Metadichol® induced expression of TLR family members in peripheral blood mononuclear cells
Main Article Content
Abstract
Introduction: Toll receptors are vital to the innate immune system. They recognize common microbial pathogen molecular patterns. The signaling pathways activated produce proinflammatory cytokines and type I interferons to start the immune response to infection. Inflammatory, autoimmune, and cancer diseases are connected to signaling abnormalities. Thus, toll receptor expression patterns and control mechanisms are essential for immune response research and treatment.
Methods: Metadichol, a nanoemulsion of long-chain alcohol, was applied to peripheral blood mononuclear cells to evaluate the expression of all ten toll receptor family members (TLR1-10), MYD88, and downstream genes (IRAK4, TRAF3, TRAF6 and TRIF). Quantitative real-time PCR measured gene expression.
Results: Toll receptors 1–10 responded as inverted U-shaped to Metadichol treatment, except Toll receptor 4. Metadichol affects TLR expression differently at low, moderate, and high dosages. Metadichol activated all 15 genes, including TLRs and downstream signalizing molecules.
Importing 15 genes into Pathway Studio produced a gene expression network analysis. Gene set enrichment analysis (GSEA) used the proprietary Elsevier pathway collection. An enriched gene list with more gene interactions than expected for a random gene collection of similar size and distribution indicated a significant biological relationship between these genes.
Conclusions: Metadichol expresses all the toll receptors, the MYD88 gene, and four other downfield genes. Since all immune cells express TLRs, this leads to a more robust solution for activating innate and adaptive immunity processes in humans.
Keywords:
Toll receptors 1-10, Metadichol, Immune responses, MYD88, IRAK4, TRAF3, TRIF, PBMCs
Article Details
How to Cite
RAGHAVAN, P. R..
Metadichol® induced expression of TLR family members in peripheral blood mononuclear cells.
Medical Research Archives, [S.l.], v. 12, n. 9, sep. 2024.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/5610>. Date accessed: 04 oct. 2024.
doi: https://doi.org/10.18103/mra.v12i9.5610.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
1.Kawasaki, T., Kawai, T., 2014. Toll-like receptor signaling pathways. Front. Immunol. 5, 461. https://doi.org/10.3389/fimmu.2014.00461.
2. Brennan, J.J., Gilmore, T.D., 2018. Evolutionary origins of Toll-like receptor signaling. Mol. Biol. Evol. 35, 1576–1587. https://doi.org/10.1093/molbev/msy050.
3. Li, J., Yang, F., Wei, F., Ren, X., 2017. The role of toll-like receptor 4 in tumor microenvironment. Oncotarget. 8, 66656–66667. https://doi.org/10.18632/oncotarget.19105.
4. Botos, I., Segal, D.M., Davies, D.R., 2011. The structural biology of Toll-like receptors. Structure. 19, 447–459. https://doi.org/10.1016/j.str.2011.02.004.
5. Kawai, T., Akira, S., 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373–384. https://doi.org/10.1038/ni.1863.5.
6. Honda, K., Taniguchi, T., 2006. IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat. Rev. Immunol. 6, 644–658. https://doi.org/10.1038/nri1900.
7. Sameer, A.S., Nissar, S., 2021. Toll-like receptors (TLRs): structure, functions, signaling, and role of their polymorphisms in colorectal cancer susceptibility. BioMed Res. Int. 2021, 1157023. https://doi.org/10.1155/2021/1157023.
8. Li, T.-T., Ogino, S., Qian, Z.R., 2014. Toll-like receptor signaling in colorectal cancer: carcinogenesis to cancer therapy. World J. Gastroenterol. 20, 17699–17708. https://doi.org/10.3748/wjg.v20.i47.17699.
9. Vijay, K., 2018. Toll-like receptors in immunity and inflammatory diseases: past, present, and future. Int. Immunopharmacol. 59, 391–412. https://doi.org/10.1016/j.intimp.2018.03.002.
10. Jin, M., Fang, J., Wang, J.-J., Shao, X., Xu, S.-W., Liu, P.-Q., Ye, W.-C., Liu, Z.-P., 2023. Regulation of Toll-like receptor (TLR) signaling pathways in atherosclerosis: from mechanisms to targeted therapeutics. Acta Pharmacol. Sin. 44, 2358–2375. https://doi.org/10.1038/s41401-023-01123-5.
11. Duan, T., Du, Y., Xing, C., Wang, H.Y., Wang, R.-F., 2022. Toll-like receptor signaling and its role in cell-mediated immunity. Front. Immunol. 13, 812774. https://doi.org/10.3389/fimmu.2022.812774.
12. El-Zayat, S.R., Sibaii, H., Mannaa, F.A., 2019. Toll-like receptors activation, signaling, and targeting: an overview. Bull. Natl. Res. Cent. 43, 187. https://doi.org/10.1186/s42269-019-0227-2.
13. Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O., Sugiyama, M., Okabe, M., Takeda, K., Akira, S., 2003. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science. 301, 640–643. https://doi.org/10.1126/science.1087262.
14. Gupta, S., Tsoporis, J.N., Jia, S.-H., dos Santos, C.C., Parker, T.G., Marshall, J.C., 2020. Toll-like receptors, associated biochemical signaling networks, and S100 ligands. Shock. 56, 167–177. https://doi.org/10.1097/shk.0000000000001704.
15. Klonowska-Szymczyk, A., Wolska, A., Robak, T., Cebula-Obrzut, B., Smolewski, P., Robak, E., 2014. Expression of Toll-like receptors 3, 7, and 9 in peripheral blood mononuclear cells from patients with systemic lupus erythematosus. Mediat. Inflamm. 2014, 381418. https://doi.org/10.1155/2014/381418.
16. Sánchez-Cuaxospa, M., Contreras-Ramos, A., Pérez-Figueroa, E., Medina-Sansón, A., Jiménez-Hernández, E., Torres-Nava, J.R., Rojas-Castillo, E., Maldonado-Bernal, C., 2016. Low expression of Toll-like receptors in peripheral blood mononuclear cells of pediatric patients with acute lymphoblastic leukemia. Int. J. Oncol. 49, 675–681. https://doi.org/10.3892/ijo.2016.3569.
17. Zhang, Y., Liang, X., Bao, X., Xiao, W., Chen, G., 2022. Toll-like receptor 4 (TLR4) inhibitors: current research and prospective. Eur. J. Med. Chem. 235, 114291. https://doi.org/10.1016/j.ejmech.2022.114291.
18. Peng, S., Li, C., Wang, X., Liu, X., Han, C., Jin, T., Liu, S., Zhang, X., Zhang, H., He, X., Xie, X., Yu, X., Wang, C., Shan, L., Fan, C., Shan, Z., Teng, W., 2016. Increased Toll-like receptors activity and TLR ligands in patients with autoimmune thyroid diseases. Front. Immunol. 7, 578. https://doi.org/10.3389/fimmu.2016.00578.
19. Shukla, N.M., Chan, M., Hayashi, T., Carson, D.A., Cottam, H.B., 2018. Recent advances and perspectives in small-molecule TLR ligands and their modulators. ACS Med. Chem. Lett. 9, 1156–1159. https://doi.org/10.1021/acsmedchemlett.8b00566.
20. Keshavarz, A., Pourbagheri‐Sigaroodi, A., Zafari, P., Bagheri, N., Ghaffari, S.H., Bashash, D., 2021. Toll‐like receptors (TLRs) in cancer; with an extensive focus on TLR agonists and antagonists. IUBMB Life. 73, 10–25. https://doi.org/10.1002/iub.2412.
21. Raghavan, P.R., US Patents: 2014; 8 722 093; 2015; 9034383 383; 2015; 9,006,292.
22. Wallace, R., 2020. Signal transduction in cognitive systems: origin and dynamics of the inverted-U/U dose-response relations. J. Theor. Biol. 504, 110377. https://doi.org/10.1016/j.jtbi.2020.110377.
23. Reding, C., Catalán, P., Jansen, G., Bergmiller, T., Wood, E., Rosenstiel, P., Schulenburg, H., Gudelj, I., Beardmore, R., 2021. The antibiotic dosage of fastest resistance evolution: gene amplifications underpinning the inverted-U. Mol. Biol. Evol. 38, 3847–3863. https://doi.org/10.1093/molbev/msab025.
24. Sourbron, J., Lagae, L., 2023. Fenfluramine: a plethora of mechanisms? Front. Pharmacol. 14, 1192022. https://doi.org/10.3389/fphar.2023.1192022.
25. Raghavan, P.R., 2023. Metadichol® a nano lipid emulsion that expresses all 49 nuclear receptors in stem and somatic cells. Arch. Clin. Biomed. Res. 7, 524–536. https://doi.org/10.26502/acbr.50170370.
26. Huang, W., Glass, C.K., 2010. Nuclear receptors and inflammation control: molecular mechanisms and pathophysiological relevance. Arterioscler. Thromb. Vasc. Biol. 30, 1542–1549. https://doi.org/10.1161/ATVBAHA.109.191189.
27. Lind, N.A., Rael, V.E., Pestal, K., Liu, B., Barton, G.M., 2022. Regulation of the nucleic acid-sensing Toll-like receptors. Nat. Rev. Immunol. 22, 224–235. https://doi.org/10.1038/s41577-021-00577-0.
28. Ito, A., Hong, C., Rong, X., Zhu, X., Tarling, E.J., Hedde, P.N., Gratton, E., Parks, J., Tontonoz, P., 2015. LXRs link metabolism to inflammation through Abca1-dependent regulation of membrane composition and TLR signaling. eLife. 4, e08009. https://doi.org/10.7554/eLife.08009.
29. Li, N., Li, Y., Han, X., Zhang, J., Han, J., Jiang, X., Wang, W., Xu, Y., Xu, Y., Fu, Y., Si, S., 2022. LXR agonist inhibits inflammation through regulating MyD88 mRNA alternative splicing. Front. Pharmacol. 13, 973612. https://doi.org/10.3389/fphar.2022.973612.
30. Kado, S., Chang, W.L.W., Chi, A.N., Wolny, M., Shepherd, D.M., Vogel, C.F.A., 2017. Aryl hydrocarbon receptor signaling modifies Toll-like receptor-regulated responses in human dendritic cells. Arch. Toxicol. 91, 2209–2221. https://doi.org/10.1007/s00204-016-1880-y.
31. Cannon, A.S., Nagarkatti, P.S., Nagarkatti, M., 2021. Targeting AhR as a novel therapeutic modality against inflammatory diseases. Int. J. Mol. Sci. 23, 288. https://doi.org/10.3390/ijms23010288.
32. Neavin, D.R., Liu, D., Ray, B., Weinshilboum, R.M., 2018. The role of the Aryl Hydrocarbon Receptor (AHR) in immune and inflammatory diseases. Int. J. Mol. Sci. 19, 3851. https://doi.org/10.3390/ijms19123851.
33. Raghavan, P.R., 2017. Metadichol ®. A novel inverse agonist of aryl hydrocarbon receptor (AHR) and NRF2 inhibitor. J. Cancer Sci. Ther. 9, 661–668. https://doi.org/10.4172/1948-5956.1000489.
34. Ma, S., Patel, S.A., Abe, Y., Chen, N., Patel, P.R., Cho, B.S., Abbasi, N., Zeng, S., Schnabl, B., Chang, J.T., Huang, W.J.M., 2022. RORγt phosphorylation protects against T cell-mediated inflammation. Cell Rep. 38, 110520.
35. Heine, H., Zamyatina, A., 2022. Therapeutic targeting of TLR4 for inflammation, infection, and cancer: a perspective for disaccharide lipid a mimetics. Pharmaceuticals. 16, 23. https://doi.org/10.3390/ph16010023.
36. Yang, Y., Lv, J., Jiang, S., Ma, Z., Wang, D., Hu, W., Deng, C., Fan, C., Di, S., Sun, Y., Yi, W., 2016. The emerging role of Toll-like receptor 4 in myocardial inflammation. Cell Death Dis. 7, e2234. https://doi.org/10.1038/cddis.2016.140.
37. Kim, H.-J., Kim, H., Lee, J.-H., Hwangbo, C., 2023. Toll-like receptor 4 (TLR4): new insight immune and aging. Immun. Ageing. 20, 67. https://doi.org/10.1186/s12979-023-00383-3.
38. Gao, W., Xiong, Y., Li, Q., Yang, H., 2017. Inhibition of Toll-like receptor signaling as a promising therapy for inflammatory diseases: a journey from molecular to nano therapeutics. Front. Physiol. 8, 508. https://doi.org/10.3389/fphys.2017.00508.
39. Raghavan, P.R., 2022. Metadichol®: a novel nanolipid formulation that inhibits SARS-CoV-2 and a multitude of pathological viruses in vitro. BioMed Res. Int. 2022, 1558860. https://doi.org/10.1155/2022/1558860.
40. Liang, H., Sathavarodom, N., Colmenares, C., Gelfond, J., Espinoza, S.E., Ganapathy, V., Musi, N., 2022. Effect of acute TLR4 inhibition on insulin resistance in humans. J. Clin. Investig. 132, e162291. https://doi.org/10.1172/JCI162291.
41. Oladiran, O., Shi, X.Q., Yang, M., Fournier, S., Zhang, J., 2021. Inhibition of TLR4 signaling protects mice from sensory and motor dysfunction in an animal model of autoimmune peripheral neuropathy. J. Neuroinflammation. 18, 77. https://doi.org/10.1186/s12974-021-02126-x.
42. Cui, W., Sun, C., Ma, Y., Wang, S., Wang, X., Zhang, Y., 2020. Inhibition of TLR4 induces M2 microglial polarization and provides neuroprotection via the NLRP3 inflammasome in Alzheimer's disease. Front. Neurosci. 14, 444. https://doi.org/10.3389/fnins.2020.00444.
43. Yesudhas, D., Gosu, V., Anwar, M.A., Choi, S., 2014. Multiple roles of toll-like receptor 4 in colorectal cancer. Front. Immunol. 5, 334. https://doi.org/10.3389/fimmu.2014.00334.
44. Davis, M.B., Vasquez-Dunddel, D., Fu, J., Albesiano, E., Pardoll, D., Kim, Y.J., 2011. Intratumoral administration of TLR4 agonist absorbed into a cellular vector improves antitumor responses. Clin. Cancer Res. 17, 3984–3992. https://doi.org/10.1158/1078-0432.CCR-10-3262.
45. Triozzi, P.L., Aldrich, W., Ponnazhagan, S., 2011. Inhibition and promotion of tumor growth with adeno-associated virus carcinoembryonic antigen vaccine and Toll-like receptor agonists. Cancer Gene Ther. 18, 850–858. https://doi.org/10.1038/cgt.2011.54.
46. Jego, G., Bataille, R., Geffroy-Luseau, A., Descamps, G., Pellat-Deceunynck, C., 2006. Pathogen-associated molecular patterns are growth and survival factors for human myeloma cells through Toll-like receptors. Leukemia. 20, 1130–1137. https://doi.org/10.1038/sj.leu.2404226.
47. Cai, Z., Sanchez, A., Shi, Z., Zhang, T., Liu, M., Zhang, D., 2011. Activation of Toll-like receptor 5 on breast cancer cells by flagellin suppresses cell proliferation and tumor growth. Cancer Res. 71, 2466–2475. https://doi.org/10.1158/0008-5472.CAN-10-1993.
48. Kaczanowska, S., Joseph, A.M., Davila, E., 2013. TLR agonists: our best frenemy in cancer immunotherapy. J. Leukoc. Biol. 93, 847–863. https://doi.org/10.1189/jlb.101250.
49. Fehri, E., Ennaifer, E., Haj Rhouma, R.B., Ardhaoui, M., Boubaker, S., 2022. TLR9 and glioma: friends or foes? Cells. 12, 152. https://doi.org/10.3390/cells12010152.
50. Li, X., Liu, D., Liu, X., Jiang, W., Zhou, W., Yan, W., Cen, Y., Li, B., Cao, G., Ding, G., Pang, X., Sun, J., Zheng, J., Zhou, H., 2012. CpG ODN107 potentiates radiosensitivity of human glioma cells via TLR9-mediated NF-κB activation and NO production. Tumor Biol. 33, 1607–1618. https://doi.org/10.1007/s13277-012-0416-1.
51. Patinote, C., Karroum, N.B., Moarbess, G., Cirnat, N., Kassab, I., Bonnet, P.-A., Deleuze-Masquéfa, C., 2020. Agonist and antagonist ligands of Toll-like receptors 7 and 8: ingenious tools for therapeutic purposes. Eur. J. Med. Chem. 193, 112238. https://doi.org/10.1016/j.ejmech.2020.112238.
52. Coussens, L.M., Werb, Z., 2002. Inflammation and cancer. Nature. 420, 860–867. https://doi.org/10.1038/nature01322.
53. Rakoff-Nahoum, S., Medzhitov, R., 2008. Toll-like receptors and cancer. Nat. Rev. Cancer. 9, 57–63. https://doi.org/10.1038/nrc2541.
54. Oosting, M., Cheng, S.-C., Bolscher, J.M., Vestering-Stenger, R., Plantinga, T.S., Verschueren, I.C., Arts, P., Garritsen, A., van Eenennaam, H., Sturm, P., Kullberg, B.-J., Hoischen, A., Adema, G.J., van der Meer, J.W.M., Netea, M.G., Joosten, L.A.B., 2014. Human TLR10 is an anti-inflammatory pattern-recognition receptor. Proc. Natl. Acad. Sci. U.S.A. 111, E4478–E4484. https://doi.org/10.1073/pnas.1410293111.
55. Fore, F., Indriputri, C., Mamutse, J., Nugraha, J., 2020. TLR10 and its unique anti-inflammatory properties and potential use as a target in therapeutics. Immune Netw. 20, e21. https://doi.org/10.4110/in.2020.20.e21.
56. Hasan, U., Chaffois, C., Gaillard, C., Saulnier, V., Merck, E., Tancredi, S., Guiet, C., Brière, F., Vlach, J., Lebecque, S., Trinchieri, G., Bates, E.E.M., 2005. Human TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through MyD88. J. Immunol. 174, 2942–2950. https://doi.org/10.4049/jimmunol.174.5.2942.
57. Torices, S., Julia, A., Muñoz, P., Varela, I., Balsa, A., Marsal, S., Fernández-Nebro, A., Blanco, F., López-Hoyos, M., Martinez-Taboada, V., Fernández-Luna, J.L., 2016. A functional variant of TLR10 modifies the activity of NFkB and may help predict a worse prognosis in patients with rheumatoid arthritis. Arthritis Res. Ther. 18, 221. https://doi.org/10.1186/s13075-016-1113-z.
58. Shaw, A.C., Panda, A., Joshi, S.R., Qian, F., Allore, H.G., Montgomery, R.R., 2011. Dysregulation of human Toll-like receptor function in aging. Ageing Res. Rev. 10, 346–353. https://doi.org/10.1016/j.arr.2010.10.007.
59. Van Duin, D., Mohanty, S., Thomas, V., Ginter, S., Montgomery, R.R., Fikrig, E., Allore, H.G., Medzhitov, R., Shaw, A.C., 2007. Age-associated defect in human TLR-1/2 function. J. Immunol. 178, 970–975. https://doi.org/10.4049/jimmunol.178.2.970.
60. Kim, H.J., Kim, S.H., Je, J.H., Shin, D.Y., Kim, D.S., Lee, M.G., 2016. Increased expression of Toll‐like receptors 3, 7, 8 and 9 in peripheral blood mononuclear cells in patients with psoriasis. Experimental Dermatology. 25, 485–487. https://doi.org/10.1111/exd.12974.
61. Khanna, V., Kim, H., Zhang, W., Larson, P., Shah, M., Griffith, T.S., Ferguson, D., Panyam, J., 2021. Novel TLR 7/8 agonists for improving NK cell mediated antibody-dependent cellular cytotoxicity (ADCC). Sci. Rep. 11, 3346. https://doi.org/10.1038/s41598-021-83005-6.
62. Yang, Y., Wang, Y., Li, L., Chen, F., Zhang, P., 2017. Activation of the Toll-like receptor 8 pathway increases the immunogenicity of mesenchymal stem cells from umbilical cord. Mol. Med. Rep. 16, 2061–2068. https://doi.org/10.3892/mmr.2017.6806.
63. Vaure, C., Liu, Y., 2014. A comparative review of toll-like receptor 4 expression and functionality in different animal species. Front. Immunol. 5, 316. https://doi.org/10.3389/fimmu.2014.00316.
64. Liu, M., Zen, K., 2021. Toll-like receptors regulate the development and progression of renal diseases. Kidney Dis. 7, 14–23. https://doi.org/10.1159/000511947.
65. Kanehisa, M., 2019. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 28, 1947–1951. https://doi.org/10.1002/pro.3715.
66. Kanehisa, M., Goto, S., 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30. https://doi.org/10.1093/nar/28.1.27.
67. Nikitin, A., Egorov, S., Daraselia, N., Mazo, I., 2003. Pathway studio—the analysis and navigation of molecular networks. Bioinformatics. 19, 2155–2157. https://doi.org/10.1093/bioinformatics/btg290.
68. Sharma, A., Costantini, S., Colonna, G., 2013. The protein–protein interaction network of the human sirtuin family. Biochim. Biophys. Acta Proteins Proteom. 1834, 1998–2009. https://doi.org/10.1016/j.bbapap.2013.06.012.
69. Tomczak, A., Mortensen, J.M., Winnenburg, R., Liu, C., Alessi, D.T., Swamy, V., Vallania, F., Lofgren, S., Haynes, W., Shah, N.H., Musen, M.A., Khatri, P., 2018. Interpretation of biological experiments changes with evolution of the Gene Ontology and its annotations. Sci. Rep. 8, 5115. https://doi.org/10.1038/s41598-018-23395-2.
70. Nie, L., Cai, S.-Y., Shao, J.-Z., Chen, J., 2016. Toll-like receptors, associated biological roles, and signaling networks in non-mammals. Front. Immunol. 9, 1523. https://doi.org/10.3389/fimmu.2018.01523.
71. Raghavan, P.R., 2023. Metadichol® induced the expression of neuronal transcription factors in human fibroblast dermal cells. J. Bioinform. Syst. Biol. 6, 326–339. https://doi.org/10.26502/jbsb.5107066.
72. Raghavan, P.R., 2024a. Metadichol, a natural ligand for the expression of Yamanaka reprogramming factors in human cardiac, fibroblast, and cancer cell lines. Med. Res. Arch. 12. https://doi.org/10.18103/mra.v12i6.5323.
73. Raghavan, P.R., 2023. Metadichol treatment of fibroblasts and embryonic stem cells regulates key cardiac progenitors. Cardiol. Cardiovasc. Med. 7, 322–330. https://doi.org/10.26502/fccm.92920340.
74. Raghavan, P.R., 2024b. Metadichol®-induced expression of sirtuin 1-7 in somatic and cancer cells. Med. Res. Arch. 12. https://doi.org/10.18103/mra.v12i6.0000.
75. Raghavan, P.R., 2018. A multi gene targeting approach to treating liver diseases with Metadichol®. J. Cytokine Biol. 3, 126. https://doi.org/10.4172/2576-3881.1000126.
76. Morphy, R., Rankovic, Z., 2005. Designed multiple ligands. An emerging drug discovery paradigm. J. Med. Chem. 48, 6523–6543. https://doi.org/10.1021/jm058225d.
77. Roth, B.L., Sheffler, D.J., Kroeze, W.K., 2004. Magic shotguns versus magic bullets: selectively non-selective drugs for mood disorders and schizophrenia. Nat. Rev. Drug Discov. 3, 353–359. https://doi.org/10.1038/nrd1346.
78. Montine, T.J., Larson, E.B., 2009. Late-life dementias: does this unyielding global challenge require a broader view? JAMA. 302, 2593–2594. https://doi.org/10.1001/jama.2009.1863.
79. Morphy, R., Kay, C., Rankovic, Z., 2004. From magic bullets to designed multiple ligands. Drug Discov. Today. 9, 641–651. https://doi.org/10.1016/s1359-6446(04)03163-0.
80. Hopkins, A.L., 2008. Network pharmacology: the next paradigm in drug discovery. Nat. Chem. Biol. 4, 682–690. https://doi.org/10.1038/nchembio.118.
81. Alemán, C., Mas, R., Hernandez, C., Rodeiro, I., Cerejido, E., Noa, M., Capote, A., Menendez, R., Amor, A., Fraga, V., 1994. A 12-month study of policosanol oral toxicity in Sprague Dawley rats. Toxicol. Lett. 70, 77–87. https://doi.org/10.1016/0378-4274(94)90147-3.
82. Alemán, C.L., Ferreiro, R.M., Puig, M.N., Guerra, I.R., Ortega, C.H., Capote, A., 1994b. Carcinogenicity of policosanol in sprague dawley rats: a 24 month study. Teratog. Carcinog. Mutagen. 14, 239–249. https://doi.org/10.1002/tcm.1770140505.
83. Alemán, C.L., Puig, M.N., Elias, E.C., Ortega, C.H., Guerra, I.R., Ferreiro, R.M., Briñis, F., 1995. Carcinogenicity of policosanol in mice: an 18-month study. Food Chem. Toxicol. 33, 573–578. https://doi.org/10.1016/0278-6915(95)00026-x.
2. Brennan, J.J., Gilmore, T.D., 2018. Evolutionary origins of Toll-like receptor signaling. Mol. Biol. Evol. 35, 1576–1587. https://doi.org/10.1093/molbev/msy050.
3. Li, J., Yang, F., Wei, F., Ren, X., 2017. The role of toll-like receptor 4 in tumor microenvironment. Oncotarget. 8, 66656–66667. https://doi.org/10.18632/oncotarget.19105.
4. Botos, I., Segal, D.M., Davies, D.R., 2011. The structural biology of Toll-like receptors. Structure. 19, 447–459. https://doi.org/10.1016/j.str.2011.02.004.
5. Kawai, T., Akira, S., 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373–384. https://doi.org/10.1038/ni.1863.5.
6. Honda, K., Taniguchi, T., 2006. IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat. Rev. Immunol. 6, 644–658. https://doi.org/10.1038/nri1900.
7. Sameer, A.S., Nissar, S., 2021. Toll-like receptors (TLRs): structure, functions, signaling, and role of their polymorphisms in colorectal cancer susceptibility. BioMed Res. Int. 2021, 1157023. https://doi.org/10.1155/2021/1157023.
8. Li, T.-T., Ogino, S., Qian, Z.R., 2014. Toll-like receptor signaling in colorectal cancer: carcinogenesis to cancer therapy. World J. Gastroenterol. 20, 17699–17708. https://doi.org/10.3748/wjg.v20.i47.17699.
9. Vijay, K., 2018. Toll-like receptors in immunity and inflammatory diseases: past, present, and future. Int. Immunopharmacol. 59, 391–412. https://doi.org/10.1016/j.intimp.2018.03.002.
10. Jin, M., Fang, J., Wang, J.-J., Shao, X., Xu, S.-W., Liu, P.-Q., Ye, W.-C., Liu, Z.-P., 2023. Regulation of Toll-like receptor (TLR) signaling pathways in atherosclerosis: from mechanisms to targeted therapeutics. Acta Pharmacol. Sin. 44, 2358–2375. https://doi.org/10.1038/s41401-023-01123-5.
11. Duan, T., Du, Y., Xing, C., Wang, H.Y., Wang, R.-F., 2022. Toll-like receptor signaling and its role in cell-mediated immunity. Front. Immunol. 13, 812774. https://doi.org/10.3389/fimmu.2022.812774.
12. El-Zayat, S.R., Sibaii, H., Mannaa, F.A., 2019. Toll-like receptors activation, signaling, and targeting: an overview. Bull. Natl. Res. Cent. 43, 187. https://doi.org/10.1186/s42269-019-0227-2.
13. Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O., Sugiyama, M., Okabe, M., Takeda, K., Akira, S., 2003. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science. 301, 640–643. https://doi.org/10.1126/science.1087262.
14. Gupta, S., Tsoporis, J.N., Jia, S.-H., dos Santos, C.C., Parker, T.G., Marshall, J.C., 2020. Toll-like receptors, associated biochemical signaling networks, and S100 ligands. Shock. 56, 167–177. https://doi.org/10.1097/shk.0000000000001704.
15. Klonowska-Szymczyk, A., Wolska, A., Robak, T., Cebula-Obrzut, B., Smolewski, P., Robak, E., 2014. Expression of Toll-like receptors 3, 7, and 9 in peripheral blood mononuclear cells from patients with systemic lupus erythematosus. Mediat. Inflamm. 2014, 381418. https://doi.org/10.1155/2014/381418.
16. Sánchez-Cuaxospa, M., Contreras-Ramos, A., Pérez-Figueroa, E., Medina-Sansón, A., Jiménez-Hernández, E., Torres-Nava, J.R., Rojas-Castillo, E., Maldonado-Bernal, C., 2016. Low expression of Toll-like receptors in peripheral blood mononuclear cells of pediatric patients with acute lymphoblastic leukemia. Int. J. Oncol. 49, 675–681. https://doi.org/10.3892/ijo.2016.3569.
17. Zhang, Y., Liang, X., Bao, X., Xiao, W., Chen, G., 2022. Toll-like receptor 4 (TLR4) inhibitors: current research and prospective. Eur. J. Med. Chem. 235, 114291. https://doi.org/10.1016/j.ejmech.2022.114291.
18. Peng, S., Li, C., Wang, X., Liu, X., Han, C., Jin, T., Liu, S., Zhang, X., Zhang, H., He, X., Xie, X., Yu, X., Wang, C., Shan, L., Fan, C., Shan, Z., Teng, W., 2016. Increased Toll-like receptors activity and TLR ligands in patients with autoimmune thyroid diseases. Front. Immunol. 7, 578. https://doi.org/10.3389/fimmu.2016.00578.
19. Shukla, N.M., Chan, M., Hayashi, T., Carson, D.A., Cottam, H.B., 2018. Recent advances and perspectives in small-molecule TLR ligands and their modulators. ACS Med. Chem. Lett. 9, 1156–1159. https://doi.org/10.1021/acsmedchemlett.8b00566.
20. Keshavarz, A., Pourbagheri‐Sigaroodi, A., Zafari, P., Bagheri, N., Ghaffari, S.H., Bashash, D., 2021. Toll‐like receptors (TLRs) in cancer; with an extensive focus on TLR agonists and antagonists. IUBMB Life. 73, 10–25. https://doi.org/10.1002/iub.2412.
21. Raghavan, P.R., US Patents: 2014; 8 722 093; 2015; 9034383 383; 2015; 9,006,292.
22. Wallace, R., 2020. Signal transduction in cognitive systems: origin and dynamics of the inverted-U/U dose-response relations. J. Theor. Biol. 504, 110377. https://doi.org/10.1016/j.jtbi.2020.110377.
23. Reding, C., Catalán, P., Jansen, G., Bergmiller, T., Wood, E., Rosenstiel, P., Schulenburg, H., Gudelj, I., Beardmore, R., 2021. The antibiotic dosage of fastest resistance evolution: gene amplifications underpinning the inverted-U. Mol. Biol. Evol. 38, 3847–3863. https://doi.org/10.1093/molbev/msab025.
24. Sourbron, J., Lagae, L., 2023. Fenfluramine: a plethora of mechanisms? Front. Pharmacol. 14, 1192022. https://doi.org/10.3389/fphar.2023.1192022.
25. Raghavan, P.R., 2023. Metadichol® a nano lipid emulsion that expresses all 49 nuclear receptors in stem and somatic cells. Arch. Clin. Biomed. Res. 7, 524–536. https://doi.org/10.26502/acbr.50170370.
26. Huang, W., Glass, C.K., 2010. Nuclear receptors and inflammation control: molecular mechanisms and pathophysiological relevance. Arterioscler. Thromb. Vasc. Biol. 30, 1542–1549. https://doi.org/10.1161/ATVBAHA.109.191189.
27. Lind, N.A., Rael, V.E., Pestal, K., Liu, B., Barton, G.M., 2022. Regulation of the nucleic acid-sensing Toll-like receptors. Nat. Rev. Immunol. 22, 224–235. https://doi.org/10.1038/s41577-021-00577-0.
28. Ito, A., Hong, C., Rong, X., Zhu, X., Tarling, E.J., Hedde, P.N., Gratton, E., Parks, J., Tontonoz, P., 2015. LXRs link metabolism to inflammation through Abca1-dependent regulation of membrane composition and TLR signaling. eLife. 4, e08009. https://doi.org/10.7554/eLife.08009.
29. Li, N., Li, Y., Han, X., Zhang, J., Han, J., Jiang, X., Wang, W., Xu, Y., Xu, Y., Fu, Y., Si, S., 2022. LXR agonist inhibits inflammation through regulating MyD88 mRNA alternative splicing. Front. Pharmacol. 13, 973612. https://doi.org/10.3389/fphar.2022.973612.
30. Kado, S., Chang, W.L.W., Chi, A.N., Wolny, M., Shepherd, D.M., Vogel, C.F.A., 2017. Aryl hydrocarbon receptor signaling modifies Toll-like receptor-regulated responses in human dendritic cells. Arch. Toxicol. 91, 2209–2221. https://doi.org/10.1007/s00204-016-1880-y.
31. Cannon, A.S., Nagarkatti, P.S., Nagarkatti, M., 2021. Targeting AhR as a novel therapeutic modality against inflammatory diseases. Int. J. Mol. Sci. 23, 288. https://doi.org/10.3390/ijms23010288.
32. Neavin, D.R., Liu, D., Ray, B., Weinshilboum, R.M., 2018. The role of the Aryl Hydrocarbon Receptor (AHR) in immune and inflammatory diseases. Int. J. Mol. Sci. 19, 3851. https://doi.org/10.3390/ijms19123851.
33. Raghavan, P.R., 2017. Metadichol ®. A novel inverse agonist of aryl hydrocarbon receptor (AHR) and NRF2 inhibitor. J. Cancer Sci. Ther. 9, 661–668. https://doi.org/10.4172/1948-5956.1000489.
34. Ma, S., Patel, S.A., Abe, Y., Chen, N., Patel, P.R., Cho, B.S., Abbasi, N., Zeng, S., Schnabl, B., Chang, J.T., Huang, W.J.M., 2022. RORγt phosphorylation protects against T cell-mediated inflammation. Cell Rep. 38, 110520.
35. Heine, H., Zamyatina, A., 2022. Therapeutic targeting of TLR4 for inflammation, infection, and cancer: a perspective for disaccharide lipid a mimetics. Pharmaceuticals. 16, 23. https://doi.org/10.3390/ph16010023.
36. Yang, Y., Lv, J., Jiang, S., Ma, Z., Wang, D., Hu, W., Deng, C., Fan, C., Di, S., Sun, Y., Yi, W., 2016. The emerging role of Toll-like receptor 4 in myocardial inflammation. Cell Death Dis. 7, e2234. https://doi.org/10.1038/cddis.2016.140.
37. Kim, H.-J., Kim, H., Lee, J.-H., Hwangbo, C., 2023. Toll-like receptor 4 (TLR4): new insight immune and aging. Immun. Ageing. 20, 67. https://doi.org/10.1186/s12979-023-00383-3.
38. Gao, W., Xiong, Y., Li, Q., Yang, H., 2017. Inhibition of Toll-like receptor signaling as a promising therapy for inflammatory diseases: a journey from molecular to nano therapeutics. Front. Physiol. 8, 508. https://doi.org/10.3389/fphys.2017.00508.
39. Raghavan, P.R., 2022. Metadichol®: a novel nanolipid formulation that inhibits SARS-CoV-2 and a multitude of pathological viruses in vitro. BioMed Res. Int. 2022, 1558860. https://doi.org/10.1155/2022/1558860.
40. Liang, H., Sathavarodom, N., Colmenares, C., Gelfond, J., Espinoza, S.E., Ganapathy, V., Musi, N., 2022. Effect of acute TLR4 inhibition on insulin resistance in humans. J. Clin. Investig. 132, e162291. https://doi.org/10.1172/JCI162291.
41. Oladiran, O., Shi, X.Q., Yang, M., Fournier, S., Zhang, J., 2021. Inhibition of TLR4 signaling protects mice from sensory and motor dysfunction in an animal model of autoimmune peripheral neuropathy. J. Neuroinflammation. 18, 77. https://doi.org/10.1186/s12974-021-02126-x.
42. Cui, W., Sun, C., Ma, Y., Wang, S., Wang, X., Zhang, Y., 2020. Inhibition of TLR4 induces M2 microglial polarization and provides neuroprotection via the NLRP3 inflammasome in Alzheimer's disease. Front. Neurosci. 14, 444. https://doi.org/10.3389/fnins.2020.00444.
43. Yesudhas, D., Gosu, V., Anwar, M.A., Choi, S., 2014. Multiple roles of toll-like receptor 4 in colorectal cancer. Front. Immunol. 5, 334. https://doi.org/10.3389/fimmu.2014.00334.
44. Davis, M.B., Vasquez-Dunddel, D., Fu, J., Albesiano, E., Pardoll, D., Kim, Y.J., 2011. Intratumoral administration of TLR4 agonist absorbed into a cellular vector improves antitumor responses. Clin. Cancer Res. 17, 3984–3992. https://doi.org/10.1158/1078-0432.CCR-10-3262.
45. Triozzi, P.L., Aldrich, W., Ponnazhagan, S., 2011. Inhibition and promotion of tumor growth with adeno-associated virus carcinoembryonic antigen vaccine and Toll-like receptor agonists. Cancer Gene Ther. 18, 850–858. https://doi.org/10.1038/cgt.2011.54.
46. Jego, G., Bataille, R., Geffroy-Luseau, A., Descamps, G., Pellat-Deceunynck, C., 2006. Pathogen-associated molecular patterns are growth and survival factors for human myeloma cells through Toll-like receptors. Leukemia. 20, 1130–1137. https://doi.org/10.1038/sj.leu.2404226.
47. Cai, Z., Sanchez, A., Shi, Z., Zhang, T., Liu, M., Zhang, D., 2011. Activation of Toll-like receptor 5 on breast cancer cells by flagellin suppresses cell proliferation and tumor growth. Cancer Res. 71, 2466–2475. https://doi.org/10.1158/0008-5472.CAN-10-1993.
48. Kaczanowska, S., Joseph, A.M., Davila, E., 2013. TLR agonists: our best frenemy in cancer immunotherapy. J. Leukoc. Biol. 93, 847–863. https://doi.org/10.1189/jlb.101250.
49. Fehri, E., Ennaifer, E., Haj Rhouma, R.B., Ardhaoui, M., Boubaker, S., 2022. TLR9 and glioma: friends or foes? Cells. 12, 152. https://doi.org/10.3390/cells12010152.
50. Li, X., Liu, D., Liu, X., Jiang, W., Zhou, W., Yan, W., Cen, Y., Li, B., Cao, G., Ding, G., Pang, X., Sun, J., Zheng, J., Zhou, H., 2012. CpG ODN107 potentiates radiosensitivity of human glioma cells via TLR9-mediated NF-κB activation and NO production. Tumor Biol. 33, 1607–1618. https://doi.org/10.1007/s13277-012-0416-1.
51. Patinote, C., Karroum, N.B., Moarbess, G., Cirnat, N., Kassab, I., Bonnet, P.-A., Deleuze-Masquéfa, C., 2020. Agonist and antagonist ligands of Toll-like receptors 7 and 8: ingenious tools for therapeutic purposes. Eur. J. Med. Chem. 193, 112238. https://doi.org/10.1016/j.ejmech.2020.112238.
52. Coussens, L.M., Werb, Z., 2002. Inflammation and cancer. Nature. 420, 860–867. https://doi.org/10.1038/nature01322.
53. Rakoff-Nahoum, S., Medzhitov, R., 2008. Toll-like receptors and cancer. Nat. Rev. Cancer. 9, 57–63. https://doi.org/10.1038/nrc2541.
54. Oosting, M., Cheng, S.-C., Bolscher, J.M., Vestering-Stenger, R., Plantinga, T.S., Verschueren, I.C., Arts, P., Garritsen, A., van Eenennaam, H., Sturm, P., Kullberg, B.-J., Hoischen, A., Adema, G.J., van der Meer, J.W.M., Netea, M.G., Joosten, L.A.B., 2014. Human TLR10 is an anti-inflammatory pattern-recognition receptor. Proc. Natl. Acad. Sci. U.S.A. 111, E4478–E4484. https://doi.org/10.1073/pnas.1410293111.
55. Fore, F., Indriputri, C., Mamutse, J., Nugraha, J., 2020. TLR10 and its unique anti-inflammatory properties and potential use as a target in therapeutics. Immune Netw. 20, e21. https://doi.org/10.4110/in.2020.20.e21.
56. Hasan, U., Chaffois, C., Gaillard, C., Saulnier, V., Merck, E., Tancredi, S., Guiet, C., Brière, F., Vlach, J., Lebecque, S., Trinchieri, G., Bates, E.E.M., 2005. Human TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through MyD88. J. Immunol. 174, 2942–2950. https://doi.org/10.4049/jimmunol.174.5.2942.
57. Torices, S., Julia, A., Muñoz, P., Varela, I., Balsa, A., Marsal, S., Fernández-Nebro, A., Blanco, F., López-Hoyos, M., Martinez-Taboada, V., Fernández-Luna, J.L., 2016. A functional variant of TLR10 modifies the activity of NFkB and may help predict a worse prognosis in patients with rheumatoid arthritis. Arthritis Res. Ther. 18, 221. https://doi.org/10.1186/s13075-016-1113-z.
58. Shaw, A.C., Panda, A., Joshi, S.R., Qian, F., Allore, H.G., Montgomery, R.R., 2011. Dysregulation of human Toll-like receptor function in aging. Ageing Res. Rev. 10, 346–353. https://doi.org/10.1016/j.arr.2010.10.007.
59. Van Duin, D., Mohanty, S., Thomas, V., Ginter, S., Montgomery, R.R., Fikrig, E., Allore, H.G., Medzhitov, R., Shaw, A.C., 2007. Age-associated defect in human TLR-1/2 function. J. Immunol. 178, 970–975. https://doi.org/10.4049/jimmunol.178.2.970.
60. Kim, H.J., Kim, S.H., Je, J.H., Shin, D.Y., Kim, D.S., Lee, M.G., 2016. Increased expression of Toll‐like receptors 3, 7, 8 and 9 in peripheral blood mononuclear cells in patients with psoriasis. Experimental Dermatology. 25, 485–487. https://doi.org/10.1111/exd.12974.
61. Khanna, V., Kim, H., Zhang, W., Larson, P., Shah, M., Griffith, T.S., Ferguson, D., Panyam, J., 2021. Novel TLR 7/8 agonists for improving NK cell mediated antibody-dependent cellular cytotoxicity (ADCC). Sci. Rep. 11, 3346. https://doi.org/10.1038/s41598-021-83005-6.
62. Yang, Y., Wang, Y., Li, L., Chen, F., Zhang, P., 2017. Activation of the Toll-like receptor 8 pathway increases the immunogenicity of mesenchymal stem cells from umbilical cord. Mol. Med. Rep. 16, 2061–2068. https://doi.org/10.3892/mmr.2017.6806.
63. Vaure, C., Liu, Y., 2014. A comparative review of toll-like receptor 4 expression and functionality in different animal species. Front. Immunol. 5, 316. https://doi.org/10.3389/fimmu.2014.00316.
64. Liu, M., Zen, K., 2021. Toll-like receptors regulate the development and progression of renal diseases. Kidney Dis. 7, 14–23. https://doi.org/10.1159/000511947.
65. Kanehisa, M., 2019. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 28, 1947–1951. https://doi.org/10.1002/pro.3715.
66. Kanehisa, M., Goto, S., 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30. https://doi.org/10.1093/nar/28.1.27.
67. Nikitin, A., Egorov, S., Daraselia, N., Mazo, I., 2003. Pathway studio—the analysis and navigation of molecular networks. Bioinformatics. 19, 2155–2157. https://doi.org/10.1093/bioinformatics/btg290.
68. Sharma, A., Costantini, S., Colonna, G., 2013. The protein–protein interaction network of the human sirtuin family. Biochim. Biophys. Acta Proteins Proteom. 1834, 1998–2009. https://doi.org/10.1016/j.bbapap.2013.06.012.
69. Tomczak, A., Mortensen, J.M., Winnenburg, R., Liu, C., Alessi, D.T., Swamy, V., Vallania, F., Lofgren, S., Haynes, W., Shah, N.H., Musen, M.A., Khatri, P., 2018. Interpretation of biological experiments changes with evolution of the Gene Ontology and its annotations. Sci. Rep. 8, 5115. https://doi.org/10.1038/s41598-018-23395-2.
70. Nie, L., Cai, S.-Y., Shao, J.-Z., Chen, J., 2016. Toll-like receptors, associated biological roles, and signaling networks in non-mammals. Front. Immunol. 9, 1523. https://doi.org/10.3389/fimmu.2018.01523.
71. Raghavan, P.R., 2023. Metadichol® induced the expression of neuronal transcription factors in human fibroblast dermal cells. J. Bioinform. Syst. Biol. 6, 326–339. https://doi.org/10.26502/jbsb.5107066.
72. Raghavan, P.R., 2024a. Metadichol, a natural ligand for the expression of Yamanaka reprogramming factors in human cardiac, fibroblast, and cancer cell lines. Med. Res. Arch. 12. https://doi.org/10.18103/mra.v12i6.5323.
73. Raghavan, P.R., 2023. Metadichol treatment of fibroblasts and embryonic stem cells regulates key cardiac progenitors. Cardiol. Cardiovasc. Med. 7, 322–330. https://doi.org/10.26502/fccm.92920340.
74. Raghavan, P.R., 2024b. Metadichol®-induced expression of sirtuin 1-7 in somatic and cancer cells. Med. Res. Arch. 12. https://doi.org/10.18103/mra.v12i6.0000.
75. Raghavan, P.R., 2018. A multi gene targeting approach to treating liver diseases with Metadichol®. J. Cytokine Biol. 3, 126. https://doi.org/10.4172/2576-3881.1000126.
76. Morphy, R., Rankovic, Z., 2005. Designed multiple ligands. An emerging drug discovery paradigm. J. Med. Chem. 48, 6523–6543. https://doi.org/10.1021/jm058225d.
77. Roth, B.L., Sheffler, D.J., Kroeze, W.K., 2004. Magic shotguns versus magic bullets: selectively non-selective drugs for mood disorders and schizophrenia. Nat. Rev. Drug Discov. 3, 353–359. https://doi.org/10.1038/nrd1346.
78. Montine, T.J., Larson, E.B., 2009. Late-life dementias: does this unyielding global challenge require a broader view? JAMA. 302, 2593–2594. https://doi.org/10.1001/jama.2009.1863.
79. Morphy, R., Kay, C., Rankovic, Z., 2004. From magic bullets to designed multiple ligands. Drug Discov. Today. 9, 641–651. https://doi.org/10.1016/s1359-6446(04)03163-0.
80. Hopkins, A.L., 2008. Network pharmacology: the next paradigm in drug discovery. Nat. Chem. Biol. 4, 682–690. https://doi.org/10.1038/nchembio.118.
81. Alemán, C., Mas, R., Hernandez, C., Rodeiro, I., Cerejido, E., Noa, M., Capote, A., Menendez, R., Amor, A., Fraga, V., 1994. A 12-month study of policosanol oral toxicity in Sprague Dawley rats. Toxicol. Lett. 70, 77–87. https://doi.org/10.1016/0378-4274(94)90147-3.
82. Alemán, C.L., Ferreiro, R.M., Puig, M.N., Guerra, I.R., Ortega, C.H., Capote, A., 1994b. Carcinogenicity of policosanol in sprague dawley rats: a 24 month study. Teratog. Carcinog. Mutagen. 14, 239–249. https://doi.org/10.1002/tcm.1770140505.
83. Alemán, C.L., Puig, M.N., Elias, E.C., Ortega, C.H., Guerra, I.R., Ferreiro, R.M., Briñis, F., 1995. Carcinogenicity of policosanol in mice: an 18-month study. Food Chem. Toxicol. 33, 573–578. https://doi.org/10.1016/0278-6915(95)00026-x.