alterations of NADPH oxidase. In fact, macrophages cultivated on immobilized IgG bound and internalized zymosan and responded to PMA with production of superoxide anion normally. Desensitization was not specific for casein-elicited macrophages because both resident peritoneal and Corynebacterium parvum-activated macrophages underwent desensitization if cultivated for 24 hr on immobilized immune complexes. Desensitization on immobilized IgG was maximal after 24 hr, lasted up to 3 days in culture, and was reversed by detaching macrophages from the IgG surface and further cultivating them in normal tissue culture plastic. Scavengers of products of the oxygen metabolism such as superoxide dismutase and catalase and inhibitors of arachidonic acid metabolism such as indomethacin and nordihydroguaiaretic acid did not prevent desensitization. In addition, the zymosan-stimulated release of arachidonic acid was suppressed after cultivation on immobilized IgG for 24 hr; also in this case, the response to PMA was conserved. Contrary to cultivation on immobilized IgG, cultivation of macrophages on fragments derived from C3 was not accompanied by desensitization of the response to zymosan. These results indicate that although the interaction of Fc receptors with their ligands does not impair binding and uptake of zymosan, alterations in the sequence of signals which leads to the activation of the oxygen metabolism can occur, causing a complete dissociation between phagocytosis and stimulation of the oxygen metabolism.
Animals↗
Soluble interleukin-2 receptors in the sera of patients with hairy cell leukemia: relationship with the effect of recombinant alpha-interferon therapy on clinical parameters and natural killer in vitro activity.
In this study we provide evidence that the sera of patients with hairy cell leukemia (HCL) contain a factor that can prevent the binding of a monoclonal antibody specific for interleukin-2 receptor (IL-2R) to its target. This factor corresponds to the soluble form of IL-2R (sIL-2R), as assessed by a specific enzyme-linked immunosorbent assay test, and appears to be released by neoplastic hairy cells. The serum sIL-2R levels were very high at diagnosis and significantly reduced during recombinant alpha-interferon (rIFN alpha 2) therapy. Values of sIL-2R appeared to be inversely related to the natural killer in vitro function displayed by peripheral blood mononuclear cells from the same patients. The presence of sIL-2R in the serum of patients with HCL might be involved in the impairment of cell-mediated immunity observed in these patients and could represent a valuable marker for monitoring different phases of the disease and for modulating IFN therapy.
Antibodies, Monoclonal↗
Measurement of NADPH oxidase activity in detergent lysates of human and mouse macrophage monolayers.
An assay to measure NADPH oxidase activity in detergent lysates of macrophage monolayers is described. The addition of a reaction mixture containing appropriate concentrations of disrupting detergents, NADPH as oxidase substrate and cytochrome c as electron acceptor, to macrophages monolayers permits the reliable detection of a superoxide dismutase-sensitive NADPH-dependent cytochrome c reductive activity. This activity is strictly substrate dependent and NADH could not substitute for NADPH. The NADPH-dependent superoxide anion-forming activity (NADPH oxidase) was investigated in different populations of human and mouse macrophages. NADPH oxidase was activated by stimulation of macrophages with phorbol-myristate acetate and activity levels correlated with ability of intact cells to produce superoxide anion. The optimal conditions for assay of NADPH oxidase were investigated and the assay was used to measure the kinetic properties of the NADPH oxidase. The assay permits investigations of the enzymatic basis of oxidative metabolism in macrophages cultivated as adherent cells without any requirements for recovery of the cells in suspension and subcellular fractionation.
Animals↗
Gamma interferon is able to enhance the oxidative metabolism of human neutrophils.
The oxidative metabolism of human neutrophils has been studied after incubation of the cells with recombinant interferon-y. Neutrophils incubated for 2-4 hours with 2-50 U/ml recombinant interferon-y undergo a higher respiratory burst measured both as Oz consumption and Oz- production when stimulated with formyl-methionyl-leucyl-phenylalanine, Concanavalin A or phorbol myristate acetate. The potentiating effect of interferon-y requires more than one hour of incubation, is optimal at 20-50 U/ml and depends on the presence of serum in the incubation medium. The interferon effect depends on new protein synthesis. Cycloheximide at doses which do not alter the respiratory response of normal cells completely prevents the potentiating effect of interferon.
Concanavalin A↗
Molecular basis of macrophage activation. Expression of the low potential cytochrome b and its reduction upon cell stimulation in activated macrophages.
The expression of the novel b-type cytochrome, which is part of the superoxide anion (O2-)-generating system in phagocytes, has been investigated in population of mouse peritoneal macrophages heterogeneous in their capability to produce O2-). Reduced minus oxidized difference spectra of intact cells showed the appearance of a b-type cytochrome with major peaks in the alpha region at 558 to 559 nm and in the gamma region at 426 to 428 nm. Resident peritoneal macrophages, as well as thioglycollate broth-elicited and Corynebacterium Parvum-activated macrophages and neutrophils expressed about 50 pmol cytochrome b/10(7) cells. In intact macrophages and neutrophils, Na-dithionite reduced greater than 75% of the cytochrome b measurable in disrupted cells. No correlation was found between capability to produce O2-) by different population of macrophages and their content of cytochrome b. When stimulated in strictly anaerobic conditions with phorbol myristic acetate, macrophages activated in vivo by i.p. injection of Corynebacterium Parvum reduced approximately 40% of their total cytochrome b. In resident peritoneal macrophages that produced five times lower amounts of O2-, cytochrome b reduction was instead undetectable. Potentiometric properties of cytochrome b was investigated in macrophage subcellular particles. Both resident and Corynebacterium Parvum-activated macrophages revealed the presence of b chromophores with very low potentials of -255 and -244 mV, respectively, whose content was not different in the two populations. These results show that resident and activated macrophages express the same amount of cytochrome b, but upon stimulation with PMA, activated macrophages recruit a higher number of cytochrome b molecules in parallel with an enhanced production of O2-.
Animals↗
Monoclonal antibodies to a particulate superoxide-forming system stimulate a respiratory burst in intact guinea pig neutrophils.
Monoclonal rat antibodies were produced against a subcellular preparation of phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig neutrophils that retains NADPH-oxidase activity. Two antibodies, 1A10.4 and IG4, were isolated that bind to a surface antigen restricted to guinea pig neutrophils from bone marrow and peritoneal exudate and to macrophages and that trigger a respiratory burst in neutrophils in the presence of cytochalasin B. Intact antibody 1A10.4, subclass IgG2c, can trigger superoxide anion release directly; F(ab')2 fragments of 1A10.4 and intact IG4 require further cross-linking by F(ab')2 fragments of anti-rat immunoglobulin antibody. Both antibodies recognize the same antigen, a proteolipid of apparent molecular mass 10 kDa. Immunoprecipitation of solubilized oxidase activity with 1A10.4 brings down this activity as part of a macromolecular complex. Surface expression of the antigen is increased on treatment of cells with both PMA and cytochalasin B. 1A10.4 also triggers release of the granule enzyme beta-glucuronidase. Triggering of a respiratory burst by the antibodies appears distinct from the PMA and fMet-Leu-Phe signalling systems. These studies indicate that the antigen defined by antibodies 1A10.4 and IG4 becomes associated with the superoxide anion-generating system of neutrophils but may play a more general role in signal transduction in phagocytic cells.
Animals↗
Partial purification of the superoxide-generating system of macrophages. Possible association of the NADPH oxidase activity with a low-potential (-247 mV) cytochrome b.
NADPH oxidase activity was solubilized by detergent treatment of subcellular particles obtained from guinea-pig peritoneal macrophages stimulated with phorbol myristate acetate. Gel filtration of the material containing the NADPH oxidase activity gave two peaks of proteins, one of which eluted with the void and the other with the included volume of an AcA 22 column. The material eluted in the void volume contained more than 50% of the NADPH oxidase activity and less than 10% of the NAD(P)H cytochrome c reductase activity. A b-type cytochrome with peaks of absorption at 558, 528 and 426 nm was also enriched in the fraction which contained the NADPH oxidase activity. The distribution of flavoproteins as revealed by the measurement of FAD was different from that of NADPH oxidase and cytochrome b, and followed the elution profile of NADH cytochrome c reductase. Studies in subcellular particles showed that the b cytochromes of mitochondria and endoplasmic reticulum reduced by selective biochemical means accounted for only a minor part of the total b-type cytochromes and that the new cytochrome b previously described in neutrophils is the major chromophore also in macrophages. Oxidation-reduction midpoint potential of the partially purified cytochrome b was shown to be -247 mV. Association of cytochrome b with the NADPH oxidase activity and its very low Em7.0 makes it a suitable candidate to be part of the superoxide-generating system also in macrophages.
Animals↗
Activation by gamma interferon of human macrophage capability to produce toxic oxygen molecules is accompanied by decreased Km of the superoxide-generating NADPH oxidase.
Capability to release superoxide anion in response to phorbol myristate acetate by intact cells has been compared with Kinetic properties of NADPH oxidase by lysates of human monocytes and monocyte-derived macrophages. Maturation of monocytes in vitro is accompanied by substantial decrease of the capability to release superoxide anion in response to phorbol myristate acetate. Exposure of mature macrophages to recombinant interferon gamma enhances respiratory burst activity up to 3-4 fold. Modifications of NADPH oxidase accompany changes in the ability to release superoxide anion. The affinity of the oxidase for its substrate is higher in monocytes and gamma interferon treated macrophages, while Vmax is not changed.
Humans↗
Mechanism of production of toxic oxygen radicals by granulocytes and macrophages and their function in the inflammatory process.
The paper deals with 1) the features of the respiratory burst (increase of the respiration with production of O2 metabolites, O2-, H2O2, OH) of the inflammatory cells; 2) the factors responsible for its activation; 3) the methods for its measurement; 4) the molecular events which take place at the level of the plasma membrane following the interaction between the stimuli and the cell surface (the Ca++ changes, the modification of membrane potential, the activation of phospholipid turnover) and the hypothesis of the activation of the protein kinase C; 5) the nature of the NADPH oxidase whose activation is responsible for the respiratory burst and the production of O2 metabolites; 6) the defensive, toxic, proinflammatory and modulatory effects due to the reactivity of the oxygen metabolites.
Animals↗
Activation of mouse macrophages causes no change in expression and function of phorbol diesters' receptors, but is accompanied by alterations in the activity and kinetic parameters of NADPH oxidase.
Mouse peritoneal macrophages activated in vivo by the injection of Corynebacterium parvum release larger amounts of superoxide anion (O2-) than macrophages from control mice when stimulated with phorbol myristate acetate (PMA). The biochemical bases for this enhanced response of activated macrophages have been investigated by studying the expression and function of receptors for the stimulant, and the activity of the enzyme NADPH oxidase which is responsible for the production of O2- in leucocytes. Studies of binding of phorbol dibutyrate, an agent closely related to PMA, showed that the affinity constants (Kds) and the number of binding sites were the same in resident and activated peritoneal macrophages. The activity of the NADPH oxidase was, however, different in the two macrophage populations which differ in their capacity to release O2-. NADPH oxidase activity was studied in macrophage monolayers after lysis with deoxycholate. The main features of this activity were as follows: stimulation of macrophages with PMA or zymosan caused an increase in NADPH-dependent O2- production; NADPH oxidase activity in the lysates followed the same dose-response curve for different concentrations of PMA as O2- release by intact macrophages; O2- release by intact macrophages could be fully accounted for by NADPH-dependent O2- production by macrophage lysates; activity was strictly substrate-specific, in that NADH could not substitute for NADPH; after stimulation with PMA or zymosan, NADPH oxidase activity was higher in lysates of C. parvum-activated macrophages than in lysates of resident macrophages; NADPH oxidase activities of activated and resident macrophages differed markedly in their kinetic parameters. The NADPH oxidase of macrophages activated by C. parvum or trehalose dimycolate of mycobacterial origin displayed a five to seven times lower Km compared to the enzyme in resident macrophages.
Animals↗
Desensitization of macrophages to stimuli which induce secretion of superoxide anion. Down-regulation of receptors for phorbol myristate acetate.
The ability of cultivated mouse peritoneal macrophages (M phi) to release superoxide anion (O-2) after repeated stimulation by phorbol myristate acetate (PMA) or serum-treated zymosan (STZ) has been studied. After a maximal first stimulus bacillus Calmette-Guérin (BCG)-activated M phi released high levels of O-2, 2-fold more than thioglycollate-elicited M phi and the response ceased within 4 h. Both populations either responded again to a second challenge or displayed a refractory state which varied in duration and selectivity. Desensitization by STZ pretreatment was transient and selective whereas PMA could render M phi refractory for 3 days to PMA alone or to both agents, depending on the amount of PMA used and the conditions of stimulation. PMA induced a selective loss of specific saturable receptors for [3H]phorbol dibutyrate, a closely related agent, and receptor activity recovered with the ability to release O-2. Loss of receptors did not account for concomitant loss of the response to STZ after nonselective deactivation. Such M phi were fully viable and able to endocytose various soluble and particulate ligands vigorously, but without stimulation of the hexose monophosphate shunt or release of O-2. Our studies indicate that M phi activities can be profoundly altered by prior stimulation, that specific receptors play a role in ligand-induced desensitization and that agents such as PMA can selectively eliminate the cells' ability to generate a second respiratory burst.
Animals↗