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(-/-)gammac(-/-) 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(-/-)gammac(-/-) 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.

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.

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.

Effect of surgeon and hospital characteristics on outcome after pyloromyotomy.

BACKGROUND: Previous studies have suggested that the outcome after pyloromyotomy is improved with increased surgeon experience. Others have proposed that infants with pyloric stenosis are best treated by specialty-trained pediatric surgeons or at children's hospitals. HYPOTHESIS: Surgeon and hospital characteristics affect complications, length of stay, and hospital charges after pyloromyotomy. DESIGN: Data for a nationally representative sample of infants (n = 1277) who underwent pyloromyotomy in 2000 in the United States were obtained from the Kids' Inpatient Database. Surgeon and hospital volumes were stratified into quintiles. Multivariate analyses were performed to analyze the impact of surgeon and hospital volume on length of stay, charges, and major operative complications using models that accounted for the hierarchical structure of patient-, surgeon-, and hospital-level covariates. RESULTS: No association between surgeon volume and either length of stay or charges was observed. Higher surgeon volume, however, was associated with fewer complications (P<.001). Surgeons with the highest volume had a 90% lower risk of complications than those with the lowest volume. Higher hospital volume was associated with shorter length of stay (P<.001). No association between hospital volume and either charges or risk of complications was observed. CONCLUSIONS: Higher surgeon and hospital volumes are associated with better outcome among infants who are treated for pyloric stenosis. Identification of aspects of medical and surgical treatment that account for this finding may lead to improvement in the outcome of infants undergoing pyloromyotomy.

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

Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity.

Early, intermediate, and late effects of a surgical skills "boot camp" on an objective structured assessment of technical skills: a randomized controlled study.

BACKGROUND: Surgical interns enter residency with variable technical abilities and many feel unprepared to perform necessary procedures. We hypothesized that interns exposed to a preinternship intensive surgical skills curriculum would demonstrate improved competency over unexposed colleagues on a test of surgical skills and that this effect would persist throughout internship. STUDY DESIGN: We designed a 3-day intensive skills "boot camp" with simulation-based training on 10 topics. Interns were randomized to an intervention group (boot camp) or a control group (no boot camp). All interns completed a survey including demographic information, previous experience, and comfort with basic surgical skills. Both groups completed a clinical skills assessment focused on 4 topics: chest tube insertion, central line placement, wound closure, and the Fundamentals of Laparoscopic Surgery peg transfer task. We assessed both groups immediately (month 0), early postcurriculum (month 1), and late postcurriculum (month 6). RESULTS: Fifteen participants were in the intervention group and 13 were in the control group. Before boot camp, mean comfort levels were similar for the groups. All participants had minimal prior experience. Competency for chest tube insertion and central line placement were considerably higher for the boot camp group at months 0 and 1, although much of this difference disappeared by month 6. There was no substantial difference between the 2 groups in the Fundamentals of Laparoscopic Surgery peg transfer and wound closure skills. CONCLUSIONS: A surgical skills boot camp accelerates the learning curve for interns in basic surgical skills as measured by a technical skills examination for some skills, although these improvements diminished over time. This can augment traditional training and translate into fewer patient errors.

Cell permeant peptide analogues of the small heat shock protein, HSP20, reduce TGF-beta1-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.

Use of organotypic coculture to study keloid biology.

BACKGROUND: Keloids are pathologic scars afflicting a large segment of our population and for which there is no definitive therapy. The lack of an animal model for keloid formation has hampered study. We developed an in vitro organotypic skin model to simulate normal keloid biology, which may allow us to study keloid formation without an animal model. METHODS: Normal (NFs) and keloid (KFs) human fibroblasts were cultured in a collagen matrix to create a 3-dimensional dermal structure. Normal human keratinocytes (NKs) were cultured as a second layer on top and exposed to an air-fluid interface to allow differentiation into a mature keratinocyte layer. The organotypic skin was maintained for 28 days in Dulbecco's modified eagle medium with 10% fetal calf serum. Samples were collected, processed, sectioned, stained with hematoxylin and eosin, and then measured for qualitative analysis. alpha-smooth-muscle actin was also evaluated by immunoblotting. RESULTS: KF/NK organotypic skin showed increased collagen deposition, based on significantly denser collagen staining, with increased dermal thickness compared with NF/NK organotypic skin. We saw increased contracture in the KF/NK construct, and this correlated with increased organization of alpha-smooth-muscle actin fibers in the dermal layer of KF/NK organotypic skin compared with NF/NK skin. CONCLUSIONS: We have shown that coculture of KFs with keloid keratinocytes leads to an increased collagen production and dermal contracture compared with NFs and NKs, consistent with known keloid behavior. Given the lack of an animal model, we believe that organotypic skin culture can serve as a surrogate to study keloid formation.

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.

The integrin alpha5beta1 regulates alphavbeta3-mediated extracellular signal-regulated kinase activation.

BACKGROUND: Integrin-mediated cell migration is essential for wound repair. Previous studies have shown that the interaction between integrins and the extracellular matrix (ECM) can initiate intracellular signaling pathways to regulate cell movement. Both the focal adhesion kinase (FAK) and the extracellular signal-regulated kinase/activated mitogen-activated protein kinase (ERK/MAPK) signaling pathways are required for efficient cell migration. Our previous work has shown that co-expression of the integrin alpha5beta1 inhibits alphavbeta3-mediated cell migration. We hypothesized that alpha5beta1 may regulate cell migration by modulating these alphavbeta3-mediated intracellular signaling events. METHODS: CHO B3 (alphavbeta3+) and B3C5 (alphavbeta3+/alpha5beta1+) cells were monitored by flow cytometry to determine integrin expression. Cells were allowed to migrate on fibrinogen (FBG)-coated transwells, with or without PD98059, an inhibitor of the ERK activator, mitogen-activated protein kinase kinase (MEK). Fixation, staining, and cell counting were used to quantify cell migration. Cells adherent to FBG were lysed and analyzed for FAK and ERK/MAPK activation by immunoblotting followed by image analysis densitometry. All experiments were repeated in triplicate. RESULTS: Treatment with PD98059 significantly decreased alphavbeta3-mediated cell migration on FBG (P = 0.0001) to a level comparable to untreated B3C5 cells. Following adhesion to FBG, B3 cells demonstrated a marked increase in ERK/MAPK activation compared to B3C5 cells. However, no significant difference was detected in FAK activation. CONCLUSION: Signaling through the ERK/MAPK pathway is required for efficient alphavbeta3-mediated migration on FBG. Inhibition of alphavbeta3-mediated migration by the integrin alpha5beta1 correlates with altered intensity and duration of ERK/MAPK activation, but not FAK activation, in response to adhesion. This suggests a mechanism for the regulatory effect of alpha5beta1 on alphavbeta3-mediated cell migration.

De novo expression of the integrin alpha5beta1 regulates alphavbeta3-mediated adhesion and migration on fibrinogen.

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin alphavbeta3 in the absence of alpha5beta1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, alphavbeta3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that alphavbeta3 exists in a higher affinity state in these cells. De novo expression of wild-type alpha5beta1 negatively regulates alphavbeta3-mediated adhesion and migration. This effect is not seen with expression of a chimeric alpha5beta1 integrin in which the cytoplasmic portion of the alpha5 integrin subunit is replaced by the cytoplasmic portion of the alpha4 integrin. In addition, it does not require ligation of alpha5beta1 by fibronectin. Cells that express a constitutively active beta3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of alpha5beta1 expression. These data provide additional evidence of "cross-talk" between the integrins alpha5beta1 and alphavbeta3, and support the idea that alpha5beta1 regulates alphavbeta3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in alpha5beta1 expression in vivo may modulate activation of alphavbeta3 to influence its adhesive function.