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>

Clinical efficacy of switching to 2nd generation of tyrosine kinase inhibitor on CML patients at poor responses to imatinib / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was to investigate the timing and clinical efficacy of switching to the 2nd generation of tyrosine kinase inhibitor (TKI) for CML patients at poor response to imatinib (dissatifed efficacy or intolerance).</p><p><b>METHODS</b>The therapeatic efficacy and side reaction of switched 2nd TKI in patients with newly diagnsed CML-CP who poorly responded to imatinib were observed, anong them 3 cases were intolerant, 6 cases did not acquire satisfied efficacy.</p><p><b>RESULTS</b>After switching to 2nd generation TKI, 3 patients with intolerance achieved complete cytogenetic remission (CCyR) in 3 months, and major molecular remission (MMR) in 3-6 months. All of them achieved optimal efficacy according to European Leukemia Network (ELN), but the pleural effusion appeared in 1 case after use of 2nd generation of TKI for 3 months, and the dadatinib was stoped temporally, and the curative efficacy still was maintained. Among 6 cases with poor efficacy by treatment with imatinib, 2 cases with BCR/ABL mutation progressed after switching 2nd generation of TKI, out of them 1 case with poor tolerance progeressed to the accelerated phase, but was cured by haploidentical allogeneic hematopoictic stem cell transplantation, 1 case progressed to blastic crisis and died of serious infection; the another 4 cases achieved MMR in 3-12 months after using 2nd generation of TKI, and maintained CMR for 12-36 months.</p><p><b>CONCLUSION</b>CML-CP patients without the optimal response to imatinib should be treated by switching to 2nd generation of TKI as soon as possible, and thereby patients may acquired satisfactory therapentic efficacy.</p>

A case report of refractory pleural effusion associated with dasatinib in the treatment of chronic myeloied leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the clinical features and prognosis of pleural effusion associated with dasatinib in treatment of chronic myeloid leukemia(CML).</p><p><b>METHODS</b>A 49 year old mal patient with CML who suffered pleural effusion(grade 3) associated with dasatinib was analyzed and summarized.</p><p><b>RESULTS</b>the patient achieved complete molecular response(CMR) after treating with dasatinib 100 mg once daily for 3 months. However, the symptom of chest distress occured in the patient after dasatinib treatment for 6 months, the chest CT scan showed bilateral pleural effusion(grade 3), the pleural effusion related with dasatinib was diagnosed, therefore the diuretic and steriod drugs were given, thoracocentesis was also used to relieve the symptom, after treatment for 5 weeks the pleural effusion disappeared, but the pleural effusion recurred when the patient taken dasatinib again, thus the dasatimib was permanently discontinued, but the patient was in CMR. Six months later, the patient began to take Imatinib (first TKI) 300 mg/d, good effects were achieved and no serious adverse effects were observed. Up to now, the patient still is in CMR for 20 months.</p><p><b>CONCLUSION</b>In the treatment of CML, appropriate TKI should be chose according to basic disease, and pleural effusion is one of the most common adverse effects during the therapy with dasatinib, close monitoring and timely intervention are necessary. For these patients who were intolerable to recieve the dasatinib, the conversion to another TKI may acquire satisfactory curative effect with tolerance of patients.</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.

Research advances on microchimerism / 中国实验血液学杂志

The microchimerism is a status of the microcell or DNA of an individual in another one with genetic differences. Taking an overall view about the discovery and research of the microchimerism, it was found that although the study of the microchimerism emphasizes the formation, origin, distribution, type, relationship to disease and several other aspects, the objects of the study are always the microchimerism that obtained naturally. As it is known to all, the microchimerism can also be produced in some clinical treatment, such as in the transplant and transfusion, but compared with the microchimerism gained naturally, obviously, the study for the iatrogenic microchimerism formed in the treatment is not elaborate enough. The curative effect of micro transplantation, a new technique for leukemia treatment, is obvious, but its mechanism is unclear, whether that is related to microchimerism still needs further research. This review summarizes the study history and perspective of the microchimerism so as to provide some ideas for studying the action mechanism of microchimerism in micro transplantation.

Risk factors analysis of cytomegalovirus infection after nonmyeloablative allogeneic peripheral blood stem cell transplantation / 中国实验血液学杂志

The aim of this study was to analyze the risk factors of cytomegalovirus (CMV) infection and CMV disease after nonmyeloablative allogeneic hematopoietic stem cell transplantation(NST) and develop a rational strategy for the diagnosis, monitoring and preemptive treatment of CMV infection. The Clinical data of 80 patients undergoing NST from November 2009 to November 2012 in the hospital 307 were retrospectively analyzed. The cytomegalovirus load in peripheral blood of patients was detected by using RT-PCR. The results indicated that the incidence of CMV infection was 77.5% (62/80), and the median time for the positive CMV-DNA firstly detected by RT-PCR was day 35 (17-133) after NST. The total of 100-day cumulative incidence of CMV disease was 11.3%(9/80) after early preemptive therapy. Both univariate and multivariate analysis showed thymoglobulin (ATG) used in preconditioning regimen, other herpesvirus infection and fungal infection in medical history before NST were the risk factors of CMV infection after NST.Univariate analysis revealed that CMV viremia and ATG used in preconditioning regimen were the risk factors for CMV disease, while the same result was not found in the multivariate analysis.The incidence of CMV infection in patients with II-IV grade of aGVHD was 91.3%,while the incidence of CMV infection in patients with 0-1 grade of aGVHD was 71.9% (P = 0.06), it seems that II-IV grade of aGVHD was not the risk factor of CMV infection for NST. It is concluded that the ATG used in preconditioning regimen may increase the incidence of both CMV infection and CMV disease after NST. CMV infection easily accompanies by other herpesvirus infection and fungal infection.Therefore other herpesvirus infection and fungal infection should be attentively monitored and prevented after trans-plantation.

Prophylactic G-CSF mobilized donor lymphocytes infusion after non-myeloablative stem cell transplantation prevents relapse in patients with high-risk leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the anti-leukemia effects of prophylactic G-CSF mobilized donor lymphocytes infusion (pG-DLI) and its relationship with the incidence of graft-versus-host disease (GVHD) in high-risk leukemia patients with non-myeloablative stem cell transplantation (NST).</p><p><b>METHODS</b>12 patients with high-risk leukemia were analyzed, including Ph⁺ acute lymphocytic leukemia (n=1), acute leukemia (AL) with persistent non-complete remission (n=2), acute myeloid leukemia (AML) with central nervous system (CNS) relapse (n=3), hybrid AL (n=1), secondary AML evolving from myelodysplastic syndrome (MDS/AML) (n=2), chronic myeloid leukemia in accelerated phase (CML-AP) (n=1), CML in blastic phase (CML-BP) (n=2). All patients received non-myeloablative conditioning and pG-DLIs were administered 30-40 days post transplantation if no signs of GVHD were present. The percentage of donor cell chimera was analyzed by short tandem repeat-polymerase chain reaction (STR-PCR) just before and after pG-DLI. The incidence of leukemia relapse and GVHD were observed.</p><p><b>RESULTS</b>12 high-risk leukemia patients with a median age of 38 (range: 29-52) years received G-DLI at a median interval of 35 (32-40) days. The median numbers of infused mononuclear cells (MNCs), CD34⁺, and CD3⁺ cells/kg recipient body weight was 2.3×10⁸/kg, 1.7×10⁶/kg, and 0.6×10⁸/kg, respectively. 10 of 12 patients had full donor chimera before pG-DLIs and conversion from mixed to full donor chimera occurred in the other 2 patients shortly after pG-DLI. Grade II acute GVHD (aGVHD) was observed in only 2 patients and chronic GVHD (cGVHD) developed in 6 patients, including involvement of skin (n=3), skin and intestine (n=2), liver (n=1). 1 patient died of cGVHD. With a median follow-up of 40 (24-64) months, 7 patients are alive in remission, with 3-year actuarial overall survival (OS) and disease-free survival (DFS) rates of the same 58.3%.</p><p><b>CONCLUSION</b>Our findings indicate that pG-DLI after NST does not increase the risk of aGVHD, but could enhance the capacity graft-vs-leukemia and prevent relapse in high-risk leukemia patients.</p>

Haploidentical nonmyeloablative allogeneic peripheral blood stem cell transplantation for treatment of refractory or relapsed leukemia: long-term follow-up / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the therapeutic effect and major complications of haploidentical nonmyeloablative allogeneic peripheral blood stem cell transplantation (NST) for refractory or relapsed leukemia.</p><p><b>METHODS</b>The results of 30 patients, including 14 cases of acute myeloid leukemia (AML), 11 cases of acute lymphoblastic leukemia (ALL), 5 case of chronic myelogenous leukemia (CML) (accelerated and blastic phase) with refractory or relapsed leukemia (RF/RL) who underwent haploidentical NST from August 2000 to April 2009 were analyzed. The conditioning regimen consisted of fludarabine (flu), antithymocyte globulin (ATG), cyclophosphamide (CTX), total body irradiation (TBI) and cytarabine (Ara-C) or myleran (Bu). Graft-versus-host disease (GVHD) prevention programmes consisted of Cyclosporine (CsA), mycophenolate mofetil (MMF), CD25 monoclonal antibody combined with mesenchymal stem cells (MSC).</p><p><b>RESULTS</b>Twenty six cases of patients were full donor engraftment and 4 cases mixed chimerism into full donor chimerism. The average duration of neutrophil >0.5×10⁸/L after NST was 11 (9-16) days, and platelet >20×10⁸/L 17 (12-60) days. Upon follow-up of 16 to 120 months, 12-month transplant-related mortality (TRM) was 46.7%, acute Ⅱ-Ⅳgraft-versus-host disease (aGVHD) incidence was 40.0%. The probability of 3-year disease relapse, EFS and overall survival (OS) rates were 16.7%, 46.2% and 50.0% respectively.</p><p><b>CONCLUSION</b>Haploidentical NST could improve OS and EFS of refractory or relapsed leukemia and reducce TRM to some extent.</p>

Abnormality of blood coagulation indexes in patients with de novo acute leukemia and its clinical significance / 中国实验血液学杂志

To explore hemorrhage risk and the clinical significance of abnormal change of prothrombin time (PT), activated partial thromboplastin time (APTT), plasma fibrinogen (FIB), plasma thrombin time (TT) and d-dimer (D-D) in de novo acute leukemia (except for APL), the different bleeding manifestations of 114 cases of de novo acute leukemia with different coagulation indexes were analyzed retrospectively. The correlation between these blood coagulation indexes and the possible correlative clinical characteristics were analysed, including age, sex, type of acute leukemia, initial white blood cell(WBC) and platelet(Plt) count, the proportion of blast cells in bone marrow and cytogenetic abnormality of patients at diagnosis. The results indicated that the incidence of abnormal blood coagulation was as high as 78.1% for de novo AL patients. These patients with 5 normal blood coagulation indexes may have mild bleeding manifestation, but the more abnormal indexes, the more severe bleeding. Both PT and D-D were sensitive indexes for diagnosis of level II bleeding. Incidence of abnormal blood coagulation significantly correlates with the proportion of blast cells in bone marrow (χ(2) = 4.184, OR = 1.021, P < 0.05) and more with D-D (P < 0.01), while age, sex, type of AL, WBC count, Plt count and abnormality of cytogenetics did not correlate with abnormal blood coagulation. It is concluded that the coagulation and fibrinolysis are abnormal in most patients with de novo acute leukemia. More abnormal indexes indicate more severe bleeding, and both PT and D-D are sensitive indexes for diagnosis of level II bleeding. Higher proportion of blast cells in bone marrow predicts higher incidence of abnormal blood clotting. Acute leukemia with elderly age, high white blood cell count and adverse cytogenetics do not predict severer abnormal blood clotting. Detection of PT, APTT, TT, FIB, and D-D may help to judge whether the patients are in a state of hypercoagulability or disseminated intravenous coagulation, which will provide experiment evidences for early intervention and medication.

Changes of Th1/Th2/Th17 in patients received non-myeloablative haploidentical hematopoietic stem cell transplantation detected by flow cytometric bead array / 中国实验血液学杂志

This study was purposed to investigate the changes of Th1/Th2/Th17 in patients received non-myeloblastic haploidentical hematopoietic stem cell transplantation (NAHSCT). The levels of IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ, as well as IL-17 level were determined by flow cytometric bead array (CBA) in samples from 18 patients underwent allo-peripheral NAHSCT at different time points before and after transplantation. The results showed that all cytokines changed obviously after transplantation, and their serum levels were higher than that before transplantation. The expression levels of IL-2, IL-4 and IL-17 changed early, and their obviously up-regulation was found after transplantation. The expression levels of IL-6, IL-10 and TNF-α changed significantly, and were high as compared with that before transplantation. The change of INF-γ serum level was observed late, its rising occurred at week 4 after transplantation. The expression of all cytokines kept increasing during 4 weeks after transplantation and peaked at week 4. It is concluded that the serum levels of all cytokines from the patients after NAHSCT increased significantly, in which the levels of IL-2, IL-4 and IL-17 increased early, but the level of INF-γ changed late. The detection of cytokines is helpful for deep understanding the pathophysiologic mechanism of transplant-related complications.

Different changes of serum cytokines following HLA-identical and HLA haploidentical non-myeloablative allogeneic hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was aimed to investigate the expression difference of serum cytokines in 20 patients receiving HLA-identical nonmyeloablative allogeneic hematopoietic stem cell transplantation (iNAHSCT) and HLA-haploidentical nonmyeloablative allogeneic hematopoietic stem cell transplantation (hiNAHSCT). IL-2, IL-4, IL-6, IL-10, TNF-α, γ-IFN and IL-17 were detected by flow cytometric bead array before and on week 1, 2, 4 after transplantation respectively. The results showed that the IL-2 level was found to be up-regulated at week 1 and 2 after transplantation in iNAHSCT group and in hiNAHSCT group respectively, but there was no difference between these two groups (P > 0.05). The γ-IFN levels was up-regulated at week 4 after transplantation in above-mentioned two groups, but no difference was found between these two groups. The IL-4 level increased at week 2 and 1 after transplantation in iNAHSCT and hiNAHCT groups respectively, but the IL-4 level in iNAHSCT group was higher than that in hiNAHSCT group. The IL-6 level rose at week 1 and 2 after transplant in above mentioned groups respectively, and reached to peak level at week 4 after transplantation, but IL-6 level in hiNAHSCT was higher than that in iNAHSCT group (P < 0.02). The IL-10 level was up-regulated at week 1 and 2 in iNAHSCT and hiNAHSCT groups respectively, but the IL-10 level in iNAHSC was higher than that in hiNAHSCT group. The TNF-α level was up-regulated at week 1 in hiNAHSCT group, but at week 2 in iNAHSCT group after transplantation. The TNF-α level in hiNHASCT group was higher than that in iNAHSCT group (P < 0.01). The IL-17 level was up-regulated at week 1 and week 4 after transplantation in hiNAHSCT and iNAHSCT groups respectively, the IL-17 level in hiNAHSCT group was high as compared with that in iNAHSCT group. It is concluded that the serum cytokine levels are obviously up-regulated in iNAHSCT and hiNHASCT groups, and reach to peak level at week 4 after transplantation. The IL-6, TNF-α and IL-17 level up-regulated significantly in hiNAHSCT group, but the IL-4 and IL-10 level up-regulated significantly in iNAHSCT.

NK cell surface receptors and their research progress - review / 中国实验血液学杂志

Natural killer (NK) cells are important immune cells in human body, which occupy an important place in adoptive immunotherapy for patients with malignancies due to their capacity of killing tumor cells without MHC limitation, as well as separating graft versus leukemia (GVL) and graft versus host disease (GVHD). Recent studies showed that different kinds of NK cell-surface receptors have been found, which transmit inhibiting signals or activating signals, the balance between them determines the functional status of NK cells. Researchers have focused on the study of NK cell-surface receptors recently in order to improve application of NK cells for adoptive immunotherapy. This review summaries the current advancement about NK cell-surface receptors and their clinical significance.

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.

Role of G-CSF in the proliferation, differentiation and cell cycle distribution of mouse thymocytes after acute radiation / 中国实验血液学杂志

This study was purposed to investigate the effect of G-CSF on the proliferation, differentiation, and cell cycle distribution of thymocytes in sublethally irradiated mice. Female BALB/c mice were exposed to 6.0 Gy γ-ray irradiation and then randomly divided into control and G-CSF treatment group. In the treatment group rhG-CSF 100 µg/(kg·d) was given subcutaneously for 14 continuous days and to make sure the first injection was given within 1 hour after irradiation. Cell cycle distribution and apoptosis of thymocytes were detected within 72 hours after irradiation. Subpopulations of CD4(-)CD8(-) cells and sequential changes in the distribution of CD4(+)CD8(+), CD8(+)CD4(-), CD8(-)CD4(+) cells were detected by a three-color flow cytometry during a four-weeks period after irradiation. The results showed that in G-CSF treatment group marked increase of cells in G(0)/G(1) phase (G-CSF vs control: 82.0 ± 5.0% vs 75.9 ± 2.8%) (p < 0.05) and a decrease of cells in S phase (G-CSF vs control: 10.2 ± 4.8% vs 15.7 ± 2.3%) (p < 0.05)could be observed as early as 6 hours after irradiation, but G-CSF seems have no evident effects on the cells in G(2)/M phase. G-CSF could also protect thymocytes against apoptosis. 6 and 12 hours after irradiation the apoptosis rates of thymic cells in G-CSF treatment group were 11.5 ± 2.4% and 15.5 ± 3.3% respectively, while in the control group the apoptosis rates were 16.5 ± 2.2% and 22.6 ± 0.7% respectively. Comparison between the two group demonstrated significant difference (p < 0.05). CD4(-)CD8(-) double negative thymocytes (DN)can be defined as DN1-4 according to their maturation. G-CSF treatment resulted in a significant increase in DN1 thymocytes and promoted their proliferation and differentiation to a more mature DN3 and DN4 stage. G-CSF could enhance the recovery of CD4(+)CD8(+) thymocytes and mitigate their relapse during reconstitution. The percentage of CD4(+)CD8(+) thymocytes in the G-CSF treatment group 28 days after irradiation was significantly higher than that of the control group (71.0 ± 6.3% vs 25.5 ± 6.3%) (p < 0.05). It is concluded that G-CSF has a positive effects on the thymic cell cycle distribution, proliferation and differentiation, which may contribute to the reconstitution of central immune system after acute irradiation.

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>