Longitudinal SARS-CoV-2 seroprevalence in Portugal and antibody maintenance 12 months after infection.

During the COVID-19 pandemic, Portugal has experienced three distinct SARS-CoV-2 infection waves. We previously documented the prevalence of SARS-CoV-2 immunity, measured by specific antibodies, in September 2020, 6 months after the initial moderate wave. Here, we show the seroprevalence changes 6 months later, up to the second week of March 2021, shortly following the third wave, which was one of the most severe in the world, and 2 months following the start of the vaccination campaign. A longitudinal epidemiological study was conducted, with a stratified quota sample of the Portuguese population. Serological testing was performed, including ELISA determination of antibody class and titers. The proportion of seropositives, which was 2.2% in September 2020, rose sharply to 17.3% (95% CI: 15.8-18.8%) in March 2021. Importantly, circulating IgG and IgA antibody levels were very stable 6 months after the initial determination and up to a year after initial infection, indicating long-lasting infection immunity against SARS-CoV-2. Moreover, vaccinated people had higher IgG levels from 3 weeks post-vaccination when compared with previously infected people at the same time post-infection.

The Proinflammatory Soluble CD40 Ligand Is Associated with the Systemic Extent of Stable Atherosclerosis.

Background and objectives: Polyvascular atherosclerosis is frequent and associated with a high cardiovascular risk, although the mechanisms regulating the atherosclerosis extent to single or multiple arterial territories are still poorly understood. Inflammation regulates atherogenesis and soluble CD40 ligand (sCD40L) is an inflammatory mediator associated with the presence of single-territorial atherosclerosis. We assessed whether the sCD40L expression is associated with the atherosclerosis extent to single or multiple arterial territories and with the atherosclerosis severity in different territories. Materials and Methods: We prospectively enrolled 94 participants with no atherosclerosis (controls, n = 26); isolated coronary atherosclerosis (group 1, n = 20); coronary and lower extremity (LE) atherosclerosis (group 2, n = 18); coronary and carotid atherosclerosis (group 3, n = 12); and coronary, LE, and carotid atherosclerosis (group 4, n = 18). Serum sCD40L levels were quantified. Results: The sCD40L levels (ng/mL, mean (standard deviation)) were 4.0 (1.5), 5.6 (2.6), 7.2 (4.2), 5.9 (3.7), and 5.1 (2.4) in controls and groups 1 to 4, respectively (ANOVA p = 0.012). In nonrevascularized patients, the sCD40L levels were significantly higher in group 2 than in group 1 and were correlated with the number of LE diseased segments. Prior LE bypass surgery was associated with lower sCD40L levels. Coexistence of coronary and LE atherosclerosis was independently associated with the sCD40L levels. Conclusions: The sCD40L levels were increased in stable atherosclerosis, particularly in polyvascular coronary and LE atherosclerosis. The number of LE diseased segments and prior LE revascularization were associated with sCD40L expression. To our knowledge, these are novel data, which provide insights into the mechanisms underlying multi-territorial atherosclerosis expression. sCD40L may be a promising noninvasive tool for refining the stratification of the systemic atherosclerotic burden.

Association between miR-146a and Tumor Necrosis Factor Alpha (TNF-α) in Stable Coronary Artery Disease.

Background and Objectives: Tumor necrosis factor alpha (TNF-α) is proatherogenic and associated with the risk of acute ischemic events, although the mechanisms that regulate TNF-α expression in stable coronary artery disease (SCAD) are not fully understood. We investigated whether metabolic, inflammatory, and epigenetic (microRNA (miRNA)) markers are associated with TNF-α expression in SCAD. Materials and Methods: Patients with SCAD were prospectively recruited and their metabolic and inflammatory profiles were assessed. TNF-α levels were assessed using an enzyme-linked immunosorbent assay. The relative expression of six circulating miRNAs associated with the regulation of inflammation and/or atherosclerosis was determined. Results: Of the 24 included patients with the mean age of 65 (9) years, 88% were male, and 54% were diabetic. The TNF-α levels were (median (interquartile range)) 1.0 (0.7-1.1) pg/mL. The percentage of glycosylated hemoglobin (r = 0.418, p = 0.042), serum triglyceride levels (r = 0.429, p = 0.037), and C-reactive protein levels (r = 0.407, p = 0.048) were positively correlated with TNF-α levels. Of the candidate miRNAs, miR-146a expression levels were negatively correlated with TNF-α levels (as indicated by r = 0.500, p = 0.035 for correlation between delta cycle threshold (ΔCt) miR-146a and TNF-α levels). In multivariate analysis, serum triglyceride levels and miR-146a expression levels were independently associated with TNF-α levels. miR-146 expression levels were not associated with metabolic or other inflammatory parameters and were negatively correlated with the number of coronary vessels with obstructive disease (as indicated by r = 0.556, p = 0.017 for correlation between ΔCt miR-146a and number of diseased vessels). Conclusions: miR-146a expression levels were negatively correlated with TNF-α levels in patients with SCAD, irrespective of other metabolic or inflammatory markers, and with the severity of coronary artery disease. The results add to the knowledge on the role of miR-146a in TNF-α-based inflammation in SCAD and support future research on the potential therapeutic use of miR-146a in such a clinical scenario.

Acetylcholinesterase conformational states influence nitric oxide mobilization in the erythrocyte.

In the human erythrocyte, band 3 protein mediates nitric oxide (NO) translocation and its effects are strongly related to phosphorylated/dephosphorylated intracellular states. The metabolism of NO could change in the presence of acetylcholinesterase (AChE). Therefore, the present study was designed to assess the effect of conformational changes in AChE (via N-19 and C-16 antibodies) and enzymatic inhibition/activation of protein kinase C (PKC) in erythrocyte NO mobilization in vitro. Our results show that by inhibiting PKC with cheletrine, impaired erythrocyte NO efflux and s-nitrosoglutathione (GSNO) levels were verified, while PKC's activation by Phorbol 12-myristate 13-acetate had the opposite effect. Those results demonstrate the influence of 4.1R complex and band 3 protein level of phosphorylation on NO efflux and GSNO concentration mediated by PKC inhibition/activation. In addition, the present study shows evidence that conformational changes in AChE promoted by incubation with N-19 and C-16 antibodies alter the enzyme's functional connection to acetylcholine (ACh) (AChE-ACh complex) in an irreversible manner, resulting in impaired GSNO concentration and NO efflux from the erythrocyte. Novel insight into NO metabolism in the erythrocyte is brought with the presented findings allowing new possibilities of modulating NO delivery, possibly involving PKC and AChE conformational alterations in combination.

Soluble CD40 ligand expression in stable atherosclerosis: A systematic review and meta-analysis.

BACKGROUND AND AIMS: The role of inflammation in atherosclerosis development and expression in different arterial territories is unclear. Soluble CD40 ligand (sCD40L) mediates inflammation and atherogenesis. Through a systematic review and meta-analysis, we assessed whether sCD40L was dysregulated in stable atherosclerosis, irrespective of the diseased arterial territory, and whether this dysregulation differed according to the specific territory. METHODS: Systematic literature searches were performed in MEDLINE, Cochrane Library, Web of Science, and Embase for studies reporting circulating sCD40L levels in individuals with and without stable atherosclerosis. sCD40L levels were compared using random-effects meta-analysis, weighted by the inverse variance method (study protocol: PROSPERO CRD42020181392). RESULTS: Fifty-four studies (59 estimates) including 7705 patients and 7841 controls were analyzed. sCD40L levels were found to be increased in patients with atherosclerosis, irrespective of the territory (standardized mean difference [SMD] 0.43, 95% CI 0.29-0.57; 59 estimates; χ2 heterogeneity p < 0.001; I2 = 92%). SMD was greatest in carotid atherosclerosis (SMD 0.58, 95% CI 0.30-0.86; 17 estimates), followed by coronary (SMD 0.43, 95% CI 0.24-0.62; 33 estimates), lower extremity (SMD 0.26, 95% CI -0.02-0.54; 7 estimates), and renal atherosclerosis (SMD -0.07, 95% CI -2.77-2.64; 2 estimates) (χ2 heterogeneity p < 0.001; I2 ≥ 80% for all). Subgroup analysis revealed that sCD40L levels were increased in clinical, but not subclinical, atherosclerosis. CONCLUSIONS: sCD40L levels were increased in stable atherosclerosis, particularly in the carotid and coronary territories. These novel data support sCD40L as a marker of systemic atherosclerosis, possibly with differential roles in specific territories.

Cigarette Smoking, miR-27b Downregulation, and Peripheral Artery Disease: Insights into the Mechanisms of Smoking Toxicity.

Cigarette smoking is a risk factor for the development of peripheral artery disease (PAD), although the proatherosclerotic mediators of cigarette smoking are not entirely known. We explored whether circulating microRNAs (miRNAs) are dysregulated in cigarette smokers and associated with the presence of PAD. Ninety-four participants were recruited, including 58 individuals without and 36 with PAD, 51 never smokers, 28 prior smokers, and 15 active smokers. The relative expression of six circulating miRNAs with distinct biological roles (miR-21, miR-27b, miR-29a, miR-126, miR-146, and miR-218) was assessed. Cigarette smoking was associated with the presence of PAD in multivariate analysis. Active smokers, but not prior smokers, presented miR-27b downregulation and higher leukocyte, neutrophil, and lymphocyte counts; miR-27b expression levels were independently associated with active smoking. Considering the metabolic and/or inflammatory abnormalities induced by cigarette smoking, miR-27b was independently associated with the presence of PAD and downregulated in patients with more extensive PAD. In conclusion, the atheroprotective miR-27b was downregulated in active smokers, but not in prior smokers, and miR-27b expression was independently associated with the presence of PAD. These unreported data suggest that the proatherogenic properties of cigarette smoking are mediated by a downregulation of miR-27b, which may be attenuated by smoking cessation.

Circulating miRNAs Are Associated with the Systemic Extent of Atherosclerosis: Novel Observations for miR-27b and miR-146.

The mechanisms that regulate the systemic extent of atherosclerosis are not fully understood. We investigated whether the expression of circulating miRNAs is associated with the extent of stable atherosclerosis to a single territory or multiple territories (polyvascular) and with the severity of atherosclerosis in each territory. Ninety-four participants were prospectively recruited and divided into five age- and sex-matched groups: presenting no atherosclerosis, isolated coronary atherosclerosis, coronary and lower extremity atherosclerosis, coronary and carotid atherosclerosis, and atherosclerosis of the coronary, lower extremity, and carotid territories. The expression of six circulating miRNAs with distinct biological roles was assessed. The expression of miR-27b and miR-146 differed across groups (p < 0.05), showing a decrease in the presence of atherosclerosis, particularly in the three territories. miR-27b and miR-146 expression decreased in association with a higher severity of coronary, lower extremity, and carotid atherosclerosis. Polyvascular atherosclerosis involving the three territories was independently associated with a decreased miR-27b and miR-146 expression. Both miRNAs presented an area under the curve of ≥0.75 for predicting polyvascular atherosclerosis involving the three territories. To conclude, miR-27b and miR-146 were associated with the presence of severe polyvascular atherosclerosis and with the atherosclerosis severity in each territory. Both are potential biomarkers of severe systemic atherosclerosis.

Inflammation is associated with the presence and severity of chronic coronary syndrome through soluble CD40 ligand.

INTRODUCTION: Inflammation contributes to the initiation and progression of atherosclerosis, although the underlying inflammatory pathways are not entirely known. Specifically, the role of the proinflammatory soluble CD40 ligand (sCD40L) on the expression of chronic coronary syndrome (CCS) is not completely understood. We evaluated whether sCD40L expression is associated with the presence of CCS and with the clinical and anatomical severity of CCS. METHODS: We prospectively recruited 94 participants, assigned to two groups matched by age and sex, without coronary artery disease (n=26) and with CCS (n=68). Clinical, laboratory and anatomical data were prospectively collected, and serum levels of sCD40L were measured. RESULTS: In patients with CCS, classic cardiovascular risk factors were more prevalent, and the sCD40L levels, leukocyte and neutrophil counts, and neutrophil/lymphocyte ratio, but not the C-reactive protein levels, were significantly higher than those in controls. sCD40L was independently associated with the presence of obstructive coronary artery disease in multivariate analysis. Regarding CCS severity, sCD40L levels showed a significant stepwise increase with increasing angina severity (ANOVA P=0.001). In addition, sCD40L was independently associated with the anatomical severity of coronary artery disease, as assessed by the Gensini score. Among patients with CCS, those with previous coronary artery bypass grafting (n=23) had lower sCD40L levels than patients waiting for revascularization (n=45) [4.3 (2.1) ng/mL vs. 6.8 (3.5) ng/mL, P=0.001]. CONCLUSIONS: The expression of the proinflammatory sCD40L was associated with the presence of CCS and reflected the clinical and anatomical severity of CCS. In addition, we describe for the first time the association between prior CABG and reduced sCD40L levels in patients with CCS.

Serial changes in oxidized low-density lipoprotein associated with culprit vessel in ST-elevation myocardial infarction--a promising marker?

The aim of the present study was to investigate variations in oxidized LDL (oxLDL) at the onset of acute myocardial infarction (AMI) and over the recovery period, exploring their relationship with coronary disease severity. A follow-up of 50 AMI patients was evaluated against 25 healthy volunteers (reference group). The AMI patients were evaluated at three time points: at admission before the administration of IIb/IIIa inhibitors and angioplasty, and two and 40 days after intervention. Plasma oxLDL concentrations were measured by ELISA. oxLDL was found to be significantly higher in AMI patients in the acute phase relative to reference levels, decreasing progressively over the recovery period. The results also demonstrated that oxLDL levels were decreased in patients with the left circumflex artery (LCX) as culprit vessel compared to the left anterior descending coronary (LAD) or right coronary artery (RCA). The results highlight a significant increase in oxLDL concentration related to coronary artery disease severity, as conditions such as LCX lesions are usually associated with a favorable prognosis, contrasting with LAD-associated conditions that can compromise large areas of myocardium. The results thus suggest that oxLDL may constitute a promising marker in assessment of AMI evolution.

Influence of dyslipidemia and smoking on redox markers in humans--a critical study.

UNLABELLED: Experimental research indicates that oxidative processes play a role in susceptibility to a large number of diseases. A better understanding of the parameters affecting redox balance could delay and even prevent such processes. OBJECTIVE: The present study aims to investigate blood parameters associated with antioxidant systems in a Portuguese population for the first time, taking into consideration gender, age range, lipid profile and smoking habits as influencing factors. DESIGN AND PARTICIPANTS: One hundred and eighty-three healthy Portuguese subjects of both genders were recruited from the metropolitan area of Lisbon. The group consisted of individuals aged from 20 to 70 years, who gave their informed consent before participating in the study. All subjects were screened to determine eligibility, which was based on a clinical report. Subjects were considered eligible if they had no acute or chronic illness and were not taking any drugs or dietary supplements that could compromise the values of the studied parameters. The subjects were then divided into different subgroups according to gender, age range, lipid profile and smoking habits. METHODS: Whole blood glutathione peroxidase activity and serum albumin, transferrin and uric acid were determined using commercially available kits. Superoxide dismutase activity in erythrocytes and thiobarbituric acid reactive substances in serum were measured using methods published elsewhere. RESULTS: Glutathione peroxidase activity was not affected by any of the studied variables, but superoxide dismutase activity decreased with smoking. Albumin levels remained unchanged under all conditions. Hyperlipidemia was associated with higher lipid peroxidation as well as higher uric acid levels. Gender was the strongest predictor for transferrin, total iron binding capacity and uric acid variations. Finally, a multivariate statistical model clearly separated genders and lipid profile and genders and smoking. CONCLUSIONS: The present study suggests that hyperlipidemia and smoking should be considered important selection criteria in epidemiological studies focusing on oxidative stress and on the atherosclerotic process.

Circulating microRNA profiles in different arterial territories of stable atherosclerotic disease: a systematic review.

AIMS: Atherosclerosis is associated with altered circulating microRNA profiles. It is yet unclear whether the expression of these potential biomarkers differs according to the location of atherosclerosis. We assessed whether atherosclerosis of different arterial territories, except the coronary, is associated with specific circulating microRNA profiles. METHODS: A systematic search in PubMed, Web of Science, Embase, and Cochrane Library was carried out using a retrieval strategy including MESH and non-MSH terms. Eligible studies have compared circulating microRNA profiles between individuals with and without stable atherosclerotic disease of large or medium size arteries. The review protocol was registered in PROSPERO database (reference CRD42017073846). RESULTS: Eighteen studies were selected for qualitative synthesis: ten focused on carotid, six on lower limbs, and two on renal arteries atherosclerosis, none reporting on other locations. A common microRNA profile to different atherosclerotic disease locations was identified, including deregulation of miR-21, miR-30, miR-126, and miR-221-3p. Specific microRNA profiles for each territory were also identified, with consistency across studies, such as deregulation of miR-21 and miR-29 in carotid atherosclerosis, and let 7e, miR-27b, miR-130a, and miR-210 in lower limbs atherosclerosis. The robustness of the results was very high for let 7e, miR-29, miR-30, considering both the adjustment of microRNA expression for baseline variables and the replication of results in different studies (miR-29 in carotid, let 7e in lower limbs, and miR-30 in carotid and lower limbs atherosclerosis). Globally, the deregulated microRNAs are associated with control of angiogenesis, endothelial cell function, inflammation, cholesterol metabolism, oxidative stress and extracellular matrix composition. CONCLUSIONS: A common microRNA profile to different atherosclerotic disease locations and specific microRNA profiles for each territory were identified. These findings may provide insights into pathophysiology and be useful for selecting potential biomarkers for clinical practice. To the best of our knowledge, no systematic data on this subject has been reported.

Myocardial infarction before and after the age of 45: Possible role of platelet receptor polymorphisms.

INTRODUCTION: We examined the potential role of polymorphisms of the platelet genes GP1BA (rs2243093, rs6065 and VNTR), ITGB3 (rs5918), ITGA2 (rs938043469) and P2RY12 (rs2046934, rs6801273 and rs6798347) as risk factors for myocardial infarction (MI). METHODS: The study population was divided into three groups: controls (n=235), MI at age ≤45 years (MI ≤45, n=44), and MI at age >45 years (MI >45, n=78). The control group was further divided into two subgroups (control ≤45 and >45), and subgroups including only men were also considered for statistical analysis. Polymorphisms were detected by polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: Regarding non-genetic risk factors, the control group differed statistically from the MI ≤45 group (p<00.5) in terms of smoking, hypertension, diabetes and obesity, and from the MI >45 group (p<0.05) in terms of hypertension, diabetes, obesity, family history of thrombosis and high cholesterol. For the studied ITGA2 polymorphism, a statistical difference was found when MI >45 was compared with the control group, with a higher risk of MI in the TT genotype (OR 2.852; 95% CI: 1.092-7.451; p=0.032). In the GP1BA rs6065 polymorphism, a statistically significant difference was found between control ≤45 only men and MI ≤45 only men, with a higher risk in the CT genotype (OR 5.568; 95% CI: 1.421-21.822; p=0.016), despite the low numbers included. The other polymorphisms studied did not show any statistically significant correlations. CONCLUSION: There is a statistically significant association between the TT genotype of the ITGA2 rs938043469 polymorphism and increased risk for MI >45.

Variations in inflammatory markers in acute myocardial infarction: a longitudinal study.

Inflammation is now considered a key component of atherosclerosis, from fatty streak formation to plaque rupture, subsequent thrombosis, and progressive mechanical and dynamic obstruction. Rupture of the arterial plaque's fibrous cap exposes tissue factors present in the necrotic core, triggering inflammatory signaling, cell adhesion, and the coagulation cascade that eventually leads to thrombus. Cytokines and adhesion molecules are key components of these events that contribute to the development of an atherosclerotic plaque. The cytokine TNF-alpha and intercellular adhesion molecule-1 (ICAM-1) are indicators of basal inflammation, while the soluble forms of adhesion molecules such as CD40L and P-selectin indicate the extent of platelet activation. This study reports on the follow-up of 17 patients with confirmed acute myocardial infarction (AMI group) undergoing angioplasty and a matched control group of 16 patients without coronary artery disease as verified by coronary angiography. Patients from the AMI group were assessed at the onset of the acute coronary syndrome, within 24 h, before the administration of glycoprotein IIb/IIIa inhibitors and coronary angioplasty, and during the recovery period, two and 40 days after intervention. For both groups, clinical characteristics were documented and serum concentrations of soluble CD40L, P-selectin, ICAM-1, TNF-alpha, and conventional biochemical indicators were analyzed. For AMI patients, these indicators were recorded at study entry and during follow-up. Concentrations of cytokines and adhesion molecules were measured using commercial immunoassay (ELISA) kits. Significant variations in sP-selectin were observed relative to the control group. Immediately after myocardial infarction, sP-selectin levels rose markedly, followed by a sharp decrease two days later. After 40 days of recovery, sP-selectin levels rose again, returning to the initial values. Variations in sCD40L levels were not significant relative to controls. However, sCD40L concentrations tended to fall until the second day after infarction, followed by a rise, and by the 40th day of recovery levels were slightly higher than controls. Unlike sCD40L and sP-selectin, consistently higher levels of TNF-alpha relative to controls were observed, which were only significant after 40 days of recovery. No significant variations were observed for ICAM-1 serum concentrations in the AMI group. The variations observed demonstrate the role of inflammatory markers in AMI progression and highlight the importance of systemic inflammation in disease evolution. The increased concentration of sP-selectin at infarction onset is evidence of thrombosis and platelet activation. Later, during the recovery period when hemodynamic variables are returning to stability in part due to medication, rises in circulating levels of sCD40L and cytokines such as TNF-alpha may reflect the role of these molecules in the recovery of endothelial and myocardial tissues.

Systemic markers of the redox balance and apolipoprotein E polymorphism in atherosclerosis: the relevance for an integrated study.

Prospective studies have demonstrated that an imbalance between oxidative damage and antioxidative protection can play a role in the development and progression of atherosclerosis. Also, genotypes with the apolipoprotein E epsilon4 allele have been associated with an increase risk for this pathology. Based on this knowledge, the aim of this study was to evaluate indicators of the redox balance, trace elements, and apolipoprotein E allelic profile in subjects from the Lisbon population with clinically stable atherosclerosis, at risk for atherosclerotic events, and in healthy subjects for comparison. The activities of superoxide dismutase in erythrocytes and glutathione peroxidase in whole blood, plasma total thiols, and serum ceruloplasmin were kept unchanged among the three groups. Serum alpha- tocopherol was increased in atherosclerotic patients. Total malondialdehyde in serum and protein carbonyls in plasma, which are indicators of lipid and protein oxidative damage, respectively, reached their highest values in risk subjects. The concentrations of potassium and calcium, in plasma and in blood cells, were slightly elevated in patients and might reflect an electrolytic imbalance. Regarding the apolipoprotein E polymorphism, atherosclerotic patients had an increased incidence of the high-risk genotypes for atherogenesis (epsilon3/epsilon4 and epsilon4/epsilon4). A multivariate model applied to the general population using most of the parameters clearly separated the three groups at study (i.e., the healthy group from the steady-state group of risk disease and from the atherosclerotic one). As shown by us, the usefulness of biochemical and complementary genetic markers is warranted for a better knowledge on atherosclerosis molecular basis.

Effect of oxidized LDL on erythrocyte nitric oxide metabolism.

AIMS: Oxidized low density lipoprotein (ox-LDL) has been reported as an inhibitor of nitric oxide (NO)-mediated dilatation in microcirculation. Oxidized LDL effect on NO metabolism of erythrocytes is not known. Therefore, this study aims to evaluate the effect of ox-LDL on erythrocytes NO metabolism. METHODS: The effect of different concentrations of human purified ox-LDL (25, 50 and 100 µg/mL) on NO metabolism was evaluated on blood of healthy subjects. RESULTS: An inhibitory effect of higher concentrations of ox-LDL on erythrocyte NO efflux levels was verified. Concentrations of NO efflux from erythrocytes were lower as consequence of treatments with 50 µg/mL ox-LDL treatment (1.6±0.27 nM) and 100 µg/mL ox-LDL treatment (1.3±0.22 nM) than control (1.9±0.28 nM). Opposite, ox-LDL incubation has a positive effect on GSNO content of erythrocytes. That effect is proportional to concentrations of ox-LDL treatments (10.8±1.4 nM for 25 µg/mL, 12.9±1.5 nM for 50 µg/mL and 12.1±1.9 nM for 100 µg/mL) and is significant relative to control (8.56±0.76 µM) and ACh (8.9±0.52 µM) aliquots. CONCLUSIONS: Presence of oxidized LDL in erythrocyte NO metabolism induces a decrease of NO efflux amount and an increase on intra-erythrocyte GSNO concentrations. These results suggest a role of ox-LDL in mobilization of NO between NO derivatives molecules in dependence of oxidized LDL concentration. An anti - reactive nitrogene role can be attributed to ox-LDL for its contribution in the erythrocyte scavenged ability for nitric oxide.

An ex vivo study of nitric oxide efflux from human erythrocytes in both genders.

INTRODUCTION: Acetylcholinesterase (AChE) is located on outer surface of erythrocyte membrane. Gender-related differences in erythrocyte AChE enzyme activity had been verified in young adults. It is also known that binding of acetylcholine (ACh) with AChE on erythrocyte membrane initiates a signal transduction mechanism that stimulates nitric oxide (NO) efflux. AIMS: This ex vivo study was done to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females. METHODS: We included 66 gender age-matched healthy donors (40-60 years old). We performed quantification of erythrocyte NO efflux from erythrocytes and of the membrane AChE enzyme activity. RESULTS: There are no significant differences in NO efflux from erythrocytes between men and women. Regarding AChE enzyme activity values, in this range of age, no differences between genders were obtained. However, the values of AChE enzyme activity in the third quartile of NO efflux values were significantly higher (p < 0.05) in women than in men. CONCLUSIONS: The efflux of NO from erythrocyte of healthy humans did not change with gender. For the same range of values of NO efflux from erythrocytes, in both gender, it was verified higher values of AChE enzyme activity in women.

Soluble CD40 ligand profiles in patients with septic shock.

AIM: Soluble CD40 ligand (sCD40L) has been considered as a marker of thrombosis and inflammation in several diseases, including sepsis. Recent studies challenge this view and point to a role of sCD40L in vascular and endothelial function. An indication of that association in sepsis has not been obtained so far. Therefore, herein we evaluated association between sCD40L and markers of hemorheology and inflammation on context of septic shock. METHODS: Time-changes of sCD40L levels over 72 hours of Intensive Care Unit (ICU) internment were assessed in 22 patients with septic shock and compared with 36 healthy volunteers. Association of sCD40L levels with erythrocyte deformability and aggregation (as markers of hemorheology), plasma concentrations of haemoglobin (Hb, as markers of endothelial function) and white blood cells (WBC) count (as marker of low-grade inflammation) were assessed in patients with septic shock. RESULTS: At ICU admission, sCD40L concentrations in patients with septic shock were lower (p = 0.024) than levels of healthy volunteers. However, sCD40L did not change over 72 hours of internment (F = 2.1, p = 0.137). Soluble CD40L levels in patients with septic shock at ICU admission correlate with concentrations of Hb (r = 0.61, p = 0.00) and WBC count (r = 0.63, p = 0.00), but not to erythrocyte deformability (r≥0.157, p≤0.235) and aggregation (r≥-0.109, p≤0.192). CONCLUSIONS: These results seem to highlight a possible association of sCD40L to endothelial function and inflammation in septic shock context.

Erythrocyte nitric oxide in glaucoma patients - ex vivo study.

BACKGROUND: Glaucoma is an optic neuropathy associated with vascular dysregulation and increased intra-ocular pressure (IOP). Timolol is used as treatment for reducing IOP, by limiting aqueous humour production. Increased NOS expression as well as decreased levels of nitric oxide (NO) metabolites, and high activity of erythrocyte acetylcholinesterase (AChE) were observed in primary open angle glaucoma patients. OBJECTIVE: This ex vivo study aims to evaluate timolol effect in NO efflux and its derivatives in glaucoma patient's erythrocytes. METHODS: Venous blood from 15 glaucoma patients was collected. Erythrocyte suspensions were incubated with the AChE modulators acetylcholine (ACh) and timolol at 10 µM. Erythrocyte NO efflux and S-nitrosoglutathione (GSNO) concentration were measured. RESULTS: No significant differences were obtained in erythrocyte NO efflux and GSNO concentration in response to ACh or timolol when compared with the untreated erythrocytes of glaucoma patients. When comparing the same incubation conditions for erythrocyte suspensions between glaucoma patients and healthy subjects, those from glaucoma patients showed higher NO efflux in presence and absence of timolol, and higher values of GSNO in the presence of timolol. CONCLUSIONS: We demonstrated that erythrocytes from glaucoma patients have similar availability to release NO both in absence and presence of timolol, and have higher GSNO values in presence of timolol.

Identification of erythrocyte biomarkers in amyotrophic lateral sclerosis.

INTRODUCTION: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. It has been hypothesised that red blood cells (RBCs) may be involved in the disease process by the release of damaging molecules. OBJECTIVE: The aim of this ex vivo study is to compare RBCs biochemical and hemorheological parameters between ALS patients and healthy donors to identify novel biomarkers of the ALS disease. METHODS: We included 82 ALS patients and 40 gender age-matched healthy donors. We performed quantification of erythrocyte aggregation and deformability, nitric oxide (NO) efflux from RBCs, acetylcholinesterase (AChE) enzyme activity and intraerythrocytic concentration of nitrite, nitrate and S-nitrosogluthatione (GSNO). RESULTS: Erythrocyte deformability and AChE activity were increased in patients with ALS in comparison to healthy donors. NO efflux from RBCs and concentration of intraerythrocytic nitrite were lower in ALS patients. In patients, we found that for higher NO range of values the respiratory function is worse and that for higher AChE range of values the RBCs nitrite content increase. CONCLUSION: The results of the present study indicate that NO efflux from RBCs and RBCs AChE should be further explored as potential biomarkers for ALS.

Stratification of ST-elevation myocardial infarction patients based on soluble CD40L longitudinal changes.

Involvement of soluble CD40 ligand (sCD40L) in thrombosis and inflammation on the context of coronary artery disease is currently being revised. In that perspective, we had studied the association of sCD40L with markers of platelet activation and markers of endothelial and vascular function. On that cohort, a stratification of patients with acute myocardial infarction (AMI) 1 month after percutaneous coronary intervention (PCI) was observed based on concentrations of sCD40L. The study intended to identify the groups of AMI patients with different profiles of sCD40L concentrations and verify how medication, clinical evolution, biochemical data, and markers of regulation of endothelial function at genetic (endothelial nitric oxide synthase polymorphisms) and post-transcriptional levels (circulating microRNAs) affect sCD40L serum levels. Lower quartiles of sCD40L (<2.3 ng/mL) were associated with higher concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP), high frequency of G894T polymorphism, and altered expression of a set of microRNAs assumed to be involved in the regulation of endothelial and cardiac function and myocardium hypertrophy, relative to patients in sCD40L upper quartiles. A characteristic sCD40L variation pattern in STEMI patients was identified. Low levels of sCD40L 1 month after PCI distinguish STEMI patients with worse prognosis, a compromised cardiac healing, and a persistent endothelial dysfunction, as given by the association between sCD40L, NT-proBNP, G894T polymorphism, and specific profile of miRNA expression. These results suggest sCD40L could have a prognostic value in STEMI patients.