Objective To investigate how culture duration affects the expression of immune membrane proteins in human monocyte-derived dendritic cells (DCs) and their exosomes (DEXs). Methods Human monocytes were induced with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) to differentiate into DCs and were subsequently matured with tumor necrosis factor α(TNF-α). Exosomes were isolated by ultracentrifugation, and DEXs were identified by transmission electron microscopy and Amnis imaging flow cytometry, which were also used to quantify the expression of immune membrane proteins on DCs and DEXs. Results On the 10th day of culture, DCs displayed high surface expression of CD11c, CD80, CD86, major histocompatibility complex class I (MHC-I), and MHC-II. Expression peaked at day 18(CD11c: 78.66%±20.33%, CD80: 76.41%±10.02%, CD86: 96.43%±0.43%, MHC-I: 84.71%±2.96%, MHC-II: 80.01%±7.03%). After day 24, the overall expression showed a declining trend, with statistically significant differences observed for all markers except CD80 and MHC-II. By day 30, 80% of the DCs still expressed CD80, CD86, and MHC-II. The expression of immune membrane proteins on DEX surfaces also reached its peak on day 18, followed by an overall decline with prolonged culture time, with statistically significant differences observed for all markers except CD80. Correlation analysis revealed a significant positive relationship between the expression levels of immune membrane proteins on DC and DEX surfaces (CD11c: r=0.98; CD80: r=0.65; CD86: r=0.82; MHC-I: r=0.86; MHC-II: r=0.93). Conclusion Human monocyte-derived DCs in vitro express high expression of immune membrane proteins and maintain stable expression over a specific period. The exosomes secreted by these cells similarly demonstrate high surface expression of immune membrane proteins, with temporal trends aligned with those of the parent DCs.
Humans ; Dendritic Cells/immunology* ; Exosomes/immunology* ; Monocytes/metabolism* ; Cells, Cultured ; Time Factors ; B7-1 Antigen/metabolism* ; Membrane Proteins/immunology* ; Cell Culture Techniques/methods* ; B7-2 Antigen/metabolism* ; Cell Differentiation ; CD11c Antigen/metabolism* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology*

OBJECTIVES: To investigate the associations between Tau protein deposition and brain biochemical metabolites detected by proton magnetic resonance spectroscopy (¹H-MRS) in patients with advanced Alzheimer's disease (AD). METHODS: From April, 2022 to December, 2024, 64 Tau-positive AD patients and 29 healthy individuals underwent ¹⁸F-APN-1607 PET/MR and simultaneously acquired multi-voxel ¹H-MRS in the Department of Nuclear Medicine, Nanjing First Hospital. Visual analysis and voxel-based analysis of PET/MR data were performed to investigate the Tau protein deposition patterns in AD patients. Valid voxels within the ¹H-MRS field of view were selected, and their standardized uptake value ratio (SUVr) in PET and metabolite levels of N-acetylaspartate (NAA), choline (Cho), creatine (Cr), NAA/Cr, and Cho/Cr were recorded. The Tau-positive (Tau⁺) voxels and Tau-negative (Tau^-) voxels of the AD patients were compared for PET and ¹H-MRS parameters, and the correlations between the metabolites and Tau PET SUVr within Tau⁺ voxels were analyzed. RESULTS: Significant Tau protein deposition were observed in the AD patients, involving mainly the bilateral frontal lobes (30.07%), parietal lobes (29.96%), temporal lobes (21.07%), and occipital lobes (15.89%). A total of 1422 valid voxels in AD group (including 994 Tau⁺ and 428 Tau^- voxels) and 814 voxels in the control group were selected. The AD patients showed significantly decreased NAA level and increased SUVr compared with the control group (P<0.05). Subgroup analyses revealed that Tau⁺ voxels had higher SUVr and lower Cr and Cho/Cr than Tau^- voxels (P<0.05). Compared with the control group, Tau⁺ voxels exhibited higher SUVr and lower Cr (P<0.05), while Tau^- voxels showed lower NAA (P=0.004). No significant differences were found in Cho or NAA/Cr among the subgroups (P>0.05). Within Tau⁺ voxels, NAA, Cho, and Cr were negatively correlated with SUVr (P<0.001). CONCLUSIONS The patients with progressive AD have significant Tau protein deposition in the brain, which is correlated with alterations in metabolite levels. Decreased NAA is more prominent in early or pre-tau deposition stages, while Cr changes is more significant in the regions with Tau protein deposition, suggesting the potential of NAA and Cr as biomarkers for Tau protein deposition in AD for disease monitoring and treatment evaluation.
Humans ; Alzheimer Disease/diagnostic imaging* ; Aspartic Acid/metabolism* ; tau Proteins/metabolism* ; Creatine/metabolism* ; Brain/metabolism* ; Biomarkers/metabolism* ; Positron-Emission Tomography ; Male ; Female ; Proton Magnetic Resonance Spectroscopy ; Choline/metabolism* ; Aged ; Middle Aged

Purpose To investigate the diagnostic value of 18F-FDOPA PET/CT imaging and semi-quantitative analysis platform for the diagnosis of Parkinson's disease(PD).Materials and Methods There were 27 healthy controls and 56 clinically diagnosed PD patients,including 33 early PD(Hoehn-Yahr class Ⅰ-Ⅱ)and 23 advanced PD(Hoehn-Yahr class Ⅲ-Ⅳ),underwent 18F-FDOPA PET imaging in Nanjing First Hospital,Nanjing Medical University were consecutively enrolled from January 2018 to December 2019.The striatal to occipital ratio(SORs)in radioactivity was calculated by HERMES BRASS platform,thereby completing the semi-quantitative analysis of the brain based on regions of interest and observing the asymmetry of the striatal subregions in early-stage PD and late-stage PD patients.Using artificial intelligence techniques to perform principal component analysis on the SORs of the striatal subregions in PD group and healthy control group,the degree of data aggregation and the distinguishability between groups were observed.Results The SORs was significantly reduced in the whole caudate,anterior,posterior putamen and striatum of advanced PD patients(t=9.02-11.72,P<0.000 1).The area under the curve was 0.952,0.973,0.995 and 0.982,respectively.Compared with the healthy control group,the loss of striatal asymmetry index(mean)in each subregion of the striatum in early PD group was caudate(7.61±5.50)%,anterior putamen(11.43±8.97)%,posterior putamen(17.17±11.63)%,and whole striatum(10.65±7.46)%,respectively.The uptake of 18F-FDOPA in the striatum of PD patients was significantly reduced,and the most obvious loss of early PD patients was contralateral posterior putamen,with a decrease of 34%.Conclusion The platform semi-quantitative analysis of 18F-FDOPA PET/CT images provides objective semi-quantitative values for early diagnosis and differential diagnosis of PD.Asymmetry in the striatum,especially in the putamen,may be an important parameter for early diagnosis of PD..

Objective To analyze the dynamic changes to disease activity,colonic inflammation,histopathology,and serum cytokine levels in mice with chronic ulcerative colitis(UC).Methods For UC induction,2.5％dextran sodium sulfate solution was provided ad libitum for 5 days,and to model remission,tap water was supplied for another 5 days in one induction cycle.Disease activity index(DAI),colon length,and pathological changes to colon tissue were determined.The levels of myeloperoxidase(MPO)in colon tissue and of cytokines such as IL-1 β in serum and colon were detected.Results During the three cycles,disease activity was aggravated and colon length shortened in mice during the induction periods,both of which were relieved during the remission periods.The blood appeared was observed in the stool was earlier in cycles 2 and 3.The number of mice with stool blood increased,and their body weight decreased by a small amount briefly,then recovered rapidly.The degree of histopathological damage to the colon and MPO content in cycles 1 and 3 increased in the induction periods and decreased in the remission periods,with the magnitude of change smaller than that of the change in DAI values;and they increased in the remission period of cycle 2.During induction,the spleen index and serum levels of IL-1β,IL-6,and IL-17A increased continuously and were higher than those in the control group at the end of the experiment.Levels of TNF-α were increased in the induction periods and decreased in the remission periods,and the trend in IL-10 change was similar to that of TNF-α.TGF-β content increased and then decreased and was higher than that in the control group at the end of cycle 3.The colon contents of IL-1β,IL-6,and IL-17A showed similar trends of increasing and then decreasing,but there was no significant change in colon TNF-α.The concentration of IL-10 decreased during the induction periods and increased during the remission periods.Conclusions During the induction of chronic UC in mice,the symptoms of hematochezia and systemic inflammatory reactions gradually increased,and the mice showed an increase in tolerance and ability to resist mortality,weight loss,and histopathological injury to the colon.The onset and remission of colonic histopathological damage lags behind symptomatic changes,and there is a gradual shift in colonic inflammation to a pattern dominated by polymorphonuclear neutrophils(PMN)activation.

COVID-19 ; Coronavirus Nucleocapsid Proteins ; Humans ; Nucleocapsid Proteins ; Phosphoproteins ; SARS-CoV-2

Objective:To establish a rapid method for detecting the activity of 2019-nCoV2.Methods:The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection system was screened after propidium monoazide (PMA) treatment and exposure to heat-inactivated 2019-nCoV samples, and the PMA pretreatment conditions were optimized to establish the 2019-nCoV PMA-RT-qPCR detection method . The established method was used to detect virus inactivated by different temperatures and chlorine-containing disinfectant, to evaluate its effect in detecting virus activity.Results:For the PMA-RT-qPCR assay, 200 μmol/L of PMA, 10 min of incubation time, 15 min of exposure time, and the CDC ORF1ab detection system were selected; there was no significant difference in the result of PMA-RT-qPCR and direct RT-qPCR for the active virus; the Ct values of PMA-RT-qPCR for virus inactivated by 95 ℃ and chlorine-containing disinfectant were higher than that of control groups at different dilutions; only partial dilutions of 70 ℃ and 56 ℃ heat-inactivated virus had higher Ct values than control groups. Conclusions:The established PMA-RT-qPCR for 2019-nCoV activity detection method has a good detection effect on the virus inactivated by 95 ℃ heat and chlorine disinfectant, and provides an auxiliary means for judging the infectivity of the virus in the sample.

Objective: To analyze the clinical manifestations and results of etiological examinations of 17 elderly patients with influenza A (H1N1) viral pneumonia, and to understand the clinical features of pneumonia and molecular characteristics of influenza A (H1N1) virus infection in the elderly. Methods: The elderly patients with pneumonia who were hospitalized in the Department of Respiratory Diseases of Nanjing First Hospital from January 2018 to March were enrolled. The cases were confirmed by nucleic acid examination for influenza virus and the clinical data were collected. After the amplification of the whole genome of influenza virus, the high throughput sequencing and bioinformatics analysis were performed. Results: The mean age of the 17 enrolled patients was 73.8±10.8. All of them had at least 1 underlying disease, and 7 cases had co-infection. Respiratory symptoms and fever were the most prominent clinical manifestations. Lesions in both lungs were found in 76.5% of the patients. The result of high throughput sequencing showed that all the viruses were highly homologous to the vaccine strain, and the HA gene belonged to the 6B.1 subgroup. Furthermore, three variations of antigenic locus (H138Y, S74R and S164T in HA) and a drug-resistant variation (H275Y in NA) were detected in the circulating strains. Conclusions Elderly patients with influenza A (H1N1) virus pneumonia often have underlying diseases and are prone to have co-infection. The molecular characteristics of the virus and the variation of key amino acid loci should be closely monitored in order to provide evidence for epidemic prevention and clinical antiviral treatment.

Adaptor Proteins, Signal Transducing ; chemistry ; genetics ; Crystallography, X-Ray ; Humans ; Nuclear Proteins ; chemistry ; genetics ; Protein Structure, Quaternary ; Protein Structure, Secondary ; Structure-Activity Relationship ; X-linked Nuclear Protein ; chemistry ; genetics

To prepare the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 for use as RNA standard and control in RNA virus detection, the genes coding the coat protein and maturase of E.coli bacteriophage MS2 were amplified by PCR and then cloned into prokaryotic expression vector pET32a to construct the intermediate vector pET32a-MS2. In addition, the gene sequences coding hemagglutinin (HA), neuraminidase(NA) and M protein of the H5N1 virus were also cloned separately to the down-stream of plasmid pET32a-MS2, thus constructing the prokaryotic expression vectors pET32a-NS2-HA, pET32a-MS2-NA and pET32a-MS2-M. These recombinant plasmids were then transformed separately to E.coli BL21(DE3) with induction by IPTG. to express the virus-like particles. The virus-like particles observed under electron microscopy were identified by RT-PCR ,while their stability was confirmed by real-time RT-PCR. In this way, the virus-like particles were successively constructed and identified through PCR amplification, enzymolysis identification and sequencing analysis. These virus-like particles observed under electron microscopy appeared to be circular in shape with a diameter of about 50 nm. Their stability was proved to be rather good. From these observations, it is apparent that these virus-like particles can be used as RNA standard and quality control in the detection of avian influenza virus H5N1.

Objective To prepare and study the immunogenicity of hepatitis B virus surface anti-gen (HBsAg)-tetanus toxoid (TT) conjugate vaccine. Methods Tr was activated by cyangen bromide and reacted with adipic acid dihydrazide, then HBsAg-TT conjugate was prepared by carbediimide. Conjugate, HBsAg or hepatitis B vaccine was injected subcutaneously into mice. Anti-HBsAg and HBsAg-specific T cell response elicited by these immunogens were assayed. Results New HBsAg-TT conjugate elicited higher levels of anti-HBsAg and HBsAg positive conversion rates after the immunization than did HBsAg alone or hepatitis B vaccine. Conjugate induced mesdy antibodies of the IgG2a subclass, while HBsAg alone or hepa-titis B vaccine mainly elicited anti-HBsAg in the IgG1 subclass. The number of IFN-γand IL-2 secreting T cells induced by conjugate was also significantly higher than that did by HBsAg or hepatitis B vaccine. Con-clusion This study indicated new HBsAg-TT conjugate can induce both stronger humoral and TH1 type of cellular immune response.