Effects of MHC II, ICOSL, IL-2 neutralisation on IFN-γ production in the tripartite cultures.

<p>Individually, anti-MHC II (A), anti-ICOSL (B), anti-IL-2 (C) and Orencia (D) did not affect IFN-γ production. However, when all treatments were combined they did reduce the endothelial cell-mediated enhancement of IFN-γ (E). uRBC  =  unparasitised red blood cells. Control  =  no PBMC, uRBC or iRBC. MHC II, ICOSL and IL-2 neutralising antibodies, and Orencia, were incubated with the tripartite cultures for 24 h. Supernates were then harvested and analysed for IFN-γ. Columns represent means of two separate experiments.</p

Effect of caspase-1 inhibition on IFN-γ and IL-1β production in co-cultures of PBMC and iRBC.

<p>YVAD did not affect IFN-γ production but did inhibit that of IL-1β. uRBC = unparasitised red blood cells. Control  =  no PBMC, uRBC or iRBC. YVAD (50 µmol/L), a caspase-1 inhibitor, was pre-incubated with PBMC and HBEC for 30 min prior to addition of the iRBCs. At 24 h, supernates were analysed for IFN-γ and IL-1β. Columns represent values from a single experiment.</p

Ratio of IL-10:IFN-γ, IL-10:TNF and IL-10:CXCL10 protein in the presence and absence of HBEC.

<p>Columns and vertical bars represent means ± SEM of three experiments. Statistical significance (***p<0.001) was assessed using ANOVA and the Bonferroni post hoc test.</p

HBEC-dependent induction of ICAM-1 and CXCL10 in tripartite co-cultures is inhibited by IFN-γ neutralising antibody.

<p>A. IFN-γ neutralising antibody inhibits ICAM-1 mRNA induction in tripartite cultures. B. IFN-γ neutralising antibody inhibits expression of CXCL10 mRNA and protein in the tripartite cultures. uRBC  =  unparasitised red blood cells. Control  =  no PBMC, uRBC or iRBC. Columns and horizontal bars represent means ± SEM of three experiments. Two-way ANOVA showed that the values contained significant differences among the groups (***p<0.001), using ANOVA and the Bonferroni post hoc test.</p

Effect of endothelial cells on cytokine production in PBMC/iRBC co-cultures.

<p>A. Significant alterations in IFN-γ and IL-10 (mRNA and protein) production in PBMC/iRBC (<i>P. falciparum</i> strain 3D7) co-culture in the presence of endothelial cells (HBEC-5i). B. TNF protein production was significantly decreased in the presence of HBEC-5i. uRBC =  unparasitised red blood cells. Control  =  no PBMC, uRBC or iRBC. Columns and horizontal bars represent means ± SEM of nine experiments. Two-way ANOVA showed significant differences among the groups (*p<0.05; ***p<0.001), identified using the Bonferroni post hoc test.</p

Effect of NK depletion on IFN-γ production in tripartite cultures.

<p>A. NK cell depletion strategy. B. IFN-γ protein production was decreased by NK cell depletion. uRBC  =  unparasitised red blood cells. Control  =  no PBMC, uRBC or iRBC. “PBMC N (NK depleted)” co-cultures contained PBMC that had been depleted of 99% NK cells stained with CD16, CD56 and NKp46 prior. “PBMC N” refers to co-cultures in which PBMC had not been depleted of NK cells. Co-cultures were 24 h, following which supernates were harvested and analysed for IFNγ. Columns represent means of two separate experiments.</p

Primer sequences used.

<p>Primer sequences used.</p

Effect on immunomodulator expression of the physical separation of cellular components of the tripartite system.

<p>The enhancement of IFN-γ (A), CXCL10 (B) and ICAM-1 (C) production was perturbed when PBMC were physically separated from HBEC. iRBCs, iRBCs + PBMCs or PBMCs were isolated in the upper chamber of the transwell insert, whereas HBEC were consistently retained in the bottom chamber. Columns and horizontal bars represent means ± SEM of three experiments. One way ANOVA showed significant differences among the groups (*p<0.05; **p<0.01; ***p<0.001), using Tukey's post hoc test.</p

Characterization of nSSL before and after GC loading.

<p>Values shown in each category are the average±SD for at least 10 formulations (13 empty nSSL, 27 nSSL-BMS, and 17 nSSL-MPS formulations). No significant differences were observed when comparing size according to intensity, number, or volume. No significant differences were observed when comparing nSSL-MPS and nSSL-BMS. PdI: an indication of size distribution variance between different batches prepared. A low PdI (<0.2) indicates that the sample is monodispersed.% drug encapsulated  = 100×([drug]/[lipid]_{after Dowex anion exchanger})/([drug]/[lipid]_{after dialysis}).</p

Survival rates after early treatment with 10 mg/kg free or nSSL-encapsulated MPS (upper graph) or BMS (lower graph).

<p>Representative results for ICR mice infected with PbA are presented. Arrows denote treatment administration. ECM prevention is reflected in longer survival times, due to the development of severe anemic malaria, and as a result creation of a survival time-window for anti-plasmodial administration. Significant differences in survival were seen between non-treated and nSSL-MPS-treated groups (p = 0.01) and between non-treated mice and mice administered free or nSSL-BMS (p<0.01).</p