Although DNA vaccination is now a promising strategy against hepatitis B virus (HBV) infection, this approach has relatively modest antiviral effect, indicating that immunosuppressive mechanisms may occur in the long-term established infection. In this study, we studied the immunogenicity and anti-HBV efficiency of a combination of HBV surface (HBsAg) and core (HBcAg) DNA vaccine, enhanced by heat shock protein (HSP) gp96 or HSP70 and mediated by in vivo electroporation. Immunization with gp96 adjuvanted HBsAg/HBcAg DNA formulation induced potent T cell and antibody immunity against HBsAg and HBcAg. Notably, treatment with gp96 or HSP70 as adjuvant resulted in reduction of Treg populations by around 20%. Moreover, compared with nonimmunized control mice, immunization with gp96 or HSP70 adjuvanted DNA vaccine dramatically decreased serum HBsAg and viral DNA levels, and HBcAg expression in liver. These results may therefore provide an effective strategy for designing gp96-based DNA vaccine for immunotherapy of chronic HBV infection.
Adjuvants, Immunologic ; Animals ; Electroporation ; HSP70 Heat-Shock Proteins ; immunology ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Hepatitis B, Chronic ; prevention & control ; Immunization ; Membrane Glycoproteins ; immunology ; Mice ; Mice, Transgenic ; Vaccines, DNA ; immunology

Secretory anti-gp96 scFv fragment was expressed in Pichia pastoris to obtain a small molecule antibody that specifically recognizes heat shock protein gp96. The gp96-scFv fragment gene was synthesized and cloned to Pichia pastoris expression plasmid pPICZa-A. Pichia pastoris X33 was electroporated with the linearized recombinant expression vector, and expression of gp96-scFv fragment was induced by methanol. The His-tagged recombinant protein was then purified by affinity chromatography and analyzed with SDS-PAGE and Western blotting assays. The biological activities of recombinant gp96-scFv fragment were determined by Western blotting, Immunofluorescence, ELISA and FACS assays. The gp96-scFv fragment was expressed successfully in Pichia pastoris. About 50 mg of recombinant protein could be purified from 1 liter of the Pichia pastoris culture supernatant. Its molecular weight was about 15 kDa. The gp96-scFv fragment could specifically bind to gp96 protein by Western blotting, immunofluorescence, ELISA and FACS analyses. Pichia pastoris-expressed gp96-scFv fragment specifically recognizes gp96 protein, which could be used for Western blotting, Immunofluorescence, ELISA and FACS analyses.
Blotting, Western ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Membrane Glycoproteins ; immunology ; Pichia ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; Single-Chain Antibodies ; biosynthesis

Objective:To summarize the best evidence for the management of lower urinary tract dysfunction (LUTD) in total hysterectomy patients, so as to provide evidence-based basis for clinical practice.Methods:According to the "6S" pyramid model, literature related to the management of LUTD in total hysterectomy patients was successively searched from guide websites, evidence-based websites, professional websites and comprehensive databases. The search deadline was from the establishment of the databases to March 31, 2023. Two researchers evaluated the quality of the included literature, extracted evidence and recommended the level of evidence.Results:A total of 14 articles were included, including one clinical decision, two evidence summaries, three guidelines, one expert consensus and seven systematic evaluations. A total of 25 pieces of evidence were summarized from four aspects, such as symptom assessment, urinary tract management, symptom intervention and health education.Conclusions:Medical staff should manage lower urinary tract dysfunction in patients undergoing total hysterectomy based on evidence-based evidence to prevent or reduce the occurrence of lower urinary tract dysfunction in patients.

Objective:To establish a mice model of inflammatory bowel disease (IBD) induced by dextran sulfate sodium (DSS), and to analyze the changes in intestinal inflammation and macrophage subsets at different stages, so as to find a new target for the treatment of IBD.Methods:Thirty male C57BL/6 mice of 6-8 weeks were randomly divided into control group, activation stage group and resolution stage group.The latter 2 groups were given 25 g/L DSS for 5 consecutive days to establish the IBD model.After 5 days, the mice were given filtered and sterilized water and sacrificed on the 10 th and 15 th day, respectively.Colon inflammation in mice was evaluated, including body weight, disease activity index (DAI) score, changes in colon length, histopathology and histopathological score.Then the expression levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β in colon tissues were detected by quantitative real-time PCR(qPCR). Finally, the changes of intestinal macrophage subsets were detected by flow cytometry. Results:The colon inflammation of mice in the activation stage group was significantly more severe than that in the control group, while the colon inflammation of mice in the resolution stage group was reduced.The colon length of mice in the activation stage group was (5.94±0.40) cm, which was significantly shorter than that in the control group [(7.25±0.29) cm], and the situation was slightly improved in the resolution stage with the colon length of [(6.87±0.95) cm], and the differences were statistically significant (all P<0.05). The mRNA expression levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in the activation stage were 53.40±6.58, 117.69±30.78 and 2.52±0.25, respectively, which were significantly higher than those in the control group (1.00±0.13, 1.00±0.39, 1.00±0.10); the mRNA expression levels of IL-1β, IL-6 and TNF-α in the resolution stage were 2.51±0.13, 5.43±0.51 and 1.73±0.14, respectively, which were significantly lower than those in the activation stages(all P<0.05). The expression level of anti-inflammatory cytokine TGF-β in the resolution stage was 2.41±0.17, which was significantly higher than that in the activation stage (0.94±0.12), and the diffe-rence was statistically significant ( P<0.05). During the progression of IBD, there were 3 groups of macrophages in the lamina propria of intestinal mucosa of mice, of which the number of F4/80 lowCD 64-MHCⅡ - macrophage subset with the lowest maturity increased significantly in the activation stage of IBD, accounting for (10.68±4.62)%, and it decreased and returned to the normal level in the resolution stage, accounting for (4.63±1.06)%, and the difference was statistically significant ( P<0.05). Conclusions:Macrophages play an important role in the progression of IBD, the hindrance of maturation and development may be the main cause of inflammatory injury in the activation stage of IBD, and the transformation of macrophage subsets may become a new target for the treatment of IBD.

In the original publication Fig. 1D and supplementary material is incorrect. The correct figure and supplementary material is provided in this correction.

Recently we have established a new culture condition enabling the derivation of extended pluripotent stem (EPS) cells, which, compared to conventional pluripotent stem cells, possess superior developmental potential and germline competence. However, it remains unclear whether this condition permits derivation of EPS cells from mouse strains that are refractory or non-permissive to pluripotent cell establishment. Here, we show that EPS cells can be robustly generated from non-permissive NOD-scid Il2rg mice through de novo derivation from blastocysts. Furthermore, these cells can also be efficiently generated by chemical reprogramming from embryonic NOD-scid Il2rg fibroblasts. NOD-scid Il2rg EPS cells can be expanded for more than 20 passages with genomic stability and can be genetically modified through gene targeting. Notably, these cells contribute to both embryonic and extraembryonic lineages in vivo. More importantly, they can produce chimeras and integrate into the E13.5 genital ridge. Our study demonstrates the feasibility of generating EPS cells from refractory mouse strains, which could potentially be a general strategy for deriving mouse pluripotent cells. The generation of NOD-scid Il2rg EPS cell lines permits sophisticated genetic modification in NOD-scid Il2rg mice, which may greatly advance the optimization of humanized mouse models for biomedical applications.

One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem (ES) cells, which is used to produce gene-targeted mice for wide applications in biomedicine. However, a major bottleneck in this approach is that the robustness of germline transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing, which impairs the efficiency and robustness of mouse model generation. Recently, we have established a new type of pluripotent cells termed extended pluripotent stem (EPS) cells, which have superior developmental potency and robust germline competence compared to conventional mouse ES cells. In this study, we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage. Based on gene targeting in mouse EPS cells, we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation, which could be accomplished in approximately 2 months. Importantly, using this approach, we successfully constructed mouse models in which the human interleukin 3 (IL3) or interleukin 6 (IL6) gene was knocked into its corresponding locus in the mouse genome. Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting, which have great application potential in biomedical research.