Journal: International Journal of Molecular Sciences

Article Title: Are There Differences in Inflammatory and Fibrotic Pathways between IPAF, CTD-ILDs, and IIPs? A Single-Center Pilot Study

doi: 10.3390/ijms232315205

Figure Lengend Snippet: Estimated marginal means for TGF-β1 by group membership.

Article Snippet: Below we present names of the assays and their detection limits: Interleukin 8 (IL-8) ELISA kit RD194558200R, BioVendor-Laboratorní medicína a.s. (Brno, Czech Republic): 12 pg/mL; Human TGF-β1 ELISA kit 650.010.096, Diaclone SAS (Besançon, France): 8.6 pg/mL; Human Surfactant Protein D ELISA kit RD194059101, BioVendor-Laboratorní medicína a.s. (Brno, Czech Republic): 0.1 ng/mL; Surfactant-Associated Protein A (SPA) ELISA kit SEA890Hu, Cloud-Clone Corp (Wuhan, China): 46.88 pg/mL; Enzyme-Linked Immunosorbent Assay Kit Chemokine (C-X-C motif) Ligand 1 (CXCL1) SEA041Hu, Cloud-Clone Corp (Wuhan, China): 6.1 pg/mL.

Techniques:

Journal:

Article Title: TGF-?-dependent suppressive function of Tregs requires wild-type levels of CD18 in a mouse model of psoriasis

doi: 10.1172/JCI34916

Figure Lengend Snippet: (A) Representative pictures of cluster formation of allogeneic DCs with Tregs derived from either Cd18wt mice or Cd18hypo mice are shown. Original magnification, ×40. (B) Cluster formation between allogeneic DCs and Tregs of different genotypes from Cd18wt mice and Cd18hypo mice was assessed by counting aggregated clusters/HPF in 100 randomly selected HPFs. Cluster formation with allogeneic DCs was substantially reduced for Tregs derived from Cd18hypo mice compared with Tregs from Cd18wt control mice. **P = 0.0029, using Student’s t test. (C) Increased neutralizing mAb against CD18 resulted in decreased proliferative response of specific allogeneic Tregs in MLRs. Numbers on the top left of C and F indicate the percentage of CFSE-labeled proliferating cells. Numbers on the top right of C and F indicate the percentage of undivided CFSE-labeled cells. (D) Increased TGF-β1 expression by Cd18wt Tregs was observed in MLRs. Neutralizing mAb against CD18 in MLRs resulted in a dramatic decrease in TGF-β1 expression compared with isotype-matched control antibody. Gray region, TGF-β1 expression; white region, normal goat IgG control for TGF-β1 staining. Numbers on the top of D indicate the percentage of CFSE-labeled proliferating cells. CD4+CD25+CD127– Tregs were purified from 4 pooled spleens of Cd18wt PL/J mice and cocultured with irradiated allogeneic DCs in the presence of 500 units/ml recombinant murine IL-2 and various concentrations of anti-CD18 mAb (E), or anti-mouse CD11a mAb (F), or isotype-matched IgG for 7 days. Tregs were then separated from allogeneic DCs by CD11c MACS beads, extensively washed 3 times with PBS, and mixed at a ratio of 1:4 with Cd18wt Tresp cells. After 3 days of culture, cells were harvested and analyzed by flow cytometry. One representative experiment out of 3 or 4 independent experiments is shown.

Article Snippet: Two hundred and fifty micrograms of TGF-β neutralizing mAb (clone 1D11; BioExpress) or isotype control IgG was injected intraperitoneally into recipient mice 1 day after a transfer of 1 × 10 6 Cd18 wt Tregs for 3 consecutive days.

Techniques: Derivative Assay, Control, Labeling, Expressing, Staining, Purification, Irradiation, Recombinant, Flow Cytometry

Journal:

Article Title: TGF-?-dependent suppressive function of Tregs requires wild-type levels of CD18 in a mouse model of psoriasis

doi: 10.1172/JCI34916

Figure Lengend Snippet: (A) Cryosections from affected Cd18hypo mice were double stained with CD25-FITC and TGF-β1–Cy3 mAbs. Original magnification, ×20. (B) FACS analysis of pooled DLNs from affected Cd18hypo mice using TGF-β1 and CD25 mAbs. (C and D) Seven days after adoptive transfer of MACS-sorted Cd18wt Tregs into affected Cd18hypo mice, skin sections were stained with antibody against TGF-β1 and CD18. The overlay (yellow) of CD18-positive Cd18wt Tregs (green) and TGF-β1–expressing cells (red) indicate that most of the Cd18wt Tregs express TGF-β1 in the skin (C) and skin DLNs (D) after transfer into Cd18hypo mice. The dotted line indicates the border between epidermis and dermis. Three independent experiments were performed in total. (E) A total of 1 × 106 Tregs from either Cd18wt or Cd18hypo mice were labeled with CFSE and adoptively transferred into affected Cd18hypo mice. At day 4 after adoptive Tregs transfer, FACS analysis was performed to measure TGF-β1 expression of CFSE-labeled Cd18wt Tregs (left panel) or Cd18hypo Tregs (right panel) from skin DLNs of affected recipients. Gray region, TGF-β1 expression; white region, normal goat IgG control for TGF-β1 staining. Numbers on the top of B and E indicate the percentage of CFSE-labeled proliferating cells. (F) Following adaptive transfer of 1 × 106 Cd18wt Tregs, 250-μg TGF-β1–neutralizing mAb (left panel) or isotype control IgG (right panel) were injected intraperitoneally into affected Cd18hypo recipients. Repetitive injection of TGF-β neutralizing antibody or isotype control IgG after adoptive transfer of Cd18wt Tregs was performed till the end of treatment (21 days). Original magnification, ×40 (C and D). **P = 0.002, using Student’s t test.

Article Snippet: Two hundred and fifty micrograms of TGF-β neutralizing mAb (clone 1D11; BioExpress) or isotype control IgG was injected intraperitoneally into recipient mice 1 day after a transfer of 1 × 10 6 Cd18 wt Tregs for 3 consecutive days.

Techniques: Staining, Adoptive Transfer Assay, Expressing, Labeling, Control, Injection