Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells / 中华血液学杂志

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

Effect of Dichloromethane Extraction Phase of Patrinia Scabiosaefolia Fisch. Stem on Proliferation and Differentiation of K562 Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of dichloromethane extraction phase of ethanol extract from stem of Patrinia scabiosaefolia Fisch.(DPSS) on proliferation and differentiation of K562 cells and its related mechanism.@*METHODS@#MTT assay was used to detect the effects of DPSS at 0, 25, 50, 100 and 200 μg/ml on the proliferation of K562 cells at 24, 48 and 72 hours. Flow cytometry was used to analyze the changes of cell cycle and apoptosis at 24 and 48 hours. Wright-Giemsa staining was used to observe the morphological changes of K562 cells. The cell surface antigens CD33 and CD11b were detected by flow cytometry.@*RESULTS@#The proliferation of K562 cells treated with different concentrations of DPSS was inhibited in a time-dose dependent manner (r=-0.96). Cell cycle analysis showed that with the increase of DPSS concentration, cells in G2/M phase increased (r=0.88), and cells were blocked in G2/M phase. Flow cytometry results showed that with the apoptosis rate of K562 cells was the highest when treated with 200 μg/ml DPSS for 48 h. Morphological observation showed that the K562 cell body increased, the amount of cytoplasm increased, the ratio of nucleus to cytoplasm decreased, and the nuclear chromatin was rough after DPSS treatment. Cell differentiation antigen, CD33 and CD11b, were positively expressed after treated with DPSS.@*CONCLUSION@#DPSS can induce apoptosis through cell cycle arrest, inhibit the proliferation of K562 cells, and induce K562 cells to differentiate into monocytes, which has a potential anti-leukemia effect.

Evaluation of the value of indirect immunofluorescence on salt-split skin in the diagnosis of bullous pemphigoid / 中华皮肤科杂志

Objective:To evaluate the value of indirect immunofluorescence on salt-split skin (IIF-SSS) in the diagnosis of bullous pemphigoid (BP) .Methods:A single-center clinical retrospective study was conducted. Totally, 163 patients with newly diagnosed BP were collected from Hospital of Dermatology, Chinese Academy of Medical Sciences from January 2013 to January 2019, so were 404 controls, including 161 with pemphigus, 67 with eczema, 26 with drug eruption, 23 with erythema multiforme, 18 with prurigo nodularis, etc. Blood samples were collected before the treatment, and IIF-SSS, BP180 NC16A enzyme-linked immunosorbent assay (ELISA) and direct immunofluorescence (DIF) assay were performed to evaluate the value of IIF-SSS in the diagnosis of BP. Measurement data were compared by using t test and Mann-Whitney test, and enumeration data were compared by using chi-square test and Fisher′s exact test or McNemar test. Results:The number of cases positive for IIF-SSS, BP180 NC16A ELISA and DIF assay was 160, 153 and 127 respectively in the BP group, and 0, 18 and 26 respectively in the control group. The sensitivities of IIF-SSS, BP180 NC16A ELISA and DIF assay for the diagnosis of BP were 98.15%, 93.86% and 77.91% respectively, and their specificities were 100%, 95.54% and 93.56% respectively. There was strong consistency in the diagnosis of BP between IIF-SSS and DIF (Kappa coefficient= 0.767, P < 0.001) . Conclusion:IIF-SSS has relatively high sensitivity and specificity for the diagnosis of BP, and can serve as a routine method for diagnosing BP.

Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects / 中华血液学杂志

Objective: To construct chimeric antigen receptor (CAR) T cells targeting CD52 (CD52 CAR-T) and validate the effect of CD52 CAR-T cells on CD52-positive leukemia. Methods: A second-generation CD52-targeting CAR bearing 4-1BB costimulatory domain was ligated into a lentiviral vector through molecular cloning. Lentivirus was prepared and packaged by 293 T cells with a four-plasmid system. Fluorescein was used to label cell surface antigens to evaluate the phenotype of CD52 CAR-T cells after infection. Flow cytometry and ELISA were used to evaluate the specific cytotoxicity of CD52 CAR-T cells to CD52-positive cell lines in vitro. Results: ①A pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP expressing plasmid was successfully constructed and used to transduce T cells expressing a novel CD52-targeting CAR. ②On day 6, CD52-positive T cells were almost killed by CD52-targeted CAR-T post lentivirus transduction [CD52 CAR-T (4.48 ± 4.99) %, vs Vector-T (56.58±19.8) %, P=0.011]. ③T cells transduced with the CAR targeting CD52 showed low levels of apoptosis and could be expanded long-term ex vivo. ④The CD52 CAR could promote T cell differentiation into central and effector memory T cells, whereas the proportion of T cells with a CD45RA(+) effector memory phenotype were reduced. ⑤CD52 CAR-T cells could specifically kill CD52-positive HuT78-19t cells but had no killing effect on CD52-negative MOLT4-19t cells. For CD52 CAR-T cells, the percentage of residual of HuT78-19t cells was (2.66±1.60) % at an the E:T ratio of 1∶1 for 24 h, while (56.66±5.74) % of MOLT4-19t cells survived (P<0.001) . ⑥The results of a degranulation experiment confirmed that HuT78-19t cells significantly activated CD52 CAR-T cells but not MOLT4-19t cells[ (57.34±11.25) % vs (13.06± 4.23) %, P<0.001]. ⑦CD52 CAR-T cells released more cytokines when co-cultured with HuT78-19t cells than that of vector-T cells [IFN-γ: (3706±226) pg/ml, P<0.001; TNF-α: (1732±560) pg/ml, P<0.01]. Conclusions: We successfully prepared CD52 CAR-T cells with anti-leukemia effects, which might provide the foundation for further immunotherapy.

Preparation of CD33 targeted bispecific- and trispecific-T cell engagers and their cytotoxicity on leukemia cells / 中华血液学杂志

Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.

Effects of interferon regulatory factor 9 on the biological phenotypes in PML-RARα-induced promyelocytic leukemia / 中华血液学杂志

Objective: To investigate the prognostic significance of interferon regulatory factor 9 (IRF9) expression and identify its role as a potential therapeutic target in acute promyelocytic leukemia (APL) . Methods: The gene expression profile and survival data applied in the bioinformatic analysis were obtained from The Cancer Genome Atlas and Beat acute myeloid leukemia (AML) cohorts. A dox-induced lentiviral system was used to induce the expression of PML-RARα (PR) in U937 cells, and the expression level of IRF9 in U937 cells treated with or without ATRA was examined. We then induced the expression of IRF9 in NB4, a promyelocytic leukemia cell line. In vitro studies focused on leukemic phenotypes triggered by IRF9 expression. Results: ①Bioinformatic analysis of the public database demonstrated the lowest expression of IRF9 in APL among all subtypes of AML, with lower expression associated with worse prognosis. ②We successfully established a PR-expression-inducible U937 cell line and found that IRF9 was downregulated by the PR fusion gene in APL, with undetectable expression in NB4 promyelocytic cells. ③An IRF9-inducible NB4 cell line was successfully established. The inducible expression of IRF9 promoted the differentiation of NB4 cells and had a synergistic effect with lower doses of ATRA. In addition, the inducible expression of IRF9 significantly reduced the colony formation capacity of NB4 cells. Conclusion: In this study, we found that the inducible expression of PR downregulates IRF9 and can be reversed by ATRA, suggesting a specific regulatory relationship between IRF9 and the PR fusion gene. The induction of IRF9 expression in NB4 cells can promote cell differentiation as well as reduce the colony forming ability of leukemia cells, implying an anti-leukemia effect for IRF9, which lays a biological foundation for IRF9 as a potential target for the treatment of APL.

Functional investigation of chimeric antigen receptor T cells targeting LMP1 antigen / 中华血液学杂志

Objective: This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies. Methods: To generate LMP1 CAR-T cells, a plasmid expressing LMP1 CAR was created using molecular cloning technology, and T cells were infected with LMP1 CAR lentivirus. The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed. Results: ① LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified. ② The LMP1 CAR-expressing plasmid was created, and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system, with an infection efficiency of more than 80% . ③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells. The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture, but there was no significant killing effect on Ramos, which are LMP1-negative lymphoma cells. ④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h, the degranulation effect was enhanced. The proportion of CD107a(+) T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group [ (13.25±2.94) % vs (1.55±0.05) % , t=3.972, P=0.017]. ⑤After coculture with LMP1-positive lymphoma cells, the proportion of CD69(+) and CD25(+) T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group [ (7.40±0.41) % vs (3.48±0.47) % , t=6.268, P=0.003; (73.00±4.73) % vs (57.67±2.60) % , t=2.842, P=0.047]. ⑥After coculture with LMP1-positive lymphoma cells, cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group [interferon-gamma: (703±73) ng/L vs (422±87) ng/L, t=2.478, P=0.068; tumor necrosis factor-alpha: (215±35) ng/L vs (125±2) ng/L, t=2.536, P=0.064]. Conclusion: In this study, we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell. Simultaneously, we created an LMP1 CAR-expressing plasmid and obtained LMP1 CAR-T cells by infecting T cells with a lentivirus packaging system. Furthermore, we demonstrated that LMP1 CAR-T cells could specifically kill LMP1-positive tumor cells in vitro. The degranulation and activation effects of LMP1 CAR-T cells were enhanced after coculture with LMP1-positive tumor cells, indicating a potential clinical application.

Linear IgA bullous dermatosis / 中华皮肤科杂志

Linear IgA bullous dermatosis (LABD) is a rare autoimmune subepidermal blistering disease. Currently, researches on LABD are still limited, and most are case reports. This review summarizes research advance in etiology and pathogenesis, clinical and histopathological manifestations, diagnosis and treatment of LABD.

Clinical and immunoserological features of 7 cases of anti-p200 pemphigoid / 中华皮肤科杂志

Objective:To analyze clinical and immunoserological features of patients with anti-p200 pemphigoid.Methods:Clinical data were collected from patients with confirmed anti-p200 pemphigoid in Hospital of Dermatology, Chinese Academy of Medical Sciences from January 2015 to October 2021, and their clinical and immunoserological characteristics were retrospectively analyzed.Results:Seven patients with anti-p200 pemphigoid were included. Indirect immunofluorescence on salt-split skin (IIF-SSS) showed that serum IgG antibodies of the 7 patients were located in the dermis of the salt-split skin, and Western blot analysis with dermal extracts as substrates revealed a protein band with a relative molecular mass of 200 000. Four patients presented with classic bullous pemphigoid-like skin lesions, 2 initially presented with eczematous lesions, and 1 presented with linear IgA bullous dermatosis-like skin lesions. Circulating IgG antibodies could recognize the recombinant laminin γ1 C-terminal region in 6 cases. Four patients received different doses of systemic glucocorticoids, 1 of whom was resistant to high-dose systemic glucocorticoids (equivalent to 1.4 mg·kg -1·d -1 prednisone) ; 2 responded well to minocycline and dapsone; 1 was lost to follow-up. Four patients achieved complete remission and discontinued the treatment at a mean follow-up of 22.5 months; 2 received complete remissiona on minimal therapy at a mean follow-up of 8 months. Conclusion:Patients with anti-p200 pemphigoid presented with heterogeneous clinical manifestations, and the recombinant C-terminal fragment of laminin γ1 can serve as a reliable antigen substrate for the detection of autoantibodies in patients with anti-p200 pemphigoid; some patients can eventually achieve complete remission off treatment.

Analysis of clinical characteristics of 64 cases of livedoid vasculopathy / 中华皮肤科杂志

Objective:To analyze clinical features and distribution patterns of livedoid vasculopathy lesions, especially obvious livedo reticularis and purpuric lesions.Methods:Clinical data were collected from 64 patients with confirmed livedoid vasculopathy in Hospital of Dermatology, Chinese Academy of Medical Sciences from July 2017 to October 2020, and analyzed retrospectively.Results:Among the 64 patients, 23 were males and 41 were females, aged 13 - 54 years; their age at onset ranged from 7 to 51 years, and 48 developed livedoid vasculopathy before the age of 25 years; the course of disease ranged from 6 months to 10 years. Livedoid vasculopathy occurred or worsened in summer in 49 patients, and skin lesions mainly manifested as necrotic irregular purpura, purpuric dermatosis-like erythema, irregular ulcers, livedo reticularis, telangiectasia, irregular white atrophic scars and pigmentation. Among the 64 patients, ulcers and necrotic purpura were mostly irregular, and occurred on the dorsum of the foot and around the ankle. A total of 40 patients presented with purpuric dermatosis-like lesions, including 32 with pigmented purpura and 4 with telangiectatic purpura. Besides, numbness, tingling and other symptoms of nerve terminal damage occurred in 4 patients.Conclusion:Clinical manifestations of livedoid vasculopathy are diverse, and differential diagnosis is important for patients with generalized livedo reticularis, purpuric dermatosis-like lesions and symptoms such as numbness.

Correlation of anti-desmoglein conformational epitope antibody and anti-acetylcholine receptor antibody titers with disease activity of pemphigus vulgaris / 中华皮肤科杂志

Objective:To investigate the correlation of related antibody titers in serum of patients with pemphigus vulgaris (PV) with disease severity and activity.Methods:A total of 24 patients with active PV were collected, who firstly visited Hospital for Skin Diseases, Chinese Academy of Medical Sciences from 2012 to 2015. Pemphigus disease area index (PDAI) was evaluated in the patients with PV at active and stable stages, and serum samples were collected. Enzyme-linked immunosorbent assay (ELISA) was conducted to determine titers of pathogenic anti-desmoglein (Dsg) conformational epitope antibodies, total anti-Dsg antibodies and anti-acetylcholine receptor (AChR) antibody in serum samples. Measurement data were compared by using t test, enumeration data were compared by using Fisher′s exact test, and correlations were analyzed by using Pearson correlation analysis. Results:Among the patients with active PV, there was no significant difference between the anti-Dsg1 antibody titers (611.4 ± 136.8) and anti-Dsg1 conformational epitope antibody titers (585.5 ± 134.7, t = 0.13, P = 0.89) , but the anti-Dsg3 antibody titers (708.6 ± 130.7) were significantly higher than the anti-Dsg3 conformational epitope antibody titers (297.2 ± 54.4, t = 2.90, P < 0.01) . In addition, both the anti-Dsg1 antibody titers and anti-Dsg1 conformational epitope antibody titers were positively correlated with PDAI scores in the patients with active PV (both r = 0.54, P < 0.01) ; PDAI scores were not correlated with the anti-Dsg3 antibody titers ( r = 0.11, P = 0.62) , but positively correlated with the anti-Dsg3 conformational epitope antibody titers ( r = 0.53, P < 0.01) . Among the 20 patients with stable PV, the serum titers of anti-Dsg1 antibodies and anti-Dsg1 conformational epitope antibodies significantly decreased compared with those at their first visit; anti-Dsg3 antibody titers significantly decreased in only 7 patients, and 13 patients still had high titers of anti-Dsg3 antibodies, including 6 with declined anti-Dsg3 conformational epitope antibody titers, and 5 converted from anti-AChR antibody-positive to anti-AChR antibody-negative. Conclusions:Both the anti-Dsg1 antibody and anti-Dsg1 conformational epitope antibody titers can reflect the disease activity of PV. The disease activity was not consistent with anti-Dsg3 antibody titers in some patients, and the anti-Dsg3 conformational epitope antibody or anti-AChR antibody may facilitate evaluating the disease activity of PV.

Oxidative Damage of Bone Marrow Stromal Cells Caused by Chemotherapy Drugs / 中国实验血液学杂志

OBJECTIVE@#To explore the oxidative damage of OP9 cells induced by daunorubicin (DNR) treatment.@*METHODS@#The TMRM probe was used to detect mitochondrial membrane potential by flow cytometry; the reactive oxygen species (ROS) was determined by flow cytometry DCFDA probe; the real-time PCR was used to detect the molecular expression of antioxidant enzyme，glutathione peroxidase (GPX) in OP9 cells; the expression of γ-H2AX was determined by flow cytometry.@*RESULTS@#Compared with normal OP9 cells, the positive rate of TMRM in DNR-treated OP9 cells decreased by 56.7% (P＜0.05); the positive rate of DCFDA in DNR-treated OP9 cells increased by 3.52 times (P＜0.01). Compared with normal OP9 cells, DNR-treated OP9 cells showed a decrease in the expression of GPX4 by 44.22% (P＜0.001); the expression of GPX7 decreased by 65.7% (P＜0.001); the expression of GPX8 decreased by 24.7% (P＜0.001); the positive rate of γ-H2AX in DNR-treated OP9 cells increased (P＜0.05).@*CONCLUSION@#After DNR treatment, mitochondrial membrane potential of OP9 cells decreases; the level of reactive oxygen species increases; the expression of glutathione peroxidase (GPX) molecules decreases significantly; genomic instability increases obviously; the oxidative damage of cells increased.

Effect of Chemotherapeutic Drug-Induced Damage of Bone Marrow Stroma Cells on Normal Hematopoietic Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of damage of bone marrow stroma cells induced by chemotherapeutic drug on the function of normal hematopoitic cells.@*METHODS@#Senescence cells were detected by flow cytometry after SA-β-gal staining; real-time PCR was used to detect the expression of a serial molecules in bone marrow stromal cell line OP9 cells; the expression of γ-H2AX was determined by flow cytometry after histone γ-H2AX staining; the colony forming ability of hematopoietic cells was tested by colony formation assay.@*RESULTS@#The percentage of senescence cells in OP9 cells after DNR treatment was 2.24 times as much as that in untreated OP9 cells (P<0.05). Compared with normal OP9 cells, the expression levels of IL-6 and TNF-alpha in DNR-treated OP9 cells increased by 2.73 times (P<0.01) and 0.56 times (P<0.01), and the expression levels of N-cadherin, alpha smooth muscle actin (alpha-SMA), angiopoietin1 (Angpt1) and osteopontin (OPN) decreased by 69.54%（P<0.01），63.90%（P<0.01），87.41%（P<0.01）and 42.78%（P<0.01）respectively. After the co-culture with DNR-treated OP9 cells, the colony formation of normal hematopoietic cells decreased by 47.10% than that co-cultured with untreated OP9 cells (P< 0.05), meanwhile, the percentage of γ-H2AX+ cells in normal hematopoietic cells increased by 2.19 times (P<0.05).@*CONCLUSION@#After treatment with DNR, the senescence cell number of OP9 cells sgnificantly increases; the expression of TNF-α and IL-6 is up-regulated, while the expression of α-SMA, Angpt-1 and OPN is down-regulated as compared with normal OP9 cells. In addition, after co-culture of DNR-treated OP9 cells with normal hematopoietic cells, the colony formation ability of hematopoietic cells decreases and the genome instability of hematopoietic cells increases as compared with normal hematopoietic cells.

Evaluation of the value of Western blot analysis based on human epidermal extracts prepared by thermal separation in the diagnosis of bullous pemphigoid / 中华皮肤科杂志

Objective To prepare human epidermal extracts by thermal separation,and to evaluate the value of epidermal extract-based Western blot analysis in the diagnosis of bullous pemphigoid (BP).Methods Human epidermal extracts were prepared by thermal separation from circumcised foreskins of healthy males.Serum samples were obtained from 22 inpatients with BP and 25 inpatients without BP in Hospital for Skin Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College between January 2015 and August 2017.These serum samples were subjected to Western blot analysis with epidermal extracts as substrates,as well as to BP180-NC16A enzyme-linked immunosorbent assay (ELISA).Statistical analysis was carried out using chi-square test and Fisher's exact test with the SPSS22.0 software.Results The sensitivities of epidermal extract-based Western blot analysis and BP 180-NC16A ELISA in the diagnosis of BP were 86.36％ (95 ％ CI:64.03％-96.41％) and 95.45％ (95％ CI:75.11％-99.76％) respectively (~ =1.10,P =0.294),and the specificities were 100％ (95％ CI:83.42％-100％) and 92％ (95％ CI:75.11％-99.76％) respectively (x2 =20.8,P =0.149).Epidermal extract-based Western blot analysis in the 22 patients with BP showed a protein band with relative molecular mass (RMM) of 230 000 in 4 patients,a protein band with RMM of 180 000 in 18,a protein band with RMM of 120 000 in 1,and a protein band with RMM of 97 000 in 1.The BP180-NC16A ELISA showed that the antibody titers were more than 50 U/ml in the BP patients with protein bands of RMM of 180 000.Conclusions The epidermal extract-based Western blot analysis mainly showed the protein band with RMM of 180 000 in the patients with BP.The sensitivity of the epidermal extract-based Western blot analysis was lower than that of the BP180-NC16A ELISA,and the epidermal extract-based Western blot analysis tends to be negative when the titer of the autoantibody is low.

Associations of anti-desmoglein antibodies with clinical phenotypes and disease activity in pemphigus patients and their change patterns / 中华皮肤科杂志

Objective To investigate associations of anti-desmoglein (Dsg1 and Dsg3) antibodies detected by enzyme-linked immunosorbent assay (ELISA) with clinical phenotypes and disease activity in pemphigus patients,and to explore their change patterns.Methods A total of 111 patients with pemphigus were enrolled from Hospital for Skin Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College between January 2015 and January 2018.ELISA was performed to detect serum levels of anti-Dsg1 and anti-Dsg3 antibodies in these patients with different clinical types of pemphigus at different stages,including onset stage,control stage (no new erythema or vesicles occurred in the last 2 or more weeks,and primary lesions began to regress),maintenance stage (the condition had been stable for ≥ 1 month,and treatment was maintained with a low dose of glucocorticoids [prednisone equivalent of ＜ 15 mg/d]),and recurrence stage,and the change patterns of serum levels of anti-Dsg1 and anti-Dsg3 antibodies were analyzed.Statistical analysis was carried out with SPSS 22 software by using oneway analysis of variance for the comparison among groups,and least significant difference (LSD)-t test for multiple comparisons.Results At the disease onset stage,control stage,maintenance stage and recurrence stage,92,53,33,and 9 patients respectively completed the detection.Among the 92 patients with initial onset of pemphigus,the positive rates of anti-Dsg1 and anti-Dsg3 antibodies were 100％ and 2.77％ respectively in 36 patients with pemphigus foliaceus,20％ and 80％ respectively in 10 with mucosaldominant pemphigus vulgaris,and 97.82％,95.65％ respectively in 46 with mucocutaneous pemphigus vulgaris.The serum levels of anti-Dsg1 antibodies in the patients with pemphigus foliaceus significantly differed among the disease onset stage,control stage,maintenance stage and recurrence stage (137.43 ±77.74,13.94 ± 14.81,21.50 ± 58.33,121.13 ± 86.89 U/ml,respectively),the serum levels of anti-Dsg3 antibodies in the patients with mucosal-dominant pemphigus vulgaris also significantly differed among the above clinical stages (125.61 ± 94.81,34.5 ± 16.26,0.6,258 U/ml,respectively),and the serum levels of anti-Dsg1 and anti-Dsg3 antibodies in patients with mucocutaneous pemphigus vulgaris both significantly differed among the above clinical stages(anti-Dsg1 antibody:115.39 ± 70.62,15.74 ± 25.10,3.62 ± 12.09,78.60 ± 92.25 U/ml,respectively;anti-Dsg3 antibody:137.98 ± 81.25,58.14 ± 63.46,29.26 ± 64.70,136.9 ± 101.47 U/ml,respectively).Additionally,the serum levels of anti-Dsg1 antibodies in the patients with pemphigus foliaceus,as well as the serum levels of anti-Dsg3 antibodies in the patients with mucosaldominant pemphigus vulgaris and those with mucocutaneous pemphigus vulgaris,were both significantly lower at the disease control stage and maintenance stage than at the disease onset stage and recurrence stage (all P ＜ 0.05).During the treatment,epitope spreading occurred in 2 patients,and high-titer anti-Dsg antibodies were observed in 4 patients at the stable stage.Conclusion Anti-Dsg antibody spectrum is associated with clinical phenotypes of pemphigus,and its serum levels measured by ELISA can be applied to disease activity monitoring and evaluation of therapeutic efficacy.

Sex difference in aversive stimulation is associated with the activity of lateral habenula in mice / 西安交通大学学报(医学版)

Objective: To explore the difference in response to aversive stimulation and the expression of CaMK-2β in the lateral habenula between male and female mice. Methods: Male and female mice were given the non-condition stimulation with electric shock associated with the condition-stimulation with the light and tone context. After 24 hours, the mice were placed into the stimulation context and their responses were recorded and analyzed. At 48 hours after non-condition stimulation, the effects on extinction were observed in the male and female mice. Immunohistochemical method was used to detect the number of CaMK-2β positive cells in the lateral habenula nucleus. The expression of CaMK-2β protein in the lateral habenula was detected by Western blot. Results: Female mice were more sensitive to context cue compared with the male mice. Similarly, the female mice were more tolerant to extinction than their male counterparts. Consistently, at 48 hours after aversive stimulation, the CaMK-2β-positive cells in lateral habenula of female mice outnumbered those of the male mice. Additionally, the expression of CaMK-2β in habenula protein was higher in the female mice than in the male mice after aversive stimulation. Conclusion: Male and female mice showed different responses to the same stress stimulation, which determined the consolidation to stress information. The sex difference in aversive stimulation may contribute to the expression of CaMK-2β in the lateral habenula.

Anatomy and three-dimensional reconstruction study of SSL area / 中国实用妇科与产科杂志

OBJECTIVE: Use cadavers and three-dimensional MRA models to study the anatomical relationship between the sacrospinous ligament(SSL)and its adjacent vessels and nerves.METHODS: Totally 24 female cadavers provided by Anatomy Department of Southern Medical University from September 2017 to September 2018 were dissected,and 30 MRA data collected by Gynecology Department of Nanfang Hospital of Southern Medical University from January 2015 to December 2018 were selected to reconstruct to measure the relevant application parameters.RESULTS: The medial structure of the pudendal canal was the pudendal nerve,and the horizontal distance between the right pudendal nerve and the sciatic spine was(1.51 ± 0.35)cm. The thickness of the thickest point of the right coccygeus-sacrospinousligament(CSSL)was(1.03±0.23)cm and the horizontal distance between the point and the right sciatic spine was(2.81±0.55)cm;the vertical distance from where the right inferior gluteal artery is out of pelvis cavity to the horizontal line of sciatic spine was(2.43±0.95)cm,and the distance between the vertical point and the sciatic spine was(1.83±0.83)cm. In anatomy and MRA model,none of the inferior gluteal artery went dorsally through the SSL,and the sciatic nerve was away from the SSL.There was no obvious vascular nerve traveling on the pelvic surface of SSL.CONCLUSION: The point which is 2.81 cm away from the right sciatic spine of the SSL at the horizonal level may be the best suspension point for SSLF.

Diagnosis and treatment of skin diseases manifesting as desquamative gingivitis / 中华皮肤科杂志

Skin diseases manifesting as desquamative gingivitis (DG) can be divided into recurrent DG-and chronic DG-related skin diseases,including oral lichen planus,mucosal pemphigoid,pemphigus vulgaris and so on.A thorough medical history,detailed oral and histopathological examinations and serum immunological tests can be helpful for correct diagnosis of DG-related skin diseases.The treatment of DG-related skin diseases includes topical and systemic therapies.It is necessary to individualize treatment protocols due to treatment response.During the treatment of DG,oral hygiene should be strengthened,secondary fungal and bacterial infections should be avoided,and attention should be paid to the protection of oral cavity and periodontal tissues.

Indirect immunofluorescence on salt-split skin and bullous pemphigoid 180 NC16a enzyme-linked immunosorbent assay in the diagnosis of bullous pemphigoid / 中华皮肤科杂志

Objective To evaluate the value of indirect immunofluorescence on salt-split skin (IIF-SSS) and bullous pemphigoid 180 N C 16a enzyme-linked immunosorbent assay (BP 180 N C 16a-ELISA) in the diagnosis of bullous pemphigoid (BP).Methods Serum samples were collected from 174 BP patients and 129 controls,who were enrolled from Institute of Dermatology of Chinese Academy of Medical Sciences and Peking Union Medical College between January 2015 and August 2017,and subjected to IIF-SSS and BP180 NC16a-ELISA.Direct immunofluorescence (DIF) test was performed in 25 cases of BP,and its sensitivity for the diagnosis of BP was compared with that of IIF-SSS and BP180 NC16a-ELISA.Results The sensitivities for IIF-SSS and BP180 NC16a-ELISA were 93.67％ and 96.55％ respectively,and the specificities for IIF-SSS and BP180 NC16a-ELISA were 100％ and 96.12％ respectively.IIF-SSS was weakly correlated with BP180 NC16a-ELISA with a correlation coefficient of 0.147.There was no significant difference in the sensitivity between the serological diagnostic methods (IIF-SSS and BP180 NC 16a-ELISA) and DIF.Conclusion Serological diagnostic methods show high specificity and sensitivity in the diagnosis of BP,and are worthy of clinical promotion and application.