9a3cd6ff5b5b2d6129dd25bd5e1ef5e9 pmc_11640336.pdf cd20e3fcd33ee1b887dad024848b3d66a4e5a713 pmc_11640336.pdf 36ebfa8d98670aa7d286e8f76d7ec0375cb9d4848dd30827f8e1c01b44ec3a15 pmc_11640336.pdf Title: Development of a Simple IFN- Release Whole Blood Assay for the Assessment of Leishmania infantum Specific Cellular Immunity in Dogs Subject: Canine leishmaniosis (CanL) is a zoonotic disease caused by Leishmania infantum, where increased interferon-gamma (IFN-) levels are associated with controlling the infection and mild to moderate disease. Therefore, monitoring IFN- concentrations is essential for monitoring the immune responses in CanL. This study compared a faster, cost-effective IFN- release whole blood assay in tubes (WBA-T) with a standardized version (WBA-S) in 41 dogs at different states of L. infantum infection. WBA-T was performed at 24, 48, and 72 h of incubation with three conditions: blood, blood with L. infantum-soluble antigen (LSA), and blood with mitogen ConA. WBA-S was performed in plates, with blood diluted and incubated for five days using the same conditions. Supernatants (WBA-S) or plasma (WBA-T) were harvested for IFN- measurement by ELISA. No significant differences were observed in terms of IFN- concentration between WBA-T and WBA-S under LSA conditions. However, the 48 h incubation period during WBA-T showed the highest median of IFN- concentration compared to other incubation periods and WBA-S. The IFN- concentrations under ConA stimulation in WBA-S were significantly higher than in WBA-T at all incubation times studied. In conclusion, WBA-T stimulated with LSA at 48 h incubation time was shown to be the most appropriate for assessing IFN- production. Keywords: canine; ELISA; immune response; leishmaniosis; cytokine Author: Anna Sára Molnár, Andrea Murillo-Picco, Clara Jiménez-Fortunato and Laia Solano-Gallego Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Sat Nov 30 07:16:56 2024 CET ModDate: Sat Nov 30 07:18:47 2024 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 316692 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AKXRSZ+EURB10 Type 1 Builtin yes yes yes 10 0 GIFXFT+VnURWPalladioL-Bold Type 1 Custom yes yes yes 15 0 OQIVQT+EURM10 Type 1 Builtin yes yes yes 21 0 FUCDTL+URWPalladioL-Bold Type 1 Custom yes yes yes 26 0 UYMHFE+URWPalladioL-Roma Type 1 Custom yes yes yes 32 0 CXZARO+URWPalladioL-Ital Type 1 Custom yes yes yes 37 0 JSSGKG+URWPalladioL-BoldItal Type 1 Custom yes yes yes 42 0 FKHRKJ+VnURWPalladioL Type 1 Custom yes yes yes 47 0 RUWDWW+EURM7 Type 1 Builtin yes yes yes 52 0 OHXQCJ+CMSY10 Type 1 Builtin yes yes yes 89 0 GEWEQC+PazoMath-Italic Type 1 Builtin yes yes yes 97 0 LIXCEK+wasy9 Type 1 Builtin yes yes yes 108 0 ESSRQF+VnURWPalladioL-Italic Type 1 Custom yes yes yes 128 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-02-15 02:46:16 CET RepresentationInformation: pmc_11640336.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-02-12 02:33:11 CET Size: 316692 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 303 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Dogs and Sampling Destination: subsection.2.1 Item: Title: Whole Blood Assay (WBA) Destination: subsection.2.2 Item: Title: IFN- Enzyme-Linked Immunosorbent Assay (ELISA) Destination: subsection.2.3 Item: Title: Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies Against L. infantum Antigen Destination: subsection.2.4 Item: Title: Statistical Analysis Destination: subsection.2.5 Item: Title: Results Destination: section.3 Children: Item: Title: Clinical and Immunological Characteristics of Dogs Destination: subsection.3.1 Item: Title: Comparison Between WBA-S and WBA-T After Stimulation with LSA Destination: subsection.3.2 Item: Title: Comparison Between WBA-S and WBA-T After Stimulation with ConA Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Development of a Simple IFN- Release Whole Blood Assay for the Assessment of Leishmania infantum Specific Cellular Immunity in Dogs Author: Anna Sára Molnár, Andrea Murillo-Picco, Clara Jiménez-Fortunato and Laia Solano-Gallego Subject: Canine leishmaniosis (CanL) is a zoonotic disease caused by Leishmania infantum, where increased interferon-gamma (IFN-) levels are associated with controlling the infection and mild to moderate disease. Therefore, monitoring IFN- concentrations is essential for monitoring the immune responses in CanL. This study compared a faster, cost-effective IFN- release whole blood assay in tubes (WBA-T) with a standardized version (WBA-S) in 41 dogs at different states of L. infantum infection. WBA-T was performed at 24, 48, and 72 h of incubation with three conditions: blood, blood with L. infantum-soluble antigen (LSA), and blood with mitogen ConA. WBA-S was performed in plates, with blood diluted and incubated for five days using the same conditions. Supernatants (WBA-S) or plasma (WBA-T) were harvested for IFN- measurement by ELISA. No significant differences were observed in terms of IFN- concentration between WBA-T and WBA-S under LSA conditions. However, the 48 h incubation period during WBA-T showed the highest median of IFN- concentration compared to other incubation periods and WBA-S. The IFN- concentrations under ConA stimulation in WBA-S were significantly higher than in WBA-T at all incubation times studied. In conclusion, WBA-T stimulated with LSA at 48 h incubation time was shown to be the most appropriate for assessing IFN- production. 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