Antigen characterization of vector-borne and cultured metacyclic trypomastigotes of Trypanosoma cruzi

Metacyclic trypomastigotes ol the CL strain of Trypanosoma cruzi obtained from triatomid vectors and from axenic cultures were comparatively analysed as to their antigen make-up and immunogenic characteristics. They were found to be similar by the various parameters examined. Thus, sera of mice immunized with either one of the two metacyclic types precipitated a 82Kd surface protein from 131I-labeled culture metacyclics. Sera of mice protected against acute T. cruzi infection by immunization with killed culture metacyclics of a different strain (G) recognized, by immunoblotting, a 77Kd protein in both types of CL strain metacyclics. A monoclonal antibody raised against G strain metacyclics, and specific for metacyclic stages of this strain, reacted with both CL strain metacyclic types. Both metacyclic forms were similarly Iysed by various anti-T. cruzi sera, in a complement-mediated reaction.

Sensibility of different larval stages of Schistosoma mansoni to the Larvae Disappearing Reaction (LDR) in murine schistosomiasis

The delay produced by drug, in the process of cercaria-schistosomulum transformation, was used to verify the sensibility of different larval stages to the host cell immune responses, in vivo. The peritoneal cavity of mice, a model used for in vivo observations, was choiced for the experiments. As well characterized schistosomules, cercariae and larvae in the process of transformation were coated and arrested by host cells, and could not be recovered by simple saline washings. After 10-²M EDTA saline washings, they were released alive, with good vitality and movements. Thus, different kind of larvae in the process of adaptation of the cercaria to the host are strongly coated by immune cells, but these fail to kill the invading organisms, at least during a few hours after invasion.

Growth curves of Leishmania braziliensis braziliensis Promastigotes and surface antigen expression before and after adaptation to Schneider's drosophila medium as assessed by anti-Leishmania human sera

Leishmania braziliensis braziliensis(MHOM/BR/75/M2903) was grown in Schneider's Drosophila medium. In one set of experiments promastigotes were already adapted to the medium by means of serial passages whereas in the second cells were grown in a biphasic medium and transfered to the liquid. Growth was more abundant for culture medium adapted cells; degenerate cells in small numbers as well as dead ones were present from day 5 for promastigotes adapted to liquid medium and from day 3 for newly adapted cells. Synthesis of surface antigens differed according to length of cell culture as assessed by the titer of five mucocutaneous leishmaniasis sera on subsequent days. Five days of culture for cells already adapted to the culture medium and 3 days for newly adapted ones were judged to be the best for the preparation of immunofluorescence antigens.

Histological observations on Montenegro's reaction in man

The Montenegro skin test is widely used as a diagnostic method for American cutaneous leishmaniasis (ACL) but little is known about the histological changes that occur in the skin after administration of the antigen. This report is based on histological studies of biopsied material obtained, from inoculation sites, 48 hours after individuals had been given intradermal injections with a standardized Montenegro antigen. The material examined was obtained from four distinctly different test groups: naturally infected patients with parasitologically proved ACL and with positive Montenegro's reaction; individuals without previous history of ACL and not previously tested with Montenegro antigen; participants in anti-ACL vaccine trials who developed positive reactions to Montenegro antigen after vaccination; other participants in vaccine trials who had negative Montenegro responses after vaccination or had served as controls in the trials. The histological pictures of each group are described and discussed. Histologically, the reactions of vaccinated individuals were indistinguishable from those with naturally acquired infections.

Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Aggregatory behaviour of platelets incubated with subcellular fractions of normal and chagasic human syncytiotrophoblast

The surface of human syncytiotrophoblast does not induce maternal blood platelet aggregation even though it is not an endothelium. It can be surmised that as occurs in endothelial injury the subcellular components of the syncytiotrophoblast may have pro-or antiaggregatory activity. During congenital Chagas' disease which is associated to trophoblast lesions, platelets may play a role in the development of T. cruzi-induced placentitis. In the present work the aggregatory behaviour of normal human blood platelets was recorded after their challenging with subcellular fractions of syncytiotrophoblast isolated from normal and chagasic women. Nuclear, Mitochondrial, Microsomal and Supernatant fractions isolated from normal and chagasic syncytiotrophoblast failed to induce per se any aggregatory reaction on platelets. When samples of platelet-rich plasma (PRP) were preincubated with normal and chagasic nuclear fractions and then stimulated with collagen at threshold level (CT-PRP) an inhibition of the aggregatory response was observed. Treatment of CT-PRP with normal and chagasic mitochondrial fractions induced inhibition of platelet aggregation whereas only chagasic fraction reduced latency time. Microsornal fraction from normal placentas showed no significant effects on platelet aggregation. It is concluded that subcellular fractions of normal human syncytiotrophoblast do not exhibit any effect on platelet aggregation, whereas those subcellular fractions enriched in intracellular membrane components isolated from chagasic placentas inhibit platelet aggregation.

Detection of hepatitis B virus DNA by the polymerase chain reaction in anti-HBE positive chronic hepatitis B patients

Detection of HBV-DNA by PCR was compared with other serological markers (HBsAg, HBeAg and anti-HBe) in a series of49 Chronic Hepatitis B patients, including 12 with a spontaneous clearance of HBsAg. None of these HBsAg negative cases were PCR positive, but 33/37 (89.2%) HBsAg positive cases were PCR positive (p < 0.0001). Among HBsAg positive samples, nine cases were HBeAg positive and anti-HBe negative, all of them PCR positive. Other 3 patients were HBeAg and anti-HBe positive and these cases were also found PCR positive. A third group included 21 patients anti-HBe positive and HBeAg negative: 19 of them were PCR positive and 2 were PCR negative. The last 4 cases were HBeAg and anti-HBe negative, two of them were PCR positive. The detection of anti-HBe viremic cases in the present series suggest that preC variants could occur in our country. In conclusion, the integrated phase o f chronic hepatitis B seems to be less frequent than it was assumed, when only HBeAg or dot blot hybridization techniques were used. The new term "low replication phase" might favorably replace the former "integrated phase".

Schistosoma mansoni: identification of a 46KDa antigen of the schistosomular surface by monoclonal antibody

An IgG2a subclass monoclonal antibody, C6G9, was obtained by immunization of BALB/c mice with Schistosoma mansoni egg antigens. With this monoclonal antibody, it was possible to identify a schistosomular antigen with a molecular weight of 46 kilodaltons (KDa), and its expression being evaluated by means of indirect immunofluorescence. The antigen persisted in the integument of the developing schistosomulum, for at least 96 hours post-transformation. The monoclonal antibody also reacted with the cercaria surface, but not with that of adult worm. The C6G9 was also able to mediate significant levels of cytotoxicity in the presence of complement for newly transformed schistosomula.

Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American Cutaneous Leishmaniasis

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100ºC for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

Differentiation of pathogenic and non-pathogenic leptospires by means of the polymerase chain reaction

A polymerase chain reaction was carried out to detect pathogenic leptospires isolated from animals and humans in Argentina. A double set of primers (G1/G2, B64-I/B64-II), described before, were used to amplify by PCR a DNA fragment from serogroups belonging to Leptospira interrogans but did not allow to detect saprophytic strains isolated from soil and water (L. biflexa). This fact represents an advantage since it makes possible the differentiation of pathogenic from non-pathogenic leptospires in cultures. The sensitivity of this assay has been determined, allowing to detect just only 10 leptospires in the reaction tube. Those sets of primers generated either a 285 bp or 360 bp fragment, depending on the pathogenic strain

Histoplasmin reaction. Comparison of a polysaccharide antigen to the filtrate antigen

This work was planned by taking into account all the knowledge accumulated from the immunological study of paracoccidioidomycosis. It aimed at comparing a polysaccharide antigen from Histoplasma capsulatum to a classic histoplasmin with the help of intradermal tests of delayed type of hypersensitivity. Tests were applied to 115 individuals in Santo Amaro, a town in the state of São Paulo. Positive results using classic histoplasmin were obtained in 46.0% cases whereas positive results using the polysaccharide antigen at its hightest concentration were obtained in 51.30% cases. The major conclusion in this investigation is that it is possible to use the polysaccharide antigen as histoplasmin instead of the filtrate antigen

Detection of mycoplasmas in urethral swabs from HIV-1 infected patients and control individuals using culture techniques and polymerase chain reaction

The objective of the present study was to determine the prevalence of certain mycoplasma species, i.e., Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma penetrans, in urethral swabs from HIV-1 infected patients compared to swabs from a control group. Mycoplasmas were detected by routine culture techniques and by the Polymerase Chain Reaction (PCR) technique, using 16SrRNA generic primers of conserved region and Mycoplasma penetrans specific primers. The positivity rates obtained with the two methods were comparable. Nevertheless, PCR was more sensitive, while the culture techniques allowed the quantification of the isolates. The results showed no significant difference (p < 0.05) in positivity rates between the methods used for mycoplasma detection.