Lymphoscintigraphy for sentinel lymph node mapping in Japanese patients with malignant skin neoplasms of the lower extremities: comparison with previously investigated Japanese lymphatic anatomy.

OBJECTIVES: Lymph nodes (LN) and lymphatic drainage were identified by lymphoscintigraphy using 99(m)Tc-phytate in order to map the sentinel lymph nodes (SLNs) in patients with malignant skin neoplasms of the lower extremities, and to compare the results with an atlas of Japanese lymphatic anatomy. METHODS: Sentinel lymphoscintigraphs of 18 patients with malignant skin neoplasms of the lower extremities (9 men, 9 women; age range 45-84 years, mean age 66 years) were analyzed retrospectively, and the LNs detected were identified as SLNs or secondary nodes. RESULTS: The patterns of lymphatic drainage were divided into three different categories: (1) initial drainage into inguinal LN without visualization of popliteal LNs (inguinal type), (2) initial drainage into popliteal LNs and then into intrapelvic LNs (popliteal type), and (3) initial drainage into both popliteal and inguinal LNs (inguinal and popliteal type). More than half of the cases were the inguinal and popliteal type, as both inguinal and popliteal LNs were identified as SLNs. In the cases in which the hallux and its surrounding area were injected, all were the inguinal type and popliteal LNs were not visualized. In one case, only dynamic images detected lymphatic drainage without visualization of popliteal LNs. In contrast to the previously published literature on Japanese lymphatic anatomy, SLN lymphatic drainage from the skin of the lower extremities was wide and overlapping in many areas. However, in agreement with currently accepted anatomy, only the great saphenous lymphatic vessel drained the skin of the hallux and its surrounding area. The present results suggest that it is important to confirm lymphatic drainage in order to identify SLNs in the lower extremities. CONCLUSIONS: The patterns of lymphatic drainage from the skin of the foot were divided into three different categories. In contrast to previously published Japanese lymphatic anatomy, lymphatic drainage from the skin of the lower extremities was wide and overlapping in many areas. However, only the great saphenous lymphatic vessel drained the skin of the hallux and its surrounding area in agreement with currently accepted Japanese lymphatic anatomy. It is important to confirm lymphatic drainage to identify SLNs in the lower extremities.

Localized pagetoid reticulosis (Woringer-Kolopp disease) in early childhood.

Localized pagetoid reticulosis, also known as Woringer-Kolopp disease, is an uncommon cutaneous lymphoproliferative disorder classified as a solitary variant of mycosis fungoides. We describe a case of localized pagetoid reticulosis that occurred in early childhood. A 6-year-old boy presented with an erythematous plaque on the left thigh. His parents had first noted brownish erythema of his thigh a few months after birth. The size and elevation of the plaque gradually increased. Physical examination showed well-demarcated reddish plaque, 6.2 x 2.3 cm, with central erosion. Histopathological examination showed an epidermotropic infiltrate of medium atypical lymphocytes with hyperchromatic and pleomorphic nuclei. The atypical cells infiltrated as individual cells or clusters in the epidermis. Immunohistologically, the phenotype of the atypical cells was CD3(+), CD4(-), CD8(+), CD45RO(+), CD20(-), CD30(-), and CD79a(-). We discuss the characteristics of this rare disease, including the differential diagnoses.

A randomized double-blind trial of intravenous immunoglobulin for pemphigus.

BACKGROUND: Pemphigus is a rare life-threatening intractable autoimmune blistering disease caused by IgG autoantibodies to desmogleins. It has been difficult to conduct a double-blind clinical study for pemphigus partly because, in a placebo group, appropriate treatment often must be provided when the disease flares. OBJECTIVE: A multicenter, randomized, placebo-controlled, double-blind trial was conducted to investigate the therapeutic effect of a single cycle of high-dose intravenous immunoglobulin (400, 200, or 0 mg/kg/d) administered over 5 consecutive days in patients relatively resistant to systemic steroids. METHODS: We evaluated efficacy with time to escape from the protocol as a novel primary end point, and pemphigus activity score, antidesmoglein enzyme-linked immunosorbent assay scores, and safety as secondary end points. RESULTS: We enrolled 61 patients with pemphigus vulgaris or pemphigus foliaceus who did not respond to prednisolone (> or =20 mg/d). Time to escape from the protocol was significantly prolonged in the 400-mg group compared with the placebo group (P < .001), and a dose-response relationship among the 3 treatment groups was observed (P < .001). Disease activity and enzyme-linked immunosorbent assay scores were significantly lower in the 400-mg group than in the other groups (P < .05 on day 43, P < .01 on day 85). There was no significant difference in the safety end point among the 3 treatment groups. LIMITATION: Prednisolone at 20 mg/d or more may not be high enough to define steroid resistance. CONCLUSION: Intravenous immunoglobulin (400 mg/kg/d for 5 d) in a single cycle is an effective and safe treatment for patients with pemphigus who are relatively resistant to systemic steroids. Time to escape from the protocol is a useful indicator for evaluation in randomized, placebo-controlled, double-blind studies of rare and serious diseases.

Effect of a novel ascorbic derivative, disodium isostearyl 2-O-L-ascorbyl phosphate, on normal human dermal fibroblasts against reactive oxygen species.

The novel amphiphilic vitamin C derivative disodium isostearyl 2-O-L-ascorbyl phosphate (VCP-IS-2Na), which has a C(18) alkyl chain attached to the stable ascorbate derivative sodium L-ascorbic acid 2-phosphate (VCP-Na), was evaluated for reduction of cell damage induced by oxidative stress, ultraviolet A (UVA), ultraviolet B (UVB), and H(2)O(2); stimulation of collagen synthesis against UVA irradiation; and inhibition of matrix metalloproteinase-1 (MMP-1) activity induced by UVA in human normal dermal fibroblasts. VCP-IS-2Na pretreatment resulted in significant protection against cell damage induced by UVB, UVA, and H(2)O(2). The amount of type I collagen following UVA irradiation was increased by treatment with VCP-IS-2Na in a concentration-dependent manner. These effects of VCP-IS-2Na were superior to those of L-ascorbic acid (vitamin C, VC) and VCP-Na. On the other hand, VCP-IS-2Na suppressed 65% of the excess MMP-1 irradiated UVA, and VC and VCP-Na slightly suppressed it.

Up-regulation of hyaluronan synthase genes in cultured human epidermal keratinocytes by UVB irradiation.

Hyaluronan controls keratinocyte proliferation and regeneration. We examined effect of UV on the expression of hyaluronan synthases (HASs) and hyaluronidases in cultured normal human newborn foreskin epidermal keratinocytes, NHEK(F). HAS3 mRNA was expressed predominantly and HAS2 mRNA expressed in lesser amounts and both were up-regulated after a single irradiation with moderate UVB but hyaluronidases was unchanged. Increased accumulation of hyaluronan in the culture medium mirrored the UVB-induced increase in the mRNA levels of HAS3 and HAS2. Unexpectedly, hyaluronan derived from UVB-irradiated and non-irradiated cells had identical size distribution. Increased expression of KGF and IL-1beta was detected just prior to the increase of HAS3 and HAS2 mRNAs after UVB irradiation. Antibody-neutralization study revealed that KGF and/or IL-1beta were at least involved in the up-regulation of HAS3 and HAS2 expressions. UVB-irradiated cells may enhance hyaluronan production to maintain homeostasis through up-regulation of HAS3 and HAS2 genes via cytokine response mechanism.

Epistatic connections between microphthalmia-associated transcription factor and endothelin signaling in Waardenburg syndrome and other pigmentary disorders.

Waardenburg syndrome (WS) is an inherited sensorineural deafness condition in humans caused by melanocyte deficiencies in the inner ear and forelock. Mutation of microphthalmia-associated transcription factor (MITF) is known to produce WS type IIA whereas mutations of either endothelin (EDN) or its receptor endothelin receptor B (EDNRB) produce WS type IV. However, a link between MITF haploinsufficiency and EDN signaling has not yet been established. Here we demonstrate mechanistic connections between EDN and MITF and their functional importance in melanocytes. Addition of EDN to cultured human melanocytes stimulated the phosphorylation of MITF in an EDNRB-dependent manner, which was completely abolished by mitogen-activated protein kinase kinase inhibition. The expression of melanocyte-specific MITF mRNA transcripts was markedly augmented after incubation with EDN1 and was followed by increased expression of MITF protein. Up-regulated expression of MITF was found to be mediated via both the mitogen-activated protein kinase-p90 ribosomal S6 kinase-cAMP response element-binding protein (CREB) and cAMP-protein kinase A-CREB pathways. In addition, EDNRB expression itself was seen to be dependent on MITF. The functional importance of these connections is illustrated by the ability of EDN to stimulate expression of melanocytic pigmentation and proliferation markers in an MITF-dependent fashion. Collectively these data provide mechanistic and epistatic links between MITF and EDN/EDNRB, critical melanocytic survival factors and WS genes.

Differential regulation of karyopherin alpha 2 expression by TGF-beta1 and IFN-gamma in normal human epidermal keratinocytes: evident contribution of KPNA2 for nuclear translocation of IRF-1.

Despite a number of studies on signal transduction in epidermal keratinocytes, very little is known about how signals move from the cytosol to the nucleus during the course of keratinocyte proliferation and differentiation. In this study, we first compared the expression patterns of the karyopherin alpha (KPNA) subtypes, and found that KPNA2, KPNA3, and KPNA4 were the major subtypes in both normal human epidermal keratinocytes (NHEKs) and normal human dermal fibroblasts (NHDFs). Stimulation with either transforming growth factor (TGF)-beta1 or IFN-gamma for 24 hours resulted in the downregulation of KPNA2 expression specifically in NHEK at both the mRNA and protein levels. Interestingly, IFN-gamma, but not TGF-beta1, specifically downregulated KPNA2 expression at the promoter level, suggesting differential regulation of KPNA2 expression by IFN-gamma and TGF-beta1. We then demonstrated that KPNA2 physically bound to IFN regulatory factor-1 (IRF-1), a transcription factor induced by IFN-gamma, and induced nuclear translocation of IRF-1 in NHEKs. We finally performed microarray and quantitative real-time PCR analysis for the mRNA expression pattern of NHEK with either overexpression or knockdown of KPNA2, and indicated KPNA2 involvement for various epidermal gene regulations such as involucrin. Our data suggest that KPNA2 may play an important role in the signal-transduction pathways that regulate epidermal proliferation and differentiation.

Cytokine modulation of retinoic acid-inducible gene-I (RIG-I) expression in human epidermal keratinocytes.

BACKGROUND: Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH/D box family proteins and designated as a putative RNA helicase, which plays various roles in gene expression and cellular functions in response to a variety of RNA viruses. OBJECTIVE: The present study was designed to investigate the effects of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha on RIG-I expression in human keratinocytes, and the expression of RIG-I in skin lesions of psoriasis vulgaris, in which IFN-gamma and TNF-alpha are considered to be involved in its pathogenesis. METHODS: The mRNA and protein expression of RIG-I was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Immunohistochemical staining of RIG-I was examined on psoriatic skin section. RESULTS: The levels of RIG-I mRNA and protein were upregulated in HaCaT keratinocytes in the presence of IFN-gamma or TNF-alpha. Immunohistochemically, RIG-I was detected in the cytoplasm of the spinous and basal layers of psoriatic skin. CONCLUSION: Our results suggest that RIG-I might operate not only as a RNA helicase but also as a mediator of the cytokine network in the inflammatory skin diseases, such as psoriasis vulgaris.

Interferon-gamma down-regulates expression of the 230-kDa bullous pemphigoid antigen gene (BPAG1) in epidermal keratinocytes via novel chimeric sequences of ISRE and GAS.

The 230-kDa bullous pemphigoid antigen (BPAG1) is an integral component of hemidesmosomes. We have previously reported that interferon-gamma (IFNgamma) inhibits the transcription of the BPAG1 gene (1). Here we investigated the target sequences of IFNgamma-signal transduction pathway in the BPAG1 promoter in epidermal keratinocytes. Transient transfections with 5'-deletion constructs of BPAG1 promoter-luciferase reporter gene plasmids in cultured normal human epidermal keratinocytes (NHEK) allowed us to narrow the DNA region containing IFNgamma inhibitory element (IGIE) to between -1 and -89, upstream from the transcription initiation site (+1). Homology search in this region identified a chimeric sequence, consisting of IFN-stimulated responsive element (ISRE) with a partial 7-bp sequence of IFNgamma activation site (GAS), as identified in the guanylate-binding protein (GBP) gene, inserted at its center. Functional analysis of IGIE, inserted in front of the heterologous thymidine kinase promoter, indicated that IGIE acts as a down-regulatory element of the promoter through IFNgamma-dependent signal pathway. Transient transfection studies with BPAG1 promoter-reporter gene constructs containing mutated IGIE (with TT to GG transversions in the region of 5'ISRE, GAS, and 3'ISRE) demonstrated that disruption of the ISRE sequences, but not GAS, markedly suppressed the BPAG1 basal promoter activity and resulted in attenuated IFNgamma response in keratinocytes. Our findings provide novel insight into the mechanism of IFNgamma regulation in keratinocyte differentiation and proliferation.

A case of bullous dermatitis artefacta possibly induced by a deodorant spray.

Dermatitis artefacta is one of a spectrum of factitious diseases etiologically responsible for skin lesions denied by patients. These factors often make it difficult to identify the causative agents of the condition. Herein, we report a case of bullous dermatitis artefacta in a 12-year-old girl, for which a deodorant spray was suspected as the probable cause. Pathological examination revealed subepidermal blistering with full-thickness necrosis of the epidermis, suggesting a thermo- or cryo-induced injury. Psychological testing demonstrated her immaturity and dependence. In searching for the causative agent, we suspected a deodorant spray as a blister-inducing agent. We succeeded in reproducing a similar blister lesion on the volunteer's healthy skin using the same spray. Psychiatric involvement significantly complicates the treatment of factitious diseases, including dermatitis artefacta. Cooperation among dermatologists, psychiatrists and the patient's family members is required for ensuring a favorable prognosis.

Human metallothionein gene expression is upregulated by beta-thujaplicin: possible involvement of protein kinase C and reactive oxygen species.

Recently, we discovered that beta-thujaplicin (BT) induces metallothionein (MT) expression in mouse keratinocytes, both in vivo and in vitro. However, the molecular mechanisms by which BT exerts its biological effects have not been elucidated. The purpose of this study is to explore the signal transduction pathway involved in the MT mRNA induction by BT. Using a HaCaT keratinocyte cell line, Northern blotting was performed for analyzing the human MT-IIA mRNA expression levels in combination with BT and a number of protein kinase (PK) inhibitors including H7, HA1004 and a PKC-specific inhibitor chelerythrin. CAT assays with the MT-IIA gene promorter-CAT construct were conducted for examining the transcriptional regulation by BT of MT. A free radical scavenger N-acetylcysteine (NAC) was used for analyzing a role of oxidative stress for the MT gene induction by BT. BT increased MT-IIA gene transcript levels and CAT activity in a dose-dependent fashion in HaCaT cells. The increase in MT-IIA mRNA levels and CAT activity were completely suppressed by H7 but not by HA1004. In addition, chelerythrin prevented BT-inducible MT-IIA promoter activation. Furthermore, NAC suppressed BT-inducible MT-IIA promoter activation. These results demonstrate that BT is a potent activator of the MT-IIA gene promoter and that PKC activation and reactive oxygen species are implicated in BT-inducible MT-IIA gene expression. BT may be a useful tool for dissecting the signal transduction pathway mediating MT-IIA promoter activation.

A case of photocontact urticaria induced by photodynamic therapy with topical 5-aminolaevulinic acid.

Photodynamic therapy (PDT) with topical application of 5-aminolaevulinic acid (ALA) is a promising new treatment option for the management of various cutaneous malignancies. Generally, topical ALA-based PDT has relatively insignificant adverse effects of transient character; these include itching, stinging or burning pain and slight to moderate erythema. We describe the first case of photocontact urticaria induced by topical ALA-based PDT for the treatment of unilesional mycosis fungoides. Although the first treatment session resulted merely in mild erythema, the second PDT caused marked urticaria corresponding to the PDT-applied area with an intolerable stinging sensation. A photopatch test demonstrated that black light and visible light irradiation after topical ALA provoked an urticarial reaction in the patient's uninvolved skin. These observations suggested an allergic pathogenesis for the wheal formation induced by PDT with topical ALA in this case. Photocontact urticaria should be considered as a possible adverse effect in ALA-based PDT.

Successful treatment of lymphadenosis benigna cutis with topical photodynamic therapy with delta-aminolevulinic acid.

BACKGROUND: Photodynamic therapy (PDT) using topical delta-aminolevulinic acid (ALA) has been used for nonmelanoma skin cancers, including malignant cutaneous T-cell lymphomas. Moreover, PDT has been tried for benign inflammatory or infectious skin lesions. OBJECTIVE: To evaluate the effects of ALA-PDT on skin lesions of lymphadenosis benigna cutis (LABC). PATIENTS AND METHODS: Two 16-year-old females with solitary and infiltrated nodules were treated 5 times with topical ALA-PDT. RESULTS: Both patients responded well and showed dramatic clinical and histopathological improvement without visible scars. CONCLUSION: The results confirm that topical ALA-PDT is an effective and safe modality for the treatment of LABC, and that such treatment may be cosmetically beneficial.

Sugar micro needles as transdermic drug delivery system.

We designed and fabricated an array of sugar micro needles of the length ranging from 150 micro m to 2 mm for transdermic delivery of drugs. Micro needles were molded out of maltose mixed with pharmaceutical material, being expected bio-degradable in the human skin. To test basic tolerance to the healthy human skin, a clinical experiment was carried out for 10 healthy adult volunteers. 500 microm-needles containing 5 wt% of ascorbate-2-glycoside were inserted into the skin of the forearm and snapped off to be left in the skin. They spontaneously dissolved by hydrolysis to release ascorbate in the epidermis and the dermis. No dermatological problems were observed in terms of the International Contact Dermatitis Research Group criteria. These observations indicate that the present system is a novel approach to achieve transdermic drug delivery.

Keratinocyte-specific modulation of type VII collagen gene expression by pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-1beta).

Previous studies have shown that pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta up-regulate type VII collagen gene (COL7A1) expression in cultured dermal fibroblasts. The present study was designed to investigate the effects of TNF-alpha and IL-1beta on COL7A1 expression in epidermal keratinocytes. We demonstrated that both TNF-alpha and IL-1beta reduced COL7A1 expression in epidermal keratinocytes in an additive manner, whereas they increased COL7A1 expression in dermal fibroblasts. Thus, regulation of COL7A1 by pro-inflammatory cytokines is cell type specific. In particular, the inhibitory effects of TNF-alpha and IL-1beta occurred, at least in part, at the transcriptional level. Finally, we demonstrated that TNF-alpha and IL-1beta enhanced the TGF-beta-mediated up-regulation of COL7A1 expression in HaCaT keratinocytes, suggesting that the combination of TGF-beta and TNF-alpha or IL-1beta induces a signaling pathway that is completely different from that induced by either pro-inflammatory cytokine alone.

Clinicopathologic significance of dysadherin expression in cutaneous malignant melanoma: immunohistochemical analysis of 115 patients.

BACKGROUND: The E-cadherin-mediated cell adhesion system is frequently inactivated by multiple mechanisms and is involved in tumor progression in many types of cancer. Recently, the authors reported a novel cell membrane glycoprotein, dysadherin, which has an anti-cell-cell adhesion function and down-regulates E-cadherin. METHODS: Expression of both dysadherin and E-cadherin was investigated immunohistochemically in 115 patients with cutaneous malignant melanoma to determine the correlation between the 2 molecules and their associations with both patient survival and the clinicopathologic features of the tumors. RESULTS: Dysadherin and E-cadherin were expressed at the cell membranes of melanoma cells. Fifty-two percent of the tumors showed dysadherin immunopositivity, and 91% of the tumors showed reduced E-cadherin immunopositivity. There was no significant inverse correlation between dysadherin expression and E-cadherin expression. Increased dysadherin expression was significantly correlated with nodular subtype (P = 0.042), Clark level (P < 0.001), tumor thickness (P < 0.001), ulceration (P = 0.008), lymph node metastasis (P < 0.001), high TNM classification (P < 0.001), and poor patient survival (P < 0.001). Multivariate analysis of patient survival revealed that increased dysadherin expression was a significant predictor of poor survival (P < 0.001). CONCLUSIONS: Thus, increased expression of dysadherin was a significant indicator of poor prognosis in patients with cutaneous malignant melanoma.

IFN-gamma and TNF-alpha are involved in urushiol-induced contact hypersensitivity in mice.

Contact hypersensitivity (CHS) is a cutaneous T-cell-mediated immunological reaction to applied haptens. Activated antigen-specific T cells release several cytokines and chemokines followed by the recruitment of inflammatory cells and skin damage. CD8+ T cells and CD4+ T cells have been involved in the establishment of previously described CHS. In this study, we investigated the induction of CHS by urushiol in mice. Maximum swelling in mouse ears was elicited 24 h after challenge with urushiol on day 9 of sensitization. IFN-gamma, TNF-alpha and IFN-gamma-inducible protein 10 (IP-10) mRNA were expressed after challenge of the antigen in urushiol-sensitized mice, but not in unsensitized mice. IFN-gamma knockout (KO) mice and TNF-alpha KO mice failed to elicit CHS with urushiol. Contact hypersensitivity and expressions of IFN-gamma, TNF-alpha and IP-10 mRNA were markedly suppressed in CD4+ and CD8+ cell-depleted mice. These results suggest that IFN-gamma, TNF-alpha, and possibly IP-10, play a critical role in CHS induced by urushiol, depending on both CD4+ T cells and CD8+ T cells.