Denis Raichvarg

Effects of alpha-1 acid glycoprotein on human polymorphonuclear neutrophils: influence of glycan microheterogeneity.

The biological functions of alpha-1 acid glycoprotein (AGP) are poorly understood but appear to depend on glycan microheterogeneity. Variations of AGP glycan structure (in terms of concanavalin A (ConA) reactivity) have been observed during the inflammatory process. We studied these modifications in AGP from patients with chronic renal impairment and investigated the effects of AGP microheterogeneity on healthy polymorphonuclear leukocyte (PMN) chemotaxis and oxidative metabolism. AGP was extracted by a two-step procedure from sera from ten patients with various degrees of renal impairment, selected according to AGP glycan heterogeneity determined by crossed immunoaffinity electrophoresis with ConA. AGP (0.5 g/l) significantly inhibited the chemotactic response of PMN to formyl-methionyl-leucyl-phenylalanine (10(-7) mol/l) and complement fraction C5a, regardless of ConA reactivity. AGP also inhibited superoxide anion generation in response to phorbol myristate acetate (10(-7) mol/l). After stimulation by opsonized zymosan (1 g/l), the effect of AGP appeared to depend on its glycan structure (r = 0.70, P < 0.05), decreasing with ConA non-reactivity. These data suggest that AGP can down-regulate neutrophil responsiveness, an effect that depends in part on its glycan microheterogeneity. Alterations of AGP microheterogeneity in various pathological states, particularly renal failure, may be related to the inflammatory process.

Modulation of human polymorphonuclear neutrophil functions by α1acid glycoprotein

Abstractα1-Acid glycoprotein (α1-AGP), a naturally occurring human plasma protein and acute-phase reactant, was extracted by a two-step procedure from sera collected from four healthy men. Its activity was testedin vitro on human polymorphonuclear (PMN) functions (migration, aggregation, O2− generation). α1,-AGP was not chemoattractant but inhibited the PMN response to the chemoattractant formylmethionyl-leucyl-phenylalanine without affecting spontaneous migration (Boyden and agarose methods of assessment). At concentrations between 0.15 and 0.45 mg/ml, α1AGP exerted an aggregating effect with a maximal effective concentration of 0.3 mg/ml. α1-AGP inhibited superoxide generation by PMNs stimulated either by opsonized zymosan or phorbol myristate acetate. This inhibition varied according to the intensity of the stimulation. At low stimulus concentrations, a dose-dependent inhibition of membrane-associated PMN responsiveness to soluble or particulate stimuli was observed. These findings suggest that α1-AGP may be able to prevent PMN activation in the course of inflammatory processesin vivo.

Influence of Uremia on Polymorphonuclear Leukocytes Oxidative Metabolism in End-Stage Renal Disease and Dialyzed Patients

The oxygen (O2) consumption, and superoxide anion (O2-.) and hydrogen peroxide (H2O2) production by polymorphonuclear leukocytes (PMNs) were investigated in 5 end-stage renal disease patients, before and after the 1st, 4th and 10th dialysis sessions. Resting values of O2-. production and O2 consumption were not significantly different from values for PMNs from normal subjects. After stimulation by opsonized zymosan or phorbol myristate acetate, the three parameters measured were significantly (p less than 0.001) enhanced in comparison with healthy control values. Cross-incubation studies showed a lack of effect of patient plasma on O2-. production by stimulated control cells: PMN oxidative metabolism would therefore appear to be increased in these patients. The anomalies observed probably arise via a mechanism involving a cellular dysfunction resulting from the renal disease, rather than from the presence of a plasma factor.

Characterization of lipoproteins during human cholestasis

We have characterized changes in lipoproteins from cholestatic individuals and reproduced them by incubating lipoproteins from healthy individuals with cholic acid. The cholestatic patients showed an increase in low density lipoprotein (LDL) (>85%), with a smaller proportion of esterified cholesterol, and a fall in high density lipoprotein (HDL) (<10%), with a larger proportion of phospholipids. The protein composition of cholestatic HDL1 was characterized by a smaller proportion of apo A (I, II) and a prominent apo E fraction (39% vs. 9%). These changes involved an increase in degree of molecular packing (order) of HDL1. The addition of cholic acid to serum from healthy individuals altered the lipoprotein distribution, with an increase in LDL, the disappearance of HDL2 and HDL3 and the appearance of HDL1. These HDL1 were characterized by increased phospholipid and reduced apo AI fractions. They also showed a lower density and appeared as spherical particles in contrast to cholestatic HDL. Incubation of healthy HDL with cholic acid in vitro reproduces some of the alteration observed in cholestatic HDL.

Effects exerted by RU 41740 on oxidative metabolism and migration of rat polymorphonuclear leukocytes collected after induction of one acute non specific inflammatory reaction

The activity of RU 41740, a glycoprotein extract from Klebsiella pneumoniae, endowed with immuno-modulating properties, has been investigated on polymorphonuclear (PMN) leukocyte functions. This report deals with the effect of RU 41740 on oxidative metabolism (assessed by chemiluminescence, 02 consumption and O2- production) and on chemotaxis and random migration (using agarose and Boyden chamber techniques). PMNs were collected from the rat pleural cavity after induction of one acute inflammatory reaction (pleurisy induced by injection of a suspension of calcium pyrophosphate crystals). Experiments were performed in parallel after in vivo treatment or incubation in vitro. RU 41740 enhanced PMN oxidative metabolism and inhibited PMN chemotaxis while random migration was only affected using agarose assay at high concentration. This effect on PMN migration was observed with at least two attractants. These observations have been obtained either after incubation in vitro or administration in vivo. The minimal effective dose was 1 mg/kg in vivo and 0.1 microgram/ml in vitro. These data suggest that RU 41740 acts directly on PMN membrane receptors.

Effect of an immunomodulating agent, RU 41740, on polymorphonuclear responsiveness after burn injury

An impairment of polymorphonuclear leukocyte (PMN) functions has been described following burn trauma. It was thus of interest to investigate the effect of RU 41740, an agent known to stimulate these cells, on rat PMN functions after burn injury. In the present study the responsiveness to classical stimuli of PMN from untreated burned rats was approximately 40% lower than healthy control values. In vitro treatment with RU 41740 increased oxidative metabolism of PMNs from burned and healthy rats. The effect was dose-related but was most striking in the case of PMNs from healthy rats. Significant differences were obtained with concentrations higher than 1μg/ml for healthy rats but only 10μg/ml for burned rats. In vivo treatment with RU 41740 also led to an enhancement of PMN oxidative metabolism on both burned and healthy rats. The maximal effective dose was 10 mg/kg/day in both cases. In contrast, 25 and 50 mg/kg/day doses inhibited PMN oxidative metabolism.

The immunomodulating agent RU 41740 complexed with very-low-density lipoproteins enhances human polymorphonuclear neutrophil oxidative metabolism in vitro

Added to human serum in vitro, RU 41740, an immunomodulating agent extracted from Klebsiella pneumoniae, binds selectively to lipoproteins containing apolipoprotein B (low-density lipoproteins and very-low-density lipoproteins, VLDL) and, at higher concentrations, to lipoproteins containing apolipoprotein A (high-density lipoproteins). The fact that lipoproteins modulate polymorphonuclear neutrophil (PMN) functions led us to suspect that the VLDL-RU 41740 complex might affect PMN functions. In this study, the effect of this complex on PMN superoxide generation was measured in the presence and absence of the classical stimulants formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate. The VLDL-RU 41740 complex enhanced the stimulating effect of VLDL on quiescent PMN, but not following stimulation with formyl-methionyl-leucyl-phenylalanine. In contrast, it partially counteracted the inhibiting effect exerted by VLDL alone on phorbol myristate acetate stimulation. Such a complex might be formed in vivo during RU 41740 therapy and constitute an important feature in the immunostimulating properties of the drug.

In vitro and in vivo effects produced by the immunomodulating agent ru 41740 on human polymorphonuclear leukocytes in the elderly

The activity of RU 41740, a glycoprotein extract from Klebsiella pneumoniae has been investigated on some polymorphonuclear (PMN) functions. Chemotaxis, random migration and oxidative metabolism (assessed by chemiluminescence, O2 consumption and O2- generation) were studied in parallel. PMN were collected from adult and aged human volunteers. Experiments were performed either in vitro or in vivo in a double blind placebo assay. In both PMN populations RU 41740 enhanced oxidative metabolism either in in vivo or in vitro experiments. However, a higher and dose-related activity was observed on PMN collected from the younger subjects whereas maximal effective concentration was reached earlier with PMN collected from aged subjects. RU 41740 did not modify random migration but inhibited chemotaxis of PMN collected from the younger population in a dose-related manner. These data corroborated previous results observed on PMN collected from various animal species and suggested an interaction of RU 41740 on PMN membrane. Moreover drug-induced macrophage and lymphocyte stimulation might also explain, at least in part, the in vivo effects described in this study. Thus RU 41740 could partly account for the protective effects exerted against bacterial and fungal infections through its activity on PMN functions.

Effects of Storage on the Pyruvate-Lactate System and Random Migration of Human Granulocytes

Abstract. This work demonstrates a time‐dependent loss of polymorphonuclear neutrophilic leukocyte (PMN) functions during the first 48 h with only slight lysis and release of cellular LDH. It confirms that storage decreases ATP levels in PMNs and significantly increases intracellular lactate and pyruvate. These data correlated with an inhibition of PMN migratory function and a considerable decrease in chemiluminescence response.

Human polymorphonuclear leukocyte metabolism and lipoperoxidation during adult respiratory distress syndrome treated by extracorporeal carbon dioxide removal

Abstract Circulating polymorphonuclear leukocyte (PMNs) oxidative metabolism and lipoperoxidation were evaluated in patients with the adult respiratory distress syndrome (ARDS) treated with low-frequency positive-pressure ventilation and extracorporeal carbon dioxide removal. Chemiluminescence (CL) of resting PMNs from ARDS patients was significantly enhanced relative to controls ( P P P P 2− production was observed after pre-incubation with normal and ARDS serum. Plasma malondialdehyde (MDA) and α 1 proteinase inhibitor-elastase levels were significantly increased in ARDS patients plasma ( P P r = 0.824; P r = 0.46; P = 0.056). Our results show that oxygen metabolism and plasma elastase levels in circulating PMNs from ARDS patients are significantly enhanced. Furthermore, ARDS PMN functions are not enhanced by exogenous stimuli. No correlation between PMN functions and peroxidation was found in ARDS sera. These findings confirm that PMNs are primed during ARDS, but free radical production seems to be only one of the events responsible for the increased lipoperoxidation.