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Objective: To explore the efficiency and toxicity of allo-geneic hematopoietic stem cell transplantation (Allo-HSCT) for refractory acute lymphocytic leukemia. Patients and Methods: Thirty-seven patients of refractory acute lymphocytic leukemia received allo-geneic hematopoietic stem cell transplantation containing 18/37 cases from HLA-identical siblings donors, 8/37 cases from HLA-identical unrelated donors and 11/37 cases from haplo-identical donors of the mother. The tolerance, transplant related complications, survival rate were observed and analyzed. A myeloablative conditioning regimen included TBI and Cyclophosphamide. Cyclosporine A combined MTX were used to prevent graft versus host disease (GVHD) and G-CSF was administered after Allo-HSCT. Result: All 37 patients tolerated the therapy well and Slight or moderate mucositis was happened. Nineteen patients had light abnormal liver function and two had renal disfunction lightly, one patient had epilepsy during the therapy. Fatal infection complications were occurred in 6 patients (including idiopathic pneumonia in 2 patients). Severe acute GVHD was presented in 5 patients. Chronic GVHD was occurred in 16 patients. Hemorrhagic cystitis was happened in 1 patient, and 9 patients relapsed again after transplantation. Venoocclusive liver disease, host versus graft disease, substantia alba encephalopathy, fatal hemorrhage, secondary tumor and dysphrenia were not occured. Hematopoietic reconstitution obtained on days 12 to 25(day 17 on average). In the follow-up duration of 9 to 55 months (34 months of mid position), most of these patients had a complete chimerical state. Twenty of 37 patients are alive (54.1%). Disease free survival is 48.6%. Conclusion: Allo-HSCT is an effective therapy in the treatment of refractory acute lymphocytic leukemia. Relapse again after transplantation, fatal infection, and severe acute GVHD are the main reasons of failure.

Objective: To explore the possible immune pathophysiology of CD4+T regulatory (CD4+Treg) in acquired aplastic anemia (AA). Methods: According to the markers on membrane, cytokines, and effective mechanisms of CD4+Treg, the levels of CD4+CD25+Treg,CD4+CTLA-4+Treg,CD4+ICOS+Treg,CD4+PD-1+Treg,CD3+CD8−IL-10+Treg,CD3+CD8−TGF-β1+Treg,CD3+CD8−IL-Treg in the bone marrow of 23 cases with AA at active phase, 10cases with AA at recovery phase, 15 normal controls, were measured, and the relationship between CD4+Treg and priming immune factor-CD28 or effective immune factor-IFN-γ were also evaluated respectively. Results: [circ1]In the group of AA at active phase, at recovery phase, and normal controls, CD4+CD25+Treg: (3.36±2.05)%, (3.49±1.71)%, (2.40±0.77)%, CD4+CTLA-4+ Treg: (1.43±0.67)%, (3.46±2.26)%, (2.45±1.30)%, CD4+ICOS+Treg: (2.04±1.79)%, (2.95±2.23)%, (2.37±1.68)%, CD4+PD-1+Treg: (1.40±1.32)%, (2.34±0.87)%, (2.07±1.64)%, CD3+CD8−IL-10+Treg: (2.14±1.19)%, (2.37±1.20)%, (2.04±1.25)%, CD3+CD8−TGF-β1+Treg: (1.83±0.99)%, (1.52±0.73)%, (2.15±1.17)%, CD3+CD8−IL-4+Treg: (2.46±1.65)%, (2.53±1.61)%, (1.99±1.27)%; costimulatory of CD3+CD4+CD28+ was: (39.84±10.89)%, (22.72±9.08)%, 31.40±10.83)%, the Th1cells CD3+CD8−IFN-γ+ were: (11.13±4.96)%, (5.39±4.29)%, (4.21±2.11)%. [circ2] Contrast to normal controls, while CD4+CTLA-4+Treg of AA at active phase deceased markedly (p=0.05); [circ3] Contrast to normal controls, the ratio of membrane costimulatory of CD28/ICOS CD28/CTLA-4, CD28/PD-1 were all increased significantly (p Conclusion: [circ1] In AA, the positive regulatory costimulatory increased while the negative regulatory costimulatory did not change or decreased, which shifted the immune balance to intensification. [circ2] Not only at priming stage but also at effective stage, the immune strengthened factors were higher than attenuated factors in AA.

Objective To explore the relationship among CD4+CD25+ regulatory T cell, the expression of FOXP3 mRNA levels of donor bone marrow (BM) and graft versus host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods Thirty patients undergoing allo-HSCT were enrolled in this study. The levels of donor CD4+CD25+ regulatory T cell were compared in the patients with GVHD and those without GVHD by immnofluorescence and flowcytometry. The donor BM expressions of Foxp3 mRNA levels were analyzed by using reverse transcriptase polymerase chain reaction (RT-PCR). Results All patients achieved engraftment. ANC ≥0.5×109/L and PLT ≥ 20×109/L were at day 14(range, 12–15)and day 18 (range, 15–25) respectively. With a median follow-up of 12.8 (8–16) months, Acute GVHD occurred in 15 of 30 patients(50.0%) with gradeII–IV Acute GVHD (40.0%). Chronic GVHD developed in 6 (extensive 2, limited 4) out of 30 patients (20.0%). The levels of donor CD4+CD25+ regulatory T cell pre-and pro-mobilization were (2.67±0.38)% and (5.01±1.33)% respectively, no difference were observed (P>0.05). Patients with I–II aGVHD demonstrated higher donor-type CD4+CD25+ immune regulatory T cell level than those with III–IV aGVHD; Patients with III–IV aGVHD showed much lower donor-type CD4+CD25+ immune regulatory T cell level than those without GVHD; no difference were observed between patients withI–II aGVHD and those without aGVHD. The transcripts of FOXP3 donor BM were detected in seven donors of thirty recipients who received allo-HSCT. Five patients had no GVHD after transplantation;two developedIaGVHD. Donor BM Foxp3 transcripts were never detected in patients with II–IV aGVHD. Conclusion Donor-type CD4+CD25+ immune regulatory T cell level was relative to recipient II–IV aGVHD. Up-grade donor-type CD4+CD25+ immune regulatory T cell level could reduce the incidence of aGVHD. Donor BM FOXP3 transcripts were detected in patients without serious aGVHD after transplantation.

Objective: To explore the possible immune annormality of CD28 costimulatory super family and CD4+T regulatory (CD4+Treg) in myelodysplastic syndrome (MDS). Methods: Gated by CD3 and SSC, the changes of CD28, CTLA-4, PD-1, and CD25, on CD3+CD4+ cells were evaluated in 38 new diagnosed cases with MDS(RA 17, RAS 1, RAEB 14, RAEB-t 6) and 11 normal controls respectively by directive immune labels. Results: [circ1]In the group of MDS, the CD3+CD4+CD28+ was 79.82%±8.99% ( RA 78.65%±10.4%, RAEB/RAEB-t 80.10%±7.28% respectively), was lower than 89.56%±3.06% of normal controls (P Conclusion: [circ1] In MDS, the immune priming factor-CD28 decreased while the suppressive factor-CTLA-4 increased. [circ2] The negative immune CD4+ regulatory increased in MDS, and was predominance in immune balance. [circ3] Along with progression of MDS, negative regulatory factors was enforced, which promoted clone of MDS expansion.

Many European groups have described that mutations at exon-12 of the nucleophosmin (NPM1) gene are the most frequent genetic lesion in acute myeloid leukemia (AML) (45.7%~61.7% of all adult AMLs with normal karyotype). This outstanding discovery provides a promising minimal residual disease (MRD) marker for AMLs with normal karyotype. To clarify the prevalence and the clinical profile of NPM1 mutations in Chinese patients with AML, we analyzed a cohort of 156 newly diagnosed adult AMLs for this mutation. Genomic DNAs were prepared from bone marrow samples of these patients. NPM1 exon 12 mutations were detected using direct sequencing or fragment analysis of DNA-PCR products. NPM1 mutations were present in 28.2% of the overall population, including 1/1(100%) of M0, 11/27(40.7%) of M1, 11/46(23.9%) of M2, 0/29(0%) of M3, 2/9(22.2%) of M4, 18/39(46.2%) of M5 and 1/5(20.0%) of M6. NPM1 gene mutations were more prevalent in patients with a normal karyotype (37 of 90; 41.1%), when compared with patients with karyotypic abnormalities (7 of 66; 10.6%; P

After stimulated by GM-CSF, Akt and IkB kinase (IKK) can be phosphorylated by activated PIK3-kinase. Activated IKK phosphorylate IkB, making NF-kB released and translocate to nucleus. Granulocyte-macrophage colony-stimulating factor (GM-CSF) restrain TF-1 cell mitochondria pathway apoptosis through PI3K pathway, so we asked if NF-κB is the only way in this process. All group were plused TF-1 cells 2.5X105/250μl, group A plused hGM-CSF (100ng/ml ) 50μl; group B plused hGM-CSF (100ng/ml) 50 μl and SN50 (50 μmol/L) 20μl; group C plused hGM-CSF 50μl and SN50 200μl. After cultured for 48 hours, all groups were dyed with Annexin and PI, analyzed in FCM. At the same time all groups were dyed by diluted MitoCapture to analyze mitochondria membrane electrical potential. Drop experimental group B 10μl on slips, so as group C, then observe it under Fluorescence microscopes FL1 channel. Group A cells that have normal mitochondria membrane electrical potential can form poly-MitoCapture in it, and inspired red fluorescence. Group C cells that have monomer MitoCapture was inspired green fluorescence. The red fluorescence intensity of control, group A, B, C means is 368.93, 1137.59, 1033.61 and 618.37 respectively. Group A and B retained a statistically significant difference with control (P

CXCR4, receptor of the chemokine stromal derived factor-1 (SDF-1), is expressed on CD34+ cells, and has been implicated in the process of CD34+ cell migration and homing. We studied the mobilization of CD34/CXCR4 cells and the plasma levels of SDF-1 and sgp130 in 22 patients, 11 acute Leukemia, 5 non-Hodgkin’s lymphoma, and 6 multiply myloma respectably, receiving cyclophosphamide (Cy) and plus G-CSF, or Mitoxantrone(Mit) and Cytarabine plus G-CSF for peripheral blood stem cell (PBSC) mobilization and autotransplantation. We observed lower plasma levels of SDF-1 in PBSCs compared with premobilized PB and bone marrow samples. The average levels of SDF-1 and sgp130 were 24.67±5.58ng/ml and 106.2±16.4ng/ml respectively while the level of SDF-1 as well as sgp130 decreased to 14659±2.11ng/ml(p

Background. It’s not surprising that the traditional Chinese herbal remedy is yielding useful drug leads. The mixture composed of thirteen Chinese medicinal plants---jieduweikang(JDWK) pill including Qingdai, Qinghao, Huangchi, Baihuasheshecao, Banzhilian and so on has been shown the clinical antitumor effects in China. We here report the mechanism investigation of JDWK pill cytotoxicity to leukemia cell line HL-60. Methods By using the method of serum pharmacology, the serum containing the JDWK pill was collected from rats. The MTT and tumor colony formation were used to determine the effects of JDWK pill on proliferation of HL-60 cells. NBT reduction, DNA electrophoresis, flow cytometry and RT-PCR were used to analyze the differentiation and apoptosis of HL-60 cells after treating with JDWK pill. Elisa was used to assay the secretion of VEGF from HL-60 cells. Results The liquid growth and colony formation of HL-60 cells were evidently inhibited by the serum containing JDWK pill and the cells were shown characteristic DNA “ladder” on agarose gel electrophoresis and more than 20% apoptotic cells in DNA histogram as well as the downregulation of bcl-2 gene related to apoptosis in a concentration-dependent manner, compared with the control. NBT reduction assay indicated that JDWK pill with low concentration may induce myeloid terminal differentiation of HL-60 cells and the level of VEGF was obviously decresed. Conclusion The JDWK pill can exerts the its anti-proliferative effect on the leukemic HL-60 cells through inducing apoptosis relating to the downregulation of Bcl-2 gene and anti-angiogenic effects with higher concentration and inducing myeloid differentiation with lower concentration.

Unrelated donor HSCT and haploidentical related donor HSCT have been evaluated as alternative transplant options for the approximately 70% of patients without an HLA-identical sibling donor. To compare the clinical outcomes between Non-TCD unrelated donor HSCT and Non-TCD haploidentical HSCT, we studied 55 patients with high-risk or advanced leukemia who underwent Non-TCD HSCT from unrelated or haploidentical related donors from June 2001 to May 2006. Group A including 25 patients received HLA-matched unrelated donor HSCT, group B, including the other 30 patients, received HLA-haploidentical family donor HSCT. 20 recipient/donor pairs were allele matched and 5 pairs were 1–2 alleles disparity mismatched in the group A, HLA-haploidentical family donors in the group B included mother (22 cases), sibling(5 cases) and offspring (3 cases). Patients with myeloid leukemia were conditioned with the regimen consisting of Simustine (MeCCNU) 250mg/m2×1day, Ara-c 4g/ m2×2days, busulfan (Bu) 4mg/kg×3days, and cyclophosphamide (Cy)1.8g/m2×2days, patients with lymphoblastic leukemia were conditioned with the regimen consisting of MeCCNU 250mg/m2×1day, total-body irradition(TBI) 8Gy×1day, Ara-c 4g/ m2×2days, and Cy 1.8g/m2×2days. 15 patients received Non-TCD bone marrow transplantation (BMT), and 10 patients received Non-TCD peripheral blood stem cell transplantation (PBSCT) in the group A. 17 patients received G-CSF-primed bone marrow grafts that had not been depleted ex vivo of T cells, and 13 patients received G-CSF-primed bone marrow grafts plus G-CSF-mobilized peripheral blood stem cell without ex vivo T cell depletion in the group B. Prophylaxis against GVHD was performed with cyclosporine (CSP), short-term methotrexate (MTX), and mycophenolate mofetil (MMF), some patients received the combination of CSP, MTX and MMF plus antithymocyte globulin (ATG). When GVHD developed, methylprednisolone(MP) was given at first, if the response was poor, anti-CD25 monoclonal antibody was given to the patients as quickiy as possible and CSP was replaced with tacrolimus. All patients of the group A and 29 patients of the group B were engrafted successfully. Acute GVHD grades III-IVoccurred in 10 and 11 patients in the group A and B, respectively(the cumulaitive incidence 40% vs 37.9%, P>0.05). 2 patients relapsed in each group (the actuarial probilities of relapse 8% vs 6%, P>0.05). The 2-year probabilities of event-free survival(EFS) for the group A and B were (58.7±5.9)% and (42.2±2.0)%, respectively (P>0.05). 10 patients of the group A and 17 of the group B died of transplantation- related disease. The primary causes of death included severe acute GVHD and pulmonary infection. These results suggested that both Non-TCD unrelated donor HSCT and Non-TCD haploidentical related donor HSCT are effective treatments for patients with refractory or high-risk hematologic malignancies, the high transplantation related mortality due to GVHD and infection is still a major challenge.

Severe neurotoxicity following treatment with itraconazole and vincristine has been reported in 25 patients. Here we presented two adult acute lymphoblastic leukemia (ALL) patients developed neurotoxicity after treated with itraconazole and another vinca alkaloids, vindesine. Case 1: A 20-year-old male was diagnosed as having ALL and achieved complete remission (CR) after induction chemotherapy with DVP. He was treated with Hyper-CVAD for consolidation. Because he has a history of pulmonary invasive fungal infection, antifungal prophylaxis with itraconazole solution was started. Nine days after itraconazole was started, some symptom and signs of paralytic ileus were presented. He was diagnosed to have paralytic ileus secondary to neurotoxicity caused by itraconazole and vindesine. After the itraconazole was stopped, his paralytic ileus was improved rapidly. Case 2: A 37-year-old female was diagnosed as having ALL. A diagnosis of B-cell/T-cell biphenotypic acute leukemia was established based on the results of flow cytometry analysis. She was commenced on induction chemotherapy as for ALL (dexamethasone, Idarubicin, and vindesine). The patient presented as pulmonary infection with fever, cough, and expectoration in day 9. Emperical antifungal treatment with itraconazole injection was initiated. Five days after itraconazole injection was started, some symptom of neurotoxicity including limbs anesthesia, and paralytic ileus were presented. After the itraconazole and vindesine were discontinued, these symptoms were resolved. Given the interaction of itraconazole with vinca alkaloids leading to severe and even potentially fatal toxicities, the combination use of these drugs should be avoided.

We analyzed donor chimerism(DC) in unfractionated(UF) and CD3+ cells from a secondary acute myeloid leukemia patient who relapsed after reduced-intensity allogeneic hematopoitic stem cell transplantation(RIST) and had undergone donor lymphocyte infusion(DLI). Sequential and quantitative chimerism was performed by multiplex PCR amplification of STR markers (STR-PCR). The peripheral blood(PB) every 3 days and bone marrow every week were collected for chimerism analysis. The patient presented hematological relapse 26 months after the first RIST. There were 31.5% blast cell in the bone marrow smear and presented additional abnormal chromosome [44,XY,-7,dic(16,17)]. While UF donor chimerism from PB and CD3+ fraction donor chimerism from PB and BM were always full donor chimerism (range, 96.4%~98.1%). However UF chimerism from BM was 64.9% at the same time point. Afterwards the patient received 4 times of dose-escalated donor lymphocytes infusion (DLI) with CD3+ cell from 1*107/kg to 1.0*108/kg. Unfortunately the patient hasn’t response to this therapy. The blast cell in BM smear augment from 31.5% to 51.5%. UF chimerism from BM ranged from 55.2 to 46.5%. But UF DC from PB and CD3+ fraction DC from PB and BM ranged from 95.5% to 98%. In conclusion, Our result suggest that in this AML patient the donor T-cell chimerism and unfractionated chimerism in PB can not predict relapse and response to donor lymphocyte infusion. and the reason is not clear.

Objective To explore the efficiency and safety of Low-dose Cytosine arabinoside and aclarubicin in combination with granulocyte colony-stimulating factor (CAG regimen) for patients with refractory and relapsed adult acute lymphocytic leukemia (ALL). Methods We treated 10 patients of refractory and relapsed adult ALL with CAG regimen (median age 28 years, range 17–58, male 5, female 5). The regimen consisted of low-dose cytosine arabinoside (10mg/m2 q12h, day1to14), aclarubicin (10mg/day, day1to8), and granulocyte colony-stimulating factor (G-CSF) (200 μg/m2/day). Result 8/10 patients (80%) achieved complete remission (CR) after one course, including 4 out of 5 refractory and 4 out of 5 relapsed patients. 1 patient who relapsed after the first course, then achieved CR again after the second course. 6 of the 8 complete remission received consolidation therapy with the CAG regimen or other regimens. Toxic effects were very rare and mainly consisted of neutropenia and thrombocytopenia due to myelosuppression; 8 of all patients had neutropenia or thrombocytopenia that exceeded WHO grade II. However, severe non-hematologic toxicity included nausea and vomiting (WHO grade ≥III) was characteristically rare. Conclusion CAG regimen is an effective and safety in the treatment of refractory and relapsed ALL.

Objective The exact reason of aplastic anemia is still unknown. Recently more and more researchers realized that immune factors play an important role in aplastic anemia. In this study, we detected the expression of CD28 on different subgroups of T cells in patients with aplastic anemia and furthermore studied the effect of cyclosporine A to CD28 and FLIP on these cells. Method 21 patients and 15 healthy controls were investigated. The Expression of CD4CD28 and CD8CD28 on patients’ T cells was detected with FCM; patients’ CD8+T cells were purified with Mini MACR system, then the expression of FLIP, caspase-8 in CD8+T cells were analyzed by RT-PCR; we also detected the expression of CD28, FLIP and caspase-8 in patients’ CD8+T cells after incubation with cyclosporine A. Result: The percentage of CD8+CD28−T cells in the patients is significantly higher than that of normal controls (30.45±5.26% versus 19.32±4.72%, p Conclusion: The increased expression of FLIP in patients’ CD8+T cells is related to the increased percentage of CD8+CD28−T cells in patients’ peripheral blood. Cyclosporine A can down regulate the FLIP expression and correct the abnormal percentage of CD8+CD28−T cells in patients with aplastic anemia.

Background Hemorrhagic cystitis (HC) is a frequent and sometimes life-threatening complication after hematopoietic stem cell transplantation (HSCT). Many therapeutic approaches have been performed to control HC, but effective treatment for severe HC is still limited. This study investigated the role of selective embolization of the vesical arteries in patients with severe HC following hematopoietic stem cell transplantation (HSCT). Methods Eight patients with late onset HC receiving HSCT were enrolled in the study, All patients underwent allogeneic transplantation and cyclophosphamide for preconditioning, including grade 3 or 4 HC, no improvement to routine management. A retrograde arterial catheterization of the femoral artery by seldinger’s technique was achieved in order to perform digital angiography of the aorto-iliac sector. Selective catheterization of the hypogastric arteries with a standard angiographic catheter was performed to localize the vesical vessels and confirm their bleeding site. Embolization of these arteries was carried out with gelatin sponge. Results Eight patients with severe HC were treated with selective embolization of the vesical arteries. Five patients achieved complete response, with hematuria and symptoms ceased after 1–3 days of treatment. Two patients achieved partial response, with diminution of hematuria or presence of microscopic hematuria with no symptoms. One patient obtained no response to treatment. Conclusion We investigated eight patients, among more than 280 patients treated in our hospital by allogeneic hematopoietic stem cell transplantation, in which HC was severe enough to require embolization. Our study showed that selective embolization of the vesical arteries is an effective treatment option in patients with severe HC following HSCT.

objective: To investigate the efficacy of unrelated donor hematopoietic stem cell transplantation(URD-HSCT) for high-risk leukemia. Methods: From 2001 to March, 2006, 24 patients of high-risk leukemia underwent HSCT with unrelated donor. Patients between 6 and 49 years of age(median age 21.5 years) There were 16 unrelated donor’s grafts came from Buddhist Tzu Chi Stem Cells center in Taiwan, and 8 unrelated grafts came from Red Cross Society of China Hematopoietic Stem Cell Marrow Donor Program Administration Center. Fifteen patients received bone marrow transplantation and another 9 patients received peripheral blood stem cell transplantation. Eighteen patients were fully matched with their donors for HLA loci A and B as well as for HLA-DRB1. Four of 24 patients had 1 molecular loci mismatch and 2 of 24 patients had 2 molecular locus mismatch. The conditioning regimen consisted of modified BU/CY or modified total body irradiation(TBI) plus CY. Eleven patients received cyclosporine (CSA), short-term methotrexate(MTX) and mycophenolate mofetil (MMF) as the regimen of prophylaxis of GVHD. Ten patients received CSA, MTX and MMF plus ATG/ALG for GVHD prophylaxis. The combination of CSA, MTX, MMF,ATG/ALG and CD25 monoclonal antibody for preventing GVHD in 3 patients. Results: The incidence and severity of regimen-related toxicity were mild. The median number of MNC and CD34 positive cell was 4.18×108/kg and 7.75×106/kg respectively. The median time of the engraftment of neutrophil and platelet was 13 days and 19 days posttransplant respectively. The incidence of infection posttransplant was 70%(17/24). The complicated pneumonitis occurred frequently (13/17, 76%), The incidence of grade I-II and grade III-IV acute GVHD was 33% and 41% respectively. Limited chronic GVHD occurred in 25% patients and extensive cGVHD presented in 31% patients. Median follow up was 16 (range 1~57) months after transplantation. 75% patients achieved full donor chimerism(FDC) detected by STR-PCR and FISH. While 25% patients presented mixed chimerism(MC) and 2 of them converted to unstable MC. Decreasing values of donor chimerism were detected prior to the relapse of disease. Furthermore the treatment related mortality(TRM) was 33%(8/24), The incidence of relapse was 8%. Until now 14 patients are still alive. The median overall survival time were 26 months and 5-years expected survival was 47.7%. Conclusion: URD-HSCT can be an effective and curable approach for leukemia with higher incidence of GVHD and infection. The treatment for the severe and deadly GVHD and pulmonary infection are still major problems need to be resolved in the future.

Objective In order to compare the functions of ADP in platelet aggregation and platelet membrane surface glycoproteins expression after thrombin receptors activation, then to investigate the role of ADP in thrombin signal transmission. Methods Peptide SFLLRN (PAR1-AP, TRAP) and AYPGKF(PAR4-AP)were used for stimulating platelet at different time points (0~30minute) in 12 normal subjects, then the alterations of platelet aggregation and GPIbα, P-selectin were analyzed in the involvement of ADP inhibitor, ApyraseVII, which was observed by aggregometer and flowcytometry respectively. Results Either PAR1 or PAR4 peptide can lead to platelet activation, inducing absolute platelet aggregation, together with a persistent increase of P-selectin and reversible interlisation of GPIb. Platelet aggregation was partly inhibited by ApyraseVII and showed a reversible curve upon PAR1 activation, and no change upon PAR4 activation. In addition, ApyraseVII accelerated the return of GPIb to platelet surface in PAR1 pathway, produced weak effect on GPIbα internalisation, … ltogether with little effect on GPIb alteration in PAR4 pathway. Meanwhile, no change of P-selectin was obtained in the involvement of Apyrase for both peptides. Conclusion All the results confirm a critical role of ADP in thrombin signal transmission through the process of PAR1 pathway. ADP induces platelet aggregation and slows down the restoration of GPIb to platelet surface.

The clinical and experimental results indicated that the T cell immune reconstitution was slow after hematopoietic stem cell transplantation. This study was To analyze the molecular characteristics of T lymphocytic clones during immune reconstitution in leukemia patients after allogeneic hematopoietic stem cell transplantation(Allo-HCST)by investigating complementarity determining region (CDR3) repertoires of T cell receptor β chain variable region (TCRVB) and made it possible to understand the relationship between the express of TCRVB and immune reconstitution. Reverse transcriptase-polymerase chain reaction(RT-PCR) amplified 24 subfamily genes of TCRVB from peripheral blood lymphocytes (PBLs) of twenty-four patients with leukemia underwent three kinds of Allo-HSCT(haploidentical bone marrow transplantation, matched sibling bone marrow transplantation and matched-unrelated PBSCT), five normal donors as control. The PCR products were further analyzed by genescane to evaluate the clonality of VB subfamily. The monoclonal bands which associated with GVHD and CMV infection were obtained through denaturation polyacrylamide gel electrophoresis and sequenced. Compared the sequences of TCRVB CDR3 gene repertoire with other sequences associated with GVHD or CMV infection which had been reported. +2~+19months after transplantation, for nine patients among them which underwent haploidentical bone marrow transplantation, there were 6~14 VB subfamilies expressed with 33% 15 VB subfamilies expressed, which 45%of them were polyclones. In the 5 patients of matched-unrelated PBSCT, there were 10~15 VB families expressed, with 45% the polyclone expression. For ten patients underwent matched sibling bone marrow transplantation, 10~16 VB subfamilies expressed and more than 48% were polyclones. Monoclones and oligoclones existed in 24 VB subfamilies, no common monoclone VB subfamilies expressed. According to the usage of VB family and polyclonal expression, Immune reconstitution in patients which underwent haploidentical BMT was later than that of other two groups (p0.05). After Allo-HSCT, two patients detected TCRVB in +2m and +3m and found that it has tendency of adding to use VB subfamilies and increasing to express CDR3 polymorphism. Using biology-information tool, compared 23 TCRVB CDR3 molecules which related to GVHD and CMV infection and found that, different cases in the same VB subfamilies may share similarity in amino acid motif, while in different VB subfamilies none of clones appeared to share the same amino acid motif. In 1.5 years after AHSCT, the usage of TCRVB subfamilies still restricted. T-cell immune reconstitution in patients which underwent haploidentical BMT was later than that of other two groups. TCRVB CDR3 molecules which related to GVHD and CMV infection showed that different cases in the same VB subfamilies may share similarity in amino acid motif, while in different VB subfamilies none of clones appeared to share the same amino acid motif.

Haploidentical transplantation (H-SCT) has been an alternative allogenetic transplant option for the high risk lukemia patients who do not have an HLA-identical related donor. The high dose of stem cell infusion and intensive GVHD prophylaxis treatment were applied in H-SCT to overcome the haploidentical major histo compatibility barrier and to achieve engraftment. We speculated that the dynamics of donor chimerism are different from conventional myeloablative allogeneic bone marrow transplantation(allo-BMT). Methods Quantitative analysis of chimerism were performed by multiplex PCR amplification of STR markers (STR-PCR) in unfractionated nucleated cells from bone marrow or whole blood of 30 high risk leukemia patients who received H-SCT(14 females,16 males; median age 25 years). Eighteen of 30 patients received G-CSF primed bone marrow transplantation and another 12 patients received G-CSF primed bone marrow and peripheral blood stem cell transplantation. The conditioning regimen consisted of modified BU/CY or modified total body irradiation(TBI) plus CY. One patient received cyclosporine (CSA), short-term methotrexate(MTX) and mycophenolate mofetil (MMF) as the regimen of prophylaxis of GVHD. Twenty-four patients received CSA, MTX and MMF plus ATG/ALG for GVHD prophylaxis. The combination of CSA, MTX, MMF, ATG/ALG and CD25 monoclonal antibody for preventing GVHD in 5 patients. Results: All of the patients except one showed initial donor engraftment and just one patient presented primary graft failure. The early chimerism and its kinetics after transplantation showed that the donor chimerism became dominant (DC 84.1% >60%) by day +3, which preceded the detection of hematologic engraftment by a median of 9 days. On day +7, donor cells of majority patients had achieved stable engraftment. The median chimerism was 92.4%;The probability of achieving full donor chimerism (FDC) in PB was 56.3% on day +14, 61.5% on day +28;Comparing with the group of standard allo-BMT, median quantity of CD34+ cells and CD3+ T lymphocyte cells of H-SCT group were 2 and 3 times higher. Furthermore GVHD prophylaxis was more intensive with ATG/ALG and MMF in H-SCT group. The median percentage of donor chimerism of H-SCT was significantly higher than that of BMT on the day of +7d, +14d and +1month. Meanwhile there were difference in the rates of FDC among H-SCT and BMT(61.5% VS 20%, p90%), and another 10 patients achieved stable FDC(DC ≥95%). Conclusion: In the early stage after transplantation, the engraftment of donor cells of H-SCT was more rapidly and completely than that of conventional BMT owing to the high quantity of donor CD34+ cells, T lymphocyte cells and the more intensive immunosuppressive therapy.

Nowadays it is widely used as glucocorticoids, ortho-oxybenzoic acid, immunosuppressive agents and biological agents for Crohn’s Disease. But all these agents can only control the symptoms rather than curing the diseases. About 70% patients need surgery in the course. 5 patents of Crohn’s Diseases have received AHSCT in our department since 2004. These patients, including 3 male and 2 female, with a mean age of 32 (range, 23~46), were final confirmed by enteroscope and histology. The mean CDAI of these patients was 213 points (range, 170~256), and the mean interval between invasion and transplantation was 63 months (range, 27~132 months). All the patients had mild or severe diarrhea and abdominal pain, one of them have a 6cm×7cm lump touchable in the abdominal region, while one of them complicated with vesicorectal fistula. These patients were mainly treated with glucocorticoids, SASP, cyclophosphamide (CTX), et al, which didn’t control the condition. Mobilization program was CTX 2g/m2 for 2 days and G-CSF 5~10 μg/kg/d on the 5th day of medication. Leukapheresis was initiated when WBC>5.0×109/L. The collections of CD34+ cells from two cases were over 4×106/kg, and ClinMACs cell separation system was used to purify the CD34+ cells and to deplete T cells. Two collections of CD34+ cells were 1.3×106/kg and 2.8×106/kg, respectively, and then were kept in −80°. And the other case failed in the collection, and then was treated with bone marrow transplantation. The conditioning programs were MC (CTX 60mg/kg -2, -1d;melphalan 140mg/m2 -2d) for one case, and CTX 50mg/kg×4d for the other 4 cases. 2 of the 3 patients without in vitro purification of CD34+ cells to deplete T cells were treated with rabbit antithymocyte globulin 5 mg/kg on day-2 and day -1 for T cell depletion. The mean CD34+ cells infused was 1.4×106/kg(range, 1.3×106/kg~2.8×106/kg). Neutrophile granulocyteand>0.5×109/L occurred at day +10~+12, while platelet>2.0×109/L occurred at day +9~+11. Fever occurred in all patients in the granulocytopenia phase, and lasted from 1 to 3 weeks. One of the patients complicated with ESBL+ ichoremia, and was cured with antibiotics. After the transplantation, 2 patients were treated with prednisone 10mg/d as maintenance therapy, and the other 3 stopped using antidiarrheal and glucocorticoids. 4 patients got free of abdominal pain and diarrhea in the following 2~3 months, and the other did in 1 year. The average body weight of the patients increased 11.2 kg (range, 3~20kg) 3 months after transplantation. The mean follow-up was 16 months. 2 patients have been maintaining remission until now. The lump in the abdomen of one of these 2 patient decreased from 6×7cm to 5×5cm, which was resected 19 months after transplantation. And this patient is still in remission now. 3 patients relapsed after 4~8 months of remission. One of them relapsed 5months after transplantation and died complicated with peritonitis in 7 months. One patient relapsed in 8 months with abdominal discomfort, mild diarrhea, and decreasing of body weight. The other complicated with rectovesical fistula showed closure of syrinx by barium enema, and suffered abdominal discomfort, mild diarrhea, and decreasing of body weight too 4 months after transplantation, then relapsed with rectovesical fistula 8 months after transplantation. HSCT for Crohn’s disease is safe and effective. However, it has a comparative higher relapse rate.

The clinical and experimental results indicated that the T cell immune reconstitution was slow after haploidentical hematopoietic stem cell transplantation and existed high rate of GVHD and infection. This study was to analyze the molecular characteristics of T cell clones during immune reconstitution in leukemia patients after haploidentical bone marrow transplantation (H-BMT). Reverse transcriptase-polymerase chain reaction (RT-PCR) amplified 24 subfamily genes of TCRVB from peripheral blood lymphocytes of nine leukemia patients underwent H-BMT, five normal donors as control. The PCR products were further analyzed by genescane to evaluate the clonality of VB subfamily and characteristics of CDR3 and usage rate in VB subfamily. The monoclonal bands which associated with GVHD obtained and sequenced. Compared the sequences of TCRVB CDR3 gene repertoire with other sequences associated with GVHD had been reported before. In +10~+19m, the usage of TCRVB subfamilies still restricted. It can detected that some VB subfamilies were missing, others expanded in monoclonal or oligoclonal. 4 patients in stable condition showed that 9~14 VB subfamilies expressed and more than 50 percent were polyclone. In other 5 patients which have GVHD or CMV-pp65(+), the usage of TCRVB decreased obviously(p

Targeted therapy with the Bcr-Abl tyrosine kinase inhibitor Imatinib Mesylate can induce high response rates in chronic myelogenous leukemia (CML) patients. However, evidences that discontinuation of imatinib mesylate inevitably exerts rapid loss of response and some patients with imatinib mesylate monotherapy virtually occur potential relapse suggest that the modification of treatment strategy is critical. We have previously demonstrated that the combination of trisenox (arsenic trioxide) with the tyrosine kinase inhibitor imatinib mesylate or genistein appears to induce markedly more cell apoptosis than imatinib mesylate alone through downregulating Bcl-XL and Bcr-Abl in Bcr-Abl gene transfected HL-60 cells. We here report the preliminary results of a pilot study comparing imatinib mesylate plus trisenox with imatinib mesylate alone for frontline treatment of CML patients. Up to date 56 patients were enrolled in this clinical trail. All patients (required to be 18 years or older with Bcr-Abl positive CML in chronic phase within three month of diagnosis) were divided into two groups, i.e., monotherapy group and combined therapy group. 42 patients entering monotherapy received imatinib mesylate 400mg daily and 14 patients entering combined therapy imatinib mesylate 400mg daily plus trisenox 10mg daily for one week and then twice a week. We compared treatment results of both groups including complete hematologic response(CHR), major/complete cytogenetic response(MCR/CCR),--defined as 1–35% Ph+ and 0% Ph+ metaphases respectively and major/complete molecular response(MMR/CMR),--defined as ≥ 3 log reduction and negative expression in Bcr-Abl transcript numbers assayed by RQ-PCR respectively. The median follow-up for patients in both groups lasted 36 months. In the combined therapy group, the median age of patients is 42 years old (range, 22–61), the CHR, MCR, CCR, MMR and CMR is 92.8%, 64.3%, 42.9%, 35.7% and 21.4%, respectively. While in the imatinib mesylate monotherapy group, the median age of patients is 46 years old (range, 23–65), the CHR, MCR, CCR, MMR and CMR is 85.7%, 59.5%, 40.5%, 33.3% and 19%, respectively. Although combination therapy of imatinib with trisenox is not significantly superior to imatinib mesylate monotherapy in efficacy, no resistance case happens in the combination therapy group, in the imatinib mesylate monotherapy group, imatinib resistance occurs in 4 patients. In addition, the safety and tolerability of a combination of imatinib mesylate and trisenox is good. This result indicates that the combination of imatinib and trisenox to treat chronic myeloid leukemia may be promising in avoiding the occur of imatinib resistance.

Objective Hemorrhagic cystitis(HC) is a common complication of allogeneic stem cell transplantation occurring in 7–52% of transplant recipients. The severity of HC can range from mild hematuria to life-threatening bleeding with urinary tract obstruction or renal insufficiency. Severe HC is often refractory to the conventional therapy. Currrent treatment modalities for severe HC include selective embolization of the hypogastric arteries and hyperbaric oxygen. We studied 6 patients with refractory severe HC to investigate the therapeutic value of embolization in severe HC following allogeneic stem cell transplantation. Methods 6 patients underwent allogeneic stem cell transplantation developed severe HC with a median onset time of 33 d after transplatation. All these patients had been resistant to conservative managements and received the treatment of selective embolization of hypogastric arteries. Results 8 times of embolizations were performed for 6 patients. The HC resolved in 4 patients and the HC improved in 1 patient, yielding a response rate of 83%. One patient didn’t respond to this measure. For the patients with response, macroscopic hematuria disappeared 7 to 10 days after the treatments and microscopic hematuria vanished 20 to 30 days after the intervention therapy. Both procedures were tolerated well and no severe adverse effects were observed. Conclusion It suggest that selective embolization of hypogastric arteries seems to be a safe and effective treatment for severe HC. This measure can be proposed for patients undergoing refractory and persistent severe HC.

We aimed to evaluate the effects of arsenic trioxide (ATO, Trisenox) on STI571 (Gleevec, imatinib mesylate)-induced apoptosis of a chronic myelogenous leukemia (CML) cell line, K562 cells. Cell prolifration and colony-forming assays were performed to determine the cytotoxicity of ATO alone and in combination with STI571. Apoptosis was analyzed by morphological changes, apoptosis rate and cell cycles. An Elisa assay was used to detect the levels of cytosolic cytochrome c (cyt c) and caspase-3 in K562 cells exposed to ATO and STI571 at graded concentrations. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assay the transcriptional levels of Bcl-XL and Bcr-Abl genes in K562 cells. Results showed that both the colony-forming assay and cell proliferation assay demonstrated additive to synergistic effects of ATO on STI571-induced apoptosis in K562 cells, a Bcr-Abl positive cell line. Caspase-3 was activated during apoptosis and there was an increase in cytosolic accumulation of cytochrome c. Treatment of K562 cells with STI571 alone led to down-regulation of transcriptional levels of Bcl-XL at 12 hours and Bcr-Abl at 96 hours after drug administration. Treatment with ATO alone only led to reduce the mRNA levels of Bcl-XL, but not Bcr-Abl. Combined treatment with ATO and STI571 down regulated the transcripts of Bcl-XL at 12 hours and Bcr-Abl 72 hours after drug administration. We conclude that ATO enhanced cytotoxic and proapoptotic actions of STI571 could be mediated by the down-regulation of Bcr-Abl and Bcl-XL genes in K562 cells. Therefore ATO in combination with STI571 could be a promising therapy for CML.

Objective: To explore the efficiency and side-effects of basiliximab in the treatment of steroid-refractory acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Patients and Methods: Basiliximab, a chimeric interleukin-2 receptor antagonist, was evaluated in twenty-eight patients with steroid-refractory acute graft-versus-host disease after Allo-HSCT. Patients were transplanted from a related (n=9) or unrelated (n=6) HLA- identical donor or haplo-identical donor(n=13) because of chronic myelogenous leukemia (chronic phase n=11, accelerate phase n=2), acute myeloid leukemia (n=8), high risk acute lymphocytic leukemia (n=6), high risk non-Hodgkin’s lymphoma (n=1). Basiliximab was given at a dose of 20mg on the day after steroid-refractory acute GVHD had developed, and was repeated on day 4 in all these cases. The efficiency and side-effects of basiliximab were observed at the same time. Basiliximab was repeated again 4 weeks later in some cases of having response. Result: None had infusion-associated or other side-effects after the treatment with basiliximab. Twenty-two of 28 patients (78.57%) responded to basiliximab, 15/28(53.57%) had a complete response (CR) of acute GVHD and 7/28 (25.00%) had a partial response (PR). Six of 28 patients (21.43%) did not respond. Chronic GVHD developed in 15/24 evaluable patients. Only 9/24 evaluable patients have no chronic GVHD and all had a complete response before. Conclusion: Basiliximab is safe and effective in the treatment of steroid-refractory acute graft-versus-host disease (GVHD) after allogeneic stem cell transplantation(Allo-SCT).

Non-T-cell-depleted maternal HLA-haploidentical hematopoietic stem cell transplantations(SCT) have been reported feasible based on the hypothesis that long-term fetomaternal microchimerism(FMC) is linked with hyporesponsiveness to inherited paternal antigens(IPAS),but the hypothesis is still in debate due to lack of direct evidence. To determine whether the existence of such microchimerism affect the outcome of maternal HLA-haploidentical SCT, we investigated the outcomes of 18 patients (median age 19) with hematologic malignancies (ALL 8, AML 6, CML 3, NHL 1) who underwent haploidentical SCT from mothers with the same regime from July 2003 to January 2005. Donor was treated with granulocyte colony stimulating factor subcutaneously at a dose of 300μg per day for 5 days before bone marrow harvesting. Leukaphereses were performed using a continuous-flow blood cell separator if CD34+ cells in the bone marrow harvest were less than 4.0×106 cells/kg. Patients with CML or AML in CR received 7.5Gy TBI based standard-intensity regimen combined with cyclophosphamide plus cytarabine. The remaining received non-TBI standard-intensity regimen consisting of cytarabine, busulfan and cyclophosphamide. Antithymocyte globulin(ATG) at a dosage of 2.5mg/kg/day on days −5 to −3 was given as an additional immunosuppressive measure in all patients. To prevent GVHD, patients also received cyclosporin at a dose of 3mg/kg/day as a continuous infusion from day −10, 30mg/kg/day mycophenolate mofetil starting on day −10, and short-term methotrexate administered on days 1, 3, 6, 11 at doses of 15, 10, 10, and 10mg/m2. Among all these patients, microchimerism were confirmed with nested polymerase chain reaction using sequence-specific primers(PCR-SSP) typing for HLA in 10 donor-recipient pairs. In the 2 categories as to whether the microchimeric status of the donor is positive or negative, they had similar characteristics in the background factors such as recipient sex, type of conditioning regime, stem cell source, and disparity of HLA except recipients of positive group had more CML. Engraftment was obtained in all patients. As of august 1, 2005, 5 patients died (1 of relapse, 4 of complication), other 13 patients were alive and free of disease. The 2-year overall survival for the whole cohort was 58%. Although there was a survival advantage for pairs with microchimerism over pairs without it, the influence on outcome was not statistically significant (P=0.9, log-rank test). The cumulative incidences of grade Ò/Ô acute graft-versus-host disease (GVHD) were 39% (95%CI, 17%–64%). Also no significant difference was observed between the two groups. Extensive chronic GVHD developed in 5 of 13 patients who could be evaluated. These results indicate maternal HLA-haploidentical hematopoietic SCT is a acceptable option for patients who lack immediate access to a conventional stem cell source, but the presence of fetomaternal microchimerism may not correlate to the existence of immunological tolerance between mother and offspring.

Objective: To study the distribution of CMV glycoprotein B (gB) genotypes in the patients with CMV infection after allo-HSCT Methods: Seventy-four patients undergoing allo-HSCT were enrolled in this study. After allo-HSCT, all the patients were detected for CMV gB DNA in peripheral blood by haplo-nested PCR methods;The CMV gB DNA positive samples were screened, which were sufficient for enzyme digestion. Glycoprotein B typing was performed through digesting PCR products with RsaIand HinfI;The sequences were mensurated with ABI PRISM 377 XL DNA Sequencer. Results: The PCR products of thirty-eight patients were digested with enzyme. the distribution of gB genotypes was as follows:I(gB1) 19/38 (50.0%), II(gB2) 3/38 (7.89%),III(gB3) 14/38 (36.84%), and unclassification 2/38 (5.27%). Conclusion: The distribution of gB genotypes was different in the patients with CMV infection after allo-HSCT; There may be exiting new genotype.

Objectives To explore the subsets and function of T helper (Th) in bone marrow of the patients with immune related pancytopenia(IRP). Methods The CD4+ cells producing IFN-γ or IL-4 in cytoplasm were defined as Th1 or Th2 cells respectively. All these cells in bone marrow were measured from 16 normal controls, 25 untreated IRP patients; The mRNA expressions of IL-4, IL-10, IFN-γ and IL-2 genes in unstimulated bone marrow mononuclear cells(BMMNC)from 25 untreated IRP patients, 10 normal controls were measured by reverse transcription polymerase chain reaction (RT-PCR). Results The percentage of Th1 cells, Th2 cells and ratio of Th1/Th2 in bone marrow of normal controls was: 0.42%, 0.24%, 1.57 respectively, and the percentage of Th1 cells in untreated patients with IRP was 0.58%, which was not markedly different from the that of normal controls(t=0.903, P>0.05). But the percentage of Th2 cells of the patients with IRP was significantly higher than that of normal controls(t=4.673, P Conclusions The percentage of Th2 cells increased in the patients with IRP, and the balance of Th1/Th2 shifted to Th2. And the expression of Th2 type cytokines was more frequent in IRP. The imbalance of subtypes of Th lymphocytes and hyperfunction of Th2 lymphocytes might play important roles in the pathogenic mechanism of IRP, which lead to more B lymphocytes and producing autoantibodies consistenly.

Objective: To compare the dendritic cells reconstitution after different allogeneic hematopoietic stem cell transplantation in early time Methods: From June 2004 to March 2005, Twenty-eight patients undergoing allo-HSCT were enrolled in this study. There were 16 patients who undergone normal HSCT, 8 patients who undergone Haploidentical HSCT and 4 patients who undergone Nonmyeloablative HSCT. Three-colour flow cytometry was applied to study the alteration of the percentage and number in circulating peripheral blood dendritic cells subsets on day 14,day 30,day 60 after transplantation among three distinct type HSCT. Results: The dendritic cells subsets number of myeloablative HSCT patients were very low. No difference was observed in the kinetics of DC1 (Lin−HLA-DR+CD11c+)and DC2 (Lin−HLA-DR+CD123+) reconstitution between the normal HSCT group and Haploidentical HSCT group patients(p Conclusion: The early reconstitution of dendritic cells in Nonmyeloablative HSCT patients is earlier than the patients who undergone myeloablative HSCT. The early reconstitution of dendritic cells in normal HSCT and Haploidentical HSCT patients were later than others.

Objective To explore whether the dynamics and outcome of chimerism are different between allogenic bone marrow transplantation (allo-BMT) and nonmyeloablative stem cell transplantation (NST) these two kinds of transplant strategies in order to define factors that significantly influence the early engraftment of donor stem cells. Methods A retrospective comparative study was performed between 20 patients undergoing BMT and 18 patients receiving NST. There are several differences between NST and standard allo-BMT, such as conditioning regimens, the source and the quantity of hematopoietic stem cells and the protocal of GVHD prophylaxis. But component of two groups patients were similar. Sequential monitoring of chimerism were performed in 38 patients in different periods post transplantation using multiple short tandem repeat(STR) amplification by fluorescence labeling polymerase chain reaction(PCR) combined with a capillary electrophoresis. Results The median age of recipient, median quantity of MNC, CD34+ cells and T lymphocyte cells of NST were significantly higher compared with the BMT. The median time for absolute neutrophils recovery of NST(without G-CSF) was similar to that of BMT. While compared to BMT, NST lead to faster engraftment of platelets. The establishment of full donor chimerism (FDC) of NST was much more earlier than that of BMT (1 month VS 3 months, p=0.01). In the early post-transplant period(up to day 30), there was an apparent increase in the incidence of full donor chimerism (FDC) following NST compared with BMT(38.9% VS 20%, p=0.019). Whereas a significantly lower incidence of mixed chimerism(MC) occurred in recipients of NST compare to BMT.(61.2% VS 80%, p=0.02). It suggest that in the early stage after transplantation, the engraftment of donor cells of NST was more rapid than that of BMT. The major relevant factor causing full donor chimerism formation seems to be the high quantity of donor CD34+ cells and T lymphocyte cells. Fludarabine-based reduced-intensity conditioning didn’t result in a significant delay of donor cell engraftment compared to standard conditioning regimen. The incidence of chronic GVHD was much higher in group of NST than that of BMT group(80% VS 50%, p=0.007). This finding was related to that patients of NST group received the higher doses of CD34+ cells. Conclusion Factors that significantly influence the early kinetics of donor chimerism and donor cell engraftment after transplantation were the quantity of stem cell and T lymphocyte cell of grafts.

objective: To investigate the effect of non-T cell depleted HLA haploidentical bone marrow transplantation(BMT) to patients with advanced malignant hematologic disease. Method: 26 patients with advanced malignant hematologic disease received non-T cell depleted haploidentic BMT. Donors included group A: offspring(5cases), sibling(3cases) and group B: mother(18cases). Hematologic Stem Cells(HSC) mobilization, collection and infusion: donors received G-CSF(300ug/12h×15d) from −5d, bone marrow cells were collected at −2d, PBSC were collected at −1d if CD34+ cells less than 4×106/kg of recipient’s body weight. The average of CD34+ cells were 5.91(3.66~9.29) ×106/kg of recipient’s body weight, the average of MNC were 10.05(2.10~29.70) ×108/kg of recipient’s body weight. Preparative regimens: recipients were prepared for transplantation with different conditioning regimens according to the type of disease. Myeloid leukemia regimen: MeCCNU 250mg/m2 −10d, Ara-C 2g/m2 q12h, from −9d to −8d, Bu 4mg/kg, from −7d to −5d, Cy 1.8g/m2.d, from −4d to −3d. Lymphoblastic leukemia regimen: MeCCNU 250mg/m2 −8d, Total-body irradiation(TBI) 8Gy −7d, Ara-C 2g/m2 q12h, from −6d to −5d, Cy 1.8g/m2.d, from −4d to −3d. All the patients were given hydration, alkalization, and given mesna to prevent hemorrhage cystitis. GVHD prophylaxis: All patients received GVHD prophylaxis consisting of Cyclosporine(CSA), Methotrexate(MTX), Mycophenolate mofetil(MMF) and antithymocyle globulin(ATG). When GVHD developed, methylprednisolone(MP) was given at first, if the response was poor, anti-CD25 antibody was given to the recipients as quickly as possible and CSA was replaced with tacrolimus. [Results] All patients achieved sustained, full-donor-type engraftment. CD34+ cells in graft were (5.91±1.34)×106/kg of recipient’s body weight, all patients attained successful neutrophil recovery at a median of (12.2±2.82) days (range, 10 to 20 days), recovery of platelet counts of more than 20×109/L was observed at a median of (20.73±7.62) days (range, 13 to 44 days). All the patients detected 99.9%~100% donor chimerism with FISH, recipient blood type of red blood cells transferred to donor blood type, STR showed donor chimerism exceeded 93%. 23 patients developed aGVHD in 26, the respective number of patients who developed grade I, II, III or IV aGVHD was 7, 7, 2, 7, the cumulative incidence of grade II–IV aGVHD was 65.4% and grade III–IV aGVHD was 34.6%. 12 patients developed cGVHD, the incidence was 46.2%. As of July 30 2005, 10 patients died, owing to pulmonary infection in 7, septicemia and HC in 1, leukoencephalopathy in 1, and recurrent disease in 1 case. 16 patients were alive and disease free with 2 years disease-free survival probabilities of 49.04%, 6 patients died in group A with 1 year disease-free survival probabilities of 25%, 4 patients died in group B with 2 years disease-free survival probabilities of 62.5%, p=0.0012 with Log-Rank test Conclusion HLA-haploidentical BMT is a effective treatment for advanced malignant hematologic disease. Donor from mother is superior to the donor from sibling and offspring with lower aGVHD incidence, lower mortality and longer survival.

Objective To explore the relationship among CD4+CD25+ regulatory T cell, the expression of FOXP3 mRNA levels of donor BM and GVHD in allogeneic hematopoietic stem cell transplantation(allo-HSCT). Methods (1)Thirty patients undergoing allo-HSCT were enrolled in this study. The levels of donor CD4+CD25+ regulatory T cell were compared in the patients with GVHD and those without GVHD by immnofluorescence and flowcytometry. (2)The donor BM expressions of FOXP3 mRNA levels were analyzed by using reverse transcriptase polymerase chain reaction (RT-PCR). Results (1)All patients achieved engraftment. ANC ≥ 0.5′109•L−1and Plt ≥ 20′109•L−1 were at day 14(range, 12~15)and day 18 (range, 15~25) respectively. Acute GVHD occurred in 15 of 30 patients(50.0%) with gradeII°-IV° aGVHD (40.0%). Chronic GVHD developed in 6 (extensive 2, limited 4) out of 30 patients (20.0%). 3 patients relapsed.(2)The levels of CD4+CD25+ regulatory T cell pre-and pro-mobilization were (2.67±0.38) % and (5.01± 1.33)% respectively, no difference were observed(P>0.05).(3)Patients with I°~II° aGVHD demonstrated higher donor-type CD4+CD25+ immune regulatory T cell level than those with III°~IV° aGVHD; Patients with III°~IV° aGVHD showed much lower donor-type CD4+CD25+ immune regulatory T cell level than those without GVHD; no difference were observed between patients withI°~II° aGVHD and those without aGVHD.(4)The transcripts of FOXP3 donor BM were detected in seven donors of thirty recipients who received allo-HSCT. Three relapsed after transplantation;two developedI°aGVHD; and the other two had no GVHD. Donor BM FOXP3 transcripts were never detected in patients with II°~IV° aGVHD. Conclusion(1)Donor-type CD4+CD25+ immune regulatory T cell level was relative to recipient II°~IV° aGVHD. Up-grade donor-type CD4+CD25+ immune regulatory T cell level could reduce the incidence of aGVHD.(2)Donor BM FOXP3 transcripts were detected in patients without serious aGVHD after transplantation.

Objective To study the quantity and ratio of Th1, Th2 cells in the bone marrow of patients with MDS;The tumor burden that Th1 cells bear in the patients with MDS. Evaluation of the correlation between the ratio of the blast cells and the number of the Th1 cells in the bone marrow in the patients with MDS. Methods By FACS, the quantity and ratio of IFN-γ producing CD4+ T cell (Th1) and IL-4 producing CD4+ T cell (Th2) cells in the bone marrow were detected in 21 patients with MDS,18 normal controls and 13 patients with SAA respectively;The karyotypes of 18 patients with MDS and 15 normal controls were detected, The burden,(the number of the cells with anormal karyotypes /the number of all the detected cells) that Th1 cells bear in the patients with MDS and that in normal controls were analyzed;The correlation between the ratio of the blast cells in the bone marrow and the number of the Th1 cells in the patients with MDS was also analyzed. Results The percentages of Th1 cells and Th2 cells and ratio of Th1/Th2 in the bone marrow of normal controls were: (0.48±0.10)%,(0.24±0.19)%,2.31±0.76 respectively, while those of the patients with MDS were (0.36±0.11)%,(0.76±0.35)% and 0.51±0.13. The percentage of Th1 cells of patients with MDS was reduced and the ratio of Th1/Th2 of them was significantly lower than that of normal controls(p Conclusion The immune function of T lymphocytes in MDS is abnormal: the balance between Th1 and Th2 cells is broken. Th1 cell, the most important element for antitumorigenesis, is decreased. Comparing with the burden of the malignant clone, the number of Th1 cells of the patients with MDS is overwhelmingly scarce. With descending of the number of Th1 cells in the bone marrow of the patients with MDS, the number of the blast cells contrarily grows. MDS is a heterogeneous group of neoplastic disease accompanied with hypofunction of the T lymphocytes mediating immune surveillance. All these hint that during the treat of clearing the malignant clone of MDS, enhancement of the T cell fuction should been done.

Objective Monitoring of minimal residual disease (MRD) and cellular chimerism in patients with hematopoietic malignancies after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Methods From May 2001 to June 2005, seventy four patients were received allo-HSCT. Including 50 Males and 24 females. 41 patients received sibling HLA-matched BMT, 7 patients received un-related BMT, 9 patients received Nonmyeloablative stem cell transplantation (NST), 14 patients received related haploidentical transplantation and 3 patients received allo-PBSCT. Among them, 45 patients were diagnosed with CML, 13 patients with AML, 14 patients with ALL, one patient with Multiple myeloma and one patient with malignant lymphoma. Chimerism and MRD were monitored using X and Y specific centromeric probes or gene probes for BCR/ABL, MLL and AML1/ETO by fluorescence in situ hybridization (FISH),1000 cells were analysised. Results Among 18 patients, received sex-matched transplant, we did not found the former chromosome rearrangements in 14 patients after transplantation, MRD were detected in 17% and 10% of cells in two patients, MRD were decreased from 10% to 1% of cells after the reduction of the dose of immunotherapy in one patient four month later, the patient was still in remission one year after transplantation. Another patient died of sever GVHD after the reduction of immunotherapy. 2 patients were found to have the former chromosomal rearrangement 1 and 4 month after transplantation respectively who did not achieve remission after chemotherapy and died 3 and 5 months respectively after transplantation. Over 99% donor chimerisms were found in 44 patients on day 25, donor cells were at a low level (96.2%~98.7) in 7 patients on day 25, and increased over 99% on day 180, they were in remission without relapse. The donor chimerisms decreased gradually in 6 patients, host cells were found over 20 cells, 3 patients showed cytogenetic or hematologic bone marrow relapse, two patients died of sever GVHD after the reduction of cyclosporine A, Over 99% donor chimerisms was achieved in one patient. Conclusion FISH could play a pivotal role in the detection of MRD and chimerism. It is helpful to the evaluation of graft and relapse, to the guide of implement of early immunotherapy.

Objective To explore the inducing differentiation and apoptosis in a novel human acute monocytic leukemic cell line SHI-1. Methods Cell morphological analysis, NBT reduction test, the expression of CD11b and CD14, cell cycle analysis and binding of annexinV tested by FCM, DNA electrophoresis were performed to evaluate whether tributyrin (TB), arsenic trioxide (As2O3), and 12-O-tetradecanoyl-phorbol-13 acetate (TPA) could induce the differentiation and apoptosis in the SHI-1 cell line. Results The results of morphological analysis, NBT reduction test, and FCM assay showed that TB could induce differentiation of SHI-1 cells. The SHI-1 cell line presented typical apoptotic evidences including morphology features, DNA ladders and the increased apoptotic rate determined by FCM. Moreover, the SHI-1 cell line presented typical apoptotic morphology after being treated with As2O3. The apoptotic rate of the SHI-1 cell line treated with As2O3 was increased determined by FCM. The expression level of CD14 was upregulated while that of CD11b was unchanged in the SHI-1 cell line. Most of the SHI-1 cells adhered to the flask after being treated with TPA for 24 hours. Conclusions TB may produce dual effects of differentiation and apoptosis. The cell line could be induced to differentiation partially or apoptosis after being treated with 0.5μM, 1.0μM, and 2.0μM As2O3. TPA could induce differentiation and adhesion of the SHI-1 cell line.

Platelets play a crucial role in thrombosis, platelet activation induces an alteration of platelet membrane glycoproteins between the platelet surface and surface connected canalicular system (SCCS). In an attempt to better understand this process and assess its significance in the study of thrombosis disease, we used flowcytometry for detecting the redistribution of antigens on the platelet surface following activation. The expression of platelet membrane glycoproteins (GP) Ib, IIIa and P-selectin as well as the platelet agglutination induced by ristocetin were measured in 20 normal subjects, whose platelets were stimulated by the peptide SFLLRNP(TRAP, thrombin receptor activated peptide) at different time points (0~30minute). The same antigens were also analyzed in whole blood in 30 patients with cerebral infarction. As expected, a stable increase of P-selectin was obtained from 2 minute upon TRAP activation, while a reversible reduction of GPIbα and agglutinability by ristocetin were showed at the same time, with a maximum decrease at 2 minute, then followed by a return of GP Ibα to platelet surface. In addition, the platelet membrane GPIIIa was not significantly changed in the initial stage after platelet stimulation and increased by degrees 10 minutes later. Compared with those in normal group, there was an apparent increase of P-selectin and a lower expression of GPIb in the patients with cerebral infarction, while GPIIIa was not significantly changed in all the samples. In summary, TRAP can mediate platelet activation, which leads to the redistribution of GPIb and GPIIIa in a time-dependent manner, together with the release of P-selectin. Meanwhile, the change of activation-dependent antigens on the platelet surface could be an important index reflecting the platelet activation in vivo.

Objective To evaluate the quantity and functional changes of subsets of Th cells in the bone marrow of severe aplastic anemia (SAA) patients and the relationship between these changes and the patient hematopoietic function. Methods By FACS, the quantity and ratio of Th1 cells and Th2 cells, the percentage of CD3+CD8+ cells in the bone marrow were detected in 24 patients with SAA at active phase, 15 patients with SAA at recovery phase, and 16 normal controls respectively. By radioimmunoassay, the levels of cytokines of Th1 type (TNF-α), or Th2 type (IL-4) in the serum of 20 patients with SAA at active phase, 12 patients with SAA at recovery phase, 16 normal controls were measured respectively. The relationships between CD3+CD8+ cells, TNF-α and Ret, ANC were evaluated; the relationships between Th1 cells and CD3+CD8+ cells, TNF-α or Ret, ANC were evaluated; the relationships between IL-4, balance of Th1/Th2 and Ret, ANC were also evaluated. Results The percent of Th1 cells, Th2 cells, and ratio of Th1/Th2 in bone marrow of patients with SAA at active phase was: 4.87±2.64%, 0.41±0.26%, 21.22±5.07, were higher than those of normal controls: 0.42±0.30% (p0.05); the percent of CD3+CD8+ cells decreased from 32.32±18.69% at active phase to 13.76±2.96% at recovery phase significantly (p Conclusion The formation of bone marrow failure in SAA might be caused not only by the increase of Th1 cells, Th1 type effective cells and cytokines, but also by unsufficient compensation of Th2 cells and Th2 type cytokines, which shifted the balance of Th1/Th2 to Th1.

Objective In order to investigate the value of sequential and quantitative analysis of chimerism to predict relapse or graft rejection after allogeneic stem cell transplantation, to determine the optional time point of adoptive immunotherapy and to estimate the efficacy of adoptive immunotherapy. Methods 70 patients who received HLA compatible allo-HSCT were evaluated. Peripheral blood and bone marrow were collected before and after transplantation in different period. Serial and quantitative analysis of donor chimerism (DC) both prior to and following adoptive immunotherapy were performed by multiplex PCR amplification of STR markers (STR-PCR) and capillary electrophoresis with fluorescence detection. Results 13 of 70 patients relapsed at median time of 6months post transplant (4/13 patients developed hematologic relapse and 9/13 patients appeared cytogenetic or molecular relapse respectively). 2 of 70 patients experienced graft rejection. Before the time of relapse or graft rejection, STR-PCR indicated the decreasing DC in all 15 patients, at levels ranging from 24.8%~86.2%. The declining value of donor chimerism (DC< 90%) was detected in four patients 26 days before relapse or graft rejection diagnosed clinically. Adoptive immunotherapy was used to treat 15 patients(9 CML, 5 AML, 1 MDS). A response to immunotherapy was achieved in 6 patients(6 CML) following withdrawal of CsA and in a further 4 patients(2 CML, 1 M2, 1 MDS) after additional donor lymphocyte infusion(DLI). In these patients, the value of DC increased with convertion to a predominant donor profile (>90%) or converted to stable full donor chimerism(FDC)(6 patients) or stable mixed chimerism(MC)(4 patients) shortly after immunotherapy. All these 10 patients who responsed to immunotherapy developed acute or chronic GVHD. While in the patients without response, the level of DC decreased persistently or declined after transient increasing. Three patients without response received second DLI but remain failed to therapy. Conclution The results demonstrate that the decreasing value of DC can identify the patients who have high risk of relapse or graft failure and can be used to guide adoptive immunotherapy implementation at early stage. The sequential and quantitative monitoring of DC has been shown to be a valuable tool to determine the optional time point of immunotherapy and to early evaluate the efficacy of treatment. Furthermore, it can present a rational basis for treatment intensification in patients who did not respond to first-line DLI treatment

Multiple myeloma(MM) is a monoclonal expansion of malignant cells with a plasmablast-plasma cell morphology that is almost exclusively localized to the bone marrow, except at the final stages of disease, when they proliferate in the extramedullary area. The mechanisms of the selective homing of MM cells to the bone marrow compartment are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 contribute to stem cell homing and play a role in trafficking of leukemic cells. In this study we have investigated expression and biological behavior of SDF-1/CXCR4 in MM-derived cell lines and primary MM cells. FACS and RT-PCR analysis was used to study the expression of CXCR4 and ICAM-1(CD54) on the surface of MM cells from 4 IL-6 dependant cell lines (XG1,XG2,XG6 and XG7) and 25 freshly isolated tumor samples from patients with diagnosed MM. Mononuclear cells were purified by positive selection of magnetical and FACS sorting. Chemotaxis assay through transwell bore polycaronate and ELISA assay were employed to monitor the SDF-1, IL-6, and sICAM-1 levels. We found that[circ1]Fresh MM cells and MM cell lines expressed various levels of functional CXCR4 ranging from 23.1% to 77.7%,which was correlated with the in vitro migration ability of MM cells[(23.2±1.08)%, P

Objective: To establish quick and reliable diagnostic methods for detecting the cytomegavirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to evaluate their clinical value in early diagnosis of CMV infection Methods: (1) From March 2001 to December 2003, Seventy-four patients undergoing allo-HSCT were enrolled in this study. After allo-HSCT, all the patients were detected for CMV gB DNA in peripheral blood by haplo-nested PCR methods and 35 of 74 patients for CMV PP65 antigen in peripheral blood by the fluorescent immunohistochemistry (FIHT) assay. The positive rate and correspondence of these two methods were evaluated. Results: (1) In 35 of 74 patients who were tested for CMV infection by two assays at the same time, the CMV-viremia was first detected at a median of 36 days after transplantation by FIHT assay and 26 days by haplo-nested PCR assay during the follow-up time of 90~720 days. The median time of positive persistence was 19 days by FIHT assay and 28 days by haplo-nested PCR assay. The significant difference of them was observed (P0.05). The infection rate of CMV in the NST group was significantly higher than in R-PBSCT and/or R-BMT group (P0.05). Both CMV-antigen and CMV-DNA were detected in all seven patients undergoing haploidentical hematopoietic stem cell transplantation (Hi-HSCT). Conclusion: (1) Both FIHT and haplo-nested PCR assays were quick and reliable diagnostic methods for detecting the CMV infection after allo-HSCT and their correspondence was good. However CMV infection could be found earlier by haplo-nested PCR assay than by FIHT assay. (2) The infection rate of CMV is high after allo-HSCT and might be different in different types of allo-HSCT.

Objective To measure the changes of subsets of dendritic cells 1 (DC1) in the bone marrow of severe aplastic anemia (SAA) patients and evaluate the relationships between the CD11c+CD83+cells and Th1 cells, CD3+CD8+ cells or hematopoietic function. Methods By FACS, the quantity and ratio of CD11c+CD1a+ cells, CD11c+CD83+ cells, Th1 cells, and CD3+CD8+ cells in the bone marrow of SAA patients and normal controls were detected respectively. The relationships between CD3+CD8+ cells and Ret or ANC, between Th1 cells and CD3+CD8+ cells, Ret or ANC, between CD11c+CD83+ cells, and Th1 cells, CD3+CD8+ cells, Ret or ANC were evaluated. Results In normal control’s bone marrow, the percentages of Th1 cells, CD11c+CD1a+ cells, CD11c+CD83+ cells and ratio of CD11c+CD83+ /CD11c+CD1a+ was 0.42±0.30%, 0.38±0.29%, 0.37±0.32% and 1.07±0.10 respectively. In the untreated SAA patient’s bone marrow, they were 4.87±0.54%, 1.73±0.24%, 3.38±0.56% and 2.21±0.32 respectively, and increased markedly(p0.05). The percentages of CD3+CD8+ cells of untreated SAA patients was 32.32±10.22%, and that of recovering SAA patients decreased to 13.67%±5.24 significantly (p Conclusion Both immature DC1 and activated DC1 increased in the bone marrow of SAA patients, and the balance of subsets of DC1 shifted from stable form to active one, which might promote Th0 cells to polarize to Th1 cells, then cause the over-function of T lymphocytes and hematopoietic failure in SAA.

Objective: To investigate the feasibility and efficacy of DAC (decitabine) bridge therapy followed by allo-HSCT (allogeneic hematopoietic stem cell transplantation) in patients with MDS (myelodysplastic syndrome) and AML (acute myeloid leukemia). Methods: Seven patients with MDS and 12 patients with AML received DAC bridge therapy followed by allo-HSCT. Results: With DAC bridge therapy, 4 MDS patients achieved complete remission/marrow complete remission and 3 remained stable disease before allo-HSCT. After successful engraftment attained in all the seven MDS patients, six survived without disease, one received donor lymphocyte infusion and obtained complete remission after relapse and eventually died of pneumonia. Of 12 AML patients, 6 achieved complete remission after DAC bridge therapy; 5 survived without disease, one still survived but having disease, and 6 had died. The rates of acute and chronic GVHD were 31.6% and 21.1%, respectively. The two-year overall survival rate and the two-year cumulative recurrence rate were 57.9% and 36.2% after allo-HSCT, respectively. The two-year cumulative recurrence-free rate was 23.6% after allo-HSCT. Conclusion: DAC regimen can be safely and efficiently administrated to bridge time to allo-HSCT in patients with MDS/AML. DOI:10.3781/j.issn.1000-7431.2013.03.010