Description: The utility of 18 F-FDG PET/CT in patients with nasal-type natural killer (NK)/T-cell lymphoma has not been established. Therefore, we evaluated the role of 18 F-FDG PET/CT for determining cancer staging by comparing its results to those of conventional staging methods (CSMs) (physical examination, CT with intravenous contrast, biopsies from primary sites, and bone marrow examinations) in patients with nasal-type NK/T-cell lymphoma. Methods: In this study, 52 consecutive patients (34 men, 18 women; mean age, 49.4 y) with newly diagnosed nasal-type NK/T-cell lymphoma were studied. Anatomic regions ( n = 1,300; 16 nodal and 9 extranodal regions per patient) were assessed with an 18 F-FDG PET/CT scan and with CSMs, and each anatomic region was classified as positive or negative for malignancy. Biopsy and clinical follow-up, including additional imaging studies, were used as the gold standard for diagnosis. Results: Of the 59 nodal and 71 extranodal anatomic regions that were truly positive for malignancy, 18 F-FDG PET/CT detected 58 nodal and 69 extranodal. CSMs, however, detected only 44 of the nodal and 61 of the extranodal anatomic regions that were positive for malignancy (nodal comparison of PET/CT vs. CSMs, P 〈 0.001; extranodal comparison of PET/CT vs. CSMs, P = 0.008). PET/CT scans exhibited a significantly better sensitivity (97.7% vs. 80.7%, P 〈 0.001) than CSMs for the detection of malignant lesions. PET/CT findings altered the original staging category for 12 patients (21.2%) and affected treatment planning in 23 cases (44.2%). Conclusion: Our study demonstrated that 18 F-FDG PET/CT scanning is a valuable modality for staging and treatment planning in patients with nasal-type NK/T-cell lymphoma.

Description: The usefulness of 18 F-FDG PET in gastric cancer recurrence is limited by low sensitivity. Given that detectability by PET is dependent on the tumor’s metabolic characteristics, we tested whether the performance of PET for gastric cancer recurrence is enhanced in patients with 18 F-FDG–avid primary tumors. Methods: Three hundred sixty-eight patients with advanced gastric cancer underwent 18 F-FDG PET/CT for initial staging and for recurrence surveillance after curative surgery. On initial PET/CT, primary tumors were 18 F-FDG–avid if they displayed focal uptake with an SUV max 4 or more. Follow-up 18 F-FDG PET/CT was evaluated for recurrent disease. Results: On initial PET/CT, the primary tumor was 18 F-FDG–avid in 236 of 368 (64.1%) and nonavid in 132 patients (35.9%). During follow-up for 18.9 ± 13.3 mo, 72 patients (19.6%) had recurrence. Of the 63 PET scans with recurrence, 42 (66.7%) and 21 (33.3%) were scans of patients with 18 F-FDG–avid and nonavid primary tumors, respectively. PET sensitivity was higher in scans of patients with 18 F-FDG–avid than nonavid tumors for all recurrences (81.0% vs. 52.4%; P = 0.018) and nonanastomosis site recurrences (82.1% vs. 47.4%; P = 0.006). The sensitivity for detecting peritoneal recurrence was also higher for the avid tumor group. PET specificity was similarly high (97.1% and 97.5%) for both groups. Adding cell type and Lauren classification to tumor 18 F-FDG avidity further enhanced PET sensitivity. Conclusion: Surveillance 18 F-FDG PET/CT after resection of gastric cancer has significantly higher sensitivity in patients with 18 F-FDG–avid primary tumors and may have greater value in this group.

Description: Somatic heterozygous mutations of the DNA methyltransferase gene DNMT3A occur frequently in acute myeloid leukemia and other hematological malignancies, with the majority (~60%) of mutations affecting a single amino acid, Arg882 (R882), in the catalytic domain. Although the mutations impair DNMT3A catalytic activity in vitro, their effects on DNA methylation in cells have not been explored. Here, we show that exogenously expressed mouse Dnmt3a proteins harboring the corresponding R878 mutations largely fail to mediate DNA methylation in murine embryonic stem (ES) cells but are capable of interacting with wild-type Dnmt3a and Dnmt3b. Coexpression of the Dnmt3a R878H (histidine) mutant protein results in inhibition of the ability of wild-type Dnmt3a and Dnmt3b to methylate DNA in murine ES cells. Furthermore, expression of Dnmt3a R878H in ES cells containing endogenous Dnmt3a or Dnmt3b induces hypomethylation. These results suggest that the DNMT3A R882 mutations, in addition to being hypomorphic, have dominant-negative effects.

Description: 11 C-LY2795050 is a novel -selective antagonist PET tracer. The in vitro binding affinities ( K i ) of LY2795050 at the -opioid (KOR) and μ-opioid (MOR) receptors are 0.72 and 25.8 nM, respectively. Thus, the in vitro KOR/MOR binding selectivity is about 36:1. Our goal in this study was to determine the in vivo selectivity of this new KOR antagonist tracer in the monkey. Methods: To estimate the ED 50 value (dose of a compound [or drug] that gives 50% occupancy of the target receptor) of LY2795050 at the MOR and KOR sites, 2 series of blocking experiments were performed in 3 rhesus monkeys using 11 C-LY2795050 and 11 C-carfentanil with coinjections of various doses of unlabeled LY2795050. Kinetic modeling was applied to calculate regional binding potential ( BP ND ), and 1- and 2-site binding curves were fitted to these data to measure 11 C-LY2795050 binding selectivity. Results: The LY2795050 ED 50 at MOR was 119 μg/kg based on a 1-site model for 11 C-carfentanil. The 1-site binding model was also deemed sufficient to describe the specific binding of 11 C-LY2795050 at KOR. The ED 50 at KOR estimated from the 1-site model was 15.6 μg/kg. Thus, the ED 50 ratio for MOR:KOR was 7.6. Conclusion: The in vivo selectivity of 11 C-LY2795050 for KOR over MOR is 7.6. 11 C-LY2795050 has 4.7-fold-lower selectivity at KOR over MOR in vivo as compared with in vitro. Nevertheless, on the basis of our finding in vivo, 88% of the PET-observed specific binding of 11 C-LY2795050 under baseline conditions will be due to binding of the tracer at the KOR site in a region with similar prevalence of KOR and MOR. 11 C-LY2795050 is sufficiently selective for KOR over MOR in vivo to be considered an appropriate probe for studying the KOR with PET.

Description: Regulation of lipogenesis by pathophysiological factors in the liver and skeletal muscle is well understood; however, regulation in the kidney is still unclear. To elucidate nutritional regulation of lipogenic factors in the kidney, we measured the renal expression of lipogenic transcriptional factors and enzymes during fasting and refeeding in chow-fed and high-fat-fed mice. We also examined the regulatory effect of the liver X receptor (LXR) on the expression of lipogenic factors. The renal gene expression of sterol regulatory element-binding protein (SREBP)-1c and fatty acid synthase (FAS) was reduced by fasting for 48 h and restored by refeeding, whereas the mRNA levels of forkhead box O (FOXO)1/3 were increased by fasting and restored by refeeding. Accordingly, protein levels of SREBP-1, FAS, and phosphorylated FOXO1/3 were reduced by fasting and restored by refeeding. The patterns of lipogenic factors expression in the kidney were similar to those in the liver and skeletal muscle. However, this phasic regulation of renal lipogenic gene expression was blunted in diet-induced obese mice. LXR agonist TO901317 increased the lipogenic gene expression and the protein levels of SREBP-1 precursor and FAS but not nuclear SREBP-1. Moreover, increases in insulin-induced gene mRNA and nuclear carbohydrate-responsive element binding protein (ChREBP) levels were observed in the TO901317-treated mice. These results suggest that the kidney shows flexible suppression and restoration of lipogenic factors following fasting and refeeding in lean mice, but this is blunted in obese mice. LXR is involved in the renal expression of lipogenic enzymes, and ChREBP may mediate the response.

Description: Neutrophil extracellular traps (NETs; webs of DNA coated in antimicrobial proteins) are released into the vasculature during sepsis where they contribute to host defense, but also cause tissue damage and organ dysfunction. Various components of NETs have also been implicated as activators of coagulation. Using multicolor confocal intravital microscopy in mouse models of sepsis, we observed profound platelet aggregation, thrombin activation, and fibrin clot formation within (and downstream of) NETs in vivo. NETs were critical for the development of sepsis-induced intravascular coagulation regardless of the inciting bacterial stimulus (gram-negative, gram-positive, or bacterial products). Removal of NETs via DNase infusion, or in peptidylarginine deiminase-4–deficient mice (which have impaired NET production), resulted in significantly lower quantities of intravascular thrombin activity, reduced platelet aggregation, and improved microvascular perfusion. NET-induced intravascular coagulation was dependent on a collaborative interaction between histone H4 in NETs, platelets, and the release of inorganic polyphosphate. Real-time perfusion imaging revealed markedly improved microvascular perfusion in response to the blockade of NET-induced coagulation, which correlated with reduced markers of systemic intravascular coagulation and end-organ damage in septic mice. Together, these data demonstrate, for the first time in an in vivo model of infection, a dynamic NET–platelet–thrombin axis that promotes intravascular coagulation and microvascular dysfunction in sepsis.

Description: Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) and mitochondria play a critical role in growth factor-induced switch from a quiescent to an angiogenic phenotype in endothelial cells (ECs). However, how highly diffusible ROS produced from different sources can coordinate to stimulate VEGF signaling and drive the angiogenic process remains unknown. Using the cytosol- and mitochondria-targeted redox-sensitive RoGFP biosensors with real-time imaging, here we show that VEGF stimulation in human ECs rapidly increases cytosolic RoGFP oxidation within 1 min, followed by mitochondrial RoGFP oxidation within 5 min, which continues at least for 60 min. Silencing of Nox4 or Nox2 or overexpression of mitochondria-targeted catalase significantly inhibits VEGF-induced tyrosine phosphorylation of VEGF receptor type 2 (VEGFR2-pY), EC migration and proliferation at the similar extent. Exogenous hydrogen peroxide (H 2 O 2 ) or overexpression of Nox4, which produces H 2 O 2 , increases mitochondrial ROS (mtROS), which is prevented by Nox2 siRNA, suggesting that Nox2 senses Nox4-derived H 2 O 2 to promote mtROS production. Mechanistically, H 2 O 2 increases S36 phosphorylation of p66Shc, a key mtROS regulator, which is inhibited by siNox2, but not by siNox4. Moreover, Nox2 or Nox4 knockdown or overexpression of S36 phosphorylation-defective mutant p66Shc(S36A) inhibits VEGF-induced mtROS, VEGFR2-pY, EC migration, and proliferation. In summary, Nox4-derived H 2 O 2 in part activates Nox2 to increase mtROS via pSer36-p66Shc, thereby enhancing VEGFR2 signaling and angiogenesis in ECs. This may represent a novel feed-forward mechanism of ROS-induced ROS release orchestrated by the Nox4/Nox2/pSer36-p66Shc/mtROS axis, which drives sustained activation of angiogenesis signaling program.

Description: Alveolar epithelial cell (AEC) apoptosis and inadequate repair resulting from "exaggerated" lung aging and mitochondrial dysfunction are critical determinants promoting lung fibrosis. α-Klotho, which is an antiaging molecule that is expressed predominantly in the kidney and secreted in the blood, can protect lung epithelial cells against hyperoxia-induced apoptosis. We reasoned that Klotho protects AEC exposed to oxidative stress in part by maintaining mitochondrial DNA (mtDNA) integrity and mitigating apoptosis. We find that Klotho levels are decreased in both serum and alveolar type II (AT2) cells from asbestos-exposed mice. We show that oxidative stress reduces AEC Klotho mRNA and protein expression, whereas Klotho overexpression is protective while Klotho silencing augments AEC mtDNA damage. Compared with wild-type, Klotho heterozygous hypomorphic allele ( kl/+ ) mice have increased asbestos-induced lung fibrosis due in part to increased AT2 cell mtDNA damage. Notably, we demonstrate that serum Klotho levels are reduced in wild-type but not mitochondrial catalase overexpressing ( MCAT ) mice 3 wk following exposure to asbestos and that EUK-134, a MnSOD/catalase mimetic, mitigates oxidant-induced reductions in AEC Klotho expression. Using pharmacologic and genetic silencing studies, we show that Klotho attenuates oxidant-induced AEC mtDNA damage and apoptosis via mechanisms dependent on AKT activation arising from upstream fibroblast growth factor receptor 1 activation. Our findings suggest that Klotho preserves AEC mtDNA integrity in the setting of oxidative stress necessary for preventing apoptosis and asbestos-induced lung fibrosis. We reason that strategies aimed at augmenting AEC Klotho levels may be an innovative approach for mitigating age-related lung diseases.

Description: Kappa-opioid receptors (KOR) are believed to be involved in the pathophysiology of depression, anxiety disorders, drug abuse, and alcoholism. To date, only 1 tracer, the KOR agonist 11 C-GR103545, has been reported to be able to image KOR in primates. The goal of the present study was to synthesize the selective KOR antagonist 11 C-LY2795050 and evaluate its potential as a PET tracer to image KOR in vivo. Methods: The in vitro binding affinity of LY2795050 was measured in radioligand competition binding assays. Ex vivo experiments were conducted using microdosing of the unlabeled ligand in Sprague–Dawley rats and in wild-type and KOR knockout mice, to assess the ligand’s potential as a tracer candidate. Imaging experiments with 11 C-LY2795050 in monkeys were performed on the Focus-220 scanner with arterial blood input function measurement. Binding parameters were determined with kinetic modeling analysis. Results: LY2795050 displays full antagonist activity and high binding affinity and selectivity for KOR. Microdosing studies in rodents and ex vivo analysis of tissue concentrations with liquid chromatography–tandem mass spectrometry identified LY2795050 as an appropriate tracer candidate able to provide specific binding signals in vivo. 11 C-LY2795050 was prepared in an average yield of 12% and greater than 99% radiochemical purity. In rhesus monkeys, 11 C-LY2795050 displayed a moderate rate of peripheral metabolism, with approximately 40% of parent compound remaining at 30 min after injection. In the brain, 11 C-LY2795050 displayed fast uptake kinetics (regional activity peak times of 〈20 min) and an uptake pattern consistent with the distribution of KOR in primates. Pretreatment with naloxone (1 mg/kg, intravenously) resulted in a uniform distribution of radioactivity. Further, specific binding of 11 C-LY2795050 was reduced by the selective KOR antagonist LY2456302 in a dose-dependent manner. Conclusion: 11 C-LY2795050 displayed favorable pharmacokinetic properties and binding profiles in vivo and therefore is a suitable ligand for imaging the KOR in primates. This newly developed KOR antagonist tracer has since been advanced to PET imaging of KOR in humans and constitutes the first successful KOR antagonist radiotracer.

Description: In contrast to the conventional belief that systemic arteries dilate under hypoxia, we found that α-adrenergic contraction of rat deep femoral artery (DFA) is largely augmented by hypoxia (HVC DFA ) while hypoxia (3% P o 2 ) alone had no effect. HVC DFA was consistently observed in both endothelium-intact and -denuded vessels with partial pretone by phenylephrine (PhE) or by other conditions (e.g., K + channel blocker). Patch-clamp study showed no change in the membrane conductance of DFA myocytes by hypoxia. The RhoA-kinase inhibitor Y27632 attenuated HVC DFA . The nitric oxide synthase inhibitor [nitro- l -arginine methyl ester ( l -NAME)] and soluble guanylate cyclase inhibitor [oxadiazole quinoxalin (ODQ)] strongly augmented the PhE-pretone, while neither of the agents had effect without pretone. NADPH oxidase type 4 (NOX4) inhibitors (diphenylene iodonium and plumbagin) also potentiated PhE-pretone, which was reversed by NO donor. No additive HVC DFA was observed under the pretreatment with l -NAME, ODQ, or plumbagin. Western blot and immunohistochemistry analysis showed that both NOX4 and endothelial nitric oxide synthase (eNOS) are expressed in smooth muscle layer of DFA. Various mitochondria inhibitors (rotenone, myxothiazol, and cyanide) prevented HVC DFA . From the pharmacological data, as a mechanism for HVC DFA , we suggest hypoxic inhibition of eNOS in myocytes. The putative role of NOX4 and mitochondria requires further investigation. The HVC DFA may prevent imbalance between cardiac output and skeletal blood flow under emergent hypoxia combined with increased sympathetic tone.