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Objective The aim is to utilize CRISPR/Cas9 gene editing technology to construct Dmd gene mutant mice with a point mutation in exon 23 of the Dmd gene. Subsequently, the phenotypic changes of the mice in muscles and immune systems are analyzed and verified, providing an evaluation model for Duchenne muscular dystrophy and other related diseases.MethodsBased on the sequence characteristics of exon 23 of the Dmd gene, small guide RNA (sgRNA) was designed and synthesized. Cas9 mRNA, sgRNA fragments, and oligo donor DNA were microinjected into fertilized eggs of C57BL/6J mice. After transferring the fertilized eggs to surrogate mice, F0 generation mice were born. After mating with F0 generation mice, offspring mice were obtained, and Dmd gene positive mutant (DmdMu/+) mice were obtained after genotype identification. Male hemizygous DmdMu/+(DmdMu/Y) mice were selected for phenotype validation. The body weight of live 3- and 9-month-old mice were recorded. Muscle tension was evaluated through the grid test. Hearts and semitendinosus muscles were collected, and the histopathological changes were observed using HE staining. Further, the expression of Dmd protein in muscle tissue of 9-month-old mice was analyzed by Western blotting.An acute inflammation model was established in DmdMu/Y mice using lipopolysaccharide induction. Peripheral blood from the submandibular vein was collected, and the changes in the proportion of neutrophils and monocytes were detected by flow cytometry.Results The results of genome sequencing and Western blotting confirmed the successful construction of Dmd gene point mutant mice (DmdMu/+ mice). Dmd protein expression was not detected in skeletal muscle and myocardium of DmdMu/+ mice, and it was significantly reduced compared to wild-type C57BL/6J mice (P<0.05). Compared with wild-type mice of the same background, DmdMu/Y mice at 3 and 9 months of age showed significant weight loss (P<0.01) and decreased muscle tension (P<0.05). 9-month-old DmdMu/Y mice exhibited significant pathological changes in skeletal muscle and myocardium, including widening of intermuscular space. Under normal condition, compared with wild-type mice, the proportion of neutrophils and monocytes in the peripheral blood of 3-month-old DmdMu/Y mice was significantly lower than that of wild-type mice (P<0.01). After lipopolysaccharide stimulation, the proportion of neutrophils in peripheral blood of 3-month-old DmdMu/Y mice remained significantly lower compared to that of wild-type mice (P<0.01). The proportion of neutrophils in peripheral blood of 9-month-old DmdMu/Y mice significantly decreased after lipopolysaccharide induction (P<0.01), with a trend of change observed in monocytes between groups.Conclusion The successful construction of the Dmd gene mutant mouse model has confirmed the vital function of Dmd gene in maintaining normal muscle tissue morphology and muscle tone. It preliminarily indicated that Dmd gene deletion could significantly reduce the proportion of neutrophils in peripheral blood, offering a new perspective for the study of immune system alterations in Duchenne muscular dystrophy patients.
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Objective:To observe the clinical efficacy of Qigui Tangtongning Granules in the treatment of diabetic peripheral neuropathy (DPN) with qi deficiency and blood stasis.Methods:Prospective cohort study. A total of 80 DPN patients with Qi deficiency and blood stasis in Endocrinology Department of the First Affiliated Hospital of Anhui University of Chinese Medicine from May 2021 to May 2022 who met the inclusion criteria were divided into 2 groups by random number table method, with 40 cases in each group. The control group was treated with epalrestat on the basis of routine hypoglycemia, and the treatment group was treated with Qigui Tangtongning Granules on the basis of control group. Both groups were treated for 8 weeks. TCM syndromes were scored before and after treatment. Disease severity was assessed using the Toronto Clinical Scoring System (TCSS). The motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) of median nerve and common peroneal nerve were detected by electromyography/induced potentiometer. Serum CRP, TNF-α and IL-6 were detected by ELISA, fasting blood glucose (FPG) and two hours post-meal blood glucose (2 hPG) were detected by automatic biochemical analyzer, and glycosylated hemoglobin (HbA1c) was detected by automatic HBA1C analyzer. Adverse reactions were recorded and clinical efficacy was evaluated.Results:The total effective rate was 95.0% (38/40) in the treatment group and 77.5% (31/40) in the control group, the difference between the two groups was statistically significant ( χ2=5.17, P=0.023). After treatment, the TCM syndrome score and TCSS score of the treatment group were significantly lower than those in the control group ( t=-3.19 and -7.63, P<0.01); Median nerve SNCV [(47.90±4.51) m/s vs. (44.76±3.72) m/s, t=3.40], MNCV [(53.79±3.65) m/s vs. (51.32±4.25) m/s, t=2.79] and common peroneal nerve SNCV [(44.21±2.08) m/s vs. (40.51±2.49) m/s, t=7.23], MNCV [(44.63±4.72) m/s vs. (41.36±4.87) m/s, t=3.05] were significantly higher than those in the control group ( P<0.01); FPG [(5.05±0.63) mmol/L vs. (7.05±1.23) mmol/L, t=-9.17], 2 hPG [(9.10±1.64) mmol/L vs. (12.19±2.61) mmol/L, t=-6.35], HbA1c [(6.79±0.90) % vs. (7.22±1.02) %, t=-2.02] were significantly lower than those in the control group ( P<0.01 or P<0.05); TNF-α [(15.75±5.44) ng/L vs. (32.01±5.33) ng/L, t=-13.51], hs-CRP [(2.58±0.80) mg/L vs. (3.79±1.04) mg/L, t=-5.83], IL-6 [(18.20±4.92) ng/L vs. (29.97±5.18) ng/L, t=-10.41] were significantly lower than those in the control group ( P<0.01). No obvious adverse reactions were observed in 2 groups during treatment. Conclusion:Qigui Tangtongning Granules combined with conventional Western medicine can improve nerve conduction velocity, reduce inflammation and improve clinical efficacy in DPN patients with Qi-deficiency and blood-stasis syndrome.
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Objective:To investigate clinicopathological features of hypopigmented mycosis fungoides (HMF) and hypopigmented interface T-cell dyscrasia (HITCD) .Methods:A total of 41 patients with cutaneous hypopigmented lymphoproliferative diseases, who had complete clinicopathological data, were collected from Department of Dermatology, the Third People′s Hospital of Hangzhou from January 2015 to September 2020, and the clinicopathological and immunophenotypic features were analyzed. Comparisons of normally distributed measurement data were carried out using t test, comparisons of categorical data using Chi-square test or Fisher′s exact test, and comparisons of ranked data between 2 groups using rank-sum test. Results:All of the 41 patients clinically presented with irregular hypopigmentation, some of which was accompanied by erythema or furfuraceous scales. In terms of pathological features, 21 patients showed infiltration and aggregation of atypical lymphoid cells in the epidermis, which was consistent with typical pathological features of mycosis fungoides, and they were diagnosed with HMF; 20 patients showed vacuolar degeneration of the basal layer, accompanied by infiltration of lymphoid cells and mild epidermotropism, and they were diagnosed with HITCD. All immune cells expressed T-cell phenotype, and epidermal lymphocytes expressed a CD8-dominated phenotype in 14 (67%) cases of HMF and 13 (65%) of HITCD. In the epidermis, the total number of lymphocytes was significantly higher in the HMF group than in the HITCD group ( t= 1.81, P= 0.012) ; in the dermis, the number of CD4 + lymphocytes and CD8 + lymphocytes, and the total number of lymphocytes were all significantly higher in the HMF group than in the HITCD group ( t= 2.64, 1.51, 2.60, P= 0.012, 0.002, 0.001, respectively) . All patients were treated with narrow-band ultraviolet B radiation. Among 34 patients who completed the follow-up, 30 achieved complete clearance of skin lesions without recurrence, including all patients with HITCD, and 4 with HMF achieved partial regression of the lesions. Conclusions:Compared with HMF, HITCD presents different pathological characteristics and benign biological behaviors. Thus, HITCD should be distinguished from HMF as an independent disease. Phototherapy alone is effective for the treatment of HITCD.
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Objective:To identify genes interacting with the ubiquitin-conjugating enzyme E2S (UBE2S) in melanoma, and to explore the molecular mechanisms regulating the biological behavior of melanoma cells by UBE2S.Methods:Cultured A375 melanoma cells were divided into 2 groups to be transfected with LV-UBE2S-RNAi (14011-1) -containing lentivirus (gene knockdown group) and a negative control lentivirus CON053 (negative control group), respectively, and the UBE2S gene-knockdown cell line and negative control cell line were established. RNA was extracted from the two cell lines, purified, fragmented, and hybridized with GeneChip probes, followed by washing and dyeing, and then microarray data were obtained. Ingenuity pathway analysis (IPA) software was used to further analyze the gene expression profiling data to identify potential genes interacting with UBE2S. Western blot analysis was performed to verify the downstream molecules regulated by UBE2S. A two-tailed t test was used to screen for differentially expressed genes. Results:A total of 512 differentially expressed genes with a|fold change| > 2 and a P value < 0.05 were screened out between the gene knockdown group and negative control group, including 247 upregulated genes and 265 downregulated genes. IPA of differentially expressed genes revealed that interferon signaling and liver X receptor (LXR) /retinoid X receptor (RXR) activation pathways were significantly activated, while eukaryotic initiation factor-2 (EIF2) signaling and nuclear factor-κB (NF-κB) signaling pathways were inhibited. Among the upstream regulators, IFNA2 was predicted to be strongly activated, and NF-κB (complex) to be strongly inhibited. Based on the above bioinformatics analysis results, it was suggested that the UBE2S gene may exert some effects in melanoma cells by regulating the expression of IFITM1, STAT1, ISG15 and TNFRSF11B genes. Western blot analysis showed that IFITM1 protein was not expressed in A375 cells before and after the UBE2S gene knockdown. After the UBE2S gene knockdown, ISG15 protein expression was upregulated by 19.94 times, STAT1 protein expression was upregulated by 1.47 times, and TNFRSF11B protein expression was downregulated by 79.1%. Conclusion:UBE2S may interact with STAT1, ISG15 and TNFRSF11B to regulate the biological behavior of melanoma cells.
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BackgroundMales and females differ in their immunological responses to foreign pathogens. However, most of the current COVID-19 clinical practices and trials do not take sex as consideration. MethodsWe performed an unbiased sex-based comparative analysis for the clinical outcomes, peripheral immune cells, and SARS-CoV-2 specific antibody levels of 1,558 males and 1,499 females COVID-19 patients from a single center. The lymphocyte subgroups were measured by Flow cytometry. Total antibody, Spike protein (S)-, receptor binding domain (RBD)-, and nucleoprotein (N)-specific IgM and IgG levels were measured by chemiluminescence. ResultsWe found that the mortality and ICU admission rates were approximately 2-fold higher in males than that in females (P<0.005). Survival analysis revealed that sex is an independent prognostic factor for COVID-19 (Hazard ratio=2.2, P=0.003). The concentration of inflammatory factors in peripheral blood was significantly higher in males. Besides, the renal and hepatic abnormality induced by COVID-19 was more common in males during the hospitalization. The analysis of lymphocyte subsets revealed that the percentage of CD19+ B cell and CD4+ T cell was significantly higher in females (P<0.001) during hospitalization, indicating the stronger humoral immunity in females than males. Notably, the protective IgG sharply increased and reached a peak in the fourth week after symptom onset in females, while gradually increased and reached a peak in the seventh week in males. ConclusionsThe unfavorable prognosis of male COVID-19 patients may result from the weak humoral immunity and indolent antibody responses during SARS-CoV-2 infection and recovery. Early medical intervention and close monitoring are important, especially for male COVID-19 patients. Hormonal or convalescent plasma therapy may help improve the immunity of males to fight against SARS-CoV-2 infection.
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Deciphering the dynamic changes of antibodies against SARS-CoV-2 is essential for understanding the immune response in COVID-19 patients. By comprehensively analyzing the laboratory findings of 1,850 patients, we describe the dynamic changes of the total antibody, spike protein (S)-, receptor-binding domain (RBD)-, and nucleoprotein (N)- specific IgM and IgG levels during SARS-CoV-2 infection and recovery. Our results indicate that the S-, RBD-, and N- specific IgG generation of severe/critical COVID-19 patients is one week later than mild/moderate cases, while the levels of these antibodies are 1.5-fold higher in severe/critical patients during hospitalization (P<0.01). The decrease of these IgG levels indicates the poor outcome of severe/critical patients. The RBD- and S-specific IgG levels are 2-fold higher in virus-free patients (P<0.05). Notably, we found that the patients who got re-infected had a low level of protective antibody on discharge. Therefore, our evidence proves that the dynamic changes of antibodies could provide an important reference for diagnosis, monitoring, and treatment, and shed new light on the precise management of COVID-19.
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Objective:To analyze consistency between histopathological and reflectance confocal microscopy (RCM) characteristics of early-stage mycosis fungoides (MF) , and to evaluate the value of RCM in assisting the pathological diagnosis of early-stage MF and the feasibility of dynamic monitoring of treatment response with RCM.Methods:From January 2014 to January 2018, 40 cases of clinically suspected MF were collected from Department of Dermatology, Third People′s Hospital of Hangzhou, including 26 males and 14 females, and their age was 47.0 ± 17.6 years. According to the summarized RCM characteristics of early-stage MF, biopsy sites were preliminarily located, and then a histopathological examination was performed. The RCM and pathological features of MF were compared. In addition, a combination therapy with narrowband ultraviolet B and interferon was performed in patients with confirmed MF. Targeted lesions were followed up with RCM for 9 months, and then therapeutic efficacy was evaluated.Results:Among the 40 cases of clinically suspected MF, 8 were preliminarily diagnosed as typical MF, 18 as suspected MF, and 14 were excluded according to the RCM characteristics; according to the pathological features, 12 could be diagnosed as typical MF, 14 as suspected MF, and 14 were excluded. Consistency analysis showed that the kappa coefficient between RCM classification and pathological diagnosis was 0.848 ( P < 0.01) . The consistency of epidermal infiltration of mildly refractive cells was the highest between RCM and pathological findings (kappa coefficient = 1, P = 0.005) , followed by dermal fibrosis at the erythema stage (kappa coefficient = 0.714, P = 0.035) . The RCM characteristics of MF gradually returned to normal during treatment, but atypical lymphocytes still existed when clinical lesions completely regressed. Conclusion:RCM can be used for pathological localization of suspected MF lesions in the early stage, and for dynamic monitoring of therapeutic efficacy in MF.
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Objective To determine the expression of ubiquitin-conjugating enzyme E2S (UBE2S) in malignant melanoma (MM),and to evaluate its effect on the biological behavior of melanoma cells.Methods Immunohistochemical study was performed to determine the UBE2S expression in 128 primary MM tissue chips,64 metastatic MM tissue chips,16 non-tumor tissue chips (8 paralesional normal skin tissues and 8 normal epidermal tissues).Real-time quantitative RCR was conducted to determine the UBE2S mRNA expression in the melanoma cell lines A375,MUM-2B and MUM-2C.The melanoma cell lines A375 and MUM-2B were divided into 2 groups separately:interference group transfected with a lentiviral vector carrying UBE2S RNA interference sequence,and control group transfected with a lentiviral vector carrying control sequence.After 72 hours,real-time quantitative RCR was performed to determine the UBE2S mRNA expression in the melanoma cell lines A375 and MUM-2B.Caspase-3/7 activity in the groups was assessed by using kits,and cell apoptosis and cell cycle distribution were detected by flow cytometry.The effect of UBE2S knockdown on the migratory and invasive abilities of and N-cadherin expression in A375 cells were evaluated by Transwell assay and Western blot analysis respectively.Statistical analysis was carried out with SPSS 22.0 software by using independent sample t-test for the comparison of normally distributed data between two groups,chi-square test for enumeration data,MannWhitney U test for the comparison of non-normally distributed data,and Spearman's coefficient for assessment of the correlation of UBE2S expression with T staging of melanoma.Results UBE2S was highly expressed in 98 (51.0％) MM tissues,but lowly expressed in 16 non-tumor tissues,and the UBE2Sexpression rate significantly differed between the above two kinds of tissues (x2 =11.905,P ＜ 0.01).UBE2S expression was negatively correlated with T staging of melanoma (ρ =-0.210,P =0.043).The relative mRNA expression of UBE2S significantly differed among the A375,MUM-2B,and MUM-2C cells (F =817.228,P ＜ 0.01).After UBE2S knockdown,the caspase-3/7 activity was significantly up-regulated in the A375 interference group (t =17.572,P ＜ 0.01) and MUM-2B interference group (t =24.552,P ＜0.01) compared with the A375 and MUM-2B control groups respectively.Compared with the control group,the A375 interference group showed significantly increased proportion of A375 cells at G1 phase (t =7.365,P ＜ 0.01),decreased proportion at S phase (t =-9.190,P ＜ 0.01),and no change in the proportion of A375 cells at G2/M phase (t =-0.227,P ＞ 0.05).The MUM-2B interference group showed significantly increased proportions of MUM-2B cells at G1 (t =12.676,P ＜ 0.01) and G2/M phases (t =13.045,P ＜0.01),but significantly decreased proportion at S phase (t =-15.718,P ＜ 0.01) compared with the control group.Transwell assay revealed decreased migratory and invasive abilities of A375 cells in the interference group compared with the control group (t =-35.727,-125.000,P ＜ 0.05,＜ 0.01,respectively).Western blot analysis showed down-regulated expression of N-cadherin protein in A375 cells in the interference group compared with the control group.Conclusions UBE2S is over-expressed in melanoma tissues,whose expression is associated with the T staging of melanoma.Knockdown of UBE2S affects the apoptosis,cell cycle,migration and invasion of melanoma cells,and may promote the metastasis of MM cells by regulating N-cadherin expression.