Establishment of Mouse Model of H-2 Haploidentical Hematopoietic Stem Cell Transplantation from Double Donors / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish a new mouse model of H-2 haploidentical stem cell transplantation from double donors (DHSCT) and compare with conventional haploidentical hematopoietic stem cell transplantation (HSCT) so as to alleviate transplant-related complications.</p><p><b>METHODS</b>The recipients CB6F1 of conventional HSCT group were pretreated by 8 Gy total body irradiation(TBI), and received 3×10donor (male C57) spleen mononuclear cells (spMNC) mobilized by G-CSF within 2 hours after TBI. Recipients CB6F1 of D-HSCT groups accepted 2 Gy TBI, and received total 12×10spMNC mobilized by G-CSF from 2 donors within 2 hours after TBI, each donor donated 6×10cells. According to the different strains and sex of donors, DHSCT were divided into 3 groups: in group A, the stem cells were from male C57 and female BALB/c; in group B, stem cells were from male C57 and male BALB/c, while the stem cells in group C were from male C57 and male C3H. Hematopoietic reconstruction, engraftment, GVHD and survival were observed among these 4 groups.</p><p><b>RESULTS</b>The nadir of white blood cell count after conventional HSCT were lower than 1×10/L and lasted for 3 to 5 days, while not less than 3×10/L after D-HSCT among either group A, B or C. The complete chimerism (CC) in conventional HSCT group was achieved quickly within only 1 week in peripheral blood. Mixed chimerism (MC) in peripheral blood was found within the first week after DHSCT among either group A, B or C, and transformed into stable CC within the second week eventually. Both GVHD morbidity and mortality of conventional HSCT were 100% at 34th day after transplantation.Among DHSCT groups,the overall GVHD morbidity and mortality at 34th day after transplant were 49.6% and 50%(P<0.01,P<0.05), respectively,and 60.4% and 81.2% at 50th day after transplant. Overall survival of 50 days was 50.9% that indicated a long survival in such mice DHSCT. The differences of hematopoietic reconstruction, donor cell engraftment, GVHD incidence, GVHD mortality and OS were not statistically significant among group A, B and C(P>0.05).</p><p><b>CONCLUSION</b>A new mouse model of H-2 haploidentical peripheral blood stem cell transplantation from double donors (DHSCT) has been successfully established by reducing conditioning intensity and increasing graft cell numbers from double haploidentical donors without GVHD prophylaxis. DHSCT successfully achieved stable complete chimerism, less GVHD morbidity and mortality and longer OS without hematopoietic suppression. This study provides experimental evidence for clinical application of HLA haploidentical peripheral blood stem cell transplantation from double donors.</p>

Biological Characteristics of Microvesicles Secreted by Human Peripheral Blood Hematopoietic Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of microvesicles(MV) isolated from human peripheral blood hematopoietic stem cells(PB-HSC) on immune regulation and hematopoiesis.</p><p><b>METHODS</b>PB-HSCs were separated by density-gradient centrifugation and cultrued. The supernatants of PB-HSC at 48 h were harvested for isolation and purification of MV by using ultracentrifugation. The electron microscopy was used to observe the morphology of MV. The protein level in MV was quantified through bicinchoninic acid(BCA) protein assay. Flow cytometry was used to detect the immunophenotype of MV. Human peripheral blood mononuclear cells(PB-MNC) were isolated from healthy donor and treated with isolated MV. After being co-cultured for 12 h, confocal microscopy was used to observe the action mode of MV on PB-MNC. After being co-cultured for 48 h, the levels of IL-2, IL-6, IL-8, IL-10, IFN-γ and TNF-α were detected by ELISA. Flow cytometry was used to detect the changes of T cell subsets and the activation of T cell subsets as well as intracellular cytokine staining after co-culture for 48 h. The methylcellulose was used to assess the hematopoiesis-supportive function of MV as well as co-cultured supernatants.</p><p><b>RESULTS</b>The eletron microscopy revealed that MV were elliptical membrane vesicles. The protein amount in MV ranges from 29 to 110 µg. Flow cytometry showed that MV expressed mix markers on the surface, especially highly expressed MV specific marker CD63(85.86%) and hematopoietic stem cell marker CD34(33.52%). After being co-cultured for 12 h, confocal microscopy showed that MV were merged with PB-MNC. After being co-cultured for 48 h, ELISA showed that the secretion of cytokines IL-6,IL-8, IL-10 as well as TNF-α was increased while the level of IL-2 and IFN-γ was not changed much. The results of flow cytometry showed that there was no significant change in T cell subsets and T cell activation. Staining of intracellular factor showed that IL-8 was increased significantly in CD11ccells. The colony-forming experiments revealed that MV and the co-cultured supernatants could facilitate the colony formation.</p><p><b>CONCLUSION</b>MV isolated from PB-HSC have immune-regulatery function and can prornote hematopoiesis.</p>

Effect of Infusion of Recipient Spleen Cells at Different Time after Murine Haploidentical Hematopoietic Stem Cell Transplantation on Graft Versus Host Disease / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of infusing G-CSF mobilized recipient spleen cells at different time after haploidentical stem cell transplantation(HSCT) on graft-versus-host disease (GVHD) in mice and its possible mechanism.</p><p><b>METHODS</b>Forty mice after HSCT were randomly divided into 4 groups (n=10): GVHD positive control group (control group), 1st d recipient cell infusion group after transplantation (+1 d group), 4th d recipient cell infusion group after transplantation(+4 d group), 7th d recipient cell infusion group after transplantation(+7 d group). The mice in control group were injected the normal saline of same equivalent with experimental group which were given the same amount of G-CSF-mobilized recipient spleen cells. The general manifestation and pathological change of GVHD were observed. The expression changes of CD3CD4, CD3CD8cell subsets and FasL in peripheral blood were detected by flow cytometry.</p><p><b>RESULTS</b>The incidence of GVHD was significantly decreased in +4 d group and the median survival time was longer than 60 days, which was significantly higher than that of control group (24 d), +1 d group (21 d), +7 d group (28 d). (P<0.01, P<0.01, P<0.01). The Fasl expression of peripheral blood T lymphocytes in +4 d group were significantly lower than that in the other 3 groups(P<0.05).</p><p><b>CONCLUSION</b>The +4 d infusion of G-CSF mobilized recipient spleen cells on 4th day after haploidentical HSC transplantation can inhibit the expression of FasL in donor T lymphocytes, and significantly reduce the incidence of GVHD.</p>

Biological characteristics of Microvesicles Derived from Bone Marrow Mesenchymal Stem Cells and Their Capacities Supporting ex vivo Expansion of Hematopoietic Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the biological characteristics of microvesicles(MV) derived from bone marrow mesenchymal stem cells (BM-MSC) and their capability supporting ex vivo expansion of hematopoietic stem cells(HSC).</p><p><b>METHODS</b>The MV from cultured BM-MSC supernatant were isolated by multi-step differential velocity contrifugation; the morphological characteristics of MV were observed by electron microscopy with negative staining of samples; the protein level in MV was detected by using Micro-BCA method; the surface markers on MV were analyzed by flow cytometry. The peripheral blood HSC(PB-HSC) were isolated after culture and mobilization; the experiment was diveded into 2 group: in MV group, the 10 mg/L MV was given, while in control group, the same volume of PBS was given; the change of PB-HSC count was observed by cell counting; the change of surface markers on PB-HSC was detected dynamically by flow cytometry; the cell colony culture was used to determin the function change of PB-HSC after co-culture with MV.</p><p><b>RESULTS</b>MSC-MVs are 20-100 nm circular vesicles under electron microscope. About 10 µg protein could be extracted from every 1×10MSC. The flow cytometry showed that CD63 and CD44 were positive with a rate of 96.0% and 50.2%, while the HLA-DR, CD34, CD29 and CD73 etc were negative. When being co-cultured with GPBMNC for 2 days, the cell number of MV groups was 1.49±0.15 times of the control group (P>0.05). When being co-cultured for 4 days, the cell number of MV groups was 2.20±0.24 times of the control group(P<0.05). The CD34cell number of MV groups was 1.76±0.30 times the control group after culture for 2 day and 1.95±0.20 times after culture for 4 day.</p><p><b>CONCLUSION</b>The MV has been successfully extracted from MSC culture supernatant by multi-step differential velocity centrifugation. MSC-MV can promote HSC expansion in vitro.</p>

Expression of WT1 Gene in Bone Marrow of Patients with Acute Myeloid Leukemia and Its Influence on Prognosis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression level of WT1 gene in bone marrow of patients with acute myeloid leukemia (AML) and its relationship with prognosis.</p><p><b>METHODS</b>The copy numbers of WT1 and internal reference gene in bone marrow samples from 75 newly diagnosed AML patients were detected by using real-time quantitative PCR. The gene WT1 expression level was determined by the ratio of the copy numbers of WT1 to reference gene. And the clinical characteristics, the complete remission (CR) rate after induction chemotherapy, 2-year overall survival (OS) rate and event-free survival (EFS) rate were calculated and analysed.</p><p><b>RESULTS</b>The expression level of WT1 did not significantly correlate with common clinical parameters such as age, sex, molecular abnormality, FAB classification and risk stratification. The CR rate in the high WT1 expression group before treatment was 65.4%, which was lower than that of 93.9% in the low expression group (χ2=8.25, P<0.01). The 2-year overall survival rate and event-free survival rate of the two groups were statistically significantly different (P<0.05), and the OS and EFS rates in high WT1 expression group were lower than those in low expression group. After the induction chamotheropy for about 1, 3 month and 6 months, the 2-year OS rate significantly increased in patients with decrease of WT1 gene expression level by one log or more (P<0.05).</p><p><b>CONCLUSION</b>The expression level of WT1 gene in bone marrow may be an effective marker to evaluate therapy efficacy and prognosis for AML patients (non APL).</p>

Efficacy of Nonmyeloablative Allogeneic Hematopoietic Stem Cells for 14 Case of Severe Acquired Aplastic Anemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic efficacy of nonmyeloablative allogeneic hematopoietic stem cells transplantation for severe acquired aplastic anemia (SAA).</p><p><b>METHODS</b>Fourteen patients with severe acquired aplastic anemia received nonmyeloablative allogeneic hematopoietic stem cells transplantation from HLA matched sibling donors, among them 8 cases were dagnosed as SAA-I, 6 cases were diagnosed as SAA-II. The conditioning regimen consisted of fludarabine (FIUD), cyclophosphamide (CTX) and anti-thymocyte globulin (ATG/ALG). The prophylaxis for graft-versus-host disease (GVHD) was performed with cyclosporine (CsA) combined with mycophenolate mofetil (MMF) or tacrolimus (FK506).</p><p><b>RESULTS</b>All the patients gained a quick successfully engraftment of donor hametopoietic cells. The mean recovery time for neutrophil and platelet was 9 d and 13 d respectively. All the patients have acquired a full donor chimerism before 14 d. There were only 2 cases of GVHD: one out of them was acute skin GVHD (grade I) at day 70 after transplantation and the other was chronic liver GVHD (grade I) in 1 years after transplantation, the GVHD more than degree II did not coccur in all patients, 9 patients with bacterial and fungal mixed infection and (or) virus infection were observed, and improved after anti-infection therapy. The median follow-up time were 54.5 months (ranged between 5-144 months), and 12 patients remain disease-free survival currently, only 2 patients died of fungal infectin.</p><p><b>CONCLUSION</b>Transplantation of nonmyeloablative allogeneic hematopoietic stem cell is safe and effective for the treatment of severe acquired aplastic, but the prevention, treatment and monitoring of infection need to be enhance.</p>

Establishment and identification of a H-2 completely mismatched microtransplantation model of leukemia mouse / 中国实验血液学杂志

This study was purposed to establish and identify a H-2 completely mismatched microtransplantation model of leukemia mouse. The recipients were female BALB/c mice, while donors were male C57BL/6J mice. Recipients were inoculated intravenously with 1×10(6) of WEHI-3 cells, a cell line of myelomonocytic leukemia. Donors received 100 µg/kg G-CSF mobilization through hypodermic injection, every 12 hours, and it last 5 days. Chemotherapy regimens was MA (mitoxantrone+cytarabine), and it last 4 days. Recipients were given chemotherapy conditioning without GVHD prophylaxis after inoculation of leukemic cells for 2 days, and within 8 hours after last chemotherapy received donor mobilized spleen mononuclear cells (sMNC). The number of sMNC was (3, 6, 12) ×10(7), respectively. The early death rate, recovery level of WBC in peripheral blood and leukemia load were compared between chemotherapy and microtransplantation groups. The donor chimerism was detected by RT-PCR. From the clinical manifestation and pathological features, the GVHD in recipients was evaluated. The results showed that the early mortality in chemotherapy group was 25%, meanwhile those in the (3, 6, 12)×10(7) groups were 16.67%, 8.33%, 8.33%, respectively. The(3, 6)×10(7) groups has a stronger hematopoietic recovery capability than that in chemotherapy and 12×10(7) groups (P < 0.05) . There were more leukemic cells in chemotherapy mice than that in microtransplantation mice (P < 0.01) , and (12, 6)×10(7) groups had lower leukemia load than that in 3×10(7) group (P < 0.05) . No signs of GVHD were observed in microtransplantation mice. The donor microchimerism could be discovered at eraly 2 weeks after donor cell transfusion. It is concluded that a H-2 completely mismatched microtransplantation model of leukemia mouse has been successfully established, and it will provide a experimental base for studying microtransplantation in clinic.

Expression of NOV and BNIP3 gene in mouse myelomonocytic leukemia and its significance / 中国实验血液学杂志

This study was aimed to investigate the expression level of NOV and BNIP3 mRNA in mice myelomonocytic leukemia (AML-M(4)) and its significance. The mice were inoculated intravenously with myelomonocytic leukemia cells of WEHI-3, and divided randomly into chemotherapy group and control (untreated) group. Bone marrow samples were then collected from both groups at different times. The NOV and BNIP3 mRNA expression were detected by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and the relationship between these expression levels and clinical significance in leukemia incidence and progression were analyzed with β-actin as the housekeeping gene. The results showed that the mean values of NOV and BNIP3 increased gradually from 2 weeks after inoculation and achieved highest level at death in control group. Expression level of NOV increased from 1.85E-05 before inoculation to 3.57E-02 at death (p < 0.05), and BNIP3 from 3.44E-03 to 3.48E-02. While 2 gene expression in the chemotherapy group decreased quickly to 2.51E-05 and 1.58E-03 (p < 0.05) respectively after chemotherapy, which were close to the level before inoculation (p > 0.05). The 2 gene expressions again rose at relapse, and difference of expression level between 2 group at death were no statistically significant (p > 0.05). It is concluded that the expression of NOV and BNIP3 in leukemia AML-M(4) is significantly higher than that in normal controls, of which high level expression is an important factor in the development of leukemia. Close relation between the therapeutic effect and expression level of these two genes suggests the great value in prognostic evaluation and MRD detection.

Relationship between WT1-specific T-cell subsets and graft-versus-host disease after nonmyeloablative allogeneic transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the relationship between WT1-induced T-cell subsets and graft-versus-host disease (GVHD) after nonmyeloablative allogeneic hematopoietic stem cell transplantation (NST).</p><p><b>METHODS</b>Peripheral blood mononucleated cells (PBMCs) from 19 patients who expressed WT1 and developed GVHD after NST were simulated by WT1126-134 peptide in vitro, and proportions of WT1-induced-T-cell subsets (Tc1, Tc2, Th1, Th2 cells) before and after transplant were detected by intracellular cytokine staining (ICCS) assay. WT1-specific CD8(+) CTLs of 14 patients with HLA-A*0201 were detected by HLA-A*0201/WT1 pentamer.</p><p><b>RESULTS</b>(1) 17 of 19 patients developed GVHD, among whom proportions of Tc1 and Th1 cells, achieved peak value in 16 patients at occurrence of GVHD (P = 0.039); (2) The peak proportions of Tc1 and Th1 cells in patients with aGVHD above grade II were higher than those with grade I, but being no statistical difference (P = 0.900 and P = 0.140, respectively); (3) The peak proportion of Th1 cells (P = 0.004), but not Tc1 cells (P = 0.060) in patients with extensive cGVHD was significantly higher than that in patients with limited one; (4) Proportions of Tc1, Th1 and WT1(+)CD8(+)CTL in patients without GVHD were similar to those in patients with Grade I aGVHD, but lower than those in aGVHD above grade II.</p><p><b>CONCLUSION</b>GVHD promotes the generation of WT1-induced GVL effect, and the intensity of the latter maybe correlated with the intensity of GVHD, especially cGVHD. Th1 cells play a more important role in the enhancement of WT1-induced GVL effect in extensive cGVHD patient than in limited cGVHD patients.</p>

Application of HLA-A*0201/WT1 pentamer combined with intracellular IFNgamma+ staining in detecting circulating WT1 specific T cells in leukemia / 中国实验血液学杂志

This study was purposed to investigate the value of combination of pentamer and intracellular IFNgamma staining in the qualitative and quantitative detection of circulating antigen-specific T cells. WT1 expressions in 14 HLA-A*0201+ patients and their matched donors were detected by RT-PCR, and circulating WT1 specific T cells were assayed by HLA-A*0201/WT1 pentamer combined with intracellular IFNgamma+ staining. The results showed that the low level of WT1 expression was found only in 2 cases out of 14 donors, but different levels of WT1 expression could be observed in all leukemic patients. The WT1+CD8+ CTL and WT1+IFNgamma+ cells did not detected in all 14 donors, but WT1+CD8+ CTL cells in 2 patients and WT1+IFNgamma+ cells in 3 patients could be detected before transplantation respectively, there was no significant difference between them, while the WT1+CD8+ CTL cells and WT1+IFNgamma+ cells both could be detected in all 14 patients after transplantation, the positive detection rate after transplantation was obviously higher than that before transplantation. The WT1+CD8+ and WT1+ IFNgamma+ cells could be detected within 30 days after transplantation, but the positive detection rate of WT1+IFNgamma+ cells was higher than that of WT1+CD8+ CTL cells (p=0.014). The median peak value of WT1+CD8+ CTL cells was 0.18% in 14 patients, and the median peak value of WT1+IFNgamma+ cells was 0.83% in 14 patients, the later was significantly higher than former. The median peak time of WT1+CD8+ CTL cells was 75 days after transplantation, while the WT1+IFNgamma+ cells was 105 days after transplantation, there was no significant difference between them. It is concluded that pentamer and intracellular IFNgamma staining may effectively detect circulating WT1 specific T cells in leukemic patients, and the combination of these two methods profit to the exact qualitation and quantitation of circulating antigen-specific T cells.

Establishment and evaluation of quantitative analysis method for donor chimerism of mice / 中国实验血液学杂志

This study was aimed to explore the effectiveness of sequential and quantitative detection method for analysing donor chimerism (DC). In order to simulate a mouse model of haploidentical stem cell transplantation, C57BL/6 male mice were used as donors, while CB6F1 female mice were used as recipients and were divided into 2 groups. The two groups of recipients were irradiated with 2 Gy and 6 Gy from (⁶⁰CO gamma-ray source respectively, and then were inoculated intravenously with bone marrow cells (BMCs) and spleen mononuclear cells (SPMNCs). Quantitative analyses of DC were performed with real-time PCR or flow cytometry (FCM) on different days after transplantation. The results showed that real-time PCR and FCM both have advantages and disadvantages in the detection of DC. When DC amount in group of 6 Gy was > 90% with stable macrochimerism for more than 3 months, the efficacy of detection by FCM was well; while the DC amount in group of 2 Gy was below 10% and gradually transformed to different forms of microchimerism until disappeared, in which condition the use of real time-PCR was more appropriate. It is concluded that FCM can detect macrochimerism with high accuracy but would fail when DC amount is less than 1% due to sensitivity limitation, while the real time-PCR is more sensitive for detecting microchimerism but lack of accuracy for detecting macrochimerism. Combination of the two methods can afford sensitive and accurate tool for quantitative analysis of chimerism in mouse model of transplantation and adoptive immunotherapy.