Our previous works disclosed the contributing role of macrophage migration inhibitory factor (MIF) and dopaminergic inhibition by lysine dimethyltransferase G9a/Glp complex in peripheral nerve injury-induced hypersensitivity. We herein propose that the proinflammatory cytokine MIF participates in the regulation of neuropathic hypersensitivity by interacting with and suppressing the descending dopaminergic system. The lumbar spinal cord (L-SC) and ventral tegmental area (VTA) are two major locations with significant upregulation of MIF after chronic constriction injury (CCI) of the sciatic nerve, and they display time-dependent changes, along with a behavioral trajectory. Correspondingly, dopamine (DA) content shows the reverse characteristic change to MIF with a time-dependent curve in post-surgical behavior. The levels of both MIF and DA are reversed by the MIF tautomerase inhibitor ISO-1, and a negative relationship exists between MIF and DA. The reversed role of ISO-1 also affects tyrosine hydroxylase expression. Furthermore, CCI induces Th promoter CpG site methylation in the L-SC and VTA areas, and this effect could be abated by ISO-1 administration. G9a/SUV39H1 and H3K9me2/H3K9me3 enrichment within the Th promoter region following CCI in the L-SC and VTA was also decreased by ISO-1. In cultured dopaminergic neurons, rMIF enhanced the recruitment of G9a and SUV39H1, followed by an increase in H3K9me2/H3K9me3. These molecular changes correspondingly exhibited alterations in Th promoter CpG site methylation and pain behaviors. In summary, MIF functions as a braking factor in curbing dopaminergic descending inhibition in peripheral nerve injury-induced hypersensitivity by mediating Th gene methylation through G9a/SUV39H1-associated H3K9 methylation.

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 evaluate the effectiveness of swallowing training supplemented with neuromuscular electrical stimulation to provide a reference for clinical treatment and further study.Methods Reports of randomized and controlled trials of surface neuromuscular electrical stimulation in treating post-stroke dysphagia were sought in the Cochrane library,the PubMed and Embase databases,the Cumulative Index to Nursing and Allied Health Literature (CINAHL),and also in the ProQuest,PsycARTICLES,CBMdisc,China National Knowledge Infrastructure (CNKI),CQVIP database and Wanfang databases.All of the literature found was evaluated by 2 researchers according to predefined inclusion and exclusion criteria and the data were extracted and combined.Then meta-analysis was performed using version 5.3 of the RevMan software package.Results Eleven randomized and controlled trials involving 576 patients were included in the meta-analysis.Together,the data showed that swallowing training supplemented by neuromuscular electrical stimulation is significantly more effective than swallowing training alone in improving swallowing function.It reduces the risk of aspiration and improves quality of life.It does not,however,generally shorten the pharyngeal transmit time.Conclusions Swallowing training supplemented with neuromuscular electrical stimulation is a promising approach for treatment of post-stroke dysphagia and warrants further study.

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.

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 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.

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.

< 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 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.