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Objective:To determine the expression of transglutaminase 2 (TGM2) in peripheral blood mononuclear cells (PBMCs) from patients with atopic dermatitis (AD), and to analyze its correlation with AD-related inflammatory factors and disease severity.Methods:A total of 29 AD patients and 15 healthy controls were collected from the First Affiliated Hospital of Fujian Medical University from July 2020 to January 2021. Ten milliliters of peripheral blood samples were collected from each subject, so was the clinical information, including age, gender, course of disease, eosinophil counts, basophil counts, total IgE levels, Scoring AD index (SCORAD), etc. PBMCs were isolated by density gradient centrifugation. Fluorescence-based quantitative PCR was performed to determine the mRNA expression of TGM2 and AD-related inflammatory factors (interleukin [IL]-1β, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17, thymic stromal lymphopoietin [TSLP], P2RX7 [purinergic receptor P2X, ligand-gated ion channel, 7], etc.) in PBMCs from 29 AD patients and 15 healthy controls, and flow cytometry to determine TGM2 protein expression on PBMCs. Mann-Whitney U test was used to analyze differences between groups, and Spearman correlation analysis to evaluate the correlation. Results:The relative mRNA expression of TGM2 in PBMCs did not differ between the AD group and control group ( M[ Q1, Q3]: 0.509 [0.325, 0.958] vs. 0.475 [0.328, 1.051], U = 210.50, P = 0.872). Compared with the control group, the AD group showed significantly decreased IL-4 mRNA expression (0.171[0.049, 0.449] vs. 0.824 [0.397, 1.378], P < 0.001), but significantly increased mRNA expression of IL-8 and IL-13 ( P = 0.011, 0.006, respectively). Spearman correlation analysis showed that the mRNA expression level of TGM2 in PBMCs was positively correlated with the mRNA expression levels of IL-4 and P2RX7 in the AD group ( rs = 0.42, 0.40, P = 0.024, 0.034, respectively), while there were no correlations between TGM2 mRNA expression and AD severity-related indicators (all P>0.05), such as age (21[16, 29] years), course of disease (4[1,10] years), eosinophil counts (0.33[0.18, 0.65] × 10 9/L), basophil counts (0.04[0.03, 0.06] × 10 9/L], SCORAD scores (60.5[46.98, 66.13] points), and serum total IgE levels (373 [40, 1 815] IU/ml). The relative protein expression levels of TGM2 on the surface of PBMCs did not differ between the AD group and control group (54.9 [47.6, 62.8] vs. 55.55 [51.5, 60.25], U = 112.00, P = 0.922) ], and no correlations were observed between the protein expression of TGM2 on PBMCs and AD severity-related indicators in the AD group (all P > 0.05) . Conclusion:No significant differences were observed in TGM2 mRNA expression in PBMCs or TGM2 protein expression on the surface of PBMCs between the AD patients and healthy controls, and there were no correlations between the TGM2 mRNA and protein expression and AD severity.
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Objective:To investigate the immunoregulatory effects of lentivirus-mediated microRNA (miR)-31-5p overexpression on peripheral blood T helper cell 17 (Th17) in a rabbit model of autoimmune dry eye.Methods:The miR-31-5p recombinant lentiviral vector was constructed.Lentivirus overexpressing miR-31-5p and its control virus were packaged.The concentration measurement and lentiviral titer determination were carried out.A rabbit model of autoimmune dry eye was established and the peripheral blood mononuclear cells (PBMC) of the rabbits were isolated.PBMC infected with miR-31-5p and negative control lentivirus particles were assigned as the miR-31-5p overexpression group and control group, respectively.The miR-31-5p expression level was detected using quantitative real-time PCR (qRT-PCR). Then PBMC in the two groups were co-cultured with γ-ray irradiated lacrimal gland epithelial cells.The expressions of Th17 cell related transcription factor retinoic acid-receptor-related orphan receptor C (RORC) and interleukin-17 (IL-17) mRNA, IL-1β, IL-6 and IL-23 were determined by qRT-PCR.The IL-17 protein expression level was detected by Western blot.The use and care of animals complied with Regulation for the Administration of Affair Concerning Experiment Animals by State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Tianjin Medical University Eye Hospital (No.TJYY20201221036).Results:The construction of the miR-31-5p recombinant lentiviral vector was verified by DNA sequencing.The lentiviral titer of lentivirus overexpressing miR-31-5p and control lentivirus particles was 3.82×10 7 TU/ml and 3.50×10 7 TU/ml, respectively.The miR-31-5p relative expression level of PBMC was significantly increased in miR-31-5p overexpression group in comparison with control group, showing a statistically significant difference ( t=-9.696, P<0.001). When PBMC were co-cultured with lacrimal gland epithelial cells in vitro, the relative expression levels of RORC and IL-17 mRNA in miR-31-5p overexpression group were 0.33±0.03 and 0.28±0.09, which were significantly decreased in comparison with 1.00±0.00 and 1.00±0.00 in control group, with statistically significant differences between them ( t=46.256, 13.810; both at P<0.05). The relative expression level of IL-17 protein in miR-31-5p overexpression group was significantly reduced than control group ( t=4.977, P=0.008). The relative expression levels of IL-1β, IL-6 and IL-23 mRNA were significantly lower in miR-31-5p overexpression group than control group ( t=220.076, 6.641, 13.271; all at P<0.05). Conclusions:The overexpression of miR-31-5p can inhibit the Th17-immune response via down-regulating the expression of IL-6, IL-1β and IL-23.
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Objective To investigate the predictive and diagnostic value of absolute value and function of different lymphocyte subsets in evaluating the risk of early viral infection after kidney transplantation. Methods Ninety-five kidney transplant recipients were enrolled in this prospective observational cohort study, and divided into the stable group (n=77) and infection group (n=18) according to postoperative immune status. Peripheral blood samples were collected for flow cytometry before operation, and 2 weeks, 1 month, 2 months and 6 months after operation. The dynamic changes of the absolute values of CD4+T cells, CD8+T cells and natural killer (NK) cells were compared between two groups. The function of lymphocyte subsets in two groups was evaluated by detecting the proportion of interferon (IFN)-γ+CD4+T cells, IFN-γ+CD8+T cells and IFN-γ+NK cells. The value of the absolute values and function of lymphocyte subsets in predicting and diagnosing viral infection in the early stage after kidney transplantation was evaluated. Results During viral infection, the absolute values of CD4+T cells, CD8+T cells and NK cells in the infection group were at a relatively low level. At 2 months after operation, the absolute values of CD4+T cells and NK cells in the infection group were lower than those in the stable group. At 6 months after operation, the absolute values of CD4+T cells and CD8+T cells in the infection group were significantly lower compared with those in the stable group (all P < 0.05). During viral infection, the proportion of IFN-γ+CD4+T cells, IFN-γ+CD8+T cells and IFN-γ+NK cells in the infection group were all at a relatively low level, especially that of IFN-γ+CD8+T cells decreased most significantly. At postoperative 2 months, the proportion of IFN-γ+CD8+T cells and IFN-γ+NK cells in the infection group was significantly higher than those in the stable group. At 6 months after operation, the proportion of IFN-γ+CD4+T cells and IFN-γ+CD8+T cells in the infection group was significantly higher than those in the stable group (all P < 0.05). Logistic regression analysis showed that the increasing proportion of IFN-γ+CD8+T cells and IFN-γ+NK cells was correlated with the increasing risk of viral infection at 2 months after operation (both P < 0.05). The receiver operating characteristic (ROC) curve demonstrated that the diagnostic value of absolute values of lymphocyte subsets combined with IFN-γ secretion function for viral infection in the immunocompromised recipients was significantly higher than that of absolute values of lymphocyte subsets alone (P < 0.05). Conclusions Dynamic monitoring of the changes of absolute values and function of lymphocyte subsets provides critical reference value for the prediction, diagnosis and medication guidance of viral infection.
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Currently, extracellular concentration measurement is the major approach of therapeutic drug monitoring (TDM) of clinical immunosuppressant in organ transplantation. Its correlation with the efficacy of immunosuppressant remains elusive. With widespread application of liquid chromatography, the detection technology of intracellular concentration of immunosuppressant is gradually mature. Theoretically, it may more accurately reflect the efficacy of immunosuppressant due to that the level of drug exposure in target cells can be directly measured. In this article, the history and present situation of the determination of intracellular concentration of immunosuppressant were summarized, and the association between the determination methods of intracellular concentration of immunosuppressant and drug efficacy was emphatically analyzed. Detection of intracellular concentration of immunosuppressant possesses better application value in clinical practice, which is worthy of promotion in clinical settings.
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Occult hepatitis C infection (OCI) is defined as HCV RNA not detected in serum or plasma but in hepatocytes and peripheral blood mononuclear cells (PBMCs). OCI exists in general population and voluntary blood donors, and its infectivity and risk of transmission by transfusion has been confirmed. HCV RNA in PBMCs could not be detected in plasma or serum by blood screening in transfusion services, neither by enzyme-linked immunosorbent assay nor by nucleic acid amplification testing. OCI has become a potential threat to transfusion safety, therefore effective detection technologies and transmission blocking strategies need to be further developed.
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Objective: To detect the expression levels of miR-194-5p and magnesium transporter protein 1 (MagTl) in peripheral blood mononuclear cells (PBMCs) of the patients with chronic hepatitis B virus (HBV) infection, and to investigate their possible clinical significances. Methods: The clinical materials of 40 healthy subjects (healthy control group). 40 cases of asymptomatic hepatitis B carriers (HBV carrier group). 40 patients with mild-moderate chronic hepatitis B (mild-moderate CHB group) and 40 patients with severe chronic hepatitis B (severe CHB group) were collected. The peripheral blood of the subjects in various groups was collected and the PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation. The expression levels of miR-194-5p and MagTl mRNA in PBMCs of the subjects in various groups were detected by RT-PCR assay, the expression levels of MagTl protein in PBMCs of the subjects in various groups were detected by Western blotting method, the levels of Mg in peripheral blood and PBMCs of the subjects in various groups were detected, and the expression levels of CD6+ T lymphocyte surface molecules programmed death receptor 1 (PD-1) and natural killer cell receptor 2 group D molecule (NKG2D) in PBMCs of the subjects in various groups were analyzed by flow cytometry. The miR-194-5p mimic (miR-194-5p mimic group) and miR-jNC (miR-NC group) were transfected into the 293T cells, and then the dual luciferase reporter gene assay was used to detect the luciferase activities in 293T cells in two groups. Results: Compared with healthy control group, the expression levels of MagTl mRNA and protein, the expression levels of CD8 + T lymphocyte surface molecule NKG2D and the levels of Mg in PBMCs of the patients in HBV carrier group, mild-moderate CHB group and severe CHB group were decreased ( P<-0. 05) . the expression levels of miR-194-5p mRNA in PBMCs and CD8 + T lymphocyte surface molecule PD-1 of the patients were increased (P<.0. 05). Compared with HBV carrier group and mild-moderate CHB group, the expression levels of MagTl mRNA and protein, the expression level of CD8+ T lymphocyte surface molecule NKG2D and the level of Mg in PBMCs of the patients in severe CHB group were decreased ( P<0. 05). and the expression levels of miR-194-5p mRNA in PBMCs and CD8 + T lymphocyte surface molecule PD-1 of the patients were increased (P<0. 05). Compared with HBV carrier group, the expression levels of MagTl mRNA and protein, the expression level of CD8+ T lymphocyte surface molecule NKG2D and the level of Mg in PBMCs of the patients in mild-moderate CHB group were decreased ( P
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Objective To analyze the differences in the expression levels of the lncRNA MALAT1, NEAT, NEAT2 in peripheral blood mononuclear cell (PBMC) from tuberculosis patients and healthy controls. Methods We detected the lncRNA expression levels in PBMC from 79 tuberculosis patients and 82 healthy controls by quantitative reverse transcription polymerase chain reaction, and analyzed the correlation between lncRNA expression levels and some clinical features and laboratory indicators in tuberculosis patients. Results The expression levels of MALAT1, NEAT1 in PBMC of tuberculosis patients were significantly higher than healthy controls (Z=-4.386, P<0.001; Z=-10.175, P<0.001). There was no significant difference in the expression of NEAT2 between tuberculosis patients and healthy controls (Z=-0.203，P=0.839). The correlation results of lncRNA levels and some clinical features, laboratory indicators in tuberculosis patients suggested that the NEAT2 level in PBMC of newly treated tuberculosis patients was higher than recurrent tuberculosis patients, while the NEAT2 level in PBMC of sputum smear positive tuberculosis patients was lower than that of sputum smear negative tuberculosis patients (all P<0.05). There was a negative correlation between MALAT1 level and erythrocyte sedimentation rate (rs=-0.256, P=0.034). Conclusion MALAT1 and NEAT1 are abnormally expressed in PBMC of tuberculosis patients, and may be involved in the pathogenesis of pulmonary tuberculosis.
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Objective To investigate the regulating function of human gingival mesenchymal stem cell (GMSC) on the proliferation and differentiation of B cells and its underlying molecular mechanism. Methods GMSC were isolated and B cells were isolated from peripheral blood. GMSC or fibroblasts were co-cultured with B cells in vitro and assigned into the GMSC group and fibroblast group. The proliferation of B cells was detected in two groups. The expression of IgG1 and IgM in the cell supernatants was measured between two groups. The secretion of interleukin (IL)-6, Perforin, interferon (IFN)-γ and tumor necrosis factor (TNF)-α was compared between two groups. The expression levels ofIL-10 and transforming growth factor (TGF)-β in B cells were detected between two groups. The expression of PC-1 in B cells was measured in two groups. The signaling pathway involved with the regulating effect of GMSC on B cell function was investigated. The regulating effect of GMSC on the role of B cells in activating T cell function was assessed. Results Compared with the fibroblast group, the proliferation of B cells was significantly weakened in the GMSC group (P < 0.05). Co-culture of GMSC and B cells significantly inhibited the secretion of IgG1 and IgM from B cells and the secretion ofIL-6, Perforin, IFN-γ and TNF-α (all P < 0.05). Compared with the fibroblast group, the secretion of IL-10 and TGF-βwas significantly higher in the GMSC group (both P < 0.05). The expression level of PC-1 in the GMSC group was significantly down-regulated (P < 0.05). After adding ALK5, an inhibitor of TGF-β receptor, the inhibitory effect of GMSC upon B cells was significantly weakened (P < 0.05). Compared with the fibroblast group, the ability of B cells to activate and proliferate T cells was significantly attenuated in the GMSC group (P < 0.05). Conclusions GMSC can inhibit B cells and their mediated immune responses. The activation of B cells and other related functions can be suppressed through the TGF-β signaling pathway.
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OBJECTIVE@#To investigate the functional pathways enriched and differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs) of patients with gram-positive and gram-negative sepsis.@*METHODS@#Dataset GSE9960 obtained from NCBI GEO database containing PBMC samples from 16 non-infectious systematic inflammatory response syndrome (SIRS) patients, 17 gram-positive septic patients and 18 gram-negative septic patients were included in the study. Functional pathway annotations were conducted by gene set enrichment analysis and weighted gene co-expression network analysis. DEGs were filtered and master DEGs were then validated in PBMCs of gram-positive septic, gram-negative septic and non-infectious SIRS patients.@*RESULTS@#The enriched gene sets in gram-positive sepsis and gram-negative sepsis were significantly different. The results indicated the opposite co-expression networks in SIRS and gram-negative sepsis, and the entirely different co-expression networks in gram-positive and gram-negative sepsis. Furthermore, we validated that @*CONCLUSIONS@#The results indicate that there are differences in the mechanism and pathogenesis of gram-positive and gram-negative sepsis, which may provide potential markers for sepsis diagnosis and empirical antimicrobial therapy.
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Humans , Biomarkers/analysis , Gene Expression Profiling , Gram-Negative Bacterial Infections/physiopathology , Gram-Positive Bacterial Infections/physiopathology , Leukocytes, Mononuclear/pathology , Sepsis/physiopathologyABSTRACT
Objective@#To investigate expressions of Toll-like receptor(TLR)-2, TLR-4 and TLR-6 in peripheral blood mononuclear cells (PBMC) and serum immunoglobulin G (IgG) and IgM levels in pediatric idiopathic nephrotic syndrome (INS). The correlation between Toll-like receptors (TLR-2, TLR-4 and TLR-6) and serum immunoglobulin levels (IgG and IgM) will be proven in the pathogenesis of childhood INS in active stage (AS) and remission stage (RS).@*Methods@#Forty-two INS patients (experimental group, 32 boys, 10 girls) and 29 healthy individuals (healthy control group, 19 boys, 10 girls) were enrolled in the present study from June, 2017 to October, 2018 in the First Affiliated Hospital of Xinxiang Medical University. There were no significant differences in gender (χ2=0.966, P=0.326) and age (t=-0.139, P=0.89) between 2 groups.TLR-2 mRNA, TLR-4 mRNA and TLR-6 mRNA expressions of PBMC were evaluated by using real-time PCR in both INS children with AS and RS and healthy children.The blood samples were drawn to detect by way of immunoturbidimetry serum immunoglobulin (IgG and IgM) levels in both INS children with AS and healthy children.The correlation between Toll-like receptors (TLR-2 mRNA, TLR-4 mRNA, TLR-6 mRNA) and serum immunoglobulin (IgG, IgM) levels were analyzed by using spearman correlation analysis.@*Results@#The expression levels of TLR-2 mRNA in INS children with AS, RS and healthy children were 1.85±0.30, 1.00±0.23 and 0.85±0.12, respectively, and the expression levels of TLR-4 mRNA were 1.24±0.27, 0.80±0.23 and 0.68±0.09, respectively, while the expression levels of TLR-6 mRNA were 1.35±0.23, 0.92±0.19 and 0.79±0.15, respectively, so the differences were statistically significant(F=198.453, 67.013, 82.405, all P<0.01). The serum IgG level(2.90±0.89) g/L was lower in INS children with AS compared with the controls(9.21±1.88) g/L.However, the serum IgM level (2.87±0.96) g/L was higher in INS children with AS when compared with the controls(1.48±0.30), and the differences were statistically significant(t=-16.810, 8.701, all P<0.05). No significant correlation was noted between Toll-like receptors(TLR-2 mRNA, TLR-4 mRNA, TLR-6 mRNA) and serum immunoglobulin levels (IgG, IgM) (all P>0.05).@*Conclusions@#The results indicate that Toll-like receptors (TLR-2, TLR-4 and TLR-6) and immunoglobulin (IgG, IgM) might play a role in the pathogenesis of pediatric INS via distinct pathway.
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Objective@#To confirm expression alteration of long non-coding RNA(lncRNA) in peripheral blood mononuclear cells (PBMCs) of generalized anxiety disorder(GAD) patients and anti-anxiety treatment effects on aberrant expression of lncRNAs.@*Methods@#Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed in 80 GAD patients and 40 healthy participants to confirm 10 aberrant lncRNAs screened by microarray expression profiling.And 26 out of all the 80 GAD patients were recruited for lncRNA expression level testing and Hamilton Anxiety Scale(HAMA) assessments before and after 6 weeks’ treatment.@*Results@#Six of ten lncRNAs selected by array profiling (lncRNA4(7.44±2.26), lncRNA5(6.83±2.28), lncRNA6(8.09±2.30), lncRNA8(9.10±2.36), lncRNA9(7.66±2.12), lncRNA10(7.34±2.12)) were verified by qRT-PCR that the lncRNA expression levels were significantly up regulated in GAD patients compared with healthy controls(Z=-3.022--1.996, P<0.05 or 0.01), and lncRNA4(9.73±2.53), lncRNA6(9.91±2.01), lncRNA8(10.48±1.68), lncRNA9(9.02±1.58), lncRNA10(9.04±2.08) were down regulated significantly after 6 weeks’ anti-anxiety treatment(Z=-3.180--2.530, P<0.05 or 0.01) along with signicant reduction of total HAMA score (11.19±8.37), dimension scores of somatic anxiety(5.31±4.76), psychic anxiety(5.88±3.82) (t=5.502-5.971, P<0.01). The alterations of lncRNA4, lncRNA6, lncRNA8, lncRNA9, lncRNA10 were positively correlated with that of HAMA total score and psychic anxiety score(r=0.39-0.69, P<0.05 or 0.01), and alteration of lncRNA6, lncRNA8, lncRNA10 had positive correlation with that of somatic anxiety score(r=0.44-0.59, P<0.01).@*Conclusion@#The expression level of lncRNA4, lncRNA5, lncRNA6, lncRNA8, lncRNA9, lncRNA10 are up-regulation in PBMCs of GAD patients and anti-anxiety treatment can reverse the expression level of lncRNAs. Alteration of lncRNA expression has osculatory association with improvement of anxious symptom.
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Objective To confirm expression alteration of long non-coding RNA( lncRNA) in pe-ripheral blood mononuclear cells (PBMCs) of generalized anxiety disorder( GAD) patients and anti-anxiety treatment effects on aberrant expression of lncRNAs. Methods Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed in 80 GAD patients and 40 healthy participants to con-firm 10 aberrant lncRNAs screened by microarray expression profiling. And 26 out of all the 80 GAD patients were recruited for lncRNA expression level testing and Hamilton Anxiety Scale(HAMA) assessments before and after 6 weeks’ treatment. Results Six of ten lncRNAs selected by array profiling (lncRNA4(7. 44± 2. 26),lncRNA5(6. 83±2. 28),lncRNA6(8. 09±2. 30),lncRNA8(9. 10±2. 36),lncRNA9(7. 66±2. 12), lncRNA10(7. 34±2. 12)) were verified by qRT-PCR that the lncRNA expression levels were significantly up regulated in GAD patients compared with healthy controls ( Z=-3. 022--1. 996,P<0. 05 or 0. 01),and lncRNA4(9. 73 ± 2. 53),lncRNA6 ( 9. 91 ± 2. 01), lncRNA8 ( 10. 48 ± 1. 68), lncRNA9 ( 9. 02 ± 1. 58), lncRNA10(9. 04 ± 2. 08) were down regulated significantly after 6 weeks’ anti-anxiety treatment ( Z=-3. 180--2. 530,P<0. 05 or 0. 01) along with signicant reduction of total HAMA score (11. 19±8. 37),di-mension scores of somatic anxiety(5. 31±4. 76),psychic anxiety(5. 88±3. 82) (t=5. 502-5. 971,P<0. 01). The alterations of lncRNA4,lncRNA6,lncRNA8,lncRNA9,lncRNA10 were positively correlated with that of HAMA total score and psychic anxiety score(r=0. 39-0. 69,P<0. 05 or 0. 01),and alteration of lncRNA6, lncRNA8,lncRNA10 had positive correlation with that of somatic anxiety score(r=0. 44-0. 59,P<0. 01). Conclusion The expression level of lncRNA4,lncRNA5,lncRNA6,lncRNA8,lncRNA9,lncRNA10 are up-regulation in PBMCs of GAD patients and anti-anxiety treatment can reverse the expression level of lncRNAs. Alteration of lncRNA expression has osculatory association with improvement of anxious symptom.
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Objective To investigate the expression of sialic acid-binding immunoglobulin-like-lectin-1 (Siglec-1)in the peripheral blood mononuclear cell (PBMC) of patients with autoimmune thyroiditis ( AIT) and its relationship with AIT. To explore the moduratory role of activated Siglec-1 on the differentiation of T cells and the promotion of in flammation after PBMC culture. Methods The peripheral whole blood and serum samples were collected from 30 AIT patients with normal thyroid function and 30 sex-and age-matched controls. The expression of sSiglec-1 in serum was detected by ELISA. The expression of Siglec-1 in PBMC was detected by RT-PCR and WB. The expression of Siglec-1 in CD14+ monocytes and the proportion of Th1 and Th17 cells in each group were detected by flow cytometry. The PBMC in AIT or control was stimulated with NaI in the presence or absence of LPS for 72 h. The expression of Siglec-1 in CD14+ monocytes and the proportion of Th1 and Th17 cells were detected by flow cytometry. Results sSiglec-1 in serum, Siglec-1 mRNA, and Siglec-1 protein in AIT patients'PBMC were higher than those in control group ( P＜0. 01). The expression of Siglec-1 in CD14+ monocytes by flow cytometry and differentiation of Th1 and Th17 cells were significantly higher than that in control group ( both P＜0. 01). The expression of Siglec-1 in control and AIT patients was up-regulated by 5×10-5 mmol/L to 1×10-2 mmol/L stimulated with NaI in the presence or absence of LPS for 72 h (P＜0.01), but the differentiation of Th1 and Th17 cells was up-regulated only in patients (P＜0.01), and in a dose-dependent manner. Conclusion Elevated Siglec-1 expression in PBMCs and monocytes can potentially serve as a biomarker for AIT. Iodine may affect Th1 and Th17 cell differentiation by activating Siglec-1 to adjust the AIT immune response.
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OBJECTIVE@#Garden cress (Lepidium sativum L.) is an important herb in traditional medicine used to improve production of breast milk in women and semen in men. In the present research the authors evaluated its ability to destroy leukemic cancer (Jurkat E6-1) cells, using the alkaloid extract of this plant.@*METHODS@#Constituents of the alkaloid extract were analyzed by gas chromatography-mass spectrometry (GC-MS) and their cytotoxicity in leukemic cancer cells and healthy peripheral blood mononuclear cells (PBMCs) was assessed. Cell death via apoptosis was confirmed by DNA laddering, caspase-3 activity, annexin V-fluorescein isothiocyanate and mitochondrial toxicity assays. The specific course of gene activation in treated cells was determined through quantitative polymerase chain reaction (qPCR).@*RESULTS@#GC-MS analysis identified six alkaloids and proto-alkaloids, namely, benzyl isothiocyanate (1), 2-ethoxy-4H-3,1-benzoxazin-4-one (2), (4R)-2-(2-aminophenyl)-4-phenyloxazoline (3), 5-acetyl-1,2-dihydro-6-methyl-2-oxo-4-phenyl-3-pyridinecarbonitrile (4), benzo[b][1,8]-naphthyridin-5(10H)-one,2,4,7-trimethyl (5) and 1,4-diaminoanthraquinone (6), in the alkaloid extract of L. sativum. Of these, compound 1 was previously identified in the seeds of L. sativum. Exposure to the alkaloid extract caused death of Jurkat E6-1 cells, with median lethal concentration (LC) of 75.25 µg/mL. However, the alkaloid extract also showed a nontoxic and proliferative (1.6-fold) effect in healthy PBMCs. Further experiments performed with Jurkat cells at LC and sub-LC doses demonstrated DNA fragmentation, activation of caspase-3 and time-dependant phosphatidylserine translocation (apoptosis) from inner to outer cell membranes. Cell toxicity and assessment of adenosine triphosphate level, together with using qPCR to evaluate expression profile of major apoptosis genes, revealed that apoptosis may be induced by disruption in the mitochondrial outer membrane potential, through activation of extrinsic and intrinsic apoptosis pathways in Jurkat cells.@*CONCLUSION@#The ability of the alkaloid extract of L. sativum seeds to induce apoptosis indicates a potential pharmacological use in cancer chemotherapy. The separation of individual active compounds and further in-depth exploration of the molecular mechanism of apoptosis may lead to novel chemotherapeutic compounds in our future antineoplastic research.
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Objective To investigate expressions of Toll-like receptor (TLR)-2,TLR-4 and TLR-6 in peripheral blood mononuclear cells (PBMC) and serum immunoglobulin G (IgG) and IgM levels in pediatric idiopathic nephrotic syndrome (INS).The correlation between Toll-like receptors (TLR-2,TLR-4 and TLR-6) and serum immunoglobulin levels (IgG and IgM) will be proven in the pathogenesis of childhood INS in active stage (AS) and remission stage (RS).Methods Forty-two INS patients (experimental group,32 boys,10 girls) and 29 healthy individuals (healthy control group,19 boys,10 girls) were enrolled in the present study from June,2017 to October,2018 in the First Affiliated Hospital of Xinxiang Medical University.There were no significant differences in gender (x2 =0.966,P =0.326) and age (t =-0.139,P =0.89) between 2 groups.TLR-2 mRNA,TLR-4 mRNA and TLR-6 mRNA expressions of PBMC were evaluated by using real-time PCR in both INS children with AS and RS and healthy children.The blood samples were drawn to detect by way of immunoturbidimetry serum immunoglobulin (IgG and IgM) levels in both INS children with AS and healthy children.The correlation between Toll-like receptors (TLR-2 mRNA,TLR-4 mRNA,TLR-6 mRNA) and serum immunoglobulin (IgG,IgM) levels were analyzed by using spearman correlation analysis.Results The expression levels of TLR-2 mRNA in INS children with AS,RS and healthy children were 1.85 ± 0.30,1.00 ± 0.23 and 0.85 ± 0.12,respectively,and the expression levels of TLR-4 mRNA were 1.24 ± 0.27,0.80 ± 0.23 and 0.68 ± 0.09,respectively,while the expression levels of TLR-6 mRNA were 1.35 ± 0.23,0.92 ± 0.19 and 0.79 ± 0.15,respectively,so the differences were statistically significant (F =198.453,67.013,82.405,all P ＜ 0.01).The serum IgG level(2.90 ± 0.89) g/L was lower in INS children with AS compared with the controls (9.21 ± 1.88) g/L.However,the serum IgM level (2.87 ± 0.96) g/L was higher in INS children with AS when compared with the controls(1.48 ± 0.30),and the differences were statistically significant(t =-16.810,8.701,all P ＜0.05).No significant correlation was noted between Toll-like receptors (TLR-2 mRNA,TLR-4 mRNA,TLR-6 mRNA) and serum immunoglobulin levels (IgG,IgM) (all P ＞ 0.05).Conclusions The results indicate that Toll-like receptors (TLR-2,TLR-4 and TLR-6) and immunoglobulin (IgG,IgM) might play a role in the pathogenesis of pediatric INS via distinct pathway.
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Objective To investigate the microRNA ( miRNA ) expression level of peripheral blood mononuclear cell ( PBMC) in autoimmune diabetes mellitus ( ADM) which includes type 1 diabetes mellitus ( T1DM) and latent autoimmune diabetes in adults ( LADA ) , T2DM patients, and matched healthy individuals. Methods Patients of T1DM, LADA, and T2DM were recruited in the Second Xiangya Hospital of Central South University from January 2015 to December 2016. The subjects were divided into two groups. The first group was used for high-throughput screening of differentially expressed microRNAs. The second group was used to validate the expression of miR-142-5p and miR-143-3p by real-time quantitative polymerase chain reaction (RT-qPCR). Results (1)The different miRNA expression patterns of PBMC were found among T1DM patients, LADA patients, T2DM patients, and health individuals. ( 2) Compared with T2DM patients and healthy controls, LADA and T1DM patients had down-regulated PBMC miR-142-5p expression, and up-regulated miR-143-3p expression. (3)RT-qPCR validation showed that the expression of miR-142-5p in LADA patients was significantly lower than that in T2DM patients (0.30±0.24 vs 1.33 ± 1.29, P<0.05) . The expression of miR-143-3p in T1DM and LADA was higher than that in T2DM and health individuals. However, no significant differences were found. Conclusion The miRNA expression patterns are different in the PBMC of T1DM patients, LADA patients, T2DM patients, and healthy individuals; the abnormal expressions of miR-142-5p and miR-143-3p may participate in the development of ADM by affecting apoptosis and immune cell differentiation.
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Objective:To analyze the expression of TNF-related apoptosis-inducing factor TRAIL,c-FLIP and caspase-8 in peripheral blood mononuclear cells of patients with rheumatoid arthritis at different period,in order to provide a experimental basis that treating and looking for a more effective way and method.Methods:mRNA expression of TRAIL,c-FLIP and caspase-8 were detected by Real-time PCR from peripheral blood mononuclear cells;TRAIL,c-FLIP and caspase-8 were checked by Western blot from peripheral blood mononuclear cells.Results:The mRNA expression level of TRAIL,c-FLIP and caspase-8 from PBMC at different groups of RA:TRAIL mRNA in group M was 3.26±0.78 which was much higher than healthy controls(1.30±0.20) (P=0.028),and those of group L and group H (1.56±0.37 and 1.83±0.26 respectively) was slightly higher than controls(P<0.05).C-FLIP mRNA in low activity group (L),middle activity group (M) and high activity group (H) were 1.27±0.28,1.32±0.34 and 1.93±0.40 re-spectively,which was higher than that of healthy controls (1.08 ± 0.12) (P=0.035,0.034,0.030).The relative level caspase-8 mRNA in group L,group M,group H were(2.77 ±0.97),(4.52 ± 0.85),and(2.13 ± 0.44)which was higher than healthy controls (1.04±0.13) (P=0.023,0.012,0.032).The protein level of TRAIL,c-FLIP and caspase-8 in PBMC from different groups of RA patients:The expression of TRAIL in group M was significantly increased than group L,H and control(P<0.05) with no difference in group L.c-FLIP protein in all protein expressed in group M quantity highest,significantly higher than other groups.Caspase-8 expression in the group M and H was significantly enhanced than control (P=0.003,0.001 ) with no difference in group L (P> 0.05). Conclusion:During the different active stages of RA,in peripheral blood mononuclear cells,the expression of TRAIL,c-FLIP and caspase-8 are increased overall trend,possible can provide certain experimental reference for clinical therapy.
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Objective To explore the pathogenesis of ovarian cancer by investigating the function of Toll-like receptor 1 (TLR1),TLR2 and TLR6 in peripheral blood mononuclear cell(PBMC) from patients with ovarian cancer.Methods PBMC,SK OV 3 co culture system and anti-TLR1,anti-TLR2,anti-TLR6 mAb blocking experiment were used to explore the relationship between TLR1,TLR2 or TLR6 signaling and inflammation in ovarian cancer.Quantitative real time PCR was used to measure interleukin(IL)-1β,IL-6,IL-8,and tumor necrosises factor(TNF)-α in the PBMC.MyD88,TRAF6,TANK,NF-κB and P-NFκB were observed by Western blotting.Results In the PBMC and SK-OV-3 coculture system,we found the activation of TLR signaling pathways,including significantly increased MyD88,TRAF6,TANK and P-NF-κB levels following cocultured with SK-OV-3 in PBMC from ovarian cancer patients.PBMC derived from ovarian cancer patients led to a increase in IL-1β,IL-6 and IL-8 mRNA levels after 24 hours of co-incubation with SK-OV-3 (Fold=1.74,Fold=1.92,Fold=1.65,P＜0.05),though there was no difference of TNF-a mRNA expression.In contrast to the ovarian cancer patients,coculture of PBMC derived from benign diseases controls and healthy normal controls decreased IL-1β at the mRNA level (Fold=0.71,P＜0.05;Fold=0.72,P＜0.05),furthermore the expression of MyD88,TRAF6,TANK,P-NF-κB,and NF-κB showed no changes.PBMC which treated with anti-TLR1,anti-TLR2 or-TLR6 mAb could inhibite inflammatory IL-1β (Fold=0.16,Fold=0.31,Fold=0.29,P＜0.05) and IL-6 (Fold=0.14,Fold=0.20,Fold=0.28,P＜0.05).Conclusion TLR1/TLR2/TLR6 in PBMC of ovarian cancer patients participate in the recognition of the factors.
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Objective To investigate the diagnostic value of plasma ,peripheral blood mononuclear cell (PB-MC) and throat swab EB virus (SBV) load in the patients with infectious mononucleosis (IM ) aged over 16 years old .Methods The detection results in 130 patients with suspected IM aged over 16 years old of 3 differ-ent samples of plasma ,peripheral blood mononuclear cell(PBMC) and throat swab EB virus(SBV) load were analyzed retrospectively .Results Among 61 cases of IM verified by clinic and laboratory ,16 cases of IM were detected by plasma sample ,the detection rate of EBV-DNA was 26 .22％ ;in 31 cases of PBMC detection ,the EBV-DNA detection rate was 61 .29％ ;in 31 cases of throat swab detection ,the EBV-DNA detection rate was 83 .78％ .The EBV-DNA detection rate of the combined detection with one indicator or multiple indicators positive of plasma ,PBMC and throat swab as the basis was 100 .00％ .Conclusion The combined detection of plasma ,PBMC and throat swab can improve the positive rate in the patients with IM aged over 16 years old , w hich can provide more bases for pathogenic diagnosis and treatment for clinic .
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Objective To explore the surface morphological and biomechanical properties differences of peripheral blood mononuclear cells (PBMCs) between groups of patients with mitochondrial diabetes caused by mt.3243A ＞ G mutation and healthy controls.Methods 2 milliliters blood were obtained from each subject of the mitochondrial diabetes group (n =5) and the control group (n =5).The PBMCs were separated from the blood using the standard Ficoll-Hypaque density-gradient centrifugation method and detected by atomic force microscope (AFM).Results The morphological analysis revealed that compared with control group,the PBMCs of diabetic patients tended to have a lower cell height (0.73 ± 0.24μm vs 2.49 ± 1.17μm,P =0.011) and a much rougher cell membrane (Ra:161.8 ± 33.2nm vs 66.4 ± 16.3 nm,P =0.000;Rq:202.2 ± 40.9nm vs 85.4 ± 17.1 nm,P =0.000).The adhesion force distribution was nearly three times higher in PBMCs of diabetic patients than that of the control group (779.6 ± 190.0pN vs 161.1 ± 83.1 pN,P =0.000).The Young's modulus of PBMCs was significantly increased in diabetic patients (421.4 ± 140.0kPa vs 138.3 ± 77.2kPa,P ＜ 0.01),indicating that diabetic PBMCs were stiffer than control cells.Conclusion Our study demonstrated the surface morphological and biomechanical properties changes in mitochondrial diabetes caused by mt.3243A ＞ G mutation at PBMCs level,which was beneficial to the better understanding of the pathophysiological mechanisms of mitochondrial diabetes associated with mt.3243A ＞ G mutation.