Supplemental material for Pigs are useful for the molecular study of bone inflammation and regeneration in humans

<p>Supplemental material for Pigs are useful for the molecular study of bone inflammation and regeneration in humans by Freja Lea Lüthje, Kerstin Skovgaard, Henrik Elvang Jensen and Louise Kruse Jensen in Laboratory Animals</p

Additional file 1: of Characterization and differentiation of equine experimental local and early systemic inflammation by expression responses of inflammation-related genes in peripheral blood leukocytes

Time-table of clinical examinations performed during the 24-h experimental period. All procedures were carried out in a similar manner for both systemically and locally injected (LI) horses, except only LI horses underwent lameness evaluation. PIH: post-injection hour. (DOCX 14 kb

Characterization of pig AGP by 2D electrophoresis and 2D blotting.

<p>A: Pig AGP 2-D electrophoresis, influence of sample preparation conditions, from left to right: reduced sample, non-reduced sample, non-denatured sample. Close-up from gels with pH gradient 2.5–5, individual pig serum sample (B16/221). Mw markers: 30,43,67,94 kDa (from bottom). Arrow: Pig AGP isoforms. B: The reaction of MAb 1.62 with non-purified pig PAGP isoforms by 2-D electrophoresis. Individual pig serum sample (Aus), non-reducing, complete gel/blot with IPG pH 2.5–5 in the first dimension, left: silver-stained, right: blot probed with MAb 1.62. Mw markers: 14, 20, 30, 43, 67, 94 kDa (from bottom). Arrow: Pig AGP isoforms C: Close-up of 2-D electrophoresis of two individual pig sera (827 µg/ml (left), 1692 µg/ml (right)), silver-stained (top), blotted and stained by RuBPS (general protein stain)(middle), and the same blot subsequently probed with MAb 1.62 (bottom). Non-reducing, pH gradient 2.5–5. Arrow: Pig AGP isoforms.</p

Serum concentrations of pigAGP during the acute phase response.

<p>Serum concentration of pig AGP (left) and pig haptoglobin (right) at different days post infection after experimental <i>Streptococcus suis</i> (A), <i>Actinobacillus pleuropneumoniae</i> (haptoglobin data not included) (B), and <i>Staphylococcus aureus</i> (C) infection and after aseptic inflammation (D). Note: In the <i>Staphylococcus aureus</i> experiment, only two infected pigs were sampled at 48 hours.</p

Hepatic expression of pig AGP gene during acute infection.

<p>A: Relative expression levels of pig AGP (left) and pigMAP (right) (mean of controls (CTRL, N = 6) set to 1) at 24 hours after experimental infection with <i>Actinobacillus pleuropneumoniae</i> serotype 6 (Ap6) and serotype 2 (Ap2), respectively, as indicated. Values for all individual animals are shown. Error bars depict SEM. Analysis was done on liver tissue samples by qPCR (see text). P<0.01: **, not significant: NS. B: Relative expression levels (mean of controls (CTRL, N = 2) set to 1) in <i>Staphylococcus aureus</i> liver samples 30 (N = 3), 36 (N = 2) and 48 (N = 2) hours after i.v. infection with the bacterium as determined by qPCR, pig AGP (left), pig MAP (middle) and haptoglobin (right). Controls received sterile isotonic saline and were euthanized at 48 hours. Values for individual animals are shown. Error bars depict SEM.</p

Characterization of pig AGP by SDS PAGE and Western blotting.

<p>A: Left panel: Silver-stained SDS PAGE, from the left: Salted-out pooled pig serum supernatant; purified pig AGP; purified pig AGP after sialidase treatment. Right panel: Western blot with the same samples probed with rabbit anti human AGP (DAKO). Arrow: Position of pig AGP in salted-out serum supernatant. B: Western blot probed with anti human AGP, from the left: Purified pig AGP; purified AGP after sialidase treatment; purified AGP after sialidase and PNGase F treatment; buffer control for PNGase F treatment. C: Western blot of pooled pig serum probed with antiserum (1/500) from mouse immunized with purified pig AGP (see text)(representative example). D: Western blot probed with MAb 1.62, from the left: Pooled pig serum; purified pig AGP; purified pig AGP after sialidase treatment.</p

ELISA quantification of pigAGP.

<p>A: Titration of purified pig AGP, pig AGP standard (Saikin Kagaku Institute Ltd.), and two individual pig sera in competitive MAb 1.62 based ELISA. B: Serum concentrations of pig AGP in newborn piglets and in 1-month old piglets (Landrace, Duroc, Yorkshire crossbreds, N = 31). Bars indicate mean and SEM. C: Serum concentrations of pig AGP in different pig breeds and rearing conditions (individual samples), mean and SEM shown. DD: Duroc (2 months, herd) LL: Landrace (2 months, herd) YY: Yorkshire (2 months, herd) Ossabaw minipigs, Experimental stables (14–16 months of age) Göttingen minipigs, Experimental stables (41–47 months of age) L/Y: Landrace/Yorkshire crossbreds (experimental stables, 8–9 months of age) Conventional herd (5 months, D/L/Y cross bred production pigs) SPF herd (5 months, D/L/Y cross bred production pigs).</p

Additional file 1: of Activation of innate immune genes in caprine blood leukocytes after systemic endotoxin challenge

Gene name, gene abbreviations and primer sequences used in the real time qPCR analysis. For previously untested primer assays, two primer pairs were designed for each transcript. (DOCX 22 kb

Venn diagram comparing differentially expressed miRNAs identified by and qPCR and RNAseq.

<p>Overlap of miRNAs found to be differentially expressed at one or more post-challenge time points by RNAseq (yellow) and qPCR (blue). †miRNAs assayed by qPCR but not detected by sequencing; *miRNAs detected by sequencing, but not assayed by qPCR. When a known porcine (ssc) sequence for a given miRNA was not available in miRBase (v. 21), human (hsa) or mouse (mmu) names are applied in accordance with the homolog that best matched the novel porcine miRNA discovered in the RNAseq data, and these homolog sequences were likewise used for qPCR primer design.</p

IFN-λ and microRNAs are important modulators of the pulmonary innate immune response against influenza A (H1N2) infection in pigs

<div><p>The innate immune system is paramount in the response to and clearance of influenza A virus (IAV) infection in non-immune individuals. Known factors include type I and III interferons and antiviral pathogen recognition receptors, and the cascades of antiviral and pro- and anti-inflammatory gene expression they induce. MicroRNAs (miRNAs) are increasingly recognized to participate in post-transcriptional modulation of these responses, but the temporal dynamics of how these players of the antiviral innate immune response collaborate to combat infection remain poorly characterized. We quantified the expression of miRNAs and protein coding genes in the lungs of pigs 1, 3, and 14 days after challenge with swine IAV (H1N2). Through RT-qPCR we observed a 400-fold relative increase in IFN-λ3 gene expression on day 1 after challenge, and a strong interferon-mediated antiviral response was observed on days 1 and 3 accompanied by up-regulation of genes related to the pro-inflammatory response and apoptosis. Using small RNA sequencing and qPCR validation we found 27 miRNAs that were differentially expressed after challenge, with the highest number of regulated miRNAs observed on day 3. In contrast, the number of protein coding genes found to be regulated due to IAV infection peaked on day 1. Pulmonary miRNAs may thus be aimed at fine-tuning the initial rapid inflammatory response after IAV infection. Specifically, we found five miRNAs (ssc-miR-15a, ssc-miR-18a, ssc-miR-21, ssc-miR-29b, and hsa-miR-590-3p)–four known porcine miRNAs and one novel porcine miRNA candidate–to be potential modulators of viral pathogen recognition and apoptosis. A total of 11 miRNAs remained differentially expressed 14 days after challenge, at which point the infection had cleared. In conclusion, the results suggested a role for miRNAs both during acute infection as well as later, with the potential to influence lung homeostasis and susceptibility to secondary infections in the lungs of pigs after IAV infection.</p></div