Summary The technique of counterimmunoelectrophoresis (CIE) has been adapted for detection of serum precipitins to calf thymus (CT) DNA in patients with SLE, discoid LE, miscellaneous connective tissue and infectious diseases, and control populations. Of seventy‐eight LE patients, 58% demonstrated anti‐ss DNA precipitins, and 20% exhibited anti‐ds DNA precipitins. Good correlation was noted between the presence of ss DNA precipitins and ss DNA binding values determined by the more sensitive ammonium sulphate precipitation assay. Depressed total serum haemolytic complement activity in CH 50 u/ml was noted in 64% of sera exhibiting ss DNA precipitins and 38% of those with negative ss DNA precipitins. There was a strong association, however, between ds DNA precipitins and depressed serum complement levels. Although less sensitive than primary binding assays, CIE can be used as a rapid and simple screening test for detection of circulating anti‐native and denatured CT DNA precipitins. CT DNA serum precipitins are present in a significantly higher percentage of SLE patients when compared with other disease states and normal control populations.

<h4>Objective</h4>Lupus nephritis (LN) treatment faces the challenge of balancing effective immunosuppression with systemic safety. To address this, we aimed to develop a biomimetic nanoplatform capable of simultaneously targeting multiple pathogenic pathways in LN, thereby achieving potent immunomodulation without broad toxicity.<h4>Methods</h4>A "smart immune decoy" (RAPA@MEX-PL) was engineered by encapsulating rapamycin (RAPA) in mesenchymal stromal cell-derived exosomes (MEX) and coating the surface with a cationic polylysine (PLL) corona. The platform was designed to concurrently: (1) the polylysine corona potently scavenges cell-free DNA (cfDNA) to quench TLR9-mediated inflammation, (2) the MEX core mediates the repolarization of macrophages from an M1 to an M2 phenotype, and (3) localized RAPA release provides durable mTOR inhibition, synergistically rebalancing autoimmune responses. Renal targeting, immunomodulatory activity, and systemic safety were evaluated in lupus-prone mouse models.<h4>Results</h4>In lupus-prone mice, RAPA@MEX-PL demonstrated precise accumulation in renal tissue, leading to a significant reduction in auto-antibody levels and resolution of glomerular inflammation. The platform concurrently addressed three key pathogenic pathways-cfDNA scavenging, macrophage repolarization and mTOR inhibition-resulting in synergistic rebalancing of autoimmune responses. Notably, it circumvented the metabolic side effects typically associated with systemic RAPA administration.<h4>Conclusions</h4>The RAPA@MEX-PL nanoplatform represents a targeted and effective immunotherapeutic strategy for LN, capable of achieving sufficient immunosuppression without systemic toxicity. These findings emphasize its potential as a favorable candidate for the therapy for autoimmune diseases. Materials and Methods Material DSPE-PEG-PLL, Cy5-rapamycin, and DSPE-PEG-FITC were ordered from Ruixi Biotechnology Co., Ltd (Xian, China). DMEM/F12 and fetal bovine serum (FBS) were ordered from Gibco. Phosphate-buffered saline (PBS, pH 7.4) and DMEM were commercially available from the Servicebio Biology Co., Ltd (Wuhan, China). Iscove's Modified Dulbecco's was ordered from Thermo Fisher Scientific (USA). CpG 2006 and its Cy5.5-labeled counterpart were sourced from Genscript China. A 100 μg/mL stock solution of unlabeled CpG was prepared by diluting the compound in PBS. The Annexin V-APC apoptosis kit was acquired from Elabscience. The Bradford Protein Content Assay Kit was sourced from Beyotime Biotechnology Co., Ltd. Additionally, the CCK-8 assay kit, Calcein AM, propidium iodide (PI), Actin-Tracker Green (a microfilament-specific dye), Lysotracker Red DNA-99, 2’, 7’-Dichlorodihydrofluorescein diacetate (DCHF-DA) (for ROS detection), and anti-fade mounting medium with DAPI were all procured from Beyotime Biotechnology Co., Ltd. Rapamycin (RAPA, 10 mM*1 mL in DMSO), calf thymus DNA, Polyethylene glycol 35000 (PEG), puromycin aminonucleoside (PAN), Pam3Cys-Ser-(Lys) 4 (Pam3CSK4), lipopolysaccharide (LPS) and Interferon-gamma (IFN-γ) were obtained from MedChemExpress. Antibodies used for immunofluorescence analysis, including pS6K p62 and COL4A3 were obtained from Proteintech, p4EBP and LC3B were obtained from Zenbio. Anti-dsDNA antibody levels (FineTest), enzyme-linked immunosorbent assays (MultiSciences). Cells and culture of primary cells MSCs were supplied by Taisheng Corp (Nanjing) and cultivated in DMEM/F-12 complete medium. Mouse podocyte clone-5 (MPC-5) was obtained from the Servicebio (Wuhan) and incubated in RPMI 1640 media. RAW264.7 was obtained from the Servicebio (Wuhan) and incubated in DMEM high glucose media. Invitrogen (USA) was the source of Ramos Blue™ reporter cells cultured in IMDM complete medium (serum inactivated). Evaluation of cfDNA-binding efficiency of engineered exosomes Separate solutions of calf thymus DNA and ethidium bromide (EtBr) were prepared in PBS at 1 mg/mL each. A reaction mixture was prepared by combining the following components: calf thymus DNA (4 μL), EtBr (4 μL), specified volumes of RMP, RM, MEX, or PEG, and either FBS or PBS (16 μL). The final volume was adjusted to 160 μL with PBS. This enhanced binding affinity was evident under both physiologically mimetic conditions—Phosphate Saline Buffer (PBS, pH 7.4) and a serum-rich environment (10% Fetal bovine serum, FBS)—highlighting its potential robustness in vivo (Figure 3 B). However, the neutral polymer (PEG) and exosome alone groups had almost no cfDNA scavenging ability. This potent DNA-binding capacity was further visually confirmed by agarose gel electrophoresis. The intensity of the CpG-ODN2006 (a cfDNA mimic) band was markedly diminished following incubation with RMP, and nearly absent compared to the MP or RM groups ( Figure S2 ). Thus, these three mechanisms are not isolated; rather, they form a multidimensional and mutually reinforcing synergistic therapeutic network, operating from the circulatory system to renal tissue and down to specific cells. Conclusion In summary, this study developed an engineered exosome-based multifunctional nanoplatform with dual functions of clearance of circulating cfDNA and targeted delivery of RAPA for the treatment of LN. By combining exosomes, RAPA and PLL, we successfully constructed three composite systems, including MP, RM and RMP, and systematically evaluated their biocompatibility, DNA clearance ability and TLR inhibition effect. During the process of manuscript preparation, image generation, data collection, and data analysis, no artificial intelligence tools were used at all. Data availability statement The data on which the results of this study are based are available in the supplementary material of this article. References Associated Data Supplementary Materials Supplementary materials and methods, figures. Data Availability Statement The data on which the results of this study are based are available in the supplementary material of this article.

Cytochrome P450 2U1 (CYP2U1) is an extrahepatic monooxygenase that metabolizes both endogenous fatty acids and xenobiotic substrates. Because of its high expression in both healthy and cancerous thymus tissues, this study investigated CYP2U1-mediated biotransformation of 2 tumor-targeting tyrosine kinase inhibitors, sorafenib and sunitinib, both are commonly prescribed for thymus cancers. Recombinantly expressed CYP2U1 and CYP2D6 were incorporated into nanodiscs, and their metabolism of sorafenib and sunitinib was investigated. Liquid chromatography-tandem mass spectrometry metabolite profiling revealed that both CYP2U1 and CYP2D6 nanodiscs oxidized sorafenib to generate sorafenib N-oxide and both enzymes catalyzed the dealkylation of sunitinib to form N-desethyl sunitinib. Spectroscopic studies (UV-Visible and fluorescence) confirmed favorable binding interactions of CYP2U1 and CYP2D6 to both drugs tested. Molecular dynamics simulations demonstrated binding of sorafenib and sunitinib in the CYP2U1 active site and identified key interactions between the drug and key residues at the enzyme's active site. Rates of metabolite formation were quantified by targeted metabolomics, and inhibition of tyrosine kinase was assessed by ELISA assay. Both the parent compound sorafenib and metabolite sorafenib N-oxide showed similar inhibitory effects on cellular migration in HepG2 cells. The metabolite sorafenib N-oxide was approximately twice as potent as the parent compound in inhibiting cancer cell migration. In contrast, N-desethyl sunitinib failed to show similar extent of inhibition. Together, these findings highlight the potential role of extrahepatic CYP2U1 in the local metabolism of tyrosine kinase inhibitors and suggest that CYP2U1-mediated transformations directly influence antitumor efficacy at thymic tumor sites. SIGNIFICANCE STATEMENT: Understanding the interactions between cytochrome P450 2U1 and cytochrome P450 2D6 in nanodiscs and thymus tumor-targeting drugs, sorafenib and sunitinib, led to discovery of new bioactive metabolites that carry differential anticancer properties compared with their parent compounds.