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The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium, where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the public domain of NIH, the Molecular Libraries Probe Production Centers Network library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was done in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC50 < 10 μmol/L for KLF5 inhibition and EC50 > 10 μmol/L for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized on the basis of computational, Western blot, and cell viability analyses. Both of these compounds, and two newly synthesized structural analogs of CID 5951923, significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results show the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5. Mol Cancer Ther; 10(11); 2043–51. ©2011 AACR.

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Objective— We aimed to develop and validate a model of angioplasty and stenting in mice that would allow investigation of the response to stent injury using genetically modified mouse strains. Methods and Results— Aortic segments from either C57BL/6 wild-type or atherosclerotic ApoE-KO mice underwent balloon angioplasty alone or balloon angioplasty and stenting with a 1.25×2.5 mm stainless steel stent. Vessels were carotid-interposition grafted into genetically identical littermate recipients and harvested at 1, 7, 14, or 28 days. In wild-type mice, stenting generated an inflammatory vascular injury response between days 1 to 7, leading to the development of neointimal hyperplasia by day 14, which further increased in area by day 28 leading to the development of in-stent stenosis. Uninjured vessels and vessels injured by balloon angioplasty alone developed minimal neointimal hyperplasia. In stented ApoE-KO mice, neointimal area at 28 days was 30% greater compared with wild-type mice. Conclusions— By reproducing important features of human stenting in atherosclerotic mice, we provide the potential to investigate molecular pathways and evaluate novel therapeutic targets for stent injury and restenosis.

OBJECTIVES: Optical coherence tomography (OCT) is a high resolution imaging technique used to assess superficial atherosclerotic plaque morphology. Utility of OCT may be enhanced by contrast agents targeting molecular mediators of inflammation. METHODS AND RESULTS: Microparticles of iron oxide (MPIO; 1 and 4.5 μm diameter) in suspension were visualized and accurately quantified using a clinical optical coherence tomography system. Bound to PECAM-1 on a plane of cultured endothelial cells under static conditions, 1 μm MPIO were also readily detected by OCT. To design a molecular contrast probe that would bind activated endothelium under conditions of shear stress, we quantified the expression (basal vs. TNF-activated; molecules μm(-2)) of VCAM-1 (not detected vs. 16 ± 1); PECAM-1 (132 ± 6 vs. 198 ± 10) and E-selectin (not detected vs. 46 ± 0.6) using quantitative flow cytometry. We then compared the retention of antibody-conjugated MPIO targeting each of these molecules plus a combined VCAM-1 and E-selectin (E+V) probe across a range of physiologically relevant shear stresses. E+V MPIO were consistently retained with highest efficiency (P < 0.001) and at a density that provided conspicuous contrast effects on OCT pullback. CONCLUSION: Microparticles of iron oxide were detectable using a clinical OCT system. Assessment of binding under flow conditions recommended an approach that targeted both E-selectin and VCAM-1. Bound to HUVEC under conditions of flow, targeted 1 μm E+V MPIO were readily identified on OCT pullback. Molecular imaging with OCT may be feasible in vivo using antibody targeted MPIO.

Objectives: Our aim was to determine the effects of high-dose (2 g) nicotinic acid (NA) on progression of atherosclerosis and measures of vascular function.Background: NA raises high-density lipopr...

Our aim was to determine the effects of high-dose (2 g) nicotinic acid (NA) on progression of atherosclerosis and measures of vascular function.NA raises high-density lipoprotein cholesterol (HDL-C) and reduces low-density lipoprotein cholesterol and is widely used as an adjunct to statin therapy in patients with coronary artery disease. Although changes in plasma lipoproteins suggest potential benefit, there is limited evidence of the effects of NA on disease progression when added to contemporary statin treatment.We performed a double-blind, randomized, placebo-controlled study of 2 g daily modified-release NA added to statin therapy in 71 patients with low HDL-C (40 mg/dl) and either: 1) type 2 diabetes with coronary heart disease; or 2) carotid/peripheral atherosclerosis. The primary end point was the change in carotid artery wall area, quantified by magnetic resonance imaging, after 1 year.NA increased HDL-C by 23% and decreased low-density lipoprotein cholesterol by 19%. At 12 months, NA significantly reduced carotid wall area compared with placebo (adjusted treatment difference: -1.64 mm(2) [95% confidence interval: -3.12 to -0.16]; p = 0.03). Mean change in carotid wall area was -1.1 +/- 2.6 mm(2) for NA versus +1.2 +/- 3.0 mm(2) for placebo. In both the treatment and placebo groups, larger plaques were more prone to changes in size (r = 0.4, p = 0.04 for placebo, and r = -0.5, p = 0.02 for NA).In statin-treated patients with low HDL-C, high-dose modified-release NA, compared with placebo, significantly reduces carotid atherosclerosis within 12 months. (Oxford Niaspan Study: Effects of Niaspan on Atherosclerosis and Endothelial Function; NCT00232531).

Nine rabbits were given subcutaneous doses of 7.5 mg of gentamicin/kg every 24 hr, and nine were given 2.5 mg of gentamicin/kg every 8 hr for three weeks or longer. Renal function was evaluated by monitoring of the concentration of creatinine in serum and the rate of clearance of gentamicin from serum. In the animals receiving the 8-hr divided dosage, a deterioration of renal function as early as day 7 was demonstrated by the rate of clearance of gentamicin, and deterioration by day 21 was evidenced by the concentration of creatinine in serum; the renal function of the animals receiving the single daily dose was unchanged. There was no relationship between peak or trough concentrations of gentamicin in serum and the development of nephrotoxicity. The animals receiving the 8-hr dosages had a significantly longer daily duration of exposure to concentrations of greater than or equal to 0.1, greater than or equal to 1.0, greater than or equal to 2.0, and greater than or equal to 3.0 microng of gentamicin/ml in serum than did animals receiving the daily dosage.

One hundred and eleven strains of Pseudomonas aeruginosa isolated from clinical material were studied for susceptibility to carbenicillin. Of the strains, 80% were inhibited by 125 μg/ml or more. The combination of carbenicillin and gentamicin was shown to have inhibitory and bactericidal synergistic effect on 15 of 16 strains of P. aeruginosa tested. There was poor correlation between the single-disc sensitivity method and the tube dilution method.

The in vitro activity of 6-[ d -α-(3 guanylureido)-phenylacetamido]-penicillanic acid (BL-P1654) was evaluated against 117 clinical isolates of Pseudomonas aeruginosa , many of which were known to be resistant to both gentamicin and carbenicillin. BL-P1654 was two to eight times more active than carbenicillin against P. aeruginosa . However, all 28 highly carbenicillin-resistant isolates (minimal inhibitory concentration [MIC] > 500 μg/ml) were also highly resistant to BL-P1654. When an MIC of 32 μg/ml or less and a zone of inhibition of 12 mm or more by a 10-μg disk were used as criteria indicating susceptibility to BL-P1654, the false-resistance rate by the disk test was 10.6% and the false-susceptibility rate was 4.2%. The combination of BL-P1654 and gentamicin was synergistic against 45 of 70 isolates of P. aeruginosa tested, but synergism was demonstrated against only 4 of 24 highly gentamicin-resistant (MIC > 63 μg/ml), 1 of 12 highly BL-P1654-resistant (MIC > 250 μg/ml) isolates, and none of nine isolates highly resistant to both of these antibiotics.

The in vitro activity of BL-S640, a 7-(2-aryl-2-aminoacetamido)-3-(heterocyclic-thiomethyl) cephalosporin, was evaluated against 338 clinical isolates of Enterobacteriaceae in comparison with ampicillin, cephalothin, cefazolin, and cephalexin. Against Escherichia coli , BL-S640 was as active as cefazolin and more active than ampicillin, cephalothin, and cephalexin. BL-S640 was as effective as the other cephalosporins tested and far more active than ampicillin against Klebsiella and was more active than cephalexin against Proteus mirabilis and the indole-positive Proteus . The majority of Enterobacter, Serratia , and Citrobacter were resistant to ampicillin and all the cephalosporins tested. With rare exceptions, the zone of inhibition by the BL-S640 30-μg disk was either larger or the same as the zone obtained by the cephalothin 30-μg disk in the Kirby-Bauer disk susceptibility test.

Levels of ampicillin and amoxicillin in sera and skin window fluid of 22 volunteers were studied. Higher levels of amoxicillin than of ampicillin were attained. Approximately 20% of both antibiotics circulated in a protein-bound form, and the ratio of the concentration in skin window fluid to that in serum were comparable. This finding confirms our previous studies of penicillins and cephalosporins, in which an inverse relationship between protein binding and diffusion of these antibiotics into the skin window fluid was demonstrated.