ObjectiveTo construct an Enterococcus faecium （Ef）-based recombinant Ef-PA0057 vaccine of Pseudomonas aeruginosa（Pa）and to study its protective immune mechanism in mice. MethodsThe PA0057 gene was cloned from the genomic DNA of PA01 strain ATCC9027 by PCR and inserted into pGEX-1λT to construct pGEX-PA0057. The recombinant plasmid was electroporated into TX0016 strain to construct rEf-PA0057 vaccine. The plasmid was extracted from rEf for PCR. The rEf vaccine was expressed through IPTG induction， and the expression of protein was analyzed by SDS-PAGE and Western blot. BALB/c mice were immunized intragastrically with 5×10⁸ CFU rEf-PA0057 vaccine 3 times per week for 3 weeks. 4 weeks after the first immunization， mice were challenged intranasally with 5×10⁷ CFU of PA01 strain. 2 weeks after challenge， mice were sacrificed， and their lungs were separated. Bacteria in lungs were incubated and colonies were counted. Sera were collected at 0， 4， and 6 weeks after the first immunization. The IgG and its subclasses， and IgE were detected by ELISA. ResultsThe 900 bp PA0057 gene was successfully cloned by PCR. PCR showed that PA0057 gene was amplified when the extracted plasmid from rEf as template； the relative molecular mass （Mr） of the expressed PA0057-GST fusion protein was approximately 58 ku， detected by SDS-PAGE. The amount of the expressed protein was 18% of the total bacterial proteins. Western blot showed that the target protein could be recognized by Pa sera. The colony numbers of lung tissue in rEf-PA0057 vaccine group， blank vector group and Ef control group were （0.297±0.011）×10⁸ CFU， （7.576±0.206）×10⁸ CFU and （7.551±0.185）×10⁸ CFU， respectively， the difference was statistically significant （P0.01）. The levels of IgG， IgG1， IgG2b， IgG3 and IgE increased. At the same time point， there was a significant difference compared with the two control groups （P0.01）. ConclusionThe rEf-PA0057 vaccine is successfully constructed. It may induce mice to produce humoral response against challenge with PA01.

IgG4-related sclerosing cholangitis (IgG4-SC) is an IgG4-related disease charac-terized by bile duct wall thickening and luminal stricture. Its pathogenesis involves complex multi-dimensional regulation, including immune imbalance, genetic epigenetic regulation, microbial ecolo-gical abnormalities, and activation of fibrotic pathways. Due to the lack of specificity in clinical mani-festations, IgG4-SC is easily misdiagnosed as biliary tract diseases such as cholangiocarcinoma and primary sclerosing cholangitis in the early stage, leading to diagnostic difficulties. At present, gluco-corticoids are the preferred treatment option. Although most patients can quickly recover in the early stages, high recurrence rates and hormone dependence limit the long-term effectiveness of treatment. The authors combine the latest research progress at home and abroad to deeply analyze the pathogenesis, clinical manifestations, and diagnosis of IgG4-SC, focusing on treatment strategies and cutting-edge advances, in order to provide reference and inspiration for clinical practice.

IgG4-related sclerosing cholangitis (IgG4-SC) is an IgG4-related disease charac-terized by bile duct wall thickening and luminal stricture. Its pathogenesis involves complex multi-dimensional regulation, including immune imbalance, genetic epigenetic regulation, microbial ecolo-gical abnormalities, and activation of fibrotic pathways. Due to the lack of specificity in clinical mani-festations, IgG4-SC is easily misdiagnosed as biliary tract diseases such as cholangiocarcinoma and primary sclerosing cholangitis in the early stage, leading to diagnostic difficulties. At present, gluco-corticoids are the preferred treatment option. Although most patients can quickly recover in the early stages, high recurrence rates and hormone dependence limit the long-term effectiveness of treatment. The authors combine the latest research progress at home and abroad to deeply analyze the pathogenesis, clinical manifestations, and diagnosis of IgG4-SC, focusing on treatment strategies and cutting-edge advances, in order to provide reference and inspiration for clinical practice.

BACKGROUND:It was found that the ligands and receptors of Notch are both cell membrane surface proteins,which are important proteins to mediate intercellular communication,and the Notch signaling pathway plays a crucial regulatory role in the proliferation and differentiation of mesenchymal stem cells. OBJECTIVE:To review the regulatory mechanism of the Notch signaling pathway on the proliferation and differentiation of mesenchymal stem cells,summarize and clarify the research advance in how the Notch signaling pathway regulates the proliferation and differentiation of mesenchymal stem cells,and provide theoretical support for the future use of stem cells to treat various related diseases. METHODS:By using the computer,the first author searched the relevant studies involving Notch signaling pathway regulation of mesenchymal stem cell proliferation and differentiation on CNKI,Wanfang,VIP,PubMed,Web of Science,and Nature databases with Chinese search terms"mesenchymal stem cells,Notch,Notch signaling pathway,proliferation,differentiation"and the English search terms"mesenchymal stem cells,MSC,Notch,Notch signaling pathway,proliferation,differentiation".Part of the literature was searched in combination with the literature tracing method.Finally,87 articles were included in the review analysis. RESULTS AND CONCLUSION:(1)Notch signaling pathway is a conserved signaling pathway in multicellular organisms,which plays an important role in regulating cell differentiation,proliferation,apoptosis,and the cell cycle by mediating communication between neighboring cells through receptor-ligand binding.(2)Mesenchymal stem cells are a class of adult stem cells with self-proliferative and multi-directional differentiation potential,which can be regulated by external signaling pathways to affect their proliferation and differentiation.Notch signaling pathway,as one of them,when Notch ligands are activated,the Notch proteins will undergo two protein hydrolysis cleavages to release Notch intracellular structural domain NICD,which then enters the nucleus and thus promotes the transcription of target genes to regulate the proliferation and differentiation of mesenchymal stem cells from different sources,such as bone marrow,adipose,and umbilical cord.However,the specific mechanisms that regulate the proliferation and differentiation of mesenchymal stem cells from different tissue sources of the same species are different.(3)The Notch signaling pathway can regulate the differentiation of mesenchymal stem cells into different target cells,but due to different target cells,the expression levels of receptors or ligands in the Notch signaling pathway vary.(4)Clinical targeting of the Notch signaling pathway to promote mesenchymal stem cells for the treatment of various refractory diseases,such as aplastic anemia,severe joint injuries,ischemic strokes,and myocardial infarctions,has a promising application.(5)By exploring the Notch signaling pathway via regulating the expression levels of its receptors and ligands in bone marrow mesenchymal stem cells from rat,mouse,and human,it can be found that the Notch signaling pathway expression levels in the proliferation and differentiation of mesenchymal stem cells from different species origins are also different.(6)The role of mesenchymal stem cells in tissue engineering has been gradually highlighted due to their advantages of safety,low immune rejection,and wide therapeutic prospects.The Notch signaling pathway regulates the proliferation and differentiation of mesenchymal stem cells with a wide range of influencing factors,and subsequent studies should further optimize the influencing factor variables and explore the standardized studies of regulating the proliferation and differentiation of mesenchymal stem cells.

Objective:To explore the short-term and long-term effects of pregnancy with aortic dissection (AD) on newborns.Methods:In this retrospective cohort study, 31 live births of pregnant women with AD admitted to Beijing Anzhen Hospital, Capital Medical University from June 2010 to June 2022 were selected as the AD group.Among them, 19 preterm births were in the preterm AD group.Additionally, 100 neonates delivered by pregnant women without pregnancy complications were selected as the normal control group, and 50 preterm infants delivered by pregnant women without pregnancy complications were selected as the preterm control group during the same period.The differences between the AD group and the normal control group, and between the preterm AD group and the preterm control group were compared using the independent samples t-test, Chi-square test, and Fisher′s exact test.Patients in the AD group were followed up for 5 years. Results:(1) The incidence rates of small for gestational age (SGA) (12.90% vs. 1.00%), neonatal respiratory distress syndrome (NRDS) (16.13% vs. 2.00%), digestive tract hemorrhage (16.13% vs. 1.00%), nervous system disease (16.13% vs. 1.00%), multiple organ dysfunction syndrome (MODS) (12.90% vs. 0), premature infant (61.29% vs. 5.00%), asphyxia (35.48% vs. 1.00%), cardiopulmonary resuscitation (64.52% vs. 1.00%), respiratory support (38.71% vs. 2.00%), transfer to the department of pediatrics within 24 hours of birth (90.32% vs. 13.00%), caesarean section (100% vs. 25.00%), and low birth weight infant (38.71% vs. 2.00%) in the AD group were significantly higher than those in the normal control group, while the gestational age[(34.90±2.80) weeks vs. (38.87±1.50) weeks], length [(45.65±3.56) cm vs. (50.17±0.81) cm], and weight [(2 456.77±634.02) g vs. (3 383.90±413.05) g] were significantly less than those in the normal control group, and the differences were statistically significant (all P<0.05).(2) The gestational age [(33.26±2.31) weeks vs. (34.80±1.29) weeks], length [(43.89±3.36) cm vs. (46.64±2.89) cm], and weight [(2 158.95±558.92) g vs. (2 418.20±431.20) g] in the preterm AD group were less than those in the preterm control group, while the incidence rates of NRDS (26.32% vs. 4.00%), digestive tract hemorrhage (21.05% vs. 4.00%), nervous system disease (26.32% vs. 6.00%), MODS (21.05% vs. 2.00%), caesarean section (100% vs. 52.00%), cardiopulmonary resuscitation (73.68% vs. 8.00%), respiratory support (63.16% vs. 10.00%), and asphyxia (47.37% vs. 2.00%) were significantly higher than those in the preterm control group, and the differences were statistically significant (all P<0.05).(3) Twenty-seven cases in the AD group survived, and 4 cases were lost to follow-up.Among them, 9 cases had abnormal physical development, 3 cases had eye disease, and 1 case was mentally retarded.None of them underwent genetic detection. Conclusions:Neonates born to mothers with AD in pregnancy have poor physical development, high incidence of complications, and reduced quality of life, and need long-term monitoring and follow-up in their growth and development.

Objective:To construct Lactococcus lactis (LL) based EmⅡ/3 vaccine of Echinococcus multilocularis and observe its expression efficiency. Methods:EmⅡ/3 gene was obtained through PCR amplification using the pCD-EmⅡ/3 as a template, the gene was cloned into pMG36e to construct pMG36e-EmⅡ/3 and transformed into Escherichia coli BL21 (DE3) competent cell, the recombinant plasmid was identified by double restriction endonuclease digestion, then was electroporated into LL MG1363 to construct recombinant (r) LL-EmⅡ/3 vaccine. After screening for roxithromycin resistance, the plasmid was extracted for PCR identification, and the expression was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Results:The pMG36e-EmⅡ/3 recombinant plasmid was identified by double restriction endonuclease digestion, and there was an approximately 3 600 bp carrier band and a 1 759 bp EmⅡ/3 gene band. The plasmid extracted from roxithromycin resistant rLL strain was used as a template, and 1 759 bp EmⅡ/3 gene was amplified by PCR; SDS-PAGE demonstrated that the rLL-EmⅡ/3 vaccine could express EmⅡ/3 protein with a relative molecular weight of 66 × 10 3, and the expressed protein accounted for about 20% of the total bacteria protein after 3 days of culture; Western blot showed that the expressed protein could be recognized by mouse serum infected with alveolar hydatid cyst. Conclusion:We have successfully constructed the rLL-EmⅡ/3 vaccine of Echinococcus multilocularis, which expresses Em Ⅱ/3 protein with specific antigenicity.

Objective:To construct a recombinant Efs-EmⅡ/3-Em14-3-3 vaccine against Echinococcus multilocularis (Em) using Enterococcus faecalis (Efs) as a vector, and investigate its antigenicity. Methods:The recombinant plasmid pGEX-EmⅡ/3-Em14-3-3 was transformed into Efs ATCC47077 strain using electroporation method, and the recombinant Efs-EmⅡ/3-Em14-3-3 vaccine was constructed. The plasmid was extracted for PCR amplification and identification. The recombinant Efs-EmⅡ/3-Em14-3-3 vaccine was expressed through isopropyl-β-D-thiogalactoside (IPTG) induction, and the recombinant protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and the proportion of expressed proteins in the total proteins of the bacteria was analyzed by thin layer scanning technology.Results:Using the plasmid extracted from recombinant Efs bacteria as a template, the EmⅡ/3-Em14-3-3 fusion gene with a size of about 2 554 bp could be amplified by PCR. The relative molecular mass ( Mr) of the expressed EmⅡ/3-Em14-3-3 fusion protein was approximately 119 × 10 3 by SDS-PAGE; after 5 h induction by IPTG, the expression level of target protein was high, accounting for about 9% of the total bacterial protein. Western blotting showed that the expressed protein could be recognized by mouse serum infected with alveolar hydatid cyst. Conclusion:The recombinant Efs-EmⅡ/3-Em14-3-3 vaccine is successfully constructed, and the expressed fusion protein shows specific antigenicity.

Objective:To construct a recombinant vaccine of Schistosoma japonicum (Sj) mediated by Enterococcus faecalis (Efs, rEfs-Sj26GST vaccine), and to study the expression of Sj26GST-GST fusion protein in the recombinant vaccine. Methods:The recombinant plasmid pGEX-Sj26GST was transformed into the susceptible strain Efs ATCC47077 by electroporation to construct rEfs-Sj26GST vaccine, and the plasmid was extracted for PCR identification. After induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), the products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results:After PCR identification, a 676 bp fragment was amplified, which was consistent with the length of Sj26GST amplification fragment. SDS-PAGE analysis showed that the relative molecular mass was 52 × 10 3, which was consistent with the band of Sj26GST-GST fusion protein. Western blot results showed that the Sj26GST-GST fusion protein expressed by rEfs-Sj26GST vaccine could be specifically recognized by the serum of Sj infected patients. Conclusion:The rEfs-Sj26GST vaccine is successfully constructed, and the Sj26GST-GST fusion protein expressed by recombinant vaccine can be specifically recognized by the serum of Sj infected patients.

Objective To construct Lactococcus lactis(LL)-based recombinant LL-Eg95(rLL-Eg95)vaccine for Echinococcus granulosus(Eg)and to examine its expression efficiency.Methods Eg95 gene was obtained by PCR from the template of pCD-Eg95.Then,pMG36e was inserted in the Eg95 gene after double cleaving with restriction endonucleases Xba Ⅰ and Hind Ⅲ to construct recombinant plasmid pMG36e-Eg95,which was transformed into E.coli BL2(DE3)competent cells.The recombinant plasmid was extracted and identified by double restriction endonuclease digestion and was then electroporated into LL MG1363 to construct rLL-Eg95 vaccine.Then,the plamid was extracted and identified by PCR.Results Examination of the recombinant plasmid by double restriction endonuclease digestion showed that the segment was of the expected length.PCR showed that 471 base pairs of Eg95 gene were amplified when the plasmid extracted from roxithromycin-resistant recombinant LL was used as the template.Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)showed that the relative molecular mass of the Eg95 protein expressed was approximately 16.5×103 and that the amount of the expressed protein was 17％of the total bacterial proteins.Western blot findings suggested that the expressed protein could be recognized by mice serum infected with hydatid cyst.Conclusion The rLL-Eg95 vaccine was successfully constructed,expressing Eg95 protein that has specific antigenicity.

Objective:To investigate clinical efficacy and safety of dupilumab in the treatment of atopic dermatitis (AD) .Methods:An ambispective study was conducted on 123 AD patients treated with dupilumab in Department of Dermatology, the Second Xiangya Hospital of Central South University from July 2020 to March 2022, clinical data were collected, and efficacy and safety of dupilumab were evaluated. Primary outcomes included scores of eczema area and severity index (EASI) , patient-oriented eczema measure (POEM) , peak pruritus numerical rating scale (NRS) and dermatology life quality index (DLQI) before and after 4-, 8-, 12- and 16-week treatment, and adverse reactions and events were recorded. Comparison of scores before and after treatment was performed using paired t test or repeated measures analysis of variance, Mann-Whitney U test was used for the comparison of efficacy among patients with different types of skin lesions or different IgE levels, and multiple regression model based on robust standard errors was used to analyze factors influencing the efficacy. Results:Among the 123 AD patients, 107 were enolled into the efficacy analysis, and 85 (79.44%) completed at least 4 weeks of treatment, including 6 (7.06%) achieving EASI75 and 23 (27.06%) achieving EASI50, and the EASI, NRS, POEM, DLQI scores (10.41 ± 6.72, 4.12 ± 1.74, 8.60 ± 4.29, 7.81 ± 4.38, respectively) significantly decreased compared with those before treatment (18.08 ± 10.69, 7.21 ± 2.01, 16.88 ± 5.74, 12.95 ± 5.95, respectively; all P < 0.001) in the 85 patients. Among the 107 patients, 47 (43.93%) completed at least 16 weeks of treatment. Among the 47 patients, 23 (82.14%) of 28 adults and 17 (89.47%) of 19 adolescents and children achieved 75% or greater improvement in EASI score; the EASI, NRS, POEM and DLQI scores before the treatment all significantly differred from those 4, 8, 12, 16 weeks after the treatment (all P < 0.001) , and all the scores were significantly lower at weeks 4, 8, 12 and 16 than at the previous adjacent time points (all P < 0.05) . At week 4 during the treatment, the EASI improvement rate was significantly lower in the AD patients with prurigo nodularis than in those without ( U = 151.00, P = 0.006) , while there was no significant difference in the EASI improvement rate between the AD patients with xeroderma and those without ( P > 0.05) ; at week 16 during the treatment, there was no significant difference in the EASI improvement rate between patients with prurigo nodularis or xeroderma and those without (both P > 0.05) . Multiple regression analysis based on robust standard errors at week 16 showed that the improvement degree in the EASI score was not correlated with the type of skin lesions ( β = 3.20, P = 0.075) , but correlated with age ( β = -0.22, P = 0.030) , whether patients were in adulthood ( β = 9.54, P = 0.049) , immediate family history ( β = 7.46, P = 0.017) ; the improvement degree in the NRS score was correlated with the type of skin lesions ( β = 0.55, P = 0.032) , age ( β = -0.04, P = 0.033) , weight ( β = -0.05, P = 0.020) , whether patients were in adulthood ( β = 2.06, P = 0.003) and whether patients received combined treatment with antihistamines ( β = -1.91, P = 0.001) . Adverse reactions: among the 123 patients, 6 (4.88%) developed conjunctivitis, and 2 (1.63%) developed facial erythema. Adverse events: vitiligo-like changes occurred on the right forehead of 1 patient, and 3 patients discontinued the treatment with dupilumab due to Henoch-Sch?nlein purpura, distal axonal damage in peripheral nerves in both upper limbs, and epilepsy, respectively. The causal relationship between these adverse events and dupilumab was unclear. Conclusion:Dupilumab is effective in the treatment of AD with high overall safety, and can serve as a new treatment option for AD patients with an unsatisfactory response to traditional treatment.