Central and Effector Memory Human CD4+ and CD8+ T Cells during Cutaneous Leishmaniasis and after In Vitro Stimulation with Leishmania (Viannia) braziliensis Epitopes

Cutaneous Leishmaniasis (CL) is a Neglected Tropical Disease characterized by skin ulcers caused by Leishmania spp. protozoans and there is no safe and effective vaccine to reduce its negative consequences. In a previous work by our group, we identified T cell epitopes of Leishmania (Viannia) braziliensis which stimulated patients' T cells in vitro. In the present work, the peptides were tested as two pools for their ability to rescue memory T cells during natural infection by Leishmania. We analyzed the frequency of central memory (TCM, CD45RA-CD62L+) and effector memory (TEM, CD45RA + CD62L-) cells during active CL and post-treatment. In parallel, we investigated cell proliferation levels and the cytokines produced after stimulation. Interestingly, we observed higher frequencies (%) in CD4+ TEM during CL, and CD8+ TEM and CD8+ TCM during CL and post-treatment. Cell proliferation was increased, and a significant difference in expression was observed on T-bet and RORγT. Besides that, IFN-γ, IL-2, and IL-10 were detected in patient samples. Collectively, this dataset suggests that during CL there is an increase in the frequency of TCM and TEM, especially in the CD8 compartment. These results indicate a potentially immunogenic profile of the peptide pools, which can support the development of anti-Leishmania formulations.

Antifungal Effect and Inhibition of the Virulence Mechanism of D-Limonene against Candida parapsilosis.

Yeasts from the Candida parapsilosis complex are clinically relevant due to their high virulence and pathogenicity potential, such as adherence to epithelial cells and emission of filamentous structures, as well as their low susceptibility to antifungals. D-limonene, a natural compound, emerges as a promising alternative with previously described antibacterial, antiparasitic, and antifungal activity; however, its mechanisms of action and antivirulence activity against C. parapsilosis complex species have not been elucidated. Therefore, in the present study, we aimed to evaluate the antifungal and antivirulence action, as well as the mechanism of action of D-limonene against isolates from this complex. D-limonene exhibited relevant antifungal activity against C. parapsilosis complex yeasts, as well as excellent antivirulence activity by inhibiting yeast morphogenesis and adherence to the human epithelium. Furthermore, the apoptotic mechanism induced by this compound, which is not induced by oxidative stress, represents an important target for the development of new antifungal drugs.

Thiophene-thiosemicarbazone derivative (L10) exerts antifungal activity mediated by oxidative stress and apoptosis in C. albicans.

Reactive oxygen species (ROS) cause cell damage and death. To reverse these effects, cells produce substances such as reduced glutathione (GSH) that serve as substrates for antioxidant enzymes. One way to combat microbial resistance includes nullifying the effect of glutathione in microbial cells, causing them to die from oxidative stress. The compound 2-((5-nitrothiophen-2-yl)methylene)-N-(pyridin-3-yl) hydrazine carbothioamide (L10) is a new thiophene-thiosemicarbazone derivative with promising antifungal activity. The aim of this study was to evaluate its mechanism of action against Candida albicans using assays that evaluate its effects on redox balance. Treatment with L10 promoted significant changes in the minimum inhibitory concentration (MIC) values in ascorbic acid and GSH protection tests, the latter increasing up to 64-fold of the MIC. Using nuclear magnetic resonance, we demonstrated interaction of L10 and GSH. At concentrations of 4.0 and 8.0 µg/mL, significant changes were observed in ROS production and mitochondrial membrane potential. The cell death profile showed characteristics of initial apoptosis at inhibitory concentrations (4.0 µg/mL). Transmission electron microscopy data corroborated these results and indicated signs of apoptosis, damage to plasma and nuclear membranes, and to mitochondria. Taken together, these results suggest a possible mechanism of action for L10 antifungal activity, involving changes in cellular redox balance, ROS production, and apoptosis-compatible cellular changes.

Lippia sidoides and Lippia origanoides essential oils affect the viability, motility and ultrastructure of Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered a public health problem. The current chemotherapy for this illness causes serious side effects and its use in the chronic phase of the disease is still controversial. In this regard, the investigation of novel therapeutic strategies remains a priority. The essential oils (EOs) from aromatic plants emerge as a promising source of bioactive compounds. In a previous work we reported the trypanocidal activity of the essential oils from the medicinal plants Lippia sidoides (LSEO) and Lippia origanoides (LOEO) against T. cruzi. Herein, we aimed to further investigate, in more details, the mode of action of LSEO and LOEO on the different developmental stages of this parasite. We showed that Lippia sidoides (LSEO) and Lippia origanoides (LOEO) induced a significant reduction in the percentage of macrophages infected by T. cruzi and in the number of intracellular parasites. Ultrastructural analysis showed that the treatment with both oils caused morphological changes consistent with loss of viability and cell death. The reduced staining with calcein and the increase in the proportion of HE-positive cells also demonstrated that LSEO and LOEO caused loss of parasite viability and membrane integrity. A considerable decrease in Rhodamine 123 and an increase in fluorescence intensity of MitoSox in LOEO were indicative of loss of mitochondrial potential and generation of reactive oxygen species, which ultimately lead to parasite death. Moreover, the optical tweezer analysis indicated that LOEO was more effective in reducing the motility of the epimastigotes. Taken together, our results demonstrated that the LSEO and LOEO are active against T. cruzi and constitute a promising drugs for the therapy of Chagas disease.

Novel indol-3-yl-thiosemicarbazone derivatives: Obtaining, evaluation of in vitro leishmanicidal activity and ultrastructural studies.

Parasitic diseases still represent serious public health problems, since the high and steady emergence of resistant strains is evident. Because parasitic infections are distributed predominantly in developing countries, less toxic, more efficient, safer and more accessible drugs have become desirable in the treatment of the infected population. This is the case of leishmaniasis, an infectious disease caused by a protozoan of the genus Leishmania sp., responsible for triggering pathological processes from the simplest to the most severe forms leading to high rates of morbidity and mortality throughout the world. In the search for new leishmanicidal drugs, the thiosemicarbazones and the indole fragments have been identified as promising structures for leishmanicidal activity. The present study proposes the synthesis and structural characterization of new indole-thiosemicarbazone derivatives (2a-j), in addition to performing in vitro evaluations through cytotoxicity assays using macrophages (J774) activity against forms of Leishmania infantum and Leishmania amazonensis promastigote as well as ultrastructural analyzes in promastigotes of L. infantum. Results show that the indole-thiosemicarbazone derivatives were obtained with yield values varying from 32.09 to 94.64%. In the evaluation of cytotoxicity, the indole-thiosemicarbazone compounds presented CC50 values between 53.23 and 357.97 µM. Concerning the evaluation against L. amazonensis promastigote forms, IC50 values ranged between 12.31 and  > 481.52 µM, while the activity against L. infantum promastigotes obtained IC50 values between 4.36 and 23.35 µM. The compounds 2d and 2i tested against L. infantum were the most promising in the series, as they showed the lowest IC50 values: 5.60 and 4.36 respectively. The parasites treated with the compounds 2d and 2i showed several structural alterations, such as shrinkage of the cell body, shortening and loss of the flagellum, intense mitochondrial swelling and vacuolization of the cytoplasm leading the parasite to cellular unviability. Therefore, the indole-thiosemicarbazone compounds are promising because they yield considerable synthesis, have low cytotoxicity to mammalian cells and act as leishmanicidal agents.

Adoptive Transfer of Bone Marrow-Derived Monocytes Ameliorates Schistosoma mansoni -Induced Liver Fibrosis in Mice.

Liver diseases are a major health problem worldwide leading to high mortality rates and causing a considerable economic burden in many countries. Cellular therapies as potential treatments for liver diseases have proven beneficial in most of the conditions. In recent years, studies involving therapy with bone marrow cells have been implemented to promote liver regeneration and to reduce hepatic fibrosis, however identifying the cell population present in the bone marrow that is responsible for hepatic improvement after therapy is still necessary. The aim of the present study was the evaluation of the therapeutic efficacy of monocytes obtained from bone marrow in fibrosis resulting from S. mansoni infection in C57BL/6 mice. Monocytes were isolated by immunomagnetic separation and administered to the infected animals. The effects of treatment were evaluated through morphometric, biochemical, immunological and molecular analyzes. Monocyte therapy promoted reduction of liver fibrosis induced by S. mansoni infection, associated with a decrease in production of inflammatory and pro-fibrogenic mediators. In addition, monocyte infusion caused downregulation of factors associated with the M1 activation profile, as well as upregulation of M2reg markers. The findings altogether reinforce the hypothesis that the predominance of M2reg macrophages, producers of immunosuppressive cytokines, may favor the improvement of hepatic fibrosis in a preclinical model, through fibrous tissue remodeling, modulation of the inflammatory response and fibrogenesis.

Bone marrow-derived monocyte infusion improves hepatic fibrosis by decreasing osteopontin, TGF-ß1, 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-1ß, in addition to pro-fibrotic factors, such as IL-13, transforming growth factor-ß1 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.

In vitro evaluation of cytotoxicity and leishmanicidal activity of phthalimido-thiazole derivatives.

It is estimated that the worldwide prevalence of leishmaniasis is around 12 million individuals in 80 countries, with 400,000new cases per year. In the search for new leishmanicidal agents, the hybrid phthalimido-thiazoles have been identified as an important scaffold for drug design and discovery. The present study thus reports the in vitro activity of a series of phthalimido-thiazole derivatives. Cytotoxicity against a strain of L. infantum, Vero cells, J774 macrophages and peritoneal macrophages was evaluated, as well as nitric oxide (NO) production. Activity against amastigote and promastigote forms of L. infantum and microscopic changes in the parasite and intracellular targets of the parasite were achieved. The results show that the compounds arising from hybridization of phthalimide and 1,3-thiazole exhibit promising leishmanicidal activity. Compounds 2j and 2m were the most potent of the series tested and the parasites treated with these compounds exhibited ultrastructural changes, such as cell body shrinkage, loss of cellular membrane integrity, vacuolization of cytoplasm, membrane profiles surrounding organelles and swelling of mitochondria. The data showed that these compounds reduced the survival of intracellular amastigotes and presented low toxicity for mammalian cells. The compounds produced increased NO production compared to untreated cells in non-infected macrophages. Treated promastigote forms showed an increase in the number of cells stained with propidium iodide. The compounds brought about significant changes in mitochondrial membrane potential. According to the present study, phthalimido-thiazole compounds exhibit leishmanicidal activity and could be used to develop novel antileishmaniasis drugs and explore potential molecular targets.

Phthalimido-thiazoles as building blocks and their effects on the growth and morphology of Trypanosoma cruzi.

Chagas disease is a parasitic infection caused by protozoan Trypanosoma cruzi that affects approximately 6-7 million people worldwide. Benznidazole is the only drug approved for treatment during the acute and asymptomatic chronic phases; however, its efficacy during the symptomatic chronic phase is controversial. The present work reports the synthesis and anti-T. cruzi activities of a novel series of phthalimido-thiazoles. Some of these compounds showed potent inhibition of the trypomastigote form of the parasite at low cytotoxicity concentrations in spleen cells, and the resulting structure-activity relationships are discussed. We also showed that phthalimido-thiazoles induced ultrastructural alterations on morphology, flagellum shortening, chromatin condensation, mitochondria swelling, reservosomes alterations and endoplasmic reticulum dilation. Together, these data revealed, for the first time, a novel series of phthalimido-thiazoles-structure-based compounds with potential effects against T. cruzi and lead-like characteristics against Chagas disease.

Effect of neonatal malnutrition on expression of nitric oxide synthase enzyme, production of free radicals and in vitro viability of alveolar macrophages infected with methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

OBJECTIVE: Evaluate the effects of neonatal malnutrition on the microbicidal response and viability of in vitro macrophages infected with Staphylococcus aureus sensitive/resistant to methicillin. METHODS: Male Wistar rats (n = 24) were divided into two distinct groups: nourished (rats breast-fed by mothers undergoing diet with 17% casein) and malnourished (rats breast-fed by mothers undergoing diet with 8% casein). Macrophages were recovered after surgical tracheostomy procedure by collecting bronchoalveolar lavage. Four systems were established: negative control, composed only by phagocytes; positive control, macrophages plus lipopolysaccharide; and two test systems, macrophages plus Staphylococcus aureus sensitive and resistant to methicillin. Plates were incubated at 37 °C for 24 h. After this period, tests for the analysis of cell viability and microbicidal response were performed. In the statistical analysis, the Student's t and ANOVA tests were used, accepting p < 0.05. RESULTS: The neonatal malnutrition impaired the animals' body weight. There was a lower expression of the inducible nitric oxide enzyme (iNOS), nitric oxide production, and viability of macrophages infected with methicillin-resistant Staphylococcus aureus. However, increased production of superoxide anion in the malnourished group was detected. CONCLUSION: Neonatal malnutrition focusing on critical periods of development promoted lower expression of iNOS, nitric oxide production, cell viability, and exacerbated reactive oxygen species production. The high levels of reactive oxygen species may favor the onset of serious and systemic infections with fatal outcome if associated with methicillin-resistant Staphylococcus aureus.

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.

Immunomodulatory effects of pCramoll and rCramoll on peritoneal exudate cells (PECs) infected and non-infected with Staphylococcus aureus.

Peritoneal exudate cells (PECs) play important roles in host defense against Staphylococcus aureus and other pathogens. In this study we evaluated the potentials of native (pCramoll or Cramoll 1,4) and recombinant (rCramoll) lectins from seeds of Cratylia mollis as immunomodulatory tools on mice PECs infected and non-infected with S. aureus. Both lectins significantly enhanced nitric oxide, superoxide and cytokines (IL-1ß, IL-6, IFN-γ and TNF-α). pCramoll and rCramoll downregulated the induction of TNF-α and IL-6 and upregulated the expression of IL-1ß, IFN-γ in S. aureus infected PECs. Phagocytic activity of S. aureus was also enhanced in 27.1% and 22.47% by pCramoll and rCramoll, respectively. Our results showed that pCramoll induced stronger effects than rCramoll, which could be explained by the different hemagglutinating activities of C. mollis isolectins and nature fragmentation, although the biologic meaning should be studied in detail using in vivo models. Future works will be focused on the molecular mechanisms involved in these actions, using in vitro and in vivo models, to support the use of these lectins as biotechnological tool in immunological studies.

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.

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.

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.

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.