Cytotoxic response of sinusoidal endothelial cells to polymorphonuclear leukocytes and its potential implication in hypoxia-reoxygenation injury.

AIMS: Interactions between polymorphonuclear leukocytes (PMN) and sinusoidal endothelial cells (SEC) may contribute to ischemia-reperfusion injury. The aim of the study was to determine the influence of PMN hypoxia-reoxygenation and degranulation, on SEC toxic response. METHODS: PMNs collected from rat pleural cavity underwent hypoxia- reoxygenation or N-formyl-methionyl-leucyl-phenylalanine (fMLP) degranulation treatment, and were then separated from their conditioned medium. Rat SECs were incubated either with PMNs in coculture or with their conditioned medium, for 210 min. Oxidative metabolism in PMNs was measured by chemiluminescence. LDH release and elastase activity were measured in SEC supernatants. RESULTS: PMN-conditioned medium induced an increase in LDH release in SECs. Hypoxia-reoxygenation of PMNs induced an increase in their chemiluminescent response without increasing the cytotoxic effect of their conditioned medium. By contrast, the cytotoxic effect of conditioned medium was increased following PMN treatment with fMLP. In the latter case, cytotoxicity was combined with a rise in the elastase activity released in the supernatants, but was not reduced by inhibitors of elastase or of other proteases. CONCLUSIONS: The results indicate that toxic products are released, at least in part through degranulation, by PMNs, and induce cytotoxicity in SECs. This mechanism may contribute to SEC injury during hypoxia-reoxygenation.

[Crystallochemistry of copper (II) and zinc (II) chelates by nonsteroidal antiinflammatory drugs]. / Cristallochimie des complexes à visée thérapeutique du cuivre (II) et du zinc (II) par des anti-inflammatoires non-stéroïdiens.

Copper(II) and zinc(II) chelates by some non-steroidal antiinflammatory drugs NSAIDs (niflumic acid, indomethacin) and 3,5-diisopropylsalicylic acid (DIPS) were characterized by single X-ray diffraction methods. Copper(II) complexes by these two types of chelates are binuclear compounds, with Cu(2)(DIPS)(4)L(2) or Cu(2)(AINS)(4)L(2) formula (L=axial non-NSAID ligand such as diethylether, dimethylsulfoxide DMSO). In zinc(II) complex by DIPS, the metal ion is tetrahedrally coordinated and the corresponding compound is mononuclear with Zn(DIPS)(2)(DMSO)(2) formula. These copper(II) and zinc(II) complexes were found to be more active than their parent drugs from the antiinflammatory and anticonvulsant properties. It was pointed out that the Cu(2)(DIPS)(4)L(2) complexes (L=diethylether, N,N-dimethylformamide) exhibited no rotorod toxicity when examined for anticonvulsant activity using the seizure produced by maximal electroshock, following oral administration to rats.

Low-temperature crystal structures of tetrakis-mu-3,5-diisopropylsalicylatobis-dimethylformamidodico pper(II) and tetrakis-mu-3,5-diisopropylsalicylatobis-diethyletheratodicopp er(II) and their role in modulating polymorphonuclear leukocyte activity in overcoming seizures.

Two binuclear copper(II) complexes of 3,5-diisopropylsalicylic acid were characterized by single crystal X-ray diffraction methods and examined for anti-inflammatory activity using activated polymorphonuclear leukocytes and for anticonvulsant activities using electroshock and metrazol models of seizures. These complexes were crystallized from dimethylformamide (DMF) or diethylether. Tetrakis-mu-3,5-diisopropylsalicylatobis-dimethylformamidodicop per(II) [Cu(II)2(3,5-DIPS)4(DMF)2] I is in space group P 1; a = 10.393 (2), b = 11.258 (2), c = 12.734 (2) A, alpha = 96.64 (2), beta = 92.95 (2), gamma = 94.90 (2) degrees; V = 1471.7 (4) A3; Z = 1. Tetrakis-mu-3,5-diisopropylsalicylatobis-etheratodicopper(II ) [Cu(II)2(3,5-DIPS)4(ether)2] II is in space group P 1; a = 10.409 (3), b = 11.901 (4), c = 12.687 (6) A, alpha = 91.12 (5), beta = 90.84 (5), gamma = 100.90 (4) degrees; V = 1542 (1) A3; Z = 1. The structure of I was determined at 140 K from 4361 unique reflections (I > 2sigma(1)) and refined on F2 to R1 = 0.04 and wR2 = 0.09. The structure of II was determined at 180 K from 4605 unique reflections (I > 2sigma(I)) and refined on F2 to R1 = 0.05 and wR2 = 0.13. Each compound is a crystallographically centrosymmetric binuclear complex with Cu atoms bridged by four 3,5-diisopropylsalicylate ligands related by a symmetry center [Cu-Cu(i): 2.6139 (9) A in I and 2.613 (1) in II]. The four nearest O atoms around each Cu atom form a nearly rectangular planar arrangement with the square pyramidal coordination completed by the dimethylformamide (or diethylether) oxygen atom occupying an apical position, at a distance of 2.129 (2) A in I and 2.230 (3) A in II. Each Cu atom is displaced towards the DMF (or diethylether) ligand, by 0.189 A in I and 0.184 A in II, from the plane of the four O atoms. The crystal structures of I and II are essentially similar to each other, except for the DMF or diethylether accommodation. Many disorder phenomena were found in the crystal structure of I. Copper(II)2(3,5-DIPS)4(DMF)2 inhibited polymorphonuclear leukocyte (PMNL) oxidative metabolism in vitro. This effect was concentration related and significant for concentrations higher than 10 microg or 0.68 nmol/ml. Copper(II)2(3,5-DIPS)4(DMF)2 was more active than the parent ligand, 3,5-DIPS, as has been demonstrated with copper complexes of other non-steroidal anti-inflammatory drugs. The DMF and diethylether ternary complexes of Cu(II)2(3,5-DIPS)4 were found to have anticonvulsant activity in the maximal electroshock model of grand mal epilepsy in doses ranging from 26 to 258 micromol/kg of body mass following intraperitoneal, subcutaneous, or oral treatment. The DMF ternary complex was also found to be effective in the subcutaneous injection of metrazol model of petit mal epilepsy. We conclude that both ternary copper complexes are lipophilic and bioavailable, capable of facilitating the inflammatory response to brain injury and causing the subsidence of this response in bringing about remission of these disease states.

[Caveolae membrane domains, specialized transmembrane exchange zones implicated in cell signalling]. / Des groupements lipidiques membranaires (caveolae), zones d'échanges transmembranaires privilégiées à l'origine de signaux intracellulaires.

Caveolae are small pockets or invaginations localized at the plasma membrane. They are enriched in glycosphingolipids, cholesterol, sphingomyelin and lipid-enchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4 C. Caveolins are the principal protein components of caveolae and play an important structural role in the formation of caveolae membranes. Numerous molecules involved in cell signalling have been identified in caveolae, suggesting that these structures may serve to compartimentalize, modulate and integrate signalling events at the cell surface. Depletion of membrane cholesterol disrupts the formation and function of caveolae, suggesting that these membrane microdomains are involved in a range of biological processes. Moreover, exposure of endothelial cells to high levels of cholesterol upregulates the caveolin abundance in caveolae, and decreases nitric oxide synthesis, suggesting that this may be an early event in atherogenesis. Alteration in the expression of caveolin genes has also been implicated in human diseases such as cancers, diabetes, Alzheimer's disease and muscular distrophy.

Copper(II) complexes of a nonsteroidal anti-inflammatory drug niflumic acid. Synthesis, crystal structure of tetrakis-mu-(2-[3-(trifluoromethyl) phenyl]aminonicotinato)bis(dimethylsulfoxide)-dicopper(II) complex at 190 K. Anti-inflammatory properties.

The synthesis and characterization of three complexes with a potent nonsteroidal anti-inflammatory drug niflumic acid {2-[3-(trifluoromethyl)phenyl]aminonicotinic acid} with formula [Cu(niflumato)2L] (L = H2O, DMSO = dimethylsulfoxide, DMF = N,N-dimethylformamide) were investigated. The crystal and molecular structure of the {Cu(niflumato)2(DMSO)}2 was reported. Crystallographic data are as follows: monoclinic system, space group P2(1)/n, Z = 2, a = 11.1318(8), b = 17.513(2), c = 15.336(1) A, beta = 103.316(8) degrees, V = 2909.4(4) A3. The structure was refined to R = 0.030 and wR = 0.037 for 3702 reflections with I > sigma (I). It consists of centrosymmetric binuclear units with the Cu-Cui (symmetry code i: 1-x, -y, 1-z) distance between two centrosymmetrically related ions of 2.6272(5) A. Each Cu(II) ion in [Cu2(DMSO)2(mu-niflumato)4] is coordinated to an apical dimethylsulfoxide O atom on the one hand and to the equatorial carbonyl and carboxylic O atoms of two crystallographically independent niflumate moieties and their centrosymmetric counterparts on the other hand. In spite of the low-temperature (190 K) crystal measurements, one L-CF3 grouping exhibits some disorder. The biological activities of these complexes were compared to that of niflumic acid. Niflumic acid and its various copper complexes significantly inhibited polymorphonuclear leukocyte (PMNL) oxidative metabolism, as assessed by chemiluminescence and O2- generation measurement. This effect was dose-dependent. All copper complexes exerted a similar inhibiting effect which was always significantly higher than that exerted by the parent drug.

Ornithine alpha-ketoglutarate counteracts the decrease of liver cytochrome P-450 content in burned rats.

The effect of ornithine alpha-ketoglutarate (OKG) on cytochrome P-450 enzyme activities was studied in a well-defined model of injury (burn followed by fasting then subsequent hypocaloric diet) administered to young rats for 3 d. Hepatic microsomes were prepared by ultracentrifugation and levels of cytochromes P-450 were determined spectrophotometrically. The activities of ethoxy-resorufin-O-deethylase (EROD), benzyloxy-resorufin-O-dealkylase (BROD), and erythromycin demethylase were measured as markers of P-450 1A, 2A, and 3A isotypes respectively. The level of total hepatic microsomal proteins (8 mg/mL) remained constant. The level of cytochrome P-450 (1.14+/-0.08 nmol/mg microsomal proteins) was decreased by a hypocaloric diet (23%, P = 0.003) and burn further enhanced this phenomenon (15%, P = 0.03). Both healthy and burned rats receiving OKG showed the same level of cytochrome P-450 as the rats fed ad libitum. OKG supplementation counteracted the enhancement (40%) of EROD activity induced by hypocaloric diet but did not influence BROD and erythromycin demethylase activities. OKG sustained cytochrome P-450 levels in rats fed a hypocaloric diet, even after burning. These findings indicate that OKG may favor drug metabolism in this injured population.

Copper-indomethacinate associated with zwitterionic phospholipids prevents enteropathy in rats: effect on inducible NO synthase.

Intestinal toxicity exerted by indomethacin was compared to that induced by copper-indomethacinate, free or associated to zwitterionic phospholipids. A single high dose of indomethacin (15 or 20 mg/kg), copper-indomethacinate (15 or 20 mg/kg), or copper-indomethacinate liposomes or nanocapsules (15 mg/kg) was orally administered. Then 24 hr later jejunoileal tissue was taken for macroscopic observation, ex vivo nitrite production, and determination of myeloperoxydase and iNOS activities. Antiinflammatory activity of the drugs was investigated using the carrageenan-induced paw edema model. Indomethacin induced penetrating ulcerations of the intestine that were maximal at hour 24. Copper-indomethacinate induced significantly less ulceration than indomethacin with no significant difference in MPO and iNOS activities. The injurious action of indomethacin on the small intestine was further reduced when copper-indomethacinate was administered as the phospholipid-associated state while similar anti-inflammatory action was observed on rat paw edema. The antiulcerogen effect of copper-indomethacinate seems to be linked to its free radical scavenging effect without any modification of nitric oxide release.

Effect of piracetam on polyphosphoinositide metabolism, cytosolic calcium release, and oxidative burst in human polymorphonuclear cells: interaction with fMLP-induced stimulation.

We investigated the action of piracetam on human polymorphonuclear leukocyte (PMN) responsiveness in vitro. We first studied phosphoinositide metabolism and calcium release with and without fMLP (formyl-methionyl-leucyl-phenylalanine) stimulation. Piracetam at concentrations from 10(-4) to 10(-2) M induced a slight increase in inositol 1,4,5-trisphosphate (IP3) release and phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. At concentrations above 10(-3) M, piracetam sensitized PMNs to subsequent stimulation by fMLP used at subliminal concentrations (10(-9) and 10(-8) M), inducing a significant increase in IP3 release and PIP2 breakdown similar to that obtained with cells stimulated by the highest effective concentrations of fMLP (10(-7) and 10(-6) M). In the same way, piracetam greatly enhanced calcium release induced by weak concentrations of fMLP. However, piracetam had no effect on oxidative metabolism. We then studied the binding of (3H)fMLP to the PMN membrane in the presence of various concentrations of piracetam. We were not able to demonstrate an obvious action of piracetam either on receptor recruitment or on receptor affinity to fMLP. The difference between the actions of piracetam on phosphoinositide metabolism and calcium release on the one hand and oxidative burst on the other could be explained by an uncoupling of the triggering and activating effects of piracetam on PMNs. The enhancement by piracetam of intracellular cyclic AMP levels rapidly induced termination of the PMN response and accounted for the lack of effect on superoxide production. Thus, piracetam was able to modulate human PMN reactivity and in particular to exert a "priming effect" (rather due to structural modifications of the membrane), which might be of importance in infectious episodes given the absence of deleterious actions such as oxygen free radical production leading to tissue injury.

Reactive oxygen species induce apoptosis of synoviocytes in vitro. Alpha-tocopherol provides no protection.

Reactive oxygen species (ROS) are released during the inflammation of the synovial membrane associated with cartilage degradation in osteoarthritis. In this work, we exposed synoviocytes to superoxide anions at concentrations that may cause either apoptosis or necrosis. We studied membrane organization, dehydrogenase mitochondrial activity and nuclear morphology and integrity, to determine the nature of the death process initiated by superoxide anions and tried to counteract ROS effects with alpha-tocopherol. We found that oxidative stress caused synoviocytes to undergo a process of cell death of an apoptotic nature rather than necrotic. Mitochondrial injury occurred at an early stage, and the FITC-annexin-V-positive/propidium iodide-positive cells occurred later than the metabolic changes. DNA strand breaks were evident at 8 h and nuclear condensation at 24 h. No LDH activity was detected in culture supernatants. In our experimental conditions, alpha-tocopherol had little effect on stress damage; the antioxidant properties of this molecule did not affect the apoptosis caused by superoxide anions.

Crystal structure of 180 degree K of bis-3, 5-diisopropylsalicylatobisdimethylsulfoxidozinc(II) and the inhibition of seizures and polymorphonuclear leukocyte chemiluminescence.

Dimethylsulfoxide (DMSO) formed a ternary complex when mixed with a Zn-3, 5-diisopropylsalicylate complex of unknown structure. The structure of this new ternary complex was characterized in an initial effort to understand the nature of this compound. Since the original complex is known to have anticonvulsant activity, the new ternary complex was also examined for anticonvulsant activity. The original complex was examined for inhibition of the polymorphonuclear leukocyte (PMNL) respiratory burst in an effort to mechanistically account for zinc complex mediated anticonvulsant activity. Dissolving the structurally unknown complex in DMSO gave crystals of a characterizable complex with an empirical formula C30H46O8S2Zn. Crystallographic data: P 1, Z = 2, a = 8.06(1), b = 12.452(2), c = 17.951(2) A, alpha = 74.42(l), beta = 77.07(1), gamma = 89.50(1) degree. The structure was refined to R = 0.03, RW = 0.04 for 3815 independent reflections with I > 2 sigma(I). This complex is mononuclear, with two 3,5-diisopropylsalicylate ligands and two bonded DMSO ligands, Zn(II)(3,5-DIPS)2(DMSO)2, Zn(II) is coordinate covalently bonded to four O atoms in a strongly distorted tetrahedral arrangement. Each DMSO ligates via its sulfoxide O atom while each 3,5-diisopropylsalicylate ligand is monodentate The non-ligating carbonyl O atom of each 3,5-DIPS is free except for an intramolecular hydrogen bond from the hydroxy group of the same ligand. Both 3,5-DIPS acid and Zn(II)(3,5-DIPS)2(DMSO)2 were examined for anticonvulsant activity in the Maximal Electroshock (MES) and Metrazol (MET) models of seizures and found to prevent both types of seizures. The Zn complex was qualitatively and quantitatively more effective than treatment with the free ligand. The influence of a Zn 3,5-DIPS complex and of the ligand 3,5-DIPS on PMNL oxidative metabolism was also studied to help understand the mechanism of anticonvulsant activity of these compounds. A dose-related and significant decrease in chemiluminescent (CL) response to opsonized Zymosan was observed, and the Zn complex was significantly more effective than the free ligand. It is concluded that mononuclear Zn complexes have anticonvulsant activity in Grand Mal and Petit Mal models of seizure possibly due to inhibition of the synthesis of superoxide or down-regulation of Nitric Oxide Synthase in activated phagocytic cells of the central nervous system.

Dermaseptin, a peptide antibiotic, stimulates microbicidal activities of polymorphonuclear leukocytes.

Dermaseptin (DRs S1), a 34-amino acid residue cationic antimicrobial peptide was studied for its effects on the production of reactive oxygen species (respiratory burst) and exocytosis of polymorphonuclear leukocytes (PMN). Treatment of PMN with DRs S1 (10-100 nM) stimulated significant production of reactive oxygen species (approximately a 2-fold increase relative to control) and release of myeloperoxidase. In addition, low DRs S1 concentrations (1-10 nM) primed the stimulation of respiratory burst induced by zymosan particles. In contrast to the native peptide, a dermaseptin fragment without either the COOH-terminal (DRs 1-10) or NH2 terminal (DRs 16-34) portion was inactive. The DRs S1-induced respiratory burst was inhibited by a selective protein kinase C inhibitor, GF 109203X, and was associated with early signalling events such as a rapid and transient elevation of cytosolic-free calcium concentration and phospholipase D activity. These data provide the first evidence of stimulating and priming properties of a peptide antibiotic on microbicidal activities of neutrophils, suggesting a potential role of dermaseptin in modulating host-defense mechanisms.

In Vivo Nitric Oxide Synthase Inhibitors Can Be Deprived of This Activity: Unexpected Influence of the Tetrachloroplatinate(II) Counteranion. Crystal Structures of Bis(S-Methyl-Isothiouronium)-N,N'-Bis(3-Guanidinopropyl)Piperazinium and Hexamidinium Tetrachloroplatinates(II) Salts.

The synthesis and crystal structures of bis(S-methylisothiouronium) (MSTUH)(+), N,N'-bis((3- guanidinopropyl)piperazinium (PipeC3GuaH4)(4+) and hexamidinium (HexaH2)(2+) tetrachloro platinate(ll) salts ( called hereafter PtMSTU, PtPipeC3Gua and PtHexa respectively ) were investigated. These compounds contain the "amidine" function ( - C(=NH)NH(2) ) in which the H atoms supplied by the acid have become attached to the imino group of each terminal amidino function. Moreover, in PtPipeC3Gua, the nitrogen atoms of the chair-piperazine moiety are also protonated. The influence of tetrachloroplatinate(ll) counteranion ( versus sulfate, nitrate and diisethionate ) in the in vivo nitrite inhibition by the (MSTUH)(+), (PipeC3GuaH4)(4+) and (HexaH2)(2+) cations was investigated. The three tetrachloroplatinate(ll) salts, unexpectedly, do not inhibit significantly the in vivo nitrite production in comparison with the other salts (sulfate, nitrate and diisethionate and their corresponding previous countercations) which exhibit NO synthase inhibition, especially bis(S-methylisothiouronium) sulfate, a selective and potent inducible NO synthase (iNOS) inhibitor commonly used as standard.

Ternary Copper(II) Complexes With Indomethacin, a Potent Non-Steroidal Antiinflammatory Drug. Crystal Structure of Bis (Dimethylformamide)-Tetrakis[1-(4-Chlorobenzoyl)-5-Methoxy-2-Methyl-1-H-Indole-3-Acetato]Dicopper(II). Antiinflammatory Properties and Prevention of Gastrointestinal Side Effects by Nanocapsules.

Two ternary copper(ll) complexes of indomethacin [1-(4-chlorobenzoyl)-5-methoxy-2- methyl-1-H-indole-3-acetic acid] called hereafter lndo, were prepared and characterized by single crystal X-ray diffraction. The first complex Cu(2)(Indo)(4)(DMF)(2) I crystallizes in space group P-1 (a = 10.829(2), b = 13.379(2), c = 16.491(3) A; alpha = 105.58(2), beta = 101.06(2), gamma = 106.96(2) degrees ; V= 2104.6(6) A(3), Z= 1). The title molecule is a centrosymmetric binuclear complex, with Cu atoms bridged by the carboxylate moieties of four indomethacinate ligands. The four nearest O atoms around each Cu atom form a square planar arrangement with the square pyramidal coordination completed by the O atom of N,N'-dimethylformamide. Daily administration for seven days of 1 mg/kg of indomethacin, I and I encapsulated into liposomes induces a weak inflammation of rat gastrointestinal tract. I was less inflammatory than indomethacin but the better protection was brought by encapsulation of the compound. This might be of interest in sustained therapies of chronic inflammatory diseases.

In vivo inhibition of nitric oxide synthase by bisisothiouronium and bisguanidinium salts.

The ability of two S,S'-(alkane-1,omega-diyl) bisisothiouronium dibromides, three N,N'-(alkane-1, omega-diyl) bis guanidinium dinitrates and N,N'-bis (3-guanidinopropyl)piperazine dinitrate to inhibit constitutive (i.e. endothelial and neuronal forms) and inducible forms of nitric oxide synthases has been evaluated in vivo. These compounds, synthesized by two of us (J. C. L. and C. S.), have been tested in vivo; they were administered simultaneously with an irritant (carrageenan lambda) into the pleural cavity. The amount of nitrites collected 0.5 and 7 hours after this injection can be considered as an indicator of nitric oxide (NO) production. According to previous data, the first harvesting time can be related to activation of constitutive NO synthases and the second to activation of inducible NO synthases. These substances significantly inhibited nitrite production as did 2-methyl-2-thiopseudourea sulphate, previously described as a potent inhibitor of NO synthases and considered as the reference compound. The inhibiting effect varied according to the chemical structure of the compounds. Results were significantly different from controls at 0.5 h only with the S,S'-(octane-1,8-diyl) bisisothiouronium dibromide and the S,S'-(nonane-1,9-diyl) bisisothiouronium dibromide at the highest concentration, N,N'-(heptane-1,7-diyl) bisguanidinium dinitrate and N,N'-bis (3-guanidinopropyl)piperazine dinitrate. At 7 h, all the results were significantly different from controls, with a major effect observed with N,N'-(heptane-1,7-diyl) bisguanidinium dinitrate. The most active substances exerted similar effects to the reference substance.

Active and alpha-1 proteinase inhibitor complexed leukocyte elastase levels in crevicular fluid from patients with periodontal diseases.

Human leukocyte elastase is present in large amounts in the crevicular fluid of patients with periodontal disease and was considered as a putative biological marker of the evolution of such diseases. The aim of this work was to measure spectrophotometrically amounts of active elastase (AE) and elastase complexed to alpha 1 proteinase inhibitor (E-alpha 1-PI) in gingival crevicular fluid obtained, from patients suffering from rapidly progressive periodontitis (RPP group) or adult periodontitis (AP group) with different probing depths (3 to 5 mm and > 6 mm). AE and E-alpha 1-PI concentrations were negligible in healthy individuals. AE, but not E-alpha 1-PI, concentration appears to vary significantly with the probing depth in patients suffering either from rapidly progressive or adult periodontitis. No correlations were found between levels of AE and E-alpha 1-PI in the different groups of patients. AE concentration seems to be a marker of periodontal diseases in relation with probing depth.

Effects of human low-density lipoproteins on superoxide production by formyl-methionyl-leucyl-phenylalanine activated polymorphonuclear leukocytes.

Neutrophils play a major role in the host defence by producing reactive oxygen species. These products are liberated by activated cells and are known to cause endothelial cell injury and damage. The present study shows that low-density lipoproteins increase superoxide anion production by twofold in polymorphonuclear leukocytes stimulated by formyl-Met-Leu-Phe in vitro. Moreover, LDL induced a large increase in phosphoinositides and cytosolic-free calcium. Data from experiments performed on neutrophils treated with pertussis toxin, staurosporine, propranolol or niflumic acid suggest that modulation of phospholipase D and A2 activities could be involved in the modification by LDL of leukocyte response to formyl-Met-Leu-Phe. LDL lipid moiety could play a key role in their action on polymorphonuclear functions because cholesterol was exchanged between lipoproteins and cells that can modify membrane fluidity and interact with the formyl-Met-Leu-Phe receptor.

Effects of plasma lipoproteins on the production of superoxide anion by human polymorphonuclear leukocytes in vitro.

Polymorphonuclear leukocytes (PMN) generate highly reactive oxygen derived free radicals that may cause lipoprotein lipid oxidation and so contribute to the pathogenesis of atherosclerosis. On the other hand it has been shown that lipoproteins can alter cell functions in vitro. We therefore studied the effects of atherogenic lipoproteins, VLDL and LDL, on the production of superoxide anion by human PMN in the presence or absence of formyl-methionyl-leucyl-phenylalanine (fMLP). VLDL and LDL stimulate PMN superoxide production and potentialize PMN stimulation by fMLP. The lipid moiety of the lipoproteins might be mainly involved in these effects. The binding of radio-labelled fMLP to its specific membrane receptor was significantly enhanced in the presence of VLDL and only slightly in the presence of LDL. The study of the signal transduction suggests that modulation of phospholipase D and A2 activities could be involved in the modification by LDL of PMN response to fMLP.

Effect of an encapsulated anti-elastase compound on experimental gingival inflammation in the rat.

An animal (rat) model of gingival injury ("impaction") induced a gingival inflammatory reaction, which was characterized by a breakdown of gingival collagen and the elastic network, as well as a significant increase of gingival elastase. The present study was conducted to investigate whether ceramides, sphingolipids composed of sphingosine N-acyl-linked to fatty acids, a chemical structure with antielastase properties, could counteract the development of such an inflammatory process. The ceramides used in these experimental series were extracted from wheat and characterized. The main fatty acids were 16:0, 18:1, 18:2, and the sphingoid moiety was phytosphingosine. Inhibition of elastase by ceramides was demonstrated in vitro and the concentration necessary to inhibit 50% of elastase activity was 41 mg/l using the synthetic substrate methoxysuccinyl-alanine-alanine-proline-valine-p-nitroanilide (MeOSuc-AlaAlaProValpNA). However, this anti-elastase activity was not observed in vivo in our animal model of gingival inflammation. A glycosaminoglycan (Heparin), recognized as a potent inhibitor of elastase, was entrapped in ceramides. A local treatment of impacted gingivae by encapsulated heparin led to a dose-related decrease of the elastase level in gingival extracts. Encapsulation in ceramides potentiated the effect exerted by heparin alone. This inhibitory effect of encapsulated heparin on elastase suggested a vector effect of these amphipathic molecules.

Kinetic evaluation of nitric oxide production in pleural exudate after induction of two inflammatory reactions in the rat.

NO generation in the course of two acute, non immune, inflammatory reactions (pleurisy induced by rat isologous serum and carrageenan) was assessed by means of nitrite measurement in pleural exudate from 0.5 to 24 h. NO release varied time-dependently, similarly for the two inflammatory reactions. A first, but transient, peak was reached in 30 min while a second peak, more sustained, began at the fourth hour and was maximum at the tenth. Kinetic evolution of NO release was consistent with activation, in a first step, of a constitutive NO synthase probably from endothelial origin (inhibited by 2-Methyl-2-Thiopseudourea sulfate but not by dexamethasone) and with activation, in a second wave, of inducible NOS from endothelial and exudative cells. NO release was potentiated by administration per os of L-Arginine and seems to be involved in the evolution of acute inflammatory reactions and oxygen metabolite production.

Immunomodulatory effects of ornithine alpha-ketoglutarate in rats with burn injuries.

OBJECTIVE: To investigate the influence of enterally administered ornithine alpha-ketoglutarate (OKG) on muscular amino acid content, eicosanoid release, and polymorphonuclear leukocyte responsiveness after induction of burn injury in rats. DESIGN: Experimental trial. MATERIALS AND METHODS: Four groups of rats were considered: (1) healthy rats that received a standard diet supplemented with 5 g/kg per day of OKG; (2) rats with burn injuries that received the same nutrition as group 1; (3) healthy rats that received standard diet supplemented with glycine in an isonitrogenous amount relative to OKG; and (4) rats with burn injuries that received the same nutrition as group 3. The thymus and 1 skeletal muscle were weighed. The oxidative metabolism of pleural polymorphonuclear leukocytes was measured by means of superoxide generation (O2-) and the chemiluminescent response to opsonized zymosan. Prostaglandin E2 and 6-keto-prostaglandin F1 alpha were measured in the supernatants of pleural and peritoneal cells. RESULTS: The weights of the thymus and the muscle from healthy rats were similar. Those of rats from group 4 were significantly lower (P < .05), whereas those of rats from group 2 were not. Metabolism of OKG led to enhanced amounts of arginine and glutamine in skeletal muscle. The metabolic bursts of polymorphonuclear leukocytes from healthy rats were similar. Those of glycine-treated rats with burn injuries were significantly depressed (P < .05), whereas those of the OKG-treated group were not. Pleural and peritoneal cells from the rats with burn injuries that received OKG generated significantly more prostaglandins (P < .01) than did cells from the other groups of rats. CONCLUSION: Ornithine alpha-ketoglutarate administered to rats with burn injuries displays immunomodulatory properties that can enhance host-defense mechanisms in animals that are affected by a severe injury.