Close-up observations on the spawning behavior of a captive Japanese flying squid (Todarodes pacificus).

The spawning behavior of a Japanese flying squid (Todarodes pacificus) is described based on up-close observation of a captive female. The squid was first transferred from a 10-ton tank to a polystyrene plastic box containing 45 liters of seawater. About one hour later, the mantle-contraction rate increased rapidly, followed by a brief convulsion of the mantle and arms and a whitening of the body. The mantle contractions become shallow and rapid, and several seconds later, semitransparent jelly presumably from the nidamental glands emerged from the funnel and passed between the ventral pair of arms. Approximately 90 seconds after the egg mass first emerged, the female began ejecting oocytes through the funnel into the egg mass using rapid, powerful mantle contractions. Soon after the oocytes were ejected, translucent strands (presumably sperm) emanated from the buccal membrane. The female continued to eject oocytes for approximately two minutes, after which the mantle convulsed, and the mantle-contraction rate decreased slowly for about one minute until the contractions stopped. The squid died soon afterwards.

Observations on the spawning behavior, egg masses and paralarval development of the ommastrephid squid Todarodes pacificus in a laboratory mesocosm.

The spawning behavior of ommastrephid squids has never been observed under natural conditions. Previous laboratory observations of Japanese flying squid (Todarodes pacificus) suggest that pre-spawning females might rest on the continental shelf or slope before they ascend above the pycnocline to spawn, and that the egg masses might settle in the pycnocline. Here, two mesocosm experiments were conducted in a 300 m(3) tank that was 6 m deep to investigate this hypothesis. In the first experiment, a thermocline (2.5-3.5 m) was established in the tank by creating a thermally stratified (17-22°C) water column. In the second experiment, the temperature was uniform (22°C) at all depths. Prior to spawning, females did not rest on the tank floor. In the stratified water column, egg masses remained suspended in the thermocline, but in an unstratified water column, they settled on the tank bottom, collapsed and were infested by microbes, resulting in abnormal or nonviable embryos. Eleven females spawned a total of 18 egg masses (17-80 cm in diameter), indicating that females can spawn more than once when under stress. Paralarvae hatched at stage 30/31 and survived for up to 10 days, allowing us to observe the most advanced stage of paralarvae in captivity. Paralarvae survived after consumption of the inner yolk, suggesting they might have fed in the tank.

Vaccinia virus strain LC16m8 defective in the B5R gene keeps strong protection comparable to its parental strain Lister in immunodeficient mice.

BACKGROUND: Attenuated vaccinia virus strain, LC16m8, defective in the B5R envelope protein gene, is used as a stockpile smallpox vaccine strain in Japan against bioterrorism: the defect in the B5R gene mainly contributes to its highly attenuated properties. METHODS: The protective activity of LC16m8 vaccine against challenge with a lethal dose of vaccinia Western Reserve strain was assessed in wild-type and immunodeficient mice lacking CD4, MHC class I, MHC class II or MHC class I and II antigens. RESULTS: The immunization with LC16m8 induced strong protective activity comparable to that of its parent strain, Lister (Elstree) strain, in wild-type mice from 2 days to 1 year after vaccination, as well as in immunodeficient mice at 2 or 3 weeks after vaccination. These results implicated that the defect in the B5R gene hardly affected the potential activity of LC16m8 to induce innate, cell-mediated and humoral immunity, and that LC16m8 could be effective in immunodeficient patients. CONCLUSION: LC16m8 with truncated B5 protein has an activity to induce immunity, such as innate immunity and subsequent cell-mediated and humoral immunity almost completely comparable to the activity of its parental strain Lister.

Safety of attenuated smallpox vaccine LC16m8 in immunodeficient mice.

Freeze-dried live attenuated smallpox vaccine LC16m8 prepared in cell culture has been the sole smallpox vaccine licensed in Japan since 1975 and was recently recommended as a WHO stockpile vaccine. We evaluated the safety of recently remanufactured lots of LC16m8 using a series of immunodeficient mouse models. These models included suckling mice, severe combined immunodeficiency disease (SCID) mice, and wild-type mice treated with cyclosporine. LC16m8 showed extremely low virulence in each of the three mouse models compared with that of its parental strains, Lister and LC16mO. These results provide further evidence that LC16m8 is one of the safest replication-competent smallpox vaccines in the world and may be considered for use in immunodeficient patients.

Considerations for non-clinical safety studies of therapeutic peptide vaccines.

Guidelines for non-clinical studies of prophylactic vaccines against infectious diseases have been published widely, but similar guidelines for therapeutic vaccines, and especially therapeutic peptide vaccines, have yet to be established. The approach to non-clinical safety studies required for therapeutic vaccines differs from that for prophylactic vaccines due to differences in the risk-benefit balance and the mechanisms of action. We propose the following guidelines for non-clinical safety studies for therapeutic peptide vaccines. (i) Since the main safety concern is related to the immune response that might occur at normal sites that express a target antigen, identification of these possible target sites using in silico human expression data is important. (ii) Due to the strong dependence on HLA, it is not feasible to replicate immune responses in animals. Thus, the required non-clinical safety studies are characterized as those detecting off-target toxicity rather than on-target toxicity.

Pharmacokinetics, distribution, and excretion of 125I-labeled human plasma-derived-FVIIa and -FX with MC710 (FVIIa/FX mixture) in rats.

INTRODUCTION: MC710 is a mixture agent consisting of plasma-derived activated factor VII (FVIIa) and factor X (FX) at a weight ratio of 1:10 developed as a novel bypassing agent for the management of the bleeding of hemophilia patients with inhibitors. The pharmacokinetics, distribution, and excretion of (125)I-labeled-FVIIa ((125)I-FVIIa) and -FX ((125)I-FX) were studied in male rats after a single intravenous administration of (125)I-FVIIa or (125)I-FX combined with MC710. METHODS: (125)I-FVIIa or (125)I-FX was administered intravenously with MC710 to male rats in a single dosage (FVIIa 0.4 mg and FX 4 mg/kg body weight) and radioactivity and antigen levels in plasma were quantified for 24h. Urine and feces were sampled to study the excretion of radioactivity during 168 h after dosing. Whole-body autoradiography was performed to evaluate the qualitative distribution of radioactivity 168 h after dosing. RESULTS AND CONCLUSIONS: The half-life (t(1/2)α and t(1/2)ß) of radioactivity and FVIIa antigen were 0.704 and 6.27 h, and 0.496 and 1.66 h, respectively and the area under the plasma concentration-time curve (AUC(0-∞)) of radioactivity and FVIIa antigen were 17,932 and 8671 ng·h/mL, respectively. The t(1/2) of radioactivity and FX antigen were 4.06 and 3.05 h, respectively, and the AUC(0-∞) of radioactivity and FX antigen were 320,143 and 395,794 ng·h/mL, respectively. About 80% of the administered dose of radioactivity was excreted in urine and feces by 168 h after administration. Tissue distribution experiments showed that FVIIa- and FX-related (125)I accumulated in bone and bone marrow, and disappeared slowly.

Smallpox vaccine safety is dependent on T cells and not B cells.

The licensed smallpox vaccine, ACAM2000, is a cell culture derivative of Dryvax. Both ACAM2000 and Dryvax are administered by skin scarification and can cause progressive vaccinia, with skin lesions that disseminate to distal sites. We have investigated the immunologic basis of the containment of vaccinia in the skin with the goal to identify safer vaccines for smallpox. Macaques were depleted systemically of T or B cells and vaccinated with either Dryvax or an attenuated vaccinia vaccine, LC16m8. B cell depletion did not affect the size of skin lesions induced by either vaccine. However, while depletion of both CD4(+) and CD8(+) T cells had no adverse effects on LC16m8-vaccinated animals, it caused progressive vaccinia in macaques immunized with Dryvax. As both Dryvax and LC16m8 vaccines protect healthy macaques from a lethal monkeypox intravenous challenge, our data identify LC16m8 as a safer and effective alternative to ACAM2000 and Dryvax vaccines for immunocompromised individuals.

The predictivity of preliminary embryo-fetal development (EFD) studies: results of a retrospective survey in Japanese pharmaceutical companies.

To explore the predictivity of dose range-finding (DRF) studies, we conducted asurvey by sending out questionnaires to 72 Japanese pharmaceutical companies.The survey yielded data for 108 and 85 compounds for which any embryo-fetaldevelopment (EFD) toxicities were observed in the definitive studies in rodentsand non-rodents, respectively. As a result of the analysis, 83% of studies inrodents and 80% in non-rodents showed EFD effects in the DRF studies. Whenfocusing on teratogenicity, 91% of studies in rodents and 100% in non-rodentswere judged "positive" in the DRF studies when all EFD toxicities were used asmarkers. When the effects of both the rodent and non-rodent studies wereevaluated together, the combination predictive value in the DRF studies was 96%for EFD toxicants and 100% for teratogens. To evaluate the influence of theexamination items, the predictive value was analyzed using 54 compounds forwhich full examinations (external, visceral and skeletal examination) wereconducted in both rodent and non-rodent DRF studies. When the results werejudged by including or excluding skeletal and visceral examinations results,the predictive values were not significantly different. In conclusion, theresults of this survey showed that a pair of the DRF studies in the rodents andnon-rodents is useful to increase the predictivity of DRF studies. In additionthe inclusion of observations such as fetal survival, body weight and externalexamination into the DRF studies are important to predict effects in thedefinitive studies.

Postinfection passive transfer of KD-247 protects against simian/human immunodeficiency virus-induced CD4+ T-cell loss in macaque lymphoid tissue.

BACKGROUND: Preadministration of high-affinity humanized anti-HIV-1 mAb KD-247 by passive transfer provides sterile protection of monkeys from heterologous chimeric simian/human immunodeficiency virus infection. METHODS: Beginning 1 h, 1 day, or 1 week after simian/human immunodeficiency virus-C2/1 challenge (20 50% tissue culture infective dose), mature, male cynomolgus monkeys received multiple passive transfers of KD-247 (45 mg/kg) on a weekly basis for approximately 2 months. Concentrations and viral loads were measured in peripheral blood, and CD4 T-cell counts were examined in both peripheral blood and various lymphoid tissues. RESULTS: Pharmacokinetic examination revealed similar plasma maintenance levels ranging from 200 to 500 microg/ml of KD-247 in the three groups. One of the six monkeys given KD-247 could not maintain these concentrations, and elicitation of anti-KD-247 idiotype antibody was suggested. All monkeys given KD-247 exhibited striking postinfection protection against both CD4 T-cell loss in various lymphoid tissues and atrophic changes in organs compared with control group animals treated with normal human immunoglobulin G. The KD-247-treated groups were also partially protected against plasma viral load elevation in peripheral blood samples, although the complete protection previously reported with preadministration of this mAb was not achieved. CONCLUSION: Postinfection passive transfer of humanized mAb KD-247 with strong neutralizing capacity against challenged virus simian/human immunodeficiency virus-C2/1 protected CD4 T cells in lymphoid organs.

Anti-V3 humanized antibody KD-247 effectively suppresses ex vivo generation of human immunodeficiency virus type 1 and affords sterile protection of monkeys against a heterologous simian/human immunodeficiency virus infection.

In an accompanying report (Y. Eda, M. Takizawa, T. Murakami, H. Maeda, K. Kimachi, H. Yonemura, S. Koyanagi, K. Shiosaki, H. Higuchi, K. Makizumi, T. Nakashima, K. Osatomi, S. Tokiyoshi, S. Matsushita, N. Yamamoto, and M. Honda, J. Virol. 80:5552-5562, 2006), we discuss our production of a high-affinity humanized monoclonal antibody, KD-247, by sequential immunization with V3 peptides derived from human immunodeficiency virus type 1 (HIV-1) clade B primary isolates. Epitope mapping revealed that KD-247 recognized the Pro-Gly-Arg V3 tip sequence conserved in HIV-1 clade B isolates. In this study, we further demonstrate that in vitro, KD-247 efficiently neutralizes CXCR4- and CCR5-tropic primary HIV-1 clade B and clade B' with matching neutralization sequence motifs but does not neutralize sequence-mismatched clade B and clade E isolates. Monkeys were provided sterile protection against heterologous simian/human immunodeficiency virus challenge by the passive transfer of a single high dose (45 mg per kg of body weight) of KD-247 and afforded partial protection by lower antibody doses (30 and 15 mg per kg). Protective neutralization endpoint titers in plasma at the time of virus challenge were 1:160 in animals passively transferred with a high dose of the antibody. The antiviral efficacy of the antibody was further confirmed by its suppression of the ex vivo generation of primary HIV-1 quasispecies in peripheral blood mononuclear cell cultures from HIV-infected individuals. Therefore, KD-247 promises to be a valuable tool not only as a passive immunization antibody for the prevention of HIV infection but also as an immunotherapy for the suppression of HIV in phenotype-matched HIV-infected individuals.

Aerobic catechol oxidation catalyzed by a bis(mu-oxo)dimanganese(III,III) complex via a manganese(II)-semiquinonate complex.

A 3,5-di-tert-butyl-1,2-semiquinonato (DTBSQ) adduct of Mn(II) was prepared by a reaction between Mn(II)(TPA)Cl(2) (TPA = tris(pyridin-2-ylmethyl)amine) and DTBSQ anion and was isolated as a tetraphenylborate salt. The X-ray crystal structure revealed that the complex is formulated as a manganese(II)-semiquinonate complex [Mn(II)(TPA)(DTBSQ)](+) (1). The electronic spectra in solution also indicated the semiquinonate coordination to Mn. The exposure of 1 in acetonitrile to dioxygen afforded 3,5-di-tert-butyl-1,2-benzoquione and a bis(mu-oxo)dimanganese(III,III) complex [Mn(III)(2)(mu-oxo)(2)(TPA)(2)](2+) (2). The reaction of 2 with 3,5-di-tert-butylcatechol (DTBCH(2)) quantitatively afforded two equivalents of 1 under anaerobic conditions. The highly efficient catalytic oxidation of DTBCH(2) with dioxygen was achieved by combining the above two reactions, that is, by constructing a catalytic cycle involving both manganese complexes 1 and 2. It was revealed that dioxygen is reduced to water but not to hydrogen peroxide in the catalytic cycle.

Non-clinical and phase I clinical trials of a Vero cell-derived inactivated Japanese encephalitis vaccine.

The safety and effectiveness of a Vero cell-derived inactivated Japanese encephalitis (JE) vaccine were compared with those of a current JE vaccine in non-clinical studies and a phase I clinical trial. The single-dose toxicity study showed no toxicity of either the current JE vaccine or the investigational Vero cell-derived JE vaccine. In a local irritation study, the degree of irritation caused by both vaccines was determined to be the same as that induced by normal saline. To investigate genotoxicity, a chromosomal aberration test was conducted and the results were negative. Both JE vaccines were administered to a group of 30 subjects who were seronegative (neutralizing antibody titer <10(1)) for JEV virus (Beijing-1 Strain). Each subject was subcutaneously inoculated twice at an interval of 1-4 weeks, followed by an additional booster inoculation 4-8 weeks later, and clinical reactions and serological responses were subsequently investigated. Adverse drug reactions of local reaction, headache and malaise were mild, occurring at a rate of 6.7 and 20.0% after administration of the Vero cell-derived JE vaccine and the current JE vaccine, respectively. The seroconversion rate after three doses of both JE vaccines was 100%, while the geometric mean titer for the Vero cell-derived and current JE vaccines was 10(2.35) and 10(2.03), respectively. These results suggest that the safety and effectiveness of the Vero cell-derived inactivated JE vaccine are equal to those of the currently available conventional vaccine in humans, and that the Vero cell-derived vaccine could be a useful second-generation JE vaccine.