Scielo RSS <![CDATA[Memórias do Instituto Oswaldo Cruz]]> vol. 113 num. 6 lang. en <![CDATA[SciELO Logo]]> <![CDATA[Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of <em>Streptococcus agalactiae</em> in human endothelial cells]]> BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis. <![CDATA[Single nucleotide polymorphisms of cytokine-related genes and association with clinical outcome in a Chagas disease case-control study from Brazil]]> BACKGROUND The severity of chronic chagasic cardiomyopathy (CCC), the most frequent clinical outcome of Chagas disease (CD), has been associated with cytokine-enriched heart tissue inflammation, and high serum levels of transforming growth factor (TGFβ), interferon-gamma (IFNγ), and tumour necrosis factor (TNF). Conversely, increased interleukin (IL)-10 serum concentrations have been associated with asymptomatic CD. Cytokines and cytokine-related gene polymorphisms may control cytokine expression and have been proposed to contribute to CCC outcomes. OBJECTIVES We evaluated the association of 13 cytokine-related genes (TGFB: rs8179181, rs8105161, rs1800469; IL10: rs1800890, rs1800871, rs1800896; IFNG: rs2430561; TNF: rs1800629; BAT1: rs3853601; LTA: rs909253, rs2239704; TNFR1: rs767455; TNFR2: rs1061624) with risk and progression of CCC. FINDINGS Four hundred and six seropositive patients from CD endemic areas in the state of Pernambuco, north-eastern Brazil, were classified as non-cardiopathic (A, 110) or cardiopathic (mild, B1, 163; severe, C, 133). We found no evidence of TGFB, IL10, TNF, or TNFR1/2 gene polymorphisms associated with CCC risk or progression. Only BAT1 rs3853601 −22G carriers (B1 vs. C: OR = 0.5; p-value = 0.03) and IFNG rs2430561 +874AT (A vs. C: OR = 0.7; p-value = 0.03; A vs. B1+C: OR = 0.8; p-value = 0.02) showed a significant association with protection from cardiopathy in a logistic regression analysis with adjustment for gender and ethnicity; however, the association disappeared after performing adjustment for multiple testing. A systematic review of TNF rs1800629 −308G&gt;A publications included five studies for meta-analysis (534 CCC and 472 asymptomatic patients) and showed no consensus in pooled odds ratio (OR) estimates for A allele or A carriers (OR = 1.4 and 1.5; p-values = 0.14 and 0.15, respectively). In CD patients, TNF serum levels were increased, but not affected by the TNF rs1800629 −308A allele. MAIN CONCLUSIONS Our data suggest no significant contribution of the analysed gene variants of cytokine-related molecules to development/severity of Chagas' heart disease, reinforcing the idea that parasite/host interplay is critical to CD outcomes. <![CDATA[Entomological investigation of Japanese encephalitis outbreak in Malkangiri district of Odisha state, India]]> BACKGROUND A severe outbreak of Japanese encephalitis (JE) and acute encephalitis syndrome (AES) with high case fatality was reported from Malkangiri district of Odisha state, India during September to November 2016 affecting 336 children with 103 deaths. OBJECTIVES The purpose of this study was to investigate the outbreak in the light of entomological determinants. METHODS Entomological investigation was carried out in 48 villages from four mostly affected Community Health Centres (CHCs) of Malkangiri district. Dusk collections of resting adults was done in villages from indoor and outdoor sites to record the density of mosquito species, including the known JE vectors, feeding behaviour, parity, dusk index and infection status with JE virus (JEV). FINDINGS The per man hour density and dusk index of JE vector species varied from 2.5 to 24.0 and 0.81 to 7.62, respectively in study villages. A total of 1136 mosquitoes belonging to six vector species were subjected to PCR and one pool of Culex vishnui was found to be positive for JEV. CONCLUSION The JE transmission in Malkangiri district was confirmed. Thorough screening of human blood samples of JE/AES suspected cases and JE vector mosquitoes for the presence of JEV during rainy season every year is recommended. <![CDATA[Analysis of the immunological biomarker profile during acute Zika virus infection reveals the overexpression of CXCL10, a chemokine linked to neuronal damage]]> BACKGROUND Infection with Zika virus (ZIKV) manifests in a broad spectrum of disease ranging from mild illness to severe neurological complications and little is known about Zika immunopathogenesis. OBJECTIVES To define the immunologic biomarkers that correlate with acute ZIKV infection. METHODS We characterized the levels of circulating cytokines, chemokines, and growth factors in 54 infected patients of both genders at five different time points after symptom onset using microbeads multiplex immunoassay; comparison to 100 age-matched controls was performed for statistical analysis and data mining. FINDINGS ZIKV-infected patients present a striking systemic inflammatory response with high levels of pro-inflammatory mediators. Despite the strong inflammatory pattern, IL-1Ra and IL-4 are also induced during the acute infection. Interestingly, the inflammatory cytokines IL-1β, IL-13, IL-17, TNF-α, and IFN-γ; chemokines CXCL8, CCL2, CCL5; and the growth factor G-CSF, displayed a bimodal distribution accompanying viremia. While this is the first manuscript to document bimodal distributions of viremia in ZIKV infection, this has been documented in other viral infections, with a primary viremia peak during mild systemic disease and a secondary peak associated with distribution of the virus to organs and tissues. MAIN CONCLUSIONS Biomarker network analysis demonstrated distinct dynamics in concurrence with the bimodal viremia profiles at different time points during ZIKV infection. Such a robust cytokine and chemokine response has been associated with blood-brain barrier permeability and neuroinvasiveness in other flaviviral infections. High-dimensional data analysis further identified CXCL10, a chemokine involved in foetal neuron apoptosis and Guillain-Barré syndrome, as the most promising biomarker of acute ZIKV infection for potential clinical application. <![CDATA[<em>Trypanosoma cruzi</em> XRNA granules colocalise with distinct mRNP granules at the nuclear periphery]]> BACKGROUND Eukaryotic ribonucleoprotein (RNP) granules are important for the regulation of RNA fate. RNP granules exist in trypanosomatids; however, their roles in controlling gene expression are still not understood. XRNA is a component of granules in Trypanosoma brucei but has not been investigated in Trypanosoma cruzi. OBJECTIVES This study aimed to investigate the TcXRNA dynamic assembly and its interaction with RNP components under conditions that affect the mRNA availability. METHODS We used in vitro metacyclogenesis of T. cruzi to observe changes in RNP granules during the differentiation process. TcXRNA expression was analysed by Western blot and immunofluorescence. Colocalisation assays were performed to investigate the interaction of TcXRNA with other RNP components. FINDINGS TcXRNA is constantly present during metacyclogenesis and is localised in cytoplasmic granules. TcXRNA does not colocalise with TcDHH1 and TcCAF1 granules in the cytoplasm. However, TcXRNA granules colocalise with mRNP granules at the nuclear periphery when mRNA processing is inhibited. MAIN CONCLUSIONS TcXRNA plays a role in mRNA metabolism as a component of mRNP granules whose assembly is dependent on mRNA availability. TcXRNA granules colocalise with distinct RNP granules at the nuclear periphery, suggesting that the perinuclear region is a regulatory compartment in T. cruzi mRNA metabolism. <![CDATA[<em>Scedosporium apiospermum, Scedosporium aurantiacum, Scedosporium minutisporum</em> and <em>Lomentospora prolificans</em>: a comparative study of surface molecules produced by conidial and germinated conidial cells]]> BACKGROUND Scedosporium/Lomentospora species are opportunistic mould pathogens, presenting notable antifungal resistance. OBJECTIVES/METHODS We analysed the conidia and germinated conidia of S. apiospermum (Sap), S. aurantiacum (Sau), S. minutisporum (Smi) and L. prolificans (Lpr) by scanning electron microscopy and exposition of surface molecules by fluorescence microscopy. FINDINGS Conidia of Sap, Smi and Sau had oval, ellipsoidal and cylindrical shape, respectively, with several irregularities surrounding all surface areas, whereas Lpr conidia were rounded with a smooth surface. The germination of Sap occurred at the conidial bottom, while Smi and Sau germination primarily occurred at the centre of the conidial cell, and Lpr germination initiated at any part of the conidial surface. The staining of N-acetylglucosamine-containing molecules by fluorescein-labelled WGA primarily occurred during the germination of all studied fungi and in the conidial scars, which is the primary location of germination. Calcofluor white, which recognises the polysaccharide chitin, strongly stained the conidial cells and, to a lesser extent, the germination. Both mannose-rich glycoconjugates (evidenced by fluoresceinated-ConA) and cell wall externally located polypeptides presented distinct surface locations and expression according to both morphotypes and fungal species. In contrast, sialic acid and galactose-containing structures were not detected at fungal surfaces. MAIN CONCLUSIONS The present study demonstrated the differential production/exposition of surface molecules on distinct morphotypes of Scedosporium/Lomentospora species. <![CDATA[Effect of vegetation on cutaneous leishmaniasis in Paraná, Brazil]]> BACKGROUND Cutaneous leishmaniasis (CL) is endemic in the state of Paraná, Brazil. OBJECTIVE This study aimed at analysing the influence of the remaining native vegetation on the prevalence of CL in Paraná. METHODS Global testing was used for spatial autocorrelation along with simultaneous autoregressive model (SAR). The regression was based on the CL coefficient (cases/100,000 inhabitants) as a function of the percentage of natural vegetation cover, altitude, total number of cases, and spatial density (SD) per km2; the location data of the Paraná state municipalities and the detection coefficient (DC) (cases/100,000 inhabitants) of autochthonous cases of CL were obtained from the SINAN in 2012 and 2016. Data on the remaining forests were collected from the Fundação SOS Mata Atlântica and Instituto Nacional de Pesquisas Espaciais. FINDINGS The spatial regression of DC revealed statistical significance for SD (Z = 24.1359, p &lt; 0.05, 2012-2013; Z = 24.0817, p &lt; 0.05, 2013-2014; Z = 33.4824, p &lt; 0.05, 2014-2015; and Z = 27.1515, p &lt; 0.05, 2015-2016. CONCLUSIONS CL cases are reported in areas with native vegetation, such as in riparian forests. However, vegetation is not the only variable that influences the incidence of CL. <![CDATA[Detection of <em>bla</em><sub>NDM-1</sub> in <em>Stenotrophomonas maltophilia</em> isolated from Brazilian soil]]> This study reports the presence of the blaNDM-1 gene in an isolate of Stenotrophomonas maltophilia obtained from a Brazilian soil, inside an IncA/C plasmid with ~ 45 Kb. To the best of our knowledge, this is the second report in the world and the first in Brazil of NDM-producing bacterium isolated from soil. <![CDATA[An <em>in silico</em> pipeline to filter the <em>Toxoplasma gondii</em> proteome for proteins that could traffic to the host cell nucleus and influence host cell epigenetic regulation]]> Toxoplasma gondii uses epigenetic mechanisms to regulate both endogenous and host cell gene expression. To identify genes with putative epigenetic functions, we developed an in silico pipeline to interrogate the T. gondii proteome of 8313 proteins. Step 1 employs PredictNLS and NucPred to identify genes predicted to target eukaryotic nuclei. Step 2 uses GOLink to identify proteins of epigenetic function based on Gene Ontology terms. This resulted in 611 putative nuclear localised proteins with predicted epigenetic functions. Step 3 filtered for secretory proteins using SignalP, SecretomeP, and experimental data. This identified 57 of the 611 putative epigenetic proteins as likely to be secreted. The pipeline is freely available online, uses open access tools and software with user-friendly Perl scripts to automate and manage the results, and is readily adaptable to undertake any such in silico search for genes contributing to particular functions. <![CDATA[Follow up of a robust meta-signature to identify Zika virus infection in <em>Aedes aegypti</em>: another brick in the wall]]> The mosquito Aedes aegypti is the main vector of several arthropod-borne diseases that have global impacts. In a previous meta-analysis, our group identified a vector gene set containing 110 genes strongly associated with infections of dengue, West Nile and yellow fever viruses. Of these 110 genes, four genes allowed a highly accurate classification of infected status. More recently, a new study of Ae. aegypti infected with Zika virus (ZIKV) was published, providing new data to investigate whether this “infection” gene set is also altered during a ZIKV infection. Our hypothesis is that the infection-associated signature may also serve as a proxy to classify the ZIKV infection in the vector. Raw data associated with the NCBI/BioProject were downloaded and re-analysed. A total of 18 paired-end replicates corresponding to three ZIKV-infected samples and three controls were included in this study. The nMDS technique with a logistic regression was used to obtain the probabilities of belonging to a given class. Thus, to compare both gene sets, we used the area under the curve and performed a comparison using the bootstrap method. Our meta-signature was able to separate the infected mosquitoes from the controls with good predictive power to classify the Zika-infected mosquitoes.