Prognostic factors in methylprednisolone pulse therapy for alopecia areata.

Many treatments induce remission in patients with alopecia areata. Systemic steroids, for example, are effective in the treatment of severe alopecia areata but have many side-effects. To avoid these side-effects, high-dose bolus infusions of methylprednisolone have been used to treat severe alopecia areata. The purpose of this study was to evaluate the prognostic factors associated with pulse therapy and to establish proper indications for methylprednisolone pulse therapy. Seventy patients with severe alopecia areata were treated i.v. with methylprednisolone on 3 consecutive days. All of the patients had rapid and extensive hair loss with the bald area exceeding 50% of the scalp. Seventy percent of the patients showed terminal hair growth and 41.4% showed complete responses with acceptable cosmetic outcomes. The prognostic factors that influenced successful outcome were the disease duration before treatment and the type of alopecia areata. Based on these two factors, a good response was obtained for all types of alopecia areata with a duration of 3months or less before treatment and for the plurifocal type of alopecia areata with a duration of 4-6months. Methylprednisolone pulse therapy is indicated for those alopecia areata patients who fall within our good response group.

Analysis of familial factors using the basic and specific (BASP) classification in Korean patients with androgenetic alopecia.

BACKGROUND: The mechanisms and inheritance of androgenetic alopecia (AGA) have yet to be elucidated. Several clinical studies suggest that a predisposition to AGA is affected by a variety of paternal and/or maternal hereditary factors. No previous study has addressed the association of AGA with family history based on the pattern of hair loss. OBJECTIVES: The purpose of this study was to investigate paternal and/or maternal genetic influences in each type of hair loss pattern using the basic and specific (BASP) classification and to explore whether the morphology of AGA tends to be inherited in family members. METHODS: Between October 2007 and September 2008, 1220 Korean participants, 998 male and 222 female, were classified according to the BASP classification at 13 university dermatologic centers. Information was collected using a standard questionnaire and BASP classification sheet. RESULTS: Parental influences on anterior hairline shape in men were predominantly from the paternal side, whereas these effects were less notable in women. In the absence of a family history, statistical analysis showed a higher frequency for early-onset AGA than late-onset AGA (Pearson χ(2)P < .05). Basic types of hair loss had a higher degree of inheritance from the paternal side of the family, regardless of specific type. LIMITATIONS: The evaluation of hair loss pattern and family history was done by the patients. CONCLUSIONS: Familial factors affecting the morphology of AGA differ between male and female individuals, and for each type of BASP classification.

Expression of N-terminal truncated desmoglein 3 (deltaNDg3) in epidermis and its role in keratinocyte differentiation.

During a search for keratinocyte differentiation-related genes, we obtained a cDNA fragment from the 5'-untranslated region of a previously identified splicing variant of desmoglein 3 (Dg3). This transcript encodes a protein of 282 amino acids, which corresponds to the N-terminal truncated intracellular domain of Dg3 (deltaNDg3). Northern blot analysis detected a 4.6-kb transcript matching the predicted size of deltaNDg3 mRNA, and Western blot analysis with an antibody raised against the Dg3 C-terminus (H-145) detected a 31-kDa protein. Increased deltaNDg3 expression was observed in differentiating keratinocytes by RT-PCR and Western blot analysis, suggesting that deltaNDg3 is indeed a differentiation-related gene product. In immunohistochemical studies of normal and pathologic tissues, H-145 antibody detected the protein in the cytoplasm of suprabasal layer cells, whereas an antibody directed against the N-terminal region of Dg3 (AF1720) reacted with a membrane protein in the basal layer. In addition, deltaNDg3 transcript and protein were upregulated in psoriatic epidermis, and protein expression appeared to increase in epidermal tumors including Bowen's disease and squamous cell carcinoma. Moreover, overexpression of deltaNDg3 led to increased migration and weakening of cell adhesion. These results suggest that deltaNDg3 have a role in keratinocyte differentiation, and that may be related with tumorigenesis of epithelial origin.

Pitx2, a beta-catenin-regulated transcription factor, regulates the differentiation of outer root sheath cells cultured in vitro.

BACKGROUND: Beta-catenin exerts its crucial role in hair follicle development and hair growth cycle. Although the importance of Wnt/beta-catenin is well recognized, the downstream effectors of beta-catenin have not been clearly elucidated yet. OBJECTIVE: The aim of this study is to identify the beta-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells. METHODS: We transduced ORS cells with adenovirus harboring the expression cassette for constitutive active form of beta-catenin, then performed cDNA microarray. RESULTS: Overexpression of beta-catenin led to the upregulation of hair cell differentiation markers such as keratin 16 and 17. In addition, the expression of Pitx2, a bicoid-type homeodomain transcription factor, was also increased by overexpression of beta-catenin in ORS cells cultured in vitro. To investigate the potential role of Pitx2, we made the recombinant adenovirus expressing Pitx2, then transduced into the cultured ORS cells. Interestingly, Pitx2 induced the expression of keratin 16 and 17, indicating that Pitx2 activates ORS cells towards the follicular differentiation pathway preferentially. CONCLUSION: Our results implicate the potential importance of Pitx2 as a beta-catenin downstream modulator in hair growth control.

Isolated Langerhans cell histiocytosis of the vulva in an infant.

Langerhans cell histiocytosis affecting only the vulva of a child is very rare. We report a 1-year-old female infant with isolated Langerhans cell histiocytosis presenting as pruritic papules confined to the vulva, confirmed by histopathology with immunohistochemical staining.

A case of isolated plexiform neurofibroma in a patient with myasthenia gravis.

We report a case of an isolated plexiform neurofibroma occurring in a patient with myasthenia gravis. A 48-year-old man presented with asymptomatic skin-colored nodules on the tip of his 4th finger. Microscopically, a plexiform neurofibroma was identified located in the dermis that appeared to originate from small superficial nerves. He had a 20-year history of treated myasthenia gravis; otherwise, his personal and family histories were unremarkable. Given that myasthenia gravis is a disorder of the peripheral nerves, plexiform neurofibromas could be associated with myasthenia gravis. However, the development of an isolated plexiform neurofibroma in a case of myasthenia gravis has not yet been reported. The occurrence of a neurofibromas in a patient with myasthenia gravis suggests a link in the pathogenesis of these two diseases.

Angiolymphoid hyperplasia with eosinophilia that was possibly induced by vaccination in a child.

Angiolymphoid hyperplasia with eosinophilia (ALHE) is a rare benign vasoproliferative disease of an unknown cause involving the skin or subcutaneous tissue of the head and neck, and particularly around the ear. It predominantly affects Caucasian adults during the third and fourth decades and it very rarely occurs in children. We experienced a case of ALHE in a 2-year-old Korean boy who had a firm, pruritic, skin-colored, subcutaneous nodule on his right upper arm. The histopathological findings were compatible with ALHE and they showed prominent vascular changes with epitheloid or histiocytoid endothelial cells surrounded by inflammatory cells, including a large proportion of eosinophils. This unusual distribution of the lesion and the young age of the patient may be associated with vaccination.

Expression of neutrophil gelatinase-associated lipocalin in calcium-induced keratinocyte differentiation.

In a previous search for the differentially expressed genes in keratinocyte differentiation, we identified neutrophil gelatinase-associated lipocalin (NGAL) as a calcium-induced gene. In this study, we further verified the expression of NGAL in cultured keratinocytes as well as in several skin diseases. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and ELISA clearly showed that NGAL expression was markedly increased in calcium-induced keratinocyte differentiation in vitro. However, in our previous report, NGAL expression was not detected in normal skin tissue except for hair follicle by in situ hybridization and immunohistochemistry, indicating the difference of cell status between in vitro and in vitro conditions. Interestingly, NGAL expression was highly increased in psoriasis-like inflammatory disorders (lichen planus and pityriasis rubura pilaris) and skin cancers (keratoacanthoma and squamous cell carcinoma), implying that NGAL may be related with the epidermal hyperplasia. Collectively, these results reveal the potential importance of NGAL in the maintenance of skin homeostasis.

Induction of synapse associated protein 102 expression in cyclosporin A-stimulated hair growth.

Cyclosporin A (CsA) has been used as a potent immunosuppressive agent for inhibiting the graft rejection after organ transplantation. However, CsA provokes lots of side effects including hirsutism, the phenomenon of abnormal hair growth in the body. In the present study, we investigated the hair growth stimulating effect of CsA using in vivo and in vitro test models. When topically applied on the back skin of mice, CsA induced fast telogen to anagen transition. In contrast, CsA had no effect on the growth of human hair follicle tissues cultured in vitro, indicating that it might not have the mitogenic effect on hair follicles. To identify the genes related with CsA-induced hair growth, we performed differential display RT-PCR. Among the genes obtained, the expression of synapse associated protein 102 (SAP102) was verified using competitive RT-PCR. The result showed that the expression of SAP102 was significantly induced by CsA treatment in the back skin of C57BL/6 mice. However, the increase of SAP102 mRNA was also seen in spontaneous anagen mice, suggesting that induction of SAP102 is one event of the anagen hair growth response regardless of how the growth state was induced. SAP102 was not expressed in cultured human hair outer root sheath and dermal papilla cells. Immunohistochemistry analysis showed that CsA induced the expression of SAP102 in perifollicular region of mouse anagen hair. Together, these results suggest that SAP102 is one of hair-cycle-dependent genes, whose expression is related with the anagen progression.

Establishment of type II 5alpha-reductase over-expressing cell line as an inhibitor screening model.

Dihydrotestosterone (DHT) is the most potent male hormone that causes androgenetic alopecia. The type II 5alpha-reductase is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitors for type II 5alpha-reductase may improve the pathophysiologic status of androgenetic alopecia. In this study, we attempted to establish the reliable and convenient screening model for type II 5alpha-reductase inhibitors. After transfection of human cDNA for type II 5alpha-reductase into HEK293 cells, the type II 5alpha-reductase over-expressing stable cells were selected by G418 treatment. RT-PCR and Western blot analyses confirmed that type II 5alpha-reductase gene was expressed in the stable cells. In in vitro enzymatic assay, 10 microg of stable cell extract completely converted 1 microCi (approximately 0.015 nmol) of T into DHT. The type II 5alpha-reductase activity was inhibited by finasteride in a dose-dependent manner, confirming the reliability of screening system. In cell culture condition, 2 x 10(5) of stable cells completely converted all the input T (approximately 0.03 nmol) into DHT by 4h incubation, demonstrating that the stable cell line can be used as a cell-based assay system. Using this system, we selected the extracts of Curcumae longae rhizoma and Mori ramulus as the potential inhibitors for type II 5alpha-reductase. These results demonstrate that the type II 5alpha-reductase over-expressing stable cell line is a convenient and reliable model for screening and evaluation of inhibitors.

Novel synthetic ceramide derivatives increase intracellular calcium levels and promote epidermal keratinocyte differentiation.

Ceramide is an important constituent of stratum corneum lipids, which act as both physical barriers and signal modulators. We synthesized several ceramide derivatives and investigated their effects on keratinocyte differentiation. RT-PCR and Western blotting showed that the novel synthetic ceramide derivatives K6PC-4 [N-(2,3-dihydroxypropyl)-2-hexyl-3-oxo-decanamide], K6PC-5, [N-(1,3-dihydroxypropyl-2-hexyl-3-oxo-decanamide] and K6PC-9 (N-ethanol-2-hexyl-3-oxo-decanamide) [corrected] These ceramide derivatives elicited a rapid transient increase in intracellular calcium levels, which were measured using laser scanning confocal microscopy. In addition, K6PC-4, K6PC-5, and K6PC-9 stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In a reconstituted epidermis model, K6PC-4, K6PC-5, and K6PC-9 significantly increased keratin 1 expression in the suprabasal layer. These results indicate that these novel synthetic ceramide derivatives have the potential to promote keratinocyte differentiation, suggesting that the lipid molecules are applicable for treating skin diseases involving abnormal keratinocyte differentiation.

A new classification of pattern hair loss that is universal for men and women: basic and specific (BASP) classification.

BACKGROUND: Pattern hair loss (PHL) can be classified into several patterns. Currently, the Hamilton-Norwood classification system for men and the Ludwig grade system for women are commonly used to describe patterns of hair loss. However, these pre-existing classifications have some limitations. OBJECTIVE: To establish an acceptable, universal, and accurate standard of both male and female pattern hair loss and to report its use in determining the incidence of PHL. METHODS: We developed a new classification system (BASP classification) and then applied this system to classify the types of PHL. The BASP classification was based on observed patterns of hair loss. The basic (BA) types represent the shape of the anterior hairline, and the specific types (SP) represent the density of hair on distinct areas (frontal and vertex). There are four basic types (L, M, C, and U) and two specific types (F and V). The final type is decided by the combination of the assigned basic and specific types. Between November 2004 and June 2005, 2213 Korean subjects, comprised of 1768 males and 445 females, were classified according to the BASP classification at 13 university dermatologic centers nationwide throughout South Korea, as a multicenter study of the Korean Hair Research Society. RESULTS: For both sexes, the majority of patients enrolled in the study were in the third and fourth decade of life (65.1% of males and 56.68% of females). In males, the older group as well as the younger group in the study were more likely to have little recession of the frontal hairline (classified as type M1 approximately 2) and diffuse thinning over the top of scalp (type F1 approximately 2). The women in the study developed typical female PHL. LIMITATIONS: The subjects of our study were mostly outpatients and some inpatients who complained about hair loss, not the general population of Korea. CONCLUSION: The BASP classification is a new stepwise, systematic, and universal classification system for PHL, regardless of sex.

Anti-angiogenic effect of tetraacetyl-phytosphingosine.

In a search for the wound healing accelerators, we found that tetraacetyl-phytosphingosine (TAPS), a sphingolipid metabolite produced by phytosphingosine acetylation, has significant inhibitory potential on healing of rabbit ear wound. As angiogenesis is fundamental to proper wound healing, we examined the effect of TAPS on angiogenesis using human umbilical vein endothelial cells cultured in vitro. TAPS markedly decreased vascular endothelial growth factor (VEGF)-induced chemotactic migration and capillary-like tube formation. Recognizing its inhibitory potential on angiogenesis, we further investigated the action mechanism of TAPS. TAPS significantly inhibited VEGF-induced proteolytic enzyme production, including matrix metalloproteinase-2, urokinase-type plasminogen activator and plasminogen activator inhibitor-1. TAPS also suppressed VEGF-induced phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In addition, TAPS abolished VEGF-induced intracellular calcium increase, measured using laser scanning confocal microscopy. Together, these results suggest that TAPS exerts its inhibitory action on angiogenesis through the inhibition of mitogen-activated protein kinase activation and intracellular calcium increase, thereby affecting the process of wound healing negatively.

Effect of thioredoxin reductase 1 on glucocorticoid receptor activity in human outer root sheath cells.

Alopecia areata (AA) is a common disease of patchy hair loss on the scalp that can progress to cover the entire scalp and eventually the entire body. Intralesional injection of corticosteroids is the first-line therapy for adult patients, however some patients do not respond to glucocorticoid treatment effectively. To delineate the molecular mechanism underlying glucocorticoid insensitivity, we examined the expression of glucocorticoid receptor (GR) and thioredoxin reductase 1 (TrxR1). In some case of glucocorticoid-resistant AA patients, the expression of TrxR1 was decreased in outer root sheath (ORS). We then investigated the effect of TrxR1 on GR activity using recombinant adenoviruses. Overexpression of TrxR1 markedly increased GR activity in ORS cells cultured in vitro. In addition, TrxR1 protected GR activity against H(2)O(2). Finally, TrxR1-enhanced GR activity was significantly inhibited by the overexpression of dominant negative form of Trx (Trx(C32S/C35S)). These results suggest that decreased TrxR1 may be one putative cause for glucocorticoid resistance in AA, through the impact on intracellular redox system.

Sphingosylphosphorylcholine-induced interleukin-6 production is mediated by protein kinase C and p42/44 extracellular signal-regulated kinase in human dermal fibroblasts.

BACKGROUND: Sphingosylphosphorylcholine (SPC) has been reported as a novel lipid mediator that exerts various actions on wound healing process. OBJECTIVE: The aim of this study is to evaluate the involvement of interleukin-6 (IL-6) in SPC-induced wound healing acceleration. METHODS: We performed immunohistochemical analysis to demonstrate the IL-6 induction by SPC. To analyze the signaling events, skin fibroblasts were treated with SPC, and then RT-PCR, ELISA and Western blot analyses were carried out. RESULTS: SPC markedly induced interleukin-6 (IL-6) expression in rabbit ear wound. SPC also induced IL-6 expression at both the mRNA and protein levels in human dermal fibroblasts cultured in vitro. SPC rapidly phosphorylated p42/44 extracellular signal-regulated kinase (ERK). Pretreatment with PD 98059, a specific MAPK kinase 1/2 inhibitor, markedly suppressed SPC-induced IL-6 expression in a dose-dependent manner. Protein kinase C (PKC) activation by phorbol myristate acetate (PMA) potentiated IL-6 mRNA expression, whereas PKC inhibition by bisindolylmaleimide blocked SPC-induced p42/44 ERK phosphorylation and IL-6 expression. Over-expression of PKCalpha markedly induced the IL-6 expression and p42/44 ERK activation. CONCLUSION: These results suggest that SPC-induced IL-6 production is mediated by PKC-dependent p42/44 ERK activation in human dermal fibroblasts cultured in vitro.

Molecular cloning and expression of human keratinocyte proline-rich protein (hKPRP), an epidermal marker isolated from calcium-induced differentiating keratinocytes.

We isolated a human gene encoding keratinocyte proline-rich protein (hKPRP). hKPRP gene is located in the region of epidermal differentiation complex on chromosome 1q21, and its approximately 2.5 kb mRNA encodes 579 amino acid protein with high proline content (18%). The mRNA level of hKPRP was markedly increased at both 7 and 14 d after treatment with 1.2 mM calcium in cultured normal human epidermal keratinocytes. In situ hybridization demonstrated that hKPRP was expressed in upper granular layer of normal epidermis with characteristic intermittent pattern. In psoriatic lesion, hKPRP expression was increased as compared with normal skin and showed continuous pattern. Immunohistochemical analysis also confirmed the expression of hKPRP at the protein level. Western blot analysis showed that hKPRP protein of approximately 70 kDa size was significantly increased by calcium in a time-dependent manner. In mouse tissue blot assays, the expression of KPRP was detected in stomach and skin tissues, and began at 17.5 embryonic days. Additionally, hKPRP expression was detected in the periderm of human fetal skin from 16 wk estimated gestational age. Together, these results suggest that hKPRP is an epidermal marker expressed in stratified squamous epithelia and has a potential role in keratinocytes differentiation.