[The cytogenetic characteristics of 178 acute myeloid leukemia patients].

OBJECTIVE: To explore the cytogenetic characteristics of acute myeloid leukemia (AML) patients. METHODS: The karyotype analysis was performed in 178 AML using the short-term culture of bone marrow cell and G-banding technique. RESULTS: Among the 178 patients, 171 had enough metaphases for analysis and 128 (74.9%) had clonal karyotypic abnormalities. Twenty-seven patients were secondary to myelodysplastic syndrome (MDS-AML), with 25 (92.6%) patients carrying clonal karyotypic abnormalities. Among the remaining 144 patients of de novo AML, 103 (71.5%) had clonal karyotypic abnormalities. The rate of abnormal clonal karyotype was higher in MDS-AML than that of de novo AML (P = 0.021). Among the 171 patients, 41 (24.0%) were in favorable risk group, 80(46.8%) in intermediate risk group and 50 (29.2%) in adverse risk group. t(15;17) was the most common chromosomal aberration. The majority intermediate risk chromosomal aberration was normal karyotype. The most common cytogenetic abnormality among adverse group was a complex karyotype. Adverse cytogenetic aberrations, such as -5/5q-, -7/7q-, frequently occurred in conjunction with one another as part of a complex karyotype. Totally 75 patients were 60 years or older, among them, 16.0% were in favorable risk group, 48.0% in intermediate risk group and 36.0% in adverse risk group. Among 96 younger patients, 30.2% were in favorable risk group, 45.8% in intermediate risk group and 24.0% in adverse risk group. The rate of favorable risk chromosomal aberration was lower in elder patients than in younger (P = 0.031). The rate of adverse risk chromosomal aberration and the rate of monosomal karyotype were higher in MDS-AML than in de novo AML patients (P < 0.001). CONCLUSIONS: The most common favorable, intermediate and adverse chromosomal aberrations were t(15;17), normal karyotype and complex karyotype respectively. The karyotype was poor in MDS-AML and elder AML patients.

[The cytogenetic and molecular genetic study of 81 multiple myeloma patients].

OBJECTIVE: To explore the cytogenetic characteristics of multiple myeloma (MM) patients, to evaluate the effect of a long-term culture stimulated by cytokines on cytogenetic study of MM, and to investigate the clinical detection value of RB1 and P53 deletion in interphase plasma cells by using fluorescence in situ hybridization (FISH). METHODS: Karyotype analysis was performed in 81 MM patients by using the short-term culture of bone marrow cell and G-banding technique. Among the 81 MM patients, 28 patients used two culture methods: one was the short-term culture and the other was to culture cells for 6 days with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) (40 µg/L) and IL-6 (10 µg/L). RB1 and P53 deletion were detected on interphase plasma cells by using FISH in 31 patients. RESULTS: Among the 81 patients, 75 had enough metaphases for analysis. Among the 75 patients, 31 (41.3%) had clonal karyotypic abnormalities including 4 numeric abnormalities, 11 structural abnormalities and 16 both abnormalities. Among the 28 patients using two culture methods, the clonal karyotypic abnormalities were detected in 6 patients (25.0%) in the group of cultured for 24 hours, and 14 patients (51.9%) in 6-day culture group with a significant difference (P = 0.026). RB1 deletion and P53 deletion were detected in 10 patients (32.3%) and 11 patients (35.5%), respectively, with both RB1 and P53 deletions be detected in 5 patients (16.1%). CONCLUSIONS: More than half of the tested MM patients have both numeric and structural chromosome abnormalities. The karyotype analysis using banding technique is basic cytogenetic study. Extended culture in the presence of IL-6 and GM-CSF could improve the efficiency of cytogenetic analysis to MM. Interphase FISH is a sensitive method of clinical application significance to detect the gene deletion of MM.

[Telomere length and telomerase expression activity in mononuclear cells of patients with chronic lymphocytic leukemia].

This study was aimed to detect the telomere length and the telomerase expression activity in patients with chronic lymphocytic leukemia (CLL), and investigate their relation to prognosis of CLL. The telomere length and the telomerase expression activity of peripheral blood and / or bone marrow mononuclear cells were examined by Tel-FISH, a semi-quantitative method and by TRAP-ELISA respectively; the expressions of ZAP70 and CD38 were detected by flow cytometry. The results showed that comparing the telomere length in different stages, there was a tendency that the telomere became prolonged when the stage raised up. There was statistical significant difference between Rai stages III-IV and stage 0, Rai stages III-IV and stages I-II, Binet stage C and stage A, Binet stage C and stage B; while no statistical significant difference existed between Rai stage 0 and stages I-II, Binet stage A and stage B. The telomere length in ZAP70 negative group was found similar as in ZAP70 positive group. The telomere length in CD38 positive group was shorter than that in CD38 negative group, but there was no statistical difference between them. Comparing the telomerase expression activity between different stages, there was a tendency that it increased when the stages went up; comparing the telomerase expression activity at different Rai stages, it increased at the higher stages. One case of CLL demonstrated that telomerase expression did not show at remission stage, but was found at relapse stage, which suggested that telomerase expression may relate to prognosis of disease. It is concluded that the telomerase length is in relation to Rai and Binet stage, which was shorter at higher stage than that at lower stage and intermediate stage. It seemed that the telomerase expression activity increased at higher stages. The expression of telomerase in mononuclear cells is stable and not influenced by treatment.

[Changes of pathogens and susceptibility to antibiotics in hematology ward from years 2001 to 2005].

The purpose of this study was to determine the changes of pathogens in hematological ward and susceptibility of patients received chemotherapy to antibiotics. The pathogens were taken from blood, urine and sputum of patients who accepted chemotherapy from years 2001 to 2005, then were isolated and identified. The susceptibility test was performed by disk diffusion method. The results showed that the total of 418 strains were detected. Gram-negative bacteria were the most common of nosocomial infection. Pseudomonas aeruginosa, Enterobacter cloacae, E. coli account for the most of Gram negative- bacteria infection and most resistant to broad-spectrum penicillin, Acinetobacter baumannii showed a trend of increase. The ratios of gram positive bacteria and fungi were increased slowly, mainly as Enterococcus and Candida. Enterococcus is the most common cause of Gram-positive bacterial infection. Vancomycin resistance did not occur. It is concluded that Gram-negative bacteria are main cause of nosocomial infection in patients with hematological malignancies. Gram positive bacteria and fungi had been more frequent. Strains resistant to antimicrobial agents increase.

[A clinical study of 103 idiopathic thrombocytopenic purpura].

OBJECTIVE: To investigate the prevalence by age, response to different therapies and outcome in newly diagnosed idiopathic thrombocytopenic purpura (ITP). METHODS: ITP patients who were hospitalized from July 1992 to December 2006 and followed up with telephone were retrospectively analyzed. RESULTS: 103 patients with ITP were investigated. The time of follow-up was between 2 months to 15years. Male:female = 35:68. The rate of patients over 60 years old was 34.0% (35/103). 59 patients were sensitive to adrenocorticosteroid and 4 patients under going splenectomy achieved a normal platelet count. In those immunosuppressive agents: including vincristine, cyclophosphamide, azathioprine and cyclosporin A(CsA) used in the present series, CsA was shown to be more effective. It could increase the platelet count when given together with prednisolone, the effective rate was 81.3% (26/32). Severe side effects like kiney function failure were not found in CsA treated patients so the use of geug in ITP would be recommended. There were 2, 1 and 1 ITP patients progressing respectively to Sjogren' s syndrome, systemic lupus erythematosus and lymphoma. 7 patients died, 1 patient died of cerebral bleeding, 2 brain infarction, 3 malignant neoplasm and 1 nephrosis The refractory rate of ITP is 17.2% (10/58). CONCLUSIONS: The morbidity in older people is high. The mortal bleeding in ITP is low. Treatment should be tailored to the individual patient.

[Long-term expansion of T cell progenitors by JAK3 gene transduced primitive hematopoietic cells in vitro].

OBJECTIVE: To explore whether activation of the JAK3-signaling pathway can stimulate long-term expansion of the earliest T cell progenitors from transduced primitive hematopoietic cells and evaluate their potential ability of committed differentiation. METHODS: A retrovirus vector (RV) containing JAK3 gene, two binding sites for chemical inducers of dimerization (AP20187), and green fluorescence protein (GFP), MGI-F(2)JAK3, was constructed. The RV vector MGI-F(2)JAK3 was then transduced into murine bone marrow hematopoietic cells. The transduced murine bone hematopoietic marrow cells were divided equally into four groups blank control group (No drug group), AP20187 group (added with AP20187 only), SCF group (added with stem cell factor only), and AP20187 + SCF group. Cytometry was used to detect the GFP marker and observe the survival of cells. The murine bone hematopoietic marrow cells expanded for 50 days were divided into 2 groups: one group was washed to remove the cytokines to observe their survival, and AP20187 + SCF was added into the culture fluid of other group. Cytometry and CELLQuest v3.1 software analysis were used to analyze the phenotype of the cells of AP20187 + SCF after 50 days' expansion. The C-kit(hi) CD44(+)CD25(-)TN cells after 50 days' expansion were further cultured under the condition of SCF + IL7 + IL3 for 5 days. The differentiation rate of Thy1.2 positive cells was observed by cytometry. Five Ly5.2 mice underwent radiation of the dose of 600 cGy, and then injected with the expanded cells into the thymus. Three weeks later the mice were killed and their thymus glands were taken out to prepare suspension of single cells to undergo cytometry to observe the proportions of GFP positive CD3 and CD4 mature T lymphocytes. RESULTS: One week later the cells of the No Drug group all died, the cells of the AP20187 group and SCF group died 2 - 4 weeks later, however, the cells of the AP20187 + SCF continued to grow and expanded up to 10(12)-fold after 50 days' culture. The cells of the AP20187 + SCF group with the cytokines washed died 2 more weeks later, and those with the cytokines washed and added with AP20187 + SCF continued to grow. The phenotype of the expanded proportion was identified as the earliest T cell progenitors expressing C-kit(hi) CD44(+) CD25(-)TN (triple negative). These progenitors could differentiate into Thy1.2 + T cells in the presence of SCF + IL-7 + IL-3 culture condition. 32% - 96% of the mice thymus cell were GFP positive, 5% +/- 0.8% of the thymus cells were GFP + CD(3) double positive, and 11.0% +/- 2.1% of the T lymphocytes were GFP + CD4(+) double positive. CONCLUSION: AP20187 combined with SCF mediating the activation of JAK3 signaling can dramatically expand the earliest T cell progenitors subpopulation, and acquires the capacity to induce the differentiation into T mature cells. This system may help understand the T cell biology and provide a fundamental basement for gene therapy to immunodeficiency disease in the future.

[Construction of fusion gene between IgGHV and IL-2 as IgHV nucleic acid vaccine against lymphoma].

The purpose of this study was to construct the IgHV and IL-2 coexpressed vector. The IgHV gene fragments were obtained from the peripheral blood of patients with lymphoma, and were cloned into eukaryotic expression vector. Meanwhile, the gene fragments of IgHV linked with gene of IL-2 were inserted into pcDNA3.0 to form a fusion gene of IgHV-IL-2. Then fusion genes were transfected into COS cells by Lipofectin and the expression of IL-2 was detected by ELISA. The results showed that the IgHV/pcDNA3.0 expression vector was successfully constructed. The 3' end of IgHV was linked to IL-2 gene, and IL-2 could be correctly expressed. In conclusion, the expression vector of IgHV-IL-2 can express IL-2 correctly in COS cells.

[Promotion of in vitro long term expansion of primitive multipotential hematopoietic cells by transduced JAK2 gene].

OBJECTIVE: To explore whether activation of the JAK2-signaling pathway can stimulate long-term expansion and regulation of hematopoietic stem cells/multipotential hematopoietic progenitor cells (HSC/MHPC), and evaluate their potential ability of committed differentiation. METHODS: A retrovirus vector (RV) which contains JAK2 gene and two binding sites for a chemical inducers of dimerization (AP20187) was constructed. JAK2 can be dimerized by adding AP20187. Female C57BL/6 mice were euthanized and marrow cells were harvested. The RV vector was then transduced into murine bone marrow cells. Following transduction, Transduced cells were divided equally into four groups as follow: (1) No drug group, (2) AP2018 alone group, (3) SCF + Flt3-Ligand group, (4) AP20187 + SCF + Flt3-Ligand group. The expanded cells were further analyzed by phenotype, committed differentiation, progenitor colony assay as well as colony forming unite-spleen. RESULTS: Only the group of AP20187 combined SCF and Flt3-Ligand have a significant sustained outgrowth. The cells reached at 10(19) fold on the day 80. The phenotype of expanded cells were checked by flow cytometry at various time points after two months in vitro culture and we repeated them in five separate experiments. Sca-1 was consistently expressed at the levels in 52% approximately 98%, while 56% approximately 69% of cells expressed c-kit, 40% approximately 85% expressed CD34, About 12% approximately 46% expressed B220, 6% approximately 17% expressed Gr1, 0% approximately 20% expressed TER119, 5% approximately 36% expressed CD41, 35% approximately 46% expressed CD11b and none expressed CD3. The expanded cells could differentiate into granulocytes, macrophages, erythocytes and megakaryocytes under different cytokines combination. They were also capable of forming BFU-E, CFU-GM, CFU-Mix and IL-7 responsive B-lymphoid colonies in methylcellulose colonies assay. Colonies forming unites-Spleen were obtained when the expanded cells injected into lethally irradiated mice. CONCLUSION: The JAK2-mediated transgenic hematopoietic cells could be expanded and regulated in long-term period, and they are capable of maintaining multipotential differentiation. This research is setting up a fundamental basement for cell therapy in the future.

[Long term regulated expansion and committed differentiation of JAK2 gene transfected hematopoietic stem/progenitor cells in vitro].

OBJECTIVE: To explore the feasibility of regulated expansion and committed differentiation potential of JAK2 gene modified hematopoietic stem/progenitor cells in vitro. METHODS: A murine stem cell virus (MSCV) based retroviral vector MGI-F2Jak2, which encodes a green fluorescent protein (GFP) and a fusion protein containing two copies of modified FK506 binding protein (F36v) linked tyrosine kinase JAK2 was cloned. F36v served as a high-affinity binding site for dimerizer AP20187. GpE + 86 packaging cell was transfected with this vector. Bone marrow cells from C57BL/6 mice were transduced by co-cultured with irradiated (1500 cGy) GpE + 86 producer clone for 48 h. Transduced marrow cells were expanded in X-VIVO 15 and divided into four groups as follows: (1) control group; (2) AP20187 alone group; (3) SCF alone group and (4) AP20187 + SCF group. The phenotypes of the expanded cells were analyzed by directly phycoerythrin-labeled anti-Sca1, c-kit, CD(34), Gr1, CD(11b), TER119, CD(41), B220 and CD(3) monoclonal antibodies for flow cytometry. Committed differentiation, progenitor colony assay and spleen colony forming units (CFU-S) were further evaluated. RESULTS: A significant sustained outgrowth of transduced marrow cells was obtained only in the AP20187 + SCF group. Cells expanded up to 10(14)-fold after 80 days culture. The doubling time was about 30 hs. The phenotypes of the expanded cells were homogeneously strong positive for CD(34), c-kit and Sca1, while were almost negative for Gr1, CD(11b), TER119, CD(41), B220 and CD(3). Functional assay demonstrated that the expanded cells had multipotential to differentiate into granulocyte, macrophage, erythrocyte, or B-cells under different cytokines combinations. A prominent megakaryocytic differentiation was observed when cultured with SCF/Tpo/IL-11 combination. The expanded cells were also capable of forming BFU-E, CFU-GM and CFU-Mix in methylcellulose colony assay. The expanded cells over three months could still form CFU-S. CONCLUSIONS: AP20187 combined SCF mediated activation of JAK2 signaling domain can dramatically expand hematopoietic stem/progenitor cells, and the expanded cells can be regulated and committed to differentiate into multilineage cells. This system may provide important insights into stem cell biology and may be promising for gene and cell therapy.