Objective To discuss application value for detecting sentinel lymphatic node(SLN) in patients with breast mass by subcutaneous injection SonoVue. Methods Forty-two cases with breast masses who were detected by conventional techniques were injected subcutaneously with 1.25 ml Sono Vue to the borderline of breast tumor at each 3,6,9, 12 points. The changes of contrast-enhanced ultrasound (CEUS) of the ipsilateral axillary SLN were observed and the start time of enhancement of lymphatic channel and lymph nodes, the position and numbers of enhancement of the lymph nodes, the time of regression were recorded. Compared with Methylene blue and surgicalbiopsy, the accuracy of detection of SLN by CEUS were evaluated. Results SLN were successfully detected in 30 of 42 patients, the detection rate was 71.4%. The total number of detected SLN was 40.Five nodes were detected in 5 of 10 (50%)patients with benign masses. Thirty-five nodes were detected in 25 of 32 (78.1%) patients with breast cancer. Fifty-seven SLN were successfully detected in 39 of 42 patients with Methylene blue, the accurate rate was 92.9%. Seven of 10 patients with benign masses and all 32 patients with breast cancer were Methylene blue positive. There were total 64 SLN found, including 11 with benign masses and 53 with lymph nodes metastasis of breast cancer cells. Conclusions The subcutaneous injection SonoVue is effective to detect SLN in patients with breast mass and apply clinical value for forecasting whether the lymph node metastases in patients with breast cancer.

Objective To investigate the role of T follicular helper ( Tfh) cells in the immuno-pathogenesis of persistent immune thrombocytopenic purpura ( pITP) .Methods Twenty children with pITP and twenty healthy controls were enrolled in this study .The proportion of CD4+CXCR5+ICOShigh PD-1high T ( cTfh) cells and the expression of ICOSL on CD 19+B cells in peripheral blood of the patients and healthy subjects were analyzed by flow cytometry .The expressions of Bcl-6, c-Maf, IL-21 and ICOSL at mRNA level were detected by real-time PCR.The plasma concentrations of IL-2, IL-6 and IL-21 were determined by ELISA.Results (1) Compared with the healthy controls , the proportions of cTfh cells increased signifi-cantly in patients with pITP [(17.45±9.04) %vs.(7.57±2.57) %, P<0.05], but decreased with the treatment of dexamethasone (DEX) for 7 days [(5.93±1.64) %vs.(17.45±9.04) %, P<0.05].(2) The expression of Bcl-6, c-Maf and IL-21 at mRNA level in patients with pITP were higher than those in healthy controls (P<0.05).(3) Compared with healthy controls, the expression of ICOSL at mRNA and protein levels in CD19+B cells were significantly up-regulated in patients with pITP (P<0.05), which showed no significant changes after treatment with DEX (P>0.05).(4) The plasma concentration of IL-21 was remarkably elevated in patients with pITP , regardless of DEX treatment (P<0.05).But the plasma concentrations of IL-2 and IL-6 showed no significant changes compared with control group (P>0.05).Con-clusion The immunopathogenesis of persistent immune thrombocytopenic purpura might be associated with the hyper-activation of Tfh cells caused by excessive expression of ICOSL and IL-21.The persistent high expression of ICOSL and IL-21 might be one of the important factors resulting in the recurrence of pITP in children .

Objective To investigate the effects of the intravenous immunoglobulin (IVIG) on the expression of FcγR Ⅱ b and Toll-like receptor 4 (TLR4) in children with Kawasaki disease (KD).Methods 25 children with KD and 15 healthy controls were enrolled in this study.The levels of TLR4 expression on monocytes were assessed by flow cytometry.Real-time PCR was performed to detect the transcription levels of FcγR Ⅱ b,TLR4 signaling molecules(MyD88,TRAF-6,TAK1) and cytokines(IL-1β,IL-6,TNF-α) in monocytes.The concentrations of TNF-α in plasma was determined by enzyme-linked immunosorbent assay (ELISA).Results (1) Compared with healthy controls,the expression of TLR4 and the signaling molecules in the patients were significantly up-regulated but decreased after treatment with IVIG (P＜0.05).(2)The levels of FcγR Ⅱb expression from the patients were higher than those from healthy controls,and decreased after treatment with IVIG(P＜0.05).(3) The levels of cytokine factor expression such as IL-1β,IL-6 and TNF-α in the patients were found to be higher than those in healthy controls (P＜0.05) ; plasma concentrations of TNF-α were significantly elevated during acute KD (P＜0.05).(4)There were significantly correlations to follow between FcγR Ⅱb and the levels of cytokine,TLR4 expression in monocytes (P ＜0.05).Conclusion IVIG can up regulate FcγR Ⅱb expression and down regulate TLR4 expression which might inhibit the inflammatory response.

Objective To investigate the mechanism of disturbed immunological function induced by Epstein-Barr virus(EBV) infection through evaluating NKG2D expressions in NK cells and CD8+ T lymphocytes from children with infectious mononucleosis.Methods Twenty-nine children with infectious mononucleosis (IM) and twenty-five age-matched healthy children were enrolled in the study.NKG2D/NKG2A expressions in NK cells and CD8+T lymphocytes,and NKG2D ligand MHC Ⅰ chain-related molecules A(MICA) and UL-16binding proteins (ULBP-1) expressions on CD14+ mononuclear cells (MC) were analyzed by flow cytometry.The concentrations of cytokines such as IL-7,IL-12,IL-15,IFN-γ,TGF-β and soluble MICA(sMICA) in plasma were determined by enzyme-linked immunosorbent assay (ELISA).Results (1) Compared with the control group,NKG2D expression was significantly decreased in both NK cells and CD8+T lymphocytes from children with IM (P＜0.05),especially in the three cases of suspected EVB-HLH children.(2)There was no difference in the expression of MICA and ULBP-1 in CD14+ MC between the children with IM and the normal control(P＞0.05).(3)The concentrations of IL-15 and TGF-β in plasma were lower than those of the control group (P＜0.05),while the levels of IL-7,IL-12,IFN-γ and sMICA were up-regulated(P＜0.05) in children with IM.(4)NKG2A expression in NKcells in children with IM was significantly increased as compared with healthy controls(P＜0.05),however,there was no differences in the expression of NKG2A in CD8+ T lymphocytes between the two groups (P＞0.05).Conclusion Remarkable down-regulated expressions of NKG2D in NK cells and CD8+T lymphocytes might be one of the factors causing disturbed immunological function in children with EBV infection,which might have a close relationship with abnormal cytokine milieu of IL-15 and IL-12or high concentration of sMICA.

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.

ObjectiveTo study the alteration mechanism of T follicular helper (Tfh) cells in patients with Kawasaki disease (KD).MethodsTwenty children with KD and the same number of agematched healthy subjects were studied.The proportion of CD4+CXCRS+ICOS+ T in peripheral blood was analyzed by flow cytometry.Real-time PCR was performed to detect the level of Tfh transcriptional factor( Bcl6) and its inhibitor( Blimp-1 ).The plasma concentration of IL-4 and IL-21 were determined by ELISA.ResultsThe proportion of CD4+CXCR5+ICOS+ T in patients with KD was significantly higher than healthy controls [ (2.6±0.6) ％ vs ( 1.8±0.7 ) ％,P＜0.05 ].Transcription levels of Bcl-6 were significantly elevated in patients with KD( P＜0.05),its inhibitor Blimp-1 were found to be down-regulated during acute phase of KD compared with healthy controls (P ＜ 0.05 ).The significant increase of IL-4 and IL-21 plasma concentrations were detected in patients with KD(P＜0.05),in comparison with healthy controls.ConclusionThe over activity of Tfh might be correlated with immune dysregulation in Kawasaki disease.Dysregulation of Bcl-6/Blimp-1,altered microenvironment of IL-4 and IL-21 might be correlated with the abnormal activity of Tfh cells.

Objective To investigate the changes and roles of Fc gamma receptors (FcγR) expressed on monocytes in the immune pathogenesis of Henoch-Schonlein purpura(HSP).Methods Thirty children of HSP and 15 health controls were enrolled in this study.The expressions of FcγR Ⅰ and FcγRⅢ on monocytes were determined by flow cytometry,and real-time PCR was performed to detect the transcription levels of FcγR Ⅱ a,FcγR Ⅱ b,cytokines(IL-1 β,IL-6,TNF-α,IFN-α),chemokine(IP-10,RANTES,iNOS),and BLyS/April in monocytes isolated by microbeads.The plasma concentrations of IL-4,IL-10 and TNF-α were analyzed by enzyme-linked immunosorbent assay (ELISA).Results (1) The expressions of FcγR Ⅰ and FcγR Ⅲ on monocytes in patients with HSP were significantly up-regulated compared with healthy controls.Transcription level of FcγR Ⅱ a on monocytes in patients with acute HSP was found to be higher than that in healthy controls while the inhibitory FcγR Ⅱ] b mRNA was significant down-regulated(P＜0.05),which resulted in a higher ratio of FcR Ⅱ a/Ⅱ b in patients with acute HSP.(2) The expressions of cytokine/chemokines factor such as IL-1 β,IL-6,TNF-α,IP-10,RANTES,and iNOS in patients with HSP was detected to be higher than those in healthy controls(P＜0.05).In addition,expression levels of BLyS/April were up-regulated during acute HSP(P＜0.05),the positive correlations were observed between the FcγR Ⅱ a/FcγR Ⅱ b and the cytokine/chemokines factor in monocytes(P＜0.05).(3) Plasma concentrations of IL-4,IL-10 and TNF-α were significantly elevated during acute HSP(P＜0.05),and a negative correlation was observed between concentrations of TNF-α and the mRNA level of FcγR Ⅱb in monocytes.Conclusion The abnormal expression of the cytokines and the imbalance of stimulatory and inhibitory FcγR of monocyte in acute HSP.

Objective To investigate the effects of sIL-2R in plasma on regulatory T cells (Treg) in children with Kawasaki disease ( KD ) .Methods Thirty-three children with KD and fourteen age-matched healthy children were enrolled in this study .The proportions of CD4+CD25+Foxp3+Treg cells in pe-ripheral blood and mean fluorescence intensity (MFI) of phosphorylated-STAT5 (pSTAT5) protein in CD4+CD25+T cells were analyzed by flow cytometry .The concentrations of sIL-2R, IL-2, IL-7 and IL-15 in plas-ma were measured by cytometric bead array ( CBA ) .Real-time PCR was performed to detect the gene ex-pressions of Foxp3, GITR, CTLA4, IL-2Rα, IL-2Rβand IL-2Rγat mRNA level as well as the expressions of IL-17A and ROR-γt at mRNA level in CD4+CD25-T cells.Results (1) Compared with the control group, the proportions of CD4+CD25+Foxp3+Treg cells in peripheral blood from patients with KD and the expressions of associated factors including Foxp 3, GITR and CTLA-4 at mRNA level were significantly down-regulated (P<0.05).However,the expressions of IL-17A and ROR-γt at mRNA level in Th17 cells were markedly up-regulated (P<0.05), which could be recovered to some extent after treatment with IVIG (P<0.05).(2)The expressions of pSTAT5 protein in CD4+CD25+T cells from patients with acute KD were sig-nificantly decreased (P<00.5 ), but increased with IVIG intervention (P<0.05).(3)The concentrations of sIL-2R in plasma were elevated during acute KD (P<0.05), but decreased after treatment with IVIG (P<0.05).Moreover, KD patients with coronary artery lesion ( KD-CAL+) presented a high level of sIL-2R than those without coronary artery lesion (KD-CAL-) (P<0.05), but there was no significant difference in the concentrations of IL-2, IL-7 and IL-15 in plasma between two groups (P>0.05).(4)The expressions of IL-2Rαand IL-2Rβat mRNA level in CD4+CD25+T cells from patients with acute KD were lower than those of the healthy subjects (P<0.05), but up-regulated to some extent with IVIG treatment (P<0.05).There was no significant change in the expression of IL-2Rγat mRNA level (P>0.05).The concentrations of sIL-2R in plasma were negatively correlated with the expressions of IL-2Rβand Foxp3 at mRNA level and pSTAT5 at protein level (P<0.05).The expression of pSTAT5 protein had positive correlation with the ex-pression of Foxp3 at mRNA level (P<0.05).Conclusion Aberrant IL-2/STAT5 signaling pathway media-ted by significantly increased concentration of sIL-2R in plasma might be one of the factors leading to down-regulation of Treg cells in patients with acute KD .

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.