Mechanism of action and efficacy of RX-111, a thieno[2,3-c]pyridine derivative and small molecule inhibitor of protein interaction with glycosaminoglycans (SMIGs), in delayed-type hypersensitivity, TNBS-induced colitis and experimental autoimmune encephalomyelitis.

OBJECTIVE AND DESIGN: Elucidate the mechanism of action of the small molecule inhibitor of protein binding to glycosaminoglycans, RX-111 and assay its anti-inflammatory activity in animal models of inflammatory disease. MATERIALS: The glycosaminoglycan, heparin, was used in the mechanism of action study of RX-111. Human T lymphocytes and umbilical vein endothelial cells were used to assay the in vitro activity of RX-111. Mouse and rat models of disease were used to assay the anti-inflammatory activity of RX-111 in vivo. METHODS: Circular dichroism and UV/Vis absorption spectroscopy were used to study the binding of RX-111 to the glycosaminoglycan, heparin. T lymphocyte rolling on endothelial cells under shear flow was used to assay RX-111 activity in vitro. Delayed-type hypersensitivity (DTH) and tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in mice and experimental autoimmune encephalomyelitis (EAE) in rats were used to assay anti-inflammatory activity of RX-111 in vivo. RESULTS: RX-111 was shown to bind directly to heparin. It inhibited leukocyte rolling on endothelial cells under shear flow and reduced inflammation in the mouse model of DTH. RX-111 was efficacious in the mouse model of inflammatory bowel disease, TNBS-induced colitis and the rat model of multiple sclerosis, EAE. CONCLUSIONS: RX-111 exercises its broad spectrum anti-inflammatory activity by a singular mechanism of action, inhibition of protein binding to the cell surface GAG, heparan sulfate. RX-111 and related thieno[2,3-c]pyridine derivatives are potential therapeutics for the treatment of inflammatory and autoimmune diseases.

Gene expression in mouse preimplantation embryos affected by apoptotic inductor actinomycin D.

The aim of this study was to test the effect of actinomycin D on the expression of selected genes and to elucidate possible components of its apoptotic pathway in mouse embryos. Selected mRNAs and Trp53 protein were examined in blastocysts cultured for 24 h in vitro with or without the presence of a high concentration of actinomycin D. In all tested genes, the relative quantities of mRNA were significantly lower in treated blastocysts than in controls. The mRNA quantities of H2afz, Actb, Bax, Bad and Bcl2 were reduced at a similar rate, but the decreases in Bcl2l2 and Trp53 mRNA were significantly greater. Treatment with actinomycin D also changed the ratio between the mRNA levels of some pro-apoptotic and anti-apoptotic genes: the Bad/Bcl2l2 and the Bax/Bcl2l2 ratios were on average 4.39 and 2.66 times higher in the treated embryos than in the controls, respectively. Generally, treatment led to developmental arrest and significant increase in the incidence of cells with typical apoptotic features. However, its effect on Trp53 protein expression was not significant. The results suggest that mechanisms beyond the apoptotic effect of actinomycin D might include specific changes in the expression of pro-apoptotic and anti-apoptotic genes, shifting the expression ratio in favor of the pro-apoptotic ones. The results also show that the role of Trp53 is probably not crucial in this apoptotic pathway.

Apoptotic processes and DNA cytosine methylation in mouse embryos arrested at the 2-cell stage.

The present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were cultured in vitro and treated with chemicals that cause developmental arrest and apoptosis (alpha-amanitin, actinomycin D, TNF-alpha). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.

Effects of a combination of thyme and oregano essential oils on TNBS-induced colitis in mice.

We examined the anti-inflammatory effects of the combination of thyme and oregano essential oil dietary administered at three concentrations (0.4% thyme and 0.2% oregano oils; 0.2% thyme and 0.1% oregano oils; 0.1% thyme and 0.05% oregano oils) on mice with TNBS-induced colitis. Treatment of colitic animals with the essential oils decreased the mRNA levels of pro-inflammatory cytokines IL-1beta, IL-6, GM-CSF, and TNFalpha, especially after application of the medium dose. The medium dose of the essential oils significantly lowered the amount of IL-1beta and IL-6 proteins too. Moreover, administration of the medium dose decreased the mortality rate, accelerated the body weight gain recovery, and reduced the macroscopic damage of the colonic tissue. Our results indicate that combined treatment with appropriate concentrations of thyme and oregano essential oils can reduce the production of proinflammatory cytokines, and thereby attenuate TNBS-induced colitis in mice.