The long-term outcome of reduced-intensity allogeneic stem cell transplantation from a matched related or unrelated donor, or haploidentical family donor in patients with leukemia: a retrospective analysis of data from the China RIC Cooperative Group.

This study compared 6-year follow-up data from patients undergoing reduced-intensity conditioning (RIC) transplantation with an HLA-matched related donor (MRD), an HLA-matched unrelated donor (MUD), or an HLA-haploidentical donor (HID) for leukemia. Four hundred and twenty-seven patients from the China RIC Cooperative Group were enrolled, including 301 in the MRD, 79 in the HID, and 47 in the MUD groups. The conditioning regimen involved fludarabine combined with anti-lymphocyte globulin and cyclophosphamide. Graft-versus-host disease (GVHD) prophylaxis was administered using cyclosporin A (CsA) and mycophenolate mofetil (MMF). Four hundred and nineteen patients achieved stable donor chimerism. The incidence of stage II-IV acute GVHD in the HID group was 44.3 %, significantly higher than that in the MRD (23.6 %) and MUD (19.1 %) groups. The 1-year transplantation-related mortality (TRM) rates were 44.3, 17.6, and 21.3, respectively. Event-free survival (EFS) at 6 years in the HID group was 36.7 %, significantly lower than that of the MRD and MUD groups (59.1 and 66.0 %, P < 0.001 and P = 0.001, respectively). For advanced leukemia, the relapse rate of the HID group was 18.5 %, lower than that of the MRD group (37.5 %, P = 0.05), but the EFS at 6 years was 31.7 and 30.4 % (P > 0.05), respectively. RIC transplantation with MRD and MUD had similar outcome in leukemia which is better than that with HID. RIC transplantation with HID had lower relapsed with higher TRM and GVHD rate, particularly in advanced leukemias. RIC transplantation with MRD and MUD had similar outcomes in leukemia and they were better than those with HID. RIC transplantation with HID had a lower relapse rate but higher TRM and GVHD rates, particularly in cases of advanced leukemia.

[Intertemporal allocation and cost of forest carbon sequestration in China under the carbon neutrality target]. / ç¢³ä¸­å’Œç›®æ ‡ä¸‹ä¸­å›½æ£®æž—å›ºç¢³é‡è·¨æœŸåˆ†é åŠæˆæœ¬.

The situations are complex and variant in the three stages of "carbon emission peak", "rapid reduction of carbon emission" and "deep decarbonization for carbon neutrality" in China's carbon neutralization roadmap. Forest carbon sequestration is an important means to achieve the goal of carbon neutralization in China. Its intertemporal allocation is a vital way to balance industrial emission reduction and forest carbon sequestration, reduce the cost of carbon neutrality, and gradually achieve the goal of carbon neutrality based on optimal cost. Based on the cost optimization allocation theory, we simulated the cost change process of three stages of carbon neutralization in China by quoting the theory of marginal carbon sequestration cost and combining with the existing domestic marginal abatement cost theory. The results showed that annual forest carbon sequestrations with the optimal cost in China was 20 million t, 775 million t and 1.982 billion t respectively in the three stages of "carbon emission peak", "rapid reduction of carbon emission" and "deep decarbonization for carbon neutrality", accounting for 1.8%, 17.5%, and 37.6% of the total emission reduction in each period. Compared with the way relying only on industrial emission reduction, forest carbon sequestration under the optimal cost design reduced the total cost by 48, 79136, and 909253 million US$ in the three stages of carbon neutralization, respectively. Due to the limited cost advantage of forest carbon sequestration, industrial emission reduction should be emphasized in the "carbon emission peak" stage. In the "rapid reduction of carbon emissions" stage, the cost advantage of forest carbon sequestration will be increasingly prominent. In the stage of "deep decarbonization for carbon neutrality", it is necessary to fully exploit the cost advantage of forest carbon sequestration to achieve the goal of "zero carbon" to avoid the risk of high costs, especially for industries with high decarbonization cost or that will never be completely decarbonized. The optimal cost design for forest carbon sequestration can save 988.437 billion US $ in carbon-neutral costs.

[Arsenic trioxide combined with buthionine sulfoximine enhances apoptosis in multidrug-resistant human leukemia K562/ADM cells in vitro].

OBJECTIVE: To investigate the apoptosis-inhancing effect of the combination of arsenic trioxide (As2O3 ) and buthionine sulfoximine (BSO) on multidrug-resistant human leukemic K562/ADM cells, to compare the effect of As2O3 alone and As2O3 combined with BSO and As2O3 alone, and to determine the effect of intracellular GSH content on this treatment. METHODS: As2O3 was used in a dose of 0.5 micromol/L, 2.0 micromol/L and 5.0 micromol/L, respectively, and BSO was used in a dose of 100 micromol/L in the culture of multidrug-resistant human leukenic K562/ADM cells. The cell proliferation activity was assessed with MTT assay. The cell apoptosis was detected by flow cytometry using Annexin-V and propidium iodide (PI) staining. Intracellular GSH content was measured using glutathione assay kit by spectrophotometry. RESULTS: After the GSH contents were reduced by the combination of arsenic in clinic dose (0.5, 2 micromol/L) and BSO (100 micromol/L), respectively, the K562/ADM cell proliferation activity was obviously inhibited and the cell apoptosis-inducing effect was advanced in 24 hours. In 48 and 72 hours, the effect of the combination group (clinic dose arsenic group) was significantly stronger than that of clinic dose arsenic alone group and the high dose arsenic alone group. CONCLUSION: The cell apoptosis-inducing effect of arsenic in combination of BSO on multidrug resistant human leukemia K562/ADM cells is significantly enhanced in comparison with that of arsenic alone. The reduction of intracellular glutathione content is closely correlated with this apoptosis-enhancing effect.

[Clinical Study of Patients with Myelodysplastic Syndrome Transformed into Acute Myeloid Leukemia].

OBJECTIVE: To study the biological characteristics and treatment response of patients with myelodysplastic syndrome (MDS) transformed into acute leukemia(AL). METHODS: Using WHO standard method, the clinical characteristics of patients with MDS into AML were retrospectively analyzed, the related factors influencing the MDS into AML and the treatment response of patients were analyzed. RESULTS: Twenty-six cases (17%) of MDS were transformed into AL among 153 cases of middle and high risk MDS, all of which were AML. The median time of transformation from MDS into AML was 4 months (1-29),and these cases transformed into AML-M2, M4, M5 and M6. In these 26 cases of AML patients, the varying degrees of anemia symptom appeared, 2 cases were with marrow infiltration, 18 cases (69.2%) were with abnormal chromosome karyotype. Compared with karyotype before transformation into AML, the abnormal karyotype in 9 cases had been conversed (new karyotype or disappear once of existing karyotype). Total efficiency of individualized treatment for MDS transformed into AML was 80%. This treatment could improve the patients quality of life. CONCLUSION: Middle and high risk MDS patients are prone to be tranformed into AML. Multiple factors are involved in the transformation of MDS into AML. These patients showed the special biological characteristics and poorer prognosis. Demethylation treatment is helpful to achieve a good near-term curative effect.

[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.

[Detection of abnormal numbers of chromosome 8 with interphase fluorescence in situ hybridization in hematologic malignancies].

OBJECTIVE: To explore the value of interphase fluorescence in situ hybridization (FISH) in the detection of abnormal numbers of chromosome 8 in patients with hematologic malignancies. METHODS: Conventional cytogenetics(CC) and interphase FISH using chromosome 8 centromere specific probe were simultaneously carried out to detect the abnormal numbers of chromosome 8 in eight acute myeloid leukemia cases with CC unveiled abnormal numbers of chromosome 8, ten chronic myeloid leukemia cases in accelerated phase or blast crisis, and three normal individuals. RESULTS: Nine cases that displayed trisomy 8 by means of CC were confirmed by FISH. Among them, Case 5 only displayed diploidy 8, trisomy 8 and tetrasomy 8 by CC, at the same time, FISH confirmed the presence of trisomy 8 and tetrasomy 8 and also revealed a low percentage of a pentasomy 8 clone. Case 3 and Case 17 had each only one cell with trisomy 8 by means of CC, and this could not determine whether they had the trisomy 8 clone, yet FISH confirmed the existence of trisomy 8 clone. Case 9 did not display trisomy 8 by CC, but FISH revealed the existence of trisomy 8 clone. In the other cases, the percentages of trisomy 8 cells determined by FISH were close to or significantly lower than those by CC, but for Case 16 where the percentage of trisomy 8 cells by FISH was significantly higher than that by CC. CONCLUSION: Interphase FISH is a useful method for the detection of abnormal numbers of chromosome 8, especially in the patients with normal or unsure karyotype or with less and bad metaphases. It is an important complement to CC.

[The role of intercellular adhesion molecule-1 in binding of acute myeloid leukemic blasts cells to human umbilical vein endothelial cells].

OBJECTIVE: To evaluate the adhesion of acute myeloid leukemia (AML) blasts to human umbilical vein endothelial cells (HUVECs) and the roles of intercellular adhesion molecule-1 (ICAM-1) and its ligand lymphocyte function associated antigen-1 (LFA-1) in binding of leukemic blasts to HUVECs. METHODS: AML blasts attachment to unactivated or tumor necrosis factor alpha (TNF alpha) activated-endothelial cell monolayers was investigate in vitro; The adhesion of leukemic blasts co cultured with unactivated endothelial cells under static conditions at 37 degrees C for 24 hours was observed;The binding of neutrophils and unactivated endothelial cell monolayers exposed to supernatant of blasts were tested. ICAM-1 on endothelial cell surface and sICAM-1 of endothelial cell supernatant were determined by flow cytometry and ELISA detection. We also observed the adhesion of leukemic blasts in the presence of the adhesion blocking mAbs anti-ICAM-1 and anti-LFA-1. RESULTS: This study has shown that the blast cells attached to unactivated endothelium was little (24.33 +/- 2.87)% and increased after exposure of endothelium to TNF alpha (81.87 +/- 4.08)% (n = 21, P < 0.001); The binding of blasts to endothelium also increased significantly after 24 hours co cultured with unactivated HUVEC (82.06 +/- 7.05)%, (n = 21, P < 0.001); The adhesion of neutrophils and unactivated endothelial cell monolayers exposed to supernatant of blasts was increased significantly (83.99 +/- 3.86)%, (n = 21, P < 0.001). Lower levels of ICAM-1 and sICAM-1 expression was detected on unactivated HUVECs (55.81 +/- 4.11)%, (0.839 +/- 0.236) microg/L respectively. Treatment of HUVECs with AML blasts supernatant for 24 hours increased the expression of ICAM-1 (65.36 +/- 5.97)%, (1.424 +/- 0.469) microg/L respectively (n = 21, P < 0.05) and anti-ICAM-1 and anti-LFA-1 significantly inhibited the adhesion of AML blasts attachment to TNF alpha activated-endothelial cell monolayers (20.12 +/- 1.73)%, (n = 10, P < 0.001). CONCLUSION: Our results indicate that leukemic blasts have the ability to generate some factors that stimulate endothelial cell to secrete ICAM-1 ane to promote their own adhesion to vascular endothelium, interaction of ICAM-1 and its ligand LFA-1 has a key role in adhesion of leukemic blasts and HUVECs.

[Construction of fusion gene vaccine of WT1 multi-epitope fused with stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and its expression and immunogenicity].

This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.

[Expression of FLT3 internal tandem duplication in pediatric patients with acute myeloid leukemia and its correlation with multidrug resistance].

This study was aimed to investigate the expression of FLT3 internal tandem duplication (FLT3-ITD) in pediatric patients with acute myeloid leukemia (AML) and to analyse the clinical features of patients with mutations and the relation of FLT3-ITD with multidrug resistance gene 1 (mdr1). RT-PCR was used to determine the expressions of FIT3-ITD and mdr1 gene in bone marrow samples from 81 new diagnosed pediatric patients with AML, the cytogenetics and immunophenotypes of bone marrow cells were routinely examined. The results indicated that the FLT3-ITDs were detected in 8 out of 81 pediatric patients (9.88%) and all mutations detected were hybrid, while less frequently this mutation was detected in adult patients. Although they were irrelevant with sex and immunophenotypes, the mutations seemed predominant in older pediatric patients. The leukocyte counts and bone marrow blast cell counts in pediatric patients with FLT3-ITD at diagnosis were higher than those in pediatric patients without FLT3-ITD (p = 0.001 and p = 0.041 respectively), but the normal chromosomes were found in most pediatric patients with FLT-ITD. The patients with FLT3-ITD had lower induction remission rate (only 25%), but the patients without FLT3-ITD had higher remission rate (76.1%). According results detected by RT-PCR, the mdr1 gene was found in 27 pediatric patients, but only 3 out of 8 pediatric patients with FLT3-ITD were detected to express both FLT3-ITD and mdr1, which suggests unrelation between FLT3-ITD occurrence and mdr1 expression. It is concluded that the FLT3-ITD is frequent mutation in pediatric patients with AML, the prognosis is worse and the induction remission rate is lower in these patients, but the FLT3-ITD not relates with the mdr1, which suggests that the common MDR modulators may be un effective for therapy of the patients with FLT3-ITD.

[Multicenter report of nonmyeloablative allogeneic stem cell transplantation for hematologic diseases].

OBJECTIVE: To observe the treatment effect and toxicity of nonmyeloablative allogeneic stem cell transplantation (NST) for hematologic diseases. METHODS: A total of 243 hematologic diseases patients received HLA-identical NST were enrolled in this study from 9 transplant centers of NST Cooperative Group in China. Nonmyeloablative conditioning regimen was based on fludarabine (Flud), rabbit anti-human thymocyte globulin (ATG), cyclophosphamide (CTX) (FAC), and plus cytarabine or busulfan (BU) etc. Graft-versus-host disease (GVHD) prophylaxis included cyclosporin A (CsA) and mycophenolate mofetil (MMF). RESULTS: Among the 243 patients, 219 (90.1%) achieved full donor chimerism (FDC), 2(0.8%) engraftment failure. 78 (32.1%) had mixture chimerism (MC) at 4 weeks after NST, out of which 56 switched to FDC, 16 remained MC and 6 (2.5%) developed graft rejection. The incidence of acute GVHD was 34.2%, including 6.6% of grade III-IV acute GVHD. Chronic GVHD developed in 78 (32.1%) patients. The follow-up durations were 3 - 99 months, 162 (66.7%) were still alive and the overall survival rates were 76.5%, 73.9%, 70.7%, and 27.8% for MDS/SAA, chronic myeloid leukemia, acute leukemia at first remission, and refractory or relapsed leukemia, respectively. CONCLUSIONS: The nonmyeloablative allogeneic stem cell transplantation based on FAC conditioning results in sustained engraftment and mild aGVHD, providing a new feasible curative therapy for hematology diseases.

[Immunoregulatory effect of mesenchymal stem cells on active T lymphocytes].

This study was purposed to explore the immunoregulatory effects of human bone marrow mesenchymal stem cells (MSCs) on active T lymphocytes in vitro and the new strategy to prevent graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mononuclear cells from human peripheral blood cells were isolated and cultured in the presence of phytohemagglutinin (PHA) (final concentration was 10 microg/ml) for different times. The ability of T lymphocyte proliferation and activation was measured by (3)H-Thyramine incorporation. The expressions of CD3(+)CD4(+), CD3(+)CD8(+), CD4(+)CD25(+) and CD4(+)CD152(+) on T cells were detected by FCM after coculture for 72 hours. Experiment was divided into 4 groups: A group as control (no added MSCs), B group (actived T cells + 2 x 10(4) MSCs), C group (actived T cells + 4 x 10(4) MSCs), D group (actived T cells + 8 x 10(4) MSCs). The results showed that the ability of T lymphocyte proliferation in the same PHA concentration increased with prolonging of time. ability of T lymphocyte proliferation was strongest when culturing for 48 hours (p < 0.01); the expressions of CD44, CD105, CD29 and FIK1 of MSCs were positive, expressions of CD33, CD34, CD45 and HLA-DR were negative. MSCs inhibited T lymphocyte proliferation and the inhibitory effect depended on the amount of MSCs. CD3(+)CD8(+), CD4(+)CD25(+) and CD4(+)CD152(+) T cells cocultured with MSCs increased obviously and CD3(+)CD4(+) expression significantly decreased, as compared with control group (p < 0.01). It is concluded that the MSCs inhibit T lymphocyte proliferation induced by mitogen (PHA), and perform their immunosuppressive function by up-regulation of CD3(+)CD8(+), CD4(+)CD25(+) and CD4(+)CD152(+) expressions and down-regulation of CD3(+)CD4(+) expression.

[Detection of tumor load in chronic myeloid leukemia during treatment with transplantation by conventional cytogenetics, nested-RT-PCR and FISH].

This study was purposed to investigate the sensitivity and specificity of conventional cytogenetics (CC), nested-reverse transcriptase polymerase chain reaction (nested-RT-PCR) and dual-color/dual-fusion fluorescence in situ hybridization (D-FISH) technique in monitoring the tumor load of chronic myeloid leukemia (CML) during treatment with transplantation. CC, nested-RT-PCR and interphase D-FISH were simultaneously carried out to detect the tumor load of 7 CML patients during treatment with non-myeloablative allogeneic stem cell transplantation (allo-NSCT). 40 specimens from 7 CML patients before and after allo-NSCT were analyzed. The results showed that 29 specimens were Ph (+) with different positive ratio and 3 specimens with lower cells were not analyzed by CC. 36 specimens were bcr/abl mRNA (+) by RT-PCR. 4 specimens from case 1 at 12, 18, 26 and 38 months after allo-NSCT were Ph (-) and bcr/abl mRNA (-), 4 Ph (-) bcr/abl (+) specimens containing 2 from case 1 at 9 and 10 months after allo-NSCT, 1 from case 2 at 15 months after allo-NSCT, 1 from case 3 at 12 months after allo-NSCT showed 5.4%, 0%, 16.5% and 1.5% bcr/abl (+) cells by FISH. 3 specimens with lower cells containing 2 from case 5 at 20 and 60 days after allo-NSCT and 1 from case 7 at 40 days after allo-NSCT were analyzed by FISH and showed 55.0%, 27.5% and 73.5% bcr/abl (+) cells. The Ph (-) bcr/abl (-) specimen from case 1 at 12 months post-allo-NSCT showed 0% bcr/abl (+) cells by FISH. It is concluded that CC can be used as a basic tool to monitor the change of tumor load in CML during treatment. When specimen with lower cells can not be analyzed by CC in early period after allo-NSCT, or result of CC can not evaluate precisely dynamic change of tumor load and when tumor load in treated patient are lower to Ph (-) by CC while bcr/abl mRNA (+) by RT-PCR, FISH must be used to detect precisely tumor load and monitor dynamic change of it. More sensitive RT-PCR is used to monitor tumor load when it is lower to bcr/abl (-) by FISH during treatment.

[The effect of arsenic trioxide (As2O3) combined with BSO on K562/ADM cell and its mechanisms].

OBJECTIVE: To investigate the apoptosis-induction, P-glycoprotein (P-gp) and mdr1 mRNA inhibition effects of arsenic trioxide (As2O3) and buthionine sulfoximine (BSO) on multidrug-resistant cell line K562/ADM cells, and to determine the relationship between intracellular GSH content and arsenic effect. METHODS: K562/ADM cells were treated with arsenic (0.5, 2.0, 5.0 micromol/L) alone or combined with BSO (100 micromol/L). The cell proliferating capacity was assessed with MTT assay, and cell apoptosis by Annexin V and propidium iodide (PI) staining. Intracellular GSH contents were measured using a glutathione assay kit by spectrophotometry. P-gp expression was determined by flow cytometry, and mdr1 mRNA expression by semi-quantitative RT-PCR. RESULTS: The GSH contents in K562/ADM cell was (81.13 +/- 3.91) mg/g protein. After the GSH contents were degraded by BSO, the K562/ADM cell proliferating capacity was obviously inhibited and the cells were induced apoptosis in 24 hours by the combination of clinic dose arsenic group (0.5, 2.0 micromol/L) and BSO (100 micromol/L). The cell apoptosis rates at 48 hours in arsenic alone group and combination group were (59.29 +/- 6.01)% and (65.06 +/- 8.29)%, and at 72 hours were (82.15 +/- 9.28)% and (92.72 +/- 9.41)% retrospectively. At 48 hours, the mdr1 mRNA inhibition effect of the combination group was obviously stronger than that of high dose arsenic alone group. At 72 hours, the P-gp inhibition effect of the combination group (clinic dose arsenic group, 0.5, 2.0 micromol/L) was obviously stronger than that of high dose arsenic alone group (5.0 micromol/L). CONCLUSION: The intracellular GSH contents are closely correlated with the arsenic effect. The combination of conventional dose arsenic and BSO significantly induces K562/ADM cell apoptosis and inhibits P-gp and mdr1 mRNA expression in the cells.

[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.

[Fluorescence quantitative PCR detection of WT1 gene expression in peripheral blood of patients with acute leukemias and its clinical implications].

To elucidate the expression of WT1 in all types of leukemias and its implications for monitoring minimal residual disease in patients with acute leukemia, the peripheral blood from 55 leukemia patients and 10 normal voluteer was detected by using FQ-RT-PCR. Follow-up monitoring of WT1 expression of peripheral blood was performed for 20 patients with acute leukemia. The results showed that the expression of WT1 gene in all types of leukemias was significantly higher than that in normal control (P < 0.001). For ANLL and ALL patients, the survival time in the group of WT1 6.8 x 10(-3), (P = 0.027). Follow-up detection of the expression of WT1 in peripheral blood samples from 20 acute leukemia patients, 7 cases relapsed after complete remission has been done. In 5 of 7 relapsed patients, the expression of WT1 had obviously increased about 2 - 3 months before clinical relapse became apparent. It is concluded that the established FQ-RT-PCR method is accurate and specific. The expression of WT1 gene is relatively high in all types of leukemias compared with normal peripheral blood cells, the higher WT1 expression may associate with poor prognosis in acute leukemia, and the dynamics of WT1 level correlate with the disease status. The quantitative assessment of WT1 expression in peripheral blood samples by FQ-RT-PCR may be a useful tool for monitoring minimal residual disease.

Coexistence of tetrasomy 8 and trisomy 8 in acute promyelocytic leukemia (AML-M3) with t(15;17)(q22;q12).

This study was purposed to characterize the first case of acute promyelocitic leukemia (AML-M(3a)) with t(15;17), trisomy 8 and tetrasomy 8, and explore its characteristics of morphology, cytogenetics, molecular biology, immunology and clinical features. Morphological changes of peripheral blood and bone marrow smears were observed under microscope. Chromosome specimen was prepared by 24 h short-term culture of bone marrow cell, RHG-banding technique was used for karyotypic analysis. PML-RARa fusion gene transcript was detected by nested-reverse transcription-polymerase chain reaction (nested RT-PCR). Interphase fluorescence in situ hybridization (FISH) using chromosome 8 centromere specific probe were carried out to detect abnormal numbers of chromosome 8. Immunophenotypic analysis was performed by flow cytometry. The results showed that peripheral blood smear revealed 65% promyelocyte, and bone marrow aspirate was hypercellular with 72.4% promyelocyte, moderately basophilic cytoplasm with numerous azurophilic granules. Karyotype analysis demonstrated 48, XY, +8, +8, t(15;17)(q22;q12) [16]/47, XY, +8, t(15;17)(q22;q12) [3]/46, XY, t(15;17)(q22;q12) [1]. RT-PCR assay revealed PML-RARa fusion gene transcript (+). FISH showed that the percentages of cells exhibiting 1, 2, 3, 4, 5, 6 green fluorescence signals were 0.5, 7, 19, 55, 18 and 0.5, respectively. This confirmed the presence of tetrasomy 8 and trisomy 8 and also revealed a low percentage of a pentasomy 8 clone. Immunophenotypes of the blasts displayed that CD13 (96.2%), CD33 (55.9%), CYMPO (93.5%) were positive. All the lymphoid markers tested were negative. The patient survival time was just 10 days. It is concluded that tetrasomy 8 is secondary cytogenetic event after t(15;17) in this case. It may be a consequence of clonal evolution of trisomy 8. t(15;17) AML-M(3) with tetrasomy 8 heralds a poor prognosis.

[Clinical studies on expressions of Fas and mdr-1 in acute leukemia and their correlations].

To explore the Fas and mdr-1 expression and their correlation in multidrug resistance (MDR), Fas and mdr-1 expressions of bone marrow from 59 patients with newly diagnosed AL before therapy and after complete remission were detected by direct immunofluorescence with flow cytometry and semi-quantitative RT-PCR, respectively. The results showed that in newly diagnosed AL patients, Fas expression in AML was higher than in ALL (P < 0.05), mdr-1 expression in AML and ALL had no difference (P > 0.05), the expressions of Fas and mdr-1 had correlation (r = -0.282, P < 0.05) in AL; the results of simple-variable and multivariable COX survival factor model analysis suggested that Fas and mdr-1 expressions were prognostic factors for the effect of therapy and survival. Log rank test, comparing the groups of Fas(+) with Fas(-), mdr-1(+) with mdr-1(-), demonstrated that the CR rates and median remission time of every two groups had significant difference. It is concluded that in AL, Fas and mdr-1 expressions have high correlation with the effect of treatment, Fas expression probably is one of the favorable prognostic factors, mdr-1 is an unfavorable prognostic and less effective factor.

[Construction of expression vector for recombinant annexin II and characteristics of its fibrinolysis].

The study was designed to investigate annexin II resulting in molecular pathological mechanism of the primary fibrinolysis and establish annexin II vector model for further research on disturbance of coagulation. A target gene was amplified from human umbilical vein endothelial cells (HUVEC) by RT-PCR. Annexin II gene fragment was purified and ligated with molecular biological recombinant technology. The recombinant of plasmid annexin II was transfected into HL-60 cells and its distribution in the cell and structure characteristics of annexin II protein were evaluated by multi-photon excitation laser scanning microscope. By means of flow cytometry (FCM) and Werstern blot technique, the protein expression was qualitatively and quantitatively analyzed. Transfected cells were treated in vitro with annexin II antisense oligonucleotide (AS) targeting to the start site of annexin II cDNA. The results showed that the recombinant pZeoSV2(+)/ANN II was constructed successfully and expressed in HL-60 cells. The protein expression was distributed on the surface of cell by fluorescence assay. After transfection for 48 hours, the cells occurred higher level of expression. The level of the plasmin was significantly enhanced in the present of annexin II. The FCM and Western blot analysis showed the annexin II expression was similar both in transiently and stably transfected in HL-60 cells. Annexin II antisense oligonucletide and McAb significantly inhibited the activity of plasminogen. It was concluded that annexin II plays an important role in the fibrinotysis. Annexin II vector was defined as a expression tool for further studying fibrinolysis and coagulopathy in malignant disease.

[Non-myeloablative allogeneic stem cell transplantation in patients with hematologic malignancies].

In order to investigate the clinical efficacy of non-myeloablative allogeneic stem cell transplantation (allo-NST) and related technology in patients with hematologic malignancies, twenty-six cases of hematological malignancies (10 AL, 14 CML, 2 MM patients) received NST following conditioning regimens with fludara + cyclophosphamide + ATG (14 cases) and busulfan or melphalan + cyclophosphamide + ATG (12 cases), G-CSF (600 micro g/d) or G-CSF (300 micro g/d) + GM-CSF (300 micro g/d) were used for mobilizing peripheral blood stem cell. A combination of cyclosporine A (CsA) and methotrexate (MTX) was administered for GVHD prophylaxis. Patients will be eligible for donor lymphocyte infusion (DLI) or donor stem cell infusion (DSI) given in graded increments according to the chimeric formation and clinical reaction. Generally the dose of the first infusion was 1 x 10(7)/kg at 4th week post-transplantation. The engraftment analysis 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 22 patients (84.62%) were engrafted, among which 18 patients were full donor chimerism (FDC) up to now. Acute GVHD occurred in 3/26 cases (11.54%). Chronic GVHD was diagnosed in 6 of 26 (23.07%) evaluable patients. The incidence of infection and hemorrhage was low and slight. It is concluded that allo-NST is a safe and effective therapeutic method for hematologic malignancies, but the related technology such as selection of indication, conditioning regimen and transplantation immunotherapy should be studied further.