RESUMO
Dialkylphosphates (DAPs), metabolites of organophosphate (OP) pesticides, are widely distributed in the environment and are often used as biomarkers of OP exposure. Recent reports indicate that DAPs may be genotoxic, both in vitro and in vivo. We have examined the genotoxicity of the methylated DAPs dimethyldithiophosphate (DMDTP) and dimethylphosphate (DMTP) and the ethylated DAPs diethyldithiophosphate (DEDTP) and diethylphosphate (DETP), in comparison with their parental compounds, malathion and terbufos, respectively, in bone marrow polychromatic erythrocytes (PCE) of male and female Balb/c mice. We also compared DNA damage (comet assay) induced by DMDTP and dimethyl phosphate (DMP) in human cell lines. Both DMDTP and DMP caused DNA damage in peripheral blood mononuclear cells, HeLa cells, and the hepatic cell lines HepG2 and WRL-68. In the in vivo micronucleus assay, methylated and ethylated DAPs increased micronucleated PCE cells in both male and female mice. Female mice were more susceptible to DNA damage. In comparison to their parental compounds, methylated DAPs, particularly DMTP, were more genotoxic than malathion; DEDTP, DETP, and terbufos were similar in potency. These results suggest that DAPs may contribute to DNA damage associated with OP pesticide exposure.
Assuntos
Inseticidas , Praguicidas , Masculino , Feminino , Humanos , Animais , Camundongos , Malation/toxicidade , Camundongos Endogâmicos BALB C , Leucócitos Mononucleares/química , Células HeLa , Compostos Organofosforados/toxicidade , Organofosfatos/toxicidade , Dano ao DNA , Células da Medula Óssea/metabolismo , Praguicidas/toxicidade , Exposição AmbientalRESUMO
Down syndrome (DS), caused by trisomy of chromosome 21 (HSA21), results in a broad range of phenotypes. However, the determinants contributing to the complex and variable phenotypic expression of DS are still not fully known. Changes in microRNAs (miRNAs), short non-coding RNA molecules that regulate gene expression post-transcriptionally, have been associated with some DS phenotypes. Here, we investigated the genome-wide mature miRNA expression profile in peripheral blood mononuclear cells (PBMCs) of children with DS and controls and identified biological processes and pathways relevant to the DS pathogenesis. The expression of 754 mature miRNAs was profiled in PBMCs from six children with DS and six controls by RT-qPCR using TaqMan® Array Human MicroRNA Cards. Functions and signaling pathways analyses were performed using DIANA-miRPath v.3 and DIANA-microT-CDS software. Children with DS presented six differentially expressed miRNAs (DEmiRs): four overexpressed (miR-378a-3p, miR-130b-5p, miR-942-5p, and miR-424-3p) and two downregulated (miR-452-5p and miR-668-3p). HSA21-derived miRNAs investigated were not found to be differentially expressed between the groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed potential target genes involved in biological processes and pathways pertinent to immune response, e.g., toll-like receptors (TLRs) signaling, Hippo, and transforming growth factor ß (TGF-ß) signaling pathways. These results suggest that altered miRNA expression could be contributing to the well-known immunological dysfunction observed in individuals with DS.
Assuntos
Síndrome de Down , MicroRNAs , Síndrome de Down/genética , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Transdução de Sinais/genéticaRESUMO
ABSTRACT Introduction: Fabry disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme α-galactosidase A (α-GAL). Reduced α-GAL activity leads to progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. The recent report of increased expression of CD77 in blood cells of patients with FD indicated that this molecule can be used as a potential marker for monitoring enzyme replacement therapy (ERT). Objective: The purpose of this study was to evaluate the CD77 levels throughout ERT in FD patients (V269M mutation). Methods: We evaluated the fluctuations in PBMC (peripheral blood mononuclear cell) membrane CD77 expression in FD patients undergoing ERT and correlated these levels with those observed in different cell types. Results: A greater CD77 expression was found in phagocytes of patients compared to controls at baseline. Interestingly, the variability in CD77 levels is larger in patients at baseline (340 - 1619 MIF) and after 12 months of ERT (240 - 530 MIF) compared with the control group (131 - 331 MFI). Furthermore, by analyzing the levels of CD77 in phagocytes from patients throughout ERT, we found a constant decrease in CD77 levels. Conclusion: The increased CD77 levels in the phagocytes of Fabry carriers together with the decrease in CD77 levels throughout ERT suggest that measuring CD77 levels in phagocytes is a promising tool for monitoring the response to ERT in FD.
RESUMO Introdução: A doença de Fabry (DF) é um distúrbio causado por mutações no gene que codifica a enzima lisossômica α-galactosidase A (α-GAL). A redução da atividade de α-GAL leva ao acúmulo progressivo de globotriaosilceramida (Gb3), também conhecida como CD77. O recente relato de aumento da expressão de CD77 em células sanguíneas de pacientes com DF indicou que essa molécula pode ser utilizada como um potencial marcador para o monitoramento da terapia de reposição enzimática (TRE). Objetivo: O objetivo deste estudo foi avaliar os níveis de CD77 ao longo da TRE em pacientes com DF (mutação V269M). Métodos: Foram avaliadas as flutuações na expressão de CD77 nas membranas das CMSP (células mononucleares do sangue periférico) em pacientes com DF submetidos à TRE e correlacionados com aqueles observados em diferentes tipos de células. Resultados: Uma maior expressão de CD77 foi encontrada em fagócitos de pacientes em comparação aos controles no início do estudo. Curiosamente, a variabilidade nos níveis de CD77 é maior em pacientes no início do estudo (340 - 1619 MIF) e após 12 meses de TRE (240 - 530 MIF) em comparação com o grupo controle (131 - 331 MFI). Além disso, analisando os níveis de CD77 em fagócitos de pacientes ao longo da TRE, encontramos uma diminuição constante nos níveis de CD77. Conclusão: O aumento nos níveis de CD77 nos fagócitos de portadores de Fabry, juntamente com a diminuição nos níveis de CD77 ao longo da TRE, sugerem que medir os níveis de CD77 nos fagócitos é uma ferramenta promissora para monitorar a resposta à TRE na DF.
Assuntos
Humanos , Masculino , Feminino , Adulto , Adulto Jovem , Triexosilceramidas/biossíntese , Leucócitos Mononucleares/metabolismo , Doença de Fabry/tratamento farmacológico , Doença de Fabry/sangue , alfa-Galactosidase/uso terapêutico , Terapia de Reposição de Enzimas , Triexosilceramidas/análise , Leucócitos Mononucleares/químicaRESUMO
INTRODUCTION: Fabry disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme α-galactosidase A (α-GAL). Reduced α-GAL activity leads to progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. The recent report of increased expression of CD77 in blood cells of patients with FD indicated that this molecule can be used as a potential marker for monitoring enzyme replacement therapy (ERT). OBJECTIVE: The purpose of this study was to evaluate the CD77 levels throughout ERT in FD patients (V269M mutation). METHODS: We evaluated the fluctuations in PBMC (peripheral blood mononuclear cell) membrane CD77 expression in FD patients undergoing ERT and correlated these levels with those observed in different cell types. RESULTS: A greater CD77 expression was found in phagocytes of patients compared to controls at baseline. Interestingly, the variability in CD77 levels is larger in patients at baseline (340 - 1619 MIF) and after 12 months of ERT (240 - 530 MIF) compared with the control group (131 - 331 MFI). Furthermore, by analyzing the levels of CD77 in phagocytes from patients throughout ERT, we found a constant decrease in CD77 levels. CONCLUSION: The increased CD77 levels in the phagocytes of Fabry carriers together with the decrease in CD77 levels throughout ERT suggest that measuring CD77 levels in phagocytes is a promising tool for monitoring the response to ERT in FD.
Assuntos
Terapia de Reposição de Enzimas , Doença de Fabry/sangue , Doença de Fabry/tratamento farmacológico , Leucócitos Mononucleares/metabolismo , Triexosilceramidas/biossíntese , alfa-Galactosidase/uso terapêutico , Adulto , Feminino , Humanos , Leucócitos Mononucleares/química , Masculino , Triexosilceramidas/análise , Adulto JovemRESUMO
Pregnancy-induced hypertension (PIH) causes significant maternal and fetal morbidity and mortality. A decreased number of regulatory T (Treg) cells is associated with the pathogenesis of PIH. The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical to normal pregnancy (NP) by promoting Treg cell development. However, the relationship between PD-1/PD-L1 and Treg differentiation in PIH has not been fully elucidated. In this study, venous blood was obtained from 20 NP and 58 PIH patients. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood. The levels of Treg-related cytokines (TGF-ß, IL-10, and IL-35) in serum and PBMCs were measured by ELISA. The percentage of Treg cells in PBMCs was assessed by flow cytometry. The mRNA levels of Treg-specific transcription factor Foxp3 in PBMCs, and PD-1 and PD-L1 in Treg cells were detected by qRT-PCR. The protein levels of PD-1 and PD-L1 in Treg cells were evaluated by western blot. The serum levels of TGF-ß, IL-10, IL-35, and Foxp3 mRNA expression and CD4+CD25+ Treg cell percentage in PBMCs were decreased in PIH. Furthermore, a significant increase of PD-1 in Treg cells was found in PIH compared with NP. In addition, PD-L1 Fc, an activator of PD-1/PD-L1 pathway, increased Treg cell percentage, enhanced Foxp3 mRNA expression, and elevated levels of TGF-ß, IL-10, and IL-35 in PBMCs. However, anti-PD-L1 mAb exerted a reverse effect. These findings revealed that PD-L1 Fc had a favorable effect on Treg cell differentiation, indicating a potential therapeutic value of PD-1/PD-L1 pathway for PIH treatment.
Assuntos
Apoptose , Antígeno B7-H1/metabolismo , Hipertensão Induzida pela Gravidez/metabolismo , Interleucina-10/metabolismo , Interleucinas/metabolismo , Leucócitos Mononucleares/química , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Western Blotting , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Gravidez , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Pregnancy-induced hypertension (PIH) causes significant maternal and fetal morbidity and mortality. A decreased number of regulatory T (Treg) cells is associated with the pathogenesis of PIH. The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical to normal pregnancy (NP) by promoting Treg cell development. However, the relationship between PD-1/PD-L1 and Treg differentiation in PIH has not been fully elucidated. In this study, venous blood was obtained from 20 NP and 58 PIH patients. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood. The levels of Treg-related cytokines (TGF-β, IL-10, and IL-35) in serum and PBMCs were measured by ELISA. The percentage of Treg cells in PBMCs was assessed by flow cytometry. The mRNA levels of Treg-specific transcription factor Foxp3 in PBMCs, and PD-1 and PD-L1 in Treg cells were detected by qRT-PCR. The protein levels of PD-1 and PD-L1 in Treg cells were evaluated by western blot. The serum levels of TGF-β, IL-10, IL-35, and Foxp3 mRNA expression and CD4+CD25+ Treg cell percentage in PBMCs were decreased in PIH. Furthermore, a significant increase of PD-1 in Treg cells was found in PIH compared with NP. In addition, PD-L1 Fc, an activator of PD-1/PD-L1 pathway, increased Treg cell percentage, enhanced Foxp3 mRNA expression, and elevated levels of TGF-β, IL-10, and IL-35 in PBMCs. However, anti-PD-L1 mAb exerted a reverse effect. These findings revealed that PD-L1 Fc had a favorable effect on Treg cell differentiation, indicating a potential therapeutic value of PD-1/PD-L1 pathway for PIH treatment.
Assuntos
Humanos , Feminino , Gravidez , Leucócitos Mononucleares/química , Interleucinas/metabolismo , Interleucina-10/metabolismo , Apoptose , Hipertensão Induzida pela Gravidez/metabolismo , Antígeno B7-H1/metabolismo , Ensaio de Imunoadsorção Enzimática , Leucócitos Mononucleares/metabolismo , Estudos de Casos e Controles , Western Blotting , Fator de Crescimento Transformador beta/metabolismo , Linfócitos T Reguladores/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Oxidative stress and inflammation play a role in the physiopathology of insulin resistance, diabetes and cardiovascular disease. A single high-fat, high-carbohydrate (HFHC) meal induces an increase in inflammatory and oxidative stress markers in peripheral blood mononuclear cells (PBMC). Previous studies have shown that orange juice is able to prevent this response by inhibiting toll like receptors (TLR) expression and endotoxemia. Our goal was to study the proteome response in PBMC after the consumption of a HFHC meal consumed with water, orange juice or an isocaloric beverage (water with glucose). Twelve healthy individuals completed the protocol in a crossover design, and blood samples were obtained before and 1, 3, and 5 h after consumption. Proteomic profile, glucose, insulin, lipid and cytokines levels were investigated. The glycemic and insulinemic response was higher when the meal was consumed with glucose, while there was no difference in the response between water and orange juice. Proteome analysis in PBMC was carried out using TMT ten-plex. A total of 3813 proteins, originating from 15â¯662 peptides were identified. Three proteins showed significantly altered expression in the three treatments: apolipoprotein A-II, ceruloplasmin and hemopexin. When the HFHC meal was consumed with water there was an increase in some inflammatory pathways such as the Fc-gamma receptor dependent phagocytosis and the complement cascade, but the immune system as a whole was not significantly altered. However, when the meal was consumed with glucose, the immune system was up regulated. Among the pathways induced after 3 h were those of the adaptive immune system and cytokine signaling. Five hours after the meal, pathways of the complement cascade and classical antibody mediated complement activation were up regulated. When the meal was consumed with orange juice there was an up regulation of proteins involved in signal transduction, DNA replication and cell cycle. The promyelocytic leukemia protein (PML) showed a 28.2-fold increase. This protein was down regulated when the meal was consumed with water. Regarding the immune system, several of the pathways induced by glucose were down regulated when the meal was consumed with orange juice: proteins involved with the adaptive immune system and cytokine signaling. Therefore, we have shown that orange juice can not only suppress diet induced inflammation, but also regulate the expression of proteins such as PML, which may play a key role in the regulation of metabolism.
Assuntos
Citrus sinensis , Sucos de Frutas e Vegetais , Leucócitos Mononucleares/química , Adulto , Estudos Cross-Over , Carboidratos da Dieta/farmacologia , Gorduras na Dieta/farmacologia , Feminino , Glucose/farmacologia , Humanos , Sistema Imunitário/efeitos dos fármacos , Inflamação/etiologia , Masculino , Refeições , Pessoa de Meia-Idade , ProteômicaRESUMO
An open-label pharmacokinetics (PK) clinical trial was conducted to comparatively assess the PK and explore the pharmacodynamics (PD) of miltefosine in children and adults with cutaneous leishmaniasis (CL) in Colombia. Sixty patients, 30 children aged 2 to 12 years and 30 adults aged 18 to 60 years, were enrolled. Participants received miltefosine (Impavido) at a nominal dose of 2.5 mg/kg/day for 28 days. Miltefosine concentrations were measured in plasma and peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry of samples obtained during treatment and up to 6 months following completion of treatment, when therapeutic outcome was determined. Fifty-two patients were cured, 5 pediatric patients failed treatment, and 3 participants were lost to follow-up. Leishmania (Viannia) panamensis predominated among the strains isolated (42/46; 91%). Noncompartmental analysis demonstrated that plasma and intracellular miltefosine concentrations were, overall, lower in children than in adults. Exposure to miltefosine, estimated by area under the concentration-time curve and maximum concentration, was significantly lower in children in both the central and intracellular compartments (P < 0.01). Leishmania persistence was detected in 43% of study participants at the end of treatment and in 27% at 90 days after initiation of treatment. Clinical response was not dependent on parasite elimination. In vitro miltefosine susceptibility was similar for Leishmania strains from adults and children. Our results document PK differences for miltefosine in children and adults with cutaneous leishmaniasis that affect drug exposure and could influence the outcome of treatment, and they provide bases for optimizing therapeutic regimens for CL in pediatric populations. (This study has been registered at ClinicalTrials.gov under identifier NCT01462500.).
Assuntos
Antiprotozoários/farmacocinética , Leishmania braziliensis/efeitos dos fármacos , Leishmania guyanensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Fosforilcolina/análogos & derivados , Adolescente , Adulto , Antiprotozoários/sangue , Antiprotozoários/farmacologia , Área Sob a Curva , Criança , Pré-Escolar , Esquema de Medicação , Feminino , Humanos , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania guyanensis/crescimento & desenvolvimento , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/parasitologia , Leucócitos Mononucleares/química , Leucócitos Mononucleares/parasitologia , Masculino , Pessoa de Meia-Idade , Testes de Sensibilidade Parasitária , Fosforilcolina/sangue , Fosforilcolina/farmacocinética , Fosforilcolina/farmacologia , Resultado do TratamentoRESUMO
INTRODUCTION: Vitamin D3 (VD3) has been described as a modulator of immune system cells, including dendritic cells (DC). Previous studies have shown its importance in in vitro generation of tolerogenic DC, which have a similar function and phenotype to that of CD141 dermal DCs that produce IL-10 and induce (LTreg) CD4+ T regulator cells. OBJECTIVE: This paper presents a study that compares the phenotype and cytokines produced by DC generated in presence and absence of VD3, which were matured with lipopolysaccharide (LPS), and their ability to induce LTreg from naïve allogeneic CD4+ T cells. MATERIALS AND METHODS: In order to compare them, peripheral blood mononuclear cells were isolated to select monocytes CD14+ T cells and differentiate them in vitro from DC in the presence and absence of VD3, and to mature them with LPS. Phenotype and cytokine levels were also analyzed in the culture supernatants. Dendritic cells were then co-cultured with naïve allogeneic CD4+ T cells and the frequencies of LTreg were determined (naïve-activated). RESULTS: The results showed that unstimulated DC generated with VD3 kept the CD14. When activated with LPS, they expressed lower levels of C83, CD83 and CD86; HLA-DR; higher amounts of IL-1ß, IL-8, IL-10, and tended to lessen IL-6, IL-12p70 and TGF-ß1, compared to DCs not treated with VD3. The frequency of naïve LTreg was similar, although immature DC generated with VD3 tended to induce activated LTregs. CONCLUSION: Based on these results, it is possible to conclude that DCs generated with VD3 and treated with LPS presented a 'semi-mature' phenotype, and were able to secrete pro-inflammatory and anti-inflammatory cytokines. Besides, they did not increase their capacity to promote the polarization of naïve allogenic CD4+ T cells towards LTregs.
Assuntos
Colecalciferol/farmacologia , Citocinas/biossíntese , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Interleucina-10/metabolismo , Interleucina-8/metabolismo , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/química , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Linfócitos T Reguladores/imunologia , Células Cultivadas , Colecalciferol/metabolismo , Células Dendríticas/citologia , Humanos , Interleucina-10/imunologia , Interleucina-10/fisiologia , Interleucina-8/imunologia , Interleucina-8/fisiologia , Leucócitos Mononucleares/química , Leucócitos Mononucleares/fisiologia , Lipopolissacarídeos/metabolismo , Linfócitos T Reguladores/fisiologiaRESUMO
BACKGROUND: Although the anti-HIV-1 effects of vitamin D (VitD) have been reported, mechanisms behind such protection remain largely unexplored. METHODS: The effects of two precursor forms (cholecalciferol/calciol at 0.01, 1 and 100 nM and calcidiol at 100 and 250 nM) on HIV-1 infection, immune activation, and gene expression were analyzed in vitro in cells of Colombian and Italian healthy donors. We quantified levels of released p24 by enzyme-linked immunosorbent assay, of intracellular p24 and cell-surface expression of CD38 and HLA-DR by flow cytometry, and mRNA expression of antiviral and immunoregulatory genes by real-time reverse transcription-polymerase chain reaction. RESULTS: Cholecalciferol decreased the frequency of HIV-1-infected p24CD4 T cells and levels of p24 in supernatants in a dose-dependent manner. Moreover, the CD4CD38HLA-DR and CD4CD38HLA-DR subpopulations were more susceptible to infection but displayed the greatest cholecalciferol-induced decreases in infection rate by an X4-tropic strain. Likewise, cholecalciferol at its highest concentration decreased the frequency of CD38HLA-DR but not of CD38HLA-DR T-cell subsets. Analyzing the effects of calcidiol, the main VitD source for immune cells and an R5-tropic strain as the most frequently transmitted virus, a reduction in HIV-1 productive infection was also observed. In addition, an increase in mRNA expression of APOBEC3G and PI3 and a reduction of TRIM22 and CCR5 expression, this latter positively correlated with p24 levels, was noted. CONCLUSIONS: VitD reduces HIV-1 infection in T cells possibly by inducing antiviral gene expression, reducing the viral co-receptor CCR5 and, at least at the highest cholecalciferol concentration, by promoting an HIV-1-restrictive CD38HLA-DR immunophenotype.