Shiro Nakaike

Involvement of IL‐31 on scratching behavior in NC/Nga mice with atopic‐like dermatitis

Abstract:  Pruritus is an important symptom in atopic dermatitis (AD), but the major pruritogen have not been identified. NC/Nga mice, spontaneously develop an eczematous AD‐like skin lesion when kept under conventional conditions, but not under specific pathogen‐free (SPF) conditions, have been thought to be an animal model for AD. In this study, to determine whether newly identified cytokine, IL‐31, may be involved in pruritus of AD, we examined the IL‐31 expression in spontaneous dermatitis model which showed itch‐associated long‐lasting (over 1.5 s duration) scratching behavior and compared with that of hapten‐induced contact dermatitis model without itch‐associated long‐lasting scratching behavior, using NC/Nga mice. In NC/Nga mice cohabited with NC/Nga mice which developed severe dermatitis for 2 weeks (conventional NC/Nga mice), the numbers of long‐lasting scratching counts were significantly increased. Yet in 2,4,6‐trinitrochlorobenzene (TNCB)‐sensitized and challenged mice (TNCB‐applied NC/Nga mice), no significant increase in long‐lasting scratching counts was observed. In conventional NC/Nga mice with long‐lasting scratching behavior, expression of IL‐31 mRNA was increased, while in TNCB‐applied NC/Nga mice without long‐lasting scratching behavior, the expression of IL‐31 mRNA were unchanged. There was a good correlation between the scratching counts and expression of IL‐31 mRNA in conventional NC/Nga mice, but not so in TNCB‐applied NC/Nga mice. These results suggest that IL‐31 causes the itch‐associated scratching behavior in conventional NC/Nga mice, an experimental animal model for AD.

Critical role for OX40 ligand in the development of pathogenic Th2 cells in a murine model of asthma

Bronchial asthma is characterized by massive infiltration of eosinophils and airway hyperreactivity (AHR), which are caused by overproduction of Th2 cytokines (IL‐4, IL‐5, and IL‐13) by allergen‐specific T cells. We recently demonstrated a critical contribution of OX40 ligand (OX40L) to the development of Th2‐mediated experimental leishmaniasis. In this study, we have examined the role of OX40L in the development of Th2‐mediated pulmonary inflammation by utilizing OX40L‐deficient mice and a neutralizing anti‐OX40L mAb in a murine model of asthma. Sensitization and airway challenge with ovalbumin in wild‐type BALB/c mice induced a typical allergic asthma characterized by AHR, accumulation of eosinophils, increased mucus production, and high levels of Th2 cytokines in the lung.All these asthmatic responses were not induced in OX40L‐deficient BALB/c mice. Administration of neutralizing anti‐OX40L mAb in wild‐type BALB/c mice during the sensitization period also abolished the induction of asthmatic responses. In contrast, administration of anti‐OX40L mAb during the challenge period did not inhibit the asthmatic responses. These results indicate a critical role for OX40L in the induction phase, which leads to the development of pathogenic Th2 cells, but not in the effector phase, which includes migration and activation of pathogenic Th2 cells in the lung.

Effect of a New Inhibitor of the Synthesis of 20-HETE on Cerebral Ischemia Reperfusion Injury

Background and Purpose— Arachidonic acid that is released following cerebral ischemia can be metabolized to 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a potent vasoconstrictor that may contribute to ischemic injury. This study examined the effects of blockading the synthesis of 20-HETE with TS-011 on infarct size after transient occlusion of the middle cerebral artery (MCAO) of rats and after thromboembolic stroke in monkeys. Methods— Rats were treated with TS-011 or vehicle at various times after MCAO. Infarct size was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining and plasma levels of 20-HETE were determined by liquid chromatography mass spectrometry (LC/MS). The effect of TS-011 on infarct size was also studied in monkeys after introduction of a clot into the internal carotid artery. Results— Plasma levels of 20-HETE increased after MCAO in rats. TS-011 (0.01 to 1.0 mg/kg per hour) reduced infarct volume by 40%. Chronic administration of TS-011 for 7 days reduced neurological deficits after MCAO in rats. TS-011 given in combination with tissue plasminogen activator also improved neurological outcome in the stroke model in monkeys. Conclusion— These results suggest that blockade of the formation of 20-HETE with TS-011 may be useful for the treatment of ischemic stroke.

A Critical Role for Mouse CXC Chemokine(s) in Pulmonary Neutrophilia During Th Type 1-Dependent Airway Inflammation

Ag-specific Th1 and Th2 cells have been demonstrated to play a critical role in the induction of allergic diseases. Here we have investigated the precise mechanisms of Th1-induced airway inflammation. Airway inflammation was induced in BALB/c mice by transfer of freshly induced OVA-specific Th1 or Th2 cells followed by OVA inhalation. In this model, both Th1 and Th2 cells induced airway inflammation. The former induced neutrophilia in airways, whereas the latter induced eosinophilia. Moreover, we found that Th1 cells induced more severe airway hyperresponsiveness (AHR) than Th2 cells. The eosinophilia induced by Th2 cell infusion was almost completely blocked by administration of anti-IL-5 mAb, but not anti-IL-4 mAb. In contrast, Th1-induced AHR and pulmonary neutrophilia were inhibited by the administration of anti-human IL-8R Ab, which blocks the function of mouse CXC chemokine(s). These findings reveal a critical role of mouse CXC chemokine(s) in Th1-dependent pulmonary neutrophilia and AHR.

Modulation of scratching behavior by silencing an endogenous cyclooxygenase-1 gene in the skin through the administration of siRNA

RNA interference (RNAi) is rapidly becoming a major tool that is revolutionizing research in the bioscience and biomedical fields. To apply the RNAi technique in vivo, it is crucial to develop appropriate methods of guiding the short interfering RNA (siRNA) molecules to the right tissues and cells. Here, we demonstrate an efficient method for performing gene knockdown in the body skin using the in vivo electro‐transduction of siRNA. Using this method, we examined whether the targeted silencing of the cyclooxygenase (COX) gene in the skin could modulate the scratching behavior of an atopic dermatitis mouse model.

Continuous inhibition of 20-HETE synthesis by TS-011 improves neurological and functional outcomes after transient focal cerebral ischemia in rats

TS-011, a potent and selective inhibitor of 20-HETE synthesis, has been described as providing significant benefits in animal stroke models. However, no studies have investigated changes in brain 20-HETE levels after cerebral ischemia. Also lacking are studies of TS-011 pharmacodynamics with respect to brain 20-HETE levels that may explain the benefits of TS-011 in animal models of ischemic stroke. The present study sought to explore changes in 20-HETE levels in brain tissue, as well as in plasma, after a 90-min episode of transient focal cerebral ischemia. Pharmacodynamics of TS-011 were also examined. Then, we evaluated the long-term effects of TS-011 when administered as in this pharmacodynamics study. The major findings of the present study are as follows: (1) brain 20-HETE levels increased significantly within 7.5h after MCAO; (2) TS-011 at doses of 0.1 and 0.3mg/kg administered at regular 6-h intervals appeared to reduce brain 20-HETE levels continuously; (3) TS-011 when administered as in this pharmacodynamics study improved long-term neurological and functional outcomes. These findings strongly suggest that 20-HETE plays an important role in the development of neurological and functional deficits after focal cerebral ischemia and that TS-011 may provide benefits in patients suffering ischemic stroke.

Efficient induction of chromosome-type aberrations by topoisomerase II inhibitors closely associated with stabilization of the cleavable complex in cultured fibroblastic cells

Eukaryotic topoisomerase II (Topo-II) inhibitors such as etoposide, adriamycin and mitoxantrone, which commonly stabilize the cleavable complex of the enzyme and DNA, have been found to efficiently induce chromosome-type aberrations (mainly breaks and exchanges) in cultured Chinese hamster lung fibroblastic cells (CHL cells). To clarify whether the induction of chromosome-type aberrations is mediated by stabilization of the cleavable complex, the present study investigated (1) the correlation between the induction of chromosome-type aberrations and the amount of cleavable complex formed; and (2) the ATP dependence of the Topo-II inhibitor-induced chromosome-type aberrations due to the ATP requirement of cleavable complex formation by Topo-II. First, in cells treated with the Topo-II inhibitors, (etoposide, adriamycin) and aclarubicin, an antagonist of the inhibitor of cleavable complex formation, the frequency of chromosome-type aberrations decreased dose-dependently with aclarubicin, in contrast to an increase of chromatid-type aberrations. The formation of the cleavable complex was further established by a proteinase K/SDS precipitation assay for cleaved double-strand DNA in a cell-free system and in CHL cells. Results from both experiments showed that aclarubicin caused a dose-dependent suppression of the accumulation of the cleavable complex induced by etoposide, which corresponded particularly well to the reduction of chromosome-type aberrations in etoposide-treated cells. In ATP-depleted cells simultaneously treated with etoposide and dinitrophenol (DNP), chromosome-type aberrations were reduced as compared with DNP-untreated cells, in contrast to an increase of chromatid exchanges in the cells. This means that etoposide-induced chromosome-type aberrations in ATP-depleted cells may be attributable to incompleteness of Topo-II activities to form DNA double-strand breaks. The present findings indicate that the stabilization of the cleavable complex on Topo-II is closely associated with the induction of chromosome-type aberrations.

2-Acylimino-3H-thiazoline derivatives : A novel template for platelet GPIIb/IIIa receptor antagonists

In the course of our research for the low-molecular weight RGD peptide mimics, we have found that a rigid 2-acylimino-3H-thiazoline structure is suitable for the peptide backbone mimics. Introduction of amidinophenyl and beta-alanine moiety as arginine and aspartic acid side-chain surrogates to this backbone mimic resulted in a highly potent fibrinogen receptor antagonist 2-(4-amidinobenzoylimino)-3,4-dimethyl-N-(2-carboxyethyl)-3H-thiazoline-5-carboxamide (7c), namely PS-028 (Ki = 46.5 +/- 5.8 microM).

Mechanism of action of aragusterol a (YTA0040), a potent anti-tumor marine steroid targeting the G1 phase of the cell cycle

Aragusterol A (YTA0040), isolated from the Okinawan marine sponge of the genus Xestospongia, is a potent anti‐tumor marine steroid that possesses a unique structural component. This compound showed broad‐spectrum anti‐proliferative activity against a panel of 14 human cancer cell lines (IC50 = 0.01–1.6 μM). P‐glycoprotein–mediated, multidrug‐resistant cells showed cross‐resistance to YTA0040 cells, whereas cisplatin‐resistant non‐small‐cell lung‐cancer (NSCLC) sublines showed a collateral sensitivity to YTA0040. In transplantable murine tumor models, YTA0040 displayed a broad spectrum and high degree of anti‐tumor activity when administered i.p. or p.o. (life span T/C = 135–234%). In P388 murine leukemia cells, YTA0040 caused dose‐ and time‐dependent suppression of nucleic acid and protein synthesis, with protein synthesis being more potently and rapidly inhibited than nucleic acid synthesis. Flow‐cytometric analysis revealed that YTA0040 blocked the entry of human NSCLC‐derived A549 cells into S phase, leading to arrest in the G1 phase of the cell cycle. Western blot analysis demonstrated that YTA0040 caused a dose‐dependent decrease in the levels of expression of hyperphosphorylated pRb and cyclin A in A549 cells. The level of p53 protein expression was decreased by YTA0040 treatment. A higher concentration of YTA0040 down‐regulated the levels of expression of CDK2, CDK4, cyclin D1 and cyclin E. These findings indicated that YTA0040 arrested human NSCLC cells in late G1 phase of the cell cycle through inhibition of pRb phosphorylation. Inhibition of pRb phosphorylation by YTA0040 resulted from down‐regulation of levels of expression of the CDKs and cyclins involved in the G1/S transition and not from induction of p53 and/or the CDK inhibitor p21. Int. J. Cancer 88:810–819, 2000.

Scratching behavior in spontaneous- or allergic contact-induced dermatitis in NC/Nga mice.

Abstract:  NC/Nga mice have pathological and behavioral features similar to those seen in human atopic dermatitis. There are two known dermatitis models in NC/Nga mice, one being spontaneous‐induced dermatitis under conventional conditions and the other 2,4,6‐trinitrochlorobenzene (TNCB)‐induced allergic contact dermatitis. However, there are significant differences in time course on development of dermatitis. We studied the role of scratching behavior (sign of itch) on the development of dermatitis on spontaneous‐ and TNCB‐induced dermatitis. We measured scratching counts, transepidermal water loss (TEWL), and skin inflammation score, under conventional conditions or by applying 5% TNCB once a week for 6 weeks in NC/Nga mice. In spontaneous‐induced dermatitis, scratching counts increased with the passage of time. The scratching counts were significantly increased only 1 week after housing the mice under conventional conditions, but no changes were observed in cases of TNCB‐induced dermatitis. In spontaneous‐induced dermatitis, TEWL and skin‐inflammation score were gradually increased, time‐dependently. On the other hand, in TNCB‐induced dermatitis, these dependent values rapidly increased and reached a maximum only after 24 h TNCB application. These data suggest that pathogenesis of spontaneous‐ and allergic contact‐induced dermatitis was clearly different. It will be of major interest to identify the pruritic mediators causing profound scratching behavior and scratching‐induced aggravation of inflammation in the spontaneous‐induced dermatitis, as opposed to the inflammatory mediators that cause contact allergic dermatitis without major scratching.