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We present a comprehensive multi-omic analysis of the EPISTOP prospective clinical trial of early intervention with vigabatrin for pre-symptomatic epilepsy treatment in Tuberous Sclerosis Complex (TSC), in which 93 infants with TSC were followed from birth to age 2 years, seeking biomarkers of epilepsy development. Vigabatrin had profound effects on many metabolites, increasing serum deoxycytidine monophosphate (dCMP) levels 52-fold. Most serum proteins and metabolites, and blood RNA species showed significant change with age. Thirty-nine proteins, metabolites, and genes showed significant differences between age-matched control and TSC infants. Six also showed a progressive difference in expression between control, TSC without epilepsy, and TSC with epilepsy groups. A multivariate approach using enrollment samples identified multiple 3-variable predictors of epilepsy, with the best having a positive predictive value of 0.987. This rich dataset will enable further discovery and analysis of developmental effects, and associations with seizure development in TSC., (© 2023. The Author(s).)

Atherosclerosis is associated with a haemostatic imbalance characterized by excessive activation of pro-inflammatory and pro-coagulant pathways. Non-vitamin K antagonists oral anticoagulant (NOACs) may reduce the incidence of cardiovascular events, cerebral ischemia, thromboembolic events and atherosclerosis. Chronic inflammation, vascular proliferation and the development of atherosclerosis is also influenced by 25-hydroxycholesterol (25-OHC). The aim of the study was to assess the effect of rivaroxaban and dabigatran on the messenger RNA (mRNA) expression of anti-inflammatory cytokines transforming growth factor β (TGF-β), interleukin (IL)-37, IL-35 as well as of pro-inflammatory cytokines IL-18 and IL-23, in endothelial cells damaged by 25-OHC. Human umbilical vascular endothelial cells (HUVECs) were treated with 25-OHC (10 μg/mL), rivaroxaban (100, 500 ng/mL), dabigatran (100, 500 ng/mL), 25-OHC + rivaroxaban, and 25-OHC + dabigatran. The mRNA expression of TGF-β, IL-37, IL-35 subunits EBI3 and p35, IL-18, and IL-23 was analysed using real-time polymerase chain reaction (PCR). The results showed that 25-OHC decreased TGF-β and IL-37 mRNA expression and increased EBI3, p35, IL-18, IL-23 mRNA expression in endothelial cell as compared to an untreated control (P < .05). Messenger RNA expression of TGF-β and IL-37 significantly increased following stimulation with rivaroxaban and dabigatran as compared to an untreated control (P < .01). In HUVECs pre-treated with oxysterol, rivaroxaban and dabigatran increased mRNA expression of TGF-β, IL-37 and decreased mRNA expression of EBI3, p35, IL-23 and IL-18 as compared to 25-OHC (P < .01). Our finding suggests that both rivaroxaban and dabigatran inhibit the inflammatory activation caused by oxysterol in vitro., (© 2022 John Wiley & Sons Australia, Ltd.)

Recent data showed that dabigatran can reduce not only procoagulatory effects but also block proinflammatory stimuli by inhibiting the expression of cytokines and chemokines and reducing thrombin-induced endothelial permeability. The aim of our study was to assess the effect of dabigatran on the integrity and inflammatory properties of endothelial cells stimulated by 25-hydroxycholesterol (25-OHC, oxysterol). HUVECs (Human Umbilical Vein Endothelial Cells) were stimulated with 25-hydroxycholesterol 10 µg/ml, dabigatran 100 ng/ml or 500 ng/ml and 25-hydroxycholesterol + dabigatran (100 ng/ml, 500 ng/ml). HUVEC integrity and permeability was measured in the RTCA-DP xCELLigence system and by the paracellular flux system. The mRNA expression of ICAM-1, VEGF, IL-33, MCP-1 and TNF-α was analyzed by Real-time PCR. Cell apoptosis and viability was measured by flow cytometry. VEGF protein concentration was assessed in supernatants by ELISA. VE-cadherin expression in endothelial cells was evaluated by confocal microscopy. Pre-stimulation of HUVECs with 25-OHC decreased endothelial cell integrity (p < 0.001) and increased the expression of IL-33, ICAM-1, MCP-1, VEGF, TNF-α mRNA (p < 0.01) compared to unstimulated controls. Following stimulation of HUVECs with dabigatran 100 ng/ml or 500 ng/ml restored HUVEC integrity interrupted by 25-OHC (p < 0.001). In HUVECs pre-stimulated with oxysterol, dabigatran stimulation decreased mRNA expression of the proinflammatory cytokines IL-33 and TNF-α, chemokines MCP-1 ICAM-1 and VEGF (p < 0.01). Dabigatran 500 mg/ml+ 25-OHC increased the endothelial expression of VE-cadherin as compared to 25-OHC (p < 0.01). Our findings suggest that dabigatran stabilizes the endothelial barrier and inhibits the inflammation caused by oxysterol., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Rivaroxaban is one of the direct factor Xa inhibitors. Its function in the inactivated coagulation cascade is unclear. The aim of the study was to assess the effect of rivaroxaban on the endothelial integrity and inflammatory properties of endothelial cells stimulated by 25-hydroxycholesterol (25-OHC)., Methods: HUVECs were stimulated with 25-OHC, rivaroxaban and 25-OHC+ rivaroxaban. HUVEC integrity and permeability were measured using the xCELLigence system and paracellular flux assay. The mRNA expression of tissue factor, ICAM-1, VEGF, IL-33, MCP-1, TNF-α was analyzed in the real-time PCR. Apoptosis and viability were measured by flow cytometry. The VEGF protein concentration was assessed by ELISA. The confocal microscope was used to evaluate the expression of VE-cadherin in endothelial cells., Results: 25-OHC decreased endothelial cell integrity and increased the mRNA expression of IL-33, tissue factor, ICAM-1, MCP-1, VEGF, TNF-α as compared to unstimulated controls. Following the stimulation with rivaroxaban, HUVEC restored integrity disrupted by 25-OHC ( p < .01). In HUVECs pre-stimulated with oxysterol, rivaroxaban decreased mRNA expression of IL-33, TNF-α, chemokines MCP-1, ICAM-1, VEGF and tissue factor ( p < .01). Rivaroxaban 100 mg/ml+25-OHC increased the VE-cadherin expression in endothelium as compared to 25-OHC ( p < .05)., Conclusion: Our finding suggests that rivaroxaban may restore the endothelial barrier and inhibit the inflammatory activation caused by oxysterol in vitro .

Objective: To study the association between timing and characteristics of the first electroencephalography (EEG) with epileptiform discharges (ED-EEG) and epilepsy and neurodevelopment at 24 months in infants with tuberous sclerosis complex (TSC)., Methods: Patients enrolled in the prospective Epileptogenesis in a genetic model of epilepsy - Tuberous sclerosis complex (EPISTOP) trial, had serial EEG monitoring until the age of 24 months. The timing and characteristics of the first ED-EEG were studied in relation to clinical outcome. Epilepsy-related outcomes were analyzed separately in a conventionally followed group (initiation of vigabatrin after seizure onset) and a preventive group (initiation of vigabatrin before seizures, but after appearance of interictal epileptiform discharges [IEDs])., Results: Eighty-three infants with TSC were enrolled at a median age of 28 days (interquartile range [IQR] 14-54). Seventy-nine of 83 patients (95%) developed epileptiform discharges at a median age of 77 days (IQR 23-111). Patients with a pathogenic TSC2 variant were significantly younger (P-value .009) at first ED-EEG and more frequently had multifocal IED (P-value .042) than patients with a pathogenic TSC1 variant. A younger age at first ED-EEG was significantly associated with lower cognitive (P-value .010), language (P-value .001), and motor (P-value .013) developmental quotients at 24 months. In the conventional group, 48 of 60 developed seizures. In this group, the presence of focal slowing on the first ED-EEG was predictive of earlier seizure onset (P-value .030). Earlier recording of epileptiform discharges (P-value .019), especially when multifocal (P-value .026) was associated with higher risk of drug-resistant epilepsy. In the preventive group, timing, distribution of IED, or focal slowing, was not associated with the epilepsy outcomes. However, when multifocal IEDs were present on the first ED-EEG, preventive treatment delayed the onset of seizures significantly (P-value <.001)., Significance: Early EEG findings help to identify TSC infants at risk of severe epilepsy and neurodevelopmental delay and those who may benefit from preventive treatment with vigabatrin., (© 2021 International League Against Epilepsy.)

Objective: Epilepsy develops in 70 to 90% of children with tuberous sclerosis complex (TSC) and is often resistant to medication. Recently, the concept of preventive antiepileptic treatment to modify the natural history of epilepsy has been proposed. EPISTOP was a clinical trial designed to compare preventive versus conventional antiepileptic treatment in TSC infants., Methods: In this multicenter study, 94 infants with TSC without seizure history were followed with monthly video electroencephalography (EEG), and received vigabatrin either as conventional antiepileptic treatment, started after the first electrographic or clinical seizure, or preventively when epileptiform EEG activity before seizures was detected. At 6 sites, subjects were randomly allocated to treatment in a 1:1 ratio in a randomized controlled trial (RCT). At 4 sites, treatment allocation was fixed; this was denoted an open-label trial (OLT). Subjects were followed until 2 years of age. The primary endpoint was the time to first clinical seizure., Results: In 54 subjects, epileptiform EEG abnormalities were identified before seizures. Twenty-seven were included in the RCT and 27 in the OLT. The time to the first clinical seizure was significantly longer with preventive than conventional treatment [RCT: 364 days (95% confidence interval [CI] = 223-535) vs 124 days (95% CI = 33-149); OLT: 426 days (95% CI = 258-628) vs 106 days (95% CI = 11-149)]. At 24 months, our pooled analysis showed preventive treatment reduced the risk of clinical seizures (odds ratio [OR] = 0.21, p = 0.032), drug-resistant epilepsy (OR = 0.23, p = 0.022), and infantile spasms (OR = 0, p < 0.001). No adverse events related to preventive treatment were noted., Interpretation: Preventive treatment with vigabatrin was safe and modified the natural history of seizures in TSC, reducing the risk and severity of epilepsy. ANN NEUROL 2021;89:304-314., (© 2020 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder with a high risk of early-onset epilepsy and a high prevalence of neurodevelopmental comorbidities, including intellectual disability and autism spectrum disorder (ASD). Therefore, TSC is an interesting disease model to investigate early biomarkers of neurodevelopmental comorbidities when interventions are favourable. We investigated whether early EEG characteristics can be used to predict neurodevelopment in infants with TSC. The first recorded EEG of 64 infants with TSC, enrolled in the international prospective EPISTOP trial (recorded at a median gestational age 42 4/7 weeks) was first visually assessed. EEG characteristics were correlated with ASD risk based on the ADOS-2 score, and cognitive, language, and motor developmental quotients (Bayley Scales of Infant and Toddler Development III) at the age of 24 months. Quantitative EEG analysis was used to validate the relationship between EEG background abnormalities and ASD risk. An abnormal first EEG (OR = 4.1, p -value = 0.027) and more specifically a dysmature EEG background (OR = 4.6, p -value = 0.017) was associated with a higher probability of ASD traits at the age of 24 months. This association between an early abnormal EEG and ASD risk remained significant in a multivariable model, adjusting for mutation and treatment (adjusted OR = 4.2, p -value = 0.029). A dysmature EEG background was also associated with lower cognitive ( p -value = 0.029), language ( p -value = 0.001), and motor ( p -value = 0.017) developmental quotients at the age of 24 months. Our findings suggest that early EEG characteristics in newborns and infants with TSC can be used to predict neurodevelopmental comorbidities., (Copyright © 2020 De Ridder, Lavanga, Verhelle, Vervisch, Lemmens, Kotulska, Moavero, Curatolo, Weschke, Riney, Feucht, Krsek, Nabbout, Jansen, Wojdan, Domanska-Pakieła, Kaczorowska-Frontczak, Hertzberg, Ferrier, Samueli, Benova, Aronica, Kwiatkowski, Jansen, Jóźwiak, Van Huffel and Lagae.)

Objective: To evaluate the relationship between age at seizure onset and neurodevelopmental outcome at age 24 months in infants with TSC, as well as the effect on neurodevelopmental outcome of early versus conventional treatment of epileptic seizures with vigabatrin (80-150 mg/kg/day)., Methods: Infants with TSC, aged ≤4 months and without previous seizures were enrolled in a prospective study and closely followed with monthly video EEG and serial standardized neurodevelopmental testing (Bayley Scales of Infant Development and Autism Diagnostic Observation Schedule)., Results: Eighty infants were enrolled. At the age of 24 months testing identified risk of Autism Spectrum Disorder (ASD) in 24/80 children (30.0%), and developmental delay (DD) in 26/80 (32.5%). Children with epilepsy (51/80; 63.8%) had a higher risk of ASD (P = 0.02) and DD (P = 0.001). Overall, no child presented with moderate or severe DD at 24 months (developmental quotient < 55). In 20% of children abnormal developmental trajectories were detected before the onset of seizures. Furthermore, 21% of all children with risk of ASD at 24 months had not developed seizures at that timepoint. There was no significant difference between early and conventional treatment with respect to rate of risk of ASD (P = 0.8) or DD (P = 0.9) at 24 months., Interpretation: This study confirms a relationship between epilepsy and risk of ASD/DD. However, in this combined randomized/open label study, early treatment with vigabatrin did not alter the risk of ASD or DD at age 2 years., (© 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Postprandial hypertriglyceridemia is an independent risk factor for cardiovascular disease. The aim of this study was to assess the effects of a single fat-rich meal on barrier functions and inflammatory status on human umbilical vascular endothelial cells (HUVECs), furthermore we assess the effects of mixture of palmitic acid and 25-hydroxycholesterol (PA +25OHCH) on integrity of endothelial cells and their inflammatory properties. HUVECs were induced with serum of healthy volunteers taken before, and 3 h after, the consumption of a meal with a standardized daily required dose of fats. In addition, endothelial cells were induced with PA +25OHCH (800 μM/L+10 μg/mL). Total cholesterol, triglycerides (TGs), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, high sensitivity c-reactive protein, and glucose were measured at fasting and postprandially. HUVEC integrity was measured in the RTCA-DP xCELLigence system. mRNA expression of interleukin (IL)-33, IL-32, intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), CX3C-chemokine, vascular endothelial growth factor (VEGF) occludin, and VE-cadherin was analyzed by real-time polymerase chain reaction. Viability and apoptosis were assessed in flow cytometry. The level of VEGF and IL-33 in fasting and postprandial serum was assessed by enzyme-linked immunosorbent assay (ELISA). Three hours after consumption of a fatty meal, all patients displayed increased levels of TGs and Toll-like receptors (110 ± 37 mg/dL versus 182 ± 64 mg/dL P  < 0.05) (24 ± 11 mg/dL versus 42 ± 14 mg/dL P  < 0.05). Postprandial serum and PA +25OHCH caused >20% decrease of HUVEC integrity than fasting serum ( P  < 0.001). HUVEC disintegration was accompanied by a decrease of occludin mRNA expression as compared with fasting serum ( P  < 0.05). The fatty meal affected neither VE-cadherin mRNA expression nor its apoptosis ( P  > 0.05). Mixture of PA +25OHCH caused decrease of VE-cadherin mRNA expression as compared with fasting serum ( P  < 0.01). PA +25OHCH did not affect HUVEC apoptosis ( P  > 0.05). Postprandial serum and PA +25OHCH caused increase of IL-33, MCP-1, ICAM-1, IL-32, VEGF, and CX3C-chemokine mRNA expression as compared with fasting serum ( P  < 0.05). Moreover, level of VEGF in fatty serum was significantly higher ( P  < 0.001). Postprandial lipemia after a single fatty meal may destabilize the endothelial barrier and initiate inflammatory processes.

Atherogenesis is associated with chronic gut infections; however, the mechanisms are not clear. The aim of the study was to determine whether lipopolysaccharide of E. coli (E. coli LPS) may affect endothelial barrier and modify IL-10 expression in dendritic cells (DCs). Human umbilical vein endothelial cells (HUVECs) and monocyte-derived DCs were treated with E. coli LPS, apolipoprotein B100 (ApoB100) and 7-ketocholesterol (7-kCH) - harmful oxidized form of cholesterol. The effect of E. coli LPS, 7-kCH and ApoB100 on the barrier functions of HUVECs in real-time cell electric impedance sensing system (RTCA-DP) was assessed. Furthermore, the effect of 7-kCH and ApoB100 on barrier functions of HUVECs co-cultured with DCs previously treated with LPS was analyzed. Both E. coli LPS and 7-kCH decreased barrier functions of HUVECs and reduced tight junction protein mRNA expression, whereas ApoB100 increased endothelial barrier. In DCs, ApoB100 and E. coli LPS decreased IL-10 mRNA expression. In HUVECs co-cultured with DCs treated with LPS and subsequently pulsed with ApoB100 or 7-kCH, IL-10 mRNA expression was lower. E. coli LPS-exposed DCs diminished the protective effect of ApoB100 on endothelial integrity and led to the decrease in occludin mRNA expression. LPS potentially derived from gut microflora may destabilize endothelial barrier together with oxidized cholesterol and intensify the immunogenicity of ApoB100., (© 2019 APMIS. Published by John Wiley & Sons Ltd.)

Background: FoxP3 is a marker of human T regulatory cells (Tregs), which are supposed to play an important role in the pathophysiology of atherosclerosis. Interleukin 10 (IL-10) is a cytokine with pleiotropic, immunoregulatory properties, produced mostly by Tregs and B regulatory cells. Due to their anti-inflammatory action, both Tregs and IL-10 are believed to inhibit plaque development and decrease atherosclerosis progression. The effect of hypolipidemic drugs - statins or ezetimibe - on FoxP3-positive Tregs and anti-inflammatory cytokines, such as IL-10, is still unclear., Objectives: The objective of the study was to investigate the effects of 3 different therapies of equivalent hypolipidemic activity: atorvastatin, rosuvastatin, and combination therapy of atorvastatin and ezetimibe on FoxP3-Tregs transcription factor and IL-10 mRNA expression in peripheral blood mononuclear cells (PBMCs) from patients with stable coronary artery disease (CAD)., Material and Methods: Sixty-five patients with diagnosed CAD participated in the study. They were randomly assigned to 3 therapeutic groups: atorvastatin at a dose of 40 mg/day (A40 group); rosuvastatin 20 mg/day (R20 group); and atorvastatin 10 mg/day combined with ezetimibe 10 mg/day (A10+E10 group). After 1 month and 6 months of therapy, the mRNA expression for FoxP3 and IL-10 in PBMCs was evaluated using real-time polymerase chain reaction (RT-PCR) and lipid parameters., Results: An improvement in lipid parameters was observed in each of the groups studied; however, hypolipidemic treatment did not induce any change in FoxP3 and IL-10 mRNA expression. After 6 months, an increase in FoxP3 mRNA expression was noted in A40 group as compared to R20 group., Conclusions: None of the therapies of equal hypolipidemic efficacy affected FoxP3 and IL-10 mRNA expression in patients with stable CAD.

Autism spectrum disorder (ASD) is highly prevalent in subjects with Tuberous Sclerosis Complex (TSC), but we are not still able to reliably predict which infants will develop ASD. This study aimed to identify the early clinical markers of ASD and/or developmental delay (DD) in infants with an early diagnosis of TSC. We prospectively evaluated 82 infants with TSC (6-24 months of age), using a detailed neuropsychological assessment (Bayley Scales of Infant Development-BSID, and Autism Diagnostic Observation Schedule-ADOS), in the context of the EPISTOP (Long-term, prospective study evaluating clinical and molecular biomarkers of EPIleptogenesiS in a genetic model of epilepsy-Tuberous SclerOsis ComPlex) project (NCT02098759). Normal cognitive developmental quotient at 12 months excluded subsequent ASD (negative predictive value 100%). The total score of ADOS at 12 months clearly differentiated children with a future diagnosis of ASD from children without ( p = 0.012). Atypical socio-communication behaviors ( p < 0.001) were more frequently observed than stereotyped/repetitive behaviors in children with ASD at 24 months. The combined use of BSID and ADOS can reliably identify infants with TSC with a higher risk for ASD at age 6-12 months, allowing for clinicians to target the earliest symptoms of abnormal neurodevelopment with tailored intervention strategies.

Aim: The proteins accumulated in the meconium reflect the intrauterine environment and are naturally excreted by a neonate. The identification and classification of individual meconium proteins may be a valuable source of information about physiological and pathological processes in utero., Methods: Proteomic analysis was used to study the protein composition in pooled 50 serial meconium portions from 10 neonates. The proteins were classified based on the gene ontology database. The amounts and relative number of proteins (%) in the identified categories and their subcategories were assessed., Results: A total of 946 proteins identified in meconium, including 430 represented by two or more peptides were classified into three categories: biological process (n = 401), molecular function (n = 386) and cellular component (n = 422). The highest number of proteins (>25% of the total) was found in the subcategories: developmental processes, signaling, transport, response to stimulus, regulation, metabolic processes, ion binding, extracellular region, membrane and cytoplasm., Conclusion: The composition of meconium proteins identified in this study may be a rich source of new biomarkers for use in neonatology with a potential to predict later development., (© 2018 Japan Society of Obstetrics and Gynecology.)

Introduction: Atherosclerosis leading to coronary artery disease (CAD) is a chronic inflammatory condition. Interleukin 35 (IL-35) released by regulatory T cells (Tregs) has been found to be associated with CAD in the Chinese population. However, nothing is known about the relation between IL-35 concentrations and cholesterol levels. The aim of the study was to assess the levels of IL-35 in CAD patients and healthy subjects from a Caucasian population, and to analyze the relationship between IL-35 and the levels of total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, left ventricular ejection fraction (LVEF), sex and postmenopausal status., Material and Methods: Thirty-one patients with CAD and 30 healthy controls were included in the study. Levels of plasma IL-35 were analyzed by ELISA. The LVEF was assessed by transthoracic echocardiographic examination. Plasma levels of cholesterol fractions and C-reactive protein (CRP) were assessed by immunoenzymatic methods., Results: The CAD patients had higher levels of IL-35 as compared to healthy controls (58.1 ±16.6 pg/ml vs. 5.35 ±3.35 pg/ml; p < 0.001). IL-35 levels negatively correlated with total and LDL cholesterol concentrations ( R = -0.31, p < 0.01) and positively correlated with HDL cholesterol in men ( R = 0.53, p < 0.01). In women, IL-35 levels negatively correlated with LVEF ( R = -0.29, p < 0.05) and positively with the duration of postmenopausal status ( R = 0.55, p < 0.01)., Conclusions: These results suggest a possible association between high levels of IL-35 and CAD., Competing Interests: The authors declare no conflict of interest.

Background: IL-22 is expressed at barrier surfaces, which suggests its critical role in the maintenance of normal barrier homeostasis and tissue repair. IL-22 can both promote pathological inflammation and prevent the destruction of tissues. The functional outcomes of IL-22 on vascular smooth muscle cells, which are shown to regulate immune processes within the vascular wall and which are involved in certain pathologies, have not been analyzed., Objectives: The effect of IL-22 on the expression of novel antiand pro-inflammatory and barrier disrupting cytokines, apoptosis and the expression of adhesive molecules in human primary aortic smooth muscle cells (AoSMC) was investigated., Material and Methods: Human AoSMC were induced with IL-22 for 24 h in vitro. The influence of IL-22 on IL-35 subunits EBI3 and p35, IL-33, IFN-γ and VEGF mRNA expression in Ao-SMC were assessed using real-time PCR. Additionally, the surface expression of ICAM-1 and apoptosis of AoSMC were analyzed in the flow cytometer., Results: IL-22 caused a 2- and 3-fold increase of mRNA expression of the EBI3 and p35 IL-35 subunits, and a 40% decrease of IL-33 mRNA expression in AoSMC. Additionally, IL-22 decreased ICAM-1 expression on the surface of AoSMC by 30%. However, IL-22 affected neither IFN-γ and VEGF mRNA expression in AoSMC nor their apoptosis and viability., Conclusions: Our data suggest that IL-22, which is released by Th22 and NK cells, may be an agent affecting the inflammatory response of AoSMC, and thus it may regulate immune homeostasis of the vascular wall.

Background: Human vascular endothelial function and integrity may be regulated by many non-specific factors. However, the potential influence of specific antigens via an IgE-mediated mechanism remains unknown. The aim of the study was to determine the expression of the IgE receptors FcεRI and FcεRII in the human vascular endothelium and to assess their relevance in the IgE-mediated regulation of endothelial integrity., Material/methods: FcεRI and FcεRII expression in human umbilical vein endothelial cells (HUVEC) was genetically assessed by PCR with respective primers and sequencing. HUVEC were cultured with IL-4, and changes in FcεRI and FcεRII mRNA expression were analyzed by real-time PCR. Changes in the integrity of endothelium pre-treated with anti-BSA-DNP IgE following exposure to the specific BSA-DNP antigen was assessed using the Real-time Cell Electric Impedance Sensing system (RTCA-DP)., Results: PCR and sequencing revealed the expression of FcεRI and FcεRII receptors in the human vascular endothelium. IL-4 caused respective 2- and 3-fold increases in FcεRI and FcεRII mRNA expression. Exposure of endothelium pre-treated with anti-BSA-DNP IgE to specific BSA-DNP antigen led to a 20% increase of endothelial integrity (p<0.05) after 24 hours, but only in cells pre-incubated with IL-4., Conclusions: The presence of FcεRI and FcεRII may allow the human vascular endothelium to respond to a specific antigen by increasing its integrity via an IgE-mediated mechanism.

The low-grade inflammation present in obese visceral adipose tissue impairs glucose metabolism, and contributes to the development of insulin resistance and weight gain. Immune processes occurring in response to the deposition of cholesterol within the vascular walls support atherosclerotic plaque growth and contribute to the cardiovascular complications. In both the obese adipose tissue and the atherosclerotic plaque, the Th1-type immune environment dominates over the Th2/Treg-type due to the overproduction of pro-inflammatory cytokines (IFN-γ, IL-6, TNF-α) and the deficiency of Th2-type processes and interleukins (IL-4, IL-5, IL-10, IL-13). So far, Th2 cells and eosinophils have been considered as the main providers of Th2-type mediators and the basis of Th2-type immunity in tissues. However, recently discovered innate lymphoid type 2 cells (ILC2s), which infiltrate lean visceral adipose tissue and the vascular wall, are believed to orchestrate local Th2-like immune responses. Upon activation by tissue-derived IL-33 and IL-25, ILCs2 secrete mostly IL-4, IL-5, IL-9 and IL-13: cytokines responsible for the accumulation of eosinophils and polarization of alternatively-activated macrophages, which altogether create the beneficial anti-inflammatory and metabo-regulatory environment in the adipose tissue and the vascular wall. Consequently, ILC2s-orchestrated immune environment seems to prevent obesity and atherosclerosis. Thus, ILCs2 appear to be the emerging immune regulators of immune and metabolic homeostasis in both adipose tissue and the vascular wall., (Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

The vascular endothelium forms a barrier that controls flow of solutes and proteins and the entry of leukocytes into tissue. Injured tissue releases IL-33, which then alarms the immune system and attracts Th2 cells, thus increasing local concentration of IL-4. The aim of the study was to assess the influence of IL-33 and IL-4 on barrier functions of the human endothelium, expression of tight and adherent junction proteins, apoptosis and adhesive molecule surface expression in human endothelium in order to describe the mechanism of this effect. IL-33 and IL-4 decreased endothelial integrity and increased permeability. When added together, both cytokines lowered the endothelial integrity twice as much as used alone. This effect was accompanied by the down-regulation of occludin and VE-cadherin mRNA expression. Additionally, IL-4, but not IL-33, induced cell apoptosis. Both IL-33 and IL-4 showed the additive potency to down-regulate VE-cadherin mRNA expression. IL-33, unlike IL-4, increased the surface expression of ICAM-1, but not PECAM-1 in endothelial cells. Our results indicate that IL-33 may reversibly destabilize the endothelial barrier, thus accelerating the supply with immunomodulators and assisting leukocytes to reach wounded tissue. However, extended and less-controlled down-regulation of endothelial barrier, which may be a consequence of IL-33-initiated, but in fact IL-4-induced apoptosis of endothelial cells, may be deleterious and may eventually lead to the aggravation of inflammatory processes and the prolongation of tissue dysfunction., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Interleukin-35 (IL-35) is a novel immunomodulatory cytokine produced by CD4+ 25+ foxp3+ regulatory T-cells (T regs). Vascular smooth muscle cells (VSMCs) are involved in local immune homeostasis and certain chronic inflammatory pathologies. The effect of IL-35 on electrical impedance reflecting tissue integrity, the surface expression of ICAM-1 and mRNA expression of IL-32, as well as apoptosis in human primary aortic smooth muscle cells (Ao-SMCs) was investigated., Methods: The influence of IL-35 on Ao-SMC integrity was assessed with the real-time cell electric impedance sensing system (RTCA-DP) based on normalized Cell Index (nCI). Additionally, Ao-SMCs were stimulated with IL-35 in order to assess ICAM-1 surface expression and apoptosis in flow cytometer. IL-32 mRNA expression was measured using real-time PCR., Results: We found that the nCI of Ao-SMCs induced with IL-35 was lower after 12, 24 and 48h of incubation than the nCI of unstimulated cells. IL-35 slightly enhanced ICAM-1 surface expression and increased IL-32 mRNA expression in Ao-SMCs after 24h of induction. However, IL-35 did not affect Ao-SMC apoptosis, necrosis or viability., Conclusion: Our data suggest that IL-35 may be an agent affecting the inflammatory properties of AoSMCs and thus it may regulate immune homeostasis of the vascular wall. Hence, IL-35 may be a novel player affecting Ao-SMC-controlled arterial wall immune homeostasis., (Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

The overexpression or amplification of the human epidermal growth factor receptor 2 gene (HER2/neu) is associated with high risk of brain metastasis (BM). The identification of patients at highest immediate risk of BM could optimize screening and facilitate interventional trials. We performed gene expression analysis using complementary deoxyribonucleic acid-mediated annealing, selection, extension and ligation and real-time quantitative reverse transcription PCR (qRT-PCR) in primary tumor samples from two independent cohorts of advanced HER2 positive breast cancer patients. Additionally, we analyzed predictive relevance of clinicopathological factors in this series. Study group included discovery Cohort A (84 patients) and validation Cohort B (75 patients). The only independent variables associated with the development of early BM in both cohorts were the visceral location of first distant relapse [Cohort A: hazard ratio (HR) 7.4, 95 % CI 2.4-22.3; p < 0.001; Cohort B: HR 6.1, 95 % CI 1.5-25.6; p = 0.01] and the lack of trastuzumab administration in the metastatic setting (Cohort A: HR 5.0, 95 % CI 1.4-10.0; p = 0.009; Cohort B: HR 10.0, 95 % CI 2.0-100.0; p = 0.008). A profile including 13 genes was associated with early (≤36 months) symptomatic BM in the discovery cohort. This was refined by qRT-PCR to a 3-gene classifier (RAD51, HDGF, TPR) highly predictive of early BM (HR 5.3, 95 % CI 1.6-16.7; p = 0.005; multivariate analysis). However, predictive value of the classifier was not confirmed in the independent validation Cohort B. The presence of visceral metastases and the lack of trastuzumab administration in the metastatic setting apparently increase the likelihood of early BM in advanced HER2-positive breast cancer.

Introduction: The aim of this study was to evaluate the effect of melatonin on blood pressure in patients with essential hypertension receiving medical treatment and with type 2 diabetes in good metabolic control., Material and Methods: The study lasted 8 weeks. Patients were equipped with a 24-hour ambulatory blood pressure monitor and took melatonin (3 mg a day in the evening) for 4 weeks. The patients were divided into four groups: group 1 (n = 32) including dippers, group 2 (n = 34) non-dippers treated with melatonin; and two control groups: group 3 (n = 28) including dippers and group 4 (n = 30) non-dippers treated without melatonin. After 4 weeks patients took melatonin for the next 4 weeks (5 mg a day). In each visit were analyzed: systolic, diastolic and mean blood pressure in both day and night time., Results: We observed that 29.5% non-dippers (n = 10) treated with melatonin in a dose of 3 mg/day achieved features of dippers compared to control group (p < 0.05). Five mg of melatonin per day restored normal diurnal blood pressure rhythm in 32.4% non-dippers (n = 11, p < 0.05). In non-dippers treated with melatonin significant decreases of diastolic, systolic and mean night blood pressure values (p < 0.05) were observed., Conclusions: More than 30% of non-dippers with type 2 diabetes treated with melatonin were restored to the normal circadian rhythm of blood pressure. The effect of melatonin in both doses (3 mg and 5 mg) was significant for non-dippers only and included nocturnal systolic, diastolic and mean arterial pressure.

The intestinal epithelium is exposed to oxygenated cholesterol products present in foodstuffs. In vitro studies demonstrate the effect of oxysterols on cytokine release by intestinal cells cultured alone. However, physiologically, the response of the intestinal epithelium to external agents occurs in the presence of dendritic cells (DCs). The aim of the study was to analyze the effect of 7-ketocholesterol on the barrier functions and IL-10 mRNA expression of Caco-2 cells in the presence of DCs, and secondly, on IL-10 mRNA expression in DCs. Caco-2 cells were co-cultured with monocyte-derived dendritic cells and induced with 7-ketocholesterol in a transwell system. DCs did not affect the transepithelial electrical resistance (TER) of the Caco-2 cell monolayer, but increased IL-10 mRNA expression in Caco-2 cells. 7-ketocholesterol decreased the TER of Caco-2 cells co-cultured with DCs and diminished IL-10 mRNA expression in Caco-2 cells induced by the presence of DCs. IL-10 mRNA expression fell in DCs co-cultured with Caco-2 cells after treatment with 7-ketocholesterol. Oxidized cholesterols present in gut mucosa may contribute to the decrease of epithelial barrier functions and the inappropriate development of an inflammatory response to food compounds., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Gene expression profiling is one of the most explored methods for studying cancers and microarray data repositories have become a rich and important resource. The most common human cancers develop in organs that are walled by smooth muscles. The only method of sample extraction free of unintentional contamination with surrounding tissue is microdissection. Nevertheless, such an approach is implemented infrequently. In the light of the above, there is a possibility of smooth muscle contamination in a large portion of publicly available data. In this study, 2292 publicly available microarrays were analysed to develop a simple screening method for detecting smooth muscle contamination. Microarray Inspector software was used to perform the tests since it has the unique ability to use many selected genes and probesets in a single group as a tissue definition. Furthermore, the test was dataset-independent. Two strategies of tissue definition were explored and compared. The first one depended on Tissue Specific Genes Database (TiSGeD) and BioGPS web resources, which themselves were based on meta-analysis of thousands of microarrays. The second method was based on a differential gene expression analysis of a few hundred preselected arrays. The comparison of the two methods proved the latter to be superior. Among the tested samples of undefined contamination, nearly half were identified to possibly contain significant smooth muscle traces. The obtained results equip researches with a simple method of examining microarray data for smooth muscle contamination. The presented work serves as an example of how to create definitions when searching for other possible contaminations.

Objective and Design: The damage of barrtier tissues, such as the vascular endothelium and intestinal epithelium, may lead to disturbances of local immune homeostasis. The aim of the study was to assess and compare the effect of oxidized cholesterols (7-ketocholesterol and 25-hydroxycholesterol) on the barrier properties of human primary aortic endothelium (HAEC) and intestinal epithelium Caco-2 cells using a realtime cell electric impedance sensing system (RTCA-DP)., Materials and Methods: HAEC and Caco-2 cells were stimulated with 7-ketocholesterol and 25-hydroxycholesterol by the RTCA-DP system. Apoptosis was assessed by flow cytometry and cell monolayer morphology was assessed under a light microscope., Results: 7-ketocholesterol decreased impedance (nCI) in both the endothelium and epithelium. However, the decrease was more profound in the endothelium. Similarly, although 25-hydroxycholesterol decreased nCI in both the endothelium and epithelium, the effect was weaker than that of 7-ketocholesterol, which caused extensive damage to the endothelial monolayer, while 25-hydroxycholesterol caused partial damage and did not affect the epithelial monolayer. 7-ketocholesterol, but not 25-hydroxycholesterol, increased endothelial cell apoptosis and decreased the viability of endothelial cells. However, 7-ketocholesterol and 25-hydroxycholesterol decreased epithelial cell apoptosis and increased viability., Conclusion: Oxidized cholesterols destroy the HAEC, but not the Caco-2 epithelial barrier, via cell apoptosis dependent on the site of oxidation. Damage to the endothelium by oxidized cholesterol may disrupt local homeostasis and provide open access to inner parts of the vascular wall for lipids, other peripheral blood-derived agents, and immune cells, leading to inflammation and atherogenesis.

For many years atherosclerosis was believed to be the passive accumulation of cholesterol in vessel walls. Today the picture is more complex, as immune processes occur in atherogenesis. Considerable attention is focused on the particular role of adaptive immune responses orchestrated by T cell subsets. Since the role of Th1/Th2 balance and Th1 cell domination in atherogenesis is already known, the involvement of regulatory T lymphocytes and recently described Th17 cells raises new concerns. On one hand, each of these cells may specifically drive responses of vascular wall tissues and immune cells; however, they are subject to the control of a plethora of tissue- and pathogen-derived agents. Due to ineffective tissue regeneration, remodeling of the vascular wall occurs. The understanding of the immune regulatory network gives perspectives of innovative immunomodulatory therapies of atherosclerosis and the prevention of its complications, such as coronary artery disease.

Microarray technology changed the landscape of contemporary life sciences by providing vast amounts of expression data. Researchers are building up repositories of experiment results with various conditions and samples which serve the scientific community as a precious resource. Ensuring that the sample is of high quality is of utmost importance to this effort. The task is complicated by the fact that in many cases datasets lack information concerning pre-experimental quality assessment. Transcription profiling of tissue samples may be invalidated by an error caused by heterogeneity of the material. The risk of tissue cross contamination is especially high in oncological studies, where it is often difficult to extract the sample. Therefore, there is a need of developing a method detecting tissue contamination in a post-experimental phase. We propose Microarray Inspector: customizable, user-friendly software that enables easy detection of samples containing mixed tissue types. The advantage of the tool is that it uses raw expression data files and analyses each array independently. In addition, the system allows the user to adjust the criteria of the analysis to conform to individual needs and research requirements. The final output of the program contains comfortable to read reports about tissue contamination assessment with detailed information about the test parameters and results. Microarray Inspector provides a list of contaminant biomarkers needed in the analysis of adipose tissue contamination. Using real data (datasets from public repositories) and our tool, we confirmed high specificity of the software in detecting contamination. The results indicated the presence of adipose tissue admixture in a range from approximately 4% to 13% in several tested surgical samples.

Purpose: A diet rich in berries is believed to play a distinct role in the prevention of metabolic diseases associated with obesity. So far, there have been no published clinical observations evaluating the influence of Aronia melanocarpa on hemostasis. The aim of our study was to investigate the effects of A. melanocarpa extract (AM) supplementation on platelet aggregation, clot formation, and lysis in patients with metabolic syndrome (MS)., Methods: Middle-aged non-medicated subjects with MS (n = 38) and 14 healthy volunteers were included in this study. Patients with MS were treated with 100 mg of AM three times daily for 2 months., Results: We observed a significant reduction in the concentration of TC, LDL-C, and TG after AM supplementation. Beneficial changes in coagulation parameters were also observed. After 1 month of AM administration, we noticed significant inhibition of platelet aggregation. However, this effect became less pronounced after 2 months of supplementation. In the case of coagulation induced by endogenic thrombin, a significant decrease in the overall potential for coagulation was induced after 1 or 2 months of supplementation. Moreover, after 1 month of AM extract supplementation, we observed a beneficial reduction in the overall potential for clot formation and fibrinolysis., Conclusions: We observed the normalization of hemostasis parameters in MS patients after both 1 and 2 months of AM administration. After 1 month of AM supplementation, we found favorable changes in regards to the overall potential for plasma clotting, clot formation, and lysis, as well as in the lipid profiles of subjects.

Background: The prevalence of hypertension is growing at an alarming rate. Increasing attention is being focussed on the oxidative stress accompanying this disease. In this study we examined the impact of this disease on some parameters of erythrocytes and human blood plasma., Material/methods: We examined the impact of hypertension on some parameters of erythrocytes and human plasma. The study involved 13 patients with hypertension and 19 healthy subjects. We determined lipid peroxidation, SH groups concentration, antioxidants enzymes activity, ATPase activity, total antioxidant capacity, total cholesterol level and erythrocyte membrane fluidity., Results: We found an increased level of lipid peroxidation and the concentration of SH groups in membrane proteins in patients with hypertension, and a decrease in the activity of catalase and superoxide dysmutase. No changes were observed in glutathione peroxidase and ATPase activity, level of total antioxidant capacity, total cholesterol level and fluidity of erythrocyte membranes., Conclusions: These results suggest the existence of an impaired oxidative balance in hypertensive human erythrocytes.