Disialoganglioside GD2 is widely expressed among neuroblastomas, melanomas, small-cell lung carcinoma, sarcomas and brain tumors. Immunity directed against this antigen may have anti-tumor utility. Since GD2 is poorly immunogenic, anti-idiotypic antibodies may serve as alternative tumor vaccines. Monoclonal antibody 3F8, a murine IgG3 specific for GD2, has shown excellent tumor-targeting ability in vitro and in vivo. LOU/CN rats were immunized with 3F8 and their spleens were used in somatic-cell hybridization, using SP2/0, P3 and Y3 as fusion partners. Six anti-idiotypic (anti-id) MAbs (C2D8, Idio-2, AIG4, C2H7, C4E4, A2A6) were selected based on their reactivity with 3F8 and non-reactivity with murine IgG3 myelomas. Specificity of each anti-id was demonstrated by using various ELISA: (i) lack of direct binding to solid phase myelomas and serum proteins; (ii) inability of other myelomas to inhibit anti-id binding to 3F8; (iii) absence of cross-reactivity of other myelomas to solid-phase anti-id; (iv) lack of inhibition by anti-id of binding of other ganglioside antibodies to their antigens. Antigen specificity was further examined by inhibition of binding of 3F8 to GD2 on immuno-thin-layer chromatography, and by inhibition of 3F8 immunostaining of neuroblastoma cell lines. These 6 antibodies were demonstrated to be distinct, in view of their cross-reactivity, fusion partners and relative strength of binding to 3F8. Anti-GD2 antibodies were induced after immunization with these anti-id antibodies in C57Bl/6 mice. These rat anti-3F8-idiotypic antibodies with exquisite specificity for anti-GD2 antibodies may be useful in vaccine construction.
Certain immunophenotypes in multiple myeloma (MM), including CD56 and CD117, have been reported to be associated with overall survival (OS). However, previous reports have ignored the impact of different treatment regimens and the long-term prognostic value of immunophenotyping in MM when treated with novel agents, including thalidomide and bortezomib, in the absence of transplantation for autologous stem cell transplantation and allo-hematopoietic stem cell transplantation. To further understand the long-term prognostic value of immunophenotyping in MM, when treated with bortezomib combined with thalidomide-based regimens without transplantation, 80 patients who were newly diagnosed between January 2007 and December 2015, were analyzed retrospectively. In contrast to previous studies, no significant survival time difference was observed between CD56+/CD117+ and CD56-/CD117- groups. Multivariate analysis suggested that human leukocyte antigen-antigen D-related (HLA-DR)+ was independently associated with shorter OS and progression-free survival (PFS), while CD117+ was an independent prognostic factor for decreased PFS. In addition, the myeloma prognostic index (MPI), defined by HLA-DR+, age ≥65 years and international staging system stage III, was suitable for risk stratification of patients treated with novel agents for OS and PFS. The results of the current study suggested that HLA-DR+ patients had a shorter OS and PFS and CD117+ patients had shorter PFS. HLA-DR+ or CD117+ was sufficient to affect survival. Evaluating these markers may reveal valuable prognostic factors for MM in patients receiving bortezomib combined with thalidomide-based regimens without autologous stem cell transplantation and allo-hematopoietic stem cell transplantation). MPI may describe an accessible tool to predict the prognosis of patients with MM.
Background E1A-associated 300-kDa protein (P300), an endogenous histone acetyltransferase, contributes to modifications of the chromatin landscape of genes involved in multiple cardiovascular diseases. Ferroptosis of vascular smooth muscle cells (VSMCs) is a novel pathological mechanism of aortic dissection. However, whether P300 regulates VSMC ferroptosis remains unknown. Methods Cystine deprivation (CD) and imidazole ketone erastin (IKE) were used to induce VSMC ferroptosis. Two different knockdown plasmids targeting P300 and A-485 (a specific inhibitor of P300) were used to investigate the function of P300 in the ferroptosis of human aortic smooth muscle cells (HASMCs). Cell counting kit-8, lactate dehydrogenase and flow cytometry with propidium iodide staining were performed to assess the cell viability and death under the treatment of CD and IKE. BODIPY-C11 assay, immunofluorescence staining of 4-hydroxynonenal and malondialdehyde assay were conducted to detect the level of lipid peroxidation. Furthermore, co-immunoprecipitation was utilized to explore the interaction between P300 and HIF-1α, HIF-1α and P53. Results Compared with normal control, the protein level of P300 was significantly decreased in HASMCs treated with CD and IKE, which was largely nullified by the ferroptosis inhibitor ferrostatin-1 but not by the autophagy inhibitor or apoptosis inhibitor. Knockdown of P300 by short-hairpin RNA or inhibition of P300 activity by A-485 promoted CD- and IKE-induced HASMC ferroptosis, as evidenced by a reduction in cell viability and aggravation of lipid peroxidation of HASMCs. Furthermore, we found that hypoxia-inducible factor-1α (HIF-1α)/heme oxygenase 1 (HMOX1) pathway was responsible for the impacts of P300 on ferroptosis of HASMCs. The results of co-immunoprecipitation demonstrated that P300 and P53 competitively bound HIF-1α to regulate the expression of HMOX1. Under normal conditions, P300 interacted with HIF-1α to inhibit HMOX1 expression, while reduced expression of P300 induced by ferroptosis inducers would favor HIF-1α binding to P53 to trigger HMOX1 overexpression. Furthermore, the aggravated effects of P300 knockdown on HASMC ferroptosis were largely nullified by HIF-1α knockdown or the HIF-1α inhibitor BAY87-2243. Conclusion Thus, our results revealed that P300 deficiency or inactivation facilitated CD- and IKE-induced VSMC ferroptosis by activating the HIF-1α/HMOX1 axis, which may contribute to the development of diseases related to VSMC ferroptosis.
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