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

OBJECTIVEThis study examined the expressions of plasma stromal cell derived factor-1 (SDF-1) and CXCR4 mRNA in peripheral blood mononuclear cells (PBMC) of children with Kawasaki disease (KD) and aimed to explore the significance of SDF-1 and CXCR4 mRNA in KD.

METHODSFifty-six children with KD (12 cases complicated by coronary artery lesions) and 60 age and gender-matched healthy children (normal controls) were enrolled in this study. Plasma SDF-1 levels and CXCR4 mRNA expression in PBMC were measured using ELISA and real-time quantitative PCR at the acute and convalescence stages of KD.

RESULTSPlasma SDF-1 levels (1833 +/- 395 ng/L vs 1126 +/- 408 ng/L; P < 0.05) and the CXCR4 mRNA expression in PBMC (6.57 +/- 2.81 vs 2.58 +/- 1.01; P < 0.01) in KD patients were significantly higher than those in normal controls at the acute stage. Both plasma SDF-1 levels and CXCR4 mRNA expression in KD patients decreased significantly at the convalescence stage, but nevertheless remained higher than those in the normal controls. The patients with concomitant coronary artery lesions showed higher CXCR4 mRNA levels than without at the acute stage (8.19 +/- 2.39 vs 6.13 +/- 2.77; P < 0.05).

CONCLUSIONSPlasma SDF-1 concentration and CXCR4 mRNA expression in PBMC increased in KD patients. CXCR4 mRNA might be involved in the development of coronary artery lesions in KD.

Chemokine CXCL12 ; Chemokines, CXC ; blood ; Child, Preschool ; Female ; Humans ; Infant ; Leukocytes, Mononuclear ; metabolism ; Male ; Mucocutaneous Lymph Node Syndrome ; blood ; RNA, Messenger ; blood ; Receptors, CXCR4 ; genetics

OBJECTIVETo study the expression of stromal cell derived factor 1(SDF-1)/CXCR4 and their association with clinicopathologic features and lymph node metastasis in invasive breast carcinoma.

METHODSThe expression of SDF-1 was studied by immunohistochemistry and in-situ hybridization. Immunohistochemical study for CXCR4 was also performed. The correlation with various clinicopathologic parameters was analyzed.

RESULTS(1) SDF-1 was mainly expressed in tumor cells and the level of its expression (both membranous and cytoplasmic) in lymph node-positive group was higher than that in lymph node-negative group (P = 0.033). Only cytoplasmic expression correlated with the number of positive lymph node involved by metastasis, TNM tumor stage, histologic grade, tumor dimension and estrogen receptor status (P < 0.05). (2) SDF-1 protein was also detected in the endothelial cells, although its mRNA was rarely detected. SDF-1 staining in lymphatics was associated with positive lymph node (P = 0.005) and SDF-1 staining in blood vessels correlated with stromal lymphocytic reaction (P = 0.001). The extent of nodal involvement was higher in the group with positive SDF-1 staining in blood vessels and with prominent lymphocytic reaction than that in other groups with one or neither of the two features (P < 0.05). (3) On the other hand, CXCR4 was mainly expressed in tumor cells (both nuclear and cytoplasmic); and the level of its expression in lymph node-positive group was higher than that in lymph node-negative group (P = 0.005). Only cytoplasmic expression correlated with the number of positive lymph node involved by metastasis, TNM tumor stage, histologic grade, tumor dimension and HER2 status (P < 0.05). The nuclear expression of CXCR4 was only correlated with progesterone receptor status (P < 0.01). The cytoplasmic expression CXCR4 also positively correlated with SDF-1 expression (P = 0.001).

CONCLUSIONSSDF-1 and CXCR4 can serve as biomarkers for diagnosis and prediction of lymph node metastasis, as well as potential therapeutic targets in invasive breast carcinoma. The difference in localization and staining patterns may also carry different significance.

Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Chemokine CXCL12 ; genetics ; metabolism ; Chemokines, CXC ; metabolism ; Female ; Humans ; Immunohistochemistry ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; pathology ; Middle Aged ; Receptor, ErbB-2 ; genetics ; metabolism ; Receptors, CXCR4 ; genetics ; metabolism

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

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

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

Animals ; Chemokines, CXC ; genetics ; Enzyme Activation ; Intercellular Signaling Peptides and Proteins ; genetics ; Lipopolysaccharides ; pharmacology ; Lung ; enzymology ; Male ; Mitogen-Activated Protein Kinases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Respiration, Artificial ; Tidal Volume ; Tumor Necrosis Factor-alpha ; genetics

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

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