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: Inflammatory bowel disease (IBD) is a chronic inflammatory disease with increasing incidence and prevalence worldwide. Here we investigated the newly synthesized jasmonate analogue 2-hydroxyethyl 5-chloro-4,5-didehydrojasmonate (J11-Cl) for its anti-inflammatory effects on intestinal inflammation. First, to test whether J11-Cl can activate peroxisome proliferator-activated receptors (PPARs), we performed docking simulations because J11-Cl has a structural similarity with anti-inflammatory 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the endogenous ligands of PPARγ. J11-Cl bound to the ligand binding domain of PPARγ in the same manner as 15d-PGJ2 and rosiglitazone, and significantly increased transcriptional activity of PPARγ. In animal experiments, colitis was significantly reduced in mice with J11-Cl treatment, determined by analyses of survival rate, body weight changes, clinical symptoms, and histological evaluation. Moreover, J11-Cl decreased production of pro-inflammatory cytokines including IL-6, IL-8, and G-CSF as well as chemokines including chemokine (C-C motif) ligand (CCL)20, chemokine (C-X-C motif) ligand (CXCL)2, CXCL3, and chemokine (C-X3-C motif) ligand 1 (CX3CL1) in colon tissues, and LPS or TNF-α-stimulated macrophages and epithelial cells. In contrast, production of anti-inflammatory cytokines including IL-2 and IL-4 as well as the proliferative factor, GM-CSF, was increased by J11-Cl. Furthermore, inhibition of MAPKs and NF-κB activation by J11-Cl was also observed. J11-Cl reduced intestinal inflammation by increasing the transcriptional activity of PPARγ and modulating inflammatory signaling pathways. Therefore, our study suggests that J11-Cl may serve as a novel therapeutic agent against IBD.

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: Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori.

Description: Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.

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.