[Clinical Characteristics and Prognosis Analysis of Pediatric Severe Aplastic Anemia].

OBJECTIVE: To analyze the clinical characteristics and factors affecting prognosis in children with severe aplastic anemia (SAA). METHODS: Two hundred and five children with SAA treated in our department from January 2008 to April 2018 were selected, and the clinical characteristics and factors affecting prognosis were retrospectively analyzed. RESULTS: Among 205 SAA children, the effective rate (CR+PR) at 3, 6 and 12 months after immunosuppressive therapy (IST) treatment was 50.9%, 59.0% and 73.9%, respectively, and 5-year overall survival rate was 93.1%±2.0%. Univariate analysis showed that 5-year overall survival rate of SAA children of spontaneous delivery was higher than that of cesarean section (P=0.039), while multivariate analysis showed that birth way had no significant influence on 5-year overall survival rate (P>0.05). The response rate at 3 months after IST of children with a recent history of decoration before SAA onset was higher than those without history of decoration (P<0.05). CONCLUSION: Most of the SAA children can achieve high response rate and overall survival rate. Patients with recent history of home/school decoration may be the factor affecting hematological response after 3 months of IST, but have no influence on long-term overall survival.

[Analysis of the Different Therapeutic Effects in 21 Ph+ Acute Lymphoblastic Leukemia Children].

OBJECTIVE: To analyze the outcomes of the children suffered from philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) treated with tyrosine kinase inhibitor (TKI) plus chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: 21 cases of firstly diagnosed Ph+ALL patients aged <12 year treated with Chinese Childhood Leukemia Group ALL 2008 (CCLG-ALL 2008) protocol form January 2008 and April 2015 were retrospectively analyzed．The patients were divided into two groups, one group was TKI+ chemotherapy group, the other group was allo-HSCT group. RESULTS: Among 21 patients, 17 were male and 4 were female with a median age of 8 years old (range, 4-12 years), the median follow-up time was 30 moths (range, 10-133 months). All the patients were treated with chemotherapy induced by the high-risk project of CCLG-ALL 2008. Among 14 patients treated with TKI plus chemotherapy, nine patients achieved complete remission. During 3 months after treatment, patients without complete molecular response or with the second complete remission and intensity desire of transplantation were treated with allo-HSCT, among 9 patients with allo-HSCT, six patients achieved long term survival. CONCLUSION: At TKI era, TKI combined with strong chemotherapy can make Ph+ ALL children achieve 5 years event-free survival as campared those treated with allo-HSCT. However, for the patients without complete molecular response persistently and relapsed they can still benefit from allo-HSCT.

[The Correlation of Minimal Residual Disease with Prognosis in TCF3-PBX1+ Acute Lymphoblastic Leukemia in Children].

OBJECTIVE: To investigate the consistency between FCM and PCR on the detecting of MRD in TCF3-PBX1+ ALL, and to investigate the prognosis value of these 2 methods. METHODS: 55 cases of paediatric TCF3-PBX1+ ALL patients from April 2008 to April 2015 were enrolled and analyzed. The FCM and PCR was used to detect the MRD in 239 bone marrow samples of 55 patients. All statistical analyses were carried out by using SPSS software version 16. RESULTS: Among the 55 children with TCF3-PBX1+ ALL, there were 30 male and 25 female. The median age was 5 (1-14) years. 20 patients relapsed during follow-up. The MRD results from PCR and FCM showed a strong correlation between both methods (K=0.774, P<0.001). There was no significant difference in 5-years DFS and OS between the patients in PCR+ and PCR- groups on day 15 or day 33. The 5 year DFS rate between the patients in FCM- and FCM+ was 63.9%±7.0% and 0; the 5 year OS rate was 66.5%±7.9% and 0. Combined with the result of FCM and PCR, at the d 33 of treatment, the 5-year DFS rate in FCM-/PCR- and single positive group was 65.4%±7.2% and 25.0%±15.3% (P<0.01). CONCLUSION: The detection result of MRD in TCF3-PBX1 detect by FCM and PCR shows better consistency. MRD positivity detected by FCM at the end of induction therapy (day 33) predicts a high risk of relapse in TCF3-PBX1 ALL patients.

Inhibition of red blood cell development by arsenic-induced disruption of GATA-1.

Anemia is a hematological disorder that adversely affects the health of millions of people worldwide. Although many variables influence the development and exacerbation of anemia, one major contributing factor is the impairment of erythropoiesis. Normal erythropoiesis is highly regulated by the zinc finger transcription factor GATA-1. Disruption of the zinc finger motifs in GATA-1, such as produced by germline mutations, compromises the function of this critical transcription factor and causes dyserythropoietic anemia. Herein, we utilize a combination of in vitro and in vivo studies to provide evidence that arsenic, a widespread environmental toxicant, inhibits erythropoiesis likely through replacing zinc within the zinc fingers of the critical transcription factor GATA-1. We found that arsenic interacts with the N- and C-terminal zinc finger motifs of GATA-1, causing zinc loss and inhibition of DNA and protein binding activities, leading to dyserythropoiesis and an imbalance of hematopoietic differentiation. For the first time, we show that exposures to a prevalent environmental contaminant compromises the function of a key regulatory factor in erythropoiesis, producing effects functionally similar to inherited GATA-1 mutations. These findings highlight a novel molecular mechanism by which arsenic exposure may cause anemia and provide critical insights into potential prevention and intervention for arsenic-related anemias.

[CD20 is not a poor prognostic factor for childhood B-lineage acute lymphoblastic leukemia with high white blood cell count].

OBJECTIVE: To study the significance of CD20 combined with white blood cell (WBC) count at diagnosis in the prognosis assessment in children with B-lineage acute lymphoblastic leukemia (ALL). METHODS: A retrospective analysis was performed on the medical data of 821 B-ALL children who were treated with CCLG-ALL2008 regimen from April 2008 to April 2015. Their survival status was followed up. RESULTS: Among the 821 children, 547 (66.6%) were negative, while 274 (33.4%) were positive for CD20 expression. Among 694 children with WBC<50×109/L (lower WBC count), the 5-year EFS rates were 65.9%±3.2% and 77.3%±2.0% for CD20 positive and negative patients respectively (P=0.001); the 5-year OS rates were 78.3%±2.9% and 87.5%±1.6% for CD20 positive and negative patients respectively (P=0.005); CD20 positive expression was an independent risk factor for EFS (HR=1.634, P=0.001) and OS (HR=1.761, P=0.005). Among 127 children with WBC>50×109/L (higher WBC count), the 5-year EFS rates was 64.3%±7.7% and 53.7%±5.5% for CD20 positive and negative patients respectively (P=0.135); the 5-year OS rate was 81.4%±6.4% and 58.6%±5.6% for CD20 positive and negative patients respectively (P=0.022); CD20 positive expression was an independent protective factor for OS (HR=0.367, P=0.016). CONCLUSIONS: In children with B-ALL who are treated with CCLG-ALL2008 regimen, those with CD20 positive expression in lower WBC count at diagnosis have a poor prognosis; however, those with CD20 positive expression in higher WBC count at diagnosis have a better long-time survival.

Propofol induces apoptosis by activating caspases and the MAPK pathways, and inhibiting the Akt pathway in TM3 mouse Leydig stem/progenitor cells.

Propofol is an anesthetic agent moderating GABA receptors in the nervous system. A number of studies have demonstrated that propofol exerts a negative effect on neural stem cell development in the neonatal mouse hippocampus. However, to the best of our knowledge, there is no study available to date illustrating whether neonatal exposure to propofol affects Leydig stem/progenitor cell development for normal male reproductive development and functions, and the regulatory mechanism remains elusive. In the present study, TM3 cells, a mouse Leydig stem/progenitor cell line, was treated with propofol. The data illustrated that propofol significantly reduced TM3 cell viability. TM3 subG1 phase cell numbers were significantly increased by propofol assayed by flow cytometric analysis. Annexin V/PI double staining assay of the TM3 Leydig cells also demonstrated that propofol increased TM3 cell apoptosis. In addition, cleaved caspase­8, ­9 and ­3 and/or poly(ADP­ribose) polymerase (PARP) were significantly activated by propofol in the TM3 cells. Furthermore, the expression levels of phospho­JNK, phospho­ERK1/2 and phospho­p38 were activated by propofol in the TM3 cells, indicating that propofol induced apoptosis through the mitogen­activated protein kinase (MAPK) pathway. Additionally, propofol diminished the phosphorylation of Akt to increase the apoptosis of TM3 cells. On the whole, the findings of the present study demonstrate that propofol induces TM3 cell apoptosis by activating caspases and MAPK pathways, as well as by inhibiting the Akt pathway in TM3 cells. These findings illustrate that propofol affects the viability of Leydig stem/progenitor cells possibly related to the development of the male reproductive system.

[Association of cerebrospinal fluid status with prognosis in children with acute lymphoblastic leukemia].

OBJECTIVE: To study the clinical features of central nervous system infiltration-positive (CNSI+) children with acute lymphoblastic leukemia (ALL) based on flow cytometry, as well as the association of such clinical features with prognosis. METHODS: A retrospective analysis was performed for the clinical data of 66 CNSI+ children with ALL treated from April 2008 to June 2013. Clinical features, laboratory examination results and prognosis were compared between the children in different chemotherapy stages (induction stage and consolidation/maintenance stage). RESULTS: Among the 66 CNSI+ children, 50 were in the induction stage and 16 in the consolidation/maintenance stage. Compared with the CNSI+ children in the induction stage, the CNSI+ children in the consolidation/maintenance stage had a significantly higher proportion of children with the genes associated with good prognosis based on the results of molecular biology (P<0.05), as well as a significantly higher recurrence rate (P<0.05). Recurrence was observed in 21 CNSI+ ALL children, among whom 10 were in the induction stage and 11 were in the consolidation/maintenance stage. Compared with the children experiencing recurrence in the induction stage, the children experiencing recurrence in the consolidation/maintenance stage had a significantly higher proportion of children with recurrence of the central nervous system and bone marrow (P<0.05), as well as significantly higher proportion of biochemical positive rate of cerebrospinal fluid (P<0.05). The children in the induction stage had a significantly higher recurrence-free survival rate than those in the consolidation/maintenance stage (P<0.001), while there was no significant difference in overall survival rate between the two groups (P>0.05). CONCLUSIONS: In children with ALL, CNSI+ has a marked effect on recurrence-free survival rate in different chemotherapy stages, but has no obvious effect on overall survival rate. CNSI+ patients in the consolidation/maintenance stage have a higher recurrence.

[Therapeutic Outcomes of 28 ALL Patients with MLL Gene Rearrangement].

OBJECTIVE: To analyze the therapeutic outcomes of acute lymphoblastic leukemia(ALL) patients with MLL gene rearrangement. METHODS: Clinical outcomes of 28 ALL patients aged less than 12 years old with MLL gene rearrangement treated with Chinese Childhood Leukemia Group ALL 2008 (CCLG -ALL 2008) protocol from January 2008 to April 2015 were retrospectively analyzed. RESULTS: Among 28 patients, 14 were boys and 14 were girls, median age was 36(4-144) months and median follow-up period was 12.5 (ranged 2-106) months, and 10 were infants. All patients were given induction chemotherapy and the high-risk project of CCLG-ALL 2008. At the last time of follow-up, five patients gave up and were loss to follow-up. The 5 years OS and EFS were 52.2% and 47.8% respectively for the other 23 patients. The 5 years OS and EFS(28.6% and, 88.9%), (P=0.012), (28.6%, 77.8%), (P=0.048) rate of WBC≥50×109/L and ＜50×109/L groups at first vistit showed a significantly different. Patients with good response to predisone, less than 12 months， sex， CD10- showed low trend of 5 years OS and EFS, however there was no statistical difference (P＞0.05). CONCLUSION: The 5 years OS is 52.2% and 5 years EFS is 47.8% for those ALL patients with MLL gene rearrangement. The WBC≥50×109/L at first visit is adverse prognostic factor.

[The Outcome of Severe Aplastic Anemia Children Treated with Reduced Dose of Cyclophosphamide Combined Cyclosporine A].

OBJECTIVE: To analyze the clinical efficacy and side effects of reduced-dose of cyclophosphamide combined cyclosporine A for severe aplastic anemia(SAA) children. METHODS: Ten pediatric patients with SAA from January 2008 to May 2012 were enrolled. All the patients were treated with reduced dose of cyclophosphamide combined cyclosporine A. The dose of cyclophosphamide was 30 mg/(kg·d)×4 d, the dose of cyclosporine A gradually increased >15 mg/L accroding to the blood concentration. RESULTS: The median follow-up time of the 10 pediatric patients was 100 months (6-126 months). Among 10 children with SAA, 4 cases achieved complete response(CR), 3 cases obtained partial response (PR) and the overall response rate was 70%, the remaining 3 cases showed no response (NR). One refractory patient treated by cyclophosphamide was progressed to paroxysmal nocturnal hemoglobinuria(PNH) at 25 months and was dead at 42 months after therapy. CONCLUSION: The results show that reduced-dose cyclophosphamide (30 mg/kg·d for 4 consecutive days) combinated with CsA (initial dose 4 mg/kg·d, and drugvallery concentration >150 ng/ml) can make 7 of 10 children with severe aplastic anemia achieve complete response or partial response, and this regimen may be the second line regimen selected for some SAA children.

[Clinical features and gene mutation spectrum in children with sideroblastic anemia].

OBJECTIVE: To study the clinical features and gene mutation spectrum of children with sideroblastic anemia (SA) and the clinical value of targeted next-generation sequencing in the molecular diagnosis of children with SA. METHODS: Clinical data were collected from 36 children with SA. Targeted next-generation sequencing was used to detect mutations in SA-related pathogenic genes and genes associated with heme synthesis and mitochondrial iron metabolism. The association between genotype and clinical phenotype was analyzed. RESULTS: Of the 36 patients, 32 had congenital sideroblastic anemia (CSA) and 4 had myelodysplastic syndrome with ring sideroblasts (MDS-RS). Mutations in CSA-related genes were detected in 19 children (19/36, 53%), among whom 9 (47%) had ALAS2 mutation, 4 (21%) had SLC25A38 mutation, and 6 (32%) had mitochondrial fragment deletion. No pathogenic gene mutation was detected in 4 children with MDS-RS. Among the 19 mutations, 89% (17/19) were known mutations and 11% (2/19) were novel mutations. The novel mutation of the ALAS2 gene c.1153A>T(p.I385F) was rated as "possibly pathogenic" and the novel mutation of the SLC25A38 gene c.175C>T(p.Q59X) was rated as "pathogenic". CONCLUSIONS: ALAS2 and SLC25A38 gene mutations are commonly seen in children with CSA, but mitochondrial gene fragment deletion also accounts for a relatively high proportion. For children with hypoplastic anemia occurring in infancy, mitochondrial disease should be considered.

[Long-term clinical effect of the CCLG-ALL2008 regimen in treatment of childhood acute lymphoblastic leukemia with different molecular biological features].

OBJECTIVE: To study the long-term clinical effect of the CCLG-ALL2008 regimen in the treatment of children newly diagnosed with acute lymphoblastic leukemia (ALL) with different molecular biological features. METHODS: A total of 940 children who were newly diagnosed with ALL were enrolled in this study. The children were treated with the CCLG-ALL2008 regimen. A retrospective analysis was performed for the long-term outcome of ALL children with different molecular biological features. RESULTS: Among the 940 children with ALL, there were 570 boys and 370 girls, with a median age of onset of 5 years (range 1-15 years) and a median follow-up time of 65 months (range 3-123 months). The complete response (CR) rate was 96.7%, the predicted 10-year overall survival (OS) rate was 76.5%±1.5%, and the event-free survival (EFS) rate was 62.6%±3.0%. After CR was achieved after treatment, the overall recurrence rate was 21.9%. The children with positive ETV6-RUNX1 had the lowest recurrence rate and were prone to late recurrence, and those with positive MLL rearrangement had the highest recurrence rate and were prone to early recurrence. The children with positive ETV6-RUNX1 had a significantly higher predicted 10-year OS rate than those with positive TCF3-PBX1, BCR-ABL, or MLL rearrangement and those without molecular biological features (P<0.05). The children with positive ETV6-RUNX1 had a significantly higher predicted 10-year EFS rate than those with positive BCR-ABL or MLL rearrangement (P<0.05). CONCLUSIONS: Molecular biological features may affect the long-term prognosis of children with ALL, and positive MLL rearrangement and BCR-ABL fusion gene are indicators of poor prognosis. Children with positive ETV6-RUNX1 fusion gene have the highest long-term survival rate.

[Clinical features and prognosis of children with acute lymphoblastic leukemia and different platelet levels].

OBJECTIVE: To study the association of platelet level at diagnosis with prognosis in children with acute lymphoblastic leukemia (ALL). METHODS: A total of 892 children with ALL who underwent chemotherapy with the CCLG-ALL 2008 regimen were enrolled. According to the platelet count at diagnosis, these children were divided into normal platelet count group (platelet count ≥100×109/L; n=263) and thrombocytopenia group (platelet count <100×109/L; n=629). The thrombocytopenia group was further divided into (50- <100)×109/L (n=243), (20- <50)×109/L (n=263), and <20×109/L (n=123) subgroups. The association of clinical features (sex, age, immunophenotype, and molecular biology) with event-free survival (EFS) and overall survival (OS) was analyzed. RESULTS: Compared with the thrombocytopenia group, the normal platelet count group had significantly lower positive rate of MLL gene rearrangement and recurrence rate (P<0.05), as well as a significantly higher 10-year EFS rate (P<0.05). There was no significant difference in 10-year OS between the two groups (P>0.05). The normal platelet count group still had a significantly higher 10-year EFS rate than the thrombocytopenia group after the children with MLL gene rearrangement were excluded (P<0.05), and there was still no significant difference in 10-year OS between the two groups (P>0.05). The <20×109/L subgroup had significantly lower 10-year EFS and OS rates than the normal platelet count group, the (50- <100)×109/L subgroup, and the (20- <50)×109/L subgroup (P<0.05). After the children with MLL gene rearrangement were excluded, the <20×109/L subgroup still had significantly lower 10-year EFS and OS rates than the normal platelet count group, the (50-<100)×109/L subgroup, and the (20- <50)×109/L subgroup (P<0.05). CONCLUSIONS: ALL children with MLL gene rearrangement often have the clinical manifestation of thrombocytopenia. Platelet level at diagnosis is associated with the prognosis of ALL children. The children with normal platelet count have a low recurrence rate and good prognosis, and those with a platelet count of <20×109/L have the worst prognosis.

Propofol may increase caspase and MAPK pathways, and suppress the Akt pathway to induce apoptosis in MA­10 mouse Leydig tumor cells.

In the western world, there is an increasing trend of occurrence in testicular cancer. Treatment of malignant testicular cancer is primarily combined surgery with various chemical drugs. Propofol has been frequently used as an anesthetic and sedative induction agent, which could modulate different γ­aminobutyric acid receptors in the central nervous system. Studies demonstrated that propofol activates endoplasmic reticulum stress to induce apoptosis in lung cancer. However, it remains elusive whether propofol regulates caspase and/or mitogen­activated protein kinase (MAPK) pathways to induce apoptosis in Leydig tumor cells. In the present study, MA­10 mouse Leydig tumor cells were treated with propofol, and possible signal pathways associated with apoptosis were investigated. Results demonstrated that increasing dosage of propofol (300­600 µM) for 24 h significantly decreased cell viability in MA­10 cells (P<0.05). In flow cytometry analysis, the amount of sub­G1 phase cell numbers in MA­10 cells was significantly increased by propofol (P<0.05). Additionally, Annexin V/propidium iodide double staining further confirmed that propofol could induce MA­10 cell apoptosis. Furthermore, cleaved caspase­8, ­9 and ­3, and/or poly(ADP­ribose) polymerase were significantly activated following treatment of propofol in MA­10 cells. In addition, c­Jun N­terminal kinase, extracellular signal­regulated kinase 1/2, and p38 were significantly activated by propofol in MA­10 cells (P<0.05), indicating that propofol may induce apoptosis through the MAPK pathway. Additionally, propofol diminished the phosphorylation of Akt to activate apoptosis in MA­10 cells. In conclusion, propofol may induce MA­10 cell apoptosis by activating caspase and MAPK pathways, and inhibiting the Akt pathway in MA­10 cells, demonstrating that propofol may be a potential anticancer agent against Leydig cell cancer.

[Mitoxantrone-cytarabine-etoposide induction therapy in children with acute myeloid leukemia: a single-center study of complications and clinical outcomes].

OBJECTIVE: To investigate the complications and clinical outcome of children with acute myeloid leukemia (AML) undergoing mitoxantrone-cytarabine-etoposide (MAE) induction therapy. METHODS: A total of 170 children with AML were given MAE induction therapy, and the complications and remission rate were analyzed after treatment. RESULTS: The male/female ratio was 1.33:1 and the mean age was 7.4 years (range 1-15 years). Leukocyte count at diagnosis was 29.52×109/L [range (0.77-351)×109/L]. Of all children, 2 had M0-AML, 24 had M2-AML, 2 had M4-AML, 48 had M5-AML, 3 had M6-AML, 7 had M7-AML, 69 had AML with t(8;21)(q22;q22), and 15 had AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22). The most common complication was infection (158/170, 92.9%). Among these 158 patients, 22 (13.9%) had agranulocytosis with pyrexia (with no definite focus of infection), and 136 (86.1%) had definite focus of infection (including bloodstream infection). Other complications included non-infectious diarrhea, bleeding, and drug-induced hepatitis. Treatment-related mortality was observed in 10 children, among whom 8 had severe infection, 1 had multiple organ failure, and 1 had respiratory failure. Remission rate was evaluated for 156 children and the results showed a complete remission rate of 85.3%, a partial remission rate of 4.5%, and a non-remission rate of 10.3%. CONCLUSIONS: Induction therapy with the MAE regimen helps to achieve a good remission rate in children with AML after one course of treatment. Infection is the main complication and a major cause of treatment-related mortality.

Midazolam activates caspase, MAPKs and endoplasmic reticulum stress pathways, and inhibits cell cycle and Akt pathway, to induce apoptosis in TM3 mouse Leydig progenitor cells.

BACKGROUND: Midazolam (MDZ) has powerful hypnosis, amnesia, anti-anxiety and anticonvulsant effects. Studies have shown that prenatally developmental toxicity of diazepam can be observed in many organs/tissues. However, it remains elusive in male reproductive system. MATERIALS AND METHODS: TM3 mouse Leydig progenitor cell line was used to determine whether MDZ has any unfavorable effects. RESULTS: Midazolam significantly decreased cell viability in dose- and time-dependent manners in TM3 cells. In flow cytometry analysis, midazolam significantly increased subG1 phase cell numbers, and annexin V/PI double staining assay further confirmed that MDZ induced apoptosis in TM3 cells. Moreover, MDZ significantly induced the expression of caspase-8 and -3 proteins and the phosphorylation of JNK, ERK1/2 and p38. Besides, MDZ didn't activate Akt pathway in TM3 cells. Furthermore, the expressions of p-EIF2α, ATF4, ATF3 and CHOP were induced by midazolam, suggesting that midazolam could induce apoptosis through endoplasmic reticulum (ER) stress in TM3 cells. Additionally, the expressions of cyclin A, cyclin B and CDK1 were inhibited by midazolam through the regulation of p53 in TM3 cells, indicating that midazolam could regulate cell cycle to induce apoptosis. CONCLUSION: Midazolam could activate caspase, MAPKs and ER stress pathways and impede Akt pathway and cell cycle to induce apoptosis in TM3 mouse Leydig progenitor cells.

Low level arsenite exposures suppress the development of bone marrow erythroid progenitors and result in anemia in adult male mice.

Epidemiological studies report an association between chronic arsenic (As) exposure and anemia in men, and women who are predisposed to anemia. The purpose of these studies was to determine whether a 60 d drinking water exposure of adult male C57BL/6J mice to 0, 100, and 500ppb arsenite (As+3) results in anemia due to alterations in erythroid progenitor cell development in the bone marrow. Exposure to 500ppb As+3 for 60 d resulted in a reduction of mean corpuscular hemoglobin (MCH) levels, but did not significantly alter red blood cell (RBC) counts, hemoglobin (Hgb) levels, mean corpuscular Hgb concentrations (MCHC), or mean corpuscular volumes (MCV). Attenuation of burst-forming unit-erythroid (BFU-E) colony formation was observed in bone marrow cells of mice exposed to 500ppb As+3. The differentiation of late-stage bone marrow erythroblasts as defined by CD71 and Ter119 surface marker expression was reduced with the 500ppb As+3 exposure. Mice exposed to 500ppb As+3 also had elevated serum levels of erythropoietin (EPO). Collectively, these results show that exposure to low levels of As+3 attenuate the development of early BFU-E cells and reduce the differentiation of late-stage erythroblasts. This suppression of bone marrow erythropoiesis may be a contributing factor to the mild hypochromic anemia observed in 500ppb As+3 exposed mice.

[Clinical characteristics of clonal evolution after immunosuppressive therapy in children with severe/very severe aplastic anemia].

OBJECTIVE: To evaluate the clinical characteristics and risk factors of clonal evolution after immunosuppressive therapy (IST) in children with severe/very severe aplastic anemia (SAA/VSAA). METHODS: The clinical data of 231 children with newly-diagnosed SAA/VSAA who received IST were retrospectively studied. The incidence and risk factors of clonal evolution after IST were analyzed. RESULTS: The 5-year overall survival rate of the 231 patients was 82.7%. Except for 18 cases of early deaths, 213 patients were evaluated for IST efficacy. Among the 231 patients, cytogenetic abnormalities for at least two chromosome metaphase were detectable in 14 (7.4%) patients, and PNH clones were detectable in either peripheral red blood cells or neutrophils for 95 patients. Among the 213 patients evaluated for IST efficacy, 15 patients experienced clonal evolution after IST. Five patients had PNH and trisomy 8 which were defined as favorable progressions, and ten patients experienced monosomy 7 and MDS/AML as unfavorable progressions. The 5-year accumulative incidence of favorable and unfavorable progression were (2.2±2.2)% and (4.8±3.3)%, respectively. Until the last follow-up, 100% (5/5) of patients with favorable progressions and 50% (5/10) of patients with unfavorable progressions survived. WBC>3.5×109/L, CD3+T cell percentage>80%, dosage of antithymocyte globulin >3.0 mg/(kg·d) and no response to IST were related to unfavorable progressions by univariate analysis. Cox multivariate analysis revealed that an increased CD3+T cell percentage (>80%) and no response to IST were independent risk factors for unfavorable progressions. CONCLUSIONS: The children with SAA/VSAA who have an increased CD3+T cell percentage at diagnosis or have no response to IST are in high risks of unfavorable progressions.

Midazolam regulated caspase pathway, endoplasmic reticulum stress, autophagy, and cell cycle to induce apoptosis in MA-10 mouse Leydig tumor cells.

PURPOSE: Midazolam is widely used as a sedative and anesthetic induction agent by modulating the different GABA receptors in the central nervous system. Studies have also shown that midazolam has an anticancer effect on various tumors. In a previous study, we found that midazolam could induce MA-10 mouse Leydig tumor cell apoptosis by activating caspase cascade. However, the detailed mechanism related to the upstream and downstream pathways of the caspase cascade, such as endoplasmic reticulum (ER) stress, autophagy, and p53 pathways plus cell cycle regulation in MA-10 mouse Leydig tumor cells, remains elusive. METHODS: Flow cytometry assay and Western blot analyses were exploited. RESULTS: Midazolam significantly decreased cell viability but increased sub-G1 phase cell numbers in MA-10 cells (P<0.05). Annexin V/propidium iodide double staining further confirmed that midazolam induced apoptosis. In addition, expressions of Fas and Fas ligand could be detected in MA-10 cells with midazolam treatments, and Bax translocation and cytochrome c release were also involved in midazolam-induced MA-10 cell apoptosis. Moreover, the staining and expression of LC3-II proteins could be observed with midazolam treatment, implying midazolam could induce autophagy to control MA-10 cell apoptosis. Furthermore, the expressions of p-EIF2α, ATF4, ATF3, and CHOP could be induced by midazolam, indicating that midazolam could stimulate apoptosis through ER stress in MA-10 cells. Additionally, the expressions of cyclin A, cyclin B, and CDK1 could be inhibited by midazolam, and the phosphorylation of p53, P27, and P21 could be adjusted by midazolam, suggesting that midazolam could manage cell cycle through the regulation of p53 pathway to induce apoptosis in MA-10 cells. CONCLUSION: Midazolam could induce cell apoptosis through the activation of ER stress and the regulation of cell cycle through p53 pathway with the involvement of autophagy in MA-10 mouse Leydig tumor cells.

[Significance of PAX5 deletion in childhood B-lineage acute lymphoblastic leukemia without reproducible chromosomal abnormalities].

OBJECTIVE: To identify the incidence of PAX5 deletion in childhood B-lineage acute lymphoblastic leukemia (B-ALL) without reproducible chromosomal abnormalities and to investigate the association between PAX5 abnormalities and prognosis of ALL. METHODS: Multiplex ligation-dependent probe amplification was used to determine the copy numbers of PAX5 gene in children newly diagnosed with B-ALL without reproducible chromosomal abnormalities between April 2008 and April 2013 and controls (children with non-hematologic diseases or tumors). The patients were classifiied into deletion group and non-deletion group based on the presence of PAX5 deletion. RESULTS: Eighteen (21%) out of 86 children with B-ALL had PAX5 deletion. The deletion group had a significantly higher total white blood cell count at diagnosis than the non-deletion group (P=0.001). The Kaplan-Meier analysis demonstrated that the deletion group had a significantly lower disease-free survival (DFS) rate than the non-deletion group (0.69±0.12 vs 0.90±0.04; P=0.017), but there was no significant difference in the overall survival rate between the two groups (P=0.128). The Cox analysis showed that PAX5 deletion was a risk factor for DFS (P=0.03). CONCLUSIONS: PAX5 deletion is an independent risk factor for DFS in B-ALL children without reproducible chromosomal abnormalities.

Evaluation of Toxicity in Mouse Bone Marrow Progenitor Cells.

Development of blood cells through hematopoiesis occurs in the bone marrow (BM), and can be adversely impacted by various substances and/or conditions ranging from known therapeutic, intentionally administered xenobiotics to unintentional food additives and exposure to environmental chemicals. The principles underlying the techniques for evaluating toxicity to BM progenitors (erythroid, myeloid, and lymphoid) exploit changes in the normal hematopoietic process, biochemical cell surface and intracellular markers, as well as components of the BM microenvironment. Toxicological investigations following in vivo exposures of mice or in vitro exposures of mouse primary BM cell cultures allow the assessment of the developmental and functional integrity of BM cells, cell population shifts, and adverse biochemical effects due to toxicity. Colony forming unit (CFU) assays and flow cytometry are indispensable techniques in these toxicity studies.