MEMBRANE FRACTIONS FROM Strongyloides venezuelensis IN THE IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS: USE OF SIX DIFFERENT ANTIGENIC FRACTIONS FROM Strongyloides venezuelensis PARASITIC FEMALES

SUMMARY The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis.

Dot enzyme-linked immunosorbent assay (dot-ELISA) for schistosomiasis diagnosis using dacron as solid-phase

Dacron and nitrocellulose were evaluated as matrices for the dot enzyme linked immunosorbent assay (dot-ELISA) for schistosomiasis and compared to indirect immunofluorescence (IMF). Titration of sera from 18 schistosomiasis patients against soluble worm antigen preparation (SWAP) was carried out and sera from healthy individuals from non-endemic areas were used as controls. The IMF was less sensitive than the dot-ELISAs, although the difference was not statistically significant (p > 0.05). The dot-ELISA based on nitrocellulose was as sensitive as that using dacron. Stability did not differ between nitrocellulose and dacron. Specificity was lower when dacron was used than when nitrocellulose was used, although the difference was not statistically significant (p > 0.05). In conclusion, this work showed that nitrocellulose and dacron performed similarly in dot-ELISA, suggesting that they may be used alternatively in population surveillance in endemic areas.

Trypanosoma cruzi in marsupial didelphids (Philander frenata and Didelhis marsupialis): differences in the humoral immune response in natural and experimental infections

Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients.

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

Carbohydrate-rich high-molecular-mass antigens are strongly recognized during experimental Histoplasma capsulatum infection

INTRODUCTION: During histoplasmosis, Histoplasma capsulatum soluble antigens (CFAg) can be naturally released by yeast cells. Because CFAg can be specifically targeted during infection, in the present study we investigated CFAg release in experimental murine histoplasmosis, and evaluated the host humoral immune response against high-molecular-mass antigens (hMMAg. >150 kDa), the more immunogenic CFAg fraction. METHODS: Mice were infected with 2.2x10(4) H. capsulatum IMT/HC128 yeast cells. The soluble CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg circulating immune complexes (CIC) levels were determined by enzymelinked immunosorbent assay, at days 0, 7, 14, and 28 post-infection. RESULTS: We observed a progressive increase in circulating levels of CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg CIC after H. capsulatum infection. The hMMAg showed a high percentage of carbohydrates and at least two main immunogenic components. CONCLUSIONS: We verified for the first time that hMMAg from H. capsulatum IMT/HC128 strain induce humoral immune response and lead to CIC formation during experimental histoplasmosis.

Antigens of worms and eggs showed a differentiated detection of specific IgG according to the time of Schistosoma mansoni infection in mice

INTRODUCTION: The correlation between the immunological assay and the antibody titer can offer a tool for the experimental analysis of different phases of the disease. METHODS: Two simple immunological assays for Schistosoma mansoni in mice sera samples based on specific IgG detection for worms soluble antigens and eggs soluble antigens were standardized and evaluated in our laboratory. Fifty mice were used in negative and positive groups and the results obtained by enzyme-linked immunosorbent assays (ELISA) assays were compared with the number of worms counted and the IgG titers at different times of infection. RESULTS: Data showed that ELISA using adult worm antigens (ELISA-SWAP) presented a satisfactory correlation between the absorbance value of IgG titers and the individual number of worms counted after perfusion technique (R²=0.62). In addition, ELISA-SWAP differentially detected positive samples with 30 and 60 days post infection (p=0.011 and 0.003, respectively), whereas ELISA using egg antigens (ELISA-SEA) detected samples after 140 days (p=0.03). CONCLUSIONS: These data show that the use of different antigens in immunological methods can be used as potential tools for the analysis of the chronological evolution of S. mansoni infection in murine schistosomiasis. Correlations with human schistosomiasis are discussed.

Acute schistosomiasis diagnosis: a new tool for the diagnosis of schistosomiasis in a group of travelers recently infected in a new focus of Schistosoma mansoni

IntroductionThe diagnosis of schistosomiasis mansoni on early stages of infection is important to prevent late morbidity. A simple, cheap, sensitive and specific assay for routine diagnosis of schistosome infection based on the detection of specific IgG for schistosomula tegument antigens (ELISA-SmTeg) was developed by our group.MethodsWe describe here an acute outbreak involving a travel group of 80 individuals from a non-endemic area of the State of Minas Gerais, Brazil. These individuals were in contact with a freshwater pool where Biomphalaria glabrata was found. Results obtained from our new methodology were compared to IgG antibody titers against soluble worm antigenic preparation (SWAP) by ELISA and, also to parasitological examination, nuclear magnetic resonance and clinical findings.ResultsELISA-SmTeg was capable of detecting 64 positive cases among the 80 individuals participating at the survey with a positivity ratio of 80% and a higher sensitivity than ELISA-SWAP that was only sensitive for 56% of positive cases. Besides, a significant correlation was found for the severity of the infection and the specific IgG titers against SmTeg.ConclusionsOur data showed that ELISA-SmTeg might serve as the initial diagnostic tool for acute stages of the infection in community-based helminth control programs or for the surveillance of individuals from non-endemic areas.

Micorrizas arbusculares del sur de la Amazonia colombiana y su relación con algunos factores fisicoquímicos y biológicos del suelo

La presencia de micorrizas arbusculares en la vegetación de la Amazonia mejora la nutrición de las plantas en suelos de baja fertilidad. Este trabajo evaluó la presencia natural de hongos micorrícicos de tipo arbuscular (HMA) en suelos ácidos de textura franco-arcillosa a arcillosa del sur de la Amazonia colombiana bajo bosque, rastrojo joven, y praderas establecidas, a dos profundidades diferentes. Fue estudiada la presencia de HMA (riqueza y abundancia de esporas) relacionado con la acidez, la capacidad de intercambio catiónico, el carbono orgánico, el fósforo total, las fracciones de fósforo soluble y fijado al aluminio, hierro y calcio, y el ADN total del suelo. Se detectaron diferencias significativas, en el contenido de ADN total y el número de esporas respecto a la profundidad de muestreo. El pH presentó un efecto significativo sobre el contenido de ADN y el número de esporas de HMA. El contenido de ADN en el suelo se vio afectado por las concentraciones de fosfatos de aluminio, mientras la esporulación de HMA fue afectada por las concentraciones de fosfatos de hierro del suelo. Así, el número de esporas de HMA en suelos de la Amazonia se ve afectada por la profundidad, el pH y por el tipo de fosfatos minerales presentes.

Seasonal and spatial variations in limnological conditions of a floodplain lake (lake Catalão) connected to both the Solimões and Negro Rivers, Central Amazonia

Lakes play an important role in biogeochemical, ecological and hydrological processes in the river-floodplain system. The aim of this study was to evaluate the dynamics of the limnological conditions of Catalão Lake, an Amazon floodplain lake. Thus, some of the main limnological environment variables (O2, temperature, pH, nutrient, electrical conductivity) of the Catalão Lake were analyzed under temporal and spacial scales. The study was conducted between November/2004 and August/2005. Sampling excursion were carried out every three months; one excursion for each of the four different hydrological periods (low water, rising water, high water and falling water). Sampling points were chosen so that it could be obtained a gradient of the distance from Negro River. Limnological profiles in Catalão Lake showed generally acidic to slightly alcaline water, with low levels of dissolved oxygen and low concentrations of soluble reactive phosphorous. The Negro River seems to exert the main influence during the rising water period, while the Solimões River is the principal controlling river during peak water. The Principal Component Analysis (PCA) grouped the seasonal collections by hydrological period, showing the formation of a north-south spatial gradient within the lake in relation to the limnological variables. Multivariate dispersion analysis based on distance-to-centroid method demonstrated an increase in similarity over the course of the hydrological cycle, as the lake was inundated in response to the flood pulse of the main river channels. However, the largest spatial homogeneity in the lake was observed in the epilimnion layer, during the falling water period. The daily analysis of variation indicated an oligomitic pattern during the years in which the lake was permanently connected to the Negro River. Although Catalão Lake receives large quantities of both black water from the Negro River and sediment-filled water from the Solimões River, the physical and chemical characteristics of the lake are more similar to those of the Solimões (várzea lake) than the Negro (blackwater lake).

Plasma levels of immunoinflammatory markers in De Novo coronary atherosclerosis and coronary restenosis postangioplasty

OBJECTIVE: To compare circulating plasma levels of immunoinflammatory markers in patients with known de novo coronary artery disease and patients with postangioplasty restenosis. METHODS: Using enzymatic immunoabsorbent assay, we measured plasma levels of soluble interleukin-2 receptosr, tumor necrosis factor alpha, and soluble tumor necrosis alpha receptors I and II in 11 patients with restenosis postcoronary angioplasty (restenosis group), in 10 patients with primary atherosclerosis (de novo group) who were referred for coronary angiography because of stable or unstable angina, and in 9 healthy volunteers (control group). Levels of soluble interleukin-2 receptors were significantly higher in the de novo group compared with that in the restenosis and control groups. Levels were also higher in the restenosis group compared with that in the control group. Plasma levels of tumor necrosis alpha and receptor levels were significantly higher in the de novo group compared to with that in the restenosis and control groups, but levels in the restenosis group were not different from that in the controls. CONCLUSION: Coronary artery disease, either primary or secondary to restenosis, is associated with significant immunoinflammatory activity, which can be assessed by examining the extent of circulating plasma levels of inflammatory markers. Moreover, patients with de novo lesions appear to have increased inflammatory activity compared with patients with restenosis.

Cytokines and troponin-I in cardiac dysfunction after coronary artery grafting with cardiopulmonary bypass

OBJECTIVE: The association between cytokines and troponin-I with cardiac function after cardiac surgery with cardiopulmonary bypass remains a topic of continued investigation. METHODS: Serial measurements, within 24h following surgery, of tumor necrosis factor-alpha, its soluble receptors, and troponin-I were performed in patients with normal ejection fraction undergoing coronary artery bypass grafting. Ejection fraction was measured by radioisotopic ventriculography preoperatively, at 24h and at day 7 postoperatively. RESULTS: Of 19 patients studied (59±8.5 years), 10 (group 1) showed no changes in ejection fraction, 53±8% to 55±7%, and 9 (group 2) had a decrease in ejection fraction, 60±11% to 47±11% (p=0.015) before and 24h after coronary artery bypass grafting, respectively. All immunological variables, except tumor necrosis factor-alpha soluble receptor I at 3h postoperation (5.5± 0.5 in group 1 versus 5.9±0.2 pg/ml in group 2; p=0.048), were similar between groups. Postoperative troponin-I had an inverse correlation with ejection fraction at 24h (r= -0.44). CONCLUSIONS: Inflammatory activity, assessed based on tumor necrosis factor-alpha and its receptors, appears to play a minor role in cardiac dysfunction after cardiac surgery. Troponin I levels are inversely associated with early postoperative ejection fraction.

Comparison of the effect of two HMG CoA reductase inhibitors on LDL susceptibility to oxidation

OBJECTIVE: To study the differences between fluvastatin and pravastatin regarding LDL susceptibility to oxidation, plasma levels of total cholesterol (TC), HDL-C, LDL-C and triglycerides (TG) in hypercholesterolemic patients with established coronary heart disease (CHD). METHODS: A double-blind randomized parallel study was conducted that included 41 hypercholesterolemic outpatients with CHD treated at the Instituto de Cardiologia do Rio Grande do Sul. The inclusion criteria were LDL-C above 100 mg/dL and triglycerides below 400 mg/dL based on 2 measures. After 4 weeks on a low cholesterol diet, those patients that fullfilled the inclusion criteria were randomized into 2 groups: the fluvastatin group (fluvastatin 40 mg/day) and the pravastatin group (pravastatin 20 mg/day), for 24 weeks of treatment. LDL susceptibility to oxidation was analyzed with copper-induced production of conjugated dienes (Cu2+) and water-soluble free radical initiator azo-bis (2'-2'amidinopropanil) HCl (AAPH). Spectroscopy nuclear magnetic resonance was used for determination of lipids. RESULTS: After 24 weeks of drug therapy, fluvastatin and pravastatin significantly reduced LDL susceptibility to oxidation as demonstrated by the reduced rate of oxidation (azo and Cu) and by prolonged azo-induced lag time (azo lag). The TC, LDL-C, and TG reduced significantly and HDL-C increased significantly. No differences between the drugs were observed. CONCLUSION: In hypercholesterolemic patients with CHD, both fluvastatin and pravastatin reduced LDL susceptibility to oxidation.

Envelhecimento das aguardentes

1 - Colour, by itself, does not constitute a solid ground for judging of the age of a brandy because the more or less pronounced colour it acquires through aging can also be obtained by the addition of oack essence to newly distilled brandy. 2 - Urder the same conditions, colour intensity of a brandy wiU depend upon the nature of the wood and the condition of the storage. 3 - In accordance with the experimental results obtained by the present writers it rests no doubt that fermentation facility ferment resistence, produce and quality of the brendy all are factors depending upon the variety of the sugar cane. In addition, the authors presume that the variety of sugar cane has also influence upon the alteration of composition of the brandy submitted to aging. 4 - All aging phenomena of the brandy are accompanied by volume decreasing, what happens in a slow and continuous manner depending upon storage and environment conditions 5 - During brandy aging the alcoholic degree is greatly af- fected by evaporation, increasing or decreasing in accordance to the hygrometric state of the air and the teriperature in the place where the tuns are stored. 6 - The specific weight of the brandy is inversely proportio- nal to its alcoholic degree, but directly proportional to the extracts since the latter indicates the amount of dissolved residues. 7 - Brandy which shows high specific weight together with high alcoholic degree cannot be considered as aged. It may, however, be takens for brandy artificially coloved in order to conceal its actual age. 8 - The amount of extracts increases with aging, since it is the result of the solvent action of the brandy upon the soluble extractive substances of the wood. Notwithstanding that the extract, considered alone, has no value in determining the age of a brandy, since nothing easier is ohan to nake it change artificially. 9 -During aging the brandy get acidity in physiological as well as in physical way, but never by the action of microorganisms. 10 - The estturs produced during aging by the action of acids upon alcohols are the mean factors of the savour (bouquet) of a brandy and therefore every thing shall be done tor fhr estherification of a preserved brandy being not limited. 11 - Aeration increases esther formation, reduces the aging- time and turn better the taste qualities of the brandy. 12 - Due to the great proportion of high alcohols ordinarily found in the brandy, their analytical discrimination will be greatly important. 13 - The high alcohols are not responsable for the disastrous consequences of the alcoholism, but the high percentage of uthyl alcohol present in the brandy. 14 - The aldehydes appear always in high rate in the brazilian brandys in consequence of some intermediary products of the oxydation of the alcohols being left in the brandys during aging. 15 - The age has little or no influence on the quantity of phurphurol present in a brandy whose amount varies greatly the manner in which the wines to be distilled are treated. Wines centrifugalized or filtered before distillation always give rise to brandys poorer in phurphurol as compared with those distilled without these treatments. 16 - Though greatly variable, brandys of good qualities generally show a high residues coefficient, never under 200 mmg 17 - Lusson - Rocques oxydation coefficient cannot be indis- criminately applied to any brandy class, being, on the contrary, specifically destined to cognacs.