Biomedical subjects

Yuji Shirakata

Publications and source records attributed to Yuji Shirakata.

28 records · Page 2Linked to original sources

PubMed2003

Identification of the human IAI.3B promoter element and its use in the construction of a replication-selective adenovirus for ovarian cancer therapy.

Little is known concerning promoters or gene therapy specific for ovarian cancer. To explore the potential use of IAI.3B isolated from ovarian cancer cells in gene therapy for ovarian cancer, we identified the promoter region of the IAI.3B gene and created a replication-selective adenovirus, AdE3-IAI.3B, driven by the promoter. Transient transfection experiments showed that the DNA segment located between -1816 and -1 bp resulted in preferential expression in ovarian cancer cells with negligible expression in squamous cell carcinoma and normal cells. The promoter activity of IAI.3B was almost the same as that of cytomegalovirus and an order of magnitude higher than those of midkine and cyclooxygenase-2 in ovarian cancer cells. AdE3-IAI.3B replicated as efficiently as the wild-type adenovirus and caused extensive cell killing in a panel of ovarian cancer cells in vitro. In contrast, squamous cell carcinoma and normal cells were not able to support AdE3-IAI.3B replication. In animal studies, AdE3-IAI.3B administered to flank and i.p. xenografts of ovarian cancer cells led to a significant therapeutic effect. These results demonstrate the usefulness of the IAI.3B promoter for generation of ovarian cancer-specific adenoviral vectors and provide a potential for the development of ovarian cancer-specific oncolytic viral therapies.

Adenoviridae↗

PubMed2003

Essential roles of perforin in antigen-specific cytotoxicity mediated by human CD4+ T lymphocytes: analysis using the combination of hereditary perforin-deficient effector cells and Fas-deficient target cells.

Although the cytotoxic mechanisms of murine CTLs have been investigated extensively using various mutant and knockout mice, those of human CTLs, especially CD4+ CTLs, are still obscure. To clarify the roles of perforin in Ag-specific cytotoxicity mediated by human CD4+ CTLs, alloantigen-specific and HSV-specific human CD4+ T lymphocyte bulk lines and clones were established from a patient with hereditary perforin deficiency and her healthy father, and their cytotoxic activities were investigated. Alloantigen-specific CD4+ T lymphocytes expressing perforin exerted cytotoxicity against Fas-negative as well as Fas-positive allogeneic B lymphoblastoid cell lines established from members of a family with hereditary Fas deficiency. Perforin-deficient, but not perforin-expressing, CD4+ T lymphocytes failed to show strong cytotoxicity against HSV-infected autologous B lymphoblastoid cells. Perforin-deficient CD4+ T lymphocytes could exert relatively low level cytotoxicity against allogeneic IFN-gamma-treated keratinocytes. Although cytotoxicity mediated by perforin-expressing CD4+ CTLs was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, cytotoxicity against IFN-gamma-treated keratinocytes mediated by perforin-deficient CD4+ T lymphocytes was inhibited only partially by concanamycin A, but was inhibited significantly by antagonistic anti-Fas Ab and anti-Fas ligand Ab. The combination of perforin-deficient effector T lymphocytes and Fas-negative target cells used in the present study provides a novel experimental system for studying the detailed mechanisms of human CTL-mediated cytotoxicity. The present data demonstrate that perforin-negative CD4+ CTLs can exert cytotoxicity against Fas-sensitive target cells; however, perforin plays essential roles in Ag-specific cytotoxicity mediated by human CD4+ as well as CD8+ CTLs.

B-Lymphocytes↗

PubMed2003

Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro.

BACKGROUND: thymus and activation-regulated chemokine (TARC)/CCL17 is a CC chemokine that selectively attracts Th2-type lymphocytes. Immunohistochemical analyses have revealed that TARC is expressed in the epidermal keratinocytes of atopic dermatitis (AD), suggesting TARC involvement in the pathogenesis of the disease. However, keratinocyte TARC production has been described only in the transformed keratinocyte cell line HaCaT. OBJECTIVE: to examine TARC production in normal human epidermal keratinocytes (NHEK) in vitro. METHODS: the expression of TARC mRNA and protein were examined in NHEK and HaCaT cells stimulated with various cytokines. RESULTS: stimulation with inflammatory cytokines, including interleukin (IL)-1, IL-4, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and tumor necrosis factor (TNF)-alpha failed to induce TARC mRNA expression in NHEK. However, stimulation with IFN-gamma and TNF-alpha together enhanced expression slightly. ELISA analysis failed to detect TARC protein in NHEK culture supernatant, even following stimulation with IFN-gamma and TNF-alpha. In contrast, HaCaT cells produced TARC protein even without stimulation of cytokines. CONCLUSION: these results indicate that production of TARC by HaCaT cells is a phenomenon specific to the cell line and the observation on TARC in HaCaT cells can not be generalized. NHEK do not produce TARC protein in vitro.

Cells, Cultured↗

PubMed2003

So-called biological dressing effects of cultured epidermal sheets are mediated by the production of EGF family, TGF-beta and VEGF.

BACKGROUND: Cultured epidermal sheet (CES) grafts accelerate wound healing as a result of so-called biological dressing effect, which is thought to be mediated by various growth factors. However, the profile of growth factor expression in CESs is unclear. OBJECTIVE: To investigate whether CESs produce growth factors along with stratification we investigated the production of growth factors and their regulation in CESs. METHODS: CESs conditioned medium was harvested and the concentration of TGF-alpha, TGF-beta1, TGF-beta2, and VEGF was measured using ELISA. The mRNA of EGF family, TGF-beta family and VEGF was detected by Northern blot or RNase protection assay. RESULTS: The concentration of TGF-alpha was 100 pg/ml in the monolayer culture, but dramatically increased to 600 pg/ml 2 days after stratification. It decreased to baseline, and then gradually increased to 300 pg/ml in the presence of EGF and remained at that level until day 20. TGF-beta1 increased from 50 to 400 pg/ml after stratification, and remained at that level day 20. TGF-beta2 was undetectable in the monolayer culture, but dramatically increased to 200 pg/ml 2 days after stratification. Unlike TGF-beta1, TGF-beta2 gradually increased over time after stratification. VEGF increased with stratification from 500 to 1500 pg/ml. The addition of EGF upregulated EGF family, TGF-beta, and VEGF production in CESs, as confirmed by ELISA, Northern blot, and RNase protection assay. CONCLUSION: These results indicate that so-called biological dressing effect of CESs is mediated by production of the EGF family, TGF-beta, and VEGF. Our results also demonstrate the ability of EGF to enhance growth factor production in CESs.

Biological Dressings↗

PubMed2003

Suppressor of cytokine signaling 1/JAB and suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 negatively regulate the signal transducers and activators of transcription signaling pathway in normal human epidermal keratinocytes.

The suppressor of cytokine signaling/cytokine-inducible SH2 containing proteins are cytokine inducible and are negative regulators of the signal transducers and activators of the transcription signaling pathway. We investigated the mechanism regulating signal transducers and activators of transcription and the suppressor of cytokine signaling/cytokine-inducible SH2 containing protein family in keratinocytes, one of the major target cells for cytokines. Suppressor of cytokine signaling 1 mRNA was upregulated 3 h post-interferon gamma, and a 8.1-fold increase in the suppressor of cytokine signaling 1 mRNA occurred 48 h post-interferon gamma. The suppressor of cytokine signaling 3 mRNA was also upregulated from 1 h post-interferon gamma, and a 6.7-fold increase in the suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 mRNA occurred between 6 and 12 h post-interferon gamma. Interleukin-6 exposure for 1 h enhanced the expression of the suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 mRNA, but the suppressor of cytokine signaling 1/JAB mRNA was not induced by interleukin-6. Interleukin-4 upregulated the suppressor of cytokine signaling 1/JAB and cytokine-inducible SH2 containing protein 1 mRNA, with 3.4-fold and 5.1-fold increases in mRNA observed at 1 h post-interleukin-4, respectively. In contrast, epidermal growth factor, which phosphorylates signal transducers and activators of transcription 3, did not influence the level of the suppressor of cytokine signaling/cytokine-inducible SH2 containing protein family mRNA expression. Transfection of an adenovirus vector expressing the suppressor of cytokine signaling 1/JAB completely inhibited interferon gamma-dependent signal transducers and activators of transcription 1 phosphorylation and interleukin-4-dependent signal transducers and activators of transcription 6 phosphorylation. Transfection of adenovirus vector expressing the suppressor of cytokine signaling 1/JAB did not inhibit interleukin-6-dependent signal transducers and activators of transcription 3 phosphorylation-several reports show that the suppressor of cytokine signaling 1/JAB is a potent inhibitor of signal transducers and activators of transcription 3 signaling in the myeloid leukemia M1 cell. Transfection of the adenovirus vector expressing suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 completely inhibited interleukin-6-dependent signal transducers and activators of transcription 3 phosphorylation and partially inhibited interferon gamma-dependent signal transducers and activators of transcription 1 phosphorylation. Transfection of the adenovirus vector expressing suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3, however, did not inhibit interleukin-4-dependent signal transducers and activators of transcription 6 phosphorylation. Transfection of the adenovirus vector expressing cytokine-inducible SH2 containing protein 1 had no effect on signal transducers and activators of transcription 1, 3, and 6 signaling in normal keratinocytes. Therefore, the relationship between signal transducers and activators of transcription and suppressor of cytokine signaling is unique in the keratinocytes, and the suppressor of cytokine signaling regulates cytokine signals in these cells.

Antineoplastic Agents↗

PubMed2003

Keratinocyte growth inhibition by high-dose epidermal growth factor is mediated by transforming growth factor beta autoinduction: a negative feedback mechanism for keratinocyte growth.

The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor beta is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor beta contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor beta1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor beta1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor beta1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor beta-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor beta type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor beta acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor beta1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth.

Activin Receptors, Type I↗

PubMed2003

Severe palmo-plantar hyperkeratosis in Koebner epidermolysis bullosa simplex.

A 22-year-old Japanese woman with Koebner epidermolysis bullosa simplex presented with severe palmo-plantar hyperkeratosis. Skin biopsy specimens from the palm revealed hyperkeratosis and acanthosis with blistering in the basal cell layer. An electron microscopic study showed cytolysis of basal cells and clumping of tonofilaments forming thick bundles and perinuclear electron-dense bodies. Hand contractures were released and covered with full thickness autografts or split thickness autografts. The donor site was successfully treated with cultured epidermal autografts.

Adult↗

PubMed2002

Phosphatidylinositol 3-kinase is a key regulator of early phase differentiation in keratinocytes.

The survival and growth of epithelial cells depend on adhesion to the extracellular matrix. Because epidermal keratinocytes differentiate as they leave the basement membrane, an adhesion signal may regulate the initiation of differentiation. Phosphatidylinositol 3-kinase (PI3K) is a fundamental signaling molecule that regulates the adhesion signal. Transfection of a dominant negative form of PI3K into keratinocytes using an adenovirus vector resulted in significant morphological changes comparable to differentiation and the induction of differentiation markers, keratin (K) 1 and K10. In turn, transfection with the constitutively active form of PI3K almost completely abolished the induction of K1 and K10 by differentiation in suspension cultures using polyhydroxyethylmethacrylate-coated dishes. PI3K activity was lost in suspension culture, except by cells bearing the constitutively active form of PI3K. These data demonstrate that blockade of PI3K results in differentiation and that activation of PI3K prevents differentiation. Furthermore, expression of the dominant negative form of PI3K significantly inhibited keratinocyte adhesion to the extracellular matrix and reduced the surface expression of alpha(6) and beta(1) integrins in suspension culture. Moreover, expression of the active form of PI3K restored the mRNA levels of adhesion molecules that were reduced in suspension culture, including alpha(3), alpha(6), and beta(1) integrins, BP180, and BP230. In conclusion, loss of PI3K activity results in keratinocytes leaving the basement membrane and the initiation of a "default" differentiation mechanism.

Cell Differentiation↗

PubMed2002

BP180 ELISA using bacterial recombinant NC16a protein as a diagnostic and monitoring tool for bullous pemphigoid.

Bullous pemphigoid (BP) is an acquired autoimmune subepidermal blistering disease against hemidesmosomal cytoplasmic BP230 and transmembrane BP180 proteins. Epitope mapping studies have shown that the membrane-proximal noncollagenous (NC) 16a domain of BP180 harbors clusters of antigenic sites recognized by the vast majority of BP sera. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) using bacterial recombinant NC16a protein and evaluated its clinical benefit for diagnosis and monitoring disease activity. Fifty four (84.4%) of 64 sera from BP patients were positive, while only one (1.1%) of 91 sera from collagen disease patients and five (1.5%) of 336 sera from normal control barely exceeded the cut-off value. None of 69 pemphigus vulgaris sera and none of 42 pemphigus foliaceus sera exceeded the cut-off value. Thus, the sensitivity and specificity of NC16a ELISA were 84.4 and 98.9%, respectively. The correlation between ELISA scores and disease activity along the time course was examined using seven BP patients. NC16a ELISA scores tended to fluctuate in parallel with the disease activity along the time course and reflected the disease activity much better than indirect immunofluorescence. These findings indicate that NC16a ELISA will be a valuable tool not only for the diagnosis of patients with BP but also for the monitoring of the disease activity.

Adult↗

PubMed2002

Differential effects of desmoglein 1 and desmoglein 3 on desmosome formation.

The desmoglein plays an important part in the formation of desmosomes. We constructed recombinant adenoviruses containing desmoglein 1 and desmoglein 3 derivatives partly lacking the extracellular domain (desmoglein 1DeltaEC and desmoglein 3DeltaEC, respectively), and full-length desmoglein 1 and desmoglein 3 and studied the involvement of desmoglein 1 and desmoglein 3 in desmosome formation. During low-level expression of desmoglein 3DeltaEC in transduced HaCaT cells, keratin insertion at cell-cell contact sites was only partially inhibited and desmoplakin was partially stained at cell-cell contact sites. Low-level expression of desmoglein 1DeltaEC, however, resulted in complete inhibition of keratin insertion at the cell-cell contact sites, and desmoplakin was stained in perinuclear dots. These results indicate the dominant-negative effect of desmoglein 1DeltaEC on desmosome formation was stronger than that of desmoglein 3DeltaEC. Desmoglein 1DeltaEC coprecipitated plakoglobin to approximately the same extent as desmoglein 3DeltaEC. Therefore, we conclude that the dominant-negative effect of desmoglein 1DeltaEC is not simply due to plakoglobin sequestration. On the other hand, during low-level expression of full-length desmoglein 3 and desmoglein 1, they both colocalized with desmoplakin. During high-level expression, however, keratin insertion at cell-cell contact sites was inhibited in desmoglein 1 but not in desmoglein 3, and desmoplakin was stained at cell-cell contact sites in desmoglein 3 but not in desmoglein 1. These data suggest desmoglein 1 and desmoglein 3 expressed at low level were incorporated into desmosome but at high-level expression, desmoglein 1 disrupted desmosomes but desmoglein 3 did not. Our findings provide biologic evidence that desmoglein 1 and desmoglein 3 play a different functional role in cell-cell adhesion of keratinocytes.

Cadherins↗