Groups 2a and 2b did not demonstrate statistically significant increases of IL-4 secretion in response to PHA compared to untreated and metal challenged lymphocytes

Likewise, metal challenge did not result in increases in IL-4 secretion.<p><b>Copyright information:</b></p><p>Taken from "Th1 type lymphocyte reactivity to metals in patients with total hip arthroplasty"</p><p>http://www.josr-online.com/content/3/1/6</p><p>Journal of Orthopaedic Surgery and Research 2008;3():6-6.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2275232.</p><p></p

Groups 2a and 2b demonstrate statistically significant increases in IL-2 secretion in response to PHA compared to untreated and metal challenged lymphocytes

Group 2a (metal-reactive) subjects demonstrated a statistically significant increase (>2 fold) of IL-2 secretion in response to Chromium than did Group 2b subjects (non-reactive) or untreated control lymphocytes of the same group. Note * = p < 0.05, t-test.<p><b>Copyright information:</b></p><p>Taken from "Th1 type lymphocyte reactivity to metals in patients with total hip arthroplasty"</p><p>http://www.josr-online.com/content/3/1/6</p><p>Journal of Orthopaedic Surgery and Research 2008;3():6-6.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2275232.</p><p></p

Groups 2a and 2b demonstrate statistically significant increases in lymphocyte proliferation in response to PHA relative to non-stimulated controls

Group 2a (metal-reactive) subjects demonstrated a statistically significant increase (>5 fold) in lymphocyte proliferation in response to Chromium than did Group 2b subjects (non-reactive). Note * = p < 0.03 (PHA and all other challenge conditions) and ** = p < 0.03, t-test compared to all other metal and group values.<p><b>Copyright information:</b></p><p>Taken from "Th1 type lymphocyte reactivity to metals in patients with total hip arthroplasty"</p><p>http://www.josr-online.com/content/3/1/6</p><p>Journal of Orthopaedic Surgery and Research 2008;3():6-6.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2275232.</p><p></p

Groups 2a and 2b demonstrate statistically significant increases in IFN-gamma secretion in response to PHA compared to untreated and metal challenged lymphocytes

Group 2a (metal-reactive) subjects demonstrated a statistically significant increase (>2 fold) of IFN-gamma secretion in response to Chromium than did Group 2b subjects (non-reactive) or untreated control lymphocytes of the same group. Note * = p < 0.05, t-test.<p><b>Copyright information:</b></p><p>Taken from "Th1 type lymphocyte reactivity to metals in patients with total hip arthroplasty"</p><p>http://www.josr-online.com/content/3/1/6</p><p>Journal of Orthopaedic Surgery and Research 2008;3():6-6.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2275232.</p><p></p

Cobalt Alloy Implant Debris Induces Inflammation and Bone Loss Primarily through Danger Signaling, Not TLR4 Activation: Implications for DAMP-ening Implant Related Inflammation - Fig 6

<p>Hematoxylin and eosin staining of C57BL/6 mouse calvarial tissue and bone thickness 10 d post-op that either received (n = 1/5 represented per group): (A) sham-surgery (sterile PBS), showing no signs of inflammation or significant osteolysis with remaining bone thickness at 193.8 um or (B) 2 mg/mouse calvaria of endotoxin-free Cobalt-alloy particles, with inflammatory infiltrate into the calvarial bone identified by arrows and osteolysis with remaining bone thickness at 101.8 um.</p

An in vivo murine calvarial model of particle-induced osteolysis demonstrates Cobalt-alloy debris co-challenge with TLR4 induces osteolysis but not more than Cobalt alloy alone.

<p>C57BL/6 12 wk old male mice either received (n = 5 per group): (1) sham-surgery (sterile PBS), (2) 2 mg/mouse calvaria of endotoxin-free Cobalt-alloy particles, (3) 5 μg/mL LPS or (4) Cobalt-alloy/LPS+. 10 days later, calvaria were retrieved and analyzed by microCT. (A) Representative images (n = 1/5) and (B) Graphical representation of the percentage decrease in bone thickness relative to sham controls (average of n = 5 per group). (C) IL-1β and TNF-α cytokine production by isolated peritoneal male C57BL/6 macrophages after 20 h of <i>in vitro</i> challenge (performed in triplicate). Note: * <i>p<0</i>.<i>05</i>.</p

TLR4 LPS and Cobalt-alloy particles induce THP-1 macrophage secretion of IL-1β and TNF-α in a dose dependant manner.

<p>IL-1β and TNF-α cytokine production was assessed after THP-1 differentiated macrophages were challenged with (A-B) increasing concentrations of TLR4 agonist LPS and (C-D) increasing dose of Cobalt-alloy (particles to cells ratio) for 20 h and was quantified by ELISA. Cobalt induced significantly less IL-1β and TNF-α than TLR agonist LPS. Note: * indicates <i>p</i><0.05 respective to control and ** represents significance at p<0.01 respective to 0ng/mL.</p

Blocking TLR4 does not significantly decrease IL-1β and TNF-α response of human primary monocyte/macrophages to Cobalt-alloy particles.

<p>(A) IL-1β and (B) TNF-α secreted by human primary monocytes/macrophages (n = 5) challenged with Cobalt-alloy particles (particles:cell = 5:1), LPS or Alum (NLRP3 inflammasome activator) for 20 h, with or without ZVAD-FMK (caspase-1 inhibitor) or PAb TLR4 antibody and was quantified by ELISA. Cytokine levels with use of PAb TLR4 are represented as percent increase as compared to respective control cells and averaged as a group. Note: * <i>p<0</i>.<i>05</i> to each treatment groups respective control values.</p

Cobalt Alloy Implant Debris Induces Inflammation and Bone Loss Primarily through Danger Signaling, Not TLR4 Activation: Implications for DAMP-ening Implant Related Inflammation - Fig 9

<p><b>A general schematic showing the effects of Cobalt alloy particulate on macrophages acting directly and indirectly on three major pro-inflammatory innate immune pathways</b>: 1) general toxins (such as inducing hypoxia like cell responses), 2) as danger associated molecular patterns, DAMPs (inflammasome induced activation), and 3) interacting with the pathogen associated molecular pattern (PAMP) pathway of TLR4.</p

TLR4 agonist LPS and Cobalt-alloy particles in THP-1 Macrophages induce significantly elevated secretion of IL-1β, TNF-α and IL-10, while only Cobalt particles significantly increase IL-1β without concomitant IL-10 increase.

<p>(A) IL-1β (B) TNF-α <b>(C)</b> and IL-10 cytokine production was assessed after THP-1 differentiated macrophages were challenged with Cobalt-alloy particles (particles:cell = 5:1), Cobalt-alloy/LPS+, and LPS (100ng/mL) for 20 h and was quantified by ELISA. <b>(D)</b> THP-1 macrophages were challenged with Co-alloy particles and incubated with 15μg/ml DQ ovalbumin simultaneously for 4 hrs and subsequently were fixed and evaluated for the presence of large pools of dispersed DQ ovalbumin fluorescence co-localized with particles, indicative of lysosomal destabilization. Note: * p<0.05 compared to control macrophages.</p