Aviad Keren

The p38 MAPK signaling pathway: a major regulator of skeletal muscle development.

Skeletal muscle development is regulated by extracellular growth factors that transmit largely unknown signals into the cell affecting the muscle-transcription program. One intracellular signaling pathway activated during the differentiation of myogenic cell lines is p38 mitogen-activated protein kinase (MAPK). As a result of modifying the activity of p38 in myoblasts, the pathway proved essential for the expression of muscle-specific genes. P38 affects the activities of transcription factors from the MyoD and MEF2 families and participates in the remodeling of chromatin at specific muscle-regulatory regions. P38 cooperates with the myogenic transcription factors in the activation of a subset of late-transcribed genes, hence contributing to the temporal expression of genes during differentiation. Recent developmental studies with mouse and Xenopus embryos, substantiated and further extended the essential role of p38 in myogenesis. Evidence exists supporting the crucial role for p38 signaling in activating MEF2 transcription factors during somite development in mice. In Xenopus, p38 signaling was shown to be needed for the early expression of Myf5 and for the expression of several muscle structural genes. The emerging data indicate that p38 participates in several stages of the myogenic program.

What causes alopecia areata

The pathobiology of alopecia areata (AA), one of the most frequent autoimmune diseases and a major unsolved clinical problem, has intrigued dermatologists, hair biologists and immunologists for decades. Simultaneously, both affected patients and the physicians who take care of them are increasingly frustrated that there is still no fully satisfactory treatment. Much of this frustration results from the fact that the pathobiology of AA remains unclear, and no single AA pathogenesis concept can claim to be universally accepted. In fact, some investigators still harbour doubts whether this even is an autoimmune disease, and the relative importance of CD8+ T cells, CD4+ T cells and NKGD2+ NK or NKT cells and the exact role of genetic factors in AA pathogenesis remain bones of contention. Also, is AA one disease, a spectrum of distinct disease entities or only a response pattern of normal hair follicles to immunologically mediated damage? During the past decade, substantial progress has been made in basic AA‐related research, in the development of new models for translationally relevant AA research and in the identification of new therapeutic agents and targets for future AA management. This calls for a re‐evaluation and public debate of currently prevalent AA pathobiology concepts. The present Controversies feature takes on this challenge, hoping to attract more skin biologists, immunologists and professional autoimmunity experts to this biologically fascinating and clinically important model disease.

Autoimmune Disease Induction in a Healthy Human Organ: A Humanized Mouse Model of Alopecia Areata

Abbreviations: AA, alopecia areata; IP, immune privilege; NK, natural killer; PBMCs, peripheral blood mononuclear cells; PHA, phytohemagglutinin

Abnormal interactions between perifollicular mast cells and CD8+ T-cells may contribute to the pathogenesis of alopecia areata

Alopecia areata (AA) is a CD8+ T-cell dependent autoimmune disease of the hair follicle (HF) in which the collapse of HF immune privilege (IP) plays a key role. Mast cells (MCs) are crucial immunomodulatory cells implicated in the regulation of T cell-dependent immunity, IP, and hair growth. Therefore, we explored the role of MCs in AA pathogenesis, focusing on MC interactions with CD8+ T-cells in vivo, in both human and mouse skin with AA lesions. Quantitative (immuno-)histomorphometry revealed that the number, degranulation and proliferation of perifollicular MCs are significantly increased in human AA lesions compared to healthy or non-lesional control skin, most prominently in subacute AA. In AA patients, perifollicular MCs showed decreased TGFβ1 and IL-10 but increased tryptase immunoreactivity, suggesting that MCs switch from an immuno-inhibitory to a pro-inflammatory phenotype. This concept was supported by a decreased number of IL-10+ and PD-L1+ MCs, while OX40L+, CD30L+, 4–1BBL+ or ICAM-1+ MCs were increased in AA. Lesional AA-HFs also displayed significantly more peri- and intrafollicular- CD8+ T-cells as well as more physical MC/CD8+ T-cell contacts than healthy or non-lesional human control skin. During the interaction with CD8+ T-cells, AA MCs prominently expressed MHC class I and OX40L, and sometimes 4–1BBL or ICAM-1, suggesting that MC may present autoantigens to CD8+ T-cells and/or co-stimulatory signals. Abnormal MC numbers, activities, and interactions with CD8+ T-cells were also seen in the grafted C3H/HeJ mouse model of AA and in a new humanized mouse model for AA. These phenomenological in vivo data suggest the novel AA pathobiology concept that perifollicular MCs are skewed towards pro-inflammatory activities that facilitate cross-talk with CD8+ T-cells in this disease, thus contributing to triggering HF-IP collapse in AA. If confirmed, MCs and their CD8+ T-cell interactions could become a promising new therapeutic target in the future management of AA.

The PDE4 inhibitor, apremilast, suppresses experimentally induced alopecia areata in human skin in vivo

Jungersted JM, Hellgren LI, Jemec GB, Agner T.. Lipids and skin barrier function – a clinical perspective. Contact Dermat 2008;58:255–62. Ishikawa J, Narita H, Kondo N, Hotta M, Takagi Y, Masukawa Y, et al. Changes in the ceramide profile of atopic dermatitis patients. J Invest Dermatol 2010;130:2511–4. van Smeden J, Janssens M, Kaye EC, Caspers PJ, Lavrijsen AP, Vreeken RJ, et al. The importance of free fatty acid chain length for the skin barrier function in atopic eczema patients. Exp Dermatol 2014;23:45–52. Joo KM, Nam GW, Park SY, Han JY, Jeong HJ, Lee SY, et al. Relationship between cutaneous barrier function and ceramide species in human stratum corneum. J Dermatol Sci 2010;60:47–50. Joo KM, Choi D, Park YH, Yi CG, Jeong HJ, Cho JC, et al. A rapid and highly sensitive UPLC-MS/MS method using pre-column derivatization with 2-picolylamine for intravenous and percutaneous pharmacokinetics of valproic Kyung-Mi Joo, Jee-Hyun Hwang, SeungJin Bae, Dong-Ho Nahm, Hae-Sim Park, Young-Min Ye**, Kyung-Min Lim* AmorePacific RD College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea; Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, Republic of Korea

Treating fat grafts with human endothelial progenitor cells promotes their vascularization and improves their survival in diabetes mellitus.

Background: Bone marrow–derived endothelial progenitor cells are required for vascularization of a fat graft to form a functional microvasculature within the graft and to facilitate its integration into the surrounding tissues. Organ transplantation carries a high risk of graft loss and rejection in patients with diabetes mellitus because endothelial progenitor cell function is impaired. The authors investigated the influence of endothelial progenitor cell treatment on the phenotype and survival of human fat grafts in immunocompromised mice with experimentally induced diabetes mellitus. Methods: The authors injected 1 ml of human fat tissue into the scalps of 14 nondiabetic and 28 diabetic immunocompromised mice, and then treated some of the grafts with endothelial progenitor cells that were isolated from the blood of a human donor. The phenotype of the endothelial progenitor cell–treated fat grafts from the 14 diabetic mice was compared with that of the untreated fat grafts from 14 nondiabetic and 14 diabetic mice, 18 days and 15 weeks after fat transplantation. Determination of graft phenotype included measurements of weight and volume, vascular endothelial growth factor levels, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase, and caspase 3 expression levels, and histologic analysis of the extent of vascularization. Results: The untreated grafts from the diabetic mice were fully resorbed 15 weeks after fat transplantation. The phenotype of endothelial progenitor cell–treated fat grafts from the diabetic mice was similar to that of the untreated fat grafts from the nondiabetic mice. Conclusion: Endothelial progenitor cell treatment of transplanted fat can increase the survival of a fat graft by inducing its vascularization and decreasing the extent of apoptosis.

Neural ectoderm-secreted FGF initiates the expression of Nkx2.5 in cardiac progenitors via a p38 MAPK/CREB pathway

Vertebrate heart development is derived from paired primordia of anterior dorsolateral mesoderm expressing Nkx2.5 and GATA4 transcription factors. Yet growth factors and intracellular pathways specifying heart precursor gene expression are poorly understood. In the present work, we investigated the signaling events initiating Nkx2.5 expression in Xenopus laevis. We describe here that fibroblast growth factor (FGF) initiates the expression of Nkx2.5 without affecting GATA4. At gastrula, FGF3 is expressed in anterior neural ectoderm, and results presented here indicate that this tissue is involved in the induction of Nkx2.5 expression in neighboring lateral tissues. Further studies indicate that the intracellular p38 MAPK and the CREB transcription factor function downstream of FGF to initiate Nkx2.5 expression. Activation of the p38 MAPK pathway and of the CREB protein is both necessary and sufficient for the initial expression of Nkx2.5. Therefore, we would like to suggest that FGF expressed in anterior neural ectoderm is a major inducer of Nkx2.5 expression in neighboring cells. In these cells, FGF activates an intracellular p38 MAPK signaling pathway and its downstream target, the CREB transcription factor, all participating in the expression of Nkx2.5 in cardiac progenitors.

A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus.

Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/beta-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.

A new humanized mouse model for alopecia areata

Although alopecia areata (AA) is not life threatening, it may lead to severe psychological disturbances, reducing the quality of life in all ages. Thus, a new animal model is needed for shedding more light onto the pathogenesis of this cell-mediated, organ-specific autoimmune disease to identify more effective therapeutic strategies. Recently, we succeeded in developing a new humanized mouse model of AA, which includes transplantation of healthy human scalp skin obtained from normal volunteers on to severe-combined immunodeficient mice. This is followed by intradermal injection of either autologous or allogeneic peripheral blood mononuclear cells, which had been cultured with high dose of IL-2 and enriched for natural killer group 2D-positive (NKG2D+) and CD56+ cells. This protocol leads to rapid and predictable development of focal hair loss, with all the characteristic clinical, histological, and immunohistochemical features of AA. This humanized mouse AA model underscores the functional importance of NKG2D+ and CD56+ cells in AA pathogenesis and promises to be instrumental for identifying novel AA treatment strategies.

Hybrid wound dressings with controlled release of antibiotics: Structure-release profile effects and in vivo study in a guinea pig burn model.

Over the last decades, wound dressings have evolved from a crude traditional gauze dressing to tissue-engineered scaffolds. Many types of wound dressing formats are commercially available or have been investigated. We developed and studied hybrid bilayer wound dressings which combine a drug-loaded porous poly(dl-lactic-co-glycolic acid) top layer with a spongy collagen sublayer. Such a structure is very promising because it combines the advantageous properties of both layers. The antibiotic drug gentamicin was incorporated into the top layer for preventing and/or defeating infections. In this study, we examined the effect of the top layers structure on the gentamicin release profile and on the resulting in vivo wound healing. The latter was tested on a guinea pig burn model, compared to the neutral non-adherent dressing material Melolin® (Smith & Nephew) and Aquacel® Ag (ConvaTec). The release kinetics of gentamicin from the various studied formulations exhibited burst release values between 8% and 38%, followed by a drug elution rate that decreased with time and lasted for at least 7 weeks. The hybrid dressing, with relatively slow gentamicin release, enabled the highest degree of wound healing (28%), which is at least double that obtained by the other dressing formats (8-12%). It resulted in the lowest degree of wound contraction and a relatively low amount of inflammatory cells compared to the controls. This dressing was found to be superior to hybrid wound dressings with fast gentamicin release and to the neat hybrid dressing without drug release. Since this dressing exhibited promising results and does not require frequent bandage changes, it offers a potentially valuable concept for treating large infected burns.