Evaluation of serum procalcitonin concentration in the ICU following severe burn.

The goal of the study was to analyse plasma procalcitonin (PCT) concentrations during infectious events of burns in ICU. Clinical and laboratory data were collected at admission and twice a week in burned patients admitted with a total body surface area (TBSA) >20%. Procalcitonin was determined using both a semi-quantitative detection (PCT-Q) and a quantitative immunoluminometric method (PCT-Lumi). A total of 359 time points in 25 consecutive patients with 40+/-17% (20-86%) TBSA burned, defined as a procalcitonin concentration associated with an inflammatory status according to society critical care medicine definition, were made. The principal site of infection was the respiratory tract (84% of patients required mechanical ventilation). PCT-Lumi values corresponded to the four semi-quantitative ranges of PCT-Q and statistically reflected the simultaneously observed inflammatory status (Kruskall-Wallis test). The area under the receiver operating characteristic curve for C-reactive protein (CRP) was higher than those for PCT and white blood cell (WBC) count, but this difference was not significant. The optimum PCT cut-off value was 0.534 ng/ml with sensitivity and specificity of 42.4% and 88.8%, respectively. However, PCT does not appear to be superior to C-reactive protein (CRP) and white blood count (WBC) as diagnosis marker of sepsis in burns. PCT is not sufficient to diagnose and to follow infection in burns admitted in ICU.

Alterations of antioxidant trace elements (Zn, Se, Cu) and related metallo-enzymes in plasma and tissues following burn injury in rats.

To improve the nutritional support for burn patients, we evaluated the alterations of selenium, zinc and copper (Se, Zn and Cu) and their possible contributions to an unbalanced antioxidant response to burn injury. These trace elements and the related antioxidant enzymes, glutathione peroxidase (GPx) and superoxide dismutase (SOD), were studied both in plasma (or serum) and tissues of 20% total body surface area (TBSA) burned rats for 10 days. While plasma Se and serum Zn levels significantly decreased 6 h after burn injury, serum Cu levels increased after 1 day and remained elevated the following 9 days. Selenium levels increased in kidney but decreased progressively in liver. The hepatic Zn and Cu concentrations followed a biphasic increase following burn injury. During the first day, GPx activity decreased in plasma and remained unchanged in the organs, except for a moderate diminution in the liver. Liver Cu/Zn SOD activity increased from 6 h to 4 days. In summary, following burn injury, copper and zinc were redistributed to the liver and selenium to the kidney with non-detectable changes in the muscle and brain. Changes in antioxidant enzyme activities following burn injury were significant mainly in the plasma. Early combined antioxidant supplementation to maintain and restore antioxidant status in burn patients requires further study.

Production of procalcitonin (PCT) in non-thyroidal tissue after LPS injection.

Procalcitonin (PCT) is one of the precursors in the synthesis of calcitonin in thyroidal C-cells and other neuroendocrine cells. PCT, among other calcitonin precursors, is elevated in the serum of many conditions associated with a systemic inflammatory response syndrome, even in the absence of the thyroid gland. The aim of our study was to identify PCT-producing extrathyroidal tissues in primate sepsis. In order to induce PCT production, we treated four olive baboons ( papio cynocephalus anubis) with the endotoxin lipopolysaccharide (LPS) from s. typhimurium. We found an increase in serum PCT 3 to 5 hours after LPS injection to levels of 0.2 ng/ml, attaining a peak above 4 ng/ml PCT at 10 hours. In contrast, the untreated baboon had no detectable circulating PCT in the serum. In one animal, additional LPS boosting after 24 hours did not increase serum PCT further. Soluble proteins were extracted from different organs, fractionated by C18 extraction, and PCT was measured in an immunoluminometric assay (ILMA), which was specifically developed for this study. PCT concentrations above 0.2 ng/g of wet tissue were found in a variety of organs in LPS treated baboons, but not in the control baboon. Organs and tissues with the highest PCT concentration included liver, kidney, aorta, fat, ovaries, bladder and adrenal gland. RT-PCR confirmed an extrathyroidal origin of PCT. Importantly, CT-mRNA expression was found in liver, lung, kidney, adrenal, colon, skin, spleen, brain and pancreas. In conclusion, our data confirm previous findings in hamsters, indicating an extrathyroidal origin for PCT in sepsis. Our primate model offers a valuable tool for further investigation of PCT's pathophysiological role and its immunoneutralization as a therapy for sepsis.

[Apoptosis and gamma rays]. / Apoptose et rayonnement gamma.

Gamma radiation can induce cell death in lymphocytes. Apoptosis is characterized by numerous morphological, biochemical and molecular modifications measurable using various methods. Some radioprotectors have antioxidant properties and are able to inhibit radiation-induced DNA fragmentation and caspase activation. There are several caspases that cleave proteolytically many proteins and trigger phosphatidylserine externalization recognized by phagocytes. Three main proteins are involved in the regulation of radiation-induced apoptosis: p53, Fas and Bcl-2. The pharmacological regulation of cell death is discussed in order to investigate the subsequent effects related to cell regeneration following radiation injury.

Role of nitric oxide in depressed lymphoproliferative responses and altered cytokine production following thermal injury in rats.

Immunodeficiency follows extensive burns. We investigated some underlying mechanisms in rats, 10 days after a full-thickness skin burn affecting 20% of total body surface area. In both normal and burned rats the splenocyte proliferative response to Con A was linearly and negatively correlated with nitric oxide (NO) production. In all burned rats, the proliferative response was depressed by more than 80% and NO production corresponded to a nitrite concentration above 20 microM. Proliferative responses in burned rats were fully restored in the presence of 250 microM NG-monomethyl-L-arginine (NMMA). A time course study of NO production in response to Con A, LPS, anti-CD3, and IFN-gamma showed that splenic macrophages from burned rats responded to direct and indirect stimuli more rapidly and more intensively than normal macrophages. In the second part of this work, the effect of the overproduction of NO on the synthesis of immunoregulatory and proinflammatory cytokines was investigated. Although it was inhibited, IFN-gamma production by splenocytes from burned rats remained sufficient for NO synthase induction and was restored by NMMA. Concomitantly, IL-2 concentration was enhanced but returned to normal in the presence of NMMA. TNF production was halved after burn injury and NMMA partially restored it. In contrast, IL-6 production was enhanced and increased further in the presence of NMMA. Therefore, cytokines were differently affected by burn injury and variously regulated by NO.

Diet, antioxidant status, and smoking habits in French men.

The aim of this study was to assess the association between smoking, food consumption, and antioxidant vitamin intake and plasma indexes of oxidative stress and antioxidant defenses in French adults. Food and nutrient intakes of 459 healthy men aged 23-57 y were estimated by the diet history method and analyzed by smoking status. Plasma alpha-tocopherol, ascorbic acid, and carotenoids were measured as antioxidants and malondialdehyde, protein Schiff bases, and autoantibodies against malondialdehyde-protein adducts as oxidative stress indexes. Smokers ate less fruit and vegetables than nonsmokers, leading to lower vitamin E, vitamin C, and carotene intakes, even after adjustment for age, education, and marital status. Unlike vitamin E, plasma ascorbic acid and beta-carotene concentrations were reduced in smokers compared with nonsmokers and were inversely related to cigarette consumption. This difference remained significant after adjustment for alcohol and dietary intakes. Among the measured oxidative stress indexes, only Schiff base concentration was positively related to the number of cigarettes smoked. In our sample of French men, smoking had an adverse effect on antioxidant status; vitamin intakes were reduced in smokers and plasma antioxidant indexes were altered independently of dietary intakes. As in other countries, in France smokers require particular attention in terms of public health intervention.

Radiation-induced apoptosis in thymocytes: inhibition by diethyldithiocarbamate and zinc.

Apoptosis is a process of physiological cell death characterized by DNA fragmentation, chromatin condensation, loss of membrane asymmetry, mitochondrial alterations and cell lethality. In the present study, apoptosis induced in thymocytes by gamma irradiation is evaluated by flow cytometry, by a diphenylamine colorimetric method and by gel electrophoresis. Treatment of thymocytes with diethyldithiocarbamate or zinc shows that these compounds can inhibit radiation-induced apoptosis. Moreover, a synergistic effect is observed by using combinations of both compounds: ZnSO4 potentiates the effect of diethyldithiocarbamate at concentrations at which the compounds used separately show a low efficacy. A study of kinetics shows that addition of 1 microM diethyldithiocarbamate + 50 microM ZnSO4 (the most efficient combination) after irradiation can decrease DNA fragmentation even when it is added 2-3 h after irradiation. However, 1 microM diethyldithiocarbamate + 50 microM ZnSO4 cannot prevent the radiation-induced loss of membrane asymmetry and the decrease in alteration of the mitochondrial membrane as measured by binding of merocyanine 540 and uptake of rhodamine 123, respectively.

[Oxidative stress and apoptosis]. / Stress oxydatif et apoptose.

Programmed cell death or apoptosis is a process characterized by several morphological, biochemical and molecular events in response to physiological or pathological stimuli, such as gamma radiation. Free radicals being involved in many physiological and pathological processes, the aim of this study is to investigate the literature about the involvement of oxidative pathway during apoptotic process. As reported by several authors, the literature is abundant in this field and show the complexity of the network in which numerous molecules can regulate cell death and proliferation. However, reactive oxygen intermediate species (ROls) seem to play an important role in the induction of apoptosis as underlined by several reports. Regulation of cellular redox status may appear to be a key component which determines cell proliferation or apoptosis.

["Autoantibodies" and cell aging. Does malonic dialdehyde constitute an immunological marker of cell aging?]. / "Autoanticorps" et sénescence cellulaire. Le dialdéhyde malonique constitue-t-il un marqueur immunologique du vieillissement cellulaire?

Malondialdehyde (MDA), a lipid peroxidation product, reacts with free amino groups of proteins to give amino-3-iminopropene (AIP) bridges which are immunogenic. Antibodies reacting specifically with MDA-crosslinked proteins preexist in the sera of healthy human subjects, and we hypothesize that they are implicated in processes of senescent cells recognition and elimination. Results obtained by immunofluorescence and flow cytometry show an immunological response against structures arising on cell membranes from MDA reaction with free amino groups of proteins. Such structures might also be implicated in autoimmunity.

DNA fragmentation induced in lymphocytes by gamma irradiation or dexamethasone: inhibition by diethyldithiocarbamate (DTC), potentiated by zinc.

Apoptosis is a process of physiological cell death characterized by DNA fragmentation, chromatin condensation, loss of membrane asymmetry and cell lethality. In the present study, apoptosis induced in thymocytes by dexamethasone or gamma irradiation is evaluated by flow cytometry, gel electrophoresis and other techniques. Treatment of thymocytes with DTC or zinc shows that these products can inhibit radiation- or dexamethasone-induced apoptosis. Moreover, a synergistic effect is observed by using associations of both products (5 microM DTC + 50 microM ZnSO4): ZnSO4 potentiates the effect of DTC at concentrations for which the molecules used separately show a low efficacy. These results indicate that DNA fragmentation induced by dexamethasone or irradiation in thymocytes share some identical mechanisms.

Polyclonal antibodies against malondialdehyde-modified proteins: characterization and application in study of in vitro lipid peroxidation of cellular membranes.

In order to develop an immunofluorescent technique able to detect the cellular damage induced by lipid peroxidation, we produced rabbit polyclonal antibodies (AbAIP) against malondialdehyde (MDA)-modified lysozyme. These antibodies specifically recognized all the MDA-treated proteins tested, but not native proteins or proteins treated by other aldehydes. Moreover, we showed by an enzyme-linked immunosorbent assay (ELISA) that the amount of immunoreactive protein in MDA-treated erythrocytes was proportional to the concentration of MDA added, suggesting that this ELISA measurement may represent a quantitative determination of peroxidative protein alterations. In addition, when coupled to an indirect fluorochrome antibody conjugate [fluorescein isothiocyanate (FITC)], these antibodies permitted precise localization of the modified proteins within the membranes of peroxidized cells.

Immunological approach to investigating membrane cell damages induced by lipoperoxidative stress. Application to far UV-irradiated erythrocytes.

Oxygen-reactive species are being described as agents responsible for cell degeneration mechanisms resulting from membrane, enzyme, and nuclear alterations. Lipid peroxidation on its own is considered to be one of the consequences of the free radicals attack, and among the different reactive aldehydes that can be formed from the decomposition of lipid peroxides, the most extensively assayed have been malondialdehyde (MDA). However, the different techniques currently used for MDA assay (HPLC, GLC) are barely sensitive enough to follow its production at the cellular level. In order to develop an immunofluorescent technique able to detect cellular damages provoked by lipoperoxidation, polyclonal antibodies against lysozyme modified by MDA treatment have been raised in rabbits. We show that this immunserum recognizes specifically all the MDA-treated proteins tested, but not the intact proteins or the proteins treated by other aldehydes. Moreover, we demonstrate using an ELISA technique that the amount of immunoreactive proteins in MDA-treated membrane erythrocytes is proportional to the concentration of MDA applied, suggesting that this assay may represent a quantitative method of determination of lipoperoxidative alterations. In addition, when coupled to an indirect fluorophore antibody (FITC), the immunserum allows a precise location of these modified proteins within the membranes of erythrocytes in which lipid peroxidation was initiated by far UV irradiation. In summary, the interest of this work is to provide an immunological probe that can precociously detect membrane damages induced by MDA, regardless of the cell type and pro-oxidant (physiological or pathological) conditions.

[Immunologic relevance of malonic dialdehyde]. / Incidences immunitaires du dialdéhyde malonique.

Free radicals are involved in many physiological and pathological processes, including oxidation of polyunsaturated fatty acids, i.e. lipoperoxidation. Among the different by-products of this lipoperoxidation, malonic dialdehyde (MDA) has been one of the most studied. It reacts with the primary amino groups of biological molecules, especially those of proteins, with formation of 1-amino-3-imino-propene (AIP) bridges. Studies undertaken in our laboratory about the immunological relevance of MDA have first shown the existence of antibodies (AcAIP) recognizing epitopes containing AIP bridges, and reacting specifically with MDA-modified proteins but not with the corresponding native ones. These antibodies occur even under physiological conditions, independently of any disease or any stimulation induced by antigen injection. However, their seric level may be modified in pathologies involving an increased lipoperoxidation and/or an inflammatory process (cardiovascular pathology, diabetes, alcoholism). Injections of a MDA-protein adduct to rabbits or mice stimulates AcAIP production, even if the protein contained in the adduct in an autologous one, but such injections may also result in production of other antibodies. Other results obtained in our laboratory indicate that AcAIP are involved in elimination of senescent or lipoperidized erythrocytes, and probably also in discarding other cells having undergone oxidative stress, at least the circulating ones. The physiological and pathological implications of MDA and AcAIP have lead us to formulate hypotheses about their more general role within the immune system, and to try to situate them in this system. Of course, these hypotheses remain to be assessed by further experimentations.

Recognition and elimination of senescent erythrocytes: implication of antibodies specific for malonic dialdehyde-protein adducts, as demonstrated by flow cytometry.

Many different hypotheses have been formulated about the mechanisms of specific recognition of senescent red blood cells (RBC). It is usually assumed that novel epitopes appear on RBC membranes during ageing and are responsible for recognition of aged RBC by antibodies, which is followed by binding to mononuclear phagocytes and then phagocytosis. But these age-related epitopes have not so far been identified. Lipoperoxidation is known to produce aldehydes, among which malonic dialdehyde (MDA). This dialdehyde reacts with primary amino groups of biological molecules, producing 1-amino-3-imino propene (AIP) bridges, and we had previously shown that sera of healthy mammals contain antibodies recognizing epitopes containing AIP bridges (AbAIP). Lipoperoxidation is responsible for many age-related damages in RBC membrane, and we tried in the present work to determine whether age-related epitopes responsible for recognition of aged RBC were not derived from lipoperoxidation. Using flow cytometry techniques, we demonstrated that some of the epitopes recognized by immunoglobulins which bind to aged RBC contain AIP bridges, and that some of these RBC-bound immunoglobulins are AbAIP. Consequently, AbAIP/AIP bridges interactions appear to play a role in recognition and elimination of senescent RBC.

Immunization of mice with proteins reacted with malonic dialdehyde (MDA): comparison between autologous and heterologous modified proteins.

In previous experiments, rabbits were injected with heterologous proteins reacted with MDA, and produced antibodies cross-reacting with other MDA-modified proteins (MPr), but not with the corresponding native ones (Pr). It was concluded that these antibodies (AbAIP) recognized epitopes including 1-amino-3-imino-propene (AIP) bridges resulting from reactions of MDA with primary amino groups of proteins. In the present work, mice were injected with autologous MDA-modified albumin (MAI) or with heterologous MPr. Mice immunized with MAI developed an immune response leading to an increased production of AbAIP, which clearly indicates that such a response may occur even with an autologous MPr.

Effect of alcohol consumption on blood antioxidant nutrients and oxidative stress indicators.

The effects of alcohol consumption on plasma concentrations of antioxidant vitamins (alpha-tocopherol and ascorbic acid), selenium, and markers of oxidative stress, especially malondialdehyde (MDA) and autoantibodies directed to MDA adducts to proteins (Ig-NH2-MDA) were investigated in a large population of 417 supposedly healthy men who consumed only low or moderate amounts of alcohol as compared with 102 alcoholic patients without severe liver disease, who were studied both before and after 21 d of withdrawal treatment. Plasma concentrations of alpha-tocopherol, ascorbic acid, and selenium were lower in alcoholics than in men who drank low amounts of alcohol (P < or = 0.001), whereas MDA and Ig-NH2-MDA were higher (P < or = 0.001). Plasma concentrations of alpha-tocopherol and selenium remained unchanged after the withdrawal period, whereas ascorbic acid (P < or = 0.01), MDA, and Ig-NH2-MDA concentrations decreased (P < or = 0.001). Adjustment of data for circulating lipids and nutritional intake suggests a specific effect of alcohol on antioxidant vitamins, independent of nutritional status.

Naphtyl methyl imidazoline (NMI) membranes interactions: NMR study and evaluation of possible radioprotective consequences.

The interactions of the radioprotective molecule Naphtyl methyl imidazoline (NMI) with membranes have been studied using biophysical and biochemical methods. 1H, 2H and 31P-NMR methods showed a strong interaction with the deep part of the layer of model membranes inducing the formation of a new membrane compartment. These properties are not involved in the radioprotection effect of NMI which is more probably due to its vasoconstrictive properties.

Restoration of postburn impaired lymphocyte responsiveness by nonsteroidal anti-inflammatory drugs is independent of prostaglandin E2 inhibition.

Prostaglandin E2 (PGE2) has been implicated in postburn immunosuppression, which is responsible for septic complications. In the present work, seven non-steroidal anti-inflammatory drugs (NSAIDs), differing by their capacity to inhibit the cyclooxygenase pathway, were compared for their ability to restore T lymphocyte proliferative responses evaluated 4 days after thermal injury in rats. Salicylic acid, 5-aminosalicylic acid, and niflumic acid, given daily, fully restored spleen cell responses to concanavalin A (Con A) and phytohemagglutinin. These drugs were active only at doses that were below the anti-inflammatory doses and did not modify normal spleen cell responses. In these conditions, indomethacin slightly restored lymphocyte reactivity, whereas acetylsalicylic acid, ketoprofen, and piroxicam were ineffective. PGE2 production by Con A-stimulated spleen cells from untreated burned rats and after treatment with niflumic acid or 5-aminosalicylic acid did not correlate with the intensity of the proliferative response. Indomethacin, niflumic acid, and 5-aminosalicylic acid were added in vitro to spleen cells from normal and burned rats, at concentrations from 10(-7) to 10(-4) M. PGE2 production was strongly depressed by indomethacin and niflumic acid and not modified by 5-aminosalicylic acid. The proliferative response of normal spleen cells was depressed in a concentration-dependent manner by niflumic acid and slightly inhibited at the highest concentrations of indomethacin. In contrast, indomethacin concentration dependently restored the burn-impaired proliferative response, whereas niflumic acid further depressed it and 5-aminosalicylic acid had no effect. These results demonstrate that only some NSAIDs are able to restore T lymphocyte reactivity impaired after thermal injury and that this property is not related to inhibition of PGE2 production.

High dosage vitamin E effect on oxidative status and serum lipids distribution in streptozotocin-induced diabetic rats.

This study was performed to determine whether vitamin E supplementation in streptozotocin-induced diabetic rats treated by insulin could reduce serum oxidation markers (malondialdehyde: MDA, Schiff bases, anti-protein-MDA adduct antibodies) and modulate lipid changes. After 10 weeks, diabetes induced in rats a significant increase in Schiff bases (P < 0.006) and anti-protein-MDA adduct antibodies (P < 0.01). These alterations were accompanied by a significant rise in serum free fatty acids (225%), triglycerides (35%), and phospholipids (30%) and changes in fatty acid distribution in these fractions and in cholesterol esters. Vitamin E supplementation in diabetic rats reduced Schiff bases and anti-protein-MDA adduct antibodies and tended to restore the fatty acid profile close to control rats without decreasing quantitatively serum lipids enhanced by diabetes. Concerning fatty acids, vitamin E chiefly reduced stearic acid (C18:0) in free fatty acids, cholesterol esters, and phospholipids and cancelled the decrease in low molecular triglycerides observed in diabetic rats. Furthermore, vitamin E maintained the ratio of monounsaturated and polyunsaturated fatty acids, particularly with respect to oleic acid (C18:1), dihomo-gamma-linolenic acid (C20:3 n-6), eicosapentaenoic (C20:5 n-3), and docosapentaenoic acid (C22:5 n-3), in serum phospholipids. These changes observed in vitamin E supplemented rats, compared to vitamin E-untreated diabetic rats, could favor prevention of accelerated atherogenesis. Particularly, the decrease of serum peroxides and enhancement in phospholipid fatty acids (C20:3 n-6, C20:5 n-3, and C22:5 n-3) could induce the preferential formation of prostaglandins (PGE1, PGI2, PGI3) which are protective in cardiovascular diseases.