Additional file 2: of Selection of reliable reference genes for the normalisation of gene expression levels following time course LPS stimulation of murine bone marrow derived macrophages

Table S3. Assessment of RNA quality. Spectrophotometry analysis of the quality of RNA used in all qRT-PCRs. (DOCX 17 kb

Protective effects of immunization with C-1 peptidases SmCB1 and FhCL1 alone and in combination with SG3PDH/PRX-MAP on male and female worm burdens and parasitological parameters.

<p>Mice (10–12/group) were immunized with SmCB1 or FhCL1 combined with rSG3PDH/PRX-MAP and challenged 2 (panels 1–3) or 3 (panels 4 and 5) weeks after the last immunization.</p>*<p>Percent reduction was evaluated by the formula: % reduction = mean number in infected controls−mean number in infected, immunized mice/mean number in infected controls×100.</p>a<p><i>P</i> values as calculated by Student 2-tailed <i>t</i>-test. NS = not significant.</p

Infection with <i>Fasciola hepatica</i> does not inhibit IL-12 production early in infection with <i>Toxoplasma gondii.</i>

<p>BALB/c mice were infected with <i>F. hepatica</i> 5 days prior to infection with <i>T. gondii</i>. Mice infected with either <i>F. hepatica</i> alone or <i>T. gondii</i> alone were used as controls. On day 4 post <i>T. gondii</i> infection (day 9 post <i>F. hepatica</i> infection) the peritoneal cavity was rinsed with PBS and the resulting lavage was spun at 500 <i>g</i> to pellet the exudate cells. The supernatant was retained to measure the level of IL-12 being secreted into the peritoneal cavity by cytokine ELISA (B). Adherent peritoneal exudate cells (PEC) were cultured for 48 h and supernatants assayed for secretion of IL-12 by cytokine ELISA (A). Serum was collected by cardiac puncture and levels of IL-12 determined by cytokine ELISA (C). The results represent the mean±SE of one experiment with five animals per group. ^a represents groups where the values obtained were significantly different to values obtained for the <i>T. gondii</i>-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the <i>F. hepatica</i>-only infected group (<i>P</i><0.05, Mann-Whitney non-parametric test).</p

Cysteine peptidases vaccine potential.

<p>Mice were immunized 1x (a) or 2x (b–e) with 10 (a–d) or 20 (e) µg active (a–e) or inactive (d,e) cysteine peptidase/mouse/injection, alone (a–e) or in a mixture (d,e), and exposed 15 days later to 120 (a–c) or 140 (d,e) <i>S. mansoni</i> cercariae. Columns represent mean worm burden in 7–14 mice/group, and vertical bars denote SD about the mean. <i>P</i> values and percent reduction in worm burden as compared to control mice are shown above the columns of the test groups. Differences in mean worm burden between mice immunized with active and inactive cysteine peptidases (d,e) are highly significant (<i>P</i><0.0001).</p

Recovery of lung-stage schistosomules from unimmunized and immunized mice 6 days after the challenge infection.

<p>For each experiment, lung-stage larvae were recovered from 2–3 mice per group, and counted on an individual mouse basis. Mean number ± SD around the mean of larvae retrieved from 2–6 repeat groups is shown. NS = not significant compared to PBS controls, as assessed using the Student 2-tailed <i>t</i>-test.</p>*<p>Immunization of mice with SG3PDH or PRX-MAP alone or emulsified with Freund's, alum, or Allison' adjuvant failed to elicit changes in number of lung-stage larvae recovered from immunized mice compared to adjuvant controls (data not shown).</p

Human antibody isotype responses.

<p>A total of 50 patients (18–20 year-old) with confirmed schistosomiasis (100–400 eggs/gram stool) were tested for serum antibody IgM, IgG1, IgG2 (sera diluted 1∶250), IgG4, IgA1/A2, and IgE (sera diluted 1∶25) reactivity to recombinant SmCB, FhCL, or SG3PDH (250 ng/well), or SEA (1 µg/well) in duplicate wells. Patients with mean absorbance values higher than the mean absorbance values of 14 sex- and age-matched, parasite-free, control donors are considered responders.</p

Mice infected with both <i>Toxoplasma gondii</i> and <i>Fasciola hepatica</i> produce both anti-<i>T. gondii</i> IgG2a and anti-<i>F. hepatica</i> IgG1 indicating a Th1 response to the <i>T. gondii</i> infection and a Th2 response to the <i>F. hepatica</i> infection.

<p>BALB/c mice were infected with <i>F. hepatica</i> (Fh) 5 days prior to infection with <i>T. gondii</i> (A,C) or <i>T. gondii</i> 3 days prior to infection with <i>F. hepatica</i> (B,D). Mice infected with either <i>F. hepatica</i> alone or <i>T. gondii</i> alone or uninfected mice were used as controls. Serum was collected by cardiac puncture on: (A,C) day 10 post <i>T. gondii</i> infection (day 15 post <i>F. hepatica</i> infection) or (B,D) day 14 post <i>T. gondii</i> infection (day 11 post <i>F. hepatica</i> infection). <i>T. gondii</i>-specific IgG1 and IgG2a (A,B) and <i>F. hepatica</i>-specific IgG1 and IgG2a (C,D) antibodies were measured by ELISA. The results represent the mean±SE of one experiment with five animals (A,C) or eight animals (B,D) per group. ^a indicates groups where the values obtained were significantly different to values obtained for the <i>T. gondii</i>-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the <i>F. hepatica</i>-only infected group (<i>P</i><0.05, Mann-Whitney non-parametric test).</p

Adjuvant effect of SmCB and FhCL mixture.

<p>Mice were immunized 1x (a) or 2x (b) with rSG3PDH+PRX-MAP alone or combined with SmCB+FhCL, and challenged 3 weeks later with 120 cercariae of <i>S. mansoni</i>. Columns represent mean worm burden in 8–12 mice/group, and vertical bars denote SD about the mean. <i>P</i> values and percent reduction in worm burden and as compared to control mice are shown above the columns of the test groups.</p

<i>Toxoplasma gondii</i> suppresses <i>Fasciola hepatica</i>-driven non-specific Th2 responses in co-infected mice.

<p>Mice were infected with <i>F. hepatica</i> 5 days prior to infection with <i>T. gondii</i> to allow time for a Th2 response to be induced. Mice infected with either <i>F. hepatica</i> alone or <i>T. gondii</i> alone were used as controls. Fifteen days post <i>F. hepatica</i> infection (day 10 post <i>T. gondii</i> infection) spleen cells were stimulated <i>in vitro</i> with PMA plus anti-CD3 and medium alone was included as a negative control. Levels of the Th1-associated cytokine IFN-γ (A) and the Th2-associated cytokines, IL-4 and IL-5 (B, C) were assessed in supernatants 3 days later. Cytokine concentrations represent mean±SE after subtraction of background control values with medium only for five mice per group. ^a represents groups where the values obtained were significantly different to values obtained for the <i>T. gondii</i>-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the <i>F. hepatica</i>-only infected group (<i>P</i><0.05, Mann-Whitney non-parametric test).</p

<i>Toxoplasma gondii</i> infection suppresses the recruitment of cells to the site of infection normally associated with <i>F. hepatica</i> infection.

<p>(A) BALB/c mice were infected with <i>F. hepatica</i> (Fh) then 5 days later were infected with <i>T. gondii</i> (Tg) or (B) mice were infected with <i>T. gondii</i> 3 days prior to infection with <i>F. hepatica</i>. Mice infected with either <i>F. hepatica</i> alone or <i>T. gondii</i> alone or uninfected mice were used as controls. On days 10 (A) and 14 (B) post infection <i>T. gondii</i> (days 15 and 11 post <i>F. hepatica</i> infection respectively) peritoneal exudate cells were recovered from the peritoneal cavity by flushing with PBS. Total cell numbers were determined by counting using a Neubauer slide. Microscopic analysis showed macrophages to be the dominant cell type in all the mice making up 89–91% of cells; other cells present included lymphocytes (4–5%) and neutrophils (5–6%). Results are presented as mean±SE for five animals per group (A) or eight animals per group (B). ^a represents groups where the values obtained were significantly different to values obtained for the <i>T. gondii</i>-only infected mice and ^b represents groups where the values obtained were significantly different to those obtained for the <i>F. hepatica</i>-only infected group (<i>P</i><0.05, Mann-Whitney non-parametric test).</p