Treatment using vanillin-derived synthetic molecules incorporated into polymeric micelles is effective against infection caused by Leishmania amazonensis species.

Treatment against leishmaniasis presents problems, mainly due to the toxicity of the drugs, high cost, and the emergence of resistant strains. A previous study showed that two vanillin-derived synthetic molecules, 3s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], presented antileishmanial activity against Leishmania infantum, L. amazonensis, and L. braziliensis species. In the present work, 3s and 3t were evaluated to treat L. amazonensis-infected mice. Molecules were used pure or incorporated into Poloxamer 407-based micelles. In addition, amphotericin B (AmpB) and its liposomal formulation, Ambisome®, were used as control. Animals received the treatment and, one and 30 days after, they were euthanized to evaluate immunological, parasitological, and biochemical parameters. Results showed that the micellar compositions (3s/Mic and 3t/Mic) induced significant reductions in the lesion mean diameter and parasite load in the infected tissue and distinct organs, as well as a specific and significant antileishmanial Th1-type immune response, which was based on significantly higher levels of IFN-γ, IL-12, nitrite, and IgG2a isotype antibodies. Drug controls showed also antileishmanial action; although 3s/Mic and 3t/Mic have presented better and more significant parasitological and immunological data, which were based on significantly higher IFN-γ production and lower parasite burden in treated animals. In addition, significantly lower levels of urea, creatinine, alanine transaminase, and aspartate transaminase were found in mice treated with 3s/Mic and 3t/Mic, when compared to the others. In conclusion, results suggest that 3s/Mic and 3t/Mic could be considered as therapeutic candidates to treat against L. amazonensis infection.

New synthetic molecules incorporated into polymeric micelles used for treatment against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) presents problems, mainly related to drug toxicity, high cost and/or by emergence of resistant strains. In the present study, two vanillin synthetic derivatives, 3 s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3 t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], were evaluated as therapeutic candidates in a murine model against Leishmania infantum infection. Molecules were used pure (3 s and 3 t) or incorporated into Poloxamer 407-based micelles (3 s/M and 3 t/M) in the infected animals, which also received amphotericin B (AmpB) or Ambisome® as control. Results showed that 3 s/M and 3 t/M compositions induced a Th1-type immune response in treated animals, with higher levels of IFN-γ, IL-2, TNF-α, IL-12, nitrite, and IgG2a antibodies. Animals presented also low toxicity and significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as compared as control groups mice, with the evaluations performed one and 30 days after the application of the therapeutics. In conclusion, preliminary data suggest that 3 s/M and 3 t/M could be considered for future studies as therapeutic agents against VL.

The long-lasting Ascaris suum antigens in the lungs shapes the tissue adaptation modifying the pulmonary architecture and immune response after infection in mice.

Ascariasis is the most prevalent helminth affecting approximately 819 million people worldwide. The acute phase of Ascariasis is characterized by larval migration of Ascaris spp., through the intestinal wall, carried to the liver and lungs of the host by the circulatory system. Most of the larvae subsequently transverse the lung parenchyma leading to tissue injury, reaching the airways and pharynx, where they can be expectorated and swallowed back to the gastrointestinal tract, where they develop into adult worms. However, some larvae are trapped in the lung parenchyma inciting an inflammatory response that causes persistent pulmonary tissue damage long after the resolution of infection, which returns to tissue homeostasis. However, the mechanism by which chronic lung disease develops and resolves remains unknown. Here, using immunohistochemistry, we demonstrate that small fragments and larval antigens of Ascaris suum are deposited and retained chronically in the lung parenchyma of mice following a single Ascaris infection. Our results reveal that the prolonged presence of Ascaris larval antigens in the lung parenchyma contributes to the persistent immune stimulation inducing histopathological changes observed chronically following infection, and clearly demonstrate that larval antigens are related to all phases of tissue adaptation after infection: lung injury, chronic inflammation, resolution, and tissue remodeling, in parallel to increased specific humoral immunity and the recovery of lung function in mice. Additional insight is needed into the mechanisms of Ascaris antigen to induce chronic immune responses and resolution in the host lungs following larval migration.

In vitro evaluation of antileishmanial activity, mode of action and cellular response induced by vanillin synthetic derivatives against Leishmania species able to cause cutaneous and visceral leishmaniasis.

The treatment against leishmaniasis presents problems, mainly due to their toxicity of the drugs, high cost and/or by the emergence of parasite resistant strains. In this context, new therapeutics should be searched. In this study, two novel synthetic derivatives from vanillin: [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] or 3s and [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde] or 3t, were evaluated regarding their antileishmanial activity against distinct parasite species able to cause cutaneous and visceral leishmaniasis. Results showed that compounds 3s and 3t were effective against Leishmania infantum, L. amazonensis and L. braziliensis promastigote and amastigote-like forms, showing selectivity index (SI) of 25.1, 18.2 and 22.9, respectively, when 3s was used against promastigotes, and of 45.2, 7.5 and 15.0, respectively, against amastigote-like stage. Using the compound 3t, SI values were 45.2, 53.0 and 80.0, respectively, against promastigotes, and of 35.9, 46.0 and 58.4, respectively, against amastigote-like forms. Amphotericin B (AmpB) showed SI values of 5.0, 7.5 and 15.0, respectively, against promastigotes, and of 3.8, 5.0 and 7.5, respectively, against amastigote-like stage. The treatment of infected macrophages and inhibition of the infection upon pre-incubation with the molecules showed that they were effective in reducing the infection degree and inhibiting the infection in pre-incubated parasites, respectively, as compared to data obtained using AmpB. The mechanism of action of 3s and 3t was evaluated in L. infantum, revealing that both 3s and 3t altered the parasite mitochondrial membrane potential leading to reactive oxygen species production, increase in lipid corps and changes in the cell cycle, causing the parasite' death. A preliminary assay using the cell culture supernatant from treated and infected macrophages showed that 3s and 3t induced higher IL-12 and lower IL-10 values; suggesting the development of an in vitro Th1-type response in the treated cells. In this context, data indicated that 3s and 3t could be considered therapeutic agents to be tested in future studies against leishmaniasis.

Synthesis of vanillin derivatives with 1,2,3-triazole fragments and evaluation of their fungicide and fungistatic activities.

Vanillin is the main component of natural vanilla extract and is responsible for its flavoring properties. Besides its well-known applications as an additive in food and cosmetics, it has also been reported that vanillin can inhibit fungi of clinical interest, such as Candida spp., Cryptococcus spp., Aspergillus spp., as well as dermatophytes. Thus, the present work approaches the synthesis of a series of vanillin derivatives with 1,2,3-triazole fragments and the evaluation of their antifungal activities against Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, Cryptococcus gattii, Trichophyton rubrum, and Trichophyton interdigitale strains. Twenty-two vanillin derivatives were obtained, with yields in the range of 60%-91%, from copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction between two terminal alkynes prepared from vanillin and different benzyl azides. In general, the evaluated compounds showed moderate activity against the microorganisms tested, with minimum inhibitory concentration (MIC) values ranging from 32 to >512 µg mL-1 . Except for compound 3b against the C. gattii R265 strain, all vanillin derivatives showed fungicidal activity for the yeasts tested. The predicted physicochemical and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties for the compounds indicated favorable profiles for drug development. In addition, a four-dimensional structure-activity relationship (4D-SAR) analysis was carried out and provided useful insights concerning the structures of the compounds and their biological profile. Finally, molecular docking calculations showed that all compounds bind favorably at the lanosterol 14α-demethylase enzyme active site with binding energies ranging from -9.1 to -12.2 kcal/mol.

Circulating Nestin-GFP+ Cells Participate in the Pathogenesis of Paracoccidioides brasiliensis in the Lungs.

Multiple infectious diseases lead to impaired lung function. Revealing the cellular mechanisms involved in this impairment is crucial for the understanding of how the lungs shift from a physiologic to a pathologic state in each specific condition. In this context, we explored the pathogenesis of Paracoccidioidomycosis, which affects pulmonary functioning. The presence of cells expressing Nestin-GFP has been reported in different tissues, and their roles as tissue-specific progenitors have been stablished in particular organs. Here, we explored how Nestin-GFP+ cells are affected after lung infection by Paracoccidioides brasiliensis, a model of lung granulomatous inflammation with fibrotic outcome. We used Nestin-GFP transgenic mice, parabiosis surgery, confocal microscopy and flow cytometry to investigate the participation of Nestin-GFP+ cells in Paracoccidioides brasiliensis pathogenesis. We revealed that these cells increase in the lungs post-Paracoccidioides brasiliensis infection, accumulating around granulomas. This increase was due mainly to Nestin-GPF+ cells derived from the blood circulation, not associated to blood vessels, that co-express markers suggestive of hematopoietic cells (Sca-1, CD45 and CXCR4). Therefore, our findings suggest that circulating Nestin-GFP+ cells participate in the Paracoccidioides brasiliensis pathogenesis in the lungs.

Immunological underpinnings of Ascaris infection, reinfection and co-infection and their associated co-morbidities.

Human ascariasis is the most common and prevalent neglected tropical disease and is estimated that ~819 million people are infected around the globe, accounting for 0.861 million years of disability-adjusted life years in 2017. Even with the existence of highly effective drugs, the constant presence of infective parasite eggs in the environment contribute to a high reinfection rate after treatment. Due to its high prevalence and broad geographic distribution Ascaris infection is associated with a variety of co-morbidities and co-infections. Here, we provide data from both experimental models and humans studies that illustrate how complex is the interaction of Ascaris with the host immune system, especially, in the context of reinfections, co-infections and associated co-morbidities.

Vaccination with chimeric protein induces protection in murine model against ascariasis.

An estimated 400 million people are infected by parasites of the genus Ascaris and the existing control measures are inefficient. Vaccine development using B cell antigens is a promising strategy for increased protection against this parasite. The present study aimed at developing a chimeric protein capable of conferring protection against infection by Ascaris sp. For this purpose, we performed B-cell epitope predictions on previously described vaccine candidate proteins from Ascaris suum and the corresponding peptides were used to construct a chimeric protein. Female BALB / c mice were immunized subcutaneously in three doses at 10 day intervals with a vaccine formulation comprised of the chimeric protein together with monophosphoryl lipid A (MPLA). Control groups included protein alone, MPLA, or PBS. After challenge infection, animals vaccinated with chimeric protein plus MPLA showed a reduction of 73.54% of larval load in the lung compared to control group animals. Animals immunized with chimeric protein plus MPLA also display higher IgG response and a reduction in lung inflammation. Our study highlights how chimeric proteins containing more than one B cell epitope can enhance immune protection against helminthic infection and offer new approaches to the development of Ascaris vaccines.

ACKR2 contributes to pulmonary dysfunction by shaping CCL5:CCR5-dependent recruitment of lymphocytes during influenza A infection in mice.

Inflammation triggered by influenza A virus (IAV) infection is important for viral clearance, induction of adaptive responses, and return to lung homeostasis. However, an exaggerated immune response, characterized by the overproduction of chemokines, can lead to intense lung injury, contributing to mortality. Chemokine scavenger receptors, such as ACKR2, control the levels of CC chemokines influencing the immune responses. Among the chemokine targets of ACKR2, CCL5 is important to recruit and activate lymphocytes. We investigated the role of ACKR2 during IAV infection in mice. Pulmonary ACKR2 expression was increased acutely after IAV infection preceding the virus-induced lung dysfunction. ACKR2-knockout (ACKR2-/-) mice were protected from IAV, presenting decreased viral burden and lung dysfunction. Mechanistically, the absence of ACKR2 resulted in augmented airway CCL5 levels, secreted by mononuclear and plasma cells in the lung parenchyma. The higher chemokine gradient led to an augmented recruitment of T and B lymphocytes, formation of inducible bronchus-associated lymphoid tissue and production of IgA in the airways of ACKR2-/- mice post-IAV. CCL5 neutralization in ACKR2-/- mice prevented lymphocyte recruitment and increased bronchoalveolar lavage fluid protein levels and pulmonary dysfunction. Finally, CCR5-/- mice presented increased disease severity during IAV infection, displaying increased neutrophils, pulmonary injury and dysfunction, and accentuated lethality. Collectively, our data showed that ACKR2 dampens CCL5 levels and the consequent recruitment of CCR5+ T helper 1 (Th1), T regulatory cells (Tregs), and B lymphocytes during IAV infection, decreasing pathogen control and promoting lung dysfunction in wild type mice. Therefore, ACKR2 is detrimental and CCR5 is protective during IAV infection coordinating innate and adaptive immune responses in mice.

Zirconium catalyzed synthesis of 2-arylidene Indan-1,3-diones and evaluation of their inhibitory activity against NS2B-NS3 WNV protease.

A simple and efficient Knoevenagel procedure for the synthesis of 2-arylidene indan-1,3-diones is herein reported. These compounds were prepared via ZrOCl2·8H2O catalyzed reactions of indan-1,3-dione with several aromatic aldehydes and using water as the solvent. The 2-arylidene indan-1,3-diones were obtained with 53%-95% yield within 10-45 min. The synthesized compounds were evaluated as inhibitors of the NS2B-NS3 protease of West Nile Virus (WNV). It was found that hydroxylated derivatives impaired enzyme activity with varying degrees of effectiveness. The most active hydroxylated derivatives, namely 2-(4-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (14) and 2-(3,4-dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (17), were characterized as noncompetitive enzymes inhibitors, with IC50 values of 11 µmol L-1 and 3 µmol L-1, respectively. Docking and electrostatic potential surfaces investigations provided insight on the possible binding mode of the most active compounds within an allosteric site.

Immunocytochemistry Improving the Diagnosis of Trichomonas vaginalis Infections.

The aim of this study was to evaluate the immunocytochemistry (ICC) to diagnose trichomoniasis, particularly asymptomatic infections. By culture serial dilutions, ICC was able to detect 1 trophozoite/mL, while the culture was positive up to 100 trophozoites/mL. The ICC in vivo detection capability was assessed in vaginal secretions of mice experimentally infected and in vaginal swabs from asymptomatic HIV-positive pregnant women compared with culture. All vaginal secretion samples from mice were positive according to both methods. Swabs from fifty-five asymptomatic women were positive in four (7.27%) of them by culture. Beyond these four, another ten (25.45%) women were positive by immunocytochemistry, proving their higher sensitivity (p = 0.002), noticing 3.5 times more positives. ICC had better performance in both successive dilutions as in asymptomatic women, showing higher sensitivity and specificity. In this way, its facility of execution and cost-effectiveness support its practicality, as a routine procedure to diagnose trichomoniasis not only when the parasite load is lower but probably in all clinical scenarios.

New insights into the immunopathology of early Toxocara canis infection in mice.

BACKGROUND: Nematodes of the genus Toxocara are cosmopolitan roundworms frequently found in dogs and cats. Toxocara spp. can accidentally infect humans and cause a zoonosis called human toxocariasis, which is characterized by visceral, ocular or cerebral migration of larval stages of the parasite, without completing its life cycle. In general, chronic nematode infections induce a polarized TH2 immune response. However, during the initial phase of infection, a strong pro-inflammatory response is part of the immunological profile and might determine the outcome and/or pathology of the infection. METHODS: Parasitological aspects and histopathology during larval migration were evaluated after early T. canis experimental infection of BALB/c mice, which were inoculated via the intra-gastric route with a single dose of 1000 fully embryonated eggs. Innate immune responses and systemic cytokine patterns (TH1, TH2, TH17 and regulatory cytokines) were determined at different times after experimental challenge by sandwich ELISA. RESULTS: We found that experimental infection with T. canis induced a mix of innate inflammatory/TH17/TH2 responses during early infection, with a predominance of the latter. The TH2 response was evidenced by significant increases in cytokines such as IL-4, IL-5, IL-13 and IL-33, in addition to increasing levels of IL-6 and IL-17. No significant increases were observed for IL-10, TNF-α or IFN-γ levels. In parallel, parasitological analysis clearly revealed the pattern of larval migration through the mouse organs, starting from the liver in the first 24 h of infection, reaching the peak in the lungs on the 3rd day of infection and finally being found numerously in the brain after 5 days of infection. Peripheral leukocytosis, characterized by early neutrophilia and subsequent eosinophilia, was remarkable during early infection. The tissue damage induced by larvae was evidenced by histopathological analysis of the organs at different time points of infection. In all of the affected organs, larval migration induced intense inflammatory infiltrate and hemorrhage. CONCLUSION: In conclusion, these new insights into early T. canis infection in mice presented here enabled a better understanding of the immunopathological events that might also occur during human toxocariasis, thus contributing to future strategies of diagnosis and control.

Evaluation of the Schistosoma mansoni Y-box-binding protein (SMYB1) potential as a vaccine candidate against schistosomiasis.

Schistosomiasis is a neglected tropical disease, and after malaria, is the second most important tropical disease in public health. A vaccine that reduces parasitemia is desirable to achieve mass treatment with a low cost. Although potential antigens have been identified and tested in clinical trials, no effective vaccine against schistosomiasis is available. Y-box-binding proteins (YBPs) regulate gene expression and participate in a variety of cellular processes, including transcriptional and translational regulation, DNA repair, cellular proliferation, drug resistance, and stress responses. The Schistosoma mansoni ortholog of the human YB-1, SMYB1, is expressed in all stages of the parasite life cycle. Although SMYB1 binds to DNA or RNA oligonucleotides, immunohistochemistry assays demonstrated that it is primarily localized in the cytoplasm of parasite cells. In addition, SMYB1 interacts with a protein involved in mRNA processing, suggesting that SMYB1 functions in the turnover, transport, and/or stabilization of RNA molecules during post-transcriptional gene regulation. Here we report the potential of SMYB1 as a vaccine candidate. We demonstrate that recombinant SMYB1 stimulates the production of high levels of specific IgG1 antibodies in a mouse model. The observed levels of specific IgG1 and IgG2a antibodies indicate an actual protection against cercariae challenge. Animals immunized with rSMYB1 exhibited a 26% reduction in adult worm burden and a 28% reduction in eggs retained in the liver. Although proteins from the worm tegument are considered optimal targets for vaccine development, this study demonstrates that unexposed cytoplasmic proteins can reduce the load of intestinal worms and the number of eggs retained in the liver.

Exercise attenuates pulmonary injury in mice with bleomycin-induced pulmonary fibrosis.

Human idiopathic pulmonary fibrosis (IPF) is a disease with unknown etiology and poor prognosis in which patients present a decrease in functional exercise tolerance and quality of life. At present, no treatment which can improve the prognosis of this disease is available. Many biomarkers of pulmonary fibrosis have been studied, and surfactant protein A (SP-A) expression is considered a specific marker of lung disease. This study aimed to investigate the influence of exercise training on exercise endurance capacity and murine-lung lesions induced by bleomycin (BLM). Thirty-four male Balb/c mice were subdivided into four groups: control sedentary (C-SED), bleomycin-treated sedentary (BLM-SED), control exercised (C-EXE) and bleomycin-treated exercised (BLM-EXE). Mice received 6.25 U/kg of BLM or saline via intratracheal instillation. After adaptation in a swimming pool, the animals started training one hour per day, with 60% of maximum load obtained in exercise endurance capacity assessment, five days/week for four weeks. The lungs were collected 48 h after the second endurance capacity assessment, fixed in buffered formalin and embedded in paraffin. Sections were analyzed using histochemical and immunohistochemical reactions for digital morphometry of pulmonary fibrosis, type I collagen, SP-A and type II pneumocytes (PII). The exercise endurance capacity of groups C-EXE (9.20 ± 0.81 min) and BLM-EXE (8.40 ± 0.82 min) increased significantly when compared with groups C-SED (5.84 ± 0.4 min) and BLM-SED (5.67 ± 0.60 min). The amounts of connective tissue, type I collagen, PII and SP-A increased significantly in the BLM-SED group. Exercise training significantly attenuated this response as observed in the BLM-EXE group. The present study shows that exercise training can prevent the decline of exercise endurance capacity and attenuate the progression of IPF.

Susceptibility to Entamoeba histolytica intestinal infection is related to reduction in natural killer T-lymphocytes in C57BL/6 mice.

Entamoeba histolytica is a protozoan that causes amoebiasis. Recent studies demonstrated that natural killer T lymphocytes (NKT) are critical for preventing the development of amoebic liver abscess. In spite of that, there are only a handful of studies in the area. Herein, we explored the role of NKT cells in E. histolytica infection using C57BL/6 wild-type and CD1(-/-) mice. Animals were inoculated with E. histolytica and sacrificed 48 hours later to collect caecum samples that were used for quantitative analyses of lesions, trophozoites, NK1.1(+) T lymphocytes and expression of the mucus protein MUC-2 by immunohistochemistry technique. Quantitative analyses confirmed that the frequency of NK1.1(+) T cells was significantly lower in samples from C57BL/6 CD1(-/-) mice as compared to their wild type (WT) counterparts. The extension of necrotic mucosa was larger and the number of trophozoites higher in Entamoeba (Eh)-infected CD1(-/-) mice when compared with Eh-infected WT mice. In mice from both groups, non-infected (CTRL) and Eh-infected CD1(-/-), there was a reduction in the thickness of the caecal mucosa and in the MUC-2-stained area in comparison with CTRL- and Eh-WT mice. Our results showed that NKT lymphocytes contribute to resistance against Entamoeba histolytica infection and to the control of inflammation in the colitis induced by infection. The presence of a normal epithelial layer containing appropriate levels of mucus had also a protective role against infection.

Melatonin reduces the severity of experimental amoebiasis.

BACKGROUND: Melatonin has immunomodulatory effects but very little is known about its influence in protozoan infections, such as Entamoeba histolytica, which causes amoebiasis, a disease with significant morbidity and mortality. In this study, we evaluated the effects of exogenous melatonin interference in experimental amoebiasis and on interactions between human blood cells and E. histolytica trophozoites. METHODS: The effect of melatonin was investigated in models of experimental amoebiasis in hamsters and rats by evaluating the area of necrosis induced by E. histolytica. The activity of melatonin on the interactions between leukocytes and amoebae was determined by examining leukophagocytosis. For in vitro tests, polymorphonuclear and mononuclear human blood leucocytes were incubated with E. histolytica trophozoites. RESULTS: The areas of amoebic necrosis were significantly reduced in animals treated with melatonin. Melatonin treatment increased leukophagocytosis but was associated with a greater number of dead amoebae. CONCLUSIONS: These results suggest that melatonin may play a beneficial role in the control of amoebic lesions, raising the possibility that this drug may be used as an adjuvant in anti-amoebic therapy.