Effect of APN/CD13 on bestatin enhancing all-trans-retinoic acid-inducing differentiation in NB4 cells / 中国实验血液学杂志

This study was purposed to investigate the effect of aminopeptidase N/CD13 on bestatin enhancing all-trans-retinoic acid (ATRA)-inducing differentiation in NB4 cells. The nitroblue-tetrazolium (NBT) reduction assay was performed to determine the differentiation of NB4 cells, MR2 cells and primary APL blasts. The expression of P38 MAPK protein and the phosphorylation of P38 MAPK protein in NB4, MR2 and K562 cells were detected by Western blot. The results showed that pre-incubation with 5 µg/ml WM-15 blocked the enhancement effect of bestatin on differentiation of NB4 cells induced by ATRA. 5 µg/ml CD13 antibody WM-15 partly blocked the inhibition of bestatin on the phosphorylation of P38 MAPK in NB4 cells. 100 µg/ml bestatin inhibited the phosphorylation of P38 MAPK in NB4 cells and MR2 cells in a time-dependent manner. 100 µg/ml bestatin had no effect on the phosphorylation of P38 MAPK in K562 cells with low level of CD13. Bestatin could not restore the sensitivity to ATRA in ATRA-resistant primary APL blasts and MR2 cells. It is concluded that aminopeptidase N/CD13 inhibitor bestatin may enhance the differentiation-inducing activity of ATRA through inhibiting the phosphorylation of P38 MAPK in NB4 cells mediated by the cell surface APN/CD13.

Arsenic trioxide enhances the effects of bortezomib, dexamethasone on multiple myeloma cell line KM3 in vitro / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of bortezomib (Bor) alone or in combination with As(2)O(3) (ATO) and/or dexamethasone (DXM) on proliferation and apoptosis in KM3 human multiple myeloma cell line KM3.</p><p><b>METHODS</b>KM3 cells were cultured with different concentrations of Bor and ATO and/or DXM in combination or Bor, ATO, DXM alone for different times. Cell proliferation was assayed by MTT assay, and IC(50) was calculated. Cell morphology was observed with light and electric microscopy. The agarose gel electrophoresis was used to evaluate DNA content, and the flow cytometry was used to exam Annexin V-FITC/PI stain.</p><p><b>RESULTS</b>Bor, ATO and DXM inhibited KM3 cell proliferation in a time-and dose-dependent manner with the IC(50) of 0.27, 3.10 and 8.01 micromol/L, respectively. The inhibition rate of KM3 cells by Bor plus ATO and DXM was significantly higher than Bor plus ATO or DXM \[(34.51 +/- 0.51)% vs (25.39 +/- 0.90)% and (34.51 +/- 0.51)% vs (23.80 +/- 0.78)% respectively\]. Typical morphology for apoptosis and DNA ladder were observed in KM3 cell treated with 0.25 micromol/L Bor for 48 h, by Annexin V positivity. The apoptosis rate induced by Bor plus both ATO and DXM was higher than that induced by Bor plus DXM.</p><p><b>CONCLUSION</b>Bor can inhibit the proliferation and induce apoptosis of KM3 cells. Bor enhances the inhibitory effect of ATO and DXM on the growth of KM3 cell. ATO enhances the apoptosis effects of Bor and DXM on KM3 cells.</p>

Combined effects of bortezomib and daunorubicin on multiple myeloma cell KM3 in vitro / 中国实验血液学杂志

The aim of this study was to investigate the combined effects of bortezomib (Bor) and daunorubicin (DNR) or each drug alone on proliferation of human multiple myeloma cell line KM3. KM3 cells were cultured with different concentrations of Bor and DNR, Bor or DNR alone for different times. The cell proliferation was analyzed by MTT assay, and the concentration of 50% growth inhibition (IC(50)) was calculated. The results indicated that both of Bor and DNR inhibited KM3 cell proliferation in dose dependent manner. The IC(50) of both drugs were 0.27 micromol/L and 0.16 micromol/L respectively. The inhibiting rate of Bor plus DNR on KM3 cells was much higher than that of Bor (p < 0.05). It is concluded that the Bor has synergistic inhibitory effect with DNR on the growth of KM3 cell in vitro.

Candida tropicalis arthritis in a patient with acute leukemia / 中国实验血液学杂志

Candida arthritis in patient with hematological malignancy is rare. A case of Candida tropicalis arthritis of knee occurred in a patient with acute monocytic leukemia was reported during the recovery phase of post chemotherapy myelosuppression and agranulocytosis. The patient was diagnosed as Candida tropicalis arthritis of knee according to the Candida tropicalis isolated from the synovial fluid. Itraconazole and amphotericin B were intravenously injected for therapy for 4 - 5 weeks based on the susceptibility test in vitro, which showed better efficacy. But the arthritis relapsed at 4 - 6 weeks after the drug withdrawal. The curative effect was found in patient after treatment with fluconazole injection and articular cavity douching with amphotericin B for 8 weeks. In conclusion, although Candida arthritis in patient with hematological malignancy is rare, it still occurred in the patient with hypoimmunity. The treatment emphasis showed be placed on the full dosage and full treatment course of antifungal agent.

Effects of soluble M-CSF receptor on differentiation of umbilical cord blood-derived myelomonocytic hematopoietic precursors in mesenchymal stem cell microenvironment / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of soluble M-CSF receptor (sMR) on proliferation and differentiation of hematopoietic precursors derived from umbilical cord blood in mesenchymal stem cell (MSC) microenvironment.</p><p><b>METHOD</b>In group of cytokine (CK) + sMR, MSCs were used as feeder cells, mononuclear cells (MNCs) from cord blood were expanded in MSC microenvironment in presence of SCF, Flt3L, TPO, IL-6 and sMR. In CK control group, no sMR was added. MNC counting and colony forming cell (CFC) culture were performed at week 1, 2, 3 and 4.</p><p><b>RESULTS</b>1) The number of MNCs increased rapidly in both group CK and group CK + sMR (108.47 -fold and 120.67 -fold, respectively, P > 0.05). 2) CFC increased, peaked at week 3(38.1 x 10(3)) and declined rapidly at week 4(18.1 x 10(3)) in group CK, but still increased in group CK + sMR at week 4 (84 x 10(3)), the total number of CFC was higher in group CK + sMR than in group CK at week 3 and week 4 (P <0.01). 3) The erythroid CFC peaked at week 1 (5891.2 and 5635.6 for groups CK and CK + sMR, respectively), then dropped rapidly and to zero at week 3, in both group CK and group CK + sMR (P > 0. 05). 4) Myeloid CFC expanded continuously and peaked at week 3 (31.5 x 10(3)), then declined at week 4 (18.3 x 10(3)) in group CK; but still increased at week 4(80.8 x 10(3)) in group CK + sMR, being higher than that in group CK at week 3 and week 4 (P <0.01).</p><p><b>CONCLUSION</b>sMR can inhibit the differentiation of cord blood hematopoietic precursors expanded in MSC microenvironment, but the inhibition exerts only on myelomonocytic but not on erythroid precursors.</p>

Study on unrelated donor allogeneic bone marrow transplantation with Bu-CY2 conditioning regimen for myelodysplastic syndrome / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation (BMT) with unrelated donor for myelodysplastic syndrome.</p><p><b>METHODS</b>Six patients received chemotherapy regimen of busulfan (Bu) and cyclophosphamide (CY) before allogeneic BMT (Bu 4 mg . kg(-1) . d(-1), -7 d - -4 d, CY 60 mg . kg(-1) . d(-1), -3 d - -2 d). Mycophenolate mofetil combined with cyclosporin A and methotrexate was used for prevention of acute graft-versus-host disease after transplantation. Lipo prostaglandin E(1)was used in prophylactic regimen for hepatic veno-occlusive disease.</p><p><b>RESULT</b>Neutrophil count began to be higher than 0.5 x 10(9)/Lat the 18th day after BMT. Platelet count began to be higher than 20 x 10(9)/Lat the 21st day after BMT. Disease-free survival in the six patients was 27 months.</p><p><b>CONCLUSION</b>Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation with unrelated donor is an effective therapy for patients with myelodysplastic syndrome.</p>

Evaluation of interferon-gamma producing-cells using enzyme linked immunospot assay in mice model of acute graft versus host disease / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate IFN-gamma producing-cells (IFN-gamma PCs) in allogeneic mixed lymphocyte reaction (MLR) and acute graft versus host disease (aGVHD) model of mice.</p><p><b>METHODS</b>Enzyme linked immunospot assay (ELISPOT) was applied to study IFN-gamma PCs in MHC mismatched mice spleen cell MLR and aGVHD model of mice.</p><p><b>RESULT</b>IFN-gamma PCs increased significantly in MLR after allogeneic mice spleen cell stimulation. In the experimental mice aGVHD model, IFN-gamma PCs were significantly higher in the severe aGVHD group than those in the moderate aGVHD. In the moderate aGVHD group, mice with GVHD prophylaxis regimen demonstrated significantly lower level of IFN-gamma PCs, compared with those without prophylaxis. IFN-gamma PCs were significantly correlated with the GVHD clinical scores in the group with moderate aGVHD and prophylaxis regimen.</p><p><b>CONCLUSION</b>ELISPOT is a fast, sensitive and specific approach to evaluate alloresponse in allogeneic mice MLR and IFN-gamma PCs are correlated closely with the severity of aGVHD and prophylaxis regimen in the MHC-mismatched mice model.</p>

Impaired microfilament cytoskeleton rearrangement in cytomegalovirus infected cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of cytomegalovirus (CMV) infection on actin and microfilament in human embryo fibroblast cells (HF) and its relationship with CMV replication.</p><p><b>METHODS</b>Cell morphology was observed after the infection of CMV. Western-blot was used to measure the expression levels of beta-actin, G-actin and F-actin proteins. CMV immediately early antigen (CMV IE) in HF cells was analyzed by indirect immunofluorescence assay. Microfilament alteration was determined by cytoskeleton fluorescence probe.</p><p><b>RESULT</b>CMV IE was demonstrated in more than 95% of HF cells after infection, which was primarily located in nucleus. The shape of HF cells changed from thin shuttle like to round and thick ball like, even escaping from wall after infection by CMV. Compared with control group, the expression of G-actin protein increased at 24 h of CMV infection (0.941 +/-0.061 compared with 0.714 +/-0.119, P <0.05), then decreased at 72 h, 96 h respectively(0.218 +/-.035, 0.230 +/-0.055 compared with 0.714 +/-0.119, P <0.05). The levels of F-actin in infected cells gradually decreased at 24 h, 72 h and 96 h compared with control HF cells (0.256 +/-0.021, 0.127 +/-0.032, 0.026 +/-0.008 compared with 0.373 +/-0.050, P<0.05). In infected HF cells, microfilaments were found ruptured, arranged turbulently. Cells fused and fluorescence density of microfilament markedly reduced.</p><p><b>CONCLUSION</b>Cytomegalovirus can induce alteration of actins and microfilament, which may be associated with its infection, replication and reactivity in host cells.</p>

Detection, enrichment and expansion of T lymphocytes mediating alloresponse based on cytokine / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To detect, enrich and expand the cytokine secreting T lymphocytes after allogeneic PBMNCs stimulation.</p><p><b>METHODS</b>The novel cytokine secretion assay (CKSA) was applied to detect T lymphocytes secreting IFN-gamma at single cell level in human mixed lymphocytes reaction. IFN-gamma secreting T cells were enriched by means of magnetic sorting system and expanded with OKT(3), anti-CD(3)mAb and IL-2 combination. Antigen specificity of the expanded cells was confirmed using enzyme linked immunospot assay.</p><p><b>RESULTS</b>A sizable proportion of IFN-gamma secreting T lymphocytes could be detected [(1.12 +/-0.13)% compared with (0.23 +/-0.07)%] and be further enriched to (67.3 +/-10.5)%, or (93.8 +/-22.1) fold. T lymphocytes could be expanded up to 600-fold within 21-28 days and the specific IFN-gamma response of expanded cells was confirmed with stimulation of the relevant allogeneic PBMNC, which was significantly higher than the irrelevant PBMNC control.</p><p><b>CONCLUSION</b>It is feasible to detect significantly increased IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation based on the CKSA technique at single cell level and these cells can be efficiently enriched and expanded for further research.</p>

Relationship between the IFN-gamma producing cells specific for recipient and acute graft versus host disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the relationship between the IFN-gamma producing cell specific for recipient (IFN-gamma-PCSR) in allogeneic stem cell transplantation (allo-HSCT) and acute graft versus host disease (aGVHD).</p><p><b>METHODS</b>In 37 consecutive HLA-identical sibling allo-HSCT pairs, peripheral blood mononuclear cells (PBMNC) from donors before allo-HSCT and recipients after allo-HSCT were taken as responder cells (RC), and PBMNC from recipients before allo-HSCT as allogeneic stimulator cells (allo-SC) in mixed lymphocyte reaction (MLR). IFN-gamma-PCSR in PBMNC were assayed using enzyme linked immunospot assay (ELISPOT).</p><p><b>RESULTS</b>Pretransplantation frequencies of IFN-gamma-PCSR in donor PBMNC were significantly higher in aGVHD group than in non-aGVHD group (P < 0.01) and IFN-gamma PCSRs (>or= 20/2 x 10(5)RC) were significantly associated with the occurrence of grade II-IV GVHD (P < 0.05). Compared with that before allo-HSCT, IFN-gamma PCSR in PBMNC of aGVHD patients was significantly increased (P < 0.05). When PBMNC from aGVHD patients reacted with donor PBMNC, the IFN-gamma PC was significantly lower than that with recipient PBMNC before allo-HSCT. Longitudinal analysis of IFN-gamma PCSR following allo-HSCT showed that compared with that in patients without aGVHD, the IFN-gamma PCSR were significantly higher in patients with that in aGVHD on day +14 (P < 0.01) and day +28 (P < 0.01), respectively. After immunosuppressive therapy for 7 days, IFN-gamma PC declined significantly (P < 0.05).</p><p><b>CONCLUSION</b>The recipient-specific IFN-gamma PC is closely correlated with the allo-response during allo-HSCT and may be helpful for the prediction, diagnosis and monitoring of aGVHD.</p>

Experimental study of the enhancement effect of aminopeptidase N inhibitor ubenimex on the differentiation induction activity of all-trans-retinoic acid in acute promyelocytic leukemia cells and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of aminopeptidase N inhibitor ubenimex on differentiation induction of all-trans-retinoic acid (ATRA) in acute promyelocytic leukemia (APL) cells and its mechanism.</p><p><b>METHODS</b>The expression of CD11b was analyzed by flow cytometry and nitroblue-tetrazolium (NBT) reduction assay was performed to determine the cell differentiation of APL cells. The expressions of c-Myc, ERK1/2, p38MAPK protein and the phosphorylation of ERK1/2, p38MAPK protein in NB4 cells were detected by Western blot assay.</p><p><b>RESULTS</b>Ubenimex alone induced no significant changes in NBT reduction activity and CD11b expression but potentiated the differentiation induction activity of ATRA in APL cells. 100 microg/ml of ubenimex could enhance the NBT reduction activity induced by 10 nmol/L of ATRA, intensify the down-regulation of c-Myc protein expression and inhibit the phosphorylation of p38MAPK protein induced by 10 nmol/L of ATRA in NB4 cells.</p><p><b>CONCLUSIONS</b>Ubenimex could potentiate ATRA induced differentiation in APL cells, which may be correlated with the inhibition of p38 MAPK protein phosphorylation and regulation of c-Myc protein expression.</p>

Expression of survivin gene in NB4 cell line and cells of acute promyelocytic leukemia and its anti-apoptosis and clinical significance / 中国实验血液学杂志

The study was aimed to detect expression rate of survivin gene in APL cell and to explore the relationship between its expression and clinical manifestation. PML/RARalpha and survivin mRNA expression were analyzed by using reverse transcriptase polymerase chain reaction (RT-PCR) technique. The results showed: (1) the survivin gene expression was detected in NB4 cell line. By treatment with ATRA, survivin mRNA expression in NB4 cell gradually decreased along with time delay and almost could not be detected at the 72th hour. (2) the positive and negative rate of survivin mRNA expression was 67% and 33% respectively, while in all 36 cases of de novo and relapse APL patients, the PML/RAR(alpha) fusion gene expression was positive. In 22 cases at remission stage, the PML/RARalpha fusion gene expression was negative, and the positive and negative rate of survivin mRNA expression was 36% and 64% respectively. The survivin mRNA expression positive rates in the de novo group, relapse group and PML/RARalpha fusion gene L-type positive group were obviously higher than those in remission period group (P < 0.05) and were significantly lower than those in acute leukemia group (P < 0.05, < 0.001). (3) whether the survivin mRNA expression was positive or negative in 36 cases of de novo and relapse APL patients, all the 36 cases could obtain complete remission. 4 APL patients with positive expression of survivin mRNA had DIC and serious infection (one patient died). The clinical symptom showed slight skin or mucosa bleeding, fever and asthenic in the patients with negative expression of survivin mRNA. When 2 APL patients with positive expression of survivin mRNA had been treated with ATRA, induction differentiation sign in their peripheral blood and bone marrow figures was not obvious. It is concluded that the survivin gene positive expression rate is lower in acute promyelocytic leukemia than that in any other types of leukemia and is related to clinical manifestation.

Expression level of TRF1 protein in human acute leukemia and its relationship with activity of telomerase / 中国实验血液学杂志

The study was aimed to investigate the expression level of TRF1 protein in human acute leukemia and relationship between expression level of TRF1 protein and activity of telomerase. A quantitative Western blot technique was developed using anti-TRF1(33 - 277) monoclonal antibody and GST-TRF1 fusion protein as a standard to further determine the expression level of TRF1 protein in total proteins extracted from clinical specimens. 20 cases of acute leukemias were studied when 11 normal volunteer's bone marrow was used as control. The results showed that the expression level of TRF1 protein in normal bone marrow (2.217 +/- 0.461 microg/microl) was significantly higher than that in bone marrow of acute leukemia patients (0.754 +/- 0.343 microg/microl) (P < 0.01). There was no remarkable difference of expression level of TRF1 protein between ALL and ANLL (0.628 +/- 0.281 microg/microl vs 0.844 +/- 0.360 microg/microl, P > 0.05). After chemotherapy, TRF1 expression level of patients with complete remission raised (0.772 +/- 0.307 microg/microl vs 1.683 +/- 0.344 microg/microl, P < 0.01), but lower than that of normal (2.217 +/- 0.461 microg/microl, P < 0.01). TRF1 expression level of patients without complete remission was not remarkable different after chemotherapy (0.726 +/- 0.443 microg/microl vs 0.894 +/- 0.338 microg/microl, P > 0.05). TRF1 expression level of patients with complete remission was higher than that in patients without complete remession (1.683 +/- 0.344 microg/microl vs 0.894 +/- 0.338 microg/microl, P < 0.01). For all sample the telomerase activity was determined. It was confirmed that the activity of telomerase in normal bone marrow was lower than that in bone marrow of acute leukemia patients (0.125 +/- 0.078 microg/microl vs 0.765 +/- 0.284 microg/microl, P < 0.01). There was no significantly difference of expression level of TRF1 protein between ALL and ANLL (0.897 +/- 0.290 microg/microl vs 0.677 +/- 0.268 microg/microl, P > 0.05). After chemotherapy, telomerase activity of patients with complete remission reduced (0.393 +/- 0.125 microg/microl), but higher than that of normal (0.125 +/- 0.078 microg/microl, P < 0.01). It is concluded that expression level of TRF1 protein in AL patients is significantly decrese and associated with therapeutic efficaciousness and the activity of telomerase (P < 0.001).

Effect of aminopeptidase inhibitor on differentiation induction activity of all-trans retinoic acid in human acute promyelocytic leukemia NB4 cells and its mechanism / 中国实验血液学杂志

This study was purposed to investigate whether aminopeptidase inhibitor, bestatin, can potentiate all-trans retinoic acid (ATRA)-inducing differentiation in NB4 cells, and to explore its mechanism. The NB4 cells were exposed to either bestatin and ATRA alone or in combination, the morphological changes of NB4 cells were observed by optical microscopy, the CD11b expression was measured by flow cytometry, the function of defferentiation cells was analyzed by nitroblue-tetrazolium (NBT) reduction assay, the mRNA expressions of c-myc and c-EBPepsilon in NB4 cells were detected by RT-PCR, the c-Myc protein expression was determined by Western blot. The results showed that treatment with bestatin alone induced no significant changes in morphology, NBT reduction activity and CD11b expression in NB4 cells. NB4 cells incubated with 10 nmol/L ATRA plus 100 microg/ml bestatin showed more morphologic feature of metamyelocyte and band neutrophil than ATRA alone treated cells. 100 microg/ml bestatin enhanced the NBT reduction activity in NB4 cells induced by various concentrations of ATRA (10, 20, 40 nmol/L). The effects of various concentrations of ATRA in combination with 100 microg/ml bestatin were statistically different from the effect of ATRA alone (P < 0.01). From 48 to 96 hours, 100 microg/ml bestatin time-dependently increased NBT reduction in NB4 cells induced by 10 nmol/L ATRA (P < 0.01). 10 nmol/L ATRA plus 100 microg/ml bestatin for 72 hours prominently elevated CD11b expression in NB4 cells as compared with ATRA alone treated NB4 cells (P < 0.01). There was a substantial decrease in c-myc mRNA levels when 100 microg/ml bestatin was added to 10 nmol/L ATRA (P < 0.05). Various concentrations (50, 75, 100 microg/ml) of bestatin combined with 10 nmol/L ATRA down-regulated the expression of c-Myc protein, which was negatively correlated with the NBT reduction activity of NB4 cells induced by 10 nmol/L ATRA alone or plus bestatin at various concentrations (r = -0.940, P = 0.017). However, 100 microg/ml bestatin plus 10 nmol/L ATRA could not induce any significant changes in the levels of c-EBPepsilon mRNA as compared with ATRA alone treated NB4 cells. It is concluded that an aminopeptidase inhibitor bestatin can potentiate ATRA-inducing differentiation of NB4 cells, possibly by down-regulating c-myc expression in synergy with ATRA.

Impaired microfilament cytoskeletal rearrangement in cytomegalovirus infected cells / 中国实验血液学杂志

The objective of this study was to investigate the effect of cytomegalovirus (CMV) infection on actin and microfilament in human embryo fibroblast cells (HF) and to explore the possible relationship with CMV replication. The cell shape was observed by microscopy after the infection of CMV, RT-PCR assay was used to detect the mRNA expression of beta-actin gene, while Westen-blot was used to measure the level of beta-actin protein. CMV immediately early antigen (IE) in HF cells was analyzed by indirect immunofluorescence assay. Microfilament alteration was determined by cytoskeleton fluorescence probe. The results showed that CMV IE was observed in more than 95% of HF cells after infection, primarily located in nucleus. HF cells infected by CMV changed from thin shuttle shape to round and thick ball shape, even detached from wall. Beta-actin got a significant and gradual decreasing of mRNA level in time-dependent manner (P < 0.05). Compared with uninfected group, the expression of beta-actin protein decreased to (74.2 +/- 13.4)% at 96 hours after infection (P < 0.05). In infected HF cells, microfilaments were ruptured, arranged turbulently, as well as cells merged and fluorescence density of microfilament obviously reduced. It is concluded that cytomegalovirus can induce alteration of actin and microfilament, which may be helpful for CMV to infect, replicate and reactivate in host cells.

A study of the relationship between expression level of TRF1 protein and telomerase activity in human acute leukemia / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To study the expression level of TRF1 (telomeric repeat binding factor 1) protein in human acute leukemia and relationship between expression level of TRF1 protein and telomerase.</p><p><b>METHODS</b>A quantitative Western-Blot technique was developed using anti-TRF1(33-277) monoclonal antibody and GST-TRF1 purity protein as a standard to further determine the expression level of TRF1 protein in total proteins extracted from clinical specimens.</p><p><b>RESULTS</b>Bone marrow tissues of 20 acute leukemia patients were studied, 11 healthy donors' bone marrows were taken as a control. The expression level of TRF1 protein was significantly higher (P<0.01) in normal bone marrow ((2.217+/-0.462) microg/microl) than that of acute leukemia patients ((0.754+/-0.343) microg/microl). But there was no remarkable difference between ALL and ANLL patients ((0.618+/-0.285) microg/microl vs (0.845+/-0.359) microg/microl, P>0.05). After chemotherapy, TRF1 expression level of patients with complete remission elevated ((0.772+/-0.307) microg/microl vs (1.683+/-0.344) microg/microl, P<0.01), but lower than that of normal ((2.217+/-0.462) microg/microl, P<0.01). There was no significantly difference after chemotherapy ((0.726+/-0.411) microg/microl vs (0.895+/-0.339) microg/microl, P>0.05). TRF1 expression level of patients with complete remission is higher than that of patients without complete remission ((1.683+/-0.344) microg/microl vs (0.895+/-0.339) microg/microl, P<0.01). All samples were determined for telomerase activity. It was confirmed that the activity of telomerase in normal bone marrow was lower than that of acute leukemia patients ((0.125+/-0.078) microg/microl vs (0.765+/-0.284) microg/microl, P<0.01). There was no significant difference of expression level of TRF1 protein between ALL and ANLL patients ((0.897+/-0.290) microg/microl vs (0.677+/-0.268) microg/microl, P>0.05). After chemotherapy, telomerase activity of patients with complete remission decreased ((0.393+/-0.125) microg/microl), but was still higher than that of normal ((0.125+/-0.078) microg/microl, P<0.01).</p><p><b>CONCLUSION</b>The expression level of TRF1 protein has correlativity to the activity of telomerase (P<0.001).</p>

Anti-CD47 monoclonal antibody (B6H12) impairs the maturation and function of human dendritic cells / 中国实验血液学杂志

To investigate the influence and mechanisms of CD47 engagement by its soluble mAb B6H12 on the maturation and function of cultured dendritic cells (DCs), monocyte-derived DCs were propagated in granulocyte-macrophage colony stimulating factor (GM-CSF) combined with lipopolysaccharide (LPS) and interleukin (IL)-4, in the presence or absence of soluble anti-CD47 monoclonal antibodies (anti-CD47 mAbs, B6H12). The characteristic morphology of DCs was identified by using the transmission electron microscopy. Flow cytometry was used to detect the cell surface phenotypes. The concentration of IL-12 P70 in supernatant was measured by ELISA. The antigen-presenting functions of DCs were determined in one-way mixed leukocyte reaction by BrdU-ELISA. Electrophoretic mobility shift assay (EMSA) was applied to examine the activity of NF-kappaB. The results indicated that the anti-CD47 mAbs markedly suppressed the expression of CD80, CD86, CD83, CD1a and HLA-DR on the surface of DCs (P < 0.05). The data of the mixed leukocyte reaction and IL-12 P70 production were consistent with the results by flow cytometry (P < 0.01). Pre-exposure to B6H12 mAb during the development of DCs resulted in a dramatic depletion of the DNA binding activity of NF-kappaB toward nucleus protein. Moreover, such an inhibition effect seemed to be dose dependent. In conclusion, the soluble mAb B6H12 inhibits the expression of the costimulatory molecules and MHCII molecules on the DCs. The antigen-presenting function of DCs was also impaired by B6H12. And these modulations are closely related with the depletive DNA binding activity of NF-kappaB. It is suggested that the soluble B6H12 exerts a negative effect on the maturation and function of in vitro cultured DCs due to inhibition of NF-kappaB binding activity.

Effects of berbamine on K562 cells and its mechanisms in vitro and in vivo / 中国实验血液学杂志

To elucidate the antileukemi effects of berbamine and the possible molecular mechanisms in vitro and in vivo, MTT method was used to examine the effect of berbamine on K562 cell growth. The apoptosis rate was measured by flow cytometry. The mRNA expression level of BCR/ABL gene (semiquantity value) was determined by RT-PCR and the BCR/ABL protein (P210) level was detected by Western blot. The K562-bearing mice were used to reveal the therapeutic effect in vivo. The results showed that a significant time- and concentration-dependent inhibition of cell growth was found in the cells treated with berbamine. After the cells were exposed to 8.0 microg/ml berbamine for 24, 48 and 72 hours, the percentage of growth inhibition of K562 cells progressively increased by (26.63 +/- 3.57)%, (61.84 +/- 4.74)%, (75.32 +/- 1.95)%, respectively (compared with control, P < 0.01). The IC(50) (72 hours) value was 5.227 +/- 1.307 microg/ml. The apoptosis rate of K562 cells treated with 8.0 microg/ml berbamine for 24 and 72 hours increased from (29.20 +/- 3.82)% to (61.77 +/- 4.35)% (P < 0.01). Berbamine down-regulated the expression levels of bcr/abl gene and P210 in K562 cells in a time- and concentration-dependent manner. The bcr/abl expression decreased from (1.38 +/- 0.02) to (0.97 +/- 0.01) after exposure of the cells to 8.0 microg/ml berbamine for 0 and 72 hours (P < 0.01). When the cells were treated with 4.0 - 16.0 microg/ml berbamine for 24 hours, the level of P210 decreased from (0.95 +/- 0.03) to (0.63 +/- 0.01) (P < 0.01). In vivo, after treatment for 4 weeks, the tumor weight of berbamine-treated group was also lower than that of untreated group [(1.46 +/- 0.43) g vs (2.90 +/- 0.94) g, P < 0.01] and the inhibition rate was 49.66%, moreover, berbamine down-regulated the expression level of bcr/abl gene of tumor cells. It is concluded that berbamine can obviously inhibit the cell proliferation and induce apoptosis in K562 cell lines in a time- and concentration-dependent manner in vitro. The mechanisms of berbamine-induced apoptosis may be involved in down-regulation of bcr/abl gene expression and P210 level. In vivo, berbamine can aslo display a better antileukemic effect and down-regulate expression of bcr/abl gene. Berbamine extracted from Chinese herb may be a promising candidate of new drug for clinical anticancer treatment, especially for bcr-abl(+) diseases.

Expression of C II TA gene in five human malignant hematological cell lines and its significance / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the role of MHC class II Transactivator (C II TA) in expression of HLA molecules in five human malignant hematological cell lines.</p><p><b>METHODS</b>The expressions of HLA molecules and C II TA protein were detected by immunohistochemistry and flow cytometry. The expression of C II TA gene was detected by RT-PCR. The response of peripheral T cells after stimulation by Jurkat cells was detected by mixed lymphocyte reaction.</p><p><b>RESULT</b>The HLA II-positive tumor cells expressed the C II TA and IFN-gamma induced the expression of HLA I, II in tumor cells, which were able to express C II TA constitutively.</p><p><b>CONCLUSION</b>There is a correlation between the inability of the tumor cells in response to IFN-gamma for HLA expression and the deficiency in the inducible expression of C II TA.</p>

Factors regulating expression of antiapoptosis gene survivin / 中国实验血液学杂志

To explore the regulation mechanism of survivin gene, the NB4 and HL-60 cells were used in experiments, the cell culture in vitro and cell morphological observation were performed and survivin mRNA expression was detected by semi-quantitative RT-PCR. The results showed that the survivin expression in NB4 cell was positive. By treatment of 1 micromol/L ATRA, cell differentiation antigen CD11b was gradually increased ([chi = 47.002, P = 0.000) and CD33 was gradually decreased (chi = 1.614, P = 0.806) with time. Simultaneously, survivin mRNA expression was down-regulated and the cell cycle was arrested at G(0)-G(1) phase (chi = 58.566, P = 0.000). ATRA could down-regulate the survivin mRNA expression of HL-60 cell, but G-CSF, GM-CSF and PHA could up-regulate the survivin expression of HL-60 cell. The cytokine could regulate survivin expression in gene transcription level. The up-regulation of survivin expression was observed while HL-60 cell was stimulated by PHA. The survivin gene expression could be blocked by the survivin antisense oligonucleotide. The survivin mRNA expression of NB4 cell was inhibited by 100 nmol/L-1000 nmol/L survivin antisense oligonucleotide in a dose-dependent manner. The survivin mRNA expression in the NB4 cell was obviously inhibited in 600 nmol/L survivin AS-ODN groups (38%) while the AS-ODN dose increases, the inhibition rate does not descend, but was not inhibited in the control groups, liposomes groups and ODN groups. After NB4 cell was treated by survivin AS-ODN, the typical morphological changes for the apoptosis emerged in NB4 cell. These changes were not found in control groups. It is concluded that PHA, GM-CSF and G-CSF can up-regulate the survivin gene expression, but survivin AS-ODN and ATRA can down-regulate survivin gene expression. The cell cycle arrest at G(0)-G(1) phase while the survivin gene expression was down-regulated by ATRA. It suggested that the survivin gene expression is very related to cell cycle. The morphological changes of cell apoptosis can be observed when the survivin gene expression of NB4 cell was suppressed.