Amphotericin b

RK13 cells were cultivated in MEM with Earle’s salts (Eurobio) complemented with 10% foetal bovine serum (Eurobio), 1% L-glutamine (Eurobio), 100 IU/mL penicillin, 0.1 mg/mL streptomycin and 0.25 μg/mL amphotericin B (Eurobio) at 37 °C and 5% CO₂. Subsequently, 1 × 10⁶ PBMCs were seeded on RK13 monolayer cells on 6-well plates and incubated at 37 °C for 72 h. The presence of viral cytopathic effects was observed using an inverted microscope, and plates were frozen at −80 °C for viral quantitation. Eight tenfold serial dilutions of swab samples were inoculated in triplicate on RK13 monolayer cells on 96-well plates and incubated at 37 °C for 7 days. Wells with cytopathic effects were recorded, and the TCID₅₀ of each sample was determined using the Spearman–Kärber method [54 (link)].

Synoviocytes were grown from synovial tissue samples obtained from RA patients undergoing joint surgery. The patients with RA fulfilled the American College of Rheumatology criteria for RA [20 (link)]. Each individual signed an informed consent form, and the protocol was approved by the committee for the protection of persons participating in biomedical research of the Hospital of Lyon, in compliance with the Helsinki Declaration. Briefly, synovial tissue was minced in small pieces which were allowed to fix on plastic plates. Those samples were maintained in Dulbecco’s modified Eagle’s medium (Eurobio, Courtaboeuf, France) supplemented with 10 % fetal bovine serum (Life Technologies, part of Thermo Fisher Scientific, Carlsbad, CA, USA), 2 % Penicillin-Streptomycin (Eurobio), 1 % L-glutamine (Eurobio), and 1 % Amphotericin B (Eurobio) until cells grew out of the tissue and colonized the plastic dishes. After cells reached confluence, tissue pieces were removed and cells were trypsinized. Synoviocytes were used between passages 4 and 9. Each independent experiment was performed with a different batch of synoviocytes isolated from different RA donors.

Cell culture media, cell transfection opti-MEM (1×) medium, epithelial growth factor (PHG0311), fetal bovine serum (FBS), insulin transferrin selenium, Pierce BCA Protein Assay Kit (23225), Lipofectamine 3000 Transfection Reagent (L3000008), and Lipofectamine RNAiMAX Transfection Reagent (13778150) were obtained from ThermoFisher Scientific (Illkirch-Graffenstaden, France). Antibiotics (streptomycin, penicillin, and amphotericin B) and glutamine were purchased from Eurobio Scientific (Les Ulis, France). Dimethyl sulfoxide (DMSO), HMGB1 (SRP6265, a mixture of different forms), and Fibronectin (F1141) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Human S100A4 (4137-S4), S100A8 (9876-S8), S100A9 (9254-S9), and MCP1 (279-MC-010/CF) recombinant proteins were obtained from R&D systems (Noyal-Châtillon-sur-Seiche, France). RAP (RAGE inhibitor, 553031) and TAK-242 (TLR4 inhibitor, 243984-11-4) were purchased from Merck (Saint-Quentin-Fallavier, France). LightCycler 480 SYBR Green I Master (04887352001) was provided by Roche (Meylan, France).

Equine dermal fibroblasts (E. Derm, NBL-6 CCL-57™; ATCC^®, Manassas, VA, USA) were maintained in Eagle’s Minimum Essential Medium (EMEM; ATCC^®, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS) (Eurobio, Courtaboeuf, France), 100 UI/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B (Eurobio) and cultivated at 37 °C and 5% CO₂. Cells were seeded at 1.2 × 10⁴ cells/well in 96-well plates.
The EHV-4 405/76 strain (VR-2230™; ATCC^®) was used as the reference strain for the screening of the antiviral effect of compounds at an MOI of 0.23 on E. Derm cells.

Equine articular chondrocytes (eACs) were isolated from biopsies of healthy equine anterior carpus on young horses (4–6 years) as previously described [37 (link)]. eACs were cultured in high-glucose DMEM (HG-DMEM, 4.5 g/L BioWest, Nuaillé, France) supplemented with 10% of fetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL of erythromycin, and 0.25 mg/mL of amphotericin B (Eurobio Scientific, Courtaboeuf, France) at 37 °C in a humidified atmosphere containing 5% CO₂. During expansion, the medium was changed twice a week. For cell passages, at 80% confluency, chondrocytes were harvested by trypsinization with trypsin–ethylenediaminetetraacetic acid (EDTA) (Eurobio Scientific, Courtaboeuf, France), counted with trypan blue to evaluate viability, and seeded at 2 × 10⁴ cells/cm².

Mouse fibroblasts of the L929 cell line (Sigma-Aldrich) were used to evaluate the cytotoxicity of the different CS and COS molecules. Cells were cultured in culture flasks in the complete culture medium Dulbecco’s Modified Eagle’s Medium (DMEM, Eurobio Scientific) supplemented with 10% fetal calf serum (Eurobio Scientific, Les Ulis, France), 1% penicillin/streptomycin (Eurobio Scientific), 1% L-glutamine (Eurobio Scientific) and 0.1% amphotericin B (Eurobio Scientific). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. After 7 days of culture, cells were harvested at approximately 80% confluence by treatment with 0.5 g/L trypsin–0.2 g/L EDTA (Eurobio Scientific). Cell cultures were tested by PCR to determine the absence of mycoplasma.

Biopsies of synovial tissue samples have been aseptically isolated from RA and OA patients’ joints. The study was approved by the ethics committee of the hospitals of Lyon and patients signed a written informed consent form. The RA patients fulfilled the American College of Rheumatology criteria for RA (18). The synovial tissue was minced into small pieces, which were allowed to adhere to plastic plates. Those samples were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Eurobio, Courtaboeuf, FR) supplemented with 10% FBS (Life Technologies by Thermo Fischer scientific, Grand Island, NY, USA), 2% Penicillin-Streptomycin, 1% L-glutamine and 1% Amphotericin B (all Eurobio) until cells colonized the plastic dishes. At 90% confluence, tissue pieces were removed and cells were trypsinized. Synoviocytes were used between the fourth and ninth passages to ensure the cell specificity. All data are the results of at least three separate experiments (n = 3) using cells taken from three different patients for each type (OA and RA).