Thirteen-week Intravenous Toxicity Study of a Novel Humanized Anti-Human Death Receptor 5 Monoclonal Antibody, CS-1008, in Cynomolgus Monkeys

CS-1008, a humanized monoclonal antibody that is agonistic to human death receptor 5, was intravenously administered to cynomolgus monkeys twice a week for 13 weeks at 3 different dose levels (5, 15 and 42 mg/kg) in order to evaluate its potential toxicity. A control group received phosphate buffered saline containing 0.01% polysorbate 80. Each of the 4 groups consisted of 3 male and 3 female cynomolgus monkeys. No animal in any group died during the dosing period. No toxic changes in clinical signs, food consumption, body weight, electrocardiography, ophthalmology, urinalysis, hematology, blood chemistry, gross pathology, organ weights or histopathology were noted in any group during the dosing period. In the toxicokinetic analysis, the values for the maximum concentration of CS-1008 in plasma and the area under the curve generally increased with increasing dose. No clear differences in the toxicokinetic parameters or profiles were observed between the sexes. Development of anti-CS-1008 antibodies was not detected in any sample. The no-observed adverse-effect level (NOAEL) of CS-1008 in cynomolgus monkeys under the conditions of this study was concluded to be 42 mg/kg in both sexes, when administered intravenously twice a week for 13 weeks. This study supports the development of CS-1008 as a therapeutic biopharmaceutical.     

Effect of a Large Dose of Di (2-ethylhexyl) phthalate (DEHP) on Hepatic Peroxisome in Cynomolgus Monkeys (Macaca Fascicularis)

To elucidate the effect of a large dose of di (2-ethylhexyl) phthalate (DEHP), a plasticizer and peroxisome proliferator-activated receptor-α (PPARα) agonist, on hepatic peroxisomes, we orally administered 1,000 mg/kg/day, once daily, to 3 male and 4 female cynomolgus monkeys for 28 days consecutively. Light-microscopic and electron microscopic examinations of the liver were carried out in conjunction with measurement of the hepatic fatty acid β-oxidation system (FAOS), carnitine acetyltransferase (CAT) and carnitine palmitoyltransferase (CPT) activities, which are peroxisomal and/or mitochondrial enzyme activities. Electron microscopically, enlargement of the mitochondria was observed with lamellar orientation of the cristae along the major axis. Although the number of peroxisomes showed a tendency to increase when compared with those in a biopsied specimen before treatment, no abnormality in morphology was observed. A slight increase in CPT activity was noted at termination. No changes were noted in hepatic FAOS or CAT activity. In conclusion, although repeated oral treatment of cynomolgus monkeys with a large dose of DEHP induced a subtle increase in the numbers of peroxisomes with slight enlargements of the mitochondria, this low-sensitivity response to peroxisome proliferators in cynomolgus monkeys was considered to be closer to the response in humans than that in rodents.     

Cytoarchitectural differences of myoepithelial cells among goat major salivary glands

Previously, the distribution of myoepithelial cells (mecs) in the salivary glands was studied by both immunohistochemistry, and transmission electron microscopy; however, little was elucidated concerning their morphological features, especially in goats. This study was performed to investigate the correlation between the cytoarchitecture of the mecs in goat major salivary glands (parotid, mandibular, and sublingual glands) and the nature of the saliva secretion. The cytoarchitectural features of the mecs were examined by scanning and transmission electron microscopy as well as immunohistochemically. The secretory endpieces in the parotid gland are of the pure serous type, but in both the mandibular and sublingual glands they are of the mixed type. In all studied glands, the intercalated ducts were covered by mecs which, unlike the large stellate cells that surrounded the secretory endpieces, were spindle-shaped with few cytoplasmic processes. Interestingly, the mecs were found to bulge on the basal surfaces of the serous acini and intercalated ducts in all glands and to be in close contact to the seromucous tubules surface in the mandibular and sublingual glands forming a continuous network around it. In conclusion, the differences in the degree of development of the mecs as well as the number of their cytoplasmic processes may be correlated with the nature of the secretion and the number of the secretory granules. Thus these observations may have some relevance in the diagnosis of atrophy and pathogenic conditions of these glands.     

Interaction between myostatin and extracellular matrix components

Myostatin, a member of the TGF-β superfamily, is a negative regulator of skeletal muscle mass. We have recently demonstrated that decorin binds to myostatin in vitro , and that immobilized decorin within the collagen matrix prevents myostatin-mediated inhibition of myoblast proliferation. However, little is known about other ECM molecules that bind to myostatin and modulate its activity. Thus, in the present study, we investigated the interaction of several other ECM molecules with myostatin. We here show that fibromodulin, fibronectin and laminin bind to myostatin in the presence of Zn²⁺ with a dissociation constant ( K_D ) of 10⁻¹⁰∼10⁻⁸ mol/L. Fibromodulin shows the highest affinity for myostatin among them. These results suggest that these ECM molecules may modulate myostatin activity like decorin does.     

Pathogenic characters of Japanese potato strains of Ralstonia solanacearum

Ralstonia solanacearum (Rs) strains in phylotypes I and IV isolated from potato in Japan were investigated for pathogenicity on potato, tomato, eggplant, Solanum integrifolium, tobacco, groundnut, and pumpkin. The strains were divided into 17 types based on differences in their pathogenicity on the tested plants. Particularly, the pathogenicity of most phylotype I strains on eggplant was distinctly different from that of the phylotype IV strains. When nine potato varieties (included two breeding lines) were inoculated with several Rs strains, phylotype IV strains were highly virulent on the breeding lines that are regarded as resistant to phylotype I strains.     

Orally administered rutin inhibits the gene expression of Th2 cytokines in the gut and lung in aged mice

Rutin is one of the flavonoids derived from plants such as buckwheat and is well known as a powerful antioxidant. To determine whether dietary rutin could modulate mucosal immunity, we examined the gene expression of Th1/Th2 cytokines and the receptors in the gut and lung. Aged (18 months old, 18 M) C3H/HeN female mice were orally administered rutin for 10 days. The small intestine and lung were taken and analyzed by real-time PCR for gene expression. Interleukin (IL)-13 and IL-13Rα2 gene expression was significantly low (P<0.05 respectively) in the small intestine of aged rutin-fed mice. Meanwhile, there was no change in interferon γ gene expression between control and rutin-fed mice. IL-13 gene expression was also downregulated in the lung. To examine the mechanism of the inhibitory effect of rutin on Th2 cytokines in aged mice, intestinal nitric oxide synthase (NOS) expression was evaluated. Rutin inhibited inducible NOS (NOS2) gene expression, but not neuronal NOS and endothelial NOS. Gene analysis of cells collected from the small intestine by laser capture dissection revealed that NOS2 expression was significantly inhibited in crypt regions. Thus, rutin might be effective against a Th2-dominant profile through NOS2 inhibition in aged mice.     

Cynomolgus macaque CYP4 isoforms are functional, metabolizing arachidonic acid

Cytochrome P450 (CYP) is important for metabolism of not only xenobiotics such as drugs, but also endogenous compounds including arachidonic acids. CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 have been identified in cynomolgus macaque, an animal species widely used for investigation of drug metabolism due to its evolutionary closeness to human. However, their metabolic functions have not been investigated. In this study, proteins were heterologously expressed in Escherichia coli and characterized by metabolic assays using arachidonic acids as substrates that are metabolized by CYP4 isoforms in human. The results showed that all four CYPs metabolized arachidonic acids. Therefore, cynomolgus macaque CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 are functional enzymes.     

Absorption and emission of CO2 by ponded water of a paddy field

In the daytime, the CO₂ concentration in the air close to the water surface of a ponded paddy field was lowest and it increased with the distance above the water surface, while an inverse relation was observed in the nighttime. On the other hand, the pH of the ponded water changed significantly throughout a day and was expected to affect atmospheric CO₂ in the vicinity of the water surface, because the solubility of CO₂ in water depends on the pH. In this study, we investigated the relationship between the changes in the pH of the ponded water and the response of the CO₂ concentration in the air above the water. The pH of the ponded water of the paddy field increased in the daytime and decreased in the nighttime, so that the water was alkaline in the daytime and weakly acidic in the nighttime. We found that the daily changes in the atmospheric CO₂ concentration gradient almost corresponded to the daily changes in pH. The increase of the pH of the ponded water in the daytime was due to the absorption of dissolved CO₂ by photosynthetic bacteria and micro-algae within the ponded water. Furthermore, we compared the pH with RpH, defined as the pH at which the CO₂ concentration of the water is in equilibrium with that of the air, to determine whether CO₂ was absorbed by or emitted from the ponded water. In the daytime, the pH value of the ponded water was higher than that of the RpH, and the water could therefore absorb CO₂ , whereas during the nighttime, since the pH value of the ponded water was lower than that of the RpH, the water was expected to emit CO₂. These results show that the ponded water absorbed CO₂ from the air above the water surface in the daytime and emitted CO₂ in the nighttime.