Spectrophotometer is useful for assessing vitiligo and chemical leukoderma severity by quantifying color difference with surrounding normally pigmented skin.

BACKGROUND: Acquired skin hypopigmentation has many etiologies, including autoimmune melanocyte destruction, skin aging, inflammation, and chemical exposure. Distinguishing lesions from normally pigmented skin is clinically important to precisely assess disease severity. However, no gold standard assessment method has been reported. We aimed to investigate whether spectrophotometers are useful for assessing vitiligo and rhododendrol (4-(4-hydroxyphenol)-2-butanol) (Rhododenol® )-induced leukoderma disease severity by quantifying skin color. METHODS: Mexameter® MX18 and CM-700d spectrophotometer were used for assessing vitiligo/leukoderma by measuring melanin index, L*a*b* color space, and ΔE*ab value, which represents the color difference between two subjects and is calculated by the values of L*a*b*. RESULTS: MX18 and CM-700d can quantitatively distinguish vitiligo/leukoderma from normally pigmented skin based on melanin index. CM-700d consistently quantified the color of vitiligo/leukoderma lesions and surrounding normally pigmented skin in L*a*b* color spaces and ΔE*ab. ΔE*ab is well correlated with melanin index and clinical appearance. CONCLUSION: ΔE*ab has been frequently used in aesthetic dentistry; however, current study is the first to use it in the measurement of skin color. ΔE*ab seems to be a useful parameter to evaluate the color contrast between vitiligo/leukoderma and surrounding normally pigmented skin and can be used to evaluate disease severity and patient's quality of life.

Survival of HER2-positive primary breast cancer patients treated by neoadjuvant chemotherapy plus trastuzumab: a multicenter retrospective observational study (JBCRG-C03 study).

We investigated the disease-free survival (DFS) of HER2-positive primary breast cancer patients treated with neoadjuvant chemotherapy plus trastuzumab, as well as predictive factors for DFS and pathologic response. Data from 829 female patients treated between 2001 and 2010 were collected from 38 institutions in Japan. Predictive factors were evaluated using multivariate analyses. The 3-year DFS rate was 87 % [95 % confidence interval (CI) 85-90]. The pathologic complete response (pCR: ypT0/is + ypN0) rate was 51 %. The pCR rate was higher in the ER/PgR-negative patients than in the ER/PgR-positive patients (64 vs. 36 %, P < 0.001). Patients with pCR showed a higher DFS rate than patients without pCR (93 vs. 82 %, P < 0.001). Multivariate analysis revealed three independent predictors for poorer DFS: advanced nodal stage [hazard ratio (HR) 2.63, 95 % CI 1.36-5.21, P = 0.004 for cN2-3 vs. cN0], histological/nuclear grade 3 (HR 1.81, 95 % CI 1.15-2.91, P = 0.011), and non-pCR (HR 1.98, 95 % CI 1.22-3.24, P = 0.005). In the ER/PgR-negative dataset, non-pCR (HR 2.63, 95 % CI 1.43-4.90, P = 0.002) and clinical tumor stage (HR 2.20, 95 % CI 1.16-4.20, P = 0.017 for cT3-4 vs. cT1-2) were independent predictors for DFS, and in the ER/PgR-positive dataset, histological grade of 3 (HR 3.09, 95 % CI 1.48-6.62, P = 0.003), clinical nodal stage (HR 4.26, 95 % CI 1.53-13.14, P = 0.005 for cN2-3 vs. cN0), and young age (HR 2.40, 95 % CI 1.12-4.94, P = 0.026 for ≤40 vs. >40) were negative predictors for DFS. Strict pCR (ypT0 + ypN0) was an independent predictor for DFS in both the ER/PgR-negative and -positive datasets (HR 2.66, 95 % CI 1.31-5.97, P = 0.006 and HR 3.86, 95 % CI 1.13-24.21, P = 0.029, respectively). These results may help assure a more accurate prognosis and personalized treatment for HER2-positive breast cancer patients.

The effect of exemestane, anastrozole, and tamoxifen on lipid profiles in Japanese postmenopausal early breast cancer patients: final results of National Surgical Adjuvant Study BC 04, the TEAM Japan sub-study.

BACKGROUND: In this Tamoxifen Exemestane Adjuvant Multinational Japan sub-study, we evaluated the time course of changes in serum lipids in postmenopausal women with hormone-sensitive early breast cancer treated with exemestane, anastrozole, or tamoxifen for postoperative adjuvant therapy. PATIENTS AND METHODS: A total of 154 breast cancer patients were assigned to receive exemestane, anastrozole, or tamoxifen in this randomized open-label study. Serum lipid parameters including triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured during 1 year of treatment. RESULTS: TC and LDL-C rapidly decreased in patients treated with tamoxifen at 3 months. Compared with anastrozole and exemestane patients, TC and LDL-C were significantly lower at all assessment time points in tamoxifen patients (P < 0.05). TG increased in tamoxifen patients; it was significantly higher compared with exemestane patients at all assessment time points (P < 0.05). HDL-C slightly decreased in exemestane patients; it was significantly lower compared with anastrozole patients at 3 months and 1 year (P = 0.0179 and 0.0013, respectively). CONCLUSION: Changes of lipid profiles in Japanese postmenopausal women treated with tamoxifen were relatively favorable, while exemestane and anastrozole had no clinically significant effect on the serum lipids.

Variations in locoregional therapy in postmenopausal patients with early breast cancer treated in different countries.

BACKGROUND: The Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial is an international randomized trial evaluating the efficacy and safety of exemestane, alone or following tamoxifen. The large number of patients already recruited offered the opportunity to explore locoregional treatment practices between countries. METHODS: Patients were enrolled in Belgium, France, Germany, Greece, Ireland, Japan, the Netherlands, the UK and the USA. The core protocol had minor differences in eligibility criteria between countries, reflecting variations in national guidelines and practice regarding adjuvant endocrine therapy. RESULTS: Between 2001 and 2006, 9779 patients of mean(s.d.) age 64(9) years were randomized. Some 58.4 per cent had T1 tumours (range between countries 36.8-75.9 per cent; P < 0.001) and 47.3 per cent were axillary node positive (range 25.9-84.6 per cent; P < 0.001). Independent factors for type of breast surgery were country, age, tumour status and calendar year of surgery. After breast-conserving surgery, radiotherapy was given to 93.2 per cent of patients, 86.0 per cent in the USA and 100 per cent in France. Axillary lymph node dissection was performed in 82.0 (range 74.6-99.1) per cent. CONCLUSION: Despite international consensus guidelines, wide global variations were observed in treatment practices of early breast cancer. There should be further efforts to optimize locoregional treatment for breast cancer worldwide.

Effects of exemestane, anastrozole and tamoxifen on bone mineral density and bone turnover markers in postmenopausal early breast cancer patients: results of N-SAS BC 04, the TEAM Japan substudy.

BACKGROUND: Use of aromatase inhibitors in women with postmenopausal breast cancer accompanies risks of bone loss. We evaluated changes in bone mineral density (BMD) and bone turnover markers in patients treated with exemestane, anastrozole or tamoxifen for hormone-sensitive postmenopausal early breast cancer. PATIENTS AND METHODS: Sixty-eight patients enrolled in the Tamoxifen Exemestane Adjuvant Multinational Japan bone substudy were randomly assigned to receive tamoxifen, exemestane or anastrozole. During a 2-year study period, lumbar spine BMD was measured using dual-energy X-ray absorptiometry, and urinary type I collagen cross-linked N-telopeptide (NTX) and serum bone-specific alkaline phosphatase (BAP) were also measured. RESULTS: BMD at 2 years of treatment was higher in tamoxifen patients compared with exemestane and anastrozole patients; however, the intergroup difference was not significant (p = 0.2521 and p = 0.0753, respectively). BMD was higher in exemestane patients compared with anastrozole patients; however, the intergroup difference was not significant (p = 0.7059 and p = 0.8134, respectively). NTX and BAP were significantly lower in tamoxifen patients compared with exemestane and anastrozole patients at 1 and 2 years of treatment (p < 0.05). CONCLUSION: Tamoxifen may provide better bone protection compared with exemestane or anastrozole. The effect of exemestane and anastrozole on bone loss may be comparable in Japanese postmenopausal women.

Direct evidence for mutual interactions between perineuronal astrocytes and interneurons in the CA1 region of the rat hippocampus.

Recent studies have demonstrated that astrocytes express a variety of ion channels and neurotransmitter receptors and can modulate the activity of neurons. Since a single astrocyte makes tight contacts with many neighboring neuronal cells, they can provide efficient and wide modulation of neuronal networks. Here, we provide direct evidence for mutual interactions between perineuronal astrocytes and interneurons in the stratum radiatum of the rat hippocampus. Direct depolarization of a perineuronal astrocyte suppressed the excitatory postsynaptic currents in an adjacent interneuron and increased the paired-pulse ratio, indicating that perineuronal astrocytes have a suppressive effect on presynaptic elements. Moreover, perineuronal astrocyte activation modulated the directly induced firing pattern of the interneuron, with initial facilitation and subsequent suppression. Conversely, direct firing of the interneuron depolarized the membrane potential and reduced the input resistance of the perineuronal astrocyte. These results directly demonstrate the existence of bidirectional interactions between neurons and perineuronal astrocytes.

Effect of resveratrol and lipoic acid on sirtuin-regulated expression of metabolic genes in bovine liver and muscle slice cultures.

Sirtuins (Sirt) are NAD-dependent deacetylases that are activated by the antioxidants resveratrol (RSV) and lipoic acid (LA). The objective of this study was to determine in bovine liver and muscle slice cultures the effect of RSV and LA treatment on the expresssion of Sirt1, Sirt3, peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A), and the forkhead box O transcription factors FoxO1 and FoxO3 as well as other factors involved in glucose and lipid metabolism and related to Sirt activity. Tissue slices from crossbred bulls were treated during 60 min with 40 or 80 µ RSV and 30, 100, 300, or 1,000 µ LA under restricted conditions (Krebs-Ringer buffer without nutrients) and fed conditions (2.5 m propionate in combination with 1 n glucagon) for liver slices or with 0.01 µ epinephrine for muscle slices. Quantitative real-time PCR was used to analyze the expression of the mRNA for the genes studied and western blot analysis for the expression of the protein for Sirt1. Our results show that the expression of the mRNA for Sirt1 was enhanced by RSV in liver under restriction ( ≤ 0.0112) and by LA in muscle, more under restriction ( ≤ 0.0121) than after epinephrine administration ( < 0.0001). Sirt3 is affected in a dose-dependent manner by both compounds in both tissues and under both metabolic conditions ( ≤ 0.0452). The expression of the protein for Sirt1 was increased by LA in both tissues under restricted conditions ( = 0.0026 and = 0.0201, respectively) but in liver also in fed conditions ( = 0.0016). Genes involved in the antioxidant response were upregulated in both tissues. These results indicate that bovine Sirt respond differently to RSV and LA stimulation than monogastric Sirt do and that gluconeogenesis in ruminants is not related to Sirt to the same degree as in monogastric species. However, these results provide information about the possible role of Sirt in ruminant metabolism.

Long-term organ culture of rabbit skin: effect of EGF on epidermal structure in vitro.

A method is described for maintaining the epidermal structure of normal rabbit ear skin explants in organ culture for up to 12 weeks. Split-thickness skin specimens were put in diffusion chambers made of either millipore filters or bovine collagen membranes, and then submitted to a roller tube culture at 15 rpm and 36 degrees C. The culture medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FCS) + 0.4 micrograms/ml hydrocortisone. The gas used in the culture tube was air + 5% CO2. Autoradiography revealed the incorporation of [3H]-glycine into the 68-kD keratin band of explants for up to 12 weeks, indicating that normal keratinization was maintained throughout the entire culture period. The turnover time of the epidermis from basal layer to granular layer was around 7 d in both the early and late stages of culture. The addition of epidermal growth factor (EGF) to the culture caused the epidermis to become acanthotic with orthokeratosis, but with high concentrations of EGF (greater than or equal to 10 ng/ml) parakeratosis and increased proliferation of the epidermis occurred. Dexamethasone (DMS) strongly inhibited the EGF effect.

Organ culture of psoriatic lesions: appearance of granular layers in vitamin A-free culture media.

Some morphologic changes of the epidermis of psoriatic skin were observed in organ culture in the presence of absence of vitamin A. Normal and uninvolved and involved psoriatic skin areas, punch biopsied in 3-4-mm diameter specimens, were put in serum-free medium containing no vitamin A with or without delipidized fetal calf serum (FCS) and rotation cultured at 60 rpm under an atmosphere consisting of 95% O2 + 5% CO2. The involved psoriatic skin specimens showed well-developed granular layers after 1 d of culture. The values of labeling indices of 3H-thymidine (3H-TdR) incorporated into the epidermal layers of the cultured specimens of the psoriatic skin were nearly constant during culture for as long as 8 d, although the viable epidermal layer gradually became thinner. Addition of tretinoin to the culture caused uninvolved and involved psoriatic skin specimens to become parakeratotic at concentrations as low as about 2.0 x 10(-6) M and 4.0 x 10(-8) M, respectively, suggesting that the involved psoriatic epidermis is much more sensitive, in terms of keratinization, than uninvolved epidermis to the effect of tretinoin.

Effect of tamoxifen on serum lipid metabolism.

The effect of tamoxifen, an antiestrogenic agent, on lipid metabolism was studied in postmenopausal patients with breast cancer who received the drug for postoperative adjuvant treatment following mastectomy. To measure total cholesterol and triglyceride concentrations, fasting blood samples were collected before and 2 months after the initiation of tamoxifen therapy from 16 patients who satisfied the study criteria. All patients were normolipidemic before tamoxifen was administered. Control samples were obtained from hypertriglyceridemia patients who were free from breast cancer. Marked hypertriglyceridemia was observed in 3 of 16 patients after tamoxifen treatment. The activity of lipoprotein lipase and hepatic triglyceride lipase, the key enzymes of triglyceride metabolism, decreased significantly in all of 16 patients as a result of tamoxifen treatment (P = 0.008 and P = 0.007, respectively). However, the mean mass of lipoprotein lipase significantly increased (P = 0.011) after tamoxifen treatment. We therefore conclude that tamoxifen might increase inactive lipoprotein lipase. Because marked hyperlipidemia is a potent risk factor for life-threatening acute pancreatitis and arteriosclerosis, plasma lipid levels should be tested periodically during tamoxifen treatment, even if the patients are normolipidemic during the pretreatment stage.

In vitro study of the effect of raloxifene on lipid metabolism compared with tamoxifen.

OBJECTIVE: Tamoxifen and raloxifene, selective estrogen receptor modulators, decrease serum concentrations of total cholesterol; however, the effect of these drugs on triglyceride metabolism is unclear. In the present study, we investigated the in vitro effect of raloxifene on lipid metabolism and compared it with that of tamoxifen. DESIGN AND METHODS: Intracellular concentrations of total cholesterol and triglyceride in HepG2 cells were measured by an enzymatic method after tamoxifen or raloxifene treatment with or without oleic acid and with or without very low density lipoprotein. RESULTS: Intracellular concentrations of total cholesterol and triglyceride without oleic acid or very low density lipoprotein were not significantly different after treatment with tamoxifen or raloxifene. In contrast, although raloxifene with oleic acid did not increase the intracellular concentrations of triglyceride, tamoxifen treatment in the presence of oleic acid or very low density lipoprotein significantly increased (P<0.05) the triglyceride concentrations. CONCLUSION: The present study suggests that raloxifene does not increase intracellular triglyceride in the presence of oleic acid or very low density lipoprotein, in contrast to tamoxifen. Therefore, raloxifene might be safer than tamoxifen for treating patients with unstable triglyceride levels or a history of hypertriglyceridemia.

Comparative study of the cellular fatty acids of methicillin-resistant and -susceptible Staphylococcus aureus.

The cellular fatty acid compositions of 26 strains of methicillin-resistant Staphylococcus aureus (MRSA) and 17 strains of methicillin-susceptible S. aureus (MSSA) were analyzed by gas-liquid chromatography. The fatty acid compositions of the two groups were very similar with 16 identified components. The major fatty acids were Ci14 = 0, Ci15 = 0, C18 = 0 and C20 = 0. Among these fatty acids, the percentage of the Ci15 = 0 fatty acid component of MRSA strains (11.4 +/- 3.9%) was statistically higher than that of MSSA strains (6.2 +/- 2.4%) (p < 0.001). On the other hand, the percentage of the C20 = 0 fatty acid components of MRSA strains (20.2 +/- 8.8%) was statistically lower than that of MSSA strains (30.7 +/- 10.4%) (p < 0.001). The production of beta-lactamase and beta-hemolysin in both groups' strain was also unrelated to the relative amounts of the fatty acid components. These results indicated a statistical tendency for the percentage fatty acid compositions of the MRSA strains to be quantitatively different from those of the MSSA for both the Ci15 = 0 and C20 = 0 fatty acid components. Analysis of the fatty acid compositions may have an application in the differentiation of MRSA and MSSA strains.

Autocrine secretion of an EGF-like substance by a cell line (HSC-1) derived from a human skin squamous cell carcinoma.

The presence of EGF-autocrine secretion was investigated in a cell line derived from a human squamous cell carcinoma (HSC-1). The cell line was found to secrete the epidermal growth factor (EGF)-like substance. Meanwhile, normal human and uninvolved and involved psoriatic epidermal cells did not show any evidence of EGF secretion in culture. Not was any evidence of EGF secretion observed in vitro in several malignant cell lines other than HSC-1 derived from human squamous cell carcinomas, adenocarcinoma and melanoma. The addition of EGF did not stimulate, but rather inhibited, the growth of HSC-1 cells in GIT medium as well as Dulbecco's modified essential medium with low concentrations of fetal calf serum (0.5-1%) in vitro. Overexpression of EGF receptors is known to occur in HSC-1. The results suggest that HSC-1 cells exhibit autocrine secretion of the EGF-like substance but not autostimulation in anchorage-dependent cell culture.

Effect of cyclosporin on hair-existing area of nude mice.

We investigated the effect of cyclosporin, as well as minoxidil, testosterone, estradiol and corticosteroid on the hair growth on the hairy part of nude mice. Aliquots of solutions of cyclosporin and other agents were applied once per every day topically on the tails and the lower backs of 5 week-old BALB/c nude mice, for as long as 6 weeks. Cyclosporin prolonged the hair-existing phase of the hair cycle, but did not change the term of the hair cycle, i.e., the resting phase was not affected. Minoxidil, testosterone and estradiol did not influence the hair growth cycle. Combination of cyclosporin and other agents demonstrated that there was neither additive nor synergistic effect, but a high dose of corticosteroid inhibit the cyclosporin effect, as well as suppressing completely the reappearance of the growing phase.

Identification of K1/K10 and K5/K14 keratin pairs in human melanoma cell lines.

Keratin expression in cultured malignant melanoma cells has been studied only rarely. Moreover, no studies have reported of universality of keratin expression in human malignant melanoma cells. In this study, therefore, we analyzed keratin expression in eight cell lines. Using a low-salt aqueous solution without high salt and Triton X-100, as a washing buffer for keratin extraction, followed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and immunological analysis, we demonstrated keratin expression in all eight human malignant melanoma cell lines. The keratin polypeptide expressions common to all melanoma cells were K1, K5, K10 and K14. In addition, K8, K13, K17 and K18, respectively, were detected in individual cells. A measure of keratin expression universality in malignant melanoma cells may have implications regarding their invasive and metastatic behaviors, co-expressed with vimentin.

Immunotherapy for experimental rat autoimmune thyroiditis using a novel immunosuppressant, FTY720.

While autoimmune disease needs to be continuously treated via long term, administration of immunosuppressants conventional immunotherapy using drugs such as cyclosporin and tacrolimus may cause adverse effects. Recently, a novel agent, FTY720, which was structurally modified from a natural product, has been shown to have a moderate and different immunosuppressive effect from conventional drugs. In this study, we examined the effect of FTY720 on experimental autoimmune thyroiditis (EAT), which was induced in rats by neonatal thymectomy, followed by subsequent sublethal irradiation. Thyroid stimulating hormone (TSH) was measured before and at the end of drug therapy. Histological and hematological examinations were performed using the samples from sacrificed animals. High TSH levels in the animals returned to the normal range following treatment with FTY720. The severity of the thyroiditis was lower in the FTY720 group than in the control group. FTY720 markedly decreased the number of circulatory lymphocytes, and no infections episodes were observed under this treatment. Thus, FTY720 treatment might be preferred for continuous immunotherapy for autoimmune disease without adverse effects.

Comparative inhibitory effects of vitamin D3 and an analogue on normal and psoriatic epidermis in organ culture.

Recently, there have been many vitamin D3 analogues synthesized and tried in the treatment of psoriasis. In the experiments reported here we observed and compared their effects on normal and psoriatic epidermis in organ culture in vitro. We employed a new vitamin D3 analogue, 22-oxa-calcitriol (OCT), the effect of which was compared with that of calcitriol (1,25-D3). Both caused suppression of proliferation of normal and psoriatic epidermis, dependent upon concentration and culture time. Histologically, in the presence of the agents, degeneration started from the top of the epidermis downwards. This is the first report of cell degeneration as a direct effect of vitamin D. The nature of the degeneration was evaluated by electron microscopy (EM) and by the in situ nick end labeling technique (TUNEL), and these studies revealed that the degeneration involved necrosis rather than apoptosis. This in vitro method may be useful to assess the effectiveness of newly synthesized vitamin D3 analogues in the treatment of psoriasis.

Organ culture of human scalp hair follicles: effect of testosterone and oestrogen on hair growth.

Whole human scalp hair follicles were cultured. The follicles were dissected from skin pieces of normal scalp and put into 1.5 ml of incubation medium in a closed 5 ml glass tube under an atmosphere of 95% O2 and 5% CO2. The tube was rolled at 15 rpm at 36 degrees C. Remarkable hair growth was noticed for 7 to 8 days. Hair root sheaths also grew with the hair shafts. The structure of the hair bulbs was well maintained for at least 6 days, and then the hair matrix cells started to degenerate. Fetal calf serum was not essential for hair growth in vitro, but increased the growth rate slowly. Testosterone and oestrogen inhibited hair growth in vitro to a similar extent. The minimum effective doses of both hormones to suppress hair growth were around 5 ng/ml, which corresponds well to the normal plasma level of testosterone in adult males in vivo, suggesting that scalp hair growth may be critically controlled by testosterone in adult males.

Organ culture of psoriatic skin: effect of TGF-alpha and TGF-beta on epidermal structure in vitro.

Normal skin and uninvolved and involved psoriatic skin specimens were maintained in vitro in organ culture. The 3-4 mm punch-biopsied skin specimens were put freely into the culture medium with or without fetal calf serum, under an atmosphere of 95% O2 plus 5% CO2, and rotated at 60 rpm at 37 degrees C. In the serum-free culture medium (vitamin A-free) granular layers appeared in the involved psoriatic epidermis in culture. Addition of TGF-alpha caused normal skin and uninvolved and involved psoriatic skin specimens to become acanthotic and to degenerate easily almost to the full thickness of the epidermal layer in proportion to increasing concentrations of TGF-alpha as well as with the duration of the culture, but without disappearance of their granular layers. TGF-beta caused the normal skin and uninvolved psoriatic skin specimens to become thinned without disappearance of granular layers, but caused the involved psoriatic skin specimens to be thinned without appearance of granular layers in serum-containing medium or with their disappearance in the serum-free medium. TGF-beta also antagonized the acanthotic and degenerative effect of TGF-alpha. The results suggest that TGF-alpha and TGF-beta may partially be related to the induction of psoriatic epidermal lesions.

Keratin specificity analyses of eight anti-keratin monoclonal antibodies, and their immunostaining patterns in normal skin using formalin-fixed and paraffin-embedded tissue specimens.

Keratin specificity analyses of eight anti-keratin antibodies (34 beta B4 (K1), 35 beta H11 (K8), Ks 13.1 (K13), Ks 19.1 (K19), PKK1, LP34 (CK1), KL1 and AE1) using keratin protein derived from normal thigh epidermis, normal parotid gland and a human squamous cell carcinoma cell line (HSC-5) were performed, and compared with those described in the data sheets. The reactivities of LP34, KL1 and PKK1 were markedly different from those mentioned in the data sheets. The immunostaining pattern of these antibodies in normal skin using formalin-fixed and paraffin-embedded tissue specimens was also examined. The staining patterns of suprabasal keratinocytes (K1, K13, CK1 and KL1 positive), basal cells of the epidermis (PKK1 and AE1 positive), inner cells of the ducts (K8, K13, CK1, KL1 and AE1 positive) and secretory cells of sweat glands (K8, K19, PKK1, KL1 and AE1 positive), mature cells (K8 and KL1 positive) and peripheral cells (CK1, KL1 and AE1 positive) of sebaceous glands and outer root sheaths (PKK1, CK1, KL1 and AE1 positive) were specific. Thus, we conclude that the differentiation of epidermis and skin appendages is possible by immunostaining with these eight anti-keratin antibodies using formalin-fixed and paraffin-embedded tissue specimens with proper protease pretreatment.