LPS-induced chemokine production by secretions differentiated macrophages.

<p>The results for control macrophages are expressed as the median value and range, and are set at 100%. The effect of secretions is expressed as a percentage relative to the chemokine production by control cells. Results are means±SEM of at least six experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for macrophages stimulated with LPS. ND: not detectable.</p

Cytokine production in response to LPS.

<p>We measured the production of IL-12p40 (A,B) and IL-10 (C,D) by control and secretions-differentiated MØ-1 and MØ-2 in response to a range of LPS. The results, expressed in ng/ml, are means±SEM of 9–10 experiments. Open bars: control macrophages; filled bars: secretions differentiated macrophages. *p<0.05 for differences from control macrophages.</p

Light microscopy analysis of the effect secretions on the differentiation of monocytes to macrophages.

<p>Monocytes were differentiated to MØ-1 in the presence of GM-CSF (A) or in the presence of GM-CSF and 35 µg of secretions/ml (B) and their morphology evaluated. Similarly, monocytes were differentiated to MØ-2 in the presence of M-CSF (C) and in the presence of M-CSF and secretions (D). Results indicated in days are from a representative experiment.</p

Expression of surface receptors by secretions-differentiated macrophages.

<p>Results, expressed as the mean fluorescence intensity (MFI), are means±SEM of 6–11 experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for control cells. ND: not detectable.</p

LPS-induced cytokine production by secretions differentiated macrophages.

<p>The results for the control macrophages are expressed as the median value and range, and are set at 100%. The effect of secretions is expressed as a percentage relative to the cytokine production by control cells. Results are means±SEM of at least six experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for macrophages stimulated with LPS.</p

Cytokine production in response to LTA.

<p>We measured the production of IL-12p40 (A,B), TNF-α (C,D), IL-6 (E,F) and IL-10 (G,H) by control and secretions-differentiated MØ-1 and MØ-2 induced by a range of LTA. The results, expressed in ng/ml, are means±SEM of 12 experiments. Open bars: control macrophages; filled bars: secretions differentiated macrophages. *p<0.05 for differences from control macrophages.</p

LPS-induced growth factor production by secretions differentiated macrophages.

<p>The results for control macrophages are expressed as the median value and range, and are set at 100%. The effect of secretions is expressed as a percentage relative to the production of growth factors by control cells. Results are means±SEM of at least six experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for macrophages stimulated with LPS. ND: not detectable.</p

Inflammatory and Antimicrobial Responses to Methicillin-Resistant <i>Staphylococcus aureus</i> in an <i>In Vitro</i> Wound Infection Model

<div><p>Treatment of patients with burn wound infections may become complicated by the presence of antibiotic resistant bacteria and biofilms. Herein, we demonstrate an <i>in vitro</i> thermal wound infection model using human skin equivalents (HSE) and biofilm-forming methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) for the testing of agents to combat such infections. Application of a liquid nitrogen-cooled metal device on HSE produced reproducible wounds characterized by keratinocyte death, detachment of the epidermal layer from the dermis, and re-epithelialization. Thermal wounding was accompanied by up-regulation of markers for keratinocyte activation, inflammation, and antimicrobial responses. Exposure of thermal wounded HSEs to MRSA resulted in significant numbers of adherent MRSA/HSE after 1 hour, and multiplication of these bacteria over 24-48 hours. Exposure to MRSA enhanced expression of inflammatory mediators such as TLR2 (but not TLR3), IL-6 and IL-8, and antimicrobial proteins human β-defensin-2, -3 and RNAse7 by thermal wounded as compared to control HSEs. Moreover, locally applied mupirocin effectively reduced MRSA counts on (thermal wounded) HSEs by more than 99.9% within 24 hours. Together, these data indicate that this thermal wound infection model is a promising tool to study the initial phase of wound colonization and infection, and to assess local effects of candidate antimicrobial agents.</p> </div

Antimicrobial protein expression by HSEs after thermal wounding and MRSA exposure.

<p>Expression of (<b>a</b>) hBD-2 mRNA, (<b>b</b>) hBD-3 mRNA, (<b>c</b>) RNase-7 mRNA, (<b>d</b>) hBD-2 protein and (<b>e</b>) hBD-3 protein by control, thermal wounded and/or MRSA colonized HSEs. *P <0.05. Bars are represented as the median. (<b>f</b>) hBD-2 and hBD-3 expression in normal human skin, control HSEs, thermal wounded HSEs, MRSA colonized HSEs, and thermal wounded and MRSA colonized HSEs. N=7-9. Scale bars = 50 µm.</p

Morphology, basement membrane protein collagen type IV and keratin expression in control and thermal wounded HSEs.

<p>Shown are cross-sections of the paraffin embedded control and thermally wounded HSEs that were stained with either (<b>a</b>-<b>b</b>) hematoxylin and eosin (H&E), or immunolabeled for (<b>c</b>-<b>d</b>) collagen type IV, (e-f) K16, or (<b>g</b>-<b>h</b>) K17. Control HSEs (<b>a</b>,<b>c</b>,<b>e</b>,<b>g</b>) and HSEs 48 hours after thermal wounding (<b>b</b>,<b>d</b>,<b>f</b>,<b>h</b>). Arrows indicate dead epidermis and fibroblasts in the dermis. * indicates re-epithelialization. Pictures are representative for 3 NHK donors. Scale bars = 50 μm.</p