High-resolution AP-SMALDI MSI as a tool for drug imaging in Schistosoma mansoni.

Schistosoma mansoni is a parasitic flatworm causing schistosomiasis, an infectious disease affecting several hundred million people worldwide. Schistosomes live dioeciously, and upon pairing with the male, the female starts massive egg production, which causes pathology. Praziquantel (PZQ) is the only drug used, but it has an inherent risk of resistance development. Therefore, alternatives are needed. In the context of drug repurposing, the cancer drug imatinib was tested, showing high efficacy against S. mansoni in vitro. Besides the gonads, imatinib mainly affected the integrity of the intestine in males and females. In this study, we investigated the potential uptake and distribution of imatinib in adult schistosomes including its distribution kinetics. To this end, we applied for the first time atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) for drug imaging in paired S. mansoni. Our results indicate that imatinib was present in the esophagus and intestine of the male as early as 20 min after in vitro exposure, suggesting an oral uptake route. After one hour, the drug was also found inside the paired female. The detection of the main metabolite, N-desmethyl imatinib, indicated metabolization of the drug. Additionally, a marker signal for the female ovary was successfully applied to facilitate further conclusions regarding organ tropism of imatinib. Our results demonstrate that AP-SMALDI MSI is a useful method to study the uptake, tissue distribution, and metabolization of imatinib in S. mansoni. The results suggest using AP-SMALDI MSI also for investigating other antiparasitic compounds and their metabolites in schistosomes and other parasites.

Combination Anthelmintic/Antioxidant Activity Against Schistosoma Mansoni.

Schistosomiasis is a major neglected tropical disease. Treatment for schistosomiasis with praziquantel (PZQ), which is effective against the parasite, by itself is not capable to counteract infection-associated disease lesions including hepatic fibrosis. There is a pressing need for novel therapies. Due to their biological properties, antioxidant biomolecules might be useful in treating and reverting associated pathological sequelae. Here, we investigated a novel therapy approach based on a combination of anthelmintic drugs with antioxidant biomolecules. We used a host-parasite model involving Bioamphalaria glabrata and newly transformed schistosomula (NTS) of Schistosoma mansoni. For in vitro drug screening assays, was selected several antioxidants and evaluated not only antischistosomal activity but also ability to enhance activity of the anthelmintic drugs praziquantel (PZQ) and artesunate (AS). The morphological alterations induced by compounds alone/combined were assessed on daily basis using an inverted and automated microscope to quantify NTS viability by a fluorometric-based method. The findings indicated that not only do some antioxidants improve antischistosomal activity of the two anthelmintics, but they exhibit activity per se, leading to high mortality of NTS post-exposure. The combination index (CI) of PZQ + Mel (CI = 0.80), PZQ + Resv (CI = 0.74), AS + Resv (CI = 0.34), AS + NAC (CI = 0.89), VDT + Flav (CI = 1.03) and VDT + Resv (CI = 1.06) reveal that they display moderate to strong synergism. The combination of compounds with discrete mechanisms of action might provide a valuable adjunct to contribution for treatment of schistosomiasis-associated disease.

SmSak, the second Polo-like kinase of the helminth parasite Schistosoma mansoni: conserved and unexpected roles in meiosis.

Polo-like kinases (Plks) are a family of conserved regulators of a variety of events throughout the cell cycle, expanded from one Plk in yeast to five Plks in mammals (Plk1-5). Plk1 is the best characterized member of the Plk family, homolog to the founding member Polo of Drosophila, and plays a major role in cell cycle progression by triggering G2/M transition. Plk4/Sak (for Snk (Serum-inducible kinase) akin kinase) is a unique member of the family, structurally distinct from other Plk members, with essential functions in centriole duplication. The genome of the trematode parasite Schistosoma mansoni contains only two Plk genes encoding SmPlk1 and SmSak. SmPlk1 has been shown already to be required for gametogenesis and parasite reproduction. In this work, in situ hybridization indicated that the structurally conserved Plk4 protein, SmSak, was largely expressed in schistosome female ovary and vitellarium. Expression of SmSak in Xenopus oocytes confirmed its Plk4 conserved function in centriole amplification. Moreover, analysis of the function of SmSak in meiosis progression of G2-blocked Xenopus oocytes indicated that, in contrast to SmPlk1, SmSak cannot induce G2/M transition in the absence of endogenous Plk1 (Plx1). Unexpectedly, meiosis progression was spontaneously observed in Plx1-depleted oocytes co-expressing SmSak and SmPlk1. Molecular interaction between SmSak and SmPlk1 was confirmed by co-immunoprecipitation of both proteins. These data indicate that Plk1 and Plk4 proteins have the potential to interact and cross-activate in cells, thus attributing for the first time a potential role of Plk4 proteins in meiosis/mitosis entry. This unexpected role of SmSak in meiosis could be relevant to further consider the function of this novel Plk in schistosome reproduction.

CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions.

BACKGROUND: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. PRINCIPAL FINDINGS: We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. CONCLUSIONS: We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

Schistosoma mansoni specimens first described by Pirajá da Silva in Brazil (1908) re-examined by confocal laser scanning microscopy / Espécimes de Schistosoma mansoni descritos por Pirajá da Silva no Brasil (1908) reexaminados pela técnica de microscopia de varredura a laser confocal

INTRODUCTION: Pirajá da Silva made a seminal contribution to helminthology by demonstrating both schistosome eggs in feces of patients from the State of Bahia and the morphology of Schistosoma mansoni adult worms. METHODS: In this study, a microscopic investigation of the whole-mounts deposited at the Helminthological Collection of the Oswaldo Cruz Institute is presented. Confocal laser scanning microscopy was used as the main investigation technique. RESULTS: At the anterior end of the adult male, suckers with well-developed musculature and germinative cells inside the testicular lobes were observed, as well as spines located in the mid region of the male gynecophoric canal. Tegumental tubercles and transversal and longitudinal muscular bundles were observed at the dorsal surface. The female reproductive system presented a uterus lacking eggs and an elongated ovary with germinative cells. The vitellaria were restricted to the extreme posterior end of each female connected to a short vitelline duct. CONCLUSIONS: The results reported in this study demonstrate that the characteristic studied here are similar to those previously reported, using fresh worms. Moreover, this study also highlights the importance of deposits of specimens in helminthological collections, which further permit revisiting whole-mounts in such institutions.

INTRODUÇÃO: Pirajá da Silva fez contribuição magnífica à helmintologia ao descrever ovos de Schistosoma mansoni nas fezes de um paciente, no Estado da Bahia e a morfologia de vermes adultos. MÉTODOS: Neste estudo, apresentamos uma avaliação microscópica das lâminas montadas e depositadas na Coleção Helmintológica do Instituto Oswaldo Cruz. A técnica empregada nesta nova análise foi a microscopia de varredura a laser confocal. RESULTADOS: Na parte anterior dos vermes adultos machos, observamos ventosas com musculatura bem desenvolvida e células germinativas dentro dos lobos testiculares. Visualizamos, também, espinhos localizados na região mediana do canal ginecóforo. Na superfície dorsal, encontramos tubérculos e feixes musculares transversais e longitudinais. Em relação ao aparelho reprodutivo feminino, pudemos distinguir um ovo no interior do útero e o ovário alongado com células germinativas. As glândulas vitelínicas estavam restritas à parte posterior das fêmeas conectadas por um ducto vitelínico curto. CONCLUSÕES: As características morfológicas são similares as estudadas anteriormente por Pirajá da Silva com vermes frescos. Além disso, este estudo demonstra a importância de se depositar espécimes nas coleções helmintológicas abrindo possibilidade de novos estudos com estas lâminas.

Confocal laser scanning microscopy for detection of Schistosoma mansoni eggs in the gut of mice.

BACKGROUND: The gold standard for diagnosing Schistosoma mansoni infections is the detection of eggs from stool or biopsy specimens. The viability of collected eggs can be tested by the miracidium hatching procedure. Direct detection methods are often limited in patients with light or early infections, whereas serological tests and PCR methods fail to differentiate between an inactive and persistent infection and between schistosomal species. Recently, confocal laser scanning microscopy (CLSM) has been introduced as a diagnostic tool in several fields of medicine. In this study we evaluated CLSM for the detection of viable eggs of S. mansoni directly within the gut of infected mice. METHODOLOGY/PRINCIPAL FINDINGS: The confocal laser scanning microscope used in this study is based on the Heidelberg Retina Tomograph II scanning laser system in combination with the Rostock Cornea Module (image modality 1) or a rigid endoscope (image modality 2). Colon sections of five infected mice were examined with image modalities 1 and 2 for schistosomal eggs. Afterwards a biopsy specimen was taken from each colon section and examined by bright-field microscopy. Visualised eggs were counted and classified in terms of viability status. CONCLUSIONS/SIGNIFICANCE: We were able to show that CLSM visualises eggs directly within the gut and permits discrimination of schistosomal species and determination of egg viability. Thus, CLSM may be a suitable non-invasive tool for the diagnosis of schistosomiasis in humans.

Development of Schistosoma mansoni worms in mice analyzed by bright field and confocal microscopy

The blood flukes of mammals (Digenea: Schistosomatidae) are among trematodes unique whose adult worms have separeted sexes which are dissimilar in appearance. The developmental features, growth and organogenesis of Schistosoma mansoni were studied in Swiss Webster mice by a digital system for image analysis and confocal microscopy. Data so far obtained showed two phases with significative morphological changes at 3-4 weeks post-infection, and a gradual similar development onwards in the reproductive system and tegument. Our male-dependent phase demonstrated that mating occurs before sexual maturing. At week three, the majority of male worms (59 percent) had formed the gynaecophoric canal although testicular lobes and tegumental tubercles were absent. By this time, 33 percent females had an incipient ovary (without cellular differentiation). At week four, 77.2 percent males presented testicular lobes with few germinative cells while 26 percent had developing tegumental tubercles. The immature ovary was observed in 69 percent females. Suckers followed different pattern of growth between male and females. The size of oral and ventral suckers from six-week-old male worms grew abruptly (3.0 fold) more than that of three-week-old. In female worms, maximum growth was attained at week four, reducing in size thereafter. From sixth week onwards, all specimens showed the fully developed reproductive system. Probably, these features are morphological traits which schistosome has experienced from hermaphrodite to dioecy.

Comparison between morphological and staining characteristics of live and dead eggs of Schistosoma mansoni

Schistosoma mansoni eggs are classified, according to morphological characteristics, as follows: viable mature and immature eggs; dead mature and immature eggs, shells and granulomas. The scope of this study was to compare the staining characteristics of different morphological types of eggs in the presence of fluorescent labels and vital dyes, aiming at differentiating live and dead eggs. The eggs were obtained from the intestines of infected mice, and put into saline 0.85 percent. The fluorescent labels were Hoechst 33258 and Acridine Orange + Ethidium Bromide and vital dyes (Trypan Blue 0.4 percent and Neutral Red 1 percent). When labelled with the probe Hoechst 33258, some immature eggs, morphologically considered viable, presented fluorescence (a staining characteristic detected only in dead eggs); mature eggs did not present fluorescence, and the other types of dead eggs, morphologically defined, showed fluorescence. As far as Acridine Orange + Ethidium Bromide are concerned, either the eggs considered to be live, or the dead ones, presented staining with green color, and only the hatched and motionless miracidium was stained with an orange color. Trypan Blue was not able to stain the eggs, considered to be dead but only dead miracidia which had emerged out of the shell. Neutral Red stained both live and dead eggs. Only the fluorescent Hoechst 33258 can be considered a useful tool for differentiation between dead and live eggs.

Comparison between morphological and staining characteristics of live and dead eggs of Schistosoma mansoni.

Schistosoma mansoni eggs are classified, according to morphological characteristics, as follows: viable mature and immature eggs; dead mature and immature eggs, shells and granulomas. The scope of this study was to compare the staining characteristics of different morphological types of eggs in the presence of fluorescent labels and vital dyes, aiming at differentiating live and dead eggs. The eggs were obtained from the intestines of infected mice, and put into saline 0.85%. The fluorescent labels were Hoechst 33258 and Acridine Orange + Ethidium Bromide and vital dyes (Trypan Blue 0.4% and Neutral Red 1%). When labelled with the probe Hoechst 33258, some immature eggs, morphologically considered viable, presented fluorescence (a staining characteristic detected only in dead eggs); mature eggs did not present fluorescence, and the other types of dead eggs, morphologically defined, showed fluorescence. As far as Acridine Orange + Ethidium Bromide are concerned, either the eggs considered to be live, or the dead ones, presented staining with green color, and only the hatched and motionless miracidium was stained with an orange color. Trypan Blue was not able to stain the eggs, considered to be dead but only dead miracidia which had emerged out of the shell. Neutral Red stained both live and dead eggs. Only the fluorescent Hoechst 33258 can be considered a useful tool for differentiation between dead and live eggs.

Molecular characterization and immunolocalization of Schistosoma mansoni ATP-diphosphohydrolase.

Schistosoma mansoni, a human parasite that constitutes a major health problem in developing countries, escapes from host defenses and survives in the human bloodstream. Here, we report the cloning of a S. mansoni ATP-diphosphohydrolase ortholog (SmATPDase1). Southern blots indicated that in S. mansoni it is a single-copy gene. RT-PCR revealed that SmATPDase1 is expressed in five stages of the parasite life cycle, namely cercaria, schistosomula, adults, eggs, and miracidia. Using confocal microscopy, SmATPDase1 protein was immunolocalized on the external surface in all stages, except eggs, being conspicuously present in adults. ATPDase, which is present on the outer surface of endothelial cells lining human blood vessels, has been implicated in thromboregulation by promoting ADP hydrolysis and inhibition of platelet aggregation. The presence of an ATPDase ortholog on the surface of S. mansoni suggests that the enzyme might play a role in the escape from host defenses that would involve platelet activation.

T1/ST2 expression is enhanced on CD4+ T cells from schistosome egg-induced granulomas: analysis of Th cell cytokine coexpression ex vivo.

Th cells are categorized into subsets based on the cytokine production of in vitro-differentiated Th populations. For in vivo-differentiated Th subsets, little is known about the heterogeneity of cytokine production in single cells. We recently described a molecule, T1/ST2, that is preferentially expressed on the surface of Th2 cells. Here we combined high-gradient magnetic cell separation with four-color single-cell cytometry to analyze simultaneously three intracellular cytokines and T1/ST2 surface expression on CD4+ cells from lungs containing granulomas induced by Schistosoma mansoni eggs. T1/ST2 was highly up-regulated on CD4+ T cells from hepatic granulomas and granulomatous lungs. T1/ST2+ cells from granulomatous lungs preferentially produced type 2 cytokines ex vivo. In the total CD4+ population, coexpression of type 1 and type 2 cytokines occurred frequently. However, such coproduction was drastically reduced in T1/ST2+ cells compared with T1/ST2- cells. Coexpression of type 1 and type 2 cytokines was also rare in cells simultaneously producing two cytokines of one type. These findings indicate that individual CD4+ T cells in vivo have different levels of commitment to a certain Th phenotype. Coexpression of two type 2 cytokines or production of one type 2 cytokine together with surface expression of T1/ST2 indicate advanced commitment to the Th2 phenotype.

Infection of B-cell-deficient mice by the parasite Schistosoma mansoni: demonstration of the participation of B cells in granuloma modulation.

The contribution of B lymphocytes to immunity towards the parasite Schistosoma mansoni has been investigated in a mouse strain rendered genetically B-cell deficient (the muMT mouse). These studies demonstrated that T cells primed in vivo in B-cell-deficient mice proliferate less efficiently in vitro in response to parasite antigenic extracts except at 10 weeks of infection. In addition, analysis of the cytokine profiles (IL-2, IL-4, IL-5 and IFN-gamma), investigated using RT-PCR, showed that spleens of muMT animals displayed a predominant Th1-like profile compared to control, B-cell-intact infected mice. This showed that B cells, either per se or through their secretions, are involved in the in vivo generation and/or maximal expansion of Th2-type T lymphocytes during the course of murine S. mansoni infection. Interestingly, the data showed that B-cell-deficient mice display an increased hepatic fibrosis at 10 weeks postinfection (p.i.), whereas they behaved like infected controls, with regard to the other assessed parasitological parameters (e.g. worm burden estimation). This demonstrated that even if B lymphocytes are not essential for the development of the general immune response towards S. mansoni in the mouse, they may nevertheless be involved in the correct immunoregulation of the granulomatous reaction around the eggs.

Histoarchitecture of schistosomal granuloma development and involution: morphogenetic and biomechanical approaches

The authors present morphogenetic and biomechanical approaches on the concept of the Schistosoma mansoni granulomas, considering them as organoid structures that depend on cellular adhesion and sorting, forming rearrangement into hierarchical concentric layers, creating tension-dependent structures, aiming to acquire round form, since this is the minimal energy form, in which opposing forces pull in equally from all directions and are in balance. From the morphogenetic point of view, the granulomas function as little organs, presenting maturative and involutional stages in their development with final disappearance (pre-granulomatous stages, subdivided in: weaky and/or initial reactive and exudative; granulomatous stages: exudative-productive, productive and involutional). A model for the development of granulomas was suggested according to the following stages: encapsulating, focal hystolysis, fiber production, orientation and compacting and involution and desintegration. The autors concluded that schistosomal granuloma is not a tangled web of individual cells and fibers, but an organized structure composed by host and parasite components, which is not formed to attack the miracidia, but functions as an hybrid interface between two different phylogenetic beings.

Control of ciliary activities of Schistosoma mansoni miracidia using Triton-extracted parasites.

Extraction with 0.04% (w/v) Triton X-100 for 3 min removed the cell membrane from the locomotory cilia of Schistosoma mansoni miracidia while leaving the motile apparatus apparently intact. Immediately after Triton-extracted miracidia were treated by the reactivating solution containing ATP and magnesium ions (Mg2+) at pH 8.1, nearly 100% of Triton-extracted miracidia showed the ciliary beating and swam forward in a manner resembling that of a normal miracidium. In the standard reactivating solution (2 mM ATP, 2 mM Mg2+, pH 8.1), Triton-extracted miracidia swam at a speed of 580 microns/sec; the comparable value for live miracidia in dechlorinated tap water was 2,200 microns/sec. The swimming velocity of Triton-extracted miracidia was dependent on ATP and Mg2+ concentration, pH, and salinity. In a solution containing 0.9% NaCl, Triton-extracted miracidia were not reactivated. Among the nucleotides tested, only ATP was found to induce a significant amount of ciliary motility. In terms of divalent cation specificity, only Mg2+ was capable of producing normal motility. Barium and calcium ions (at 0.5 mM CaCl2) also were capable of activating ciliary motility but were less effective stimulants than Mg2+. However, in 1 mM CaCl2, no ciliary reactivation was observed and cilia became detached from the body surface of the miracidia. Vanadium inhibited ATP-reactivated ciliary beating of Triton-extracted miracidia.

Host-related DNA sequences are localized in the body of schistosome adults.

The localization of host (mouse)-related DNA sequences in the adults and cercariae of Schistosoma japonicum and S. mansoni was examined by in situ hybridization using 32P-labelled probes. The hybridization signals to the sequences of the mouse type C and type A retroviruses were clearly observed in the subtegumental layer and inner tissues of S. japonicum adults. In contrast, it was hard to find any signals to these sequences in S. mansoni adults. Distinct signals to the env-specific region of the mouse ecotropic type C retrovirus were observed in the subtegumental layer and inner tissues of S. mansoni adults. No signal to the sequence of the mouse type B retrovirus was found on the sections of either schistosome adult. The signals to the sequence of the genomic clone SmM51 derived from male S. mansoni were found in the tissues of both the schistosome adults. The signals to the sequences of the mouse type A and env-specific region of ecotropic type C retroviruses were not seen in the cercariae of each species, whereas the signals to the sequence of SmM51 were detected in S. mansoni cercariae. These observations suggest that host-related DNA sequences might be incorporated unequally into the schistosome genome during development in the final host.

Immunoreactivity to the pancreatic polypeptide family in the nervous system of the adult human blood fluke, Schistosoma mansoni.

The presence and distribution of neuropeptides belonging to the pancreatic polypeptide family have been demonstrated by an indirect immunofluorescence technique in the nervous systems of adult male and female Schistosoma mansoni. Seven antisera of differing regional specificity to pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY) were employed on both whole-mount and cryostat-sectioned material. Positive immunoreactivity (IR) was obtained with all antisera except an N-terminally-directed antiserum to NPY. In the CNS, immunoreactivity was restricted to cell bodies and nerve fibres in the anterior ganglia, central commissure and dorsal and ventral nerve cords of both sexes, whereas, in the PNS, positive-IR was present in the plexuses innervating the subtegumental musculature and the oral and ventral suckers. Intense immunoreactivity was observed in a plexus of nerve fibres and cell bodies in the lining of the gynaecophoric canal and in fine nerve fibres innervating the dorsal tubercles of the male. In contrast, in the female, strong immunoreactivity was evident in nerve plexuses innervating the lining of the ovovitelline duct and in the wall of the ootype, but most notably in a cluster of cells in the region of Mehlis' gland. Results suggest that molecules with C-terminal homology to the PP-family are present in S. mansoni. These peptides would appear to be important regulatory molecules in the parasite's nervous system and may play a role in the control of egg production.