calcimycin and Acute-Disease

IV magnesium (Mg2+) has been proposed as an emergent treatment for acute asthma exacerbations. Recent studies have focused on the effects of Mg2+ on bronchial smooth muscle, yet asthma is primarily an inflammatory disease.. To assess the effects of Mg2+ on the neutrophil respiratory burst of adult patients with asthma.. A prospective, blind study of volunteer adult asthmatic patients was performed. The patients' polymorphonuclear neutrophils (PMNs) were isolated, purified, and placed into phosphate-buffered saline with the following test conditions: concentrations of magnesium chloride (MgCl2) added: 0 mmol MgCl2, 1 mmol MgCl2 (low), and 10 mmol MgCl2 (high) both with and without the calcium (Ca2+) ionophore A23187 (0.1 mmol). PMNs were activated using N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10 mumol), and the production of superoxide (O2-) was measured by the spectrophotometric reduction of cytochrome c.. Mg2+ reduced activated PMN O2- production compared with that for no Mg2+ (1.0 +/- 0.1 nmol O2-/5 x 10(5) PMN/min) in both low (-0.52* +/- 0.3 nmol O2-/5 x 10(5) PMN/min) and high (-0.76* +/- 0.3 nmol O2-/5 x 10(5) PMN/min; *p < 0.05) concentrations. The addition of A23187 increased O2- production in both the high (0.53* +/- 0.02 nmol O2-/5 x 10(5) PMN/min) and the low (1.5* +/- 0.6 nmol O2-/5 x 10(5) x 10(5) PMN/min) Mg2+ groups, with no change in the control group (1.2 +/- 0.2 nmol O2-/10(5) PMN/min).. In clinically relevant concentrations, Mg2+ attenuates the neutrophil respiratory burst in adult asthmatic patients. Mg2+ appears to affect PMNs by interfering with extracellular Ca2+ influx. Mg2+ may have a beneficial anti-inflammatory effect in asthmatic individuals.

Topics: Acute Disease; Adult; Asthma; Calcimycin; Calcium Channels; Double-Blind Method; Drug Therapy, Combination; Female; Humans; Inflammation; Ionophores; Magnesium Chloride; Male; Middle Aged; Prospective Studies; Respiratory Burst

Lipoxin A(4) (LXA(4)) is a structurally and functionally distinct natural product called an eicosanoid, which displays immunomodulatory and anti-inflammatory activity but is rapidly metabolized to inactive catabolites in vivo. A previously described analogue of LXA(4), methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), was shown to have a poor pharmacokinetic profile after both oral and intravenous administration, as well as sensitivity to acid and light. The chemical stability of the corresponding E,E,E-trien-11-yne analogue, 3, was improved over 2 without loss of efficacy in the mouse air pouch model of inflammation. Careful analysis of the plasma samples from the pharmacokinetic assays for both 2 and 3 identified a previously undetected metabolite, which is consistent with metabolism by beta-oxidation. The formation of the oxidative metabolites was eliminated with the corresponding 3-oxatetraene, 4, and the 3-oxatrien-11-yne, 5, analogues of 2. Evaluation of 3-oxa analogues 4 and 5 in calcium ionophore-induced acute skin inflammation model demonstrated similar topical potency and efficacy compared to 2. The 3-oxatrien-11-yne analogue, 5, is equipotent to 2 in an animal model of inflammation but has enhanced metabolic and chemical stability and a greatly improved pharmacokinetic profile.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents, Non-Steroidal; Calcimycin; Dermatitis, Contact; Drug Stability; Ionophores; Lipoxins; Male; Mice; Mice, Inbred BALB C; Oxidation-Reduction; Phenyl Ethers; Stereoisomerism; Structure-Activity Relationship

As shown elsewhere, cultured acute myeloid leukaemia blasts acquire certain characteristics of dendritic cells upon stimulation with cytokines and calcium ionophore. The ability of leukaemia-derived dendritic-like cells to express immune costimulatory molecules and dendritic cell marker CD83 has been extensively investigated. Although migratory capacity is a major attribute of dendritic cells, the ability of in vitro modified blasts for adhesion, chemotaxis and homing remain elusive. In the present paper, we show that after stimulation with calcium ionophore acute myeloid leukaemia blasts as well as normal dendritic cell precursors demonstrate increased capacity of binding fibronectin and denatured collagen. The expression pattern of integrins on dendritic-like leukaemic cells in general closely resembles that of monocyte-derived dendritic cells, however, variation in cell properties isolated from blood of individual patients are observed.

Topics: Acute Disease; Adult; Antigens, CD; Calcimycin; CD83 Antigen; Cell Adhesion; Dendritic Cells; Female; Fibronectins; Humans; Immunoglobulins; Integrins; Ionophores; Leukemia, Myeloid; Leukocytes, Mononuclear; Male; Membrane Glycoproteins; Middle Aged; Protein Binding

The ability of acute myeloid leukaemia (AML) cells to acquire dendritic cell (DC)-like characteristics in vitro with a rapid culture method based either on the phorbol ester PMA or calcium ionophores has been studied in comparison to conventional AML-DC cultures with the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha), interleukin-3 (IL-3), SCF, FLT3-L and IL-4. In all AML patients, antigen-presenting cells (APC) could be generated from leukaemic cells in 2 days by incubation with PMA or calcium ionophore (A23187 or ionomycin) in the presence as well as in the absence of IL-4. In 30 out of 36 patients APC could be generated after 2 weeks of culture in cytokine-enriched medium. AML-APC cultured with PMA or calcium ionophores immunophenotypically and functionally were at a more mature stage than those cultured in cytokine-enriched medium. The most mature APC were generated by calcium ionophore A23187 plus IL-4, as evidenced by the higher expression of CD40, CD80, CD86 and HLA-DR. Autologous T cell mediated cytotoxicity towards AML blast cells in vitro was observed in 2 cases tested. The persistence of cytogenetic abnormalities confirmed the leukaemic origin of the AML-APC. The generation of AML-APC was possible from freshly isolated as well as cryopreserved material. Our data show that generation of sufficient AML-APC by A23187 plus IL-4 is feasible, for vaccination purposes, in approximately 70% of AML specimens, offering a time-saving and cost-effective approach in preparing anti-leukaemia vaccines.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antigen-Presenting Cells; Antigens, CD; Antigens, Neoplasm; Bone Marrow Cells; Calcimycin; Calcium; Cancer Vaccines; Cell Culture Techniques; Cryopreservation; Cytotoxicity, Immunologic; Feasibility Studies; Female; HLA-DR Antigens; Humans; Interleukin-4; Ionophores; Leukemia, Myeloid; Lymphocyte Culture Test, Mixed; Male; Middle Aged; Neoplastic Cells, Circulating; Neoplastic Stem Cells; Reproducibility of Results; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Time Factors; Tissue Preservation; Tumor Cells, Cultured; Vaccination

Diffusion magnetic resonance imaging (MRI) provides a surrogate marker of acute brain pathology, yet few studies have resolved the evolution of water diffusion changes during the first 8 hours after acute injury, a critical period for therapeutic intervention. To characterize this early period, this study used a 17.6-T wide-bore magnet to measure multicomponent water diffusion at high b-values (7 to 8,080 s/mm(2)) for rat hippocampal slices at baseline and serially for 8 hours after treatment with the calcium ionophore A23187. The mean fast diffusing water fraction (Ffast) progressively decreased for slices treated with 10-microM/L A23187 (-20.9 +/- 6.3% at 8 hours). Slices treated with 50-micromol/L A23187 had significantly reduced Ffast 80 minutes earlier than slices treated with 10-microM/L A23187 (P < 0.05), but otherwise, the two doses had equivalent effects on the diffusion properties of tissue water. Correlative histologic analysis showed dose-related selective vulnerability of hippocampal pyramidal neurons (CA1 > CA3) to pathologic swelling induced by A23187, confirming that particular intravoxel cell populations may contribute disproportionately to water diffusion changes observed by MRI after acute brain injury. These data suggest diffusion-weighted images at high b-values and the diffusion parameter Ffast may be highly sensitive correlates of cell swelling in nervous issue after acute injury.

Topics: Acute Disease; Animals; Brain Injuries; Brain Ischemia; Calcimycin; Diffusion; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Edema; Hippocampus; Ionophores; Male; Neurons; Organ Culture Techniques; Rats; Rats, Long-Evans

The signal transduction pathway through which tumour necrosis factor (TNF) induces apoptosis in leukaemic cells may involve activation of cytosolic phospholipase A(2) (cPLA(2)). The steroids dexamethasone (Dex) and 1,25(OH)(2) D(3) both render U937 leukaemic cells resistant to TNF-induced apoptosis. In this study, we found that Dex inhibited both spontaneous and TNF-induced activation of cPLA(2). Dex had no direct effect on cellular cPLA(2) levels, but facilitated cPLA(2) degradation upon subsequent stimulation of cells with TNF. In addition, Dex increased synthesis of the endogenous cPLA(2) inhibitor lipocortin 1 (LC1). An antisense oligonucleotide to LC1 could completely abrogate Dex-induced resistance to the cytotoxic action of TNF. Constitutive LC1 levels were relatively higher in myeloid leukaemic blasts showing resistance to TNF than TNF-sensitive myeloid leukaemic cell lines. Our data suggest that Dex confers the resistance of U937 cells to TNF-induced apoptosis by upregulating intracellular levels of LC1 and by facilitating a negative-feedback loop, which is activated upon stimulation with TNF. High constitutive levels of LC1 in leukaemic blasts may protect them against immune-mediated killing.

Topics: Acute Disease; Annexin A1; Antineoplastic Agents, Hormonal; Apoptosis; Arachidonic Acid; Calcimycin; Cell Line; Cytosol; Dexamethasone; Enzyme Activation; Enzyme Inhibitors; Feedback; Humans; Ionophores; Leukemia; Leukemia, Monocytic, Acute; Leukemia, Myeloid; Oligonucleotides, Antisense; Phospholipases A; Signal Transduction; Stimulation, Chemical; Tumor Necrosis Factor-alpha

The deposition of complement components within grafts, complement consumption, and prolongation of graft function by complement inactivation imply a pivotal role for complement in xenograft hyperacute rejection. The current investigations examined the endothelial production of vasoactive substances in pulmonary arteries during simulated hyperacute rejection.. Canine pulmonary arteries were suspended in organ chambers and exposed to either autologous canine serum for 90 minutes or heterologous porcine serum for 30, 60, or 90 minutes. Following serum exposure, the vessels were allowed a one-hour equilibration in buffered crystalloid solution. Dose-response curves were obtained with acetylcholine, sodium nitroprusside, and calcium ionophore A23187 following contraction with phenylephrine (10(-6) M) in the presence of indomethacin (10(-5) M). Receptor-dependent, endothelial-dependent relaxations to acetylcholine (10(-9)-10(-4) M) were impaired with 30-, 60-, or 90-minute porcine serum exposure when compared to vessels exposed to autologous canine serum (n = 10, 7, 9, respectively; p < .05; 2-way ANOVA). Receptor-independent, endothelial-dependent relaxations to calcium inophore (10(-9)-10(-6) M) were significantly impaired at 60- and 90-minute porcine exposures only (n = 7, 8; p < .05). Endothelial-independent relaxations to sodium nitroprusside (10(-9)-10(-4) M) were not impaired with either canine or porcine serum exposure. Oxyhemoglobin (10(-6) M) abolished acetylcholine-mediated relaxations, indicating that nitric oxide was the predominant mediator.. Simulated hyperacute xenograft rejection impairs endothelium-dependent relaxation of canine pulmonary arteries. Both basal and stimulated production of nitric oxide is impaired by heterologous serum exposure and, subsequently, complement activation. Reduced production of nitric oxide may explain, in part, the vasospasm and thrombosis of xenografts during hyperacute rejection.

Topics: Acetylcholine; Acute Disease; Animals; Blood; Calcimycin; Dogs; Dose-Response Relationship, Drug; Endothelium, Vascular; Graft Rejection; In Vitro Techniques; Indomethacin; Ionophores; Nitric Oxide; Nitroprusside; Phenylephrine; Pulmonary Artery; Swine; Transplantation, Heterologous; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

These studies tested whether fetal artery reactivity is sensitive to both acute changes in oxygen levels (in vitro) and chronic changes (in utero).. Pregnant guinea pigs near term were exposed to either normoxia or hypoxia (12% oxygen) for 4 or 7 days. The effect of decreasing PO (2 ) in vitro (acute hypoxia) on relaxation in response to acetylcholine, A23187, sodium nitroprusside, and 8-bromo-cyclic guanosine monophosphate was measured in isolated carotid arteries from normoxic fetuses. In separate experiments relaxation in response to acetylcholine and sodium nitroprusside of endothelially intact and denuded fetal arteries from fetuses exposed to normoxic conditions and long-term (4 and 7 days) hypoxic conditions was measured in the presence and absence of nitro-L -arginine (10(-4) mol/L).. Acute hypoxia inhibited endothelium-dependent relaxation in response to acetylcholine and A23187, increased sensitivity to sodium nitroprusside, but had no effect on relaxation in response to 8-bromo-cyclic guanosine monophosphate. Chronic hypoxia (4 but not 7 days) inhibited maximal relaxation of arteries in response to acetylcholine but not relaxation of arteries in response to sodium nitroprusside with respect to relaxation seen in arteries from normoxic fetuses. Nitro-L -arginine attenuated the differences between normoxic and hypoxic fetuses in acetylcholine response.. Hypoxia may alter relaxation of fetal arteries by decreasing the availability of oxygen for nitric oxide production and causing vascular adaptations related to altered nitric oxide release.

Topics: Acetylcholine; Acute Disease; Animals; Arteries; Calcimycin; Chronic Disease; Cyclic GMP; Disease Models, Animal; Female; Guinea Pigs; Hypoxia; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Pregnancy; Vasodilator Agents

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.

Topics: Acute Disease; Animals; Antibodies, Monoclonal; Blood Platelets; Calcimycin; Chromatography, Gel; Humans; Immunoglobulin Fab Fragments; Mice; Platelet Glycoprotein GPIIb-IIIa Complex; Recombinant Fusion Proteins; Thrombin; Thromboplastin; Thrombosis

The production of platelet-activating factor (PAF) by inflammatory cells is regulated by lyso-PAF acetyltransferase, and the activity of this enzyme is increased in neutrophils of stable asthmatic patients. The aim of this investigation was to determine whether acetyltransferase activity is further upregulated in asthmatic patients experiencing acute symptoms. A radioenzymatic assay was used to measure the enzymatic affinity constant (Km) and maximal enzymatic activity (Vmax) for acetyltransferase from unstimulated and Ca2+ ionophore (A23187)-stimulated neutrophils from 16 patients with acute asthma, and the measurement was repeated at the time of discharge (n=9) and after recovery from the acute episode (n=13). During acute asthma, Km (median 93.8 (interquartile range 64.1-109.7) microM) was lower than that measured in nonasthmatic subjects in a previous study using identical methods (155.1 (122.2-179.9) microM; p=0.0001), and in 10 out of 13 acute patients Km for unstimulated neutrophils increased following recovery. In A23187-stimulated neutrophils, Km during acute asthma (84.3 (73.6-100.2) microM) and at discharge (83.9 (83.1-94.8) microM) were similar, but Km after recovery was increased (115.0 (95.6-119.5) microM; p=0.02). The change in Km following stimulation with A23187 was also significantly less during acute asthma than previously measured in nonasthmatic subjects (p=0.003). Although Vmax during acute asthma (12.9 (interquartile range 10.5-22.5) nmol x min(-1) x mg(-1) protein) did not differ significantly from that at discharge (14.4 (12.3-20.4) nmol x min(-1) x mg(-1)) or after recovery (17.3 (12.3-18.4) nmol x min(-1) x mg(-1)), both median Km and Vmax tended to be lowest during acute asthma and increase at discharge and after recovery. An increase in lyso-PAF acetyltransferase activity alone may not account for increased systemic PAF concentrations during acute asthma. However, the reduction in the enzymatic affinity constant and its smaller change following in vitro stimulation suggest that alterations in the affinity of acetyltransferase for acetylcoenzyme A (CoA) and in the regulation of enzyme activity may be occurring during acute asthma.

Topics: Acetyltransferases; Acute Disease; Adult; Aged; Asthma; Calcimycin; Female; Humans; In Vitro Techniques; Ionophores; Male; Middle Aged; Neutrophils; Up-Regulation

To explore the possible role of platelet-activating factor (PAF) in the pathogenesis of bronchial asthma, circulating PAF and in vitro production of PAF were studied.. Radioimmunoassay kits were used in 15 children with acute asthmatic attacks, in 25 newly diagnosed asthmatic children, in 25 good and 18 poor responders to immunotherapy, and in 18 healthy controls.. The results demonstrated the following: (1) PAF was present in the blood of healthy controls. (2) New patients had much higher circulating PAF than did healthy controls (p < 0.005), and the circulating PAF decreased after immunotherapy in good (p < 0.005) but not in poor responders. (3) The circulating PAF increased up to 20 times that of healthy controls during acute asthmatic attacks. (4) The spontaneous and allergen-stimulated secretion of PAF were markedly increased in new patients and decreased to normal after successful immunotherapy (p < 0.005). (5) No increased spontaneous and allergen-stimulated production of PAF was found during acute attacks, but granulocytes from those patients still produced the greatest amount of PAF when stimulated with calcium ionophore A23187. (6) Although a major portion of allergen-induced PAF was secreted, less than 10% of ionophore-induced PAF was secreted.. The findings that the circulating PAF increased markedly during acute asthmatic attacks and the enhanced in vivo and in vitro productions of PAF decreased to normal after successful immunotherapy strongly suggest that PAF may be involved in the pathogenesis of bronchial asthma.

Topics: Acute Disease; Animals; Asthma; Calcimycin; Child; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Granulocytes; Humans; Immunotherapy; Mites; Osmolar Concentration; Platelet Activating Factor; Reference Values

The role that second messengers play in pulmonary vasoconstriction is not understood. The purpose of this study was to directly measure inositol phosphates in isolated pulmonary arterial preparations before and during norepinephrine (NE) stimulation and acute hypoxia. Rat main pulmonary arteries were isolated and incubated with myo-[3H]-inositol. After incubation, control tissue was stimulated with 0.5 microM NE or 30 mM KCl. Test preparations were precontracted with 30 mM KCl and then exposed to hypoxia. Samples were homogenized and applied to a high-pressure liquid chromatography column for analysis of inositol phosphates. Results show that inositol trisphosphate (IP3) increases twofold at 5 s following NE stimulation. Thirty micromolars of KCl results in a slight but significant increase in IP3 formation at 5 min following the stimulation. Phentolamine inhibits the KCl-induced increase in IP3 formation, whereas A23187 has no effect on IP3 levels. Hypoxia caused a biphasic contraction in the precontracted isolated rat pulmonary artery. IP3 levels did not change during the hypoxic period. In conclusion, NE causes a rapid increase in IP3 formation consistent with the time course of production of an excitation-contraction coupling second messenger. However, inositol trisphosphate is not involved in the signal transduction pathway leading to pulmonary arterial contraction induced by hypoxia.

Topics: Acute Disease; Animals; Calcimycin; Chromatography, High Pressure Liquid; Hypoxia; Inositol 1,4,5-Trisphosphate; Male; Norepinephrine; Potassium Chloride; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasoconstriction

The subcutaneous sponge implant model of acute inflammation in the rat has been evaluated as a suitable test system for evaluating the potential anti-inflammatory efficacy of 5-lipoxygenase inhibitors. The inflammatory parameters measured were exudate volume and leukocyte recruitment. Specific radioimmunoassays were used to measure (1) 5-lipoxygenase (LPO) and cyclo-oxygenase (CO) activity in exudate leukocytes stimulated ex vivo with A23187, and (2) the LTB4 and PGE2 content of inflammatory exudate. The NSAIDs flurbiprofen and indomethacin inhibited cell recruitment, exudate volume and CO activity with ED50S of approximately 1 mg per kg p.o. but failed to inhibit LPO activity at 10 mg per kg p.o. Nafazatrom (Bayer 6575), quercetin and NDGA, which inhibit LPO activity in vitro, were inactive against all parameters when dosed at 100 mg per kg p.o. The "mixed inhibitors" BW755C and phenidone were approximately equipotent inhibitors of LPO activity but BW755C was 10 times more potent than phenidone against CO activity. BW755C was also greater than 10 times more potent at inhibiting cell recruitment and exudate volume than phenidone suggesting that the anti-inflammatory efficacy of the mixed inhibitors reflect their potency against CO rather than LPO activity. Time course studies demonstrated that the inhibitor effects of BW755C and phenidone on leukocyte recruitment reflected a reduction in the PGE2 but not the LTB4 content of the inflammatory exudate. Polyester sponges soaked in high concentrations of LTB4 caused only a modest (2-fold) increase in leukocyte recruitment whilst physiological levels were inactive. The results taken together suggest that CO products make a major contribution to leukocyte recruitment in this model whilst the LPO product LTB4 has little role. This model therefore is of little value for evaluating the anti-inflammatory efficacy of 5-lipoxygenase inhibitors. Moreover, the rat would appear to be unsuitable for evaluating the role of LTB4 in acute inflammation.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Acute Disease; Animals; Blood Proteins; Calcimycin; Chemotaxis, Leukocyte; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Flurbiprofen; Indomethacin; Inflammation; Leukocytes; Leukotriene B4; Lipoxygenase Inhibitors; Male; Prostaglandins E; Pyrazoles; Pyrazolones; Rats; Rats, Inbred Strains; Skin