TNF-α In Peripheral Blood Mononuclear Cells Research Articles

AIM2 and NLRC4-driven inflammasome activation in adult-onset Still's disease and the preliminary therapeutic effect exploration of carboxyamidotriazole.

This study aimed to explore the changes of four major inflammasomes in adult-onset Still's disease (AOSD) and preliminarily evaluate the therapeutic effect of carboxyamidotriazole (CAI), which has previously been reported to have the significant anti-inflammatory activity. The mRNA expressions of proinflammatory cytokines and inflammasome components in peripheral blood mononuclear cells (PBMCs) from AOSD patients and healthy controls (HC) were determined by reverse transcription-quantitative PCR. Poly(dA:dT)-induced AIM2 inflammasome and flagellin-induced NLRC4 inflammasome activation models were established in bone marrow-derived macrophages (BMDMs). The levels of cytokines in serum and culture supernatants were measured by ELISA method. The serum levels of IL-1β, IL-6, and TNF-α in AOSD patients were significantly higher than those in HC. However, the mRNA expressions of IL-1β, IL-6, IL-18, and TNF-α in PBMCs did not differ markedly in AOSD patients in comparison with HC. Significantly increased mRNA levels of AIM2, NLRC4, ASC, and caspase-1 were observed in patients with AOSD when compared with HC, while NLRP1 and NLRP3 showed no change in AOSD samples. In addition, CAI treatment could significantly reduce the levels of IL-1β, IL-6, and TNF-α secreted by AOSD PBMCs and inhibit AIM2 and NLRC4 inflammasomes activation in BMDMs. Increased levels of proinflammatory cytokines in AOSD might be associated with NLRC4 and AIM2 inflammasomes activation. CAI is likely to have the therapeutic potential for AOSD by inhibiting NLRC4 and AIM2 inflammasomes activation and reducing the proinflammatory cytokines and worthy of further investigation. These results provide new ideas for elucidating the pathogenesis of AOSD and providing specific targeted therapy. Key points • Significantly higher mRNA levels of AIM2 and NLRC4 inflammsome signaling were observed in AOSD patients compared with health controls, indicating that AIM2 and NLRC4 inflammsome activation might be related to the increased proinflammatory cytokines in AOSD. • CAI treatment markedly reduced the secretion levels of cytokines IL-1β, IL-6, and TNF-α in AOSD PBMCs and inhibited AIM2 and NLRC4 inflammasome activation.

Preparation of the sphingolipid fraction from mycelia of Cordyceps sinensis and its immunosuppressive activity

Ethnopharmacological relevanceCordyceps sinensis (CS) is an herbal tonic in traditional Chinese medicine and is used to treat a wide range of disorders, including immune, kidney, respiratory, lung and cardiovascular diseases, in China. Most studies are focused mainly on nucleotides and polysaccharides from CS and consider them to be the main active ingredients, while other ingredients are often disregarded. Hundreds of sphingolipids have been identified from CS and showed inhibitory effects on mouse splenic lymphocytes. Aim of the studyThis study aimed to establish a method for preparing a fraction of sphingolipids from the mycelial powder of CS and evaluate its immunosuppressive activity. Materials and methodsFraction of sphingolipids (Fr-SPLs) were prepared by silica gel chromatography and reversed-phase chromatography. Its components were identified and quantified by Quadrupole-Orbitrap UHPLC–MS/MS. PBMCs were prepared from human blood, and splenic lymphocytes, B cells, and T cells were prepared from mouse spleens. The inhibitory effect of Fr-SPLs on cell viability was evaluated by CCK-8 assay. PBMC apoptosis and the ratio of CD4+ T cells and CD8+ T cells were quantified by flow cytometry analysis. The expression of IL-2, IL-10, and TNF-α in PBMCs was detected by ELISA kits. ResultsA fraction containing 84.83% of sphingolipids (SPLs) was prepared from the mycelia of CS and named Fr-SPLs. 15 SPLs were identified from the Fr-SPLs. Fr-SPLs significantly inhibited the viability of human peripheral blood mononuclear cells (PBMCs) with an IC50 value of 9.82 μg/mL and promoted PBMC apoptosis in a dose-dependent manner. Moreover, Fr-SPLs inhibited the viability of mouse splenocytes, as well as that of B cells and T cells derived from splenocytes. Furthermore, Fr-SPLs reduced the production of IL-2, IL-10, and TNF-α in PBMCs. ConclusionsFr-SPLs show immunosuppressive activity, and this study will be useful for preparing immunosuppressive components from CS and its mycelia for hyperimmune disease.

Marked up-regulation of anti inflammatory cytokine gene expression in the peripheral blood mononuclear cells of postpartum cows with endometritis

This study was designed to determine the mRNA expression profile of inflammatory and anti-inflammatory cytokines in peripheral blood mononuclear cells (PBMC) of endometritic and non-endometritic cows to find out a possible marker for diagnosis of endometritis. Cows (21) including 12 endometritic, and 9 normal (non-endometritic) that did not develop uterine diseases postpartum (pp) were selected. Expression profile (mRNA) of cytokines, viz. IL-1β, TNFα, IL-8 and IL-4 in endometritic cows relative to non-endometritic was studied in PBMC isolated from the blood of the cows collected within 4 to 5 weeks pp. Fold difference (n-fold) in cytokine gene expression in the PBMC of endometritic cows relative to normal was calculated using relative quantification method (2-ΔΔCt). The relative fold change in gene expression for IL-1 was significantly lower in endometritic animals (0.31) than in cows that did not develop endometritis. Significant up-regulation was noted for the proinflammatory cytokine TNFα (9.53 fold and anti-inflammatory cytokine IL-4 (90.09) in cows suffering from endometritis relative to nonendometritic cows. Up-regulation of chemokine IL-8 gene (6.25 fold; was also observed in endometritic compared to non-endometritic cows. Highest and significant up-regulation of mRNA expression for IL-4 followed by TNFα in PBMC of endometritic cows recorded in the study may indicate the development of endometritis in postpartum cows.

The Expression of Toll-Like Receptor 4 mRNA in PBMCs Is Upregulated in Smokers and Decreases Upon Smoking Cessation.

BackgroundStudies have shown in vitro that cigarette smoke condensate stimulates monocytes to express toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule 1 (ICAM-1), and enhances their adhesion to the endothelium. However, the same effects of cigarette smoking have not been explored in vivo. This study is to investigate the effect of cigarette smoking and smoking cessation on their mRNA expression in human peripheral blood mononuclear cells (PBMCs).MethodsA group of 97 smokers and 62 nonsmokers were enrolled. The RNA from PBMCs was assessed with real-time polymerase chain reaction (PCR) to determine the levels of ICAM-1, TNF-α, and TLR4. The same markers in PBMCs of 87 quitters were examined before and at one week, one month, and two months after smoking cessation.ResultsOf the 97 smokers, 85 (87.6%) were males, and 30 (48.4%) of the nonsmokers were males (p < 0.0001). The mean (SD) age of the smokers was 43.24 (10.89) years, which was younger than 43.45 (11.41) years of nonsmokers (p < 0.0001). The incidence of cardiovascular diseases was 13.4% in smokers, which was higher than 1.6% in nonsmokers (p < 0.05). Both ICAM-1 and TNF-α mRNA levels in PBMCs were higher among the smokers (p < 0.0001). In addition, TLR4 mRNA levels in PBMCs were statistically elevated in the smokers (p < 0.0001) comparing with those in the nonsmokers. The mRNA levels of TLR4 and TNF-α in PBMCs decreased in those who had quit smoking for 2 months (p < 0.0001).ConclusionsICAM-1, TNF-α, and TLR4 mRNA expression levels in PBMCs increased in smokers and decreased after being on a smoking cessation program for 2 months. This finding suggested that TLR4 expression may mediate the atherogenic inflammatory process induced by smoking.

Abstract PO044: RTX-321, an allogeneic red blood cell-based artificial antigen presenting cell, expressing MHC I-peptide, 4-1BBL and IL-12, engages primary human HPV-specific T cells and boosts other general immune responses

Abstract The high-risk human papillomavirus (HPV) strain 16 accounts for ~ 70% of all cervical cancers and 80% of head and neck cancers associated with HPV infection., Despite available therapies, there remains a critical need for new treatment options for advanced HPV 16-associated cancers. To address this, we have genetically engineered red blood cells to create an allogeneic artificial antigen presenting cell (APC), RTX-321, that expresses an HPV E7 peptide bound to MHC I (HLA-A*02:01), 4-1BBL and IL-12 on the cell surface to mimic the biology of T cell APC interactions. RTX-321 is designed to enhance both the quantity and quality of endogenous tumor-specific T cells. In this study, we evaluated the mechanisms through which RTX-321 promotes anti-tumor immune responses, particularly its ability to engage and activate HPV 16-specific CD8+ T cells in the context of mixed immune populations in peripheral blood mononuclear cells (PBMCs) and to modulate other key immune cell types in PBMCs. Primary CD8+ T cells were engineered to express the HPV E7 TCR, resulting in HPV E7-specific CD8+ T cells (E7 TCR-T). To determine whether RTX-321’s immune modulatory activities are dependent on the presence of antigen-specific T cells, we examined the effect of RTX-321 on PMBCs admixed with E7 TCR-T as compared to PBMCs alone. RTX-321 selectively expanded HPV E7-specific CD8+ T cells from the PBMC mixture in a dose-dependent manner. RTX-321 preferentially upregulated activation markers and effector molecules, and promoted effector memory on HPV E7-specific CD8+ T cells, compared to HPV independent CD8+ T cells. In addition to these HPV antigen-specific responses, modulations on other immune cells were observed with RTX-321 treatment. RTX-321 induced activation marker upregulation on general CD8+ T cells, as well as NK cell expansion, activation and effector molecule upregulation, independent of the presence of E7 TCR-T. In PBMCs, RTX-321 increased proinflammatory cytokine and chemokine secretion, including IFNγ, TNFα and CXCL10. The addition of E7 TCR-T cells further increased the production of IFNγ and TNFα in PBMCs, suggesting its potential role in further promoting the immune response by secreting effector molecules. Overall, our data indicate that RTX-321 not only engages HPV 16 antigen-specific CD8+ T cells, but also other key immune cell populations of the adaptive and innate immune systems to promote a broad and robust anti-tumor response. An Investigational New Drug application for RTX-321 for the treatment of patients with HPV 16-positive solid tumors is planned by the end of 2020. Citation Format: Mengyao Luo, Shamael S. Dastagir, Xuqing Zhang, Andrea Schmidt, Beatriz Marques, Timothy J. Lyford, Billy Blanco, Laurence A. Turka, Thomas J. Wickham, Tiffany F. Chen. RTX-321, an allogeneic red blood cell-based artificial antigen presenting cell, expressing MHC I-peptide, 4-1BBL and IL-12, engages primary human HPV-specific T cells and boosts other general immune responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO044.

Plasma ST6GAL1 regulates IgG sialylation to control IgA nephropathy progression.

Background:Our previous study revealed that plasma levels of a-2,6-sialyltransferase 1 (ST6GAL1) were increased in patients with IgA nephropathy (IgAN). ST6GAL1 catalyzes terminal sialylation of IgG to shift the antibody effector function to the anti-inflammatory pattern. However, the role of plasma ST6GAL1 in the progression of IgAN and underlying mechanisms are still unknown.Methods:A total of 180 IgAN patients were included. The kidney outcomes were defined as the eGFR decline or proteinuria remission. Peripheral blood mononuclear cells (PBMCs) were either stimulated with purified sialylated IgG (SA-IgG) or with non-sialylated IgG (NSA-IgG) from IgAN patients to detect the levels of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) in supernatant.Results:Compared with the lower ST6GAL1 (reference), the risk of eGFR decline decreased for the higher ST6GAL1 group after adjustment for baseline eGFR, systolic blood pressure (SBP), and proteinuria. The results showed that patients with higher ST6GAL1 levels had a higher rate of proteinuria remission. ST6GAL1, expressed as a continuous variable, was a protective factor for eGFR decline and proteinuria remission. An in vitro study showed that the administration of recombinant ST6GAL1 (rST6GAL1) decreased the levels of IL-6 and TNF-α in PBMCs. Furthermore, the administration of rST6GAL1 resulted in the enrichment of SA-IgG in a concentration-dependent manner. In addition, as compared to control, purified SA-IgG-treated PBMCs showed a significant decrease in the expression of IL-6 and TNF-α.Conclusion:Our study indicated that elevated ST6GAL1 was associated with a slower progression of IgAN, which may play a protective effect by increasing IgG sialylation to inhibit the production of proinflammatory cytokines in PBMCs.

Association between cytokines and two circulating micro-RNAs and development of premature ventricular contractions-induced cardiomyopathy.

Objective(s):Recent progress in understanding the pathogenesis of premature ventricular contraction (PVC)-induced cardiomyopathy (PIC) has suggested a key role for inflammation. The aim of this study was to evaluate the expression of messenger RNAs (mRNAs) and the protein production of interleukin-6 (IL-6), IL-10, tumor necrosis factor alpha (TNF-α) and interferon-γ (IFN-γ) and two circulating micro-RNAs related to inflammation and cardiovascular disease; miR-155 and miR-146.Materials and Methods:The study population was comprised 25 patients with PIC and 25 patients with normal left ventricular ejection fraction despite frequent PVCs. TNF-α, IL-6, IL10, and IFN-γ levels were evaluated in peripheral blood mononuclear cells (PBMCs) by flow cytometry and their mRNAs were assessed by real time PCR. We analyzed circulating levels of these cytokines by enzyme linked immunosorbent assay (ELISA). Two circulating micro-RNAs, miR-155 and miR-146a, were also investigated.Results:The flow cytometry findings showed that the median fluorescence intensity (MFI) of antibodies reacted with the IL-6 and TNF-α were higher in PIC group than the control group (P-value<0.001). In ELISA, the levels of IL-6 (P-value<0.001) and TNF-α (P-value <0.001) and in RT-PCR the relative expression levels of IL-6 (P-value<0.001) and TNF-α (P-value<0.001) were significantly higher in the PIC group. The relative expression levels of miR-155 and miR-146a were not significantly different between 2 groups (P-value>0.05).Conclusion:In our patients with PIC, there was an elevation in the expression levels of IL-6 and TNF-α in PBMCs. This finding may provide further insights into the inflammatory pathways involved in PIC.

Levels of TNF-α in PBMC (Peripheral Blood Mononuclear Cells) Induced by Recombinant Non Structural 1 Protein of Dengue Virus Serotype-2 in vitro

Levels of TNF-α in PBMC (Peripheral Blood Mononuclear Cells) Induced by Recombinant Non Structural 1 Protein of Dengue Virus Serotype-2 in vitro

KFL2 participates in the development of ulcerative colitis through inhibiting inflammation via regulating cytokines.

Ulcerative colitis (UC) is a kind of chronic inflammatory bowel diseases that seriously endangers human health. The pathogenesis of UC is closely related to the intestinal immune response. Cytokines exert a key role in the regulation of intestinal inflammatory and immune responses. Abnormalities in the function and quantity of various cytokines or imbalance of inflammatory factors and immune factors would lead to UC development. We aimed to investigate whether Kruppel-like transcription factor 2 (KFL2) participates in the development of ulcerative colitis by regulating inflammation, so as to provide a new direction for the clinical treatment. 40 UC patients were enrolled in this study, including 20 patients with mild ulcerative colitis (MUC) and 20 with severe ulcerative colitis (SUC). 20 normal end of intestinal tissues surgically resected from patients with colorectal cancer in the same period were selected as the control group. Hematoxylin-eosin (HE) staining was used to detect the inflammatory infiltration of intestinal mucosa tissues. Expressions of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-α) in peripheral blood mononuclear cells (PBMCs) of each group were detected by qRT-PCR (quantitative Real-Time Polymerase Chain Reaction). Immunohistochemistry was performed to observe the infiltration of IL-6 and TNF-α in intestinal mucosal tissues. Protein and mRNA levels of KLF2 in PBMCs of each group were detected by Western blot and qRT-PCR, respectively. The relationship between the mRNA level of KLF2 in PBMCs and expressions of IL-6, IL-8, IL-10, TNF-α were analyzed using qRT-PCR. Inflammatory cells and cytokines were infiltrated in the intestinal mucosa of UC patients. IL-6, IL-8, IL-10, and TNF-α were overexpressed in PBMCs of UC patients than those of controls. Protein and mRNA levels of KLF2 in PBMCs of UC patients were remarkably lower than those of controls, which were more significant in SUC patients. Meanwhile, KLF2 was closely related to expressions of IL-6, IL-8, IL-10, and TNF-α in PBMCs of UC patients. KLF2 was downregulated in PBMCs of UC patients than that of normal controls, which participated in the inflammatory response of UC by regulating expressions of IL-6, IL-8, IL-10, and TNF-α. KLF2 may suggest new treatments for ulcerative colitis.

MIF promotes a differential Th1/Th2/Th17 inflammatory response in human primary cell cultures: Predominance of Th17 cytokine profile in PBMC from healthy subjects and increase of IL-6 and TNF-α in PBMC from active SLE patients

Macrophage migration Inhibitory Factor (MIF) is a cytokine associated with the pathogenesis of autoimmune inflammatory diseases. There is evidence that MIF functions in a positive-feedback-loop with proinflammatory cytokines and could perpetuate the inflammatory process in Systemic Lupus Erythematosus (SLE).The aim of this study was to assess the effect of recombinant-human-MIF (rhMIF) on the expression of Th1, Th2 and Th17 cytokines in Peripheral Blood Mononuclear Cells (PBMC) from Healthy Subjects (HS) and SLE patients. The PBMC were isolated from SLE patients classified according to the 1997 SLE ACR criteria and HS donors; all subjects included were women from an unrelated Mexican-Mestizo population. The PBMC isolated were stimulated with rhMIF, LPS and ISO-1 in different combinations; Th1, Th2 and Th17cytokine profiles levels were determined by MAGPIX Bio-plex assay in supernatants from cell cultures. We observed in supernatants of PBMCs from HS treated with rhMIF a predominance of Th17 cytokine profile with an increase of IL-17A, IL-17F and IL-21 versus PBMCs from SLE patients, which showed an inflammatory profile represented by increase of IL-6 cytokine. According to SLE remission/activity presented at enrollment in the study (Mex-SLEDAI index), the PBMC from active SLE patients showed higher levels of TNF-α and IL-6 versus PBMC from remission SLE patients. In conclusion, our results suggest that MIF can induce a differential inflammatory response in physiological and pathological conditions with a predominance of a Th17 cytokine profile in PBMC from HS and an increase in TNF-α and IL-6 expression in PBMC from active SLE patients.

Recombinant human tumor necrosis factor receptor type II: IgG Fc fusion protein combined with methotrexate for the treatment of psoriasis and their effects on levels of interleukin-17A and tumor necrosis factor-α

Objective To evaluate the effect of recombinant human tumor necrosis factor receptor type Ⅱ: IgG Fc fusion protein (rhTNFR:Fc, trade name Etanercept) combined with methotrexate on levels of interleukin-17A (IL-17A) and tumor necrosis factor-α (TNF-α) in the serum and mononuclear cells of patients with moderate to severe plaque psoriasis. Methods A total of 30 patients with moderate to severe plaque psoriasis were enrolled from Department of Dermatology of Tenth People′s Hospital of Tongji University between August 2014 and February 2016, and then were randomly and equally divided into Etanercept group and Etanercept+ methotrexate group. The treatment lasted 24 weeks. Fifteen healthy blood donors served as healthy control group. Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR were performed to measure the serum levels and mRNA expression of IL-17A and TNF-α, respectively, in the patients of the above two groups before and after the treatment. Results Before the treatment, the serum levels of IL-17A and TNF-α, as well as the mRNA expression of IL-17A and TNF-α in peripheral blood mononuclear cells (PBMCs) , were all significantly higher in all the patients than in the healthy controls (all P < 0.05) . After the treatment, compared with the Etanercept group, the Etanercept+ methotrexate group showed significantly lower serum levels of IL-17A (142.67 ± 14.82 vs. 163.54 ± 23.18,P < 0.05) and TNF-α (70.07 ± 25.02 vs. 91.98 ± 14.62, P < 0.05) , as well as lower mRNA expression of IL-17A (1.12 ± 0.33 vs. 1.56 ± 0.77, P < 0.05) and TNF-α in PBMCs (2.50 ± 1.04 vs. 3.61 ± 2.14, P < 0.05) . Conclusion Etanercept combined with methotrexate is superior to Etanercept alone in the treatment of psoriasis, and can reduce treatment duration and improve therapeutic effect, likely by down-regulating the expression of IL-17A and TNF-α. Key words: Psoriasis; Receptors, tumor necrosis factor, type II; Recombinant fusion proteins; Methotrexate; Interleukin-17; Tumor necrosis factor-alpha

The anti-inflammatory and pro-resolution effects of aspirin-triggered RvD1 (AT-RvD1) on peripheral blood mononuclear cells from patients with severe asthma

Asthma is an inflammatory disease that is characterized by a predominance of eosinophils and/or neutrophils in the airways. In the resolution of inflammation, lipid mediators such as resolvin D1 (RvD1) and its epimer aspirin-triggered RvD1 (AT-RvD1) are produced and demonstrate anti-inflammatory and pro-resolution effects. In experimental models such as airway allergic inflammation induced by ovalbumin in mice, RvD1 and AT-RvD1 alleviate some of the most important phenotypes of asthma. Here, we demonstrated the effects of AT-RvD1 on peripheral blood mononuclear cells (PBMCs) from healthy individuals and patients with severe asthma stimulated with lipopolysaccharide (LPS) or Dermatophagoides pteronyssinus (DM). AT-RvD1 (100nM) reduced the concentration of TNF-α in PBMCs from healthy individuals and patients with severe asthma stimulated with LPS or DM. In addition, AT-RvD1 lowered the production of IL-10 only in PBMCs from patients with severe asthma stimulated with LPS. These effects were associated in part with decreasing NF-κB activation. Moreover, AT-RvD1 significantly increased phagocytosis of apoptotic neutrophils by monocytes from patients with severe asthma. In conclusion, AT-RvD1 demonstrated both anti-inflammatory and pro-resolution effects in PBMCs from patients with severe asthma and could become in the future an alternative treatment for asthma.

Budesonide increases TLR4 and TLR2 expression in Treg lymphocytes of allergic asthmatics

BackgroundReduced innate immunity responses as well as reduced T regulatory activities characterise bronchial asthma. ObjectivesIn this study the effect of budesonide on the expression of TLR4 and TLR2 in T regulatory lymphocyte sub-population was assessed. MethodsTLR4 and TLR2 expression in total peripheral blood mononuclear cells (PBMC), in CD4+/CD25+ and in CD4+/CD25− was evaluated, by flow cytometric analysis, in mild intermittent asthmatics (n = 14) and in controls (n = 11). The in vitro effects of budesonide in modulating: TLR4 and TLR2 expression in controls and in asthmatics; IL-10 expression and cytokine release (IL-6 and TNF-α selected by a multiplex assay) in asthmatics were also explored. ResultsTLR4 and TLR2 were reduced in total PBMC from asthmatics in comparison to PBMC from controls. CD4+CD25+ cells expressed at higher extent TLR2 and TLR4 in comparison to CD4+CD25− cells. Budesonide was able to increase the expression of TLR4, TLR2 and IL-10 in CD4+/CD25highly+ cells from asthmatics. TLR4 ligand, LPS induced Foxp3 expression. Budesonide was also able to reduce the release of IL-6 and TNF-α by PBMC of asthmatics. ConclusionsBudesonide potentiates the activity of Treg by increasing TLR4, TLR2 and IL-10 expression. This event is associated to the decreased release of IL-6 and TNF-α in PBMC treated with budesonide. These findings shed light on new mechanisms by which corticosteroids, drugs widely used for the clinical management of bronchial asthma, control T lymphocyte activation.

Increased expression of IL-37 in patients with Graves' disease and its contribution to suppression of proinflammatory cytokines production in peripheral blood mononuclear cells.

BackgroundIntreleukin-37 (IL-37), a member of IL-1 family, is primarily an anti-inflammatory cytokine, which reduces systemic and local inflammation. However, the expression and role of IL-37 in Graves' disease (GD) remains unknown. This study aims to measure the levels of serum and peripheral blood mononuclear cells (PBMCs) IL-37 in patients with Graves' disease and to examine its association with disease activity. Furthermore, we investigate the effect of IL-37 on proinflammatory cytokines involved in the pathogenesis of GD.MethodsThe expressions of IL-37, TNF-α, IL-6, and IL-17 mRNA in peripheral blood mononuclear cells (PBMCs) of 40 patients with Graves' disease were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR), and the levels of IL-37, TNF-α, IL-6, and IL-17 in serum were detected by enzyme-linked immunoassay (ELISA). The correlation of serum IL-37 levels with cytokines and disease activity in Graves' disease patients were investigated. The expressions of cytokines TNF-α, IL-6, and IL-17 in PBMCs under recombinant IL-37 stimulation were determined by RT-PCR and ELISA respectively.ResultsThe levels of IL-37, TNF-α, IL-6, and IL-17 in PBMCs and serum were significantly increased in patients with GD compared with healthy controls (HC). Serum IL-37 were closely correlated with TNF-α, IL-6, IL-17, thyrotropin (TSH), free thyroxine (FT4),free triiodothyronine (FT3) and thyrotropin receptor antibody (TRAB). GD patients with active disease showed higher IL-37 mRNA and serum protein levels compared with those with inactive disease as well as HC. Moreover, IL-37 suppressed the production of IL-6, IL-17 and TNF-α in PBMCs of patients with GD.ConclusionsIncreased level of IL-37 in patients with GD are associated with TNF-α, IL-6, IL-17 and disease activity, and it plays a protective role against inflammatory effect in GD by inhibiting the production of proinflammatory cytokines. Thus, IL-37 may provide a novel research target for the pathogenesis and therapy of GD.

AB0237 Positive Correlation of ULK1 and MMP-9 versus Negative Correlation of Mtor and Tnfα Baseline Gene Expressions in the Peripheral Blood with the Disease Activity and Joint Destruction Indices Registered in Rheumatoid Arthritic Patients after Treatment

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia, mononuclear cell infiltration, bone erosion and joint destruction. We have shown recently the involvement of genes responsible for...

Subjects with familial hypercholesterolemia are characterized by an inflammatory phenotype despite long-term intensive cholesterol lowering treatment

ObjectiveThe atherosclerotic process is driven by elevated Low-density lipoprotein (LDL)-cholesterol in combination with enhanced inflammatory responses. Several mediators participate in this complex inflammatory network including members of the tumour necrosis factor (receptor) superfamily. Familial hypercholesterolemia (FH) is associated with increased risk of developing premature atherosclerosis. Statin treatment may normalize LDL-cholesterol levels, but it is not known if the inflammatory responses are normalized in statin-treated FH patients. MethodsIn long-term statin-treated FH subjects (n = 33) and healthy controls (n = 10) the expression of tumour necrosis factor (receptor) superfamily related genes in peripheral blood mononuclear cells (PBMC) were analyzed by real-time quantitative RT-PCR. TNFα release was measured in PBMC from patients and controls by immunoassay. ResultsIn FH patients with normal LDL-cholesterol, after a median of 17 years of statin treatment, our major findings were: (i) Gene expression of CD137, LIGHT (lymphotoxins inducible expression, competes with HSV glycoprotein D for HVEM, a receptor expressed on T-lymphocytes), HVEM (Herpesvirus entry mediator), the two TNFα Receptors (TNFR1 and TNFR2), TNF related apoptosis inducing ligand (TRAIL) and CD40 were increased in PBMC from FH patients compared to controls. (ii) The release of TNFα in PBMC from FH patients, in response to LPS was increased compared to controls. (iii) PBMC from FH patients had enhanced spontaneous release of TNFα when incubated in the presence of control serum and in particular in the presence of FH serum. ConclusionDespite long-term statin therapy, an increased expression of several TNF related genes in PBMC isolated from FH patients was observed. Our findings may implicate a pathogenic role for inflammation and TNF related molecules in FH, and these findings suggest the possibility that novel treatment modalities beyond that of statins and lipid lowering drugs may be useful in FH subjects.

Idiopathic inflammatory myopathies – potential role of the immunoexpression of interleukin-35 in muscle biopsy and peripheral blood mononuclear cells

Background Interleukin-35 (IL-35) is a newly described hetero-dimeric cytokine of the IL-12 family. Studies of immunopathogenesis in idiopathic inflammatory myopathies (IIM) are of interest for future biological therapies. Objective To analyze expression of IL-35 in muscle biopsies and to study its effects on peripheral blood mononuclear cells (PBMC) in vitro to assess its pathogenetic and diagnostic value. Materials and methods Immunoexpression of IL-35 subunits (p35, EBI3) was studied immunohistochemically in 23 muscle biopsies of IIM (12 dermatomyositis, 11 polymyositis), 10 noninflammatory myopathies and 10 controls. PBMCs were stimulated with human recombinant IL-35 protein or TNF-α in vitro. Taq-Man RT-PCR was performed to analyze gene expression after 6hours and ELISA assays were used to confirm the protein secretion after 24 hours of stimulation. Results We showed localization of both IL-35 subunits in B-cells, T-cells and macrophages of the inflammatory infiltrates in IIM. No immunoreactivity was observed in controls. We found that gene expression of both IL-35 subunits were dose- and time-dependently induced by TNF-α in PBMCs. Conclusion Our data suggest pro-inflammatory properties of IL-35 and its potential role in the pathogenesis of IIM. The immunohistochemical expression of IL-35 might be also of diagnostic help in muscle biopsy.

TLR7/TLR8 Activation Restores Defective Cytokine Secretion by Myeloid Dendritic Cells but Not by Plasmacytoid Dendritic Cells in HIV-Infected Pregnant Women and Newborns

Mother-to-child transmission (MTCT) of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting in an increased number of HIV-exposed uninfected infants. The consequences of HIV infection on the innate immune system of both mother-newborn are not well understood. In this study, we analyzed peripheral blood and umbilical cord blood (CB) collected from HIV-1-infected and uninfected pregnant women. We measured TNF-α, IL-10 and IFN-α secretion after the stimulation of the cells with agonists of both extracellular Toll-like receptors (TLRs) (TLR2, TLR4 and TLR5) and intracellular TLRs (TLR7, TLR7/8 and TLR9). Moreover, as an indicator of the innate immune response, we evaluated the responsiveness of myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) to TLRs that are associated with the antiviral response. Our results showed that peripheral blood mononuclear cells (PBMCs) from HIV-1-infected mothers and CB were defective in TNF-α production after activation by TLR2, TLR5, TLR3 and TLR7. However, the TNF-α response was preserved after TLR7/8 (CL097) stimulation, mainly in the neonatal cells. Furthermore, only CL097 activation was able to induce IL-10 and IFN-α secretion in both maternal and CB cells in the infected group. An increase in IFN-α secretion was observed in CL097-treated CB from HIV-infected mothers compared with control mothers. The effectiveness of CL097 stimulation was confirmed by observation of similar mRNA levels of interferon regulatory factor-7 (IRF-7), IFN-α and TNF-α in PBMCs of both groups. The function of both mDCs and pDCs was markedly compromised in the HIV-infected group, and although TLR7/TLR8 activation overcame the impairment in TNF-α secretion by mDCs, such stimulation was unable to reverse the dysfunctional type I IFN response by pDCs in the HIV-infected samples. Our findings highlight the dysfunction of innate immunity in HIV-infected mother-newborn pairs. The activation of the TLR7/8 pathway could function as an adjuvant to improve maternal-neonatal innate immunity.

Immunomodulatory and hemagglutinating activities of acidic polysaccharides isolated from Combretum racemosum

Extracts of leaves of different species of the genus Combretum have been used historically to treat a variety of medicinal problems. However, little is known about the active components conferring therapeutic properties to these extracts. In the present studies, we evaluated biochemical properties and immunomodulatory activity of polysaccharides isolated from the leaves of Combretum racemosum. Water-soluble polysaccharides from leaves of C. racemosum were extracted and fractionated by DEAE-cellulose and Diaion HP-20 to obtain a Diaion-bound fraction, designated Combretum polysaccharide-acidic bound or CP-AB, which was eluted with methanol, and an unbound fraction, designated as CP-AU. Molecular weight determination, sugar analysis, and other physical and chemical characterization of the fractions were performed. Fraction CP-AU (mol. weight 5.0kDa) contained type II arabinogalactan and had potent immunomodulatory activity, inducing the production of interleukin (IL)-1β, -6, -10, and tumor necrosis factor-α (TNF-α) by human peripheral blood mononuclear cells (PBMC) and MonoMac-6 monocytic cells. Likewise, intraperitoneal administration of CP-AU increased in vivo serum levels of IL-6 and monocyte chemoattractant protein-1 (MCP-1) in mice. CP-AU-induced secretion of TNF-α in PBMC was prevented by Toll-like receptor 4 (TLR4) antagonist LPS-RS. Treatment with CP-AU induced phosphorylation of Akt2, Akt3, GSK-3β, HSP27, mTOR, and all p38 MAPK isoforms (α, β, δ, and γ), as well as stimulation of AP-1/NF-κB transcriptional activity. In addition, CP-AU effectively agglutinated erythrocytes from several species, including human, mouse, and rabbit. In contrast, fraction CP-AB was inactive in all biological tests, including cytokine production and hemagglutination. These data suggest that at least part of the beneficial therapeutic effects reported for the water extracts of leaves from C. racemosum are due to modulation of leukocyte functions.

TLR4 and RAGE: Similar routes leading to inflammation in type 2 diabetic patients

Aim The present study investigates the interaction of TLR4 and RAGE with their respective ligands as inducers of the inflammatory markers IL-6 and TNF-α. Also, the reactivity of peripheral blood mononuclear cells (PBMNC) from type 2 diabetic (T2D) patients and non-diabetic healthy controls (ND) were comparatively studied. Methods Concentrations of IL-6 and TNF-α were measured by sandwich Elisa, using kits supplied by Assay Designs (Ann Arbor, MI, USA). PBMNC from T2D and ND were incubated in the presence or absence of LPS, anti-TLR4 or anti-RAGE for 72 hours at 37° C under 5% CO 2. The final volume was adjusted to 300 μL in DMEM supplemented with 10% fetal bovine serum. After incubation, the cells were centrifuged, the supernatant collected and the cytokines measured. Results PBMNC from T2D were more sensitive to innate immune stimulation with LPS and monoclonal agonist anti-TLR4 than were cells from ND. The actions of LPS, anti-TLR4 and anti-RAGE potentiated the production of IL-6 and TNF-α in both groups. The simultaneous activation of monoclonal anti-RAGE and anti-TLR4 suggests that both antibodies used different receptors on the cell surface, but converged on the same PBMNC signaling metabolic pathways. This simultaneous activation induced a higher production of IL-6 and TNF-α in PBMNC from the T2D patients than from the ND subjects. Conclusion Our results clearly show an exacerbation of innate immunity in PBMNC with T2D that was possibly hyperglycaemia-induced. These data, when analyzed together, suggest the importance of innate immunity in the pathogenesis of T2D.