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1.
Chinese journal of integrative medicine ; (12): 42-51, 2024.
Article in English | WPRIM | ID: wpr-1010290

ABSTRACT

OBJECTIVE@#To obtain detailed understanding on the gene regulation of natural compounds in altering prognosis of head and neck squamous cell carcinomas (HNSC).@*METHODS@#Gene expression data of HNSC samples and peripheral blood mononuclear cells (PBMCs) of HNSC patients were collected from Gene Expression Omnibus (GEO). Differential gene expression analysis of GEO datasets were achieved by the GEO2R tool. Common differentially expressed gerres (DEGs) were screened by comparing DEGs of HNSC with those of PBMCs. The combination was further analyzed for regulating pathways and biological processes that were affected.@*RESULTS@#Totally 110 DEGs were retrieved and identified to be involved in biological processes related to tumor regulation. Then 102 natural compounds were screened for a combination such that the expression of all 110 commonly DEGs was altered. A combination of salidroside, ginsenoside Rd, oridonin, britanin, and scutellarein was chosen. A multifaceted, multi-dimensional tumor regression was showed by altering autophagy, apoptosis, inhibiting cell proliferation, angiogenesis, metastasis and inflammatory cytokines production.@*CONCLUSIONS@#This study has helped develop a unique combination of natural compounds that will markedly reduce the propensity of development of drug resistance in tumors and immune evasion by tumors. The result is crucial to developing a combinatorial natural therapeutic cocktail with accentuated immunotherapeutic potential.


Subject(s)
Humans , Leukocytes, Mononuclear , Head and Neck Neoplasms/drug therapy , Squamous Cell Carcinoma of Head and Neck/drug therapy , Immunotherapy , Prognosis
2.
Chinese Journal of Lung Cancer ; (12): 56-64, 2024.
Article in Chinese | WPRIM | ID: wpr-1010110

ABSTRACT

BACKGROUND@#With the rise of multicolor flow cytometry, flow cytometry has become an important means to detect the immune microenvironment of lung cancer, but most of them are used to detect the proportion of cell subsets or the function of major cell subsets, and they cannot be detected at the same time. Therefore, a reliable 21-color flow cytometry protocol was established to detect the immune cell subsets in human non-small cell lung cancer (NSCLC) tumor tissues.@*METHODS@#Cell membrane surface antibodies cluster of differentiation (CD)45, CD3, CD19, CD4, CD8, programmed cell death 1 (PD-1), CD39, CD103, CD25, CD127, chemokine receptor 8 (CCR8), CD56, CD11c, human leukocyte antigen (HLA)-DR, CD38, CD27, CD69, CD62L, CD45RA, CCR7 and nucleic acid dye L/D were used to develop the protocol. Firstly, antibody titration experiments, voltage optimization, subtraction of one color staining and single color staining experiments were carried out for each antibody, and after the experimental conditions and detection schemes were determined, the feasibility of the scheme was verified by using peripheral blood mononuclear cells (PBMCs) specimens of six healthy adult volunteers. Tumor tissue samples from 6 NSCLC patients were tested and analyzed.@*RESULTS@#The established 21-color flow cytometry protocol was used to detect the tumor tissue samples of 6 NSCLC patients, and the proportion of each cell subset in lung cancer tissue, as well as the immunophenotype and differentiation of the main cell population, were analyzed.@*CONCLUSIONS@#The successfully established 21-color flow cytometry protocol is suitable for the detection of PBMCs and NSCLC tissue samples, which provides an effective new idea for monitoring the immune microenvironment status in lung cancer.


Subject(s)
Adult , Humans , Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/pathology , Flow Cytometry , Leukocytes, Mononuclear/pathology , Lung/pathology , Tumor Microenvironment
3.
Journal of Biomedical Engineering ; (6): 79-86, 2023.
Article in Chinese | WPRIM | ID: wpr-970676

ABSTRACT

This study aims to clarify host factors of IFN treatment in the treatment of chronic hepatitis B (CHB) patients by screening the differentially expressed genes of IFN pathway CHB patients with different response to interferon (IFN) therapy. Three cases were randomly selected in IFN-responding CHB patients (Rs), non-responding CHB patients (NRs) and healthy participants, respectively. The human type I IFN response RT 2 profiler PCR array was used to detect the expression levels of IFN-related genes in peripheral blood monocytes (PBMCs) from healthy participants and CHB patients before and after Peg-IFN-α 2a treatment. The results showed that more differentially expressed genes appeared in Rs group than NRs group after IFN treatment. Comparing with healthy participants, IFNG, IL7R, IRF1, and IRF8 were downregulated in both Rs and NRs group before IFN treatment; CXCL10, IFIT1, and IFITM1 were upregulated in the Rs; IL13RA1 and IFI35 were upregulated in the NRs, while IFRD2, IL11RA, IL4R, IRF3, IRF4, PYHIN1, and ADAR were downregulated. The expression of IL15, IFI35 and IFI44 was downregulated by 4.09 ( t = 10.58, P < 0.001), 5.59 ( t = 3.37, P = 0.028) and 10.83 ( t = 2.8, P = 0.049) fold in the Rs group compared with the NRs group, respectively. In conclusion, IFN-response-related gene array is able to evaluate IFN treatment response by detecting IFN-related genes levels in PBMC. High expression of CXCL10, IFIT1 and IFITM1 before treatment may suggest satisfied IFN efficacy, while high expression of IL13RA1, IL15, IFI35 and IFI44 molecules and low expression of IFRD2, IL11RA, IL4R, IRF3, IRF4, PYHIN1 and ADAR molecules may be associated with poor IFN efficacy.


Subject(s)
Humans , Healthy Volunteers , Hepatitis B, Chronic/genetics , Immunotherapy , Interleukin-15 , Leukocytes, Mononuclear , Nuclear Proteins , Oligonucleotide Array Sequence Analysis/methods , Interferons/therapeutic use , Treatment Outcome
4.
Journal of Peking University(Health Sciences) ; (6): 975-981, 2023.
Article in Chinese | WPRIM | ID: wpr-1010156

ABSTRACT

OBJECTIVE@#To investigate the regulatory effect of interferon-α (IFN-α) on the apoptosis and killing function of CD56dimCD57+ natural killer (NK) cells in systemic lupus erythematosus (SLE) patients, and to explore the specific mechanism.@*METHODS@#A total of sixty-four newly treated SLE patients and sixteen healthy controls (HC) enrolled in the Second Hospital of Dalian Medical University were selected as the research subjects. And the gene expression levels of molecules related to NK cell-killing function were detected by real-time quantitative polymerase chain reaction. CD56dimCD57+ NK cells were co-cultured with the K562 cells, and the apoptotic K562 cells were labeled with Annexin-Ⅴ and 7-amino-actinomycin D. Peripheral blood mononuclear cells were treated with 20, 40, and 80 μmol/L hydrogen peroxide (H2O2), and treated without H2O2 as control, the expression level of perforin (PRF) was detected by flow cytometry. The concentration of IFN-α in serum was determined by enzyme linked immunosorbent assay. The expression levels of IFN-α receptors (IFNAR) on the surface of CD56dimCD57+ NK cells were detected by flow cytometry, and were represented by mean fluorescence intensity (MFI). CD56dimCD57+ NK cells were treated with 1 000 U/mL IFN-α for 24, 48 and 72 h, and no IFN-α treatment was used as the control, the apoptosis and the expression levels of mitochondrial reactive oxygen species (mtROS) were measured by flow cytometry and represented by MFI.@*RESULTS@#Compared with HC(n=3), the expression levels of PRF1 gene in peripheral blood NK cells of the SLE patients (n=3) were decreased (1.24±0.41 vs. 0.57±0.12, P=0.05). Compared with HC(n=5), the ability of peripheral blood CD56dimCD57+ NK cells in the SLE patients (n=5) to kill K562 cells was significantly decreased (58.61%±10.60% vs. 36.74%±6.27%, P < 0.01). Compared with the control (n=5, 97.51%±1.67%), different concentrations of H2O2 treatment significantly down-regulated the PRF expression levels of CD56dimCD57+ NK cells in a dose-dependent manner, the 20 μmol/L H2O2 PRF was 83.23%±8.48% (n=5, P < 0.05), the 40 μmol/L H2O2 PRF was 79.53%±8.56% (n=5, P < 0.01), the 80 μmol/L H2O2 PRF was 76.67%±7.16% (n=5, P < 0.01). Compared to HC (n=16), the serum IFN-α levels were significantly increased in the SLE patients (n=45) with moderate to high systemic lupus erythematosus disease activity index (SLEDAI≥10) [(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]. Meanwhile, compared with HC (n=6), IFNAR1 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=6) were increased (MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05), and compared with HC (n=6), IFNAR2 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=7) were increased (MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05). Compared with control (n=6), the stimulation of IFN-α (n=6) significantly promoted the apoptosis of CD56dimCD57+ NK cells (20.48%±7.01% vs. 37.82%±5.84%, P < 0.05). In addition, compared with the control (n=4, MFI: 1 049±174.5), stimulation of CD56dimCD57+ NK cells with IFN-α at different times significantly promoted the production of mtROS in a time-dependent manner, 48 h MFI was 3 437±1 472 (n=4, P < 0.05), 72 h MFI was 6 495±1 089 (n=4, P < 0.000 1), but there was no significant difference at 24 h of stimulation.@*CONCLUSION@#High serum IFN-α level in SLE patients may induce apoptosis by promoting mtROS production and inhibit perforin expression, which can down-regulate CD56dimCD57+ NK killing function.


Subject(s)
Humans , Interferon-alpha/metabolism , Perforin/metabolism , Leukocytes, Mononuclear/metabolism , Hydrogen Peroxide/metabolism , Interferon-gamma/metabolism , CD56 Antigen/metabolism , Killer Cells, Natural/metabolism , Lupus Erythematosus, Systemic
5.
Protein & Cell ; (12): 668-682, 2023.
Article in English | WPRIM | ID: wpr-1010765

ABSTRACT

Although the development of COVID-19 vaccines has been a remarkable success, the heterogeneous individual antibody generation and decline over time are unknown and still hard to predict. In this study, blood samples were collected from 163 participants who next received two doses of an inactivated COVID-19 vaccine (CoronaVac®) at a 28-day interval. Using TMT-based proteomics, we identified 1,715 serum and 7,342 peripheral blood mononuclear cells (PBMCs) proteins. We proposed two sets of potential biomarkers (seven from serum, five from PBMCs) at baseline using machine learning, and predicted the individual seropositivity 57 days after vaccination (AUC = 0.87). Based on the four PBMC's potential biomarkers, we predicted the antibody persistence until 180 days after vaccination (AUC = 0.79). Our data highlighted characteristic hematological host responses, including altered lymphocyte migration regulation, neutrophil degranulation, and humoral immune response. This study proposed potential blood-derived protein biomarkers before vaccination for predicting heterogeneous antibody generation and decline after COVID-19 vaccination, shedding light on immunization mechanisms and individual booster shot planning.


Subject(s)
Humans , COVID-19 Vaccines , Leukocytes, Mononuclear , Proteomics , COVID-19/prevention & control , Vaccination , Antibodies , Antibodies, Viral , Antibodies, Neutralizing
6.
Journal of Southern Medical University ; (12): 280-286, 2023.
Article in Chinese | WPRIM | ID: wpr-971526

ABSTRACT

OBJECTIVE@#To investigate the changes in percentage of GATA3+ regulatory T (Treg) cells in patients with allergic rhinitis (AR) and mouse models.@*METHODS@#The nasal mucosa specimens were obtained from 6 AR patients and 6 control patients for detection of nasal mucosal inflammation. Peripheral blood mononuclear cells (PBMC) were collected from 12 AP patients and 12 control patients to determine the percentages of Treg cells and GATA3+ Treg cells. In a C57BL/6 mouse model of AR, the AR symptom score, peripheral blood OVA-sIgE level, and nasal mucosal inflammation were assessed, and the spleen of mice was collected for detecting the percentages of Treg cells and GATA3+ Treg cells and the expressions of Th2 cytokines.@*RESULTS@#Compared with the control patients, AR patients showed significantly increased eosinophil infiltration and goblet cell proliferation in the nasal mucosa (P < 0.01) and decreased percentages of Treg cells and GATA3+ Treg cells (P < 0.05). The mouse models of AR also had more obvious allergic symptoms, significantly increased OVA-sIgE level in peripheral blood, eosinophil infiltration and goblet cell hyperplasia (P < 0.01), markedly lowered percentages of Treg cells and GATA3+ Treg cells in the spleen (P < 0.01), and increased expressions of IL-4, IL-6 and IL-10 (P < 0.05).@*CONCLUSION@#The percentage of GATA3+ Treg cells is decreased in AR patients and mouse models. GATA3+ Treg cells possibly participate in Th2 cell immune response, both of which are involved in the occurrence and progression of AR, suggesting the potential of GATA3+ Treg cells as a new therapeutic target for AR.


Subject(s)
Animals , Mice , Humans , Cytokines/metabolism , Disease Models, Animal , GATA3 Transcription Factor , Inflammation , Leukocytes, Mononuclear/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Nasal Mucosa/metabolism , Ovalbumin , Rhinitis, Allergic/therapy , T-Lymphocytes, Regulatory , Th2 Cells/metabolism
7.
Chinese Medical Journal ; (24): 2694-2705, 2023.
Article in English | WPRIM | ID: wpr-1007701

ABSTRACT

BACKGROUND@#Previous studies have examined the bulk transcriptome of peripheral blood immune cells in acquired immunodeficiency syndrome patients experiencing immunological non-responsiveness. This study aimed to investigate the characteristics of specific immune cell subtypes in acquired immunodeficiency syndrome patients who exhibit immunological non-responsiveness.@*METHODS@#A single-cell transcriptome sequencing of peripheral blood mononuclear cells obtained from both immunological responders (IRs) (CD4 + T-cell count >500) and immunological non-responders (INRs) (CD4 + T-cell count <300) was conducted. The transcriptomic profiles were used to identify distinct cell subpopulations, marker genes, and differentially expressed genes aiming to uncover potential genetic factors associated with immunological non-responsiveness.@*RESULTS@#Among the cellular subpopulations analyzed, the ratios of monocytes, CD16 + monocytes, and exhausted B cells demonstrated the most substantial differences between INRs and IRs, with fold changes of 39.79, 11.08, and 2.71, respectively. In contrast, the CD4 + T cell ratio was significantly decreased (0.39-fold change) in INRs compared with that in IRs. Similarly, the ratios of natural killer cells and terminal effector CD8 + T cells were also lower (0.37-fold and 0.27-fold, respectively) in the INRs group. In addition to several well-characterized immune cell-specific markers, we identified a set of 181 marker genes that were enriched in biological pathways associated with human immunodeficiency virus (HIV) replication. Notably, ISG15 , IFITM3 , PLSCR1 , HLA-DQB1 , CCL3L1 , and DDX5 , which have been demonstrated to influence HIV replication through their interaction with viral proteins, emerged as significant monocyte marker genes. Furthermore, the differentially expressed genes in natural killer cells were also enriched in biological pathways associated with HIV replication.@*CONCLUSIONS@#We generated an atlas of immune cell transcriptomes in HIV-infected IRs and INRs. Host genes associated with HIV replication were identified as markers of, and were found to be differentially expressed in, different types of immune cells.


Subject(s)
Humans , Acquired Immunodeficiency Syndrome , Transcriptome/genetics , HIV , HIV Infections/genetics , Leukocytes, Mononuclear/metabolism , CD4-Positive T-Lymphocytes/metabolism , Virus Replication , Membrane Proteins/metabolism , RNA-Binding Proteins/metabolism
8.
Chinese Journal of Biotechnology ; (12): 347-358, 2023.
Article in Chinese | WPRIM | ID: wpr-970379

ABSTRACT

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4+ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4+ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.


Subject(s)
Cattle , Animals , Cytokines , BCG Vaccine/metabolism , Tumor Necrosis Factor-alpha/metabolism , Interleukin-2 , Flow Cytometry/methods , Chemokine CXCL10/metabolism , Leukocytes, Mononuclear , CD4-Positive T-Lymphocytes/metabolism , Tuberculosis , Antibodies, Monoclonal/metabolism
9.
Chinese Journal of Oncology ; (12): 56-63, 2023.
Article in Chinese | WPRIM | ID: wpr-969806

ABSTRACT

Objective: To investigate the effect of long non-coding RNA urothelial carcinoma-associated 1 (UCA1) gene on the proliferation, migration, apoptosis and immune escape of endometrial cancer cells and its molecular mechanism. Methods: Endometrial cancer tissues and adjacent normal tissues of patients with endometrioid adenocarcinoma who underwent total or partial hysterectomy in Henan Provincial People's Hospital from 2017 to 2019 were collected. The expressions of UCA1 and miR-204-5p were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the cell proliferation, migration and apoptosis were detected by cell counting kit 8 (CCK8) method, Transwell method, flow cytometry, and dual-luciferase reporter assay was used to explore the target relationship between UCA1 and miR-204-5p. HEC-1A-sh-NC or HEC-1A-sh-UCA1 cells were co-cultured with peripheral blood mononuclear cells (PBMC) or cytokine-induced killer cells in vitro to explore the role of UCA1 in immune escape. Results: The expression level of UCA1 in endometrial cancer tissue (17.08±0.84) was higher than that in adjacent normal endometrial tissue (3.00±0.37), and the expression level of miR-204-5p (0.98±0.16) was lower than that in adjacent normal endometrial tissue (2.00±0.20, P<0.05). Pearson correlation analysis showed that the expression of miR-204-5p was negatively correlated with the expression of UCA1 (r=-0.330, P=0.030). The expressions of UCA1 and miR-204-5p were associated with the International Federation of Gynecology and Obstetrics stage of endometrial cancer, lymph node metastasis and vascular invasion (P<0.05). The relative ratio of absorbance (0.58±0.11) and the number of cell migration [(199.68±18.44)] in the sh-UCA1 group were lower than those in the sh-NC group (1.24±0.17 and 374.76±24.83), respectively. The apoptosis rate of sh-UCA1 group [(28.64±7.80)%] was higher than that of sh-NC group [(14.27±4.38)%, P<0.05]. After different ratios of effector cells and target cells were cultured, the cell survival rate of HEC-1A-sh-UCA1 group was lower than that of HEC-1A-sh-NC group, and the difference was statistically significant (P<0.05). UCA1 had a binding site for miR-204-5p. The relative ratio of absorbance (1.74±0.08) and the number of cell migration (426.00±18.00) cells in the UCA1+ anti-miR-204-5p group were higher than those in the control group [1.00±0.03 and (284.00±8.00) cells, respectively]. The apoptosis rate of UCA1+ anti-miR-204-5p group [(5.42±0.93)%] was lower than that of control group [(14.82±1.48)%, P<0.05]. HEC-1A-sh-UCA1 cells could induce higher interferon gamma (IFN-γ) expression when co-cultured with PBMC, and the levels of IFN-γ expression in PHA group and PHA+ pre-miR-204-5p group cells were 2.42±0.49 and 1.88±0.26, which were higher than that in the PHA+ pre-NC group (0.85±0.10, P<0.05). When co-cultured with cytokine-induced killer cells (different ratios) in vitro, the HEC-1A-sh-UCA1 group and the HEC-1A-pre-miR-204-5p group had lower survival rates than that in the HEC-1A-pre-miR-204-5p group. In the HEC-1A-pre-NC group, the differences were statistically significant (P<0.05). Conclusion: UCA1/miR-204-5p may play an important role in human endometrial cancer.


Subject(s)
Female , Humans , MicroRNAs/metabolism , RNA, Long Noncoding/genetics , Leukocytes, Mononuclear , Carcinoma, Transitional Cell , Antagomirs , Cell Line, Tumor , Urinary Bladder Neoplasms , Cell Proliferation , Endometrial Neoplasms/genetics , Apoptosis/genetics , Cell Movement/genetics , Gene Expression Regulation, Neoplastic
10.
Journal of Experimental Hematology ; (6): 699-706, 2023.
Article in Chinese | WPRIM | ID: wpr-982119

ABSTRACT

OBJECTIVE@#To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1).@*METHODS@#Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC50) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay.@*RESULTS@#The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC50 of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G2/M phase also decreased, while G0/G1 phase increased in cell cycle.@*CONCLUSION@#The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.


Subject(s)
Humans , Leukocytes, Mononuclear/metabolism , Apoptosis , Cell Line, Tumor , Lymphoma , Thymidine Kinase/pharmacology , Doxorubicin/pharmacology , Cell Division , RNA, Messenger/genetics
11.
Journal of Experimental Hematology ; (6): 553-561, 2023.
Article in Chinese | WPRIM | ID: wpr-982094

ABSTRACT

OBJECTIVE@#To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies.@*METHODS@#Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy.@*RESULTS@#After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05).@*CONCLUSION@#The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.


Subject(s)
Humans , Fetal Blood , Killer Cells, Natural , T-Lymphocytes , Leukocytes, Mononuclear/metabolism , Cell Proliferation , CD56 Antigen/metabolism
12.
Chinese Journal of Cellular and Molecular Immunology ; (12): 633-637, 2023.
Article in Chinese | WPRIM | ID: wpr-981910

ABSTRACT

Objective To identify the relationship between nephritis activity, autophagy and inflammation in patients with SLE. Methods Western blot analysis was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3) and P62 in peripheral blood mononuclear cells (PBMCs) of SLE patients with lupus nephritis and non-lupus nephritis patients. Tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) in the serum of SLE patients were determined by ELISA. The correlation between LC3II/LC3I ratio and SLE disease activity score (SLEDAI), urinary protein, TNF-α and IFN-γ levels was analyzed by Pearson method. Results The expression of LC3 was increased and P62 was decreased in SLE patients. TNF-α and IFN-γ were increased in the serum of SLE patients. LC3II/LC3I ratio was positively correlated with SLEDAI (r=0.4560), 24 hour urine protein (r=0.3753), IFN-γ (r=0.5685), but had no correlation with TNF-α (r=0.04 683). Conclusion Autophagy is found in PBMCs of SLE, and the autophagy is correlated with renal damage and inflammation in patients with lupus nephritis.


Subject(s)
Humans , Tumor Necrosis Factor-alpha/metabolism , Leukocytes, Mononuclear/metabolism , Autophagy-Related Proteins/metabolism , Lupus Nephritis/urine , Kidney , Interferon-gamma/metabolism , Inflammation/metabolism , Lupus Erythematosus, Systemic/metabolism
13.
Chinese Medical Journal ; (24): 815-821, 2023.
Article in English | WPRIM | ID: wpr-980878

ABSTRACT

BACKGROUND@#Immunotherapies such as adoptive immune cell infusion and immune-modulating agents are widely used for cancer treatment, and the concomitant symptoms, including cytokine release syndrome (CRS) or immune-related adverse events (irAEs), are frequently reported. However, clinical manifestations induced by mismatched donor granulocyte colony-stimulating factor mobilized peripheral blood mononuclear cell (GPBMC) infusion in patients receiving microtransplant (MST) have not yet been well depicted.@*METHODS@#We analyzed 88 cycles of mismatched GPBMC infusion in patients with acute myeloid leukemia receiving MST and 54 cycles of chemotherapy without GPBMC infusion as a comparison. Clinical symptoms and their correlation with clinical features, laboratory findings, and clinical response were explored.@*RESULTS@#Fever (58.0% [51/88]) and chills (43.2% [38/88]) were the significant early-onset symptoms after GPBMC infusion. Patients possessing less human leukocyte antigen-matching loci with the donor or those with unrelated donors experienced more chills (3 [2-5] loci vs. 5 [3-5] loci, P  = 0.043 and 66.7% [12/18] vs. 37.1% [26/70], P  = 0.024). On the other hand, those with decreased CD4 + /CD8 + T-cell ratio developed more fever (0.8 [0.7-1.2] vs. 1.4 [1.1-2.2], P  = 0.007). Multivariable analysis demonstrated that younger patients experienced more fever (odds ratio [OR] = 0.963, 95% confidence interval [CI]: 0.932-0.995, P  = 0.022), while patients with younger donors experienced more chills (OR = 0.915, 95% CI: 0.859-0.975, P  = 0.006). Elevated ultra-sensitive C-reactive protein levels in the absence of cytokine storm were observed following GPBMC infusion, which indicated mild and transient inflammatory response. Although no predictive value of infusion-related syndrome to leukemia burden change was found, the proportion of host pre-treatment activated T cells was positively correlated with leukemia control.@*CONCLUSIONS@#Mismatched GPBMC infusion in MST induced unique infusion-related symptoms and laboratory changes, which were associated with donor- or recipient-derived risk factors, with less safety and tolerance concerns than reported CRS or irAEs.


Subject(s)
Humans , Leukocytes, Mononuclear , Hematopoietic Stem Cell Transplantation/adverse effects , Leukemia, Myeloid, Acute/therapy , Unrelated Donors , Granulocyte Colony-Stimulating Factor , Graft vs Host Disease
14.
Chinese Journal of Preventive Medicine ; (12): 923-928, 2023.
Article in Chinese | WPRIM | ID: wpr-985497

ABSTRACT

To establish and identify induced pluripotent stem cells (iPSCs) derived from patients with Aicardi-Goutières syndrome (AGS) with TREX1 gene 667G>A mutation, and obtain a specific induced pluripotent stem cell model for Aicardi-Goutières syndrome (AGS-iPSCs). A 3-year-old male child with Aicardi-Goutieres syndrome was admitted to Zhongshan People's Hospital in December 2020. After obtaining the informed consent of the patient's family members, 5 ml peripheral blood samples from the patient were collected, and mononuclear cells were isolated. Then,the peripheral blood mononuclear cells(PBMCs) were transduced with OCT3/4, SOX2, c-Myc and Klf4 by using Sendai virus, and PBMCs were reprogrammed into iPSCs. The pluripotency and differentiation ability of the cells were identified by cellular morphological analysis, real-time PCR, alkaline phosphatase staining (AP), immunofluorescence, teratoma formation experiments in mice. The results showed that the induced pluripotent stem cell line of Aicardi-Goutieres syndrome was successfully constructed and showed typical embryonic stem-like morphology after stable passage, RT-PCR showed mRNA expression of stem cell markers, AP staining was positive, OCT4, SOX2, NANOG, SSEA4, TRA-1-81 and TRA-1-60 pluripotency marker proteins were strongly expressed. In vivo teratoma formation experiments showed that iPSCs differentiate into the ectoderm (neural tube like tissue), mesoderm (vascular wall tissue) and endoderm (glandular tissue). Karyotype analysis also confirmed that iPSCs still maintained the original karyotype (46, XY). In conclusion, induced pluripotent stem cell line for Aicardi-Goutières syndrome was successfully established using Sendai virus, which provided an important model platform for studying the pathogenesis of the disease and for drug screening.


Subject(s)
Animals , Male , Mice , Child, Preschool , Cell Differentiation , Induced Pluripotent Stem Cells/pathology , Leukocytes, Mononuclear , Teratoma/pathology
15.
Chinese Critical Care Medicine ; (12): 586-591, 2023.
Article in Chinese | WPRIM | ID: wpr-982637

ABSTRACT

OBJECTIVE@#To investigate the effect of circulating exosomes (EXO) on T cell function in patients with sepsis.@*METHODS@#Plasma EXO were obtained by ultracentrifugation from 10 patients with sepsis admitted to the emergency intensive care unit of Guangdong Provincial People's Hospital Affiliated to Southern Medical University. Transmission electron microscopy observation, nanoparticle tracking analysis (NTA), and Western blotting were used to detect EXO markers to identify their characteristics. Furthermore, peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of 5 healthy volunteers, primary T cells were sorted by magnetic beads and expanded in vitro. After 24 hours of intervention with different doses (0, 1, 2.5, 5, 10 mg/L) of circulating EXO in patients with sepsis, T-cell activity was assessed using a cell counting kit-8 (CCK-8). The expression of T cell activation indicators CD69 and CD25 were observed using flow cytometry. Additional evaluations were performed on immunosuppressive indicators including the expression of programmed cell death 1 (PD-1) in CD4+ T cells and the proportion of regulatory T cell (Treg).@*RESULTS@#The identification results confirmed that the successful isolation of EXO from the plasma of sepsis patients. The expression level of circulating EXO in sepsis patients was higher than that in healthy control group (mg/L: 48.78±5.14 vs. 22.18±2.25, P < 0.01). After 24 hours of intervention with 5 mg/L of plasma EXO from sepsis patients, T cells activity began to show suppression [(85.84±0.56)% vs. (100.00±0.00)%, P < 0.05]. As the dosage increased, after 24 hours of intervention with 10 mg/L of EXO, T cells activity was significantly suppressed [(72.44±2.36)% vs. (100.00±0.00)%, P < 0.01]. Compared with the healthy control group, after T cells intervention with plasma EXO from sepsis patients, the expression of early activation marker CD69 was significantly reduced [(52.87±1.29)% vs. (67.13±3.56)%, P < 0.05]. Meanwhile, there was an upregulation of PD-1 expression in T cells [(57.73±3.06)% vs. (32.07±0.22)%, P < 0.01] and an increase in the proportion of Treg [(54.67±1.19)% vs. (24.60±3.51)%, P < 0.01]. However, the expression of the late activation marker CD25 remained stable [(84.77±3.44)% vs. (85.93±2.32)%, P > 0.05].@*CONCLUSIONS@#Circulating EXO in sepsis patients induce T cell dysfunction, which may be a novel mechanism lead to immunosuppression in sepsis.


Subject(s)
Humans , Leukocytes, Mononuclear , Exosomes/metabolism , Programmed Cell Death 1 Receptor/metabolism , T-Lymphocytes, Regulatory/metabolism , Sepsis/metabolism
16.
Journal of Central South University(Medical Sciences) ; (12): 829-836, 2023.
Article in English | WPRIM | ID: wpr-982353

ABSTRACT

OBJECTIVES@#This study aims to investigate the genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells (PBMCs) in patients with systemic sclerosis (SSc) with interstitial lung disease (ILD), and to analyze the effects of DNA methylation on Wnt/β-catenin and chemokine signaling pathways.@*METHODS@#PBMCs were collected from 19 patients with SSc (SSc group) and 18 healthy persons (control group). Among SSc patients, there were 10 patients with ILD (SSc with ILD subgroup) and 9 patients without ILD (SSc without ILD subgroup). The genome-wide DNA methylation and gene expression level were analyzed by using Illumina 450K methylation chip and Illumina HT-12 v4.0 gene expression profiling chip. The effect of DNA methylation on Wnt/β-catenin and chemokine signal pathways was investigated.@*RESULTS@#Genome-wide DNA methylation analysis identified 71 hypermethylated CpG sites and 98 hypomethylated CpG sites in the SSc with ILD subgroup compared with the SSc without ILD subgroup. Transcriptome analysis distinguished 164 upregulated genes and 191 downregulated genes in the SSc with ILD subgroup as compared with the SSc without ILD subgroup. In PBMCs of the SSc group, 35 genes in Wnt/β-catenin signaling pathway were hypomethylated, while frizzled-1 (FZD1), mitogen-activated protein kinase 9 (MAPK9), mothers against DPP homolog 2 (SMAD2), transcription factor 7-like 2 (TCF7L2), and wingless-type MMTV integration site family, member 5B (WNT5B) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of dickkopf homolog 2 (DKK2), FZD1, MAPK9 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). In PBMCs of the SSc group, 38 genes in chemokine signaling pathway were hypomethylated, while β-arrestin 1 (ARRB1), C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 16 (CXCL16), FGR, and neutrophil cytosolic factor 1C (NCF1C) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of ARRB1, CXCL10, CXCL16 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05).@*CONCLUSIONS@#There are differences in DNA methylation and transcriptome profiles between SSc with ILD and SSc without ILD. The expression levels of multiple genes in Wnt/β- catenin and chemokine signaling pathways are upregulated, which might be associatea with the pathogenesis of SSc.


Subject(s)
Humans , DNA Methylation , Transcriptome , beta Catenin , Leukocytes, Mononuclear , Ligands , DNA , RNA, Messenger/genetics
17.
Journal of Experimental Hematology ; (6): 1797-1803, 2023.
Article in Chinese | WPRIM | ID: wpr-1010040

ABSTRACT

OBJECTIVE@#To investigate the effect of miR-125b on T cell activation in patients with aplastic anemia (AA) and its molecular mechanism.@*METHODS@#A total of 30 AA patients were enrolled in department of hematology, Binzhou Medical University Hospital from January 2018 to October 2021, as well as 15 healthy individuals as healthy control (HC) group. Peripheral blood mononuclear cells (PBMCs) were isolated, in which the levels of miR-125b and B7-H4 mRNA were detected by RT-qPCR. Immunomagnetic beads were used to separate naive T cells and non-naive T cells from AA patients and healthy people to detect the levels of miR-125b and B7-H4 mRNA. Lentivirus LV-NC inhibitor and LV-miR-125b inhibitor were transfected into cells, and T cell activation was detected by flow cytometry. The dual-luciferase reporter gene assay was used to detect the targetting relationship between miR-125b and B7-H4. RT-qPCR and Western blot were used to detect the levels of miR-125b, CD40L, ICOS, IL-10 mRNA and B7-H4 protein.@*RESULTS@#Compared with HC group, the expression of miR-125b was up-regulated but B7-H4 mRNA was down-regulated in PBMCs of AA patients (P <0.05), and the proportions of CD4+CD69+ T cells and CD8+CD69+ T cells in PBMCs of AA patients were higher (P <0.05). The expression of miR-125b was significantly up-regulated but B7-H4 mRNA was down-regulated in both naive T cells and non-naive T cells of AA patients (P <0.05), and non-naive T cells was more significant than naive T cells (P <0.05). Compared with NC inhibitor group, the expression of miR-125b was significantly decreased, the expression level of CD69 on CD4+ and CD8+ T cells in PBMCs was also significantly decreased, while the luciferase activity was significantly increased after co-transfection of miR-125b inhibitor and B7-H4-3'UTR-WT in the miR-125b inhibitor group (P <0.05). Compared with NC inhibitor group, the mRNA and protein levels of B7-H4 were significantly increased in the miR-125b inhibitor group (P <0.05). Compared with miR-125b inhibitor+shRNA group, the expression levels of CD69 on CD4+ and CD8+ T cells were significantly increased, and the levels of CD40L, ICOS and IL-10 mRNA were also significantly increased in the miR-125b inhibitor+sh-B7-H4 group (P <0.05).@*CONCLUSION@#MiR-125b may promote T cell activation by targetting B7-H4 in AA patients.


Subject(s)
Humans , Anemia, Aplastic/genetics , CD40 Ligand/metabolism , Interleukin-10 , Leukocytes, Mononuclear/metabolism , Luciferases , MicroRNAs/genetics , RNA, Messenger/metabolism , Lymphocyte Activation , T-Lymphocytes/metabolism
18.
Journal of Experimental Hematology ; (6): 1523-1530, 2023.
Article in Chinese | WPRIM | ID: wpr-1010003

ABSTRACT

OBJECTIVE@#To explore the effect of human bone marrow mesenchymal stem cells (MSCs) with ectopic high OCT4 expression on T-cell proliferation, activation and secretion in vitro.@*METHODS@#Peripheral blood mononuclear cells were isolated from healthy children. Anti-CD3 and anti-CD28 monoclonal antibodies were used to activate T lymphocytes, which were stimulated by interleukin (IL)-2 for one week in vitro. Then MSCs with ectopic high OCT4 expression (MSC-OCT4) were co-cultured with activated T lymphocytes. After one week of co-culture, the supernatant was collected and the levels of Th1/Th2 cytokines [IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ] were determined by flow cytometry. The lymphocytes after one week of co-culture were collected and counted by Countstar software. After the proportions of activated/inactivated T cell subsets were determined by flow cytometry, the absolute lymphocyte counts were calculated and expressed as mean ± standard deviation.@*RESULTS@#Compared with control T cell alone culture group, the proliferation of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells were significantly inhibited in MSC group and MSC-OCT4 group. Compared with MSC, MSC-OCT4 could inhibit CD3+CD8+ T cell proliferation better (P =0.049), and mainly inhibited early T cell activation. Compared with control T cell alone culture group, the levels of IL-2 and INF-γ were significantly down-regulated both in MSC group and MSC-OCT4 group.After co-culture with T cells for one week, the level of IL-6 significantly increased in MSC group and MSC-OCT4 group compared with that before co-culture. Compared with control MSC group, MSC-OCT4 group had higher viable cell numbers after 1 week of co-culture (P =0.019), and could resist the inhibition of proliferation by higher concentration of mitomycin C.@*CONCLUSION@#Both MSC and MSC-OCT4 can inhibit the proliferation and activation of IL-2-stimulated T cells in vitro. After overexpression of OCT4, MSC has better proliferation ability in vitro and can inhibit the proliferation of CD3+CD8+ T cells more effectively, which may have a better and more lasting immunosuppressive ability to regulate the balance of Th1/Th2.


Subject(s)
Child , Humans , Bone Marrow Cells , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation , Cells, Cultured , Interleukin-2 , Interleukin-6/metabolism , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation , Mesenchymal Stem Cells , Tumor Necrosis Factor-alpha/metabolism
19.
Chinese Journal of Cellular and Molecular Immunology ; (12): 1100-1107, 2023.
Article in Chinese | WPRIM | ID: wpr-1009461

ABSTRACT

Objective To investigate the expressions of IL-18, IL-18 binding protein isoform a (IL-18BPa) and IL-18 receptor α (IL-18Rα) in blood CD4+ Th2 cells of patients with allergic rhinitis (AR) and the effects of allergens on their expressions. Methods Blood samples of AR patients and healthy control subjects (HCs) were collected. Peripheral blood mononuclear cells (PBMCs) and CD4+ T cells sorted by immunomagnetic beads were stimulated by crude extract of Artemisia sieversiana wild allergen (ASWE), Platanus pollen (PPE) and house dust mite extract (HDME). Flow cytometry was used to detect the expression of IL-18, IL-18BPa and IL-18Rα in CD4+ Th2 cells, and BioPlex was used to detect the level of plasma IL-4 and analyze its correlation with the proportion of IL-18+ Th2 cells. Results Compared with HCs, the proportion of IL-18+ cells was increased in Th2 cells of AR patients; MFI of IL-18 was increased, while that of IL-18Rα was decreased. Moreover, allergens induced IL-18 and IL-18Rα expression in sorted CD4+ Th2 cells of HCs and induced IL-18Rα in that of AR patients. Additionally, elevated plasma IL-4 level was found in AR patients, which was moderately correlated with the percentage of IL-18+ Th2 cells. Conclusion Allergens may be involved in the pathogenesis of AR by inducing expression of IL-18 in peripheral blood CD4+ Th2 cells.


Subject(s)
Humans , Th2 Cells , Interleukin-18/metabolism , Up-Regulation , Leukocytes, Mononuclear/metabolism , Interleukin-4/metabolism , Rhinitis, Allergic/metabolism , Allergens , Cytokines/metabolism
20.
Chinese Journal of Cellular and Molecular Immunology ; (12): 1094-1099, 2023.
Article in Chinese | WPRIM | ID: wpr-1009460

ABSTRACT

Objective To investigate the effect of calcitonin gene-related peptide (CGRP) on the regulation of group 2 innate lymphoid cells (ILC2) in the peripheral blood of patients with allergic rhinitis (AR). Methods Peripheral blood mononuclear cells (PBMCs) were extracted from normal healthy individuals and AR patients, then stimulated with CGRP, interleukin 33 (IL-33) and CGRP combined with IL-33 for 3 days, with blank stimulus as control. The percentage of ILC2 in the four groups was measured by flow cytometry. After being sorted, ILC2 was given to CGRP, IL-33 and CGRP combined with IL-33 stimulation for 3 days, with blank stimulus as control. The percentage of IL-5 and IL-13 positive cells in ILC2 was detected by flow cytometry, and the levels of IL-5 and IL-13 in ILC2 supernatant were measured by ELISA. Results The percentage of ILC2 in the peripheral blood of AR patients was significantly higher than that of the control group. The levels of IL-5+ILC2 and IL-13+ILC2 were significantly increased by IL-33 single stimulation after culturing PBMCs. After adding IL-33 combined with CGRP stimulation, the levels of IL-5+ILC2 and IL-13+ILC2 in PBMCs were significantly reduced; after CGRP single stimulation, the levels of IL-5+ILC2 and IL-13+ILC2 in PBMCs were further decreased. After ILC2 was sorted and cultured, the levels of IL-5+ILC2 and IL-13+ILC2 showed significant increase after IL-33 single stimulation. The levels of IL-5+ILC2 and IL-13+ILC2 were decreased by IL-33 and CGRP co-stimulation, and they were further reduced after CGRP single stimulation. Compared to IL-33 single stimulation, IL-5 and IL-13 levels dropped significantly due to the IL-33 and CGRP co-stimulation. The levels of IL-5 and IL-13 were further reduced by CGRP single stimulation. Conclusion CGRP inhibits the proliferation and activation of peripheral blood ILC2 in AR and exert anti-inflammatory effects in AR.


Subject(s)
Humans , Calcitonin Gene-Related Peptide/pharmacology , Leukocytes, Mononuclear , Immunity, Innate , Interleukin-33/pharmacology , Interleukin-13 , Lymphocytes , Interleukin-5/pharmacology , Rhinitis, Allergic , Cell Proliferation
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