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1.
Adv Exp Med Biol ; 1141: 203-240, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571166

RESUMO

Transporters play an important role in the absorption, distribution, metabolism, and excretion (ADME) of drugs. In recent years, various in vitro, in situ/ex vivo, and in vivo methods have been established for studying transporter function and drug-transporter interaction. In this chapter, the major types of in vitro models for drug transport studies comprise membrane-based assays, cell-based assays (such as primary cell cultures, immortalized cell lines), and transporter-transfected cell lines with single transporters or multiple transporters. In situ/ex vivo models comprise isolated and perfused organs or tissues. In vivo models comprise transporter gene knockout models, natural mutant animal models, and humanized animal models. This chapter would be focused on the methods for the study of drug transporters in vitro, in situ/ex vivo, and in vivo. The applications, advantages, or limitations of each model and emerging technologies are also mentioned in this chapter.


Assuntos
Proteínas de Membrana Transportadoras , Projetos de Pesquisa , Animais , Transporte Biológico , Linhagem Celular , Interações de Medicamentos , Humanos , Técnicas In Vitro , Projetos de Pesquisa/tendências
2.
Adv Exp Med Biol ; 1164: 35-46, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576538

RESUMO

We have characterized two highly tumorigenic and metastatic basal B TNBC cell lines, XtMCF and LmMCF, with the additional values of having the normal and early-stage counterparts of them. This model allows the study of the evolution of TNBC, and investigates molecular pathways at different stages of transformation and progression in a relatively constant genetic background. This constitutes an ideal model for developing targeted therapy in two important fields in cancer biology which are the epithelial mesenchymal transition (EMT) and cancer stem cells (CSC).


Assuntos
Linhagem Celular Tumoral , Modelos Biológicos , Neoplasias de Mama Triplo Negativas , Transição Epitelial-Mesenquimal , Humanos , Técnicas In Vitro , Células-Tronco Neoplásicas
3.
Braz Oral Res ; 33: e042, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31508725

RESUMO

This study evaluated the cytotoxicity and biocompatibility of a new bioceramic endodontic sealer (i.e., Sealer Plus BC) in comparison with those of MTA Fillapex and AH Plus. L929 fibroblasts were cultured and Alamar Blue was used to evaluate cell viability of diluted extracts (1:50, 1:100, and 1:200) from each sealer at 24 h. Polyethylene tubes that were filled with material or empty (as a control) were implanted in the subcutaneous tissue of rats. The rats were killed after 7 and 30 d (n = 8), and the tubes were removed for histological analysis. Parametric data was analyzed using a one-way ANOVA test, and nonparametric data was analyzed via the Kruskal-Wallis test followed by the Dunn test (p < 0.05). A reduction in cell viability was observed in the extracts that were more diluted for Sealer Plus BC when compared to that of Control and AH Plus (p < 0.05). However, the 1:50 dilution of the Sealer Plus BC was similar to that of the Control (p > 0.05). Conversely, more diluted extracts of MTA Fillapex (1:200) and AH Plus (1:100 and 1:200) were similar to the Control (p > 0.05). Histological analysis performed at 7 d did not indicate any significant difference between tissue response for all materials, and the fibrous capsule was thick (p > 0.05). At 30 d, Sealer Plus BC was similar to the Control (p > 0.05) and MTA Fillapex and AH Plus exhibited greater inflammation than the Control (p < 0.05). The fibrous capsule was thin for the Control and for most specimens of Sealer Plus BC and AH Plus. Thus, Sealer Plus BC is biocompatible when compared to MTA Fillapex and AH Plus, and it is less cytotoxic when less-diluted extracts are used.


Assuntos
Cimentos para Ossos/química , Hidróxido de Cálcio/química , Cerâmica/química , Materiais Restauradores do Canal Radicular/química , Compostos de Alumínio/química , Animais , Materiais Biocompatíveis , Cimentos para Ossos/farmacologia , Cimentos para Ossos/toxicidade , Compostos de Cálcio/química , Hidróxido de Cálcio/farmacologia , Hidróxido de Cálcio/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Combinação de Medicamentos , Resinas Epóxi/química , Fibroblastos/efeitos dos fármacos , Técnicas In Vitro , Inflamação , Masculino , Teste de Materiais , Óxidos/química , Ratos Wistar , Materiais Restauradores do Canal Radicular/toxicidade , Silicatos/química , Tela Subcutânea/patologia
4.
Braz Oral Res ; 33: e009, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31508728

RESUMO

This study investigates the color correspondence of resin cements and try-in pastes, and the color stability of bonded lithium disilicate ceramic disks. Resin composite disks were fabricated (n = 36) to serve as the background for lithium disilicate disks prepared in two thicknesses (0.5 and 1.0 mm, n = 18 each). Two brands were used for try-in and cement procedures: Variolink Veneer and AllCem Veneer. For baseline, water was applied between the ceramic disks and their respective backgrounds to achieve the control group. This set was subjected to color measurement using an intraoral measurement device (T0). The try-in was inserted between background and ceramic, and this set was subjected to color measurement (T1). After adhesive procedures, the ceramic disk was placed under cement, and color measurement was performed with uncured cement (T2) and 24 h after light-curing (T3). Each set was immersed in distilled water and thermal-cycled, with color measurement being performed after 10,000 (T4) and 20,000 (T5) cycles. Color differences were calculated by CIELab (rEab) and CIEDE2000 (rE00). Data were analyzed by two-way ANOVA for repeated measurements and Tukey's test (α=5%). There was color correspondence of try-in and resin cement for the Variolink system, regardless of the ceramic thickness (p > 0.05). For the AllCem system, the thickness significantly influenced the color measurement (p < 0.001). The Variolink system also demonstrated color stability after 20,000 thermal cycles with rEab < 3.46 and rE00 < 2.25. It was concluded that the color correspondence between a try-in and its respective cement may vary according to resin cement composition.


Assuntos
Cor , Porcelana Dentária/química , Cimentos de Resina/química , Cerâmica/química , Facetas Dentárias , Técnicas In Vitro , Teste de Materiais , Pomadas/química
5.
J Biol Regul Homeost Agents ; 33(3 Suppl. 1): 51-61, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31538450

RESUMO

This study is to compare the cleaning effectiveness of two Ni-Ti files systems. Thirty single-rooted human teeth were selected and two NiTi rotary systems were used. Group A: canal shaping with ProTaper® Universal (Dentsply Tulsa Dental Specialties, Tulsa, OK) (PTU); Group B: (n=15) canal shaping with Mtwo Ni-Ti instruments (Sweden and Martina, Padova, Italy) and apical finishing with Mtwo Apical Ni-Ti instruments (Sweden and Martina, Padova, Italy). The amount of debris and smear layer were quantified on a basis of a numerical evaluation scale. The data established for scoring the debris and the smear layer was recorded separately and statistically analysed using the Kruskal-Wallis test. No significant differences were found for debris. Mtwo instruments resulted in significantly less smear layer (P less than 0.05) compared with ProTaper® Universal. Under the conditions of this study, Mtwo resulted in significantly less smear layer compared with canal preparation with ProTaper® Universal.


Assuntos
Desenho de Equipamento , Microscopia Eletrônica de Varredura , Preparo de Canal Radicular/instrumentação , Camada de Esfregaço , Humanos , Técnicas In Vitro , Titânio
6.
J Biol Regul Homeost Agents ; 33(3 Suppl. 1): 161-167, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31538463

RESUMO

Osseointegrated dental implants showed elevated success rates on the long-term treatment in the last ten years. However, the risk of peri-implantitis and implant failure is the main complication of implantology. The presence of a micro gap at the implant-abutment connection (IAC) allows microorganisms to penetrate and colonize the inner part of the implant leading to biofilm accumulation and consequently to peri-implantitis development. Some chemical devices (CD) has been studied to reduce bacterial penetration at IAC level but no one have been demonstrated to be effective for this purpose. Aim of the present study is to evaluate the effectiveness of a new chemical formulation STCX-1, placed in the internal part of dental implants for killing bacteria present in the IAC. To identify the antibacterial power of SXTC-1 at interface between implant-abutment connection, the passage of genetically modified Escherichia coli across IAC was evaluated. A total of eight implants were used (Edierre Implant System, Edierre SpA, Genova, Italy). The inner side of four out of the eight implants were firstly contaminated with few microliters of pure bacteria, subsequently were treated with SXTC-1 for few second and finally, the antibacterial was replaced with Lysogeny Broth (LB) and antibiotics without bacteria. The remaining four implants were not treated with SXTC-1 and just filled with LB with antibiotics. Bacteria viability was determined by measuring their Optical Density (OD) at 600nm. The analysis revealed that, in untreated implants, bacteria grew (internally and externally) for the first 48 hours, but subsequently they started to dye. In treated implants, instead, bacteria grew just in the space surrounding the device suggesting that, even if bacteria were able to get into, they immediately died thanks to the presence of SXTC-1. The STCX-1 liquid formulation have been demonstrated to be an adjuvant CD effective for prevention of of bacterial colonization at IAC level.


Assuntos
Dente Suporte/microbiologia , Projeto do Implante Dentário-Pivô , Implantes Dentários/microbiologia , Infiltração Dentária/prevenção & controle , Escherichia coli , Humanos , Técnicas In Vitro , Viabilidade Microbiana , Peri-Implantite/prevenção & controle
7.
Bol. latinoam. Caribe plantas med. aromát ; 18(5): 480-491, sept. 2019. ilus, tab
Artigo em Inglês | LILACS | ID: biblio-1008273

RESUMO

In the present study, we investigated the antiproliferative activity of essential oil from leaves of Melissa officinalis L. grown in Southern Bosnia and Herzegovina. In vitro evaluation of antiproliferative activity of the M. officinalis essential oil was carried out on three human tumor cell lines: MCF-7, NCI-H460 and MOLT-4 by MTT assay. M. officinalis essential oil was characterized by high percentage of monoterpenes (77,5%), followed by the sesquiterpene fraction (14,5%) and aliphatic compounds (2,2%). The main constituents of the essential oil of M. officinalis are citral (47,2%), caryophyllene oxide (10,2%), citronellal (5,4%), geraniol (6,6%), geranyl acetate (4,1%) and ß- caryophyllene (3,8%). The essential oil showed significant antiproliferative activity against three cancer cell lines, MOLT-4, MCF-7, and NCI-H460 cells, with GI50 values of <5, 6±2 and 31±17 µg/mL, respectively. The results revealed that M. officinalis L. essential oil has a potential as anticancer therapeutic agent.


En el presente estudio, investigamos la actividad antiproliferativa del aceite esencial de las hojas de Melissa officinalis L. cultivadas en el sur de Bosnia y Herzegovina. La evaluación in vitro de la actividad antiproliferativa del aceite esencial de M. officinalis se llevó a cabo en tres líneas celulares de tumores humanos: MCF-7, NCI-H460 y MOLT-4 utilizando el ensayo de MTT. El aceite esencial de M. officinalis se caracterizó por un alto porcentaje de monoterpenos (77,5%), seguido de la fracción sesquiterpénica (14,5%) y compuestos alifáticos (2,2%). Los principales constituyentes del aceite esencial de M. officinalis fueron citral (47,2%), óxido de cariofileno (10,2%), citronelal (5,4%), geraniol (6,6%), acetato de geranilo (4, 1%), y ß-cariofileno (3,8%). El aceite esencial mostró una actividad antiproliferativa significativa contra las líneas celulares de cáncer MOLT-4, MCF-7 y NCI-H460, con valores GI50 de <5, 6±2 y 31±17 µg/mL, respectivamente. Los resultados revelaron que el aceite esencial de M. officinalis L. tiene potencial como agente terapéutico contra el cáncer.


Assuntos
Óleos Voláteis/farmacologia , Melissa , Antineoplásicos/farmacologia , Sesquiterpenos/análise , Técnicas In Vitro , Óleos Voláteis/química , Células Tumorais Cultivadas , Folhas de Planta , Monoterpenos/análise , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Antineoplásicos/química
8.
J Agric Food Chem ; 67(35): 9805-9811, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31407895

RESUMO

Stachydrine (STA) is a constituent of citrus fruits and Leonurus heterophyllus Sweet. In the present study, we established that STA caused acute endothelium-dependent relaxation. The vascular action of STA was mediated by nitric oxide (NO) via cyclic guanosine monophosphate. Mechanistically, STA activated AMP-activated protein kinase (AMPK), protein kinase B/Akt, and endothelial NO synthase (eNOS) in vascular endothelial cells (ECs). AMPK inhibition by compound C blocked STA-induced Akt/eNOS phosphorylation, suggesting that AMPK is the upstream of Akt and eNOS. Inhibition of Akt by MK2206 blocked STA-stimulated eNOS phosphorylation without altering AMPK phosphorylation. Furthermore, we showed that STA activated AMPK via the induction of liver kinase B1 phosphorylation. These results indicated that STA can induce eNOS phosphorylation and vasorelaxation by regulating the interplay between AMPK and Akt pathways in ECs. These findings further highlighted the potential of STA as a nutritional factor in the beneficial effects of fruit intake in preventing the endothelial dysfunction-related metabolic cardiovascular diseases.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aorta Torácica/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Prolina/análogos & derivados , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasodilatadores/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatologia , Bovinos , Citrus/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Técnicas In Vitro , Leonurus/química , Masculino , Óxido Nítrico Sintase Tipo III/genética , Fosforilação/efeitos dos fármacos , Prolina/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Sprague-Dawley , Vasodilatação/efeitos dos fármacos
9.
J Enzyme Inhib Med Chem ; 34(1): 1474-1480, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31414611

RESUMO

The discovery of allosteric modulators is a multi-disciplinary approach, which is time- and cost-intensive. High-throughput screening combined with novel computational tools can reduce these factors. Thus, we developed an enzyme activity assay, which can be included in the drug discovery work-flow subsequent to the in-silico library screening. While the in-silico screening yields in the identification of potential allosteric modulators, the developed in-vitro assay allows for the characterisation of them. Candida rugosa lipase (CRL), a glyceride hydrolysing enzyme, has been selected for the pilot development. The assay conditions were adjusted to CRL's properties including pH, temperature and substrate specificity for two different substrates. The optimised assay conditions were validated and were used to characterise Tropolone, which was identified as an allosteric modulator. In conclusion, the assay is a reliable, reproducible, and robust tool, which can be streamlined with in-silico screening and incorporated in an automated high-throughput screening workflow.


Assuntos
Lipase/metabolismo , Miniaturização , Regulação Alostérica , Candida/enzimologia , Cristalografia por Raios X , Estabilidade Enzimática , Ensaios de Triagem em Larga Escala , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Limite de Detecção , Lipase/química , Reprodutibilidade dos Testes , Especificidade por Substrato , Temperatura Ambiente
10.
Cell Physiol Biochem ; 53(2): 388-399, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31403269

RESUMO

BACKGROUND/AIMS: Doxorubicin, a chemotherapy drug used successfully for years, could induce cardiotoxicity. Euterpe oleracea Mart. (açai) is a fruit high in antioxidant properties. The aim of this study was to evaluate doxorubicin-induced cardiotoxicity prevention after açai administration. METHODS: A total of 64 male Wistar rats were allocated into 4 groups: control (C), açai (A), doxorubicin (D) and açai-doxorubicin (DA). Rats received regular chow (C and D groups) or chow supplemented with açai 5% (A and DA groups) for 4 weeks. Subsequently, rats received doxorubicin 20 mg/kg (D and DA groups) or saline (C and A groups). Euthanasia was performed 48 hours after doxorubicin injection. Left ventricular function was evaluated by echocardiography in vivo and by isolated heart study ex vivo. Oxidative stress, myocardial metabolism and nitric oxide metabolite were evaluated by spectrophotometry, MMP-2 activity by zymography and caspase-3 and Bcl-2 protein expression by Western blot. RESULTS: Doxorubicin induced decreases in body weight, food and water ingestion. We observed decreases in left ventricular fractional shortening in rats treated with doxorubicin. Additionally, the same rats showed lower +dP/dt and -dP/dt during isolated heart study than those who did not receive doxorubicin. Doxorubicin injection increased caspase-3 protein expression, myocardium lipid hydroperoxide concentration, MMP-2 activity, phosphofructokinase and lactate dehydrogenase activity, and decreased ß-hydroxyacyl-CoA dehydrogenase, pyruvate dehydrogenase, citrate synthase, complex I, complex II and ATP synthase activity in myocardium. Açai supplementation improved left ventricular fractional shortening, decreased myocardium lipid hydroperoxide concentration, MMP-2 activity, and improved ß-hydroxyacyl-CoA dehydrogenase, phosphofructokinase, citrate synthase, complex II and ATP synthase enzymatic activities. We did not observe differences in nitric oxide metabolite concentrations between groups. CONCLUSION: Doxorubicin induced left ventricular dysfunction, increases in oxidative stress, changes in myocardium metabolism and MMP-2 activation. Açai supplementation was able to prevent these alterations.


Assuntos
Doxorrubicina/toxicidade , Euterpe/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Suplementos Nutricionais , Ecocardiografia , Euterpe/metabolismo , Cardiopatias/etiologia , Cardiopatias/prevenção & controle , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Miocárdio/metabolismo , Óxido Nítrico/sangue , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Função Ventricular Esquerda/efeitos dos fármacos
11.
Br J Anaesth ; 123(4): 519-530, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31262508

RESUMO

BACKGROUND: Respiratory complications after surgery are associated with morbidity and mortality. Acute lung injury can result from the systemic inflammatory response after acute kidney injury. The mechanisms behind this remote injury are not fully understood. In this study, a renal transplantation model was used to investigate remote lung injury and the underlying molecular mechanisms, especially the role of osteopontin (OPN). METHODS: In vitro, human lung epithelial cell line (A549) and monocyte/macrophage cell line (U937) were challenged with tumour necrosis factor-alpha (TNF-α) in combination with OPN. In vivo, the Fischer rat renal grafts were extracted and stored in 4°C University of Wisconsin preserving solution for up to 16 h, and transplanted into Lewis rat recipients. Lungs were harvested on Day 1 after grafting for further analysis. RESULTS: Renal engraftment was associated with pathological changes and an increase in TNF-α and interleukin-1 beta in the lung of the recipient. OPN, endoplasmic reticulum (ER) stress, and necroptosis were increased in both the recipient lung and A549 cells challenged with TNF-α. Exogenous OPN exacerbated lung injury and necroptosis. Suppression of OPN through siRNA reduced remote lung injury by mitigation of ER stress, necroptosis, and the inflammatory response. CONCLUSIONS: Renal allograft transplant triggers recipient remote lung injury, which is, in part, mediated by OPN signalling. This study may provide a molecular basis for strategies to be developed to treat such perioperative complications.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Transplante de Rim/efeitos adversos , Osteopontina/farmacologia , Complicações Pós-Operatórias/prevenção & controle , Animais , Apoptose , Células Cultivadas , Modelos Animais de Doenças , Humanos , Técnicas In Vitro , Masculino , Necrose , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew
12.
Life Sci ; 233: 116685, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31348947

RESUMO

AIMS: The present study aimed to investigate the effects of laser irradiation on the growth factors and cell apoptosis of in vitro cultured infant hemangioma endothelial cells. MAIN METHODS: Endothelial cells of infant hemangioma were cultured in vitro and irradiated using a variable pulse width 1064 nm Nd:YAG laser and intense pulsed light (IPL), the expression of VEGF, VEGFR-2, bFGF and their mRNAs before and after irradiation were measured by ELISA, western blot, RT-PCR and flow cytometry, and changes in the apoptotic rate of endothelial cells in hemangioma were monitored. KEY FINDINGS: The mRNA and protein expressions of VEGF, VEGFR-2, bFGF in hemangioma endothelial cells were inhibited by both Nd:YAG laser and ILP compared to the control cells. The apoptotic rates of hemangioma endothelial cells were also decreased after both laser irradiation treatments in comparison to the blank group. The differences were statistically significant. SIGNIFICANCE: Laser irradiation treats hemangioma not only through a selective photothermal mechanism, but also through cytokine signaling pathways.


Assuntos
Apoptose/efeitos da radiação , Células Endoteliais/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Hemangioma/metabolismo , Hemangioma/patologia , Terapia a Laser/métodos , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hemangioma/radioterapia , Humanos , Técnicas In Vitro , Lactente , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
13.
Yakugaku Zasshi ; 139(7): 989-992, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31257257

RESUMO

The molecular basis underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) has not been fully elucidated. The protein-misfolding cyclic amplification (PMCA) technique, which can amplify PrPSc in vitro with the use of intermittent sonication, mimics the process of in vivo PrPSc replication. Accumulating evidence suggests that co-factors other than PrP may play a crucial role in the faithful replication of PrPSc. In conventional PMCA, brain homogenates (BHs) from normal animals are used as the PrPC substrate. Since BHs contain many impurities, it is difficult to identify the co-factors using conventional PMCA. Thus, we developed a modified PMCA system using baculovirus and insect cell-derived recombinant PrP as a substrate (insect cell PMCA; iPMCA). We demonstrated that nucleic acids and glycosaminoglycans (GAGs) such as heparan sulfate (HS) or its analogue heparin (HP) are critical for PrPSc amplification in iPMCA. Of note, the addition of HS or HP restored the conversion efficiency in iPMCA under nucleic acid-depleted conditions. Moreover, the iPMCA products were infectious and preserved the strain properties of the input seed PrPSc. These data suggest that not only nucleic acids but also some GAGs play an important role in facilitating faithful replication of prions, at least in vitro.


Assuntos
Baculoviridae/genética , Insetos/genética , Proteínas Priônicas/química , Animais , Sistema Livre de Células , Glicosaminoglicanos , Heparina , Heparitina Sulfato , Técnicas In Vitro , Ácidos Nucleicos , Proteínas Recombinantes/química
14.
Yakugaku Zasshi ; 139(7): 999-1005, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31257259

RESUMO

Pathological α-synuclein (αSyn) has been shown to retain the ability to propagate as prions in humans and animals. However, the molecular basis underlying the prion-like properties of αSyn remains poorly understood. We examined whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, exhibit prion-like seeding activity in vitro using the real-time quaking-induced conversion (RT-QuIC) seeding assay. Brain tissues from cases of diffuse neocortical DLB yielded a 50% seeding dose of 107.3-109.8/g brain. The RT-QuIC assay could discriminate between DLB and other neurological and neurodegenerative disorders, suggesting its potential applicability for differential diagnosis. Insoluble aggregates of αSyn>250 kDa detected only in DLB brain tissues by Western blotting analysis were specifically phosphorylated at Ser129. Therefore, we postulated that Ser129-phosphorylated insoluble aggregates of αSyn have prion-like seeding activity. However, insoluble aggregates of recombinant human αSyn (rSyn) with increased ß-sheet structures showed little seeding activity in either the phosphorylated or nonphosphorylated state. In contrast, prefibrillar oligomers of rSyn showed seeding activity both with and without phosphorylation. The findings of the present study suggested that soluble oligomeric αSyn, but not the fully fibrillary form, is a seeding species in vitro.


Assuntos
Encéfalo/metabolismo , Agregação Patológica de Proteínas , alfa-Sinucleína/metabolismo , Humanos , Técnicas In Vitro , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/metabolismo , Príons/metabolismo , Dobramento de Proteína
15.
Bone Joint J ; 101-B(7): 779-786, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31256663

RESUMO

AIMS: The aim of this study was to estimate the 90-day risk of revision for periprosthetic femoral fracture associated with design features of cementless femoral stems, and to investigate the effect of a collar on this risk using a biomechanical in vitro model. MATERIALS AND METHODS: A total of 337 647 primary total hip arthroplasties (THAs) from the United Kingdom National Joint Registry (NJR) were included in a multivariable survival and regression analysis to identify the adjusted hazard of revision for periprosthetic fracture following primary THA using a cementless stem. The effect of a collar in cementless THA on this risk was evaluated in an in vitro model using paired fresh frozen cadaveric femora. RESULTS: The prevalence of early revision for periprosthetic fracture was 0.34% (1180/337 647) and 44.0% (520/1180) occurred within 90 days of surgery. Implant risk factors included: collarless stem, non-grit-blasted finish, and triple-tapered design. In the in vitro model, a medial calcar collar consistently improved the stability and resistance to fracture. CONCLUSION: Analysis of features of stem design in registry data is a useful method of identifying implant characteristics that affect the risk of early periprosthetic fracture around a cementless femoral stem. A collar on the calcar reduced the risk of an early periprosthetic fracture and this was confirmed by biomechanical testing. This approach may be useful in the analysis of other uncommon modes of failure after THA. Cite this article: Bone Joint J 2019;101-B:779-786.


Assuntos
Artroplastia de Quadril/instrumentação , Fraturas do Quadril/prevenção & controle , Prótese de Quadril/efeitos adversos , Fraturas Periprotéticas/prevenção & controle , Complicações Pós-Operatórias/prevenção & controle , Desenho de Prótese/efeitos adversos , Reoperação/estatística & dados numéricos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Artroplastia de Quadril/métodos , Fenômenos Biomecânicos , Criança , Feminino , Seguimentos , Fraturas do Quadril/etiologia , Fraturas do Quadril/cirurgia , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Fraturas Periprotéticas/etiologia , Fraturas Periprotéticas/cirurgia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Sistema de Registros , Fatores de Risco , Resultado do Tratamento , Adulto Jovem
16.
Nat Commun ; 10(1): 2430, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160583

RESUMO

Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients.


Assuntos
Tecido Adiposo/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Células-Tronco/citologia , Adipócitos/patologia , Adipogenia/efeitos dos fármacos , Tecido Adiposo/patologia , Adolescente , Idade de Início , Animais , Anexina A2/metabolismo , Estudos de Casos e Controles , Disferlina/genética , Venenos Elapídicos/toxicidade , Feminino , Fibrose , Humanos , Técnicas In Vitro , Masculino , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Índice de Gravidade de Doença , Células-Tronco/efeitos dos fármacos , Tiofenos/farmacologia , Adulto Jovem
17.
Am J Vet Res ; 80(7): 663-669, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31246122

RESUMO

OBJECTIVE: To evaluate effects of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors on the production of tumor necrosis factor-α (TNF-α) by interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of horses as an in vitro model of inflammation in horses. SAMPLE: 1,440 samples of PBMCs from 6 healthy research horses. PROCEDURES: From heparinized whole blood samples, PBMC cultures were obtained. An initial dose-response trial on 48 PBMC samples from 2 horses (24 samples each) was used to determine concentrations of IFN-γ and LPS for use as low- and high-level stimulation concentrations. Seventy-two PBMC samples from 6 horses were assigned equally to 1 of 4 PARP1 inhibition categories: no PARP1 inhibitor (PARP1 inhibition control); 2-((R)-2-methylpyrrolidin-2-yl)-1H-benzimidazole-4-carbozamide dihydrochloride (ABT888);4-(3-(1-(cyclopropanecarbonyl)piperazine-4-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (AZD2281); or N-(6-oxo-5,6-dihydrophenanthridin-2-yl) -N,N-dimethylacetamide hydrochloride (PJ34). Samples of PBMCs from each horse and each PARP1 inhibition category were then assigned to 1 of 3 levels of IFN-γ and LPS stimulation: none (control), low stimulation, or high stimulation. After a 24-hour incubation period, a TNF-α ELISA was used to measure TNF-α concentration in the supernatant. Results were compared across treatments and for each horse. Data were analyzed with repeated-measures ANOVA. RESULTS: Median TNF-α concentration was significantly lower for PJ34-treated, high-level stimulated PBMCs than for PARP1 inhibition control, high-level stimulated PBMCs; however, no other meaningful differences in TNF-α concentration were detected among the inhibition and stimulation combinations. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that PJ34 PARP1 inhibition may reduce TNF-α production in horses, a potential benefit in reducing inflammation and endotoxin-induced damage in horses.


Assuntos
Cavalos/sangue , Interferon gama/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Modelos Animais de Doenças , Doenças dos Cavalos/fisiopatologia , Técnicas In Vitro , Inflamação/fisiopatologia , Leucócitos Mononucleares/enzimologia , Lipopolissacarídeos/farmacologia
18.
Am J Vet Res ; 80(7): 625-630, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31246126

RESUMO

OBJECTIVE: To determine the amount of negative pressure generated by syringes of various sizes with and without an attached thoracostomy tube and whether composition of thoracostomy tubes altered the negative pressure generated. SAMPLE: Syringes ranging from 1 to 60 mL and 4 thoracostomy tubes of various compositions (1 red rubber catheter, 1 polyvinyl tube, and 2 silicone tubes). PROCEDURES: A syringe or syringe with attached thoracostomy tube was connected to a pneumatic transducer. Each syringe was used to aspirate a volume of air 10 times. Negative pressure generated was measured and compared among the various syringe sizes and various thoracostomy tubes. RESULTS: The negative pressure generated decreased as size of the syringe increased for a fixed volume across syringes. Addition of a thoracostomy tube further decreased the amount of negative pressure. The red rubber catheter resulted in the least amount of negative pressure, followed by the polyvinyl tube and then the silicone tubes. There was no significant difference in negative pressure between the 2 silicone tubes. The smallest amount of negative pressure generated was -74 to -83 mm Hg. CONCLUSIONS AND CLINICAL RELEVANCE: Limited data are available on the negative pressure generated during intermittent evacuation of the thoracic cavity. For the present study, use of a syringe of ≥ 20 mL and application of 1 mL of negative suction volume resulted in in vitro pressures much more negative than the currently recommended pressure of -14.71 mm Hg for continuous suction. Additional in vitro or cadaveric studies are needed.


Assuntos
Tubos Torácicos/veterinária , Pressão , Sucção/veterinária , Seringas/veterinária , Toracostomia/veterinária , Técnicas In Vitro , Estudos Prospectivos , Sucção/métodos , Toracostomia/métodos
19.
Niger J Clin Pract ; 22(6): 812-816, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31187767

RESUMO

Aim: The purpose of this study was to compare the marginal and internal fit of Co-Cr and titanium ceramic crowns fabricated with 2 different techniques: CAD/CAM milling and traditional casting (TC). Materials and Methods: Ten traditional casting of Co-Cr (TCC), 10 CAD/CAM milling of Co-Cr (MC), 10 traditional casting of titanium (TCT), 10 CAD/CAM milling of titanium ceramic crowns (MT) were fabricated. Silicone replicas were obtained to measure internal gap volume, marginal gap and internal adaptation that was evaluated at 3 regions: axial wall, axio-occlusal angle, and occlusal surface. Measurements were made with a X-ray micro computerized tomography (micro-CT) and analyzed with Bonferroni and Dunnet T3 post-hoc tests (α = 0.05). Results: One-way ANOVA revealed no statistically significant differences among the groups for measurements at the marginal gap (P > 0.05). At axial wall region the mean values of TCT group were higher than those of groups but only statistically not significant for TCC group (P < 0.05). TCC group statistically gives better results than MC group in axio-occlusal and occlusal regions (P < 0.05). The mean measurement of internal gap volume were 20.59 ± 0.83 mm3 for TCC, 22.73 ± 0.82 mm3 for MC, 22.83 ± 1.11 mm3 for TCT and 20.51 ± 1.16 mm3 for MT. Mean internal gap volume values MT group were smaller than those of groups but only statistically not significant for TCC group (P > 0.05). Conclusion: All groups performed similar marginal adaptation. The cement film thickness at axio-occlusal angle point and occlusal region were higher for MC crowns.


Assuntos
Cerâmica , Adaptação Marginal Dentária , Planejamento de Prótese Dentária/métodos , Ligas de Cromo , Cobalto , Projeto Auxiliado por Computador , Coroas , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Titânio
20.
Nat Commun ; 10(1): 2450, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164648

RESUMO

Tumor-associated myeloid cells regulate tumor growth and metastasis, and their accumulation is a negative prognostic factor for breast cancer. Here we find calcium/calmodulin-dependent kinase kinase (CaMKK2) to be highly expressed within intratumoral myeloid cells in mouse models of breast cancer, and demonstrate that its inhibition within myeloid cells suppresses tumor growth by increasing intratumoral accumulation of effector CD8+ T cells and immune-stimulatory myeloid subsets. Tumor-associated macrophages (TAMs) isolated from Camkk2-/- mice expressed higher levels of chemokines involved in the recruitment of effector T cells compared to WT. Similarly, in vitro generated Camkk2-/- macrophages recruit more T cells, and have a reduced capability to suppress T cell proliferation, compared to WT. Treatment with CaMKK2 inhibitors blocks tumor growth in a CD8+ T cell-dependent manner, and facilitates a favorable reprogramming of the immune cell microenvironment. These data, credential CaMKK2 as a myeloid-selective checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer.


Assuntos
Neoplasias da Mama/imunologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/imunologia , Carcinoma/imunologia , Neoplasias Mamárias Animais/imunologia , Células Mieloides/imunologia , Evasão Tumoral/imunologia , Microambiente Tumoral/imunologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linfócitos T CD8-Positivos/imunologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Proliferação de Células , Quimiocinas/imunologia , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Evasão Tumoral/genética
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