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

OBJECTIVETo obtain a specific antagonist of CXCR4, SDF-1P2G54 by mutating SDF-1 second proline (P) into glycin (G) and removing the α-helix of its C-terminal.

METHODSSDF-1p2g54 gene amplified by PCR was inserted into the vector pET-30a (+) and transformed into Escherichia coli (E. coli) strain BL21. After IPTG induction of E. coli, the expressed recombinant protein was purified with nickel-affinity chromatography column under denaturing conditions and refolded with gradient dilution and ultra-filtration. The chemotactic effect of SDF-1P2G54 on Jurkat cells and its antagonistic effect against SDF-1 were determined by transwell assay; flow cytometry was used to assay the ability of SDF-1P2G54 to induce calcium influx and CXCR4 internalization in MOLT4 cells.

RESULTSThe recombinant protein SDF-1P2G54 completely lost the functions to activate CXCR4 or to induce transmembrane migration of Jurkat cells and calcium influx in MOLT4 cells, but maintained a high affinity to CXCR4. SDF-1P2G54 effectively inhibited the chemotactic effect of wild-type SDF-1 to Jurkat cells, and induced rapid CXCR4 internalization in MOLT4 cells.

CONCLUSIONSDF-1P2G54 is a new antagonist of CXCR4 with a potential value as an effective inhibitor of HIV-1 infection, cancer metastasis or other major diseases.

Cell Line ; Chemokines, CXC ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Mutant Proteins ; biosynthesis ; genetics ; Receptors, CXCR4 ; antagonists & inhibitors ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVEChemokine receptor CXCR4 and its ligand stromal-derived factor 1 alpha (SDF-1alpha) have been paid increasing attention for their involvement in megakaryocytic hematopoiesis. It has been revealed in recent years that they can induce mature and immature megakaryocytes (MKs) to migrate through bone marrow endothelial cells (BMEC) by increasing the affinity of MKs for BMEC. Thus MKs maturity and eventual release of platelet from MKs ensues. While maturity disturbance of MKs and impaired production of platelets have been regarded as the main pathogenesis of ITP, the mechanism of which still remains unclear. Therefore, a clear understanding of the levels of CXCR4 and SDF-1alpha within bone marrow in children with ITP will help us to elucidate further the mechanism of ITP as well as to provide direct theoretical evidence for predicting treatment effect and evaluating prognosis.

METHODSBone marrow were aspirated from 28 children with AITP and 12 normal children. Percoll density gradient and immunomagnetic beads method were used to purify megakaryocytes from the bone marrow. The immune cytochemistry was used to detect CXCR4 on megakaryocytes. The levels of SDF-1alpha were detected by ELISA. SPSS10.0 statistical software was used to deal with the experimental data.

RESULTSBefore the treatment in children with AITP, both the CXCR4 expression on megakaryocytes and the SDF-1alpha level in bone marrow plasma were markedly decreased compared with the normal controls (P < 0.05). As to the cases who were sensitive to the high-dose intravenous immunoglobulin (HDIVIgG), the CXCR4 and SDF-1alpha levels were much higher in children after the treatment than those before the treatment (P < 0.05). In 6 cases insensitive to HDIVIgG, before the treatment the CXCR4 level was much lower than the children sensitive to HDIVIgG (P < 0.05).

CONCLUSIONSThe low levels of CXCR4/SDF-1alpha system in bone marrow may be one of the factors which contribute to the maturity disturbance of megakaryocytes and disturbance of platelets production in AITP, while decreased CXCR4/SDF-1alpha system may be caused by the effect of autoantibody against platelet. The mechanism of HDIVIgG in the treatment of AITP may involve in the increasing expression of CXCR4/SDF-1alpha system. The level of CXCR4 on megakaryocytes may play a certain role in predicting the treatment effect of immunoglobulin.

Adolescent ; Bone Marrow ; metabolism ; Chemokine CXCL12 ; Chemokines, CXC ; blood ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Humans ; Infant ; Ligands ; Megakaryocytes ; metabolism ; Purpura, Thrombocytopenic, Idiopathic ; blood ; Receptors, CXCR4 ; biosynthesis

OBJECTIVETo investigate the effect of stromal cell derived factor-1alpha(SDF-1alpha) expression and its receptor CXCR4 on the biological behavior of multiple myeloma (MM) cells and on the expression of soluble intercellular adhesion molecule 1 (ICAM-1).

METHODSFACS analysis was used to study the expression of ICAM-1 (CD(54)) and CXCR4 on the surface of MM cells. Chemotaxis assay through transwell bore polycaronate and ELISA assay were employed to monitor the soluble ICAM-1 level.

RESULTS(1) Fresh MM cells expressed variable levels of functional CXCR4 [(50.4 +/- 27.3)%], which was correlated with the in vitro ability of transwell migration of MM cells [(23.6 +/- 17.2)%, P < 0.01]. (2) SDF-1alpha could up-regulate the expression of ICAM-1 on MM cells. Furthermore, the serum level of sICAM-1 was correlated with the expression of CXCR4 on MM cells.

CONCLUSIONSDF-1alpha/CXCR4 plays an important role on the biological behavior of MM cells via mediating the effect of adhesion molecules.

Adult ; Aged ; Cell Movement ; Chemokine CXCL12 ; Chemokines, CXC ; biosynthesis ; Female ; Humans ; Intercellular Adhesion Molecule-1 ; biosynthesis ; Male ; Middle Aged ; Monocytes ; metabolism ; pathology ; Multiple Myeloma ; metabolism ; pathology ; Receptors, CXCR4 ; biosynthesis ; physiology ; Tumor Cells, Cultured ; Up-Regulation

The study was aimed to explore the expression of stromal cell derived factor-1alpha (SDF-1alpha) and its receptor CXCR4, and their relationship with the extramedullary infiltration in acute lymphoblastic, grannulocytic and monocytic leukemia. 66 cases of acute leukemia included 31 cases of acute lymphoblatic leukemia (ALL), 20 cases of acute grannulocytic leukemia (M(2)) and 15 cases of acute monocytic leukemia (M(4)+M(5)). There were 41 cases with extramedullary infiltration and 25 cases without-extramedullary infiltration. Enzyme-linked immunoabsorbent assay (ELISA) and flow cytometry were used to determine expression of SDF-1alpha and CXCR4 respectively on leukemia cells in peripheral blood and bone marrow of different groups. The results showed that average plasma level of SDF-1alpha in the ALL, M(4)+M(5), M(2) patients and the normal control were 1317.87 +/- 220.76, 1339.79 +/- 187.06, 1063.70 +/- 190.74, 1908.34 +/- 135.55 (pg/ml) respectively. The average levels in the ALL, M(4)+M(5) and M(2) patients groups were lower than those in normal control group. Both levels in ALL and M(4)+M(5) patient groups were higher than that in M(2) patient group. The average levels of SDF-1alpha in patient group with extramedullary infiltration and patient groups without-extramedullary infiltration were 1252.49 +/- 263.12, 1234.91 +/- 185.50 (pg/ml) respectively. The former seemed as if higher than the latter, but without statistical significance. The MFI of CXCR4 expression in ALL, M(4)+M(5), M(2) patient group were 78.47 +/- 33.96, 67.21 +/- 24.29, 41.66 +/- 17.18, respectively. CXCR4 expression in ALL and M(4)+M(5) patient groups were higher than that in M(2) patient group (P > 0.05). There was no significant difference between the ALL and M(4)+M(5) patient group (P > 0.05). The MFI of CXCR4 expression in patients with extramedullary infiltration and patients without extramedullary infiltration were 81.72 +/- 27.63, 36.94 +/- 11.86 respectively. The former was higher than the latter (P < 0.05). It is concluded that the higher expression of CXCR4 on acute lymphoblatic and monocytic leukemia cells may be one of the molecular mechanisms of extramedullary infiltration in both kinds of leukemia. The average plasma levels of SDF-1alpha decreased in leukemia patients and this decrease not related to the extramedullar infiltration, which may be due to the SDF-1alpha local expression in the organ infiltrated.
Acute Disease ; Adolescent ; Adult ; Chemokine CXCL12 ; Chemokines, CXC ; biosynthesis ; genetics ; Child ; Child, Preschool ; Female ; Humans ; Leukemia ; metabolism ; pathology ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Leukemic Infiltration ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; Receptors, CXCR4 ; biosynthesis ; genetics ; Stromal Cells ; metabolism

A DNA fragment encoding mouse B Lymphocyte Chemoattractant (BLC, MV10Kda) was obtained by PCR. The amplified fragment was inserted into prokaryotic expression vector PQE30. Recombinant protein was expressed in E. Coli XL-1 blue and purified by affinity chromatography on a nickel-nitrilotriacetic acid gel matrix. Then it was identified by sequence analysis and Western blot analysis. The fragment inserted into prokaryotic expression vector PQE30 was identified to be BLC gene fragment by sequence analysis. And a specfic band was shown by Western blot analysis. These findings provide the evidence that the recombinant protein obtained and purified in this study using gene engineering method is mouse B Lymphocyte Chemoattractant.
Animals ; Chemokine CXCL13 ; Chemokines, CXC ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; Genetic Vectors ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sequence Analysis, DNA

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

OBJECTIVETo investigate the effects of nitric oxide (NO) on reperfusion injury following pancreaticoduodenal transplantation in rats.

METHODSThe homologous male Wistar rat model of heterotopic total pancreaticoduodenal transplantation was used. The L-arginine (L-Arg) group received intravenous injection of L-Arg 5 minutes before and after reperfusion at a dose of 200 mg/kg while the N-Nitro-L-Arginine methyl ester (L-NAME) group received intravenous injection of L-NAME at a dose of 10 mg/kg, and control group received saline. The amount of NO in the pancreas graft was measured. Serum concentration of cytokine-induced neutrophil chemoattractant (CINC) determined by enzyme-linked immunosorbant assay, expression of CINC mRNA detected by Northern blot assay, and myeloperoxidase (MPO) activity in the pancreas graft were measured. Histological observation was performed.

RESULTSThe amount of NO in the L-Arg group was higher than in the control group, while in the L-NAME group was lower than in the control group (P < 0.05). The peak of serum CINC concentration occurred 3 hours after reperfusion with significant difference among groups. Expression peak of CINC mRNA in the pancreas graft occurred 3 hours after reperfusion. The expression level in the L-Arg group was lower than in the control group, the L-NAME group was higher than control group (P < 0.05). MPO activity in the L-Arg group obviously decreasd compared with other groups. The pancreas inflammation was ameliorated in L-Arg group, and pancreas damage was aggravated in L-NAME group.

CONCLUSIONSL-Arg can increase the amount of NO and inhibit the elevation of CINC, CINC mRNA expression, and early neutrophil accumulation in the transplanted pancreas. NO has protective effects on the ischemia/reperfusion injury of pancreaticoduodenal transplantation.

Animals ; Arginine ; pharmacology ; Chemokines, CXC ; biosynthesis ; genetics ; Duodenum ; transplantation ; Male ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; metabolism ; Pancreas Transplantation ; Peroxidase ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism

OBJECTIVETo investigate the pathophysiological role of CXCL16 in immunological liver injury induced by Bacille de Calmette et Guerin (BCG) and lipopolysaccharides (LPS).

METHODSImmunological liver injury was induced by BCG and LPS in mice, and the expression of CXCL16 was detected in the liver tissues by real-time quantitative PCR and immunohistochemical examination. The relationship of the expression of CXCL16 and the extent of hepatic necrosis was investigated histopathologically and immunohistochemically. Mononuclear cells were isolated from the liver tissues and their numbers were counted; T lymphocytes populations in the liver tissue were also analyzed with FACS.

RESULTSThe immunological liver injury model was successfully created. Up-regulation of CXCL16 in injured livers correlated with the extent of liver injury and the amountmononuclear cell infiltrations.

CONCLUSIONThese findings suggest that up-regulation of CXCL16 was closely correlated with liver injury extent during the immunological liver injury induced by BCG-LPS in mice, and intrahepatic recruitment of specific lymphocytes might be an important mechanism of liver injury.

Animals ; Chemical and Drug Induced Liver Injury ; Chemokine CXCL16 ; Chemokine CXCL6 ; Chemokines, CXC ; biosynthesis ; genetics ; Lipopolysaccharides ; Liver Diseases ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mycobacterium bovis ; Receptors, Scavenger ; biosynthesis ; genetics

This study investigated the production of stromal cell-derived factor-1 (SDF-1) and the expression of CXCR4 in human bone marrow endothelial cells (BMECs). Human BMEC cell line BMEC-1 cells expressed SDF-1 mRNA, and conditioned medium induced chemoattraction of CD34+ cells. Migration was not inhibited by pretreating the input cells with pertussis toxin, indicating that the chemoattractive activity was not dependent on SDF-1. Three-day culture of BMEC-1 and primary human BMEC cells produced 1,710+/-204 and 1,050+/-153 pg/mL SDF-1alpha, respectively, which was much less than primary human BM stromal cells (29,536+/-532 pg/ mL). By immuno-histochemistry, CXCR4 was detected in the endothelial cells lining sinusoids, arterioles, and venules in the bone marrow. However, cultured BMECs and BMEC-1 cells did not express CXCR4 on their surfaces. These results indicate that BMECs produce and release small amounts of SDF-1 and express CXCR4 in vivo only.
Antigens, CD34/biosynthesis ; Bone Marrow Cells/*metabolism ; Cell Movement ; Cells, Cultured ; Chemokines, CXC/*biosynthesis ; Chemotaxis ; Culture Media, Conditioned/pharmacology ; Endothelial Cells/*metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Hematopoietic Stem Cells/metabolism ; Human ; Immunohistochemistry ; Pertussis Toxin/pharmacology ; RNA, Messenger/metabolism ; Receptors, CXCR4/*biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Support, Non-U.S. Gov't ; Time Factors ; Umbilical Veins/cytology