Prostaglandin E 2 (PGE 2 ) synthesis modulates the response to radiation injury in the mouse intestinal epithelium through effects on crypt survival and apoptosis; however, the downstream signaling events have not been elucidated. WT mice receiving 16,16-dimethyl PGE 2 (dmPGE 2 ) had fewer apoptotic cells per crypt than untreated mice. Apoptosis in Bax -/-mice receiving 12 Gy was approximately 50% less than in WT mice, and the ability of dmPGE 2 to attenuate apoptosis was lost in Bax -/-mice. Positional analysis revealed that apoptosis in the Bax -/-mice was diminished only in the bax-expressing cells of the lower crypts and that in WT mice, dmPGE 2 decreased apoptosis only in the bax-expressing cells. The HCT-116 intestinal cell line and Bax -/-HCT-116 recapitulated the apoptotic response of the mouse small intestine with regard to irradiation and dmPGE 2 . Irradiation of HCT-116 cells resulted in phosphorylation of AKT that was enhanced by dmPGE 2 through transactivation of the EGFR. Inhibition of AKT phosphorylation prevented the reduction of apoptosis by dmPGE 2 following radiation. Transfection of HCT-116 cells with a constitutively active AKT reduced apoptosis in irradiated cells to the same extent as in nontransfected cells treated with dmPGE 2 . Treatment with dmPGE 2 did not alter bax or bcl-x expression but suppressed bax translocation to the mitochondrial membrane. Our in vivo studies indicate that there are bax-dependent and bax-independent radiation-induced apoptosis in the intestine but that only the bax-dependent apoptosis is reduced by dmPGE 2 . The in vitro studies indicate that dmPGE 2 , most likely by signaling through the E prostaglandin receptor EP 2 , reduces radiation-induced apoptosis through transactivation of the EGFR and enhanced activation of AKT and that this results in reduced bax translocation to the mitochondria.

Dysfunction of macrophages (MΦs) in diabetic wounds impairs the healing. MΦs produce anti-inflammatory and pro-resolving neuroprotectin/protectin D1 (NPD1/PD1, 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid); however, little is known about endogenous NPD1 biosynthesis by MΦs and the actions of NPD1 on diabetic MΦ functions in diabetic wound healing. We used an excisional skin wound model of diabetic mice, MΦ depletion, MΦs isolated from diabetic mice, and mass spectrometry-based targeted lipidomics to study the time course progression of NPD1 levels in wounds, the roles of MΦs in NPD1 biosynthesis, and NPD1 action on diabetic MΦ inflammatory activities. We also investigated the healing, innervation, chronic inflammation, and oxidative stress in diabetic wounds treated with NPD1 or NPD1-modulated MΦs from diabetic mice. Injury induced endogenous NPD1 biosynthesis in wounds, but diabetes impeded NPD1 formation. NPD1 was mainly produced by MΦs. NPD1 enhanced wound healing and innervation in diabetic mice and promoted MΦs functions that accelerated these processes. The underlying mechanisms for these actions of NPD1 or NPD1-modulated MΦs involved 1) attenuating MΦ inflammatory activities and chronic inflammation and oxidative stress after acute inflammation in diabetic wound, and 2) increasing MΦ production of IL10 and hepatocyte growth factor. Taken together, NPD1 appears to be a MΦs-produced factor that accelerates diabetic wound healing and promotes MΦ pro-healing functions in diabetic wounds. Decreased NPD1 production in diabetic wound is associated with impaired healing. This study identifies a new molecular target that might be useful in development of more effective therapeutics based on NPD1 and syngeneic diabetic MΦs for treatment of diabetic wounds.

Severe burns are some of the most challenging problems in clinics and still lack ideal modalities. Mesenchymal stem cells (MSCs) incorporated with biomaterial coverage of burn wounds may offer a viable solution. In this report, we seeded MSCs to a biodegradable hybrid hydrogel, namely ACgel, that was synthesized from unsaturated arginine-based poly(ester amide) (UArg-PEA) and chitosan derivative. MSC adhered to ACgels. ACgels maintained a high viability of MSCs in culture for 6 days. MSC seeded to ACgels presented well in third-degree burn wounds of mice at 8 days postburn (dpb) after the necrotic full-thickness skin of burn wounds was debrided and filled and covered by MSC-carrying ACgels. MSC-seeded ACgels promoted the closure, reepithelialization, granulation tissue formation, and vascularization of the burn wounds. ACgels alone can also promote vascularization but less effectively compared with MSC-seeded ACgels. The actions of MSC-seeded ACgels or ACgels alone involve the induction of reparative, anti-inflammatory interleukin-10, and M2-like macrophages, as well as the reduction of inflammatory cytokine TNFα and M1-like macrophages at the late inflammatory phase of burn wound healing, which provided the mechanistic insights associated with inflammation and macrophages in burn wounds. For the studied regimens of these treatments, no toxicity was identified to MSCs or mice. Our results indicate that MSC-seeded ACgels have potential use as a novel adjuvant therapy for severe burns to complement commonly used skin grafting and, thus, minimize the downsides of grafting.

Aim To assess the impact of the 5-fluorouracil (5-FU) drug-pathway genes on cytotoxicity, and determine whether loss-of-function analyses coupled with functional assays can help prioritize pharmacogenomic candidate genes. Materials & methods Dose-response experiments were used to quantify the phenotype of sensitivity to 5-FU following the specific knockdown of genes selected from the 5-FU PharmGKB drug pathway in three human colorectal cell lines. Changes in sensitivity were considered significant if the IC50 for shRNA-exposed cells were three standard deviations outside the mean IC50 for control-treated cells. Results Of the 24 genes analyzed, 13 produced significant changes on the phenotype of sensitivity to 5-FU (DHFR, DPYS, DTYMK, DUT, FPGS, GGH, NME1, NT5C, RRM1, TYMS, UCK2, UNG and UMPS). Conclusion The RNAi screening strategy enabled prioritization of the genes from the 5-FU drug pathway. Further validation of the genes credentialed in this study should include gene activity or expression and mutation analyses of clinical samples.