Imaging Pulmonary NF-kappaB Activation and Therapeutic Effects of MLN120B and TDZD-8

NF-κB activation is a critical signaling event in the inflammatory response and has been implicated in a number of pathological lung diseases. To enable the assessment of NF-κB activity in the lungs, we transfected a luciferase based NF-κB reporter into the lungs of mice or into Raw264.7 cells in culture. The transfected mice showed specific luciferase expression in the pulmonary tissues. Using these mouse models, we studied the kinetics of NF-κB activation following exposure to lipopolysaccharide (LPS). The Raw264.7 cells expressed a dose-dependent increase in luciferase following exposure to LPS and the NF-κB reporter mice expressed luciferase in the lungs following LPS challenge, establishing that bioluminescence imaging provides adequate sensitivity for tracking the NF-κB activation pathway. Interventions affecting the NF-κB pathway are promising clinical therapeutics, thus we further examined the effect of IKK-2 inhibition by MLN120B and glycogen synthase kinase 3 beta inhibition by TDZD-8 on NF-κB activation. Pre-treatment with either MLN120B or TDZD-8 attenuated NF-κB activation in the pulmonary tissues, which was accompanied with suppression of pro-inflammatory chemokine MIP-1ß and induction of anti-inflammatory cytokine IL-10. In summary, we have established an imaging based approach for non-invasive and longitudinal assessment of NF-κB activation and regulation during acute lung injury. This approach will potentiate further studies on NF-κB regulation under various inflammatory conditions

Pharmacokinetics and pharmacodynamics of the novel monobactam LYS228 in a neutropenic murine thigh model of infection

Objectives: The neutropenic murine thigh infection model and a dose-fractionation approach were used to determine the pharmacokinetic/pharmacodynamic (PK/PD) relationship of LYS228, a novel monobactamantibiotic with activity against Enterobacteriaceae including carbapenem-resistant strains. Methods: Mice (n"4 per group) were inoculated with Enterobacteriaceae strains via intramuscular injection. Two hours post-bacterial inoculation, treatment with LYS228 was initiated. Animals were euthanized with CO2 24 h after the start of therapy and bacterial counts (log10 cfu) per thigh were determined. PK parameters were calculated using free (f) plasma drug levels. Results: Following a dose-fractionation study, non-linear regression analysis determined that the predominant PK/PD parameter associated with antibacterial efficacy of LYS228 was the percentage of the dosing interval that free drug concentrations remained above the MIC (%fT.MIC). In a dose-dependent manner, LYS228 reduced the thigh bacterial burden in models established with Enterobacteriaceae producing b-lactamase enzymes of all classes (e.g. ESBLs, NDM-1, KPC, CMY-2 and OXA-48). The range of the calculated static dose was 86-649 mg/kg/ day for the isolates tested, and the magnitude of the driver of efficacy was 37-83%fT.MIC. %fT.MIC was confirmed as the parameter predominantly driving efficacy as evidenced by a strong coefficient of determination (r2"0.68). Neutrophils had minimal impact on the effect of LYS228 in the murine thigh infection model. Conclusions: LYS228 is efficacious in murine thigh infection models using b-lactamase-producing strains of Enterobacteriaceae, including those expressing metallo-b-lactamases, ESBLs and serine carbapenemases, with the PK/PD driver of efficacy identified as%T.MIC

Kinetics of NF-κB activation.

<p>TNFAIP3-luc2 reporter transfected mice were challenged with LPS (1 mg/kg, intratracheally, n = 11) or TNFα (1 µg/mouse, intravenously, n = 4) or saline (n = 4). Mice were imaged at 0 (prior to injection), 3, 6, and 24 hours after injection. Bioluminescent images from one representative mouse per group (A) and quantification of lung signals (B) are shown. Data presented as mean ± SEM.</p

Impact of ageing on presentation and outcome of mitral regurgitation due to flail leaflet: a multicentre international study

Define the impact of age at diagnosis on degenerative mitral regurgitation (MR) prognosis.Methods and resultsThe Mitral regurgitation International DAtabase (MIDA) is a multicentre registry of MR due to flail leaflets including 862 patients (65 \ub1 12 years) diagnosed by echocardiography. The 498 older patients ( 6565 years at diagnosis) were compared with the 364 younger (<65) with regard to presentation and the outcome was compared with that expected in the general population. Older vs. younger patients had MR of similar severity and ventricular overload but presented with more MR consequences and incurred higher mortality [risk ratio (rr) 95% confidence interval (95% CI) 4.7 (2.5-10.0), P < 0.001] independently of co-morbidity. Compared with expected survival [relative risk (95% confidence interval)], excess mortality, non-significant in younger patients [1.1 (0.6-2.0), P = 0.65], was prominent in older patients [1.4 (1.2-1.7), P < 0.001]. Compared with expected, excess heart failure (HF) occurred in younger [9.3 (6.5-13.3), P < 0.0001) and in older patients [6.7 (5.6-8.1), P < 0.0001]. Excess atrial fibrillation (AF) was even higher in younger [6.9 (4.5-10.6), P < 0.0001] than in older patients [3.5 (2.6-4.7), P < 0.0001; P < 0.001 for comparison between age groups]. Subsequent excess mortality [rr (95% CI)] was associated with occurrence of HF and/or AF in both age groups [13.5 (7.4-24.6), P < 0.001]. Mitral surgery was associated with reduced long-term mortality in older patients and lower rate of HF in both the age groups (all P < 0.01).ConclusionsBoth older and younger patients incurred excess risk of complications. Older patients suffered excess mortality, AF, and HF, whereas younger incurred excess morbidity linked to subsequent long-term excess mortality. The excess risks of uncorrected degenerative MR should be considered in deliberating surgical management, which significantly reduced mortality in older patients and HF in younger patient

In vitro comparison of NF-κB reporters.

<p>RAW264.7 cells were transiently transfected with NF-κB reporters. Cells were treated with LPS at 0–1 µg/ml concentrations overnight and imaged with IVIS after adding luciferin. Quantification of luciferase signal (A). Fold change of cells treated with 1 µg/ml LPS compared to vehicle treated cells (B). Data presented as mean ± SEM; n = 4 wells per reporter construct and LPS concentration of 1 µg/ml.</p

Effect of IKK2 inhibition on NF-κB induction in vivo.

<p>At 2 weeks after in vivo gene delivery with the NF-κB2-luc2 reporter, mice (n = 7) were pre-treated with MLN120B at 300 mg/kg orally or vehicle control 16 and 1-hour prior to LPS delivery. Mice were imaged immediately before LPS injection (T = 0) and at 4 hours. Bioluminescence images (A) and quantification of lung signals (B) are shown. Cytokines measured in bronchial lavage fluid as specified (C). Data presented as standard box and whisker plots, *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney U test.</p

Effect of anti-inflammatory agents on NF-κB induction in vitro.

<p>The effect of MLN120B (A) and TDZD-8 (B) on luciferase induction by LPS in TNFAIP3-luc2, NF-κB2-luc2 and IL8-luc2 transfected RAW264.7 cells are shown. All the compounds were tested at 1–100 µM concentrations. The data are presented as percentage change over vehicle treated cells. Data presented as mean ± SEM; n = 4 wells per reporter construct and LPS concentration.</p

Effect of glycogen synthase kinase beta inhibition on NF-κB induction in vivo.

<p>NF-κB2-luc2 reporter transfected mice (n = 6 per group) were pre-dosed with either TDZD-8 (10 mg/kg, i.p.), or DMSO vehicle control 16 and 1-hour prior to LPS challenge. Mice were imaged immediately before LPS injection (T = 0) and at 3 hours post injection. Another group of mice was not treated with LPS and served as negative controls (n = 3). Bioluminescence images (A) and quantification of lung signals (B) are shown. Cytokines measured in bronchial lavage fluid as specified (C). Data presented as standard box and whisker plots, *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney U test.</p