{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T17:40:13Z","timestamp":1760118013512,"version":"build-2065373602"},"reference-count":29,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2024,12,24]],"date-time":"2024-12-24T00:00:00Z","timestamp":1734998400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Brazilian Council for the Scientific and Technologic Development Agency (CNPq)","award":["457664\/2013\u20134","303999\/2016\u20130"],"award-info":[{"award-number":["457664\/2013\u20134","303999\/2016\u20130"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Necroptosis is a regulated form of cell death implicated in several pathological conditions, including viral infections. In this study, we investigated the expression and correlation of necroptosis markers MLKL, RIP1 and RIP3 in human liver tissue from fatal cases of yellow fever (YF) using immunohistochemistry (IHC). The liver samples were obtained from 21 YF-positive individuals and five flavivirus-negative controls with preserved liver parenchymal architecture. The cases underwent histopathological analysis, followed by tissue immunostaining with the immunohistochemical method of streptavidin\u2013biotin peroxidase. Using the in situ method, we evaluated the centrilobular zone (Z3), midzonal zone (Z2), periportal zone and portal tract (PT) of human liver parenchyma with markers for necroptosis, RIPK1, RIPK3 and MLKL. A quantitative analysis revealed a significantly higher expression of MLKL, RIP1 and RIP3 in the liver parenchyma of YF cases compared to controls in different zones (Z3, Z2, Z1) and portal tracts (PTs) of the liver, especially in zone 2. Immunostaining confirmed the localization of MLKL, RIP1 and RIP3 in hepatocytes and inflammatory infiltrates, highlighting their involvement in the pathogenesis of YF. A Pearson correlation analysis demonstrated significant correlations among necroptosis markers, which indicates their coordinated regulation during YF-induced liver injury.<\/jats:p>","DOI":"10.3390\/v17010003","type":"journal-article","created":{"date-parts":[[2024,12,24]],"date-time":"2024-12-24T04:45:49Z","timestamp":1735015549000},"page":"3","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Analysis of MLKL, RIP1 and RIP3 Immunostaining Markers in Human Liver Tissue from Fatal Yellow Fever Cases: Insights into Necroptosis"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1586-9887","authenticated-orcid":false,"given":"Vanessa do Socorro Cabral","family":"Miranda","sequence":"first","affiliation":[{"name":"Departmento of Pathology, Evandro Chagas Institute, Ministry of Health, Ananindeua 67030-000, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8391-2694","authenticated-orcid":false,"given":"Luiz Fabio Magno","family":"Falc\u00e3o","sequence":"additional","affiliation":[{"name":"Departmento of Pathology, State University of Para, Belem 66050-540, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hellen Thais","family":"Fuzii","sequence":"additional","affiliation":[{"name":"Tropical Medicine Center, Federal University of Para, Belem 66055-240, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3085-566X","authenticated-orcid":false,"given":"Marcos Luiz Gaia","family":"Carvalho","sequence":"additional","affiliation":[{"name":"Departmento of Pathology, Evandro Chagas Institute, Ministry of Health, Ananindeua 67030-000, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7055-6851","authenticated-orcid":false,"given":"Jeferson da Costa","family":"Lopes","sequence":"additional","affiliation":[{"name":"Departmento of Pathology, Evandro Chagas Institute, Ministry of Health, Ananindeua 67030-000, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9153-1234","authenticated-orcid":false,"given":"Arnaldo Jorge Martins","family":"Filho","sequence":"additional","affiliation":[{"name":"Departmento of Pathology, Evandro Chagas Institute, Ministry of Health, Ananindeua 67030-000, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4607-9346","authenticated-orcid":false,"given":"Ana Cecilia Ribeiro","family":"Cruz","sequence":"additional","affiliation":[{"name":"Departmento of Pathology, Evandro Chagas Institute, Ministry of Health, Ananindeua 67030-000, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1932-9976","authenticated-orcid":false,"given":"Raimunda do Socorro da Silva","family":"Azevedo","sequence":"additional","affiliation":[{"name":"Departmento of Pathology, Evandro Chagas Institute, Ministry of Health, Ananindeua 67030-000, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jorge Rodrigues de","family":"Sousa","sequence":"additional","affiliation":[{"name":"Departmento of Pathology, Evandro Chagas Institute, Ministry of Health, Ananindeua 67030-000, PA, Brazil"},{"name":"Departmento of Pathology, State University of Para, Belem 66050-540, PA, Brazil"},{"name":"Tropical Medicine Center, Federal University of Para, Belem 66055-240, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mayumi Duarte","family":"Wakimoto","sequence":"additional","affiliation":[{"name":"Evandro Chagas National Institute of Infectious Diseases (INI-FIOCRUZ), Oswaldo Cruz Foundation, Rio de Janeiro 21040-360, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6603-5527","authenticated-orcid":false,"given":"Pedro Fernando da Costa","family":"Vasconcelos","sequence":"additional","affiliation":[{"name":"Departmento of Pathology, Evandro Chagas Institute, Ministry of Health, Ananindeua 67030-000, PA, Brazil"},{"name":"Departmento of Pathology, State University of Para, Belem 66050-540, PA, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6267-9966","authenticated-orcid":false,"given":"Juarez Ant\u00f4nio Sim\u00f5es","family":"Quaresma","sequence":"additional","affiliation":[{"name":"Departmento of Pathology, Evandro Chagas Institute, Ministry of Health, Ananindeua 67030-000, PA, Brazil"},{"name":"Departmento of Pathology, State University of Para, Belem 66050-540, PA, Brazil"},{"name":"Tropical Medicine Center, Federal University of Para, Belem 66055-240, PA, Brazil"},{"name":"Evandro Chagas National Institute of Infectious Diseases (INI-FIOCRUZ), Oswaldo Cruz Foundation, Rio de Janeiro 21040-360, RJ, Brazil"},{"name":"Department of Infectious Disease, School of Medicine, Sao Paulo University, Sao Paulo 01246-930, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1016\/j.cll.2010.01.001","article-title":"Yellow fever: A reemerging threat","volume":"30","author":"Gardner","year":"2010","journal-title":"Clin. Lab. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1578","DOI":"10.3201\/eid1009.040197","article-title":"Genetic Divergence and Dispersal of Yellow Fever Virus, Brazil","volume":"10","author":"Vasconcelos","year":"2004","journal-title":"Emerg. Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"960","DOI":"10.1001\/jama.300.8.960","article-title":"100 Years of Discovery the Etiology of Yellow Fever: An Additional Note","volume":"36","author":"Staples","year":"2008","journal-title":"JAMA Class"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"6999","DOI":"10.1128\/JVI.75.15.6999-7008.2001","article-title":"Phylogenetic and Evolutionary Relationships among Yellow Fever Virus Isolates in Africa","volume":"75","author":"Mutebi","year":"2001","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Couto-Lima, D., Madec, Y., Bersot, M.I., Campos, S.S., Motta, M.D.A., Dos Santos, F.B., Vazeille, M., Da Costa Vasconcelos, P.F., Louren\u00e7o-De-Oliveira, R., and Failloux, A.B. (2017). Potential Risk of Re-Emergence of Urban Transmission of Yellow Fever Virus in Brazil Facilitated by Competent Aedes Populations. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-05186-3"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1590\/S0037-86822003000200012","article-title":"Febre amarela: A doen\u00e7a e a vacina, uma hist\u00f3ria inacabada [Yellow fever: The disease and the vaccine, an unfinished history]","volume":"36","author":"Vasconcelos","year":"2003","journal-title":"Rev. Soc. Bras. Med. Trop."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e35","DOI":"10.1590\/s1678-9946201961035","article-title":"Kidney Involvement in Yellow Fever: A Review","volume":"61","author":"Lopes","year":"2019","journal-title":"Rev. Inst. Med. Trop. Sao Paulo"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Melo, J.M., Falc\u00e3o, L.F.M., da Ponte, L.C.T., Silva, C.C., Martins, L.C., Chiang, J.O., Martins Filho, A.J., Franco, E.C.S., Duarte, M.I.S., and Sousa, J.R. (2022). Emergence of New Immunopathogenic Factors in Human Yellow Fever: Polarisation of the M1\/M2 Macrophage Response in the Renal Parenchyma. Viruses, 14.","DOI":"10.3390\/v14081725"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1143","DOI":"10.1038\/ni.2159","article-title":"Programmed necrosis: Backup to and competitor with apoptosis in the immune system","volume":"12","author":"Han","year":"2011","journal-title":"Nat. Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Okamoto, T., Suzuki, T., Kusakabe, S., Tokunaga, M., Hirano, J., Miyata, Y., and Matsuura, Y. (2017). Regulation of apoptosis during flavivirus infection. Viruses, 9.","DOI":"10.3390\/v9090243"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.virol.2005.09.058","article-title":"Revisiting the liver in human yellow fever: Virus-induced apoptosis in hepatocytes associated with TGF-\u03b2, TNF-\u03b1 and NK cells activity","volume":"345","author":"Quaresma","year":"2006","journal-title":"Virology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"aaf2154","DOI":"10.1126\/science.aaf2154","article-title":"Programmed necrosis in inflammation: Toward identification of the effector molecules","volume":"352","author":"Wallach","year":"2016","journal-title":"Science"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1709","DOI":"10.1038\/cdd.2014.77","article-title":"Distinct roles of RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necroptosis","volume":"21","author":"Wu","year":"2014","journal-title":"Cell Death Differ."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Weber, K., Roelandt, R., Bruggeman, I., Estornes, Y., and Vandenabeele, P. (2018). Nuclear RIPK3 and MLKL contribute to cytosolic necrosome formation and necroptosis. Commun. Biol., 1.","DOI":"10.1038\/s42003-017-0007-1"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Wu, Y., Zheng, Z., Cao, X., Yang, Q., Norton, V., Adini, A., Maiti, A.K., Adini, I., and Wu, H. (2021). RIP1\/RIP3\/MLKL Mediates Myocardial Function Through Necroptosis in Experimental Autoimmune Myocarditis. Front. Cardiovasc. Med., 8.","DOI":"10.3389\/fcvm.2021.696362"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Ol\u00edmpio, F.A., Falc\u00e3o, L.F.M., Carvalho, M.L.G., da Costa Lopes, J., Mendes, C.C.H., Filho, A.J.M., da Silva, C.A.M., Miranda, V.D.S.C., dos Santos, L.C., and da Silva Vilacoert, F.S. (2022). Endothelium Activation during Severe Yellow Fever Triggers an Intense Cytokine-Mediated Inflammatory Response in the Liver Parenchyma. Pathogens, 11.","DOI":"10.3390\/pathogens11010101"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/j.actatropica.2005.03.003","article-title":"Reconsideration of histopathology and ultrastructural aspects of the human liver in yellow fever","volume":"94","author":"Pagliari","year":"2005","journal-title":"Acta Trop."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.trstmh.2006.02.019","article-title":"Hepatocyte lesions and cellular immune response in yellow fever infection","volume":"101","author":"Quaresma","year":"2007","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1016\/j.mehy.2006.01.060","article-title":"Midzonal lesions in yellow fever: A specific pattern of liver injury caused by direct virus action and in situ inflammatory response","volume":"67","author":"Quaresma","year":"2006","journal-title":"Med. Hypotheses"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1177\/29.4.6166661","article-title":"Use of Avidin-Biotin-Peroxidase Complex (ABC) in Immunoperoxidase Techniques: A comparison between ABC and unlabeled antibody (PAP) procedures","volume":"29","author":"Hsu","year":"1981","journal-title":"J. Histochem. Cytochem."},{"key":"ref_21","first-page":"727","article-title":"Molecular mechanisms of necroptosis: An ordered cellular explosion","volume":"14","author":"Vandenabeele","year":"2013","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"E961","DOI":"10.1073\/pnas.1613305114","article-title":"Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner","volume":"114","author":"Conos","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1186\/s12974-018-1235-0","article-title":"Necroptosis: A regulated inflammatory mode of cell death","volume":"15","author":"Dhuriya","year":"2018","journal-title":"J. Neuroinflammation"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"E5944","DOI":"10.1073\/pnas.1806973115","article-title":"Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNF\u03b1-mediated apoptosis","volume":"115","author":"Amin","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1038\/nature00858","article-title":"Release of chromatin protein HMGB1 by necrotic cells triggers inflammation","volume":"418","author":"Scaffidi","year":"2002","journal-title":"Nature"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"20388","DOI":"10.1073\/pnas.0908698106","article-title":"Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome","volume":"106","author":"Iyer","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1007\/s10495-005-6075-6","article-title":"Morphological criteria to distinguish cell death induced by apoptotic and necrotic treatments","volume":"10","author":"Rello","year":"2005","journal-title":"Apoptosis"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Verburg, S.G., Lelievre, R.M., Westerveld, M.J., Inkol, J.M., Sun, Y.L., and Workenhe, S.T. (2022). Viral-mediated activation and inhibition of programmed cell death. PLoS Pathog., 18.","DOI":"10.1371\/journal.ppat.1010718"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1146\/annurev-pathol-052016-100247","article-title":"Necroptosis: Mechanisms and relevance to disease","volume":"12","author":"Galluzzi","year":"2017","journal-title":"Annu. Rev. Pathol. Mech. Dis."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/1\/3\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:58:59Z","timestamp":1760115539000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/1\/3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,12,24]]},"references-count":29,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1]]}},"alternative-id":["v17010003"],"URL":"https:\/\/doi.org\/10.3390\/v17010003","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2024,12,24]]}}}