Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation.

AIM: The phosphorylation of histone H2AX, a novel tumor suppressor protein, is involved in regulation of cancer cell apoptosis. The aim of this study was to examine whether H2AX phosphorylation was required for resveratrol-induced apoptosis of human chronic myelogenous leukemia (CML) cells in vitro. METHODS: K562 cells were tested. Cell apoptosis was analyzed using flow cytometry, and the phosphorylation of H2AX and other signaling proteins was examined with Western blotting. To analyze the signaling pathways, the cells were transfected with lentiviral vectors encoding H2AX-wt or specific siRNAs. RESULTS: Treatment of K562 cells with resveratrol (20-100 µmol/L) induced apoptosis and phosphorylation of H2AX at Ser139 in time- and dose-dependent manners, but reduced phosphorylation of histone H3 at Ser10. Resveratrol treatment activated two MAPK family members p38 and JNK, and blocked the activation of another MAPK family member ERK. Pretreatment with the p38 inhibitor SB202190 or the JNK inhibitor SP600125 dose-dependently reduced resveratrol-induced phosphorylation of H2AX, which were also observed when the cells were transfected with p38- or JNK-specific siRNAs. Overexpression of H2AX in K562 cells markedly increased resveratrol-induced apoptosis, whereas overexpression of H2AX-139m (Ser139 was mutated to block phosphorylation) inhibited resveratrol-induced apoptosis. K562 cells transfected with H2AX-specific siRNAs were resistant to resveratrol-induced apoptosis. CONCLUSION: H2AX phosphorylation at Ser139 in human CML cells, which is regulated by p38 and JNK, is essential for resveratrol-induced apoptosis.

Imatinib induces H2AX phosphorylation and apoptosis in chronic myelogenous leukemia cells in vitro via caspase-3/Mst1 pathway.

AIM: Histone H2AX is a novel tumor suppressor and its phosphorylation at the C terminus (Ser139 and Tyr142) is required for tumor cell apoptosis. The aim of the present study was to elucidate the mechanisms underlying imatinib-induced C-terminal phosphorylation of H2AX in chronic myelogenous leukemia cells in vitro. METHODS: BCR-ABL-positive K562 cells were used. Microscopy, Western blotting and flow cytometry were used to study the signaling pathways that regulate imatinib-induced H2AX phosphorylation and the apoptotic mechanisms. RESULTS: Treatment of K562 cells with imatinib (1-8 µmol/L) induced phosphorylation of H2AX at Ser139 and Tyr142 in time- and dose-dependent manners. In contrast, imatinib at the same concentrations did not affect H2AX acetylation at Lys 5, and the acetylated H2AX maintained a higher level in the cells. Meanwhile, imatinib (1-8 µmol/L) activated caspase-3 and its downstream mammalian STE20-like kinase 1 (Mst1), and induced apoptosis of K562 cells. The caspase-3 inhibitor Z-VAD (40 µmol/L) reduced imatinib-induced H2AX phosphorylation at Ser139 and Tyr142 and blocked imatinib-induced apoptosis of K562 cells. Imatinib (4 µmol/L) induced expression of Williams-Beuren syndrome transcription factor (WSTF), but not wild-type p53-induced phosphatase 1 (Wip1) in K562 cells. CONCLUSION: The caspase-3/Mst1 pathway is required for H2AX C-terminal phosphorylation at Ser139 and Tyr142 and subsequent apoptosis in Bcr-Abl-positive K562 cells induced by imatinib.

Haploidentical hematopoietic stem cell transplantation in child hematologic malignancies with G-CSF-mobilized marrow grafts without T-cell depletion: a single-center report of 45 cases.

In this report, the authors describe a protocol for haploidentical bone marrow transplantation in children who received G-CSF-mobilized bone marrow grafts without T-cell depletion from HLA-mismatched parents. Forty-two of 45 patients achieved complete donor hematopoietic engraftment; the medium time for neutrophil and platelet recovery was 17 and 19 days, respectively. Three died of early transplantation-associated complications; other causes of death included relapse (11 cases), fungal pneumonia (5), and acute graft-versus-host disease (2). The total disease-free survival rate longer than 2 years was 53.3%. These data suggest that haploidentical hematopoietic transplantation is an alterative strategy for children who lack immediate access to HLA-matched sources.

[Analysis of Risk Factors and Therapeutic Strategies for Relapse of Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To analyze the risk factors of patients with relapsed leukemia after allogeneic hematopoietic stem cell transplantation, and to explore the therapeutic strategies for recurrence. METHODS: The Cox proportional hazard regression model was used for univariate and multivariate analysis of transplantation-related index, a single center retrospective study of clinical data of 202 cases of leukemia received allo-HSCT from March 2004 to October 2014 had been conducted to screen the risk factors for recurrence after transplantation. RESULTS: In the leukemia patients received allo-HSCT, 68 cases relapsed. The relapse rate was 33.6%. The median time of relapse was 4(1.5-26 ) months. Univariate analysis indicated that there were 5 risk factors related with the disease relapse(P<0.05), including the type of disease, extramedullary disease prior to transplant, the course of induced remission, the status of disease at HSCT and chronic graft versus host disease(cGVHD). Multivariate analysis showed that extramedullary disease prior to transplant(RR=2.622, 95%CI 1.139-6.037), the course of induced remission(RR=1.156, 95%CI 0.682-1.957), cGVHD (RR=1.728,95%CI 0.999-2.991) were independent risk factors for relapse of the patients received transplantation. Treatment strategies for the relapsed patients included withdraw immunosuppressant, donor lymphocyte infusion, systemic chemotherapy and local radiotherapy, targeted therapy, and second transplantation. Individualized choice was needed according to the relapsed site. The relapse-related mortality was 25.2%. CONCLUSION: The relapsed patients with leukemia after allo-HSCT have poor prognosis, early interference has good effect. The evaluation and prevention of risk factors before transplantation is even more important.

[Unrelated Donor Peripheral Blood Stem Cell Transplantation Combined with Umbilical Cord Mesenchymal Stem Cells in Patients with Hematologic Malignancies].

OBJECTIVE: To explore the safety and efficiency of unrelated donor peripheral blood stem cells (URD-PBSC) transplantation combined with umbilical cord mesenchymal stem cells (UC-MSC). METHODS: The clinical data of 49 patients received unrelated donor peripheral blood stem cells transplantation (URD-PBSCT) for treating hematologic malignancies were retrospectively evaluated, including 12 ANLL, 17 ALL, 18 CML and 2 MDS. Out of them, 22 patients received the URD-PBSCT combined with UC-MSC and 27 patients received only URD-PBSCT. The average number of infusing UC-MSC was 1.0 × 106/kg in the UC-MSC+URD-PBSCT group. RESULTS: As compared with URD-PBSCT group, in UC-MSC+URD-PBSCT group the median recovery time of neutrophilc granulocytes was shorter (12 d vs 15 d) (P = 0.041), the incidence and severity of chronic graft versus host disease (cGVHD) were lower (20.0% vs 51.9%) (P = 0.026) (5.0% vs 33.3%) (P = 0.040), the incidence of CMV infection after transplantation was higher (81.8% vs 51.9%) (P = 0.028). In addition to these, the differences were not statistically significant in term of implantation level, PLT reconstitution, aGVHD, lung infection, hemorrhagic cystitis, 1-year relapse and survival between the 2 groups (P > 0.05). CONCLUSION: The transplantation of URD-PBSC combined with UC-MSC is effective and safe. The speed of neutrophils reconstitution is faster. The incidence and severity of cGVHD are lower, but the attention should be paid to prevent the CMV infection.

[Influence of calcineurin on apoptosis of pre-B lymphocytes and the leukemia cells derived from pre-B lymphocytes].

This study was aimed to compare the differential expressions of calcineurin (PP2B, PP3) in the mouse Pre-B cell lines (S9) and the tumor cell lines (S4C2) derived from pre-B lymphocytes, and to clarify its possible mechanism involving in the leukemia cell apoptosis. The quantitative real-time PCR was used to detect the differential expressions of H2AX-associated phosphakinase ATM, ATR, DNA-PKs, JNK1, P38 and the γ-H2AX-related phosphatase PP1, PP2A, calcineurin, PP4, PP6, PP5 between S9 and S4C2 cell lines. CCK-8 assay and flow cytometry were used to detect the effect of imatinib (IM) and cyclosporine A (CsA) on cytotoxicity and apoptosis of 2 cell lines. The Western blot was used to detect the effects of 2 drugs on apoptosis of S9 and S4C2 cell lines. The results showed that the expression level of calcineurin gene in the leukemia cell S4C2 was about 3.5 times of that in S9 cells, while the expression of other genes in these 2 kinds of cells was not significantly different. The apoptosis and toxicity of IM and CsA on S4C2 cells was significantly stronger than that on S9 cells. The expression level of calcineurin in S4C2 cells was higher than that in S9 cells.When CsA inhibited the calcineurin activity, the expression of DNA damage marker γ-H2AX in S9 cells was significantly lower than that in S4C2 cells,while the expression level of γ-H2AX between the two cell lines was no significantly different after treatment with imatinib, the expression level of γ-H2AX in S9 cells was lower than that in S4C2 cells when the two drugs were combined. It is concluded that the calcineurin plays a role of anti-apoptosis in B leukemic cells, cyclosporine A can promote the leukemia cell apoptosis.

[Allogeneic hematopoietic stem cell transplantation for chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia].

This study was purposed to explore the therapeutic efficacy and influencing factors of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with chronic myelomonocytic leukemia (CMML) and in patients with juvenile myelomonocytic leukemia (JMML). The clinical data of 3 cases of CMML and 2 cases of JMML underwent allo-HSCT were analysed in term of multiparameter. The results showed that the hematopoietic stem cells in 5 patients grafted successfully. One case of JMML died of pulmonary disease, other 4 cases survive without disease. The analysis found that the disease burden before transplant, chromosome karyotype, acute GVHD II-IV and poor risk cytogenetics all associated with the relapse rate and disease-free survival rate of CMML. The low intensity conditioning regimen was better than myeloablative conditioning regimen. Type of donor and source of stem cells did not statistically and significantly affect OS and RFS. The splenectomy before allo-HSCT as well as spleen size at time of the alloHSCT did not influence on posttransplantation outcome of JMML. However, cord blood HSCT for JMML patients delayed hematologic recovery as compared to that of bone marrow or peripheral blood HSCT. The age, GVHD, HbF level played an important role in leukemia replace. It is concluded that the allogeneic hematopoietic stem cell transplantation is a curative regimen for CMML and JMML, but there also is a serial problems to be resolved.

[Treatment of leukemia with immunized donor cell infusion after nonmyeloablative haploidentical bone marrow transplantation].

This study was purposed to investigate the therapeutic effects of early transfusion of immunized donor lymphocytes after haploidentical transplantation by means of mouse model of nonmyeloablative haploidentical bone marrow transplantation. CB6F1 female mouse was served as recipient and C57BL/6 male mouse was served as donor. Each CB6F1 female mouse was subjected to intravenous transfusion with 1×10(6) erythroleukemia (EL9611) cells at day 4 before transplantation, followed with intraperitoneal injection of Ara-C (0.015 g) respectively at day 2 and day 1, then conditioned for BMT with TBI (450 cGy) at day 1 before transplantation. After conditioning (day 0), each of recipients was transplanted with 6×10(7) mixture of bone marrow and spleen cells from the C57BL/6 mice, and was infused with 6 × 10(7) immunized donor lymphocytes at day 15 after transplantation. All treated animals were evaluated for survival, development of leukemia and aGVHD. The donor CD3(+) cell chimerism and sex determining region Y gene (SRY)in recipients were monitored periodically after transplantation. The results showed tht all mice with only inoculation of 10(6) EL9611 cells survived for 15 ± 1 days (n = 4); all mice of other groups obtained the varying degrees of implantation. SRY could be detected at day 30 and 60 after transplantation. The chimerism of donor CD3(+) cells in mixed bone marrow transplantation (MT) group at day 14, 30 and 60 respectively reached 17.95% ± 12.03%, 37.34% ± 2.78% and 47.06% ± 6.1%. In donor lymphocyte infusion (DLI) group it reached 69.78% ± 12.62%, 75% ± 15.97%, 83.41% ± 16.07% at day 30, 45 and 60 after transplantation. The mice of MT and DLI group survived for 66.66 ± 1.47 days and 78.2 ± 7.82 days. It is concluded that the high tumor burden before transplantation can affect donor cell engraftment and prognosis.Early post-transplanted infusion of immunized lymphocytes from donor can help to improve the therapeutic efficacy and survival.

[Detection of the expression of NK ligands in acute leukemia cell lines by real-time PCR].

OBJECTIVE: To detect the expression profile of NK ligands in acute leukemia cell lines and investigate the differential expression pattern between acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). METHODS: Using quantitative real-time PCR, 23 NK ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, HLA-E, HLA-G, CD48, NBTA, HLA-F, LLT-1, PVR, Nectin2, CD72, CD80, ICAM-1, LFA-3, CRACC, Fas, DR4, DR5, TNFR1) were detected in 6 acute leukemia cell lines, including 3 ALL cell lines (CEM, Jurkat T, Reh) and 3 AML cell lines (HL-60, KG-1a, NB4), respectively. Independent-samples t test analysis was performed to determine statistical significance. RESULTS: Using ß-actin as reference gene, the relative expression results showed that the expression of 4 NK ligands between ALL and AML is significantly different. Specifically, the level of ULBP-2 is higher in ALL (CEM: 1, Jurkat T: 0.617, Reh: 0.246) than that in AML (HL-60: 0.000, KG-1a: 0.003, NB4: 0.000)(P = 0.047). However, the expressions of CD48, PVR(PVR-1, PVR-2) and DR4 is higher in AML (HL-60: 13.987, 4.403, 10.334, 8.711; KG-1a: 5.387, 2.900, 7.315, 4.512; NB4: 7.763, 3.248, 7.049, 6.127) than that in ALL (CEM: 1, 1, 1, 1; Jurkat T: 2.035, 1.553, 3.888, 0.449; Reh: 1.559, 0.000, 0.000, 1.304) (P = 0.044, 0.014, 0.014, 0.011). And there're no significant differences between the rest 19 NK ligands. CONCLUSIONS: ULBP-2, CD48, PVR and DR4 might play an important role in the distinct mechanisms in leukemogenesis between ALL and AML and could be potential targets for diagnosis and treatment.

[Veto T cells enhance engraftment allogeneic hematopoietic stem cells].

Veto activity was defined as the capacity of specifically downregulating cytotoxic T-precursor cell (CTL-p) directed against antigens of the veto cells themselves but not against third-party antigens. Many studies have shown that the most potent veto cells are CD8(+) cytotoxic T lymphocytes (CD8(+)CTLs). Effectively, CD8(+)CTLs of donor origin can facilitate engraftment of donor's stem cells by eliminating host-alloreactive T lymphocytes. In this article, effect mechanisms, depletion of GVH ex vivo, application in vivo, synergistic enhancement with rapamycin and regulatory T cells, and anti-tumor effect in the hematopoietic stem cell transplantation are summarized.

[Expression of NKG2D and NKG2A with their ligands MHC-I A/B and HLA-E in acute leukemia patients and its significance].

This study was aimed to explore the difference of NK cell receptor NKG2D and NKG2A expression on NK cells and CD3(+) T cells and their ligand MHC-I A/B (major histocompatibility complex class I-related chains A/B) and HLA-E expression in leukemia cells, as well as its immunological significance. Flow cytometry was used to detect the killing rate of NK92 cells to 8 leukemia cell lines, and the expression of NKG2D and NKG2A on NK cells and CD3(+) T cells as well as their ligand MHC-I A/B and HLA-E expression on leukemia cells. The results indicated that the NK92 showed different killing activity to different leukemia cell lines. The positive expression rate of NKG2D and NKG2A on NK cells and CD3(+) T cells in ALL patients was no significantly different from that in AML patients (p > 0.05), but positive expression rate of MHC-I A/B and HLA-E in ALL patients was obviously higher than that in AML patients (p < 0.05). It is concluded that there is difference of immune cell function between ALL and AML patients, this difference may be associated with the expression difference of NKG2D and NKG2A ligands on leukemia cells while does not associated with the killing and inhibiting receptors expressed on NK cells and CD3(+) T cells.

[Allogeneic hematopoietic stem cell transplantation in 6 patients with myelodysplastic syndrome].

This study was purposed to explore the efficacy of hematopoietic reconstitution and survival of patients with myelodysplastic syndrome (MDS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT without T lymphocyte depletion was used in 6 patients with MDS from November 1999 to June 2007. 4 cases out of them received allo-PBSCT from HLA matched sibling donors with conditioning regimen of cyclophosphamide (CTX) and Bu. Graft versus host disease (GVHD) was prevented by the administration of immunosuppressive drugs of cyclosporine A (CsA) and short-course MTX. 2 patients received haploidentical allogeneic bone marrow transplantation (hi-alloBMT) after preconditioning with cytosine arabinoside (Ara-C), CTX and total body irradiation (TBI) with a linear accelerator. GVHD was prevented by the administration of immunosuppressive drugs including CSA, short-course MTX, MMF, anti-CD25 monoclonal antibody and ATG. The results showed that all of the patients were engrafted successfully. The median time of granulocyte recovery exceeding 0.5 x 10(9)/L and platelets exceeding 20 x 10(9)/L were days 15 and 20.3 respectively, and 100% donor hematological cells were detected by cytogenetic analysis. All patients did not experience serious acute graft-versus-host disease (aGVHD). During 18 - 108 months of following-up, 2 cases died of pulmonary complication and of relapse; the other 4 cases survive in a disease-free situation. In conclusion, allo-HSCT was an effective approach for the treatment of MDS.

[Efficacy of anti-CD25 monoclonal antibody used in treating steroid-resistant acute graft-versus-host disease following haploidentical bone marrow transplantation].

The aim of this study was to investigate the efficacy and feasibility of anti-CD25 monoclonal antibody (basiliximab) in treating steroid-refractory acute graft-versus-host disease (aGVHD) following haploidentical bone marrow transplantation (hiBMT). 15 cases who developed II-IV grade steroid-resistant aGVHD after haploidentical BMT were treated by intravenous injection of basiliximab at a dose of 20 mg on days 1 and 4. In those patients not achieving CR after 1 week, basiliximab injection was repeated. The results showed that 8 cases (53.33%) got complete response (CR). Out of them 4 cases have been still in disease-free survival, 2 cases have been in survival with limited cGVHD, 2 cases died from pulmonary infection; 3 cases (65%) got partial response (PR), out of whom 1 case has been still in disease-free survival, one died from GVHD and infection, and another one died from pulmonary infection; 4 cases without response died from GVHD, pulmonary infection and cardiac failure. Overall response rate was 73.3% and long-term survival rate was 46. 7%. There were no infusion-associated side-effects after treatment with basiliximab. It is concluded that the anti-CD25 monoclonal antibody is efficacious and feasible for steroid-refractory grade II-IV aGVHD after hiBMT, but the overall survival rate is low. Infection is the main cause of death. Thereby, it is especially important to strengthen environmental protection and prevent infection.

Haploidentical hematopoietic stem-cell transplantation for non-Hodgkin lymphoma with bone marrow involvement.

Here, we report the preliminary results of haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) with granulocyte-colony-stimulating factor (G-CSF) mobilized bone marrow grafts without T-cell depletion for 10 patients with refractory non-Hodgkin lymphoma accompanied by bone marrow involvement. Eight patients received a conditioning regimen consisting of high-doses of cytarabine and cyclophosphamide with total body irradiation, whereas two cases were preconditioned with busulfan, thiotepa, and cyclophosphamide. All patients had rapid hematopoietic engraftment with the mean time for neutrophil and platelet recovery being 16.6 days and 19.2 days, respectively. Three cases died within 6 months after transplantation from severe acute graft-versus-host disease, fungal infection, or relapse. The others are currently alive in complete remission at a median follow-up of 60.71 months (range: 44-81 months). The results here suggest that haplo-HSCT might provide an opportunity of myeloablative therapy for refractory lymphoma with marrow infiltration.

[Haploidentical allogeneic bone marrow stem cell transplantation combined with peripheral blood stem cells for therapy of leukemia].

The purpose of this study was to investigate the feasibility and clinical outcome of granulocyte colony stimulating factor (G-CSF)-mobilized haploidentical bone marrow transplantation combined with peripheral blood stem cells (hiBM+PBSCT) for therapy of leukemia. 125 leukemia patients underwent G-CSF primed haploidentical stem cell transplantation without ex-vivo T cell depletion. All haploidentical donors were injected with G-CSF at dose of 5 microg/(kg.d) for 7 days. The patients were divided into groups A and B. 29 patients in group A underwent hiBM+PBSCT at 7th and 8th days of mobilization in donors with G-CSF respectively; 96 patients in group B underwent hiBMT. All patients received the same GVHD prophylaxis regimen, the clinical outcomes were investigated. The results showed that all patients except one CML-myelofibrosis patient achieved trilineage engraftment. Engraftment median times were 15 and 19 days for neutrophil and platelet in group A respectively, while engraftment median times were 18 and 23 days for neutrophil and platelet in group B respectively. The incidences of grade II-IV aGVHD were 31.03% in group A and 12.5% in group B respectively (p<0.05). The incidences of grade III-IV aGVHD was 13.79% and 10.41% in group A and group B (p>0.05). The aGVHD-related death incidence was 3.45% and 5.21% in group A and group B (p>0.05). The incidence of grade II-IV cGVHD was 48.2% and 35.4% in group A and group B respectively (p>0.05). The incidence of extensive cGVHD was 23.3% and 15.6% in group A and group B respectively (p>0.05). The disease relapse rate was 6.8% (2/29) and 18.75% (18/96) in group A and group B respectively (p<0.05). It is concluded that the G-CSF-mobilized allogeneic haploidentical BM plus peripheral blood HSCT without T cell depletion provides a rapid and sustained engraftment without increase of severe GVHD, furthermore, the relapse rate of disease is reduced remarkably, thus this method can be used in clinic.

[Detection of bcr/abl fusion gene changes in patients with chronic myeloid leukemia after allo-HSCT by real-time quantitative reverse transcription polymerase chain reaction and its significance].

This study was aimed to detect the changes of bcr/able gene level in ph+ CML patients at different stages after allo-HSCT by real-time quantitative PCR and to evaluate the significance of this detection. The serial detection of bcr/abl fusion gene levels in 21 cases of CML treated with allo-HSCT was performed by RQ-PCR. The results showed that the bcr/able fusion gene could not be detected in 7 out 21 CML cases with positive fusion gene after allo-HSCT, while the bcr/abl fusion gene of different levels could be detected in 14 cases within 1-6 months. Dynamic detection indicated that the bcr/abl fusion gene levels in 9 cases were lower with relative value 0.0074%-0.088% and then could not be detected within 3-7 months after allo-HSCT. The bcr/abl fusion gene levels in 5 cases diagnosed as molecular relapse were between 0.077%-75%. The bcr/abl fusion gene levels in 1 out of 5 cases were 0.95%, 1.5%, and 0.16% in month 1, 2 and 3, respectively, and turned to negative in the month 4 without any treatment after allo-HSCT. 2 cases received the donor peripheral blood stem cell infusion, and then their bcr/abl mRNA levels could not be detected in bone marrow. Another 2 cases developed to the hematologic relapse, 1 out of 2 cases reached CR again after infusion of donor peripheral blood stem cells and chemotherapy, the other one died. It is concluded that serial quantifications of bcr/abl mRNA levels by RQ-PCR are reliable and can be used to detect the MRD, to monitor the outcome and to predict the relapse.

[Clinical investigation on treatment of children leukemia with non-T-cell depleted haploidentical bone marrow transplantation].

To investigate the efficacy and feasibility of parent non-T cell depleted haploidentical bone marrow transplants (haploidentical BMT) for children with leukemia, the efficacy of haploidentical BMT was evaluated in 8 leukemia children (1.9-9 years) received hematopoietic stem cell transplantation, donors were their parents with HLA-mismatched for two or three loci. Five children were pre-conditioned with a myeloablative regimen consisting of high-dose cytarabine (Ara-C), cyclophosphamide (CY) and total body irradiation. Busulfan (BU), Ara-C and CY were used for preconditioning regimen in other three children. The donors were given G-CSF prior to marrow harvest and the non-T-cell depleted grafts were used. A combination of CsA, MTX, ATG, mycophenolate mofetil (MMF) and CD25 monoclonal antibody were used for GVHD prophylaxis. The results showed that rapid engraftment was observed in all cases after transplantation by cytogenetic evidence. The mean time of neutrophil count exceeded 0.5 x 10(9)/L and the mean time of platelet count exceeded 20 x 10(9)/L were 16 and 17 days after transplantation respectively. Incidence of lethal aGVHD was lower, II-III acute aGVHD was found only in one out of eight patients. Chronic GVHD was observed in five patients, 4 from which showed local cGVHD, one developed extensive cGVHD. During the follow-up of 33 months (range 7-56 months), two patients died from relapsed leukemia, including one relapsed as donor-origin leukemia. Disease-free survival was achieved in the remaining six patients. No death occurred during the follow-up of 6 months. It is concluded that above-mentioned preconditioning regimen and GVHD prophylaxis procedure in non-T-cell depleted bone marrow transplantation from HLA-mismatched parents are effective approaches and safe strategy for the treatment of children leukemia.

[Application of fibrotic bronchoscopy in the diagnosis of pulmonary diffuse infiltration following bone marrow transplantation].

In order to evaluate the diagnostic value of fibrotic bronchoscopy (FB) in the pulmonary infiltration following bone marrow transplantation (BMT), 18 patients with pulmonary complications after BMT from November 2003 to March 2006 were performed with FB. Bronchoalveolar lavage (BAL) and brushing were performed in patients who had received short-term empirical therapy without good response, and transbronchial lung biopsy (TBLB) was carried out in 3 cases. The results showed that 9 out of 10 cases with pulmonary infection, including bacterial pneumonia (n = 3), aspergillosis (n = 2), pneumocystis carinii pneumonia (n = 3) and viral infection (n = 1) were diagnosed by using FB. One case was diagnosed as tuberculosis after open lung biopsy following negative results from twice BAL. 2 out of 8 cases were diagnosed by TBLB as noninfectious pulmonary complications. In conclusion, FB, especially with BAL, is a safe and useful procedure for the evaluation of pulmonary complications, which is particularly suitable for diagnosis of pulmonary infection after BMT. Furthermore, TBLB should be recommended in order to avoid open lung biopsy, if the patients tolerate the operation.

[Correlation between CD34+CD61+ megakaryocyte precursors and platelet engraftment in allogeneic hematopoietic stem cell transplantation].

This study was purposed to investigate the correlation between the dose infused megakaryocytic precursors (CD34+, CD34+CD61+) and recovery time of platelet count following an allogeneic PBSCT and/or BMT through quantitative detection of CD34+ and its subpopulation in peripheral blood and BM mobilized by G-CSF. 24 patients with various hematologic malignancies received PBSCT/BMT from their HLA matched or unrelated donors and haploidentical siblings in April-December 2007. 20 evaluated patients were divided into 2 groups according to different transplant schemes. HLA matched group received PBSCT regime and haploidentical group received PBSCT combined with BMT. CD34+CD61+ subpopulations in sample from patients receiving PBSCT/BMT were measured by flow cytometry immediately or storage over night. The results showed that the median number of infused CD34+, CD34+CD61+ and CD34-CD61+ cells in haploidentical group were 6.24x10(6)/kg (1.53-20.48), 66.19x10(4)/kg (8.16-493.83), and 34.38x10(6)/kg (14.71-109.16) respectively, in HLA matched group those were 4.88x10(6)/kg (1.00-8.24), 14.16x10(4)/kg (11.63-96.87), and 13.50x10(6)/kg (1.74-35.61), respectively. Median days of ANCs>0.5x10(9)/L and platelets>20x10(9)/L were 18.5 (11.0-29.0) days and 16.5 (9.0-35.0) days in haploidentical group respectively; in HLA matched group those were 14.5 (9.0-24.0) and 10.5 (6.0-37.0) respectively. A significance difference of median days for ANC engraftment presented between two groups (p=0.048). There was no significant difference of time for platelet engraftment between 2 groups. For patients with CD34+ cell dose>2x10(6)/kg there was significant difference of time of platelet engraftment between HLA matched and haploidentical groups (p=0.006). The number of CD34+CD61+ cells infused in 12 haploidentical patients or in 8 HLA matched patients were much better correlated with the time of platelet recovery up to 20x10(9)/L than that of number of CD34+ cells infused in total 20 patients (r=-0.768 and p=0.004 for haploidentical CD34+CD61+ cells, r=-0.747 and p=0.033 for HLA matched CD34+ CD61+ cells, r=-0.449 and p=0.047 for CD34+ cells). There was an inverse correlation between the number of infused CD34+ CD61+ cells and time of platelet engraftment. Therefore, as the number of CD34+ CD61+ cells increased, duration of platelet engraftment (time to reach platelet count of 20x10(9)/L) shortened significantly. It is concluded that the determining the number of megakaryocytic precursor by flow cytometry may predict the platelet reconstitutive capacity of the allogeneic hematopoietic stem cell transplantation, which is in haploidentical PBSCT and in BMT.