Peroxiredoxins from Trypanosoma cruzi: virulence factors and drug targets for treatment of Chagas disease?

Cytosolic and mitochondrial Trypanosoma cruzi tryparedoxin peroxidases belong to the family of 2-Cys peroxiredoxins. These enzymes play an essential role as antioxidants by their peroxidase and peroxynitrite reductase activities. TXNPx are key components of the trypanosomatid peroxide detoxification pathways. The aim of this work was to determine the role of TXNPx as virulence factors in the parasite, and whether these enzymes are good candidates for drug design. We observed that peroxiredoxins are not highly abundant proteins expressed at similar levels throughout the T. cruzi life cycle. In order to study the role of c-TXNPx and m-TXNPx in invasion and infectivity, parasites overexpressing TXNPx were produced, and infection experiments were carried out using phagocytic and non-phagocytic cells. Parasites overexpressing peroxiredoxins showed a significant increase in infectivity with respect to the control ones. The results presented in this work point out that the T. cruzi peroxiredoxins are important in survival, replication and differentiation of T. cruzi and could constitute virulence factors. Moreover, their expression in the infective forms of the life cycle and their low intracellular concentration make them good candidates to become targets for drug design.     

IL-10 and IFN-γ gene expression in chronic Chagas disease patients after in vitro stimulation with recombinant antigens of Trypanosoma cruzi

Along with several other aspects of Chagas disease, the mechanisms responsible for the different clinical outcomes observed in chronic infected individuals have not yet been clarified. It is believed that the host immune response to the parasite plays an important role in the development of the pathology. Therefore, the aim of this study was to evaluate the relationship between IL-10 and IFN-γ gene expression profile, after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with Trypanosoma cruzi recombinant antigens CRA (cytoplasmatic repetitive antigen) and FRA (flagellar repetitive antigen), and the clinical forms of chronic Chagas disease. Twenty patients with the cardiac form of the disease (CARD), of whom 10 had the mild cardiac form (CARD 1) and 10 the severe cardiac form (CARD 2), and 20 patients with the indeterminate form (IND), were selected at the Chagas Disease Unit of the Oswaldo Cruz University Hospital, University of Pernambuco, Recife, Pernambuco, Brazil. The PBMCs of these individuals were cultured in the presence of CRA or FRA for 3 days and IL-10 and IFN-γ gene expression was evaluated by detection of its messenger RNA using Real Time Quantitative PCR. Although no significant difference was observed between the groups of individuals studied, we found that most patients with IND displayed high levels of IFN-γ gene expression, while the majority of patients with CARD 1 presented high levels of IL-10. The results of this study thus highlight the important role that inflammatory cytokines play in patients with the IND group controlling for parasite replication, and that anti-inflammatory cytokines play in determining susceptibility to progression to symptomatic clinical forms of the disease.     

Pre-Columbian Chagas disease in Brazil: Trypanosoma cruzi I in the archaeological remains of a human in Peruaçu Valley, Minas Gerais, Brazil

We evaluated the presence and distribution of Trypanosoma cruzi DNA in a mummy presenting with megacolon that was dated as approximately 560 +/- 40 years old. The mummy was from the Perua?u Valley in the state of Minas Gerais, Brazil. All samples were positive for T. cruzi minicircle DNA, demonstrating the presence and broad dissemination of the parasite in this body. From one sample, a mini-exon gene fragment was recovered and characterized by sequencing and was found to belong to the T. cruzi I genotype. This finding suggests that T. cruzi I infected humans during the pre-Columbian times and that, in addition to T. cruzi infection, Chagas disease in Brazil most likely preceded European colonization.     

Trypanosoma cruzi infection of human induced pluripotent stem cell-derived cardiomyocytes: an in vitro model for drug screening for Chagas disease

Chagas disease, caused by Trypanosoma cruzi, is an important global public health problem which, despite partial efficacy of benznidazole (Bz) in acute phase, urgently needs an effective treatment. Cardiotoxicity is a major safety concern for conduction of more accurate preclinical drug screening platforms. Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) are a reliable model to study genetic and infectious cardiac alterations and may improve drug development. Herein, we introduce hiPSC-CM as a suitable model to study T. cruzi heart infection and to predict the safety and efficacy of anti-T. cruzi drugs.     

Identification by Q-PCR of Trypanosoma cruzi lineage and determination of blood meal sources in triatomine gut samples in México

Triatomine vectors were collected on human dwellings in Michoacán México. Blood meal sources were identified by real time polymerase chain reaction (Q-PCR) using DNA extracted from triatomine guts. The assay was performed with one only specific primer set to amplify a fragment of the mitochondrial 12S ribosomal gene from vertebrate species. Also Trypanosoma cruzi parasites were detected in triatomine gut samples by microscopy and the positive infection was tested in mice. In addition T. cruzi discrete taxonomic units (DTUs) were identified by Q-PCR with two sets of primers that amplify the mini-circle region (miniexon) and 18S ribosomal mitochondrial gene. The sequences obtained from 18S ribosomal gene amplifications confirmed the presence of T. cruzi I and II lineages, and provide evidence of the presence of lineage TcIII and TcIV.     

Epidemiology of Chagas disease in Jaguaruana, Ceará, Brazil. I. Presence of triatomines and index of Trypanosoma cruzi infection in four localities of a rural area

In order to assay the triatomine infestation and domiciliation in the rural area of Jaguaruana district, state of Ceará, Brazil, we studied, from November 2000 to April 2002, 4 localities comprising 158 domiciles as a whole, with an average of 4 inhabitants/house, who are dwelling in there for more than 7 years. Most houses have tile-covered roofs and the walls built with plaster-covered bricks (57%), followed by bricks without plaster (33%), and mud walls (7.5%). A total of 3082 triatomines were captured from different locations, according to the following capture plan: (a). intradomiciles: 238 Triatoma brasiliensis, 6 T. pseudomaculata, 9 Rhodnius nasutus, and 2 Panstrongylus lutzi; (b). peridomiciles (annexes): 2069 T. brasiliensis, 223 T. pseudomaculata, 121 R. nasutus, and 1 P. lutzi; (c). wild, in carnauba palms (Copernicia prunifera): 413 R. nasutus. From the captured triatomines, 1773 (57.5%) were examined. The natural index of Trypanosoma cruzi infection ranged from 10.8% to 30.2% (average of 17%), depending on the species and the location from where the triatomines were captured.     

The challenge of Chagas’ disease: Has the human pathogen,Trypanosoma cruzi,learned how to modulate signaling events to subvert host cells?

Chagas’ disease, caused by Trypanosoma cruzi, is an urgent and highly prevalent danger that is endemic to Latin America, and which the research community continues to ignore. Each year, Chagas’ disease kills more people in Latin America compared to any other parasite-borne disease, including malaria. In addition, between 15 and 18 million people worldwide are afflicted with this potentially lethal disease. Despite these devastating numbers, less than 0.5% of worldwide research and development for neglected diseases was aimed at Chagas’ disease. The aim of this review is to draw the attention of biotechnologists to the intriguing parasite that causes Chagas’ disease, which is T. cruzi. Additionally, we would also like to convince the community that basic science research can have a profound impact on the diagnosis and treatment of Chagas’ disease. In this review, we introduce distinct features of T. cruzi such as its complex life cycle (e.g. the potentially infective extracellular amastigote form), its genome and genomics, as well as proteomic analysis of this parasite. Notably, the PIK pathway has been widely acknowledged as an excellent target for drug discovery to combat this pathogen. Furthermore we also describe how the identification and characterization of PIK genes can aid in neutralizing Trypanosoma infections.     

Serum heat shock protein 70 and oxidized LDL in patients with type 2 diabetes: does sex matter?

Several studies suggest that the response to various stressors differs between the sexes. We aimed to study serum HSP70 and levels of oxidized-LDL (ox-LDL) as markers of oxidative stress in men and women with type 2 diabetes. We quantified serum HSP70 and levels of ox-LDL in three cohorts; patients with newly diagnosed diabetes, patients with long-standing diabetes and normal controls. The cohort of patients with newly diagnosed diabetes was followed up for 3 months under glucose-lowering therapy with metformin. Our findings showed that serum HSP70 level was increased in women with long-standing diabetes in comparison with men. HSP70 did not decrease after glucose lowering therapy in women with newly diagnosed diabetes, but it did decrease in men. There was no significant difference on ox-LDL between men and women in any of the studied cohorts. It decreased significantly in the cohort of patients with newly diagnosed diabetes after treatment, regardless of sex. There was no significant correlation between HSP70 and ox-LDL in any of the studied cohorts except among normal women. We suggest that diabetes induces an immune response and impairs cellular defense mechanisms against oxidative stress more commonly in women with type 2 diabetes than in men.     

Distribution and infection of triatomines (Hemiptera: Reduviidae) by Trypanosoma cruzi in the state of Michoacán, Mexico

An entomological study of triatomine species was carried out to assess their prevalence in 10 localities of the state of Michoacán, Mexico. Entomological indices were calculated to estimate the risk for vector-borne transmission of Trypanosoma cruzi to the human population in this area. Four triatomine species (Triatoma barberi, Triatoma dimidiata, Meccus pallidipennis and Meccus longipennis) were collected from the study area. This is the first report of M. longipennis and T. dimidiata in Michoacán. M. pallidipennis was significantly (p < 0.05) more abundant than any of the other species collected in the study area. Infection indices were greater than 50% for each of the four collected triatomine species. Significantly more triatomines were collected from intradomiciliary areas than from peridomiciliary or sylvatic areas. Infestation, crowding and density indices were low, whereas colonisation indices were high in five localities. The current vectorial conditions in the study area require continuous entomological and serological surveillance to diminish the risk of T. cruzi transmission to human populations.     

Metal chelatorNNN’ N’-tetrakis-(2-pyridylmethyl)ethylene diamine inhibits the induction of heat shock protein 70 synthesis by heat in cultured keratinocytes

Heat shock protein (HSP) synthesis results from various types of injury, including heat shock (HS) and some oxidants. The intracellular signals leading to HSP synthesis are not yet fully elucidated. We have studied the influence ofNNN’N’-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a metal chelator known to induce cellular zinc and copper deprivation, on resistance to heat and on hsp70 synthesis in HaCaT keratinocytes. TPEN was shown to sensitize HaCaT cells to heat shock. The effect of TPEN was neutralized by equimolar Zn²⁺. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blotting characterization of hsp70, it was shown that cultured HaCaT cells constitutively express the inducible form of hsp70. The application of TPEN alone slightly increases the level of hsp70 but inhibits its induction by HS. This inhibitory effect is related to metal deprivation, because it is eliminated when Cu²⁺ or Zn²⁺ ions are supplied together with TPEN. These results suggest that these metals are involved in the expression by keratinocytes of a stress protein which has a protective action against environmental stress.     

Selection of HLA-B57-associated Gag A146P mutant by HLA-B∗48:01-restricted Gag140-147-specific CTLs in chronically HIV-1-infected Japanese

We previously showed the possibility that Gag A146P, which is an escape mutant from HLA-B157-restricted CTLs, was selected by HLA-B148:01-restricted Gag138–147(LI10)-specific CTLs in a Japanese cohort in which HLA-B157 individuals were not detected. We herein demonstrated Gag140–147(GI8) to be the optimal epitope rather than LI10 and that GI8-specific T cells failed to recognize the A146P mutant virus-infected cells. The sequence analysis of Gag146 in 261 chronically HIV-1-infected Japanese showed the accumulation of the A146P mutation in HLA-B148:01⁺ individuals. These findings together indicate that the A146P mutant is accumulating in Japanese by selection by GI8-specific CTLs.     

Exact break point of a 50?kb deletion 8?kb centromeric of the HLA-A locus with HLA-A*24:02: the same deletion observed in other A*24 alleles and A*23:01 allele

In a structural aberration analysis of patients with arthritis mutilans, a 50?kb deletion near the HLA-A locus with HLA-A*24:02 allele was detected. It was previously reported that HLA-A*24:02 haplotype harbored a large-scale deletion telomeric of the HLA-A gene in healthy individuals. In order to confirm that the deletion are the same in patients with arthritis mutilans and in healthy individuals, and to identify the break point of this deletion, the boundary sequences across the deletion in A*24:02 was amplified by polymerase chain reaction (PCR) as a 3.7?kb genomic fragment and subjected to nucleotide sequence determination. A comparison of these genomic sequences with those of the non-A*24:02 haplotype revealed that the deleted genomic region spanning 50?kb was flanked by 3.7?kb repetitive element-rich segments homologous to each other on both sides in non-A*24. The nucleotide sequences of the PCR products were identical in patients with arthritis mutilans and in healthy individuals, revealing that the deletion linked to A*24:02 is irrelevant to the onset of arthritis mutilans. The deletion was detected in all other A*24 alleles so far examined but not in other HLA-A alleles, except A*23:01. This finding, along with the phylogenic tree of HLA-A alleles and the presence of the 3.7?kb highly homologous segments at the boundary of the deleted genomic region in A*03 and A*32, may suggest that this HLA-A*24:02-linked deletion was generated by homologous recombination within two 3.7?kb homologous segments situated 50?kb apart in the ancestral A*24 haplotype after divergence from the A*03 and A*32 haplotypes.     

Theiler’s murine encephalomyelitis virus infection induces a redistribution of heat shock proteins 70 and 90 in BHK-21 cells,and is inhibited by novobiocin and geldanamycin

Theiler’s murine encephalomyelitis virus (TMEV) is a positive-sense RNA virus belonging to the Cardiovirus genus in the family Picornaviridae. In addition to other host cellular factors and pathways, picornaviruses utilise heat shock proteins (Hsps) to facilitate their propagation in cells. This study investigated the localisation of Hsps 70 and 90 in TMEV-infected BHK-21 cells by indirect immunofluorescence and confocal microscopy. The effect of Hsp90 inhibitors novobiocin (Nov) and geldanamycin (GA) on the development of cytopathic effect (CPE) induced by infection was also examined. Hsp90 staining was uniformly distributed in the cytoplasm of uninfected cells but was found concentrated in the perinuclear region during late infection where it overlapped with the signal for non-structural protein 2C within the viral replication complex. Hsp70 redistributed into the vicinity of the viral replication complex during late infection, but its distribution did not overlap with that of 2C. Inhibition of Hsp90 by GA and Nov had a negative effect on virus growth over a 48-h period as indicated by no observable CPE in treated compared to untreated cells. 2C was detected by Western analysis of GA-treated infected cell lysates at doses between 0.01 and 0.125 μM, suggesting that processing of viral precursors was not affected in the presence of this drug. In contrast, 2C was absent in cell lysates of Nov-treated cells at doses above 10 μM, although CPE was evident 48 hpi. This is the first study describing the dynamic behaviour of Hsps 70 and 90 in TMEV-infected cells and to identify Hsp90 as an important host factor in the life cycle of this virus.     

Comparative analysis by polymerase chain reaction amplified minicircles of kinetoplast DNA of a stable strain of Trypanosoma cruzi from São Felipe, Bahia, its clones and subclones: possibility of predominance of a principal clone in this area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of S?o Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicircles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment length polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%. This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.     

Escape in one of two cytotoxic T-lymphocyte epitopes bound by a high-frequency major histocompatibility complex class I molecule,Mamu-A*02: a paradigm for virus evolution and persistence?

It is now accepted that an effective vaccine against AIDS must include effective cytotoxic-T-lymphocyte (CTL) responses. The simian immunodeficiency virus (SIV)-infected rhesus macaque is the best available animal model for AIDS, but analysis of macaque CTL responses has hitherto focused mainly on epitopes bound by a single major histocompatibility complex (MHC) class I molecule, Mamu-A*01. The availability of Mamu-A*01-positive macaques for vaccine studies is therefore severely limited. Furthermore, it is becoming clear that different CTL responses are able to control immunodeficiency virus replication with varying success, making it a priority to identify and analyze CTL responses restricted by common MHC class I molecules other than Mamu-A*01. Here we describe two novel epitopes derived from SIV, one from Gag (Gag(71-79) GY9), and one from the Nef protein (Nef(159-167) YY9). Both epitopes are bound by the common macaque MHC class I molecule, Mamu-A*02. The sequences of these two eptiopes are consistent with the molecule's peptide-binding motif, which we have defined by elution of natural ligands from Mamu-A*02. Strikingly, we found evidence for the selection of escape variant viruses by CTL specific for Nef(159-167) YY9 in 6 of 6 Mamu-A*02-positive animals. In contrast, viral sequences encoding the Gag(71-79) GY9 epitope remained intact in each animal. This situation is reminiscent of Mamu-A*01-restricted CTL that recognize Tat(28-35) SL8, which reproducibly selects for escape variants during acute infection, and Gag(181-189) CM9, which does not. Differential selection by CTL may therefore be a paradigm of immunodeficiency virus infection.