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Extracellular vesicles (EVs) are under evaluation as therapeutics or as vehicles for drug delivery. Preclinical studies of EVs often use mice or other animal models to assess efficacy and disposition. However, as most EVs under evaluation are derived from human cells, they may elicit immune responses which may contribute to toxicities or enhanced EV clearance. Furthermore, EVs from different cell sources or EVs comprising various cargo may differ with respect to immunogenicity or toxicity. To assess EV-induced immune response and toxicity, we dosed C57BL/6 mice with EVs intravenously and intraperitoneally for 3 weeks. EVs were harvested from wild type or engineered HEK293T cells which were modified to produce EVs loaded with miR-199a-3p and chimeric proteins. Blood was collected to assess hematology, blood chemistry, and immune markers. Spleen cells were immunophenotyped, and tissues were harvested for gross necropsy and histopathological examination. No signs of toxicity were observed, and minimal evidence of changes in immune markers were noted in mice dosed with engineered, but not with wild type EVs. This study provides a framework for assessment of immunogenicity and toxicity that will be required as EVs from varying cell sources are tested within numerous animal models and eventually in humans.

A promotional role for androgen receptor (AR) signaling in hepatocellular carcinogenesis is emerging. In pre-clinical models, including diethylnitrosamine- (DEN-) induced hepatocellular carcinoma (HCC), anti-androgen therapies delay hepatocarcinogenesis. However, pharmacologic anti-androgen therapy in advanced HCC patients fails, suggesting that AR plays a role in HCC onset. This study aims to characterize AR expression and function throughout DEN-induced liver inflammation and carcinogenesis and evaluate the efficacy of prophylactic AR antagonism to prevent hepatocarcinogenesis. We demonstrate that pharmacologic AR antagonism with enzalutamide inhibits hepatocellular carcinogenesis. With enzalutamide treatment, we observe decreased CYP2E1 expression, reducing DEN-induced hepatocyte death and DNA ethyl-adducts. AR protein expression analyses show that DEN causes an initial upregulation of AR in portal fibroblasts and leukocytes, but not hepatocytes, suggesting that hepatocyte-autonomous AR signaling is not essential for DEN-induced carcinogenesis. Ablating androgen signaling by surgical castration reduced pre-carcinogen Kupffer cell populations but did not alter DEN-mediated immune cell recruitment nor AR expression. In this study, we identified that anti-androgen interventions modulate mutagenic DNA adducts, tumour initiation, and immune cell composition. Additionally, we find that AR expression in hepatocytes is not present during nor required for early DEN-mediated carcinogenesis.

Background Additional efficacious immunomodulatory treatment is needed for the management of immune‐mediated disease in horses. Mycophenolate mofetil (MMF) is an immunosuppressive drug that warrants assessment as a viable therapeutic agent for horses. Hypothesis/Objectives To evaluate the pharmacokinetics (PK) of multiple‐day oral dosing of MMF in healthy horses and to determine the tolerability of this dosing regimen. Animals Six healthy Standardbred mares. Methods Horses received MMF 10 mg/kg PO q12h for 7 days in the fed state. Serial sampling was performed over 12 hours on Days 1 and 7 with trough samples collected every 24 hours, immediately before morning drug administration. Noncompartmental PK analyses were performed to determine primary PK parameters, followed by calculation of geometric means and coefficients of variation. A CBC, serum biochemical profile, physical examination, and fecal scoring were used to assess dose tolerability. Results Seven days of treatment resulted in a mycophenolic acid (MPA) area under the curve (AUC0‐12) of 12 594 h × ng/mL (8567‐19 488 h × ng/mL) and terminal half‐life (T1/2) of 11.3 hours (7.5‐15.9 hours), yielding minor metabolite accumulation in all horses treated. Salmonellosis was detected in the feces of 2 horses by Day 7, and all horses developed myelosuppression, hyperbilirubinemia, hyporexia, decreased gastrointestinal motility, and decreased fecal output by the seventh day of treatment. Conclusion and Clinical Importance Administration of MMF at 10 mg/kg PO q12h resulted in hematologic and clinical toxicity within 1 week of treatment. A decreased MMF dose, frequency, or both is needed to avoid colic. Drug monitoring should include frequent hemograms, serum biochemical profiles, and strict biosecurity protocols.

Bioactive components from dietary supplements such as curcumin may represent attractive agents for cancer prevention or treatment. DNA methylation plays a critical role in acute myeloid leukemia (AML) development, and presents an excellent target for treatment of this disease. However, it remains largely unknown how curcumin, a component of the popular Indian spice turmeric, plays a role in DNA hypomethylation to reactivate silenced tumor suppressor genes and to present a potential treatment option for AML. Here we show that curcumin down-regulates DNMT1 expression in AML cell lines, both in vitro and in vivo, and in primary AML cells ex vivo. Mechanistically, curcumin reduced the expression of positive regulators of DNMT1, p65 and Sp1, which correlated with a reduction in binding of these transcription factors to the DNMT1 promoter in AML cell lines. This curcumin-mediated down-regulation of DNMT1 expression was concomitant with p15(INK4B) tumor suppressor gene reactivation, hypomethylation of the p15(INK4B) promoter, G1 cell cycle arrest, and induction of tumor cell apoptosis in vitro. In mice implanted with the human AML MV4-11 cell line, administration of curcumin resulted in remarkable suppression of AML tumor growth. Collectively, our data indicate that curcumin shows promise as a potential treatment for AML, and our findings provide a basis for future studies to test the clinical efficacy of curcumin - whether used as a single agent or as an adjuvant - for AML treatment.

Triphala churna (THL) is a combination of three fruits that has been used for many years in India for the treatment of various diseases. There are now reports which indicate that THL can inhibit growth of malignant tumors in animals. However, the mechanisms by which THL mediates its anti-tumor actions are still being explored. Because vascular endothelial growth factor-A (VEGF) induced angiogenesis plays a critical role in the pathogenesis of cancer, we therefore investigated whether tumor inhibitory effects of THL or its active constituents are through suppression of VEGF actions. We herein report that THL and chebulinic (CI) present in THL can significantly and specifically inhibit VEGF induced angiogenesis by suppressing VEGF receptor-2 (VEGFR-2) phosphorylation. These results are of clinical significance as these inexpensive and non-toxic natural products can be used for the prevention and treatment of diseases where VEGF induced angiogenesis has an important role.

The study of chloroplast genome evolutionary dynamics provides critical insights into plant adaptive evolution and phylogenetic relationships. This research conducted a systematic comparative analysis of chloroplast genomes across 35 species within the Rutaceae family. All genomes displayed the typical quadripartite structure, with sizes ranging from 155 to 161 kb, GC contents between 38.17% and 38.83%, and gene counts varying from 122 to 144. Structural conservation was high across species, with variations mainly localized at the boundaries of inverted repeat (IR) regions. AT-rich mononucleotide simple sequence repeats (SSRs) were dominant and primarily distributed in non-coding regions. Collinearity analysis revealed high sequence conservation alongside lineage-specific rearrangements. Relative synonymous codon usage (RSCU) analysis revealed significant heterogeneity among species, with values ranging from 0.386 to 1.797. ENC-GC3s, GC3-GC12, and PR2 analyses indicated a marked deviation from neutral evolution. Selection pressure analysis indicated strong purifying selection (Ka/Ks < 0.2) acting on photosynthetic system genes, while certain genes (e.g., matK , rpl20 ) exhibited signals of positive selection, highlighting adaptive evolutionary features in specific genomic regions. Phylogenetic reconstruction placed Murraya paniculata within a clade containing other Murraya species, closely related to Citrus and Clausena , reflecting morphological and biogeographic patterns. This study provides a molecular framework for taxonomic revision in Rutaceae and enhances understanding of chloroplast genome evolution in the family.

Rapid urbanization and increasing land scarcity have made urban agriculture and efficient space utilization critical directions in modern agriculture. Ougan, a fruit tree valued for both its economic and ecological benefits, holds significant promise for dwarfing cultivation techniques. In this study, a root-irrigation method was used to apply paclobutrazol at various concentrations (200, 500, 1000, 1500, and 2000 mg/L) to Ougan seedlings, with a control group for comparison. Growth parameters include an average daily increase of plant height, stem girth, new branches, and new branch girth, as well as physiological indices such as leaf SPAD values, leaf nitrogen content, net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate, were measured during both spring and summer growth periods. The results demonstrate that PBZ exerts a distinct concentration-dependent regulatory effect on Ougan growth: higher concentrations significantly inhibited plant height while promoting increases in stem diameter, with several parameters exhibiting a unimodal response. Short-term (spring) PBZ application enhanced certain photosynthetic parameters, such as net photosynthetic rate and stomatal conductance; however, prolonged exposure (summer) resulted in a decline in photosynthetic efficiency and overall leaf physiological status. Through comprehensive evaluation using principal component analysis and PLS-SEM, the 500 mg/L PBZ treatment was identified as achieving the optimal balance between growth inhibition and the maintenance of photosynthetic and nutritional status, closely approximating the ideal dwarfing effect. This study elucidates the complex regulatory effects of PBZ on the growth, photosynthesis, and carbon assimilation of Ougan through natural climate, providing robust technical parameters and theoretical support for future dwarf cultivation practices. These findings facilitate the development of dwarf fruit trees into bonsai vegetation, demonstrating significant horticultural application potential.

Concentrative nucleoside transporters (CNTs) are active nucleoside influx systems, but their in vivo roles are poorly defined. By generating CNT1 knockout (KO) mice, here we identify a role of CNT1 in the renal reabsorption of nucleosides. Deletion of CNT1 in mice increases the urinary excretion of endogenous pyrimidine nucleosides with compensatory alterations in purine nucleoside metabolism. In addition, CNT1 KO mice exhibits high urinary excretion of the nucleoside analog gemcitabine (dFdC), which results in poor tumor growth control in CNT1 KO mice harboring syngeneic pancreatic tumors. Interestingly, increasing the dFdC dose to attain an area under the concentration-time curve level equivalent to that achieved by wild-type (WT) mice rescues antitumor efficacy. The findings provide new insights into how CNT1 regulates reabsorption of endogenous and synthetic nucleosides in murine kidneys and suggest that the functional status of CNTs may account for the optimal action of pyrimidine nucleoside analog therapeutics in humans. Concentrative nucleoside transporters (CNTs) are cellular nucleoside influx systems, but their in vivo roles are poorly defined. By generating CNT1 knockout (KO) mice, here the authors show a role of CNT1 in the renal reabsorption of endogenous and synthetic nucleosides.

BackgroundCancer cachexia is a multifactorial syndrome characterized by irreversible loss of skeletal muscle and adipose tissue resulting from increased catabolism and metabolic activity.1Patients with cachexia are generally resistant to immune checkpoint inhibitor (ICI) therapy while also displaying elevated monoclonal antibody (mAb) catabolic clearance (CL), and this increased CL serves as a prognostic indicator of overall survival, independent of dose received.2 3 Increased ICI CL is also replicated in the murine Lewis lung carcinoma (LLC) model of cancer cachexia but is absent in the non-cachectic MC38 colon adenocarcinoma model.4 Elevated ICI CL is independent of mAb variable region and target binding, suggesting an important role for the Fc constant region and Fc domain binding to Fc receptors (FcRs). FcRs (FcRn + FcgRs) also play a critical role in both the pharmacokinetics (PK) and efficacy of ICI therapy.5 6 We sought to understand cachexia-associated changes in leukocyte FcR expression as it pertains to ICI PK and efficacy.MethodsMass cytometry time-of-flight (CyTOF) was used to assess circulating immune cells and FcR expression in peripheral blood mononuclear cells (PBMCs) from cancer patients and in mouse splenocytes. Healthy C57BL/6 mice and mice with LLC or MC38 tumors were used to investigate the effect of cachexia on FcR expression. PBMCs were obtained from patients receiving either pembrolizumab, nivolumab, or combination nivolumab and ipilimumab for the treatment of non-small cell lung cancer or renal cell carcinoma. Cachexia in these patients was assessed using L3 CT image analysis of skeletal muscle index (SMI).7 ResultsMany cachexia associated changes in leukocyte FcR expression were observed. Both FcgRI8 and FcgRIIb9 10 play a role in ICI PK and efficacy, and both illustrate a general inverse trend between expression and cachexia severity with less expression in cachectic patients and animals. We also observed trends between FcR expression and ICI CL that could potentially explain observed differences in CL in clinical patients. Cachectic mice displayed decreased circulating CD8+ T cells, which was also supported in humans in which patients displaying low SMI had lower levels of CD8+ T cells at the time of ICI therapy initiation.ConclusionsMany studies have highlighted the importance of Fc and FcR interaction on the therapeutic activity and PK of ICIs, however no studies have characterized expressional changes in FcRs as a function of disease state. Here, we highlight how disease state can mediate FcR expressional changes in patterns that can potentially explain altered ICI PK and efficacy.ReferencesBaracos VE, et al. Cancer-associated cachexia. Nat Rev Dis Primers, 2018;4:17105.Shiroyama T, et al. Impact of sarcopenia in patients with advanced non-small cell lung cancer treated with PD-1 inhibitors: A preliminary retrospective study. Sci Rep, 2019;9(1):2447.Turner DC, et al. Pembrolizumab Exposure-Response Assessments Challenged by Association of Cancer Cachexia and Catabolic Clearance. Clin Cancer Res, 2018;24(23):5841–5849.Castillo AMM, et al. Murine cancer cachexia models replicate elevated catabolic pembrolizumab clearance in humans. JCSM Rapid Commun, 2021;4(2):232–244.Challa DK, et al. Neonatal Fc receptor expression in macrophages is indispensable for IgG homeostasis. MAbs, 2019;11(5):848–860.Dahan R, et al. FcgammaRs Modulate the Anti-tumor Activity of Antibodies Targeting the PD-1/PD-L1 Axis. Cancer Cell, 2015;28(3):285–95.Mourtzakis M, et al. A practical and precise approach to quantification of body composition in cancer patients using computed tomography images acquired during routine care. Appl Physiol Nutr Metab, 2008;33(5):997–1006.Zhang T, et al. The binding of an anti-PD-1 antibody to FcgammaRIota has a profound impact on its biological functions. Cancer Immunol Immunother, 2018;67(7):1079–1090.Arlauckas SP, et al. In vivo imaging reveals a tumor-associated macrophage-mediated resistance pathway in anti-PD-1 therapy. Sci Transl Med, 2017;9(389).Oldham RJ, et al. FcgammaRII (CD32) modulates antibody clearance in NOD SCID mice leading to impaired antibody-mediated tumor cell deletion. J Immunother Cancer, 2020;8(1).Ethics ApprovalAnimal experiments were conducted according to protocols approved by The Ohio State University Institutional Animal Care and Use Committee (protocol #2017A00000117). Human studies were approved by The Ohio State University Cancer Institutional Review Board, approval #2020C0048.