[Quantitative proteomics and bioinformatics analyses of human coronary artery endothelial cell injury induced by Kawasaki disease].

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.

[Effect of Paidu Baoshen Pill on renal fibrosis in 5/6 nephrectomized rats].

OBJECTIVE: To observe the anti-renal fibrosis effect of Paidu Baoshen Pill (PBP) on 5/6 nephrectomized rats and to explore its mechanism. METHODS: 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 ß1 (TGF-ß1), 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-ß1 and FN was detected by immunohistochemical method. mRNA expression levels of TGF-ß1 and FN were detected using real time fluorescent quantitative PCR. RESULTS: 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-ß1, PC III, Col IV, LN, and FN increased; the residual renal pathological lesion was serious; expression levels of TGF-ß1, TGF-ß1, 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-ß1, 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-ß1, TGF-ß1, 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). CONCLUSIONS: PBP had the effect of anti-renal fibro- sis in 5/6 nephrectomized rats. Down-regulating expression levels of TGF-ß1, and FN from gene transcription and protein translation levels might be one of its mechanisms.

[Expression of leukocyte-associated Ig-like receptor-1 in children with immune thrombocytopenia].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[Alterations of CD4+CXCR5+Tfh cells and its transcription regulatory factors in children with asthma].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[Clinical investigation and mutation analysis of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia].

OBJECTIVE: To analyze the clinical features and SLC25A13 gene mutations of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Investigation of the change in CD4⁺ T cell subset in children with Henoch-Schonlein purpura.

Helper T (Th) cells comprising Th1, Th2, Th17, and Treg are involved in the pathogenesis of various vascular inflammations, and information about Th cells in Henoch-Schonlein purpura (HSP) is still controversial. The aim of our study was to investigate the changes in CD4(+) T cell subsets and their roles in the pathogenesis of HSP. Thirty children with diagnosis of HSP and thirty age-matched healthy controls were enrolled in this study. Real-time PCR was used to evaluate the mRNA expression levels of transcriptional factors and cytokines of CD4(+) T cells. Proportions of Th1, Th2, Th17, and Treg cells in peripheral blood were detected by flow cytometry. Plasma cytokine concentrations were measured by ELISA. The proportions of Th2 and Th17 cells increased significantly in children with acute HSP (P < 0.05), while there were no significant differences between HSP and healthy controls regarding the proportions of Treg cells and Th1 cells (P > 0.05). mRNA levels of transcriptional factors and cytokines of Th2 and Th17 cells were significantly up-regulated (P < 0.05), while the differences were not significant as to those of Th1 and Treg cells (P > 0.05). Plasma concentrations of IL-17A, IL-4, and IL-6 in patients with HSP were found to be much higher than those of the control group (P < 0.05), and no differences between IFN-γ, IL-12, and TGF-ß were detected between the two groups (P > 0.05). Presence of higher proportions of Th2 and Th17 cells in patients with HSP could be closely correlated with aberrant creation of antibody and development of vessel vasculitis. The changes in cytokine milieu in peripheral blood may play an important role in the derangement of CD4(+) T cell subset.

[Analysis of clinical features and GCDH gene mutations in four patients with glutaric academia type I].

OBJECTIVE: To review clinical features of four male patients with glutaric academia type I and screen glutaryl-CoA dehydrogenase (GCDH) gene mutations. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Utilization of high-resolution melting analysis to screen patients with neonatal intrahepatic cholestasis caused by citrin deficiency].

OBJECTIVE: To assess the feasibility of high-resolution melting (HRM) analysis for screening patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). METHODS: 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. RESULTS: 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. CONCLUSION: HRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.

[SLC25A13 gene analysis in neonates with intrahepatic cholestasis caused by citrin deficiency].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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 ofï¼»1638ins23+IVS16ins3kbï¼½was 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%). CONCLUSIONS: 851del4 is the major mutation type in Chinese patients with NICCD.

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells.

AIM: Somatostatin receptor subtype 2 (SSTR2) is the principal mediator of somatostatin's (SST) antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved. METHODS: SSTR2 expression levels were determined by qRT-PCR in several tumor cell lines. Then, a plasmid pIRES2-EGFP-SSTR2 (pSIG) was constructed and stably transfected into MCF-7 cells (MCF-7/pSIG). After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor (EGFR) expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT. RESULTS: The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G(1)/S cell cycle arrest. Furthermore, the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSIG cells. CONCLUSION: Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G(1)/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.Acta Pharmacologica Sinica (2009) 30: 1053-1059; doi: 10.1038/aps.2009.59.

[Role of transduce molecules and modulatory factors of signal pathways of Toll-like receptor in inflammatory response of children with sepsis].

OBJECTIVE: To investigate the role of transduce molecules and modulatory factors of signal pathways of Toll-like receptors (TLRs) in aberrant inflammatory response in children with sepsis. METHODS: Peripheral blood mononuclear cell (PBMC) and serum were obtained from 10 children with sepsis, 13 children with severe sepsis and 17 age-matched healthy children as controls. Real-time polymerase chain reaction (real-time PCR) were used to evaluate the mRNA expression levels of TLR2, TLR4, myeloid differentiation primary response gene 88(MyD88), interleukin-1 receptor-associated kinase 4 (IRAK-4), tumor necrosis factor receptor-associated factor 6 (TRAF6), TAK1-binding protein 2 (TAB2) transforming growth factor-beta-activating kinase 1 (TAK1), interleukin-1 receptor-associated kinase 3 (IRAK-M), zinc finger protein inhibiting nuclear factor-KappaB (Triad3A), protein associated with Tlr4 (PRAT4B) and signal-transducing adaptor protein-2 (STAP2), and the levels of mRNA expression and production of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and IL-6] were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with the healthy control group, the levels of gene expression and protein of pro-inflammatory cytokines (TNF-alpha, IL-1 beta and IL-6), the levels of gene expression of signal pathway molecules of TLRs (TLR2, TLR4, MyD88, TRAF6, IRAK4, TAK1 and TAB2) and the levels of gene expression of positive modulators of TLRs pathways (PRAT4B and STAP2) were significantly increased in sepsis group and severe sepsis group (all P < 0.01), and the levels in severe sepsis group were even higher than those in sepsis group (all P < 0.01). The mRNA expression levels of negative modulators of TLRs pathways (IRAK-M and Triad3A) were significantly elevated in sepsis group compared with those in healthy control group (all P < 0.01), but the mRNA expression levels of the negative modulators in severe sepsis group were significantly lowered compared with those in sepsis group (all P < 0.01). CONCLUSION: Aberrant expression of signal pathway molecules and the modulators in TLRs signaling might play an important role on production and development of abnormal inflammatory response in children with sepsis.

Preliminary investigation on the abnormal mechanism of CD4+FOXP3+CD25high regulatory T cells in pediatric B-cell acute lymphoblastic leukemia.

The current study aimed to investigate the changes and regulatory mechanism of cluster of differentiation (CD)4+CD25high forkhead box protein 3 (Foxp3+) regulatory T cells (Tregs) in childhood B-cell acute lymphocytic leukemia (B-ALL). A total of 18 children with B-ALL and 15 age-matched healthy children were included. Reverse-transcription quantitative polymerase chain reaction was used to evaluate the mRNA levels of Foxp3, cytotoxic T-lymphocyte associated protein 4 (CTLA4), glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte activation gene 3 (LAG3), interleukin (IL)-2 receptor (R)ß/γ, IL-6Rα/ß, mothers against decapentaplegic homolog (Smad)3/4 and runt-related transcription factor (RUNX)1/3 in CD4-positive cells. The concentration of cytokines in plasma were measured using a cytometric bead array. Additionally, the proportion of CD4+CD25highFoxp3+ Tregs and levels of associated proteins was analyzed using flow cytometry. The results demonstrated that the proportion of CD4+CD25highFoxp3+ and expression of Foxp3 in children with B-ALL was significantly higher compared with healthy controls (P<0.05) and that transcription levels of CTLA4, GITR and LAG3 were also significantly elevated (P<0.05). Compared with healthy controls, the expression of IL-2Rα/ß and its downstream molecule phosphorylated signal transducer and activator of transcription 5 (pSTAT5) in CD4-positive cells significantly increased (P<0.05); however, no significant difference of IL-2Rγ levels was identified between the two groups. Correlation analysis demonstrated a significant positive correlation between the expression of phosphorylated (p) signal transducer and activator of transcription factor (STAT)5 and CD4+CD25highFoxp3+ Tregs in children with B-ALL (r=0.17; P<0.05). The plasma concentration of TGF-ß, the expression of its receptor TGF-ßRI/II and downstream molecules Smad3/4 were significantly upregulated in children with B-ALL (P<0.05), whereas the expression of RUNX1/3 was lower compared with healthy controls (P<0.05). Furthermore, the expression of Smad3 and RUNX1 was positively correlated with CD4+CD25highFoxp3+ Tregs in children with B-ALL (r=0.87 and 0.60, respectively; P<0.05). Additionally, the expression of pSTAT3 in CD4-positive cells decreased significantly in pediatric patients with B-ALL when compared with healthy controls; however, plasma concentrations of IL-6 was significantly higher (P<0.05). Furthermore, a negative correlation was identified between pSTAT3 and CD4+CD25highFoxp3+ Tregs in pediatric patients with B-ALL (r=-0.39; P<0.05). However, no significant differences in IL-6Rα/ß expression were identified between the two groups. The results demonstrated that the excessive activation of IL-2/pSTAT5 and TGF-ß/Smad signaling, and insufficiency of pSTAT3 may be correlated with increased CD4+CD25highFoxp3+ Tregs in pediatric B-ALL.

The effects of ketogenic diet on the Th17/Treg cells imbalance in patients with intractable childhood epilepsy.

PURPOSE: The ketogenic diet (KD) is an effective treatment for intractable epilepsy (IE), however the therapeutic mechanism is still unclear. This study was designed to investigate T helper type 17/regulatory T cell (Th17/Treg) levels in children with IE and age-matched healthy controls following KD. METHOD: Circulating levels of Th17/Treg cells were analyzed by flow cytometry. Plasma concentration of interleukin (IL)-17 was measured by cytometric bead array assay. Real-time PCR was performed to measure mRNA levels of mTOR, HIF1α and Th17/Treg associated factors in purified CD4(+)CD25(+) T and CD4(+)CD25(-) T cells. RESULTS: By one-way ANOVA, the proportion of circulating Th17 cells and expression of IL-17A and RORγt were significantly higher (P<.05), while the proportion of circulating Tregs and expression of Foxp3, GITR, CTLA-4 were significantly lower (P<.05) in IE patients than healthy subjects. However, these alternations were reversed following KD (P<.05). In CD4(+)CD25(+) T and CD4(+)CD25(-) T cells mTOR and HIF1α expression were significantly higher in IE patients (P<.05), however KD reduced mTOR and HIF1α expression (P<.05). The plasma IL-17A concentrations were higher in IE patients than controls (P<.05). KD partially reduced IL-17A levels (P<.05). CONCLUSION: Our results suggest that Th17/Treg imbalance is characteristic of childhood IE, and may contribute to IE pathogenesis. KD treatment is able to correct this imbalance, probably via inhabiting the mTOR/HIF-1α signaling pathway.

Preliminary study of a new pathological evolution-based clinical hepatolithiasis classification.

AIM: To investigate clinical features, treatment strategies and outcomes of patients with hepatolithiasis (HL) undergoing surgical treatment, using a new clinical classification. METHODS: Sixty-eight HL patients were hospitalized and treated surgically from August 2011 to December 2012 and they were classified into four HL types according to pathological evolution of the disease. These four HL types included type I primary type (defined as no previous biliary tract surgery), type II inflammatory type (with previous biliary tract surgery and cholangitis), type III mass-forming type (HL complicated by hepatic mass-forming lesion), and type IV terminal type (with secondary biliary cirrhosis and resultant portal hypertension). The perioperative data including general information, imaging data, postoperative complications, and immediate and final stone clearance rate were obtained and analyzed. RESULTS: In all 68 patients, the proportion of HL type I-IV was 50% (34/68), 36.8% (25/68), 10.3% (7/68) and 2.8% (2/68), respectively. Abdominal pain was the main clinical manifestation in type I (88.2%), fever was predominant in type II (52.0%), the malignancy rate in type III was high (71.4%), and portal hypertension and spleen enlargement were common in type IV (2/2, 100.0%). Liver resection rate for types I-III was 79.4%, 72.0% and 71.4%, respectively. The overall incidence of postoperative complications was 23.5% (16/68). There were no perioperative deaths. The average length of hospital stay was 12.7±7.3 d. Immediate and final stone clearance rate was 73.5% (50/68) and 89.7% (61/68), respectively. Fifty-nine of 68 patients (86.8%) were followed- up for >1 year after surgery, and 96.6% of these patients (57/59) had a good quality of life according to a criterion recommended for postoperative evaluation of quality of life. CONCLUSION: The pathological evolution-based clinical classification of HL has a role in optimizing treatment strategy, and patients can benefit from this classification when it is used properly.

Effects of carbon dioxide and nitrogen on adhesive growth and expressions of E-cadherin and VEGF of human colon cancer cell CCL-228.

AIM: To study the effects of carbon dioxide on the metastatic capability of cancer cells, and to compare them with that of nitrogen. METHODS: The colon cancer cell CCL-228 was treated with 100 % carbon dioxide or nitrogen at different time points and then cultured under normal condition. Twelve hours after the treatment, the survival rates of suspension cells and the expressions of e-cadherin and VEGF were examined. RESULTS: After 60 min of carbon dioxide and longer time of nitrogen treatment, the suspended cells increased and the expression of e-cadherin decreased while the expression of VEGF was enhanced significantly. And the effects of nitrogen were similar to, but weaker than, those of carbon dioxide. CONCLUSION: Carbon dioxide may improve the metastatic capability of cancer cells and its effects are significantly stronger than that of nitrogen. A sequential use of carbon dioxide and nitrogen in pneumoperitoneum may take the advantage of both gases.

[Variations of the zinc finger protein 161 gene in Chinese with or without high myopia].

To investigate the association between variations of ZFP161 gene and high myopia, A total of 204 probands with simple high myopia(< or = -6.0 dipoters) were collected while 116 normal persons from different families without high myopia or related disease were used as controls. Genomic DNA was prepared from the peripheral leucocytes. The coding sequences of ZFP161 gene in 320 subjects were analyzed by using exon-by-exon PCR-heteroduplex-SSCP analysis. Identification of the Variations by cloning and sequencing, combinated with controls and family analysis, was used to disclose the correlation between ZFP161 gene and high myopia. A mutation of ZFP161 gene was identified as an insertion of AT before the 58th nucleotide of intron 1 (IVS1 58-59)(1/204) and a variation of ZFP161 gene was identified as a heterozygous C to A of the 168th nucleotide in exon 2 (Codon56, GCC-->GCA, Ala56Ala). Ala56Ala is a non-sense mutation identified in 5 of the 204 patients and 3 of 116 controls. No evidence shows that these variations are responsible for high myopia.

[Antisense hTERT inhibits gene expression and functional activity of telomerase in leukemia cell lines].

OBJECTIVE: To explore the inhibitory effects of gene expression and functional activity of telomerase in leukemia cell lines by in vitro antisense hTERT treatment. METHODS: An antisense hTERT eukaryotic expression vector was constructed by using gene recombination technique, targeting the 5' end mRNA sequence of the telomerase catalytic subunit. The vector expression in leukemia cell lines (HL60 and K562) was achieved by transfection using the SuperFect transfection reagent (Qiagen). After transfection, ectopic expression of the telomerase catalytic subunit was analyzed by quantitative fluorescence real-time RT-PCR, and cellular apoptosis and cell cycle parameters were evaluated by flow cytometry respectively. RESULTS: An antisense pcDNA-hTERT eukaryotic expression vector was successfully constructed. Leukemia cell lines transfected with antisense hTERT constructed displayed a significant inhibition of gene expression of telomerase and its activity in vitro, as compared with the result of the control groups (without transfection and vector control). CONCLUSION: In-vitro antisense hTERT expression may down-regulate the gene expression and biological activity of telomerase in leukemia cells, suggesting a possibility of gene therapy against human malignancy through the telomerase-targeted molecular mechanism.

[Antisense human telomerase reverse transcriptase inhibits leukemia cell proliferation in vitro].

OBJECTIVE: To study the inhibitory effect of antisense human telomerase reverse transcriptase (hTERT) on leukemia cell proliferation in vitro. METHODS: Sense and antisense hTERT eukaryotic expression vectors previously constructed were transfected into leukemia cell line HL(60) using SuperFect transfection reagent (Qiagen) to obtain HL(60)-s and HL(60)-as, and the G418-resistant colonies were identified for the presence of hTERT insert by PCR with T7 and pcDNA3.1/BGH reverse primers. Endogenous hTERT mRNA expression and telomerase activity were then detected by quantitative real-time RT-PCR and telomerase associated protein -silver staining in each cell line. MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were also employed to analyze the changes in proliferation capacity of leukemia cell in vitro and apoptosis of the tumor cells induced by antisense hTERT. RESULTS: Antisense hTERT remarkably reduced endogenous hTERT mRNA expression (P<0.01) and down-regulated telomerase activity in HL(60), as compared with the blank control and sense hTERT. After 25 passages of the 3 cell lines, a 7-day cell growth curve and the numbers (size) of soft agar colony formation showed that the proliferation rates and the anchorage-independent growth ability of HL(60)-as cells were significantly decreased in comparison with HL(60) and HL(60)-s cells, but a significant increase in apoptosis of HL(60)-as cells occurred as determined by flow cytometry. CONCLUSIONS: Antisense hTERT can obviously inhibit leukemia cell growth and proliferation in vitro, and this telomerase-targeted molecular biotherapy may be achieved by apoptosis pathway through down-regulation of hTERT mRNA and telomerase activity.

[Effect of Astragalus membranaceus on baroreflex sensitivity in spontaneously hypertensive rats].

OBJECTIVE: To observe the chronicity decompression effect of Astragalus Membranaceus(AM) and evaluate the effect on baroreflex sensitivity (BRS). METHOD: Nineteen spontaneously hypertensive rats(SHR) were randomly divided into four groups. The AM groups were intraperitoneally administered with AM parenteral solution 0.9 mL, 1.2 mL and 1.8 mL respectively and the control group was not given AM for eight weeks. Then the change of blood pressure was observed successivly. After eight weeks, BRS were also determined. At last, the difference of blood pressure and BRS among the groups were compared. RESULT: Blood pressure in the control group became higher and higher frome the third week to the eighth week, but the other SHR admistered with AM showed no changein blood pressure level. We also found that the BRS in AM group was higher than that in the control group(P < 0.01). CONCLUSION: AM can promote the BRS in SHR.

Berberine protects endothelial progenitor cell from damage of TNF-α via the PI3K/AKT/eNOS signaling pathway.

Endothelial progenitor cells (EPCs) dysfunction is closely correlated with the coronary artery injury induced by Kawasaki disease (KD). The level of tumor necrosis factor-α (TNF-α) elevated significantly in acute phase of KD which can damage the functions of EPCs. The aim of this study was to investigate whether berberine (BBR) can protect EPCs from the inhibition caused by TNF-α via the PI3K (Phosphatidyl Inositol 3-kinase) /AKT (Serine/threonine protein kinase B) /eNOS (endothelial Nitric Oxide synthase) signaling pathway. The cell proliferative ability of EPCs was determined by MTT (methyl thiazolyl tetrazolium) assays. Nitric oxide (NO) level was determined in supernatants. The mRNA level of eNOS, PI3K and AKT were measured by Real Time-Polymerase Chain Reaction (RT-PCR), and the protein levels of eNOS, phospho-eNOS (p-eNOS), Akt, phospho-Akt (p-Akt) and PI3K were analyzed using Western-blot. The results demonstrated that TNF-α inhibits the proliferative ability of EPCs. However, BBR improves the proliferative activity of EPCs inhibited by TNF-α. Blockade of PI3K by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) and blockade of eNOS by l-NAME (NG-Nitroarginine Methyl Ester) attenuates the effect of BBR. BBR can increase the level of PI3K/Akt/eNOS mRNA and the protein level of PI3K, p-Akt, eNOS and p-eNOS, which can be blocked by PI3K inhibitor (LY294002) and eNOS inhibitor (l-NAME). Therefore, we concluded that impaired EPCs proliferation could be reversed by BBR via the PI3K/AKT/eNOS signaling pathway.