cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites

Water Balloon

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Two Naomis

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Transaortic TAVR and Mitral Repair Under Deep Hypothermic Circulatory Arrest in a Porcelain Aorta Patient

Vascular and valvular calcifications, commonly seen in renal patients, increase operative mortality and can preclude conventional valvular management. We show a novel approach to treat aortic stenosis and degenerative mitral regurgitation under hypothermic circulatory arrest in a hemodialysis patient with aortic, mitral disease and porcelain aorta with surgical and transcatheter contraindications

Sildenafil impairs mature GIE filterability.

<p><b>A</b>. Stage V GIE were harvested by magnetic isolation and incubated at 37°C for 30 min with 100 μM sildenafil, or 0.1% DMSO (Control). The total intracellular cAMP concentration was measured on aliquots of 6.10⁶ cells in duplicate wells. The assay was carried out five times. Error bars denote the standard error of the mean. **Highly significant differences in retention rates compared to control (DMSO) (<i>P <</i> 0.01). <b>B.</b> Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C for 30 min with 100μM sildenafil, or 0.1% DMSO (Control). <b>C</b>. Differential interference contrast images of paraformaldehyde-fixed GIE, as they flow through the microsphilters. <b>D</b>. Retention rates in microsphilters of stage V GIE (light grey) and uninfected red blood cells (uRBC, dark grey) pre-incubated 30 min at 37°C with different concentrations of sildenafil. Error bars denote the standard error of the mean. Outliers are shown as open circles. *** and **Highly significant differences in retention rates compared to control (**<i>P <</i> 0.01; ***<i>P <</i> 0.001); ns: non-significant differences in retention rates compared to control. n: number of experiments.</p

<i>Pf</i>PKA-mediated phosphorylation contributes to immature GIE stiffness.

<p><b>A.</b> Retention in microsphilters of stage III GIEs from the B10 clone pre-incubated 30 min to 1 h at 37°C with 10 μM H89, 10 μM KT5720, 10 μM PKI-m, 10 μM compound 2, 10 μM GGTI-298 or 0,1% DMSO (Control). Error bars denote the standard error of the mean. *** and ** Highly significant differences in retention rates (*** <i>P</i> < 0.001; **<i>P <</i> 0.01); ns: non-significant differences in retention rates compared to control; n: number of experiments. Outliers are shown as open circles. <b>B.</b> Retention in microsphilters of stages III GIEs from the B10 clone and the pHL-<i>pfpka-r</i> clone cultivated with and without pyrimethamine for 15 generations. The pHL-<i>pfpka-r</i> clone was pre-incubated 15 min at 37°C with 100μM 8Br-cAMP. Error bars denote the standard error of the mean. ***Highly significant differences in retention rates (<i>P <</i> 0.001); ns: non-significant differences in retention rates; n: number of experiments. Outliers are shown as open circles. <b>C</b>. Western-blot analysis of <i>Pf</i>PKA-R expression in stage III GIE from the B10 clone and the pHL-<i>pfpka-r</i> clone cultivated in presence (+ pyr) or absence (- pyr) of pyrimethamine. Immunoblots were probed with rabbit polyclonal antibodies directed against <i>Pf</i>PKA-R and with a mAb directed against <i>Pf</i>HSP70 to normalize expression. Black star indicates the expected size for <i>Pf</i>PKA-R (50.8 kDa); Red star indicates <i>Pf</i>PKA-R with post-translational modifications. The experiment has been performed seven times. Error bars denote the standard error of the mean. *Significant differences in phosphorylation signal (*<i>P</i> < 0.05); ns: non-significant differences in phosphorylation signal. <b>D</b>. Quantitation of signal intensities in panel C using Quantity One software (BioRad). Analysis shows a 1.6-fold increase in <i>Pf</i>PKA-R expression in the pHL-<i>pfpka-r</i> clone (+ pyr) compared to B10. Decrease of <i>Pf</i>PKA-R expression in absence of pyrimethamine (- pyr) indicates a loss of episomal expression of the <i>Pf</i>PKA-R protein. <b>E.</b> Immunofluorescence analysis of stage III GIE from the B10 clone and the pHL-<i>pfpka-r</i> clone cultured for 15 generations in the presence (+ pyr) or absence (- pyr) of pyrimethamine. Infected erythrocytes were stained with anti-<i>Pf</i>PKA-R antibodies followed by anti-rabbit Alexa 594-conjugated IgG. Pictures were taken under identical exposure conditions. The bars represent 2 μm.</p

Viability of 3D7 stage V GIE treated with different inhibitors.

<p>3D7 Stage V GIE were treated with the inhibitors for the indicated times, then washed and their viability evaluated with the colorimetric pLDH assay either immediately (time 0) or after 72 h incubation at 37°C, as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref056" target="_blank">56</a>]. The data are expressed as percent of untreated controls and are the mean of two experiments in quadruplicate.</p><p>Viability of 3D7 stage V GIE treated with different inhibitors.</p

GIE filterability is dependent on cAMP concentration.

<p><b>A.</b> cAMP concentration drops in mature GIE. The total intracellular cAMP concentration was measured in stage III and V GIE using a competitive immunoassay for the quantitative determination of cAMP. GIE were harvested by magnetic isolation and aliquots of 6.10⁶ cells were assayed in duplicate wells. The assay was carried out three times. Error bars denote the standard error of the mean. **Highly significant difference compared to stage III GIE (<i>P <</i> 0.01). <b>B</b>. Western-blot analysis of PKA-R expression in MACS-purified stage III and stage V GIE (5.10e6 parasites/lane). Immunoblots were probed with rabbit polyclonal antibodies directed against <i>Pf</i>PKA-R and with a mAb directed against <i>Pf</i>HSP70 to normalize expression. Quantity One (BioRad) analysis shows that <i>Pf</i>PKA-R levels were not significantly different between stage III and stage V. <b>C</b>. Retention rates in microsphilters of stage III GIE (dark grey), stage V GIE (light grey) and uninfected red blood cells (uRBC, black) pre-incubated 15 min at 37°C with different concentrations of 8Br-cAMP. Error bars denote the standard error of the mean. Outliers are shown as open circles. **Highly significant differences in retention rates compared to control without 8Br-cAMP (<i>P <</i> 0.01); ns: non-significant differences in retention rates compared to control; n: number of experiments.</p

The effect of PDE inhibitors on particulate fractions isolated from <i>P</i>. <i>falciparum</i> parasites.

<p>IC₅₀ values (the concentration of compound that gave 50% inhibition of native PDE activity) are mean ± SEM in μM and were calculated using the Prism software package (GraphPad Software, Inc.). Percentage inhibition data were fitted to a sigmoidal dose-response curve using non-linear regression. Bovine control PDE column displays cAMP dependent activity. All TbPDE1 and most Human values obtained from Kunz, <i>et al</i>.[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref058" target="_blank">58</a>].</p><p>*Value obtained from Tang, <i>et al</i>.[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref059" target="_blank">59</a>].</p><p>>200, sufficient inhibition was not observed for an IC₅₀ calculation.</p><p>Tb: <i>Trypanosama brucei</i>.</p><p>The effect of PDE inhibitors on particulate fractions isolated from <i>P</i>. <i>falciparum</i> parasites.</p