Secretome of brain microvascular endothelial cells promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage.

Cell-based therapeutic strategies have been proposed as an alternative for brain and blood vessels repair after stroke, but their clinical application is hampered by potential adverse effects. We therefore tested the hypothesis that secretome of these cells might be used instead to still focus on cell-based therapeutic strategies. We therefore characterized the composition and the effect of the secretome of brain microvascular endothelial cells (BMECs) on primary in vitro human models of angiogenesis and vascular barrier. Two different secretome batches produced in high scale (scHSP) were analysed by mass spectrometry. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used as well as in vitro models of EC monolayer (CMECs) and blood-brain barrier (BBB). Cells were also exposed to oxygen-glucose deprivation (OGD) conditions and treated with scHSP during reoxygenation. Protein yield and composition of scHSP batches showed good reproducibility. scHSP increased CD34+-EC proliferation, tubulogenesis, and migration. Proteomic analysis of scHSP revealed the presence of growth factors and proteins modulating cell metabolism and inflammatory pathways. scHSP improved the integrity of CMECs, and upregulated the expression of junctional proteins. Such effects were mediated through the activation of the interferon pathway and downregulation of Wnt signalling. Furthermore, OGD altered the permeability of both CMECs and BBB, while scHSP prevented the OGD-induced vascular leakage in both models. These effects were mediated through upregulation of junctional proteins and regulation of MAPK/VEGFR2. Finally, our results highlight the possibility of using secretome from BMECs as a therapeutic alternative to promote brain angiogenesis and to protect from ischemia-induced vascular leakage.

25-Hydroxycholesterol attenuates tumor necrosis factor alpha-induced blood-brain barrier breakdown in vitro.

Intracellular cholesterol metabolism is regulated by the SREBP-2 and LXR signaling pathways. The effects of inflammation on these molecular mechanisms remain poorly studied, especially at the blood-brain barrier (BBB) level. Tumor necrosis factor α (TNFα) is a proinflammatory cytokine associated with BBB dysfunction. Therefore, the aim of our study was to investigate the effects of TNFα on BBB cholesterol metabolism, focusing on its underlying signaling pathways. Using a human in vitro BBB model composed of human brain-like endothelial cells (hBLECs) and brain pericytes (HBPs), we observed that TNFα increases BBB permeability by degrading the tight junction protein CLAUDIN-5 and activating stress signaling pathways in both cell types. TNFα also promotes cholesterol release and decreases cholesterol accumulation and APOE secretion. In hBLECs, the expression of SREBP-2 targets (LDLR and HMGCR) is increased, while ABCA1 expression is decreased. In HBPs, only LDLR and ABCA1 expression is increased. TNFα treatment also induces 25-hydroxycholesterol (25-HC) production, a cholesterol metabolite involved in the immune response and intracellular cholesterol metabolism. 25-HC pretreatment attenuates TNFα-induced BBB leakage and partially alleviates the effects of TNFα on ABCA1, LDLR, and HMGCR expression. Overall, our results suggest that TNFα favors cholesterol efflux via an LXR/ABCA1-independent mechanism at the BBB, while it activates the SREBP-2 pathway. Treatment with 25-HC partially reversed the effect of TNFα on the LXR/SREBP-2 pathways. Our study provides novel perspectives for better understanding cerebrovascular signaling events linked to BBB dysfunction and cholesterol metabolism in neuroinflammatory diseases.

Sexual risk behavior and knowledge about sexually transmitted infections in a university population in Sorocaba, Brazil / Comportamento sexual de risco e conhecimento sobre infecções sexualmente transmissíveis em uma população universitária na cidade de Sorocaba/SP

INTRODUCTION: Sexually transmitted infections (STIs) are a major public health problem to which young people are highly exposed and knowledge about vulnerabilities that affect them is needed. OBJECTIVE: To evaluate the knowledge about STIs and sexual behavior of a university population in the city of Sorocaba/SP. METHODS: A descriptive, cross-sectional study was conducted with data collection realized by an online application with qualitative and quantitative characteristics. RESULTS: Four hundred and seventy-seven (477) university students from different areas of knowledge were analyzed. The majority pointed to the beginning of sexual life between 15 and 18 years old. Information about sex education was obtained mainly through parents and/ or guardians, while little additional knowledge was obtained after entering higher education. Biological and Health Sciences students achieved a higher score on the knowledge questionnaire and were less likely (0.391) to contract STIs when compared to Applied Social Sciences or Engineering students (2.8 and 2.9 more likely, respectively). CONCLUSION: Students who demonstrated greater knowledge about STIs and acquired more information on the subject during graduation were less likely to become infected, suggesting that campaigns aimed at the university public are essential for the prevention and control of these pathogens.

INTRODUÇÃO: As infecções sexualmente transmissíveis (IST) são um grande problema de saúde pública, ao qual os jovens apresentam alta exposição, sendo necessário um maior conhecimento sobre as vulnerabilidades que os acometem. OBJETIVO: Avaliar o conhecimento sobre as IST e o comportamento sexual de uma população universitária na cidade de Sorocaba/SP. MÉTODOS: Realizou-se um estudo descritivo, de corte transversal, com a coleta de dados realizada por meio de aplicação online de questionário com características qualitativas e quantitativas. RESULTADOS: Quatrocentos e setenta e sete (477) universitários de diferentes áreas de conhecimento foram avaliados. A maioria dos relatos apontou para o início da vida sexual entre 15 e 18 anos. As informações sobre educação sexual foram obtidas principalmente por intermédio dos pais e/ou responsáveis, enquanto pouco conhecimento adicional foi obtido após o ingresso no Ensino Superior. Estudantes de Ciências Biológicas e da Saúde alcançaram o maior score no questionário sobre conhecimento e apresentaram chances menores (0,391) de contrair IST, quando comparados aos estudantes de Ciências Sociais Aplicadas ou Engenharias (2,8 e 2,9 mais chances, respectivamente). CONCLUSÃO: Os estudantes que demonstraram maior conhecimento sobre as IST e que adquiriram mais informações sobre o tema durante a graduação apresentaram chances menores de se infectar, o que sugere que campanhas destinadas ao público universitário são essenciais para a prevenção e o controle desses patógenos.

Bile acid-activated receptors in innate and adaptive immunity: targeted drugs and biological agents.

Bile acid-activated receptors (BARs) such as a G-protein bile acid receptor 1 and the farnesol X receptor are activated by bile acids (BAs) and have been implicated in the regulation of microbiota-host immunity in the intestine. The mechanistic roles of these receptors in immune signaling suggest that they may also influence the development of metabolic disorders. In this perspective, we provide a summary of recent literature describing the main regulatory pathways and mechanisms of BARs and how they affect both innate and adaptive immune system, cell proliferation, and signaling in the context of inflammatory diseases. We also discuss new approaches for therapy and summarize clinical projects on BAs for the treatment of diseases. In parallel, some drugs that are classically used for other therapeutic purposes and BAR activity have recently been proposed as regulators of immune cells phenotype. Another strategy consists of using specific strains of gut bacteria to regulate BA production in the intestine.

TNFα Activates the Liver X Receptor Signaling Pathway and Promotes Cholesterol Efflux from Human Brain Pericytes Independently of ABCA1.

Neuroinflammation and brain lipid imbalances are observed in Alzheimer's disease (AD). Tumor necrosis factor-α (TNFα) and the liver X receptor (LXR) signaling pathways are involved in both processes. However, limited information is currently available regarding their relationships in human brain pericytes (HBP) of the neurovascular unit. In cultivated HBP, TNFα activates the LXR pathway and increases the expression of one of its target genes, the transporter ATP-binding cassette family A member 1 (ABCA1), while ABCG1 is not expressed. Apolipoprotein E (APOE) synthesis and release are diminished. The cholesterol efflux is promoted, but is not inhibited, when ABCA1 or LXR are blocked. Moreover, as for TNFα, direct LXR activation by the agonist (T0901317) increases ABCA1 expression and the associated cholesterol efflux. However, this process is abolished when LXR/ABCA1 are both inhibited. Neither the other ABC transporters nor the SR-BI are involved in this TNFα-mediated lipid efflux regulation. We also report that inflammation increases ABCB1 expression and function. In conclusion, our data suggest that inflammation increases HBP protection against xenobiotics and triggers an LXR/ABCA1 independent cholesterol release. Understanding the molecular mechanisms regulating this efflux at the level of the neurovascular unit remains fundamental to the characterization of links between neuroinflammation, cholesterol and HBP function in neurodegenerative disorders.

Effects of slime toy poisoning in children and teenagers / Efeitos da intoxicação por slime em crianças e adolescentes

Abstract Objective: The aim of this study was to identify which types of skin reactions are associated with slime toys and which of their ingredients are most frequently involved in cases of poisoning. Data source: Between January and July 2021, articles were selected using PubMed, SciELO, and LILACS databases. The following descriptors were used: (dermatitis OR rash OR eczema OR inflammation) AND slime. Inclusion criteria were articles available in full, in either Portuguese, English, or Spanish, published between January 2000 and July 31, 2021, and articles reporting cases of contact dermatitis or eczema potentially or directly attributed to slime toys. Articles not meeting these criteria and duplicate texts in the databases were excluded. Data synthesis: In total, 65 publications were identified, of which 16 were included in this review. This resulted in a total of 22 children (2 males, 20 females), aged between 4 and 13 years, who were reportedly intoxicated by slime toys, most of these being linked to homemade preparations. Studies reported the occurrence of contact or allergic dermatitis on hands, fingers, nails, forearms, and cheeks. The most allergenic and/or irritant ingredients included liquid detergent and soap. Additionally, patch tests identified positive reactions to methylisothiazolinone and methylchloroisothiazolinone, the preservatives used by chemical industries on preparation of glue, soap, detergents, etc. Conclusions: Although slime toys might be important for improving motor development and parental relationships, homemade slime toy recipes include several allergenic and irritant ingredients which might be exposed to vulnerable children and cause intoxications. Therefore, homemade slime toys preparations should be used cautiously and under the supervision of adults.

Resumo Objetivo: Identificar quais tipos de reações de pele e ingredientes do brinquedo slime estão frequentemente envolvidos em relatos de intoxicação. Fontes de dados: Entre janeiro e julho de 2021, ocorreu a seleção dos artigos, utilizando-se as bases de dados: United States National Library of Medicine (PubMed), Scientific Electronic Library Online (SciELO) e Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS). Foram utilizados os seguintes descritores: (dermatitis OR rash OR eczema OR inflammation) AND slime. Incluíram-se artigos disponíveis na íntegra, em português, inglês ou espanhol, publicados entre janeiro de 2000 e 31 julho de 2021, que relatassem casos de crianças e adolescentes que apresentaram reação cutânea após a manipulação do brinquedo slime. Foram excluídos artigos sem aderência ao tema e textos duplicados nas bases de dados. Síntese dos dados: Identificaram-se 65 publicações, sendo 16 utilizadas para a elaboração desta revisão. Isso resultou no total de 22 crianças (duas do sexo masculino, 20 do feminino), com idades entre quatro e 13 anos, que teriam sido intoxicadas por slime, a maioria dos casos ligado a preparações caseiras. Estudos relataram a ocorrência de dermatite de contato ou alérgica nas mãos, dedos, unhas, antebraços e bochechas. Os ingredientes mais alergênicos e/ou irritantes foram detergentes líquidos e sabão. Ademais, o patch test identificou reações positivas para metilisotiazolinona e metilcloroisotiazolinona, que são conservantes utilizados em produtos como cola, sabão, detergente, etc. Conclusões: Ainda que o brinquedo slime seja importante para o desenvolvimento motor e das relações parentais, receitas caseiras incluem vários ingredientes alergênicos e irritantes, que podem ser expostos a crianças vulneráveis e causar intoxicações. Sendo assim, as preparações do slime devem ser feitas com cautela e sob supervisão de adultos.

Effects of slime toy poisoning in children and teenagers.

OBJECTIVE: The aim of this study was to identify which types of skin reactions are associated with slime toys and which of their ingredients are most frequently involved in cases of poisoning. DATA SOURCE: Between January and July 2021, articles were selected using PubMed, SciELO, and LILACS databases. The following descriptors were used: (dermatitis OR rash OR eczema OR inflammation) AND slime. Inclusion criteria were articles available in full, in either Portuguese, English, or Spanish, published between January 2000 and July 31, 2021, and articles reporting cases of contact dermatitis or eczema potentially or directly attributed to slime toys. Articles not meeting these criteria and duplicate texts in the databases were excluded. DATA SYNTHESIS: In total, 65 publications were identified, of which 16 were included in this review. This resulted in a total of 22 children (2 males, 20 females), aged between 4 and 13 years, who were reportedly intoxicated by slime toys, most of these being linked to homemade preparations. Studies reported the occurrence of contact or allergic dermatitis on hands, fingers, nails, forearms, and cheeks. The most allergenic and/or irritant ingredients included liquid detergent and soap. Additionally, patch tests identified positive reactions to methylisothiazolinone and methylchloroisothiazolinone, the preservatives used by chemical industries on preparation of glue, soap, detergents, etc. CONCLUSIONS: Although slime toys might be important for improving motor development and parental relationships, homemade slime toy recipes include several allergenic and irritant ingredients which might be exposed to vulnerable children and cause intoxications. Therefore, homemade slime toys preparations should be used cautiously and under the supervision of adults.

Pioglitazone Attenuates the Effects of Peripheral Inflammation in a Human In Vitro Blood-Brain Barrier Model.

Biological mediators secreted during peripheral chronic inflammation reach the bloodstream and may damage the blood-brain barrier (BBB), triggering central nervous system (CNS) disorders. Full-fledged human BBB models are efficient tools to investigate pharmacological pathways and mechanisms of injury at the BBB. We here employed a human in vitro BBB model to investigate the effects of either plasma from inflammatory bowel disease (IBD) patients or tumor necrosis factor α (TNFα), a cytokine commonly released in periphery during IBD, and the anti-inflammatory role of pioglitazone, a peroxisome proliferator-activated receptor γ agonist (PPARγ). The BBB model was treated with either 10% plasma from healthy and IBD donors or 5 ng/mL TNFα, following treatment with 10 µM pioglitazone. Patient plasma did not alter BBB parameters, but TNFα levels in plasma from all donors were associated with varying expression of claudin-5, claudin-3 and ICAM-1. TNFα treatment increased BBB permeability, claudin-5 disarrangement, VCAM-1 and ICAM-1 expression, MCP1 secretion and monocyte transmigration. These effects were attenuated by pioglitazone. Plasma from IBD patients, which evoked higher BBB permeability, also increased ICAM-1 expression, this effect being reversed by pioglitazone. Our findings evidence how pioglitazone controls periphery-elicited BBB inflammation and supports its repurposing for prevention/treating of such inflammatory conditions.

Secretome of endothelial progenitor cells from stroke patients promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage.

BACKGROUND: Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier. METHODS: Two different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs-CMECs) and blood-brain barrier (BBB; brain-like ECs-BLECs). Cells were treated with scCM (5 µg/mL) or protein-free endothelial basal medium (scEBM-control). CMECs or BLECs were exposed (6 h) to oxygen-glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control-5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h). RESULTS: The analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity. CONCLUSION: Our results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage.

Formyl Peptide Receptors and Annexin A1: Complementary Mechanisms to Infliximab in Murine Experimental Colitis and Crohn's Disease.

Non-responsiveness to anti-TNF-α therapies presents relevant rates in inflammatory bowel disease patients, presenting the need to find biomarkers involved in therapeutic efficacy. Herein, we demonstrate that higher levels of colonic formyl peptide receptor 1 and annexin A1 correlate with histological recovery in Crohn's disease patients under remission. Using the dextran sulfate sodium colitis model in mice, we suggest that infliximab induces annexin A1 expression and secretion in activated intestinal leukocytes. Conversely, this mechanism might stimulate epithelial formyl peptide receptors, inducing wound healing and consequent histological remission. Our data indicate that assessing intestinal expressions of formyl peptide receptors and annexin A1 might provide precious information on the disease activity and responsiveness to infliximab in inflammatory bowel disease patients.

Evaluation of a human iPSC-derived BBB model for repeated dose toxicity testing with cyclosporine A as model compound.

The blood-brain barrier (BBB) is a highly restrictive barrier that preserves central nervous system homeostasis and ensures optimal brain functioning. Using BBB cell assays makes it possible to investigate whether a compound is likely to compromise BBBs functionality, thereby probably resulting in neurotoxicity. Recently, several protocols to obtain human brain-like endothelial cells (BLECs) from induced pluripotent stem cells (iPSCs) have been reported. Within the framework of the European MSCA-ITN in3 project, we explored the possibility to use an iPSC-derived BBB model to assess the effects of repeated dose treatment with chemicals, using Cyclosporine A (CsA) as a model compound. The BLECs were found to exhibit important BBB characteristics up to 15 days after the end of the differentiation and could be used to assess the effects of repeated dose treatment. Although BLECs were still undergoing transcriptional changes over time, a targeted transcriptome analysis (TempO-Seq) indicated a time and concentration dependent activation of ATF4, XBP1, Nrf2 and p53 stress response pathways under CsA treatment. Taken together, these results demonstrate that this iPSC-derived BBB model and iPSC-derived models in general hold great potential to study the effects of repeated dose exposure with chemicals, allowing personalized and patient-specific studies in the future.

Central nervous system delivery of molecules across the blood-brain barrier.

Therapies targeting neurological conditions such as Alzheimer's or Parkinson's diseases are hampered by the presence of the blood-brain barrier (BBB). During the last decades, several approaches have been developed to overcome the BBB, such as the use of nanoparticles (NPs) based on biomaterials, or alternative methods to open the BBB. In this review, we briefly highlight these strategies and the most recent advances in this field. Limitations and advantages of each approach are discussed. Combination of several methods such as functionalized NPs targeting the receptor-mediated transcytosis system with the use of magnetic resonance imaging-guided focused ultrasound (FUS) might be a promising strategy to develop theranostic tools as well as to safely deliver therapeutic molecules, such as drugs, neurotrophic factors or antibodies within the brain parenchyma.

Control of expression and activity of peroxisome proliferated-activated receptor γ by Annexin A1 on microglia during efferocytosis.

Annexin A1 (AnxA1) is a protein secreted by phagocytic cells which plays a pivotal role on the resolution of inflammation by enhancing phagocytosis carried out by phagocytes. Which factors and intracellular mechanisms are linked to such actions exerted by AnxA1 are yet to be completely understood. In order to investigate such, BV2 microglial cells were transfected with plasmids aimed at down-modulating AnxA1 expression and also treated with exogenous recombinant rAnxA1; gene and protein expression of proliferated-activated receptor γ (PPARγ) and CD36, STAT6 phosphorylation and phagocytosis of apoptotic neurons were investigated. Down-modulating AnxA1 in BV2 cells impaired gene and protein expression of PPARγ, effects reversed by treatment with recombinant AnxA1 (rAnxA1). Lower levels of CD36 were also verified in AnxA1 down-modulated BV2 cells. AnxA1-mediated phagocytosis of apoptotic cells was abrogated due to blockade of PPARγ activation, and in AnxA1 down-modulated cells exogenous AnxA1 failed to exert any effects on phagocytosis. Lower levels of STAT6/pSTAT6 in AnxA1 down-modulated BV2 cells suggest the involvement of this transcription factor with PPARγ and CD36 synthesis and actions. Data here shown suggest that there is a probable connection between AnxA1, PPARγ, and CD36, which must all act in association in order for efferocytosis to occur properly. AnxA1-mediated phosphorylation of STAT6 is probably involved with intracellular pathways involving PPARγ and CD36 actions. These data evidence that PPARγ/CD36 play a role on AnxA1-mediated efferocytosis in microglial cells. SIGNIFICANCE OF THE STUDY: The findings of this work provide evidence that the glucocorticoid-mediated protein annexin A1 modulates PPARγ expression and that PPARγ is important for annexin A1-mediated efferocytosis. Only recently the interaction between these two factors has begun to be explored, and knowledge on associated cell mechanisms are still scarce. Elucidating how annexin A1 and PPARγ interact with one another provides basis for further research aimed at understanding molecular pathways and cell signaling events involved with these factors, expanding existing knowledge on the anti-inflammatory effects of such factors.

Estrogen Promotes Pro-resolving Microglial Behavior and Phagocytic Cell Clearance Through the Actions of Annexin A1.

Local production of estrogen rapidly follows brain tissue injury, but the role this hormone plays in regulating the response to neural damage or in the modulation of mediators regulating inflammation is in many ways unclear. Using the murine BV2 microglia model as well as primary microglia from wild-type and annexin A1 (AnxA1) null mice, we have identified two related mechanisms whereby estradiol can modulate microglial behavior in a receptor specific fashion. Firstly, estradiol, via estrogen receptor ß (ERß), enhanced the phagocytic clearance of apoptotic cells, acting through increased production and release of the protein AnxA1. Secondly, stimulation of either ERß or the G protein coupled estrogen receptor GPER promoted the adoption of an anti-inflammatory/pro-resolving phenotype, an action similarly mediated through AnxA1. Together, these data suggest the hypothesis that locally produced estrogen acts through AnxA1 to exert powerful pro-resolving actions, controlling and limiting brain inflammation and ultimately protecting this highly vulnerable organ. Given the high degree of receptor selectivity in evoking these responses, we suggest that the use of selective estrogen receptor ligands may hold therapeutic promise in the treatment of neuroinflammation, avoiding unwanted generalized effects.

Direct effects of poly(ε-caprolactone) lipid-core nanocapsules on human immune cells.

Aim: Poly(ε-caprolactone) lipid-core nanocapsules (LNCs) are efficient drug carriers and drug-free LNCs display therapeutic effects, inhibiting tumor growth and neutrophil activities. Herein, we investigated the direct actions of LNCs on human immune cells, to guide their therapeutic application. Materials & methods: LNC's uptake, cytokine release, cell migration, proliferation and intracellular pathways under inflammatory stimulation were investigated. Results & conclusion: LNCs quickly penetrated leukocytes without cytotoxicity; inhibited mitogen-induced lymphocyte proliferation, cytokine release and leukocyte migration under inflammatory stimulation, which were associated with inhibition of the MAP kinase pathway and intracellular calcium influx. Hence, we showed LNCs as a down-regulatory agent on immune cells, suggesting that either the particles themselves or their application as a drug carrier can halt non-desired inflammatory processes.

Mechanisms of the effectiveness of poly(ε-caprolactone) lipid-core nanocapsules loaded with methotrexate on glioblastoma multiforme treatment.

PURPOSE: The low penetration of drugs across the blood-brain barrier (BBB) compromises the delivery of chemotherapeutic agents to the brain parenchyma and contributes to the poor prognosis of glioblastoma multiforme (GBM). We investigated the efficacy of methotrexate-loaded lipid-core nanocapsules (MTX-LNC) administered by the oral route to treat murine GBM, its ability to cross the BBB, and the mechanisms of MTX-LNC uptake by cultured GL261 glioma and BV2 microglia cells. MATERIALS AND METHODS: Female C57B/6 mice were used in intravital microscopy assays to investigate the penetrance of rhodamine B-label MTX-LNC (RhoB/MTX-LNC) in the brain after oral or IV administration, and to evaluate the BBB integrity. Intracranial implantation of GL261 cells was undertaken to induce a murine GBM model, and the effectiveness of oral MTX or MTX-LNC treatments (started on Day 10 of GBM, every 2 days for 12 days) was quantified by tumor size, body weight, and leukogram. Pharmacological blockade of endocytic pathways was done to investigate the mechanisms of MTX-LNC uptake by cultured GL261 and microglia BV2 cells by using fluorescence microscopy. The effect of MTX-LNC or MTX on GL261 and BV2 proliferation was evaluated to compare the cytotoxicity of such compounds. RESULTS: RhoB/MTX-LNC was detected in brain parenchyma of mice after IV or oral administration, without any damage on BBB. Oral treatment with MTX-LNC reduced tumor volume and prevented weight loss and leukopenia in comparison to MTX-treated mice. MTX-LNC uptake by GL261 is caveolae-dependent, whereas endocytosis of MTX-LNC by BV2 occurs via phagocytosis and macropinocytosis. Both MTX-LNC and MTX reduced GL261 and BV2 proliferation; however, MTX-LNC showed higher efficacy in the inhibition of glioma proliferation. CONCLUSION: Together, we infer that the higher ability of MTX-LNC to cross the BBB and be captured by cancer and immune brain cells by different mechanisms is responsible for the higher efficacy of oral MTX-LNC treatment in GBM.

The restorative role of annexin A1 at the blood-brain barrier.

Annexin A1 is a potent anti-inflammatory molecule that has been extensively studied in the peripheral immune system, but has not as yet been exploited as a therapeutic target/agent. In the last decade, we have undertaken the study of this molecule in the central nervous system (CNS), focusing particularly on the primary interface between the peripheral body and CNS: the blood-brain barrier. In this review, we provide an overview of the role of this molecule in the brain, with a particular emphasis on its functions in the endothelium of the blood-brain barrier, and the protective actions the molecule may exert in neuroinflammatory, neurovascular and metabolic disease. We focus on the possible new therapeutic avenues opened up by an increased understanding of the role of annexin A1 in the CNS vasculature, and its potential for repairing blood-brain barrier damage in disease and aging.

Long-term in vivo polychlorinated biphenyl 126 exposure induces oxidative stress and alters proteomic profile on islets of Langerhans.

It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 µg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early ß-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2.

L-Arginine Transport and Nitric Oxide Production in Kinin Receptor B1-/- Endothelial Cells.

Kinins are important vasoactive peptides, but the role of the B1 receptor subtype in the vascular control is poorly understood. This study analyzed the nitric oxide (NO) release, L-arginine (L-Arg) uptake and the expression of the cationic amino acid transporter (CAT) -1 in endothelial cells obtained from B1 receptor knockout (B1-/-) and wild type (WT) mice. NO production was assessed through a fluorescent dye in living cells stimulated with acetylcholine. L-Arg uptake was determined indirectly in the culture medium by HPLC, in the presence or absence of the CAT-1 blocker N-ethylmaleimide (NEM). CAT-1 mRNA levels and protein expression were determined by qPCR and western blot, respectively. NO release was significantly reduced in B1-/- when compared to WT cells. This result was accompanied by a decreased rate in the L-Arg uptake by B1-/- cells. Incubation with NEM impaired the L-Arg uptake in WT, but had no effect in B1-/- cells. Protein expression and mRNA levels for CAT-1 were reduced in B1-/- in comparison to WT cells. These findings suggest an important role of the endothelial B1 receptor in the vascular control by interfering with CAT-1 expression, L-Arg uptake and NO release.