Targeted delivery of cytokine therapy to rheumatoid tissue by a synovial targeting peptide.

OBJECTIVES: The synovial endothelium targeting peptide (SyETP) CKSTHDRLC has been identified previously and was shown to preferentially localise to synovial xenografts in the human/severe combined immunodeficient (SCID) mouse chimera model of rheumatoid arthritis (RA). The objective of the current work was to generate SyETP-anti-inflammatory-cytokine fusion proteins that would deliver bioactive cytokines specifically to human synovial tissue. METHODS: Fusion proteins consisting of human interleukin (IL)-4 linked via a matrix metalloproteinase (MMP)-cleavable sequence to multiple copies of either SyETP or scrambled control peptide were expressed in insect cells, purified by Ni-chelate chromatography and bioactivity tested in vitro. The ability of SyETP to retain bioactive cytokine in synovial but not control skin xenografts in SCID mice was determined by in vivo imaging using nano-single-photon emission computed tomography-computed tomography (nano-SPECT-CT) and measuring signal transducer and activator of transcription 6 (STAT6) phosphorylation in synovial grafts following intravenous administration of the fusion protein. RESULTS: In vitro assays confirmed that IL-4 and the MMP-cleavable sequence were functional. IL-4-SyETP augmented production of IL-1 receptor antagonist (IL-1ra) by fibroblast-like synoviocytes (FLS) stimulated with IL-1ß  in a dose-dependent manner. In vivo imaging showed that IL-4-SyETP was retained in synovial but not in skin tissue grafts and the period of retention was significantly enhanced through increasing the number of SyETP copies from one to three. Finally, retention correlated with increased bioactivity of the cytokine as quantified by STAT6 phosphorylation in synovial grafts. CONCLUSIONS: The present work demonstrates that SyETP specifically delivers fused IL-4 to human rheumatoid synovium transplanted into SCID mice, thus providing a proof of concept for peptide-targeted tissue-specific immunotherapy in RA. This technology is potentially applicable to other biological treatments providing enhanced potency to inflammatory sites and reducing systemic toxicity.

High-throughput high-volume nuclear imaging for preclinical in vivo compound screening§.

BACKGROUND: Preclinical single-photon emission computed tomography (SPECT)/CT imaging studies are hampered by low throughput, hence are found typically within small volume feasibility studies. Here, imaging and image analysis procedures are presented that allow profiling of a large volume of radiolabelled compounds within a reasonably short total study time. Particular emphasis was put on quality control (QC) and on fast and unbiased image analysis. METHODS: 2-3 His-tagged proteins were simultaneously radiolabelled by 99mTc-tricarbonyl methodology and injected intravenously (20 nmol/kg; 100 MBq; n = 3) into patient-derived xenograft (PDX) mouse models. Whole-body SPECT/CT images of 3 mice simultaneously were acquired 1, 4, and 24 h post-injection, extended to 48 h and/or by 0-2 h dynamic SPECT for pre-selected compounds. Organ uptake was quantified by automated multi-atlas and manual segmentations. Data were plotted automatically, quality controlled and stored on a collaborative image management platform. Ex vivo uptake data were collected semi-automatically and analysis performed as for imaging data. RESULTS: >500 single animal SPECT images were acquired for 25 proteins over 5 weeks, eventually generating >3500 ROI and >1000 items of tissue data. SPECT/CT images clearly visualized uptake in tumour and other tissues even at 48 h post-injection. Intersubject uptake variability was typically 13% (coefficient of variation, COV). Imaging results correlated well with ex vivo data. CONCLUSIONS: The large data set of tumour, background and systemic uptake/clearance data from 75 mice for 25 compounds allows identification of compounds of interest. The number of animals required was reduced considerably by longitudinal imaging compared to dissection experiments. All experimental work and analyses were accomplished within 3 months expected to be compatible with drug development programmes. QC along all workflow steps, blinding of the imaging contract research organization to compound properties and automation provide confidence in the data set. Additional ex vivo data were useful as a control but could be omitted from future studies in the same centre. For even larger compound libraries, radiolabelling could be expedited and the number of imaging time points adapted to increase weekly throughput. Multi-atlas segmentation could be expanded via SPECT/MRI; however, this would require an MRI-compatible mouse hotel. Finally, analysis of nuclear images of radiopharmaceuticals in clinical trials may benefit from the automated analysis procedures developed.

Ambient visual information confers a context-specific, long-term benefit on memory for haptic scenes.

We investigated the effects of indirect, ambient visual information on haptic spatial memory. Using touch only, participants first learned an array of objects arranged in a scene and were subsequently tested on their recognition of that scene which was always hidden from view. During haptic scene exploration, participants could either see the surrounding room or were blindfolded. We found a benefit in haptic memory performance only when ambient visual information was available in the early stages of the task but not when participants were initially blindfolded. Specifically, when ambient visual information was available a benefit on performance was found in a subsequent block of trials during which the participant was blindfolded (Experiment 1), and persisted over a delay of one week (Experiment 2). However, we found that the benefit for ambient visual information did not transfer to a novel environment (Experiment 3). In Experiment 4 we further investigated the nature of the visual information that improved haptic memory and found that geometric information about a surrounding (virtual) room rather than isolated object landmarks, facilitated haptic scene memory. Our results suggest that vision improves haptic memory for scenes by providing an environment-centred, allocentric reference frame for representing object location through touch.

Quantitative Accuracy of Low-Count SPECT Imaging in Phantom and In Vivo Mouse Studies.

We investigated the accuracy of a single photon emission computed tomography (SPECT) system in quantifying a wide range of radioactivity concentrations using different scan times in both phantom and animal models. A phantom containing various amounts of In-111 or Tc-99m was imaged until the activity had decayed close to background levels. Scans were acquired for different durations, employing different collimator pinhole sizes. VOI analysis was performed to quantify uptake in the images and the values compared to the true activity. The phantom results were then validated in tumour-bearing mice. The use of an appropriate calibration phantom and disabling of a background subtraction feature meant that absolute errors were within 12% of the true activity. Furthermore, a comparison of in vivo imaging and biodistribution studies in mice showed a correlation of 0.99 for activities over the 200 kBq to 5 MBq range. We conclude that the quantitative information provided by the NanoSPECT camera is accurate and allows replacement of dissection studies for assessment of radiotracer biodistribution in mouse models.

Visual and haptic representations of scenes are updated with observer movement.

Scene recognition has been found to be sensitive to the orientation of the scene with respect to the stationary observer. Recent studies have shown, however, that observer movement can compensate for changes in visual scene orientation, through a process of spatial updating. Here we investigated whether spatial updating in scene recognition is affected by the encoding or learning modality by examining whether observer movement can also compensate for orientation changes in haptic scene recognition. In experiment 1, we replicated previously reported effects of observer movement on visual scene recognition. In experiment 2, we used the same apparatus as in experiment 1 but here participants were required to learn and recognize the scenes using touch alone. We found a cost in recognition performance with changes in scene orientation relative to the stationary observer. However, when participants could move around the scene to recognize the new orientation, then this cost in recognition performance disappeared. Thus, we found that spatial updating applies to recognition in both the visual and haptic modalities, both of which intrinsically encode the spatial properties of a scene.