Objective To investigate the effect of immunoglobulin G (IgG) in bullous pempbigoid (BP) blister fluid on the secretion of chemokines by human keratinocytes. Methods IgG was obtained from the blister fluid of patients with bullous pemphigoid and sera of normal human controls, then purified by sequential precipitation with caprylic acid and ammonium sulfate. The immunological activity of blister fluid was tested before and after the purification by BP180 ELISA kit. Keratinocytes were isolated from the foreskin tissue of yong adults, and subjected to primary culture. After 3 passages, the primary keratinocytes were harvested and subcultured in the presence of purified IgG of 0.5, 1, 2, 4 and 8 g/L, respectively, for 24 hours, or IgG of 4 g/L for 3, 6, 12, 24 hours, respectively, followed by the detection of levels of eotaxin, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 in the supemate of keratinocytes by ELISA. Results The valence of IgG remained unchanged after the purification with caprylic acid and ammonium sulfate. Compared with IgG from the sera of normal controls, that from bullous pemphigoid blister fluid sig- nificantly enhanced the secretion of IL-8 by keratinocytes in a time- and dose-dependent manner (both P < 0.01 ). Neither eotaxin nor MCP-1 was detected in the supemate of control IgG-treated, BP IgG-treated or untreated keratinocytes. Condusions The IgG in BP blister fluid has been proved to stimulate the secretion of IL-8 by cultured human keratinocytes, which may be involved in the pathogenesis of BP.

Objective To investigate the role of AP-1 in the pathogenesis of chronic idiopathic urticaria (CIU). Methods By using immunomagnetic separation technology, peripheral blood basophils were isolated from 10 CIU patients and 10 normal human controls followed by the extraction of nuclear protein from the basophils. TransAMTM AP-1 family kit was used to detect the DNA binding activity changes of AP-1 family transcription factors in basophils, and Western blotting to detect the expression of P-c-jun protein. Results There were some differences in the DNA binding activity of AP-1 family transcription factors in basophils between CIU patients and normal controls. The DNA binding activity of Phospho-c-jun, c-fos, Fos-B, Jun-B and Jun-D factors was increased in CIU patients compared with the controls, and the increase in that of P-c-jun and Jun-D was statistically significant (both P < 0.05). There was an insignificant decrease in the DNA binding activity of Fra-1 factor in the CIU patients compared with the controls (P > 0.05). The P-c-jun (Ser73) protein expression was higher in CIU patients than that in the controls (0.527 ± 0.312 vs. 0.435 ± 0.042, P < 0.05),whereas there was no significant difference in the P-c-jun (Ser63) protein expression level. Conclusion Some changes in DNA binding activity of AP-1 and overexpression of P-c-jun (Ser73) protein in basophils may be involved in the pathogenesis of CIU.

Objective To study the level of expression of RAR?/RXR?mRNA in skin lesions of patients with psoriasis vulgaris.Methods The skin biopsies were collected fro mskin lesions in 20cases of psoriasis vulgaris and 10normal controls.Expression o f RAR?/RXR?mRNA was detected by RT -PCR.Results Expression levels of RXR?mRNA were 0.19760?0.0933in epiderm ides from psoriatic lesions,which were markedly lower than those in normal controls(0.5867?0.0132)(P0.05).Conclusion The results indicate that decreased expression of RXR?mRNA might be related to the pathogen esis of psoriasis.

Objective To study level of the expression of RAR? mRNA in the skin lesions of patients with psoriasis vulgaris. Methods The biopsies were taken from skin lesions in 20 patients with psoriasis vulgaris and the skin of 10 normal controls, and levels of RAR? mRNA were investigated with RT-PCR. Results The level of RAR?mRNA was significanlly lower in the epidermis of patients with psoriasis vulgaris than that in the control(P

Objective To explore the expression of neutrophil adhesion molecule CD11b,and plasma level of elastase in patients with anaphylactoid purpura during different clinical phases and their correlation with disease activity.Methods A total of 20 patients with anaphylactoid purpura were recruited into this study,along with 20 normal human controls.Two blood samples were collected from each patients at the first visit(active phase)and after 3～5 weeks of treatment(remission phase).The expression of CD11b was measured by flow cytometry in 12 patients and normal controls,and plasma levels of elastase by ELISA in 20 patients and normal controls.Results Increased CD11b expression and elastase level were noted in patients in active phase compared with those in patients in remission phase(3367.25±434.57 vs 2569.33±411.06.13.98±2.05 vs 4.29±0.80.both P＜0.01).No significant difference was found in CD11b expression between patients in remission phase and normal controls(P＞0.05).while the elastase level was higher in patients in remission phase than in normal controls(4.29±0.80 vs 3.67±0.54.P＜0.05).In active phase of anaphylactoid purpura,the expression of CD11b was positively correlated with the plasma level of elastase(r=0.73,P＜0.01),while no correlation was noticed between them in remission phase(r=0.20,P=0.54).Conclusion Peripheral neutrophils are activated in anaphylactoid purpura,which seems to be more obvious in active phase than in remission phase.

Objective To investigate the mRNA expression of Fc gamma RⅡA(FcγR ⅡA)on polymorphonuclear neutrophils(PMN)from patients with Beh(c)et's disease(BD).Methods Twenty-five patients with active BD and 20 healthy human controls were included in this study.Blood samples were obtained from all patients with active BD before treatment,from 15 patients with inactive BD after treatment and from healthy controls.PMN were isolated.The FcγR Ⅱ A mRNA expression on PMNs was detected by RT-PCR,and plasma myeloperoxidase (MPO)activity which represented neutrophil activation,was measured spectrophotometricaily.Results The relative expression level of FcγR Ⅱ A on PMN and plasma MPO aetivity were 1.80 1±0.829 and 32±5 U/L.respectively,in patients with active BD,0.820±0.625 and 27±4 U/L,respectively,in those with inactive BD,and 0.745 ±0.931 and 29±5 U/L,respectively,in normal controls;the differences were significant in the two parameters between the patients with active and inactive BD (both P＜0.01),while no statistical difference was observed between inactive patients and normal human controls(P＞0.05).There was a positive eorrelation between the expression level of FcγR Ⅱ A on PMN and plasma MPO activity in patients with BD(r=0.39,P＜0.0 1).Conclusions The mRNA expression of FcγR ⅡA on neutrophils is up-regulated in patients with active BD.It is likely that FcγR Ⅱ A is involved in the activation of neutrophils in BD.

Objective To investigate the effect ofpeptidoglycan from Staphylococcus aureus on the release of several chemokines including intedeukin 8 (IL-8), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage-derived chemokine (MDC) by normal human epidermal keratinocytes (KCs) and the role of Toll-like receptor 2 (TLR2) in this process. Methods KCs were derived from the foreskin of a healthy boy and propagated. After 2 - 4 passages, KCs were collected and treated with various concentrations (3, 10, 30 and 100 mg/L) of peptidoglycan for 24 hours or with peptidoglycan of 100 mg/L for varying durations (3, 6, 12, 36 hours). A fi'action of KCs were pretreated with functional grade purified anti-TLR2 monoclonal antibody before the treatment with peptidoglycan of 100 mg/L. After additional 12-hour culture following the treatment, enzyme linked immunosorbent assay was used to detect the level of IL-8, RANTES and MDC in culture supernatants of KCs. Results KCs spontaneously released IL-8 and RANTES. Peptidoglycan increased the production of IL-8 but decreased that of RANTES by KCs. The levels of IL-8 were 209.96 ± 10.31 ng/L, 250.28 ± 9.52 ng/L, 285.11 ± 10.28 ng/L, 359.40 ± 6.93 ng/L in KCs treated with peptidoglycan of 3, 10, 30, 100 mg/L, respectively, compared to 135.41 ± 14.37 ng/L in untreated KCs (all P < 0.05). On the contrary, a significant decrement was seen in the secretion of RANTES by KCs treated with peptidoglycan of 10, 30, 100 mg/L compared with untreated KCs (110.72 ± 8.51 ng/L, 90.50 ±2.45 ng/L, 49.89 ± 13.74 ng/L vs 149.94 ± 18.71 ng/L, all P < 0.05). The monoclonal antibody to TLR-2 could markedly suppress the promotion of IL-8 production by peptidoglycan, but had no obvious influence on the inhibition of RANTES production by peptidoglycan. MDC could not be detected in the culture super-natants of KCs with or without peptidoglycan stimulation. Conclusion Peptidoglycan could inhibit RANTES secretion but induce IL-8 production by KCs likely via TLR2.

Objective To detect the mRNA and protein expressions of desmoglein 1 (DSG1) and DSG3 in different types of keratinocytes (KCs).Methods Two keratinocyte cell lines HaCaT and A431,as well as primary keratinocytes from human abdomenal skin served as the object of this study.Direct immunofluorescence assay was performed to observe and quantify the expressions of DSG1 and DSG3,and quantitative PCR (qPCR) to determine the mRNA expressions of DSG1 and DSG3,in these cells.Results Both DSG1 and DSG3 were expressed in all the three types of keratinocytes,and the fluorescence intensity of DSG1 and DSG3 in HaCaT cells was higher than that in primary keratinocytes but lower than that in A431 cells.Similarly,all the keratinocytes expressed DSG1 and DSG3 mRNA,with the relative expression levels of DSG1 and DSG3 mRNA in primary keratinocytes being 291.7％ and 237.4％ of those in HaCaT cells respectively (both P ＜ 0.01),and those in A431 cells being 0.1％ and 18.8％ of those in HaCaT cells respectively (both P ＜ 0.05).Conclusions HaCaT cells,A431 cells and primary keratinocytes all can be used for the study of DSG1 and DSG3,of which,A431 cells show the strongest expressions of DSG1 and DSG3,and primary keratinocytes display the highest expressions of DSG1 and DSG3 mRNAs.

ObjectiveTo investigate the correlation between the methylation status of HLA class Ⅰ genes(HLA-A, -B and -C) in psoriatic epidermis and disease severity in patients with psoriasis vulgaris. MethodsDNA specimens were obtained from the lesional and nonlesional epidermis of 46 patients with psoriasis vulgaris and from the normal skin of 28 human controls. Methylation specific PCR (MSP) was conducted to detect the methylation status of CpG islands in the promoter region of HLA-A, -B and -C genes. The severity of psoriasis was evaluated by psoriasis area and severity index(PASI) scores. ResultsThe percentage of promoter methylation of HLA-B and HLA-C genes was 4.35％(2/46) and 21.74％(10/46), respectively in nonlesional epidermis, 4.35％ (2/46) and 4.35％ (2/46), respectively in lesional epidermis from these patients. No methylation was observed for the promoter of HLA-A, -B or -C gene in the normal control epidermis or for that of HLA-A gene in the nonlesional or lesional epidermis from the patients. The frequency of HLA-C gene promoter methylation in the nonlesional epidermis was significantly higher than that in the lesional epidermis and control epidermis, but was uncorrelated to the disease severity. No significant difference was observed for the methylation frequency of HLA-A or -B gene promoter among the three groups of specimens. Conclusion Abnormal methylation of HLA-C gene promoter is observed in patients with psoriasis vulgaris.

Objective To investigate the expression of CD200 in peripheral lymphocytes and hair follicles from patients with alopecia areata (AA).Methods Flow cytometry was used to detect the expression of CD200 in peripheral lymphocytes from 43 patients with AA and 43 healthy controls.Immunohistochemistry was carried out to quantify the expression of CD200 and cytokeratin 15(CK15,a marker for basal cells of the outer root sheath) in resected scalp specimens from 8 patients with AA and 8 healthy controls.Differences in the expression of CD200 and CK15 between the patients and controls were assessed by independent-samples t test and rank sum test.Data were processed by the software SPSS17.0.Results The percentage of CD200-expressing cells in peripheral blood lymphocytes and T lymphocytes was significantly lower in the patients with AA than in the healthy controls (5.73％ ± 3.46％ vs.12.01％ ± 4.90％,8.85％ ± 4.80％ vs.12.31％ ± 3.12％,t =6.865,3.964,respectively,both P ＜ 0.05).However,no significant difference was observed in the percentage of CD200-expressing cells in peripheral blood B lymphocytes between the patients and controls (74.68％ ± 8.12％ vs.75.75％ ± 9.45％,t =0.570,P ＞ 0.05).Further more,the patients showed a lower expression of CD200 (P ＜ 0.05),but a similar expression of CK15 (P ＞ 0.05) in hair follicles compared with the controls.Conclusion The decrease in CD200 expression in peripheral lymphocytes and hair follicles may be involved in the pathogenesis of AA.