Daphne P. Ly

Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271

The question of whether tumorigenic cancer stem cells exist in human melanomas has arisen in the last few years. Here we show that in melanomas, tumour stem cells (MTSCs, for melanoma tumour stem cells) can be isolated prospectively as a highly enriched CD271+ MTSC population using a process that maximizes viable cell transplantation. The tumours sampled in this study were taken from a broad spectrum of sites and stages. High-viability cells isolated by fluorescence-activated cell sorting and re-suspended in a matrigel vehicle were implanted into T-, B- and natural-killer-deficient Rag2−/−γc−/− mice. The CD271+ subset of cells was the tumour-initiating population in 90% (nine out of ten) of melanomas tested. Transplantation of isolated CD271+ melanoma cells into engrafted human skin or bone in Rag2−/−γc−/− mice resulted in melanoma; however, melanoma did not develop after transplantation of isolated CD271− cells. We also show that in mice, tumours derived from transplanted human CD271+ melanoma cells were capable of metastatsis in vivo. CD271+ melanoma cells lacked expression of TYR, MART1 and MAGE in 86%, 69% and 68% of melanoma patients, respectively, which helps to explain why T-cell therapies directed at these antigens usually result in only temporary tumour shrinkage.

Cell Permeant Peptide Analogues of the Small Heat Shock Protein, HSP20, Reduce TGF-β1-Induced CTGF Expression in Keloid Fibroblasts

A growing body of evidence suggests the involvement of connective tissue growth factor (CTGF) in the development and maintenance of fibrosis and excessive scarring. As the expression of this protein requires an intact actin cytoskeleton, disruption of the cytoskeleton represents an attractive strategy to decrease CTGF expression and, consequently, excessive scarring. The small heat-shock-related protein (HSP20), when phosphorylated by cyclic nucleotide signaling cascades, displaces phospho-cofilin from the 14-3-3 scaffolding protein leading to activation of cofilin as an actin-depolymerizing protein. In the present study, we evaluated the effect of AZX100, a phosphopeptide analogue of HSP20, on transforming growth factor-beta-1 (TGF-beta1)-induced CTGF and collagen expression in human keloid fibroblasts. We also examined the effect of AZX100 on scar formation in vivo in dermal wounds in a Siberian hamster model. AZX100 decreased the expression of CTGF and type I collagen induced by TGF-beta1, endothelin, and lysophosphatidic acid. Treatment with AZX100 decreased stress fiber formation and altered the morphology of human dermal keloid fibroblasts. In vivo, AZX100 significantly improved collagen organization in a Siberian hamster scarring model. Taken together, these results suggest the potential use of AZX100 as a strategy to prevent excessive scarring and fibrotic disorders.

Comparison of aesthetic breast reconstruction after skin-sparing or conventional mastectomy in patients receiving preoperative radiation therapy.

Many options exist for the surgical treatment of breast cancer in terms of tumor extirpation and reconstruction. Skin-sparing mastectomy (SSM) with immediate reconstruction offers patients a superior result, but this can be jeopardized by preoperative radiotherapy. We compared the outcomes of reconstruction after SSM or conventional mastectomy (CM) in the previously irradiated breast. We evaluated 41 patients over an 8-year period, who were divided into 3 categories: preoperative radiotherapy prior to SSM (n = 8), CM after preoperative radiation therapy (n = 9), and no chest wall irradiation prior to SSM (n = 20). The first group demonstrated significantly higher frequency of native flap compromise and capsular contracture formation than the other 2 groups. SSM with TRAM or latissimus with implant reconstruction is an esthetically optimal option for the treatment of patients without previous radiotherapy. However, for patients with preoperative chest wall radiation, TRAM flap reconstruction was superior to latissimus flap with implant after SSM.

Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation

humanEGFR physically interacts with humanEGF by fluorescence‐activated cell sorting (View Interaction 1, 2, 3, 4, 5, 6, 7)

Unfolded Protein Response Regulation in Keloid Cells

BACKGROUND Keloids are a common form of pathologic wound healing characterized by excessive production of extracellular matrix. The unfolded protein response (UPR) is a cellular response to hypoxia, a component of the wound microenvironment, capable of protecting cells from the effects of over-accumulation of misfolded proteins. Since keloids have hypersecretion of extracellular matrix, we hypothesized that keloid fibroblasts (KFs) may have enhanced activation of the UPR compared with normal fibroblasts (NFs). METHODS KFs and NFs were placed in a hypoxia chamber for 0, 24, and 48h. We also used tunicamycin to specifically up-regulate the UPR. UPR activation was assayed by PCR for xbp-1 splicing and by immunoblotting with specific antibodies for the three UPR transducers. Nuclear localization of XBP-1 protein in KFs was confirmed by immunofluorescence. RESULTS There is increased activation of XBP-1 protein in KFs compared with NFs following exposure to hypoxia. Pancreatic ER kinase (PERK) and ATF-6, two other pathways activated by the UPR, show comparable activation between KFs and NFs. We confirmed that there is enhanced activation of XBP-1 by demonstrating increased nuclear localization of XBP-1 using immunofluorescence. CONCLUSION In contrast to our initial hypothesis that keloids would have broad activation of the UPR, we demonstrate here that there is a specific up-regulation of one facet of the UPR response. This may represent a specific molecular defect in KFs compared with NFs, and also suggests modulation of the UPR can be used in wound healing therapy.

Comparative Effectiveness of Two Ultrasound-Guided Regional Block Techniques for Surgical Anesthesia in Open Unilateral Inguinal Hernia Repair

Transversus abdominis plane (TAP) and ilioinguinal/iliohypogastric (II/IH) nerve blocks have been described as analgesic adjuncts for inguinal hernia repair, but the efficacy of these techniques in providing intraoperative anesthesia, either individually or together, is not known. We designed this retrospective cohort study to test the hypothesis that combining TAP and II/IH nerve blocks (“double TAP” technique) results in greater accordance between the preoperative anesthetic plan and actual anesthetic technique provided when compared to TAP alone. Based on this study, double TAP may be preferred for patients undergoing open inguinal hernia repair who wish to avoid general anesthesia.