Establishment and Applications of Double-Fluorescent Protein Allo-Transplantation Mice Model / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish allo-transplantation model by using mRFP⁺ to eGFP⁺ transgenic mice and to observe the distribution of donor cells and donor-recipient cellular interaction in the bone marrow after semi-solid decalcification (SSD).</p><p><b>METHODS</b>After myeloablative irradiation, C57BL/6 female eGFP⁺ transgenic mice were infused with (5 × 10⁶) bone marrow cells from FVB male donor mice through tail vein. The control group was infused with PBS. Then the general conditions, engraftment level, hematopoietic recovery, incidence of GVHD and survival of recipients were evaluated after transplantation. In the recovery process, SSD was used to treat the femora before observing the cells distribution, morphology and interaction by confocal microscopy directly or after making frozen section.</p><p><b>RESULTS</b>WBC of recipient eGFP⁺ mice was recovered on (20 ± 3.07) d, (93.94 ± 1.59)% in peripheral cells were RFP⁺ cells (n = 10), GVHD happened in 4 of 10 mice within 1 month. During SSD, the hard components were replaced gradually and RFP⁺ cells could be seen mainly in the bone trabecula and surrounded by eGFP⁺ cells under confocal microscope, their interactions could be further observed clearly in bone marrow microenvironment in three-dimensional reconstruction.</p><p><b>CONCLUSION</b>The double fluorescent allo-transplantation mouse model successfully established, by means of our novel protocol named SSD, the donor and recipient cell location and their interaction can be visually observed, which provides the basis for clinical studies on the distribution and homing of donor cells, and some related explorations after transplantation.</p>

Anti-apoptotic effect of Astragalus Polysaccharide on myeloid cells / 中国实验血液学杂志

This study was aimed to assess the effect of Astragalus Polysaccharide (ASPS) on in-vitro hematopoiesis. CFU-GM assays were used to determine the effect of ASPS and thrombopoietin (TPO) on granulocytic-monocyte progenitor cells. The CFU assays were also used to investigate the effect of ASPS on the proliferation of HL-60 cells.HL-60 cells were cultured with serum-free RPMI 1640 medium and treated with or without of different concentrations of ASPS. After 72 h incubation, the number of cells were counted.In addition, the caspase-3 and JC-1 expression was determined by flow cytometry with Annexin V/PI double staining. The results showed that ASPS (100, 200 µg/ml) and TPO (100 ng/ml) significantly promoted CFU-GM formation in vitro. Various concentrations of ASPS and TPO also promoted the colony formation of HL-60 cells, the largest effect of ASPS was observed at a concentration of 100 µg/ml. There were no synergistic effects between TPO and ASPS on cellular proliferation. The results also showed that ASPS significantly protected HL-60 cells from apoptosis in condition of serum-free medium culture, suppressed caspase 3 activation, and reduced the cell apoptosis. It is concluded that ASPS can significantly promote the formation of bone marrow CFU-GM and the proliferation of HL-60 cells, the optimal concentration of ASPS is at 100 µg/ml. In the absence of serum inducing apoptosis, ASPS also significantly reduced the apoptosis of HL-60 cells via suppressing the activation of caspase-3.

Simultaneous analysis of telomere length and cell surface antigen in leukemia by multicolor Flow-FISH / 中国实验血液学杂志

This study was purposed to explore the feasibility of simultaneous analysis of telomere length and cell surface antigen by multicolor Flow-FISH to assess minimal residual disease (MRD) in leukemia. The telomere length in 34 leukemia patients versus 20 normal controls was compared by using Flow-FISH, and the relationship between telomere length and therapeutic effect and prognosis was analyzed preliminarily. As for those patients with follow-up samples, the changes of telomere length combined with surface antigen in different courses of disease were observed by multicolor Flow-FISH. The results indicated that the telomere length of de novo patients was significantly shorter than that of controls except the patients in chronic myeloid leukemia-chronic phase (CML-CP). The shorter telomere, the lower complete remission (CR) rates were observed in acute leukemia cases and the shorter duration of CP before onset of blast phase (BP) occurred in CML cases. The acute leukemia patients showed longer telomere and fewer cells expressed the related antigen after CR. The telomere length of cases with continued CR remained at normal level during remission, and there was no increased expression of the specific antigen. However, the telomere of relapsed cases shortened again after relapse with elevated specific antigen expression. In the relapsed cases, the telomere of related antigen positive cells shortened ahead of telomere length change of the whole cells and morphologic change of bone marrow cells. It is concluded that analysis of telomere length by flow-FISH manifests the significance for monitoring disease conditions, estimating prognosis and guiding therapy in all kinds of leukemia. The simultaneous analysis of telomere length and cell surface antigen by multicolor flow-FISH may monitor abnormal clone or clonal evolution to predict recurrence more sensitively and specifically, and may provide a promising and widely applicable method for monitoring MRD in leukemia.

CD56 and CD11b antigen expressions in patients with acute monocytic leukemia and the clinical implications / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expressions of cell surface differentiation antigen CD56 and CD11b antigen in acute monocytic leukemic (AML-M(5)) cells and their clinical significance.</p><p><b>METHODS</b>A total of 113 cases of de nove adult AML-M(5) were examined genetically and immunologically using G-banding technique, interphase fluorescence in situ hybridization (I-FISH) and flow cytometry immunophenotyping, and the results were analyzed in relation to their clinical data.</p><p><b>RESULTS</b>Of the 113 cases, the expression rates of CD56 and CD11b was 28.32% and 73.45%, respectively. The CD56(+) patients had high CD11b expression, and the expression levels of CD11b and CD56 were positively correlated (P<0.05). The incidence of karyotypic abnormalities was 48.57% (55 cases) in these patients, including 25 (22.12%) with 11q23 aberrations. Twenty-five cases were positive for MLL gene abnormalities as found by I-FISH analysis. Compared with the patients positive for both CD56 and CD11b, those negative for both CD56 and CD11b showed increased peripheral blood white blood cell (WBC) count and also increased blast and progenitor cells in the bone marrow (P<0.05); the former patients often had karyotypic abnormalities, commonly involving 11q23 aberrations (P<0.05), whereas the latter patients presented more likely with extramedullary infiltration and refractory leukemia (P<0.01) with lowered complete remission rate and shortened median survival time (P<0.01). CD56-positive patients were more likely to have karyotypic abnormalities and refractory leukemia than CD11b-postive patients (P<0.05), but the peripheral blood WBC counts, bone marrow blast and progenitor cells, extramedullary infiltration, complete remission rate or median survival time showed no significant differences between them (P>0.05).</p><p><b>CONCLUSION</b>AML-M(5) patients with CD56 positivity and high expression of CD11b often have aberrant karyotypes, commonly involving 11q23/MLL gene abnormality. These patients frequently develop extramedullary infiltration and refractory leukemia often with poor prognosis.</p>

Clinical characteristics and outcomes of 59 patients with acute lymphoblastic leukemia positive for BCR/ABL / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the clinical characteristics and outcomes of BCR/ABL-positive acute lymphoblastic leukemia (BCR/ABL360888725-ALL) and screen the prognostic factors for BCR/ABL360888725-ALL.</p><p><b>METHODS</b>From January 2001 to May 2008, 59 patients (median age of 32 years ranging from 3 to 69 years) with the diagnosis of BCR/ABL360888725-ALL by fluorescence in situ hybridization received induction chemotherapy with VDLP-/+Ara-C regimen. The patients who failed to respond to the chemotherapy received subsequent consolidation chemotherapy with imatinib (400-800 mg/day) (17 cases) or allogeneic hematopoietic stem cell transplantation (allo-HSCT) (16 cases).</p><p><b>RESULTS</b>Of the 59 patients, 32 (58.3%) achieved complete remission (CR) after the first induction cycle. In patients with peripheral white blood cell (WBC) count <30=10(9)/L, 30-99.9(9)/L and > or =100(9)/L, the CR rates were 75.0% (18/24), 56.3% (9/15) and 26.3% (5/19) (P=0.006), and the overall survival probability of 2 years ( OSs of 2-yrs) was 24.7%, 22.5% and 21.1%, respectively (P=0.180). According to the FAB classification, 56 cases were divided into L1, L2 and biphenotypic acute leukemia (BAL) subgroups, and their CR rates were 66.7% (6/9), 63.2% (24/38) and 22.2% (2/9) (P=0.029), with OSs of 2-yrs of 22.2%, 27.0% and 22.0%, respectively (P=0.623). In terms of immunophenotype grouping by EGIL, the patients with ALL, myeloid antigen-positive ALL and BAL had CR rates of 61.1% (11/18), 60.6% (20/33) and 12.5% (1/8) (P=0.039), and the OSs of 2-yrs of 22.7%, 21.0% and 18.8%, respectively (P=0.643). In 55 patients with known karyotype, the CR rates were 71.4%(5/7), 70.8% (17/24) and 37.5% (9/24) in normal, sole t(9;22) abnormality, t(9;22) with additional abnormalities groups (P=0.046), with the OSs of 2-yrs of 42.9%, 34.0% and 7.3%, respectively (P=0.000). The patients complicated by septicemia had significantly lower OSs of 2-yrs than those without septicemia (0% vs 38.8%, P=0.005). The OSs of 2-yrs were significantly higher in patients with consolidation chemotherapy with imatinib than those without (48.0% vs 11.2%, P=0.001), and allo-HSCT was associated with significantly higher OSs of 2-yrs than exclusive chemotherapy (54.2% and 8.5%, P=0.000).</p><p><b>CONCLUSION</b>BCR/ABL360888725-ALL with WBC> or =100 x 10(9)/L, presence of BAL diagnosed by FAB or FACM, t(9;22) with additional chromosome abnormalities all adversely affect the treatment results, and additional chromosome abnormalities and septicemia are associated with lower OSs of 2-yrs. Imatinib treatment and allo-HSCT can both improve the OSs of 2-yrs of the patients with BCR/ABL(+)-ALL.</p>

Effects of graft compositions on hematopoietic reconstitution and graft-versus-host disease in related donor peripheral blood stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of graft composition on hematopoietic reconstitution and graft-versus-host disease (GVHD) in HLA-identical related donor peripheral blood stem cell transplantation (PBSCT) for hematological malignancies.</p><p><b>METHODS</b>The relationship between the number of graft composition and their hematopoietic reconstitution and GVHD in 107 patients with hematological malignancies undergoing HLA-identical related donor PBSCT was retrospectively analyzed.</p><p><b>RESULTS</b>None of the graft composition numbers had correlation with the time of neutrophil reconstitution. There was a negative correlation between the number of mononuclear cells (MNCs) or CD34+ cells and the time of platelet reconstitution (P < 0.05) with the absolute correlation coefficients below 0.4, and so did between the number of CD34+ or CD34+ CD38- cells and the development of acute GVHD (P < 0.05) and their absolute correlation coefficients. Each lymphocyte subset number had no correlation with acute GVHD. There was a positive correlation between the number of CD25+ CD4+, CD3+ or CD4+ CD3+ cells or CD4+ /CD8+ ratio and the development of chronic GVHD (P < 0.05). And the correlation coefficients all exceeded 0.4, more over, CD25+ CD4+ cells number reached up to 0.78. CD34+, CD34+ CD38- cells number had no correlation with chronic GVHD.</p><p><b>CONCLUSION</b>In HLA-identical related donor PBSCT, further increasing infused MNC, CD34+, CD34+ CD38- cells can no more accelerated hematopoietic reconstitution after these cells attained their threshold values, otherwise the incidence of cGVHD might increase due to the increase of the accompanied lymphocytes.</p>

Role of membrane-bound human leukocyte antigen G in inducing immune tolerance after allogeneic hematopoietic stem cell transplantation / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the role of human leukocyte antigen G (HLA-G) in the better effect of allogenetic bone marrow transplantation than that of peripheral blood stem cell transplantation.</p><p><b>METHODS</b>Flow cytometry was used to detect the expression of membrane-bound HLA-G (mHLA-G) on donor peripheral blood (PBC) or bone marrow (BM) mononuclear cells. The levels of soluble HLA-G (sHLA-G) in the plasma and bone marrow fluid were determined using enzyme-linked immunosorbent assay (ELISA) before and after granulocyte colony-stimulating factor (G-CSF) mobilization.</p><p><b>RESULTS</b>The mean levels of mHLA-G after G-CSF mobilization in the PBC and BM were significantly higher than that before G-CSF mobilization (P=0.001 and 0.000), but the plasma levels of sHLA-G showed no significant changes after the mobilization (P=0.279). The mean levels of sHLA-G in the BM fluid significantly increased (P=0.002) to a level higher than that in the PBC after G-CSF mobilization (P=0.004).</p><p><b>CONCLUSION</b>HLA-G plays an important role in immune tolerance after hematopoietic stem cell transplantation with G-CSF mobilization.</p>

STI571 enhances the effect of arsenic trioxide and Velcade on bcr/abl+-CD34+ cell proliferation and apoptosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of STI571, arsenic trioxide (As2O3) and Velcade, used alone or in combination, on the proliferation and apoptosis of bcr/abl+-CD34+ cells.</p><p><b>METHODS</b>bcr/abl+-CD34+ cells isolated from the bone marrow of patients with chronic myeloid leukemia (CML) were treated for 96 h with STI571, As2O3 and Velcade either alone and in combination, and the cell proliferation and apoptosis were analyzed by CCK-8 assay and flow cytometry. The morphological changes of the apoptotic cells were observed by Hoechst33342 staining and fluorescent microscope. The inhibitory effects of the drugs on normal CD34+ cells were also observed.</p><p><b>RESULTS</b>Low-concentration STI571, As2O3 or Velcade all dose-dependently inhibited bcr/abl+-CD34+ cell proliferation without obvious apoptosis-inducing effects. STI571 at 0.25-2 micromol/L combined with As2O3 at 2.5 micromol/L and with Velcade at 15 nmol/L both significantly increased the cell inhibition and apoptosis rates, showing obvious additive or synergistic effects of the drugs without further enhancement of normal CD34+ cell inhibition.</p><p><b>CONCLUSION</b>Combination with STI571 enhances the effects of As2O3 and Velcade on bcr/abl+-CD34+ cells, suggesting the potential clinical value of this regimen.</p>

Effect of G-CSF on the proliferation and differentiation of bcr/abl(+)-CD34+ cells from CML patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of granulocyte colony-stimulating factor (G-CSF) on the proliferation and differentiation of bcr/abl(+)-CD34+ cells.</p><p><b>METHODS</b>bcr/abl(+)-CD34+ cells were isolated from bone marrow of chronic myelocytic leukemia (CML) patients and were treated with 0, 10, 100, 1000 ng/ml of G-CSF for 48, 96, 144 hs. CD34 cells from normal bone marrow were used as controls. Cell proliferation was determined by trypan blue dye exclusion, cell-cycle and antigen differentiation were determined by flow cytometry and cell morphology was observed under light microscope.</p><p><b>RESULTS</b>The number of bcr/abl(+)-CD34+ cells was increased obviously in all groups. After cultured for 48 and 96 h, the number of bcr/abl(+)-CD34+ cells at G-CSF 10 ng/ml group was significantly higher than that in G-CSF 0 ng/ml group (P < 0.05) , the number of normal CD34 cells was increased only in the presence of G-CSF. After cultured for 48, 96 and 144 h, the cell number in G-CSF 100 ng/ml group was significantly higher than that in G-CSF 0 ng/ml group (P < 0.05, P < 0.01, P < 0.01, respectively). After cultured for 144 h, the cell percentages in G0/G1 phase for bcr/abl(+)-CD34+ cells in G-CSF 10, 100, 1000 ng/ml groups were significantly less than that in G-CSF 0 ng/ml group (P < 0. 05), and that for normal CD34 cells in G-CSF 10, 100, 1000 ng/ml groups were significantly less than that of G-CSF 0 ng/ml group after cultured for 48 and 96 h. The expressions of CD34 on bcr/abl(+)-CD34+ cells and normal CD34+ cells were decreased along with the culture duration, accompanied by the expression of CD33 and CD13 increased first and decreased later, which was not correlated with the concentration of G-CSF. Both bcr/abl(+)-CD34+ cells and normal CD34+ cells showed mature morphology along with proliferation and differentiation.</p><p><b>CONCLUSIONS</b>G-CSF promotes proliferation of both bcr/abl(+)-CD34+ cells and normal CD34+ cells, but not necessary for the former, and the former differentiates more rapidly than the latter does, but both was independent of G-CSF.</p>

In vitro effect of STI571 on expression of c-kit in bone marrow cells from patients with acute non-lymphocytic leukemia / 中国实验血液学杂志

This study was designed to explore the influence of STI571, a tyrosine kinase inhibitor, on the expression of c-kit in the bone marrow cells from patients with acute non-lymphocytic leukemia (ANLL). The cells were exposed to various concentration of STI571 for 72 hours, the expression of c-kit mRNA and CD117 was assayed by RT-PCR and flow cytometry, respectively. The results showed that STI571 treatment induced concentration-dependent decrease of c-kit and CD117 expression, which was significant lower than that in group before treatment and untreated control groups (P < 0.05) and 0.1 micro mol/L STI571 group was significantly higher than that in 10 micro mol/L group (P < 0.05). It is concluded that STI571 has an obvious effect on the restraint of c-kit in ANLL cells.

Study of murine hematopoietic stem/progenitor cell mobilized by recombinant human interleukin 11 combination with granulocyte-colony stimulating factor / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the recombinant human interleukin 11 (rhIL-11) in combination with granulocyte-colony stimulating factor (rhG-CSF) for mobilizing peripheral blood stem/progenitor cells in C57BL/6 mice.</p><p><b>METHODS</b>rhIL-11,250 micro g x kg(-1) x d(-1) per mouse alone or in combination with rhG-CSF 250 micro g x kg(-1) x d(-1) per mouse was administered to C57BL/6 mice from day 1 to 7. The changes of peripheral white blood cell count (WBC), platelet counts (BPC) and hematopoietic stem/progenitor cells yields were observed.</p><p><b>RESULTS</b>The results showed that rhIL-11 alone or in combination with rhG-CSF resulted in increase in absolute numbers of WBC, BPC, CD(34)(+) cells, and CFU-GM, CFU-E, CFU-MK yields in peripheral blood more than those of control (P < 0.001). The yields of CFU-MK was significantly more than that of rhG-CSF group (P < 0.001).</p><p><b>CONCLUSION</b>rhIL-11 alone or in combination with G-CSF could significantly mobilize hematopoietic stem/progenitor cells from bone marrow into peripheral blood.</p>