{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:40:34Z","timestamp":1760150434397,"version":"build-2065373602"},"reference-count":33,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2023,12,5]],"date-time":"2023-12-05T00:00:00Z","timestamp":1701734400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"4Life Research LLC","award":["PO # 52856"],"award-info":[{"award-number":["PO # 52856"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Repeated exposure to pathogens leads to evolutionary selection of adaptive traits. Many species transfer immunological memory to their offspring to counteract future immune challenges. Transfer factors such as those found in the colostrum are among the many mechanisms where transfer of immunologic memory from one generation to the next can be achieved for an enhanced immune response. Here, a library of 100 plants with high protein contents was screened to find plant-based proteins that behave like a transfer factor moiety to boost human immunity. Aqueous extracts from candidate plants were tested in a human peripheral blood mononuclear cell (PBMC) cytotoxicity assay using human cancerous lymphoblast cells\u2014with K562 cells as a target and natural killer cells as an effector. Plant extracts that caused PBMCs to exhibit enhanced killing beyond the capability of the colostrum-based transfer factor were considered hits. Primary screening yielded an 11% hit rate. The protein contents of these hits were tested via a Bradford assay and Coomassie-stained SDS-PAGE, where three extracts were confirmed to have high protein contents. Plants with high protein contents underwent C18 column fractionation using methanol gradients followed by membrane ultrafiltration to isolate protein fractions with molecular weights of &lt;3 kDa, 3\u201330 kDa, and &gt;30 kDa. It was found that the 3\u201330 kDa and &gt;30 kDa fractions had high activity in the PBMC cytotoxicity assay. The 3\u201330 kDa ultrafiltrates from the top two hits, seeds from Raphanus sativus and Brassica juncea, were then selected for protein identification by mass spectrometry. The majority of the proteins in the fractions were found to be seed storage proteins, with a low abundance of proteins involved in plant defense and stress response. These findings suggest that Raphanus sativus or Brassica juncea extracts could be considered for further characterization and immune functional exploration with a possibility of supplemental use to bolster recipients\u2019 immune response.<\/jats:p>","DOI":"10.3390\/molecules28247961","type":"journal-article","created":{"date-parts":[[2023,12,5]],"date-time":"2023-12-05T11:27:06Z","timestamp":1701775626000},"page":"7961","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Discovery of Transfer Factors in Plant-Derived Proteins and an In Vitro Assessment of Their Immunological Activities"],"prefix":"10.3390","volume":"28","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5009-1586","authenticated-orcid":false,"given":"Mesfin","family":"Yimam","sequence":"first","affiliation":[{"name":"Unigen Inc., 2121 South State Street, Suite #400, Tacoma, WA 98405, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Teresa","family":"Horm","sequence":"additional","affiliation":[{"name":"Department of Biology, Pacific Lutheran University, 12180 Park Ave. S, Tacoma, WA 98447, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shengxin","family":"Cai","sequence":"additional","affiliation":[{"name":"Unigen Inc., 2121 South State Street, Suite #400, Tacoma, WA 98405, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandria","family":"O\u2019Neal","sequence":"additional","affiliation":[{"name":"Seagen Pfizer, 21717 30th Dr SE, Bothell, WA 98021, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ping","family":"Jiao","sequence":"additional","affiliation":[{"name":"Unigen Inc., 2121 South State Street, Suite #400, Tacoma, WA 98405, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mei","family":"Hong","sequence":"additional","affiliation":[{"name":"Unigen Inc., 2121 South State Street, Suite #400, Tacoma, WA 98405, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thida","family":"Tea","sequence":"additional","affiliation":[{"name":"Unigen Inc., 2121 South State Street, Suite #400, Tacoma, WA 98405, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qi","family":"Jia","sequence":"additional","affiliation":[{"name":"Unigen Inc., 2121 South State Street, Suite #400, Tacoma, WA 98405, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,12,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1098\/rstb.2008.0137","article-title":"Maternal transfer of antibodies in vertebrates: Trans-generational effects on offspring immunity","volume":"364","author":"Hasselquist","year":"2009","journal-title":"Philos. Trans. R. Soc. Lond. B Biol. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1172\/JCI103075","article-title":"The transfer in humans of delayed skin sensitivity to streptococcal M substance and to tuberculin with disrupted leukocytes","volume":"34","author":"Lawrence","year":"1955","journal-title":"J. Clin. Investig."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1016\/j.arcmed.2020.06.016","article-title":"Transfer Factor: Myths and Facts","volume":"51","author":"Macias","year":"2020","journal-title":"Arch. Med. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"908","DOI":"10.1056\/NEJM197304262881712","article-title":"Does transfer factor act specifically or as an immunologic adjuvant?","volume":"288","author":"Bloom","year":"1973","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Medina-Rivero, E., Vallejo-Castillo, L., Vazquez-Leyva, S., P\u00e9rez-S\u00e1nchez, G., Favari, L., Velasco-Vel\u00e1zquez, M., Estrada-Parra, S., Pav\u00f3n, L., and P\u00e9rez-Tapia, S.M. (2016). Physicochemical Characteristics of TransferonTM Batches. Biomed. Res. Int., 2016.","DOI":"10.1155\/2016\/7935181"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/0008-8749(83)90145-4","article-title":"A new basis for the immunoregulatory activities of transfer factor An arcane dialect in the language of cells","volume":"82","author":"Lawrence","year":"1983","journal-title":"Cell. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1080\/08923970600928072","article-title":"In vitro antibacterial activity of bovine dialyzable leukocyte extract","volume":"28","year":"2006","journal-title":"Immunopharmacol. Immunotoxicol."},{"key":"ref_8","first-page":"1","article-title":"Non-specific immunostimulatory effects of transfer factor","volume":"8","author":"Vetvicka","year":"2020","journal-title":"Int. Clin. Pathol. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1447","DOI":"10.1093\/plcell\/koac041","article-title":"Thirty years of resistance: Zig-zag through the plant immune system","volume":"34","author":"Ngou","year":"2022","journal-title":"Plant Cell"},{"key":"ref_10","first-page":"e1475175","article-title":"Plant peptides in plant defense responses","volume":"13","author":"Hu","year":"2018","journal-title":"Plant Signal. Behav."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2095","DOI":"10.1105\/tpc.15.00440","article-title":"The plant peptidome: An expanding repertoire of structural features and biological functions","volume":"27","author":"Tavormina","year":"2015","journal-title":"Plant Cell"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"943","DOI":"10.1093\/mp\/ssu050","article-title":"Connecting growth and defense: The emerging roles of brassinosteroids and gibberellins in plant innate immunity","volume":"7","year":"2014","journal-title":"Mol. Plant"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Othman, E.M., Fathy, M., Bekhit, A.A., Abdel-Razik, A.H., Jamal, A., Nazzal, Y., Shams, S., Dandekar, T., and Naseem, M. (2021). Modulatory and Toxicological Perspectives on the Effects of the Small Molecule Kinetin. Molecules, 26.","DOI":"10.3390\/molecules26030670"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"15522","DOI":"10.1038\/s41598-017-15481-8","article-title":"Plant-to-plant communication triggered by systemin primes anti-herbivore resistance in tomato","volume":"7","author":"Coppola","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4997","DOI":"10.1093\/jxb\/ery294","article-title":"The role of antimicrobial peptides in plant immunity","volume":"69","author":"Campos","year":"2018","journal-title":"J. Exp. Bot."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.peptides.2014.02.003","article-title":"The use of versatile plant antimicrobial peptides in agribusiness and human health","volume":"55","author":"Sousa","year":"2014","journal-title":"Peptides"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"677","DOI":"10.3389\/fpls.2014.00677","article-title":"Peptides and small molecules of the plant-pathogen apoplastic arena","volume":"5","author":"Mott","year":"2014","journal-title":"Front. Plant Sci."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Barbosa Pelegrini, P., Del Sarto, R.P., Silva, O.N., Franco, O.L., and Grossi-de-Sa, M.F. (2011). Antibacterial peptides from plants: What they are and how they probably work. Biochem. Res. Int., 2011.","DOI":"10.1155\/2011\/250349"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1054","DOI":"10.1016\/j.cell.2018.07.017","article-title":"Innate Lymphoid Cells: 10 Years On","volume":"174","author":"Vivier","year":"2018","journal-title":"Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1007\/s00262-016-1792-y","article-title":"Selection and expansion of natural killer cells for NK cell-based immunotherapy","volume":"65","author":"Becker","year":"2016","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2930","DOI":"10.1002\/eji.200425146","article-title":"Comparative analysis of NK cell subset distribution in normal and lymphoproliferative disease of granular lymphocyte conditions","volume":"34","author":"Pascal","year":"2004","journal-title":"Eur. J. Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"S55","DOI":"10.1038\/d41586-019-03640-y","article-title":"How plants and insects inherit immunity from their parents","volume":"575","author":"Owens","year":"2019","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1859","DOI":"10.1104\/pp.110.157263","article-title":"Tobacco mosaic virus infection results in an increase in recombination frequency and resistance to viral, bacterial, and fungal pathogens in the progeny of infected tobacco plants","volume":"153","author":"Kathiria","year":"2010","journal-title":"Plant Physiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1010","DOI":"10.4161\/psb.4.11.9755","article-title":"Plant defensins: Defense, development and application","volume":"4","author":"Stotz","year":"2009","journal-title":"Plant Signal. Behav."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1016\/j.peptides.2009.01.018","article-title":"Plant defensins--prospects for the biological functions and biotechnological properties","volume":"30","author":"Gomes","year":"2009","journal-title":"Peptides"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"85","DOI":"10.2174\/1389203053027575","article-title":"Defensins--components of the innate immune system in plants","volume":"6","author":"Lay","year":"2005","journal-title":"Curr. Protein Pept. Sci."},{"key":"ref_27","first-page":"573","article-title":"Small cysteine-rich antifungal proteins from radish: Their role in host defense","volume":"7","author":"Terras","year":"1995","journal-title":"Plant Cell"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1584\/jpestics.D12-033","article-title":"Purification and cDNA cloning of a defensin in Brassica juncea, its functional expression in Escherichia coli, and assessment of its antifungal activity","volume":"38","author":"Sagehashi","year":"2013","journal-title":"J. Pestic. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2513","DOI":"10.1016\/j.febslet.2009.07.004","article-title":"The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans","volume":"15","author":"Aerts","year":"2009","journal-title":"FEBS Lett."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1120","DOI":"10.1016\/j.peptides.2005.01.003","article-title":"Sesquin, a potent defensin-like antimicrobial peptide from ground beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase","volume":"26","author":"Wong","year":"2005","journal-title":"Peptides"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1391","DOI":"10.1007\/s00299-007-0329-4","article-title":"Arabidopsis thaliana plants expressing human beta-defensin-2 are more resistant to fungal attack: Functional homology between plant and human defensins","volume":"26","author":"Aerts","year":"2007","journal-title":"Plant Cell Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1265","DOI":"10.1074\/mcp.M500061-MCP200","article-title":"Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein","volume":"4","author":"Ishihama","year":"2005","journal-title":"Mol. Cell. Proteom."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Somanchi, S.S., McCulley, K.J., Somanchi, A., Chan, L.L., and Lee, D.A. (2015). A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0141074"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/28\/24\/7961\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:33:45Z","timestamp":1760132025000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/28\/24\/7961"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,12,5]]},"references-count":33,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2023,12]]}},"alternative-id":["molecules28247961"],"URL":"https:\/\/doi.org\/10.3390\/molecules28247961","relation":{},"ISSN":["1420-3049"],"issn-type":[{"type":"electronic","value":"1420-3049"}],"subject":[],"published":{"date-parts":[[2023,12,5]]}}}