a819dc4daa61e595e4a6643aae368462 vetsci_a2020v7n4p179.pdf baac9e361977af10024a4883c2e9b3d5a1fd54e5 vetsci_a2020v7n4p179.pdf 3c1b3eb2d5cef0813b4cf06052cf37ea2dc86bdc4249c79882d4be079a5962bc vetsci_a2020v7n4p179.pdf Title: The Effects of Polyhexamethylene Biguanide (PHMB) and TLR Agonists Alone or as Polyplex Nanoparticles against Leishmania infantum Promastigotes and Amastigotes Subject: Dogs are the main reservoir for Leishmania infantum, manifesting from a subclinical to a fatal disease. Limited treatments are available, although new antiparasitics and immunomodulators are pursued. Polyhexamethylene biguanide (PHMB) has a broad antimicrobial spectrum, including antiparasitic activity. Here, we evaluated the potential for Toll-like receptor agonists (TLRa) and PHMB alone, and as polyplex nanoparticles containing PHMB and TLR4 or TLR9 agonists, to selectively kill L. infantum. Susceptibility of L. infantum promastigotes to PHMB, miltefosine, and allopurinol was performed, and the half-maximum inhibitory concentrations (IC50) were determined. Then, DH-82 cells were infected and treated with PHMB alone or combined with TLR4a (MPLA-SM) or TLR9a (CpG ODNs) and allopurinol alone. The IC50 values of L. infantum promastigotes were PHMB (1.495 M), miltefosine (9.455 M), and allopurinol (0.124 M). After infection, treated DH-82 cells displayed a lower percentage (p = 0.0316), intensity (p = 0.0002), and index of infection (p = 0.0022) when compared to non-treated cells. PHMB induced lower percentage of infection alone (p = 0.043), in combination with TLR9a (p = 0.043), and with TLR4a (p = 0.0213). Supernatants were collected and used to measure TNF- and IL-6 levels. Increased TNF- was observed after PHMB plus TLR4a, relative to uninfected and infected untreated macrophages (p = 0.043). PHMB combined with TLR4a shows promise as a potential anti-L. infantum drug combination, as well as inducer of proinflammatory response, as demonstrated by decreased infection and increased TNF- production. Keywords: TLR4 agonist; TLR9 agonist; PHMB; Leishmania infantum; canine; macrophages Author: Pamela Martínez-Orellana, Marta Baxarias, Liam Good and Laia Solano-Gallego Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Sun Nov 15 11:40:04 2020 CET ModDate: Mon Nov 16 04:01:15 2020 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 7628404 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- EYHMMV+VnURWPalladioL Type 1 Custom yes yes no 10 0 RDORHG+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 HTNPDL+Rpxi Type 1 Builtin yes yes no 20 0 BEYGLR+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 WOZBHT+Rpxb Type 1 Builtin yes yes no 28 0 XAWCZX+URWPalladioL-BoldItal Type 1 Custom yes yes no 32 0 GMWNWY+URWPalladioL-Roma Type 1 Custom yes yes 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DH-82 Cell Line Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Intracellular L. infantum Amastigotes Susceptibility Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Percentage of L. infantum-Infected Macrophages, Intensity of Infection and Infection Index Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Sandwich ELISA for the Determination of TNF- and IL-6 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Children: Item: Title: Polyplex Nanoparticles Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Promastigotes and Macrophage Drugs Susceptibility Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Percentages of Infected Macrophages Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Intensity of Infection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Infectivity Index Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: TNF- and IL-6 Concentrations on Supernatant of DH-82 Treated Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Info: Title: The Effects of Polyhexamethylene Biguanide (PHMB) and TLR Agonists Alone or as Polyplex Nanoparticles against Leishmania infantum Promastigotes and Amastigotes Author: Pamela Martínez-Orellana, Marta Baxarias, Liam Good and Laia Solano-Gallego Subject: Dogs are the main reservoir for Leishmania infantum, manifesting from a subclinical to a fatal disease. Limited treatments are available, although new antiparasitics and immunomodulators are pursued. Polyhexamethylene biguanide (PHMB) has a broad antimicrobial spectrum, including antiparasitic activity. Here, we evaluated the potential for Toll-like receptor agonists (TLRa) and PHMB alone, and as polyplex nanoparticles containing PHMB and TLR4 or TLR9 agonists, to selectively kill L. infantum. Susceptibility of L. infantum promastigotes to PHMB, miltefosine, and allopurinol was performed, and the half-maximum inhibitory concentrations (IC50) were determined. Then, DH-82 cells were infected and treated with PHMB alone or combined with TLR4a (MPLA-SM) or TLR9a (CpG ODNs) and allopurinol alone. The IC50 values of L. infantum promastigotes were PHMB (1.495 M), miltefosine (9.455 M), and allopurinol (0.124 M). After infection, treated DH-82 cells displayed a lower percentage (p = 0.0316), intensity (p = 0.0002), and index of infection (p = 0.0022) when compared to non-treated cells. PHMB induced lower percentage of infection alone (p = 0.043), in combination with TLR9a (p = 0.043), and with TLR4a (p = 0.0213). Supernatants were collected and used to measure TNF- and IL-6 levels. Increased TNF- was observed after PHMB plus TLR4a, relative to uninfected and infected untreated macrophages (p = 0.043). PHMB combined with TLR4a shows promise as a potential anti-L. infantum drug combination, as well as inducer of proinflammatory response, as demonstrated by decreased infection and increased TNF- production. 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