Number of brain cysts and survival of mice immunized with microneme proteins and infected with <i>T</i>. <i>gondii</i>.

<p>Mice immunized on days 0, 15, and 30 with the indicated preparations of TgMICs or with the vehicle (PBS) were challenged after one month (day 60) with <i>T</i>. <i>gondii</i> infection, provoked by gavage with cysts of the ME49 strain. (A) Cyst numbers were counted from whole brain homogenates of mice, harvested one month after challenge with 40 cysts. The results are expressed as means ± SD for each group. Significance is denoted as *p < 0.05, compared to the PBS group. (B) Survival curves of mice that were challenged with 80 cysts of the ME49 strain and observed daily for mortality. Data are representative of six experiments with similar results.</p

Cladogram analysis of TgMIC1, TgMIC4, and TgMIC6 of the <i>T</i>. <i>gondii</i> isolates.

<p>Dendrograms grouping microneme proteins of <i>T</i>. <i>gondii</i> isolates showing similarity of approximately 99% according to their sequences. (A) TgMIC1. (B) TgMIC4. (C) TgMIC6. Amino acids sequences were obtained from available sequence database at NCBI, and the alignment was performed using MEGA 6.06 software.</p

SDS-PAGE and western blot analysis of native and recombinant microneme proteins.

<p>SDS-PAGE of recombinant proteins (panels A, B, and C, Coomassie Blue stained) or native (panel D, silver-stained) proteins. Heterologous expression was noted in <i>E</i>. <i>coli</i> (DE3) and recombinant proteins were detected in inclusion bodies. Lane 1: protein expression before induction. Lane 2: protein expression after induction. Lane 3: purified and refolded histidine-tagged recombinant proteins, displayed apparent molecular masses of 70-kDa (TgMIC1, panel A), 80-kDa (TgMIC4, panel B), and 30-kDa (TgMIC6, panel C). Panel E shows the electrophoretical profile of the Lac+ fraction, composed of the native proteins TgMIC1 (53-kDa) and TgMIC4 (68-kDa). Lane M: Molecular mass markers. Reactivity of recombinant and native microneme proteins with anti-Lac+ mouse serum was examined by western blot (Panel E), developed with peroxidase-conjugated goat anti-mouse IgG.</p

Specific humoral responses elicited by immunization of mice with microneme proteins.

<p>The reactivity of immunoglobulins anti-STAg was determined by ELISA in serum samples collected from both immunized (TgMICs) and control (PBS) mice 15 days after the last antigen injection. Each point/bar represents the average absorbance ± SD of the serum samples from 4 animals. (A) Absorbance provided by the reaction of serum IgG with STAg. (B) Absorbance provided by the reaction of serum IgG1 and IgG2a (diluted 1:25) with STAg. The average absorbance ± SD generated by the reaction of serum IgG1 or IgG2a from each group of immunized mice was significantly higher than the corresponding values provided by control mice, with the exception of the TgMIC6-immunized group, whose results were not significantly different of those of the control group. Three independent experiments were performed, and data from one representative experiment is shown. Asterisks represent statistical significant differences (*p < 0.05) between IgG2b and IgG1 for each group of mice (Bonferroni’s t test).</p

Experimental Protocol.

<p><b>(A)</b> In the first experimental procedure mice were subcutaneously (s.c.) vaccinated with microneme proteins emulsified in Freund’s complete adjuvant. Mice were boosted at the same dose and regimen on day 15 and 30 after first injection, now emulsified in Freund’s incomplete adjuvant. Fifteen days after the last injection, blood and spleen samples were collected to assess serum IgG, <i>in vitro</i> T cell proliferation, and cytokine concentrations. <b>(B)</b> One month after the last immunization procedure, the mice were orally infected with 80 cysts of the ME49 strain and the mortality was monitored daily for 1 month. To evaluate the tissue cyst burden, the brain of the mice infected with 40 cysts was removed 1 month after the challenge and the mean number of cysts per brain was determined. Additionally, blood samples from mice challenged with 40 cysts were collected 30 days after <i>T</i>. <i>gondii</i> challenge and the serum cytokine levels were analyzed.</p

Specific cell-mediated immune responses elicited by immunization with microneme proteins.

<p>Spleen cells were harvested from immunized (indicated as TgMICs) and control mice (PBS) on day 15 post the last antigen injection and cultured in the presence of medium only, STAg (10 μg/ml), or Concanavalin A (2 μg/ml) for 72 h. (A) Proliferation of spleen cells was measured by [3H]-thymidine incorporation assay. Each bar represents the average of four mice per group and is representative of three independent experiments. Statistical significance is denoted as *p < 0.05 compared to the control group. (B–D) Cytokine concentration was measured by ELISA in the supernatant of spleen cell cultures. Panels show the IL-12 (B), IFNγ(C), and IL-10 (D) concentrations. Each bar represents the mean ± SD of triplicate samples and the results are representative of three independent experiments. Statistical significance is denoted as *p < 0.05 compared to PBS-inoculated mice; # p < 0.05 compared to non-stimulated cells of the same group.</p

LPC activates JNK and p38, but not ERK, in macrophages.

<p>Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of different concentrations of LPC mix (Sigma) for 20 min at 37 °C in a 5% CO₂ atmosphere, and the cytoplasm content was homogenized and assayed as follows. The Phospho-MAPK array was used for analysis of enzymatic activation (<b>A</b>). The reaction was visualized with the enhanced chemiluminescent system and subjected to densitometric analysis (***, p< 0.001, ANOVA). Protein levels of the phosphorylated MAPKs JNK (<b>B</b>), p38 (<b>C</b>) and ERK (<b>D</b>) were determined by Western blot. Data is the mean ± S.E. of two different experiments.</p

LPC triggers IL-8 production through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected and stimulated as described on Figure 1. After 20 hours of incubation, IL-8 production was measured by the ELISA assay. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC inhibits LPS-induced ERK activation.

<p>Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of 1 µg/mL LPS or in the presence or absence of the indicated concentrations of LPC (Sigma) at 37 °C in a 5% CO₂ atmosphere (<b>A</b>, <b>D</b>, <b>E</b>). In parallel HEK 293A cells with TLR constructs as indicated (<b>B</b>, <b>C</b>). Each group received expression constructs for TLR4 (<b>B</b>) or both TLR2 and TLR1 (<b>C</b>), as well as MD-2, CD14 and CD36 plasmids. The cells were then incubated in the absence or presence of 100 ng/mL LPS or 1 nM Pam3CSK4 (P3C) and 10 or 100 µM of LPC, for 40 min at 37 °C in a 5% CO₂ atmosphere. After incubation either macrophages or HEK cells were homogenized, the protein levels was determined and samples evaluated by Western blot with the use of antibodies against p-ERK (<b>A</b>, <b>B</b>, <b>C</b>), p-JNK (<b>D</b>) and p-P38 (<b>E</b>). Loading controls were run with the use of antibodies raised towards actin. Experiments were performed at least two times with different animals and samples.</p

LPC triggers NF-қB activation through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected in three different groups. Groups A and B received expression constructs for TLR4 (<b>A</b>) or TLR2 and TLR1 (<b>B</b>). Both also received MD-2, CD14, and CD36 constructs and the ELAM-1-firefly luciferase and β-actin-<i>Renilla</i> luciferase reporter plasmids. The third group (<b>C</b>) received only the empty vector pDisplay and the luciferase reporter plasmids. Groups A and B were separately stimulated with 0.1, 1, 10, 100 and 200 µM of different types of LPC (Sigma; C14:0, C16:0, C18:0, and C18:1), 100 ng/mL of LPS and 1 nM of Pam3CSK4 (P3C). Group C was stimulated with LPS, Pam3Cys or 0.1, 1, 10 and 100 µM of LPC (C16:0). The agonists were diluted in DMEM medium with 10% bovine fetal serum. After 4 h of incubation, luciferase activity was measured and expressed as the ratio of NF-қB-dependent firefly luciferase activity to the control <i>Renilla</i> luciferase activity. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p