In vitro, the levels of TopoIIa protein expression correlate with response to etoposide but also multiple molecular events namely DNA-PK and MDR also play a role in cell sensitivity to etoposide. That we did not find a high expression of TopoIIa in clinical specimens further suggests the mechanisms underlying treatment response are complex.

Integrins have become a target for novel therapeutic strategies against malignant gliomas. Cilengitide, a synthetic Arg-Gly-Asp (RGD)-motif peptide, interferes with ligand binding to avb3 and avb5 integrins and is currently investigated in clinical trials. Integrins may also be involved in the activation of transforming growth factor (TGF)-b, a mediator of invasiveness and immune escape of glioma cells. Using flow cytometry, we demonstrate that the target integrins of cilengitide are expressed not only in glioblastoma blood vessels, but also by tumor cells. After exposure of glioma cells to cilengitide, we noticed reduced phosphorylation of Smad2 in most glioma cell lines, including stem-like glioma cells. Phophorylation of Smad2, but not cilengitide-induced detachment, is rescued by addition of recombinant TGF-b. Administration of cilengitide to glioma cells results in reduced TGF-b-mediated reporter gene activity. Furthermore, exposure to cilengitide leads to decreased TGF-b 1 and TGF-b 2 mRNA and protein expression. These effects are mimicked by blocking av, b3 or b5 antibodies or by silencing of integrins av, b3, b5 or b8 using RNA interference. Treatment of mice bearing experimental LN-308 glioma xenografts with cilengitide results in reduced pSmad2 levels. Taken together, cilengitide may exert anti-invasive and immune stimulatory activity in human glioblastoma patients by its anti-TGF-b properties.

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The intestinal tract is full of proteins ranging in antigenicity from inert food antigens to potential pathogens and bacterial toxins. A major focus of mucosal immunology is to better understand how these antigens are acquired and introduced to the immune system. Using intravital two-photon imaging we recently showed that in conventionally housed mice, small intestinal goblet cells, but not colonic goblet cells, act as a route of antigen delivery to intestinal dendritic cells by forming goblet cell-associated antigen passages (GAPs), in a mechanism linked to goblet cell secretion. In contrast to conventionally housed mice, we observed that GAPs were present in the colon of germfree mice, mice given antibiotics, and, surprisingly, neonatal mice. We found that the activation of p44/p42 MAPK and EGFR in colonic goblet cells correlated with the presence of luminal microbiota and the ability to sense the flora via TLRs. Inhibition of either p42/p44 MAPK or EGFR allowed GAP formation to occur in the colon of SPF housed mice, and colonic GAPs in germfree mice were blocked by activation of the EGFR. These observations indicate that sensing the enteric flora inhibits colonic GAP formation. This regulation of antigen delivery would allow sampling of luminal antigens when the environment is permissive, such as in the homeostatic small intestine, but would prevent sampling luminal antigens in a hostile environment, such as the high microbial load in the colon or during infection.