Journal: Journal of Molecular Histology

Article Title: Interleukin-1 Receptor-Associated Kinase 3 Attenuates Chondrocyte Senescence and Osteoarthritis via Inhibition of the TLR7/9–NF-κB Axis

doi: 10.1007/s10735-026-10804-4

Figure Lengend Snippet: illustrates the role of IRAK3 in modulating TLR7/9 signaling and reducing inflammation in chondrocytes. AT791 is a TLR7 and TLR9 inhibitor. The treatment concentration is 3 µM and the treatment time is 48 h. The TLR9 agonist used is ODN 1826, with a concentration of 1 µg/ml and a treatment time of 48 h. The TLR7 agonist used is Resiquimod, with a concentration of 1 µM and a treatment time of 48 h. Both were purchased from MedChemExpress, HY-124,603, HY-146,245 and HY-13,740. Panels A and B show Western blot analysis of TLR7 and TLR9 expression following IRAK3 knockdown and overexpression, respectively. Panels C through E present ELISA results indicating levels of IL-1β, IL-6, and TNF-α after IRAK3 knockdown. Similarly, panels F through H display ELISA findings for IL-1β, IL-6, and TNF-α levels following IRAK3 overexpression. In panel I, chondrocytes were treated with TLR7/9 inhibitors and agonists, and β-galactosidase staining was performed on senescent chondrocytes for statistical analysis. (* p < 0.05, ** p < 0.01)

Article Snippet: The treatment concentration is 3 μM and the treatment time is 48 h. The TLR9 agonist used is ODN 1826, with a concentration of 1 μg/ml and a treatment time of 48 h. The TLR7 agonist used is Resiquimod, with a concentration of 1 μM and a treatment time of 48 h. Both were purchased from MedChemExpress, HY-124,603, HY-146,245 and HY-13,740.

Techniques: Concentration Assay, Western Blot, Expressing, Knockdown, Over Expression, Enzyme-linked Immunosorbent Assay, Staining

Journal: Vaccine

Article Title: Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release

doi: 10.1016/j.vaccine.2014.02.027

Figure Lengend Snippet: Adjuvant encapsulation in nanoparticles (SVP) results in suppressed TNFα induction in murine cells in vitro. SVP containing TLR7/8 agonist R848 (A, B) or TLR9 agonists PO-CpG 1826 (C) or PS-CpG1826 (D) were added to J774 cells (A, C) or fresh mouse splenocyte cultures (B, D) in parallel with free adjuvants in triplicates. The amount of TNF-α in culture supernatants was measured 6 h (splenocytes) or 16 h (J774) after incubation. Representative results out of two separate experiments with groups of three mice are shown.

Article Snippet: Phosphorothioate (PS) or phosphodiester (PO) forms of CpG-1826 (5′-TCCATGACGTTCCTGACGTT-3′) were purchased either from Enzo Life Sciences or from Oligo Factory (Holliston, MA, USA).

Techniques: In Vitro, Incubation

Journal: Vaccine

Article Title: Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release

doi: 10.1016/j.vaccine.2014.02.027

Figure Lengend Snippet: Higher humoral immunogenicity of SVP-encapsulated antigen coupled with free TLR9 agonist PS-CpG or with SVP-encapsulated TLR9 agonist PO-CpG. Mice (5/group) were immunized (s.c., hind limb) with the indicated SVP or combinations of free antigen and PS-CpG 1826. Antibody titers were determined at times indicated. Mice were immunized either two (A, C) or three (B) times with 2-week intervals between immunizations. OVA (A, B) and prostatic acid phosphatase (PAP) (C) proteins were evaluated as test antigens, either in free form or encapsulated in SVP. In Panel B, the total dose of both free OVA and free PS-CpG 1826 was five times greater than that administered in the SVP-treated groups. In Panel C, the amount of free PAP used was 50 times higher than that of SVP-encapsulated PAP, and the amount of free PS-1826 was two times higher than used in SVP-encapsulated form (C). No adjuvant effect of free PO-1826 was detected (data not shown).

Article Snippet: Phosphorothioate (PS) or phosphodiester (PO) forms of CpG-1826 (5′-TCCATGACGTTCCTGACGTT-3′) were purchased either from Enzo Life Sciences or from Oligo Factory (Holliston, MA, USA).

Techniques:

Journal: Vaccine

Article Title: Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release

doi: 10.1016/j.vaccine.2014.02.027

Figure Lengend Snippet: Nanoparticle encapsulation of both antigen and TLR9 agonist PO-CpG (adjuvant) results in a higher local cellular immune response than utilization of free antigen and adjuvant. Groups of 2–3 mice were injected (s.c., hind limb, once or twice with 2-wk interval) with a combination of SVP-encapsulated OVA and SVP-encapsulated PO-CpG 1826, free OVA admixed with free PS-CpG 1826, or PBS as indicated. The amount of free OVA (50 μg) was ten times higher and the amount of free PS-CpG 1826 (20 μg) was five times higher than that present within SVP (5 and 4 μg, respectively). Tissues were taken at 5 days after the last immunization and restimulated in vitro for 7 days with mitomycin-treated E.G7-OVA cells. LN cells were stained with surface marker antibodies and analyzed by flow cytometry. Top row – popliteal LN cultures (initiated 5 days after a single immunization), bottom row – splenocyte cultures (initiated 5 days after prime-boost immunization). Representative flow cytometry dot plots from two separate experiments are shown. The numbers indicate the percentage of CD8 + T cells that are specific for the OVA SIINFEKL peptide (percent share of CD8 + CD19 − cells).

Article Snippet: Phosphorothioate (PS) or phosphodiester (PO) forms of CpG-1826 (5′-TCCATGACGTTCCTGACGTT-3′) were purchased either from Enzo Life Sciences or from Oligo Factory (Holliston, MA, USA).

Techniques: Injection, In Vitro, Staining, Marker, Flow Cytometry

Journal: Vaccine

Article Title: Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release

doi: 10.1016/j.vaccine.2014.02.027

Figure Lengend Snippet: Focused local cytokine induction by SVP-encapsulated, but not free TLR9 agonist CpG. Mice were injected s.c. in a single hind limb with either SVP-PS-CpG 1826 or free PS-CpG 1826. At the times indicated, popliteal LNs from the injection side (ipsilateral, I) and from the opposite side (contralateral, C) were taken and incubated in vitro overnight. Culture supernatants were collected and analyzed for cytokine presence by ELISA. A – IFN-γ, B – IL-12(p40), C – IL-1β. Average of two mice per group per time-point is shown.

Article Snippet: Phosphorothioate (PS) or phosphodiester (PO) forms of CpG-1826 (5′-TCCATGACGTTCCTGACGTT-3′) were purchased either from Enzo Life Sciences or from Oligo Factory (Holliston, MA, USA).

Techniques: Injection, Incubation, In Vitro, Enzyme-linked Immunosorbent Assay