RESUMO
ETHNOPHARMACOLOGY RELEVANCE: Ayahuasca is a tea produced through decoction of Amazonian plants. It has been used for centuries by indigenous people of South America. The beverage is considered to be an ethnomedicine, and it is traditionally used for the treatment of a wide range of diseases, including neurological illness. Besides, some scientific evidence suggests it may be applicable to Parkinson's disease (PD) treatment. Thus, Ayahuasca deserves in depth studies to clarify its potential role in this disease. AIM OF THE STUDY: This study aimed to use an untargeted metabolomics approach to evaluate the neuroprotective potential of the Ayahuasca beverage, the extracts from its matrix plants (Banisteriopsis caapi and Psychotria viridis), its fractions and its main alkaloids on the viability of SH-SY5Y neuroblastoma cells in an in vitro PD model. MATERIAL AND METHODS: The cytotoxicity of Ayahuasca, crude extracts, and fractions of B. caapi and P. viridis, as well as neuroprotection promoted by these samples in a 6-hydroxydopamine (6-OHDA)-induced neurodegeneration model, were evaluated by the MTT assay at two time-points: 48 h (T1) and 72 h (T2). The main alkaloids from Ayahuasca matrix plants, harmine (HRE) and N,N-dimethyltryptamine (DMT), were also isolated and evaluated. An untargeted metabolomics approach was developed to explore the chemical composition of samples with neuroprotective activity. Ultra-Performance Liquid Chromatography coupled to Electrospray Ionisation and Time-of-Flight (UPLC-ESI-TOF) metabolome data was treated and further analysed using multivariate statistical analyses (MSA): principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The metabolites were dereplicated using the Dictionary of Natural Products and an in house database. The main alkaloids were also quantified by UPLC-MS/MS. RESULTS: The samples did not cause cytotoxicity in vitro and three of samples intensely increased cell viability at T1. The crude extracts, alkaloid fractions and HRE demonstrated remarkable neuroprotective effect at T2 while the hydroalcoholic fractions demonstrated this neuroprotective effect at T1 and T2. Several compounds from different classes, such as ß-carbolines and monoterpene indole alkaloids (MIAs) were revealed correlated with this property by MSA. Additionally, a total of 2419 compounds were detected in both ionisation modes. HRE showed potent neuroprotective action at 72 h, but it was not among the metabolites positively correlated with the most efficacious neuroprotective profile at either time (T1 and T2). Furthermore, DMT was statistically important to differentiate the dataset (VIP value > 1), although it did not exhibit sufficient neuroprotective activity by in vitro assay, neither a positive correlation with T1 and T2 neuroprotective profile, which corroborated the MSA results. CONCLUSION: The lower doses of the active samples stimulated neuronal cell proliferation and/or displayed the most efficacious neuroprotection profile, namely by preventing neuronal damage and improving cell viability against 6-OHDA-induced toxicity. Intriguingly, the hydroalcoholic fractions exhibited enhanced neuroprotective effects when compared to other samples and isolated alkaloids. This finding corroborates the significance of a holistic approach. The results demonstrate that Ayahuasca and its base plants have potential applicability for PD treatment and to prevent its progression differently from current drugs to treat PD.
Assuntos
Antiparkinsonianos/farmacologia , Banisteriopsis/química , Metabolômica , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Psychotria/química , Antiparkinsonianos/isolamento & purificação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Etnofarmacologia , Humanos , Análise dos Mínimos Quadrados , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/isolamento & purificação , Oxidopamina/toxicidade , Extratos Vegetais/isolamento & purificação , Polissacarídeos , Análise de Componente Principal , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coli BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an αßαßαß topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (1H-15N HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies.
Assuntos
Proteínas Fúngicas/imunologia , Nucleotidiltransferases/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Trifosfato de Adenosina/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Citidina Trifosfato/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Escherichia coli/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologia , Adulto JovemRESUMO
OBJECTIVES: Verify the in-vitro effect of triiodothyronine (T3) on the chondrogenic differentiation of female rat bone marrow mesenchymal stem cells (BMMSCs) over several time periods and at several doses. METHODS: CD54 + /CD73 + /CD90 + BMMSCs from Wistar female rats were cultured in chondrogenic medium with or without T3 (0.01; 1; 100; 1000 nm). At seven, 14 and 21 days, the cell morphology, chondrogenic matrix formation and expression of Sox9 and collagen II were evaluated. KEY FINDINGS: The dose of 100 nm did not alter the parameters evaluated in any of the periods studied. However, the 0.01 nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and expression of Sox9 and collagen II in at least one of the evaluated periods; the 1 nm T3 dose also improved the chondrogenic potential by increasing the chondrogenic matrix formation and the expression of collagen II in at least one of the evaluated periods. The 1000 nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and Sox9 expression in at least one of the evaluated periods. CONCLUSIONS: T3 has a dose-dependent effect on the differentiation of BMMSCs from female rats.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Animais , Células Cultivadas , Condrócitos/citologia , Colágeno Tipo II/genética , Relação Dose-Resposta a Droga , Feminino , Células-Tronco Mesenquimais/citologia , Ratos , Ratos Wistar , Fatores de Transcrição SOX9/genética , Fatores de Tempo , Tri-Iodotironina/administração & dosagemRESUMO
Tumor cells capture the signaling pathways used by normal tissue to promote their own survival and dissemination and among them, the NF-κB and MAPK pathways (ERK, JNK and p38). MAPK activation has ambiguous effects on tumor cell fate depending on cell type, cancer stage and the engaged MAPK isoforms. A synthetic peptide named LyeTx II, derived from the venom of the Brazilian spider Lycosa erythrognatha, was capable of increasing MDA-MB-231 aggressive breast cancer cell proliferation as indicated by MTT and BrdU (5-bromo-2'-deoxyuridine) incorporation assay and cell migration. A correlation has been established between the accelerated proliferation and migration observed in the presence of LyeTx II and the upregulation of p38 MAPK phosphorylation. The use of the selective inhibitor of p38α/ß (SB203580) abrogated the peptide effect in MDA-MB-231 cells. Besides, an augment of the canonical NF-κB pathway activation considered as crucial in cancer progression was noted after cell incubation with LyeTx II. Importantly, activation of p38 and NF-κB pathways was dependent on TAK1 activity. Together, these data suggest that TAK1-p38 pathway may represent an interesting target for treatment of aggressive breast cancers.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Peptídeos/farmacologia , Venenos de Aranha/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Fosforilação , Piridinas/farmacologia , Regulação para CimaRESUMO
BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. OBJECTIVE: The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. METHODS: To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. RESULTS: The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. CONCLUSION: The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological samples in order to validate this method and then generate a diagnostic kit for Paracoccidioidomycosis.
Assuntos
Proteínas Fúngicas/genética , Paracoccidioides/isolamento & purificação , Paracoccidioidomicose/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Brasil , Simulação por Computador , Genoma Fúngico , Humanos , Paracoccidioides/genética , Paracoccidioidomicose/microbiologia , Patentes como Assunto , Reação em Cadeia da Polimerase em Tempo Real/economia , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, ß-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and ß-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and ß-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and ß-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells.
Assuntos
Núcleo Celular/metabolismo , Receptores ErbB/metabolismo , Transporte Ativo do Núcleo Celular , Betacelulina/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Neoplasias/metabolismo , Fosforilação , Fator de Crescimento Transformador alfa/metabolismoRESUMO
O objetivo deste estudo foi comparar o potencial osteogênico das células tronco mesenquimais extraídas da medula óssea (CTM-MO) com as do tecido adiposo (CTM-AD) de cães adultos. As células foram caracterizadas fenotipicamente quanto à expressão de CD29, CD90, CD34 e CD45 e submetidas à diferenciação adipogênica e condrogênica por 21 dias e osteogênica por 7, 14 e 21 dias. Foram constituídos quatro grupos: 1) CTM-MO em meio osteogênico, 2) CTM-MO em meio basal, 3) CTM-AD em meio osteogênico e 4) CTM-AD em meio basal. Aos 7, 14 e 21 dias de diferenciação osteogênica as culturas foram submetidas às avaliações da conversão de MTT em formazan, da atividade da fosfatase alcalina (FA), da síntese de colágeno e de matriz mineralizada, avaliação do número de células por campo e foram quantificados os transcritos gênicos para osterix, sialoproteina óssea (BSP), osteonectina (ON) e osteocalcina (OC). Tanto as células extraídas da medula óssea quanto do tecido adiposo mostraram elevada expressão de marcadores para células tronco e baixa expressão de marcadores de células hematopoiéticas (menor que 2%). Além disso, foram capazes de se diferenciar em osteoblastos, condrócitos e adipócitos. As CTM-AD submetidas à diferenciação osteogênica mostraram maior conversão do MTT em formazan que as CTM-MO, sob mesmas condições aos 7 e 21 dias. O número de células por campo, a atividade da FA, a síntese de colágeno e de matriz mineralizada foram superior nas CTM-AD em diferenciação, em relação às CTM-MO sob as mesmas condições, em todos os tempos estudados. As expressões de osterix, BSP e OC foram predominantemente superiores nas CTM-MO diferenciadas, mas a expressão de ON foi superior nas CTM-AD diferenciadas aos 7, 14 e 21 dias. Conclui-se que as CTM-AD apresentam maior potencial osteogênico que as CTM-MO quando extraídas de cães adultos.(AU)
The aim of this study was to compare the osteogenic potential of mesenchymal stem cells obtained from bone marrow (BM-MSC) with those extracted from adipose tissue (AT-MSC) of adult dogs. The cells were phenotypically categorized according to the expression of CD29, CD90, CD34 and CD45, and submitted to adipogenic and chondrogenic differentiation for 21 days and osteogenic differentiation for 7, 14 and 21 days. Four groups were formed: BM-MSC in osteogenic medium (1), BM-MSC in basal medium (2), AT-MSC in osteogenic medium (3) and ATMSC in basal medium (4). On days 7, 14 and 21 of osteogenic differentiation, the cultures were submitted to evaluations of MTT conversion in formazan, of alkaline phosphatase activity (AP), of collagen and mineralized matrix synthesis, evaluation of the number of cells per field and there was quantification of the gene transcripts for osterix, bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC). Both the cells obtained from bone marrow and those from adipose tissue showed high expression of stem cells markers and low expression of hematopoietic cells markers (lower than 2%). Besides, they were able to differentiate into osteoblasts, chondrocytes and adipocytes. AT-MSC submitted to osteogenic differentiation showed higher MTT conversion in formazan than BM-MSC, under the same conditions on days 7 and 21. The number of cells per field, the AP activity, the collagen and mineralized matrix synthesis were higher in AT-MSC en differentiation, in relation to BM-MSC under the same conditions in all evaluated times. Expressions of osterix, BSP and OC were predominantly higher in differentiated BMMSC, however the expression of ON was higher AT-MSC differentiated on days 7, 14 and 21. In conclusion, AT-MSC present higher osteogenic potential than BM-MSC when extracted from adult dogs.(AU)
Assuntos
Animais , Cães , Tecido Adiposo/citologia , Células da Medula Óssea , Osteogênese , Células-Tronco , Regeneração ÓsseaRESUMO
O objetivo deste estudo foi comparar o potencial osteogênico das células tronco mesenquimais extraídas da medula óssea (CTM-MO) com as do tecido adiposo (CTM-AD) de cães adultos. As células foram caracterizadas fenotipicamente quanto à expressão de CD29, CD90, CD34 e CD45 e submetidas à diferenciação adipogênica e condrogênica por 21 dias e osteogênica por 7, 14 e 21 dias. Foram constituídos quatro grupos: 1) CTM-MO em meio osteogênico, 2) CTM-MO em meio basal, 3) CTM-AD em meio osteogênico e 4) CTM-AD em meio basal. Aos 7, 14 e 21 dias de diferenciação osteogênica as culturas foram submetidas às avaliações da conversão de MTT em formazan, da atividade da fosfatase alcalina (FA), da síntese de colágeno e de matriz mineralizada, avaliação do número de células por campo e foram quantificados os transcritos gênicos para osterix, sialoproteina óssea (BSP), osteonectina (ON) e osteocalcina (OC). Tanto as células extraídas da medula óssea quanto do tecido adiposo mostraram elevada expressão de marcadores para células tronco e baixa expressão de marcadores de células hematopoiéticas (menor que 2%). Além disso, foram capazes de se diferenciar em osteoblastos, condrócitos e adipócitos. As CTM-AD submetidas à diferenciação osteogênica mostraram maior conversão do MTT em formazan que as CTM-MO, sob mesmas condições aos 7 e 21 dias. O número de células por campo, a atividade da FA, a síntese de colágeno e de matriz mineralizada foram superior nas CTM-AD em diferenciação, em relação às CTM-MO sob as mesmas condições, em todos os tempos estudados. As expressões de osterix, BSP e OC foram predominantemente superiores nas CTM-MO diferenciadas, mas a expressão de ON foi superior nas CTM-AD diferenciadas aos 7, 14 e 21 dias. Conclui-se que as CTM-AD apresentam maior potencial osteogênico que as CTM-MO quando extraídas de cães adultos.(AU)
The aim of this study was to compare the osteogenic potential of mesenchymal stem cells obtained from bone marrow (BM-MSC) with those extracted from adipose tissue (AT-MSC) of adult dogs. The cells were phenotypically categorized according to the expression of CD29, CD90, CD34 and CD45, and submitted to adipogenic and chondrogenic differentiation for 21 days and osteogenic differentiation for 7, 14 and 21 days. Four groups were formed: BM-MSC in osteogenic medium (1), BM-MSC in basal medium (2), AT-MSC in osteogenic medium (3) and ATMSC in basal medium (4). On days 7, 14 and 21 of osteogenic differentiation, the cultures were submitted to evaluations of MTT conversion in formazan, of alkaline phosphatase activity (AP), of collagen and mineralized matrix synthesis, evaluation of the number of cells per field and there was quantification of the gene transcripts for osterix, bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC). Both the cells obtained from bone marrow and those from adipose tissue showed high expression of stem cells markers and low expression of hematopoietic cells markers (lower than 2%). Besides, they were able to differentiate into osteoblasts, chondrocytes and adipocytes. AT-MSC submitted to osteogenic differentiation showed higher MTT conversion in formazan than BM-MSC, under the same conditions on days 7 and 21. The number of cells per field, the AP activity, the collagen and mineralized matrix synthesis were higher in AT-MSC en differentiation, in relation to BM-MSC under the same conditions in all evaluated times. Expressions of osterix, BSP and OC were predominantly higher in differentiated BMMSC, however the expression of ON was higher AT-MSC differentiated on days 7, 14 and 21. In conclusion, AT-MSC present higher osteogenic potential than BM-MSC when extracted from adult dogs.(AU)
Assuntos
Animais , Cães , Osteogênese , Células-Tronco Adultas , Tecido Adiposo/citologia , Células da Medula Óssea , Achados Morfológicos e Microscópicos , Regeneração ÓsseaRESUMO
Intraocular delivery systems have been developed to treat many eye diseases, especially those affecting the posterior segment of the eye. However, ocular toxoplasmosis, the leading cause of infectious posterior uveitis in the world, still lacks an effective treatment. Therefore, our group developed an intravitreal polymeric implant to release clindamycin, a potent anti-Toxoplasma antibiotic. In this work, we used different techniques such as differential scanning calorimetry, thermogravimetry, X-ray diffraction, scanning electron microscopy, and fourier-transform infrared spectroscopy to investigate drug/polymer properties while manufacturing the delivery system. We showed that the lyophilization, hot molding process, and sterilization by gamma irradiation did not change drug/polymer physical-chemistry properties. The drug was found to be homogeneously dispersed into the poly lactic-co-glycolic acid (PLGA) chains and the profile release was characterized by an initial burst followed by prolonged release. The drug profile release was not modified after gamma irradiation and non-covalent interaction was found between the drug and the PLGA. We also observed the preservation of the drug activity by showing the potent anti-Toxoplasma effect of the implant, after 24-72 h in contact with cells infected by the parasite, which highlights this system as an alternative to treat toxoplasmic retinochoroiditis.
Assuntos
Antiprotozoários/administração & dosagem , Clindamicina/administração & dosagem , Raios gama , Temperatura Alta , Ácido Láctico , Ácido Poliglicólico , Toxoplasma/efeitos dos fármacos , Corpo Vítreo , Varredura Diferencial de Calorimetria , Linhagem Celular , Liofilização , Humanos , Microscopia Eletrônica de Varredura , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Espectroscopia de Infravermelho com Transformada de Fourier , Termogravimetria , Difração de Raios XRESUMO
Objective: Evaluate the effect of in vitro triiodothyronine (T3) on the reduced osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) of adult rats with osteoporosis compared with BMMSCs of young and adult rats without osteoporosis. Methods: groups were tested: BMMSCs of young rats; BMMSCs of adult rats without osteoporosis; BMMSCs of adult rats with osteoporosis without T3 and BMMSCs of adult rats with osteoporosis treated with T3 (0.01, 1, 100 and 1000 nM). Alkaline phosphatase activity, MTT reduction, mineralized nodules and gene expression for collagen, osteocalcin, sialoprotein, osteopontin and BMP-2 were evaluated. Results: Osteoporosis increased the alkaline phosphatase activity and reduced the formation of mineralized nodules and expression of collagen and osteopontin in at least one of the observed time points. However, the T3 treatment of BMMSCs of rats with osteoporosis altered these parameters. Conclusion: It was concluded that doses of T3, 0.01 and 1000 nM had a positive effect promoted by increased osteogenic matrix synthesis and collagen expression in at least one of the evaluated time points compared to BMMSCs of rats with osteoporosis without treatment. However, T3 was unable to reach the osteogenic potential of the MSCs of healthy young rats.(AU)
Assuntos
Cobaias , Ratos , Osteoporose/veterinária , Medula Óssea , Hormônios Tireóideos/uso terapêutico , Osteogênese , Células-Tronco , Técnicas de Cultura de Células/veterináriaRESUMO
Objective: Evaluate the effect of in vitro triiodothyronine (T3) on the reduced osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) of adult rats with osteoporosis compared with BMMSCs of young and adult rats without osteoporosis. Methods: groups were tested: BMMSCs of young rats; BMMSCs of adult rats without osteoporosis; BMMSCs of adult rats with osteoporosis without T3 and BMMSCs of adult rats with osteoporosis treated with T3 (0.01, 1, 100 and 1000 nM). Alkaline phosphatase activity, MTT reduction, mineralized nodules and gene expression for collagen, osteocalcin, sialoprotein, osteopontin and BMP-2 were evaluated. Results: Osteoporosis increased the alkaline phosphatase activity and reduced the formation of mineralized nodules and expression of collagen and osteopontin in at least one of the observed time points. However, the T3 treatment of BMMSCs of rats with osteoporosis altered these parameters. Conclusion: It was concluded that doses of T3, 0.01 and 1000 nM had a positive effect promoted by increased osteogenic matrix synthesis and collagen expression in at least one of the evaluated time points compared to BMMSCs of rats with osteoporosis without treatment. However, T3 was unable to reach the osteogenic potential of the MSCs of healthy young rats.
Assuntos
Cobaias , Ratos , Hormônios Tireóideos/uso terapêutico , Medula Óssea , Osteogênese , Osteoporose/veterinária , Células-Tronco , Técnicas de Cultura de Células/veterináriaRESUMO
BACKGROUND: The objective of the present study was to evaluate the effect of the ionic product (IP) of BG60S on osteoblastic activity. The following media groups were created: DMEM, which is formed by osteoblasts in basal medium; IP DMEM, which is formed by osteoblasts in IP with basal medium; OST, which is formed by osteoblasts in osteogenic medium; and IP OST, which is formed by osteoblasts in IP with osteogenic medium. The osteoblasts were cultivated in an incubator at 37 °C and 5 % CO2 for 7, 14 and 21 days. After each period, the alkaline phosphatase (AP) activity, mineralised area per field and expression of osterix (OSX), bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC) were evaluated by reverse transcription (RT)-PCR. RESULTS: The IP significantly increased the AP activity in the IP DMEM group at 7 and 14 days and reduced the AP activity in the IP OST group at 14 and 21 days relative to their respective controls (DMEM and OST). The groups that received the IP displayed a significant increase in the percentage of mineralised area per field and more advance maturation of the extracellular matrix relative to those that did not receive IP. The IP significantly increased the expression of OSX, BSP and ON in osteoblast cultures maintained in IP DMEM compared with the control (DMEM) for the majority of studied periods. In osteogenic medium, IP also significantly increased OSX, BSP, ON and OC expression compared with the control (OST) for the majority of studied periods. CONCLUSIONS: The IP of BG60S alters the gene expression of canine osteoblasts, favouring the synthesis and mineralisation of the extracellular matrix.
Assuntos
Técnicas de Cultura de Células/veterinária , Cerâmica , Cães , Osteoblastos , Fosfatase Alcalina/metabolismo , Animais , Cálcio/metabolismo , Meios de Cultura , Regulação da Expressão Gênica , Silício/metabolismoRESUMO
INTRODUCTION: Human adipose tissue-derived stem cells (hASCs) are attractive cells for therapeutic applications and are currently being evaluated in multiple clinical trials. Prior to their clinical application, hASCs must be expanded ex vivo to obtain the required number of cells for transplantation. Fetal bovine serum is the supplement most widely used for cell culture, but it has disadvantages and it is not safe for cell therapy due to the risks of pathogen transmission and immune reaction. Furthermore, the cell expansion poses a risk of accumulating genetic abnormalities that could lead to malignant cell transformation. In this study, our aim was to evaluate the proliferation pattern as well as the resistance to spontaneous transformation of hASCs during expansion in a xeno-free culture condition. METHODS: hASCs were expanded in Dulbecco's modified Eagle's medium supplemented with pooled allogeneic human serum or fetal bovine serum to enable a side-by-side comparison. Cell viability and differentiation capacity toward the mesenchymal lineages were assessed, along with immunophenotype. Ki-67 expression and the proliferation kinetics were investigated. The expression of the transcription factors c-FOS and c-MYC was examined with Western blot, and MYC, CDKN2A, ERBB2 and TERT gene expression was assessed with quantitative PCR. Senescence was evaluated by ß-gal staining. Karyotype analysis was performed and tumorigenesis assay in vivo was also evaluated. RESULTS: The hASCs expanded in medium with pooled allogeneic human serum did not show remarkable differences in morphology, viability, differentiation capacity or immunophenotype. The main difference observed was a significantly higher proliferative effect on hASCs cultured in pooled allogeneic human serum. There was no significant difference in C-FOS expression; however, C-MYC protein expression was enhanced in pooled allogeneic human serum cultures compared to fetal bovine serum cultures. No difference was observed in MYC and TERT mRNA levels. Moreover, the hASCs presented normal karyotype undergoing senescence, and did not form in vivo tumors, eliminating the possibility that spontaneous immortalization of hASCs had occurred with pooled allogeneic human serum. CONCLUSIONS: This complete characterization of hASCs cultivated in pooled allogeneic human serum, a suitable xeno-free approach, shows that pooled allogeneic human serum provides a high proliferation rate, which can be attributed for the first time to C-MYC protein expression, and showed cell stability for safe clinical applications in compliance with good manufacturing practice.
Assuntos
Tecido Adiposo/citologia , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Células Cultivadas , Humanos , Cariotipagem , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo , Soro/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Telomerase/genética , Telomerase/metabolismoRESUMO
Human adipose-derived stem cells (hASCs) are an attractive cell source for therapeutic applicability in diverse fields for the repair and regeneration of damaged or malfunctioning tissues and organs. There is a growing number of cell therapies using stem cells due to their characteristics of modulation of immune system and reduction of acute rejection. So a challenge in stem cells therapy is the delivery of cells to the organ of interest, a specific site. The aim of this paper was to investigate the effects of a supramolecular assembly composed of single-walled carbon nanotubes (SWCNT), molecular magnets (lawsone-Co-phenanthroline), and a synthetic peptide (FWYANHYWFHNAFWYANHYWFHNA) in the hASCs cultures. The hASCs were isolated, characterized, expanded, and cultured with the SWCNT supramolecular assembly (SWCNT-MA). The assembly developed did not impair the cell characteristics, viability, or proliferation. During growth, the cells were strongly attached to the assembly and they could be dragged by an applied magnetic field of less than 0.3 T. These assemblies were narrower than their related allotropic forms, that is, multiwalled carbon nanotubes, and they could therefore be used to guide cells through thin blood capillaries within the human body. This strategy seems to be useful as noninvasive and nontoxic stem cells delivery/guidance and tracking during cell therapy.
Assuntos
Imãs/química , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Nanotubos de Carbono/química , Peptídeos/química , Tecido Adiposo/citologia , Separação Celular/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Transplante de Células-Tronco Mesenquimais , Nanotubos de Carbono/toxicidade , Naftoquinonas/química , Fenantrolinas/químicaRESUMO
BACKGROUND: The Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach. CONCLUSIONS/SIGNIFICANCE: We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.
Assuntos
Paracoccidioides/genética , Interferência de RNA , Técnicas de Silenciamento de Genes , Genômica , Humanos , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Transformação GenéticaRESUMO
Root lesions may affect both dorsal and ventral roots. However, due to the possibility of generating further inflammation and neuropathic pain, surgical procedures do not prioritize the repair of the afferent component. The loss of such sensorial input directly disturbs the spinal circuits thus affecting the functionality of the injuried limb. The present study evaluated the motor and sensory improvement following dorsal root reimplantation with fibrin sealant (FS) plus bone marrow mononuclear cells (MC) after dorsal rhizotomy. MC were used to enhance the repair process. We also analyzed changes in the glial response and synaptic circuits within the spinal cord. Female Lewis rats (6-8 weeks old) were divided in three groups: rhizotomy (RZ group), rhizotomy repaired with FS (RZ+FS group) and rhizotomy repaired with FS and MC (RZ+FS+MC group). The behavioral tests electronic von-Frey and Walking track test were carried out. For immunohistochemistry we used markers to detect different synapse profiles as well as glial reaction. The behavioral results showed a significant decrease in sensory and motor function after lesion. The reimplantation decreased glial reaction and improved synaptic plasticity of afferent inputs. Cell therapy further enhanced the rewiring process. In addition, both reimplanted groups presented twice as much motor control compared to the non-treated group. In conclusion, the reimplantation with FS and MC is efficient and may be considered an approach to improve sensory-motor recovery following dorsal rhizotomy.
RESUMO
Spiders of the Loxosceles genus represent a risk to human health due to the systemic and necrotic effects of their bites. The main symptoms of these bites vary from dermonecrosis, observed in the majority of cases, to occasional systemic hemolysis and coagulopathy. Although the systemic effects are well characterized, the mechanisms of cell death triggered by the venom of these spiders are poorly characterized. In this study, we investigated the cell death mechanisms induced by the whole venom of the spider Loxosceles similis in human skin fibroblasts. Our results show that the venom initiates an apoptotic process and a caspase cascade involving the initiator caspase-9 and the effector caspases-3, -6, and -7.
Assuntos
Caspases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Venenos de Aranha/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Cultura Primária de Células , Pele/citologia , Pele/efeitos dos fármacos , Pele/enzimologia , Aranhas/fisiologiaRESUMO
SMYB1 is a Schistosoma mansoni protein highly similar to members of the Y-box binding protein family. Similar to other homologues, SMYB1 is able to bind double- and single-stranded DNA, as well as RNA molecules. The characterization of proteins involved in the regulation of gene expression in S. mansoni is of great importance for the understanding of molecular events that control morphological and physiological changes in this parasite. Here we demonstrate that SMYB1 is located in the cytoplasm of cells from different life-cycle stages of S. mansoni, suggesting that this protein is probably acting in mRNA metabolism in the cytoplasm and corroborating previous findings from our group that showed its ability to bind RNA. Protein-protein interactions are important events in all biological processes, since most proteins execute their functions through large supramolecular structures. Yeast two-hybrid screenings using SMYB1 as bait identified a partner in S. mansoni similar to the SmD3 protein of Drosophila melanogaster (SmRNP), which is important in the assembly of small nuclear ribonucleoprotein complexes. Also, pull-down assays were conducted using immobilized GST-SMYB1 proteins and confirmed the SMYB1-SmRNP interaction. The interaction of SMYB1 with a protein involved in mRNA processing suggests that it may act in processes such as turnover, transport and stabilization of RNA molecules.
Assuntos
Proteínas de Helminto/metabolismo , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismo , Schistosoma mansoni/metabolismo , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Transporte Biológico , Citoplasma/metabolismo , Feminino , Biblioteca Gênica , Proteínas de Helminto/genética , Imuno-Histoquímica , Masculino , RNA de Helmintos/genética , RNA Mensageiro/genética , Coelhos , Schistosoma mansoni/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Doxorubicin (Dox) is one of the most effective chemotherapeutic agents; however, it causes dose-dependent cardiotoxicity. Evaluation of left ventricular function relies on measurements based on M-mode echocardiography. A new technique based on quantification of myocardial motion and deformation, strain echocardiography, has been showed promising profile for early detection of cardiac dysfunction. Different therapy strategies, such as flavonoid plant extracts and stem cells, have been investigated to improve heart function in toxic cardiomyopathy. This work aimed to assess early cardiac function improvement after treatments with either flavonoid extract from Camellia sinensis or mesenchymal stem cells in Dox cardiotoxicity using strain echocardiography. Twenty Wistar rats were randomly assigned to four groups. They received water (control, Dox, Dox + stem cells) or 100 mg/kg C. sinensis extract (Dox + C. sinensis) via gavage, daily, for four weeks. Animals also received saline (control) or 5 mg/kg doxorubicin (Dox, Dox + C. sinensis, Dox + stem cells) via intraperitoneal injection, weekly, for four weeks. Stem cells were injected (3 × 106 cells) through tail vein prior the beginning of the experiment (Dox + stem cells). Animals were evaluated by hematological, electrocardiography, echocardiography, and histopathological examinations. Dox cardiotoxicity was only diagnosed with strain echocardiography, detecting a decrease in ventricular function. C. sinensis extract did not prevent ventricular dysfunction induced by Dox. However, strain echocardiography examination revealed that Dox cardiotoxicity was significantly suppressed in rats treated with stem cells. In conclusion, strain echocardiography was able to detect precocity signs of heart failure and stem cell therapy showed cardioprotection effect against Dox cardiotoxicity.
RESUMO
Cardiovascular diseases are the number one cause of death globally and are projected to remain the single leading cause of death. Treatment options abounds, although efficacy is limited. Recent studies attribute discrete and ephemeral benefits to adult stem cell therapies, indicating the urge to improve stem cell based-therapy. In this study, we show that priming mesenchymal stem cells (MSC) towards cardiomyogenic lineage enhances their beneficial effects in vivo as treatment option for acute phase myocardial infarction. MSC were primed using cardiomyogenic media for 4 days, after which peak expression of key cardiomyogenic genes are reached and protein expression of Cx-43 and sarcomeric α-actinin are observed. MSC and primed MSC (pMSC) were characterized in vitro and used to treat infarcted rats immediately after left anterior descending (LAD) occlusion. Echocardiography analysis indicated that MSC-treated myocardium presented discrete improvement in function, but it also showed that pMSC treatment lead to superior beneficial results, compared with undifferentiated MSC. Seven days after cell injection, MSC and pMSC could still be detected in the myocardium. Connexin-43 expression was quantified through immunoblotting, and was superior in pMSC, indicating that this could be a possible explanation for the superior performance of pMSC therapy.