Infection of Galleria mellonella (Lepidoptera) Larvae With the Entomopathogenic Fungus Conidiobolus coronatus (Entomophthorales) Induces Apoptosis of Hemocytes and Affects the Concentration of Eicosanoids in the Hemolymph

Apoptosis and autophagy, the mechanisms of programmed cell death, play critical roles in physiological and pathological processes in both vertebrates and invertebrates. Apoptosis is also known to play an important role in the immune response, particularly in the context of entomopathogenic infection. Of the factors influencing the apoptotic process during infection, two of the lesser known groups are caspases and eicosanoids. The aim of this study was to determine whether infection by the entomopathogenic soil fungus Conidiobolus coronatus is associated with apoptosis and changes in caspase activity in the hemocytes of Galleria mellonella larvae, and to confirm whether fungal infection may affect eicosanoid levels in the host. Larvae were exposed for 24 h to fully grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24 group) or 24 h later (F48 group). Apoptosis/necrosis tests were performed in hemocytes using fluorescence microscopy and flow cytometry, while ELISA tests were used to measure eicosanoid levels. Apoptosis and necrosis occurred to the same degree in F24, but necrosis predominated in F48. Fungal infection resulted in caspase activation, increased PGE1, PGE2, PGA1, PGF2α, and 8-iso-PGF2α levels and decreased TXB2 levels, but had no effect on TXA2 or 11-dehydro-TXB2 concentrations. In addition, infected larvae demonstrated significantly increased PLA2 activity, known to be involved in eicosanoid biosynthesis. Our findings indicate that fungal infection simultaneously induces apoptosis in insects and stimulates general caspase activity, and this may be correlated with changes in the concentrations of eicosanoids.

Comparison of Lp-PLA2 Activity, Lp-PLA2 Mass and Lp-PLA2 mRNA in Acute Coronary Syndrome Patients

Background: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk predictor for cardiovascular diseases (CVD). Generally, plasma Lp-PLA2 was thought to be secreted by circulatory inflammatory cells. Lp-PLA2 mRNA expression of PBMC may also be a risk predictor. Methods: A total of 104 subjects angiographically verified ACS patients were enrolled, including 73 unstable angina pectoris (UAP) patients and 31 acute myocardial infarction (AMI) patients. Plasma lipids, Lp-PLA2 activity and Lp-PLA2 mass were measured. Lp-PLA2 mRNA expression of PBMC was relatively quantified by real-time fluorescence PCR. Results: Plasma Lp-PLA2 activity was increased in AMI patients compared to UAP patients (395.21±145.91 vs. 328.53±127.03 U/L, p=0.024). Lp-PLA2 mass of AMI patients was also higher than UAP patients (136.43±45.46 vs. 119.16±44.19 ng/mL, p=0.093), while PBMC mRNA expression was not statistically different [1.07 (0.74, 1.57) vs. 0.88(0.49, 1.99), p=0.453]. Comparing Lp-PLA2 mRNA by groups, Lp-PLA2 mRNA level was higher in male ACS patients and smoking ACS patients (p=0.008, p=0.048, respectively). Multivariate logistic regression analysis showed that Lp-PLA2 activity was an AMI risk predictor (OR=5.224, 95% CI 1.687-16.181, p=0.004), after smoking, systolic blood pressure, diabetes and hyperlipidemia were adjusted. Recurrent ACS patients were older (p=0.035), but they showed lower levels of Lp-PLA2 mass and Lp-PLA2 activity (p=0.014, p=0.045, respectively), compared to primary ACS patients. Conclusion: Smoking may be an important regulatory factor for Lp-PLA2 mRNA expression in PBMC. Among three Lp-PLA2 indexes, Lp-PLA2 activity was the best marker indicating AMI risk, while Lp-PLA2 mass maybe play better role as a predictor in avoiding ACS recurrence.

Diabetes status modifies the long-term effect of lipoprotein-associated phospholipase A2 on major coronary events

Aims/hypothesis Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity has an independent prognostic association with major coronary events (MCE). However, no study has investigated whether type 2 diabetes status modifies the effect of Lp-PLA2 activity or inhibition on the risk of MCE. We investigate the interaction between diabetes status and Lp-PLA2 activity with risk of MCE. Subsequently, we test the resulting hypothesis that diabetes status will play a role in modifying the efficacy of an Lp-PLA2 inhibitor. Methods A retrospective cohort study design was utilised in two study populations. Discovery analyses were performed in the Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS) cohort based in Scotland, UK. Participants were categorised by type 2 diabetes control status: poorly controlled (HbA1c ≥ 48 mmol/mol or ≥6.5%) and well-controlled (HbA1c < 48 mmol/mol or <6.5%) diabetes (n = 7420). In a secondary analysis of the Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy (STABILITY) trial of Lp-PLA2 inhibitor (darapladib) efficacy, 15,828 participants were stratified post hoc by type 2 diabetes diagnosis status (diabetes or no diabetes) at time of recruitment. Lp-PLA2 activity was then divided into population-specific quartiles. MCE were determined from linked medical records in GoDARTS and trial records in STABILITY. First, the interaction between diabetes control status and Lp-PLA2 activity on the outcome of MCE was explored in GoDARTS. The effect was replicated in the placebo arm of STABILITY. The effect of Lp-PLA2 on MCE was then examined in models stratified by diabetes status. This helped determine participants at higher risk. Finally, the effect of Lp-PLA2 inhibition was assessed in STABILITY in the higher risk group. Cox proportional hazards models adjusted for confounders were used to assess associations. Results In GoDARTS, a significant interaction between increased Lp-PLA2 activity (continuous and quartile divided) and diabetes control status was observed in the prediction of MCE (p < 0.0001). These effects were replicated in the placebo arm of STABILITY (p < 0.0001). In GoDARTS, stratified analyses showed that, among individuals with poorly controlled diabetes, the hazards of MCE for those with high (Q4) Lp-PLA2 activity was 1.19 compared with individuals with lower (Q1–3) Lp-PLA2 activity (95% CI 1.11, 1.38; p < 0.0001) and 1.35 (95% CI 1.16, 1.57; p < 0.0001) when compared with those with the lowest activity (Q1). Those in the higher risk group were identified as individuals with the highest Lp-PLA2 activity (Q4) and poorly controlled diabetes or diabetes. Based on these observations in untreated populations, we hypothesised that the Lp-PLA2 inhibitor would have more benefit in this higher risk group. In this risk group, Lp-PLA2 inhibitor use was associated with a 33% reduction in MCE compared with placebo (HR 0.67 [95% CI 0.50, 0.90]; p = 0.008). In contrast, Lp-PLA2 inhibitor showed no efficacy in individuals with low activity, regardless of diabetes status, or among those with no baseline diabetes and high Lp-PLA2 activity. Conclusions/interpretation These results support the hypothesis that diabetes status modifies the association between Lp-PLA2 activity and MCE. These results suggest that cardiovascular morbidity and mortality associated with Lp-PLA2 activity is especially important in patients with type 2 diabetes, particularly those with worse glycaemic control. Further investigation of the effects of Lp-PLA2 inhibition in diabetes appears warranted. Data availability STABILITY trial data are available from clinicaltrials.gov repository through the GlaxoSmithKline clinical study register https://clinicaltrials.gov/ct2/show/NCT00799903. GoDARTS datasets generated during and/or analysed during the current study are available following request to the GoDARTS Access Managements Group https://godarts.org/scientific-community/. Graphical abstract

Association of PAF and its metabolic enzymes with GGT and the Fatty Liver Index in healthy volunteers

Background: Platelet-activating factor (PAF) is a lipid inflammatory mediator implicated in liver disease. Its main biosynthetic enzymes are cytidine diphosphate (CDP)-choline:1-alkyl-2-acetyl-sn-glycerol-cholinephosphotransferase (PAF-CPT) and acetyl-coenzyme A: lyso-PAF-acetyltransferases (Lyso-PAF-AT), while PAF acetylhydrolase (PAF-AH) and lipoprotein-associated phospholipase A2 (Lp-PLA2) degrade PAF. Objective: To explore the relation of PAF metabolism with liver diseases and non-alcoholic fatty liver disease, as reflected by the fatty liver index (FLI). Methods: In 106 healthy volunteers, PAF concentration, the activity of its metabolic enzymes, and gamma-glutamyl transferase (GGT) were measured in whole blood, leukocytes, and serum, respectively, and the FLI was calculated. Partial correlations and linear regression models were used. Results: In males, serum GGT activity was positively correlated with abdominal fat (as assessed by analysis of a manually defined region of interest in dual-energy X-ray absorptiometry), triacylglycerols, bound-PAF, and Lp-PLA2, while the FLI was positively correlated with Lp-PLA2 activity. In females, serum GGT activity was negatively associated with high-density lipoprotein cholesterol (HDL-C) (age-adjusted correlations, all p<0.05). Lp-PLA2 was a significant determinant of serum GGT activity in males after controlling for age, low-density lipoprotein cholesterol (LDL-C), and abdominal fat. The addition of bound-PAF in the model significantly increased the explained variance of serum GGT activity (total variance explanation 30%). Conclusions : Bound-PAF and Lp-PLA2 activity predicted serum GGT activity, while Lp-PLA2 was also related to FLI. Our findings shed light on the metabolic pathways linking Lp-PLA2 to other atherosclerosis and/or oxidative markers, such as HDL-C, LDL-C, GGT, and FLI, and underline the important role of PAF.

Lipoprotein-Associated Phospholipase A2: A Novel Contributor in Sjögren’s Syndrome-Related Lymphoma?

BackgroundB-cell non-Hodgkin’s lymphoma (B-NHL) is one of the major complications of primary Sjögren’s syndrome (SS). Chronic inflammation and macrophages in SS minor salivary glands have been previously suggested as significant predictors for lymphoma development among SS patients. Lipoprotein-associated phospholipase A2 (Lp-PLA2)—a product mainly of tissue macrophages—is found in the circulation associated with lipoproteins and has been previously involved in cardiovascular, autoimmune, and malignant diseases, including lymphoma.ObjectiveThe purpose of the current study was to investigate the contributory role of Lp-PLA2 in B-NHL development in the setting of primary SS.MethodsLp-PLA2 activity in serum samples collected from 50 primary SS patients with no lymphoma (SS-nL), 9 primary SS patients with lymphoma (SS-L), and 42 healthy controls (HC) was determined by detection of [3H]PAF degradation products by liquid scintillation counter. Moreover, additional sera from 50 SS-nL, 28 SS-L, and 32 HC were tested for Lp-PLA2 activity using a commercially available ELISA kit. Lp-PLA2 mRNA, and protein expression in minor salivary gland (MSG) tissue samples derived from SS-nL, SS-L patients, and sicca controls (SC) were analyzed by real-time PCR, Western blot, and immunohistochemistry.ResultsSerum Lp-PLA2 activity was significantly increased in SS-L compared to both SS-nL and HC by two independent methods implemented [mean ± SD (nmol/min/ml): 62.0 ± 13.4 vs 47.6 ± 14.4 vs 50.7 ± 16.6, p-values: 0.003 and 0.04, respectively, and 19.4 ± 4.5 vs 15.2 ± 3.3 vs 14.5 ± 3.0, p-values: &lt;0.0001, in both comparisons]. ROC analysis revealed that the serum Lp-PLA2 activity measured either by radioimmunoassay or ELISA has the potential to distinguish between SS-L and SS-nL patients (area under the curve [AUC]: 0.8022, CI [95%]: 0.64–0.96, p-value: 0.004 for radioimmunoassay, and AUC: 0.7696, CI [95%]: 0.66–0.88, p-value: &lt;0.0001, for ELISA). Lp-PLA2 expression in MSG tissues was also increased in SS-L compared to SS-nL and SC at both mRNA and protein level. ROC analysis revealed that both MSG mRNA and protein Lp-PLA2 have the potential to distinguish between SS-nL and SS-L patients (area under the curve [AUC] values of 0.8490, CI [95%]: 0.71–0.99, p-value: 0.0019 and 0.9444, CI [95%]: 0.79–1.00, p- value: 0.0389 respectively). No significant difference in either serum Lp-PLA2 activity or MSG tissue expression was observed between SS-nL and HC.ConclusionsLp-PLA2 serum activity and MSG tissue mRNA/protein expression could be a new biomarker and possibly a novel therapeutic target for B-cell lymphoproliferation in the setting of SS.

Traumatic Brain Injury Impairs Systemic Vascular Function Through Disruption of Inward-Rectifier Potassium Channels

Trauma can lead to widespread vascular dysfunction, but the underlying mechanisms remain largely unknown. Inward-rectifier potassium channels (Kir2.1) play a critical role in the dynamic regulation of regional perfusion and blood flow. Kir2.1 channel activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid that is degraded by phospholipase A2 (PLA2) in conditions of oxidative stress or inflammation. We hypothesized that PLA2–induced depletion of PIP2 after trauma impairs Kir2.1 channel function. A fluid percussion injury model of traumatic brain injury (TBI) in rats was used to study mesenteric resistance arteries 24 hours after injury. The functional responses of intact arteries were assessed using pressure myography. We analyzed circulating PLA2, hydrogen peroxide (H2O2), and metabolites to identify alterations in signaling pathways associated with PIP2 in TBI. Electrophysiology analysis of freshly-isolated endothelial and smooth muscle cells revealed a significant reduction of Ba2+-sensitive Kir2.1 currents after TBI. Additionally, dilations to elevated extracellular potassium and BaCl2- or ML 133-induced constrictions in pressurized arteries were significantly decreased following TBI, consistent with an impairment of Kir2.1 channel function. The addition of a PIP2 analog to the patch pipette successfully rescued endothelial Kir2.1 currents after TBI. Both H2O2 and PLA2 activity were increased after injury. Metabolomics analysis demonstrated altered lipid metabolism signaling pathways, including increased arachidonic acid, and fatty acid mobilization after TBI. Our findings support a model in which increased H2O2-induced PLA2 activity after trauma hydrolyzes endothelial PIP2, resulting in impaired Kir2.1 channel function.

Changes in Lipid Profile of Keratinocytes from Rat Skin Exposed to Chronic UVA or UVB Radiation and Topical Application of Cannabidiol

UV radiation is a well-established environmental risk factor known to cause oxidative stress and disrupt the metabolism of keratinocyte phospholipids. Cannabidiol (CBD) is a phytocannabinoid with anti-inflammatory and antioxidant effects. In this study, we examined changes in the keratinocyte phospholipid profile from nude rat skin exposed to UVA and UVB radiation that was also treated topically with CBD. UVA and UVB radiation promoted up-regulation of phosphatidylcholines (PC), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE) and down-regulation of sphingomyelin (SM) levels and enhanced the activity of phospholipase A2 (PLA2) and sphingomyelinase (SMase). Application of CBD to the skin of control rats led to down-regulation of SM and up-regulation of SMase activity. After CBD treatment of rats irradiated with UVA or UVB, SM was up-regulated and down-regulated, respectively, while ceramide (CER) levels and SMase activity were down-regulated and up-regulated, respectively. CBD applied to the skin of UV-irradiated rats down-regulated LPC, up-regulated PE and phosphatidylserines (PS) and reduced PLA2 activity. In conclusion, up-regulation of PS may suggest that CBD inhibits their oxidative modification, while changes in the content of PE and SM may indicate a role of CBD in promoting autophagy and improving the status of the transepidermal barrier.