Changes in circadian gene cryptochrome 2 expression in mouse models of psoriasis and HaCaT cells and their underlying mechanisms / 中华皮肤科杂志

Objective:To investigate changes in circadian gene cryptochrome 2 (CRY2) expression in mouse models of psoriasis and HaCaT cells, and to explore underlying mechanisms.Methods:Imiquimod-induced mouse model experiment: 12 C57BL/6 female mice were randomly and equally divided into imiquimod group receiving topical imiquimod treatment for 5 consecutive days and control group receiving no treatment; these mice were sacrificed on day 6, skin tissues were resected from the back of mice, and immunofluorescence staining was performed to determine the CRY2 expression in the epidermis. HaCaT cell transfection experiment: HaCaT cells with small interfering RNA (siRNA) -mediated knockdown of CRY2 served as siRNA-CRY2 group, and siRNA-NC group as control group; 5-ethynyl-2′-deoxyuridine (EdU) staining was performed to evaluate the proliferative activity of the HaCaT cells, real-time fluorescence-based quantitative PCR (qPCR) to determine the mRNA expression of chemokines in the HaCaT cells, and Western blot analysis to determine phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2) . Tumor necrosis factor-α (TNF-α) -stimulated animal and cell experiments: 12 C57BL/6 female mice were randomly and equally divided into TNF-α group subcutaneously injected with TNF-α solution in the ear for 6 days, and phosphate buffered saline (PBS) group subcutaneously injected with the same amount of PBS; the mice were sacrificed on day 7, skin tissues were resected from the ear of mice, and immunofluorescence staining was conducted to determine the CRY2 expression in the epidermis; CRY2-knockdown HaCaT cells stimulated with 50 ng/ml TNF-α for 12 hours served as siRNA-CRY2 + TNF-α group, and siRNA-NC + TNF-α group as control group; qPCR was performed to determine the mRNA expression of chemokines in HaCaT cells in the above groups. Statistical analysis was carried out by using two-independent-sample t test. Results:Immunofluorescence staining showed that the CRY2 protein expression was significantly lower in the mouse dorsal epidermis in the imiquimod group (0.94 ± 0.23) than in the control group (2.30 ± 0.25, t = 3.99, P = 0.016) . Compared with the siRNA-NC group, the siRNA-CRY2 group showed significantly increased proportions of EdU-positive cells (48.13% ± 10.97% vs. 38.23% ± 0.81%, t = 5.00, P = 0.007) , mRNA expression levels of chemokines CXCL1 and CXCL8, as well as significantly increased phosphorylated (p) -ERK1/2 protein expression levels (all P < 0.05) , while there were no significant differences in the CCL20 mRNA expression or ERK1/2 protein expression between the two groups (both P > 0.05) . Immunofluorescence staining showed significantly decreased CRY2 protein expression level in the mouse ear epidermis in the TNF-α group (0.37 ± 0.34) compared with the PBS group (2.04 ± 0.17, t = 4.38, P = 0.012) ; the relative mRNA expression levels of chemokines CXCL1, CXCL8, and CCL20 in HaCaT cells were significantly higher in the siRNA-CRY2 + TNF-α group than in the siRNA-NC + TNF-α group (all P < 0.05) . Conclusion:CRY2 was markedly underexpressed in psoriasis, which might promote the proliferation of keratinocytes and expression of chemokines CXCL1, CXCL8 and CCL20, and TNF-α might be an upstream cytokine that could downregulate CRY2 expression.

Tumor necrosis factor α-mediated low expression of fatty acid desaturase 2 in psoriasis / 中华皮肤科杂志

Objective:To investigate the expression of fatty acid desaturase 2 (FADS2) in psoriatic skin lesions, as well as its regulatory factors.Methods:FADS2 expression in psoriatic skin lesions was analyzed by using the dataset GDS4602 in Gene Expression Omnibus (GEO) database. Skin tissues were obtained from the back of 5 C57BL/6 mouse models of imiquimod-induced psoriasis, normal skin of 4 patients without psoriasis or other immune skin diseases, lesions of 4 patients with psoriasis before and after 10-week treatment with infliximab, as well as lesions of 3 patients with psoriasis before and after 12-week treatment with secukinumab in Shanghai Skin Disease Hospital. FADS2 expression was determined by both immunohistochemical staining and Western blot analysis in the epidermis of mouse skin tissues, and by immunohistochemical staining in that of human skin tissues. In vitro cultured human immortalized keratinocytes (HaCaT) were divided into several groups to be treated with 50 ng/ml tumor necrosis factor-α (TNF-α) alone for 0, 6, 12 and 24 hours respectively, 200 ng/ml interleukin-17A (IL-17A) alone for 0, 6 and 12 hours respectively, or treated with 50 ng/ml TNF-α and 5 μmol/L BAY 11-7082 (a nuclear factor-κB pathway inhibitor) for 6 hours (TNF-α+ BAY 11-7082 6 h group) , and the cells receiving normal culture served as the control group. After the above treatment, real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were conducted to determine the mRNA and protein expression of FADS2 respectively. Statistical analysis was carried out by using one-way analysis of variance and t test. Results:Analysis of the dataset GDS4602 showed that the FADS2 mRNA expression was significantly lower in the lesional and non-lesional skin tissues from the patients with psoriasis (0.656 ± 0.475, 1.503 ± 1.062, respectively) than in the normal skin tissues (2.035 ± 1.226; F = 55.17, 3.07, P < 0.001, = 0.012, respectively) , and was significantly lower in the lesional skin tissues than in the non-lesional skin tissues from the patients with psoriasis ( F = 26.27, P < 0.001) . Western blot analysis and immunohistochemical staining both showed significantly decreased FADS2 protein expression in the mouse skin tissues in the imiquimod group (gray-value ratio: 0.463 ± 0.172; fluorescence intensity: 21.840 ± 3.125) compared with the normal control group (gray-value ratio: 1.000, t = 7.00, P = 0.002; fluorescence intensity: 30.720 ± 6.850, t = 3.15, P = 0.035) . Compared with the skin lesions before treatment, the FADS2 protein expression significantly increased in the skin lesions from the patients with psoriasis after 10-week treatment with infliximab (43.775± 3.342 vs. 27.950 ±1.218, t = -6.95, P = 0.006) , but was not significantly changed in the skin lesions from the patients with psoriasis after 12-week treatment with secukinumab (28.667 ± 3.402 vs. 31.933 ± 2.987, t = 2.72, P = 0.113) . qPCR revealed that the FADS2 mRNA expression significantly decreased in HaCaT cells in the TNF-α 6 h group and TNF-α 12 h group compared with the TNF-α 0 h group ( P = 0.002, 0.003, respectively) , while there was no significant change in the FADS2 mRNA expression in the IL-17A 6 h group and IL-17A 12 h group compared with the IL-17A 0 h group ( P = 0.849, 0.961, respectively) . The FADS2 mRNA expression significantly decreased in HaCaT cells in the TNF-α 6 h group (0.682 ± 0.132) compared with the control group (1.000, t = 4.82, P = 0.017) , but significantly increased in the TNF-α + BAY 11-7082 6 h group (1.541 ± 0.525) compared with the TNF-α 6 h group ( t = -3.58, P = 0.037) . Western blot analysis revealed significantly decreased FADS2 protein expression in HaCaT cells in the TNF-α 24 h group compared with the TNF-α 0 h group ( F = 6.24, P = 0.013) . Conclusion:FADS2 expression was downregulated in psoriatic lesions, which may be related to TNF-α.

Efficacy and safety of infliximab in the treatment of severe plaque psoriasis and its effect on the expression of programmed cell death-1 and programmed cell death ligand-1 / 中华皮肤科杂志

Objective:To investigate the efficacy and safety of infliximab in the treatment of severe plaque psoriasis and its effect on the expression of programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) in psoriatic lesions.Methods:A total of 17 patients with severe plaque psoriasis were enrolled from Shanghai Skin Disease Hospital from February 2019 to April 2019, and were treated with intravenous drips of infliximab at a dose of 5 mg/kg at weeks 0, 2, 6, 14, 22, 30, 38 and 46. Efficacy was evaluated by using the psoriasis area and severity index (PASI) score at weeks 2, 6, 10, 14, 22, 30, 38, 46 and 52, and adverse events were recorded during the trial. Real-time PCR was performed to determine the expression of PD-1 and PD-L1 in skin tissues of 8 volunteer controls, as well as in skin lesions of 14 patients with plaque psoriasis before treatment and 5 patients with plaque psoriasis after 10-week treatment, and immunofluorescence assay to measure the expression of PD-1 and PD-L1 in skin tissues of 5 volunteers and 5 patients with psoriasis. The independent sample t-test was used to compare the expression of PD-1 and PD-L1 in skin tissues between the patients with plaque psoriasis and controls, and paired t-test to compare the expression of PD-1 and PD-L1 in the skin lesions of patients before and after infliximab treatment. Results:After 2, 6, 10, 14, 22, 30, 38, 46 and 52 weeks of infliximab treatment, the proportion of patients with plaque psoriasis achieving PASI75 was 1/17, 6/16, 9/16, 10/16, 15/15, 14/15, 13/14, 11/13 and 10/11, respectively. Antinuclear antibody staining turned positive in 12 patients, which was the most common adverse reaction, and 1 patient experienced an infusion reaction, which was the most severe adverse reaction. Before the treatment, the expression of PD-1 and PD-L1 (1.111 ± 0.391, 0.902 ± 0.169, respectively) was significantly higher in the skin lesions of patients with psoriasis than in the skin tissues of controls (0.620 ± 0.225, t=3.116, P=0.007; 0.474 ± 0.360, t=3.208, P=0.006, respectively) ; after infliximab treatment, the expression of PD-1 and PD-L1 (0.570 ± 0.230, 0.150 ± 0.050, respectively) in the improved skin lesions was significantly lower than that in the corresponding lesions before the treatment (1.238 ± 0.414, t=3.107, P=0.036; 0.966 ± 0.184, t=8.423, P=0.001, respectively) . Conclusions:Infliximab is effective and safe for the treatment of plaque psoriasis, but monitoring is necessary during treatment. The expression of PD-1 and PD-L1 is aberrantly upregulated in plaque psoriasis lesions, and decreased after infliximab treatment, suggesting that PD-1/PD-L1 may be involved in inflammation regulation in psoriasis.

Various acupuncture therapies for lateral epicondylitis: A bayesian network meta-analysis / 中国组织工程研究

BACKGROUND: Acupuncture therapy plays a very important role in the treatment of lateral epicondylitis. However, due to the diverse characteristics of acupuncture therapy, the current research mostly focuses on a simple comparison between acupuncture therapy and conventional blocking therapy. OBJECTIVE: To compare the efficacy and visual analogue scale score of different acupuncture therapies for lateral epicondylitis using a Bayesian network meta-analysis. METHODS: Randomized controlled trials on acupuncture therapy for lateral epicondylitis included in PubMed, The Cochrane Library, CNKI, VIP, and WanFang were searched. The search time was from inception until October 2019 in each database. Two researchers independently screened and extracted data according to the inclusion criteria, and then evaluated the quality of the literature. Direct meta-analysis and network meta-analysis of data were performed using ADDIS 1.16.8 software. RESULTS AND CONCLUSION: A total of 2 318 lateral epicondylitis patients were included in 32 randomized controlled trials/controlled clinical trials, concerning 6 treatment measures, including warming needle, fire needle, electroacupuncture, filiform needle acupuncture, Fu’s acupuncture, fire needle plus filiform needle. Network meta-analysis results show that: in terms of efficiency, warming needles are better than electroacupuncture, warming needles are better than filiform needles, fire needles are better than warming needles, fire needles are better than electroacupuncture, fire needles are better than filiform needles, Fu’s acupuncture is better than electroacupuncture, Fu’s acupuncture is better than filiform needles, and fire needle plus filiform needle is better than filiform needles alone. In terms of the visual analogue scale score, warming needles are better than electroacupuncture, warming needles are better than filiform needles, fire needles are better than electroacupunture, fire needles are better than filiform needles, Fu’s acupuncture is better than electroacupuncture, Fu's acupuncture is better than filiform needles, fire needle plus filiform needle is better than electroacupuncture. Efficiencies rank from the best to the worst: Fu’s electroacupuncture>fire needle>fire needle plus filiform needle>warming needle>electroacupuncture needle>filiform needles. The visual analogue scale scores rank as follows: Fu’s acupuncture>fire needle+filiform needle>fire needle>warming needle>filiform needle>electroacupuncture. The direct meta-analysis results are highly consistent with the network meta-analysis results, indicating that there is consistency between the direct and indirect comparison, that is, transitive. In the clinical treatment of lateral epicondylitis, Fu’s acupuncture can be preferentially selected, but each acupuncture therapy has advantages and disadvantages. In clinical practice, the appropriate treatment should be selected in accordance with the actual situation and dialectical Chinese medicine.

Guidance on the use of biologic agents in psoriatic patients during COVID-19 outbreak / 中华皮肤科杂志

The COVID-19 outbreak may have some impact on the use of biologics in psoriatic patients because immunosuppressive effects of biologics may potentially alter the susceptibility of patients to the virus, deteriorate the condition of infected patients or even change the prognosis of infection. According to currently available recommendations from international psoriasis academic organizations and specialists, as well as specific situation in China, the authors provide some guidance on the use of biologics for psoriatic patients undergoing or planning to undergo treatment with biologics, those with low or high risk of infection, and for those with or without COVID-19 infection, so as to provide references for clinical practice.

Psoriasis comorbidities / 中华皮肤科杂志

Psoriasis is an immune-mediated chronic inflammatory disease,whose occurrence is closely associated with genes,immunity,environment and other factors.Classic comorbidities associated with psoriasis mainly include cardiovascular diseases,obesity and inflammatory bowel diseases.Emerging data have shown some other diseases closely associated with psoriasis,including metabolic syndrome,chronic renal diseases,tumors,infections,and so on.This review summarizes psoriasis-associated comorbidities that have been reported in studies,and analyzes the possible co-pathogenesis of these diseases and psoriasis.

Effects of Osmotic Pressure on Biomechanical Properties and Immune Functions of Immature Dendritic Cells / 医用生物力学

Objective To explore the effects of osmotic pressure on biomechanical properties and immune function of immature dendritic cells (imDCs) from mechanobiological viewpoint. Methods After treated with different osmotic pressures, the cell viability of imDCs was detected using cell counting kit-8 (CCK-8). The changes in morphology of imDCs were observed under laser scanning confocal microscope. Cell electrophoresis was applied to detect the changes in cell electrophoresis mobility. The membrane fluidity of the cells was detected by fluorescence polarization method, and the expression changes of immune-related molecules were detected by real-time fluorescent quantitative PCR (qPCR). The phagocytic ability of the cell was detected by flow cytometry. ResultsBoth hyperosmosis and hypoosmosis could remodel the cyoskeletonof cells, even induce apoptosis. The electrophoresis mobility of the hypoosmosis group was significantly higher than that of the normal osmolarity group, while that of the hyperosmosis group was lower than that of the normal osmolarity group (P<0-05). Fluorescence polarization results showed that both hyperosmosis and hypoosmosis could significantly decrease the membrane fluidity of cells (P<0-05). The results of qPCR detection showed that both hyperosmosis and hypoosmosis could significantly increase the expression of CCR7, CD40, CD205, CD11a, CD11c on the surface of DCs, and the phagocytosis of cell was increased (P<0-05). Conclusions Hypertonic and hypotonic stress can influence biomechanical properties of imDCs and expression of immune-related molecules. The research findings are important for further understanding the immune regulation function of DCs.

Comparison of fluorescent staining versus KOH wet-mount microscopy for detection of superficial fungal infection / 中华皮肤科杂志

Objective To compare the diagnostic value of fluorescent staining versus KOH wet-mount microscopy in detecting superficial fungal infection.Methods Totally,600 specimens from cases of clinically diagnosed superficial fungal infections and 102 from cases of clinically diagnosed Malassezia infection (including 54 cases of pityriasis versicolor and 48 cases of Malassezia folliculitis) were collected from the dermatology clinic of Tenth People's Hospital of Tongji University between July 2017 and February 2018.These specimens were subjected to fluorescent staining and KOH wet mount separately followed by direct microscopy,and the positive rate and average time for slide reading were compared between the two methods.Culture served as the gold standard method,and the missed diagnosis rate was compared between the two methods.Statistical analysis was carried out using chi-square test or Fisher's exact test for comparing enumeration data,and paired t test for comparing emeasurement data.Results Of the 600 specimens from clinically diagnosed superficial fungal infection cases,fungi were detected in 546 (91.00％) and 489 (81.50％) by fluorescent staining and KOH wet-mount microscopy respectively (x2 =22.83,P ＜ 0.05).Fluorescent staining showed significantly shorter average reading time (73.67 ± 13.56 s)compared with KOH wet-mount microscopy (87.12 ± 15.83 s,t =14.60,P ＜ 0.05).Among the 54 specimens from pityriasis versicolor cases,fluorescent staining and KOH wet-mount microscopy positive results in 51 (94.44％) and 50 (92.59％) specimens respectively (adjusted x2 =0,P ＞ 0.05),with the average reading time being 38.36 ± 8.79 s and 41.25 ± 15.67 s respectively (t =1.14,P ＞ 0.05).Of the 48 specimens from Malassezia infection cases,43 (89.58％) and 11 (22.92％) specimens were detected to be positive for fungi by fluorescent staining and KOH wet-mount microscopy respectively (x2 =43.34,P ＜ 0.05),and fluorescent staining showed shorter average reading time (42.14 ± 12.61 s) compared with KOH wet-mount microscopy (103.56 ± 9.48 s,t =17.83,P ＜ 0.05).Among the 600 specimens from superficial fungal infection cases,culture yielded fungi in 479.Moreover,476 specimens were found positive by fluorescent staining,and 3 were found negative (0.63％),while KOH wet-mount microscopy showed 465 positive results and 14 negative results (2.92％).There was a significant difference in the missed diagnosis rate between the two methods (x2 =7.25,P ＜ 0.05).Conclusion Compared with KOH wet-mount microscopy,fluorescent staining can increase the detection rate,reduce missed diagnosis rate and shorten reading time.

Diagnosis value of age-specific Procalcitionin and C-reactive protein on early-onset neonatal sepsis / 实用医学杂志

Objective To compare different diagnosis values of age-specific procalcitionin (PCT) and C-reactive protein (CRP) on early-onset neonatal sepsis (EONS) during the first 72h of life.Methods From September 2008 to December 2015,96 neonates (including 2 confirmed sepsis and 94 clinical sepsis) without severe complications were chosen as the EONS group and 170 non-infectious newborns as the control group.A total of 605 blood samples were collected from all 266 newborns.Serum concentration of PCT and CRP were measured in both the EONS group and the control group at each age over the first 72 h of life.The diagnostic value of PCT and CRP within 1 ～ 12 h and 13 ～ 24 h and 25 ～ 48 h and 49 ～ 72 h of life was evaluated by calculating the cut-off values,sensitivity,specificity,and the area under the receiver operating characteristic curve (ROC).Results PCT and CRP levels of neonates within each age in the EONS group were significantly higher than those in the control group during the first 72h of life.(all P ＜ 0.05).Within 1 ～ 12 h,13 ～ 24 h,25 ～ 48 h and 49 ～ 72 h of life,the cutoff value of PCT was 0.45 μg/L (sensitivity 84.2％,specificity 74.4％),1.885 μg/L (sensitivity 73.5％,specificity 75％),0.995 μg/L (sensitivity 82.4％,specificity 74.1％) and 0.51 μg/L (sensitivity 83.3％,specificity 79.2％) respectively;that of CRP3 185 mg/L (sensitivity 68.4％,specificity 82.1％),6.29 mg/L (sensitivity 58.8％,specificity 89.7％),8.615 mg/L (sensitivity 54.3％,specificity 93.9％) and 10.27 mg/L (sensitivity 59.1％,specificity 100％) respectively,and the area under ROC of PCT for the diagnosis of EONS was 0.767,0.754 and 0.755,and 0.8 respectively;that of CRP 0.773,0.8,0.815 and 0.789 respectively.Conclusions There are age-specific cut-off values of PCT and CRP in the diagnosis of EONS without severe complications during the first 72 h of life.Both PCT and CRP are moderately accurate for the diagnosis of EONS.PCT may be more helpful for the early diagnosis of EONS for its higher sensitivity but CRP presents higher specificity.

Recombinant human tumor necrosis factor receptor type Ⅱ : IgG Fc fusion protein combined with methotrexate for the treatment of psoriasis and their effects on levels of interleukin-17A and tumor necrosis factor-α / 中华皮肤科杂志

Objective To evaluate the effect of recombinant human tumor necrosis factor receptor type Ⅱ:IgG Fc fusion protein (rhTNFR:Fc,trade name Etanercept) combined with methotrexate on levels of interleukin-17A (IL-17A) and tumor necrosis factor-α (TNF-α) in the serum and mononuclear cells of patients with moderate to severe plaque psoriasis.Methods A total of 30 patients with moderate to severe plaque psoriasis were enrolled from Department of Dermatology of Tenth People's Hospital of Tongji University between August 2014 and February 2016,and then were randomly and equally divided into Etanercept group and Etanercept + methotrexate group.The treatment lasted 24 weeks.Fifteen healthy blood donors served as healthy control group.Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR were performed to measure the serum levels and mRNA expression of IL-17A and TNF-α,respectively,in the patients of the above two groups before and after the treatment.Results Before the treatment,the serum levels of IL-17A and TNF-ct,as well as the mRNA expression of IL-17A and TNF-α in peripheral blood mononuclear cells (PBMCs),were all significantly higher in all the patients than in the healthy controls (all P ＜ 0.05).After the treatment,compared with the Etanercept group,the Etanercept + methotrexate group showed significantly lower serum levels of IL-17A (142.67 ± 14.82 vs.163.54 ± 23.18,P ＜ 0.05) and TNF-α (70.07 ± 25.02 vs.91.98 ± 14.62,P ＜ 0.05),as well as lower mRNA expression of IL-17A (1.12 ± 0.33 vs.1.56 ± 0.77,P ＜ 0.05) and TNF-α in PBMCs (2.50 ± 1.04 vs.3.61 ± 2.14,P ＜ 0.05).Conclusion Etanercept combined with methotrexate is superior to Etanercept alone in the treatment of psoriasis,and can reduce treatment duration and improve therapeutic effect,likely by down-regulating the expression of IL-17A and TNF-α.

Clinical features of 70 cases of psoriatic arthritis / 中华皮肤科杂志

Objective To investigate the prevalence of psoriatic arthritis (PsA) in patients with psoriasis and its clinical features.Methods A cross-sectional study was conducted among patients diagnosed with psoriasis from January 2014 to January 2015.Through a questionnaire survey,the diagnosis of PsA was confirmed according to the Classification Criteria for Psoriatic Arthritis (CASPAR) in patients with suspected PsA.Clinical data were collected from patients with newly and previously diagnosed PsA.Statistical analysis was carried out by t test for two-group comparisons,one-way analysis of variance (ANOVA) for multi-group comparisons,and chi-square test for comparisons of rates.All the statistical tests were two-sided.Results Totally,1 062 outpatients with psoriasis were enrolled into this study,and 125 were suspected to have PsA.According to the CASPAR,70 (6.59％) patients were finally diagnosed with PsA,with the ratio of male to female being 2.1 ∶ 1,and 45 of them (64.29％) were newly diagnosed.Psoriasis vulgaris lesions were observed in 50 (71.43％) patients with PsA,and were the most common type of skin lesions in patients with PsA.There were 5 clinical types of PsA in these patients,including asymmetrical oligoarthritis (23 cases,32.86％),symmetric polyarthritis (19 cases,27.14％),distal interphalangeal predominant arthritis (10 cases,14.29％),vertebral or sacroiliac arthropathy (7 cases,10.00％),and arthritis mutilans (11 cases,15.71％),with some overlap among these clinical types.As relatively distinctive manifestations of PsA,dactylitis and enthesitis were observed in 14 (20.00％) and 8 cases (11.43％) respectively.In addition,43 (61.43％) cases had nail involvements.Conclusion To master clinical features of PsA and to diagnose it early are of great significance for long-term prognosis of PsA patients.

Expressions of activation antigens CD69 and HLA-DR in peripheral blood mononuclear cells and skin lesions of patients with psoriasis vulgaris / 中华皮肤科杂志

Objective To investigate changes in expressions of activation antigens CD69 and HLA-DR in CD3+T lymphocytes in peripheral blood and skin lesions in patients with psoriasis vulgaris. Methods Peripheral blood samples were obtained from 20 patients with psoriasis vulgaris and 20 healthy controls, and skin specimens from the lesions of 15 out of the 20 patients and 10 healthy controls. Flow cytometry was performed to quantify the expressions of CD69 and HLA-DR in peripheral blood CD3+T cells, and an immunohistochemical study to measure the expression of HLA-DR in skin specimens. Statistical analysis was carried out by a two-sample t-test and Pearson correlation analysis with the SPSS 19.0 software. Results Compared with the healthy controls, the patients with psoriasis vulgaris showed increased expression rates of CD69 (4.70%± 1.90%vs. 1.56%± 0.95%, t=6.629, P<0.01)and HLA-DR (8.97%± 1.79% vs. 3.02% ± 1.15%, t= 6.204, P< 0.01)in peripheral blood. Pearson correlation analysis revealed that the percentage of CD3+HLA-DR+cells in peripheral blood was positively correlated with the psoriasis area and severity index (PASI)score (r=0.5626, P<0.05). The expression rate of HLA-DR was significantly higher in the dermis (64.87%± 17.31%vs. 19.80%± 5.69%, t=7.916, P<0.01), but lower in the epidermis(11.80%± 5.55%vs. 27.40%± 8.61%, t=5.479, P<0.01)in the psoriatic specimens compared with the control specimens. Immunohistochemically, HLA-DR was widely expressed in the dermis of psoriatic lesions, but mainly distributed around blood vessels in the control skin. Conclusions There is an aberrant activation of CD3+T cells in peripheral blood and inflammatory cells in skin lesions in patients with psoriasis vulgaris, and the percentage of CD3 +HLA-DR+ cells in peripheral blood is correlated with the severity of psoriasis vulagaris.

Effects of resina draconis on Toll-like receptor-4/nuclear factor-kappaB and dendritic cell phenotypes in colitis rats / 中国组织工程研究

BACKGROUND:Dendritic cel s can regulate the immunological reaction in the intestinal tract, this functional deficit may induce inflammatory bowel disease. Tol-like receptor-4/nuclear factor-κB pathway is highly involved in this reaction. OBJECTIVE:To establish experimental colitis model in rats, to observe effects of resina draconis on dendritic cel s and Tol-like receptor-4/nuclear factor-κB expression in rats with experimental colitis, and to explore its action mechanism. METHODS:A total of 44 male Sprague-Dawley rats were randomly assigned to four groups (n=11):blank control group, model group, resina draconis group, 5-aminosalicylic acid treatment group. With the exception of blank control group, 2,4,6-trinitrobenzenesulfonic acid-induced ulcerative colitis models were established in the model group, resina draconis group and 5-aminosalicylic acid treatment group. After the models were successful y established, the rats in the resina draconis and 5-aminosalicylic acid treatment groups were intragastrical y treated with resina draconis [(0.75 g(kg·d)] and 5-aminosalicylic acid [100 mg(kg·d)] respectively for 10 days. RESULTS AND CONCLUSION:Disease activity index, macroscopic colonic damage score and histopathological score were significantly decreased in the resina draconis group compared with the model group (P<0.05). Symptoms and tissue damages were obviously lessened in the 5-aminosalicylic acid treatment and resina draconis groups compared with the model group. Expression rates of CD80 and CD86, as wel as expression levels of Tol-like receptor-4 and nuclear factor-κB were significantly higher in the model group compared with the blank control group, resina draconis group and 5-aminosalicylic acid treatment group (P<0.05, P<0.01). Tol-like receptor-4 and nuclear factor-κB expression was significantly lower in the resina draconis group than that in the 5-aminosalicylic acid treatment group. Experimental findings indicate that, resina draconis can partial y relieve experimental colitis symptoms in rats and effectively inhibit the activation of dendritic cel s in the mesenteric lymph node. Resina draconis can relieve enteric inflammatory reaction by suppressing the expression of Tol-like receptor-4 and nuclear factor-κB in rats.

Evaluation of long-term-siRNA treatment with HBV transgene mice on inhibit replication of hepatitis B virus / 中国免疫学杂志

Objective:To investigation of the long-term-siRNA treatment with HBV transgene mice on inhibit replication of hepatitis B virus .Methods:The constructed siRNA expressed vectors was transfected HBV transgene mice by hydrodynamics -based in-jection via vena caudalis .Different groups were set including:specificity siRNA groups ( pSilencer5.1/C2,pSilencer4.1/C2,pSilenc-er3.1/C2),PBS group and negative vector group (n=10).The effect was observed in different periods (6 d,21 d,1 months,3 months, 6 months and 9 months after injection ) .HBsAg was analyzed by Chemiluminescence method , HBV-DNA was analyzed by real time quantitative PCR ( RQ-PCR) .Results:Compared with the PBS group , specificity siRNA groups showed decreased levels of HBsAg and HBV-DNA (P0.05).Conclu-sion:The siRNA based on the expression vector can suppress the expression and replication of HBV in HBV transgene mice .The inhi-bition effects of long-term-siRNA treatment was specific .

Preparation of a monoclonal antibody against fusion protein preM/EIII of WNV / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare a monoclonal antibody (mAb) against the fusion protein preM/EIII of West Nile virus (WNV) for clinical detection of WNV.</p><p><b>METHODS</b>Sp2/0 cells were fused with the spleen cells of BALB/c mice immunized with the recombinant fusion protein preM/EIII expressed in E. coil to obtain the hybridoma cell line that secreted preM/EIII mAb. The hybridoma cells were injected into the peritoneal cavity of BALB/c mice and the ascites was collected and purified. The specificity and titer of the obtained mAb were determined using ELISA and Western blotting.</p><p><b>RESULTS</b>One hybridoma cell line secreting preM/EIII mAb, named ME1, was obtained. The titer of the purified mAb was 10(-6). Identified as a mAb of the Ig subclass IgG1, ME1 was capable of specific reactions with preM/EIII protein and WNV without cross-reactions with other viruses such as JEV, SLEV, YFV and DENV. The accuracy of clinical testing of MNV with ME1 was 97.78%.</p><p><b>CONCLUSION</b>The mAb against preM/EIII obtained have a high specificity and accuracy in clinical testing of MNV and can be used in clinical diagnosis of MNV infection.</p>

Effects of rhPTH (1-34) on the proliferation and cell cycle of human keratinocyte HaCaT cells induced by tumor necrosis factor-α / 中华皮肤科杂志

Objective To investigate the influence of recombinant human parathyroid hormone [rhPTH (1-34)]on the proliferation of HaCaT cells induced by tumor necrosis factor-α (TNF-α) in vitro.Methods Cultured HaCaT cells were treated with various concentrations of rhPTH (1-34) for different durations after incubation with recombinant human TNF-α of 10 g/L for 24 hours.MTT assay and flow cytometry were performed to detect the proliferation and cell cycle of HaCaT cells,respectively.Results As contrast phase microscopy showed,the growth of HaCaT cells was inhibited by rhPTH (1-34) along with a decrease in the growth speed.MTT assay showed a suppressed proliferation of HaCaT cells after being treated with rhPTH (1-34) of 0.05,0.2,0.8,3.2 and 12.8 pmol/L for 36 and 48 hours (P＜ 0.01 or 0.05).The percentage of cells at G1 phase in HaCaT cells markedly increased (all P ＜ 0.01 ),while that at S phase declined (all P ＜ 0.01 )after 48-hour treatment with rhPTH(1-34) of 0.2,0.8,3.2 and 12.8 μ mol/L.Conclusions rhPTH(1-34) has an obvious inhibitive effect on the proliferation of HaCaT cells induced by TNF-α in vitro,and the effect is in a dose-dependent manner.

Detection of clostridium perfringens with qPCR in mouse models and a clinical case / 中国医师杂志

Objective To detect of clostridium perfringens by qPCR in mouse models and a clinical case in order to offer early diagnosis.Methods 40 Kunming mice were randomly grouped and intramuscular injected clostridium perfringens type A in leg 0.1 ml(3.5 × 109cfu/ml or 3.5 × 108cfu/ml or 3.5× 107cfu/ml,diluted with saline),while control group was injected with 9% sodium chloride 0.1ml.The mouse models and a clinical case were detected by qPCR.Results The death rate of 3.5 × 109,3.5 × 108,3.5 × 107cfu/ml and the blank group were 90%,70%,10% and 0% after intramuscular injection for 72 h spectively.The mean Ct values among these groups were 21.21 ±2.69,28.45 ±2.74,32.49 ±2.87 and 0.00 ± 0.00(P ＜ 0.05).The Ct values of the patient were 30.67 and 30.44.Conclusions Cclostridium perfringens could be successful identified with qPCR in mouse models when the mice still did not show any symptoms.

Expressions of interleukin (IL)-22 and related cytokines in the sera and peripheral blood mononuclear cells (PBMCs) of patients with psoriasis / 中华皮肤科杂志

Objective To investigate the expressions and significance of IL-22 and related cytokines (IL-23pl9 and IL-6) in sera and PBMCs of patients with psoriasis. Methods Sera and PBMCs were obtained from the venous blood samples from 58 patients with psoriasis vulgaris and 20 normal human controls. The PBMCs were subjected to culture for 5 hours followed by the collection of cells and culture supernatant. Then,quantitative real-time RT-PCR was used to examine the mRNA expressions of IL-22, IL-23pl9 and IL-6 in PBMCs, enzyme-linked immunosorbent assay (ELJSA) to detect the level of IL-22 protein in the sera and culture supernatant of PBMCs. Results In the patients with psoriasis and controls, the relative expression level in PBMCs was 4.48 ± 2.64 and 2.35 ± 0.91 respectively for IL-22 mRNA, 6.07 ± 4.09 and 2.61 ± 1.46 respectively for IL-23pl9 mRNA, 3.87 ± 1.49 and 1.48 ± 0.62 respectively for IL-6 mRNA; significant differences were observed between the two groups in all the above parameters (all P < 0.01). ELISA revealed that the level of IL-22 protein in the patients and controls was (86.23 ± 25.58) ng/L and (43.67 ± 14.82) ng/L respectively in the sera (P< 0.01), (119.11 ± 21.51) ng/L and (57.70 ± 13.17) ng/L respectively in the culture supernatant of PBMCs (P< 0.01). Conclusion There is an overexpression of IL-22 in the PBMCs and sera of patients with psoriasis, implying that IL-22 is involved in the pathogenesis of psoriasis.

Serum levels of alpha-fetoprotein and alpha-fetoprotein variants in decompensated cirrhosis patients following intrahepatic transplantation of peripheral blood stem cells via portal vein / 中国组织工程研究

BACKGROUND:Transplantation of autologous stem cells for treatment of liver cirrhosis has been widely reported.But up to now,there exist some concerns for clinical physicians,including relationship between stem cells and post-transplantation prognosis/turnover of liver cirrhosis,directed differentiation of stem cells in the impaired liver,and malignant phenotype.OBJECTIVE:To dynamically monitor the serum levels of alpha-fetoprotein(AFP)and AFP variants(AFP-L3)in decompensated cirrhosis patients following intrahepatic transplantation of peripheral blood stem calls via portal vein and evaluate the safety of this treatment method.METHODS:A total of 44 decompensated cirrhosis patients who underwent intrahepatic transplantation of peripheral blood stem cells via portal vein in the 309 Hospital of Chinese PLA in April 2007 were included in this study.Prior to and after surgery,serum levels of AFP and AFP-L3 were detected by chemiluminescence.Through the use of a positive criterion for liver cirrhosis,i.e.,the proportion of AFP-L3 in AFP[AFP-L3(%)]≥10%,and the relationship between decompensated cirrhosis treatment using stem cells transplantation and the malignant phenotype of liver cancer were analyzed.RESULTS AND CONCLUSION:At 2 months after surgery,serum level of AFP showed a transient increase.There was no significant difference in AFP-L3(%)between prior to and after surgery(P>0.05).No significant difference in AFP-L3-positve rate,as well as AFP-L3(%),existed among patients with different serum level of AFP.These findings indicate that clinical symptoms and liver function of decompensated cirrhosis patients recovered to some extent after transplantation of peripheral blood stem cells via portal vein.Results regarding serum level of AFP-L3,a serological marker of liver cancer,did not demonstrate the appearance of malignant biological phenotype.

Diagnostic role of Golgi protein 73 and its gene determination in primary hepatic carcinoma / 中华检验医学杂志

Objective To explore the diagnostic value of GP-/3 protein in gene detection in the patient of primary hepatic carcinoma, to discuss the joint roles of serum GP73 and AFP, and provide a novel method for the diagnosis for PHC and screening for high-risk population. Methods ELISA was used to detect the serum level of GP73 and AFP in 73 cases of PHC, 13 cases of hepatic cirrhosis, 32 cases of hepatitis and 62 cases of health people. SYBR Green real time fluorescence quantitative PCR was used to detect the relative value of GP73 mRNA in the peripheral blood cells of each group. Comparative Ct method was used to evaluate the relative expression levels. Eight cases of normal liver tissues and 8 cases of PHC tissues were detected at the same time to compare the relative expression levels. Results Kruskal-Wallis test showed that the serum levels of GP73 and AFP had significant differences between four groups(H value were 89. 6 and 52.0, P ＜ 0. 01) and the whole blood GP73 mRNA had no significant differences(H =4. 33, P ＞ 0. 05). Mann-Whitney test showed that the serum levels of GP73 had significant differences among PHC groups[166. 7 (162. 7-231.8) μg/L] and liver cirrhosis[57. 3 (46. 6-113. 6) μg/L], hepatitis[29. 6(26. 2-54. 5) μg/L], health group[25.1 (20. 8-29. 4) μg/L] (U value were 246, 297, 349, P ＜ 0. 01).The A FP levels of the four groups were 380. 9 (258.5-503.2) μg/L, 3.8 (1.3-14. 5) μg/L, 5. 1 (2. 4-7. 8)μg/L and 2. 8(2. 2-5.7) μg/L. It also showed significant differences (U value were 246,419 and 790,P ＜0. 01). The GP73 mRNA expression of PHC liver tissues(12. 64) was significant higher than normal liver tissues (1.00). The critical values for GP73 and AFP was determined to be 123. 2 μg/L and 10. 6 μg/L through the 8OC curves. Under the critical value the sensitivity of GP73 and AFP were 65.8% and 53.4% ,and the specificity of CP73 and AFP were 95.3% and 92. 5% respectively. Joint detection could increase the sensitivity up to 79. 5%, and achieve the high specificity of 90. 7%. Conclusions As a new diagnositic marker of primary hepatic carcinoma, GP73 protein has the very good sensitivity and specificity. The GP73 mRNA in the whole blood sample could not be used for the diagnosis of PHC. But it woule be a good molecular marker for diagnosis of PHC in the liver tissue sample. The joint detection of GP73 and AFP could improve PHC diagnostic performance, and provide an effective approcach to the PHC high-risk screening.