Humans ; Haploinsufficiency ; NF-kappa B/genetics* ; Mutation ; Tumor Necrosis Factor alpha-Induced Protein 3/genetics*

OBJECTIVE: To analyze the correlation of EBV infection with expression of TNF-α-inducing protein 3 gene and A20 protein in Hodgkin lmphoma. METHODS: The clinical data and pathological specimens of 65 cases of Hodgkin's lymphoma in our hospital were analyzed retrospectively, and the tissue chips were made for the rich area of the tumor cells. The latent membrane protein 1 encoded by EBV was measured by immunohistochemical staining, and the RNA encoded by EBV was measured by in situ hybridization to analyze the infection state. The gene expression of tumor necrosis factor.α-induced protein 3 was detected by fluorescence in situ hybridization, and the expression of A20 protein encoded by EBV was detected by immunohistochemical staining. The obtained data were processed by SPSS 23.0 version statistical software. RESULTS: The positive rate of latent membrane protein 1 was 26.15% (17/65), the positive rate of EBV encoded RNA was 26.15% (17/65), and the coincidence rate was 100.00%. In 65 patients, A20 protein expression was lost in 18 cases (27.69%), and 14 cases (21.54%) showed homozygous or heterozygous deletion of tumor necrosis factorα protein 3 gene. Only 1 case showed A20 loss combined with homozygous deletion of TNFα inducible protein 3. Correlation analysis showed that EBV infection did not significantly relate with expression loss of A20 protein and the gene deletion of TNF-α inducing protein 3 (P>0.05). CONCLUSION The expression loss of A20 protein and gene detection of TNFα inducing protein 3 are found in both EBV negative and positive patients with Hodgkin's lymphoma, however the results of immunohistochemical staining and fluorescence in situ hybridization are not complete consistant, the reason may closely relate with the technical factors.
Epstein-Barr Virus Infections ; Herpesvirus 4, Human ; Hodgkin Disease ; Humans ; In Situ Hybridization ; In Situ Hybridization, Fluorescence ; Retrospective Studies ; Tumor Necrosis Factor alpha-Induced Protein 3 ; genetics ; Viral Matrix Proteins

OBJECTIVETo establish the method for cotransferring human A20 gene and human heme oxygenase-1 (HO-1) gene into the isolated rat islets using lentiviral transfection system, and to study the protective effect of A20 and HO-1 protein against the apoptosis induced by cycloheximide (CHX) and TNF-α, and finally to explore the underlying mechanism.

METHODThe A20 gene and HO-1 gene were cloned and inserted into the lentiviral transfection system. The efficacy of gene transfer was measured by the intensity of the enhanced green fluorescent protein (EGFP) fluorescence-positive islets. Western blot was applied to verify the expression of the A20 and HO-1 genes. To induce apoptosis in vitro, the isolated islets were treated with CHX+TNF-α, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the fluorescence-activated cell sorting (FACS) methods were used to evaluate the apoptosis of the islet cells and Western blot was used to detect caspase-3 activation.

RESULT(1) A20 and HO-1 genes were introduced into the isolated islets by lentiviral transfection, both of the genes were highly expressed in the islets after 96 hours culture detected by Western blot method. (2) The insulin levels in the cell culture medium from A20 and/or HO-1 transgenic islets were significantly higher than that in non-transgenic controls (P < 0.01). (3)After CHX + TNF-alpha treatment, the cell culture medium insulin concentration in the A20 gene transfected group [(93.58 ± 4.12)µg/ml], HO-1 gene transfected group [(88.98 ± 4.77) µg/ml ] and A20/HO-1 co-transfected group [(103.33 ± 3.16) µg/ml] were significantly higher than that in the EGFP group [(9.03 ± 0.65) µg/ml ] and the control group [(8.86 ± 0.38) µg/ml] (P < 0.001). Minimum expression level of the activated caspase-3 was found in the A20/HO-1 co-transfected group.

CONCLUSIONThe lentiviral gene transfer system was an efficient and stable gene transfer vector, the over-expressed A20 and HO-1 protein delivered via lentivirus could preserve rats' islets function and act against the apoptosis induced by CHX and TNF-&alpha;.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Flow Cytometry ; Genetic Vectors ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Insulin ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Islets of Langerhans ; drug effects ; enzymology ; physiology ; Lentivirus ; genetics ; Male ; Nuclear Proteins ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transfection ; methods ; Tumor Necrosis Factor alpha-Induced Protein 3 ; Tumor Necrosis Factor-alpha ; pharmacology

BACKGROUNDTumor necrosis factor-induced protein 3 (TNFAIP3) gene has been shown important in cardiac remodeling. The aim of the present study was to investigate whether the variants of TNFAIP3 gene are associated with left ventricular hypertrophy (LVH) in hypertensive patients.

METHODSFour representatives of all the other single nucleotide polymorphisms (SNPs) in TNFAIP3 gene were tested for association with hypertrophy in two independent hypertensive populations (n = 2120 and n = 324).

RESULTSWe found that only the tag SNP (rs5029939) was consistently lower in the hypertensives with cardiac hypertrophy than in those without cardiac hypertrophy in the two study populations, indicating a protective effect on LVH (odds ratio (OR) (95% confidence interval (CI)) 0.58 (0.358 - 0.863), P = 0.035; OR (95%CI) = 0.477 (0.225 - 0.815), P < 0.05, respectively). Multiple regression analyses confirmed that the patients with G allele of rs5029939 had less thickness in inter-ventricular septum, left ventricular posterior wall, relative wall thickness and left ventricular mass index than did those with CC allele in the hypertensive patients in both study populations (all P < 0.01).

CONCLUSIONThese findings indicate that the SNP (rs5029939) in the TNFAIP3 gene may serve as a novel protective genetic marker for the development of LVH in patients with hypertension.

Adult ; Aged ; Cross-Sectional Studies ; DNA-Binding Proteins ; Echocardiography ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Hypertension ; genetics ; Hypertrophy, Left Ventricular ; genetics ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; Phenotype ; Polymorphism, Single Nucleotide ; genetics ; Tumor Necrosis Factor alpha-Induced Protein 3

BACKGROUNDA20, also known as tumor necrosis factor alpha induced protein 3 (TNFaip3), is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B (NF-kappaB) activity and prevents tumor necrosis factor (TNF)-mediated programmed cell death. NF-kappaB is a transcription factor that regulates expression of genes involved in cell proliferation, cell survival and anti-apoptosis. Several studies have implicated that the NF-kappaB signal pathway is associated with angiogenesis and clinico-pathological process of adenoid cystic carcinoma (ACC) of the salivary glands.

METHODSThe ability of overexpression of A20 to influence the biological behavior and invasion of ACC cells was examined. The cells were stably transfected with full-length A20 cDNA. Stable gene transfer was verified by realtime-polymerase chain reaction (PCR) and Western blot analysis. The change of cell biological behavior was examined by methyl thiazolyl tetrazolium (MTT) and NF-kappaB luciferase reporter assay and the invasion of the cells was examined by a Matrigel invasion chamber.

RESULTSpEGPFN3-A20 gene was stably transferred into ACC-2 cells and overexpressed. When cells were treated with TNFalpha, the NF-kappaB activity of ACC-2-A20 cells could be down-regulated about 46.32% in contrast to ACC-2-GFP cells (P < 0.05). A20 potently inhibited growth of A20 transfectant ACC-2-A20 compared with control vector transfected groups and the ACC-2 empty control group (P < 0.05). The ACC-2-A20 cells showed significantly reduced ability to invade through Matrigei-coated filters compared to ACC-2-GFP and ACC-2 cells. The inhibition rate was up to 71.05% (P < 0.05).

CONCLUSIONSA20 gene transfer is associated with decreased tumor invasion, in part via the down-regulation of NF-kappaB expression, providing evidence for a potential application of A20 in designing a treatment modality for salivary gland cancers such as ACC.

Carcinoma, Adenoid Cystic ; pathology ; therapy ; Cell Line, Tumor ; DNA-Binding Proteins ; Genetic Therapy ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; NF-kappa B ; antagonists & inhibitors ; Neoplasm Invasiveness ; Nuclear Proteins ; genetics ; Salivary Gland Neoplasms ; pathology ; therapy ; Transfection ; Tumor Necrosis Factor alpha-Induced Protein 3

This study aims to investigate the therapeutic effect of alcohol extract of root and root bark of Toddalia asiatica(TAAE) on collagen-induced arthritis(CIA) in rats through phosphatidylinoinosidine-3 kinase/protein kinase B(PI3K/Akt) signaling pathway. To be specific, CIA was induced in rats, and then the rats were treated(oral, daily) with TAAE and Tripterygium Glycoside Tablets(TGT), respectively. The swelling degree of the hind leg joints was scored weekly. After 35 days of administration, the histopathological changes were observed based on hematoxylin and eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the levels of cytokines [tumor necrosis factor-α(TNF-α), interleukin(IL)-6)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed to detect the apoptosis of synoviocytes in rats. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)-associated X(Bax), Bcl-2, and caspase-3 and pathway-related proteins phosphoinositide 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), and p-Akt. RT-qPCR was conducted to examine the mRNA levels of Bax, Bcl-2, caspase-3, TNF-α, IL-6, and IL-1β and pathway-related proteins PI3K, p-PI3K, Akt, and p-Akt. TAAE can alleviate the joint swelling in CIA rats, reduce serum levels of inflammatory cytokines, improve synovial histopathological changes, promote apoptosis of synoviocytes, and inhibit synovial inflammation. In addition, RT-qPCR and Western blot results showed that TAAE up-regulated the level of Bax, down-regulated the level of Bcl-2, and activated caspase-3 to promote apoptosis in synoviocytes. TAAE effectively down-regulated the protein levels of p-PI3K and p-Akt. In this study, TAAE shows therapeutic effect on CIA in rats and reduces the inflammation. The mechanism is that it suppresses PI3K/Akt signaling pathway and promotes synoviocyte apoptosis. Overall, this study provides a new clue for the research on the anti-inflammatory mechanism of TAAE and lays a theoretical basis for the better clinical application of TAAE in the treatment of inflammatory and autoimmune diseases.
Rats ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Caspase 3/genetics* ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Plant Bark ; Anti-Inflammatory Agents/therapeutic use* ; Arthritis, Experimental/chemically induced* ; Inflammation/drug therapy* ; Cytokines/metabolism* ; Proto-Oncogene Proteins c-bcl-2 ; Apoptosis