The CRK14 gene encoding a cysteine-rich receptor-like kinase is implicated in the regulation of global proliferative arrest in Arabidopsis thaliana.

Global proliferative arrest (GPA) is a phenomenon in monocarpic plants in which the activity of all aboveground meristems generally ceases in a nearly coordinated manner after the formation of a certain number of fruits. Despite the fact that GPA is a biologically and agriculturally important event, the underlying molecular mechanisms are not well understood. In this study, we attempted to elucidate the molecular mechanism of GPA regulation by identifying the gene responsible for the Arabidopsis mutant fireworks (fiw), causing an early GPA phenotype. Map-based cloning revealed that the fiw gene encodes CYSTEIN-RICH RECEPTOR-LIKE KINASE 14 (CRK14). Genetic analysis suggested that fiw is a missense, gain-of-function allele of CRK14. Since overexpression of the extracellular domain of CRK14 resulted in delayed GPA in the wild-type background, we concluded that CRK14 is involved in GPA regulation. Analysis of double mutants revealed that fiw acts downstream of or independently of the FRUITFULL-APETALA2 (AP2)/AP2-like pathway, which was previously reported as an age-dependent default pathway in GPA regulation. In addition, fiw is epistatic to clv with respect to GPA control. Furthermore, we found a negative effect on WUSCHEL expression in the fiw mutants. These results thus suggest the existence of a novel CRK14-dependent signaling pathway involved in GPA regulation.

Lit-LAMP-DNA-vaccine for shrimp allergy prevents anaphylactic symptoms in a murine model.

Allergen-specific immunotherapy (AIT) is a promising therapeutic approach to food allergy but requires optimization in terms of both efficacy and safety due to the risk of undesired anaphylactic reactions. Here, we investigated the potential of a single DNA plasmid vaccine (Lit-LAMP-DNA-vaccine) encoding multivalent shrimp antigens (Lit v (Litopenaeus vannamei; Whiteleg shrimp) 1, Lit v4, and Lit v3) and a lysosomal-associated membrane protein (LAMP) as the next generation of AIT for patients with allergy. We first confirmed the expression of the LAMP-1-Lit v1-Lit v4-Lit v3 fusion protein in human cells transfected with the Lit-LAMP-DNA-vaccine, and the induction of anti-Lit v1, Lit v3, and Lit v4 IgG2a antibody production as well as Th1 response in Lit-LAMP-DNA-vaccine-treated mice. Next, we established an anaphylaxis model in mice epicutaneously sensitized with a crude shrimp protein extract (SPE) and investigated both the efficacy of Lit-LAMP-DNA-vaccine, and the difference in the mechanism of action (MOA) from oral immunotherapy (OIT). In the mouse shrimp allergy model, Lit-LAMP-DNA-vaccine potently suppressed anaphylactic reactions and mast cell activation with robust antigen-specific IgG2a production. The IgG1:IgG2a ratio was significantly lower than that of OIT. This suppressive effect was also confirmed by plasma transfer from mice previously vaccinated with the Lit-LAMP-DNA-vaccine. These results suggest that this Lit-LAMP-DNA-vaccine may represent a promising therapeutic strategy for human shrimp allergy which acts via the efficient induction of antigen-specific IgG with antagonism.

PGE2-EP2/EP4 signaling elicits immunosuppression by driving the mregDC-Treg axis in inflammatory tumor microenvironment.

Active inflammation generally promotes immune activation. However, in the tumor microenvironment (TME), active inflammation occurs in parallel with immunosuppression, and both contribute to tumor growth. Why inflammation does not lead to immune activation in TME remains unclear. In this study, using the immune checkpoint inhibitor-insensitive mouse cancer model and single-cell RNA sequencing, we show that PGE2-EP2/EP4 signaling simultaneously promotes active inflammation by inducing expression of the NF-κB genes in myeloid cells and elicits immunosuppression by driving the mregDC (mature DC enriched in immunoregulatory molecules)-Treg (regulatory T cell) axis for Treg recruitment and activation in the tumor. Importantly, the EP2/EP4 expression level is strongly correlated with the gene signatures of both active inflammation and the mregDC-Treg axis and has significant prognosis value in various human cancers. Thus, PGE2-EP2/EP4 signaling functions as the key regulatory node linking active inflammation and immunosuppression in TME, which can be targeted by EP2 and EP4 antagonists for cancer therapeutics.

Peficitinib improves bone fragility by recovering bone turnover imbalance in arthritic mice.

Peficitinib, a pan-JAK inhibitor, is known to suppress the activation of fibroblast-like synoviocytes (FLSs) and thereby reduces joint inflammation associated with rheumatoid arthritis (RA). However, the effect on osteoporosis in RA remains to be elucidated. In this study, the effect of peficitinib or etanercept on joint inflammation, and consequently decreased bone mineral density (BMD) was evaluated in mice with collagen-induced arthritis (CIA). Additionally, the effect on RANKL production from osteoblasts differentiated from the mesenchymal stem cells of RA patients was evaluated. Administration of peficitinib for established CIA ameliorated arthritis and improved BMD in the femoral metaphysis, but not in the femoral diaphysis. Conversely, etanercept suppressed an increase in synovial inflammatory markers but did not improve arthritic conditions or the reduction of BMD in either region. All elevated bone formation and bone resorption markers were decreased with peficitinib but only partially decreased with etanercept. Furthermore, production of RANKL by human osteoblasts was suppressed by peficitinib but enhanced by etanercept. Unlike etanercept, peficitinib is thought to increase BMD by ameliorating the high bone turnover associated with RA states, resulting in improvement of bone fragility. Our data provide evidence that peficitinib would be expected to show efficacy for osteoporosis associated with RA.

Characterization of ASP8374, a fully-human, antagonistic anti-TIGIT monoclonal antibody.

The T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) is a validated immune checkpoint protein expressed on memory CD4+T-cellls, Tregs, CD8+T-cell and natural killer (NK) cells. ASP8374 is a fully human monoclonal immunoglobulin (Ig) G4 antibody designed to block the interaction of TIGIT with its ligands and inhibit TIGIT signaling. ASP8374 exhibited high affinity binding to TIGIT and increased interferon (IFN)-γ production of cultured peripheral blood mononuclear cells (PBMCs) in a titratable manner. When used in combination with pembrolizumab, an anti-programmed death-1 (PD-1) antibody, ASP8374 induced higher T-cell activation in vitro than either treatment alone. An anti-mouse TIGIT antibody surrogate, mSEC1, displayed anti-tumor efficacy in an MC38 syngeneic mouse tumor model alone and in combination with an anti-programmed death-ligand 1 (PD-L1) antibody. In an additional syngeneic mouse tumor model (CT26), while mSEC1 alone did not demonstrate anti-tumor efficacy, mSEC1 combined with an anti-PD-1 antibody enhanced anti-tumor efficacy above that of the anti-PD-1 antibody alone. These data provide evidence that ASP8374 has therapeutic potential for advanced malignancies.

Role of JAK-STAT signaling in the pathogenic behavior of fibroblast-like synoviocytes in rheumatoid arthritis: Effect of the novel JAK inhibitor peficitinib.

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLS) play a crucial role in the pathogenesis of RA. RA-FLS display passive pro-inflammatory responses and self-directed aggressive responses, such as pro-inflammatory mediator production, reduced apoptosis and formation of a thickened synovial lining. Evidence suggests a role for Janus kinase (JAK)-signal transducer and transcriptional activator (STAT) signaling in the passive response but the aggressive behavior of RA-FLS is poorly understood. The pharmacologic effects of the novel JAK inhibitor, peficitinib, on cytokine-induced intracellular signaling and self-directed aggressive behavior of RA-FLS (e.g., increased expression of apoptosis-resistant genes and sodium nitroprusside-induced apoptosis) were investigated and compared with approved JAK inhibitors. RA-FLS assembly to form a lining-like structure and pro-inflammatory mediator production was investigated in three-dimensional (3D)-micromass culture. Peficitinib inhibited STAT3 phosphorylation in RA-FLS following induction by interferon (IFN)-α2b, IFN-γ, interleukin (IL)-6, oncostatin M, and leukemia inhibitory factor in a concentration-related manner, and was comparable to approved JAK inhibitors, tofacitinib and baricitinib. Peficitinib and tofacitinib suppressed autocrine phosphorylation of STAT3 and expression of apoptosis-resistant genes, and promoted cell death. In 3D-micromass culture, peficitinib reduced multi-layered RA-FLS cells to a thin monolayer, an effect less pronounced with tofacitinib. Both compounds attenuated production of vascular endothelial growth factor-A, matrix metalloproteinases, IL-6 and tumor necrosis factor superfamily-11. This study confirmed the pathogenic role of uncontrolled JAK-STAT signaling in the aggressive and passive responses of RA-FLS that are critical for RA progression. The novel JAK inhibitor peficitinib suppressed the pro-inflammatory behavior of RA-FLS, accelerated cell death and abrogated thickening of the synovium.

Integrin, alpha9 subunit blockade suppresses collagen-induced arthritis with minimal systemic immunomodulation.

Integrin, alpha9 subunit (hereinafter, alpha9) has been identified as a novel putative therapeutic target for rheumatoid arthritis (RA). Support for this target comes from the observations that alpha9 is overexpressed both in the joints of RA patients and in animal models of arthritis. In the experimental models, the increase in alpha9 expression precedes the onset of arthritic symptoms. The current study presents data on the pharmacological profile of an anti-alpha9 antibody in a collagen-induced arthritis (CIA) mouse model. Administration of an alpha9-blocking antibody in CIA mice suppressed the development of arthritis and significantly decreased plasma level of activated fibroblast-like synoviocyte (FLS)-derived biomarkers without reducing the formation of anti-type II collagen antibodies. While anti-alpha9 antibody administration significantly suppress the accumulation of immune cells in arthritic joints it had no effect on immune cell number in the spleen. Furthermore, in non-arthritic mice, alpha9 had no inhibitory effect in either a mixed lymphocyte reaction (MLR) or in a delayed type hypersensitivity (DTH) reaction. These results suggest that blocking alpha9 exerts its anti-arthritic effect through suppression of FLS-activation via a non-immune mediated mechanism. Finally, therapeutic administration of anti-alpha9 antibody alleviated established arthritis in CIA mice. Our data provide evidence that alpha9 blockade is a promising therapy for joint inflammation with minimal systemic immunomodulation.

Constitutive Activation of Integrin α9 Augments Self-Directed Hyperplastic and Proinflammatory Properties of Fibroblast-like Synoviocytes of Rheumatoid Arthritis.

Despite advances in the treatment of rheumatoid arthritis (RA), currently approved medications can have significant side effects due to their direct immunosuppressive activities. Additionally, current therapies do not address residual synovial inflammation. In this study, we evaluated the role of integrin α9 and its ligand, tenascin-C (Tn-C), on the proliferative and inflammatory response of fibroblast-like synoviocytes (FLSs) from RA patients grown in three-dimensional (3D)-micromass culture. FLSs from osteoarthritis patients, when grown in the 3D-culture system, formed self-directed lining-like structures, whereas FLSs from RA tissues (RA-FLSs) developed an abnormal structure of condensed cellular accumulation reflective of the pathogenic features of RA synovial tissues. Additionally, RA-FLSs grown in 3D culture showed autonomous production of proinflammatory mediators. Predominant expression of α9 and Tn-C was observed in the condensed lining, and knockdown of these molecules abrogated the abnormal lining-like structure formation and suppressed the spontaneous expression of matrix metalloproteinases, IL-6, TNFSF11/RANKL, and cadherin-11. Disruption of α9 also inhibited expression of Tn-C, suggesting existence of a positive feedback loop in which the engagement of α9 with Tn-C self-amplifies its own signaling and promotes progression of synovial hyperplasia. Depletion of α9 also suppressed the platelet-derived growth factor-induced hyperplastic response of RA-FLSs and blunted the TNF-α-induced expression of matrix metalloproteinases and IL-6. Finally, α9-blocking Ab also suppressed the formation of the condensed cellular lining by RA-FLSs in 3D cultures in a concentration-related manner. This study demonstrates the central role of α9 in pathogenic behaviors of RA-FLSs and highlights the potential of α9-blocking agents as a nonimmunosuppressive treatment for RA-associated synovitis.

Amelioration of collagen-induced arthritis in mice by a novel phosphodiesterase 7 and 4 dual inhibitor, YM-393059.

YM-393059 is a novel phosphodiesterase (PDE) 7 and PDE4 dual inhibitor that inhibits PDE7A with high potency (IC50=14 nM) and PDE4 with moderate potency (IC50=630 nM). It inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha production in mice with an ED50 value of 2.1 mg/kg [Yamamoto, S., Sugahara, S., Naito, R., Ichikawa, A., Ikeda, K., Yamada, T., Shimizu, Y., 2006. The effects of a novel phosphodiesterase 7A and -4 dual inhibitor, YM-393059, on T-cell-related cytokine production in vitro and in vivo. Eur. J. Pharmacol. 541, 106-114.]. In this study, we investigated the therapeutic potential of YM-393059 for the treatment of rheumatoid arthritis in several animal models. YM-393059 was found to inhibit LPS-induced interleukin (IL)-1beta production in mice with an ED50 value of 16.6 mg/kg, but it had only a slight effect on IL-6 production. YM-393059 and cyclosporine significantly suppressed arthritis development at doses of 30-100 mg/kg and 20 mg/kg, respectively, in the mice collagen-induced arthritis model. YM-393059 (100 mg/kg) significantly inhibited increases in the serum immunoglobulin G level that occurred in response to autoantigenic collagen in arthritic mice, whereas cyclosporine (20 mg/kg) did not. In contrast, cyclosporine completely suppressed the acute rejection of cardiac allografts in rats, whereas YM-393059 did not, even at a dose of 100 mg/kg. YM-393059 potently inhibited proinflammatory cytokine production and selectively suppressed the response to the autoantigen without affecting the response to alloantigens. These results suggest that YM-393059 is an attractive compound for the treatment of autoimmune disorders such as rheumatoid arthritis.

Pharmacological profile of a novel phosphodiesterase 7A and -4 dual inhibitor, YM-393059, on acute and chronic inflammation models.

YM-393059 is a novel phosphodiesterase (PDE) 7A and PDE4 dual inhibitor that inhibits both Th1 [interleukin (IL)-2 and interferon-gamma] and Th2 (IL-4) cytokines in vitro [Yamamoto, S., Sugahara, S., Naito, R., Ichikawa, A., Ikeda, K., Yamada, T., Shimizu, Y., 2006. The effects of a novel phosphodiesterase 7A and -4 dual inhibitor, YM-393059, on T-cell-related cytokine production in vitro and in vivo. Eur. J. Pharmacol. 541, 106-114]. To characterize the pharmacological profile of YM-393059, its effects on several acute and chronic inflammation models were examined. In acute inflammation models, YM-393059 significantly suppressed the delayed-type hypersensitivity reaction to sheep red blood cells in mice with an ED(50) value of 17.1 mg/kg. YM-393059 failed to suppress paw edema in the carrageenin-induced edema model in rats. These pharmacological effects were similar to those of cyclosporine, a typical T-cell immunosuppressant. However, YM-393059, but not cyclosporine, significantly inhibited zymosan-induced neutrophil accumulation in mice with an ED(50) value of 25.7 mg/kg. In mouse toluene-2,4-diisocyanate-induced contact dermatitis, a chronic inflammation model, YM-393059 and cyclosporine significantly suppressed ear edema at doses of 30 and 20 mg/kg, respectively. In this model, YM-393059 also tended to reduce the serum immunoglobulin E antibody level, whereas cyclosporine dramatically potentiated it. These results suggest that YM-393059 inhibits both Th1- and Th2-cell-dependent reactions and also the function of neutrophils.

The effects of a novel phosphodiesterase 7A and -4 dual inhibitor, YM-393059, on T-cell-related cytokine production in vitro and in vivo.

YM-393059, (+/-)-N-(4,6-dimethylpyrimidin-2-yl)-4-[2-(4-methoxy-3-methylphenyl)-5-(4-methylpiperazin-1-yl)-4,5,6,7-tetrahydro-1H-indol-1-yl]benzenesulfonamide difumarate, is a novel phosphodiesterase (PDE) inhibitor that inhibited the PDE7A isoenzyme with a high potency (IC50=14 nM) and PDE4 with a moderate potency (IC50=630 nM). In a cell-based assay, YM-393059 was found to inhibit anti-CD3 antibody, Staphylococcal enterotoxin B, and phytohaemagglutinin-induced interleukin (IL)-2 production in mouse splenocytes with IC50 values ranging from 0.48 to 1.1 microM. It also inhibited anti-CD3 antibody-induced interferon (IFN)-gamma and IL-4 production in splenocytes with IC50 values of 1.8 and 2.8 microM, respectively. YM-393059's inhibition of anti-CD3 antibody-stimulated cytokine (IL-2, IFN-gamma, and IL-4) production was 20- to 31-fold weaker than that of YM976, a selective PDE4 inhibitor. However, orally administered YM-393059 and YM976 inhibited anti-CD3 antibody-induced IL-2 production equipotently in mice. In addition, YM-393059 inhibited lipopolysaccharide-induced tumor necrosis factor-alpha production in vivo more potently than IL-2 (ED50 values of 2.1 mg/kg and 74 mg/kg). In contrast to YM976, YM-393059 did not shorten the duration of alpha2-adrenoceptor agonist-induced sleep in mice, which is a model for the assessment of the typical side effects caused by PDE4 inhibitors, nausea and emesis. YM-393059 is a novel and attractive compound for the treatment of a wide variety of T-cell-mediated diseases.

Kinins are involved in the development of allergic nasal hyperresponsiveness in guinea pigs.

We evaluated roles of kinins in allergen-induced nasal blockage and sneezing, and development of nasal hyperresponsiveness to leukotriene D4 in a Japanese cedar pollen-induced allergic rhinitis model of guinea pigs. Sensitised guinea pigs were repeatedly challenged by pollen inhalation once every week. Neither a bradykinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin nor a bradykinin B2 receptor antagonist, icatibant suppressed allergen-induced sneezing and nasal blockage. However, development of nasal hyperresponsiveness to leukotriene D4 was significantly suppressed by them. The amount of bradykinin in nasal cavity lavage fluid was immediately increased after the challenge. In non-sensitised animals, hyperresponsiveness to leukotriene D4 was developed by a bradykinin B2 receptor agonist, bradykinin, but not by a bradykinin B1 receptor agonist, des-Arg10-kallidin, while in the sensitised-challenged animal, both agonists developed hyperresponsiveness. In conclusion, the nasal hyperresponsiveness appeared to be induced by kinins produced in response to the antigen challenge through activation of not only bradykinin B2 but also B1 receptors.