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: Although the role of IL-7 and IL-7R has been implicated in the pathogenesis of rheumatoid arthritis (RA), the majority of the studies have focused on the effect of IL-7/IL-7R in T cell development and function. Our novel data, however, document that patients with RA and greater disease activity have higher levels of IL-7, IL-7R, and TNF-α in RA monocytes, suggesting a feedback regulation between IL-7/IL-7R and TNF-α cascades in myeloid cells that is linked to chronic disease progression. Investigations into the involved mechanism showed that IL-7 is a novel and potent chemoattractant that attracts IL-7R + monocytes through activation of the PI3K/AKT1 and ERK pathways at similar concentrations of IL-7 detected in RA synovial fluid. To determine whether ligation of IL-7 to IL-7R is a potential target for RA treatment and to identify their mechanism of action, collagen-induced arthritis (CIA) was therapeutically treated with anti–IL-7 Ab or IgG control. Anti–IL-7 Ab treatment significantly reduces CIA monocyte recruitment and osteoclast differentiation as well as potent joint monocyte chemoattractants and bone erosion markers, suggesting that both direct and indirect pathways might contribute to the observed effect. We also demonstrate that reduction in joint MIP-2 levels is responsible for suppressed vascularization detected in mice treated with anti–IL-7 Ab compared with the control group. To our knowledge, we show for the first time that expression of IL-7/IL-7R in myeloid cells is strongly correlated with RA disease activity and that ligation of IL-7 to IL-7R contributes to monocyte homing, differentiation of osteoclasts, and vascularization in the CIA effector phase.

Description: LPSs are widely used to stimulate TLR4, but their effects on ion channels in immune cells are poorly known. In THP-1 cells and human blood monocytes treated with LPS, inwardly rectifying K + channel current (I Kir,LPS ) newly emerged at 1 h, peaked at 4 h (–119 ± 8.6 pA/pF), and decayed afterward (–32 ± 6.7 pA/pF at 24 h). Whereas both the Kir2.1 and Kir2.2 mRNAs and proteins were observed, single-channel conductance (38 pS) of I Kir,LPS and small interfering RNA–induced knockdown commonly indicated Kir2.2 than Kir2.1. LPS-induced cytokine release and store-operated Ca 2+ entry were commonly decreased by ML-133, a Kir2 inhibitor. Immunoblot, confocal microscopy, and the effects of vesicular trafficking inhibitors commonly suggested plasma membrane translocation of Kir2.2 by LPS. Both I Kir,LPS and membrane translocation of Kir2.2 were inhibited by GF109203X (protein kinase C [PKC] inhibitor) or by transfection with small interfering RNA–specific PKC. Interestingly, pharmacological activation of PKC by PMA induced both Kir2.1 and Kir2.2 currents. The spontaneously decayed I Kir,LPS at 24 h was recovered by PI3K inhibitors but further suppressed by an inhibitor of phosphatidylinositol(3,4,5)-trisphosphate (PIP 3 ) phosphatase (phosphatase and tensin homolog). However, I Kir,LPS at 24 h was not affected by Akt inhibitors, suggesting that the decreased phosphatidylinositol(4,5)-bisphosphate availability, that is, conversion into PIP 3 by PI3K, per se accounts for the decay of I Kir,LPS . Taken together, to our knowledge these data are the first demonstrations that I Kir is newly induced by TLR4 stimulation via PKC-dependent membrane trafficking of Kir2.2, and that conversion of phosphatidylinositol(4,5)-bisphosphate to PIP 3 modulates Kir2.2. The augmentation of Ca 2+ influx and cytokine release suggests a physiological role for Kir2.2 in TLR4-stimulated monocytes.

Description: Our aim was to examine the impact of TLR5 ligation in rheumatoid arthritis (RA) and experimental arthritis pathology. Studies were conducted to investigate the role of TLR5 ligation on RA and mouse myeloid cell chemotaxis or osteoclast formation, and in addition, to uncover the significance of TNF-α function in TLR5-mediated pathogenesis. Next, the in vivo mechanism of action was determined in collagen-induced arthritis (CIA) and local joint TLR5 ligation models. Last, to evaluate the importance of TLR5 function in RA, we used anti-TLR5 Ab therapy in CIA mice. We show that TLR5 agonist, flagellin, can promote monocyte infiltration and osteoclast maturation directly through myeloid TLR5 ligation and indirectly via TNF-α production from RA and mouse cells. These two identified TLR5 functions are potentiated by TNF-α, because inhibition of both pathways can more strongly impair RA synovial fluid–driven monocyte migration and osteoclast differentiation compared with each factor alone. In preclinical studies, flagellin postonset treatment in CIA and local TLR5 ligation in vivo provoke homing and osteoclastic development of myeloid cells, which are associated with the TNF-α cascade. Conversely, CIA joint inflammation and bone erosion are alleviated when TLR5 function is blocked. We found that TLR5 and TNF-α pathways are interconnected, because TNF-α is produced by TLR5 ligation in RA myeloid cells, and anti–TNF-α therapy can markedly suppress TLR5 expression in RA monocytes. Our novel findings demonstrate that a direct and an indirect mechanism are involved in TLR5-driven RA inflammation and bone destruction.

Description: Although ingenol 3,20-dibenzoate (IDB) is known as a selective novel protein kinase C (PKC) agonist, its biologic actions and underlying mechanisms remain incompletely understood. In this study, we identified IDB as a proliferative agent for an erythropoietin (EPO)-dependent cell line, UT-7/EPO, through the screening of a natural compound library. To clarify the underlying mechanism of IDB’s EPO-like activities, we thoroughly analyzed the mutual relation between EPO and IDB in terms of in vitro and in vivo activities, signaling molecules, and a cellular receptor. IDB substantially induced the proliferation of UT-7/EPO cells, but not as much as EPO. IDB also lessened the anemia induced by 5-fluorouracil in an in vivo mouse model. Interestingly, IDB showed a synergistic effect on EPO at low concentration, but an antagonistic effect at higher concentration. Physical interaction and activation of PKCs by IDB- and EPO-competitive binding of IDB to EPO receptor (EPOR) explain these synergistic and antagonistic activities, respectively. Importantly, we addressed IDB’s mechanism of action by demonstrating the direct binding of IDB to PKCs, and by identifying EPOR as a novel molecular target of IDB. Based on these dual targeting properties, IDB holds promise as a new small molecule modulator of EPO-related pathologic conditions.

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: 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: Connexin 43 (Cx43) deficiency was found to increase mortality in a mouse model of bacterial peritonitis, and Cx43 is upregulated in macrophages by LPS treatment. In this study, we characterized a novel signaling pathway for LPS-induced Cx43 expression in RAW264.7 cells and thioglycolate-elicited peritoneal macrophages (TGEMs). LPS alone or LPS-containing conditioned medium (CM) upregulated Cx43. Overexpression or silencing of Cx43 led to the enhancement or inhibition, respectively, of CM-induced TGEM migration. This response involved the inducible NO synthase (iNOS)/focal adhesion kinase (FAK)/Src pathways. Moreover, CM-induced migration was compromised in TGEMs from Cx43 +/– mice compared with TGEMs from Cx43 +/+ littermates. Cx43 was upregulated by a serum/glucocorticoid-regulated kinase 1 (SGK) activator and downregulated, along with inhibition of CM-induced TGEM migration, by knockdown of the SGK gene or blockade of the SGK pathway. LPS-induced SGK activation was abrogated by Torin2, whereas LPS-induced Cx43 was downregulated by both Torin2 and rapamycin. Analysis of the effects of FK506 and methylprednisolone, common immunosuppressive agents following organ transplantation, suggested a link between these immunosuppressive drugs and impaired macrophage migration via the Cx43/iNOS/Src/FAK pathway. In a model of Escherichia coli infectious peritonitis, GSK650349-, an SGK inhibitor, or Torin2-treated mice showed less accumulation of F4/80 + CD11b + macrophages in the peritoneal cavity, with a delay in the elimination of bacteria. Furthermore, following pretreatment with Gap19, a selective Cx43 hemichannel blocker, the survival of model mice was significantly reduced. Taken together, our study suggested that Cx43 in macrophages was associated with macrophage migration, an important immune process in host defense to infection.

Description: Cerivastatin (CER) was withdrawn from the world market because of lethal rhabdomyolysis. Coadministrations of CER and cyclosporine A (CsA) or gemfibrozil (GEM) have been reported to increase the CER blood concentration. CsA is an inhibitor of organic anion transporting polypeptide (OATP)1B1 and CYP3A4, and GEM and its glucuronide (GEM-glu) inhibit OATP1B1 and CYP2C8. The purpose of this study was to describe the transporter-/enzyme-mediated drug-drug interactions (DDIs) of CER with CsA or GEM based on unified physiologically based pharmacokinetic (PBPK) models and to investigate whether the DDIs can be quantitatively analyzed by a bottom-up approach. Initially, the PBPK models for CER and GEM/GEM-glu were constructed based on the previously reported standard protocols. Next, the drug-dependent parameters were optimized by Cluster Newton Method. Thus, described concentration-time profiles for CER and GEM/GEM-glu agreed well with the clinically observed data. The DDIs were then simulated using the established PBPK models with previously obtained in vitro inhibition constants of CsA or GEM/GEM-glu against the OATP1B1 and cytochrome P450s. DDIs with the inhibitors were underestimated compared with observed data using the geometric means of reported values. To search for better described parameters within the range of in vitro values, sensitivity analyses were performed for DDIs of CER. Using the in vitro parameter sets selected by sensitivity analyses, these DDIs were well reproduced, indicating that the present PBPK models were able to describe adequately the clinical DDIs based on a bottom-up approach. The approaches in this study would be applicable to the prediction of other DDIs involving both transporters and metabolic enzymes.

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.