Sternal wound environment induces opioid peptide expression in PMN: presence of opioid inducing IL-4 and IL-10 in sternal wound environment.

<p><b>A,</b> HL-60 cells were differentiated to PMN and then treated with varying dilutions (0–25%, v/v, 24 h) of wound fluid (WF) derived from sternal wounds. WF was sterile filtered and added directly to the PMN culture medium. POMC mRNA levels in WF treated PMN was measured using qPCR. Data are mean ± SD (n = 3).*, p<0.05 compared to 0% treated cells. <b>B</b>, HL-60 cells were differentiated to PMN and then were treated with 100 ng/ml of IL-10 or IL-4 for 48 h. The expression of POMC transcripts were measured using qPCR. Data are mean ± SD (n = 3). * p<0.05 compared to control. Both IL-4 and IL-10 that are abundantly present in WF significantly induced POMC mRNA expression. <b>C</b>, Levels of IL-4 and IL-10 in wound fluid (WF) and plasma were determined using ELISA at the indicated time points after surgery. The cytokine levels were normalized to the total albumin level in the fluid or plasma. Data are mean ± SD (n = 5). * <i>p</i><0.01 compared to plasma.</p

Increased endogenous opioid peptide levels in wound fluid at late inflammatory phase.

<p>Wound fluid (WF) was obtained from sternal wounds. Opioid peptide β-endorphin (END) and met-enkephalin (ENK) levels in WF was determined using ELISA at the indicated time points after surgery. The peptide levels were normalized to total albumin level in the fluid. Data are mean±SD (n = 5).* <i>p</i><0.05 compared to 24 h post-surgery.</p

Cells from WF show activated PMN phenotype: increased CD66b and decreased CD43 expression.

<p>The WF and PB cells were subjected to flow cytometry analysis following immunostaining with CD66b or CD43. <b>A&C,</b> representative flowcytometry histograms of CD66b and CD43; <b>B&D,</b> quantification of CD66b and CD43 flow cytometry analysis. Data presented as mean±SD, n = 4. *<i>p</i><0.05 compared to PB.</p

Increased CD177 expression in PMN derived from WF.

<p>Cells from WF and PB were immunostained with CD177 followed by flow cytometry or cytospin analysis. Representative <b>A</b>, scatter plot and <b>B</b>, histogram and <b>C</b>, quantification from flow cytometry analysis showing cells with low (lo) or high (hi) expression of CD177. <b>D</b>, Representative images of cytospun cells derived from WF or PB. The cells were immunostained with CD177 (green) and DAPI (nucleus, red). Scale bar = 10 µm.</p

Increased endogenous opioid peptide expression in wound-site PMN.

<p>WF derived cells were isolated from sternal wounds. <b>A</b>, mRNA expression of pro-opiomelanocortin (POMC) or preproenkephalin (PENK), the precursors of END and ENK, respectively, were determined in WF and peripheral blood (PB) PMN using quantitative PCR (qPCR). Data are mean±SD (n = 3).* <i>p</i><0.05 compared to PB; <b>B</b>, <b><i>Left</i></b>: Representative images of cytospun cells derived from WF or PB. Cells were immunostained with anti-β-Endorphin (β-END; red) and DAPI (nucleus, blue). Scale bar = 20 µm. <b><i>Right:</i></b> zoomed images of white box shown in corresponding left panels. Scale bar = 5 µm. <b>C</b>, Intracellular opioid peptide β-endorphin (END) and met-enkephalin (ENK) levels in WF derived PMN determined using ELISA at the indicated time points after surgery. The peptide levels were normalized to total cellar protein levels. Data are mean±SD (n = 3).* <i>p</i><0.05 compared to 24 h post-surgery.</p

Fluid and cell collection from sternal wound environment.

<p><b>A</b>, image of the sternal wound before closure where the Blake drain is yet to be placed; <b>B</b>, after closure of the sternum, the surgeon placed a Blake drain over the sternum, and then closed the wound in layers. The Blake drain was connected to a heparinized J-VAC bulb suction reservoir for wound fluid collection.</p

Presence of staphylococci within the infected debrided wound tissues.

<p>Representative confocal microscopy images of debrided tissue using immunofluorescence staining (debrided tissues was counterstained red with Phalloidin). Note large aggregates of staphylococci (intense green granular stain) colonizing the debrided tissues of infected sternal wound (lower panels), while tissues taken from a non-infected sternal wound during resternotomy (upper panels) show no colonization with staphylococci. Scale bar = 50 µm, 400x magnification. (SWI: sternal wound infection). Right panel is the zoom of the dashed boxed area in the left panel (scale bar = 20 µm).</p

Confocal laser scanning microscopy images showing three dimensional presence of staphylococci in infected debrided wound tissue.

<p>Z-stack image created by merging serial scans of thick tissue section (20 µm), viewed under 600x magnification confocal laser scanning microscopy. Showing in the <i>x/y</i> plane clumps of staphylococci colonizing the debrided tissues (red), while the <i>x/z</i> and <i>y/z</i> planes display the depth of the colonization throughout the tissue section. Three-dimensional orthogonal projections of z-stack image in panel (oriented in two different planes) showing of staphylococci aggregates biomass within the debrided tissues.</p

MRSA Strain USA300 biofilm exhibits enhanced tolerance to tobramycin when grown as a biofilm on surgical wires.

<p>USA300 was used to inoculate in vitro wells containing sections of wire. Planktonic bacteria and wire-associated biofilms were challenged with 10 ug/ml of tobramycin for 2 hours. Bacteria tolerant to antibiotic challenge were enumerated using viability plating and compared to untreated parallel controls. Percent survivability of triplicate cultures is represented. nd, not detected, ns, not significant. Data are mean±SD (n = 3), *p<0.05 compared to untreated planktonic (Mann Whitney test).</p

Demographic characteristics of patients (n = 9) and SWI status.

<p>M, male, F, female; AKI, acute kidney disease; BMI, body mass index, CAD, coronary artery disease; CGH, coronary heart disease; DM, diabetes mellitus; END, endocarditis; GERD, gastro esophageal reflux disease; HTN, hypertension; HTN- P, Pulmonary hypertension; HLD, hyperlipidemia; RD, renal dysfunction; COPD, chronic obstructive pulmonary disease; PVD, peripheral vascular disease; OSA, obstructive sleep apnea; RHD, rheumatic heart disease; CABG, coronary artery bypass graft; MVR, mitral valve replacement; LVAD, left ventricular assisted device; PM, pace maker; RV, right ventricle; N, negative; MSSA, Methicillin-sensitive <i>Staphylococcus aureus</i>; MRSA, Methicillin-resistant <i>Staphylococcus aureus;</i> SVT, supraventricular tachycardia.</p