Comparative analysis of Decitabine intensified BUCY2 and BUCY2 conditioning regimen for high-risk MDS patients undergoing allogeneic hematopoietic stem cell transplantation.

The optimal conditioning regimen for high-risk myelodysplastic syndrome (MDS) patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains elusive. This study aimed to explore the anti-leukemic efficacy and toxicity of Decitabine (Dec, 20 mg/m2/day, day -11 to -7) intensified BUCY2 vs. traditional regimen in high-risk MDS population. We retrospectively evaluated 93 consecutive high-risk MDS patients undergoing allo-HSCT in our institution, comparing discrepancies in clinical characteristics and outcomes between cases using Dec-intensified BUCY2 (n = 52) and traditional BUCY2 regimen (n = 41). Three-year cumulative incidence of relapse after Dec-intensified BUCY2 conditioning was remarkably lower than that of patients using BUCY2 regimen (20.2% vs. 39.0%, p = 0.034). Overall survival and disease-free survival at 3 years for Dec-intensified BUCY2 group were 70.2% and 64.9%, respectively, which were significantly improved when compared with BUCY2 group (51.1% and 43.9%, p = 0.031 and p = 0.027). Furthermore, overall survival and disease-free survival for MDS cases receiving cytoreduction therapy were dramatically better than patients in non-cytoreduction group (p = 0.041, p = 0.047). In summary, the Dec-intensified conditioning regimen could be effective and feasible, providing prominent recurrence control with moderate toxicity for high-risk MDS patients. These patients might also benefit from pre-transplant cytoreductive therapeutic schedules. Larger randomized controlled trials are still needed to further confirm these conclusions.

Giardia lamblia mimicking acute graft versus host disease after allogeneic hematopoietic stem cell transplantation: A case report.

RATIONALE: As the major complications post allogeneic hematopoietic stem cell transplantation (allo-HSCT), gastrointestinal disorders were most commonly ascribed to acute graft-versus-host disease (aGVHD) and opportunistic infections. Though Giardia lamblia (G lamblia) is the most common waterborne parasite of intestinal infection worldwide, seldom has it been reported in a patient with acute severe aplastic anemia after allo-HSCT. PATIENT CONCERNS: A 23-year-old male with severe aplastic anemia developed diarrhea, abdominal cramps, bloating, nausea, vomiting, fever, weight loss, and fatigue after allo-HSCT. DIAGNOSIS: Stool examinations for ova and parasites showed Giardia trophozoites and cysts. INTERVENTIONS: Methylprednisolone was stopped and the patient was intravenously treated with a 7-day course of metronidazole (500 mg, tid.). Simultaneously, cyclosporine (5 mg/kg) was continually utilized for suspicious gut GVHD. OUTCOMES: The Giardia lamblia in stool turned negative and his symptoms were resolved after the 7-day course. LESSONS: Incorporating non-invasive monitoring of stool examination for ova and parasites in the follow-up algorithm for post-HSCT patients can expedite clinical decision-making in the differential diagnoses for aGVHD even in the non-endemic area. Metronidazole therapy can be well-tolerated in HSCT patients with giardiasis.

Ruxolitinib add-on in corticosteroid-refractory graft-vs-host disease after allogeneic stem cell transplantation: Results from a retrospective study on 38 Chinese patients.

BACKGROUND: Graft-vs-host disease (GVHD) is a major cause of mortality after allogeneic hematopoietic stem cell transplantation. Some patients have steroid-refractory (SR) GVHD. AIM: To evaluate the effect and safety of ruxolitinib add-on in the treatment of patients with SR acute (a) and chronic (c) GVHD. METHODS: We retrospectively analyzed 38 patients administered ruxolitinib add-on to standard immunosuppressive therapy for SR-aGVHD or SR-cGVHD following allogeneic hematopoietic stem cell transplantation. Ruxolitinib was administered 5-10 mg/d depending on disease severity, patient status, and the use of anti-fungal drugs. Overall response rate, time to best response, malignancy relapse rate, infection rate, and treatment-related adverse events were assessed. RESULTS: The analysis included 10 patients with SR-aGVHD (grade III/IV, n = 9) and 28 patients with SR-cGVHD (moderate/severe, n = 24). For the SR-aGVHD and SR-cGVHD groups, respectively: Median number of previous GVHD therapies was 2 (range: 1-3) and 2 (1-4); median follow-up was 2.5 (1.5-4) and 5 (1.5-10) mo; median time to best response was 1 (0.5-2.5) and 3 (1-9.5) mo; and overall response rate was 100% (complete response: 80%) and 82.1% (complete response: 10.7%) with a response observed in all GVHD-affected organs. The malignancy relapse rates for the SR-aGVHD and SR-cGVHD groups were 10.0% and 10.7%, respectively. Reactivation rates for cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus, respectively, were 30.0%, 10.0%, and 0% for the SR-aGVHD group and 0%, 14.3%, and 7.1% for the SR-cGVHD group. CONCLUSION: Ruxolitinib add-on was effective and safe as salvage therapy for SR-GVHD.

Monitoring and Analysis of Chinese Chronic Myeloid Leukemia Patients Who Have Stopped Tyrosine Kinase Inhibitor Therapy.

Discontinuation of tyrosine kinase inhibitor (TKI) therapy after achieving a persistent deep molecular response (DMR) is an urgently needed treatment goal for chronic myeloid leukemia (CML) patients and has been included in the National Comprehensive Cancer Network (NCCN) guidelines (version 2.2017) for CML. Indeed, various studies have confirmed the feasibility of discontinuing TKI therapy. In this study, we analyzed data from 45 CML patients who had discontinued TKI therapy. Univariate analysis was performed to predict factors that were potentially related to treatment-free remission (TFR) and identify the differences between early relapse and late relapse. Out of the 45 patients, 20 exhibited molecular relapse after a median follow-up of 18 months (range, 1-54 months), and the estimated TFR at 24 months was 40%. The univariate analysis revealed that a high Sokal score and interruptions or dose reductions during TKI treatment were the only baseline factors associated with poor outcomes. Our results indicate that TKI discontinuation could be successfully put into practice in China.

Efficacy and Safety of Generic Dasatinib as a Second-line Treatment for Patients with Chronic Myeloid Leukemia: a Multicenter Retrospective Study in Hubei Province, China.

Dasatinib is a second-generation tyrosine kinase inhibitor (TKI) and it could be used as a second-line treatment for patients with chronic myeloid leukemia (CML). Yinishu, a generic dasatinib made in China, was approved by the China Food and Drug Administration in 2013 and it costs much less than the patented dasatinib SPRYCEL. The present study aimed to examine the efficacy and safety of Yinishu as a second-line treatment for CML by comparing the baseline clinical characteristics, rates of adverse events and efficacy between Yinishu and SPRYCEL groups. The results showed that there were no significant differences in the rates of optimal response between Yinishu and SPRYCEL for patients who started second-line treatment because of treatment failure. For patients who started second-line treatment because of intolerance of first-line treatment, their levels of BCR-ABL1/ABL1 on the international scale (BCR-ABLIS) was maintained very low throughout the course of Yinishu treatment. Drug-related adverse events occurred with the same frequency in these two groups. It was confirmed that Yinishu was effective and safe as a secondline treatment for CML patients. Yinishu may be more suitable for patients who are economically unable to pay for the patented dasatinib SPRYCEL.

Decitabine for relapsed acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation.

Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a main question on treatment failure. Current strategies for management that usually include salvage chemotherapy, donor lymphocytic infusion and second transplantation. Our study assessed the efficacy of decitabine (DAC) for treating patients with acute lymphoblastic leukemia (ALL) who relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We retrospectively analyzed the outcomes of 12 patients with relapsed ALL after allo-HSCT who received DAC therapy. Nine patients received DAC combined with chemotherapy and donor stem cell infusion, and 3 patients received single- agent DAC. Ten of the 12 patients achieved complete remission (CR), 1 achieved a partial remission (PR), and 1 had no response (NR) after treatment at the latest follow-up (LFU), the median survival was 11.2 months (range, 3.8-34, 7 months). The 1- and 2-year overall survival (OS) rates were 50% (6/12) and 25% (3/12), respectively. Five patients were still alive; 4 had maintained CR and 1 was alive with disease. Patients with Philadelphia chromosome-positive ALL had higher survival rate than patients with Philadelphia chromosome-negative ALL (57.1% vs. 20%). No aggravated flares of graft-versus-host disease (GVHD) were observed during DAC treatment. Therefore, DAC may be a promising therapeutic agent for ALL recurrence after allo-HSCT.

Decitabine for Relapsed Acute Lymphoblastic Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation / 华中科技大学学报（医学）（英德文版）

Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a main question on treatment failure.Current strategies for management that usually include salvage chemotherapy,donor lymphocytic infusion and second transplantation.Our study assessed the efficacy of decitabine (DAC) for treating patients with acute lymphoblastic leukemia (ALL) who relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT).We retrospectively analyzed the outcomes of 12 patients with relapsed ALL after allo-HSCT who received DAC therapy.Nine patients received DAC combined with chemotherapy and donor stem cell infusion,and 3 patients received single-agent DAC.Ten of the 12 patients achieved complete remission (CR),1 achieved a partial remission (PR),and 1 had no response (NR) after treatment at the latest follow-up (LFU),the median survival was 11.2 months (range,3.8-34,7 months).The 1-and 2-year overall survival (OS) rates were 50％ (6/12) and 25％ (3/12),respectively.Five patients were still alive;4 had maintained CR and 1 was alive with disease.Patients with Philadelphia chromosome-positive ALL had higher survival rate than patients with Philadelphia chromosome-negative ALL (57.1％ vs.20％).No aggravated flares of graft-versus-host disease (GVHD) were observed during DAC treatment.Therefore,DAC may be a promising therapeutic agent for ALL recurrence after allo-HSCT.

Angiogenic factors are associated with development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-ß in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-ß promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.

Umbilical cord blood-derived mesenchymal stem cells ameliorate graft-versus-host disease following allogeneic hematopoietic stem cell transplantation through multiple immunoregulations.

Although mesenchymal stem cells (MSCs) are increasingly used to treat graft-versus-host disease (GVHD), their immune regulatory mechanism in the process is elusive. The present study aimed to investigate the curative effect of third-party umbilical cord blood-derived human MSCs (UCB-hMSCs) on GVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their immune regulatory mechanism. Twenty-four refractory GVHD patients after allo-HSCT were treated with UCB-hMSCs. Immune cells including T lymphocyte subsets, NK cells, Treg cells and dendritic cells (DCs) and cytokines including interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were monitored before and after MSCs transfusion. The results showed that the symptoms of GVHD were alleviated significantly without increased relapse of primary disease and transplant-related complications after MSCs transfusion. The number of CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells decreased significantly, and that of NK cells remained unchanged, whereas the number of CD4(+) and CD8(+) Tregs increased and reached a peak at 4 weeks; the number of mature DCs, and the levels of TNF-α and IL-17 decreased and reached a trough at 2 weeks. It was concluded that MSCs ameliorate GVHD and spare GVL effect via immunoregulations.

[Modified method for whole bone marrow adherent culture of human bone marrow mesenchymal stem cells].

This study was aimed to investigate a more convenient and efficient method to cultivate the human bone marrow mesenchymal stem cells by means of natural erythrocyte sedimentation principle, based on the whole bone marrow adherent method. The bone marrow was cultured with a six-well plate instead of the flasks.Firstly, the bone marrow specimen was cultivated with the MSC complete medium for 48 h, then the upper RBC-free supernatant layer was drawn and placed into the new wells to isolate MSC. Inverted microscope was used to observe the cell morphology and to record the adherent time of first cell passage, first passaging time. The traditional whole bone marrow adherent method was used as the control. The cell cycle and cell surface markers were detected by flow cytometry,and the differentiative capacity of MSC into osteocyte and adipocyte was identified by alkaline phosphatase kit and oil red O, respectively. Besides, the proliferative curve of P1,P3,P5 of BMSC was depicted by counting method. The results showed that MSC cultured by the modified method highly expressed CD90, CD105, CD13, CD44 and lowly expressed CD14, CD45, CD34. Concerning the cell cycle feature, it was found that most of the cells were in G0/G1 phase (88.76%) , followed by G2/M phase (3.04%) and S phase (8.2%), which was in accordance with stem cell cycle characteristics. The proliferative curve showed a typical "S" type, and both the oil red O and alkaline phosphatase staining of MSC were positive. Compared with the traditional method, the modified method had the advantage of high adherence rate (P = 0.0001) and shorter passaging time for the first passage (P = 0.001), with the statistically significant difference. It is concluded that there is a large number of adherent, active and suspended MSC in the RBC-free supernatant layer after the culture of bone marrow for 48 h. Isolating MSC by the modified method is more convenient and efficient than the traditional whole bone marrow adherent method.

[Exogenous VEGF promotes hematopoietic stem cell mobilization].

This study was aimed to investigate the effect of exogenous VEGF on hematopoietic stem cell mobilization and immune system. The C57BL/6J mice were randomly divided into the normal control group, VEGF short-term group (5 d) and VEGF long-term group (27 d). Mice in the experimental group were injected ip with VEGF (100 ng/d); mice in control group were injected ip with PBS. The white blood cell (WBC) count and the ratio of lymphocyte in the peripheral blood at different time point were assayed by hemacytometer. The percentage of hematopoietic stem cell (HSC), lymphocyte subgroup, regulatory T cell (Treg), myeloid-derived suppressor cells (MDSC) in the peripheral blood and spleen of different groups were detected by flow cytometry. The morphological changes of spleen and spleen index of mice in the control and long-term group were observed by microscopy. The results showed that the absolute number of WBC in the peripheral blood of mice significantly increased after injection of VEGF, and the peak value was at day 3. The percentage of Lin(-)Sca-1(+)CD117(+) cells in the peripheral blood and spleen of the long-term group were significantly higher than that in the normal control group (P < 0.05). The spleen of the mice in VEGF long-term group was larger than that of the control group, the spleen index also increased (P < 0.05), and remarkable extramedullary hematopoietic signs were found in the HE stained sections. There was no significant change in the total ratio of lymphocytes in the peripheral blood after injection, but the percentage of CD3(+) cells and the CD3(+)/B220(+) ratio in the long-term group deceased; the percentages of Treg and Gr-1(+)CD11b(+) MDSC in the experimental groups increased (P < 0.05), which more significantly increased in the long-term group than that in the short-term group (P < 0.05). It is concluded that the exogenous VEGF promotes hematopoietic stem cell mobilization, and at same time up-regulates the many kinds of suppressive immune cell levels which leads to changes of immuno-function.

Amplification of functional myeloid-derived suppressor cells during stem cell mobilization induced by granulocyte colony-stimulation-factor.

The effects of granulocyte colony-stimulation-factor (G-CSF) on stem cell mobilization and its impact on the amplification of myeloid-derived suppressor cells (MDSCs) of donor mice were examined. A mouse model of stem cell mobilization was established by consecutive subcutaneous injection of 100 µg/kg G-CSF for 5 days. The blood from the donor mice was routinely examined during mobilization. Stem cells and MDSCs were analyzed by flow cytometry. The immunosuppressive molecules derived from MDSCs in serum and spleen, including hydrogen dioxide (H2O2) and nitric oxide (NO), and the activity of nitric oxide synthase (NOS) were determined during the mobilization. Apoptosis of T lymphocytes was assessed by using Annexin-V/PI. During stem cell mobilization, the number of lymphocytes and white blood cells in the peripheral blood was increased, and peaked on the 4th day. The number of stem cells in G-CSF-treated mice was significantly greater than that in controls (P<0.01). The expansions of MSDCs were also observed after G-CSF mobilization, with a more notable rate of growth in the peripheral blood than in the spleen. The activity of NOS and the production of NO were increased in the donor mice, and the serum H2O2 levels were approximately 4-fold greater than the controls. Consequently, apoptosis of T lymphocytes was increased and showed a positive correlation with the elevated percentage of MDSCs. It was concluded that G-CSF could provide sufficient peripheral blood stem cells for transplantation. Exogenous administration of G-CSF caused the accumulation of MDSCs in the peripheral blood and the spleen, which could lead to apoptosis of T lymphocytes and may offer a new strategy for the prevention and treatment of graft versus host disease.

Analysis of seizure risk factors after allogeneic hematopoietic stem cell transplantation: a 8 case report and literature review.

The clinical characteristics of patients with seizures after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were analyzed. A total of 8 cases of seizures after allo-HSCT were investigated. Clinical data of these cases were studied retrospectively. Of 159 cases subjected to allo-HSCT, seizure occurred in 8 cases during 29-760 days after transplantation, median survival time was 46 days, and there were 6 cases of tonic-clonic seizure. The incidence of seizure after matched unrelated HSCT was higher than that after related HSCT (P=0.017). Of 7 cases treated with cyclosporine A (CsA), 4 cases obtained high blood levels of CsA. In addition, hyponatremia was diagnosed in 5 cases. Abnormal electroencephalogram and brain MRI findings were found in some cases. During 20 days after seizure, 2 cases died due to infection and graft-versus-host disease (GVHD), respectively. It was suggested that multiple factors are associated with seizures after allo-HSCT. Rapid identification and correction of the causative factors are very important to prevent permanent central nervous system damage and reduce the mortality.

Infusions of recipient-derived cytokine-induced killer cells of donor origin eradicated residual disease in a relapsed leukemia patient after allo-hematopoietic stem cell transplantation.

A female patient diagnosed with acute myelocytic leukemia M5a (AML-M5a) relapsed 986 days after her allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an unrelated male donor with matched human leukocyte antigen (HLA). Three re-induction chemotherapies were administered, and partial remission was achieved. The patient was given repetitive infusion of cytokine-induced killer (CIK) cells expanded from recipient peripheral mononuclear cells of full donor chimerism due to loss of contact of quondam donor for donor lymphocyte infusion (DLI) and rejection of second transplantation. The patient achieved complete cytogenetical remission. This strategy might overcome the obstacle of donor unavailability and present an appealing new therapeutic alternative to donor-recruited adoptive immunotherapy for relapsed disease at post-transplantation.

[Proliferation inhibiting and apoptosis inducing effects of parthenolide on human multiple myeloma cells].

OBJECTIVE: To investigate the effect of parthenolide (PTL) on human multiple myeloma (MM) cells in vitro and its mechanism. METHODS: Human MM cells of the line PRMI8266 were cultured and treated with PTL of the concentrations of 1, 2.5, 5, 7.5, and 10 micromol/L for 24, 48, or 72 hours. MM cells treated with DMSO were used as control group. The optical density was measured so as to draw a growth curve. The cell viability was detected by MTT and trypan-blue exclusion. The apoptosis was detected by flow cytometry. AO/EB staining and Wright-Giemsa staining were used to observe the morphological changes of the cells by fluorescence microscope and light microscope respectively. The caspase-3 activity was evaluated by BD ApoAlert Caspase Colorimetric Assay Kit. RESULTS: PTL significantly inhibited the proliferation and viability of the MM cells time and dose-dependently (all P < 0.01), and significantly induced the cell apoptosis after 48 h in a dose-dependent manner (P < 0.01). The early cell apoptosis rates for PTL of the concentrations of 2, 5 and 10 micromol/L were 17.1% +/- 2.6%, 33.6% +/- 3.8%, and 40.9% +/- 3.1% respectively, all significantly higher than that of the control group (5.6% +/- 1.2%, all P < 0.01). The MM cells treated with PTL of the concentration of 5 micromol/L for 48 h showed typical cell apoptotic features. The caspase-3 activity of the MM cells was enhanced significantly by PTL in a time and dose-dependent manner (all P < 0.01). CONCLUSION: This first report of anti-proliferation and apoptosis induction effects of PTL on MM cells shows that able to significantly inhibit the proliferation and induce the apoptosis of MM cells and enhance the caspase-3 activity, PTL may be a potentially useful drug for treatment of MM.

[Expression of BLM mRNA in six tumor cell strains].

The mutations of BLM gene may result in Bloom's syndrome which includes immunodeficiency, predisposition to malignant tumors and so on, and enhances sister chromati exchange (SCE), DNA replication failure, genome instability, and increases cancer susceptibility. This study was aimed to investigate the variability of mRNA expression level and cDNA structure of BLM gene in tumor cell strains so as to look for a new cancerogenic mechanism and to find a new therapeutic target. The expression level of mRNA and the structure of cDNA of BLM gene in six tumor cell strains and the normal human bone marrow mononuclear cells were detected with RT-PCR and DNA sequencing was performed. The results indicated that these tumors cells expressed BLM mRNA higher than the normal human bone marrow mononuclear cells (P < 0.01), but no cDNA sequence abnormality of BLM gene in these tumors cells was observed. It is concluded that the increase of expressing level of BLM mRNA may play an important role in the development of these tumors.

[Recombinant adenovirus mediated hVEGF165 gene transfer promotes recovery of hematopoiesis in post-BMT mice].

To investigate the effects of vascular endothelial growth factor (VEGF) on the recovery of hematopoiesis in post-BMT mice, the recombinant adenovirus Ad-EGFP/hVEGF(165) was injected into syngeneic BMT BALB/c mice via the tail vein. At day 10, 20, 30 after BMT, the in vivo expression of hVEGF(165) was measured. At the same different time points, the numbers of WBC, Plt, RBC in peripheral blood and MNC in bone marrow were counted. By the way, the bone marrow MNCs at day 30 post-BMT were used for further CFU-S assay. The results indicated that a long-term expression of hVEGF(165) in plasma and different organs was successfully mediated by recombinant adenovirus. At each time point of post-BMT, the numbers of WBC, Plt, RBC as well as bone marrow MNC observed in the group treated with recombinant adenovirus Ad-EGFP/hVEGF(165) were lower than those of the normal control group, but were higher than those in other testing groups (P < 0.05). The number of CFU-S (21.4 +/- 2.67) formed by bone marrow MNC at day 30 after BMT reached to the normal level (19.50 +/- 2.46) (P > 0.05), which was much higher than that in other groups (P < 0.05). It is concluded that hVEGF(165) gene transfer mediated by recombinant adenovirus plays a role of promoting the recovery of hematopoiesis in post-BMT mice.

Effects of adenovirus mediated vascular endothelial growth factor gene transfer on reconstitution of hematopoiesis in post-bone marrow transplantation mice.

BACKGROUND: Bone marrow transplantation (BMT) conditioning procedure is considered as the cause of damage to bone marrow microvasculature and the delay of hematopoiesis recovery. However, hematopoiesis regulation post BMT by vascular endothelial growth factor (VEGF) has not yet been studied. In this study, adenovirus were used to investigate the effects of VEGF gene transfer on preventing damages to bone marrow microenvironment and its promotion of hematopoiesis in post-BMT mice. METHODS: Recombinant adenovirus (Ad)-enhanced green fluorescent protein (EGFP)/hVEGF165 was injected via tail vein into BALB/c mice undergoing syngeneic BMT. During the different phases post BMT, the distribution of adenovirus and the plasma levels of hVEGF were measured as well as the numbers of white blood cells (WBC), platelet (PLT) and red blood cells (RBC) in peripheral blood. At the same time, the mice were injected with Chinese ink via tail vein, following which the tibias were separated and were used for analysis of bone marrow microvasculature surface area and cellularity. RESULTS: Significant expression of EGFP and hVEGF was observed in multiple organs at different phases post BMT, and the plasma level of hVEGF was up to (866.67 +/- 97.13) pg/ml. The recovery of WBC, PLT and RBC of the group treated with recombinant adenovirus Ad-EGFP/hVEGF165 were significantly more rapid than those of other BMT groups (P < 0.05, respectively). At the 20th day post BMT, the percentage of bone marrow microvasculature surface area in group treated with VEGF [(61.2 +/- 4.0)%] returned to normal level [(62.0 +/- 5.0)%, P > 0.05]. The restoration of hematopoiesis was retarded more than that of microvasculature. The cellularity of bone marrow in each group was still lower than that of normal control [(62.3 +/- 4.0)%, P < 0.05] at the 30th day post BMT, but the percentage in group treated with VEGF at the 20th and 30th days post BMT [(46.5 +/- 5.0)% and (55.1 +/- 4.5)%] exceeded those of other BMT groups (P < 0.05, respectively). CONCLUSION: VEGF gene transfer mediated by adenovirus may protect the hematopoietic microenvironment to promote the restoration of hematopoiesis in post-BMT mice.

[Blocking the escape of leukemic cells from killing of T cell by combining anti-Fas ribozyme and CD80-IgG fusion protein].

OBJECTIVE: To study Fas expression regulation of cytotoxic T lymphocyte(CTL)via anti-Fas ribozyme, increasing of CD80 epitope on the surface of acute myelomonocytic leukemia cells by CD80-IgG fusion protein and their effects on the apoptosis and killing ability against acute myelomonocytic leukemia cells of CTL. METHODS: A hammerhead ribozyme gene targeting the Fas mRNA was synthesized and its expression vector pEGFP-RZ596 was constructed and transfected into the mouse spleen T cells via electroporation. The Fas expression on T cells was detected by RT-PCR and Western bloting. In the meantime the eukaryotic expression vector pcDNA/CD80-IgG was constructed by gene recombinant technique and transfected into ovarian cells of hamster of the line CHO. The CD80-IgG fusion protein was purified from the supernatant of G418-selected CHO cells by Protein G affinity chromatography method. Then allogeneic mixed lymphocytes culture between the mouse spleen T cells transfected with pEGFP-RZ596 and WEHI-3 cells (mouse acute myelomonocyte leukemia cell line) incubated with CD80-IgG fusion protein was performed. The apoptosis rate of the T cells was detected with annexin V-FITC. The proliferation and killing ability in vitro against WEHI-3 cells of the T cells were detected by MTT colorimetry. RESULTS: The luminance of Fas Western bloting results from the mouse spleen T cells negative control, transfected with pEGFPC1 and transfected with pEGFP-RZ596 were separately 1, 0.98 and 0.45 (P < 0.01). After being cocultured with WEHI-3 cells, which has higher expression of Fas ligand (64% +/- 3%), the apoptosis rate and the killing ability against WEHI-3 cells of the mouse spleen T cells transfected with pEGFP-RZ596 were separately 37% and 67%. Whereas that of the mouse spleen T cells negative control and transfected with pEGFPC1 were separately 88%, 84% (P < 0.01) and 32%, 31% (P <0.01). The CD80 positive expression rate of WEHI-3 cells was upregulated from 5.1% +/- 0.4% to 27.4% +/- 2.2% after these cells were preincubated with CD80-IgG fusion protein (P < 0.01). The killing ability of the mouse spleen T cells against WEHI-3 cells preincubated and not preincubated with CD80-IgG fusion protein were separately 64% and 49% (P <0.01), but that of the mouse spleen T cells, which were transfected with pEGFP-RZ596, was further promoted to 82% (P < 0.01) CONCLUSION: The apoptosis of mouse CTL inducing by FasL-Fas pathway could be avoided and the killing ability of mouse CTL against WEHI-3 cells can be significantly promoted at the same time by combining anti-Fas ribozyme and CD80-IgG fusion protein.