Endotoxin Mass Concentration in Plasma Is Associated With Mortality in a Multicentric Cohort of Peritonitis-Induced Shock

Objectives: To investigate the association of plasma LPS mass with mortality and inflammation in patients with peritonitis-induced septic shock (SS).Design: Longitudinal endotoxin and inflammatory parameters in a multicentric cohort of SS.Patients: Protocolized post-operative parameters of 187 SS patients collected at T1 (12 h max post-surgery) and T4 (24 h after T1).Intervention:Post-hoc analysis of ABDOMIX trial.Measurements and Results: Plasma concentration of LPS mass as determined by HPLC-MS/MS analysis of 3-hydroxymyristate, activity of phospholipid transfer protein (PLTP), lipids, lipoproteins, IL-6, and IL-10. Cohort was divided in low (LLPS) and high (HLPS) LPS levels. The predictive value for mortality was tested by multivariate analysis. HLPS and LLPS had similar SAPSII (58 [48.5; 67]) and SOFA (8 [6.5; 9]), but HLPS showed higher death and LPS to PLTP ratio (p < 0.01). LPS was stable in HLPS, but it increased in LLPS with a greater decrease in IL-6 (p < 0.01). Dead patients had a higher T1 LPS (p = 0.02), IL-6 (<0.01), IL-10 (=0.01), and day 3 SOFA score (p = 0.01) than survivors. In the group of SAPSII > median, the risk of death in HLPS (38%) was higher than in LLPS (24%; p < 0.01). The 28-day death was associated only with SAPSII (OR 1.06 [1.02; 1.09]) and HLPS (OR 2.47 [1; 6.11]) in the multivariate model. In HLPS group, high PLTP was associated with lower plasma levels of IL-6 (p = 0.02) and IL-10 (p = 0.05).Conclusions: Combination of high LPS mass concentration and high SAPS II is associated with elevated mortality in peritonitis-induced SS patients.

Increased Phospholipid Transfer Protein Activity Is Associated With Markers of Enhanced Lipopolysaccharide Clearance in Human During Cardiopulmonary Bypass

Introduction: Lipopolysaccharide (LPS) is a component of gram-negative bacteria, known for its ability to trigger inflammation. The main pathway of LPS clearance is the reverse lipopolysaccharide transport (RLT), with phospholipid transfer protein (PLTP) and lipoproteins playing central roles in this process in experimental animal models. To date, the relevance of this pathway has never been studied in humans. Cardiac surgery with cardiopulmonary bypass is known to favor LPS digestive translocation. Our objective was to determine whether pre-operative PLTP activity and triglyceride or cholesterol-rich lipoprotein concentrations were associated to LPS concentrations in patients undergoing cardiac surgery with cardiopulmonary bypass.Methods: A post-hoc analysis was conducted on plasma samples obtained from patients recruited in a randomized controlled trial.Total cholesterol, high density lipoprotein cholesterol (HDLc), low density lipoprotein cholesterol (LDLc), triglyceride and PLTP activity were measured before surgery. LPS concentration was measured by mass spectrometry before surgery, at the end of cardiopulmonary bypass and 24 h after admission to the intensive care unit.Results: High PLTP activity was associated with lower LPS concentration but not with inflammation nor post-operative complications. HDLc, LDLc and total cholesterol were not associated with LPS concentration but were lower in patients developing post-operative adverse events. HDLc was negatively associated with inflammation biomarkers (CRP, PCT). Triglyceride concentrations were positively correlated with LPS concentration, PCT and were higher in patients with post-operative complications.Conclusion: Our study supports the role of PLTP in LPS elimination and the relevance of RLT in human. PLTP activity, and not cholesterol rich lipoproteins pool size seemed to be the limiting factor for RLT. PLTP activity was not directly related to post-operative inflammation and adverse events, suggesting that LPS clearance is not the main driver of inflammation in our patients. However, HDLc was associated with lower inflammation and was associated with favorable outcomes, suggesting that HDL beneficial anti-inflammatory effects could be, at least in part independent of LPS clearance.

Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Ethanol Induces Extracellular Vesicle Secretion by Altering Lipid Metabolism through the Mitochondria-Associated ER Membranes and Sphingomyelinases

Recent evidence pinpoints extracellular vesicles (EVs) as key players in intercellular communication. Given the importance of cholesterol and sphingomyelin in EV biology, and the relevance of mitochondria-associated endoplasmic reticulum membranes (MAMs) in cholesterol/sphingomyelin homeostasis, we evaluated if MAMs and sphingomyelinases (SMases) could participate in ethanol-induced EV release. EVs were isolated from the extracellular medium of BV2 microglia treated or not with ethanol (50 and 100 mM). Radioactive metabolic tracers combined with thin layer chromatography were used as quantitative methods to assay phospholipid transfer, SMase activity and cholesterol uptake/esterification. Inhibitors of SMase (desipramine and GW4869) and MAM (cyclosporin A) activities were also utilized. Our data show that ethanol increases the secretion and inflammatory molecule concentration of EVs. Ethanol also upregulates MAM activity and alters lipid metabolism by increasing cholesterol uptake, cholesterol esterification and SMase activity in microglia. Notably, the inhibition of either SMase or MAM activity prevented the ethanol-induced increase in EV secretion. Collectively, these results strongly support a lipid-driven mechanism, specifically via SMases and MAM, to explain the effect of ethanol on EV secretion in glial cells.

Association between the PLTP rs4810479 SNP and Serum Lipid Traits in the Chinese Maonan and Han Populations

The association between the phospholipid transfer protein (PLTP) gene rs4810479 single-nucleotide polymorphism (SNP) and serum lipid levels is largely unknown. This investigation aimed to evaluate the relationship between the PLTP rs4810479 SNP, several environmental risk factors, and serum lipid parameters in the Chinese Maonan and Han nationalities. Polymerase chain reaction-restriction fragment length polymorphism, gel electrophoresis, and direct sequencing were employed to determine the PLTP rs4810479 genotypes in 633 Maonan and 646 Han participants. The frequencies of CC, CT, and TT genotypes and the C allele were different between Maonan and Han groups (29.07%, 53.08%, 17.85%, and 55.61% vs. 35.60%, 49.70%, 14.70%, and 60.45%, respectively, P < 0.05 ). The C allele carriers in the Maonan group had higher high-density lipoprotein cholesterol levels than the C allele noncarriers, but this finding was only found in Maonan males but not in females. The C allele carriers in Han males had lower total cholesterol and low-density lipoprotein cholesterol levels than the C allele noncarriers. Serum lipid profiles were also affected by several traditional cardiovascular risk factors in both populations. There might be an ethnic- and/or sex-specific association between the PLTP rs4810479 SNP and serum lipid traits.

3×Tg-AD Mice Overexpressing Phospholipid Transfer Protein Improves Cognition Through Decreasing Amyloid-β Production and Tau Hyperphosphorylation

Background: Phospholipid transfer protein (PLTP) belongs to the lipid transfer glycoprotein family. Studies have shown that it is closely related to Alzheimer’s disease (AD); however, the exact effect and mechanism remain unknown. Objective: To observe the effect of PLTP overexpression on behavioral dysfunction and the related mechanisms in APP/PS1/Tau triple transgenic (3×Tg-AD) mice. Methods: AAV-PLTP-EGFP was injected into the lateral ventricle to induce PLTP overexpression. The memory of 3×Tg-AD mice and wild type (WT) mice aged 10 months were assessed using Morris water maze (MWM) and shuttle-box passive avoidance test (PAT). Western blotting and ELISA assays were used to quantify the protein contents. Hematoxylin and eosin, Nissl, and immunochemistry staining were utilized in observing the pathological changes in the brain. Results: 3×Tg-AD mice displayed cognitive impairment in WMW and PAT, which was ameliorated by PLTP overexpression. The histopathological hallmarks of AD, senile plaques and neurofibrillary tangles, were observed in 3×Tg-AD mice and were improved by PLTP overexpression. Besides, the increase of amyloid-β42 (Aβ 42) and Aβ 40 were found in the cerebral cortex and hippocampus of 3×Tg-AD mice and reversed by PLTP overexpression through inhibiting APP and PS1. PLTP overexpression also reversed tau phosphorylation at the Ser404, Thr231 and Ser199 of the hippocampus in 3×Tg-AD mice. Furthermore, PLTP overexpression induced the glycogen synthase kinase 3β (GSK3β) inactivation via upregulating GSK3β (pSer9). Conclusion: These results suggest that PLTP overexpression has neuroprotective effects. These effects are possibly achieved through the inhibition of the Aβ production and tau phosphorylation, which is related to GSK3β inactivation.