Macrophages mediate colon carcinoma cell adhesion in the rat liver after exposure to lipopolysaccharide

The surgical resection of primary colorectal cancer is associated with an enhanced risk of liver metastases. Moreover, bacterial translocation or anastomic leakage during resection has been shown to correlate with a poor long-term surgical outcome, suggesting that bacterial products may contributeto the formation of metastases. Driven by these premises, we investigated the role of the bacterial product lipopolysaccharide (LPS) in the generation of liver metastases. Intraperitoneal injection of LPS led to enhanced tumor-cell adhesion to the rat liver as early as 1.5 h post-administration. Furthermore, a rapid loss of the expression of the tight junction protein zonula occludens-1 (ZO-1) was observed, suggesting that LPS disrupts the integrity of the microvasculature. LPS addition to endothelial-macrophage co-cultures damaged endothelial monolayers and caused the formation of intercellular gaps, which was accompanied by increased tumor-cell adhesion. These results suggest that macrophages areinvolved in the endothelial damage resulting from exposure to LPS. Interestingly, the expression levels of of ZO-1 were not affected by LPS treatment in rats in which liver macrophages had been depletedas well as in rats that had been treated with a reactive oxygen species (ROS) scavenger. In both settings, decreased tumor-cell adhesion was observed. Taken together, our findings indicate that LPS induces ROS release by macrophages, resulting in the damage of the vascular lining of the liver and henceallowing increased tumorcell adherence. Thus, peri-operative treatments that prevent the activation of macrophages and-as a consequence- limit endothelial damage and tumor-cell adhesion may significantly improve the long-term outcome of cancer patients undergoing surgical tumor resection

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen degradation, as measured by the release of hydroxyproline, decreased significantly on inhibition of the intracellular pool of cysteine proteinases by Ep453. This inhibitor also induced an accumulation of intracellular fibrillar collagen in fibroblasts, indicating a decreased degradation of phagocytosed collagen. The extracellular inhibitor, Ep475, had minor or no effects. Histochemical analysis using a substrate for the cysteine proteinases cathepsins B and L revealed a high level of enzyme activity, which was completely blocked in explants preincubated with a selective intracellular inhibitor of cathepsin B, Ca074-Me. Moreover, the cathepsin B inhibitor strongly affected collagen degradation, decreasing the release of hydroxyproline and increasing the accumulation of phagocytosed collagen. These effects were comparable or slightly stronger than those found with the general intracellular inhibitor (Ep453). Taken together, these data strongly suggest that intracellular cysteine proteinases, in particular cathepsin B, play an important role in the digestion of soft connective tissue collage

Electron microscopy of bone

Mineralized tissues like bone, dentin and mineralized cartilage are difficult to prepare for ultrastructural analysis. In general, the higher the level of mineralization is, the more difficult it is to obtain ultrathin sections of these tissue. Tissues with a low level of mineral, e.g. from young animals, are rather simple to prepare and sectioning is not that much of a problem. In the present chapter we describe step-by-step how to prepare mineralized tissues for ultrastructural examination