Removal of stem cell factor or addition of monoclonal anti-c-KIT antibody induces apoptosis in murine melanocyte precursors.

Previous findings indicate that the protein c-KIT and its ligand, stem cell factor (SCF) play a crucial role in the development of melanocytes from their precursors in the embryonic neural crest cells. Using a monoclonal anti-c-KIT antibody, ACK2, which is an antagonistic blocker of c-KIT function, we and colleagues demonstrated that mouse melanocytes disappeared with the injection of ACK2 during certain periods of embryonic and postnatal life. The precise mechanisms of this disappearance, however, remain unclear. Because melanocytes disappeared without any inflammation in these in vivo studies, we suspect that apoptosis was a main cause of their disappearance. In this study, to clarify the underlying mechanism, we studied whether ACK2 induces apoptosis in c-KIT-positive melanoblasts, which appear in mouse neural crest cells cultured with SCF from 9.5 d old mouse embryos. With an in situ apoptosis detection kit, a significant increase in apoptosis was detected after the removal of SCF, which further increased with the addition of ACK2 during SCF-dependent periods. The occurrence of apoptosis in the cultured cells was also demonstrated by a DNA analysis and electron microscopy. Immunohistochemical double staining confirmed that the apoptotic cells were c-KIT positive, and the electron microscopy showed that these apoptotic cells were melanocyte precursors. It was therefore demonstrated that apoptosis was induced in the SCF-dependent c-KIT-positive melanocytes in vitro when the SCF/c-KIT interaction was obstructed. These findings elucidate the mechanism of the regulation of melanocyte development, and the survival and proliferation of these precursor cells, by SCF/c-KIT interaction.

Suppressive effect of intravenous immunoglobulins on the activity of interleukin-1.

In order to study the effect of human immunoglobulin preparations for intravenous use (IVIg) on the production and activity of interleukin-1 (IL-1) derived from monocytes, we treated cultured monocytes with IVIg and examined the lymphocyte-activating factor (LAF) activity of IL-1 in the culture supernatants. The results showed that IVIg suppressed the activity from most healthy adults and some febrile children with acute respiratory disease or Kawasaki disease. Further studies revealed that intact Ig (whole molecular Ig) did not suppress the mRNA expression of IL-1 alpha or IL-1 beta in mononuclear cells, that intact Ig and pepsin-digested Ig inhibited the LAF activity of recombinant IL-1 (rIL-1) and also that intact Ig contains immunoglobulin (probably anti-IL-1 antibody) which binds with rIL-1 by dot blotting using biotin-streptavidin. These results suggest that IVIg suppresses neither IL-1 synthesis nor the release of IL-1 from monocytes but does neutralize IL-1 alpha and IL-1 beta activity by binding IL-1 proteins as an anti-IL-1 antibody.

CDw49b/CD29 integrin complex mediates the differentiation of human endothelial cells into capillary-like structures in vitro.

We have investigated different beta-1 integrins (CDw49/CD29) on human umbilical vein endothelial cells (HUVEC) with regard to their roles in modifying the morphological structure of these cells on/in matrigel. The inhibition of matrigel-induced capillary formation by antibodies against subunits of beta-1 integrins was examined quantitatively using a digital analyzer. Antibodies to CDw49b and CD29 (common beta chain) caused a marked inhibition of capillary formation (up to 70%) in a dose-dependent manner, whereas antibodies to CDw49d, CDw49e and CDw49f were less inhibitory. We also examined the appearance of HUVEC cultured in matrigel. HUVEC suspended in matrigel for 24 h formed extended cell processes which connected, resulting in the formation of a capillary network. In contrast, fibroblasts cultured in matrigel showed only bipolar extensions without cell-cell contact. After 48 h in culture in matrigel, some HUVEC showed the capillary-unit of a lumen encircled by EC which may mimic the basic putative unit in the formation of capillaries. However, HUVEC pretreated with antibodies to CDw49b and CD29 failed to form significant processes and a hollow lumen. These phenomena may illustrate the importance of endothelial cell-basement membrane matrix interaction (through integrins, especially CDw49b/CD29 complex) occurring during differentiation of endothelial cells in angiogenesis.

Enhanced expression of the high-affinity receptor for IgE (Fc(epsilon)RI) associated with decreased numbers of Langerhans cells in the lesional epidermis of atopic dermatitis.

Epidermal Langerhans cells (LCs) and the high-affinity receptor for IgE (Fc(epsilon)RI) on their surface are considered important in the pathogenesis of atopic dermatitis (AD). We investigated the numbers of epidermal LCs and their Fc(epsilon)RI expression in patients with AD and healthy controls. Biopsy specimens taken from lesional skin from 17 patients with AD, non-lesional skin from five patients with AD and normal skin from five healthy individuals were immunohistochemically stained with a monoclonal antibody against CD1a or with either of two monoclonal antibodies against two different epitopes of Fc(epsilon)RI alpha chain. Many dendritic cells were positively stained with anti-CD1a antibody in the epidermis of each skin sample, and fewer cells were stained with anti-Fc(epsilon)RI antibodies. The numbers of epidermal LCs positive for Fc(epsilon)RI were significantly increased in both lesional and non-lesional skin from AD patients compared with those in normal skin, suggesting important roles of Fc(epsilon)RI+LCs in the pathogenesis of the disease. In contrast, the numbers of total epidermal LCs (CD1a-positive) were decreased in AD lesional skin compared with those in non-lesional skin from AD patients and in normal skin from healthy subjects. Together with our finding that the numbers of epidermal LCs were negatively correlated with the clinical severity of the AD lesions, we concluded that epidermal LCs may decrease in some conditions of AD, probably in lesions with severe inflammation.

Pathogenesis of mucocutaneous lesions in Behçet's disease.

Behçet's disease (BD) is characterized by recurrent oral aphthae, skin lesions, eye lesions, and genital ulceration. To determine the pathogenesis of BD, we performed histological and immunohistochemical studies of these mucocutaneous lesions, an assay of neutrophil activity, and HLA typing. Dense dermal or subcutaneous infiltrations of polymorphonuclear cells (PMN) without leukocytoclastic vasculitis were found in 28 of 57 lesions. Immunohistochemically, deposits of C3 on the vessels were found in 12 of 31 lesions. Deposits of immunoglobulin were not found except for one of IgM. C3 deposits and PMN infiltrations were significantly related (p < 0.05). PMN activity by polarization was enhanced; however, the results did not show a significant relationship with the PMN infiltrations or the C3 deposits. The incidence of HLA-B51 was significantly high in BD, but no significant relationship was found between HLA-B51 and the results of other examinations. These results suggest that the pathogenesis of BD lesions differs from that of collagen diseases and that C3 deposits on the vessels may play an important role in the development of mucocutaneous lesions where PMN have mainly infiltrated.

Monoclonal antibody OKB19, reactive with a B-lymphoid differentiation antigen (CD19), binding to basal layer keratinocytes of normal human skin.

Monoclonal antibody (moAB) OKB19 reacts with CD19 antigen, which is the broadest lineage-specific surface marker on B-lymphocytes. In frozen tissue sections, using an immunohistochemical technique, the OKB19-positive cells in the basal layer were sharply demarcated from the negative suprabasal layers. In normal hair follicles, the OKB19 reactivity was also confined to one layer of the dermal side of the outer root sheath. However, this reactivity gradually disappeared in the lower areas. The inner surface of the lumina in the eccrine duct was weakly stained with OKB19. The basal keratinocytes were also stained with OKB19 in the lesional epidermis of the various dermatoses examined in this study, when the basal keratinocytes remained unaffected. Even in the hyperproliferative state of psoriasis, the OKB19 reactivity was confined to the basal layer. Several kinds of tumor cells derived from the skin were not stained with OKB19. No labeling was seen even in the basaloid cells of basal cell carcinoma, which are morphologically similar to basal keratinocytes. B4 and Leu-12, other monoclonal antibodies reacting with CD19, did not recognize any keratinocytes in the normal human skin. MoAB OKB19, therefore, reacts with an antigen present on basal keratinocytes and provides a probe for the isolation of the basal keratinocyte subpopulation. Thus, this antibody should be useful in studies of not only B-lymphocyte differentiation, but also normal and aberrant differentiation of the epidermal keratinocytes.

Hair growth on nude mice due to cyclosporin A.

One of the most common dermatological side effects of cyclosporin A (CsA) is dose-dependent hypertrichosis. Similar hair growth was noted in nude mice in an attempt to increase the acceptance of human xenografts with CsA in the T-cell-deficient congenitally athymic nude (nu/nu) mice. The aim of the present study was to further investigate the stimulation of hair growth on nude mice not only by oral administration of CsA but also by topical and subcutaneous administration of CsA. Young BALB/c female nude mice were treated for 3 or 4 weeks with topical, oral, or subcutaneous applications of CsA dissolved in olive oil at various doses. The hair of CsA-treated mice appeared to grow from 7 days after the treatment, even at low doses. Induced hair growth was dose-dependent and became clearly obvious 3 weeks after the treatment. The stimulation of hair growth was not restricted to the site of topical application. The distribution of the new hair depended on the natural pattern of hair growth in the mice. However, there was no hair growth in the control mice which were given only olive oil. Histological examination revealed that there were no differences in the structures of skin and hair between the control and the CsA-treated mice. Furthermore, the number of hair follicles did not remarkably increase after CsA treatment. The hair growth in the CsA-treated mice stopped after cessation of the treatment and returned to the level of the control mice on day 14 after the end of the treatment. Subsequent retreatment with CsA resulted in further regrowth of the hair.(ABSTRACT TRUNCATED AT 250 WORDS)

Hair-discoloration of Japanese elite swimmers.

To understand hair-discoloration in relation to swimming, we examined sixty-seven elite swimmers of the Japan National Swimming Team and fifty-four, age-matched subjects as controls. The incidence of hair discoloration (61%) in the swimmers' group was significantly higher than that in controls (0%) (p<0.0001). Interestingly, surface damage of the nail plates coexisted in the swimmers with the scalp-hair discoloration. The hairs picked from the eight swimmers and two age-matched individuals as controls were examined by electron microscope (EM) and EM X-ray microanalyzer. The swimmers' discolored, golden hair revealed complete disappearance of hair cuticle both by scanning EM (SEM) and transmission EM (TEM). The quantity of melanosomes in the cortex decreased, and their diameter was smaller than that of controls. In addition, irregularly shaped melanosomes with variable electron density and less electron-dense melanosomes with white haloes were frequently observed in the swimmers' golden hair. The X-ray elemental spectrograph by SEM revealed that the content of sulfur in all the swimmers' discoloured hair was lower than that in the normal controls and that the content of chlorine in the male swimmers' discoloured hair was higher than that in the female swimmers and the normal controls. The X-ray elemental microanalysis by TEM focused on melanosomes in the cortex of the cross section and detected elemental chlorine in all swimmers' golden hairs. It did not detect any element in the control hairs. The 14C-tyrosine uptake test of hairbulbs found no significant difference between the swimmers and the normal controls. These findings suggest that hair discoloration was mainly due to cuticle damage by friction with water. Hypochlorous acid in the swimming pool water can penetrate to the hair cortex through the cuticle. It can oxidize and degenerate melanosomes there.

Success in the radical resection of esophageal cancer with artificial blood infusion: case report.

Using the oxygen-carrying, so-called "artificial blood", Fluosol-DA, developed by us, we successfully performed a radical resection for esophageal cancer and a one-stage reconstruction, without any blood transfusion. The operation was performed quite safely despite a large blood loss, a stable condition was performed quite safely despite a large blood loss, a stable condition being maintained during and after the operation. Fluosol-DA has no relation to blood type and is stable in a frozen state for one year or more and can, therefore, be used conveniently and safely for treating severe and intraoperative hemorrhages. Furthermore, this preparation may well have at wide application because it greatly reduces the risk of postoperative hepatitis.

[Splenic vein occlusion due to pancreatic disease: regional portal hypertension from hemodynamic points of view].

The purpose of this study is to clarify the clinicopathophysiology of splenic vein occlusion due to pancreatic disease from hemodynamic points of view. We reviewed the angiographic findings and medical records of 82 patients who had pancreatitis, pancreatic cyst or pancreatic cancer in the pancreatic body and tail. According to the site of occlusion in 16 patients with complete splenic vein occlusion, this entity may be divided into two categories: Type A, an occlusion close to the spleen in which short-gastric system seems to be major collateral, and Type B, an occlusion distant from splenic hilum in which gastroepiploic system becomes prominent as collateral. As compared to 7 patients with incomplete splenic vein occlusion, gastric varices and splenomegaly were frequently observed with the patients having complete occlusion. Among these 16 patients, splenic arterial occlusion was superimposed in 3 patients with pancreatic cancer in whom gastric varices were not detected. Thus, clinical features of this entity must be carefully assessed according to the nature of the underlying disease. Based on these observations, three consecutive phases: Phase 1 Insiduous or latent phase, Phase 2 Collateral developing phase, Phase 3 Vanishing phase may be distinguished for splenic vein occlusion secondary to pancreatic disease.

Alkaline bismuth solution as an en bloc stain for formaldehyde-glutaraldehyde potassium permanganate fixed fungal spores.

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.

Methotrexate inhibits proliferation and regulation of the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by cultured human umbilical vein endothelial cells.

BACKGROUND: The mechanism by which a low dose of methotrexate (MTX) works to treat psoriasis is not clear. The overexpression of cell adhesion molecules on dermal vessels is important in the pathogenesis of psoriasis and is probably induced by upregulation of tumour necrosis factor (TNF)-alpha. OBJECTIVES: To determine the effects of MTX at concentrations comparable with in vivo levels after the administration of low-dose MTX to human umbilical vein endothelial cells (HUVEC) on the growth and expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). METHODS: Cell proliferation assay, immunostaining, immunoblotting, cell enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed to examine the effects of MTX on HUVEC. RESULTS: MTX inhibited the proliferation of HUVEC at 10-7 mol L-1 and 10-6 mol L-1 without showing cytotoxic effects. It also inhibited TNF-alpha-induced ICAM-1 and VCAM-1 expression by HUVEC at 10-6 mol L-1. The inhibitory effect of MTX was more pronounced on ICAM-1 expression than on VCAM-1 expression. RT-PCR analysis revealed that TNF-alpha-induced ICAM-1 gene expression was strongly downregulated by MTX. CONCLUSIONS: Low-dose MTX may act on psoriasis by suppressing the TNF-alpha-induced expression of ICAM-1 and VCAM-1 by vascular endothelial cells. Inhibition of neovascularization may be another mechanism of action of MTX.

Cutaneous extramedullary hematopoiesis in myelofibrosis.

Erythematous papulonodules that resembled a malignant lymphoma developed in a 62-year-old man. Further examination revealed that he had primary myelofibrosis with cutaneous extramedullary hematopoiesis. All three marrow elements (myeloid, erythroid, and megakaryocytic series) were present in the skin lesions. Although extramedullary hematopoiesis of the skin is a rare complication of myelofibrosis, 13 similar cases have been reported. In nine cases, including ours, all three marrow elements were present in the cutaneous lesions.

Production of interleukin-1-alpha and -beta by human peripheral polymorphonuclear neutrophils.

The mechanism of the production of interleukin-1 (IL-1) by human peripheral polymorphonuclear neutrophils (PMN) was investigated. Supernatants of PMN stimulated with 30 micrograms/ml lipopolysaccharide (LPS) were used as extracellular IL-1 and supernatants of their lysate as intracellular IL-1 source. IL-1 activity was measured by the C3H/HeJ thymocyte co-mitogenic assay. The supernatants from PMN stimulated with LPS for 72 h showed IL-1 activity which had an apparent molecular weight of 15-20 kilodaltons and pI of 5.0 and more than 8.5. It was neutralized with anti-IL-1 antibodies and it lacked IL-2 activity. Our time course study of the IL-1 assay with neutralization by anti-IL-1-alpha and -beta antibodies indicated that the extracellular IL-1-beta activity appeared predominantly in the early incubation periods, whereas alpha activity appeared predominantly in the late periods. Intracellular IL-1-alpha but not beta activity was detected mainly at the intermediate incubation periods. These data indicate that PMN stimulated with LPS produce both IL-1-alpha and -beta, and release IL-1-beta first and IL-1-alpha later.

[A metachronous and synchronous multiple colonic cancer appearing 15 years and 3 months after resection of a synchronous multiple colonic cancer].

A case of multiple colonic cancer appearing 15 years and 3 months after the resection of a synchronous multiple colonic cancer is reported. A 65-year-old man complained of abdominal pain, abdominal fullness, and diarrhea. A barium enema revealed a so-called "apple core lesion" and a filling defect, and he was diagnosed as having a multiple colonic cancer. A subtotal colectomy was performed and he has been free of any sign of recurrence since this operation. In this case, the interval between the first and the second cancer is longer than most reported cases. Thus, as a precaution, a longer follow-up is recommended for patients with a synchronous multiple colonic cancer.

Clinical, pathological, and etiologic aspects of acquired dermal melanocytosis.

To study the pathogenesis of acquired dermal melanocytosis (ADM), we reviewed the clinical, immunohistochemical, and ultrastructural features of 34 cases (female, 33, and male, 1) of ADM. The patients' ages at onset ranged from 8 to 51 years and averaged 26.8 +/- 12.7 years. There was a positive family history. Gray-brown macules were mostly recognized on the face. Not only active dermal melanocytes but also non-pigmented c-KIT- and TRP-2-positive immature melanocytes were detected in the dermis. Taken together those clinical and histological findings, activation of pre-existing immature melanocytes by sunlight, estrogen, and/or progesterone, and some other factors, may be the most likely mode of the development of ADM. Moreover, using cultured murine neural crest cells as a model of c-KIT-positive immature melanocytes, we confirmed that endothelin-1, which is produced and secreted by keratinocytes after UV-irradiation, affects melanocytes and accelerated melanogenesis.

Serum levels of soluble stem cell factor and soluble KIT are elevated in patients with atopic dermatitis and correlate with the disease severity.

BACKGROUND: Mast cell infiltration in skin lesions of atopic dermatitis (AD) is considered to play an important role in the pathogenesis of the disease. The most common factor that stimulates mast cell growth, migration and differentiation is stem cell factor (SCF), and the interaction of SCF and its receptor, KIT (tyrosine kinase transmembrane receptor), appears to be the key event in the recruitment and proliferation of mast cells. OBJECTIVES: To determine whether any altered metabolism of SCF and/or KIT is present in patients with AD. METHODS: We measured serum levels of soluble SCF (sSCF) and soluble KIT (sKIT) using enzyme-linked immunosorbent assay in 54 patients with AD, five patients with erythrodermic psoriasis vulgaris and 64 healthy individuals. RESULTS: Serum levels of both peptides in AD patients were significantly higher than those in healthy individuals, whereas patients with psoriasis vulgaris did not show any difference from healthy controls. Both sSCF and sKIT levels were positively correlated with the disease severity in AD patients, and decreased after effective treatment with topical corticosteroids. Conclusion Serum levels of sSCF and sKIT may be useful indicators for evaluation of the activity and severity of AD.

Establishment and characterization of a mouse neural crest derived cell line (NCCmelan5).

Stem cell factor (SCF) and endothelin 3 (EDN3) are both necessary for melanocyte development. We have established an immortal cell population of neural crest cells from C57BL/6 mice, cultivating them with SCF, EDN3 and 15% fetal calf serum without feeder cells, and have designated that line as C57NCC SE. C57NCC SE consists of a population of melanocytes in various stages of differentiation. We used a single-cell cloning method, in which only one cell is transferred to each new culture plate, and succeeded in establishing an immortal cell line named NCCmelan5. All NCCmelan5 cells were positive for KIT (SCF receptor), HMB45 (human melanosomal antigen), tyrosinase-related protein-1 (TYRP1), tyrosinase-related protein-2 (TYRP2), tyrosinase and endothelin receptor B (EDNRB) and all could oxidize 3,4-dihydroxyphenylalanine (DOPA) to form melanin. Measurement of their DNA content revealed that 88.6% of the cells were in the G0-G1 phase, suggesting that they retained normal DNA ploidy. Thus, NCCmelan5 cells have the characteristics of mature melanocytes except that they are immortal; these cells may prove useful to study factors that directly affect melanogenesis and melanocyte development without the influence of feeder cells. It is clear that our attempt to establish immortal cell lines from murine neural crest cells would have never been successful without the addition of SCF and EDN3, since C57NCC SE and NCCmelan5 cells require those factors to proliferate.

Keratin subunit expression in human cultured melanocytes and mouse neural crest cells without formation of filamentous structures.

The synthesis of keratin is considered to occur in epithelial and epidermal cells. Previous studies have not reported on keratin synthesis within melanocytes that derive from neural crest cells. Epithelial and neural crest cells originally develop from ectodermal tissue. We previously reported that the expression of keratin is a universal phenomenon seen in cultured melanoma cell lines, as demonstrated by two-dimensional polyacrylamide gel electrophoresis, western blot, and electron microscopy analyses. To further investigate the specificity of keratin function in melanocytic cells, we first examined the presence of keratin proteins in cultured human melanocytes, and unexpectedly found keratin subunits in melanocytes by the above-mentioned procedures. The keratin (K) subunits were composed of K1, K5, K8, K10, K14, K16, and K18, together with vimentin. Neural crest cells, which contain immature embryonic melanocytes developing from ectoderm, already expressed keratins; however, under electron microscopy, the expressed keratin did not form filamentous structures. Although the ATP synthase alpha-chain, which is expressed universally in cultured epidermal tumor cell lines, was also expressed in cultured melanocytes and neural crest cells, a novel malignant melanoma-related protein (MMRP) was absent in melanocytes and neural crest cells. We concluded that keratin subunits are present in both cells, but do not construct keratin filaments.