Objective:To establish a new method and platform for screening, identifying, and exploring a new strategy for anti-hepatitis B immunotherapy based on hepatitis B virus (HBV)-specific TCR.Methods:Peripheral blood mononuclear cells were isolated from patients with acute hepatitis B. CD3 +CD8 +CD137 +T single cells were sorted out after stimulation with the HBsAg peptide library. The α and β chains in TCRs of single cells were amplified by PCR. TCR double-chain pairing and lentiviral packaging were performed through high-throughput sequencing. Re-infected Jurkat-76-NFAT-GFP cells and the cell lines stably expressing TCR were screened. HBsAg peptide library and immortalized B lymphocytes co-cultured with J76N-TCR were used to screen HBsAg-specific TCRs. K562 cell lines stably expressing HLA-A*24:02 were established to determine epitope peptide by screening A*24:02-restricted TCR. The screened TCRs were replaced with mouse C regions and packaged with lentiviruses. Functional validation was performed on healthy human CD4 +T and CD8 +T lymphocytes following infection. Results:Stable TCR-expressing cell lines were successfully prepared based on single-cell TCRαβ double-chain amplification and pairing technology. Twenty-one TCRs were screened using immortalized B lymphocytes, resulting in nine possible HLA-A*24:02-restricted HBsAg-specific TCRs. Further screening with K562-A2402 resulted in six A*24:02-restricted HBsAg-specific TCRs with identically recognized epitope peptide. The functional determination of the two TCR clones revealed their specific recognition function for target cells expressing HBsAg.Conclusion:HLA-A*24:02-restricted HBsAg-specific TCR with recognition function for target cells expressing HBsAg was successfully obtained based on the new experimental technology system, laying an important foundation for further exploration of antiviral immunotherapy based on HBV-specific TCR.

OBJECTIVE To explore the relationship between peripheral blood interferon γ(IFN-γ)/interleukin-4(IL-4)and the short-term prognosis of children with Mycoplasma pneumoniae pneumonia(MPP),and to analyze the influencing factors for the short-term prognosis of children with MPP.METHODS A total of 170 children with MPP admitted to the hospital from Jan.2021 to Jan.2024 were selected(MPP group).Based on the condition 28 days after treatment,they were divided into a poor prognosis group(n=49)and a good prognosis group(n=121).Clinical data of the children were collected,and the levels of peripheral blood interleukin-4(IL-4)and inter-feron-γ(IFN-γ)were measured to calculate the IFN-γ/IL-4 ratio.Multivariate Logistic regression model was used to analyze the factors affecting the short-term prognosis of children with MPP.RESULTS In the poor prognosis group,the duration of antibiotic use,the proportion of pleural effusion,the proportion of extrapulmonary compli-cations,and the levels of peripheral blood IL-4 and IFN-γ were higher than those in the good prognosis group,while the IFN-γ/IL-4 ratio was lower than that in the good prognosis group(P＜0.05).Logistic regression re-sults showed that persistent fever,prolonged antibiotic use and the occurrence of extrapulmonary complications were risk factors for the short-term prognosis of children with MPP(P＜0.05),and a high IFN-γ/IL-4 ratio was a protective factor(P＜0.05).CONCLUSION Persistent fever,prolonged antibiotic use and extrapulmonary com-plications are risk factors for the short-term prognosis of children with MPP,and high IFN-γ/IL-4 values is a pro-tective factor.

OBJECTIVE To explore the relationship between peripheral blood interferon γ(IFN-γ)/interleukin-4(IL-4)and the short-term prognosis of children with Mycoplasma pneumoniae pneumonia(MPP),and to analyze the influencing factors for the short-term prognosis of children with MPP.METHODS A total of 170 children with MPP admitted to the hospital from Jan.2021 to Jan.2024 were selected(MPP group).Based on the condition 28 days after treatment,they were divided into a poor prognosis group(n=49)and a good prognosis group(n=121).Clinical data of the children were collected,and the levels of peripheral blood interleukin-4(IL-4)and inter-feron-γ(IFN-γ)were measured to calculate the IFN-γ/IL-4 ratio.Multivariate Logistic regression model was used to analyze the factors affecting the short-term prognosis of children with MPP.RESULTS In the poor prognosis group,the duration of antibiotic use,the proportion of pleural effusion,the proportion of extrapulmonary compli-cations,and the levels of peripheral blood IL-4 and IFN-γ were higher than those in the good prognosis group,while the IFN-γ/IL-4 ratio was lower than that in the good prognosis group(P＜0.05).Logistic regression re-sults showed that persistent fever,prolonged antibiotic use and the occurrence of extrapulmonary complications were risk factors for the short-term prognosis of children with MPP(P＜0.05),and a high IFN-γ/IL-4 ratio was a protective factor(P＜0.05).CONCLUSION Persistent fever,prolonged antibiotic use and extrapulmonary com-plications are risk factors for the short-term prognosis of children with MPP,and high IFN-γ/IL-4 values is a pro-tective factor.

Objective:To establish a new method and platform for screening, identifying, and exploring a new strategy for anti-hepatitis B immunotherapy based on hepatitis B virus (HBV)-specific TCR.Methods:Peripheral blood mononuclear cells were isolated from patients with acute hepatitis B. CD3 +CD8 +CD137 +T single cells were sorted out after stimulation with the HBsAg peptide library. The α and β chains in TCRs of single cells were amplified by PCR. TCR double-chain pairing and lentiviral packaging were performed through high-throughput sequencing. Re-infected Jurkat-76-NFAT-GFP cells and the cell lines stably expressing TCR were screened. HBsAg peptide library and immortalized B lymphocytes co-cultured with J76N-TCR were used to screen HBsAg-specific TCRs. K562 cell lines stably expressing HLA-A*24:02 were established to determine epitope peptide by screening A*24:02-restricted TCR. The screened TCRs were replaced with mouse C regions and packaged with lentiviruses. Functional validation was performed on healthy human CD4 +T and CD8 +T lymphocytes following infection. Results:Stable TCR-expressing cell lines were successfully prepared based on single-cell TCRαβ double-chain amplification and pairing technology. Twenty-one TCRs were screened using immortalized B lymphocytes, resulting in nine possible HLA-A*24:02-restricted HBsAg-specific TCRs. Further screening with K562-A2402 resulted in six A*24:02-restricted HBsAg-specific TCRs with identically recognized epitope peptide. The functional determination of the two TCR clones revealed their specific recognition function for target cells expressing HBsAg.Conclusion:HLA-A*24:02-restricted HBsAg-specific TCR with recognition function for target cells expressing HBsAg was successfully obtained based on the new experimental technology system, laying an important foundation for further exploration of antiviral immunotherapy based on HBV-specific TCR.

Clinically,pancreatic cancer is a highly aggressive tumor,and neurotropic growth is an important biological feature of pancreatic cancer.Nerve invasion brings great pain burden to patients,and it seriously affects the quality of life and the will to survive of patients.The"three-step analgesia principle"for the management of cancer pain proposed by World Health Organization(WHO)is a traditional therapeutic regimen for cancer pain.However,because of its obvious toxic side effects,poor efficacy,easy addiction,easy drug resistance,non-standard medication of clinical physicians,etc.,the"three-step analgesia principle"is unable to meet the needs of the patient's condition..In recent years,with the development of interventional technology and the development of extensive clinical trials,the interventional means,which is regarded as the"fourth step"of cancer pain management,has achieved great clinical effect,it includes various therapeutic methods and imaging-guided techniques such as neural destruction(denervation),125I particle implantation,patient-controlled analgesic pump technology,implantation of intrathecal drug infusion system,etc.,and clinical practice has proved that these techniques have significant clinical efficacy and they can provide a convenient,safe and effective treatment method for HCC patients.

Objective·To explore the heterogeneity in single-cell genomics between human-induced pluripotent stem cell(iPSC)-derived macrophages(IPSDM)and human peripheral blood-derived macrophages(PBDM).Methods·iPSCs were differentiated into IPSDMs in vitro using a feeder-free and serum-free protocol.The expression of cluster of differentiation antigen 14(CD14)and monocyte-macrophage marker genes in IPSDMs was analyzed using flow cytometry and real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR),respectively.Single-cell sequencing was then performed on IPSDMs.Simultaneously,the single-cell sequencing dataset GSE126085 was downloaded from the Gene Expression Omnibus database as a reference dataset for PBDMs.Sequencing data for both IPSDMs and PBDMs were processed and analyzed using the seurat package in R software,with PBDMs annotated using the singleR package.A reference dataset was constructed with highly variable genes from PBDMs,and the highly variable genes of IPSDMs were projected onto the PBDM dataset using the scmap package to infer IPSDMs cell identities based on variable gene similarity.IPSDMs were annotated using cell-type annotation tools and referenced against relevant studies.The expression distribution of macrophage marker genes was compared between IPSDMs and PBDMs.Differentially expressed genes(DEGs)between IPSDMs and PBDMs were identified using the seurat package,and their potential biological functions were explored through Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses.Results·Suspended IPSDMs were obtained after 29 d of in vitro differentiation.Flow cytometry and RT-qPCR confirmed that approximately 23.1%of IPSDMs expressed CD14,and IPSDMs exhibited higher expression of monocyte-macrophage marker genes compared to the U-937 cell line.All cells in the PBDM dataset were annotated as macrophages.After constructing a scmap reference dataset using PBDMs,59.8%of IPSDMs were annotated as macrophages through mapping their highly variable genes to the PBDM dataset.The remaining 40.2%of IPSDM cells could not be matched to the variable genes of PBDMs.Further manual annotation of IPSDMs revealed a composition of 97.15%macrophages,2.71%hematopoietic precursor cell-like cells,and 0.14%dendritic cells.When comparing the expression of macrophage markers,both IPSDMs and PBDMs highly expressed the classical macrophage marker CD68 gene,while IPSDMs exhibited higher expression of markers associated with tissue-resident macrophages.GO analysis of DEGs showed enrichment in the molecular functions such as ubiquitin-like protein ligase binding,cellular components such as the nuclear speck and nuclear envelope,and biological processes such as the regulation of translation.KEGG pathway enrichment indicated that the DEGs between IPSDMs and PBDMs might be related to various intracellular pathogen infections.Conclusion·Human IPSDMs and PBDMs exhibit certain similarities and heterogeneity at the single-cell transcriptional level.Transcriptomic analysis indicates that IPSDMs display more characteristics of tissue-resident macrophages.The DEGs between IPSDMs and PBDMs are potentially associated with intracellular infection immunity.

Objective·To explore the heterogeneity in single-cell genomics between human-induced pluripotent stem cell(iPSC)-derived macrophages(IPSDM)and human peripheral blood-derived macrophages(PBDM).Methods·iPSCs were differentiated into IPSDMs in vitro using a feeder-free and serum-free protocol.The expression of cluster of differentiation antigen 14(CD14)and monocyte-macrophage marker genes in IPSDMs was analyzed using flow cytometry and real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR),respectively.Single-cell sequencing was then performed on IPSDMs.Simultaneously,the single-cell sequencing dataset GSE126085 was downloaded from the Gene Expression Omnibus database as a reference dataset for PBDMs.Sequencing data for both IPSDMs and PBDMs were processed and analyzed using the seurat package in R software,with PBDMs annotated using the singleR package.A reference dataset was constructed with highly variable genes from PBDMs,and the highly variable genes of IPSDMs were projected onto the PBDM dataset using the scmap package to infer IPSDMs cell identities based on variable gene similarity.IPSDMs were annotated using cell-type annotation tools and referenced against relevant studies.The expression distribution of macrophage marker genes was compared between IPSDMs and PBDMs.Differentially expressed genes(DEGs)between IPSDMs and PBDMs were identified using the seurat package,and their potential biological functions were explored through Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses.Results·Suspended IPSDMs were obtained after 29 d of in vitro differentiation.Flow cytometry and RT-qPCR confirmed that approximately 23.1%of IPSDMs expressed CD14,and IPSDMs exhibited higher expression of monocyte-macrophage marker genes compared to the U-937 cell line.All cells in the PBDM dataset were annotated as macrophages.After constructing a scmap reference dataset using PBDMs,59.8%of IPSDMs were annotated as macrophages through mapping their highly variable genes to the PBDM dataset.The remaining 40.2%of IPSDM cells could not be matched to the variable genes of PBDMs.Further manual annotation of IPSDMs revealed a composition of 97.15%macrophages,2.71%hematopoietic precursor cell-like cells,and 0.14%dendritic cells.When comparing the expression of macrophage markers,both IPSDMs and PBDMs highly expressed the classical macrophage marker CD68 gene,while IPSDMs exhibited higher expression of markers associated with tissue-resident macrophages.GO analysis of DEGs showed enrichment in the molecular functions such as ubiquitin-like protein ligase binding,cellular components such as the nuclear speck and nuclear envelope,and biological processes such as the regulation of translation.KEGG pathway enrichment indicated that the DEGs between IPSDMs and PBDMs might be related to various intracellular pathogen infections.Conclusion·Human IPSDMs and PBDMs exhibit certain similarities and heterogeneity at the single-cell transcriptional level.Transcriptomic analysis indicates that IPSDMs display more characteristics of tissue-resident macrophages.The DEGs between IPSDMs and PBDMs are potentially associated with intracellular infection immunity.

Objective:To establish a disk (CD) microfluidic chip detection platform for the rapid detection of CALR-1 and CALR-2 mutations in patients with cerebral infarction, and summarize its clinical application value.Methods:Based on microfluidic technology and loop mediated isothermal amplification technology, a CD microfluidic chip detection platform for simultaneous detection of CALR-1 and CALR-2 gene mutations were established, and the sensitivity, specificity, repeatability and accuracy of the platform were verified. A total of 124 patients with cerebral infarction treated in Huashan Hospital, Shanghai Medical College, Fudan University from November 2019 to March 2021 were prospectively selected into the experimental group; and 80 healthy subjects were included in the control group. The CALR-1 and CALR-2 gene mutations in anticoagulant peripheral blood samples were detected by the CD microfluidic chip. Each chip could detect 4 samples at the same time and synchronously detect 3 indexes of each sample. The detection results could be obtained after isothermal amplification for 40 min. At the same time, sequencing method was used to verify the test results, and the consistency of the results of the two detection methods was compared.Results:Using this CD microfluidic chip platform, the synchronous amplification of 3 indexes in the sample could be completed within 40 min without the need of thermal circulation, and the whole detection process of the sample could be completed within 60 min. For samples with a high concentration of target nucleic acid, typical positive signals could be visualized after amplification for 10 min, and the test results would be available within 30 minutes after receiving the samples. The detection sensitivity of CD microfluidic chip method for CALR-1 and CALR-2 mutation load concentration was 1.0% and 0.5% respectively. Nonspecific amplification was not observed for the non-target nucleic acid samples, indicating the high specificity of this method. The coincidence rates of intra and inter batch repeatability were 100% (20/20) respectively. Two samples with CALR gene mutation were found in the cerebral infarction group, both of which were CALR-1 mutations (L367fs*46). There was no CALR-1 or CALR-2 mutation in the control group. The detection results of CD microfluidic chip method were completely consistent with the sequencing verification results (100% [204/204]).Conclusions:The CD microfluidic chip method could be used for the detection of CALR-1 and CALR-2 gene mutations in clinical samples of patients with cerebral infarction. This method has the advantages of high detection sensitivity, good detection specificity, fast detection speed and high detection flux, which is helpful to clarify the etiology of patients with cerebral infarction.

ObjectiveTo investigate the effect of the expression of HBcAg in hepatocytes on the serum level of HBcAb and seroconversion of HBeAg after antiviral therapy with nucleos(t)ide analogues (NUCs). MethodsSerum samples and liver tissue paraffin sections were collected from 101 chronic hepatitis B (CHB) patients who received antiviral therapy with NUCs in Nanfang Hospital and Panyu Central Hospital from January 2015 to June 2018. ELISA was used to measure the serum level of HBcAb, and immunohistochemistry was used to measure the expression of HBcAg in the liver. The GEO database (GSE96851) was analyzed to obtain differentially expressed genes in the liver of patients with HBcAg-positive hepatitis. The two-independent-samples t test was used for comparison of continuous data between two groups; the multiple-independent-samples nonparametric Kruskal-Wallis H test was used for comparison of continuous data between multiple groups, and Dunnett method was used for further comparisons; the chi-square test was used for comparison of categorical data between groups. ResultsThe expression pattern of HBcAg in hepatocytes was classified as absent expression, nuclear expression, cytoplasmic expression, and nuclear/cytoplasmic expression, and according to expression level, HBcAg expression was classified as grades Ⅰ, Ⅱ, Ⅲ, and Ⅳ expression. HBeAg seroconversion rates after 96 weeks of antiviral therapy were 5.88%, 16.67%, 22.73%, and 24.24%, respectively, in the patients with absent expression, nuclear expression, cytoplasmic expression, and nuclear/cytoplasmic expression (χ2=4753, P=0.037), and HBeAg seroconversion rates after 96 weeks of antiviral therapy were 5.88%, 13.04%, 27.59%, and 26.67%, respectively, in the patients with grade Ⅰ, Ⅱ, Ⅲ, and Ⅳ expression (χ2=6.580, P=0.016). There were significant differences in the serum levels of HBcAb-IgM and total HBcAb between the patients with absent expression, nuclear expression, cytoplasmic expression, and nuclear/cytoplasmic expression of HBcAg (HBcAb-IgM: H=9.760, P=0.021; total HBcAb: H=21.46, P＜0.001), and there were also significant differences in the serum levels of HBcAb-IgM and total HBcAb between the patients with grade Ⅰ, Ⅱ, Ⅲ, and IV expression of HBcAg (HBcAb-IgM: H=18.80, P＜0.001; total HBcAb: H=26.03, P＜0.001). The analysis of differentially expressed genes in the liver showed that the expression of antibody-related genes was upregulated in the liver of patients with HBcAg-positive acute liver failure. ConclusionThe expression pattern and level of HBcAg in the cytoplasm of hepatocytes are associated with serum HBcAb, and the measurement of HBcAg may help to predict the efficacy of antiviral therapy with NUCs.

Acute respiratory failure due to acute hypoxemia is the major manifestation in severe coronavirus disease 2019 (COVID-19). Rational and effective respiratory support is crucial in the management of COVID-19 patients. High-flow nasal cannula (HFNC) has been utilized widely due to its superiority over other non-invasive respiratory support techniques. To avoid HFNC failure and intubation delay, the key issues are proper patients, timely application and improving compliance. It should be noted that elder patients are vulnerable for failed HFNC. We applied HFNC for oxygen therapy in severe and critical ill COVID-19 patients and summarized the following experiences. Firstly, to select the proper size of nasal catheter, to locate it at suitable place, and to confirm the nose and the upper respiratory airway unobstructed. Secondly, an initial ow of 60 L/min and 37℃ should be given immediately for patients with obvious respiratory distress or weak cough ability; otherwise, low-level support should be given first and the level gradually increased. Thirdly, to avoid hypoxia or hypoxemia, the treatment goal of HFNC should be maintained the oxygen saturation (SpO) above 95% for patients without chronic pulmonary disease. Finally, patients should wear a surgical mask during HFNC treatment to reduce the risk of virus transmission through droplets or aerosols.
Aged ; Betacoronavirus ; isolation & purification ; Cannula ; Coronavirus Infections ; therapy ; Humans ; Oxygen ; administration & dosage ; Pandemics ; Pneumonia, Viral ; therapy