Changes in the myeloperoxidase and acid phosphatase activities in human peripheral blood neutrophils with the cells stimulated in vitro]

In four series of experiments human peripheral blood neutrophils were found capable of synthetizing the active forms of such enzymes as myeloperoxidase and acid phosphatase after stimulation with opsonized zymosan, and the optimum conditions for testing the synthetizing activity of neutrophils were established. The examination of 39 practically healthy donors revealed an approximate equilibrium between the synthesis of the active forms of the enzyme and its excretion from the cell after this cell was activated. Considerable changes in the enzymatic activity of peripheral blood neutrophils were found in patients with yersiniosis, chronic herpes, chronic bronchitis and AIDS-associated complex. The possibility of a deficient enzymatic activity in some patients was suggested.     

Regulation of Free Radical Processes by Delta-Sleep Inducing Peptide in Rat Tissues under Cold Stress

An intraperitoneal injection of an exogenous delta-sleep inducing peptide (DSIP) at a dose of 12 g/100 g body weight shifted the prooxidant–antioxidant balance of free radical process (FRP) in tissues and erythrocytes of rats: the activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) and the concentrations of antioxidants (reduced glutathione in particular) increased. The DSIP stimulated the myeloperoxidase activity in blood neutrophils and had no effect on the activity of xanthine oxidase, a prooxidant enzyme, in the brain and liver. Cold stress displaced the prooxidant–antioxidant balance by increasing the xanthine oxidase activity in tissues and decreasing the myeloperoxidase activity in blood neutrophils; it also inhibited the enzyme antioxidant activities in tissues and erythrocytes that was neutralized by an increased ceruloplasmin activity in blood plasma and by an elevated level of antioxidants in rat blood and tissues. Preliminary administration of DSIP to animals exposed to cold stress restored the prooxidant–antioxidant balance: it normalized the myeloperoxidase activity in blood neutrophils, decreased the xanthine oxidase activity, and increased the activity of antioxidant enzymes in tissues and erythrocytes restoring the antioxidant level. The molecular regulation mechanism of free radical processes by DSIP in tissues under stressful conditions is discussed.     

The effect of salmonella infection on the functional activity of the polymorphonuclear leukocytes]

Experiments on noninbred white mice have revealed that in the animals infected with S. moscow secondary immunodeficiency develops, which is manifested by a significant decrease in the activity of the bactericidal system of peripheral blood granular leukocytes. Simultaneously, the content of myeloperoxidase in the blood neutrophils of infected mice decreases 1.4 times and the content of lysozyme in these neutrophils decreases 2 times. Such changes are the consequence of an increase in the secretory activity of cells, occurring in the process of the development of Salmonella infection.     

Alkaptonuria is a novel human secondary amyloidogenic disease

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.     

Role of redox imbalance and cytokines in mediating oxidative damage and disease progression of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disorder wherein the contributory role of oxidative stress has been established in the synovial fluid. As availability of synovial fluid is limited, this study aimed to evaluate in the peripheral blood of patients with RA, the relationship if any, between the extent of oxidative stress in terms of generation of reactive oxygen species (ROS) in neutrophils, plasma NADPH oxidase and myeloperoxidase activity with markers of oxidative damage, circulating cytokines and disease activity score (DAS28). In patients with RA, neutrophils in peripheral blood demonstrated an enhanced generation of ROS, coupled with depletion of free radical scavenging activity. Furthermore, the NADPH oxidase and myeloperoxidase activity was enhanced as were markers of damage. There was a positive correlation between the DAS 28 and generation of ROS, NADPH oxidase and myeloperoxidase activity as also with oxidative stress mediated protein carbonylation. Patients with RA demonstrated an increase in proinflammatory (IL-17, IL-23, and IFN-γ) and some anti-inflammatory (IL-4, IL-5, and TGF-β) cytokines. Although the levels of IL-17 correlated positively with generation of ROS, myeloperoxidase, markers of protein damage and DAS28, IL-23 correlated positively only with protein damage, and negatively with free radical scavenging activity. Importantly, incubation of neutrophils from healthy donors with plasma or SF from patients with RA translated into an enhanced generation of ROS, along with an elevation of intracellular proinflammatory cytokines. Taken together, in patients with RA, circulating neutrophils mediated a shift in the oxidant/antioxidant balance favouring the former, which translated into protein damage and contributed towards disease progression.     

Analysis of human neutrophil granule protein composition in chronic myeloid leukaemia by immuno-electron microscopy

Summary In this study immuno-electron microscopy was used to assay, semi-quantitatively, the granule contents of elastase, lactoferrin, lysozyme and myeloperoxidase in human peripheral blood neutrophils from 13 chronic myeloid leukaemia patients in the chronic phase of the disease and from normal non-smoking donors. The fixation conditions that adequately preserved the antibody binding capacities of these antigens and reasonably preserved the ultrastructure of the neutrophils were selected by light-microscopic immunoperoxidase cytochemistry on cytospin smears. Immunogold cytochemistry on LR White resin sections localised elastase and myeloperoxidase to the primary granules, lactoferrin to the secondary granules and lysozyme to both types of granule. When applicable, peroxidase cytochemistry was combined with immunogold staining making it easier to distinguish the primary from the secondary granules. A comparison of the immunolabelling density values obtained for the leukaemic and normal states revealed no significant abnormalities in the immunoreactivity patterns for any of these neutrophil granule antigens in the leukaemic patients. All 13 patients gave normal immunostaining reactivities for these neutrophil granule proteins. Consequently the distribution patterns of these proteins, as shown in this study, cannot be used as indices in distinguishing chronic myeloid leukaemic neutrophils from normal neutrophils.     

Lymphocyte and neutrophil cytochemistry in the dynamics of different forms of diphtheria in adults]

The cytochemical study of lymphocytes and neutrophils isolated from fractionated blood of diphtheria patients and carriers has revealed that a decrease in the activity of lymphocyte succinic dehydrogenase and myeloperoxidase can be observed at all periods of the disease and in all its forms. A decrease in the activity of lymphocyte nonspecific esterase has been noted only in patients with toxic and subtoxic diphtheria and a decrease in the activity of neutrophil alkaline phosphatase, in carriers. The analysis of correlations between the parameters of five enzymes under study (lymphocyte succinic dehydrogenase, lymphocyte acid phosphatase, lymphocyte non-specific esterase, myeloperoxidase, neutrophil alkaline phosphatase) and enzymatic rosette parameters has been made. The analysis has revealed an essential increase in the number of correlations in comparison with donors, changes in the qualitative nature of these correlations and sometimes the reversion of the correlations. Carriers have shown the greatest number of correlations. By the end of the terms of observations no restoration of normal correlations has been observed.     

Lipid peroxidation and the myeloperoxidase activity of neutrophilic leukocytes in shereshevsky?-Turner syndrome

The level of lipid peroxides, activity and thermostability of myeloperoxidase have been studied in neutrophil extracts from the peripheral blood of patients with the Shereshevsky-Turner syndrome and in normal subjects. A statistically significant rise in the level of lipid peroxides and respective decrease in the activity and thermostability of myeloperoxidase have been revealed in the patients as compared with the control group. These findings point to functional inferiority of neutrophils. It is probable that at the basis of the disorders seen there lie an abnormal set of sex chromosomes and upset hormonal regulation determined by the abnormality.     

Cytochrome c oxidase, Cu,Zn-superoxide dismutase, and ceruloplasmin activities in copper-deficient bovines

The activity of several cuproenzymes in relation to the immune system was examined in serum and blood cells from bovines with molybdenum-induced copper deficiency. Five female cattle were given molybdenum (30 ppm) and sulfate (225 ppm) to induce experimental secondary copper deficiency. Ceruloplasmin activity was determined in serum. The Cu,Zn-superoxide dismutase and cytochrome c oxidase activities were measured in peripheral blood lymphocytes, neutrophils, and monocyte-derived macrophages. Copper deficiency was confirmed from decreased serum copper levels and the animals with values less than 5.6 μmol/L were considered deficient. The content of intracellular copper decreased between 40% and 70% in deficient cells compared with the controls. In copper-deficient animals, the serum ceruloplasmin activity decreased to half of the control value. Both of them, the Cu,Zn-superoxide dismutase and the cytochrome c oxidase activities, undergo a significant reduction in leukocytes, showing differences among diverse cell populations. We concluded that the copper deficiency alters the activity of several enzymes, which mediate antioxidant defenses and ATP formation. These effects may impair the cell immune functionality, affecting the bactericidal capacity and making the animals more susceptible to infection.     

Oxidative inactivation of myeloperoxidase released from human neutrophils.

Within 1 min of stimulation of human neutrophils by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B, myeloperoxidase (together with other granule enzymes) was secreted and detected extracellularly. In contrast with the other granule constituents assayed (vitamin B12-binding protein and beta-glucuronidase), the activity of released myeloperoxidase rapidly decreased, so that, by 10 min after stimulation, only about 5% of the total cellular activity was detected. This inactivation was shown to be dependent on oxidant generation during the respiratory burst, since inactivation was not observed (a) after stimulation of anaerobic suspensions or (b) after release from neutrophils from a patient with chronic granulomatous disease; purified myeloperoxidase was rapidly inactivated after incubation with H2O2, presumably owing to the formation of an inactive enzyme-H2O2 complex. These results show that experiments designed to assess the role of myeloperoxidase in neutrophil functions which utilize assays based on peroxidase activity will grossly underestimate this enzyme if oxidant generation during the respiratory burst has also been activated.     

Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils

It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase — 4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC₅₀ = 20 μM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p < 0.01) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a “vicious circle” of neutrophil activation at the inflammatory site.     

The secondary structure of human amyloid deposits as determined by circular dichroism spectroscopy

The deposition of amyloid fibrils has been associated with a diversity of pathologies including plasma cell dyscrasias, chronic inflammatory diseases, and several types of neurological diseases including Alzheimer's disease. Using circular dichroism spectroscopy, the secondary structure of a human amyloid protein deposit from a patient with plasma cell (light chain)-associated amyloidosis (amyloidosis AL) has been determined. The protein contains 52% beta structure, which is consistent with these depositions arising from the aberrant catabolism of immunoglobulin light chains, which are rich in beta sheets. The protein was also found to contain 20% alpha-helix, suggesting that partial refolding may occur during amyloidogenesis.     

Functional and metabolic activity of peripheral blood neutrophils in acute viral hepatitis B and C

The functional metabolic activity of peripheral blood neutrophils in acute virus hepatitis B (VHB) and/or virus hepatitis C (VHC) was evaluated. 48 patients were examined; of these, VHB was diagnosed in 28 patients and VHC was diagnosed in 9 patients and the mixed form of virus hepatitis (VHB + VHC), in 11 patients. Determination of adhesive capacity of neutrophils, production of superoxidase anion in the nitro blue tetrazolium (NBT) test, activity of myeloperoxidase (MPO) and acidic phosphatase (AP), the amount of cation proteins (CP) was made. Most pronounced functional dysbalance of neutrophil leukocytes and considerable changes in biochemical characteristics of the activity of the infectious process in patients with the mixed form of virus hepatitis were established. These data demonstrated that in acute virus hepatitis B and C at the peak of the disease such characteristics of the functional activity of neutrophils as results of the NBT test, the activity of MPO and AP, as well as the amount of CP, were highly informative.     

The role of neutrophil myeloperoxidase in the development of inflammation induced by thermal skin burns

Luminol-dependent chemiluminescence (CL) of blood neutrophils stimulated with phorbol-12- myristate-13-acetate (PMA) and myeloperoxidase (MPO) activity of neutrophils and plasma have been investigated in children (n = 16) during the early period (1?7 days) after thermal skin burns exceeding 20% of total body surface. The CL level of stimulated neutrophils was higher in burn patients than in healthy children of the reference group (p lt; 0.01). Increased neutrophil MPO activity was found in 40% of patients, while increased plasma MPO activity was detected in 57% of patients. The albumin fraction isolated from plasma of burn patients increased the PMA-stimulated CL response of blood from healthy donors. These results suggest that the acute inflammatory response to the thermal burn causes neutrophil activation and MPO release into plasma. MPO-mediated modification of plasma proteins, particularly albumin, may stimulate neutrophil activation and provoke further inflammatory response of the body to the thermal injury.     

Features of regulating delta sleep-inducing peptide by free radical processes in tissue and erythrocyte membranes in intact animals and under stress

In activity the comparative analysis of metabolic effects delta--sleep inducing peptide (DSIP) in tissues and erythrocytes of intact rats and under cold stress is conducted. The regulation effect of DSIP in attitude of free radical processes will be realised through modulation the prooxidant--antioxidant balance: both for intact animal, and at stress. Exogenous DSIP increases the antioxidant system activity in tissues of brain, liver and blood in standard conditions and under cold stress. The anti-stress effect of DSIP is directed as on increase of power endogenic enzymatic antioxidant system activity, specially glutathione peroxidase activity, and not enzymatic of antioxidant protection. The DSIP renders different influence on activity of prooxidant enzymes: for intact animal boosts the myeloperoxidase activity in blood neutrophils, not rendering essential influencing on the xanthine oxidase activity in tissues of brain, liver and activates the myeloperoxidase activity, depresses the xanthine oxidase activity for rats at stress. The membranotropic effect of DSIP in the norm and under stress is connected to increase of stability of protein--lipid interplays. The membranostabilizing effect of DSIP in conditions of stress is characterized decrease of polarity of lipid phase and negative surface charge of erythrocyte membranes, modified in course of lipid peroxidation.     

Superoxide production by rat neutrophils in the oleic acid model of lung injury

The purpose of this study was to investigate the superoxide anion (O2-)-generating capacity of neutrophils isolated from rats at various stages of oleic acid(OA)-induced lung injury. Neutrophils were collected from blood, bronchoalveolar lavage (BAL), and peritoneal cavity (glycogen induced) after OA administration. Control neutrophils were collected from the blood of normal animals as a representative of nonprimed cells that produce low levels of O2-. A second control was the glycogen-elicited peritoneal neutrophil of normal animals which represented primed cells that produce enhanced levels of O2-. The ability of the neutrophils to produce O2- was evaluated by using both myristate acetate and opsonized zymosan as stimulants. Neutrophils isolated from blood and BAL from OA-injured lungs produced low levels of O2- and resembled closely the circulating, nonprimed neutrophil. Myeloperoxidase levels were measured in plasma and BAL and were found to be elevated in BAL of OA-injured animals. The inability of neutrophils to produce high levels of O2- and the elevation of myeloperoxidase suggest that neutrophils present in the lung may have degranulated in response to prior activation and are therefore incapable of further superoxide production.     

Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide

It is well known that catalase is transformed to nitric oxide-Fe2+-catalase by hydrogen peroxide (H2O2) plus azide. In this report, we show that myeloperoxidase is also inactivated by H2O2 plus azide. Utilizing this system, we studied the presence and source of intracellular H2O2 generated by activated neutrophils. Stimulation of neutrophils with phorbol myristate acetate (PMA, 100 ng/ml) plus azide (5 mM) for 30 min completely inactivated intragranular myeloperoxidase and reduced cytosolic catalase to 35% of resting cells. This intracellular inactivation of heme enzymes did not occur in normal neutrophils incubated with either PMA or azide alone or in neutrophils from patients with chronic granulomatous disease (CDG) which cannot produce H2O2 in response to PMA. Incubation of neutrophils with azide and a H2O2 generating system (glucose-glucose oxidase) inactivated 41% of neutrophil myeloperoxidase. Glutathione-glutathione peroxidase (GSH-GSH peroxidase), an extracellular H2O2 scavenger, totally protected neutrophil myeloperoxidase from inactivation by azide plus glucose-glucose oxidase. In addition, when a mixture of normal and CGD cells was stimulated with PMA in the presence of azide, 90% of the myeloperoxidase in CGD neutrophils was inactivated. Therefore, H2O2 released extracellularly from activated neutrophils can diffuse into cells. In contrast, myeloperoxidase in normal polymorphonuclear leukocytes stimulated with PMA in the presence of azide and GSH-GSH peroxidase was 75% inactivated. Thus, the results indicate that a GSH-GSH peroxidase-insensitive pool of H2O2 is also generated, presumably at the plasma membrane, and this pool of H2O2 can undergo direct internal diffusion to inactivate myeloperoxidase.     

Plasminogen activator in nephrotic syndrome

Plasminogen activators were studied in blood urine in 207 patients with nephrotic syndrome of different etiological forms. The blood plasminogen activator activity was decreased in chronic glomerulonephritis, SLE, systemic vasculities as result of great level of inhibitors (L2M), penetration of enzymes to abdominal and pleural transudates, excretion to urine. The blood plasminogen activator activity and urokinase level in chronic glomerulonephritis was dependent on the degree of nephrotic syndrome. The plasminogen activator in amyloidosis was sharply elevated because of permanent irritability of endothelial wall by amyloid mass. Venous occlusion caused the release of plasminogen activator to blood only in more favourable clinical course of nephrotic syndrome.     

Chemotactic factor inactivation by stimulated human neutrophils mediated by myeloperoxidase-catalyzed methionine oxidation

Leukocyte chemoattractants were inactivated when exposed to human neutrophils and either ingestible particles or phorbol esters. Loss of biologic activity was time- and temperature-dependent, required physiologic concentrations of viable neutrophils and a halide, and was inhibited by azide or catalase. Neutrophils from patients with either hereditary myeloperoxidase deficiency or chronic granulomatous disease failed to inactivate the chemoattractants unless purified myeloperoxidase or H2O2, respectively, was added. Susceptibility to inactivation by neutrophils correlated with the presence of methionine in the attractant. Loss of chemotactic activity was blocked by low concentrations of methionine and by higher concentrations of other reducing agents, but was unaffected by oxidized methionine. Paper chromatography demonstrated that exposure of a formyl-methionyl peptide chemotactic factor to either the cellfree myeloperoxidase system or stimulated neutrophils resulted in its conversion to a molecular species whose location in the chromatographs was identical to that of the peptide containing oxidized methionine. Thus, stimulated human neutrophils inactivate peptide chemoattractants by secretion of myeloperoxidase and H2O2, which combine with halides to form oxidants that react with a critical methionine residue. We suggest that myeloperoxidase-catalyzed oxidation of thioethers may constitute an inflammatory control mechanism as well as a general means of modifying the functional properties of biologic mediators.     

Reaction of peripheral blood leukocytes to wound processes during phytohemagglutinin therapy in rabbits with different hypersensitivity of delayed type

Altogether 165 rabbits were distributed into strongly and poorly reacting groups in terms of delayed type hypersensitivity (DTH) to phytohemagglutinin (PHA). It was established that leukocyte response to wound process correlated with the intensity of DTH. The strongly reacting animals manifested of DTH. The strongly reacting animals manifested an adequate response (leukocytosis, neutrophilosis, nomocytosis, lymphocytosis, a decrease in myeloperoxidase and enhancement of phagocytic activity of neutrophils), while the poorly responding animals an in adequate one (passivity of the leukocytic parameters with the exception of monocytosis). Application of the liniment containing PHA (3.3 micrograms per g base, Difco, USA) does not change the response in the strongly reacting animals and essentially corrects all the leukocytic parameters in the poorly reacting animals with the exception of phagocytic activity of neutrophils. From the leukocytic response pattern a conclusion is made about multiple defect of the leukocytic system under poor DTH to PHA and about the leading role of monocytes in correcting the defect by PHA.