Objective To establish a method to quantitatively assess melanosome transfer with incorporation of 14C-thiouracil (TU) into nascent melanin. Methods To characterize whether 14C-TU was exclusively incorporated into melanin-producing cells, the same number of mouse melan-a or SP-1 keratinocytes were labeled with 14C-TU for 12 hours and 48 hours, respectively, followed by the measurement of radioactivity. Mouse melan-a melanocytes were pre-labeled with 1 Ci/mL 14C-TU, and cocultured with mouse SP1 keratinocytes to develop an assay system for melanosome transfer to keratinocytes. Following co-culture, the keratinocytes with transferred radioactivity were separated from melanocytes at different time points via two times of differential trypsinization. Transferred radioactivity in keratinocytes, denoting the amount of melanosome transfer, was measured with liquid scintillation counting. Meanwhile, the effects of forskolin, a PKA activator, and nicotinamide on melanosome transfer were also investigated with this assay system.Results The incorporated radioactivity in melan-a cells was 66- or 80-fold as high as that in SP-1 cells,indicating that 14C-TU would be a suitable tracer for melanosome transfer in co-culture with keratinocytes. A purity of 84.5% was achieved for keratinocytes with transferred radioactivity by twice differential trypsinization.As shown by this assay, there was an approximately 0.67-fold decrease in melanosome transfer with the treatment of 1 g/L nicotinamide and 2.3-fold increase with 20μmol/L forskolin treatment. After coculture with SP1 cells for 8-12 hours, melan-a cells developed well-extending dendrites with detectable melanosome transfer, while no proliferation of melan-a cells induced by forskolin was seen. Conclusion An optimized protocol for selective incorporation of 14C-TU into nascent melanin has been successfully applied to the quantitative measurement of melanosome transfer from melanocytes to keratinocytes induced by forskolin or nicotinamide.

< 0.05; 8.9% vs 0.1%, x2 = 8.23, P< 0.05). Conclusion Low concentration (0.1%) of DMSO could enhance the effect of ALA-PDT on HaCaT cells.

Objective To investigate the effects of ciprofloxacin on dermal collagen synthesis and profibrotic gene expressions in an experimental mouse model of scleroderma induced by bleomycin. Methods Experimental mouse models of scleroderma were established by subcutaneous injection of bleomycin into the dorsal skin of 15 BALB/c mice for 4 consecutive weeks. Then, the mouse models were randomly and equally divided into 3 groups to be topically treated with 1% ciprofloxacin cream (ciprofloxacin group), 2.5% asiaticoside cream (asiaticoside group)and cream vehicle (model group)respectively for 5 consecutive weeks. Five mice firstly injected with sterile phosphate buffered saline (PBS)for 4 weeks then topically treated with cream vehicle for 5 weeks served as the blank control group. After the 5-week topical treatment, all the mice were sacrificed, skin specimens were resected from the dorsal skin of them, and subjected to HE staining and Masson staining. Further more, an immunohistochemical assay was performed to measure the expressions of type I collagen (COL-1), matrix metalloproteinase-1 (MMP1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1), semi-quantitative reverse transcription PCR to quantify the expressions of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGFβ1)and Smad3 genes, and alkaline hydrolysis-spectrophotometry to determine the level of hydroxyproline in skin. Statistical analysis was carried out by one-way analysis of variance and the least significant difference(LSD)test with the SPSS 17.0 software. Results Compared with the blank control group, the model group showed increased dermal thickness at injection sites (432.76 ± 93.74 μm vs. 301.69 ± 79.47 μm, P < 0.01). Masson staining revealed thick and dense collagen bundles in an irregular arrangement in the dermis in the model group, which was consistent with dermal fibrosis in scleroderma. The total content of collagen and staining intensity of COL-1, MMP1 and TIMP1 were all significantly decreased in the ciprofloxacin group and asiaticoside group compared with the model group (F = 1628.54, 33.29, 84.82, 224.81, respectively, all P < 0.01), while no significant changes were observed in dermal thickness (both P > 0.05). Moreover, compared with cream vehicle, asiaticoside down-regulated the expressions of the three profibrotic genes(CTGF, TGFβ1 and Smad3)to different extents (all P < 0.05), while ciprofloxacin only inhibited the expressions of TGFβ1 and Smad3 genes (both P < 0.05)with no significant effect on CTGF gene expression (P > 0.05). Conclusion Ciprofloxacin may counteract dermal fibrosis by inhibiting the TGFβ1/Smad3 pathway and modulating the unbalanced expressions of MMP1 and TIMP1.

Objective To investigate the susceptibility ofepidermal cells to ultraviolet A(UVA)-induced apoptosis in dopachrome tautomerase knockout Dct~(-/-) mice versus wildtype C57BL/6J mice.Methods High titer of anti-Ro/SSA-positive sera collected from three patients with SLE and typical cutaneous phntosensitivity were intraperitoneally injected into both Dct~(-/-) and wildtype mice,which were then chronically exposed to UVA irradiation at a single dose of 10 J/cm~2 three times a week for two weeks.Then,UVA-irradiated tail skin was excised from each mouse,embedded with paraffin,cut into 4 to 5-μm sections followed by hematoxylin/eosin staining and terminal deoxynucleotidyl transferase nick end labeling(TUNEL),respectively,for the counting of sunburn cells(SBC) and apoptotie cells.Results After chronic UVA exposure,the number of SBC and TUNEL-positive cells per 100 epithelial cells was significantly higher in serum-injected Dct~(-/-) mice than in serum-injected wildtype mice(14±1.0 vs 7±-0.6,62±2.7 vs 30 ±1.6,both P<0.05).A significant decrease was also observed in the number of SBC (6 ±0.9 per 1 00 epithelial cells)and TUNEL-positive cells (42±2.5 per 100 epithelial cells)in uninjected Dct~(-/-) mice compared with those of serum-injcoted Dct~(-/-) mice(both P<0.05).Conclusions The deficiency of Dct gene increases the susceptibility of epidermal cells to UVA-induced apoptosis under the presence of anti-Ro/SSA antibody,which potentially contributes to the develop-ment of anti-Ro/SSA antibody-mediated photosensitivity in SLE.

In the bulge region of the hair follicle, a densely and concentrically packed cell mass is encircled by the arrector pili muscle (APM), which offers a specilized microenvironment (niche) for housing heterogeneous adult stem cells. However, the detailed histological architecture and the cellular composition of the bulge region warrants intensive study and may have implications for the regulation of hair follicle growth regulation. This study was designed to define the gene-expression profiles of putative stem cells and lineage-specific precursors in the mid-portions of plucked hair follicles prepared according to the presence of detectable autofluorescence. The structure was also characterized by using a consecutive sectioning technique. The bulge region of the hair follicle with autofluorescence was precisely excised by employing a micro-dissection procedure. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to identify the gene expression profiles specific for epithelial, melanocyte and stromal stem cells in the bulge region of the hair follicle visualized by autofluorescence. The morphology and its age-dependent changes of bulge region of the hair follicles with autofluorescence segment were also examined in 9 scalp skin specimens collected from patients aged 30 weeks to 75 years, by serial sectioning and immuno-staining. Gene expression profile analysis revealed that there were cells with mRNA transcripts of Dct(Hi)Tyrase(Lo)-Tyrp1(Lo)MC1R(Lo)MITF(Lo)/K15(Hi)/NPNT(Hi) in the bulge region of the hair follicle with autofluorescence segments, which differed from the patterns in hair bulbs. Small cell-protrusions that sprouted from the outer root sheath (ORS) were clearly observed at the APM inserting level in serial sections of hair follicles by immunohistological staining, which were characteristically replete with K15+/K19+expressing cells. Likewise, the muscle bundles of APM positive for smooth muscle actin intimately encircled these cell-protrusions, and the occurrence frequency of the cell-protrusions was increased in fetal scalp skin compared with adult scalp skin. This study provided the evidence that the cell-protrusions occurring at the ORS relative to the APM insertion are more likely to be characteristic of the visible niches that are filled with abundant stem cells. The occurrence frequency of these cell-protrusions was significantly increased in fetal scalp skin samples (128%) as compared with the scalp skins of younger (49.4%) and older (25.4%) adults (P<0.01), but difference in the frequency between the two adult groups were not significant. These results indicated that these cell-protrusions function as a niche house for the myriad stem cells and/or precursors to meet the needs of the development of hair follicles in an embryo. The micro-dissection used in this study was simple and reliable in excising the bulge region of the hair follicle with autofluorescence segments dependent on their autofluorescence is of value for the study of stem cell culture.

Objective To evaluate the effects of δ-aminolaevulinic acid-photodynamic therapy (ALA-PDT) in the treatment of primary carcinomas on the facial skin. Methods In the accordance of these tumors' sites and morphology, 14 patients with squamous cell carcinoma (SCC), 38 patients with basal cell carcinoma (BCC) and 5 patients with Bowen disease were given four to eight times of topical ALA followed by PDT. Results Ten (71.4%) SCC cases, 34 (89.5%) BCC cases and all (100%) Bowen disease cases completely recovered after ALA-PDT. The others all obtained signifi-cant improvement after final treatment. Their unaffected tissues around these tumors kept well and no scaring appeared after ALA-PDT. The recurrence rates among the completely-recovered cases were 10.0% (SCC), 11.8% (BCC) and 0% (Bowen disease), respectively, by the end of six-month's follow-up. Conclusions Topical ALA-PDT is an effective new therapeutical method with lower recur-rence rates, fewer side effects, no scar formation and excellent cosmetic results for primary carcinomas localizing on the facial skin.

Objective To study the molecular genetic polymorphism of exons 1,5, 6, 7 of HLA-C gene in Chinese population and evaluate the significance of additional sequencing based typing at exons 1,5, 6, 7 of HLA-Cw gene in clinical HLA matching, Methods A total of 324 individuals were typed at exons 2,3, 4 of HLA-C gene by sequence-based typing. If ambiguities appeared outside of exons 2 -4, we designed a total of 5 in-house sequencing primers and optimized the sequencing reaction, additional sequencing based typing at exons 1,5, 6, 7 was performed to solove the emerging ambiguities. Results In the three hundred and twenty-four samples typed by PCR-SBT at exons 2, 3 and 4 of HLA-Cw gene, 23.8 % (77/324) of the typed samples were assigned the conclusive genotype in four digital level 76. 2% (247/324) of the typed samples were given with the ambiguous allele combination results, in which 73 kinds of ambiguous allele combinations were detected. Increasing the additional sequencing analysis at exons 1, 5, 6, 7 of HIA-C gene, ten frequent ambiguities including Cw* 030201/030202, Cw* 070201/0750, Cw* 040101/0409N/0430, Cw* 0403/0409N/0430, Cw* 080101/0822 could be distinguished. ConclusionsIncreasing the sequencing anlysis at exons 1, 5, 6 and 7 of HLA-Cw gene will help to make clear the ambiguous SBT results and also improve the accuracy of HLA-Cw typing. It shows important significance in clinical histoeompatibility matching.

0.05). After treatment, the amount of urinary protein and anti-dsDNA antibodies level in leflunomide group were significantly lower than those in dexamethasone and saline groups (P

Objective To investigate the effect of CpG sequences on t he induction of anti-dsDNA antibodies and the possible mechanism. Methods Usin g a synthetic oligodeoxynucleotide (CpG-ODN) containing CpG as an adjuvant, nati ve calf thymus DNA (nCTDNA) was used as the mimic self antigen to immunize norma l BALB/c mice. About one week after immunization, anti-dsDNA antibodies were tes ted by ELISA using nCTDNA and native Escherichia coli DNA (nECDNA) as test antig ens. Results The levels of anti-dsDNA antibodies and cytokines including IL-6, IL-12, IFN- but the binding abilities were different significantly. Conclusion The CpG motif can promote the product ion of cross-reactive anti-dsDNA antibodies in normal mice.

Objective To evaluate the efficacy of embryo thymus transplantation in the treatment of lupus-like BXSB mice,study the pathogenesis of SLE in BXSB mice and the therapeutic effect of embryo thymus transplantation.Methods The embryonic thymus of CB57L mice was transplanted to50day-old male BXSB mice.Levels of proteinuria,ANA,blood urea nitrogen(BUN),blood creatinine and the deposits of immunoglobulin(Ig)in the glomerulus were detected regularly for5months,and the number of mice died of SLE was observed.Results The levels of proteinuria,ANA,BUN,blood creatinine of the5,6,7month old mice in embryo thymus transplantation group were lower than that of5month old mice in control group.The efficacy of treatment in embryo thymus transplantation group were similar to that of dexamethasone treatment group,and the SLE-caused death was reduced in these two groups.However,the embryo thymus transplantation seemed not to reduce the deposits of lg in glomerulus significantly,the deposits of lg in the glomerulus were similar in all three groups.Conclusions Embryo thymus transplantation could improve renal functions and reduce the titer of ANA.Its efficacy is similar to that of dexamethasone.Embryo thymus transplantation has a short term therapeutic effect in the treatment of lupus-like BXSB mice.The deficiency of thymus in the BXSB mice may play an important role in the pathogenesis of lupus-like mice.Embryo thymus transplantation may be a valuable approach to treat SLE.