Luis Vila

Human Apolipoprotein A-II Enrichment Displaces Paraoxonase From HDL and Impairs Its Antioxidant Properties: A New Mechanism Linking HDL Protein Composition and Antiatherogenic Potential

Apolipoprotein A-II (apoA-II), the second major high-density lipoprotein (HDL) apolipoprotein, has been linked to familial combined hyperlipidemia. Human apoA-II transgenic mice constitute an animal model for this proatherogenic disease. We studied the ability of human apoA-II transgenic mice HDL to protect against oxidative modification of apoB-containing lipoproteins. When challenged with an atherogenic diet, antigens related to low-density lipoprotein (LDL) oxidation were markedly increased in the aorta of 11.1 transgenic mice (high human apoA-II expressor). HDL from control mice and 11.1 transgenic mice were coincubated with autologous very LDL (VLDL) or LDL, or with human LDL under oxidative conditions. The degree of oxidative modification of apoB lipoproteins was then evaluated by measuring relative electrophoretic mobility, dichlorofluorescein fluorescence, 9- and 13-hydroxyoctadecadienoic acid content, and conjugated diene kinetics. In all these different approaches, and in contrast to control mice, HDL from 11.1 transgenic mice failed to protect LDL from oxidative modification. A decreased content of apoA-I, paraoxonase (PON1), and platelet-activated factor acetyl-hydrolase activities was found in HDL of 11.1 transgenic mice. Liver gene expression of these HDL-associated proteins did not differ from that of control mice. In contrast, incubation of isolated human apoA-II with control mouse plasma at 37°C decreased PON1 activity and displaced the enzyme from HDL. Thus, overexpression of human apoA-II in mice impairs the ability of HDL to protect apoB-containing lipoproteins from oxidation. Further, the displacement of PON1 by apoA-II could explain in part why PON1 is mostly found in HDL particles with apoA-I and without apoA-II, as well as the poor antiatherogenic properties of apoA-II–rich HDL.

Immediate peritoneal response to bacterial contamination during laparoscopic surgery

BackgroundSeveral studies have shown that laparoscopic surgery (LS) minimizes surgical trauma and the immune function is better preserved. Another major advantage of LS is the lower incidence of septic complications. However, several in vitro studies have shown that CO2 severely impairs macrophage physiology. In theory, this would reduce the ability to respond to peritoneal contamination. However, there is some controversy in view of the evidence of a better preserved peritoneal response to sepsis. This study analyzed the early response of the peritoneum to contamination in a CO2 ambience.MethodsA total of 192 CD-1 mice were distributed in three groups: group 1, laparotomy (LAP, n = 64); group 2, CO2 laparoscopy (CO2-LC, n = 64); and group 3, wall lift laparoscopy (WL-LC, n = 64). Mice in each group were randomized to receive 1 ml of Escherichia coli suspension (1 × 104 colony-forming units/ml) or saline. Peritoneal fluid was obtained at 1.5, 3, 6, and 12 h after surgery. Monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) were measured.ResultsMCP-1 levels were significantly greater and higher earlier in group 2 (CO2-LC) than in group 1 (LAP) (p < 0.007). Simultaneously, the increment in the traction group (WL-LC, group 3) was significantly higher (p < 0.002) than after laparotomy, with no differences in group 2 (CO2-LC). When a contamination was added to the laparotomy subgroup, there was a significant increase compared to the group without contamination (p < 0.5). MCP-1 modifications after contamination in the LAP group were statistically significant and appeared later than in the WL-LC (p < 0.002) and CO2-LC groups (p < 0.02). For IL-6, the three models presented a significant increase in the noncontaminated groups. This occurred significantly later in the LAP group. Simultaneously, the increase in IL-6 occurred earlier and was significantly higher in the WL-LC group compared to the LAP group (p < 0.003), without differences between CO2-LC and wall lift groups. Significant differences between contaminated and noncontaminated subgroups were only observed in the LC-CO2 groups. When contaminated, the traction model sustained a higher and earlier rise in IL-6 levels compared to the LAP and LC-CO2 groups (p < 0.001). For PGE2, The three models showed a significant increase in PGE2 levels in the noncontaminated groups. However, there were no significant differences between them. In the contaminated groups, there was no statistical difference between the groups.ConclusionDespite a transient impairment of the immediate peritoneal response to a septic challenge, the degree of injury with LS is lower than that with open surgery, and abdominal infection can therefore be better controlled.

Metabolism of exogenous arachidonic acid by polymorphonuclear leukocytes from psoriatic patients

SummaryWe evaluated the metabolism of exogenous arachidonic acid in polymorphonuclear leukocytes from psoriatic patients in comparison with those from normal subjects. The leukocytes were incubated with 10 ΜM labelled arachidonic acid and 5 ΜM A23187 for varying periods of time. We evaluated all the compounds derived from the 5-lipoxygenase activity, including the products of non-enzymatic transformation of leukotriene A4 and w-oxidized leukotriene B4. In the experimental conditions used, there was no significant difference in the formation of any compound at any time of incubation. These results indicate that there is no intrinsic alteration either in 5-lipoxygenase activity or in w-oxidation ability of circulating polymorphonuclear leukocytes in psoriatic patients.

Prostacyclin‐synthase expression in head and neck carcinoma patients and its prognostic value in the response to radiotherapy

Prostacyclin (PGI2) plays a role in cancer progression but the mechanism is currently poorly understood. Additionally, no data are available about the prognostic value of the PGI2 pathway in head and neck squamous cell carcinoma (HNSCC) therapy. We evaluated the expression of the PGI2 pathway in HNSCC patients. PGI2 production and PGI synthase (PGIS) expression, in terms of mRNA (RT‐PCR) and protein (immunoblotting), were lower in tumour samples than in non‐tumoural mucosa, whereas, as expected, COX‐2 expression was increased in HNSCC tumour samples. Using local control of the tumour after radiotherapy or chemoradiotherapy as a dependent variable, patients were classified into two categories of PGIS transcript levels. The high‐PGIS group had a significantly lower frequency of local and distant failure than the low‐PGIS group, and the 5‐year cancer‐specific survival was higher [90.2% (95% CI 81.0–99.4%) versus 60.5% (95% CI 44.4–76.6%)]. None of the four HNSCC cell lines analysed expressed PGIS and therefore they did not produce PGI2. However, HNSCC‐conditioned media enhanced PGI2 production in endothelial cells (ECs). The stable analogue of PGI2, carbaprostacyclin (cPGI2), exerted little effect on HNSCC cell line migration, and no effect on cell cycle distribution or proliferation rate after radiation injury was observed. Nevertheless, cPGI2 promoted EP‐4‐dependent in vitro angiogenesis. Von Willebrand factor expression (EC marker) and capillary density were significantly higher in the group of patients with high expression of PGIS. Our results indicate that PGIS expression was associated with radiotherapy efficiency. Although we do not provide direct evidence of a relationship between tumour vascularization and radiotherapy efficiency, our results suggest that the effect of PGI2 is related to its ability to promote vascularization. These results also support the concept that co‐adjuvant therapy with PGIS enhancers, such as retinoids, could have therapeutic value for HNSCC treatment. Copyright

Morphological Aspects Sodium lauryl sulfate stimulates the metabolism of endogenous arachidonic acid in epidermal cell suspensions

In guinea pigs, the elicitation of contact sensitization is conventionally measured by visual assessment of eruthematous reactions at the site of challenge; a method that is subjective and which may be difficult to interpret when coloured chemicals are used. Neither is the preferred method in mice: the evaluation of challenge-induced increases in ear thickness, without limit. We have therefore examined whether there exist opportunities to use serological markers of acute inflammation to assess the elicitation reaction in contact sensitivity. We have previously reported that challenge-induced increases in ear thickness of contact sensitized mice correlate closely with elevations in the serum concentration of2 phase proteins (APP); haptoglobin and serum amyloid A (!). As changes in the hepatic synthesis and serum concentration of APP are largely a consequence of the action ofinterleukin 6 (IL-6), we have also examined whether elicitation reactions in mice are associated with increased concentrations of this cytokine. The data reveal that there is in fact a strong correlation between plasma IL-6 and challenge-induced increases in ear thickness. Furthermore, we have observed that elicitation of contact sensitization is also associated with a significant elevation in serum histamine concentration. It is our belief that measurement of mediators of acute inflammation such as IL-6 and histamine may provide the basis of an alternative strategy for the objective evaluation of contact allergic reactions.

Exploring correlation between bone metabolism markers and densitometric traits in extended families from Spain

Osteoporosis is a common multifactorial disorder characterized by low bone mass and reduced bone strength that may cause fragility fractures. In recent years, there have been substantial advancements in the biochemical monitoring of bone metabolism through the measurement of bone turnover markers. Currently, good knowledge of the genetics of such markers has become an indispensable part of osteoporosis research. In this study, we used the Genetic Analysis of Osteoporosis Project to study the genetics of the plasma levels of 12 markers related to bone metabolism and osteoporosis. Plasma phenotypes were determined through biochemical assays and log-transformed values were used together with a set of covariates to model genetic and environmental contributions to phenotypic variation, thus estimating the heritability of each trait. In addition, we studied correlations between the 12 markers and a wide variety of previously described densitometric traits. All of the 12 bone metabolism markers showed significant heritability, ranging from 0.194 for osteocalcin to 0.516 for sclerostin after correcting for covariate effects. Strong genetic correlations were observed between osteocalcin and several bone mineral densitometric traits, a finding with potentially useful diagnostic applications. In addition, suggestive genetic correlations with densitometric traits were observed for leptin and sclerostin. Overall, the few strong and several suggestive genetic correlations point out the existence of a complex underlying genetic architecture for bone metabolism plasma phenotypes and provide a strong motivation for pursuing novel whole-genome gene-mapping strategies.

Effect of lonapalene on metabolism of exogenous arachidonic acid in human platelets.

Lonapalene was studied as a topical antipsoriatic agent on the basis of its inhibitory effect on 5-lipoxygenase. We studied the effect of lonapalene on the metabolism of exogenous arachidonic acid in washed platelet suspensions by RP-HPLC. Lonapalene was shown to inhibit platelet cyclooxygenase, with substrate diversion towards 12-HETE production. The pattern of inhibition was similar to that of indometacin, although lonapalene was about 1,000 times weaker. The dual inhibition of lonapalene and concomitant deviation of arachidonic acid metabolism may have potential implications, with regard to therapeutic indications and side effects, which deserve further study.

Keratinocytes as a cellular source of inflammatory eicosanoids

The term ‘eicosanoid’ was introduced by Corey et al. [1] and comprises a large and complex family of compounds derived from 20-carbon polyunsaturated fatty acids, among which arachidonic acid is the most biologically relevant. This group of substances includes prostaglandins, thromboxanes, leukotrienes, hydroperoxy- and hydroxyeicosatetraenoic acids (HPETEs and HETEs), hepoxilins, lipoxins, triox-ilins, nonleukotriene dihydroxyeicosatetraenoic acids (DiHETEs) and isoprostanes.

Activation of 5-Lipoxygenase in Whole Polymorphonuclear Leukocytes by Arachidonic Acid: Evidence of Cytosolic Active Enzyme

The initial enzymatic step in the biosynthesis of leukotrienes is the oxidation of arachidonic acid (AA) by means of 5-lipoxygenase (5-LO). Mammalian 5-LO has been shown to require ATP and Ca2+ for activity in enzyme preparations (1,2). Kinetic studies using 5-LO preparations from guinea pig polymorphonuclears PMN, suggested a substrate inhibition and also a product inactivation of the enzyme (3,4). In intact cells, 5-LO needs activation via an increase in cytosolic Ca2+ levels (5). The challenge with calcium ionophore A23187 of a variety of granulocytic cells results in translocation of 5-LO from the cytosol to the membrane compartment. A membrane-bound 18-kDa protein has been postulated as a receptor for 5-LO, this protein has been termed five-lipoxygenase activating protein (FLAP) (6–8). The role of FLAP as an essential activator of 5-LO is supported by three pieces of evidence: a) blockage of FLAP suppresses leukotriene formation in A23187 challenged cells (7), b) the expression of both 5-LO and FLAP is needed for leukotriene formation in response to A23187 (6), and c) when differentiation of HL-60 cells towards granulocytes is induced, both 5-LO and FLAP are expressed (8). Nevertheless, little is known about the mechanism of activation of 5-LO in response to physiological agonists. At the inflamed tissues, infiltrating PMN are closely surrounded by other cell types which can supply free AA to PMN to be subsequently transformed into leukotrienes. Actually, elevated levels of free AA have been detected in inflammatory lesions (9). AA is not only the substrate for eicosanoids biosynthesis but it is also a second messenger (10). In fact, AA mobilizes Ca2+ towards the cytosol in PMN through an apparently complex mechanism, and activation of 5-LO by AA has been reported (11,12).