Isolation and characterization of outer root sheath melanocytes of human hair follicles.

BACKGROUND: Outer root sheath melanocytes (ORSM) are not yet routinely cultured and their biology is not known in detail because of their relatively low numbers in the hair follicle and their limited proliferative capacity in in vitro culture in routine media. OBJECTIVES: To develop a method for culturing ORSM more easily and to investigate the length of telomeres and antigenic characteristics of ORSM compared with epidermal melanocytes (EM). METHODS: Hair follicles were obtained from three Korean individuals during hair transplantation surgery. Single-cell suspensions of the outer root sheath were made and cultured in melanocyte growth medium with stem cell factor. After 21 days, second-passage outer root sheath keratinocytes (ORSK) (2 x 10(4) mL(-1) MGM) were added into the culture plates. We studied the proliferation pattern, morphological and antigenic characteristics of ORSM for each passage of cultured cells, and observed ORSM telomere length. RESULTS: We established an ORSM culture method using ORSK. Two morphologically different ORSM types were obtained in the primary cultures. At the end of primary culture, ORSM appeared as whitish-cream pellets. The proliferation pattern of ORSM showed a sigmoidal shape, the accumulated numbers of population doublings showed a plateau after approximately 5 months, and senescence occurred at approximately 33 +/- 5 accumulated population doublings. The length of ORSM telomeres continued to shorten as the cells proliferated. In contrast, EM showed a marked proliferation from the early proliferation period which formed a plateau pattern towards the later period, and the number of accumulated population doublings was estimated to be 18 +/- 5 after 2 months. ORSM in the primary culture reacted variably with l-dihydroxyphenylalanine (DOPA): some cells were DOPA negative, some DOPA positive. There were some different antigenic expressions of microphthalmia-associated transcription factor (MITF) showing cytoplasmic expression in ORSM and nuclear expression in EM. By nuclear extraction and Western blotting, we showed that MITF expression of ORSM was marked in the cytoplasm and minimal in the nucleus. Antigenic expression of MITF and Bcl-2 gradually decreased with increasing passage number, whereas tyrosinase-related protein-1 expression did not change. CONCLUSIONS: Culture of ORSM requires ORSK or ORSK-related factors; ORSM have greater proliferation potential and show different MITF antigenic expression compared with EM; and the length of ORSM telomeres shortens with repeated proliferation.

Upregulation of the NNP-1 (novel nuclear protein-1, D21S2056E) gene in keloid tissue determined by cDNA microarray and in situ hybridization.

BACKGROUND: A keloid results from excessive collagen deposition, the cause of which remains elusive. A thorough understanding of the pathophysiology of keloid tissue can help determine the most appropriate treatment strategy. OBJECTIVES: To assess the differences in gene expression between keloids and adjacent normal skin in order to define the genes involved in keloid formation. METHODS: Three Korean patients with keloids underwent excision of the keloid and adjacent normal skin, which was used as the control. We investigated expression patterns of genes in the keloids and the normal skin using cDNA microarray and in situ hybridization techniques. RESULTS: Nine genes in the keloid tissue were consistently upregulated over the 2.0 ratio compared with the normal control from the cDNA microarray composed of 3063 clones: collagen type I alpha1 (NM_000088), DNA segment on chromosome 21 (unique) 2056 expressed sequence (D21S2056E, NNP-1, NM_003683), suppressor of Ty 5 homologue (NM_003169), phosphoglycerate dehydrogenase (NM_032692), adenosine triphosphate synthase beta (NM_001686), serine (or cysteine) proteinase inhibitor, clade H (heat shock protein 47, NM_001235), LIV-1 protein, oestrogen regulated (LIV-1, NM_012319), interleukin-11 receptor alpha (IL11RA, NM_004512) and carbonyl reductase 3 (CBR3, NM_001236). From the in situ hybridization study, the staining signals in the keloid tissue hybridized with anti sense probes of NNP-1 mRNA were stronger than signals in normal controls. Further, endothelial epithelium, but not the epidermis, expressed the signal equally in both keloid and normal control tissue. CONCLUSIONS: We identified nine upregulated genes in keloid tissue using cDNA microarray. Of the nine, the NNP-1 gene was confirmed by topological information using the in situ hybridization technique. We conclude that these nine genes, especially NNP-1, probably contribute either directly or indirectly to keloid formation.

Lichenoid sarcoidosis in a 3-year-old girl.

We report a case of lichenoid sarcoidosis in a 3-year-old girl. She had numerous discrete skin-colored or erythematous, infiltrated follicular papules on the buttocks and extremities since 2 months of age. Histopathologic examination showed follicular plugging and an upper dermal granulomatous infiltrate of epithelioid cells closely surrounding the follicular ducts. No acid-fast bacilli were seen in the sections examined. Chest radiograph and high-resolution computed tomography (CT) showed no hilar lymphadenopathy or pulmonary parenchymal changes. An angiotensin-converting enzyme level was elevated. The Mantoux reaction was negative and results of ophthalmologic examinations were normal. Treatment was started with triamcinolone 0.2 mg/kg and prednicarbate ointment. Some lesions healed completely and others showed residual pitting.

Kasabach-Merritt syndrome: identification of platelet trapping in a tufted angioma by immunohistochemistry technique using monoclonal antibody to CD61.

Kasabach-Merritt syndrome (KMS) consists of large and rapidly growing vascular tumors associated with thrombocytopenia, generalized petechiae, and bleeding. The cause of the thrombocytopenia is thought to be related to the trapping of platelets by the abnormal endothelium of the tumor. We report an infant with KMS that developed in association with a large tufted angioma. In this case we directly demonstrated platelet trapping in the vascular lumen of the tumor by an immunohistochemical technique using a monoclonal antibody against CD61, a marker of platelets and megakaryocytes.

Single hair grafting for the treatment of vitiligo.

BACKGROUND: Vitiligo is a disease of unknown cause, and many medical and surgical therapeutic methods are used to treat it. OBJECTIVE: Our purpose was to evaluate the effectiveness of single hair grafting in patients with vitiligo. METHODS: Single hairs were grafted into vitiliginous areas of 21 patients. The diameter of the spreading pigmentation was evaluated periodically. RESULTS: Perifollicular repigmentation around the grafted hair was observed in 15 patients (71%) within 2 to 8 weeks. The diameter of the spreading pigmentation ranged from 2 to 10 mm during a 12-month follow-up period. In cases of generalized vitiligo, perifollicular pigmentation was seen in one of four patients (25%), whereas it was observed in 14 of 17 patients (82%) with localized/segmental vitiligo. Transformation of depigmented hairs into pigmented ones occurred in five patients. CONCLUSION: Single hair grafting appears to be an effective method for treating localized/segmental vitiligo, especially on hairy parts of the skin, including the eyelids and eyebrows, and for small areas of vitiligo.

Herpes zoster in three healthy children immunized with varicella vaccine (Oka/Biken); the causative virus differed from vaccine strain on PCR analysis of the IV variable region (R5) and of a PstI-site region.

This study was undertaken to determine whether the causative virus was a vaccine-derived or wild-type virus when zoster occurred in healthy children immunized with varicella vaccine (Oka/Biken). The DNAs of clinical isolated strains and vaccine strain (Oka/Biken) were analyzed by the polymerase chain reaction with two sets of primers for the variable region IV (R5 tandem direct reiterations, TDR) and for the region with a PstI site in a middle portion of the long unique segment of the varicella zoster virus genome. Of six zoster patients after vaccination with Biken, three clinical isolates were examined and had two copies in R5 TDR and were PstI-site positive. Therefore, these strains were different from the vaccine-type strain (Oka/Biken), which had two copies in R5 TDR and was PstI-site-negative. The mean age of onset of zoster was 4 years. The mean age of vaccination was 25 months. The mean interval between vaccination and onset of zoster was 22 months. Hence the results indicate that the causative virus of zoster in healthy children immunized with varicella vaccine (Oka/Biken) was wild type and differed from the vaccine strain. Some vaccinees probably do not have protective immunity for a long time after immunization because the mean interval between vaccination and onset of zoster was 22 months.