Jackie R. Bickenbach

Genetic disorders of keratin: are scarring alopecias a sub-set?

Recent advances have challenged the prevailing view that keratins are merely passive bystanders of keratinocyte biology. With the exciting discovery that three autosomal dominant genetic skin disorders, epidermolysis bullosa simplex (EBS), epidermolytic hyperkeratosis (EHK) and palmoplantar keratoderma (PPK), are in fact disorders of keratins comes the realization that the integrity of the keratin filament network is crucial to the structural integrity of the skin. Since it has been recently established that mutations in keratins K5/K14, K1/K10 and K9 are causative for these keratinocyte disorders, it is very likely that mutations in K6 or in its obligate partner, K16 will result in disease. In order to test this we have produced transgenic mice that express a mutant K6 gene. These mice develop a progressive scarring alopecia at about 6 months of age. Later, the denuded areas developed a keratosis which was prone to infection. Ultrastructural analysis suggests that hair loss is due to the destruction of the outer root sheath. We believe that these mice are models of another keratin disorder.

HSP70 and EndoG modulate cell death by heat in human skin keratinocytes in vitro.

We examined how young and old keratinocytes died from heat stress in vitro. We found that keratinocyte cell death was not due to oxidative stress as neither Mn-SOD nor Cu-Zn-SOD was produced in either young or old heated keratinocytes. Instead, analysis of the anti-apoptotic factors, Bcl2 and HSP70, and the pro-apoptotic factors, caspase 3, caspase 8, Apaf-1, cytochrome c, AIF, and EndoG, indicated that keratinocyte cell death occurred via the caspase-independent EndoG apoptotic pathway. We found that both young and old keratinocytes died via the same pathway, and that we could specifically reduce both young and old keratinocyte death by addition of the EndoG inhibitor NEM. Further analysis suggested that the difference between young and old keratinocyte death was due to the synthesis of HSP70 protein, with the increase in response to heat more pronounced in young keratinocytes than in old keratinocytes. When we inhibited HSP70 by adding quercetin, death was increased in both young and old keratinocytes, but more so in old keratinocytes. These data suggest that old keratinocytes may die more readily than young keratinocytes when heated because they synthesize HSP70 at a lower efficiency. Such findings suggest that HSP70 production may be age-dependent.

Immunological Gene Therapy Approaches for Malignant Melanoma

Immuno-gene therapy approaches for the treatment of malignant melanoma are categorized into two major subgroups according to an active or passive immunological principle. Active immuno-gene therapy is

Human Skin Keratinocytes Can Be Reprogrammed to Express Neuronal Genes and Proteins After a Single Treatment with Decitabine

Abstract Patient-specific cell replacement therapy is fast becoming the future of medicine, requiring safe, effective methods for reprogramming a patients own cells. Previously, we showed that a single transient transfection with a plasmid encoding Oct4 was sufficient to reprogram human skin keratinocytes (HSKs), and that this transfection resulted in a decrease in global DNA methylation. In more recent work we showed that decreasing global DNA methylation using the U.S. Food and Drug Administration–approved cancer treatment drug decitabine was sufficient to induce expression of endogenous Oct4. Here we report that a single treatment with decitabine, followed by 5 days in a defined neuronal transformation medium, then 7 days in a neuronal maintenance medium is sufficient to convert HSKs into cells that change their morphology substantially, gain expression of neuronal markers, and lose expression of keratinocyte markers. Within 1 week of treatment the cells express mRNA for β3-tubulin and doublecortin, and at the end of 2 weeks express mRNA for NeuN, FOXP2, and NCAM1. Additionally, at the end of this protocol, neurofilament-1, nestin, synapsin, FOXP2, and GluR1 proteins are detectable by immunostaining. Thus, we demonstrate a simple method that begins the process for producing cells for cell replacement therapies without using exogenously introduced DNA.

Adenovirus-Mediated Ex Vivo Immunogene and in Vivo Combination Gene Therapy Strategies Induce a Systemic Anti-Tumor Immune Defense in the Mouse B16 Melanoma Model

Most of the gene therapy studies in melanoma which are ongoing or have been performed in the recent past (clinical phase I/II trials) rely on a retroviral and an in vitro/ex vivo gene transfer [reviewed in 1,2]. As an alternative, we studied a recombinant, replication-deficient adenovirus (adv, serotype 5) system for its properties to transduce murine and human melanoma cells under in vivo and in vitro (ex vivo) gene transfer conditions in preclinical models [3–5]. Adv has already been translated to the clinical field by several current trials investigating the use of adv vectors for the treatment of cystic fibrosis [6]. In contrast to retroviruses, adv can be enriched to high titers because of its particular physico-chemical stability [7,8]. Moreover, adenoviral cell transduction does not depend upon division of the host cells, and adv sequences regularly do not integrate into the host genome, resulting in the added safety feature of little significant potential for insertional mutagenesis [7,8]. With regard to retroviral vectors, much concern arose when 3 of 10 primates developed T-cell lymphomas after immunosuppressive body irradiation and subsequent transfusion of autologous bone marrow cells, which had been genetically modified by a retroviral vector in vitro, and were also contaminated with replication competent retrovirus [9]. Wild-type adv seems to be more safe as proven by broad vaccination campaigns with oral administration of enteric-coated capsules containing live, unattenuated adv resulting only rarely in side effects [10,11]. Furthermore, the serotypes 2 and 5 of adv which are currently used as non-replicating vectors are not tumorigenic in rodents, and replicating wild types cause only mild respiratory infections in humans [10].