Lysophosphatidylcholine (lysoPtdCho) is a component of oxidized low density lipoprotein, and is involved in the pathogenesis of atherosclerosis and inflammation. We studied the effects of lysoPtdCho on cytotoxicity, reactive oxygen species (ROS) production, activation of the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinases and pro-inflammatory gene expression in RAW 264.7 murine macrophage cells. When cells were exposed to lysoPtdCho with various acyl chains in a culture medium containing 10% fetal bovine serum, only 1-linoleoyl (C18:2) lysoPtdCho showed a remarkable cytotoxicity, reaching the highest level at 24 h, and elicited ROS production, suggesting that oxidative stress might be implicated in the cytotoxicity of 1-linoleoyl (C18:2) lysoPtdCho. Presumably in support of this, antioxidants such as magnolol or trolox prevented 1-linoleoyl (C18:2) lysoPtdCho-induced cytotoxicity as well as ROS production, although only partially. Furthermore, the phosphorylation of ERK 1/2 and the expression of pro-inflammatory cytokines such as IL-1beta, CCL2 and CCL5 were augmented by 1-linoleoyl (C18:2) lysoPtdCho. Meanwhile, there was no structural importance of the acyl chain for the cytotoxic action of lysoPtdCho during 10 min incubation in serum-free media. Taken together, it is suggested that in a serum-containing medium, 1-linoleoyl (C18:2) lysoPtdCho can cause a significant cytotoxicity through ROS production, probably accompanied by activation of ERK and induction of related inflammatory cytokines, in RAW 264.7 cells.

We investigated the effect of the butanol fraction from the methanol extract of butterbur (Petasites japonicus Max.) (BMP) on the plasma lipid profiles and oxidative damage of liver in mice challenged with monosodium l-glutamate (MSG). ICR mice (6-8 weeks old, male) were fed BMP (0.1% or 0.3%) for 1 week, and on day 7, MSG (4 mg/g) was administered intraperitoneally to the mice. Administration of MSG resulted in a significant decrease of antioxidant biomarkers such as total glutathione level and antioxidant enzyme activities such as glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferase (GST), and quinone reductase (NQO1), whereas the thiobarbituric acid-reactive substances (TBARS) value was increased in liver tissue. However, BMP supplementation markedly enhanced the hepatic antioxidant enzyme activities, including GPx, GR, GST, and NQO1, whereas it lowered TBARS, compared to the MSG-treated group. Moreover, BMP supplementation decreased total cholesterol, atherogenic index, and low-density lipoprotein-cholesterol, compared to the MSG-treated group. Based on these results, it was proposed that BMP improve the plasma lipid profiles and decrease the oxidative stress by up-regulating the hepatic antioxidant enzymes in mice challenged with MSG.

Interaction of paraoxonase1 (PON1) with lysophospholipids was examined with respect to activity regulation and binding property. Paraoxonase activity of purified PON1 was partially inhibited by palmitoyl-lysophosphatidyl-glycerol (palmitoyl-lysoPG) and lysophosphatidylinositol (lysoPI), which had a stimulatory effect on arylesterase and diazoxonase activities. The selective inhibition of paraoxonase activity by palmitoyl-lysoPG, characterized by noncompetitiveness and charge interaction, was also observed with HDL- or dimyristoylphosphatidylcholine (DMPC)-bound PON1. Meanwhile, lysophosphatidylcholine (lysoPC) stimulated all three activities of purified PON1, although it stimulated DMPC-bound or HDL-bound PON1 to a lesser extent. The stimulatory action of lysophospholipids was observed around their CMC, suggesting that micelle formation of lysophospholpids might be involved in the stimulation of PON1 activity. Presumably in support of this, the tryptophan fluorescence intensity of PON1 was increased by lysophospholipids at concentrations required for the stimulation of PON1 activity. Separately, lysoPC stimulation was less remarkable for DMPC-bound PON1 than for either dimyristoylphosphatidylserine (DMPS)- or dimyristoylphosphatidylglycerol-bound PON1, suggesting a tight association between PON1 and DMPC. In support of this, the stimulatory role of apolipoprotein A-I was less prominent for DMPC-bound PON1 than for DMPS-bound PON1. Taken together, these data suggest that the inhibition of paraoxonase activity by lysoPG or lysoPI may be due to binding to a site distinct from the active center, whereas the stimulation by lysophospholipid may be ascribed to the micelle formation around the lipid-associable region of PON1.

No abstract

Purpose: We investigated the pharmacology of PMC-309 in ex vivo or in vivo characterization for the development of the preclinical study. Experimental procedures: Target selectivity: it was measured by SPR assay. Human VISTA expressing CHO k1 cells or CHO k1 mock cells were used in cell binding assay. Target cell analysis: human peripheral blood mononuclear cells (PBMC) containing lymphocyte and myeloid lineage cells were used for target cells analysis with flow cytometry. In vitro T cell activity: human PBMC was employed for the evaluation of T cell activity (IRB #1041107-201703-BR-002-02) and was stimulated with or w/o PMC-309 in the presence of anti-CD3/28 Antibody. In vivo study: MC38 bearing human VISTA knock-in (KI) mice were employed for the assessment of anti-tumor activity of PMC-309. The tumor infiltrated immune cells: Immune cells in the TME were evaluated by immunohistochemistry (IHC) or flow cytometry (FACS) analysis. Statistics: All data were presented as the mean ± SEM and analyzed using one-way ANOVA followed by Dunnett’s multiple comparison tests in Graph pad prism®. ***p<0.001, **p<0.01 and *p<0.05 compared to Control group. Summary of data: PMC-309 binding to VISTA expressing cells is highly selective and the selectivity is maintained even in the low pH conditions that mimic TME. PMC-309 enhances the secretion of IFN-gamma, TNF-alpha, and IL-2 in Mixed Lymphocyte Reaction (MLR) settings. In addition, PMC-309 promoted monocyte differentiation into M1 macrophage that stimulates proinflammatory cytokine secretion of T cells. For the in vivo study, PMC-309 was intravenously administrated in VISTA-KI mice. The tumor growth rate was suppressed in accompanied by a synergistic effect with an anti-PD1 antibody. The anti-tumor activity was associated with enhanced T cell activation, increased secretion of pro-inflammatory cytokines, and increased penetration of cytotoxic T cells, but lowing immune-suppressive MDSC cells into TME as demonstrated with IHC analysis. Conclusions: PMC-309 is a fully human anti-VISTA antibody that targets human VISTA specifically. PMC-309 enhanced both T cell activation and monocyte activation when monocytes were cultured with or without T cells by targeting VISTA. PMC-309 increased the number of T cell infiltration while a decrease of MDSCs in the TME. PMC-309 in combination with chemotherapy or other IO drugs could address highly medical unmet needs from patients with drug resistance to currently available IO treatment options. Citation Format: Cheon Ho Park, Sang Soon Byun, Jin Yong An, Hyerim Han, Weon Sup Lee. PMC-309, a highly selective anti-VISTA antibody reverses immunosuppressive TME to immune-supportive TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5557.