Extracellular Hsp72 does not induce or inhibit cytokine release from pleural mesothelial cells.

<p>The ability of a highly purified, recombinant Hsp72 preparation to induce inflammatory cytokine release from pleural mesothelial cells was determined (A and B). MeT-5ACells were treated with the indicated doses of Hsp72 or PMA as a positive control, and MCP-1 (A) and VEGF (B) release measured 24 hr post-treatment by ELISA. In separate experiments, the anti-inflammatory effects of extracellular Hsp72 were examined (C and D). Cells were pre-treated with Hsp72 for 2 hr, followed by treatment with TNF-α (C) or thrombin (D) for an additional 24 hr to induce MCP-1 release. In contrast to PMA stimulated MeT-5A cells, treatment with Hsp72 had no impact on cytokine release. The data are presented as the mean ± SEM of three independent experiments performed in triplicate. NS, not significant.</p

Mesothelial cells express Hsp72 and Hsp73 on the cell surface.

<p>Non-permeabilised mesothelial cells were assessed for expression of cell surface-associated HSP70 proteins by immunocytochemistry. The constitutive Hsp73 and stress-induced Hsp72 forms were examined separately. The figures are representative of three independent experiments. Bar  = 20 μm.</p

Measures of diagnostic accuracy of pleural fluid Hsp72.

<p>Values in parentheses are 95% confidence intervals.</p><p>LR, likelihood ratio; AUC, area under ROC curve.</p>*<p>This figure represents three times the upper normal limit for serum LDH.</p

Pleural fluid Hsp72 levels in infection-related and non-infective effusions.

<p>Pleural fluids Hsp72 levels were compared among infection-related and non-infective effusions in the Spanish cohort. A) Median Hsp72 levels were significantly higher in infection-related pleural effusions compared to effusions of non-infective etiologies (p<0.0001). B) Compared to non-infective effusions, Hsp72 levels were also higher when in PPE (p<0.001) and empyema (p<0.01).</p

Baseline patient characteristics.

<p>Data are presented as median (quartile range) or n (%).</p><p>+Significantly higher than the respective values in other groups by <i>post-hoc</i> test.</p>*<p>Significantly lower than the respective values in other groups by <i>post-hoc</i> test.</p

Pleural mesothelial cells release Hsp72 in response to infection with <i>Streptococcus pneumoniae</i>.

<p>MeT-5A cells were treated with live (A and C) or heat-killed (B and D) <i>Streptococcus pneumoniae</i> 262 strain (A and B) and <i>S. pneumoniae</i> TIGR4 strain (C and D) and Hsp72 levels measured in culture supernatants at various time points up to 24 hr by ELISA. Significant release of Hsp72 was observed at all time points examined (p<0.05) and was still evident following treatment with heat-killed <i>S. pneumoniae.</i> * Denotes significantly higher than vehicle control cells (p<0.05). The data are presented as the mean ± SEM of three independent experiments performed in triplicate.</p

Hsp72 levels in peritoneal lavage are elevated following intraperitoneal injection of mice with <i>Streptococcus pneumoniae</i>.

<p>BALB/c mice were given a single intraperitoneal injection of live <i>Streptococcus pneumoniae</i> D39 strain (∼1×107 CFU in 0.1 ml of saline; n = 11) or saline as a control (n = 10). The peritoneal cavity was lavaged 7 (n = 6) and 17 hr (n = 5) post-injection with 1 ml PBS for quantification of Hsp72 protein. The results are presented as the fold-increase in Hsp72 levels over the mean Hsp72 level in mice injected with saline alone. An approximately 2- and 2.5-fold increase in Hsp72 was shown peritoneal lavage following infection with <i>S. pneumoniae</i> for 7 and 17 hr, respectively (p<0.01 for both).</p

Hsp72 levels are elevated in exudative pleural effusions.

<p>Hsp72 was measured in human samples by ELISA and levels compared between pleural fluid and serum (A), and transudates and exudates (B). A) Median Hsp72 levels were significantly elevated in pleural fluid compared to their matched serum sample (n = 20 for each group) (3.6 vs. 0.49 ng/ml; p<0.0001). B) Median Hsp72 levels were higher in exudates (n = 233) compared to transudates (n = 40) (21.2 <i>vs</i> 6.5 ng/ml, p<0.0001).</p