Katsumi Hanada

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.

Interferon-γ stimulates the expression of galectin-9 in cultured human endothelial cells

Galectin‐9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin‐9 expression in endothelial cells. Galectin‐9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon‐γ (IFN‐γ). IFN‐γ also enhanced the adhesion of human eosinophilic leukemia‐1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin‐4, which induces eotaxin expression, did not affect the expression of galectin‐9. The in situ endothelium from patients with inflammatory diseases was found to express galectin‐9. IFN‐γ‐induced production of galectin‐9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.

Keratinocyte gene therapy for systemic diseases. Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin.

This study has examined the systemic effects of a circulating gene product, human interleukin 10 (IL-10), released from transduced keratinocytes. IL-10 is an anti-inflammatory cytokine which has an inhibitory effect on contact hypersensitivity (CHS). An expression vector (phIL-10) was constructed for human IL-10 and was injected into the dorsal skin of hairless rats. Local expression of IL-10 mRNA and protein was detected by reverse-transcriptase polymerase chain reaction and immunohistochemical staining, respectively. Enzyme-linked immunosorbent assay showed that the amount of IL-10 in the local keratinocytes and in the circulation increased with the dose of phIL-10 transferred. To determine whether circulating IL-10 could inhibit the effector phase of CHS at a distant area of the skin, various doses of phIL-10 were injected into the dorsal skin of sensitized rats before challenge on the ears. Our results showed that the degree of swelling of the ears of phIL-10- treated rats was significantly lower than that in the negative control animals. These results suggest that IL-10 released from transduced keratinocytes can enter the bloodstream and cause biological effects at distant areas of the skin. This study demonstrates that it may be possible to treat systemic disease using keratinocyte gene therapy.

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 haploinsuf‐ficiency 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 ribo‐somal 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 epi‐static links between MITF and EDN/EDNRB, critical melanocytic survival factors and WS genes. Sato‐Jin, K., Nishimura, E. K., Akasaka, E., Huber, W., Nakano, H., Miller, A., Du, J., Wu, M., Hanada, K., Sawamura, D., Fisher, D. E., Imokawa, G. Epistatic connections between microphthalmia‐associated transcription factor and endothelin signaling in Waardenburg syndrome and other pigmentary disorders. FASEB J. 22, 1155–1168 (2008)

Possible role of 1,25-dihydroxyvitamin D3-induced metallothionein in photoprotection against UVB injury in mouse skin and cultured rat keratinocytes

Previously cadmium chloride was successfully used to prevent sunburn cell (SBC) induction in mouse skin in vivo and to promote human cell survival in vitro after UVB exposure, indicating a protective effect of cadmium-induced metallothionein (MT) with the property of anti-oxidant action. Although cadmium is a potent inducer of MT, the cytotoxic metal is not available for clinical use. The aim of this study is to affirm MT gene expression by the active for of vitamin D3, 1,25-dihydroxyvitamin D3, [1,25(OH)2D3] in cultured keratinocytes and examine in vivo and in vitro the photo-protective effects of 1,25(OH)2D3. Northern hybridization with human MT-IIa cDNA showed a significant increase in the gene expression of MT in the cells treated with 1,25(OH)2D3. Intraperitoneal injection and topical application of 1,25(OH)2D3 caused a significant reduction in SBC formation in mouse skin after UVB administration. The experiment showed the existence of an optimum level of 1,25(OH)2D3 for reducing photodamage. The cells exposed to 1,25(OH)2D3 showed increased tolerance (cell survival) to UVB injury. 1,25(OH)2D3-induced MT may act as a radical scavenger in oxygen-mediated UV injury including SBC formation in the skin. These results indicate that 1,25(OH)2D3 may be practically applied to humans for the purpose of photoprotection.

In vivo gene introduction into keratinocytes using jet injection

Successful keratinocyte gene therapy requires the development of efficient methods of gene transfer to keratinocytes. Jet injection of a solution containing DNA can be used to transfer genes to several tissues in vivo. In this article, we tried to introduce DNA into rat and human keratinocytes using this method. First, we fired a β-gal expression vector into rat skin at several distances using a jet injector and examined β-gal activity in the epidermal keratinocytes. The highest activity in keratinocytes was found when the plasmid was fired at 10 cm from the skin surface; the activity lessened as the firing distance became shorter than 10 cm. Next, we transplanted human skin on to a nude rat, fired the vector into the human skin from a distance of 10 cm and examined the β-gal activity. We also injected the same amount of plasmid with a needle to compare jet with needle injections. The results showed that jet injection of the naked DNA could introduce and express DNA in human keratinocytes in vivo and that jet injection exhibited much higher activity than needle injection. Jet injection of the naked DNA will provide a method for keratinocyte gene therapy in the future.

Systemic sarcoidal reaction in tattoo

Following extensive tattooing, a 31‐year‐old man developed symptoms similar to those of systemic sarcoidosis. Histological examination of the skin lesions, regional lymph nodes and the lung tissue revealed noncaseating granulomatous reaction. Uveitis was also observed, Electron microscopic examination of lung specimens revealed fragments of red tattoo granules. X‐ray microanalysis of the pigment granules observed in red skin and lung lesions showed elements of mercury, aluminium and silicon. The finding of tattoo pigments in the lung tissues has not been previously described.

Analyses of Serum Copper and Zinc Levels and Copper/Zinc Ratios in Skin Diseases

With the objective of comprehending abnormal metabolisms of the essential metals of zinc (Zn) and copper (Cu) in three groups of skin diseases, skin cancer, inflammatory diseases, and non‐inflammatory disease, we measured serum levels of Zn and Cu in 151 cases of various cutaneous manifestations and estimated the significance of the ratios between the two metals (Cu/Zn). The serum level of Zn was significantly decreased in cases of bullous pemphigoid, decubitus ulcer, and alopecia areata. The serum level of Cu was elevated in cases of psoriasis, decubitus ulcer, and skin cancer. We observed no elevation of serum Zn level or abnormally depressed serum Cu level. The Cu/Zn ratio showed significantly different values among these three groups of the diseases, suggesting the utility of measuring Cu/Zn ratios for differential diagnosis over that of determining the serum level of Zn or Cu alone. It was also demonstrated that, in each skin disease, the Cu/Zn ratio clearly reflects the severity of the progress.

Successful Treatment of Dark-colored Epidermal Nevus with Ruby Laser

The pulsed ruby laser has a selective thermolytic effect. Recently, it has been available for the treatment of superficial pigmented disorders. We studied 5 cases of epidermal nevus treated with the pulsed ruby laser. In comparison with the usual methods including electrocautery, cryotherapy and skin abrasion, ruby laser therapy is an excellent tool due to technological ease and rapid improvement. Depigmentation after treatment in 2 cases was the only side effect of this therapy. Bose cases had a dark pigmentation of the skin. Despite of the risk of discoloration, the ruby laser is one of the most effective tools for therapy of pigmented epidermal nevus.

Flagellate Mushroom (Shiitake) Dermatitis and Photosensitivity

Flagellate skin lesions occur in some patients after eating the mushroom Lentinus edodes, and they are called shiitake dermatitis in Japan. We describe a 44-year-old man with such skin lesions on his trunk after eating L. edodes, who developed as well photosensitive skin lesions on exposed areas. Skin phototesting with UVB did not show a decreased minimum erythema dose; UVA irradiation provoked an erythematous lesion with nonspecific histological changes. Analysis of the case histories of 94 Japanese patients with shiitake dermatitis revealed that 44 (47%) also developed dermatitis on the skin exposed to sunlight. UVA photodermatitis has not been emphasized in previous reports. The relationship to bleomycin flagellate dermatitis is discussed.