Objective To assess the microscopic features of lentigo by using in vivo confocal laser scanning microscopy (CLSM).Methods This study included 30 patients clinically diagnosed with solar lentigo and 10 patients with lentigo simplex.Lentigo lesions and perilesional normal skin were examined by in vivo CLSM.Tissue specimens were also obtained from lesional and perilesional normal skin and subjected to routine histopathologic examination.Results The CLSM features of lentigo were mainly observed at the dermo-epidermal junction.In all of these cases of lentigo,there was an increase in the length and number of rete ridges,with a marked hyperpigmentation of the basal layer.Several distinct microscopic patterns were observed,such as extended,irregularly shaped papillary dermis surrounded by highly refractile cells (presumed to be basal keratinocytes).Histopathologically,there was an infiltrate of a small quantity of melanophages and lymphocytes in superficial dermis.Conclusion As far as lentigo lesions are concerned,CLSM images are consistent with histopathological findings.

Objective To investigate the confocal laser scanning microscopic (CLSM) features of several hypopigmented diseases, including vitiligo, nevus depigmentosus, progressive macular hypomelanosis and amelanotic nevus. Methods A total of 131 patients with hypopigmented diseases were recruited in this study. CLSM was performed on depigmented lesions, adjacent normal appearing skin and lesion borders at the dermoepidermal junction level in these patients. Results CLSM showed that in progressive vitiligo, melanin was completely absent from some areas in vitiliginous skin, while some areas were present with residual dermal papillary rings which were less intact with decreased melanin content; the border of lesions was obscure; some dermal papillary rings lost integrity in normal appearing skin adjacent to the vitiliginous skin. There was a complete disappearance of pigment in vitiliginous skin, clear border of lesions and intact dermal papillary rings with bright reflected light in adjacent normal appearing skin in stable vitiligo. Dendrite-like melanocytes with bright reflected light were noted in recovery-stage vitiligo. The CLSM imaging of nevus depigmentosus and progressive macular hypomelanosis was similar: dermal papillary rings were intact with decreased melanin content and weak reflected light in depigmented lesions. No significant difference was observed in the structure of dermal papillary rings or melanin content between vitiliginous skin and adjacent normal-appearing skin of nevus anemicus. Conclusion Combined with clinical manifestations, CLSM may serve as an auxiliary tool to discriminate vitiligo, nevus depigmentosus, progressive macular hypomelanosis and amelanotic nevus.

ObjectiveTo observe the lesions of psoriasis vulgaris by confocal laser scanning microscopy(CLSM).MethodsThe typical lesions of 120 patients with psoriasis vulgaris were observed by CLSM,and scanning results were recorded.ResultsOf the 120 patients with psoriasis vulgaris,all (100％)had dilated and twisted capillaries in dermal papillae,113 (94.17％) acanthosis,106 (88.33％) parakeratosis,86 (71.67％) Munro microabscesses at the level of stratum corneum,71(59.17％) diminished or disappeared stratum granulosum.There were a few(1 to 2) dilated capillary lumens in papillary dermis,with no obvious reduction in the distance between dermal papillae.Some dermal papillary rings lost their integrity or brightness slightly in 24 of the 27 patients with psoriasis guttata.The height of stratum spinosum was 96.85 ± 17.88 μm (ranged:80-150 μm) in patients with psoriasis guttata,lower than that in those with plaque psoriasis (average:143.45 ± 23.19 μm,range:100-190 μm).In lesions of plaque psoriasis,the capillaries in dermal papillae were apparently dilated with hyperemia,showing a characteristic'S' shape or horseshoe appearance; the diameter of dermal papillae was increased and the distance between dermal papillae was reduced,giving a 'transverse section of lotus roots' appearance.Dermal papillary rings completely disappeared in 25 of the 29 patients with plaque psoriasis.ConclusionCLSM may serve as an aid in the diagnosis of psoriasis vulgaris.

Objective To evaluate the long-term outcome ofautologous epidermal grafting in vitiligo and its correlated factors. Methods A 1-5 year follow-up of 310 patients with vitiligo, who had been treated by autologous epidermal grafting, was carried out by questionnaires, phone interview, and clinical revisit. The relevance was estimated between the efficacy of the treatment and several factors, including the time length from the grafting to the follow up, patients' age and gender, location of lesions treated, Koebner phe-nomenon on the donor sites, and the combination therapy with narrow band-ultraviolet B (NB-UVB). Results Of the 310 patients, 161 were males and 149 females with an average age of 26.69±11.76 years; 98 had localized vitiligo, 123 segmental vitiligo, 62 scattered vitiligo, 27 generalized vitiligo. Totally, 24 recipient areas with 1266 sheets were evaluated. Among these sheets, 815 (64.38%) achieved complete repigmenta- tion, 167 obvious repigmentation, 117 some repigmentation, 167 no repigmentation, with a curative rate of 64.38% and total effective rate of 86.81%. Hyperpigmentation occurred in the recipient area of 76 (24.52%) patients, and donor area of 34 patients (10.97%), and hypopigmentation developed in the recipient area of 66 patients (21.29%). Relapse developed in the recipient areas of 30 patients, and new lesions in 12 patients (3.871%). Koebner phenomenon was noticed in the donor sites of 25 (8.064%) patients. The efficacy of autologous epidermal grafting was stable within two years after the therapy, but reduced with the time. Increased efficacy was observed in those sites with little mobililty and large fixity. For example, the best effi- cacy was observed in the legs and arms, followed by feet and hands, chest and back, face and neck, finally by waist and abdomen (P<0.01). NB-UVB exposure could improve the repigmentation. Koebner phe- nomenon in donor sites was associated with a reduced efficacy of epidermal grafting (P<0.05). Gender and age seemed unrelated to the efficacy. Conclusions Autologous epidermal grafting has a good long-term efficacy for the treatment of vitiligo, and transplanted location may be the most important factor affecting the efficacy.

Objective To investigate the dermoscopic and confocal microscopic features of Riehl's melanosis,as well as their association with histopathological findings.Methods Ten patients with a previously established diagnosis of Riehl's melanosis were recruited.The lesions of the patients were observed using dermoscopy and in vivo confocal laser scanning microscopy (CLSM),followed by histopathologic analysis.Results On dermoscopy and CLSM,all the lesions showed the following features:pseudonetwork,liquefaction degeneration of the basal cell layer,and incontinence of pigment.Conclusion Both dermoscopy and in vivo CLSM can serve as noninvasive auxiliary diagnostic tools for Riehl's melanosis.

Objective To observe the effect of hyaluronic acid on the proliferation of and tyrosinase activity in melanocytes.Methods Normal primary human melanocytes were isolated from infant foreskin tissue and cultured.Different concentrations(0 to 10 g/L)of hyaluronic acid wero added to the culture medium immediately or 8 hours after the inoculation of melanocytes.MTT assay was performed to detect the proliferation of melanocytes,and tyrosinase activity was determined to evaluate the effect of hyaluronic acid on the melanin synthesis by melanocytes.Results The proliferation level (absorbance at 490 am,A490)of melanocytes was 0.14±0.02,0.37±0.08,0.45±0.11,0.49±0.07,0.55±0.12,0.52±0.11,0.49±0.07,0.39±0.05,0.19±0.03 and 0.01 4-0.01 when treated with hyaluronic acid of 0,0.008,0.016,0.313,0.625,1.250,2.500,5.000,7.500 and 10.000 g/L,respectively.The hyaluronic acid of 0.08 to 5 g/L markedly accelerated the proliferation of melanocytes,while that of 10 g/L inhibited their proliferation.The tyrosinase activity in melanocytes was promoted by hyaluronie acid of 0.2 to 5 g/L,but suppressed by that of 10 g/L.The proliferation of melanocytes treated with hyaluronic acid immediately after the inoculation was more rapid than that treated with hyaluronic acid 8 hours after the inoculation.Conclusion The hyaluronic acid of 0.2 to 5 g/L can enhance the proliferation of and tyrosinase activity in melanocytes.

Objective To characterize the progression of segmental vitiligo.Methods Clinical data were collected using questionnaires from 387 patients with segmental vitiligo at the Department of Dermatology,Third People's Hospital of Hangzhou,between October 2011 and October 2012.The progression of segmental vitiligo was analyzed.Results Among the 387 patients,329 initially sufferred from focal vitiligo that evolved into segmental vitiligo later,58 began with segmental vitiligo.Vitiligo progressed most rapidly in the initial three months,and tended to be stable after three years in 220 (66.9％) of the 329 patients experiencing the evolution from focal to segmental vitiligo.By contrast,vitiligo developed most rapidly in the first month,and tended to be quiescent after one year in 40 (69.0％) of the 58 patients presenting with segmental vitiligo only.The condition still remained active in 27.6％ (107/387) of these patients after several years of progression.Totally,333 (86.0％) patients showed the involvement of single ganglion at the onset of vitiligo,including 173 (52.0％) patients with the involvement of trigeminal ganglion.Autologous melanocyte transplantation was conducted for 62 patients whose condition had been quiescent for more than one year,and 56 (90.3％) patients achieved more than 80％ repigmentation.Conclusions Segmental vitiligo,which tends to remain quiescent after three-year progression,seems to have a more rapid onset than focal vitiligo.Autologous melanocyte transplantation appears to be an effective treatment for segmental vitiligo in stable stage.

Objective To analyze the clinical features and evolution ot segmental vitiligo in children.Methods A standardized questionnaire was used to clinically investigate segmental vitiligo in 170 children aged ＜ or =12 years and 174 adults with an age of onset ＞ 18 years.Data analysis was done by the software SPSS16.0.Results The female to male ratio was 108:62 in the 170 child patients,significantly different from that in the adult patients (x2 =14.44,P ＜ 0.05).Localized vitiligo occurred as the initial presentation in 82.9％ (141/170) ofthe children and 76.4％ (133/174) of the adults,and progressed into segmental vitiligo in half a year in 76.5％ of the 141 children and 63.9％ of the 133 adults.Vitiligo entered the quiescent stage after half-a-year progression in 71.6％ of the children and 67.3％ of the adults with localized vitiligo as the initial presentation,and after one-month progression in 62.1％ of the children and 41.5％ of the adults with segmental vitiligo as the first presentation (x2 =8.39,P ＜ 0.01).Head and face were affected at the onset of segmental vitiligo in 44.1％ of the childhood cases and 56.9％ of the adult cases.Single nerve segments were involved at the onset in 94.7％ of the child patients and 86.8％ of the adult patients (x2 =0.04,P ＞ 0.05).The proportions of patients with white hairs,autoimmune diseases,and family history were significantly lower in the child patients than in the adult patients (x2 =15.88,5.62,6.66 respectively,all P ＜ 0.05).Conclusions Childhood-onset segmental vitiligo shows a predilection for males,usually presents as localized vitiligo at the onset,and becomes quiescent after half-a-year progression in more than 70％ of patients.

Objective To establish a model for studying CD8+ cytotoxic T lymphocyte proliferation in vitro and to screen traditional Chinese drugs (TCDs) with immunosuppressive effects.Methods Spleen tissue was isolated from mice,and made into single cell suspensions followed by separation of CD8+ T lymphocytes with specific antibodies.Then,the CD8 + T lymphocytes were seeded into anti-CD3/CD28 antibody-coated 96-well plates and cocultured with the extracts of 23 TCDs (100 mg/L) separately for 96 hours.Those ceils cultured with and without the presence of anti-CD3/CD28 antibody alone served as the positive control and negative control respectively.The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay was performed to evaluate the proliferation of cells and to select the top four TCDs with the strongest inhibitory effect.The relationship between the inhibitory effect and TCD concentrations was further assessed for the four selected TCDs.Enzyme-linked immunospot (ELISPOT) assay was carried out to estimate the influence of the four TCDs on the secretion of interferon (IFN)-γ by CD8+ T lymphocytes induced by anti-CD3/CD28 antibodies.Statistical analysis was done by nonparametric rank sum test.Results Of the 23 TCDs,14 significantly inhibited the proliferation of CD8+ T lymphocytes (all P ＜ 0.05),of which,Rhizoma Coptidis,Radix Scutellariae,Radix Aucklandiae and Rhizoma Curcumae Longae displayed the strongest inhibitory capacity with the 50％ inhibitory concentration being 25,35,50 and 60 mg/L respectively,and the 100％ inhibitory concentration being 200,100,200 and 200 mg/L respectively.The anti-CD3/CD28 antibody-induced secretion of IFN-γby CD8+ T lymphocytes was markedly suppressed by Radix Scutellariae,Radix Aucklandiae and Rhizoma Curcumae Longae at the concentration of 100 mg/L,but not by Rhizoma Coptidis at this concentration.Conclusions A model for studying the proliferation of CD8+ T lymphocytes is successfully developed in vitro,and four TCDs with strong inhibitory effects on the proliferation of CD8+ T lymphocytes have been screened out with this model.

Objective To evaluate the effect of human dermal mesenchymal stem cells (DMSCs) on the expression and secretion of interleukin (IL)-13 by perilesional CD8+ T lymphocytes from patients with vitiligo.Methods Tissue specimens were obtained from the perilesional region of six patients with active vitiligo,and CD8+ T lymphocytes were isolated from both the tissue specimens and peripheral blood of these patients.DMSCs and melanocytes were obtained from the foreskin tissue of healthy males.The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay was performed to estimate the effect of different concentrations of recombinant IL-13 on the proliferation of melanocytes,reverse transcripition-PCR and Western blotting to detect the mRNA and protein expressions of IL-13 in perilesional and peripheral blood CD8+ T lymphocytes respectively,real-time quantitative reverse transcription (RT)-PCR and enzyme-linked immunosorbent assay (ELISA) to detect the IL-13 mRNA expression in,and IL-13 protein expression in the culture supematant of,CD8+ T lymphocytes before and after coculture with DMSCs,respectively.Statistical analysis was done by t test.Results No obvious changes were observed in the proliferation of melanocytes treated with different concentrations (10,50,100,250,500 μg/L) of recombinant IL-13 for various durations (24,48,72 and 96 hours)compared with untreated melanocytes (all P ＞ 0.05).Both perilesional and peripheral blood CD8+ T lymphocytes expressed IL-13,and the expression was stronger in perilesional than in peripheral blood CD8+ T lymphocytes.A significant decrease was noted in IL-13 mRNA expression (0.100 0 ± 0.002 4 vs.0.383 2 ± 0.018 7,P ＜ 0.05) and protein level in the culture supernatant ((1 509.62 ± 48.44) ng/L vs.(5 507.98 ± 34.11) ng/L,P ＜ 0.05) of CD8+ T lymphocytes cocultured with DMSCs compared with monocultured CD8+ T lymphocytes.Conclusions There is a strong expression of IL-13 by perilesional CD8+ T lymphocytes in patients with vitiligo,which may be inhibited by DMSCs and serve as a target for the treatment of vitiligo.