Helicobacter pylori induces but survives the extracellular release of oxygen radicals from professional phagocytes using its catalase activity

Helicobacter pylori can colonize the gastric epithelium of humans, leading to the induction of an intense inflammatory response with the infiltration of mainly polymorphonuclear leucocytes (PMNs) and monocytes. These professional phagocytes appear to be a primary cause of the damage to surface epithelial layers, and probably contribute to the pathogenesis associated with persistent H. pylori infections. We have shown previously that H. pylori adheres to professional phagocytes, but is not engulfed efficiently, suggesting an antiphagocytic escape mechanism that is dependent on the pathogen's type IV secretion system. Here, we show that H. pylori induces the generation and extracellular release of oxygen metabolites as a consequence of its attachment to phagocytic cells, but is capable of surviving this response. The catalase activity of H. pylori is apparently essential for survival at the phagocytes' cell surface. Opsonization of H. pylori leads to an increased burst, and the inhibition of bacterial protein synthesis to a decreased one. Ca2+ concentration, cytoskeleton rearrangement and protein kinase C (PKC) are involved in the H. pylori-induced oxidative burst in both monocytes and PMNs. This survival phenomenon has important implications for both the persistence of this important pathogen and the host tissue damage that accompanies persistent H. pylori infection.     

Concentrations of major elements and mercury in unstimulated human saliva

The aim of this study was a preliminary assessment of a possible role of human saliva in the diagnosis of some physiological and pathological changes in oral and body functions. Reliable procedures for collection and analysis of samples were established in order to assess total concentrations of Ca, K, Mg, Na, P, and Hg in whole unstimulated saliva. Possible relationships between element concentrations and sex, age, smoking, illness conditions, or side effects resulting from the use of drugs were investigated. The effects of stimulated or unstimulated collection procedures, dental prosthesis, and amalgam fillings were also evaluated. Total concentrations of major cations and Hg in whole saliva from 33 healthy adults living in the Siena district showed a coefficient of variation ranging from 11% (P) to 53% (Na) and average values were in the same range of those previously reported for unstimulated saliva. Healthy males had significantly higher concentrations of K, Na, P, and Na/K, Na/Ca, Na/Mg, and Na/P values than females. Age, smoking, dental prosthesis, and amalgam fillings had no significant effects on the concentrations of major elements. On the contrary, concentrations of Hg were positively correlated to the number of amalgam fillings and increased at a rate of about 1.9 μg/L for each filling. No correlations were found between Hg concentrations and those of major elements. Comparisons with literature data showed a different composition (particularly for Na and Hg concentrations) between unstimulated and stimulated saliva. Samples from patients affected by idiopathic pulmonary fibrosis had significantly higher concentrations of K and the maximum value was measured in a patient affected by acute pulmonary edema. This increase was likely the result of pharmacological treatments with tricyclic antidepressants and/or saline solutions. Data reported in this study, although preliminary, contribute to the assessment of levels of major cations (some of them very little investigated) and Hg in whole unstimulated human saliva and provides consistent support for further research on the possible use of this easy accessible matrix as a diagnostic tool of body function changes.     

Studies on the initial phase of dynein ATPase activity.

Kinetic measurement of the reaction of dynein ATPase (ATP phosphohydrolase, EC 3.6.1.3) extracted from the gills of Mytilus edulis shows that in the presence of Mg2+ there is a very rapid initial liberation of Pi from the dynein-ATP system, followed by a slower liberation in the steady state. In view of following results, we have confirmed that this phenomenon is not due to the accumulation of end products, a fall in substrate concentration, nor to the presence of labile impurities in ATP but is due to the catalytic activity of dynein ATPase. 1. The replacement of native dynein by heat denatured dynein or other kinds of Mg2+-ATPase could not produce such a burst phenomenon under the same condition. 2. Both the rate of initial burst and that of steady state were proportional to enzyme content over a wide range under our standard condition. 3. Initial burst was also observed under the constant ATP level by using a ATP generate system. 4. Preincubation of dynein with Pi prior to initiation of the reaction did not eliminate the initial burst. Some properties of the initial rapid liberation of dynein ATPase were also examined. These are shown below. 5. The free ADP liberation did not show any initial burst though the Pi liberation did in the initial phase and the rate of free ADP liberation was almost equal to that of Pi liberation of the steady state. 6. Mg2+ was more effective than Ca2+ for the appearance of the initial burst while the liberation of Pi in the steady state was activated more by Ca2+ than by Mg2+. The addition of K+ in the presence of Mg2+ resulted in a marked increase of Pi liberation in the steady state but not in the initial state. 7. The activation energy of the initial burst was 9.7 kcal, which is slightly smaller than that of myosin ATPase.     

Activation of the oxidative burst in aequorin-transformed Nicotiana tabacum cells is mediated by protein kinase- and anion channel-dependent release of Ca2+ from internal stores

The source of Ca2+ involved in transducing an oxidative-burst defense signal was examined in aequorin-transformed tobacco (Nicotiana tabacum L.) cells using modulators of Ca2+ entry. Treatments that either increased or decreased the influx of Ca2+ from external stores were found to have little effect on the magnitude or kinetics of an osmotically stimulated oxidative burst. In contrast, treatments that reduced the discharge of Ca2+ from internal stores inhibited dilution-activated H2O2 production. Curiously, most of the modulators commonly employed in animal studies as internal Ca(2+)-release inhibitors were neither effective in blocking discharge of intracellular Ca2+ nor in preventing the oxidative burst. When three different biochemical elicitors of the oxidative burst were similarly examined, both the H2O2 production and Ca2+ fluxes stimulated were found to be sensitive to modulators of internal Ca2+ release, but neither was impacted by alterations in externally derived Ca2+ influx. We hypothesize, therefore, that the oxidative burst does not depend on the influx of external Ca2+, but instead may generally be mediated by the release of internal Ca2+ in a manner that depends on the proper function of kinases and anion channels. These Ca2+ pulses trigger downstream signaling events that include the activation of Ca(2+)-regulated protein kinases, which are required for stimulation of the oxidative burst.     

Salivary myeloperoxidase of young adult humans

Leukocytes, principally polymorphonuclear leukocytes (PMNs), enter the oral cavity where they release a portion of their constituents, including myeloperoxidase, into oral fluids. A greater number of PMNs in the oral cavity are associated with oral inflammation. However, the quantitative contribution of the PMN to oral fluids, including saliva, during various conditions is poorly understood. An assay method based on the adsorbance loss at 278 nm from the reaction of the myeloperoxidase product hypochlorous acid with monochlorodimedon to yield dichlorodimedon was developed for the quantitation of salivary myeloperoxidase. Myeloperoxidase was determined in supernatants of whole saliva obtained at low and moderate flow rates and in parotid saliva collected during moderate and pronounced stimulation from young adults with minimal oral inflammation. The greatest myeloperoxidase activity was in whole saliva supernatants collected at low flow rates where PMN products have an opportunity to accumulate. Lesser quantities of myeloperoxidase were found in both the whole saliva supernatants and parotid saliva obtained at the faster flow rates. Low flow rate whole saliva supernatants contained about 25% of the myeloperoxidase in the PMNs which enter the oral cavity. Myeloperoxidase is responsible for a significant portion (15-20%) of the total peroxidase activity in supernatants of whole saliva obtained at low flow rates. Preliminary results indicate that young adults with phenytoin-associated gingival overgrowth or who smoke have more myeloperoxidase activity in low flow rate whole saliva.     

Hexose metabolism in pancreatic islets. Regulation of NAD-isocitrate dehydrogenase activity.

D-Glucose causes a preferential stimulation of mitochondrial oxidative events relative to glycolysis in pancreatic islets. The possible participation of a Ca(2+)-induced activation of NAD-isocitrate dehydrogenase in this process was investigated. The activity of the enzyme in rat islet homogenates was measured through the generation of either NADH or 2-ketoglutarate. In the absence of Ca2+ and ADP, half-maximal velocities were recorded at isocitrate and NAD+ concentrations close to 1.2 and 0.5 mM, respectively. At isocitrate concentrations in the 0.15-1.5 mM range, ADP (1.0 mM) markedly increased the reaction velocity recorded in the absence of Ca2+ and conferred to the enzyme the property of being activated by Ca2+, with a Ka for Ca2+ somewhat below 1.0 microM. From these data and by comparison with the activity of 2-ketoglutarate dehydrogenase, it is proposed that activation of NAD-isocitrate dehydrogenase by such factors as ADP and Ca2+ may be required in order to match, in nutrient-stimulated islets, the rates of 2-ketoglutarate generation and oxidative decarboxylation.     

The flavonoids, quercetin and isorhamnetin 3-O-acylglucosides diminish neutrophil oxidative metabolism and lipid peroxidation.

Two natural flavonoids, quercetin and isorhamnetin 3-O-acylglucosides, were examined for their inhibitory influence on the in vitro production and release of reactive oxygen species in polymorphonuclear neutrophils (PMNs). The generation of superoxide radical, hydrogen peroxide and hypochlorous acid were measured by, respectively, cytochrome c reduction, dichlorofluorescin oxidation and taurine chlorination. Membrane lipid oxidation was studied by the thiobarbituric acid method in mouse spleen microsomes. The addition of flavonoids at the concentration range 1-100 microM inhibited PMNs oxidative metabolism and lipid peroxidation in a dose-dependent manner. The results suggest that these flavonoids suppress the oxidative burst of PMNs and protect membranes against lipid peroxidation.     

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.     

Glutamate utilization promotes meningococcal survival in vivo through avoidance of the neutrophil oxidative burst

Polymorphonuclear neutrophil leucocytes (PMNs) are a critical part of innate immune defence against bacterial pathogens, and only a limited subset of microbes can escape killing by these phagocytic cells. Here we show that Neisseria meningitidis, a leading cause of septicaemia and meningitis, can avoid killing by PMNs and this is dependent on the ability of the bacterium to acquire L-glutamate through its GltT uptake system. We demonstrate that the uptake of available L-glutamate promotes N. meningitidis evasion of PMN reactive oxygen species produced by the oxidative burst. In the meningococcus, L-glutamate is converted to glutathione, a key molecule for maintaining intracellular redox potential, which protects the bacterium from reactive oxygen species such as hydrogen peroxide. We show that this mechanism contributes to the ability of N. meningitidis to cause bacteraemia, a critical step in the disease process during infections caused by this important human pathogen.     

The levels of neutrophils oxidative burst in rheumatic disorders

We investigated the participation of the polymorphonuclear leukocytes (PMNs) as mediators of tissue destruction in 2 variants of rheumatic disorders that affect primarily the joints: the rheumatoid arthritis (RA) and the osteoarthritis (OA). We noted significant differences in the number of PMNs present at the joint level, which was low in OA and high in RA. The unstimulated and in vitro stimulated with opsonized zymozan (OZ) or ConA PMNs from the peripheral blood of these two groups of patients released a normal level of oxygen free radicals (OFR). On the contrary, the neutrophils isolated from the synovial fluid (SF) of RA patients showed a distinct feature: the unstimulated cells released high amounts of OFR, while those in vitro stimulated with OZ or ConA presented a low level of respiratory burst. In OA both in vitro unstimulated and stimulated with OZ or ConA neutrophils isolated from the SF had diminished values of OFR released. We assume that the chemiluminescence assay could detect distinct differences of the PMNs implications in RA and OA injuries and may be a laboratory test in the differential diagnosis of these rheumatic disorders.     

Effect of methacrylic monomers on phagocytes reactive oxygen species: a possible BDDMA modulating action

In the last years the studies regarding the biocompatibility of dental materials investigate, in addition to the classic cytotoxic tests, the interactions between the materials and the host cells to better explain the causes of the adverse effects observed sometimes in the clinical practice. In the present study the ability of diurethane dimethacrylate (DUDMA) and 1,4‐butanediol dimethacrylate (BDDMA) methacrylic monomers present in dental composite resins to alter the functionality of peripheral blood monocytes (PBMs) and polymorphonucleate cells (PMNs) was examined. These cells are involve in the biological response to materials and in the host ability to respond to bacteria. The results obtained suggest that the examined methacrylates induce a relevant decrease of PBMs oxidative burst whereas the basal ROS production is only slightly decreased. In PMNs DUDMA induces a decrease of both basal and stimulated ROS production. BDDMA, on the contrary, it does not alter total oxidative burst in presence of stimulus while induces a statistically significant decrease of basal ROS production. Moreover this monomer alters the reaction kinetics of stimulated ROS production. The reported finding seems to indicate that this molecule could be able to stabilize PMNs in resting state and maximize their stimulated activity. Copyright © 2008 John Wiley & Sons, Ltd.     

Neutrophil hyperpolarization in response to a chemotactic peptide

The chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP), at concentrations below 10(-9) M, elicits a sustained increase in the human neutrophil's membrane potential within 10 s of its addition. This hyperpolarization, detected with the fluorescent cationic potentiometric probes, 3,3'-dipentyloxacarbocyanine (diO-C5-(3)), and 1,1'-dipropyl-3,3,3',3'-tetramethylindocarbocyanine iodide (diI-C3-(3)), and with the anionic probe bis-(1,3-diethylthiobarbituric)trimethine oxonol (bis-oxonol), is immediately followed by a large depolarization when [fMLP] greater than 10(-9) M. By extracellular substitution of sodium ions with potassium ions or choline or by pretreatment of the cells with ionophores, we report here that the hyperpolarization is primarily dependent on an intact potassium ion gradient and is accompanied by a concurrent acidification of the cytoplasm (approximately 0.05 pH unit) Although the latter occurs simultaneously with a large, transient increase in cytosolic Ca2+ at [fMLP] greater than 10(-10) M, it occurs without a detectable increase in cytosolic Ca2+ at [fMLP] less than 10(-10) M. The hyperpolarization is neither affected nor initiated by the chemotactic peptide antagonist tert-butyloxycarbonyl-methionyl-leucyl-phenylalanine, whereas the depolarization is completely inhibited. Neutrophils isolated from patients with X-linked chronic granulomatous disease exhibit normal hyperpolarizations and cytosolic Ca2+ increases in response to chemotactic peptides but exhibit no depolarization or oxidative burst. The hyperpolarization appears earlier in the ontogeny of differentiating myeloid precursor cells than either the rise in cytosolic Ca2+ or the depolarization response. Together, these findings indicate that an increase in transmembrane potential is one of the earliest events in the neutrophil response to chemotactic peptides, coinciding temporally with increases in cytoplasmic Ca2+ and H+ concentrations but preceding detectable oxidative burst activity.     

Ferrous iron release from transferrin by human neutrophil-derived superoxide anion: effect of pH and iron saturation.

The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.     

Influence of calcium on the subcellular distribution of protein kinase C in human neutrophils. Extraction conditions determine partitioning of histone-phosphorylating activity and immunoreactivity between cytosol and particulate fractions

Activation of the neutrophil respiratory burst is thought to involve a translocation and activation of protein kinase C. We report that the presence of Ca2+ during the disruption of unstimulated human neutrophils and cytoplasts resulted in an increase in protein kinase C activity (histone phosphorylation) and immunoreactive protein kinase C species in the particulate (membrane) fraction and a reduction in such activities in the cytosol. This Ca2+-induced translocation of activity was concentration-dependent and occurred at physiologically relevant concentrations of Ca2+ (30-500 nM). The Ca2+-induced membrane association of protein kinase C could be reversed by removal of Ca2+. These findings indicate that the Ca2+ concentration of the extraction buffer can determine the subcellular distribution of protein kinase C in disrupted cells and suggest that the observed location of this enzyme activity in cell fractions may not necessarily reflect the localization in intact cells. These results also raise the possibility that the distribution of protein kinase C between cytosol and membrane is a dynamic equilibrium controlled by levels of free Ca2+. Thus, Ca2+ might regulate distribution as well as activation of protein kinase C.     

Salicylic acid glucoside acts as a slow inducer of oxidative burst in tobacco suspension culture

Salicylic acid beta-glucoside (SAG) is a storage form of a defense signal against pathogens, releasing free salicylic acid (SA), to meet the requirements in plants. Since excess SA induces locally restricted cell death following oxidative burst and Ca2+ influx in plants, the effects of SAG on cell viability, Ca2+ influx, and generation of superoxide (O2*-) were examined in suspension-cultured tobacco BY-2 cells expressing aequorin. Among SA-related chemicals tested, only SAG induced the slow and long-lasting O2*- generation, although SAG was less active in acute O2*- generation, Ca2+ influx and induction of cell death. The prolonging action of SAG is likely due to gradual release of SA and the data suggested that a peroxidase-dependent reaction is involved. Notably, pretreatment with low-dose SA (50 micromu) enhanced the response to SAG by 2.5-fold. There are four possible secondary messengers in early SA signaling detectable in the BY-2 culture, namely O2*-, H2O2, Ca2+ and protein kinase (PK). If these messengers are involved in the low-dose SA-dependent priming for SAG response, they should be inducible by low-dose SA. Among the four SA-inducible signaling events, PK activation was excluded from the low-dose SA action since a much higher SA dose (> 0.4 mmu) was required for PK activation.     

Biphasic regulation of leukocyte superoxide generation by nitric oxide and peroxynitrite

Activation of the NADPH oxidase-derived oxidant burst of polymorphonuclear leukocytes (PMNs) is of critical importance in inflammatory disease. PMN-derived superoxide (O(2)) can be scavenged by nitric oxide (NO( small middle dot)) with the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the effects and mechanisms by which NO( small middle dot) and ONOO(-) modulate the PMN oxidative burst. Therefore, we directly measured the dose-dependent effects of NO( small middle dot) and ONOO(-) on O(2) generation from human PMNs stimulated with phorbol 12-myristate 13-acetate using EPR spin trapping. Pretreatment with low physiological (microm) concentrations of NO( small middle dot) from NO( small middle dot) gas had no effect on PMN O(2) generation, whereas high levels (> or =50 microm) exerted inhibition. With ONOO(-) pretreatment, however, a biphasic modulation of O(2) generation was seen with stimulation by microm levels, but inhibition at higher levels. With the NO( small middle dot) donor NOR-1, which provides more sustained release of NO( small middle dot) persisting at the time of O(2) generation, a similar biphasic modulation of O(2) generation was seen, and this was inhibited by ONOO(-) scavengers. The enhancement of O(2) generation by low concentrations of ONOO(-) or NOR-1 was associated with activation of the ERK MAPKs and was blocked by their inhibition. Thus, low physiological levels of NO( small middle dot) present following PMN activation are converted to ONOO(-), which enhances O(2) generation through activation of the ERK MAPK pathway, whereas higher levels of NO( small middle dot) or ONOO(-) feed back and inhibit O(2) generation. This biphasic concentration-dependent regulation of the PMN oxidant burst by NO( small middle dot)-derived ONOO(-) may be of critical importance in regulating the process of inflammation.     

Hyperoxia alters effect of calcium on rat alveolar macrophage superoxide production

The effect of hyperoxia on the Ca2+ dependence of stimulated superoxide anion radical (O2-.) production (the respiratory burst) of rat alveolar macrophages was investigated. Enhancement of the concanavalin A (con A)-stimulated respiratory burst by extracellular Ca2+ was suppressed by O2 exposure. Similarly, the inhibitory effect of verapamil on the con A-stimulated respiratory burst was reduced by O2 exposure. O2 exposure also inhibited con A stimulation that was independent of Ca2+ entry. Exposure to O2 also caused a decline in O2-. production stimulated by either A23187 or phorbol myristate acetate (PMA). With A23187 stimulation, extracellular Ca2+ was essential for either air-exposed (control) or O2-exposed cells. With PMA, stimulation was independent of extracellular Ca2+ for either air or O2-exposed macrophages and verapamil did not inhibit. Free intracellular Ca2+ concentration ([Ca2+]i) was measured in control and O2-exposed alveolar macrophages. Hyperoxic exposure did not alter [Ca2+]i in unstimulated cells. In controls, con A stimulated an immediate increase in [Ca2+]i followed by a rapid decrease and a second rise and fall. The second elevation was suppressed by verapamil or ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid or O2 exposure. The results of both the respiratory burst assays and measurement of con A-stimulated changes in [Ca2+]i suggest that Ca2+ entry involved in stimulus-response coupling is suppressed in cellular O2 toxicity.     

Incidence of oropharyngeal candidosis in stem cell transplant (SCT) patients

Eighty persons with haematological malignancies receiving stem cell transplantation (SCT) were examined over a 24 months period. Hyposalivation, a common complaint in patients treated by intensive chemotherapy and radiotherapy can predispose to oral candidal colonisation as well. This study was focused on correlation between the fungal colonisation of the oral cavity and the total unstimulated saliva flow rate of 80 patients with haematological malignancies before and after stem cell transplantation and in addition, on their oral health state. Despite the fact that prior to being involved in the transplantation programme, the patients were subjected to dental examination and decayed teeth were found in 20 out of 80 patients (25%). From the 2233 different oropharyngeal specimens fungi were isolated before conditioning from 16 patients (20%), and during aplasia from 19 patients (23.7%). Objective xerostomia (unstimulated total saliva flow rate < or = 0.1 ml/min) was detected in 28 patients (35%). Stem cell transplant patients with pretransplant mouth dryness had higher incidence of Candida albicans and other fungal colonisation than those with normal saliva secretion.     

Advanced glycosylated end products-mediated activation of polymorphonuclear neutrophils in diabetes mellitus and associated oxidative stress

Two important consequences of hyperglycemia in diabetes are development of oxidative stress and formation of advanced glycation end products (AGE) which are known to be associated with diabetic complications. Relationship between AGE formation and development of oxidative stress (OS) is yet to be established. In the present study, the involvement of AGE in PMN-mediated ROS generation and the associated OS were investigated in type 2 diabetic mellitus (DM) patients. We assessed OS parameters (serum MDA, FRAP and GSH), PMN oxidative functions (respiratory burst and superoxide production) and total serum AGE in 90 subjects divided equally in three groups--control group, Group I consisting of type 2 diabetic patients without microvascular complications and Group II consisting of type 2 diabetic patients with microvascular complications. PMNs isolated from both groups (I and II) exhibited higher level of respiratory burst (RB) and produced increased amount of superoxide anion as compared to the controls. The increase was more pronounced in diabetes with complications, as compared to those without. Serum malondialdehyde (MDA) level was elevated, whereas glutathione (GSH) and ferric reducing ability of plasma (FRAP) levels were significantly reduced in diabetes as compared to the controls, suggesting the presence of oxidative stress in DM. A positive correlation between PMN oxidative function and OS parameters suggested the involvement of PMN in the development of OS in DM. Serum AGE level was also elevated in diabetic groups as compared to the controls. Further, the positive correlation between serum AGE level and PMN oxidative function suggested the involvement of AGE in increased RB and generation of reactive oxygen species (ROS) by resting diabetic PMN. The results of the study indicate that AGE-PMN interaction possibly upregulates NADPH oxidase, leading to enhanced ROS generation and thus contributes to the pathogenesis in diabetes.     

Mechanism of polymorphonuclear leukocyte activation by myristate. Involvement of calcium ion and protein kinase C

The stimulative effects of myristate on the superoxide generation and depolarization of membrane potential of polymorphonuclear leukocytes (PMN) are particularly strong, yet myristate does not affect the intracellular free Ca2+ level ([Ca2+]i) in the presence of 1 microM free calcium in calcium-EGTA buffer. The half maximum concentration of myristate was 10 microM. Myristate inhibited the transitory changes in [Ca2+]i induced by formylmethionyl-leucyl-phenylalanine (FMLP), but stimulated further the FMLP-induced superoxide generation; these effects are similar to those of phorbol myristate acetate (PMA). The myristate-induced superoxide generation was partially inhibited by H-7, a specific inhibitor of protein kinase C. Myristate stimulated the activity of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in a concentration-dependent manner in the presence of 10(-6) M Ca2+. The Ka was 100 microM. These results suggested that there is no relation between the superoxide generation and the [Ca2+]i change in PMNs and that the effects of myristate are similar to those of PMA against PMN.