Combined Assessment of Serum Alpha-Synuclein and Rab35 is a Better Biomarker for Parkinson's Disease

BACKGROUND AND PURPOSE: It is essential to develop a reliable predictive serum biomarker for Parkinson's disease (PD). The accumulation of alpha-synuclein (αSyn) and up-regulated expression of Rab35 participate in the etiology of PD. The purpose of this investigation was to determine whether the combined assessment of serum αSyn and Rab35 is a useful predictive biomarker for PD. METHODS: Serum levels of αSyn or Rab35 were determined in serum samples from 59 sporadic PD patients, 19 progressive supranuclear palsy (PSP) patients, 20 multiple system atrophy (MSA) patients, and 60 normal controls (NC). Receiver operating characteristics (ROC) curves were calculated to determine the diagnostic accuracy of αSyn or/and Rab35 in discriminating PD patients from NC or atypical parkinsonian patients. RESULTS: The levels of αSyn and Rab35 were increased in PD patients. The serum level of Rab35 was positively correlated with that of αSyn in PD patients. Compared to analyzing αSyn or Rab35 alone, the combined analysis of αSyn and Rab35 produced a larger area under the ROC curve and performed better in discriminating PD patients from NC, MSA patients, or PSP patients. When age was dichotomized at 55, 60, 65, or 70 years, the combined assessment of αSyn and Rab35 for classifying PD was better in the group below the cutoff age than in the group above the cutoff age. CONCLUSIONS: Combined assessment of serum αSyn and Rab35 is a better biomarker for discriminating PD patients from NC or atypical parkinsonian patients, and is a useful predictive biomarker for younger sporadic PD patients.

One-step multiplex RT-PCR for identifying common fusion transcripts in childhood acute lymphoblastic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the efficiency of one-step multiplex RT-PCR for identifying four common fusion transcripts (TEL/AML1, E2A/PBX1, MLL/AF4 and BCR/ABL) in children with acute lymphoblastic leukemia (ALL).</p><p><b>METHODS</b>Total RNA was extracted from bone marrow samples of 76 children who were newly diagnosed with ALL between January 2003 and December 2010. These RNAs were analyzed for TEL/AML1, E2A/PBX1, MLL/AF4 and BCR/ABL by one-step multiplex RT-PCR or common nested-multiplex PCR. The PCR products were confirmed by DNA sequencing.</p><p><b>RESULTS</b>TEL/AML1 was found in 12 cases (the length of products was 298 bp in 9 cases and 259 bp in 3 cases), E2A/PBX1 was found in 3 cases (the length of products was 373 bp), BCR/ABL was found in 1 case (the length of products was 2 124 bp), and MLL/AF4 was found in 7 cases (the length of products was 427 bp in 1 case and 673 bp in 6 cases) using one-step multiplex RT-PCR combined with DNA sequencing. The results were consistent with those using common nested-multiplex PCR.</p><p><b>CONCLUSIONS</b>One-step multiplex RT-PCR may be another alternative for detection of common fusion transcripts in children with ALL.</p>

Analysis of MUT gene mutations in a patient with isolated methylmalonic acidemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the clinical features and mutation of MUT gene in a Chinese patient with isolated methylmalonic acidemia.</p><p><b>METHODS</b>The clinical characteristics and laboratory tests data were collected. Genomic DNA was extracted from peripheral blood leukocytes. The 13 exons and their flanking sequences of the MUT gene were amplified with polymerase chain reaction and subjected to direct DNA sequencing.</p><p><b>RESULTS</b>The patient has featured failure to thrive, lethargy, seizure, hypotonia, severe ketoacidosis and hyperammonemia. Tandem mass results showed reduction of multiple acylcarnitine. Urine organic acid testing showed pronounced increase in methylmalonate excretion. Homocysteine was normal. The patient showed no response to vitamin B12 treatment. The above results suggested that the patient had isolated methylmalonic acidemia. DNA sequencing analysis confirmed that the patient has carried two MUT gene mutations, c.755dupA and a novel mutation c.944dupT.</p><p><b>CONCLUSION</b>Inherited metabolic disease screening plays an important role in the diagnosis of clinical diseases. However, to confirm the results will need gene mutation analysis.</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>

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

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

Expression of pattern recognition receptor in patients of EV71 infection / 中华微生物学和免疫学杂志

Objective To investigate the expression of pattern recognition receptor (PRR)of immune cells from the patients with enterovirus 71 ( EV 71 ) infection and the changes of cytokines. Methods Seventy-one patients and 20 age-matched healthy children were enrolled in the study. The patients were divided into three groups according to the critical degree: 20 mild patients, 25 severe patients and 26 critical patients. Real-time PCR were used to evaluate the levels of retinoic acidinducible gene Ⅰ ( RIG- Ⅰ ), melanoma differentitation-associated gene 5 ( MDA5 ) and cytokines IL-12, IFN-α mRNA expression in peripheral blood mononuclear cells (PBMC). The proportions of Toll like receptors(TLRs) on monocytes/macrophages (MC) , myloiddentritic cells (mDC) and plasmacytoiddentritic cells (pDC) were analyzed by flow cytometry. The concentrations of plasma cytokines( IL-12, IFN-α) were measured by ELISA. Results ( 1 ) The proportion of TLR7 is the unique TLR which is increased in mild patients and it was significantly elevated in MC, mDC and pDC in severe group (P＜0.05), TLR2, TLR3 and TLR4 also had an enhanced expression in MC and mDC. The enhanced expression of TLRs mentioned above had a decreased trend in critical patients. (2)Transcription levels of RIG- Ⅰ and MDA5 was significantly elevated in EV71 infected children.(3)The expression levels of DC-associated cytokines gene( IL-12 and IFN-α) have an increased trend in mild cases and these cytokines were remarkably increased in severe patients (P＜0.05), whereas decreased in critical cases (P＜0.05). ConclusionTLR7 might be the main PRR recognizing EV71 in immune cells and RIG-Ⅰ/MDA5 might also take part in the recognition of EV71. The exogenous or endogenous ligands released from bacterial infection and cell destruction could result in the activation of TLR2 or TLR4, inducing the inflammatory response.

Effects of intravenous immunoglobulin and aspirin treatment on the functions of circulating endothelial progenitor cells in children with Kawasaki disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effects of intravenous immunoglobulin (IVIG) and aspirin treatment on the functions of circulating endothelial progenitor cells (EPCs) in children with Kawasaki disease (KD) and possible mechanisms.</p><p><b>METHODS</b>Blood samples were obtained in 10 children with KD before and 7 days after the treatment by IVIG and aspirin. MTT method, modified Boyden chamber method and cell culture plate adhesion method were used to assess the functions of EPCs, including proliferation, adhension and migration activities. The plasma levels of tumor necrosis factor-α (TNF-α) and high-sensitivity C reactive protein (hs-CRP) were also measured.</p><p><b>RESULTS</b>The functions of circulating EPCs 7 days after IVIG and aspirin treatment were significantly improved. IVIG and aspirin treatment significantly reduced plasma TNF-α and hs-CRP concentrations. There was a significant linear regression relationship between the reduced plasma TNF-α and hs-CRP levels and the increased functions of circulating EPCs.</p><p><b>CONCLUSIONS</b>IVIG and aspirin treatment can improve the functions of circulating EPCs, possibly through reducing plasma concentrations of TNF-α and hs-CRP.</p>

Decreased circulating endothelial progenitor cell function: relationship with serum concentrations of high-sensitivity C-reactive protein in children with Kawasaki disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the function of circulating endothelial progenitor cells and its relationship with serum concentrations of high-sensitivity C-reactive protein (Hs-CRP) in children with Kawasaki disease.</p><p><b>METHODS</b>Ten children with Kawasaki disease and ten healthy children as a control group were enrolled. The peripheral mononuclear cells were induced into endothelial progenitor cells using Dulbecco's Modified Eagle Medium containing vascular endothelial growth factor and basic fibroblast growth factor. The proliferative ability, migratory ability and adhesive ability of endothelial progenitor cells were assessed by MTT methods, modified Boyden chamber methods and cell culture plate adhesion method, respectively. The concentrations of serum Hs-CRP were measured by latex enhanced turbidimetric immunoassay.</p><p><b>RESULTS</b>The proliferative ability, migratory ability and adhesive ability of endothelial progenitor cells in the Kawasaki disease group were significantly lower than those in the control group (P<0.01). The serum concentrations of Hs-CRP in the Kawasaki disease group were significantly higher than those in the control group (87.1+/-30.2 mg/L vs 5.3+/-3.4 mg/L; P<0.01). The function of circulating endothelial progenitor cells was negatively correlated with serum concentrations of Hs-CRP in the Kawasaki disease group.</p><p><b>CONCLUSIONS</b>The function of circulating endothelial progenitor cells is decreased in children with Kawasaki disease, which may be associated with the abnormal expression of inflammatory mediators.</p>

Investigation of the change of CD4~+ T cell subset from children with type 1 diabetes / 中华微生物学和免疫学杂志

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.

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>

The imbalance of CD4~+ CD25~+ Foxp3~(high) regulatory T cells/Thl7 cells in infant with sepsis / 中华微生物学和免疫学杂志

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.

Changes of the CD4+ CD25+regulatory T cells in infant with sepsis / 中华急诊医学杂志

Objective To study the change of CD4+ CD25+ Foxp3high regulatory T cells (Treg cells) and the molecules associated with Treg cells in different immune status in infant with sepsis, and to further clarity the pathogenesis of disturbed immune function in infant with sepsis. Method Totally 36 sepsis infants admitted in In-tensive Care Unit of Shenzhen Children' s Hospital from May 2007 to November 2007 and 16 age-matched healthy infants were collected for prospective study, after excluding autoimmune disease, immunodeficiency, inherited metabolic disorders, tumor, and drug-treatment that could affect immune function during lately 6 months. The study was approved by Ethics Committee of Shenzhen Children's Hospital. The 36 infants with sepsis were divided into two groups according to expression levels of HLA-DR in CD14-positive cells: DR-H group was defined as patients with HLA-DR > 30%, while DR-L group was defined as patients with HLA-DR < 30%. Expression levels of HLA-DR in CD14-positive cells and the proportion of Treg cells were analyzed by flow cytometry. Real-time PCR were used to evaluate the mRNA levels of Foxp3, CTLA-4,GITR, and IL-10 in CD4-posidve ceils. Statistical analysis was performed by one-way Anova. There was statistical difference with P < 0.05. Results The proportion of Treg cells in DR-L group was found to be significantly higher than that in healthy control or DR-H group (P <0.05).Compared with healthy control group or DR-H group, transcriptional levels of Foxp3, CTLA-4 and IL-10were significantly increased in DR-L group (P <0.05). The levels of GITR mRNA in DR-L group were detected to be higher than those in DR-H group (P < 0.05). Conclusions Aberrant increased proportion of Treg cells may be associated with suppressed immune status in infant with sepsis.

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>

Roles of CD8+ CD28- T regulatory cells in acute infectious mononucleosis in children / 中华儿科杂志

<p><b>OBJECTIVE</b>To explore the role of CD(8)(+)CD(28)(-) T regulatory cells (Tr) in the immunological pathogenesis of acute infection with Epstein-Barr virus in children.</p><p><b>METHODS</b>The present study enrolled 25 children with infectious mononucleosis (IM) and 25 age-matched healthy children. Flow cytometric analysis was performed to detect the percentage of CD(3)(+), CD(3)(+)CD(4)(+), CD(3)(+)CD(8)(+), CD(8)(+)CD(28)(+) by determining the ratio of positive cells in lymphocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR were used to analyze IL-6, IL-10, IFN-gamma expression in CD(8)(+)CD(28)(-) Tr cells and ILT-3, ILT-4 expression in monocytes/macrophages.</p><p><b>RESULTS</b>The proportions of CD(8)(+)CD(28)(-)T cells in children with acute-phase IM was significantly higher than those in the controls (P < 0.01). The expression level of IL-6, IL-10, IFN-gamma, ILT-3, ILT-4 mRNA significantly increased compared to those of the controls (P < 0.01).</p><p><b>CONCLUSION</b>The CD(28) expressed on CD(8)(+) T cells in vivo is gradually lost with age and CD(8)(+)CD(28)(-) cells increase up 50% to adult. EBV can directly infect B cells, trigger CD(8)(+) CTL response and destroy the target cells to cause serious immunopathological lesion. Therefore we speculate that the expansion of CD(8)(+)CD(28)(-) Tr cells in children with IM may be an adaptive immune response to avoid serious inflammation and autoimmune reactions.</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>

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>

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>

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.