OBJECTIVE: To explore the expression and clinical significance of chemokine CXCL10 and CXCR3 in hepatocellular carcinoma (HCC). METHODS: The expression and prognostic of CXCL10 and CXCR3 in HCC tumor tissues and non-tumor tissues were analyzed in two different publicly available databases the Cancer Genome Atlas (TCGA) and Liver Cancer Institute (LCI). In addition, quantitative real-time PCR (qPCR) was used to detect the mRNA expression of CXCL10 and CXCR3 in 45 HCC clinical samples with HBV infection background. Pearson correlation and Spearman rank correlation were used to determine the correlation between the expression level of CXCL10 and CXCR3 in tumor and non-tumor tissues. RESULTS: In TCGA database, the expression of CXCL10 in HCC tumor tissues was significantly higher than that in non-tumor tissues (nonpaired samples: 3.379±2.081 vs. 2.213±2.274, P<0.001; paired samples: 3.159±2.267 vs. 2.213±2.274, P=0.018). Similarly in LCI datebase (7.625±1.683 vs. 7.287±1.328, P=0.009). And higher CXCL10 expression was significantly associated with a better prognosis in the patients with HCC both in TCGA and LCI database (P=0.107, P=0.002). In TCGA database, the expression of CXCR3 in HCC tumor tissues was significantly higher than that in non-tumor tissues (nonpaired samples: -0.906±1.697 vs. -1.978±1.629, P<0.001; paired samples: -1.329±1.732 vs. -1.978±1.629, P=0.037), while lower in LCI database (3.989±0.339 vs. 4.074±0.309, P=0.003). In both databases, higher CXCR3 expression was significantly associated with a better prognosis in the HCC patients (P=0.004, P=0.014). Furthermore, in TCGA database, the expression level of CXCL10 and CXCR3 was positively correlated both in HCC tumor tissues and matched non-tumor tissues (r=0.584, P<0.001; r=0.776, P<0.001). The qPCR assay showed that the expression of CXCL10 in HBV-related HCC tumor tissues was significantly higher than those in normal liver tissues [0.479(0.223, 1.094) vs. 0.131(0.106, 0.159), P=0.010], and the expression in HBV-related non-tumor tissues was also significantly higher than those in normal liver tissues [0.484(0.241, 0.846) vs. 0.131(0.106, 0.159), P<0.001]. The same was true as CXCR3 [0.011(0.006, 0.019) vs. 0.002(0.001, 0.004), P=0.004; 0.016(0.011, 0.021) vs. 0.002(0.001, 0.004), P<0.001]. However there was no significant difference of CXCL10 and CXCR3 between tumor tissues and matched non-tumor tissues (P=1.000, P=0.374). CONCLUSION Expression of CXCL10 was up-regulated in HCC tissues, expression of CXCR3 was down-regulated in HBV-related HCC tissues, and the higher expression of both genes was correlated with better overall survival in HCC patients.
Adult ; Carcinoma, Hepatocellular/metabolism* ; Chemokine CXCL10/metabolism* ; Humans ; Liver Neoplasms/metabolism* ; Prognosis ; Receptors, CXCR3/metabolism*

Interferon gamma-inducible protein 10, a member of the family of CXC chemokines, is secreted by interferon gamma-stimulated, monocytes, endothelial cells and keratinocytes. Interferon gamma-inducible protein 10 plays an important role in recruiting activated T cells into sites of tissue inflammation. In this experiment, PCR products of Interferon gamma-inducible protein 10 were cloned into prokaryote expression vector pET 32(a) to generate recombinant pET-IP10 with S-Tag at the N-terminus, and expressed successfully in E. coli BL21 (DE3). The total expressed products amounted to 25.3% in all bacterion proteins. pET-IP10 mainly formed inclusion body in E. coli. Soluble recombinant protein accounted for 20% among IP-10 fusion protein. The soluble recombinant proteins were purified by using S-Tag affinity chromatography effectively with purity of over 90%. The chemotaxis biological activity of purified Interferon gamma-inducible protein 10 could specifically exhibit the directional migration of stimulated T cells at concentration of 100 ng/ml. The results indicated that the strategy we used in this experiment was effective for recombinant Interferon gamma-inducible protein 10 production with biological activity.
Chemokine CXCL10 ; biosynthesis ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Recombinant Proteins ; biosynthesis ; T-Lymphocytes ; cytology ; Thioredoxins ; biosynthesis

Animals ; Chemokine CCL20 ; Chemokine CXCL10 ; Chemokines, CC ; biosynthesis ; Chemokines, CXC ; biosynthesis ; Liver ; metabolism ; Liver Transplantation ; Macrophage Inflammatory Proteins ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Transplantation, Homologous

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

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

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

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

OBJECTIVETo detect the disparity of three cytokines interleukin-6 (IL-6), interferon-inducible protein 10 (IP-10) and interleukin-17 (IL-17) in peripheral blood (PB) and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA).

METHODSerum concentrations of the three cytokines were measured in 27 patients with 13 systemic-onset JIA (sJIA), 14 polyarticular JIA (pJIA) and 28 healthy controls using enzyme-linked immunosorbent assay (ELISA). Nineteen patients with no marked arthritis symptom or only temporary arthralgia were enrolled in probable sJIA group. SF from 18 patients with 7 sJIA, 11 pJIA were examined for cytokine levels.

RESULT(1) The statistically significant difference in serum IL-6 was detected between sJIA and healthy control group [28.0(4.2-59.2) ng/L vs. 12.3 (2.1-13.8) ng/L, P < 0.05], but no significant difference between probable sJIA and healthy control group [11.8(7.7-39.2) ng/L vs. 12.3 (2.1-13.8) ng/L, P > 0.05] was found. There were statistically significant differences between sJIA group and healthy control group in serum concentrations of IL-17 [14.0(9.8-34.3) ng/L vs. 9.8 (7.9-16.2) ng/L, P < 0.05], yet compared to healthy control group, no significant difference in concentration level of IL-17 was found in pJIA Group [14.2(9.9-16.9) ng/L vs. 9.8(7.9-16.2) ng/L, P > 0.05].(2) In sJIA and pJIA SF, the median IP-10 level was significantly higher compared to respective PB levels [619.7 (160.9, 873.1) ng/L vs. 64.8 (27.4-111.9) ng/L;660.9 (401.9, 1349.8) ng/L vs. 97.4 (41.9-222.1) ng/L, P < 0.01, respectively], but there was only significant difference in IL-17 between pJIA SF and PB [22.9 (17.1, 45.8) ng/L vs. 14.2 (9.9-16.9) ng/L, P < 0.01].

CONCLUSIONIL-6 may play more important role in the pathogenesis of sJIA. Moreover, IL-6 may be the biomarker associated with arthritis in early JIA stage. Both autoinflammation and autoimmune response may be involved in the pathogenesis of sJIA. IL-17 enrichment may only occur in local joint, the levels of IL-17 in PB may not be significantly increased. The prominent expression gradient between SF and PB of IP-10 maybe the basis of performing chemotaxis and further causing joint damage.

Adolescent ; Arthritis, Juvenile ; blood ; immunology ; metabolism ; Case-Control Studies ; Chemokine CXCL10 ; blood ; metabolism ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interleukin-17 ; blood ; metabolism ; Interleukin-6 ; blood ; metabolism ; Knee Joint ; metabolism ; Male ; Synovial Fluid ; immunology ; metabolism

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4⁺ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4⁺ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.
Cattle ; Animals ; Cytokines ; BCG Vaccine/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-2 ; Flow Cytometry/methods* ; Chemokine CXCL10/metabolism* ; Leukocytes, Mononuclear ; CD4-Positive T-Lymphocytes/metabolism* ; Tuberculosis ; Antibodies, Monoclonal/metabolism*

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