Serum Concentrations of IgM, IgG and IgA Antibodies 24 Hours post-AMI.

<p>A subgroup of patients (n = 10) were evaluated 24 hours post-AMI for the presence of circulating IgM, IgG and IgA anti-MAA antibody levels (Figure 3A) and the total serum IgM, IgG, and IgA concentrations (Figure 3B). Results are expressed as relative mg/L or g/L of Human IgM, IgG, and IgA using a standard curve. *P<0.01 significantly different comparing AMI and 24 hours post-AMI.</p

Relative Serum Concentrations of anti-MDA LDL and anti-MAA LDL IgG Antibody are not Different in Individuals with Coronary Artery Disease (CAD) and in Individuals who Present with an Acute Myocardial Infarction (AMI).

<p>CAD patients were grouped in the following categories; control patients (n = 82), patients with chest pain and CAD (Non-Obstructive CAD, n = 40), patients presenting with AMI (n = 42), and patients with significant Multi-Vessel Obstructive CAD requiring coronary bypass grafting (n = 72). Serum anti-MDA LDL (Figure 2A) and anti-MAA LDL (Figure 2B). There is no significant difference in serum antibody levels when comparing all study groups (p>0.5).</p

MAA-Adduction and its Implications.

<p>MAA-Adduction and its Implications.</p

Light and Confocal Microscopy of MAA in the Culprit AMI Aspirated Tissue.

<p>Panel A and B illustrates a Masson’s Trichrome staining at low (20X) and high magnification (80X) with panel B as the inset box of panel A. Panel C is the rabbit IgG isotype control stain. Panel D illustrates the rabbit anti-MAA staining with Cy3 reporter (80X). Note the absence of collagen or fibrosis and the presence of cholesterol clefts in Panel A which are typical of an atheroma. Also note the localization of MAA in Panel D (white arrows) to cellular vacuolization and necrosis as noted by the arrows on the Masson’s Trichrome in Panel B (black arrows).</p

Relative Serum Concentrations of anti-MAA IgM, IgG and IgA Antibodies are Increased in Individuals with Coronary Artery Disease (CAD) and in Individuals who Present with an Acute Myocardial Infarction (AMI).

<p>CAD patients were grouped in the following categories; control patients (n = 82), patients with chest pain and CAD (Non-Obstructive CAD, n = 40), patients presenting with AMI (n = 42), and patients with significant Multi-Vessel Obstructive CAD requiring coronary bypass grafting (n = 72). Serum anti-MAA antibodies were evaluated for the isotypes IgM (Figure 1A), IgG (Figure 1B), and IgA (Figure 1C). *P<0.001 significantly increased compared to controls. #P<0.03 significantly increased compared to Non-Obstructive CAD. $P<0.003 significantly increased compared to Multi-Vessel Obstructive CAD.</p

Patient demographics.

a<p>P<0.01 significantly increased compared to control.</p>b<p>P<0.05 significantly increase compared to Non-Obstructive and Acute AMI.</p>c<p>P<0.001 significantly increased compared to control.</p>d<p>P<0.01 significantly increased compared to control.</p>e<p>P<0.001 significantly increased compared to control, Non-Obstructive CAD, and Obstructive Multi-Vessel CAD.</p>f<p>P = 0.03 significantly decreased compared to control, Non-Obstructive CAD, and acute.</p>g<p>P<0.02 significantly decreased compared to control, acute.</p><p>*#Control subjects reported they were healthy with no medical problems or medications.</p><p>Patient demographics.</p

Repetitive inhalant organic dust extract (ODE)-induced airway inflammatory responses were reduced in TLR2 and TLR4 KO mice.

<p>WT, TLR2 KO, and TLR4 KO mice were <i>i</i>.<i>n</i>. treated with ODE daily or saline for 3 weeks whereupon animals were euthanized 5 hrs following final exposure. Neutrophil recruitment (<b>A</b>), IL-6 (<b>B</b>), TNF-α (<b>C</b>), IL-1β (<b>D</b>), CXCL1 (<b>E</b>), and CXCL2 (<b>F</b>) were determined in bronchoalveolar lavage fluid. Bar graphs represent the mean with standard error bars shown (N = minimum of 6 mice/group from 3 independent studies). Statistical significance denoted by asterisks (*p<0.01, **p<0.01, ***p<0.001) as compared to respective saline treatment group. Line denotes statistical significance (#p<0.05, ##p<0.01, ###p<0.001) of WT vs. TLR2 and TLR4 KO mice.</p

Organic dust extract (ODE) promotes osteoclastogenesis <i>in vitro</i> with WT and TLR2 KO, but not TLR4 KO, bone marrow macrophages.

<p>Bone marrow derived cells from C57BL/6 WT mice (<b>A, top panel</b>), TLR2 KO mice (<b>B, middle panel</b>), and TLR4 KO (<b>C, bottom panel</b>) were cultured with M-CSF and RANKL and ± ODE (0.5%). In side-by-side experiments, cells were also cultured with a TLR4 agonist (lipopolysaccharide; LPS; 10 ng) or TLR2 agonist (peptidoglycan; PGN; 1 μg) as additional controls. Membrane receptor activator of NF-κB ligand (mRANKL) expression was determined by flow cytometry analysis. A representative contour plot from each experimental group is shown with a rightward shift demonstrating gating of positive mRANKL expression after exclusion of debris. Bar graphs show the mean with standard error bars of percentage of mRANKL expression (P2 gate). N = 6/group from three separate studies ran in duplicate. Statistical significance denoted by asterisks (***p<0.001) vs. unstimulated control.</p

Inhalant exposure with organic dust extract (ODE) increases osteoclast precursor populations (OCP) in murine bone marrow cells.

<p>WT mice were <i>i</i>.<i>n</i>. treated with saline or ODE daily for three weeks whereupon mice were euthanized and bone marrow cells were collected and analyzed by flow cytometry. After exclusion of debris and dead cells, triple negative (TN) cells were gated based upon CD45R^-CD3^-CD11b^lo phenotype with <b>panel A</b> depicting the frequency of TN cells as a percentage of live cells in a bar graph. <b>Panel B</b> depicts a representative dot plot of TN cells expressing CD115 with associated frequency distribution shown in bar graph. Next, representative dot plots with frequency distribution of osteoclast precursor populations: TN CD115+CD117+ (<b>panel C</b>), and TN CD115+CD117+CD27+ (<b>panel D)</b> are shown. All bar graphs represent mean percentage with SEM bars. N = minimum of 6 mice/group from three independent experiments. Statistical significance denoted as asterisks (*p<0.05) vs. saline.</p

Loss of trabecular bone demonstrated in WT and TLR2 KO, but not TLR4 KO, mice treated repetitively with inhalant ODE.

<p>WT, TLR2 KO, and TLR4 KO animals were <i>i</i>.<i>n</i>. treated daily with saline or ODE for 3 weeks. A representative three-dimensional (3D) reconstructed image from region of interest of proximal tibia from one mouse per treatment group (minimum of 6 mice/group from 3 independent studies).</p