Summary of the treatment protocols used.

<p><b>First protocol</b>: BALB/c mice received 4 weekly injections. Animals were infected and sacrificed 30 or 60 days later. <b>Second protocol</b>: BALB/c and B10.A mice were infected intratracheally. Mice received 4 weekly vaccine doses and animals were sacrificed 1 week after the last injection. <b>Third protocol</b>: B10.A mice were infected i.t. and one month after infection, they were immunized with the DNA vaccine. The animals were sacrificed one month after the last injection, six months after infection.</p

Cytokines in lung homogenates of B10.A mice infected i.t. with <i>P. brasiliensis</i> Pb 18 yeasts and submitted to gene immunization for 5 months.

<p>Mice were sacrificed 1 month after the last dose.</p

Immunoprophylactic treatment of PCM.

<p>Gene immunization was initiated 30 days before fungal challenge. CFUs are from lungs of BALB/c mice infected intratracheally with 3×10⁵ yeast cells and subjected to immunization with vectors containing P10 (pP10) and/or IL-12 (pIL-12) DNA insert. Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 30 (▪) and 60 (□) days after infection. Each bar represents the average counts and standard deviations of CFUs in lungs from 10 animals in each group. Experiments were carried out in triplicate with similar results. <b>**</b><i>p</i>≤0.0001, comparing vector with and without insert; ^##<i>p</i>≤0.0001, .comparing untreated and other groups.</p

Therapeutic treatment of PCM.

<p>Gene immunization started 30 days after infection. CFUs are from lungs of BALB/c (□) and B10.A(▪) mice infected intratracheally with 3×10⁵ yeast cells and subjected to immunization with vectors containing P10 (pP10) or IL-12 (pIL-12) DNA inserts. Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 60 days after infection. Each bar represents the average counts and standard deviations of CFU in lungs from 10 animals in each group. Experiments were performed three times and similar results were achieved. <b>*</b><i>p</i>≤0.05, <b>**</b><i>p</i>≤0.005, comparing vector with and without insert; ^##<i>p</i>≤0.005, comparing untreated and other groups.</p

Histopathology of lungs from intratracheally infected B10.A mice.

<p>Animals were infected with <i>P. brasiliensis</i> for one month, treated with or without vectors carrying P10 or IL-12 DNA inserts according to protocol 3, and sacrificed 6 months after the initial infection. Infected mice treated with (<b>A</b>) control pcDNA3, (<b>B</b>) pP10, (<b>C</b>) pIL-12 DNA, and (<b>D</b>) P10 and IL-12 DNA. Gomori staining; original magnification, 40×.</p

Long term therapeutic treatment of experimental PCM.

<p>Gene immunization started 30 days after infection and mice were sacrificed 6 months after infection. CFUs were counted in lungs of B10.A mice infected intratracheally with 3×10⁵ yeast cells and immunized with vectors containing the insert encoding P10 (pP10) or IL-12 (pIL-12). Control mice were inoculated with PBS or with vector without insert. Each bar represents the average counts and standard deviations of CFU in lungs from 5 to 10 animals in each group. Experiments were carried out in triplicate, with similar results. <b>**</b><i>p</i>≤0.001, comparing vector with and without insert; ^##<i>p</i>≤0.001, comparing untreated and other groups.</p

Activity of C16:0-PAF and different <i>T. cruzi</i> LPC species on the aggregation of rabbit platelets.

<p>Platelet aggregation assays were performed as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003077#s2" target="_blank">Materials and Methods</a>, using synthetic 16:0-PAF and C16:0-, C18:0-, C18:1-LPC, and purified C18:2-LPC. Control platelets or platelets pre-treated for 30 min with 10 µM WEB 2086 were assayed in the absence or presence of 1 µM C16:0-PAF or the LPC species at 10 µM (C16:0-LPC, C18:0-LPC, C18:1-LPC, and C18:2-LPC) and 100 µM (C18:1-LPC). Each lipid was tested in duplicate as indicated by black and blue curves in each graph.</p

Proposed molecular structures of the three major LPC species of <i>T. cruzi</i>.

<p>Fragment ions obtained from the MSⁿ analysis of <i>T. cruzi</i> ion species at <i>m/z</i> 530, 528, and 526 are indicated. The tandem fragmentation of C16:0-PAF standard is included as a reference.</p

Distances between the nitrogen, phosphorous, and oxygen atoms in functional groups of C16:0-PAF and each LPC species.

<p>^<i>a</i>This refers to the nitrogen of the amino group, the phosphorous of the phosphate group, and the oxygen at <i>sn</i>-1, linking the glycerol backbone to the carbonyl group of the acyl chain.</p

Full ESI-LIT-MS spectra of <i>T. cruzi</i> phospholipids.

<p>(<b>A</b>) MS1 spectra of lipids obtained in the Folch lower phase prior to fractionation. Lipid samples from all <i>T. cruzi</i> stages were diluted in methanol containing 5 mM LiOH and analyzed by direct infusion in an LTQXL ESI-LIT-MS (positive-ion mode, MS+). Note that the region of spectrum corresponding to LPAF, LPC, and PAF species in Epi, Meta, and TCT has been magnified for better visualization. (<b>B</b>) MS1 spectra of phospholipids obtained by SPE followed by POROS R1 fractionation. Lipids eluted in 25% n-propanol were diluted in methanol containing 5 mM LiOH and analyzed as above. Since the same initial total number of cells (5×10⁸) was used for lipid fractionation from each parasite stage, all spectra were normalized. Magnification of the MS range where PAF and LPC species would be found is indicated (insets). Epi, epimastigote; Meta, metacyclic trypomastigote; ICA, intracellular amastigote; TCT, tissue culture-derived trypomastigote. <i>m/z</i>, mass to charge ratio.</p