Beyond cones: an improved model of whisker bending based on measured mechanics and tapering.

The sense of touch is represented by neural activity patterns evoked by mechanosensory input forces. The rodent whisker system is exceptional for studying the neurophysiology of touch in part because these forces can be precisely computed from video of whisker deformation. We evaluate the accuracy of a standard model of whisker bending, which assumes quasi-static dynamics and a linearly tapered conical profile, using controlled whisker deflections. We find significant discrepancies between model and experiment: real whiskers bend more than predicted upon contact at locations in the middle of the whisker and less at distal locations. Thus whiskers behave as if their stiffness near the base and near the tip is larger than expected for a homogeneous cone. We assess whether contact direction, friction, inhomogeneous elasticity, whisker orientation, or nonconical shape could explain these deviations. We show that a thin-middle taper of mouse whisker shape accounts for the majority of this behavior. This taper is conserved across rows and columns of the whisker array. The taper has a large effect on the touch-evoked forces and the ease with which whiskers slip past objects, which are key drivers of neural activity in tactile object localization and identification. This holds for orientations with intrinsic whisker curvature pointed toward, away from, or down from objects, validating two-dimensional models of simple whisker-object interactions. The precision of computational models relating sensory input forces to neural activity patterns can be quantitatively enhanced by taking thin-middle taper into account with a simple corrective function that we provide.

Direct visualization of heterogeneous extravascular distribution of trastuzumab in human epidermal growth factor receptor type 2 overexpressing xenografts.

PURPOSE: The high molecular weight and binding affinity of trastuzumab, a monoclonal antibody in use for treatment of breast cancers overexpressing human epidermal growth factor receptor type 2 (HER2), in combination with microenvironmental factors, may limit its distribution and efficacy. We assessed and mapped the distribution of systemically given, unlabeled trastuzumab at micrometer resolution in tumor xenografts using immunohistochemistry. EXPERIMENTAL DESIGN: Mice bearing MDA-435/LCC6(HER2) xenografts were given single doses of 4 or 20 mg/kg unlabeled trastuzumab with tumor harvest at various time points thereafter; bound trastuzumab was imaged directly in tumor cryosections using fluorescently tagged antihuman secondary antibodies. Combinations of additional markers, including HER2, 5-bromo-2-deoxyuridine, CD31, DioC(7)(3), desmin, and collagen IV were also mapped on the same tumor sections. RESULTS: Distribution of trastuzumab in MDA-435/LCC6(HER2) tumors is found to be heterogeneous, with tumor margins saturating more thoroughly in doses and times analyzed. Considerable intervessel heterogeneity is also seen. For example, in unsaturated tissues, there remain perfused vessels without any trastuzumab in addition to vessels with a few layers of positively stained perivascular cells, in addition to vessels with bound drug up to 150 microm away. This heterogeneity is independent of HER2 expression, microvessel density, and perfusion. A slightly greater proportion of vessels were associated with pericytes in sections with greater trastuzumab saturation, but this would not adequately account for observed heterogeneous trastuzumab distribution. CONCLUSIONS: Complete penetration of trastuzumab in tumor tissue was not seen in our study, leaving the possibility that inadequate distribution may represent a mechanism for resistance to trastuzumab.

Irinophore C, a novel nanoformulation of irinotecan, alters tumor vascular function and enhances the distribution of 5-fluorouracil and doxorubicin.

PURPOSE: To examine the antitumor effects of Irinophore C, a nanopharmaceutical formulation of irinotecan, on the tissue morphology and function of tumor vasculature in HT-29 human colorectal tumors. EXPERIMENTAL DESIGN: Fluorescence microscopy was used to map and quantify changes in tissue density, tumor vasculature, hypoxia, and the distribution of Hoechst 33342, a perfusion marker, and the anticancer drug, doxorubicin. Noninvasive magnetic resonance imaging was used to quantify Ktrans, the volume transfer constant of a solute between the blood vessels and extracellular tissue compartment of the tumor, as a measure of vascular function. Following treatment with Irinophore C, 19F magnetic resonance spectroscopy was used to monitor the delivery of 5-fluorouracil (5-FU) to the tumor tissue, whereas scintigraphy was used to quantify the presence of bound [14C]5-FU. RESULTS: Irinophore C decreased cell density (P = 8.42 x 10(-5)), the overall number of endothelial cells in the entire section (P = 0.014), tumor hypoxia (P = 5.32 x 10(-9)), and K(trans) (P = 0.050). However, treatment increased the ratio of endothelial cells to cell density (P = 0.00024) and the accumulation of Hoechst 33342 (P = 0.022), doxorubicin (P = 0.243 x 10(-5)), and 5-FU (P = 0.0002) in the tumor. Vascular endothelial growth factor and interleukin-8, two proangiogenic factors, were down-regulated, whereas the antiangiogenic factor TIMP-1 was up-regulated in Irinophore C-treated tumors. CONCLUSIONS: Irinophore C treatment improves the vascular function of the tumor, thereby reducing tumor hypoxia and increasing the delivery and accumulation of a second drug. Reducing hypoxia would enhance radiotherapy, whereas improving delivery of a second drug to the tumor should result in higher cell kill.

The Sensorimotor Basis of Whisker-Guided Anteroposterior Object Localization in Head-Fixed Mice.

Active tactile perception combines directed motion with sensory signals to generate mental representations of objects in space. Competing models exist for how mice use these signals to determine the precise location of objects along their face. We tested six of these models using behavioral manipulations and statistical learning in head-fixed mice. Trained mice used a whisker to locate a pole in a continuous range of locations along the anteroposterior axis. Mice discriminated locations to ≤0.5 mm (<2°) resolution. Their motor program was noisy, adaptive to touch, and directed to the rewarded range. This exploration produced several sets of sensorimotor features that could discriminate location. Integration of two features, touch count and whisking midpoint at touch, was the simplest model that explained behavior best. These results show how mice locate objects at hyperacute resolution using a learned motor strategy and minimal set of mentally accessible sensorimotor features.

Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs.

Various methods have been explored to enhance antibody-based cancer therapy. The use of multivalent antibodies or fragments against tumor antigens has generated a great deal of interest, as various cellular signals, including induction of apoptosis, inhibition of cell growth/survival, or internalization of the surface molecules, can be triggered or enhanced on extensive cross-linking of the target/antibody complex by the multivalent form of the antibody. The goal of the studies reported here was to develop multivalent antibody constructs via grafting of antibody molecules onto liposome membranes to enhance antibody activity. Using trastuzumab and rituximab as examples, up to a 25-fold increase in the antibody potency in cell viability assay was observed when the antibodies were presented in the multivalent liposome formulation. Key cell survival signaling molecules, such as phosphorylated Akt and phosphorylated p65 nuclear factor-kappaB, were down-regulated on treatment with multivalent liposomal trastuzumab and liposomal rituximab, respectively. Potent in vivo antitumor activity was shown for liposomal trastuzumab. The data presented here showed the potential of liposome technology to enhance the therapeutic effect of antibodies via a mechanism that modulates cell survival through clustering of the target/antibody complex.

Characterization of cationic liposome formulations designed to exhibit extended plasma residence times and tumor vasculature targeting properties.

Cationic liposomes exhibit a propensity to selectively target tumor-associated blood vessels demonstrating potential value as anti-cancer drug delivery vehicles. Their utility however, is hampered by their biological instability and rapid elimination following i.v. administration. Efforts to circumvent rapid plasma elimination have, to date, focused on decreasing cationic lipid content and incorporating polyethylene glycol (PEG)-modified lipids. In this study we wanted to determine whether highly charged cationic liposomes with surface-associated PEG could be designed to exhibit extended circulation lifetimes, while retaining tumor vascular targeting properties in an HT29 colorectal cancer xenograft model. Cationic liposomes prepared of DSPC, cationic lipids (DODAC, DOTAP, or DC-CHOL), and DSPE-PEG(2000) were studied. Our results demonstrate that formulations prepared with 50 mol% DODAC or DC-CHOL, and 20 mol% DSPE-PEG(2000) exhibited circulation half-lives ranging from 6.5 to 12.5 h. Biodistribution studies demonstrated that DC-CHOL formulations prepared with DSPE-PEG(2000) accumulated threefold higher in s.c. HT29 tumors than its PEG-free counterpart. Fluorescence microscopy studies suggested that the presence of DSPE-PEG(2000) did not adversely affect liposomal tumor vasculature targeting. We show for the first time that it is achievable to design highly charged, highly pegylated (20 mol% DSPE-PEG(2000)) cationic liposomes which exhibit both extended circulation lifetimes and tumor vascular targeting properties.

Non-invasive evaluation of tumour hypoxia in the Shionogi tumour model for prostate cancer with 18F-EF5 and positron emission tomography.

OBJECTIVE: To evaluate hypoxia non-invasively in androgen-dependent (AD), regressing (6-days after castration, RG) and androgen-independent (AI) Shionogi tumours, using the radiolabelled tracer for hypoxia, 18F-EF5, and positron emission tomography (PET). MATERIALS AND METHODS: Groups of mice bearing AD, RG and AI Shionogi tumours were co-injected with 18F-EF5 and unlabelled EF5. The mice were imaged non-invasively with PET to examine the accumulation of 18F-EF5 in hypoxic regions of the tumour. The tumours were subsequently placed in a gamma-counter, or disaggregated for flow cytometry, to determine the levels of 18F-EF5 and the percentage of hypoxic cells present in the tumour, respectively. RESULTS: The mean (sd) levels of hypoxia in AD Shionogi tumours decreased significantly 6 days after androgen ablation as measured by flow cytometry, from 17.1 (4.77) to 1.74 (0.46)% (P=0.003). There were no significant differences in the levels of 18F-EF5 in the tissue between AD and RG tumours using region-of-interest analysis of PET images or gamma-counting, although the differences were significant when measured by flow cytometry. However, mean (sd) levels of hypoxia in AI Shionogi tumours were significantly higher than in AD tumours regardless of the analysis method; PET, 10.5 (4.93)x10(-5)) Bq/cm2 (P=0.017), flow cytometry, 42.98 (3.35)% (P<0.001), well count, 6.81 (1.17)x10(4) and 13.1 (1.99)x10(4) cpm/g, for AD and AI tumours, respectively (P<0.001). CONCLUSIONS: Differences in hypoxia between AD and AI, but not RG, Shionogi tumours can be detected non-invasively with 18F-EF5 and PET. As prostate tumours are hypoxic and the oxygen levels can change with androgen ablation, noninvasive imaging of hypoxia with PET and 18F-EF5 might ultimately have a prognostic and/or diagnostic role in the clinical management of the disease.

Genome-wide expression analyses of Campylobacter jejuni NCTC11168 reveals coordinate regulation of motility and virulence by flhA.

We examined two variants of the genome-sequenced strain, Campylobacter jejuni NCTC11168, which show marked differences in their virulence properties including colonization of poultry, invasion of Caco-2 cells, and motility. Transcript profiles obtained from whole genome DNA microarrays and proteome analyses demonstrated that these differences are reflected in late flagellar structural components and in virulence factors including those involved in flagellar glycosylation and cytolethal distending toxin production. We identified putative sigma(28) and sigma(54) promoters for many of the affected genes and found that greater differences in expression were observed for sigma(28)-controlled genes. Inactivation of the gene encoding sigma(28), fliA, resulted in an unexpected increase in transcripts with sigma(54) promoters, as well as decreased transcription of sigma(28)-regulated genes. This was unlike the transcription profile observed for the attenuated C. jejuni variant, suggesting that the reduced virulence of this organism was not entirely due to impaired function of sigma(28). However, inactivation of flhA, an important component of the flagellar export apparatus, resulted in expression patterns similar to that of the attenuated variant. These findings indicate that the flagellar regulatory system plays an important role in campylobacter pathogenesis and that flhA is a key element involved in the coordinate regulation of late flagellar genes and of virulence factors in C. jejuni.