Soluble Phospholipase A2 Research Articles

Distinguishing Dynamic Ca2+ Signals Originating from Different Entry Pathways in Microvascular Endothelial Cells

Background Pseudomonas aeruginosa ExoU infection causes formation of an infectious prion like tau proteinopathy in lung microvasculature, leading to respiratory endothelial cell injury. ExoU, a soluble phospholipase A2, disrupts the host cell membrane phospholipid chains, producing arachidonic acid (AA), and lysophosphatidic acid. A synthetic AA molecule has been shown to potently inhibit –cellular calcium channel transients through AA metabolites. Calcium transients have been linked to autophagy whereby the host endothelial cell entraps misfolded proteins. Although the mechanisms inherent to the cytotoxicity of ExoU infection are not clear, recent findings support an important role for AA metabolites. It is suspected that specific Ca2+ dynamics drive autophagy and inhibition of these transients by AA metabolites contributes to the pathologic signaling of Pseudomonas aeruginosa ExoU infection. The current project aims to evaluate whether our automated detection and analysis algorithm can effectively discern dynamic Ca2+ signals originating from distinct dynamic membrane ion channels. Method Microvascular endothelial cells (MVC's) were seeded and grown in 35mm glass-bottomed dishes. Cells were loaded with the fluorescent calcium indicator Cal-520 and assessed via confocal microscopy for calcium signals. Signals were recorded before and after treatment with either the 4α-PDD, which solicits Ca2+ entry through membrane TRPV4 channels, or the SERCA inhibitor thapsigargin (TG), which solicits Ca2+ entry through membrane store-operated Ca2+ channels. All image sequences were analyzed for frequency, duration, and amplitude of events using ImageJ and customized calcium signal detection software LC Pro. Data were expressed as mean ± SEM. Group data were subjected to Mann-Whitney U statistical method. Results We observed that TG treatments elicited Ca2+ signals with significant increase in mean amplitude of 7 ± 0.17 (F/F0) from mean amplitude of 4α-PDD treatment which was 5 ± 0.43 (F/F0) (p<0.05, n=3). The mean frequency of TG treatment was 1.46 ± 0.12 events/second, whereas mean frequency of 4α-PDD treatment was found to be 0.1 ± 0.04 events/second (p<0.05, n=3). Also, the mean event duration was 150 ± 17.56 seconds when subjected to TG as opposed to 4α-PDD treatment which elicited a mean event duration of 30 ± 7.12 seconds (p<0.05, n=3). Conclusion In these experiments, we were able to discern specific Ca2+ signaling profiles resulting from stimulation of two distinct Ca2+ entry pathways. Thus, application of automated dynamic signal tracking may provide a useful approach for distinguishing distinct Ca2+ entry mechanisms in future studies.

Mucosal expression of defensin-5, soluble phospholipase A2 and lysozyme in the intestine in a rat model of acute liver failure and its relationship to intestinal bacterial translocation

To study the expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in the intestine in a rat model of acute liver failure and its relationship with intestinal bacterial translocation (BT). Sprague–Dawley (SD) rats were divided into two groups. The experimental group was divided into five subgroups according to the lapsing time after the model was established, which were designated accordingly as 8 h, 16 h, 24 h, 48 h, and 72 h groups. Acute liver failure (ALF) model was induced by intraperitoneal injection of 10% d -galactosamine. The homogenates of mesenteric lymph nodes (MLNs), liver and spleen from each group were cultured in agar to determine the bacterial outgrowth. The mRNA expression of RD-5, sPLA2, lysozyme and the protein expression of sPLA2, lysozyme were determined. No bacteria grew in the organ cultures from the control group while experimental groups had positive cultures. Expression of the RD-5 and sPLA2 mRNA in the experimental groups gradually increased at early time points and peaked 16 h after induction of ALF, then progressively decreased. The mRNA expression of lysozyme in the experimental group peaked at 8 h after ALF induction, then progressively decreased. Similar results were obtained with Western blot and immunohistochemical staining. The immune barrier function of the ileal mucosa in the rat model of acute liver failure was compromised as demonstrated by the decreased expression of RD-5, sPLA2 and lysozyme in Paneth cells along with increased intestinal bacterial translocation. Estudiar la expresión de defensina-5 (RD-5), fosfolipasa A2 soluble (sPLA2) y lisozima en el intestino de un modelo de rata con insuficiencia hepática aguda y su relación con la traslocación bacteriana (TB) intestinal. Se dividieron ratas Sprague-Dawley® (SD) en 2 grupos. El grupo experimental se dividió en 5 subgrupos según el tiempo transcurrido desde que se estableció el modelo, y se designaron en consecuencia como grupos de 8, 16, 24, 48 y 72 h. El modelo de insuficiencia hepática aguda (IHA) se indujo mediante inyección intraperitoneal de D-galactosamina al 10%. Se cultivaron homogeneizados de ganglios linfáticos mesentéricos (GLM), hígado y bazo de cada grupo en agar para determinar la proliferación bacteriana. Se determinaron la expresión de ARNm de RD-5, sPLA2 y lisozima, y la expresión de proteínas de sPLA2 y lisozima. En los cultivos de órganos del grupo de control no creció ninguna bacteria, mientras que los grupos experimentales presentaron cultivos positivos. La expresión del ARNm de RD-5 y sPLA2 en los grupos experimentales aumentó gradualmente en los primeros momentos y alcanzó el máximo 16 h después de la inducción de la IHA, para después disminuir de forma progresiva. La expresión de lisozima en el grupo experimental alcanzó el valor máximo 8 h después de la inducción de la IHA y después disminuyó progresivamente. Se obtuvieron resultados similares con la inmunoelectrotransferencia y la tinción inmunohistoquímica. La función de barrera inmunológica de la mucosa ileal en el modelo de rata de insuficiencia hepática aguda se vio afectada, como lo demuestra la disminución de la expresión de RD-5, sPLA2 y lisozima en las células de Paneth junto con el aumento de la translocación bacteriana intestinal.

Mucosal expression of defensin-5, soluble phospholipase A2 and lysozyme in the intestine in a rat model of acute liver failure and its relationship to intestinal bacterial translocation

IntroductionTo study the expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in the intestine in a rat model of acute liver failure and its relationship with intestinal bacterial translocation (BT). Patients and methodsSprague–Dawley (SD) rats were divided into two groups. The experimental group was divided into five subgroups according to the lapsing time after the model was established, which were designated accordingly as 8h, 16h, 24h, 48h, and 72h groups. Acute liver failure (ALF) model was induced by intraperitoneal injection of 10% d-galactosamine. The homogenates of mesenteric lymph nodes (MLNs), liver and spleen from each group were cultured in agar to determine the bacterial outgrowth. The mRNA expression of RD-5, sPLA2, lysozyme and the protein expression of sPLA2, lysozyme were determined. ResultsNo bacteria grew in the organ cultures from the control group while experimental groups had positive cultures. Expression of the RD-5 and sPLA2 mRNA in the experimental groups gradually increased at early time points and peaked 16h after induction of ALF, then progressively decreased. The mRNA expression of lysozyme in the experimental group peaked at 8h after ALF induction, then progressively decreased. Similar results were obtained with Western blot and immunohistochemical staining. DiscussionThe immune barrier function of the ileal mucosa in the rat model of acute liver failure was compromised as demonstrated by the decreased expression of RD-5, sPLA2 and lysozyme in Paneth cells along with increased intestinal bacterial translocation.

Bee Venom Soluble Phospholipase A2 Exerts Neuroprotective Effects in a Lipopolysaccharide-Induced Mouse Model of Alzheimer's Disease via Inhibition of Nuclear Factor-Kappa B.

Neuroinflammation is important in the pathogenesis and development of Alzheimer’s disease (AD). In the AD brain, microglial activation and upregulation of pro-inflammatory mediators both induce amyloid beta (Aβ) accumulation. Regulatory T cells (Tregs) and nuclear factor-kappa B (NF-κB) signaling have been implicated in AD development through their effects on neuroinflammation and microglial activation. The bee venom soluble phospholipase A2 (bv-sPLA2) enzyme is known to exert anti-inflammatory and anti-immune effects. Here, we investigated the inhibitory effects of bv-sPLA2 on memory deficiency in a lipopolysaccharide (LPS)-induced mouse model of AD. We examined whether bv-sPLA2 (0.02, 0.2, and 2 mg/kg by i.p. injection three times for 1 week) could inhibit neuroinflammation and memory impairment in LPS-treated mice (250 μg/kg by i.p. injection daily for 1 week). We also assessed the effects of bv-sPLA2 administration (0.01, 0.1, and 1 μg/ml) on LPS (1 μg/ml)-treated microglial BV-2 cells. In the LPS-injected mouse brain, sPLA2 treatment rescued memory dysfunction and decreased Aβ levels, through the downregulation of amyloidogenic proteins, and decreased the expression of inflammatory proteins and pro-inflammatory cytokines. Moreover, the LPS-mediated increase in inflammatory protein expression was attenuated bv-sPLA2 treatment in BV-2 cells. Treatment with bv-sPLA2 also downregulated signaling by NF-κB, which is considered to be an important factor in the regulation of neuroinflammatory and amyloidogenic responses, both in vivo and in vitro. Additionally, co-treatment with NF-κB (5 μM) and bv-sPLA2 (0.1 μg/ml) exerted more marked anti-inflammatory effects, compared to bv-sPLA2 treatment alone. These results indicate that bv-sPLA2 inhibits LPS-induced neuroinflammation and amyloidogenesis via inhibition of NF-κB.

Derivation and Validation of a Biomarker-Based Clinical Algorithm to Rule Out Sepsis From Noninfectious Systemic Inflammatory Response Syndrome at Emergency Department Admission: A Multicenter Prospective Study.

To derive and validate a predictive algorithm integrating a nomogram-based prediction of the pretest probability of infection with a panel of serum biomarkers, which could robustly differentiate sepsis/septic shock from noninfectious systemic inflammatory response syndrome. Multicenter prospective study. At emergency department admission in five University hospitals. Nine-hundred forty-seven adults in inception cohort and 185 adults in validation cohort. None. A nomogram, including age, Sequential Organ Failure Assessment score, recent antimicrobial therapy, hyperthermia, leukocytosis, and high C-reactive protein values, was built in order to take data from 716 infected patients and 120 patients with noninfectious systemic inflammatory response syndrome to predict pretest probability of infection. Then, the best combination of procalcitonin, soluble phospholipase A2 group IIA, presepsin, soluble interleukin-2 receptor α, and soluble triggering receptor expressed on myeloid cell-1 was applied in order to categorize patients as "likely" or "unlikely" to be infected. The predictive algorithm required only procalcitonin backed up with soluble phospholipase A2 group IIA determined in 29% of the patients to rule out sepsis/septic shock with a negative predictive value of 93%. In a validation cohort of 158 patients, predictive algorithm reached 100% of negative predictive value requiring biomarker measurements in 18% of the population. We have developed and validated a high-performing, reproducible, and parsimonious algorithm to assist emergency department physicians in distinguishing sepsis/septic shock from noninfectious systemic inflammatory response syndrome.

Characterization of Phospholipase A2 Enzyme Activity in Serum of the Komodo Dragon (Varanus komodoensis)Soluble phospholipase A2 (sPLA2) is an enzyme found in the peripheral circulation of vertebrates which has significant immunological activity. This en

Soluble phospholipase A2 (sPLA2) is an enzyme found in the peripheral circulation of vertebrates which has significant immunological activity. This enzyme exerts immune activity by the hydrolysis of fatty acids from the sn-2 position of membrane glycerophospholipids of microbes, thus compromising membrane integrity and casing eventual lysis. We utilized membrane fatty acids labeled with a fluorescent probe (BODIPY) at the sn-2 position fatty acid to label the membranes. Incubation of different volumes of serum from Komodo dragons with BODIPY-labeled bacteria resulted in liberation of labeled fatty acid in a serum volume-dependent manner. This cleavage of fatty acid occurred rapidly, with a biphasic production of fluorescent product. An immediate accumulation of product was noted, which increased steadily for a 30-minute period, followed by a slower hydrolysis between 30 and 180 min. The activity was temperature-dependent, with low activities observed at 5oC and a linear increase up to 40oC. The liberation for fatty acid was inhibited by p-bromophenacyl bromide, a specific phospholipase A2 (PLA2) inhibitor, in a concentration-dependent manner, indicating that the activity was due to the presence of sPLA2.

Red wine intake but not other alcoholic beverages increases total antioxidant capacity and improves pro-inflammatory profile after an oral fat diet in healthy volunteers

IntroductionDifferent alcoholic beverages exert different effects on inflammation and oxidative stress but these results are controversial and scanty in some aspects. We analyze the effect of different alcoholic beverages after a fat-enriched diet on lipid profile, inflammatory factors and oxidative stress in healthy people in a controlled environment. MethodsWe have performed a cross-over design in five different weeks. Sixteen healthy volunteers have received the same oral fat-enriched diet (1486kcal/m2) and a daily total amount of 16g/m2 of alcohol, of different beverages (red wine, vodka, brandy or rum) and equivalent caloric intakes as sugar with water in the control group. We have measured the levels of serum lipids, high sensitivity C-reactive protein (hsCRP), tumor necrosis factor α (TNFα), interleukin 6 (IL-6), soluble phospholipase A2 (sPLA2), lipid peroxidation (LPO) and total antioxidant capacity (TAC). ResultsRed wine intake was associated with decreased of mean concentrations of hsCRP, TNFα and IL-6 induced by fat-enriched diet (p<0.05); nevertheless, sPLA2 concentrations were not significantly modified. After a fat-enriched diet added with red wine, TAC increased as compared to the same diet supplemented with rum, brandy, vodka or the control (water with sugar) (p<0.05). ConclusionsModerate red wine intake, but not other alcoholic beverages, decreased pro-inflammatory factors and increased total antioxidant capacity despite a fat-enriched diet intake in healthy young volunteers.

Red wine intake but not other alcoholic beverages increases total antioxidant capacity and improves pro-inflammatory profile after an oral fat diet in healthy volunteers

IntroductionDifferent alcoholic beverages exert different effects on inflammation and oxidative stress but these results are controversial and scanty in some aspects. We analyze the effect of different alcoholic beverages after a fat-enriched diet on lipid profile, inflammatory factors and oxidative stress in healthy people in a controlled environment. MethodsWe have performed a cross-over design in five different weeks. Sixteen healthy volunteers have received the same oral fat-enriched diet (1486kcal/m2) and a daily total amount of 16g/m2 of alcohol, of different beverages (red wine, vodka, brandy or rum) and equivalent caloric intakes as sugar with water in the control group. We have measured the levels of serum lipids, high sensitivity C-reactive protein (hsCRP), tumor necrosis factor α (TNFα), interleukin 6 (IL-6), soluble phospholipase A2 (sPLA2), lipid peroxidation (LPO) and total antioxidant capacity (TAC). ResultsRed wine intake was associated with decreased of mean concentrations of hsCRP, TNFα and IL-6 induced by fat-enriched diet (p<0.05); nevertheless, sPLA2 concentrations were not significantly modified. After a fat-enriched diet added with red wine, TAC increased as compared to the same diet supplemented with rum, brandy, vodka or the control (water with sugar) (p<0.05). ConclusionsModerate red wine intake, but not other alcoholic beverages, decreased pro-inflammatory factors and increased total antioxidant capacity despite a fat-enriched diet intake in healthy young volunteers.

Expression of intestinal defensin-5, soluble phospholipase A2 and lysozyme and the relation to bacterial translocation in rat models of acute liver failure

To study the intestinal expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in acute liver failure (ALF) using rat models, and to determine the relation of these expressions to intestinal bacterial translocation. Forty-eight healthy male Sprague-Dawley rats were divided into a control group (n=8) and a model group (n=40; intraperitoneal injection of 10% D-galactosamine). The model group was further divided into five subgroups according to the time lapse after model establishment (8, 16, 24, 48, and 72 hours). At the end of the experiments, homogenates of mesenteric lymph nodes, liver and spleen were cultured in agar for bacterial outgrowth.Hematoxylin-eosin stained sections of liver and terminal ileum were examined under an optical microscope to assess pathological changes. mRNA expression of RD-5, sPLA2 and lysozyme in the terminal ileum was determined by reverse transcription-polymerase reaction (RT-PCR), and protein expression of sPLA2 and lysozyme from the same anatomic location was determined by western blotting and immunohistochemistry. Means between groups were compared with one-way analysis of variance. ALF was successfully induced in the D-galactosamine injected rats. No bacteria grew in the organ cultures from the control group, while 8.3%, 37.5% and 58.3% of the rats in the 24-, 48-and 72-hour model groups showed positive cultures. Despite this, the structure of the terminal ileum from the rats in the 72-hour model group was nearly intact, without obvious necrosis of mucosal epithelial cells. Expression of RD-5 and sPLA2 mRNA in the model groups gradually increased at early time points and peaked at 16 hours after induction of ALF (1.291+/-0.153 and 1.131+/-0.128), which was significantly higher than that detected in the control group (0.725+/-0.116 and 0.722+/-0.112, t=69.25, 95.71, all P<0.01). After that, the expression of RD-5 and sPLA2 mRNA progressively decreased, and by 72 hours after the induction of ALF, the expression (0.415+/-0.104 and 0.425+/-0.076) was significantly lower than that of the control group (t=31.55 and 44.98, all P<0.01). Lysozyme mRNA expression in the model group peaked at 8 hours after ALF induction (1.211+/-0.107), which was higher than that of the control group at this time point (0.853+/-0.093), and by 72 hours after ALF induction it declined to 0.704+/-0.103, which was significantly lower than that of the control group (t=9.224; all P=0.009). In addition, at 72 hours after ALF induction the protein expression of both lysozyme and sPLA2 was significantly lower in the model group (0.327+/-0.086 and 0.382+/-0.057) than in the control group (0.583+/-0.121 and 0.650+/-0.093, t=12.28 and 15.83, P=0.004 and 0.001). Similar results were obtained with immunohistochemical staining. The function of the ileal mucosal immune barrier in the rat model of acute liver failure decreased, along with decreases in expression of RD-5, sPLA2 and lysozyme in the Paneth cells.At the same time, the rate of organ bacterial translocation increased without obvious injury to the intestinal mucosa structure.

Ultrastructural and histochemical study on the Paneth cells in the rat ascending colon.

Paneth cells (PCs) contribute to the host defense against indigenous bacteria in the small intestine. We found Paneth cell-like cells (PLCs) in the rat ascending colon, but the nature of PLCs is never clarified. Therefore, the present study aimed to clarify the cytological characteristics of PLCs and discuss their cellular differentiation. PLCs were localized in the bases of intestinal crypts, especially follicle-associated intestinal crypts in proximal colonic lymphoid tissue, but were very seldom found in the ordinary intestinal crypts of the ascending colon. PLCs possessed specific granules with highly electron-dense cores and haloes, as well as PCs in the small intestine. The secretory granules of PLCs were positive for PAS reaction, lysozyme and soluble phospholipase A2, but negative for Alcian blue staining, β-defensin-1 and -2, as well as the ones of PCs. Furthermore, intermediate cells possessing both the PLC-specific granules and the mucus granules similar to those of goblet cells (GCs) were occasionally found in the vicinity of PLCs. Intermediate cells ranged from goblet cell-like cells rich in mucus granules to PLC-like cells with few mucus granules. The cellular condensation and fragmentation were exclusively found in PLCs but never seen in intermediate cells or GCs. The PLCs, which were identified as PC, were suggested to be transformed from GCs through intermediate cells and finally to die by apoptosis in intestinal crypts of proximal colonic lymphoid tissue in the rat ascending colon.

Poster #S140 THE INFLUENCE OF AHI1 VARIANTS ON THE DIAGNOSIS AND TREATMENT OUTCOME IN SCHIZOPHRENIA

Changes in endothelium functions during ischemia are thought to be of importance in numerous pathological conditions, with, for instance, an increase in the release of inflammatory mediators like prostaglandins. Here, we showed that hypoxia increases phospholipase A2 (PLA2) activity in human umbilical vein endothelial cells. Both basal PLA2 activity and PG synthesis are sensitive to BEL and AACOCF3, respectively, inhibitors of calcium-independent PLA2 (iPLA2) and cytosolic PLA2 (cPLA2), while OPC, an inhibitor of soluble PLA2 (sPLA2) only inhibited the hypoxia-induced AA release and PGF2α synthesis. Hypoxia does not alter expression of iPLA2, sPLA2 and cPLA2 and cycloheximide did not inhibit PLA2 activation, indicating that hypoxia-induced increase in PLA2 activity is due to activation rather than induction. However, mRNA levels for sPLA2 displayed a 2-fold increase after 2 hr incubation under hypoxia. BAPTA, an intracellular calcium chelator, partially inhibited the AA release in normoxia and in hypoxia. Direct assays of specific PLA2 activity showed an increase in sPLA2 activity but not in cPLA2 activity after 2 hr hypoxia. Taken together, these results indicate that the hypoxia-induced increase in PLA2 activity is mostly due to the activation of sPLA2.

Peculiar Composition of Epithelial Cells in Follicle-Associated Intestinal Crypts of Peyer’s Patches in the Rat Small Intestine

ABSTRACTThe epithelial cell composition was investigated in the follicle-associated intestinal crypt (FAIC) of rat Peyer’s patches. The epithelium of the FAIC mainly consisted of columnar epithelial cells, goblet cells and Paneth cells. The characteristics of secretory granules in Paneth cells and goblet cells of both the FAIC and ordinary intestinal crypts (IC) were almost the same in periodic acid-Schiff (PAS) reaction, Alcian blue (AB) staining and the immunohistochemical detection of lysozymes and soluble phospholipase A2. Both goblet cells and Paneth cells were markedly less frequent on the follicular sides than on the anti-follicular sides of the FAIC. Goblet cells were also markedly less frequent in the follicle-associated epithelium (FAE) than in the ordinary intestinal villi (IV). Indigenous bacteria were more frequently adhered to FAE than to follicle-associated intestinal villi or IV. These findings suggest that the host defense against indigenous bacteria is inhibited on the follicular sides of FAIC, which might contribute to the preferential settlement of indigenous bacteria on the FAE; they also suggest that differentiation into secretory cells is inhibited in the epithelium of the follicular sides of FAIC, so that differentiation into M cells might be admitted in the FAE of rat Peyer’s patches. Furthermore, intermediate cells possessing characteristics of both Paneth cells and goblet cells were rarely found in the FAIC, but not in the IC. This finding suggests that the manner of differentiation into Paneth cells in the FAIC differs from that in the IC.

1,25(OH)2D3 suppresses COX-2 up-regulation and thromboxane production in placental trophoblast cells in response to hypoxic stimulation

In this study, we determined if vitamin D could inhibit oxidative stress-induced thromboxane production by placental trophoblasts. Trophoblast isolated from normal placentas were stimulated with CoCl2, a hypoxic mimicking agent, with or without pretreatment of 1,25(OH)2D3. Soluble phospholipase-A2, metabolites of thromboxane-A2 and prostacyclin, and 8-isoprostane were measured. Expression of cyclooxygenase-1 (COX-1), COX-2, and heme oxygenase-1 (HO-1) were determined. We found that pretreatment of trophoblasts with 1,25(OH)2D3 significantly reduced 8-isoprostane and the ratio of thromboxane-A2 to prostacyclin production, and blocked COX-2 expression induced by CoCl2. These results provide evidence of the beneficial effects of vitamin D on placental trophoblasts.

Glomérulopathies extramembraneuses idiopathiques et secondaires

Antibodies to neutral endopeptidase, a podocyte protein, are responsible for rare alloimmune neonatal membranous nephropathy that develops in children from neutral endopeptidase-deficient mothers. Neutral endopeptidase was the first podocyte antigen described in human membranous nephropathy. PLA2R1, the type-M receptor of soluble phospholipase A2, is a major target antigen in so-called idiopathic membranous nephropathy in adults. Antibodies to PLA2R1 are detected in 60 to 80% of patients before immunosuppressive treatment, and are only occasionally found in secondary membranous nephropathy. To date, they have not been detected in other pathological conditions and in healthy individuals. PLA2R1 and HLA-DQA1 gene variants defined by single nucleotide polymorphisms are strongly associated with idiopathic membranous nephropathy in patients of white ancestry, and can thus be considered as predisposing genes. In addition to their diagnostic value, anti-PLA2R1 antibodies can be used to monitor treatment. Immunization against cationic bovine serum albumin is a cause of early childhood membranous nephropathy. This finding points to a possible role of food and environmental antigens in membranous nephropathy. The newly identified antigen-antibody systems should be considered as molecular signatures challenging the uniform histological definition and having a major impact on patient care in a near future.

Accumulation of bioactive lipids during storage of blood products is not cell but plasma derived and temperature dependent

Bioactive lipids (lysophosphatidylcholines [lysoPCs]) accumulating during storage of cell-containing blood products are thought to be causative in onset of transfusion-related acute lung injury through activation of neutrophils. LysoPCs are thought to be derived from cell membrane degradation products such as phosphatidylcholines (PC) by partial hydrolysis of PC, a process that is catalyzed by phospholipase A(2) (PLA(2) ). We investigated the underlying mechanisms of lysoPC generation and its contribution to in vitro neutrophil-priming capacity during storage of red blood cells (RBCs), platelet (PLTs) concentrates, and cell-free plasma. Blood from healthy volunteers was drawn, processed, and stored according to Sanquin Blood Bank protocols. Storage of RBCs in saline-adenine-glucose-mannitol (SAGM) did not result in accumulation of lysoPCs or neutrophil-priming capacity. Replacement of SAGM by plasma as RBC storage medium caused elevated lysoPC levels on Day 0, which did not further increase during storage. Cell-free plasma stored at 22°C showed accumulation of lysoPCs during storage, which was not present at 4°C. Addition of a soluble PLA(2) or cytosolic PLA(2) inhibitor did not prevent accumulation of lysoPCs in plasma. In PLTs, lysoPC accumulation during storage was plasma dependent, but lysoPCs did not explain the observed neutrophil-priming effect as preventing accumulation of lysoPCs by removing the plasma fraction did not prevent the neutrophil-priming capacity. Accumulation of lysoPCs during storage is not cell but plasma derived and storage temperature dependent and does not explain the neutrophil-priming effect of aged products.

A lytic mechanism based on soluble phospholypases A2 (sPLA2) and β-galactoside specific lectins is exerted by Ciona intestinalis (ascidian) unilocular refractile hemocytes against K562 cell line and mammalian erythrocytes

Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca 2+-dependent cytotoxic activity toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations were separated (B1–B6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In addition the separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture. Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysins can be released into a culture medium. The B5 activity was blocked by d-Galactose, α-Lactose, Lactulose, LacNAc, thiodigalactoside (TDG), l-Fucose, d-Mannose, d-Glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca 2+-dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2 could be responsible for changes and large alterations of the target cell membrane. An apoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.

Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-alpha, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.

Secretory phospholipase A2 type IIA: a regulator of immune function in atherosclerosis?

Soluble phospholipase A2 type IIA (sPLA2-IIA) is an acute-phase reactant that is markedly increased in inflammatory disorders including cardiovascular disease. Inflammatory cytokines such as interleukins IL-6 and IL-1β, interferon (IFN)-γ, and tumour necrosis factor (TNF)-α increase its expression in vascular smooth muscle cells and hepatocytes, two cell types largely responsible for its elevations in plasma. sPLA2-IIA is also highly expressed in atherosclerotic lesions and associated with smooth muscle cells, macrophages, and glycoaminoglycans,1 where it exerts proatherogenic effects in part by modifying low-and high-density lipoproteins (LDL and HDL). It hydrolyses the sn -2 ester bond in the glyceroacyl phospholipids present in both LDL and HDL and cell membranes, releasing fatty acids and lysophospholipids. sPLA2-IIA can also exert effects that are independent of its hydrolytic functions. It elevates IL-6 production by macrophages2 and contributes to elevations in cyclooxygenase-2 expression by mast cells:3 two cell types with significant roles in atherosclerosis. Thus, sPLA2-IIA is thought to promote atherosclerosis by hydrolysing lipoproteins and regulating macrophage and mast cell responses via non-hydrolytic mechanisms. In this issue of Cardiovascular Research , Ibeas et al. 4 provide additional new and somewhat unexpected insights as to how … *Corresponding author. Tel: +61 3 8532 1190; fax: 61 3 8532 1100. E-mail address : alex.bobik{at}bakeridi.edu.au

Abstract 4998: Elevated Oxidized Phospholipids on Apolipoprotein B-100 Particles Predict 6-Year Cardiovascular Events in the Epic-Norfolk Study: Potentiation of Risk with Lipoprotein-Associated (Lp-PLA 2 ) and Soluble Phospholipase A2 (sPLA 2 ) Activity

Objective: We aimed to assess whether oxidized phospholipids on apoB-100 particles (OxPL/apoB) predict future coronary heart disease (CHD) in the prospective EPIC-Norfolk cohort of apparently healthy men and women aged 45–79 years followed up 1993–2007. Methods and Results: Cases who developed CHD during follow-up were matched to controls who remained free of cardiovascular disease and who matched to cases by sex, age and enrollment time. OxPL/apoB levels were measured with antibody E06 in 905 cases and 1809 controls. Subjects in the highest quartile of OxPL/apoB at entry in the study had significantly higher risk of future CHD events (OR 1.72, 95%CI 1.32–2.24, p&lt;0.0001) compared to those in the lowest quartile. This relationship was present in both men and women and was also observed after adjustment for sex, age, diabetes, smoking, blood pressure, LDL-C, HDL-C and Framingham Risk Score (FRS). The risk of future CHD events was significantly potentiated by elevated activity levels of sPLA 2 and Lp-PLA 2 . Subjects in the highest tertiles of both OxPL/apoB and LpPLA2 (OR 2.22 [1.51–3.27]) or OxPL/apoB and sPLA 2 (OR 4.34 [2.84 – 6.64]) had significantly higher risk of future CHD than subjects in the lowest tertiles (p&lt;0.0001 for both). Area under receiver operating curves revealed significantly increased values by adding OxPL/apoB and sPLA 2 to traditional risk factors and the FRS. Strong inverse associations were noted between the OxPL/apoB and VLDL and IDL size, CETP mass and RANK Ligand. Conclusion: Elevated OxPL/apoB levels are a strong, independent predictor of CHD events. The predictive values is further enhanced by elevated activity levels of sPLA 2 and Lp-PLA 2 , which mediate breakdown of OxPL present on lipoproteins and within atherosclerotic lesions. This study links several pathophysiologically related oxidative biomarkers in the prediction of CHD events and suggests novel approaches to predicting CHD risk in apparently health men and women.

Uteroglobin: A Steroid-Inducible Immunomodulatory Protein That Founded the Secretoglobin Superfamily

Blastokinin or uteroglobin (UG) is a steroid-inducible, evolutionarily conserved, secreted protein that has been extensively studied from the standpoint of its structure and molecular biology. However, the physiological function(s) of UG still remains elusive. Isolated from the uterus of rabbits during early pregnancy, UG is the founding member of a growing superfamily of proteins called Secretoglobin (Scgb). Numerous studies demonstrated that UG is a multifunctional protein with antiinflammatory/ immunomodulatory properties. It inhibits soluble phospholipase A(2) activity and binds and perhaps sequesters hydrophobic ligands such as progesterone, retinols, polychlorinated biphenyls, phospholipids, and prostaglandins. In addition to its antiinflammatory activities, UG manifests antichemotactic, antiallergic, antitumorigenic, and embryonic growth-stimulatory activities. The tissue-specific expression of the UG gene is regulated by several steroid hormones, although a nonsteroid hormone, prolactin, further augments its expression in the uterus. The mucosal epithelia of virtually all organs that communicate with the external environment express UG, and it is present in the blood, urine, and other body fluids. Although the physiological functions of this protein are still under investigation, a single nucleotide polymorphism in the UG gene appears to be associated with several inflammatory/autoimmune diseases. Investigations with UG-knockout mice revealed that the absence of this protein leads to phenotypes that suggest its critical homeostatic role(s) against oxidative damage, inflammation, autoimmunity, and cancer. Recent studies on UG-binding proteins (receptors) provide further insight into the multifunctional nature of this protein. Based on its antiinflammatory and antiallergic properties, UG is a potential drug target.