Humans ; Melanocytes ; Melanosis ; Fibroblasts ; Skin

It has long been noted that dermabrasion, plum-blossom needling or micro-needling, and even ablative fractional CO 2 laser can effectively induce repigmentation of vitiligo lesions resistant to conventional ultraviolet B phototherapy. In addition to trauma-induced increase in the transdermal absorption of drugs, these treatments also initiate wound repair and activate melanocytes in hair follicles or epidermis. Basal layer keratinocytes, vascular endothelial cells and fibroblasts in the wound margins can secrete chemokine CXCL12 to recruit melanocytes or melanocyte stem cells expressing chemokine receptors CXCR4/CXCR7 in the hair follicle bulge or around the skin lesions to move towards the vitiliginous area. This review summarizes progress in repigmentation of vitiligo lesions induced by therapeutic skin trauma.

Objective:To investigate the expression and distribution of human dermal papillary fibroblasts (Fp) , reticular fibroblasts (Fr) , and myofibroblasts (MFB) in keloid tissues.Methods:Keloid tissues were collected from 15 outpatients (including 8 males and 7 females) aged 20-50 years, who were diagnosed in the Department of Dermatology, Renmin Hospital of Wuhan University from May to December 2019. Normal skin tissues were taken from 15 age-matched women who underwent mammoplasty, and served as controls. The distribution of fibroblast activation protein (FAP) , CD90 and alpha-smooth muscle actin (α-SMA) was observed in the keloid tissues and normal skin tissues by dual immunofluorescence staining. Furthermore, fibroblasts were isolated from 3 normal skin and 3 keloid tissue samples, and subjected to primary culture. Subsequently, the fibroblasts were treated with 10 ng/ml transforming growth factor-β1 (TGF-β1) for 48 hours in vitro, during which, changes in fibroblast phenotypes were observed in the 2 groups. Fluorescence-based quantitative RT-PCR and Western blot analysis were performed to determine the mRNA and protein expression of FAP, CD90 and α-SMA. Measurement data were compared between 2 groups by using t test. Results:Immunofluorescence staining of the normal skin tissues revealed that FAP +/CD90 - fibroblasts were predominantly distributed in the superficial dermis, FAP -/CD90 + fibroblasts in the deep dermis, and CD90 + cells hardly expressed α-SMA; however, a large number of FAP + fibroblasts and CD90 + fibroblasts were observed in the deep keloid tissues, and many CD90 + fibroblasts also expressed α-SMA. Dual immunofluorescence staining showed that normal tissue-derived fibroblasts hardly expressed α-SMA, and keloid-derived fibroblasts expressed α-SMA. The fluorescence intensity of α-SMA + cells significantly increased in the normal tissue-and keloid-derived fibroblasts after 24-hour treatment with TGF-β1 (21.058 ± 0.709, 27.112 ± 0.097, respectively) compared with that in the corresponding untreated fibroblasts (11.312 ± 0.636, 21.306 ± 0.464, t=22.430, 13.370, respectively, both P < 0.05) . RT-PCR and Western blot analysis showed that the mRNA and protein expression of FAP, CD90 and α-SMA significantly increased in the keloid-derived fibroblasts after 48-hour treatment with TGF-β1 (mRNA: 92.610 ± 3.667, 1.366 ± 0.105, 3.240 ± 0.141; protein: 0.652 ± 0.073, 1.046 ± 0.119, 0.946 ± 0.117, respectively) compared with the untreated keloid-derived fibroblasts (all P < 0.05) . Conclusion:CD90 + Fr aberrantly proliferated in the deep dermis of keloid tissues, suggesting that directional intervention in aberrantly proliferating FAP -/CD90 + Fr in the deep dermis may promote the efficacy for keloids.

Objective To determine the expression of cathepsin L2 (CTSL2)and evaluate its activity in skin lesions of seborrheic keratosis (SK),to observe the ultrastructural changes of melanosomes in the skin lesions of SK,and to estimate the effect of CTSL2 on the degradation of melanosomes.Methods Twenty patients with SK were enrolled from the Department of Dermatology,Renmin Hospital of Wuhan University.The lesional tissue and the perilesional normal skin were biopsied from each patient.Among 15 of the 20 patients,hematoxylin and eosin (HE)staining and Fontana-Masson silver staining were performed to observe the distribution of melanin granules,transmission electron microscopy (TEM)was conducted to observe the ultrastructural changes of melanosomes,and immunohistochemical staining was performed to estimate the cellular proliferative activity.RT-PCR and fluorogenic substrate cleavage assay were performed in the other 5 patients to determine the mRNA expression of CTSL2 and evaluate its activity,respectively.Sucrose density gradient ultracentrifugation was performed to isolate and purify melanosomes from the retinal pigment epithelium (RPE) harvested from a discarded eyeball of a 35-year old male patient with informed consent.The purified melanosomes were incubated with epidermal lysates of SK lesions,and TEM was used to observe the changes in the membrane structure of melanosomes.Statistical analysis was carried out by paired t test,and a P value ＜ 0.05 was considered statistically significant.Results A large number of melanin granules were deposited in SK lesions,while the linear deposition of melanin granules was only seen in the basal layer of the normal skin.TEM showed that the percentage of damaged melanosomes was much higher in the normal skin (49.00％ ± 4.00％) than in the SK lesions (24.33％ ± 3.06％)(t =8.49,P ＜ 0.05).RT-PCR revealed that the mRNA expression and activity of CTSL2 were both significantly lower in the SK lesions than in the normal skin (mRNA:0.35 ± 0.09 vs.0.43 ± 0.08,t =3.17,P ＜ 0.05;activity:17.46 ± 0.45 vs.28.78 ± 0.58,t =34.29,P ＜ 0.05).Moreover,TEM also showed that the percentage of damaged melanosome was lower in the SK lesion lysate-treated group (32.33％ ± 4.93％) than in the normal skin lysate-treated group (43.00％ ± 2.65％,t =3.30,P ＜ 0.05).Conclusion Decreased expression of CTSL2 in the SK lesions can affect the degradation of melanosomes by keratinocytes.However,whether CTSL2 directly takes part in the pathogenesis of SK or not is still needed to be further confirmed.

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 evaluate the efficacy and safety of olopatadine hydrochloride for the treatment of chronic idiopathic urticaria (CIU). Methods A multicentre, double-blind, randomized, parallel-group, controlled clinical trial was conducted. A total of 144 patients with CIU from 3 research centers were enrolled into this study, and randomly and equally divided into a test group and a control group. The test group administrated olopatadine hydrochloride 5 mg twice a day for 28 consecutive days, while the control group administrated levocetirizine hydrochloride 5 mg in the forenoon and a placebo tablet of olopatadine hydrochloride 5 mg in the afternoon for 28 consecutive days. The symptom score reducing index(SSRI)served as the primary outcome, and global assessment score for efficacy and total response rates as the secondary outcome. Results Totally, 137 patients completed the trial, including 70 in the test group and 67 in the control group. As intention-to-treat analysis showed, there were no significant differences in the total response rate between the test group and control group on day 7 (64.29% (45/70)vs. 56.72%(38/67), P > 0.05), 14(82.86%(58/70)vs. 74.63%(50/67), P > 0.05), or 28(87.14%(61/70)vs. 77.61%(52/67), P >0.05)after start of treatment. The SSRI was significantly higher in the test group than in the control group after 4 weeks of treatment(82.67% ± 22.70% vs. 70.51% ± 32.07%, P < 0.05). In addition, no significant difference was observed in the incidence of adverse reactions between the test group and control group(33.80%(24/71)vs. 27.94%(19/68), P > 0.05), and adverse reactions mainly included lethargy, dry mouth, fatigue, etc. Conclusion Olopatadine hydrochloride is effective and safe for the treatment of CIU.

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.
Adult ; Adult Stem Cells ; cytology ; Aged ; Hair Follicle ; cytology ; Humans ; Middle Aged ; Young Adult

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 assess the relationships among the autologous serum-induced skin wheal-andflare reaction,ex vivo serum-induced basophil histamine release, and serum levels of IgG anti-FcεRI autoantibodies in patients with chronic idiopathic urticaria (CIU).Methods Sixty patients with CIU collected from the Renmin Hospital of Wuhan University were recruited for this study.Sera were obtained from the subjects,and ASST was performed in all of the subjects.The results of ASST were determined according to a recommended criterion described by Sabroe et al,and the positive results were further subclassified into wheal plus flare (W+F) pattern and wheal-only (W) pattern,negative results into flare-only (F) pattern and no response pattern.Enzyme linked immunosorbent assay was used to quantify the content of histamine released by autologous serum-induced basophils and serum levels of IgG anti-FcεRI autoantibodies.Results Of the 60 patients,19 (31.7％)were positive for ASST,including 16 (84.2％) presenting W+F pattern and 3 presenting W pattern; 41 were negative for ASST,including 3 (7.3％) giving F pattern and 38 giving no response pattern.The histamine release rate was significantly higher in ASST-positive patients than in ASST-negative patients (33.38％ ± 9.83％ vs.4.06％ ±1.44％,t =5.13,P＜ 0.01),and was nearly twice as high as that in basophils induced by 10 μmol/L formylmethionyleucylphenylalanine (Fmlp).The serum levels of IgG anti-FcεRI autoantibodies were high in only patients giving (W+F) pattern (757.64 ± 168.99 ng/L),but low in the normal human controls (43.25 ± 16.63 ng/L).Conclusions The positive ASST result of wheal plus flare pattern is associated with high serum levels of IgG anti-FcεRI autoantibodies,and is suggestive of a clinical diagnosis of autoimmune chronic urticaria (ACU).

Objective To establish a primary culture of human melanocytes from tiny skin sheets harvested by using a suction blister method, to carry out a serial subcultivation of the melanocytes with human amniotic membrane (AM) as a scaffold, and to observe the influence of AM on the adhesion, proliferation and dendrite development of melanocytes. Methods Tiny skin sheets were collected from the flexual forearm or lower abdomen of a healthy male volunteer by a suction blister method and melanocytes in the skin sheet were counted following Dopa staining under a microscope. The trypsinized skin sheets were scraped with a scalpel to harvest melanocytes which were subjected to a primary culture. Then, the melanocytes were inoculated onto fresh or cryopreserved AM followed by a culture for various durations (4, 8 and 12 days). The morphology and dendrite development of melanocytes were visualized under an inverted microscope after dopa-staining, cell viability evaluated by MTT assay, the adhesion to AM examined by hematoxylin and eosin (HE) staining protocol. Results The density of melanocytes was 1543.1±13.3 cells per mm2 and 857.4±101.7 cells per mm2 in skin sheets obtained from the forearm flexure and lower abdomen of the volunteer, respectively. A skin sheet of about 25.1 mm2 from approximately two blister roof was required to ensure the success of primary culture of melanocytes within 1 month. After culture on fresh or cryopreserved AM for 4, 8, and 12 days, most melanocytes were bi-polar with extended slender dendrites compared with those cultured in common cell culture medium. HE staining showed that melancytes adhered and were evenly distributed on the basement membrane of AM. MTT assay showed that the AM inhibited the proliferation of melanocytes, and no statistical difference was observed in the inhibitory effect between fresh AM and cryopreserved AM (P＞ 0.05). Conclusions Enriched with melanocyes, flexural forearm is a preferable donor site to offer skin sheets for primary culture of melanocytes. Human AM could improve the adhesive growth and dendrite development of melanocytes, and may serve as a promising bioscaffold for in vitro expansion of melanocytes.