Comparison of interferon-gamma, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor for priming leukocyte-mediated hyphal damage of opportunistic fungal pathogens.
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Proinflammatory cytokines have been proposed as adjunctive therapeutic agents to enhance the host immune response during infections caused by opportunistic fungi. The study compared the differential in vitro priming effects of interferon-gamma (IFN-gamma), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on hyphal damage of opportunistic fungi mediated by isolated neutrophils (polymorphonuclear leukocytes, PMNL) and buffy coat cells (polymorphonuclear leukocytes/peripheral blood mononuclear cells, PMNL/PBMC) from healthy donors. IFN-gamma (1000 U/mL) effectively primed both PMNL and PMNL/PBMC for enhanced hyphal damage of Aspergillus fumigatus, Fusarium solani, and Candida albicans. G-CSF (100 ng/mL) increased hyphal damage mediated by both PMNL and PMNL/PBMC against F. solani, and GM-CSF (100 ng/mL) augmented the antifungal activity of PMNL/PBMC against hyphal forms of both F. solani and C. albicans. IFN-gamma may be superior to G-CSF or GM-CSF for enhancing the microbicidal activity of PMNL and PMNL/PBMC against opportunistic fungi. (+info)
Multicenter phase III trial to evaluate CD34(+) selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma.
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High-dose chemotherapy followed by autologous transplantation has been shown to improve response rates and survival in multiple myeloma and other malignancies. However, autografts frequently contain detectable tumor cells. Enrichment for stem cells using anti-CD34 antibodies has been shown to reduce autograft tumor contamination in phase I/II studies. To more definitively assess the safety and efficacy of CD34 selection, a phase III study was completed in 131 multiple myeloma patients randomized to receive an autologous transplant with either CD34-selected or unselected peripheral blood progenitor cells after myeloablative therapy. Tumor contamination in the autografts was assessed by a quantitative polymerase chain reaction detection assay using patient-specific, complementarity-determining region (CDR) Ig gene primers before and after CD34 selection. A median 3.1 log reduction in contaminating tumor cells was achieved in the CD34 selected product using the CEPRATE SC System (CellPro, Inc, Bothell, WA). Successful neutrophil engraftment was achieved in all patients by day 15 and no significant between-arm difference for time to platelet engraftment occurred in patients who received an infused dose of at least 2.0 x 10(6) CD34(+) cells/kg. In conclusion, this phase III trial demonstrates that CD34-selection of peripheral blood progenitor cells significantly reduces tumor cell contamination yet provides safe and rapid hematologic recovery for patients receiving myeloablative therapy. (+info)
Missense mutations in the gp91-phox gene encoding cytochrome b558 in patients with cytochrome b positive and negative X-linked chronic granulomatous disease.
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Chronic granulomatous disease (CGD) is a disorder of host defense due to genetic defects of the superoxide (O2-) generating NADPH oxidase in phagocytes. A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the oxidase. The X-linked form of the disease is due to defects in the gp91-phox gene. We report here biochemical and genetic analyses of patients with typical and atypical X-linked CGD. Immunoblots showed that neutrophils from one patient had small amounts of p22-phox and gp91-phox and a low level of O2- forming oxidase activity, in contrast to the complete absence of both subunits in two patients with typical CGD. Using polymerase chain reactions (PCR) on cDNA and genomic DNA, we found novel missense mutations of gp91-phox in the two typical patients and a point mutation in the variant CGD, a characteristic common to two other patients with similar variant CGD reported previously. Spectrophotometric analysis of the neutrophils from the variant patient provided evidence for the presence of heme of cytochrome b558. Recently, we reported another variant CGD with similar amounts of both subunits, but without oxidase activity or the heme spectrum. A predicted mutation at amino acid 101 in gp91-phox was also confirmed in this variant CGD by PCR of the genomic DNA. These results on four patients, including those with two variant CGD, are discussed with respect to the missense mutated sites and the heme binding ligands in gp91-phox. (+info)
Rho-kinase in human neutrophils: a role in signalling for myosin light chain phosphorylation and cell migration.
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The role of a Rho-associated coiled-coil forming kinase in migration of neutrophils has been investigated. Rho-associated coiled-coil forming kinase I was expressed in human neutrophils. Chemotactic peptide led to a Rho-associated coiled-coil forming kinase-dependent increase in phosphorylation of myosin light chain. This was determined with the help of an antibody directed against serine 19-phosphorylated myosin light chain and an inhibitor of Rho-associated coiled-coil forming kinase (Y-27632). Y-27632 suppressed myosin light chain phosphorylation and chemotactic peptide-induced development of cell polarity and locomotion with similar potency (ED50 0.5-1.1 microM). The data strongly suggest that a Rho-associated coiled-coil forming kinase isoform, activated in human neutrophils exposed to chemotactic peptide, is important for motile functions of these cells. (+info)
Neutrophils sense flow-generated stress and direct their migration through alphaVbeta3-integrin.
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During inflammation neutrophils are recruited from the blood onto the surface of microvascular endothelial cells. In this milieu the presence of soluble chemotactic gradients is disallowed by blood flow. However, directional cues are still required for neutrophils to migrate to the junctions of endothelial cells where extravasation occurs. Shear forces generated by flowing blood provide a potential alternative guide. In our flow-based adhesion assay neutrophils preferentially migrated in the direction of flow when activated after attachment to platelet monolayers. Neutralizing alphaVbeta3-integrin with monoclonal antibodies or turning the flow off randomized the direction of migration without affecting migration velocity. Purified, immobilized alphaVbeta3-integrin ligands, CD31 and fibronectin, could both support flow-directed neutrophil migration in a concentration-dependent manner. Migration could be randomized by neutralizing alphaVbeta3-integrin interactions with the substrate using antibodies or Arg-Gly-Asp-containing peptide. These results exemplify mechanical signal transduction through integrin-ligand interactions and reveal a guidance system that was hitherto unknown in neutrophils. In more general terms, it demonstrates that cells can use integrin molecules to "sample" their physical microenvironment through adhesion and use this information to modulate their behavior. (+info)
Role of nitric oxide-derived oxidants in vascular injury from carbon monoxide in the rat.
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Studies were conducted with rats to investigate whether exposure to CO at concentrations frequently found in the environment caused nitric oxide (NO)-mediated vessel wall changes. Exposure to CO at concentrations of 50 parts per million or higher for 1 h increased the concentration of nitrotyrosine in the aorta. Immunologically reactive nitrotyrosine was localized in a discrete fashion along the endothelial lining, and this was inhibited by pretreatment with the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). The CO-induced elevations of aortic nitrotyrosine were not altered by neutropenia or thrombocytopenia, and CO caused no change in the concentration of endothelial NOS. Consequences from NO-derived stress on the vasculature included an enhanced transcapillary efflux of albumin within the first 3 h after CO exposure and leukocyte sequestration that became apparent 18 h after CO exposure. Oxidized plasma low-density lipoprotein was found immediately after CO exposure, but this was not inhibited by L-NAME pretreatment. We conclude that exposure to relatively low CO concentrations can alter vascular status by several mechanisms and that many changes are linked to NO-derived oxidants. (+info)
S-nitrosoglutathione enhances neutrophil DNA fragmentation and cell death.
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Enhancing the clearance of neutrophils by enhancing apoptotic cell death and macrophage recognition may be beneficial in acute lung injury. Exogenous nitric oxide gas depresses neutrophil oxidative functions and accelerates cell death (A. H. Daher, J. D. Fortenberry, M. L. Owens, and L. A. Brown. Am. J. Respir. Cell Mol. Biol. 16: 407-412, 1997). We hypothesized that S-nitrosoglutathione (GSNO), a physiologically relevant nitric oxide donor, could also enhance neutrophil DNA fragmentation. Neutrophils were incubated for 2-24 h in the absence and presence of GSNO (dose range 0.1-5 mM) and evaluated for cell death by a fluorescent viability/cytotoxicity assay. Neutrophil DNA fragmentation was assessed by cell death detection ELISA and by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling assay. Neutrophil oxidative function was also determined. Incubation with GSNO increased cell death at 2, 4, and 24 h. GSNO incubation for 24 h significantly increased DNA fragmentation in a dose-dependent fashion at 0.5 (median 126% of control value; P = 0.002) and 5 mM (185% of control value; P = 0.002) by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling and at 0.5 mM by ELISA (164% of control value; P = 0.03). The apoptosis-to-total cell death ratio increased with increasing GSNO concentration (P < 0.05). Effects were mitigated by coincubation with superoxide dismutase. Five millimolar GSNO decreased overall superoxide generation and O2 consumption but not when adjusted for dead neutrophils. GSNO significantly enhances cell death and neutrophil DNA fragmentation in a dose-dependent fashion. (+info)
Mediators of anaphylaxis but not activated neutrophils augment cholinergic responses of equine small airways.
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Neutrophilic inflammation in small airways (SA) and bronchospasm mediated via muscarinic receptors are features of chronic obstructive pulmonary disease in horses (COPD). Histamine, serotonin, and leukotrienes (LTs) are reported to be involved in the exacerbation of COPD, and currently, histamine has been shown to increase tension response to electrical field simulation (EFS) in equine SA. We tested the effects of these mediators and the effects of activated neutrophils on the cholinergic responses in SA. Histamine, serotonin, and LTD4 had a synergistic effect on EFS responses and only an additive effect on the tension response to exogenous ACh or methacholine. Atropine and TTX entirely eliminated the EFS-induced tension response in the presence of all three inflammatory mediators, indicating that augmentation of the EFS response applies only to the endogenous cholinergic response. Neutrophils isolated from control and COPD-affected horses were activated by zymosan, producing 18.1 +/- 2.3 and 25.0 +/- 2.3 nmol superoxide. 10(6) cells-1. 30 min-1, respectively. However, in contrast to the profound effect of mediators, incubation of SA for over 1 h in a suspension of up to 30 x 10(6) zymosan-treated neutrophils/ml did not significantly affect EFS responses of SA isolated from either control or COPD-affected horses. We conclude that in equine SA 1) the endogenous cholinergic responses are subject to strong facilitation by inflammatory mediators; 2) activated neutrophils do not affect cholinergic responses in SA; and 3) in acute bouts of equine COPD, histamine, LTD4, and serotonin (mediators primarily associated with type I allergic reaction) rather than mediators derived from neutrophils most likely contribute to increased cholinergic airway tone. (+info)