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.     

Establishment of an in vitro culture model to study milk production and the blood–milk barrier with bovine mammary epithelial cells

This study attempted to establish a culture model to recreate the milk production pathway in bovine mammary epithelial cells (BMECs). BMECs were isolated from Holstein cows (nonlactating, nonpregnant, and parous) and were stored by cryopreservation. To separate the apical and basolateral compartments, BMECs were cultured on a cell culture insert with a collagen gel in the presence of bovine pituitary extract and dexamethasone to induce milk production and tight junction (TJ) formation. The culture model showed the secretion of the major milk components, such as β‐casein, lactose, and triglyceride, and formed less‐permeable TJs in BMECs. Moreover, the TJs were distinctly separated from the apical and basolateral membranes. Glucose transporter‐1, which transports glucose into the cytoplasm through the basolateral membrane, localized in the lateral membrane of BMECs. Toll‐like receptor‐4, which binds to lipopolysaccharide in the alveolar lumen in mastitis, localized in the apical membrane. Beta‐casein was mainly localized near the Golgi apparatus and the apical membrane. Moreover, milk components were almost secreted into the upper chamber of the cell culture insert. These findings indicate that this model has clear cell polarity as well as in vivo and is effective to study of milk production and the blood–milk barrier in lactating BMECs.     

Energy partitioning in cultured juvenile Pacific bluefin tuna,Thunnus orientalis (Temminck & Schlegel, 1844)

The characteristics of dietary utilization, energy conversion efficiency, metabolic rate and energy partitioning were measured for cultured Pacific bluefin tuna (PBT) juveniles fed on an artificial diet. Thirty‐one juveniles (1.1 ± 0.3 g BW) were stocked into each of two 2500 L tanks to measure oxygen consumption (?O₂), swimming speed, digestibility and growth performance. ?O₂ elevated until 2.5 ± 0.3 times of pre‐feeding level within 1.5 h after feeding, except for the first feeding, and returned to pre‐feeding level within 3 h. Swimming speed fluctuation was corresponded with the ?O₂ fluctuation, and both parameters were stable from 02:00 to 06:00 and also during the whole day for starved fish. These indicate that feeding has strong influence on their metabolic rate. Energy partitioning for faecal, urinary and branchial, heat increment and voluntary activity, standard metabolism, and retained energy were calculated to be 17.2%, 5.9%, 14.9%, 41.3% and 20.7% of total ingested energy, respectively. The results indicate that, unlike other fish, juvenile PBT distribute large amount of energy for maintenance, which allows only a little proportion of ingested energy available for growth.     

Simultaneous steroids measurement in dogs with hyperadrenocorticism using a column-switching liquid chromatography-tandem mass spectrometry method

We developed an analytical method using an on-line column-switching liquid chromatography with triple quadrupole mass spectrometry (LC/MS/MS) for quantifying multiple steroids in serum. Using the developed method, we evaluated the serum concentration of nine steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 21-deoxycortisol, deoxycorticosterone, progesterone, 17α-OH-progesterone and aldosterone) in dogs with hyperadrenocorticism (HAC). Serum was mixed with stable isotope internal standards and thereafter purified by the automated column-switching system. The limit of detection ranged 2–16 pg/ml for nine steroids. In the baseline samples, five steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, and 17α-OH-progesterone) were detected in all dogs. The concentrations of cortisone, 11-deoxycortisol, and 17α-OH-progesterone in dogs with HAC (n=19) were significantly higher those in dogs without HAC (n=15, P<0.02). After the adrenocorticotropic hormone stimulation test, six steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 17α-OH-progesterone, and deoxycorticosterone) were above the limit of quantification in all dogs. Cortisol, corticosterone, cortisone, and deoxycorticosterone concentrations of dogs with HAC were significantly higher than those of dogs without HAC (P<0.02). In addition, 11-deoxycortisol and 17α-OH-progesterone concentration was higher in dogs with HAC than in dogs without HAC (P=0.044 and P=0.048, respectively). The on-line column-switching LC/MS/MS would be feasible for measuring multiple steroids in dog serum. The results suggest that cortisone, 11-deoxycortisol, and 17α-OH-progesterone would be related to HAC. Further studies are warranted to assess the clinical feasibility of steroid profile in dogs with HAC.     

Relationship between Mucosal Barrier Function of the Oviduct and Intestine in the Productivity of Laying Hens

The mucosa of the intestine and oviduct of hens are susceptible to pathogens. Pathogenic infections in the mucosal tissues of laying hens lead to worsened health of the host animal, decreased egg production, and bacterial contamination of eggs. Therefore, better understanding of the mechanisms underlying mucosal barrier function is needed to prevent infection by pathogens. In addition, pathogen infection in the mucosal tissue generally causes mucosal inflammation. Recently, it has been shown that inflammation in the oviduct and intestinal tissue caused by disruption of the mucosal barrier function, can affect egg production. Therefore, it is vitla to understand the relationship between mucosal barrier function and egg production to improve poultry egg production. This paper reviews the studies on (1) oviductal mucosal immune function and egg production, (2) intestinal inflammation and egg production, and (3) improvement of mucosal immune function by probiotics. The findings introduced in this review will contribute to the understanding of the mucosal barrier function of the intestine and oviduct and improve poultry egg production in laying hens.     

Estimation of kelp forest, <Emphasis Type="Italic">Laminaria</Emphasis> spp., distributions in coastal waters of the Shiretoko Peninsula,Hokkaido, Japan,using echosounder and geostatistical analysis

Sustainable management of the kelp forests of the Shiretoko Peninsula, a World Natural Heritage site, is necessary due to kelp’s ecological and economic importance. The objectives of this study were to estimate the area of kelp forests and to clarify their spatial characteristics in coastal waters of the Shiretoko Peninsula. Data on the presence/absence and thickness of kelp forests were collected via acoustic observation on transects over about 80 km using an echosounder at 200 kHz. Acoustic data were geostatistically interpolated, and the areas covered by kelp forests were estimated. Differences in kelp distribution between the eastern and western sides of the peninsula were compared. The total area of kelp forest was 3.88 km² (eastern area: 3.49 km²; western area: 0.39 km²). The range of thickness of the kelp forests was 34–91 cm. Many kelp forests in the eastern area were thick (>78 cm) and distributed continuously, while kelp forests in the western area were sparsely distributed.     

Mitigation of nitrous oxide (N2O) emission from swine wastewater treatment in an aerobic bioreactor packed with carbon fibers

Mitigation of nitrous oxide (N₂O) emission from swine wastewater treatment was demonstrated in an aerobic bioreactor packed with carbon fibers (CF reactor). The CF reactor had a demonstrated advantage in mitigating N₂O emission and avoiding NOx (NO₃ + NO₂) accumulation. The N₂O emission factor was 0.0003 g N₂O‐N/gTN‐load in the CF bioreactor compared to 0.03 gN₂O‐N/gTN‐load in an activated sludge reactor (AS reactor). N₂O and CH₄ emissions from the CF reactor were 42 g‐CO₂ eq/m³/day, while those from the AS reactor were 725 g‐CO₂ eq/m³/day. The dissolved inorganic nitrogen (DIN) in the CF reactor removed an average of 156 mg/L of the NH₄‐N, and accumulated an average of 14 mg/L of the NO₃‐N. In contrast, the DIN in the AS reactor removed an average 144 mg/L of the NH₄‐N and accumulated an average 183 mg/L of the NO₃‐N. NO₂‐N was almost undetectable in both reactors.     

Evaluation of a recombinant measles virus expressing hepatitis C virus envelope proteins by infection of human PBL-NOD/Scid/Jak3null mouse

In this study, we infected NOD/Scid/Jak3null mice engrafted human peripheral blood leukocytes (hu-PBL-NOJ) with measles virus Edmonston B strain (MV-Edm) expressing hepatitis C virus (HCV) envelope proteins (rMV-E1E2) to evaluate the immunogenicity as a vaccine candidate. Although human leukocytes could be isolated from the spleen of mock-infected mice during the 2-weeks experiment, the proportion of engrafted human leukocytes in mice infected with MV (10³–10⁵ pfu) or rMV-E1E2 (10⁴ pfu) was decreased. Viral infection of the splenocytes was confirmed by the development of cytopathic effects (CPEs) in co-cultures of splenocytes and B95a cells and verified using RT-PCR. Finally, human antibodies against MV were more frequently observed than E2-specific antibodies in serum from mice infected with a low dose of virus (MV, 10⁰–10¹ pfu, and rMV-E1E2, 10¹–10² pfu). These results showed the possibility of hu-PBL-NOJ mice for the evaluation of the immunogenicity of viral proteins.     

Identification of bovine dendritic cell phenotype from bovine peripheral blood

Dendritic cells (DCs) are professional antigen presenting cells, which initiate primary immune responses and also play an important role in the generation of peripheral tolerance. There is no reliable method established for the isolation of bovine peripheral blood DCs, and furthermore, the phenotypes and the functions of bovine DCs are still not fully clear. In the present study, we have attempted to identify bovine peripheral blood DCs by negative-selection. In bovine peripheral blood mononuclear cells (PBMC), we have newly characterized the phenotype of DCs, which is CD11c+/CD172a+. These cells display features of myeloid type DCs. In the thymic medulla, CD11c+/CD172a+ cells were also present and CD1+/CD172a+ cells were additionally detected as a population of DCs. The data suggest that one of the bovine DCs phenotypes from PBMC is derived from myeloid lineages lacking a CD1 molecule, which then drift to several tissues, and that they then may express a CD1 molecule upon their functional differentiation.     

Nondestructive detection of cracks near the surface of wooden boards by dynamic heat dissipation

A method to detect cracks near the surface of wooden boards has been developed where the change in surface temperature of the wood sample is monitored using an infrared camera following momentary heating by a flash. Cylindrical holes simulating cracks were drilled into the wood samples, and blackbody paint was painted onto the surface of the samples to assist flash absorption. This method uses the dynamic heat transport process from the blackbody paint to the surface of the wood sample to the cracks over a short timescale. The theoretical foundations of the observation method were outlined, and the technique was verified in experiments with samples of Cercidiphyllum japonicum, Western red cedar, Japanese cedar, Balsa, and medium-density fiberboard. The developed technique is able to detect the presence of holes located near the surface of some samples. However, this method could not detect the presence of holes in Balsa nor holes located deeper than 1 mm from the surface of the sample. A theoretical analysis of these phenomena was provided to help interpreting them.