1.The relationship between histone acetylation modification and IFN-gamma responsive gene regulation.
Jin-jun GUO ; Ying HUANG ; Jun ZHANG ; Ai-long HUANG
Chinese Journal of Hepatology 2006;14(7):525-528
OBJECTIVESTo study the role of histone modification in the regulation of IFN-gamma-activated gene using chromatin immunoprecipitation technique.
METHODSReal-time PCR was used to measure the mRNA level of interferon-gamma-inducible protein 10 (IP-10) in Hela cells. Chromatin immunoprecipitation combined with Real-time PCR was used to check the histone H4 acetylation level at IFN-stimulated response element (ISRE) locus of IP-10 gene.
RESULTSIP-10 was strongly activated by IFN-gamma. The histone H4 deacetylation happened at the ISRE locus when IP-10 was induced by IFN-gamma. The activation of IP-10 and the deacetylation of histone H4 at the ISRE site induced by IFN-gamma were inhibited or blocked by histone deacetylase (HDAC) inhibitor trichostatin A (TSA).
CONCLUSIONThe histone H4 deacetylation at the ISRE site is related with the activation of IP-10 by IFN-gamma.
Acetylation ; Chemokine CXCL10 ; metabolism ; Gene Expression Regulation ; HeLa Cells ; Histone Deacetylases ; genetics ; metabolism ; Histones ; genetics ; metabolism ; Humans ; Interferon-gamma ; genetics ; metabolism ; RNA, Messenger ; genetics
2.Influence of avian influenza virus NS1 protein on the expression of IP-10 in BEAS-2B cells.
Xiao-Jun JIA ; Jian-Fang ZHOU ; Jing-Yu WANG ; Jie DONG ; Hong BO ; Zi LI ; Kui-Biao LI ; Yu LAN ; Yue-Long SHU
Chinese Journal of Experimental and Clinical Virology 2008;22(3):183-185
OBJECTIVETo investigate the influence of avian influenza virus (AIV) NS1 protein on the expression of interferon-inducible protein 10 (IP-10).
METHODSNSI gene from virus A/Anhui/1/2005 (H5N1), NS1 gene inserted with 80-84 amino acids from virus A/Anhui/1/2005 (H5N1) and NS1 gene from virus A/Puerto Rico/8/1934 (H1N1) were cloned into the eukaryotic expression vector pEGFP-N1, and transfected into BEAS-2B cells, IP-10 expression level in transfected cells was detected by flow cytometry.
RESULTSCompared with the control group pEGFP-N1, expression of these three different NS1 genes can down-regulate the expression of IP-10 in BEAS-2B cells, but there is no significant difference as to the lower level among them.
CONCLUSIONNS1 protein of A/Anhui/1/2005 (H5N1) can down-regulate the expression level of IP-10, but this may not clarify its relationship with the virulence of AIV.
Cell Line ; Chemokine CXCL10 ; genetics ; metabolism ; Down-Regulation ; Gene Expression ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; metabolism ; Influenza, Human ; genetics ; metabolism ; virology ; Viral Nonstructural Proteins ; genetics ; metabolism
3.Construction of a eukaryotic expression vector of the gene encoding rat interferon-gamma-inducible protein and its expression in NIH 3T3 cells.
Yu-jie ZHAO ; Yuan LIN ; Ming-yuan LI ; Hong LI ; Zhong-hua JIANG
Journal of Southern Medical University 2009;29(4):615-618
OBJECTIVETo construct an expression vector of the gene encoding rat interferon-gamma-inducible protein (IP-10) and identify its expression in NIH 3T3 cells.
METHODSIP-10 gene was amplified by polymerase chain reaction (PCR) and inserted into the eukaryotic expression vector pcDNA3.1(+). After identification by PCR, restriction endonuclease digestion and sequence analysis, the recombinant expression vector pcDNA3.1(+)-IP-10 was transfected into NIH 3T3 cells via liposome. Immunofluorescence method was used to confirm the expression of pcDNA3.1(+)-IP-10 in the transfected NIH 3T3 cells. The expression of IP-10 protein in the supernatant of the transfected cells was examined by Western blotting.
RESULTSPCR, restriction endonuclease digestion and sequence analyses confirmed successful construction of the recombinant vector pcDNA3.1(+)-IP-10. Immunofluorescence assay identified the expression of pcDNA3.1(+)-IP-10 in NIH 3T3 cells, and the expression of IP-10 protein was detected by Western blotting in the supernatant of the transfected cells.
CONCLUSIONA eukaryotic expression vector pcDNA3.1(+)-IP-10 has been successfully constructed, which provides the basis for investigating the therapeutic effect of IP-10 on Th1 type autoimmune disease.
Animals ; Chemokine CXCL10 ; genetics ; metabolism ; DNA Restriction Enzymes ; metabolism ; Fluorescent Antibody Technique ; Gene Expression ; Genetic Vectors ; genetics ; Mice ; NIH 3T3 Cells ; Plasmids ; genetics ; Rats ; Transfection ; methods
4.Construction of a recombinant adenovirus vector for interferon-gamma-inducible protein 10 and the adenovirus preparation.
Zi-yun SHAO ; Zhi-feng LIU ; Yi PENG ; Jia XU ; Peng DENG ; Yong JIANG
Journal of Southern Medical University 2006;26(11):1552-1555
OBJECTIVETo construct a recombinant adenovirus vector for expressing interferon-gamma-inducible protein 10 (IP-10) by homogenous bacterial recombination.
METHODSIP-10 gene was cloned into the shuttle plasmid pAdTrack-CMV that contained the coding sequence of enhanced green fluorescent protein (EGFP). The shuttle plasmid was then transformed into E. coli BJ5183 with pAdEasy-1 vector by chemical transformation. The recombinant adenovirus vector pAd/IP-10 was identified by enzyme digestion with Pac I and the linearized plasmid was transfected into HEK293 cells.
RESULTSThe positive clones were identified with enzyme digestion and polymerase chain reaction (PCR) and were further verified by DNA sequencing. The recombinant adenovirus of high titration was obtained after transfection and packaging in HEK293 cells.
CONCLUSIONA recombinant adenovirus vector for expression of IP-10 has been constructed successfully and high-titer active adenovirus is obtained for functional study of IP-10 protein.
Adenoviridae ; genetics ; Cell Line ; Chemokine CXCL10 ; genetics ; metabolism ; Cloning, Molecular ; Defective Viruses ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Virus Cultivation ; methods
5.Benzylideneacetophenone derivatives attenuate IFN-gamma-induced IP-10/CXCL10 production in orbital fibroblasts of patients with thyroid-associated ophthalmopathy through STAT-1 inhibition.
Sung Hee LEE ; Seul Ye LIM ; Ji Ha CHOI ; Jae Chul JUNG ; Seikwan OH ; Koung Hoon KOOK ; Youn Hee CHOI
Experimental & Molecular Medicine 2014;46(6):e100-
The aim of the present study was to identify a new candidate anti-inflammatory compound for use in the active stage of thyroid-associated ophthalmopathy (TAO). Benzylideneacetophenone compound JC3 [(2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-l-one] was synthesized based on a structural modification of yakuchinone B, a constituent of the seeds of Alpinia oxyphylla, which belongs to the ginger family (Zingiberaceae), has been widely used in folk medicine as an anti-inflammatory phytochemical. Orbital fibroblasts were primarily cultured from patients with TAO, and the potential of JC3 to suppress the interferon (IFN)-gamma-induced protein (IP)-10/CXCL10 production in these cells was determined. IFN-gamma strongly increased the level of IP-10/CXCL10 in orbital fibroblasts from patients with TAO. JC3 exerted a significant inhibitory effect on the IFN-gamma-induced increase in IP-10/CXCL10 in a dose-dependent manner; its potency was greater than that of an identical concentration of yakuchinone B with no toxicity to cells at the concentration range used. Moreover, the constructed dimer and trimer polystructures of JC3, showed greater potency than JC3 in suppressing the IFN-gamma-induced production of IP-10/CXCL10. JC3 significantly attenuated the IP-10/CXCL10 mRNA expression induced by IFN-gamma, and a gel-shift assay showed that JC3 suppressed IFN-gamma-induced DNA binding of signal transducer and activator of transcription-1 (STAT-1) in TAO orbital fibroblasts. Our results provide initial evidence that the JC3 compound reduces the levels of IP-10/CXCL10 protein and mRNA induced by IFN-gamma in orbital fibroblasts of TAO patients. Therefore, JC3 might be considered as a future candidate for therapeutic application in TAO that exerts its effects by modulating the pathogenic mechanisms in orbital fibroblasts.
Cells, Cultured
;
Chalcone/chemical synthesis/*pharmacology
;
Chemokine CXCL10/genetics/*metabolism
;
Diarylheptanoids/chemistry/pharmacology
;
Fibroblasts/*drug effects/metabolism
;
Graves Ophthalmopathy/*metabolism
;
Humans
;
Interferon-gamma/*metabolism
;
Orbit/cytology
;
RNA, Messenger/genetics/metabolism
;
STAT1 Transcription Factor/genetics/*metabolism
6.Expression of monocyte chemoattractant protein-1 in the pancreas of mice.
Dong LI ; Su-wen ZHU ; Dong-juan LIU ; Guo-liang LIU ; Zhong-yan SHAN
Chinese Medical Journal 2005;118(15):1269-1273
BACKGROUNDType 1 diabetes has been recognized as an organ specific autoimmune disease owing to the immune destruction of pancreatic islet beta cells in genetically susceptible individuals. In both human and rodent models of type 1 diabetes, such as nonobese diabetic (NOD) mice, biobreeding rats, the disease has a distinct stage characterized by immune cells infiltrating in the pancreas (insulitis). The major populations of infiltrating cells are macrophages and T lymphocytes. Therefore, immune cell infiltration of pancreatic islets may be a crucial step in the pathogenesis of type 1 diabetes. Monocyte chemoattractant protein-1 can specifically attract monocytes in vivo. Interferon induced protein-10 has chemoattractant effects on the activated lymphocytes. In this study, we analysed the expression of monocyte chemoattractant protein-1 in the pancreas of mice and interferon inducible protein-10 mRNA in the pancreas of NOD mice, and discussed their possible role in the pathogenesis of type 1 diabetes.
METHODSThe immunohistochemical method and immunoelectronmicroscopy were used to evaluate the expression of monocyte chemoattractant protein-1 in the pancreas of NOD mice and BALB/c mice. RT-PCR was used to evaluate the expression of monocyte chemoattractant protein-1 and interferon inducible protein mRNA in NOD mice.
RESULTSMonocyte chemoattractant protein-1 was positive in the pancreas of NOD mice, whereas negative in the pancreas of BALB/C mice. RT-PCR showed that monocyte chemoattractant protein-1 and interferon inducible protein-10 mRNA could be found in the pancreas of NOD mice. Immunoelectronmicroscopy demonstrated that monocyte chemoattractant protein-1 was produced by beta cells and stored in the cytoplasm of the cells.
CONCLUSIONSPancreatic islet beta cells produce monocyte chemoattractantprotein-1 in NOD mice. Monocyte chemoattractant protein-1 may play an important part in the pathogenesis of type 1 diabetes by attracting monocytes/macrophages to infiltrate pancreatic islets.
Animals ; Chemokine CCL2 ; analysis ; genetics ; Chemokine CXCL10 ; Chemokines, CXC ; genetics ; Diabetes Mellitus, Type 1 ; etiology ; metabolism ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Mice, Inbred NOD ; Microscopy, Immunoelectron ; Pancreas ; chemistry ; RNA, Messenger ; analysis
7.Correlationship between chemokines and oxidative stress in chronic hepatitis B.
Jing-Wei WANG ; Li-Yuan WANG ; Zhen-Hua ZHAO ; Cheng-Bao WANG ; Xing LIU ; Li-Mei HUANG ; Kai WANG
Chinese Journal of Experimental and Clinical Virology 2012;26(4):246-249
OBJECTIVEThe aim of this study is to investigate the possible associations of chemokines IP-10, Rantes and oxidative stress in chronic hepatits B (CHB).
METHODS70 CHB patients and 10 healthy controls were enrolled in the study. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of IFN-gamma-inducible protein-10 (IP-10) and regulated on activation normal T-cell-expressed and secreted (Rantes) and oxidative stress parameters (glutathione, GSH; glutathione disulfide, GSSG). Correlationship were analyzed by Spearman's rank correlation.
RESULTThe levels of IP-10 and Rantes were higher in CHB patients than healthy controls, and strong positive associations were found between IP-10/Rantes and alanine aminotransferase (ALT). The levels of GSH and GSH/GSSG were lower in CHB patients than healthy controls, and GSH and GSH/GSSG were negatively correlated with ALT. The levels of IP-10 and Rantes were negatively correlated with GSH and GSH/GSSG respectively.
CONCLUSIONStrong associations were found between chemokines and oxidative stress which participated in the pathogenesis of CHB.
Adult ; Alanine Transaminase ; blood ; Chemokine CCL5 ; blood ; Chemokine CXCL10 ; blood ; Female ; Glutathione ; blood ; Glutathione Disulfide ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; blood ; metabolism ; virology ; Humans ; Male ; Malondialdehyde ; blood ; Middle Aged ; Oxidative Stress
8.Abnormal interferon-inducible protein-10 expression in the labial glands of patients with Sjogren's syndrome.
Wei ZHOU ; Yi DONG ; Yan ZHAO ; Fu-lin TANG
Acta Academiae Medicinae Sinicae 2003;25(5):603-607
OBJECTIVETo investigate whether interferon-inducible protein 10 (IP-10) is involved in the inflammatory process of the labial gland of patients with Sjogren's Syndrome (SS).
METHODSForty-nine patients performed labial gland biopsy, the number of lymphocytes in the biopsy tissues was calculated and the IP-10 was detected by the methods as following: 39 biopsied labial tissues were examined by RT-PCR, among them, 21 were from primary SS, 5 from secondary SS and 13 from other diseases. With RT-PCR, the IP-10 and beta-actin were co-amplified with specific primers. The gel-fractioned and ethidium bromide amplification products were then analyzed by densitometry. The expression of IP-10 was semi-quantificated by IP-10/beta-actin ratio. Twenty-one samples were examined by immunohistochemistry with specific goat anti-IP-10 antibody, 10 of them from primary SS, 3 from secondary SS, 8 from other diseases. 11 out of 21 samples were examined by both RT-PCR and immunohistochemistry.
RESULTSThe expression of IP-10 mRNA was significantly up-regulated in labial glands of patients with SS compared with other diseases (IP-10/beta-actin ratio was 0.329 +/- 0.157 vs 0.099 +/- 0.059, P < 0.01). The number of lymphocyte infiltration foci in labial glands of patients with SS correlated to the IP-10/beta-actin ratio (r = 0.657, P < 0.05). Ductal epithelial cells and some of the infiltrating lymphocytes were stained by anti-IP-10 antibody by immunohistochemistry in 8 of the primary SS (8/10), all of the secondary SS (3/3) and one with primary biliary sclerosis (1/8). The expression of IP-10 protein detected by immunohistochemistry was consistent with that of mRNA detected by RT-PCR.
CONCLUSIONSIP-10 is abnormally highly expressed in the labial glands of patients with SS and positively relates to the lymphocyte infiltration. It thus suggests chemokine IP-10 may be one of the important molecules attracting the lymphocytes to the minor salivary glands to form the lymphocytic foci of Sjogren's Syndrome.
Adult ; Chemokine CXCL10 ; Chemokines, CXC ; biosynthesis ; genetics ; Epithelial Cells ; metabolism ; Female ; Humans ; Immunohistochemistry ; Lip ; metabolism ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Salivary Glands, Minor ; metabolism ; Sjogren's Syndrome ; metabolism ; Up-Regulation
9.An expression plasmid encoding recombinant immunotoxin IP10-DT390 suppresses the experimental autoimmune encephalomyelitis.
Wenjie CHEN ; Hong LI ; Yi JIA ; Mingyan LI ; Zhonghua JIANG ; Meili LÜ ; Lin ZHANG
Journal of Biomedical Engineering 2007;24(5):1118-1122
Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS); it serves as a model for the human multiple sclerosis (MS). In mice, EAE is mediated by T cells specific for various myelin basic proteins which migrate from the periphery to the CNS. In search of a way to prevent the induction and progression of EAE, we observed the effects of recombinant immunotoxin IP10-DT390 on blocking or eliminating the active T cells in the EAE model. In this paper is presented an experimental gene therapy-based model in which the mice were made resistant to EAE induction by plasmid DNA encoding recombinant immunotoxin that was injected into the leg muscles of mice. The new immuno-biological construct could selectively impair autoreactive T-cell homing while the duration of clinical signs is shorter, and the new construct would not affect other components of the immune response. These data demonstrated the effectiveness of the constructs in the treatment of EAE and suggested its usefulness in the treatment of other autoimmune diseases.
Animals
;
Chemokine CXCL10
;
biosynthesis
;
genetics
;
therapeutic use
;
Diphtheria Toxin
;
biosynthesis
;
genetics
;
therapeutic use
;
Encephalomyelitis, Autoimmune, Experimental
;
immunology
;
pathology
;
therapy
;
Female
;
Genetic Therapy
;
Immunoglobulin Fragments
;
biosynthesis
;
genetics
;
therapeutic use
;
Immunotoxins
;
genetics
;
metabolism
;
therapeutic use
;
Mice
;
Mice, Inbred C57BL
;
Receptors, CXCR3
;
metabolism
;
Recombinant Fusion Proteins
;
biosynthesis
;
genetics
;
therapeutic use
;
Recombinant Proteins
;
biosynthesis
;
genetics
;
therapeutic use
;
T-Lymphocytes
;
immunology
;
Transfection
10.Pentoxifylline attenuates cigarette smoke-induced overexpression of CXCR3 and IP-10 in mice.
Zheng WANG ; Yan-Wei CHEN ; Jin-Nong ZHANG ; Xiao-Fei HU ; Mei-Jun PENG
Chinese Medical Journal 2012;125(11):1980-1985
BACKGROUNDCigarette smoke-induced emphysema is associated with overexpression of the chemokine receptor CXCR3 and its ligands. Previously, we have demonstrated that pentoxifylline (PTX) alleviated cigarette smoke-induced emphysema. The aim of this study was to determine if the overexpression of CXCR3 and its ligand interferon-inducible protein-10 (IP-10) that was elicited by smoke exposure were attenuated by PTX.
METHODS(1) The study in vitro: a given number of RAW264.7 macrophages with decreasing concentrations of PTX in the culture medium were challenged with cigarette smoke extract (CSE); (2) The study in vivo: male BALB/c mice were randomized into four groups, i.e., sham-smoke, smoke only, smoke with 2 mg/kg PTX, and smoke with 10 mg/kg PTX. The smoke exposure time was 90 minutes once a day, 6 days a week for 16 weeks. PTX was given intraperitoneally before each episode of smoke exposure. Interferon (IFN)-γ and IP-10 in broncho-alveolar lavage fluid (BALF) and in culture medium were measured by enzyme-linked immunosorbent assay (ELISA). IP-10 mRNA in lung tissue was assessed by RT-PCR. CXCR3 positive cells in lung sections were visualized by immunochemistry staining.
RESULTSUp-regulation of IFN-γ and IP-10 in the culture medium of macrophages elicited by CSE was inhibited by PTX in a dose-dependent manner. Chronic cigarette smoke exposure led to overexpression of IFN-γ and IP-10 in BALF, upregulation of IP-10 mRNA and increased infiltration of CXCR3(+) cells into lung parenchyma. Administration of PTX decreased the level of IFN-γ from (6.26 ± 1.38) ng/ml to (4.43 ± 0.66) ng/ml by low dose PTX or to (1.74 ± 0.28) ng/ml by high dose PTX. IP-10 was reduced from (10.35 ± 1.49) ng/ml to (8.19 ± 0.79) ng/ml by low dose PTX or to (7.51 ± 0.60) ng/ml by high dose PTX. The expression of IP-10 mRNA was also down-regulated (P < 0.05). But only with a high dose of PTX was the ratio of CXCR3(+) cells decreased; 15.2 ± 7.3 vs. 10.4 ± 1.8 (P < 0.05).
CONCLUSIONPTX attenuates cigarette smoke-induced overexpression of chemokine receptor CXCR3 and its ligand IP-10, which is relevant to its inhibitory effect on pulmonary emphysema.
Animals ; Cell Line ; Chemokine CXCL10 ; genetics ; metabolism ; Gene Expression ; drug effects ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred BALB C ; Pentoxifylline ; pharmacology ; therapeutic use ; Pulmonary Emphysema ; drug therapy ; genetics ; metabolism ; Random Allocation ; Receptors, CXCR3 ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Smoking ; adverse effects