RESUMO
Neutrophils are polymorphonuclear leukocytes that play a key role in the organism defense. These cells enroll in a range of actions to ensure pathogen elimination and orchestrate both innate and adaptative immune responses. The main physiological structures of neutrophils are their storage organelles that are essential since the cells activation and participate in all their functions. The storage organelles are divided into 2 types: granules and secretory vesicles. The granules are subdivided into azurophilic, specific and gelatinase. The granules are distinguished by their protein content, and since they play an important role on the neutrophil function, the knowledge of the proteins stored in these organelles can help to better understand these cells. Some proteins are present in high abundance and are used as markers for each storage organelle. These proteins are myeloperoxidase (MPO) for azurophil granules, neutrophil gelatinase associated with lipocalin-2 (NGAL) and lactoferrin (LTF) for specific granules, matrix metalloproteinase-9 (MMP9) for gelatinase granules and alkaline phosphatase (AP) for secretory vesicles. The isolation of neutrophils granules, however, is challenging and the existing procedures rely on large sample volumes, about 400 mL of peripheral blood or 3 x 108 neutrophils, not allowing for multiple biological and technical replicates. Therefore, the aim of this study was to develop a miniaturized neutrophil granules isolation method and to use biochemical assays, mass spectrometry-based proteomics and a machine learning approach to investigate the protein content of the neutrophils storage organelles. With that in mind, 40 mL of the peripheral blood of three apparently healthy volunteers were collected. The neutrophils were isolated, disrupted using nitrogen cavitation and organelles were fractionated with a discontinuous 3-layer Percoll density gradient. The presence of granules markers in each fraction was assessed using western blot , gelatin zymography and enzymatic assays. The isolation was proven successful and allowed for a reasonable separation of all neutrophils storage organelles in a gradient of less than 1 mL, about 37 times smaller than the methodsdescribed in the literature. Moreover, mass spectrometry-based proteomics identified 369 proteins in at least 3 of the 5 samples, and using a machine learning strategy, the localization of 140 proteins was predicted with confidence. Furthermore, this study was the first to investigate the proteome of neutrophil granules using technical and biological replicates, creating a reliable database for further studies. In conclusion, the developed miniaturized method is reproducible, cheaper, and reliable. In addition, it provides a resource for further studies exploring neutrophil granules protein content and mobilization during activation with different stimuli
Neutrófilos são leucócitos polimorfonucleares que possuem papel fundamental na defesa do organismo. Essas células desempenham diversas ações a fim de assegurar a eliminação de um patógeno e, além disso, orquestram a resposta imune inata e adaptativa. O conjunto composto pelos grânulos de armazenamento e as vesículas secretórias compõe a principal estrutura fisiológica dos neutrófilos. Estes componentes são essenciais desde a ativação celular, participando de todas as funcionalidades desta célula. Os grânulos são subdivididos em azurófilos, específicos e gelatinase. Eles podem ser distinguidos por meio de seu conteúdo proteico e, como são importantes na funcionalidade dos neutrófilos, identificar quais proteínas são armazenadas nestas organelas é imprescindível para entender melhor essa célula como um todo. Algumas proteínas, estão presentes de forma abundante e, portanto, são utilizadas como marcadores dos grânulos. Tais proteínas são mieloperoxidase (MPO) para os grânulos azurófilos, gelatinase de neutrófilo associada a lipocalina (NGAL) e lactoferrina (LTF) para os específicos, metaloproteinase de matrix 9 (MMP9) para os grânulos de gelatinase e fosfatase alcalina (AP) para as vesículas secretórias. Isolar estas estruturas, no entanto, é desafiador visto que os protocolos existentes na literatura utilizam grandes volumes de amostra, cerca de 400 mL de sangue ou 3 x 108 neutrófilos, para apenas um isolamento, impedindo a realização de replicatas técnicas e biológicas. Desta forma, o objetivo do presente estudo foi desenvolver um protocolo miniaturizado de isolamento dos grânulos neutrofílicos e utilizar métodos bioquímicos, de proteômica e machine learning para investigar o conteúdo proteico destas estruturas celulares. Para isto, 40 mL de sangue periférico de três voluntários aparentemente saudáveis foi coletado. Os neutrófilos foram então isolados, lisados com cavitação de nitrogênio e o fracionamento subcelular foi realizado baseado em um gradiente descontínuo de 3 camadas de Percoll. O método de isolamento foi avaliado através da investigação dos marcadores utilizando western blotting (WB), zimografia de gelatina e ensaios enzimáticos em cada fração coletada. O isolamento demonstrou-se eficiente e permitiu uma ótima separação dos grânulosem um gradiente menor que 1 mL, cerca de 37 vezes menor que os métodos atualmente descritos na literatura. Além disso, a análise proteômica foi capaz de identificar 369 proteínas presentes em pelo menos 3 das 5 réplicas investigadas e, utilizando ferramentas de machine learning, 140 proteínas foram classificadas como pertencentes a um dos tipos de grânulos ou vesícula secretória com alto nível de confiabilidade. Por fim, o presente estudo foi o primeiro a investigar o proteoma dos grânulos utilizando replicatas técnicas e biológicas, criando e fornecendo uma base de dados robusta que poderá ser utilizada em estudos futuros. Conclui-se, portanto, que a metodologia miniaturizada desenvolvida é eficaz, reprodutível e mais barata, além de permitir estudos mais complexos e profundos sobre o proteoma dos grânulos dos neutrófilos em diferentes momentos celulares, tais como quando ativados via estímulos distintos
Assuntos
Proteômica/instrumentação , Metodologia como Assunto , Neutrófilos/classificação , Espectrometria de Massas/métodos , Cavitação , Western Blotting/instrumentação , Gelatinases/análise , Fosfatase Alcalina/efeitos adversos , Aprendizado de Máquina/classificaçãoRESUMO
Neutrophil gelatinase- associated lipocalin (NGAL) is a protein molecule predominantly expressed in the distal nephron after the occurrence of renal injury. Unlike the serum creatinine and the glomerular filtration rate, which are the renal function markers, the increased levels of NGAL, both in serum and urine, are closely linked to the structural injury to the nephron. Clinical studies indicate that a few hours after the occurrence of acute renal injury, the serum and urinary levels of NGAL are already significantly elevated, while the serum creatinine levels and renal clearance only undergo significant changes between 24-48h after injury. Thus, the use of renal function markers, usually assessed in the clinical practice, they may have some limitations also hindering the implementation of early measures aimed at protecting the kidneys. This literature review aims at examining the biological aspects and the applications of NGAL measurement in some clinical conditions, including kidney failure, kidney diseases and renal ischemia.(AU)
Lipocalina associada à gelatinase neutrofílica (NGAL) é uma molécula proteica predominantemente expressa na parte distal do néfron após a ocorrência de lesão renal. Diferentemente da creatinina sérica e da taxa de filtração glomerular, que são marcadores de função renal, os níveis aumentados de NGAL, tanto no soro quanto na urina, estão intimamente ligados a lesões estruturais do néfron. Os estudos clínicos indicam que poucas horas após a ocorrência da lesão renal aguda os níveis séricos e urinários de NGAL já se apresentam significativamente elevados, enquanto os níveis séricos de creatinina e a sua depuração renal apenas sofrem alterações significativas entre 24-48h após a lesão. Assim, a utilização de marcadores de função renal, usualmente avaliados na prática clínica, pode apresentar algumas limitações além de dificultar a aplicação de medidas precoces que visam a proteção renal. Esta revisão da literatura tem por objetivo analisar os aspectos biológicos e as aplicações da mensuração de NGAL em algumas condições clínicas, incluindo injúria renal, nefropatias e isquemia renal.(AU)
Assuntos
Lipocalinas/análise , Biomarcadores , Gelatinases/análise , Neutrófilos , Rim/lesões , Néfrons/fisiologia , Nefropatias , Prognóstico , IsquemiaRESUMO
Neutrophil gelatinase- associated lipocalin (NGAL) is a protein molecule predominantly expressed in the distal nephron after the occurrence of renal injury. Unlike the serum creatinine and the glomerular filtration rate, which are the renal function markers, the increased levels of NGAL, both in serum and urine, are closely linked to the structural injury to the nephron. Clinical studies indicate that a few hours after the occurrence of acute renal injury, the serum and urinary levels of NGAL are already significantly elevated, while the serum creatinine levels and renal clearance only undergo significant changes between 24-48h after injury. Thus, the use of renal function markers, usually assessed in the clinical practice, they may have some limitations also hindering the implementation of early measures aimed at protecting the kidneys. This literature review aims at examining the biological aspects and the applications of NGAL measurement in some clinical conditions, including kidney failure, kidney diseases and renal ischemia.
Lipocalina associada à gelatinase neutrofílica (NGAL) é uma molécula proteica predominantemente expressa na parte distal do néfron após a ocorrência de lesão renal. Diferentemente da creatinina sérica e da taxa de filtração glomerular, que são marcadores de função renal, os níveis aumentados de NGAL, tanto no soro quanto na urina, estão intimamente ligados a lesões estruturais do néfron. Os estudos clínicos indicam que poucas horas após a ocorrência da lesão renal aguda os níveis séricos e urinários de NGAL já se apresentam significativamente elevados, enquanto os níveis séricos de creatinina e a sua depuração renal apenas sofrem alterações significativas entre 24-48h após a lesão. Assim, a utilização de marcadores de função renal, usualmente avaliados na prática clínica, pode apresentar algumas limitações além de dificultar a aplicação de medidas precoces que visam a proteção renal. Esta revisão da literatura tem por objetivo analisar os aspectos biológicos e as aplicações da mensuração de NGAL em algumas condições clínicas, incluindo injúria renal, nefropatias e isquemia renal.
Assuntos
Biomarcadores , Gelatinases/análise , Lipocalinas/análise , Neutrófilos , Néfrons/fisiologia , Rim/lesões , Isquemia , Nefropatias , PrognósticoRESUMO
We evaluated the frequency of enterococci from food and found 95.2% of positivity, being E. faecium and E. faecalis the most frequent species. High-level streptomycin resistance was observed, as well as gelatinase and hemolysis activity, showing the potential role of environmental strains as reservoir of virulence and resistance traits.
Assuntos
Enterococcus/classificação , Enterococcus/isolamento & purificação , Contaminação de Alimentos , Microbiologia de Alimentos , Brasil , Farmacorresistência Bacteriana , Enterococcus/fisiologia , Gelatinases/análise , Hemólise , Prevalência , Fatores de Virulência/análiseRESUMO
The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the ß-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and ß-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.
Assuntos
Proteínas de Bactérias/genética , Enterococcus faecalis/genética , Microbiologia de Alimentos , Infecções por Bactérias Gram-Positivas/microbiologia , Fatores de Virulência/genética , Biofilmes/crescimento & desenvolvimento , Brasil , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/fisiologia , Gelatinases/análise , Hemólise , HumanosRESUMO
The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.
Assuntos
Humanos , Proteínas de Bactérias/genética , Enterococcus faecalis/genética , Microbiologia de Alimentos , Infecções por Bactérias Gram-Positivas/microbiologia , Fatores de Virulência/genética , Brasil , Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/fisiologia , Gelatinases/análise , HemóliseRESUMO
We evaluated the frequency of enterococci from food and found 95.2% of positivity, being E. faecium and E. faecalis the most frequent species. High-level streptomycin resistance was observed, as well as gelatinase and hemolysis activity, showing the potential role of environmental strains as reservoir of virulence and resistance traits.
Assuntos
Enterococcus/classificação , Enterococcus/isolamento & purificação , Contaminação de Alimentos , Microbiologia de Alimentos , Brasil , Farmacorresistência Bacteriana , Enterococcus/fisiologia , Gelatinases/análise , Hemólise , Prevalência , Fatores de Virulência/análiseRESUMO
The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.(AU)
Assuntos
Humanos , Proteínas de Bactérias/genética , Enterococcus faecalis/genética , Microbiologia de Alimentos , Infecções por Bactérias Gram-Positivas/microbiologia , Fatores de Virulência/genética , Brasil , Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/fisiologia , Gelatinases/análise , HemóliseRESUMO
We evaluated the frequency of enterococci from food and found 95.2% of positivity, being E. faecium and E. faecalis the most frequent species. High-level streptomycin resistance was observed, as well as gelatinase and hemolysis activity, showing the potential role of environmental strains as reservoir of virulence and resistance traits.(AU)
Assuntos
Enterococcus/classificação , Enterococcus/isolamento & purificação , Contaminação de Alimentos , Microbiologia de Alimentos , Brasil , Farmacorresistência Bacteriana , Enterococcus/fisiologia , Gelatinases/análise , Hemólise , Prevalência , Fatores de Virulência/análiseRESUMO
OBJECTIVES: To determine the phenotypic and molecular characteristics of Enterococcus faecalis recovered from primary endodontic infections in Brazilian patients. METHODS: Twenty isolates of E. faecalis recovered from 43 Brazilian patients with primary endodontic infections were identified by biochemical profiling (API20Strep) and 16S rDNA sequencing. Antimicrobial susceptibility was ascertained by agar dilution, using the recommended protocol of the Clinical and Laboratory Standards Institute (CLSI). PCR with validated primers was used to detect genes associated with antibiotic resistance and specific virulence factors. RESULTS: All isolates were deemed susceptible to penicillin G, erythromycin and vancomycin. However, nine isolates had a minimum inhibitory concentration of 4µg/mL to vancomycin (the resistance breakpoint). Fourteen isolates (70% of isolates) were also resistant to tetracycline with MICs of >64µg/mL. PCR products for tetracycline resistance genes were detected in test isolates, while erythromycin and vancomycin resistance genes were not evident. Gelatinase, aggregation substance and enteroccocal surface protein genes were detected in 20, 18 and 12 isolates, respectively. CONCLUSIONS: Endodontic E. faecalis isolates exhibit high level of resistance to tetracycline, an antibiotic that has use in local treatment of dental infections. This opens up a much-needed debate on the role and efficacy of this antibiotic for oral infections. Furthermore, these isolates were shown to possess genes that could contribute to pathogenicity in the pulp cavity.
Assuntos
Doenças da Polpa Dentária/microbiologia , Farmacorresistência Bacteriana , Enterococcus faecalis/fisiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/análise , Técnicas Bacteriológicas , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Eritromicina/farmacologia , Gelatinases/análise , Perfilação da Expressão Gênica , Proteínas de Membrana/análise , Testes de Sensibilidade Microbiana , Penicilina G/farmacologia , Fenótipo , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Resistência a Tetraciclina , Vancomicina/farmacologia , Virulência , Fatores de Virulência/análiseRESUMO
INTRODUCTION: The aim of this study was to compare the gelatinolytic activity of matrix metalloproteinase (MMP)-2 and MMP-9 and the expression of tissue inhibitor of matrix metalloproteinase (TIMP)-2 and myeloperoxidase protein (MPO) in clinically healthy human pulp and inflamed pulp tissue specimens. METHODS: Twenty dental pulps clinically diagnosed as inflammatory tissues and 20 healthy pulp tissues from enclosed third molars were harvested and evaluated. The gelatinolytic activity for MMP-2 and MMP-9 was assessed by using the zymography technique, TIMP-2 gene expression was evaluated using the enzyme-linked immunosorbent assay, and MPO was determined using the MPO assay. RESULTS: Data showed increased levels of MMP-9, active MMP-2, TIMP-2, and MPO in inflammatory pulp tissues compared with healthy tissues (P < .05). No statistical difference could be observed for pro-MMP-2 (P > .05). CONCLUSIONS: Although all samples were associated with MMP-2 expression, the active form of this MMP was observed only in inflamed pulps. Inflamed pulps showed an up-regulation of MMP-9, TIMP-2, and MPO.
Assuntos
Polpa Dentária/enzimologia , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Peroxidase/análise , Pulpite/enzimologia , Inibidor Tecidual de Metaloproteinase-2/análise , Exposição da Polpa Dentária/enzimologia , Precursores Enzimáticos/análise , Ensaio de Imunoadsorção Enzimática , Gelatinases/análise , Humanos , Inibidores de Proteases/análise , Regulação para CimaRESUMO
Samples of sewage from a university hospital and a chemistry technical school were analysed for the percentage of bacterial tolerance to chromium (Cr), silver (Ag) and mercury (Hg). Additionally, we investigated the effect of these metals on pigmentation and on some enzymatic activities of the metal tolerant strains isolated, as well as antimicrobial resistance in some metal tolerant Enterobacteriaceae strains. Tolerance to Cr was observed mainly in Gram positive bacteria while in the case of Ag and Hg the tolerant bacteria were predominately Gram negative. Hg was the metal for which the percentage of tolerance was significantly higher, especially in samples from the hospital sewage (4.1%). Mercury also had the most discernible effect on color of the colonies. Considering the effect of metals on the respiratory enzymes, one strain of Ag-tolerantBacillus sp. and one of Hg-tolerant P. aeruginosa were unable to produce oxidase in the presence of Ag and Hg, respectively, while the expression of gelatinase was largely inhibited in various Gram negative strains (66% by Cr). Drug resistance in Hg-tolerant Enterobacteriaceae strains isolated from the university hospital sewage was greater than 80%, with prevalence of multiple resistance, while the Ag-tolerant strains from the same source showed about 34% of resistance, with the predominance of mono-resistance. Our results showed that, despite the ability of metal tolerant strains to survive and grow in the presence of these elements, the interactions with these metals may result in metabolic or phisiological changes in this group of bacteria.
Assuntos
Humanos , Águas Residuárias/análise , Resistência a Medicamentos , Infecções por Enterobacteriaceae , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/metabolismo , Gelatinases/análise , Metais Pesados/isolamento & purificação , Metais Pesados/metabolismo , Oxirredutases/análise , Pigmentos Biológicos/análise , Ativação Enzimática , Hospitais , Prevalência , Amostras de ÁguaRESUMO
Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the Instituto Antártico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities present in 161 microorganisms (120 bacteria, 31 yeasts and 10 filamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20°C. Most of yeasts and bacteria grew better at 20°C than at 4°C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identified by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum.
Assuntos
Bactérias/enzimologia , Bactérias/isolamento & purificação , Microbiologia Ambiental , Fungos/enzimologia , Fungos/isolamento & purificação , Amilases/análise , Regiões Antárticas , Bactérias/classificação , Bactérias/genética , Celulase/análise , Análise por Conglomerados , Temperatura Baixa , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Fúngico/química , DNA Fúngico/genética , Fungos/classificação , Fungos/genética , Gelatinases/análise , Lipase/análise , Filogenia , Análise de Sequência de DNARESUMO
AIM: To investigate the effects of root canal sealers on the cytotoxicity and gelatinolytic activity of matrix metalloproteinases (MMPs) in human fibroblasts. METHODOLOGY: Human fibroblasts (MRC5, 3×10(5) cells per well) were incubated directly or indirectly with AH Plus, Endomethasone N, Pulp Canal Sealer EWT or Sealapex for 30 min, 1, 4 or 24 h (time-points). The cytotoxicity of all root canal sealers was determined by counting viable cells using the trypan blue exclusion assay. Supernatants of cell cultures incubated with root sealers directly or indirectly were collected after each time-point to determine the levels of MMP-2 and MMP-9 gelatinolytic activity by gelatin zymography. Data were analysed using anova and the Tukey's tests. RESULTS: Cells secreted MMP-2 after periods of 4 and 24 h; however, there were no significant differences between the sealers. Secretion of gelatinases was elevated by root canal sealers in direct contact with the cell monolayer when compared to indirect contact (P < 0.05). At the time-points tested, no gelatinolytic activity could be detected in the control group without the sealers. The cytotoxicity results revealed that all sealers were cytotoxic in both contact forms. Sealapex had the lowest cytotoxicity and AH Plus the most cytotoxicity. CONCLUSIONS: All root canal sealers induced the expression of MMP-2 in MRC5 fibroblasts. AH Plus had the highest cytotoxicity amongst the tested sealers, but all were associated with cytotoxic effects.
Assuntos
Fibroblastos/efeitos dos fármacos , Gelatinases/efeitos dos fármacos , Materiais Restauradores do Canal Radicular/toxicidade , Regulação para Cima/efeitos dos fármacos , Hidróxido de Cálcio/química , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Corantes , Dexametasona/química , Combinação de Medicamentos , Resinas Epóxi/química , Fibroblastos/enzimologia , Formaldeído/química , Gelatinases/análise , Humanos , Hidrocortisona/química , Teste de Materiais , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Materiais Restauradores do Canal Radicular/química , Salicilatos/química , Propriedades de Superfície , Timol/análogos & derivados , Timol/química , Fatores de Tempo , Azul Tripano , Cimento de Óxido de Zinco e Eugenol/químicaRESUMO
FUNDAMENTO: Insuficiência cardíaca (IC) causada por Doença de Chagas (DC) é uma cardiomiopatia inflamatória progressiva que afeta milhões de pessoas na América Latina. Estudos com modelos de camundongo de IC devido à DC indicam que o transplante de células mononucleares derivadas da medula óssea (TCDMO) pode reduzir a inflamação, fibrose e melhorar a função miocárdica. OBJETIVO: O propósito desse estudo foi avaliar, pela primeira vez em seres humanos, a segurança e a eficácia de TCDMO no miocárdio de pacientes com IC devido à DC. MÉTODOS: Um total de 28 pacientes com IC devido à DC (média de idade de 52,2 ± 9,9 anos) com classe funcional NYHA III e IV foram submetidos à TCDMO através de injeção coronariana. Os efeitos na fração de ejeção do ventrículo esquerdo (FEVE), capacidade funcional, qualidade de vida, arritmias e parâmetros bioquímicos, imunológicos e neuro-humorais foram avaliados. RESULTADOS: Não houve complicações diretamente relacionadas ao procedimento. A FEVE foi 20,1 ± 6,8 por cento e 28,3 ± 7,9 por cento, p < 0,03 a nível basal e 180 dias após o procedimento, respectivamente. No mesmo período, melhoras significantes foram observadas na classe funcional NYHA (3,1 ± 0,3 para 1,8 ± 0,5; p < 0,001), qualidade de vida (50,9 ± 11,7 para 25,1 ± 15,9; p < 0,001), e no teste de caminhada de seis minutos (355 ± 136 m para 437 ± 94 m; p < 0,01). Não houve alterações nos marcadores de ativação imune ou neurohormonais. Nenhuma complicação foi registrada. CONCLUSÃO: Nossos dados sugerem que a injeção intracoronariana de células derivadas da medula óssea é segura e potencialmente efetiva em pacientes com IC devido à DC. A extensão do benefício, entretanto, parece ser discreta e precisa ser confirmada em estudos clínicos maiores, randomizados, duplo-cegos, controlados com placebo.
BACKGROUND: Heart failure due to Chagas' disease (HFCD) is a progressive inflammatory cardiomyopathy that affects millions of individuals in Latin America. Studies using mice models of HFCD indicate that bone marrow mononuclear cell transplantation (BMCT) may reduce inflammation, fibrosis, and improve myocardial function. OBJECTIVE: The purpose of this study was to evaluate, for the first time in humans, the safety and efficacy of BMCT to the myocardium of patients with HFCD. METHODS: A total of 28 HFCD patients (mean age 52.2 ± 9.9 years) with NYHA class III and IV were submitted to BMCT through intracoronary injection. Effects on the left ventricle ejection fraction (LVEF), functional capacity, quality-of-life, arrhythmias, biochemical, immunological, and neuro-humoral parameters, were evaluated. RESULTS: There were no complications directly related to the procedure. LVEF was 20.1 ± 6.8 percent and 28.3 ± 7.9 percent, p < 0.03 at baseline and 180 days after the procedure, respectively. In the same period, significant improvements were observed in the NYHA class (3.1 ± 0.3 to 1.8 ± 0.5; p < 0.001), quality-of-life (50.9 ± 11.7 to 25.1 ± 15.9; p < 0.001), and in the six-minute walking test (355 ± 136 m to 437 ± 94 m; p < 0,01). There were no changes in markers of immune or neurohormonal activation. No complications were registered. CONCLUSION: Our data suggest that the intracoronary injection of BMCT is safe and potentially effective in patients with HFCD. The extent of the benefit, however, appears to be small and needs to be confirmed in a larger randomized, double blind, placebo controlled clinical trial.
FUNDAMENTO: La insuficiencia cardíaca (IC), causada por la enfermedad de Chagas (EC), es una cardiomiopatía inflamatoria progresiva que afecta a millones de personas en Latinoamérica. Estudios con modelos experimentales de IC en razón de la EC, nos indican que el transplante de células mononucleares derivadas de la médula ósea (TCMO), puede reducir la inflamación y la fibrosis, mejorando así la función miocárdica. OBJETIVO:El objetivo de este estudio fue evaluar, por primera vez en seres humanos, la seguridad y la eficacia del TCMO en el miocardio de pacientes con IC debido a la EC. MÉTODOS:Fueron estudiados un total de 28 pacientes con IC debido a la EC (con edad promedio 52,2 ± 9,9 años), en clases funcionales III y IV (NYHA), al TCMO por medio de una inyección coronaria. Se evaluaron los efectos en la fracción de eyección del ventrículo izquierdo (FEVI), capacidad funcional, calidad de vida, arritmias y parámetros bioquímicos, inmunológicos y neurohumorales. RESULTADOS:No se registraron complicaciones relacionadas directamente con el procedimiento. La FEVI pasó de 20,1 ± 6,8 por ciento para 28,3 ± 7,9 por ciento, p < 0,03, cuando se comparó con el período basal y 180 días después del procedimiento, respectivamente. En el mismo período, también se observaron mejorías en la clase funcional NYHA promedio (3,1 ± 0,3 para 1,8 ± 0,5; p < 0,001), puntuación de calidad de vida de Minnesota (50,9 ± 11,7 para 25,1 ± 15,9; p < 0,001), y en el test de esfuerzo de seis minutos (355 ± 136 m para 437 ± 94 m; p < 0,01). No hubo alteraciones en los marcadores de activación inflamatoria o neurohormonales. Ninguna complicación fue registrada. CONCLUSIÓN:Nuestros datos sugieren que la inyección intracoronaria de las células derivadas de la médula ósea es segura y potencialmente efectiva en pacientes con IC debido a la EC. La extensión del beneficio, sin embargo, parece ser discreta, y necesita ser confirmada en los ensayos clínicos randomizados, doble ciegos, controlados con placebo.
Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Transplante de Medula Óssea , Cardiomiopatia Chagásica/cirurgia , Insuficiência Cardíaca/cirurgia , Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/imunologia , Cardiomiopatia Chagásica/complicações , Fluorimunoensaio , Gelatinases/análise , Insuficiência Cardíaca/etiologia , Monocinas/análise , Qualidade de Vida , Volume Sistólico/fisiologia , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: Heart failure due to Chagas' disease (HFCD) is a progressive inflammatory cardiomyopathy that affects millions of individuals in Latin America. Studies using mice models of HFCD indicate that bone marrow mononuclear cell transplantation (BMCT) may reduce inflammation, fibrosis, and improve myocardial function. OBJECTIVE: The purpose of this study was to evaluate, for the first time in humans, the safety and efficacy of BMCT to the myocardium of patients with HFCD. METHODS: A total of 28 HFCD patients (mean age 52.2 ± 9.9 years) with NYHA class III and IV were submitted to BMCT through intracoronary injection. Effects on the left ventricle ejection fraction (LVEF), functional capacity, quality-of-life, arrhythmias, biochemical, immunological, and neuro-humoral parameters, were evaluated. RESULTS: There were no complications directly related to the procedure. LVEF was 20.1 ± 6.8% and 28.3 ± 7.9%, p < 0.03 at baseline and 180 days after the procedure, respectively. In the same period, significant improvements were observed in the NYHA class (3.1 ± 0.3 to 1.8 ± 0.5; p < 0.001), quality-of-life (50.9 ± 11.7 to 25.1 ± 15.9; p < 0.001), and in the six-minute walking test (355 ± 136 m to 437 ± 94 m; p < 0,01). There were no changes in markers of immune or neurohormonal activation. No complications were registered. CONCLUSION: Our data suggest that the intracoronary injection of BMCT is safe and potentially effective in patients with HFCD. The extent of the benefit, however, appears to be small and needs to be confirmed in a larger randomized, double blind, placebo controlled clinical trial.
Assuntos
Transplante de Medula Óssea , Cardiomiopatia Chagásica/cirurgia , Insuficiência Cardíaca/cirurgia , Adulto , Idoso , Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/imunologia , Cardiomiopatia Chagásica/complicações , Feminino , Fluorimunoensaio , Gelatinases/análise , Insuficiência Cardíaca/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Monocinas/análise , Projetos Piloto , Qualidade de Vida , Volume Sistólico/fisiologia , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
AIMS: The current research was aimed at comparing extracellular proteolytic activities and zymogram profiles among Aeromonas spp. METHODS AND RESULTS: Extracellular proteases of 47 strains of Aeromonas were analyzed by substrate (casein and gelatin) co-polymerized SDS-PAGE, and caseinolytic activity was determined using azocasein. Large variation on caseinolytic activity was evidenced. In general, the caseinolytic activity of Aeromonas hydrophila strains was significantly higher than that of other Aeromonas species. Several caseinolytic and gelatinolytic profiles were detected in Aeromonas. Cluster analysis allowed separating Aeromonas strains in four and three groups, based on their caseinolytic and gelatinolytic profiles, respectively. Although not specific patterns were evident, most Aer. hydrophila strains were clustered together and differed from Aeromonas caviae strains. The main caseinases of Aer. hydrophila were a serine protease with an apparent molecular weight (AMW) of 56 kDa and a metalloprotease with AMW of 22 kDa. Gelatinase profiles were characterized by the presence of high molecular weight metalloproteases (84 and 93 kDa), although the most active enzyme was a serine protease with AMW of 56 kDa. Other new caseinases and gelatinases were detected in specific Aeromonas strains. CONCLUSIONS: Aeromonas strains exhibited several extracellular proteolytic profiles, with a larger inter than intraspecific variation. Moreover, zymogram analyses allowed identifying new caseinases and gelatinases in Aeromonas. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the intra- and interspecific variation of proteolytic profiles in Aeromonas determined by zymogram analysis, including the detection of new caseinases and gelatinases in this genus.
Assuntos
Aeromonas/enzimologia , Gelatinases/análise , Metaloendopeptidases/análise , Caseínas/metabolismo , Análise por Conglomerados , Eletroforese em Gel de Poliacrilamida , Gelatina/metabolismo , Gelatinases/metabolismo , Metaloendopeptidases/metabolismo , Especificidade da EspécieRESUMO
Se evaluó el efecto de la concentración de hidróxido de calcio (Ca(OH)2) y tiempo de cocción del grano de maíz sobre las propiedades fisicoquímicas y reológicas durante la nixtamalización tradicional. Se observó que a mayor concentración de Ca(OH)2 y mayor tiempo de cocción del grano, la temperatura de gelatinización aumentó significativamente. El maíz nixtamalizado con 2 g/100g de Ca(OH)2 y 60 min de tiempo de cocción, presentó mayor absorción de calcio, 0.152 g/100g, mostrando un aumento significativo respecto a la concentración inicial (500 g/100g). Todas las muestras presentaron un patrón de difracción de rayos X de almidón tipo A, con diferencias en la intensidad de la difracción. La viscosidad aparente del nixtamal disminuyó considerablemente al utilizar condiciones de procesamiento más altas. Se encontró que la capacidad de retención de agua aumentó con la temperatura.
The effect of calcium hydroxide (Ca(OH)2) concentration and cooking time on physicochemical and pasting properties of maize, during traditional nixtamalization was evaluated. It was observed that the higher the Ca(OH)2 concentration and cooking time the higher the gelatinization temperature. Grain samples treated with 2 g/100g of Ca(OH)2 and 60 min of cooking time presented higher calcium absorption (0.152 g/100g) showing a significant increment (500 g/100g). Results of X-ray diffraction showed that all the samples had X-ray diffraction pattern of starch type A, showing differences in the intensity of the diffraction as indicated by the percentage of cristallinity. The apparent viscosity of the nixtamal considerably decreased when the processes conditions were higher. Water retention capacity increased with temperature.
Assuntos
Amido/análise , Fenômenos Químicos/métodos , Hidróxido de Cálcio , Gelatinases/análise , Zea mays , Análise de AlimentosAssuntos
Proteínas de Bactérias/genética , Enterococcus faecalis/citologia , Enterococcus faecalis/genética , Gelatinases/análise , Infecções por Bactérias Gram-Positivas/microbiologia , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/fisiologia , Genótipo , Humanos , FenótipoRESUMO
Several colubrid snakes produce venomous oral secretions. In this work, the venom collected from Venezuelan opisthoglyphous (rear-fanged) Philodryas olfersii snake was studied. Different proteins were present in its venom and they were characterized by 20% SDS-PAGE protein electrophoresis. The secretion exhibited proteolytic (gelatinase) activity, which was partially purified on a chromatography ionic exchange mono Q2 column. Additionally, the haemorrhagic activity of Philodryas olfersii venom on chicken embryos, mouse skin and peritoneum was demonstrated. Neurotoxic symptoms were demonstrated in mice inoculated with Philodryas olfersii venom. In conclusion, Philodryas olfersii venom showed proteolytic, haemorrhagic, and neurotoxic activities, thus increasing the interest in the high toxic action of Philodryas venom.