Arcus, and he has a patent filed by his institution related to the use of tumor mutation burden to predict response to immunotherapy (PCT/US2015/062208), which has received licensing fees from PGDx. R. Korn serves as CMIO of ImaginAb. A. Mascioni is employed by ImaginAb. W. Le serves as VP of Operations at ImaginAb. I. Wilson serves as CEO of ImaginAb. M. Gordon receives consulting fees from ImaginAb and Imaging Endpoints. A. Wu receives consulting fees from ImaginAb. G. Ulaner receives consulting fees from ImaginAb and is a member of the Scientific Advisory Board for ImaginAb. J. Wolchock has equity in Tizona Pharmaceuticals, Adaptive Biotechnologies, Imvaq, Beigene, Linneaus, Apricity, Arsenal IO, and Georgiamune. M. Postow receives grant/research support from ImaginAb. N. Pandit-Taskar has served as a consultant for or been on an advisory board and has received honoraria for ImaginAb, and receives grant/research support from ImaginAb. No other potential conflict of interest relevant to this article was reported.

Self-assembling heparin nanoparticles have attracted much attention as promising drug carriers for various drugs, genes and imaging agents. In the present investigation, we found that heparin nanoparticles are selective Toll-like receptor 4 (TLR-4) antagonists and have a much greater anti-inflammatory effect than native heparin. More specifically, we developed self-assembling nanoparticles composed of glycol-split heparin/D-erythro-sphingosine conjugates (NAHNP), characterized their physicochemical properties and anti-inflammatory effect in vitro. Unlike native heparin, NAHNP significantly inhibited lipopolysaccharide-induced activation of MyD88-dependent NF-κB signaling pathway and production of pro-inflammatory cytokines such as TNF-alpha from mouse macrophages with IC50 = 0.019 mg/mL. Furthermore, we investigated the structure-activity relationship of the conjugates and identified the length of attached alkyl chains of d-erythro-sphingosine to be critical for anti-inflammatory effect. Decrease in alkyl chain length of NAHNP resulted in loss of inhibitory activity. In line with these findings, 6-O-sulfate groups of D-glucosamine residue were essential for effective inhibition, while removal of 2-O-sulfo and 3-O-sulfo groups as well as replacement of N-sulfo groups with N-acetyl did not alter anti-inflammatory activity. Therefore, NAHNP would be a promising candidate in acute and chronic inflammatory disorders, in addition to the nature of a drug carrier.

Background Perioperative infections, particularly surgical site infections pose significant morbidity and mortality. Phagocytosis is a critical step for microbial eradication. We examined the effect of commonly used anesthetics on macrophage phagocytosis and its mechanism. Methods The effect of anesthetics (isoflurane, sevoflurane, propofol) on macrophage phagocytosis was tested using RAW264.7 mouse cells, mouse peritoneal macrophages, and THP-1 human cells. Either opsonized sheep erythrocytes or fluorescent labeled Escherichia coli were used as phagocytic objects. The activation of Rap1, a critical protein in phagocytosis was assessed using the active Rap1 pull-down and detection kit. To examine anesthetic binding site(s) on Rap1, photolabeling experiments were performed using azi-isoflurane and azi-sevoflurane. The alanine scanning mutagenesis of Rap1 was performed to assess the role of anesthetic binding site in Rap1 activation and phagocytosis. Results Macrophage phagocytosis was significantly attenuated by the exposure of isoflurane (50% reduction by 1% isoflurane) and sevoflurane (50% reduction by 1.5% sevoflurane), but not by propofol. Photolabeling experiments showed that sevoflurane directly bound to Rap1. Mutagenesis analysis demonstrated that the sevoflurane binding site affected Rap1 activation and macrophage phagocytosis. Conclusions We showed that isoflurane and sevoflurane attenuated macrophage phagocytosis, but propofol did not. Our study showed for the first time that sevoflurane served as a novel small GTPase Rap1 inhibitor. The finding will further enrich our understanding of yet-to-be determined mechanism of volatile anesthetics and their off-target effects. The sevoflurane binding site was located outside the known Rap1 functional sites, indicating the discovery of a new functional site on Rap1 and this site would serve as a pocket for the development of novel Rap1 inhibitors.

Dezocine could potentially be used to alleviate opioid dependence. Due to the unique molecular target profile different from buprenorphine, it might have important value in studying the mechanisms of morphine dependence and developing novel therapeutic approaches.

Volatile anesthetics have been in clinical use for a long period of time and are considered to be promiscuous by presumably interacting with several ion channels in the central nervous system to produce anesthesia. Because ion channels and their existing evolutionary analogues, ion transporters, are very important in various organisms, it is possible that volatile anesthetics may affect some bacteria. In this study, we hypothesized that volatile anesthetics could affect bacterial behaviors. We evaluated the impact of anesthetics on bacterial growth, motility (swimming and gliding) and biofilm formation of four common bacterial pathogens in vitro. We found that commonly used volatile anesthetics isoflurane and sevoflurane affected bacterial motility and biofilm formation without any effect on growth of the common bacterial pathogens studied here. Using available Escherichia coli gene deletion mutants of ion transporters and in silico molecular docking, we suggested that these altered behaviors might be at least partly via the interaction of volatile anesthetics with ion transporters.

Sepsis remains a significant health care burden, with high morbidities and mortalities. Patients with sepsis often require general anesthesia for procedures and imaging studies. Knowing that anesthetic drugs can pose immunomodulatory effects, it would be critical to understand the impact of anesthetics on sepsis pathophysiology. The volatile anesthetic sevoflurane is a common general anesthetic derived from ether as a prototype. Using a murine sepsis model induced by cecal ligation and puncture surgery, we examined the impact of sevoflurane on sepsis outcome. Different from volatile anesthetic isoflurane, sevoflurane exposure significantly improved the outcome of septic mice. This was associated with less apoptosis in the spleen. Because splenic apoptosis was largely attributed to the apoptosis of neutrophils, we examined the effect of sevoflurane on FasL‐induced neutrophil apoptosis. Sevoflurane exposure significantly attenuated apoptosis. Sevoflurane did not affect the binding of FasL to the extracellular domain of Fas receptor. Instead, in silico analysis suggested that sevoflurane would bind to the interphase between Fas death domain (DD) and Fas‐associated DD (FADD). The effect of sevoflurane on Fas DD‐FADD interaction was examined using fluorescence resonance energy transfer (FRET). Sevoflurane attenuated FRET efficiency, indicating that sevoflurane hindered the interaction between Fas DD and FADD. The predicted sevoflurane binding site is known to play a significant role in Fas DD‐FADD interaction, supporting our in vitro and in vivo apoptosis results.—Koutsogiannaki, S., Hou, L., Babazada, H., Okuno, T., Blazon‐Brown, N., Soriano, S. G., Yokomizo, T., Yuki, K. The volatile anesthetic sevoflurane reduces neutrophil apoptosis via Fas death domain‐Fas‐associated death domain interaction. FASEB J. 33, 12668–12679 (2019). http://www.fasebj.org

It has been recently shown that Toll-like receptor4 mediated nuclear factor κB (TLR4-NF-κB) signaling plays a critical role in the pathogenesis of rheumatoid arthritis mediated by pro-inflammatory cytokines in arthritic synovium. Here we evaluate the therapeutic potential of glycol-split non-anticoagulant heparin/d-erythro-sphingosine nanoparticles (NAHNPs), which have shown strong inhibitory effect against TLR4 induced inflammation, in an experimental arthritis model. NAHNP significantly inhibited the production of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β in lipopolysaccharide (LPS)-induced primary mouse macrophages and DC2.4 dendritic cell line. The nanoparticles were administered to type II collagen-induced arthritis (CIA) mice by intraarticular injections once per day starting from onset of the disease symptoms. Treatment with NAHNP had a potent suppressive effect in CIA mice, observed with a decrease in arthritis score and footpad swelling. The animals treated with NAHNP significantly reduced levels of IgG1 and IgG2a antibodies against bovine type II collagen. Levels of proinflammatory cytokines--e.g., TNF-α, IL-6 and IL-1β in knee joints and sera were significantly inhibited compared to control mice. Moreover, nuclear localization of RelA in knee joints was significantly inhibited in NAHNP treatment, indicating down-regulation of the NF-κB signaling pathway. In addition, histological examination revealed significant suppression of inflammatory cell infiltration, joint destruction and synovial proliferation in synovium compared with control mice. These results suggest that selective inhibition of TLR4-NF-κB signaling with lipid modified heparin derivatives composited to nanostructures provides an effective therapeutic approach to inhibit chronic inflammation in an animal model of rheumatoid arthritis.

Immune checkpoint inhibitors (ICIs) have been effective in treating a subset of refractory solid tumors, but only a small percentage of treated patients benefit from these therapies. Thus, there is a clinical need for reliable tools that allow for the early assessment of response to ICIs, as well as a preclinical need for imaging tools that aid in the future development and understanding of immunotherapies. Here we demonstrate that CD69, a canonical early-activation marker expressed on a variety of activated immune cells, including cytotoxic T cells and Natural Killer (NK) cells, is a promising biomarker for the early assessment of response to immunotherapies. We have developed a positron emission tomography (PET) probe by radiolabeling a highly specific CD69 monoclonal antibody, H1.2F3, with Zirconium-89, [89Zr]-DFO-H1.2F3. [89Zr]-DFO-H1.2F3 detected changes in CD69 expression on primary mouse T cells in vitro and detected activated immune cells in a syngeneic tumor immunotherapy model. In vitro uptake studies with [89Zr]-DFO-H1.2F3 showed a 15-fold increase in CD69 expression for activated primary mouse T cells, relative to untreated resting T cells. In vivo PET imaging showed that tumors of ICI-responsive mice had greater uptake than the tumors of nonresponsive and untreated mice. Ex vivo biodistribution, autoradiography, and immunohistochemistry analyses supported the PET imaging findings. These data suggest that the CD69 PET imaging approach detects CD69 expression with sufficient sensitivity to quantify immune cell activation in a syngeneic mouse immunotherapy model and could allow for the prediction of therapeutic immune responses to novel immunotherapies.