Cytometric analysis, genetic manipulation and antibiotic selection of the snail embryonic cell line Bge from Biomphalaria glabrata, the intermediate host of Schistosoma mansoni.

The invertebrate cell line, Bge, from embryos of the snail Biomphalaria glabrata, remains to date the only established cell line from any species of the Phylum Mollusca. Since its establishment in 1976 by Eder Hansen, few studies have focused on profiling its cytometrics, growth characteristics or sensitivity to xenobiotics. Bge cells are reputed to be challenging to propagate and maintain. Therefore, even though this cell line is a noteworthy resource, it has not been studied widely. With growing interest in functional genomics, including genetic transformation, to elucidate molecular aspects of the snail intermediate hosts responsible for transmission of schistosomiasis, and aiming to enhance the convenience of maintenance of this molluscan cell line, we deployed the xCELLigene real time approach to study Bge cells. Doubling times for three isolates of Bge, termed CB, SL and UK, were longer than for mammalian cell lines - longer than 40 h in complete Bge medium supplemented with 7% fetal bovine serum at 25 °C, ranging from ∼42 h to ∼157 h when 40,000 cells were seeded. To assess the potential of the cells for genetic transformation, antibiotic selection was explored. Bge cells were sensitive to the aminonucleoside antibiotic puromycin (from Streptomyces alboniger) from 5 μg/ml to 200 ng/ml, displaying a half maximal inhibitory concentration (IC50) of ∼1.91 μg/ml. Sensitivity to puromycin, and a relatively quick kill time (<48 h in 5 μg/ml) facilitated use of this antibiotic, together with the cognate resistance gene (puromycin N-acetyl-transferase) for selection of Bge cells transformed with the PAC gene (puroR). Bge cells transfected with a plasmid encoding puroR were partially rescued when cultured in the presence of 5 μg/ml of puromycin. These findings pave the way for the development of functional genomic tools applied to the host-parasite interaction during schistosomiasis and neglected tropical trematodiases at large

Influence of 'Trichobilharzia regenti' (Digenea: Schistosomatidae) on the defence activity of 'Radix lagotis' (Lymnaeidae) haemocytes

Radix lagotis is an intermediate snail host of the nasal bird schistosome Trichobilharzia regenti. Changes in defence responses in infected snails that might be related to host-parasite compatibility are not known. This study therefore aimed to characterize R. lagotis haemocyte defence mechanisms and determine the extent to which they are modulated by T. regenti. Histological observations of R. lagotis infected with T. regenti revealed that early phases of infection were accompanied by haemocyte accumulation around the developing larvae 2–36 h post exposure (p.e.) to the parasite. At later time points, 44–92 h p.e., no haemocytes were observed around T. regenti. Additionally, microtubular aggregates likely corresponding to phagocytosed ciliary plates of T. regenti miracidia were observed within haemocytes by use of transmission electron microscopy. When the infection was in the patent phase, haemocyte phagocytic activity and hydrogen peroxide production were significantly reduced in infected R. lagotis when compared to uninfected counterparts, whereas haemocyte abundance increased in infected snails. At a molecular level, protein kinase C (PKC) and extracellular-signal regulated kinase (ERK) were found to play an important role in regulating these defence reactions in R. lagotis. Moreover, haemocytes from snails with patent infection displayed lower PKC and ERK activity in cell adhesion assays when compared to those from uninfected snails, which may therefore be related to the reduced defence activities of these cells. These data provide the first integrated insight into the immunobiology of R. lagotis and demonstrate modulation of haemocyte-mediated responses in patent T. regenti infected snails. Given that immunomodulation occurs during patency, interference of snail-host defence by T. regenti might be important for the sustained production and/or release of infective cercariae

Evaluation of informative materials on leishmaniasis distributed in Brazil: criteria and basis for the production and improvement of health education materials Avaliação de material informativo sobre leishmanioses distribuído no Brasil: critérios e subsídios para a elaboração e o aperfeiçoamento de materiais educativos para a saúde

Based on categories related to structure, content, language, and illustrations, the present study provides an evaluation of the quality of educational materials on leishmaniasis available to health services in Brazil. The 18 publications evaluated consisted of four handbooks, four guided studies, four booklets, and six leaflets. Of the total publications assessed, nine were produced by the Brazilian National Health Foundation (FUNASA), five by State and Municipal Health Departments jointly with FUNASA, and one by the Pan-American Health Organization. The evaluations were also performed by three professionals: a physician specialized in leishmaniasis, a parasitologist, and an information/communications expert. The publications failed to specify key items such as target public, objective, and bibliography. The illustrations, especially in the booklets and leaflets, failed to clarify the text, portrayed biased concepts, and omitted credits and scale. According to this study, informative materials on leishmaniasis distributed in Brazil present major limitations which jeopardize the quality of information they contain.<br>Baseado nas categorias estrutura, conteúdo, linguagem e ilustrações, o presente estudo mostra uma avaliação da qualidade da informação de material informativo sobre leishmanioses disponível para os serviços de saúde no Brasil. Dos 18 exemplares analisados, quatro eram manuais, quatro estudos dirigidos, quatro cartilhas e seis folhetos. Nove exemplares foram produzidos pela Fundação Nacional de Saúde (FUNASA), cinco por Secretarias de Saúde, três por Secretarias em colaboração com a FUNASA e um pela Organização Pan-Americana da Saúde/Organização Mundial da Saúde. Para a avaliação foram convidados: um médico especialista na doença, um parasitologista e um especialista da área de informação. Verificou-se nas publicações, ausência de itens como: público alvo, objetivo e bibliografia. Em ilustrações, as imagens, especialmente nas cartilhas e folhetos, não elucidavam o texto e chamou a atenção a presença de idéias preconceituosas, a ausência de crédito à autoria das ilustrações e de escala. O material informativo avaliado apresentou limitações que comprometeram a informação. É importante que os órgãos de saúde pública estejam atentos à qualidade do material educativo produzido, por ser este recurso pedagógico valioso na construção do conhecimento transformador

Regulation of <i>Schistosoma mansoni</i> Development and Reproduction by the Mitogen-Activated Protein Kinase Signaling Pathway

<div><p>Background</p><p>Protein kinases are proven targets for drug development with an increasing number of eukaryotic Protein Kinase (ePK) inhibitors now approved as drugs. Mitogen-activated protein kinase (MAPK) family members connect cell-surface receptors to regulatory targets within cells and influence a number of tissue-specific biological activities such as cell proliferation, differentiation and survival. However, the contributions of members of the MAPK pathway to schistosome development and survival are unclear.</p><p>Methodology/Principal Findings</p><p>We employed RNA interference (RNAi) to elucidate the functional roles of five <i>S. mansoni</i> genes (SmCaMK2, SmJNK, SmERK1, SmERK2 and SmRas) involved in MAPK signaling pathway. Mice were injected with post-infective larvae (schistosomula) subsequent to RNAi and the development of adult worms observed. The data demonstrate that SmJNK participates in parasite maturation and survival of the parasites, whereas SmERK are involved in egg production as infected mice had significantly lower egg burdens with female worms presenting underdeveloped ovaries. Furthermore, it was shown that the c-fos transcription factor was overexpressed in parasites submitted to RNAi of SmERK1, SmJNK and SmCaMK2 indicating its putative involvement in gene regulation in this parasite's MAPK signaling cascade.</p><p>Conclusions</p><p>We conclude that MAPKs proteins play important roles in the parasite <i>in vivo</i> survival, being essential for normal development and successful survival and reproduction of the schistosome parasite. Moreover SmERK and SmJNK are potential targets for drug development.</p></div

Primer sequences.

<p>Primer sequences.</p

Survival of the parasite after RNAi of MAPKs <i>in vitro</i> and subsequent transfer into mice.

<p>Schistosomula were treated with GFP, SmJNK, SmCaMK2, and SmERK1 dsRNAs for two days and then injected into mice. After 37 days adult worms were recovered and counted. Each symbol in the chart represents worm counts from one mouse and the horizontal lines are median values per treatment group. Data were generated from 3 independent experiments and all treatments were statistical analyzed using Mann-Whitney U-test within each experiment (<i>P</i>≤0.05). The asterisk indicates a significance value of <i>P</i><0.022 for RNAi of SmJNK relative to the GFP control.</p

Transcript levels of target genes in schistosomula 2, 4, and 7 days after exposure to dsRNA.

<p>Bar graph indicating the relative steady-state transcript levels of SmCaMK2 (red), SmJNK (green), SmRas (orange), SmERK-2 (blue), and SmERK1 (purple) genes after 2, 4, and 7 days after dsRNA exposure. For each dsRNA tested, data are represented as mean fold-differences (+/−SE) relative to GFP control (1.00 – dashed line). Transcript levels were determined by qPCR and data analyzed using the ΔΔCt method <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002949#pntd.0002949-Koressaar1" target="_blank">[24]</a>, followed by statistical analysis using the Mann-Whitney U-test. Data were generated from 3 independent experiments, each one in duplicate, and all the data shown is statistically different from GFP controls.</p

Hepatic egg counts after RNAi of MAPKs <i>in vitro</i> and subsequent transfer of parasites into mice.

<p>Schistosomula were treated with GFP, SmJNK, SmCaMK2 and SmERK1 dsRNAs for two days <i>in vitro</i> and then injected into mice. After 37 days of parasite eggs per liver digest were counted. Each symbol in the chart represents worm counts from one mouse and the horizontal lines are median values per treatment group. Data were generated from 3 independent experiments and all treatments were statistical analyzed using Mann-Whitney <i>U</i>-test within each experiment (P≤0.05). The asterisk indicates a significance value of SmJNK and SmERK <i>P</i><0.0072 relative to the GFP control.</p

Morphology of adult female worms after RNAi of SmERK1/2 <i>in vitro</i> and subsequent transfer of parasites into mice.

<p>Adult 37-day-old worms were fixed and stained, and visualized by confocal microscopy as described in the text. A - mean of females' ovary area (µM²) of SmERK-knockdown and control showing a significant size reduction; B and C show normal worms treated with GFP dsRNA where the ovary (OV) with immature and mature oocytes, an egg (EG) and the vitelloduct (VD) are visible; D, E and F show morphological changes in worms treated with SmERK dsRNA where the ovary (D) present no mature oocytes (D) or even when mature oocytes are visible (E) an unexpected phenotype (a lot of oocytes) is observed in the uterus (UT) (F). The eggs shoud be fully formed in the uterus as showing in (C). Statistical analyses were performed using Mann-Whitney U-test, P≤0.05; n = 5).</p

Morphology of adult worms after RNAi of SmJNK <i>in vitro</i> and subsequent transfer of parasites into mice.

<p>Adult 37-day-old worms were fixed and stained, and visualized by confocal microscopy as described in the text. A, B and C show normal worms treated with GFP dsRNA, whereas D, E and F show morphological changes in worms treated with SmJNK dsRNA. A and B - the tubercules (TB) are highlighted on the tegument; C – female worm ovary (OV) showing immature and mature oocytes; D – muscular structure of a worm without tubercules; E- subtegumentar lesion (SL); F- immature ovary.</p