Enhanced Autophagy Suppresses Proplatelet Formation in Pediatric Immune Thrombocytopenia / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of enhanced autophagy in megakaryocyte to proplatelet formation in children with immune thrombocytopenia(ITP).@*METHODS@#Giemsa staining and immunofluorescence staining were used to observe megakaryocyte morphology and proplatelet formation, Western blot was used to determine the expression of cytoskeleton protein and autophagy related protein. Autophagr regulation drugs Rap or 3-MA was used to regulate autophagy of megakaryocytes.@*RESULTS@#Some vacuole-like structures was found in ITP megakaryocytes of the children, the expression of LC3II/I (ITP 1.32±0.18； Ctrl 0.49±0.16，P<0.05) and Atg5-Atg12 (ITP 0.69±0.17； Ctrl 0.12±0.08，P<0.05) was significantly higher in ITP children as compared with those in control group. The immu- nofluorescence staining showed that the cytoskeleton arrangement in megakaryocytes of ITP children was abnormal, and the phosphorylation of myosin light chain was also increased(ITP 0.74±0.09, Ctrl 0.05±0.02，P<0.05). In vitro, inducer or inhibitor of autophagy could regulate the production of proplatelet and the expression of cell cycle related protein, including CyclinD1（Veh 1.08±0.12; Rap 0.46±0.04; Rap+3-MA 0.70±0.03）, CyclinD2（Veh 0.47±0.04; Rap 0.27±0.04; Rap+3-MA 0.41±0.03）, P21（Veh 0.15±0.01; Rap 0.04±0.01; Rap+3-MA 0.05±0.01）.@*CONCLUSION@#Enhanced autophagy is the key factor of poor proplatelet formation in megakaryocytes of ITP children.

Clinical and laboratory features of pediatric acute myeloid leukemia with inversion of chromosome 16 / 中华儿科杂志

<p><b>OBJECTIVE</b>To evaluate the clinical and laboratory features of pediatric inv(16) acute myeloid leukemia (AML) retrospectively.</p><p><b>METHOD</b>Dual color fluorescence in situ hybridization (D-FISH) using a LSI CBFβ inv(16) break apart probe labeled by Spectrum red and Spectrum green was performed in 15 acute myeloid leukemia cases, including 13 cases with or without abnormal eosinophils but with positive core binding factor β (CBFβ)-MYH11 fusion transcript detected by RT-PCR, and 2 cases with trisomy 8 (+8). The results were compared with the morphology, immunophenotype, karyotype and RT-PCR.</p><p><b>RESULT</b>Morphologically, 12 cases were diagnosed as M(4)EO, 2 as M(4), and 1 as M(2a). Immunophenotypically, all 13 AML cases with inv(16) showed positive expression of CD(13) and CD(33), but without the expression of any lymphoid lineage antigens. Karyotyping analysis with G-banding detected inv(16) in 10 AML cases, including 9 M(4)EO cases and 1 M(2a), but only 5 positive cases were detected using R-banding technique. Among them, 2 cases had simultaneous +8 and trisomy22 (+22), one had +22 only in addition to inv(16). D-FISH revealed a CBFβ-MYH11 rearrangement in 13 cases of AML with positive RT-PCR results, and the mean positive rate of cell detection was 55.15% (range 37.0% - 86.0%). The complete remission rate (CR) and median survival period in this series of inv(16) AML were 81.5%and 11 months, respectively, of whom, 8 cases were still in CR. Relapse and karyotypic evolution were seen in case 5 with +8, +22 in addition to inv(16).</p><p><b>CONCLUSION</b>AML with inv(16) is a special subtype. Most cases belong to M(4)EO. Its prognosis is good in general, but it seems to be an unfavorable feature for AML with inv(16) and +8, +22 simultaneously, especially with karyotypic evolution. For detection of inv(16), G-banding technique is evidently superior to R-banding technique. D-FISH combined with RT-PCR are more sensitive and reliable than chromosome banding analysis.</p>

Clinical significance of dynamic monitoring the minimal residual disease in childhood B-lineage acute lymphoblastic leukemia by multiparameter flow cytometry / 中国实验血液学杂志

This study was aimed to explore the clinical significance of monitoring level of minimal residual disease (MRD) at different time point in B-lineage childhood acute lymphoblastic leukemia (B-ALL). Two hundred and six children with B-ALL were enrolled in this study from Augest 2008 to September 2011 in our hospital. MRD levels were detected by flow cytometry at day 15, 33 and week 12 after initial chemotherapy. The event-free survival (EFS) for patients based on MRD levels measured at different stages of chemotherapy were compared by Kaplan Meier analyses. The results showed that out of 206 cases 196 cases achieved complete remission (CR) after induction therapy (CR rate 95.1%), the 1- and 3-year EFS rate were (92.7 ± 1.8)% and (78.7 ± 3.7)%, respectively, and the 3-year EFS rate was (85.6 ± 4.9)% in standard risk group, (82.1 ± 5.8)% in intermediate risk group and (58.1 ± 9.2)% in high risk group, there was significant statistical difference between above mentioned 3 groups (P < 0.001). The MRD analysis at different time points showed that the higher the MRD level, the lower the 3-year EFS rate of children with ALL, in which the 3-year EFS rate of MRD ≥ 10(-2) at day 15, MRD ≥ 10(-3) at day 33 and MRD ≥ 10(-3) at week 12 were significantly lower. The MRD ≥ 10(-3) at week 12 was proven to be an independent predictor by multivariate Cox proportional-hazards regression model. The 3-year EFS rate for patients with MRD < 10(-3) and MRD ≥ 10(-3) at week 12 were (86.3 ± 4.1)% vs (55.8 ± 9.1)% (P < 0.05); 8 relapsed among 98 cases with negative MRD (MRD < 10(-4)) at day 33, 19 relapsed among 108 cases with positive MRD at day 33 between the two groups for recurrence rate has significant difference (P < 0.05). It is concluded that dynamically monitoring MRD by multi-parameter flow cytometry can precisely evaluate treatment response, judge treatment outcome and predict relapse in childhood B-ALL. The MRD 10(-2) at day 15, MRD 10(-3) at day 33 and MRD 10(-3) at week 12 should be considered as the best cut-off. MRD ≥ 10(-3) at week 12 was proven to be an independent factor of poor prognosis.

Clinical and experimental studies of childhood acute myeloid leukemia with 11q23/MLL rearrangements / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore clinical and experimental features of 28 cases of childhood acute myeloid leukemia (AML) with 11q23/MLL gene rearrangements.</p><p><b>METHODS</b>Karyotypes of 234 cases of de novo childhood AML were analyzed using short-term culture of bone marrow cells and R-banding. The fusion transcripts involving MLL gene and partial tandem duplication of MLL (MLL-PTD) were detected by multiple reverse transcription polymerase chain reaction (RT-PCR) assay. Two cases with 11q23 translocation by karyotypic analysis but with negative result of multiple RT-PCR were studied with MLL-dual-color fluorescence in situ hybridization (D-FISH).</p><p><b>RESULTS</b>R-banding karyotypic analysis has revealed 20 cases with 11q23 translocation (14 cases with M5, 4 cases with M4, 2 cases with M2), including 12 cases with t(9;11)(p22;q23), 3 cases with t(1;11)(q21;q23), 2 cases with t(6;11)(q27;q23), 1 case with t(11;19)(q23;p13), 1 with t(5;11)(q31;q23), and 1 with t(X;11)(q24;q23). Eighteen cases with 11q23 translocation having fusion transcripts involving MLL genes were confirmed with multiple RT-PCR; 2 cases showed negative results, but they were confirmed to have MLL rearrangements by D-FISH. MLL-PTD was also detected in 8 cases (4 cases M5, 2 cases M4, M2 and M6, one case each) from the other childhood AML cases. The total incidence of 11q23/MLL gene rearrangements was 11.97% (28/234), and most of patients(85.7%, 24/28) were M4/M5. The complete remission (CR) rate after treatment for the 28 cases with MLL rearrangements was 53.8%, the difference was significant by statistics (P< 0.05) compared with 90.5% for the control group (M4/M5 childhood AML with other karyotypic abnormalities or normal karyotype). Of them, 2 cases receiving intensive chemotherapy survived for 81 and 66 months, respectively, 4 cases receiving allogeneic stem cell transplantation survived for 21, 20, 16 and 11 months, respectively, and are still alive with CR. The medium survival (MS) time for 28 cases with 11q23/MLL rearrangements was 11 months, whereas the MS for control group was 15 months. The difference was not statistically significant(P> 0.05).</p><p><b>CONCLUSION</b>The 11q23/MLL rearrangements is highly correlated with the occurrence of monocytic leukemia (M4 and M5). The 11q23 translocation and MLL-PTD are mutually exclusive, though both are indicative of poor prognosis. Intensive chemotherapy and allogeneic stem cell transplantation may ameliorate the clinical outcome. Multiple RT-PCR combined with karyotypic analysis and D-FISH are useful for screening the 11q23/MLL rearrangements in childhood AML.</p>

Analysis of clinical features and prognostic significance of childhood T-lineage acute lymphoblastic leukemia / 中国实验血液学杂志

This study was aimed to explore the clinical features and prognosis outcome of childhood T-cell acute lymphoblastic leukemia (T-ALL). The clinical data of 38 cases of newly diagnosed T-ALL from Jan 2005 to Aug 2010 were analyzed retrospectively, and 78 cases of B-ALL with intermediate and high risk were collected as control group, then the sensitive rate of patients to prednisone pretreatment, complete remission (CR) rate at day 33 after induction chemotherapy, relapse rate and 3-year event-free survival (EFS) were compared between T-ALL and B-ALL children. The results showed that no significant statistic difference were found in distribution of age, infiltration of liver, spleen and lymph nodes as well as central nervous system disease, chromosome abnormality, expression level of fusion gene and so on between T-ALL and B-ALL groups (p > 0.05), but there were significant differences in sex and number of cases with WBC count ≥ 50 × 10(9)/L between them (p < 0.05). The sensitive rate of T-ALL and B-ALL patients to prednisone pretreatment was 51.9% and 89.3% respectively (p < 0.05). The ratio failed to achieve CR at day 33 after induction chemotherapy was 15.4% and 8.1% in the two groups (p > 0.05). The relapse rate of T-ALL and B-ALL cases was 30.8% (8/26) and 14.9% (11/74) respectively (p > 0.05). The time from CR to relapse was (9.78 ± 3.48) month and (21.28 ± 14.32) month (p < 0.05). The 3 year EFS of T-ALL cases with intermediate and high risk was (37.5 ± 17.1)% and (22.2 ± 9.8)%, while 3 year EFS of B-ALL cases was (66.7 ± 7)% and (51.7 ± 9.3)% respectively (p < 0.05) according to Kaplan-Meier survival curve. It is concluded that as compared with B-ALL cases, the male ratio and initial WBC count are higher, moreover the early response to prednisone pretreatment and 3 year EFS are poor in T-ALL cases, the prognosis outcome is poor also.

Preliminary study of gene expression profile associated with risk classification of childhood patients with acute lymphoblastic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore genes associated with risk classification of childhood acute lymphoblastic leukemia (ALL) by gene chip technology.</p><p><b>METHODS</b>Group A and B were both composed of three newly diagnosed ALL cases with standard risk. After re-evaluation, group B was relegated to high-risk. The control group was composed of three idiopathic thrombocytopenic purpura (ITP) patients. The gene expression profiles of group A and B were studied by Illumina Human-6 Beadchip. Eighty-two ALL patients were selected as the experimental group and 21 with normal bone marrow as control group for real-time quantitative RT-PCR (RQ-PCR).</p><p><b>RESULTS</b>(1) There were 19 genes expressed differently between group B and A, including 14 up-regulated as ABCC4 and BCL11A, 5 down-regulated genes as TOP2A. (2) ABCC4 and BCL11A were validated by RQ-PCR and their expression level was higher in the high risk group than in the standard risk group (P < 0.05). The gene expression level in the group A and B was higher than that in the normal control group (P < 0.01). TOP2A was also validated by RQ-PCR and its expression level in the high risk group was lower than that in the standard risk group (P < 0.05). The gene expression level in the groups A and B was lower than that in the normal control group and the difference was statistically significance (P < 0.01). (3) There was a significant difference in the expression level of ABCC4 between the remission and unremission patients (P < 0.05). There was no significant difference in the expression level of BCL11A between different clinical indicators (P > 0.05). There was significant difference in the expression level of TOP2A between remission and prednisone good responder groups (P < 0.05).</p><p><b>CONCLUSIONS</b>Fourteen genes studied were involved in the pathogenesis and drug resistance mechanism in childhood ALL patients. Investigation of gene expression profile will be helpful for predicting drug resistance, prognosis, early intervention and target therapy in childhood ALL.</p>

Prevalence and clinical features of human metapneumovirus infection in hospitalized pediatric patients with respiratory tract infection in Suzhou area / 中华儿科杂志

<p><b>OBJECTIVE</b>To determine whether human metapneumovirus (hMPV) was circulating in Suzhou area and the epidemiology and clinical features associated with hMPV infection.</p><p><b>METHOD</b>Samples were collected from January 2006 to December 2007; respiratory specimens were tested for the presence of hMPV by reverse-transcription polymerase Chain reaction (RT-PCR). PCR products of hMPV N gene from some patients were randomly selected for sequencing analysis, and the sequences of the nucleotides and deduced amino acids were compared with those in the GenBank.</p><p><b>RESULT</b>Of the 4702 patients screened, 8% had evidence of hMPV infection. The positive rate in 2006 and 2007 was 8.4% and 7.6%, respectively. The positive rates detected during January to March, November and December were higher. The median age of patients was 22. 56 months. The infected children were diagnosed as having upper respiratory tract infection (3.2%), laryngitis (2.1%), bronchiolitis (27.1%), pneumonia (55.9%), and asthma exacerbation (11.7%). Sequence analysis of these hMPV N genes showed 99%-100% homology with the registered sequence in GenBank.</p><p><b>CONCLUSION</b>(1) hMPV accounted for a significant proportion of respiratory tract infection in infants and children. (2) hMPV prevailed predominantly in the winter and spring time. (3) Clinically, hMPV infection can not be discriminated from the infection with other respiratory tract viruses.</p>

Epidemiological study on respiratory syncytial virus and its bronchopneumonia among children in Suzhou / 中华预防医学杂志

<p><b>OBJECTIVE</b>To probe the epidemiological trend of respiratory syncytial virus (RSV) and cellular immunological change of RSV bronchopneumonia among children in Suzhou in the past five years.</p><p><b>METHODS</b>10,205 children with acute respiratory tract infection from January 2001 to December 2005 were enrolled into the study. Nasopharyngeal aspirates were obtained from the respiratory tract by aseptic vacuum aspiration. Direct immuno-fluorescence assay was employed to detect seven kinds of virus antigens including RSV antigen. CD3, CD4, CD8, CD19, CD16 and CD56 in peripheral blood mononuclear cells of 30 patients with RSV bronchopneumonia (1.5-24.0 months old group) were analyzed by flow cytometry analysis, and 15 normal infants (1.5-24.0 months old group) were enrolled as control group.</p><p><b>RESULTS</b>The annual positive rate of RSV was 24.94%, 25.83%, 24.05%, 25.39% and 27.30% respectively from 2001 to 2005. It also found that the peak season for RSV infection was spring or winter (January to March or November to December). The positive rate of RSV was significantly higher in 1-12 months old group than that in > 12 months old group (chi2 = 97.320, P < 0.01), as well as the groups between 1-12 months old (chi2 = 7.804, P < 0.05, the highest positive rate was occurred at 3-6 months old group). The positive rate of RSV was significantly higher in boys than that in girls (chi2 = 9.693, P < 0.01). The percentages of CD3+, CD4+, CD8+ and NK (CD16 + 56)+ cells were significantly lower in RSV bronchopneumonia than those in control group (t = 3.199, P < 0.01; t = 2.215, P < 0.05; t = 2.619, P < 0.05 and t = 5.240, P < 0.01, respectively). While the percentage of CD19+ cells was significantly elevated in RSV bronchopneumonia than that in control group (t = 2.875, P < 0.01).</p><p><b>CONCLUSION</b>RSV infection is of obvious seasonal changes. The younger the patient, the higher positive rates of RSV infection is, while and the cellular immunity function is lower. The effective measures for preventing RSV infection are important, especially for the infants. Further investigation is necessary to understand the causes of the variations for RSV infections between boys and girls.</p>

Effect of bacillus calmette-guerin treatment on airway inflammation and T regulatory cells in mice with asthma / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Previous studies have shown that bacillus calmette-guerin (BCG) can deviate TH2 response toward TH1 response, resulting in a suppressive effect on the development of asthma/atopy. This study examined the effect of BCG treatment on regulatory T cells in asthmatic mice to investigate the possible mechanism.</p><p><b>METHODS</b>Kunming mice were sensitized and challenged with ovalbumin (OVA) to establish asthmatic models. Asthmatic mice were injected intradermally with BCG five days before and after sensitization. After 24 hrs of last challenge, bronchoaveolar lavage fluid (BALF) and peripheral blood were collected . The total cells and eosinophils were counted in the BALF. The percentage of CD4(+) CD25(+) in peripheral blood was detected with flow cytometry. Single spleen cell suspension was prepared and cultured in 1640 medium for 48 hrs and then the cytokine IL-10 level in the supernatant was determined using ELISA. The mice which were challenged with normal saline were used as the Normal control group.</p><p><b>RESULTS</b>The number of total cells and eosinophils in BALF in asthmatic mice [(27.27 +/- 5.36) x 10(7)/L and (6.59 +/- 1.32) x 10(7)/L respectively] were more than in the Normal control group [(1.52 +/- 0.36) x 10(7)/L and zero respectively] (P < 0.01). The number of total cells and eosinophils in BALF in asthmatic mice were reduced after BCG treatment [(13.71 +/- 3.17) x 10(7)/L and (1.43 +/- 0.37) x 10(7)/L respectively] (P < 0.01). The percentage of CD4(+) CD25(+) in peripheral blood of asthmatic mice [(11.59 +/- 1.33)%] was noticeably lower than that of the Control group [(13.66 +/- 1.68)%] (P < 0.01), but increased significantly in asthmatic mice after BCG treatment [(14.40 +/- 2.70)%] (P < 0.05). The IL-10 level in spleen cell supernatant in the BCG-treated group (7.79 +/- 1.34 pg/mL) also increased compared with that in the untreated asthmatic mice (5.54 +/- 0.66 pg/mL) (P < 0.01).</p><p><b>CONCLUSIONS</b>BCG can markedly inhibit the airway inflammation in asthmatic mice possibly by promoting the production of regulatory T cells.</p>

Therapeutic effectiveness of CCLG-97 protocol on standard-risk childhood acute lymphoblastic leukemia / 中华儿科杂志

<p><b>OBJECTIVE</b>With the improvement of the diagnosis and treatment, the complete remission (CR) rate and the survival rate of childhood acute lymphoblastic leukemia have been increased in the recent 10 years. The objective of this study was to analyze the outcomes of 119 standard-risk childhood acute lymphoblastic leukemia (SR-ALL) patients, and explore how to improve the survival rate in ALL.</p><p><b>METHODS</b>A total of 119 patients aged 14 months to 15 years were diagnosed as SR-ALL according to the Suggestion of Diagnosis And Treatment for Childhood Acute Leukemia-1993. Among them, seventy-nine were boys and 40 were girls. All of the patients were treated with the CCLG-97 protocol and were followed up for a period of 20 approximately 78 months.</p><p><b>RESULTS</b>The complete remission rate reached 97.4% in four-week induction. Twenty-one patients were out of follow-up, comprising 63%, 14%, 10%, 8% and 5% of all subjects in 1998, 1999, 2000, 2001 and 2002, respectively. The overall survival rates were 93.3%, 90.2%, 88.0%, 85.0%, 85.0% and 85.0% in 1 year, 2 years, 3 years, 4 years and 5 years, respectively. Relapses occurred in 13 patients (13.8%). Among 9 isolated hematologic relapses, 5 patients (56%) were given irregular therapy, 2 did not reach CR within 4 weeks and relapsed 2 years later, 2 accepted regular therapy, 1 was of hypodiploidy and 1 T-ALL. Isolated central nervous system (CNS) relapse occurred in 4 patients (4.3%). Fifteen patients (12.6%) died, 5 of whom (4.2%) died of complications.</p><p><b>CONCLUSION</b>Reinforcing administration and regular therapy are important to improve the long-term survival rate in childhood ALL. The clinical classification should be adjusted with the improvement of diagnostic methods. CCLG-97 protocol decreased the rate of the relapses in SR-ALL and didn't increase the rate of therapy-related death. High-dose methotrexate should be used in therapy and its dosage, usage and individualized therapeutic regimen should be further studied.</p>

Clinical and laboratory studies on childhood acute leukemia with 11q23 abnormalities / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the interrelations among morphology, immunology, cytogenetics and clinical outcome in childhood acute leukemia with 11q23 abnormalities.</p><p><b>METHODS</b>Eighteen patients with 11q23 abnormalities, from 320 childhood acute leukemia patients, were retrospectively analysed for cell morphology, flow cytometry, immunophenotyping, R-banding karyotype as well as clinical features and prognosis. Twenty cases of childhood AL with normal karyotype during the same period were used as control.</p><p><b>RESULTS</b>The incidence of 11q23 abnormalities in our childhood acute leukemia patients was 5.63% including 14 acute lymphoblastic leukemia (ALL) and 4 acute myeloid leukemia (AML). Of 16 cases immunophenotypically tested, 13 expressed lymphoid antigens and 3 CD(34) and other myeloid antigens. Karyotype analysis disclosed the following abnormalities: t(4; 11)(q21; q23) in 6 cases, t(10; 11)(p13; q23) in 3, t(11; 19)(q23; p13) in one and del(11)(q23) in 6. The complete remission rate for these patients with 11q23 abnormalities was comparable to that of the control (72.2% vs 80.0%, P > 0.05), while the mortality rate in the former was significantly higher than that in the latter (61.1% vs 25.0%, P < 0.05).</p><p><b>CONCLUSIONS</b>11q23 abnormalities were mainly seen in childhood ALL and acute monocytic leukemia with unique prognostic features.</p>