OBJECTIVE To investigate the impact of lysosomal associated membrane protein 2b(Lamp2b)modification on the mucosal immune efficacy of engineered exosome-based vaccines.METHODS In vitro experiments:The murine dendritic cell line DC2.4 was transfected with a plasmid encoding the Lamp2b-RBD fusion protein.Real-time quantitative PCR and Western blotting were employed to assess Lamp2b-RBD expressions,flow cytometry was used to evaluate the proportion of Lamp2b-RBD-positive cells,and immunofluorescence staining was performed to determine their membrane localization.Exosomes were isolated via ultracentrifugation,and their morphology and particle size distribution were examined using transmission electron microscopy and nanoparticle tracking analysis.Western blotting was applied to confirm exosomal marker proteins[cluster of differentiation 9(CD9),CD63,ALG-2-interacting protein X(Alix),and Golgi marker GM130]and Lamp2b-RBD expression.In vivo experiments:① Female BALB/c mice were divided into the Lamp2b-RBD-Exo group and the lipid nanoparticle(LNP)group,and administered intratracheally for mucosal immunization.Pulmonary reten-tion was assessed by immunofluorescence staining.② Female BALB/c mice were divided into three groups:placebo group(PBS group),Lamp2b-RBD-Exo intratracheal administration group,and Lamp2b-RBD-Exo intramuscular injection group(im).Immunizations were performed on days 0 and 14,and on days 7 and 21.The titers of RBD-specific immunoglobulin G(IgG)in serum and RBD-specific IgA and IgG antibodies in bronchoalveolar lavage fluid were determined by enzyme-linked immunosor-bent assay(ELISA).RESULTS In vitro experiments:Lamp2b-RBD-positive cells accounted for 71.16％.Lamp2b-RBD mRNA levels were upregulated 1 979-fold compared with controls,with Lamp2b-RBD proteins localized on the cell membrane.Purified engineered exosomes displayed regular morphology,expressed CD9,CD63,and Alix but not GM130,had an average diameter of approximately 124 nm,and carried 3 009 pg of RBD protein per 1×109 exosomes.In vivo experiments:At 4 h after administra-tion,fluorescence signals were observed in the lung tissues of both the Lamp2b-RBD-Exo and LNPs groups.At 24 h,the fluorescence signal in the LNPs group shifted to the liver,while in the Lamp2b-RBD-Exo group,the fluorescence expanded from the trachea to the bronchioles and lung tissue,showing significantly better distribution and retention capacity than the LNPs group.Seven days after immuniza-tion,both the Lamp2b-RBD-Exo and Lamp2b-RBD-Exo(im)groups induced RBD-specific IgG antibody titers.At 21 days after immunization,Lamp2b-RBD-Exo elicited a higher level of RBD-specific immune response,with serum IgG titers reaching 1∶8 100 and bronchoalveolar lavage fluid(BALF)IgA titers reaching 1∶300.No RBD-specific IgA antibody titers were detected in the BALF of the Lamp2b-RBD-Exo(im)group.CONCLUSION Lamp2b-RBD modification enables efficient RBD protein loading and enhances pulmonary retention of engineered exosomes,thereby inducing potent antigen-specific mucosal immune responses.

Objective:To dynamically assess liver fibrosis using magnetic resonance elastography (MRE) and explore factors associated with fibrosis reversal in patients with metabolic dysfunction-associated steatohepatitis (MASH).Methods:This study included data from patients diagnosed with MASH by liver biopsy who underwent at least two MRE examinations. Patients were divided into a fibrosis reversal group and a non-reversal group according to whether MRE values decreased by 20% during follow-up. Differences in clinical data between the groups were compared using analysis of variance, the Kruskal-Wallis test, and the chi-square test. Univariate and multivariate logistic regression analyses were used to explore independent risk factors for fibrosis reversal in MASH.Results:A total of 46 cases were included in this study (mean age 50.1±12.3 years, BMI 26.1±3.1 kg/m2). Among them, the reversal group accounted for 26.1%. The rate of decrease in MRI proton density fat fraction (PDFF) was significantly higher in the reversal group (-50.0% vs. -8.1%, P=0.001) than in the non-reversal group between the two MRE examinations. The reversal group showed a more significant change rate of decreases in fasting insulin (-37.3% vs. -3.6%, P=0.011), insulin resistance index (-38.6% vs. -6.5%, P=0.044), and ALP (-24.9% vs. 0, P=0.004). Multivariate logistic regression analysis indicated that the rate of change in MRI PDFF was an independent predictor of fibrosis reversal ( OR=0.96, 95% CI: 0.92-1.00, P=0.046). Conclusion:A decrease in MRI proton density fat fraction levels is independently associated with liver fibrosis reversal in MASH, suggesting that intervention targeting liver fat content may be an effective treatment strategy.

Objective:To analyze the risk factors for symptomatic cerebral infarction (SCI) in patients with spontaneous intracerebral hemorrhage (ICH).Methods:A total of 337 patients with spontaneous ICH admitted to the stroke center of Zhengzhou People’s Hospital were consecutively collected from May 2019 to March 2023. Patients were divided into the SCI group and without SCI group according to whether the presence of new neurological deficits consistent with diffusion-weighted imaging hyperintense lesions distant from the hematoma. Magnetic resonance was used to quantify cerebral small vessel disease imaging markers and the degree of cerebral artery stenosis (CAS). SPSS 25.0 software was used for data analysis. Univariate and multivariable Logistic regression models were adopted to assess risk factors associated with concomitant SCI. Subgroup analysis stratified by mean arterial pressure (MAP) tertiles was performed.Results:Compared with patients without SCI, patients with SCI have more histories of ischemic stroke, higher rates of complication of pneumonia and deep venous thrombosis, higher baseline systolic blood pressure, greater MAP change, higher periventricular white matter hyperintensities score and deep white matter hyperintensities (DWMHs) score, and more severe CAS (all P<0.05). Multivariable regression analysis showed that, the degree of CAS ( B=1.095, OR=2.989, 95% CI=1.645-5.432, P<0.001) and DWMHs score ( B=0.789, OR=2.201, 95% CI=1.163-4.166, P=0.015) were risk factors for ICH patients with SCI. Compared with patients with mild CAS, the proportion of SCI significantly increased in patients with moderate and severe CAS in the maximum MAP change group (both P<0.05). Compared with patients with mild DWMHs, the proportion of SCI in patients with severe DWMHs in the maximum MAP change group significantly increased ( P=0.002). Conclusion:Severe CAS and DWMHs are independent risk factors for SCI after ICH, especially with greater fluctuations in MAP change.

Objective:To analyze the risk factors for symptomatic cerebral infarction (SCI) in patients with spontaneous intracerebral hemorrhage (ICH).Methods:A total of 337 patients with spontaneous ICH admitted to the stroke center of Zhengzhou People’s Hospital were consecutively collected from May 2019 to March 2023. Patients were divided into the SCI group and without SCI group according to whether the presence of new neurological deficits consistent with diffusion-weighted imaging hyperintense lesions distant from the hematoma. Magnetic resonance was used to quantify cerebral small vessel disease imaging markers and the degree of cerebral artery stenosis (CAS). SPSS 25.0 software was used for data analysis. Univariate and multivariable Logistic regression models were adopted to assess risk factors associated with concomitant SCI. Subgroup analysis stratified by mean arterial pressure (MAP) tertiles was performed.Results:Compared with patients without SCI, patients with SCI have more histories of ischemic stroke, higher rates of complication of pneumonia and deep venous thrombosis, higher baseline systolic blood pressure, greater MAP change, higher periventricular white matter hyperintensities score and deep white matter hyperintensities (DWMHs) score, and more severe CAS (all P<0.05). Multivariable regression analysis showed that, the degree of CAS ( B=1.095, OR=2.989, 95% CI=1.645-5.432, P<0.001) and DWMHs score ( B=0.789, OR=2.201, 95% CI=1.163-4.166, P=0.015) were risk factors for ICH patients with SCI. Compared with patients with mild CAS, the proportion of SCI significantly increased in patients with moderate and severe CAS in the maximum MAP change group (both P<0.05). Compared with patients with mild DWMHs, the proportion of SCI in patients with severe DWMHs in the maximum MAP change group significantly increased ( P=0.002). Conclusion:Severe CAS and DWMHs are independent risk factors for SCI after ICH, especially with greater fluctuations in MAP change.

OBJECTIVE To investigate the impact of lysosomal associated membrane protein 2b(Lamp2b)modification on the mucosal immune efficacy of engineered exosome-based vaccines.METHODS In vitro experiments:The murine dendritic cell line DC2.4 was transfected with a plasmid encoding the Lamp2b-RBD fusion protein.Real-time quantitative PCR and Western blotting were employed to assess Lamp2b-RBD expressions,flow cytometry was used to evaluate the proportion of Lamp2b-RBD-positive cells,and immunofluorescence staining was performed to determine their membrane localization.Exosomes were isolated via ultracentrifugation,and their morphology and particle size distribution were examined using transmission electron microscopy and nanoparticle tracking analysis.Western blotting was applied to confirm exosomal marker proteins[cluster of differentiation 9(CD9),CD63,ALG-2-interacting protein X(Alix),and Golgi marker GM130]and Lamp2b-RBD expression.In vivo experiments:① Female BALB/c mice were divided into the Lamp2b-RBD-Exo group and the lipid nanoparticle(LNP)group,and administered intratracheally for mucosal immunization.Pulmonary reten-tion was assessed by immunofluorescence staining.② Female BALB/c mice were divided into three groups:placebo group(PBS group),Lamp2b-RBD-Exo intratracheal administration group,and Lamp2b-RBD-Exo intramuscular injection group(im).Immunizations were performed on days 0 and 14,and on days 7 and 21.The titers of RBD-specific immunoglobulin G(IgG)in serum and RBD-specific IgA and IgG antibodies in bronchoalveolar lavage fluid were determined by enzyme-linked immunosor-bent assay(ELISA).RESULTS In vitro experiments:Lamp2b-RBD-positive cells accounted for 71.16％.Lamp2b-RBD mRNA levels were upregulated 1 979-fold compared with controls,with Lamp2b-RBD proteins localized on the cell membrane.Purified engineered exosomes displayed regular morphology,expressed CD9,CD63,and Alix but not GM130,had an average diameter of approximately 124 nm,and carried 3 009 pg of RBD protein per 1×109 exosomes.In vivo experiments:At 4 h after administra-tion,fluorescence signals were observed in the lung tissues of both the Lamp2b-RBD-Exo and LNPs groups.At 24 h,the fluorescence signal in the LNPs group shifted to the liver,while in the Lamp2b-RBD-Exo group,the fluorescence expanded from the trachea to the bronchioles and lung tissue,showing significantly better distribution and retention capacity than the LNPs group.Seven days after immuniza-tion,both the Lamp2b-RBD-Exo and Lamp2b-RBD-Exo(im)groups induced RBD-specific IgG antibody titers.At 21 days after immunization,Lamp2b-RBD-Exo elicited a higher level of RBD-specific immune response,with serum IgG titers reaching 1∶8 100 and bronchoalveolar lavage fluid(BALF)IgA titers reaching 1∶300.No RBD-specific IgA antibody titers were detected in the BALF of the Lamp2b-RBD-Exo(im)group.CONCLUSION Lamp2b-RBD modification enables efficient RBD protein loading and enhances pulmonary retention of engineered exosomes,thereby inducing potent antigen-specific mucosal immune responses.

Objective:To dynamically assess liver fibrosis using magnetic resonance elastography (MRE) and explore factors associated with fibrosis reversal in patients with metabolic dysfunction-associated steatohepatitis (MASH).Methods:This study included data from patients diagnosed with MASH by liver biopsy who underwent at least two MRE examinations. Patients were divided into a fibrosis reversal group and a non-reversal group according to whether MRE values decreased by 20% during follow-up. Differences in clinical data between the groups were compared using analysis of variance, the Kruskal-Wallis test, and the chi-square test. Univariate and multivariate logistic regression analyses were used to explore independent risk factors for fibrosis reversal in MASH.Results:A total of 46 cases were included in this study (mean age 50.1±12.3 years, BMI 26.1±3.1 kg/m2). Among them, the reversal group accounted for 26.1%. The rate of decrease in MRI proton density fat fraction (PDFF) was significantly higher in the reversal group (-50.0% vs. -8.1%, P=0.001) than in the non-reversal group between the two MRE examinations. The reversal group showed a more significant change rate of decreases in fasting insulin (-37.3% vs. -3.6%, P=0.011), insulin resistance index (-38.6% vs. -6.5%, P=0.044), and ALP (-24.9% vs. 0, P=0.004). Multivariate logistic regression analysis indicated that the rate of change in MRI PDFF was an independent predictor of fibrosis reversal ( OR=0.96, 95% CI: 0.92-1.00, P=0.046). Conclusion:A decrease in MRI proton density fat fraction levels is independently associated with liver fibrosis reversal in MASH, suggesting that intervention targeting liver fat content may be an effective treatment strategy.

Pan-immune inflammation value (PIV) is a comprehensive immune inflammatory biomarker based on complete blood cell counts, which has been proven to predict treatment response and survival outcomes for different types of tumors. However, the predictive value of the PIV varies in different strategies for tumor treatment. This paper aims to systematically review the latest progress of PIV in predicting survival outcomes and tumor prognosis for immunotherapy, radiotherapy, targeted therapy, endocrine therapy, surgical treatment and neoadjuvant therapy, and analyze its existing challenges and issues, as well as look forward to its future development direction and application prospects.

Objective To investigate the correlation between pre-treatment pan-immune inflammation value (PIV) and clinicopathological features in esophageal squamous cell carcinoma (ESCC) patients with postoperative adjuvant radiotherapy and evaluate its value in prognosis assessment combined with T stage. Methods A retrospective analysis was conducted on data of 85 ESCC patients with postoperative adjuvant radiotherapy in the Department of Radiation Oncology of the Affiliated Hospital of Yangzhou University from January 2019 to January 2023. The receiver operating characteristic (ROC) curve was drew to obtain the optimal cut-off value of PIV and other immune-inflammatory biomarkers. The area under the curve (AUC) and clinical applicability of PIV and other immune-inflammatory biomarkers were compared based on the ROC curve and decision curve analysis (DCA). According to the optimal cut-off value, patients were divided into high PIV group and low PIV group, and the correlation between PIV level and clinicopathological features of ESCC was evaluated. Kaplan-Meier method was used for survival analysis, the Cox proportional hazards model was used for multivariate analysis, and a risk stratification model combining PIV and T stage was established by recursive partitioning analysis (RPA). Results The optimal cut-off value of pre-treatment PIV was determined as 187.22 based on the ROC curve. The AUC of PIV was 0.679, which was greater than 0.640, 0.583, 0.656 and 0.644 of the other four immune-inflammatory biomarkers such as the systemic immune-inflammation index (SII), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), and neutrophil-to-lymphocyte ratio (NLR). The 85 patients were divided into low PIV group (< 187.22, n=48) and high PIV group (≥187.22, n=37). The level of PIV was significantly correlated with tumor diameter (P < 0.05). The 3-year overall survival (OS) (75.6% versus 30.6%, P < 0.001) and 3-year disease-free survival (DFS) (56.1% versus 21.0%, P < 0.001) were significantly higher in the low PIV group than the high PIV group. Tumor diameter, T stage and PIV were independent factors affecting OS in ESCC patients (P < 0.05), and T stage and PIV were independent factors affecting DFS in ESCC patients (P < 0.05). A new staging system with three risk groups was established by the RPA stratification model based on T stage and PIV, which further improved the predictive value of prognosis compared with T stage or PIV alone. Conclusion Pre-treatment PIV is helpful in predicting the prognosis of ESCC patients with postoperative adjuvant radiotherapy, and the combination of PIV and T stage can improve the predictive value.

Objective:To investigate the value of a nomogram predicting the outcome of intracerebral hemorrhage (ICH) based on clinical characteristics and diffusion-weighted imaging (DWI) of hyperintense lesions.Methods:A case-control study. Consecutive patients, aged 30-88(59±13) years old, with ICH were recruited at the Stroke Center of Zhengzhou People′s Hospital from January 2018 to August 2021. Patients were divided into a group with DWI lesions and a group without DWI lesions depending on whether there were DWI hyperintense lesions distant from the hematoma. Prognosis was evaluated at 90 days via the modified Rankin Scale (mRS). Univariate and multivariable logistic regression models were used to identify independent predictors of a poor ICH outcome (mRS score≥4), and a nomogram model was developed. The performance of the nomogram was validated via the area under the receiver operating characteristic curve (AUC) and a calibration chart.Results:Of the 303 patients included in the study, 24.8% presented with DWI lesions; 17.5% with asymptomatic DWI lesions and 7.3% with symptomatic DWI lesions. Poor outcomes were significantly more frequent in the group with DWI lesions than in the group without DWI lesions ( χ2=21.32, P<0.001). In multivariable regression analysis, age [odds ratio ( OR)=1.032, 95% confidence interval ( CI) 1.002-1.063, P=0.035], hematoma volume ( OR=1.050, 95% CI 1.011-1.090, P=0.012), hematoma location ( OR=3.839, 95% CI 1.248-11.805, P=0.019), DWI lesions ( OR=3.955, 95% CI 1.906-8.206, P<0.001), and baseline NIHSS scores ( OR=1.102, 95% CI 1.038-1.170, P=0.001) were independent predictors of a poor outcome. In subgroup analysis patients with asymptomatic DWI lesions had a 3-fold greater risk of a poor outcome compared to those without DWI lesions ( OR=3.135, 95% CI 1.382-7.112, P=0.006), and patients with symptomatic DWI lesions had a 7-fold greater risk of a poor outcome compared to those without DWI lesions ( OR=7.126, 95% CI 2.279-22.277, P=0.001). A nomogram model was established based on the independent predictors for a poor outcome. The AUC of the nomogram was 0.846 (95% CI 0.795-0.898), and a calibration chart indicated good consistency between values predicted by the nomogram and actual observed values. Conclusions:DWI lesions are an independent risk factor for a poor outcome in patients with ICH-particularly symptomatic DWI lesions. A nomogram model based on clinical characteristics and DWI lesions exhibited good efficacy when predicting the outcome of ICH.

Objective:To investigate the associations between small diffusion-weighted imaging (DWI) hyperintensities lesions and total cerebral small vessel disease (cSVD) burden and the influence on prognosis in patients with acute intracerebral hemorrhage (ICH).Methods:Consecutive patients with acute spontaneous ICH from January 2018 to June 2021 were recruited in the Stroke Center of Zhengzhou People′s Hospital. Magnetic resonance imaging was performed to quantify DWI hyperintensities lesions and cSVD imaging markers, including white matter hyperintensities, enlarged perivascular spaces, lacunes and cerebral microbleeds, which were calculated for the total cSVD burden (0-4 points). The prognosis was assessed with the modified Rankin Scale (mRS) at discharge and 90-day. Multivariable Logistic regression models were adopted to explore the associations between DWI lesions and total cSVD burden and clinical outcome.Results:Of 283 included patients, 59 (20.8%) had small DWI lesions, 32 (11.3%) had multiple lesions. They were mostly punctate, mainly located in the cortical and subcortical regions, and scattered in multiple vascular territories. With the increase of cSVD burden, the number of DWI lesions gradually increased. Spearman correlation analysis showed that the total cSVD burden was positively correlated with the number of DWI lesions ( r=0.21, P<0.001). In multivariable regression analyses, the total cSVD burden was independently associated with DWI lesions ( OR=1.63, 95% CI 1.23-2.15, P=0.001). The 90-day poor outcome (mRS scores≥4) in patients with DWI lesions was significantly higher than those without DWI lesions (39.3% vs 16.3%, χ 2=14.38, P<0.001), while there was no statistically significant difference in the poor outcome of discharge between the two groups (26.5% vs 17.7%, χ 2=3.06, P=0.080). With the increase in the number of DWI lesions, the 90-day poor outcome increased significantly (trend chi-squared test χ 2=11.50, P=0.001). Multivariable analyses showed that DWI lesions ( OR=4.39, 95% CI 1.92-10.03, P<0.001) and their number ( OR=1.42, 95% CI 1.06-1.90, P=0.018) were independently associated with the 90-day poor outcome. Conclusions:Higher total cSVD burden is an independent risk factor for small DWI lesions in patients with ICH. Small DWI lesions were independently associated with the 90-day poor outcome, but not with the discharge outcome.