Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case

Figure 8

In contrast to impure mu rSAA3, RP-HPLC-purified mu rSAA3 (mu rSAA3_pur) does not chemoattract neutrophils in vivo. (a) Female NMRI mice were injected i.p. with 100 μl of PBS (Co), mu rSAA3 (10 or 100 ng) or RP-HPLC-purified mu rSAA3_pur (10 or 100 ng; 3-5 mice per group). After 2 h, mice were sacrificed and peritoneal lavages were performed. Total cell counts in peritoneal lavages were determined and the percentage of neutrophils (Ly-6G⁺CD11b⁺) was quantified via flow cytometry. (b) Female NMRI mice were injected i.p. with 100 μl of PBS (Co) or LPS (2 or 10 pg; 4-8 mice per group). After 2 h, mice were sacrificed and peritoneal lavages were performed. Total cell counts in peritoneal lavages were determined, and cytospins were prepared for differential leukocyte counts by 2 individuals independently. (a and b) Data represent the mean percentage (black bars) or absolute (hatched bars) of derived from 1 (a) or 1-3 (b) independent experiment(s). Statistically significant recruitment of neutrophils compared to PBS-injected mice, determined by the Mann-Whitney test, is indicated by asterisks (; ). (c) Human CD14⁺ monocytes derived from buffy coats from healthy individuals were stimulated with LPS (500 ng/ml), mu rSAA3 (10 or 100 ng/ml), mu rSAA3_pur (10 or 100 ng/ml) or were left untreated (Co). After 2 h, supernatants were collected and levels of CXCL8 were determined via a specific ELISA. Data represent the mean production of derived from 4 independent experiments. Statistically significant differences from untreated control cells, determined by the Mann-Whitney test, are indicated by asterisks ().

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