Journal: Investigative Ophthalmology & Visual Science

Article Title: Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa -Induced Keratitis

doi: 10.1167/iovs.16-19134

Figure Lengend Snippet: MicroRNA-183/96/182 is expressed in mouse Mϕ and PMNs ( A , B ) and in human PBMCs and PMNs ( C , D ). It is inactivated in the miR-183/96/182 ko mice ( A , B ). n = 3 for each group. **** P < 0.0001.

Article Snippet: Human PMNs and PBMCs were purchased from Zen-Bio, Inc. (Research Triangle Park, NC, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Cutaneous kinase activity correlates with treatment outcomes following PI3K delta inhibition in mice with experimental pemphigoid diseases

doi: 10.3389/fimmu.2022.865241

Figure Lengend Snippet: Parsaclisib impairs immune complex-induced neutrophil activation in vitro . Human polymorphonuclear leukocytes (PMNs) were isolated from healthy blood donors and activated by immobilized immune complexes (IC) consisting of recombinant human COL7 (hCOL7E-F) and anti-hCOL7E-F IgG1 in the presence or absence of parsaclisib at an 8-fold concentration range. Relative reactive oxygen species (ROS) release was detected by a luminescence-based assay. (A) Representative example of one donor showing the relative luminescence after IC-stimulation over the time. (B) Area under the curve (AUC, cumulative values) of luminescence, n=12. (C) PMNs were isolated from murine femurs and activated using immobilized ICs consisting of recombinant murine COL7 (mCOL7 C ) and anti-mCOL7 C -IgG in the presence or absence of parsaclisib, n=4. (D) ROS were generated enzymatically by myeloperoxidase, catalase, and glucose oxidase, n=4. The ROS scavenger N-acetylcysteine (NAC) was used as assay control. (E) Chemotaxis of freshly isolated PMNs was induced by IL-8 in the presence of parsaclisib using a Boyden chamber assay. The attracted cell number during a time period of 60 minutes is shown as AUC. (F) Human PMNs were stimulated with immobilized ICs in the presence/absence of parsaclisib. To exclude toxicity, the amount of propidium iodide (PI)- and Annexin V-positive cells after IC stimulation was identified. (G) Cryosections of human skin were incubated with IgG isolated from EBA patients, followed by the addition of PMNs, isolated from healthy donors (cryosection assay). Split formation along the dermal epidermal junction (DEJ, dotted line) was analyzed as percentage of the whole DEJ. Asterisks indicate split formation. (A-F) Data was normalized to positive control (either IC- or IL-8-stimulated PMNs). Solvent control was always added to positive and negative controls. (B-G) Data are shown as Tukey’s box-and-whisker plots. ANOVA on ranks (Kruskal-Wallis) was applied followed by a Dunn´s multiple comparison test, (B) n=12, (C) n=2-4, (D, F) n=4, (E) n=5, (G) n=6 (for detailed information see attached raw data table), *p<0.05, **p<0.01.

Article Snippet: Following published protocols ( , ), cryosections of human skin, that was obtained from elective plastic surgery, were incubated with immunoapheresis materials or IgG isolated from serum of two EBA patients using protein G columns, of EBA patients for 1 h at 37°C, followed by addition of human PMNs isolated by dextran sedimentation (Carl Roth, Karlsruhe, Germany) of freshly collected, heparinized blood from healthy volunteers (n=3).

Techniques: Activation Assay, In Vitro, Isolation, Recombinant, Concentration Assay, Luminescence Assay, Generated, Control, Chemotaxis Assay, Boyden Chamber Assay, Incubation, Cryosection Assay, Positive Control, Solvent, Whisker Assay, Comparison