Efficacy of Posaconazole for Primary Prophylaxis in the Induction Therapy of Childhood Acute Lymphoblastic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of posaconazole in the primary prevention of invasive fungal disease (IFD) in the induction therapy of childhood acute lymphoblastic leukemia (ALL).@*METHODS@#From August 2018 to November 2020, 144 pediatric patients with ALL treated in Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University were selected, 88 cases received fluconazole as IFD prophylaxis (fluconazole prophylaxis group), 56 cases received posaconazole as IFD prophylaxis (posaconazole prophylaxis group). The incidence of IFD and treatment-related adverse reactions between the two groups were compared, and the safety of posaconazole was evaluated.@*RESULTS@#The incidence of IFD in the fluconazole prophylaxis group was 20.4% (18/88), and in the posaconazole prophylaxis group was 7.1% (4/56). The incidence of IFD between the two groups was statistically significant different(P=0.030). There was no serious adverse reactions in the two groups. The incidence of mild adverse reactions in the posaconazole prophylaxis group (23.2%) was lower than that in the fluconazole prophylaxis group(39.8%), and the difference was statistically significant (P=0.039). There were 12 cases died in the fluconazole prophylaxis group and 4 in the posaconazole prophylaxis group, while no significant difference in the overall survival rate between the two groups (P=0.281).@*CONCLUSION@#The effect of posaconazole in the primary prophylaxis of IFD is better and incidence of adverse reactions is lower than fluconazole. Posaconazole can be tolerated, and expected to become the first-line primary prophylaxis drug for IFD during the induction remission therapy of childhood ALL.

Analysis of Clinical Characteristics and Prognosis in Children with Acute Megakaryoblastic Leukemia without Down Syndrome / 中国实验血液学杂志

OBJECTIVE@#To analyze the clinical characteristics and treatment effects of children with acute megakaryoblastic leukemia without down syndrome (non-DS-AMKL).@*METHODS@#The clinical data of 19 children with non-DS-AMKL treated in the Pediatric Hematology Ward in Sun Yat-sen Memorial Hospital of Sun Yat-sen University from May 2008 to April 2018 were analyzed retrospectively. The clinical characteristics, laboratory test and treatment methods of the children were concluded. All patients were followed up to evaluate the effect of treatment.@*RESULTS@#The 19 cases of children included nine male and ten female, the median age of onset was 2 years old. The clinical manifestations showed nonspecific. The median white blood cell of peripheral blood was 15.88×10@*CONCLUSION@#Non-DS-AMKL was rare in children and difficult to be diagnosed. Determination of MICM classification as early as possible was helpful for diagnosis, and genetic testing played an important role for diagnosis and prognosis evaluation. Early hematopoietic stem cell transplantation in patients with CR after chemotherapy might be an effective way to cure AMKL.

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.

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>

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>

Relation of Treg and iNKT cell reconstruction with aGVHD after allogeneic hematopoietic stem cell transplantation in children / 中国实验血液学杂志

This study was aimed to explore the relation of Treg and invariant natural killer T (iNKT) cell reconstruction with acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children. According to the occurrence or absence of aGVHD, 29 pediatric patients who underwent allo-HSCT were firstly divided into two groups non-aGVHD and aGVHD group,then those patients with aGVHD were divided into steroid effective group and steroid resistant group according to their reaction to the steroid treatment. Flow cytometry was used to detect the frequency of Treg cells and iNKT cells in the peripheral blood of the recipients at different time after allo-HSCT(d 15, d 30, d 60, d 90, the time of aGVHD onset and two weeks after steroid treatment). The result showed that the frequencies of Treg cells and the iNKT/T ratio on day 15 in non-aGVHD group were significantly higher than those in the aGVHD group (P < 0.05). It is concluded that a combined monitoring strategy of Treg and iNKT cell reconstruction early after allo-HSCT may facilitate the diagnosis and treatment of aGVHD in children.

Acute leukemia child with ocular Kaposi's sarcoma after hematopoietic stem cell transplantation: a case report and literatures review / 中华血液学杂志

<p><b>OBJECTIVE</b>To summarize clinical features of eye Kaposis' sarcoma ( KS ) in leukemia child after peripheral blood stem cell transplantation (PBSCT).</p><p><b>METHODS</b>One 13 years-old child with acute lymphoblastic leukemia (ALL) and negative HIV test who developed KS restricted in right conjunctiva, cornea and sclera after successful allogeneic PBSCT was reviewed retrospectively.</p><p><b>RESULTS</b>The child suffered from T cell type ALL. He received immunosuppressive treatment after PBSCT, and had once extensive herpes zoster restricted in skin. Seven months after PBSCT, he had blurred vision with right eye and slowly neoplasm formed in cornea and conjunctiva. Pathological examination confirmed KS with changes like capillary hemangioma, atypical fusiform cell, typical immunochemistry and positive immunofluorescent result of HHV8. He received excision of lump of cornea, conjunctiva, sclera and transplantation of cornea and sclera. Antiviral therapy was given together with anti-infection, prevention of cornea rejection and biotherapy. He kept right eye and hand-move eyesight, survived without GVHD or recurrence of ALL and KS.</p><p><b>CONCLUSION</b>This was the first ocular KS case in ALL child after PBSCT, without correlation with HIV infection. Complete excision combined with biotherapy was safe and effective for single ocular lesions.</p>

Distribution and interaction of bone marrow mesenchymal stem cells and cord blood CIK/NK cells infused via different ways at different time periods in NOD/SCID mice / 中国实验血液学杂志

The study was aimed to explore the distribution and interaction mechanism of human bone marrow mesenchymal stem cells (MSC) and cord blood cytokine-induced killer (CIK)/natural killer (NK) cells infused via different ways at different times in NOD/SCID mice. 5 microl 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DiI) dye(red) was added in suspension of MSC per ml, and 1 microl carboxyfluorescein diacetate, succinimidyl ester(CFDA SE) dye(green) was added in suspension of CIK/NK cells per ml. The amounts of MSC and CIK/NK cells infused in each 6 NOD/SCID mouse were 1 x 10(6) (0.1 ml) and 1 x 10(7) (0.1 ml) respectively. All mice were divided into 4 groups, each group consisted of 6 mice. Group A: MSC (intravenous infusion, iv) + CIK/NK cells (iv) at the same time, group B: MSC (iv) + CIK/NK cells (iv) at 48 hours after infusion of MSC; group C: MSC (intramedullary infusion, im) + CIK/NK cells (iv) at the same time; group D: MSC (im) + CIK/NK cells (iv) at 48 hours after infusion of MSC. 3 NOD/SCID mice were sacrificed per batch at 24 hours and 48 hours after infused CIK/NK cells. Frozen sections of liver, spleen, lung and kidney were prepared, and then followed by counting the amounts of red and green fluorescence cells under fluorescence microscope, and calculating the ratio of MSC to CIK/NK cells for reflecting the interaction of MSC and CIK/NK cells in mice, and for showing the suppressive intensity of MSCs on CIK/NK cells. The results showed that the sums of average ratios of MSC to CIK/NK cells in lung, liver and spleen of group A and B were higher than that in group C and D at 24 hours and 48 hours respectively after infusing CIK/NK cells. The sum of average ratios of MSC to CIK/NK cells in group A was slightly higher than that in group B at 24 hours and 48 hours after infusing CIK/NK cells, but there was no significant difference between them. The sum of average ratios of MSC to CIK/NK cells in lung, liver and spleen in group C was slightly lower than that in group D at 24 hours after infusing CIK/NK cells, but reversed at 48 hours later and there was no significant difference between them. The sums of average ratios of MSC to CIK/NK cells in lung, liver and spleen in group A, B, C and D were all higher than those in kidney at 24 and 48 hours respectively after infusing CIK/NK cells. It is concluded, the MSC and CIK/NK cells may interact if they are infused via the same way and at the same time, the location where the suppression of MSC on CIK/NK cells occur in vivo may be reticulo-endothelial systems in lungs and livers.

Unrelated allogeneic umbilical cord blood transplantation: present status, problems and countermeasures / 中国实验血液学杂志

As unrelated allogeneic umbilical cord blood transplantation (UCBT) has been developed for 20 years already since 1988, more than ten thousands cases have cumulatively undergone UCBT over the world. A huge number of clinical data confirmed that UCBT had unique characters with low rate of severe GVHD. The efficacy and data on TRM, relapse and EFS of allogeneic UCBT with HLA 0-1 mismatched are similar to those in HLA matched BMT. UCBT has become the optimal choice for source of hematopoietic stem cells for allogeneic stem cell transplant especially when HLA-matched or haploidentical donors are not available in time. In most developed countries, unrelated allogeneic UCBT developed successively, and in recent years HLA mismatched UCBT with double units performed in adults increased even more rapidly than in children. Another recent trend of UCBT has been extending to treat some non-malignant but refractory diseases in pediatrics, such as severe combined immunodeficiency, thalassemia major, bone marrow failure syndrome and metabolic disorders. The clinical successful practice of double units for cord blood transplantation inspires to ponder over questions remaining mystery. What is the conflict like between two mismatched donor cells in vivo, which does not spoil the whole transplantation but enable the patient to be engrafted successfully without any increment of the dosage by the sum of two doses together? How can they both be taken at the same time firstly by the recipient, but why does only one predominate later? What are the factors enable the donor cells of the winner to sustain? With the references of the international experiences, how to solve the clinical encountered problems, perspective of unrelated allogeneic UCBT and proper strategies to be enacted are reviewed.

The cytotoxicity of CIK/NK cells stimulated by K562-DC fusion vaccines in NOD/SCID mice model for human erythroleukemia / 中华血液学杂志

<p><b>OBJECTIVES</b>To study the in vivo efficacy and the safety of cord blood derived CIK/NK cells stimulated by K562-dendritic cells (DC) fusion vaccines in NOD/SCID mice model for human erythroleukemia.</p><p><b>METHODS</b>DC and CIK /NK cells were both derived from cord blood mononuclear cells. DC were fused with inactivated K562 leukemia cell by PEG to produce K562-DC fusion vaccines. K562-DC fusion vaccines were co-cultured with CIK/NK cells to prepare K562-DC fusion vaccine stimulated CIK/NK cells. NOD/SCID mice were inoculated with 1 x 10(6) K562 cells. 24 hours later, 1 x 10(7) vaccines stimulated CIK/ NK cells and 1 x 10(7) CIK/NK cells were transfused into the NOD/SCID mice. NOD/SCID mice without inoculation of K562 cells were used as control group. CD13 and CD56 positive cells were assayed by flow cytometry.</p><p><b>RESULTS</b>All the leukemia NOD/SCID mice without therapy died within 39 days, tumor was found in 5 of 8 mice. One of 8 leukemia mice treated with K562-DC fusion vaccines stimulated CIK/NK cells died at the 65th day, the anti-tumor response rate was 87.5%. Two of the leukemia mice treated with CIK/NK cells died at the 56th and 65th day respectively, the anti-tumor response rate was 75%. There was no significant difference in survival time between these two groups, and both survivals were longer than that of the control group. There was no significant difference in CD13 positive cells in the survival mice between these two groups, and both of that were less than that of the control mice. There was no significant difference in CD56 positive cells between the two treated groups and the control group.</p><p><b>CONCLUSIONS</b>Cord blood derived CIK/ NK cells stimulated by inactivated tumor cells retain the cytotoxicity and do not develop tumor in vivo.</p>

BMP-4 and VEGF promote development of hematopoietic stem cells during the embryoid body formation from embryonic stem cells / 中国实验血液学杂志

The objective of this study was to explore the effects of BMP-4 and VEGF on the development of primary hematopoietic stem cells during the differentiation of embryonic stem cells (ESCs) into embryoid body (EB). Murine E14 ESCs were seeded into semisolid methylcellulose-based medium for EB formation. According to added or not cytokines, experiments were divided into: (1) group of spontaneous differentiation without cytokine as control; (2) group of BMP-4 in different concentrations (0, 5, 15, 25 and 50 ng/ml); (3) group of BMP-4 combined with VEGF; (4) group of VEGF alone. EBs were collected on days 3, 6, 9, 12, 15, and the proportion of Flk-1(+) cells were assayed by flow cytometry. The results showed that in the different BMP-4 concentration groups, the proportions of Flk-1(+) cells were significantly different, and it reached the peak values in 25 ng/ml BMP-4 group as 6.51 +/- 1.02% at day 3 and 7.70 +/- 1.12% at day 6 respectively, which were statistically higher than those in control group without-BMP-4 and in 5 ng/ml BMP-4 group (p < 0.05). When BMP-4 was used in combination with VEGF, Flk-1(+) cells went to peak proportion value at day 9 as 27.53 +/- 8.14%, which was statistically higher than that in spontaneous differentiation group as 8.77 +/- 2.35% (p < 0.05) and VEGF treatment group as 11.21 +/- 2.23% (p < 0.05). It is concluded that BMP-4 in combination with VEGF can promote Flk-1(+) cells genesis during EB formation in vitro, which provides experimental evidence for researches on directed differentiation of ESCs into hematopoietic stem cells simulating the microenvironment in vivo.

Analysis of lymphocyte immune abnormality in 52 cases of children idiopathic aplastic anemia / 中国实验血液学杂志

This study was aimed to investigate the characteristics of lymphocyte immune abnormality in children idiopathic aplastic anemia (IAA) in order to explore the immune pathogenesis of childhood IAA. The phenotypes of lymphocytes, the ratios of Th1/Th2 and Tc1/Tc2, the levels of CD25(+) and CD4(+)CD25(+) T lymphocytes in peripheral blood of IAA patients were measured at the onset of disease by flow cytometry and were compared with that in normal controls. The influences of those immunological indicators on prognosis of IAA were also analyzed. The results showed that there were 40 cases of severe aplastic anemia (SAA) and 12 cases of mild aplastic anemia (MAA). The levels of CD3(+) CD8(+) T cells in SAA group and MAA group were significantly higher than those in controls (p < 0.05). The levels of CD3(+) and CD3(+) CD4(+) T cells in MAA group were lower than that in SAA group (p < 0.05), but there were no difference was compared with control group. No differences of the levels of CD3(-)CD19(+) T cells were between the both SAA and MAA groups and the control group. The levels of CD3(-)CD56(+) T cells in SAA group and MAA groups were lower significantly than that in control group. As compared to control group, the levels of Th1 and Tc1 in SAA group and MAA groups increased significantly (p < 0.05), and the ratios of Th1/Th2 and Tc1/Tc2 in SAA group and MAA groups increased significantly (p < 0.05). The level of Th2 increased in SAA group. As compared to MAA group, the levels of Th1 and Tc1 and the ratios of Th1/Th2 and Tc1/Tc2 in SAA group increased significantly (p < 0.05). The levels of CD25(+) T lymphocyte in SAA group and MAA group increased significantly (p < 0.05), and were higher than that in normal controls, but levels of CD4(+)CD25(+) T lymphocyte and ratio of CD4(+)CD25(+)/CD4(+) in SAA group and MAA group had no significant difference. It is concluded that the abnormal lymphocyte immune function exist in the onset of childhood IAA. The polarization of Th1/Th2 and Tc1/Tc2 shifts to Th1 and Tc1 cells. These changes closely relate to severity of the disease. There is high level of CD25(+) T lymphocyte in children IAA. These changes reveal that abnormality of immune function plays an important role at the onset of childhood idiopathic aplastic anemia.

Experiment study of efficacy on hematopoietic reconstitution and GVHD prophylaxis after mesenchymal cell infused by intra-bone marrow cavity or intravenous in rat BMT models / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the in vivo distribution of mesenchymal stem cells (MSCs) after administrated by intra-bone marrow (IBM) or intravenous (i.v.), and compare the effects on hematopoiesis reconstitution and GVHD in rat BMT models.</p><p><b>METHODS</b>(1) MSCs from male Wistar rats marked with CFSE were injected into the bone marrow cavity (IBM) or the vein (i.v.) of recipient rats, and observed the distribution of MSCs in vivo. (2) Allogeneic BMT model of Fischer344 rats (RT1A(1)) to Wistar rats (RT1A(u)) was established. The recipient rats were exposed to 8 Gy of gamma irradiation 1 day before transplantation. The 6 groups were (1) IBM group [IBM-injection of MSCs + IV-injection of bone marrow cells (BMC)]; (2) IV group (i.v.-injection of MSCs (i.v.) + i.v.-injection of BMC); (3) BMT group (only i.v.-injection of BMC); (4) MSCs control group (only i.v.-injection of MSC); (5) normal control group and (6) irradiation control group.</p><p><b>RESULTS</b>(1) After i.v.-injection, large numbers of the MSCs lodged in lungs while small numbers in the peripheral blood, liver, thymus and spleen, and a few marked MSCs could be seen in bone marrow. After IBM injection, most cells distributed in long bones and those lungs were less than that in i.v. group. (2) Co-transplantation of MSCs (IBM/IV) could accelerate the recovery of hematopoiesis, including the recovery of WBC, hemoglobin and platelet, and in IBM-injection was more effective in the recovery of hematopoiesis than that in i.v. group. (3) Incidence rate of GVHD in BMT group was 42% (3/7), and no GVHD occurred in co-transplantation groups. (4) Recovery of CFU-Mix and CFU-MSCs could be seen at 21st and 30th day after transplantation in co-transplantation groups, and IBM-injection was more effective than i.v.-injection.</p><p><b>CONCLUSION</b>(1) IBM-injection results in most MSCs distributed in long bones. (2) MSCs improve the survival rate after BMT. (3) Co-transplantation of MSCs accelerates the recovery of hematopoiesis and reduces the morbidity of GVHD. (4) MSC promotes reconstitution of hematopoietic cells and bone marrow MSCs in recipient rates and the effects of MSCs administrated via IBM is more effective than via i.v.</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>

Screening and cloning the genes differentially expressed in human embryonic AGM-derived stromal cells / 中国实验血液学杂志

To screen and separate the genes differentially expressed in human embryonic aorta-gonad-mesonephros (AGM)-derived stromal cells, a subtracted library was generated through the suppression subtractive hybridization using the cDNA of human embryonic AGM-derived stromal cells as target and human fetal liver (FL)-derived stromal cells as drivers. Then a high though screening technique, gene chip, was used to screen the differentially expressed genes in the established subtractive library. Approximately 18 of the resulting subtracted cDNA clones were partially sequenced and analyzed by blastn in the GenBank database. The results showed that 211 Clones were selected and identified from the established subtractive library, the positive ratio was amount to 76.4%. 18 over-expressed genes were screened by gene chip with more than a 5-fold difference expression levels between AGM and FL-derived stromal cells, and were selected to sequence, results of sequencing indicated that the 18 sequences was compared to known sequences in the GenBank database, and among the sequenced clones, 14 sequences were considered as part of the known genes, and 4 sequences representing previously unknown genes. The known genes were reported to involve the regulation of cell migration, cell differentiation, cell proliferation, cell cycle, signal transduction, and angiogenesis. Most of these genes have not been reported to relate to the haematogenesis in ontogeny. It is concluded that many genes both known and unknown are differentially expressed in human embryonic aorta-gonad-mesonephros-derived stromal cells. Discovery of these genes provides a solid foundation to elucidate the mechanism of haematogenesis in ontogeny.

Effects of human umbilical cord blood mesenchymal stem cells on the expansion of CD34+ cells from umbilical cord blood / 中华儿科杂志

<p><b>OBJECTIVE</b>The previous studies indicated that mesenchymal stem cells (MSCs) either from umbilical cord blood (UCB) or from bone marrow (BM) had the same biological characteristics and the function of secreting hematopoietic growth factors (HGFs). The present study aimed to understand the effects of human UCB MSCs on the expansion of CD(34)(+) cells from UCB.</p><p><b>METHODS</b>1. Human UCB CD(34)(+) cells were incubated in the system containing UCB MSCs, HGFs and serum free medium. 2. The surface markers (CD(34)(+), CD(34)(+)CD(38)(-), CD(34)(+)CD(3)(+), CD(34)(+)CD(19)(+), CD(34)(+)CD(33)(+), CD(34)(+)CD(41a)(+)) on expanded UCB cells were examined by flow cytometry on the 6th and 12th days. 3. The expanded and unexpanded cells were cultured in semi-solid culturing system and checked for colony forming units of granulocyte and macrophage (CFU-GM), erythroid burst-forming unit (BFU-E), colony forming units of granulocyte- erythrocyte-megakaryocyte-macrophage (CFU-Mix) and colony forming units of high-proliferative potential (CFU-HPP).</p><p><b>RESULTS</b>1. The expansion folds of CD(34)(+)CD(38)(-) cells from UCB MSCs + HGFs groups on the 6th and 12th days were 159.43 and 436.68, respectively. Interestingly, the percentage of CD(34)(+)CD(38)(-) cells declined in HGFs group after expanding for 12 days, but it rose to 9.98% in the UCB MSCs + HGFs group. 2. Colony forming capacity of expanded UCB cells showed that the folds of CFU-Mix and CFU-HPP of UCB MSCs + HGFs group increased from day 6 to day 12, but the folds decreased in the HGFs group. 3. From day 0 to day 12, CD(34)(+)CD(33)(+) cells and CD(34)(+)CD(41a)(+) cells were amplified gradually, but CD(34)(+)CD(19)(+) and CD(34)(+)CD(3)(+) cells decreased gradually, and in UCB MSCs + HGFs group this phenomenon was more significant than that in HGFs group.</p><p><b>CONCLUSION</b>1. UCB MSCs containing system not only has the ability to expand the primitive HSCs but also has the ability to sustain the proliferation of HSCs. 2. UCB MSCs containing system amplified mainly myeloid and megakaryocytoid progenitor subsets. These may have clinical significance in reducing infection and hemorrhage.</p>

Prophylaxis and treatment of chronic graft versus host disease / 中华儿科杂志

<p><b>OBJECTIVE</b>Chronic graft versus host disease (cGVHD) is the most common late complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and it represents the major cause of mortality in long-term survivors. Over the past decade, although conventional therapy has achieved complete responses in approximately 50% of patients, the prophylaxis and treatment of cGVHD are still not satisfactory. In the late years, utilization of new immunosuppressant such as tacrolimus (FK506), mycophenolate mofetil (MMF) on cGVHD improved the curative effects. This study tried to analyze the results of combination of methylprednisolone (MP), MMF and FK506 or cyclosporine A (CSA) as immunosuppressive therapies for cGVHD and to explore the effective regimen for children.</p><p><b>METHODS</b>Forty-five patients received allo-HSCT. Among them 32 received UCBT and 13 received PBSCT. The conditional regimen mainly consisted of busalphan, cyclophosphamide, antihuman thymocyte globulin, fludarabin, melphalan, thiotepa and total lymph node irradiation. Prophylaxis of GVHD consisted of CSA, MP and MMF. Patients with cGVHD received a regimen with combination of MP, MMF and FK506 or CSA.</p><p><b>RESULTS</b>Seventeen out of 32 patients who received UCBT were engrafted. while 9 out of 13 patients who received PBSCT were engrafted. Nine cases of the 30 engrafted patients developed cGVHD (morbidity 30%). Among the 17 patients who received UCBT, 3 developed cGVHD (18%). Among the 13 patients who received PBSCT, 6 developed cGVHD (46%). Six cGVHD continued from aGVHD (6/9). One patient was given CSA plus MMF, and 8 were given three-drug regimen with MP, MMF and FK506. The overall response rate was 100%. Two patients died of CMV-IP or septicemia (mortality 20%). Seven (78%) patients survived (event free survival, EFS) longer than 3 years. The side effects included hepatotoxicity, nephrotoxicity, hypertension, articular capsulitis and arrhythmia. The main complication and the major causes of death were infection.</p><p><b>CONCLUSION</b>The incidence of cGVHD is low in children. The incidence of cGVHD after PBSCT is higher than that after UCBT. aGVHD is a highly dangerous factor. Combined therapy of MP plus MMF and FK506 or CSA is safe and effective for the treatment of cGVHD in children.</p>