DNA G-quadruplexes in the genome of Trypanosoma cruzi as potential therapeutic targets for Chagas disease: Dithienylethene ligands as effective antiparasitic agents.

Chagas disease is caused by the parasite Trypanosoma cruzi and affects over 7 million people worldwide. The two actual treatments, Benznidazole (Bzn) and Nifurtimox, cause serious side effects due to their high toxicity leading to treatment abandonment by the patients. In this work, we propose DNA G-quadruplexes (G4) as potential therapeutic targets for this infectious disease. We have found 174 PQS per 100,000 nucleotides in the genome of T. cruzi and confirmed G4 formation of three frequent motifs. We synthesized a family of 14 quadruplex ligands based in the dithienylethene (DTE) scaffold and demonstrated their binding to these identified G4 sequences. Several DTE derivatives exhibited micromolar activity against epimastigotes of four different strains of T. cruzi, in the same concentration range as Bzn. Compounds L3 and L4 presented remarkable activity against trypomastigotes, the active form in blood, of T. cruzi SOL strain (IC50 = 1.5-3.3 µM, SI = 25-40.9), being around 40 times more active than Bzn and displaying much better selectivity indexes.

Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients.

BACKGROUND: Antigen specificity and IgG subclass could be significant in the natural history of Chagas' disease. The relationship between the different stages of human Chagas' disease and the profiles of total IgG and its subclasses were thus analysed here; they were directed against a crude T. cruzi extract and three recombinant antigens: the T. cruzi kinetoplastid membrane protein-11 (rKMP-11), an internal fragment of the T. cruzi HSP-70 protein 192-433, and the entire Trypanosoma rangeli HSP-70 protein. METHODS: Seventeen Brazilian acute chagasic patients, 50 Colombian chronic chagasic patients (21 indeterminate and 29 cardiopathic patients) and 30 healthy individuals were included. Total IgG and its subtypes directed against the above-mentioned recombinant antigens were determined by ELISA tests. RESULTS: The T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins were able to distinguish both acute from chronic chagasic patients and infected people from healthy individuals. Specific antibodies to T. cruzi crude antigen in acute patients came from IgG3 and IgG4 subclasses whereas IgG1 and IgG3 were the prevalent isotypes in indeterminate and chronic chagasic patients. By contrast, the specific prominent antibodies in all disease stages against T. cruzi KMP-11 and T. rangeli HSP-70 recombinant antigens were the IgG1 subclass. CONCLUSION: T. cruzi KMP-11 and the T. rangeli HSP-70 recombinant proteins may be explored together in the immunodiagnosis of Chagas' disease. Polarising the IgG1 subclass of the IgG response to T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins could have important biological effects, taking into account that this is a complement fixing antibody.

Changes in saliva of dogs with canine leishmaniosis: A proteomic approach.

In the present study, a quantitative proteomic approach to study changes in saliva proteins associated with canine leishmaniosis (CanL) was performed. For this, canine salivary proteins were analysed and compared between dogs before (T0) and after (T1) experimental infection with Leishmania infantum by high-throughput label-based quantitative LC-MS/MS proteomic approach and bioinformatic analysis of the in silico inferred interactome protein network was created from the initial list of differential proteins. More than 2000 proteins were identified, and of the 90 differentially expressed proteins between T0 and T1, 12 were down-regulated with log2 fold change lower than -0.5849, and 19 were up-regulated with log2 fold change greater than 0.5849. This study provides evidence of changes in salivary proteome that can occur in canine leishmaniosis and revealed biological pathways in saliva modulated in canine leishmaniosis with potential for further targeted research.

Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide.

BACKGROUND: Celiac disease is an immune-mediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Immunotoxic gluten peptides that are recalcitrant to degradation of digestive enzymes appear to trigger celiac syndromes. A 33-mer peptide from alpha-2 gliadin has been identified as a principal contributor to gluten immunotoxicity. A gluten-free diet is the usual first therapy for celiac disease patients; therefore, the characterization and quantification of the toxic portion of the gluten in foodstuffs is crucial to avoid celiac damage. OBJECTIVE: We aimed to develop immunologic assays as a novel food analysis tool for measuring cereal fractions that are immunotoxic to celiac disease patients. DESIGN: The design focused on the production of monoclonal antibodies against the gliadin 33-mer peptide and the development of enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis with the use of novel antibodies. RESULTS: A sandwich ELISA method showed a detection limit for wheat, barley, and rye of <1 ppm prolamine. However, the method required a sample that was > or =1 order of magnitude greater for the detection of low-toxic oats, and there was no signal with the safe cereals maize and rice. A competitive ELISA method was also developed for detection of the toxic peptide in hydrolyzed food, which had a detection limit of <0.5 ppm gliadin. CONCLUSIONS: Both ELISAs designed for use with the toxic gliadin 33-mer peptide suggested a high correlation between the presence of the peptide and the amount of cereal that was toxic to celiac disease patients. The sensitivity was significantly higher than that of equivalent methods recognizing other gluten epitopes.

Cellular location of KMP-11 protein in Trypanosoma rangeli.

We describe the localization of the KMP-11 protein in the Trypanosoma rangeli parasite determined by immunoelectron microscopy using a monoclonal antibody generated against the Trypanosoma cruzi KMP-11 protein. The data reported herein show that the T. rangeli KMP-11 protein is mainly accumulated in the parasite cytoplasm, the coat, the flagellum, and the flagellar pocket. The high degree of sequence homology between the KMP-11 proteins from both parasites suggests that the KMP-11 protein from T. rangeli, like that of T. cruzi, could also be associated with the parasite cytoskeleton.

[Expression of markers on dendritic cells from chronic chagasic patients stimulated with the KMP-11 protein and the K1 peptide from Trypanosoma cruzi]. / Expresión de marcadores en células dendríticas de pacientes chagásicos crónicos estimuladas con la proteína KMP-11 y el péptido K1 de Trypanosoma cruzi.

INTRODUCTION: The kinetoplastid membrane protein 11, KMP-11, from Trypanosoma cruzi elicits humoral and cellular immunity in mice that protects them from infection against further parasite challenge. OBJECTIVE: To characterize the expression of surface markers on dendritic cells from chronic chagasic patients and healthy individuals, in response to the KMP-11 protein from Trypanosoma cruzi and its N-terminal peptide K1. MATERIALS AND METHODS: Monocyte-derived dendritic cells from seven chronic chagasic patients and seven healthy individuals were stimulated with the KMP-11 protein and the K1 peptide. Seven days after culturing, the CD83, CD86, and HLA-DR membrane expression as well as the production of cytokines were evaluated by flow cytometry. RESULTS: Neither KMP-11 protein nor K1 peptide elicited the expression of the maturation marker CD83 on dendritic cells of patients or healthy control individuals. Dendritic cells from chronic chagasic patients exposed to K1 and LPS at the same time presented a significant reduction in CD86 and CD83 membrane expression in contrast to the cells exposed to LPS alone, whereas dendritic cells from healthy individuals did not show this behavior. The secretion of interleukin-12 was decreased in the cultures of dendritic cells from chronic chagasic patients but not from healthy controls. CONCLUSIONS: KMP-1 1 protein does not affect the maturation of dendritic cells, but in the presence of LPS the K1 peptide leads to a decreased expression of CD86 and CD83 as well as interleukin-12 production, This phenomenon may be associated with an impaired T cell stimulation.

[Chromosomal localization of the KMP-11 genes in the KP1(+) and KP1(-) strains of Trypanosoma rangeli]. / Localización cromosómica de los genes KMP-11 en cepas KP1(+) y KP1 (-) de Trypanosoma rangeli.

Genes encoding for the KMP-11 protein were localized on the chromosomes of Trypanosoma rangeli. These genes were located in two chromosomes of 3,100 and 3,400 kb in the KP1(-) strain whereas in the KP1(+) H14 and Choachí strains, the genes are located in a chromosome of 1,600 kb. The Choachí strain presents an additional band of 1,400 kb. In the Shubacbarina and Munanta strains of Trypanosoma cruzi, the KMP-11 genes are located on a chromosomal band of 1,490 kb. Therefore, the chromosomal localization of the KMP-11 genes presents a potential tool to differentiate among these parasites.

Rabbit serum against K1 peptide, an immunogenic epitope of the Trypanosoma cruzi KMP-11, decreases parasite invasion to cells.

KMP-11 is a highly conserved protein of Trypanosoma cruzi implicated in parasite's motility. Here we show that K1, a peptide derived from KMP-11, induced polyclonal antibodies capable of decreasing T. cruzi infection in vitro. Rabbit sera rose against K1 peptide showed recognition of the recombinant protein by ELISA and Western blot and also of the native protein in both epimastigotes and trypomastigotes as evaluated by immunofluorescence test and flow cytometry. Invasion assays showed a significant reduction of trypomastigotes infection of eukaryotic cells when parasites were pre-incubated with anti-K1 rabbit serum. Computational modeling predicted that the K1 sequence conserved its α-helical configuration into the protein, and some of the amino acid residues appear accessible for recognition by antibodies in vivo. Taken together, these results support the idea that the K1 peptide induces antibodies than can have a potential role in protective immunity in Chagas disease.

Monocyte-derived dendritic cells from chagasic patients vs healthy donors secrete differential levels of IL-10 and IL-12 when stimulated with a protein fragment of Trypanosoma cruzi heat-shock protein-70.

We analyzed the effect of the truncated heat-shock protein 70 from Trypanosoma cruzi on maturation of human dendritic cells (DCs) derived from monocytes of peripheral blood mononuclear cells from healthy donors and chagasic patients. The results show that the T-HSP70 is capable of maturing human DCs inducing an increase in the expression level of the CD83, CD86 and human leukocyte antigen-DR surface markers, as well as in the secretion of interleukin (IL)-12, tumor necrosis factor-alpha (TNF-alpha) and IL-6 cytokines. Results also show the existence of a differential functional activity of matured DCs from chagasic patients vs healthy donors in response to T-HSP70 protein and to HSP-70-derived A72 peptide, as only T-HSP70-matured DCs from chagasic patients have an enhanced secretion of IL-10 and a reduced secretion of IL-12. Moreover, the addition of A72 peptide to immature DCs from chagasic patients induced an increase in the percentage of cells expressing CD83 and CD86 molecules regarding to the expression level observed by cells from healthy donors. These findings suggest that T. cruzi HSP70 protein may induce a specific maturation profile on chagasic patients' DCs, which would favor the persistence of the parasite in the human host.

Immunogenicity of HSP-70, KMP-11 and PFR-2 leishmanial antigens in the experimental model of canine visceral leishmaniasis.

Zoonotic visceral leishmaniasis (ZVL) is a parasitic disease caused by Leishmania infantum/L. chagasi that is emerging as an important medical and veterinary problem. Dogs are the domestic reservoir for this parasite and, therefore, the main target for controlling the transmission to humans. In the present work, we have evaluated the immunogenicity of the Leishmania infantum heat shock protein (HSP)-70, paraflagellar rod protein (PFR)-2 and kinetoplastida membrane protein (KMP)-11 recombinant proteins in dogs experimentally infected with the parasite. We have shown that peripheral blood mononuclear cells (PBMC) from experimentally infected dogs proliferated in response to these recombinant antigens and against the soluble leishmanial antigen (SLA). We have also quantified the mRNA expression level of the cytokines induced in PBMC upon stimulation with the HSP-70, PFR-2 and KMP-11 proteins. These recombinant proteins induced an up-regulation of IFN-gamma. HSP-70 and PFR-2 also produced an increase of the TNF-alpha transcripts abundance. No measurable induction of IL-10 was observed and low levels of IL-4 mRNA were produced in response to the three mentioned recombinant antigens. Serum levels of specific antibodies against HSP-70, PFR-2 and KMP-11 recombinant proteins were also determined in these animals. Our study showed that HSP-70, KMP-11 and PFR-2 proteins are recognized by infected canines. Furthermore, these antigens produce a Th1-type immune response, suggesting that they may be involved in protection. The identification as vaccine candidates of Leishmania antigens that elicit appropriate immune responses in the canine model is a key step in the rational approach to generate a vaccine for canine visceral leishmaniasis.