Antimicrobial peptides in frog poisons constitute a molecular toxin delivery system against predators.

Animals using toxic peptides and proteins for predation or defense typically depend on specialized morphological structures, like fangs, spines, or a stinger, for effective intoxication. Here we show that amphibian poisons instead incorporate their own molecular system for toxin delivery to attacking predators. Skin-secreted peptides, generally considered part of the amphibian immune system, permeabilize oral epithelial tissue and enable fast access of cosecreted toxins to the predator's bloodstream and organs. This absorption-enhancing system exists in at least three distantly related frog lineages and is likely to be a widespread adaptation, determining the outcome of predator-prey encounters in hundreds of species.

AAV9 delivered bispecific nanobody attenuates amyloid burden in the gelsolin amyloidosis mouse model.

Gelsolin amyloidosis is a dominantly inherited, incurable type of amyloidosis. A single point mutation in the gelsolin gene (G654A is most common) results in the loss of a Ca2+ binding site in the second gelsolin domain. Consequently, this domain partly unfolds and exposes an otherwise buried furin cleavage site at the surface. During secretion of mutant plasma gelsolin consecutive cleavage by furin and MT1-MMP results in the production of 8 and 5 kDa amyloidogenic peptides. Nanobodies that are able to (partly) inhibit furin or MT1-MMP proteolysis have previously been reported. In this study, the nanobodies have been combined into a single bispecific format able to simultaneously shield mutant plasma gelsolin from intracellular furin and extracellular MT1-MMP activity. We report the successful in vivo expression of this bispecific nanobody following adeno-associated virus serotype 9 gene therapy in gelsolin amyloidosis mice. Using SPECT/CT and immunohistochemistry, a reduction in gelsolin amyloid burden was detected which translated into improved muscle contractile properties. We conclude that a nanobody-based gene therapy using adeno-associated viruses shows great potential as a novel strategy in gelsolin amyloidosis and potentially other amyloid diseases.

Comparison of Several White Matter Tracts in Feline and Canine Brain by Using Magnetic Resonance Diffusion Tensor Imaging.

Recently, we published a first anatomical diffusion tensor imaging (DTI) atlas regarding white matter tracts in the canine brain. The purpose of this study was to show the significance of DTI in the revelation of the white matter fibres in the feline brain (i.e., to obtain an anatomical DTI atlas of images) and to descriptively compare these to previously obtained white matter fibre images of the canine brain. DTI MR Images of four cats euthanized for reasons other than neurological disorders were obtained with a 3 T system. Combined fractional anisotropic (FA) and directional maps were obtained within the hour after death. An experienced anatomist tracked white matter tracts of clinical relevance using the scanner software. After validation of these tracts, we compared relevant neurological connections between the cat and the dog. Comparison of cerebral structures between different species is easier when the three dimensional anatomy is visualized by using DTI. 3D rendered DTI images clearly show major differences in neurological architecture between cats and dogs for example, the more important space occupying role of the limbic system, and the less diffuse, less nodular, less pronounced and thinner fibre bundles in the feline brain compared to the canine brain (except for the cerebellum different parts connecting fibres passing through the brainstem which are pronouncedly developed). Anat Rec, 300:1270-1289, 2017. © 2017 Wiley Periodicals, Inc.

Non-Invasive Imaging of Amyloid Deposits in a Mouse Model of AGel Using 99mTc-Modified Nanobodies and SPECT/CT.

PURPOSE: Gelsolin amyloidosis (AGel), also known as familial amyloidosis, Finnish type (FAF), is an autosomal, dominant, incurable disease caused by a point mutation (G654A/T) in the gelsolin (GSN) gene. The mutation results in loss of a Ca2+-binding site in the second gelsolin domain. Subsequent incorrect folding exposes a cryptic furin cleavage site, leading to the formation of a 68-kDa C-terminal cleavage product (C68) in the trans-Golgi network. This C68 fragment is cleaved by membrane type 1-matrix metalloproteinase (MT1-MMP) during secretion into the extracellular environment, releasing 8- and 5-kDa amyloidogenic peptides. These peptides aggregate and cause disease-associated symptoms. We set out to investigate whether AGel-specific nanobodies could be used to monitor amyloidogenic gelsolin buildup. PROCEDURES: Three nanobodies (FAF Nb1-3) raised against the 8-kDa fragment were screened as AGel amyloid imaging agents in WT and AGel mice using 99mTc-based single-photon emission computed tomography (SPECT)/X-ray tomography (CT), biodistribution analysis, and immunofluorescence (IF). The quantitative characteristics were analyzed in a follow-up study with a Nb11-expressing mouse model. RESULTS: All three nanobodies possess the characteristics desired for a 99mTc-based SPECT/CT imaging agent, high specificity and a low background signal. FAF Nb1 was identified as the most potent, based on its superior signal-to-noise ratio and signal specificity. As a proof of concept, we implemented 99mTc-FAF Nb1 in a follow-up study of the Nb11-expressing AGel mouse model. Using biodistribution analysis and immunofluorescence, we demonstrated the validity of the data acquired via 99mTc-FAF Nb1 SPECT/CT. CONCLUSION: These findings demonstrate the potential of this nanobody as a non-invasive tool to image amyloidogenic gelsolin deposition and assess the therapeutic capacity of AGel therapeutics currently under development. We propose that this approach can be extended to other amyloid diseases, thereby contributing to the development of specific therapies.

A standardized method for in vivo mouse pancreas imaging and semiquantitative ß cell mass measurement by dual isotope SPECT.

PURPOSE: In order to evaluate future ß cell tracers in vivo, we aimed to develop a standardized in vivo method allowing semiquantitative measurement of a prospective ß cell tracer within the pancreas. PROCEDURES: 2-[(123)I]Iodo-L-phenylalanine ([(123)I]IPA) and [Lys(40)([(111)In]DTPA)]exendin-3 ([(111)In]Ex3) pancreatic uptake and biodistribution were evaluated using SPECT, autoradiography, and an ex vivo biodistribution study in a controlled unilaterally nephrectomized mouse ß cell depletion model. Semiquantitative measurement of the imaging results was performed using [(123)I]IPA to delineate the pancreas and [(111)In]Ex3 as a ß cell tracer. RESULTS: The uptake of [(123)I]IPA was highest in the pancreas. Aside from the kidneys, the uptake of [(111)In]Ex3 was highest in the pancreas and lungs. Autoradiography showed only uptake of [(111)In]Ex3 in insulin-expressing cells. Semiquantitative measurement of [(111)In]Ex3 in the SPECT images based on the delineation of the pancreas with [(123)I]IPA showed a high correlation with the [(111)In]Ex3 uptake data of the pancreas obtained by dissection. A strong positive correlation was observed between the relative insulin positive area and the pancreas-to-blood ratios of [(111)In]Ex3 uptake as determined by counting with a gamma counter and the semiquantitative analysis of the SPECT images. CONCLUSIONS: [(123)I]IPA is a promising tracer to delineate pancreatic tissue on SPECT images. It shows a high uptake in the pancreas as compared to other abdominal tissues. This study also demonstrates the feasibility and accuracy to measure the ß cell mass in vivo in an animal model of diabetes.

Comparison of the gene transfer efficiency of mRNA/GL67 and pDNA/GL67 complexes in respiratory cells.

Complexes between mRNA and GL67:DOPE:DMPE-PEG5000 (GL67) liposomes were formulated and characterized. Subsequently, the in vitro and in vivo expression characteristics of mRNA/GL67 complexes and pDNA/GL67 complexes, each produced at their optimal ratio, were compared in respiratory cells. Transfection of A549 cells with mRNA/GL67 complexes resulted in a much faster expression than after transfection with pDNA/GL67 complexes. The percentage of GFP-positive cells after mRNA and pDNA transfection peaked after 8 and 24 h, respectively. At these time points the percentage of GFP-positive cells was two times higher after mRNA transfection than after pDNA transfection. Furthermore, the efficacy of mRNA/GL67 complexes was independent of the cell cycle. This was in sharp contrast with pDNA/GL67 complexes that caused only a weak expression in nondividing cells. This confirms that the nuclear barrier is a crucial obstacle for pDNA but not for mRNA. Finally, mRNA/GL67 and pDNA/GL67 complexes encoding luciferase were administered intranasally to the lungs of mice. The mRNA/GL67 complexes did not give rise to a measurable luciferase expression in the murine lungs. In contrast, a detectable bioluminescent signal was present in the lungs of mice that received the pDNA/GL67 complexes. We showed that mRNA/GL67 complexes have a lower stability in biological fluids. Consequently, this may be an explanation for their lower performance in vivo.

Preliminary in vivo evaluation of [131I]-2-iodo-D-phenylalanine as a potential radionuclide therapeutic agent in R1M-fluc rhabdomyosarcoma tumor-bearing NuNu mice using bioluminescent imaging.

BACKGROUND: Carrier-added [(123)I]-2-iodo-D-phenylalanine (CA [(123)I]-2-I-D-Phe) was previously found to have a preferential retention in tumors with a high tumor background contrast in animal models. A previous human dosimetry study demonstrated a favorable biodistribution and radiation burden in human subjects. AIM: The aim of this study was to investigate the potential of CA [(131)I]-2-I-D-Phe as an agent for radionuclide therapy. METHODS: Sixty (60) nude athymic mice were inoculated subcutaneously with firefly luciferase-transduced R1M rhabdomyosarcoma cells. The mice in the therapy group were injected intravenously (i.v.) with 148 MBq [(131)I]-2-I-D-Phe (432 GBq/mmol) in kit solution. Controls were injected with kit solution without radioactivity, with physiological saline, or with 148 MBq [(131)I](-) in physiological saline. Tumor growth was quantified using bioluminescent imaging and caliper measurements. RESULTS: [(131)I]-2-I-D-Phe clearly reduced tumor growth in the treated mice compared with the control groups. A tumor growth-rate reduction of at least 33% was found for mice receiving a therapeutic dose. There were no serious adverse side-effects of the therapy. CONCLUSIONS: In conclusion, i.v. injection of CA 148 MBq [(131)I]-2-I-D-Phe specifically reduces tumor growth in athymic nude mice without relevant side-effects on the animals' health.

Notch signaling as gatekeeper of rat acinar-to-beta-cell conversion in vitro.

BACKGROUND & AIMS: Exocrine acinar cells in the pancreas are highly differentiated cells that retain a remarkable degree of plasticity. After isolation and an initial phase of dedifferentiation in vitro, rodent acinar cells can convert to endocrine beta-cells when cultured in the presence of appropriate factors. The mechanisms regulating this phenotypic conversion are largely unknown. METHODS: Using rat acinar cell cultures, we studied the role of Notch signaling in a model of acinar-to-beta-cell conversion. RESULTS: We report a novel lectin-based cell labeling method to demonstrate the acinar origin of newly formed insulin-expressing beta-cells. This method allows for specific tracing of the acinar cells. We demonstrate that growth factor-induced conversion of adult acinar cells to beta-cells is negatively regulated by Notch1 signaling. Activated Notch1 signaling prevents the reexpression of the proendocrine transcription factor Neurogenin-3, the key regulator of endocrine development in the embryonic pancreas. Interfering with Notch1 signaling allows modulating the acinar cell susceptibility to the differentiation-inducing factors. Its inhibition significantly improves beta-cell neoformation with approximately 30% of acinar cells that convert to beta-cells. The newly formed beta-cells mature when transplanted ectopically and are capable of restoring normal blood glycemia in diabetic recipients. CONCLUSIONS: We report for the first time an efficient way to reprogram one third of the acinar cells to beta-cells by adult cell type conversion. This could find application in cell replacement therapy of type 1 diabetes, provided that it can be translated from rodent to human models.

Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D: -luciferin: effect on intensity, time kinetics and repeatability of photon emission.

INTRODUCTION: In vivo bioluminescence imaging (BLI) is a promising technique for non-invasive tumour imaging. D: -luciferin can be administrated intraperitonealy or intravenously. This will influence its availability and, therefore, the bioluminescent signal. The aim of this study is to compare the repeatability of BLI measurement after IV versus IP administration of D: -luciferin and assess the correlation between photon emission and histological cell count both in vitro and in vivo. MATERIALS AND METHODS: Fluc-positive R1M cells were subcutaneously inoculated in nu/nu mice. Dynamic BLI was performed after IV or IP administration of D: -luciferin. Maximal photon emission (PE(max)) was calculated. For repeatability assessment, every acquisition was repeated after 4 h and analysed using Bland-Altman method. A second group of animals was serially imaged, alternating IV and IP administration up to 21 days. When mice were killed, PE(max) after IV administration was correlated with histological cell number. RESULTS: The coefficients of repeatability were 80.2% (IV) versus 95.0% (IP). Time-to-peak is shorter, and its variance lower for IV (p < 0.0001). PE(max) was 5.6 times higher for IV. A trend was observed towards lower photon emission per cell in larger tumours. CONCLUSION: IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden.