Background and purpose:Cytotoxic T lymphocyte (CTL) plays a vital role in the process of anti-tumor immunology. The aim of this study was to investigate whether changes in concentration of IL-2 (50, 200 and 1 000 U/mL) would affect the sub-population and cytotoxic function of cells cultivated by peptide-specific CTL induction systemin vitro and also observe whether using the concentration of IL-2 at a range of 50-1 000 U/mL isbeneifcial to regulatory cells (Tregs) enrichment.Methods:Peripheral blood from 10 healthy donors and 10 cancer patients that were HLA-A2 positive, were collected in the study. HLA-A2 restricted CTL epitope P321 (ILIGETIKI) derived from COX-2 pulsed with different concentrations of IL-2 were used to induce peptides-speciifc CTLin vitro. Flow cytometry was performed to analyze the proliferative capability, the proportion of different T-cell subsets, and secretion of perforin, granzyme B and IFN-γ. IFN-γ secretion was assessed by ELISpot assay.Results:High concentration of IL-2 increased the proliferative activity. The percentage of CD4+ T cells of cancer patient group was signiifcantly higher than that of healthy donor group, while the percentage of CD8+ T cells of cancer patient group was signiifcantly lower than that of healthy donor group. And there was no signiifcant difference in the percentages of CD4+ T cells, CD8+ T cells and Tregs among groups with different IL-2 concentrations. No difference was seen in cytokine (perforin, granzyme B, IFN-γ) secretion capacity of CD8+ T cells. ELISpot study revealed that high-dose IL-2 resulted in the increasing of IFN-γ secretion.Conclusion:The sub-population and the function of cells cultured by peptide-speciifc CTL induction systemin vitro are not affected by different concentrations of IL-2. Furthermore, high concentrations of IL-2 (50-1 000 U/mL) do not provide the enrichment for Tregs. Higher concentration of IL-2 is likely to cause high secretion of IFN-γ in ELISpot assay. In order to exclude the distraction of NK cells or NKT cells, the concentration of 50 U/mL is better choice.

Objective:To investigate the role of folliculin in apoptosis of and chemokine secretion by melanocytes mediated by interferon-γ (IFN-γ) .Methods:Normal primary melanocytes were isolated from circumcised foreskin tissues from a healthy male child, and primary vitiliginous melanocytes were isolated from normally pigmented suction-blistered epidermis from patients with vitiligo after suction blister epidermal grafting. Western blot analysis was performed to determine the folliculin protein expression in normal primary melanocytes, primary vitiliginous melanocytes and a human primary melanocyte line PIG1. PIG1 cells stimulated with 10 ng/ml IFN-γ for 48 hours served as induction group, and untreated PIG1 cells served as control group. Real-time quantitative RCR (qRT-PCR) was performed to determine the mRNA expression of folliculin, autophagy-related microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ and Beclin genes, and Western blot analysis to determine the protein expression of folliculin, Beclin1 and LC3Ⅱ/Ⅰ, as well as phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) in the above cells. Furthermore, the melanocytes stimulated with 10 ng/ml IFN-γ for 48 hours were divided into several groups: negative control group infected with an empty lentiviral vector, folliculin inhibition group infected with a folliculin-inhibiting lentivirus, autophagy enhancement group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with a mTOR inhibitor, autophagy inhibition group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with an AMPK inhibitor. Then, flow cytometry was conducted to detect apoptosis of PIG1 cells, and enzyme-linked immunosorbent assay to measure the concentration of chemokines CXCL10 and CCL20 in the culture supernatant of PIG1 cells in the above groups. Measurement data were compared among multiple groups by using one-way analysis of variance, and multiple comparisons were carried out by using least significant difference- t test. Results:The relative protein expression level of folliculin significantly differed among the normal primary melanocytes (0.850 ± 0.120) , primary vitiliginous melanocytes (1.507 ± 0.170) and PIG1 cells (0.697 ± 0.130; F = 50.09, P < 0.001) , and was significantly higher in the primary vitiliginous melanocytes than in the normal primary melanocytes and PIG1 cells ( t = 4.06, 5.89, respectively, both P < 0.01) . Compared with the control group, the induction group showed significantly increased relative mRNA and protein expression levels of folliculin (both P < 0.01) , but significantly decreased relative mRNA and protein expression levels of LC3Ⅱ and Beclin (all P < 0.01) ; moreover, the induction group showed significantly decreased LC3Ⅱ/Ⅰ levels (0.72 ± 0.02) and AMPK phosphorylation levels (0.714 ± 0.023) in the PIG1 cells compared with the control group (1.13 ± 0.02, 1.176 ± 0.002, t = 7.34, 6.67, respectively, both P < 0.01) , but significantly increased mTOR phosphorylation levels (1.051 ± 0.023) compared with the control Group (0.451 ± 0.016, t = 3.81, P = 0.009) . There were significant differences in the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 among the control group, induction group and other treatment groups (all P < 0.001) ; specifically, the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 were significantly higher in the induction group than in the control group, lower in the folliculin inhibition group than in the negative control group, lower in the autophagy enhancement group than in the folliculin inhibition group, and higher in the autophagy inhibition group than in the folliculin inhibition group (all P < 0.05) . Conclusions:Folliculin is highly expressed in vitiliginous melanocytes. Folliculin expression and downstream signaling pathways are regulated by IFN-γ, and folliculin may participate in IFN-γ-mediated melanocyte apoptosis and chemokine secretion via regulating autophagy.

Objective:To evaluate the barrier-free level of village clinics of a city in the aging era, for references in improving the barrier-free medical environment for the elderlies.Methods:The village dectors of 156 village clinics in 12 counties of a city were selected as the objects of a questionnaire survey from Dec.2020 to Jan.2021, with 6 evaluation indexes established, each set with 1-10 points. This questionnaires was used to survey the barrier-free construction levels of village clinics. The data were statistically analyzed by rank sum ratio and quadrant graph model.Results:The barrier-free buildings of such clinics scored 32.86 points in average. Among the six indexes, the barrier-free access and doors(7.21 points) scored the highest, and the barrier-free parking space(4.71points) and toilets(3.28 points) scored the lowest. All the counties of the city fell into four categories, including 2 counties with high degree of aging-high level of accessibility, 4 counties with low degree of aging-high level of accessibility, 4 counties with low degree of aging-low level of accessibility, and 2 counties with high degree of aging-low level of accessibility, according to the quadrant graph model built based on the aging degrees and the barrier-free levels of village clinics.Conclusions:The barrier-free level of the village clinics in a city needs to be further improved, and the barrier-free transformation of parking space and toilets should also be strengthened in the future. The government should take different measures in view of conditions of each county to improve the accessibility of village clinics and facilitate the health care-seeking of elderlies.

Objective:To investigate the effect of human tumor suppressor folliculin (FLCN) on the expression of melanocyte chemokines (MC) mediated by immune factors in vitiligo.Methods:The MC of vitiligo patients that received autologous melanocyte transplantation in the Department of Dermatology, Hangzhou Third People′s Hospital from January to April 2019 were collected. The blister fluid of the white spot and the normal part was taken. Western blot was used to analyze the expression difference of MC and FLCN protein in normal, vitiligo patients and that induced by immune factors; FLCN shRNA lentivirus was constructed by shRNA and transfected into normal MC (FLCN shRNA MC) to interfere with the expression of silenced FLCN gene. The effect of immune factors on chemokines in FLCN shRNA MC was detected by ELISA.Results:The results of Western blot showed that FLCN protein was highly expressed in melanocytes of vitiligo patients, immune factors stimulated FLCN protein expression in normal melanocytes significantly increased ( t=1.27; P<0.001), chemokine CXCL10 and CCL20 also significantly increased ( t=104.53 and 60.21, respectively; P<0.001). The expression of FLCN in FLCN shRNA MC was significantly decreased ( F=1.95, P<0.001); and the high expression of CXCL10 and CCL20 induced by immune factors was significantly inhibited ( F=93.676 and 74.096, all P<0.001). Conclusions:Immune factors can stimulate the expression of CXCL10 and CCL20, which are closely related to vitiligo, while FLCN is a key protein involved in immune factors inducing melanocyte chemokine expression.

Patients with abdominal surgery history always present different degrees of abdominal adhesion. In the past, it was regarded as a relative contraindication of laparoscopic surgery. With the development of minimally invasive concept, reoperative minimally invasive surgery is proposed in clinic to avoid huge trauma caused by multiple open surgeries. In June 2022, a laparoscopic assisted ileostomy reduction was performed for a patient with history of multiple abdominal injuries in Shanghai Changzheng Hospital. Minimally invasive achieved after huge trauma, and it maximized the benefit to the patient.

Objective:To revise the family resilience assessment scale (FRAS) in patients with stroke and to evaluate its psychometric properties and applicability.Methods:FRAS was authorized and translated into Chinese.The scale items were revised based on the interview results of 13 patients with stroke and 11 caregivers and the opinions of 20 experts in related fields.A convenience sampling method was used to sample neurology and neurosurgery inpatients from 2 tertiary hospitals in Hangzhou twice from June to November 2022, with 325 questionnaires (sample 1) distributed the first time for item analysis and exploratory factor analysis, and 285 questionnaires (sample 2) distributed the second time for confirmatory factor analysis, criterion validity and reliability test.Data analysis was conducted by SPSS 26.0 and AMOS 24.0.Results:The exploratory factor analysis extracted 6 common factors(family beliefs, family spirit, family connection, family resources, family communication, family collaboration) with 32 items.Confirmatory factor analysis showed that the model fit well( χ2/ df=2.67, RMSEA=0.025, CFI=0.98, GFI=0.90, IFI=0.98, RMR=0.031). The cronbach's α coefficient of the total scale was 0.96, and 0.82-0.92 for the dimensions.The 2-week retest reliability was 0.99.The total scale score was positively correlated with the validity scale (family resilience scale) ( r=0.882, P<0.001). Conclusion:The family resilience scale for patients with stroke has good reliability and validity and can be used to assess the family resilience of patients with stroke.

Objective To investigate the clinical features,diagnosis,treatment,and prognosis of patients with liver injury caused by Periploca forrestii Schltr.Methods A retrospective analysis was performed for the general data,clinical manifestations,and laboratory examinations of 22 patients who were diagnosed with liver injury caused by Periploca forrestii Schltr.from November 2014 to December 2015,and their clinical type was determined according to the classification criteria of drug-induced liver injury recommended by the Council for Intemational Organizations of Medical Sciences.Results There were 12 female and 10 male patients.The mean medication time ranged from 1 week to 2 months,and as for biochemical markers,there were mainly abnormalities in alanine aminotransferase (ALT),aspartate aminotransferase (AST),total bilirubin (TBil),alkaline phosphatase (ALP),and gamma-glutamyl transpeptidase (GGT).ALT and AST increased in all the patients,with mean levels of 676.68±481.11 U/L and 527.36±361.14 U/L,respectively;TBil increased to a mean level of 170.26±147.30 μmol/L in 19 patients;ALP increased to a mean level of 135.61±59.26 U/L in 13 patients;GGT increased to a mean level of 195.65±138.48 U/L in 20 patients.As for clinical typing,18 patients had liver cell injury,none had cholestasis,3 had a mixed type,and 1 had an unclassified type.One patient died and all the other patients fully recovered.Conclusion Periploca forrestii Schltr.had complex constituents,and liver injury caused by this drug is mainly liver cell injury.The pathogenesis of liver injury caused by Periploca forrestii Schltr.is presumed to be related to patients' idiosyncratic reaction to its constituents.

Objective:To clarify the connotation of clown care by conceptual analysis of clown care, so as to provide references for clinical nursing work.Methods:Literature related to clown care was searched in PubMed, EBSCO, Embase, CNKI, Wanfang Database and VIP, and the search period was from the establishment of the databases until August 31, 2022. Based on the inclusion and exclusion criteria, literature was screened. The Rodgers evolutionary concept analysis method was adopted to analyze the relevant contents of clown care from aspects of development history, definition and evolution, conceptual attributes, typical cases, antecedents, consequences and identification of related concepts.Results:A total of 463 articles were retrieved, and 94 were finally included, including 8 Chinese articles and 86 English articles. At present, there was no unified and comprehensive definition of clown care. It included 5 conceptual attributes, such as support relationship, individuation, interaction, flexibility and empowerment. The antecedents included demographic, clown, and social factors, and the consequences mainly included the effect on the patient's health status, negative emotions of medical staff, clown care skills and creativity.Conclusions:Clarifying the concept of clown care can provide clear conceptual support for the research on clown care, and provide references for the construction of scientific clown care program.

Objective:To investigate the disease control rate and its influencing factors in patients with progressive non-segmental vitiligo after combined treatment with systemic compound betamethasone injection (CBI) .Methods:A retrospective analysis was conducted on the patients with progressive non-segmental vitiligo, who visited and were treated with CBI in the Department of Dermatology, Hangzhou Third People′s Hospital from October 2022 to April 2023. The disease control rate was analyzed after 3-month treatment. Effects of clinical factors such as disease onset characteristics, duration, disease condition and treatment methods on the disease control rate were analyzed. Chi-square test was used for comparisons of enumeration data between groups, and logistic regression analysis was conducted to analyze factors influencing the efficacy.Results:A total of 145 progressive non-segmental vitiligo patients treated with CBI were collected, including 56 males and 89 females, aged 14 - 67 (35.43 ± 11.54) years. Among the 18 patients having received an intramuscular injection of CBI, 10 (55.6%) obtained stable condition; among the 105 having received 2 injections of CBI, 60 (59.0%) obtained stable condition; among the 22 having received 3 or 4 injections of CBI, 12 (54.5%) obtained stable condition. The overall disease control rate after treatment was 57.9% (84 cases). The disease control rate was significantly higher in the patients with the lesional area < 1% body surface area (BSA) (42/47, 89.4%) than in those with the lesional area ≥ 1% BSA (42/98, 42.9%; P < 0.001), significantly higher in the patients with disease duration ≤ 2 years (32/41, 78.0%) than in those with disease duration > 2 years (52/104, 50.0%; P < 0.05), and significantly higher in the patients treated with CBI combined with phototherapy (33/44, 75.0%) than in those treated with CBI alone (21/44, 47.7%; χ2 = 6.90, P = 0.009), but significantly lower in the patients with special clinical markers (Koebner phenomenon, trichrome vitiligo, confetti-like depigmentation, inflammatory vitiligo, etc., 4/21, 19.9%) than in those without special clinical markers (80/124, 64.5%, P < 0.001). Among the patients with the lesional area ≥ 1% BSA and receiving 2 injections of CBI, the disease control rate was also significantly higher in the patients treated with CBI combined with phototherapy (21/36, 58.3%) than in those treated with CBI alone (12/37, 32.4%; χ2 = 4.94, P = 0.026). There was no significant difference in the disease control rate after the treatment between the patients with first-onset and reccurrent vitiligo, between those with and without predisposing factors, between those with and without family history, among those with different vitiligo disease activity scores, among those with different number of injections, as well as among those with different treatment intervals (all P > 0.05). Multivariate logistic regression analysis showed that the lesional area ( OR = 8.11, 95% CI: 2.74 - 24.04), disease duration ( OR = 0.26, 95% CI: 0.07 - 0.99), having or not having special clinical markers ( OR = 6.37, 95% CI: 1.72 - 23.57), and whether or not receiving combined phototherapy ( OR = 0.34, 95% CI: 0.15 - 0.77) were factors influencing the efficacy (all P < 0.05) . Conclusion:CBI may be suitable for the treatment of mild to moderate progressive vitiligo, especially for patients with lesional area < 1% BSA, while not for those with lesional area > 5% BSA, and combining phototherapy may improve the control rate of progressive vitiligo.

Objective To explore the effect of blended teaching model based on "Rain Class" in general practitioner training.Methods Random sampling method was adopted in which totally 132 general practitioners were divided into two groups,66 in experimental group,66 in the control group.Blended teaching method and traditional teaching method was implemented in the experimental group and the control group,respectively.At the end of the course,unified examination questions were used to evaluate the teaching effect,and questionnaires were used to investigate the students' evaluation of the "rain classroom" teaching method.Results The final exam score of experimental group was significantly higher than that of control group [(82.95 ± 12.86) vs.(60.45 ± 18.91),P=0.000].Further stratification analysis was conducted according to educational background,and the average score of low education group in the observation group was significantly higher than that of high education group [(88.06 ± 9.12) vs.(49.66 ± 13.88),P=0.000].55.3％ of the general practitioners in experimental group were very satisfied with "Rain Class" teaching,34.8％ satisfied,and only 9.9％ of the general practitioners were dissatisfied.Conclusion Blended teaching model could help to promote the exchange and interaction between students and teachers and improve learning efficiency and clinical ability.