Expanded polyglutamine in the Machado-Joseph disease protein induces cell death in vitro and in vivo.

Recently, we identified a novel gene, MJD1, which contains an expanded CAG triplet repeat in Machado-Joseph disease. Here we report the induction of apoptosis in cultured cells expressing a portion of the MJD1 gene that includes the expanded CAG repeats. Cell death occurs only when the CAG repeat is translated into polyglutamine residues, which apparently precipitate in large covalently modified forms. We also created ataxic transgenic mice by expressing the expanded polyglutamine stretch in Purkinje cells. Our results demonstrate the potential involvement of the expanded polyglutamine as the common aetiological agent for inherited neurodegenerative diseases with CAG expansions.

Failure of parturition in mice lacking the prostaglandin F receptor.

Mice lacking the gene encoding the receptor for prostaglandin F2alpha (FP) developed normally but were unable to deliver normal fetuses at term. Although these FP-deficient mice showed no abnormality in the estrous cycle, ovulation, fertilization, or implantation, they did not respond to exogenous oxytocin because of the lack of induction of oxytocin receptor (a proposed triggering event in parturition), and they did not show the normal decline of serum progesterone concentrations that precedes parturition. Ovariectomy at day 19 of pregnancy restored induction of the oxytocin receptor and permitted successful delivery in the FP-deficient mice. These results indicate that parturition is initiated when prostaglandin F2alpha interacts with FP in ovarian luteal cells of the pregnant mice to induce luteolysis.

Prostaglandin D2 as a mediator of allergic asthma.

Allergic asthma is caused by the aberrant expansion in the lung of T helper cells that produce type 2 (TH2) cytokines and is characterized by infiltration of eosinophils and bronchial hyperreactivity. This disease is often triggered by mast cells activated by immunoglobulin E (IgE)-mediated allergic challenge. Activated mast cells release various chemical mediators, including prostaglandin D2 (PGD2), whose role in allergic asthma has now been investigated by the generation of mice deficient in the PGD receptor (DP). Sensitization and aerosol challenge of the homozygous mutant (DP-/-) mice with ovalbumin (OVA) induced increases in the serum concentration of IgE similar to those in wild-type mice subjected to this model of asthma. However, the concentrations of TH2 cytokines and the extent of lymphocyte accumulation in the lung of OVA-challenged DP-/- mice were greatly reduced compared with those in wild-type animals. Moreover, DP-/- mice showed only marginal infiltration of eosinophils and failed to develop airway hyperreactivity. Thus, PGD2 functions as a mast cell-derived mediator to trigger asthmatic responses.

Blocking effects of synthetic trypsin inhibitor (camostat) on pancreatic carcinogenesis in hamsters initiated with N-nitrosobis(2-oxopropyl)amine.

The effects of concomitant administration of a synthetic trypsin inhibitor (camostat) on pancreatic carcinogenesis in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP) were investigated. Thirty-two female Syrian golden hamsters were given weekly 10 mg/kg s.c. injections of BOP for 5 weeks while simultaneously receiving a 500 ppm camostat diet (BOP + camostat group). Additional groups of 30 animals received either the s.c. injections of BOP (BOP group), or the 500 ppm camostat diet (camostat group) during the same 5-week period. Thirty weeks after the first BOP administration, the incidence of pancreatic adenocarcinomas in the BOP + camostat group was significantly lower than in the group administered BOP only (p < 0.05). Similarly, the total numbers of pancreatic adenocarcinomas or dysplastic lesions were significantly decreased in the BOP + camostat group as compared with the BOP group (p < 0.01). None of the animals receiving camostat alone developed any adenocarcinomas or dysplastic lesions of the pancreas. The results of the present experiments clearly show that camostat can inhibit induction of hamster pancreatic ductal neoplasms when administered simultaneously with BOP.

Involvement of cysteinyl leukotrienes in biphasic increase of nasal airway resistance of antigen-induced rhinitis in guinea pigs.

We examined the effect of a specific cysteinyl leukotriene (LT) receptor antagonist, 4-oxo-8-[4-(4-phenylbutoxy)benzoylamino]-2-(tetrazol-5-yl)-4 H-1-benzopyran hemihydrate (pranlukast), on a novel model of allergic rhinitis induced by repeated intranasal ovalbumin challenge in actively sensitized guinea pigs. Repeated intranasal ovalbumin challenge caused a biphasic increase of nasal airway resistance, peaking 0.5 and 4 h after the final challenge. The early-phase response was accompanied by an increase in sneezing and nasal secretion, while that in the late phase was associated with edema and eosinophil infiltration of the nasal mucosa. Analysis of nasal lavage fluid showed that cysteinyl LTs increased in both phases. Pranlukast, when administered 1 h before every ovalbumin challenge, dose-dependently suppressed the increase of nasal airway resistance in the early- and late phase with evidence of histopathological improvements in the late phase. Pranlukast, however, failed to suppress sneezing and nasal secretion. We suggest that cysteinyl LTs play an important role in allergic rhinitis especially in the nasal obstruction due to edema of the nasal mucosa membrane.

Intratumoral tumor necrosis factor induction and tumor growth suppression by ONO-4007, a low-toxicity lipid A analog.

BACKGROUND: ONO-4007 is a lipid A analog with low toxicity. MATERIALS AND METHODS: The antitumor activity and tumor necrosis factor (TNF)-inducing activity of ONO-4007 were compared with those of lipopolysaccharide (LPS) in WKAH rats bearing KDH-8 hepatoma cells. RESULTS: Weekly injections of ONO-4007 (3 and 10 mg/kg i.v.) suppressed tumor growth, but LPS (0.01 and 0.1 mg/kg i.v.) did not. A single injection of ONO-4007 (3 and 10 mg/kg i.v.) into tumor-bearing rats induced higher levels of endogenous TNF production in tumor tissues than LPS (0.001, 0.01 and 0.1 mg/kg i.v.). Repeated injections of LPS caused a reduction of TNF production in tumor tissues, whereas the reduction by ONO-4007 was less remarkable than that by LPS. Intratumoral injections of anti-rat TNF-alpha antibody attenuated the antitumor effect of ONO-4007. CONCLUSION: The antitumor effect of ONO-4007 is more pronounced than that of LPS and the effect is mediated by TNF produced in tumor tissues.

Effects of nitrofurazone on spermatogenesis and reproductive toxicity in male rats--part of a collaborative work to determine optimal administration period and endpoints.

To determine an appropriate administration period and sensitive end-points for the evaluation of effects on male fertility, male Sprague-Dawley rats were orally given nitrofurazone, a model compound, at doses of 12.5, 25, or 50 mg/kg/day for 4 weeks, or at doses of 12.5 or 25 mg/kg/day for 9 weeks before mating with untreated females. Copulation and fertility indices were decreased, and pregnancy did not result at doses of 25 mg/kg/day and over with both dosing periods. An increase in preimplantation loss, and decreases in implants and live fetuses were observed with 12.5 mg/kg/day after 9-weeks dosing. However, no reproductive endpoints were affected by the same dose level for 4-weeks. Sperm head count was reduced at doses of 25 mg/kg/day and over with both dosing periods. Histopathology revealed tubular degeneration and interstitial cell hyperplasia at doses of 25 mg/kg/day and over after both periods of dosing. Moreover, failure of spermiation in tubular epithelia was also detected in the 12.5 mg/kg groups. These results suggest that 4-weeks premating exposure is sufficient for evaluation of the effects of nitrofurazone on mate fertility, and the most sensitive endpoint in this 4-week premating-dose study is a histopathological change.

Spontaneous tumors in F-344/Jcl rats.

The age-related incidence of spontaneously occurring neoplastic and non-neoplastic lesions in untreated F-344/Jcl rats, used as controls in carcinogenicity testing, were studied from the histological examination of tissues from 469 males and 354 females. The incidence of spontaneous tumors was 83.2% in the males and 71.2% in the females. The most common neoplasms were leukemia (males: 24.3%, females: 24.0%), pituitary adenoma (males: 16.0%, females: 45.2%), pheochromocytoma (males: 14.7%, females: 7.3%), testicular interstitial cell tumor (males: 79.1%), and uterine endometrial stromal polyp(females: 16.4%). The incidence of other tumors of almost all the organs and/or tissues was low. Non-neoplastic lesions generally increased with advancing age of the animals.

[Toxicity studies of landiolol hydrochloride (ONO-1101) (1). Single intravenous toxicity study in rats and dogs].

Single dose toxicity studies of landiolol hydrochloride (ONO-1101), a novel ultra short acting beta-blocker, were conducted in Sprague-Dawley (SD) rats and beagle dogs. ONO-1101 was administered intravenously at a dose level of 37.5, 75, 150 or 300 mg/kg to rats of both sexes and 25, 50 or 100 mg/kg to male dogs. In the rat study, 5/6 males in the 150 mg/kg group and all animals in the 300 mg/kg group died during or right after administration. Survivors in the 150 mg/kg group showed temporal hypoactivity, bradypnea, dyspnea, tremor, loss of righting reflex and reddish lacrimation up to 5 min after injection. One male in the 150 mg/kg group had a tendency of suppression on body weight gain. No effects on clinical signs and body weight gain were seen in the 75 mg/kg group or lower. Necropsy findings showed only red tear in the majority of the decedents. In the dog study, all animals died within 6 min after administration in the 100 mg/kg group, showed ataxic gait, rolling and tachypnea followed by bradypnea and gasping/apnea. Incontinence of urine, defecation and vocalization were also seen in each one of two animals before death. Temporal hypoactivity was seen 1 min after administration in the 50 mg/kg group. No clinical signs were seen in the 25 mg/kg group. ONO-1101 did not affect bodyweight or food consumption. Necropsy findings of the decedents showed no abnormalities. It is indicated that the minimum lethal doses are 150 mg/kg in rats and 100 mg/kg in dogs.

[Reproductive and developmental toxicity studies of landiolol hydrochloride (ONO-1101) (3). Teratogenicity study in rabbits].

A teratogenicity study of landiolol hydrochloride (ONO-1101), a novel ultra short acting beta-blocker, was conducted in KAR:NZW rabbits. ONO-1101 was administered intravenously at a dose level of 0 (control), 25, 50 or 100 mg/kg/day to pregnant rabbit from day 6 to 18 of gestation to examine the effects on dams and fetuses. Death occurred on 3 animals in the 100 mg/kg/day group and one animal in the 50 mg/kg/day group during the treatment period. Hypoactivity, bradypnea/apnea and clonic convulsion after administration was observed in animal dosed 100 mg/kg/day. No maternal effects were seen on body weight, food consumption, necropsy findings or organ weights. External, skeletal and visceral findings revealed no adverse effects of ONO-1101 on fetuses. From the above results, it is estimated that the no-toxic dose level of ONO-1101 under these experimental conditions is 25 mg/kg/day for dams and 100 mg/kg for fetuses.

[Reproductive and developmental toxicity studies of landiolol hydrochloride (ONO-1101) (4). Perinatal and postnatal study in rats].

A perinatal and postnatal study of landiolol hydrochloride (ONO-1101), a novel ultra short acting beta-blocker, was conducted in Sprague-Dawley(SD) rats. ONO-1101 was administered intravenously at a dose level of 0 (control), 25, 50 or 100 mg/kg/day from day 17 of gestation to day 20 after parturition to examine the effects on pregnancy, delivery, lactation and the effects on postnatal growth and development of offspring. In the 100 mg/kg/day group, hypoactivity, reddish lacrimation, clonic convulsion and bradypnea/apnea were observed after administration and 5 animals died in the treatment period, and body weight on day 21 and food consumption on day 14-21 of dam at weaning were lower than control group. In the 50 mg/kg/day group, reddish lacrimation was occasionally seen in some animals. ONO-1101 had no effects on pregnancy, delivery, lactation and necropsy findings or organ weights of dams. In the 100 mg/kg/day group, viability of offspring on day 4 after birth decreased and body weight gain of the suckling suppressed, but those changes recovered after weaning. On the skeletal examination of offspring culled on day 4 after birth, decrease in the mean number of osiffied phalanges of hindpaw and increase in the incidence of unossified talus bone were seen in the 100 mg/kg/day group, however, no delay of ossification was found form the weanling. There were no influence of ONO-1101 on external differentiation, functional, behavioral or learning abilities or reproductive performance in offspring. From the above results, it is estimated that the no-toxic dose level of ONO-1101 under these experimental conditions is 50 mg/kg/day for dam and offspring.

[Reproductive and developmental toxicity study of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na (1). Fertility study in rats].

Fertility study of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na), a novel neutrophil elastase inhibitor, was conducted in Sprague-Dawley (SD) rats. ONO-5046.Na was administered intravenously at doses of 18.75, 37.5 and 75 mg/kg/day to male rats from 64 prior to mating, through the mating period and until necropsy, and to female rats from 15 days prior to mating until Day 7 of gestation, in order to examine its effects on fertility and reproductive performance of males and females and the development of their fetuses. There were no changes attributable to ONO-5046.Na in general signs, body weight, food consumption or autopsy findings in males and females. No drug-related changes were observed in estrous cycles, copulation and fertility indices in males and females. Pituitary weight of dams was decreased in each of the ONO-5046.Na treated groups, but no histopathological changes were observed in the pituitary. In the cesarean section findings in dams, ONO-5046.Na had no effects on the number of corpola lutea, the number of live fetuses, the implantation ratio, the resorbed and dead fetus ratio, fetal or placental weight, or the incidences of external, skeletal or visceral anomalies of the fetuses. From these results, it is considered that the NOAEL of ONO-5046.Na is 75 mg/kg/day for general and reproductive toxicity in males and females and for developmental toxicity in their fetuses.

[Reproductive and developmental toxicity study of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na) (2). Study for effects on pre- and postnatal development in rats, including maternal function].

Prenatal and postnatal toxicity of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na), a novel inhibitor of human neutrophil elastase, was studied in Sprague-Dawley (SD) rats. ONO-5046.Na was injected intravenously at doses of 0, 18.75, 37.5 and 75 mg/kg/day to pregnant rats from day 7 of pregnancy to day 20 after delivery. All pregnant rats were allowed to deliver naturally for postnatal examination of their offspring. No adverse effects on dams were observed in clinical signs, body weight change, food consumption, pregnant, delivery or lactating performances. ONO-5046.Na did not affect the postnatal development of offspring, including birth index, survival index, physical and functional development, motor activity, emotionality, learning ability and reproductive performance. From these results, it is considered that the NOAEL of ONO-5046.Na is 75 mg/kg/day for dams and their offspring.

[Toxicity study of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na) (4). 6-month repeated dose intravenous toxicity study in rats with 1-month recovery test].

A 6-month repeated dose toxicity study with 1-month recovery test of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na), a novel neutrophil elastase inhibitor, was conducted in Sprague-Dawley (SD) rats. The rats of both sexes were administered ONO-5046.Na intravenously at a daily dose of 0 (vehicle control), 18.75, 37.5 or 75 mg/kg. ONO-5046.Na did not affect clinical signs, body weight, food consumption, opthalmology, urinalysis, hematology, blood chemistry, organ weight, necropsy or histopathology at any dose. These results indicate that the NOAEL of ONO-5046.Na in rats is 75 mg/kg/day for both sexes in this study.

Megakaryocytic emperipolesis in the rat bone marrow induced by lipopolysaccharide.

Megakaryocytic emperipolesis was observed in the rat bone marrow when lipopolysaccharide (LPS) was administrated intravenously at a daily dose of 0.5 mg/kg body weight for 4 weeks. Emperipolesis was significantly increased in the LPS-treated group (3.54%) as compared with the control group (1.35%). Histologically, several types of hematocytes were engulfed within megakaryocytes, but the host cells and entering hematocytes showed no degenerative alterations. Morphologically, most megakaryocytes showing emperipolesis were classified into the mature type indicating high platelet producting ability but platelet counts in the peripheral blood were markedly reduced in the LPS-treated group (63.7 x 10(4)/mm3) as compared with the control group (128.4 x 10(4)/mm3). These results suggest that the emperipolesis is a phenomenon closely related to the marked reduction in the number of platelets and accelerated platelet production of megakaryocytes.

Morphological observations of megakaryocytic emperipolesis in the bone marrow of rats treated with lipopolysaccharide.

A morphological examination was performed on megakaryocytic emperipolesis containing neutrophils in the rat bone marrow induced by intravenous administration of lipopolysaccharide (LPS). Ultrastructurally, the engulfed neutrophils appeared to lie in the demarcation membrane system (DMS) of megakaryocytes. Intact neutrophils generally migrated from DMS into the lumen of vascular sinus across the endothelium of sinus wall. However, some neutrophils showed characteristics of apoptosis, and DNA fragmentation was detected in these cells by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling method. Thus, the cell death of neutrophils engulfed by megakaryocytes was thought to be apoptosis. The present results suggest that megakaryocytes contribute to random selection of an excess of neutrophils in the bone marrow of rats treated with LPS.

Chordoma with osseous element in a F344 rat.

Chordoma occurred in the sacrococcygeal region of the vertebral column of a 97-week old male control F344 rat. Macroscopically, it was a mass (20 mm in diameter) protruding into the retroperitoneum and the cut surface was white to dark-reddish and showed a gelatinous appearance. Histologically, the tumor was composed of physaliphorous and stellate cells growing in nests or cords and divided into lobules by a thin fibrous stroma. The tumor cells locally invaded adjacent tissues, but no metastasis to other organs was noted. Centrally, the tumor often had islands of bone that entrapped physaliphorous cells and the bone tissue consisted of well-differentiated osteocytes. Based on these findings, this tumor was diagnosed as chordoma with osseous element.

Adhesion molecule LFA-1/ICAM-1 influences on LPS-induced megakaryocytic emperipolesis in the rat bone marrow.

To investigate the effect of adhesion molecules on the occurrence of megakaryocytic emperipolesis of neutrophils, we examined the expression of lymphocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) in the bone marrow of lipopolysaccharide (LPS)-treated rats (experiment I) and the occurrence of megakaryocytic emperipolesis in anti-LFA-1 antibody-treated rats (experiment II). In experiment I, rats were injected with LPS intravenously at a daily dose of 0.5 mg/kg for 3 days. ICAM-1 was intensely stained on megakaryocytes in LPS treated rats, as detected by flow cytometric analysis. ICAM-1 was immunostained in the megakaryocytes showing emperipolesis. LFA-1 was immunostained in the neutrophils engulfed by megakaryocytes. In experiment II, rats received anti-LFA-1 antibody intravenously at a single dose of 3 mg/kg. One hour after treatment, rats were given LPS intravenously as a single dose of 0.5 mg/kg. The incidence of megakaryocytic emperipolesis was markedly lower in the anti-LFA-1 antibody + LPS group than in the LPS alone group. These findings suggest that the occurrence of megakaryocytic emperipolesis is partly dependent on adhesion molecules via LFA-1/ICAM-1.

Restoration of immune responses in tumor-bearing mice by ONO-4007, an antitumor lipid A derivative.

ONO-4007, a synthetic lipid A derivative, has been found to exhibit potent antitumor activity in several animal models. In the present study, we examined the effects of ONO-4007 on delayed-type hypersensitivity (DTH) reaction and antibody production in Meth A sarcoma-bearing BALB/c mice. The DTH reaction to sheep red blood cells (SRBC) and the IgG production against keyhole limpet hemocyanin (KLH), were depressed in tumor-bearing mice as well as in normal mice given Mitomycin C (MMC). However, ONO-4007 restored these immune responses to normal levels. In addition, in vitro studies showed that ONO-4007 induced the production of tumor necrosis factor-alpha (TNF-alpha) in splenic adherent cells of tumor-bearing mice more than those in normal mice. Though ONO-4007 alone had little effect on the induction of IL-2 production in spleen cells, it augmented the Concanavalin A (Con A)-stimulated IL-2 production. Moreover, ONO-4007 had a mitogenic effect on spleen cells. These results suggest that ONO-4007 may improve immunocompetence in tumor-bearing hosts and contribute to the induction of antitumor immunity and prevention of bacterial infections.

ONO-4007, an antitumor lipid A analog, induces tumor necrosis factor-alpha production by human monocytes only under primed state: different effects of ONO-4007 and lipopolysaccharide on cytokine production.

ONO-4007 is a synthetic lipid A analog that exhibits strong antitumor activity in several animal models via intratumoral production of tumor necrosis factor (TNF). In the present study the cytokine-inducing effect of ONO-4007 was investigated in human monocytes that were freshly isolated or had been incubated for 3 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor. ONO-4007 induced slight production of TNF-alpha, Interleukin (IL)-1 beta, IL-6 and IL-12 in fresh monocytes but strongly induced TNF-alpha production in GM-CSF-treated monocytes. Monocytes treated with macrophage colony-stimulating factor were also primed to produce TNF-alpha in response to ONO-4007. In the production of IL-1 beta, IL-6 and IL-12, GM-CSF did not show a priming effect. In contrast to ONO-4007, lipopolysaccharide (LPS) induced significant amounts of all these cytokines in fresh monocytes. In whole blood, ONO-4007 failed to induce TNF-alpha, whereas LPS and LA-15-PP (Escherichia coli-type lipid A) strongly induced TNF-alpha production. In the GM-CSF-treated monocytes, both elimination of serum from the culture medium and anti-CD14 antibody treatment attenuated LPS-induced TNF-alpha production but not ONO-4007-induced TNF-alpha production. This study shows that ONO-4007 activates human monocytes/ macrophages to release TNF-alpha only in a primed state and suggests that ONO-4007 would activate these cells via different pathways from LPS. These differences could mean that ONO-4007 has potent antitumor activity with lower toxicity than LPS.