IMD-4690, a Novel Specific Inhibitor for Plasminogen Activator Inhibitor-1, Reduces Allergic Airway Remodeling in a Mouse Model of Chronic Asthma via Regulating Angiogenesis and Remodeling-Related Mediators

Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.     

Viruses Roll the Dice: The Stochastic Behavior of Viral Genome Molecules Accelerates Viral Adaptation at the Cell and Tissue Levels

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1–20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence–tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell.     

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.     

Skeletal Metastasis of Unknown Primary Origin at the Initial Visit: A Retrospective Analysis of 286 Cases

Background

Skeletal metastasis is a common metastatic event for several carcinomas, and the treatment for skeletal metastasis of unknown primary (SMUP) are a critical issue in cancer therapy. Making a diagnosis of the primary site is the most crucial step in the treatment of SMUP; however, the procedures are sometimes difficult and time-consuming, and the primary site often remains unknown. Therefore, to establish optimal diagnostic strategies and elucidate the overall survival rates of SMUP, we conducted this retrospective study.

Methods

We retrospectively analyzed the clinical data for 286 SMUP cases from a total of 2,641 patients with skeletal metastases who were treated between 2002 and 2014 at our initiations.

Results

The primary sites were identified in 254/286 patients (88.8%), while 32 (11.2%) primary sites were not detected by our diagnostic strategies. Lung cancer was identified in 72 (25.2%) cases, and was the most frequently observed primary lesion. The median survival time of the SMUP patients was 20.0 months, while the median survival times of solitary bone metastasis cases and multi-bone metastasis cases were 39.0 months and 16.0 months, respectively. The median survival times of prostate cancer cases was over 120 months, that of patients with primary lung cancers was 9.0 months and the median survival time of cases who were finally diagnosed with an unknown primary was 11.0 months.

Conclusions

We believe that our study would contribute to establishing an optimal strategy for diagnosing the primary site in SMUP patients, and our data provide definite indications for the survival times for different SMUP situations.     

Endoplasmic reticulum stress-inducible protein, Herp, enhances presenilin-mediated generation of amyloid beta-protein.

Presenilin (PS) is essential for the gamma-cleavage required for the generation of the C terminus of amyloid beta-protein (Abeta). However, the mechanism underlying PS-mediated gamma-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of gamma-cleavage. Herp was originally identified as a homocysteine-responsive protein, and its expression is up-regulated by endoplasmic reticulum stress. Herp is an endoplasmic reticulum-localized membrane protein that has a ubiquitin-like domain. Here, we report that a high expression of Herp in cells increases the level of Abeta generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated Abeta generation, possibly through its binding to PS. Immunohistochemical analysis of a normal human brain section with an anti-Herp antibody revealed the exclusive staining of neurons and vascular smooth muscle cells. Moreover, the antibody strongly stained activated microglia in senile plaques in the brain of patients with Alzheimer disease. Taken together, Herp could be involved in Abeta accumulation, including the formation of senile plaques and vascular Abeta deposits.     

Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS

POU5F1 (more commonly known as OCT4/3) is one of the stem cell markers, and affects direction of differentiation in embryonic stem cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stem cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stem cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and cell surface analysis revealed that the cells are subcategorized into the group of human embryonic stem cells and embryonal carcinoma cells. These results imply that cells of mesenchymal origin can be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide us with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity.     

Discovery of novel spiro-piperidine derivatives as highly potent and selective melanin-concentrating hormone 1 receptor antagonists

Optimization of high-throughput screening hit 1a led to the identification of a novel spiro-piperidine class of melanin-concentrating hormone 1 receptor (MCH-1R) antagonists. Compound 3c was identified as a highly potent and selective MCH-1R antagonist, which has an IC₅₀ value of 0.09 nM at hMCH-1R. The synthesis and structure–activity relationships of the novel spiro-piperidine MCH-1R antagonists are described.     

CYP710A genes encoding sterol C22-desaturase in <Emphasis Type="Italic">Physcomitrella patens</Emphasis> as molecular evidence for the evolutionary conservation of a sterol biosynthetic pathway in plants

We have characterized cytochromes P450, CYP710A13, and CYP710A14, as the sterol C22-desaturase in the moss Physcomitrella patens. GC–MS analyses demonstrated that P. patens accumulated stigmasterol as the major sterol (56–60% of total sterol) and sitosterol to a lesser extent (8–12%); this sterol profile contrasts with those in higher plants accumulating stigmasterol as a minor component. Recombinant CYP710A13 and CYP710A14 proteins prepared using a baculovirus/insect cell system exhibited the C22-desaturase activity with β-sitosterol to produce stigmasterol, while campesterol and 24-epi-campesterol were not accepted as the substrates. The K _m values for β-sitosterol of CYP710A13 (1.0 ± 0.043 μM) and CYP710A14 (2.1 ± 0.17 μM) were at comparable levels of those reported with higher plant CYP710A proteins. In Arabidopsis T87 cells over-expressing CYP710A14, stigmasterol contents reached a level 20- to 72-fold higher than those in the basal level of T87 cells, confirming the C22-desaturase activity of this P450 enzyme. The occurrence of the end-products together with the enzymes involved in the last step of the pathway substantiated the presence of an entire sterol biosynthetic pathway in P. patens, providing evidence for the conservation of the sterol biosynthetic pathway through the evolutionary process of land plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.