BackgroundIn vivo imaging of orally administered lactic acid bacteria (LAB) and commensal bacteria in mice is shown to provide information on the spatial and temporal distribution of bacteria in the gastrointestinal tract. The bacteria can be detected and monitored using bioluminescence or near-infrared fluorescence.ResultsFluorescence imaging of bacteria was established by expressing the infrared fluorescent protein IRFP713 in Lactococcus lactis, Lactobacillus plantarum and Escherichia coli. All three bacterial species were monitored in live mice and no major differences in transit time were observed. Bacteria passed through the stomach and small intestine in 1 h and the majority were secreted from the large intestine after 6–8 h. Intestinal localization of bacteria was confirmed by imaging the isolated intestines and culturing the intestinal content. The use of fluorescence tomography for spatial localization of fluorescent bacteria has been established. The expression of an additional infrared fluorescent protein IRFP682 enabled concomitant detection of two bacterial populations in live mice.ConclusionsThe present work provides a methodological basis for future studies of probiotic and theranostic actions of LAB in mouse disease models.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0376-4) contains supplementary material, which is available to authorized users.

a b s t r a c tInnate immune responses are tightly regulated to avoid excessive activation and subsequent inflammatory damage to the host, and interleukin-10 (IL-10) plays a crucial role in preventing inflammation. Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases. In stefin B-deficient bone marrow-derived macrophages (BMDMs), we detected an increase in the induction of the LPS-induced pro-inflammatory signal nitric oxide (NO) but decreased IL-10 expression. The phosphorylation of ERK and p38 MAP-kinases was significantly decreased in stefin B-deficient macrophages, as was STAT-3 phosphorylation. These findings show that stefin B influences the expression of anti-inflammatory IL-10 in response to the TLR4 agonist LPS.

Cysteine cathepsins often contribute to cancer progression due to their overexpression in the tumour microenvironment and therefore present attractive targets for non-invasive diagnostic imaging. However, the development of highly selective and versatile small molecule probes for cathepsins has been challenging. Here, we targeted tumour-associated cathepsin B using designed ankyrin repeat proteins (DARPins). The selective DARPin 8h6 inhibited cathepsin B with picomolar affinity (Ki = 35 pM) by binding to a site with low structural conservation in cathepsins, as revealed by the X-ray structure of the complex. DARPin 8h6 blocked cathepsin B activity in tumours ex vivo and was successfully applied in in vivo optical imaging in two mouse breast cancer models, in which cathepsin B was bound to the cell membrane or secreted to the extracellular milieu by tumour and stromal cells. Our approach validates cathepsin B as a promising diagnostic and theranostic target in cancer and other inflammation-associated diseases.

Gene therapy with Plasmid AMEP (antiangiogenic metargidin peptide) has recently been studied as a potential targeted therapy for melanoma. This plasmid is designed to downregulate α5β1 and αvβ3 integrins. In our study, electroporation was used as a nonviral delivery system. We investigated the antiangiogenic and direct antitumor effectiveness of this gene therapy on low and highly metastatic B16 melanoma variants. In vitro, the antiangiogenic effectiveness as determined by tube formation assay on endothelial cells was predominantly dependent on AMEP expression levels. In vivo, antitumor effectiveness was mediated by the inhibition of proliferation, migration and invasion of melanoma cells and correlated with the expression of integrins on tumor cells after intratumor delivery. In addition, reduced metastatic potential was shown. Intramuscular gene electrotransfer of Plasmid AMEP, for AMEP systemic distribution, had no antitumor effect with this specific preventive treatment protocol, confirming that direct tumor delivery was more effective. This study confirms our previous in vitro data that the expression levels of integrins on melanoma cells could be used as a biomarker for antitumor effectiveness in integrin-targeted therapies, whereas the expression levels of AMEP peptide could be a predictive factor for antiangiogenic effectiveness of Plasmid AMEP in the treatment of melanoma.

Cysteine cathepsins are proteases that, in addition to their important physiological functions, have been associated with multiple pathologies, including cancer. Cystatin C (CstC) is a major endogenous inhibitor that regulates the extracellular activity of cysteine cathepsins. We investigated the role of cystatin C in mammary cancer using CstC knockout mice and a mouse model of breast cancer induced by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium. We showed that the ablation of CstC reduced the rate of mammary tumor growth. Notably, a decrease in the proliferation of CstC knockout PyMT tumor cells was demonstrated ex vivo and in vitro, indicating a role for this protease inhibitor in signaling pathways that control cell proliferation. An increase in phosphorylated p-38 was observed in CstC knockout tumors, suggesting a novel function for cystatin C in cancer development, independent of the TGF-β pathway. Moreover, proteomic analysis of the CstC wild-type and knockout PyMT primary cell secretomes revealed a decrease in the levels of 14-3-3 proteins in the secretome of knock-out cells, suggesting a novel link between cysteine cathepsins, cystatin C and 14-3-3 proteins in tumorigenesis, calling for further investigations.