The pathophysiology of the acquired extracellular matrix alterations, like non-enzymatic glycosylation (NEG) of proteins secondary to diabetes or ageing, is not well characterized, particularly considering its relationship with leukocytes. We have analysed the influence of collagen NEG on the fundamental function of phagocytic cells, i.e. the production of reactive oxygen species (ROS). To this aim, we considered the activity of polymorphonuclear leukocytes and monocytes incubated in the presence of both non-glycated and glycated collagen. ROS production was monitored by chemi-luminescence (CL), a standardized and sensitive assay of phagocytes oxidative metabolism. All experiments were performed in triplicate on collagen-coated and glycated collage-coated vials. Results showed that PMNs ROS metabolism appeared unrelated to the glycation state of the substrate. Conversely, data regarding zymosan-induced CL by monocytes indicated a significant and intriguing decrease in reactive oxygen metabolism, which appeared greatly compromised by the glycation state of the matrix (monocytes in collagen, 197.4 +/- 31.2 vs. monocytes in glycated collagen, 138.0 +/- 20.4; p < 0.001 expressed as counts/cell/60 min). These data highlight the different role of polymophonuclear and monocytic phagocytes in the pathophysiology of the acquired extracellular matrix alteration secondary to non-enzymatic glycosylation (NEG) of proteins.

Collagen↗

PubMedDate not supplied

Impaired reactive oxygen metabolism of phagocytic leukocytes in NIDDM patients. A role for non-enzymatic glycosylation of collagen.

Non-enzymatic glycosylation (NEG) of collagen has been previously shown to significantly influence the reactive oxygen metabolism (ROM) of phagocytic cells in healthy subjects. Considering the role of NEG in the pathophysiology of diabetes, we have further analysed the oxidative metabolism of polymorphonuclear cells (PMNs) and monocytes in 23 patients with non-insulin dependent diabetes mellitus in order to better elucidate a possible pathogenic role of NEG of the extracellular matrix in long-term complications of diabetes. Experiments were performed in triplicate on native-collagen and glycated-collagen coated vials, using a chemiluminescence (CL) assay. Results show that PMNs from diabetic patients display a significant increased basal and zymosan-induced CL activity with respect to controls that are not related to the glycation state of the substrate. Conversely, the CL activity of monocytes induced by zymosan shows a decrease in diabetic patients with respect to healthy volunteers (p < 0.05). Moreover, monocyte CL was reduced by the glycated matrix, both in healthy volunteers and in diabetic subjects (p < 0.05 and p < 0.01, respectively). These data highlight a complex role of phagocytic leukocytes in the pathophysiology of extracellular matrix alterations secondary to NEG that are typically present in clinical conditions such as diabetes or ageing.

Collagen↗

PubMedDate not supplied

Effect of aqueous extract of cigarette smoke on peripheral blood polymorphonuclear leukocytes chemiluminescence.

Cigarette smoke induces a vast cohort of deleterious effects on biological structures. In the present paper, the effect of aqueous extract of cigarette smoke on the activity of polymorphonuclear leukocytes was studied. Although the aqueous extract of cigarette smoke inhibits the luminol oxidation catalysed by horseradish peroxidase, it strongly interacts with polymorphonuclear leukocytes and inhibits their phorbol-induced chemiluminescence in the presence of either luminol or lucigenin. The results indicate that at least some of the components of the aqueous extract of cigarette smoke may strongly interfere with polymorphonuclear cells, contributing to the deleterious effects of smoke products.

Acridines↗

PubMedDate not supplied

Effect of homocysteine on polymorphonuclear leukocyte activity and luminol-dependent chemiluminescence.

Homocysteine is a non-protein-forming sulphur amino acid that plays an important role in remethylation and trans-sulphuration processes. In recent years, a high plasma homocysteine concentration has been implied as a possible pathophysiological factor in atherosclerosis and artery and deep vein thrombosis, probably through generation of H(2)O(2), enhanced platelet activity and increased production of macrophage-derived tissue factor. Furthermore, an increase of polymorphonuclear leukocyte (PMN) activity mediated by homocysteine-generated H(2)O(2) has also been reported. Because some preliminary experimental results in our laboratory did not confirm this effect of homocysteine on PMNs, we investigated the effect of homocysteine on the activity of PMNs, measured by their luminol-dependent chemiluminescence. Moreover, we also studied the effect of homocysteine in a luminol-hypochlorite chemiluminescent system. Our results clearly indicate that homocysteine at micromol/L concentrations (10-100 micromol/L) slightly inhibits neutrophil chemiluminescence, while it strongly inhibits the luminescence of the luminol-hypochlorite system. Therefore, the hypothesis that homocysteine induces an increase of H(2)O(2)-mediated neutrophil activity is not supported and, probably, the common opinion that views the H(2)O(2) generated by homocysteine as a possible mechanism for cardiovascular damage should be reconsidered.

Homocysteine↗

PubMedDate not supplied

Effect of aqueous cigarette smoke extract on the chemiluminescence kinetics of polymorphonuclear leukocytes and on their glycolytic and phagocytic activity.

Water-soluble extracts of cigarette smoke are easily formed in some body compartments, such as saliva or fluid lining alveolar spaces, and can act on both cellular and extracellular compartments. In this paper we have analysed the effect of aqueous smoke extract on some metabolic and functional aspects of polymorphonuclear leukocytes. In particular, the following cellular aspects were studied: chemiluminescence, glycolysis, membrane fluidity and microscopic interaction with zymosan particles. While chemiluminescence and glycolytic activity are highly inhibited, no effect of smoke extract on membrane fluidity was observed. Moreover, the response of luminol-dependent chemiluminescence was significantly delayed, while that of lucigenin-dependent chemiluminescence was anticipated. Furthermore, the phagocytic ability of neutrophils pretreated with aqueous smoke extract was also significantly hindered. All these results might indicate that the finely tuned activity of polymorphonuclear leukocytes is somehow hampered by the aqueous extract of cigarette smoke in a way which makes these cells less effective against bacteria and more noxious towards surrounding tissues.

2-Naphthylamine↗

PubMedDate not supplied

Effect of cigarette smoke extract on the polymorphonuclear leukocytes chemiluminescence: influence of a filter containing glutathione.

Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many compounds formed by cigarette burning, ranging from particulate materials to water solutes and gaseous extracts, are considered to be noxious agents, and many biochemical and molecular mechanisms have been proposed for the toxic effects of cigarette smoke. The oral cavity and the upper respiratory tract represent the first contact areas for smoke compounds; even a single cigarette can produce marked effects on some components of the oral cavity, either chemical compounds, such as glutathione and enzymes, or cellular elements, such as polymorphonuclear leukocytes. Several studies suggest a protective role of glutathione against the noxious effects of tobacco smoke; the sulphydril groups of glutathione, in fact, could react with some smoke products, such as unsaturated aldehydes, leading to the formation of harmless intermediate compounds and simultaneously preventing the inactivation of metabolically essential molecules, such as some enzymes. In this paper we analyse the effect of a filter containing glutathione on the respiratory burst of polymorphonuclear leukocytes exposed to aqueous extract of cigarette smoke, measuring their chemiluminescence activity. The results of this paper indicate that the GSH-containing filter has a likely protective effect against the inhibition of cigarette smoke extract on polymorphonuclear leukocyte activity.

Filtration↗

PubMedDate not supplied

Separation of reduced and oxidized glutathione by micellar electrokinetic capillary chromatography.

The separation of reduced and oxidized glutathione at an absolute sensitivity of about 100 pg by micellar electrokinetic capillary chromatography without derivatization is described. The time required for the separation is less than 10 min (the time between two following injections is about 15 min). The separation is characterized by high efficiency and good reliability. A partition mechanism is responsible for the high resolution observed. The method was utilized for the analysis of commercial preparations of glutathione and a good agreement with the expected results was obtained; the oxidation of the commercial glutathione in solution was easily analysed.

Electrophoresis↗

PubMedDate not supplied

Exogenous fructose-1,6-bisphosphate is a metabolizable substrate for the isolated normoxic rat heart.

Isolated rat hearts were perfused by the recirculating Langendorff mode under normoxic conditions for 60 min. The Krebs-Ringer buffer was supplemented with 10 mM glucose + 12 IU/l insulin and either [U-14C]-fructose-1,6-bisphosphate (together with 5 mM cold fructose-1,6-bisphosphate) or [U-14C]-fructose (together with 5 mM cold fructose). At the end of perfusion, gaseous 14CO2, 14CO2 trapped in the perfusates, 14C-lactate output and tissue 14C-lactate were assayed in both groups of hearts. Analysis of high-energy compounds, glycogen, lactate, and pyruvate was also performed on the neutralized perchloric acid extracts of the freeze-clamped hearts. Data obtained from the 14C catabolites, originating from the metabolism of the radiolabeled substrates, indicated that the isolated normoxic rat heart metabolizes an 8.5 times higher amount of fructose-1,6-bisphosphate (7.07 mumoles/min/g d.w.) than of fructose (0.83 mumoles/min/g d.w.). CrP, CrP/Cr, glycogen, and total lactate in both tissue and perfusate were significantly higher in fructose-1,6-bisphosphate-perfused hearts. The overall indication is that fructose-1,6-bisphosphate can be taken up in its intact form by myocytes and successively metabolized to support their energy demand, and that its effects on myocardial performance and metabolism should be attributed to the molecule itself rather than to its eventual degradation products.

Animals↗

PubMedDate not supplied

The relevance of malondialdehyde as a biochemical index of lipid peroxidation of postischemic tissues in the rat and human beings.

By using a recently developed ion-pairing high-performance liquid chromatographic method for the direct determination of malondialdehyde (MDA) and several other acid-soluble low-mol-wt compounds (ascorbate, oxypurines, nucleosides, nicotinic coenzymes, high-energy phosphates), the variations of tissue and plasma MDA as a function of ischemia and reperfusion were determined in the rat (isolated Langendorff-perfused hearts and short-term incomplete cerebral ischemia) and in human beings (patients suffering from acute myocardial infarction subjected to fibrinolysis). In the rat, the data obtained indicate that, contrary to what had been previously reported in literature, MDA is not present either in control heart or in control brain. Oxygen deprivation induces the production of a low, but detectable amount of MDA in both heart and brain, whereas reperfusion causes a marked increase of MDA in both tissues. In human beings, plasma MDA was deeply affected only in patients suffering from acute myocardial infarction with successful thrombolysis, thus indicating the occurrence of oxygen radical-mediated tissue injury also in humans. On the whole, these results suggest that MDA is a valid biochemical marker of lipid peroxidation of postischemic tissues, which however needs a reliable analytical technique for its determination.

Analysis of Variance↗

PubMedDate not supplied

Incomplete cerebral ischemia in the rat provokes increase of tissue and plasma malondialdehyde.

Short-term incomplete cerebral ischemia was induced in the rat by bilaterally clamping for 5 min the common carotid arteries; subsequent reperfusion of 10 min was obtained by removing carotid occlusion. At the end of ischemia or reperfusion, animals were sacrificed by decapitation. A control group was represented by sham-operated rats. Peripheral venous blood samples were withdrawn from the femoral vein from rats subjected to cerebral reperfusion 5 min before ischemia, at the end of ischemia, and 10 min after reperfusion. A highly sensitive HPLC method for the direct determination of malondialdehyde, oxypurines, and nucleosides was used on 200 microL of brain tissue and plasma extracts. Incomplete cerebral ischemia induced the appearance of a significant amount of tissue malondialdehyde (undetectable in control animals) and a decrease of ascorbic acid. A further 6.6-fold increase of malondialdehyde and a 18.5% decrease of ascorbic acid occurred after 10 min of reperfusion. Plasma malondialdehyde, which was present in minimal amount before ischemia, significantly increased after 5 min of ischemia, being strikingly augmented after 10 min of reperfusion. A similar trend was observed for oxypurines and nucleosides. From these data, it can be affirmed that tissue concentrations of malondialdehyde and ascorbic acid, and plasma levels of malondialdehyde, oxypurines, and nucleotides, reflect both the oxygen radical-mediated tissue injury and the depression of energy metabolism, thus representing early biochemical markers of short-term incomplete brain ischemia and reperfusion in the rat.

Animals↗

PubMedDate not supplied

A two-dimensional electrophoresis preliminary approach to human hepatocarcinoma differentiation induced by PPAR-agonists.

Adopting biochemical and proteomic approaches, we investigated the effect of some PPAR-agonists, a new class of differentiating agents, on human hepatocellular carcinoma Hep-G2 cell line. Cancer differentiation was assayed by checking albumin, transferrin and alpha-fetoprotein synthesis. Cell metabolism was studied by NMR spectroscopy of cell culture supernatants and by evaluation of mitochondrial respiratory chain enzyme activities. The two dimensional electrophoresis approach was employed to analyze modifications in the expression of cellular proteins linked to cell phenotype differentiation in the attempt to identify potential diagnostic and prognostic biomarkers of hepatocellular carcinoma. Results indicate that PPAR-agonists are able to act as differentiating inducers in human hepatocellular carcinoma Hep-G2 cell line as well as to inhibit mitochondrial respiratory chain Complex I, provoking a selective derangement of cellular oxidative metabolism. Lastly, two dimensional electrophoresis showed interesting modifications in the pattern of expression of cellular proteins that confirm biochemical data (increase in albumin and transferrin, decrease of alpha-fetoprotein synthesis) and, moreover, emphasize the meaning of these data by the increase of spots indicatively ascribed to HSP70 and catalase.

Bezafibrate↗

PubMedDate not supplied

[Studies on diffusion of HAA in a resuscitation center].

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Antibody Formation↗

PubMedDate not supplied

Increased levels of IGF-1 and beta2-microglobulin in epithelial lining fluid of preterm newborns developing chronic lung disease. effects of rhG-CSF.

UNLABELLED: Insulin-like growth factor-1 (IGF-1) is involved in regulating the Th-1/Th-2 balance, favoring the development of the Th-2 compartment which enhances fibrosis, one of the main characteristics of Chronic Lung Disease (CLD) in premature newborns. Limited data is available concerning a possible association between early epithelial lining fluid (ELF) concentrations of IGF-1 (total and free forms), IGF-binding protein-3 (IGFBP-3), beta2-microglobulin and subsequent development of CLD in preterm neonates. If neutropenic, preterm neonates are frequently treated with recombinant human granulocyte colony stimulating factor (rhG-CSF). The objective of the study was to correlate ELF concentrations of IGF-1 and beta2 microglobulin during the first week of life both in non-neutropenic and in rhGCSF-treated neutropenic preterm neonates, with subsequent development in CLD. Thirty preterm neonates with Respiratory Distress Syndrome (6 with neutropenia) were studied. Eleven out of 24 non-neutropenic preterm infants (46%) and all of the six neutropenic subjects (100%) developed CLD. With the exception of first day values, there was a clear similarity in the behaviors of assayed molecules between non-neutropenic and neutropenic patients developing CLD. Non-neutropenic patients without CLD showed significantly lower values of free IGF-1 and beta2M both on days 1 and 3. Total IGF-I and cell counts were different only on the 3rd day. CONCLUSIONS: 1) the mechanisms leading to CLD might be mediated by high levels of IGF-family molecules soon after birth 2) beta2M could be a marker of increased bronchoalveolar lavage fluid cellularity with potential inflammatory properties 3) G-CSF treatment induces an increased synthesis of IGF-1 molecules by cells recruited in the lung, with possible enhancement of the fibrogenic mechanisms.

Bronchoalveolar Lavage Fluid↗