TiO2-coated luminescent porous silicon micro-particles as a promising system for nanomedicine.

Porous silicon (pSi) is a sponge-like material obtained by electrochemical etching of a crystalline silicon wafer. Due to quantum confinement effects, this material is photoluminescent and this is a fundamental property from the perspective of bioimaging applications. Limitations in nanomedicine to the use of photoluminescent pSi structures are mainly due to optical quenching in an aqueous environment and to the adverse effects of reactive groups introduced by etching procedures. In this work, we exploited an inorganic TiO2 coating of pSi microparticles by Atomic Layer Deposition (ALD) that resulted in optical stability of pSi particles in a biological buffer (e.g. PBS). The use of a rotary reactor allows deposition of a uniform coating on the particles and enables a fine tuning of its thickness. The ALD parameters were optimized and the photoluminescence (PL) of pSi-TiO2 microparticles was stabilized for more than three months without any significant effect on their morphology. The biocompatibility of the coated microparticles was evaluated by analyzing the release of cytokines and superoxide anion (O2 -) by human dendritic cells, which play an essential role in the regulation of inflammatory and immune responses. We demonstrated that the microparticles per se are unable to significantly damage or stimulate human dendritic cells and therefore are suitable candidates for nanomedicine applications. However, a synergistic effect of the microparticles with bacterial products, which are known to stimulate immune-response, was observed, indicating that a condition unfavorable to the use of inorganic nanomaterials in biological systems is the presence of infection diseases. These results, combined with the proved PL stability in biological buffers, open the way for the use of pSi-TiO2 microparticles as promising materials in nanomedicine, but their ability to increase immune cell activation by other agonists should be considered and even exploited.

Orange and blue luminescence emission to track functionalized porous silicon microparticles inside the cells of the human immune system.

Porous silicon micro-particles (micro-pSi) with size in the range of 1-10 µm are obtained by etching of silicon wafers followed by sonication. The derivatization of the micro-pSi surface by wet chemistry (silylation and coupling with a diamine) yields an interface, which exposes negative (carboxylic) or positive (amine) groups at pH 7.4. The surface modification, beyond the introduction of groups for the drug loading by covalent or electrostatic interactions, stabilizes the intense orange luminescence characteristic of the silicon nano-crystallites. Derivatization by amines introduces also a second emission in the blue region, which follows a different excitation pathway and can be attributed to the interface defects. The micro-pSi are efficiently internalized by human dendritic cells and do not show any toxic effect even at a concentration of 1 mg mL-1. The intrinsic luminescence of the differently functionalized micro-pSi is preserved inside the cells and permits the selective and efficient tracking of the microparticles without using molecular tags and thus leaving the organic coating available for the interaction with the drug. The results obtained suggest that the functionalized micro-pSi are an efficient platform for simultaneous imaging and delivery of therapeutic agents to the disease site.

Oxidative stress in scleroderma: maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH oxidase complex pathway.

OBJECTIVE: To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc). METHODS: Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O2- and H2O2 released from fibroblasts were estimated by the superoxide dismutase (SOD)-inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti-p47phox-specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by 3H-thymidine incorporation. Northern blot analysis was used to study alpha1 and alpha2 type I collagen gene expression. RESULTS: Unstimulated skin fibroblasts from SSc patients released more O2- and H2O2 in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component of NADPH oxidase, p47phox, was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin-1beta (IL-1beta), platelet-derived growth factor type BB (PDGF-BB), transforming growth factor beta1 (TGFbeta1), and H2O2. Treatment of normal and SSc fibroblasts with tumor necrosis factor a (TNFalpha), IL-2, IL-4, IL-6, IL-10, interferon-alpha (IFNalpha), IFNgamma, granulocyte-macrophage colony-stimulating factor (GM-CSP), G-CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL-1 receptor antagonist, or antibodies blocking the effect of TGFbeta1, PDGF-BB, and other agonists (IL-4, IL-6, TNFalpha, CTGF). In contrast, treatment of SSc fibroblasts with the membrane-permeant antioxidant N-acetyl-L-cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down-regulation of alpha1(I) and alpha2(I) collagen messenger RNA. CONCLUSION: The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase-like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O2-, H2O2, IL-1beta, TGFbeta1, PDGF-BB, IL-4, IL-6, TNFalpha, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.

A 1.1-kb duplication in the p67-phox gene causes chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency that is caused by a functional defect of the NADPH oxidase of phagocytes, and that leads to severe recurrent infections. CGD results from the absence or the dysfunction of various components of NADPH oxidase, and autosomal recessive CGD with the lack of p67-phox (A67 CGD) is the rarest form of the disease. Identifying familiar mutations in subjects with A67 CGD provides the most reliable method of detecting carriers and is the basis for prenatal diagnosis. In the present study, we report the detailed characterization of the first duplication in the p67-phox gene identified in a 30-year-old patient affected by systemic aspergillosis attributable to p67-phox deficiency. We show that this new mutation involving exons 9 and 10 is the result of a tandem duplication of approximately 1.1 kb, which resulted from the juxtaposition of intron 8 to intron 10. We have sequenced both the junction fragment of this duplication and the corresponding wild-type regions and have found that the breakpoint regions in intron 8 and in intron 10 show limited homology. Our result suggests that this interchange arose as an illegitimate recombination event. As in other non-homologous rearrangements previously reported, the duplication breakpoints are located within the sequence motif 5'-CCAG-3' and its complement 5'-CTGG-3'.

Mechanisms of expression of NADPH oxidase components in human cultured monocytes: role of cytokines and transcriptional regulators involved.

Human blood monocytes lose their capability to produce microbicidal oxidants during culture. We report that this process is associated with decreased gp91phox, p22phox and p47phox expression, release of PU.1 and CP-1 from gp91phox promoter, and PU.1 from p47phox promoter. However, in presence of IFN-gamma or TNF-alpha, the superoxide anion (O(2)(-)) production, the p47phox, gp91phox and p22phox expression, and the binding of PU.1 and CP-1 to DNA are maintained at the high levels observed in blood monocytes. To clarify the role of PU.1 in the expression of NADPH oxidase components, oligonucleotides competing for PU.1-DNA binding were added to cultured monocytes. These oligonucleotides abrogated the maintenance of gp91phox and p22phox expression by IFN-gamma and TNF-alpha, but did not inhibit the effect of these cytokines on p47phox expression and O(2)(-) production. Our results indicate that in monocytes the IFN-gamma- and TNF-alpha-induced expression of gp91phox and p22phox, but not p47phox, requires the binding of PU.1 to gp91phox promoter. However, the preservation of O(2)(-) production by IFN-gamma and TNF-alpha is unrelated to their effect on gp91phox and p22phox expression.

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor.

Helicobacter pylori infection induces the appearance of inflammatory infiltrates, consisting mainly of neutrophils and monocytes, in the human gastric mucosa. A bacterial protein with neutrophil activating activity (HP-NAP) has been previously identified, but its role in infection and immune response is still largely unknown. Here, we show that vaccination of mice with HP-NAP induces protection against H. pylori challenge, and that the majority of infected patients produce antibodies specific for HP-NAP, suggesting an important role of this factor in immunity. We also show that HP-NAP is chemotactic for human leukocytes and that it activates their NADPH oxidase to produce reactive oxygen intermediates, as demonstrated by the translocation of its cytosolic subunits to the plasma membrane, and by the lack of activity on chronic granulomatous disease leukocytes. This stimulating effect is strongly potentiated by tumor necrosis factor alpha and interferon gamma and is mediated by a rapid increase of the cytosolic calcium concentration. The activation of leukocytes induced by HP-NAP is completely inhibited by pertussis toxin, wortmannin, and PP1. On the basis of these results, we conclude that HP-NAP is a virulence factor important for the H. pylori pathogenic effects at the site of infection and a candidate antigen for vaccine development.

Induction of functional IL-8 receptors by IL-4 and IL-13 in human monocytes.

IL-8 and related Glu-Leu-Arg (ELR+) CXC chemokines are potent chemoattractants for neutrophils but not for monocytes. IL-13 and IL-4 strongly increased CXCR1 and CXCR2 chemokine receptor expression in human monocytes, macrophages, and dendritic cells. The effect was receptor- and cell type-selective, in that CCRs were not increased and no augmentation was seen in neutrophils. The effect was rapid, starting at 4 h, and concentration dependent (EC50 = 6.2 and 8.3 ng/ml for CXCR1 and CXCR2, respectively) and caused by new transcriptional activity. IL-13/IL-4-treated monocytes showed increased CXCR1 and CXCR2 membrane expression. IL-8 and related ELR+ chemokines were potent and effective chemotactic agents for IL-13/IL-4-treated monocytes, but not for untreated mononuclear phagocytes, with activity comparable to that of reference monocyte attractants, such as MCP-1. In the same cells, IL-8 also caused superoxide release. Macrophages and dendritic cells present in biopsies from Omenn's syndrome and atopic dermatitis patients, two Th2 skewed pathologies, expressed IL-8 receptors by immunohistochemistry. These results show that IL-13 and IL-4 convert IL-8 and related ELR+ chemokines, prototypic neutrophil attractants, into monocyte chemotactic agonists, by up-regulating receptor expression. Therefore, IL-8 and related chemokines may contribute to the accumulation and positioning of mononuclear phagocytes in Th2-dominated responses.

beta-amyloid activates the O-2 forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer's disease.

The deposition of beta-amyloid in the brain is the key pathogenetic event in Alzheimer's disease. Among the various mechanisms proposed to explain the neurotoxicity of beta-amyloid deposits, a new one, recently identified in our and other laboratories, suggests that beta-amyloid is indirectly neurotoxic by activating microglia to produce toxic inflammatory mediators such as cytokines, nitric oxide, and oxygen free radicals. Three findings presented here support this mechanism, showing that beta-amyloid peptides (25-35), (1-39), and (1-42) activated the classical NADPH oxidase in rat primary culture of microglial cells and human phagocytes: 1) The exposure of the cells to beta-amyloid peptides stimulates the production of reactive oxygen intermediates; 2) the stimulation is associated with the assembly of the cytosolic components of NADPH oxidase on the plasma membrane, the process that corresponds to the activation of the enzyme; 3) neutrophils and monocytes of chronic granulomatous disease patients do not respond to beta-amyloid peptides with the stimulation of reactive oxygen intermediate production. Data are also presented that the activation of NADPH oxidase requires that beta-amyloid peptides be in fibrillary state, is inhibited by inhibitors of tyrosine kinases or phosphatidylinositol 3-kinase and by dibutyryl cyclic AMP, and is potentiated by interferon-gamma or tumor necrosis factor-alpha.

Monocytes of patients wiht systemic sclerosis (scleroderma spontaneously release in vitro increased amounts of superoxide anion.

It has been suggested that toxic oxygen free radicals can be involved in the pathogenesis of systemic sclerosis (scleroderma) (SSc). Because the cells that contribute to the generation of free radicals are not known, our aim was (i) to evaluate the ability of unmanipulated and phorbol 12-myristate 13-acetate-stimulated monocytes and polymorphonucleate neutrophils of SSc patients to generate superoxide anion (O2*-); and (ii) to investigate whether the O2*- produced by these cells involved the activation of nicotinamide-adenine dinucleotide diphosphate oxidase biochemical pathway. Employing the superoxide dismutase-inhibitable reduction of cytochrome c to evaluate the generation of O2*-, unmanipulated monocytes of SSc patients generated more O2*- than primary Raynaud's phenomenon patients and normal control monocytes (p = 0.0001), and the release was higher in patients with diffuse cutaneous involvement and 5 y or less disease duration (p = 0.02). The involvement of nicotinamide-adenine dinucleotide diphosphate oxidase in the enhanced 02*- production was demonstrated by the finding that the cytosolic components of the enzyme, p47phox and p67phox, were both translocated to the plasma membrane of enriched but otherwise unmanipulated monocytes of SSc patients. The involvement of mitochondrial oxidases was excluded by the lack of inhibition of O2*- production when monocytes were incubated in the presence of rotenone, a mitochondrial oxidase inhibitor. Upon stimulation with phorbol 12-myristate 13-acetate, monocytes of SSc patients produced more O2*- than controls. In SSc patients untreated polymorphonucleate neutrophils generated significantly less O2*- than monocytes (p = 0.0001) and only slightly more than polymorphonucleate neutrophils of primary Raynaud's phenomenon patients and normal controls (p = 0.03). In conclusion, we demonstrate that in patients with scleroderma, unmanipulated and phorbol 12-myristate 13-acetate-stimulated monocytes release in vitro increased amounts of superoxide anion through the activation of nicotinamide-adenine dinucleotide diphosphate oxidase and, thus, contribute to the oxidative stress found in this disease.

Nicotinamide-adenine dinucleotide phosphate oxidase assembly and activation in EBV-transformed B lymphoblastoid cell lines of normal and chronic granulomatous disease patients.

This paper deals with the mechanisms of activation of NADPH oxidase investigated using EBV-transformed human B lymphoblastoid cell lines (B cells) from normal subjects and from patients affected by X-linked chronic granulomatous disease (CGD). The results reported are as follows. 1) In normal B cells, the NADPH oxidase components p67phox, p40phox, p22phox, and gp91phox were less expressed than in polymorphonuclear neutrophils. 2) In normal B cells stimulated with PMA, p47phox, p67phox, and p40phox translocated to the membranes as occurs in polymorphonuclear neutrophils. 3) In CGD, B cells expressing p22phox in the absence of gp91phox, p47phox, p67phox, and p40phox did not translocate to the membranes after stimulation with PMA. 4) In PMA-stimulated B cells from an X91+ CGD patient in which p22phox was normally expressed and gp91phox was present but lacked five amino acids, translocation of p47phox to the membranes was unaffected, but p67phox and p40phox were poorly translocated, and the production of O2- was greatly reduced with respect to that by normal B cells. Taken together, these findings indicate that 1) a low expression of some NADPH oxidase components may represent the molecular basis of the low production of O2- in B lymphocytes; 2) the cytosolic components of NADPH oxidase cannot bind to p22phox on the membranes in the absence of gp91phox; 3) p47phox can translocate to the membranes independently of p67phox and p40phox; and 4) gp91phox may have a role in mediating and/or stabilizing the binding of p67phox and p40phox to the membranes of activated cells.

Identification of a double mutation (D160V-K161E) in the p67phox gene of a chronic granulomatous disease patient.

In neutrophils of a chronic granulomatous disease (CGD) patient with a lack of p67phox the mRNA for p67phox was present in normal amount and size. This mRNA was reverse transcribed, and the coding region was analyzed by single-strand conformation polymorphism analysis. Direct DNA sequencing allowed the identification of a A479-to-T and A481-to-G substitution in exon 5 of the p67phox gene resulting in a double nonconservative amino acid change 160Lys-to-Glu and 161Asp-to-Val (D160V-K161E). This defect was found in the genomic DNA of this patient in heterozygous state and does not correspond to those previously found in other cases of CGD lacking the p67phox.

Mechanisms of stimulation of the respiratory burst by TNF in nonadherent neutrophils: its independence of lipidic transmembrane signaling and dependence on protein tyrosine phosphorylation and cytoskeleton.

This study concerns the controversial problem of whether the TNF-alpha (TNF) induces a respiratory burst in human neutrophils in suspension. The results have shown that in these cells TNF induces a classical respiratory burst. In fact, the production of oxygen free radicals 1) is linked to the translocation of NADPH oxidase components from cytosol to the plasma membrane, 2) does not take place in neutrophils from a patient lacking the cytochrome b558, and 3) does not involve other sources such as mitochondrial respiratory chain or xanthine oxidase. Signal transduction studies have demonstrated that this respiratory burst 1) is not accompanied by calcium transients, stimulation of phosphoinositide turnover, and phospholipase D activity (moreover, this burst is associated with the stimulation of the activity of phospholipase A2, but not of sphingomyelinase); 2) is strictly dependent on activation of tyrosine kinases, which is functional to the translocation to the plasma membrane of the cytosolic NADPH oxidase component rac; and 3) is dependent on the integrity of the cytoskeleton because it is completely suppressed by cytochalasin B. The integrity of the cytoskeleton is required for a full translocation of all the NADPH oxidase components and for an optimal activation of tyrosine kinases, but not for phospholipase A2 activation. Taken together, these findings demonstrate that TNF activates the NADPH oxidase through stimulation of tyrosine kinases, whose function is cytoskeleton-dependent, and raise the problem of whether the activation of this respiratory burst involves signals arising from TNF-activated beta2 integrins.

Tyrosine phosphorylation and subcellular redistribution of p125 ras guanosine triphosphatase-activating protein in human neutrophils stimulated with FMLP.

In this paper, we show that the p125 ras guanosine triphosphatase-activating protein (p125 GAP) is present in the cytosol of human neutrophils and is transiently tyrosine phosphorylated and translocated to the membranes upon cell activation with formyl-methionyl-leucyl-phenylalanine (FMLP). When concanavalin A (ConA) or phorbol 12-myristate 13-acetate (PMA), which both induced a long-lasting respiratory burst, were used as stimuli, tyrosine phosphorylation and translocation of p125 GAP did not occur.

Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox.

On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox, p47phox, p40phox, as well as the Ras-related G-proteins Rac1 and Rac2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b, forming a functional complex responsible for the production of oxygen radicals in phagocytes. In this paper we show that (a) in neutrophils from a patient with a form of chronic granulomatous disease (CGD) in which p67phox is absent, p47phox and Rac2, but not p40phox and Rac1 were translocated from the cytosol to the membrane on stimulation with formylmethionyl-leucylphenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA); (b) in neutrophils from a patient with a form of CGD in which p47phox is absent, p67phox, p40phox and Rac1 failed to associate with the membrane on stimulation with fMLP or PMA, whereas Rac2 was translocated as in normal neutrophils. We also show that in neutrophils from a patient lacking p67phox, the amount of cytosolic p40phox was decreased by about 40%. These findings indicate that, on neutrophil stimulation, p67phox mediates the translocation of p40phox and Rac1 from the cytosol to cell membranes and that Rac2 associates with the membranes independently of p47phox and p67phox.

Translocation of p190rho guanosine triphosphatase-activating protein from cytosol to the membrane in human neutrophils stimulated with different agonists.

In this paper, we investigated the subcellular distribution of p190rho guanosine triphosphatase-activating protein (p190 GAP) in human neutrophils stimulated with different agonists. The results show that in neutrophils treated with formyl-methionyl-leucyl-phenylalanine (FMLP) (1) p190 GAP was translocated from the cytosol to the membranes; (2) the translocation of p190 GAP took place only at doses of FMLP that induced the translocation of rac 1 and rac 2 and the activation of the NADPH oxidase; and (3) the kinetic of translocation of p190 GAP paralleled that of rac 1 and rac 2. However, when the agonist was concanavalin A (ConA) or phorbol 12-myristate 13-acetate (PMA), rac 1 and rac 2, but not the p190 GAP, were translocated.

Tumor necrosis factor triggers redistribution to a Triton X-100-insoluble, cytoskeletal fraction of beta 2 integrins, NADPH oxidase components, tyrosine phosphorylated proteins, and the protein tyrosine kinase p58fgr in human neutrophils adherent to fibrinogen.

Tumor necrosis factor (TNF) triggers cell spreading, release of granule constituents, and production of toxic oxygen derivatives in human neutrophils adherent to fibrinogen. This response requires cytoskeleton reorganization and is dependent on expression of beta 2 integrins. We analyzed distribution of distinct proteins in Triton X-100-soluble and insoluble fractions in neutrophils adherent to fibrinogen. We found that stimulation of adherent neutrophils with TNF causes the redistribution to a Triton-insoluble fraction of alpha-actinin, beta 2 integrins, and the four components whose assembly constitutes an active NADPH oxidase: the gp91-phox, p22-phox, p47-phox, and p67-phox proteins. Redistribution of these different proteins to a Triton-insoluble fraction took relatively long times and was maximal after about 30 min of stimulation with TNF. Prevention of actin polymerization with cytochalasin B hampered the TNF-induced redistribution of these proteins from a Triton-soluble to an insoluble fraction. In addition, tyrosine phosphorylated proteins and the protein tyrosine kinase p58fgr were recovered in this Triton-insoluble fraction. These findings show that stimulated, beta 2 integrin-dependent adhesion of neutrophils to fibrinogen is accompanied by redistribution to cytoskeletal structures of (1) beta 2 integrins, that is, neutrophil receptors for fibrinogen; (2) proteins involved in neutrophil effector functions, that is, components of NADPH oxidase; and (3) tyrosine phosphorylated proteins and the protein tyrosine kinase p58fgr, molecules that are potentially involved in the formation of a submembranous signaling complex.

Role of 55- and 75-kDa TNF receptors in the potentiation of Fc-mediated phagocytosis in human neutrophils.

Human neutrophils respond with an increased phagocytosis when exposed to TNF. Two types of TNF receptors have been identified, namely 55 kDa (TR55) and 75 kDa (TR75). We addressed the problem of the role of these receptors in the priming effect of TNF. By using monoclonal antibodies (MoAbs) directed either against TR55 or TR75, we have shown that 1) only TR55 is the signaling receptor for the potentiation of Fc-mediated phagocytosis and upregulation of beta 2-integrin CD11b/CD18; 2) TR75 may control the function of TR55 by regulating the binding of TNF to the signaling receptor.

Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes.

NADPH oxidase is the enzyme complex responsible for the production of oxygen radicals in phagocytes. On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox and p47phox, as well as the Ras-related G-protein rac 2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b to form a functional complex. Besides rac 2, rac 1 G-protein is also involved in the activation of the NADPH oxidase, but, to date, it has not been documented whether it is also translocated in activated neutrophils. In this paper we show that: (a) in neutrophils stimulated with formylmethionyl-leucylphenylalanine, concanavalin A or phorbol 12-myristate 13-acetate, both rac 1 and rac 2 are translocated from cytosol to the membranes; (b) in neutrophils from a patient with a form of chronic granulomatous disease in which p67phox is absent, rac 2 and p47phox were translocated as in normal neutrophils on stimulation with the above agonists, but rac 1 failed to be translocated from the cytosol to the membranes. This is the first demonstration that, in activated neutrophils, rac 1 is translocated from the cytosol to the membranes and this translocation requires p67phox. These results, coupled with those showing that rac 2 is not translocated in activated neutrophils lacking p47phox [El Benna, Ruedi and Babior (1994) J. Biol. Chem. 269, 6729-6734], may suggest that the assembly of the cytosolic components of NADPH oxidase on the plasma membrane takes place through selective coupling of activated rac 1 and rac 2 with p67phox and p47phox respectively.

Pentoxifylline as a supportive agent in the treatment of cerebral malaria in children.

In an open, randomized, controlled therapeutic trial, 56 children with cerebral malaria (CM) were randomly assigned to receive standard quinine regimen with or without pentoxifylline (10 mg/kg/day by continuous intravenous infusion). Pentoxifylline exerted an inhibitory effect on the synthesis of tumor necrosis factor (TNF), a possible mediator of CM. The 26 children who received pentoxifylline had significantly shorter comas than controls (median, 6 vs. 46 h; P < .001) Pentoxifylline recipients showed a trend toward a lower mortality, with a borderline significant difference (P = .055). The better outcome in the pentoxifylline group was associated with a decline in TNF serum levels on the third day of treatment in a few subjects that was not seen in controls. While alternative or concurrent mechanisms of action may be of some relevance, larger double-blind trials are needed to determine whether pentoxifylline has a therapeutic role in CM.

Interleukin-10 decreases tyrosine phosphorylation of discrete lipopolysaccharide-induced phosphoproteins in human granulocytes.

Although Interleukin-10 (IL-10) has been recently shown to modulate lipopolysaccharide (LPS)-induced release of cytokines in human granulocytes, the intracellular signalling pathways of LPS have been only partially defined, while those of IL-10 remain unknown. The present study shows that LPS induces an increase in tyrosine phosphorylation of a discrete number of proteins, in a time- and concentration-dependent manner. In addition, IL-10 negatively influenced protein tyrosine phosphorylation in LPS-treated human polymorphonuclear leukocytes (PMN). The effect of IL-10 was evident only after 60 min LPS-stimulation and was detected by analysing either cell lysates or lysates which were previously immunoprecipitated with anti-phosphotyrosine antibodies. Amongst the tyrosine phosphoproteins mostly affected by IL-10 in LPS-stimulated cells were the species with molecular weights ranging from 46 to 49 kDa. The identity and possible function of these proteins remain unknown. Taken together, our results suggest that tyrosine phosphorylation may constitute one of the intracellular events that mediate LPS and IL-10 responses in granulocytes.