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

LPC inhibits NF-қB translocation, iNOS expression, and NO production in LPS-stimulated macrophages.

<p>Peritoneal macrophages from BALB/cmice were incubated in the absence or presence of 1 µg/mL LPS and different concentrations of LPC (Sigma) at 37 °C in a 5% CO₂ atmosphere. After 1 h of incubation, NF-қB translocation (<b>A</b>) was assayed by Western blot analysis. After 24 hours, NO production (<b>B</b>) was assayed by measuring the amount of nitrite in the culture supernatant using the Griess reagent, and iNOS expression (<b>C</b>) was determined by Western blot analysis followed by densitometry (lower panel). Data is the mean ± S.D. of three different experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</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

Glycolipids from <i>T. rangeli</i> and <i>T. cruzi</i> suppress NOS expression.

<p><i>Rhodnius</i> were injected with either Tr GIPL or Tc GIPL and three days later salivary glands were dissected, homogenized and NOS expression evaluated by Western blotting. A. Western blotting against NOS in salivary glands obtained from control and insects injected with Tr GIPLs. B. Western blotting against NOS in salivary glands obtained from control and insects injected with Tc GIPLs.</p

NADPH-diaphorase activity of NOS in <i>Rhodnius prolixus</i> salivary glands after a blood meal and the expression of NOS.

<p>A. Salivary glands were dissected in different days after blood feeding and evaluated for NOS NAPDH-diaphorase activity. Salivary glands were assayed in 10 mM Tris-HCl pH 8,0, 0,05 M NaCl, 0,1%, Triton X-100, 1 mM CaCl₂, 5 µM FAD, 1 mM NADPH and 0,5 mg/mL MTT. MTT reduction was followed at 540 nm for 30 min at 37°C. Also samples were obtained and NOS content evaluated by Western blotting. Each point is the average and SE of 05 different experiments. B. Immunoblotting using an anti-NOS antibody. Blottings were developed with the use of a secondary antibody conjugated to alkaline phosphatase in the presence of the substrate Western Blue. Molecular mass markers are indicated at the left. C. Upper panel<b>,</b> total RNA from the salivary glands at different days after feeding was isolated and cDNA was synthesized. Samples were then analyzed by semi-quantitative PCR with temperatures of 55, 72 and 94°C for 27 cycles with primers for NOS. Lower panel, analysis of 18 S RNA levels. In this case reaction occurred for 25 cycles. The products of reactions shown on panels C were separated on agarose gel 1.4% stained with ethidium bromide and photographed under ultraviolet light. Molecular mass standards are indicated at the left.</p

Chemical composition of GIPL purified from <i>T</i>. <i>rangeli</i> (Tr GIPL).

a<p>Determined by GC as trimethylsilyl derivatives of methylglycosides.</p>b<p>Determined by GC and GC-MS as fatty acid methyl esters (FAMEs).</p>c<p>Determined by GC and GC-MS after N-acetylation and trimethylsilylation.</p

Infection with <i>T. rangeli</i> reduces the NOS activity and the levels of NOS protein in the salivary glands of <i>R. prolixus</i>.

<p>A. <i>Rhodnius</i> were dissected 7 days after control injection of water or <i>T. rangeli</i> and assayed for NADPH-diaphorase activity. Results from three experiments were evaluated statistically using the Student t test (* p<0.05). B. Salivary gland extracts from control or <i>T. rangeli-</i>injected insects were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with primary antibody anti-NOS and then with an anti-rabbit antibody conjugated to alkaline phosphatase. This experiment was performed three times. Tr, <i>Trypanosoma rangeli</i> cells evalutated for NOS blotting. N, salivary glands from non-injected insects. C, control salivary glands from insects injected with water. I, Salivary glands from <i>T. rangeli</i>-injected insects. C. NADPH-diaphorase activity was measured in salivary gland extracts of salivary glands three days after injection with 100 ng of glycolipids from either <i>T. rangeli</i> (Tr GIPL), <i>P. serpens (</i>Ps GIPL<i>)</i> or <i>T. cruzi</i> eGPI-mucin (Tc Mucin). The experiment was performed three times and analyzed by ANOVA (* p<0.05).</p

Glycolipid-mediated suppression of NO synthesis occurs through the manipulation of intracellular phosphorylation-dephosphorylation circuits.

<p>Intracellular circuits of protein-phosphorylation and dephosphorylation were evaluated through different assays. A. Salivary glands obtained from either control or <i>T. rangeli</i>-infected insects were dissected three days after the injection, homogeneized and phosphorylated in the presence of ³²P-ATP followed by SDS-gel electrophoresis and autoradiograph. B. A similar experiment was conducted with salivary glands isolated from insects injected with Tr GIPL or Tc GIPL. C. Following a blood meal on rabbit ear salivary glands were dissected at different points in time. Total protein phosphatase activity was followed during the refilling cycle of salivary glands using pNPP as substrate. Data is the mean ± S.E. of three different experiments. D. Insects were injected with Tr GIPL and evaluated for protein phosphatase activity in the presence and in the absence of SO. The fraction of enzyme activity inhibited by SO in control and Tr GIPL-injected insects is shown. Data is the mean ± S.E. of three different experiments.</p