Characterization of Innate Responses Induced by PLGA Encapsulated- and Soluble TLR Ligands In Vitro and In Vivo in Chickens.

Natural or synthetic Toll-like receptor (TLR) ligands trigger innate responses by interacting with distinct TLRs. TLR ligands can thus serve as vaccine adjuvants or stand-alone antimicrobial agents. One of the limitations of TLR ligands for clinical application is their short half-life and rapid clearance from the body. In the current study, encapsulation of selected TLR ligands in biodegradable poly(D,L-lactide-co-glycolide) polymer nanoparticles (PLGA NPs) was examined in vitro and in vivo as a means to prolong innate responses. MQ-NCSU cells (a chicken macrophage cell line) were treated with encapsulated or soluble forms of TLR ligands and the resulting innate responses were evaluated. In most cases, encapsulated forms of TLR ligands (CpG ODN 2007, lipopolysaccharide and Pam3CSK4) induced comparable or higher levels of nitric oxide and cytokine gene expression in macrophages, compared to the soluble forms. Encapsulated CpG ODN, in particular the higher dose, induced significantly higher expression of interferon (IFN)-γ and IFN-β until at least 18 hr post-treatment. Cytokine expression by splenocytes was also examined in chickens receiving encapsulated or soluble forms of lipopolysaccharide (a potent inflammatory cytokine inducer in chickens) by intramuscular injection. Encapsulated LPS induced more sustained innate responses characterized by higher expression of IFN-γ and IL-1β until up to 96 hr. The ability of TLR ligands encapsulated in polymeric nanoparticles to maintain prolonged innate responses indicates that this controlled-release system can extend the use of TLR ligands as vaccine adjuvants or as stand-alone prophylactic agents against pathogens

Comparative Susceptibility of Madin–Darby Canine Kidney (MDCK) Derived Cell Lines for Isolation of Swine Origin Influenza A Viruses from Different Clinical Specimens

Madin–Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75–31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5–7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-β, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples

Physical properties of PLGA NPs encapsulating different classes of TLR ligands.

<p>Physical properties of PLGA NPs encapsulating different classes of TLR ligands.</p

Nitric oxide (NO) production from chicken macrophages (six replicates/group) stimulated with encapsulated and soluble forms of three TLR ligands, measured by Griess reagent system.

<p>(A). LPS; (B). Pam3CSK4, and (C). CpG ODN. Significance (<i>P</i> <0.05) between delivery systems (encapsulated and soluble TLR ligands) within a treatment dose was determined. P-LPSHi—encapsulated high dose LPS; P-LPSlo—encapsulated low dose LPS; LPSHi—soluble high dose LPS; LPSlo—soluble low dose LPS. P-PamHi—encapsulated high dose Pam3CSK4; P-Pamlo—encapsulated low dose Pam3CSK4; PamHi—soluble high dose Pam3CSK4; Pamlo—soluble low dose Pam3CSK4. Similar designations were made for CpG ODN. * indicates significant difference.</p

Genes and primer sequences used for real-time PCR.

<p>Genes and primer sequences used for real-time PCR.</p

Quantification of IFN-γ (A, B), IL-1β (C, D) and IL-8 (E, F) expression in spleen of chickens intramuscularly injected with encapsulated and soluble LPS.

<p>At 3, 18, 48 and 96 hr post-injection, spleens were collected and the relative expression of cytokine genes was determined. Bars represent mean fold expression of cytokines in treated chickens compared to the control chickens. Cytokine gene expressions were compared between delivery systems (encapsulated and soluble) within a dose and at a given time point. * indicates significant difference at <i>P</i> <0.05.</p

Expression profiles of IFN-γ for (A). LPS; (B). Pam3CSK4, and (C). CpG ODN.

<p>Chicken macrophages (six replicates/group) were treated with encapsulated and soluble forms of the three TLR ligands and mean fold expression of IFN-γ in treated groups were compared to the cell culture medium treated group. Significance (<i>P</i> <0.05) was tested between delivery systems (encapsulated and soluble TLR ligands) within a dose and at a given time point. * indicates significant difference.</p

Expression level of IFN-β by stimulation with CpG ODN.

<p>Chicken macrophages (six replicates/group) were treated with encapsulated and soluble CpG ODN. Mean fold expression of IFN-β in treated groups were compared to the cell culture medium treated group. Significance (<i>P</i> <0.05) was tested between delivery systems within a dose and at a given time point. * indicates significant difference.</p

IL-1β mRNA expression in chicken macrophages stimulated with (A). LPS; (B). Pam3CSK4; and (C) CpG ODN.

<p>Chicken macrophages (six replicates/group) were treated with encapsulated and soluble forms of the three TLR ligands and mean fold expression of IL-1β in treated groups were compared to the cell culture medium treated group. Significance (<i>P</i> <0.05) was tested between delivery systems within a dose and at a given time point. * indicates significant difference.</p