In this contribution we report on the design of new versatile fluorescent linkers (L1-L3, L1N) suitable for the covalent assembly of specific recognition motifs to bind biologically relevant targets such as HER2 receptors. The central fluorescent molecular platform emits in red and represents a key building block for the synthesis of conjugates and their subsequent analyses. The presence of amino groups enables straightforward functionalization with bifunctional PEG linkers (NHS, MI) of variable length for further directional bioconjugation. To demonstrate the value of this approach, the linkers were conjugated with chemically synthesized affibodies (AfB, ZHER2:2891(M9Nle/D37E)), bearing a unique cysteine at N-terminal or C-terminal end. Indeed, this AfB binds the human epidermal growth factor receptor 2 (HER2), which is often overexpressed in different carcinomas, and it is therefore considered as a tumour marker in bioimaging and drug delivery systems. Seven AfB conjugates-three mono-AfBs (N1, N1N, C1) and four di-AfBs (N2, N3, C2, C3)-were obtained through MI-thiol conjugation. The binding of fluorescent mono- and di-affibody (AfB) constructs was evaluated using flow cytometry to demonstrate their ability to interact with the HER2-expressing SKOV3 cells in relation with their structural properties. All tested conjugates specifically bound to HER2 receptors present on SKOV3 cells with nanomolar affinities, and only small differences between constructs were detected. The presence of our fluorescent conjugates bound to the cell surface was visualized using confocal microscopy. In addition, the internalization mechanism of AfB constructs in SKOV3 cells was investigated due to the measurement of their fluorescence following trypsin treatment. In conclusion, our innovative synthetic approach with the integration of a versatile fluorescent platform offers new perspectives for monitoring molecular therapeutics inside cell and for tuning multivalent conjugates for cancer immunotherapy.

This paper provides an overview of past and new experimental studies with kHz helium plasma jets and floating-electrode dielectric barrier discharge (DBD) in the context of biomedical applications with the aim to discuss the interaction of plasma jet with skin tissue models. The key motivation is to summarize and address perspectives on the understanding of the mode of action of nonthermal plasma from surface delivery to the biological response of deeper and deeper tissues. First, the significant impact of the target exposed to the plasma jet on the plasma characteristics is documented, highlighting the importance of considering this interaction for in vivo studies. Next, cell permeabilization was first reported and translated to the study of plasma jet permeation of reconstructed epidermis, human explants, and human tissues. Strong analogies are observed in all three substrates, demonstrating a potent but transient modulation of surface features and skin barrier function for a few minutes following a brief plasma exposure time of a few tens of seconds. Interestingly, the modulation of reactive oxygen and nitrogen species generation, so-called RONS, with the variation of the pulse repetition rate of the plasma jet shows no direct correlation with the permeation efficiency. This questions the role of RONS alone in the mode of action of nonthermal plasma for biological response in the few hundreds of microns to the few millimeter tissue layers as was previously also questioned for subcutaneous action of plasma in wound and tumor in vivo treatments. Finally, the combinative role of RONS with electrical factors (charging, current, electric field) is hypothesized and supported with the investigation of deeper living skin tissue oxygenation and vasodilation.

The skin is made up of different layers with various gradients, which maintain a complex microenvironment, particularly in terms of oxygen levels. However, all types of skin cells are cultured in conventional incubators that do not reproduce physiological oxygen levels. Instead, they are cultured at atmospheric oxygen levels, a condition that is far removed from physiology and may lead to the generation of free radicals known to induce skin ageing. This review aims to summarize the current literature on the effect of physiological oxygen levels on skin cells, highlight the shortcomings of current in vitro models, and demonstrate the importance of respecting skin oxygen levels. We begin by clarifying the terminology used about oxygen levels and describe the specific distribution of oxygen in the skin. We review and discuss how skin cells adapt their oxygen consumption and metabolism to oxygen levels environment, as well as the changes that are induced, particularly, their redox state, life cycle and functions. We examine the effects of oxygen on both simple culture models and more complex reconstructed skin models. Finally, we present the implications of oxygen modulation for a more therapeutic approach.

PDF - 1037KB, Effect of the IFP1.4 and mCherry expression on the B16F10 cells proliferation.

PDF - 2157KB, Characterization of the transfected clones for msVEGFR2 and mVEGF-A production.

PDF - 55KB, Shortening of the minCMV promoter.

Keratinocytes prevent skin photoaging by ensuring the defence against oxidative stress, an excessive production of reactive oxygen species (ROS). They are localized within the epidermis where the oxygen level (1-3% O2), named physioxia, is low compared to other organs. Oxygen is essential for life but also generates ROS. Most of the in vitro studies on keratinocyte antioxidant capacities are performed under atmospheric oxygen, named normoxia, which is very far from the physiological microenvironment, thus submitting cells to an overoxygenation. The present study is aimed at investigating the antioxidant status of keratinocyte grown under physioxia in both 2D and 3D models. First, we show that the basal antioxidant profiles of keratinocytes display important differences when comparing the HaCaT cell line, primary keratinocytes (NHEK), reconstructed epidermis (RHE), and skin explants. Physioxia was shown to promote a strong proliferation of keratinocytes in monolayers and in RHE, resulting in a thinner epidermis likely due to a slowdown in cell differentiation. Interestingly, cells in physioxia exhibited a lower ROS production upon stress, suggesting a better protection against oxidative stress. To understand this effect, we studied the antioxidant enzymes and reported a lower or equivalent level of mRNA for all enzymes in physioxia conditions compared to normoxia, but a higher activity for catalase and superoxide dismutases, whatever the culture model. The unchanged catalase amount, in NHEK and RHE, suggests an overactivation of the enzyme in physioxia, whereas the higher amount of SOD2 can explain the strong activity. Taken together, our results demonstrate the role of oxygen in the regulation of the antioxidant defences in keratinocytes, topic of particular importance for studying skin aging. Additionally, the present work points out the interest of the choice of both the keratinocyte culture model and the oxygen level to be as close as possible to the in situ skin.

PDF - 2705KB, Expression of VEGFRs on the surface of B16F10-msVEGFR2 melanoma clone 13.3 and 16.4.

PDF - 3176KB, Characterization of B16F10 wt and msVEGFR2 expressing clones 13.3 and 16.4 tumors in vivo.

PDF - 150KB, Supplementary Materials and Methods and legends for Supplementary Figures 1 through 6.

PDF - 86KB, Construction maps for the pIFP1.4-HREmsVEGFR2 and pHREmsVEGFR2 vector,

Pathologic angiogenesis directly responds to tumour hypoxia and controls the molecular/cellular composition of the tumour microenvironment, increasing both immune tolerance and stromal cooperation with tumour growth. Myo-inositol-trispyrophosphate (ITPP) provides a means to achieve stable normalization of angiogenesis. ITPP increases intratumour oxygen tension (pO(2)) and stabilizes vessel normalization through activation of endothelial Phosphatase-and-Tensin-homologue (PTEN). Here, we show that the tumour reduction due to the ITPP-induced modification of the tumour microenvironment by elevating pO(2) affects the phenotype and properties of the immune infiltrate. Our main observations are as follows: a relative change in the M1 and M2 macrophage-type proportions, increased proportions of NK and CD8(+)T cells, and a reduction in Tregs and Th2 cells. We also found, in vivo and in vitro, that the impaired access of PD1(+)NK cells to tumour cells is due to their adhesion to PD-L1(+)/PD-L2(+) endothelial cells in hypoxia. ITPP treatment strongly reduced PD-L1/PD-L2 expression on CD45+/CD31+ cells, and PD1(+) cells were more numerous in the tumour mass. CTLA-4(+) cell numbers were stable, but level of expression decreased. Similarly, CD47(+) cells and expression were reduced. Consequently, angiogenesis normalization induced by ITPP is the mean to revert immunosuppression into an antitumor immune response. This brings a key adjuvant effect to improve the efficacy of chemo/radio/immunotherapeutic strategies for cancer treatment.

The melanocortin 1 receptor (MC1R) is a G-protein coupled receptor (GPCR) which plays a major role in controlling melanogenesis. A large body of evidence indicates that GPCRs are part of large protein complexes that are critical for their signal transduction properties. Among proteins which may affect MC1R signaling, neurofibromin (Nf1), a GTPase activating protein (GAP) for Ras, is of special interest as it regulates adenylyl cyclase activity and ERK signaling, two pathways involved in MC1R signaling. Moreover, mutations in this gene encoding Nf1 are responsible for neurofibromatosis type I, a disease inducing hyperpigmented flat skin lesions. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer experiments we demonstrated a physical interaction of Nf1 with MC1R. In particular, the GAP domain of Nf1 directly and constitutively interacts with MC1R in melanocytes. Pharmacologic and genetic approaches revealed that the GAP activity of Nf1 is important to regulate intracellular signaling pathways involved in melanogenesis and, consequently, melanogenic enzyme expression and melanin production. These finding shed new light on the understanding and cure of skin pigmentation disorders.

Advances in medicine, and particularly those concerning dermatology and microbial decontamination of biological surfaces, have recently led several laboratories to take an interest in the applications of atmospheric pressure cold plasmas to (e.g. [1][2][3]), a field in which the use of physical principles (e.g. weak current, light, ultrasound, lasers) is more widespread. At the same time, there are already plasma-based systems for skin treatments on the market, many of which use thermal effect mediated by plasmas whose action, linked to local energy delivery and increase in temperature [4], is fundamentally different from that of non-equilibrium cold plasmas. In addition, the treatment using thermal plasmas, usually involve damages to healthy skin (mostly erythema and micro-burns) which should not be sought in the case of daily cosmetic treatments. Considering the above, it appeared important to discuss what could be transferred from achievements in cold medicine to cosmetics, to clarify the contours of what plasma cosmetics can be and to appreciate what are the market needs in this sector. That also extends to decontamination and to packaging for cosmetic products as used medicine sources can also serve to induce surface modifications of interest in this domain. To this end, the first meeting on Plasma Cosmetic Science (IMPCS1) was organized in Orleans, France, in November 2019. In this presentation, after recalling the general context, we will address the main themes dealt with during this meeting and we will set out the main lessons learned from the presentations and discussions concerning the future opportunities for Plasma Cosmetics. IMPCS1 was supported by Le Studium and Region Centre-Val de Loire. The authors belongs to the Plasma Cosmetic Consortium financed by the Le Studium Loire Valley Institute for Advanced Studies References [1] E. Robert, G. Busco, C. Grillon and J-M Pouvesle, “Potential of low temperature atmospheric pressure sources in cosmetic”, COSMINNOV 2016, Orleans, France, May 25 2016 [2] K.Y. Baik “Application of atmospheric pressure treated water for hair loss therapy” ISPB 2017, Jeju, Korea, June 27 2017 [3] Th von Woedtke, H-S. Metelmann and K-D Weltmann, “Plasma in cosmetic applications: possibilities and boundary conditions”, ISPB 2018, Incheon, Korea, July 25 2018 [4] Foster, K. W et al, “Advances in skin regeneration. Journal of Cosmetic Dermatology, 7: 169-179 (2008)