Synthesis and activity of benzimidazole N-Acylhydrazones against Trypanosoma cruzi, Leishmania amazonensis and Leishmania infantum.

In this study, we present the design, synthesis, and cytotoxic evaluation of a series of benzimidazole N-acylhydrazones against strains of T. cruzi (Y and Tulahuen) and Leishmania species (L. amazonensis and L. infantum). Compound (E)-N'-((5-Nitrofuran-2-yl)methylene)-1H-benzo[d]imidazole-2-carbohydrazide demonstrated significant activity against both trypomastigote and amastigote forms (Tulahuen strain), with an IC50/120 h of 0.033 µM and a selectivity index (SI) of 7680. This represents a potency 46 times greater than that of benznidazole (IC50/120 h = 1.520 µM, SI = 1390). Another compound (E)-N'-(2-Hydroxybenzylidene)-1H-benzo[d]imidazole-2-carbohydrazide showed promising activity against both trypomastigote and amastigote forms (Tulahuen strain), with an IC50/120 h of 3.600 µM and an SI of 14.70. However, its efficacy against L. infantum and L. amazonensis was comparatively lower. These findings provide valuable insights for the development of more effective treatments against Trypanosoma cruzi.

Synthesis of 4-(4-chlorophenyl)thiazole compounds: in silico and in vitro evaluations as leishmanicidal and trypanocidal agents.

Neglected tropical diseases are a diverse group of communicable pathologies that mainly prevail in tropical and subtropical regions. Thus, the objective of this work was to evaluate the biological potential of eight 4-(4-chlorophenyl)thiazole compounds. Tests were carried out in silico to evaluate the pharmacokinetic properties, the antioxidant, cytotoxic activities in animal cells and antiparasitic activities were evaluated against the different forms of Leishmania amazonensis and Trypanosoma cruzi in vitro. The in silico study showed that the evaluated compounds showed good oral availability. In a preliminary in vitro study, the compounds showed moderate to low antioxidant activity. Cytotoxicity assays show that the compounds showed moderate to low toxicity. In relation to leishmanicidal activity, the compounds presented IC50 values that ranged from 19.86 to 200 µM for the promastigote form, while for the amastigote forms, IC50 ranged from 101 to more than 200 µM. The compounds showed better results against the forms of T. cruzi with IC50 ranging from 1.67 to 100 µM for the trypomastigote form and 1.96 to values greater than 200 µM for the amastigote form. This study showed that thiazole compounds can be used as future antiparasitic agents.

A Flow Cytometry-Based Serological Assay to Detect Visceral Leishmaniasis in HIV-Infected Patients.

Visceral Leishmaniasis (VL) is a severe parasitic disease that has emerged as an important opportunistic condition in HIV-infected patients and whose control is impaired by inaccurate identification. This is mainly due to the serological tests used for VL having a reduced performance in cases of VL-HIV coinfection due to a low humoral response. In this situation, however, a positive test has even greater diagnostic value when combined with the clinical status. This study aimed to evaluate the application and performance of flow cytometry to detect anti-Leishmania infantum antibodies in HIV-infected patients. Sera from VL/HIV coinfected patients, characterized using "gold standard" techniques, were compared with sera from healthy controls plus sera from HIV-infected individuals. The flow cytometry results were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). A ROC curve analysis of a serum titration indicated a PPFP of 1.26% as being the cutoff point to segregate positive and negative results. At the 1:2,048 dilution, with 89% sensitivity and 83% specificity, flow cytometry showed greater sensitivity in relation to the serological tests evaluated. Futhermore, flow cytometry was the only assay that positively identified all VL-HIV patients with quantified HIV load. Together, these findings suggest that flow cytometry may be used as an alternative serological approach for VL identification and as a tool to characterize the humoral response against Leishmania infantum in HIV-infected patients.

Case Report: Severe Visceral Leishmaniasis in a Patient with HIV Coinfection Undergoing Treatment for Erythema Nodosum Leprosum.

We report a case of visceral leishmaniasis (VL)/HIV coinfection in a patient undergoing regular antiretroviral therapy and treatment with thalidomide for erythema nodosum leprosum. He presented at a health service with high fever, chills, asthenia, pale skin, lower limb edema, hepatomegaly, and splenomegaly. Visceral leishmaniasis was confirmed by direct examination, and serological and molecular tests. Serum levels of Th1/Th2 cytokines were measured. The patient began treatment with liposomal amphotericin B, with good clinical response; however, VL recurred 6 months later. Treatment was reinitiated, maintaining secondary prophylaxis with liposomal amphotericin B. The patient showed clinical improvement with important recovery of CD4+ T-lymphocyte count.

Assessment of Leishmania cell lines expressing high levels of beta-galactosidase as alternative tools for the evaluation of anti-leishmanial drug activity.

Leishmaniasis, caused by protozoa belonging to the genus Leishmania, is an important public health problem found in >90 countries and with still limited options for treatment. Development of new anti-leishmanial drugs is an urgent need and the identification of new active compounds is a limiting factor that can be accelerated through large scale drug screening. This requires multiple steps and can be expensive and time consuming. Here, we propose an alternative approach for the colorimetric assessment of anti-Leishmania drug activity that can be easily scaled up. L. amazonensis and L. infantum cell lines were generated having the ß-galactosidase (ß-gal) gene integrated into their chromosomal 18S rRNA (ssu) locus. Both cell lines expressed high levels of ß-gal and had their growth easily monitored and quantified colorimetrically. These two cell lines were then evaluated as tools to assess drug susceptibility and their use was validated through in vitro assays with Amphotericin B, which is routinely used against leishmaniasis. ß-gal expression was also confirmed through flow-cytometry, another method of phenotypic detection. With these recombinant parasites, an alternative in vitro model of drug screening against cutaneous and visceral leishmaniasis is now available.

Human leukocyte antigen-G 3' untranslated region polymorphism +3142G/C (rs1063320) and haplotypes are associated with manifestations of the American Tegumentary Leishmaniasis in a Northeastern Brazilian population.

While the role of cytokine genes has been well documented in the context of Leishmania (Viannia) braziliensis infection, no studies have addressed the influence of human leukocyte antigen-G (HLA-G) in susceptibility/resistance to American Tegumentary Leishmaniasis (ATL). Here, we evaluated the influences of HLA-G, IL-10, TNF-A and IFN-G in the susceptibility and clinical manifestations of ATL. DNA of 114 ATL patients and 346 healthy individuals were sequenced for well-documented polymorphisms in HLA-G 3' untranslated region (UTR), in IL-10 and TNF-A promoters and in IFN-G intron 1. Soluble HLA-G (sHLA-G) and cytokine levels were evaluated by ELISA and flow cytometry, respectively. Analyses were performed using GraphPad and R-package software. Individuals bearing HLA-G +3142G/G showed an association with increased risk for ATL, whereas those carrying the HLA-G +3142C/G and one copy of UTR6 haplotype, showed an association with decreased risk for ATL. sHLA-G was overexpressed in "susceptible" patients compared to the "resistant'' one, and also in patients bearing +3142G/G genotype. From these results, HLA-G +3142G/G may be considered as genotype of susceptibility and UTR6 as marker of protection to ATL. Our findings showed a participation of HLA-G in the pathogenesis of the ATL.

Performance evaluation of anti-fixed Leishmania infantum promastigotes immunoglobulin G (IgG) detected by flow cytometry as a diagnostic tool for visceral Leishmaniasis.

Visceral Leishmaniasis (VL) is a severe disease, caused by the protozoans Leishmania infantum and L. donovani that is widely diagnosed using serological tools. These, however, have limitations in performance that limit their use for the correct identification of the cases. This study aimed to evaluate the performance of flow cytometry with fixed parasites for VL diagnosis, comparing it with four other serological tests. Samples from two endemic VL regions in Brazil, diagnosed by direct examination (DG1) and by at least two or one standard serological test (DG2 and DG3, respectively), as well as patients with chronic Chagas' disease (CG1) and healthy controls (CG2) were used in this study. The flow cytometry results were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). Using a 1:4096 serum dilution, a ROC curve analysis of the serum titration on flow cytometry has indicated a PPFP of 2% as the cutoff point to segregate positive and negative results. In the present study, flow cytometry had the best performance for DG1 (sensitivity of 96%) while rK39 (imunocromagraphic rapid test) and DAT (Direct agglutination test) were also associated with high sensitivity and specificity. The substantial agreement and kappa indexes observed suggested similar performances between these two tests and flow cytometry. IFAT (Immunofluorescent antibody test) and ELISA (Enzyme-linked immunosorbent assay) had lower performances and the lower values of agreement with flow cytometry. Together, these findings suggest that although adjustments are needed in order to reduce cross reactivity with other trypanosomatids, flow cytometry has the potential to be a safe serological alternative for the diagnosis of VL.

Selective cytotoxic and genotoxic activities of 5-(2-bromo-5-methoxybenzylidene)-thiazolidine-2,4-dione against NCI-H292 human lung carcinoma cells.

BACKGROUND: Thiazolidine-2,4-dione ring system is used as a pharmacophore to build various heterocyclic compounds aimed to interact with biological targets. In the present study, benzylidene-2,4-thiazolidinedione derivatives (compounds 2-5) were synthesized and screened against cancer cell lines and the genotoxicity and cytotoxicity of the most active compound (5) was investigated on normal and lung cancer cell line. METHODS: For in vitro cytotoxic screening, the MTT assay was used for HL60 and K562 (leukemia), MCF-7 (breast adenocarcinoma), HT29 (colon adenocarcinoma), HEp-2 (cervix carcinoma) and NCI-H292 (lung carcinoma) tumor cell lines and Alamar-blue assay was used for non-tumor cells (PBMC, human peripheral blood mononuclear cells) were used. Cell morphology was visualized after Giemsa-May-Grunwald staining. DNA content, phosphatidylserine externalization and mitochondrial depolarization were measured by flow cytometry. Genotoxicity was assessed by Comet assay. RESULTS: 5-(2-Bromo-5-methoxybenzylidene)-thiazolidine-2,4-dione (5) presented the most potent cytotoxicity, especially against NCI-H292 lung cancer cell line, with IC50 value of 1.26µg/mL after 72h incubation. None of the compounds were cytotoxic to PBMC. After 48h incubation, externalization of phosphatidylserine, mitochondrial depolarization, internucleosomal DNA fragmentation and morphological alterations consistent with apoptosis were observed in NCI-H292 cells treated with compound (5). In addition, compound (5) also induced genotoxicity in NCI-H292 cells (2.8-fold increase in damage index compared to the negative control), but not in PBMC. CONCLUSION: Compound 5 presented selective cytotoxic and genotoxic activity against pulmonary carcinoma (NCI-H292 cells).

Antitumor and immunomodulatory activities of thiosemicarbazones and 1,3-Thiazoles in Jurkat and HT-29 cells.

Cancer remains a high incidence and mortality disease, causing around 8.2 million of deaths in the last year. Current chemotherapy needs to be expanded, making research for new drugs a necessary task. Immune system modulation is an emerging concept in cancer cell proliferation control. In fact, there are a number of mechanisms underlying the role immune system plays in tumor cells. In this work, we describe the structural design, synthesis, antitumor and immunomodulatory potential of 31 new 1,3-thiazole and thiosemicarbazone compounds. Cisplatin was used as anticancer drug control. Cytotoxicity against J774A.1 macrophages and antitumor activity against HT-29 and Jurkat cells was determined. These 1,3-thiazole and thiosemicarbazone compounds not only exhibited cytotoxicity in cancer cells, but were able to cause irreversible cancer cell damage by inducing necrosis and apoptosis. In addition, these compounds, especially pyridyl-thiazoles compounds, regulated immune factors such as interleukin 10 and tumor necrosis factor, possible by directing immune system in favor of modulating cancer cell proliferation. By examining their pharmacological activity, we were able to identify new potent and selective anticancer compounds.

American tegumentary leishmaniasis: mRNA expression for Th1 and Treg mediators are predominant in patients with recent active disease.

Besides the Th1×Th2 paradigm, Treg and Th17 cytokines may play a role in the response to American tegumentary leishmaniasis. Considering the sensitivity and accuracy of qPCR and the lack of studies using this approach, we evaluated mRNA expression for IFN-γ, TNF-α, IL-4, IL-10, IL-6, IL-17A, IL-22, TGF-ß, Foxp3 and RORC in peripheral blood mononuclear cells (PBMC) from patients with active disease, after stimulation with L. (V.) braziliensis soluble or insoluble fractions. Our results show that the antigens promoted specific mRNA expression related to the immune response in patients with ATL, and the insoluble fraction seems to stimulate the immune response in a higher intensity. The pro-inflammatory response was also fueled by IFN-γ and TNF-α, probably due to the active disease. IL-4, in certain way, seems to regulate this response along with IL-10 that may be produced by Treg cells, which are supposedly present in the patients' samples due the evidenced expression of Foxp3, in the presence of AgIns. In contrast, down-regulated RORC suggests that the significant levels of IL-6 expressed in response to AgSol were not able to induce an expressive Th17 profile along with TGF-ß, which might have predominantly contributed to the development of a regulatory profile in the active disease.

Thiosemicarbazones as Aedes aegypti larvicidal.

A set of aryl- and phenoxymethyl-(thio)semicarbazones were synthetized, characterized and biologically evaluated against the larvae of Aedes aegypti (A. aegypti), the vector responsible for diseases like Dengue and Yellow Fever. (Q)SAR studies were useful for predicting the activities of the compounds not included to create the QSAR model as well as to predict the features of a new compound with improved activity. Docking studies corroborated experimental evidence of AeSCP-2 as a potential target able to explain the larvicidal properties of its compounds. The trend observed between the in silico Docking scores and the in vitro pLC50 (equals -log LC50, at molar concentration) data indicated that the highest larvicidal compounds, or the compounds with the highest values for pLC50, are usually those with the higher docking scores (i.e., greater in silico affinity for the AeSCP-2 target). Determination of cytotoxicity for these compounds in mammal cells demonstrated that the top larvicide compounds are non-toxic.

Induction of cancer cell death by apoptosis and slow release of 5-fluoracil from metal-organic frameworks Cu-BTC.

This study aimed to evaluate the mechanism associated with cytotoxic activity displayed by the drug 5-fluorouracil incorporated in Cu-BTC MOF and its slow delivery from the Cu-BTC MOF. Structural characterization encompasses elemental analysis (CHNS), differential scanning calorimetry (DSC), thermogravimetric analysis (TG/DTG), Fournier transform infrared (FIT-IR) and X-ray diffraction (XRD) was performed to verify the process of association between the drug 5-FU and Cu-BTC MOF. Flow cytometry was done to indicate that apoptosis is the mechanism responsible for the cell death. The release profile of the drug 5-FU from Cu-BTC MOF for 48 hours was obeisant. Also, the anti-inflammatory activity was evaluated by the peritonitis testing and the production of nitric oxide and pro-inflammatory cytokines were measured. The chemical characterization of the material indicated the presence of drug associated with the coordination network in a proportion of 0.82 g 5-FU per 1.0 g of Cu-BTC MOF. The cytotoxic tests were carried out against four cell lines: NCI-H292, MCF-7, HT29 and HL60. The Cu-BTC MOF associated drug was extremely cytotoxic against the human breast cancer adenocarcinoma (MCF-7) cell line and against human acute promyelocytic leukemia cells (HL60), cancer cells were killed by apoptosis mechanisms. The drug demonstrated a slow release profile where 82% of the drug was released in 48 hours. The results indicated that the drug incorporated in Cu-BTC MOF decreased significantly the number of leukocytes in the peritoneal cavity of rodents as well as reduced levels of cytokines and nitric oxide production.

Investigation on the pharmacological profile of antimony(III) complexes with hydroxyquinoline derivatives: anti-trypanosomal activity and cytotoxicity against human leukemia cell lines.

Complexes [Sb(QN)(2)Cl] (1), [Sb(QC)(2)Cl] (2) and [Sb(QI)(2)Cl] (3) were obtained with 8-hydroxyquinoline (HQN), 5-chloro-8-hydroxyquinoline (HQC) and 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol, HQI). The quinoline derivatives and their antimony(III) complexes were evaluated for their anti-trypanosomal activity as well as for their cytotoxicity against HL-60 and Jurkat human leukemia cell lines. Upon coordination to antimony(III) the anti-trypanosomal activity of HQC and HQI increases, the highest improvement being observed for complex (3), which was the most active among all studied compounds against both epimastigote and trypomastigote forms of Trypanosoma cruzi. All quinoline derivatives proved to be cytotoxic against both leukemia cell lineages. Upon coordination to antimony(III) the cytotoxicity of HQN improved against Jurkat leukemia cells. While SbCl(3) proved to be cytotoxic against HL-60 cells, it was not active against Jurkat cells. However, its coordination to the quinoline derivatives resulted in complexes with significant cytotoxicity against Jurkat cells.

Cellular immune response profile in patients with American tegumentary leishmaniasis prior and post chemotherapy treatment.

In this study, we have the objective of evaluating the lymphoproliferative response and determining interferon (IFN)-gamma and interleukin (IL)-10 cytokine production in the peripheral blood mononuclear cells (PBMC) of patients with American tegumentary leishmaniasis prior and post 12 months of chemotherapy treatment with meglumine antimoniate compared with the PBMC of noninfected donors. Lymphoproliferation, such as cytokine production, was evaluated through in vitro stimulus with the soluble antigenic fraction from Leishmania (Viannia) braziliensis promastigotes (1.25 microg/ml) and Concanavalin A (2.5 microg/ml). Patients showed a significant lymphoproliferative response prior and post treatment compared with the control group. Similar result, prior to chemotherapy treatment, was observed in IFN-gamma and IL-10 production when patients were compared with the control group. After chemotherapy treatment, PBMC lymphoproliferative response of the patients revealed an increase, whereas patients have shown a decrease in IFN-gamma levels and an increase in IL-10, although without statistical difference. These results may indicate that the patients produced a specific cellular response to the soluble antigenic fraction suggesting that besides Th1 and Th2 dichotomy, immunological regulation mechanisms with the participation of memory T cells and regulatory T cells could be present in the clinical evolution of these patients. This understanding will allow the study and identification of new L. (V.) braziliensis molecules potentially candidates to vaccines.

Cellular immune response from Chagasic patients to CRA or FRA recombinant antigens of Trypanosoma cruzi.

We propose to analyze the relation between the cellular immune response of Chagas' disease patients after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with recombinant antigens cytoplasmatic repetitive antigen (CRA) or flagellar repetitive antigen (FRA) of T. cruzi and the chronic clinical forms of disease. Cells were stimulated using phytohemagglutinin, CRA, FRA, or a soluble antigen of Epimastigota (Ag-Epi) for 24 hr, 72 hr, or 6 days. The proliferation of cells was evaluated after 6 days of culture by quantification of incorporated 3H-thymidine. Cytokines were measured in the supernatants obtained after 24 hr (tumor necrosis factor [TNF]-alpha and interleukin [IL]-4), 72 hr (IL-10), and 6 days (interferon [IFN]-gamma) using enzyme-linked immunosorbent assay (ELISA). Cells of the Chagas patients stimulated with the recombinant antigens exhibited higher proliferation responses compared with that of non-Chagas (NC) individuals. However, when proliferation was compared between patients with the cardiac form (CF) or indeterminate form (IF), it was not possible to establish a difference in the response. So far as the cytokines secreted in the culture supernatants after stimulation in vitro with T. cruzi antigens were concerned, the results showed that CRA, as well as Epi-Ag, were able to stimulate the production of TNF-alpha and IFN-gamma in Chagas patients as compared with NC individuals. However, the cytokine levels after stimulation with the T. cruzi antigens were not different between the patients with CF and IF. CRA was capable of inducing a T helper type 1 (Th1) immune response, with elevated production of TNF-alpha and IFN-gamma in Chagas patients that are carriers of CF and IF clinical forms.

Assessment of a DNA vaccine encoding an anchored-glycosylphosphatidylinositol tegumental antigen complexed to protamine sulphate on immunoprotection against murine schistosomiasis.

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44%. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.

Assessment of a DNA vaccine encoding an anchored-glycosylphosphatidylinositol tegumental antigen complexed to protamine sulphate on immunoprotection against murine schistosomiasis

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44 percent. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.

Humoral and cellular immune responses in BALB/c and C57BL/6 mice immunized with cytoplasmic (CRA) and flagellar (FRA) recombinant repetitive antigens, in acute experimental Trypanosoma cruzi infection.

In previous studies, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins induced specific humoral and cellular immune responses in susceptible and resistant mice in the absence of Trypanosoma cruzi infection with a significant induction of the Interferon-gamma (IFN-gamma) production in those animals. In this follow-up paper, the immunostimulatory and protective effects of these proteins were evaluated by immunizing with CRA or FRA antigens, BALB/c and C57BL/6 mice and challenging with a T. cruzi (Y strain). Both proteins induced humoral response with high levels of IgG isotypes as well as cellular immunity with high levels of IFN-gamma when compared to controls. However, the lymphocyte proliferative response was minimal. The survival rate at 30 days post-infection was significant in CRA (60%) or FRA (50%)--immunized BALB/c mice and CRA (83.3%)--immunized C57BL/6 mice. Taken as a whole these findings indicate that CRA and FRA are immunogenic and potentially important for protective immunity.

Antibody isotype responses in Balb/c mice immunized with the cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi.

In the present report we analyzed the levels of IgG1, IgG2a, IgG2b and IgG3 isotypes from Balb/c mice immunized with cytoplasmic repetitive antigen (CRA), and flagellar repetitive antigen (FRA) of Trypanosoma cruzi. The immunization was done by subcutaneous route three times (20 days apart) and the analysis was performed 14 days after each treatment. CRA-immunized mice produced high levels of all IgG isotypes, mainly IgG3 and IgG1. FRA-immunization elicited only high levels of IgG1.