Imaging of Single Antigens, Antibodies, and Specific Immunocomplex Formation by Scanning Force Microscopy

The most sensitive analytical techniques available today for detecting immuno assay complexes are radio or enzyme immuno analytical techniques, by which quantities of 107-108 analyte molecules can be detected. With the introduction of scanning force microscopy, a new method for detecting biological processes became available. Here, we examine the feasibility of using scanning force microscopy as a biosensitive tool. We demonstrate that single or multiple rabbit anti-human serum albumin molecules form complexes with preadsorbed single human serum albumin molecules on mica. However, no interaction is observed between human immunoglobulin G molecules and preadsorbed single albumin molecules; only separate antigens and antibodies are observed at random positions on the mica. This shows the ability of scanning force microscopy to act as a biosensor for detection of immunocomplexes, and to act as a very powerful tool to study molecule-surface interactions in general

A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells.

Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2

Cytokine and Chemokine Recovery Is Increased by Colloid Perfusates during Dermal Microdialysis

Cytokines and chemokines play important roles in cell signalling, and microdialysis is a promising tool for monitoring these inflammation markers ex vivo. Therefore, the collecting of these mediators at the highest concentrations possible is crucial. Depending on the size of the mediator of interest, the collection of these high molecular mass molecules has thus far been difficult due to their low recovery, even when using high cut-off (100 kDa) microdialysis membranes. This study aimed to optimize the recovery of various cytokines and chemokines by validating the use of different perfusates in cutaneous microdialysis, and comparing intravenous (i.v.) colloids, crystalloids, and a lipid emulsion formulations that are approved for i.v. applications. Methods: In vitro and in vivo recovery experiments using six recombinant cytokines varying in molecular size (interleukin-2 (15 kDa), interleukin-6 (20.5 kDa), interleukin-8 (8 kDa), interleukin-12p70 (70 kDa), TNF-α (17.5 kDa), and vascular endothelial growth factor (VEGF) (38 kDa)) were performed in the presence of different perfusates for i.v. applications: Ringer’s lactate, dextran 60 kDa, hydroxyethyl starch 70 kDa, and hydroxyethyl starch 200 kDa solutions as well as a lipid emulsion formulation. Recovery was determined through (i) microdialysis of cytokines and chemokines in Ringer’s lactate solution or human serum in vitro, and (ii) retrodialysis of excised porcine and human skin cadavers in vitro and porcine skin in vivo. Furthermore, we used skin trauma (catheter insertion) and Ultraviolet B irradiation of 3 × 3 cm2 skin areas to sample cytokines and chemokines in vivo and compared the amounts that were obtained using crystalloid and colloid perfusates. All the cytokines and chemokines within the dialysates were quantified through a flow cytometry-based bead array assay. Results: Overall, recovery was strongly increased by the colloids, particularly hydroxyethyl starch 70 kDa, in vitro, ex vivo, and in vivo. When compared with the recovery achieved using Ringer’s lactate, this increase was most effective for proteins ranging from 8 to 20.5 kDa. Hydroxyethyl starch 70 kDa significantly increased the recovery of interleukin (IL)-8 in human serum in vitro when compared with Ringer’s lactate. More cytokines and chemokines were recovered using colloids compared with crystalloids. However, the increase in recovery values was lower for IL-12p70 and VEGF. Conclusions: Regarding the dialysate volumes and final dialysate concentrations, colloid perfusates are overall superior to crystalloid perfusates, such as Ringer’s lactate, when sampling cytokines and chemokines, resulting in higher recoveries. However, the sampling of high-molecular-mass cytokines during microdialysis remains challenging, and experimental in vitro data are not completely comparable with data obtained ex vivo or in vivo

Expression of Potential Dermal Progenitor Cell Markers in the Tumour and Stroma of Skin Adnexal Malignant and Benign Tumours

Stem cells are multipotent cells that maintain the skin epidermis including skin appendages such as hair follicle, sebaceous glands, and sweat glands. There is evidence that reciprocal signalling between the epidermis and the dermis plays an important role in skin development, homeostasis, wound repair, and skin cancer. The origin of skin cancer that derive from skin appendages is still controversial, including basal cell carcinoma and even more of rare tumours such as sebaceous carcinomas and whether those tumours originate from resident tissue stem cells. To investigate whether markers reported to label dermal progenitor cells are preserved in the tumour including the tumour stroma of skin adnexal tumours, we tested 45 human basal cell carcinomas, including superficial, nodular, adenoid, infiltrating, and sclerosing types, and further 38 human tumours of skin appendages including 13 sebaceous adenomas and carcinomas, 20 eccrine sweat gland tumours, and 5 pilomatricomas, syringomas, and hair follicle tumours for the expression of the potential dermal and epidermal cell markers CRABP1, Nestin, and Ephrin B2 and compared these findings with healthy, age-related human epidermis. We detected that CRABP1, Nestin, and Ephrin B2 are expressed in the intratumoural stroma as well as the tumour invasive front of skin tumours of appendages and BCCs

p53 activity contributes to defective interfollicular epidermal differentiation, in hyperproliferative murine skin

This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/bjd.14048Background-\ud The role of p53 in the pathogenesis of skin diseases such as plaque-type psoriasis has long been questioned but never resolved. \ud \ud Objectives- \ud In this study we set out to determine the contribution of p53 activity to defective interfollicular epidermal skin differentiation in a murine hyperproliferative skin model. \ud \ud Methods-\ud We used the tamoxifen-inducible K14MycER mouse model which exhibits abnormal epidermal differentiation in response to high MYC activity, crossed with p53 knock-out mice. \ud \ud Results-\ud We show that genetic deletion of p53 leads to improvements in granular layer formation. Furthermore, we show that p53 activity regulates down-stream expression of Keratin 6a, Pparb/d and Pparg and is regulated upstream by retinoic acid signalling-dependent mechanisms. \ud \ud Conclusion-\ud We conclude aberrant non-apoptotic p53 activity contributes, in-part, to abnormal differentiation and granular layer defects.This work was supported by the Prof. Fiona M. Watt via the MRC, Wellcome Trust, CRUK, EU FP7 programme, the University of Cambridge, Hutchison Whampoa Ltd. This work was also supported by A/ Prof. Ian M. Smyth and Monash University

Epidermal barrier defects link atopic dermatitis with altered skin cancer susceptibility

Atopic dermatitis can result from loss of structural proteins in the outermost epidermal layers, leading to a defective epidermal barrier. To test whether this influences tumour formation, we chemically induced tumours in EPI-/- mice, which lack three barrier proteins-Envoplakin, Periplakin, and Involucrin. EPI-/- mice were highly resistant to developing benign tumours when treated with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The DMBA response was normal, but EPI-/- skin exhibited an exaggerated atopic response to TPA, characterised by abnormal epidermal differentiation, a complex immune infiltrate and elevated serum thymic stromal lymphopoietin (TSLP). The exacerbated TPA response could be normalised by blocking TSLP or the immunoreceptor NKG2D but not CD4+ T cells. We conclude that atopy is protective against skin cancer in our experimental model and that the mechanism involves keratinocytes communicating with cells of the immune system via signalling elements that normally protect against environmental assaults

Rewiring of an Epithelial Differentiation Factor, miR-203, to Inhibit Human Squamous Cell Carcinoma Metastasis

SummaryMetastatic colonization of distant organs underpins the majority of human-cancer-related deaths, including deaths from head and neck squamous cell carcinoma (HNSCC). We report that miR-203, a miRNA that triggers differentiation in multilayered epithelia, inhibits multiple postextravasation events during HNSCC lung metastasis. Inducible reactivation of miR-203 in already established lung metastases reduces the overall metastatic burden. Using an integrated approach, we reveal that miR-203 inhibits metastasis independently of its effects on differentiation. In vivo genetic reconstitution experiments show that miR-203 inhibits lung metastasis by suppressing the prometastatic activities of three factors involved in cytoskeletal dynamics (LASP1), extracellular matrix remodeling (SPARC), and cell metabolism (NUAK1). Expression of miR-203 and its downstream effectors correlates with HNSCC overall survival outcomes, indicating the therapeutic potential of targeting this signaling axis

c-MYC-Induced Sebaceous Gland Differentiation Is Controlled by an Androgen Receptor/p53 Axis

SummaryAlthough the sebaceous gland (SG) plays an important role in skin function, the mechanisms regulating SG differentiation and carcinoma formation are poorly understood. We previously reported that c-MYC overexpression stimulates SG differentiation. We now demonstrate roles for the androgen receptor (AR) and p53. MYC-induced SG differentiation was reduced in mice lacking a functional AR. High levels of MYC triggered a p53-dependent DNA damage response, leading to accumulation of proliferative SG progenitors and inhibition of AR signaling. Conversely, testosterone treatment or p53 deletion activated AR signaling and restored MYC-induced differentiation. Poorly differentiated human sebaceous carcinomas exhibited high p53 and low AR expression. Thus, the consequences of overactivating MYC in the SG depend on whether AR or p53 is activated, as they form a regulatory axis controlling proliferation and differentiation

BLIMP1 Is Required for Postnatal Epidermal Homeostasis but Does Not Define a Sebaceous Gland Progenitor under Steady-State Conditions

SummaryB-lymphocyte-induced nuclear maturation protein 1 (BLIMP1) was previously reported to define a sebaceous gland (SG) progenitor population in the epidermis. However, the recent identification of multiple stem cell populations in the hair follicle junctional zone has led us to re-evaluate its function. We show, in agreement with previous studies, that BLIMP1 is expressed by postmitotic, terminally differentiated epidermal cells within the SG, interfollicular epidermis, and hair follicle. Epidermal overexpression of c-Myc results in loss of BLIMP1+ cells, an effect modulated by androgen signaling. Epidermal-specific deletion of Blimp1 causes multiple differentiation defects in the epidermis in addition to SG enlargement. In culture, BLIMP1+ sebocytes have no greater clonogenic potential than BLIMP1− sebocytes. Finally, lineage-tracing experiments reveal that, under steady-state conditions, BLIMP1-expressing cells do not divide. Thus, rather than defining a sebocyte progenitor population, BLIMP1 functions in terminally differentiated cells to maintain homeostasis in multiple epidermal compartments