Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: HAL hepatotoxicity and the induced inflammatory response depend on ovarian hormones. (A) For each mouse, the stage of estrous cycle was determined by vaginal cytology analysis before treatment with HAL (5 mmol/kg, ip). Plasma ALT activity was measured 12 h after HAL administration (n = 3–5 per group). P, proestrus; E/M, estrus/metestrus; D, diestrus. *significantly different from other groups. (B) Plasma ALT activity was evaluated 12 h after vehicle (VEH) or HAL (15 mmol/kg, ip) administration in OVX or SHAM mice (n = 3–5 per group). *significantly different from respective VEH control; #significantly different from HAL-treated SHAM mice. (C) Plasma IFN-γ concentration was evaluated in at various times after HAL administration (n = 5–6 per group). #significantly different from time-matched male group; *significantly different from sex-matched 6 h group. (D) IFN-γ concentration was evaluated 12 h after HAL treatment in SHAM and OVX mice (n = 4 per group). *significantly different from SHAM group.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Activity Assay, Concentration Assay

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: IFN-γ KO mice are protected from developing severe HAL hepatotoxicity. Female WT BALB/cJ (WT) and IFN-γ KO mice were treated with HAL (15 mmol/kg, ip), and plasma and liver samples were collected at various times. (A) Plasma ALT activity was evaluated 8 and 12 h after HAL treatment (n = 5–6 per group). *significantly different from HAL-treated WT mice. (B) Immunoblot detection of TFA protein adducts in liver homogenates (n = 3 per group). (C) Hematoxylin and eosin liver sections from HAL-treated WT and IFN-γ KO mice 30 h after treatment. Labeled in picture are central vein (CV) and portal triad (PT). Images were photographed at ×200 magnification.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Activity Assay, Western Blot, Labeling

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: HMGB-1 release and the response to HAL in Tlr4Lps-d mice. (A) Plasma HMGB-1 concentration was evaluated at various times after HAL treatment (15 mmol/kg, ip) in male and female mice (n = 6 per group). VEH-treated animals had plasma HMGB1 concentrations < 5 pg/ml. #significantly different from sex-matched 6 h time point. *significantly different from time-matched male and all other female groups. (B and C) Female WT BALB/cBYJ (WT) mice and Tlr4Lps-d mice were treated with HAL (15 mmol/kg, ip). Plasma ALT activity and IFN-γ concentration were evaluated 24 h after HAL treatment (n = 4–5 per group). *significantly different from WT controls.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Concentration Assay, Activity Assay

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: KC-depleted mice and CD1d KO and RAGNULL mice develop severe HAL-induced liver injury. Control- or clodronate liposome-pretreated mice were given VEH or HAL (15 mmol/kg, ip), and plasma and liver samples were collected 24 h later. Plasma ALT activity (A) and IFN-γ concentration (B) were evaluated (n = 4–6 per group). WT BALB/cJ (WT), NKT-deficient mice (CD1d KO), or T- and B cell–deficient mice (RAGNULL) were treated with HAL (15 mmol/kg, ip). (C) Plasma ALT activity was evaluated in WT and CD1d KO mice 12 and 24 h after HAL administration (n = 5 per group). (D) Plasma ALT activity was evaluated in HAL-treated WT and RAGNULL mice 12 h after HAL administration (n = 5 per group). (E and F) Hematoxylin and eosin liver sections taken 24 h after HAL treatment of CD1d KO and RAGNULL mice. Labeled in picture are central vein (CV) and portal triad (PT). Arrowheads identify areas of necrosis. Images were photographed at ×200X magnification.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Activity Assay, Concentration Assay, Labeling

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: HAL-induced hepatotoxicity depends on NK cell activity. Mice treated with IgG or anti-AsGM1 were given HAL (15 mmol/kg, ip) as described in Methods section, and plasma samples were collected at 12 and 24 h. Plasma ALT activity (A) and IFN-γ concentration (B) (n = 4–6 per group). *significantly different from time-matched controls. Plasma ALT activity (C) and IFN-γ concentration (D) in WT and BALBPrf1 mice 12 h after HAL administration (n = 4 per group). *significantly different from WT mice.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Activity Assay, Concentration Assay

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: Proposed mechanism of innate immune-mediated severe HAL-induced liver injury. (A) In the absence of stress stimulation, self-proteins such as H2Dd are expressed on the plasma membrane of hepatocytes and RAE-1 is not; this condition keeps NK cells quiescent. (B) When hepatocytes are exposed to HAL, intracellular TFA adducts form (1). This induces a stress response in hepatocytes (2) that alters the surface NK receptor ligands (3) and activates NK cells. Activated NK cells release IFN-γ as well as the contents of cytotoxic granules (4), such as perforin and granzyme B, which contribute to hepatocellular necrosis (5). Damaged hepatocytes release endogenous danger signals, such as HMGB-1 (6). These endogenous danger signals are ligands for TLR4 (7), and the resultant signals are involved in a positive feedback loop that further activates NK cells as well as recruits polymorphonuclear leukocytes (PMNs) (Scaffidi et al., 2002) that participate (8) in the progression of injury (Dugan et al., 2010).

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques:

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: Nature Microbiology

Article Title: Evolution of enhanced innate immune suppression by SARS-CoV-2 Omicron subvariants

doi: 10.1038/s41564-023-01588-4

Figure Lengend Snippet: a – g , Calu-3 infection with 2,000 E copies per cell of Delta (yellow, Ο), BA.1 (blue, Ο), BA.2 (blue, Δ), BA.4 (purple, O) and BA.5 (purple, Δ), n = 3: mean viral E copies at 2 h.p.i. across three independent experiments ( a ); viral replication over time measured by RT–qPCR for intracellular E copies per microgram RNA ( b ); infection levels measured by nucleocapsid expression (% N+ by flow cytometry) ( c ); expression of IFNB , CXCL10 , IFIT1 , IFIT2 , RSAD2 , MX1 , MX2 and DDX58 in infected cells over time ( d ); IFNβ ( e ) and CXCL10 ( f ) secretion from infected Calu-3 cells measured by ELISA at 48 h.p.i.; rescue of viral replication by JAK1-inhibitor ruxolitinib in Calu-3 cells at 48 h.p.i., where relative infection levels are shown across three independent experiments determined by E copies per microgram RNA normalized to the median infection level of the untreated control ( g ). h – k , Primary bronchial HAEs were infected with the indicated variants at 1,500 E copies per cell: viral replication measured by intracellular E copies at 72 h.p.i. ( h ) and viral release into apical washes over time ( i ), with three biological replicates shown; expression of IFNB , CXCL10 , IFIT1 , IFIT2 , DDX58 and RSAD2 in HAEs at 72 h.p.i., with six biological replicates shown ( j ); intracellular viral E copies in HAEs in the presence or absence of 5 μM ruxolitinib at 72 h.p.i., with three biological replicates shown ( k ). For a , one-way analysis of variance (ANOVA) with Bonferroni post-test was used. n.s., not significant at P > 0.05 for all comparisons. For b – h and j , one-way ANOVA and Dunnett’s post-test were used. For i , two-way ANOVA with a Bonferroni post-test was used. For k , one-tailed unpaired Student’s t -test was used. Replicate measurements from one of three independent experiments. Fold change over mock is shown. Mean ± s.e.m. or individual datapoints are shown. h.p.i., hours post infection.

Article Snippet: IFNβ, IFNλ1/IFNλ3 and CXCL10 were measured using Human IFN-β Quantikine ELISA Kit, Human IL-29/IL-28B (IFNλ1/IFNλ3) DuoSet ELISA or Human CXCL10/IP-10 DuoSet ELISA reagents (Bio-Techne R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Quantitative RT-PCR, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: The Journal of Clinical Investigation

Article Title: CDK12/13 inactivation triggers STING-mediated antitumor immunity in preclinical models

doi: 10.1172/JCI193745

Figure Lengend Snippet: ( A ) Enrichment of the indicated pathways in Myc-CaP cells treated with YJ1206 at 1 μM for 24 hours. Adj., adjusted. ( B ) Immunoblot of the indicated proteins in Myc-CaP cells treated with YJ1206 at increasing concentrations for 4 hours, or siRNAs targeting Cdk12 and/or Cdk13 . Nontargeting siRNA was used as control. GAPDH was used as a loading control. ( C ) Top: Analysis of the indicated gene expression by RT-qPCR in Myc-CaP cells treated with YJ1206 at 1 μM for 15 hours, or siRNAs targeting Cdk12 and/or Cdk13 . Nontargeting siRNA was used as control. Bottom left: IFN-β ELISA results in Myc-CaP cells treated as described above. Bottom right: Flow cytometry assessing surface MHC-I expression in Myc-CaP cells treated as described above. ( D ) Immunoblot of the noted proteins in B16-F10 cells treated with YJ1206 at increasing concentrations for 4 hours. ( E and F ) Flow cytometry median fluorescence intensity (MFI) quantifications of surface MHC-I ( E ) or PD-L1 ( F ) in the indicated cells treated with YJ1206 at 1 μM or 3 μM for 15 hours. ( G ) Quantification of immunofluorescence DNA/RNA hybrid (red) staining in Myc-CaP cells treated with 1 μM YJ1206 for 4 hours or siRNA targeting Cdk12 and/or Cdk13 , with/without RNase H. Representative images are in . Nontargeting siRNA was used as control. Forty (siRNA treatment) or 20 (YJ1206 or DMSO treatment) cells were used per data point. Data in C , E , and F are displayed as mean ± SD of triplicate experiments. Data in G are presented as box-and-whisker plots, with the median (center line), 25th–75th percentiles (box), and minimum to maximum values (whiskers). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by 2-tailed t test. NS, not significant. Bonferroni’s correction was applied for multiple comparisons in C and E – G .

Article Snippet: IFN-β ELISA (R&D Systems, DY8234-05 and DY008B) and cGAMP ELISA (Cayman Chemical, 501700) kits were used per the manufacturer’s protocols.

Techniques: Western Blot, Control, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Fluorescence, Immunofluorescence, Staining, Whisker Assay

Journal: The Journal of Clinical Investigation

Article Title: CDK12/13 inactivation triggers STING-mediated antitumor immunity in preclinical models

doi: 10.1172/JCI193745

Figure Lengend Snippet: ( A and B ) Representative images ( A ) or quantification ( B ) of immunofluorescence assessing PCNA-RNAPII PLA foci in Myc-CaP cells treated with siRNA targeting Cdk12 and/or Cdk13 , or YJ1206 at 3 μM for 4 hours, with or without DRB or triptolide treatment. ( C – E ) Representative images ( C ) or quantification ( D and E ) of dsDNA ( C , left, and D ) and ssDNA ( C , right, and E ) in Myc-CaP cells treated with 3 μM YJ1206 for 4 hours, with or without DRB treatment. Scale bars: 5 μm. ( F ) ELISA measuring cGAMP levels in Myc-CaP cells treated with YJ1206 at the indicated concentrations. ( G ) Immunoblot of the noted proteins in Myc-CaP cells treated with YJ1206 at 1 μM, with or without DRB for 4 hours. Data in D and E are presented as box-and-whisker plots, with the median (center line), 25th–75th percentiles (box), and minimum to maximum values (whiskers). Data are displayed as mean ± SEM in F of triplicate experiments. One hundred cells were used per data point in B , D , and E . **** P < 0.0001 by 2-tailed t test. NS, not significant. Bonferroni’s correction was applied for multiple comparisons.

Article Snippet: IFN-β ELISA (R&D Systems, DY8234-05 and DY008B) and cGAMP ELISA (Cayman Chemical, 501700) kits were used per the manufacturer’s protocols.

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Whisker Assay