Identification of novel functional sequence variants in the gene for peptidase inhibitor 3

BACKGROUND: Peptidase inhibitor 3 (PI3) inhibits neutrophil elastase and proteinase-3, and has a potential role in skin and lung diseases as well as in cancer. Genome-wide expression profiling of chorioamniotic membranes revealed decreased expression of PI3 in women with preterm premature rupture of membranes. To elucidate the molecular mechanisms contributing to the decreased expression in amniotic membranes, the PI3 gene was searched for sequence variations and the functional significance of the identified promoter variants was studied. METHODS: Single nucleotide polymorphisms (SNPs) were identified by direct sequencing of PCR products spanning a region from 1,173 bp upstream to 1,266 bp downstream of the translation start site. Fourteen SNPs were genotyped from 112 and nine SNPs from 24 unrelated individuals. Putative transcription factor binding sites as detected by in silico search were verified by electrophoretic mobility shift assay (EMSA) using nuclear extract from Hela and amnion cell nuclear extract. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by χ(2 )goodness-of-fit test. Haplotypes were estimated using expectation maximization (EM) algorithm. RESULTS: Twenty-three sequence variations were identified by direct sequencing of polymerase chain reaction (PCR) products covering 2,439 nt of the PI3 gene (-1,173 nt of promoter sequences and all three exons). Analysis of 112 unrelated individuals showed that 20 variants had minor allele frequencies (MAF) ranging from 0.02 to 0.46 representing "true polymorphisms", while three had MAF ≤ 0.01. Eleven variants were in the promoter region; several putative transcription factor binding sites were found at these sites by database searches. Differential binding of transcription factors was demonstrated at two polymorphic sites by electrophoretic mobility shift assays, both in amniotic and HeLa cell nuclear extracts. Differential binding of the transcription factor GATA1 at -689C>G site was confirmed by a supershift. CONCLUSION: The promoter sequences of PI3 have a high degree of variability. Functional promoter variants provide a possible mechanism for explaining the differences in PI3 mRNA expression levels in the chorioamniotic membranes, and are also likely to be useful in elucidating the role of PI3 in other diseases

Atividade antagônica in vitro de Trichoderma spp. ao fungo Lasiodiplodia theobromae.

Lasiodiplodia spp., são fungos de ampla distribuição geográfica, típicos de regiões tropicais e temperadas, conhecidos pelo seu polifagismo, sendo patógenos de uma extensa lista de plantas hospedeiras, a maioria frutíferas de clima tropical. Fungos deste gênero são capazes de causar diferentes sintomas nas plantas infectadas, incluindo a seca- descendente (die-back), cancro e lesões em diferentes partes da planta, além de incitar a morte de mudas e enxertos. Em relação a fungos do gêneroPDF. 155_11

Atividade antagônica de Trichoderma spp. ao fungo Lasiodiplodia theobromae.

Lasiodiplodia spp., são fungos de ampla distribuição geográfica, típicos de regiões tropicais e temperadas, conhecidos pelo seu polifagismo, sendo patógenos de uma extensa lista de plantas hospedeiras, a maioria frutíferas de clima tropical. Fungos deste gênero são capazes de causar diferentes sintomas nas plantas infectadas, incluindo a secadescendente (die-back), cancro e lesões em diferentes partes da planta, além de incitar a morte de mudas e enxertos.Em paralelo aconteceram também os seguintes eventos: V Seminário de Pesquisa do Recôncavo da Bahia; V Seminário Estudantil de Pesquisa da UFRB; V Seminário da Pós-Graduação da UFRB; II Seminário Regional de Pesquisa da EBDA; 5ª Jornada Científica da Embrapa Mandioca e Fruticultura; VIII Seminário Estudantil de Pesquisa e Extensão da FAMAM; Semana de Ciência, Tecnologia e Inovação no Agronegócio; Fórum de Gestores de Iniciação Científica e Tecnológica da Bahia; II Simpósio Baiano de Defesa Agropecuária; I Semana de Educação Tutorial da UFRB

Patógenos associados a doenças de plantas ornamentais na região metropolitana de Salvador, Bahia.

O mercado de flores e plantas ornamentais está se expandido na Bahia, sendo grande a diversidade de espécies cultivadas. Pode-se considerar que, atualmente, a produção de flores e plantas ornamentais é uma importante alternativa de trabalho e renda para as mais diversas classes da população baiana.PDF. 157_11

Clínica fitopatológica da EBDA ? identificação de agentes etiológicos de doenças em diversos hospedeiros.

As plantas cultivadas são acometidas por diversas doenças principalmente em agroecossistemas simplificados, podendo causar grandes perdas na produção e prejuízos aos agricultores. Neste sentido, é recomendada a resolução dos problemas relacionados à ocorrência de doenças de plantas, com base, frequentemente, na conservação dos recursos naturais e no aumento da diversidade biológica, de modo a atender a demanda crescente de práticas mais sustentáveis para a agricultura, sem riscos à saúde humana.PDF. 156_11

Patógenos associados a doenças de plantas ornamentais na região metropolitana de Salvador.

O mercado de flores e plantas ornamentais está se expandido na Bahia, sendo grande a diversidade de espécies cultivadas. Pode-se considerar que, atualmente, a produção de flores e plantas ornamentais é uma importante alternativa de trabalho e renda para as mais diversas classes da população baiana.Em paralelo aconteceram também os seguintes eventos: V Seminário de Pesquisa do Recôncavo da Bahia; V Seminário Estudantil de Pesquisa da UFRB; V Seminário da Pós-Graduação da UFRB; II Seminário Regional de Pesquisa da EBDA; 5ª Jornada Científica da Embrapa Mandioca e Fruticultura; VIII Seminário Estudantil de Pesquisa e Extensão da FAMAM; Semana de Ciência, Tecnologia e Inovação no Agronegócio; Fórum de Gestores de Iniciação Científica e Tecnológica da Bahia; II Simpósio Baiano de Defesa Agropecuária; I Semana de Educação Tutorial da UFRB

Silver nanoparticles impair retinoic acid-inducible gene I mediated mitochondrial anti-viral immunity by blocking the autophagic flux in lung epithelial cells

Silver nanoparticles (AgNPs) are microbicidal agents which could be potentially used as alternative to antivirals to treat human infectious diseases, especially Influenza virus infection where antivirals have generally proven unsuccessful. However, concerns about the use of AgNPs on humans arise from their potential toxicity, although mechanisms are not well-understood. We show here, in the context of Influenza virus infection of lung epithelial cells, that AgNPs down-regulated Influenza induced-CCL-5 and -IFN-β release (two cytokines important in anti-viral immunity) through RIG-I inhibition, while enhancing IL-8 production, a cytokine important for mobilizing host antibacterial responses. AgNPs activity was independent of coating and was not observed with gold nanoparticles. Down-stream analysis indicated that AgNPs disorganized the mitochondrial network and prevented the anti-viral IRF-7 transcription factor influx into the nucleus. Importantly, we showed that the modulation of RIG-I-IRF-7 pathway was concomitant with inhibition of either classical or alternative autophagy (ATG-5- and Rab-9 dependent, respectively), depending on the epithelial cell type used. Altogether, this demonstration of a AgNPs-mediated functional dichotomy (down-regulation of IFN-dependent anti-viral responses and up-regulation of IL-8 -dependent antibacterial responses) may have practical implications for their use in the clinic

Trappin-2/Elafin Modulate Innate Immune Responses of Human Endometrial Epithelial Cells to PolyI∶C

BACKGROUND: Upon viral recognition, innate and adaptive antiviral immune responses are initiated by genital epithelial cells (ECs) to eradicate or contain viral infection. Such responses, however, are often accompanied by inflammation that contributes to acquisition and progression of sexually transmitted infections (STIs). Hence, interventions/factors enhancing antiviral protection while reducing inflammation may prove beneficial in controlling the spread of STIs. Serine antiprotease trappin-2 (Tr) and its cleaved form, elafin (E), are alarm antimicrobials secreted by multiple cells, including genital epithelia. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated whether and how each Tr and E (Tr/E) contribute to antiviral defenses against a synthetic mimic of viral dsRNA, polyinosine-polycytidylic acid (polyI:C) and vesicular stomatitis virus. We show that delivery of a replication-deficient adenovector expressing Tr gene (Ad/Tr) to human endometrial epithelial cells, HEC-1A, resulted in secretion of functional Tr, whereas both Tr/E were detected in response to polyI:C. Moreover, Tr/E were found to significantly reduce viral replication by either acting directly on virus or through enhancing polyI:C-driven antiviral protection. The latter was associated with reduced levels of pro-inflammatory factors IL-8, IL-6, TNFα, lowered expression of RIG-I, MDA5 and attenuated NF-κB activation. Interestingly, enhanced polyI:C-driven antiviral protection of HEC-Ad/Tr cells was partially mediated through IRF3 activation, but not associated with higher induction of IFNβ, suggesting multiple antiviral mechanisms of Tr/E and the involvement of alternative factors or pathways. CONCLUSIONS AND SIGNIFICANCE: This is the first evidence of both Tr/E altering viral binding/entry, innate recognition and mounting of antiviral and inflammatory responses in genital ECs that could have significant implications for homeostasis of the female genital tract

Diagnostic importance of pulmonary interleukin-1beta and interleukin-8 in ventilator-associated pneumonia.

BACKGROUND: Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP. METHODS: A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age- and sex-matched volunteers. Growth of pathogens at >10(4) colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from "non-VAP". Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves. RESULTS: Seventy-two patients had recoverable lavage-24% had VAP. BALF interleukin-1beta (IL-1beta), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1alpha were significantly higher in the VAP group (all p<0.005). Using a cut-off of 10 pg/ml, BALF IL-1beta generated negative likelihood ratios for VAP of 0.09. In patients with BALF IL-1beta <10 pg/ml the post-test probability of VAP was 2.8%. Using a cut-off value for IL-8 of 2 ng/ml, the positive likelihood ratio was 5.03. There was no difference in cytokine levels between patients with sterile BALF and those with growth of <10(4) cfu/ml. CONCLUSIONS: BALF IL-1beta and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations

Neutrophil elastase reduces secretion of secretory leukoproteinase inhibitor (SLPI) by lung epithelial cells: role of charge of the proteinase-inhibitor complex

<p>Abstract</p> <p>Background</p> <p>Secretory leukoproteinase inhibitor (SLPI) is an important inhibitor of neutrophil elastase (NE), a proteinase implicated in the pathogenesis of lung diseases such as COPD. SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes <it>in vitro</it>.</p> <p>Methods</p> <p>Respiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling.</p> <p>Results</p> <p>We observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration.</p> <p>Conclusion</p> <p>These data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon <it>in vitro</it>, which may correlate with changes observed <it>in vivo</it>.</p