Quantitative proteomics and bioinformatics analyses of human coronary artery endothelial cell injury induced by Kawasaki disease / 中国当代儿科杂志

OBJECTIVE@#To study the biomarkers for human coronary artery endothelial cell (HCAEC) injury induced by Kawasaki disease (KD) using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics.@*METHODS@#HCAECs cultured with the serum of children with KD were used as the KD group, and those cultured with the serum of healthy children was used as the healthy control group. The iTRAQ technique was used to measure the expression of proteins in two groups. The data on proteins were analyzed by bioinformatics. Western blot was used for the validation of protein markers.@*RESULTS@#A total of 518 significantly differentially expressed proteins were identified (with an absolute value of difference fold of >1.2, P<0.05). The gene ontology analysis showed that the differentially expressed proteins were significantly enriched in biological processes (including cellular processes, metabolic processes, and biological regulation), cellular components (including cell parts, cells, and organelles), and molecular functions (including binding, catalytic activity, and molecular function regulators). The KEGG analysis showed that the proteins were significantly enriched in the signaling pathways of ribosomes, PI3K-Akt signaling pathway, and transcriptional dysregulation in cancer. The PPI network showed that the top 9 protein markers in relation density were PWP2, MCM4, MCM7, MCM5, MCM3, MCM2, SLD5, HDAC2, and MCM6, which were selected as the protein markers for coronary endothelial injury in KD. Western blot showed that the KD group had significantly lower expression levels of the protein markers HDAC2, PWP2, and MCM2 than the healthy control group (P<0.05).@*CONCLUSIONS@#The serum of children with KD significantly changes the protein expression pattern of HCAECs and affects the signaling pathways associated with the cardiovascular system, which provides a new basis for the pathophysiological mechanism and therapeutic targets of KD.

Resources of Orchidaceae Medicinal Plants in Jiangxi Province / 中国实验方剂学杂志

To provide theoretical basis for protection and rational use of medicinal plants resources of Orchidaceae,we investigated and studied the existing species,distribution,utilization and resources of Orchidaceae medicinal plants in Jiangxi province. Orchidaceae medicinal plants in different areas of Jiangxi province were investigated in different seasons by means of field investigation,route investigation and folk interview. Orchidaceae medicinal plants collected from field investigation as well as Orchidaceae specimens stored in the herbariums of Jiangxi scientific research institutes were studied and identified. The existing species,distribution location,quantity,medicinal value and resource utilization of Orchidaceae medicinal plants in Jiangxi province were studied and analyzed. Orchidaceae medicinal plants in herbal literatures were consulted for their category,medicinal use and other information. Relevant modern research literatures on Orchidaceae medicinal plant resources were consulted. There were 93 species of Orchidaceae medicinal plants in 37 genera in Jiangxi province, and 19 of them were new species of Orchidaceae,including 6 species in Dendrobium alone. The number of medicinal genera accounted for 71.2% of Orchidaceae genera in Jiangxi province,20.1% of Orchidaceae genera in China,76.9% of Orchidaceae species in Jiangxi province and 31.2% of Orchidaceae medicinal species in China. There are abundant resources of Orchidaceae medicinal plants in Jiangxi province,and many species of Orchidaceae medicinal plants have a high medicinal value and ornamental value. However,with the overexploitation and utilization of Orchidaceae medicinal plant resources,some wild Orchidaceae medicinal plant resources are facing exhaustion,and need to urgently strengthen scientific management and protection.

Effect of Paidu Baoshen Pill on renal fibrosis in 5/6 nephrectomized rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the anti-renal fibrosis effect of Paidu Baoshen Pill (PBP) on 5/6 nephrectomized rats and to explore its mechanism.</p><p><b>METHODS</b>Totally 50 SD male healthy rats were randomly divided into the normal control group (n = 10), the sham-operation group (n = 10), and the nephrectomy model group (n = 30) according to the proportion of 1:1:3. Rats in the sham-operation group had their renal capsule isolated without nephrectomy. Rats in the nephrectomy model group had their kidneys 5/6 nephrectomized. Then 24 h urine was collected and 24 h urinary protein (24 h UP) detected. Serum blood urea nitrogen (BUN) and serum creatitine (SCr) were also tested. According to the SCr level 30 rats of the model group were further randomly divided into the model group, the PBP group, and the Niaoduqing Granule (NG) group, 10 in each group. Rats in the PBP group and the NG group were respectively administered with PBP (at the daily dose of 1.0 g/kg) and NG (at the daily dose of 3.33 g/kg) by gastrogavage (they were dissolved in distilled water). At the same time, 2 mL distilled water was administered by gastrogavage to rats in the normal control group, the sham-operation group, and the nephrectomy model group, once daily for 4 successive weeks. Mental conditions, activities, hair color, shape of stool, and the body weight were observed during administration. After 4 weeks, urine was collected to detect 24 h UP. Blood was sampled to detect SCr, BUN, transforming growth factor β1 (TGF-β1), type III procollagen (PC III), collagen type IV (Col IV), laminin (LN), and fibronectin (FN). After rats were killed, their left remnant renal tissues were collected for pathological examinations. The protein expression quantity of TGF-β1 and FN was detected by immunohistochemical method. mRNA expression levels of TGF-β1 and FN were detected using real time fluorescent quantitative PCR.</p><p><b>RESULTS</b>There was no statistical difference in the above indices between the normal control group and the sham-operation group (P > 0.05). Compared with the sham-operation group, rats' general condition was poorer in the model group, their body weight grew slower, and 24 h UP increased; serum levels of BUN, SCr, TGF-β1, PC III, Col IV, LN, and FN increased; the residual renal pathological lesion was serious; expression levels of TGF-β1, TGF-β1, mRNA, FN, and FN mRNA increased in the renal tissue (all P < 0.01). Compared with the model group, rats' general condition was better, their body weight grew faster, 24 h UP reduced (P < 0.05), blood levels of BUN and SCr decreased significantly (P < 0.01), serum levels of TGF-β1, PC III, CoL IV, LN, and FN decreased (P < 0.05, P < 0.01); the residual renal pathological lesion was attenuated in the PBP group and the NG group; expression levels of TGF-β1, TGF-β1, mRNA, FN, and FN mRNA decreased (P < 0.01). Compared with the NG group, blood levels of SCr and FN, and expression levels of FN and FN mRNA decreased more in the PBP group (P < 0.05).</p><p><b>CONCLUSIONS</b>PBP had the effect of anti-renal fibro- sis in 5/6 nephrectomized rats. Down-regulating expression levels of TGF-β1, and FN from gene transcription and protein translation levels might be one of its mechanisms.</p>

Expression of leukocyte-associated Ig-like receptor-1 in children with immune thrombocytopenia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the expression of leukocyte-associated Ig-like receptor-1(LAIR-1) in children with immune thrombocytopenia (ITP), in order to explore the possible role of LAIR-1 in the pathogenesis of childhood ITP.</p><p><b>METHODS</b>Expression levels of LAIR-1 on CD4(+) T cells, CD8(+) T cells and CD19(+)CD20(+) B cells of peripheral blood were measured in 40 children with ITP by flow cytometry. Serum level of solubility LAIR-1 (sLAIR-1) was measured using ELISA. Real-time PCR was used to measure LAIR-1 mRNA expression. Thirty-two healthy children served as the control group.</p><p><b>RESULTS</b>The percentages of CD19(+)CD20(+) B cells in the ITP group were significantly higher than in the control group (P<0.05). In contrast, the percentage of CD4(+) T cells in the ITP group was significantly lower than in the control group (P<0.05). The expression levels of LAIR-1 on CD4(+) T cells and CD8(+) T cells were significantly lower in the ITP group than in the control group (P<0.05). Serum sLAIR-1 level and LAIR-1 mRNA expression in the ITP group significantly increased compared with the control group (P<0.05).</p><p><b>CONCLUSIONS</b>LAIR-1 expression on CD4(+) and CD8(+) T cells decreases and serum sLAIR-1 level increases in children with ITP, suggesting that LAIR-1 may play an important role in immune imbalance in these children.</p>

Alterations of CD4+CXCR5+Tfh cells and its transcription regulatory factors in children with asthma / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the alterations of follicular T helper cells (CD4(+)CXCR5(+)Tfh cells, Tfh) on circulating T lymphocytes in children with asthma, and to study the expression of transcription regulatory factors BCL-6 and BLIMP-1 mRNA.</p><p><b>METHODS</b>Sixty-four children with asthma and 25 healthy controls were enrolled in this study. On the basis of the disease, the children with asthma were classified into acute phase group (n=36) and remission phase group (n=28). The flow cytometry was used to detect the proportion of CD4(+)CXCR5(+)Tfh cells on CD4(+)T lymphocytes. Real-time PCR was performed to detect the levels of BCL-6 mRNA and BLIMP-1 mRNA. The double -antibody Sandwich ELISA was used to detect plasma concentrations of total IgE, IL-2, IL-6 and IL-21.</p><p><b>RESULTS</b>The proportion of CD4(+)CXCR5(+)Tfh cells was significantly higher in the acute group than in the control group and the remission group (P<0.05). Transcription levels of BCL-6 mRNA were significantly higher, while the inhibitory factors BLIMP-1 mRNA was significantly lower in the acute group than in the remission group and control group (P<0.05). The plasma concentration of IL-6 in the acute group increased significantly compared with the control group (P<0.05). Plasma concentrations of total IgE and IL-21 increased significantly, in contrast, plasma IL-2 concentration decreased significantly in the acute group, compared with the control group and the remission group (P<0.05). Correlation analysis showed that both IL-21 and IL-6 concentrations were positively correlated with the proportion of CD4(+)CXCR5(+)Tfh cells (r=0.76, r=0.46 respectively; P<0.05), while IL-2 level was negatively correlated with the proportion of Tfh cells (r=-0.68, P<0.05).</p><p><b>CONCLUSIONS</b>The abnormal proportion of CD4(+)CXCR5(+)Tfh cells might be involved in the immunological pathogenesis of acute asthma in children. The increased expression of BCL-6 mRNA and decreased expression of BLIMP-1 mRNA as well as the alterations of plasma total IgE, cytokines IL-2, IL-6 and IL-21 in microenvironment might be account for the increased proportion of CD4(+)CXCR5(+)Tfh cells in children with acute asthma.</p>

Change of Toll-like receptors in children with acute Epstein-barr virus infection / 中华实用儿科临床杂志

Objective To investigate the possible role of Toll-like receptors (TLRs) in immunological pathogenesis of Epstein-barr virus(EBV) infection.Methods Fifteen children with acute EBV infection,18 children with infectious mononucleosis (IM),and 25 age-matched healthy children were enrolled in the study.Reverse-transcription polymerase chain reaction and real-time PCR were used to evaluate the levels of TLR2,TLR3,TLR7,TLR9,myeloid differentiation factor 88 (MyD88),tumor necrosis factor receptor-associated factor 6 (TRAF6),TGF-β activated kinase 1 (TAK1),TIR domain-containing adaptor protein inducing interferon β (TRIF),tumor necrosis factor receptor-associated factor 3 (TRAF3),TBK1,IL-1β,TNF-α,IFN-α and IFN-β mRNA expression in peripheral blood mononuclear cells(PBMC).The plasma concentrations of cytokines such as IL-12 and interferon gamma (IFN-γ) were determined by enzyme-linked immunosorbent assay.Results 1.Compared with healthy control group,the expression levels of TLR2,TLR3,TLR7,TLR9,MyD88,TRAF6,TAK1,TRIF,TRAF3 and TBK1 mRNA were up-regulated significantly from the children with acute EBV infection and IM (all P ＜ 0.05),there was no difference between the children with acute EBV infection and IM(all P ＞ 0.05).2.The levels of cytokines expression such as IL-1β,TNF-α,IFN-α and IFN-β in children with acute EBV infection were higher than those of the healthy control group (all P ＜0.05).3.The plasma concentrations of IL-12 and IFN-γwere up-regulated (all P ＜ 0.05) in children with acute EBV infection.Conclusion Imbalance of antiviral/inflammatory response resulting from the aberrant activation of TLRs may be one of the factors causing disturbed immunological function in vivo infected by EBV.

Role of Th17 cell and CD4+CD25+ regulatory T cells in inflammatory response in neonatal sepsis / 中华实用儿科临床杂志

Objective To investigate the roles of Thl7 cells and CD4 + CD25 + regulatory T cells in inflammatory response in neonatal sepsis.Methods Twenty children with neonatal sepsis (neonatal sepsis group) and 16 healthy neonates (healthy control group)were studied.Flow cytometric analysis (FCM) was performed to detect the percentage of CD4 + CD25 + Treg cells subpopulation.Real-time transcription-polymerase chain reaction (Real-time PCR) was used to analyze interleukin-17A (IL-17A),IL-17F,the transcription factor retinoid-related orphan nuclear receptor γt (ROR-γt),the forkhead/winged-helix protein 3 (Foxp3) and cytokines IL-6,transforming growth factor beta (TGF-β) expression in CD4 + T cell.Expressions of proinflammatory cytokines [IL-1β,IL-6,IL-10,tumor necrosis factor α (TNF-α)] were measured by using enzyme-linked immunosorbent serologic assay.Results Compared with healthy control group:1.The proportions of CD4 + CD25 + Treg cells in neonatal sepsis group were significantly higher [(15.33 ± 2.68) ％ vs (2.96 ± 0.56) ％,P ＜ 0.01].The mRNA expression of transcription factor Foxp3 in neonatal sepsis group showed similar tendency[(42.76 ± 10.83) × 10-4 vs (22.34 ±4.17) × 10-4,P ＜0.01].2.Expression levels of IL-17A and IL-17F were significantly up-regulated in neonatal sepsis group[IL-17A:(13.56 ±3.21) × 10-6 vs (4.76 ±1.39) ×10-6,P＜0.01 ;IL-17F:(7.62 ±1.45) ×10-4 vs (1.89 ±0.48) ×10-4,P＜0.01] and the expression levels of the transcription factor ROR-γt in CD4 + T cells were significantly increased in neonatal sepsis group [(9.22 ± 1.79) × 10-5 vs (2.84 ±0.56) × 10-5,P ＜0.01].3.Expressions of proinflammatory cytokines (IL-1β,IL-6,IL-8,TNF-α) in neonatal sepsis group were higher than those in control group [IL-1β:(2977.36 ± 653.97) pg/L vs (480.52 ± 120.36) pg/L,P ＜ 0.01 ; IL-6:(3143.82 ± 775.08) pg/L vs (393.78 ± 96.55) pg/L,P ＜ 0.01) ; IL-10:(3216.98 ± 678.43) pg/L vs (326.11 ± 62.45) pg/L,P ＜ 0.01 ; TNF-α:(3582.24 ± 876.13) pg/L vs (1233.68 ± 289.39) pg/L,P ＜ 0.01].Conclusion Aberrant activation of Th17 cell and CD4 + CD25 + Treg cell might be involved in pathogenesis in neonatal sepsis.

Clinical investigation and mutation analysis of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the clinical features and SLC25A13 gene mutations of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia.</p><p><b>METHODS</b>The patient was subjected to physical examination and routine laboratory tests. Blood amino acids and acylcarnitines, and urine organic acids and galactose were analyzed respectively with tandem mass spectrometry and gas chromatographic mass spectrometry. SLC25A13 gene mutation screening was conducted by high resolution melt (HRM) analysis.</p><p><b>RESULTS</b>The petechiae on the patient's face and platelet count (27×10(9)/L, reference range 100×10(9)/L-300×10(9)/L) supported the diagnosis of immunologic thrombocytopenic purpura (ITP). Laboratory tests found that the patient have abnormal coagulation, cardiac enzyme, liver function and liver enzymes dysfunction. Tandem mass spectrometry also found methionine to be increased (286 μmol/L, reference ranges 8-35 μmol/L). The patient did not manifest any galactosemia, citrullinemia and tyrosinemia. Analysis of SLC25A13 gene mutation found that the patient has carried IVS16ins3kb, in addition with abnormal HRM result for exon 6. Direct sequencing of exon 6 revealed a novel mutation c.495delA. The same mutation was not detected in 100 unrelated healthy controls. Further analysis of her family has confirmed that the c.495delA mutation has derived from her farther, and that the IVS16ins3kb was derived from her mother.</p><p><b>CONCLUSION</b>The clinical features and metabolic spectrum of citrin deficiency can be variable. The poor prognosis and severity of clinical symptoms of the patient may be attributed to the novel c.495delA mutation.</p>

Utilization of high-resolution melting analysis to screen patients with neonatal intrahepatic cholestasis caused by citrin deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the feasibility of high-resolution melting (HRM) analysis for screening patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).</p><p><b>METHODS</b>Based on previous studies on SLC25A13 gene in Chinese patients with NICCD, four hotspot mutations (851del4, 1638ins23, IVS6+5G>A and IVS16ins3kb) were selected. Results of the HRM analysis was validated using 50 negative controls and 20 patients with NICCD whose genotypes were confirmed previously by direct sequencing. With the established protocol, 171 suspected patients were enrolled. Samples with abnormal melting curves were further validated by DNA sequencing.</p><p><b>RESULTS</b>HRM analysis can accurately determine the genotypes of all negative controls and patients. The sensitivity and specificity of the technique reached 100% (70/70). The melting curves of samples with the same genotype were highly reproducible. In 171 suspected patients, seven NICCD patients were detected by HRM. Identified mutations have included one case of 851del4 homozygote, one case of IVS6+5G>A heterozygote, 3 cases of 851del4 heterozygotes, one case of [IVS6+5G>A]+[ 851del4] and one case of [1638ins23+IVS16ins3kb]+[1638ins23]. All mutations were subsequently confirmed by DNA sequencing.</p><p><b>CONCLUSION</b>HRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.</p>

Analysis of clinical features and GCDH gene mutations in four patients with glutaric academia type I / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To review clinical features of four male patients with glutaric academia type I and screen glutaryl-CoA dehydrogenase (GCDH) gene mutations.</p><p><b>METHODS</b>The 4 patients underwent brain computer tomography (CT) and magnetic resonance imaging (MRI) analyses. Blood acylcarnitine and urine organic acid were analyzed with tandem mass spectrometry and gas chromatographic mass spectrometry. Genomic DNA was extracted from peripheral blood samples. The 11 exons and flanking sequences of GCDH gene were amplified with PCR and subjected to direct DNA sequencing.</p><p><b>RESULTS</b>All patients have manifested macrocephaly, with head circumference measured 50 cm (14 months), 47 cm (9 months), 46 cm (5 months) and 51 cm (14 months), respectively. Imaging analyses also revealed dilation of Sylvian fissure and lateral ventricles, frontotemporal atrophy, subarachnoid space enlargement and cerebellar vermis abnormalities. All patients had elevated glutarylcarnitine (5.8 umol/L, 7.5 umol/L, 8.3 umol/L and 7.9 umol/L, respectively) and high urinary excretion of glutaric acid. Seven mutations were identified among the patients, among which c.146_149del4, IVS6-4_Ex7+4del8, c.508A>G (p.K170E), c.797T>C (p.M266T) and c.420del10 were first discovered.</p><p><b>CONCLUSION</b>Macrocephaly and neurological impairment are the most prominent features of glutaric academia type I. Blood tandem mass spectrometry and urine gas chromatographic mass spectrometry analysis can facilitate the diagnosis. The results can be confirmed by analysis of GCDH gene mutations.</p>

SLC25A13 gene analysis in neonates with intrahepatic cholestasis caused by citrin deficiency / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) which resulted from mutation in SLC25A13 gene can present transient intrahepatic cholestasis, low birth weight, growth retardation, hypoproteinemia and so on. This study aimed to identify the mutation type of NICCD patients by DNA sequencing.</p><p><b>METHODS</b>Twenty children diagnosed as NICCD were consented to enroll in this study. PCR assays were performed to amplify the eighteen exons and its flanking sequences of SLC25A13 gene, which were defined as the upstream and downstream 50 bp from starting and ending site of the exons. Then the PCR products were purified and followed by automated DNA sequencing. The IVS16ins3kb mutation was detected by nested PCR and RT-PCR.</p><p><b>RESULTS</b>Seven genetic variations of SLC25A13, termed as 851del4, 1638ins23, IVS16ins3kb, IVS6+5G>A, c.775C>T (p.Q259X), c.1505C>T (p.P502L) and c.1311C>T (p.C437C), were identified in the subjects, of which c.775C>T (p.Q259X), c.1505C>T (p.P502L) and c.1311C>T (p.C437C) were reported for the first time in NICCD patients. And a compound mutation of［1638ins23+IVS16ins3kb］was also identified. In 20 patients with NICCD, 6 patients were 851del4 homozygotes, 7 patients were compound heterozygotes, and 7 patients were heterozygotes of single mutation. 851del4 was the major mutation type (64%), followed by 1638ins23 (15%), IVS16ins3kb (12%) and IVS6+5G>A (6%).</p><p><b>CONCLUSIONS</b>851del4 is the major mutation type in Chinese patients with NICCD.</p>

Changes of immune function in patients with 2009 influenza A (H1N1) / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the alteration of immune function and possible immunopathogenesis in the children with 2009 influenza A (H1N1) infection.</p><p><b>METHOD</b>Sixty patients with 2009 influenza A (H1N1) infection hospitalized in Shenzhen Children's Hospital between November 1, 2009 and January 10, 2010 and 20 age-matched healthy children were enrolled in this study. The patients were divided into two groups according to the severity of influenza A infection: 35 mild cases (mild pneumonia) and 25 severe cases (severe pneumonia, acute encephalopathy associated with influenza A, and 3 died from acute necrotizing encephalopathy with influenza A infection). Real-time PCR was used to evaluate the expression levels of pattern recognition receptor (PRRs), retinoic acid induced gene I/melanoma differentiation associated gene 5 (RIG/MDA5), Toll-like receptors (TLRs) and TLRs signaling molecules, and negative-regulator. Three color fluorescent and flow cytometry were used to investigate the apoptosis of CD3(+), CD4(+), CD8(+) and CD19(+) cells. Plasma cytokines (IL-1β, IL-6, TNF-α, IFN-γ, IFN-α, IL-10) concentrations were measured by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULT</b>(1) The expression levels of RIG/MDA5, TLR2, 4 were much higher in the patients with influenza A infection, especially severe cases [TLR2 (9.69 ± 3.15) × 10(-2) vs. (3.96 ± 0.83) × 10(-2), t = 10.16, P < 0.05; TLR4 (10.23 ± 2.85) × 10(-2) vs. (7.46 ± 2.18) × 10(-2), t = 3.76, P < 0.05]. The expression levels of TLRs signal transduction molecules like MyD88 and TRAM also increased. (2) The cell counts of CD3(+), CD4(+), CD8(+) T cells and NK cells were markedly lower in the patients with influenza A infection compared to the NC group [CD3(+)(1.22 ± 0.38) × 10(9)/L vs.(3.59 ± 1.10) × 10(9)/L, t = 9.21, P < 0.05]. (3) Plasma concentrations and the mRNA expression of TNF-α, IL-6, and IL-1β were elevated in mild cases, while declined in severe cases [TNF-α (6.42 ± 1.76) × 10(-2) vs. (9.05 ± 2.51) × 10(-2), t = 4.55, P < 0.05]. Plasma concentrations of IFN-α/IFN-β were up-regulated gradually with the aggravation of the disease, especially in severe cases. Compared with healthy controls, the expression of IFN-I inducible gene IP-10, RANTES, or iNOS was significantly higher in children with mild [IP-10 (20.52 ± 6.09) × 10(-2) vs.(1.18 ± 0.34) × 10(-2), t = 18.74, P < 0.05], and relatively lower in severe cases. (4) The apoptosis of CD3(+), CD4(+), CD8(+) and NK cells significantly increased in the patients with influenza A infection than those in NC group [CD3(+)(32.90 ± 7.66)% vs. (20.21 ± 6.58)%, t = 6.21, P < 0.05]. Compared with healthy controls, the expression levels of apoptosis-related gene like TRAIL and CASPASE-3 significantly increased in the patients with influenza A infection. (5) The expression levels of negative regulator of SOCS1, SOCS3, IRAK-M, TRAF4 and FLN29 were significantly increased in the patients with influenza A, especially in severe cases than those in NC group (P < 0.05).</p><p><b>CONCLUSION</b>Immune function changed with the severity of the disease. The mild cases presented systemic immune activation status, while critically ill cases presented mixed immune activation and immunosuppression status.</p>

Changes of immune function in patients with enterovirus 71 infection / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the association of changes in immune function with enterovirus 71 (EV71) cases with different severity of the disease.</p><p><b>METHOD</b>Forty-six EV71-infected patients and 12 age-matched healthy children were enrolled in this study. The patients were divided into four groups according to critical degree of enterovirus 71 infection: hand-foot-and-mouth disease (HFMD); central nervous system disease (CNSD); autonomic nervous system dysregulation (ANSD) and pulmonary edema (PE). We analyzed CD14+ monocyte HLA-DR expression, lymphocyte immunophenotypes, the proportion of CD4+CD25+ Foxp3high regulatory T cells (Treg cells) and Th17 cells, cytokines (IL-1beta, TNF-alpha, IL-10, TGF-beta, IL-6, IL-17A), evaluated the mRNA levels of Foxp3 and ROR-gammat, and serum immunoglobulin and complements.</p><p><b>RESULT</b>(1) Serum concentrations of IL-1beta and TNF-alpha elevated in mild cases, while declined in severe cases, and were lower in PE group (P<0.05). Serum concentrations of IL-10 and IL-10/TNF-alpha ratio gradually raised with the aggravation of the disease, and higher in PE group (P<0.05). (2) Circulating CD14+ monocyte HLA-DR expression, CD3+T cells, CD4+T cells, CD8+T cells, and NK cells gradually decreased, and lower in PE group (P<0.05). There was no significant difference in B cells, immunoglobulin and complement among the four groups. (3) The proportion of CD4+CD25+ Foxp3high Treg cells, mRNA level of Foxp, and serum concentrations of TGF-beta gradually decreased with the aggravation of the disease, while the proportion of Th17 cells, serum concentrations of IL-17A, mRNA level of ROR-gammat, and IL-6 gradually increased with the aggravation.</p><p><b>CONCLUSION</b>Immune function changed with different illness phases. The mild cases presented systemic inflammatory response syndrome status, while critically ill cases presented compensatory anti-inflammatory response syndrome or mixed antagonist response status. Immunoregulatory treatment of patients with EV71 infection should emphasize different methods at different stage and individualization.</p>

Changes and significance for regulatory factors for signal pathways of Toll-like receptors in immunological pathogenesis of Kawasaki disease / 中华儿科杂志

<p><b>OBJECTIVE</b>Many clinical evidences and epidemiologic data in the past suggested that Kawasaki disease (KD) is correlated with an acute immune dysfunction caused by infection. In our preliminary study, Toll-like receptor 4 signal pathway, which could activate nuclear transcription factor-kappaB and induce excessive product of proinflammatory cytokines, chemokines and co-stimulatory molecules, was observed to be significantly activated during acute phase of Kawasaki disease. But the causative factors and regulatory mechanism are still unknown. In this study, the authors further investigated the changes and significances of regulatory factors for signal pathway of Toll-like receptors (TLRs) in immunological pathogenesis of Kawasaki disease.</p><p><b>METHODS</b>Forty-eight children with KD, sixteen children with infectious disease (ID) and sixteen age-matched healthy children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the expression levels of regulatory and effective factors in toll-like receptor 4 (TLR4) signal pathways and proinflammatory factors in peripheral blood monocyte/macrophage (MC). The expression of TLR4 protein in MC was analyzed by flow cytometry.</p><p><b>RESULTS</b>(1) Expression levels of TLR4, MD-2, MyD88, IRAK-4, TRAF6, TAK1, TAB1 and TAB2 mRNA in KD group were elevated significantly during acute phase (P < 0.05). (2) Transcription levels of regulatory factors PRAT4B and STAP2 in patients with KD or ID were found to be higher than those in the healthy volunteers (P < 0.05), but no significant differences in these parameters were detected between KD patients and ID patients (P > 0.05). Transcription levels of regulatory factors such as FLN29, RP105 and MD-1 were up-regulated to some extents and expression level of DAP12 mRNA in KD patients were found to be lower than that in normal controls (P < 0.05), while all of the four regulatory factors were found to be lower than those in ID patients (P < 0.05). Expressions of proinflammatory cytokines such as L-1beta, IL-6 and TNF-alpha in KD patients were significantly higher than those in ID patients (P < 0.05). (3) Stimulation with lipopolysaccharide (LPS) elevated remarkably the expressions of regulatory factors PRAT4B and STAP2 in KD patients or healthy volunteers (P < 0.05). All of the four negative-regulatory factors were found to be significantly up-regulated after stimulation with LPS in controls (P < 0.05). No responses to LPS were observed in expression of FLN29, RP105 and MD-1 mRNA in KD patients (P > 0.05), except for increased transcription of DAP12. (4) The levels of PRAT4B and STAP2 mRNA in KD patients with coronary artery lesion (KD-CAL(+)) were detected to be higher than those in KD patients without coronary artery lesion (KD-CAL(-)) during acute phase (P < 0.05), while those of FLN29, RP105 and MD-1 in KD-CAL(+) group were lower than that in the latter (P < 0.05). No significant difference in DAP12 mRNA expression level was detected between the two groups (P > 0.05). Expressions of proinflammatory cytokines and TLR4 protein on surface of CD14-positive cells in KD-CAL(+) group were found to be higher than those in KD-CAL(-) group [(11.9 +/- 2.4)% vs. (6.5 +/- 1.7)%, P < 0.05].</p><p><b>CONCLUSION</b>Disturbance of negative-regulatory factors may be one of the factors causing aberrant immunological function in KD.</p>

Expression and significance of BLyS/April in patients with acute idiopathic thrombocytopenic purpura / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the roles of B-lymphocyte stimulator/ a proliferation-inducing ligand (BLyS/April) in immunological pathogenesis of idiopathic thrombocytopenic purpura (ITP).</p><p><b>METHODS</b>Thirty ITP children and 30 age-matched healthy children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the mRNA levels of BLyS/ April, receptors for BLyS/April (BR3, BCMA and TACI) and cytokines in ITP patients. Flow cytometry was performed to measure relative mean fluorescence intensity (relative MFI) for platelet-associated immunoglobulin G (PAIgG).</p><p><b>RESULTS</b>(1) The transcription levels of BLyS/April in monocytes/macrophage [(8.30 +/- 2.31) x 10(-1) and (7.51 +/- 1.93) x 10(-3), respectively] were significantly up-regulated in acute ITP compared with that in healthy controls [(3.95 +/- 1.04) x 10(-1) and (3.08 +/- 0.82) x 10(-3), respectively] (P < 0.0.1). (2) Expression levels of the BLyS/April receptors BR3, BCMA and TACI mRNA were remarkably raised during acute phase of ITP (P < 0.01). (3) The mRNA levels of cytokines, including IL-4, IL-5, IL-6, IL-10 and IL-15, were significantly higher in acute phase ITP than in healthy controls (P < 0.01). (4) The mRNA levels of IL-10 and IFN-alpha were significantly elevated in acute phase of ITP. (5) Relative MFI of acute phase ITP patients (67.4 +/- 28.1) was higher than that in healthy controls (19.5 +/- 8.5) (P < 0.01), and there was a significant positive correlation between relative MFI and BLyS/April as well as their receptors (BR3, BCMA and TACI) (r = 0.56, 0.53, 0.62, 0.70, 0.45, respectively, P < 0.01), relative MFI in ITP patients decreased after treatment.</p><p><b>CONCLUSION</b>Over-expression of BLyS/April may be one of factors contributed to the immunological dysfunction in ITP.</p>

Change of MyD88-independent signal transduction of Toll-like receptor 4 in immunological pathogenesis of Kawasaki disease / 中华儿科杂志

<p><b>OBJECTIVE</b>Kawasaki disease (KD) is an acute febrile, multi-system endangeitis, which is mainly found in early childhood. Its etiology is still unknown. A great deal of clinical evidence and epidemiologic data suggest that KD is correlated with an acute immune dysfunction caused by infection. Many evidences in the past suggested that over-expression of proinflammatory cytokines, co-stimulatory molecules and chemokines, which were observed in KD, may contribute to the pathologic lesion of vascular endothelial cells. But the causative factors are still unknown. Toll-like receptor is a type I trans-membrane protein which could recognize ligands of pathogenic microbes, induce interferon beta (IFN-beta) and promote gene transcription of proinflammatory cytokines, co-stimulatory molecules and chemokines. This study was designed to investigate the role of MyD88-independent signal transduction of Toll-like receptor 4 in immunological pathogenesis of KD.</p><p><b>METHODS</b>Thirty-two children with KD and 16 age-matched healthy children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the mRNA levels of Toll-like receptor 4 and the molecules such as Toll-IL-1-receptor domain containing adaptor inducing IFN-beta (TRIF), TRIF-related adaptor molecule (TRAM), TANK-binding kinase 1 (TBK-1), IFN-beta, interferon-gamma-inducible protein 10 (IP-10), regulated on activation normal T cells expressed and secreted (RANTES), inducible nitric oxide synthase (iNOS) and suppressor of cytokine signaling 1 (SOCS-1) in monocytes/macrophages (MC), which participate in MyD88-independent signal transduction of toll-like receptors. Expression of costimulatory molecules such as CD40 in MC was analyzed by flow cytometry. Methylation-specific PCR was performed to analyze the methylation status of cytosine-phosphate-guanine (CpG) motif in SOCS-1 gene.</p><p><b>RESULTS</b>(1) Compared with healthy controls, transcription levels of the molecules such as TLR4, TRIF, TRAM, TBK-1 and IFN-beta, were significantly up-regulated during acute phase of KD (P < 0.05), and down-regulated after treatment with intravenous immunoglobulin therapy. (2) Expression of iNOS and chemokines such as IP10 and RANTES in MC during acute phase of KD was remarkably elevated (P < 0.05), and down-regulated to some extents after treatment with intravenous immunoglobulin therapy. (3) Expression of costimulatory molecule CD40 in MC increased significantly during acute phase of KD [(6.19 +/- 2.25)% vs. (2.00 +/- 1.37)%, P < 0.05], while the protein levels of CD40 in KD-coronary artery lesion (CAL)(+) group was found to be significantly higher than that of KD-CAL-group [KD-CAL, (9.63 +/- 2.96)% vs. (4.12 +/- 1.91)%, P < 0.05]. (4) Expression levels of SOCS-1 mRNA were significantly up-regulated during acute phase of KD [(4.31 +/- 0.83) x 10(-3) vs. (1.09 +/- 0.23) x 10(-3), P < 0.05], and the levels of SOCS-1 gene in KD-CAL(+) group was found to be significantly lower than that of KD-CAL(-) group [(5.73 +/- 1.04) x 10(-3) vs (1.94 +/- 0.46) x 10(-3), P < 0.05]. (5) The CpG island of SOCS-1 DNA in KD patients was remarkably demethylated [(26.9 +/- 8.6)% vs (5.9 +/- 1.4)%, P < 0.05], and demethylation levels of SOCS-1 in KD-CAL(-) group were higher than that in KD-CAL+ group [(35.1 +/- 10.3)% vs. (13.2 +/- 3.7)%, P < 0.05].</p><p><b>CONCLUSION</b>Aberrant activation of MyD88-independent pathways of Toll-like receptor 4 may be one of the factors causing disturbed immunological function in KD.</p>

Role of regulatory T cells in pathogenesis of Henoch-Schonlein purpura in children / 中华儿科杂志

<p><b>OBJECTIVE</b>Henoch-Schonlein purpura (HSP) is one of the most common small vessel forms of autoimmune vasculitis in children. Immunologic derangement including humoral and cellular immunity disequilibrium and proinflammatory factors dysfunction are involved in the acute stage of HSP. Recently data revealed that regulatory T cells (Tr) play a pivotal role in preventing development of autoimmune and allergic diseases. This study aimed to explore the role of Tr cells in pathogenesis of HSP and the factors affecting development of Tr cells.</p><p><b>METHODS</b>Twenty patients with HSP and 20 age-matched healthy children were enrolled into this study. Flow cytometric (FCM) analysis was performed to detect the percentage of regulatory T cells subpopulation (including CD(4+)CD(25+) Tr, Tr1 and Th3) and helper T cells subpopulation (Th1 and Th2). Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were used to analyze Foxp3 expression in peripheral blood mononuclear cell (PBMC).</p><p><b>RESULTS</b>Compared with healthy control subjects, the proportions of Th2 cell in patients with HSP were significantly higher (P < 0.05), and the ratio of Th1/Th2 was remarkably decreased (P < 0.05). The proportions of three subpopulation of regulatory T cells including CD(4+)CD(25+) Tr, Tr1, Th3 in patients with HSP were all significantly lower than those of controls (P < 0.05). The mRNA expression of Foxp3 in patients with HSP showed similar tendency (P < 0.001).</p><p><b>CONCLUSIONS</b>The decrease of three subpopulations of regulatory T cells might be involved in pathogenesis of HSP and associated with the decreased expression of Foxp3 gene.</p>

The role of activation of toll-like receptors in immunological pathogenesis of Kawasaki disease / 中华儿科杂志

<p><b>OBJECTIVE</b>A great deal of clinical evidence and epidemiologic data suggest that Kawasaki disease (KD) is correlated with an acute regulating imbalance of immunology. Lots of evidences in the past suggested that nuclear transcription factor-kappaB and preinflammation factors were up-regulated significantly in patients with KD. But the causative factors are still unknown. Toll-like receptors (TLRs) is a type I trans-membrane protein which could recognize ligands of pathogen microbes, activate the nuclear transcription factor-kappaB and promote gene transcription of pre-inflammation factors and co-stimulatory molecules on cell surface. Aberrant activation of signal pathway of TLRs could interfere with autoimmune tolerance and cause autoimmune diseases. The study was designed to investigate the role of signal transduction of TLRs on immunological pathogenesis of KD.</p><p><b>METHODS</b>Sixteen children with KD and 16 age-matched health children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the levels of TLRs 1 - 10, MD-2, MyD88, IL-1beta, IL-6 and IL-8 mRNA expressions in peripheral blood mononuclear cells, and expressions of TLRs 2, 4 and co-stimulatory molecules such as CD80 and CD86 in monocyte/macrophage were analyzed by flow cytometry.</p><p><b>RESULTS</b>(1) Compared with control group, the protein and mRNA levels of TLR4 in KD group were up-regulated significantly [(Real-time PCR: 325.22 +/- 50.34 vs. 2.20 +/- 0.23, P < 0.01); (flow cytometry: 15.96% +/- 5.94% vs. 3.21% +/- 0.62%, P < 0.01)], the difference being not significant as to other TLRs. (2) Transcriptional levels of MD-2 and Myd88 were significantly up-regulated in acute phase of KD (P < 0.01), and down-regulated after the treatment with intravenous gamma globulin therapy. (3) Expressions of co-stimulatory molecules and cytokines in monocyte/macrophage during acute phase of KD were higher than those of control group (P < 0.01).</p><p><b>CONCLUSION</b>Expressions of TLRs 4, MD-2 and Myd88 were up-regulated during acute phase in KD, suggesting that aberrant activation of TLRs 4 might be one of the initiating factors of immune aberrance in KD.</p>

Changes of Signal Transduction of Toll-Like Receptors in Children with Acute Idiopathic Thrombocytopenic Purpura / 实用儿科临床杂志

Objective To investigate the role of signal transduction of toll-like receptors(TLRs)in immunological pathogenesis in children with acute idiopathic thrombocytopenic purpura(ITP).Methods Thirty children with actue ITP and 30 age-matched healthy children were studied.Real-time fluorescent quantitative PCR was used to evaluate the levels of TLR 1-10 and signal transducing molecules,and cytokines associated with TLRs,such as IL-1?,granulocyte-macrophage colony-stimulating factor(GM-CSF),macrophage colony-stimulating factor(M-CSF)and IFN-?/? mRNA.Expressions of co-stimulatory molecules such as CD40,CD80 and CD86 in mo-nocyte/macrophage(MC)was analyzed by flow cytometry.Results Compared with healthy control group,the mRNA levels of TLR3,7,8 and 9 in ITP group were significantly up-regulated(Pa0.05).Transcription le-vels of MyD88-dependent and-independent pathway molecules such as MD-2,MyD88,IRAK-4,TRAF6,TAK1,TRIF,TRAM,TBK-1 and IFN-? were significantly up-regulated in acute ITP(Pa0.05).Conclusion Aberrant activation of toll-like receptors signaling may be one of the initiating factors of immune dysfunction in children with acute ITP.

Screening for Causative Genes Involved in Children with Minimal Change Nephritic Syndrome / 实用儿科临床杂志

Objective To screen for the causative genes involved in the occurrence and development of minimal changes nephritic syndrome(MCNS) and to furtherly assist the genetic diagnosis and treatment of MCNS.Methods Human genome U133 Array Set from Affymetrix Inc was used to evaluate gene expression patterns in peripheral blood mononuclear cells(PBMC) isolated from 7 children with primary MCNS and 7 age-matched health volunteers.Reverse transcription-polymerase chain reaction(RT-PCR) and real-time PCR were performed to identify the findings of gene chip.Results Of 33 000 genes detected,969 genes showed significant difference between children with(MCNS) and healthy volunteers;552 genes were up-regulated,while 417 genes down-regulated significantly.Findings from RT-PCR and real-time PCR were consistent with those of gene chip.Conclusions Gene chip of expression patterns is a powerful method to detect expression difference of genes correlated with MCNS.Occurrence and development of MCNS can be a complicated process that many correlative genes may participate in.