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ALTERATION IN THE CYTOKINE SECRETION PATTERN IN T CELLS OF PATIENTS WITH CYSTIC FIBROSIS CAUSED BY DNA METHYLTRANSFERASE INHIBITOR 5-AZACITIDINE.

Cystic fibrosis (CF) is the autosomal-recessive disorder caused by mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The Airway inflammation plays a central role in the progression of CF disease. Cystic fibrosis characterized by the overproduction of the pro-inflammatory cytokines and reduced expression of anti-inflammatory cytokines. Although the mechanisms of abnormal cytokine expression is still poorly understood, altered epigenetic regulations in T cells might contribute. In the present study we examined the expression of IFN-γ and IL-10 by CF T cells prior to and following 5-azaC treatment. In addition we investigated DNMTs levels in nuclear extracts of CD4+ T cells derived from CF and non-CF individuals. Seven CF patients (age: 5-12 years) were included in the study and compared to six age-matched healthy subjects (age: 6- 13 years). CD4+ T cells were isolated from PBMC using CD4 MicroBead kit (Miltenyi Biotec GmbH) and were cultured in RPMI 1640 medium at 37°C with 5% CO2, in presence or absence of 5-azacytidine. Concentrations of IL-10 and γ-INF in CD4+ T Cells were measured by ELISA (eBoiscience, san Diego, CA, USA). In our study we showed that 5 Azacytidine alters nuclear levels of DNMT 3a as well as modulates cytokine levels in CD4+ T cells derived from CF patients. After 5-azaC treatment secretion of IFN-γ was significantly decreased in CF T cells, while amount of IL-10 was elevated by ~2.5 times compared to untreated controls (P<0.05). In summary, data presented in this report demonstrates that epigenetic mechanisms such as DNA methylation may be considered as a one of the potential therapeutic target in a treatment of Cystic Fibrosis.

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Human Leukocyte Antigen-G Inhibits the Anti-Tumor Effect of Natural Killer Cells via Immunoglobulin-Like Transcript 2 in Gastric Cancer.

Human leukocyte antigen-G (HLA-G) plays an important role in inhibiting natural killer (NK) cell function and promoting immune escape. However, the specific mechanism of HLA-G on NK in gastric cancer (GC) remains not well understood. This study investigated the expression of HLA-G in GC and the role of HLA-G-effected NK cells in GC progression.

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Generation and Characterization of a Bispecific Antibody Targeting Both PD-1 and c-MET.

Bispecific antibodies, BsAbs, are molecules with the ability to bind to two different epitopes on the same or different antigens. c-MET, cellular-mesenchymal to epithelial transition factor, is deregulated in many types of human malignancies. Abnormal c-MET activation in cancer correlates with poor prognosis. PD-1, programmed death-1, is an additional inhibitory receptor expressed by T cells. Blocking the interactions between PD-1 and PD-L1 has emerged as a promising immunotherapy for treating cancer.

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Serum IFN-λ3 Levels Correlate with Serum Hepatitis C Virus RNA Levels in Symptomatic Patients with Acute Hepatitis C.

Recent genome-wide association studies demonstrated that 2 single nucleotide polymorphisms (SNPs), upstream of the interferon-λ (IFNL) 3 gene, are associated with the spontaneous clearance of hepatitis C virus (HCV) in symptomatic patients with acute hepatitis C (AHC). Although these 2 SNPs, rs8099917 and rs12979860, have established their significant roles in the innate immunity response to spontaneously clear HCV in patients with AHC, the detailed mechanisms of their roles remain largely unknown.

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Role of S100A9 in the development of neutrophilic inflammation in asthmatics and in a murine model.

S100A9 is an endogenous danger signal that promotes and exacerbates the neutrophilic inflammatory response. To investigate the role of S100A9 in neutrophilic asthma, S100A9 levels were measured in sputum from 101 steroid-naïve asthmatics using an ELISA kit and the levels were significantly correlated with percentages of neutrophils in sputum. Intranasal administration of recombinant S100A9 markedly increased neutrophil numbers at 8h and 24h later with concomitant elevation of IL-1β, IL-17, and IFN-γ levels. Treatment with an anti-S100A9 antibody restored the increased numbers of neutrophils and the increased airway resistance in OVA/CFA mice toward the levels of sham-treated mice. Concomitantly, the S100A9 and neutrophil elastase double positive cells were markedly reduced with attenuation of IL-1β, IL-17, and IFN-γ levels by the treatment with the anti-S100A9 antibody. Our data support a role of S100A9 to initiate and amplify the neutrophilic inflammation in asthma, possibly via inducing IL-1β, IL-17 and IFN-γ.

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Expression and role of interleukin-9 in Vogt-Koyanagi-Harada disease.

Vogt-Koyanagi-Harada (VKH) disease is a systemic autoimmune disease that can lead to blindness. This study was designed to investigate whether interleukin (IL)-9 plays a role in the development of VKH disease.

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[Inhibitory effect of bispecific antibody targeting IL-12 p40 and TNF-α simultaneously on psoriasis in mice].

Objective To construct bispecific antibodies, which can block interleukin 12 (IL-12)/IL-23 p40 subunit and tumor necrosis factor α (TNF-α) simultaneously, and identify their biological function and inhibitory effect on psoriasis formation in mice. Methods Based on the sequences of adalimumab and ustekinumab, three kinds of bispecific antibodies were designed, named BiAU003, BiAU022 and BiAU023. The specificity and binding capacity of bispecific antibodies were determined by ELISA. After co-treated with bispecific antibodies and TNF-α, the level of endothelial leukocyte adhesion molecule-1 (ELMA-1) labeled with fluorescein isothiocyanate (FITC) in human umbilical vein endothelial cells (HUVECs) were examined by flow cytometry. Human peripheral blood mononuclear cells (PBMCs) were purified and cultured in the medium containing IL-2 and IL-12 in the absence or presence of bispecific antibodies. Commercial ELISA kit was used to detect interferon γ (IFN-γ) concentration in the supernatant. BALB/c mice were used for psoriasis model construction through injection of IL-12 and TNF-α subcutaneously. Then they were treated with the bispecific antibodies. Psoriasic skin was measured in thickness and scale under microscopy after H&E staining. Results The three kinds of bispecific antibodies could specifically recognize IL-12/23 p40 and TNF-α protein, and inhibit IFN-γ secretion and the expression of ELAM-1 protein. Data also indicated that bispecific antibodies inhibited the formation of psoriasic skin, and showed an equal or superior effect to control antibody drug. Conclusion The novel bispecific antibodies, BiAU003, BiAU022 and BiAU023, can serve as an antagonist of TNF-α and IL-12/23 p40, and have a blocking effect on mouse psoriasis formation.

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Biochemical and functional analysis of corticotropin releasing factor purified from an aqueous extract of human placenta used as wound healer.

Human placental extract constitutes of innumerable therapeutically important components mostly used in wound healing arising from the skin and burn injuries. However, there is still some bioactive present in the placental extracts yet to be characterized to better under the complex process of wound healing mediated by the placental extract. In this study, the presence of corticotropin releasing factor (CRF) in an aqueous extract of human placenta was detected and quantified by dot blot and CRF-ELISA immunoassay kit respectively. Subsequently, it was purified by immuno-affinity chromatography and quantified as 0.45±0.05μg of CRF per ml of placental extract where its molecular weight found to be 4.78kDa by MALDI-TOF. To study functional analysis of CRF, an in vitro WI-38 lung fibroblast cell scratch wound model was used which indicated proliferation, motility of cells after treatment with purified CRF. Moreover, reduction in apoptosis rate of cells during closure of wound was observed from microscopy studies and FACS analysis. Also, Antalarmin, an antagonist of CRF type 1 receptor inhibited the wound closure potency of the purified component. Faster healing of wound with an elevation of IL-6 and TGF-β during early stages of repair by placental CRF was observed on excision rat model. The process of healing was accompanied by the decrease in the level of TNF-α and IFN-γ.

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[Identification and evaluation of T cell epitopes of Rv0585c from ].

To investigate the human T cell epitopes of () Rv0585c protein antigen and their immunogenicity and provide evidence for the development of specific tuberculosis immune diagnostic techniques and tuberculosis vaccine. We synthesized peptides from Rv0585c protein antigen predicted by TE-predict and IEDB human T cell epitope prediction tool. The cellular immunoreactivity of the predicted peptides was evaluated through ELISpot assay with the peripheral blood monouclear cells (PBMC) of clinical tuberculosis patients. In animal experiments, BALB/c mice were respectively immunized with high dose (100 μg/mice) and low dose (50 μg/mice) of the peptides of Rv0585c, at the same time, high dose (50 μg/mice) and low dose (20 μg/mice) of Ag85B protein were used in positive control group. The levels of IFN-γ, IL-2, IL-4 and IL-10 were tested with ELISA kit respectively. By means of bioinformatics technique, 66 human T cell epitopes of Rv0585c were predicted, from which9 peptides concentrated epitopes were synthesized for the animal immune experiments. Peptides P10110, P10112 and P10117 were confirmed to be antigenic. The sensitivity and specificity of P10110, P10112 and P10117 were 14.00, 12.00, 6.00 and 100.00, 100.00, 97.96 respectively when they were used as diagnostic reagents of tuberculosis. The sensitivity and specificity were 22.00 and 97.96 when the epitopes were combined together. The results of animal immunity test showed that high levels of cytokines IFN-γ, IL-2, IL-4 and IL-10 were induced by high and low dose of P10110, and high levels of IFN-γ、IL-2 and IL-10 were induced by high and low dose of P10112, which were much higher than that in negative controls, respectively (<0.001). Rv0585c, including its human T cell epitopes, has good immunogenicity and immunoreactivity, stimulating the body to produce a stronger cellular immune response and has better potential application value in cellular diagnosis of tuberculosis and the development of new type of tuberculosis vaccine.

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Increase of Soluble Programmed Cell Death Ligand 1 in Patients with Chronic Hepatitis C.

To determine whether the soluble programmed cell death ligand 1 (sPD-L1) levels in patients with chronic hepatitis C (CHC) are associated with the clinical features of the disease and the efficacy of treatment, including interferon (IFN)-α. We investigated the sPD-L1 levels in the sera of 80 genotype 1b Japanese patients with CHC who underwent 12 weeks of telaprevir (TVR)- or simeprevir (SMV)-based triple therapy followed by 12 weeks of dual therapy with pegylated IFN-α plus ribavirin. Serum was also obtained from 22 patients with chronic hepatitis B (CHB) and from 10 healthy donors (HC). The sPD-L1 levels were measured using an ELISA kit. In addition, we examined the PD-L1 expression on the cell surface of immortalized hepatocytes (HPT1) after incubation with cytokines, including IFN-γ. The pretreatment serum sPD-L1 levels were significantly increased in patients with CHC (median 109.3 pg/ml, range 23.1-402.3) compared with patients with CHB (69.2 pg/ml, 15.5-144.8; <0.001) and HC (100.3 pg/ml, 40.1-166.6; = 0.039). No significant differences in the sustained virological response (SVR) rates were found between the TVR- (85.0%, n=40) and SMV-treated (80.0%, n=40) groups, and the pretreatment levels of serum sPD-L1 were not significantly different between patients who achieved SVR (105.0 pg/ml, 23.1-402.3) and non-SVR patients (133.5 pg/ml, 39.9-187.2; = 0.391). The pretreatment level of sPD-L1 was positively correlated with the alanine aminotransferase and alpha-fetoprotein levels (R = 0.082, = 0.016, and R = 0.149, = 0.002, respectively). Although immortalized hepatocytes do not express PD-L1, we confirmed that PD-L1 expression was induced after stimulation with IFN-γ. In this study, we first found that sPD-L1 was increased in patients with CHC. Our results indicate that the level of serum sPD-L1 might be associated with the progression of CHC and the generation of hepatocellular carcinoma.

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