Calculating rabies virus neutralizing antibodies titres by flow cytometry

The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton & Dickinson FACSCalibur® flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity .

CO-INFECTION OF DENGUE VIRUS BY SEROTYPES 1 AND 4 IN PATIENT FROM MEDIUM SIZED CITY FROM BRAZIL

SUMMARY The natural co-infection with dengue virus can occur in highly endemic areas where different serotypes have been observed for many years. We report one case of DENV-1/DENV-4 co-infection in human serum detected by molecular tests. Phylogenetic analysis of the sequences obtained indicated the presence of genotype V and II for DENV-1 and DENV-4, respectively.

USE OF MUELLER-HINTON BROTH AND AGAR IN THE GERM TUBE TEST

Candida albicans is often isolated from clinical samples, thus its presumptive differentiation from other species of the same genus can be based on its ability to form the germ tube in human serum. Nevertheless, there are two other species that share this characteristic: C. dubliniensis and C. africana. The aim of this study was to compare four different substrates to perform the germ tube (GT) test. The Candida spp. isolates were identified using a manual system (135 C. albicans, 24 C. tropicalis and one C. dubliniensis). The germ tube test was performed with fresh, previously frozen serum and Mueller-Hinton (MH) broth and agar. GT was observed in 96% (130/136) of the isolates through the fresh serum technique, 94% (128/136) through previously frozen serum, 92% (125/136) in MH agar, and 90% (122/136) in MH broth. The sensitivity of each test was higher than 90%, with 100% specificity. Both the MH agar and broth were able to identify the true positives, and false positives were not found. However, some C. albicans isolates were not identified. MH agar and broth may be used in laboratory for the rapid presumptive identification of C. albicans, as an alternative method for germ tube test.

Determination of germ tube, phospholipase, and proteinase production by bloodstream isolates of Candida albicans

Introduction Candida albicans is a commensal and opportunistic agent that causes infection in immunocompromised individuals. Several attributes contribute to the virulence and pathogenicity of this yeast, including the production of germ tubes (GTs) and extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate GT production and phospholipase and proteinase activities in bloodstream isolates of C. albicans. Methods One hundred fifty-three C. albicans isolates were obtained from blood samples and analyzed for GT, phospholipase, and proteinase production. The assays were performed in duplicate in egg yolk medium containing bovine serum albumin and human serum. Results Detectable amounts of proteinase were produced by 97% of the isolates, and 78% of the isolates produced phospholipase. GTs were produced by 95% of the isolates. A majority of the isolates exhibited low levels of phospholipase production and high levels of proteinase production. Conclusions Bloodstream isolates of C. albicans produce virulence factors such as GT and hydrolytic enzymes that enable them to cause infection under favorable conditions.

Analysis of the chemical components of hydatid fluid from Echinococcus granulosus

Introduction The aim of this study was to explore the environment of Echinococcus granulosus (E. granulosus) protoscolices and their relationship with their host. Methods Proteins from the hydatid-cyst fluid (HCF) from E. granulosus were identified by proteomics. An inductively coupled plasma atomic emission spectrometer (ICP-AES) was used to determine the elements, an automatic biochemical analyzer was used to detect the types and levels of biochemical indices, and an automatic amino acid analyzer was used to detect the types and levels of amino acids in the E. granulosus HCF. Results I) Approximately 30 protein spots and 21 peptide mass fingerprints (PMF) were acquired in the two-dimensional gel electrophoresis (2-DE) pattern of hydatid fluid; II) We detected 10 chemical elements in the cyst fluid, including sodium, potassium, calcium, magnesium, copper, and zinc; III) We measured 19 biochemical metabolites in the cyst fluid, and the amount of most of these metabolites was lower than that in normal human serum; IV) We detected 17 free amino acids and measured some of these, including alanine, glycine, and valine. Conclusions We identified and measured many chemical components of the cyst fluid, providing a theoretical basis for developing new drugs to prevent and treat hydatid disease by inhibiting or blocking nutrition, metabolism, and other functions of the pathogen.

Imunidade e permeabilidade celular

After going through the more important theories on cellular permeability, researches were undertaken with the purpose of proving the actual influence of the various degrees of cellular permeability on the phenomena of organic resistance against infections, and on the production of antibodies. Three groups of substances known to have action on cellular permeability were used; the first consisting of the following permeable substances: testos-terona, acetylcholine, and the spreading-factor of the staphyloccocus. The second group included substances which help in developing low cellular permeability: atropin, adrenalin and calcium. Finally, the third group consisted of a substance which helps to maintain normal permeability: cortin (an extract of the suprarenal cortex). In order to study the process developed by these elements with regard to organic resistance against infections, adult mice were inoculated with the following germs: K. pneumoniae, P. aeruginosa, S. enteriditis and D. pneumoniae, in the smallest possible amount capable of starting a mortal sep infection in approximately 24 hours, exception made of D. pneumonias which causes death in 48 hours. The animals were divided into groups of 10, a before taking the injections containing the germs, they were given the sub lances under observation, through their peritoneum of intramuscularly. T. animals that died were autopsied and blood was taken from their hearts an aseptic process so as not to introduce extraneous organisms. For the purpose of determining the development of antibodies (hem lysins, precipitins and aglutinins), rabbits were used, which had been prep ously immunized by a treatment consisting of 6 intravenous injections of polyvolent antigen made of sheep blood cells, fresh human serum, and of suspension of S. enteriditis. It was concluded that: Cellular permeability plays a very important part in the development infections. Permeable substances help the development of germ infections. Substances helping to develop low permeability proved not to have any influence worth mentioning. Substances helping to maintain normal permeability, such as coffin, it crease resistance against infections. The different substances used which have action on cellular permeability had no influence worth mentioning on the development of certain ant bodies (hemolysins, precipitins and aglutinins). It was admitted that the phenomena under study relative to resistance against infections are closely connected to the dynamics of the cellular elements, which circumstance is basically dependent on the permeability of Citations of cells.

Analysis of toxoplasma gondii proteins after Triton X-114 solubilization and hidropholic chromotograhy

The distribution of the surface proteins of toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Twopolypeptides with 15,000 and 70,000 distributed on both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities.two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of t. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.

The use of ferromagnetic dacron as solid-phase in enzyme immunoassays

Ferromagnetic dacron is proposed as an alternative solid-phase for magnetic enzyme immunoassays. Human serum albumin (HSA) was covalentlyimmobilized onto ferromagnetic dacron and as enzyme immunoassay was developed using anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used as the enzymatic label and substrates, respectively. Best results were observed when particles of 63-100 µm (diameter) and 10 µg of immobilized antigen were used. Positive reactions were detected until dilutions of1:51200 of immune sera. Its reproducibility was similar to standard ELISA. Disruption of the immunocomplexes formed and recuperation of the immobilized antigen in other immunoassays also proved to be reliable.

Primary isolation of spotted fever group rickettsiae from Amblyomma cooperi collected from Hydrochaeris hydrochaeris in Brazil

This paper reports the first isolation of a spotted fever group rickettsia from an Amblyomma cooperi ixodid collected from a capybara (Hydrochaeris hydrochaeris) in an endemic area of spotted fever in the County of Pedreira, State of São Paulo, Brazil. Isolation was performed in Vero cell culture and submitted to immunofluorescence, using antibody from Rickettsia rickettsii-positive human serum.

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.

First detection of Rio Negro virus (Venezuelan equine encephalitis complex subtype VI) in Córdoba, Argentina

Rio Negro virus (RNV) (Venezuelan equine encephalitis subtype VI) circulates only in Argentina; in northern provinces, isolates have been obtained from mosquitoes and rodents since 1980 and have been associated with acute febrile illness in humans. However, no studies of RNV have been performed in the central area of the country. We carried out molecular and serological detection of RNV in Córdoba, a province of the central part of the country, in mosquitoes and humans, respectively. One mosquito pool tested positive for alphavirus RNA by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR). Subsequent sequencing determined that this alphavirus grouped with RNV. Serological studies detected antibodies to RNV in one human serum sample, which was obtained during the same period that RNV was detected using the aforementioned molecular methods. This is the first report of RNV circulation in the central area of Argentina, indicating an expansion of its original distribution. These results highlight the importance of strengthening surveillance procedures in endemic areas, as well as in new regions where RNV may emerge.

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen

The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.

Determinação eletroanalítica de corante reativo presente como contaminante em proteínas purificadas por cromatografia de afinidade

Procion Green HE-4BD is a reactive dye currently used in affinity purification, and commonly present as a contaminant in the final biological preparation. An assay method is described to determine trace amounts of the dye in the presence of human serum albumin(HSA) and leakage from agarose as affinity sorbent by cathodic stripping voltammetry. The proposed method is based on the reductive peak at -0.55V in B-R buffer pH 3 (E=0V and t= 240s), obtained when samples of HSA 2% (m/v) containing dye concentrations in sodium hydroxide pH 12 are submitted to a heating time of 330 min at 80 ºC. Linear calibration curves can be obtained for RG19 dye concentrations from 5x10-9 mol L-1 to 8 x10-8 mol L-1. The detection limit (3sigma) is 1x10-9 mol L-1.

Stability-indicating RP-HPLC method for simultaneous determination of gatifloxacin and flurbiprofen in binary combination

A stability-indicating RP-HPLC method is presented for determination of gatifloxacin and flurbiprofen in binary combination. Gatifloxacin, flurbiprofen and their degradation products were detected at 254 nm using a BDS Hypersil C8 (250 X 4.6 mm, 5 µm) column and mixture of 20 mM phosphate buffer (pH 3.0) and methanol 30:70 v/v as mobile phase. Response was linear over the range of 15-105 mg mL-1 for gatifloxacin (r² > 0.998) and of 1.5-10.5 mg mL-1 for flurbiprofen (r² > 0.999). The developed method efficiently separated the analytical peaks from degradation products (peak purity index > 0.9999). The method developed can be applied successfully for determination of gatifloxacin and flurbiprofen in human serum, urine, pharmaceutical formulations, and their stability studies.

INVESTIGAÇÃO DA SUPRESSÃO DE FLUORESCÊNCIA DE SORO ALBUMINA BOVINA E HUMANA POR COMPLEXO DE RUTÊNIO

The binding of [RuCl2(L)] (L = N,N-bis(7-methyl-2-pyridylmethylene)-1,3-diiminopropane) to bovine and human serum albumin was investigated by the fluorescence quenching technique. The comparison of the quenching effect of serum albumin fluorescence by ruthenium complex allowed the estimation of subdomain IB in BSA and subdomain IIA in HSA as the binding sites for this complex. The results of fluorescence titration revealed that ruthenium complex quenches the intrinsic fluorescence of BSA through a dynamic quenching mechanism, while HSA has a static quenching mechanism. The thermodynamic parameters indicated that hydrophobic forces played a major role in the binding of ruthenium complex to proteins. The process of binding was a spontaneous process in which Gibbs free energy change was negative.