Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab.

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, Sprycel), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (EGFR) and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo and HCT116) cell lines and two KRAS wild-type cell lines (SW48 and CaCo2). In vitro, using poly-D-lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab, whereas KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the ß-catenin pathway, the mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation, as measured by Ki67, and higher rates of apoptosis, as measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.

Macrophage inflammatory protein-1alpha (not T helper type 2 cytokines) is associated with severe forms of respiratory syncytial virus bronchiolitis.

It has been suggested that the pathogenesis of respiratory syncytial virus (RSV) infection is related to the development of T helper (Th) type 2 cytokine responses. The presence of Th1 and Th2 cytokines and the chemokines macrophage inflammatory protein (MIP)-1alpha and monocyte chemotactic protein (MCP)-1 were assessed by ELISA in nasopharyngeal secretions of infants with RSV infection. Infants with mild bronchiolitis had increased Th1 cytokines and reduced Th2 cytokines, compared with infants with upper respiratory tract illness alone. Severe bronchiolitis was characterized by a more balanced Th1-Th2 response that did not differ from that of infants with upper respiratory tract illness alone. In contrast, MIP-1alpha was markedly increased in infants with severe bronchiolitis. MIP-1alpha and MCP-1 levels also were inversely related to oxygen saturation (P<.005). Thus, the severity of RSV bronchiolitis appears to be related more to chemokine release than to Th2 cytokine production.

Food processing by animals: do beavers leach tree bark to improve palatability?

Beavers store and consume tree parts in the bodies of water where they live. We examined whether such soaking renders food more palatable by leaching out undesirable compounds. In experiment 1, saplings of red maple, Acer rubrum (RM), were first soaked in a pond for periods of 2, 18, and 36 days, then offered to free-ranging beavers. Soaking for two days rendered RM slightly more acceptable to beavers. To further examine the time window around two days, RM sticks were soaked in distilled water in the laboratory for 1, 2, 4, and 6 days before presenting them to beavers (experiment 2). In experiment 3, twigs of three species were placed on land. Beavers placed RM in the water for 1 to 3 days before consuming the twigs. In experiment 4, sticks were provided in the water at Cranberry Lake Biological Station (CLBS). Most quaking aspen (QA) was consumed during the first night, and most witch hazel, Hamamelis virginiana (WH), during the third night. At Allegany State Park (ASP), no such difference was found. Twigs were provided in the water in experiment 5. At ASP, WH was taken after three days in the water, and at CLBS little WH was consumed, and only during the third night. A meta-analysis of all experiments shows that relatively more WH is consumed after two days than any other species. Experiment 6 traced the time beavers left their own harvested branches in the water. Unlike other tree species, WH remained in the water for two to four days before being consumed. Experiment 7 measured the phenolics leached into water from RM twigs and small pieces of bark soaked for 10 and 8 days, respectively. Shredded bark lost 50-60% of leachable phenolics into the water, and twigs 70-80%. We conclude that beavers can use water to leach undesirable compounds from their food. Although this effect was not robust, our study is the first of its kind.

Development of an ELISA to measure soluble CD163 in biological fluids.

CD163 is a monocyte/macrophage restricted transmembrane glycoprotein and a member of the scavenger receptor cysteine-rich (SRCR) family of proteins. SRCR proteins are typically associated with the immune system. The regulation of CD163 by cytokines and glucocorticoids suggests that it plays a role in inflammatory processes. While CD163 is expressed as a membrane-bound protein, it has been shown to be actively shed from the surface of monocytes in a protease-dependent fashion when cells are stimulated with a phorbol ester. To better elucidate the function and biological importance of CD163, we have developed a solid-phase sandwich enzyme linked immunosorbant assay (ELISA) for the detection of soluble CD163 in biological fluids. This assay has good repeatability both within and between runs (coefficients of variation (CVs) of 3.2% and 7.1% or better, respectively). While detection of CD163 was inhibited by ethylenediamine tetraacetic acid (EDTA), CD163 immunoreactivity was not altered by the addition of heparin or hemoglobin. This report details the development of this novel assay for soluble CD163 and provides the first evidence of CD163 immunoreactivity in normal plasma and serum samples.