Uneven distribution pattern and increasing numbers of mesenchyme cells during development in the starfish, Asterina pectinifera.

During development, the embryos and larvae of the starfish Asterina pectinifera possess a single type of mesenchyme cell. The aim of this study was to determine the patterns of behavior of mesenchyme cells during the formation of various organs. To this end, we used a monoclonal antibody (mesenchyme cell marker) to identify the distribution patterns and numbers of mesenchyme cells. Our results revealed the following: (i) mesenchyme cell behavior differs in the formation of different organs, showing temporal variations and an uneven pattern of distribution; and (ii) mesenchyme cells continue to be generated throughout development, and their numbers are tightly regulated in proportion to total cell numbers.

Surgical punctal occlusion with a high heat-energy releasing cautery device for severe dry eye with recurrent punctal plug extrusion.

PURPOSE: To report the rate of recanalization and the efficacy of punctal occlusion surgery with a high heat-energy-releasing cautery device in patients with severe dry eye disease and recurrent punctal plug extrusion. DESIGN: Prospective, interventional case series. METHODS: Seventy puncta from 44 eyes of 28 dry eye patients underwent punctal occlusion with thermal cautery. All patients had a history of recurrent punctal plug extrusion. A high heat-energy-releasing thermal cautery device (Optemp II V; Alcon Japan) was used for punctal occlusion surgery. Symptom scores, best-corrected visual acuity, fluorescein staining score, rose bengal staining score, tear film break-up time, and Schirmer test values were compared before and 3 months after the surgery. Rate of punctal recanalization also was examined. RESULTS: Three months after surgical cauterization, symptom score decreased from 3.9 ± 0.23 to 0.56 ± 0.84 (P < .0001). Logarithm of the minimal angle of resolution best-corrected visual acuity improved from 0.11 ± 0.30 to 0.013 ± 0.22 (P = .003). Fluorescein staining score, rose bengal staining score, tear film break-up time, and the Schirmer test value also improved significantly after the surgery. Only 1 of 70 puncta recanalized after thermal cauterization (1.4%). CONCLUSIONS: Punctal occlusion with the high heat-energy-releasing cautery device not only was associated with a low recanalization rate, but also with improvements in ocular surface wetness and better visual acuity.

Interferometry in the evaluation of precorneal tear film thickness in dry eye.

PURPOSE: To compare tear film thickness between normal subjects and aqueous tear deficiency dry eye patients by tear interferometry. DESIGN: Prospective case-control study. METHODS: Central precorneal tear film thickness was measured noninvasively using an interference thin-film thickness measurement device (Quore MSPA1100; Mamiya-OP). Tear film thickness of 14 eyes from 14 normal subjects and of 28 eyes from 28 aqueous tear deficiency dry eye patients were compared along with noninvasively measured tear meniscus height, DR-1 (Kowa) dry eye severity grading, fluorescein and rose bengal staining scores, tear film break-up time, and Schirmer test results. Among dry eye patients, 13 eyes underwent punctal occlusion, and tear film thickness was compared before and after the surgery. RESULTS: Tear film was significantly thinner in dry eye patients (2.0 ± 1.5 µm) than normal subjects (6.0 ± 2.4 µm; P < .0001). Tear film thickness showed good correlation with other dry eye examinations. After punctal occlusion, tear film thickness increased significantly from 1.7 ± 1.5 µm to 4.9 ± 2.8 µm (P = .001) with the improvement of tear meniscus height, fluorescein and rose bengal staining scores, tear film break-up time, and Schirmer test values. CONCLUSIONS: Interferometric tear film thickness measurement revealed impaired precorneal tear film formation in aqueous tear deficiency dry eyes and was useful for showing the reconstruction of tear film after punctal occlusion surgery. Interferometry of precorneal tear film may be helpful for the evaluation of aqueous tear deficiency in conjunction with other dry eye examinations.

Noninvasive interference tear meniscometry in dry eye patients with Sjögren syndrome.

PURPOSE: To compare noninvasive tear meniscus height (NI-TMH) using a tear interference device in normal subjects and dry eye patients with Sjögren syndrome (SS), and to investigate the applicability of this new method before and after the punctal occlusion procedure. DESIGN: Prospective case control study. METHODS: Tear meniscus was visualized noninvasively using a tear interference device (Tearscope plus, Keeler, Windsor, United Kingdom). Tear interference image was captured with digital video camera (SP-321, JFC Sales Plan Co, Tokyo, Japan) attached to the slit-lamp. Lower lid margin NI-TMH was measured using image analysis software. NI-TMH of 28 eyes from 17 normal subjects and 46 eyes from 27 aqueous tear deficiency (ATD) dry eye patients with SS were compared. The change of NI-TMH three weeks after the successful punctal occlusion was examined in 11 eyes of eight dry eye subjects. RESULTS: Tear meniscus was well visualized with the tear interference device in all cases. Lower lid margin NI-TMH was 0.22 +/- 0.065 mm in normal subjects, and 0.13 +/- 0.042 mm in SS subjects, respectively (P < .0001). After the punctal occlusion, lower lid margin NI-TMH increased significantly from 0.12 +/- 0.026 mm to 0.42 +/- 0.21 mm (P = .001). CONCLUSIONS: NI-TMH was substantially lower in SS subjects and also significantly improved after punctal occlusion. This method is expected to be helpful in the diagnosis and in the evaluation of the efficacy of punctal occlusion in ATD dry eyes such as SS.

Establishment of a kit-negative cell line of melanocyte precursors from mouse neural crest cells.

We previously established a mouse neural crest cell line named NCCmelb4, which is positive for Kit and negative for tyrosinase. NCCmelb4 cells were useful to study the effects of extrinsic factors such as retinoic acids and vitamin D(3) on melanocyte differentiation, but in order to study the development of melanocytes from multipotent neural crest cells, cell lines of melanocyte progenitors in earlier developmental stages are needed. In the present study, we established an immortal cell line named NCCmelb4M5 that was derived from NCCmelb4 cells. NCCmelb4M5 cells do not express Kit and are immortal and stable in the absence of Kit ligand. They are positive for melanocyte markers such as tyrosinase-related protein 1 and DOPAchrome tautomerase and they contain stage I melanosomes. Interestingly, glial fibrillary acidic protein, which is a marker for glial cells, is also positive in NCCmelb4M5 cells, while NCCmelb4 cells are negative for this protein. Immunostaining and a cell ELISA assay revealed that 12-O-tetradecanoylphorbol 13-acetate (TPA) and cholera toxin (CT) induce Kit expression in NCCmelb4M5 cells. Real-time polymerase chain reaction analysis also demonstrated the induction of Kit mRNA by TPA and CT. Microphthalmia-associated transcription factor mRNA is simultaneously enhanced by the same treatment. Kit induced by TPA/CT in NCCmelb4M5 cells disappeared after the cells were subcultured and incubated without TPA/CT. These findings show that NCCmelb4M5 cells have the potential to differentiate into Kit-positive melanocyte precursors and may be useful to study mechanisms of development and differentiation of melanocytes in mouse neural crest cells.

Effects of ultraviolet light on melanocyte differentiation: studies with mouse neural crest cells and neural crest-derived cell lines.

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L-3,4-dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT- or DOPA-positive cells between the UV-irradiated cultures and the non-irradiated cultures. We then examined the effects of UV light on KIT-positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase-positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with alpha-melanocyte-stimulating hormone (alpha-MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase-negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal-regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as alpha-MSH and/or endothelin-1.

Differentiation of murine melanocyte precursors induced by 1,25-dihydroxyvitamin D3 is associated with the stimulation of endothelin B receptor expression.

The effects of 1,25-dihydroxyvitamin D3 on the differentiation of immature melanocyte precursors were studied. The NCC-/melb4 cell line is an immature melanocyte cell line established from mouse neural crest cells. 1,25-Dihydroxyvitamin D3 inhibited the growth of NCC-/melb4 cells at concentrations higher than 10(-8) m. That growth inhibition was accompanied by the induction of tyrosinase and a change in L-3,4-dihydroxyphenylalanine reactivity from negative to positive. Electron microscopy demonstrated that melanosomes were in more advanced stages after 1,25-dihydroxyvitamin D3 treatment. In primary cultures of murine neural crest cells, L-3,4-dihydroxyphenylalanine-positive cells were increased after 1,25-dihydroxyvitamin D3 treatment. These findings indicate that 1,25-dihydroxyvitamin D3 stimulates the differentiation of immature melanocyte precursors. Moreover, immunostaining and reverse transcription-polymerase chain reaction analysis revealed that endothelin B receptor expression was induced in NCC-/melb4 cells following treatment with 1,25-dihydroxyvitamin D3. The induction of endothelin B receptor by 1,25-dihydroxyvitamin D3 was also demonstrated in neural crest cell primary cultures, but not in mature melanocytes. The expression of microphthalmia-associated transcription factor was induced in NCC-/melb4 cells treated with 1,25-dihydroxyvitamin D3 and endothelin 3, but not by 1,25-dihydroxyvitamin D3 alone, suggesting that endothelin 3 may stimulate the expression of the microphthalmia-associated transcription factor gene after binding to the endothelin B receptor induced by 1,25-dihydroxyvitamin D3. These findings suggest a regulatory role for vitamin D3 in melanocyte development and melanogenesis, and may also explain the working mechanism of vitamin D3 in the treatment of vitiligo.

Transforming growth factor beta1 regulates melanocyte proliferation and differentiation in mouse neural crest cells via stem cell factor/KIT signaling.

Stem cell factor is essential to the migration and differentiation of melanocytes during embryogenesis based on the observation that mutations in either the stem cell factor gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Stem cell factor is also required for the survival of melanocyte precursors while they are migrating towards the skin. Transforming growth factor beta1 has been implicated in the regulation of both cellular proliferation and differentiation. NCC-melb4, an immortal cloned cell line, was cloned from a mouse neural crest cell. NCC-melb4 cells provide a model to study the specific stage of differentiation and proliferation of melanocytes. They also express KIT as a melanoblast marker. Using the NCC-melb4 cell line, we investigated the effect of transforming growth factor beta1 on the differentiation and proliferation of immature melanocyte precursors. Immunohistochemically, NCC-melb4 cells showed transforming growth factor beta1 expression. The anti-transforming growth factor beta1 antibody inhibited the cell growth, and downregulated the KIT protein and mRNA expression. To investigate further the activation of autocrine transforming growth factor beta1, NCC-melb4 cells were incubated in nonexogenous transforming growth factor beta1 culture medium. KIT protein decreased with anti-transforming growth factor beta1 antibody concentration in a concentration-dependent manner. We concluded that in NCC-melb4 cells, transforming growth factor beta1 promotes melanocyte precursor proliferation in autocrine and/or paracrine regulation. We further investigated the influence of transforming growth factor beta1 in vitro using a neural crest cell primary culture system from wild-type mice. Anti-transforming growth factor beta1 antibody decreased the number of KIT positive neural crest cell. In addition, the anti-transforming growth factor beta1 antibody supplied within the wild-type neural crest explants abolished the growth of the neural crest cell. These results indicate that transforming growth factor beta1 affect melanocyte precursor proliferation and differentiation in the presence of stem cell factor/KIT in an autocrine/paracrine manner.