The imbalanced profile and clinical significance of T helper associated cytokines in bone marrow microenvironment of the patients with acute myeloid leukemia.

BACKGROUND: Immunological disorder has shown to be related to the pathogenesis of acute myeloid leukemia (AML). The microenvironment of AML is immunosuppressive, favoring the survival of malignant hematopoietic cells. However, the systematic research on AML abnormal immune microenvironment, especially the T helper (Th) cells imbalance, remains unsettled. DESIGN AND METHODS: The levels of cytokines in bone marrow plasma including Th1-associated cytokine (IFN-γ), Th2-associated cytokine (IL-4), Th17-associated cytokines (IL-17, IL-6, TGF-ß, and IL-21), regulatory T cell (Treg)-associated cytokines (IL-35 and IL-10) and Th22-associated cytokine (IL-22) were examined by enzyme-linked immunosorbent assay (ELISA) in AML patients and controls. The relative expression levels of IL-4, IL-10, and IL-21 mRNA were analyzed by real time polymerase chain reaction (PCR). RESULTS: Significant differences on cytokine levels tested were observed among the AML newly-diagnosed (ND) patients, AML patients in complete remission (CR) and controls except IL-21 and IL-35. In AML-ND group IFN-γ level was positively correlated with IL-21 or IL-22 level. Additionally, significant associations were observed between IL-17, IL-21 and some clinical characteristics. CONCLUSION: Our results showed that many cytokines were abnormal in AML bone marrow microenvironment. The dysregulation of Th subsets cytokines is thought to contribute to the pathogenesis of AML.

[Monitoring of plasma concentration of imatinib mesylate in patients with chronic myeloid leukemia].

OBJECTIVE: To analyze the clinical efficacy of imatinib mesylate (IM) for Ph-positive or BCR-ABL positive chronic myeloid leukemia (CML) to couple the trough plasma concentrations (C mins) of IM with clinical responses and adverse events (AEs). METHODS: One hundred and one CML patients received IM therapy, and Cmins of IM were determined in 30 patients. RESULTS: (1) Cumulative complete hematological response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR) and negative BCR/ABL fusion gene rates were 96.6%, 86.5%, 77.5% and 47.2%, respectively, in CML-CP patients. In accelerated and blastic phases (AP and BC) patients, CHR, MCyR, CCyR and negative BCR-ABL fusion gene rates were 58.3%, 25.0%, 25.0%, 8.3%, respectively. (2) Mean Cmins of IM was significantly higher in the CCyR at 1 year [(1472 +/- 482) microg/L] group than in the non-CCyR at 1 years group [(1067 +/- 373) microg/L] (P < 0.05), and higher in the MMR at 1 year group than in the non-MMR at 1 years group [(1624 +/- 468) microg/L vs (1137 +/- 404) microg/L, P < 0.05]. CONCLUSION: IM significantly improves cytogenetic and molecular response, event-free survival, and overall survival for patients with Ph-positive CML. The Cmins of IM exerts a significant impact on clinical response (CCyR and MMR at 1 year).

[Synergistic effect of DNA methylation inhibitor and histone deacetylase inhibitor on RASSF1A gene expression in U266 cells.].

OBJECTIVE: To investigate the effect of DNA methylation in combination with histone deacetylase inhibitor on transcription regulation of Ras associated domain family gene 1(RASSF1A) tumor suppressor gene and the molecular biological behaviors in U266 cells. METHODS: The U266 cells were treated with different doses of 5-Aza-2'-deoxycytidine (5-Aza-CdR) and Valproate (VPA) each alone or in combination. Methylation-specific PCR (MSP) was used to detect CpG island methylation in RASSF1A promoter. Quantitative real-time reverse transcription polymerase chain reaction (RQ-PCR) was used to examine the expression of RASSF1A gene in U266 cells. MTT was used for cell proliferation. Cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The methylation of RASSF1A gene promoter was detected in U266 cells, while there was little RASSF1A gene expressing in the control group. The demethylation effect could be detected in the 5-Aza-CdR treated and combined treatment groups but no in the VPA group. The expression level of RASSF1A was induced by 5-Aza-CdR in a concentration-dependent manner while VPA had no such effect. The expression level of RASSF1A mRNA was increased significantly in the combined treatment group. Higher growth inhibition and apoptosis effects were found in 5-Aza-CdR and VPA combination group than that in 5-Aza-CdR or VPA alone group (P < 0.05). After treatment with 5-Aza-CdR or VPA alone for 72 h, more cells were arrested in G(0)/G(1) phase as conpared with control group (P < 0.05), and even more cells were so arrested in combined treatment group (P < 0.05). CONCLUSION: DNA methylation and histone deacetylase inhibitor can synergistically induce demethylation of the RASSF1A gene, re-express RASSF1A gene silenced in U266 cells, inhibit the proliferation of U266 cells and induce cell apoptosis.

[Effect of CD44 gene silence on multi-drug resistance reversal and biologic activity in K562/A02 cells].

This study was aimed to investigate the effect of CD44 gene silence on the drug resistance and biologic activity of human multidrug resistant leukemia cell line K562/A02. The oligonucleotides of CD44 gene were designed according to related data of GenBank, double-stranded DNA was produced by annealing, and was inserted into pGCsilencerU6/Neo/GFP vector. The resultant recombinant plasmid pGCsiRNA-CD44 was transfected into K562/A02 cell line. Expressions of CD44, mdr-1 and blc-2 mRNA were assayed by real time RT-PCR. The 50% inhibitory concentration (IC50) of doxorubicin (ADM) for K562/A02 cell line was determined by MTT method. Cell cycle was determined by flow cytometry. The morphology of apoptotic cells was examined by Hochst 33258 staining. The results indicated that the siRNA eukaryotic plasmid directing at CD44 gene could effectively silence the CD44 gene of K562/A02 cells; as compared with control group, the CD44 expression in K562/A02 cells transfected with 4 pGCsiRNA-CD44 plasmids was obviously inhibited, while the inhibition of CD44 expression in cells transfected with siCD44-1 was strongest. After being transfected with pGCsiRNA-CD44, the expression of CD44 mRNA in K562/A02 cells reduced by 64.1% (p<0.05), at the same time the expression of mdr-1 and bcl-2 mRNA in pGCsiRNA-CD44-transfected K562/A02 cells reduced by 25.6% and 50.8% respectively. IC50 of K562/A02 cells after transfection decreased to (8.77+/-1.63) microg/ml and was obviously lower than that of control (17.97+/-1.61) microg/ml (p<0.01). After transfection for 48 hours, the ratio of K562/A02 cells in G0/G1 increased by 10.7%, and the cells displayed karyopyknosis, nuclear margination and apoptotic bodies. It is concluded that the siRNA plasmid specifically targeting CD44 gene can remarkably down-regulate the expression of CD44 gene, inhibit K562/A02 cell proliferation, induce its apoptosis and effectively reverse the multidrug resistance of K562/A02 cells.

[Significance of CD4+ CD25+ CD127(low) regulatory T cells and notch1 pathway in the pathogenesis of aplastic anemia].

OBJECTIVE: To quantify the CD4+ CD25+ CD127(low) regulatory T cell (Treg), the expression levels of forkhead/winged helix transcription factor FOXP3 and Notch1 mRNA in aplastic anemia (AA) patients before and after treatment, and explore the significance of Treg in pathogenesis of AA. METHOD: CD4+ CD25+ and CD4+ CD25+ CD127(low) T cells in peripheral blood were examined with FACS in 29 AA patients at active phase, 14 at recovery phase, 11 at unrecovery phase, and 15 normal controls. The levels of FOXP3 mRNA and Notch1 mRNA expression were detected with RT-PCR, and the correlations between Treg, FOXP3 mRNA and Notchl mRNA were analyzed. RESULTS: The percentages of peripheral activated CD4+ CD25+ T cells in AA patients at active phase (4.3 +/- 0.7)% and unrecovery phase (4.2 +/- 0.6)% were significantly higher than those in normal controls (2.4 +/- 0.8)% (P < 0.05). The proportion of these cells in AA patients at recovery phase was reduced to (2.6 +/- 0.7)% (P < 0.05), being no difference from that in control group. The number of CD4+ CD25+ CD127(low) T cells in AA patients at active phase (2.4 +/- 1.2)% and unrecovery phase (2.5 +/- 1.1)% was decreased significantly compared with those in normal controls (7.1 +/- 2.7)% (P < 0.01) and in AA patients at recovery phase (5.3 +/- 1.0)% (P < 0.01), there was no difference between the latter two groups. In active phase AA patients, the levels of FOXP3 mRNA and Notchl mRNA (0.260 +/- 0.011 and 0.018 +/- 0.005, respectively) were lower than that in control group (1.307 +/- 0.011 and 0.308 +/- 0.028, respectively) (P < 0.01 and P < 0.01). After treatment, the levels significantly increased to 1.287 +/- 0.012 and 0.281 +/- 0.013 (P < 0.01 and P < 0.01), but there was no difference with that of normal controls (P > 0.05). CD4+ CD25+ CD2(low) T cells and FOXP3 were positively related with Notchl (P < 0.01) in AA patients. CONCLUSION: The decreased number and suppressive activity of CD4 CD25+ CD127(low) Treg cells in the peripheral blood of AA patients cause over-activation of autoreactive T cells and suppression of haematopoiesis. One of the mechanisms maybe the reduced expression of Notch1 in the target cells.

[The effect of selectively inhibiting the nuclear factor kappaB activation on survivin expression in leukemic cells].

OBJECTIVE: To investigate the highly specific proteasomal inhibitor MG132-induced apoptosis and its effect on nuclear factor (NF)-kappaB activation and survivin expression in leukemic K562 cell line. METHODS: leukemic cells of the line K562 were cultured and divided into 2 groups: treatment group, undergoing co-incubation with MG132 of the concentrations of 2, 4, 6, and 8 micromol/L respectively for 24 hours, and control group without treatment of MG132. Apoptosis was detected by examination of cell morphology and flow cytometry. Survivin expression and NF-kappaB activation were analyzed by immunocytochemistry and Western blotting. RESULTS: MG132 induced apoptosis of the K562 cells dose-dependently. Both survivin and NF-kappaB were highly expressed in the K562 cells. Compared with the control group, K562 cell treated with MG132 at the concentrations of 2, 4, 6, and 8 micromol/L for 24 hours showed the decrease of NF-kappaB activation to 75.0% +/- 3.7%, 59.9% +/- 5.3%, 45.4% +/- 5.7%, and 25.0% +/- 4.2% respectively, and decrease of survivin expression to 90.9% +/- 10.1%, 66.7% +/- 5.2%, 45.4% +/- 5.7%, and 30.3% +/- 6.6% respectively. Downregulation of survivin expression was closely correlated with the inhibition of NF-kappaB activation (Pearson correlation coefficient = 0.989, P < 0.01). CONCLUSION: MG132 induces apoptosis of leukemic cells, and effectively inhibits the NF-kappaB activation accompanied by the downregulation of survivin expression.

[Prophylactic effect of matrix metalloproteinase inhibitor on acute graft-versus-host disease in a murine model].

OBJECTIVE: To explore the prophylactic effect of tissue inhibitor of matrix metalloproteinase (TIMP) on acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in a murine model. METHODS: The murine model of aGVHD after allo-HSCT was established by using female C57BL/b (H-2b) mouse as the donor, and male BALB/c (H-2b) as the recipient. After allo-HSCT, recipient mice were divided into 3 groups. For aGVHD prophylaxis group A was given TIMP (2 mg/d) , group B CsA (5 mg x kg(-1) x d (-1)) was given, and group C nothing. Physical signs, mean survival time (MST), peripheral blood counts and aGVHD histopathology were observed. RESULTS: Mice in group C developed typical aGVHD and 100% of mortality, with a MST of 8 days, and those in group A and B had longer survival, the MST being (4.8 +/- 1.4) d and (4.3 +/- 0.9) d respectively, with no statistical difference in peripheral blood count between these two groups. Mice in group A showed less severe signs. CONCLUSIONS: TIMP markedly prolongs MST of allo-HSCT recipients, delays the onset of aGVHD signs, and has no adverse effect on hematopoiesis reconstitution.

[Effect of anti-CD49d monoclonal antibody on mobilization of CD34 positive cell into peripheral blood in mice].

To study the mobilization of peripheral blood stem cells (PBSC) with anti-CD49d monoclonal antibody and try to find a new method for mobilization of PBSC, anti-CD49d McAb, rhG-CSF and combination of anti-CD49d McAb with rhG-CSF were administered subcutaneously to mice separately, the count of white blood cells (WBC) and percentage of CD34(+) cells in peripheral blood of donor mice were dynamically observed, CD34 positive cells obtained by above methods were transfused to recipient mice. The results showed that the count of WBC and percentage of CD34(+) cells in peripheral blood of donor mice elevated significantly after the administration of anti-CD49d McAb, rhG-CSF or combination of anti-CD49d McAb with rhG-CSF. The most effective method for mobilization is the combination of rhG-CSF with anti-CD49d McAb. Reconstitution of hematopoiesis was successful in all group recipient mice after transplantation. Most rapid hematopoietic recovery was observed in recipient mice by rhG-CSF plus anti-CD49d McAb for mabilization. In conclusion, anti-CD49d McAb is effective and synergistic with rhG-CSF in mobilization of CD34 positive cells from bone marrow into peripheral blood.