Trypanosoma cruzi: explant organ cultures from mice with chronic Chagas' disease

Explants of 13 different organs obtained from C3H/HEN, Swiss-Webster, and C57Bl/6 mice chronically infected with Trypanosoma cruzi (Y strain) were cocultivated with mouse embryo fibroblasts to determine the organs that contain T. cruzi during the chronic infection. Explant cultures frequently yielded T. cruzi as late as 12 months after infection. Spleen and skeletal muscle were most frequently positive; heart cultures were rarely positive in any mouse strain. C3H/HEN mice had significantly more cultures positive than Swiss-Webster mice, as expected from relative susceptibility of C3H/HEN mice to acute infection. In contrast, C57Bl/6 mice, relatively resistant to acute infection, had significantly more cultures positive at 12 months of infection than Swiss-Webster mice. Also, C57Bl/6 mice had a significant increase in the number of positive cultures at 12 months of infection compared to 6 months of infection. These results show that organisms can be recovered routinely from some tissues during the chronic infection, that murine susceptibility to infection should differentiate between acute and chronic infection, and that C57Bl/6 mice may lose control of infection during the chronic infection.     

Longitudinal study, by xenodiagnosis, of parasitemia in patients with chronic Trypanosoma cruzi infection]

A 2-5 years follow-up of parasitemia, by the use of xenodiagnosis (XD) was carried out in nine patients with chronic T. cruzi infection who proceeded from chagasic endemic areas of Chile. The patients (mean age 55 years) were hospitalized in the chronic section of a psychiatry institution sited in a permanent triatomines free urban area. Clinical examination, X-rays images (cardiovascular, esophagus and colon) and electrocardiogram resulted normal in all the patients. Basic study unit of parasitemia was a XD box which contained 7 nymphs III of Triatoma infestans which was used in a serial XD consisting in the simultaneous application of a pair of boxes a day during three consecutive days, making a total of six boxes (42 nymphs). The minimal time of duration of infection (M.D.I.) for each patient was estimated that this was adquired close to hospitalization. The M.D.I. varied between 6 and 45 years. The global positivity of XD boxes ranged between 6.3 and 84.7%, being in three patients lower than 12% and in six patients higher than 52%. In considering the yield of XD it is important to stress that during all the study of the nine patients with chronic chagasic infection 1282 XD boxes were applied resulting positive 582 (45.4%). At the end of the follow-up all patients received specific treatment for chagasic infection with nifurtimox at the daily dose of 10 mg/kg of body weight during 60 days. According to the result, two main conclusions arise: 1.--Serial XD has a high efficiency for detecting, evaluate and evolve parasitemia in patients with chronic chagasic infection. 2.--Parasitemia may present low, medium or high levels in different individuals and has a variable and fluctuating character.     

Trypanosoma cruzi clonal diversity and the epidemiology of Chagas' disease

Chagas' disease is caused by the protozoan Trypanosoma cruzi and it has a variable clinical outcome. The basis for this variability relies in part on the complexity of the parasite population consisting of multiple clones displaying distinct biological properties. A major current challenge is to correlate parasite genetic variability with pathogenesis.     

Trypanosoma cruzi: antibodies to a MAP-like protein in chronic Chagas' disease cross-react with mammalian cytoskeleton

Trypanosoma cruzi lambda gt 11 library from epimastogote derived mRNA was screened with human chagasic sera or sera from chronically infected mice. Strong reactive recombinants were detected with both sera. Two recombinant clones were studied in more detail and shown to be composed of the same 114-bp repetitive sequence coding for a 38 amino acid repetition. This repetition is the same size and shares greater than 60% homology with the reported T. brucei microtubule associated protein (MAP) p320. The insert of one of these clones, K1-7 (228 bp), was subcloned into pMSgt11 and the soluble recombinant polypeptide expressed. Antibodies against the K1-7 fusion polypeptide recognized a major 110-kDa band from cytoskeleton. Anti K1-7 monospecific antibodies detected several cytoskeletal proteins from 3T3 fibroblasts and bovine brain microtubule preparations. Reciprocally, anti-MAP1b monoclonal antibodies raised against bovine brain microtubule reacted with the K1-7 polypeptide on Western blots. The protein identified by K1-7 antibodies may be one of the parasite molecules associated to molecular mimicry.     

Trypanosoma cruzi: a possible control of transfusion-induced Chagas' disease by phenolic antioxidants

The following phenolic antioxidant food additives were evaluated against Trypanosoma cruzi epimastigotes: BHT, BHA, gallic acid and its methyl, propyl, octyl, and lauryl esters, 2,4-di-tert-butyl-6-(4-methoxybenzyl)-phenol, 4,4'-isopropilidenediphenol, and protocatechuic acid and its ethyl ester. The inhibition of the respiration; the changes in motility, shape, and lysis of the parasites; and the human blood hemolysis caused by these chemicals were studied. Human blood samples experimentally contaminated with 2000 or 150,000 trypomastigotes per milliliter were freed of parasites after treatment for 24 hr at 4 degrees C with 5 or 10 mM BHT (2,6-di-tert-butyl-4-hydroxytoluene), respectively. Consequently, BHT and other phenolic compounds deserve further study to determine their role in preventing the transmission of Chagas' disease by blood transfusion.     

Immunohistochemical detection of Trypanosoma cruzi in tissues of mice with experimental Chagas' disease

We compared two immunoperoxidase procedures for the identification of Trypanosoma cruzi antigens in wax sections of infected mouse tissues. Both an indirect immunoperoxidase and an avidin-biotinated horseradish peroxidase complex (ABC) procedure gave identical patterns of localization. The ABC procedure could be used with greater dilutions of antisera than the indirect procedure. Individual organisms were clearly identified in the absence of inflammation at scanning magnifications. In addition, fragments of parasites were identified in inflammatory lesions. Therefore, this technique should be valuable in experimental and clinical situations when the identification of few organisms or fragments of organisms is required.     

Trypanosoma cruzi: 4-aminopyrazolopyrimidine in the treatment of experimental Chagas' disease

An allopurinol metabolite, 4-aminopyrazolopyrimidine, was tested on two different strains of mice (NMRI-IVIC and C57Bl/6J) that had been infected 4 days earlier with the virulent Ya strain of Trypanosoma cruzi. Low doses of 4-aminopyrazolopyrimidine (0.125-0.500 mg/kg body wt/day) for 10 days induced a significant reduction in parasitemia (direct counts and subinoculation experiments) and increased survival time (without any evidence of toxicity) compared with untreated animals. When tested in vitro, 4-aminopyrazolopyrimidine was sixfold more active than allopurinol as a trypanostatic drug. The low therapeutic doses of 4-aminopyrazolopyrimidine suggest that this drug may be useful in the treatment of acute Chagas' disease.     

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop

Abstract A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease. 2 Attempts to upgrade the reliability and the efficacy of xenodiagnosis in chronic Chagas' disease

In order to upgrade the reliability of xenodiagnosis, attention has been directed towards population dynamics of the parasite, with particular interest for the following factors: 1. Parasite density which by itself is not a research objective, but by giving an accurate portrayal of parasite development and multiplication, has been incorporated in screening of bugs for xenodiagnosis. 2. On the assumption that food availability might increase parasite density, bugs from xenodiagnosis have been refed at biweekly intervals on chicken blood. 3. Infectivity rates and positives harbouring large parasite yields were based on gut infections, in which the parasite population comprised of all developmental forms was more abundant and easier to detect than in fecal infections, thus minimizing the probability of recording false negatives. 4. Since parasite density, low in the first 15 days of infection, increases rapidly in the following 30 days, the interval of 45 days has been adopted for routine examination of bugs from xenodiagnosis. By following the enumerated measures, all aiming to reduce false negative cases, we are getting closer to a reliable xenodiagnostic procedure. Upgrading the efficacy of xenodiagnosis is also dependent on the xenodiagnostic agent. Of 9 investigated vector species, Panstrongylus megistus deserves top priority as a xenodiagnostic agent. Its extraordinary capability to support fast development and vigorous multiplication of the few parasites, ingested from the host with chronic Chagas' disease, has been revealed by the strikingly close infectivity rates of 91.2% vs. 96.4% among bugs engorged from the same host in the chronic and acute phase of the disease respectively (Table V), the latter comporting an estimated number of 12.3 x 10(3) parasites in the circulation at the time of xenodiagnosis, as reported previously by the authors (1982).     

Trypanosoma cruzi isolates from Mexican and Guatemalan acute and chronic chagasic cardiopathy patients belong to Trypanosoma cruzi I

Trypanosoma cruzi is classified into two major groups named T. cruzi I and T. cruzi II. In the present work we analyzed 16 stocks isolated from human cases and four isolated from triatomines from diverse geographical origins (Mexico and Guatemala). From human cases four were acute cases, six indeterminates, and six from chronic chagasic cardiophatic patients with diagnosis of dilated cardiomyopathy established based on the left-ventricular end systolic dimension and cardiothoracic ratio on chest X-radiography and impaired contracting ventricle and different degree conduction/rhythm aberrations. DNA samples were analyzed based on mini-exon (ME) polymorphism, using a pool of three oligonucleotide for the amplification of specific intergenic region of T. cruzi ME gene. All the Mexican and Guatemalan isolates regardless their host or vector origin generated a 350 bp amplification product. In conclusion T. cruzi I is dominant in Mexico and Guatemala even in acute and chronic chagasic cardiopathy patients. To our knowledge, this is the first study describing predominance of T. cruzi I in human infection for North and Central America.     

Identification of antigens of Trypanosoma cruzi which induce antibodies during experimental Chagas' disease

Experiments were conducted to identify antigens of Trypanosoma cruzi (Brazil strain) to which antibodies are directed during the course of experimental Chagas' disease in C3H(He) (susceptible) and C57BL/6J (resistant) female mice. An extract of culture forms of the parasite was subjected to SDS-polyacrylamide gel electrophoresis, transferred to a solid phase matrix of nitrocellulose and used as antigens to detect antibodies in the sera of infected mice. Reactive antibodies were detected using an avidin-biotin peroxidase test. Two antigens were consistently detected with sera of normal, uninfected C57BL/6 and C3H(He) mice (51,000 and 44,000; and 53,000 and 46,000 daltons, respectively). A total of 32 antigens with m.w. of 230,000 to 25,000 daltons reacted with antibodies in sera of C3H mice infected for 25 days. Both the number of antigens detected and intensity of reactions increased with time of infection in C3H mice. An early (day 5), rapid, although weak response was observed to antigens of 85,000, 56,000, 53,000, 46,000 and 41,000 daltons. Throughout infection intense responses to antigens of 75,000, 67,000, 45,000, 41,000 and 36,000 daltons were detected. A similar number of components (a total of 34) with m.w. of 210,000 to 20,000 daltons were detected as being antigenic during the course of T. cruzi infection of C57BL/6 mice. A high number of antigens (25) was observed early in infection of C57BL/6 mice by day 10, including components with m.w. of 90,000, 85,000 and 70,000 daltons. Only minor changes were detected, however, after day 20 until day 120, when increases in the number of antigens and the intensity of certain reactions (e.g., antigens of 75,000, 46,000 and 26,000 daltons) were detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ～1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.     

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop.

A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

Isolation and characterization of paraflagellar proteins from Trypanosoma cruzi

Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and characterized in this work. These two polypeptides designated PAR 1 and PAR 2, respectively, co-purified during each step of the isolation procedure and were found to be located exclusively in T. cruzi flagella by indirect immunofluorescence. A pre-embedding immunoelectron microscopy procedure, with a gold-tagged secondary antibody, permitted direct identification of PAR 2 as a component of the T. cruzi paraflagellar rod. PAR 1 and PAR 2 were found to be immunologically distinct and showed no cross-reactivity with actin, tubulin, intermediate filament proteins, or other proteins present in mammalian cells. The results presented indicate that PAR 1 and PAR 2 are the major components of T. cruzi paraflagellar filaments, and that these filaments have no counterpart in mammalian cells.     

Humoral autoimmune response in Chagas' disease: Trypanosoma cruzi ribosomal antigens as immunizing agents

Abstract Molecular expression cloning techniques have revealed that patients with chronic Chagas' heart disease (cChHD) present a strong humoral response against the cloned C-terminal portions of the four Trypanosoma cruzi ribosomal P proteins TcP1, TcP2α (TcP2b), TcP2β (TcPJL5), and TcP0. This protein family presents several features that may be important in the immunopathology of Chagas disease. Their exposed location on the ribosome, and the amplification of their parasite-specific, Ser free C-terminal domain, generate a strong anti-parasite P response that may induce anti-P autoimmunity. Evidences indicate that the serological pattern of the anti-P response from chagasic patients may be the consequence of a chronic immunization with T. cruzi ribosomal antigens.     

Isolation and characterization of a reservosome fraction from Trypanosoma cruzi

Resistance to different antibiotics was found in 26 of the 30 strains analyzed, more than 70% of the strains analyzed were resistant to carbenicillin and ampicillin and a significant correlation was found between the resistance to both antibiotics. Plasmids were found in 80% of the strains analyzed, and 11 different plasmid profiles were observed. The most common profile obtained had only a 21.2-kbp plasmid, a significant correlation was found between the presence of this plasmid and resistance to carbenicillin, although some exceptions could be detected. Plasmids were cured from a cephalothin resistant strain and reintroduced into the plasmid-free cell and into Escherichia coli DH5alpha, both strains gained resistance to this antibiotic.