Journal: Clinical Epigenetics

Article Title: The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry

doi: 10.1186/s13148-017-0312-z

Figure Lengend Snippet: RRx-001 induced IFN response through upregulation of type I and III IFN expression and JAK/STAT pathway. Cells were transiently (24 h) treated with 0.5 μM RRx-001 or 0.5 μM 5-AZA and subsequently maintained in drug-free medium for an additional 7 days. RRx-001 induced a significant increase in type I IFN (IFN-β) ( a ) and type III IFN (IL-29/IL-28B) ( b ) secretion into culture medium by HCT 116 cells as measured by ELISA. Transcript levels of IL29 / IL28A were also increased as determined by qPCR ( c ). ISGs ( IFI27 , IFi44 , IFI44L , and IFI6 ) were upregulated by 5-AZA and RRx-001 but blocked by the JAK/STAT inhibitor ruxolitinib (rux) at 2 μM concentration ( d ). Expression of ISGs ( IRF7 , ISG15 , DDX58 , and OASL ) was also upregulated in HCT 116 cells cultured in conditioned medium containing secreted IFNs induced by 5-AZA and RRx-001 as determined by qPCR ( e )

Article Snippet: Levels of type I IFN (IFN-β) and type III IFN (IL-29/IL-28B) were determined using a VeriKine Human IFN-beta ELISA Kit (PBL Assay Science, Piscataway Township, NJ, USA) and a Human IL-29/IL-28B (IFN-lambda 1/3) DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to manufactures’ instructions, respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: Cancers

Article Title: Intravenous Oncolytic Vaccinia Virus Therapy Results in a Differential Immune Response between Cancer Patients.

doi: 10.3390/cancers14092181

Figure Lengend Snippet: Figure 1. Peripheral immune response to Pexa-Vec infusion. (A) Trial schema showing timing of virus infusion and peripheral blood sample collection. Differential IFN (B) and inflammatory cytokine (C) response to Pexa-Vec in Responder (black; n = 4) and Exhausted (white; n = 5) patients. Data is shown as fold-change from baseline (D1 pre); *** q-value < 0.001, **** q-value < 0.0001.

Article Snippet: IFN-β was measured using the VeriKine-HS Human Interferon Beta ELISA Kit for plasma (R&D Systems, Abingdon, UK), as per manufacturer’s instructions.

Techniques: Virus

Journal: Virus research

Article Title: SARS-CoV-2 omicron sub-lineages differentially modulate interferon response in human lung epithelial cells.

doi: 10.1016/j.virusres.2023.199134

Figure Lengend Snippet: Fig. 1. (A) Secreted IFN-β was assessed in Calu-3 cells infected (MOI=0.01) with Wuhan-1 SARS-CoV-2 or different Omicron sub-lineages by enzyme-linked immunoassay (ELISA) at 48h and 72h post-infection (p.i.). Negative control (Ctr-) was represented by uninfected Calu-3 cells. Quantitative evaluation was per formed, based on relative standard curves. Results are reported as mean concentration (pg/ml) ± standard deviations (SD) from at least three independent ex periments (n≥3). (B) The effects of SARS-CoV-2 lineages on IFN-β modulation were evaluated at transcriptional level in Calu-3. Total RNA was purified from mock- infected (Ctr-) or infected (MOI=0.01) cells collected at different times post infection. Specific IFN-β mRNA content was detected by quantitative reverse- transcription polymerase chain reaction (RT-qPCR). RNaseP gene expression was used for relative quantification based on 2-ΔΔCt method. At least three (n≥3) in dependent experiments were performed and representative data are presented as mean values ± standard deviations. With respect to Wuhan-1 reference strain, significance was determined using unpaired, two-tailed Student’s t-test as p<0.0005, ***; p<0.005, **, p<0.05, *, p>0.05, n.s.

Article Snippet: IFN-β quantification was assessed by VeriKine-HS Human IFN Beta TCM ELISA Kit (PBL assay science, NJ, USA), following manufacturer’s instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Negative Control, Concentration Assay, Purification, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression, Quantitative Proteomics, Two Tailed Test

Journal: Virus research

Article Title: SARS-CoV-2 omicron sub-lineages differentially modulate interferon response in human lung epithelial cells.

doi: 10.1016/j.virusres.2023.199134

Figure Lengend Snippet: Fig. 4. Inhibitory activity of SARS-COV-2 ORF6 protein variants. (A) HEK-293T cells were co-transfected with the IFN-β promoter-mediated firefly luciferase (pIFN- β) reporter plasmid in combination with wild-type, Δ61 and the D61L or M58R ORF6 inactive mutant expressing plasmids or with empty vector (Ctr-). At 36h post- transfection, cells were poly(I:C)-stimulated by transfection. Firefly and Renilla luciferase activities were evaluated at 48h post-transfection. Three (n=3) independent experiments were performed. Representative data are presented as mean fold change of relative luminescence unit (RLU) ± standard deviations (SD). (B) The production of IFN-β was tested by enzyme-linked immunoassay (ELISA) in supernatant of wild-type, M58R, D61L or Δ61 ORF6 expressing A549 cells. Negative control (Ctr-) was represented by A549 cells transfected with empty plasmid alone. Quantitative evaluation, based on relative standard curves, was performed. Results are reported as mean concentration (pg/ml) ± standard deviations (SD) from at least three separate experiments (n>3). (C) IFN-β expression was evaluated in A549 cells expressing different ORF6 mutants by specific IFN-β mRNA quantification using quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Ribonu clease P (RNaseP) gene expression was used for relative quantification based on 2-ΔΔCt method. With respect to Wuhan-1 reference strain, significance was deter mined using unpaired, two-tailed Student’s t-test as p<0.0005, ***; p<0.005, **; p<0.05, *; p>0.05, n.s.

Article Snippet: IFN-β quantification was assessed by VeriKine-HS Human IFN Beta TCM ELISA Kit (PBL assay science, NJ, USA), following manufacturer’s instructions.

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Mutagenesis, Expressing, Enzyme-linked Immunosorbent Assay, Negative Control, Concentration Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression, Quantitative Proteomics, Two Tailed Test

Journal: bioRxiv

Article Title: MNS induces antiviral protection and suppresses inflammation

doi: 10.64898/2026.02.11.705318

Figure Lengend Snippet: (A-C) IL1B mRNA (qPCR) and protein levels (ELISA) in RAW 264.7 cells (A), BMDMs (B), and THP1 cells (C) following treatment with DMSO or MNS and infection with indicated viruses. (D-G) Il1b expression (qPCR) and protein levels (ELISA) in liver tissues from C57BL/6J mice under viral challenge and MNS treatment. (H) MNS markedly downregulated the expression of Il1b , Il6 , Tnf α, Ptgs2 , Nos2 mRNA in RAW264.7 cells. (I) MNS significantly reduced the expression of COX2, iNOS, p-IκBα, p-P65 at the protein level. (J) MNS treatment led to a significant reduction in mortality among LPS-challenged mice (n=13). (K) The expression level of Il1b in heart, spleen, lung, kidney, and liver was quantified by RT-qPCR. Values are presented as mean ± SEM. Significance was indicated as follows: N.S., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Mouse cells were assayed with a mouse IL1B ELISA kit (Boster, EK2285), and human primary cells were assayed with a human IL1B ELISA kit (Boster, EK2286), following the manufacturers’ instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Expressing, Quantitative RT-PCR