Objective To observe the changes in proliferative activity of and autophagosome formation in human HaCaT keratinocytes and primary keratinocytes after different doses of ultraviolet B (UVB) radiation,and to assess the potential relationship between proliferation impairment and autophagosome formation.Methods Both cultured HaCaT cells and primary keratinocytes from human foreskin were irradiated with different doses (5,10,20and 40 mJ/cm2) of UVB.Those receiving no irradiation served as the control.After additional 12-hour culture,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of cells,monodansylcadaverin (MDC) staining to detect autophagosomes in cells.The number of autophagosome-positive or negative cells was counted using inverted fluorescence microscopy.Results UVB radiation induced a significant decrease in the proliferation of keratinocytes,especially in that of HaCaT cells.The proliferative activity expressed as the absorbance value at 490 nm was significantly lower in HaCaT cells (1.367 ± 0.035,1.173 ± 0.034 and 0.873 ±0.025 vs.1.519 ± 0.022,all P＜ 0.01) and primary keratinocytes (0.782 ± 0.012,0.773 ± 0.021 and 0.725 ± 0.031 vs.0.887 ± 0.035,all P ＜ 0.05) irradiated with UVB of 10,20 and 40 mJ/cm2 than in the unirradiated control cells.Significant differences were also observed in the proliferative activity among HaCaT cells irradiated with UVB of 10,20 and 40 mJ/cm2.The proportion of autophagosome-positive cells was increased after irradiation with UVB of 5,10 and 20 mJ/cm2,but decreased after irradiation with UVB of 40 mJ/cm2 in keratinocytes,especially in the primary keratinocytes.In detail,the proportion of autophagosome-positive cells was 22.69％ ± 2.15％,28.10％ ± 2.92％ and 22.92％ ± 2.61％ in HaCaT cells irradiated with UVB of 10,20 and 40 mJ/cm2 respectively,significantly higher than that in the unirradiated cells (10.18％ ± 1.50％,chi-square test for trends:x2 =27.48,P ＜ 0.01).No significant changes were observed in the proportion of autophagosome-positive cells in primary keratinocytes after irradiation with UVB of 5,10 and 20 mJ/cm2,but a marked decrease was found after irradiation with UVB of 40 mJ/cm2 compared with unirradiated keratinocytes (chi-square test for trends:x2 =6.86,P ＜ 0.01).Conclusions UVB radiation (10-40 mJ/cm2) decelerates the proliferation of HaCaT cells and primary keratinocytes in a dosedependent manner,and primary keratinocytes seem to be more resistant to UVB damage than HaCaT cells.Low to moderate doses (5-20 mJ/cm2) of UVB promote autophagosome formation in HaCaT cells in a dose-dependent manner,and exert no significant influence on that in primary keratinocytes; however,UVB of 40 mJ/cm2 suppresses autophagosome formation in keratinocytes,especially in primary keratinocytes.

Objective To investigate the immunogenicity of immunodominant cytotoxic T lymphocyte epitope E749-57 of human papilloma virus (HPV) 16 oncoprotein E7 chaperoned by heat shock protein (HSP)110. Methods Mouse HSP110 gene was cloned into prokaryotic expression vector pQE-80L for the expression of HSP110 protein, which was purified using Ni-NTA column. SDS-PAGE and Western-blot were conducted to confirm the purified mHSP110 protein, which was subsequently incubated with E749-57 peptide under heat shock condition, and high-performance liquid chromatography (HPLC) was used to evaluate the binding efficiency of the recombinant protein and E749-57 peptide. Twenty mice were divided into 4 groups to be immunized with mHSP110 protein, E749-57 peptide, mHSP110-E749-57 complex and phosphate buffered saline (PBS),respectively. Two weeks after the last immunization, spleen cells were collected from the immunized mice and divided into 2 parts: one were stimulated by E749-57 peptide followed by the detection of CD8+ INF-γ+ T cells with flow cytometry; the other one were subjected to MTT analysis for the estimation of cell proliferation. The mHSP110-E749-57 complex was also used to immunize TC-1 tumor bearing mice to observe its anti-tumor effect.Results The full-length 2577 bp-sized mHSP110 gene was amplified from mouse liver cDNA and cloned into pQE-80L vector. Direct sequencing confirmed the correctness of the cloning. SDS-PAGE and Western-blot demonstrated the successful purification of mHSP110. HPLC assay showed that the purified mHSP110 protein could bind with E749-57 to form a relatively stable protein complex. The percentage of IFN-γ+ CD8+ T cells in and proliferation index of spleen cells from the complex-immunized mice were statistically higher than those from the other 3 groups of mice. Moreover, the complex could obviously inhibit the growth of TC-1 tumor in mice. Conclusion The mHSP110-E749-57 complex could enhance the generation of specific cytotoxic T lymphocytes and exert anti-tumor effects in mice.

[Objective] To observe the effects of autophagy induced by different doses of ultraviolet B (UVB) irradiation on the apeptosis in human skin fibroblasts.[Methods] Skin fibroblasts were isolated from the circumcision specimen of a 23-year-old healthy male,and subjected to a primary culture.After 3 to 10 passages,the cells were collected and applied in the following experiment.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of some fibroblasts treated with different concentrations (0,0.5,2.0,5.0and 10.0 mmol/L) of 3-methyladenine (3-MA).To qualitatively and quantitatively detect the autophagy in fibroblasts treated with different concentrations of 3-MA and in fibroblasts treated with 3-MA of 0.5 mmol/Lfollowing UVB irradiation,monodansylcadaverine (MDC) staining was carried out,and immunofluorescence was used to detect the expression of microtubule-associated protein 1 light chain (LC3).Some fibroblasts were classified into 8 groups to remain untreated,be irradiated with UVB of 30,50 and 100 mJ/cm2 alone,treated with 3-MA of 0.5 mmol/L alone,or treated with 0.5 mmol/L 3-MA following irradiation with UVB of 30,50 and 100 mJ/cm2,respectively,then,cell apoptosis was qualitatively detected by Hoechst and propidium iodide (PI)staining,and quantitatively detected by flow cytometry with annexin V and PI.[Results] The percentage of autophagic cells was (63.037 ± 5.876) ％ in fibroblasts treated with starvation condition,significantly decreased to (34.425 ± 5.183) ％ in fibroblasts treated with 3-MA of 0.5 mmol/L.The expression of LC3 showed a gradually increasing trend from untreated fibroblasts,to fibroblasts irradiated with UVB of 30,50 and 100 mJ/cm2,while the increase was attenuated by the 4-hour treatment with 3-MA immediately after the irradiation.Compared with the other concentrations,the 3-MA of 0.5 mmol/L showed the least influence on the viability of fibroblasts.The addition of 3-MA of 0.5 mmol/L increased the percentage of cells both positive for Hoechst and PI staining in fibroblasts irradiated with UVB of 50 mJ/cm2,but decreased that in fibroblasts irradiated with UVB of 100 mJ/cm2.Similarly,the percentage of middle and late apoptotic cells was significantly higher in fibroblasts irradiated with UVB of 50 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 50 mJ/cm2alone ((10.933 ± 0.839) ％ vs.(7.267 ± 0.473) ％,t =5.20,P＜ 0.05),but lower in fibroblasts irradiated with UVB of 100 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 100 mJ/cm2alone ( (7.100 ± 0.781 ) ％ vs.( 1 0.133 ± 0.681 ) ％,t =6.29,P ＜ 0.05 ).[Conclusion]s The irradiation with UVB of 50 mJ/cm2 may protect fibroblasts by inducing autophagy and suppressing apoptosis,while the high level of autophagy induced by UVB of 100 mJ/cm2 may lead to autophagic cell death in fibroblasts.

Objective: To analyze clinical features and prognosis of 15 children with hydroa vacciniforme-like lymphoproliferative disorder (HVLPD) . Methods: Clinical features, histopathological findings, treatment outcomes in 15 patients, who were diagnosed with HVLPD in the Department of Dermatology, Children′s Hospital of Chongqing Medical University from 2014 to 2018, were retrospectively analyzed. Results: Among the 15 patients, 7 were males and 8 were females. The age of onset ranged from 2 to 13 years, and the average age was 6.5 years. Main clinical manifestations included facial edema, and papules, blisters, ulcers and crusts repeatedly occurring on both the sun-exposed parts such as the face and limbs and non-sun-exposed parts such as the trunk. Of the 15 patients, 13 had intermittent fever, 13 had hepatosplenomegaly, and 15 had lymphadenectasis. Moreover, kidney failure occurred in 1 patient, hemophagocytic syndrome occurred in 2, and lymphoma occurred in 1. Laboratory examination showed that 15 patients were all positive for Epstein-Barr virus (EBV) -immunoglobulin G (IgG) antibody, and all negative for EBV-IgM antibody. Additionally, high replication of EBV DNA was detected. Histopathological study of skin lesions showed mild to dense perivascular and periadnexal infiltration of lymphocytes in the dermis and subcutaneous tissue. Immunohistochemistry study showed positive staining for CD4 and CD8 in 13 of 15 patients, for CD56 in 7 of 15 patients, and for CD3 in 12 of 13 patients. Moreover, 9 of 11 cases were detected positive for T-cell intracellular antigen-1, 8 cases were all detected positive for granzyme B, and the Ki-67 proliferation index ranged from 3% to 50% in 11 of 12 cases. The treatment protocols were similar among the 15 patients. After the treatment, 2 patients showed stable disease condition, and 10 experienced relapse. During the follow-up, 2 patients developed hemophagocytic syndrome, 1 developed lymphoma, and the 3 patients finally died of the worsening condition after chemotherapy. Conclusions HVLPD is closely associated with chronic active EBV infection. Its clinical symptoms can be relieved after immunomodulatory therapies such as glucocorticoids, gamma globulin and interferon, and the clinical manifestations, treatment outcomes and prognosis vary greatly among different patients.

Objective:To construct a serine protease inhibitor Kazal type-5 (Spink5) conditional knockout mouse model, and to identify its phenotype.Methods:B cell-specific Spink5 conditional knockout mice of genotype Mb1 cre/+Spink5 floxp/floxp were constructed by using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9) technology, and served as the knockout group. Mice of genotype Mb1 +/+Spink5 floxp/floxp served as the control group. The mice of genotype Mb1 cre/+Spink5 floxp/floxp or Mb1 +/+Spink5 floxp/floxp were sacrificed when they were 4 to 6 weeks old, splenic mononuclear cells were isolated, and B lymphocytes and non-B lymphocytes were sorted by flow cytometry and fluorescence-activated cell sorting. Genotype identification was performed by PCR, and protein expression of lymphoepithelial Kazal-type-related inhibitor (LEKTI) was determined by Western blot analysis. Skin tissues were resected from the mice, and subjected to hematoxylin-eosin staining for measuring the epidermal thickness. Immunofluorescence staining was performed to determine fluorescence intensity of LEKTI protein in the mouse skin tissues. Paired t test or two-independent-sample t test was used for comparisons between groups. Results:Genotype identification results demonstrated that the stable B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed. Western blot analysis revealed that the relative protein expression of LEKTI in the B lymphocytes in the knockout group was 0.01 ± 0.02, which was significantly lower than that in the non-B lymphocytes in the knockout group (0.66 ± 0.11, t = 9.99, P < 0.001) , and that in the B lymphocytes in the control group (1.08 ± 0.13, t = 13.78, P < 0.001) . Among 39 mice in the knockout group, 4 presented with dry skin and scattered scaly hypertrophic maculopapules. The epidermal thickness of the lesional skin tissues in the knockout group was 90.42 ± 21.31 μm, significantly higher than that of the non-lesional skin tissues in the knockout group (29.71 ± 3.63 μm, t = 5.05, P = 0.002) and that of normal skin tissues in the control group (12.42 ± 2.21 μm, t = 6.74, P < 0.001) . Immunofluorescence staining showed no significant difference in the fluorescence intensity of LEKTI protein among the lesional skin tissues (46.21 ± 1.21) , non-lesional skin tissues (46.62 ± 2.13) in the knockout group and normal skin tissues in the control group (47.69 ± 1.71, P > 0.05) . Conclusion:The B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed, which provides a basis for further exploring mechanisms underlying skin barrier defects and immune dysfunction in Netherton syndrome.

Objective:To analyze and summarize clinical and pathological characteristics of clear cell papulosis (CCP) .Methods:Clinical data were collected from 57 children with CCP, who were diagnosed in Department of Dermatology, Children′s Hospital of Chongqing Medical University from November 2014 to March 2022, and their clinical and pathological characteristics were analyzed.Results:Among the 57 patients, 30 were males and 27 were females; their age ranged from 6 months to 7 years and 10 months; the age at onset ranged from 1 to 74 months, and 39 (68.4%) experienced onset of disease in infancy; the course of disease ranged from 1 to 84 months. Skin lesions mainly manifested as millet- to soybean-sized roundish white macules or short bar-like white macules measuring 1 - 2 cm in length. Most patients (49 cases, 85.9%) had skin lesions less than 20 in number. The most common location of skin lesions was the lower abdomen (35 cases, 61.4%), followed in turn by chest or upper abdomen (34 cases, 59.6%), anterior axilla or armpit (17 cases, 29.8%), and vulva (11 cases, 19.3%). Short bar-like white macules predominated on the chest or upper abdomen (23/34, 65.7%) and anterior axilla or armpit (10/17), roundish white macules predominated on the lower abdomen (30/35, 85.7%), and the vulvar lesions all manifested as roundish white macules. Histopathological findings showed that slightly larger cells with abundant clear cytoplasm were scattered in or above the basal layer of the epidermis, and these cells were positive for cytokeratin 7, carcinoembryonic antigen, and periodic acid-Schiff staining.Conclusion:CCP mainly occurred in young children, roundish white macules predominated on the lower abdomen and vulva, and short bar-like white macules predominated on the chest or upper abdomen, as well as on the anterior axilla or armpit, indicating that CCP has strongly identifiable clinical features.

Objective To investigate clinical features of hair collar sign in children and their implications.Methods Children with confirmed hair collar signs were collected from Department of Dermatology,Children's Hospital of Chongqing Medical University between January 2014 and December 2017.The morphology,distribution and quantity of skin lesions and auxiliary examination results were analyzed retrospectively in 15 patients with complete clinical records.Results The center of skin lesions manifested as a round cystic lesion or a slightly depressed atrophic patch or plaque with lack of hair on the surface of the lesions.The lesions were surrounded by thick and long hair in 13 patients.The skin lesions occurred on the parietal lobe in 9 patients,and on occipital or temporal region in other patients.Of the 15 patients,14 had solitary lesions,and 1 had multiple lesions.Six patients were complicated by nevus flammeus.Brain magnetic resonance imaging examination of 11 patients revealed abnormalities in 2,including 1 with potential skull defect and brain tissue bulging,and 1 with cystic changes on the outer side of the cranial plate.B-mode ultrasound of the brain was performed in 4 patients,and no abnormality was found.Conclusions The skin lesions of hair collar sign are various,and most are solitary lesions.It mostly occurs on the parietal lobe,and is often complicated by nevus flammeus.This sign may suggest the presence of central nervous system malformation in patients.