Human alphaherpesvirus 2 (HSV-2) is the main pathogen resulting human genital herpes, which poses a major threat to the socio-economic development, while there is no effective vaccine. In this study, we developed a novel lipopolyplex (LPP)-delivered mRNA vaccine expressing the HSV-2 envelope glycoprotein gD and evaluated its immunogenicity in mice. The mRNA vaccine was prepared from the genetically modified gD mRNA synthesized in vitro combined with the LPP delivery platform and it was named gD-ORI mRNA. The expression of gD antigen in the mRNA vaccine was validated in vitro by Western blotting and indirect immunofluorescence assay, then the immune responses induced by this mRNA vaccine in mice were evaluated. The immunization with gD mRNA alone induced strong humoral and cellular immune responses in mice. Robust and long-lasting gD-specific IgG antibodies were detected in the mouse serum after booster immunization with gD-ORI mRNA. The immunized mice exhibited a Th1/Th2 balanced IgG response and robust neutralizing antibodies against HSV-2, and a clear dose-response relationship was observed. The gD-specific IgG antibodies were maintained in mice for a long time, up to 18 weeks post-booster immunization. At the same time, multifunctional gD-specific CD4⁺ and CD8⁺ T cells in vaccinated mice were detected by intracellular cytokine staining (ICS). This novel gD-expressing mRNA vaccine delivered by LPP induces strong and long-lasting immune responses in mice post booster immunization and has a promising prospect for development and application. This study provides scientific evidence and reference for the development of a new mRNA vaccine for HSV-2.
Animals ; Herpesvirus 2, Human/genetics* ; Viral Envelope Proteins/genetics* ; Mice ; Herpes Genitalis/immunology* ; RNA, Messenger/immunology* ; Female ; Mice, Inbred BALB C ; Antibodies, Viral/blood* ; mRNA Vaccines/immunology* ; Antibodies, Neutralizing/blood* ; Humans

Objective:To accurately ascertain the whole genome sequencing and the composition of open reading frames (ORFs) of non-replicating Tiantan strain of vaccinia virus (NTV) using next-generation long-read sequencing technology.Methods:NTV, obtained from our laboratory stock, was amplified and purified on chicken embryo fibroblast cells(CEFs), and the full-length genomic nucleic acid of NTV was extracted. The PacBio HiFi sequencing platform was utilized for de novo assembly to obtain the complete genomic sequence of NTV. Using a homology annotation strategy, we identified its ORF composition and compared it with known non-replicating vaccinia virus strains. Results:The total length of NTV′s genome was 171 729 bp, with a GC content of 33%. Its unique inverted terminal repeat (ITR) region comprised hairpin structures, two tandem repeat regions, and three non-repeat regions. NTV contained 166 ORFs, with major differences observed in the ITR and its surrounding regions when compared to MVA-BN and NYVAC. These three strains shared a common set of 138 ORFs. NTV encoded six unique ORFs related to virus evasion of host antiviral response.Conclusions:This study accurately determines the whole genome sequencing and ORFs composition of NTV, and reveals its similarities and differences with other replication-deficient vaccinia virus strains, which pave a way for the development and application of the next generation of monkeypox vaccines and novel viral vectors.

Objective:To construct a novel respiratory syncytial virus (RSV) vaccine based on a recombinant influenza virus vector and evaluate its immune protective effects in mice.Methods:A recombinant H1N1 influenza A virus (IAV) expressing the extracellular domain (Gecto) of RSV A2 G protein was constructed and rescued, named as PR8NAGecto/WSN. After in vitro verification of the Gecto expression and PR8NAGecto/WSN growth kinetics, a single dose of PR8NAGecto/WSN was used to immunize BALB/c mice through intranasal administration to evaluate the efficacy of PR8NAGecto/WSN by assessing humoral (IgG, neutralizing antibody), mucosal (IgA) and cellular immunity (IFN-γ ELISPOT). Four weeks after immunization, the mice were challenged with RSV A2 or RSV B9320 to evaluate the protective effects of PR8NAGecto/WSN by analyzing mouse body weight changes, lung tissue virus titers and pathological changes. Results:A single-dose intranasal immunization with PR8NAGecto/WSN induced robust humoral, mucosal and cellular immunity in mice. Moreover, the mice in the immunized group had lower lung virus loads and mild lung pathological damages following the challenge with RSV A or RSV B subtype as compared with the control group.Conclusions:A single-dose intranasal immunization with PR8NAGecto/WSN induces robust immunity and provide protection against RSV A and B challenges in mice. This study provides new ideas and reference for the development of novel mucosal vaccines against RSV.

Objective:To compare the trueness of incisal guidance of implant-supported single crowns designed by patient-specific motion(PSM)with that designed by average-value virtual articulator(AVA).Methods:The study had recruited 12 participants with complete dentition and stable incisal guidance.An intraoral scanner was used to scan digital casts and record two types of patient-specific mo-tion(data only including protrusive movement,and data including protrusive movement and lateral pro-trusive movement).The lingual surfaces of the maxillary incisors which guided the protrusive movement was selected and elevated to create a reference cast.A maxillary central incisor of original casts was vir-tually extracted and implanted to generate a working cast.The Dental system software program was used to design implant-supported single crowns with the anatomical coping design method.The incisal guidance was designed by different methods.The incisal guidance in control group was designed by the average-value virtual articulator.The incisal guidance in experiment groups was designed by the patient-specific motion only including protrusive movement(PSM1)and with the patient-specific motion including protru-sive movement and lateral protrusive movement(PSM2).The incisal guidance of prosthesis designed by these 3 methods were compared with the original incisal guidance in Geomagic Control 2015(3DSystem,America).The measurements included:Average of positive values,ratio of positive area and maximum value reflecting supra-occlusion;average of negative values,ratio of negative area and minimum value re-flecting over-correction;and root mean square reflecting overall deviation.Results:Statistical data were collected using the median(interquartile range)method.The average of positive values,ratio of positive area and average of negative values of the PSM2 group were smaller than those of the control group[8.0(18.8)μm vs.37.5(47.5)μm;0vs.7.2％(38.1％);-109.0(63.8)μm vs.-66.5(64.5)μm],and the ratio of negative area of PSM2 group was larger than those of the control group[52.9％(47.8％)vs.17.3％(45.3％)],with significant differences(P all＜0.05).The ratio of positive area[0.1％(7.0％)]and average of negative values[-97.0(61.5)μm]of PSM1 group,were smaller than those of the control group,and the ratio of negative area[40.7％(39.2％)]of the PSM1 group was larger than that of the control group,with significant differences(P＜0.05).The average of positive values[20.0(42.0)μm]and ratio of positive area of PSM1 group was larger than that of the PSM2 group with significant differences(P＜0.05).Conclusion:To establish the incisor guidance of implant-supported single crowns,compared with the average-value virtual articulator and the patient-specific motion only including protrusive movement,the patient-specific motion including protrusive movement and lateral protrusive movement is more conducive to reducing the protrusive interference of prosthesis and improving the occlusal fit.

Objective·To investigate the safety and feasibility of early goal-directed mobilization in the recovery of gastrointestinal function after pancreaticoduodenectomy.Methods·The non-contemporaneous controlled studies were conducted.Subjects who underwent pancreaticoduodenectomy were included.From Sep 2022 to May 2023,forty patients were selected as the control group,and forty patients were selected from June 2023 to February 2024 as the experimental group.The general clinical data of the two groups were collected.The control group was treated with the nursing routine after pancreaticoduodenectomy,and there were no specific requirements for the time and goal of early activity.The experimental group had daily activity goals established for early mobilization,which were performed by the patients and their families,while the rest of their care was identical to that of the control group.The main index of effectiveness evaluation was the time of first flatus and first defecation,and the secondary indexes included the time of first getting out of bed,the time of oral drinking,the time of the gastric tube removal,and the postoperative levels of K+,Na+,and Cl-on the 3rd day of the postoperative period.Safety evaluations included chyle leak,postoperative pancreatic fistula,biliary leak and delayed gastric emptying,postoperative hemorrhage,unplanned reoperation,unplanned extubation,falls and death.Results·There was no statistically significant difference in the general clinical data of the patients in the 2 groups.After the implementation of early goal-directed mobilization,the time of first flatus was advanced from(3.95±1.68)d to(2.88±0.91)d(t=-3.560,P=0.001),and the time of first defecation was advanced from(4.90±1.61)d to(3.80±1.30)d(t=-3.352,P=0.001).The time of first getting out of bed was advanced from(5.18±1.77)d to(2.30±0.88)d(t=-9.205,P＜0.001),and the time of oral drinking was advanced from(4.10±1.89)d to(2.73±1.20)d(t=-3.883,P＜0.001).Significant differences were also observed in postoperative day 3 Na+(t=-2.745,P=0.008)and Cl-(t=-2.033,P=0.045)levels.Conclusion·Early goal-directed activity programs are safe and effective in promoting the recovery of gastrointestinal function after pancreaticoduodenectomy.

Objective To explore the underlying mechanism of β2 adrenergic receptor(ADRB2)in fibrogenesis during wound healing.Methods Non-specific ADRB2 gene knockdown adeno-associated virus(AAV-ADRB2 group,6 mice)and control virus(AAV-NC group,6 mice)was injected randomly into the back skin of 12 mice for 21 days,a full-thickness skin defected wound healing murine model was established.Wound healing rates were recorded at the 1st,3rd,5th,and 7th day after operation.Histological examinations by H-E staining,Masson staining,and immunohistochemistry were conducted to observe wounded skin tissue structure,fibrosis,and α-SMA protein expression;quantitative PCR was employed to analyze ADRB2 and matrix metalloproteinase(MMP)mRNA levels;Western blotting was utilized to assess the protein expression levels of COL1A1,COL3A1,TGF-β1,and Smad3.Results On postoperative day 5 and 7,the wound healing rate of the AAV-ADRB2 group significantly decreased(P＜0.05),accompanied by a series pathological changes,including thickened epidermis,exaggerated inflammation,reduced fibroblast count,and inhibited collagen deposition;the α-SMA expression showed a significant decrease(P＜0.05),and the ratio of COL1A1 to COL3A1 decreased(P＜0.05);ADRB2 mRNA levels significantly decreased(P＜0.01),while MMP-1 and MMP-8 mRNA levels increased(P＜0.01);the protein levels of TGF-β1 and Smad3 exhibited a significant decrease(P＜0.05).Conclusions ADRB2 knockdown reduced fibrosis during wound healing and degenerated connective tissue content around the wound bed by inhibiting the TGF-β1/Smad3 signaling pathway,which leads to an increase in MMP mRNA levels and a decrease in the ratio of type Ⅰ to type Ⅲ collagen.

Objective:To provide reference for clinical diagnosis and treatment of hepatic cystic echinococcosis by analyzing the clinical characteristics of these patients.Methods:Clinical data of 15 patients with hepatic cystic echinococcosis hospitalized in the Second Hospital of Lanzhou University from April 22, 2019 to March 5, 2022 were collected. The general data, clinical manifestations, abdominal imaging results, treatment and outcome of the included patients were retrospectively analyzed.Results:The age of 15 patients with hepatic cystic echinococcosis ranged from 39 to 78 years, with a median age of 51 years. Among the 15 patients, 12 were farmers, two were freelance workers, and one was unknown. Eleven patients presented with abdominal pain and fatigue, one patient presented with obstructive jaundice, and three patients whom were found by physical examination did not complain of obvious discomfort. Echinococcoid cysts were located at the right lobe of the liver in eight patients, at the left lobe of the liver in five patients, and with multiple lesions in the left and right lobes of the liver in two patients. Two patients received conservative therapy. All the 13 patients who received surgical treatment recovered without complications, such as biliary fistula and subphrenic infection. The clinical symptoms including abdominal pain and fatigue were relieved significantly after surgery. The hospital stay were four to 23 days. All patients were administrated with albendazole (400 mg once daily).Conclusions:Hepatic cystic echinococcosis is more common in the right lobe of the liver, with atypical clinical symptoms. Preoperative imaging diagnosis and postoperative pathological examination are necessary for diagnosis. Surgery is the most effective treatment at present, and albendazole should be taken regularly in all patients.

Objective To investigate the effect of SMAD family member 3(SMAD3) silenced by small interfering RNA (siRNA) on macrophage polarization and transforming growth factor β1 (TGF-β1)/ SMAD family signaling pathway in rheumatoid arthritis (RA). Methods RA macrophages co-cultured with rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were used as a cell model. TGF-β1 was used to stimulate macrophages, and SMAD3-specific siRNA (si-SMAD3) and negative control siRNA (si-NC) were transfected into human RA macrophages co-cultured in Transwell^TM chamber. The expression of SMAD3 mRNA was detected by real-time fluorescence quantitative PCR, and the expression of TGF-β1, SMAD3 and SMAD7 protein was detected by Western blot analysis. The contents of TGF-β1 and IL-23 in cell culture supernatant were determined by ELISA. Cell proliferation was detected by CCK-8 assay. Transwell^TM chamber was used to measure cell migration. Results Compared with the model group and the si-NC group, the expression of TGF-β1, SMAD3 mRNA and protein in RA macrophages decreased significantly after silencing SMAD3. In addition, the secretion of IL-23 decreased significantly, and the cell proliferation activity and cell migration were inhibited, with high expression of SMAD7. Conclusion Knockdown of SMAD3 can promote M2 polarization and SMAD7 expression in RA macrophages.
Humans ; Arthritis, Rheumatoid/genetics* ; Interleukin-23 ; Macrophages ; RNA, Messenger ; RNA, Small Interfering/genetics* ; Smad7 Protein/genetics* ; Transforming Growth Factor beta1/genetics* ; Smad3 Protein/genetics* ; Gene Silencing

Objective To evaluate the effectiveness of comprehensive echinococcosis control measures with emphasis on management of infectious source in Sichuan Province from 2010 to 2022, so as to provide insights into formulation of future control interventions. Methods Data pertaining to comprehensive echinococcosis control measures with emphasis on management of infectious source and echinococcosis surveillance in Sichuan Province from 2010 to 2022 were collected. The effectiveness of comprehensive echinococcosis control measures with emphasis on management of infectious source was evaluated with prevalence of human echinococcosis, detection of newly diagnosed echinococcosis patients, prevalence of Echinococcus infection in domestic dogs, prevalence of cystic echinococcosis in livestock, prevalence of alveolar echinococcosis in small mammals and awareness of echinococcosis control knowledge, and Spearman’s rank correlation analysis. Results The prevalence of human echinococcosis reduced from 1.08% in 2010 to 0.40% in 2022 in Sichuan Province (χ² = 1 482.97, P < 0.05), with a reduction from 0.30% to 0.02% in the detection of newly diagnosed echinococcosis cases (χ² = 2 776.41, P < 0.05), a reduction from 15.87% to 0.46% in the prevalence of Echinococcus infection in domestic dogs (χ² = 20 823.96, P < 0.05), a reduction from 8.05% to 1.07% in the prevalence of cystic echinococcosis in livestock (χ² = 1 296.02, P < 0.05), and the awareness of echinococcosis control knowledge increased from 50.65% to 95.24% (χ² = 34 938.63, P < 0.05); in addition, there was a year-specific prevalence rate of alveolar echinococcosis in small mammals (χ² = 164.07, P < 0.05). Spearman’s rank correlation analysis revealed that the detection of newly diagnosed echinococcosis cases correlated positively with the prevalence of Echinococcus infections in domestic dogs (r_s = 0.823, P < 0.05) and the prevalence of cystic echinococcosis in livestock (r_s = 0.795, P < 0.05), and correlated negatively with the awareness of echinococcosis control knowledge (r_s = − 0.918, P < 0.05), and the prevalence of Echinococcus infection in domestic dogs correlated positively with the prevalence of cystic echinococcosis in livestock (r_s = 0.753, P < 0.05) and negatively with the awareness of echinococcosis control knowledge (r_s = −0.747, P < 0.05); however, there was no correlation between the prevalence of Echinococcus infections in domestic dogs and the prevalence of alveolar echinococcosis in small mammals (r_s = −0.750, P > 0.05). Conclusions The comprehensive echinococcosis control measures with emphasis on management of infectious source had achieved remarkable effectiveness in Sichuan Province; however, the transmission chain of echinococcosis has not been interrupted. Reinforced comprehensive echinococcosis control measures with emphasis on management of infectious source and sustained tracking evaluation of the effectiveness are recommended in Sichuan Province.