Ginsenoside Rd inhibits IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Preparation and Assessment of IL1RAP Monoclonal Antibody / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To prepare and identify human monoclonal antibody against IL-1 receptor accessory protein (IL1RAP), which is a new identified surface marker for leukemia stem cells (LSC).</p><p><b>METHODS</b>BALB/c mice were inoculated intraperitoneally with hybridoma cells (3H6E10, 10D8A7) and their ascites were collected. The monoclonal antibody against hu-IL1RAP specifically was purified from ascites, the nondenaturing-PAGE, ELISA and Western blot were used to detect the purity, titer and sensitivity of antibody.</p><p><b>RESULTS</b>Two purified antibodies were obtained and named as 3H6E10 McAb and 10D8A7 McAb, whose purity was 95% and 94% respectively. The titer of two purified monoclonal antibodies was 1 : 81000 and specific conjugation of IL1RAP purified protein and endogenous protein from normal people and leukemia patients with purified antibodies were confirmed.</p><p><b>CONCLUSION</b>The purified monoclonal antibodies which can specifically bind to hu-IL1RAP are successfully prepared, thus providing novel way to effectively clear LSC in the future.</p>

Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs).</p><p><b>METHODS</b>The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells.</p><p><b>RESULTS</b>The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired.</p><p><b>CONCLUSION</b>Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.</p>

Construction of hybridoma cells with IL1RAP as a new marker for leukemia stem cells and detection of its monoclonal antibody / 中国实验血液学杂志

This study was aimed to prepare and identify human monoclonal antibody against IL-1 receptor accessory protein (IL1RAP), which is a new identified surface marker for leukemia stem cell (LSC), BALB/c mice were immunized with recombinant hu-IL1RAP and the spleen cells from immunized mice were fused with SP2/0 myeloma cells by conventional hybridoma technique. Positive hybridoma cells were selected and cultured. ELISA and Western blot were used to detect the type, titer and sensitivity of antibody. Peripheral blood mononuclear cells were isolated and used to test the antibody specificity. The results showed that 8 hybridoma cell lines able to stably secrete IL1RAP monoclonal antibodies were obtained and named 3H6E10, 4B6A6, 8G11B5, 9E9F2, 10D8A7, 1C7H7, 1D7G11 and 2D3D3 respectively. These monoclonal antibodies belonging to IgG1/κ type could specifically bind to IL1RAP from peripheral blood mononuclear cells. It is concluded that the hybridoma cell lines with stable secretion of IL1RAP monoclonal antibodies is successfully constructed, thus providing novel ways to effectively clear LSC in the future.