Table 1.
Insulin glargines employed in this study.
Fig 1.
Insulin glargine (Glaritus) lots 32 and 96 induce heightened innate activity in the MIMIC® PTE.
MIMIC® PTE cultures were treated with different batches of insulin glargines at 30 nM (5 U/ml) for 48 hours. Thereafter, the culture supernatant were collected and evaluated for the secretion of different cytokines by multiplex assay. The plotted values represent mean ± SEM (pg/ml) of IL-8 secretion (A) and IL-6 secretion (B) for 12–14 donors. (C) Graphical representation of Pearson correlation analysis using confidence intervals (CI) of IL-8 and IL-6 secretion induced by all insulin glargine products. **, p<0.01; ****, p<0.001; -, No treatment; L+R, LPS+R848; Bas, Basalin; B, Bonglixan; HI, Human Insulin (Insuman). Lot numbers represent the last two digits of the lot numbers shown in Table 1.
Table 2.
Statistical analysis of IL-8 and IL-6 secretion: Glaritus versus Lantus.
Fig 2.
Removal of insulin glargine from Glaritus lots 32 and 96 abrogates innate immune activity induced by these formulations.
Insulin glargines were incubated overnight with the L6B10 anti-insulin mAb and then processed through a 30 kDa filter. (A) Pre- and post-filtration samples were analyzed by a 12% reducing SDS-PAGE gel; the insulin glargine band is observed at 6–10 kDa. (B) Pre- and post-filtration samples were analyzed by 48-hour MIMIC® PTE assay, then multiplex assay for IL-8 production. Data from 19 healthy donors were plotted as % IL-8 secretion over no-treatment control. (C) MIMIC® PTE cultures were treated with preservative and non-preservative insulins at doses of 30, 50, and 500 nM and then examined for IL-8, IL-6, and MCP-1 secretion by multiplex assay. Data is plotted as mean ± SEM (pg/ml) from 8–13 healthy donors. ****, p <0.001.
Fig 3.
The innate response induced by insulin glargine in MIMIC® PTE assays is largely driven by insulin signaling through the IRA/IRB and MEK pathways.
MIMIC® PTE cultures were incubated with the indicated treatments immediately following PBMC application. (S961 is the insulin receptor AB antagonistic peptide.) 1 hour later, 30 nM (5 U/ml) insulin glargines were added to the wells and incubated for 48 hours. Thereafter, the culture supernatants was collected and analyzed for IL-8 secretion by multiplex assay. Data represented as mean ± SEM of 8–12 healthy donors. Lot numbers represent the last two digits of the lot numbers shown in Table 1.
Fig 4.
Glaritus lots 32 and 96 show differential by-product profiles by SEC-HPLC analysis.
(A) 500 ng insulin glargines were analyzed by 3–12% gradient Bis-Tris Native-PAGE under non-reducing conditions followed by silver staining. Molecular markers were used as size standards. Images were taken with a Kodak GL 1500 Imaging system and the insulin glargine band was observed at 6–10 kDa. (B) Representative SEC-HPLC analysis of Glaritus lots 32 and 96 compared with the originator lot 06. Arrows indicate the presence of distinct peaks observed between the originator and non-originator insulin glargines.