Evaluation of mechanical and geometric accuracy of two different image guidance systems in radiotherapy.

AIM: To assess the mechanical and the geometric accuracy of two different clinically used image guidance systems in radiotherapy for a period of 6 months. BACKGROUND: With the image guidance procedures being routine in the clinical radiotherapy department, the quality assurance tests for these systems become essential. The mechanical and geometric accuracy of these systems are crucial since it directly affects patient treatment set-up and delivery. MATERIALS AND METHODS: We have assessed the mechanical and the geometric accuracy of two different image guidance systems (MV and kV based), being used clinically for a period of 6 months. The quality assurance tests such as imager positioning/repositioning, imaging and treatment beam isocentre coincidence, imager mechanical alignment, image scaling, geometric accuracy of cone beam computed tomography system, automatic image registration and offset calculation accuracy were assessed in this period. RESULTS: It was found that both systems were mechanically and geometrically accurate within ±2 mm in this period. CONCLUSION: The quality assurance tests for MV based image guidance system were simple compared to kV based systems. We recommend performing periodic quality assurance tests to verify the integrity of both image guidance systems.

Tumor-promoting role of TGFß1 signaling in ultraviolet B-induced skin carcinogenesis is associated with cutaneous inflammation and lymph node migration of dermal dendritic cells.

Transforming growth factor beta 1 (TGFß1) is a pleiotropic cytokine in the skin that can function both as a tumor promoter and suppressor in chemically induced skin carcinogenesis, but the function in ultraviolet B (UVB) carcinogenesis is not well understood. Treatment of SKH1 hairless mice with the activin-like kinase 5 (ALK5) inhibitor SB431542 to block UVB-induced activation of cutaneous TGFß1 signaling suppressed skin tumor formation but did not alter tumor size or tumor cell proliferation. Tumors that arose in SB-treated mice after 30 weeks had significantly reduced percentage of IFNγ(+) tumor-infiltrating lymphocytes compared with control mice. SB431542 blocked acute and chronic UVB-induced skin inflammation and T-cell activation in the skin-draining lymph node (SDLN) and skin but did not alter UVB-induced epidermal proliferation. We tested the effect of SB431542 on migration of skin dendritic cell (DC) populations because DCs are critical mediators of T-cell activation and cutaneous inflammation. SB431542 blocked (i) UVB-induced Smad2 phosphorylation in dermal DC (dDC) and (ii) SDLN and ear explant migration of CD103(+) CD207(+) and CD207(-) skin DC subsets but did not affect basal or UV-induced migration of Langerhans cells. Mice expressing a dominant-negative TGFß type II receptor in CD11c(+) cells had reduced basal and UVB-induced SDLN migration of CD103(+) CD207(+) and CD207(-) DC subsets and a reduced percentage of CD86(high) dDC following UVB irradiation. Together, these suggest that TGFß1 signaling has a tumor-promoting role in UVB-induced skin carcinogenesis and this is mediated in part through its role in UVB-induced migration of dDC and cutaneous inflammation.

The TGFß1 pathway is required for NFκB dependent gene expression in mouse keratinocytes.

The transforming growth factor-beta 1 (TGFß1) and NFκB pathways are important regulators of epidermal homeostasis, inflammatory responses and carcinogenesis. Previous studies have shown extensive crosstalk between these pathways that is cell type and context dependent, but this has not been well-characterized in epidermal keratinocytes. Here we show that in primary mouse keratinocytes, TGFß1 induces NFκB-luciferase reporter activity that is dependent on both NFκB and Smad3. TGFß1-induced NFκB-luciferase activity was blocked by the IκB inhibitor parthenolide, the IκB super-repressor, a dominant negative TGFß1-activated kinase 1 (TAK1) and genetic deletion of NFκB1. Coexpression of NFκB p50 or p65 subunits enhanced NFκB-luciferase activity. Similarly, inhibition of the TGFß1 type I receptor with SB431542 or genetic deletion of Smad3 blocked TGFß1 induction of NFκB-luciferase. TGFß1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic IκB levels or nuclear translocation of NFκB subunits, although EMSA showed rapid NFκB nuclear binding activity that could be blocked by SB431542 treatment. TNFα, a well characterized NFκB target gene was also induced by TGFß1 and this was blocked in NFκB+/- and -/- keratinocytes and by the IκB super-repressor. To test the effects of the TGFß1 pathway on a biologically relevant activator of NFκB, we exposed mice and primary keratinocytes in culture to UVB irradiation. In primary keratinocytes UVB caused a detectable increase in levels of Smad2 phosphorylation that was dependent on ALK5, but no significant increase in SBE-dependent gene expression. Inhibition of TGFß1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNFα message. Similarly, UVB induction of TNFα mRNA was blocked in skin of Tgfb1+/- mice. These studies demonstrate that intact TGFß1 signaling is required for NFκB-dependent gene expression in mouse keratinocytes and skin and suggest that a convergence of these pathways in the nucleus rather than the cytoplasm may be critical for regulation of inflammatory pathways in skin by TGFß1.

Gambogic acid covalently modifies IkappaB kinase-beta subunit to mediate suppression of lipopolysaccharide-induced activation of NF-kappaB in macrophages.

GA (gambogic acid) is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi with a long history of use as a complementary and alternative medicine. The antitumour activity of GA has been well demonstrated and is thought to arise partly from the associated anti-inflammatory activity. Recent studies have indicated that the antitumour activity of GA is mediated by its ligation of TfR1 (transferrin receptor-1). Since the cellular expression of TfR1 is down-regulated by LPS (lipopolysaccharide), we hypothesized that an alternative pathway exists in immune cells, such as macrophages, where GA could mitigate the expression of pro-inflammatory genes. Here we demonstrate that GA inhibits the LPS-dependent expression of NF-kappaB (nuclear factor kappaB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-kappaB-luciferase reporter and gel-shift analyses revealed that GA strongly blocked the activation of NF-kappaB induced by LPS, whereas 9,10-dihydro-GA, which lacks the reactive alpha,beta-unsaturated carbonyl group, was ineffective. Moreover, GA was able to decrease nuclear p65 levels in RAW264.7 macrophages, where the expression of TfR1 was down-regulated by RNA interference. in vitro kinase assays coupled with interaction studies using biotinylated GA as well as proteomic analysis demonstrated that IKKbeta [IkappaB (inhibitory kappaB) kinase-beta], a key kinase of the NF-kappaB signalling axis, was covalently modified by GA at Cys-179, causing significant inhibition of its kinase activity. Taken together, these results demonstrate the potent anti-inflammatory activity of GA.