Intra-articular kinetics of a cartilage targeting cationic PEGylated protein for applications in drug delivery.

OBJECTIVES: Cartilage targeting cationic glycoprotein Avidin was PEGylated to synthesize a multi-arm Avidin (mAv) nano-construct with high drug loading content. Here we investigate mAv biodistribution and kinetics over a 7-day period following intra-articular (IA) administration in rat knee joints. METHODS: Labeled mAv was injected into healthy rat knees, and joint tissues (articular cartilage, menisci, ligaments, tendons, fat pad) were harvested following sacrifice at 6 h, 1, 4 and 7 days. Its IA biodistribution and retention were measured using fluorescence microscopy. Tissue localization was compared in young vs old rats by immunohistochemistry. mAv chondrotoxicity and immune response were evaluated to determine safe carrier dose limits. RESULTS: mAv penetrated through the full thickness of rat cartilage and other joint tissues within 6 h, remaining detectable within most joint tissues over 7 days. Intra-tissue uptake correlated strongly with tissue GAG concentration, confirming the dominant role of electrostatic interactions between positively charged mAv and the negatively charged aggrecan proteoglycans. mAv was uptaken by chondrocytes and also penetrated the osteocyte lacuno-canalicular system of peri-articular bone in both young and old rats. mAv did not cause cytotoxicity at concentrations up to 300 µM but elicited a dose dependent immunogenic response. CONCLUSIONS: mAv's ability to target a variety of joint tissues, chondrocytes, and peri-articular osteocytes without sequestration in synovial fluid makes it a versatile carrier for delivering a wide range of drugs for treating a broad class of musculoskeletal diseases. Drugs can be conjugated using simple aqueous based avidin-biotin reaction, supporting its clinical prospects.

Lentiviral-based reporter constructs for profiling chondrogenic activity in primary equine cell populations.

Successful clinical translation of mesenchymal stem cell (MSC)-based therapies for cartilage repair will likely require the implementation of standardised protocols and broadly applicable tools to facilitate the comparisons among cell types and chondroinduction methods. The present study investigated the utility of recombinant lentiviral reporter vectors as reliable tools for comparing chondrogenic potential among primary cell populations and distinguishing cellular-level variations of chondrogenic activity in widely used three-dimensional (3D) culture systems. Primary equine MSCs and chondrocytes were transduced with vectors containing combinations of fluorescent and luciferase reporter genes under constitutive cytomeglavirus (CMV) or chondrocyte-lineage (Col2) promoters. Reporter activity was measured by fluorescence imaging and luciferase assay. In 3D cultures of MSC aggregates and polyethylene glycol-hyaluronic acid (PEG-HA) hydrogels, transforming growth factor beta 3 (TGF-ß3)-mediated chondroinduction increased Col2 reporter activity, demonstrating close correlation with histology and mRNA expression levels of COL2A1 and SOX9. Comparison of chondrogenic activities among MSC populations using a secretable luciferase reporter revealed enhanced chondrogenesis in bone-marrow-derived MSCs relative to MSC populations from synovium and adipose tissues. A dual fluorescence reporter - enabling discrimination of highly chondrogenic (Col2-GFP) cells within an MSC population (CMV-tdTomato) - revealed marked heterogeneity in differentiating aggregate cultures and identified chondrogenic cells in chondrocyte-seeded PEG-HA hydrogels after 6 weeks in a subcutaneous implant model - indicating stable, long-term reporter expression in vivo. These results suggested that lentiviral reporter vectors may be used to address fundamental questions regarding chondrogenic activity in chondroprogenitor cell populations and accelerate clinical translation of cell-based cartilage repair strategies.

Harnessing extracellular vesicles to direct endochondral repair of large bone defects.

Large bone defects remain a tremendous clinical challenge. There is growing evidence in support of treatment strategies that direct defect repair through an endochondral route, involving a cartilage intermediate. While culture-expanded stem/progenitor cells are being evaluated for this purpose, these cells would compete with endogenous repair cells for limited oxygen and nutrients within ischaemic defects. Alternatively, it may be possible to employ extracellular vesicles (EVs) secreted by culture-expanded cells for overcoming key bottlenecks to endochondral repair, such as defect vascularization, chondrogenesis, and osseous remodelling. While mesenchymal stromal/stem cells are a promising source of therapeutic EVs, other donor cells should also be considered. The efficacy of an EV-based therapeutic will likely depend on the design of companion scaffolds for controlled delivery to specific target cells. Ultimately, the knowledge gained from studies of EVs could one day inform the long-term development of synthetic, engineered nanovesicles. In the meantime, EVs harnessed from in vitro cell culture have near-term promise for use in bone regenerative medicine. This narrative review presents a rationale for using EVs to improve the repair of large bone defects, highlights promising cell sources and likely therapeutic targets for directing repair through an endochondral pathway, and discusses current barriers to clinical translation. Cite this article: E. Ferreira, R. M. Porter. Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 2018;7:263-273. DOI: 10.1302/2046-3758.74.BJR-2018-0006.

Sustained intra-cartilage delivery of low dose dexamethasone using a cationic carrier for treatment of post traumatic osteoarthritis.

Disease-modifying osteoarthritis drugs (DMOADs) should reach their intra-tissue target sites at optimal doses for clinical efficacy. The dense, negatively charged matrix of cartilage poses a major hindrance to the transport of potential therapeutics. In this work, electrostatic interactions were utilised to overcome this challenge and enable higher uptake, full-thickness penetration and enhanced retention of dexamethasone (Dex) inside rabbit cartilage. This was accomplished by using the positively charged glycoprotein avidin as nanocarrier, conjugated to Dex by releasable linkers. Therapeutic effects of a single intra-articular injection of low dose avidin-Dex (0.5 mg Dex) were evaluated in rabbits 3 weeks after anterior cruciate ligament transection (ACLT). Immunostaining confirmed that avidin penetrated the full cartilage thickness and was retained for at least 3 weeks. Avidin-Dex suppressed injury-induced joint swelling and catabolic gene expression to a greater extent than free Dex. It also significantly improved the histological score of cell infiltration and morphogenesis within the periarticular synovium. Micro-computed tomography confirmed the reduced incidence and volume of osteophytes following avidin-Dex treatment. However, neither treatment restored the loss of cartilage stiffness following ACLT, suggesting the need for a combinational therapy with a pro-anabolic factor for enhancing matrix biosynthesis. The avidin dose used caused significant glycosaminoglycan (GAG) loss, suggesting the use of higher Dex : avidin ratios in future formulations, such that the delivered avidin dose could be much less than that shown to affect GAGs. This charge-based delivery system converted cartilage into a drug depot that could also be employed for delivery to nearby synovium, menisci and ligaments, enabling clinical translation of a variety of DMOADs.

Drug-induced photosensitivity: new insights into pathomechanisms and clinical variation through basic and applied science.

Drug-induced photosensitivity occurs when a drug is capable of absorbing radiation from the sun (usually ultraviolet A) leading to chemical reactions that cause cellular damage (phototoxicity) or, more rarely, form photoallergens (photoallergy). The manifestation varies considerably in presentation and severity from mild pain to severe blistering. Despite screening strategies and guidelines in place to predict photoreactive drugs during development there are still new drugs coming onto the market that cause photosensitivity. Thus, there is a continuing need for dermatologists to be aware of the different forms of presentation and the culprit drugs. Management usually involves photoprotection and cessation of drug treatment. However, there are always cases where the culprit drug is indispensable. The reason why some patients are susceptible while others remain asymptomatic is not known. A potential mechanism for the phototoxic reactions is the generation of reactive oxygen species (ROS), and there are a number of reasons why some patients might be less able to cope with enhanced levels of ROS.

Rapid and reliable healing of critical size bone defects with genetically modified sheep muscle.

Large segmental defects in bone fail to heal and remain a clinical problem. Muscle is highly osteogenic, and preliminary data suggest that autologous muscle tissue expressing bone morphogenetic protein-2 (BMP-2) efficiently heals critical size defects in rats. Translation into possible human clinical trials requires, inter alia, demonstration of efficacy in a large animal, such as the sheep. Scale-up is fraught with numerous biological, anatomical, mechanical and structural variables, which cannot be addressed systematically because of cost and other practical issues. For this reason, we developed a translational model enabling us to isolate the biological question of whether sheep muscle, transduced with adenovirus expressing BMP-2, could heal critical size defects in vivo. Initial experiments in athymic rats noted strong healing in only about one-third of animals because of unexpected immune responses to sheep antigens. For this reason, subsequent experiments were performed with Fischer rats under transient immunosuppression. Such experiments confirmed remarkably rapid and reliable healing of the defects in all rats, with bridging by 2 weeks and remodelling as early as 3-4 weeks, despite BMP-2 production only in nanogram quantities and persisting for only 1-3 weeks. By 8 weeks the healed defects contained well-organised new bone with advanced neo-cortication and abundant marrow. Bone mineral content and mechanical strength were close to normal values. These data demonstrate the utility of this model when adapting this technology for bone healing in sheep, as a prelude to human clinical trials.

Review of the 94th Annual Meeting of the British Association of Dermatologists, Glasgow 2014.

This is a review of the 94th Annual Meeting of the British Association of Dermatologists, held in Glasgow from 1 to 3 July 2014. The conference covered some of the latest developments in the treatment of atopic dermatitis, psoriasis and cancer, a follow-up on the methylisothiazolinone contact allergy epidemic, advances in genetically inherited disorders and somatic mutations underlying birth marks. In addition, there was an international perspective on vitiligo, leprosy and HIV, and a session discussing the regulatory process behind pharmaceutical development.

A review of UVA-mediated photosensitivity disorders.

A number of skin conditions are characterised by photosensitivity to UVA. Some of these are exclusively UVA-mediated conditions, while others include UVA in the action spectrum which also include UVB and/or visible light. This review aims to describe this diverse range of conditions for non-dermatologist scientists with an interest in this topic. As such, clinical details, including treatments, are brief and succinct. Recent advances in understanding the pathogenesis of these conditions is highlighted.

Interleukin-1beta and tumor necrosis factor alpha inhibit chondrogenesis by human mesenchymal stem cells through NF-kappaB-dependent pathways.

OBJECTIVE: The differentiation of mesenchymal stem cells (MSCs) into chondrocytes provides an attractive basis for the repair and regeneration of articular cartilage. Under clinical conditions, chondrogenesis will often need to occur in the presence of mediators of inflammation produced in response to injury or disease. The purpose of this study was to examine the effects of 2 important inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), on the chondrogenic behavior of human MSCs. METHODS: Aggregate cultures of MSCs recovered from the femoral intermedullary canal were used. Chondrogenesis was assessed by the expression of relevant transcripts by quantitative reverse transcription-polymerase chain reaction analysis and examination of aggregates by histologic and immunohistochemical analyses. The possible involvement of NF-kappaB in mediating the effects of IL-1beta was examined by delivering a luciferase reporter construct and a dominant-negative inhibitor of NF-kappaB (suppressor-repressor form of IkappaB [srIkappaB]) with adenovirus vectors. RESULTS: Both IL-1beta and TNFalpha inhibited chondrogenesis in a dose-dependent manner. This was associated with a marked activation of NF-kappaB. Delivery of srIkappaB abrogated the activation of NF-kappaB and rescued the chondrogenic response. Although expression of type X collagen followed this pattern, other markers of hypertrophic differentiation responded differently. Matrix metalloproteinase 13 was induced by IL-1beta in a NF-kappaB-dependent manner. Alkaline phosphatase activity, in contrast, was inhibited by IL-1beta regardless of srIkappaB delivery. CONCLUSION: Cell-based repair of lesions in articular cartilage will be compromised in inflamed joints. Strategies for enabling repair under these conditions include the use of specific antagonists of individual pyrogens, such as IL-1beta and TNFalpha, or the targeting of important intracellular mediators, such as NF-kappaB.

Use of genetically modified muscle and fat grafts to repair defects in bone and cartilage.

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human bone morphogenetic protein-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess bone healing, the genetically modified ("gene activated") tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as 10 days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed bone were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal bone and cartilage more expeditiously, and at lower cost, than is presently possible.

Ex vivo adenoviral transfer of bone morphogenetic protein 12 (BMP-12) cDNA improves Achilles tendon healing in a rat model.

The aim of our study was to evaluate the histological and biomechanical effects of BMP-12 gene transfer on the healing of rat Achilles tendons using a new approach employing a genetically modified muscle flap. Biopsies of autologous skeletal muscle were transduced with a type-five, first-generation adenovirus carrying the human BMP-12 cDNA (Ad.BMP-12) and surgically implanted around experimentally transected Achilles tendons in a rat model. The effect of gene transfer on healing was evaluated by mechanical and histological testing after 1, 2, 4 and 8 weeks. One week after surgery, the maximum failure load of the healing tendons was significantly increased in the BMP-12 group, compared with the controls, and the tendon stiffness was significantly higher at 1, 2 and 4 weeks. Moreover, the size of the rupture callus was increased in the presence of BMP-12 and there was evidence of accelerated remodeling of the lesion in response to BMP-12. Histological examination showed a much more organized and homogeneous pattern of collagen fibers at all time points in lesions treated with the BMP-12 cDNA muscle graft. Both single fibrils and the collagen fibers had a greater diameter, with a higher degree of collagen crimp than the collagen of the control groups. This was confirmed by sirius red staining in conjunction with polarized light microscopy, which showed a higher shift of small yellow-green fibers to strong yellow-orange fibers after 2, 4 and 8 weeks in the presence of BMP-12 cDNA. There was also an earlier shift from fibroblasts to fibrocytes within the healing tendon, with less fat cells present in the tendons of the BMP-12 group compared with the controls. Treatment with BMP-12 cDNA-transduced muscle grafts thus produced a promising acceleration and improvement of tendon healing, particularly influencing early tissue regeneration, leading to quicker recovery and improved biomechanical properties of the Achilles tendon. Further development of this approach could have clinical applications.

A simple, lanthanide-based method to enhance the transduction efficiency of adenovirus vectors.

Based upon the powerful bridging and charge-masking properties of lanthanide cations (Ln3+), we have investigated their use to improve the transduction efficiency of adenovirus vectors. Using a luciferase marker gene, it was possible to increase transgene expression by the murine mesenchymal stem cell line C3H10T(1/2) by up to four log orders when using very low multiplicities of infection in conjunction with Ln3+; La3+ was superior to Gd3+, Y3+ and Lu3+ in this regard. All Ln3+ were more effective than Ca2+. Flow cytometry, using a green fluorescent protein marker gene, confirmed that La3+ increased both the percentage of transduced cells and the level of transgene expression per cell. Transduction of primary cultures of a variety of different mesenchymal cells from human, rabbit, bovine and rat sources, as well as gene transfer to synovium and muscle in vivo, was also greatly enhanced. Our findings suggest that this lanthanide-based method holds much promise for expediting both experimental and clinical applications of gene transfer with adenoviral vectors.

Functional improvement of mutant keratin cells on addition of desmin: an alternative approach to gene therapy for dominant diseases.

A major challenge to the concept of gene therapy for dominant disorders is the silencing or repairing of the mutant allele. Supplementation therapy is an alternative approach that aims to bypass the defective gene by inducing the expression of another gene, with similar function but not susceptible to the disrupting effect of the mutant one. Epidermolysis bullosa simplex (EBS) is a genetic skin fragility disorder caused by mutations in the genes for keratins K5 or K14, the intermediate filaments present in the basal cells of the epidermis. Keratin diseases are nearly all dominant in their inheritance. In cultured keratinocytes, mutant keratin renders cells more sensitive to a variety of stress stimuli such as osmotic shock, heat shock or scratch wounding. Using a 'severe' disease cell culture model system, we demonstrate reversion towards wild-type responses to stress after transfection with human desmin, an intermediate filament protein normally expressed in muscle cells. Such a supplementation therapy approach could be widely applicable to patients with related individual mutations and would avoid some of the financial obstacles to gene therapy for rare diseases.

Human keratin 8 mutations that disturb filament assembly observed in inflammatory bowel disease patients.

We have identified miss-sense mutations in keratin 8 in a subset of patients with inflammatory bowel disease (Crohn disease and ulcerative colitis). Inflammatory bowel diseases are a group of disorders that are polygenic in origin and involve intestinal epithelial breakdown. We investigated the possibility that these keratin mutations might contribute to the course of the disease by adversely affecting the keratin filament network that provides mechanical support to cells in epithelia. The mutations (Gly62 to Cys, Ile63 to Val and Lys464 to Asn) all lie outside the major mutation hotspots associated with severe disease in epidermal keratins, but using a combination of in vitro and cell culture assays we show that they all have detrimental effects on K8/K18 filament assembly in vitro and in cultured cells. The G62C mutation also gives rise to homodimer formation on oxidative stress to cultured intestinal epithelial cells, and homodimers are known to be polymerization incompetent. Impaired keratin assembly resulting from the K8 mutations found in some inflammatory bowel disease patients would be predicted to affect the maintenance and re-establishment of mechanical resilience in vivo, as required during keratin cytoskeleton remodeling in cell division and differentiation, which may lead to epithelial fragility in the gut. Simple epithelial keratins may thus be considered as candidates for genes contributing to a risk of inflammatory bowel disease.

Functional analysis of keratin components in the mouse hair follicle inner root sheath.

BACKGROUND: Recently, a family of novel type I keratins of the inner root sheath of the hair follicle were discovered, increasing the number of keratins known to be expressed in the hair follicle. The mouse database shows three keratins that are possible orthologues of these inner root sheath keratins. The sequences of these keratins include rather unusual changes to a highly conserved motif at the end of the alpha-helical rod domain of the proteins, thought to be important in filament assembly. OBJECTIVES: To investigate whether these keratins are expressed in the inner root sheath and to determine whether they assemble normally. METHODS: To investigate this, polyclonal antibodies were raised for immunolocalization of the keratins and their cDNAs were cloned for transfection into cultured cells. RESULTS: At least two of these keratins were expressed in the inner root sheath but the timing of expression of the different keratins was variable. Transfection of the relevant cDNAs into cells in culture indicated that these keratins were capable of integrating into existing keratin networks without disruption, but that de novo filament assembly with the type II inner root sheath keratin, mK6irs, was poor. CONCLUSIONS: These results provide further evidence of the complexity of keratin expression in the three concentric layers of the inner root sheath.

Alopecia in a novel mouse model RCO3 is caused by mK6irs1 deficiency.

Reduced coat 3 (Rco3) is a new spontaneous autosomal recessive mutation with defects in hair structure and progressive alopecia. Here we describe chromosomal mapping and molecular identification of the Rco3 mutation. The murine Rco3 locus maps to a 2-Mb interval on chromosome 15 encompassing the keratin type II gene cluster. Recently, mK6irs1 was described as a type II keratin expressed in Henle's and Huxley's layer of the murine inner root sheath. Genomic sequencing revealed a 10-bp deletion in exon 1 of mK6irs1 resulting in a frameshift after 58 amino acid residues and, therefore, the absence of 422 carboxy-terminal amino acid residues containing the complete alpha-helical rod domain. Henle's and Huxley's layers show no immunoreactivity with mK6irs1-specific antibodies and the absence of intermediate filament formation in electron microscopic images. These results indicate that the expression of functional mK6irs1 is indispensable for intermediate filament formation in the inner root sheath and highlights the importance of the keratinization of the inner root sheath in the normal formation of the hair shaft.

Keratin K6irs is specific to the inner root sheath of hair follicles in mice and humans.

BACKGROUND: Keratins are a multigene family of intermediate filament proteins that are differentially expressed in specific epithelial tissues. To date, no type II keratins specific for the inner root sheath of the human hair follicle have been identified. OBJECTIVES: To characterize a novel type II keratin in mice and humans. METHODS: Gene sequences were aligned and compared by BLAST analysis. Genomic DNA and mRNA sequences were amplified by polymerase chain reaction (PCR) and confirmed by direct sequencing. Gene expression was analysed by reverse transcription (RT)-PCR in mouse and human tissues. A rabbit polyclonal antiserum was raised against a C-terminal peptide derived from the mouse K6irs protein. Protein expression in murine tissues was examined by immunoblotting and immunofluorescence. RESULTS: Analysis of human expressed sequence tag (EST) data generated by the Human Genome Project revealed a fragment of a novel cytokeratin mRNA with characteristic amino acid substitutions in the 2B domain. No further human ESTs were found in the database; however, the complete human gene was identified in the draft genome sequence and several mouse ESTs were identified, allowing assembly of the murine mRNA. Both species' mRNA sequences and the human gene were confirmed experimentally by PCR and direct sequencing. The human gene spans more than 16 kb of genomic DNA and is located in the type II keratin cluster on chromosome 12q. A comprehensive immunohistochemical survey of expression in the adult mouse by immunofluorescence revealed that this novel keratin is expressed only in the inner root sheath of the hair follicle. Immunoblotting of murine epidermal keratin extracts revealed that this protein is specific to the anagen phase of the hair cycle, as one would expect of an inner root sheath marker. In humans, expression of this keratin was confirmed by RT-PCR using mRNA derived from plucked anagen hairs and epidermal biopsy material. By this means, strong expression was detected in human hair follicles from scalp and eyebrow. Expression was also readily detected in human palmoplantar epidermis; however, no expression was detected in face skin despite the presence of fine hairs histologically. CONCLUSIONS: This new keratin, designated K6irs, is a valuable histological marker for the inner root sheath of hair follicles in mice and humans. In addition, this keratin represents a new candidate gene for inherited structural hair defects such as loose anagen syndrome.

Actin cytoskeleton role in the structural response of epithelial (MDCK) cells to low extracellular Ca2+.

Kinetic and stereometric assessment of the mechanical responses of epithelial cells to variations in the concentration of extracellular Ca2+ was carried out in vivo at the single cell level. Continuous monitoring of individual MDCK cells in subconfluent cultures attested to an intense, immediately relaxable, and cytochalasin D-sensitive contraction, equivalent to that seen in confluent monolayers following depletion of external Ca2+ (<0.1 mM). Increasingly greater and less readily reversible contractions were performed upon repeated stimulation with short-term cycles of alternating normal (30 min) and low Ca2+ (30 min) media. Constriction of a narrow horizontal girdle corresponding in position to the major ring-like bundle of actin filaments eventually develops into a deep lateral furrow in intensely contracted cells. Substantial membrane infolding in the contracted state is indicated also by stereometric estimates of apparent bounding surface area. Irrespective of the contracted or relaxed cell condition, rhodamine-phalloidin labeling showed a marginal position of the ring-like bundle of microfilaments and other components of the actin cytoskeleton. These results suggest, contrary to prevalent views, that the actin-myosin system stays associated to the cortex and retains contractile capability in epithelial cells deprived of external Ca2+. Hence, the mechanical responses to variations of Ca2+ may be an overstrained expression of a physiological mechanism.

K15 expression implies lateral differentiation within stratified epithelial basal cells.

Keratins are intermediate filament proteins whose expression in epithelial tissues is closely linked to their differentiated state. The greatest complexity of this expression is seen in the epidermis and associated structures. The critical basal (proliferative) cell layer expresses the major keratin pair, K5 and K14, but it also expresses an additional type I keratin, K15, about which far less is known. We have compared the expression of K15 with K14 in normal, pathological, and tissue culture contexts; distinct differences in their expression patterns have been observed that imply different regulation and function for these two genes. K15 appears to be preferentially expressed in stable or slowly turning over basal cells. In steady-state epidermis, K15 is present in higher amounts in basal cells of thin skin but in lower amounts in the rapidly turning over thick plantar skin. Although remaining high in basal cell carcinomas (noninvasive) it is suppressed in squamous cell carcinomas (which frequently metastasize). Wounding-stimulated epidermis loses K15 expression, whereas K14 is unchanged. In cultured keratinocytes, K15 levels are suppressed until the culture stratifies, whereas K14 is constitutively expressed throughout. Therefore, unlike K14, which appears to be a fundamental component of all keratinocytes, K15 expression appears to be more tightly coupled to a mature basal keratinocyte phenotype.