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A major advance of drug discovery and targeted therapy directed to cancer cells may be achieved by exploitation and immunomodulation of their unique biological property. This re-view summarizes our efforts to develop novel chemo-thermo-immuno-therapy (CTI therapy) by conjugating a melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP: amine analog of tyrosine), with magnetite nanoparticles (MNP). In our approach, NPrCAP provides a unique drug delivery system (DDS) because of its selective incorporation into melanoma cells. It also functions as a melanoma-targeted therapeutic drug because of its production of highly reactive free radicals (melanoma-targeted chemotherapy). Moreover, utilization of MNP is a platform to develop thermo-immunotherapy because of heat shock protein (HSP) generation upon exposure to an alternating magnetic field (AMF). The feasibility of our approach was successfully shown in experimental in vivo and in vitro mouse melanoma models and in preliminary clinical trials to a limited number of advanced melanoma patients.

Melanoma is highly metastatic, but the mechanism of melanoma cell migration is still unclear. We found that melanoma cells expressed the nicotinamide adenine dinucleotide–dependent protein deacetylase SIRT1 in the cytoplasm. Cell membrane extension and migration of melanoma cells were inhibited by SIRT1 inhibitors or SIRT1 knockdown, whereas SIRT1 activators enhanced elongation of protrusion and cellular motility. In B16F1 cells, growth factor stimulation induced lamellipodium extension, a characteristic feature at the leading edge of migrating cells, and SIRT1 was found in the lamellipodium. SIRT1 inhibitor nicotinamide (NAM) or SIRT1 small interfering RNAs suppressed the lamellipodium extension by serum or platelet-derived growth factor (PDGF). The lamellipodium formation by dominant-active Rac1 was also inhibited by NAM, a SIRT1 inhibitor. NAM inhibited the accumulation of phosphorylated Akt at the submembrane by serum or PDGF. Using fluorescence resonance energy transfer, we found that NAM impaired PDGF-dependent increase in the phosphatidylinositol-3,4,5-trisphosphate level at the leading edge. NAM inhibited the abdominal metastasis of transplanted B16F1 melanoma cells in C57BL6/J mice and improved survival. Finally, SIRT1-knockdown B16F1 cells showed significantly reduced metastasis in transplanted mice compared with that in control B16F1 cells. These results indicate that SIRT1 inhibition is a strategy to suppress metastasis of melanoma cells.

Induced pluripotent stem (iPS) cells have the ability to differentiate into multiple cell types in the body and have an unlimited growth potential. However, iPS cell-derived melanocytes produced by existing protocols have significant limitations in developing novel strategies for regenerative medicine and cell therapies of pigmentation disorders in humans because they involve culture in media containing fetal bovine serum and nonphysiological agents. In this study, we established an in vitro approach to generate iPS cell-derived human melanocytes that have higher proliferation rates and increased melanin production compared with melanocytes prepared by previously reported approaches. Importantly, our iPS cell-derived human melanocytes are prepared in fetal bovine serum-free culture conditions that do not contain any nonphysiological agents. We designed two original methods, transferring black colonies by pipette and recovering black cell pellets from centrifuged medium, and numerous human iPS cell-derived melanocytes proliferated in gelatinous dishes coated with Matrigel after 12 days. We also succeeded in inducing melanin pigmentation in the nude mouse skin in vivo using those human iPS cell-derived melanocytes. We propose that this method using iPS cells established from T cells in the blood of normal human volunteers could be applied clinically to develop regenerative medicine and cell therapies for various forms of human pigmentation disorders.

Exploitation of biological properties unique to cancer cells may provide a novel approach to overcome difficult challenges to the treatment of advanced melanoma. In order to develop melanoma-targeted chemothermoimmunotherapy, a melanogenesis substrate, N-propionyl-4-S-cysteaminylphenol (NPrCAP), sulfur-amine analogue of tyrosine, was conjugated with magnetite nanoparticles. NPrCAP was exploited from melanogenesis substrates, which are expected to be selectively incorporated into melanoma cells and produce highly reactive free radicals through reacting with tyrosinase, resulting in chemotherapeutic and immunotherapeutic effects by oxidative stress and apoptotic cell death. Magnetite nanoparticles were conjugated with NPrCAP to introduce thermotherapeutic and immunotherapeutic effects through nonapoptotic cell death and generation of heat shock protein (HSP) upon exposure to alternating magnetic field (AMF). During these therapeutic processes, NPrCAP was also expected to provide melanoma-targeted drug delivery system.

Background N -propionyl-4- S -cysteaminylphenol (NPr-4- S -CAP) is selectively incorporated into melanoma cells and degrades them. However, it remains unclear whether NPr-4- S -CAP can induce cell death associated with the induction of host immune responses and tumor suppression in vivo. Objective To examine the molecular mechanism of NPr-4- S -CAP-mediated cytotoxicity toward melanoma cells and to test whether NPr-4- S -CAP can suppress transplanted primary and secondary B16F1 melanomas. Methods Cytotoxicity and apoptosis of melanoma cells were assessed by cell counting, flow cytometry, and detection of reactive oxygen species (ROS) and apoptotic molecules. NPr-4- S -CAP-associated host immunity was studied using a B16F1 mouse melanoma model through the application of CD4- and CD8-specific antibodies and tetramer assay. Results NPr-4- S -CAP suppressed growth of pigmented melanoma cells associated with an increase of intracellular ROS, activation of caspase 3 and DNA fragmentation, suggesting that NPr-4- S -CAP mediated ROS production, eliciting apoptosis of melanoma cells. Growth of transplanted B16F1 melanomas was inhibited after the consecutive intratumoral injections of NPr-4- S -CAP, and the tumor growth after rechallenge of B16F1 was significantly suppressed in the treated mice. This suppression occurred when the treated mice were given the anti-CD4 antibody, but not the anti-CD8 antibody. Tetramer assay demonstrated increased TYRP-2-specific CD8 + T cells in the lymph node and spleen cells prepared from NPr-4- S -CAP-treated B16F1-bearing mice. Conclusions These suggest that NPr-4- S -CAP induces apoptosis in melanoma cells through ROS production and generates CD8 + cell immunity resulting in the suppression of rechallenged B16F1 melanoma.

How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase-related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild-type (Rab7WT), constitutively active (Rab7Q67L) or dominant-negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio-distribution of melanosomal proteins between Rab7WT-expressing cells and mutant Rab7-expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.

A 21-year-old woman with recurrent skin lesions present since December 1992 was seen at the University of Alberta hospitals in 1994. The skin lesions consisted of pruritic, painful, erythematous, “beefy” plaques on the trunk and extremities and also of vesicles, bullae, and pustules on the ankles and hands. She experienced numerous flare-ups of her eruption that required hospital admission on five separate occasions. Each episode was associated with sweats, chills, and dizziness. Headaches, arthralgias, myalgias, and generalized fatigue occurred on occasion. Previous skin biopsies suggested Wells' syndrome. Detailed investigations did not reveal any underlying etiology. The patient was a single woman who had recently moved from New Brunswick to live with her aunt and two cousins in Edmonton and to find employment. She had previously been a healthy young woman until her episodes first began in December 1992. There was a personal history of childhood atopic dermatitis, but she denied any other manifestations of atopy. Her mother had recently passed away at the age of 37 from Hodgkin's lymphoma, otherwise the family history was unremarkable. At the time of this admission she was not taking any medications; however, during the previous months in New Brunswick she had been using numerous medications including prednisone, dapsone, colchicine, and an oral contraceptive for a functional ovarian cyst. Prednisone, up to 60 mg/day, had initially appeared beneficial for her skin lesions, but later her symptoms became resistant. In addition, she suffered from many side-effects induced by chronic corticosteroid usage, inciuding biurred vision, weight gain, striae, and irreguiar menstruai periods. She had not been on corticosteroids for the past 6 weeks, and none of the other medications were of benefit. In fact, she had suffered a severe idiosyncratic reaction to dapsone consisting of high fever, abdominai pain, and abnormal liver parameters. There was no significant travel history and she denied any drug abuse or HIV risk factors, the patient was an animal lover and often played for hours at a time with her aunt's dog and two cats. By coincidence, in New Brunswick she had also owned a dog and two cats. A review of systems was unremarkable. On physical examination, she was a moderately obese young woman who appeared cushingoid, but otherwise well. She was afebrile. Examination of the skin revealed multiple vesicles, bullae, and pustules on her hands and fingers, involving both dorsal and palmar surfaces and extending to her wrists (Fig. 1). Some of the lesions had broken open forming overlying crusts. A few bullae had become hemorrhagic. There was an obvious foul-smelling odor emanating from the lesions. Multiple small excoriations were present bilaterally on the shins and two erythematous, edematous papules were seen on the abdomen. A live flea was found in her suprapubic area. No other fleas were found. The rest of the general examination was unremarkable. Pertinent laboratory investigations revealed an absolute eosinophilia of 2700/mm3 (24% eosinophilia) with a white blood count (WBC) of 11,400/mm”. Swabs of hand lesions were culture-positive for multiple aerobes and anaerobes: 3+ growth of Staphylococcus aureus, 3+ growth of Haemophilus parainfluenza, 1 + growth of Coxiella oxytoca, 2+ growth of Streptococcus viridans, and 4-i- growth of mixed anaerobes. Virologic tests were negative. Examination of a skin biopsy specimen (Fig. 2) revealed multiple flame figures in the dermis with surrounding and interspersed eosinophils. Numerous eosinophils, / lymphocytes, and histiocytes were also present around small blood vessels. There were degenerative changes of the upper epidermis with spongiosis, suggestive of early intraepidermal vesicle formation. Direct immunofluorescence on skin specimens was negative. Skin prick testing with flea antigen was positive for immediate hypersensitivity, but negative for a delayed reaction after 48 h. On further questioning, the patient admitted that she had played with a cat just prior to her admission, which, unbeknownst to her at the time, was flea-infested. Her aunt strongly denied any fleas in the home environment, despite her pets. No other household members complained (5f insect bites or skin symptoms. The patient's skin improved remarkably with hospitalization and treatment with antibiotics; no new lesions developed while in hospital and she was discharged 12 days later. She continued to experience recurrent, sporadic eruptions that resolved without corticosteroids, but was later lost to follow-up when she returned home to New Brunswick.

To clarify the mechanisms underlying declines in wound contraction caused by basic fibroblast growth factor (bFGF) and the role of autologous fibroblasts in modulating wound healing, we have examined the expression of alpha-smooth muscle actin (alpha-SMA) and apoptosis in a model of wound healing using collagen sponges with and without bFGF (1 microg) and/or fibroblasts (1 x 10(6) cells/cm(2)) applied to experimentally produced full-thickness skin wounds in rats (n=10 for each group). At 7 days postoperatively, wounds filled with a fibroblast-seeded collagen sponge (fibroblast-seeded group) displayed a greater area of collagen sponge and a smaller area of fibroblasts compared with control wounds filled with collagen sponge alone (control group). Therefore, seeding of fibroblasts in the dermal substitute might retard degradation of the collagen sponge, inhibiting fibroblast infiltration into the substitute. By day 14, wounds filled with bFGF-treated collagen sponge without fibroblast seeding (bFGF group) displayed decreased alpha-SMA expression and significantly increased apoptosis compared with other wounds. Double staining revealed that apoptosis in alpha-SMA-positive fibroblastic cells was significantly increased in the bFGF group, suggesting that bFGF treatment is a potent stimulator of myofibroblast apoptosis. Furthermore, morphometric analysis demonstrated the significant decrease in the level of wound contraction and the degree of mature collagen bundle formation in the bFGF group by day 42. The bFGF group also showed increased bFGF expression in macrophages by day 28. These results suggest that bFGF administration to an artificial dermis promotes apoptosis of alpha-SMA-positive fibroblastic cells and inhibits alpha-SMA expression in the treated wound, thus reducing wound contraction.

Polymerase chain reaction(PCR)を用いると帯状疱疹患者の末梢血リンパ球からも水痘帯状疱疹ウイルス(varicella-zoster virus: VZV)核酸が検出される。本研究では，札幌医大附属病院皮膚科に入院しビダラビン(アラセナ®A)600mg/日を5日間投与された帯状疱疹患者10例よりリンパ球を調製し，PCRによって末梢血リンパ球のVZV gene 29およびgene 62のDNAおよびRNAの検出を行った。検討した10例のうち基礎疾患のある重症帯状疱疹の3例と中等症の1例は補体結合反応値が128倍と高値を示し，さらにPCRによりVZV DNAおよびRNAが検出された。しかし，これら4症例とも入院7～10日後(第14病日)には末梢血リンパ球からウイルス核酸は検出されなかった。他の中等症の1例と軽症5例の末梢血リンパ球からはVZV gene 29あるいは62のDNAとRNAは検出されなかった。患部皮疹が潰瘍化した重症の1例を除き，いずれの症例も3週間以内に上皮化し2～4週間後の帯状疱疹関連神経痛はVASスコアで0/5あるいは1/5まで低下した。これらの結果より，重症例ではウイルス抗体価が上昇し末梢血リンパ球にVZV遺伝子が検出され少なくとも一部のウイルス遺伝子が発現しているが，ビダラビン600mg/日，5日間の投与によって発症2週間後には末梢血よりウイルスが駆逐されると考えられた。

Psoralen plus ultraviolet A (PUVA) photochemotherapy is widely used for the therapy of mycosis fungoides (MF). Clinical progression of MF is often associated with an increase in the size of tumour cells known as transformation. We report two patients with CD30+ large cell transformation that appeared after low-dose PUVA therapy for MF. Clinical data, histopathology, immunohistopathology and T-cell receptor gene rearrangement were studied. Nodules consisted of atypical large cells that expressed CD30. Monoclonal rearrangement of T-cell receptors was observed in one case. Low-dose PUVA therapy may be associated with CD30+ large cell transformation in patients with MF.

Patients with familial atypical multiple mole and melanoma (FAMMM) [so-called familial dysplastic naevus syndrome (FDNS)] have a high risk for the development of malignant melanoma. The underlying gene defect has an autosomal dominant inheritance with variable expression and incomplete penetrance. Fibroblasts derived from FAMMM patients have high sensitivity to UVC and mutagens, e.g. 4-nitroquinoline-1-oxide. We were interested in identifying how the combination of inherent sensitivity to UV light and abnormal melanin synthesis interacts in the development of melanoma in FAMMM patients. Intermediates of melanin synthesis produce free radicals that are toxic to cells. Atypical moles (dysplastic naevi) are engaged in the biosynthesis of abnormal melanin pigments. This study examined whether there was any abnormal melanin pigmentation or cell damage after the ectopic expression of tyrosinase in fibroblasts from FAMMM patients when compared with fibroblasts from normal subjects. Fibroblasts from FAMMM patients (3012T and 3072T) were associated with a higher sensitivity than normal human fibroblasts to the toxicity of UVB. When cells were infected with tyrosinase-expressing adenovirus (Ad-HT) and irradiated with UVB, FAMMM fibroblasts showed higher tyrosinase activity, produced more melanin pigments and were degraded more significantly than normal human fibroblasts. Western blot analysis revealed that Ad-HT-infected 3072T produced a larger amount of tyrosinase protein than did Ad-HT-infected normal fibroblasts after UVB irradiation. Our findings suggest: (1) that FAMMM fibroblasts have an unknown machinery which enhances tyrosinase expression by UVB irradiation; and (2) that the resulting increase in melanin synthesis affects the cytotoxicity of UVB to FAMMM fibroblasts. All of these processes may be involved in the genomic instability and development of melanoma in FAMMM patients.

Background: malignant melanoma has one of the highest rates of metastasis. Unlike other solid cancers, no sensitive tumor markers or laboratory tests that can provide information of the risk of metastasis and predict the prognosis have yet been established. Objective: the study was done to establish a RT-PCR sensitive and specific enough to detect melanocyte-specific transcripts from peripheral blood cells. Methods: peripheral white blood cells were collected from 30 healthy donors and 43 melanoma patients. Melanocyte-specific tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) were selected as targets of RT-PCR. The sensitivity of detection using SK-mel-23 melanoma cells and rates of false-positiveness using non-melanoma blood were compared between single-step PCR and nested PCR. Analysis of melanoma blood samples was carried out by the single-step PCR. Results: the nested RT-PCR amplified the TYR and TYRP1 sequences from 14 and 2 of the 30 healthy bloods, respectively. However, the single-step RT-PCR did not amplify TYR or TYRP1 sequences from any of the healthy controls. Our single-step RT-PCR detected 1.7 and 0.8 SK-mel-23 melanoma cells per ml blood for TYR and TYRP1, respectively. Overall, TYR mRNA was detected in 15 of the 43 melanoma patients (34.9%), and TYRP1 mRNA in 16 of the 43 (37.2%). The specificities of detection of TYR and TYRP1 were 83.3 and 88.9%, respectively. Conclusion: our single-step RT-PCR for TYR and TYRP1 mRNAs is more specific than the previous nested RT-PCR for TYR and applicable to detect circulating melanoma cells.

A multicenter randomized controlled study was conducted to assess the long-term efficacy and safety of cyclosporin A therapy for psoriasis using either a continuous or an intermittent regimen. Initially, both regimens consisted of 3-5 mg/kg/day administration of CyA. Once remission was obtained, CyA dose was maintained between 0.5 and 3 mg/kg/day under the continuous regimen, while under the intermittent regimen, CyA dose was tapered off and, when necessary, topical corticosteroids were used until relapse occurred. Thirty-one patients were followed for at least 48 months (mean follow-up period: 55.9+/-4.6 months): 15 received continuous therapy, and 16 received intermittent therapy. With both regimens, the PASI (Psoriasis Area and Severity Index) score was maintained at 5-12 points throughout the follow-up period. The score was decreased by more than 70% from baseline with both regimens: the responses between them were not significantly different. However, overall control of psoriasis, as assessed from the averaged PASI score, was better in the patients receiving continuous therapy. Although the overall frequency of adverse reactions was similar for the two regimens, cancer occurred in two patients on continuous therapy (gastric cancer and hepatocellular carcinoma in one patient each). We could not, however, definitely attribute the cancers in the two patients to continuous therapy itself. There was a significantly higher incidence of renal impairment in elderly patients receiving either regimen when compared with younger patients. In conclusion, CyA administered to psoriasis patients under both regimens exhibited long-term efficacy and tolerability. Despite a lower overall efficacy, it seems proper to conclude that intermittent therapy is more useful than continuous therapy due to the occurrence of malignancies with continuous therapy. Further investigation is required to determine whether intermittent therapy is really safer than continuous therapy, and, if so, how it should be designed to minimize long-term adverse reactions and achieve overall control comparable to that of continuous CyA therapy.

We have recently identified the association of Rab7 in melanosome biogenesis and proposed that Rab7 is involved in the transport of tyrosinase-related protein 1 from the trans-Golgi network to melanosomes, possibly passing through late-endosome-delineated compartments. In order to further investigate the requirement of Rab7-containing compartments for vesicular transport of tyrosinase family proteins, we expressed tyrosinase and tyrosinase-related protein by recombinant adenovirus and analyzed their localization in human amelanotic melanoma cells (SK-mel-24) in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infection of the recombinant adenoviruses carrying tyrosinase (Ad-HT) and TRP-1 (Ad-TRP-1) resulted in the enhancement of tyrosinase activity and melanin production compared to a single infection of Ad-HT. In the Ad-HT-infected SK-mel-24 cells many of the newly synthesized tyrosinase proteins were colocalized in lysosomal lgp85-positive granules of the entire cytoplasm, whereas in the presence of Rab7N125I the colocalization of tyrosinase and lgp85 proteins was decreased markedly in the distal area of the cytoplasm. In the Ad-TRP-1-infected SK-mel-24 cells, TRP-1, which is reported to be present exclusively in melanosomes, was detected throughout the cytoplasm, but not colocalized in prelysosomal (early endosomal) EEA-1 granules. In the presence of Rab7N125I, however, TRP-1 was retained in the EEA-1-positive granules. Our findings indicate that the dominant-negative mutant of Rab7 impairs vesicular transport of tyrosinase and TRP-1, suggesting that the transport of these melanogenic proteins from the trans-Golgi network to maturing melanosomes requires passage through endosome-delineated compartments.

Melanoma cells rarely contain mutant p53 and hardly undergo apoptosis by wild-type p53. By using recombinant adenoviruses that express p53 or p53-related p51A or p73beta, we tested their apoptotic activities in melanoma cells. Yeast functional assay revealed a mutation of p53 at the 258th codon (AAA [K] instead of GAA [E]) in one cell line, 70W, out of six human melanoma cell lines analyzed (SK-mel-23, SK-mel-24, SK-mel-118, TXM18, 70W, and G361). Adenovirus-mediated transfer of p53, p51A, and/or p73beta suppressed growth and induced apoptotic DNA fragmentation of SK-mel-23, SK-mel-118, and 70W cells. Interestingly, p51A induced DNA fragmentation in them more significantly than p53 and p73beta. By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family members. Apoptotic Bax and antiapoptotic Bcl-2 were not significantly upregulated or downregulated by expression of p53, p51A, or p73beta, except for p53-expressing 70W cells, which contained a larger amount of Bax protein than LacZ-expressing cells. Activation of caspase-3 was demonstrated only in p51A-expressing SK-mel-118 cells. We show here that p51A can mediate apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and p73beta. It is also suggested that in melanoma cells (i) cellular target protein(s) other than Bcl-2 and Bax might be responsible for induction of p51A-mediated apoptosis and (ii) caspase-3 is not always involved in the apoptosis by p53 family members.

Cytokines, in particular IL-4 and IL-5, regulate IgE synthesis and eosinophil activation in atopic dermatitis (AD). To elucidate whether the serum levels of IL-4 and IL-5 are related to the serum IgE level, eosinophilia, or clinical severity of the disease, 25 cases with AD were studied. Blood samples were isolated from two groups of donors: 1) patients with AD (n = 25); 2) non-allergic individuals (NA, n = 20) with serum IgE levels below 100 IU/ml and with blood eosinophil counts below 250/microliter. Each parameter was evaluated at least twice in AD patients at the beginning of the study and after 4, 8 or 12 weeks of treatment. IL-4 was hardly detected in AD and NA, but IL-5 was increased (> 10 pg/ml) in most cases (22/25) of AD group with 513.6 pg/ml as the mean. AD with normal serum IgE levels exhibited increased levels of IL-5, whereas AD with high serum IgE levels did not necessarily have elevated IL-5 levels. The IL-5 level tended to change in parallel with the clinical severity in each AD case, although the level itself was not correlated with the clinical severity per se. A significant decrease of IL-5 was observed in AD when the clinical severity decreased. Eosinophils also decreased along with the improvement of AD, whereas the serum level of IgE did not change during the observation period. Our results suggest that IL-5 is involved in the regulation of clinical courses of AD and that its kinetics at the serum level reflects the clinical activity of AD.

21歳,男性。中背部右側に自覚症状を欠く11×7mmのドーム状に隆起する黒色結節を認め,単純切除術を施行。病理組織学的に結節型悪性黒色腫,tumor thickness 5.0mm, level 4であった。過去10年間に当院で経験した悪性黒色腫113例の統計では,結節型黒色腫(以下NM)は若年層男性の体幹に,表在拡大型(以下SSM)は若年層女性の下腿に発症率が高かった。又,足底,体幹の非露出部では他部位と比較して男性の発症が,一方顔面,四肢の露出部では女性の発症が多かった。

疼痛·そう痒を伴う各種皮膚疾患患者34名(男性20名,女性14名,平均年齢53.9歳)を対象に0.025%カプサイシン軟膏を外用し,その有用性を検討した。各疾患ごとの有効率は,帯状疱疹後神経痛(PHN)10例:67%,尋常性乾癬10例:67%,結節性痒疹2例:59%,慢性光線過敏性皮膚炎(CAD)3例:96%,アトピー性皮膚炎3例:41%,肥厚性瘢痕のそう痒7例:66%,肥厚性瘢痕の疼痛3例:75%であった。全症例の平均有効率は68%であった。カプサイシン軟膏外用後の刺激感·灼熱感は全症例でみられたが,症状の強い症例に対しては5%テーカイン軟膏を事前塗布することで対処した。その他の副作用として外用局所の紅斑:15%,流涙:3%を認めたが,他に問題となる副作用はみられなかった。以上より,カプサイシン軟膏は帯状疱疹後神経痛に有用であることが再確認されたとともに,慢性疼痛·そう痒を伴う各種皮膚疾患に対する局所療法としても有効であると考えられた。

Background: Sun-induced malignancies (basal cell and squamous cell carcinomas) are common in oculocutaneous albinism, however, the incidence of malignant melanoma is a topic of controversy. Objective: We have reviewed the literature and report a case of a woman with oculocutaneous albinism with an amelanotic melanoma of the anterior chest wall. Results: There are only 26 previously reported cases (both case reports and African albino population studies) in 25 patients in the literature. A 27th case with immunohistochemical and ultrastructural evaluation is presented. Conclusions: It appears that melanoma, a malignancy for which sun exposure and light colouration are felt to be major risk factors, has a low incidence among a population that is both hypopigmented and often exposed to high levels of ultraviolet light. This low incidence is poorly understood and frequently disputed.

Sulfur-containing tyrosine analogs such as 4-S-cysteami- nylphenol (4-S-CAP) and its N-acetyl derivative, N-acetyl-4-S-CAP, are tyrosinase substrates and can cause selective cytotoxicity or cell death of melanocytes and melanoma cells. It is not clear, however, if the cytotoxicity derives from a cytostatic or cytocidal effect. The latter can also be either apoptotic or necrotic. This paper summarizes our attempt to clarify the nature of melanocytotoxicity and cell death by using a new derivative of 4-S-CAP, N-propionyl-4-S-CAP (NPr-CAP). The i.p. administration of NPr-CAP caused marked depigmentation of black hair follicles in C57 mice. At 12h postadministration of NPr-CAP, follicular melanocytes showed histochemical and morphologic features indicative of apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining and electron microscopy. The agarose gel electrophoresis of DNA from drug-treated melan a2 cells, an immortal melanocyte line of C57 black mice, showed the nucleosomal DNA ladder pattern. NPr-CAP caused irreversible cytotoxicity in melan a2 and the effect was inhibited by a tyrosinase inhibitor, phenylthiocarbamide. The tyrosinase-mediated cytotoxicity of NPr-CAP was further confirmed by the decreased viability of COS 7 monkey-kidney cells, which expressed a level of high tyrosinase activity through transfection of human tyrosinase cDNA. NPr-CAP, however, also transiently inhibited the proliferation of melan c cells, a control tyrosinase-negative albino melanocyte line, and vector-transfected COS 7 cells. Thus, the major process of NPr-CAP-mediated melanocytotoxicity involves cytocidal apoptosis associated with active tyrosinase. In addition, there is transient, nontyrosinase-mediated cytostatic cytotoxicity.

This study characterizes the induction of melanogenesis and the expression of tyrosinase, tyrosinase-related protein (TRP) and lysosome-associated membrane protein (LAMP) gene families in the cultured melanocyte lines of non-agouti mice with four major genetic loci, i.e. melan-a2 (black, wild type), melan-b (brown, TRP-1 mutation), melan-s (black, piebaldism mutation) and melan-c (white, tyrosinase mutation) in response to repeated exposure to ultraviolet (UV) B (5 mJ/cm2, 7 consecutive days). Electron microscopy showed that new melanogenesis was induced in melan-a2, melan-s and melan-b melanocytes. Melan-a2, melan-s and melan-b showed an almost twofold increase in tyrosinase activity and gene expression with increased synthesis of melanosomes, although melan-b showed a minimum increase in tyrosinase activity. There was a twofold upregulation of LAMP-1 mRNA but no alteration in LAMP-2 and LAMP-3 mRNA expression in melan-a2, while there was no alteration in LAMP-1 mRNA expression but increased expression of LAMP-2 and LAMP-3 mRNA in melan-s, LAMP-3 showing a higher increase. Melan-b cells showed the same gene expression of LAMP-1, LAMP-2 and LAMP-3 as that of non-UV exposed cells. All three lines, however, exhibited simultaneously cell death, melan-b reaching the highest rate of cell death (96.5%). In contrast, melan-c, which did not have any tyrosinase activity with failure of melanogenesis induction, expressed all the mRNAs of the tyrosinase and LAMP gene families, but was not associated with any significant melanocyte death. Our study indicated: (i) that melanogenesis induction and melanocyte death are two photobiological processes occurring simultaneously after repeated UVB exposure, (ii) that in response to an upregulation of tyrosinase mRNA and enzymic activity, there was a co-ordinated upregulation of the LAMP-1 gene in wild type melan-a2, while no upregulation was found in melan-s and melan-b mutants, and (iii) that UV-induced melanocyte death is related to the upregulation of the tyrosinase gene, induction of new melanogenesis and mutation of the TRP-1 gene in immortal murine melanocytes.

Vitiligo is a common idiopathic acquired or inherited disease with loss of normal melanin pigments and functioning melanocytes from otherwise healthy looking skin. Clinically, vitiligo can be grouped into several unique types (Figs. 1, 2). The localized type includes focal and segmental vitiligo. Generalized types are vitiligo vulgaris, acrofacial vitiligo, and universal vitiligo. A genetic predisposition is considered. 13,46 The leukoderma of vitiligo does not contain any functioning melanocytes. 35 Pathogenesis of vitiligo leukoderma is still unknown, but currently three major theories have been proposed. 11 One is the neural theory, that presupposes that there is a chemical mediator liberated at the nerve endings that destroys the melanocytes or inhibits the production of melanin pigments. A second theory proposes that melanocytes are destroyed either by themselves through generation of melanin precursors (or metabolites) or by surrounding keratinocytes that liberate chemicals that cause oxidative stresses. In normal melanocytes there is some defense or scavenging mechanism against these cytotoxic melanin precursors or chemicals generating oxidative stresses, but there is a defect in this defense process (mechanism) in vitiligo melanocytes (Fig. 3). 26 The third theory is the autoimmune hypothesis, which speculates that vitiligo is an autoimmune disease. Vitiligo patients develop autoantibodies against melanocytes. 9,39 In metastatic melanoma patients, depigmented macules similar or identical to vitiligo leukoderma can be developed by destruction of melanocytes through the interaction with cytolytic T-lymphocytes, which are activated by tyrosinase gene family proteins 7,56 and other melanogenesis-associated proteins. 4

Background N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD) is a toxic tyrosinase substrate developed to treat melanoma. Objective We investigated the effect of NPCMD on innate immune responses in monocytes. Methods CD14+ monocytes and a monocytic cell line, THP-1, were stimulated with NPCMD in vitro. Cytokines in the culture supernatants were determined by ELISA and flow cytometry. Results NPCMD stimulated CD14+ monocytes and THP-1 cells to secrete TNFα, IL-6 and IL-8, but not IL-10 or IL-12. TNFα secretion from THP-1 cells stimulated with NPCMD was inhibited by addition of an anti-TLR4 mAb in culture. Moreover, NPCMD stimulated production of pro-IL-1β in CD14+ monocytes and monocytic cell line THP-1 cells and activated the NLRP3-inflammasome, resulting in production of mature IL-1β. Use of ASC and NLRP3-deficient THP-1 cell lines established involvement of the NLRP3 inflammasome in an IL-1β secretion in treatment with NPCMD. Inhibition of IL-1β secretion by an endocytosis inhibitor, cytochalasin B, and a lysosomal enzyme cathepsin B inhibitor, CA-074 Me, suggested the involvement of lysosomal rupture and leakage of cathepsin B into the cytosol in NLRP3 activation by NPCMD. Conclusion The immunopotentiating effect of NPCMD mediated by TLR4 and NLRP3 inflammasome activation could be useful for eliciting effective adaptive immune responses against melanoma and other tumors.

Pigmented follicular cyst is a rare disorder which typically presents as a pigmented papule on the head or neck and which, histologically, exhibits terminally differentiated, pigmented hair shafts in an epidermoid cyst. We report a 22-year-old man with the multiple variant of this disorder. Clinically he had numerous brown-blue to flesh-coloured, domed-shaped papules, on the anterior chest and abdomen, of 10 years duration. Histologically, hybrid cysts exhibiting trichilemmal and epidermoid keratinization were seen. The cysts contained numerous pigmented, terminally differentiated hair shafts and, embedded in the wall of one cyst, was a sebaceous gland. The condition of multiple pigmented follicular cysts, is thought to represent a distinct subtype within the spectrum of multiple pilosebaceous cystic disorders.

Summary It has been shown previously that N-acetyl-4-S-cysteaminylphenol (N-Ac-4-S-CAP) is a tyrosinase Substrate and a potent depigmenting agent of dark skin and black hair. The present study evaluated the depigmenting potency of an acetyl derivative of N-Ac-4-S-CAP. N-2,4-acetoxyphenyl thioethyl acetamide (NAP-TKA) in the skin and hair. We tested for (i) in vitro metabolites in the skin after topical application. and (ii) in vivo depigmenting potency in the skin and hair. We found that NAPTEA was stable in water. but was converted to N-Ac-4-S-CAP after topical application to human skin. Therefore. although NAP-TEA was not a tyrosinase substrate. it could react with tyrosinase after being converted to N-Ac-4-S-CAP by 0-deacetylation in vivo. NAP-TEA produced marked depigmentation of dark skin (Yucatan pig) after daily topical application. When given by intraper-itoneal injection. it resulted in complete loss of hair colour (white) grown at the epilated site in adnlt C57 black mice after daily administration for l0 days, and incomplete loss of coat colour (silver grey) in newborn C57 black mice after a single administration, The depigmentation of the skin and hair was reversible, Splil-dopa preparalion and eieclron microsnipy indicated thal lliis depigmentation is primarily related to (i) a marked decrease in the number of functioning melanocytes and melanized melanosomes, (ii) a decrease in the number of melanosomes transferred to keratinocytes, and (iii) selective degeneration/inactivation of melanocytes. and deposition of melanin-like malerial in the Golgi cisternae. coated vesicles and melanosomes. where tyrosinase is reported to be located. We propose the NAP-TEA is converted in vivo to N-Ac-4-.S-CAP which. via interaction with tyrosinase. causes reversible depigmentation of the skin and hair.

The pathogenesis of Fox-Fordyce disease has been reported to be hyperkeratosis and obstruction of the upper hair follicle, where the duct of the apocrine sweat gland opens. We report a case of Fox-Fordyce disease with full clinical manifestation. It appeared to be caused by the obstruction of intraepidermal apoeccrine sweat ducts by apoeccrine secretory cells detached and released from the secretory epithelium. A 24-year-old woman visited our clinic with intensely pruritic papules on axillae, mammary areolae, and pubic areas. Histopathologic examination revealed an obstruction of the sweat duct in the epidermis, which opened directly to the skin surface. The closing substance of the duct was an aggregate of epithelial cells, probably derived from the secretory portion. In the dermis, the secretory cells of apocrinelike sweat glands had been detaching from the secretory epithelia. These findings suggest that Fox-Fordyce disease can occur by the mechanism in which apoeccrine secretory cells obstruct sweat ducts. (J Am Acad Dermatol 2003;48:453-5.)

53歳,男性。1999年に左下腿,2002年に右下腿の皮疹が出現。臨床的に,表面凹凸不整な褐色の肉芽腫様浸潤性局面があり,多数の瘻孔が開口していた。病理組織では,表皮索の延長,角質を含む嚢腫の形成,真皮·皮下組織の慢性炎症像等を認め,慢性膿皮症と診断した。自験例では両下腿に限局して病変が認められ,陰股部,腋窩,項部,頭部等,慢性膿皮症の好発部位には同皮疹は認められなかった。

Melanin synthesis is a biologic property unique to the melanocyte. It is highly elevated in malignant melanoma with the production of both eumelanin (brown/black pigment) and pheomelanin (yellow/red pigment), dihydroxyindole (DHI) and cysteinyldopa (CD), respectively, being major precursors. Melanin metabolites are often released in the urine of patients with disseminated melanoma metastasis (melanuria). To establish a better method for the detection of occult melanoma this study compares the plasma levels of a pheomelanin metabolite, 5-S-CD, and a eumelanin metabolite, 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C), in melanoma and non-melanoma patients and correlates them with tumor thickness and melanoma metastasis. We found a) that the normal plasma levels of 5-S-CD and 6H5M12C are less than 2.22 ng/ml and 1.04 ng/ml, respectively; b) that the group with the normal 6H5MI2C plasma level does not have any metastasis, whereas a normal 5-S-CD level is seen in both non-melanoma and melanoma patients with and without metastasis; c) that a high plasma 6H5MI2C level is seen in all melanoma patients with tumor thickness more than 3.0 mm regardless of the presence or absence of metastasis, whereas in thinner melanoma patients this is seen only in positive metastasis group; and d) that all melanoma patients with positive metastases showed a high plasma 6H5MI2C level (more than 1.75 ng/ml). We conclude that the measurement of plasma levels of melanin metabolites provides a method for detecting occult melanoma metastasis and estimating the prognosis of melanoma patients, plasma 6H5MI2C level being more sensitive and reliable than that of 5-S-CD, and its increased level being a high risk factor.

Using melanotic cells (SK-MEL-23 and G361) and amelanotic cells (C32 and SK-MEL-24) of human melanoma, this study examined whether UV-B irradiation has a direct stimulatory effect on the expression of genes involved in melanogenesis. Our initial screening of methylthiazol tetrazolium (MTT)-formazan formation assay indicated a low dose of ultraviolet (UV)-B irradiation, 2.5 and 5.0 mJ/cm2, can metabolically stimulate these cells. Repeated exposure of UV-B at 5.0 mJ/cm2 for seven consecutive days resulted in increased tyrosinase activity and melanin synthesis in SK- MEL-23 and G361 cells, but not in C32 and SK-MEL-24 cells. On reverse-transcription – polymerase chain reaction and immunoprecipitation studies, the two melanotic cell lines exhibited upregulated expression of mRNA and antigenic epitopes of tyrosinase, tyrosinase-related protein (TRP-1; gp75/HMSA-5), and lysosomal membrane associated protein (Lamp-1). The amelanotic cell line, C32, expressed the tyrosinase gene and protein constitutively but revealed no active tyrosinase or melanin synthesis even after UV-B exposure. Another amelanotic cell line, SK-MEL-24, exhibited no expression of tyrosinase gene and protein before and after UV-B exposure and, therefore, no melanin synthesis. Both C32 and SK-MEL-24 showed no gene or protein expression of TRP-1 before or after UV exposure, but upregulation of the Lamp-1 gene and protein expressions after exposure.We conclude that tyrosinase is the key enzyme responsible for UVB-induced melanogenesis. Both TRP-1 and Lamp-1 act together in melanogenesis, TRP-1 being essential and necessary. There is no change in the expression of CD63 lysosomal membrane protein at either the mRNA or protein level.

A 50-year-old Japanese male visited our clinic in April 1999 with a 2-year history of self-healing, reddish papules on his right palm. On examination, there were grouped erythematous papules, 2–4 mm in size, which formed a relatively well-circumscribed erythematous plaque. A biopsy specimen showed a wedge-shaped, dense dermal infiltrate consisting of variously sized mononuclear lymphoid cells mixed with few large CD30-positive cells and inflammatory cells, suggesting the diagnosis of regional lymphomatoid papulosis (LyP). Analysis of the T cell receptor gene revealed a polyclonal pattern on lesional skin. Only 5 cases of LyP presenting in a regional distribution have been reported previously. Although the etiology of localized LyP remains unknown, considering that 2 of 5 reported patients developed widespread lesions regional LyP may be the initial presentation of typical LyP.

This report introduces some aspects of our current basic research Focus on the unique metabolic pathways within the melanocyte. Using this approach, we hope to gain a better understanding of the pathophysiology of malignant melanoma and develop early laboratory diagnostic tests for this disease. Specifically, we will discuss that 1) the synthesis of pheomelanin is markedly increased in malignant melanoma and dysplastic melanocytic nevi; 2) high levels of metabolites of pheomelanin and eumelanin can be detected in the urine and blood of patients with metastasic melanoma; 3) this release of melanin metabolites appears to correlate with tumor thickness and tumor load, including the extent of metastasis; 4) the synthesis of melanosomal proteins also becomes aberrant in malignant melanoma; and 5) this abnormal me-lanosome synthesis can be utilized in the identification of antigenic epitopes that are uniquely expressed in malignant melanoma. We believe that this synthesis and secretion of abnormal melanin pigment and melanosomal proteins (human melanosome-specific antigen) would be useful for the development of early laboratory diagnostic and monitoring tools for malignant melanoma. In addition, we also report the detection of pheomelanin component in “normal” unex-posed skin; however, the relative amount of pheomelanin in the skin does not reflect hair color (e.g., red hair). The nature of this pheomelanin component in the skin needs to be further clarified.

Human malignant melanoma represents a difficult therapeutic challenge to both medical scientists and practicing physicians. However, the biologic uniqueness of the tumor may provide opportunities for exploitation in therapeutics. This study proposed to undertake a systemic approach to the chemotherapy of malignant melanoma based upon the uniqueness of pigment-cell metabolic pathway pertaining to conversion of tyrosine and dopa with subsequent formation of melanin by tyrosinase and its related enzymes. The sulphur homologue of tyrosine, cysteinylphenol (CP), its amine derivative, cysteaminylphenol (CAP), and their N-acetyl and α-methyl derivatives have been synthesized and tested in in vivo and in vitro melanocytotoxicity and antimelanoma effects. These phenolic thioethers (PTEs) and phenolic thioether amine (amides) (PTEAs), which are substrates of tyrosinase, showed significant cytotoxicity that is selective to melanocytes and melanoma cells. Most previous attempts to impair the melanin pathway as a therapeutic strategy have been of limited success because they have been directed to catecholic compounds that are unstable and insufficient in lethality at physiologically tolerable doses. By contrast, our approach relies on phenolic compounds, PTEs and PTEAs, which are more stable than catechols and become toxic only after oxidation by tyrosinase. We found PTEA as the most promising agent for the future development of chemotherapeutic agents. The possible biologic, chemical, and pharmacologic reactions of these synthetic compounds within the melanoma cells are studied and discussed. J Invest Dermatol 100;231S–238S, 1993

A case of a 17-year-old Japanese-Canadian boy with a 2-year history of skin lesions relevant to confluent and reticulated papillomatosis (CRP) is reported with electron microscopic findings. He had scattered skin lesions of brownish-colored, rough-surfaced, fine-scaled patches and plaques distributed on the nape, shoulders, and both flanks, extending to the chest and back. Importantly, they showed reticular or confluent arrangement. There was no family history of the same skin lesions. The histology showed epidermal papillomatosis and mild acanthosis with a marked hyperkeratosis. Electron microscopically, the skin lesions consisted of the following abnormal findings: (1) a marked alteration of cornified cell structures showing snake coil-like, or triangle-like stacks, (2) a marked increase in the number of lamellar granules in the granular layer, (3) an increased number of melanosomes in the horny layers, and (4) no significant fine structural changes of epidermal melanocytes. This is the first electron microscopic report on CRP suggesting that the pathophysiology of this disease is related to abnormal keratinocyte differentiation.

Melanocortin-1 receptor (MC1R) and its ligands, a-melanocyte stimulating hormone (aMSH) and agouti signaling protein (ASIP), regulate switching between eumelanin and pheomelanin synthesis in melanocytes. Here we investigated biological effects and signaling pathways of ASIP. Melan-a non agouti (a ⁄ a) mouse melanocytes produce mainly eumelanin, but ASIP combined with phenylthiourea and extra cysteine could induce over 200-fold increases in the pheomelanin to eumelanin ratio, and a tan-yellow color in pelletted cells., Moreover, ASIP-treated cells showed reduced proliferation and a melanoblast-like appearance, seen also in melanocyte lines from yellow (Ay ⁄ a and Mc1re ⁄ Mc1re) mice. However ASIP-YY, a C-terminal fragment of ASIP, induced neither biological nor pigmentary changes. As, like ASIP, ASIP-YY inhibited the cAMP rise induced by aMSH analog NDP-MSH, and reduced cAMP level without added MSH, the morphological changes and depigmentation seemed independent of cAMP signaling. Melanocytes genetically null for ASIP mediators attractin or mahogunin (Atrnmg-3J ⁄ mg-3J or Mgrn1md-nc ⁄ md-nc) also responded to both ASIP and ASIP-YY in cAMP level, while only ASIP altered their proliferation and (in part) shape. Thus, ASIP–MC1R signaling includes a cAMP-independent pathway through attractin and mahogunin, while the known cAMP-dependent component requires neither attractin nor mahogunin., Parts of the research were supported by Wellcome Trust program grants 064583 and 078327 to D.C.B. and E.V.S.; the Japan Society for the Promotion of Science KAKENHI (grants 20790808 to T.H. and 18591262 and 20591357 to K.W. and S.I.); a Grant-in-Aid from the Japanese Ministry of Health, Labour and Welfare (K.J.), the Spanish Ministry of Education and Science BFU2006-12185 (L.M.), the South West Academic Network (A.J.D. and E.V.S.), and NIH grant DK064265 (B.Y. and G.L.M.).

A magnetite nanoparticle, NPrCAP/M, was produced for intracellular hyperthermia treatment of melanoma by conjugating N-propionyl-cysteaminylphenol (NPrCAP) with magnetite and used for the study of selective targeting and degradation of melanoma cells. NPrCAP/M, like NPrCAP, was integrated as a substrate in the oxidative reaction by mushroom tyrosinase. Melanoma, but not non-melanoma, cells incorporated larger amounts of iron than magnetite from NPrCAP/M. When mice bearing a B16F1 melanoma and a lymphoma on opposite flanks were given NPrCAP/M, iron was observed only in B16F1 melanoma cells and iron particles (NPrCAP/M) were identified within late-stage melanosomes by electron microscopy. When cells were treated with NPrCAP/M or magnetite and heated to 43 degrees C by an external alternating magnetic field (AMF), melanoma cells were degraded 1.7- to 5.4-fold more significantly by NPrCAP/M than by magnetite. Growth of transplanted B16 melanoma was suppressed effectively by NPrCAP/M-mediated hyperthermia, suggesting a clinical application of NPrCAP/M to lesional therapy for melanoma. Finally, melanoma cells treated with NPrCAP/M plus AMF showed little sub-G1 fraction and no caspase 3 activation, suggesting that the NPrCAP/M-mediated hyperthermia induced non-apoptotic cell death. These results suggest that NPrCAP/M may be useful in targeted therapy for melanoma by inducing non-apoptotic cell death after appropriate heating by the AMF.

There are rapidly increasing opportunities for dermatologists to see patients suffering from retrovirus infections. The HTLV-I was the first class of human oncogenic retrovirus that was found in cultured cells of a patient with skin manifestations similar or identical to those of CTCL (MF). It was soon recognized as the agent causing ATLL. The skin manifestations, histopathology, and immunophenotypes of ATLL share many similarities with MF and SS. Both HTLV-I and HIV-I (HTLV-III) cause immunodeficiency with an increased susceptibility to opportunistic infections. Persistent generalized lymphadenopathies are the initial manifestations of most of the HIV infections. The incidence of lymphoid malignancies is expected to become much higher as the life span of AIDS patients is prolonged. They can have both B-cell and T-cell non-Hodgkin lymphomas, although the incidence of the latter (B-cell lymphoma) is still much higher than that of the former. All human retroviruses are transmitted in similar ways.

This study evaluates the expression of human melanosome specific antigen-1 and -2 (HMSA-1, -2) by dysplastic melanocytic nevus (DMN) and its significance by (a) immunostaining pattern, (b) other immunohistochemical markers (e.g., S-100), and (c) mesenchymal responses (e.g., lymphocytic infiltration). From 31 patients with DMN syndrome, 55 clinically characteristic and histologically (HE) proven dysplastic nevi were assessed for HMSA expression. Approximately 70% of DMN lesions expressed the HMSA antigens; similar gross staining patterns were seen with both monoclonal antibodies (MoAbs). The expression of HMSA-2 was invariably associated with that of HMSA-1, but the converse did not apply. Two general staining patterns with MoAbs HMSA were appreciated: (a) uniform staining of the epidermal melanocytes in the DMN lesion (type I) and (b) nonuniform staining, with the epidermal melanocytes at the periphery of the DMN lesion reacting most intensely (type II). Although anti-S-100 antibody demonstrated a similar sensitivity (67%), it lacked specificity and did not distinguish different types of DMN, as compared with MoAbs HMSA. There was no correlation between these observed staining patterns and common histological features of DMN. There was a poor correlation between lymphocytic infiltrate (as judged by immunostaining for pan-T, Ti/h, Tc/s, and pan-B markers) and heterogeneity of immunostaining. We conclude that HMSA-1 and -2 are able to delineate two types of DMN and possibly bind different epitopes of the same antigen. Their potential usefulness in predicting malignant transformation of DMN has yet to be established.

The epidermal melanin unit (EMU) denotes the symbiotic relationship between a melanocyte and a pool of associated keratinocytes. We propose to show that alterations in the biology of the EMU are the main determinant of the different patterns of intraepidermal growth of melanocytes in lentigo maligna melanoma (LMM) and superficial spreading melanoma (SSM). They also appear to affect the biosynthesis of melanin and melanosomes during malignant transformation. Findings in histochemical studies with monoclonal antibodies generated against melanosomal proteins to produce different stains of melanocytes of normal skin, dysplastic melanocytic nevi (DMN), common melanocytic nevi (CMN), LMM, and SSM have led to the suggestion that the altered melanosome synthesis is a main phenotype in the pathophysiology in neoplastic transformation of melanocytes. Altered melanin synthesis may also affect the carcinogenesis in malignant melanoma: pheomelanin is increased in malignant melanoma and DMN, but not in normal skin and CMN. Pheomelanin and its precursors could aid the malignant transformation of melanocytes through the generation of mutagenic ultraviolet photoproducts in familial DMN syndrome.

In order to identify a means to reduce scar formation of the skin after incision, this study examined the effect of local administration of basic fibroblast growth factor (bFGF) in humans. bFGF was administered to a sutured wound immediately after an operation. The drug was injected once into the dermis of the margins of wounds using a 27G needle or rinsing after performing dermostitches. The lengths of the treated wounds varied from 1 to 32 cm, and the subjects were 2-86 years old. Sutured wounds after excision of skin tumors from the face, trunk, and limbs and sutured wounds such as those at the donor sites of full-thickness skin grafts were treated with low-dose bFGF injections (0.1 microg/cm wound; Group 2), high-dose bFGF injections (1 microg/cm wound; Group 3), and rinsed with high-dose bFGF (1 microg/cm wound; Group 4). No patient treated with bFGF had hypertrophic scars, while some patients had hypertrophic or very wide scars in the control group (Group 1), and the ratios of minimum scarring of Group 2 (p

One of the specific skin lesions occurring after Bacille Calmette-Guerin (BCG) vaccination is generalized tuberculid-like eruptions, which occur rarely, but have a tendency to heal spontaneously. Their pathogenesis and relationship to "true" tuberculids are poorly understood. This report presents a case of a 6-month-old girl who developed generalized papulonecrotic tuberculid-like eruptions after BCG vaccination. The skin lesions healed spontaneously in 3 months. Culture of blood, gastric juice and reverse transcription polymerase chain reaction (RT-PCR) of papulonecrotic skin biopsies were all negative for Mycobacterium tuberculosis. Histopathology of papulonecrotic eruptions revealed marked epidermal necrosis, perivascular lymphocytic infiltrates and epidermotropic infiltration of lymphocytes showing markers of CD3(+) lymphocytes (90-95% of all infiltrating cells), CD4(+) (40-50%), CD8(+) (40-50%), and CD45RO(+) (70%). In contrast, the BCG vaccination site revealed intradermal granuloma with epithelioid cells, occasional giant cells and infiltration of lymphocytes consisting of CD3(+) (60-70%), CD4(+) (40-50%), CD8(+) (30-40%), CD45RO(+) (40%), CD79a(+) (30-40%), and CD20(+) (20-30%). Our patient did not reveal any signs indicative of tuberculosis. Papulonecrotic lesions were therefore called papulonecrotic tuberculid-like eruptions, rather than tuberculids, that occurred after BCG vaccination and appeared to derive from a hypersensitive reaction mediated by immune lymphocytic infiltration.

Summary Background In obtain images of skin tumors non-invasively with real-time, confocal laser scanning microscope (CLSM) is introduced. Objective Reconstructed images of given horizontal sections were converted into three-dimensions using the data set of a large number of tomograms in the horizontal directions. Methods To develop the multiplaner reconstruction images of skin tumors in vertical directions and three-dimensionally reconstructed images of tumors will be obtained from the continuously collected horizontal image data sets. Results Three-dimensional analyses of the skin tumors from reconstructed images of the CLSM scanning have provided the information as to their physiological characteristics as well as the extent of deep invasion in real-time with non-invasive manner. High performance three-dimensional conversion software was effective in displaying three-dimensional construction of skin tumors. Conclusion The CLSM scanning images followed by three-dimensional reconstruction using them can provide the real-time and non-invasive diagnoses of skin tumors and analyze the radial growth phase of tumors and the three-dimensional growth characteristics.

A case of multiple osteomyelitides due to Mycobacterium avium (M. avium) infection with osteosclerotic bone lesions is reported. A 67-year-old male had been suffering from persistent fever and back pain since October 1999, and 20.0-2.5 mg prednisolone per day was prescribed for continuous inflammatory symptoms in January 2000. Six months later, computed tomography revealed osteosclerotic lesions in the left femur and thoracic vertebrae, but no skin lesion associated with mastocytosis or internal malignancy was identified. In September of 2002, a dome-shaped, soft subcutaneous tumor developed on the upper sternum. Histopathological findings revealed subcutaneous adipose tissue with several foci of tiny abscesses. Two weeks later, creamy pus was discharged through a draining sinus at the center of the wound. M. avium was demonstrated in the pus by Zeel-Nielsen staining and microplate hybridization.

Idiopathic acquired generalized anhidrosis is a very rare disease of unknown pathogenesis. We report a 25-year-old man with acquired generalized anhidrosis due to occlusion of the coiled ducts. He did not have sweat secretion over the entire surface of the body, including the palms and soles. Sweat-inducing stimuli provoked tingling pain on the skin. Pilocarpine iontophoresis on the forearm did not induce sweat secretion. Neurological examination did not reveal any abnormality in the central or peripheral nervous system. Skin biopsy showed that the coiled ducts were occluded by an amorphous eosinophilic substance. This amorphous eosinophilic substance was positive with periodic acid-Schiff (PAS) staining and was resistant to digestion by diastase. Electron microscopy demonstrated that the coiled ducts were completely occluded by an amorphous substance. The substance occluding the coiled ducts contained fibrous structures. These findings suggested that the acquired generalized anhidrosis in this patient was caused by occlusion of the coiled ducts by a PAS-positive substance probably derived from dark cell granules.