The effect of Spondias mombin L. against Strongyloides venezuelensis: An in vitro approach.

Strongyloidiasis is a neglected tropical disease mainly caused by the nematode parasite Strongyloides stercoralis. Current treatment consists in the administration of ivermectin or, alternatively, albendazole (or analogues). Concerns regarding these drugs' irregular cure rates and side effects, raise a need for therapeutic alternatives. In this study, we tested the in vitro effect of Spondias mombin L. ethanolic extract against the laboratory model for strongyloidiasis, Strongyloides venezuelensis. The ethanolic extract was further fractionated and each fraction was also tested. Tested fractions were analyzed through thin layer chromatography and gas chromatography (GC/MS). Our results showed that S. mombin extract and fractions had a better in vitro effect than ivermectin, particularly fraction 4 which showed the better results causing 100% mortality in 4 h after exposure to an extract concentration of 400 µg/mL of RPMI medium and caused 100% mortality 12 h after exposure to an extract concentration of 50 µg/mL. Scanning electron microscopy showed that this fraction caused both wrinkling and peeling of the parasites cuticle, whilst ivermectin only caused wrinkling. GC/MS showed a high percentage of monoaromatic phenolic lipids (3-R phenol and 3-R1 phenol), which were likely responsible for the anti-Strongyloides effect. The use of polyvinylpyrrolidone reduced the efficiency, thus raising a need for alertness when using this excipient. Our results suggest that S. mombin is a potential source of compounds that could be used for stongyloidiasis treatment.

Sesquiterpenes evaluation on Schistosoma mansoni: Survival, excretory system and membrane integrity.

The main challenge in schistosomiasis control has been the emergence of drug-resistant parasites. Since the 1970's, praziquantel (PZQ) is the single drug for treatment. This fact highlights the importance to research news chemotherapeutic agents. In the last years, S. mansoni excretory system and tegument have been major targets for drug development. The purpose of this study was to evaluate the effect of sesquiterpenes, alpha-humulene and trans-caryophyllene on S. mansoni survival, excretory system and membrane integrity, after in vitro exposure. The in vitro studies, showed that sesquiterpenes reduced egg production and motor activity of worms at sublethal concentrations, and caused death in a concentration-dependent manner (100 and 200µg/mL). Tegumental analysis by Scanning Electron Microscopy (SEM), showed tegument damage. Additionally, it was possible to observe lesions, evidenced by intense marking trough Hoechst probe, in the tegument and suckers of worms exposed to 200µg/mL. In this study, we also showed that resorufin is only capable of identifying the interaction of sesquiterpenes in males excretory system, Pgp expression and inferring that females are more tolerant to treatments. Thus, the present study results contribute to an understanding of alpha-humulene and trans-caryophyllene effect over these targets, contributing for the development of schistosomicidal drugs.

Early developmental stages of Ascaris lumbricoides featured by high-resolution mass spectrometry.

Ascaris lumbricoides is responsible for a highly disseminated helminth parasitic disease, ascariosis, a relevant parasitosis that responds for great financial burden on the public health system of developing countries. In this work, metabolic fingerprinting using high-resolution mass spectrometry (HRMS) was employed to identify marker molecules from A. lumbricoides in different development stages. We have identified nine biomarkers, such as pheromones and steroidal prohormones in early stages, among other molecules in late development stages, making up four molecules for fertilized eggs, four marker molecules for first larvae (L1) and one marker molecule for third larvae (L3). Therefore, our findings indicate that this approach is suitable for biochemical characterization of A. lumbricoides development stages. Moreover, the straightforward analytical method employed, with almost no sample preparation from a complex matrix (feces) using high-resolution mass spectrometry, suggests that it is possible to seek for an easier and faster way to study animal molding processes.

Morphological Characteristics of Schistosoma mansoni PZQ-Resistant and -Susceptible Strains Are Different in Presence of Praziquantel.

Schistosomiasis is one of the most common human parasitic diseases whose socioeconomic impact is only surpassed by malaria. Praziquantel (PZQ) is the only drug commercially available for the treatment of all schistosome species causing disease in humans. However, there has been stronger evidences of PZQ-resistance on Schistosoma mansoni and thus it is very important to study the phenotypic characteristics associated with it. The aim of this study was to evaluate morphological alterations in S. mansoni PZQ-resistant adult worms and eggs, by comparing a PZQ- resistant strain obtained under PZQ drug pressure with a PZQ-susceptible strain. For this, scanning electronic microscopy was used to assess tegumental responsiveness of both strains under PZQ exposure, and optical microscopy allowed the monitoring of worms and eggs in the presence of the drug. Those assays showed that PZQ-susceptible worms exposed to the drug had more severe tegumental damages than the resistant one, which had only minor alterations. Moreover, contrary to what occurred in the susceptible strain, resistant worms were viable after PZQ exposure and gradually regaining full motility after removal of the drug. Eggs from resistant strain parasites are considerably smaller than those from susceptible strain. Our results suggest that there might be a difference in the tegument composition of the resistant strain and that worms are less responsive to PZQ. Changes observed in egg morphology might imply alterations in the biology of schistosomes associated to PZQ-resistance, which could impact on transmission and pathology of the disease. Moreover, we propose a hypothetical scenario where there is a different egg tropism of the S. mansoni resistant strain. This study is the first comparing two strains that only differ in their resistance characteristics, which makes it a relevant step in the search for resistance determinants.

Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni.

Finding specific molecular targets and the mechanism of action of praziquantel in the treatment of schistosomiasis remains a challenging task. Our efforts were focused on obtaining further information on worm composition before and after exposure to praziquantel in the treatment of schistosomiasis to elucidate the potential sites of action of this drug. Evidence indicates that the lipid bilayer is changed by treatment with praziquantel. Following this rationale, we employed a mass spectrometry imaging-based approach that helped to characterise lipids in specific locations, which are directly involved in the biochemical pathways of the BH strain of Schistosoma mansoni, as well as differentiating the molecular response that each worm sex presents in vivo. Our findings demonstrated significant differences between the chemical markers found in adult worms before and after praziquantel exposure, especially in phospholipids, which were predominantly identified as chemical markers in all samples. Results also indicate that distinct molecular pathways in both male and female worms could be differentially affected by praziquantel treatment. These data shine new light on the mechanism of action of praziquantel, taking a further step towards its full understanding.

Screening the life cycle of Schistosoma mansoni using high-resolution mass spectrometry.

Schistosomiasis is a common tropical disease caused by Schistosoma species Schistosomiasis' pathogenesis is known to vary according to the worms' strain. Moreover, high parasitical virulence is directly related to eggs release and granulomatous inflammation in the host's organs. This virulence might be influenced by different classes of molecules, such as lipids. Therefore, better understanding of the metabolic profile of these organisms is necessary, especially for an increased potential of unraveling strain virulence mechanisms and resistance to existing treatments. In this report, direct-infusion electrospray high-resolution mass spectrometry (ESI(+)-HRMS) along with the lipidomic platform were employed to rapidly characterize and differentiate two Brazilian S. mansoni strains (BH and SE) in three stages of their life cycle: eggs, miracidia and cercariae, with samples from experimental animals (Swiss/SPF mice). Furthermore, urine samples of the infected and uninfected mice were analyzed to assess the possibility of direct diagnosis. All samples were differentiated using multivariate data analysis, PCA, which helped electing markers from distinct lipid classes; phospholipids, diacylglycerols and triacylglycerols, for example, clearly presented different intensities in some stages and strains, as well as in urine samples. This indicates that biochemical characterization of S. mansoni may help narrowing-down the investigation of new therapeutic targets according to strain composition and aggressiveness of disease. Interestingly, lipid profile of infected mice urine varies when compared to control samples, indicating that direct diagnosis of schistosomiasis from urine may be feasible.

Anthelmintic activity in vitro and in vivo of Baccharis trimera (Less) DC against immature and adult worms of Schistosoma mansoni.

Although its efficiency against all Schistosoma species, praziquantel (PZQ) shows low efficacy against schistosomula and juvenile stages. The potential for development of resistance to PZQ has justified the search for new alternative chemotherapies. In this scenario, studies to new formulations, more comprehensive and without adverse effects, are being conducted. One viable and promising treatment is the study of medicinal plants as a new approach to the experimental treatment for Schistosomiasis. Amongst all the variety of the medicinal species studied, we can highlight Baccharis trimera (Less) DC, known as "Carqueja-amarga". This paper not only describes the effect of crude dichloromethane extract (DE) and aqueous fraction (AF) obtained from B. trimera, in vitro but also is the first one that investigates the in vivo efficacy of B. trimera against schistosomula, juvenile and adult worms of Schistosoma mansoni BH strain. In the experiment, mice were treated with DE, AF and PZQ (40 and 200mg/kg) over the period of larval development (3 and 30 post-infection; pi), and adult worms (60days post-infection; pi). The in vitro results show that the DE and AF effects are dose-dependents, being the 130µg/mL the most effective one in a shorter period of incubation. The exposure of the in vitro samples over adult parasites were able to inhibit 100% of the oviposition in females. Likewise caused the mortality of the parasites with morphological alterations on the tegument, on the suckers, oral and acetabulum, in both males and females after 6-72h of exposure. Additionally, the in vivo treatments against juvenile and adult infection were more effective compared to the control group untreated. Administrations of AF and DE in day 30pi (juvenile worms) show female worm total burden reductions of 75% and 68% respectively. At the same period of infection reductions of respectively 98% and 97% egg/g in the faeces were seen. In relation to the different egg developmental stages (oogram), the results showed significant reductions, due to the reduction in the number of worms, especially the females. In conclusion, B. trimera exhibits major schistosomicidal effects in vivo against immature and adult worms of S. mansoni, opening up perspectives for future researches on substance or compound isolation and the elucidation of its mechanisms of action.

Mass spectrometry imaging: a new vision in differentiating Schistosoma mansoni strains.

Schistosomiasis is a neglected disease with large geographic distribution worldwide. Among the several different species of this parasite, S. mansoni is the most common and relevant one; its pathogenesis is also known to vary according to the worms' strain. High parasitical virulence is directly related to granulomatous reactions in the host's liver, and might be influenced by one or more molecules involved in a specific metabolic pathway. Therefore, better understanding the metabolic profile of these organisms is necessary, especially for an increased potential of unraveling strain virulence mechanisms and resistance to existing treatments. In this report, MALDI-MSI and the metabolomic platform were employed to characterize and differentiate two Brazilian S. mansoni strains: males and females from Belo Horizonte (BH) and from Sergipe (SE). By performing direct analysis, it is possible to distinguish the sex of adult worms, as well as identify the spatial distribution of chemical markers. Phospholipids, diacylglycerols and triacylglycerols were located in specific structures of the worms' bodies, such as tegument, suckers, reproductive and digestive systems. Lipid profiles were found to be different both between strains and males or females, giving specific metabolic fingerprints for each group. This indicates that biochemical characterization of adult S. mansoni may help narrowing-down the investigation of new therapeutic targets according to worm composition, molecule distribution and, therefore, aggressiveness of disease.

In vitro evaluation of permeation, toxicity and effect of praziquantel-loaded solid lipid nanoparticles against Schistosoma mansoni as a strategy to improve efficacy of the schistosomiasis treatment.

Solid lipid nanoparticles (SLN) are a promising drug delivery system for oral administration of poorly-water soluble drugs because of their capacity to increase the solubility of drug molecules when loaded in their lipid matrices, with the resulting improvement of the drug bioavailability. In the present work, we have developed praziquantel (PZQ)-loaded SLN and explored the biological applications of this system for intestinal permeation of PZQ. The effect in vitro on Schistosoma mansoni culture and the cytotoxicity in HepG2 line cell were also evaluated. The results showed a significant decrease in the intestinal absorption of PZQ loaded in SLN compared to free PZQ, suggesting that the SLN matrix could act as reservoir system. In culture of S. mansoni, we observed that PZQ-loaded SLN were more effective than free PZQ, leading the death of the parasites in less time. The result was proportional to doses of PZQ (25 and 50 µg mL⁻¹) and lipid concentration. Regarding cytotoxicity, the encapsulation of PZQ into SLN decreased the toxicity in HepG2 cells in comparison to the free PZQ. From the obtained results, PZQ-loaded SLN could be a new drug delivery system for the schistosomiasis treatment especially in marginalized communities, improving the therapeutic efficacy and reducing the toxic effects of PZQ.

Schistosoma mansoni: in vitro schistosomicidal activity of essential oil of Baccharis trimera (less) DC.

Schistosomiasis is a chronic parasitic disease caused by the trematode species Schistosoma mansoni. Chemotherapy is the only immediate recourse to minimize the prevalence and incidence of this disease worldwide. At present, praziquantel (PZQ) is the drug of choice for the treatment of all forms of schistosomiasis. However, dependence on a single drug is concern because some strains can become resistant. In this context, medicinal plants become potential candidates as sources of new drug prototypes. This study provides findings on the schistosomicidal activity of the essential oil of Baccharis trimera in in vitro assays. During the assays parameters such as motility of adult worms, oviposition, morphological changes on the tegument and especially the mortality rate of adult worms of the BH strain were evaluated. The assays, which were carried out with four concentrations - 24, 48, 91 and 130 µg/mL - of the essential oil, have shown a promising activity regarding the parameters under study. It was possible to notice a significant decline in the motility of the worms and a mortality rate of 100% 30 h after they had been exposed to the essential oil in the concentration of 130 µg/mL. Male worms were more susceptible, producing a dose-response effect within a smaller exposition period than female worms. In what refers to morphological changes, the essential oil of B. trimera induced a peeling on the tegument surface, as well as the destruction of tubercles and spines, which resulted in smooth areas on the body surface. The essential oil also caused tegument destruction in female worms, in addition to destruction of the oral and acetabular suckers. It is the first time that the schistosomicidal activity has been reported for essential oil of B. trimera (less) DC.