Supplemental Material, DS1_VET_10.1177_0300985818784162 - Pathological Findings in the Pituitary Glands of Dogs and Cats

<p>Supplemental Material, DS1_VET_10.1177_0300985818784162 for Pathological Findings in the Pituitary Glands of Dogs and Cats by Laura Polledo, Guy C. M. Grinwis, Peter Graham, Mark Dunning, and Kerstin Baiker in Veterinary Pathology</p

QPCR primers for genes of interest (based on Microarray analyses) and reference genes (efficiencies varied between 95.0 and 104.8%).

<p>Genes identified using microarray as being significantly different comparing Soft Tissue Histiocytic Sarcoma (STHS) and Visceral Histiocytic Sarcoma (VHS): <i>C-type lectin domain family 12, member A (CLEC12A); C-C motif chemokine 5 Precursor (Small-inducible cytokine A5) (CCL5_CANFA); Asporin (ASPN); CD9 molecule (CD9);Transketolase-like 1, transcript variant 1 (TKTL1); Complement component 6, transcript variant 1(C6); S100 calcium binding protein A12 (S100A12); Immunoglobulin J polypeptide (IGJ); S100 calcium binding protein A8 (S100A8); Phytanoyl-CoA dioxygenase, peroxisomal like (PHYH)</i><u>Reference genes primers for q PCR:</u><i>Hypoxanthine phosphoribosyltransferase (HPRT), Ribosomal protein S19 (RPS19) ribosomalprotein L8 (RPL8), Signal recognition particle receptor (SRPR), Ribosomal protein L13, (RPL13), glucuronidase, beta (GUSB), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Beta-2-Microglobulin (B2MG), 40S ribosomal protein S5 (RPS5), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-2-microglobulin (B2MG), Ribosomal protein S5 (RPS5).</i></p

Quantitative PCR results.

<p>The upregulation or downregulation of selected genes in STHS (Soft Tissue Histiocytic Sarcoma) and VHS (Visceral Histiocytic Sarcoma). The thick black line represents the median (50th percentile) and also the first and third quartile (25th and 75th percentile respectively) are displayed.Three genes are significantly differentially expressed: <i>C6</i> was up-regulated when comparing the more aggressive visceral histiocytic sarcoma to the soft tissue form, and <i>CLEC12A</i> and <i>CCL5</i> were down-regulated when comparing the more aggressive visceral histiocytic sarcoma to the soft tissue histiocytic sarcoma. Abbreviations<i>: C-type lectin domain family 12, member A (CLEC12A); C-C motif chemokine 5 Precursor (Small-inducible cytokine A5) (CCL5_CANFA); Asporin (ASPN); CD9 molecule (CD9);Transketolase-like 1, transcript variant 1 (TKTL1); Complement component 6, transcript variant 1(C6); S100 calcium binding protein A12 (S100A12); Immunoglobulin J polypeptide (IGJ); S100 calcium binding protein A8 (S100A8); Phytanoyl-CoA dioxygenase, peroxisomal like (PHYH).</i></p

Histopathology and immunohistochemistry of pituitary tissue specimens.

<p>Histopathology and immunohistochemistry of pituitary tissue specimens.</p

Microarray- based heatmap of 11 genes.

<p><i>C-type lectin domain family 12, member A (CLEC12A); C-C motif chemokine 5 Precursor (Small-inducible cytokine A5) (CCL5_CANFA); Asporin (ASPN); CD9 molecule (CD9);Transketolase-like 1, transcript variant 1 (TKTL1); Complement component 6, transcript variant 1(C6); S100 calcium binding protein A12 (S100A12); Immunoglobulin J polypeptide (IGJ); S100 calcium binding protein A8 (S100A8); Phytanoyl-CoA dioxygenase, peroxisomal like (PHYH).</i></p

Clinical characteristics of the patients included in the study.

<p>Clinical characteristics of the patients included in the study.</p

CD200-deficiency and sex determine the outcome of influenza A virus infection.

<p>Naïve or influenza A virus-infected mice were sampled at indicated time points. <b>A</b> Relative amounts of viral RNA in the lungs were determined by qRT-PCR. <b>B</b> IFNα concentration in the BAL fluid was determined by ELISA. The dotted line indicates the detection limit. <b>C, D</b> Body weight of infected female (<b>C</b>) and male (<b>D</b>) <i>Cd200^−/−</i> mice (filled symbols) and WT mice (open symbols) as percentage of the weight at day of infection. <b>E</b> Quantification of neutrophil numbers in BAL fluid by differential cell count. <b>F</b> Quantification of the total protein content in BAL fluid. Concentrations of KC (IL-8) (<b>G</b>), IL-6 (<b>H</b>) and TNFα (<b>I</b>) in the BAL fluid were measured by ELISA. In all panels mean ± SEM is shown, statistical significance was calculated with Mann-Whitney test. * = p<0.05, ** = p<0.01.</p

Sanger sequencing results of the 14-3-3 binding site of canine ubiquitin specific protease 8 (USP8).

<p>Representation of protein (A) and DNA (B) USP8 sequence consensus around the 14-3-3 binding motif (red rectangle) between <i>Homo sapiens</i> (region corresponding to the amino-acids 715–720) and <i>Canis lupus familiaris</i> (region corresponding to the amino-acids 713–718). Sanger sequencing of directly (C) and cloning PCR (D) amplified DNA showed no mutations present in the 14-3-3 binding motif (red rectangle).</p

CD200-deficiency and sex determine the outcome of MHV infection.

<p><b>A</b> Male and female WT (open bars) and <i>Cd200^−/−</i> (gray bars) mice (n = 7 per group) were intraperitoneally inoculated with CoV (MHV-EFLM). At day 4 after infection mice were injected intraperitoneally with luciferin and were subjected to BLI. Integrated light intensity is shown. Results are representative of three independent experiments. Uninfected mice did not give a BLI signal. <b>B</b> Quantification of the data in (A). <b>C</b> Four days after infection mice were sacrificed, RNA was isolated from livers and fold-difference in viral RNA was measured by qRT-PCR. <b>D</b> Livers from mice 4 days post-infection were fixed and stained with hematoxylin and eosin. Cellular infiltration foci were quantified in the whole liver sections, (n = 7). In (B–D) mean ± SEM is shown. Statistical significance was calculated with a Mann-Whitney test. ns = not significant. * = p<0.05. ** = p<0.01. *** = p<0.005. <b>E</b> Representative example of single foci from livers of mice 4 days after infection, (n = 7). Similar results were obtained in 3 independent experiments.</p

Stimulation of CD200R directly inhibits TLR mediated NFκB activity.

<p><b>A</b> HEK 293 T cells were transiently transfected with TLR7, NF-κB luciferase reporter and LAIR-1-CD200R chimera or signaling-defective LAIR-1-CD200R-FFF constructs. Twenty-four hours later cells were stimulated with control or anti-LAIR-1 antibody. Forty-eight hours after transfection cells were stimulated with the TLR7 ligand imiquimod (3.0 µg/ml). Seventy-two hours after transfection cells were harvested, luciferase activity, and total protein content were determined. Protein normalized, luciferase activity from 3 independent experiments is shown. <b>B</b> HEK 293 T cells with stable expression of TLR7 were transiently transfected with a NF-κB luciferase reporter construct or an IFNβ luciferase reporter construct and a LAIR-1-CD200R chimera. TLR7 stimulation and CD200R crosslinking was performed as in (A). Protein normalized with luciferase activity from 3 independent experiments is shown. Mean ± SEM is shown. Statistical significance was calculated with Mann-Whitney test. ns = not significant. * = p<0.05.</p