Objective To investigate the methylation status of Foxp3 gene and its roles in immunological pathogenesis of Kawasaki disease(KD). Methods Thirty children with KD and eighteen agematched healthy children consented to participate in this study. Quantitative methylation specific polymerase chain reaction(MSQP) was used to assess the methylation status of Foxp3 promoter and regulatory T cells specific demethylated region(TSDR) in CD4+ T cells. The proportion of CD4+ CD25 + Foxp3 + regulatory T (Tr) cells was analyzed by flow cytometry. Transcriptional levels of CD4+ CD25 + Foxp3 + Tr associating genes (Foxp3, CTLA4, GITR, LAG3 and CCR8 ) and Foxp3-dependent molecules (UBD and LGAIS3)were measured by real-time PCR. Results ( 1 ) Demethylation level of Foxp3 promoter in CD4 + T cells from patients with KD was lower significantly than that of health subjects( P ＜ 0.01 ), and increased significantly after treated with intravenous gamma globulin therapy(IVIG) (P ＜ 0.01 ). No difference of demethylation level of TSDR region was observed among all groups ( P ＞ 0. 05 ). ( 2 ) The proportion of CD4 + CD25 + Foxp3 + Tr in peripheral blood from patients with KD , as well as mRNA levels of Foxp3 gene in CD4+ T cells, was significantly lower than those of health subjects ( P ＜0. 01 ), and increases significantly after IVIG therapy (P ＜0.01 ). Significant positive correlations between demethylation level of Foxp3 promoter and the proportion of CD4 + CD25 + Foxp3 + Tr , or expression levels of Foxp3 in CD4 + T cells, were observed during acute phase of KD (CD4+CD25+ Foxp3+ Tr: r=0.76, P＜0. 01; Foxp3: r=0.89, P＜0. 01). (3)Transcription level of CD4+ CD25+ Foxp3+ Tr associating factors, such as CTLA4, GITR, LAG3 and CCR8, was significantly down-regulated in acute phase of KD(P＜0. 01 ), and up-regulated to some extent after treated with IVIG(P ＜0. 01 ). Expression levels of Foxp3-dependent molecules UBD and LGALS3 in CD4 + T cells decreased significantly during acute phase of KD (P ＜ 0.01 ), and basically recovered to the levels of health subjects( P ＜ 0.01 ). Conclusion Decrease of demethylation level of Fopx3 promoter is correlated with immune dysfunction in Kawasaki disease.

Objective To investigate the effect of SOCS1 and SOCS3 hypomethylation on homeostasis of Th1/Th2 in Kawasaki disease(KD). Methods Thirty-six children with KD and sixteen age-matched healthy children consented to participate in this study. Protein concentration of IL-6 in plasma was measured by ELISA. Transcriptional levels of SOCS1, SOCS3, T-bet, IFN-γ, GATA3 and IL-4 were assessed by realtime PCR. The proportion of Th1 and Th2 cells, and mean fluorescence intensity(MFI)for phosphorylated STAT3(pSTAT3)protein in CD4+ T cells was analyzed by flow cytometry. A quantitative methylation specific PCR based on SYBR Green was used to evaluate methylation status of CpG islands in SOCSl exon2, and three potential binding sites for STAT3 in 5'-untraslated region(5'-UTR)of SOCS3 in CD4+T cells. Comparisons between groups were performed with t-test. Results ①Compared with healthy volunteers, plasma IL-6 concentration[(51.8±16.3)pg/ml vs(8.6±2.0)pg/ml, respectively]and MFI for pSTAT3[(52±14)vs(10±4), respectively]in CD4+ T cells were elevated significantly during acute phase of KD(P＜0.05), and the two items in KD patients with coronary artery lesion(KD-CAL+)were found to be higher than those in KD patients without coronary artery lesion(KD-CAL-)[IL-6:(87.2±27.4)pg/ml vs(36.2±12.8)pg/ml, P＜0.05; pSTAT3 MFI:(75±15)vs(42±11), P＜0.05]. ② The proportions of Th1 and Th2 cells and transcription levels of Th-associating factors(T-bet, IFN-γ, GATA3 and IL-4)in CD4+ T cells increased significantly in acute KD(P＜0.05), while the rate of Thl div Th2 in KD patients was found to be lower than that in normal controls(P＜0.05). In addition, the proportions of Th1 and Th2 cells and expressions levels of Th-associating factors in KD-CAL+ group were higher than those in KD-CAL-group, as well as the rate of Thl div Th2 cells in KD -CAL+ group were lower than that in KD-CAL- group(P＜0.05). ③ The mRNA levels of SOCSl and SOCS3 in CD4+ T cells increased significantly during acute phase of KD(P＜0.05), while the two items in KDCAL+ group were lower than those in KD-CAL- group(P＜0.05). Furthermore, CpG islands in SOCSl exon2 and the third potential binding site for STAT3 in SOCS3 5'-UTR were hypomethylated in acute KD, while those in healthy volunteers were fully demethylated(P＜0.05). Demethylation levels of the two items mentioned above in the KD-CAL+ group were lower than those in the KD-CAL-group(P＜0.05). CpG islands in the other two binding sites for STAT3 in SOCS3 5'-UTR were fully demethylated among all the groups(P＞0.05).Conclusion Relative insufficiency of SOCS1 and SOCS3 expression caused by hypomethylation may be one contributing factor for the imbalance of Th1/Th2 in KD.

ObjectiveTo investigate the role of signal transduction of TLRs in the Henoch-Schonlein purpura (HSP). Methods Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the levels of TLRs(1～10), MyD88, TRAF6, TRIF, IFN-α, IFN-β, IL-6, IL-1β, TNF-α, IP-10, RANTES,iNOS, Blys/April mRNA expression in peripheral blood mononuclear cells and their expression levels were compared using t test., while the concentration of plasma cytokines such as Blys、IFN-α、IFN-β、IL-6、IL-1、TNF-α was measured by enzyme-linked immunosorbent assay(ELISA).Expression levels of those genes were compared using t test. Results①Compared with the control group, the expression levels of TLR1, TLR2,TLR6, TLR5, TLR3, TLR7, TLR9 mRNA were up-regulated significantly(P＜0.01), while no difference of TLR4 was detected (P＞0.05).②Transcription levels of MyDg8(2.47±1.06) vs(0.73±0.22), TRAF6 (2.54±0.72)×10-3vs(0.70±0.20)×10-3, TRIF(3.18±0.86)×10-3vs(0.93±0.35)×10-3 were significantly up-regulated in acute phase of HSP (P＜0.01).③The levels of IFN-α and IFN-β protein and mRNA were remarkable increased (P＜0.01).④ The expression of cytokine/chemotactic factor such as IL-6, IL-1β, IP-10, RANTES,iNOS was higher than that of the control group(p＜0.01), while TNF-αdid not change in children with HSP (P＞0.05). ⑤ It was detected that the expression of Blys/April was higher than that of the control group(P＜0.01). ConclusionExpressions of TLR1, TLR2, TLR6, TIR5, TLR3, TLR7, TLR9, MyD88, TRAF6, and TRIF are up-regulated during acute phase of HSP, suggesting that aberrant activation of TLRs triggered by microbes may be one of the initiating factors of immune aberrance in HSP. Over expression of cytokine/chemotactic factor or Blys/April owning to the aberrant activation of TLRs, may be correlated with immunological pathogenesis of HSP.

Objective To investigate the role of IL-6/STAT3 signaling in Th17/Tr imbalance of Kawasaki disease(KD). Methods Forty-eight children with KD and eighteen age-matched healthy children were consented to participate in this study. Protein concentration of IL-6 in plasma was measured by ELISA. Transcriptional levels of IL-17A, IL-17F, RORγt, Foxp3, SOCS1 and SOCS3 were assessed by real-time PCR. The proportion of CD4+CD25+Foxp3+ regulatory T(Tr) cells and mean fluorescence intensity(MFI) for phosphorylated-STAT3(pSTAT3) protein in CD4+ T cells was analyzed by flow cytometry. A quantitative methylation specific PCR based on SYBR Green was used to evaluate methylation status of CpG islands in SOCS1 exon2, three potential bind sites for STAT3 in 5'-untraslated region(5'-UTR) of SOCS3 in CD4+ T cells. Results (1)Compared with healthy volunteers, plasma IL-6 concentration and MFI for pSTAT3 in CD4+ T cells were elevated significantly during acute phase of KD[IL-6:(54.02±20.58) pg/ml vs (8.72±2.06) pg/ml, P<0.05;pSTAT3 MFI:(55.41±15.08) vs (9.35±3.76), P<0.05], and the two items in KD patients with coronary artery lesion (KD-CAL+) were found to be higher than those in KD patients without coronary artery lesion (KD-CAL-)[IL-6:(84.76±29.35) pg/ml vs (38.65±13.76) pg/ml, P<0.05;pSTAT3 MFI:(72.36±16.81) vs (46.93±13.57), P<0.05]. (2)Transcription levels of IL-17A, IL-17F and RORγt in patients with KD were significantly elevated (P<0.05) while the proportion of CD4+CD25+Foxp3+ Treg and expression levels of Foxp3 were detected to be lower than those in normal controls (P<0.05). The mRNA levels of IL-17A, IL-17F and RORγt in KD-CAL+ group were higher than those in KD-CAL- group(P<0.05), as well as expression level of Foxp3 were found to be lower in KD-CAL+ group(P<0.05). (3)The mRNA levels of SOCS1 and SOCS3 in CD4+ T cells increased significantly during acute phase of KD(P<0.05), while the two items in KD-CAL+ group were lower than those in KD-CAL- group(P<0.05). Furthermore, CpG islands in SOCS1 exon2 and the third potential bind site for STAT3 in SOCS3 5'-UTR were hypomethylated in acute KD, while those in healthy controls were fully demethylated(P<0.05). Demethylation levels of SOCS1 exon2 and the third potential bind site for STAT3 in SOCS3 5'-UTR in KD-CAL+ group were lower than those in KD-CAL- group(P<0.05). CpG islands in the other two bind sites for STAT3 in SOCS3 5'-UTR were fully demethylated among all the groups(P>0.05). ConclusionAberrant activation of IL-6/STAT3 signaling caused by hypomethylation of SOCS1 and SOCS3 might be one contributing factor to unbalance of Th17/Tr in KD.

Objective To further explore the pathogenesis of disturbed adaptive immune response in infants with sepsis. Methods Forty-eight infants with sepsis and 26 age-matched healthy infants were enrolled in this study. The HLA-DR expression of CD14~+ monocyte, the proportion of CD4~+ CD25~+ Foxp3~(high) Tr cells and the proportion of Thl7 cells were measured by flow cytometry. Cytokines (IL-1β, IL-6, TNF-α, IL-10, TGF-β and IL-17A) were measured by ELISA. Real-time PCR were used to evaluate the mRNA levels of Foxp3, ROR-γt in CD4-positive cells and IL-17A. Forty-eight infants with sepsis were divided into two groups according to HLA-DR expression of CD14~+ monocyte: DR-H group ( ＞ 30% ) and DR-L group ( ＜ 30% ). Results The ratio of IL-10/TNF-α in DR-L group was higher than that in healthy control or DR-H group(P ＜0.05). The proportion of CD4~+ CD25~+ Foxp3~(high) Tr cells and mRNA expression of transcription factor Foxp3 in DR-L group was found to be significantly higher than that in healthy control or DR-H group(P＜0.05). The proportion of Thl7 cells, Serum concentration of IL-17A, the mRNA expression of IL-17A and transcription factor ROR-γt were significantly increased in DR-H group and DR-L group (P ＜ 0.05) , while there is no significant difference between DR-H and DR-L group( P ＞0.05). Serum levels of Th17-inducing cytokine such as IL-1β, IL-6 were significantly elevated in DR-H group and DR-L group (P＜0.05), while there is no significant difference between DR-H and DR-L group( P＞0.05). Serum level of CD4~+ CD25~+ Foxp3~(high) Tr-inducing cytokine TGF-p in DR-L group was higher than that in DR-H or healthy control group(P＜0. 05). Conclusion Over-activation of Th17 cells may be one of the factors causing aberrant increase of pro-inflammatory cytokine/chemotatic factor in infant with sepsis. The imbalance of CD4~+ CD25~+ Foxp3~(high) Tr cells/Th17 cells may be contributed to the pathogenic mechanism of mixed antagonist response syndrome ( MARS) in infant with sepsis. The changes of cytokine environment in infants with sepsis may be one of the factors causing the imbalance of CD4~+ CD25~+ Foxp3~(high) Tr cells/Th17 cells.

Objective To investigate the changes of CD4~+ T cell subset in the role of immuno-pathogenesis of type 1 diabetes mellitus(T1DM). Methods Real-time PCR was used to evaluate the ex-pression levels of transcriptional factors (T-bet, GATA-3, Foxp3, ROR-γt), cytokines (IFN-γ, IL-4, IL-10, IL-17A) and negative regulatory factors (CTLA-4, GITR) mRNA from CD4~+ T cells. The proportions of Th1, Th2, Tr and Th17 cells in peripheral blood were detected by flow cytometric analysis. Plasma cyto-kine (IFN-γ, IL-4, TGF-β and IL-6) concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Results (1) The proportions of Th1 cells in peripheral blood from children with T1DM were siguificanfly increased than that of healthy controls, and proportions of Th2 were decreased (P < 0.01). There were no significant differences between diabetic patients and healthy controls regarding the proportions of Tr cells and Th17 cells(P >0.05). (2) Transcription levels of T-bet and IFN-γ mRNA were significantly up-regulated, while GATA3 and IL-4 were significantly down-regulated in children with T1DM. The mRNA expression levels of Tr negativity regulatory factors such as IL-10, CTLA-4 and GITR were lower in CD4~+ T cells from children with TIDM compared with the controls(P <0.01). There were no statistically differences to be observed in mRNA expression levels of ROR-γt and IL-17A genes between two groups(P > 0.05).(3) In comparison with controls, serum concentrations of IFN-γ or IL-4 were remarkable increased or de-creased respectively (P < 0. 01), while TGF-β and IL-6 did not change in children with T1DM (P > 0.05). Conclusion The Th1/Th2 imbalance might be play an important role in immunopathogenesis of T1DM. Functional deficiency of Tr cell might further exacerbate Th1/Th2 imbalance and lead to disturbance of cellu-lar immune response.

BACKGROUND: Acute renal insufficiency (ARI) usually occurred following liver transplantation due to the surgical trauma and the application of immunosuppressant, which lack of unified diagnostic criteria. OBJECTIVE: To investigate the experience of diagnosis and treatment of ARI following liver transplantation.DESIGN, TIME AND SETTING: The experiment was performed at the 458 Hospital of Chinese PLA from January 2004 to December 2006.PARTICIPANTS: A total of 37 cases received liver transplantation, including 35 males and 2 females, aged 37-67 years, mean aged (48.5±8.9) years. All cases were divided into the liver cancer group (n=16) and liver cirrhosis group (n=21). The liver cirrhosis group included 16 cases with posthepatitic type B cirrhosis, 4 with posthepatitic type C cirrhosis, and 1 with alcoholic cirrhosis. All these cases were in decompensation stage. The final diagnosis was performed by pathological examination. METHODS: The removal of kidney and construction of blood outflow tract was achieved by modified piggy-back liver transplantation. The arterial blood gas analysis, blood routine examination, renal function and liver function were examined more than twice per day. The cephalosporins, Fluconazole and ganciclovir or vancomycin were used for 5-7 days to prevent infections.MAIN OUTCOME MEASURES: The incidence rate of acute ARI, clinical features and outcomes of patients were observed.RESULTS: ARI developed in 19 patients with liver transplantation, 5 patients died, 14 patients recovered in 2-3 weeks. The incidence of ARI following liver transplantation was associated with infection, bleeding shock, respiratory failure and acute respiratory distress syndrome (P < 0.05). CONCLUSION: The incidence of ARI following liver transplantation was 51.35%, with 26.32% mortality rate. The early diagnosis and treatment are the key steps for increasing successful rate of ARI treatment following liver transplantation.

OBJECTIVESepsis is the major cause of death in pediatric intensive care unit (PICU). The clinical manifestations of early sepsis is very similar to systemic inflammatory response syndrome (SIRS) caused by non-infectious reason. This study aimed to investigate the expression of miRNA and inflammatory cytokines in plasma in pediatric sepsis patients and its clinical significance.

METHODForty children with sepsis seen in Shenzhen children's hospital PICU from April 2012 to March 2013 were enrolled in this study, the median age was 0.75 (0.52, 1.90) years; 27 were males and 13 females, of whom 16 had severe sepsis. We selected 20 postsurgical patients with SIRS and 15 healthy children as a control group. The expression levels of plasma miR-21, miR-125b, miR-132, miR-146a, miR-155 and miR-223 were detected by real-time quantitative PCR (qRT-PCR). The predictive value of miRNA, PCT and CRP for sepsis were evaluated by Receiver operating characteristic curve (ROC). TNF-α and IL-10 levels in plasma detected by Cytometric Beads Array (CBA). Quantitative data of normal distribution was compared with ANOVA among the three groups and LSD-t test between two groups. To non-normal distribution of data, multiple comparisons among three groups were conducted by Kruskal-Wallis H test and differences between two groups were assessed by Mann-Whitney U test for post hoc analysis.

RESULTThere were no significant differences between the age and gender of each group. Expression of miR-21, miR-125b, miR-132 and miR-155 in plasma had no significant difference in each group (all P > 0.05). MiR-146a and miR-223 levels in sepsis were upregulated compared with SIRS group and control group [(5.7 ± 3.5)×10(-5) vs. (2.4 ± 1.6)×10(-5) and (2.6 ± 1.2)×10(-5), (12.5 ± 7.7)×10(-4) vs. (8.3 ± 3.4)×10(-4) and (5.3 ± 2.2)×10(-4), all P < 0.01], expression levels of miR-223 in SIRS increased as compared to control group (P < 0.01). MiR-146a levels in severe sepsis were higher than those of the general sepsis [ (7.1 ± 3.3)×10(-5) vs. (4.6 ± 2.6)×10(-5), P < 0.01]. CRP and PCT levels are all higher in sepsis and SIRS groups than control group (all P < 0.01). The area under ROC curve (AUC) of miR-146a, miR-223, PCT and CRP to predict sepsis were 0.815 (95%CI: 0.708-0.922), 0.678(95%CI: 0.537-0.818), 0.706 (95%CI: 0.571-0.842) and 0.588 (95%CI: 0.427-0.748). Expression levels of IL-10 and IL-10/TNF-α in sepsis were upregulated compared with and SIRS group and the control group (all P < 0.01). There was a positive correlation between miR-146a, miR-223 and IL-10 and IL-10/TNF-α (r = 0.545, 0.305, 0.562, 0.373, all P < 0.01).

CONCLUSIONThe expression levels of miR-146a and miR-223 in plasma in pediatric patients with sepsis was significantly upregulated, and had a positive correlation with IL-10 and IL-10/TNF-α, which may be used as early diagnostic markers and can reflect the severity of condition to a certain degree.

Biomarkers ; blood ; Calcitonin ; blood ; Case-Control Studies ; Female ; Humans ; Infant ; Infant, Newborn ; Intensive Care Units, Pediatric ; Interleukin-10 ; blood ; Male ; MicroRNAs ; blood ; Prognosis ; ROC Curve ; Sepsis ; blood ; diagnosis ; Severity of Illness Index ; Systemic Inflammatory Response Syndrome ; blood ; diagnosis ; Tumor Necrosis Factor-alpha ; blood