Comparison between a soluble antigen-based ELISA and IFAT in detecting antibodies against Babesia canis in dogs

O presente trabalho estudou um ensaio imunoenzimático (ELISA) indireto para a detecção de anticorpos anti-Babesia canis no soro de cães, tendo a Reação de Imunofluorescência Indireta (RIFI), como teste de referência O antígeno utilizado no ELISA do presente estudo consistiu em uma preparação antigênica solúvel de merozoítas B. canis e as diluições ótimas do antígeno, soros e conjugado foram determinadas por titulação em bloco, utilizando soros de referência positivos e negativos. A preparação antigênica solúvel de B. canis ótima foi de 10 µg.mL-1, com soros de referência positivos e negativos em uma única diluição de 1:100, e conjugado a 1:4.000. Um total de 246 amostras séricas foram colhidas em cães, durante a campanha de vacinação anti-rábica em Jaboticabal, São Paulo, Brasil e a presença de anticorpos anti-B. canis foi avaliada pelo ELISA e RIFI. Nestas condições, a média de absorbância dos soros de referência negativos foi de 0,129 ± 0,025, resultando em um ponto de corte de 0,323 (Nível de ELISA 3) e a média da absorbância dos soros de referência positivos foi de 2,156 ± 1,187. As amostras com sorologia positiva para B. canis por ELISA e RIFI foram 67,89% (n = 167) e 59,35% (n = 146), respectivamente. Os resultados obtidos sugerem que o ELISA descrito revelou-se um teste sorológico eficaz no diagnóstico da babesiose canina.

Role of neurokinin-1 expressing neurons in the locus coeruleus on ventilatory and cardiovascular responses to hypercapnia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Up-converter nanophosphor Y2O2S:Er,Yb aminofunctionalized containing or not spherical silica conjugated with BSA

This work reports on the study of the nanophosphor. Y2O2S:Er(2%),Yb(1%) obtained from polymeric resin to be evaluated as fluorescent label with Suitable features to conjugate with bio-molecules for bioassay up-converting phosphor technology (UPT) application A conjugation protocol between bovine serum albumin (BSA) and the aminofunctionalized nanophosphor containing or not spherical silica was established UV-vis results indicated an effective conjugation between nanophosphor particles and the protein up-conversion measurements under 980 nm excitation performed for samples before and after aminofunctionalization showed that nanophosphor particles luminescence features keep unchanged in all cases All results suggest that the adapted protocol is feasible to provide a nanoparticle-protein effective conjugation preserving nanophosphor optical features The presence of spherical silica can be considered advantageous to increase conjugation efficiency Therefore. the developed procedure is applicable for future conjugations between the chosen nanophosphor and the streptavidin protein chat takes part in the well known self-recognition system avidin-biotin. (C) 2009 Elsevier B.V All rights reserved.

Immobilization of streptavidin in sol-gel films: Application on the diagnosis of hepatitis C virus

Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45 V versus Ag/AgCl using H2O2 and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease. (c) 2006 Elsevier B.V. All rights reserved.

Immunosensor for the diagnosis of Chagas' disease

Trypanosoma cruzi proteins from epimastigote membranes, herein referred as antigens, have been used for the construction of an amperometric immunosensor for serological diagnosis of Chagas' disease. The proteins used had a molecular mass ranging from 30 to 100 kDa. The gold electrode was treated with cysteamine and glutaraldehyde prior to antigen immobilization. Antibodies present in the serum of patients with Chagas' disease were captured by the immobilized antigens and the affinity interaction was monitored by chronoamperometry at a potential of -400 mV (versus Ag pseudo-reference electrode) using peroxidase-labeled IgG conjugate and hydrogen peroxide, iodide substrate. The incubation time to allow maximum antigen-antibody and antibody-peroxidase-labeled IgG interactions was 20 min with a reactivity threshold at -0.104 mu A. (c) 2004 Elsevier B.V. All rights reserved.

Antimicrobial resistance of isolated Streptococcus pneumoniae in a hospital of the Brazilian public system

Streptococcus pneumoniae is the predominant bacterial agent that affects the human population with pneumonia. This disease is an important cause of death in the elderly and the children under five years old. In this study, 29 strains of invasive S. pneumoniae were isolated from 29 patients of pneumonia, bacteremia and meningitis in the laboratory of the Municipal Hospital in Paulinia, Brazil, from May 2006 to October 2007. Patients' age ranged from 8 months old to 60 years old. These strains of S. pneumoniae were isolated from blood, pleural fluid and cerebrospinal fluid (CSF) of patients. After typing of encapsulated strains of S. pneumoniae through quellung reaction, their resistance to antimicrobial agents was gauged through Disc Diffusion Technique followed by determination of minimum inhibitory concentration (MIC). Among the 29 strains analyzed, 23 were methicillin-sensitive and six were methicillin-resistant and penicillin intermediate resistant. No strain presented full resistance to penicillin. Serotyping was performed only in two samples, which belonged to serotype 18. Our data may alert ambulatory regarding the incidence of pneumococcal strains resistant to the most common drugs due to inappropriate use of antimicrobials and also collaborate to the elaboration of pneumococcal conjugate vaccines specific to each region.

Condicionamento estrutural das zonasa de cisalhamento da região de Forquilha, domínio Ceará Central: uma abordagem integrada de sensoriamento remoto e geologia estrutural

The studied region, named Forquilha and localized in northwestern Central Ceará domain (northern portion of Borborema Province), presents a lithostratigraphic framework constituted by paleoproterozoic metaplutonics, metasedimentary sequences and neoproterozoic granitoids. The metasedimentary rocks of Ceará group occupy most part of the area. This group is subdivided in two distinct units: Canindé and Independência. Canindé unit is represented basically by biotite paragneisses and muscovite paragneisses, with minor metabasic rocks (amphibolite lens). Independência sequence is composed by garnetiferous paragneisses, sillimanite-garnet-quartz-muscovite schists and quartz-muscovite schists, pure or muscovite quartzites and rare marbles. At least three ductile deformation events were recognized in both units of Ceará group, named D1, D2 and D3. The former one is interpreted as related to a low angle tangential tectonics which mass transport is southward. D2 event is marked by the development of close/isoclinal folds with a N-S oriented axis. Refolding patterns generated by F1 and F2 superposition are found in several places. The latest event (D3) corresponds to a transcurrent tectonics, which led to development of mega-folds and several shear zones, under a transpressional regime. The mapped shear zones are Humberto Monte (ZCHM), Poço Cercado (ZCPC) and Forquilha (ZCF). Digital image processing of enhanced Landsat 7-ETM+ satellite images, combined with field data, demonstrate that these penetrative structures are associated with positive and negative geomorphologic patterns, distributed in linear and curvilinear arrangements with tonal banding, corresponding to the ductile fabric and to crests. Diverse color composites were tested and RGB-531 and RGB-752 provided the best results for lineament analysis of the most prominent shear zones. Spatial filtering techniques (3x3 and 5x5 filters) were also used and the application of Prewitt filters generated the best products. The integrated analysis of morphological and textural aspects from filtered images, variation of tonalities related to the distribution of geologic units in color composites and the superposition over a digital elevation model, contributed to a characterization of the structural framework of the study area. Kinematic compatibility of ZCHM, ZCPC, ZCF shear zones, as well as Sobral-Pedro II (ZCSPII) shear zone, situated to the west of the study area, was one of the goal of this work. Two of these shear zones (ZCHM, ZCPC) display sinistral movements, while the others (ZCSPII, ZCF) exhibit dextral kinematics. 40Ar/39Ar ages obtained in this thesis for ZCSPII and ZCPC, associated with other 40Ar/39Ar data of adjacent areas, indicate that all these shear zones are related to Brasiliano orogeny. The trend of the structures, the opposite shear senses and the similar metamorphic conditions are fitted in a model based on the development of conjugate shear zones in an unconfined transpression area. A WNW-ESE bulk shortening direction is infered. The geometry and kinematic of the studied structures suggest that shortening was largely accommodated by lateral extrusion, with only minor amounts of vertical stretch

Estudo geológico e geofísico da falha de carnaubais, Bacia Potiguar-RN, e implicações neotectônicas

The segment of Carnaubais Fault located in the southeasthern portion of Guamaré Graben (Potiguar Basin) was studied. Several structures were detected and some of them strongly suggest that the last movements in Carnaubais Fault are of Neotectonic age. The study comprises an integrated interpretation of geologic, geomorphologic and geophysical data (gravimetry, magnetometry, resistivity, and self potential methods). According to the size of the studied areas, two approaches were used in this research. The first approach is of a regional nature and was conducted in an area, hereafter named Regional Area, having approximately 6,000 km2 and localized in the northern portion of Rio Grande do Norte state, around Macau city. The second approach comprises detailled studies of two small areas inside the Regional Area: the Camurupim and São Bento areas. Gravimetric and topographic data were used in the Regional Area. A separation into regional and residual components were conducted both on gravimetric and topographic data. The interpretation of the residual component of the gravimetric data allows a precise mapping of the borders of the Guamaré Graben. The regional component features of the topographic data are controlled by the pair of conjugate faults composed by the Carnaubais Fault (NE direction) and the Afonso Bezerra Fault (NW direction). On the other hand, the residual component of the topographic data shows that river valleis of NW direction are sharply interrupted where they intersect Carnaubais Fault. This fact is interpreted as an evidency that the last significant moviments occured in the Carnaubais Fault. Geologic, geomorphologic and geophysical data (magnetometry, resistivity, and self potential methods) were used in the Camurupim Area. The geologic mapping allows to identify five lithophacies unities. The first two unities (from base to top) were interpreted as composing a marine (or transitional) depositional sequency while the other were interpreted is composing a continental depositional sequence. The two sequences are clearly separated of an erosional discordance. The unities grouped in the marine sequence are composed by calcarenites (Unity A) and mudstones (Unity B). Unity A was deposited in a shalow plataform while Unity B, in a tidal flat. The unities grouped in the continental sequence are composed of conglomerate (Unity C) and sandstones (Unities D and E). Unities C and D are fluvial deposits while unity E is an eolian deposit. Unities A and B can be stratigraphycally correlated with Guamaré Formation. Unities C and D present three possible correlations. They may be correlated with Tibau Formation; or with Barreiras Formation; or with a clastic sediment deposit, commonly found in some rivers of Rio Grande do Norte state, and statigraphycally positioned above Barreiras Formation. Based on the decrease of the grain sizes from base to top both on unities C and D, it is proposed that these unities are correlated with the clastic sediment above mentioned. In this case, these unities would have, at least, Pleistocenic age. Finally, it is proposed that Unity E represent an eolian deposit that sufferred recent changes (at least in the Quaternary). The integrated interpretation of hydrographic, morphologic and geophysical data from Camurupim Area shows that Carnaubais Fault is locally composed by a system of several paralel subvertical faults. The fault presenting the larger vertical slip controls the valley of Camurupim river and separates the area in two blocks; in the nothern block the top of the Jandaira limestone is deeper than in the southern block. In addition, at least one of the faults in the northern block is cutting the whole sedimentary section. Because unities C , D, and/or E may be of Quaternary age, tectonic moviments possibly occured in Carnaubais Fault during this period. Detailled geologic mapping were conducted in beachrocks found in São Bento Area. This area is located at the intersection of the coast line with the Carnaubais Fault. The detected structures in the beachrocks are very similar to those caused by fragile deformations. The structures mapped in the beachrocks are consistent with a stress field with maximun compressional stress in E-W direction and extensional stress in the N-S direction. Since the Carnaubais Fault has a NE direction, it is optimally positioned to suffer tectonic movements under the action of such stress field. In addition, the shape of the coastal line appear to be controlled by the Carnaubais Fault. Furthemore, the observed structures in Camurupim Área are consistent with this stress field. These facts are interpreted as evidences that Carnaubais Fault and beachrocks suffered coupled tectonic movements. These moviments are of Neotectonic age because the beachrocks present ages less than 16,000 years

Familial cancer: depressed NK-cell cytotoxicity in healthy and cancer affected members

Este estudo se reporta às funções de células natural killer (NK), como adesão, lise e citotoxicidade e de subpopulações de células T em uma família com alta prevalência de pacientes com câncer e que apresentaram: glioblastoma, leucemia mielóide crônica, osteoblastoma, melanoma e carcinomas gástrico, pancreático e cólon retal. Quinze membros dessa família foram estudados, sendo 13 sadios, acompanhados por 5 anos e dois com câncer: glioblastoma e leucemia mielóide crônica. Duas pessoas sadias, no momento da avaliação, desenvolveram posteriormente osteoblastoma mandibular ou melanoma maligno. Como controle, foram avaliados 19 indivíduos saudáveis de faixa etária equivalente. A determinação de linfócitos T CD3+ e de suas subpopulações CD4+ e CD8+ foi realizada empregando-se anticorpos monoclonais e a atividade citotóxica de células NK, avaliada pelo teste de single-cell contra células alvo da linhagem eritroleucêmica K562. Os resultados mostraram que as percentagens de células T totais (CD3+), da subpopulação CD4+ e da relação CD4/CD8 foram significativamente menores nos indivíduos da família estudada em comparação aos valores observados no grupo controle. em todos os membros dessa família a percentagem de formação de conjugados entre células NK-células alvo foi inferior ao valor mínimo observado nos controles. Essa alteração poderia estar relacionada a defeito na expressão de moléculas de adesão, presentes na membrana de células NK, como provável causa das alterações funcionais dessas células. A herança dos mecanismos determinantes desta deficiência pode ser um fator de risco, com valor prognóstico para o desenvolvimento de cancer.

An acqueous extract of Bidens pilosa L. protects liver from cholestatic disease: experimental study in young rats

OBJETIVO: Testar o efeito hepatoprotetor do extrato aquoso de Bidens pilosa L. (EBP) na doença hepática induzida pela ligadura e ressecção do ducto biliar comum (LRDBC) em ratos jovens. MÉTODOS: Estudamos ratos Wistar com 21º. dia de vida (P21) divididos em quatro grupos de 10 animais, Grupo SA: operação simulada e água; Grupo SD: operação simulada e EBP (160mg de folhas frescas/100g de peso corporal/dia); Grupo LA: LRDBC e água e Grupo LD: LRDBC e EBP diariamente. O tempo de sono por pentobarbital (TSP), aspartato (AST) e alanina (ALT) aminotransferase foram determinadas após o sacrifício (P70). O Score de Ruwart (SR) para fibrose hepática foi atribuído para cada animal. Foi realizada análise de variância com dois fatores e pelo teste de Tukey para comparações múltiplas ou por teste não paramétrico. RESULTADOS: Houve diferença estatisticamente significativa entre LA e LD nas variáveis: TSP (médias LA=390; LD=173), AST (médias LA=8, LD=5), ALT (medianas LA=2; LD=1) e SR (medianas LA=2; LD=1). CONCLUSÃO: EBP poderá ser empregado na fitoterapia da doença hepática induzida pela colestase obstrutiva crônica, pois protege a função hepática, diminui a velocidade de necrose e a intensidade da fibrose hepática na obstrução biliar extra-hepática.

Anti-leishmania antibodies in cerebrospinal fluid from dogs with visceral leishmaniasis

Visceral leishmaniasis in Brazil is caused by Leishmania (Leishmania) chagasi and the dog is its most important reservoir. The clinical features in dogs include loss of weight, lymphadenopathy, renal failure, skin lesions, fever, hypergammaglobulinemia, hepatosplenomegaly, anemia, and, rarely, neurological symptoms. Most infected animals develop active disease, characterized by high anti-leishmania antibody titers and depressed lymphoproliferative ability. Antibody production is not primarily important for protection but might be involved in the pathogenesis of tissue lesions. An ELISA test was used to determine if there is an association between neurological symptoms and the presence of anti-L. chagasi antibodies in cerebrospinal fluid (CSF). Thirty serum and CSF samples from symptomatic mixed breed dogs (three with neurological symptoms) from a region of high incidence of visceral leishmaniasis in Brazil were examined for antibody using total parasite antigen and anti-dog IgG peroxidase conjugate. A high level of L. chagasi antibodies was observed in sera (mean absorbance ± SD, 1.939 ± 0.405; negative control, N = 20, 0.154 ± 0.074) and CSF (1.571 ± 0.532; negative control, N = 10, 0.0195 ± 0.040) from all animals studied. This observation suggests that L. chagasi can cause breakdown of filtration barriers and the transfer of antibodies and antigens from the blood to the CSF compartment. No correlation was observed between antibody titer in CSF and neurological symptoms.

Crystallization, preliminary X-ray analysis and Patterson search of a new aspartic protease isolated from human urine

Aspartic protease (EC 3.4.23) make up a widely distributed class of enzymes in animals, plants, microbes and, viruses. In animals these enzymes perform diverse functions, which range from digestion of food proteins to very specific regulatory roles. In contrast the information about the well-characterized aspartic proteases, very little is known about the corresponding enzyme in urine. A new aspartic protease isolated from human urine has been crystallized and X-ray diffraction data collected to 2.45 Angstrom resolution using a synchrotron radiation source. Crystals belong to the space group P2(1)2(1)2(1) the cell parameters obtained were a=50.99, b=75.56 and c=89.90 Angstrom. Preliminary analysis revealed the presence of one molecule in the asymmetric unit. The structure was determined using the molecular replacement technique and is currently being refined using simulated annealing and conjugate gradient protocols.

Parameter degeneracies in neutrino oscillation measurement of leptonic CP and T violation

The measurement of the mixing angle theta(13), sign of Deltam(13)(2), and the CP or T violating phase delta is fraught with ambiguities in neutrino oscillation. In this paper we give an analytic treatment of the paramater degeneracies associated with measuring the nu(mu)-->nu(e) probability and its CP and/or T conjugates. For CP violation, we give explicit solutions to allow us to obtain the regions where there exist twofold and fourfold degeneracies. We calculate the fractional differences, (Deltatheta/(θ) over bar), between the allowed solutions which may be used to compare with the expected sensitivities of the experiments. For T violation we show that there is always a complete degeneracy between solutions with positive and negative Deltam(13)(2) which arises due to a symmetry and cannot be removed by observing one neutrino oscillation probability and its T conjugate. Thus there is always a fourfold parameter degeneracy apart from exceptional points. Explicit solutions are also given and the fractional differences are computed. The biprobability CP/T trajectory diagrams are extensively used to illuminate the nature of the degeneracies.

Quantizing a relativistic free-particle on the light front: some subtle aspects

We use the light-front machinery to study the behavior of a relativistic free particle and obtain the quantum commutation relations from the classical Poisson brackets. We argue that their usual projection onto the light-front coordinates from the covariant commutation relations show that there is an inconsistency in the expected correlation between canonically conjugate variables time x(+) and energy p(-). This incompatibility between canonical conjugate variables in the light front is discussed in the context of Poisson brackets and a suggestion is made on how to avoid it.

Tunneling time through a barrier using the local value of a time operator

A time for a quantum particle to traverse a barrier is obtained for stationary states by setting the local value of a time operator equal to a constant. This time operator, called the tempus operator because it is distinct from the time of evolution, is defined as the operator canonically conjugate to the energy operator. The local value of the tempus operator gives a complex time for a particle to traverse a barrier. The method is applied to a particle with a semiclassical wave function, which gives, in the classical limit, the correct classical traversal time. It is also applied to a quantum particle tunneling through a rectangular barrier. The resulting complex tunneling time is compared with complex tunneling times from other methods.