Figure 2.
Transfection with p47phoxmRNA to correct AR CGD granulocytes. Correction of AR CGD p47phox-deficient patient cells with p47phox mRNA. Granulocyte-enriched apheresis products from 3 different AR p47phox-CGD patients were transfected by EP with p47phox mRNA for each condition shown. (A) Effect of p47phox mRNA concentrations at EP on cell viability up to 120 hours post-EP at a constant cell number of 5 × 108 cells per milliliter in EP mix (n = 3 ± standard deviation [SD]; control = non-EP). (B) Effects of varying cell concentrations in EP mix on cell viability up to 120 hours post-EP at 200 or 300 µg of mRNA per milliliter. (C) Representative FACS dot plot analyses of granulocyte-rich apheresis product at times indicated after EP treatment of p47phox-deficient AR CGD patient 5 × 108 cells per milliliter with 400 µg of mRNA per milliliter in EP mix (treated) or non-EP (not treated) control. Top left panel of side scatter (SSC) by forward scatter (FSC) shows gate that captures both mature and immature granulocytes in the granulocyte-rich apheresis product used for analyses shown in the other panels. For the not treated and treated 4-hour and 24-hour groups, the top panels show p47phox expression and the bottom panels show phorbol 12-myristate 13-acetate (PMA)–stimulated increase of DHR fluorescence as a measure of oxidase activity, where the percentage of cells in the positive gates is shown at the top of each panel and the mean fluorescence intensity (MFI) as a measure of expression per positive cell is shown at the bottom of each panel. (D) The kinetics of p47phox protein expression shown as a percentage of p47phox+ cells over 120 hours in culture post-EP. For each sample, 5 × 108 cells per milliliter were treated at the indicated p47phox mRNA concentrations in the EP mix (n = 3 ± SD; control = non-EP). (E) The kinetics of NADPH oxidase activity shown as percentage of DHR+ cells over 120 hours in culture post-EP for the same preparations as in panel D (n = 3 ± SD; control = non-EP). Using 2-way ANOVA analysis, no effect on viability due to cell concentrations (F = 0.241, P = .627) or p47phox mRNA concentrations (F = 1.252, P = .307) was observed.

Transfection with p47phoxmRNA to correct AR CGD granulocytes. Correction of AR CGD p47phox-deficient patient cells with p47phox mRNA. Granulocyte-enriched apheresis products from 3 different AR p47phox-CGD patients were transfected by EP with p47phox mRNA for each condition shown. (A) Effect of p47phox mRNA concentrations at EP on cell viability up to 120 hours post-EP at a constant cell number of 5 × 108 cells per milliliter in EP mix (n = 3 ± standard deviation [SD]; control = non-EP). (B) Effects of varying cell concentrations in EP mix on cell viability up to 120 hours post-EP at 200 or 300 µg of mRNA per milliliter. (C) Representative FACS dot plot analyses of granulocyte-rich apheresis product at times indicated after EP treatment of p47phox-deficient AR CGD patient 5 × 108 cells per milliliter with 400 µg of mRNA per milliliter in EP mix (treated) or non-EP (not treated) control. Top left panel of side scatter (SSC) by forward scatter (FSC) shows gate that captures both mature and immature granulocytes in the granulocyte-rich apheresis product used for analyses shown in the other panels. For the not treated and treated 4-hour and 24-hour groups, the top panels show p47phox expression and the bottom panels show phorbol 12-myristate 13-acetate (PMA)–stimulated increase of DHR fluorescence as a measure of oxidase activity, where the percentage of cells in the positive gates is shown at the top of each panel and the mean fluorescence intensity (MFI) as a measure of expression per positive cell is shown at the bottom of each panel. (D) The kinetics of p47phox protein expression shown as a percentage of p47phox+ cells over 120 hours in culture post-EP. For each sample, 5 × 108 cells per milliliter were treated at the indicated p47phox mRNA concentrations in the EP mix (n = 3 ± SD; control = non-EP). (E) The kinetics of NADPH oxidase activity shown as percentage of DHR+ cells over 120 hours in culture post-EP for the same preparations as in panel D (n = 3 ± SD; control = non-EP). Using 2-way ANOVA analysis, no effect on viability due to cell concentrations (F = 0.241, P = .627) or p47phox mRNA concentrations (F = 1.252, P = .307) was observed.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal