13 research outputs found
Spermidine restores dysregulated autophagy and polyamine synthesis in aged and osteoarthritic chondrocytes via EP300: response to correspondence by Borzi et al
Nociceptive Sensitizers Are Regulated in Damaged Joint Tissues, Including Articular Cartilage, When Osteoarthritic Mice Display Pain Behavior
OBJECTIVE: Pain is the most common symptom of osteoarthritis (OA), yet where it originates in the joint and how it is driven are unknown. The aim of this study was to identify paināsensitizing molecules that are regulated in the joint when mice subjected to surgical joint destabilization develop OAārelated pain behavior, the tissues in which these molecules are being regulated, and the factors that control their regulation. METHODS: Tenāweekāold mice underwent sham surgery, partial meniscectomy, or surgical destabilization of the medial meniscus (DMM). Painārelated behavior as determined by a variety of methods (testing of responses to von Frey filaments, cold plate testing for cold sensitivity, analgesiometry, incapacitance testing, and forced flexion testing) was assessed weekly. Once painārelated behavior was established, RNA was extracted from either whole joints or microdissected tissue samples (articular cartilage, meniscus, and bone). Reverse transcriptionāpolymerase chain reaction analysis was performed to analyze the expression of 54 genes known to regulate pain sensitization. Cartilage injury assays were performed using avulsed immature hips from wildātype or genetically modified mice or by explanting articular cartilage from porcine joints preinjected with pharmacologic inhibitors. Levels of nerve growth factor (NGF) protein were measured by enzymeālinked immunosorbent assay. RESULTS: Mice developed painārelated behavior 8 weeks after undergoing partial meniscectomy or 12 weeks after undergoing DMM. NGF, bradykinin receptors B1 and B2, tachykinin, and tachykinin receptor 1 were significantly regulated in the joints of mice displaying painārelated behavior. Little regulation of inflammatory cytokines, leukocyte activation markers, or chemokines was observed. When tissue samples from articular cartilage, meniscus, and bone were analyzed separately, NGF was consistently regulated in the articular cartilage. The other pain sensitizers were also largely regulated in the articular cartilage, although there were some differences between the 2 models. NGF and tachykinin were strongly regulated by simple mechanical injury of cartilage in vitro in a transforming growth factor Ī²āactivated kinase 1ā, fibroblast growth factor 2ā, and Src kinaseādependent manner. CONCLUSION: Damaged joint tissues produce proalgesic molecules, including NGF, in murine OA
Demonstration of the concept that a bacterial mimetic insult can cause EV release.
<p>THP-1 cells were treated with RPMI (vehicle) or LPS (0.1 ĀµM) and incubated overnight and samples were collected and centrifuged to remove the cells. The <sub>supernatants</sub> were collected and split into two equal fractions: non-ultracentrifuged (EV-rich ā left side) and ultracentrifuged (EV-<sub>deficient ā right side</sub>). The samples were pre-treated with vehicle (DMSO, 0.1%, V/V) or P2X<sub>7</sub> antagonist (AZ 11645373; 10<sup>ā7</sup> M). Samples were incubated for one hour and then treated with vehicle (PBS) or exogenous ATP<i>Ī³S</i> (10<sup>ā3</sup> M). The samples were then incubated for a further 4 hours prior to ELISA assessment for cytokines (A: IL-1Ī², B: IL-18, C: TNFĪ±, D: MMP-9). The data is shown as mean +/ā S.E.M.</p
Demonstration that LPS-induced release of EVs can enhance IL-1Ī² and neutrophil levels and change disease phenotype in model known to have increased levels of ATP.
<p>Mice (nā=ā8 per treatment group) were exposed to either room air (control) or CS (3R4F cigarettes) using a negative pressure system. Mice were subjected to 2 periods of CS exposure (500 ml/minute) per day (4 hours apart) for 3 consecutive days. On the morning of the third challenge day, the mice were exposed to aerosolised vehicle of endotoxin free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Animals were culled and BALF and lung tissue samples were collected 24 hours after LPS treatment. IL-1Ī² levels were measured in the BALF and neutrophil numbers were determined in the BALF and lung tissue. In separate BALF samples collected from parallel smoke or LPS driven challenges ATP levels were measured (Panel A). Data shown as mean +/ā S.E.M. (A: ATP # ā=āPā=ā0.0023, Mann-Whitney; B: IL-1Ī² # ā=āPā=ā0.0009, Mann-Whitney; C: BALF neutrophil number, # ā=āPā=ā0.0431, Students T test; D: lung tissue neutrophil number; # ā=āPā=ā0.0006, Mann-Whitney).</p
Human translation data: exogenous ATP increases IL-1Ī²/IL-18 level in samples collected from LPS challenged healthy subjects.
<p>Healthy subjects were challenged with inhaled LPS and BALF was collected 6(10<sup>ā3</sup> M) and incubated for 4 hours; cytokine release was analysed by ELISA. Panel A shows the paired IL-1Ī² data. Panel B, C and D represents the levels of IL-1Ī², IL-18 and TNFĪ±, respectively. Data shown as mean +/ā S.E.M. Statistical analysis using a paired T-test.</p
Determining whether the ATP/P2X<sub>7</sub> axis is central to the exacerbation response <i>in vivo.</i>
<p>Mice (nā=ā8 per group) were challenged with the aerosolised vehicle of endotoxin-free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Four hours later the mice were intranasally dosed with saline (2 ml/kg) or ATPĪ³s (0.001 mg/kg) whilst under light anaesthesia (4% isoflurane in oxygen). The mice received oral vehicle or P2X<sub>7</sub> inhibitor, A438079, 30 minutes prior to the ATP challenge, 4 hours after the challenge and 1 hour prior to cull. Twenty four hours after the LPS exposure the mice were culled and lavaged. IL-1Ī² (A) and neutrophil (B) numbers were measured in the BALF. Data shown as mean +/ā S.E.M. An unpaired T-test was used for the statistical analysis. * ā=āPā=ā0.0378 (Panel A); * ā=āPā=ā0.0162 (Panel B).</p
Determining if a bacterial mimetic (LPS) can cause the release of EVs in the lung ā Signalling.
<p>Mice (nā=ā6 per group) were challenged with the aerosolised vehicle of endotoxin-free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Animals were sacrificed and BALF obtained 6 hours after challenge. The samples were then centrifuged (900 g) to remove the white blood cells and then pre-treated with inhibitors (P2X<sub>7</sub> antagonist A 438079 (10<sup>ā6</sup> M) or caspase-1 inhibitor VX 765 (10<sup>ā7</sup> M)) and incubated for 1 hour. Samples were then treated with vehicle (PBS) or ATP<i>Ī³S</i> (10<sup>ā3</sup> M), and incubated for a further 4 hours and subsequent cytokine release was analysed by ELISA. Data shown as mean +/ā S.E.M. (A: IL-1Ī², B: TNFĪ±). * ā=āPā=ā0.0138 (One way ANOVA followed by a Bonferroni's Multiple Comparison test).</p
Determining if a bacterial mimetic (LPS) can cause the release of EVs in the lung ā Nanosight imaging.
<p>Mice were challenged with the aerosolised vehicle of endotoxin-free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Animals were sacrificed and BALF obtained 6 hours after challenge. The samples were then centrifuged (900 g) to remove the white blood cells and debris. The presence of EVs was imaged using Nanosight technology.</p
Determining if a bacterial mimetic (LPS) can cause the release of EVs in the lung ā Cytokine release.
<p>Mice were challenged with the aerosolised vehicle of endotoxin-free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Animals were sacrificed and BALF obtained 6 hours after challenge. The samples were centrifuged (900 g) to remove the white blood cells and debris, and then treated with vehicle (PBS) or ATP<i>Ī³S</i> (10<sup>ā3</sup> M), and incubated for a further 4 hours and subsequent cytokine release was analysed by ELISA. Data shown as mean +/ā S.E.M. (A: IL-1Ī², B: IL-18, C: IL-1Ī±).</p
Determining if a bacterial mimetic (LPS) can cause the release of EVs in the lung ā Electron Microscopy.
<p>Mice were challenged with the aerosolised vehicle of endotoxin-free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Animals were sacrificed and BALF obtained 6 hours after challenge. The samples were then centrifuged (900 g) to remove the white blood cells and debris. The presence of EVs was imaged using EM (top panel ā vehicle, middle panels ā vehicle challenge, bottom panels ā LPS challenge).</p