Objective:To characterize the molecular evolution of coxsackievirus A10 (CVA10) strains in Anhui Province.Methods:The nucleic acids of CVA10 isolates in Anhui Province were extracted for whole-genome PCR amplification. One-generation sequencing was performed and the complete whole-genome sequences of 10 isolates were obtained. Nucleotide and amino acid sequence similarity analysis of CVA10 isolates and reference strains was performed using MegAlign in DNAStar software. MEGA 11.0 was used to classify the genotypes of CVA10 isolates and representative strains based on phylogenetic analysis of the VP1 region, and the average evolutionary differences between genotypes were calculated. BioEdit 7.2 was used to calculate the entropy of amino acid substitution in the P1-P3 region of the isolates and analyze the amino acid non-synonymous substitution sites. Recombination signals associated with the Anhui isolates were detected using RDP4 and further verified using Simplot 3.5. DnaSp6 software was selected to analyze the selection pressure of CVA10 isolates and prototype strains.Results:Based on the VP1 region, CVA10 isolates and CVA10 representative strains were categorized into A-F genotypes, and most of the CVA10 prevalent in mainland China belonged to the F genotype, with some isolates in Anhui Province being more closely related to the Yunnan isolates. The average rate of evolutionary difference in nucleotides among genotypes ranged from 18.70% to 33.70%. The isolates shared 17 non-synonymous amino acid mutation sites in the VP1 region, and amino acid substitutions in the VP1, 3A and 3C regions might affect the pathogenicity of the strains. The isolates frequently recombined with a variety of other EVA strains in the 5′UTR, 2C, 3C, 3D, and 3′UTR regions. Selection pressure analysis of the isolates showed that the isolate genes were affected by negative selection pressure.Conclusions:Genetic evolutionary analysis of CVA10 suggests that mutations and recombination with other types of EVA strains are prevalent, affecting the molecular epidemiological trend of CVA10, and that molecular surveillance of CVA10 strains in Anhui Province should continue to be strengthened.

Objective To investigate the therapeutic effect of dental pulp stem cells(DPSCs)on autoimmune hepatitis in in vivo and in vitro experiments and the related mechanism.Methods An in vitro co-culture system was used to evaluate the immunoregulatory effect of DPSCs,and 32 mice were randomly divided into healthy control group,model group,positive drug group,and DPSCs treatment group,with 8 mice in each group.The serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and inflammatory factors were measured,and HE staining was used to assess liver pathological injury.An analysis of variance was used for comparison of normally distributed continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results The in vitro experiment showed that the positive rates of CD105,CD73,and CD90 in DPSCs were 99.97%,100%,and 99.53%,respectively,while the positive rates of CD34,HLA-DR,and CD45 were 0.56%,0.17%,and 0,respectively.DPSCs significantly inhibited the proliferation of Th1 and Th17 subsets,with inhibition rates of 31.32%and 45.76%,respectively;DPSCs promoted the proliferation of Treg(CD4+CD25+FoxP3+),with a promoting rate of 52.29%.DPSCs had an inhibition rate of 93.70%on the proliferation of lymphocytes.In the mouse model of autoimmune hepatitis,compared with the model group,the DPSCs treatment group had significant reductions in the serum levels of ALT and AST,with reduction rates of 66.8%and 60.0%,respectively(t=3.321 and 2.907,P=0.007 5 and 0.017 5)and significant reductions in the inflammatory factors tumor necrosis factor-α and interleukin-1β,with reduction rates of 57.5%and 71.3%,respectively(t=2.484 and 2.796,P=0.039 8 and 0.020 6),and histopathological examination showed no significant improvement in periportal bridging necrosis(t=1.969,P=0.098).Conclusion DPSCs effectively alleviate immune-mediated liver injury through immunoregulation,which provides an experimental basis for clinical translation.

Objective To investigate the therapeutic effect of dental pulp stem cells(DPSCs)on autoimmune hepatitis in in vivo and in vitro experiments and the related mechanism.Methods An in vitro co-culture system was used to evaluate the immunoregulatory effect of DPSCs,and 32 mice were randomly divided into healthy control group,model group,positive drug group,and DPSCs treatment group,with 8 mice in each group.The serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and inflammatory factors were measured,and HE staining was used to assess liver pathological injury.An analysis of variance was used for comparison of normally distributed continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results The in vitro experiment showed that the positive rates of CD105,CD73,and CD90 in DPSCs were 99.97%,100%,and 99.53%,respectively,while the positive rates of CD34,HLA-DR,and CD45 were 0.56%,0.17%,and 0,respectively.DPSCs significantly inhibited the proliferation of Th1 and Th17 subsets,with inhibition rates of 31.32%and 45.76%,respectively;DPSCs promoted the proliferation of Treg(CD4+CD25+FoxP3+),with a promoting rate of 52.29%.DPSCs had an inhibition rate of 93.70%on the proliferation of lymphocytes.In the mouse model of autoimmune hepatitis,compared with the model group,the DPSCs treatment group had significant reductions in the serum levels of ALT and AST,with reduction rates of 66.8%and 60.0%,respectively(t=3.321 and 2.907,P=0.007 5 and 0.017 5)and significant reductions in the inflammatory factors tumor necrosis factor-α and interleukin-1β,with reduction rates of 57.5%and 71.3%,respectively(t=2.484 and 2.796,P=0.039 8 and 0.020 6),and histopathological examination showed no significant improvement in periportal bridging necrosis(t=1.969,P=0.098).Conclusion DPSCs effectively alleviate immune-mediated liver injury through immunoregulation,which provides an experimental basis for clinical translation.

Objective:To characterize the molecular evolution of coxsackievirus A10 (CVA10) strains in Anhui Province.Methods:The nucleic acids of CVA10 isolates in Anhui Province were extracted for whole-genome PCR amplification. One-generation sequencing was performed and the complete whole-genome sequences of 10 isolates were obtained. Nucleotide and amino acid sequence similarity analysis of CVA10 isolates and reference strains was performed using MegAlign in DNAStar software. MEGA 11.0 was used to classify the genotypes of CVA10 isolates and representative strains based on phylogenetic analysis of the VP1 region, and the average evolutionary differences between genotypes were calculated. BioEdit 7.2 was used to calculate the entropy of amino acid substitution in the P1-P3 region of the isolates and analyze the amino acid non-synonymous substitution sites. Recombination signals associated with the Anhui isolates were detected using RDP4 and further verified using Simplot 3.5. DnaSp6 software was selected to analyze the selection pressure of CVA10 isolates and prototype strains.Results:Based on the VP1 region, CVA10 isolates and CVA10 representative strains were categorized into A-F genotypes, and most of the CVA10 prevalent in mainland China belonged to the F genotype, with some isolates in Anhui Province being more closely related to the Yunnan isolates. The average rate of evolutionary difference in nucleotides among genotypes ranged from 18.70% to 33.70%. The isolates shared 17 non-synonymous amino acid mutation sites in the VP1 region, and amino acid substitutions in the VP1, 3A and 3C regions might affect the pathogenicity of the strains. The isolates frequently recombined with a variety of other EVA strains in the 5′UTR, 2C, 3C, 3D, and 3′UTR regions. Selection pressure analysis of the isolates showed that the isolate genes were affected by negative selection pressure.Conclusions:Genetic evolutionary analysis of CVA10 suggests that mutations and recombination with other types of EVA strains are prevalent, affecting the molecular epidemiological trend of CVA10, and that molecular surveillance of CVA10 strains in Anhui Province should continue to be strengthened.

Due to limited options and modalities for the etiological treatment of autoimmune liver diseases, it is urgent to seek new therapeutic methods for liver autoimmune diseases. As the most common source of cells for stem cell therapy, mesenchymal stem cells (MSCs) play an important role in regulating innate and adaptive immune responses and have been widely used in clinical trials for the treatment of autoimmune diseases and inflammatory diseases. Recent experimental and clinical studies have shown that MSCs and MSC-EVs can inhibit the activation and proliferation of a variety of liver proinflammatory cells (such as Th1, Th17, and M1 macrophages), regulate the differentiation of different subsets of T and B cells, reduce the secretion of proinflammatory cytokines, and promote the proliferation of anti-inflammatory cells, thereby playing an immunoregulatory role. This article reviews the clinical trials of MSCs and MSC-EVs in the treatment of autoimmune liver diseases and their mechanism in regulating immune function and promoting hepatocyte regeneration and briefly describes the potential application and limitations of MSCs and MSC-EVs in clinical practice.

Objective : To analyze the epidemiological characteristics and pathogen spectrum of hand，foot mouth disease (HFMD) in Anhui province from 2015 to 2022，and to provide scientific evidence for prevention and control measures of HFMD． Methods : The surveillance data of hand，foot and mouth disease in Anhui province from 2015 to 2022 were analyzed by descriptive epidemiology. Real-time PCR was used to detect and classify HFMD samples. Results : A total of 650 590 HFMD cases were reported in Anhui province from 2015 to 2022，including 1 406 se- vere cases and 17 deaths．The annual reported incidence was 131. 45 /100 000．The epidemic features of“low incidence in odd years and high incidence in even years”were presented from 2015 to 2019．The incidence showed a continuous decline from 2020 to 2022．The monthly distribution showed the characteristics of bimodal epidemic，and the main peak was not obvious in 2020．Hefei，Fuyang，Bozhou，Chuzhou and Suzhou ranked the top five cities in terms of cumulative incidence．The age of onset was mainly distributed in children aged 5 years and below，accounting for 89. 26% of the total cases．The male to female ratio was 1. 48 ∶ 1．A total of 28 657 laboratory-confirmed cases had been reported from 2015 to 2022．EV71 cases accounted for 10. 57% ，Cox A16 cases accounted for 24. 90% ，and other enterovirus cases accounted for 64. 53%．The dominant pathogens showed dynamic changes in different years．Since 2018，the proportion of EV71 decreased significantly，and the proportion of other enteroviruses gradually increased to become the dominant pathogens．Among other enteroviruses，Cox A6 strain was dominant (80. 48% ) ． Conclusion This study suggests that the prevention and control of HFMD in Anhui province should be paid more attention from April to July and from October to December．The focus areas are the cities in northern Anhui and Hefei where the floating population is large．The focus of prevention and control is on children aged 5 years and below．Other enteroviruses have become the dominant pathogens of hand-foot-mouth disease in Anhui province，Cox A6 strain is dominant.

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 study the correlation between the level of T-bet expression and liver damage in peripheral plasma cells of patients with autoimmune hepatitis (AIH) in order to provide reference for the study of pathogenesis and development of diseases. Methods: The peripheral venous blood and clinical examination data of 29 cases with AIH and 6 healthy volunteers were collected. The percentage of subpopulations of peripheral blood B cells and the proportion of T-bet⁺ cells in each subgroup were detected by flow cytometry. Plasma cells (CD19⁺CD10^-CD27^hiCD38^hi), primary B cells (CD19⁺CD10^-CD27^-IgD⁺), transitional B cells (CD19⁺CD10⁺), and memory B cells (CD19⁺CD10^-CD27⁺IgD^-) were the included subsets of B cells. Serum immunoglobulin G (IgG) and alanine aminotransferase (ALT) levels, the proportion of B cells in peripheral blood subsets and IgG level, the proportion of T-bet⁺ cells in each subset and the proportion of T-bet⁺ plasma cells in each subset in B cells, the proportion of T-bet⁺ plasma cells and the level of serum ALT were analyzed for correlation analysis. Statistical analysis was performed using two independent sample t-tests and linear regression. Results: The serum IgG level of AIH patients with abnormal ALT (19.47 ± 1.039)g/L was significantly higher than that of normal ALT patients (15.5 ± 1.069)g/L, and the difference was statistically significant (t = 2.65, P < 0.05). The percentage of peripheral plasma cells in B cells of AIH patients (2.80 ± 0.14) % was higher than that of healthy volunteers (0.73 ± 0.09) %, and the difference was statistically significant (P < 0.01). The percentage of T-bet⁺ cells in peripheral plasma cells of AIH patients (23.54 ± 1.61) % was higher than that of healthy volunteers (6.59±0.59) % , and the difference was statistically significant (P < 0.01). The correlation analysis showed that the proportion of T-bet⁺ cells in peripheral plasma cells of AIH patients was positively correlated with the proportion of plasma cells to B cells (r = 0.224 7, P < 0.01), and the percentage of peripheral plasma cells to B cells was positively correlated with the level of serum IgG (r = 0.299 1, P < 0.01). Serum IgG level was correlated with the level of ALT, reflecting an indicator of liver damage (t = 2.65, P < 0.05). Conclusion The increase of T-bet expression in the peripheral plasma cells of AIH patients is associated with liver damage, which is a new mechanism of AIH pathogenesis and disease progression.