Dermoscopy of Zosteriform and Swirling Pattern Type 1 Segmental Darier Disease.

Dear Editor, Segmental Darier disease (DD) is a rare disease with around 40 described English literature cases. It is hypothesized that one of the causes of the disease is a post-zygotic somatic mutation for the calcium ATPase pump, only present in lesional skin. There are two types of segmental DD: type 1, where lesions follow Blaschko's lines unilaterally, and type 2, characterized by focal areas of increased severity in patients with generalized DD (1). Type 1 segmental DD is not easily diagnosed due to the lack of positive family history, the late onset of the disease in the third or fourth decade of life, and lack of DD-associated features. The differential diagnosis of type 1 segmental DD includes acquired papular dermatoses distributed in linear or zosteriform fashion, such as lichen planus, psoriasis, lichen striatus, or linear porokeratosis (2). We report two cases of segmental DD, of which the first case was a 43-year-old woman who presented with pruritic skin changes five years in duration and a history of seasonal aggravation. On examination, light brownish to reddish keratotic small papules were observed on the left abdomen and inframammary area, arranged in a swirling pattern (Figure 1, a). Dermoscopy showed polygonal or roundish yellowish/brown areas surrounded with whitish structureless areas (Figure 1, b). The histopathological correlations for dermoscopic brownish polygonal or round areas are hyperkeratosis, parakeratosis, and dyskeratotic keratinocytes, which were present in the biopsy specimen (Figure 1, c). The patient was prescribed 0.1% tretinoin gel, which led to marked improvement (Figure 1, d). The second case was a 62-year-old woman who presented with a flare of small red-brown papules, eroded papules, and some yellowish crusts arranged in a zosteriform pattern on the right side of the upper abdomen (Figure 2, a). Dermoscopy showed polygonal, roundish, yellowish areas surrounded with whitish and reddish structureless areas (Figure 2, b). Histopathology mainly revealed compact orthokeratosis and small foci of parakeratosis, marked granular layer with dyskeratotic keratinocytes, and foci of suprabasal acantholysis consistent with the diagnosis of DD (Figure 2, d, d). The patient was prescribed topical steroid cream and 0.1% adapalene cream, which also led to improvement. In both of our cases, a final diagnosis of type 1 segmental DD was established based on clinico-histopathologic correlation, since acantholytic dyskeratotic epidermal nevus could not have been ruled out only based on the histopathology report as it is clinically and histologically indistinguishable from segmental DD. However, the late age of onset and aggravation resulting from external factors such as heat, sunlight, and sweat supported the diagnosis of segmental DD. Although the final diagnosis of type 1 segmental DD is typically established based on clinico-histopathological correlation, we find dermoscopy particularly useful in aiding the diagnosis by eliminating differential diagnoses and being aware of their well-known dermoscopic patterns.

Pyogenic Granuloma of the Proximal Part of a Nail Unit in a Child.

Dear Editor, We present a case of proximal pyogenic granuloma in 4-year-old child. The patient presented to our Department due to a fast-growing lesion on the proximal part of the nail unit. The lesion had appeared over several weeks, and it was extremely painful for the child. On the day of the 1st visit, the lesion was not bleeding but was very painful during examination and photo-documentation. Clinically, it presented as an exogenous tumoral lesion of the proximal 1/3 of the nail, partially exulcerated with one part exhibiting coagulated hemorrhage and with uneven coloration (Figure 1). The lesion was not sharply demarcated. Dermoscopically, the majority of the lesion presented an unspecific dermoscopic structure, orange background color, and matched the criteria for a vascular lesion: few unspecific vessels and hemorrhage. The "sticky fiber" sign was also present (Figure 2). Since the lesion was fast-growing and due to the unspecific dermoscopic appearance, the child was referred to a pediatric surgeon and a complete excisional biopsy of the lesion was performed. The dermoscopy of pyogenic granuloma has been already described (1). The histology report confirmed pyogenic granuloma. Pyogenic granulomas of the nail unit are not a common finding, but our case confirms that even this location can be site of this type of benign lesion. It more commonly found in the periungual region and can be expected due to adverse effects of different kinds of systemic therapies. However, due to differential diagnosis that includes different types of tumors occurring at the nail unit, most importantly amelanotic melanoma and SCC, it is suggested to excise or take a biopsy of this type of lesion to be able to exclude aggressive tumor types, which are very rare but not impossible the in pediatric population (2). In cases of unquestionable diagnosis, several local treatments are available. Since the lesion presented a destructive nature in our case, we decided to perform excisional biopsy followed by histology, which in our case was both a diagnostic and therapeutic procedure.

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth.

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation.

Understanding the Relationship Between Smoking and Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic skin disease affecting hair follicles in intertriginous areas, characterized by deep, recurrent, painful nodules and abscesses, fistulae, sinus tracts, and scarring. With a prevalence of 1-4%, HS is not an uncommon disease. Several risk factors have been linked with the development of HS, such as genetic predisposition, smoking, and obesity, leading to the hypothesis that HS develops as a result of environmental triggers in a genetically susceptible individual. Smoking has been recognized as one of the environmental factors with the most impact on HS. This review aims to provide a comprehensive and holistic view on how smoking habits affect the incidence, severity, treatment, and pathophysiology of HS. A growing body of published literature has reported the association between smoking and HS, despite limitations in proving the causal relationship due to the retrospective design of the available studies. There is a consensus that patients with HS who are active smokers have a higher number of affected body areas than patients with HS who do not smoke or have stopped smoking. Similarly, it is recommended for patients with HS to discontinue tobacco use because of its association with weaker treatment response. Studies on the pathophysiological mechanism of smoking on the skin show that tobacco smoke with many of its chemicals as well as nicotine promote the proinflammatory cytokines found in HS lesions, activate the nicotinic acetylcholine (nAChRs) and aryl hydrocarbon receptors (AHRs), and further suppress Notch signaling pathway.

The association between clinical and laboratory findings of bullous pemphigoid and dipeptidyl peptidase-4 inhibitors in the elderly: a retrospective study.

AIM: To evaluate the association between the use of dipeptidyl peptidase-4 inhibitors (DPP4I) and clinical and laboratory findings of bullous pemphigoid (BP) in patients treated at the European Reference Network - Skin Reference Centre in Croatia. METHODS: This retrospective study enrolled 82 patients treated for BP at the Department of Dermatovenereology, University Hospital Center Zagreb from January 2015 to December 2019. Clinical features of BP, presence of comorbidities, and laboratory findings of anti-BP antibodies and eosinophilia were analyzed in three groups of BP patients: 1) diabetes mellitus (DM) type II patients treated with DPP4I, 2) DM type II patients not treated with DPP4I, and 3) non-DM type II patients. RESULTS: The average age and anti-BP180 titer were similar in all three groups. DPP4I group had a slightly lower eosinophil level in both peripheral blood (4.89%) and biopsy specimens (87.5%), but the difference was not significant. The prevalence of inflammatory BP in DPP4I group was 76.5%. DPP4I group had significantly higher percentage of patients with chronic renal failure and dementia (52.9% and 11.8%, respectively) compared with non-DPP4I DM (14.3% and 0%, respectively) and non-DM type II patients (15.7% and 0%, respectively). CONCLUSION: BP patients treated with DPP4I and those not treated with DPP4Is did not significantly differ in laboratory findings. However, DPP4I treatment was associated with an inflammatory subtype of BP and a higher prevalence of dementia and chronic renal failure. These findings warrant further research into the association of BP and DM with dementia and chronic renal failure.

Novel strategies for expansion of tooth epithelial stem cells and ameloblast generation.

Enamel is secreted by ameloblasts derived from tooth epithelial stem cells (SCs). Humans cannot repair or regenerate enamel, due to early loss of tooth epithelial SCs. Contrarily in the mouse incisors, epithelial SCs are maintained throughout life and endlessly generate ameloblasts, and thus enamel. Here we isolated Sox2-GFP+ tooth epithelial SCs which generated highly cellular spheres following a novel in vitro strategy. This system enabled analysis of SC regulation by various signaling molecules, and supported the stimulatory and inhibitory roles of Shh and Bmp, respectively; providing better insight into the heterogeneity of the SCs. Further, we generated a novel mouse reporter, Enamelin-tdTomato for identification of ameloblasts in live tissues and cells, and used it to demonstrate presence of ameloblasts in the new 3D co-culture system of dental SCs. Collectively, our results provide means of generating 3D tooth epithelium from adult SCs which can be utilized toward future generation of enamel.

Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling.

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. In situ hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.

Do We Utilize Our Knowledge of the Skin Protective Effects of Carotenoids Enough?

Due to their potential health-promoting effects, carotenoids have drawn both scientific and public attention in recent years. The primary source of carotenoids in the human skin is diet, mainly fruits, vegetables, and marine product, but they may originate from supplementation and topical application, too. In the skin, they accumulate mostly in the epidermis and act as a protective barrier to various environmental influences. Namely, the skin is exposed to numerous environmental factors, including ultraviolet radiation (UVR), air pollution, and smoking, that cause oxidative stress within the skin with consequent premature (extrinsic) aging. UVR, as the most prominent environmental factor, may cause additional detrimental skin effects, such as sunburn, DNA damage, and skin cancer. Therefore, photoprotection is the first line intervention in the prevention of premature aging and skin cancer. Numerous studies have demonstrated that carotenoids, particularly ß-carotene, lycopene, lutein, and astaxanthin, have photoprotective effects, not only through direct light-absorbing properties, but also through their antioxidant effects (scavenging reactive oxygen species), as well as by regulation of UV light-induced gene expression, modulation of stress-dependent signaling, and/or suppression of cellular and tissue responses like inflammation. Interventional studies in humans with carotenoid-rich diet have shown its photoprotective effects on the skin (mostly by decreasing the sensitivity to UVR-induced erythema) and its beneficial effects in prevention and improvement of skin aging (improved skin elasticity and hydration, skin texture, wrinkles, and age spots). Furthermore, carotenoids may be helpful in the prevention and treatment of some photodermatoses, including erythropoietic protoporphyria (EPP), porphyria cutanea tarda (PCT) and polymorphous light eruption (PMLE). Although UVR is recognized as the main etiopathogenetic factor in the development of non-melanoma skin cancer (NMSC) and melanoma, and the photoprotective effects of carotenoids are certain, available studies still could not undoubtedly confirm the protective role of carotenoids in skin photocarcinogenesis.

Hyperpigmented Mycosis Fungoides - A Rare Entity.

Dear Editor, A 67-year-old man of Kosovar-Albanian ethnic origin (skin phenotype IV) presented to our dermatology clinic with generalized hyperpigmented patches and plaques all over the body, so-called melanoerythroderma (Figure 1). The lesions, which first appeared nearly six years ago, developed gradually; they were diagnosed as mycosis fungoides (MF), and were subsequently treated only with topical corticosteroids. We performed further examinations upon admission to our department. Relevant laboratory parameters - blood cell count, LDH, urinalysis, and serum chemistry - were within normal limits. Endocrinological examination excluded Addison disease, and the patient was not receiving any drugs that could cause skin hyperpigmentation. Chest-abdomen-pelvis computed tomography (CT) scan and sternal puncture were normal. Flow cytometric immunophenotyping revealed less than 5% aberrant T-cells. Histopathology and immunohistochemistry of skin specimens revealed lichenoid infiltration of small- to medium-sized atypical T-lymphocytes within the upper dermis, epidermotropic lymphocytes with several Pautrier's microabscesses (Darier's nests), pigment incontinence, abundant melanophages in the papillary dermis (Figure 2, a, b), and the T-cell CD4+CD7-CD8+ phenotype (Figure 2, c, d). Based on the clinical picture, histopathology, and immunohistochemistry the diagnosis of hyperpigmented mycosis fungoides (MF) stage IIIA (T4N0M0B0) was established. Skin-oriented therapy (retinoids-PUVA) resulted in slight improvement. Hyperpigmented MF is a rare, uncommon, clinical variant of MF, with a predilection for dark-skinned people (1). Only a few cases of hyperpigmented MF have been reported so far, and our case being one of them (2-5). Hyperpigmented patches or/and plaques dominate the clinical picture. Hyperpigmented MF is characterized by a predominantly CD8+ epidermotropic T-cell phenotype, although different phenotypes have been reported (CD4+ or CD4-CD8-) (2). Histopathologically, interface changes, pigment incontinence and melanophages are usually found in addition to the classical findings of early MF (1). The exact mechanism of hyperpigmentation is not well understood. Hyperpigmented MF had an indolent course in most reported cases, and skin-directed therapy is therefore the treatment of choice. Although MF and its hyperpigmented variant is a lymphoma of low-grade malignancy, large-cell transformation (CD30+) of hyperpigmented MF can occur (1). These rare cases of special clinical MF variants are extremely valuable and can help us investigate and understand the pathophysiology of the disease. Treatment and close follow-up is mandatory in the hyperpigmented variant of MF.

Functionally Distinctive Ptch Receptors Establish Multimodal Hedgehog Signaling in the Tooth Epithelial Stem Cell Niche.

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238-1248.

Isolation of Dental Stem Cell-Enriched Populations from Continuously Growing Mouse Incisors.

Continuous growth of the rodent incisor is enabled by epithelial and mesenchymal stem cells (ESCs and MSCs) which unceasingly replenish enamel and dentin, respectively, that wear by persistent animal gnawing. Lineage tracing studies have provided evidence that ESCs contribute to all epithelial lineages of the tooth in vivo. Meanwhile, in the mouse incisor, MSCs continuously contribute to odontoblast lineage and tooth growth. However, in vitro manipulation of ESCs has shown little progress, mainly due to lack of appropriate protocol to successfully isolate, culture, expand, and differentiate ESCs in vitro without using the co-culture system. In this chapter we describe the isolation of the Sox2-GFP+ cell population that is highly enriched in ESCs. Isolated cells can be used for various types of analyses, including in vitro culture, single cell-related analyses, etc. Furthermore, we describe ways to obtain populations enriched in the incisor MSCs using FACS sorting of antibody-labeled cells. Easily accessible FACS sorting enables easy and relatively fast isolation of the cells labeled by the fluorescent protein.

Long-term Follow-up of a Case of Lymphomatoid Papulosis with a Benign Course.

Dear Editor,We present the case of a 40-year old male patient with lymphomatoid papulosis of a waxing and waning course on whom three biopsies were performed during a 14-year period with no change in histopathological or immunophenotypical characteristics. Lymphomatoid papulosis (LP) is a chronic, recurrent, self-healing papulonodular skin eruption with the histopathologic features of a cutaneous T-cell lymphoma but an often benign and indolent clinical course (1). It is designated as a primary, cutaneous, CD30+ lymphoproliferative disorder. The histopathologic features of LP are variable, with five main types (A-E) and several other variants (2). In most cases, LP presents with a generalized eruption of reddish-brown papules or nodules usually smaller than one cm on the trunk and limbs. Rarely, large, rapidly growing nodules may be the first manifestation of the disease (3). Patients with LP have an excellent prognosis even though they are at increased risk of developing secondary cutaneous or nodal lymphomas such as mycosis fungoides, primary cutaneous anaplastic large cell lymphoma (PC-ALCL), or Hodgkin lymphoma (4). LP-associated lymphomas develop in between 10% and, as recently reported, 52% percent of patients and may occur before, concurrent with, or after the onset of LP (4,5). Our patient was diagnosed with "conventional" type An LP 14 years earlier based on the clinicopathologic correlation. The diagnosis was confirmed a year later after excision of a rapid growing ulcerated nodule on the forearm measuring 17 mm in diameter, which was clinically suspected to be anaplastic large cell lymphoma. During these 14 years, there were only a few worrisome recurrences of the disease, which resolved spontaneously or were successfully controlled with local steroids. During a recent exacerbation, when the third biopsy was performed, the patient presented with a large number of generalized reddish-brown pruritic papules and nodules on the trunk, extremities, neck, and face, predominantly up to one cm, some among which were necrotic and excoriated (Figure 1). There were three sites of clustered papules on the trunk, groin, and neck that resembled large, infiltrated plaques larger than two cm, at a glance mimicking cutaneous lymphoma (Figure 1, b, c). There were also older residual hyper- and hypopigmentations on the skin with prominent scarring. Excisional skin biopsy of one larger papule from the abdominal plaque was performed and was not morphologically or immunophenotypically different from the previous ones (Figure 2). Immunohistochemistry showed expression of CD 30 and the phenotypic markers of T-helper lymphocytes (CD 3+/-, CD4+) by neoplastic cells (Figure 2, c, d). Associated systemic malignant lymphoma was excluded based on examination findings, normal laboratory tests, the absence of palpably enlarged lymph nodes, hepatosplenomegaly, and systemic symptoms followed with MSCT. Serology for HIV and EBV was performed and was positive for EBV EBNA, VCA IgG, and IgM, which could be associated with the exacerbation of LP. Topical corticosteroids and phototherapy were administered when needed in this 14-year period, and methotrexate in a lower dose was prescribed during the extensive generalized eruptions. All of the applied therapeutic modalities led to a partial response. LP is a self-limiting disease for which many patients do not require specific treatment. Therapy should be directed at controlling symptoms in generalized eruptions or minimizing the frequency of recurrences, but none of the available treatment options disrupt the natural history of LP or reduce the risk of developing an associated lymphoma (6). Low-dose methotrexate is the initial therapy of choice in patients with the extensive or symptomatic disease or disease involving cosmetically sensitive areas like the face or hands, which were the affected areas in our patient (6,7). There are no markers that can help predict the course of the disease in a given patient, although some indicators have been suggested (8,9). Because of this lack of markers that can help predict the course of the disease and occurrence of malignant lymphoma, patients should remain in monitoring for the rest of their life.

Atypical Nevi in a Patient After Toxic Epidermal Necrolysis.

Dear Editor, There are few literature data about nevi in patients with a history of toxic epidermal necrolysis (TEN) and little recommendations for follow-up and risks of melanoma (MM). Eruptive melanocytic nevi (EMN) is a rare phenomenon that has been associated with bullous disorders, immunosuppression, and immunodeficiency, but in some cases can occur without precipitating factors (1). The etiology is largely unknown, but there is evidence that immunosuppression might play a crucial role in nevogenesis, probably due to the inability of the immune system to inhibit melanocytic (MC) proliferation (2,3). We report the follow-up of a patient with a history of TEN who later developed atypical nevi. A 17-year-old man with a history of severe TEN two years earlier, most probably due to valproic acid and diclofenac, was referred to our Department due to atypical nevi. The patient presented with scars, scattered pigmentation (Fig. 1), and symblepharon as a consequence of TEN (Fig. 2). Most of his nevi developed in following two years after TEN. During the first visit in 2009, clinical and dermoscopic photodocumentation was performed. The patient presented with a moderate number of nevi (Fig. 3), dermoscopically subclassified as globular. One atypical MN was found on the back, with dermoscopic findings of reticular pattern and presence of suspected areas of regression (Fig. 4. a, b), and it was excised to rule out melanoma (Fig. 4, Fig. 5). The patient did not come to regular follow-up from 2009 to 2014, and presented in 2014 which was when comparative photo documentation was made. As this visit another, speckled type of newly-occurred nevus was excised. Both excised nevi were histopathologically characterized as dysplastic. Only a few references are available on nevi development after TEN. Anticonvulsives and NSAIDs, as in our case, are often involved in the etiopathogenesis of TEN (4,5). Survivors may experience a variety of long-term complications; authors reported that 19% of their patients developed new nevi after TEN (6,7). EMN develop several years after TEN as a suddenly arising large number of nevi that may resemble speckled lentiginous nevi (8). Histologically. EMN demonstrate a proliferation of MC at the dermo-epidermal junction and, if compound, in the papillary dermis, arranged mostly in nests. Junctional MC may appear slightly pleomorphic, but no significant cytological atypia or prominent pagetoid spread of MC was reported (9). EMN have been associated with a specific dermoscopic finding of a symmetrical peripheral rim of globules which represent pigmented junctional nests of MC in the periphery and are a specific feature of rapidly enlarging MC nevi (10,11). The pathogenesis of EMN is not known. The microenvironment of epidermal regeneration may have some effects on MC because MC hyperplasia develops after cutaneous trauma (observed in recurrent nevi). The cytokines and growth factors produced and secreted during epidermal regeneration might contribute to the proliferation of residual epidermal MC and subsequent nevus formation (12). Because most of the bullous disorders associated with EMN are transient, the authors believe that changes in local growth factors may also be temporary and MN remain stable without a propensity to malignant degeneration without further stimuli (13). This is corroborated by the fact that no reports of malignant change of EMN in patients with bullous disorders have been described. It is likely that the etiology and natural course of EMN differs between two main populations of patients, with EMN arising after bullous disorders being more likely to remain benign compared with those with ongoing immunosuppression, but this hypothesis has yet to be proven. The actual risk of MM in patients with EMN remains unknown. Since our patient did not have many nevi, he does not fit into the EMN category. Due to the atypical appearance of his nevi, long-term follow-up on 6-month basis is recommended.

Tatami Mats: A Source of Pitted Keratolysis in a Martial Arts Athlete?

Dear Editor, Pitted keratolysis (PK), also known as keratosis plantaris sulcatum, is a non-inflammatory, bacterial, superficial cutaneous infection, characterized by many discrete superficial crateriform ''pits'' and erosions in the thickly keratinized skin of the weight-bearing regions of the soles of the feet (1). The disease often goes unnoticed by the patient, but when it is noticed it is because of the unbearable malodor and hyperhidrosis of the feet, which are socially unacceptable and cause great anxiety to many of the patients. PK occurs worldwide, with the incidence rates varying based on the environment and occupation. The prevalence of this condition does not differ significantly based on age, sex, or race. People who sweat profusely or wash excessively, who wear occlusive footwear, or are barefoot especially in hot and humid weather are extremely prone to this condition (2). Physicians commonly misdiagnose it as tinea pedis or plantar warts. Treatment is quite simple and straightforward, with an excellent expected outcome if treated properly. We report a case of a 32-year-old male patient with skin changes of approximately one-year duration diagnosed as plantar verrucae, who was referred to our Department for cryotherapy. The patient presented with asymptomatic, malodorous punched-out pits and erosions along with hyperkeratotic skin on the heel and metatarsal region of the plantar aspect of both feet. The arches, toes, and sides of the feet were spared (Figure 1). Except for these skin changes, the patient was healthy and denied any other medical issues. He was an athlete active in martial arts and had a history of sweating of feet and training barefoot on the tatami mat for extended periods of time. The diagnosis of PK was established based on the clinical findings (crateriform pitting and malodor), negative KOH test for hyphae, and a history of prolonged sweating in addition to contact of the skin with tatami mats, which are often a source of infection if hygiene measures are not adequately implemented. Swabs could have been helpful to identify causative organisms, but they were not crucial for the diagnosis and treatment. The patient was prescribed with general measures to prevent excessive sweating (cotton socks, open footwear, and proper hygiene), antiseptic potassium permanganate foot soaks followed by clindamycin 1% and benzoyl peroxide 5% in a gel vehicle twice daily. At the one-month follow-up visit, the skin changes, hyperhidrosis, and malodor were entirely resolved (Figure 2). Pitted keratolysis is common among athletes (3,4). The manifestations of PK are due to a superficial cutaneous infection caused by several bacterial Gram-positive species including Corynebacterium species, Kytococcus sedentarius, Dermatophilus congolensis, Actynomices keratolytica, and Streptomyces that proliferate and produce proteinase and sulfur-compound by-products under appropriate moist conditions (5-7). Proteinases digest the keratin and destroy the stratum corneum, producing the characteristic skin findings, while sulfur compounds (sulfides, thiols, and thioesters) are responsible for the malodor. Athletes and soldiers who wear occlusive footwear for prolonged periods of time or even barefooted people that sweat extensively and spend time on wet surfaces such as laborers, farmers, and marine workers are more prone to this problem (3,4,8-11). Martial arts athletes are at greater risk of skin infections due to the constant physical contact that can lead to transmission of viral, bacterial, and fungal pathogens directly but also indirectly through contact with the mat and the skin flora of an another infected individual. A national survey of the epidemiology of skin infections among US high school athletes conducted by Ashack et al. supported the prevalent theory that contact sports are associated with an increased risk of skin infections. In this study, wrestling had the highest skin infection rate of predominantly bacterial origin (53.8%), followed by tinea (35.7%) and herpetic lesions (6.7%), which is consistent with other literature reporting (12). Being barefoot on the tatami mat in combination with excessive sweating and non-compliance with hygiene measures makes martial arts athletes more susceptible to skin infections, including PK. The diagnosis is clinical, by means of visual examination and recognition of the characteristic odor. Dermoscopy can be useful, revealing abundant pits with well-marked walls that sometimes show the bacterial colonies (13). Cultures, if taken, show Gram-positive bacilli or coccobacilli. Because of the ease of diagnosis on clinical findings, biopsy of pitted keratolysis is rarely performed. Skin scraping is often performed to exclude tinea pedis, which is one of the main differential diagnosis, the others including verrucae, punctate palmoplantar keratoderma, keratolysis exfoliativa, circumscribed palmoplantar hypokeratosis, and basal cell nevus syndrome. If unrecognized and left untreated, skin findings and smelly feet can last for many years. Sometimes, if unrecognized, PK can be mistreated with antifungals, or even with aggressive treatment modalities such as cryotherapy. Appropriate treatment includes keeping feet dry with adequate treatment of hyperhidrosis, preventive measures, and topical antibiotic therapy. Topical forms of salicylic acid, sulfur, antibacterial soaps, neomycin, erythromycin, mupirocin, clindamycin and benzoyl peroxide, clotrimazole, imidazoles, and injectable botulinum toxin are all successful in treatment and prevention of PK (14,15). Topical antibiotics are the first line of medical treatment, among which fusidic acid, erythromycin 1% (solution or gel), mupirocin 2%, or clindamycin are the most recommended (14). As in our case, a fixed combination of two approved topical drugs - clindamycin 1%-benzoyl peroxide 5% gel, had been already demonstrated by Vlahovich et al. as an excellent treatment option with high adherence and no side-effect (16). The combined effect of this combination showed significantly greater effect due to the bactericidal and keratolytic properties of benzoyl peroxide. Additionally, this combination also lowers the risk of resistance of causative microorganisms to clindamycin. Skin infections are an important aspect of sports-related adverse events. Due to the interdisciplinary nature, dermatologists are not the only ones who should be aware of the disease, but also family medicine doctors, sports medicine specialists, and occupational health doctors who should educate patients about the etiology of the skin disorder, adequate prevention, and treatment. Athletes must enforce the disinfecting and sanitary cleaning of the tatami mats and other practice areas. Keeping up with these measures could significantly limit the spread of skin infections that can infect athletes indirectly, leading to significant morbidity, time loss from competition, and social anxiety as well.

Mesenchymal Wnt/ß-catenin signaling limits tooth number.

Tooth agenesis is one of the predominant developmental anomalies in humans, usually affecting the permanent dentition generated by sequential tooth formation and, in most cases, caused by mutations perturbing epithelial Wnt/ß-catenin signaling. In addition, loss-of-function mutations in the Wnt feedback inhibitor AXIN2 lead to human tooth agenesis. We have investigated the functions of Wnt/ß-catenin signaling during sequential formation of molar teeth using mouse models. Continuous initiation of new teeth, which is observed after genetic activation of Wnt/ß-catenin signaling in the oral epithelium, was accompanied by enhanced expression of Wnt antagonists and a downregulation of Wnt/ß-catenin signaling in the dental mesenchyme. Genetic and pharmacological activation of mesenchymal Wnt/ß-catenin signaling negatively regulated sequential tooth formation, an effect partly mediated by Bmp4. Runx2, a gene whose loss-of-function mutations result in sequential formation of supernumerary teeth in the human cleidocranial dysplasia syndrome, suppressed the expression of Wnt inhibitors Axin2 and Drapc1 in dental mesenchyme. Our data indicate that increased mesenchymal Wnt signaling inhibits the sequential formation of teeth, and suggest that Axin2/Runx2 antagonistic interactions modulate the level of mesenchymal Wnt/ß-catenin signaling, underlying the contrasting dental phenotypes caused by human AXIN2 and RUNX2 mutations.

Mesenchymal Wnt/ß-Catenin Signaling Controls Epithelial Stem Cell Homeostasis in Teeth by Inhibiting the Antiapoptotic Effect of Fgf10.

Continuous growth of rodent incisors relies on epithelial stem cells (SCs) located in the SC niche called labial cervical loop (LaCL). Here, we found a population of apoptotic cells residing in a specific location of the LaCL in mouse incisor. Activated Caspase 3 and Caspase 9, expressed in this location colocalized in part with Lgr5 in putative SCs. The addition of Caspase inhibitors to incisors ex vivo resulted in concentration dependent thickening of LaCL. To examine the role of Wnt signaling in regulation of apoptosis, we exposed the LaCL of postnatal day 2 (P2) mouse incisor ex vivo to BIO, a known activator of Wnt/ß-catenin signaling. This resulted in marked thinning of LaCL as well as enhanced apoptosis. We found that Wnt/ß-catenin signaling was intensely induced by BIO in the mesenchyme surrounding the LaCL, but, unexpectedly, no ß-catenin activity was detected in the LaCL epithelium either before or after BIO treatment. We discovered that the expression of Fgf10, an essential growth factor for incisor epithelial SCs, was dramatically downregulated in the mesenchyme around BIO-treated LaCL, and that exogenous Fgf10 could rescue the thinning of the LaCL caused by BIO. We conclude that the homeostasis of the epithelial SC population in the mouse incisor depends on a proper rate of apoptosis and that this apoptosis is controlled by signals from the mesenchyme surrounding the LaCL. Fgf10 is a key mesenchymal signal limiting apoptosis of incisor epithelial SCs and its expression is negatively regulated by Wnt/ß-catenin. Stem Cells 2015;33:1670-1681.

Intracellular autofluorescence: a biomarker for epithelial cancer stem cells.

Cancer stem cells (CSCs) are thought to drive tumor growth, metastasis and chemoresistance. Although surface markers such as CD133 and CD44 have been successfully used to isolate CSCs, their expression is not exclusively linked to the CSC phenotype and is prone to environmental alteration. We identified cells with an autofluorescent subcellular compartment that exclusively showed CSC features across different human tumor types. Primary tumor-derived autofluorescent cells did not overlap with side-population (SP) cells, were enriched in sphere culture and during chemotherapy, strongly expressed pluripotency-associated genes, were highly metastatic and showed long-term in vivo tumorigenicity, even at the single-cell level. Autofluorescence was due to riboflavin accumulation in membrane-bounded cytoplasmic structures bearing ATP-dependent ABCG2 transporters. In summary, we identified and characterized an intrinsic autofluorescent phenotype in CSCs of diverse epithelial cancers and used this marker to isolate and characterize these cells.

Chloroquine targets pancreatic cancer stem cells via inhibition of CXCR4 and hedgehog signaling.

Pancreatic ductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells in vitro and corresponding patient-derived pancreatic cancer tissue xenografts in vivo. Intriguingly, we found that in vitro treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished in vivo tumorigenicity and invasiveness in a large panel of pancreatic cancers. In vivo treatment in combination with gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer.

Metformin targets the metabolic achilles heel of human pancreatic cancer stem cells.

Pancreatic ductal adenocarcinomas contain a subset of exclusively tumorigenic cancer stem cells (CSCs), which are capable of repopulating the entire heterogeneous cancer cell populations and are highly resistant to standard chemotherapy. Here we demonstrate that metformin selectively ablated pancreatic CSCs as evidenced by diminished expression of pluripotency-associated genes and CSC-associated surface markers. Subsequently, the ability of metformin-treated CSCs to clonally expand in vitro was irreversibly abrogated by inducing apoptosis. In contrast, non-CSCs preferentially responded by cell cycle arrest, but were not eliminated by metformin treatment. Mechanistically, metformin increased reactive oxygen species production in CSC and reduced their mitochondrial transmembrane potential. The subsequent induction of lethal energy crisis in CSCs was independent of AMPK/mTOR. Finally, in primary cancer tissue xenograft models metformin effectively reduced tumor burden and prevented disease progression; if combined with a stroma-targeting smoothened inhibitor for enhanced tissue penetration, while gemcitabine actually appeared dispensable.

Multimodal Treatment Eliminates Cancer Stem Cells and Leads to Long-Term Survival in Primary Human Pancreatic Cancer Tissue Xenografts.

PURPOSE: In spite of intense research efforts, pancreatic ductal adenocarcinoma remains one of the most deadly malignancies in the world. We and others have previously identified a subpopulation of pancreatic cancer stem cells within the tumor as a critical therapeutic target and additionally shown that the tumor stroma represents not only a restrictive barrier for successful drug delivery, but also serves as a paracrine niche for cancer stem cells. Therefore, we embarked on a large-scale investigation on the effects of combining chemotherapy, hedgehog pathway inhibition, and mTOR inhibition in a preclinical mouse model of pancreatic cancer. EXPERIMENTAL DESIGN: Prospective and randomized testing in a set of almost 200 subcutaneous and orthotopic implanted whole-tissue primary human tumor xenografts. RESULTS: The combined targeting of highly chemoresistant cancer stem cells as well as their more differentiated progenies, together with abrogation of the tumor microenvironment by targeting the stroma and enhancing tissue penetration of the chemotherapeutic agent translated into significantly prolonged survival in preclinical models of human pancreatic cancer. Most pronounced therapeutic effects were observed in gemcitabine-resistant patient-derived tumors. Intriguingly, the proposed triple therapy approach could be further enhanced by using a PEGylated formulation of gemcitabine, which significantly increased its bioavailability and tissue penetration, resulting in a further improved overall outcome. CONCLUSIONS: This multimodal therapeutic strategy should be further explored in the clinical setting as its success may eventually improve the poor prognosis of patients with pancreatic ductal adenocarcinoma.