In Vitro and In Vivo Anti-Schistosomal Activity of the Alkylphospholipid Analog Edelfosine

Background: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites.

Methodology/Principal Findings: We found APLs ranked edelfosine> perifosine> erucylphosphocholine> miltefosine for their in vitro schistosomicidal activity against adult S. mansoni worms. Edelfosine accumulated mainly in the worm tegument, and led to tegumental alterations, membrane permeabilization, motility impairment, blockade of male-female pairing as well as induction of apoptosis-like processes in cells in the close vicinity to the tegument. Edelfosine oral treatment also showed in vivo schistosomicidal activity and decreased significantly the egg burden in the liver, a key event in schistosomiasis.

Conclusions/Significance: Our data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.

Neuropeptides in Helminths: Occurrence and Distribution

Nematode neuropeptide systems comprise an exceptionally complex array of similar to 250 peptidic signaling molecules that operate within a structurally simple nervous system of similar to 300 neurons. A relatively complete picture of the neuropeptide complement is available for Caenorhabditis elegans, with 30 flp, 38 ins and 43 nlp genes having been documented; accumulating evidence indicates similar complexity in parasitic nematodes from clades I, III, IV and V. In contrast, the picture for parasitic platyhelminths is less clear, with the limited peptide sequence data available providing concrete evidence for only FMRFamide-like peptide (FLP) and neuropeptide F (NPF) signaling systems, each of which only comprises one or two peptides. With the completion of the Schmidtea meditteranea and Schistosoma mansoni genome projects and expressed sequence tag datasets for other flatworm parasites becoming available, the time is ripe for a detailed reanalysis of neuropeptide signaling in flatworms. Although the actual neuropeptides provide limited obvious value as targets for chemotherapeutic-based control strategies, they do highlight the signaling systems present in these helminths and provide tools for the discovery of more amenable targets such as neuropeptide receptors or neuropeptide processing enzymes. Also, they offer opportunities to evaluate the potential of their associated signaling pathways as targets through RNA interference (RNAi)-based, target validation strategies. Currently, within both helminth phyla, the flp signaling systems appear to merit further investigation as they are intrinsically linked with motor function, a proven target for successful anti-parasitics; it is clear that some nematode NLPs also play a role in motor function and could have similar appeal. At this time, it is unclear if flatworm NPF and nematode INS peptides operate in pathways that have utility for parasite control. Clearly, RNAi-based validation could be a starting point for scoring potential target pathways within neuropeptide signaling for parasiticide discovery programs. Also, recent successes in the application of in planta-based RNAi control strategies for plant parasitic nematodes reveal a strategy whereby neuropeptide encoding genes could become targets for parasite control. The possibility of developing these approaches for the control of animal and human parasites is intriguing, but will require significant advances in the delivery of RNAi-triggers.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.

Introdução, estabelecimento e adaptação de Bradirrizóbios simbiontes da soja em a solos brasileiros.

Rizóbios microssimbiontes da soja; Introdução de estirpes nos solos brasileiros; Adaptação das estirpes de B. japonicum / B. elkanii aos solos brasileiros; Competitividade das estirpes de B elkanii SEMIA 587 e 29 W; Competitividade das estirpes do sorogrupo SEMIA 566 de B. japonicum; Variabilidade nas estirpes de Bradyrhizobium após a introdução nos solos brasileiros; Transferência horizontal de genes entre estirpes inoculantes e rizóbios indígenas ou naturalizados nos solos brasileiros.

Traitement de la schistosomiase à S. mansoni : quelle alternative au praziquantel ?

Les schistosomiases sont des maladies parasitaires causées par des helminthes du genre Schistosoma (S.) qui touchent 200 millions de personnes dans le monde, mais restent rares chez le voyageur. Contrairement à S. heamatobium, agent de la bilharziose urinaire, S. mansoni, présent en Afrique subsaharienne, en Egypte ainsi qu'aux Antilles, au Surinam et dans le nordest du Brésil, est responsable des formes hépato-intestinales de la maladie. Les larves, vivant en eaux douces contaminées par des selles infectées, peuvent pénétrer la peau des baigneurs sans que l'individu ne s'en rende compte. Les parasites adultes s'établissent dans le système veineux digestif où ils se reproduisent et excrètent des oeufs qui migreront dans la lumière intestinale. Cette revue systématique évalue les effets des médicaments antibilharziens, utilisés seuls ou en association, pour traiter l'infection à S. mansoni.

The effect of nitrate fertilization on the distribution of exported 14c-photosynthate in nodulated soybean (Glycine max (L.) Merr.) plants

Soybean (Glycine ~ (L.) Merr. cv. Harosoy 63) plants inoculated with Rhizobium japonicum were grown in vermiculite in the presence or absence of nitrate fertilization for up to 6 weeks after planting. Overall growth of nodulated plants was enhanced in the presence of nitrate fertilization, while the extent of nodule development was reduced. Although the number of nodules was not affected by nitrate fertilization when plants were grown at a light intensity limiting for photosynthesis, at light intensities approaching or exceeding the light saturation point for photosynthesis, nitrate fertilization resulted in at least a 30% reduction in nodule numbers. The mature, first trifoliate leaf of 21 day old plants was allowed to photoassimi1ate 14C02. One hour after·· the initial exposure to 14C02, the , plants were harvested and the 14C radioactivity was determined in the 80% ethanol-soluble fraction: in. o:rider to assess· "the extent of photoassimilate export and the pattern of distribution of exported 14C. The magnitude of 14C export was not affected by the presence of nitrate fertilization. However, there was a significant effect on the distribution pattern, particularly with regard to the partitioning of 14C-photosynthate between the nodules and the root tissue. In the presence of nitrate fertilization, less than 6% of the exported 14C photosynthate was recovered from the nodules, with much larger amounts (approximately 37%) being recovered from the root tissue. In the absence of nitrate fertilization, recovery of exported 14C-photosynthate from the nodules (19 to 27%) was approximately equal to that from the root tissue (24 to 33%). By initiating- or terminating the applications of nitrate at 14 days of age, it was determined that the period from day 14 to day 21 after planting was particularly significant for the development of nodules initiated earlier. Addition of nitrate fertilization at this time inhibited further nodule development while stimulating plant growth, whereas removal of nitrate fertilization stimulated nodule development. The results obtained are consistent with the hypothesis that nodule development is inhibited by nitrate fertilization through a reduction in the availability of photosynthate to the nodules.

Hijos del parásito.

Programa emitido el 12 de enero de 1995

Integrated cytokine and metabolic analysis of pathological responses to parasite exposure in rodents

Parasitic infections cause a myriad of responses in their mammalian hosts, on immune as well as on metabolic level. A multiplex panel of cytokines and metabolites derived from four parasite-rodent models, namely, Plasmodium berghei-mouse, Trypanosoma brucei brucei-mouse, Schistosoma mansoni-mouse, and Fasciola hepatica-rat were statistically coanalyzed. 1H NMR spectroscopy and multivariate statistical analysis were used to characterize the urine and plasma metabolite profiles in infected and noninfected animals. Each parasite generated a unique metabolic signature in the host. Plasma cytokine concentrations were obtained using the ‘Meso Scale Discovery’ multi cytokine assay platform. Multivariate data integration methods were subsequently used to elucidate the component of the metabolic signature which is associated with inflammation and to determine specific metabolic correlates with parasite-induced changes in plasma cytokine levels. For example, the relative levels of acetyl glycoproteins extracted from the plasma metabolite profile in the P. berghei-infected mice were statistically correlated with IFN-γ, whereas the same cytokine was anticorrelated with glucose levels. Both the metabolic and the cytokine data showed a similar spatial distribution in principal component analysis scores plots constructed for the combined murine data, with samples from all infected animals clustering according to the parasite species and whereby the protozoan infections (P. berghei and T. b. brucei) grouped separately from the helminth infection (S. mansoni). For S. mansoni, the main infection-responsive cytokines were IL-4 and IL-5, which covaried with lactate, choline, and D-3-hydroxybutyrate. This study demonstrates that the inherently differential immune response to single and multicellular parasites not only manifests in the cytokine expression, but also consequently imprints on the metabolic signature, and calls for in-depth analysis to further explore direct links between immune features and biochemical pathways.

Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts

Schistosoma mansoni is a well-adapted blood-dwelling parasitic helminth, persisting for decades in its human host despite being continually exposed to potential immune attack. Here, we describe in detail micro-exon genes (MEG) in S. mansoni, some present in multiple copies, which represent a novel molecular system for creating protein variation through the alternate splicing of short (<= 36 bp) symmetric exons organized in tandem. Analysis of three closely related copies of one MEG family allowed us to trace several evolutionary events and propose a mechanism for micro-exon generation and diversification. Microarray experiments show that the majority of MEGs are up-regulated in life cycle stages associated with establishment in the mammalian host after skin penetration. Sequencing of RT-PCR products allowed the description of several alternate splice forms of micro-exon genes, highlighting the potential use of these transcripts to generate a complex pool of protein variants. We obtained direct evidence for the existence of such pools by proteomic analysis of secretions from migrating schistosomula and mature eggs. Whole-mount in situ hybridization and immunolocalization showed that MEG transcripts and proteins were restricted to glands or epithelia exposed to the external environment. The ability of schistosomes to produce a complex pool of variant proteins aligns them with the other major groups of blood parasites, but using a completely different mechanism. We believe that our data open a new chapter in the study of immune evasion by schistosomes, and their ability to generate variant proteins could represent a significant obstacle to vaccine development.

Transcriptome analysis of Taenia solium cysticerci using Open Reading Frame ESTs (ORESTES)

Background: Human infection by the pork tapeworm Taenia solium affects more than 50 million people worldwide, particularly in underdeveloped and developing countries. Cysticercosis which arises from larval encystation can be life threatening and difficult to treat. Here, we investigate for the first time the transcriptome of the clinically relevant cysticerci larval form. Results: Using Expressed Sequence Tags (ESTs) produced by the ORESTES method, a total of 1,520 high quality ESTs were generated from 20 ORESTES cDNA mini-libraries and its analysis revealed fragments of genes with promising applications including 51 ESTs matching antigens previously described in other species, as well as 113 sequences representing proteins with potential extracellular localization, with obvious applications for immune-diagnosis or vaccine development. Conclusion: The set of sequences described here will contribute to deciphering the expression profile of this important parasite and will be informative for the genome assembly and annotation, as well as for studies of intra- and inter-specific sequence variability. Genes of interest for developing new diagnostic and therapeutic tools are described and discussed.

Disease fingerprinting with cDNA microarrays reveals distinct gene expression profiles in lethal type 1 and type 2 cytokine-mediated inflammatory reactions

Development of polarized immune responses controls resistance and susceptibility to many microorganisms. However, studies of several infectious, allergic, and autoimmune diseases have shown that chronic type-1 and type-2 cytokine responses can also cause significant morbidity and mortality if left unchecked. We used mouse cDNA microarrays to molecularly phenotype the gene expression patterns that characterize two disparate but equally lethal forms of liver pathology that develop in Schistosoma mansoni infected mice polarized for type-1 and type-2 cytokine responses. Hierarchical clustering analysis identified at least three groups of genes associated with a polarized type-2 response and two linked with an extreme type-1 cytokine phenotype. Predictions about liver fibrosis,  apoptosis, and granulocyte recruitment and activation generated by the microarray studies were confirmed later by traditional biological assays. The data show that cDNA microarrays are useful not only for determining  coordinated gene expression profiles but are also highly effective for molecularly “fingerprinting” diseased tissues. Moreover, they illustrate the potential of genome-wide approaches for generating comprehensive views on the molecular and biochemical mechanisms regulating infectious  disease pathogenesis.

Undiagnosed and potentially lethal parasite infections among immigrants and refugees in Australia

Background: Intestinal parasite infections are a major cause of ill health in many resource-poor countries. This study compares the types and rates of these infections and their risk factors in recently arrived and long-term immigrants in Australia.

Method: Cross-sectional surveys of 127 East African and 234 Cambodian immigrants and refugees were undertaken in 2000 and 2002, respectively, to assess the burden of intestinal parasites and collect demographic information. Serum samples were assessed for eosinophilia and Strongyloides stercoralis and Schistosoma antibodies, and feces examined for ova, cysts, and parasites.

Results: Intestinal parasites were identified in 77/117 fecal samples from East African and in 25/204 samples collected from Cambodian participants. Eleven percent (14/124) of East Africans and 42% (97/230) of Cambodians had positive or equivocal serology for S stercoralis. Schistosoma serology was positive or equivocal in 15% (19/124) of East African participants.

Conclusion: Potentially serious intestinal parasite infections are common among recent and longer term immigrants despite multiple visits to health care providers. Immigrants and refugees from high-risk countries would benefit from comprehensive health checks soon after resettlement.

Variabilidade de isolados de estirpes de Bradyrhizobium ssp recomendadas para a cultura da soja

A produção da soja em escala comercial tem sido viabilizada técnica e economicamente, entre outros fatores, devido a fixação biológica do N2 por estirpes de Bradyrhizobium que podem suprir a demanda de nitrogênio desta leguminosa. No entanto, a variabilidade existente nas estirpes de Bradyrhizobium spp recomendadas para inoculação da soja tem comprometido o processo simbiótico. Variantes espontâneos isolados a partir das estirpes de B. japonicum (SEMIA 5079 e SEMIA 5080) e B. elkanii (SEMIA 587 e SEMIA 5019) foram avaliados quanto ao desempenho simbiótico (eficiência, nodulação em diferentes hospedeiros e competitividade), indução de clorose foliar em diferentes hospedeiros, morfologia colonial, habilidade de metabolização de carboidratos e tolerância à salinidade em diferentes temperaturas de incubação. Nas etapas de eficiência simbiótica e competitividade foi usada a cultivar de soja BR-16 e na avaliação da nodulação e indução de clorose foliar foram usados os seguintes hospedeiros: soja (Glycine max) (cultivares Clark e Peking), caupi (Vigna unguiculata) e guandu (Cajanus cajan). A caracterização genotípica foi realizada através da reação em cadeia da polimerase (PCR), com os oligonucleotídeos iniciadores BOX A 1-R, ERIC e RP01 Os resultados obtidos demonstraram que variantes e estirpes originais diferem quanto a características fenotípicas, e que diferenças nos perfis eletroforéticos de DNA analisados através da amplificação com os oligonucleotídeos iniciadores testados, evidenciaram a variabilidade genética presente entre as estirpes originais e os variantes selecionados. A cultivar de soja Clark, assim como caupi e guandu foram susceptíveis à rizobiotoxina produzida por estirpes e variantes de B. elkanii. Embora não se tenha verificado diferenças na nodulação em diferentes hospedeiros quando variantes ou estirpes de B. japonicum e B. elkanii foram inoculados em soja, caupi e guandu, foi observado uma simbiose eficiente para a soja (cultivares BR 16, Clark e Peking). A partir da variabilidade existente nas estirpes SEMIA 587, SEMIA 5019, SEMIA 5079 e SEMIA 5080 foram selecionados variantes eficientes e competitivos quanto à fixação de N2 em soja.

Inoculação de soja em solo submetido a diferentes sistemas de manejo

A soja (Glycine max) destaca-se pela importância econômica e pela capacidade de associação simbiótica com bactérias (Bradyrhizobium spp e Sinorhizobium spp) fixadoras do dinitrogênio. O benefício da fixação biológica do nitrogênio (FBN) é potencializado pela inoculação. Entretanto, a resposta à inoculação depende de fatores bióticos e abióticos, que variam de acordo com o sistema de manejo do solo. Os objetivos deste trabalho foram avaliar a resposta à inoculação e reinoculação da soja, além da sobrevivência e a competitividade das estirpes de B. elkanii (SEMIA 587 e SEMIA 5019) e de B. japonicum (SEMIA 5079 e SEMIA 5080) em diferentes sistemas de manejo do solo. O experimento, com diferentes sistemas de manejo do solo, foi iniciado em 2000, a partir de campo nativo, sendo avaliado nos anos agrícolas de 2000/2001 e 2002/2003. Os tratamentos consistiram de adubação orgânica, mineral e adubação mineral com irrigação, todos utilizando sistemas de preparo plantio direto, plantio reduzido e plantio convencional, com ou sem inoculação. Foram avaliados o teor de nitrogênio mineral do solo, o número e a massa de nódulos, a massa e o nitrogênio total do tecido da parte aérea e a produção dos grãos, além da ocupação nodular pelas estirpes inoculadas, avaliadas por soroaglutinação. A inoculação avaliada promoveu um aumento médio na produção de grãos de 200 kg ha-1 em 2000/2001 e a reinoculação de 125 kg ha-1 na parcela irrigada em 2002/2003. Teores de nitrogênio mineral do solo acima de 12 mg kg-1 determinaram redução no número e massa de nódulos. As estirpes SEMIA 587 e 5019 apresentaram maior sobrevivência e competitividade. As SEMIA 5079 e 5080 demonstraram maior dependência da inoculação. Os dados obtidos mostram que a sobrevivência e competitividade das estirpes são características pouco influenciadas pelo sistema de manejo do solo.

Biopolímeros como suporte para inoculantes

A utilização de inoculantes em culturas de leguminosas é uma prática conhecida e empregada há longo tempo e o substrato mais utilizado até o momento tem sido a turfa. Diversos tipos de formulações inoculantes que existem no mercado visam oferecer uma alternativa ao emprego da turfa, porém muitas apresentam baixa capacidade para manter a sobrevivência e eficiência dos rizóbios. Este trabalho avaliou a utilização de catorze misturas diferentes de polímeros naturais e/ou sintéticos como suportes para inoculantes, visando a produção de inoculantes comerciais para soja, e a capacidade de manter a sobrevivência e preservar as características de infectividade e de efetividade das estirpes SEMIA 587 de Bradyrhizobium elkanii e SEMIA 5079 de Bradyrhizobium japonicum. Avaliou-se a sobrevivência dos rizóbios nas formulações armazenadas, a capacidade de aderência das formulações em sementes, a sobrevivência dos rizóbios em sementes a 40 ºC e a eficiência dos inoculantes em plantas de soja, cultivadas em vasos com solo, submetidas a estresse hídrico e térmico. Observou-se que as misturas contendo goma xantana, jataí e guar, tanto nas formulações em gel como líquidas, podem ser usadas como veículo para inoculantes proporcionando maior proteção aos rizóbios contra as condições de dessecação e temperatura. Todos os inoculantes mantiveram a sobrevivência da população de rizóbios durante um ano de armazenamento. Os rizóbios das formulações G5, L1 e L7, que continham goma arábica, foram afetados pelo estresse hídrico e pelas temperaturas elevadas, reduzindo o número de nódulos formados. Dentre as formulações líquidas, as que continham xantana e glicerol (L2) e com adição de polivinilpirrolidona (L5) e a formulação que continha xantana, carboximetilcelulose e polivinilpirrolidona (L6), foram as mais promissoras para a formulação de inoculantes para soja.