Human bocavirus infection diagnosed serologically among children admitted to hospital with community-acquired pneumonia in a tropical region

Human bocavirus (HBoV) is a human virus associated with respiratory disease in children. Limited information is available on acute infection with HBoV among children admitted to hospital with community-acquired pneumonia in tropical regions and the current diagnosis is inadequate. The aims were to diagnose and describe acute HBoV infections among children hospitalized for community-acquired pneumonia. In Salvador, Brazil, 277 children with community-acquired pneumonia were prospectively enrolled. Paired serum samples were tested by IgG, IgM, and IgG-avidity enzyme immunoassays (EIAs) using recombinant HBoV VP2. HBoV DNA was detected in nasopharyngeal aspirates and serum by a quantitative polymerase-chain reaction (PCR). HBoV DNA was detected in nasopharyngeal aspirates of 62/268 (23%) children and 156/273 (57%) were seropositive. Acute primary HBoV infection was reliably diagnosed (bearing at least two acute markers: Positive IgM, a fourfold increase/conversion of IgG, low IgG avidity or viremia) in 21 (8%) of 273 patients, 90% of 20 had HBoV DNA in nasopharyngeal aspirates, 83% with a high DNA load. The median age of infection with HBoV was 16 months, range 5-36.Community-acquired pneumonia was confirmed radiographically in 85% of 20 patients with acute HBoV infection diagnosed serologically. HBoV DNA was found in nasopharyngeal aspirates of 42/246(17%) children without an acute primary HBoV infection and available nasopharyngeal aspirate. Four children with HBoV secondary immune responses were detected, lacking both IgM and viremia. HBoV infection was diagnosed accurately in children aged 5-36 months with community-acquired pneumonia confirmed radiographically. PCR of nasopharyngeal aspirates is not a reliable marker of acute HBoV infection. J. Med. Virol. 84:253-258, 2012. (C) 2011 Wiley Periodicals, Inc.

Thermal effects and morphological aspects of human dentin surface irradiated with different frequencies of Er:YAG laser

This study reports the effects on micromorphology and temperature rise in human dentin using different frequencies of Er:YAG laser. Sixty human dentin fragments were randomly assigned into two groups (n = 30): carious or sound dentin. Both groups were divided into three subgroups (n = 10), according to the Er:YAG laser frequency used: 4, 6, or 10 Hz (energy: 200 mJ; irradiation distance: 12 mm; and irradiation time: 20 s). A thermocouple adapted to the tooth fragment recorded the initial temperature value (degrees C); then, the temperature was measured after the end of the irradiation (20 s). Morphological analysis was performed using images obtained with scanning electron microscope. There was no difference between the temperatures obtained with 4 and 6 Hz; the highest temperatures were achieved with 10 Hz. No difference was observed between carious and sound dentin. Morphological analyses revealed that all frequencies promoted irregular surface in sound dentin, being observed more selectively ablation especially in intertubular dentin with tubule protrusion. The caries dentin presented flat surface for all frequencies used. Both substrates revealed absence of any signs of thermal damage. It may be concluded that the parameters used in this study are capable to remove caries lesion, having acceptable limits of temperature rise and no significant morphological alterations on dentin surface. Microsc. Res. Tech. 2012. (c) 2012 Wiley Periodicals, Inc.

Colistin Resistance in Escherichia coli and Salmonella enterica Strains Isolated from Swine in Brazil

Reports about acquired resistance to colistin in different bacteria species are increasing, including E. coli of animal origin, but reports of resistance in wild S. enterica of different serotypes from swine are not found in the literature. Results obtained with one hundred and twenty-six E. coli strains from diseased swine and one hundred and twenty-four S. enterica strains from diseased and carrier swine showed a frequency of 6.3% and 21% of colistin-resistant strains, respectively. When comparing the disk diffusion test with the agar dilution test to evaluate the strains, it was confirmed that the disk diffusion test is not recommended to evaluate colistin resistance as described previously. The colistin MIC 90 and MIC 50 values obtained to E. coli were 0.25 mu g/mL and 0.5 mu g/mL, the MIC 90 and MIC 50 to S. enterica were 1 mu g/mL and 8 mu g/mL. Considering the importance of colistin in control of nosocomial human infections with Gram-negative multiresistant bacteria, and the large use of this drug in animal production, the colistin resistance prevalence in enterobacteriaceae of animal origin must be monitored more closely.

A quantitative proteomic and transcriptomic comparison of human mesenchymal stem cells from bone marrow and umbilical cord vein

Human mesenchymal stem cells (hMSCs) are adult multipotent cells that have high therapeutic potential due to their immunological properties. They can be isolated from several different tissues with bone marrow (BM) being the most common source. Because the isolation procedure is invasive, other tissues such as human umbilical cord vein (UCV) have been considered. However, their interchangeability remains unclear. In the present study, total protein extracts of BM-hMSCs and UCV-hMSCs were quantitatively compared using gel-LC-MS/MS. Previous SAGE analysis of the same cells was re-annotated to enable comparison and combination of these two data sets. We observed a more than 63% correlation between proteomic and transcriptomic data. In silico analysis of highly expressed genes in cells of both origins suggests that they can be modulated by microRNA, which can change protein abundance. Our results showed that MSCs from both tissues shared high similarity in metabolic and functional processes relevant to their therapeutic potential, especially in the immune system process, response to stimuli, and processes related to the delivery of the hMSCs to a given tissue, such as migration and adhesion. Hence, our results support the idea that the more accessible UCV could be a potentially less invasive source of MSCs.

Geochemical evidence of the 8.2 ka event and other Holocene environmental changes recorded in paleolagoon sediments, southeastern Brazil

The paleoclimatic record of Jureia Paleolagoon, coastal southeastem Brazil, includes cyclic and gradual changes with different intensities and frequencies through geological time, and it is controlled by astronomical, geophysical, and geological phenomena. These variations are not due to one single cause, but they result from the interaction of several factors, which act at different temporal and spatial scales. Here, we describe paleoenvironmental evidence regarding climatic and sea level changes from the last 9400 cal yr BP at the Jureia Paleolagoon - one of the main groups of protected South Atlantic ecosystems. Geochemical evidences were used to identify anomalies from multi-proxy analyses of a paleolagoon sediment core. The anomalies of centennial scale were correlated to climate and transgression-regression cycles from the Holocene period. Decadal scale anomalous oscillations in the Quaternary paleolagoon sediments occur between 9400 and 7500 cal yr BP, correlated with long- and short-term natural events, which generated high sedimentation rates, mainly between 8385 and 8375 cal yr BP (10 cm/yr). Our results suggest that a modem-day short-duration North Atlantic climatic event, such as the 82 ka event, could affect the environmental equilibrium in South America and intensify the South American Summer Monsoon. (C) 2011 University of Washington. Published by Elsevier Inc. All rights reserved.

Tungiasis in a free-ranging jaguar (Panthera onca) population in Brazil

Tungiasis is an ectoparasitic disease caused by fleas of the genus Tunga. The disease is reported to occur mostly in human populations. In wildlife, however, the occurrence and impact of this disease remains uncertain. We captured and examined 12 free-ranging jaguars for the presence of Tunga penetrans in the Pantanal region of Mato Grosso do Sul state, Brazil. Tungiasis prevalence was 100% in the population; lesions were confined to the jaguar's paws. T. penetrans was identified based on the characteristics of the embedded fleas and the morphological identification of a collected free-living flea. The intensity and stage of infestation varied between individual animals. However, in general, all captured jaguars were in good health. The 100% prevalence of tungiasis may be related to the fact that all captures were performed during the dry season. Their high ecological requirements for space make jaguars potential disseminators of T. penetrans in the Pantanal region. Because cattle ranching and ecotourism are the main economic activities in the Pantanal, further studies should evaluate the risks of tungiasis to human and animal health. To the best of our knowledge, this is the first report of tungiasis in jaguars.

Expression of BAG-1 and PARP-1 in Precursor Lesions and Invasive Cervical Cancer Associated with Human Papillomavirus (HPV)

Cervical cancer remains persistently the second most common malignancies among women worldwide, responsible for 500,000 new cases annually. Only in Brazil, the estimate is for 18,430 new cases in 2011. Several types of molecular markers have been studied in carcinogenesis including proteins associated with apoptosis such as BAG-1 and PARP-1. This study aims to demonstrate the expression of BAG-1 and PARP-1 in patients with low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs) and invasive squamous cell carcinomas (SCCs) of the uterine cervix and to verify a possible association with HPV infection. Fifty samples of LSILs, 50 samples of HSILs and 50 samples of invasive SCCs of the uterine cervix were analyzed by immunohistochemistry for BAG-1 and PARP-1 expression. PCR was performed to detect and type HPV DNA. BAG-1 expression levels were significantly different between LSILs and HSILs (p = 0,014) and between LSILs and SCCs (p = 0,014). In regards to PARP-1 expression, we found significant differences between the expression levels in HSILs and SCCs (p = 0,022). No association was found between BAG-1 expression and the presence of HPV. However, a significant association was found between PARP-1 expression and HPV positivity in the HSILs group (p = 0,021). In conclusion our research suggests that BAG-1 expression could contribute to the differentiation between LSIL and HSIL/SCC whereas PARP-1 could be useful to the differentiation between HSIL HPV-related and SCC. Further studies are needed to clarify the molecular aspects of the relationship between PARP-1 expression and HPV infection, with potential applications for cervical cancer prediction.

Judiciary as the last resort to protect children and adolescents: intersectoral actions, investment in human resources, and structuring of services

This study aimed to identify the work developed by the Judiciary to prevent sexual violence against children and adolescents within the family. The approach to social representations in a cultural perspective was used. The field study consisted in the 1st and 2nd Court of Crimes against Children and Adolescents, at the State Supreme Court of Pernambuco, Brazil. Participant observation, semi-structured interviews, and focus group with 17 subjects were the techniques for data collection, analyzed through the interpretation of meanings, allowing the identification of the category "The Judiciary as the ultimate level" and the following subcategories: "The public policies to prevent violence" and "The structure and dynamics of Courts". This study allows the visualization of the Judiciary's limitations with regard to the full protection and absolute priority, and that the work along with the victims demands investments in structure and human resources.

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr(-)) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (similar to 64 and 59 kDa) and secreted (63-69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditionalmethods of screening high-producing recombinant cellsmay represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

RNA interference-mediated knockdown of CD49e (α5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Abstract Background The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin α5β1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Interleukin-10 production by tumor infiltrating macrophages plays a role in Human Papillomavirus 16 tumor growth

Abstract Background Human Papillomavirus, HPV, is the main etiological factor for cervical cancer. Different studies show that in women infected with HPV there is a positive correlation between lesion grade and number of infiltrating macrophages, as well as with IL-10 higher expression. Using a HPV16 associated tumor model in mice, TC-1, our laboratory has demonstrated that tumor infiltrating macrophages are M2-like, induce T cell regulatory phenotype and play an important role in tumor growth. M2 macrophages secrete several cytokines, among them IL-10, which has been shown to play a role in T cell suppression by tumor macrophages in other tumor models. In this work, we sought to establish if IL-10 is part of the mechanism by which HPV tumor associated macrophages induce T cell regulatory phenotype, inhibiting anti-tumor activity and facilitating tumor growth. Results TC-1 tumor cells do not express or respond to IL-10, but recruit leukocytes which, within the tumor environment, produce this cytokine. Using IL-10 deficient mice or blocking IL-10 signaling with neutralizing antibodies, we observed a significant reduction in tumor growth, an increase in tumor infiltration by HPV16 E7 specific CD8 lymphocytes, including a population positive for Granzyme B and Perforin expression, and a decrease in the percentage of HPV specific regulatory T cells in the lymph nodes. Conclusions Our data shows that in the HPV16 TC-1 tumor mouse model, IL-10 produced by tumor macrophages induce regulatory phenotype on T cells, an immune escape mechanism that facilitates tumor growth. Our results point to a possible mechanism behind the epidemiologic data that correlates higher IL-10 expression with risk of cervical cancer development in HPV infected women.

Assessment of the role of transcript for GATA-4 as a marker of unfavorable outcome in human adrenocortical neoplasms

Abstract Background Malignant neoplasia of the adrenal cortex is usually associated with very poor prognosis. When adrenocortical neoplasms are diagnosed in the early stages, distinction between carcinoma and adenoma can be very difficult to accomplish, since there is yet no reliable marker to predict tumor recurrence or dissemination. GATA transcription factors play an essential role in the developmental control of cell fate, cell proliferation and differentiation, organ morphogenesis, and tissue-specific gene expression. Normal mouse adrenal cortex expresses GATA-6 while its malignant counterpart only expresses GATA-4. The goal of the present study was to assess whether this reciprocal change in the expression of GATA factors might be relevant for predicting the prognosis of human adrenocortical neoplasms. Since human adrenal cortices express luteinizing hormone (LH/hCG) receptor and the gonadotropins are known to up-regulate GATA-4 in gonadal tumor cell lines, we also studied the expression of LH/hCG receptor. Methods We conducted a study on 13 non-metastasizing (NM) and 10 metastasizing/recurrent (MR) tumors obtained from a group of twenty-two adult and pediatric patients. The expression of GATA-4, GATA-6, and LH/hCG receptor (LHR) in normal and tumoral human adrenal cortices was analysed using reverse transcriptase-polymerase chain reaction (RT-PCR) complemented by dot blot hybridization. Results Messenger RNA for GATA-6 was detected in normal adrenal tissue, as well as in the totality of NM and MR tumors. GATA-4, by its turn, was detected in normal adrenal tissue, in 11 out of 13 NM tumors, and in 9 of the 10 MR tumors, with larger amounts of mRNA found among those presenting aggressive clinical behavior. Transcripts for LH receptor were observed both in normal tissue and neoplasms. A more intense LHR transcript accumulation was observed on those tumors with better clinical outcome. Conclusion Our data suggest that the expression of GATA-6 in human adrenal cortex is not affected by tumorigenesis. GATA-4 expression is more abundant in MR tumors, while NM tumors express more intensely LHR. Further studies with larger cohorts are needed to test whether relative expression levels of LHR or GATA-4 might be used as prognosis predictors.

Awareness and knowledge of HPV, cervical cancer, and vaccines in young women after first delivery in São Paulo, Brazil - a cross-sectional study

Abstract Background The success of HPV vaccination programs will require awareness regarding HPV associated diseases and the benefits of HPV vaccination for the general population. The aim of this study was to assess the level of awareness and knowledge of human papillomavirus (HPV) infection, cervical cancer prevention, vaccines, and factors associated with HPV awareness among young women after birth of the first child. Methods This analysis is part of a cross-sectional study carried out at Hospital Maternidade Leonor Mendes de Barros, a large public maternity hospital in Sao Paulo. Primiparous women (15-24 years) who gave birth in that maternity hospital were included. A questionnaire that included questions concerning knowledge of HPV, cervical cancer, and vaccines was applied. To estimate the association of HPV awareness with selected factors, prevalence ratios (PR) were estimated using a generalized linear model (GLM). Results Three hundred and one primiparous women were included; 37% of them reported that they "had ever heard about HPV", but only 19% and 7%, respectively, knew that HPV is a sexually transmitted infection (STI) and that it can cause cervical cancer. Seventy-four percent of interviewees mentioned the preventive character of vaccines and all participants affirmed that they would accept HPV vaccination after delivery. In the multivariate analysis, only increasing age (P for trend = 0.021) and previous STI (P < 0.001) were factors independently associated with HPV awareness ("had ever heard about HPV"). Conclusions This survey indicated that knowledge about the association between HPV and cervical cancer among primiparous young women is low. Therefore, these young low-income primiparous women could benefit greatly from educational interventions to encourage primary and secondary cervical cancer prevention programs.

Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients

Abstract Background Plasmodium vivax is the most widely distributed human malaria, responsible for 70–80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected. Methods A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10-30 was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology Results A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them. Conclusion These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.

Epidemiologic aspects of the malaria transmission cycle in an area of very low incidence in Brazil

Abstract Background Extra-Amazonian autochthonous Plasmodium vivax infections have been reported in mountainous regions surrounded by the Atlantic Forest in Espírito Santo state, Brazil. Methods Sixty-five patients and 1,777 residents were surveyed between April 2001 and March 2004. Laboratory methods included thin and thick smears, multiplex-PCR, immunofluorescent assay (IFA) against P. vivax and Plasmodium malariae crude blood-stage antigens and enzyme-linked immunosorbent assay (ELISA) for antibodies against the P. vivax-complex (P. vivax and variants) and P. malariae/Plasmodium brasilianum circumsporozoite-protein (CSP) antigens. Results Average patient age was 35.1 years. Most (78.5%) were males; 64.6% lived in rural areas; 35.4% were farmers; and 12.3% students. There was no relevant history of travel. Ninety-five per cent of the patients were experiencing their first episode of malaria. Laboratory data from 51 patients were consistent with P. vivax infection, which was determined by thin smear. Of these samples, 48 were assayed by multiplex-PCR. Forty-five were positive for P. vivax, confirming the parasitological results, while P. malariae was detected in one sample and two gave negative results. Fifty percent of the 50 patients tested had IgG antibodies against the P. vivax-complex or P. malariae CSP as determined by ELISA. The percentages of residents with IgM and IgG antibodies detected by IFA for P. malariae, P. vivax and Plasmodium falciparum who did not complain of malaria symptoms at the time blood was collected were 30.1% and 56.5%, 6.2% and 37.7%, and 13.5% and 13%, respectively. The same sera that reacted to P. vivax also reacted to P. malariae. The following numbers of samples were positive in multiplex-PCR: 23 for P. vivax; 15 for P. malariae; 9 for P. falciparum and only one for P. falciparum and P. malariae. All thin and thick smears were negative. ELISA against CSP antigens was positive in 25.4%, 6.3%, 10.7% and 15.1% of the samples tested for "classical" P. vivax (VK210), VK247, P. vivax-like and P. malariae, respectively. Anopheline captures in the transmission area revealed only zoophilic and exophilic species. Conclusion The low incidence of malaria cases, the finding of asymptomatic inhabitants and the geographic separation of patients allied to serological and molecular results raise the possibility of the existence of a simian reservoir in these areas.