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1.
Arq. bras. med. vet. zootec. (Online) ; 71(3): 917-928, May-June 2019. ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1011332

RESUMO

In veterinary medicine, the cell therapy is still unexplored and there are many unanswered questions that researchers tend to extrapolate to humans in an attempt to treat certain injuries. Investigating this subject in nonhuman primates turns out to be an unparalleled opportunity to better understand the dynamics of stem cells against some diseases. Thus, we aimed to compare the efficiency of bone marrow mononuclear cells (BMMCs) and mesenchymal stem cells (MSCs) from adipose tissue of Chlorocebus aethiops in induced bone injury. Ten animals were used, male adults subjected, to bone injury the iliac crests. The MSCs were isolated by and cultured. In an autologous manner, the BMMCs were infused in the right iliac crest, and MSCs from adipose tissue in the left iliac crest. After 4.8 months, the right iliac crests fully reconstructed, while left iliac crest continued to have obvious bone defects for up to 5.8 months after cell infusion. The best option for treatment of injuries with bone tissue loss in old world primates is to use autologous MSCs from adipose tissue, suggesting we can extrapolate the results to humans, since there is phylogenetic proximity between species.(AU)


Na medicina veterinária, a terapia celular ainda é inexplorada e há muitas perguntas não respondidas, o que leva os pesquisadores a uma tendência a estender a terapia para os seres humanos, na tentativa de tratar certas lesões. Investigar esse assunto em primatas não humanos revela-se uma oportunidade sem precedentes para compreender melhor a dinâmica das células-tronco contra algumas doenças. Assim, objetivou-se comparar a eficiência das células mononucleares de medula óssea (BMMCs) e das células-tronco mesenquimais (MSCs) do tecido adiposo de Chlorocebus aetiops na lesão óssea induzida. Foram utilizados 10 animais, adultos do sexo masculino, submetidos à lesão óssea nas cristas ilíacas. As MSCs foram isoladas e cultivadas; de forma autóloga, as BMMCs foram infundidas na crista ilíaca direita e as MSCs de tecido adiposo na crista ilíaca esquerda. Após 4,8 meses, a crista ilíaca direita foi totalmente reconstruída, enquanto a crista ilíaca esquerda continuou apresentando defeito ósseo evidente por até 5,8 meses após a infusão. A melhor opção para o tratamento de lesões com perda de tecido ósseo em primatas do Velho Mundo é a utilização de MSCs autólogas de tecido adiposo, sugerindo que se podem estender os resultados para seres humanos, uma vez que há proximidade filogenética entre as espécies.(AU)


Assuntos
Animais , Masculino , Células da Medula Óssea , Transplante de Células-Tronco/veterinária , Células-Tronco Mesenquimais , Terapia Baseada em Transplante de Células e Tecidos/veterinária , Chlorocebus aethiops , Modelos Animais , Ílio/lesões
2.
Mem. Inst. Oswaldo Cruz ; 114: e190150, 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1020077

RESUMO

BACKGROUND Zika virus (ZIKV) infections reported in recent epidemics have been linked to clinical complications that had never been associated with ZIKV before. Adaptive mutations could have contributed to the successful emergence of ZIKV as a global health threat to a nonimmune population. However, the causal relationships between the ZIKV genetic determinants, the pathogenesis and the rapid spread in Latin America and in the Caribbean remain widely unknown. OBJECTIVES The aim of this study was to characterise three ZIKV isolates obtained from patient samples during the 2015/2016 Brazilian epidemics. METHODS The ZIKV genomes of these strains were completely sequenced and in vitro infection kinetics experiments were carried out in cell lines and human primary cells. FINDINGS Eight nonsynonymous substitutions throughout the viral genome of the three Brazilian isolates were identified. Infection kinetics experiments were carried out with mammalian cell lines A549, Huh7.5, Vero E6 and human monocyte-derived dendritic cells (mdDCs) and insect cells (Aag2, C6/36 and AP61) and suggest that some of these mutations might be associated with distinct viral fitness. The clinical isolates also presented differences in their infectivity rates when compared to the well-established ZIKV strains (MR766 and PE243), especially in their abilities to infect mammalian cells. MAIN CONCLUSIONS Genomic analysis of three recent ZIKV isolates revealed some nonsynonymous substitutions, which could have an impact on the viral fitness in mammalian and insect cells.


Assuntos
Humanos , Animais , Aedes/virologia , Zika virus/genética , Infecção por Zika virus/virologia , Camundongos Endogâmicos BALB C , Filogenia , Cultura de Vírus , Replicação Viral , Células Vero , Brasil , Chlorocebus aethiops , Carga Viral
3.
Mem. Inst. Oswaldo Cruz ; 113(2): 102-110, Feb. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-894895

RESUMO

BACKGROUND In a screen of extracts from plants and fungi to detect antileishmanial activity, we found that the ethyl acetate extract of the fungus Nectria pseudotrichia, isolated from the tree Caesalpinia echinata (Brazilwood), is a promising source of bioactive compounds. OBJECTIVES The aims of this study were to isolate and determine the chemical structures of the compounds responsible for the antileishmanial activity of the organic extract from N. pseudotrichia. METHODS Compounds were isolated by chromatographic fractionation using semi-preparative high-performance liquid chromatography, and their chemical structures were determined by analytical and spectral data and by comparison with published data. The antileishmanial activity of the isolated compounds was evaluated in intracellular amastigote forms of Leishmania (Viannia) braziliensis expressing firefly luciferase as reporter gene, and cytotoxicity was determined in Vero and THP-1 mammalian cell lines by MTT assay. FINDINGS Fractionation of the extract yielded seven compounds: 10-acetyl trichoderonic acid A (1), 6′-acetoxy-piliformic acid (2), 5′,6′-dehydropiliformic acid (3), piliformic acid (4), hydroheptelidic acid (5), xylaric acid D (6), and cytochalasin D (7). Compounds 1, 2 and 3 are reported here for the first time. Compounds 1, 2, and 5 were more active, with IC50 values of 21.4, 28.3, and 24.8 µM, respectively, and showed low toxicity to Vero and THP-1 cells. MAIN CONCLUSIONS N. pseudotrichia produces secondary metabolites that are more toxic to intracellular amastigote forms of L. (V.) braziliensis than to mammalian cells.


Assuntos
Leishmania braziliensis/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Testes de Toxicidade , Caesalpinia/microbiologia , Sobrevivência Celular , Chlorocebus aethiops , Concentração Inibidora 50
4.
Mem. Inst. Oswaldo Cruz ; 113(4): e170332, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-894914

RESUMO

BACKGROUND Trypanosoma cruzi is a protozoan parasite and an etiological agent of Chagas disease. There is a wide variability in the clinical outcome of its infection, ranging from asymptomatic individuals to those with chronic fatal mega syndromes. Both parasite and host factors, as well as their interplay, are thought to be involved in the process. OBJECTIVES To evaluate the resistance to complement-mediated killing in two T. cruzi TcI strains with differential virulence and the subsequent effect on their infectivity in mammalian cells. METHODS Tissue-culture derived trypomastigotes of both strains were incubated in guinea pig serum and subjected to flow cytometry in order to determine their viability and complement activations. Trypomastigotes were also incubated on host cells monolayers in the presence of serum, and infectivity was evaluated under different conditions of complement pathway inhibition. Relative expression of the main parasite-specific complement receptors between the two strains was assessed by quantitative real-time polymerase chain reaction. FINDINGS In this work, we showed that two TcI strains, one with lower virulence (Ninoa) compared to the other (Qro), differ in their resistance to the lytic activity of complement system, hence causing a compromised ability of Ninoa strain to invade mammalian cells. These results correlate with the three-fold lower messenger RNA (mRNA) levels of complement regulatory protein (CRP), trypomastigote-decay acceleration factor (T-DAF), and complement C2 receptor inhibitor trispanning (CRIT) in Ninoa compared to those in Qro. On the other hand, calreticulin (CRT) mRNA and surface protein levels were higher in Ninoa strain and promoted its infectivity when the lectin pathway of the complement system was inhibited. MAIN CONCLUSIONS This work suggests the complex interplay of CRP, T-DAF, CRIT, and CRT, and the diagnostic value of mRNA levels in the assessment of virulence potential of T. cruzi strains, particularly when dealing with isolates with similar genetic background.


Assuntos
Humanos , Chlorocebus aethiops , Doença de Chagas/parasitologia , Antígenos de Protozoários/análise , Células Vero , Western Blotting
5.
Braz. j. microbiol ; 48(4): 764-768, Oct.-Dec. 2017. graf
Artigo em Inglês | LILACS | ID: biblio-889184

RESUMO

ABSTRACT Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.


Assuntos
Humanos , Animais , Doenças das Aves Domésticas/microbiologia , Aderência Bacteriana , Infecções por Clostridium/microbiologia , Infecções por Clostridium/veterinária , Clostridium perfringens/fisiologia , Células Epiteliais/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Vero , Chlorocebus aethiops , Galinhas , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/genética
6.
Mem. Inst. Oswaldo Cruz ; 112(4): 281-291, Apr. 2017. graf
Artigo em Inglês | LILACS | ID: biblio-841788

RESUMO

BACKGROUND Dengue is considered one of the world’s most important mosquito-borne diseases. MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that play an important role in the regulation of gene expression in eukaryotes. Although miRNAs possess antiviral activity against many mammalian-infecting viruses, their involvement in Dengue virus (DENV) replication remains poorly understood. OBJECTIVE To determine the role of miR-484 and miR-744 in DENV infection and to examine whether DENV infection alters the expression of both miRNAs. METHODS We used bioinformatics tools to explore the relationship between DENV and cellular miRNAs. We then overexpressed miR-484 or miR-744 in Vero cells to examine their role in DENV replication using flow cytometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and western blotting. FINDINGS We found several cellular miRNAs that target a conserved region within the 3′ untranslated region (3′ UTR) of the genome of the four DENV serotypes and found that overexpression of miR-484 or miR-744 inhibits infection by DENV-1 to DENV-4. Furthermore, we observed that DENV RNA might be involved in the downregulation of endogenous miR-484 and miR-744. CONCLUSION Our study identifies miR-484 and miR-744 as two possible restriction host factors against DENV infection. However, further studies are needed to directly verify whether miR-484 and miR-744 both have an anti-DENV effect in vivo.


Assuntos
Animais , Replicação Viral/fisiologia , Replicação Viral/genética , Chlorocebus aethiops , Regulação da Expressão Gênica/genética , Western Blotting , Reação em Cadeia da Polimerase , Biologia Computacional , Regiões não Traduzidas , Regiões não Traduzidas/fisiologia , Vírus da Dengue/fisiologia , Vírus da Dengue/genética , MicroRNAs/metabolismo , Citometria de Fluxo
7.
Mem. Inst. Oswaldo Cruz ; 112(2): 131-139, Feb. 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-841764

RESUMO

BACKGROUND Recent studies showed that essential oils from different pepper species (Piper spp.) have promising leishmanicidal and trypanocidal activities. OBJECTIVES In search for natural compounds against Trypanosoma cruzi, different forms of the parasite were incubated for 24 h at 28ºC or 4ºC with Piper aduncum essential oil (PaEO) or its main constituents linalool and nerolidol. METHODS PaEO chemical composition was obtained by GC-MS. Drug activity assays were based on cell counting, MTT data or infection index values. The effect of PaEO on the T. cruzi cell cycle and mitochondrial membrane potential was evaluated by flow cytometry. FINDINGS PaEO was effective against cell-derived (IC50/24 h: 2.8 μg/mL) and metacyclic (IC50/24 h: 12.1 μg/mL) trypomastigotes, as well as intracellular amastigotes (IC50/24 h: 9 μg/mL). At 4ºC - the temperature of red blood cells (RBCs) storage in blood banks - cell-derived trypomastigotes were more sensitive to PaEO (IC50/24 h = 3.8 μg/mL) than to gentian violet (IC50/24 h = 24.7 mg/mL). Cytotoxicity assays using Vero cells (37ºC) and RBCs (4ºC) showed that PaEO has increased selectivity for cell-derived trypomastigotes. Flow cytometry analysis showed that PaEO does not affect the cell cycle of T. cruzi epimastigotes, but decreases their mitochondrial membrane potential. GC-MS data identified nerolidol and linalool as major components of PaEO, and linalool had trypanocidal effect (IC50/24 h: 306 ng/mL) at 4ºC. MAIN CONCLUSION The trypanocidal effect of PaEO is likely due to the presence of linalool, which may represent an interesting candidate for use in the treatment of potentially contaminated RBCs bags at low temperature.


Assuntos
Animais , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Bioensaio , Óleos Voláteis/farmacologia , Piper/química , Células Vero , Testes de Sensibilidade Microbiana , Chlorocebus aethiops , Temperatura Baixa , Monoterpenos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas
8.
Braz. j. microbiol ; 47(4): 896-901, Oct.-Dec. 2016. tab, graf
Artigo em Inglês | LILACS | ID: biblio-828210

RESUMO

Abstract The study aimed to evaluate the anti-Sporothrix sp. activity of the essential oil of Origanum majorana Linn. (marjoram), its chemical analysis, and its cytotoxic activity. A total of 18 fungal isolates of Sporothrix brasiliensis (n: 17) from humans, dogs and cats, and a standard strain of Sporothrix schenckii (n: 1) were tested using the broth microdilution technique (Clinical and Laboratory Standard Institute - CLSI M27-A3) and the results were expressed in minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). The MIC50 and MIC90 of itraconazole against S. brasiliensis were 2 µg/mL and 8 µg/mL, respectively, and the MFC50 and MFC90 were 2 µg/mL and >16 µg/mL, respectively, with three S. brasiliensis isolates resistant to antifungal. S. schenckii was sensitive at MIC of 1 µg/mL and MFC of 8 µg/mL. For the oil of O. majorana L., all isolates were susceptible to MIC of ≤2.25-9 mg/mL and MFC of ≤2.25-18 mg/mL. The MIC50 and MIC90 were ≤2.25 mg/mL and 4.5 mg/mL, respectively, and the MFC50/90 values were twice more than the MIC. Twenty-two compounds were identified by gas chromatography with a flame ionization detector (CG-FID) and 1,8-cineole and 4-terpineol were the majority. Through the colorimetric (MTT) assay, the toxicity was observed in 70-80% of VERO cells between 0.078 and 5 mg/mL. For the first time, the study demonstrated the satisfactory in vitro anti-Sporothrix sp. activity of marjoram oil and further studies are needed to ensure its safe and effective use.


Assuntos
Animais , Sporothrix/efeitos dos fármacos , Óleos Voláteis/farmacologia , Antifúngicos/farmacologia , Esporotricose/microbiologia , Sporothrix/isolamento & purificação , Células Vero , Óleos Voláteis/química , Testes de Sensibilidade Microbiana , Zoonoses/microbiologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Farmacorresistência Fúngica , Antifúngicos/química
9.
Mem. Inst. Oswaldo Cruz ; 111(8): 528-531, Aug. 2016. graf
Artigo em Inglês | LILACS | ID: lil-788995

RESUMO

In Brazil, the spotted fever group (SFG) Rickettsia rickettsii and Rickettsia parkeri related species are the etiological agents of spotted fever rickettsiosis. However, the SFG, Rickettsia rhipicephali, that infects humans, has never been reported. The study of growth dynamics can be useful for understanding the infective and invasive capacity of these pathogens. Here, the growth rates of the Brazilian isolates R. rickettsii str. Taiaçu, R. parkeri str. At#24, and R. rhipicephali HJ#5, were evaluated in Vero cells by quantitative polymerase chain reaction. R. rhipicephali showed different kinetic growth compared to R. rickettsii and R. parkeri.


Assuntos
Animais , Rickettsia/crescimento & desenvolvimento , Chlorocebus aethiops , Rickettsia/classificação , Especificidade da Espécie , Fatores de Tempo , Células Vero
10.
Mem. Inst. Oswaldo Cruz ; 111(8): 532-534, Aug. 2016. graf
Artigo em Inglês | LILACS | ID: lil-788996

RESUMO

Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient’s blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible.


Assuntos
Humanos , Animais , Vírion/ultraestrutura , Zika virus/ultraestrutura , Técnicas de Cultura de Células , Chlorocebus aethiops , Genoma Viral , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Células Vero , Replicação Viral
11.
Mem. Inst. Oswaldo Cruz ; 111(6): 411-413, June 2016. graf
Artigo em Inglês | LILACS | ID: lil-784251

RESUMO

In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.


Assuntos
Animais , Aedes/ultraestrutura , Vírus da Dengue/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Aedes/virologia , Técnicas de Cultura de Células , Centrifugação/métodos , Chlorocebus aethiops , Fixadores , Indicadores e Reagentes , Células Vero/ultraestrutura
12.
Mem. Inst. Oswaldo Cruz ; 111(3): 209-217, Mar. 2016. tab, graf
Artigo em Inglês | LILACS | ID: lil-777369

RESUMO

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.


Assuntos
Animais , Antifúngicos/farmacologia , Aspergillus/química , Clima Desértico , DNA Fúngico/isolamento & purificação , Paracoccidioides/efeitos dos fármacos , Chlorocebus aethiops , Cromatografia de Fase Reversa , Sobrevivência Celular/efeitos dos fármacos , Citocalasinas/análise , Espectrometria de Massas , Cloreto de Metileno , Testes de Sensibilidade Microbiana , Filogenia , Análise de Sequência de DNA , Extração em Fase Sólida , Células Vero/efeitos dos fármacos
13.
Pesqui. vet. bras ; 36(2): 119-122, fev. 2016. graf
Artigo em Inglês | LILACS | ID: lil-777388

RESUMO

Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.


Chlorocebus aethiops é uma espécie de primata não humano frequentemente utilizados em pesquisa biomédica. Algumas pesquisas envolve esta espécie como modelo experimental para várias doenças e possível tratamento com células-tronco. A medula óssea é uma das principais fontes destas células e proporciona fácil acesso. O objetivo deste estudo foi o de padronizar o protocolo de coleta e separação de medula óssea em C. aethiops. Dez animais foram submetidos a punção de medula óssea com acesso à crista ilíaca e separação de células por gradiente de densidade. A medula óssea de C. aethiops tinha uma média de 97% de viabilidade. A partir dos resultados obtidos, podemos concluir que C. aethiops é um excelente modelo para obter e isolar células mononucleares da medula óssea, promovendo vários estudos no campo da terapia celular.


Assuntos
Animais , Medula Óssea , Chlorocebus aethiops/sangue , Leucócitos Mononucleares , Punção Espinal , Protocolos , Células-Tronco , Terapia Baseada em Transplante de Células e Tecidos/veterinária
14.
Mem. Inst. Oswaldo Cruz ; 110(8): 989-995, Dec. 2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-769835

RESUMO

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.


Assuntos
Animais , Cricetinae , Feminino , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Leptospira/imunologia , Leptospirose/prevenção & controle , Vacinação/métodos , Adjuvantes Imunológicos , Biópsia , Chlorocebus aethiops , Sequência Conservada , Ensaio de Imunoadsorção Enzimática , Imunidade Humoral/imunologia , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Rim/patologia , Leptospirose/imunologia , Pulmão/patologia , Mesocricetus , Análise de Sobrevida , Células Vero , Vacinas de DNA/imunologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/microbiologia
15.
Mem. Inst. Oswaldo Cruz ; 110(6): 745-754, Sept. 2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-763101

RESUMO

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatuscaptured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatuspools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.


Assuntos
Adolescente , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Infecções por Bunyaviridae/epidemiologia , Culex/virologia , RNA Viral/isolamento & purificação , Vírus Simbu/classificação , Distribuição Animal , Sequência de Bases , Brasil/epidemiologia , Infecções por Bunyaviridae/sangue , Chlorocebus aethiops , Culex/classificação , Surtos de Doenças , Dengue/epidemiologia , Febre/fisiopatologia , Febre/virologia , Genótipo , Orthobunyavirus/classificação , Orthobunyavirus/genética , Filogenia , Reação em Cadeia da Polimerase , Prevalência , Sorogrupo , Vírus Simbu/genética , Células Vero
16.
Braz. j. biol ; 75(2,supl): 68-74, May 2015. tab, ilus
Artigo em Inglês | LILACS | ID: lil-755012

RESUMO

Some water bodies in the Sinos River Basin (SRB) have been suffering the effects of pollution by residential, industrial and agroindustrial wastewater. The presence of cytotoxic and genotoxic compounds could compromise the water quality and the balance of these ecosystems. In this context, the research aimed to evaluate the genotoxicity and cytotoxicity of the water at four sites along the SRB (in the cities of Santo Antônio da Patrulha, Parobé, Campo Bom and Esteio), using bioassays in fish and cell culture. Samples of surface water were collected and evaluated in vitro using the Astyanax jacuhiensis fish species (micronucleus test and comet assay) and the Vero lineage of cells (comet assay and cytotoxicity tests, neutral red - NR and tetrazolium MTT). The micronucleus test in fish showed no significant differences between the sampling sites, and neither did the comet assay and the MTT and NR tests in Vero cells. The comet assay showed an increase in genetic damage in the fish exposed to water samples collected in the middle and lower sections of the basin (Parobé, Campo Bom and Esteio) when compared to the upper section of the basin (Santo Antônio da Patrulha). The results indicate contamination by genotoxic substances starting in the middle section of the SRB.

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Alguns corpos d’água da Bacia Hidrográfica do Rio dos Sinos (BHRS) vêm sofrendo os efeitos da poluição por efluentes domésticos, industriais e agroindustriais. A presença de compostos citotóxicos e genotóxicos pode comprometer a qualidade da água e o equilíbrio desses ecossistemas. Neste contexto, o objetivo do trabalho foi avaliar a genotoxicidade e a citotoxicidade da água em quatro pontos ao longo da BHRS (Santo Antônio da Patrulha, Parobé, Campo Bom e Esteio), utilizando bioensaios em peixes e em cultura celular. As amostras de água de superfície foram coletadas e avaliadas in vitro utilizando a espécie de peixe Astyanax jacuhiensis (teste de micronúcleo e ensaio cometa) e a linhagem celular tipo Vero (ensaio cometa e os testes de citotoxicidade vermelho neutro - VN e tetrazólio MTT). O teste de micronúcleos em peixes não apresentou diferenças significativas entre os pontos de coleta, assim como o ensaio cometa e os testes VN e MTT nas células Vero. O ensaio cometa demonstrou aumento nos danos genéticos em peixes expostos às amostras de água coletadas nos trechos médio e inferior da bacia (Parobé, Campo Bom e Esteio) em relação ao trecho superior da bacia (Santo Antônio da Patrulha). Os resultados indicam contaminação por substâncias genotóxicas a partir do trecho médio da BHRS.

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Assuntos
Animais , Citotoxinas/toxicidade , Mutagênicos/toxicidade , Rios/química , Qualidade da Água , Poluentes Químicos da Água/toxicidade , Brasil , Chlorocebus aethiops , Ensaio Cometa , Characidae/genética , Characidae/metabolismo , Dano ao DNA , Monitoramento Ambiental , Testes para Micronúcleos , Células Vero
17.
Int. arch. otorhinolaryngol. (Impr.) ; 19(1): 80-85, Jan-Mar/2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-741526

RESUMO

Introduction Obstructive sleep apnea syndrome affects up to 4% of middle-aged men and 2% of adult women. It is associated with obesity. Objective The objective of this article is to review the literature to determine which factors best correlate with treatment success in patients with obstructive sleep apnea syndrome treated with a mandibular repositioning appliance. Data Synthesis A search was performed of the PubMed, Cochrane, Lilacs, Scielo, and Web of Science databases of articles published from January 1988 to January 2012. Two review authors independently collected data and assessed trial quality. Sixty-nine articles were selected from PubMed and 1 from Cochrane library. Of these, 42 were excluded based on the title and abstract, and 27 were retrieved for complete reading. A total of 13 articles and 1 systematic review were considered eligible for further review and inclusion in this study: 6 studies evaluated anthropomorphic and physiologic factors, 3 articles addressed cephalometric and anatomic factors, and 4 studies evaluated variables related to mandibular repositioning appliance design and activation. All the studies evaluated had low to moderate methodologic quality and were not able to support evidence on prediction of treatment success. Conclusion Based on this systematic review on obstructive sleep apnea syndrome treatment, it remains unclear which predictive factors can be used with confidence to select patients suitable for treatment with a mandibular repositioning appliance. .


Assuntos
Animais , Evolução Biológica , Proteínas de Transporte/química , Cinesina/química , Modelos Moleculares , Microtúbulos/metabolismo , Transporte Biológico/fisiologia , Chlorocebus aethiops , Células COS , Dimerização , Transferência Ressonante de Energia de Fluorescência , Cinética , Microscopia de Fluorescência
18.
Braz. j. infect. dis ; 18(6): 581-590, Nov-Dec/2014. tab, graf
Artigo em Inglês | LILACS | ID: lil-730422

RESUMO

Purpose: The purpose of this study was to identify measles virus in Shanghai in 2012 and study the genotype trend of measles virus epidemic strains during 2000–2012. Methods: Nose and throat swab specimens were collected from 34 suspected measles cases in Shanghai. Measles virus was isolated using Vero-SLAM cells (African green monkey kidney cells/lymphoid signal activating factor-transfected African green monkey kidney cells). The 450 bp of C terminus of the N gene and the entire hemagglutinin gene sequence was amplified using RT-PCR. Phylogenetic analysis was performed by comparing the seven measles strains in Shanghai with the reference strains for H1a, H1b and D8 genotypes, as well as the Chinese measles virus vaccine strain. Results: Seven measles viruses strains were isolated from the 34 throat swap specimens. Six strains were genotype H1a, which is the predominant strain in China and one strain was genotype D8, which is the first imported strain since 2000. All these seven strains maintained most of the glycosylation sites except subtype H1a, which lost one glycosylation site. Conclusion: Since 2000, measles virus strains in Shanghai are consistent with measles virus from other provinces in China with H1a being the predominant genotype. This study is also the first report of genotype D8 strain in Shanghai. All strains maintained their glycosylation sites except H1a that lost one glycosylation site. These strains could still be neutralized by the Chinese measles vaccine. We suggest that Shanghai Center for Disease Control laboratories should strengthen their approaches to monitor measles cases to prevent further spread of imported strains. .


Assuntos
Adulto , Animais , Feminino , Humanos , Lactente , Masculino , Surtos de Doenças , Vírus do Sarampo/genética , Sarampo/epidemiologia , Chlorocebus aethiops , China/epidemiologia , Genótipo , Dados de Sequência Molecular , Sarampo/virologia , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Viral/genética , Análise de Sequência de DNA , Células Vero
19.
Braz. j. microbiol ; 45(4): 1513-1519, Oct.-Dec. 2014. ilus, tab
Artigo em Inglês | LILACS | ID: lil-741307

RESUMO

Mycoplasma spp, belongs to the class Mollicutes and is capable to produce alterations in cellular cultures causing damages to the biotechnological industry. Bioproducts generally require two essential inputs, bovine serum and cells. The study herein aims to evaluate the mycoplasma concentrations that affect the growing of BHK21 and Vero cells. The species used were: Mycoplasma orale, M. salivarium, M. arginini and M. hyorhinis, cultivated in a SP4 media. Two contamination tests were performed with BHK21 and Vero cells and one of them applied different concentrations of mycoplasma. In the first one, mycoplasma was applied at the day zero and, in the second one, the contamination was performed after the monolayer establishment. The both cellular cultures presented cytopathic effects with mycoplasma contamination, but the Vero cells suffered more damages than the BHK21 ones. It was also observed that the severity of the cytopathic effect depended on the mycoplasma specie, on the concentration and on the time of contact with the cellular culture, which evidences the importance of controlling the presence of mycoplasma in biotechnological industries.


Assuntos
Animais , Cricetinae , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Mycoplasma/crescimento & desenvolvimento , Linhagem Celular , Chlorocebus aethiops , Técnicas de Cocultura , Meios de Cultura/química
20.
Invest. clín ; 55(2): 155-167, jun. 2014. ilus, tab
Artigo em Espanhol | LILACS | ID: lil-749973

RESUMO

Estudios previos han demostrado que la adaptación de diversos virus a crecer en líneas celulares de vertebrados, conduce a la selección de variantes virales que unen al heparán sulfato (HS) con alta afinidad. En el presente trabajo se determinó la susceptibilidad de cepas del virus dengue (DENV) a la heparina hipersulfatada un análogo al HS, después de pases seriados en células BHK-21. A aislados de campo de los cuatro serotipos de DENV, se les realizaron ocho pases seriados en células BHK-21. La adaptación de los DENV al cultivo celular seleccionó variantes virales con una aumentada capacidad replicativa en células BHK-21 y una incrementada susceptibilidad a la heparina, en relación a las respectivas cepas no adaptadas, obteniéndose una inhibición de la infectividad más significativa en DENV-2 y DENV-4. Las cepas de DENV adaptadas presentaron cambios en la secuencia de aminoácidos de la proteína de envoltura (E), en particular una substitución K204R para DENV-1, N67K para DENV-2, K308R y V452A para DENV-3 y E327G para DENV-4. Estas sustituciones implicaron ganancia de residuos básicos que incrementaron la carga neta positiva de la proteína. Los resultados sugieren, que la adaptación de cepas de DENV a células BHK-21 selecciona variantes virales sensibles a la heparina y que la efectividad de este compuesto varía dependiendo de la cepa viral. Además sugieren que el HS puede jugar un papel importante en la infectividad de las cepas de DENV adaptadas al cultivo celular, a diferencia de los aislados de DENV no adaptados.


Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.


Assuntos
Animais , Cricetinae , Vírus da Dengue/efeitos dos fármacos , Heparina/farmacologia , Seleção Genética , Proteínas do Envelope Viral/genética , Aedes/citologia , Linhagem Celular , Chlorocebus aethiops , Vírus da Dengue/crescimento & desenvolvimento , Rim/citologia , Mesocricetus , Modelos Moleculares , Mutação , Mutação de Sentido Incorreto , Ligação Proteica , Conformação Proteica , RNA Viral/genética , Análise de Sequência de RNA , Células Vero , Ensaio de Placa Viral , Cultura de Vírus , Replicação Viral , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/fisiologia
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