Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films-1

automatic total cell count, user correction thereof and parasitaemia scoring; blue crosses indicate where the user has added cells to the total cell-count, and green dots indicate where the user has subtracted cells. Yellow circles indicate cells that the user has deemed infected.<p><b>Copyright information:</b></p><p>Taken from "Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films"</p><p>http://www.malariajournal.com/content/7/1/62</p><p>Malaria Journal 2008;7():62-62.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2358916.</p><p></p

Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films-5

N arrows indicate unifected RBCs, and red arrows indicate infected RBCs. In this example the user has opted not to discriminate between the different stages of infected RBC. . A scored image of a human RBC culture experimentally infected with , captured at 600× magnification. In this example the user has chosen to specifically determine the number of trophozoites present (blue arrows), and infected cells at any other stage of development have simply been scored as 'infected RBC' (red arrows).<p><b>Copyright information:</b></p><p>Taken from "Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films"</p><p>http://www.malariajournal.com/content/7/1/62</p><p>Malaria Journal 2008;7():62-62.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2358916.</p><p></p

Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films-0

N arrows indicate unifected RBCs, and red arrows indicate infected RBCs. In this example the user has opted not to discriminate between the different stages of infected RBC. . A scored image of a human RBC culture experimentally infected with , captured at 600× magnification. In this example the user has chosen to specifically determine the number of trophozoites present (blue arrows), and infected cells at any other stage of development have simply been scored as 'infected RBC' (red arrows).<p><b>Copyright information:</b></p><p>Taken from "Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films"</p><p>http://www.malariajournal.com/content/7/1/62</p><p>Malaria Journal 2008;7():62-62.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2358916.</p><p></p

The 'interphase' after automatic identification of individual cells and gridding of these ranked on various features potentially discriminating infected and uninfected cells

<p><b>Copyright information:</b></p><p>Taken from "Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films"</p><p>http://www.malariajournal.com/content/7/1/62</p><p>Malaria Journal 2008;7():62-62.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2358916.</p><p></p

Foxp3 induction is MHC class II-dependent.

<p>MHC class-II dependency was examined using 5 µg/ml neutralizing anti-HLA DR or isotype control mAb. Cells were analysed for CD25 and Foxp3 expression on CD4 T cells on day 6 (A). Culture supernatants were sampled on day 2 (IL-2) or day 6 (IL-10) for cytokine analysis by ELISA (B). Fold changes in the proportion of Foxp3^hi and Foxp3^int cells (black bars) were calculated by normalizing for each donor the percentage of each of the two cell types of CD4⁺ T cells or cytokine levels on the respective values obtained for PBMCs cultured with iRBCs in the absence of any antibody (white bars). Experiments were set up with a 2∶1 iRBC∶PBMC ratio.</p

Induction of Foxp3^hi and Foxp3^int CD4⁺CD25^hi T cells by malaria-infected RBCs is time and concentration-dependent.

<p>PBMC were cultured alone, with uninfected RBCs (nRBC) or iRBCs for 6 days at different iRBC∶PBMC ratios. Cells were analysed by flow cytometry for CD25 and Foxp3 expression in gated CD4⁺CD3⁺ lymphocytes. Dot plots show 1 representative of 12 donors examined (A). Comparative analysis of Foxp3 and isotype control staining on CD4⁺CD25⁻ T cells and CD4⁺CD25⁺ T cells in day 6 cultures revealed specific Foxp3 expression solely in the CD25⁺ population (B). Absolute numbers of different CD4 T cell populations for 4 donors (Mean+/−SEM) were calculated based on light microscopic cell counts and flow cytometry analysis on day 6 of culture of originally 2×10⁷ PBMCs co-cultured in the absence or presence of nRBCs and iRBCs (C). Data from 12 donors demonstrate iRBC concentration-dependent induction of Foxp3^hi and Foxp3^int CD4⁺CD25^hi T cells (D). A representative plot of 1 out of 7 donors shows the induction of these cells over a time course of 6 days (E).</p

Phenotype of Foxp3^hi and Foxp3^int CD4⁺CD25^hi T cells induced by malaria-infected RBCs.

<p>After 8 days of iRBC∶PBMC co-culture, immuno magnetic selection was used to isolate CD4⁺CD25^hi (black line) and CD25⁻ cells (grey filled). CD25 and Foxp3 expression in the two sorted populations was analysed by flow cytometry (A). mRNA levels for Foxp3, TGFβ1 and IL-10 were normalized on 18SrRNA levels for each donor. Data from 8 donors (Mean+/−SEM) are shown, presented as relative mRNA levels normalized on mRNA levels in CD4⁺CD25⁻ T cells for each donor (A). PBMC of 3 donors were phenotyped on day 0 prior to iRBC-PBMC co-culture set up, and after 6 days of iRBC∶PBMC co-culture (2∶1 ratio). Dot plots show the gating strategies for Foxp3 expressing cells on day 0 and day 6, respectively (B). Representative histograms depicting isotype control staining (grey filled) and specific staining for various surface markers on CD25⁻Foxp3⁻ and CD25^hi Foxp3-expressing CD4 T cells (black line) from each respective culture are shown (B).</p

Foxp3 induction requires soluble factors from monocyte-CD4 T cell interaction, but not necessarily direct contact of monocytes with converting T cells.

<p>To determine contact-dependency for converting T cells, T cells were separated from iRBC-co-cultured whole PBMC or T cells+monocytes through a 20 nm pore size transwell and analysed by flow cytometry on day 6. Bar graphs show data of 5 donors (Mean+/−SEM), analysed as fold change in the induction of CD25^hiFoxp3^hi or CD25^hiFoxp3^int CD4 T cells (A). Representative FACS plots for each condition are shown (B). Foxp3^hi∶Foxp3^int ratios were calculated for T cells in direct co-cultures as well as inside and outside the transwell (C). Foxp3^hi and Foxp3^int levels, as well as IL-2 and IL-10 levels in culture supernatants were monitored on day 2, 4, 5 and 6 and are shown for 1 out of 2 donors (D). To determine which T cell subpopulation has to interact with monocytes to induce Foxp3 expression in transwell-separated T cells, T cells that were added back to monocytes were depleted of CD4⁺, CD8⁺, or CD25⁺ cells prior to culture. Proportions of CD25^hiFoxp3^hi and CD25^hiFoxp3^int cells of CD4 T cells induced in the transwell-separated T cell compartment (on day 6) as well as IL-2 and IL-10 levels in the culture supernatant (on day 2 and 6, respectively) were measured and are shown as fold changes compared to whole T cells (E). Fold changes (black bars) were calculated by normalizing for each donor the percentage of each of the two cell types of CD4⁺ T cells or cytokine levels on the respective values obtained for monocytes+T cells directly cultured with iRBCs (A) or for cultures with whole T cells added back to monocytes within the transwell (E) (white bars). The T cell compartment analysed and depicted in each individual graph is highlighted in bold red. Experiments were set up with a 2∶1 iRBC∶PBMC ratio.</p

Cytokine levels in iRBC∶PBMC co-culture supernatants.

<p>Co-culture supernatants were harvested and assayed at different time points for IL-2, IFNγ, IL-6 and IL-10. Histograms show absolute values in pg/ml (Mean+/−SEM) for 14–18 donors (A). Total TGFβ1 levels in supernatants of untreated or iRBC-treated PBMC were determined on day 6 of co-culture and are shown as fold change for 12 donors, normalized on values from untreated PBMC (relative level = 1) for each donor (B). The role of serum-derived TGFβ1 for final TGFβ1 levels in co-culture supernatants was established in time course experiments for untreated (C) or iRBC-treated PBMC (data not shown) cultured in serum-supplemented or serum-free AIM-V. A representative histogram for supernatant TGFβ1 levels in pg/ml (Mean+/−StDev) for 1 out of 3 donors is shown (C). The proportions of Foxp3^hi and Foxp3^int CD25^hi CD4⁺ T cells in these cultures on day 6 were compared and are shown as Mean+/−SEM from 4 donors, normalized for each donor on values in serum-free cultures (D).</p

Monocytes and CD4 T cells are the main cellular source of TGFβ1 and IL-10 in iRBC∶PBMC co-cultures.

<p>On day 6 of iRBC∶PBMC co-culture (1∶5 ratio), individual cell types were separated by MACS and FACS to subsequently isolate RNA from purified populations. Relative mRNA levels (normalized on 18SrRNA) for TGFβ1 and IL-10 were determined and normalized for each donor on respective levels in total unseparated PBMC (A). The proportion of individual cell populations expressed as percent of total PBMC was determined by flow cytometry (B). Relative mRNA levels for IL-10 and TGFβ1 for each cell population were corrected for their proportion in whole PBMC by multiplying relative mRNA levels with the percentage of each population in whole PBMCs for each donor (C). Data (Mean+/−SEM) for 3 donors are shown.</p