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Supplementary Tables 1-3 from Silencing of Peroxiredoxin 2 and Aberrant Methylation of 33 CpG Islands in Putative Promoter Regions in Human Malignant Melanomas

Aberrant methylation of promoter CpG islands (CGI) is involved in silencing of tumor suppressor genes and is also a potential cancer biomarker. Here, to identify CGIs aberrantly methylated in human melanomas, we did a genome-wide search using methylation-sensitive representational difference analysis. CGIs in putative promoter regions of 34 genes (ABHD9, BARHL1, CLIC5, CNNM1, COL2A1, CPT1C, DDIT4L, DERL3, DHRS3, DPYS, EFEMP2, FAM62C, FAM78A, FLJ33790, GBX2, GPR10, GPRASP1, HOXA9, HOXD11, HOXD12, HOXD13, p14ARF, PAX6, PRDX2, PTPRG, RASD1, RAX, REC8L1, SLC27A3, TGFB2, TLX2, TMEM22, TMEM30B, and UNC5C) were found to be methylated in at least 1 of 13 melanoma cell lines but not in two cultured normal melanocytes. Among these genes, Peroxiredoxin 2 (PRDX2) was expressed in normal melanocytes, and its expression was lost in melanomas with methylation. The loss of expression was restored by treatment of melanomas with a demethylating agent 5-aza-2′-deoxycytidine. In surgical melanoma specimens, methylation of PRDX2 was detected in 3 of 36 (8%). Furthermore, immunohistochemical analysis of PRDX2 showed that disappearance of immunoreactivity tends to associate with its methylation. PRDX2 was recently reported to be a negative regulator of platelet-derived growth factor signaling, and its silencing was suggested to be involved in melanomas. On the other hand, 12 CGIs were methylated in ≥9 of the 13 melanoma cell lines and are considered as candidate melanoma biomarkers. (Cancer Res 2006; 66(12): 6080-6)

Basal cell carcinoma is the most common cutaneous malignant tumor and usually arises from the facial skin and is diagnosed as the solid tumor. However, rarely it arises from genital skin and/or proceeds with ulcerative manner. We report a case of male basal cell carcinoma arising from penile base, which showed urinary disturbance. Tumor resection including total penectomy and skin resection was performed then scrotal myofasciocutaneous flap was used to cover the defect. Scrotal myofasciocutaneous flap was useful for covering the defect in the genital lesion. Moreover, this simple closure was helpful for early postoperative ambulation especially for aged male patients.

Scratch the surface: Dehydrocondensations between carboxylates and amines by using an amphiphilic 1,3,5-triazinylammonium-based coupling agent were accelerated by the interfacial effect of micelles and emulsions of common surfactants (see figure). The reaction of carboxylates was promoted by both anionic and nonionic surfactants, and that of amines was promoted by only a nonionic surfactant. High selectivities for more lipophilic substrates were observed in micelles or emulsions. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cutaneous candidiasis is a common skin infection caused by the Candida species, especially in intertriginous areas, and neutrophils usually infiltrate histopathologically. We describe a case of secondary cutaneous candidiasis which spread extensively to the trunk and extremities and showed marked dermal eosinophilia. This case and a similar reported case suggest that Candida can sometimes cause cutaneous inflammation predominantly composed of eosinophils.

A 46-year-old man had a cystic mass on the right side of his scalp. Histological examination revealed a cystic dermal nodule composed of relatively circumscribed lobules of proliferating squamous epithelium, with atypical mitoses and dyskeratotic cells of invasive structure, which was diagnosed as proliferating tricholemmal cystic carcinoma (PTCC). Most of the cyst was composed of thick layers of highly proliferating, atypical, dedifferentiated epithelium (dedifferentiated part), which was attached to a highly proliferative but mildly differentiated part. A completely differentiated, tricholemmal cyst (TC)-like part was also attached to the main cyst, which supports the idea of PTCC beginning in a pre-existing TC. The dedifferentiated and mildly differentiated parts exhibited a high frequency of proliferating cell nuclear antigen (PCNA)-positive cells both in the basal and the suprabasal layers, while PCNA staining was almost negative in the TC-like part. Expression of cytokeratin (CK)10 and CK16 suggested disturbed epidermal differentiation in dedifferentiated part, while TC-like part showed well-differentiated trichilemmal epithelium and the mildly differentiated part was in the middle of these two.

To identify tumor-suppressor genes inactivated by aberrant methylation of promoter CpG islands (CGIs) in human malignant melanomas, genes upregulated by treatment of cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), were searched for using oligonucleotide microarrays in melanoma cell lines, HMV-I, MeWo and WM-115. Seventy-nine known genes with CGIs were identified as being upregulated (>or=16-fold), and 18 of them had methylation of their putative promoter CGIs in 1 or more of 8 melanoma cell lines. Among the 18 genes, TFPI-2, which is involved in repression of the invasive potential of malignant melanomas, was further analyzed. Its expression was repressed in a melanoma cell line with its complete methylation, and was restored by 5-aza-dC treatment. It was unmethylated in cultured neonatal normal epidermal melanocyte, and was induced by ultraviolet B. In surgical melanoma specimens, TFPI-2 methylation was detected in 5 of 17 metastatic site specimens (29%), while it was not detected in 20 primary site specimens (0%) (p=0.009). By immunohistochemistry, the 5 specimens with promoter methylation lacked immunoreactivity for TFPI-2. The results showed that TFPI-2 is silenced in human malignant melanomas by methylation of its promoter CGI and suggested that its silencing is involved in melanoma metastasis.

Necrolytic migratory erythema (NME) is an uncommon inflammatory dermatosis with a distinctive clinical and histological appearance. It shows irregular erythema, bullae, erosion, crusts and pigmentation. While it is typically associated with glucagonoma, some cases of NME without glucagonoma have been reported. Herein, we report a case of necrolytic migratory erythema associated with malabsorption 30 years after ileocolectomy. She presented erosive erythema with scale or partly flaccid bullae on her intergluteal cleft, buttock and extremities. Her laboratory data revealed essential amino acid deficiency and a slightly decreased serum zinc level, while her plasma glucagon level was low. With diagnosis of non-glucagonoma-associated NME with malabsorption due to short-bowel syndrome, she was treated and improved by i.v. amino acid supplement. Histological findings of NME include necrotic changes of keratinocytes in the upper epidermis, proliferation of those in the lower epidermis and inflammatory cell infiltration of upper dermis. We also examined the expression pattern of epidermal keratins (K6, K10) and Ki-67, one of the markers of proliferative activity, to assess the proliferation and differentiation of keratinocytes in a NME lesion by immunostaining. The findings with these immunostainings support the characteristics of HE-staining, and suggest hyponutrition may induce changing differentiation/proliferation of keratinocytes.

Background Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are angiogenic cytokines, that have been reported to elevate serum levels in patients with collagen diseases such as systemic sclerosis and dermatomyositis as well as in those with inflammatory bowel diseases. Objective In this study, the serum levels of bFGF and VEGF were measured by using enzyme-linked immunosorbent assay in 20 patients with polyarteritis nodosa (PAN; 5 with systemic PAN and 15 with cutaneous PAN) and in 20 control subjects. We also investigated the expression of bFGF and VEGF in cutaneous lesions of patients by using immunohistochemical methods. Results Basic FGF was undetectable in the serum of control subjects, but detectable levels were found in 4 of 5 patients with classical PAN and 3 of 15 patients with cutaneous PAN. The serum bFGF level in these patients with systemic PAN was significantly elevated in comparison with that in healthy control subjects. The VEGF level was 178 ± 41 pg/mL in the serum of healthy individuals. The mean VEGF level in patients with systemic or cutaneous PAN was significantly higher than that in healthy controls. Serum bFGF and VEGF levels in patients with systemic PAN were significantly elevated in comparison with those having cutaneous PAN. Immunohistochemical studies showed elevated bFGF expression on damaged endothelial cells in necrotizing vasculitis lesions. Elevated expression of bFGF was also observed on fibroblasts around the vasculitis. Some of the infiltrating cells around vasculitis lesions expressed VEGF. Limitations The study was small. Conclusions Serum bFGF and VEGF levels may be useful markers of the disease activities of PAN.

A 11-year-old female admitted to our hospital because of erythema of the face and the trunk, and a wide and dense cluster of verruca vulgaris on the right sole. She had no family history of immunodeficiency, no perinatal abnormality, no growth abnormality, or no history of severe infections. From the age of 4 years, she noticed erythema around her nose. At the age of 9 years, small erythema and papules appeared on her chest. In January, 2003, erythema around her nose and papules of the trunk spread rapidly, and she also felt fatigue and effort dyspnea. Laboratory examinations revealed near absence of serum IgG, and IgE, high serum IgM (525 mg/dl), and normal IgA and IgD. Thl/Th2 ratio was 36.9. We diagnosed her as having hyper-IgM syndrome. Histological examinations of a skin biopsy showed the infiltration composed of mainly histiocytes,and mildly atypical CD8 + T cells around the blood vessels in the dermis. We concluded her skin manifestations as reactive lymphohistiocytic infiltration at the base of immunodeficiency and durable stimulation of various antigens. Her skin manifestations improved transiently by the intravenous immunogrobulin and corticosteroids therapy.

A case of burn scar carcinoma was reported. A 65-year-old man discovered a finger-sized mass on the top of his head 4 months prior to his first visit to our department. He came with a fist-sized tumor and an ulcer of the same size beside it. He had a burn scar caused 60 years ago by an “iron” (Japanese hearth) . Histopathological examinations revealed squamous cell carcinoma. Cranial CT/MRI showed that the tumor was invading the brain. Metastasis to pleura, periaortic lymph nodes, and cervical vertebrae made curative excision difficult. During the course of his radiation therapy (total 42Gy), pressure fracture of the cervical vertebrae occurred, resulting in tetraparesis. Additional radiation of 33Gy was applied to his neck fracture, followed by chemotherapy using low-dose cisplatin (5mg/day, for 5 days/week) . Although the tumor was decreasing in size, a sudden onset of respiratory failure occurred, which resulted in his death 2 months after his first visit. [Skin Cancer (Japan) 2003; 18: 75-79]

The effects of nefiracetam on the cerebral adenylyl cyclase (AC) activity of animals with microsphere embolism-induced memory dysfunction were examined. Sustained cerebral ischemia in the right cerebral hemisphere was induced by an injection of microspheres into the right internal carotid artery of rats. To examine learning and memory function, the water maze test was performed from day 7 to day 10 after the operation. The escape latency of the microsphere-embolized (ME) rat in the water maze task was longer than that of the sham-operated (Sham) rat, suggesting that spatial memory dysfunction occurred in the ME rat. Gsalpha and Gi(1/2)alpha protein levels in the cerebral cortex, striatum and hippocampus of the ME rat, when determined on day 11, were similar to those of the Sham rats. The basal AC activity in the striatum, but not in the other two regions, of the ME rat decreased. The AC activity in the presence of 10 microM colforsin daropate (Col), a direct stimulator of AC, was increased by approximately 20-fold in sham animals and 7- to 10-fold in the ME rat. Treatment of the ME rat with 10 mg/kg/d nefiracetam p.o. from day 1 to day 10 after the operation shortened the escape latency, restored the basal AC activity in the striatum, and reversed the Col-induced increases in AC in these three regions without any changes in the cerebral Gsalpha and Gi(1/2)alpha protein levels. These results suggest that nefiracetam-mediated activation of AC activity may contribute to the improvement of memory and learning function in sustained cerebral ischemia.

Matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitors of metalloproteinases-1 (TIMP-1) are involved in tissue inflammation and fibrotic processes. We previously reported an elevated serum TIMP-1 level in patients with systemic sclerosis (SSc). We measured serum TIMP-1 and active MMP-9 levels in 62 patients with SSc, 10 patients with rheumatoid arthritis (RA) and 15 normal controls using a modified enzyme-linked immunosorbent assay. The active MMP-9 level in the serum of the patients with RA or SSc was not significantly different from that of controls. Serum MMP-9 activity in patients with diffuse cutaneous SSc was significantly decreased compared with that of limited cutaneous SSc or normal controls. The MMP-9 activity and modified-Rodnan total skin thickness score in patients with SSc were negatively correlated. Serum MMP-9 activity and TIMP-1 level in patients with SSc were not correlated. Serum MMP-9 activity might be a useful indicator of disease activity, especially skin severity, in SSc patients.

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) have been suggested to play important roles in wound healing. We investigated the effect of TGF-beta1 on the mitogenic and chemotactic activities of PDGF A-chain homodimer (PDGF-AA) and B-chain homodimer (PDGF-BB) in primary cultures of human skin fibroblasts. TGF-beta1 inhibited the growth-promoting activity of both PDGFs. Proliferative responses to basic fibroblast growth factor and epidermal growth factor were also restricted by TGF-beta1. A Boyden chamber chemotaxis assay revealed that the chemotactic migration to PDGF-AA was inhibited by TGF-beta1 pretreatment, but in contrast, the response to PDGF-BB was not affected by the same treatment. Western blot analysis showed that TGF-beta1 downregulated PDGF alpha-receptors, but not beta-receptors, indicating that the isoform-specific inhibition of chemotaxis is related to differential effects of TGF-beta1 on PDGF receptor expression. The present findings suggest that TGF-beta1 may act antagonistically towards PDGFs in humans under certain conditions, and this antagonistic nature of TGF-beta1 must be considered when it is applied to human wounds as a therapeutic agent.

We investigated the role of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in the growth regulation. Primary (PM-WK and KHm-4), recurrent primary (RPM-EP and RPM-MC), lymph node metastatic (MM-AN, MM-BP and MM-RU), and a visceral metastatic (MM-LH) melanoma cell lines were used. Reverse transcriptase-coupled polymerase chain reaction and Western blotting revealed that all expressed and produced TIMP-1 and TIMP-2 except for PM-WK, which neither expressed nor produced TIMP-1. TIMP-1 and TIMP-2 secretion levels were measured by enzyme-linked immunosorbent assay in supernatants of cells. We found that the TIMP-1 production level was correlated with the cell migration rate. Moreover, TIMP-1 enhanced the cell migration of PM-WK. The growth of the primary melanoma cell lines was stimulated by TIMP-1 and inhibited by TIMP-2. In contrast, the growth of the visceral metastatic melanoma cell line was stimulated by TIMP-2.

Inflammatory bowel disease (IBD), the precise etiology of which remains unknown, is comprised of two forms of chronic intestinal inflammation; ulcerative colitis (UC) and Crohn's disease (CD). Recent evidence increasingly suggests that IBD is the result of dysfunctional immunoregulation manifested by inappropriate production of mucosal cytokines. An abnormal microcirculatory system has also been implicated in its pathogenesis. To elucidate the mechanism of ischemic change in IBD, we assesse serum concentration levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF), and plasma level of endothelin-1 (ET-1). We also investigated the expression of VEGF, b-FGF, and transforming growth factor-beta1,2,3 (TGF-beta1,2,3) in tissue by immunostaining.Blood samples were obtained from 11 patients with UC, 11 patients with CD, and 10 patients as controls. Paraffin-embedded samples were used for an immunohistochemical study.The concentration levels (in picograms per milliliter) were as follows: for ET-1, UC: 127+/-47.0, CD: 167.3+/-35.1, and controls (asthma: 38.5+/-23.8, p0.01; diverticulitis: 40.5+/-25.6, p0.01), for b-FGF, UC: 9.2+/-1.9, CD: 9.1+/-1.5, and controls (asthma: 5.0+/-0, p0.01; diverticulitis: 5.0+/-0, p0.01), for VEGF, UC: 659.8+/-181.0, CD: 740.0+/-182.3, and controls (asthma: 193.7+/-58.7, p0.01; diverticulitis: 199.6+/-59.7, p0.01). The levels of VEGF and b-FGF were significantly higher in active IBD than those in the controls. There was a significant positive correlation among the serum levels of VEGF and b-FGF and the plasma level of ET-1; that is, elevated VEGF, b-FGF, and ET-1 levels correlated well with each other. Immunohistochemical studies showed increased venula in the submucosa and lamina propria. Overexpression of VEGF and b-FGF in endothelial cells was revealed and TGF-beta2 and TGF-beta3 were found in inflammatory cells of active IBD, but no change was observed around the vessels in the controls.It is suggested that the reciprocal reaction of these cytokines may contribute to angiogenesis in IBD b inducing intestinal ischemia through vasoconstriction.

Serum containing anti-U1RNP antibodies reacts with the nuclear matrix, the relatively insoluble component of the cell nucleus, in addition to U1RNP. In this study, we determine the serum titer and clinical correlations of antinuclear matrix antibodies in samples from patients with anti-U1RNP antibodies. The patients with anti-U1RNP antibodies were classified as having mixed connective tissue disease (MCTD, 15 patients), systemic sclerosis (SSc, 12 patients), systemic lupus erythematosus (SLE, 7 patients), and undifferentiated CTD (UCTD, 9 patients). Antinuclear matrix antibodies were detected using indirect immunofluorescence staining on HCl-treated HEp-2 cells. The antinuclear matrix antibody titer was significantly higher in serum from patients with MCTD or SSc than in serum from patients with SLE or UCTD. The antinuclear matrix antibody titer was significantly increased in serum from patients with sclerodactyly, pitting scars, contracture of the phalanges, and decreased carbon monoxide diffusion capacity. Thus, a higher titer of antinuclear matrix antibodies in serum from patients with anti-U1RNP antibodies may be associated with a clinical diagnosis of MCTD or SSc rather than a diagnosis of SLE or UCTD.

We have experienced thirty-nine cases of cutaneous malignant melanoma for twelve years from 1987 to 1998 at the Department of Dermatology, University of Tokyo. The number of patients is increasing but this phenomenon is mostly due to increase of in situ patients. We assume that because of the prevalence of knowledge on pigmented lesions, those patients consult us in early stage. [Skin Cancer (Japan) 2000; 15: 45-48]

Background: The serum levels of soluble forms of vascular cell adhesion molecule 1 (sVCAM-1) and E-selectin (sE-selectin) can be used as a marker of endothelial activation. Objective: We investigated whether the serum level of sVCAM-1 and sE-selectin in patients with localized scleroderma (LSc) was correlated with the clinical or serologic features of this disease. Methods: Serum samples from 59 patients with LSc, 20 patients with systemic sclerosis (SSc) and 29 healthy controls were examined using specific enzyme-linked immunosorbent assays. Results: The serum levels of sVCAM-1 and sE-selectin in patients with LSc were significantly higher than those of the healthy controls. The serum levels of sVCAM-1 and sE-selectin were correlated with both the number of sclerotic lesions and the number of involved areas. Conclusion: These results indicate that the serum levels of sVCAM-1 and sE-selectin may reflect the extent of the skin involvement (ie, the disease severity) and may be useful for monitoring the in vivo states of endothelial activation in LSc. (J Am Acad Dermatol 2000;42:64-9.)

Anti-histone antibodies have been detected in the sera of patients with various autoimmune diseases. The existence of anti-histone antibodies in patients with polymyositis/dermatomyositis, however, has not been reported. We found anti-histone antibodies in eight (17%) of 46 sera from patients with polymyositis/dermatomyositis by an enzyme-linked immunosorbent assay. One serum was positive for both IgG anti-histone antibodies and IgM anti-histone antibodies. Six sera were positive only for IgG anti-histone antibodies. One serum was positive only for IgM anti-histone antibodies. An indirect immunofluorescence analysis using HEp-2 cells as the substrate showed that all sera positive for anti-histone antibodies produced homogeneous nuclear fluorescence. This immunofluorescence pattern disappeared after absorption of anti-histone activity with total histones. An immunoblotting analysis demonstrated that the anti-histone antibodies were predominantly directed against histone H1 in all seven sera with IgG anti-histone antibodies. Weak reactivity with H2B and H4 were also found in three sera from the patients with polymyositis/dermatomyositis. Sera from two patients with polymyositis/dermatomyositis displayed anti-H2A and H3 activity. One of the two sera showed IgM anti-histone antibodies in the enzyme-linked immunosorbent assay reacted with H1, H2A, H2B, H3, and H4, whereas the other serum reacted with no fractions of total histones. The activity of anti-histone antibodies disappeared in immunoblotting after absorption with total histones. All of the patients with anti-histone antibodies were free from lung fibrosis or internal malignancies. Thus, our data indicate that the presence of anti-histone antibodies is classified as one of the serologic abnormalities observed in polymyositis/dermatomyositis.

We describe a patient with Sezary syndrome (SS) who was successfully treated with topical steroid and narrowband UVB. Sezary cells in peripheral blood correlated with severity of skin lesions. In addition, serum levels of CCL17 and CCL27 decreased as disease activity improved. These chemokines may be important for the pathogenesis of SS.

In the period 1994-2002, we saw 71 patients with sclerotic skin changes; 66 were diagnosed with systemic sclerosis (SSc) while five (7%) were diagnosed with pseudoscleroderma associated with various malignancies. The mean duration of disease in these five patients was significantly shorter than that of patients with SSc. The incidence of positive antinuclear antibodies, Raynaud's phenomenon or oesophageal involvement in patients with pseudoscleroderma was significantly lower than that in patients with SSc. The distributions of skin sclerosis varied in each case. Serum basic fibroblast growth factor (bFGF) levels in the five patients with pseudoscleroderma were very elevated compared with levels in controls. Elevated expression of bFGF was detected on fibroblasts of affected skin and in one lung-cancer tissue sample obtained by excision.

In mammals, transforming growth factor-beta (TGF-beta) is found in 3 highly homologous isoforms that exert their effects via heteromeric complexes of type-I and type-II receptors (TbetaR-I and TbetaR-II). TGF-beta regulates the growth and metabolism of various cell types, including keratinocytes. We have investigated the immunohistological localization of TGF-beta1, TGF-beta2, TbetaR-I and TbetaR-II in normal human skin, basal-cell carcinoma (BCC), Bowen's disease, seborrheic keratosis, eccrine poroma and eccrine spiradenoma using frozen tissue specimens. In normal human skin, the immunoreactive TGF-beta2, but not TGF-beta1, was detected predominantly in the epidermis, follicles and sebaceous glands. The epidermal expression of TbetaR-I and TbetaR-II was very weak in the majority of normal skins. In BCC, TGF-beta2 expression was markedly reduced or completely negative. In addition, TbetaR-I- and TbetaR-II-positive stromal cells were accumulated in the fibrotic stroma in some BCCs. These stromal cells were partly but moderately positive for TGF-beta1. Decreased expression of TGF-beta2 was likely to be associated with the differentiation state of BCC cells, since TGF-beta2 expression was clearly observed in the squamoid foci of BCC. In addition, no expression of TGF-beta2 was detected in the eccrine secretory portion or in eccrine spiradenoma, but it was detected in the upper eccrine ducts and in eccrine poroma.

Background Previous studies on pulmonary hypertension (PH) in systemic sclerosis and related disorders used differing patient populations, and defined PH according to different criteria. We have attempted to determine the prevalence and cause of PH in these disorders using a mainly noninvasive cardiopulmonary evaluation. Patients and methods One-hundred thirty-five Japanese patients with systemic sclerosis and related disorders were divided into proximal and distal scleroderma groups, with or without overlapping features of systemic lupus erythematosus (SLE) and polymyositis. They underwent multiple cardiopulmonary tests, including chest radiography, pulmonary function tests, and echocardiography to screen for pulmonary fibrosis and PH. Results The pulmonary artery systolic pressure was estimated by Doppler echocardiography in 80 patients (59%). PH (systolic pressure ≥40 mm Hg) was diagnosed in 28 patients by the Doppler method and in two patients by right heart catheterization (mean pressure ≥20 mm Hg). Doppler-estimated pulmonary artery pressures were significantly higher in patients with proximal scleroderma (p Conclusions These findings suggest that proximal scleroderma predisposes patients to PH mainly because of pulmonary fibrosis, but occasionally because of pulmonary arteriopathy. An overlap of SLE/polymyositis predisposes patients to PH due to the occurrence of arteriopathy.

Background: Basic fibroblast growth factor (bFGF), a cytoplasmic polypeptide growth regulator that induces endothelial cell and fibroblast proliferation, is produced by endothelial cells and skeletal muscle. Objective: We hypothesized that this factor is involved in fibrotic changes in muscle and skin in collagen diseases. Methods: The serum level of bFGF was measured in 74 patients with systemic sclerosis, 12 with systemic lupus erythematosus, 33 with dermatomyositis, 13 with Raynaud's disease, and 20 control subjects. Results: bFGF was undetectable in the serum of normal persons, but detectable levels were found in 31 of 74 patients with systemic sclerosis and 7 of 33 patients with dermatomyositis. Elevated serum bFGF level was correlated with an elevated plasma endothelin level and anticentromere antibody in patients with systemic sclerosis. An elevated serum bFGF level correlated with lung fibrosis and an elevated creatine kinase level in the patients with dermatomyositis. Conclusion: Measurement of the serum bFGF level may be useful to detect vascular damage in patients with systemic sclerosis and muscule fibrosis in patients with dermatomyositis.

Sera from patients with localized scleroderma frequently produce cytoplasmic staining by indirect immunofluorescence, although the antigen remains to be determined. We studied the prevalence, antigen specificity and associated clinical characteristics of anti-cytoplasmic antibodies in localized scleroderma. Serum samples from 60 patients with localized scleroderma were examined by indirect immunofluorescence analysis and immunoblotting. By immunofluorescence analysis on HEp-2 cell substrate, seven of 60 (12%) patients were shown to be positive for anti-cytoplasmic antibodies. Among these, six patients with generalized morphea had anti-mitochondrial antibodies as shown by immunoblotting: they showed reactivity with the E2 component of pyruvate dehydrogenase complex (PDC), with protein X, and with the E2 component of α-oxo-glutarate dehydrogenase complex, while two of them showed reactivity with PDC-E1α. One of these patients who was positive for anti-PDC-E1α antibody showed laboratory abnormalities, suggesting the presence of primary biliary cirrhosis. The age of disease onset was significantly higher in these six patients than in those without anti-mitochondrial antibodies. Furthermore, five of them were classified into generalized morphea with multiple plaque lesions but without linear lesions (multiple plaque type). These observations suggest that major antigens for anti-cytoplasmic antibodies in patients with localized scleroderma are mitochondrial enzymes, 2-oxo-acid dehydrogenase complexes. Patients with anti-mitochondrial antibodies may comprise a unique subset of localized scleroderma designated multiple plaque type of generalized morphea of older onset.

This study investigated the prevalence and clinical significance of anti-ribosomal P protein (anti-P) antibodies in patients with systemic sclerosis (SSc). Serum samples from 150 patients with SSc were examined by indirect immunofluorescence, ELISA and immunoblotting. Anti-P antibodies were detected in four (3%) patients with SSc. Three of the four patients showed SSc/SLE (systemic lupus erythematosus) overlap syndrome, but psychiatric disorders were not observed in these patients. By longitudinal immunoblotting analysis one patient, who was initially diagnosed with SSc, later developed anti-P antibodies along with clinical manifestations of SLE. Our data suggest that anti-P antibodies are uncommon in SSc and that the presence of anti-P antibodies in patients with SSc indicates an overlap with SLE.

We investigated the responses of normal and scleroderma fibroblasts to various growth factors, especially transforming growth factor-beta 1 (TGF-beta 1). The effects of various growth factors on [3H]thymidine incorporation in normal and scleroderma fibroblasts were examined. [125I]-labeled platelet-derived growth factor (PDGF)-BB binding in scleroderma and normal fibroblasts was examined both in the presence and absence of TGF-beta 1 (1 ng/ml). Cytoplasmic protein was isolated and analyzed by Western blotting. Total RNA from fibroblasts was also isolated and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primer sets. Mitogenic responses to TGF-beta 1 (0.33-1 ng/ml) in seven scleroderma fibroblast strains were significantly greater than those in normal controls. [125I]-PDGF-BB binding to scleroderma fibroblasts was increased after TGF-beta 1 stimulation. The increased response to TGF-beta 1 was shown to be mediated through PDGF-like protein induction; TGF-beta 1-treated scleroderma fibroblasts produced greater amounts of 36-kD PDGF-like protein, which was reported previously as connective tissue growth factor (CTGF), than did TGF-beta 1-treated normal fibroblasts. TGF-beta 1 treatment also upregulated PDGF-alpha receptor expression in scleroderma fibroblasts but not in normal dermal fibroblasts. mRNA expression of CTGF and PDGF-alpha receptor was correlated with the above protein expression. These observations suggest that the increased growth response to TGF-beta 1 in scleroderma fibroblasts is mediated through the induction of CTGF and PDGF-alpha receptor.

Objective. To determine the prevalence and clinical significance of anti-pyruvate dehydrogenase complex (anti-PDC) antibodies in systemic sclerosis (SSc). Methods. Serum samples from patients with limited cutaneous SSc (n = 81) or diffuse cutaneous SSc (n = 63) were examined for anti-PDC antibodies by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results. IgG- and/or IgM-isotype anti-PDC antibodies were demonstrated by ELISA in 26 of 144 patients with SSc (18%). By immunoblotting, 19 patients had IgG anti-PDC antibodies. Among these patients with IgG anti-PDC antibodies, antibody to the E1α subunit was significantly associated with the presence of laboratory abnormalities typical of primary biliary cirrhosis (PBC). Conclusion. Antibody to the E1α subunit of PDC may be a serologic indicator for the development of PBC in patients with SSc.

Background: Recently we demonstrated the presence of antihistone antibodies (AHA) in localized scleroderma. Objective: Our purpose was to determine clinical characteristics associated with AHA in patients with localized scleroderma. Methods: We examined 57 serum samples by an enzyme-linked immunosorbent assay in the following three subgroups: 15 patients with generalized morphea, 27 with linear scleroderma, and 15 with morphea, We classified the patients as having generalized morphea when they had four or more lesions on at least two areas of the body, irrespective of whether the lesions were of morphea or linear type. Results: AHA were detected in 42% of patients with localized scleroderma (24 of 57), and in 87% of patients with generalized morphea (13 of 15). The presence of AHA strongly correlated with the number of morphea lesions, the total number of lesions, and the number of involved areas of the body. However, AHA did not correlate with the presence or number of linear lesions. The presence of AHA showed a 87% sensitivity (13 of 15 patients) and a 74% specificity (31 of 42 patients) for generalized morphea. Conclusion: Our data suggest that AHA are a serologic marker for generalized morphea and that the validity of our new classification for generalized morphea is supported by the high frequency of AHA detection.

We describe a 56-year-old woman with Stewart-Treves syndrome who had severe dyspnea from a pleural effusion caused by metastatic angiosarcoma in the right lung. Tumor infiltrating lymphocytes (TIL) in the pleural effusion were cultured and expanded in vitro in the continuous presence of recombinant interleukin 2 with periodic stimulation by CD3 antibody. The expanded TIL were administered intrapleurally seven times at I- to 4-week intervals in combination with intravenous infusion of recombinant interleukin 2. A panel of T-cell clones was also obtained from TIL. Immunotherapy dramatically improved the patient's dyspnea and pleural effusion. A CD4 + T-cell clone and a CD8 + T-cell clone established from TIL had specific cytotoxicity to the tumor cells.

Objective. To determine the prevalence and clinical significance of antihistone antibodies (AHA) in systemic sclerosis (SSc). Methods. Serum samples from patients with limited cutaneous SSc (n = 44), diffuse cutaneous SSc (dcSSc; n = 48), and other SSc-related disorders (n = 22) were examined by enzyme-linked immunosorbent assay and immunoblotting for AHA. Results. AHA were demonstrated in 29% of the 92 SSc patients and in 44% of those with dcSSc. The presence of AHA correlated with severe pulmonary fibrosis in those with dcSSc. Immunoblotting revealed that the predominant antigen was histone H1. Conclusion. AHA might be a serologic indicator of the severity of pulmonary fibrosis in SSc.

Effects of transforming growth factor beta (TGF-beta) on proliferative responses to basic fibroblast growth factor (bFGF) were studied in human diploid fibroblasts cell strains derived from three different sources: adult skin, scleroderma, and newborn foreskin. All three types of cell strains were similarly responsive to TGF-beta, whereas adult skin fibroblasts were significantly more responsive to bFGF. Incubation of cells with TGF-beta prior to bFGF addition substantially increased responsiveness of adult skin fibroblasts to this latter cytokine, slightly increased that of scleroderma fibroblasts, and decreased that of foreskin fibroblasts. Modulation of bFGF receptors by TGF-beta correlated positively with these mitogenic effects. Adult skin fibroblasts showed increases of both high- and low-affinity receptors and scleroderma fibroblasts showed small increases of high-affinity receptors only, whereas foreskin fibroblasts showed no changes. Heparitinase treatment of adult skin fibroblasts during TGF-beta pre-incubation resulted in reduced bFGF binding to low-affinity receptors and reduced mitogenic response to bFGF, suggesting that the TGF-beta-stimulated increase of low-affinity receptors in these cells contributes to the observed enhanced mitogenic effects of bFGF. Abnormal responses of scleroderma fibroblasts to TGF-beta/bFGF stimulation, particularly failure to synthesize low-affinity receptors in response to TGF-beta, adds a new characteristic to the fibrotic phenotype of scleroderma fibroblasts.

Extramammary Paget's disease (EMPD) is regarded as a malignancy most likely derived from or differentiated into apocrine sweat duct and gland. The tumor cells in EMPD usually have abundant pale cytoplasm and large pleomor- phic nuclei. We present a case with EMPD where the tumor included many signet ring cells, cells with intracytoplasmic lumen formation and glandular formation. This case is interesting in that the tumor included many cells with intracytoplasmic lumen formation. Signet ring cells and glandular formation support the traditional speculation that EMPD is derived from apocrine sweat gland and intracytoplasmic lumen formation may mimic the intrafollicular apocrine duct.

29歳男子。10歳頃より日光裸露部に小色素斑が多発。11歳時, 右鼻翼に基底細胞上皮腫, 25歳時, 左角膜に扁平上皮癌を生じた。基底細胞上皮腫は局所再発傾向が強く, 難治であつた。神経症状は認めなかつた。患者由来培養皮膚線維芽細胞は10J/m2の紫外線誘発不定期DNA合成(unscheduled DNA synthesis, 以下UDS)が正常人の45%と低下しており, 相補性テストでC群色素性乾皮症(xeroderma pigmentosum, 以下XP)と診断した。同培養細胞は紫外線の致死効果に対し, 高い感受性を示したが, 他のC群患者由来の細胞と比較すると, やや感受性が低かつた。

Necrolytic migratory erythema (NME) is an uncommon inflammatory dermatosis with a distinctive clinical and histological appearance. It shows irregular erythema, bullae, erosion, crusts and pigmentation. While it is typically associated with glucagonoma, some cases of NME without glucagonoma have been reported. Herein, we report a case of necrolytic migratory erythema associated with malabsorption 30 years after ileocolectomy. She presented erosive erythema with scale or partly flaccid bullae on her intergluteal cleft, buttock and extremities. Her laboratory data revealed essential amino acid deficiency and a slightly decreased serum zinc level, while her plasma glucagon level was low. With diagnosis of non-glucagonoma-associated NME with malabsorption due to short-bowel syndrome, she was treated and improved by i.v. amino acid supplement. Histological findings of NME include necrotic changes of keratinocytes in the upper epidermis, proliferation of those in the lower epidermis and inflammatory cell infiltration of upper dermis. We also examined the expression pattern of epidermal keratins (K6, K10) and Ki-67, one of the markers of proliferative activity, to assess the proliferation and differentiation of keratinocytes in a NME lesion by immunostaining. The findings with these immunostainings support the characteristics of HE-staining, and suggest hyponutrition may induce changing differentiation/proliferation of keratinocytes.

Aberrant methylation of promoter CpG islands (CGI) is involved in silencing of tumor suppressor genes and is also a potential cancer biomarker. Here, to identify CGIs aberrantly methylated in human melanomas, we did a genome-wide search using methylation-sensitive representational difference analysis. CGIs in putative promoter regions of 34 genes (ABHD9, BARHL1, CLIC5, CNNM1, COL2A1, CPT1C, DDIT4L, DERL3, DHRS3, DPYS, EFEMP2, FAM62C, FAM78A, FLJ33790, GBX2, GPR10, GPRASP1, HOXA9, HOXD11, HOXD12, HOXD13, p14ARF, PAX6, PRDX2, PTPRG, RASD1, RAX, REC8L1, SLC27A3, TGFB2, TLX2, TMEM22, TMEM30B, and UNC5C) were found to be methylated in at least 1 of 13 melanoma cell lines but not in two cultured normal melanocytes. Among these genes, Peroxiredoxin 2 (PRDX2) was expressed in normal melanocytes, and its expression was lost in melanomas with methylation. The loss of expression was restored by treatment of melanomas with a demethylating agent 5-aza-2′-deoxycytidine. In surgical melanoma specimens, methylation of PRDX2 was detected in 3 of 36 (8%). Furthermore, immunohistochemical analysis of PRDX2 showed that disappearance of immunoreactivity tends to associate with its methylation. PRDX2 was recently reported to be a negative regulator of platelet-derived growth factor signaling, and its silencing was suggested to be involved in melanomas. On the other hand, 12 CGIs were methylated in ≥9 of the 13 melanoma cell lines and are considered as candidate melanoma biomarkers. (Cancer Res 2006; 66(12): 6080-6)

Over 125 pigmentation-related genes have been identified to date. Of those, PMEL17/GP100 has been widely studied as a melanoma-specific antigen as well as a protein required for the formation of fibrils in melanosomes. PMEL17 is synthesized, glycosylated, processed, and delivered to melanosomes, allowing them to mature from amorphous round vesicles to elongated fibrillar structures. In contrast to other melanosomal proteins such as TYR and TYRP1, the processing and sorting of PMEL17 is highly complex. Monoclonal antibody HMB45 is commonly used for melanoma detection, but has the added advantage that it specifically reacts with sialylated PMEL17 in the fibrillar matrix in melanosomes. In this study, we generated mutant forms of PMEL17 to clarify the subdomain of PMEL17 required for formation of the fibrillar matrix, a process critical to pigmentation. The internal proline/serine/threonine-rich repeat domain (called the RPT domain) of PMEL17 undergoes variable proteolytic cleavage. Deletion of the RPT domain abolished its recognition by HMB45 and its capacity to form fibrils. Truncation of the C-terminal domain did not significantly affect the processing or trafficking of PMEL17, but, in contrast, deletion of the N-terminal domain abrogated both. We conclude that the RPT domain is essential for its function in generating the fibrillar matrix of melanosomes and that the luminal domain is necessary for its correct processing and trafficking to those organelles.