Effect of Huai Qi Huang on asthma in rats and the mechanism research / 中国当代儿科杂志

OBJECTIVE@#To study the role and mechanism of action of Huai Qi Huang (HQH) in the rat model of asthma.@*METHODS@#Forty Sprague-Dawley rats were randomly divided into a control group, an asthma model group, a budesonide group, and an HQH group, with 10 rats in each group. A rat model of asthma was established by ovalbumin sensitization and challenge. The budesonide group was given budesonide aerosol 2 mg before each challenge. The HQH group was given HQH 4 g/kg dissolved in water by gavage before each challenge. Hematoxylin and eosin staining was used to observe the pathological changes of lung tissues. The percentage of eosinophils in bronchoalveolar lavage fluid (BALF) was measured. Enzyme-linked immunosorbent assay was used to determine the levels of interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), interferon gamma (INF-γ), and immunoglobulin E (IgE) in BALF. Flow cytometry was used to determine T-helper type 1 (Th1)/T-helper type 2 (Th2) ratio in peripheral blood and the spleen. RT-PCR and Western blot were used to measure the mRNA and protein expression of T-bet and GATA-3 in lung tissue.@*RESULTS@#Compared with the control group, the asthma model group showed significant increases in the degree of airway inflammation, the percentage of eosinophils in BALF, and the levels of IL-3, IL-4, IL-5 and IgE in BALF (P<0.05), however, the asthma model group showed significant reductions in the levels of IL-10 and INF-γ in BALF (P<0.05). The asthma model group had significantly lower percentage of Th1 cells but significantly higher percentage of Th2 cells in peripheral blood and the spleen compared with the control group (P<0.05). The mRNA and protein expression of T-bet in lung tissue was significantly lower, but the mRNA and protein expression of GATA-3 in lung tissue was significantly higher in the asthma group than those in the control group (P<0.05). Both HQH and budesonide significantly improved airway inflammation and the above markers in asthmatic rats (P<0.05), with comparable effects between them. However, there were still significant differences in these indices between the control group and the HQH or budesonide group (P<0.05).@*CONCLUSIONS@#HQH can reduce the airway inflammation of asthmatic rats and alleviate the symptoms of asthma, possibly by regulating the levels of related cytokines and Th1/Th2 ratio through the T-bet/GATA-3 pathway.

Pure red cell aplasia in children: a clinical analysis of 16 cases / 中国当代儿科杂志

OBJECTIVE@#To study the clinical features, treatment, and prognosis of pure red cell aplasia (PRCA) in children.@*METHODS@#A retrospective analysis was performed for the clinical data of 16 children with PRCA. The outcome and prognosis of patients treated with prednisone combined with Huaiqihuang granules versus prednisone alone were evaluated.@*RESULTS@#All the 16 children complained of symptoms of anemia including pale or sallow complexion. Of 12 children undergoing pathogen test, 7 (58%) were found to have pathogen infection, among which human cytomegalovirus was the most common. Lymphocyte subsets were measured for 7 children, among whom 5 (71%) had lymphocyte immune disorder. Six children were found to have abnormalities in immunoglobulin and complement. The 8 children treated with prednisone combined with Huaiqihuang granules had a median follow-up time of 21.5 months, among whom 1 was almost cured, 1 was relieved, and 6 were obviously improved; the median onset time of treatment was 1 month, and 2 children had disease recurrence in the course of drug reduction or withdrawal. The 8 children in the prednisone alone treatment group had a median follow-up time of 34 months, among whom 4 were almost cured, and 4 were obviously improved; the median onset time of treatment was 2.5 months, and 4 children had recurrence during drug reduction or withdrawal.@*CONCLUSIONS@#Children with PRCA usually complain of anemia-related symptoms. Laboratory tests show pathogen infection in some children with PRCA, and most of children have immune disorders. Glucocorticoids have a good therapeutic effect, but some children relapse in the course of drug reduction or withdrawal. Combined treatment with prednisone and Huaiqihuang granules may have a faster onset of action and less possibility of recurrence.

Behavioral intervention reduces unhealthy eating behaviors in preschool children via a behavior card approach / 华中科技大学学报（医学）（英德文版）

Many eating behaviors form in childhood, and some unhealthy behaviors may persist into adulthood and have potential impacts on people's health. This study evaluated the effectiveness of behavioral intervention in reducing consumption of Western fast food, sweetened beverages, fried food in preschool children, and changing parents' rewarding behaviors that encourage the consumption of the unhealthy foods. The research was a cluster randomized trial of seven kindergartens, involving 1138 children aged 3-6 years and their parents in Beijing, China. Parents and children allocated to the intervention group received two lectures and printed resources, including behavior cards, educational sheets. Children's behavior cards, applied with behavior-changing techniques, were used to intervene, and monitor behavior changes over time. Children in the control group just followed their usual health education curriculum in kindergartens. Intervention effects on food consumption behaviors were assessed by examining pre- and post-questionnaires. Of the 1138 children screened at baseline, 880 (77.3%) were measured at the end of the intervention period. The intervention lasted from March to June in 2010. The results showed that consumption of Western fast food, sweetened beverages, and fried food was decreased among the intervention group (P<0.001). Proportions of parents using Western fast food as rewards for their children were decreased (P=0.002). From March to June 2010, the frequency of each target behavior in children tended to decrease over the intervention period (P<0.001). Most parents favored regularly-delivered behavior cards or materials for behavioral intervention. In conclusion, the behavioral intervention encourages the healthier eating behaviors of children and reduces the parents' practice of using unhealthy foods as reward.

Effect of high-fat diet on cholesterol metabolism in rats and its association with Na⁺/K⁺-ATPase/Src/pERK signaling pathway / 华中科技大学学报（医学）（英德文版）

Abnormal cholesterol metabolism is associated with an elevated risk of developing atherosclerosis, hypertension, and diabetes etc. Na(+)/K(+)-ATPase was found to regulate cholesterol synthesis, distribution and trafficking. This study aimed to examine the effect of high-fat diet on cholesterol metabolism in rats and the role of Na(+)/K(+)-ATPase/Src/ERK signaling pathway in the process. Forty male SD rats were evenly divided into high-fat diet group and control group at random. Animals in the former group were fed on high-fat diet for 12 weeks, and those fed on basic diet served as control. Blood lipids, including total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), and low density lipoprotein-cholesteral (LDL-C) levels, were detected at 3, 6 and 12 weeks. The ratio of cholesterol content in cytoplasm to that in cell membrane was detected in liver tissues. RT-PCR and Western blotting were used to measure the expression of lipid metabolism-associated genes (HMG-CoA reductase and SREBP-2) after 12-week high-fat diet. Na(+)/K(+)-ATPase/Src/ERK signaling pathway-related components (Na(+)/K(+)-ATPase α1, Src-PY418 and pERK1/2) were also measured by Western blotting. The results showed that the serum TC, TG, and LDL-C levels were significantly higher in high-fat diet group than those in control group, while the HDL-C level was significantly lower in high-fat diet group at 6 weeks (P<0.01). High-fat diet led to an increase in the cholesterol content in the cytoplasm and cell membrane. The ratio of cholesterol content in cytoplasm to that in cell membrane was elevated over time. The expression of HMG-CoA reductase and SREBP-2 was significantly suppressed at mRNA and protein levels after 12-week high-fat diet (P<0.05). Moreover, high-fat diet promoted the expression of Na(+)/K(+)-ATPase α1 but suppressed the phosphorylation of Src-PY418 and ERK1/2 at 12 weeks (P<0.05). It was concluded that high-fat diet regulates cholesterol metabolism, and Na(+)/K(+)-ATPase signaling pathway is involved in the process possibly by regulating the expression of lipid metabolism-associated proteins HMG-CoA reductase and SREBP-2.

Effects of dendritic cell-activated and cytokine-induced killer cell therapy on 22 children with acute myeloid leukemia after chemotherapy / 华中科技大学学报（医学）（英德文版）

The efficiency of dendritic cell-activated and cytokine-induced killer cell (DC-CIK) therapy on children with acute myeloid leukemia (AML) after chemotherapy was investigated. Mononuclear cells were collected from children achieving complete remission after chemotherapy, cultured in vitro and transfused back into the same patient. Interleukin-2 (IL-2) was injected subcutaneously every other day 10 times at the dose of 1 × 10(6) units. Peripheral blood lymphocyte subsets and minimal residual disease (MRD) were detected by flow cytometry. Function of bone marrow was monitored by methods of morphology, immunology, cytogenetics and molecular biology. The side effects were also observed during the treatment. The average follow-up period for all the 22 patients was 71 months and relapse occurred in two AML patients (9.1%). The percentage of CD3(+)/CD8(+) cells in peripheral blood of 15 patients at the 3rd month after DC-CIK treatment (36.73% ± 12.51%) was dramatically higher than that before treatment (29.20% ± 8.34%, P < 0.05). The MRD rate was >0.1% in 5 patients before the treatment, and became lower than 0.1% 3 months after the treatment. During the transfusion of DC-CIK, side effects including fever, chills and hives appeared in 7 out of 22 (31.82%) cases but disappeared quickly after symptomatic treatments. There were no changes in electrocardiography and liver-renal functions after the treatment. MRD in children with AML can be eliminated by DC-CIK therapy which is safe and has fewer side effects.

Clinical and biological characteristics of childhood acute myeloid leukemia with EVI1 gene positive expression / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the expression of ecotropic viral integration site (EVI1) gene in childhood acute myeloid leukemia (AML) and the clinical features of EVI1-positive children with AML.</p><p><b>METHODS</b>The clinical data of EVI1-positive children with AML were collected and analyzed. RT-PCR and real-time quantitative PCR were used for qualitative and quantitative analysis of expression of EVI1. Flow cytometry (FCM) was used for determining the immunophenotypes of bone marrow cells. Multiparameter FCM was used for monitoring minimal residual disease. The karyotypes were determined.</p><p><b>RESULTS</b>Of 241 children with AML, 33 (13.7%) were positive for EVI1 expression. There were no significant differences in age at first visit as well as the white blood cell count, hemoglobin level, and platelet count in peripheral blood between EVI1-positive and EVI1-negative children with AML (P>0.05), but EVI1-positive children had a significantly increased proportion of females compared with EVI1-negative children (P<0.05). The change in EVI1 expression was not synchronous with clinical remission and the change of MRD: some children had clinical remission or negative conversion of MRD before negative conversion of EVI1, while some had negative conversion of EVI1 before clinical remission or while MRD showed positive. EVI1 gene was usually co-expressed with other fusion genes. CD33 (100%), CD38 (88%), and HLADR (76%) were highly expressed in EVI1-positive children with AML. Abnormal chromosome structure or number was found in 15 patients. Compared with EVI1-negative children, EVI1-positive children had significantly lower complete remission rates after the first course of treatment (P<0.05).</p><p><b>CONCLUSIONS</b>EVI1-positive children with AML have a poor short-term prognosis. In the development of AML, the activation of EVI1 gene is not isolated, but the result of interactions with other genes or chromosome abnormalities, and the mechanism of activation and its function need further study.</p>

Clinical characteristics of childhood leukemia with EVI1 gene and BCR/ABL gene co-expression / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the clinical characteristics of ecotopic viral integration site-1 (EVI1) and BCR/ABL positive childhood leukemia.</p><p><b>METHODS</b>Clinical data of four children with EVI1 and BCR/ABL positive leukemia and eight children with BCR/ABL positive but EVI1 negative chronic myeloid leukemia (CML) were retrospectively analyzed.</p><p><b>RESULTS</b>In the four children with EVI1 and BCR/ABL positive leukemia, two were initially diagnosed with chronic phase of CML, one with accelerated phase of CML and one with high-risk acute lymphoblastic leukemia (ALL). There were no significant differences in clinical characteristics at diagnosis between the patients with EVI1 and BCR/ABL positive leukemia and BCR/ABL positive but EVI1 negative leukemia. CD33 and CD38 were highly expressed and t(9;22) abnormality was present in all patients with EVI1 and BCR/ABL positive leukemia. Two of the 3 children with EVI1 and BCR/ABL positive CML achieved complete remission one or three months after treatment. Acquired negative status conversion occurred for EVI1 but not BCR/ABL in one CML case. The 3 children with EVI1 and BCR/ABL positive CML survived 20, 13 and 14 months, respectively, without recurrence. The child with EVI1 and BCR/ABL positive ALL failed to achieve complete remission after the first course of treatment and discontinued further treatment.</p><p><b>CONCLUSIONS</b>Co-expression of EVI1 and BCR/ABL fusion gene can be found in childhood CML and ALL. The relatively rare leukemia has not significant difference respect to clinical characteristics. Prognosis of the disease needs to be determined by clinical studies with a larger sample size.</p>

Clinical analysis of pediatric acute myeloid leukemia with EVI1 gene positive / 中华实用儿科临床杂志

Objective To analyze the clinical features and prognosis of pediatric acute myeloid leukemia (AML) with EVI1 gene positive.Methods The nested RT-PCR was performed to detect the EVI1 expression in pediatric AML patients from Jan.2009 to Dec.2011.The patients with EVI1 were investigated on clinical features,curative effects and prognosis.The differences between EVI1 (+) and EVI1 (-) patients were also analyzed.Results The frequency of EVI1 (+) expression was 15.65％ (13/83 cases)in pediatric AML,with the highest incidence in high-risk patients.EVI1 (+) was obviously associated with some unfavorable molecular genetic changes such as complex karyotype,MLL rearrangement,and monosomy 7.There were significant differences for EVI1 (+) group and EVI1 (-) group in the complete remission rate (45.5％ vs 79.3％,x2 =5.497,P ＜ 0.05) and complete remission(CR) rate after the first chemotherapy (18.2％ vs 63.8％,x2 =7.828,P ＜0.01).Although significant difference in death rate was not observed,EVI1(+) group had significantly higher early-death rate (45.5％ vs 8.6％,P ＜0.01).The EVI1(+) group also had lower 4 years event-free survival (EFS) [(21.2 ± 13.8) ％ vs (50.2 ± 9.1) ％,x2 =4.493,P ＜ 0.05] and lower 4 years overall survival(OS) [(32.4 ± 7.1) ％ vs (60.3 ± 10.9) ％,x2 =4.602,P ＜ 0.05] compared with EVI1 (-) group.But binary Logistic analysis did not identify EVI1 (+) as an independent unfavorable prognostic factor.Conclusions The pediatric AML with positive EVI1 expression had lower CR rate,higher early death rate and lower EFS.Positive EVI1 expression is related with an adverse outcome,but is not an independent poor prognostic factor.

A single-site retrospective study of pediatric arterial ischemic stroke etiology, clinical presentation, and radiologic features / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Stroke occurs upon obstruction of cerebral blood circulation and is clinically characterized by sudden onset symptoms. Advanced age is the main risk factor of stroke, but cases of pediatric stroke have been rarely reported. This study aimed to determine the etiology, clinical presentation, and radiologic features of neurological deficit for pediatric arterial ischemic stroke (PAIS).</p><p><b>METHODS</b>The medical records of 42 PAIS patients (age range: 9 months to 13 years) treated at Wuhan Children's Hospital between July 2007 and January 2011 were retrospectively reviewed. Infarction location was first determined by craniocerebral computed tomography and magnetic resonance (MR) imaging. The stenotic or occluded main cerebral arteries and/or branches were determined by MR angiography and digital subtraction angiography.</p><p><b>RESULTS</b>The majority of the 42 PAIS cases (66.7%, n = 28) were ≤ 3 years old (vs. >3 years old: 33.3%, n = 14; P<0.05), but the male: female ratio was similar in both groups (P > 0.05). The most frequently reported signs and symptoms for both age groups were limited physical activity followed by convulsions and delirium, but convulsions were more prevalent in children ≤ 3 years-old. Children > 3 years-old mainly experienced the limited physical activity symptoms, including hemiparalysis, aphasia, and ataxia. For all 42 cases, the most frequent etiologies were infections (38.1%, n = 16), iron deficiency anemia (16.7%, n = 7), and moyamoya syndrome (11.9%, n = 5). The predominant infarcts among all cases were middle cerebral artery (63.6%, n = 21) and basal ganglia (64.3%, n = 27).</p><p><b>CONCLUSIONS</b>PAIS occurs more frequently in younger children and this group most frequently presents with convulsion as the initial symptom. The overall etiologies of PAIS may be different from those of adult stroke and the involved regions may be distinguishing features of PAIS or its different forms, but more research is required.</p>

Comparisons of clinical features of chronic aplastic anemia and myelodysplastic syndrome in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>This study compared the differences in clinical features between chronic aplastic anemia (CAA) and myelodysplastic syndrome (MDS) in children in order to provide a basis for the differential diagnosis of the two diseases.</p><p><b>METHODS</b>A retrospective study of 23 cases of CAA and 9 cases of MDS from September 2007 to September 2010 was performed. The clinical data including routine blood test results, reticulocyte counts, serum lactate dehydrogenase level, serum ferritin level, cytological examination of bone marrow, bone marrow CD34+ cell counts, bone marrow chromosome and FISH test results were compared between the CAA and MDS groups.</p><p><b>RESULTS</b>Neutrophils, reticulocytes, and serum ferritin and lactate dehydrogenase levels increased in the MDS group compared with those in the CAA group. There were significant differences in bone marrow blast cell counts and dyshematopoiesis phenomena of three lines blood cells between the CAA and MDS groups. The bone marrow CD34+ cell counts and the rate of chromosomal abnormalities detected in bone marrow cytogenetic analysis in the MDS group were significantly higher than those in the CAA group.</p><p><b>CONCLUSIONS</b>There are differences in the results of laboratory examinations and morphological and cytogenetic examinations of bone marrow between the children with CAA and MDS. The differences are useful to the differential diagnosis of the two diseases.</p>

Effects of ouabain at different concentrations on growth of leukemia cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Cardiotonic steroids (CTS) can bind to Na+, K+ -ATPase to activate complex intracellular signaling cascades regulating the proliferation and apoptosis of cells. The aim of this study was to investigate the effects of ouabain at different concentrations on growth regulation in various kinds of leukemia cell lines and explore the pathogenesis of leukemia, the functions of Na+, K+ -ATPase as a signal transduction conductor and its effects on cell growth.</p><p><b>METHODS</b>Using the MTT assay, the survival rates of leukemia cell lines were observed 6, 12 and 24 hrs after treatment with 1 or 10 nmol/L ouabain. The expression of Na+, K+ -ATPase alpha1 subunit of leukemia cells was detected by Western blot.</p><p><b>RESULTS</b>The MTT results showed that ouabain at 1 nmol/L or 10 nmol/L induced proliferation of lymphocytic leukemia B95 and Jhhan cell lines, as well as megakaryocytic leukemia M07e and Meg01 cell lines. Ouabain at 1 nmol/L or 10 nmol/L increased the expression of Na+, K+ -ATPase alpha1 subunit. There were significant differences in the proliferation and the expression of Na+, K+ -ATPase alpha1 subunit of the leukemia cell lines between the ouabain treatment and the blank control groups 24 hrs after ouabain treatment (P<0.05). The proliferation effect of leukemia cell lines was in a direct proportion with the ouabain concentration and incubation time.</p><p><b>CONCLUSIONS</b>Na+, K+ -ATPase plays an important role in signal transductions. Through binding to ouabain, Na+, K+ -ATPase may regulate proliferation of leukemia cell lines of different origins. Ouabain at 1 nmol/L or 10 nmol/L may induce proliferation of lymphocytic leukemia cell lines (B95, Jhhan) and megakaryocytic leukemia cell lines (M07e, Meg01), and the proliferation effect was in a direct proportion with the concentration and incubation time of ouabain.</p>

Therapeutic effects of a combination of high-dose immunoglobulin and cyclosporine A in children with aplastic anemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the therapeutic effects of a combined immunotherapy, high-dose immunoglobulin (HDIG) plus cyclosporine A (CsA) plus prednisone (P), in children with aplastic anemia (AA) and to explore the association of peripheral blood lymphocyte subsets, peripheral blood cells and marrow CD34+ cells with therapeutic effects in AA.</p><p><b>METHODS</b>The clinical data of 46 children with AA and who received the combined immunotherapy of HDIG + CsA + P were retrospectively studied.</p><p><b>RESULTS</b>Of the 46 children with AA, 31 (67.4%) were responded to the combined immunotherapy. The binary logistic regression analysis showed low absolute neutrophil count (B=4.703, p<0.05), low percentage of peripheral blood CD4+ cells (B=0.142, p<0.05) and low ratio of peripheral blood CD4+/CD8+ (B=2.945, p<0.05)were associated with poor therapeutic effects. The ratio of CD34+/karyocytes of bone marrow in children with AA was lower than that in normal individuals, but it was not significantly related to the therapeutic effect.</p><p><b>CONCLUSIONS</b>The combined immunotherapy (HDIG+CsA+P) was effective in children with AA. The absolute neutrophilcount, the percentage of peripheral blood CD4+ and the ratio of peripheral blood CD4+/CD8+ were important prognostic factors in AA.</p>

Signal pathways of leukemia cell proliferation induced by ouabain / 中国实验血液学杂志

The aim of this study was to investigate the effects of ouabain and some specific signal pathway inhibitors on growth regulation in various kinds of leukemia cell lines and to explore the role of signal pathways participating in proliferation or apoptosis of leukemia cells induced by ouabain. By using MTT, the survival rates of leukemia cell lines were observed after utilizing ouabain and the specific signal pathway inhibitors. The expressions of Na(+), K(+)-ATPase alpha1 subunit of leukemia cells were evaluated by RT-PCR and Western blot. The results showed that low concentration of ouabain (10 nmol/L) could increase the survival rates of lymphocytic leukemia Jhhan cell line and megakaryocytic leukemia M07e cell line, and could up-regulate the expression of Na(+), K(+)-ATPase alpha1 subunit. Proliferation of these leukemia cells induced by ouabain could be inhibited by PP2 and PD98059 with different extents. It is concluded that Na(+), K(+)-ATPase plays an important role in signal transductions through binding to CTS (ouabain), and they can activate complex signal pathways regulating the growth of leukemia cells. The proliferation effects of cells promoted by ouabain are mediated by activation of Src kinase and ERK1/2 dependent signaling pathway.

Therapeutic effectiveness of the ALL-XH-99 protocol for childhood acute lymphoblastic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>The ALL-XH-99 protocol for the treatment of childhood acute lymphoblastic leukemia (ALL) has been performed in the Union Hospital for 10 years. This study aimed to evaluate the therapeutic effectiveness of the protocol for childhood ALL and to investigate the prognostic factors for childhood ALL.</p><p><b>METHODS</b>This is a retrospective study. The eligible patients were treated with the ALL-XH-99 protocol. However a minor modification based on the ALL-XH-99 protocol was performed in this study, i.e., the high-risk patients as the low- and moderate-risk patients were not administered with cranial irradiation. Event-free survival (EFS) was evaluated using the Kaplan-Meier method and the differences of the EFS among groups were compared with the log-rank test. Prognostic factors for childhood ALL were investigated by the stepwise Cox proportional hazard model.</p><p><b>RESULTS</b>One hundred fifteen patients were eligible for the ALL-XH-99 protocol clinical study. The 115 patients consisted of 62 low-risk, 12 moderate-risk and 41 high-risk patients. The overall EFS at 5 years in the 115 patients was 69.0 +/- 5.0%. The 5-year-EFS in the low-risk, moderate-risk and high-risk patients was 82.0 +/- 6.0%, 77.0 +/- 15.0% and 43.0 +/- 11.0%, respectively (P <0.01). Relapse occurred in 16 patients (13.9%) in a median time of 17 months. Without administering cranial irradiation to all of the patients, the incidence of CNS leukemia relapse (2/115, 1.7%) was not higher than that previously reported. Multivariate analysis showed that the risk degree of leukemia, the presence of t (9; 22)/bcr/abl fusion gene and leukocyte count were independent adverse prognostic factors for ALL and their hazard ratio was 1.867, 3.397 and 2.236 respectively.</p><p><b>CONCLUSIONS</b>The therapeutic effectiveness of the ALL-XH-99 protocol for childhood ALL is satisfactory, with an EFS rate comparable to that of the developed countries. t (9; 22)/bcr/abl is the most important adverse independent prognostic factor for childhood ALL. Cranial irradiation may be eliminated to reduce late adverse effects in all of ALL patients.</p>

Immortalization of rat epiphysis cartilage cells induced by simian virus 40 large T antigen gene transfection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To establish immortalized epiphysis cartilage cell strains in order to provide a stable cell resource for cell substitution and gene therapies of growth retardation.</p><p><b>METHODS</b>Plasmid pEGFP-IRES2-SV40LTag containing simian virus 40 large T antigen gene was transfected into primarily cultured epiphysis cartilage cells of the newborn rat using the lipofectin transfection method. Colonies were isolated by G418 selection and cultured to immortalized cell strains. Fibroblast growth factor receptor-3 (FGFR-3), anti-collagen type II and type X antibodies were used to identify cultured cells and to investigate the capability of differentiation of the transfected cells. SV40LTag expression in expanded cell strains was identified by RT-PCR, Southern blot and immunocytochemistry method.</p><p><b>RESULTS</b>Anti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive epiphysis cartilage cells with the capability of stable proliferation. mRNA and protein of SV40LTag were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains and named as immortalized epiphysis cartilage cells. The immortalized cells were elliptic or triangular, with two or three short axons. The immortalized epiphysis cartilage cell strains had stable biological characters.</p><p><b>CONCLUSIONS</b>SV40LTag gene transfection can immortalize epiphysis cartilage cells. The establishment of FGFR-3 positive immortalized epiphysis cartilage cell strains may provide a stable cell resource for cell substitution and gene therapies of growth retardation.</p>

Effects of ouabain at low concentrations on growth of leukemia cells / 中国实验血液学杂志

This study was aimed to investigate the effects of ouabain at low concentrations on growth regulation in various leukemia cell lines and to determine the therapeutic potential of ouabain in leukemia. By using MTT, flow cytometry (FCM), the changes in cell growth and cell cycle of leukemia cell lines were observed after treating with ouabain at low concentrations (<or=10 nmol/L). The expression of sodium pump alpha1 subunit was evaluated by Western blot. The results showed that in megakaryocytic leukemia M07e and Meg-01 cell lines, the low concentrations of ouabain (<or=10 nmol/L) inhibited the cell growth, blocked the cell cycle at G1 phase, and decreased the protein expression of sodium pump alpha1 subunit. The same concentrations of ouabain induced the proliferation of lymphocytic leukemia B95 and Jhhan cell lines, increased the percentage of cells at S phase and G2/M phase and up-regulated the expression of sodium pump alpha1 subunit. The basic expression of the sodium pump alpha1 subunit in M07e and Meg-01 was lower than that in B95 and Jhhan. It is concluded that the low concentrations of ouabain inhibit the cell growth of the megakaryocytic leukemia cell lines M07e and Meg-0, which may be related with the low expression of sodium pump alpha1 subunit of that cell lines and the negative regulation of the protein induced by ouabain.

Clinical study of human cytomegalovirus infection in colony forming unit-megakaryocyte in patients with idiopathic thrombocytopenic purpura / 中华儿科杂志

<p><b>OBJECTIVE</b>Idiopathic thrombocytopenic purpura (ITP) is a hemorrhagic disease in children with blood platelets redundant destruction caused by chaotic immunological mechanism. However, some patients with ITP with negative platelet-associated antibody and ineffective adrenal cortical hormone therapy probably have special pathogenesis. It is indicated that the human cytomegalovirus (HCMV) can incubate in haemopoietic stem cell/ancestral cell to inhibit its generation and differentiation. Therefore, the study was designed to investigate HCMV-late mRNA expression in megakaryoblast for the purpose of examining the pathogenesis of ITP and to examine the effectiveness of ganciclovir on ITP.</p><p><b>METHODS</b>Colony forming unit-megakaryocyte (CFU-MK) of 46 ITP patients with HCMV infection were incubated. Reverse transcription-polymerase chain reaction (RT-PCR) was subsequently used for HCMV-late mRNA detection. Ganciclovir therapy was given to both positive group and negative group for comparison of therapeutic effectiveness.</p><p><b>RESULTS</b>Nineteen out of 46 CFU-MK culture cell specimens with positive HCMV-DNA by PCR or positive CMV-IgM by enzyme linked immunosorbent assay (ELISA) from serum of peripheral blood showed positive for HCMV-late mRNA. While, the remaining 27 were negative. Sixteen positive responders to ganciclovir therapy were observed amongst those with positive HCMV-DNA. Whereas, only 4 positive responders to ganciclovir therapy were noticed amongst those with negative HCMV-DNA. The curative effectiveness in positive group was significantly higher than that in negative group (P < 0.01).</p><p><b>CONCLUSION</b>HCMV can directly infect CFU-MK, which might be one of the mechanisms responsible for ITP. Ganciclovir is an effective therapy resulting in an increase in thrombocyte in ITP patients whose HCMV-late mRNA was positive in their CFU-MK.</p>

Biological Activity of Cytokine-Induced Killer Cells in vitro / 实用儿科临床杂志

Objective To investigate the biological activity of cytokine-induced killer(CIK)cells in vitro.Methods Lymphocytes isolated from peripheral blood in leukemic children were induced with interferon-?(IFN-?),anti-CD3 monoclonal antibody(CD3McAb)and interleukin-2(IL-2)and co-cultured with dendritic cells(DC)to generate DC-CIK cells.The morphology and immunophenotype of these cells were determined by electron microscopy and flow cytometry,respectively.Cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the methyl thiazolyl tetrazolium (MTT) assay.Interleukin-12(IL-12),tumor necrosis factor-?(TNF-?)levels released by DC-CIK cells were quantified by enzyme-linked immunosorbent assays.Results Induced DC-CIK cells were regular,round and transparent with variable cell volume and cellular aggregation.At the 0th-4th day,its amplification was very slow,and it increased quickly at the 5th-8th day,it reached its peak amplification at the 9th-10th day,at approximately 100-fold.The main effector cells in this population were CD3+CD8+ cells and CD3+CD56+ cells.DC-CIK cells were cytotoxic to B95 cells,K562 cells and HL-60 cells,with the highest cytotoxicity towards B95 cells.The expression levels of IL-12 and TNF-? in supernatant were very high.Conclusions DC-CIK cells induced with cytokines displayed powerful amplification and strongly killing activities in vitro.It suggested that DC-CIK cells induced with cytokines may play killing activities through Th1 pathway in vitro,as a result of high secretion of Th1 cytokines,such as IL-12 and TNF-?.

Biological Characteristics and Mechanism of Killing Tumor of Cytokine-Induced Killer Cells / 实用儿科临床杂志

Objective To investigate the cytotoxicity and mechanism of killing tumor of cytokine-induced killer (CIK) cells in vitro.Methods Mononuclear cells were acquired freshly from bone marrow of children with leukemia,and the cells obstained were induced into dendritic cells by adding granulocyte-macrophage colony-stimulating,IL-4,TNF-? and other cytokines.Lymphocytes cells were isolated freshly from peripheral blood of children with leukemia by Ficoll-Hypaque density centrifugation,and the cells obstained were induced by IFN-?,IL-2 and CD3McAb.The DC cells and CIK cells were co-cultured for 10-25 days,then DC-CIK cells were obtained.Phenotypes of DC-CIK were analyzed by flow cytomery.The cytotoxicity of DC-CIK against a variety of leukemic cell lines was investigated by MTT technique.When treated with mouse-anti-human LFA-1 monoclonal antibody,the expression of GATA-3 and T -bet in the levels of mRNA and protein were mea-sured by using RT-PCR and Western Blot technique,respectively.Results In the first 0-6 days,DC-CIK induced slowly,the proliferation of DC-CIK got 100-fold at the 13th day,cells were rapidly proliferating in the first 13-21 days.The maximum proliferation of DC-CIK reached at the 22nd day.The phenotypes of CD3,CD11a,CD54,HLA-DR were expressed highly; CD3/CD56,CD25,CD28,CD69,FasL were expressed moderately on DC-CIK.The expression of CD16 was not increased.DC-CIK possessed the cytotoxicity against tumor cells of B95,Jhhan and M07e.The effect was stronger to B95,there was no significant difference when the efficiency target ratio was 12.5:1.0 or 25:1,the cytotoxicity reached about 50% and 60%,respectively,against tumor cells of B95.However,it was not obvious to Jhhan and M07e.When the efficiency target ratio was 12.5:1.0 or 25:1,the cytotoxicity reached to 27.21%,25.13%,33.05%,29.72%,respectively,against tumor cells of Jhhan and M07e.When treated with mouse-anti-human LFA-1 monoclonal antibody,the expression of GATA-3 in the level of mRNA was up-regulated(t=3.425,4.523 Pa

Study on Clinical and Immunophenotype of Acute Leukemia in Children / 实用儿科临床杂志

Objective To study the clinical significance and characteristic of acute leukemia(AL) immunophenotype expression in children.Methods Immunofluoscence labeled with three and two color monoclonal antibodies directly were used to analyze the surface antigen and cytoplastic antigen.Cytoplastic antigen was detected by flow cytometry additionally.Results Specimens from 85 patients with childhood acute leukemia were detected.1.The undifferentiated phenotype account for 1 case(1.2%);Mixed leukemia 7 cases(8.2%);Acute myeloid leukemia 18 cases(21.2%);Acute lymphoid leukemia 59 cases(69.4%).The subtypes of acute lymphoid leukemia:(B-ALL) 50 cases(84.7%),T-ALL 6 cases(10.2%),T/B 3 cases(5.1%).2.Among 59 cases of acute lymphoid leukemia,there were 31 cases ALL expression myeloid-associated antigen;There was no difference on the clinical and prognosis between the group of myeloid-associated antigen positive and the negative group.3.Of 18 patients with AML,5 patients(27.8%) expressed lymphoid antigen,CD7 was the most commonly expressed(60%);The frequency of CD_(14) expression in childhood M4,M5 was 75%.CD_(13),CD_(33) presented in 83.3% and 94.4% cases of AML,respectively.4.Among 7 cases acute mixed leukemia,B/M 6 cases,TB/M 1 case.Five cases were misdiagnosed as ALL by FAB classification.Conclusions With the multi-color flow cytometry,every acute leukemia patient was diagnosed accurately,especially on acute mixed leukemia and cross-lineage expression leukemia.Those help us choice the best treatment protocol for childhood AL.