Circulating and PBMC Lp-PLA2 Associate Differently with Oxidative Stress and Subclinical Inflammation in Nonobese Women (Menopausal Status)

BACKGROUND: This study aimed to determine the association of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity in circulation and peripheral blood mononuclear cells (PBMCs) with inflammatory and oxidative stress markers in nonobese women and according to menopausal status. Lp-PLA(2) activity, a marker for cardiovascular risk is associated with inflammation and oxidative stress. METHODOLOGY/PRINCIPAL FINDINGS: Eighty postmenopausal women (53.0±4.05 yr) and 96 premenopausal women (39.7±9.25 yr) participated in this study. Lp-PLA(2) activities, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β in plasma as well as in PBMCs were measured. Plasma ox-LDL was also measured. Postmenopausal women demonstrated higher circulating levels of ox-LDL and IL-6, as well as IL-6, TNF-α, and IL-1β in PBMCs, than premenopausal women. In both groups, plasma Lp-PLA(2) activity positively correlated with Lp-PLA(2) activity in PBMCs and plasma ox-LDL. In premenopausal women, Lp-PLA(2) activities in plasma and PBMCs positively correlated with IL-6, TNF-α, and IL-1β in PBMCs. In postmenopausal women, plasma ox-LDL positively correlated with PBMC cytokine production. In subgroup analysis of postmenopausal women according to plasma ox-LDL level (median level: 48.715 U/L), a significant increase in Lp-PLA(2) activity in the plasma but not the PBMCs was found in the high ox-LDL subgroup. Plasma Lp-PLA(2) activity positively correlated with unstimulated PBMC Lp-PLA(2) activity in the low ox-LDL subgroup (r = 0.627, P<0.001), whereas in the high ox-LDL circulating Lp-PLA(2) activity positively correlated with plasma ox-LDL (r = 0.390, P = 0.014) but not with Lp-PLA(2) activity in PBMCs. CONCLUSIONS/SIGNIFICANCE: The lack of relation between circulating Lp-PLA(2) activity and Lp-PLA(2) activity in PBMCs was found in postmenopausal women with high ox-LDL. This may indicate other sources of circulating Lp-PLA(2) activity except PBMC in postmenopausal women with high ox-LDL. We also demonstrated that circulating Lp-PLA(2) and PBMC secreted Lp-PLA(2) associate differently with markers of oxidative stress and sub clinical inflammation in nonobese women, particularly according to the menopausal states

Development of a universal chemiluminometric genotyping method for high-throughput detection of 7 LDLR gene mutations in greek population

Objectives: Familial hypercholesterolemia (FH) is caused by mutations in the LDL receptor (LDLR) gene. We report the application of a universal method with high allele discrimination properties to the simultaneous genotyping of 7 LDLR mutations in Greeks, in dry-reagent format. Design and methods: We genotyped mutations C858A, C939A, G1285A, T1352C, G1646A, G1775A, C/T81G. Unpurified amplicons from a multiplex PCR that produced fragments encompassing all 7 mutations were subjected to probe extension reactions in the presence of fluorescein-modified dCTP, and a microtiter well-based assay of extension products with a peroxidase-antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents and assigned genotypes by the signal ratio of normal-to-mutant-specific probe. Results: We standardized the method and optimised all steps for specificity. The method was validated by genotyping blindly 119 (833 genotypings). Results were fully concordant with other methods used as standards. Conclusions: This method is accurate, simple, rapid and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations. © 2007 The Canadian Society of Clinical Chemists

High-throughput microtiter well-based chemiluminometric genotyping of 15 H88 gene mutations in a dry-reagent format

Background: Hemoglobinopathies are the most common inherited diseases worldwide. Various methods for genotyping of hemoglobin, beta (HBB) gene mutations have been reported, but there is need for a high sample-throughput, cost-effective method for simultaneous screening of several mutations. We report a method that combines the high detectability and dynamic range of chemiluminescence with the high allele-discrimination ability of probe extension reactions for simultaneous genotyping of 15 HBB mutations in a high sample-throughput, dry-reagent format. Methods: We genotyped the HBB mutations IVSI-110G&gt;A, CD39C&gt;T, IVSI-1G&gt;A, IVSI-6T&gt;C, IVSII-745C&gt;G, IVSII-1G&gt;A, FSC6GAG&gt;G-G, -101C&gt;T, FSC5CCT&gt;C-, IVSI-5G&gt;A, FSC8AAG&gt;-G, -87C&gt;G, IVSII-848C&gt;A, term+6C&gt;G, and HbS (cd6GAG&gt; GTG). The method used comprises the following: (a) duplex PCR that produces fragments encompassing all 15 mutations, (b) probe extension reactions in the presence of fluorescein-modified dCTP, using unpurified amplicons, and (c) microliter well-based assay of extension products with a peroxidase- antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents to simplify the procedure and assigned the genotype by the signal ratio of the normal-to-mutant-specific probe. Results: We standardized the method by analyzing 60 samples with known genotypes and then validated by blindly genotyping 115 samples with 45 genotypes. The results were fully concordant with sequencing. The reproducibility (including PCR, probe extension reaction, and chemiluminometric assay) was studied for 20 days, and the CVs were 11%-19%. Conclusions: This method is accurate, reproducible, and cost-effective in terms of equipment and reagents. The application of the method is simple, rapid, and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations. © 2007 American Association for Clinical Chemistry

Antibiotic prophylaxis in preterm premature rupture of membranes at 24–31 weeks’ gestation: Perinatal and 2‐year outcomes in the EPIPAGE‐2 cohort

International audienceObjectiveTo compare different antibiotic prophylaxis administered after preterm premature rupture of membranes to determine whether any were associated with differences in obstetric and/or neonatal outcomes and/or neurodevelopmental outcomes at 2 years of corrected age.DesignProspective, nationwide, population-based EPIPAGE-2 cohort study of preterm infants.SettingFrance, 2011.SampleWe included 492 women with a singleton pregnancy and a diagnosis of preterm premature rupture of membranes at 24–31 weeks. Exclusion criteria were contraindication to expectant management or indication for antibiotic therapy other than preterm premature rupture of membranes. Antibiotic prophylaxis was categorised as amoxicillin (n = 345), macrolide (n = 30), third-generation cephalosporin (n = 45) or any combinations covering Streptococcus agalactiae and >90% of Escherichia coli (n = 72), initiated within 24 hours after preterm premature rupture of membranes.MethodsPopulation-averaged robust Poisson models.Main Outcome MeasuresSurvival at discharge without severe neonatal morbidity, 2-year neurodevelopment.ResultsWith amoxicillin, macrolide, third-generation cephalosporin and combinations, 78.5%, 83.9%, 93.6% and 86.0% of neonates were discharged alive without severe morbidity. The administration of third-generation cephalosporin or any E. coli-targeting combinations was associated with improved survival without severe morbidity (adjusted risk ratio 1.25 [95% confidence interval 1.08–1.45] and 1.10 [95 % confidence interval 1.01–1.20], respectively) compared with amoxicillin. We evidenced no increase in neonatal sepsis related to third-generation cephalosporin-resistant pathogen.ConclusionIn preterm premature rupture of membranes at 24–31 weeks, antibiotic prophylaxis based on third-generation cephalosporin may be associated with improved survival without severe neonatal morbidity when compared with amoxicillin, with no evidence of increase in neonatal sepsis related to third-generation cephalosporin-resistant pathogen