Blockade of EP4 by ASP7657 Modulates Myeloid Cell Differentiation In Vivo and Enhances the Antitumor Effect of Radiotherapy.

The tumor microenvironment (TME) is thought to influence the antitumor efficacy of immuno-oncology agents through various products of both tumor and stromal cells. One immune-suppressive factor is prostaglandin E2 (PGE2), a lipid mediator whose biosynthesis is regulated by ubiquitously expressed cyclooxygenase- (COX-) 1 and inducible COX-2. By activating its receptors, PGE2 induces immune suppression to modulate differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) rather than dendritic cells (DCs). Pharmacological blockade of prostaglandin E receptor 4 (EP4) causes a decrease in MDSCs, reprogramming of macrophage polarization, and increase in tumor-infiltrated T cells, leading to enhancement of antitumor immunity in preclinical models. Here, we report the effects of the highly potent EP4 antagonist ASP7657 on the DC population in tumor and antitumor immune activation in an immunocompetent mouse tumor model. Oral administration of ASP7657 inhibited tumor growth, which was accompanied by an increase in intratumor DC and CD8+ T cell populations and a decrease in the M-MDSC population in a CT26 immunocompetent mouse model. The antitumor activity of ASP7657 was dependent on CD8+ T cells and enhanced when combined with an antiprogrammed cell death-1 (PD-1) antibody. Notably, ASP7657 also significantly enhanced the antitumor efficacy of radiotherapy in an anti-PD-1 antibody refractory model. These results indicate that the therapeutic potential of ASP7657 arises via upregulation of DCs and subsequent CD8+ T cell activation in addition to suppression of MDSCs in mouse models and that combining EP4 antagonists with radiotherapy or an anti-PD-1 antibody can improve antitumor efficacy.

Effects of riluzole on psychiatric disorders with anxiety or fear as primary symptoms: A systematic review.

AIM: Previous behavioral pharmacology studies involving rodents suggested riluzole had potential to be an ideal psychotropic drug for psychiatric disorders with anxiety or fear as primary symptoms. Several clinical studies have recently been conducted. The purpose of this study was to gather information about the efficacy and tolerability of riluzole for patients with those symptoms. METHODS: We searched PubMed, PsycINFO, CINAHL, EMBASE, and the Cochrane database from inception until April 2021, and performed manual searches for additional relevant articles. This review included: (1) studies involving participants that were patients with generalized anxiety disorder (GAD), social anxiety disorder, panic disorder, obsessive-compulsive disorder (OCD), posttraumatic stress disorder (PTSD), acute stress disorder, or phobias; and (2) randomized controlled trials (RCTs) or intervention studies (e.g., single arm trials) examining the effects and safety of riluzole. RESULTS: Of the 795 identified articles, four RCTs, one RCT subgroup-analysis, and three open-label trials without control groups met the inclusion criteria. Most trials evaluated the efficacy of riluzole as an augmentation therapy with selective serotonin reuptake inhibitors and other antidepressants for PTSD, OCD, or GAD. However, there was insufficient evidence to confirm the effects of riluzole for patients with these psychiatric disorders. Most trials demonstrated adequate study quality. CONCLUSIONS: This review found insufficient evidence to confirm the effects of riluzole for psychiatric disorders with anxiety or fear as primary symptoms. It would be worthwhile to conduct studies that incorporate novel perspectives, such as examining the efficacy of riluzole as a concomitant medication for psychotherapy.

Effects of AS2819899, a novel selective PI3Kδ inhibitor, in a NZB/W F1 mouse lupus-like nephritis model.

Phosphoinositide 3-kinases generate lipid-based second messengers that control an array of intracellular signaling pathways. In particular, phosphoinositide 3-kinases delta (PI3Kδ) is expressed primarily in hematopoietic cells and plays an important role in B-cell development and function. B cells play a critical role in autoimmune diseases by producing autoantibodies. Studies have therefore increasingly focused on PI3Kδ as a therapeutic target for the treatment of inflammatory and autoimmune diseases. One such autoimmune disease is systemic lupus erythematosus (SLE). SLE is a chronic systemic autoimmune disease with repeated recurrence and remission, and autoantibodies play an important role in its pathogenesis. Here, we examined the pharmacological profile of the novel PI3Kδ selective inhibitor AS2819899 and investigated its therapeutic potential against SLE in a NZB/W F1 mouse lupus-like nephritis model, a widely-used SLE mouse model. AS2819899 prevented B and T cell activation in vitro, and inhibited antibody production in a T-cell independent de novo antibody production mouse model. In the spontaneous NZB/W F1 mouse model, AS2819899 treatment significantly reduced anti-dsDNA antibody titers and improved kidney dysfunction. Further, AS2819899 inhibited the memory recall reaction in a T-cell dependent antibody production mouse model, suggesting that AS2819899 can potentially maintain remission of SLE. Moreover, we identified a pharmacodynamics marker for AS2819899 that may be useful in clinical studies. These results indicate that AS2819899 may be an attractive therapeutic candidate for SLE, including the maintenance of remission.

Examination of beauty ingredient distribution in the human skin by time-of-flight secondary ion mass spectrometry.

In this study, the authors evaluated the distribution of low-abundance beauty ingredients in human skin tissues. The distribution of collagen tripeptide, a beauty ingredient, in the human skin was evaluated by applying multivariate curve resolution (MCR) to the time-of-flight secondary ion mass spectrometry mapping data, including reference information. The intensity of the secondary ion peaks was increased by the accumulation of secondary ion intensity in the depth direction obtained by argon cluster sputtering. Consequently, the collagen tripeptide distribution in the skin was evaluated by separating it from collagen peptide, although the conventional analysis was difficult because of the dilution of the collagen tripeptide in the skin. Additionally, the distribution of the collagen tripeptide in the skin may be determined with an autoencoder. In this study, the distribution of small amounts of beauty ingredients (the collagen tripeptide) in the skin was observed by integrating the secondary ion intensity in the depth direction with Ar cluster sputtering and applying MCR or autoencoder.

Investigating matrix effects of different combinations of lipids and peptides on TOF-SIMS data.

Matrix effects, which cause a change in ion intensity, occur in mass spectrometry methods including time-of-flight secondary ion mass spectrometry (TOF-SIMS). Matrix effects often cause large issues in quantitative analysis because secondary ions related to a particular molecule could be dramatically enhanced or suppressed regardless of the concentration. To investigate matrix effects in biological samples, the authors evaluated mixed lipid {POPC [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine, molecular weight (MW) 759.6]}, peptide [leu-enkephalin, neo-leu-enkephalin (amino acid sequence: YAGFL, MW 569.3), and neo-angiotensin II (amino acid sequence: DRVYIHAF, MW 1019.5)] samples. Matrix effect features were investigated by analyzing the concentration dependence of secondary ions in lipid-peptide mixed samples to develop a method that enables quantitative analysis using TOF-SIMS. Matrix effects depended on the lipid-peptide combination. Interestingly, some secondary ions possessed an intensity that was highly dependent on concentration.

OrbiSIMS Imaging Identifies Molecular Constituents of the Perialgal Vacuole Membrane of Paramecium bursaria with Symbiotic Chlorella variabilis.

The protist (mostly single-celled organisms), Paramecium bursaria, forms an intracellular symbiotic relationship with the single-celled algae, Chlorella variabilis, where P. bursaria provides nutrients (i.e., Ca2+, Mg2+, and K+), carbon dioxide for photosynthesis and protection from viruses, while C. variabilis provides oxygen, carbon fixation, and nutrients. Key to this successful relationship is the perialgal vacuole (PV) membrane, which surrounds C. variabilis and protects it from digestion by P. bursaria. The membrane is fragile and difficult to analyze using conventional methods therefore very little is known about the molecular composition. We used the OrbiSIMS, a new high-resolution mass spectrometer with subcellular resolution imaging, to study the compartmentalization of endosymbionts and elucidate biomolecular interactions between the host and endosymbiont. Ions from the region of interest, close to C. variabilis, and specific to the target samples containing PVs were found based on the chemical mapping and masses of the ions. We show chemical localizations of oligosaccharides in close proximity of C. variabilis endosymbionts in P. bursaria. These oligosaccharides are detected in host-endosymbiont samples containing PV membrane-bound algae and absent in free-living algae and digestive vacuole (DV) membrane-bound algae in P. bursaria.

Effects of AS2541019, a novel selective PI3Kδ inhibitor, on antibody production and hamster to rat xenotransplantation.

B cell-mediated antibodies play a critical role in protecting the body from infections; however, excessive antibody production is involved in the pathogenesis of autoimmune diseases and transplanted organ rejection. Regulation of antibody production is therefore crucial for overcoming these complications. Phosphatidylinositol-3-kinase p110δ (PI3Kδ), a member of the family of PI3K lipid kinases, is a key mediator of B cell activation and proliferation, with a small molecule PI3Kδ inhibitor having been approved for the treatment of B cell lymphoma. However, the effect of PI3Kδ inhibitors on B cell-mediated antibody production has not been clearly elucidated. In this study, we investigated the effect of the selective PI3Kδ inhibitor, AS2541019, on B cell immunity and antibody production. Our results show that AS2541019 effectively prevented B cell activation and proliferation in vitro, and that oral administration of AS2541019 resulted in significant inhibition of both T-dependent and T-independent de novo antibody production in peripheral blood. Further, in a hamster to rat concordant xenotransplant model, AS2541019 significantly prolonged graft survival time by inhibiting xenoreactive antibody production. Therefore, our study demonstrates that the selective PI3Kδ inhibitor AS2541019 inhibits antibody production through potent inhibitory effects on B cell activation, and can protect against organ dysfunction.

Evaluation of matrix effects on TOF-SIMS data of leu-enkephalin and 1,2-dioleoyl-sn-glycero-3-phosphocholine mixed samples.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is one of the most powerful methods to analyze biomolecules in biological tissues and cells because it provides detailed chemical structure information and chemical images with a high spatial resolution. However, in terms of quantitative analysis, there are issues such as matrix effects that often cause secondary ion intensity changes regardless of the actual concentration in a sample. For instance, the intensity of secondary ions related to peptides is generally suppressed when lipids coexist. Since the evaluation of biomolecules is crucial to understand biological phenomena, it is required to analyze peptides or lipids without matrix effects. Therefore, the mechanism of matrix effects regarding peptides and lipids in TOF-SIMS was investigated in this study. Leu-enkephalin (YGGFL, molecular weight of 555.3 Da) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, C44H84NO8P, molecular weight 785.6 Da) were employed to prepare model samples. Model samples contain different weight ratios of these two molecules. The intensity of secondary ions related to the peptide or the lipid was compared with control samples containing pure leu-enkephalin or DOPC. As a result, it is indicated that the intensity of DOPC related secondary ions is strongly enhanced by coexisting leu-enkephalin, while the intensity of leu-enkephalin related secondary ions is suppressed by coexisting DOPC especially in a low concentration range of the peptide.

Peptide fragmentation caused by Ar cluster ions depending on primary ion energy.

RATIONALE: Time-of-flight secondary ion mass spectrometry (TOF-SIMS) with an Ar cluster ion beam as a primary ion source provides useful information in terms of peptide analysis. It is, however, difficult to interpret the spectra. The ToF-SIMS peptide spectra obtained with Ar clusters having different energies have been investigated in order to classify the secondary ions into the peptide fragment ions and those related to contaminants or the substrate. METHODS: Three peptides having different molecular weights from 600 to 1300 u were measured with Ar cluster beams having different energies per atom from 4 to 40 eV/atom. RESULTS: In the spectra normalized to a geometric average of all the spectra, the amino acid fragment ions are distinguished from other secondary ions. In the mass range above 600 u, the peptide fragment ions increase with mass while those not related to the peptide decrease with mass. CONCLUSIONS: Energy-dependence fragmentation helps in understanding the peptide spectra. Specific peptide fragment ions of the larger peptides are likely to be detected under lower energy than energy higher than 10 eV/atom. Although it is difficult to interpret the TOF-SIMS spectra of a peptide obtained with an Ar cluster ion beam, the secondary ions can be classified by comparing those obtained with different energy Ar cluster ion beams.

Effect of novel PKCθ selective inhibitor AS2521780 on acute rejection in rat and non-human primate models of transplantation.

Selective inhibition of protein kinase Cθ (PKCθ) may be useful in inducing T cell-specific immunosuppression with a reduced rate of side effects. To our knowledge, however, no reports have been published regarding the selective inhibition of PKCθ by small-molecule compounds in animal models of allograft rejection. Here, we investigated the effect of the newly synthesized PKCθ selective inhibitor AS2521780 in mono- and combination therapies on acute rejection in ACI-to-Lewis rat cardiac and non-human primate (NHP) renal transplantation models. In the rat cardiac transplantation model, AS2521780 significantly prolonged graft survival to 14days at 10mg/kg twice daily (b.i.d.) and to 20days at 30mg/kg b.i.d. In contrast, acute rejection occurred in all recipients in the non-treated group by Days 5 or 6 post-transplantation. Significant improvements (P<0.001) in graft survival were observed following treatment with a combination of AS2521780 at 3mg/kg b.i.d. and a suboptimal dose of tacrolimus (0.02mg/kg) or mycophenolate mofetil (15mg/kg). In the NHP renal transplantation model, AS2521780 at 3mg/kg b.i.d. and tacrolimus at 1mg/kg (suboptimal dose) significantly improved graft survival compared to tacrolimus alone (P<0.05). The present study of AS2521780 in rat cardiac and NHP renal transplantation models demonstrates the potential of PKCθ as a novel drug target for organ transplantation. As AS2521780 was well tolerated and the dose of tacrolimus or mycophenolate mofetil can be reduced when used in combination with this drug, immunosuppressive regimens containing selective inhibitors of PKCθ might have good safety profiles.

ToF-SIMS observation for evaluating the interaction between amyloid ß and lipid membranes.

The adsorption behaviour of amyloid beta (Aß), thought to be a key peptide for understanding Alzheimer's disease, was investigated by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Aß aggregates depending on the lipid membrane condition though it has not been fully understood yet. In this study, Aß samples on different lipid membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were observed with ToF-SIMS and the complex ToF-SIMS data of the Aß samples was interpreted using data analysis techniques such as principal component analysis (PCA), gentle-SIMS (G-SIMS) and g-ogram. DOPC and DMPC are liquid crystal at room temperature, while DPPC is gel at room temperature. As primary ion beams, Bi3(+) and Ar cluster ion beams were used and the effect of an Ar cluster ion for evaluating biomolecules was also studied. The secondary ion images of the peptide fragment ions indicated by G-SIMS and g-ogram were consistent with the PCA results. It is suggested that Aß is adsorbed homogeneously on the liquid-crystalline-phase lipid membranes, while it aggregates along the lipid on the gel-phase lipid membrane. Moreover, in the results using the Ar cluster, the influence of contamination was reduced.

Effect of AS2521780, a novel PKCθ selective inhibitor, on T cell-mediated immunity.

T cell-mediated immunity is central to the pathogenesis of autoimmune diseases, and is a target in the development of alternative therapeutic strategies with reduced adverse effects on other cell types and organs. Protein kinase C (PKC) is a family of serine/threonine kinases, with knockout of the PKCθ isoform in mice resulting in defective T cell activation. However, the effects of selective inhibition of PKCθ by small-molecule compounds on T cell signaling are still unknown. Here, we evaluated the effect of the novel PKCθ inhibitor AS2521780 on T cell activation and joint inflammation in a rat model of arthritis. AS2521780 exerted potent inhibition of recombinant human PKCθ enzyme activity (IC50=0.48 nM), which was more than 30-fold higher than that of other PKC isoforms. Further, AS2521780 exerted little or no inhibition on other protein kinases. AS2521780 suppressed CD3/CD28-induced Interleukin-2 (IL-2) gene transcription in Jurkat T cells and proliferation of human primary T cells. AS2521780 also suppressed concanavalin A-induced cytokine production by rat splenocytes and monkey peripheral blood mononuclear cells with similar potency. Moreover, AS2521780 significantly reduced paw swelling in a dose-dependent manner in a rat model of adjuvant-induced arthritis. These results indicate that PKCθ is an attractive drug target and AS2521780 is a potential immunosuppressant for T cell-mediated autoimmune diseases.

Peptide structural analysis using continuous Ar cluster and C60 ion beams.

A novel application of time-of-flight secondary ion mass spectrometry (ToF-SIMS) with continuous Ar cluster beams to peptide analysis was investigated. In order to evaluate peptide structures, it is necessary to detect fragment ions related to multiple neighbouring amino acid residues. It is, however, difficult to detect these using conventional ToF-SIMS primary ion beams such as Bi cluster beams. Recently, C60 and Ar cluster ion beams have been introduced to ToF-SIMS as primary ion beams and are expected to generate larger secondary ions than conventional ones. In this study, two sets of model peptides have been studied: (des-Tyr)-Leu-enkephalin and (des-Tyr)-Met-enkephalin (molecular weights are approximately 400 Da), and [Asn(1) Val(5)]-angiotensin II and [Val(5)]-angiotensin I (molecular weights are approximately 1,000 Da) in order to evaluate the usefulness of the large cluster ion beams for peptide structural analysis. As a result, by using the Ar cluster beams, peptide molecular ions and large fragment ions, which are not easily detected using conventional ToF-SIMS primary ion beams such as Bi3 (+), are clearly detected. Since the large fragment ions indicating amino acid sequences of the peptides are detected by the large cluster beams, it is suggested that the Ar cluster and C60 ion beams are useful for peptide structural analysis.

A novel target-specific gene delivery system combining baculovirus and sequence-specific long interspersed nuclear elements.

Transposable elements are valuable for somatic and germ-line transformation. However, long interspersed nuclear elements (LINEs) have not been used because of poor information on the transposition mechanism. We have developed a novel gene delivery system combining baculovirus AcNPV and two silkworm LINEs, SART1 and R1, which integrate into specific sequences of telomeric repeats and 28S ribosomal DNA, respectively. When two LINEs containing the enhanced green fluorescent protein gene recombined into AcNPV were infected into fifth instar larvae of the silkworm, we observed target-specific retrotransposition of LINEs at 72h post-infection, using polymerase chain reaction amplification and sequencing. Telomere- and 28S rDNA-specific transposition occurred in all nine tissues tested, including the ovary and testis. This is the first demonstration of site-specific gene delivery in living larvae. Insertion efficiencies were dependent on the virus titer for injection and the host strains of Bombyx mori. Using this system, we successfully detected the intergeneration transmission of retrotransposed sequences. In addition, AcNPV-mediated SART1 also transposed into telomere of another lepidopteran, Orgyia recens, suggesting that this system is useful for a wide variety of AcNPV-infectious insects. Site-specific gene delivery by virus-mediated LINE will be a potential gene therapy tool to avoid harmful unexpected insertions.

Growth arrest-specific gene 6 and Axl signaling enhances gastric cancer cell survival via Akt pathway.

Activation of tyrosine kinases is an important factor during cancer development. Axl, one of the receptor tyrosine kinases, binds to the specific ligand growth arrest-specific gene 6 (Gas6), which encodes a vitamin K-dependent gamma-carboxyglutamyl protein. Although many receptor tyrosine kinases and their ligands are involved in gastric carcinogenesis, whether Gas6-Axl signaling is involved in gastric carcinogenesis has not been elucidated. The aim of this study was to investigate the expression of Gas6 and Axl in gastric cancer and also their roles during gastric carcinogenesis. mRNA and protein of Gas6 and Axl were highly expressed in a substantial proportion of human gastric cancer tissue and cell lines, and Gas6 expression was significantly associated with lymph node metastasis. With recombinant Gas6 and a decoy-receptor of Axl in vitro, we demonstrated that Gas6-Axl signaling pathway enhanced cellular survival and invasion and suppressed apoptosis via Akt pathway. Our results suggests that Gas6-Axl signaling plays a role during gastric carcinogenesis, and that targeting Gas6-Axl signaling could be a novel therapeutic for gastric cancer.

In vivo gene transfer into the honeybee using a nucleopolyhedrovirus vector.

The honeybee Apis mellifera L. is a social insect and one of the most industrially important insects. We examined whether a baculovirus-mediated retrotransposon is applicable to in vivo transfer of exogenous genes to the honeybees. Honeybee larvae and pupae were injected with two types of recombinant Autographa californica nucleopolyhedrovirus (AcNPV) vectors, one that includes the enhanced green fluorescent protein gene (egfp) as a reporter to be inserted into the honeybee genome, and another that includes the reverse transcriptase gene responsible for the insertion. Fluorescence was observed in most of the viral-injected larvae and pupae. Reverse transcription-polymerase chain reaction and immunoblotting confirmed egfp mRNA and eGFP expression in these honeybees, although egfp insertion into the honeybee genome was not confirmed. These results indicate that AcNPV vectors can be used for the transfer and transient expression of an exogenous gene in the larval and pupal honeybees.

STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells.

Signal transduction and activator of transcription 3(STAT3) signaling is constitutively activated in various tumors, and is involved in cell survival and proliferation during oncogenesis. There are few reports, however, on the role of STAT3 signaling in gastric cancer. The aim of the present study was to clarify the role of STAT3 signaling in apoptosis and cellular proliferation in gastric cancer. Here we reported that STAT3 was constitutively activated in various human gastric cancer cells and its inhibition by ectopic dominant-negative STAT3 or Janus kinase inhibitor, tyrphostin AG490, induced apoptosis. Furthermore, STAT3 inhibition markedly decreased survivin expression, and forced expression of survivin rescued AGS cells from apoptosis induced by STAT3 inhibition. Although some reports demonstrated that the PI3K/Akt pathway regulates survivin expression, inhibition of the PI3K/Akt pathway did not affect survivin expression in AGS and MKN1 cells. Finally, activated form of STAT3, Tyr-705 phospho-stat3, was found in the nucleus of cancer cells in 11 of 40 (27.5%) human gastric cancer specimens. These findings suggest that constitutively activated STAT3 signaling supports gastric cancer cell survival in association with survivin expression.2004