Low 15d-PGJ2 status is associated with oxidative stress in chronic obstructive pulmonary disease patients.

BACKGROUND: Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent nuclear receptor and highly expressed in human and rodent lungs. 15-Deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2), known for cyclopentenone prostaglandin, is the endogenous ligand of PPARγ. However, the associations among PPARγ, 15d-PGJ2 and chronic obstructive pulmonary disease (COPD) were unclear. METHODS: All 130 fasting blood samples and 40 lung specimens were obtained from COPD patients and control subjects. Serum 15d-PGJ2 was detected by ELISA. The expressions of oxidative stress indicators were measured using western blotting and PPARγ nuclei were evaluated with immunohistochemistry in lungs. The associations among serum 15d-PGJ2, pulmonary PPARγ and oxidative stress indicators, and COPD were estimated. RESULTS: Serum 15d-PGJ2 was reduced in COPD patients compared with healthy volunteers. Linear and logistic regression analysis indicated that serum 15d-PGJ2 was positively associated with pulmonary function in COPD patients. In addition, PPARγ-positive nuclei were reduced and oxidative stress indicators, included HO-1 and NOX-4, were increased in lungs of COPD patients. Further correlative analysis suggested that pulmonary function parameters was positively correlated with serum 15d-PGJ2 and pulmonary PPARγ-positive nuclei, inversely related to oxidative stress indicators in lungs of COPD patients. Pretreatment with 15d-PGJ2 obviously attenuated TNFα-induced oxidative stress in BEAS-2B cells. CONCLUSIONS: Serum 15d-PGJ2 and pulmonary PPARγ are reduced, and oxidative stress is elevated in COPD patients. Serum 15d-PGJ2 is inversely associated with oxidative stress in COPD patients.

[Effect of Low-Dose Triptolide and Sorafenib Alone and Their Combination on AML Cell Line MV411 and the Pathway of STAT5].

OBJECTIVE: To investigate the effects of inhibiting proliferation and inducing apoptosis of low-dose triptolide and sorafenib alone or in combination on FLT3-ITD+ acute myeloid leukemia cell line MV4-11 and STAT5 pathway. METHODS: The MV4-11 cells were treated with low dose triptolide(IC20) and sorafenib(IC50) alone or in combination for 48 hours. The cell proliferation and inhibition were detected by using CCK-8 kit, the cell apoptosis was detected by flow cytometry, the expression of FLT3,STAT5 in mRNA and protein levels was detected by RT-PCR and Western blot respectively. RESULTS: The treatment of MV4-11 cells with low dose triptolide and sorafenib alone and in combination for 48 hours could inhibit cell proliferation and induce cell apoptosis, moreover the inhibitory rate and apoptotic rate of MV4-11 cells in drug-combination group both were higher than those in single drug group. The mRNA expression and protein expression of FLT3,STAT5 signaling pathway in drug combination group were significantly lower than those in single drug group. CONCLUSION: Low-dose triptolide combined with sorafenib can synergistically inhibit the proliferation and induce the apoptosis of MV4-11 cells, which may be related with the inhibition of FLT3 and STAT5 pathway.

[Apoptosis-inducing effect of tetrandrine and imatinib on K562/G01 cells and its related mechanism].

This study was purposed to explore the apoptosis-inducing effect of tetrandrine (Tet) and imatinib (IM) alone or both combined on K562/G01 cells and their mechanism. MTT assay was used to detect the inhibitory effect of drugs on cell growth, flow cytometry was used to detect the cell cycle and apoptosis rate. The expression of caspase-3/BCL-2 mRNA was determined by real time-PCR, and the expression of caspase-3/BCL-2 protein was assayed by Western blot. The results showed that after being treated by 1.0 µmol/L IM or 1.5 µmol/L Tet alone and combination of these two drugs for 48 h, the inhibitory rate was (22.74 ± 0.05)%, (20.34 ± 0.57)% and (44.28 ± 0.60)%, respectively, suggesting that inhibitory effect of two drug combination was more obvious. The arrest of cell cycle at G1/S phase could be observed after Tet treatment. Early apoptosis rate was (7.81 ± 0.16) %, (14.10 ± 0.28) % respectively after being treated by combination of 1.5 µmol/L and 3.0 µmol/L Tet with 1.0 µmol/L IM. After being treated with Tet alone, FQ-PCR and Western blot showed that the expressions of caspase-3 mRNA and caspase-3 protein were up-regulated, the expressions of BCL-2 mRNA and protein were down-regulated, the effect of both drug combination was more significant. It is concluded that IM or Tet alone can induce apoptosis of K562/G01. Combination of IM with Tet shows obvious synergistic effect, mechanism of which may associate with up-regulation of caspase-3 mRNA and protein expressions, and down-regulation of BCL-2 mRNA and protein expressions.

[Triptolide inhibits proliferation and induces apoptosis of imatinib resistant K562/G01 cells].

This study was aimed to explore the inhibitory effect of triptolide on proliferation and inducing apoptosis effect of K562/G01 cells and their possible mechanism. MTT assay was used to detect the effect of imatinib or triptolide alone and their combination on K562/G01 proliferation; the cell cycle, apoptosis rate, P-gp protein expression were detected by flow cytometry (FCM); the expression of P-gp was assessed by Western blot; the BCR/ABL gene expression was assayed by real time quantitative PCR. The results showed that triptolide could enhance the effect of imatinib on proliferation inhibition and apoptosis of K562/G01, arrested the cell cycle in G1 phase, down-regulated the expression of BCR/ABL gene and P-gp protein. It is concluded that triptolide induces K562/G01 cell proliferation inhibition and apoptosis, the mechanism may be related to cell cycle arrest, decrease of P-gp protein expression, inhibition of BCR/ABL gene expression.

[Silencing of ICSBP/IRF8 expression in U226 cells and bone marrow mononuclear cells from patients with multiple myeloma].

The objective of this study was to investigate expression of interferon regulatory factors (ICSBP/IRF8) and the potential role of DNA methylation in silencing ICSBP/IRF8 gene in multiple myeloma (MM) cell line U266 and bone marrow mononuclear cells from 10 MM patients (MM-BMMNC). The bone marrow mononuclear cells from 10 healthy persons (N-BMMNC) were collected and used as normal controls. Expression of ICSBP/IRF8 gene was detected by real-time fluorescence quantitative PCR (using 2(-ΔΔCT) to calculate); DNA methylation level of the ICSBP/IRF8 gene was measured using methylation-specific PCR (using the ratio of interest gene ICSBP/IRF8 and internal reference ß-actin expression as results). The results showed that as compared with N-BMMNC the lower expression of ICSBP/IRF8 gene was found in U266 cells and MM-BMMNC, the hypermethylation of the CpG island in the ICSBP/IRF8 promoter was observed, there were significant differences between N-BMMNC and MM-BMMNC or U266 cells (P < 0.05). It is concluded that the expression of ICSBP/IRF8 gene can be silenced in the MM-BMMNC and U226 cells. As the hypermethylation of CpG island in ICSBP/IRF8 promoter is a frequent event in MM cells, the ICSBP/IRF8 gene silencing caused by DNA methylation may take part in the pathogenesis and development of MM.

[Inhibitory effect of histone deacetylase inhibitor LBH589 on multiple myeloma MM1R cells in vitro].

This study was purposed to explore the effect of a new generation of histone deacetylase inhibitor LBH589 alone or combined with bortezomib (Bor) on multiple myeloma cells (MM1R) in vitro. The effect of LBH589 (10, 20, 50 nmol/L) alone or combined with Bor (10, 20 nmol/L) on MM1R proliferation was detected by MTT method; the effect of LBH589 on cell cycle and apoptosis of MM1R cells were determined by flow cytometry; the histone H4 acetylation level of MM1R cells treated with LBH589 (10, 20, 50 nmol/L) for 24 h was analyzed by Western blot. The results showed that the LBH589 alone or combined with Bor all could inhibit the proliferation of MM1R cells in a concentration- and time-dependent manner. After MM1R cells were treated with drugs for 48 h, the cells in G(0)/G(1) phase increased, the cells in G(2)/M and S phase decreased, suggesting the arrest of cells in G(0)/G(1) phase, at the same time, the apoptosis rate of MM1R cells treated with drugs increased in a concentration-dependent manner, while the effect of LBH589 combined with Bor was more obvious than that of LBH589 alone (P < 0.001). Western blot analysis showed that the histone H4 acetylation level was enhanced in concentration-dependent manner after MM1R cells were treated with different concentrations of LBH589 for 24 h. It is concluded that the LBH589 can inhibit the proliferation of MM1R cells, block the cell cycle, induce cell apoptosis, moreover LBH589 combined with Bor has synergistic effect on MM1R cells.

[Significance of detection of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura].

OBJECTIVE: To detect the frequencies of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura (ITP) and non-immune thrombocytopenia, and to evaluate their roles in the diagnosis of ITP and their clinical significance. METHODS: The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody and platelet-specific antibody in 58 ITP patients, 33 non-ITP patients and 31 healthy controls were tested by Enzyme-linked Immunospot Assay (ELISPOT) and modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) respectively. RESULTS: The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody in ITP patients \[(6.6 ± 4.2)/10(5) PBMNC\] were significantly increased (P < 0.05) than that of the controls \[(1.3 ± 0.5)/10(5) PBMNC\] and non-immune thrombocytopenic purpura patients \[(2.2 ± 2.0)/10(5) PBMNC\]. However there was no apparent difference between the latter two groups (P > 0.05). ELISPOT had a sensitivity of 70.69%, a specificity of 90.91% for the diagnosis of ITP, the sensitivity being higher than that of modified MAIPA's (43.10%) (χ(2) = 7.03, P < 0.05). The ROC curve showed the discriminative validity of cytometric bead array was 0.886. CONCLUSION: The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody may reflect the pathogenesis of ITP. ELISPOT assay have high sensitivity and specificity than modified MAIPA for the diagnosis of ITP and the guidance for clinical therapy.

[The clinical significance of immune-related marker detection in idiopathic thrombocytopenic purpura].

OBJECTIVE: To assess the clinical significance of detecting the immune markers in idiopathic thrombocytopenic purpura (ITP). METHODS: The frequencies of circulating B cells secreting platelet-specific antibody, platelet-specific antibody, the percentage of T lymphocyte subsets, the percentage of reticulated platelet and the level of thrombopoietin in 64 ITP patients and 31 healthy controls were measured with enzyme-linked immunospot assay (ELISPOT), modified monoclonal antibody immobilization of platelet antigens assay (MAIPA), flow cytometry and sandwich enzyme-linked immunosorbent assay respectively. RESULTS: Compared with the controls [1.3 ± 0.5/10(5) peripheral blood mononuclear cell (PBMC), (0.33 ± 0.06, 0.41 ± 0.03), (22.08 ± 4.54)% and (8.19 ± 2.46)%], the frequencies of circulating B cells secreting platelet-specific antibody (7.6 ± 4.6/10(5) PBMC in acute ITP group, 5.3 ± 3.0/10(5) PBMC in chronic ITP group), platelet-specific antibody (including the anti-GPIIb/IIIa antibody, anti-GPIb/IX antibody) (0.51 ± 0.11, 0.48 ± 0.06 in acute ITP group; 0.49 ± 0.10, 0.46 ± 0.09 in chronic ITP group), the percentage of CD(8)(+) T Lymphocyte (27.09 ± 9.86)%, the percentage of reticulated platelet in ITP patients [the megakaryocyte cytosis group (24.85 ± 19.18)%, the normal megakaryocyte group (23.89 ± 18.90)%] were significantly increased (all P < 0.05). The frequencies of circulating B cells secreting platelet-specific antibody in acute ITP patients were notably increased (P < 0.05) compared to the chronic ITP patients. In T lymphocyte subsets, the percentage of CD(3)(+) T lymphocyte and CD(4)(+) T lymphocyte and the ratio of CD(4)(+)/CD(8)(+) in the patients with ITP [(60.88 ± 14.59)%, (28.41 ± 10.55)%, 1.18 ± 0.59] were notably decreased than those in the healthy controls [(69.89 ± 6.43)%, (35.38 ± 5.05)%, 1.64 ± 0.29, P < 0.05]. There was no apparent difference of the level of thrombopoietin between ITP patients with megakaryocyte cytosis (72.09 ± 41.64) and health controls (75.37 ± 26.32, P > 0.05), however, the level of thrombopoietin of ITP patients with normal megakaryocyte apparently increased (118.60 ± 70.72, P < 0.05). CONCLUSION: Detecting the frequencies of circulating B cells secreting platelet-specific antibody, platelet-specific antibody, the percentage of T lymphocyte subsets, the percentage of reticulated platelet and the level of thrombopoietin in the patients with ITP may improve the diagnosis and guide clinical therapy.

[Mechanism of C-reactive protein on proliferation of multiple myeloma U266 cells].

This study was purposed to investigate the mechanism of C-reactive protein (CRP) on proliferation of U266 cells. The human multiple myeloma cell line U266 was incubated with human CRP (0, 5, 10, 20 mg/L) for 24 hours, then the proliferation level of U266 cells was detected by using blood analyser. The mRNA expressions of survivin and HSP90alpha were examined by RT-PCR. The results showed that the proliferation ratio was increased, as compared with the control group (p<0.05); furthermore, the mRNA levels of survivin and HSP90alpha were up-regulated in proportion to the increased CRP concentrations. There was significant correlation between expression of survivin and HSP90alpha (r=0.737, p<0.0001) in incubated cells. It is concluded that CRP can stimulate the proliferation of MM cells directly by up-regulating the expression of survivin and HSP90alpha in MM cells. CRP can be regarded as a potential target for MM treatment.

[Erythrocyte native immune adhering function (ENIAF) in patients with hematopoietic and lymphoid neoplasms and its effect on NK Cell killing activity].

The objective of this study was to investigate the change of native immune adhering function (ENIAF) in self-plasma of patients with hematologic and lymphoid neoplasms and its effect on the killing activity of NK cells. The whole blood was anticoagulated with citric acid. 5 microl precipitated red blood cells and 500 microl plasma of patients or controls were directly mixed with 750 microl quantitative K562 cells at 37 degrees C for 30 minutes. One K562 cell attached by one or more erythrocytes was counted as one rosette, the ratio of rosettes was calculated. Using K562 cells as target cells, the killing activity of NK cells isolated from normal persons was detected by MTT assay, the change of the killing activity was observed after adding RBCs. The results indicated that the ratio of rosettes formed by RBCs of 21 normal controls and K562 cells was 15.3% +/- 6.4%, and the ratio of rosettes formed by RBCs of 24 patients and K562 cells was 7.6% +/- 7.0%. The ability of ENIAF in patients with hematologic and lymphoid neoplasms was significantly lower than that in healthy individuals (t = 3.61, p < 0.001). The killing rate of NK cells in peripheral blood of normal individuals was 67% - 71% without adding RBCs, and it increased by 14.7% +/- 5.2% after adding RBCs of normal controls but decreased by 4.3% +/- 7.6% with RBCs of patients. It is concluded that the ENIAF of RBCs in patients with hematopoietic and lymphoid neoplasms decreases, accompanying the reduction of the killing activity of NK cells to K562 cells, so to detect change of ENIAF may be helpful for the assessment of the immunological function of patients with hematopoietic and lymphoid neoplasms.

[Levels of intracellular IL-6 and IFN-gamma in children with acute lymphoblastic leukemia].

OBJECTIVE: To study the changes of intracellular interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) expressions in children with acute lymphoblastic leukemia (ALL) at different stages, and to examine the correlation between IL-6 and IFN-gamma in ALL children. METHODS: The levels of intracellular IL-6 and IFN-gamma in venous blood lymphocytes were detected by flow cytometry in 42 children with ALL at diagnosis and at remission stage. Twenty healthy children were used as the controls. RESULTS: The intracellular IL-6 level in ALL children at diagnosis was 81.74+/-9.31, which was much higher than that in the Control group (5.67 +/- 0.96 ) (P < 0.01). The intracellular IFN-gamma level in ALL children (1.31 +/- 0.32) was significantly lower than that in the Control group (1.46 +/- 0.49) (P < 0.01). However, the intracellular IL-6 level (27.52 +/- 3.40) decreased remarkably in ALL patients at remission stage (P < 0.01), but was still higher than that in the Control group (P < 0.01). In contrast, the intracellular IFN-gamma level (1.97 +/- 0.72) increased noticeably in ALL patients at remission stage, which was higher than that at diagnosis and the Control group (P < 0.01). A negative correlation was found between the intracellular IL-6 and the IFN-gamma levels in ALL patients (r=-0.476, P < 0.05). CONCLUSIONS: Intracellular IL-6 and IFN-gamma levels may be used as the markers for monitoring the response to treatment in ALL patients. There is a negative correlation between intracellular IL-6 and IFN-gamma levels in ALL children.

Nonmyeloablative allogeneic hematopoietic stem cell transplantation in 26 cases of hematological malignancies.

The purpose of this study was to investigate the efficacy of non-myeloablative allogeneic stem cell transplantation (allo-NST) and its related technologies in hematological malignancies. 26 patients with hematological malignancies (acute leukemia 10, chronic myeloid leukemia 14, multiple myeloma 2) received allo-NST following conditioning regimens with fludarabine/cyclophosphamide/ATG in 14 cases or busulfan or melphalan/cyclophosphamide/ATG in 12 cases prior to infusion of 2 or 3 collections of G-CSF (600 microg/d) or G-CSF (300 microg/d) plus GM-CSF (300 microg/d) mobilized blood stem cell on the fifth day. A combination of cyclosporine A (CsA) and methotrexate (MTX) was administered for GVHD prophylaxis. Patients were eligible for donor lymphocyte infusion (DLI) (or donor stem cell infusion (DSI)) given in graded increments according to the chimeric formation and clinical feature. Generally, the dose of the first infusion was 1 x 10(7)/kg in 4th week post-transplantation. The engraftment analyses included the detection of microsatellite short tandem repeats (STRs), bcr/abl fusion gene, Philadelphia chromosome, HLA-locus analysis, sex chromosome and ABO blood type or blood subtype. The results showed that out of 26 patients, 22 (84.62%) were engrafted, 18/22 were full donor chimerism (FDC) up to now. Acute GVHD occurred in 3/26 (11.54%), while chronic GVHD was diagnosed in 6 out of 26 (23.07%) patients. The incidence and degree of infection and hemorrhage were low and slight. It is concluded that NST is a safe and effective therapy for hematological malignancies, whereas related technologies such as adaptation selected, conditioning regimen and transplantation immunotherapy should be studied further.