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1.
Braz J Med Biol Res ; 52(9): e8551, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482977

RESUMO

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Fibroblastos/fisiologia , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Western Blotting , Fibroblastos/metabolismo , Imunofluorescência , Camundongos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
2.
Exp Parasitol ; 206: 107757, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31494216

RESUMO

Equine theileriosis is a severe equine disease caused by the protozoan Theileria equi, which is prevalent in tropical and subtropical areas. In this study, a recombinant equi merozoite antigen-2 (rEMA-2) of T. equi was used as an immunogen. Two groups of 10 mice each were divided into control and vaccinated groups. Sixty mares seronegative for theileriosis were divided in two groups, one vaccinated and another group as a control animal. Mice and mares of the vaccinated groups were inoculated with 150 µL of the vaccine containing 50 µg of rEMA-2 and 2 mL of the vaccine containing 200 µg of rEMA-2, respectively, at days 0 and 21. The immunogenicity of rEMA-2 was evaluated by ELISA and fluorescent antibody test (IFAT) using serum from vaccinated mice, mares and antigenicity in naturally infected horse. At every point throughout the ELISA study, there were significant differences between the vaccinated and control groups (p < 0.05). The vaccine induced 3- and 4-fold IgG increases in mice at the 14th and 28th day, respectively, compared to the control group. The horses' IgG dynamics showed a significant (p < 0.05) increase in the total IgG titer as early as day 7, which increased until day 28 at which time a more significant (p < 0.001) IgG titer was observed. In evaluating the isotypes, we observed a trend similar to that of total IgG, where IgG(T) (IgG3-5) were significantly (p < 0.05) more elevated than the other isotypes analyzed, followed by IgGb (IgG4-7) and IgGa (IgG1). Positive fluorescence was detected by IFAT, suggesting that the protein is immunogenic and conserves some epitopes identical to the native T. equi antigens present in the equine blood smear. Thus, our results suggest that rEMA-2 can be a promising vaccinal antigen.


Assuntos
Antígenos de Protozoários/imunologia , Pichia/imunologia , Theileria/imunologia , Análise de Variância , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Cavalos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Merozoítos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Distribuição Aleatória , Proteínas Recombinantes/imunologia
3.
Biol Res ; 52(1): 53, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542051

RESUMO

BACKGROUND: Oxidative stress is the hallmark of diabetic encephalopathy, which may be caused by hyperglycaemic toxicity. We aimed to discover pharmacologic targets to restore redox homeostasis. We identified the transcription factor Nrf2 as such a target. METHODS: HT22 cells were cultured in 25 or 50 mM D-glucose with various concentrations of sulforaphane (SFN) (from 1.25 to 5.0 µM). Cell viability was tested with the Cell Counting Kit-8 assay. Reactive oxygen species (ROS) production was detected with an inverted fluorescence microscope using the dichlorodihydrofluorescein-diacetate fluorescent probe. The expression of NF-E2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1) and nuclear factor-κB (NF-κB) at the mRNA and protein levels was detected by reverse transcription quantitative polymerase chain reaction and western blotting. RESULT: We found that a high glucose concentration (50 mM) increased the generation of ROS, downregulated the expression of Nrf2/HO-1 and upregulated the expression of NF-κB. Moreover, HT22 cell viability significantly decreased after culture in high-glucose medium for 24, 48 and 72 h, whereas the activation of the Nrf2/HO-1 pathway using a pharmacological Nrf2 activator abrogated this high-glucose-induced toxicity. CONCLUSION: This study suggests that the activation of the Nrf2-ARE signalling pathway might be a therapeutic target for the treatment of diabetic encephalopathy.


Assuntos
Glucose/toxicidade , Hipocampo/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/agonistas , Neuroproteção , Animais , Western Blotting , Linhagem Celular , Eletroforese em Gel de Campo Pulsado , Imunofluorescência , Hipocampo/citologia , Camundongos , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
4.
Exp Parasitol ; 205: 107748, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31442453

RESUMO

Trypanosoma cruzi (the causative agent of Chagas disease) presents a complex life cycle that involves adaptations in vertebrate and invertebrate hosts. As a protozoan parasite of hematophagous insects and mammalian hosts, T. cruzi is exposed to reactive oxygen species (ROS). To investigate the functionality of T. cruzi tartrate-resistant acid phosphatase type 5 (TcACP5), we cloned, superexpressed and purified the enzyme. Purified TcACP5 exhibited a Vmax and apparent Km for pNPP hydrolysis of 7.7 ±â€¯0.2 nmol pNP × µg-1 × h-1 and 169.3 ±â€¯22.6 µM, respectively. The pH dependence was characterized by sharp maximal activity at pH 5.0, and inhibition assays demonstrated its sensitivity to acid phosphatase inhibitors. Similar activities were obtained with saturating concentrations of P-Ser and P-Thr as substrates. The enzyme metabolizes hydrogen peroxide (H2O2) in vitro, and parasites superexpressing this enzyme were more resistant to oxidative stress promoted by H2O2. Taken together, these results suggest that TcACP5 plays a central role in phosphoryl transfer and redox reactions.


Assuntos
Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/fisiologia , Fosfatase Ácida Resistente a Tartarato/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Microscopia Confocal , Oxirredução , Especificidade por Substrato , Fosfatase Ácida Resistente a Tartarato/antagonistas & inibidores , Fosfatase Ácida Resistente a Tartarato/química , Transfecção , Trypanosoma cruzi/efeitos dos fármacos
5.
Klin Lab Diagn ; 64(7): 409-412, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31408592

RESUMO

The use of technology for preparing components for immunofluorescence reaction (RIF) in a number of clinical diagnostic laboratories (CDL) in accordance with the recommendations of the Guidelines introduced by order of the RF Ministry of Health No 87 dated March 26, 2001 does not guarantee the required standardization of RIF preparation and conduct conditions. The latter is achieved by using the appropriate industrial production reagent kits in the СDL practice.


Assuntos
Imunofluorescência/normas , Kit de Reagentes para Diagnóstico/normas , Sorodiagnóstico da Sífilis/normas , Sífilis/diagnóstico , Humanos , Treponema pallidum
6.
Braz Oral Res ; 33: e058, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31432925

RESUMO

Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.


Assuntos
Técnicas de Cultura de Células/métodos , Cemento Dentário/citologia , Adolescente , Adulto , Análise de Variância , Proteínas Morfogenéticas Ósseas/análise , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Cemento Dentário/metabolismo , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/genética , Feminino , Imunofluorescência , Expressão Gênica , Marcadores Genéticos/genética , Humanos , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Dente Molar/citologia , Fosfatos/farmacologia , Fosfoproteínas/análise , Fosfoproteínas/genética , Sialoglicoproteínas/análise , Sialoglicoproteínas/genética , Fatores de Tempo , Adulto Jovem
7.
Exp Parasitol ; 205: 107753, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31469986

RESUMO

Trypanosoma brucei causes human African trypanosomiasis and Nagana disease in cattle, imposing substantial medical and economic burden in sub-Saharan Africa. The current treatments have limitations, including the requirement for elaborated protocols, development of drug resistance, and they are prone to adverse side effects. In vitro screening of a library of 14 dinuclear-thiolato bridged arene ruthenium complexes, originally developed for treatment of cancer cells, resulted in the identification of 7 compounds with IC50 values ranging from 3 to 26 nM. Complex [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-o-Pri)3]Cl (2) (IC50 = 4 nM) and complex [(η6-p-MeC6H4Pri)2Ru2(µ2-SCH2C6H4-p-But)2(µ2-SC6H4-p-OH)]BF4(9) (IC50 = 26 nM) were chosen for further assessments. Application of complex 2 and 9 at 20 nM and 200 nM, respectively, for 4.5 h induced alterations in the trypanosome mitochondrion as evidenced by immunofluorescence employing an antibody against mitochondrial Hsp70 and Mitotracker labeling. Transmission electron microscopy of parasites taken at 2 and 4h of treatment demonstrated massive alterations in the mitochondrial ultrastructure, while other organelles and structural elements of the parasites remained unaffected. Complex 2 treated trypanosomes exhibited a distorted mitochondrial membrane, and the mitochondrial matrix was transformed into an amorphous mass with different degrees of electron densities. Complex 9 did not notably impair the integrity of the membrane, but the interior of the mitochondrion appeared either completely translucent, or was filled with filamentous structures of unknown nature. Dose- and time-dependent effects of these two compounds on the mitochondrial membrane potential were detected by tetramethylrhodamine ethyl ester assay. Thus, the mitochondrion and associated metabolic processes are an important target of dinuclear thiolato-bridged arene ruthenium complexes in T. brucei.


Assuntos
Potencial da Membrana Mitocondrial/efeitos dos fármacos , Compostos de Rutênio/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/parasitologia , Animais , Relação Dose-Resposta a Droga , Imunofluorescência , Humanos , Concentração Inibidora 50 , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Compostos de Rutênio/química , Fatores de Tempo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/ultraestrutura , Tripanossomíase Africana/sangue
8.
J Immunol Methods ; 471: 1-10, ago.2019.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IPPROD, Sec. Est. Saúde SP | ID: biblio-1016183

RESUMO

Polyclonal or monoclonal antibodies against rabies virus ribonucleoprotein (RNP) conjugated to fluorescein isothiocyanate (FITC) have been employed for Rabies virus (RABV) antigen detection by the direct fluorescent antibody test (DFA). To date, these biomolecules have been purified by traditional methods such as precipitation by ammonium sulfate or ion exchange chromatography followed by ammonium sulfate precipitation, which allows only for partial detection of the protein of interest. In this study, we aimed to purify anti-RNP polyclonal horse IgG antibodies by cation-exchange chromatography in combination with a homemade immunoaffinity chromatography on RNP immobilized (RNP-IAC). Furthermore, to evaluate the accuracy of the prepared anti-RNP IgG fluorescent antibody in diagnostic purposes, DFA was applied for RABV antigen detection in suspected brain samples of different animal species. The combination of these two techniques made it possible to obtain antibodies with high selectivity and purity. Compared with the performance of the traditional method, anti-RNP IgG antibodies purified by RNP-IAC can be obtained from a smaller volume of hyperimmune serum and with greater avidity. Furthermore, the results obtained by DFA analyses revealed that the prepared anti-RNP IgG fluorescent antibody achieved 100% diagnostic specificity and sensitivity for RABV antigen detection. Thus, two-technique chromatographic, including RNP-IAC technology could be appropriate methods for the purification of polyclonal anti-RNP IgG for the use as a diagnostic reagent for rabies.(AU)


Assuntos
Animais , Raiva/diagnóstico , Vírus da Raiva/isolamento & purificação , Ribonucleoproteínas , Cromatografia de Afinidade , Imunofluorescência , Isotiocianatos , Anticorpos Monoclonais
9.
Rev Soc Bras Med Trop ; 52: e20180199, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31390441

RESUMO

INTRODUCTION: The Jirau hydroelectric power plant built in Rondônia state has environmental impacts that could be relevant to rabies outbreaks. METHODS: Bat populations were monitored for rabies by fluorescent antibody testing and simplified fluorescent inhibition microtesting between 2010 and 2015. RESULTS: All 1,183 bats tested negative for rabies. The prevalence of rabies antibodies was 17.5% in 1,049 bats. CONCLUSIONS: The rabies antibody dosage was not reactive in samples collected before the environmental changes, and there was a progressive increase in subsequent collections that could indicate an increase in rabies virus circulation among bats and risk of a rabies outbreak.


Assuntos
Quirópteros/virologia , Vírus da Raiva/isolamento & purificação , Raiva/veterinária , Animais , Anticorpos Antivirais/isolamento & purificação , Brasil/epidemiologia , Surtos de Doenças , Imunofluorescência/métodos , Raiva/epidemiologia , Raiva/virologia , Vírus da Raiva/imunologia , Estudos Soroepidemiológicos , Fatores de Tempo
10.
Exp Parasitol ; 204: 107727, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31344389

RESUMO

BACKGROUND: Trypanosoma rangeli is a protozoan parasite that is non-virulent to the mammalian host and is morphologically and genomically related to Trypanosoma cruzi, whose proliferation within the mammalian host is controversially discussed. OBJECTIVES: We aimed to investigate the T. rangeli cell cycle in vitro and in vivo by characterizing the timespan of the parasite life cycle and by proposing a molecular marker to assess cytokinesis. METHODOLOGY: The morphological events and their timing during the cell cycle of T. rangeli epimastigotes were assessed using DNA staining, flagellum labelling and bromodeoxyuridine incorporation. Messenger RNA levels of four genes previously associated with the cell cycle of trypanosomatids (AUK1, PLK, MOB1 and TRACK) were evaluated in the different T. rangeli forms. FINDINGS: T. rangeli epimastigotes completed the cell cycle in vitro in 20.8 h. PLK emerged as a potential molecular marker for cell division, as its mRNA levels were significantly increased in exponentially growing epimastigotes compared with growth-arrested parasites or in vitro-differentiated trypomastigotes. PLK expression in T. rangeli can be detected near the flagellum protrusion site, reinforcing its role in the cell cycle. Interestingly, T. rangeli bloodstream trypomastigotes exhibited very low mRNA levels of PLK and were almost entirely composed of parasites in G1 phase. MAIN CONCLUSIONS: Our work is the first to describe the T. rangeli cell cycle in vitro and proposes that PLK mRNA levels could be a useful tool to investigate the T. rangeli ability to proliferate within the mammalian host bloodstream.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Citocinese/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Trypanosoma rangeli/citologia , Animais , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Citocinese/genética , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Citometria de Fluxo , Imunofluorescência , Hidroxiureia/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Fatores de Tempo , Trypanosoma rangeli/efeitos dos fármacos , Trypanosoma rangeli/enzimologia , Trypanosoma rangeli/genética , Tripanossomíase/parasitologia
11.
Anal Bioanal Chem ; 411(21): 5437-5444, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31300858

RESUMO

To meet the need for high-throughput immunoassays, many multiplex fluorescent immunoassays have been proposed. Most of them need different kinds of fluorescent label indicators during the test. In this work, a novel indirect ELISA-inspired dual-channel fluorescent immunoassay based on 3-mercaptopropionic acid capped CdTe/ZnS quantum dots (QDs) was constructed. The ELISA wells were coated with two kinds of antigen-QD complex. When the primary antibodies were present in a sample, they mediated the binding of a secondary antibody-DNA-gold nanoparticle complex to the antigen-QD complex. Then the gold nanoparticles quenched the fluorescence of the QDs and a decrease in fluorescence intensity was observed. Thus, the amount of primary antibody could be estimated from the decrease of fluorescence intensity. Owing to the wide absorption range and the relatively narrow emission band of the QDs, the dual-channel fluorescent immunoassay system could work at the same excitation wavelength and the emission wavelengths of each channel had no interference. As a result, two different kinds of primary antibody could be detected at the same time in one ELISA well, which simplified the operation and greatly improved the efficiency. Besides, only one type of secondary antibody needs to be added to the prepared microtiter plates, which further simplified the operation during the detection procedure. This dual-channel fluorescent immunoassay system will provide new insights into high-throughput immunodetection. Graphical abstract.


Assuntos
Compostos de Cádmio/química , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Pontos Quânticos , Sulfetos/química , Telúrio/química , Compostos de Zinco/química , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão
12.
Rev Med Chil ; 147(3): 390-394, 2019 Mar.
Artigo em Espanhol | MEDLINE | ID: mdl-31344179

RESUMO

Goodpasture Syndrome is described as a single episode disease entity. It is diagnosed with the demonstration of antiglomerular basement (anti-GBM) antibodies in plasma or renal tissue. Although the recurrence of anti-GBM disease is rare, it has been reported in up to 3% of cases. Recurrence with negative anti-GBM antibodies in plasma is even less frequent We report a 63 years old male in whom anti-GBM disease recurred without detectable anti-GBM antibodies in plasma, despite having positive antibodies at the onset.


Assuntos
Doença Antimembrana Basal Glomerular/patologia , Autoanticorpos/análise , Antibacterianos/uso terapêutico , Doença Antimembrana Basal Glomerular/diagnóstico por imagem , Doença Antimembrana Basal Glomerular/tratamento farmacológico , Biópsia , Ciclofosfamida/uso terapêutico , Imunofluorescência , Humanos , Nefropatias/patologia , Glomérulos Renais/patologia , Masculino , Pessoa de Meia-Idade , Prednisona/uso terapêutico , Recidiva , Combinação Trimetoprima e Sulfametoxazol/uso terapêutico
13.
Rev Bras Parasitol Vet ; 28(2): 221-228, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31271639

RESUMO

Geese, ducks, mallards, and swans are birds of the order Anseriformes, which are found in the wild, in zoos and parks, and raised for meat consumption. Toxoplasma gondii, Sarcocystis sp., and Neospora caninum are protozoans of several species of animals. Wild and domestic birds can serve as intermediate hosts, disseminators and potential sources of infection of these protozoa to humans through contaminated meat. The aims of this study were: (i) to perform a serological survey of T. gondii, Sarcocystis sp. and N. caninum in geese (Anser sp.) from public parks and from captivity and (ii) to compare seroprevalence between these two locations. Antibodies were detected by Immunofluorescence antibody test using the serum of 149 geese. Antibodies to Sarcocystis sp., T. gondii, and N. caninum were detected in 28.18%, 18% and 0.67% of geese, respectively; 57% of geese from urban parks and 26.53% of geese from captivity were seropositive for at least one protozoa. The results indicate environmental contamination, particularly for the occurrence of antibodies against T. gondii - a zoonosis that causes toxoplasmosis and is transmitted through oocyte ingestion. This is the first serological survey of T. gondii, Sarcocystis sp. and N. caninum in geese from urban parks in Curitiba, Brazil.


Assuntos
Anticorpos Antiprotozoários/sangue , Doenças das Aves/epidemiologia , Doenças das Aves/parasitologia , Gansos/parasitologia , Neospora/imunologia , Sarcocystis/imunologia , Toxoplasma/imunologia , Animais , Brasil/epidemiologia , Imunofluorescência , Estudos Soroepidemiológicos , População Urbana
14.
Immunity ; 50(6): 1439-1452.e5, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31178352

RESUMO

Hematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45+ AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.


Assuntos
Diferenciação Celular , Desenvolvimento Embrionário , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Animais , Biomarcadores , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Imunofluorescência , Imunofenotipagem , Inflamação/etiologia , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/metabolismo
15.
Vet J ; 249: 33-40, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31239162

RESUMO

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19-63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas. Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/µm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.


Assuntos
Doenças do Gato/metabolismo , Neoplasias da Íris/veterinária , Melanoma/veterinária , Animais , Gatos , Feminino , Imunofluorescência/veterinária , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/biossíntese , Neoplasias da Íris/metabolismo , Melanoma/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas B-raf/biossíntese , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Supressoras de Tumor/biossíntese
16.
Transplant Proc ; 51(5): 1371-1377, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31155175

RESUMO

BACKGROUND: Graft immunocomplex capture fluorescence analysis is an attractive method to detect intragraft donor-specific anti-HLA antibodies. In ABO-incompatible transplantation, anti-A and B antibodies are also considered to be important donor specific antibodies (ABO-DSA). Therefore, it is useful to monitor intragraft ABO-DSAs to assess antibody-mediated rejection. METHODS: To capture A and B antigens, anti-Band III, von Willebrand factor (VW), and plasmalemma vesicle-associated protein (PLVAP) beads were produced. The allograft specimen was homogenized in a lysis buffer. Subsequently, A and B antigens were captured by anti-Band III, VW, or PLVAP beads. The immune complexes were then detected by phycoerythrin-conjugated anti-human IgG antibodies and analyzed using a Luminex system. RESULTS: Although Band III and VW beads yielded false positives and false negatives, PLVAP beads captured A and B antigens with high sensitivity (91.7%) and specificity (100%) when an index > 1.5 was considered positive. The proximity in A and B antigens and PLVAP expression was confirmed using immunohistochemical evaluation. Furthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis supported that PLVAP is an A and B antigen carrier protein. CASE REPORT: Biopsies were conducted following an ABO-incompatible renal transplant (type A to O) and evaluated for ABO-DSA. Graft immunocomplex capture fluorescence analysis was demonstrated as follows: 3.19 (1 h, serum creatinine [s-Cr] 3.95 mg/dL, titer IgG 1:512, glomerulitis [g] 0, peritubular capillaritis [ptc] 0, complement 4d [C4d] 1); 1.8 (4 d, s-Cr 2.29 mg/dL, titer 1:256, g 0, ptc 0, C4d 3); 1.2 (22 d, s-Cr 1.58 mg/dL, titer 1:128, g 0, ptc 2, C4d 3). This result indicated that the remnant ABO-DSA were adsorbed and subsequently removed from the allograft successfully. CONCLUSIONS: This novel application could be used to detect intragraft ABO-DSAs, which could lead to a correct diagnosis and shed light on the ABO-DSA kinetics following ABO-incompatible transplantation.


Assuntos
Antígenos de Grupos Sanguíneos/análise , Imunofluorescência/métodos , Rejeição de Enxerto/imunologia , Isoanticorpos/análise , Transplante de Rim , Adulto , Biópsia , Feminino , Antígenos HLA/imunologia , Humanos , Masculino , Doadores de Tecidos , Transplante Homólogo
17.
Rev Assoc Med Bras (1992) ; 65(5): 637-646, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31166440

RESUMO

OBJECTIVE: Aplastic anemia (AA) is an immune-mediated disease that destroys hematopoietic cells through activated T lymphocytes. B lymphocyte-mediated humoral immunity also plays an important role in the pathogenesis of AA. Regulatory B cell (Breg) subpopulation, which is defined as "B10", secretes interleukin 10 (IL-10). The objective of our experiment was to investigate whether the scale-down proportion of B10 cells in AA patients may play a key role in the pathogenesis. METHODS: A total of 38 AA patients (14 SAA patients and 24 NSAA patients) and 20 healthy control subjects were included. All subjects did not suffer from autoimmune diseases or any other diseases affecting the immune system, such as infectious diseases. Bone marrow mononuclear cells (PBMCs) were isolated and analyzed by Flow cytometry (FCM) and Immunofluorescence double-labeling assay. The relationship between the relative proportions of B10 and ProB10 and their associations to AA, as well as disease severity, were assessed by common clinical indicators and then examined. RESULTS: Our analyses revealed AA patients had significantly lower proportions of peripheral B10 and B10pro compared to healthy controls. SAA patients had a substantially lower percentage of B10 cells and B10pro cells compared to NSAA patients. In addition, B10 cells and B10pro cells were negatively correlated with absolute neutrophil counts, hemoglobin levels and platelet, and absolute reticulocyte counts in AA patients. CONCLUSIONS: The present study attempted to elucidate the potential role of the scale-down proportion of B10 cells in the pathogenesis of AA.


Assuntos
Anemia Aplástica/patologia , Linfócitos B Reguladores/patologia , Adolescente , Adulto , Idoso , Anemia Aplástica/sangue , Antígenos CD19/análise , Antígenos CD19/metabolismo , Células da Medula Óssea/citologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Interleucina-10/análise , Interleucina-10/metabolismo , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Neutrófilos , Valores de Referência , Contagem de Reticulócitos , Índice de Gravidade de Doença , Adulto Jovem
18.
Int. j. morphol ; 37(2): 522-532, June 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1002254

RESUMO

Amelogenin is one of the enamel matrices secreted by ameloblasts. A mutation of the amelogenin gene can cause hereditary dental enamel defects known as amelogenesis imperfecta (AI). Since lysosome-associated membrane protein-1 (LAMP-1), -3 (LAMP-3), and 78kDa glucose-related protein (Grp78) were identified as binding proteins of amelogenin, several studies have suggested the involvement of these binding proteins with the cell kinetics of ameloblasts in normal or abnormal conditions. The purpose of this study is to investigate the distribution of these amelogenin binding proteins in the ameloblast cell differentiation of mice with a point mutation of the amelogenin gene (Amelx*). The incisors of Amelx* mice had a white opaque color and the tooth surface was observed to be rough under a scanning electron microscope. Among the sequential ameloblast cell differentiation in the Amelx* mice, the shape of ameloblasts at the transition stage was irregular in comparison to those in wild-type (WT) mice. Immunostaining of Grp78 revealed that the whole cytoplasm of the transition stage ameloblasts was immunopositive for Grp78 antibody, while only the distal part of cell was positive in the WT mice. Furthermore, in the Amelx* mice, the cytoplasm of the transition stage ameloblasts was immunopositive for LAMP-1 and LAMP-3. These results suggest that Amelx* may cause the abnormal distribution of amelogenin binding proteins in the cytoplasm of ameloblasts.


La amelogenina es una de las matrices de esmalte secretadas por los ameloblastos. Una mutación del gen de amelogenina puede causar defectos hereditarios del esmalte dental conocidos como amelogénesis imperfecta (AI). Dado que la proteína de membrana asociada a lisosoma-1 (LAMP-1), -3 (LAMP-3) y la proteína relacionada con la glucosa de 78 kDa (Grp78) se identificaron como proteína de unión a amelogenina, varios estudios han sugerido la participación de estas proteínas con la cinética celular de los ameloblastos en condiciones normales o anormales. El objetivo del estudio fue investigar la distribución de LAMP-1, LAM-3 y Grp78 durante la diferenciación celular de ameloblastos de ratones con una mutación puntual del gen de amelogenina (Amelx*). Los incisivos de los ratones Amelx* presentaron un color blanco opaco y se observó en microscopio electrónico de barrido que la superficie del diente era áspera. La diferenciación celular secuencial y la forma de los ameloblastos en la etapa de transición en los ratones Amelx* fue irregular en comparación con los ratones silvestres (RS). La inmunotinción de Grp78 reveló que todo el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para el anticuerpo Grp78, mientras que solo la parte distal de la célula fue positiva en los ratones RS. Además, en ratones Amelx*, el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para LAMP-1 y LAMP-3. Estos resultados sugieren que Amelx* puede causar distribución anormal de proteínas de unión a amelogenina en el citoplasma de los ameloblastos.


Assuntos
Animais , Camundongos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Amelogenina/metabolismo , Amelogênese Imperfeita , Proteínas de Choque Térmico/metabolismo , Microscopia Eletrônica de Varredura , Imunofluorescência , Esmalte Dentário/patologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Amelogenina/genética , Proteína 3 de Membrana Associada ao Lisossomo/metabolismo , Incisivo/patologia
19.
Exp Parasitol ; 203: 8-18, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31150653

RESUMO

Toxoplasma gondii is an important human and veterinary pathogen and the causative agent of toxoplasmosis, a potentially severe disease especially in immunocompromised or congenitally infected humans. Current therapeutic compounds are not well-tolerated, present increasing resistance, limited efficacy and require long periods of treatment. On this context, searching for new therapeutic targets is crucial to drug discovery. In this sense, recent works suggest that N-myristoyltransferase (NMT), the enzyme responsible for protein myristoylation that is essential in some parasites, could be the target of new anti-parasitic compounds. However, up to date there is no information on NMT and the extent of this modification in T. gondii. In this work, we decided to explore T. gondii genome in search of elements related with the N-myristoylation process. By a bioinformatics approach it was possible to identify a putative T. gondii NMT (TgNMT). This enzyme that is homologous to other parasitic NMTs, presents activity in vitro, is expressed in both intra- and extracellular parasites and interacts with predicted TgNMT substrates. Additionally, NMT activity seems to be important for the lytic cycle of Toxoplasma gondii. In parallel, an in silico myristoylome predicts 157 proteins to be affected by this modification. Myristoylated proteins would be affecting several metabolic functions with some of them being critical for the life cycle of this parasite. Together, these data indicate that TgNMT could be an interesting target of intervention for the treatment of toxoplasmosis.


Assuntos
Aciltransferases/metabolismo , Toxoplasma/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/efeitos dos fármacos , Aciltransferases/genética , Sequência de Aminoácidos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Fibroblastos/parasitologia , Imunofluorescência , Prepúcio do Pênis/citologia , Prepúcio do Pênis/parasitologia , Humanos , Imunoprecipitação , Masculino , Filogenia , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Toxoplasma/classificação , Toxoplasma/enzimologia , Toxoplasma/genética
20.
Anal Bioanal Chem ; 411(20): 5277-5285, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161325

RESUMO

Rapid, highly sensitive detection of tau protein and other neurodegenerative biomarkers remains a significant hurdle for diagnostic tests for Alzheimer's disease. In this work, we developed a novel tyrosinase (TYR)-induced tau aptamer-tau-tau antibody (anti-tau) sandwich fluorescence immunoassay to detect tau protein that used dopamine (DA)-functionalized CuInS2/ZnS quantum dots as the fluorophore. CuInS2/ZnS core/shell quantum dots with high luminescence, low toxicity, and excellent biocompatibility were successfully fabricated and decorated with DA through amide conjugation. Meanwhile, TYR was conjugated with anti-tau by a click reaction. When DA-functionalized CuInS2/ZnS quantum dots were added to the sandwich system, TYR catalyzed the transformation of DA to dopamine quinone, which acted as an effective electron acceptor and triggered fluorescence quenching. The fluorescence intensity of the immunoassay based on DA-functionalized CuInS2/ZnS quantum dots shows good performance in terms of linearity with the logarithm of tau protein concentration, with a linear concentration range from 10 pM to 200 nM. This work is the first to use a TYR-induced fluorescence immunoassay for the rapid detection of tau protein, paving a new way for the detection of disease biomarkers. Graphical abstract.


Assuntos
Cobre/química , Imunofluorescência/métodos , Índio/química , Monofenol Mono-Oxigenase/química , Pontos Quânticos/química , Selênio/química , Sulfetos/química , Compostos de Zinco/química , Proteínas tau/análise , Cristalografia por Raios X , Dopamina/análogos & derivados , Dopamina/química , Microscopia Eletrônica de Transmissão , Análise Espectral/métodos
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