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. 2017 Jul;140(1):e20162169. doi: 10.1542/peds.2016-2169

FIGURE 3.

FIGURE 3

Short-acting natural corticotropin therapy results in de novo formation of corticotropin-reactive neutralizing antibodies. A, The short-acting formulation of natural corticotropin used for the study patient has been prepared from purified pituitary extracts of mixed bovine, ovine, and porcine origins and contains a mixture of melanocortin peptides, including the predominant corticotropin, as indicated by gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the drug (left panel). Natural corticotropin was fractionated on SDS-PAGE gels, transferred to nitrocellulose membrane, and subjected to immunoblotting overnight with a commercial rabbit anticorticotropin antibody (positive control) or with the patient’s sera (1:1000 dilution), which were collected 1 week before (–1WK) or 2 or 6 weeks (2WK or 6WK) after starting corticotropin therapy. The blots were then developed by using an anti-rabbit secondary antibody or anti-human IgM or IgG antibodies. Bands for corticotropin were indicated. B, B16 melanoma cells were treated for 24 hours with vehicle, natural corticotropin (100 mIU/mL), or corticotropin (100 mIU/mL) premixed with an equal volume of sera collected 1 week before (–1WK) or 2 weeks (2WK) after starting corticotropin therapy. Representative phase contrast micrographs demonstrate the morphogenic effect of corticotropin in melanoma cells, highlighting the newly formed dendrites (arrowheads). C, Absolute counting of the number of cells with dendrites expressed as a percentage of the total number of cells. The cell culture experiment was repeated 6 times (n = 6); *P < .01 versus control group; #P < .01 versus ACTH group or –1WK + corticotropin group. D, Cell lysates were collected after different treatments and processed for immunoblot analysis for phosphorylated ERK (p-ERK), total ERK, phosphorylated GSK3β (p-GSK3β), and total GSK3β. E, Arbitrary units of p-ERK/ERK ratios and F, p-GSK3β/GSK3β ratios expressed as immunoblot densitometric ratios of the molecules as folds of the vehicle treated group. The cell culture experiment was repeated 6 times (n = 6); *P < .01 versus control group; #P < .01 versus –1WK + corticotropin group.