Efficacy of BMMSCs on aGVHD and Its Correlation with SerumInflammatory Cytokines in Pediatric Patients with Severe Refractory Acute Graft-Versus-Host Disease / 中国实验血液学杂志

OBJECTIVE@#To investigate the efficacy of bone marrow mesenchymal stem cells (BMMSC) on children with refractory graft-versus-host disease (GVHD) and to judge the efficacy of BMMSC by dynamically monitoring the changes of cytokines in children with GVHD before and after infusion of BMMSC, so as to provide a theoretical basis for clarifying the mechanism of BMMSC.@*METHODS@#17 children with refractory aGVHD including 7 of grade II, 6 cases of grade III and 4 cases of grade IV after allo-HSCT were enrolled. All the children with aGVHD, who received routine immunosuppressive therapy, but the state of disease not improved, were treated with immunosuppressive drugs combined with BMMSC infusion. Study endpoints included safety of BMMSC infusion, response to BMMSC, and overall response of aGVHD. The serum levels of IL-2α, IL-6, IL-10, IL-8 and TNF-α in aGVHD patients were measured by chemiluminescence before infusion of BMMSCs and Day 7, Day 14 after infusion of BMMSCs.@*RESULTS@#The cumulative median dose of BMMSCs was 5.5 (3.4-11.1) × 10/kg for average of 3.7 times, and the median time of 16.5 (4-95) days for the first infusion of MSCs. In 17 cases of refractory GVHD, 14 responded to treatment, whereas 3 patients failed. The total effective rate was 82.4% and no adverse reactions occurred. Of the 14 survived cases (82.4%), the median follow-up time was 944 (559-1245) days from the first infusion of MSCs. The levels of TNF-α in children with grade II, III and IV GVHD before treatment were 9.5±4.3 pg/ml, 16.3±10.9 pg/ml and 35.8±21.2 pg/ml respectively. The difference between grade II and IV, III and IV was statistically significant (P<0.05). Compared with the ineffective group of BMMSC infusion, the serum TNF-αlevel in the BMMSCs treatment effective group was 10.8±5.6 pg/ml vs 40.6±14.8 pg/ml (t=-3.901, P<0.05) before treatment. In the effective group of BMMSCs infusion, IL-10 20±17.4 pg/ml of day 14 was significantly higher than that 7.3±3.1 pg/ml before the treatment (t=-2.850, P<0.05), while , the serum levels of IL-2α, IL-6, IL-8, TNF-α were not statistically significantly different (P＞0.05).@*CONCLUSION@#The infusion of BMMSC is safe and effective in the treatment of refractory GVHD in children. TNF-αlevel relates with the severity of GVHD. BMMSC may play an anti-GVHD role by up regulating the level of cytokine IL-10 in vivo.

Clinical Analysis of 164 Children of Blood Disease Complicated with Invasive Fungal Disease / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical characteristics, prevention and treatment of invasive fungal disease (IFD).@*METHODS@#The clinical data of 164 patients who met the diagnostic criteria of IFD in our center from January 2012 to January 2015 were retrospectively analyzed. The incidence, clinical characteristics, related factors, treatment methods and prognosis were analyzed.@*RESULTS@#Among 1289 cases of blood diseases, 164 cases suffered from IFD with inciduce of 12.7%. The main infection sites were as followed: lung, blood and gastrointestinal tract, with incidence of 84.2%, 5.5% and 3% respectively. The funge was found in 35 cases by detection; among fungi, the detected rate of candida albicans. aspergillus and candida glabrata was more high with 51.5%, 20% and 14.3% respectively. Among 164 childen with blood deseases complicated by IFD, 36 cases gained complete remission, 97 cases gained partial remission, 10 cases were stable, 11 cases were progressive and 10 cases died, the overall effective rate reached 81.1%. The univariate analysis showed that the gramulopenia, granulocyte recovery, long-term use of corticosteroid and immuno-suppressive agents, as well as different grades of diagnosis were significant factors affecting the efficacy of antifungal therapy for blood disease children with IFD, the multivariate analysis further showed that the granulocyte recovery and diagnosis grades were independent prognostic factors affecting the therapeutic efficacy for IFD children. The overall survival rate of IFD children with 12 weeks of antifungal treatnment was 81.7%, out of which the survival rate of IFD children at 12 weeks of treatment with itraconazole, voriconazole, amphotericin B and caspofungin was 81.4%, 80%, 69.4% and 97.1% respectively, there were significant differences in survival rate between each other by long rank test. In addition of caspofungin, the other 3 kinds of drugs had toxic side effects of different degrees, but IFD children could tolerated these effects after symptomatic treatment.@*CONCLUSION@#The incidence of IFD in children with blood deseases in our hospital is 12.7%, the lung is most common infective site, moreover patogens of IFD mainly is candida. The promotion of granulocyte recovery and early stratified diagnosis can contribule to the treatment of IFD. For the IFD children with better economic condition, the caspofungin is a potent antifungal agent with high efficacy, low toxicity and better prognosis.

Efficacy and Safety of MSC Infusion in Treatment of Children with Refractory LOHC: A Clinical Study / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the curative effect and safety of menchymal stem cell infusion in treatment of children with refractory late-onset hemorrhagic cystitis(LOHC) after allogeneic HSCT.</p><p><b>METHODS</b>Thirty cases of children with refractory LOHC after allo-HSCT in our department between December 2010 and July 2016 were analyzed retrospectively, out of 30 cases 7 received MSC treatment. The used MSC of all were four-to-five generation MSC from bone marrows of third party donors, and were infused into patients with (1.87±0.456)×10/kg MSCs once a week (1-4 times in total) until the hematuria and odynuria symptoms being improved. To observe whether unfavorable reactions occurred after MSC treatment, the patients accepted daily physical examination and regular assistant examination. The cytokine levels were also measured and dynamically detected in 2 cases before and after MSC treatment.</p><p><b>RESULTS</b>In 30 children with refractory LOHC, the hematuria difficultly reached the remission after routine hydration, alkalizing and antiviral therapy, Among 25 cases who were received methylprednisolone, MTX and CTX therapy, 7 cases received MSC infusion for 1-4 times with dose of (1.87±0.456)×10/(kg·time) as a result, 7 cases of LOHC were cured. The TNF-α and IL-2R levels in 2 cases progressively decreased after MSC infusion, no occurence of fever, rash, embolism and so on were found in 7 cases received MSC infusion; the BKV detection showed that the viral load did not increase; the leukemia relapse or secondary cancer did not occure.</p><p><b>CONCLUSION</b>The MSC treatment is safe and effective for refractory LOHC after allo-HSCT.</p>

Etiological Study of Late Onset Hemorrhagic Cystitis after Allogeneic Hematopoietic Stem Cell Transplantation in Children / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To prospectively study the correlation BKV with the occurrence and development of late onset hemorrhagic cystitis (LOHC) after allogeneic hematopoietic stem cell transplantation(allo-HSCT).</p><p><b>METHODS</b>The clinical data of a total of 276 patients with allo-HSCT in our department between January 1998 and March 2016 were analyzed ratrospectvely. Quantitative Real-time PCR assay was used to prospectively monitor the BKV DNA load of the urine and plasma for 23 patients accepting allo-HSCT from August 2015 to March 2016.</p><p><b>RESULTS</b>LOHC(24.28%) occurred in 67 of 276 cases with allo-HSCT. Univariate analysis showed that age older than 6 years, different diseases, unrelated donor, pretreatment with BU, Ⅲ-Ⅳ aGVHD significantly correlated with LOHC. Multivariate analysis demonstrated that age older than 6 years (P<0.01), pretreatment with BU(P<0.05), and aGVHD of grade Ⅲ-Ⅳ （P= 0.011） were the independent risk factors for LOHC. Among 23 patients after allo-HSTC, 10 of which were positive of urine BKV, and LOHC occurred in 6 cases. The positive rate of urine BKV (85.7%)in group LOHC was significantly higher than that in the group LOHC(25.0%）(χ=5.043, P<0.01). The incidence of LOHC positively correlated with the positive rate of BKV (r=0.564, P<0.01), and the severity of LOHC positively correlated with urinary BKV load (r = 0.502, P<0.01). And 5 of 6 petriatic patients with LOHC had aGVHD. All of them were subject to the strengthened antiviral treatment, and 4 of them accepted intensive immunosuppression therapy.</p><p><b>CONCLUSION</b>Age ≥6 years old, precenditioning regieme with BU and aGVHD of grade Ⅲ-Ⅳ are independent risk factors for LOHC after allo-HSCT, the positive rate of urine BKV load positively correlates with the severity of LOHC after allo-HSCT.</p>

Clinical Charcteristics and Prognostic Analysis of 28 cases of Pediatric Myelodysplastic Syndrome / 中国实验血液学杂志

OBJECTIVE@#To analyze the clinical features and prognosis of 28 children with myelodysplastic syndrome (MDS) and to screen the high risk factors affecting the prognosis so as to provide the new ideas for standard of clinical diagnosis and therapy.@*METHODS@#The clinical data of 28 children with newly diagnosed MDS treated in our hospital from March 1994 to July 2016 were analyzed retrospectively, the features of disease onset and the results of laboratory examination were summarized, all MDS children were followed up, the prognosis and the high risk factors affecting the prognosis were evaluated.@*RESULTS@#In all 28 MDS children, the ratio of male to female was 1.8∶1, the incidence of MDS was observed in boys, while the low incidence of MDS was found in older children. The clinical manifestations were mainly the decrease of three series blood cells in 16 cases (57.14%), other cases presented simple anemia (7.1%), simple thrombocytopenia (7.1%), neutropenia with anemia (14.29%), and anemia with thrombocytopenia (14.28%).The bone marrow image showed mainly hyperplasia (82.14%), and the pathological hematopoiesis, moreover the manifistation of pathological hematopoiesis was different in forma and degree; the bone marrow biopsy showed the typical abnormal localization of immature precursor(ALIP) accepted for 33.33%; the chromosome karyotype detection showed the detected rate of chronosome abnormality was 41.18%. The median follow-up time was 1.75 years. 5 children with MDS received the hematopoietic stem cell transplantation (HSCT), among them 1 dead and 4 maintained CCR; Out of other 23 patients no-received HSCT, 7 cases given up treatment after confirmed diagnosis, 16 cases received the chemotherapy (2 cases given up treatment after CR, 5 cases transformed into AML, 3 cases relapsed, 3 cases maintained CCR), 11 cases dead, 9 cases failed to be followed up. The 5-years OS rate and EFS rate in all patients were predicted as (38.2±11.3)% and (35.3±11.3)%,respectively, among them, the OS and EFS rates of patients received the HSCT allo superior to those of patients did not received HSCT ［(80.0±17.9）% vs.(22.8±11.5）%］ (P0.05).@*CONCLUSION@#The children MDS is rare and easy to be misdiagnosis, moreover displays more high heterogeneity and poor prognosis, thereby the early diagnosis is crucial, in addition, the system of prognosis evaluation is imperative to be perfected. The HSCT may be the effective method for curative treatment of childhood MDS.

Clinical Efficacy of NOPHO-AML 2004 Regimen for Treatment of Children with Acute Myelocytic Leukemia (Non-M3) / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the efficacy and safety of NOPHO-AML 2004 chemotherapy regimen for treatment of children with acute myelocytic leukemia(non-M3).</p><p><b>METHODS</b>Thirty-three patients aged 1-13 with acute myelocytic leukemia (non-M3) were diagnosed from January 2013 to June 2017. FAB typing showed that 1 case in M0, 4 cases in M1, 12 cases in M2, 5 cases in M4, 8 cases in M5, 1 case in M6, and 2 cases in M7; Risk stratification showed that: 19 cases in standard risk, and 14 cases in high risk. All patients were treated with NOPHO-AML 2004 chemotherapy regimen. SPSS 22.0 software was used, the Kaplan-Meier survival analysis method and Cox regression model were used for statistical analysis.</p><p><b>RESULTS</b>In the first course of treatment (AIET), among 33 child patients there were 27 cases with complete remission, and 5 cases with non-remission, thus the remission rate was 81.8%. Out of the 5 child patients without remission, 4 cases reached to the complete remission after the second course (AM), and 1 case did not remission, thus the total remission rate was 96.9%.9 cases (27.3%) underwent bone marrow recurrence and the median recurrence time was 30 months after complete continuous remission. Univariate analysis showed that age and erythrocyte transfusion frequency were significant factors to affect the early treatment response; the multiple Cox regression analysis showed that: age >7, MRD positive, erythrocyte transfusion >4 times and poor response to early treatment were independent risk factors for recurrence; Allogeneic hematopoietic stem cell transplantation(HSCT) in 8 high-risk children received enhanced chemotherapy had better efficacy as compared with the chemotherapy alone. The 3-year event-free survival rate was 59.9%, and 3-year overall survival rate was 69.2%. 33 children patients experienced varying degrees of infection and myelosuppression, or drug-related gastrointestinal reactions and allergic reactions, patients were tolerable to these side reactions after active symptomatic treatment.</p><p><b>CONCLUSION</b>NOPHO-AML 2004 chemotherapy regimen has high response rate and good tolerance, early treatment response is an important factor influencing prognosis. Age and repeated red blood cell infusions are the important factors influencing the prognosis, which promote bone marrow recurrence in AML children. For the children suffered from clinical high-risk AML, the NOPHO-AML 2004 chemotherapy regimen combined with HSCT can improve the prognosis of patients.</p>

Comparison of Curative Effect between Fu Fang Huang Dai Pian and Arsenic Trioxide in Treatment of 45 Patients with Acute Promyelocytic Leukaemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical efficacy of Fu Fan Huang Dai Pian(RIF) and arsenic trioxide (ATO) regimens for treatment of children with acute promyelocytic leukemia (APL) and to explore the risk factors affecting the prognosis of patients.</p><p><b>METHODS</b>The clinical data of 45 newly diagnosed APL children admitted in our hospital from January 2004 to May 2017 were analyzed retrospectively. Among 45 APL children, 25 children were treated by chemotherapetic regimen including RIF (RIF group), another 20 children were treated by chemotherapeutic regimen including ATO (ATO group). The follow-up was performed in all APL children. The prognosis and incidence of side reactions from drugs in 2 groups were compared, and the high risk factors affecting the prognosis of patients were analyzed.</p><p><b>RESULTS</b>The median follow-up time was 49.8% months. In RIF group, no early death occured in 25 APL children; 5 cases did not achieve complete remission (CR) after induction therapy, CR rate was 88%. Out of 25 cases 2 caes relapsed, 3 cases died, 20 cases maintained contined CR (CCR), 2 cases failed to be followed-up. In ATO group, 2 cases suffered from early death, 5 cases did not achieve CR after induction therapy, CR rate was 90%, 2 caese relapsed and died, 15 cases maintained CCR, the follow-up failed in 1 caes. The 5 year- OS and EFS rate in all the patients were predicted as (82.2±6.2)% and (76.4±6.6)% respectively. The OS and EFS rate in RIF group were (86.1±7.4)% and (78.4±8.6)% respectively, which were significantly different from OS and EFS rate (76.4%±10.6%) and (74.0%±10.1%) respectively in ATO group (all P>0.05). As for the side reaction from drug, except for the cardiac damage (P<0.05), incidence of other side reactions was not significantly different between 2 groups (P>0.05). In addition, the 5 year-OS and EFS rates in APL children with CNSL were significantly lower than those in APL children without CNSL (all P<0.05), the 5 year OS and EFS rate in APL children did not reache M1 and with high risk were significantly lower than those in APL children reached M1 after induction therapy and with low and standerd risk (P<0.05 and P<0.05); the 5 year-OS and EFS rates did not correlate with age and sex.</p><p><b>CONCLUSION</b>The Fu Fang Huang Dai Pian shows the therapeutic efficacy on APL children same as ATO, moreover, no obvious enhancement in incidence of side reactions is observed, therefore, the Fu Fang Huang Dai Pian is effective and safe for treatment of APL children. The CNSL, poor respond to treatment, high risk in clinical stratification are high risk factors affecting prognosis of patients.</p>

Familial Haemophagocytic Lymphohistiocytosis Occurs in A Fetus at His Third Trimester-A Case Report / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the clinical features and pathogenetic gene mutation in a fetus at his third trimester with familial haemophagocytic lymphohistiocytosis (FHL).</p><p><b>METHODS</b>Target region sequencing and high-throughput sequencing were used to detect pathogenetic gene mutations for familial haemophagocytic lymphohistiocytosis in a late onset HLH fetus. Pathogenetic gene mutations of the patient and his parents were verified by Sanger dideoxy sequencing.</p><p><b>RESULTS</b>A male neonate, who had right pleural effusion, hepatomegaly and splenomegaly previously revealed by fetus ultrasound, was delivered at full-term by cesarean section. His clinical presentation showed recurrent fever, tachypenea, decreased breath sounds on right side, hepatosplenomegaly etc., which were gradually aggravating Lab.tests results were as follows: WBC 9.88×10/L, Hb 91 g/L, Plt 13×10/L, ALT 18 U/L,AST 69 U/L,TBIL 207.2 µmol/L, DBIL 183.5 µmol/L, TG 3.05 mmol/L, Fib 0.88 g/L, Serum ferritin 3 120 ng/ml and sIL-2R 57 420 U/ml. FCM showed that CD3CD16CD56cells reached to 3.60% in the pripheral blood. Haemophagocytes were occasionally found in the bone marrow. NK/NKT stimulation test showed a severe damage of degranulation of NK cells. Sequence analysis of genomic DNA from his peripheral blood demonstrated the compound heterozygous mutations of UNC13D gene: c.2448-13 G>A in exon26 and c.1055+1 G>A in exon12, both were pathogenetic mutations. In detailed family survey, it was confirmed that the mutation c.2448-13 G>A in exon26 was inherited from his mother and c.1055+1 G>A in exon12 from his father.</p><p><b>CONCLUSION</b>A rare case of familial haemophagocytic lymphohistiocytosis type 3 (FHL3) with late fetus onset who carried pathogenetic compound heterozygous mutations of UNC13D gene. Those neonates with recurrent fever, serous effusions and multiple organ failure should be screened for FHL. Identifying the pathogenic gene mutations laid the foundation of conceiving disease-free newborns.</p>

Antifungal azoles exacerbate vinblastine-related hyponatremia in ALL children / 中国实验血液学杂志

The purpose of this study was to investigate the clinical characteristics and the treatments of patients with vinblastine-related hyponatremia which was aggravated by azole antifungal agents in children with acute lymphoblastic leukemia(ALL). A total of 93 children treated with vinblastine in our department during April 2013 to March 2014 were enrolled in this study and were divided into 3 groups:VDLD, VDLD with azoles antifungal, VDLD with non azoles antifungal. The incidence and severity of hyponatremia were statistically analysed. The results showed that (1) the incidence of hyponatremia in VDLD group was 93.1%(67/72),100%(13/13) in VDLD with azoles antifungal group, and 75%(6/8) in VDLD with non-azoles antifungal, there was no statistically difference between these three groups. (2) Incidence of moderate to severe hyponatremia (Na<129 mmol/L) in VDLD with azoles antifungal group was(9/13,69.2%),which was significartly higher than those in VDLD group (22/72, 30.6%) and in VDLD with non azoles antifungal group (1/8, 12.5%). However, the difference between VDLD group and VDLD with non azoles antifungal group were not statistical significant. (3) the lowest serum sodium level in VDLD with azoles antifungal group (124.0 ± 8.6 mmol/L) was significantly lower than that in VDLD group (130.8 ± 3.8 mmol/L)and VDLD+non azoles antifungal group(132.9 ± 4.9 mmol/L). Otherwise, the difference was not statistically significant between VDLD group and VDLD with non azoles antifungal group. (4) four children with severe hyponatremia showed convulsions and coma which all belong to VDLD with azoles antifungal group. The children with hyponatremia were restricted intake of fluid, adjusted the liquid tension, supplied hypertonic sodium and given diuretic, the serum sodium value gradually picked up in these children. In 4-11 months' follow-up, no hyponatremia happened again in these children. It is concluded that the incident of hyponatremia in children treated with vinblastine is high, but most of them seldom showed clinical characteristics. The combination of antifungal azoles with vinblastine can increase the incidence and severity of hyponatremia. Therefore, combined administration of azole antifungals with vinblastine should be avoided.

Comparison of the anti-leukemia effect and mechanism of L-asparaginase between two different strains / 中国实验血液学杂志

This study was purposed to compare the anti-leukemic effects of E.coli-L-Asp and Erwinia-L-Asp in vitro, and to investigate their mechanism. The cell apoptosis and proliferation inhibition rate were measured by CCK-8 kit, and IC50 of two drugs was calculated by using SPSS software. Pro-apoptosis effect of E.coli-L-Asp and Erwinia-L-Asp on REH and Jurkat cell lines was also determined by flow cytometry with Annexin V/PI double staining. Concentration changes of 4 amino acids (Asn, Aspa, Gln, and Glu) before and after medication were detected by using high pressure liquid chromatography (HPLC) assay. The results showed that both REH and Jurkat cell lines were sensitive to L-Asp drugs from two different strains, and E.coli-L-Asp and Erwinia-L-Asp displayed the inhibition effect on the proliferation of Jurkat and REH cell lines in dose-dependent and time-dependent manners. The inhibition cell of proliferation and cell apoptosis in Erwinia-L-Asp group were higher than those in E.coli-L-Asp group after 24 hours (P < 0.05) . However, after treatment of REN and Jurkat cells with 2 kind of L-Asp for 48 hours, the inhibition of cell proliferation and apoptosis rates were not significantly different between the 2 L-Asp drugs (P > 0.05). The Asn in medium could be depleted by two different L-Asp drugs with low concentration. Both the two L-Asp drugs had the same capability to deplete the Asn surrounding leukemia cells (P > 0.05). The Gln in medium could be depleted by two L-Asp drugs with high concentration. The hydrolysis effect of Erwinia-L-Asp on Gln was stronger than that of E.coli-L-Asp (P < 0.05). It is concluded that in a certain range of concentrations, E.coli-L-Asp and Erwinia-L-Asp exert anti-leukemia effect in dose-dependent manner. Depletion of Gln and Asn in surrounding environment and induction of cell apoptosis are two potential mechanisms, by which leukemia cells can be killed. Erwinia-L-Asp may be chosen as the first-line drug to treat childhood ALL for its fast action and stronger hydrolysis effect on Gln.

Therapeutic efficacy evaluation of rabbit anti-thymocyte globulin combined with cyclosporine A in children with aplastic anemia / 中国实验血液学杂志

This study was aimed to investigate the therapeutic efficacy of rabbit anti-thymocyte globulin (r-ATG) combined with cyclosporine A (CsA) and to analyse the efficacy-related factors in children with aplastic anemia (AA). Twenty five AA children treated with r-ATG [3.5 mg/(kg·d)×5 days] combined with CsA were analyzed retrospectively. The lymphocyte subgroups, CD4(+)/CD8 ratio and expression of CD55, CD59 on surface of neutrophils and erythrocytes in peripheral blood were detected by direct immunofluorescence method and flow cytometry; the responsive time, effective rate, adverse effects and infections after immunosuppressive therapy (IST) were analyzed; the distribution of T-lymphocyte subgroups in IST-effective and IST-uneffective groups was compared, and therapeutic efficacy-related factors were evaluated. The results showed that the response to treatments was found in 21 out of 25 cases, the total responsive rate was 84.0%; the response time was 3 - 6 months, average of 4 months; the effective rates in month 3, 6, 9, 12 after treatment were 56.0%, 72.0%, 80.0% and 84.0% respectively. The AA children with age ≥ 5 years old, course of disease < 6 months and absolute neutrophil value ≥ 1.5 ×10(9)/L on 30 days after IST had good curative effect; the effective rate in AA children with age ≥ 5 years old, course of disease < 6 months, high or reverse ratio of CD4(+)/CD8(+) and absolute neutrophil value ≥ 1.5×10(9)/L after IST was higher than that in AA children with age < 5 years old, course of disease ≥ 6 months, normal ratio of CD4(+)/CD8(+) and absolute neutrophil value after IST < 1.5×10(9)/L (94.4% vs 57.1%, 90.4% vs 50.0%, 94.1% vs 62.5%, 94.1% vs 62.5%) (P < 0.05). The high effective rate was observed in AA children with decrease of CD55 and CD59 expression, but there was no significant difference (P > 0.05) as compared with normal expression of CD55, CD59. It is concluded that the treatment using r-ATG (3.5 mg/kg·d × 5 d) combined with CsA is a safe and effective for children with AA. Age, course of disease and absolute neutrophil value on 30 days after IST are the main factors affecting curative affect.

Analysis of one case of adolescent blastic plasmacytoid dendritic cell neoplasm / 中国实验血液学杂志

This study was purposed to summarize the clinical characteristics and laboratorial data of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in pediatric patients in order to enhance understanding this disease in diagnosis and therapy. A rare case of BPDCN in children was enrolled in this study. The blood routine test, examination of bone marrow cell morphology, histopathology and immunophenotype of the skin lesions were performed and analysed, the single cell suspensions of the biopsied skin mass were detected by flow cytometry. The results showed that tumor cells expressed CD4, CD56, CD43 and CD123, while not expressed CD19, CD20, CD3, CD8, CD13, CD11b and myeloperoxidase (MPO). According to the clinical and laboratorial features and the results from histopathological and immunophenotype examinations, BPDCN was confirmed. It is concluded that BPDCN in children is an extremely rare hematopoietic malignancy with presenting a rapidly and fatally aggressive clinical course. The diagnosis of this disease is mainly based on the clinical presentations, pathologic and immunohistochemical features. BPDCN is a highly aggressive disease, its prognosis is very poor, its pathogenesis remans still unclear. A standard treatment protocol for BPDCN has not yet been established.

Experimental study on rejection of allogeneic donor bone marrow cells in sensitized recipients / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a murine model of sensitization, and investigate the effect and mechanism of sensitization on allogeneic donor bone marrow cells (BMCs).</p><p><b>METHODS</b>Sensitized BALB/c mice were established by transfusions of allogeneic splenocytes. The donor reactive antibodies were detected by binding and complement-dependent cytotoxicity assays. After irradiation, 1 × 10(7) BMCs of C57BL/6 donor mice were injected into non-sensitized or sensitized BALB/c recipient mice. The distribution pattern of donor BMCs in peripheral blood, spleen and bone marrow of recipient mice were analyzed at different time points (2 h, 12 h and 48 h) post transplantation. Hematopoietic recovery post transplantation was assessed, and survival was monitored. Moreover, sera and splenocytes derived from non-sensitized or sensitized recipients were incubated with allogeneic BMCs in vitro, and the cytotoxic indexes were calculated in the immune experiments.</p><p><b>RESULTS</b>The binding and complement-dependent cytotoxicity assays showed that a high level of donor reactive antibodies was presented in sensitized sera. Compared with the non-sensitized recipients, the homing assay showed significantly decreased distributions of allogeneic donor BMCs in peripheral blood, spleen and femur of sensitized recipients. Non-sensitized recipients survived long term after irradiation, while all the sensitized recipients died within 12-15 days. Fourteen days post transplantation, the white blood cells and BMCs of non-sensitized recipients were (3240 ± 300) × 10(6)/L and (396 ± 27) × 10(6)/femur, respectively; while the white blood cells and BMCs of sensitized recipients were (320 ± 80) × 10(6)/L and (6 ± 2) × 10(6)/femur, respectively; the differences were statistically significant between this two groups (P < 0.05). Seven days post transplantation, the percentage of donor cells in bone marrow of non-sensitized and sensitized recipients was (48.07 ± 4.70)% and (0.77 ± 0.11)%, respectively, and the differences were statistically significant (P < 0.05). Furthermore, the white blood cells and BMCs following transplantation decreased along with time in sensitized recipients. The immune experiments of complement-dependent cytotoxicity reaction, cytotoxic T lymphocytes reaction and antibody-dependent cellular cytotoxicity showed the cytotoxic indexes were higher in sensitized group than the non-sensitized group.</p><p><b>CONCLUSION</b>A sensitized model was established by transfusions of allogeneic spleen cells. Allogeneic donor BMCs were rejected in sensitized recipients, and its mechanism might be through immune impairment pathways.</p>

Therapeutic effect of focal adhesion kinase gene silence on leukemia / 中国实验血液学杂志

This study was aimed to investigate the effects of focal adhesion kinase (FAK) gene silence on leukemia cell growth, leukemogenesis and efficacy of chemotherapy drug. Vector containing lentiviral-FAK-shRNA was constructed and transfected into BCR/ABL-BaF3 leukemic cells, the cell growth and apoptosis were detected in vitro. The effect of FAK shRNA on leukemogenesis was studied in a murine model with leukemia. The apoptosis of leukemia cells and survival of leukemic mice treated by FAK shRNA combined with drug STI571 were monitored. The results showed that FAK gene expression was knocked down by lentiviral-FAK-shRNA. FAK gene silencing inhibited leukemia cell growth in vitro. The apoptosis test results showed that the percentages of Annexin V(+) cells in vector control group and FAK shRNA group were (3.46 ± 0.56)% and (7.3 ± 0.79)%, respectively, and the difference was statistically significant (p < 0.05). The mice in vector control group died at day 21 to 27, while the mice in FAK shRNA group died between day 52 and 60, and the difference was statistically significant (p < 0.05). Moreover, FAK gene silence combined with drug STI571 could enhance the apoptosis of leukemia cells and prolong survival time of leukemic mice. It is concluded that FAK gene silence inhibits leukemogenesis and promotes efficacy of chemotherapy drug on leukemia cells, indicating FAK gene silence may be considered as a new therapeutic strategy for leukemia.

Construction and selection of the most efficient siRNA interfering plasmid specific to mouse Qa-1 gene / 中国实验血液学杂志

This study was purposed to construct three siRNA eukaryotic expression vector specific to mouse Qa-1 gene, to investigate its silencing effect on Qa-1 gene and to select the most efficient siRNA plasmid specific to mouse Qa-1 gene. Three siRNA peptides specific to mouse Qa-1 through siRNA Web design tools of Ambion company were chosed. Jingsai Company helped to complete the siRNA eukaryotic expression vector. The mouse NIH3T3 cells cultured in RPMI 1640 medium with 10% fetal bovine serum were divided into four groups: three groups of the cells were transfected with lipofectamine 2000 reagent and three different siRNA eukaryotic expression vectors, while one group cells were transfected with lipofectamine 2000 reagent alone as negative control. Cells were collected at 24, 48, 72 hours after transfection; the RNA level of Qa-1 was detected by RT-PCR, and the expression position was examined with flow cytometry analysis by using anti-Qa-1 monoclonal antibody. The results indicated that the constructed three siRNA eukaryotic expression vectors were found to be specific to mouse Qa-1 gene. The sequence analysis showed that the sequence was identical to what chosed from web tools. NIH3T3 cells in vitro were adhered in culture that cell shape appeared to change after transfection. RT-PCR and flow cytometry analysis by using anti-Qa-1 monoclonal antibody approved that both Qa-1 RNA and the expression of Qa-1 on cell surface decreased. The decreased levels in the three groups were different. At 24, 48 and 72 hours, the expression of Qa-1 on NIH3T3 cells decreased as in the following: H2-T231: 60.9%, 81.9%, 43.6%; H2-T232: 64.5%, 73.9%, 61.1%; H2-T233: 61.9%, 71.2%, 47.5%. H2-T232 was most efficient one in all three time points. It is concluded that all three siRNA eukaryotic expression vectors selected can successfully suppress the expression of the Qa-1, and from them H2-T232 is most efficient.

Establishment of sensitized animal models and their sensitization effects on engraftment of hematopoietic stem cells / 中国实验血液学杂志

This study was aimed to establish sensitized animal models, explore the changes of immune function in these sensitized recipients, and investigate effects of sensitization on the engraftment of hematopoietic stem cells (HSCs). Different doses of spleen cells (1x10(5), 1x10(6) and 1x10(6)x2 at intervals of 7 days) from C57BL/6 were infused into BALB/c, the immunity function of sensitized models was tested by complement-dependent cytotoxicity method, mixed lymphocyte reaction and ELISA. After irradiation with gamma-ray of 60Co in dose 8 Gy, sensitized mice were transplanted 1x10(7) C57BL/6 bone marrow cells via tail vein or intra-bone marrow, and survival rate was detected daily. The results showed that different levels of donor reactive antibody were induced in all sensitized models. Comparing with normal mice, profound proliferation of spleen cells were found in groups of injected 1x10(6) and 1x10(6), continuous injections at intervals of 7 days. Sensitized model received bone marrow cells of C57BL/6 via tail vein died on day 10 to 14 after transplantation, and sensitized model mice received bone marrow cells of 1x10(6)x2 at intervals of 7 days via intra-bone marrow also died within two weeks after transplantation. It is concluded that different sensitized mouse models are established by different doses of allogeneic spleen cells infusion, the changes of immune function in sensitized mice are correlative with sensitization. Donor HSCs are rejected in sensitized models, and the engraftment can not be improved by intra-bone marrow injection.

Experimental study on the effect of sensitized sera on engraftment of hematopoietic stem cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of sensitized sera on engraftment of hematopoietic stem cells (HSCs) and its mechanism.</p><p><b>METHODS</b>Bone marrow cells (BMCs) from C57BL/6 mice were incubated with sensitized sera (group I) or normal sera (group II), and then washed and transplanted into irradiated BALB/c mice. The survival analysis and engraftment evaluation of the recipients were observed. The incubated BMCs were bound with goat anti mouse IgG for and labeled with Annexin V for apoptosis detection.</p><p><b>RESULTS</b>Seven out of ten recipient mice in group I died of bone marrow failure at day 10 after transplantation, while all of those in group II were long-term survived. Engraftment assay showed recipients blood count and BMCs progressively decreased along with time passing in group I; in addition, the chimeric percentage of donor cells progressively decreased as well. The percentage of BMCs binding with goat anti mouse IgG in group I or group II were (90.3 +/- 5.1)% and (5.2 +/- 2.4)%, respectively, and the difference was statistically significant (P < 0.01). However, no significant difference was found in the apoptosis detection between the two groups (P > 0.05).</p><p><b>CONCLUSION</b>The engraftment capacity of HSCs is significantly impaired by sensitized sera, the antibodies in sensitized sera may bind to antigens expressed on HSCs but do not induce apoptosis.</p>

Effect of the serum panel reactive antibody on proliferation and differentiation of cord blood CD34+ cells in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>The low rate of engraftment in children with beta-thalassemia has seriously restricted the popularity of the hematopoietic stem cell transplantation (HSCT). Panel reactive antibody (PRA) has been regarded as one of the important factors for the success of kidney transplantation. Poly-transfusion before transplantation is associated with the production of PRA. Also PRA is produced in the children with beta-thalassemia major who need poly-transfusion for life. PRA might be one of factors inducing the low rate of engraftment in children with beta-thalassemia. This study focused on observing the effect of PRA on the proliferation, differentiation, apoptosis and necrosis of cord blood CD34(+) cells in vitro by incubating the cord blood CD34(+) cells with serum containing PRA.</p><p><b>METHOD</b>Seven samples of cord blood were collected and the HLA typing for every sample was done. Seven sera positive for PRA and seven negative sera were selected respectively. Mononuclear cells (MNCs) were obtained by Ficoll-Hypaque density gradient centrifugation. CD34(+) cells were isolated from MNCs by positive selection using an immunomagnetic separation (CD34(+) progenitor cell isolation kit and auto-MACS). The CD34(+) cells of umbilical cord blood were incubated with the serum and complement in the following groups: A (absence of serum), B (presence of PRA positive serum), C (presence of PRA positive serum and complement), D (presence of complement), and E (presence of PRA negative serum). After incubation the samples were centrifuged and the supernatant was collected for LDH detection. At the same time the CD34(+) cells were harvested for assessing the expression of Annexin V and CD95 of the CD34(+) cells by flow cytometry and also for the detection of the DNA synthesis by (3)H-TaR incorporation. Meanwhile the cells were inoculated into the methylcellulose cultural system. The proliferation and hematopoietic potential of the CD34(+) cell of cord blood by the colony formation assay were detected on the day 10.</p><p><b>RESULT</b>The concentration of LDH in group A was (20.71 +/- 2.81) U/L, which was significantly lower than that in group B (64.28 +/- 5.12) U/L and group C (84.29 +/- 4.99) U/L. The concentration of LDH in group B was significantly lower than that in group C, while there were no significant differences in the concentration of LDH among groups A, D and E (P > 0.05). The cpm in group A was (22 629 +/- 3288), which was significantly higher than that in group B (4598 +/- 2178) and group C (1626 +/- 1192). And the cpm in group B was significantly higher than that in group C. There were no significant differences in the cpm among groups A, D and E (P > 0.05). On day 10 of culture, the total colonies, granulocyte-macrophage colony forming unit (CFU-GM), mixed colony forming unit (CFU-GEMM) and erythroid burst colony forming unit (BFU-E) in group A were significantly higher than that in group B and C. The total colonies, CFU-GM and CFU-GEMM in group B were significantly higher than those in group C. No significant differences were found in the total colonies, CFU-GM, CFU-GEMM and BFU-E among groups A, D and E (P > 0.05). There were no statistically significant differences in the CD95 and Annexin V expression among all the groups (P > 0.05).</p><p><b>CONCLUSION</b>PRA could impair the membrane, decrease the DNA synthesis, and inhibit the colony formation of CD34(+) cord blood cells, which could be strengthened by the presence of the complement at the given concentration in our study. PRA had no significant influence on the apoptosis of CD34(+) cells in vitro.</p>

Risk factors and treatment of hemorrhagic cystitis in children after hematopoietic stem cell transplantation / 中华儿科杂志

<p><b>OBJECTIVE</b>Hemorrhagic cystitis (HC) is one of the common complications of hematopoietic stem cell transplantation (HSCT), which causes significant pain, prolongs hospitalization, and occasionally results in renal failure and death. This study aimed at investigating the incidence, risk factors, and outcome of HC in children post umbilical cord blood transplantation (UCBT) and peripheral blood stem cell transplantation (PBSCT).</p><p><b>METHODS</b>From October 1998 to June the clinical records of 53 pediatric patients (aged 2-18 years with median age of 7.5 years) in our HCST center who underwent UCBT (n = 37) and PBSCT (n = 16) were retrospectively analyzed. Thirty out of 53 patients were diagnosed as hereditary hemolytic anemia (56.6%), and the others as haematological malignancies (43.4%): of whom 8 had acute lymphoblastic leukemia, 12 acute myeloid leukemia, 2 chronic myeloid leukemia and 1 non-hodgkin lymphoma. Conditioning regimen varied according to disease and clinical status, however based on cyclophosphamide (CTX, 120-200 mg/kg) and busulphan (BU, 12-16 mg/kg) in the cohort. Total body irradiation (TBI) or total lymphoid irradiation was added in 7 patients respectively. The patients were divided into regular treatment group (RTG) with 15 cases who received hyperhydration, alkalinizing, diuresis and Mesna during CTX infusion and prostaglandin E1 (PGE1) group (PEG) with 38 cases who received hyperhydration, alkalinizing, diuresis and Mesna plus prostaglandin E1 (0.03 microg/kg.h). The risk factors of HC were examined by univariate and multivariate analysis.</p><p><b>RESULTS</b>In all, 11 of the 53 transplanted patients developed HC (21%) with a median onset time of day +15 (rage day +2 - +25). HC was classified as early in 4 (36%) and late in 7 (64%), and scored as grade Iin 2 cases (18%), grade II in 4 (36%) and grade III in 5 (46%). There was no significant difference between RTG and PEG in the incidence of HC, however, the incidence was much higher in the group of patients who were > or = 6 years old, positive group of graft-versus-host disease (GVHD) and group of cytomegalovirus (CMV) infection than that in the group of patients who were < 6 years of age (32% vs. 8%, P < 0.05), negative group of GVHD (35% vs. 7%, P < 0.05) and CMV non-infected group (62% vs. 13%, P < 0.05), respectively. Furthermore, by multivariate analysis, > or = 6 years old (OR = 3.53, P < 0.05) and CMV infection (OR = 4.31, P < 0.05) were significant risk factors for HC. Three of 11 patients were treated with bladder irrigation. All patients recovered from HC in a median 12.8 days (range 2-53 days).</p><p><b>CONCLUSION</b>Older age (> or = 6 years) as well as CMV infection were the risk factors of HC. Both hyperhydration and Mesna were effective in preventing HC, while addition of PGE1 could not reduce the incidence of HC. The prognosis of HC in children post HSCT was satisfactory.</p>

Ex vivo expansion of megakaryocytic progenitors from mobilized human peripheral blood / 中国实验血液学杂志

This study was aimed to investigate the effect of some culture system composed of various cytokine combinations (TPO, SCF, FL, IL-1, IL-3, IL-6) on ex vivo expansion of megakaryocytic progenitors induced from CD34+ cells of peripheral blood and to seek a optimal cytokine combination and culture time. Mononuclear cells were isolated from mobilized peripheral blood (MPB) by density gradient centrifugation over Ficoll. CD34+ cells were purified by using an immunomagnetic bead separation system. The selected CD34+ cells were seeded in Iscove's modified Kulbecco's medium (IMDM) supplemented with fetal calf serum (FCS) and various kinds of cytokines. After 15 days of culture, the content of CD41+ cells in culture system were determined by flow cytometry, and the number of megakaryocyte colony-forming unit (CFU-MK) was measured simultaneously. The results showed that after definited days of culture, the cytokine combination TPO/FL/IL-6/IL-3 was the most suitable for MPB to obtain high number of MK, and better than any other three groups (P < 0.05). The increase multiple of CD41+ cells was 93.97 +/- 17.27 on day 5 and 131.23 +/- 18.26 on day 10. On day 15, the proportion and the increase multiple of CD41+ cells decreased obviously. The expansion multiples of CFU-MK were 93.33 +/- 10.02 on day 5 and 120.67 +/- 13.01 on day 10, higher than any other groups. It is concluded that TPO/FL/IL-6/IL-3 combination was the best optimal for expansion ex vivo of megakaryocytic progenitors from MPB, and its suitable duration of culture was 10 days; a culture system for expansion ex vivo of megakaryocytic progenitors have been established in this study.