Radiosynthesis and whole-body distribution in mice of a 18 F-labeled azepino[4,3-b]indole-1-one derivative with multimodal activity for the treatment of Alzheimer's disease.

Recently, the azepino[4,3-b]indole-1-one derivative 1 showed in vitro nanomolar inhibition against butyrylcholinesterase (BChE), the ChE isoform that plays a role in the progression and pathophysiology of Alzheimer's disease (AD), and protects against N-methyl- d-aspartate-induced neuronal toxicity. Three 9-R-substituted (R = F, Br, OMe) congeners were investigated. The 9-F derivative (2a) was found more potent as BChE inhibitors (half-maximal inhibitory concentration value = 21 nM) than 2b (9-Br) and 2c (9-OMe), achieving a residence time (38 s), assessed by surface plasmon resonance, threefold higher than that of 1. To progress in featuring the in vivo pharmacological characterization of 2a, herein the 18 F-labeled congener 2a was synthesized, by applying the aromatic 18 F-fluorination method, and its whole-body distribution in healthy mice, including brain penetration, was evaluated through positron emission tomography imaging. [18 F]2a exhibited a rapid and high brain uptake (3.35 ± 0.26% ID g-1 at 0.95 ± 0.15 min after injection), followed by a rapid clearance (t1/2 = 6.50 ± 0.93 min), showing good blood-brain barrier crossing. After a transient liver accumulation of [18 F]2a, the intestinal and urinary excretion was quantified. Finally, ex vivo pharmacological experiments in mice showed that the unlabeled 2a affects the transmitters' neurochemistry, which might be favorable to reverse cognition impairment in mild-to-moderate AD-related dementias.

Theranostic Surrogacy of [123I]NaI for Differentiated Thyroid Cancer Radionuclide Therapy.

Precise dosimetry has gained interest for interpreting the response assessments of novel therapeutic radiopharmaceuticals, as well as for improving conventional radiotherapies such as the "one dose fits all" approach. Although radioiodine as same-element isotope theranostic pairs has been used for differentiated thyroid cancer (DTC), there are insufficient studies on the determination of its dosing regimen for personalized medicine and on extrapolating strategies for companion diagnostic radiopharmaceuticals. In this study, DTC xenograft mouse models were generated after validating iodine uptakes via sodium iodine symporter proteins (NIS) through in vitro assays, and theranostic surrogacy of companion radiopharmaceuticals was investigated in terms of single photon emission computed tomography (SPECT) imaging and voxel-level dosimetry. Following a Monte Carlo simulation, the hypothetical energy deposition/dose distribution images were produced as [123I]NaI SPECT scans with the use of 131I ion source simulation, and dose rate curves were used to estimate absorbed dose. For the tumor, a peak concentration of 96.49 ± 11.66% ID/g occurred 2.91 ± 0.42 h after [123I]NaI injection, and absorbed dose for 131I therapy was estimated as 0.0344 ± 0.0088 Gy/MBq. The absorbed dose in target/off-target tissues was estimated by considering subject-specific heterogeneous tissue compositions and activity distributions. Furthermore, a novel approach was proposed for simplifying voxel-level dosimetry and suggested for determining the minimal/optimal scan time points of surrogates for pretherapeutic dosimetry. When two scan time points were set to Tmax and 26 h and the group mean half-lives were applied to the dose rate curves, the most accurate absorbed dose estimates were determined [-22.96, 2.21%]. This study provided an experimental basis to evaluate dose distribution and is expected hopefully to improve the challenging dosimetry process for clinical use.

Preclinical Evaluation of a Companion Diagnostic Radiopharmaceutical, [18F]PSMA-1007, in a Subcutaneous Prostate Cancer Xenograft Mouse Model.

Several radiolabeled prostate-specific membrane antigen (PSMA)-targeted agents have been developed for detecting prostate cancer, using positron emission tomography imaging and targeted radionuclide therapy. Among them, [18F]PSMA-1007 has several advantages, including a comparatively long half-life, delayed renal excretion, and compatible structure with α-/ß-particle emitter-labeled therapeutics. This study aimed to characterize the preclinical pharmacokinetics and internal radiation dosimetry of [18F]PSMA-1007, as well as its repeatability and specificity for target binding using prostate tumor-bearing mice. In PSMA-positive tumor-bearing mice, the kidney showed the greatest accumulation of [18F]PSMA-1007. The distribution in the tumor attained its peak concentration of 2.8%ID/g at 112 min after intravenous injection. The absorbed doses in the tumor and salivary glands were 0.079 ± 0.010 Gy/MBq and 0.036 ± 0.006 Gy/MBq, respectively. The variance of the net influx (Ki) of [18F]PSMA-1007 to the tumor was minimal between scans performed in the same animals (within-subject coefficient of variation = 7.57%). [18F]PSMA-1007 uptake in the tumor was specifically decreased by 32% in Ki after treatment with a PSMA inhibitor 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). In the present study, we investigated the in vivo preclinical characteristics of [18F]PSMA-1007. Our data from [18F]PSMA-1007 PET/computed tomography (CT) studies in a subcutaneous prostate cancer xenograft mouse model supports clinical therapeutic strategies that use paired therapeutic radiopharmaceuticals (such as [177Lu]Lu-PSMA-617), especially strategies with a quantitative radiation dose estimate for target lesions while minimizing radiation-induced toxicity to off-target tissues.

Thalamic and prefrontal GABA concentrations but not GABAA receptor densities are altered in high-functioning adults with autism spectrum disorder.

The gamma aminobutyric acid (GABA) neurotransmission system has been implicated in autism spectrum disorder (ASD). Molecular neuroimaging studies incorporating simultaneous acquisitions of GABA concentrations and GABAA receptor densities can identify objective molecular markers in ASD. We measured both total GABAA receptor densities by using [18F]flumazenil positron emission tomography ([18F]FMZ-PET) and GABA concentrations by using proton magnetic resonance spectroscopy (1H-MRS) in 28 adults with ASD and 29 age-matched typically developing (TD) individuals. Focusing on the bilateral thalami and the left dorsolateral prefrontal cortex (DLPFC) as our regions of interest, we found no differences in GABAA receptor densities between ASD and TD groups. However, 1H-MRS measurements revealed significantly higher GABA/Water (GABA normalized by water signal) in the left DLPFC of individuals with ASD than that of TD controls. Furthermore, a significant gender effect was observed in the thalami, with higher GABA/Water in males than in females. Hypothesizing that thalamic GABA correlates with ASD symptom severity in gender-specific ways, we stratified by diagnosis and investigated the interaction between gender and thalamic GABA/Water in predicting Autism-Spectrum Quotient (AQ) and Ritvo Autism Asperger's Diagnostic Scale-Revised (RAADS-R) total scores. We found that gender is a significant effect modifier of thalamic GABA/Water's relationship with AQ and RAADS-R scores for individuals with ASD, but not for TD controls. When we separated the ASD participants by gender, a negative correlation between thalamic GABA/Water and AQ was observed in male ASD participants. Remarkably, in female ASD participants, a positive correlation between thalamic GABA/Water and AQ was found.

Radiosynthesis and characterization of [18F]BS224: a next-generation TSPO PET ligand insensitive to the rs6971 polymorphism.

PURPOSE: Translocator protein 18-kDa (TSPO) positron emission tomography (PET) is a valuable tool to detect neuroinflammed areas in a broad spectrum of neurodegenerative diseases. However, the clinical application of second-generation TSPO ligands as biomarkers is limited because of the presence of human rs6971 polymorphism that affects their binding. Here, we describe the ability of a new TSPO ligand, [18F]BS224, to identify abnormal TSPO expression in neuroinflammation independent of the rs6971 polymorphism. METHODS: An in vitro competitive inhibition assay of BS224 was conducted with [3H]PK 11195 using membrane proteins isolated from 293FT cells expressing TSPO-wild type (WT) or TSPO-mutant A147T (Mut), corresponding to a high-affinity binder (HAB) and low-affinity binder (LAB), respectively. Molecular docking was performed to investigate the interaction of BS224 with the binding sites of rat TSPO-WT and TSPO-Mut. We synthesized a new 18F-labeled imidazopyridine acetamide ([18F]BS224) using boronic acid pinacol ester 6 or iodotoluene tosylate precursor 7, respectively, via aromatic 18F-fluorination. Dynamic PET scanning was performed up to 90 min after the injection of [18F]BS224 to healthy mice, and PET imaging data were obtained to estimate its absorbed doses in organs. To evaluate in vivo TSPO-specific uptake of [18F]BS224, lipopolysaccharide (LPS)-induced inflammatory and ischemic stroke rat models were used. RESULTS: BS224 exhibited a high affinity (Ki = 0.51 nM) and selectivity for TSPO. The ratio of IC50 values of BS224 for LAB to that for HAB indicated that the TSPO binding affinity of BS224 has low binding sensitivity to the rs6971 polymorphism and it was comparable to that of PK 11195, which is not sensitive to the polymorphism. Docking simulations showed that the binding mode of BS224 is not affected by the A147T mutation and consequently supported the observed in vitro selectivity of [18F]BS224 regardless of polymorphisms. With optimal radiochemical yield (39 ± 6.8%, decay-corrected) and purity (> 99%), [18F]BS224 provided a clear visible image of the inflammatory lesion with a high signal-to-background ratio in both animal models (BPND = 1.43 ± 0.17 and 1.57 ± 0.37 in the LPS-induced inflammatory and ischemic stroke rat models, respectively) without skull uptake. CONCLUSION: Our results suggest that [18F]BS224 may be a promising TSPO ligand to gauge neuroinflammatory disease-related areas in a broad range of patients irrespective of the common rs6971 polymorphism.

Multimodal Imaging Probe Development for Pancreatic ß Cells: From Fluorescence to PET.

Pancreatic ß cells are responsible for insulin secretion and are important for glucose regulation in a healthy body and diabetic disease patient without prelabeling of islets. While the conventional biomarkers for diabetes have been glucose and insulin concentrations in the blood, the direct determination of the pancreatic ß cell mass would provide critical information for the disease status and progression. By combining fluorination and diversity-oriented fluorescence library strategy, we have developed a multimodal pancreatic ß cell probe PiF for both fluorescence and for PET (positron emission tomography). By simple tail vein injection, PiF stains pancreatic ß cells specifically and allows intraoperative fluorescent imaging of pancreatic islets. PiF-injected pancreatic tissue even facilitated an antibody-free islet analysis within 2 h, dramatically accelerating the day-long histological procedure without any fixing and dehydration step. Not only islets in the pancreas but also the low background of PiF in the liver allowed us to monitor the intraportal transplanted islets, which is the first in vivo visualization of transplanted human islets without a prelabeling of the islets. Finally, we could replace the built-in fluorine atom in PiF with radioactive 18F and successfully demonstrate in situ PET imaging for pancreatic islets.

Loss of Substantia Nigra Hyperintensity at 3.0-T MR Imaging in Idiopathic REM Sleep Behavior Disorder: Comparison with 123I-FP-CIT SPECT.

Purpose To examine whether the loss of nigral hyperintensity (NH) on 3.0-T susceptibility-weighted (SW) magnetic resonance (MR) images can help identify high synucleinopathy risk in patients with idiopathic rapid eye movement sleep behavior disorder (iRBD). Materials and Methods Between March 2014 and April 2015, 18 consecutively recruited patients with iRBD were evaluated with 3.0-T SW imaging and iodine 123-2ß-carbomethoxy-3ß-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane (123I-FP-CIT) single photon emission computed tomography and compared with 18 healthy subjects and 18 patients with Parkinson disease (PD). Two readers blinded to clinical diagnosis independently assessed the images. 123I-FP-CIT uptake ratios were compared by using the Kruskal-Wallis test, and intra- and interobserver agreements were assessed with the Cohen κ. The synucleinopathy conversion according to NH status was evaluated in patients with iRBD after follow-up. Results NH was intact in seven patients with iRBD and lost in 11. The 123I-FP-CIT uptake ratios were comparable between those with intact NH (mean, 3.22 ± 0.47) and healthy subjects (mean, 3.37 ± 0.47) (P = .495). The 123I-FP-CIT uptake ratios in the 11 patients with iRBD and NH loss (mean, 2.48 ± 0.44) were significantly lower than those in healthy subjects (mean, 3.37 ± 0.47; P < .001) but higher than those in patients with PD (mean, 1.80 ± 0.33; P < .001). The intra- and interobserver agreements were excellent (κ > 0.9). Five patients with iRBD and NH loss developed symptoms of parkinsonism or dementia 18 months after neuroimaging. Conclusion NH loss at 3.0-T SW imaging may be a promising marker for short-term synucleinopathy risk in iRBD. © RSNA, 2017 Online supplemental material is available for this article.

Preclinical comparison study between [18F]fluoromethyl-PBR28 and its deuterated analog in a rat model of neuroinflammation.

We designed and synthesized deuterium-substituted [18F]fluoromethyl-PBR28 ([18F]1-d2) as a novel translocator protein 18 kDa (TSPO)-targeted radioligand with enhanced in vivo stability. The comparison studies between [18F]fluoromethyl-PBR28 ([18F]1) and its deuterate analog ([18F]1-d2) were investigated in terms of in vitro binding affinity, lipophilicity and in vivo stability. In addition, the accuracies of both radioligands were determined by comparing the PET imaging data in the same LPS-induced neuroinflammation rat model. Both aryloxyanilide analogs showed similar lipophilicity and in vitro affinity for TSPO. However, [18F]1-d2 provided significantly lower femur uptake than [18F]1 (1.5 ±â€¯1.2 vs. 4.1 ±â€¯1.7%ID/g at 2 h post-injection) in an ex vivo biodistribution study. [18F]1-d2 was also selectively accumulated in the inflammatory lesion with the binding potential of the specifically bound radioligand relative to the non-displaceable radioligand in tissue (BPND = 3.17 ±â€¯0.48), in a LPS-induced acute neuroinflammation rat model, comparable to that of [18F]1 (BPND = 2.13 ±â€¯0.51). These results indicate that [18F]1-d2 had higher in vivo stability, which resulted in an enhanced target-to-background ratio compared to that induced by [18F]1.

Assessment of TSPO in a Rat Experimental Autoimmune Myocarditis Model: A Comparison Study between [18F]Fluoromethyl-PBR28 and [18F]CB251.

Overexpression of the 18-kDa translocator protein (TSPO) is closely linked to inflammatory responses in the heart, including myocarditis, which can lead to myocardial necrosis. In vivo assessment of inflammatory responses has enabled the precise diagnosis of myocarditis to improve clinical outcomes. Here, we evaluated TSPO overexpression in a rat model of experimental autoimmune myocarditis (EAM) compared to healthy rats using two TSPO radiotracers, [18F]fluoromethyl-PBR28 ([18F]1) and [18F]CB251 ([18F]2). All radiolabeling methods were successfully applied to an automated module for the reproducible preparation of TSPO radiotracers. Both radiotracers were directly compared in an EAM rat model, as well as in healthy rats to determine whether either radiotracer provides a more promising assessment of in vivo TSPO overexpression. [18F]2 provided more specific TSPO-uptake in the heart of the EAM rats (1.32-fold that of the heart-to-lung uptake ratio versus healthy controls), while [18F]1 did not show a significant difference between the two groups. Histopathological characterization revealed that a prominent positron emission tomography (PET) signal of [18F]2 in the EAM rats corresponded to the presence of a higher density of TSPO compared to the healthy controls. These results suggest that the imidazole[1,2-a]pyridine-based radiotracer [18F]2 is a sensitive tool for noninvasively diagnosing myocarditis related to inflammation of the heart muscle by assessing abnormal TSPO expression.

Human galectin-9 on the porcine cells affects the cytotoxic activity of M1-differentiated THP-1 cells through inducing a shift in M2-differentiated THP-1 cells.

BACKGROUND: In xenotransplantation, immune rejection by macrophages occurs rapidly and remains a major obstacle. Studies to control immune rejection in macrophages have been continuing to date. Recent studies have reported that human galectin-9 (hGal-9) can regulate the function of regulatory T cells (Treg), as well as cytotoxicity T cells (CTL) and natural killer cells (NK). Although the effect of hGal-9 on lymphocytes has been well studied, the relationship between hGal-9 and myeloid cells has been scarcely studied. METHODS: To confirm the decreased cytotoxic activity effect by hGal-9 in M1-differentiated THP-1 cells, we established the hGal-9 expressing transgenic porcine cell line. hGal-9 siRNA was transfected to transgenic cells and recombinant hGal-9 (rhGal-9) was treated to co-culturing condition, and then, flow cytometry assay was conducted for analyzing the cytotoxic activity of M1-differentiated THP-1 cells. Related inflammatory cytokines (IL-1ß, IL-10, TNF-α, IL-6, IL-12, IL-23, and TGF-ß) and related enzymes (iNOS and Arginase 1) were analyzed by qPCR and Western blot assay. To identify the shift in M1/M2-differentiated THP-1 cells, expression levels of CCR7, CD163, iNOS, and Arginase 1 and population of M2 marker positive cells were analyzed. RESULTS: The expression levels of pro-inflammatory cytokines in M1-differentiated THP-1 cells co-cultured with hGal-9-expressing porcine kidney epithelial cells were decreased, but not in co-cultured THP-1 cells. However, the expression levels of anti-inflammatory cytokines were also increased in co-cultured M1-differentiated THP-1 cells. The cytotoxicity effect of M1-differentiated THP-1 cells on transgenic cells was decreased while the expression levels of anti-inflammatory cytokines and M2 macrophages-related molecules were increased. M2 differentiation program was turned on while M1 program was turned down by enhancing the phosphorylation levels of Akt and PI3K and the expression level of PPAR-γ. Due to these changes, differentiation of M2 program was enhanced in cells co-cultured with hGal-9. CONCLUSIONS: These data suggested that hGal-9 has a reduction in M1-differentiated THP-1 cell cytotoxic activity-related acute immune rejection in pig-to-human xenotransplantation in addition to its role in lymphoid lineage immune cell regulation.

The potentiating effect of hTFPI in the presence of hCD47 reduces the cytotoxicity of human macrophages.

BACKGROUND: In pig-to-human xenotransplantation, hyperacute rejection of pig organs could be overcome by the production of α1,3-galactosyltransferase knockout pigs. However, macrophage-mediated acute rejection is another obstacle that needs to be overcome. Among the various candidate genes involved in acute rejection, CD47 inhibits monocyte/macrophage-mediated phagocytosis by identifying the CD47 signal regulatory protein alpha (SIRP-α) as self/non-self. Tissue factor pathway inhibitor (TFPI) is involved in the regulation of the coagulation pathway and is able to bind to another ligand of CD47, thrombospondin-1 (TSP-1). When TSP-1 binds to CD47, phagocytosis in macrophages is increased. METHODS: The 2A peptide system was used to establish pig kidney cells (PK15) simultaneously expressing human CD47 and human TFPI, and they were cultured with activated THP-1 cells. After staining with 7-aminoactinomycin D, flow cytometry analysis was carried out. TFPI siRNA analysis and recombinant human TFPI (rhTFPI) treatment were performed to determine the potentiating effect of TFPI on pig cells for activated THP-1 cells in the presence of CD47. Related inflammatory cytokines produced by activated THP-1 cells were analyzed using qPCR and Western blot technique. In addition, the tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was analyzed using immunoprecipitation and Western blot. RESULTS: hCD47/hTFPI-PK15 cells survived better than hCD47-PK15, hTFPI-PK15, or normal PK15 cells on cytotoxicity tests using activated THP-1 cells. TSP-1, derived from these activated THP-1 cells, served as a mediator for this enhancing effect, and it also played a role in activated adherent peripheral blood mononuclear cells (PBMCs). The tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was further increased in the case of co-expression of CD47/TFPI than in individual non-expression or expression of CD47 or TFPI alone. CONCLUSIONS: When hCD47 was expressed, the expression of hTFPI leaded to tyrosine phosphorylation of SIRP-α in activated THP-1 cells via hTSP-1 inhibition, and consequently, it might improve the effect of hCD47-SIRP-a signaling.

Maximum Standardized Uptake Value of (99m)Tc Hydroxymethylene Diphosphonate SPECT/CT for the Evaluation of Temporomandibular Joint Disorder.

Purpose To evaluate the diagnostic accuracy of the quantitative parameter standardized uptake value (SUV) at single photon emission computed tomography (SPECT)/computed tomography (CT) for the evaluation of temporomandibular joint (TMJ) disorder (TMD). Materials and Methods This study was approved by the institutional review board, and the need for informed consent was waived. Forty-four TMJs in 22 patients with TMD (five men and 17 women; mean age ± standard deviation, 30.0 years ± 12.1) were evaluated. The patients underwent planar bone scintigraphy and SPECT/CT 3-4 hours after injection of technetium 99m hydroxymethylene diphosphonate. The planar scintigraphy parameter of relative ratio (RR) and SPECT/CT parameters mean SUV (SUVmean) and maximum SUV (SUVmax) were compared for the visual assessment of TMD on planar scintigraphy images and for the presence of TMJ arthralgia. Group comparisons, receiver operating characteristic analysis, and Pearson correlation analysis were conducted. Results SUVmax gradually increased from normal (2.82 ± 0.73) to mild or moderately abnormal (3.56 ± 0.76, P < .05) and then to severely abnormal (4.86 ± 1.25, P < .05). However, RR and SUVmean did not vary significantly according to visual grade (P > .05). On the other hand, SUVmax was significantly greater in arthralgic TMJs (4.15 ± 1.11) than in nonarthralgic TMJs (2.97 ± 0.75, P < .001), as was SUVmean (1.63 ± 0.42 vs 1.30 ± 0.31, respectively; P = .005). However, there was no significant difference in RR (3.61 ± 0.57 vs 3.76 ± 0.68, P = .45). In receiver operating characteristic curve analyses for arthralgic TMJ, SUVmax had the greatest area under the curve (area of 0.815). Conclusion SUVmax derived from bone SPECT/CT may be useful for the evaluation of TMD. (©) RSNA, 2016 Online supplemental material is available for this article.

Prediction of neoadjuvant radiation chemotherapy response and survival using pretreatment [(18)F]FDG PET/CT scans in locally advanced rectal cancer.

PURPOSE: The aim of this study was to investigate metabolic and textural parameters from pretreatment [(18)F]FDG PET/CT scans for the prediction of neoadjuvant radiation chemotherapy response and 3-year disease-free survival (DFS) in patients with locally advanced rectal cancer (LARC). METHODS: We performed a retrospective review of 74 patients diagnosed with LARC who were initially examined with [(18)F]FDG PET/CT, and who underwent neoadjuvant radiation chemotherapy followed by complete resection. The standardized uptake value (mean, peak, and maximum), metabolic volume (MV), and total lesion glycolysis of rectal cancer lesions were calculated using the isocontour method with various thresholds. Using three-dimensional textural analysis, about 50 textural features were calculated for PET images. Response to neoadjuvant radiation chemotherapy, as assessed by histological tumour regression grading (TRG) after surgery and 3-year DFS, was evaluated using univariate/multivariate binary logistic regression and univariate/multivariate Cox regression analyses. RESULTS: MVs calculated using the thresholds mean standardized uptake value of the liver + two standard deviations (SDs), and mean standard uptake of the liver + three SDs were significantly associated with TRG. Textural parameters from histogram-based and co-occurrence analysis were significantly associated with TRG. However, multivariate analysis revealed that none of these parameters had any significance. On the other hand, MV calculated using various thresholds was significantly associated with 3-year DFS, and MV calculated using a higher threshold tended to be more strongly associated with 3-year DFS. In addition, textural parameters including kurtosis of the absolute gradient (GrKurtosis) were significantly associated with 3-year DFS. Multivariate analysis revealed that GrKurtosis could be a prognostic factor for 3-year DFS. CONCLUSION: Metabolic and textural parameters from initial [(18)F]FDG PET/CT scans could be indexes to assess tumour heterogeneity for the prediction of neoadjuvant radiation chemotherapy response and recurrence in LARC.

Loss of Nigral Hyperintensity on 3 Tesla MRI of Parkinsonism: Comparison With (123) I-FP-CIT SPECT.

BACKGROUND: The aim of this study was to investigate whether 3 Tesla susceptibility-weighted imaging can detect the alteration of substantia nigra hyperintensity in Parkinson's disease (PD), multiple system atrophy (MSA), and progressive supranuclear palsy (PSP) and to assess the concordance between the loss of nigral hyperintensity on 3 Tesla susceptibility-weighted imaging and the nigrostriatal dopaminergic degeneration indicated by (123) I-2ß-carbomethoxy-3ß-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane single photon emission computerized tomography. METHODS: Consecutive subjects with suspected parkinsonism were included, and clinical diagnosis was solidified during clinical follow-up. Two blinded neuroradiologists interpreted the nigral hyperintensity on susceptibility-weighted imaging. The performance of susceptibility-weighted imaging for detection of nigral hyperintensity loss was estimated on the basis of the clinical diagnosis and compared with single photon emission computerized tomography results. RESULTS: The study included 210 subjects (126 PD, 11 MSA, 11 PSP patients, 26 healthy controls, 36 disease controls). The presence or absence of nigral hyperintensity was accurately visualized in 112 PD, 7 MSA, and 11 PSP patients and 53 controls. We identified 16 false-negative cases and 11 false-positive cases. The sensitivity and specificity of susceptibility-weighted imaging were 88.8% and 83.6%, respectively. The concordance rate between susceptibility-weighted imaging and single photon emission computerized tomography was 86.2%. CONCLUSIONS: The loss of nigral hyperintensity on susceptibility-weighted imaging suggested nigrostriatal dopaminergic degeneration in a large portion of patients with parkinsonism, which was indicated by (123) I-2ß-carbomethoxy-3ß-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane single photon emission computerized tomography. In consideration of false-negative and -positive cases, well-designed imaging protocols should be introduced to improve the performance of nigral hyperintensity imaging. © 2016 International Parkinson and Movement Disorder Society.

Synthesis and Evaluation of Tricarbonyl (99m)Tc-Labeled 2-(4-Chloro)phenyl-imidazo[1,2-a]pyridine Analogs as Novel SPECT Imaging Radiotracer for TSPO-Rich Cancer.

The 18-kDa translocator protein (TSPO) levels are associated with brain, breast, and prostate cancer progression and have emerged as viable targets for cancer therapy and imaging. In order to develop highly selective and active ligands with a high affinity for TSPO, imidazopyridine-based TSPO ligand (CB256, 3) was prepared as the precursor. (99m)Tc- and Re-CB256 (1 and 2, respectively) were synthesized in high radiochemical yield (74.5% ± 6.4%, decay-corrected, n = 5) and chemical yield (65.6%) by the incorporation of the [(99m)Tc(CO)3(H2O)3]⁺ and (NEt4)2[Re(CO)3Br3] followed by HPLC separation. Radio-ligand 1 was shown to be stable (>99%) when incubated in human serum for 4 h at 37 °C with a relatively low lipophilicity (logD = 2.15 ± 0.02). The rhenium-185 and -187 complex 2 exhibited a moderate affinity (Ki = 159.3 ± 8.7 nM) for TSPO, whereas its cytotoxicity evaluated on TSPO-rich tumor cell lines was lower than that observed for the precursor. In vitro uptake studies of 1 in C6 and U87-MG cells for 60 min was found to be 9.84% ± 0.17% and 7.87% ± 0.23% ID, respectively. Our results indicated that (99m)Tc-CB256 can be considered as a potential new TSPO-rich cancer SPECT imaging agent and provides the foundation for further in vivo evaluation.

Characterization of monoclonal antibodies against porcine pulmonary alveolar macrophages of gnotobiotic miniature swine.

There is no sufficient porcine specific antibody available to investigate the ontogeny and development of porcine pulmonary alveolar macrophage (PAM). Therefore, several mouse anti-porcine macrophage mAbs have been produced and characterized in this study. First, the monoclonal antibody PM18-7, an IgG1, kappa isotype, bound to 91% of PAM, 6% of monocytes, and 2% of granulocytes indicating PM18-7 was found to be PAM specific. Monocyte derived macrophages could not be induced to express the PM18-7 antigen by culture. PM18-7 was immunoprecipitated with a molecule of 260 kDa under non-reducing conditions and with that of 130 kDa under reducing conditions in SDS-PAGE. Second, the monoclonal antibody PM3-15, an IgG1, kappa isotype, bound to 92% of PAM, 86% of monocytes, and 3% of granulocytes indicating PM3-15 was mononuclear phagocyte specific. PM3-15 was immunoprecipitated with a molecule of 245 kDa under non-reducing conditions and those of 150, 95 kDa under reducing conditions in SDS-PAGE. Third, the monoclonal antibody PM16-6, an IgM isotype, bound to 82% of PAM, 89% of monocytes, and 82% of granulocytes indicating PM16-6 recognizes those cells of myeloid lineage such as macrophages, monocytes and granulocyte. The antigen immunoprecipitated by PM16-6 was 120 kDa under non-reducing conditions and was 75, 25 kDa under reducing conditions. Finally, the antigen bound to PM18-7 was identified as ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1, CD203a), PM3-15 was figured out as integrin alpha M beta 2 precursor (ITGaM, CD11b) and PM16-6 was identified as Thimet oligopeptidase (THOP-1) by the LC-MS/MS protein sequencing method.

Autophagy sustains the survival of human pancreatic cancer PANC-1 cells under extreme nutrient deprivation conditions.

Pancreatic ductal adenocarcinomas are an extremely aggressive and devastating type of cancer with high mortality. Given the dense stroma and poor vascularization, accessibility to nutrients is limited in the tumor microenvironment. Here, we aimed to elucidate the role of autophagy in promoting the survival of human pancreatic cancer PANC-1 cells exposed to nutrient-deprived media (NDM) lacking glucose, amino acids, and serum. NDM inhibited Akt activity and phosphorylation of p70 S6K, and induced AMPK activation and mitochondrial depolarization. NDM also time-dependently increased LC3-II accumulation, number of GFP-LC3 puncta, and colocalization between GFP-LC3 and lysosomes. These results suggested that autophagy was progressively activated through Akt- and AMPK-mTOR pathway in nutrient-deficient PANC-1 cells. Autophagy inhibitors (chloroquine and wortmannin) or silencing of Atg5 augmented PANC-1 cell death in NDM. In cells exposed to NDM, chloroquine and wortmannin induced apoptosis and Z-VAD-fmk inhibited cytotoxicity of these inhibitors. These data demonstrate that autophagy is anti-apoptotic and sustains the survival of PANC-1 cells following extreme nutrient deprivation. Autophagy modulation may be a viable therapeutic option for cancer cells located in the core of solid tumors with a nutrient-deficient microenvironment.

NPY antagonism reduces adiposity and attenuates age-related imbalance of adipose tissue metabolism.

An orexigenic hormone, neuropeptide Y (NPY), plays a role not only in the hypothalamic regulation of appetite, but also in the peripheral regulation of lipid metabolism. However, the intracellular mechanisms triggered by NPY to regulate lipid metabolism are poorly understood. Here we report that NPY deficiency reduces white adipose tissue (WAT) mass and ameliorates the age-related imbalance of adipose tissue metabolism in mice. Gene expression involved in adipogenesis/lipogenesis was found to decrease, whereas proteins involved in lipolysis increased in gonadal WAT (gWAT) of NPY-knockout mice. These changes were associated with an activated SIRT1- and PPARγ-mediated pathway. Moreover, the age-related decrease of de novo lipogenesis in gWAT and thermogenesis in inguinal WAT was inhibited by NPY deficiency. Further analysis using 3T3-L1 cells showed that NPY inhibited lipolysis through the Y1 receptor and enhanced lipogenesis following a reduction in cAMP response element-binding protein (CREB) and SIRT1 protein expression. Therefore, NPY appears to act as a key regulator of adipose tissue metabolism via the CREB-SIRT1 signaling pathway. Taken together, NPY deficiency reduces adiposity and ameliorates the age-related imbalance of adipose tissue metabolism, suggesting that antagonism of NPY may be a promising target for drug development to prevent age-related metabolic diseases.

[(18)F]Fluoromethyl-PBR28 as a potential radiotracer for TSPO: preclinical comparison with [(11)C]PBR28 in a rat model of neuroinflammation.

To develop radiotracer for the translocator protein 18 kDa (TSPO) in vivo, N-(2-[(18)F]fluoromethoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide ([(18)F]1, [(18)F]fluoromethyl-PBR28) was prepared by incorporating of fluorine-18 into triazolium triflate-PBR28 precursor (7). The radiochemical yield of [(18)F]1 after HPLC purification was 35.8 ± 3.2% (n = 11, decay corrected). Radiotracer [(18)F]1 was found to be chemically stable when incubated in human serum for 4 h at 37 °C. Both aryloxyanilide analogs (1 and 2) behaved similarly in terms of lipophilicity and in vitro affinity for TSPO. Here, both radiotracers were directly compared in the same inflammatory rat to determine whether either radiotracer provides more promising in vivo TSPO binding. Uptake of [(18)F]1 in the inflammatory lesion was comparable to that of [(11)C]PBR28, and [(18)F]1 rapidly approached the highest target-to-background ratio at early imaging time (35 min postinjection versus 85 min postinjection for [(11)C]PBR28). These results suggest that [(18)F]1 is a promising radiotracer for imaging acute neuroinflammation in rat. In addition, our use of a triazolium triflate precursor for [(18)F]fluoromethyl ether group provides the convenient application for radiofluorination of radiotracer containing a methoxy group.