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

The stromal cell-derived factor 1 (SDF-1) interacts with its receptor CXCR4 to transduction signals, playing important roles in most physiological and pathological processes. It is reported that CXCR4 is highly expressed in many kinds of hematological malignancies and closely related to the prognosis, drug resistance and relapse of diseases. The growth of tumor cells can be inhibited by the anti-SDF-1 antibody or anti- CXCR4 antibody, supporting a new way for the therapy against hematological malignancies. Their expression in relation with prognosis and drug resistance of hematological malignancies are summarized in this review.
Chemokine CXCL12 ; biosynthesis ; genetics ; Drug Resistance, Neoplasm ; genetics ; Hematologic Neoplasms ; metabolism ; pathology ; Humans ; Prognosis ; Receptors, CXCR4 ; biosynthesis ; genetics ; Signal Transduction ; Stromal Cells ; metabolism

OBJECTIVETo evaluate the application of CXC chemokine receptor-4 (CXCR4) combined with alpha-methylacyl-CoA racemase (P504S) or P63 protein in the differential diagnosis of benign and malignant prostatic diseases.

METHODSThe EnVision immunohistochemical method was used to detect the expressions of CXCR4, P504S and P63 protein in 40 specimens of PCa not treated by any anticancer therapy and 30 specimens of BPH tissues. The correlation was analyzed between CXCR4 expression and the characteristics of PCa metastasis.

RESULTSOf the 40 cases of PCa, 33 (82.5%) were stained positive for CXCR4, 37 (92.5%) for P504S and 2 (5%) for P63 protein. Of the 30 cases of BPH, 5 (16.6%) exhibited positivity for CXCR4, 1 for P504S and all for P63. P504S + P63 showed a higher rate of correct diagnosis of PCa than either CXCR4 + P63 or P504S + CXCR4. There was a statistically significant correlation between CXCR4 expression and cancer metastasis (P < 0.05).

CONCLUSIONP504S, CXCR4 and P63 are useful tumor markers for the diagnosis and differentiation of benign and malignant prostatic diseases. CXCR4 gives a high rate of correct diagnosis when combined with P504S or P63, and has an important application value in the differential diagnosis of benign and malignant prostatic diseases.

Aged ; Aged, 80 and over ; Biomarkers, Tumor ; biosynthesis ; Diagnosis, Differential ; Humans ; Male ; Membrane Proteins ; biosynthesis ; Middle Aged ; Prostatic Hyperplasia ; diagnosis ; metabolism ; pathology ; Prostatic Neoplasms ; diagnosis ; metabolism ; pathology ; Racemases and Epimerases ; biosynthesis ; Receptors, CXCR4 ; biosynthesis

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 coreceptors CCR5 and CXCR4 expressions on the peripheral blood T lymphocytes in HIV/AIDS patients, with an analysis of their clinical value.

METHODSThirty HIV/AIDS patients and 16 normal controls were selected and flow-cytometry was used to detect the coreceptors CCR5 and CXCR4 expressions in whole blood samples taken from the patients and controls. An analysis was made of the correlation of the coreceptor expression with CD4 T cell counts and the activation of CD4+/ CD8+ T cells (HLA-DR+/CD38+).

RESULTSThe CCR5 expression on CD8+ lymphocytes and the CXCR4 expression on the CD4+/ CD8+ T lymphocytes of the HIV/AIDS group were higher than those of the normal controls (P < 0.01). The CCR5 and CXCR4 expressions on the CD4+/CD8+ T lymphocytes of the HIV/AIDS patients was significantly correlated with the activation of CD4+/CD8+ T cells (HLA-DR+/CD38+) (P < 0.05).

CONCLUSIONThe expressions of CCR5 and CXCR4 are significantly correlated with the immune system reaction to HIV and the disease progression in HIV infected patients.

Acquired Immunodeficiency Syndrome ; blood ; Adolescent ; Adult ; Case-Control Studies ; Child ; Child, Preschool ; Female ; HIV Infections ; blood ; Humans ; Male ; Middle Aged ; Receptors, CCR5 ; biosynthesis ; Receptors, CXCR4 ; biosynthesis ; T-Lymphocytes ; metabolism

OBJECTIVETo suppress the expression of CCR5 and CXCR4, the co-receptors for human immunodeficiency virus type 1 ( HIV-1), and thus inhibit HIV-1 from entering cells.

METHODSDNA fragments encoding either CCR5 or CXCR4 were amplified from healthy human peripheral blood mononuclear cells (PBMCs) by reverse transcript polymerase chain reaction (RT-PCR) and sequencing was performed. Correct fragments were inserted into Shuttle plasmid inversely, which was recombined with backbone plasmid containing homologous adenoviral genome in E. coli BJ5183. The recombinant plasmids were transfected into 293 cells in which they were packaged and amplified. Recombinant adenoviruses containing antisense RNA of CCR5 or CXCR4 were obtained and identified by RT-PCR, and the titres of them were determined by cytopathic effect (CPE) method. The U937 and MT4 cells were infected by recombinant adenoviruses containing antisense RNA of CCR5 (multiplicity of infection, MOI = 100) and CXCR4 (MOI = 200), respectively. The expression of co-receptors on infected cell was measured by fluorescence activated cell sorter at 24, 48, 72 hours and 10 days after infection. In addition, the chemotactic activity and proliferation of infected cells were detected with Boyden chamber and 3H incorporation respectively.

RESULTSWe constructed the recombinant plasmids and obtained the recombinant adenoviruses which contained antisense RNA of CCR5 or CXCR4 and were designated as pAd-antiR5 and pAd-antiX4 respectively. The titers of recombinant adenoviruses pAd-antiR5 and pAd-antiX4 were 5 x 10" PFU/ml and 7 x 10(10) PFU/ml, respectively. The expression rate of CCR5 on U937 cells decreased from 82. 10% (blank control) to 1.12% (Ad-antiR5 infected) , and that of CXCR4 on MT4 cells decreased from 42% (blank control) to 1.03% (Ad-antiX4 infected) 24 hours later. The expression rates of CCR5 on Ad-antiR5 infected U937 cells were 1.02% , 1.26% , 1.23% at 48 hours, 72 hours, and 10 days later, respectively. The expression rates of CXCR4 on Ad-antiX4 infected MT4 cells were 1.13%, 1.17%, 1.22% at 48 hours, 72 hours, and 10 days later, respectively. Moreover, the recombinant adenovirus had no effects on chemotactic activity and proliferation of the cells.

CONCLUSIONThe recombinant adenovirus containing antisense CCR5 or CXCR4 can remarkably decrease the expression of co-receptors for HIV-1 on U937 or MT4 cells without affecting their chemotactic activities and proliferative abilities.

Adenoviridae ; genetics ; Cell Line, Transformed ; Cell Proliferation ; Chemotaxis ; Down-Regulation ; Genetic Vectors ; Humans ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics ; Receptors, CCR5 ; biosynthesis ; genetics ; Receptors, CXCR4 ; biosynthesis ; genetics ; Transfection ; U937 Cells

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

This study was purposed to explore the effect of different cytokine combinations on the expansion of the mononuclear cells drived from umbilical cord blood (CB) ex vivo and expression of CXCR4 and CD49d on CD34+ cells after expansion. Human fresh CB mononuclear cells were cultured in serum-free and stroma-free medium containing different combinations of cytokine for 7 days. At day o and 7, the total cells were counted, CD34+ cells and CD34+CXCR4+, CD34+CD49d+ cells were assayed by flow cytometry, and CFU were determined. According to the different combinations of cytokine, experiments were divided into four groups: control, SF group (SCF + FL), SFT group (SCF + FL + TPO) and SFT6 group (SCF + FL + TPO + IL-6). The results showed that the SF (SF group) combination supported only low expansion of total cells, CD34+ cells and CFU. The addition of TPO in SF group restored UCB stem/progenitors expansion to a higher level than that in SF group, while there was no difference between groups SFT and SFT6 (P > 0.05). The cytokine combinations in groups SF, SFT and SFT6 all could upregulate the expression levels of CD49d and CXCR4 on expanded cord blood CD34+ cells, but there were no significant differences between groups SF, SFT and SFT6 (P > 0.05). It is concluded that SCF + FL has no strong synergistic effects on primitive hematopoietic cells. TPO plays an important role in enhancing expansion of umbilical cord blood hematopoietic cells, while IL-6 only shows a neutral effect on it. SCF + FL + TPO combination not only promotes progenitor cells expansion but also upregulates the expression of CD49d and CXCR4 on CD34+ cells from cord blood.
Antigens, CD34 ; biosynthesis ; genetics ; Cytokines ; pharmacology ; Drug Synergism ; Fetal Blood ; cytology ; Humans ; Integrin alpha4 ; biosynthesis ; genetics ; Leukocytes, Mononuclear ; cytology ; Membrane Proteins ; pharmacology ; Receptors, CXCR4 ; biosynthesis ; genetics ; Stem Cell Factor ; pharmacology ; Thrombopoietin ; pharmacology

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

This study was amied to construct CXCR4 gene modified bone marrow mesenchymal stem cells (MSCs), and investigate the effect of CXCR4 expression on MSCs migration. The retrovirus vector pMSCV-CXCR4-IRES-GFP that expresses human CXCR4 gene was cloned,the MSCs were transduced by the virus, and the expression of OXCR4 was analyzed by FACS, RT-PCR and immunofluorescence staining. The migration assay was performed using Transwell method in the presence of SDF-1. FACS results showed that 46% of the transduced MSCs were CXCR4 positive, and 57% were GFP positive. The expression of CXCR4 in MSCs was also confirmed by RT-PCR and immunostaining. The migration of MSCs was induced by SDF-1 and was strongly dependent on CXCR4 expression. The concentration of SDF-1 had effect on the migration and the transmigration rate of CXCR4 modified; the amount of MSCs was 5-fold higher than that of untransduced MSCs when the optimal concentration rose to 50 ng/ml. These data indicate that SDF-1/CXCR4 plays an important role in MSCs migration ,and the CXCR4 genetic modification approach could be applied to enhance cell homing, and engraftment in MSCs therapy.
Bone Marrow Cells ; cytology ; Cell Movement ; Cells, Cultured ; Chemokine CXCL12 ; pharmacology ; Genetic Vectors ; genetics ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Receptors, CXCR4 ; biosynthesis ; genetics ; Retroviridae ; genetics ; metabolism ; Transduction, Genetic ; Transfection