OBJECTIVETo investigate the role of CCL28 in hypoxia-induced cell migration of hepatocellular carcinoma (HCC).

METHODSResected liver tissues from 50 HCC patients were subjected to real-time (rt)-PCR analysis to evaluate the mRNA expression levels of the hypoxia-induced factor HIF-1a and the chemokine CCL28. Patient data on treatment and outcome were analyzed. The human HCC cell lines HepG2 and HCCLM3 were used to investigate effects of hypoxic conditions on HIF-1a and CCL28 expressions by rt-PCR, western blotting, and enzyme-linked immunoassay. The CCL28-mediated effects of hypoxic conditions on cell mobility and invasion were assessed by trans-well and matrigel assays, respectively, in HCCLM3 with CCL28 expression silenced by small-interfering (si)RNA transfection. Spearman's rank test was used to assess the correlation between CCL28 and effects on disease- and treatment-related factors.

RESULTSThe mRNA levels of CCL28 (0.025 +/- 0.075) were found to be strongly correlated with HIF-1a(0.065 +/- 0.098) in human clinical samples of HCC (r = 0.595, P less than 0.01), with higher expressions of both related to recurrence after surgery (P = 0.011 and 0.019, respectively). In vitro hypoxic conditions stimulated HIF-1a and CCL28 expression in a time-dependent manner in both HepG2 (HIF-1a: F = 873.5; CCL28: F = 151.6) and HCCLM3 (HIF-1a: F = 964.5; CCL28: F = 285.8) (all P less than 0.01). siRNA inhibition of CCL28 in HCCLM3 cells led to a significant reduction in hypoxia-induced invasion and migration (all P = 0.011).

CONCLUSIONChemokine CCL28 expression is up-regulated in human HCC and under in vitro hypoxic conditions, and may play an important role in hypoxia-induced HCC migration and invasion.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Hypoxia ; Cell Line, Tumor ; Chemokines, CC ; genetics ; metabolism ; Gene Silencing ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; RNA, Messenger ; genetics

To investigate the role of Interleukin-17 (IL-17), Interferon-gamma (IFN-gamma), and macrophage inflammatory protein-3 alpha (MIP-3alpha) in the pathogenesis of psoriasis, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to semi-quantitatively analyze the mRNA expression of IL-17, IFN-gamma, and MIP-3alpha in 31 psoriatic lesions and 16 normal skin tissues. The results showed that the mRNA of the three cytokines was present in all specimens. And the expression level of IL-17 mRNA in skin lesions was 1.1416 +/- 0.0591, which was significantly higher than that in normal controls (0.8788 +/- 0.0344, P<0.001). The expression levels of IFN-gamma mRNA were 1.1142 +/- 0.0561 and 0. 9050 +/- 0.0263, respectively, with significant difference (P<0.001). And the expression levels of MIP-3alpha mRNA in psoriatic lesions was 1.1397 +/- 0.0521, which was markedly higher than that in normal controls (0.8681 +/- 0.0308, P<0.001). These findings indicate that up-regulated expression of IL-17, IFN-gamma, and MIP-3alpha might be involved in the pathogenesis of psoriasis.
Adolescent ; Adult ; Aged ; Chemokine CCL20 ; Chemokines, CC ; biosynthesis ; genetics ; Female ; Humans ; Interferon-gamma ; biosynthesis ; genetics ; Interleukin-17 ; biosynthesis ; genetics ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Male ; Middle Aged ; Psoriasis ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

In order to explore the possible role of CC chemokine ligand 20 (CCL20) and its receptor CC chemokine receptor 6 (CCR6) in the pathogenesis of psoriasis, the expression levels of mRNA of them in psoriatic lesions were investigated. The skin biopsies were collected from skin lesions in 35 cases of psoriasis vulgaris and 18 normal controls. RT-PCR was used to semi-quantitatively analyze the mRNA expression of CCL20 and CCR6 in the psoriatic lesions and the normal skin tissues. The results showed that the mRNA of CCL20 and CCR6 was present in every specimen. The expression levels of CCL20 mRNA in skin lesions were 1.1397 +/- 0.0521, which were greatly higher than those in normal controls (0.8681 +/- 0.0308) (P<0.001). The expression levels of CCR6 mRNA in skin lesions were 1.1103 +/- 0.0538, significantly higher than in the controls (0.9131 +/- 0.0433, P<0.001). These findings indicate that up-regulated expression of CCL20 and CCR6 mRNA might be related to the pathogenesis of psoriasis.
Adolescent ; Adult ; Aged ; Chemokine CCL20 ; Chemokines, CC ; biosynthesis ; genetics ; Female ; Humans ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Male ; Middle Aged ; Psoriasis ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, CCR6 ; Receptors, Chemokine ; biosynthesis ; genetics

OBJECTIVETo clone human CCL3L1 cDNA and to express and purify the glutathione-S-transferase (GST) fusion protein and human CCL3L1 protein.

METHODSTotal RNA was isolated from breast cancer cell line MCF7. CCL3L1 cDNA including open reading frame was obtained by RT-PCR. PCR product was digested with EcoR I and cloned into the pGEX-4T-1 vector. The plasmids from positive clone was prepared and sequenced to confirm the CCL3L1 in correct fusion form. pGEX-4T-CCL3L1 was transfected to BL21 E. coli via isopropyl-beta-D-thiogalactoside (IPTG) induction to produce GST-CCL3L1 fusion protein, which was further detected by SDS-PAGE and Western blotting.

RESULTSAs shown and confirmed by restriction endonuclease digestion analysis, CCL3L1 was correctly inserted into pGEX-4T-1 vector. The expressed fusion protein had a relative molecular weight of approximately 34 kD.

CONCLUSIONGST-CCL3L1 fusion protein can be successfully expressed using appropriate vector.

Cell Line, Tumor ; Chemokines, CC ; biosynthesis ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; drug effects ; genetics ; metabolism ; Female ; Genetic Vectors ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Isopropyl Thiogalactoside ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

Chemokines and chemokine receptors play a role in migration of circulating leukocytes to the region of inflammation. Human LZIP is an uncharacterized transcription factor and is known to participate in leukotactin (Lkn)-1/CCL15-induced cell migration. We investigated the role of human LZIP in expression of CC chemokine receptors (CCRs) and its involvement in monocyte migration. RNase protection analysis showed that LZIP increased mRNA expression of CCR2 and CCR1 in THP-1 cells. Surface expressions of both CCR2 and CCR1 were also increased by LZIP. Results from an electrophoretic mobility shift assay showed that LZIP binds to the C/EBP element in the CCR2 promoter. LZIP also enhanced the chemotactic activities of monocyte chemoattractant protein-1/CCL2 and Lkn-1. These results suggest that LZIP regulates expression of chemokine receptors that are involved in monocyte migration.
Atherosclerosis/drug therapy/etiology ; CCAAT-Enhancer-Binding Proteins/genetics/immunology/*metabolism ; Cell Line, Tumor ; Cell Movement/drug effects/*physiology ; Chemokine CCL2/*pharmacology ; Chemokines, CC/pharmacology ; Cyclic AMP Response Element-Binding ; Humans ; Macrophage Inflammatory Proteins/pharmacology ; Monocytes/drug effects/metabolism ; Promoter Regions, Genetic ; Protein Binding ; RNA, Messenger/analysis ; Receptors, CCR1/biosynthesis/genetics ; Receptors, CCR2/*biosynthesis/genetics ; Transcriptional Activation/drug effects ; Transfection ; Transgenes

OBJECTIVETo establish determination methods of eotaxin mRNA and TNF-alpha mRNA expression in the lung tissue of mice.

METHODSEotaxin mRNA and TNF-alpha mRNA expressions were determined by semi-quantitative RT-PCR. The functional implications of eotaxin mRNA and TNF-alpha mRNA expression were examined by detecting the numbers of total leucocytes and eosinophils in bronchoalveolar lavage fluid(BALF).

RESULTEotaxin mRNA and TNF-alpha mRNA expression in lung tissue total numbers of leucocyte and numbers of eosinophil in BALF increased in sensitized mice compared with those in normal mice. Dexamethasone significantly but did not inhibit eotaxin mRNA and TNF-alpha mRNA expressions, and eosinophil infiltration in the lungs of the sensitized mice. A compound preparation of traditional Chinese medicine inhibited eotaxin mRNA and eosinophil infiltration, influenced TNF-alpha mRNA expression.

CONCLUSIONIncreased eotaxin mRNA expression in lung tissue is associated with eosinophil infiltration in BALF, which indicates that the methods of semi-quantitative RT-PCR may be useful to the study of the mechanism of antiasthmatic medicine.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Asthma ; drug therapy ; metabolism ; Bronchoalveolar Lavage Fluid ; cytology ; Chemokine CCL11 ; Chemokines, CC ; genetics ; Dexamethasone ; pharmacology ; Disease Models, Animal ; Eosinophils ; physiology ; Female ; Leukocyte Count ; Lung ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; genetics

BACKGROUNDKeshan disease (KD) is an endemic cardiomyopathy in China. The etiology of KD is still under debate and there is no effective approach to preventing and curing this disease. Young women of child-bearing age are the most frequent victims in rural areas. The aim of this study was to determine the differences between molecular pathogenic mechanisms in male and female KD sufferers.

METHODSWe extracted RNA from the peripheral blood mononuclear cells of KD patients (12 women and 4 men) and controls (12 women and 4 men). Then the isolated RNA was amplified, labeled and hybridized to Agilent human 4×44k whole genome microarrays. Gene expression was examined using oligonucleotide microarray analysis. A quantitative polymerase chain reaction assay was also performed to validate our microarray results.

RESULTSAmong the genes differentially expressed in female KD patients we identified: HLA-DOA, HLA-DRA, and HLA-DQA1 associated with spontaneous autoimmunity; BMP5 and BMP7, involved in cardiomyocyte differentiation defect; and ADAMTS 8, CCL23, and TNFSF15, implicated in anti-angiogenic activities. These genes are involved in the canonical pathways and networks recognized for the female KD sufferers and might be related to the pathogenic mechanism of KD.

CONCLUSIONOur results might help to explain the higher susceptibility of women to this disease.

ADAM Proteins ; genetics ; ADAMTS Proteins ; Adult ; Autoimmunity ; genetics ; physiology ; Bone Morphogenetic Protein 5 ; genetics ; Bone Morphogenetic Protein 7 ; genetics ; Cardiomyopathies ; genetics ; pathology ; Cell Differentiation ; genetics ; physiology ; Chemokines, CC ; genetics ; Enterovirus Infections ; genetics ; pathology ; Female ; Gene Expression Profiling ; HLA-D Antigens ; genetics ; HLA-DQ alpha-Chains ; genetics ; HLA-DR alpha-Chains ; genetics ; Humans ; Male ; Middle Aged ; Myocytes, Cardiac ; cytology ; metabolism ; Oligonucleotide Array Sequence Analysis ; Sex Factors ; Tumor Necrosis Factor Ligand Superfamily Member 15 ; genetics

A previous report by this laboratory demonstrated that bacterial iron chelator (siderophore) triggers inflammatory signals, including the production of CXC chemokine IL-8, in human intestinal epithelial cells (IECs). Microarray-based gene expression profiling revealed that iron chelator also induces macrophage inflammatory protein 3 alpha (MIP-3alpha)/ CC chemokine-ligand 20 (CCL20). As CCL20 is chemotactic for the cells involved in host adaptive immunity, this suggests that iron chelator may stimulate IECs to have the capacity to link mucosal innate and adaptive immunity. The basal medium from iron chelator deferoxamine (DFO)-treated HT-29 monolayers was as chemotactic as recombinant human CCL20 at equivalent concentrations to attract CCR6+ cells. The increase of CCL20 protein secretion appeared to correspond to that of CCL20 mRNA levels, as determined by real-time quantitative RT-PCR. The efficacy of DFO at inducing CCL20 mRNA was also observed in human PBMCs and in THP-1 cells, but not in human umbilical vein endothelial cells. Interestingly, unlike other proinflammatory cytokines, such as TNF-alpha and IL-1beta, a time-dependent experiment revealed that DFO slowly induces CCL20, suggesting a novel mechanism of action. A pharmacologic study also revealed that multiple signaling pathways are differentially involved in CCL20 production by DFO, while some of those pathways are not involved in TNF-alpha-induced CCL20 production. Collectively, these results demonstrate that, in addition to some bacterial products known to induce host adaptive immune responses, direct chelation of host iron by infected bacteria may also contribute to the initiation of host adaptive immunity in the intestinal mucosa.
Calcium/metabolism ; Cell Movement/drug effects ; Chemokines, CC/genetics/*metabolism ; Deferoxamine/*pharmacology ; Egtazic Acid/analogs & derivatives/pharmacology ; HT29 Cells ; Humans ; Immunity, Mucosal/*drug effects ; Intestinal Mucosa/*drug effects/immunology/metabolism ; Iron Chelating Agents/*pharmacology ; Macrophage Inflammatory Proteins/genetics/*metabolism ; NF-kappa B/metabolism ; Phosphoprotein Phosphatase/physiology ; Protein Transport/drug effects ; Protein-Serine-Threonine Kinases/physiology ; RNA, Messenger/genetics/metabolism ; Receptors, Chemokine/metabolism ; Research Support, Non-U.S. Gov't