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E S Umezawa

Publications and source records attributed to E S Umezawa.

At least 19 recordsLinked to original sources
PubMed2004

Chagas' disease: application of TESA-blot in inconclusive sera from a Brazilian blood bank.

BACKGROUND AND OBJECTIVES: The procedure used for screening Trypanosoma cruzi-infected blood donors by using two serological techniques has frequently led to discordant results. The TESA-blot, a confirmatory test for Chagas' disease, was applied in a survey of inconclusive sera from a Brazilian blood bank. MATERIALS AND METHODS: Four hundred and forty-eight sera, obtained from blood donors at the HRU-Fundação Hemominas, were tested by using the TESA-blot assay, a Western blotting method. Of these 448 sera, 348 had previously been determined as inconclusive for Chagas' disease owing to discordance between the indirect immunofluorescence assay (IFA) and the enzyme-linked immunosorbent assay (ELISA). RESULTS: The TESA-blot was positive for 2.87% (10/348) of the inconclusive sera, and 100% positive and negative for the sera from chagasic (n=50) and non-chagasic (n=50) donors, respectively. CONCLUSIONS: Our results clearly indicate the need to improve the diagnosis of Chagas' disease in blood banks by using new confirmatory diagnostic test(s). The TESA-blot, a new test with trypomastigote fractions of the T. cruzi Y strain, has made new approaches to the confirmation of Chagas' disease possible.

Adolescent↗
PubMed2002

Chagas' disease.

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Chagas Disease↗
PubMed2001

Chagas' disease.

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Adult↗
PubMed2001

Enzyme-linked immunosorbent assay with Trypanosoma cruzi excreted-secreted antigens (TESA-ELISA) for serodiagnosis of acute and chronic Chagas' disease.

In the present report we describe the use of Trypomastigote Excreted-Secreted Antigens (TESA) as antigen in ELISA for Chagas' disease serodiagnosis. The study was carried out on 284 patients, 164 of whom were nonchagasic subjects including individuals with leishmaniasis or other pathologies, and 120 chagasic patients, being 53 in the acute (with positive IgA and IgM antibodies to T. cruzi) and 67 in the chronic phase. TESA-ELISA showed 100% positivity in the survey of IgG antibodies in chagasic patients (acute and chronic) and 100% positivity for IgM antibodies in acute phase sera. TESA preparation does not require biochemical purification procedures and does not present the cross-reactivity of leishmaniasis sera observed when ELISA with epimastigote alkaline extract (EAE) is performed. ELISA competition assays showed that anti-T. cruzi antibodies of sera from chagasic patients that react with TESA are different from those that react with EAE. Besides, partial characterization of TESA showed that several epitopes present in this fraction are absent in EAE.

Acute Disease↗
PubMed2001

Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis.

Diagnosis of individuals infected by Trypanosoma cruzi is performed mainly by serological tests using crude antigens, which might crossreact with other infections. In the past ten years, many recombinant T. cruzi proteins and synthetic peptides have been described, and some are already on the market. Managers of laboratories and blood banks need to make decisions on a cost-benefit basis whether to include these new-generation tests. Here, we indicate antigens that are likely to prove most useful.

Animals↗
PubMed2001

Alterations of the microcirculatory network during the clearance of epimastigote forms of Trypanosoma cruzi: an intravital microscopic study.

The distribution of epimastigote forms of Trypanosoma cruzi in the microcirculatory network and the vessel alterations were observed using an intravital microscopy technique. Immediately after intravenous inoculation of 2 x 10(6) epimastigote suspension into normal mice, parasites were seen as circulating clumps, and their retention at some sites of the endothelium of venules and capillaries was observed. Injection of 2 x 10(7) and 2 x 10(8) parasite suspensions induced, respectively, intermittent or total stasis of venules and capillaries, probably via obstruction by clumping. The mobility of epimastigotes in the clumps indicates that parasites were alive in the lumen of vessels. The retention of clumps in the capillaries, although intense, could only be observed when labeled parasites were inoculated. These results suggest that the rapid clearance of epimastigote forms of T. cruzi from the blood circulation of mice may be due to the retention of parasites to the endothelium of venules and capillaries that, in turn, may facilitate phagocytosis. This may be a mechanism by which mice are able to eliminate epimastigote forms from the circulation. These findings are consistent with our previous observations showing that epimastigotes are not lysed by complement activation but are phagocytosed and destroyed by a distinct population of blood cells.

Animals↗
PubMed2000

Immunoblot analysis of trypomastigote excreted-secreted antigens as a tool for the characterization of Trypanosoma cruzi strains and isolates.

An analysis of antibody recognition of Trypanosoma cruzi exoantigens by immunoblotting revealed a unique banding pattern that seems to be characteristic of each strain or isolate. Trypomastigote excreted-secreted antigens (TESA) present in supernatants of LLC-MK2 cells infected with 5 strains and 10 isolates of T. cruzi produced 13 different immunoblotting patterns. The same bands were observed when probed with acute-phase Chagas' disease serum or with serum from a rabbit immunized with the repetitive domain of T. cruzi transialidase recombinant protein (anti-shed acute-phase antigens). Three similar patterns were observed with TESA from 3 human isolates that probably belong to the same T. cruzi strain. When clone CL Brener, clone CL-14, and CL parental strain were analyzed, the same bands were observed, although they presented different biological behavior. These results suggest that immunoblotting analysis of TESA may be a useful tool for characterization of T. cruzi strains and isolates.

Acute Disease↗
PubMed1999

Elevated anti-galactosyl antibody titers in endemic goiter.

Anti-Gal is a human polyclonal antibody that constitutes approximately 1% of the circulating immunoglobulin G (IgG), interacts specifically with the mammalian carbohydrate alpha-galactosyl epitope. Furthermore, it was found to mimic in vitro thyrotropin (TSH) effects regarding stimulation for cyclic adenosine monophosphate (cAMP) synthesis, 125I uptake, and cellular proliferation on cultured porcine thyrocytes and on Graves' disease thyrocytes, but not on normal human thyrocytes. As immune activation in sporadic and endemic goiters might play a secondary role in regulating thyrocyte proliferation and function, we evaluated anti-Gal titers in endemic goiter. Serum was obtained from 109 Chagas'-negative patients living in an endemic goiter area of Brazil (Grao Mogol, MG) and 160 controls. The patients were divided into 3 groups, according to their goiter size (World Health Organization [WHO] classification): grade 0 (group 1, n = 24), grade I-II (group 2, n = 41), and grade III-IV (group 3, n = 44). Anti-Gal was assessed by a radioimmunological procedure (results expressed as the percentage of bound radioactivity/total activity [%B/T]). The antibody titer was significantly more elevated in group 1 (mean +/- SEM: 9.27%+/-0.80%), in group 2 (mean +/- SEM: 16.17%+/-0.97%), and in group 3 (20.97%+/-1.30%) than in normal controls (6.46%+/-0.33%). Analysis of the male and female data separately for anti-Gal titer did not substantially alter these results. We concluded that the anti-Gal titer is higher in patients with endemic goiter and presented a possible relationship with the size of goiter. Whether these antibodies contribute to the pathogenesis of the disease needs further clarification.

Adult↗
PubMed1999

Evaluation of recombinant antigens for serodiagnosis of Chagas' disease in South and Central America.

The commercially available diagnostic tests for Chagas' disease employ whole extracts or semipurified fractions of Trypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas' disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzi recombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas' disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of 125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas' disease.

Animals↗
PubMed1998

Reactivity of chagasic antigal antibodies with noninfected cells treated with Trypanosoma cruzi secreted/excreted antigens.

Here, we show that antigal antibodies from Chagas' disease patients react with noninfected host cells previously treated with antigens secreted by the trypomastigote forms of Trypanosoma cruzi. With the exception of human and Old World monkey cells, which are GAL-negative, cells of all mammals express the GAL epitope (Gal alpha (1-3)Gal beta (1-4)GlcNAc-R) on their surface. Thus only the former ones develop antigal antibodies. Antigal antibodies increase during infection with T. cruzi, which expresses GAL epitopes on the surface of the infective forms. Here, we show that incubation of noninfected, GAL-negative cells with antigens shed by T. cruzi renders these cells reactive to antigal antibodies purified from chagasic sera. Neither chagasic sera depleted of antigal antibodies nor antigal antibodies purified from normal sera display reactivity with treated cells. Cell reactivity of chagasic antigal was abolished in the presence of melibiose (Gal alpha (1-6)Glc) or gal-gal (methyl 3-O-alpha-D-galactopyranosyl alpha-D-galactopyranoside). Since shedding of T. cruzi antigens can occur in vivo, these antigens may induce reactivity of chagasic antigal with noninfected human cells. The reactivity of noninfected, GAL-negative cells observed only with chagasic antigal antibodies can amplify the range of reactivity of these antibodies and consequently adds to their importance in the pathogenesis of human Chagas' disease.

Animals↗
PubMed1997

Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener.

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Grace's medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.

Animals↗
PubMed1996

Changes in isotype composition and antigen recognition of anti-Trypanosoma cruzi antibodies from acute to chronic Chagas disease.

This report describes differences in humoral immune response of acute and chronic phases of human Chagas disease. The reactivities of IgG, IgM, and IgA anti-Trypanosoma cruzi antibodies in serum samples from both groups of patients were compared by enzyme-linked immunosorbent assay (ELISA) employing either one of four antigenic fractions: mouse laminin (LAM), which reacts through Gal alpha 1-3Gal epitopes expressed on trypomastigote surface: whole intact trypomastigotes (TCT); trypomastigotes excreted/secreted antigens (TESA); and epimastigote alkaline extract (EAE). The selection of T. cruzi antigen preparations was based on their relative content of surface and internal antigens found in trypomastigote forms. The proportion of IgG reactive to carbohydrate epitopes was assessed through the decay of IgG reactivity from acute and chronic sera after m-periodate oxidation of solid-phase bound antigens. Trypomastigote and TESA antigens recognized by IgG from acute and chronic sera were also compared by immunoblotting. ELISA and immunoblotting data showed that: (1) the proportion of IgG directed to trypomastigote surface antigens was higher in acute than in chronic sera, whereas the opposite was found for internal antigens, (2) acute sera contained a higher percentage of IgG reactive to trypomastigote carbohydrate epitopes than chronic sera, and (3) anti-T. cruzi IgA was found exclusively in acute sera and led to 100% positivity when LAM, TCT, and TESA were employed as antigens. IgA ELISA with these antigens and IgG immunoblotting pattern with TESA could be useful as serological markers for the acute phase of human Chagas disease.

Animals↗
PubMed1996

Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas' disease.

Immunoblotting with trypomastigote excreted-secreted antigens (TESA blot) of Trypanosoma cruzi was evaluated as a method for diagnosis of chronic and acute phases as well as congenital (in newborn children) Chagas' disease. Serum samples from acute-phase and congenital infections were considered to be positive when they reacted with ladder-like bands of 130- to 200-kDa antigens, recognized by immunoglobulin M (IgM) and IgG antibodies, while IgG from chronic-phase sera recognized a broad band antigen of 150 to 160 kDa. Nonchagasic sera were not reactive to these antigens. The study was carried out on 512 patients, 111 of whom were nonchagasic but included cases of leishmaniasis or other pathologies, and 401 chagasic patients. The latter group comprised 361 chronic cases, 36 acute cases, and 4 congenital cases in newborn children. Among the chronic cases, 256 were from areas in which T. cruzi is endemic but which differed widely in the pathogenic expression of T. cruzi infection and in parasitemia levels. These patients at the same time showed a broad range of low, medium, and high reactivity to conventional enzyme-linked immunosorbent assays and indirect immunofluorescence serotests for Chagas' disease. For these reasons they may better represent the universe of chagasic patients than would a sample of highly reactive sera obtained from chagasic patients in a single area endemic for T. cruzi. All acute and congenital cases showed positivity in the IgM and IgG TESA blots, while chronic cases were 100% positive for IgG antibodies. In nonchagasic sera, including 30 cases of visceral and muco-cutaneous leishmaniasis, the specificity index was 1.000, and no cross-reactions were observed. The TESA blot thus seems to be useful as a sensitive and specific diagnostic assay in cases of suspected acute or congenital T. cruzi infection and as a general confirmatory test for conventional Chagas' disease serology.

Acute Disease↗
PubMed1995

High specificity of Trypanosoma cruzi epimastigote ribonucleoprotein as antigen in serodiagnosis of Chagas' disease.

We assessed the performance of an enzyme-linked immunosorbent assay (ELISA) with the Trypanosoma cruzi epimastigote ribosomal fraction (Tulahuen and Y strains) in order to improve the diagnostic specificity of the test. A total of 100 serum samples from patients with chronic Chagas' disease from Brazil and Argentina were studied. Sera from 116 patients, without Chagas' disease, including 10 with active mucocutaneous leishmaniasis and 20 with visceral leishmaniasis, were used as controls. Immunoglobulin G (IgG) antibodies against the ribosomal fraction (ribonucleoproteins [RNPs]) in the ELISA were found in 97% of samples from patients with Chagas' disease. A total of 99% of the sera from patients without the disease were negative, including sera from patients with mucocutaneous and visceral leishmaniases. The distribution of IgG isotypes in randomly chosen serum samples was determined by ELISA; IgG1 and IgG3 were predominant (100% exhibited IgG1 and 85% exhibited IgG3, and 50% also presented the IgG2 isotype. The distribution of the IgG subclasses was confirmed by the Western blot (immunoblot) technique. When total IgG was assayed by Western blot assay, no correlation was found between the pattern of serum reactivity and the clinical features of the patients with Chagas' disease. Therefore, no typical profile of polypeptide recognition could be associated with any clinical form of Chagas' disease (cardiomyopathy or megaviscera). Our results showed that sera from patients with Chagas' disease react with ribosomal antigens and display a typical profile of IgG isotypes (IgG1 plus IgG3). The RNP ELISA seems to have improved specificity compared with those of routine techniques such as the indirect immunofluorescence assay and hemagglutination because it better discriminates between patients with Chagas' disease and patients without the disease. Since sera from patients with leishmaniasis failed to show cross-reactivity with this antigen, the ELISA seems useful for detecting Chagas' disease as well as confirming the nature of sera, when it is doubtful whether the patients has Chagas' disease, by the isotype distribution of IgG.

Animals↗
PubMed1994

Trypanosoma cruzi: studies on the reactivity of antibodies bound to the surface of blood forms at the early phase of infection.

The specificity and reactivity of antibodies bound to the surface of Trypanosoma cruzi blood forms at the very early acute phase of murine infection was investigated. Surface-bound antibodies of the IgG and IgM isotypes were recovered from blood forms upon incubation at 37 degrees C. The eluted antibodies immunoprecipitated several trypomastigote surface polypeptides from 80 to 100 kDa. In contrast, for epimastigotes a very faint reactivity was detected only for antigens of 50 and 95 kDa. The shed antibodies promoted in vitro complement-mediated lysis of live blood forms and reacted with fixed trypomastigotes by immunofluorescence. Thus, blood forms are already coated with active trypomastigote-specific antibodies with a potential role in the host defense, although the low levels of serum antibodies have prevented the demonstration of humoral protection at the early stages of infection.

Animals↗
PubMed1994

In situ immunoassay for the assessment of Trypanosoma cruzi interiorization and growth in cultured cells.

Interiorization and multiplication of Trypanosoma cruzi within its host cells are usually assessed by counting parasites in fixed and stained cover slip preparations, a subjective and time-consuming method. Here we describe an immunoenzymatic assay (ELISA) for assessing the number of internalized parasites in infected LLC-MK2 seed on chamber slides (NUNC). ELISA was performed employing a rabbit polyclonal serum against trypomastigote components (MOP) and anti-rabbit IgG conjugated to peroxidase. The bottom of the chamber slide was then detached and processed for quantification of internalized parasites by the conventional method. Data analysis showed a linear relationship between optical densities and number of internalized parasites (r2 = 93.99, p < 0.001). The assay was also efficient to assess inhibition of parasite interiorization induced by the monosaccharide NAc-D-glucosamine.

Animals↗
PubMed1993

Trypanosoma cruzi: detection of a circulating antigen in urine of chagasic patients sharing common epitopes with an immunodominant repetitive antigen.

A monospecific antiserum raised to a Trypanosoma cruzi recombinant antigen, with tandem repeat of 68 amino acids, was used to screen urine samples of chagasic and nonchagasic patients. The antiserum detected a specific 150-160 kDa antigen in urine of 60% of chronic chagasic patients, but not in urine samples from nonchagasic patients and healthy control individuals. The reactivity to 150-160 kDa urinary antigen could be abolished by adsorption with the recombinant repetitive antigen. These results suggest that 150-160 kDa urinary antigen is a T. cruzi-derived antigen and specific for Chagas' disease.

Adult↗