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The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit β5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes.

Bycatch of harbour porpoises (Phocoena phocoena) by gillnets is a recognised threat to populations. To develop effective mitigation measures, understanding the mechanics of bycatch is essential. Previous studies in experimental conditions suggested foraging activity is an important factor influencing porpoises' reaction to gillnets. We acoustically observed the behaviour of wild harbour porpoises around a bottom-gillnet set-up in a commercial fishing ground, especially foraging activity. Passive acoustic event recorders (A-tags) were fixed to the ends of the gillnet, and recorded for 1 392 hours. Although harbour porpoises frequently and repeatedly appeared around the net each day, incidental bycatch occurred only three times during the observations. The stomach contents of two individuals contained mainly Ammodytes sp., which were observable around the bottom-gillnet but not targeted by the fishery. A total of 276 foraging incidents were acoustically detected, and 78.2% of the foraging activity was in the bottom layer (deeper than 25 m). Porpoises appeared around the net with more frequency on the day of a bycatch incident than on the days without bycatch. These results suggest that the harbour porpoises appeared around the bottom-gillnet to forage on fish distributed in the fishing ground, but not captured by this bottom-gillnet. Thus, porpoises face the risk of becoming entangled when foraging near a gillnet, with the probability of bycatch simply increasing with the length of time spent near the net. Bycatch mitigation measures are discussed.

Purpose To analyze the structure of the choriocapillaris in healthy eyes by using averaged en face images acquired using swept source optical coherence tomography angiography and to examine the changes in the macular profile in relation to age, sex, axial length, and choroidal thickness. Methods This prospective, cross-sectional study included 81 eyes of 81 subjects without ophthalmologic or systemic diseases who underwent a full ophthalmologic examination, including 3 × 3-mm macular optical coherence tomography angiography. Four to nine choriocapillaris en face images were registered and averaged. The averaged images were then binarized and analyzed. Results The averaged choriocapillaris images showed a continuous capillary meshwork, whereas the unaveraged images had a granular appearance. The mean total area and size of flow voids were 0.99 ± 0.20 mm2 and 567.8 ± 201.5 μm2, respectively, and these values correlated positively with age (p = 0.002, R = 0.336 and p = 0.026, R = 0.247, respectively). Age-related gains in the mean total area and flow void size were 4.20 × 10−3 mm2 and 3.07 μm2 per year, respectively. However, the mean total area and flow void size had no significant correlation with axial length, subfoveal choroidal thickness, or sex. Conclusions Multiple averaged en face swept source optical coherence tomography angiography is more effective than a single optical coherence tomography angiography scan for better visualizing the choriocapillaris. The total area and size of flow voids within a 3 × 3-mm macular area positively correlated with age. This technique can be useful for investigating the changes arising in macular diseases.

PurposeTo analyze the structure of the choriocapillaris in healthy eyes by using averaged en face images acquired using swept source optical coherence tomography angiography and to examine the changes in the macular profile in relation to age, sex, axial length, and choroidal thickness.MethodsThis prospective, cross-sectional study included 81 eyes of 81 subjects without ophthalmologic or systemic diseases who underwent a full ophthalmologic examination, including 3 × 3-mm macular optical coherence tomography angiography. Four to nine choriocapillaris en face images were registered and averaged. The averaged images were then binarized and analyzed.ResultsThe averaged choriocapillaris images showed a continuous capillary meshwork, whereas the unaveraged images had a granular appearance. The mean total area and size of flow voids were 0.99 ± 0.20 mm2 and 567.8 ± 201.5 μm2, respectively, and these values correlated positively with age (p = 0.002, R = 0.336 and p = 0.026, R = 0.247, respectively). Age-related gains in the mean total area and flow void size were 4.20 × 10-3 mm2 and 3.07 μm2 per year, respectively. However, the mean total area and flow void size had no significant correlation with axial length, subfoveal choroidal thickness, or sex.ConclusionsMultiple averaged en face swept source optical coherence tomography angiography is more effective than a single optical coherence tomography angiography scan for better visualizing the choriocapillaris. The total area and size of flow voids within a 3 × 3-mm macular area positively correlated with age. This technique can be useful for investigating the changes arising in macular diseases.

The physiological actions of orally ingested peptides on the brain remain poorly understood. This study examined the effects of 39 orally administered synthetic Tyr-containing dipeptides on the enhancement of brain norepinephrine metabolism in mice by comparing the concentration of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG). Although Tyr-Tyr administration increased blood and cerebral cortex (Cx) Tyr concentrations the most, Tyr-Trp increased Cx MHPG concentration the most. The oral administration of Tyr-Trp ameliorated a short-term memory deficit of a mouse model of cognitive dysfunction induced by amyloid beta peptide 25-35. Gene expression profiling of mouse brain using a microarray indicated that Tyr-Trp administration led to a wide variety of changes in mRNA levels, including the upregulation of genes encoding molecules involved in catecholamine metabolism. A comparative metabolome analysis of the Cx of mice given Tyr-Trp or Tyr-Tyr demonstrated that Tyr-Trp administration yielded higher concentrations of Trp and kynurenine pathway metabolites than Tyr-Tyr administration, as well as higher L-dopa levels, which is the initial product of catecholamine metabolism. Catecholamines were not significantly increased in the Cx of the Tyr-Tyr group compared with the Tyr-Trp group, despite a marked increase in Tyr. Presumably, Tyr-Trp administration enhances catecholamine synthesis and metabolism via the upregulation of genes involved in Tyr and Trp metabolism as well as metabolites of Tyr and Trp. These findings strongly suggest that orally ingested Tyr-Trp modulates the brain metabolome involved in catecholamine metabolism and contributes to higher brain function.

BackgroundRecently, attention has been focused on the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the mechanism of and as a treatment target for neuropathic and inflammatory pain. Ivabradine, a blocker of HCN channels, was demonstrated to have an effect on neuropathic pain in an animal model. Therefore, in the present study, we evaluated the effect of ivabradine on inflammatory pain, and under the hypothesis that ivabradine can directly influence inflammatory responses, we investigated its effect in in vivo and in vitro studies.MethodsAfter approval from our institution, we studied male Sprague-Dawley rats aged 8 weeks. Peripheral inflammation was induced by the subcutaneous injection of carrageenan into the hindpaw of rats. The paw-withdrawal threshold (pain threshold) was evaluated by applying mechanical stimulation to the injected site with von Frey filaments. Ivabradine was subcutaneously injected, combined with carrageenan, and its effect on the pain threshold was evaluated. In addition, we evaluated the effects of ivabradine on the accumulation of leukocytes and TNF-alpha expression in the injected area of rats. Furthermore, we investigated the effects of ivabradine on LPS-stimulated production of TNF-alpha in incubated mouse macrophage-like cells.ResultsThe addition of ivabradine to carrageenan increased the pain threshold lowered by carrageenan injection. Both lamotrigine and forskolin, activators of HCN channels, significantly reversed the inhibitory effect of ivabradine on the pain threshold. Ivabradine inhibited the carrageenan-induced accumulation of leukocytes and TNF-alpha expression in the injected area. Furthermore, ivabradine significantly inhibited LPS-stimulated production of TNF-alpha in the incubated cells.ConclusionThe results of the present study demonstrated that locally injected ivabradine is effective against carrageenan-induced inflammatory pain via HCN channels. Its effect was considered to involve not only an action on peripheral nerves but also an anti-inflammatory effect.

PURPOSE:To investigate longitudinal changes in metamorphopsia associated with branch retinal vein occlusion. METHODS:In this prospective observational case series, we included 32 eyes (32 patients) with branch retinal vein occlusion and acute macular edema. Eyes were treated as needed with intravitreal ranibizumab injections for 12 months. At baseline and 1, 6, and 12 months after initiating treatment, metamorphopsia was quantified using M-CHARTS. Retinal morphology was examined through optical coherence tomography. RESULTS:Logarithm of the minimum angle of resolution visual acuity progressively improved from 0.342 ± 0.304 (Snellen equivalent: 20/44) at baseline to 0.199 ± 0.259 (20/32) and 0.118 ± 0.195 (20/26) at 1 and 12 months, respectively (both P < 0.001). The M-CHARTS score significantly decreased from 0.63 ± 0.61 at baseline to 0.45 ± 0.50 at 1 month (P = 0.044), but no further improvement was achieved with 1 year of additional treatment (6 months: 0.47 ± 0.53 [P = 0.094] and 12 months: 0.50 ± 0.44 [P = 0.173]). Three (13.6%) of 22 eyes with baseline metamorphopsia had complete metamorphopsia resolution. At 12 months, the M-CHARTS score was correlated with baseline foveal thickness (r = 0.373, P = 0.035) and the baseline M-CHARTS score (r = 0.503, P = 0.003). A multiple regression analysis revealed that only the baseline M-CHARTS score was correlated with the 12-month M-CHARTS score (β = 0.460, P = 0.027). CONCLUSIONS:Eyes with branch retinal vein occlusion often have persistent metamorphopsia, even when visual acuity and retinal morphology improve. Metamorphopsia at 12 months was correlated with metamorphopsia and foveal thickness at baseline.

ATP and its metabolites are important for taste signaling in taste buds, and thus a clearance system for them would play critical roles in maintenance of gustatory function. A previous report revealed that mRNAs for ecto-5'-nucleotidase (NT5E) and prostatic acid phosphatase (PAP) were expressed by taste cells of taste buds, and NT5E-immunoreactivity was detected in taste cells. However, there was no information on PAP-immunoreactivity in taste buds. In this study, we examined the expression profile of PAP in rat taste buds. In the isolated rat taste buds, we detected expression of mRNA for PAP, but NT5E was not detected differing from the case of mouse ones (Dando et al., 2012, J Neuroscience). On immunohistochemical analysis, PAP-immunoreactivity was found predominantly in NTPDase2-positive type I and SNAP25-positive type III taste cells, while there were no apparent signals of it in PLC-β2-positive type II, α-gustducin-positive type II, AADC-positive type III and 5HT-positive type III ones. As for NT5E, we could not detect its immunoreactivity in rat taste buds, and co-localization of it with any taste cell markers, although mouse taste buds expressed NT5E as reported previously. These findings suggest that PAP expressed by type I and one of type III taste cells of rats may contribute to metabolic regulation of the extracellular levels of adenine nucleotides in the taste buds of circumvallate papillae, and the regulating mechanisms for adenine nucleotides in taste buds might be different between rats and mice.

CCG-1423 suppresses several pathological processes including cancer cell migration, tissue fibrosis, and the development of atherosclerotic lesions. These suppressions are caused by inhibition of myocardin-related transcription factor A (MRTF-A), which is a critical factor for epithelial-mesenchymal transition (EMT). CCG-1423 can therefore be a potent inhibitor for EMT. CCG-1423 and related compounds, CCG-100602 and CCG-203971 possess similar biological activities. Although these compounds are comprised of two stereoisomers, the differences in their biological activities remain to be assessed. To address this issue, we stereoselectively synthesized optically pure isomers of these compounds and validated their biological activities. The S-isomer of CCG-1423 rather than the R-isomer exhibited modestly but significantly higher inhibitory effects on the cellular events triggered by MRTF-A activation including serum response factor-mediated gene expression and cell migration of fibroblasts and B16F10 melanoma cells. Accordingly, the S-isomer of CCG-1423 more potently blocked the serum-induced nuclear import of MRTF-A than the R-isomer. No such difference was observed in cells treated with each of two stereoisomers of CCG-100602 or CCG-203971. We previously reported that the N-terminal basic domain (NB), which functions as a nuclear localization signal of MRTF-A, is a binding site for CCG-1423. Consistent with the biological activities of two stereoisomers of CCG-1423, docking simulation demonstrated that the S-isomer of CCG-1423 was more likely to bind to NB than the R-isomer. This is a first report demonstrating the stereospecific biological activities of CCG-1423.

Epithelial-msenchymal transition (EMT) is closely associated with cancer and tissue fibrosis. The nuclear accumulation of myocardin-related transcription factor A (MRTF-A/MAL/MKL1) plays a vital role in EMT. In various cells treated with CCG-1423, a novel inhibitor of Rho signaling, the nuclear accumulation of MRTF-A is inhibited. However, the molecular target of this inhibitor has not yet been identified. In this study, we investigated the mechanism of this effect of CCG-1423. The interaction between MRTF-A and importin α/β1 was inhibited by CCG-1423, but monomeric G-actin binding to MRTF-A was not inhibited. We coupled Sepharose with CCG-1423 (CCG-1423 Sepharose) to investigate this mechanism. A pull-down assay using CCG-1423 Sepharose revealed the direct binding of CCG-1423 to MRTF-A. Furthermore, we found that the N-terminal basic domain (NB) of MRTF-A, which acts as a functional nuclear localization signal (NLS) of MRTF-A, was the binding site for CCG-1423. G-actin did not bind to CCG-1423 Sepharose, but the interaction between MRTF-A and CCG-1423 Sepharose was reduced in the presence of G-actin. We attribute this result to the high binding affinity of MRTF-A for G-actin and the proximity of NB to G-actin-binding sites (RPEL motifs). Therefore, when MRTF-A forms a complex with G-actin, the binding of CCG-1423 to NB is expected to be blocked. NF-E2 related factor 2, which contains three distinct basic amino acid-rich NLSs, did not bind to CCG-1423 Sepharose, but other RPEL-containing proteins such as MRTF-B, myocardin, and Phactr1 bound to CCG-1423 Sepharose. These results suggest that the specific binding of CCG-1423 to the NLSs of RPEL-containing proteins. Our proposal to explain the inhibitory action of CCG-1423 is as follows: When the G-actin pool is depleted, CCG-1423 binds specifically to the NLS of MRTF-A/B and prevents the interaction between MRTF-A/B and importin α/β1, resulting in inhibition of the nuclear import of MRTF-A/B.

OBJECTIVE: Periostin, a novel matricellular protein, is recently reported to play a crucial role in tissue remodeling and is highly expressed under fibrotic conditions. This study was undertaken to assess the role of periostin in scleroderma. METHODS: Using skin from patients and healthy donors, the expression of periostin was assessed by immunohistochemistry and immunoblotting analyses. Furthermore, we investigated periostin(-/-) (PN(-/-)) and wild-type (WT) mice to elucidate the role of periostin in scleroderma. To induce murine cutaneous sclerosis, mice were subcutaneously injected with bleomycin, while untreated control groups were injected with phosphate-buffered saline. Bleomycin-induced fibrotic changes were compared in PN(-/-) and WT mice by histological analysis as well as by measurements of profibrotic cytokine and extracellular matrix protein expression levels in vivo and in vitro. To determine the downstream pathway involved in periostin signaling, receptor neutralizing antibody and signal transduction inhibitors were used in vitro. RESULTS: Elevated expression of periostin was observed in the lesional skin of patients with scleroderma compared with healthy donors. Although WT mice showed marked cutaneous sclerosis with increased expression of periostin and increased numbers of myofibroblasts after bleomycin treatment, PN(-/-) mice showed resistance to these changes. In vitro, dermal fibroblasts from PN(-/-) mice showed reduced transcript expression of alpha smooth actin and procollagen type-I alpha 1 (Col1α1) induced by transforming growth factor beta 1 (TGFβ1). Furthermore, recombinant mouse periostin directly induced Col1α1 expression in vitro, and this effect was inhibited by blocking the αv integrin-mediated PI3K/Akt signaling either with anti-αv functional blocking antibody or with the PI3K/Akt kinase inhibitor LY294002. CONCLUSION: Periostin plays an essential role in the pathogenesis of Bleomycin-induced scleroderma in mice. Periostin may represent a potential therapeutic target for human scleroderma.