Expression and Significance of BLIMP-1 in Regulatory T Cells of Children with Aplastic Anemia / 中国实验血液学杂志

OBJECTIVE@#To study the expression of B lymphocyte-induced mature protein-1 (BLIMP-1) in regulatory T cells (Tregs) of children with aplastic anemia (AA), and analyze its correlation with the number of Tregs and the levels of inhibitory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β in plasma.@*METHODS@#The peripheral blood samples of 10 newly diagnosed AA children and 10 healthy children were collected for experiment. qPCR was used to detect FOXP3 and PRDM1 mRNA expression levels. Flow cytometry was used to detect the proportion of Tregs, the expression of BLIMP-1 in Tregs, and the levels of cytokines such as IL-2, IL-17A, IL-6, interferon (IFN)-γ, IL-10 and TGF-β in plasma. Pearson correlation model was used to evaluate the relationship between the expression of BLIMP-1 in Treg and the number of Tregs, as well as the levels of IL-10 and TGF-β in plasma.@*RESULTS@#Compared with control group, the proportion of Tregs in peripheral blood of AA children was decreased significantly (P<0.001); The plasma levels of proinflammatory cytokines IL-2, IL-6 and IFN-γ in AA children were increased significantly (P=0.033, P=0.031, P=0.006), and IL-17A also was increased but the difference was not statistically significant (P=0.052), while anti-inflammatory cytokines IL-10 and TGF-β were significantly reduced (P=0.048, P=0.002). The relative expressions level of FOXP3 and PRDM1 mRNA in AA children were significantly lower than those in control group (P=0.037, P=0.016). The expression of BLIMP-1 protein in Tregs of AA children was significantly lower than that in control group (P<0.001). The expression level of BLIMP-1 protein in Tregs was positively correlated with the percentage of Tregs in lymphocytes (r=0.671, P=0.001), and was also positively correlated with the levels of IL-10 and TGF-β in plasma (r=0.500, P=0.029; r=0.486, P=0.030).@*CONCLUSION@#The expression of BLIMP-1 in Tregs of AA children is impaired, and the low expression of BLIMP-1 is related to the decrease of the number in Tregs and IL-10 and TGF-β expressions.

Relationship of Blimp-1 Hypoexpression with Pathogenesis of Aplastic Anemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the relationship of Blimp-1 hypoexpression with abnormality of Treg level and pathogenesis of aplastic anemia (AA).</p><p><b>METHODS</b>The mouse model with AA was established by adminis tration of IFN-γ combined with busulfan. The samples were collected at different day establishing AA model, and the spleen Treg number was detected, the Treg cells were sorted and expression level of prdm-1 was detected.</p><p><b>RESULTS</b>The number of Tregs in mice with AA was lower than that in control mice, moreover, the level of Treg decrease positively correlated with the AA severity (r=0.805), the higher the expression level of prdm-1, the higher the ratio of Treg/lymphocytes, showing positive correlation between them (r=0.548).</p><p><b>CONCLUSION</b>Blimp-1 expression may promote the proliferation and differentiation of Treg. The hypoexpression of Blimp-1 mediates the pathogenesis of AA and promotes progression of AA through reducing the proliferation of Treg, and decreacing the number of Treg.</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>

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>

Outcome Comparison of Pediatric Langerhans Cell Histiocytosis Treated with Modified DAL-HX83/90 or JLSG-96 Protocol / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze and compare the clinical features, treatment and prognosis of 31 children with Langerhans cell histiocytosis(LCH) treated with modified DAL-HX83/90 or JLSG-96 protocol.</p><p><b>METHODS</b>The clinical features, treatment and prognosis of 31 children with Langerhans cells admitted in our hospital from January 2005 to December 2014 were analyzed retrospectively. The outcome of patients treated with modified DAL-HX83/90 or JLSG-96 protocols were compared by using the Kaplan-Meier survival curve. Among 31 children with LCH, 19 males and 12 females, 12 younger than 2 years old, and 19 older than 2 years old. LCH usually affected skeleton system(77.4%), skin(42.0%), liver(29.0%), spleen(19.4%), hematopoietic system(12.9%). The most common misdiagnoses were upper respiratory tract infection, malignancies, focal infection, and eczema.</p><p><b>RESULTS</b>Response rate at week 6 was 76.9% in modified DAL-HX83/90 group and 94.1% in JLSG-96 group respectively, and no significant differences had been found between 2 groups. The 1-and 3-year overall survival rates of the patients treated with JLSG-96 protocol were 100% and 83.3%±15.2% respectively, while The 1-, 3-,5-year overall survival rates of those patients treated with the modified DAL-HX83/90 protocol were 70%±14.5%. The 1-and 3-year event-free rates of children treated with JLSG-96 protocol were 73.3%±11.4% and 66.7%±12.2%, respectively, while the 1-, 3-, 5-year event-free rates of those treated with modified DAL-HX83/90 protocol were 50%±15.8%, 40%±15.6% and 26.7%±15% respectively. No differences were found between the 2 groups for OS or EFS.</p><p><b>CONCLUSION</b>JLSG-96 protocol shows a better prognosis, and the risk of secondary malignancy caused by etoposide can be avoided. Early diagnosis of refractory LCH, specified grouping strategy and prolonged maintenance therapy may contribute to enhancing the EFS rates and reducing relapses.</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 between two different dose of r-ATG combined with CsA for treating children with severe aplastic anemia / 中国实验血液学杂志

This study was purposed to compare the efficacy and safety of two different doses of rabbit anti-thymocyte globulin (r-ATG) combined with cyclosporine (CsA) for treating children with severe aplastic anemia (SAA). From January 2005 to July 2010, a total of 95 children with SAA accepted intensive immunosuppressive therapy (IIST) in our department, out of them 55 cases were treated with r-ATG 2.5 mg/(kg·d) for 5 days in combination with CsA (group I) and other 40 cases were treated with r-ATG 3.5 mg/(kg·d) for 5 days in combination with CsA (group II). The responsive rate, adverse reactions, early mortality, relapse and clonal disease were analyzed retrospectively and results between the two groups were compared. Out of 95 patients 43 were boys and 52 were girls, their ages were from 1 to 16 years. The sex, age, severity and course of the disease were comparable between the two groups. The results showed that after treating for 3 and 6 months, the response of patients in group II was higher than that of patients in group I (50% vs 32.1%, P = 0.08 and 65% vs 45.3%, P = 0.059), at 9 and 12 months the response rate of patients in group II and group I did not show significant difference (70.0% vs 71.1%,P = 0.904 and 82.5% vs 80.8%,P = 0.832); at 12 months of treatment, the complete response rate of patients in group II was significantly higher than that of patients in group I (40.0% vs 23.1%,P = 0.08); at 3, 6, 9 months of treatment, the complete response rate of 2 groups showed no obvious difference. The incidence of serum disease, early infection and early mortality did not show statistical difference between two groups. There was no statistical difference in 2 year overall survival rate of two groups. In group I 39 patients were followed-up for more than 2 years, among them 3 patients relapsed, 1 patient died and 1 patient was diagnosed as acute monocytic leukemia (M5). In group II 15 patients were followed up for more than 2 years, there were no relapse, death and clonal disease. It is concluded that the r-ATG combined with CsA is an effective and safe therapeutic regimen for the SAA children. The effect of r-ATG 3.5 mg/(kg·d) is better than the 2.5 mg/(kg·d). The early safety is comparable between the two groups. However, the long-term effect, complications and survival rate need longer follow-up study to evaluate.

Hyperglycemia during chemotherapy influences the prognosis of children with acute lymphocytic leukemia / 中国实验血液学杂志

This study was aimed to explore whether hyperglycemia during chemotherapy influences the prognosis of children with acute lymphocytic leukemia (ALL). The clinical medical records of all newly diagnosed patients with ALL at SUN Yat-Sen Memorial Hospital from June 2008 to May 2012 were analyzed retrospectively. The median time of follow-up for patients was 2.6 years (range 0.08 to 4.9 years). Patients were divided to hyperglycemia and euglycemia groups according to their blood glucose concentrations during chemotherapy which contains L-asp and dexamethasone. The variables between two groups were compared using χ(2) test, the RFS and OS among two groups were compared by use of Kaplan-Meier and Cox-proportional hazard analyses. The results showed that the hyperglycemia correlated with older age (43.33% vs 19.23%, P = 0.008) and high-risk disease at diagnosis (26.62% vs 4.76%, P = 0.017) , but did not associate with sex (P = 0.059). Patients with hyperglycemia had worse OS (94.2 ± 2.9% vs 83.1 ± 6.3%, P = 0.014) and more poor RFS (64.1 ± 8.9% vs 88.6 ± 3.8%, P < 0.001) at 5 years than their counterpart. It is concluded that the incidence rate of hyperglycemia during chemotherapy correlated with older age and high-risk disease in ALL children, and the patients with hyperglycemia during chemotherapy may have poorer prognosis.

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.

Treatment of children with multiple system Langerhans cell histiocytosis by Japan Langerhans Cell Histiocytosis Study Group Protocol / 中国实验血液学杂志

The purpose of this study was to evaluate the efficiency of Japan Langerhans Cell Histiocytosis Study Group (JLSG) Protocol in treatment of children suffering from multiple system langerhans cell histiocytosis (MS-LCH). The clinical features, therapeutic response and prognosis of 11 children who were diagnosed and treated by JLSG in our department during October 2004 through October 2011 were analyzed. Among all 11 cases, 8 males and 3 females, the age at diagnosis was from 3 month to 6.5 years old with a median age of 3 years old. There were 10 cases of LCH with multi-system involvement (MS-LCH) and 1 case of single-system involvement (SS-LCH). Among those MS-LCH patients, 5 patients had risk organ involvement, and the other 5 patients did not develop risk organ involvement. All patients had been treated with JLSG protocol. The results showed that 4 cases achieved good response after 6-week induction treatment and the time of drug discontinuation were 5 - 20 months without relapse; 3 cases achieved partial response after 6-week induction treatment, among them 1 case did not relapse after discontinuation of drugs for 19 months, 1 case was still receiving maintenance treatment, 1 case abandoned induction treatment; 4 patients got no response (NR) or progressive disease after 6-week of induction treatment and were switched to salvage therapy, among them, 2 patients had stopped treatment for 2 - 20 months without relapse, 1 patient was still receiving maintenance treatment, one had changed to another therapy. It is concluded that the most of childhood LCH can be effectively controlled by immunochemical therapy based on the JLSG protocol. For children with LCH who has a poor response after 6-week induction treatment, LCH can still be well controlled if switched to salvage treatment.

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 PTPN11 mutation in children leukemia and its clinical significance / 中国实验血液学杂志

This study was aimed to explore the frequency of PTPN11 mutation in children with leukemia and its clinical significance. Genomic DNAs were extracted from peripheral leukocytes of 131 patients with leukemia, including 101 cases of ALL, 26 cases of AML, 3 cases of CML and 1 case of juvenil myelomonocytic leukemia (JMML). The sequences of PTPN11 exons 3, 8, 13 were amplified by polymerase chain reaction (PCR), and the clinical characteristics of positive patients were analyzed. The results indicated that the PTPN11 mutation was found in 10 cases (9.9%) from newly diagnosed 101 cases of ALL. Grouping the newly diagnosed ALL children by various clinical features, it was found that the PTPN11 mutation did not show associations with sex, age, white blood cell (WBC) count, prednisone test sensitivity, clinical risk and disease recurrences at the first visit (P > 0.05). PTPN11 mutations were found in 2 cases out of 26 AML patients, including one AML-M(2) and one AML-M(4). No PTPN11 mutation in 3 CML patients was found. Exon 13 mutation of PTPN11 gene was found in 1 case of JMML. It is concluded that the E76 of exon 3 is the hot spot of PTPN11 mutation in children leukemia. The novel G503E (1508G > A) mutation is detected in one JMML patient. The PTPN11 mutation does not associate with the sex, age, WBC count, prednisone sensitive test and early recurrence.

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.

Experimental study on the mechanism of the apoptosis of leukemic cells induced by valproic acid / 中华儿科杂志

<p><b>OBJECTIVE</b>To overcome the drug-resistance of tumor cells is one of the methods of improving the therapeutic results. Histone deacetylase inhibitors (HDACIs) is a novel class of chemotherapeutic agents which can induce apoptosis of tumor cells. Valproic acid (VPA) is a common drug used in the treatment of epilepsy. It has been shown that VPA has a marked HDACIs effect at the pharmaceutical level, and can induce the differentiation and apoptosis of transformed cells. But the mechanism of its effect has not been clarified. The aim of this study was to investigate the mechanism of VPA in inducing the apoptosis of leukemic cells at molecular level.</p><p><b>METHODS</b>The cell lines U937, Jurkat clone E6 - 1 (Jurkat) and BALL-1 were cultured in RPMI 1640 medium containing 20% calf serum, then divided into three groups (control group, 1 mmol/L VPA group and 1 mmol/L VPA + 1 micromol/L Pan-caspase inhibitor zVAD-fmk group). At 72 hours after the treatment, the cells were double stained with Aunexin and PI (propidium iodide) and then were analyzed with the flow cytometry (FCM) to detect apoptosis. Before and after treatment with VPA the mean fluorescence index (MFI) of Bcl-2, Bax, Bcl-xl and the levels of caspase 8, 9 and 3 were also detected with the FCM. The changes of P(44/42) mitogen activating protein kinase (MAPK) and phosphorylated P(44/42) MAPK were determined by Western blotting.</p><p><b>RESULTS</b>Seventy-two hours after the treatment, 1 mmol/L VPA induced apoptosis of U937 and Jurkat. The apoptotic rate of U937 was (75.78 +/- 4.20)% and that of Jurkat was (53.50 +/- 5.87)% (P < 0.01, vs. control group); zVAD-fmk could fully inhibit the apoptosis of U937, and the apoptotic rate was (2.89 +/- 0.36)%; while it could partly inhibit the apoptosis of Jurkat, and the apoptotic rate was (15.38 +/- 1.40)% (P < 0.01). 1 mmol/L VPA could not induce the apoptosis of BALL-1 which had a high expression level of Bcl-2. The MFI of Bcl-2, Bax and Bcl-xl in these three cell lines did not change significantly with VPA (P > 0.05). After treatment with VPA, the level of caspase 3 in U937 increased from (14.09 +/- 1.19)% to (32.30 +/- 2.47)%, and caspase 8 from (4.58 +/- 1.41)% to (86.47 +/- 3.26)% (P < 0.01), but there was no significant change in caspase 9 [(13.25 +/- 3.11)% and (10.95 +/- 1.30)%]. In Jurkat, the level of caspase 3 increased from (12.01 +/- 1.63)% to (35.56 +/- 0.27)%, and caspase 9 from (13.89 +/- 1.71)% to (75.89 +/- 4.08)% (P < 0.01 for both); no significant change was observed for caspase 8 [(5.94 +/- 1.38)% and (5.44 +/- 0.72)%]. In BALL-1, there was a slight decline in caspase 3 (P < 0.05). With the effect of VPA, levels of P(44/42) MAPK and phosphorylated P(44/42) MAPK decreased in all three cell lines (P < 0.01).</p><p><b>CONCLUSION</b>VPA could induce apoptosis of U937 through the activation of caspase 3 and 8; and it induced the apoptosis of Jurkat involving the activation of caspase 3 and 9. P(44/42) MAPK pathway also plays an important role in this course. VPA induced apoptosis of these cell lines without the alteration of Bcl-2, Bax and Bcl-xl. High level of Bcl-2 could antagonize the effect of VPA.</p>

Effects of meisoindigo on the expression of globin gene in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>The pathophysiology of beta-thalassemia is the imbalance of the alpha and non-alpha globin chain which leads to a series of clinical symptoms of hemolytic anemia. Scientists continuously try to explore gene-activated drugs to increase the level of non-alpha globin chain or decrease the level of alpha globin chain in the treatment of beta-thalassemia. To probe into the effects on globin-gene expression of meisoindigo (Me) in cultured erythroid cells derived from peripheral blood, so as to provide the theoretical basis for applying Me in the treatment of beta-thalassemia.</p><p><b>METHODS</b>By using the two-step liquid culture of erythroid progenitor cells and reverse transcription polymerase chain reaction (RT-PCR), and by using alpha mRNA as an inner control, the level of gamma mRNA and beta mRNA in cultured erythroid cells derived from peripheral blood of 11 patients with severe beta-thalassemia and 6 normal volunteers were measured under the effect of different concentration (2.5 micro mol/L, 5 micro mol/L and 10 micro mol/L) of Me.</p><p><b>RESULTS</b>(1) No statistic significance was found in the ratio of beta/alpha mRNA by Me in cultured cells from both normal individuals and beta-thalassemia. (2) Me can significantly increase the ratio of gamma/alpha mRNA and (beta + gamma)/alpha mRNA (that is non-alpha/alpha mRNA) in cultured cells from normal individuals and beta-thalassemia. The ratio of gamma/alpha mRNA was increased 0.31 - 0.45 times and the ratio of non-alpha mRNA/alpha mRNA increased 0.21 - 0.32 times in Me induced cells from normal individuals. No significant result was observed among the different concentrations of Me (2.5 micro mol/L, 5 micro mol/L and 10 micro mol/L) in normal individuals. With the increasing of Me concentrations, the ratios of gamma/alpha mRNA and alpha/alpha mRNA were increased in cultured cells from beta-thalassemia. The ratio of gamma/alpha mRNA was increased 0.33 - 1.17 times and the ratio of non-alpha/alpha mRNA increased 0.25 - 0.89 times in Me induced cells from beta-thalassemia. There was no significant difference between the concentrations of 2.5 micro mol/L and 5 micro mol/L concentration in beta-thalassemia. However, there was significant difference between the concentrations of 10 micro mol/L and the concentrations of 2.5 micro mol/L and 5 micro mol/L in beta-thalassemia. (3) The increase of the ratio of gamma/alpha mRNA and non-alpha/alpha mRNA in beta-thalassemia was higher than that in normal individual with induction by Me with a higher concentration (10 micro mol/L).</p><p><b>CONCLUSION</b>Me can raise the ratio of gamma/alpha mRNA and non-alpha/alpha mRNA in cultured erythroid cells derived from peripheral blood of both normal individual and beta-thalassemia in the level of transcription, which can improve the imbalance of the alpha and non-alpha globin chain. So Me has a latent value in the therapy of beta-thalassemia.</p>