Managerial lead of an independent school refectory - allowance organization

Phylogeny of Forkhead (FOX) transcription factors. The FKH domain of forkhead proteins of H. sapiens, D. melanogaster, E. multilocularis, H. microstoma, and published FoxQ2 homologs from other animals were aligned, and a phylogeny was estimated by Maximum Likelihood analysis (with a JTT model) using MEGA 5.2 [85]. Bootstrap support values from 1,000 replicates are indicated next to the nodes. Nodes with lower than 50 % support were collapsed. GeneDB accession codes are given for E. multilocularis and H. microstoma and Genbank accession codes are given for all other sequences. The FoxQ2 group is outlined, and the foxQ2 genes of E. multilocularis and H. microstoma are indicated by arrows. (PDF 33 kb

EmTIP: a T-cell immunomodulatory protein homologue from <i>E. multilocularis</i>.

<p>(<b>A</b>) Alignment of EmTIP with human and mouse TIPs. Bioedit software, version 7.0.9, was used to align the amino acids sequences. Accession numbers for the sequences are as follows: EmTIP, HF912277; human TIP, Q8TB96; mouse TIP, Q99KW9. Identical residues are displayed in white on black background, biochemically similar residues in black on grey background. Gaps introduced to maximize the alignment are represented by dashes. Numbers at the end of each line correspond to the amino acid numbers in each respective sequence. EmTIP has 36% identity/48% similarity with Human TIP and 34% identity/42% similarity with Mouse TIP. The N-terminal signal sequences are shown in an open solid black box. Two FG-GAP repeats within EmTIP as predicted by SMART (<a href="http://smart.embl-heidelberg.de/" target="_blank">http://smart.embl-heidelberg.de/</a>) are shown within open solid red boxes. The C-terminal transmembrane domains are depicted in an open solid blue box. (<b>B</b>) Comparative structural architecture of <i>E. multilocularis</i>, human and mouse T-cell immunomodulatory proteins. Red boxes: signal sequences; Grey boxes: FG-GAP repeats; Blue boxes: Transmembrane domains. Sequence accession numbers are as described in (<b>A</b>), with structural architecture predicted with SMART (<a href="http://smart.embl-heidelberg.de/" target="_blank">http://smart.embl-heidelberg.de/</a>).</p

Elevated secretion of IFN-γ by CD4+ T-cells activated in the presence of EmTIP <i>in vitro</i>.

<p>(<b>A</b>) Freshly isolated CD4+CD25− T-cells (2×10⁵/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in the presence of E/S products of <i>E. multilocularis</i> primary cells (PCE/S) or metacestode vesicles (MVE/S). Three days later, the T-cell supernatants were collected and probed for IFN-γ and IL-10 concentrations by Elisa. (<b>B</b>) Freshly isolated CD4+CD25− T-cells (2×10⁵/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in medium supplemented with normalized supernatants of Mock-, control- (pSecTag2), Em<i>tip</i>- (pSecTag2-Em<i>tip</i>) or Em<i>tip</i>_myc (pSecTag2-Em<i>tip</i>_myc)- transfected HEK-293T cells. Three days later, the T-cell supernatants were collected and probed for IFN-γ and IL-10 concentrations by Elisa. (<b>A, B</b>) Individual results are displayed. Bars represent the mean from 4–8 independent biological replicates assessed within 3–4 independent experiments. (<b>C</b>) Freshly isolated CD4+CD25− T-cells (2×10⁵/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in the presence of conditioned medium of primary cells (PCE/S) or metacestode vesicles (MVE/S) supplemented with total rabbit IgG or purified anti-EmTIP (anti-TIP) antibody at 30 µg/ml. Three days later, the T-cell supernatants were collected and probed for IFN-γ and IL-10 concentrations by Elisa. Individual results are displayed. Bars represent the mean from experiments with 4 biological replicates. (<b>D</b>) Freshly isolated CD4+CD25− T-cells (2×10⁵/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in medium supplemented with normalized supernatants of parasite larvae (PCE/S or MVE/S) or transfected HEK-293T cells (Mock, Control or rEmTIP). At day 3 of culture, 5 µg/ml Brefeldin A was added for an additional 6 hours. Then the cells were harvested and the intracellular IFN-γ levels were determined by flow cytometry. Numbers in upper gates indicate the percentage of IFN-γ+ CD4+ cells whereas the lower gate shows the percentage of IFN-γ+ CD4− cells. One representative experiment of 3 conducted with similar results is shown (Left) and data from all three experiments are summarized as bar graphs (Right). * (p<0.05), ** (p<0.01) (<b>D</b>) Bars represent mean ± standard deviations from three independent experiments with 3–7 biological replicates.</p

Anti-EmTIP antibody impairs <i>E. multilocularis</i> stem cell proliferation and metacestode vesicle formation <i>in vitro</i>.

<p>(<b>A</b>) Dose-response curve of the antiproliferative effect of EmTIP inhibition using affinity-purified anti-EmTIP antibody on <i>E.multilocularis</i> primary cell cultures. <i>E. multilocularis</i> stem cell proliferation was assessed by measuring the level of BrdU incorporation (see material and methods for details of the BrdU cell proliferation assay) here expressed as proliferation index. (<b>B</b>) 60 µg/ml of anti-EmTIP antibody limits <i>E. multilocularis</i> stem cell proliferation whereas an equal amount of total rabbit IgG failed to do so. (<b>C</b>) Effect of anti-EmTIP antibody (60 µg/ml) on rat hepatoma cell line proliferation. (<b>D</b>) Effect of anti-EmTIP antibody vs. rabbit IgG on the <i>de novo</i> formation of <i>E. multilocularis</i> metacestode vesicles from primary cell cultures. (<b>A, B, C</b>) Bars represent mean ± SD for 4–6 replicates of two independent experiments. (<b>D</b>) Bars stand for mean levels. Data represent 4–8 biological primary cell isolates assayed individually. ^#, statistical trend <i>p<0.1</i>;*, statistical significance <i>p<0.05</i>; NS, not significant <i>p>0.1</i>.</p

Additional file 5: of Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms

Alkaline phosphatase-based development of whole-mount in situ hybridization in H. microstoma : Wnt2 and Wnt5. Bars: 50 Οm. (PDF 1653 kb

E/S-products of <i>E. multilocularis</i> differentially affect DC maturation and the DC cytokine profile.

<p>DC were exposed to <i>E. multilocularis</i> larvae (primary cell aggregates, PC; metacestode vesicles, MC; protoscoleces, PS), separated through a transwell system, for 72 h and their maturation was assessed by flow cytometry after staining for surface markers CD11c, MHCII and CD86. (A) Representative scatter plots showing the proportion of matured DC (upper right quadrant) with respect to MHCII and CD86, gated on CD11c cells. t0 indicates maturation at the beginning of the experiment. (B) Mean fluorescence intensities of MHCII and CD86 on DC after 72 h of treatment. (C) DC supernatants were collected and analyzed for the presence of IL-12p70, IL-10 and IL-6 by ELISA. Data shown are the mean +− SD of 4 independent experiments. # : not detectable by ELISA.</p

E/S-products of <i>E. multilocularis</i> larvae induce DC Death, but fail to kill splenocytes.

<p>(A) DC were exposed to comparable amounts of parasite material (1 Unit) from each of <i>E. multilocularis</i> larval stages (primary cells, PC; metacestode vesicles, MC; protoscoleces, PS), separated through a transwell system, or (B) to primary cell aggregate-conditioned medium (PCES) for 48 h. DC viability was then assessed using the Trypan Blue exclusion test. The number of surviving DC is expressed as a percentage of the initial number of DC seeded. As a control, the percentage of viable DC at the beginning of the experiment is given (t0). Results shown are the means+− SD of 4 independent experiments. * = p<0,05. (C) Splenocytes from female C57BL/6 mice were similarly exposed to metacestode vesicles (MC), separated through a transwell system, and viability was assessed using the Trypan Blue exclusion test after 48 h and 72 h. (D) Within the splenic lymphocytes, gated with respect to the forward and side scatter, CD19+ lymphocytes were stained and CD19− lymphocytes deduced by flow cytometry and the proportions of both cell populations (CD19− and CD19+) were monitored for changees upon exposure to MC. C and D are representative of two independent experiments. (E, F) E/S-products of <i>E. multilocularis</i> larvae induce DC death via apoptosis. DC were exposed to comparable amounts of <i>E. multilocularis</i> larvae (primary cell aggregates, PC; metacestode vesicles, MC; protoscoleces, PS), separated by a transwell system, for 24 h. DC were then analyzed by flow cytometry for the expression of Cd11c, AnnexinV and 7-AAD. The proportion of apoptotic DC (Cd11c+AnnexinV+7-AAD−) was then determined. UV-treatment of DC was used as positive control for apoptosis-inducing conditions. (E) Representative plots of the proportion of AnnexinV+ 7-AAD− cells, gated on CD11c+ cells. (F) Mean percentage +− SD of apoptotic DC (Apo-DC) of 4 independent experiments. * = p<0,05.</p

E/S-products of <i>E. multilocularis</i> differentially alter DC responsiveness to LPS.

<p>DC were first exposed to <i>E. multilocularis</i> larvae (primary cell aggregates, PC; metacestode vesicles, MC; protoscoleces, PS), separated through a transwell system, for 24 h. DC were then harvested, counted and seeded at equal numbers of living cells in medium containing LPS for 48 h, prior to staining and flow cytometric detection for surface markers CD11c, MHCII and CD86. (A) Representative scatter plots showing the proportion of matured DC (upper right quadrant) with respect to MHCII and CD86, gated on CD11c cells. (B) Mean fluorescence intensities of MHCII and CD86 on DC. (C) DC supernatants were collected and analyzed for the presence of IL-12p70, IL-10 and IL-6 by ELISA. Data shown are the mean +− SD of 4 independent experiments. * = p<0,05. # : not detectable by ELISA.</p

Isolation of <i>E. multilocularis</i> larval stages.

<p>(<b>A</b>) Morphology of <i>E. multilocularis</i> primary cell aggregates (a), metacestode vesicles (b), and protoscoleces (c), that were used for co-cultivation with host DC and other immune effector cells. Primary cell aggregates, representing the oncosphere-metacestode transition complex, were devoid of mature metacestode vesicles. Metacestode vesicles exhibited a cellular germinal layer (GL) and an acellular laminated layer (LL). Protoscoleces were covered by a syncytial tegument (T). White bar = 500 µm, Black bar = 50 µm. (B) Qualitative assessment of parasite material. To ensure that no host cell contamination is present in larval material prior to use, protoscoleces (PS), metacestode vesicles (MC), and primary cells (PC) had been cultivated for one week under axenic conditions. Chromosomal DNA was isolated and subjected to PCR for the <i>E. multilocularis</i>-specific gene <i>elp</i> (upper panel) and a jird-specific β-tubulin-gene (lower panel). As a control, <i>Meriones unguiculatus</i> liver tissue (MuLT) was used. (C) Quantification of larval material. To normalize larval material for cell numbers, the relative β-actin content was used. 2000 protoscoleces (PC), 4 metacestode vesicles (5 mm in diameter), and 1/6^th of the amount of primary cells that can be isolated from 40 ml MC vesicles (PC) were used to produce cell lysates. The lysates were subsequently separated on a 12.5% acrylamide gel and subjected to Western blot analysis using an anti-β-actin antibody (upper panel). β-actin band intensity was subsequently quantified using the ImageJ <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001516#pntd.0001516-Abramoff1" target="_blank">[39]</a> analysis tool (lower panel).</p

<i>In vitro</i> immunomodulation of DC by E/S-products of <i>E. multilocularis</i> larvae.

<p><i>In vitro</i> immunomodulation of DC by E/S-products of <i>E. multilocularis</i> larvae.</p