Objective To evaluate the potential association of CD4+CD25+ T cells with alopecia areata.Methods Totally,this study enrolled 23 patients with progressive alopecia areata,25 patients with stable alopecia areata,and 25 healthy controls.Peripheral blood was isolated from these subjects followed by isolation of CD4+ CD25+ regulatory T cells,which were then cuhured with the presence of anti-CD3 and-CD28 monoclonal antibodies for four days.Subsequently,enzyme-linked immunosorbent assay was performed to measure the levels of interleukin (IL)-10 and transforming growth factor (TGF)-β1 in the culture supematant of these T cells.Results The levels of IL-10 and TGF-β1 were (31.68 ± 6.78) pg/ml and (32.29 ± 6.8) pg/ml respectively in the culture supernatant of CD4+CD25+ regulatory T cells from patients with progressive alopecia areata,significantly lower than those from the healthy controls ((57.34 ± 14.15) pg/ml and (57.43 ± 15.16) pg/ml,both P ＜ 0.05) and patients with stable alopecia areata ((52.56 ± 13.02) pg/ml and (61.75 ± 14.10) pg/ml,both P ＜ 0.05).However,no significant difference was observed in the supernatant levels of IL-10 or TGF-β1 between the healthy controls and patients with stable alopecia areata.Conclusions The secretion of IL-10 and TGF-β1 by CD4+CD25+ T cells is decreased in patients with progressive alopecia areata,which may contribute to the pathogenesis of alopecia areata.

Objective To determine the level of interleukin (IL)-10 in sera and culture supernatants of CD4+CD25+T cells from children with atopic dermatitis (AD),and to evaluate its relationship with clinical course and severity of AD.Methods Totally,46 children with AD and 31 healthy controls were included in the study.All the patients were divided into 3 groups,i.e.,mild (n =10),moderate (n =16) and severe (n =20) group,according to severity scoring of atopic dermatitis (SCORAD) score.Venous blood samples were obtained from the patients and healthy controls.CD4+CD25+ regulatory T cells were separated from the blood samples by magnetic cell sorting (MACS) system in two steps and cultured in vitro.Enzyme linked immunosorbent assay (ELISA) was conducted to quantify the level of IL-10 in sera and culture supernatants of CD4+CD25 + T cells from these subjects.Analysis of variance was carried out to compare the level of supematant and serum IL-10 between the patients and controls,and Pearson correlation analysis to assess the relationship between the level of IL-10 and SCORAD score.Results The patients with mild,moderate and severe AD showed a similar serum IL-10 level compared with the healthy controls ((43.10 ± 25.07) pg/ml,(68.40 ± 36.65) pg/ml and (55.55 ± 41.97) pg/ml vs.(58.27 ± 36.84) pg/ml,all P ＞ 0.05).The level of supernatant IL-10 secreted by CD4+CD25+ T cells from the controls was significantly higher than that from the patients with severe AD ((55.15 ± 11.15) pg/ml vs.(27.25 ± 7.01) pg/ml,P ＜ 0.05),but similar to that from the patients with mild and moderate AD ((52.96 ± 11.69) pg/ml and (49.86 ± 9.18) pg/ml,respectively,both P ＞ 0.05).The level of secreted IL-10 was negatively correlated with SCORAD score (r =－0.757,P ＜ 0.01),whereas the serum level of IL-10 showed no statistical correlation with SCORAD score.Conclusion CD4+CD25+ T cells and IL-10 may be implicated in the development of AD.

Objective To assess the associations of anti-Smith antibodies with clinical manifestations and disease activity in children with systemic lupus erythematosus (SLE).Methods According to SLE disease activity index (SLEDAI) score,72 children with SLE were divided into the active group and inactive group.An immunoblotting method was used to detect serum anti-Smith antibodies in these subjects.Chi-square test was conducted to assess the associations of anti-Smith antibodies with clinical manifestations and disease activity in these patients.Results Of these patients,28 (38.9％) were assigned into the inactive group,and 44 (61.1％) to the active group.Anti-Smith antibodies were detected in 17 (23.6％) patients,but not in the other 55 (76.4％) patients.Elevated incidence rate of kidney injury was observed in anti-Smith antibody-positive patients compared with anti-Smith antibody-negative patients (70.6％ (12/17) vs.41.8％ (23/55),P ＜ 0.05).Meanwhile,the positivity rate of anti-Smith antibodies was 31.8％ (14/44) in the active group,significantly higher than that in the inactive group (10.7％,3/28,P ＜ 0.05).Conclusions Anti-Smith antibodies are not only an important indicator for the diagnosis of SLE,but also a risk factor for disease exacerbation and kidney injury in children with SLE.

Objective To investigate the clinical feature, treatment and prognosis of severe hand-footmouth disease without typical skin lesions. Methods Clinical data from 24 patients with severe hand-footmouth disease without typical skin lesions collected from January 2010 to May 2010 were retrospectively analyzed.Results There were 17 males and 7 females among the 24 patients. Of them, 3 patients with positive EV71 showed no skin lesions at the first visit, 2 presented with only herpes of mouth, 3 with only skin rashes in gluteal regions, 5 with only skin rashes on the palms, 2 with only skin rashes of the knees, 9 with skin rashes in hands, feet and gluteal regions simultaneously. The skin rashes were sparse with an atypical distribution.Fever occurred in all the 24 patients and lasted 6 - 7 days. Nervous system was involved in all the patients,and pulmonary hemorrhage occurred in 4 patients. RT-PCR and real-time PCR showed that the pharyngeal swab was positive for EV71 in 13 patients, for CoxA16 in 2, for other enterovirus in 3, and feces samples were positive for EV71 in 9 patients, for CoxA16 in 1, for other enterovirus in 2. Five patients were positive for EV71 in both pharyngeal swab and feces samples. Four patients died of multiple organ failure, the other 20 patients were cured and discharged from hospital. Conclusions The major pathogen causing severe hand-footmouth disease is EV71 in the 24 patients; children under 2 years are liable to this disease; high fever is common with various rashes. Early recognition of atypical skin lesions and timely management are most important for the control of severe hand-foot-mouth disease and improvement in overall survival of patients with this entity.

ObjectiveTo evaluate the roles of imbalance between peripheral blood T helper 17 (Th17) cells and CD4+CD25+ regulatory T(Treg) cells in the pathogenesis of atopic dermatitis (AD).Methods Peripheral blood samples were obtained from 52 patients with AD aged 2-14 years and 30 age- and sex-matched healthy controls.Flow cytometry was performed to detect the percentage of Th17 cells and Treg cells in peripheral blood.Meanwhile,enzyme linked immunosorbent assay(ELISA) was carried out to detect the serumlevel of interleukin (IL)-6 and transforming growth factor (TGF)-β1.Results The children with AD showed a higher percentage of Th17 cells but a lower percentage of Treg cells in CD3+ T cells compared with the controls (( 1.20 ± 0.41 )％ vs.(0.54 ± 0.28)％,t =2.58,P ＜ 0.05; (2.29 ± 0.67)％ vs.(5.95 ± 0.45)％,t =15.23,P ＜ 0.01 ).Moreover,the serum level of IL-6 was significantly higher,while that of TGF-β1 was lower in patients with AD than in the controls ((5.12 ± 0.45) ng/L vs.(3.89 ± 0.38) ng/L,t =2.59,P＜ 0.05; (57.65 ± 10.78) ng/L vs. (81.18 ± 7.78) ng/L,t =5.41,P ＜ 0.01 ).ConclusionsChildren with AD experience a change in the percentage of Thl7 cells and Treg cells in peripheral blood as well as in the serum level of IL-6 and TGF-β1,and the imbalance between Th17 cells and Treg cells in peripheral blood may contribute to the development of AD.

Objective:To investigate the clinical features of patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis misdiagnosed as mental disorder, improve the early diagnosis rate and reduce misdiagnosis.Methods:The clinical data of patients with anti-NMDA receptor encephalitis diagnosed at the First Affiliated Hospital of Zhengzhou University from 2012 to 2018 were collected. Patients misdiagnosed as mental disorders were screened out. Their psychiatric symptom characteristics, disease course characteristics, imaging and laboratory findings, treatment and prognosis were retrospectively analyzed.Results:A total of 121 cases of anti-NMDA receptor encephalitis were collected, and 43 cases of mental disorders were screened out. Sixteen of the 43 patients (37.2%) had prodromal symptoms, and all the patients had psychiatric behavioral abnormalities (100%), including 32 cases (74.4%) of seizures, 13 cases (30.2%) of decreased level of consciousness, 21 cases (48.8%) of involuntary movements, 15 cases (34.9%) of decreased memory, 8 cases (18.6%) of speech dysfunction, and 8 cases (18.6%) of other neurological symptoms (central hyperventilation, autonomic dysfunction). Memory loss was observed in 15 cases (34.9%), speech dysfunction in 8 cases (18.6%), other neurological symptoms (central hypoventilation, autonomic dysfunction) in 8 cases (18.6%), and various symptoms may appear simultaneously or successively in the same patient. Thirty-eight cases had complete resolution of symptoms or only minor physical impairment, and 5 cases had recurrent admissions with mental abnormalities and seizures. The recurrence rate accounted for 11.6% (5/43).Conclusions:The clinical manifestations of anti-NMDA receptor encephalitis are complex and varied. Most of them have mental behavior abnormalities as the first symptom, which is easily misdiagnosed as mental disorder and delayed treatment will lead to prolonged disease course and poor prognosis.

Finding stable expression sites on the chromosomes of Chinese hamster ovary (CHO) cells is an effective method to solve the problem of unstable expression of CHO cells in long-term culture. Our group used lentiviral transfection to integrate the tracer gene (Zsgreen1) into the chromosome of CHO cells and found multiple potential stable expression sites. This study verified the ability of one of the sites located in the 148052-148157 bp region on chromosome NW_003614241.1 to stably express exogenous proteins.The expression of Zsgreen1 gene was first observed, and CRISPR/Cas9 technology was then used to integrate the enhanced green fluorescent protein (EGFP) gene into this site. Three strains of EGFP gene integrated cells were obtained. After 60 generations of suspension culture, the fluorescence intensity of the cells had no significant changes, which proved that this site can stably express the EGFP gene. The same method was used to construct recombinant CHO cell lines expressing the human serum albumin (HSA) gene, and was verified by Western blot that this site could express and secrete HSA. It shows that the above-mentioned sites can be integrated and can stably express exogenous proteins.

Objective:To establish an analytical method for the simultaneous determination of five fat-soluble vitamins in serum using isotope dilution ultra high performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MSMS).Methods:Fat-soluble vitamins were obtained from serum samples which collected from Shanghai Tenth People′s Hospital between April 2019 and August 2019 by the extraction method, and were detected by ID-UPLC-MSMS. The performance of the method was verified by referring to the relevant documents of the Clinical and Laboratory Standards Institute (CLSI).Results:The ID-UPLC-MSMS method for the rapid detection of various fat-soluble vitamins in serum was proposed and successfully verified. The linear range of the method: vitamin A: 25-2 500 μg/L, 25(OH)D 2: 2-200 μg/L, 25(OH)D 3: 2-200 μg/L, vitamin E: 0.25-50 mg/L, vitamin K1: 0.1-20 μg/L. The intra- and inter-assay precision standard deviations of the five analytes were within ± 15%, and the accuracy of the test results of the 25(OH)D 2 and 25(OH)D 3 standards was 96.44%-102.37%. Conclusion:The performance of ID-UPLC-MSMS method for the simultaneous determination of five fat-soluble vitamins is satisying, and the result is accurate and reliable, which suggested it can be used for the clinical sample.

Objective To evaluate the clinical efficacy and safety of 595-nm pulsed dye laser with topical timolol maleate 0.5％ solution for the treatment of superficial infantile hemangioma (IH).Methods Complete clinical data were collected from 156 infants with superficial IH,who received treatment with 595-nm pulsed dye laser combined with topical timolol maleate 0.5％ solution in the Department of Dermatology of the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University from July 2015 to July 2016,and analyzed retrospectively.Of the 156 patients,44 were males,and 112 were females,with an average age of 3.8 ± 0.7 months (range,24 days-1 year).These patients were treated with 595-nm pulsed dye laser every 5 weeks and topical application of timolol maleate solution twice a day.Each treatment with timolol maleate solution lasted 30 minutes.When the hemangioma regressed generally,the treatment with laser and timolol maleate solution was stopped.At weeks 5,10,15 and 30,the visual analogue scale (VAS) was used to evaluate the efficacy,and adverse reactions were recorded.These patients were followed up till 6 months after the end of treatment.The relationships of area and thickness of hemangioma with treatment duration,treatment sessions and VAS scores were analyzed.Results After 5-30 weeks of treatment,hemangiomas regressed to different extents,and the cure rate was 93.59％ (146/156).At weeks 5,10,15 and 30,the VAS scores were 3.12 ± 0.23,4.45 ± 0.52,5.45 ± 0.71 and 7.59 ± 1.64 respectively.Repeated-measures analysis of variance showed that the VAS scores all significantly increased over time (F =189.35,P ＜ 0.05) in the 3 groups with different initial thickness of hemangiomas (＜ 1 mm,1-3 mm,and ＞ 3 mm),and significantly differed among the above 3 groups at different time points (F =215.56,P ＜ 0.05),and the group with the initial thickness of hemangiomas ＜ 1 mm showed the highest VAS scores.The total treatment duration was significantly shorter in the group with the initial thickness of hemangiomas ＜ 1 mm (2.71 ± 0.58 months) than in those with the initial thickness of hemangiomas 1-3 mm (8.22 ± 0.67 months,P ＜ 0.05) and ＞ 3 mm (11.03 ± 0.72 months,P ＜ 0.05).The VAS scores also significantly differed among the 3 groups with different initial area of hemangiomas (＜ 3 cm2,3-9 cm2 and ＞ 9 cm2),and significantly increased over time in these groups;Kruskal-Wallis H test showed that there was a significant difference in the treatment sessions among the above 3 groups (H =10.45,P ＜ 0.01),and the group with the initial area of hemangiomas ＜ 3 cm2 showed the least treatment session.The adverse reactions were mild,and no adverse cardiovascular or respiratory events were observed.Conclusion The 595-nm pulsed dye laser combined with topical timolol maleate 0.5％ solution is effective and safe for the treatment of superficial IH.

Objective:To describe our experiences in application of the 2019 revision of "CCCG-WT-2016" for the diagnosis of Wilms tumors.Methods:Ninety-one cases of Wilms tumor diagnosed at Shanghai Children′s Medical Center from January 2015 to December 2018 were collected. All cases were reviewed by two senior pathologists, including one from China and the other from Singapore, according to the 2019 revision of "CCCG-WT-2016."Results:The specimens were obtained by core biopsy ( n=21), primary nephrectomy ( n=41), post-chemotherapy nephrectomy/resection ( n=18), or biopsy/resection of metastatic/relapse/post-chemotherapy metastatic lesion(s) ( n=11). The specimens of core biopsy and primary nephrectomy ( n=62) all had favorable histology.Twelve post-chemotherapy nephrectomy cases were subdivided into three risk groups: low risk ( n=0), intermediate risk ( n=10) and high risk ( n=2). Six post-chemotherapy resection cases were subdivided into 3 risk groups:low risk ( n=0), intermediate risk ( n=5) and high risk ( n=1). The remaining 11 cases were comprised of metastatic, relapse, and post-chemotherapy metastatic lesions. The concordance rate of the two senior pathologists was 100%(91/91). Conclusions:The 2019 revision of "CCCG-WT-2016" is clearly written and easy to use. It can serve as the basis of accurate classification for clinical treatment.