Changes of Inflammasome in Children with Immune Thrombocytopenia before and after Treatment / 中国实验血液学杂志

OBJECTIVE@#To clarify the significance of inflammasome NLRP3 in children with immune thrombocytopenia (ITP) by detecting its changes before and after treatment.@*METHODS@#Twenty children with ITP diagnosed and treated in Xuzhou Children's Hospital were enrolled as observation group, and 10 healthy children as control group. The mRNA levels of NLRP3, ASC, and Caspase-1 were measured by real-time quantitative PCR (RT-qPCR), the serum levels of IL-18, IL-1β, and high mobility group protein B1 (HMGB1) were detected by ELISA, and the protein level of NLRP3 was detected by Western blot.@*RESULTS@#In newly diagnosed ITP children, the serum levels of IL-18, IL-1β and HMGB1 significantly decreased after treatment (P<0.05). After treatment, NLRP3, ASC, and Caspase-1 mRNA levels in peripheral blood mononuclear cells were significantly lower than those before treatment (P<0.05). NLRP3 protein expression decreased significantly after treatment.@*CONCLUSION@#Expression of NLRP3 inflammasome and downstream inflammatory factors are decrease after treatment in children with ITP, which may be used as effective prognostic markers.

Role of Inflammasome in Children's Immune Thrombocytopenia / 中国实验血液学杂志

OBJECTIVE@#To explore the role and the mechanism of NLRP3 inflammasome in children's immune thrombocytopenia (ITP).@*METHODS@#Twenty-one children suffered from ITP were enrolled in ITP group, 10 healthy children were selected in control group. Peripheral blood mononuclear cells (PBMNC) were isolated from ITP children and healthy controls. The mRNA levels of NLRP3, ASC, and Caspase-1 in PBMNCs were measured by quantitative real-time PCR. Moreover, the protein level of NLRP3 in PBMNCs was detected by Western blot. The plasma IL-18 level was detected by ELISA.@*RESULTS@#The expression level of NLRP3, ASC and Caspase-1 mRNA in newly-diagnosed ITP children was dramatically higher than that in control. The plasma IL-18 level was higher than that in healthy control. Furthermore, the level of NLRP3 protein was up-regulated in ITP children.@*CONCLUSION@#The NLRP3 inflammasome and up-regulated level of IL-18 have been found in newlydiagnosed ITP patients, and they may involve in the pathogenesis of ITP.

Expression of vasoactive intestinal peptide in peripheral blood of children with hand, foot and mouth disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the expression of vasoactive intestinal peptide (VIP) in peripheral blood of children with hand, foot and mouth disease and its significance.</p><p><b>METHODS</b>According to the condition of the disease, 86 children with hand, foot and mouth disease were classified into phase 1 group (19 children) and phase 2 group (67 children). ELISA was used to measure the concentrations of plasma VIP, interferon-γ (IFN-γ), and interleukin-4 (IL-4) in peripheral blood. Flow cytometry was used to measure CD3, CD4, and CD8T lymphocyte subsets. RT-PCR was used for qualitative detection of enterovirus 71 (EV71) RNA in stool.</p><p><b>RESULTS</b>Compared with the phase 1 group, the phase 2 group had a significantly higher positive rate of EV71-RNA (P<0.05) and significantly higher serum levels of IgG, IgA, IgM, and C3 (P<0.05). The phase 2 group had significantly lower proportions of peripheral CD3, CD4, and CD8T lymphocyte subsets than the phase 1 group (P<0.05), as well as significantly lower proportion of peripheral B cells and CD4/CD8ratio than the phase 1 group (P<0.05). The phase 2 group also had a significantly lower concentration of VIP in peripheral blood than the phase 1 group (P<0.05). In the 86 children with hand, foot and mouth disease, the concentration of VIP in peripheral blood was positively correlated with the proportion of CD4T lymphocyte subset and CD4/CD8ratio (r=0.533 and 0.532 respectively; P<0.05).</p><p><b>CONCLUSIONS</b>VIP may be an important marker of the severity of hand, foot and mouth disease.</p>

Construction of lentiviral vector for mouse CXC chemokine receptor 4 gene and its expression in eukaryotic cells / 中国实验血液学杂志

This study was aimed to clone the gene coding mouse CXC chemokine receptor 4 (CXCR4), to construct the recombinant lentiviral vector carrying enhanced green fluorescence protein (EGFP) and to explore its expression in eukaryotic cells (293FT cells). The full length CXCR4 gene was cloned by RT-PCR using bone marrow cells from C57BL/6 mouse as template and inserted into PCR-Blunt vector. CXCR4 fragment was generated by digestion with restriction endonuclease and subcloned into a lentiviral vector to generate recombinant lentiviral vector LV-IRES-EGFP-CXCR4, which was co-transfected into 293FT cells together with envelope plasmid and packaging plasmid by lipofectamine 2000. Viruses were gathered and concentrated using ultracentrifuge, and then transfected into 293FT cells. Expression of EGFP was detected by fluorescent microscopy and flow cytometry (FCM). And the expression of CXCR4 protein was detected by Western blot. The results demonstrated that mouse CXCR4 gene was cloned and the lentiviral vector was successfully constructed. The lentiviral particles were correctly packaged, and the virus titers were above 10(8) TU/ml in the supernatant after concentration. Expression of EGFP was detected by fluorescent microscopy in the transfected 293FT cells, and the transfection efficacy > 95% was determined by FCM. Expression of CXCR4 protein detected by FCM and Western blot was significantly higher than that in control group. It is concluded that the CXCR4 gene along with the gene coding EGFP is successfully inserted into a lentiviral vector to construct a recombinant lentiviral vector, which can be expressed in eukaryotic cells.