Ultrasound biomicroscopy for longitudinal studies of carotid plaque development in mice: validation with histological endpoints

Atherosclerosis is responsible for the death of thousands of Americans each year. The carotid constriction model of plaque development has recently been presented as a model for unstable plaque formation in mice. In this study we 1) validate ultrasound biomicroscopy (UBM) for the determination of carotid plaque size, percent stenosis, and plaque development in live animals, 2) determine the sensitivity of UBM in detecting changes in blood flow induced by carotid constriction and 3) test whether plaque formation can be predicted from blood flow parameters measured by UBM. Carotid plaques were induced by surgical constriction in Apo E−/− mice. Arteries were imaged bi-weekly by UBM, at which time PW-Doppler measurements of proximal blood flow, as well as plaque length and percent stenosis were determined. Histology was performed 9 weeks post surgery. When compared to whole mount post-mortem measurements, UBM accurately reported carotid plaque length. Percent stenosis, based on transverse B-mode UBM measurements, correlated well with that calculated from histological sections. PW-Doppler revealed that constriction reduced maximum systolic velocity (v_max) and duration of the systolic velocity peak (t_s/t_t). Pre-plaque (2 week post-surgery) PW-Doppler parameters (v_max and t_s/t_t) were correlated with plaque length at 9 weeks, and were predictive of plaque formation. Correlation of initiating PW-Doppler parameters (v_max and t_s/t_t) with resulting plaque length established the degree of flow disturbance required for subsequent plaque development and demonstrated its power for predicting plaque development.     

Targeting tumour-initiating cells with TRAIL based combination therapy ensures complete and lasting eradication of multiple myeloma tumours in vivo

Multiple myeloma (MM) remains an incurable disease despite improvements to available treatments and efforts to identify new drug targets. Consequently new approaches are urgently required. We have investigated the potential of native tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), in combination with doxorubicin, to induce apoptotic cell death in phenotypically distinct populations of myeloma cells in vitro and in vivo. The cytotoxic potential of TRAIL alone, and in combination with DOX, was assessed in vitro in purified CD138(+) and CD138(-) cells from the MM cell lines and samples from patients with MM. Mouse xenografts obtained by implanting CD138(-) MM cells were used to assess the efficacy of TRAIL, alone and in combination with DOX, in vivo. CD138(-) cells were shown to be more resistant to the cytotoxic activity of TRAIL than CD138(+) cells and have reduced expression of TRAIL death receptors. This resistance results in preferential killing of CD 138(+) cells during exposure of MM culture to TRAIL. Furthermore, prolonged exposure results in the appearance of TRAIL-resistant CD138(-) cells. However, when TRAIL is combined with doxorubicin, this results in complete eradication of MM cells in vivo. Most importantly, this treatment successfully eliminates CD138(-) cells implicated in tumour initiation and growth maintenance. These findings may explain the failure of current therapies and offer a promising new approach in the quest to cure MM and disseminated cancers.     

Organically modified silica nanoparticles are biocompatible and can be targeted to neurons in vivo

The application of nanotechnology in biological research is beginning to have a major impact leading to the development of new types of tools for human health. One focus of nanobiotechnology is the development of nanoparticle-based formulations for use in drug or gene delivery systems. However most of the nano probes currently in use have varying levels of toxicity in cells or whole organisms and therefore are not suitable for in vivo application or long-term use. Here we test the potential of a novel silica based nanoparticle (organically modified silica, ORMOSIL) in living neurons within a whole organism. We show that feeding ORMOSIL nanoparticles to Drosophila has no effect on viability. ORMOSIL nanoparticles penetrate into living brains, neuronal cell bodies and axonal projections. In the neuronal cell body, nanoparticles are present in the cytoplasm, but not in the nucleus. Strikingly, incorporation of ORMOSIL nanoparticles into the brain did not induce aberrant neuronal death or interfered with normal neuronal processes. Our results in Drosophila indicate that these novel silica based nanoparticles are biocompatible and not toxic to whole organisms, and has potential for the development of long-term applications.     

Nilotinib and imatinib are comparably effective in reducing growth of human eosinophil leukemia cells in a newly established xenograft model

We developed a xenograft model of human Chronic Eosinophilic Leukemia (CEL) to study disease progression and remission-induction under therapy with tyrosine kinase inhibitors using imatinib and nilotinib as examples. The FIP1L1/PDGFRA+ human CEL cell lineEOL-1 was injected intravenously into scid mice, and MR imaging and FACS analysis of mouse blood samples were performed to monitor disease development and the effects of imatinib and nilotinib. Organ infiltration was analyzed in detail by immunohistochemistry after sacrifice. All animals developed CEL and within one week of therapy, complete remissions were seen with both imatinib and nilotinib, resulting in reduced total tumor volumes by MR-imaging and almost complete disappearance of EOL-1 cells in the peripheral blood and in tissues. The new model system is feasible for the evaluation of new tyrosine kinase inhibitors and our data suggest that nilotinib may be a valuable additional targeted drug active in patients with FIP1L1/PDGFRA+ CEL.     

Field longevity of a fluorescent protein marker in an engineered strain of the pink bollworm, Pectinophora gossypiella (Saunders)

The cotton pest, pink bollworm (Pectinophora gossypiella (Saunders)), is a significant pest in most cotton-growing areas around the world. In southwestern USA and northern Mexico, pink bollworm is the target of the sterile insect technique (SIT), which relies on the mass-release of sterile pink bollworm adults to over-flood the wild population and thereby reduce it over time. Sterile moths reared for release are currently marked with a dye provided in their larval diet. There are concerns, however, that this marker fails from time to time, leading to sterile moths being misidentified in monitoring traps as wild moths. This can lead to expensive reactionary releases of sterile moths. We have developed a genetically marked strain that is engineered to express a fluorescent protein, DsRed2, which is easily screened under a specialised microscope. In order to test this marker under field conditions, we placed wild-type and genetically marked moths on traps and placed them in field cages. The moths were then screened, in a double-blind fashion, for DsRed2 fluorescence at regular intervals to determine marker reliability over time. The marker was shown to be robust in very high temperatures and generally proved reliable for a week or longer. More importantly, genotyping of moths on traps by PCR screening of the moths was 100% correct. Our findings indicate that this strain--and fluorescent protein markers in general--could make a valuable contribution to SIT.     

Biological roles of the Podospora anserina mitochondrial Lon protease and the importance of its N-domain

Mitochondria have their own ATP-dependent proteases that maintain the functional state of the organelle. All multicellular eukaryotes, including filamentous fungi, possess the same set of mitochondrial proteases, unlike in unicellular yeasts, where ClpXP, one of the two matricial proteases, is absent. Despite the presence of ClpXP in the filamentous fungus Podospora anserina, deletion of the gene encoding the other matricial protease, PaLon1, leads to lethality at high and low temperatures, indicating that PaLON1 plays a main role in protein quality control. Under normal physiological conditions, the PaLon1 deletion is viable but decreases life span. PaLon1 deletion also leads to defects in two steps during development, ascospore germination and sexual reproduction, which suggests that PaLON1 ensures important regulatory functions during fungal development. Mitochondrial Lon proteases are composed of a central ATPase domain flanked by a large non-catalytic N-domain and a C-terminal protease domain. We found that three mutations in the N-domain of PaLON1 affected fungal life cycle, PaLON1 protein expression and mitochondrial proteolytic activity, which reveals the functional importance of the N-domain of the mitochondrial Lon protease. All PaLon1 mutations affected the C-terminal part of the N-domain. Considering that the C-terminal part is predicted to have an α helical arrangement in which the number, length and position of the helices are conserved with the solved structure of its bacterial homologs, we propose that this all-helical structure participates in Lon substrate interaction.     

Optical molecular imaging of multiple biomarkers of epithelial neoplasia: epidermal growth factor receptor expression and metabolic activity in oral mucosa

Biomarkers of cancer can indicate the presence of disease and serve as therapeutic targets. Our goal is to develop an optical imaging approach using molecularly targeted contrast agents to assess several centimeters of mucosal surface for mapping expression of multiple biomarkers simultaneously with high spatial resolution. The ability to image biomarker expression level and heterogeneity in vivo would be extremely useful for clinical cancer research, patient selection of personalized medicine, and monitoring therapy. In this proof-of-concept ex vivo study, we examined correlation of neoplasia with two clinically relevant biomarkers: epidermal growth factor receptor (EGFR) and metabolic activity. Two hundred eighty-six unique locations in nine samples of freshly resected oral mucosa were imaged after topically applying optical imaging agents EGF-Alexa 647 (to target EGFR) and 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (to target metabolic activity). Quantitative features were calculated from resulting fluorescence images and compared with tissue histopathology maps. The EGF-Alexa 647 signal correlated well with EGFR expression as indicated by immunohistochemistry. A classification algorithm for presence of neoplasia based on the signal from both contrast agents resulted in an area under the curve of 0.83. Regions with a posterior probability from 0.80 to 1.00 contained more than 50% neoplasia 99% (84/85) of the time. This study demonstrates a proof-of-concept of how noninvasive optical imaging can be used as a tool to study expression levels of multiple biomarkers and their heterogeneity across a large mucosal surface and how biomarker characteristics correlate with presence of neoplasia. Applications of this approach include predicting regions with the highest likelihood of disease, elucidating the role of biomarker heterogeneity in cancer biology, and identifying patients who will respond to targeted therapy.