Polymeric nanoparticles containing kojic acid induce structural alterations and apoptosis-like death in Leishmania (Leishmania) amazonensis.

Leishmaniasis encompasses a cluster of neglected tropical diseases triggered by kinetoplastid phatogens belonging to the genus Leishmania. Current therapeutic approaches are toxic, expensive, and require long-term treatment. Nanoparticles are emerging as a new alternative for the treatment of neglected tropical diseases. Silk Fibroin is a biocompatible and amphiphilic protein that can be used for formulating nanoemulsions, while kojic acid is a secondary metabolite with antileishmanial actions. Thus, this study evaluated the efficacy of a nanoemulsion, formulated with silk fibroin as the surfactant and containing kojic acid (NanoFKA), against promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. The NanoFKA had an average particle size of 176 nm, Polydispersity Index (PDI) of 0.370, and a Zeta Potential of -32.3 mV. It presented inhibitory concentration (IC50) values of >56 µg/mL and >7 µg/mL for the promastigote and amastigote forms, respectively. Ultrastructural analysis, cell cycle distribution and phosphatidylserine exposure showed that NanoFKA treatment induces apoptosis-like cell death and cell cycle arrest in L. (L.) amazonensis. In addition, NanoFKA exhibited no cytotoxicity against macrophages. Given these results, NanoFKA present leishmanicidal activity against L. (L.) amazonensis.

In Vitro Leishmanicidal Activity of Copaiba Oil and Kojic Acid Combination on the Protozoan Leishmania (Leishmania) amazonensis and Host Cell.

(1) Background: Leishmaniasis refers to a group of anthropozoonotic diseases caused by Leishmania. The major chemotherapeutic agent used for its treatment is Glucantime®®, but the search continues for new compounds that are economically viable and act on the protozoan without causing damage to the host cell. As an alternative approach, this study used a combination of copaiba oil (CO) and kojic acid (KA) to determine their in vitro action on host cells, on the Leishmania (Leishmania) amazonensis protozoan and its interaction with macrophages. (2) Methods: In vitro culture, analysis of cytokine release and microscopy assays were performed. Statistical analysis was performed with ANOVA (GraphPad Prism). (3) Results: The combination did not induce cytotoxic effects on macrophages after treatment but promoted morphological changes in the protozoan, such as nuclear alterations (apoptotic characteristics), alterations in the cellular body and an increase in the number of electrodense structures and acidocalcisomes, observed mainly at the concentrations of CO20KA50 and CO30KA50 µg/mL. We observed reductions in the intracellular amastigote number and in the production of proinflammatory cytokines, such as IL-6 and TNF-α, after treatment with CO30KA at 50 µg/mL. (4) Conclusions: We report here, for the first time, that the combination of CO and KA may be a promising approach against Leishmania (Leishmania) amazonensis.

Poly (ε-caprolactone)-Based Scaffolds with Multizonal Architecture: Synthesis, Characterization, and In Vitro Tests.

Tissue engineering is vital in treating injuries and restoring damaged tissues, aiming to accelerate regeneration and optimize the complex healing process. In this study, multizonal scaffolds, designed to mimic tissues with bilayer architecture, were prepared using the rotary jet spinning technique (RJS scaffolds). Polycaprolactone and different concentrations of alginate hydrogel (2, 4, and 6% m/v) were used. The materials were swollen in pracaxi vegetable oil (PO) (Pentaclethra macroloba) and evaluated in terms of surface morphology, wettability, functional groups, thermal behavior, crystallinity, and cytotoxicity. X-ray diffraction (XRD) showed the disappearance of the diffraction peak 2θ = 31.5° for samples from the polycaprolactone/pracaxi/alginate (PCLOA) group, suggesting a reduction of crystallinity according to the presence of PO and semi-crystalline structure. Wettability gradients (0 to 80.91°) were observed according to the deposition layer and hydrogel content. Pore diameters varied between 9.27 µm and 37.57 µm. Molecular interactions with the constituents of the formulation were observed via infrared spectra with Fourier transform (FTIR), and their influence was detected in the reduction of the maximum degradation temperature within the groups of scaffolds (polycaprolactone/alginate (PCLA) and PCLOA) about the control. In vitro tests indicated reduced cell viability in the presence of alginate hydrogel and PO, respectively.

Additive manufacturing of poly (lactic acid)/hydroxyapatite/carbon nanotubes biocomposites for fibroblast cell proliferation.

Bone tissue is one of the most important in the human body. In this study, scaffolds of poly (lactic acid) PLA reinforced with hydroxyapatite (HA) and carbon nanotubes (CNT) were manufactured, evaluating their mechanical and biological properties. HA was synthesized by wet method and characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The scaffolds were produced using additive manufacturing and characterized by optical microscopy, SEM, thermogravimetric analysis (TGA), Raman spectroscopy and biological tests. The SEM results showed that the PLA surface was affected by the incorporation of CNT. TG showed that the incorporation of HA into the polymer matrix compromised the thermal stability of PLA. On the other hand, the incorporation of CNT to the polymer and the impregnation with HA on the surface by thermal effect increased the stability of PLA/CNT scaffolds. Raman spectra indicated that HA impregnation on the surface did not modify the polymer or the ceramic. In the compression tests, PLA and PLA/CNT scaffolds displayed the best compressive strength. In the biological tests, more than 85% of the cells remained viable after 48 h of incubation with all tested scaffolds and groups with CNT in the composition disclosing the best results.

Detection and activity of MMP-2 and MMP-9 in Leishmania amazonensis and Leishmania braziliensis promastigotes.

Metalloproteinases (MMPs) are remarkable zinc-dependent endopeptidases, critical for degrading components of the extracellular matrix, thus actively influencing cell migration. Their impact on intracellular parasites, such as the enigmatic protozoan Leishmania, elicits intriguing queries. This study explores into the untapped territory of MMP-2 and MMP-9 within Leishmania spp. promastigotes. Notably, we successfully detected and quantified these MMPs, while also evaluating their activity in two distinct Leishmania species-L. amazonensis (La) and L. braziliensis (Lb)-at various growth stages and isolated from distinct clinical tegumentar disease forms. The results unveiled the presence of MMP-2 and MMP-9 in both species, albeit with distinct localization patterns. Specifically, MMP-9 exhibited significantly higher gelatinolytic activity in La when compared to Lb. Moreover, our data cleverly illustrated the presence and release of MMP-2 and MMP-9 by La and Lb promastigotes, exposing their ability to invade and migrate within a collagen matrix. This pioneering study establishes a compelling correlation between MMP-2 and MMP-9 and their potential role in the dynamics of La and Lb infection. Suggesting their potential as prognostic markers for severe leishmaniasis and promising target molecules for therapeutic interventions, this research opens new avenues for combatting this debilitating parasitic disease.

Three-dimensional architecture of Cyrilia lignieresi gametocyte-stage development inside red blood cells.

The Haemogregarinidae family (Apicomplexa: Adeleina) comprises hemoprotozoa that infect mammals, birds, amphibians, fish, and reptiles. Some morphological characteristics of the Cyrilia lignieresi have been described previously, but the parasite-erythrocyte relationship is still poorly understood. In order to understand the structural architecture of C. lignieresi-infected red blood cells, electron microscopy-based three-dimensional reconstruction was carried out using TEM as well as FIB-SEM tomography. Results showed that development of the macrogametocyte-stage inside the red blood cell is related to an increase in cleft-like structures in the host cell cytoplasm. Furthermore, other aspects related to parasite intraerythrocytic development were explored by 3D visualization techniques. We observed the invagination of a large extension of the Inner Membrane Complex (IMC) on the parasite body, which results from or induces a folding of the posterior end of the parasite. Small tubular structures were seen associated with areas related to IMC folding. Taken together, results provide new information on the remodeling of erythrocytes induced by the protozoan C. lignieresi.

AgNP-PVP-meglumine antimoniate nanocomposite reduces Leishmania amazonensis infection in macrophages.

BACKGROUND: Leishmaniasis is an infectious disease caused by parasites of the genus Leishmania and presents different clinical manifestations. The adverse effects, immunosuppression and resistant strains associated with this disease necessitate the development of new drugs. Nanoparticles have shown potential as alternative antileishmanial drugs. We showed in a previous study the biosynthesis, characterization and ideal concentration of a nanocomposite that promoted leishmanicidal activity. In the present study, we conducted a specific analysis to show the mechanism of action of AgNP-PVP-MA (silver nanoparticle-polyvinylpyrrolidone-[meglumine antimoniate (Glucantime®)]) nanocomposite during Leishmania amazonensis infection in vitro. RESULTS: Through ultrastructural analysis, we observed significant alterations, such as the presence of small vesicles in the flagellar pocket and in the extracellular membrane, myelin-like structure formation in the Golgi complex and mitochondria, flagellum and plasma membrane rupture, and electrodense material deposition at the edges of the parasite nucleus in both evolutive forms. Furthermore, the Leishmania parasite infection index in macrophages decreased significantly after treatment, and nitric oxide and reactive oxygen species production levels were determined. Additionally, inflammatory, and pro-inflammatory cytokine and chemokine production levels were evaluated. The IL-4, TNF-α and MIP-1α levels increased significantly, while the IL-17 A level decreased significantly after treatment. CONCLUSIONS: Thus, we demonstrate in this study that the AgNP-PVP-MA nanocomposite has leishmanial potential, and the mechanism of action was demonstrated for the first time, showing that this bioproduct seems to be a potential alternative treatment for leishmaniasis.

Polymorphism in the IL-1ß promoter is associated with IgG antibody response to circumsporozoite protein repeats of Plasmodium vivax.

BACKGROUND: It is well established that infection by Plasmodium vivax is a result of host-parasite interactions. In the present study, association with the IL1/IL2 cytokine profiles, anticircumsporozoite protein antibody levels and parasitic loads was evaluated in individuals naturally infected with P. vivax in an endemic area of the Brazilian Amazon. METHODS: Molecular diagnosis of P. vivax and variants was performed using the PCR-RFLP method and IL1B -511C>T, IL2 -330T>G and IL2+114T>G polymorphisms were identified using PCR-RFLP and allele-specific PCR. IL-1ß and IL-2 cytokine levels were detected by flow cytometry and circumsporozoite protein (CSP) antibodies were measured by ELISA. RESULTS: Three variants of P. vivax CSP were identified and VK247 was found to be the most frequent. However, the prevalence and magnitude of IgG antibodies were higher for the VK210 variant. Furthermore, the antibody response to the CSP variants was not associated with the presence of the variant in the infection. Significant differences were observed between the single nucleotide polymorphism (SNP) -511T>C in the IL1B gene and levels of antibodies to the VK247 and P. vivax-like variants, but there were no associations between SNPs in IL1 and IL2 genes and their plasma products. CONCLUSIONS: Individuals with the rs16944 CC genotype in the IL1ß gene have higher antibody levels to the CSP of P. vivax of VK247 and P. vivax-like variants.

Limbic Encephalitis Brain Damage Induced by Cocal Virus in Adult Mice Is Reduced by Environmental Enrichment: Neuropathological and Behavioral Studies.

We previously demonstrated, using the Piry virus model, that environmental enrichment promotes higher T-cell infiltration, fewer microglial changes, and faster central nervous system (CNS) virus clearance in adult mice. However, little is known about disease progression, behavioral changes, CNS cytokine concentration, and neuropathology in limbic encephalitis in experimental models. Using Cocal virus, we infected C57Bl6 adult mice and studied the neuroanatomical distribution of viral antigens in correlation with the microglial morphological response, measured the CNS cytokine concentration, and assessed behavioral changes. C57Bl6 adult mice were maintained in an impoverished environment (IE) or enriched environment (EE) for four months and then subjected to the open field test. Afterwards, an equal volume of normal or virus-infected brain homogenate was nasally instilled. The brains were processed to detect viral antigens and microglial morphological changes using selective immunolabeling. We demonstrated earlier significant weight loss and higher mortality in IE mice. Additionally, behavioral analysis revealed a significant influence of the environment on locomotor and exploratory activity that was associated with less neuroinvasion and a reduced microglial response. Thus, environmental enrichment was associated with a more effective immune response in a mouse model of limbic encephalitis, allowing faster viral clearance/decreased viral dissemination, reduced disease progression, and less CNS damage.

In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum.

OBJECTIVE: In the present study, we evaluated the effects of the aqueous extract of Physalis angulata root (AEPa) on Leishmania infantum proliferation, morphology, and the driving mechanism in leishmanicidal activity and modulatory action on macrophages. METHODS: L. infantum promastigotes were treated with 50 and 100 µg/mL AEPa for 72 h and then antipromastigote assay was performed by counts in a Newbauer chamber, morphological changes were analyzed by transmission electron microscopy and the mechanism of the leishmanicidal activity was detected. In addition, macrophages were infected with L. infantum and were used to evaluate anti-amastigote activity of AEPa and effects of AEPa on cytokine secretion after 72-hour treatment. RESULTS: Treatment with AEPa reduced the numbers of L. infantum promastigotes (50% inhibitory concentration (IC50) = 65.9 µg/mL; selectivity index (SI) = 22.1) and amastigotes (IC50 = 37.9 µg/mL; SI = 38.5) compared with the untreated control. Amphotericin B reduced 100% of the promastigote numbers after 72 h of treatment (IC50 = 0.2 µg/mL). AEPa induced several morphological changes and increased the production of reactive oxygen species and apoptotic death in promastigotes after treating for 72 h. AEPa (100 µg/mL) promoted tumor necrosis factor-α secretion in macrophages infected with L. infantum after 72 h of treatment, but did not induce an increase in this cytokine in noninfected macrophages. In addition, AEPa showed no cytotoxic effect on J774-A1 cells (50% cytotoxic concentration >1000 µg/mL). CONCLUSION: AEPa presented antileishmanial activity against the promastigotes and amastigotes of L. infantum without macrophage cytotoxicity; these results show that natural products such as P. angulata have leishmanicidal potential and in the future may be an alternative treatment for leishmaniasis.

Granulocyte macrophage colony-stimulating factor alone reduces Toxoplasma gondii replication in microglial culture by superoxide and nitric oxide, without IFN-γ production: a preliminary report.

In vitro studies have demonstrated that GM-CSF in combination with other stimulatory factors induces a microbicidal response that control T. gondii infection. We assessed whether GM-CSF alone can control T. gondii replication in murine microglial cultures. Microglia were collected and cultured with or without GM-CSF and the half of each group was infected with T. gondii. We determined the T. gondii infectivity, cytokines levels, NO and superoxide detection. GM-CSF alone primes microglia, which after infection induces the production of TNF-α and IL-6, leading to NO and superoxide production, without any stimulus from IL-12p70 and IFN-γ.

Medicinal plants from the Brazilian Amazonian region and their antileishmanial activity: a review.

Leishmaniasis, a neglected disease caused by Leishmania protozoans, primarily affects people in tropical and subtropical areas. Chemotherapy based on the use of pentavalent antimonials, amphotericin B, paromomycin, miltefosine and liposomal amphotericin B is currently the only effective treatment. However, adverse effects, long-term treatment and the emergence of parasite resistance have led to the search for alternative treatments. Natural products used in traditional medicine provide an unlimited source of molecules for the identification of new drugs, and the Amazon region has abundant biodiversity that includes several species of plants and animals, providing a rich source of new products and compounds. Although the literature describes numerous promising compounds and extracts for combating Leishmania protozoans, the results of such research have not been embraced by the pharmaceutical industry for the development of new drugs. Therefore, this review focused on the antileishmanial activity of extracts, isolated compounds and essential oils commonly used by the local population in the Brazilian Amazonian region to treat several illnesses and described in the literature as promising compounds for combating leishmaniasis.

Biosynthesis, characterization and leishmanicidal activity of a biocomposite containing AgNPs-PVP-glucantime.

AIM: Development of functionalized nanocomposites containing AgNPs-PVP-Glucantime® to evaluate their leishmanicidal activity as a novel method for improving the pharmacological properties of the drug Glucantime® against extracellular promastigotes and intracellular amastigotes of Leishmania amazonensis in vitro to treat cutaneous leishmaniasis. MATERIALS & METHODS: The silver nanoparticles and nanocomposites prepared containing silver nanoparticles, polyvinylpyrrolidone and different amounts of Glucantime were characterized using transmission electron microscopy, x-ray diffraction, energy-dispersive x-ray spectroscopy and ζ potential analysis; in addition, the in vitro cytotoxicity was evaluated. RESULTS: The nanocomposites showed an inhibitory effect on the cellular viability of promastigote forms, with values of 47.06, 51.71 and 65.67% for nanocomposite1, nanocomposite2 and nanocomposite3, respectively, as well as a dose-dependent decrease in the infectivity index, with values of 33.33 and 23% for nanocomposite2 and nanocomposite3, respectively. CONCLUSION: The proposed nanocomposite reveals leishmanial activity and the absence of cytotoxicity in macrophages. Further investigations will be conducted in vivo.