Diversity and genetic structure of mangaba remnants in states of northeastern Brazil.

In this study, we analyzed the genetic diversity and structure of remnants of mangaba populations in states of northeastern Brazil by applying 9 microsatellite markers previously developed to establish conservation strategies for germplasm and species preservation. Six to 20 individuals per population were analyzed, with a total of 94 individuals and 6 populations from the states of Ceará, Pernambuco, and Sergipe, Brazil. The intra-population positive fixation index (f) in all populations indicated inbreeding resulting from the lack of random mating. The mean genetic diversity index values GST, FST, and RST estimated for divergence among the 6 populations were 0.14 (P < 0.05), revealing moderate genetic differentiation. The smallest FST value (P ≥ 0.05) was observed between the Jacarecoara and Tapera populations (0.005) and the highest between the Barra dos Coqueiros and Jacarecoara populations (0.287). The Jacarecoara population was the most divergent among the populations analyzed. According to analysis of molecular variance results, the largest variation percentage resulted from variability within populations (83.18%). Bayesian clustering analysis showed the formation of 2 sets (K = 2). Our results are important for developing strategies for in situ conservation of the species, seed collection, and ex situ conservation. For both methods, conservation of the greatest possible genetic variability of the species is essential.

Physical exercise on the rat ventral prostate: steroid hormone receptors, apoptosis and cell proliferation.

Studies have investigated the effect of exercise on prostate cancer risk. However, there are still doubts regarding the correlation between physical activity and the steroid hormones with respect to the reduction of the risk for prostatic lesions. We evaluated the levels of corticosterone, dihydrotestosterone (DHT), testosterone, estradiol, and steroid hormone receptors, and investigated the relationship between apoptosis and cell proliferation in the rat ventral prostate after training. Two groups were included in this study: control and trained. The trained group was submitted to training for 13 weeks (1 week of adaptation). Two days after the last training session, all animals were euthanized, and the intermediate and distal regions of the ventral prostate were collected and processed for immunohistochemistry, Western blotting and hormonal analyses. Physical exercise increased the corticosterone plasma, DHT and testosterone. In addition, androgen receptor expression was lower and estrogen receptor (ER) α and ER ß expression were higher in the trained group. However, the trained group showed disruption of the ratio of apoptotic to proliferating cells, indicating a predominance of apoptosis. We conclude that physical exercise alters the sex hormones and their receptors and is associated with the disruption of the balance between apoptosis and cell proliferation in the rat ventral prostate.

A simple boiling-based DNA extraction for RAPD profiling of landfarm soil to provide representative metagenomic content.

Landfarm soils are employed in industrial and petrochemical residue bioremediation. This process induces selective pressure directed towards microorganisms capable of degrading toxic compounds. Detailed description of taxa in these environments is difficult due to a lack of knowledge of culture conditions required for unknown microorganisms. A metagenomic approach permits identification of organisms without the need for culture. However, a DNA extraction step is first required, which can bias taxonomic representativeness and interfere with cloning steps by extracting interference substances. We developed a simplified DNA extraction procedure coupled with metagenomic DNA amplification in an effort to overcome these limitations. The amplified sequences were used to generate a metagenomic data set and the taxonomic and functional representativeness were evaluated in comparison with a data set built with DNA extracted by conventional methods. The simplified and optimized method of RAPD to access metagenomic information provides better representativeness of the taxonomical and metabolic aspects of the environmental samples.

Aquatic Macrophytes Hosting Immature Mansonia (Mansonia) Blanchard, 1901 (Diptera, Culicidae) in Porto Velho, Rondonia State, Brazil.

High abundance of hematophagous mosquitoes of the genus Mansonia Blanchard, 1901 (Diptera: Culicidae) threatens human and domestic animal health and well-being. Knowledge of the biology of nuisance mosquito species is necessary to understand specific ecological and biological factors to enable rapid and effective monitoring measures for sustainable control programs. The establishment and dispersion of Mansonia species are associated with the occurrence of aquatic macrophytes species, which are indispensable for the development of larvae and pupae. To increase knowledge of the host plants for Mansonia immature stages in Porto Velho, Rondonia State, Brazil, specimens of four plant species, which occur across the tributaries of the Madeira River were sampled and inspected for the presence of egg batches, larvae, and pupae. A total of 1,386 larvae and pupae of Mansonia spp. were collected attached to the roots of Eichhornia crassipes (Mart.) Solms (Commelinales: Pontederiaceae), Pistia stratiotes L. (Alismatales: Araceae), and Limnobium laevigatum (Humb. and Bonpl. Ex Willd.) Heine (Alismatales: Hydrocharitaceae). The novel association of Mansonia species with L. laevigatum is presented. Egg batches of Mansonia spp. were found only on Salvinia molesta D.S. Mitch. (Salviniales: Salviniaceae). Possible differences in the roles played by E. crassipes and S. molesta in the reproductive cycle of Mansonia spp. in the surveyed area are discussed. All species of host plants including E. crassipes, P. stratiotes, S. molesta, and L. laevigatum should be considered when planning macrophyte management for the control of Mansonia species.

Isolation of Histoplasma capsulatum from bats in the urban area of São Paulo State, Brazil.

The presence of bats in caves, attics, ceilings, and roofs is important epidemiologically as they can increase the chance of human acquisition of pathogens, including Histoplasma capsulatum. Brazilian urban areas contain many species of bats, especially insectivorous bats, that are attracted by a wide range of readily available food and shelter. From August 2003 to December 2008, we analysed 2427 bats in the São Paulo State region. Homogenates of the livers and spleens of the bats were plated on specific medium to identify animals infected with H. capsulatum. The fungus was isolated from 87 bats (3·6%). The infected bats were identified as Molossus molossus (74), Nyctinomops macrotis (10), Tadarida brasiliensis (1), Molossus rufus (1) and Eumops glaucinus (1), all insectivorous species. The data presented are a relevant contribution to the epidemiology of H. capsulatum in densely populated urban areas such as in São Paulo State, especially since histoplasmosis is not included in the mandatory disease notification system.

Critical review of HCC imaging in the multidisciplinary setting: treatment allocation and evaluation of response.

Imaging has not only an established role in screening and diagnosis of hepatocellular carcinoma (HCC) in patients with chronic liver inflammatory diseases, but also a crucial importance for patient stratification and treatment allocation, as well as for assessing treatment response. In the setting of increasing therapeutic options for HCC, the Barcelona Clinic Liver Cancer (BCLC) system still remains the most appropriate way to select candidate cohorts for best treatments. This classification takes into account the imaging information on tumor burden and extension, liver function, and cancer-related symptoms, stratifying patients in five risk categories (Stages 0, A, B, C and D) associated with different treatment options. Still now, there are no clear roles for biomarkers use in treatment allocation. The increasing use of locoregional non-surgical therapies in the different stages is highly dependent on reliable evaluation of treatment response, in particular when they are used with curative intention or for downstaging at liver transplantation re-assessment. Moreover, objective response (OR) has emerged as an important imaging biomarker, providing information on tumor biology, which can contribute for further prognostic assessment. Current guidelines for OR assessment recommend only the measurement of viable tumor according to mRECIST criteria, with further classification into complete response, partial response, stable disease or progressive disease. Either computed tomography (CT) or magnetic resonance (MR) imaging can be used for this purpose, and the Liver Imaging Reporting and Data System (LI-RADS) committee has recently provided some guidance for reporting after locoregional therapies. Nevertheless, imaging pitfalls resulting from treatment-related changes can impact with the correct evaluation of treatment response, especially after transarterial radioembolization (TARE). Volume criteria and emerging imaging techniques might also contribute for a better refinement in the assessment of treatment response and monitoring. As the role of imaging deeply expands in the multidisciplinary assessment of HCC, our main objective in this review is to discuss state-of-the-art decision-making aspects for treatment allocation and provide guidance for treatment response evaluation.

Quantifying steatosis in the liver and pancreas with MRI in patient with chronic liver disease. / Cuantificación por resonancia magnética de esteatosis hepática y pancreática en hepatopatía crónica.

AIM: To compare pancreatic and hepatic steatosis quantified by proton density fat fraction (PDFF) on magnetic resonance imaging (MRI) in patients with chronic liver disease. MATERIAL AND METHODS: This cross-sectional study included 46 adult patients who underwent liver biopsy for chronic viral hepatitis (n=19) or other chronic non-alcoholic liver diseases (NALD) (n=27). Liver biopsy was used as the gold standard for diagnosing and grading hepatic steatosis. All patients underwent clinical evaluation and MRI with a multi-echo chemical shift-encoded (MECSE) gradient-echo sequence for liver and pancreas PDFF quantification. We used Spearman's correlation coefficient to determine the degree of association between hepatic PDFF and steatosis grade, and between pancreatic PDFF and steatosis grade and hepatic PDFF. To compare the chronic viral hepatitis group and the NALD group, we used t-tests for continuous or ordinal variables and chi-square tests for categorical variables. RESULTS: Hepatic PDFF measurements correlated with steatosis grades (RS=0.875, p<0.001). Pancreatic PDFF correlated with hepatic steatosis grades (RS=0.573, p<0.001) and hepatic PDFF measurements (RS=0.536, p<0.001). In the subgroup of patients with chronic NALD, the correlations remained significant between pancreatic PDFF and hepatic PDFF (RS=0.632, p<0.001) and between pancreatic PDFF and liver steatosis (RS=0.608, p<0.001); however, in the subgroup of patients with viral hepatitis these correlations were no longer significant. CONCLUSION: Pancreatic fat deposition correlates with hepatic steatosis in patients with chronic NALD, but not in those with chronic viral hepatitis.

Expression, mutation and copy number analysis of platelet-derived growth factor receptor A (PDGFRA) and its ligand PDGFA in gliomas.

BACKGROUND: Malignant gliomas are the most prevalent type of primary brain tumours but the therapeutic armamentarium for these tumours is limited. Platelet-derived growth factor (PDGF) signalling has been shown to be a key regulator of glioma development. Clinical trials evaluating the efficacy of anti-PDGFRA therapies on gliomas are ongoing. In this study, we intended to analyse the expression of PDGFA and its receptor PDGFRA, as well as the underlying genetic (mutations and amplification) mechanisms driving their expression in a large series of human gliomas. METHODS: PDGFA and PDGFRA expression was evaluated by immunohistochemistry in a series of 160 gliomas of distinct World Health Organization (WHO) malignancy grade. PDGFRA-activating gene mutations (exons 12, 18 and 23) were assessed in a subset of 86 cases by PCR-single-strand conformational polymorphism (PCR-SSCP), followed by direct sequencing. PDGFRA gene amplification analysis was performed in 57 cases by quantitative real-time PCR (QPCR) and further validated in a subset of cases by chromogenic in situ hybridisation (CISH) and microarray-based comparative genomic hybridisation (aCGH). RESULTS: PDGFA and PDGFRA expression was found in 81.2% (130 out of 160) and 29.6% (48 out of 160) of gliomas, respectively. Its expression was significantly correlated with histological type of the tumours; however, no significant association between the expression of the ligand and its receptor was observed. The absence of PDGFA expression was significantly associated with the age of patients and with poor prognosis. Although PDGFRA gene-activating mutations were not found, PDGFRA gene amplification was observed in 21.1% (12 out of 57) of gliomas. No association was found between the presence of PDGFRA gene amplification and expression, excepting for grade II diffuse astrocytomas. CONCLUSION: The concurrent expression of PDGFA and PDGFRA in different subtypes of gliomas, reinforce the recognised significance of this signalling pathway in gliomas. PDGFRA gene amplification rather than gene mutation may be the underlying genetic mechanism driving PDGFRA overexpression in a portion of gliomas. Taken together, our results could provide in the future a molecular basis for PDGFRA-targeted therapies in gliomas.

Dengue infection in mice inoculated by the intracerebral route: neuropathological effects and identification of target cells for virus replication.

Dengue is an important arboviral infection, causing a broad range symptom that varies from life-threatening mild illness to severe clinical manifestations. Recent studies reported the impairment of the central nervous system (CNS) after dengue infection, a characteristic previously considered as atypical and underreported. However, little is known about the neuropathology associated to dengue. Since animal models are important tools for helping to understand the dengue pathogenesis, including neurological damages, the aim of this work was to investigate the effects of intracerebral inoculation of a neuroadapted dengue serotype 2 virus (DENV2) in immunocompetent BALB/c mice, mimicking some aspects of the viral encephalitis. Mice presented neurological morbidity after the 7th day post infection. At the same time, histopathological analysis revealed that DENV2 led to damages in the CNS, such as hemorrhage, reactive gliosis, hyperplastic and hypertrophied microglia, astrocyte proliferation, Purkinje neurons retraction and cellular infiltration around vessels in the pia mater and in neuropil. Viral tropism and replication were detected in resident cells of the brain and cerebellum, such as neurons, astrocyte, microglia and oligodendrocytes. Results suggest that this classical mice model might be useful for analyzing the neurotropic effect of DENV with similarities to what occurs in human.

Postdural puncture headache is a risk factor for new postdural puncture headache.

The aim of this study was to determine if patients with a previous history of postdural puncture headache (PDPH) might be prone to a new episode after spinal anaesthesia. Consecutive patients (n = 258) who had had surgery under spinal anaesthesia were studied. Of 42 patients with a previous history of PDPH, eight (19.0%) developed a new PDPH episode; whereas out of 216 without a previous history of PDPH, only 15 (6.9%) presented with PDPH. Previous PDPH history indicates a higher chance of a new episode of PDPH after spinal anaesthesia. Women are more susceptible to such recurrences.

An improved extraction protocol for metagenomic DNA from a soil of the Brazilian Atlantic Rainforest.

The need for the prospecting for and identification of new biomolecules is a reality. Molecular techniques allow access to the metabolic potential of microorganisms via the isolation of DNA from environmental samples, i.e., without the application of microbial culture techniques. With its great biological diversity, the Atlantic Rainforest biome has a soil rich in organic matter, some components of which interfere negatively in the reactions necessary for the exploitation of its biotechnological potential. Here, we describe a protocol for the optimization of the treatment of soil samples before DNA extraction. The new methodology gives higher yield and quality of extracted DNA as compared with pre-existing techniques, facilitating the amplification and digestion of environmental DNA, and thus allows optimal exploitation of the genetic potential of the Atlantic Rainforest biome.

Immune Function in Critically Ill Dogs.

BACKGROUND: People with critical illness (CI) commonly develop various forms of immune dysfunction, however, there is limited information concerning immune dysfunction in dogs with CI. HYPOTHESIS: The immune response in CI dogs differs from that of healthy dogs. ANIMALS: Immunologic variables were compared between 14 dogs with CI, defined as APPLEfast score of >20 points, admitted to the University of Missouri Veterinary Health Center Small Animal Clinic Intensive Care Unit and healthy controls (n = 15). METHODS: Cohort study evaluating constitutive and lipopolysaccharide (LPS)-stimulated TNF-α, IL-6, and IL-10 production, phagocytosis of opsonized E. coli and respiratory burst capacity after opsonized E. coli or phorbol 12-myristate 13-acetate (PMA) stimulation, peripheral blood lymphocyte phenotype, and monocyte expressions of HLA-DR and TLR-4. RESULTS: Lipopolysaccharide-stimulated leukocyte TNF-α (median, Q1, Q3; CI, 49, 49, 120; control, 655, 446, 1174 pg/mL; P = < 0.001), IL-6 (median, Q1, Q3; CI, 49, 49, 64; control, 100, 49, 166 pg/mL; P = 0.029), and IL-10 (CI, 49, 49, 56; control, 96, 49, 203 pg/mL; P = 0.014) production and both E. coli (median, Q1, Q3; CI, 60.5, 43, 88.5; control, 86.6, 81, 89.2%; P = 0.047) and PMA (CI, 40, 11.7, 70; control, 93, 83, 97.6%; P = < 0.001)-stimulated respiratory burst capacity significantly decreased in CI dogs. Percentage of monocytes expressing TLR-4 greater in the CI dogs (median, Q1, Q3; CI, 46.9, 24.3, 64.2; control, 16.4, 9.4, 26.2%; P = 0.005). CONCLUSION: These findings suggest dogs with CI develop immune system alterations that result in reduced respiratory burst function and cytokine production despite upregulation of TLR-4.

Effect of calcitriol on in vitro whole blood cytokine production in critically ill dogs.

Hypovitaminosis D has been identified as a predictor of mortality in human beings, dogs, cats and foals. However, the immunomodulatory effects of vitamin D in critically ill dogs has not been evaluated. The aim of this study was to evaluate the effect of calcitriol on cytokine production from whole blood collected from critically ill dogs in vitro. Twelve critically ill dogs admitted to a veterinary intensive care unit (ICU) were enrolled in a prospective cohort study. Whole blood from these dogs was incubated with calcitriol (2×10-7M) or ethanol (control) for 24h. Subsequent to this incubation, lipopolysaccharide (LPS)-stimulated whole blood production of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 were measured using a canine-specific multiplex assay. Calcitriol significantly increased LPS-stimulated whole blood production of IL-10 and decreased TNF-α production without significantly altering IL-6 production. There was no significant difference in whole blood cytokine production capacity between survivors and non-survivors at the time of discharge from the ICU or 30days after discharge. These data suggests that calcitriol induces an anti-inflammatory phenotype in vitro in whole blood from critically ill dogs.

Evaluation of immunomodulatory effect of recombinant human granulocyte-macrophage colony-stimulating factor on polymorphonuclear cell from dogs with cancer in vitro.

The objective of this in vitro study was to evaluate the immunomodulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on polymorphonuclear cell (PMN) function in dogs with cancer. PMNs were harvested from dogs with naturally developing cancer as a pre-clinical model to evaluate the immunomodulatory effects of rhGM-CSF on PMN phagocytic and cytotoxic functions, cytokine production and receptor expression. Some aspects of cancer-related PMN dysfunction in dogs with cancer were restored following incubation with rhGM-CSF including PMN phagocytosis, respiratory burst and LPS-induced TNF-α production. In addition, rhGM-CSF increased surface HLA-DR expression on the PMNs of dogs with cancer. These data suggests that dysfunction of innate immune response in dogs with cancer may be improved by rhGM-CSF. The results of this study provided a pathophysiologic rationale for the initiation of clinical trials to continue evaluating rhGM-CSF as an immunomodulatory therapy in dogs with cancer.

Peripheral effects induced in BALB/c mice infected with DENV by the intracerebral route.

The lack of an immunocompetent animal model for dengue mimicking the disease in humans is a limitation for advances in this field. Inoculation by intracerebral route of neuroadapted dengue strains in mice is normally lethal and provides a straightforward readout parameter for vaccine testing. However, systemic effects of infection and the immune response elicited in this model remain poorly described. In the present work, BALB/c mice infected by the intracerebral route with neuroadapted DENV2 exhibited several evidences of systemic involvement. DENV-inoculated mice presented virus infective particles in the brain followed by viremia, especially in late stages of infection. Infection induced cellular and humoral responses, with presence of activated T cells in spleen and blood, lymphocyte infiltration and tissue damages in brain and liver, and an increase in serum levels of some pro-inflammatory cytokines. Data highlighted an interplay between the central nervous system commitment and peripheral effects under this experimental condition.

Detection of herpesvirus DNA by the polymerase chain reaction (PCR) in vitreous samples from patients with necrotising retinitis.

AIMS: Viral uveitis and retinitis, usually caused by herpesviruses, are common in immunosuppressed patients. The diagnosis of viral anterior uveitis and retinitis is usually clinical. The polymerase chain reaction (PCR) has been used for the diagnosis of some viral infections, especially those caused by herpesviruses. This paper reports the use of PCR in the diagnosis of viral retinitis in vitreous samples from Brazilian patients. METHODS: PCR was used for the diagnosis of necrotising retinitis in vitreous samples from patients from the Hospital São Geraldo, Universidade Federal de Minas Gerais, Brazil. The vitreous samples were collected by paracentesis and stored until analysis. Samples were analysed by PCR using specific primers designed to amplify herpes simplex virus 1 (HSV-1), varicella zoster virus (VZV), or human cytomegalovirus (HCMV). In a case of anterior uveitis, PCR was performed with a sample from the anterior chamber. RESULTS: Herpesvirus DNA was amplified in 11 of 17 samples. HCVM DNA was detected in nine samples but DNA from HSV-1 and VZV were detected only once each. CONCLUSION: These results strongly suggest that PCR could be used for a rapid complementary diagnosis of viral uveitis and retinitis. A prospective study to evaluate the PCR results, clinical evolution, and treatment is imperative to corroborate the real value of PCR in diagnosis and how it could help the clinicians' approach.

Clonal and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus (MRSA) from a Portuguese hospital over time.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from a general hospital in Oporto, Portugal, during two periods (1992-1993 and 1996-2000) were characterized by pulsed-field gel electrophoresis (PFGE) of SmaI fragments, and by hybridization of ClaI digests with mecA and Tn554 probes, discriminating the isolates in mecA::Tn554::PFGE genotypes. In addition, a representative sample of the defined genotypes was characterized by multilocus sequence typing (MLST) and SCCmec (staphylococcal cassette chromosome mec) typing, generating the corresponding ST-SCCmec types. In 1992-1993, 77% of MRSA belonged to the Iberian clone (genotype I::E::A or ST247-IA). In 1996-2000, the frequency of this clone decreased to 19% and the majority (69%) of the isolates belonged to another international clone, the Brazilian MRSA (genotype XI::B::B or ST239-IIIA). Trimethoprim/sulfamethoxazole (SXT) was confirmed to be an important phenotypic marker to distinguish the Iberian (SXT-susceptible) and the Brazilian (SXT-resistant) clones in MRSA isolates from Portugal. Our observations document major shifts in the dominant MRSA clonal types that occurred in this hospital since 1992, suggesting a selective advantage of the Brazilian relatively to the Iberian clone. In addition to these two MRSA clones that are the most frequent in Portuguese hospitals since the early 1990s, sporadic MRSA clones (representing 14% of the total) were identified and characterized.

CMV infection of liver transplant recipients: comparison of antigenemia and molecular biology assays.

BACKGROUND: CMV is a major clinical problem in transplant recipients. Thus, it is important to use sensitive and specific diagnostic techniques to rapidly and accurately detect CMV infection and identify patients at risk of developing CMV disease. In the present study, CMV infection after liver transplantation was monitored retrospectively by two molecular biology assays - a quantitative PCR assay and a qualitative NASBA assay. The results were compared with those obtained by prospective pp65 antigenemia determinations. MATERIALS AND METHODS: 87 consecutive samples from 10 liver transplanted patients were tested for CMV by pp65 antigenemia, and CMV monitor and NASBA pp67 mRNA assay. RESULTS: CMV infection was detected in all patients by antigenemia and CMV monitor, whereas NASBA assay identified only 8/10 patients with viremia. Furthermore, CMV infection was never detected earlier by molecular biology assays than by antigenemia. Only 5/10 patients with CMV infection developed CMV disease. Using a cut off value of 8 cells/50,000, antigenemia was found to be the assay that better identified patients at risk of developing CMV disease. However, the kinetics of the onset of infection detected by NASBA and CMV monitor seemed to have better identified patients at risk of developing CMV disease. Furthermore, before onset of disease, CMV pp67 mRNA was found to have similar or better negative and positive predictive values for the development of CMV disease. CONCLUSIONS: The present data, suggests that the concomitant use of antigenemia and pp67 mRNA assay gives the best identification of patients at risk of developing CMV disease.

Nutritional status: anthropometry. Euronut SENECA investigators.

In 19 towns and cities across Europe anthropometric data--body weight, height, skinfold thicknesses and circumferences--have been obtained from 2332 elderly subjects born between 1913 and 1918, according to a strictly standardized methodology. Large variations exist among research towns even within countries. For body weight, means ranged from 70.1 +/- 15.4 kg to 78.2 +/- 10.7 kg in men and from 56.8 +/- 8.1 to 71.4 +/- 11.4 kg in women. In the North European towns and cities both men and women were taller than their counterparts in the southern towns. Mean body mass index varied from 24.4 +/- 3.8 kg m-2 to 30.3 +/- 5.2 kg m-2 among men. In women the range of means was from 23.9 +/- 3.6 kg m-2 to 30.5 +/- 5.1 kg m-2. Triceps skinfold thicknesses suggest that females had more fat at subcutaneous sites than the males, and the waist-hip ratio was consistently lower in elderly women.