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Dermal papilla cells (DPCs) are one type of mesenchymal cells; they play a key role on hair follicle induction. Their hair inductivity and proliferation abilities are rapidly lost during the 2-dimensional culture. Cell senescence is induced by inadequate culture conditions and telomere shortening. We previously reported that overexpression of TERT coding telomerase reverse transcriptase and BMI1 coding human B-cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) avoided senescence of murine DPC and restored hair inductive activity.To evaluate the function of TERT and BMI1 in the human DPCs (hDPCs).Cultured hDPCs obtained from human scalp hair were transduced with TERT alone (hDP-T), BMI1 alone (hDP-B), both TERT and BMI1 (hDP-TB) and empty vector (hDP-E). The hair inductive activity of those cells was assessed by chamber assay in vivo. Gene expressions were analyzed by quantitative PCR (q-PCR).hDP-TB proliferated more than hDP-T and hDP-B in vitro and only hDP-TB showed hair inductivity in vivo. Moreover, the expressions of VCAN, CTNNB1, LEF1, FGF7 and VEGFA in hDP-TB were elevated compared to those in hDP-E.Overexpression of both TERT and BMI1 extends the life span of cultured hDPCs and ameliorates their hair inducing ability on mouse hair follicles.

Lymphatic transport plays an important role in coordinating local immune responses. However, the biologic effects of impaired lymphatic flow in vivo are not fully understood. In this study, we investigated the roles of the lymphatic system in skin carcinogenesis and psoriasis-like inflammation using k-cyclin transgenic (kCYC+/−) mice, which demonstrate severe lymphatic dysfunction. kCYC+/− mice showed augmented tumor growth in the two-stage skin carcinogenesis model as well as severe clinical scores in imiquimod-induced psoriasis-like skin inflammation compared to wild-type mice. Although mRNA levels of inflammatory cytokines in skin after topical application of 12-O-tetradecanoylphorbol-13-acetate or imiquimod were comparable between kCYC+/– and wild-type mice, protein levels of inflammatory cytokines such as interleukin (IL)-17A, IL-22, and IL-23 were significantly upregulated in kCYC+/– mice in both models. Consistently, signal transducer and activator of transcription 3 pathway and nuclear factor-κB signaling were augmented in epidermal keratinocytes in kCYC+/– mice. These results suggest that lymphatic dysfunction in kCYC+/– mice caused accumulation of inflammatory cytokines, leading to the exacerbation of two-stage skin carcinogenesis and imiquimod-induced psoriasis-like skin inflammation. These findings add insight into the clinical problems of secondary malignancies and inflammatory dermatoses that may occur with extremity lymphedema.

Peribiliary glands (PBGs) are accessory glands with mucinous and serous acini in the biliary tree. The PBG is composed of a heterogeneous cell population, such as mucus- and pancreatic enzyme-producing epithelial cells, whereas it constitutes niches for multipotential stem/progenitor cells in the human extrahepatic bile duct (EHBD). By contrast, the nature of PBGs in the mouse EHBD remains unclear. Our aim was to establish a method for isolating and characterizing PBG-constituting cells in the mouse EHBD. We found that trophoblast cell surface protein 2 (Trop2) was expressed in the luminal epithelium of mouse EHBD exclusively, but not in the PBG. On the basis of the differential expression profile of Trop2, lumen-forming biliary epithelial cells (LBECs) and PBG-constituting epithelial cells (PBECs) were separately isolated for further characterization. Gene expression analysis revealed that the isolated mouse PBECs expressed several marker genes related to human PBGs. In the colony formation assay, PBECs showed significantly higher colony formation capacity than LBECs. In the organoid formation assay, PBECs formed cystic organoid with LBEC-like phenotype. Interestingly, PBECs proliferated, accompanied by reexpression of Trop2 in vivo after bile duct ligation. Furthermore, the unique expression profile of Trop2 was conserved in human EHBD. Our findings indicate that Trop2 is a useful marker in investigating the pathophysiological roles and characteristics of mouse and human PBGs in biliary diseases.

Background Classically activated macrophages produce IL-12, IL-23, and TNF-α, whereas alternatively activated macrophages (M2 cells) produce IL-10 and express several receptors such as mannose receptor and CD163. Tumor-associated macrophages exhibit M2 phenotype, whose presence has been associated with poor prognosis in various tumors. Objectives To investigate distribution of CD163 + cells in lesional skin and serum levels of soluble CD163 (sCD163) in patients with cutaneous T cell lymphoma (CTCL), atopic dermatitis (AD), or psoriasis. Methods The numbers of CD163 + and CD68 + cells in lesional skin of CTCL, AD, or psoriasis, and in normal skin were examined by immunohistochemistry. Serum soluble CD163 (sCD163) levels were quantified by enzyme-linked immunosorbent assay. Results The numbers of CD163 + cells in lesional skin of CTCL, AD, or psoriasis were significantly larger than in normal skin. In CTCL, the numbers of CD163 + or CD68 + cells increased as more tumor cells infiltrated and they decreased after treatment with topical steroid and ultraviolet light. Moreover, CTCL patients with an increased number of CD163 + cells showed worse prognosis. Serum sCD163 levels in patients with CTCL, AD, or psoriasis were significantly higher than those in normal controls. In CTCL patients, serum sCD163 levels significantly correlated with serum soluble interleukin-2 receptor and CCL17 levels. In AD patients, serum sCD163 levels correlated with serum IgE levels. Conclusion The numbers of CD163 + cells in lesional skin and serum sCD163 levels were associated with disease progression of CTCL. Further study focusing on CD163 + cells in CTCL lesional skin would be an interesting research field.

CCL21 expression by lymphatic endothelial cells (LECs) is essential for migration of CCR7+ immune cells from skin to regional lymph nodes (LNs). We investigated the importance of mitogen-activated protein kinase (MAPK) signaling in CCL21 expression by ECs in vitro and in vivo. Normal human dermal lymphatic microvascular ECs (HMVEC-dLy) stimulated in vitro with oncostatin M (OSM) expressed high amounts of CCL21 mRNA. CCL21 protein expression by HMVEC-dLy was also markedly increased by OSM compared with unstimulated cultures. Marked phosphorylation of MAPK 44/42 was detected in HMVEC-dLy stimulated by OSM. CCL21 expression by HMVEC-dLy was blocked by a JAK inhibitor 1, JAK3 inhibitor, and U0126 (a MAPK kinase inhibitor) in vitro, all of which blocked phosphorylation of MAPK 44/42. In addition, injection of U0126 into murine skin significantly decreased CCL21 mRNA and protein expression. Moreover, injection of U0126 before sensitization decreased migration of dendritic cells to draining LNs and decreased contact hypersensitivity responses. In summary, these results suggest that the MAPK pathway is important for CCL21 expression by LECs in vitro and in vivo. Blocking MAPK signaling within skin may offer a novel approach to treatment of inflammatory skin diseases.

Induction of a desired cell type by defined transcription factors (TFs) using iPS technology can be used for cell replacement therapy. However, to overcome problems such as tumor formation, genomic insertional mutagenesis by viral transduction in the induction process needs to be avoided using alternative approaches. One approach could be the direct delivery of TF protein by a protein transduction system, whereby a protein transduction domain (PTD) is fused to facilitate the penetration of cell membrane. However, fusion proteins, including TFs, are reported to be biologically less active through the interference of PTD with proper protein folding. Here, we report a proof-of-concept study in which TF proteins fused with PTDs could be reactivated by removal of PTDs from cells. We demonstrated that Sox2 and Oct3/4 proteins fused with PTD were less active in mouse embryonic stem cells. Removal of PTD by a site-specific protease, derived from tobacco etch virus (TEV), substantially restored the functionality of these proteins, proved by enhanced rescue ability for differentiation induced by endogenous Sox2 and Oct3/4 repression. These results suggest that, by removing a PTD inside the cells, directly delivered TF proteins may exert substantially enhanced function than presently considered.

Summary Background Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by autoantibodies against the hemidesmosomal proteins, BP180 and BP230. NC16A, a non-collagenous stretch of the BP180 ectodomain is the primary target of pathogenic IgG antibodies. Whereas IgG anti-BP180 autoantibodies play a primary role in the pathogenesis, there is a growing number of data regarding the potential pathogenic roles of IgE class autoantibodies in BP. Objectives To examine the levels of IgG and IgE autoantibodies against BP180 and BP230, and to investigate mutual association and clinical relevance. Methods Sera obtained from 67 BP patients and 36 healthy donors were subjected to ELISA assays to measure serum IgG and IgE levels of anti-BP180 and anti-BP230 antibodies. Results IgG anti-BP180 antibodies were positive in 63 (94%) of 67 BP patients. IgG anti-BP230, IgE anti-BP180, and IgE anti-BP230 antibodies were found in 48 (72%), 20 (30%) and 45 (67%), respectively. IgG anti-BP180 levels were correlated with the affected areas. IgG anti-BP230 antibodies tended to increase in proportion to elongation of disease duration. IgE anti-BP230 levels showed a strong association with local eosinophil accumulation, while the levels were reversely related with the affected areas in BP. Conclusions IgE autoantibodies to BP180 and BP230 are detected at high frequencies in BP. IgE anti-BP230 antibodies may have a role in attracting eosinophils to the skin lesions.

金沢大学大学院医学系研究科血管分子科学, Background: B cells have been demonstrated to have critical roles in developing autoimmune bullous diseases. Recently identified tumor necrosis factor-like molecules, B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) are essential molecules for B cell development, survival, and proliferation. Although the functions of APRIL have not been fully evaluated, recent studies suggest that circulating levels of APRIL are increased in various autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. Objectives: To determine serum APRIL levels in patients with pemphigus vulgaris (PV) and bullous pemphigoid (BP), and compare those with clinical findings and laboratory findings. Patients/Methods: Sera from 15 PV patients, 43 BP patients, and 15 normal controls were subjected to ELISA assays to measure serum APRIL, BAFF, Dsg3, and BP180 levels. Results and conclusions: Circulating APRIL levels were significantly elevated in BP patients but not in PV patients, and correlated with serum BAFF levels. Our study revealed that serum APRIL levels tended to be increased in the quite early stage of disease. In conclusion, circulating APRIL levels may be a useful marker for early activation of autoimmune diathesis, and furthermore, an effective therapeutic target molecule in patients with BP. © 2007 Japanese Society for Investigative Dermatology.

The in vitro formation of organs and/or tissues is a major goal for regenerative medicine that would also provide a powerful tool for analyzing both the mechanisms of development and disease processes for each target organ. Here, we present a method whereby pancreatic tissues can be formed in vitro from mouse embryonic stem (ES) cells. Embryoid body-like spheres (EBSs) induced from ES cell colonies were treated with retinoic acid (RA) and activin, which are candidate regulators of pancreatic development in vivo. These induced tissues had decreased expression of the sonic hedgehog (shh) gene and expressed several pancreatic marker genes. ES cell-derived pancreatic tissue was composed of exocrine cells, endocrine cells, and pancreatic duct-like structures. In addition, the ratio of exocrine to endocrine cells in the induced tissue was found to be sensitive to the concentrations of RA and activin in the present experiment.

Systemic sclerosis (SSc) is characterized by fibrosis and autoimmmunity. Peripheral blood B cells from SSc patients specifically overexpress CD19, a critical cell-surface signal transduction molecule in B cells. CD19 deficiency in B cells also attenuates skin fibrosis in the tight-skin (TSK/+) mouse, a genetic model for SSc. Herein we analyzed two transgenic mouse lines that overexpress CD19. Remarkably, 20% increase of CD19 expression in mice spontaneously induced SSc-specific anti-DNA topoisomerase I (topo I) antibody (Ab) production, which was further augmented by 200% overexpression. In TSK/+ mice overexpressing CD19, skin thickness did not increase, although anti-topo I Ab levels were significantly augmented, indicating that abnormal CD19 signaling influences autoimmunity in TSK/+ mice and also that anti-topo I Ab does not have a pathogenic role. The molecular mechanisms for abnormal CD19 signaling were further assessed. B-cell antigen receptor crosslinking induced exaggerated calcium responses and augmented activation of extracellular signal-regulated kinase in TSK/+ B cells. CD22 function was specifically impaired in TSK/+ B cells. Consistently, CD19, a major target of CD22-negative regulation, was hyperphosphorylated in TSK/+ B cells. These findings indicate that reduced inhibitory signal provided by CD22 results in abnormal activation of signaling pathways including CD19 in TSK/+ mice and also suggest that this disrupted B cell signaling contribute to specific autoantibody production.

Recently, adult stem cells have been isolated from the skin and designated as skin-derived precursors (SKPs). These SKPs, cultured in vitro, can give rise to neurons, glia, smooth muscle cells, and adipocytes. In the current study, we confirmed the clonal expansion of SKPs using a sphere-forming culture system in a medium containing methylcellulose. Among the growth factors, only transforming growth factor-beta (TGF-beta) was revealed to uniquely facilitate the sphere formation and proliferation of the SKPs in combination with EGF and bFGF. In addition, TGF-beta did not alter phenotypical characteristics of the SKPs under sphere-forming conditions. The effect of TGF-beta on sphere formation was not observed in neural stem cells, which expressed a different set of cell surface markers from SKPs, suggesting that SKPs have distinct features. Although the number of SKPs decreased with age, TGF-beta increased the sphere colony formation and proliferation in all ages. These results suggest that SKPs maintained in the presence of TGF-beta during culture are of potential use in cell-replacement therapies employing adult tissue sources.

Despite a number of studies, the etiology of keloids remains unknown. We have investigated the response of fibroblasts derived from keloid tissue and normal adult skin to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Keloid fibroblasts were more responsive in both chemotactic and mitogenic assays to all three isoforms of PDGF than fibroblasts from normal skin. No enhanced response of the cells to either EGF or FGF was detected. The enhanced PDGF response of keloid fibroblasts appears to be mediated by elevated levels of PDGF alpha receptors, which are 4-5 times higher than those in normal human skin fibroblasts.