Sphingosine 1-Phosphate Receptor Modulator ONO-4641 Regulates Trafficking of T Lymphocytes and Hematopoietic Stem Cells and Alleviates Immune-Mediated Aplastic Anemia in a Mouse Model.

ONO-4641 is a second-generation sphingosine 1-phosphate (S1P) receptor modulator that exhibits selectivity for S1P receptors 1 and 5. Treatment with ONO-4641 leads to a reduction in magnetic resonance imaging disease measures in patients with relapsing-remitting multiple sclerosis. The objective of this study was to explore the potential impact of ONO-4641 treatment based on its immunomodulatory effects. Severe aplastic anemia is a bone marrow (BM) failure disease typically caused by aberrant immune destruction of blood progenitors. Although the T helper type 1-mediated pathology is well described for aplastic anemia, the molecular mechanisms driving disease progression remain undefined. We evaluated the efficacy of ONO-4641 in a mouse model of aplastic anemia. ONO-4641 reduced the severity of BM failure in a dose-dependent manner, resulting in higher blood and BM cell counts. By evaluating the mode of action, we found that ONO-4641 inhibited the infiltration of donor-derived T lymphocytes to the BM. ONO-4641 also induced the accumulation of hematopoietic stem cells in the BM of model mice. These observations indicate, for the first time, that S1P receptor modulators demonstrate efficacy in the mouse model of aplastic anemia and suggest that treatment with ONO-4641 might delay the progression of aplastic anemia. SIGNIFICANCE STATEMENT: ONO-4641 is a second-generation sphingosine 1-phosphate (S1P) receptor modulator selective for S1P receptors 1 and 5. In this study, we demonstrated that ONO-4641 regulates the trafficking of T lymphocytes along with hematopoietic stem and progenitor cells, leading to alleviation of pancytopenia and destruction of bone marrow in a bone marrow failure-induced mouse model mimicking human aplastic anemia.

Preventive and Therapeutic Efficacy of ONO-4641, a Selective Agonist for Sphingosine 1-Phosphate Receptor 1 and 5, in Preclinical Models of Type 1 Diabetes Mellitus.

ONO-4641, 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid (ceralifimod), is a second-generation sphingosine 1-phosphate receptor agonist selective for sphingosine 1-phosphate receptors 1 and 5, and has clinical effects in multiple sclerosis. The objective of the present study was to explore other potential indications for ONO-4641 based on its immunomodulatory effects. ONO-4641 was tested in non-obese diabetic (NOD) mice, an animal model of spontaneous type 1 diabetes mellitus, an autoimmune disease with unmet medical needs. ONO-4641 at a dose of 0.1 mg/kg prevented the onset of diabetes mellitus in NOD mice. Furthermore, ONO-4641 at doses of 0.03 and 0.1 mg/kg decreased diabetic prevalence in NOD mice after the onset of diabetes mellitus in a dose-dependent manner. Histopathological analysis demonstrated that insulin-positive areas in the islets of mice administered 0.03 and 0.1 mg/kg ONO-4641 showed a tendency of high values although they were not significantly different from the Control group, which was treated with vehicle. These observations suggest ONO-4641 may delay the onset and progression of type 1 diabetes mellitus.

A sphingosine 1-phosphate receptor agonist ameliorates animal model of vasculitis.

OBJECTIVES: Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to cell surface receptors (S1P1-5). In this study, we examined the effect of S1P1 agonist, ONO-W061, on murine Candida albicans water-soluble fraction (CAWS)-induced vasculitis. METHODS: Mice were administered ONO-W061, and the number of peripheral blood cells was counted. Vasculitis was induced by an intraperitoneal injection of CAWS. Expression of S1P receptors and CXCL1 was analyzed by quantitative RT-PCR. ONO-W061 was orally administered, and vasculitis was evaluated histologically. Number of neutrophils, macrophages and T cells in the vasculitis tissue was counted using flow cytometry. Production of chemokines from S1P-stimulated human umbilical vein endothelial cells (HUVECs) was measured by ELISA. RESULTS: Number of peripheral blood lymphocytes was decreased by ONO-W061. Expression of CXCL1 and S1P1 was enhanced in CAWS-induced vasculitis tissue. Vasculitis score, CXCL1 and number of neutrophils in the vasculitis tissue were lower in ONO-W061-treated mice. Treatment of HUVECs with S1P upregulated the production of CXCL1 and IL-8 in vitro, and this was inhibited by ONO-W061. CONCLUSIONS: ONO-W061 significantly improved CAWS-induced vasculitis. This effect may be partly exerted through the inhibited production of chemokines by endothelial cells, which in turn could induce neutrophil recruitment into inflamed vessels.

Electron States of Uniaxially Strained Graphene.

We report an experimental study of electron states and the resulting electronic transport properties of uniaxially strained graphene. For this study we developed a novel strain application method that is compatible with the planar device technology. We identify the value of the strain induced in graphene by Raman spectroscopy and show with atomic force microscopy that its topography consists of wrinkles up to 4 nm height aligned along the direction of the applied strain. Transport experiments reveal a broadening of the charge neutrality region and the convergence of Landau levels to multiple Dirac points in Landau-fan diagrams. These observations are consistent with large fluctuations of the scalar potential via the strain-induced wrinkles, which is experimentally observed for the first time.

Straining graphene using thin film shrinkage methods.

Theoretical works suggest the possibility and usefulness of strain engineering of graphene by predicting remarkable properties, such as Dirac cone merging, bandgap opening and pseudo magnetic field generation. However, most of these predictions have not yet been confirmed because it is experimentally difficult to control the magnitude and type (e.g., uniaxial, biaxial, and so forth) of strain in graphene devices. Here we report two novel methods to apply strain without bending the substrate. We employ thin films of evaporated metal and organic insulator deposited on graphene, which shrink after electron beam irradiation or heat application. These methods make it possible to apply both biaxial strain and in-plane isotropic compressive strain in a well-controlled manner. Raman spectroscopy measurements show a clear splitting of the degenerate states of the G-band in the case of biaxial strain, and G-band blue shift without splitting in the case of in-plane isotropic compressive strain. In the case of biaxial strain application, we find out the ratio of the strain component perpendicular to the stretching direction is at least three times larger than what was previously observed, indicating that shrinkage of the metal or organic insulator deposited on graphene induces both tensile and compressive strain in this atomically thin material. Our studies present for the first time a viable way to apply strain to graphene without the need to bend the substrate.

Structure-activity relationship studies of S1P agonists with a dihydronaphthalene scaffold.

Structure-activity relationship (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaphthalene scaffold was investigated. Compound 1 was modified to improve S1P(1) agonistic activity and in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P(3) agonistic activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound 6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position on the terminal benzene ring showed potent hS1P(1) agonistic activity with excellent selectivity over hS1P(3) and in vivo PLL activity in mice.

Discovery of S1P agonists with a dihydronaphthalene scaffold.

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P(1) agonistic activity as well as selectivity over S1P(3) agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P(1) receptor agonist with high selectivity against S1P(3) and enhanced efficacy in lowering peripheral lymphocyte counts in mice.

Discovery of a 1-Methyl-3,4-dihydronaphthalene-Based Sphingosine-1-Phosphate (S1P) Receptor Agonist Ceralifimod (ONO-4641). A S1P1 and S1P5 Selective Agonist for the Treatment of Autoimmune Diseases.

The discovery of 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid 13n (ceralifimod, ONO-4641), a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P1 and S1P5, is described. While it has been revealed that the modulation of the S1P1 receptor is an effective way to treat autoimmune diseases such as relapsing-remitting multiple sclerosis (RRMS), it was also reported that activation of the S1P3 receptor is implicated in some undesirable effects. We carried out a structure-activity relationship (SAR) study of hit compound 6 with an amino acid moiety in the hydrophilic head region. Following identification of a lead compound with a dihydronaphthalene central core by inducing conformational constraint, optimization of the lipophilic tail region led to the discovery of 13n as a clinical candidate that exhibited >30 000-fold selectivity for S1P1 over S1P3 and was potent in a peripheral lymphocyte lowering (PLL) test in mice (ED50 = 0.029 mg/kg, 24 h after oral dosing).

New gastric epithelial cell lines from mice transgenic for temperature-sensitive simian virus 40 large T antigen show distinct types of cell differentiation.

AIM: To develop conditionally immortalized gastric mucosal cell lines that show distinct types of cell differentiation from transgenic mice harboring temperature-sensitive simian virus 40 (tsSV40) large T antigen. METHODS: Gastric mucosal cells from the transgenic mice were cultured at a permissive temperature (33 degrees C), and proliferative cells were then cloned by colony formation. RESULTS: Eight gastric cell lines showed epithelial-like morphology and grew at 33 degrees C. Three different types of the cell lines have been established: (1) MGE12-1, MGE3-2, and MGE509 cells expressing mRNAs for pit cell markers (gastric mucin and cathepsin E); (2) MGE02, MGE503, and MGE511 cells expressing mRNAs for pit and zymogenic (pepsinogen F) cell markers, and (3) MGE507 and MGE727 cells expressing mRNAs for pit, zymogenic, and parietal (H,K-ATPase alpha-subunit) cell markers. Moreover, the TaqMan assay showed that mRNA levels of mucin, H,K-ATPase alpha-subunit, and pepsinogen F were influenced by nonpermissive temperature (39 degrees C) in MGE503 and MGE727 cells. CONCLUSION: These gastric epithelial cell lines seem to reflect different stages of development of gastric mucosal cells.

Development and characterization of conditionally immortalized gastric epithelial cell lines from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene.

Conditionally immortalized gastric epithelial cell lines were established from transgenic rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene. Gastric mucosal cells and epithelial tissues isolated from the stomach of the transgenic rats were cultured at permissive temperature (33 degrees C), and proliferative cells were cloned by colony formation. Six cell lines (designated as RGE1-01, RGE1-02, RGE1-03, RGE1-21, RGE1-22 and RGE2-01) showing epithelial-like morphology have been established. All cells grew at 33 degrees C, but did not at nonpermissive temperature (39 degrees C). High expression level of large T-antigen in the nuclei was observed at 33 degrees C, whereas the expression level was gradually decreased in a time-dependent manner at 39 degrees C. These results suggest that the temperature-sensitive growth characteristics arise as a result of a function of the tsSV40 large T-antigen. None of the cell lines were transformed as judged by anchorage-independent growth assay. Immunocytochemical findings indicated that all cells expressed epithelial cell markers including cytoskeletal (cytokeratin and actin), basement membrane (laminin and collagen type IV) and junctional complex (ZO-1 and desmoplakin I+II) proteins at 33 degrees C. All cells expressed mRNA of cathepsin E, a pit cell marker. Moreover, transepithelial resistance was observed between apical and basolateral sides in the cells. RGE1-22 cells produced prostaglandin E(2). Levels of mRNA for cathepsin E, transepithelial resistance and prostaglandin E(2) were influenced by the nonpermissive temperature. Thus, these conditionally immortalized gastric cell lines which preserve some epithelial cell characteristics will provide a useful in vitro model of gastric epithelium.