ObjectiveTo investigate the change in the activity of hepatitis B virus （HBV）-specific CD8⁺ T cells after pegylated interferon-α-2b （PEG-IFN-α-2b） therapy in HBeAg-negative patients with chronic HBV infection. MethodsA total of 53 HBeAg-negative patients with chronic HBV infection who attended The First Affiliated Hospital of Xinxiang Medical University and Tangdu Hospital of Air Force Mdical University from April 2020 to June 2022 were enrolled and treated with PEG-IFN-α-2b （180 μg/week， subcutaneous injection） antiviral therapy. The study endpoint was HBsAg clearance （course of treatment<48 weeks） or 48 weeks （course of treatment≥48 weeks）. Peripheral blood mononuclear cells were isolated at baseline and study endpoint， and peripheral blood T cell counts were measured. Enzyme-linked immunospot assay was used to measure the frequency of HBV-specific CD8⁺ T cells secreting perforin， granzyme B， and interferon-γ. A total of 17 HLA-A^*02-restricted patients were selected， and CD8⁺ T cells were purified to establish direct- and indirect-contact co-culture systems for HBV-specific CD8⁺ T cells and HepG2.2.15 cells. The level of lactate dehydrogenase in supernatant was measured to calculate the mortality rate of HepG2.2.15 cells， and the levels of HBV DNA， cytotoxic molecules， and cytokines in supernatant were also measured. Flow cytometry was used to measure the expression of apoptosis ligands， and the cytotoxicity of HBV-specific CD8⁺ T cells was evaluated. The independent samples t-test or the paired t-test was used for comparison of normally distributed continuous data between two groups， and the Mann-Whitney U test or the Wilcoxon rank-sum test was used for comparison of non-normally distributed continuous data between two groups. ResultsThe HBsAg clearance rate at study endpoint was 30.19% （16/53）. There were no significant differences in peripheral blood T cell counts （CD3⁺， CD4⁺， and CD8⁺ T cells） between baseline and study endpoint （P>0.05）. At study endpoint， there was a significant increase in the frequency of HBV-specific CD8⁺ T cells secreting perforin， granzyme B， and interferon-γ （U=177.50， t=11.90， U=186.50， all P<0.001）， and the patients with HBsAg clearance had a significantly higher frequency of such HBV-specific CD8⁺ T cells than those without HBsAg clearance （U=120.50， t=2.73， U=121.50， all P<0.01）. In the direct- and indirect-contact co-culture systems at study endpoint， HBV-specific CD8⁺ T cells induced a significant reduction in HBV DNA in the supernatant of HepG2.2.15 cells （all P<0.001） and significant increases in the secretion of interferon-γ and tumor necrosis factor-α （all P<0.05）； in the direct-contact co-culture system， HBV-specific CD8⁺ T cells induced significant increases in the mortality rate of HepG2.2.15 cells （13.62%±3.27% vs 11.39%±2.40%， t=2.27， P=0.030） and the secretion of perforin and granzyme B （t=72.50， U=52.50， both P<0.05）. In the direct- and indirect-contact co-culture systems， compared with HBV-specific CD8⁺ T cells from the patients without HBsAg clearance， the HBV-specific CD8⁺ T cells from patients with HBsAg clearance had a significantly greater reduction in HBV DNA （P<0.05） and significant increases in the secretion of interferon-γ and tumor necrosis factor-α （P<0.05）. ConclusionPEG-IFN-‍α‍-2b therapy can help to achieve a relatively high HBsAg clearance rate in HBeAg-negative patients with chronic HBV infection， and the activity of HBV-specific CD8⁺ T cells is significantly enhanced， which is closely associated with HBsAg clearance.

AIM: To investigate the mechanism of Hexue Mingmu Tablets(HXMMT)in improving diabetic retinopathy(DR)based on transcriptomics.METHODS: Zebrafish DR models were established by 3-day glucose induction(130 mmol/L)starting at 3 days post-fertilization(dpf). Larvae were randomized into four groups: control group(CG; aquaculture water), model group(MG; 130 mmol/L glucose), low-dose HXMMT treatment group(L-HX; 130 mmol/L glucose +7.5 mg/L HXMMT), and high-dose HXMMT treatment group(H-HX; 130 mmol/L glucose +75 mg/L HXMMT), with a 3-day intervention period until 6 dpf. The area and length of eyes, and body length of zebrafish were observed by stereomicroscopy, retinal morphology was observed by hematoxylin-eosin staining(HE), and retinal vessel diameter was observed under fluorescence microscope. Differentially expressed genes(DEGs)were identified by RNA-sequencing(RNA-seq)technology to further elucidate the molecular mechanism of HXMMT in improving DR in zebrafish, and the sequencing accuracy was validated through quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS: HE staining demonstrated that the intervention with HXMMT significantly improved the disordered cell arrangement, widened gaps, and thickened inner nuclear layer(INL)in ganglion cell layer GCL); retinal vascular diameter quantification revealed that the retinal vessel diameter of the MG significantly increased compared with the CG, and it was significantly changed after the intervention of HXMMT, with significant efficacy in the H-HX(P<0.05); transcriptomics profiling identified 1 470 reversed DEGs, predominantly enriched in the AMPK signaling pathway, FoxO signaling pathway, retinal developmental processes, and tight junction regulation. Technical validation confirmed strong correlation between qRT-PCR and RNA-seq data(R2=0.8571, P<0.05).CONCLUSION: HXMMT may improve retinal vascular microcirculation disorders in DR by regulating core targets including vsx1, pde6c, arr3a, plk1, fbp1b, foxo1a, pcna, and cdk1, as well as synergistically modulating processes such as retinal development in camera-type eyes, visual perception, microtubule cytoskeletal organization, tight junctions, and the AMPK signaling pathway, Foxo signaling pathway.

Objective To assess inter-observer variations(IOV)in the delineation of target volumes and organs-at-risk(OAR)for intensity-modulated radiotherapy(IMRT)of nasopharyngeal carcinoma(NPC)among physicians from different levels of cancer centers,thereby providing a reference for quality control in multi-center clinical trials.Methods Twelve patients with NPC of different TMN stages were randomly selected.Three physicians from the same municipal cancer center manually delineated the target volume(GTVnx)and OAR for each patient.The manually modified and confirmed target volume(GTVnx)and OAR delineation structures by radiotherapy experts from the regional cancer center were used as the standard delineation.The absolute volume difference ratio(△V_diff),maximum/minimum volume ratio(MMR),coefficient of variation(CV),and Dice similarity coefficient(DSC)were used to compare the differences in organ delineation among physicians from different levels of cancer centers and among the 3 physicians from the same municipal cancer center.Furthermore,the IOV of GTVnx and OAR among physicians from different levels cancer centers were compared across different TMN stages.Results Significant differences in the delineation of GTVnx were observed among physicians from different levels of cancer centers.Among the 3 physicians,the maximum values of △V_diff,MMR,and CV were 97.23%±83.45%,2.19±0.75,and 0.31±0.14,respectively,with an average DSC of less than 0.7.Additionally,there were considerable differences in the delineation of small-volume OAR such as the left and right optic nerves,chiasm,and pituitary,with average MMR>2.8,CV>0.37,and DSC<0.51.However,relatively smaller differences were observed in the delineation of large-volume OAR such as the brainstem,spinal cord,left and right eyeballs,and left and right mandible,with average△V_diff<42%,MMR<1.55,and DSC>0.7.Compared with the differences among physicians from different levels cancer centers,the differences among the 3 physicians from the municipal cancer center were slightly reduced.Furthermore,there were also differences in the delineation of target volumes for NPC among physicians from different levels cancer centers,depending on the staging of the disease.Compared with the delineation of target volumes for earlier stage patients(stages I or II),the differences among physicians in the delineation of target volumes for advanced stage patients(stages III or IV)were smaller,with average △V_diff and DSC of 98.31%±67.36%vs 69.38%±72.61%(P<0.05)and 0.55±0.08 vs 0.72±0.12(P<0.05),respectively.Conclusion There are differences in the delineation of GTVnx and OAR in radiation therapy for NPC among physicians from different levels of cancer centers,especially in the delineation of target volume(GTVnx)and small-volume OAR for early-stage patients.To ensure the accuracy of multicenter clinical trials,it is recommended to provide unified training to physicians from different levels of cancer centers and review their delineation results to reduce the effect of differences on treatment outcomes.

Objective:To evaluate the safety and efficacy of the domestic robotic surgical system for general surgery.Methods:A prospective single-center, single-arm exploratory study was conducted at Gansu Provincial People's Hospital from Jun 2022 to Oct 2023, enrolling 54 patients undergoing general surgery using domestically produced Toumai? Endoscopic Surgical Robotic System. The primary study endpoint was surgical success rate, and the secondary study endpoints were intraoperative bleeding, operative time, complications, system performance, hospitalization days.Results:In this study, robotic surgery was successfully completed in 52 patients, and in 2 patients undergoing thyroid operation it was converted to open surgery due to bleeding, with a success rate of 96%, no organ injury or death during surgery, and no system failure. The types of surgery included cholecystectomy, radical gastric cancer resection, radical colorectal cancer resection, inguinal hernia repair, partial hepatectomy, total thyroidectomy and choledocho-jejunal anastomosis.Conclusion:The study provides preliminary evidence of the safety and efficacy of the Toumai? Endoscopic Surgical Robotic System for the treatment of general surgical diseases.

Objective:To investigate the expression and clinical significance of neutrophil myeloperoxidase (MPO) in patients with MPO-antibody associated vasculitis (AAV).Methods:Thirty-six newly diagnosed MPO-AAV patients who were hospitalized in the First Affiliated Hospital, Anhui Medical University from July 2018 to June 2021 were enrolled,and 36 age and sex matched healthy subjects were selected as controls. Neutrophil MPO level was detected by flow cytometry (FCM) and MPO mRNA was tested by real time quantitative polymerase chain reaction (RT-qPCR) in all subjects. Serum complement fragment C5 (C5a) and MPO in both groups and serum MPO-anti-antineutrophilic cytoplasmic antibody(ANCA) in MPO-AAV group were measured by enzyme linked immunosorbent assay (ELISA), while the disease activity was evaluated by Birmingham vasculitis activity score-V3 (BVAS-V3).Results:Compared with the heathy control group, the expression of MPO mRNA in neutrophils, serum MPO and complement C5a in MPO-AAV group were significantly higher[MPO mRNA:30.2±11.5 vs. 1.9±0.6, P<0.001;MPO:(112.0±68.7) IU/L vs. (87.4±22.9) IU/L, P=0.01; C5a:(187.3±90.3) ng/ml vs. (107.3±31.1) ng/ml, P<0.001; respectively], while the mean fluorescence intensity (MFI) of MPO in neutrophils were significantly lower [ 1 343.3±723.4 vs. 2 868.0±1 136.5, P<0.001]. In MPO-AAV group, the expression of neutrophil MPO mRNA was positively correlated with serum MPO-ANCA and MPO levels ( r=0.537, P=0.001 and r=0.358, P=0.032; respectively). Multiple regression analysis suggested that neutrophil MPO mRNA expression was positively correlated with serum MPO-ANCA level ( β=0.695, P=0.006); neutrophil MPO level was negatively correlated with serum MPO-ANCA, MPO and complement C5a levels ( r=-0.335, P=0.046; r=-0.372, P=0.026; r=-0.577, P<0.001; respectively). Further, neutrophil MPO level was negatively correlated with serum complement C5a level ( β=-0.374, P=0.043). BVAS-V3 was positively correlated with MPO mRNA expression in neutrophils, serum MPO-ANCA, MPO and complement C5a ( r=0.598, P<0.001; r=0.599, P<0.001; r=0.537, P=0.001; r=0.415, P=0.012; respectively) and negatively correlated with MPO level in neutrophils ( r=-0.342, P=0.041). In multiple regression analysis it suggested that BVAS-V3 was positively correlated with MPO mRNA expression in neutrophils ( β=0.511, P=0.002). Conclusion:In MPO-AAV patients, MPO synthesis and release in neutrophils are both significantly increased, which might be influenced by serum MPO-ANCA and C5a, respectively. Furthermore, MPO synthesis activity in neutrophils is an independent factor related to disease activity.

Objective To analyze the correlation between clinical features and renal dysfunction in patients of acute lacunar infarction with progressive cerebral microbleeds (CMBs). Methods Two hundred and sixty-five patients with first-episode acute lacunar infarction were selected. The serum creatinine was measured within 24 h of admission and the estimated glomerular filtration rate (eGFR) was calculated. The brain MRI (including gradient-echo images) was examined within 2 d of admission and after 1 years of follow-up, respectively. The progressive CMBs was assessed with microbleeds anatomical rating scale (MARS), and the patients were divided into progressive CMBs group (progressive group, 42 cases) and non progressive CMBs group (non progressive group, 223 cases). The clinical features of 2 groups were compared and the correlation between progressive CMBs and renal dysfunction was analyzed. Results The age, 24 h pulse pressure, incidences of renal dysfunction and CMBs in progressive group were significantly higher than those in non progressive group: (69.8 ± 5.8) years vs. (61.5 ± 4.9) years, (63.3 ± 3.1) mmHg (1 mmHg=0.133 kPa) vs. (51.8 ± 4.2) mmHg, 69.0％(29/42) vs. 39.9％(89/223) and 57.1％(24/42) vs. 25.1％(56/223), and the platelet was significantly lower than that in non-progression group:(168 ± 35) ×109/L vs. (189 ± 40) ×109/L, and there were statistical differences (P<0.05 or<0.01). The Logistic regression analysis result showed that renal dysfunction and CMBs were Independent risk factors of progressive CMBs (OR = 1.571 and 1.054, 95％ CI 1.042 - 2.493 and 1.010 - 1.142, P<0.05). Conclusions The rate of renal dysfunction is higher in patients of acute lacunar infarction with progressive CMBs, and progressive CMBs are associated with renal dysfunction.

Objective To evaluate the effect of resveratrol on the activity of NADPH oxidase during acute lung injury induced by intestinal ischemia-reperfusion (I/R) in rats.Methods Thirty-two pathogenfree healthy female Sprague-Dawley rats,weighing 180-230 g,were divided into 4 groups (n =8 each) using a random number table:sham operation group (Sham group),intestinal I/R group (I/R group),vehicle group (Veh group) and resveratrol group (Res group).Intestinal I/R was produced by occlusion of the superior mesenteric artery for 75 min followed by 4 h of reperfusion.In group Res,resveratrol 15 mg/kg was intraperitoneally injected for 5 consecutive days before establishment of the model and at 15 min before ischemia.The equal volume of vehicle (0.5％ alcohol) was intraperitoneally injected in group Veh.The equal volume of normal saline was intraperitoneally injected in Sham and I/R groups.At the end of reperfusion,the rats were sacrificed and the lung was removed for microscopic examination of pathologic changes which were scored and for determination of wet/dry weight ratio (W/D ratio),malondialdehyde (MDA) content and superoxide dismutase (SOD) activity (by colorimetric method) and expression of NADPH oxidase subunits (gp91phox and p47phox) in lung tissues (by Western blot).Results Compared with group Sham,pathologic scores,W/D ratio and MDA content were significantly increased,the expression of gp91phox and p47phox was up-regulated,and the activity of SOD was decreased in I/R and Veh groups (P＜0.05).Compared with I/R and Veh groups,pathologic scores,W/D ratio and MDA content were significantly decreased,the expression of gp91phox and p47phox was down-regulated,and the activity of SOD was increased in group Res (P＜0.05).Conclusion The mechanism by which resveratrol reduces intestinal I/R-induced acute lung injury is related to inhibiting NADPH oxidase-mediated oxidative stress response of rats.

Objective To investigate the relationship between different topographic locations and neurological deteriorations (ND) in patients with acute new isolated pontine infarction.Methods One hundred sixty-eight patients with acute new isolated pontine infarction during arch 2012 to March 2016 were identified by diffusion weighted imaging (DWI) for retrospective review.Patients were divided into two groups according to their clinical symptoms:patients with ND and patients without ND.According to neuroimaging of DWI,the topographic location of pontine infarction was divided into three types:The upper,middle,and lower ones,and the correlations of ND with risk factors,laboratory examination results,clinical manifestations and different topographic locations were explored by statistical tests.Results Of 168 patients,26.8％ (45/168) were diagnosed with ND,and 73.2％ (123/168) were diagnosed without ND.Univariate analysis showed that there were differences in female ratio [62.2％ (28/45) vs 41.5％ (51/ 123)],smoking ratio [13.3％ (6/45) vs 26.0％ (32/123)],mean length of hospital stay [(22.83 ± 7.12)d vs (19.31 ± 7.65)d],ratio of worse short-term clinical outcomes [77.8％ (35/45) vs 33.3％ (41/123)],and ratio of lower pontine infarction [55.6％ (25/45) vs 26.0％ (32/123)] between two groups (P ＜ 0.05).Logistic regression analysis showed that lower pontine infarction was the independent risk factor of ND (OR =1.953,95％ CI:1.092-3.535,P =0.029).Conclusions Topographic location of lower pons lesions may be reliable predictor of ND in acute new isolated pontine infarction.

ObjectiveTo investigate the influence of hepatitis B virus (HBV) infection on intrahepatic natural killer (NK) cells and innate lymphoid cell 22 (ILC22), and to provide theoretical and experimental bases for clarifying mechanisms of HBV infection in inducing innate immune response. MethodsA total of 10 male BALC/c aged 6-8 weeks were divided into experimental group and control group, with 5 mice in each group. The mice in the experimental group were treated with the hydrodynamic injection of normal saline containing 10 μg plasmids of complete HBV genome (the volume equaled to 9% of the body weight of the mouse) via the caudal vein, and those in the control group were only treated with normal saline. The mice were scarified 4 days later, and intrahepatic lymphocytes (IHLs) were isolated. Flow cytometry was used to determine the proportions of NK cells and ILC22 subset in IHLs, and the t-test was used for comparison between groups. ResultsHydrodynamic injection of the plasmids containing complete HBV genome induced high levels of HBsAg and HBeAg in mice, with an increase in the serum level of alanine aminotransferase. After HBV infection, the experimental group showed a significant increase in the proportion of intrahepatic NK cells compared with the control group (25.90%±4.92% vs 12.98%±2.13%, t=3.811, P=0.003), while there were no significant differences in the proportions of CD127+ and CD127- NK subsets in NK cells between the two groups. Moreover, after HBV infection, the experimental group showed a significant increase in the proportion of intrahepatic NKp46+ILC22 subset compared with the control group (36.05%±6.85% vs 10.22%±3.54%, t=7.372, P＜0001); however, there was no significant difference in the proportion of NKp46-ILC22 between the two groups. ConclusionHBV infection induces increased levels of intrahepatic NK cells and NKp46+ILC22 cells and thus promotes the innate immune response in the liver.

Survivin-D53A (SVV-D53A) is a dominant-negative mutant survivin, which represents a potential promising target for cancer gene therapy. The present study was designed to determine whether SVV-D53A plasmid encapsuled by DOTAP: Chol liposome would have the anti-tumor activity against SPC-A1 lung adenocarcinoma, and to detect the possible mechanisms. In our experiment, SPC-A1 cells were transfected in vitro with SVV-D53A plasmid and examined for protein expression by Western blot, then flow cytometric analysis was used to detect apoptosis. SPC-A1 lung adenocarcinoma xenografts were established in vivo in the nude mice, which received the i. v. administrations of SVV-D53A plasmid/liposome complexes. After mice were sacrificed, the paraffin-embedded tumor tissue sections were used for proliferating cell nuclear antigen (PCNA) expression and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Compared with the control group, the mice treated with SVV-D53A plasmid had an obviously reduced tumor volume, with high level of apoptosis and decreased cell proliferation in tumor tissue. The research results proved that the administration of SVV-D53A plasmid resulted in significant inhibition of SPC-A1 cells both in vitro and in vivo. The functional mechanism is that the anti-tumor response causes and induces tumor cell apoptosis.
Adenocarcinoma ; pathology ; Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Heterografts ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Liposomes ; Lung Neoplasms ; pathology ; Mice ; Mice, Nude ; Neoplasm Proteins ; metabolism ; Neoplasm Transplantation ; Plasmids ; Transfection ; Tumor Burden