W. Farrugia

Type II phospholipase A2 in human gestational tissues: Subcellular distribution of placental immuno and catalytic activity

The aims of this study were to determine the subcellular distribution of Type II phospholipase A2 immunoactivity (irPLA2) and in vitro net PLA2 catalytic activity in human term placenta and to establish the efficacy of previously utilised homogenisation procedures with respect to the quantitative recovery of Type II PLA2 immunoreactive and in vitro net PLA2 catalytic activity. Type II PLA2 immunoactivity and PLA2 catalytic activity recovered in 900 x g supernates prepared from placental tissue (n = 3) homogenised in low ionic strength media (sucrose 0.32 M Hepes 20 mM; phosphate-buffered saline or phosphate-buffered saline containing 3 mM EGTA) was less than 10% of that recovered following homogenisation in high ionic strength medium (ammonium sulphate 10%, w/v). The subcellular distribution of Type II PLA2 immunoactivity and PLA2 catalytic activity was established by the differential centrifugation (10,000, 20,000 and 100,000 x g) of placental homogenates (n = 3). Although Type II PLA2 immunoactivity was equally distributed throughout the particulate subcellular fractions examined, PLA2 catalytic activity increased by comparison in 100,000 x g particulate material. This apparent dissociation between irType II PLA2 and catalytic activity may indicate the presence of other types of PLA2 in this fraction. The data obtained in this study indicate that previous studies which have utilised low ionic strength extractions of human gestational tissue to characterise PLA2 catalytic activity and subcellular distribution have largely excluded the contribution made by Type II PLA2. Consequently, much of the available published data on the role of PLA2 in human parturition is inadequate. A reappraisal of this enzymes contribution to the biochemical events associated with human pregnancy and labour is required.

Type II phospholipase A2 in human gestational tissues: extractable immuno- and enzymatic activity in fetal membranes

In this study, we have established the presence of immunoreactive (ir) Type II PLA2 in human amnion and choriodecidua obtained from women at term prior to the onset of labour. The content of irType II PLA2 present in 1 M NaCl extracts of choriodecidua and amnion averaged 3.5 +/- 3.1 and 10.6 +/- 5.2 ng/mg tissue protein (n = 3), respectively. PLA2 enzymatic activity present in the same tissues averaged 1.3 +/- 0.2 and 1.9 +/- 0.7 nmol phosphatidylethanolamine (PE) hydrolysed/mg tissue protein per h (n = 3), respectively. To allow intra-patient comparison of the relative distribution in gestational tissues, irType II PLA2 and PLA2 enzymatic activity was also determined in placenta obtained from the same group of women, and averaged 26.0 +/- 7.0 ng/mg tissue protein and 3.5 +/- 1.0 nmol PE hydrolysed/mg protein per h (n = 3), respectively. As has been previously reported for human placenta, the recovery of Type II PLA2 and PLA2 enzymatic activity from amnion and choriodecidua was increased between 16- and 25-fold when tissues were homogenized in high-ionic strength media (i.e., 10% (w/v) ammonium sulphate or 1 M NaCl) compared with that recovered when tissues were homogenized in low-ionic strength media (i.e., 0.32 M sucrose-20 mM Hepes). The data obtained represent the first quantitative estimates of immunoreactive Type II PLA2 in human amnion and choriodecidua, and support the conclusion that previous analyses of the PLA2 enzymatic activity present in gestational tissues have essentially excluded the contribution made by this PLA2 isozyme to net enzymatic activity. We suggest that this isozyme represents a major component of the PLA2 enzymatic activity present in human gestational tissues at term and that it contributes significantly to the phospholipid metabolism and arachidonic acid release which occurs during late pregnancy and at the time of labour.

Tumor necrosis factor-β in human pregnancy and labor

Abstract The aims of this study were to determine tumor necrosis factor-β (TNF-β) concentration profiles in peripheral venous plasma and amniotic fluid during pregnancy and at the time of labor and to characterise TNF-β mRNA expression and TNF-β release from human gestational tissues. In addition, we investigated the expression of TNF-β binding protein, lymphotoxin-β (LT-β), in human gestational tissues. The mean (±S.E.M.) TNF-β concentrations in maternal plasma (TIL, 78 ± 12 pg/ml, n = 7 vs. TNIL, 304 ± 88 pg/ml, n = 7) and amniotic fluid (TIL, 8 ± 5 pg/ml, n = 6 vs. TNIL, 73 ± 20 pg/ml, n = 20) were significantly ( P

The effect of monophosphoryl lipid A on lipopolysaccharide-induced prostaglandin E2 release in human choriodecidua

The aim of this study was to determine the effect of an inhibitor of bacterial endotoxin, monophosphoryl lipid A (MLA), on lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) formation by human choriodecidua explants incubated in vitro. LPS induced the release of PGE2 from explants in a time-and dose-dependent manner (P < 0.05, n = 5), thus establishing the efficacy of the experimental model. MLA at concentrations of 10 micrograms/ml also increased PGE2 release from explants when compared to vehicle controls (P < 0.05, n = 5). When used at a concentration that did not stimulate PGE2 release (1 microgram/ml), MLA pretreatment, coincubation or a combination of these protocols did not significantly affect LPS-induced PGE2 release. These data establish that MLA does not act by abrogating tissue LPS responsiveness. Under the conditions utilized in this study, MLA acts locally as a low potency LPS-like agent. The previously reported in vivo efficacy of systemically administered MLA may involve the partial depletion or down regulation of LPS response pathways and the subsequent development of LPS tolerance.