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1.
Einstein (Sao Paulo) ; 18: eAO5262, 2020.
Artigo em Inglês, Português | MEDLINE | ID: mdl-32130328

RESUMO

OBJECTIVE: To compare the frequency of respiratory tract infections in children treated with OM-85 BV and placebo during the 3-month therapy period, and observation for a further 3 months after treatment. METHODS: A randomized, double-blind, placebo-controlled trial was conducted with 54 children (6 months to 5 years old) with no past history of recurrent respiratory infections attending daycare center. Family members were instructed to administer one capsule per day for 10 consecutive days, for 3 months of OM-85 BV or placebo. Telephone interviews were conducted every 30 days. RESULTS: There was no significant difference in the number of respiratory infections between the groups. The mean number of respiratory tract infection in the OM-85 BV Group in the first 3 months was 0.92±0.87, and in the Placebo Group was 0.74±1.02, and at 6 months it was 1.62±1.47 and 1.03±1.34, respectively. CONCLUSION: OM-85 BV was not effective in the primary prevention of respiratory tract infections. Although most authors recommend the use of this immunostimulant in children with a history of recurrent respiratory infections, more studies are needed to define its usefulness in the primary prevention of respiratory infections in healthy children exposed to few risk factors.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Extratos Celulares/uso terapêutico , Prevenção Primária/métodos , Aleitamento Materno , Creches , Pré-Escolar , Método Duplo-Cego , Feminino , Humanos , Lactente , Masculino , Projetos Piloto , Infecções Respiratórias/tratamento farmacológico , Poluição por Fumaça de Tabaco , Resultado do Tratamento
2.
Arerugi ; 69(1): 40-47, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32051368

RESUMO

BACKGROUND: Widely accepted loading protocols for rush subcutaneous immunotherapy (rSCIT) have not been established. Our aim was to evaluate the loading protocols of rSCIT. METHODS: In the low initial dose group (33 patients), the initial dose of standardized house dust mite extract was 1 JAU or less. The target dose at the end of the rush build-up phase was 500 JAU. Next, the initial dose was increased to 10 JAU with the same target dose in the high initial dose group (18 patients). Furthermore, in the modified high initial dosage group (17 patients), the initial dose was 10 JAU but the target dose at the end of the rush phase was 300 JAU. Then, the maintenance dose of 500 JAU was administered at 9 or 10 days after rSCIT initiation. We retrospectively evaluated these protocols. RESULTS: A systemic reaction (SR) occurred in 28 out of 33 (84.8%) patients in the low initial dosage group and in 12 out of 18 (66.7%) patients in the high initial dosage group, on the other hand significantly reduced in 4 out of 17 (23.5%) patients in the modified high-dosage group. The amount of antigen reached 339.3±19.0 JAU in the low initial dosage group and 358.3±24.9 JAU in the high initial dosage group at the end of the rush phase, significantly increased 452.9±20.6 JAU in the modified high-dosage group at 9 or 10 days. CONCLUSION: In rSCIT using standardized house dust mite extract, lowering the target dose at the end of the rush phase and delaying the administration of the maintenance dose may reduce SR and increase the reached amount of antigen.


Assuntos
Alérgenos , Antígenos de Dermatophagoides/administração & dosagem , Dessensibilização Imunológica , Pyroglyphidae , Animais , Extratos Celulares/administração & dosagem , Humanos , Estudos Retrospectivos
3.
J Nat Med ; 74(1): 275-281, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31493218

RESUMO

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.


Assuntos
Anelídeos/química , Artrópodes/química , Extratos Celulares/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , RNA Ribossômico 16S/genética , RNA Ribossômico/genética , Animais , Sequência de Bases , Extratos Celulares/análise , DNA/genética , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA
4.
Asian Pac J Cancer Prev ; 20(12): 3555-3562, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31870094

RESUMO

OBJECTIVE: Breast cancer is the most frequently diagnosed cancer worldwide. The main objective of the present study was to evaluate the cytotoxic effects and mechanism of cell death induced by the extract and fractions of Vitex rotundifolia (leaves) in breast cancer cell line, T-47D. METHODS: The cytotoxicity activity was measured using MTS assay. The mode of cell death was analysed by early (phosphatidylserine externalization) and late apoptosis (DNA fragmentation). The caspases 8, 9, 3/7 and apoptotic proteins bax, bcl-2 study were done by western blot and ELISA method. RESULTS: The methanol extract was found to inhibit 50% growth of T-47D cells at the concentration of 79.43µg/ml respectively after 72hr. From seven fractions, fraction F1, F2 and F3 produced cytotoxicity effects in T-47D cell line with IC50 (72hr) < 30µg/ml. The results obtained by Annexin V/PI apoptosis detection assay and TUNEL assay suggest that active fractions of  Vitex rotundifolia induced early and late apoptosis (DNA fragmentation) in T-47D cell line. Moreover, western blot analysis and Caspase GloTM luminescent assay demonstrated that fractions F2 and F3 triggered apoptotic cell death via activation of caspases -8, -9 and -3/7 and up-regulation of  Bax and down-regulation of Bcl-2 protein.  Furthermore, chemical profiling confirms the presence of potential metabolites (vitexicarpin) in fractions of Vitex rotundifolia. CONCLUSION: Thus, the present study suggests the remarkable potential of active metabolites in fractions of Vitex rotundifolia as future cancer therapeutic agent for the treatment of breast cancer.
.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Extratos Celulares/farmacologia , Extratos Vegetais/farmacologia , Vitex/química , Neoplasias da Mama/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA , Feminino , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
5.
Science ; 366(6465): 631-637, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672897

RESUMO

Every daughter cell inherits two things from its mother: genetic information and a spatially organized complement of macromolecular complexes and organelles. The extent to which de novo self-organization, as opposed to inheritance of an already organized state, can suffice to yield functional cells is uncertain. We used Xenopus laevis egg extracts to show that homogenized interphase egg cytoplasm self-organizes over the course of ~30 minutes into compartments 300 to 400 micrometers in length that resemble cells. Formation of these cell-like compartments required adenosine triphosphate and microtubule polymerization but did not require added demembranated sperm nuclei with their accompanying centrosomes or actin polymerization. In cycling extracts with added sperm, the compartments underwent multiple cycles of division and reorganization, with mother compartments giving rise to two daughters at the end of each mitotic cycle. These results indicate that the cytoplasm can generate much of the spatial organization and cell cycle function of the early embryo.


Assuntos
Núcleo Celular/fisiologia , Citoplasma/fisiologia , Óvulo/citologia , Óvulo/fisiologia , Espermatozoides/fisiologia , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Compartimento Celular , Ciclo Celular , Extratos Celulares , Centrossomo/fisiologia , Dineínas/metabolismo , Retículo Endoplasmático/ultraestrutura , Interfase , Masculino , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mitose , Tubulina (Proteína)/metabolismo , Xenopus laevis
6.
Int J Med Mushrooms ; 21(6): 561-570, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31679228

RESUMO

The in vitro antioxidant effects of petroleum ether, ethyl acetate, and ethanol extracts isolated from Hericium coralloides were investigated. Overall, the ethyl acetate extract of H. coralloides (HcEAE) showed better antioxidant activity in vitro than the petroleum ether and ethanol extracts (HcPEE and HcETE, respectively) of H. coralloides. A comprehensive investigation of the antioxidant activity of the HcEAE in vitro indicated that it possessed superior antioxidant activity, with half maximal inhibitory concentration (IC50) values of 0.93, 1.84, 1.59, and 0.6 mg/mL against DPPH, hydroxyl, ABTS+, and superoxide (O2- ) radicals, respectively. To assess in vivo antioxidant activity, three different doses of HcEAE were orally administered in a D-galactose-induced aged mouse model. Administration of HcEAE significantly increased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and lowered the levels of malondialdehyde (MDA) in brains and sera of mice in a dose-dependent manner. A histopathology assessment indicated that the HcEAE could ameliorate the anile condition of the model mice. These results suggest that the HcEAE has potent antioxidant activity and could minimize the occurrence of age-associated disorders associated with free radicals.


Assuntos
Agaricales/química , Envelhecimento , Antioxidantes/análise , Extratos Celulares/farmacologia , Acetatos/análise , Envelhecimento/efeitos dos fármacos , Alcanos/análise , Animais , Catalase/análise , Extratos Celulares/química , Etanol , Radicais Livres/análise , Concentração Inibidora 50 , Masculino , Camundongos , Superóxido Dismutase/análise
7.
Int J Med Mushrooms ; 21(6): 571-582, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31679229

RESUMO

This study is based on the phenolic composition and the antioxidant, anticancer, antimicrobial, and antibiofilm activities of the edible mushroom Marasmius oreades from Turkey. The phenolic composition of an M. oreades ethanol extract was measured by using the Folin-Ciocalteu method, aluminium chloride colorimetry, and ultraperformance liquid chromatography. The antioxidant activity was evaluated on the basis of DPPH radical scavenging activity. The effect of the M. oreades ethanol extract was also screened in order to determine glutathione-S-transferase, glutathione peroxidase, catalase, and superoxide dismutase enzyme activities. The antimicrobial activity of the mushroom extract was evaluated by using well diffusion and was based on the minimum inhibitory concentration. In addition, the antibiofilm potential of M. oreades was analyzed against Gram-positive and -negative bacteria. Finally, the anticancer effects of the mushroom extract were tested on colon (HT-29) and breast (MCF-7 and MDA-MB-231) cancer cell lines by using the MTT assay. The results revealed that the total amount of phenolics in the ethanol extract of M. oreades was 10.990 ± 0.0007 mg gallic acid equivalent/100 g, and the total amount of flavonoids was 1.139 ± 0.0052 mg quercetin equivalent/100 g. The ultraperformance liquid chromatography results indicated that the M. oreades ethanol extract contained various phenolic compounds: catechin, ferulic, gallic acid, and vanillic acid. The M. oreades ethanol extract scavenged about 80% of DPPH free radicals. It did not show any effect on the glutathione-S-transferase, glutathione peroxidase, and catalase enzyme activities, but its maximal concentration (10 mg/mL) increased superoxide dismutase activity (8%). The ethanol extract of M. oreades showed a moderate anticancer effect on the HT-29, MCF-7, and MDA-MB-231 cell lines. Although the ethanolic extract of the mushroom did not show sufficient antibacterial activity, it presented a strong antibiofilm effect against all studied pathogenic strains at the tested concentrations.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Biofilmes/efeitos dos fármacos , Extratos Celulares/farmacologia , Marasmius/química , Bactérias/efeitos dos fármacos , Extratos Celulares/química , Linhagem Celular Tumoral , Flavonoides/análise , Ácido Gálico/análise , Células HT29 , Humanos , Células MCF-7 , Testes de Sensibilidade Microbiana , Fenóis/análise
8.
Elife ; 82019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31647414

RESUMO

The RhoGTPases are characterized as membrane-associated molecular switches that cycle between active, GTP-bound and inactive, GDP-bound states. However, 90-95% of RhoGTPases are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro. We developed methods to directly image vertebrate RhoGTPases in vivo or on lipid bilayers in vitro. Using these methods, we identified pools of active and inactive RhoGTPase associated with the membrane, found that RhoGDI can extract both inactive and active RhoGTPases, and found that extraction of active RhoGTPase contributes to their spatial regulation around cell wounds. These results indicate that RhoGDI directly contributes to the spatiotemporal patterning of RhoGTPases by removing active RhoGTPases from the plasma membrane.


Assuntos
Xenopus laevis/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Animais , Extratos Celulares , Membrana Celular/metabolismo , Citocinese , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Exocitose , Proteínas Mutantes/metabolismo , Cicatrização , Proteína cdc42 de Ligação ao GTP/metabolismo
9.
Int J Mol Sci ; 20(19)2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31574976

RESUMO

Biofouling causes major economic losses in the maritime industry. In our site study, the Bay of La Paz (Gulf of California), biofouling on immersed structures is a major problem and is treated mostly with copper-based antifouling paints. Due to the known environmental effect of such treatments, the search for environmentally friendly alternatives in this zone of high biodiversity is a priority to ensure the conservation and protection of species. The aim of this work was to link chemical ecology to marine biotechnology: indeed, the natural defense of macroalgae and sponge was evaluated against biofoulers (biofilm and macrofoulers) from the same geographical zone, and some coatings formulation was done for field assays. Our approach combines in vitro and field bioassays to ensure the selection of the best AF agent prospects. The 1st step consisted of the selection of macroalgae (5 species) and sponges (2 species) with surfaces harboring a low level of colonizers; then extracts were prepared and assayed for toxicity against Artemia, activity towards key marine bacteria involved in biofilm formation in the Bay of La Paz, and the potency to inhibit adhesion of macroorganisms (phenoloxidase assays). The most active and non-toxic extracts were further studied for biofouling activity in the adhesion of the bacteria involved in biofilm formation and through incorporation in marine coatings which were immersed in La Paz Bay during 40 days. In vitro assays demonstrated that extracts of Laurencia gardneri, Sargassum horridum (macroalgae), Haliclona caerulea and Ircinia sp. (sponges) were the most promising. The field test results were of high interest as the best formulation were composed of extracts of H. caerulea and S. horridum and led to a reduction of 32% of biofouling compared with the control.


Assuntos
Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Incrustação Biológica/prevenção & controle , Biomimética , Desenvolvimento de Medicamentos , Organismos Aquáticos , Bactérias/crescimento & desenvolvimento , Aderência Bacteriana , Biomarcadores , Extratos Celulares/química , Extratos Celulares/farmacologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Testes de Sensibilidade Microbiana , Monofenol Mono-Oxigenase/metabolismo , Alga Marinha/química
10.
Int J Mol Sci ; 20(19)2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31575049

RESUMO

A major fraction (MPT-W), eluted by deionized water, was extracted from mycelium polysaccharides of Termitomyces albuminosus (MPT), and its antioxidant, anti-fibrosis, and anti-inflammatory activities in CCl4-induced chronic liver injury mice, as well as preliminary characterizations, were evaluated. The results showed that MPT-W was a polysaccharide of α- and ß-configurations containing xylose (Xyl), fucose (Fuc), mannose (Man), galactose (Gal), and glucose (Glc) with a molar ratio of 0.29:8.67:37.89:35.98:16.60 by gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared (FT-IR) spectroscopy. Its molecular weight (Mw), obtained by high-performance gel permeation chromatography (HPGPC), was 1.30 × 105 Da. The antioxidant assays in vitro showed that MPT-W displayed scavenging free-radical abilities. Based on the data of in vivo experiments, MPT-W could inhibit TGFß1/Smad3 and NF-κB pathways; decrease the level and activity of cytochrome P4502E1 (CYP2E1), malonaldehyde (MDA) and serum enzyme; activate the HO-1/Nrf2 pathway; and increase antioxidant enzymes to protect the liver in CCl4-induced chronic liver injury mice. Therefore, MPT-W could be a potentially natural and functional resource contributing to antioxidant, hepatoprotective, and anti-inflammatory effects with potential health benefits.


Assuntos
Extratos Celulares/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Polissacarídeos Fúngicos/farmacologia , Micélio/química , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Termitomyces/química , Animais , Extratos Celulares/química , Extratos Celulares/isolamento & purificação , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Polissacarídeos Fúngicos/química , Polissacarídeos Fúngicos/isolamento & purificação , Camundongos , NF-kappa B/metabolismo , Substâncias Protetoras/química , Proteína Smad3 , Análise Espectral , Fator de Crescimento Transformador beta1/metabolismo
11.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569635

RESUMO

Poria cocos Wolf (PCW) is an edible, pharmaceutical mushroom with remarkable biological properties including anti-tumor, anti-inflammation, anti-oxidation, anti-ageing, and anti-diabetic effects. In the current study, we investigated the effects of PCW extract on hepatic steatosis under in vitro and in vivo conditions, and elucidated the underlying mechanisms. In this study, a mixture of HepG2 cells treated with free fatty acid (FFA)-palmitic and oleic acid-and high-fat diet (HFD)-fed obese mice were used; in this background, the triglyceride (TG) levels in HepG2 cells and mice liver were measured, and the expression levels of genes associated with lipogenesis, fatty acid oxidation, endoplasmic reticulum (ER) stress, and autophagy were determined. Treatment of HepG2 cells with FFA enhanced intracellular TG levels in HepG2 cells, but co-treatment with PCW significantly attenuated the TG levels. Notably, PCW significantly enhanced the phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and sterol regulatory element-binding protein-1c (SREBP-1c) in FFA-treated HepG2 cells. PCW downregulated the expression of lipogenesis-related genes, but upregulated the expression of genes associated with fatty acid oxidation. Further, PCW inhibited FFA-induced expression of ER stress markers and induced autophagy proteins. However, inhibition of AMPK significantly attenuated the beneficial effects of PCW in HepG2 cells. Moreover, PCW efficiently decreased HFD-induced hepatic TG accumulation in vivo and increased the phosphorylation of hepatic AMPK. Three compounds present in PCW including poricoic acid, pachymic acid, and ergosterol, significantly decreased FFA-induced increase in intracellular TG levels, consistent with increased AMPK phosphorylation, suggesting that poricoic acid, pachymic acid, and ergosterol are responsible for PCW-mediated amelioration of hepatic steatosis. Taken together, these results demonstrated that PCW ameliorates hepatic steatosis through the regulation of lipid metabolism, inhibition of ER stress, and activation of autophagy in an AMPK-dependent manner. This suggested that PCW can be potentially used for the treatment of hepatic steatosis.


Assuntos
Agaricales/química , Autofagia/efeitos dos fármacos , Extratos Celulares/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Poria/química , Animais , Extratos Celulares/química , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Masculino , Camundongos , Inibidores de Proteínas Quinases/farmacologia
12.
Curr Protoc Protein Sci ; 97(1): e94, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31517451

RESUMO

Site- and structure-specific quantitative N-glycoproteomics characterization of differentially expressed N-glycosylation at the intact N-glycopeptide level with distinct chromatographic separation and structure-specific fragment ions has become possible with the recent development of RPLC-pentaHILIC 2DLC separation and use of the intact N-glycopeptide search engine GPSeeker. Here we provide a detailed protocol for this GPSeeker-centered structure-specific isotopic-labeling quantitative N-glycoproteomics pipeline. The protocols include sample preparation of a 1:1 mixture of light (-CH3 )2 and heavy (-13 CD2 H)2 dimethylated intact N-glycopeptides from LO2 and HepG2 cells, RPLC-pentaHILIC 2DLC separation of the mixture, intact N-glycopeptide database search and identification using GPSeeker, and quantitation of differentially expressed intact N-glycopeptides using the quantitation module GPSeekerQuan. © 2019 by John Wiley & Sons, Inc.


Assuntos
Extratos Celulares/análise , Extratos Celulares/isolamento & purificação , Glicopeptídeos/análise , Glicopeptídeos/isolamento & purificação , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Glicosilação , Conformação Proteica , Proteômica/métodos , Ferramenta de Busca/métodos , Espectrometria de Massas em Tandem
13.
Vet Immunol Immunopathol ; 217: 109944, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31563725

RESUMO

Propagation ex vivo of mesenchymal stem cells (MSCs) requires culture medium supplementation. Fetal bovine serum (FBS) has long been the gold standard supplement, but its use is being questioned mainly due to ethical and safety issues. The use of platelet lysate (PL) as substitute of FBS has been proposed but little is known about its effects on equine MSCs characteristics including their immune profile. The aim of this work was to investigate for the first time the effect of allogenic PL on the immunogenic and immunomodulatory gene expression profile of equine bone marrow derived MSCs (eBM-MSCs) as well as on their proliferation ability, phenotype markers, and viability post-cryopreservation. The eBM-MSCs (n = 3) cultures were supplemented with 20% of allogeneic pooled concentrated PL (CPL; 591 × 103 platelets/µL) or basal PL (BPL; 177 × 103 platelets/µL) from three donors, using 10% FBS supplementation as control. The proliferative ability of eBM-MSCs under the three conditions was evaluated by calculating the cell doubling times (DT) up to passage 3 (P3) and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at P3. Viability of eBM-MSCs post-cryopreserved with CPL or FBS was assessed at 15, 30 and 60 days. The gene expression profile of eBM-MSCs was evaluated in P3 by RT-qPCR for characterization, immunogenic and immunomodulatory markers. The cells cultured in CPL had significantly higher ability to proliferate than with FBS or BPL (P < 0.001) in the MTT assay. Post-cryopreserved viability was similar between cells cultured and preserved in FBS and CPL at all time-points. Gene expression of MSC characterization markers was similar among the three conditions. The gene expression of the immunogenic markers MHC-I, MHC-II and CD40 was slightly (non-significant) increased in CPL condition compared to FBS and BPL. The CPL condition showed higher expression of the genes coding for the immunomodulatory molecules VCAM-1 (non-significant) and IL-6 (P < 0.05), and similar for COX-2; whereas iNOS and IDO were not expressed under any condition. In conclusion, the replacement of FBS by allogeneic CPL as a supplement for ex vivo propagation of eBM-MSCs provides appropriate proliferation and cryopreservation, and mildly upregulates the gene expression of immunomodulatory markers, thus constituting a potentially suitable alternative to the use of FBS. Further studies are needed to clarify the composition and effects of CPL supplementation on equine MSCs immunological profile.


Assuntos
Plaquetas/química , Células da Medula Óssea/citologia , Extratos Celulares/química , Meios de Cultura/química , Células-Tronco Mesenquimais/citologia , Animais , Células da Medula Óssea/imunologia , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Perfilação da Expressão Gênica , Cavalos , Masculino , Células-Tronco Mesenquimais/imunologia , Transcriptoma
14.
Int Arch Allergy Immunol ; 180(3): 159-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31563904

RESUMO

BACKGROUND: The dawn of the "omics" technologies has changed allergy research, increasing the knowledge and identification of new allergens. However, these studies have been almost restricted to Dermatophagoides spp. Although Blomia tropicalis has long been established as a clinically important source of allergens, a thorough proteomic characterization is still lacking for this dust mite. OBJECTIVE: To increase knowledge of B. tropicalis allergens through proteomic analysis. METHODS: Eleven in-bred lineages of B. tropicalis were obtained from 11 unique different pregnant females. Their somatic extracts were analyzed and compared with a commercially available extract by liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Considerable differences in the protein expression profiles were found among the breeds, and most of them displayed higher expression levels of major allergens than the commercially available extract. Blo t 2 was the most prominent allergenic protein in the analyzed extracts. Six identified allergens and 14 isoforms have not yet been recognized by IUIS. Conversely, 3 previously recognized B. tropicalis allergens were not found. CONCLUSIONS: The clear impact of inbreeding on allergen content shown by our study leads us to conclude that the quantification and/or identification of allergens from in-bred lines should be routinely considered for mite cultivation in order to select breeds with higher amounts of major allergens. In this sense, LC-MS/MS may be a useful method to achieve this quality control for research and commercial purposes.


Assuntos
Alérgenos/imunologia , Extratos Celulares/imunologia , Hipersensibilidade/imunologia , Feromônios/imunologia , Sarcoptidae/imunologia , Alérgenos/isolamento & purificação , Animais , Animais Endogâmicos , Variação Biológica da População , Extratos Celulares/química , Cromatografia Líquida , Feminino , Humanos , Feromônios/isolamento & purificação , Gravidez , Especificidade da Espécie , Espectrometria de Massas em Tandem , Transcriptoma
15.
Int J Mol Sci ; 20(19)2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31557796

RESUMO

Sjogren's syndrome (SS) is an autoimmune disease that manifests primarily in salivary and lacrimal glands leading to dry mouth and eyes. Unfortunately, there is no cure for SS due to its complex etiopathogenesis. Mesenchymal stem cells (MSCs) were successfully tested for SS, but some risks and limitations remained for their clinical use. This study combined cell- and biologic-based therapies by utilizing the MSCs extract (MSCsE) to treat SS-like disease in NOD mice. We found that MSCsE and MSCs therapies were successful and comparable in preserving salivary and lacrimal glands function in NOD mice when compared to control group. Cells positive for AQP5, AQP4, α-SMA, CK5, and c-Kit were preserved. Gene expression of AQP5, EGF, FGF2, BMP7, LYZ1 and IL-10 were upregulated, and downregulated for TNF-α, TGF-ß1, MMP2, CASP3, and IL-1ß. The proliferation rate of the glands and serum levels of EGF were also higher. Cornea integrity and epithelial thickness were maintained due to tear flow rate preservation. Peripheral tolerance was re-established, as indicated by lower lymphocytic infiltration and anti-SS-A antibodies, less BAFF secretion, higher serum IL-10 levels and FoxP3+ Treg cells, and selective inhibition of B220+ B cells. These promising results opened new venues for a safer and more convenient combined biologic- and cell-based therapy.


Assuntos
Extratos Celulares/farmacologia , Células-Tronco Mesenquimais/metabolismo , Animais , Apoptose , Biomarcadores , Extratos Celulares/uso terapêutico , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Ceratoconjuntivite Seca/tratamento farmacológico , Ceratoconjuntivite Seca/imunologia , Ceratoconjuntivite Seca/metabolismo , Aparelho Lacrimal/imunologia , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saliva/metabolismo , Síndrome de Sjogren/tratamento farmacológico , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Xerostomia/tratamento farmacológico , Xerostomia/imunologia , Xerostomia/metabolismo
16.
Rhinology ; 57(6): 451-459, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31403136

RESUMO

BACKGROUND: Probiotics are mainly distributed in the mucosal system and have the ability to enhance mucosal barrier function and regulate immune responses. Broncho-Vaxom (BV), as a probiotic, has been applied to patients suffering from respiratory tract infections, but its potential effectiveness in allergic rhinitis (AR) has not been evaluated in human. This study aimed to investigate the clinical efficacy of BV in patients with persistent AR and to elucidate the underlying cellular mechanisms. METHODS: Sixty patients with AR were enrolled to this study and were randomly assigned to the BV group (n=30) and the placebo group (n=30). Changes of clinical symptoms and laboratory parameters of allergic inflammation were measured at baseline visit, immediately after BV treatment, four weeks, and eight weeks after the BV treatment. RESULTS: After BV treatment, medication score in the BV group was significantly decreased compared with placebo group, along with a significant drop of the total nasal symptom score and the individual nasal symptom scores (itching score: 23.72±5.32%; nasal rhinorrhea score: 18.59±4.83%; sneezing score: 23.08±4.98%). The levels of IL-4 and IL-13 in nasal lavage were diminished remarkably while the level of INF-γ was markedly increased in the BV group. This rendered a significant reduction of the ratio of IL-4/INF-γ. Moreover, a decrease of eosinophils in nasal smear was observed after BV treatment. The BV-induced favorable changes sustained for at least four to eight weeks post BV treatment. CONCLUSION: Oral administration of BV offers remarkable and sustained efficacy in alleviating AR symptoms and may be considered as an alternative therapeutic strategy for patients with persistent AR. BV acts by improving the overall mucosal immunity via restoring and maintaining the normal Th1/Th2 cytokine balance as an underlying cellular/signaling mechanism.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Extratos Celulares/administração & dosagem , Mucosa Nasal/efeitos dos fármacos , Probióticos/uso terapêutico , Rinite Alérgica/tratamento farmacológico , Administração Oral , Citocinas/imunologia , Humanos , Mucosa Nasal/imunologia , Probióticos/farmacologia , Rinite Alérgica/imunologia , Células Th1/imunologia , Células Th2/imunologia
17.
Adv Exp Med Biol ; 1155: 471-482, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468424

RESUMO

Endothelial cell dysfunction (ECD) is a broad term, which implies dysregulation of endothelial cell functions. Several factors contribute to ECD including high blood pressure, high cholesterol levels, diabetes, obesity, hyperglycemia, and advanced glycation end products (AGEs). The highly reactive dicarbonyl methylglyoxal (MGO) is mainly formed as byproduct of glycolysis. Therefore, high blood glucose levels result in increased MGO accumulation. Taurine-rich foods are considered to protect against various diseases including vasculopathy and to exert anti-aging effects. Here, we investigated the protective effect of hot water extract of Octopus ocellatus meat (OOM), which contains high amounts of taurine, on MGO-induced cell damage in human umbilical vein endothelial cells and zebrafish embryos. Hot water extract of OOMinhibited MGO-induced cytotoxicity and DNA damage, as well as AGEs accumulation. In addition, hot water extract of OOM protected against vascular damage in zebrafish embryos. These results suggest that hot water extract of OOM possesses protective activity against MGO-induced cytotoxicity in both umbilical vein endothelial cells and zebrafish embryos. Therefore, it could be used as a dietary source of an agent for the prevention of vascular diseases.


Assuntos
Extratos Celulares/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Octopodiformes/química , Aldeído Pirúvico/toxicidade , Taurina/farmacologia , Animais , Células Cultivadas , Embrião não Mamífero/efeitos dos fármacos , Humanos , Carne , Peixe-Zebra
18.
Adv Exp Med Biol ; 1155: 483-495, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468425

RESUMO

The loss of pancreatic ß-cells plays a central role in the pathogenesis of both type 1 and type 2 diabetes, and many studies have been focused on ways to improve glucose homeostasis by preserving, expanding and improving the function of ß-cell. Elevated levels of free fatty acids such as palmitate might contribute to the loss of ß-cells. A marine squid, Loliolus beka has long been used as a food in Korea, China, Japan and Europe due to its tender meat and high taurine content. Here, we investigated the protective effects of a hot water extract of Loliolus beka meat (LBM) against palmitate toxicity in Ins-1 cells, a rat ß-cell line. Treatment with LBM extract protected against palmitate-induced cytotoxicity and scavenged overproduction of nitric oxide, alkyl, and hydroxyl radicals. In addition, LBM extract protected against palmitate-induced DNA damage and ß-cell dysfunction. These findings suggest that LBM protects pancreatic ß-cells from palmitate-induced damage. LBM could be a potential therapeutic functional food for diabetes.


Assuntos
Extratos Celulares/farmacologia , Dano ao DNA , Decapodiformes/química , Radicais Livres/efeitos adversos , Células Secretoras de Insulina/efeitos dos fármacos , Palmitatos/efeitos adversos , Taurina/farmacologia , Animais , Apoptose , Linhagem Celular , Diabetes Mellitus , Carne , Ratos
19.
Adv Exp Med Biol ; 1155: 567-581, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468432

RESUMO

Here, we investigated the hepatoprotective effect of a hot water extract from Loliolus beka gray meat (LBMH) containing plentiful taurine in H2O2-induced oxidative stress in hepatocytes. LBMH potently scavenged the 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and exhibited the good reducing power and the oxygen radical absorbance capacity (ORAC) value. Also, LBMH improved the cell viability against H2O2-induced hepatic damage in cultured hepatocytes by reducing intracellular reactive oxygen species (ROS) production. In addition, LBMH inhibited apoptosis via a reduction in sub-G1 cell population, as well as inhibition of apoptotic body formation from H2O2-induced oxidative damage in hepatocytes. Moreover, LBMH regulated the expression levels of Bax, a pro-apoptotic molecule and Bcl-2, an anti-apoptotic molecule in H2O2-treated hepatocytes. Additionally, pre-treatment with LBMH increased the expression of heme oxygenase 1 (HO-1), which is a hepatoprotective enzyme, by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) in H2O2-treated hepatocytes. Taken together, LBMH may be useful as a food ingredient for treatment of liver disease by regulating the Nrf2/HO-1 signal pathway.


Assuntos
Antioxidantes , Extratos Celulares/farmacologia , Decapodiformes/química , Hepatócitos/efeitos dos fármacos , Estresse Oxidativo , Taurina/farmacologia , Animais , Células Cultivadas , Heme Oxigenase-1/metabolismo , Hepatócitos/citologia , Humanos , Peróxido de Hidrogênio , Carne , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
20.
Adv Exp Med Biol ; 1155: 583-596, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468433

RESUMO

In this study, we evaluated the protective effects of an aqueous extract from Batillus cornutus meat (BM) against cellular oxidative damage caused by hydrogen peroxide (H2O2) in human hepatocyte, Chang cells. First, we prepared an aqueous extract of BM meat (BMW) showing the highest taurine content among free amino acid contents. BMW led to high antioxidant activity showing 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) radical scavenging activity, good reducing power and an oxygen radical absorbance capacity (ORAC) value. Also, BMW improved cell viability that was diminished by H2O2 exposure, as it reduced the generation of intracellular reactive oxygen species (ROS) in Chang cells. In addition, BMW up-regulated the production of antioxidant enzymes, such as catalase and superoxide dismutase (SOD), compared to H2O2-treated Chang cells lacking BMW. Moreover, BMW induced the expressions of nuclear Nrf2 and cytosolic HO-1 in H2O2-treated Chang cells. Interestingly, the treatment of ZnPP, HO-1 inhibitor, abolished the improvement in cell viability and intracellular ROS generation mediated by BMW treatment. In conclusion, this study suggests that BMW protects hepatocytes against H2O2-mediated cellular oxidative damage via up-regulation of the Nrf2/HO-1 signal pathway.


Assuntos
Extratos Celulares/farmacologia , Gastrópodes/química , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Transdução de Sinais , Taurina/farmacologia , Animais , Células Cultivadas , Humanos , Peróxido de Hidrogênio , Carne , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
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