In vitro modulating effect of human very-low-density lipoproteins on human polymorphonuclear leukocyte oxidative metabolism and migration.

In this work, the in vitro effects of very-low-density lipoproteins (VLDL) on human polymorphonuclear leukocyte (PMN) oxidative metabolism and migration were studied. VLDL stimulated PMN superoxide generation in absence of other stimulating agents. The effect of VLDL from normotriglyceridemic subjects was more marked than with VLDL from hypertriglyceridemic subjects. VLDL reduced in a dose-dependent manner the luminol-dependent chemiluminescence of PMN stimulated by phorbol myristate acetate (PMA) and, to a lesser degree, by opsonized zymosan. This effect was observed with VLDL concentrations found in healthy and hypertriglyceridemic patients. Superoxide anion generation was also reduced by preincubation of PMN with VLDL before stimulation with PMA but not opsonized zymosan. VLDL were not cytotoxic for PMN. The above effects appear to be an intrinsic property of VLDL and might lead to reduced PMN-mediated non-specific host defences in hypertriglyceridemic subjects.

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.

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.

Total automated method for determination of cholesterol concentration in high density lipoprotein by heparin-magnesium-albumin precipitation.

We describe an automated procedure for determination of high-density-lipoprotein cholesterol in serum by use of heparin-MgCl2-albumin reagent. 'AutoAnalyzer' II equipment was used in which high-density lipoprotein was separated from precipitate containing very-low and low-density lipoproteins by filtering on-line across a cellulose acetate membrane. Cholesterol concentration was measured by the enzymic method of Allain. Good correlation was obtained using the automated method compared to the heparin-MgCl2-albumin (r=0.99) or sodium phosphotungstate-MgCl2 procedure (r=0.97). Contamination was minimal with a contamination coefficient lower than 2.5%. The method presented was linear (0 to 2.50 mmol/1) and could be applied to lipemic samples with a coefficient of variation better than 3%. This study showed thus that this new procedure was suitable for routine work with a good rate (60 samples/h) in almost any laboratory.

Changes in human high density lipoproteins in patients with extra-hepatic biliary obstruction.

Plasma high density lipoproteins (HDL) from patients with obstruction of the common bile duct were studied by crossed immunoelectrophoresis and isoelectric focusing. All cholestatic HDL fractions were rich in phospholipids (51.5 +/- 9%) with high proportions of free cholesterol (13.8 +/- 1.5%). Moreover, crossed immunoelectrophoresis of sera against anti-Apo A revealed the presence of multiple immunoprecipitates sharply contrasting with the pattern formed by normal sera. Tandem crossed immunoelectrophoresis against anti-Apo A and anti-Apo B was performed with whole serum and with the HDL fraction from various cholestatic subjects. Crossed identity was observed for two of these precipitates, which could be explained by the decrease in HDL stability due to the detergent effect of bile salts. The most noteworthy changes found in cholestatic patients appeared to be the apolipoprotein pattern of HDL. Both Apo AI (48%) and Apo AII (5.5%) were greatly diminished and Apo E was present in remarkably high amounts (39%) with two additional isoforms (Apo E'1 and Apo E'2).

Characterisation of the main molecular forms of human fecal immunoglobulin A.

The molecular composition of fecal IgA is poorly documented, although it is of theoretical and practical importance to determine the different forms of IgA in faeces. Two main molecular forms were isolated by successive steps of ion-exchange chromatography and gel-filtration. The first consisted of secretory IgA dimers dissociating into slightly lower molecular mass forms under the influence of the electric field during electrophoresis. The other contained cleaved-IgA complexed with alpha 1-antitrypsin, that is considered to be a serum origin marker. These results confirm that secretory IgA are relatively resistant to digestive enzymes in vivo, and suggest that alpha 1-antitrypsin-bound fragments originate from serum IgA monomers. Analysis of the proportions of these forms may be of value in the investigation of gut diseases.

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.

Differential measurement of monomeric and polymeric IgA by one-directional immunoelectrodiffusion.

A technique to specifically quantify monomeric IgA and total IgA in colostrum has been developed using a modified one-dimensional immunoelectrophoretic assay. This method employed electrophoresis in antibody-containing polyacrylamide-agarose gel in the presence of a gel barrier which blocks polymeric IgA. The addition of PEG (polyethylene-glycol 6000) to the anodic gel increased the sharpness of the peaks, the height of which was proportional to the antigen concentration. This method proved to be sufficiently simple, precise, reproducible (CV less than 3%) and linear (from 20-300 mg/l) to measure the monomeric IgA: total IgA ratio rapidly (14 +/- 4.5% for 20 samples in duplicate). Immunoelectrodiffusion studies confirmed that human colostral and serum IgA standards could be used to determine directly monomeric IgA, total IgA and polymeric IgA levels (by difference) rather than to apply correction factors to estimate these IgA levels.

Superoxide dismutases in polymorphonuclear leukocytes from patients with ankylosing spondylitis or rheumatoid arthritis.

The activity of cyanide-sensitive and cyanide-insensitive superoxide dismutase (CNs- and CNi-SOD) was measured in polymorphonuclear neutrophils isolated from the blood of patients with ankylosing spondylitis (A.S.) or adults with rheumatoid arthritis (R.A.). Our purpose was to detect alterations in the protecting activity of these enzymes that might cause rheumatic lesions secondary to superoxide anion generation in the inflammatory loci. There was no difference in total SOD activity (CNs + CNi) in either A.S. or R.A. when compared to the control group. In contrast, CNi-SOD activity decreased in R.A. and A.S. and CNs-SOD activity rose significantly in A.S. only. None of the changes observed in SOD activity correlated with patient's age, erythrocyte sedimentation rate, clinical evolution of the disease or the drug doses administered. It is concluded that the reduced activity of CNi-SOD might be partly responsible for the reduced protection of the joints against oxygen-free radicals in patients with A.S. or R.A. Other factors however appear to have greater effects on the clinical evolution of these diseases.

Type IV hyperlipoproteinemia in patients with algodystrophy.

The present work studies lipid metabolism in patients with algodystrophy (AD). A correct positive correlation (r = 0.75) between the triglyceride levels and low density lipoprotein (LDL)/very low density lipoprotein (VLDL) ratio and the VLDL increase observed by gel disk electrophoresis confirm that a type IV hyperlipoproteinemia is associated with AD. In contrast, the degree of high density lipoprotein (HDL) saturation in cholesterol (HDL-cholesterol/HDL-phospholipids) and the classical atherogenous index (cholesterol/HDL-cholesterol) were not modified. The decrease of plasma post-heparin lipolytic activities (PHLA) was not significant but further studies should be performed to correlate PHLA with a reduced activity of the adipose tissue lipoprotein lipase.

Blood polymorphonuclear behavior in patients with ankylosing spondylitis.

The oxidative metabolism and chemotaxis of polymorphonuclear leukocytes (PMNs) collected from patients with ankylosing spondylitis and healthy subjects were studied in parallel. The responses to opsonized zymosan were significantly lowered considering oxygen consumption and release of superoxide anions, whereas no modification of these parameters to phorbol myristate acetate and calcium ionophore (A 23187) stimulations were observed. A seric factor was not involved but the characterization of a specific intrinsic abnormality of the PMNs needs further investigations. PMN chemotaxis, assessed by two methods performed in parallel, remained unchanged.

Individual free fatty acids and lactate uptake in the human heart during severe sepsis.

Coronary haemodynamics and myocardial metabolism of nonesterified fatty acids (NEFA) and lactate were studied in 11 patients with severe sepsis, and compared to 10 control subjects. Coronary sinus blood flow was evaluated by thermodilution. Arterial and coronary sinus blood samples were collected for the measurement of lactate and total and individual NEFA concentrations both in septic and control patients. There was an increase in lactate and total NEFA arterial concentrations with a marked increase in palmitic and linolenic acids. The uptake of the main NEFA (C14:0 to C18:2) was significantly decreased. In the control group, individual NEFA uptake was proportional to their arterial concentrations. This relationship was not observed in patients with sepsis: there was no preferential extraction of any particular NEFA. Furthermore, in patients with sepsis, myocardial oxygen consumption did not correlate with NEFA, but only with lactate uptake. Alterations in NEFA uptake were found to be constant during severe sepsis and are consistent with major disturbances in myocardial metabolism.

Free fatty acid microdetermination by gas-liquid chromatography without transmethylating effects of methylation procedure.

A fast and practicable gas-liquid chromatographic method for the quantitation of non-esterified fatty acids (C14:0-C18:2) from 100 microliter plasma is described. This technique includes extraction, purification using the solvents n-heptane and 0.5 M Na2CO3, and methylation by 0.1 M HCl-methanol. Extraction, quantification and optimal methylation conditions without transmethylation have been investigated. Reproducibility is good and results agree with values found in the literature.

Manganese-containing superoxide-dismutase deficiency in polymorphonuclear leukocytes of adults with rheumatoid arthritis.

Superoxide dismutase (SOD) is known to regulate the level of superoxide radicals inside cells. The purpose of this work was to investigate the role of SOD activity in tissue damage produced by superoxide radicals. SOD was measured in polymorphonuclear cells of patients with rheumatoid arthritis and controls. The distinct SOD activities, including manganese-containing and copper-zinc-containing enzymes, were evaluated in cytoplasma and mitochondria of human granulocytes. Except for the comparison between total SOD and cytoplasmic copper-zinc SOD, no correlation was found among the different SOD levels. Moreover, a significant decrease was observed only for cytoplasmic manganese-containing enzyme in granulocytes of adults with rheumatoid arthritis. These data confirm the necessity of evaluation of various SOD classes and suggest the interest of biochemical tests in granulocytes for early diagnosis and better comprehension of tissue damage due to inflammation.

Effect of an immunomodulating agent, RU 414740, 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 1 micrograms/ml for healthy rats but only 10 micrograms/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.

Modulation of human polymorphonuclear neutrophil functions by alpha 1-acid glycoprotein.

alpha 1-Acid glycoprotein (alpha 1-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 tested in vitro on human polymorphonuclear (PMN) functions (migration, aggregation, O2- generation). alpha 1-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, alpha 1-AGP exerted an aggregating effect with a maximal effective concentration of 0.3 mg/ml. alpha 1-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 alpha 1-AGP may be able to prevent PMN activation in the course of inflammatory processes in vivo.

Serum alpha-1 acid glycoprotein in chronic renal failure and hemodialysis.

The total concentration and concanavalin A (ConA)-dependent microheterogeneity of alpha-1 acid glycoprotein (AAG) were studied in thirty hemodialyzed uremic patients and eighteen non-dialyzed uremic patients, by comparison with healthy volunteers. Serum concentrations of AAG were significantly higher in the non-dialyzed uremic (1.27 +/- 0.47 g/l) and hemodialyzed patients (1.29 +/- 0.33 g/l) than in the volunteers (0.79 +/- 0.09 g/l). The proportions of strongly ConA-reactive AAG fractions were also higher in non-dialyzed uremic (16.7%) and hemodialyzed patients (18.5%) than in volunteers (14.1%). These data may be related to an increase in bi-antennary glycans, as observed in patients on peritoneal dialysis, together with a probable change in sialylation. AAG serum levels and microheterogeneity were similar in non-dialyzed and hemodialyzed patients and did not appear in the dialyzed patients to depend on the type of dialysis membrane used, i.e. cuprophan (CU), cellulose acetate (CA), hemophan (HE), polyacrylonitrile (PAN), and polysulfon (PS), in spite of differences in biocompatibility. In patients dialyzed with CA membranes, there was a distinct decrease in the ConA non-reactive fraction (38.0%) and an increase in ConA slightly-reactive (42.2%) and strongly-reactive (19.7%) fractions. Differences in AAG serum levels and ConA reactivity between patients dialyzed with CA and PAN membranes seem to justify further investigations of other acute-phase reactants and immunological parameters.

[Lymphocytes. Roles in cellular immunity and humoral immunity]. / Les lymphocytes Rôles dans l'immunité cellulaire et l'immunité humorale

The lymphocytes are now considered as ubiquitous cells which play a role in delayed hypersensitivity, the rejection of grafts and antibody synthesis. The lymphocytes are essential in the immune process, for they may both recognise the antibody and initiate the response. They act either by themselves or after differentiation into specialised cells and, usually, in coordination with other cells with phagocytic functions. After briefly recalling the morphology, localisation, circulation, sites of production and heterogeneity of the lymphocytes, the authors discuss their function, describe lymphocytic transformation, and the role of T lymphocytes in delayed hypersensitivity, in the rejection of grafts and the direct cytotoxic effect. Antibody synthesis with B lymphocytes and the intervention of mononucleated cells with phagocytic activity, the cooperation of T lymphocytes are developed. The exploration of lymphocytic functions includes the recognition of an antigen by the lymphocyte, fixation of marked antigens, rosette formation, the demonstration of delayed hypersensitivity (inhibition of macrophage migration, cytotoxicity, skin tests), the estimation of antibodies and measurement of lymphocyte transformation. Certain factors, due to antigen-host reaction, regulate the lymphocytic immune reaction. One may experimentally intervene by immunostimulation and immunosuppression.

[Serum phospholipids determination by an automatic enzymatic method (author's transl)]. / Dosage des phospholipides sériques par méthode enzymatique appliquée au flux continu.

The serum phospholipids determination by an automatic enzymatic method (AEM) is suggested in this study. A comparison was made between the classical chemical method, the manual enzymatic method (MEM) and the AEM one. 100 serum samples were tested in this experiment and the correlation coefficient between these methods was found to be acceptable. The result indicates a good precision and a negligible contamination. The calibration curve is linear to 5 mmol/l. Triglyceride and cholesterol concentrations, glucose and high concentrated uric acid presence do not interfere on the value measured. However, the haemolysis and a large quantity of bilirubin in sera tend to induce a variation in the phospholipids determination.

[Estimation of serum lipids: method without extraction adapted to autoanalysis (author's transl)]. / Dosage des lipides sériques: méthode sans extraction adaptée à l'autoanalyseur.

An entirely automatic method, without prior extraction, is described for the determination of total serum lipids by a nephelemetric method. The results are compared with those obtained by Canal's method. There is a good correlation between the two technics. With a similar concentration of lipid the respective quantities of cholesterol and triglyceride do not intervene in the flocculation. The respective influence of the bilirubin concentration, hemolysis, and the concentration and nature of the serum proteins is considered. This method should permit simultaneous autoanalysis of the three parameters of classical lipid investigation: total lipids, triglycerides and cholesterol.