In vitro effects of reprogramming factors on the expressions of pluripotent genes and CD34 gene in human acute promyelocytic leukemia HL-60 cells.

OBJECTIVE: Our study aims to explore the in vitro effects of reprogramming factors on the expressions of pluripotent genes and CD34 gene in HL-60 cells. METHODS: According to the construction of lentiviral vector LV-OSCK of reprogramming factors (Oct-4, Sox2, Klf4, c-Myc), 293T cells were transfected to detect virus titer. The endogenous pluripotent genes (Oct4, SOX2, c-Myc and Klf4) and CD34 mRNA and protein expressions were detected by AP staining, immunofluorescence staining, qRT-PCR and flow cytometry. RESULTS: Expressions of Oct4, SOX2, c-Myc and Klf4 were 0.220±0.013, 0.186±0.009, 0.287±0.015 and 0.153±0.007. These levels were significantly higher in the experimental group than the control and blank groups. CD34 protein expression in the experimental group was also discovered to be significantly higher than the other two groups. CONCLUSION: The reprogramming factors could increase the expressions of pluripotent genes and CD34 gene in HL-60 cells.

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells.

OBJECTIVE: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. METHODS: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. RESULTS: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. CONCLUSION: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

Adverse drug interactions as a high-risk factor for lethal post-transplant complications in Chinese population.

Metabolism of triazole antifungal agents is highly competitive to conventional post-transplant immunosuppressants like cyclosporine A (CsA) via the cytochrome P450-dependent pathway. We present the first report on lethal complications that may arise due to this type of drug interaction. A retrospective survey identified 10 of 104 cases (9.62%) that suffered life-threatening complications associated with the interaction between CsA and itraconazole or voriconazole following allogeneic hematopoietic stem cell transplantation (allo-HSCT) at our center. According to the close drug monitoring, all 10 patients experienced supratherapeutic levels of CsA even with a preemptive CsA dosage reduction and prompt dose adjustment. Six patients developed grade I to III acute graft-versus-host disease (aGVHD) and eventually died from either idiopathic pneumonia syndrome or diffuse alveolar hemorrhage; another four patients died from CSA-associated neurological complications. Impaired hepatic and renal function was noted in only one of these 10 cases. The high frequency as well as the unpredictability of severe complications lead us to suggest that triazole should always be replaced by another antifungal medication (e.g., amphotericin B or Echincandins) while patients receive CsA after HSCT, especially in the Chinese population.

Iron overload as a risk factor for poor graft function following allogeneic hematopoietic stem cell transplantation.

Hematological malignancies are increasingly treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, iron overload is a frequent adverse effect of allo-HSCT and is associated with poor prognosis. In the present study, we investigated hematopoiesis in iron-overloaded mice and elucidated the effects of iron overload on the bone marrow (BM) microenvironment. Iron-overloaded BALB/C mice were generated by injecting 20 mg/mL saccharated iron oxide intraperitoneally. Hematoxylin-eosin staining was performed to evaluate the effects of an iron overload in mice. BM cells obtained from C57BL/6 mice were transplanted into irradiated BALB/C mice (whole-body irradiation of 4 Gy, twice with a 4-hours interval) by tail vein injection. Two weeks after allo-HSCT, the hematopoietic reconstitution capacity was evaluated in recipients by colony-forming assays. Histopathological examinations showed brown-stained granular deposits, irregularly arranged lymphocytes in the liver tissues, and blue-stained blocks in the BM collected from mice received injections of high-dose saccharated iron oxide (20 mg/mL). Iron-overloaded mice showed more platelets, higher-hemoglobin (HGB) concentration, fewer granulocyte-macrophage colony-forming units (CFU-GM), erythrocyte colony-forming units (CFU-E), and mixed granulocyte/erythrocyte/monocyte/megakaryocyte colony-forming units (CFU-mix) than healthy mice. Iron-overloaded recipients presented with reduced erythrocytes and HGB concentration in peripheral blood, along with decreased marrow stroma cells, CFU-GM, CFU-E, and CFU-mix relative to healthy recipients. Taken together, our findings demonstrate that iron overload might alter the number of red blood cells after transplantation in mice by destroying the BM microenvironment, thereby affecting the recovery of BM hematopoietic function.

In vitro inducing effect of dendritic cells cotransfected with survivin and granulocyte-macrophage colony-stimulating factor on cytotoxic T cell to kill leukemic cells.

BACKGROUND: Survivin is a rather specific gene in tumor tissue. We transfected dendritic cells (DCs) with recombinant adenovirus (Ad) containing survivin gene and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and tested the inducing effect of the transfected DCs on cytotoxic T lymphocytes (CTL) to kill leukemic cells. METHODS: After derived from the peripheral, DCs was assayed by mixed leukocyte reaction (MLR) tests. Lactate dehydrogenase (LDH) release test was used to evaluate cytotoxicity of CTL. RESULTS: Expression of survivin in transfected DCs was confirmed by Western blotting analysis. GM-CSF expression was confirmed by enzyme-linked immunosorbent assay (ELISA). In MLR assay, DCs coinfected with Ad-survivin and Ad-GM-CSF induced higher allogeneic lymphocyte reaction than control DCs at ratios of 1:5, 1:10, 1:50 and 1:100. DCs coinfected with Ad-survivin and Ad-GM-CSF had much higher activity of CTL to HL-60 cells than DCs infected with Ad-survivin only, Ad-GM-CSF only, or control DCs. Levels of interleukin-12 (IL-12) and interferon gamma (IFN-gamma) in lymphocyte supernatants containing DCs coinfected with Ad-survivin and Ad-GM-CSF were significantly higher than those in the control group. CONCLUSION: DCs coinfected with Ad-survivin and Ad-GM-CSF induce much higher anti-leukemic response in vitro than those infected with either factor. Therefore, adenovirus vectors containing survivin and GM-CSF genes may be promising vaccine candidates for leukemia therapy.

Bone marrow-derived mesenchymal stem cells overexpressing MiR-21 efficiently repair myocardial damage in rats.

OBJECTIVE: We investigated the ability of bone marrow derived mesenchymal stem cells (BMSCs) overexpressing microRNA-21 (miR-21) to repair cardiac damage induced by anthracyclines in rats. METHODS: Sprague-Dawley (SD) rats of 2~3 weeks old were selected to isolate and culture BMSCs. A lentivirus harboring pLVX-miR-21 was generated and transfected into rat BMSCs. The rats were assigned into an untreated negative control group, and groups injected with adriamycin alone or with adriamycin followed by BMSCs, pLVX-BMSCs or pLVX-miR-21-BMSCs (n = 10 each). Proliferation and migration of cells were detected by cholecystokinin-8 (CCK- 8) and transwell. MiR-21 expression, mRNA expressions of B cell lymphoma 2 (Bcl2), BAX (BCL-2-associated X protein) and vascular endothelial growth factor (VEGF) were tested by qRT-PCR. Western blotting was applied to detect protein expressions of Bcl-2, Bax and VEGF. RESULTS: Using CCK- 8 and transwell assays, we found that pLVX-miR-21-BMSCs, which overexpressed miR-21, exhibited greater proliferation and migration than untransfected BMSCs or pLVX-BMSCs. Ultrasonic cardiograms and immunohistochemical analysis demonstrated that among the five groups, the pLVX-miR-21-BMSC group exhibited the most improved heart function and enhanced angiogenesis. Moreover, the pLVX-miR-21-BMSC group showed enhanced expression of Bcl-2, VEGF and Cx43 and reduced expression of Bax, BNP and troponin T. CONCLUSION: These findings suggest miR-21 overexpression enhanced the proliferation, invasiveness and differentiation of BMSCs as well as expression of key factors (Bcl-2, VEGF and Bax) essential for repairing the cardiac damage induced by anthracyclines and restoring heart function.

Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials.

BACKGROUND AND OBJECTIVES: Adult bone marrow (BM) is the major source of mesenchymal stem cells (MSC) for cell therapy. However, aspiration of BM involves invasive procedures. We isolated MSC from human full term umbilical cord tissues (UC). The biological characteristics of MSC derived from UC (UC-MSC) were further determined and compared with normal adult bone marrow-derived MSC (BM-MSC). DESIGN AND METHODS: MSC were isolated from UC by enzyme digestion and cultured in appropriate growth medium. The isolation efficiency, cell yield, colony-forming unit-fibroblast (CFU-F) frequency, growth kinetics, phenotypic characteristics, multi-lineage differentiation capacity, cytokine spectrum as well as hematopoiesis-supportive function of UC-MSC were determined and compared with those of BM-MSC. RESULTS: MSC were successfully isolated from all 36 UC and six BM samples we collected for this study. The mean number of nucleated cells isolated from UC was 1yen106/cm and the yield of adherent cells was 8.6yen105/cm. UC-MSC shared most of the characteristic of BM-MSC, including fibroblastic-like morphology, immunophenotype, cell cycle status, adipogenic and osteogenic differentiation potentials, and hematopoiesis-supportive function. The CFU-F frequency was higher in UC nucleated cells (1:1609 +/- 0.18) than in BM nucleated cells (1:35700 +/- 0.01) (p < 0.05). Furthermore, in comparison with BM-MSC, the UC-MSC had a higher proliferation capacity and lower levels of expression of CD106 and HLA-ABC (p < 0.05). Immunofluoresent and western blot assays revealed that UC-MSC had a higher percentage of neuron specific enolase-positive cells than had BM-MSC after neuronal induction. Finally, reverse transcriptase polymerase chain reaction analysis showed that UC-MSC had a cytokine spectrum very similar to that of BM-MSC, including expression of the mRNA of stem cell factor, leukemia inhibitor factor, macrophage-colony stimulating factor, Flt3-ligand, interleukin-6, vascular endothelial growth factor and stromal-derived factor-1, but UC-MCS additionally expressed mRNA of granulocyte macrophage and granulocyte colony-stimulating factors. After co-culture with CD34+ cord blood cells for 5 weeks, no significant difference in colony-forming cells was observed between the CD34+ cells/UC-MSC and CD34+ cells/BM-MSC co-cultures (p > 0.05). INTERPRETATION AND CONCLUSIONS: We have established a protocol to isolate abundant MSC from human umbilical cords with a 100% success rate. The comparative study indicates that UC is an excellent alternative to BM as a source of MSC for cell therapies.

Bone marrow stromal cells transduced with human hemangiopoietin gene support hematopoiesis in vitro.

BACKGROUND AND OBJECTIVES: The aim of this study was to construct a eukaryotic expression vector containing human hemangiopoietin (hHAPO) gene and express it in mouse bone marrow stromal cell line HESS-5, then support hematopoiesis in vitro with gene-modified HESS-5 (hHAPO-HESS-5). DESIGN AND METHODS: The polymerase chain reaction (PCR) products of HAPO were digested with BamHI and BgII. Then the HAPO gene segment obtained was again cloned into pIRES2-EGFP to construct recombinant eukaryotic expression vector HAPO-pIRES2-EGFP. The recombinant vector was identified by enzyme digestion analysis, PCR, and sequencing. HESS-5 cells were transformed by recombinant vector and positive clones were selected with G418. The expression of HAPO gene in the transformed cells was detected by studying EGFP expression, reverse transcription (RT)-PCR, and Western-blotting analysis. Support of human hematopoiesis by hHAPO-HESS-5 cells was evaluated in co-culture experiments with human CD34+ cells. RESULTS: Enzyme digestion analysis and sequencing showed that the target gene had been cloned into the recombinant vector. The expression of HAPO gene in the transformed stromal cells was demonstrated by fluoro-microscopy and RT-PCR analysis. HAPO protein was also detected in the supernatant of hHAPO-HESS-5 by Western blot analysis. As expected, stably transfected hHAPO-HESS-5 cells significantly increased in both relative and absolute numbers of CD34+ cells after 14 days of culture. The PKH26 study demonstrated that cell division was faster in CD34+ cells co-cultured with hHAPO-HESS-5 cells than in cells cocultured with vector-HESS-5 cells. The hHAPO-HESS-5 cells also supported human hematopoiesis in vitro more efficiently than did control vector-HESS-5 cells. INTERPRETATION AND CONCLUSIONS: A recombinant eukaryotic expression vector has been constructed and expressed successfully in transformed cells. The hHAPO-HESS-5 cells support rapid generation of primitive progenitor cells and maintain reconstituting ability of hematopoietic stem cells in vitro. Therefore, it would be possible to use stromal cells expressing HAPO gene as seed cells in the bone marrow transplantation.

Constitutive activation of NF-κB signaling by NOTCH1 mutations in chronic lymphocytic leukemia.

NOTCH1 mutations occur in approximately 10% of patients with chronic lymphocytic leukemia (CLL). However, the relationship between the genetic aberrations and tumor cell drug resistance or disease progression remains unclear. Frameshift deletions were detected by gene sequencing in the NOTCH1 PEST domain in three naive CLL patients. These mutations were associated with chromosomal abnormalities including trisomy 12 or 13q deletion. Of note, one of the patients developed Richter's transformation during FCR treatment. Immunofluorescent and western blot analyses revealed a markedly higher intracellular domain of NOTCH (ICN) expression in the mutated cells compared with their unmutated counterparts and normal CD19+ B lymphocytes (P<0.01 and P<0.001, respectively). In addition, strong DNA-κB binding activities were observed in the mutant cells by gel shift assays. RT-PCR analysis revealed elevated RelA mRNA expression in the mutant cells, while RelB levels were variable. Reduced levels of RelA and RelB mRNA were observed in unmutated CLL and normal B cells. Compared to unmutated CLL and normal B cells, increased apoptosis occurred in the mutant cells in the presence of GSI (ICN inhibitor) and PDTC (NF-κB inhibitor), particularly under the synergistic effects of the two drugs (P=0.03). Moreover, IKKα and IKKß, the active components in the NF-κB pathway, were markedly inhibited following prolonged treatment with GSI and PDTC. These results suggested that NOTCH1 mutations constitutively activate the NF-κB signaling pathway in CLL, which is likely related to ICN overexpression, indicating NOTCH1 and NF-κB as potential therapeutic targets in the treatment of CLL.

[Oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation].

AIM: To explore whether the oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation. METHODS: Intracellular mean fluorescence intensity was measured by flow cytometry. RESULTS: After treatment with FITC-labeled G3139 at the concentration of 0.60 mumol.L-1 for 4 h, the G3139 uptake into peripheral blood mononuclear cell and bone marrow mononuclear cell in hematological tumor patients was significantly higher than that in normal control. There was different uptake of G3139 among the malignant hematological tumor cell strains, and the uptake in cells derived from monocyte, B lymphocyte and myeloid cell was much higher than that in cells derived from T lymphocyte. After treatment with all-trans retinoic acid (ATRA), HL60 cell proliferation was markedly inhibited and the uptake of G3139 decreased significantly. CONCLUSION: Hematological tumor cells were capable of taking up oligonucleotide, and the oligonucleotide uptake in hematological tumor cells is related to its cellular species and its activation.

[Cyclooxygenase-2 expression in esophageal cancer cells and induction by mitomycin C].

OBJECTIVE: To study the relationship between COX-2 expression in esophageal cancer cell (EC/CUHK-1) and mitomycin C (MMC) treatment. METHODS: 2 mg/L of MMC was added to the well-grown EC/CUHK-1 cells cultured in RPMI-1640 including 10% FCS, and the medium was totally changed after 0.5, 1, 2, 4 and 8 h treatment, respectively. Cells were collected after another 24 h culture. Protein expression of COX-2, Bcl-2, Rb, p53 were examined by Western blot, and RT-PCR method was used to confirm the COX-2 expression in mRNA level. Cell cycle analysis for cells collected at 0, 0.5, 2, 4 and 8 h was performed on an EPICS profile analyzer. RESULTS: The cell cycle analysis showed that the percentage of apoptosis cells were (4.12 +/- 0.83)%, (1.00 +/- 0.11)%, (4.32 +/- 0.99)%, (9.46 +/- 2.11)% and (31.10 +/- 3.57)%, respectively. COX-2 mRNA expression were 2.60, 1.70, and 0.08 times, COX-2 protein expression were 2.0, 3.1 and 2.8 times, Bcl-2 protein were 3.6, 14.0 and 12.0 times, p53 protein were 1.8, 0.5 and 0.2 times, hyperphosphorylated form Rb were 8.2, 8.4 and 6.2 times, underphosphorylated form Rb were 1.8, 0.5 and 0.2 times in 0.5, 2 and 4 h after MMC treatment, respectively, as compared with the control group. CONCLUSIONS: The COX-2 expression showed coincidence up-regulation according to the MMC-induced anti-apoptosis function activation in esophageal cancer cells, and the process was at least partly associated with Rb phosphorylation and p53 accumulation. It is implied that COX-2 may be a protecting factor in MMC induced esophageal cancer cell apoptosis, and the use of COX-2 inhibitor as an enhancer for esophageal cancer chemotherapy may be reasonable.

[Bcl-2 antisense oligonucleotides suppress HL-60 cell growth in a SCID mouse mode].

OBJECTIVE: To study the effects of bcl-2 antisense phosphorothioate oligonucleotides (ASPO) on suppression of HL-60 cell growth in SCID mice and to investigate the feasibility of purging leukemia cells plus bcl-2 ASPO used in vitro. METHODS: 1 x 10(7) viable HL-60 cells were treated with 10 micro mol/L bcl-2 ASPO seven days before the intraperitoneal (IP) inoculation to the SCID mice, Treatment with sense oligonucleotides (SPO) was similar as for the controls. 35 days after the inoculation, all the SCID mice of both groups were sacrificed and their peripheral blood, bone marrow, liver and spleen were examined using half nested RT-PCR and histopathology for detecting the appearance and distribution of the HL-60 cells treated beforehand with antisense or sense oligonucleotides respectively. RESULTS: ASPO could down regulate the expression of bcl-2 resulting in both inhibition of growth and induction of apoptosis in treated HL-60 cells, which failed to develop leukemia in SCID mice at all. However, SPO treated HL-60 cells still behaved their own ways and proliferated agressively, and developed leukemia at last. CONCLUSION: The bcl-2 ASPO enables to suppress HL-60 cell growth and prevent the development of leukemia in the SCID mice. The purging leukemia cells used are seemed liable in inhibiting the development of leukemia in SCID mouse model.

[Experimental investigation of non-heart-beating donor in the rat lung transplantation].

OBJECTIVE: To explore the impact of different warm ischemia time on structure and function of the non-heart-beating donor lung and to find out the feasibility of non-heart-beating donor in rat lung transplantation. METHODS: Sixty Sprague-Dawley rats were randomly divided into 3 groups: heart-beating donor (HBD) group, non-heart-beating donor (NHBD) with 30 minutes of warm ischemia time (WIT) group and NHBD with 60 minutes of WIT group. Each group has 10 pairs (the donors and the recipients). The donor lungs of group HBD were flushed with low potassium dextran (LPD) solution at 4 degrees C after asystolia while the lungs of group NHBD-30 and group NHBD-60 remained ventilated at the room temperature for 30 and 60 minutes after asystolia and then were flushed with LPD solution. All the donor lungs remained inflated when they were stored in LPD solution at 4 degrees C for 4 hours. The recipient rat underwent left thoracotomy, and then orthotopic left lung transplantation. Followed by a right thoracotomy, the right pulmonary hilum were ligated with one-hour reperfusion and ventilation. RESULTS: All the recipients in group HBD and group NHBD-30 survived the observation period of one hour with excellent gas exchange, whereas 4 of recipients in group NHBD-60 survived for 10 minutes after the ligation of right pulmonary hilum and 3 for 20 minutes. The pulmonary compliance, ultrastructure, energy metabolite and other markers revealed no significant differences between group HBD and group NHBD-30 (P > 0.05). But the differences between group NHBD-60 and other two groups were significant (P < 0.05). CONCLUSIONS: The adoption of non-heart-beating donor could be a safe and effective method to expand the lung donor pool. The NHBD lung with 30 minutes of WIT may be suitable for lung transplantation in rat.

A novel nonradioactive CFDA assay to monitor the cellular immune response in myeloid leukemia.

BACKGROUND: Donor lymphocyte transfusion (DLT) may induce the graft-versus-leukemia (GVL) effect for patients with AML relapsed after transplant. However, AML is a highly diverse disease and the limited overall efficacy of DLT in clinical practice emphasizes the importance of identifying a specific subgroup of patients who might benefit from this treatment approach. OBJECTIVE: To monitor the cellular immune response after DLT, we developed an active specific immunization strategy using in vitro generated AML-trained T cells to induce a highly specific antileukemic T-cell response and thus established a novel nonradioactive assay system to assess the antileukemia immunity by flow cytometry, correlated with [3H]-thymidine uptake. METHODS: The myeloid blasts derived from five patients with AML relapsed post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) were first labeled with CFDA (5,6-carboxyfluorescein diacetate succinimidyl ester). To analyze the growth inhibitory potential of the donor T cells trained by AML progenitor cells, the myeloid blasts were induced to proliferate by means of a cytokine cocktail (50ng/mL of SCF; 25ng/mL of IL-3; 100ng/mL of GM-CSF; 100ng/mL of G-CSF; 2U/mL of EPO; 0.47g/L of transferrin; and 5×10(-5)mmol/L of 2-ME). The T cell mediated growth inhibitory potential was detected after 5 days by flow cytometry and correlated with [3H]-thymidine uptake. The simultaneous use of TO-PRO-dye and calibrate beads allowed not only the cell viability to be known but also allowed quantification of the effector function. RESULTS: Here, we applied a CFDA dye to track the proliferation and expansion of AML blasts in response to the cytokine cocktail in vitro. AML-trained T cells, expressed high levels of the activation markers CD25 and CD69, and were generated to recognize the leukemic progenitor cells and inhibit cytokine-induced leukemic cell proliferation, which is an active specific immunization strategy circumventing the identification of leukemia-associated antigens. The capability of proliferation inhibition of AML-trained T cells evaluated with our nonradioactive, CFDA-based assay provided comparable results with the classic [3H]-thymidine assay with an even lower ratio of effector to target cells. CONCLUSION: Taken together, the novel, nonradioactive, CFDA-based assay was a robust tool to monitor the antileukemic immune response after DLT in myeloid leukemias.

[Clinical analysis on 40 patients with chronic lymphocytic leukemia].

This study was aimed to analyze the clinical and laboratorial characteristics of patients with chronic lymphocytic leukemia (CLL), as well as their relationship with outcomes of patients. The clinical and laboratorial data of 40 CLL patients admitted from 2004 to 2010 in our hospital were analyzed retrospectively. The results indicated that the most of CLL attacked the elderly male patients with median age 66 (from 42 to 80). Flow cytometric analysis showed that 25 cases were positive for typical immunophenotype of CLL. On the other hand, all the patients clearly expressed CD19 and CD5, 7 cases (17.5%) and 14 cases (35%) were positive for the expression of CD38 and Zap70 respectively. 8 cases harbored a mutated immunoglobulin heavy-chain (VH) gene, among them 4 cases belong to VH3 family. Interphase FISH analysis showed that P53 deletion, RB1 deletion, trisomy 12 and normal chromosome were detected in 6, 3, 1, and 5 cases, respectively. The median PFS in 31 patients received treatment of fludarabine based chemotherapy was 48 months (95%CI: 39 - 57 months), among them 27 cases (87.1%) achieved CR + PR. While PFS was 14 months (95%CI: 10 - 18 months, P < 0.001) in 9 patients received other treatment regimen, out of them only 3 cases (33.3%) achieved CR + PR. Patients with normal level of serum ß2-microglobulin at diagnosis showed significantly higher overall survival (78%, 95%CI: 69% - 87%) in 36 months than those with abnormal level of serum ß2-microglobulin (47%, 95%CI: 35% - 59%, P = 0.004). Significant difference in the rate of CR + PR was noted in the Zap70 positive group (50%) and in negative group (88.5%, P = 0.006). All of 8 patients with IgVH mutation displayed CR after treatment, while 4 cases (66.7%) archived CR among 6 patients without IgVH mutation. It is concluded that CLL is characterized by high heterogeneity in both clinical features and molecular markers, which are associated with prediction of outcomes for patients. The treatment with fludarabine-based chemotherapy results in a major benefit and long survival for patients with CLL.

[A case of Richter syndrome transformed from chronic lymphocytic leukemia with karyotype aberration of trisomy 12].

This study was aimed to investigate the relationship between Richter's syndrome (RS) transformation and clinical characteristics as well as karyotype of patient with chronic lymphocytic leukemia (CLL). By the follow-up of a patient with CLL, the clinical characteristics, karyotype, treatment pattern and its effect, as well as disease progression were monitored regularly with serological test, flow cytometry and FISH technique. The results indicated that the patient typically presented with history of CLL at initial diagnosis, with expression of CD5(+), CD19(+) and CD23(+), Binet stage C, as well as karyotype aberration of trisomy 12, and poorly responded to 4 cycles of standard chemotherapy of FCR regimen. The disease progression was confirmed at 5 months with the symptoms of fever in the absence of infection, elevated lactate dehydrogenase level and rapidly enlarging lymphnodes which showed typically diffuse large B cell lymphoma by the biopsy. It is concluded that karyotype aberration of trisomy 12 is one of the risk factors for RS transformation, and treatment pattern of the patient with CLL may be associated with the transformation of RS.

[Homing characteristics of graft cells in leukemia mice after allogenic bone marrow transplantation with different conditioning regimens].

The aim of this study was to investigate the homing characteristics of bone marrow cells in leukemia mice after allogenic bone marrow transplantation with different conditioning regimens on the basis of a leukemia mouse model. Allogenic bone marrow transplantation was performed after three different kinds of conditioning regimen, including nonmyeloablative conditioning regimen (5 Gy (60)Co γ ray total body irradiation, A group), radiotherapeutic myeloablative conditioning regimen (9 Gy (60)Co γ ray total body irradiation, B group) and chemotherapeutic myeloablative conditioning regimen (large dose chemotherapy, C group). In the recipient mice, the nucleated cell number in peripheral blood, bone marrow and spleen was counted, the percentage of positive cells capable of connecting with FITC labeled anti-mouse H-2K(b) antibody was detected by flow cytometry and the homing ratio in bone marrow and spleen was calculated at 24, 48, 72, 96 h after bone marrow transplantation. The results showed that donor myeloid cells displayed homing and then mobilization (going out of home) in group A; homing, mobilization, and rehoming in group B and C, and there was a little delay of homing in the spleen in group C. In bone marrow, the homing efficiency of A group was the highest in early period and the lowest [(0.90 ± 0.09)%] in the fourth day with the mobilization of myeloid cells (P < 0.05), and the homing efficiency of B and C groups was lower in the early period and the highest [(2.17 ± 0.26)%, B group] in the fourth day with the rehoming of myeloid cells (P < 0.05). In spleen, the homing efficiency was similar to that in bone marrow and there still was a little delay in C group. It is concluded that the homing ratio is high in the early period and decrease obviously in 72 h after bone marrow of leukemia mice treated with nonmyeloablative conditioning regimen. The homing ratio is low in the early period and increases obviously in 72 h after bone marrow of leukemia mice treated with radio-or chemotherapeutic myeloablative conditioning regimens. The homing ratio does not obviously change between the early period and 72 h after bone marrow of leukemia mice treated with chemotherapeutic myeloablative conditioning regimen, and lies between group A and B.

[Comparison of the biological characteristics of serum-free and fetal bovine serum-contained medium cultured umbilical cord-derived mesenchymal stem cells].

OBJECTIVE: To compare the differences of biological characteristics between human umbilical cord-derived mesenchymal stem cells (UC-MSCs) cultured by serum-free medium or fetal bovine serum-contained complete medium to establish a xenogeneic protein-free UC-MSCs culture system. METHODS: Healthy human umbilical cord segments were digested with collagenase. UC-MSCs were cultured by serum-free MesenCult-XF medium and FBS-based αMEM complete medium, then analyzed the morphology, immunophenotype, expansion potential, lineage differentiation potential, karyotype and immunosuppression of early passages. RESULTS: The average cell diameters of UC-MSCs in suspension cultured by serum-free medium and FBS-based medium were 26 (18 - 39) µm and 35 (20 - 61) µm, respectively. Cell expansion folds with serum free medium and FBS-based medium were (5.2 ± 0.2) and (3.5 ± 0.1) respectively, in the first five passages. The expansion potential of serum-free medium cultured UC-MSCs was significantly higher than FBS-based medium cultured ones (P < 0.05). A panel of markers CD29, CD44, CD90, CD73, CD105 and HLA-ABC expressed on human UC-MSCs. Hematopoietic lineage markers CD34, CD45 and HLA-DR were not detectable on UC-MSCs. The cpm were (4.57 ± 0.14)×10(4), (2.04 ± 0.16)×10(4) and (0.42 ± 0.04)×10(4), respectively when serum-free medium cultured MSCs were added to the cultures at MSCs/T cell ratios of 1:100, 1:10 and 1:5. While the cpm was (4.57 ± 0.14)×10(4), (2.04 ± 0.16)×10(4) and (0.42 ± 0.04)×10(4), respectively when serum-free medium cultured UC-MSCs were added to the cultures. The immunosuppressive potential of serum-free medium-cultured UC-MSCs was higher than serum-contained medium cultured ones at three different MSC/T cell ratios (P < 0.05). CONCLUSION: Compare with serum-contained medium cultured early passages of UC-MSCs, the cell diameter of serum-free medium cultured UC-MSCs was smaller with higher expansion potential. No xenogeneic proteins were presented in UC-MSCs preparations when cultured with serum-free medium. Human UC-MSCs suppressed T-cell proliferation in a dose-dependent manner. The immunosuppressive potential of serum-free medium cultured UC-MSCs was higher than FBS-based medium cultured ones.

[Clinical study of umbilical cord-derived mesenchymal stem cells for treatment of nineteen patients with steroid-resistant severe acute graft-versus-host disease].

OBJECTIVE: To evaluate the safety and efficacy of umbilical cord-derived mesenchymal stem cells (MSCs) infusion in patients with steroid-resistant severe acute graft-versus-host disease (aGVHD). METHODS: A total of 19 patients with steroid-resistant severe aGVHD received MSCs infusion treatment. The treatment response, transplantation-related mortality, events associated with infusion and relapse rate were analyzed. RESULTS: Two patients with grade II, 5 patients with grade III and 12 patients with grade IV aGVHD received a total of 58 infusions of MSCs. The mean total dose of MSCs was 2.13 (range 0.60 - 7.20)×10(6) cells per kg bodyweight. Seven patients received one infusion, 2 patients received two infusions, and 10 patients received three or more infusions. Eleven patients had a complete response and 4 had a partial response and 4 had no response. No patients had side-effects during or immediately after infusions, and no MSCs related tumorigenesis was detected to date. Eleven patients survived and 8 died, 4 for aGVHD, 1 for infection and 2 for aGVHD with concomitant infection and 1 for underlying leukemia relapse. The cell viability of freshly prepared MSCs is 93% (92% - 95%) by trypan blue staining. The cell viability of programmatically frozen and thawed MSCs is 72% (70% - 74%). CONCLUSION: Infusion of umbilical cord-derived MSCs expanded in vitro is an effective therapy for patients with steroid-resistant severe aGVHD without negative impact on relapse. Freshly prepared MSCs are superior to frozen and thawed cells in terms of cell viability.