Peripheral Blood Monocular Cells Research Articles

Durable natural killer cell response after three doses of SARS-CoV-2 inactivated vaccine in HIV-infected individuals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine can induce a potent cellular and humoral immune response to protect against SARS-CoV-2 infection. However, it was unknown whether SARS-CoV-2 vaccination can induce effective natural killer (NK) cell response in people living with human immunodeficiency virus (PLWH) and healthy individuals. Forty-seven PLWH and thirty healthy controls (HCs) inoculated with SARS-CoV-2 inactivated vaccine were enrolled from Beijing Youan Hospital in this study. The effect of SARS-CoV-2 vaccine on NK cell frequency, phenotype, and function in PLWH and HCs was evaluated by flow cytometry, and the response of NK cells to SARS-CoV-2 Omicron Spike (SARS-2-OS) protein stimulation was also evaluated. SARS-CoV-2 vaccine inoculation elicited activation and degranulation of NK cells in PLWH, which peaked at 2 weeks and then decreased to a minimum at 12 weeks after the third dose of vaccine. However, in vitro stimulation of the corresponding peripheral blood monocular cells from PLWH with SARS-2-OS protein did not upregulate the expression of the aforementioned markers. Additionally, the frequencies of NK cells expressing the activation markers CD25 and CD69 in PLWH were significantly lower than those in HCs at 0, 4 and 12weeks, but the percentage of CD16 + NK cells in PLWH was significantly higher than that in HCs at 2, 4 and 12weeks after the third dose of vaccine. Interestingly, the frequency of CD16 + NK cells was significantly negatively correlated with the proportion of CD107a + NK cells in PLWH at each time point after the third dose. Similarly, this phenomenon was also observed in HCs at 0, 2, and 4 weeks after the third dose. Finally, regardless of whether NK cells were stimulated with SARS-2-OS or not, we did not observe any differences in the expression of NK cell degranulation markers between PLWH and HCs. s:SARS-CoV-2 vaccine elicited activation and degranulation of NK cells, indicating that the inoculation of SARS-CoV-2 vaccine enhances NK cell immune response.

Circulating Neoplastic-Immune Hybrid Cells Predict Metastatic Progression in Uveal Melanoma.

Simple SummaryUveal melanoma is an aggressive cancer that begins in the eye, but often spreads to distant organs when tumor cells enter the blood. Disease spreads in nearly half of uveal melanoma patients, and is fatal. We discovered a new tumor cell population in the blood stream of cancer patients that has combined tumor cell and white blood cell features. These cells, called circulating hybrid cells, can develop into metastatic tumors. Recently, we detected circulating hybrid cells in the blood of patients with uveal melanoma. In this study we determine that the number of circulating hybrid cells in the blood at time of initial treatment of uveal melanomas predicts future development of disease spread. Circulating hybrid cells have promise as a non-invasive and repeatable “liquid biopsy” for uveal melanoma patients. Therefore, study of this newly discovered cancer cell will improve understanding of the biology of disease progression in uveal melanoma.Background: Uveal melanoma is an aggressive cancer with high metastatic risk. Recently, we identified a circulating cancer cell population that co-expresses neoplastic and leukocyte antigens, termed circulating hybrid cells (CHCs). In other cancers, CHCs are more numerous and better predict oncologic outcomes compared to circulating tumor cells (CTCs). We sought to investigate the potential of CHCs as a prognostic biomarker in uveal melanoma. Methods: We isolated peripheral blood monocular cells from uveal melanoma patients at the time of primary treatment and used antibodies against leukocyte and melanoma markers to identify and enumerate CHCs and CTCs by immunocytochemistry. Results: Using a multi-marker approach to capture the heterogeneous disseminated tumor cell population, detection of CHCs was highly sensitive in uveal melanoma patients regardless of disease stage. CHCs were detected in 100% of stage I-III uveal melanoma patients (entire cohort, n = 68), whereas CTCs were detected in 58.8% of patients. CHCs were detected at levels statically higher than CTCs across all stages (p = 0.05). Moreover, CHC levels, but not CTCs, predicted 3 year progression-free survival (p < 0.03) and overall survival (p < 0.04). Conclusion: CHCs are a novel and promising prognostic biomarker in uveal melanoma.

Quercetin induces autophagy-associated death in HL-60 cells through CaMKKβ/AMPK/mTOR signal pathway.

Acute myeloid leukemia (AML) is one of the most common malignancies of the hematopoietic progenitor cell in adults. Quercetin has gained recognition over the years because of its anti-cancer effect with minimal toxicity. Herein, we aim to investigate the anti-leukemia mechanism of quercetin and to decipher the signaling pathway of quercetin in HL-60 leukemic cells. We observed that quercetin induces apoptosis and autophagic cell death, in which both pathways play an important role in suppressing the viability of leukemia cells. Phosphorylated AMPK (p-AMPK) protein expressions are lower in primary AML cells, HL-60 cells, KG-1 and THP-1 cells than in peripheral blood monocular cells. After quercetin treatment, the expression of p-AMPK is increased while the expression of p-mTOR is decreased in a dose-dependent manner. Mechanistically, compound C, an AMPK phosphorylation inhibitor, upregulates the phosphorylation of mTOR and inhibits autophagy and apoptosis in quercetin-induced HL-60 cells, while silencing of CaMKKβ inhibits the quercetin-induced phosphorylation of AMPK, resulting in increased mTOR phosphorylation. Furthermore, silencing of CaMKKβ inhibits the autophagy in HL-60 cells. Taken together, our data delineate that quercetin plays its anti-leukemia role by inhibiting cell viability and inducing apoptosis and autophagy in leukemia cells. Quercetin inhibits the phosphorylation of mTOR by regulating the activity of AMPK, thus playing a role in the regulation of autophagy and apoptosis. CaMKKβ is a potential upstream molecule for AMPK/mTOR signaling pathway, through which quercetin induces autophagy in HL-60 cells.

Participation of MAPK and PI3K in Regulation of Cytokine Secretion by Peripheral Blood Monocular Cells in Response to Escherichia coli LPS and rDer p 2 Combination.

Search for the effective approaches to treat acute inflammation caused by combination of allergens and infectious agents is an important task for public health worldwide. House dust mites Dermatophagoides pteronyssinus are the source of allergens of the Der p groups and of microbial compounds, in particular, lipopolysaccharides (LPS). LPS and Der p 2 induce secretion of pro-inflammatory cytokines via activation of kinases p38 MAPK, MEK1/2, and PI3K. Participation of these kinases in the regulation of cells response to combined exposure to LPS and Der p 2 has not been sufficiently studied. We studied the effects of kinases (p38 MAPK, MEK1/2, and PI3K) inhibition on secretion of cytokines (TNF, IL-8, and IL-6) by peripheral blood mononuclear cells (PBMC) of healthy volunteers in response to E. coli LPS and rDer p 2. Contribution of kinases to the regulation of cell response to different agents (rDer p 2 and/or LPS) was revealed. It was found that p38 MAPK plays a key role in the regulation of secretion TNF by PBMC in response to the combination of LPS and rDer p 2. MEK1/2-dependent signaling is the main pathway for the synthesis of TNF and IL-8 in response to LPS and rDer p 2. PI3K-dependent signaling negatively regulates TNF production during rDer p 2-induced cell activation, but is not involved in the response to the combination of LPS and rDer p 2. PI3K-dependent signaling in the regulation of PBMC cytokine synthesis is most pronounced in response to their activation by rDer p 2. Understanding the mechanisms of immune cell responses to combinations of inflammatory agents could facilitate the search for new intracellular targets for anti-inflammatory therapy.

Diagnostic Biomarker Hsa_circ_0126218 and Functioning Prediction in Peripheral Blood Monocular Cells of Female Patients With Major Depressive Disorder.

IntroductionAlthough major depressive diroder (MDD) has brought huge burden and challenges to society globally, effective and accurate diagnoses and treatments remain inadequate. The pathogenesis that for women are more likely to suffer from depression than men needs to be excavated as well. The function of circRNAs in pathological process of depression has not been widely investigated. This study aims to explore potential diagnostic biomarker circRNA of female patients with MDD and to investigate its role in pathogenesis.MethodsFirst, an expression profile of circRNAs in the peripheral blood monocular cells of MDD patients and healthy peripherals were established based on high-throughput sequencing analysis. In addition, the top 10 differentially expressed circRNAs were quantified by quantitative real-time PCR to explore diagnostic biomarkers. To further investigate the function of biomarkers in the pathogenesis of MDD, bioinformatics analysis on downstream target genes of the biomarkers was carried out.ResultsThere is a mass of dysregulated circRNAs in PBMCs between female MDD patients and healthy controls. Among the top 10 differentially expressed circRNAs, hsa_circ_0126218 is more feasible as a diagnostic biomarker. The expression level of hsa_circ_0126218 displayed upregulation in patients with MDD and the area under the operating characteristic curve of hsa_circ_0126218 was 0.801 (95% CI 0.7226–0.8791, p < 0.0001). To explain the competing endogenous RNA role of hsa_circ_0126218 in the pathogenesis of female MDD, a hsa_circ_0126218-miRNA-mRNA network was established. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses stated that some of the enriched pathways downstream of hsa_circ_0126218 are closely related to MDD. Moreover, we established a protein-protein network to further screen out the hub genes (PIK3CA, PTEN, MAPK1, CDC42, Lyn, YES1, EPHB2, SMAD2, STAT1, and ILK). The function of hsa_circ_0126218 was refined by constructing a verified circRNA-predicted miRNA-hub gene subnetwork.Conclusionhsa_circ_0126218 can be considered as a new female MDD biomarker, and the pathogenesis of female MDD by the downstream regulation of hsa_circ_0126218 has been predicted. These findings may help further improve the early detection, effective diagnosis, convenient monitoring of complications, precise treatment, and timely recurrence prevention of depression.

Short communication: Effect of yeast cell wall supplementation on peripheral leukocyte populations and mRNA expression of cytokines in lactating dairy cows

This study was designed to examine the effect of yeast cell wall (YCW) supplementation on peripheral leukocyte populations and mRNA expression of cytokines in lactating dairy cows. Fourteen Holstein lactating cows were assigned to 1 of 2 treatments; the control group (n = 7) were fed a total mixed ration without supplementation and cows in the YCW group (n = 7) were fed a total mixed ration supplemented with YCW (SafMannan; Phileo, Lesaffre Animal Care, Lille, France; 10 g/cow per day). Blood samples were collected 3 times during the experimental period [wk 0 (before any treatment), wk 4, and wk 8]. Peripheral leukocyte populations and cytokine mRNA expression of peripheral blood monocular cells were measured using flow cytometry and real-time PCR, respectively. Among the peripheral leukocyte populations, TcR1-N12 + and CD14+ T cells increased at wk 4, and CD4+ T cells and CD8+ T cells increased at wk 4 and wk 8 with YCW supplementation. The mRNA level of IL8 tended to be increased in the YCW group at wk 4. Expression of IL12A was lower in the YCW group than in the control group before the experiment (wk 0) but no differences were observed at later time points (wk 4 and wk 8). Expression of IL12A decreased in the control group and increased in the YCW group. Expression of CCR2 increased at wk 4, and CCL2 and CCL3 were increased at wk 8 in the YCW group. Thus, YCW supplementation increased the mRNA expression of cytokines in peripheral blood mononuclear cells of lactating dairy cows.

Myeloid related proteins are up-regulated in autoimmune thyroid diseases and activate toll-like receptor 4 and pro-inflammatory cytokines in vitro

PurposeMyeloid-related protein (MRP) family plays an important role in the promotion of cell proliferation and the production of inflammatory cytokines. We investigated the expression of MRP6, MRP8 and MRP14 in thyroid tissues, serum, and peripheral blood monocular cells (PBMCs) in patients with autoimmune thyroid diseases (AITD). MethodThe expression of MRP6, MRP8, and MRP14 was investigated using immunohistochemical staining and quantitative real-time polymerase chain reaction in the thyroid glands of 7 patients with Graves' disease (GD), 8 with Hashimoto's thyroiditis (HT), and 7 healthy controls (HC). The serum levels of MRP8/MRP14 complex and MRP6 were investigated in 30 patients with GD, 36 with HT, and 30 with HC. The mRNA expression of MRP proteins in PBMCs was also explored. PBMCs from each group were incubated with MPRs and their effect on Toll-like receptor 4(TLR4) expression and their effect on the levels of the pro-inflammatory cytokines in supernatant were analyzed upon incubating with TLR4 and signaling pathways inhibitors. ResultsSerum levels of MRP8/MRP14 and MRP6 were up-regulated in patients with AITD. In addition, mRNA expression of MRP proteins in PBMCs and the thyroid gland was markedly elevated in AITD patients. MRP6 and MPR8 promoted the secretion of TNF-α and IL-6 in cultured PBMCs, and this elevation was more pronounced in AITD patients; we also found that this up-regulation was regulated by TLR4/phosphoinositide 3-kinase/nuclear factor-κB signaling pathway. ConclusionThe expression of MRP proteins was elevated in AITD patients. Therefore, an MRP-TLR4 dependent signaling may play an important role in the pathogenesis of AITD.

Bioinformatics-based identification of potential microRNA biomarkers in frequent and non-frequent exacerbators of COPD

ObjectivesMicroRNAs (miRNAs) play essential roles in the development of COPD. In this study, we aimed to identify and validate potential miRNA biomarkers in frequent and non-frequent exacerbators of COPD patients using bioinformatic analysis.Materials and methodsThe candidate miRNA biomarkers in COPD were screened from Gene Expression Omnibus (GEO) dataset and identified using GEO2R online tool. Then, we performed bioinformatic analyses including target prediction, gene ontology (GO), pathway enrichment analysis and construction of protein–protein interaction (PPI) network. Furthermore, the expression of the identified miRNAs in peripheral blood monocular cells (PBMCs) of COPD patients was validated using quantitative real-time polymerase chain reaction (qRT-PCR).ResultsMiR-23a, miR-25, miR-145 and miR-224 were identified to be significantly downregulated in COPD patients compared with healthy controls. GO analysis showed the four miRNAs involved in apoptotic, cell differentiation, cell proliferation and innate immune response. Pathway analysis showed that the targets of these miRNAs were associated with p53, TGF-β, Wnt, VEGF and MAPK signal pathway. In healthy controls, the miR-25 and miR-224 levels were significantly decreased in smokers compared with nonsmokers (P<0.001 and P<0.05, respectively). In COPD patients, the levels of miR-23a, miR-25, miR-145 and miR-224 were associated with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages. Notably, miR-23a and miR-145 were significantly elevated in non-frequent exacerbators compared with frequent exacerbators (P<0.05), and miR-23a showed higher area under the receiver–operator characteristic curve (AUROC) than miR-145 (0.707 vs 0.665, P<0.05).ConclusionMiR-23a, miR-25, miR-145 and miR-224 were associated with the development of COPD, and miR-23a might be a potential biomarker for discriminating the frequent exacerbators from non-frequent exacerbators.

Obesity-Associated Metabolic Signatures Correlate to Clinical and Inflammatory Profiles of Asthma: A Pilot Study.

PurposeObesity is associated with metabolic dysregulation, but the underlying metabolic signatures involving clinical and inflammatory profiles of obese asthma are largely unexplored. We aimed at identifying the metabolic signatures of obese asthma.MethodsEligible subjects with obese (n = 11) and lean (n = 22) asthma underwent body composition and clinical assessment, sputum induction, and blood sampling. Sputum supernatant was assessed for interleukin (IL)-1β, -4, -5, -6, -13, and tumor necrosis factor (TNF)-α, and serum was detected for leptin, adiponectin and C-reactive protein. Untargeted gas chromatography time-of-flight mass spectrometry (GC-TOF-MS)-based metabolic profiles in sputum, serum and peripheral blood monocular cells (PBMCs) were analyzed by orthogonal projections to latent structures-discriminate analysis (OPLS-DA) and pathway topology enrichment analysis. The differential metabolites were further validated by correlation analysis with body composition, and clinical and inflammatory profiles.ResultsBody composition, asthma control, and the levels of IL-1β, -4, -13, leptin and adiponectin in obese asthmatics were significantly different from those in lean asthmatics. OPLS-DA analysis revealed 28 differential metabolites that distinguished obese from lean asthmatic subjects. The validation analysis identified 18 potential metabolic signatures (11 in sputum, 4 in serum and 2 in PBMCs) of obese asthmatics. Pathway topology enrichment analysis revealed that cyanoamino acid metabolism, caffeine metabolism, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, pentose phosphate pathway in sputum, and glyoxylate and dicarboxylate metabolism, glycerolipid metabolism and pentose phosphate pathway in serum are suggested to be significant pathways related to obese asthma.ConclusionsGC-TOF-MS-based metabolomics indicates obese asthma is characterized by a metabolic profile different from lean asthma. The potential metabolic signatures indicated novel immune-metabolic mechanisms in obese asthma with providing more phenotypic and therapeutic implications, which needs further replication and validation.

P1.07-037 Testing the Positive and Negative Immune Checkpoint on PBMCs of Patients from Initiatory to Terminative Treatment of Anti PD-1 Antibody

Recent approaches to malignant tumor have turned to immunotherapy which shows outstanding clinical efficacy, especially the agents of programmed death 1(PD-1) pathway. In this study, we try to find the relationship between the clinical benefit and the change of immune biomarkers in peripheral blood monocular cells (PBMCs) by continuous testing.

Global profiling of viral and cellular non-coding RNAs in Epstein–Barr virus-induced lymphoblastoid cell lines and released exosome cargos

The human EBV-transformed lymphoblastoid cell line (LCL), obtained by infecting peripheral blood monocular cells with Epstein–Barr Virus, has been extensively used for human genetic, pharmacogenomic, and immunologic studies. Recently, the role of exosomes has also been indicated as crucial in the crosstalk between EBV and the host microenvironment. Because the role that the LCL and LCL exosomal cargo might play in maintaining persistent infection, and since little is known regarding the non-coding RNAs of LCL, the aim of our work was the comprehensive characterization of this class of RNA, cellular and viral miRNAs, and cellular lncRNAs, in LCL compared with PBMC derived from the same donors. In this study, we have demonstrated, for the first time, that all the viral miRNAs expressed by LCL are also packaged in the exosomes, and we found that two miRNAs, ebv-miR-BART3 and ebv-miR-BHRF1-1, are more abundant in the exosomes, suggesting a microvescicular viral microRNA transfer. In addition, lncRNA profiling revealed that LCLs were enriched in lncRNA H19 and H19 antisense, and released these through exosomes, suggesting a leading role in the regulation of the tumor microenvironment.

Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep

BackgroundClassical scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Our previous bioassay studies in lambs revealed that scrapie prions could be detected in association with peripheral blood monocular cells (PBMC), B lymphocytes and platelet-rich plasma fractions. In the present study, bioassay in lambs was again used to determine if scrapie prions are associated with the other two subsets of PBMC, monocytes and T lymphocytes.ResultsPBMC, monocytes and T lymphocytes were isolated from two preclinically affected VRQ/VRQ sheep naturally infected with classical ovine scrapie and intravenously transfused into VRQ/VRQ lambs post-weaning. As determined using standard immunohistochemistry for scrapie, abnormal isoforms of prion protein were detected in lymphoid tissues of lambs inoculated with PBMC (4/4 recipient lambs), monocytes (2/5) and T lymphocytes (1/4). Prion protein misfolding activity was detected by serial protein misfolding cyclic amplification (sPMCA) in PBMC from monocyte and T lymphocyte recipient sheep in agreement with antemortem rectal biopsy results, but such prion protein misfolding activity was not detected from other recipients.ConclusionsThese findings show that scrapie prions are associated with monocytes and T lymphocytes circulating in the peripheral blood of sheep naturally infected with classical scrapie. Combined with our previous findings, we can now conclude that all three major subsets of PBMC can harbor prions during preclinical disease and thus, present logical targets for development of a sensitive assay to detect scrapie prions. In this regard, we have also demonstrated that sPMCA can be used to detect scrapie prions associated with PBMC.

Regulatory effects of deguelin on proliferation and cell cycle of Raji cells

The underlying mechanism of deguelin regulating the cell cycle in human Burkitt's lymphoma cell line Raji cells in vitro, and the cytotoxicity of deguelin to Raji cells and human peripheral blood monocular cells (PBMCs) were investigated. The effects of deguelin on the growth of Raji cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was detected through Hoechst 33258 staining. The effect of deguelin on the cell cycle of Raji cells was studied by a propidium iodide method. The expression levels of cyclin D1, P21 and pRb were examined by using Western blotting. The results showed that the proliferation of Raji cells was inhibited in the deguelin-treated group, with a 24-h IC(50) value of 21.61 nmol/L and a 36-h IC(50) value of 17.07 nmol/L. Proliferation in Raji cells was inhibited significantly by deguelin, while little change was observed in PBMCs. Deguelin induced G(2)/M arrest in Raji cells. The expression of cyclin D1, P21 and pRb was dramatically down-regulated by deguelin in a dose-dependent manner. It was concluded that deguelin could inhibit the proliferation of Raji cells by arresting the cells at G(2)/M phase and inducing the cell apoptosis. Moreover, deguelin selectively induced apoptosis of Raji cells with low toxicity to PBMCs. The antitumor effects of deguelin were related to the down-regulated expression of cyclin D1, P21 and pRb proteins.

Expression of suppressor of cytokine signaling in peripheral blood monocular cells of patients with sympathetic ophthalmia

Objective To investigate the mRNA and protein expression of suppressor of cytokine signaling (SOCS) in peripheral blood mononuclear cells(PBMC) of patients with sympathetic ophthalmia.Methods Blood samples were taken from 14 sympathetic ophthalmia patients with active uveitis,13 quiescent patients,and 15 healthy individuals.IFN-γ,IL-12 and IL-4 in the serum were measured by ELISA.PBMC were subjected to analysis of SOCS mRNA and protein expression using quantitative RT-PCR and western blot,respectively.Results The level of IFN-γ,IL-12 and IL-4 were all significantly higher in the serum of sympathetic ophthalmia patients with active uveitis than that in controls and quiescent patients(P ＜0.05).There were no significant differences between the quiescent patients and the controls(P ＞ 0.05).Compared to healthy volunteers,the expressions of cytokine inducible SH2 containing protein (CIS),SOCS1,SOCS3 and SOCS5 mRNA and protein were significantly increased.There were no significant differences in expression of CIS,SOCS1,SOCS3 mRNA and protein in quiescent patients,but SOCS5 mRNA and protein were significantly increased.Conclusion Up regulation of SOCS1 and SOCS5 expression may involve in the development of a Th1 mediated immune response in sympathetic ophthalmia patients with active uveitis.CIS and SOCS3 may play important roles in mitigating the pathogenic effects of proinflammatory cytokines.It were kept balance in quiescent patients,and up-regulation of SOCS5 may have protective functions. Key words: Suppressor of cytokine signaling; Sympathetic ophthalmia

Critical role of Th17 cells in development of autoimmune hemolytic anemia

Autoimmune hemolytic anemia (AIHA) is defined as the increased destruction of red blood cells (RBCs) in the presence of anti-RBC autoantibodies with or without complement activation. However, the underlying mechanism for the development of AIHA remains largely unclear. In this study, we carefully evaluated the potential role of Th17 cells in the development of AIHA. We found an elevated frequency of Th17 cells in patients with AIHA, which were closely correlated with their disease activity, including the level of anti-RBC IgG antibodies, hemoglobin, serum C3, and lactate dehydrogenase activity. Furthermore, we observed that interleukin (IL)-17 was also closely correlated with the disease activity in AIHA patients. To further elucidate the potential role of Th17 cells in induction of AIHA, we used the Marshall-Clarke and Playfair model of murine AIHA. Notably, we found that Th17 cells affected development of AIHA by enhancing the adaptive humoral responses. Specifically, we found that adoptive transfer of Th17 cells heightened the initial anti-rat RBC antibody responses and concomitantly increased the onset of AIHA. In addition, invivo neutralization of IL-17 abrogated the development of AIHA, while initiation of anti-rat RBC IgG responses and induction of AIHA in IL-17(-/-) mice were impaired. Our findings suggest that Th17 cells contribute to the development of AIHA, which could facilitate our better understanding of AIHA pathogenesis and provide clues to developing novel forms of immunotherapy against AIHA.

ChemInform Abstract: Synthesis and Antiinflammatory Activity of 8H‐1‐Thia‐8‐aza‐dibenzo[e,h]azulenes.

AbstractThe compounds (VI) are tested in vitro for their anti‐inflammatory activity through the inhibition of the necrosis factor alpha production (TNF‐α) in lipopolysaccharide (LPS)‐activated human peripheral blood monocular cells (hPBMC).

Effects of triptolide on RIZ1 expression, proliferation, and apoptosis in multiple myeloma U266 cells

To investigate the effects of triptolide on proliferation and apoptosis as well as on the expression of RIZ1 in the human multiple myeloma cell line U266 in vitro. The effect of triptolide on the growth of U266 cells was studied by MTT assay. Apoptosis was detected by Hoechst 33258 staining and Annexin V/PI double-labeled flow cytometry, and caspase-3 mRNA was measured by RT-PCR. Western blotting, flow cytometry and RT-PCR were used to assess the expression of RIZ1, and the location and expression of H3K9me1 were detected by confocal microscopy and Western blotting. Triptolide significantly inhibited the proliferation of U266 cells in a time- and concentration-dependent manner (the IC(50) value for a 24-h exposure was 157.19+/-0.38 nmol/L). Triptolide induced typical apoptotic morphological changes. Triptolide 40, 80, and 160 nmol/L treatment induced significant caspase-3-dependent apoptosis compared with control group (10.5%+/-1.23%, 37.9%+/-2.45%, and 40.5%+/-2.30% vs 3.8%+/-1.98%, P<0.05). Compared with peripheral blood monocular cells (PBMC) from healthy donors, the protein expression of RIZ1 in U266 cells was relatively low, but the mRNA and protein expression of RIZ1 were strikingly increased by triptolide in a concentration-dependent manner. Triptolide increased the protein expression of RIZ1 and RIZ1 methylates histone H3 lysine 9 in U266 cells. Triptolide increased the protein expression of RIZ1, inhibited the proliferation, and induced caspase-dependent apoptosis in U266 cells.

Inhibition of TNF-alpha secretion from peripheral blood monocular cells by triptolid is associated with TNF-alpha-308 gene polymorphisms in rheumatoid arthritis patients

This study examined the inhibitory effect of triptolid (TP) on tumor necrosis factor-α (TNF-α) secreted from peripheral blood monocular cells (PBMCs) and the association of the inhibitory effect with TNF-α-308 gene polymorphisms in rheumatoid arthritis (RA) patients. Gene polymorphism at A-G site 308 in the promoter region of TNF-α gene was detected in 42 RA patients by using allele specific polymerase chain reaction (AS-PCR) assay. PBMCs were harvested from these patients and treated first with lipopolysaccharides (LPS) and then with different doses of TP (1, 5.4 and 15 ng/mL). The TNF-α level in the supernatants was measured by enzyme-linked immunosorbent assay (ELISA). The results showed that TNF-α level in the supernatants of TP (1 ng/mL)-treated PBMCs was decreased by 3.80% and 4.91%, respectively, in the patients with AA and AG genotypes, when compared with those treated with LPS alone (P>0.05). Moreover, the TNF-α level in the patients with GG genotype was reduced by 20.74% (P 0.05). It was concluded that gene polymorphism at TNF-α-308 sites may relate to the secretion of TNF-α in RA patients. TP has different inhibitory effects on the secretion of TNF-α in the patients harboring different genotypes, which may be one of the reasons for individual variation in response to TP.

Expression of suppressor of cytokine signaling in peripheral blood monocular cells of patients with Vogt-Koyanagi-Harada disease

To investigate suppressor of cytokine signaling (SOCS) mRNA and protein expression in peripheral blood mononuclear cells (PBMC) of patients with Vogt-Koyanagi-Harada (VKH) disease. Blood samples were taken from 15 VKH patients with active uveitis, 17 quiescent patients and 16 healthy individuals. IFN-gamma, IL-12 and IL-4 in the serum were measured by ELISA. PBMC were subjected to analysis of SOCS mRNA and protein expression using quantitative RT-PCR and western blot, respectively. The level of IL-4 in the serum of VKH patients and in controls were (28.40 +/- 5.93) ng/L, (34.5 +/- 9.47) ng/L and (11.25 +/- 4.43) ng/L, IL-12 were (24.33 +/- 8.55) ng/L, (11.53 +/- 6.11) ng/L and (5.19 +/- 2.43) ng/L, IFN-gamma were (18.05 +/- 2.23) ng/L, (15.53 +/- 2.63) ng/L and (1.61 +/- 3.47) ng/L, respectively. The level of IFN-gamma, IL-12 and IL-4 were all significantly higher in the serum of VKH patients than in controls(P < 0.01). IL-4 in quiescent patients was higher than in active patients (P < 0.01), IL-12 and IFN-gamma were lower in quiescent patients was higher than in active patients (P < 0.01, P < 0.05). Cytokine inducible SH2 containing protein (CIS) mRNA, SOCS1 mRNA, SOCS2 mRNA, SOCS3 mRNA and SOCS5 mRNA levels in PBMC of VKH patients with active uveitis are 0.72, 4.92, 1.09, 0.75 and 1.15 folds than that in healthy volunteers, respectively. They are 1.15, 2.25, 1.40, 0.69 and 1.16 folds in static patients, respectively. Marked decreased expression of CIS protein is detected in both active and quiescent patients with no significant difference between two groups (both P < 0.01). SOCS1 protein is up-regulated significantly in active patients compares to in quiescent patients nor in healthy volunteers (P < 0.01, P < 0.05). SOCS3 protein is significantly decreased in patients than in controls (both P < 0.05). SOCS5 protein is much higher in patients than in controls (both P < 0.01), and even higher in quiescent patients than in active episode (P < 0.05). Up regulation of SOCS1 and SOCS5 expression and down-regulation of SOCS3 and CIS may correlate with the development of a Th1 mediated immune response in VKH disease. There is insidious inflammation in VKH patients with clinically quiescent uveitis, and this may be one of the causes of persistence and recurrences of uveitis.

Detection of antigen-epitope-specific cytotoxic T lymphocytes in patients with hepatitis B virus infection by enzyme linked immunospot assay

To investigate the ability of secreting interferon-gamma (IFN-gamma) of the peripheral blood monocular cells (PBMC) stimulated by hepatitis B virus (HBV)-specific cytotoxic T lymphocyte (CTL) epitopes peptides and to analyze the difference of CTL immune response in patients with HBV infection. Four HLA-A2-restricted HBV cytotoxic T lymphocyte epitopes [Tp: HBV polymerase 575-583 (FLLSLGIHL), Te1: envelope 28-39 (IPQSLDSWWTSL), Te2: envelope 183-191 (FLLTRILTI) and Tc: core 18-27 (FLPSDFFPSV)] were synthesized. Human leucocyte antigen (HLA)-A2 typing was detected by Flow cytometry. PBMCs which were isolated from patients with chronic hepatitis B(CHB), patients with chronic severe hepatitis B(CSH), subjects with past HBV infection(N1) and healthy blood donors (N2) were stimulated by the four HLA-A2-restricted HBV CTLs epitopes. Enzyme linked immunospot (ELISPOT) assay was used to detect the frequency of secreting IFN-gamma CTL in each group. (1) HLA-A2 typing: 20 of 44 patients with CHB (45.5%) were HLA-A2 positive, 10/18 (55.6%) in CSH and 6/10 (60%) in group N1 were HLA-A2 positive.10 healthy blood donors' HLA-typing was detected in the early study.(2) ELISPOT results: (1) The total responses to the four epitopes in CHB, CSH, N1 and N2 groups were 50% (10/20), 10% (1/10), 83.3% (5/6) and 10% (1/10), respectively. The response in N1 group was significantly higher than that in CSH group (chi(2) = 9.000, P = 0.008) and N2 group (chi(2) = 9.000, P = 0.008). (2) The CTL average magnitude response to Tp epitope, Te1 epitope, Te2 epitope and Tc epitope was also significantly higher in past HBV infection group (77 SFC/10(6) PBMC, 59 SFC/10(6) PBMC, 100 SFC/10(6) PBMC and 57 SFC/10(6) PBMC, respectively) than that of CSH group (10 SFC/10(6) PBMC, 0 SFC/10(6) PBMC, 0 SFC/10(6) PBMC and 20 SFC/10(6) PBMC respectively, all P < 0.01) and N2 group (15 SFC/10(6) PBMC, 0 SFC/10(6) PBMC, 22 SFC/10(6) PBMC and 30 SFC/10(6) PBMC respectively, all P < 0.01). This study indicates that the T cell immune response to HBV-specific epitopes might be detected either in patient with chronic HBV infection or with previous HBV infection. This response should be much higher in patients with past HBV infection, even the virus had been cleared for long time. These results demonstrate that HBV-specific CTL might play an important role in the clearance of the virus.