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: International Journal of Molecular Sciences

Article Title: Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

doi: 10.3390/ijms21239083

Figure Lengend Snippet: Effect of plasma pre-clearing on DNA recovery and detection of EV markers in PA precipitated material. ( A ) Plasma from six NSCLC donors was either not pre-cleared, or pre-cleared at 3000× g or 17,000× g for 15 min. The post-centrifugation pellet was retained, resuspended in nuclease-free water and DNA was extracted. Peptide affinity (PA) precipitation was performed on equivalent volumes of either non-precleared or pre-cleared plasma and DNA (PA-DNA) was isolated using the Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). Cell-free DNA (cf-DNA) was obtained from equivalent volumes of plasma using the same DNA isolation kit for comparison ( n = 6; * p < 0.05). A representative overlay of the DNA profiles of ( B ) PA-DNA or ( C ) cf-DNA from plasma (Donor #1) using either no pre-clearing or 3000× g or 17,000× g pre-clearing is shown. ( D ) A representative western blot ( n = 3) of Vn96 PA precipitated material from 1 mL of plasma from donors with benign lung disease or NSCLC is shown. Canonical EV markers CD63, CD9, HSC70, and flotillin-1 were detected using specific antibodies. A vehicle control sample (without Vn96) was included as a negative control (−). In addition, calnexin, apolipoprotein A1 (Apo-A1), and albumin, which are common co-contaminants of EV isolations from plasma, were also detected using specific antibodies. Plasma protein lysate was included as a positive control for non-EV-associated plasma proteins.

Article Snippet: DNA Isolation: DNA was isolated directly from up to 0.5 mL of plasma (cf-DNA) using the Plasma/Serum Cell-Free Circulating DNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Centrifugation, Isolation, DNA Purification, DNA Extraction, Comparison, Western Blot, Control, Negative Control, Positive Control