PBMC (0.2×106) from neonates and their mothers were stimulated in 96-well flat-bottomed microtitre plates (Nunc, Denmark) with 0.1% (v/v) plasma, consisting of a pool of Ro/SSA and La/SSB positive plasma from three neonates and three mothers, respectively. The cell cultures were supplemented with 500 U/mL IntronA (Schering-Plough, Bloomfield, New Jersey, USA) and supernatant from necrotic cells prepared by freeze-thaw cycles as previously described,22 (link) and incubated for 20 hours at 37°C with 5% CO2. IFNα in the supernatants was analysed by DELFIA immunoassay.21 (link)
Intron a
Manufactured by Merck & Co
Sourced in United States
Intron A is a laboratory product manufactured by Merck & Co. It is a recombinant form of human interferon alfa-2b, a protein involved in the immune system's response to viral infections and other biological processes. The core function of Intron A is to provide a standardized source of this protein for use in research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Immunikin, by Boehringer Ingelheim (3 mentions)
96 well f plates, by Corning (1 mentions)
Imukin, by Boehringer Ingelheim (1 mentions)
Fluorokinemap, by Bio-Rad (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Il 12, by R&D Systems (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Boehringer Ingelheim (1 mentions)
Dulbecco s modified, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Montanide isa 51, by Seppic (1 mentions)
Lyse fix buffer, by BD (1 mentions)
Lsrfortessa x 20 instrument, by BD (1 mentions)
Stain buffer, by BD (1 mentions)
Perm buffer 2, by BD (1 mentions)
9 protocols using intron a
1
Neonatal Immune Response to Autoantibodies
2
Interferon-Induced Antiviral Assays
3
Cytokine Release from Whole Blood Cells
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Whole blood was diluted 1:5 in RPMI into 96-well F plates (Corning) and activated by single stimulation with IL-12 (20 ng ml−1; R&D Systems), PHA (10 μg ml−1; Sigma-Aldrich), LPS (1 μg ml−1) List Biochemicals, IFN-γ (2 × 10 IU ml−1, Imukin, Boehringer Ingelheim), IFN-α (2 × 103 IU ml−1, Intron A, Schering Plough, UK) or using co-stimulations as indicated. Supernatants were taken at 24 h. Cytokines were measured using standard ELISA according to the manufacturer's recommendations (IFN-γ, Pelikine, Sanquin, NL), or multiplexed (TNFα, IL-12, IL-10, IL-6, R+D Systems Fluorokinemap) on a Luminex analyser (Bio-Plex, Bio-Rad, UK). Data were statistically analysed by the two-tailed Mann–Whitney test using Prism 6 (GraphPad Software).
4
Dermal Fibroblast Immunoblotting Protocol
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Dermal fibroblasts from patient II:3 and three healthy controls were obtained by standard methods and cultured in Dulbecco’s Modified Eagle’s Medium supplemented by 10% foetal calf serum and 1% penicillin/streptomycin (DMEM-10). Human recombinant IFNα2b (Intron-A, Schering-Plough, USA) and IFNγ (Immunikin, Boehringer Ingelheim, Germany) were used at 1000 IU/ml. Immunoblotting was carried out as previously described (20 (link)). Antibodies used are described in Table 1
5
Vero Cell Culture and Cytokine/Inhibitor Treatments
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Vero cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% foetal calf serum (FCS; Gibco), 1% penicillin/streptomycin (Gibco) and 1% L-glutamine (Gibco). All cells were incubated in a humidified atmosphere with 5% CO2 at 37°C. Cytokines/inhibitors were used at the following concentrations: human recombinant IFN-α2b (1000 IU/ml; Intron A, Schering-Plough, USA); IFN-γ (1000 IU/ml; Immunikin, Boehringer Ingelheim, Germany) and Ruxolitinib (10 μM; Calbiochem, USA).
6
Peptide Vaccine for Melanoma Patients
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Thirty-four HLA-A*0201, stage III/IV melanoma patients were enrolled in a single-center, open-label, randomized phase II study after tumor resection (EudraCT number 2008-008211-26). The clinical-pathological characteristics of the patient cohort, the study design, the primary and secondary objectives, the inclusion/exclusion criteria, the randomization method, the treatment and clinical follow-up are detailed in (15 (link)). Briefly, 17 patients (arm 1) received and intradermal injection of Melan-A/MART-126-35 (A27L) (ELAGIGILTV) and NY-ESO-1157-165 (SLLMWITQC) GMP-grade peptides (Polypeptide Laboratories, Strasbourg, France) emulsified with Montanide ISA-51 (Seppic, Italy) in combination with subcutaneous injection of 6 MU IFN-α2b (IntronA®, Schering-Plough, USA). The immunization regimen for patients in arm 1 (n=17) consisted of 6 cycles (every 21 days) of two vaccine doses (7 days apart). Patients in arm 2 (n=17) received the same treatment of arm 1, preceded (1 day before each vaccination cycle) by an intravenous infusion of 800 mg/m2 dacarbazine (Deticene by Sanofi-Aventis, France).
7
Reduced-Dose BCG and IFN for Bladder Cancer
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As previously reported, treatment commenced 3–8 weeks after either transurethral resection of bladder tumor (TURBT) or confirmatory cystoscopy, biopsy, or positive cytology. BCG failure patients were administered 1/3 standard dose of either TICE (Organon Teknika, Roseland, NJ) or Connaught (Aventis-Pasteur, Swiftwater, PA) strain BCG per physician preference mixed directly with 50 million units (MU) IFN (Intron A, Schering-Plough, Kenilworth, NJ). Patients who experienced toxicity during induction were moved onto a dose-reduced protocol (66% reduction with identical timing) after a 2-week rest period. Additional 2-week treatment delays were allowed for repeat episodes of intolerance, as long as the entire induction cycle was completed within 10 weeks of initiation. All patients without further recurrence then received reduced dose maintenance therapy consisting of 3 “mini cycles” (3 weekly BCG instillations) at 3, 9, and 15 months after the end of the induction cycle. Patients began bladder surveillance 4–6 weeks after induction and had repeat assessments every 3 months for 2 years.
8
IFNα/γ-induced STAT1 phosphorylation
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Fresh whole blood cells were left unstimulated or stimulated with 10 ng/ml of recombinant IFNα2β (Intron A, Schering-Plough) or IFNγ (BD Pharmigen) for 10 min at 37°C. Anti-CD3 and anti-CD14 (all from Becton Dickinson) staining was performed for 20 min at 4°C. Cells where then fixed with Lyse/Fix Buffer 10 min at 37°C and further incubated for 10 min at RT with FcBlock (1:200) in Stain Buffer (all from Becton Dickinson). After permeabilization with Perm Buffer II (BD PhosFlow), samples were stained with antibodies against phosphorylated Tyrosine (701) STAT1 (pSTAT1) or isotype control antibody for 20 min at 4°C. Monocytes were gated based on CD14 expression; NK cells were gated based on CD3–CD56dimCD16+ subset. All antibodies were purchased from BD Biosciences. Samples were run on a BD LSRFortessa X−20 instrument (BD Biosciences) and data were analyzed with FlowJo software, version 8.3 (Tree Star).
9
Antiviral Combination for Viral Inhibition
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Ribavirin (ICN Pharmaceuticals, Quebec) and interferon alpha 2b (IFN; Intron-A Ò , Schering-Plough, Kenilworth, NJ) were used to inhibit viral replication. Ribavirin (100 lM) and interferon (1000 U/ml) were added to the differentiation medium starting at the time of the inoculation until the end of the infection experiment.
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