Journal: PLoS ONE

Article Title: Dobrava-Belgrade Hantavirus from Germany Shows Receptor Usage and Innate Immunity Induction Consistent with the Pathogenicity of the Virus in Humans

doi: 10.1371/journal.pone.0035587

Figure Lengend Snippet: A ) 100 µl of indicated Vero E6-derived virus stocks or medium (NC, negative control) were exposed to UV irradiation. Amount of IFN-λ1 present in virus stocks was measured by ELISA. Abbreviation “ucf” means that the virus stock was purified by the ultracentrifugation procedure. Data are presented as the mean between three independently prepared stocks ± SD from the mean B ) GRW/Aa virus stock (∼50 µl) or recombinant proteins (rhIFN-λ1 or rhIFN-β) were preincubated with 1 µg of corresponding blocking antibody (anti-IFN-λ1 or anti-IFN-β). One hour after incubation the mixture was added on the top of A549 cells. Four days post infection MxA mRNA expression was measured by qPCR. NC (negative control), untreated A549 cells. Experiment was performed three times. Data are presented as the mean ± SD of the mean.

Article Snippet: The amount of IFN-λ1 was measured by ELISA using the human IL29 DuoSet ELISA Development System (R&D Systems, DY1598), following manufacturer instructions and modifications from recently described protocol .

Techniques: Derivative Assay, Virus, Negative Control, Irradiation, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Blocking Assay, Incubation, Infection, Expressing

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: Production of IFN-α/β, IFN-λ, and TNF-α after Stimulation with Viruses in IRAK-4-Deficient Blood Cells PBMCs from controls and/or from IRAK-4-deficient patients P2, P3, or P7 were left unstimulated or stimulated with various intact and UV-inactivated viruses. (A) IFN-β mRNA levels were analyzed by reverse transcription and real-time quantitative PCR 24 hr after stimulation with intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for the controls from three independent individuals, each tested once. The patients were each tested once. (B and C) IFN-α secretion (B) and IFN-λ secretion (C) were measured by ELISA after 24 hr of stimulation. Means and standard deviations (SD) were calculated for the controls from 14 independent individuals, each tested once. (D) IFN-α secretion by control PBMCs was measured by ELISA after 24 hr of stimulation by intact or UV-inactivated viruses. Means and standard deviations (SD) were calculated for the controls from seven independent individuals, each tested once. (E) TNF-α secretion was measured by ELISA after 24 hr of stimulation by two intact viruses. Means and standard deviations (SD) for controls were calculated from two independent individuals.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: IFN-β, IFN-λ, and IL-6 Induction in IRAK-4-Deficient Fibroblasts upon Stimulation by Poly(I:C), IL-1β, TNF-α, and Viruses Induction of IFN-β, IFN-λ, and IL-6 in response to poly(I:C) (50 μg/ml), IL-1β (20 ng/ml), TNF-α (10 ng/ml), and viral stimulations in IRAK-4-deficient (P1, P2, or P4) and control fibroblasts. (A and B) IFN-β mRNA induction, as determined by Q-PCR 2 hr after stimulation (A) and IFN-β, as determined by ELISA 24 hr after stimulation (B). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (C) IFN-β and IFN-λ as determined by ELISA 24 hr after stimulation by poly(I:C), following prior treatment with IFN-α2b (10 5 U/ml for 12 hr). This experiment is representative of two independent experiments. (D) IL-6, as determined by ELISA 24 hr after stimulation. Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (E) IFN-β mRNA induction 24 hr after stimulation by seven intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Infection