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1.
Anal Chem ; 95(6): 3255-3266, 2023 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-36735349

RESUMO

Accurate reconstruction of metabolic pathways is an important prerequisite for interpreting metabolomics changes and understanding the diverse biological processes in disease models. A tracer-based metabolomics strategy utilizes stable isotope-labeled precursors to resolve complex pathways by tracing the labeled atom(s) to downstream metabolites through enzymatic reactions. Isotope enrichment analysis is informative and achieved by counting total labeled atoms and acquiring the mass isotopologue distribution (MID) of the intact metabolite. However, quantitative analysis of labeled metabolite substructures/moieties (MS2 fragments) can offer more valuable insights into the reaction connections through measuring metabolite transformation. In order to acquire the isotopic labeling information at the intact metabolite and moiety level simultaneously, we developed a method that couples hydrophilic interaction liquid chromatography (HILIC) with Zeno trap-enabled high-resolution multiple reaction monitoring (MRMHR). The method enabled accurate and reproducible MID quantification for intact metabolites as well as their fragmented moieties, with notably high sensitivity in the MS2 fragmentation mode based on the measurement of 13C- or 15N-labeled cellular samples. The method was applied to human-induced pluripotent stem cell-derived neurons to trace the fate of 13C/15N atoms from D-13C6-glucose/L-15N2-glutamine added to the media. With the MID analysis of both intact metabolites and fragmented moieties, we validated the pathway reconstruction of de novo glutathione synthesis in mid-brain neurons. We discovered increased glutathione oxidization from both basal and newly synthesized glutathione pools under neuronal oxidative stress. Furthermore, the significantly decreased de novo glutathione synthesis was investigated and associated with altered activities of several key enzymes, as evidenced by suppressed glutamate supply via glucose metabolism and a diminished flux of glutathione synthetic reaction in the neuronal model of rotenone-induced neurodegeneration.


Assuntos
Metabolômica , Rotenona , Humanos , Isótopos de Carbono/química , Cromatografia Líquida/métodos , Metabolômica/métodos , Neurônios/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo/métodos
2.
J Autoimmun ; 116: 102563, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33189487

RESUMO

Regulatory T cells (Tregs) have suppressive functions and play an important role in controlling inflammation and autoimmunity. The migratory capacity of Tregs determines their location and their location determines whether they inhibit the priming of naïve lymphocytes in lymphoid tissues or the effector phase of immune responses at inflamed sites. Tregs generated or expanded in vitro are currently being tested in clinics for the treatment of autoimmune disorders, however, little is known about the factors controlling their migration towards therapeutically relevant locations. In this study, we have modulated Treg dynamics using Toll-like receptor (TLR) agonists. Dynamic imaging with confocal and two-photon microscopy revealed that Tregs generated in vitro and stimulated with P3C (a TLR2 agonist) but not with R848 (a TLR7 agonist) or LPS (a TLR4 agonist) showed enhanced cell migration within splenic white pulp or draining lymph node when transferred into mice intravenously or into the footpad, respectively. In summary, our data demonstrate that Tregs are more motile in response to direct TLR stimulation in particular towards TLR2 signals. This may have implications for efficient clinical Treg induction protocols.


Assuntos
Movimento Celular/imunologia , Linfócitos T Reguladores/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Imidazóis/farmacologia , Camundongos Endogâmicos C57BL , Microscopia Confocal/métodos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Receptor 2 Toll-Like/agonistas , Receptor 7 Toll-Like/agonistas
3.
J Immunol ; 201(5): 1400-1411, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30030325

RESUMO

Glucose-derived mannose is a common component of glycoproteins, and its deficiency leads to a severe defect in protein glycosylation and failure in basic cell functions. In this work, we show that mannose metabolism is essential for IFN-γ production by mouse Th1 cells. In addition, we demonstrate that the susceptibility of Th1 cells to glycolysis restriction depends on the activation conditions and that under diminished glycolytic flux, mannose availability becomes the limiting factor for IFN-γ expression. This study unravels a new role for glucose metabolism in the differentiation process of Th1 cells, providing a mechanistic explanation for the importance of glycolysis in immune cell functions.


Assuntos
Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Glicólise/imunologia , Interferon gama/imunologia , Manose/imunologia , Células Th1/imunologia , Animais , Camundongos , Células Th1/citologia
4.
Mediators Inflamm ; 2020: 9175083, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32801998

RESUMO

RESULTS: In patients, an increase in the population of Th17-secreting cells negatively correlated with the abundance of both IFN-γ-producing and T regulatory as well as suppressor cells, regarding all the phenotypes studied. Although a strong dependence of the PB Th1 cell compartment on the duration of the disease was observed, it was limited to the subgroup of patients with macular edema only. The frequency of B regulatory cells was unchanged compared to controls. CONCLUSIONS: In pars planitis, the alterations in lymphocyte cell distribution affect primarily the T cell repertoire. The imbalance in PB Th1/Th17/Treg cells creates proinflammatory conditions, strengthening the suggestion that the immune background may play a role in pars planitis pathogenesis. Also, circulating Th1 level may be of potential clinical relevance in terms of prediction of a more severe course of the disease.


Assuntos
Interleucina-17/metabolismo , Pars Planite/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th1/metabolismo , Adolescente , Adulto , Idoso , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Células Th17/metabolismo , Adulto Jovem
5.
Eur J Pharm Biopharm ; 188: 137-146, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37196874

RESUMO

In this study, high energy ball milling and nano spray drying were used to prepare amorphous solid dispersions of bosentan in copovidone for the first time. In particular, the impact of this polymer on the bosentan amorphization kinetics was investigated. Copovidone was shown to facilitate the amorphization of bosentan upon ball milling. As a result, bosentan was dispersed in copovidone at the molecular level, forming amorphous solid dispersions, regardless of the ratio of the compounds. The similarity between the values of the adjustment parameter that describes the goodness of fit of the Gordon-Taylor equation to the experimental data (K = 1.16) and that theoretically calculated for an ideal mixture (K = 1.13) supported these findings. The kind of coprocessing method determined the powder microstructure and the release rate. The opportunity to prepare submicrometer-sized spherical particles using nano spray drying was an important advantage of this technology. Both coprocessing methods allowed the formation of long-lasting supersaturated bosentan solutions in the gastric environment with maximum concentrations reached ranging from four (11.20 µg/mL) to more than ten times higher (31.17 µg/mL) than those recorded when the drug was vitrified alone (2.76 µg/mL). Moreover, this supersaturation lasted for a period of time at least twice as long as that of the amorphous bosentan processed without copovidone (15 min vs. 30-60 min). Finally, these binary amorphous solid dispersions were XRD-amorphous for a year of storage under ambient conditions.


Assuntos
Pirrolidinas , Composição de Medicamentos/métodos , Bosentana , Solubilidade , Pirrolidinas/química
6.
J Chromatogr A ; 1708: 464342, 2023 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-37696124

RESUMO

The importance of lipids seen in studies of metabolism, cancer, the recent COVID-19 pandemic and other diseases has brought the field of lipidomics to the forefront of clinical research. Quantitative and comprehensive analysis is required to understand biological interactions among lipid species. However, lipidomic analysis is often challenging due to the various compositional structures, diverse physicochemical properties, and wide dynamic range of concentrations of lipids in biological systems. To study the comprehensive lipidome, a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)-based screening method with 1200 lipid features across 19 (sub)classes, including both nonpolar and polar lipids, has been developed. HILIC-MS/MS was selected due to its class separation property and fatty acyl chain level information. 3D models of class chromatographic retention behavior were established and evaluations of cross-class and within-class interferences were performed to avoid over-reporting these features. This targeted HILIC-MS/MS method was fully validated, with acceptable analytical parameters in terms of linearity, precision, reproducibility, and recovery. The accurate quantitation of 608 lipid species in the SRM 1950 NIST plasma was achieved using multi-internal standards per class and post-hoc correction, extending current databases by providing lipid concentrations resolved at fatty acyl chain level. The overall correlation coefficients (R2) of measured concentrations with values from literature range from 0.64 to 0.84. The applicability of the developed targeted lipidomics method was demonstrated by discovering 520 differential lipid features related to COVID-19 severity. This high coverage and targeted approach will aid in future investigations of the lipidome in various disease contexts.


Assuntos
COVID-19 , Lipidômica , Humanos , Espectrometria de Massas em Tandem , Pandemias , Reprodutibilidade dos Testes , Cromatografia Líquida , Gravidade do Paciente , Lipídeos
7.
Front Immunol ; 13: 1055805, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36741366

RESUMO

Background: Regulatory T (Treg) cells have emerged as key players in the maintenance of immune homeostasis. Although significant progress has been made in recent years to define the Treg surface markers involved with or identifying their suppressive function, there remains much to be elucidated, and many questions persist. This study determined the expression of surface markers on human peripheral Treg cells and conventional T (Tconv) cells in a steady state and after activation to gain insight into their mechanism of action and more precisely characterize this regulatory population in humans. Methods: To screen Treg and Tconv cells, peripheral blood mononuclear cells (PBMCs) were isolated from volunteers, stained with a commercially available lyophilized antibody array comprising 371 surface antigens, and analyzed by flow cytometry. To compare Treg cells with activated Tconv cells, PBMCs were stimulated with PMA and further stained similar to freshly isolated cells. Results: Treg and Tconv cells were positive for 135 and 168 of the 371 antigens, respectively. Based on the frequency distribution, all of the most highly expressed markers identified were shared by both Treg and Tconv cells and participate in T cell activation, act as costimulatory and signaling molecules, or exhibit adhesion and migratory functions. Additionally, we identified several differences in marker expression between Treg and Tconv cells, with most found in the expression of co-stimulatory (ICOS, GITR, 4-1BB) and co-inhibitory (TIGIT, CTLA-4) molecules, as well as chemokine receptors (CXCR4, CXCR5, CCR4, CCR5, CCR7, CCR8, and CXCR7). Furthermore, post-activation expression of surface molecules identified molecules capable of discriminating Treg cells from activated Tconv cells (GITR, 4-1BB, TIGIT, CD120b, and CD39); however, almost all of these markers were also expressed in a small fraction of activated Tconv cells. Conclusions: These results offer insight into the biology of Tregs and contribute to their accurate identification and characterization in variety of immunological diseases as well as physiological processes.


Assuntos
Leucócitos Mononucleares , Linfócitos T Reguladores , Humanos , Transdução de Sinais/fisiologia , Citometria de Fluxo , Ativação Linfocitária
8.
Int J Pharm ; 622: 121846, 2022 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-35609831

RESUMO

New clinical indications for an orphan drug bosentan are prompting the improvement of the drug formulation. Since bosentan is available as monohydrate, the information on its anhydrous form together with the assessment of its glass forming ability is necessary when developing enabling formulations. The aim of this research was, therefore, to analyze the phenomena occurring upon dehydration and amorphization of bosentan. The anhydrous form was obtained by a thermal treatment of the monohydrate and characterized for the first time using DSC and XRD. Two stable amorphous forms were prepared by cooling of the melt and high energy ball milling (Tg = 82 °C). The chemical stability of milled bosentan was evaluated using ATR-IR and 1H NMR as well. The kinetics of bosentan amorphization was established. It was stated that bosentan could be easily amorphized. Importantly, even if the system was semiamorphous, there was no recrystallization while heating. The concentration-time curves recorded in biorelevant media, confirmed the beneficial effect of amorphization on the dissolution of bosentan. Yet, the amorphous form recrystallized into the monohydrate form in the gastric milieu. This phenomenon was accompanied by a reversible color change from yellow, which is typical of bosentan glass, to creamywhite that is characteristic of the crude crystalline drug.


Assuntos
Desidratação , Reposicionamento de Medicamentos , Bosentana , Composição de Medicamentos , Estabilidade de Medicamentos , Humanos , Cinética , Solubilidade , Difração de Raios X
9.
Data Brief ; 43: 108451, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35864881

RESUMO

A mechanical activation of the solid particles upon high-energy ball milling may considerably change the physicochemical properties of pharmaceutical compounds, including the morphology, particle size distribution, thermal properties, and surface interactions with water vapour upon gaseous phase hydration. Assessment of these changes is crucial for optimizing the manufacturing process of enabling drug products. In this article, we provide a detailed characterization of binary co-milled solid dispersions composed of tadalafil and Soluplus using a laser diffraction method, differential scanning calorimetry (DSC), gravimetric measurements and solid state 1H- NMR spectroscopy. The data presented in this article is directly related to our previously published research article. They complement information on the impact that both formulation and process variables may have on the properties of these binary powder formulations.

10.
J Hazard Mater ; 433: 128812, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35398796

RESUMO

A new adsorbent that combines mineral vermiculite with the yeast Saccharomyces cerevisiae, was used for Cd2+ removal. The influence of vermiculite presence on the toxic effects of Cd2+ to Saccharomyces cerevisiae yeast was evaluated as a function of the microorganisms' respiratory activity (CO2 production). The Cd2+ toxicity increased with prolonged exposure time reaching the LC50 value of 857 and 489 mg L-1 after 30 and 120 min, respectively. The yeast managed to bioaccumulate 25.0 ± 0.6 mg g-1 of Cd2+ at the initial Cd2+ concentration of 741.9 mg L-1; the maximum Cd2+ adsorption capacity of vermiculite reached 25 ± 5 mg g-1. The addition of the mineral decreased the cations toxic effect; the LC20 value in vermiculite absence attained approximately 200 mg L-1 after 30 min and decreased to 80 mg L-1 after 2 h, while in the bio-mineral system it was at the level of 435 ± 50 mg L-1 without a significant change in time. The mineral provided a superior living environment for the yeast by removing part of the cations, releasing essential microelements and providing a protective, clay hutch-like habitat for the cells.


Assuntos
Metais Pesados , Poluentes Químicos da Água , Purificação da Água , Adsorção , Silicatos de Alumínio , Reatores Biológicos , Cádmio , Cátions , Concentração de Íons de Hidrogênio , Minerais , Saccharomyces cerevisiae , Poluentes Químicos da Água/toxicidade
12.
FEBS J ; 287(23): 5096-5113, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32160399

RESUMO

Refsum disease (RD) is an inborn error of metabolism that is characterised by a defect in peroxisomal α-oxidation of the branched-chain fatty acid phytanic acid. The disorder presents with late-onset progressive retinitis pigmentosa and polyneuropathy and can be diagnosed biochemically by elevated levels of phytanate in plasma and tissues of patients. To date, no cure exists for RD, but phytanate levels in patients can be reduced by plasmapheresis and a strict diet. In this study, we reconstructed a fibroblast-specific genome-scale model based on the recently published, FAD-curated model, based on Recon3D reconstruction. We used transcriptomics (available via GEO database with identifier GSE138379), metabolomics and proteomics (available via ProteomeXchange with identifier PXD015518) data, which we obtained from healthy controls and RD patient fibroblasts incubated with phytol, a precursor of phytanic acid. Our model correctly represents the metabolism of phytanate and displays fibroblast-specific metabolic functions. Using this model, we investigated the metabolic phenotype of RD at the genome scale, and we studied the effect of phytanate on cell metabolism. We identified 53 metabolites that were predicted to discriminate between healthy and RD patients, several of which with a link to amino acid metabolism. Ultimately, these insights in metabolic changes may provide leads for pathophysiology and therapy. DATABASES: Transcriptomics data are available via GEO database with identifier GSE138379, and proteomics data are available via ProteomeXchange with identifier PXD015518.


Assuntos
Aminoácidos/metabolismo , Biomarcadores/análise , Fibroblastos/patologia , Metaboloma , Proteoma , Doença de Refsum/patologia , Transcriptoma , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Doença de Refsum/genética , Doença de Refsum/metabolismo
13.
Kardiol Pol ; 67(7): 769-73, 2009 Jul.
Artigo em Polonês | MEDLINE | ID: mdl-19650000

RESUMO

A case of a 29-year-old woman 18 days after delivery with catastrophic antiphospholipid syndrome secondary (CAPS) due to undiagnosed systemic lupus erythematosus, leading to cardiogenic shock is reported. Laboratory evaluation revealed increased anticardiolipin antibodies, lupus anticoagulant, antinuclear antibody and thrombocytopenia. Left ventricular ejection fraction was 20%, neurologic deficit and acute renal failure were also present. Cardiac involvement is common in CAPS, but cardiomyopathy due to microvascular thrombosis is rare. CAPS should be considered as a cause of acute heart failure in a women with systemic lupus erythematosus. In the presented case early therapy with anticoagulants, steroids, immunoglobulins and plasmaferesis was beneficial.


Assuntos
Síndrome Antifosfolipídica/complicações , Síndrome Antifosfolipídica/terapia , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/terapia , Choque Cardiogênico/complicações , Choque Cardiogênico/terapia , Adulto , Síndrome Antifosfolipídica/diagnóstico , Síndrome Antifosfolipídica/etiologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Choque Cardiogênico/diagnóstico , Choque Cardiogênico/etiologia , Resultado do Tratamento
14.
J Immunol Methods ; 474: 112665, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31525366

RESUMO

Mucosal tissues are enriched in γδ T lymphocytes, which maintain epithelial homeostasis, however, the homeostatic mechanisms are still incompletely understood. To elucidate their role in the tissue integrity governance within the female genital mucosa we employed flow cytometry, which is a powerful tool used for the characterization of tissue-resident immune cells, however, often requiring cell release upon tissue enzymatic disaggregation. Here, we analyzed the impact of various proteolytic enzymes in their ability to effectively isolate viable immune cells from the reproductive system of non-pregnant mice. Murine vaginas and uteri were digested using commercially available enzyme blends (liberases) and single enzymes (dispase II and collagenase IV). Among tested enzymes, liberases released the highest number of cells from digested tissues while dispase II and collagenase IV led to a significant decrease in the number of isolated live cells. Also, liberases had only minor detrimental effects on cell viability and detection of CD45, CD3ε, γδ TCR and CD11c positive cells. We found that a single liberase blend called Liberase TL was the most suited for the analysis of γδ T cells in the reproductive tract. By examining two distinct phases of the estrous cycle - estrus and diestrus, characterized by high and low epithelial stratification, respectively, we showed that higher numbers of γδ T lymphocytes were present in the latter cycle phase in vagina and uterus. Interestingly, the diestrus-associated increase in γδ T lymphocyte number was also observed in reproductive tract draining lumbar lymph nodes but not in more distant, inguinal lymph nodes. Our data indicate that enzymes used for reproductive mucosa digestion have profound effects on the cell viability and isolation efficiency, which consequently influence the phenotypic and quantitative analysis of immune cells.


Assuntos
Separação Celular , Imunidade nas Mucosas , Mucosa/imunologia , Peptídeo Hidrolases/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Útero/imunologia , Vagina/imunologia , Animais , Colagenases/metabolismo , Diestro/imunologia , Endopeptidases/metabolismo , Estro/imunologia , Feminino , Citometria de Fluxo , Contagem de Linfócitos , Camundongos Endogâmicos C57BL , Mucosa/citologia , Fenótipo , Termolisina/metabolismo , Útero/citologia , Vagina/citologia
15.
Biochim Biophys Acta Mol Basis Dis ; 1865(2): 360-370, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30385409

RESUMO

Flavin adenine dinucleotide (FAD) and its precursor flavin mononucleotide (FMN) are redox cofactors that are required for the activity of more than hundred human enzymes. Mutations in the genes encoding these proteins cause severe phenotypes, including a lack of energy supply and accumulation of toxic intermediates. Ideally, patients should be diagnosed before they show symptoms so that treatment and/or preventive care can start immediately. This can be achieved by standardized newborn screening tests. However, many of the flavin-related diseases lack appropriate biomarker profiles. Genome-scale metabolic models can aid in biomarker research by predicting altered profiles of potential biomarkers. Unfortunately, current models, including the most recent human metabolic reconstructions Recon and HMR, typically treat enzyme-bound flavins incorrectly as free metabolites. This in turn leads to artificial degrees of freedom in pathways that are strictly coupled. Here, we present a reconstruction of human metabolism with a curated and extended flavoproteome. To illustrate the functional consequences, we show that simulations with the curated model - unlike simulations with earlier Recon versions - correctly predict the metabolic impact of multiple-acyl-CoA-dehydrogenase deficiency as well as of systemic flavin-depletion. Moreover, simulations with the new model allowed us to identify a larger number of biomarkers in flavoproteome-related diseases, without loss of accuracy. We conclude that adequate inclusion of cofactors in constraint-based modelling contributes to higher precision in computational predictions.


Assuntos
Coenzimas/metabolismo , Flavoproteínas/metabolismo , Genoma Humano , Deficiência Múltipla de Acil Coenzima A Desidrogenase/metabolismo , Trifosfato de Adenosina/metabolismo , Biomarcadores/metabolismo , Flavina-Adenina Dinucleotídeo/deficiência , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Modelos Biológicos , Proteoma/metabolismo
16.
J Chromatogr A ; 1608: 460413, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31395359

RESUMO

Recent advances in metabolomics have enabled larger proportions of the human metabolome to be analyzed quantitatively. However, this usually requires the use of several chromatographic methods coupled to mass spectrometry to cover the wide range of polarity, acidity/basicity and concentration of metabolites. Chemical derivatization allows in principle a wide coverage in a single method, as it affects both the separation and the detection of metabolites: it increases retention, stabilizes the analytes and improves the sensitivity of the analytes. The majority of quantitative derivatization techniques for LC-MS in metabolomics react with amines, phenols and thiols; however, there are unfortunately very few methods that can target carboxylic acids at the same time, which contribute to a large proportion of the human metabolome. Here, we describe a derivatization technique which simultaneously labels carboxylic acids, thiols and amines using the reagent dimethylaminophenacyl bromide (DmPABr). We further improve the quantitation by employing isotope-coded derivatization (ICD), which uses internal standards derivatized with an isotopically-labelled reagent (DmPABr-D6). We demonstrate the ability to measure and quantify 64 central carbon and energy-related metabolites including amino acids, N-acetylated amino acids, metabolites from the TCA cycle and pyruvate metabolism, acylcarnitines and medium-/long-chain fatty acids. To demonstrate the applicability of the analytical approach, we analyzed urine and SUIT-2 cells utilizing a 15-minute single UPLC-MS/MS method in positive ionization mode. SUIT-2 cells exposed to rotenone showed definitive changes in 28 out of the 64 metabolites, including metabolites from all 7 classes mentioned. By realizing the full potential of DmPABr to derivatize and quantify amines and thiols in addition to carboxylic acids, we extended the coverage of the metabolome, producing a strong platform that can be further applied to a variety of biological studies.


Assuntos
Carbono/química , Carbono/metabolismo , Aminas/química , Aminas/metabolismo , Aminoácidos/análise , Brometos/química , Carbono/urina , Ácidos Carboxílicos/análise , Ácidos Carboxílicos/metabolismo , Linhagem Celular , Cromatografia Líquida/métodos , Humanos , Metaboloma , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos
17.
J Clin Med ; 7(11)2018 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-30356025

RESUMO

Endocrine disorders, including equine metabolic syndrome (EMS), are a serious issue in veterinary medicine and horse breeding. Furthermore, EMS was shown to affect the cytophysiological properties of adipose-derived stem cells, reducing their therapeutic potential. However, it was shown that those cells can be rejuvenated while using a combination of two chemicals: 5-azacytydine (AZA) and resveratrol (RES). In the present study, we decided to evaluate the immunomodulatory properties of AZA/RES-treated adipose-derived stem cells (ASC) isolated from EMS horses (ASCEMS). Thus, we co-cultured ASC with peripheral blood mononuclear cells (PBMC) and RAW264.7 macrophages. Most attention was placed on regulatory T lymphocytes (TREG), as well as the messenger RNA (mRNA) and protein levels of several cytokines (tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-10, and IL-1ß). Moreover, we also investigated the expression of genes related to auto- and mitophagy in both PBMCs and ASCs. PBMCs were obtained from healthy and EMS-suffering individuals and were co-cultured with ASCs that were isolated from healthy and EMS horses cultured in control conditions and with AZA/RES. We discovered that cells treated with AZA/RES increase the TREG number while co-cultured with PBMCs. Moreover, the co-culture of PBMCs with AZA/RES-treated ASCEMS induced mitophagy in PBMCs. Furthermore, ASCEMS pre-treated with AZA/RES displayed anti-inflammatory properties, as decreased levels of TNF-α, nitric oxide (NO), and IL-6 were observed in those cells in comparison with their untreated counterparts in the co-culture with RAW264.7 macrophages. In summary, we demonstrated that ASCEMS treated with AZA/RES displayed increased anti-inflammatory properties, and was able to regulate and activate the TREG-related anti-inflammatory response.

18.
Environ Sci Pollut Res Int ; 25(17): 17066-17076, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29637454

RESUMO

Removal of emerging pollutants, such as pharmaceuticals, from wastewater is a challenge. Adsorption is a simple and efficient process that can be applied. Clays, which are natural and low-cost materials, have been investigated as adsorbent. In this work, raw vermiculite and its three modified forms (expanded, base, and acid/base treated) were tested for removal of a widely used antidepressant, venlafaxine. Adsorption kinetics followed Elovich's model for raw vermiculite while the pseudo-2nd order model was a better fit in the case of other materials. Equilibrium followed Langmuir's model for the raw and the acid/base-treated vermiculite, while Redlich-Peterson's model fitted better the expanded and the base-treated materials. The adsorption capacity of vermiculite was significantly influenced by the changes in the physical and chemical properties of the materials caused by the treatments. The base-treated, raw, and expanded vermiculites showed lower maximum adsorption capacities (i.e., 6.3 ± 0.5, 5.8 ± 0.7, 3.9 ± 0.2 mg g-1, respectively) than the acid/base-treated material (33 ± 4 mg g-1). The acid/base-treated vermiculite exhibited good properties as a potential adsorbent for tertiary treatment of wastewater in treatment plants, in particular for cationic species as venlafaxine due to facilitation of diffusion of the species to the interlayer gallery upon such treatment. Graphical abstract ᅟ.


Assuntos
Ácidos/química , Silicatos de Alumínio/química , Cátions/química , Cloridrato de Venlafaxina/química , Águas Residuárias/química , Adsorção , Cinética , Cloridrato de Venlafaxina/análise , Águas Residuárias/análise
19.
Viruses ; 10(10)2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30261662

RESUMO

(1) Background: Tannic acid is a plant-derived polyphenol showing antiviral activity mainly because of an interference with the viral adsorption. In this work, we tested whether the modification of silver nanoparticles with tannic acid (TA-AgNPs) can provide a microbicide with additional adjuvant properties to treat genital herpes infection. (2) Methods: The mouse model of the vaginal herpes simplex virus 2 (HSV-2) infection was used to test immune responses after treatment of the primary infection with TA-AgNPs, and later, after a re-challenge with the virus. (3) Results: The mice treated intravaginally with TA-AgNPs showed better clinical scores and lower virus titers in the vaginal tissues soon after treatment. Following a re-challenge, the vaginal tissues treated with TA-AgNPs showed a significant increase in the percentages of IFN-gamma+ CD8+ T-cells, activated B cells, and plasma cells, while the spleens contained significantly higher percentages of IFN-gamma+ NK cells and effector-memory CD8+ T cells in comparison to NaCl-treated group. TA-AgNPs-treated animals also showed significantly better titers of anti-HSV-2 neutralization antibodies in sera; and (4) Conclusions: Our findings suggest that TA-AgNPs sized 33 nm can be an effective anti-viral microbicide to be applied upon the mucosal tissues with additional adjuvant properties enhancing an anti-HSV-2 immune response following secondary challenge.


Assuntos
Antivirais/farmacologia , Genitália Feminina/virologia , Herpes Genital/tratamento farmacológico , Herpesvirus Humano 2/imunologia , Prata/farmacologia , Taninos/farmacologia , Taninos/uso terapêutico , Animais , Anticorpos Neutralizantes/imunologia , Antivirais/química , Antivirais/uso terapêutico , Feminino , Herpes Genital/imunologia , Herpes Genital/virologia , Herpesvirus Humano 2/química , Imunidade nas Mucosas/imunologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Prata/química , Taninos/química
20.
Chemosphere ; 173: 107-115, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28107708

RESUMO

Additional treatment with NaOH of acid activated vermiculite results in even higher increase in the adsorption capacity in comparison to samples modified only in acidic solution (first step of activation) with respect to raw material. Optimization of treatment conditions and adsorption capacity for two cationic dyes (methylene blue (MB) and astrazon red (AR)), also as binary mixture, was evaluated. The capacity, based on column studies, increased from 48 ± 2 to 203 ± 4 mg g-1 in the case of methylene blue and from 51 ± 1 to 127 ± 2 mg g-1 in the case of astrazon red on starting and acid-base treated material, respectively. It was shown that adsorption mechanism changes for both cationic dyes after NaOH treatment and it results in decrease of adsorption rate. In binary mixtures methylene blue is bound stronger by adsorbent and astrazon red may be removed in initial stage of adsorption. Extensive studies on desorption/regeneration process proved high efficiency in recyclable use of all materials. Although cation exchange capacity decreases due to acid treatment, after base treatment exchange properties are used more efficiently. On the other hand, increased specific surface area has less significant contribution into the adsorption potential of studied materials. Obtained adsorbents worked efficiently in 7 adsorption-regeneration cycles and loss of adsorption capacity was observed only in two first cycles.


Assuntos
Ácidos/química , Álcalis/química , Silicatos de Alumínio/química , Cátions/química , Cátions/metabolismo , Corantes/química , Corantes/metabolismo , Adsorção , Corantes/análise , Azul de Metileno/análise
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