Therapeutic effects of novel sphingosine-1-phosphate receptor agonist W-061 in murine DSS colitis

Although IL-17 is a pro-inflammatory cytokine reportedly involved in various autoimmune inflammatory disorders, its role remains unclear in murine models of colitis. Acute colitis was induced by 2.5% dextran sodium sulfate (DSS) treatment for 5 days. A novel sphingosine-1-phosphate receptor agonist W-061, a prototype of ONO-4641, was orally administered daily, and histopathological analysis was performed on the colon. The number of lymphocytes and their cytokine production were also evaluated in spleen, mesenteric lymph node, Peyer's patch and lamina propria of the colon. Daily administration of W-061 resulted in improvement of DSS-induced colitis, and significantly reduced the number of CD4+ T cells in the colonic lamina propria. Numbers of both Th17 and Th1 cells were reduced by W-061 treatment. W-061, however, had no influence on the number of Treg cells in lamina propria. Thus, Th17 and Th1 cells in lamina propria were thought to be the key subsets in the pathogenesis of DSS-induced colitis. In conclusion, W-061 may be a novel therapeutic strategy to ameliorate acute aggravation of inflammatory bowel diseases.     

Unfolding of the amyloid β-peptide central helix: mechanistic insights from molecular dynamics simulations

Alzheimer's disease (AD) pathogenesis is associated with formation of amyloid fibrils caused by polymerization of the amyloid β-peptide (Aβ), which is a process that requires unfolding of the native helical structure of Aβ. According to recent experimental studies, stabilization of the Aβ central helix is effective in preventing Aβ polymerization into toxic assemblies. To uncover the fundamental mechanism of unfolding of the Aβ central helix, we performed molecular dynamics simulations for wild-type (WT), V18A/F19A/F20A mutant (MA), and V18L/F19L/F20L mutant (ML) models of the Aβ central helix. It was quantitatively demonstrated that the stability of the α-helical conformation of both MA and ML is higher than that of WT, indicating that the α-helical propensity of the three nonpolar residues (18, 19, and 20) is the main factor for the stability of the whole Aβ central helix and that their hydrophobicity plays a secondary role. WT was found to completely unfold by a three-step mechanism: 1) loss of α-helical backbone hydrogen bonds, 2) strong interactions between nonpolar sidechains, and 3) strong interactions between polar sidechains. WT did not completely unfold in cases when any of the three steps was omitted. MA and ML did not completely unfold mainly due to the lack of the first step. This suggests that disturbances in any of the three steps would be effective in inhibiting the unfolding of the Aβ central helix. Our findings would pave the way for design of new drugs to prevent or retard AD.     

Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats

Background

Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy.

Methods and Results

Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed.

Conclusions

These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.     

Characterization of PD-1/PD-L1 immune checkpoint expression in soft tissue sarcomas

Inhibitors of the programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint system are used for treating various malignancies. However, evidence on their use in soft tissue sarcomas (STS) is limited. This study aimed to retrospectively investigate the relationship between the expression of PD-1/PD-L1 and related antigens in STS, and their association with clinical characteristics. Immunostaining for CD4, CD8, PD-1, PD-L1, IL-2, and IFN-γ was performed using pathological specimens harvested at the time of biopsy from 10 patients with undifferentiated pleomorphic sarcoma (UPS), nine with myxofibrosarcoma (MFS), and three with malignant peripheral nerve sheath tumor (MPNST) who were treated at our hospital. Subsequently, the positive immunostaining cell rates were calculated. We also examined the correlation between each immune positive cell rate and age, tissue grade, size, and maximum standardized uptake (SUV-max) values. The 3-year event-free survival (EFS) and overall survival (OS) rates were compared between the positive and negative groups (positive rate >10%; negative <10%) for various immune stains. The positive rates were also compared between the presence and absence of events groups. There was positive staining for the immune checkpoint molecules in every STS type except for PD-1 in MPNST. CD4, CD8, and PD-1 stained lymphocytes in close proximity to the tumor in adjacent tissue sections. A positive correlation was observed between the positive cell rates of each immune component including inflammatory cytokines such as IL-2 and IFN-γ. Additionally, the clinical features positively correlated with the positive PD-1/PD-L1 expression rates. No significant differences in the 3-EFS and OS rates were observed between the PD-1/PD-L1 positive and negative groups. Our results suggest that an inducible immune checkpoint mechanism may be involved in UPS, MFS, and MPNST.Key words: Immune checkpoint inhibitors, PD-1/PD-L1, soft tissue sarcoma, programmed death-1, programmed death-ligand 1     

The docking protein FRS2alpha is an essential component of multiple fibroblast growth factor responses during early mouse development

The docking protein FRS2alpha is a major mediator of fibroblast growth factor (FGF) signaling. However, the physiological role of FRS2alpha in vivo remains unknown. In this report, we show that Frs2alpha-null mouse embryos have a defect in anterior-posterior (A-P) axis formation and are developmentally retarded, resulting in embryonic lethality by embryonic day 8. We demonstrate that FRS2alpha is essential for the maintenance of self-renewing trophoblast stem (TS) cells in response to FGF4 in the extraembryonic ectoderm (ExE) that gives rise to tissues of the placenta. By analyzing chimeric embryos, we found that FRS2alpha also plays a role in cell movement through the primitive streak during gastrulation. In addition, experiments are presented demonstrating that Bmp4 expression in TS cells is controlled by mitogen-activated protein kinase-dependent FGF4 stimulation. Moreover, both the expression of Bmp4 in ExE and activation of Smad1/5 in epiblasts are reduced in Frs2alpha-null embryos. These experiments underscore the critical role of FRS2alpha in mediating multiple processes during embryonic development and reveal a potential new link between FGF and Bmp4 signaling pathways in early embryogenesis.     

Taxonomic relationships within the pan-oriental narrow-mouth toad Microhyla ornata as revealed by mtDNA analysis (Amphibia, Anura, Microhylidae)

A molecular phylogenetic survey was conducted using mtDNA sequences of 12S and 16S rRNA, and cyt-b genes to examine taxonomic relationships among populations of the Pan-Oriental microhylid, Microhyla ornata, from India, Bangladesh, Thailand, Laos, China, Taiwan, and the Ryukyu Archipelago of Japan. Two discrete clades are recognized within this species, one consisting of populations from India and Bangladesh, and the other encompassing the remaining populations. In the latter clade, populations from the Ryukyu Archipelago are clearly split from the rest (populations from Taiwan and the continent) with considerable degrees of genetic differentiations. Each of the three lineages is judged to represent a good species, and the name Microhyla ornata is restricted to the South Asian populations. For the populations from Taiwan and a wide region from China to Southeast Asia, the name Microhyla fissipes should be applied, whereas the Ryukyu populations are most appropriately referred to as Microhyla okinavensis, although further substantial genetic differentiations are recognized among some island group populations within this last species.     

Inhibition of ACh-stimulated exocytosis by NSAIDs in guinea pig antral mucous cells: autocrine regulation of mucin secretion by PGE2

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 microM) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 microM; an inhibitor of the EP1-EP4 prostaglandin receptors) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89, 20 microM; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE(2) (1 microM) prevented the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-1) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). ACh and ionomycin increased PGE(2) release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in [Ca(2+)](i) activates Ca(2+)-regulated exocytotic events and PGE(2) release mediated by COX-1. The released PGE(2) induces the accumulation of cAMP, which enhances the Ca(2+)-regulated exocytosis. The autocrine mechanism mediated by PGE(2) maintains the high-level mucin release from antral mucous cells during ACh stimulation.