Santiago Aguiar Espellet Soares, André Murilo de Souza Marques, José Rodrigues do Carmo-Neto, Clayson Moura Gomes, Grazzielle Guimarães de Matos, Milton Adriano Pelli de Oliveira
{"title":"利什曼原虫感染巨噬细胞固定和染色与培养中恢复原马鞭毛体方法的比较研究","authors":"Santiago Aguiar Espellet Soares, André Murilo de Souza Marques, José Rodrigues do Carmo-Neto, Clayson Moura Gomes, Grazzielle Guimarães de Matos, Milton Adriano Pelli de Oliveira","doi":"10.1007/s11686-025-00990-8","DOIUrl":null,"url":null,"abstract":"<div><h3>Purpose</h3><p>Infection of macrophages is a mandatory step for <i>Lesihmania</i> to promote mammalian infection and the evaluation of parasites proliferating inside macrophages reveals important information about parasites virulence and leishmanicidal drugs. Here we compare macrophage phagocytosis ability and amastigote proliferation of <i>L. major</i> or <i>L. braziliensis</i> by two different methods: fixation and staining of infected macrophages or recovery of promastigotes in culture.</p><h3>Methods</h3><p>Promastigote parasites were used to infect thioglycolate-elicited BALB/c mice peritoneal macrophages. Phagocytosis was evaluated at 3 h after infection and amastigote proliferation was evaluated directly or indirectly at 3, 6, and 9 days after infection.</p><h3>Results</h3><p>Phagocytosis of <i>L. major</i> and <i>L. braziliensis</i> by murine macrophages was respectively 54.38 ± 10.41% and 62.54 ± 19.01%, according to the fixation and staining method. The infection index (product of the percentage of infected cells X the number of parasites per cell) obtained by fixation and staining method showed proliferation of <i>L. major</i> inside macrophage from 108.42 ± 25.57 (3 h) to 510.09 ± 99.13 (9 days) and decrease of the number of <i>L. braziliensis</i> from 223.01 ± 58.46 (3 h) to 101.37 ± 20.06 (9 days). The parasites recovered/ mL in culture increased for <i>L. major</i> infected macrophage from 2,38 × 10<sup>6</sup> ± 2,31 × 10<sup>6</sup> (3 h) to 16,8 × 10<sup>6</sup> ± 8,3 × 10<sup>6</sup> (9 days) and decreased from 8.43 × 10<sup>6</sup> ± 6.8 × 10<sup>6</sup> (3 h) to 0.19 × 10<sup>6</sup> ± 0.21 × 10<sup>6</sup> (9 days) for <i>L. braziliensis</i> infected macrophage. The fixation and staining method allowed to observe that both parasite species proliferated inside of some macrophages, while other macrophages maintain the parasite number unaltered or even kill the parasite.</p><h3>Conclusion</h3><p>Both methods showed that <i>L. major</i> proliferates in vitro inside of murine macrophages while <i>L. braziliensis</i> are mostly eliminated by these cells. Only fixation and staining allowed to identify <i>L. braziliensis</i> susceptible macrophages in vitro.</p></div>","PeriodicalId":6932,"journal":{"name":"Acta Parasitologica","volume":"70 1","pages":""},"PeriodicalIF":1.5000,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Comparison of the Methods Fixation and Staining of Infected Macrophages with Recovery of Promastigotes in Culture to Evaluate Phagocytosis and Amastigote Proliferation of Leishmania sp. in vitro\",\"authors\":\"Santiago Aguiar Espellet Soares, André Murilo de Souza Marques, José Rodrigues do Carmo-Neto, Clayson Moura Gomes, Grazzielle Guimarães de Matos, Milton Adriano Pelli de Oliveira\",\"doi\":\"10.1007/s11686-025-00990-8\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Purpose</h3><p>Infection of macrophages is a mandatory step for <i>Lesihmania</i> to promote mammalian infection and the evaluation of parasites proliferating inside macrophages reveals important information about parasites virulence and leishmanicidal drugs. Here we compare macrophage phagocytosis ability and amastigote proliferation of <i>L. major</i> or <i>L. braziliensis</i> by two different methods: fixation and staining of infected macrophages or recovery of promastigotes in culture.</p><h3>Methods</h3><p>Promastigote parasites were used to infect thioglycolate-elicited BALB/c mice peritoneal macrophages. Phagocytosis was evaluated at 3 h after infection and amastigote proliferation was evaluated directly or indirectly at 3, 6, and 9 days after infection.</p><h3>Results</h3><p>Phagocytosis of <i>L. major</i> and <i>L. braziliensis</i> by murine macrophages was respectively 54.38 ± 10.41% and 62.54 ± 19.01%, according to the fixation and staining method. The infection index (product of the percentage of infected cells X the number of parasites per cell) obtained by fixation and staining method showed proliferation of <i>L. major</i> inside macrophage from 108.42 ± 25.57 (3 h) to 510.09 ± 99.13 (9 days) and decrease of the number of <i>L. braziliensis</i> from 223.01 ± 58.46 (3 h) to 101.37 ± 20.06 (9 days). The parasites recovered/ mL in culture increased for <i>L. major</i> infected macrophage from 2,38 × 10<sup>6</sup> ± 2,31 × 10<sup>6</sup> (3 h) to 16,8 × 10<sup>6</sup> ± 8,3 × 10<sup>6</sup> (9 days) and decreased from 8.43 × 10<sup>6</sup> ± 6.8 × 10<sup>6</sup> (3 h) to 0.19 × 10<sup>6</sup> ± 0.21 × 10<sup>6</sup> (9 days) for <i>L. braziliensis</i> infected macrophage. The fixation and staining method allowed to observe that both parasite species proliferated inside of some macrophages, while other macrophages maintain the parasite number unaltered or even kill the parasite.</p><h3>Conclusion</h3><p>Both methods showed that <i>L. major</i> proliferates in vitro inside of murine macrophages while <i>L. braziliensis</i> are mostly eliminated by these cells. Only fixation and staining allowed to identify <i>L. braziliensis</i> susceptible macrophages in vitro.</p></div>\",\"PeriodicalId\":6932,\"journal\":{\"name\":\"Acta Parasitologica\",\"volume\":\"70 1\",\"pages\":\"\"},\"PeriodicalIF\":1.5000,\"publicationDate\":\"2025-02-07\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Acta Parasitologica\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s11686-025-00990-8\",\"RegionNum\":3,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"PARASITOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Acta Parasitologica","FirstCategoryId":"3","ListUrlMain":"https://link.springer.com/article/10.1007/s11686-025-00990-8","RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"PARASITOLOGY","Score":null,"Total":0}
Comparison of the Methods Fixation and Staining of Infected Macrophages with Recovery of Promastigotes in Culture to Evaluate Phagocytosis and Amastigote Proliferation of Leishmania sp. in vitro
Purpose
Infection of macrophages is a mandatory step for Lesihmania to promote mammalian infection and the evaluation of parasites proliferating inside macrophages reveals important information about parasites virulence and leishmanicidal drugs. Here we compare macrophage phagocytosis ability and amastigote proliferation of L. major or L. braziliensis by two different methods: fixation and staining of infected macrophages or recovery of promastigotes in culture.
Methods
Promastigote parasites were used to infect thioglycolate-elicited BALB/c mice peritoneal macrophages. Phagocytosis was evaluated at 3 h after infection and amastigote proliferation was evaluated directly or indirectly at 3, 6, and 9 days after infection.
Results
Phagocytosis of L. major and L. braziliensis by murine macrophages was respectively 54.38 ± 10.41% and 62.54 ± 19.01%, according to the fixation and staining method. The infection index (product of the percentage of infected cells X the number of parasites per cell) obtained by fixation and staining method showed proliferation of L. major inside macrophage from 108.42 ± 25.57 (3 h) to 510.09 ± 99.13 (9 days) and decrease of the number of L. braziliensis from 223.01 ± 58.46 (3 h) to 101.37 ± 20.06 (9 days). The parasites recovered/ mL in culture increased for L. major infected macrophage from 2,38 × 106 ± 2,31 × 106 (3 h) to 16,8 × 106 ± 8,3 × 106 (9 days) and decreased from 8.43 × 106 ± 6.8 × 106 (3 h) to 0.19 × 106 ± 0.21 × 106 (9 days) for L. braziliensis infected macrophage. The fixation and staining method allowed to observe that both parasite species proliferated inside of some macrophages, while other macrophages maintain the parasite number unaltered or even kill the parasite.
Conclusion
Both methods showed that L. major proliferates in vitro inside of murine macrophages while L. braziliensis are mostly eliminated by these cells. Only fixation and staining allowed to identify L. braziliensis susceptible macrophages in vitro.
期刊介绍：
Acta Parasitologica is an international journal covering the latest advances in the subject.
Acta Parasitologica publishes original papers on all aspects of parasitology and host-parasite relationships, including the latest discoveries in biochemical and molecular biology of parasites, their physiology, morphology, taxonomy and ecology, as well as original research papers on immunology, pathology, and epidemiology of parasitic diseases in the context of medical, veterinary and biological sciences. The journal also publishes short research notes, invited review articles, book reviews.
The journal was founded in 1953 as "Acta Parasitologica Polonica" by the Polish Parasitological Society and since 1954 has been published by W. Stefanski Institute of Parasitology of the Polish Academy of Sciences in Warsaw. Since 1992 in has appeared as Acta Parasitologica in four issues per year.
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