Involvement of a phospholipase D in the mechanism of action of granulocyte-macrophage colony-stimulating factor (GM-CSF): priming of human neutrophils in vitro with GM-CSF is associated with accumulation of phosphatidic acid and diradylglycerol.

The generation of diradylglycerol (DRG) and phosphatidic acid (PdtOH) was investigated in neutrophils primed with granulocyte-macrophage colony-stimulating factor (GM-CSF). Mass accumulation of DRG and PdtOH was measured using reversed-phase high performance liquid chromatography and thin layer chromatography, respectively. GM-CSF had no direct effect on the levels of PdtOH and DRG, but it increased PdtOH generation and the late phase of DRG accumulation in human neutrophils stimulated with FMLP. The elevation of the mass of PdtOH peaked approximately 100 s and clearly preceded that of DRG, which peaked at 150 s. The diacylglycerol kinase inhibitor R59022 enhanced the sustained increase in DRG but did not produce a parallel inhibition in PdtOH production. GM-CSF was without effect on the level of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and did not affect the liberation of Ins(1,4,5)P3 induced by FMLP. These findings exclude the involvement of the PtdIns(4,5)P2-specific phospholipase C/diacylglycerol pathway in the sustained phase of DRG accumulation. The early (30-s) appearance of PdtOH clearly suggests that GM-CSF enhanced FMLP receptor-linked phospholipase D (PLD) generation of PdtOH. PLD was assessed more directly by formation of labeled phosphatidylethanol (PEt) through PLD capacity of catalyzing a trans-phosphatidylation in presence of ethanol. The formation of PEt associated with a concomitant decrease in PdtOH directly demonstrated that the mechanism by which GM-CSF enhances PdtOH production is activation of a PLD active on phosphatidylcholine. This study provides evidence that the mechanism of action of GM-CSF involves upregulation of PLD activity leading to enhanced generation of PdtOH and DRG in FMLP-stimulated neutrophils. These findings may provide the basis for several of the priming effects of GM-CSF.

Fusion Imaging to Guide Thoracic Endovascular Aortic Repair (TEVAR): A Randomized Comparison of Two Methods, 2D/3D Versus 3D/3D Image Fusion.

PURPOSE: To compare the accuracy of two-dimensional (2D) versus three-dimensional (3D) image fusion for thoracic endovascular aortic repair (TEVAR) image guidance. MATERIALS AND METHODS: Between December 2016 and March 2018, all eligible patients who underwent TEVAR were prospectively included in a single-center study. Image fusion methods (2D/3D or 3D/3D) were randomly assigned to guide each TEVAR and compared in terms of accuracy, dose area product (DAP), volume of contrast medium injected, fluoroscopy time and procedure time. RESULTS: Thirty-two patients were prospectively included; 18 underwent 2D/3D and 14 underwent 3D/3D TEVAR. The 3D/3D method allowed more accurate positioning of the aortic mask on top of the fluoroscopic images (proximal landing zone error vector: 1.7 ± 3.3 mm) than was achieved by the 2D/3D method (6.1 ± 6.1 mm; p = 0.03). The 3D/3D image fusion method was associated with significantly lower DAP than the 2D/3D method (50.5 ± 30.1 Gy cm2 for 3D/3D vs. 99.5 ± 79.1 Gy cm2 for 2D/3D; p = 0.03). The volume of contrast medium injected was significantly lower for the 3D/3D method than for the 2D/3D method (50.6 ± 22.9 ml vs. 98.4 ± 47.9 ml; p = 0.002). CONCLUSION: Higher image fusion accuracy and lower contrast volume and irradiation dose were observed for 3D/3D image fusion than for 2D/3D during TEVAR. LEVEL OF EVIDENCE: II, Randomized trial.

Onyx Migration Into the Anterior Spinal Artery During Lumbar Artery Embolisation: an Adverse Event.

INTRODUCTION: The impact of sequential lumbar and intercostal artery occlusion on the risk of spinal cord ischaemia was evaluated; however, an adverse event (paraplegia) was encountered, which resulted in study interruption. Investigations were carried out to understand the reasons for the paraplegia. REPORT: To develop a porcine model of spinal cord ischaemic preconditioning prior to extensive thoraco-abdominal aneurysm endovascular aortic repair, the lumbar arteries were selectively embolised with Onyx 5 days prior to an extended thoracic aortic stent graft. Six pigs were used in this preliminary work. Four cases of paraplegia secondary to accidental migration of Onyx to the anterior spinal artery from the lumbar arteries are reported. Histological analysis confirmed severe spinal ischaemic injury and the presence of Onyx particles in the anterior spinal artery. DISCUSSION: Onyx is used for lumbar artery embolisation in type II endoleak treatment after endovascular aortic repair, and while migration in lumbar arteries is frequent, the risk of spinal cord ischaemia has never been described. The current study demonstrates the risk of paraplegia following Onyx migration to the anterior spinal artery from the lumbar artery in an experimental model. Thus, Onyx treatment for type II endoleaks from lumbar arteries should be used cautiously.

Crystal-induced neutrophil activation. III. Inflammatory microcrystals induce a distinct pattern of tyrosine phosphorylation in human neutrophils.

The activation of human neutrophils by monosodium urate and calcium pyrophosphate dihydrate crystals is believed to play a critical role in the pathogenesis of arthritides such as acute gout and pseudogout, respectively. In this study, we investigated the potential involvement of tyrosine phosphorylation in microcrystal-mediated activation of human neutrophils. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that triclinic monosodium urate and calcium pyrophosphate dihydrate crystals stimulated a time- and concentration-dependent tyrosine phosphorylation of at least five proteins (pp130, 118, 80, 70, and 60). While phosphoprotein (pp) 118 and pp70 were the major phosphorylated substrates, pp70 was the dominant one in reactivity with antiphosphotyrosine antibodies. When the temporal patterns, as well as the levels of tyrosine phosphorylation for both types of crystals were compared, monosodium urate crystals were found to be more potent activators than calcium pyrophosphate dihydrate crystals. The tyrosine phosphorylation patterns induced by microcrystals differed from those stimulated by other soluble (FMLP, C5a, or leukotriene B4) or particulate (unopsonized latex beads or zymosan) agonists which stimulated preferentially the tyrosine phosphorylation of pp118. The ratio of the intensities of pp118 and pp70 were specific of the stimulation with microcrystals when compared to those observed with the other soluble or particulate agonists. Colchicine, a drug used specifically in the treatment of gout and pseudogout, inhibited microcrystal-induced tyrosine phosphorylation, while beta- and gamma-lumicolchicine were without effect. On the other hand, colchicine failed to inhibit FMLP-induced tyrosine phosphorylation. Furthermore, while colchicine inhibited the activation of the NADPH oxidase by microcrystals, it, on the other hand, enhanced the production of superoxide anions by FMLP. Taken together, these results (a) demonstrate that tyrosine phosphorylation is involved in the mechanism of activation of human neutrophils induced by microcrystals; and (b) suggest, on the basis of the characteristics of the observed patterns of tyrosine phosphorylation, that this response may be specific to the microcrystals and relevant to their phlogistic properties.

Crystal-induced neutrophil activation. IV. Specific inhibition of tyrosine phosphorylation by colchicine.

We recently demonstrated that pathologically relevant inflammatory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, potently stimulate a characteristic protein tyrosine phosphorylation pattern in human neutrophils that differed from that observed in response to other soluble or particulate agonists. In this study, the effects of colchicine on protein tyrosine phosphorylation induced by MSU and CPPD crystals in human blood neutrophils were investigated. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that colchicine dose-dependently inhibited the tyrosine phosphorylation of all the proteins phosphorylated in response to MSU and CPPD crystals. Other microtubule-disruptive agents such as vinblastine, nocodazole, and colcemid also inhibited crystal-induced protein tyrosine phosphorylation while lumicolchicine and trimethylcolchicinic acid were without effect. Indomethacin and phenylbutazone were similarly without effect on microcrystal-induced tyrosine phosphorylation. Colchicine, as well as the other active alkaloids, failed to inhibit the protein tyrosine phosphorylation elicited by FMLP, C5a, leukotriene B4, and unopsonized zymosan. Overall, these results demonstrate that colchicine specifically and significantly inhibits the protein tyrosine phosphorylation induced by MSU and CPPD crystals and suggest that its effects are associated, at least in part, with its interaction with microtubules. Furthermore, the use of microtubule-disrupting drugs demonstrate that the mechanisms implicated in the induction of protein tyrosine phosphorylation by microcrystals differed from those involved in response to other soluble or particulate agonists.

[Radiation-induced carotid stenosis: A personnalized approach]. / Sténose carotidienne post-irradiation cervicale : une approche personnalisée.

INTRODUCTION: Surgical treatment of radio-induced carotid stenosis (RICS) is challenging and burdened by an elevated risk of local complications. Carotid artery stenting (CAS) may be a suitable alternative. The best approach is yet to be defined. We reviewed the results of both techniques following selection based on better-suitability characteristics (anatomic and clinical). METHODS: We retrospectively reviewed 38 patients treated for 43 RICS from a group of 1230 patients who had carotid interventions between 2008 and 2015 (5 bilateral). Primary endpoints were morbidity and mortality at 30 days (transient ischemic attack, stroke, myocardial infarction, or death). Secondary endpoints were technical success, wound complications, cranial nerve injury (CNI), restenosis (≥50%) and recurrent symptoms. RESULTS: RICS was symptomatic in 6 patients in the OR group and 3 in the CAS group. Lesions in the OR group were longer (P=0.02) and more calcified (P=0.08). Technical success rate was 100%. Cranial nerve injury rate was 14.2% (3/21). All injuries were completely resolved within several weeks. In the CAS group, technical success rate was 95% (21/22) with the one failure due to a residual stenosis exceeding 30%. Periprocedural stroke rates were 0% and 4.5% in the OR and CAS groups respectively (0/21 vs 1/22, P=0.32). There were no early deaths. Mean follow-up was 19.1 months (3-75). The restenosis rate was 9.5% (2/21) in the OR group and 9% (2/22) in the CAS group. CONCLUSION: Our results do not support a preferred treatment strategy. The choice of treatment should be considered on an individual basis.

Biotin uptake: influx, efflux and countertransport in Escherichia coli K12.

Biotin uptake by Escherichia coli K12 has been reinvestigated. The vitamin uptake is an active process depending on energy and inhibited by uncouplers. The kinetic parameters (Km = 0.27 microM, Vmax = 6.8 pmol/min per mg dry cells) are close to those previously determined for a biotin-dependent strain E. coli C162 (Piffeteau, A., Zamboni, M. and Gaudry, M. (1982) Biochim. Biophys. Acta 688, 29-36). By use of biotin p-nitrophenyl ester, an affinity label of the biotin transport system, it was shown, under conditions of steady state, that the efflux of biotin is not energy dependent and is mainly mediated by a diffusion mechanism. Reexamination of the regulation of the biotin transport by biotin, revealed that only 50% of the biotin uptake system is under control by the vitamin.

Biotin transport by a biotin-deficient strain of Escherichia coli.

Biotin uptake has been investigated using an Escherichia coli biotin requiring auxotroph grown under biotin-deficient conditions. This strain accumulated biotin in the free and bound form. In agreement with a previous report by O. Prakash and M.A. Eisenberg (J. Bacteriol. 120 (1974) 785-791), the biotin entry proved to be an active process which depended on an energy source and was inhibited in the presence of uncouplers. The kinetic parameters have been determined (KM = 0.05 microM, Vmax = 7 pmol/min per mg dry weight). The pool of free biotin could be readily exchanged with external biotin and decreased to a very low level in the absence of an energy source. The use of several biotin analogues revealed that this transport system was quite specific for biotin: slight modifications, for instance in the valeric chain, lowered drastically the affinity for the carrier.

Effect of reduced vitamin K esters on vitamin K-dependent carboxylation.

The hypothesis of a vitamin K hydroquinone hemicarbonate as intermediate of vitamin K-dependent carboxylation of glutamic residues, has been examined, by testing several vitamin K hydroquinone esters as inhibitors of the reaction. Among the esters that have been synthetized, a monoacetate proved to be an inhibitor. Kinetic analysis shows that the inhibition is no competitive with respect to vitamin K.

Rapid priming of calcium mobilization and superoxide anion production in human neutrophils by substimulatory concentrations of phorbol esters: a novel role for protein kinase C and tyrosine phosphorylation in the up-modulation of signal transduction.

The modulatory influences of phorbol esters on the functional responsiveness of human peripheral blood neutrophils have been investigated. These studies focused on measurements of the levels of cytoplasmic free calcium and of tyrosine phosphorylation as well as on their ability to mount an oxidative response. Short incubation times (< 1 min) with low concentrations of phorbol esters (5-50 nM) were shown to enhance the above indices of neutrophil responsiveness to chemotactic factors such as fMet-Leu-Phe and leukotriene B4. On the other hand, a time- and concentration-dependent inhibition of calcium mobilization and superoxide production was also observed. The effects of the phorbol esters were stereo-specific and were antagonized by a novel protein kinase C inhibitor (RO 318220) but were not affected by the oxidative burst inhibitor diphenyleneiodonium. Pre-incubation of the cells with phorbol 12,13-dibutyrate (PDBu) altered in a concentration-dependent manner the tyrosine phosphorylation pattern stimulated by fMet-Leu-Phe. In addition, the tyrosine kinase inhibitor erbstatin inhibited the priming of the mobilization of calcium induced by PDBu. These data demonstrate the rapidity of the effects of the activation of protein kinase C, their potential to modulate positively the early events of the excitation-response coupling sequence and the complexity of the functional interrelationships among the various cellular activation pathways available to human neutrophils and other non-muscle cells.

Priming of calcium mobilization in human neutrophils by granulocyte-macrophage colony-stimulating factor: evidence for an involvement of phospholipase D-derived phosphatidic acid.

Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidic acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.

Evidence for the involvement of tyrosine kinases in the locomotory responses of human neutrophils.

Erbstatin, a recently described inhibitor of tyrosine kinases, has been used to examine the potential role of tyrosine phosphorylation in human neutrophil locomotion. Preincubation of human neutrophils with erbstatin inhibited both spontaneous and directed migration induced by chemotactic factors such as formylmethionylleucylphenylalanine (fMet-Leu-Phe) and leukotriene B4. The decreased migratory responses were correlated with an inhibition of adherence of neutrophils to serum-coated surfaces. Erbstatin did not, however, affect the adherence of human neutrophils to uncoated surfaces. These results indicated that the inhibitory effects of erbstatin were specific and not due to a generalized alteration of the surface of human neutrophils. To elucidate the mechanism of the inhibitory effect of erbstatin on adherence properties, the expression of the leukocyte integrin Mo1 was studied. Erbstatin induced a small but significant increase in the expression of Mo1, but decreased the stimulation of the expression of Mo1 elicited by fMet-Leu-Phe. These results suggest that mechanisms in addition to alteration of the number of surface integrins are involved in the inhibition of neutrophil adherence and locomotion by erbstatin.

Paradoxical effects of colchicine on the activation of human neutrophilis by chemotactic factors and inflammatory microcrystal.

Neutrophil activation by chemotactic factors and by inflammatory microcrystals is accompanied by increases in protein tyrosine phosphorylation and by the activation of the NADPH oxidase. The addition of colchicine inhibited both responses induced by triclinic monosodium urate or calcium pyrophosphate crystals. On the other hand, colchicine enhanced the tyrosine phosphorylation of specific protein in neutrophils stimulated by chemotactic factor and augmented the production of superoxide anions induced by these same agonists. The effects of colchicine were shared by other anti-microtubule agents (nocodazole and vinblastine) but not by its inactive analogue beta-lumicolchicine, trimethylcolchicinic acid, indomethacin, or phenylbutazone. Furthermore, the (enhancing as well as inhibitory) effects of colchicine on tyrosine phosphorylation and superoxide anion production were reversed by taxol. Finally, in human cytoplasts colchicine again inhibited microcrystal-stimulated tyrosine phosphorylation but did not change chemotactic factor-stimulated phosphorylation. These data strongly support the hypothesis that microtubule-related mechanisms are involved in the modulation of the tyrosine phosphorylation response in human neutrophils, and suggest that a relationship may exist between the augmentation of tyrosine phosphorylation and of the stimulation of the NADPH oxidase induced by chemotactic factors.

The active site region of the vitamin K-dependent carboxylase includes both the amino-terminal hydrophobic and carboxy-terminal hydrophilic domains of the protein.

In order to localize the active site of the vitamin K-dependent carboxylase, we developed an affinity probe containing the propeptide and the first two carboxylatable glutamate residues conserved in many native substrates. This probe crosslinked to both the hydrophobic amino-terminal and hydrophilic carboxy-terminal domains of the carboxylase, in contrast with previous work which localized both the catalytic and the propeptide binding site within the amino-terminal hydrophobic domain. Amino acid analysis revealed that the mass of an amino-terminal fragment is seriously underestimated by SDS-PAGE. Reanalysis of the published data in light of this information suggests that a portion of the propeptide binding site resides within the carboxy-terminal hydrophilic domain.

Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates.

Activation of various cell types by different agonists is known to stimulate a transient increase in the level of tyrosine phosphorylation of certain cellular proteins. Such phosphorylation is essential for mediating signalling by these agonists. The preservation of the tyrosine phosphorylation of proteins in lysates has proven to be a difficult task in neutrophils because of their large arsenal of proteases and phosphatases. Here we describe a technique that we found useful for preserving the tyrosine phosphorylation of cellular proteins. The technique depends on the denaturing lysis of neutrophils followed by the removal of the denaturing agents using Sephadex columns. Preparing neutrophil lysates by this technique has proven to be reliable in terms of maintaining the stability of the tyrosine phosphorylated proteins of various molecular weights and their subsequent immunoprecipitation and identification.

Vitamin K dependent carboxylation: synthesis and biological properties of tetrazolyl analogues of pentapeptidic substrates.

The pentapeptides Phe-Leu-X-Glu-Val and Phe-Leu-X-X-Val, where X = 4-(5H-tetrazolyl)-2-aminobutyric acid (tetrazolyl analogue of glutamic acid), were synthesized by addition of tri-n-butyltin azide to the corresponding nitrile-containing peptides. These tetrazolyl peptides and a dinitrile precursor were tested as possible substrates and inhibitors of the vitamin K dependent carboxylation. Phe-Leu-X-Glu-Val was carboxylated (40% of the reference peptide Phe-Leu-Glu-Glu-Val, Km = 20 mM) exclusively on the glutamyl residue, whereas the dinitrile precursor and Phe-Leu-X-X-Val were not carboxylated. The latter was a competitive inhibitor with an affinity (Ki = 3 mM) close to that of the reference peptide (Km = 3 mM).

Protective effect of indomethacin against chemotactic deactivation of human neutrophils induced by formylated peptide.

The effects of the nonsteroidal antiinflammatory drug indomethacin on the parameters relating to the migration and respiratory burst of human polymorphonuclear leukocytes (PMN) were studied in an attempt to clarify the mechanism of this drug's action on PMN. At various concentrations below 200 micrograms/ml, indomethacin partially inhibited the spontaneous migration of PMN but did not alter the directional migration induced by C5a-activated serum. In the presence of N-formyl-methionyl-leucyl-phenylalanine (FMLP) as chemoattractant, directed PMN migration was either inhibited or stimulated by indomethacin, depending on FMLP concentration. When PMN migration was induced by the optimal and suboptimal FMLP concentrations of 10(-7) and 10(-8) M, indomethacin inhibited this migration, but when the high FMLP concentration of 10(-6) M depressed this migration by chemotactic deactivation, indomethacin restored it to its maximum. Both the inhibitory and stimulatory effects of indomethacin on FMLP-induced PMN migration were due to changes in the migration speed. Indomethacin also inhibited FMLP-induced changes in the shape of floating PMN, and in respiratory burst, as well as specific FMLP binding to PMN. In contrast, indomethacin did not alter the PMN respiratory burst induced by phorbol myristate acetate or C5a-activated serum. These data show that indomethacin is able to prevent the loss of PMN chemokinetic activity induced by formylated peptides and suggest that it might be useful for investigating the mechanism of peptide-induced chemotactic deactivation.

Biotin dependent carboxylase activities in normal human and multicarboxylase deficient patient fibroblasts: relationship to the biotin content of the culture medium.

Three mitochondrial carboxylase activities can be depleted in skin fibroblasts from patients with the neonatal form of multiple carboxylase deficiency when the biotin content of the culture medium is lowered to 25 nmol/l. On the other hand this depletion can be achieved in control fibroblasts or in fibroblasts from patients with the late onset form of the deficiency when avidin (50 U/l) is added to the culture medium. The kinetics of the carboxylase activity decrease are nevertheless identical for control and for both types of fibroblasts. After depletion, control and late onset form fibroblasts recover their carboxylase activities at the same rate, whereas fibroblasts with the neonatal form of deficiency need longer times or higher concentrations of biotin to restore their carboxylase activities. These results are consistent with previous hypotheses concerning the origin of both forms of the deficiency. In addition, this technique provides a convenient access to human apocarboxylases, i.e. substrates for in vitro holocarboxylase synthetase investigation.