Four micrometer sections were stained with hematoxylin and eosin, and analyzed by two researchers blinded for group identity. To score lung inflammation and damage, the entire lung surface was analyzed till with respect to the following variables: interstitial inflammation, endothelialitis, bronchitis, edema, pleuritis, and thrombus formation, as described previously [22]. Each variable was graded on a scale of 0 to 4 (0, absent; 1, mild; 2, moderate; 3, severe; 4, very severe). The total histopathology score was expressed as the sum of the scores for all variables.In vitro studies on the antimicrobial effects of plasma-derived ATFor further analysis of the potential antimicrobial effect of plasma-derived AT we compared cell-free supernatants from BALF of infected animals which were treated with AT (BALF-AT) with BALF from infected placebo-treated rats (BALF-none).

To assess the antimicrobial effect of BALF-AT and BALF-none samples, these samples were added to inoculum suspension and incubated. To assess direct antibacterial activity of plasma-derived AT, inoculum suspension was added to dilutions of plasma-derived AT in phosphate-buffered tryptic soy broth (TSB) medium and incubated.Innate host immune responses can lead to the expression of cationic antimicrobial peptides (e.g., defensins) [23]. Such peptides can be inhibited by nonspecific binding to negatively charged molecules. We hypothesized plasma-derived AT administration to lead to lower levels of coagulation/inflammation, and therefore to lower concentrations of extracellular protein and coagulation products in the lung, and hence, to less inhibition of cationic antimicrobial peptides.

To test whether plasma-derived AT treatment leads to less inhibition of cationic antimicrobial peptides inoculum suspensions were incubated with BALF-AT with or without sodium polyanetholsulphonate (SPS).Statistical analysisAll in vivo data are expressed as mean �� standard deviation or median with interquartile ranges, as appropriate. Comparisons between the experimental groups and the saline-treated placebo group were performed using one-way analysis of variance (ANOVA) or Kruskal-Wallis test, followed by post-hoc Dunnett’s or Dunn’s tests, depending on data distribution. A P value less than 0.05 was considered statistically significant. The in vitro data was log-transformed.

Then a one-way ANOVA was performed for separate time points to determine the significance of the difference in growth followed by Bonferroni post-testing. Statistical analyses were performed with SPSS 16.0 (SPSS, Chicago, IL, USA) and Prism 4.0 (GraphPad Software, San Diego, CA, USA).ResultsPneumoniaAfter instillation of S. pneumoniae, typical clinical symptoms of illness (pilo-erection, Anacetrapib decreased activity, arched back, decreased food and water intake, and increased respiratory rate) did not occur until about 20 hours later.