Neutrophil elastase ameliorates matrix metalloproteinase-9 to promote lipopolysaccharide-induced acute lung injury in mice

PURPOSE:To investigate the regulatory roles of neutrophil elastase (NE) and matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice.METHODS:To construct LPS-induced ALI mouse models, wild-type C57BL/6 mice were administered 5.0 mg/kg of LPS through endotracheal, and/or 1.0 mg/kg of ONO-5046, and/or 20.0 mg/kg of chemically modified tetracycline-3 (CMT-3) by gavage. The levels of MMP-9, tissue inhibitor of metalloprotease-1, interleukin (IL)-6 were detected by real time RT-PCR at 6 h, 24 h and 48 h, and tumor necrosis factor (TNF), lung wet-dry weight ratio, white blood cell (WBC) count and polymorphonuclear (PMN) count in bronchoalveolar lavage fluid (BALF) were tested at 48 h after administration. The 5-day survival analysis of the ALI mice was also performed.RESULTS:Both ONO-5046 and CMT-3, regardless of being used individually or combined, significantly reduced the levels of MMP-9, IL-6, and TNF in lung tissue as well as in BALF, and the WBC and PMN count in BALF. Combined treatment with ONO-5046 and CMT-3 remarkably improved the survival rate of ALI mice.CONCLUSION:Neutrophil elastase synergizes with matrix metalloproteinase-9 to promote and regulate the release of inflammatory mediators and the infiltration of inflammatory cells, consequently affecting the survival of lipopolysaccharide-induced acute lung injury mice.(AU)

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish / Caracterização molecular de fatores de virulência em Aeromonas hydrophila obtidas de peixes

Multiple factors can be involved in the virulence processes of Aeromonas hydrophila. The objective of the present paper was to verify the presence of aerolysin, hidrolipase, elastase and lipase virulence genes through the polymerase chain reaction (PCR) in A. hydrophila isolates obtained from fish of the São Francisco River Valley, and to evaluate virulence according to the presence of these genes in Nile tilapia fingerlings. One hundred and fourteen isolates from the bacteria were used. DNA was heat extracted and PCR undertaken using specific primers described in the literature. For in vivo tests Nile tilapia fingerlings were used. From the PCR tests, negative isolates for all genes tested were selected, positive isolates for two genes (aerolysin and elastase) and positive for the four genes tested. These were inoculated at a concentration of 10(8) UFC/ml into the tilapias, considered as treatments; another group of animals was used as control (with inoculation of saline solution). In all, 12 distinct standards regarding the presence of virulence factors in isolates from A. hydrophila, were observed. Of the 114 isolates analyzed, 100 (87.72%) presented at least one of the virulence factors under study. The virulence factors were widely distributed among the A. hydrophila isolates. Aerolysin was the most frequent virulence factor present in the isolates analyzed. A. hydrophila led to the mortality of the Nile tilapia fingerlings, regardless of the absence or quantity of virulence genes tested.(AU)

Múltiplos fatores podem estar envolvidos nos processos de virulência de Aeromonas hydrophila. O objetivo do presente trabalho foi verificar a presença dos genes de virulência aerolisina, hidrolipase, elastase e lipase, utilizando a reação em cadeia da polimerase (PCR), em isolados de Aeromonas hydrophila obtidos de peixes do Vale do São Francisco e, avaliar sua virulência de acordo com a presença desses genes de virulência em alevinos de tilápia do Nilo. Cento e quatorze isolados foram utilizados. O DNA foi termoextraído e a PCR realizada com a utilização de inciadores específiicos descritos pela literatura. Para os testes in vivo alevinos de tilápia do Nilo foram utilizados. Segundo os resultados da PCR, foram selecionados isolados negativos para todos os genes de virulência testados, isolados positivos para dois genes de virulência, (aerolisina e elastase) e positivos para os quatro genes avaliados. Esses isolados foram inoculados na concentração de 108 UFC/ml nos alevinos e foram considerados como tratamentos e, um outro grupo de animais foi utilizado como grupo controle (com inoculação de solução salina). Ao fiinal, 12 padrões distintos, em relação a presença dos fatores de virulência nos isolados de A. Hydrophila, foram observados. Dentre os 114 isolados analisados, 100 (87,72%) apresentaram pelo menos um dos genes de virulência sob estudo. Os fatores de virulência foram amplamente distribuídos entre os isolados de A. hydrophila. A aerolisina foi o fator de virulência mais frequente nos isolados analisados. A. hydrophila levou à mortalidade dos alevinos de tilápia do Nilo, independente da ausência ou quantidade de genes de virulência testados.(AU)

Quercetin inhibits neutrophil extracellular traps release and their cytotoxic effects on A549 cells, as well the release and enzymatic activity of elastase and myeloperoxidase / Quercetina inibe a liberação das armadilhas extracelulares dos neutrófilos e a atividade tóxica delas sobre células A549

Neutrophil extracellular traps (NETs) were first reported as a microbicidal strategy for activated neutrophils. Through an immunologic response against several stimuli, neutrophils release their DNA together with proteins from granules, nucleus, and cytoplasm (e.g., elastase and myeloperoxidase). To date, NETs have been implicated in tissue damage during intense inflammatory processes, mainly when their release is dependent on oxygen radical generation. Flavonoids are antioxidant and anti-inflammatory agents; of these, quercetin is commonly found in our daily diet. Therefore, quercetin could exert some protective activity against tissue damage induced by NETs. In our in vitro assays, quercetin reduced NETs, myeloperoxidase (MPO), and elastase release from neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). The activity of these enzymes also decreased in the presence of quercetin. Quercetin also reduced the cytotoxic effect of NETs on alveolar cells (A549 cell line). Further, in silico assays indicated favorable interactions between quercetin and NET proteins (MPO and elastase). Overall, our results demonstrate that quercetin decreases deleterious cellular effects of NETs by reducing their release from activated neutrophils, and diminishing the enzymatic activity of MPO and elastase, possibly through direct interaction.

As armadilhas extracelulares de neutrófilos (NETs) foram relatadas pela primeira vez como uma estratégia microbicida para neutrófilos ativados. Por meio de uma resposta imunológica contra vários estímulos, os neutrófilos liberam seu DNA ligado a proteínas de grânulos, núcleo e citoplasma (por exemplo, elastase e mieloperoxidase). Até o momento, os NETs têm sido implicadas em danos aos tecidos durante processos inflamatórios intensos, principalmente quando sua liberação depende da geração de radicais de oxigênio. Os flavonóides são agentes antioxidantes e anti-inflamatórios, e destes, a quercetina é comumente encontrada em nossa dieta diária. Portanto, a quercetina pode exercer alguma atividade protetora contra o dano tecidual induzido por NETs. Em nossos ensaios in vitro, a quercetina reduziu a liberação das NETs, mieloperoxidase (MPO) e elastase a partir de neutrófilos estimulados com forbol 12-miristato 13-acetato (PMA). A atividade dessas enzimas também foi diminuída na presença de quercetina. A quercetina também reduziu o efeito citotóxico dos NETs sobre células alveolares (linha celular A549). Além disso, os ensaios in silico indicaram interações favoráveis entre a quercetina e as proteínas da NET (MPO e elastase). No geral, nossos resultados demonstram que a quercetina diminui os efeitos deletérios das NETs, reduzindo sua liberação à partir de neutrófilos ativados e diminuindo a atividade enzimática de MPO e elastase, possivelmente por meio de interação direta.

Proteases (caseinase and elastase), hemolysins, adhesion and susceptibility to antimicrobials of Stenotrophomonas maltophilia isolates obtained from clinical specimens

Forty-six S. maltophilia isolates obtained from hospital clinical specimens were studied for protease (caseinase and elastase) production, hemolytic activity, adhesion to HEp-2 cells, plastic and glass. Susceptibility to antimicrobial agents was also evaluated. The majority of isolates were obtained from respiratory tract secretions of patients using medical devices. All the isolates grown overnight were able to hydrolyze casein at 30ºC and 37ºC. After 72h, all the isolates hydrolyzed elastase at 30ºC and 40 isolates (87%) at 37ºC. Most of the isolates presented hemolytic activity after 96h of incubation at both temperatures. Rabbit blood showed the hightest hemolytic activity, after 96h 61% and 98% of tested isolates presented beta-hemolysis at 30ºC and 37ºC, respectively. All isolates were susceptible to trimethoprim-sulfametoxazole and were resistant to most beta-lactams tested. By the dilution method, S. maltophilia showed a high susceptibility to ticarcillin-clavulanate and a lower susceptibility to ciprofloxacin than the agar diffusion. The isolates showed adhesion to HEp-2 cells, plastic and glass. The proteolytic activities and adhesion to inanimate surfaces detected in S. maltophilia can be related to the pathogenesis of this bacterium and/or medical device colonization which favors the development of nosocomial infections.

Quarenta e seis amostras de S. maltophilia obtidas de amostras clínicas foram estudadas quanto à produção de protease (caseinase e elastase), atividade hemolítica, adesão a células HEp-2, ao plástico e ao vidro. A sensibilidade aos agentes antimicrobianos também foi avaliada. A maioria das amostras foi obtida de secreções do trato respiratório de pacientes em uso de "dispositivos" médicos. Todas as amostras foram capazes de hidrolisar a caseína após o crescimento a 30ºC e 37ºC por 16-18hs. Após 72 hs, todas as amostras apresentaram atividade hemolítica após 96hs de incubação em ambas as temperaturas. A maior atividade hemolítica foi verificada com o sangue de coelho; após 96hs, 61% e 98% das amostras apresentaram b-hemólise a 30ºC e 37ºC, respectivamente. Todas as amostras foram sensíveis ao sulfametoxazol-trimetoprim e resistentes à maioria dos antimicrobianos b-lactâmicos testados. Através do método de diluição em ágar, S. maltophilia mostrou uma alta sensibilidade à ticarcilina-ácido clavulânico e uma menor sensibilidade à ciprofloxacina do que pelo método de difusão em ágar. As amostras mostraram adesão às células HEp-2, ao plástico e ao vidro. A atividade proteolítica e adesão a superfícies inanimadas detectadas em S. maltophilia podem estar relacionadas à patogênese desta bactéria. A colonização de "dispositivos" médicos favorece o desenvolvimento de infecções hospitalares.