Objective The aim of this study was to clarify the role and mechanism of Pseudomonas aeruginosa vaccine subunit OprI in the fusion protein vaccine rePO(PcrV-OprI).Methods The in vitro stability of rePO,PcrV and OprI at 4 ℃,25 ℃,and 37 ℃ was examined.After immunizing mice with rePO,OprI and PcrV,respectively,the specific antibody potency in serum and the proportion of cells secreting IFN-γ and IL-4 in the spleen were examined;Additionally,detection of the levels of protein uptake by DC2.4 cells in vitro using laser confocal microscopy and flow cytometry,and their ability to promote the maturation of mouse bone marrow-derived dendritic cells(BMDC).Results The heat stability of fusion protein rePO was significantly better than that of PcrV.The induced anti-PcrV IgG and anti-OprI IgG potency of rePO was significantly higher than that of monomeric PcrV and OprI.Additionally,the number of cells secreting IFN-γ and IL-4 induced by immunization with rePO was significantly higher than that of PcrV and OprI.The uptake rate of fusion protein rePO by DC2.4 cells was significantly higher than that of PcrV and OprI.Furthermore,rePO promoted the maturation of mouse BMDC more effectively than PcrV and OprI.Conclusion OprI in the fusion protein rePO can significantly improve its thermal stability and immunogenicity,which lays the foundation for the successful development of Pseudomonas aeruginosa vaccine.

Background and Amis:Gallbladder cancer(GBC)is the most common malignant tumor of the biliary tract,accounting for approximately 80%-95%of biliary tract cancers.This type of tumor has a poor prognosis,and currently,there are no effective tools for evaluating the prognosis of GBC.Therefore,this study was performed to investigate the factors influencing the prognosis of GBC patients to provide a reference for clinical practice.Methods:The clinical data and follow-up information from 160 GBC patients treated in the Hunan Provincial People's Hospital from January 2018 to January 2024 was retrospectively conducted.The clinicopathologic characteristics of GBC patients were analyzed.Kaplan-Meier and Log-rank tests were used to calculate and compare the differences in overall survival(OS)among GBC patients with different clinicopathologic characteristics and treatment methods.Multivariate analysis using Cox regression was performed to identify independent prognostic factors for GBC.Results:Among the 160 patients,113 were females and 47 were males.The median age of the patients was 62 years,with the main clinical manifestations being abdominal pain/distention(55.63%),jaundice(40.63%),appetite loss(30.62%),and weight loss(19.38%).Serum tumor markers,including CA19-9,CA125,CEA,and CA724,were elevated in 58.75%,30.63%,30.00%,and 20.63%of GBC patients,respectively.Clinical stage classification revealed that 139 patients(86.87%)had stage Ⅲ/Ⅳ disease,132 patients(82.5%)had T3/T4 stage,91 patients(56.87%)had N1/N2 stage,and 54 patients(33.75%)had M1 stage.The pathological type of GBC was predominantly adenocarcinoma(92.50%),with the majority classified as moderately differentiated(19.38%),moderately to poorly differentiated(34.37%),and poorly differentiated(24.37%).Neural or vascular invasion was present in 29.37%and 21.25%of patients,respectively.Univariate analysis showed that diabetes,jaundice,liver function(Child-Pugh classification),tumor marker levels(CA19-9,CA125,CEA,CA724,CYFRA 21-1),clinical TNM stage,degree of differentiation,vascular or neural invasion,surgical treatment,and other treatments(chemotherapy,immunotherapy,targeted therapy,traditional Chinese medicine,etc.)were significantly associated with the prognosis of GBC patients(all P<0.05).Multivariate Cox regression analysis revealed that diabetes,elevated CA125,and TNM stage were independent risk factors for poor prognosis in GBC,while chemotherapy-based drug treatment was an independent protective factor(all P<0.05).Conclusion:Diabetes,elevated CA125,TNM staging,and treatment methods are closely related to the prognosis of GBC patients.Targeted treatment strategies should be developed for patients with risk factors,and surgery or chemotherapy-based drug therapy should be prioritized to improve patient prognosis.

Background and Amis:Gallbladder cancer(GBC)is the most common malignant tumor of the biliary tract,accounting for approximately 80%-95%of biliary tract cancers.This type of tumor has a poor prognosis,and currently,there are no effective tools for evaluating the prognosis of GBC.Therefore,this study was performed to investigate the factors influencing the prognosis of GBC patients to provide a reference for clinical practice.Methods:The clinical data and follow-up information from 160 GBC patients treated in the Hunan Provincial People's Hospital from January 2018 to January 2024 was retrospectively conducted.The clinicopathologic characteristics of GBC patients were analyzed.Kaplan-Meier and Log-rank tests were used to calculate and compare the differences in overall survival(OS)among GBC patients with different clinicopathologic characteristics and treatment methods.Multivariate analysis using Cox regression was performed to identify independent prognostic factors for GBC.Results:Among the 160 patients,113 were females and 47 were males.The median age of the patients was 62 years,with the main clinical manifestations being abdominal pain/distention(55.63%),jaundice(40.63%),appetite loss(30.62%),and weight loss(19.38%).Serum tumor markers,including CA19-9,CA125,CEA,and CA724,were elevated in 58.75%,30.63%,30.00%,and 20.63%of GBC patients,respectively.Clinical stage classification revealed that 139 patients(86.87%)had stage Ⅲ/Ⅳ disease,132 patients(82.5%)had T3/T4 stage,91 patients(56.87%)had N1/N2 stage,and 54 patients(33.75%)had M1 stage.The pathological type of GBC was predominantly adenocarcinoma(92.50%),with the majority classified as moderately differentiated(19.38%),moderately to poorly differentiated(34.37%),and poorly differentiated(24.37%).Neural or vascular invasion was present in 29.37%and 21.25%of patients,respectively.Univariate analysis showed that diabetes,jaundice,liver function(Child-Pugh classification),tumor marker levels(CA19-9,CA125,CEA,CA724,CYFRA 21-1),clinical TNM stage,degree of differentiation,vascular or neural invasion,surgical treatment,and other treatments(chemotherapy,immunotherapy,targeted therapy,traditional Chinese medicine,etc.)were significantly associated with the prognosis of GBC patients(all P<0.05).Multivariate Cox regression analysis revealed that diabetes,elevated CA125,and TNM stage were independent risk factors for poor prognosis in GBC,while chemotherapy-based drug treatment was an independent protective factor(all P<0.05).Conclusion:Diabetes,elevated CA125,TNM staging,and treatment methods are closely related to the prognosis of GBC patients.Targeted treatment strategies should be developed for patients with risk factors,and surgery or chemotherapy-based drug therapy should be prioritized to improve patient prognosis.

Objective To establish a mouse model of bronchiectasis with acute infection and evaluate the immunogenicity and protective effect of a self-assembling Pseudomonas aeruginosa(PA)nanoparticle vaccine rePO-FN based on fusion of PcrV-OprI(rePO)protein with self-assembling ferritin(Ferritin).Methods ① SPF-grade female C57BL/6 mice(aged 6～8 weeks,weighing 18～20 g)were randomly allocated into normal saline group,and low-,medium-and high-dose elastase groups(n=6).A mouse model of bronchiectasis was established via intratracheal instillation of different doses of elastase(30 μL of normal saline containing 0.65,1.30 and 2.60 IU elastase)for 3 consecutive days.At 14 and 21 d after modeling,ELISA and HE staining were performed respectively to detect the concentration of IL-6 and to observe pathological changes in lung tissue in order to confirm the modeling.② A recombinant plasmid encoding the gene of fusion protein rePO-FN was constructed and expressed in E.coli.The target protein was purified via affinity chromatography and renatured to obtain the desired protein.The physicochemical properties of the rePO-FN protein were characterized using SDS-PAGE protein gel electrophoresis,dynamic light scattering,molecular sieve chromatography,and transmission electron microscopy.③ C57BL/6J mice were randomly divided into PBS group,rePO group,rePO-FN group,and Ferritin group(n=10).The mice in the above groups were immunized intramuscularly with 100 μL PBS buffer alone or containing 10 μg of corresponding proteins on days 0,7,and 14.ELISA was used to measure the specific antibodies in serum.In 7 d after the final immunization,an acute PA infection model was used to compare the survival rates and bacterial colonization among the PBS,rePO,and rePO-FN groups.After establishing a bronchiectasis model by intratracheal instillation of 2.60 IU of elastase in C57BL/6J mice as described above,the mice were randomly divided into bronchiectasis PBS group,bronchiectasis rePO group,and bronchiectasis rePO-FN group(n=10).Immunization was conducted at the same dose and procedure as described above,in 21 d after bronchiectasis modeling.At the 7th d after the final immunization,an acute PA infection model was used to compare the survival rates and bacterial colonization among the groups.Results ①Repeated intratracheal instillation of elastase significantly increased the concentration of IL-6 in the lung tissue when compared to the content of the normal saline group(P＜0.05).Pathological observations revealed varying degrees of bronchial wall destruction,alveolar fusion,edema,neutrophil infiltration,and hemorrhage,with the severity increasing with elastase dose,which confirming successful establishment of the mouse model of bronchiectasis.② Well-dispersed rePO-FN nanoparticles were successfully prepared,with an average particle size of 91.28 nm,a Zeta potential of approximately-6.5 mV,and a polydispersity index(PDI)of 0.306.Molecular sieve chromatography determined the elution volume of rePO-FN protein to be 8.80 mL,corresponding to a molecular weight of approximately 1 400 kDa.③ Under acute PA XN-1 strain infection,the survival rate of the rePO-FN immunization group and the bronchiectasis rePO-FN immunization group were significantly higher than that of the PBS control group(P＜0.05).Additionally,bacterial colonization in the lung tissues was significantly lower in the rePO-FN immune group and the bronchiectasis rePO-FN immune group under acute PA XN-1 strain infection than that in the rePO group and the bronchiectasis rePO group(P＜0.05).Conclusion Our vaccine rePO-FN can effectively trigger a strong humoral immune response and provide significant protection against PA infection in a mouse bronchiectasis model.

Objective The aim of this study was to clarify the role and mechanism of Pseudomonas aeruginosa vaccine subunit OprI in the fusion protein vaccine rePO(PcrV-OprI).Methods The in vitro stability of rePO,PcrV and OprI at 4 ℃,25 ℃,and 37 ℃ was examined.After immunizing mice with rePO,OprI and PcrV,respectively,the specific antibody potency in serum and the proportion of cells secreting IFN-γ and IL-4 in the spleen were examined;Additionally,detection of the levels of protein uptake by DC2.4 cells in vitro using laser confocal microscopy and flow cytometry,and their ability to promote the maturation of mouse bone marrow-derived dendritic cells(BMDC).Results The heat stability of fusion protein rePO was significantly better than that of PcrV.The induced anti-PcrV IgG and anti-OprI IgG potency of rePO was significantly higher than that of monomeric PcrV and OprI.Additionally,the number of cells secreting IFN-γ and IL-4 induced by immunization with rePO was significantly higher than that of PcrV and OprI.The uptake rate of fusion protein rePO by DC2.4 cells was significantly higher than that of PcrV and OprI.Furthermore,rePO promoted the maturation of mouse BMDC more effectively than PcrV and OprI.Conclusion OprI in the fusion protein rePO can significantly improve its thermal stability and immunogenicity,which lays the foundation for the successful development of Pseudomonas aeruginosa vaccine.