SAIF

Vivanco et al.
The purpose of this study was to compare computed tomography density (rCT) obtained using typical clinical computed tomography scan parameters to ash density (rash), for the prediction of densities of femoral head trabecular bone from hip fracture patients. An experimental study was conducted to investigate the relationships between rash and rCT and between each of these densities and rbulk and rdry . Seven human femoral heads from hip fracture patients were computed tomography–scanned ex vivo, and 76 cylindrical trabecular bone specimens were collected. Computed tomography density was computed from computed tomography images by using a calibration Hounsfield units–based equation, whereas rbulk , rdry and rash were determined experimentally. A large variation was found in the mean Hounsfield units of the bone cores (HUcore) with a constant bias from rCT to rash of 42.5 mg/cm3. Computed tomography and ash densities were linearly correlated (R2 = 0.55, p \ 0.001). It was demonstrated that rash provided a good estimate of rbulk (R2 =0.78, p \ 0.001) and is a strong predictor of rdry (R2 = 0.99, p \ 0.001). In addition, the rCT was linearly related to rbulk (R2 = 0.43, p \ 0.001) and rdry (R2 = 0.56, p \ 0.001). In conclusion, mineral density was an appropriate predictor of rbulk and rdry, and rCTwas not a surrogate for rash. There were linear relationships between rCT and physical densities; however, following the experimental protocols of this study to determine rCT, considerable scatter was present in the rCT relationships.

Vivanco et al.
Although it is widely known that bone tissue responds to mechanical stimuli, the underlying biological control is still not completely understood. The purpose of this study was to validate required methods necessary to maintain active osteocytes and minimize bone tissue injury in an ex vivo three-dimensional model that could mimic in vivo cellular function. The response of 22 bovine trabecular bone cores to uniaxial compressive load was investigated by using the ZETOS bone loading and bioreactor system while perfused with culture medium for 21 days. Two groups were formed, the ‘‘treatment’’ group (n = 12) was stimulated with a physiological compressive strain (4000me) in the form of a ‘‘jump’’ wave, while the ‘‘control’’ group (n = 10) was loaded only during three measurements for apparent elastic modulus on days 3, 10, and 21. At the end of the experiment, apoptosis and active osteocytes were quantified with histological analysis, and bone formation was identified by means of the calcium-binding dye, calcein. It was demonstrated that the treatment group increased the elastic modulus by 61%, whereas the control group increased by 28% (p\0.05). Of the total osteocytes observed at the end of 21 days, 1.7% (60.3%) stained positive for apoptosis in the loaded group, whereas 2.7% (60.4%) stained positive in the control group. Apoptosis in the center of the bone cores of both groups at the end of 21 days was similar to that observed in vivo. Therefore, the three-dimensional model used in this research permitted the investigation of physiological responses to mechanical loads on morphology adaptation of trabecular bone in a controlled defined load and chemical environment.

Swanson et al.
BACKGROUND: 5-Aminolevulinic acid (5-ALA)-induced tumor fluorescence aids brain tumor resections but is not approved for routine use in the United States. We developed and describe testing of 2 novel fluorescent, cancer-selective alkylphosphocholine analogs, CLR1501 (green) and CLR1502 (near infrared), in a proof-of-principle study for fluorescence-guided glioma surgery.
OBJECTIVE: To demonstrate that CLR1501 and CLR1502 are cancer cell-selective fluorescence agents in glioblastoma models and to compare tumor-to-normal brain (T:N) fluorescence ratios with 5-ALA.
METHODS: CLR1501, CLR1502, and 5-ALA were administered to mice with magnetic resonance imaging-verified orthotopic U251 glioblastoma multiforme- and glioblastoma stem cell-derived xenografts. Harvested brains were imaged with confocal microscopy (CLR1501), the IVIS Spectrum imaging system (CLR1501, CLR1502, and 5-ALA), or the Fluobeam near-infrared fluorescence imaging system (CLR1502). Imaging
and quantitative analysis of T:N fluorescence ratios were performed.
RESULTS: Excitation/emission peaks are 500/517 nm for CLR1501 and 760/778 nm for CLR1502. The observed T:N ratio for CLR1502 (9.28 6 1.08) was significantly higher (P ,.01) than for CLR1501 (3.51 6 0.44 on confocal imaging; 7.23 6 1.63 on IVIS imaging) and 5-ALA (4.81 6 0.92). Near-infrared Fluobeam CLR1502 imaging in a mouse xenograft model demonstrated high- contrast tumor visualization compatible with surgical applications.
CONCLUSION: CLR1501 (green) and CLR1502 (near infrared) are novel tumor-selective fluorescent agents for discriminating tumor from normal brain. CLR1501 exhibits a tumor-to-brain fluorescence ratio similar to that of 5-ALA, whereas CLR1502 has a superior tumor-to-brain fluorescence ratio. This study demonstrates the potential use of CLR1501 and CLR1502 in fluorescence-guided tumor surgery.

Peng et al.
The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the N-lysine acetylation of ER-resident and ER-transiting proteins. A point-mutation (S113R) in AT-1 has been associated with a familial form of spastic paraplegia. Here, we report that AT-1S113R is unable to form homodimers in the ER membrane and is devoid of acetyl-CoA transport activity. The reduced influx of acetyl-CoA into theERlumen results in reduced acetylation ofERproteins and an aberrant form of autophagy. Mice homozygous for the mutation display early developmental arrest. In contrast, heterozygous animals develop to full term, but display neurodegeneration and propensity to infections, inflammation, and cancer. The immune and cancer phenotypes are contingent on the presence of pathogens in the colony, whereas the nervous system phenotype is not. In conclusion, our results reveal a previously unknown aspect of acetyl-CoA metabolism that affects the immune and nervous systems and the risk for malignancies.

Osting et al.
Intraoperative magnetic resonance imaging (MRI) has been proposed as a method to optimize intracerebral targeting and for tracking infusate distribution in gene therapy trials for nervous system disorders. We thus investigated possible effects of two MRI contrast agents, gadoteridol (Gd) and galbumin (Gab), on the distribution and levels of transgene expression in the rat striatum and their effect on integrity and stability of recombinant adeno-associated virus (rAAV) particles. MRI studies showed that contrast agent distribution did not predict rAAV distribution. However, green fluorescent protein (GFP) immunoreactivity revealed an
increase in distribution of rAAV5-GFP, but not rAAV2-GFP, in the presence of Gd when compared with viral vector injected alone. In contrast, Gab increased the distribution of rAAV2-GFP not rAAV5-GFP. These observations pointed to a direct effect of infused contrast agent on the rAAV particles. Negative-stain electron microscopy (EM), DNAase treatment, and differential scanning calorimetry (DSC) were used to monitor rAAV2 and rAAV5 particle integrity and stability following contrast agent incubation. EMs of rAAV2-GFP and rAAV5-GFP particles pretreated with Gd appear morphologically similar to the untreated sample; however,
Gab treatment resulted in surface morphology changes and aggregation. A compromise of particle integrity was suggested by sensitivity of the packaged genome to DNAase treatment following Gab incubation but not Gd for both vectors. However, neither agent significantly affected particle stability when analyzed by DSC. An increase in Tm was observed for AAV2 in lactated Ringer’s buffer. These results thus highlight potential interactions between MRI contrast agents and AAV that might affect vector distribution and stability, as well as the stabilizing effect of lactated Ringer’s solution on AAV2.

Leystra et al.
Aberrations in the phosphoinositide 3-kinase (PI3K) signaling pathway play a key role in the pathogenesis of numerous cancers by altering cellular growth, metabolism, proliferation, and apoptosis. Mutations in the catalytic domain of PI3K that generate a dominantly active kinase are commonly found in human colorectal cancers and have been thought to drive tumor progression but not initiation. However, the effects of constitutively activated PI3K upon the intestinal mucosa have not been previously studied in animal models. Here, we show that the expression of a dominantly active form of the PI3K protein in the mouse intestine results in hyperplasia and advanced neoplasia. Mice expressing constitutively active PI3K in the epithelial cells of the distal small bowel and colon rapidly developed invasive adenocarcinomas in the colon that spread into the mesentery and adjacent organs. The histologic characteristics of these tumors were strikingly similar to invasive mucinous colon cancers in humans. Interestingly, these tumors formed without a benign polypoid intermediary, consistent with the lack of aberrant WNT signaling observed. Together, our findings indicate a noncanonical mechanism of colon tumor initiation that is mediated through activation of PI3K. This unique model has the potential to further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification.

Hong et al.
Purpose High tumor microvessel density correlates with a poor prognosis in multiple solid tumor types. The clinical gold standard for assessing microvessel density is CD105 immunohistochemistry on paraffin-embedded tumor specimens. The goal of this study was to develop an 89Zr-based PET tracer for noninvasive imaging of CD105 expression.
Methods TRC105, a chimeric anti-CD105 monoclonal antibody, was conjugated to p-isothiocyanatobenzyldesferrioxamine (Df-Bz-NCS) and labeled with 89Zr. FACS analysis and microscopy studies were performed to compare the CD105 binding affinity of TRC105 and Df-TRC105. PET imaging, biodistribution, blocking, and ex-vivo histology studies were performed on 4T1 murine breast tumor-bearing mice to evaluate the pharmacokinetics and tumor-targeting of 89Zr-Df-TRC105. Another chimeric antibody, cetuximab, was used as an isotype-matched control. Results FACS analysis of HUVECs revealed no difference in CD105 binding affinity between TRC105 and Df-TRC105, which was further validated by fluorescence microscopy. 89Zr labeling was achieved with high yield and specific activity. Serial PET imaging revealed that the
4T1 tumor uptake of 89Zr-Df-TRC105 was 6.1±1.2, 14.3±1.2, 12.4±1.5, 7.1±0.9, and 5.2±0.3 %ID/g at 5, 24, 48, 72, and 96 h after injection, respectively (n=4), higher than all organs starting from 24 h after injection, which provided excellent tumor contrast. Biodistribution data as measured by gamma counting were consistent with the PET findings. Blocking experiments, control studies with 89Zr-Df-cetuximab, and ex-vivo histology all confirmed the in vivo target specificity of 89Zr-Df-TRC105.
Conclusion We report here the first successful PET imaging of CD105 expression with 89Zr as the radiolabel. Rapid, persistent, CD105-specific uptake of 89Zr-Df-TRC105 in the 4T1 tumor was observed.

Hilmer et al. Lobeline is a potential smoking cessation drug with affinity for the α4β2 nicotinic acetylcholine receptor and may inhibit the blood–brain barrier (BBB) amine transporter. The goal of this work was to use PET imaging to evaluate the effects of lobeline on the kinetic properties of [18F]nifene in the rat brain. Methods: Direct α4β2* competition of lobeline with [18F]nifene was evaluated using imaging experiments with both displacing and blocking doses of lobeline (1 mg/kg, i.v.) given between two injections of [18F]nifene separated by 50 min. Inhibition of the BBB amine transporter was examined using a separate imaging protocol with three injections of [18F]nifene, first at baseline, then following (−)nicotine blocking, and finally following lobeline blocking. Results: Rapid displacement of [18F]nifene was observed in the α4β2*-rich thalamus following lobeline administration, suggesting direct competition of the drug at α4β2* sites. Slight decreases in BBB transport of [18F]nifene were observed when the α4β2* system was first saturated with (−)nicotine and then given lobeline. This perturbation may be due to inhibition of the BBB amine transporter by lobeline or reductions in blood flow. Significant cerebellar displacement of [18F]nifene was found following the administration of both lobeline and (−)nicotine, indicating detectable specific binding in the rat cerebellum. Conclusion: The competition of lobeline with [18F]nifene is largely dominated at the α4β2* binding site and only small perturbations in BBB transport of [18F]nifene are seen at the 1 mg/kg dose. Similar experiments could be used to study other drugs as therapeutic agents for smoking cessation with PET.

Hafeez et al. We present here first time that Plumbagin (PL), a medicinal plant-derived 1,4-naphthoquinone, inhibits the growth and metastasis of human prostate cancer (PCa) cells in an orthotopic xenograft mouse model. In this study, human PCa PC-3M-luciferase cells (2
106) were injected into the prostate of athymic nude mice. Three days post cell implantation, mice were treated with PL (2 mg/kg body wt. i.p. five days in a week) for 8 weeks. Growth and metastasis of PC-3M-luciferase cells was examined weekly by bioluminescence imaging of live mice. PL-treatment significantly ( p ¼ 0.0008) inhibited the growth of orthotopic xenograft tumors. Results demonstrated a significant inhibition of metastasis into liver ( p ¼ 0.037), but inhibition of metastasis into the lungs ( p ¼ 0.60) and lymph nodes ( p ¼ 0.27) was not observed to be significant. These results were further confirmed by histopathology of these organs. Results of histopathology demonstrated a significant inhibition of metastasis into lymph nodes ( p ¼ 0.034) and lungs ( p ¼ 0.028), and a trend to significance in liver ( p ¼ 0.075). None of the mice in the PL-treatment group showed PCa metastasis into the liver, but these mice had small metastasis foci into the lymph nodes and lungs. However, control mice had large metastatic foci into the lymph nodes, lungs, and liver. PL-caused inhibition of the growth and metastasis of PC-3M cells accompanies inhibition of the expression of: 1) PKCε, pStat3Tyr705, and pStat3Ser727, 2) Stat3 downstream target genes (survivin and BclxL), 3) proliferative markers Ki-67 and PCNA, 4) metastatic marker MMP9, MMP2, and uPA, and 5) angiogenesis markers CD31 and VEGF. Taken together, these results suggest that PL inhibits tumor growth and metastasis of human PCa PC3-M-luciferase cells, which could be used as a therapeutic agent for the prevention and treatment of human PCa.

Haffeez et al. Protein kinase C epsilon (PKCe), a novel PKC isoform, is overexpressed in prostate cancer (PCa) and correlates with disease aggressiveness. However, the functional contribution of PKCe to development or progression of PCa remained to be determined. Here we present the first in vivo genetic evidence that PKCe is essential for both the development and metastasis of PCa in the transgenic mouse model of prostate adenocarcinoma (TRAMP). Heterozygous or homozygous genetic deletions of PKCe in FVB/N TRAMP inhibited PCa development and metastasis as analyzed by positron emission tomography/computed tomography, tumor weight determinations, and histopathology. We also examined biomarkers associated with tumor progression in this model, including markers of survival, proliferation, angiogenesis, inflammation, and metastatic progression. To find clues about the genes regulated by PKCe and linked to the Stat3 signaling pathway, we carried out focused PCR arrays of JAK/STAT signaling in excised PCa tissues from PKCe wild-type and nullizygous TRAMP mice. Notably, PKCe loss was associated with significant downregulation of proliferative and metastatic genes C/EBPb (CCAAT/enhancer binding protein b), CRP (C-reactive protein), CMK, EGFR (epidermal growth factor receptor), CD64, Jun B, and gp130. Taken together, our findings offer the first genetic evidence of the role of PKCe in PCa development and metastasis. PKCe may be potential target for prevention and/or treatment of PCa.