Nestin is expressed in HMB-45 negative melanoma cells in dermal parts of nodular melanoma.

Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells can differentiate into neurons, glia, keratocytes, smooth muscle cells and melanocytes in vitro. These pluripotent nestin-expressing stem cells are keratin 15 (K15)-negative, suggesting that they are in a relatively undifferentiated state. Recent studies suggest that the epithelial stem cells are important in tumorigenesis, and nestin expression is thought to be important in tumorigenesis. In the present study, we examined the expression of the hair follicle and neural stem cell marker nestin, as well as S-100 and HMB-45, in melanoma. Nestin immunoreactivity was observed in the HMB-45-negative melanoma cells in all five cases of amelanotic nodular melanomas. Moreover, nestin immunoreactivity was observed in the dermal parts in seven of 10 cases of melanotic nodular melanomas. Especially, nestin immunoreactivity was observed in the HMB-45-negative melanoma cells in the dermal parts of all 10 cases of HMB-45-negative amelanotic and melanotic nodular melanomas. On the other hand, nestin expression was negative in 10 of 12 cases of superficial spreading melanoma. These results suggest that nestin is an important marker of HMB-45-negative melanoma cells in the dermal parts of patients with nodular melanoma.

Th2 immune response plays a critical role in the development of nickel-induced allergic contact dermatitis.

BACKGROUND: The precise roles of T helper (Th)1-type and Th2-type cytokine responses in nickel (Ni)-induced allergic contact dermatitis have not yet been clearly defined. We investigated the involvement of Th2 cytokines in Ni-induced contact hypersensitivity reaction using GATA-3 transgenic (Tg) mice. METHODS: A Ni-titanium (Ti) alloy was implanted under the skin of GATA-3 Tg mice. A Ni solution was then injected 1 month after sensitization. The ear swelling response was measured at several time points after the injection; the cytokine levels in the skin were measured at 48 h after injection, and the serum levels of IgE were measured 1 month after injection. In addition, purified CD4+ splenic cells obtained from the GATA-3 Tg mice sensitized with the Ni-Ti alloy were infused into Rag-2(-/-) mice, and the ear swelling response of these mice after a further challenge with Ni solution was also measured. RESULTS: Marked ear swelling and elevated serum IgE levels and skin tissue levels of IL-4 were observed in Ni-Ti-sensitized GATA-3 Tg mice. The Rag-2(-/-) mice transfused with the CD4+ splenic cells from the Ni-Ti alloy sensitized GATA-3 Tg mice showed a significantly more pronounced ear swelling response than the control mice. CONCLUSION: We confirmed the participation of Th2-type immune reactions in Ni-induced allergy using GATA-3 Tg mice.

Prognostic significance of the hair follicle stem cell marker nestin in patients with malignant melanoma.

Nestin is an intermediate filament protein, and serves as a hair follicle stem cell and neural stem cell marker. Recent studies have suggested that nestin expression is also important for tumorigenesis. Previous reports from our laboratory have revealed that nestin is a marker of HMB-45-negative melanoma cells in dermal invasive lesions of nodular malignant melanoma. The present study examines nestin expression in malignant melanoma and investigates the relationship between nestin expression and prognosis in patients. We immunohistochemically stained 78 formalin-fixed and paraffin-embedded malignant melanomas for nestin, HMB-45 and S100 reactivity. We found that nestin, HMB-45 and S100 protein were detected in 56.5%, 88.4% and 100% of malignant melanomas, respectively. The 5-year survival rate of stage I and II nestin-positive cases was significantly decreased compared to the nestin-negative cases (p < 0.05). In addition, the 5-year survival rate exceeded 80% in nestin-negative malignant melanomas at all stages of tumor development. We conclude that nestin expression may be a predictor of poor prognosis in patients with malignant melanoma.

Case of follicular mucinosis: nestin-expression in mucin-producing cells.

A 29-year-old Japanese man had an asymptomatic, solitary, indurated, erythematous plaque measuring 30 mm x 30 mm on his jaw that had been present for a month. The skin lesion had follicular hyperkeratosis, and lacked hair. A skin biopsy specimen showed a dense perifollicular infiltration composed of lymphocytes, with an admixture of eosinophils in the full thickness of the dermis. The hair follicles and sebaceous glands had reticular epithelial degeneration by mucoid material of the outer root sheath and sebaceous epithelium. The mucoid material stained with Alcian blue at pH 2.5. The clinical and histological features were consistent with the diagnosis of follicular mucinosis. On immunohistochemistry, the outer root sheath cells with reticular epithelial degeneration were nestin-positive and keratin 15-negative. These results suggest that the outer root sheath cells with reticular epithelial degeneration come from the nestin-positive, multipotent, hair follicle stem cells.

Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral-nerve injury: an advantageous alternative to ES and iPS cells.

The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral-, plasmid- or transposon-mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K-15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary-acidic-protein (GFAP)-positive Schwann cells and promoted the recovery of pre-existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells.

Expression of the hair stem cell-specific marker nestin in epidermal and follicular tumors.

Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells give rise to the outer-root sheath. Nestin-expressing hair follicle stem cells that are negative for the keratinocyte marker keratin 15 (K15) can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Recent studies suggest that the epithelial stem cells are important in tumorigenesis. In this study, we immunohistochemically examined the expression of three hair follicle stem cell and progenitor cell markers, nestin, K15, and CD34, in normal human epidermis and hair follicles and in epidermal and follicular tumors, trichilemmoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). In normal human skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34. The hair follicle cells below the sebaceous glands were also positive for nestin and K15 and negative for CD34. The outer-root sheath cells under this area could be divided into three parts: an upper part of the outer-root sheath cells that was partially positive for nestin and positive for K15 and negative for CD34; a middle part that was CD34-positive and K15-negative; and a lower part that was positive for K15 and negative for CD34. In the tumor tissues, nestin immunoreactivity was observed in trichilemmoma but not in BCC. Also, immunoreactivity for K15 was strong in BCC and weak in trichilemmoma, and SCC was negative for nestin and partially positive for K15. No CD34 immunoreactivity was observed in any of the cases. These results suggested that trichilemmoma originates in the nestin-positive/K15-positive/CD34-negative outer-root sheath cells below sebaceous glands, BCC tumor cells from the more mature nestin-negative/K15-positive/CD34-negative outer-root sheath cells, and SCC from the nestin-negative/K15-positive/CD34-negative keratinocytes of the basal cell layer in the epidermis.

Recombinant Mycobacterium leprae protein associated with entry into mammalian cells of respiratory and skin components.

BACKGROUND: The transmission of Mycobacterium leprae, the causative pathogen of leprosy, has been postulated to occur mainly through upper respiratory route rather than skin-to-skin contact via minor injuries. The M. leprae genome contains mce1A gene, which encodes a putative mammalian cell entry protein. However, to date, there have been no functional analyses of the M. leprae mce1A gene product. OBJECTIVE: The aim of this study was to elucidate a possible relationship between this transmission mechanism and the mce1A gene product. METHODS: We analyzed the cell uptake activity in vitro of polystyrene latex beads coated with a purified recombinant (r-) protein expressed by a 849-bp locus within the mce1A gene. RESULTS: The r-protein promoted uptake of the beads into human nasal epithelial cells derived from nasal polyps, human bronchial epithelial cell line, normal human dermal fibroblasts, normal human microvascular endothelial cells and normal human keratinocytes cultured at 0.01 mM extracellular calcium concentration [Ca]; no uptake occurred with keratinocytes cultured at 1.2mM [Ca]. CONCLUSION: These results suggest that the mce1A gene product can mediate M. leprae entry into respiratory epithelial cells as their natural target cells, which may be the main mode of transmission. Endothelial cells, on the other hand, may serve as the reservoir of the bacilli for long-term infection. The M. leprae Mce1A protein has potential important implications for mode of transmission and pathogenesis of leprosy.

Vesicle disruption, plasma membrane bleb formation, and acute cell death caused by illumination with blue light in acridine orange-loaded malignant melanoma cells.

Acridine orange (AO), a weakly basic fluorescent dye, is permeable to plasma and vesicle membranes and preferentially remains in intracellular acidic regions. Using fluorescence microscopy, we observed dynamic changes in AO-loaded cultured malignant melanoma cells during illumination with blue light. Immediately after the start of the illumination, the successive disruption of vesicles was observed as a flash of fluorescence, and shortly after that, blebs were formed on the plasma membrane. These cells died within 5 min. Vesicle disruption was completely inhibited when cells were treated with the vacuolar H(+)-ATPase inhibitor bafilomycin A1 followed by loading with AO, but not when bafilomycin A1 was treated after AO loading. Thus, the filling of AO in the vesicle, which is driven by vacuolar H(+)-ATPase, is initially required for vesicle disruption. In contrast, bafilomycin A1 did not prevent plasma membrane blebbing, indicating that the blebs are formed independently of the vesicle disruption. Acute cell death was inhibited by treatment with bafilomycin A1 before but not after AO loading. Thus, AO- and blue light-induced acute cell death is associated with vesicle disruption rather than bleb formation. Both the vesicle disruption and the formation of plasma membrane blebs were inhibited by removal of oxygen from the cell environment and by singlet oxygen scavengers, sodium azide, ascorbic acid, and L-histidine, but not inhibited by the hydroxyl radical scavenger dimethyl thiourea. Acute cell death was also prevented by singlet oxygen scavengers but not by dimethyl thiourea. Thus, these phenomena are likely caused at least in part by the generation of singlet oxygen. The photosensitive features of plasma and vesicle membranes observed in the present study may be based on the use of the photodynamic effect, such as cancer therapy.