Maria Carolina Accioly Brelaz de Castro

2-Pyridyl thiazoles as novel anti-Trypanosoma cruzi agents: structural design, synthesis and pharmacological evaluation.

The present work reports on the synthesis, anti-Trypanosoma cruzi activities and docking studies of a novel series of 2-(pyridin-2-yl)-1,3-thiazoles derived from 2-pyridine thiosemicarbazone. The majority of these compounds are potent cruzain inhibitors and showed excellent inhibition on the trypomastigote form of the parasite, and the resulting structure-activity relationships are discussed. Together, these data present a novel series of thiazolyl hydrazones with potential effects against Chagas disease and they could be important leads in continuing development against Chagas disease.

Immunomodulatory response of Cramoll 1,4 lectin on experimental lymphocytes

Cramoll 1,4 is a lectin extracted from Cratylia mollis Mart. seeds that has shown antitumor and lymphocyte mitogenic activities in other studies. The aim of this work was to investigate, in vitro, the immunomodulatory activity of Cramoll 1,4 on experimental cultures of mice lymphocytes through cytotoxic assays, nitric oxide (NO) concentrations and IL‐10 and IFN‐γ production. Cramoll 1,4 did not show cytotoxic activity at 1–25 μg/mL concentrations, similar results were observed with concanavalin A (Con A) and phytohemagglutinin (PHA) lectins. The minimum production of IL‐10 was observed in splenocytes cultivated with Con A, PHA and Cramoll 1,4 lectins. However, splenocytes treated with Cramoll 1,4 showed higher IFN‐γ production in comparison with PHA and Con A (p < 0.05 for both). Production of NO was effectively suppressed in murine cells stimulated with the lectins and was only detected after 72 h for PHA in relation to non‐stimulated lymphocytes (p < 0.05). Cramoll 1,4 was not toxic to murine lymphocytes, induced Th1 response through IFN‐γ production and showed antiinflammatory activity through NO suppression. Therefore, Cramoll 1,4 can be considered a lectin with immunomodulatory activity. Copyright

Ruthenium complexes endowed with potent anti-Trypanosoma cruzi activity: Synthesis, biological characterization and structure–activity relationships

Although effective against epimastigotes (proliferative form) and of low cytotoxicity in mammals, the aryl-4-oxothiazolylhydrazones (ATZ) display only limited activity against trypomastigotes (bloodstream form) of Trypanosoma cruzi. Considering the metal complexation approach with bioactive ligands as one possible strategy for improving the biological efficacy of ATZ, a set of eight new ruthenium-ATZ complexes (RuCl(2)ATZCOD, COD is 1,5-cyclooctadiene) were prepared, chemically and biologically characterized, including in vitro assays against epimastigotes and trypomastigote forms of the parasite and also assessment of cytotoxicity in mammals. Two of these complexes presented antitrypanosomal activity at non-cytotoxic concentrations on mammalian cells and of higher potency than its metal-free ligands, while the metallic precursor [RuCl(2)COD(MeCN)(2)] showed only moderate antitrypanosomal activity. Comparative analysis between the ruthenium complexes and metal-free ligands demonstrated the usefulness of this approach, with the establishment of new SAR data. Additional pharmacological tests, including a DNA bond assay, gave rise to the proposal of a single preliminary explanation for the molecular origin of the bioactivity.

Conformational restriction of aryl thiosemicarbazones produces potent and selective anti-Trypanosoma cruzi compounds which induce apoptotic parasite death

Chagas disease, caused by Trypanosoma cruzi, is a life-threatening infection leading to approximately 12,000 deaths per year. T. cruzi is susceptible to thiosemicarbazones, making this class of compounds appealing for drug development. Previously, the homologation of aryl thiosemicarbazones resulted in an increase in anti-T. cruzi activity in comparison to aryl thiosemicarbazones without a spacer group. Here, we report the structural planning, synthesis and anti-T. cruzi evaluation of new aryl thiosemicarbazones (9a-x), designed as more conformationally restricted compounds. By varying substituents attached to the phenyl ring, substituents were observed to retain, enhance or greatly increase the anti-T. cruzi activity, in comparison to the nonsubstituted derivative. In most cases, hydrophobic and bulky substituents, such as bromo, biphenyl and phenoxyl groups, greatly increased antiparasitic activity. Specifically, thiosemicarbazones were identified that inhibit the epimastigote proliferation and were toxic for trypomastigotes without affecting mouse splenocytes viability. The most potent anti-T. cruzi thiosemicarbazones were evaluated against cruzain. However, inhibition of this enzyme was not observed, suggesting that the compounds work through another mechanism. In addition, examination of T. cruzi cell death showed that these thiosemicarbazones induce apoptosis. In conclusion, the structural design executed within the series of aryl thiosemicarbazones (9a-x) led to the identification of new potent anti-T. cruzi agents, such as compounds (9h) and (9r), which greatly inhibited epimastigote proliferation, and demonstrated a toxicity for trypomastigotes, but not for splenocytes. Mechanistically, these compounds do not inhibit the cruzain, but induce T. cruzi cell death by an apoptotic process.

Cytokines and NO in American tegumentary leishmaniasis patients: profiles in active disease, after therapy and in self-healed individuals

Studies suggest the influence of immune response on the successful treatment of American tegumentary leishmaniasis (ATL), and indicate the existence of protective immunity in self-healed patients. Thus, the aim of this work was to quantify interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL-) 10, IL-17, IL-22 and nitric oxide (NO) in culture supernatants of PBMC from patients with active disease (AD), after treatment (AT), and from self-healed (SH) and healthy subjects (CT), in response to Leishmania (Viannia) braziliensis insoluble antigen (AgIns). All groups of patients produced IFN-γ, indicating a predominant proinflammatory profile. AD and AT patients presented TNF-α levels, with a slight increase after therapy, whereas it was weakly quantified in SH. Interestingly, NO secretion was significant in these individuals, whereas IL-17 appeared in low levels and seems to be regulated by NO. Although IL-22 was detected in AD, its role is still questionable. The presence of IL-10 in all groups of patients suggests that the cytokine plays distinct roles in the disease. These results indicate that specific cellular immunity takes part against Leishmania, but with some similarities between the different clinical states herein described; these mediators seem to be necessary for the cure to occur.

American Tegumentary Leishmaniasis: Cytokines and Nitric Oxide in Active Disease and After Clinical Cure, With or Without Chemotherapy

The influence of immune response on the treatment of American tegumentary leishmaniasis is pointed by several authors, and the existence of protective immunity in self‐healed patients (SH) is also suggested. Thus, interferon‐gamma (IFN‐γ), tumour necrosis factor‐alpha (TNF‐α), interleukin (IL‐) 10, IL‐17, IL‐22 and nitric oxide (NO) production was determined in PBMC culture supernatants from patients with active disease (AD) and after therapy, SH patients and healthy subjects, in response to the soluble antigen of Leishmania (Viannia) braziliensis. It was demonstrated that, during the active disease, there is a predominance of IFN‐γ and TNF‐α, indicating a proinflammatory phase of the response; IL‐17 is also highlighted at this clinical state. Also, TNF‐α was slightly increased in patients after therapy. NO secretion was noticed in SH individuals, while IL‐17 appeared in low levels in these patients and seems to be regulated by NO. The presence of IL‐10 was observed in all groups of patients. From this study, we can suggest that in the active disease and after clinical cure, with or without chemotherapy, specific cellular immunity takes part against Leishmania, but with some similarities between the clinical states. Thus, it indicates that the mediators herein described are necessary for the cure to occur.

Trypanosoma cruzi Cell Death Induced by the Morita-Baylis-Hillman Adduct 3-Hydroxy-2-Methylene-3-(4-Nitrophenylpropanenitrile).

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a serious health concern due to the lack of effective vaccines or satisfactory treatment. In the search for new compounds against this neglected disease, we have previously demonstrated that the compound 3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile) (MBHA3), derived from the Morita-Baylis-Hillman reaction, effectively caused a loss of viability in both the epimastigote and trypomastigote forms. However, the mechanisms of parasite death elicited by MBHA3 remain unknown. The aim of this study was to better understand the morphophysiological changes and the mechanism of cell death induced by MBHA3 treatment on T. cruzi. To perform this analysis, we used confocal microscopy and flow cytometry to monitor the fluorescent probes such as annexin-V/propidium iodide (AV/PI), calcein-AM/ethidium homodimer (CA/EH), acridine orange (AO) and rhodamine 123 (Rho 123). Lower concentrations of MBHA3 led to alterations in the mitochondrial membrane potential and AO labeling, but did not decrease the viability of the epimastiogote forms, as determined by the CA/EH and AV/PI assays. Conversely, treatment with higher concentrations of MBHA3 led to extensive plasma membrane damage, loss of mitochondrion membrane potential, DNA fragmentation and acidification of the cytoplasm. Our findings suggest that at higher concentrations, MBHA3 induces T. cruzi epimastigote death by necrosis in a mitochondrion-dependent manner.

Comparison of flow cytometry and indirect immunofluorescence assay in the diagnosis and cure criterion after therapy of American tegumentary leishmaniasis by anti-live Leishmania (Viannia) braziliensis immunoglobulin G

The aim of this study was to compare the techniques of indirect immunofluorescence assay (IFA) and flow cytometry to clinical and laboratorial evaluation of patients before and after clinical cure and to evaluate the applicability of flow cytometry in post-therapeutic monitoring of patients with American tegumentary leishmaniasis (ATL). Sera from 14 patients before treatment (BT), 13 patients 1 year after treatment (AT), 10 patients 2 and 5 years AT were evaluated. The results from flow cytometry were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). The 1:256 sample dilution allowed us to differentiate individuals BT and AT. Comparative analysis of IFA and flow cytometry by ROC (receiver operating characteristic curve) showed, respectively, AUC (area under curve)=0.8 (95% CI=0.64-0.89) and AUC=0.90 (95% CI=0.75-0.95), demonstrating that the flow cytometry had equivalent accuracy. Our data demonstrated that 20% was the best cut-off point identified by the ROC curve for the flow cytometry assay. This test showed a sensitivity of 86% and specificity of 77% while the IFA had a sensitivity of 78% and specificity of 85%. The after-treatment screening, through comparative analysis of the technique performance indexes, 1, 2 and 5 years AT, showed an equal performance of the flow cytometry compared with the IFA. However, flow cytometry shows to be a better diagnostic alternative when applied to the study of ATL in the cure criterion. The information obtained in this work opens perspectives to monitor cure after treatment of ATL.

pCramoll and rCramoll as New Preventive Agents against the Oxidative Dysfunction Induced by Hydrogen Peroxide

Oxidative stress plays an important role in the induction of cell death and is associated with various pathologic disorders; therefore, the search for natural products that attenuate the effects produced by oxidant agents is greatly increased. Here, the protective effects of native lectin from Cratylia mollis seeds (pCramoll) and recombinant Cramoll 1 (rCramoll) against H2O2-induced oxidative stress in Vero cells were evaluated. Both lectins significantly attenuated the H2O2-induced cytotoxicity in a concentration-dependent way. The maximum protective effects were 96.85 ± 15.59% (rCramoll) and 59.48 ± 23.44% (pCramoll). The Live/Dead analysis showed a reduction in the percentage of dead cells from 65.04 ± 3.29% (H2O2) to 39.77 ± 2.93% (pCramoll) and 13.90 ± 9.01% (rCramoll). The deleterious effects of H2O2 on cell proliferation were reduced to 10.83% (pCramoll) and 24.17% (rCramoll). Lectins treatment attenuated the excessive superoxide production, the collapse of the mitochondrial membrane potential, and the lysosomal and DNA damage in H2O2-treated cells. In conclusion, our results suggest that pCramoll and rCramoll blocked H2O2-induced cytotoxicity through decreasing reactive oxygen species, restoring the mitochondrial potential, preventing the lysosomal damage and DNA fragmentation, and thus promoting cell survival and proliferation.

Bone marrow-derived monocyte infusion improves hepatic fibrosis by decreasing osteopontin, TGF-β1, IL-13 and oxidative stress

AIM To evaluate the therapeutic effects of bone marrow-derived CD11b+CD14+ monocytes in a murine model of chronic liver damage. METHODS Chronic liver damage was induced in C57BL/6 mice by administration of carbon tetrachloride and ethanol for 6 mo. Bone marrow-derived monocytes isolated by immunomagnetic separation were used for therapy. The cell transplantation effects were evaluated by morphometry, biochemical assessment, immunohistochemistry and enzyme-linked immunosorbent assay. RESULTS CD11b+CD14+ monocyte therapy significantly reduced liver fibrosis and increased hepatic glutathione levels. Levels of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-6 and IL-1β, in addition to pro-fibrotic factors, such as IL-13, transforming growth factor-β1 and tissue inhibitor of metalloproteinase-1 also decreased, while IL-10 and matrix metalloproteinase-9 increased in the monocyte-treated group. CD11b+CD14+ monocyte transplantation caused significant changes in the hepatic expression of α-smooth muscle actin and osteopontin. CONCLUSION Monocyte therapy is capable of bringing about improvement of liver fibrosis by reducing oxidative stress and inflammation, as well as increasing anti-fibrogenic factors.