Study on blocking the leukemia immune escape after BMT by Fas-Fas ligand pathway / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>To investigate if bone marrow transplantation (BMT) with bone marrow mononuclear cells (BMMCs) transducted with murine soluble Fas gene (sFas) using adenovirus vector could block the immune escape of leukemia cells eliminate the residual leukemia cells and reduce their relapse.</p><p><b>METHODS</b>The recombinant adenovirus vector with murine sFas, adsFas, and the control vector adEGFP were constructed using homologous recombination between two plasmids in Escherichia coli. BMT was carried out after the BMMCs were infected with Adenoviruses. The mice models of leukemia/lymphoma were constructed by inoculating female C57BL/6 mice (H-2b) with 10(5) EL4 cells/mouse through caudal vein. Donors of bone marrow grafts were syngeneic male mice. BMMCs were infected with AdsFas or AdEGFP 24 hours before (Group D or E). The following three groups were simultaneously used: Group A, no BMMCs transplanted; Group B, transplanted with BMMCs not infected with adenoviruses; Group C, only transfusing EL4 cells, neither irradiation nor BMT. The hematopoietic reconstitution, generation of leukemia/lymphoma and the survival rate were observed in all groups after BMT.</p><p><b>RESULTS</b>The adenovirus vectors were successfully constructed. The titre of virus after purification was up to 2.5 x 10(11) pfu/ml. Spleen indices examined 11 days after BMT were not obviously different among Group B, D and E (P > 0.05), but indices in Group A were significantly lower than those in the latter three groups (P < 0.01). Counts of leukocytes and platelets on +30 day showed mice were reconstituted satisfactorily in Group B and D, but very low in Group C and E. The Y-chromosomes existed 2 months after BMT and examination of bone marrow cytology showed that Group B and D were almost normal, but Group C and E had plenty of lymphoblast-like tumor cells. Tumors were obviously observed in the mice of Group C and E by histopathological examination, but the mice in Group B and D were normal. The survival rates were 0 (0/4) in Group A, 100% in Group B (6/6) and D (16/16), 12.5% (2/16) in Group C and 6.25% (1/16) in Group E respectively. It is demonstrated that, in contrast with the control (Group EGFP), survival rate was significantly increased in the sFas Group (P < 0.01).</p><p><b>CONCLUSIONS</b>The transfer of sFas gene by adenovirus changed the prognosis state of leukemia/lymphoma mice after auto-BMT. The transduction of sFas might block the effect of the immune escape of EL4 cells through FasL. These results could thus provide a new direction to find a way to treat the leukemia and its recurrence after BMT.</p>

Role of apoptosis in cryoinjury of cord blood hematopoietic stem/progenitor cells and its mechanism / 中国实验血液学杂志

To investigate the role and mechanism of apoptosis in cryoinjury of cord blood hematopoietic stem/progenitor cells, apoptosis of CD34(+) cells, mitochondrial membrane potential (MMP) and Fas antigen expression were detected by flow cytometry (FCM), the Bcl-2 protein expression was detected by immunohistochemistry, caspase-3 expression was determined by Western blot and caspase-3 activity analysis, colony-forming units (CFU) was performed by semi-solid methylcellulose culture. The results showed that when cells were store at -196 degrees C for 2 weeks or 4 weeks, apoptotic cells increased, gel electrophoresis displayed typical DNA ladder, and CFU decreased by 25.2% and 30.1%. The value of MMP reduced and expression of Bcl-2 protein was down-regulated during the freeze-thaw process, but the Fas antigen expression was not effected. However, only the 32 kD inactive caspase-3 proenzyme was detected in freshly isolated CD34(+) cells. After freeze-thaw, caspase-3 was activated and a cleavage of 20 kD protein was detected. Cryopreserved cells showed a 1.2-fold and 1.5-fold increase in caspase-3 activity, respectively. It is concluded that apoptosis plays an important role in cryoinjury of cord blood hematopoietic stem/progenitor cells, which triggers a mitochondrial apoptotic pathway that is caspase-dependent but does not require death receptors, where caspase-3 is the key effector.

Effect of angiotensin II on cord blood CD34(+) cells expansion in vitro / 中国实验血液学杂志

Angiotensin II (Ang II) is one active substance of renin-angiotensin system. In order to explore the effect of Ang II combined with various cytokines on proliferation and differentiation of cord blood CD34(+) cells, in vitro experiments of cell cultures of Ang II with or without cytokines were taken place. The results showed that Ang II stimulated both BFU-E and CFU-GM expansion. The numbers of BFU-E and CFU-GM raised with increase of Ang II concentrations ranged from 0.01 - 0.1 micro mol/L. In semi-solid culture assay, Ang II stimulated CFU-GM production but no effect on BFU-E occurred. The multiple number of CFU-GM increased from 2.3 +/- 0.8 to 7.8 +/- 1.9 times when Ang II was added into SCF + G-CSF + GM-CSF + IL-3 combination. Similarly, the multiple number of BFU-E increased from 3.1 +/- 1.8 to 9.2 +/- 2.3 times when Ang II was combined with SCF + EPO + TPO + IL-3. In conclusion, Ang II stimulated cord blood hematopoietic stem/progenitor cell expansion in vitro the in presence of various cytokines.

A new method for construction of EGFP-labled recombinant adenovirus containing hVEGF(165) and its property in vitro / 中国实验血液学杂志

By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labled recombinant adenovirus vector containing hVEGF(165) was generated quickly and its property was studied in vitro. First, hVEGF(165) coding sequence was subcloned into the shuttle plasmid pAdTrack-CMV, then linearized and cotransferred with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ(5183). After positive kanamycin-resistant colony was picked up, the recombinant adenoviral plasmid was identified by restriction analysis with PacI and transfected into HEK 293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF(165). The further amplified recombinant adenoviruses were purified by CsCl banding at 32,000 rpm for 18 to 24 hours. Electron microscopy and PCR were performed for testing the recombinant adenovirus. The results showed that the purified particles were homogenous hexagon with a high titer up to 2 x 10(12)pfu/ml. An amplified band of 540 bp fragment demonstrated the successful insert of hVEGF(165). Under fluorescence microscopy, the expression of EGFP was easily detected in HEK 293 and other target cells. The maximal stimulating effect on the proliferation of hUVEC was obtained when the given multiplicity of infection (MOI) of Ad-EGFP/hVEGF(165) was 100. The rate of EGFP positive mouse bone marrow mononuclear cells analysed by flow cytometry was 27.3% after 24 hour-incubation with Ad-EGFP/hVEGF(165) (MOI = 100), and the expression of hVEGF(165) protein in the conditioned medium was 1385 +/- 332 pg/10(6) cells. It is concluded that the construction of adenovirus vector by homologous recombination in bacteria using AdEasy system can be quickly and easily performed, and the prepared high titer of Ad-EGFP/hVEGF(165) is an efficient helpful vector to transfer genes into target cells, all of which make the further in vivo experiments with VEGF(165) possible.

Experimental study on blocking immune escape of leukemia cells in the recipient after bone marrow transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate whether murine soluble Fas gene transfected marrow graft could block the immune escape of leukemia cells, so as to eliminate the residual leukemia cells and reduce relapse after bone marrow transplantation (BMT).</p><p><b>METHODS</b>The murine leukemia/lymphoma models were established by inoculating female C57BL/6 mice (H-2b) with 10(5) EL4 cells/mouse through caudal vein. Donors of BM grafts were C57BL/6 male mice. Bone marrow mononuclear cells (BMMCs) were transfected with sFas or EGFP by adenovirus (adsFas or adEGFP) 24 hours before BMT (group D or E). The following three groups were set simultaneously: group A, no BMMCs transplanted; group B, BMMCs transplanted with no adenoviruses transfection; group C, EL4 cells transfusion only. Hematopoietic reconstitution, generation of leukemia/lymphoma and the survival rate were observed in all the groups after BMT.</p><p><b>RESULTS</b>The spleen indices examined 11 days after BMT were not obviously different among group B, D and E (P > 0.05), but in group A were significantly lower than those in the groups B, D, E (P < 0.01). The leukocyte and platelet counts on day 30 after BMT were recovered in group B and D, but were very low in group C and E. The Y-chromosomes appeared 2 months after BMT. Bone marrow pictures in group B and D were almost normal, but in group C and E had plenty of lymphoblast-like tumor cells. Tumors were obviously revealed in the mice of group C and E by histopathology examination, but did not in group B and D. The survival rate was 0 in group A, 100% in group B and D, 12.5% in group C and 6.25% in group E. Compared with that in group E, the survival was significantly increased in the sFas group (P < 0.01).</p><p><b>CONCLUSIONS</b>Graft transfected with sFas gene prolonged the post-BMT survival of leukemia/lymphoma mice. The transfection of sFas might block the effect of the immune escape of EL4 cells through FasL.</p>

Study on blocking the tumor immune escape by Fas ligand pathway / 中国实验血液学杂志

The expression of Fas ligand (FasL) on the membrane of many kinds of leukemia or solid tumor cells played an important role in the immune escape of tumor cells. This study was aimed to know if the soluble Fas (sFas), expressed by adenovirus, could block the immune escape of tumor cells by FasL pathway. The two recombinant adenoviral vectors, AdsFas with murine soluble Fas gene and AdEGFP with enhanced GFP protein gene, were constructed by homologous recombination between two plasmids in Escherichia coli with the AdEasy adenovirus vector system. The viruses were propagated and purified by two times ultracentrifugation. Their titres were detected by plaque assays. The expressed protein was evaluated by Western blot analysis. Then the tumor EL4 cells were infected with AdsFas and AdEGFP respectively. The apoptosis ratio of the target cells-YAC-1 cells induced by EL4 cells was respectively detected by (3)H-thymidine ((3)H-TdR) labeling. The results showed that the recombinant adenoviral vectors AdsFas and AdEGFP were successfully obtained. The titres of viruses purified by two times ultracentrifugation were up to 10(11) pfu/ml by plaque assays. The sFas protein was highly expressed in the target cells by Western blot analysis. After the EL4 cells were transfected with the adenoviruses AdsFas, the apoptosis rate of YAC-1 cells in the sFas transfection group (respectively 6%, 7% and 9% when the effector:target (E:T) was 3:1, 10:1 and 30:1) was obviously lower than that in the control group (respectively 28%, 37% and 45%), P < 0.01. But when the EL4 cells were transfected with AdEGFP, the apoptosis rate of YAC-1 cells (respectively 30%, 36% and 48%) was similar to the control group, P > 0.05. In conclusion, the transfer of sFas by adenovirus could inhibit the apoptosis of Fas(+) cells-YAC-1 cells induced by tumor EL4 cells. It showed that the transduction of sFas could block the effect of the immune escape of EL4 cells through FasL in vitro. These results thus provide a new direction to find a way to treat tumors.

Study on expression and function of fas ligand in human myeloid leukemia cells / 中国实验血液学杂志

To determine whether the Fas receptor-Fas ligand (FasR-FasL) system, which triggers apoptosis in sensitive cells, is an important mechanism of cytotoxicity in myeloid leukemia. FasL expression was investigated in myeloid leukemia cells and its upregulation by a combination of IL-2 and INF-gamma, +/- as well as its function of inducing Jurkat cells to apoptosis mainly by flow cytometry (FCM). Results showed that leukemia cells expressed more FasL (3.59 +/- 1.05)% than that expressed in the healthy individuals (0.36% +/- 0.16)%, P < 0.001 and the FasL was upregulated (7.78 +/- 3.40)%, P < 0.01 when treated with IL-2 and IFN-gamma. Leukemia cells were co-cultivated with Jurkat cells for 24 hours. Then Jurkat cells were labeled with FITC-annexin V and PE-CD3 to assess apoptosis by FCM. The leukemia cells, which had been incubated with IL-2 and IFN-gamma, induced more Jurkat cells to apoptosis than the ones that freshly isolated from the peripheral blood mononuclear cells, which raised the figure from (8.28 +/- 1.61)% to (10.73 +/- 2.16%). And the supernatant derived from the former killed more Jurkat cells than the latter. It was concluded that human myeloid leukemia cells expressed high levels of functional FasL that can kill Jurkat T-cells by apoptosis. FasR-FasL sys tem could play a role in the "immune escape" and relapse of the leukemia. The induction of apoptosis through the Fas pathway might be a novel and effective approach for leukemia immunotherapy.

Retroviral vector-mediated in vitro expression of human soluble fas / 中国实验血液学杂志

Leukemic cells from patients expressed high level FasL cause apoptosis of autologous activated T cells via the Fas/FasL pathway. To investigate the role of soluble Fas (sFas) in reversing this process, a retroviral-mediated expression vector pLXIN-sFas was established. A retroviral-mediated expression system of human sFas was established in vitro and the biological activity of the expression product sFas was observed. To obtain the soluble Fas cDNA, the specific part of the full-length Fas cDNA was deleted by multiple PCR. After pLXIN-sFas packaged by PA317 cells, it was transferred into the target cell COS-7. The quantity of the sFas was (2.2 +/- 0.7) micro g/ml in supernatant of cultured COS-7 cells, and it could greatly inhibit apoptosis of Jurket cells induced by anti-Fas antibody. Our results suggested that the recombinant is able to express the target proteins in vitro and it has the perfect biological activity.