Induction of single- and double-strand breaks in plasmid DNA by 100-1500 eV electrons

PURPOSE: To investigate the induction of DNA strand breaks by electrons with energies ranging from 0.1 to 1.5 keV. MATERIALS AND METHODS: Dry supercoiled plasmid DNA was irradiated with electrons of energies ranging from 0.1 to 1.5 keV and the results were compared with those obtained by gamma-irradiation of the same plasmid in solution. For electron irradiation, the plasmid was deposited on a gold substrate under a controlled atmosphere to minimize contamination of the DNA film. Electron bombardments were performed under ultra-high vacuum conditions (UHV 10(-9) torr). DNA damage was detected by gel electrophoresis followed by quantitation of the DNA bands by fluorescence or by hybridization with a radioactive probe. RESULTS: Electrons with energies from 0.1 to 1.5 keV induced single, double and multiple double-strand breaks in supercoiled plasmid DNA. For equal doses, we observed a marked increase in the efficiency of induction of double- and multiple-strand breaks in supercoiled DNA as a function of electron energy. In contrast to gamma-irradiation, the formation of small DNA fragments by electrons did not seem to be related to the production of the linear form of the plasmid. CONCLUSIONS: Electrons within the energy; range of the secondary electrons generated by high-energy ionizing radiation induce single, double and multiple double-strand breaks in DNA. Problems associated with low-energy electron irradiation experiments and dose calculations in thin films are also discussed.     

An estradiol matrix transdermal system for the prevention of postmenopausal bone loss

OBJECTIVE: The aim of this study was to evaluate the efficacy, safety, and tolerability of 2 years' application of an estradiol matrix transdermal system for the prevention of postmenopausal bone loss. METHODS: In this multicenter, randomized, placebo-controlled, parallel-group study, 261 surgically or naturally postmenopausal women were randomized to apply the estradiol matrix transdermal system (0.025, 0.0375, 0.05, or 0.1 mg/d) or matching placebo twice a week for 2 years. The study was double blind with respect to treatment (active vs placebo) but not to the dose levels of active treatment (because of the differing sizes and shapes of the patches). In addition to receiving the assigned treatment, the 100 nonhysterectomized women received 2.5 mg medroxyprogesterone acetate daily throughout the study. RESULTS: The evaluable group (n = 259) had a mean age of 52 years and a mean duration of menopause of 32 months. Following 2 years of treatment, there were significant differences in favor of estradiol between all doses of the estradiol matrix transdermal system and placebo in terms of the percentage change from baseline in the bone mineral density (BMD) of the L1-L4 anteroposterior lumbar spine (0.1 and 0.05 mg/d, P < 0.001; 0.0375 mg/d, P = 0.024; 0.025 mg/d, P = 0.002). Percentage changes from baseline in the BMD of the femoral neck after 2 years of treatment also consistently demonstrated the efficacy of the estradiol matrix transdermal system compared with placebo (all, P < or = 0.044). The estradiol matrix transdermal system was well tolerated. CONCLUSION: The estradiol matrix transdermal system was effective in preventing postmenopausal bone loss at dosages of 0.025 to 0.1 mg/d, and had a safety profile consistent with the known effects of estrogen/progestin.     

Induction of single- and double-strand breaks in plasmid DNA by 100–1500 eV electrons

Purpose : To investigate the induction of DNA strand breaks by electrons with energies ranging from 0.1 to 1.5 keV. Materials and methods : Dry supercoiled plasmid DNA was irradiated with electrons of energies ranging from 0.1 to 1.5keV and the results were compared with those obtained by gamma-irradiation of the same plasmid in solution. For electron irradiation, the plasmid was deposited on a gold substrate under a controlled atmosphere to minimize contamination of the DNA film. Electron bombardments were performed under ultra-high vacuum conditions (UHV 10 9 torr). DNA damage was detected by gel electrophoresis followed by quantitation of the DNA bands by fluorescence or by hybridization with a radioactive probe. Results : Electrons with energies from 0.1 to 1.5keV induced single, double and multiple double-strand breaks in supercoiled plasmid DNA. For equal doses, we observed a marked increase in the efficiency of induction of double- and multiple-strand breaks in supercoiled DNA as a function of electron energy. In contrast to γ-irradiation, the formation of small DNA fragments by electrons did not seem to be related to the production of the linear form of the plasmid. Conclusions : Electrons within the energy range of the secondary electrons generated by high-energy ionizing radiation induce single, double and multiple double-strand breaks in DNA. Problems associated with low-energy electron irradiation experiments and dose calculations in thin films are also discussed.     

Therapeutic arthritis research and gastrointestinal event trial of lumiracoxib - study design and patient demographics

BACKGROUND: Cyclooxygenase-2 selective inhibitors were developed in order to reduce the incidence of life-threatening gastrointestinal ulcer complications compared with non-selective non-steroidal anti-inflammatory drugs. Previous outcomes studies have, variously, lacked power to investigate this endpoint, focused on broader outcomes, or been too small to quantify the influence of aspirin. AIM: To evaluate lumiracoxib, a novel cyclooxygenase-2 selective inhibitor, vs. non-selective non-steroidal anti-inflammatory drugs in an outcomes study of considerably increased size. This paper describes the study's methodology. METHODS AND PATIENTS: The Therapeutic Arthritis Research and Gastrointestinal Event Trial was a randomized, double-blind, 52-week study of lumiracoxib 400 mg once daily (two to four times the recommended dose for osteoarthritis) versus naproxen 500 mg twice daily or ibuprofen 800 mg three-times daily in patients with osteoarthritis. Randomization was stratified for low-dose aspirin use and age (< or = 64, 65-74, > or= 75 years). The study was powered to investigate upper gastrointestinal ulcer complications (primary endpoint) in patients not taking aspirin and in the overall study population; other endpoints included cardiovascular, renal and hepatic measures. CONCLUSIONS: Therapeutic Arthritis Research and Gastrointestinal Event Trial was designed to provide definitive answers concerning the gastrointestinal safety of lumiracoxib, addressing the controversial issues arising from outcomes studies with other cyclooxygenase-2 selective inhibitors.     

Selective Activation of Adenosine A2A Receptors on Immune Cells by a CD73-Dependent Prodrug Suppresses Joint Inflammation in Experimental Rheumatoid Arthritis

Adenosine A(2A) receptor (A(2A)R) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A(2A)R agonists (prodrugs) that require the presence of ecto-5'-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5'-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 ((19)F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A(2A)R. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A(2A)R expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A(2A)R agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation.     

T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon-γ and is inhibited by transforming growth factor-β

It was observed in vitro and in vivo that both interferon (IFN)-γ and interleukin (IL)-12 can promote the development of T helper type 1 (T_H1) cells. Since IL-12 was shown to be a costimulator for the production of IFN-γ by T or natural killer (NK) cells, IL-12 might play only an indirect role in T_H1 differentiation by providing IFN-γ which represents the essential differentiation factor. Using anti-CD3 monoclonal antibody (mAb) for activation of naive CD4⁺ T cells in the absence of accessory cells we could demonstrate that costimulation by IFN-γ alone results only in marginal T_H1 development. Similarly, IL-12 in the absence of IFN-γ is only a poor costimulator for inducing differentiation towards the T_H1 phenotype. Our data indicate that both cytokines are required to allow optimal T_H1 development and that IL-12 has a dual role, it promotes differentiation by direct costimulation of the T cells and also enhances the production of IFN-γ which serves as a second costimulator by an autocrine mechanism. Another cytokine that was reported to favor T_H1 differentiation in certain experimental systems is transforming growth factor (TGF)-β. With naive CD4⁺ T cells employed in this study TGF-β strongly inhibited the production of IFN-γ triggered by IL-12 as well as the IL-12-induced T_H1 development. When TGF-β was combined with anti-IFN-γ mAb for neutralization of endogenous IFN-γ the T_H1-inducing capacity of IL-12 was completetly suppressed.