Roferon a

Peripheral blood mononuclear cells (MNCs) were obtained by density gradient centrifugation (Ficoll-Paque, Sigma–Aldrich) of heparinized whole blood samples. DCs were generated by culturing the plastic-adherent MNC fraction in 6-well plates (Nunclon, Denmark) in RPMI-1640 medium (Sigma–Aldrich) supplemented with 0.3 mg/ml L-glutamine, 5 mM HEPES buffer, 100 μg/ml gentamicin, and 5% fetal calf serum (FCS, Sigma–Aldrich) in the presence of recombinant human GM-CSF (40 ng/ml, Sigma–Aldrich) and rIFN-α (Roferon-A, 1000 U/ml, Roche, Switzerland) for 4 days at 37°С in a 5% СО₂ atmosphere (IFNα-DCs). The resulting DCs were then stimulated with 10 μg/ml lipopolysaccharide (LPS E. coli 0114: B4, Sigma–Aldrich) as a maturation stimulus for an additional 24 h. In some experiments, DHEAS (Sigma–Aldrich, 10⁻⁶ М) was added to the DC culture along with LPS. The viability of IFN-DCs, as determined by Trypan blue exclusion, was at least 93–95% in all cases.

Resting CD4+ T cells were purified from two healthy blood donors by Ficoll gradient separation followed by negative selection and magnetic separation using the human CD4+ T Cell enrichment kit supplemented with anti-HLA-DR, anti-CD25 and anti-CD69 (Stem Cell Technologies). Cells (10⁶) were incubated for 24 hours, either with medium only, or with 100 or 1000 IU/ml interferon α (Roferon-A, Roche) or interferon γ (R&D Systems), or with 2 μg anti-CD3/1 μg anti-CD28 and 100 U/ml recombinant interleukin-2 (R&D Systems) to mimic TCR stimulation. After 24 hour incubation, total RNA extraction was performed using Illustra RNAspin mini isolation kit (GE Healthcare) and further processed for mRNA-Seq library preparation (TruSeq RNA sample prep kit, Illumina). 100 bp paired-end sequencing was performed using Illumina HiSeq2000. About 140 mio read pairs were obtained for the samples. Sequencing reads were quality filtered before alignment using Cutadapt [56 ] and PrInseq [57 (link)]. Cleaned sequencing reads were aligned to a build genome using SOAPsplice. Gene expression was assessed by determining the number of mapped reads per gene using HTSeq-count tool, followed by DESeq R package normalization. An additional normalization step taking into account the gene coding length was performed so that the resulting counts could be compared across samples for a given gene, and also between genes.

Calu-3 cells were pre-incubated with 1% human serum in the presence or absence of 200–400 IU/ml IFN-α2a (Roferon®-A, Roche) or 20–50 ng/ml IFN-ω (PeproTech). After 24 h, IFN and serum were removed and cells were infected with SARS-CoV-2 at a multiplicity of infection 0.01. Virus inoculum was removed after 1 h, cells were washed with PBS, and 100 µl medium was added per well. Twenty-four hours post-infection, cell culture supernatant was collected for viral RNA quantification by RT-PCR and infectious titer determination by plaque assay.

HEK293T cells were cultured in Arg/Lys-free DMEM, supplemented with light (R0K0), medium (R6K4) or heavy (R10K8) amino acids, as described (33 ). 1 × 10⁷ cells were transfected with 10 μg plasmid DNA using Lipofectamine 2000 (ThermoFisher). After 24 h, media was replaced to contain 1000 U/ml human interferon α-2a (Roferon-A, Roche) for a further 16 h. Cells were harvested in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X100) containing 1:200 Protease Inhibitor Cocktail Set III (Merck) and 1:200 Benzonase nuclease (Sigma-Aldrich). Lysates were normalized to 3 mg/ml of protein before incubation with anti-FLAG-M2 affinity gel (Sigma) at 4°C for 18 h. Beads were washed three times in Tris-buffered saline, then resuspended in 2× SDS-sample buffer (Invitrogen) and boiled for 5 min to elute bound proteins.

Beta-blockers were used as first-line therapy for symptomatic treatment (G1: A, B, D, H, E) of patients with destructive thyrotoxicosis and GD when tachycardia occurred as an adverse reaction to antithyroid drug (ATD) therapy. ATDs, methimazole or propylthiouracil were applied for at least 6 months to achieve remission (G2: I). Radioiodine (RAI) was used after 1 year of ineffective therapy, when ATD therapy was contraindicated (agranulocytosis, thrombocytopenia, transaminasemia) and also in the case of thyroid cancer (G1: H). Substitution therapy with l-thyroxine was administered to compensate for hypothyroidism in HT (subgroup: O; G3) or after RIT (G3: P or subgroup P; G3 (use one)). The patients with IIT received IFN-α-2b (Roferon-A, Hoffman-LaRoche Inc., Basel, Switzerland) or pegylated-IFN-α-2b in a combination with ribavirin (Rebetol; Schering-Plough, Germany) (G1: E), a similar combination which was used in the cases of AM (G1: F, G).