Antiproliferative activity on Trypanosoma cruzi (Y strain) of the triterpene 3ß,6ß,16ß-trihidroxilup-20 (29)-ene isolated from Combretum leprosum.

Chagas disease infects approximately seven million people worldwide. Benznidazole is effective only in the acute phase of the disease, with an average cure rate of 80% between acute and recent cases. Therefore, there is an urgent need to find new bioactive substances that can be effective against parasites without causing so many complications to the host. In this study, the triterpene 3ß-6ß-16ß-trihydroxilup-20 (29)-ene (CLF-1) was isolated from Combretum leprosum, and its molecular structure was determined by NMR and infrared spectroscopy. The CLF-1 was also evaluated in vitro and in silico as potential trypanocidal agent against epimastigote and trypomastigote forms of Trypanosoma cruzi (Y strain). The CLF-1 demonstrated good results highlighted by lower IC50 (76.0 ± 8.72 µM, 75.1 ± 11.0 µM, and 70.3 ± 45.4 µM) for epimastigotes at 24, 48 and 72 h, and LC50 (71.6 ± 11.6 µM) for trypomastigotes forms. The molecular docking study shows that the CLF-1 was able to interact with important TcGAPDH residues, suggesting that this natural compound may preferentially exert its effect by compromising the glycolytic pathway in T. cruzi. The ADMET study together with the MTT results indicated that the CLF-1 is well-absorbed in the intestine and has low toxicity. Thus, this work adds new evidence that CLF-1 can potentially be used as a candidate for the development of new options for the treatment of Chagas disease.Communicated by Ramaswamy H. Sarma.

Zoonotic parasites infecting free-living armadillos from Brazil.

Armadillos are specialist diggers and their burrows are used to find food, seek shelter and protect their pups. These burrows can also be shared with dozens of vertebrate and invertebrate species and; consequently, their parasites including the zoonotics. The aim of this study was to diagnose the presence of zoonotic parasites in four wild-caught armadillo species from two different Brazilian ecosystems, the Cerrado (Brazilian savanna) and the Pantanal (wetland). The investigated parasites and their correspondent diseases were: Toxoplasma gondii (toxoplasmosis), Trypanosoma cruzi (Chagas disease), Leishmania spp., (leishmaniasis), Paracoccidioides brasiliensis (Paracoccidioidomicosis) and Mycobacterium leprae (Hansen's disease). Forty-three free-living armadillos from Pantanal and seven road-killed armadillos from the Cerrado were sampled. Trypanosoma cruzi DTU TcIII were isolated from 2 out of 43 (4.65%) armadillos, including one of them also infected with Trypanosoma rangeli. Antibodies anti-T. gondii were detected in 13 out of 43 (30.2%) armadillos. All seven armadillos from Cerrado tested positive for P. brasiliensis DNA, in the lungs, spleen, liver fragments. Also, by molecular analysis, all 43 individuals were negative for M. leprae and Leishmania spp. Armadillos were infected by T. cruzi, T. rangeli, P. brasiliensis and presented seric antibodies to T. gondii, highlighting the importance of those armadillos could have in the epidemiology of zoonotic parasites.

Triatomines: Trypanosomatids, Bacteria, and Viruses Potential Vectors?

Triatominae bugs are the vectors of Chagas disease, a major concern to public health especially in Latin America, where vector-borne Chagas disease has undergone resurgence due mainly to diminished triatomine control in many endemic municipalities. Although the majority of Triatominae species occurs in the Americas, species belonging to the genus Linshcosteus occur in India, and species belonging to the Triatoma rubrofasciata complex have been also identified in Africa, the Middle East, South-East Asia, and in the Western Pacific. Not all of Triatominae species have been found to be infected with Trypanosoma cruzi, but the possibility of establishing vector transmission to areas where Chagas disease was previously non-endemic has increased with global population mobility. Additionally, the worldwide distribution of triatomines is concerning, as they are able to enter in contact and harbor other pathogens, leading us to wonder if they would have competence and capacity to transmit them to humans during the bite or after successful blood feeding, spreading other infectious diseases. In this review, we searched the literature for infectious agents transmitted to humans by Triatominae. There are reports suggesting that triatomines may be competent vectors for pathogens such as Serratia marcescens, Bartonella, and Mycobacterium leprae, and that triatomine infection with other microrganisms may interfere with triatomine-T. cruzi interactions, altering their competence and possibly their capacity to transmit Chagas disease.

The role of primary infection of Schwann cells in the aetiology of infective inflammatory neuropathies.

Numerous different pathogens are responsible for infective peripheral neuropathies and this is generally the result of the indirect effects of pathogen infection, namely anti pathogen antibodies cross reacting with epitopes on peripheral nerve, auto reactive T cells attacking myelin, circulating immune complexes and complement fixation. Primary infection of Schwann cells (SC) associated with peripheral nerve inflammation is rare requiring pathogens to cross the Blood Peripheral Nerve Barrier (BPNB) evade anti-pathogen innate immune pathways and invade the SC. Spirochetes Borrelia bourgdorferi and Trepomema pallidum are highly invasive, express surface lipo proteins, but despite this SC are rarely infected. However, Trypanosoma cruzi (Chaga's disease) and Mycobacterium leprae. Leprosy are two important causes of peripheral nerve infection and both demonstrate primary infection of SC. This is due to two novel strategies; T. cruzi express a trans-silalidase that mimics host neurotrophic factors and infects SC via tyrosine kinase receptors. M. leprae demonstrates multi receptor SC tropism and subsequent infection promotes nuclear reprogramming and dedifferentiation of host SC into progenitor stem like cells (pSLC) that are vulnerable to M. leprae infection. These two novel pathogen evasion strategies, involving stem cells and receptor mimicry, provide potential therapeutic targets relevant to the prevention of peripheral nerve inflammation by inhibiting primary SC infection.

Alelos HLA-DRB1 en pacientes infectados con patógenos intracelulares en Rosario, Santa Fe / HLA-DRB1 alleles in patients infected with intracellular pathogens in Rosario, Santa Fe

En el desarrollo de la respuesta inmune a patógenos intracelulares participa el elevado polimorfismo de las moléculas HLA de clase II. El objetivo de este trabajo fue establecer la participación de los alelos HLA-DRB1 en personas con infección con Trypanosoma cruzi (T. cruzi) o con Mycobacterium leprae (M. leprae). Se estudiaron 252 individuos de la ciudad de Rosario, divididos en: 86 personas seropositivas para T cruzi (sin compromiso cardiológico de relevancia), 85 pacientes con diagnóstico de Lepra y 81 individuos controles, sin evidencia de patologías. El ADN genómico fue extraído de sangre periférica utilizando el método de salting out y empleado como templado para amplificar por PCR el segundo exón polimórfico de HLA-DRB1. Los alelos fueron tipificados mediante la técnica de PCR­-SSOP. La comparación de frecuencias mostró prevalencia de los alelos DRB1 *0409 y DRB1 *1503 en los individuos seropositivos para T. cruzi con respecto al grupo control. Por otra parte, el análisis estadístico indicó una disminución significativa del alelo DRB1 *1103 en pacientes con esta tripanosomiasis. Al examinar las frecuencias observamos en el grupo de pacientes con Lepra un aumento significativo de los alelos DRB1 *1401 y DRB1 *1406. Además observamos que las proporciones de los alelos DRB1 *0808 y DRB1 *1103 en los enfermos son significativamente inferiores con respecto al grupo control. Los alelos HLA DRB1 podrían actuar solos o en combinación con otros genes para conferir susceptibilidad o resistencia a estas infecciones en la población de Rosario, Argentina.

In the development of the immune response to intracellular pathogens implicated the high polymorphism of HLA class II molecules. The aim of this study was to establish the involvement of the HLA-DRB1 alleles in infected subjects with T. cruzi or leprosy patients in Rosario, Argentina. We studied 252 individuals who divided into: 86 positive people for T. cruzi without cardiac damage, 85 patients diagnosed with leprosy and controls 81 individuals without evidence of disease. Genomic DNA was extracted from peripheral blood using the standard salting out method and used as a template to amplify by the PCR the polymorphic second exon of the HLA­-DRB1. PCR products were hybridized separately with sequence-specifics oligonucleotides (SSOP). Statistical analysis indicated that of increased frequencies of DRB1 *0409, and DRB1 *1503 in individuals with Chagas' disease. DRB1 *1103 allele was prevalence in the group control and could be associated with resistance to the presence of trypanosomiasis. DRB1 *1401 and DRB1 *1406 alleles were significantly more prevalent in leprosy patients, whereas a decreased frequency of DRB1 *0808 and DRB1 *1103 alleles was found, by comparison with the group control. The HLA-DRB1 alleles could act alone or in combination with other genes to confer differential susceptibility and also protection to these diseases in Rosario, Argentina.

Prevalence and incidence density of Mycobacterium leprae and Trypanosoma cruzi infections within a population of wild nine-banded armadillos.

A total of 415 wild 9-banded armadillos from the East Atchafalaya River Levee (Point Coupee, LA) were collected over 4 years to estimate the incidence and prevalence of Mycobacterium leprae and Trypanosoma cruzi and to discern any relationship between the 2 agents. M. leprae infections were maintained at a high steady prevalence rate year to year averaging 19%. T. cruzi antibody prevalence remained relatively low, averaging 3.9%, and varied markedly between years. Prevalence rates were independent, with only 3 armadillos coinfected with both agents. M. leprae incidence density ranged from 0.47 to 3.5 cases per 1,000 animal-days, depending on case definition, confirming active intense transmission of M. leprae among armadillos. No incident T. cruzi cases were found. These infections seem to occur independently and may be used in comparisons to understand better factors that may influence transmission of these agents.

Prevalence and incidence density of Mycobacterium leprae and trypanosoma cruzi infections within a population of wild nine-banded armadillos

A total of 415 wild 9-banded armadillos from the East Atchafalaya River Levee (Point Coupee, LA) were collected over 4 years to estimate the incidence and prevalence of Mycobacterium leprae and Trypanosoma cruzi and to discern any relationship between the 2 agents. M. leprae infections were maintained at a high steady prevalence rate year to year averaging 19 per cent. T. cruzi antibody prevalence remained relatively low, averaging 3.9 per cent, and varied markedly between years. Prevalence rates were independent, with only 3 armadillos coinfected with both agents. M. leprae incidence density ranged from 0.47 to 3.5 cases per 1,000 animal-days, depending on case definition, confirming active intense transmission of M. leprae among armadillos. No incident T. cruzi cases were found. These infections seem to occur independently and may be used in comparisons to understand better factors that may influence transmission of these agents.

Fragmentación en geles de sds-poliacrilamida e inmunotransferencia para la detección de antigenos de una cepa de trypanosoma cruzi aislada en el país y su evaluación con el método de microelisa

Por ser el Trypanosoma cruzi un protozoario de estructura bioquímica compleja presenta en su cubierta celular externa antígenos específicos de su especie y antígenos de reacción cruzada con otros microorganismos. El trypanosoama cruzi comparte el 60 por ciento de sus antígenos solubles con el trypanosoma rangeli,el 30 por ciento con lesihmania donovani y leishmania mexicana y algunos determinantes antigénos con micobacterium tuberculoso. En forma adicional evidencias ecperimentales indican que las cepas de trypanosoma cruzi aisladas a partir de diferentes áreas geográficas de Latinoamérica, se comportan de manera distinta una de otra en el campo inmunológico, clínico y farmacológico. En este estudio demostramos que en el trypanosoma cruzi existen moléculas antigénicamente específicas del parásito, las mismas que al ser utilizadas en el inmunodiagnóstico incrementa la especificiadad de la prueba. En tanro que la sensibilidad mejora al utilizar este antígeno con la técnica de microELISA, la misma que detecta cantidades de anticuerpos en el rango de manogram,os-picogramos. Al realizar el fraccionamiento de las diferentes proteínas estructurales en geles SDS-poliacrilamida de una cepa de trypanosoma cruzi aislada en el país, luego de la subsiguiente inmunotransferencia de esta electroforesis a papel de nitrocelulosa, se logró seleccionar las fracciones antigénicas de 63, 76, 80 y 91 Kd que son reconocidas específicamente por sueros de pacientes con enfermedad de chagas en su fase crónica.Con estos antígenos se estandarizó el inmunodiagnóstico con la prueba de micro elisa. En este inmunoensayo se puede diferenciar con un amplio rango de diferencia las densidades ópticas (D:O) de los sueros provenientes de pacientes chagásicos (D.O 1,797), en relación a los sueros provenientes de pacientes chagásicos (D.O. 1797), en relación a los sueros provenientes de pacientes con leishmaniasis D.O 0.0099),lepra (D.O 0.012), tuberculosis (D.O 0.092), sífilis (D.O 0.067), toxoplasmosis (D.O 0.024). Quedando demostrado la especificidad de las bandas seleccionadas para reconocer sueros de pacientes chagásicos. En forma adicional, en relación a estas moléculas, es importante determinar su naturaleza bioquímica y su papel biológico en la fisiología de trypanonoma cruzi y en la relación huésped-parásito con miras de buscar nuevas estrategias de lucha frente a la enfermedad de chagas.

The flagellar fraction of Trypanosoma cruzi depleted of an immunosuppressive antigen enhances protection to infection and elicits spontaneous T cell responses.

The flagellar fraction (FF) of Trypanosoma cruzi can be separated by immunoaffinity chromatography in two fractions with balanced but opposite immunological effects. The immunoaffinity purified fraction has immunosuppressive activity mediated at least partially by TGF-beta (Hansen et al., submitted). Here we report that the fraction depleted of immunosuppresive antigens (FT) administered with iscom-matrix as adjuvant provides enhanced protection to an infection challenge in immunized mice. In vitro, the FT but not the FF stimulated resident peritoneal cells to produce IL-1 and IL-6. In immunized mice, the FT elicited higher levels of antigen-specific IgG2a than the FF as well as broader recognition of T. cruzi antigens. Splenocytes from mice immunized with FT proliferated spontaneously in vitro and secreted TH1 and TH2 cytokines. The protection provided by FT correlates with its capacity to enhance the secretion of IFN-gamma. We postulate that immunosuppressive antigens present in the FF prevent the development of memory cells secreting IFN-gamma through a TGF-beta dependent mechanism.

Cellular immune response to common mycobacterial antigens in subjects seropositive for Trypanosoma cruzi.

The immune response is impaired in the silent stage of Chagas' disease. We used quadruple skin-testing with new tuberculins in 37 adults who were symptom-free but seropositive for Trypanosoma cruzi and in 37 matched seronegative controls. Whereas 19% of controls responded to common mycobacterial antigens, none of the Chagas' seropositive group responded to them (p < 0.006), demonstrating specificity in their unresponsiveness. The enhanced tuberculin reactivity after BCG vaccination in the control group was suppressed in seropositive subjects (p < 0.002). Selective loss of response to common mycobacterial antigens may have implications for the autoimmune pathology of Chagas' disease, and for susceptibility to tuberculosis, leprosy, and HIV disease.

Serodiagnosis of Chagas' disease by enzyme-linked immunosorbent assay using two synthetic peptides as antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against Trypanosoma cruzi. Two synthetic T. cruzi peptides, TcD and PEP2, were used. The specificity and sensitivity of the peptide ELISA were determined with 260 serum samples from individuals living in an area in which Chagas' disease is endemic. ELISAs were performed with the peptides singly or in combination. The evaluation of these tests showed that 168 (93.8%) of 179 serum samples from T. cruzi-infected patients were positive when TcD peptide was used as antigen; 164 (91.6%) samples were positive with PEP2, and 178 (99.4%) samples were positive when the two peptides were combined. Thus, the sensitivity of the ELISA using the two peptides exceeded 99%. The specificity was evaluated by using a panel of 118 serum samples that included samples from 81 individuals living in an area of endemicity with negative serology for Chagas' disease and from 37 patients from areas in which T. cruzi was not endemic but with other pathologies, such as leishmaniasis, tuberculosis, and leprosy. Only two false-positive serum samples were found in this group of individuals, giving a test specificity of more than 98%. Because these peptides can be synthesized and are very stable at room temperature, the use of such reagents can improve the standardization and reproducibility of ELISAs for the serodiagnosis of T. cruzi infection.

Trypsin-treated and coomassie blue-stained epimastigote antigen in a microagglutination test for Chagas' disease.

A microagglutination test using trypsin-treated and Coomassie blue-stained Trypanosoma cruzi epimastigote antigen was adapted for the diagnosis of Chagas' disease. When incorporated in the test, 2-mercaptoethanol treatment of chagasic sera had no influence on antibody titer. In contrast, titers in sera from patients with visceral leishmaniasis, African trypanosomiasis, and autoimmune disorders, subjected to similar treatment, showed remarkable decline. Accordingly, a lower cut-off point for Chagas' disease serological negativity could be taken resulting in a higher sensitivity (95.6%); the specificity was 94.7%. Similar specificities were obtained with Leishmania donovani chagasi and L. d. donovani antigens applied to homologous visceral leishmaniasis and heterologous Chagas' sera. Of 316 nonchagasic sera, only 3 with leptospirosis and 1 with leprosy showed seropositive titers prior to and after 2-mercaptoethanol treatment.

Presenca de inibidor específico de fixaçào de complemento : Em antígenos preparados de trypanosoma cruzi / Presence of specific complement-fixation inhibitor in antigens prepared from trypanosoma cruzi

Tritation performed by the quantitative complement fixation method on sera from parasitologically confirmed Chagas' disease eases using an aqueous of a methylic extract of Tripanosoma cruzi as antigen, revealed a marked discrepancy in titers. The serum titer was expressed by the slope of the regresion line between amounts of serum and number of complement units needed for 50 per cent hemolysis

In every case, the highest titers were obtained with the methylic antigen. It was observed also that the aqueous antigen, when added to the methylic antigen, produced a significant drop in titer. Everything suggested the presence of an hapten, able to combine specifically with Chagasic antibodies to form immune complexes which do not fix complement

Since the inhibiter was soluble in water and almost insoluble in methanol, it could be isolated as an aqueous extract from trypanosomes previously treated by methanol. This hapten gave very weak complement fixation reactions with Chagasic sera. However, when added to the methylic antigen, a strong inhibition was observed. This inhibition was observed only with Chagasic sera, and not with sera from cases of syphilis, brucellosis, leprosy or schistosomiasis

An antigen free of inhibiter can be prepared by treating the dried trypanosomes, previously delipidized by benezen, with water saturated with chloroform. The extract trypanosomes are dried again and ...(AU)

Presenca de inibidor específico de fixação de complemento : Em antígenos preparados de trypanosoma cruzi / Presence of specific complement-fixation inhibitor in antigens prepared from trypanosoma cruzi

Tritation performed by the quantitative complement fixation method on sera from parasitologically confirmed Chagas' disease eases using an aqueous of a methylic extract of Tripanosoma cruzi as antigen, revealed a marked discrepancy in titers. The serum titer was expressed by the slope of the regresion line between amounts of serum and number of complement units needed for 50 per cent hemolysis

Since the inhibiter was soluble in water and almost insoluble in methanol, it could be isolated as an aqueous extract from trypanosomes previously treated by methanol. This hapten gave very weak complement fixation reactions with Chagasic sera. However, when added to the methylic antigen, a strong inhibition was observed. This inhibition was observed only with Chagasic sera, and not with sera from cases of syphilis, brucellosis, leprosy or schistosomiasis

An antigen free of inhibiter can be prepared by treating the dried trypanosomes, previously delipidized by benezen, with water saturated with chloroform. The extract trypanosomes are dried again and ...(AU)

In every case, the highest titers were obtained with the methylic antigen. It was observed also that the aqueous antigen, when added to the methylic antigen, produced a significant drop in titer. Everything suggested the presence of an hapten, able to combine specifically with Chagasic antibodies to form immune complexes which do not fix complement