Effects of Notch1 signaling on histone acetylation of Foxp3 gene in children with B-cell precursor acute lymphoblastic leukemia / 中华微生物学和免疫学杂志

Objective:To investigate the effects of Notch1 signaling on histone acetylation of Foxp3 gene and its roles in regulating regulatory T (Treg) cells in children with acute B-cell precursor lymphoblastic leukemia (BCP-ALL).Methods:Blood samples were collected form 38 children with BCP-ALL before treatment and 15 age-matched healthy children (control group). Flow cytometry was performed to detect the proportion of peripheral blood CD4 + CD25 hiFoxp3 + Treg cells and the expression of Foxp3, cytotoxic lymphocyte antigen 4 (CTLA4), glucocorticoid-induced tumor necrosis factor receptor (GITR), CD39 and Notch1 at protein level. Histone 4 acetylation (H4Ac) at Foxp3 gene promoter and the binding abilities of Foxp3 gene promoter to NICD1 and p300 in CD4 + T cells were measured by chromatin immunoprecipitation. Quantitative real-time PCR was performed to detect the expression of Foxp3, presenilin 1 (PSEN1), mastermind-like transcriptional coactivator 1 (MAML1), SKI-interacting protein (SKIP), F-box and WD40 domain protein 7 (FBXW7), glycogen synthase kinase-3 beta (GSK3β) and IKAROS at mRNA level in CD4 + T cells. The concentrations of TGF-β and IL-10 in plasma were evaluated by ELISA. Results:(1) The proportion of peripheral blood CD4 + CD25 hiFoxp3 + Treg cells, the expression of differentiation- and function-associated molecules (Foxp3, CTLA4, GITR and CD39) and the concentrations of TGF-β and IL-10 in plasma were higher in the BCP-ALL group than in the control group ( P<0.05). (2) In children with acute BCP-ALL, H4Ac at Foxp3 promoter and the binding abilities of Foxp3 gene promoter to NICD1 and p300 were significantly increased as compared with those in control group( P<0.05). The binding abilities of Foxp3 gene promoter to NICD1 and p300 were positively correlated with the expression of Foxp3 at mRNA level ( r=0.58 and 0.46, both P<0.05). After competitive inhibition, the three aforementioned indexes in the acute BCP-ALL group were significantly lower than those in untreated group ( P<0.05); the binding ability of Foxp3 gene promoter to NICD1 in the control group was also significantly lower than that in untreated control group ( P<0.05), but no statistical differences in the other two indexes were found between the control groups with or without treatment ( P>0.05). ⑶ Compared with the control group, the expression of Notch1, PSEN1, MAML1 and SKIP in CD4 + T cells were elevated significantly ( P<0.05), while the transcription level of negative regulatory factor FBXW7 was decreased remarkably in children with acute BCP-ALL ( P<0.05). No statistical differences in the expression of GSK3β or IKAROS were found between the two groups ( P>0.05). Conclusions:Overactivation of Notch1 signaling caused by low expression of FBXW7 might be the key factor resulting in histone 4 hyperacetylation at foxp3 gene promoter and Treg cell dysfunction in children with acute BCP-ALL.

Clinical phenotype, laboratory examination, gene diagnosis and literature review for 3 children with phytosterolemia / 临床检验杂志

Objective@#To study the characteristics of clinical diagnosis and treatment for 3 children with phytosterolemia. @*Methods@#The different clinical manifestations of 3 children with phytosterolemia were retrospectively reviewed. The case 1 and case 2, who were 7 years and 2 months old twin sisters, hospitalized for frequent epistaxis and abdominal pain. The case 3, who was 5 years and 7 months old male, came to the hospital for cutaneous xanthoma. The phytosterol levels in serum of the children were analyzed by gas chromatography-mass spectrometry, and the second generation sequencing method was used to analyze the disease-causing gene. Sanger sequencing method was used to verify the ABCG5 gene mutation and parental source. @*Results@#(1) The case 1 and case 2 showed moderate anemia, raised reticulocytes, total bilirubin and indirect bilirubin as well as splenomegaly. The blood smear showed that there were more irregular red blood cells, such as oral red blood cells, increased large/giant platelets, and ristomycin-induced platelet aggregation test was decreased. The urine routine examination indicated that there was bleeding in the urinary system. The results of blood lipid test were almost normal. The case 3 showed mild anemia with normal shape of erythrocyte and normal size of spleen. The large/giant platelets increased. The results of platelet aggregation test, bilirubin and urine routine examination were in normal range, but the levels of total cholesterol and low-density lipoprotein cholesterol increased significantly. (2) The levels of serum phytosterol were significantly increased in all the 3 children. (3) Two heterozygous mutations were detectable in ABCG5 gene of case 1 and 2 which were complex heterozygous mutation, i.e., c.9041G＞A and c.751C＞T. The variations were from their father and mother respectively. In case 3, only one homozygous mutation was detectable in ABCG5 gene which originated from their parents. @*Conclusion@#When the child showed increased large/giant platelets, hemolytic anemia, erythrocytosis or xanthoma of skin and rised total cholesterol and low-density lipoprotein cholesterol at first visit, the possibility of phytosterolemia should be considered. The blood phytosterol content and gene detection should be carried out as early as possible in order to treat early and improve prognosis.

X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection, and neoplasia: report of a family and literature review / 中华儿科杂志

Objective@#To investigate the clinical features and genetic characteristics of cases with X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN).@*Methods@#Characteristics of clinical material, immunological data and gene mutation of two cases with XMEN in the same family in China were retrospectively analyzed. The related reports literature were searched by using search terms'MAGT1 gene’or'XMEN’.@*Results@#The proband, a 2-year-eight-month old boy, was admitted due to 'Urine with deepened color for two days and yellow stained skin for one day’. He had suffered from recurrent upper respiratory tract infection and sinusitis previously. Hemoglobin level was 38 g/L. The absolute count of reticulocytes was 223.2×109/L. Urobilinogen level was 38 μmol/L (3-16 μmol/L). Coomb's test was positive. Both total (77.2 μmol/L) and indirect bilirubin (66 μmol/L) levels were elevated. There was an inverted CD4+/CD8+T cell ratio (0.89). The gene sequencing results showed MAGT1 gene c.472delG, p.D158Mfs*6 mutation. His 1-year-6-month old brother, was also identified to have MAGT1 gene c.472delG, p.D158Mfs*6 mutation.The younger brother mainly suffered from recurrent upper respiratory tract infection, accompanied by an inverted CD4+/CD8+T cell ratio (0.45), an elevated ratio and number of total B cells (45.7%). A total of 7 reports were retrieved including 11 male cases caused by MAGT1 gene mutation. These 11 cases were characterized by EBV viremia (11 cases), recurrent upper respiratory tract infection, otitis media or sinusitis (10 cases), secondary neoplasia diseases (8 cases), reduction of CD4+/CD8+ T cell ratio (7 cases),and autoimmune thrombocytopenia or hemolytic anemia (2 cases).@*Conclusion@#XMEN often manifests as male onset, recurrent upper respiratory tract infection, otitis media or sinusitis, EBV viremia, lymphoproliferative disease or lymphoma, autoimmune diseases and reduction of CD4+/CD8 +T cell ratio. NKG2D expression in NK cells is significantly reduced, and gene sequencing analysis shows a pathogenic mutation in MAGT1 gene.

Expression of leukocyte-associated immunoglobulin-like receptor on Treg cells in children with immune thrombocytopenia / 中华实用儿科临床杂志

Objective To observe the expression of leukocyte-associated immunoglobulin(Ig)-like receptor-1 (LAIR-1) on Treg cells in children with immune thrombocytopenia (ITP) and the level of soluble LAIR-1 (sLAIR-1),LAIR-2 in peripheral blood,and to discuss the possible role of LAIR in the pathogenesis of childhood ITP.Methods The levels of LAIR-1 on Treg cells of peripheral blood were measured in 36 children with ITP by using flow cytometry.Plasma levels of sLAIR-1 and LAIR-2 were measured by adopting enzyme-linked immunosorbent assay(ELISA).Real-time PCR was used to measure both LAIR-1 mRNA and LAIR-2 mRNA.Twenty-eight healthy children served as the healthy control group.Results The expression of Treg cells in children with ITP was significantly lower than that in the healthy control group [(2.05 ± 0.85) ％ vs (3.04 ± 1.03) ％,t =4.198,P ＜ 0.001].The expression rate of LAIR-1 on Treg cells and tyrosine phosphatase-2 (SHP-2) in children with ITP group had no statistically significant difference with the healthy control group [(71.18 ± 13.36) ％ vs (67.69 ± 13.07)％,t=1.045,P＞0.05;(1.20 ± 0.97) ％ vs (0.85 ±0.66)％;t=1.718,P＞0.05].The levels of sLAIR-1 and LAIR-2 in plasma in children with ITP group were increased significantly than those in the healthy control group [(20.53 ±4.32) μg/L vs (17.51 ± 5.15) μg/L,t =2.424,P ＜0.05;(5.83 ± 1.08) μg/L vs (5.19 ± 1.24) μg/L,t =2.267,P ＜ 0.05].The LAIR-1 mRNA expression level in children with ITP group was significantly increased compared with the healthy control group (t =2.851,P ＜ 0.05),but not the LAIR-2 mRNA expression level (t =1.715,P ＞ 0.05).Conclusions The expression of Treg cells in children with ITP is decreased,and it may be associated with the onset of ITP,and it may suggest that LAIR-1 does not play a leading role in Treg cells when ITP occurrs.And the levels of sLAIR-1,LAIR-2 in plasma are both increased,suggesting that LAIR-1,LAIR-2 may be one of the factors of immune disorders in children with ITP.

Liver and heart iron deposition status in patients with β thalassemia major: a multicenter study / 中华儿科杂志

<p><b>OBJECTIVE</b>To observe the status of iron deposition in patient with β thalassemia major, and to formulate appropriate treatment strategies.</p><p><b>METHOD</b>The data of status of transfusion and chelation in 135 patients aged from 6 years and 4 months to 17 years and 11 months with β thalassemia major were collected and analyzed. Serum ferritin levels were determined and cardiac and hepatic iron deposition was determined using MRI T2(*) technology.</p><p><b>RESULT</b>Of the 135 cases studied, 66 were male, and 69 were female, their average age was 12.1 years. Serum ferritin (SF) was determined for 111 cases, it varied from 1 086.8 µg/L to 15 011.5 µg/L. Among them, 16 cases had SF level <2 000 µg/L (14.5%) , in 41 cases SF were between 2 000 and 4 000 µg/L (36.0%) ;in 54 cases SF >4 000 µg/L (48.7%) . Liver MRI T2(*) results showed that in only 8 cases (5.9%) iron content in the liver was in normal range, 19 cases (14.9%) showed mild liver iron deposition;34 (25.2%) moderate and 74 (54.8%, the youngest one was only 6 years and 4 months of age) had severe iron deposition respectively. Cardiac MRI T2(*) showed that in 89 cases (65.9%) iron content in the heart was in normal range;19 cases (14.1%) had mild cardiac iron deposition and 27 (20.0%) presented severe iron deposition (the youngest one was only 9 years and 3 months of age) . SF level was obviously related to liver and cardiac iron deposition (MRI T2(*)) r and P value were -0.284, 0.003 and -0.374, 0.000 respectively. In 108 cases regular transfusion and chelation were delayed due to financial problem. The late and insufficient dosage administered and irregular chelation caused the higher SF level and the severe iron deposition.</p><p><b>CONCLUSION</b>The survival status of β thalassemia major in China is worrisome. Majority of them had not received regular transfusion and chelation. Liver and cardiac iron deposition occur early and had a high incidence.</p>

Changes of CD4 +CD25highFoxp3 +regulatory T cells and their significance in childhood B-cell acute lymphocytic leukemia / 中华微生物学和免疫学杂志

Objective To investigate the changes of CD4 +CD25highFoxp3 +regulatory T (Treg) cells and their significance in immune escape of childhood B-cell acute lymphocytic leukemia ( B-ALL ) . Methods Forty-two children with B-ALL and twenty-eight age-matched healthy children were enrolled in this study.Flow cytometry analysis was performed to evaluate the proportion of CD 4 +CD25high Foxp3 +Treg cells as well as CD4 +CD25high ICOS+Foxp3 +and CD4 +CD25high ICOS-Foxp3 +subsets in peripheral blood samples.The expression of associated molecules including IL-10, TGF-β, IL-35, TGF-βRII, ICOS and CD28 at protein level were also measured by flow cytometry analysis .The transcription level of Smad3/4, TIEG1 and Itch by CD4 +T cells were determined by quantitative real-time PCR.The concentration of TGF-βin plasma was detected by enzyme-linked immunosorbent assay.Results (1)The proportion of CD4 +CD25highFoxp3 +Treg cells in children with B-ALL were significantly higher than those of health subjects (P0.05).(3)The concentra-tion of TGF-βin plasma from children with B-ALL were higher than those from control group [ ( 25 .83 ± 12.65) ng/ml vs (8.59 ±5.73) ng/ml, P<0.05].The expression of TGF-βRII and its associated mole-cules (Smad3/4, TIEG1 and Itch) by CD4 +T cells were significantly up-regulated.Moreover, an increased expression of ICOS and CD28 by CD4 +CD25highFoxp3 +Treg cells were also observed in children with B-ALL (P<0.05).Conclusion The hyper-activity of TGF-β, ICOS and CD28 signaling might be closely associ-ated with the increased proportion of CD4 +CD25high Foxp3 +Treg cells and the imbalance of its subsets in children with B-ALL.

Alteration of T follicular helper cells in children with persistent immune thrombocytopenic purpura / 中华微生物学和免疫学杂志

Objective To investigate the role of T follicular helper ( Tfh) cells in the immuno-pathogenesis of persistent immune thrombocytopenic purpura ( pITP) .Methods Twenty children with pITP and twenty healthy controls were enrolled in this study .The proportion of CD4+CXCR5+ICOShigh PD-1high T ( cTfh) cells and the expression of ICOSL on CD 19+B cells in peripheral blood of the patients and healthy subjects were analyzed by flow cytometry .The expressions of Bcl-6, c-Maf, IL-21 and ICOSL at mRNA level were detected by real-time PCR.The plasma concentrations of IL-2, IL-6 and IL-21 were determined by ELISA.Results (1) Compared with the healthy controls , the proportions of cTfh cells increased signifi-cantly in patients with pITP [(17.45±9.04) %vs.(7.57±2.57) %, P0.05).(4) The plasma concentration of IL-21 was remarkably elevated in patients with pITP , regardless of DEX treatment (P0.05).Con-clusion The immunopathogenesis of persistent immune thrombocytopenic purpura might be associated with the hyper-activation of Tfh cells caused by excessive expression of ICOSL and IL-21.The persistent high expression of ICOSL and IL-21 might be one of the important factors resulting in the recurrence of pITP in children .

Alteration of Fc gamma receptors in childhood acute immune thrombocytopenia / 中华微生物学和免疫学杂志

ObjectiveTo investigate the alteration of Fc gamma receptors expressed on monocytes in childhood acute immune thrombocytopenia (aITP).Methods27 patients with aITP and 25 health controls were enrolled in this study.The expression of FcγR Ⅰ and FcγRⅢ on monocytes surface was determined by flow cytometry.Real-time PCR was performed to detect the levels of FcγR Ⅱ a and FcγR.Ⅱ b mRNA.The plasma concentration of IL-4 and IFN-γwas analyzed by ELISA.ResultsThe expression of FcγR I and FcγRⅢ on monocytes was significantly highet in ITP pationts than thatinhealth control group.Transcription levels of FcγR Ⅱ a mRNA and the ratio of FcγR Ⅱ a/Ⅱ b mRNA on monocytes were significantly elevated,while the inhibitory FcγR Ⅱ b mRNA was significant lower in aITP patients (P＜0.05) ; in comparison with healthy controls. After dexamethasone treatment,the stimulatory FcγR Ⅰ,FcγRⅢ and FcγR Ⅱ a mRNA were significantly decreased while inhibitory FcγR Ⅱ b mRNA was increased.(2)The higher levels of plasma IFN-y and lower IL-4 were to be found in aITP patients compared with healthy controls.Correlation analysis showed that the level of plasma IFN-γwas positively correlated with the elevated expression of FcγR Ⅱ a mRNA on monocytes,while the level of IL-4 was positively correlated with FcγRⅡ b mRNA.Conclusion The over expression of inhibitory FcγRs and the down-regulation of inhibitory FcγR Ⅱb mRNA on monocytes,and the imbalance of stimulatory and inhibitory FcγR might be involve in immunological pathogenesis of the acute ITP.The alteration levels of plasma pro-inflammatory cytokine IFN-γand anti-inflammatory cytokine IL-4 might be involved in imbalance expression of FcγR in acute ITP.Dexamethasone therapy could shift the balance between stimulatory and inhibitory FcγR toward inhibitory FcγR Ⅱ b in aITP patients.

Diagnosis and treatment progress of children's acute myeloid leukemia / 国际儿科学杂志

Acute mveloid leukemia(AML) is a heterogeneous group of leukemias that arise from clonal transformation of hematopoietic precursors.This review summarizes classification of pediatric AML,more precise risk-group stratification,ongoing phase Ⅲ studies and minimal residual diseasse monitoring.In addition,we discuss the opportunities for innovative chemotherapy drugs in refractory AML and relapsed AML,such as gemtuzumab ozogamicin,liposomal daunorubicin,clofarabine,cladribine and lmatinib.Finally,the roles of allogeneic hematopoietic stem cell transplantation in pediatric AML are also discussed.

A study on the influential factors of G6PD/6PGD specific value assay in the heterozygotes of G6PD gene variants in female patients / 国际检验医学杂志

Objective To improve the detection of G6PD heterozygotes in female patients by the optimum experimental factors of G6PD/6PGD specific value assay.Methods (1)Identifying the mutations of G6PD gene with the use of amplified refractory mutation system (ARMS).(2)Measuring the G6PD/6PGD specific value.Results According to the data analysed by statistics and ROC curve, the optimum experimental factors included that the incubation temperature was 37℃,the substrate concentrations were 0.78 mmol/L G6PNa2 and 0.195mmol/L NADP+, the reaction time was 10min.Conclusion The optimum experimental factors of G6PD/6PGD specific value assay may be used to improve the detection rate of G6PD heterozygotes in female patients.