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Canine parvovirus type 2a (CPV-2a) and type 2b (CPV-2b) have recently been isolated from cats throughout the world, and CPV-2b strain FP84 has been reported to be virulent in domestic cats. Although live feline panleucopenia virus (FPLV) vaccines protect domestic cats from CPV infection, the efficacy of inactivated FPLV vaccines has not been established. In this study, two domestic cats were vaccinated with a commercial inactivated FPLV vaccine and challenged with CPV-2b strain FP84 isolated from a domestic cat. The cats were protected against CPV-2b strain FP84 infection and their clinical signs were suppressed, although the two unvaccinated cats showed the typical clinical signs of parvovirus infection.

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An international collaborative study of a quantitative colorimetric method for determination of formaldehyde in veterinary vaccines was conducted on a series of replicate, blinded veterinary vaccine products by 15 laboratories in three regions: North America, Europe and Japan. Participants conducted determinations using a modification of a method from the European Pharmacopoeia, a colorimetric method based on the reaction of formaldehyde with methylbenzothiazolone hydrazone hydrochloride. For this study, three licensed vaccine products containing formaldehyde were revialed, randomly numbered, tested for uniformity and distributed by one of the participating laboratories through regional coordinators to collaborators. One of the revialed products was spiked with a known amount of formaldehyde and included in the test series. Results along with all raw data were returned to the distributing laboratory for consolidation and statistical treatment. For the modified method spike recovery was 101% and reproducibility (inter-laboratory variation expressed as relative standard deviation) ranged from 18.0 to 8.0% for respective formaldehyde concentrations of 0.28 to 1.07 g/l. Based on the study, the method was proposed by the Biologicals Working Group of the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) as a candidate for the VICH Guideline standard method for residual formaldehyde.

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EPR imaging by using an acyl-protected hydroxylamine, 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP), in the head of a living rat after kainic acid (KA)-induced epileptic seizures was performed. ACP is a stable non-radical compound, but is easily deprotected with intracellular esterase to yield a hydroxylamine, which is oxidized by intracellular oxidative stress to yield an EPR-detectable nitroxide radical. From in vivo image data, the average values of EPR signal intensity from the hippocampus, striatum, and cerebral cortex were computed. There was no significant difference in cortical signal intensity between the control and KA-treated rats. The signal intensities from the hippocampus and striatum for the KA-treated rats were significantly higher than those for the control. The in vitro study showed that almost the same quantity of ACP moved into all regions of the brain of the control and KA-treated rats. These findings indicate that following a KA-induced seizure, the oxidative stress in the hippocampus and striatum is enhanced, but not so in the cerebral cortex.

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Recently, we developed an in vivo temporal electron paramagnetic resonance (EPR) imaging technique to be applied to the brain of a rat, into which a blood-brain barrier (BBB)-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) was injected intraperitoneally. This imaging technique made it possible to measure decay rates of a nitroxide radical in multiple regions of the brain simultaneously. Using this technique, the half-life of PCAM was estimated from the exponential decay of the signal intensity derived from the temporal EPR images in the hippocampus and cerebral cortex of rats after a kainic acid (KA)-induced seizure. The hippocampal half-life of PCAM after KA-induced seizures was significantly prolonged (p < .01), whereas the prolongation of the cortical half-life was not significant. These findings suggest that following a KA-seizure, the intrahippocampal ability to reduce the nitroxide radical is impaired, but the ability is intact in the cerebral cortex. This is the first in vivo quantitative EPR imaging study that has a bearing on the pathogenesis of KA-induced seizures in the brain.

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The results of the autopsy of a 38-year-old female with mixed connective tissue disease who had suffered from painful subcutaneous calcification in her buttocks and extremities for 14 years and died from rapidly progressive pulmonary hypertension are reported. On autopsy, her heart and lungs revealed changes of severe pulmonary hypertension with intimal thickening and plexiform lesions in the small pulmonary arteries which had resulted in the collapse of both lungs and caused marked dilatation and hypertrophy of the right ventricle of the heart. Microscopic examinations of the subcutaneous calcified tissues indicated that the calcification may have been caused by repeated panniculitis.

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The purpose of this study was to propose reference values, from a viewpoint of prognostic significance, for blood pressure (BP) measured at home with a semiautomated device (home BP measurement) to differentiate normotension and hypertension. We obtained home BP measurements for 1,913 population-based subjects aged 40 years and over in a rural Japanese community and followed up their survival for a mean duration of 5.0 years. There were 141 deaths during the follow-up period. The association between baseline BP values and the overall mortality was examined by Cox proportional hazards regression model, adjusted for age, gender, and the use of antihypertensive medication. The results indicated that the predictive power of home BP level for subsequent mortality was stronger than that of casual screening BP. There was a linear association between home systolic BP and mortality. The association between home diastolic BP and mortality was nonlinear and well approximated with the secondary degree equation of diastolic BP values. Based on this relation, we propose that the reference value for hypertension is 137/84 mm Hg, and normotension is below 137 mm Hg for home systolic BP and between 66 and 83 mm Hg for home diastolic BP. Home diastolic BP below 66 mm Hg should be considered as low diastolic blood pressure. In this population, home systolic BP of 137 mm Hg and home diastolic BP of 84 mm Hg corresponded to the 80th and 87th percentiles, respectively. Then, 29% of the subjects were classified as having hypertension, 52% as normotension, and 19% as low diastolic blood pressure. All previous studies proposing reference values for home BP measurement, derived from cross-sectional observations, were based on the statistical distribution of home BP values. The reference value must, however, be the one that best predicts the risk for morbidity and mortality from hypertension-related complications. This is the first report proposing reference values for home BP measurement based on prognostic criteria.

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OBJECTIVE: To compare the prediction of mortality by ambulatory blood pressure monitoring and screening blood pressure measurements in a general population. DESIGN: A prospective cohort study. PATIENTS AND METHODS: We obtained blood pressure data for 1542 subjects (565 men and 977 women) aged > or = 40 years who were followed up for up to 8.1 years (mean 5.1 years). Subjects were subdivided into five groups according to their ambulatory and screening blood pressure levels. The prognostic significance of blood pressure for mortality was examined by the Cox proportional hazards regression model. RESULTS: The association between blood pressure level and mortality was more distinctive for the ambulatory blood pressure than it was for the screening blood pressure. The risk of cardiovascular mortality increased significantly for the highest quintiles of 24 h ambulatory blood pressure, whereas there was no significant association between the screening blood pressure and the cardiovascular mortality. When both 24 h and screening blood pressure values were included in the Cox model, only the systolic ambulatory blood pressure was related significantly to the increased risk of cardiovascular mortality. CONCLUSIONS: The ambulatory blood pressure had a stronger predictive power for mortality than did the screening blood pressure. This appears to have been the first study of the prognostic significance of ambulatory blood pressure monitoring versus screening blood pressure measurements in a general population.

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We performed in vivo ESR-CT (electron spin resonance-computed tomography) on rats' heads, in which the blood-brain barrier-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) was injected intraperitoneally, using a rapid scan ESR-CT system operating at 700 MHz. In a spatiotemporal study we found that different regions of the brain showed differences in the rate of decay of the radical. Repeated injection of PCAM gave clear ESR-CT images of the brain. We think that the present method is useful for evaluating the capacity to eliminate exogenous free radicals in some parts of the brain.

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A competitive enzyme-linked immunosorbent assay (c-ELISA) was developed as an alternative to the viral neutralization (VN) test for rapid and simple detection of antibodies to rabies virus. The competitor antibody in the c-ELISA was a biotinylated monoclonal antibody to the nucleoprotein of rabies virus. Initial comparisons showed a high correlation between titers obtained with the VN test and the c-ELISA (n = 88, r = 0.90), indicating that the c-ELISA could be used as a reliable substitute for the VN test. To evaluate the immune status of Japanese dogs to rabies virus, a total of 1,019 serum samples were collected from domestic dogs in 1994 and tested for antibodies with the c-ELISA. Overall, 84.8% of the dogs had antibodies against rabies virus, indicating that the vaccination strategy for preventing rabies outbreaks in domestic dogs is probably sufficient in Japan. Dogs receiving final vaccinations a year or more previously were 48.3 and 90.3% positive for antibodies when vaccinated once only or two or more times, respectively. This suggests that almost all dogs vaccinated twice or more remain seropositive for over 1 year in Japan.

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The mortality rate of stroke has decreased dramatically over the past 30 years in Japan, but the prevalence of cerebral infarction has increased, mainly because of the higher incidence of nonfatal cerebral infarction in the elderly. Significant advances in the development of antihypertensive drugs over the last 30 years have contributed to the decrease in stroke mortality, but antihypertensive treatment appears to relate to an increase in the prevalence of cerebral infarction in the elderly. We investigated the risks of low BP levels determined by ambulatory BP monitoring and home BP measurements to clarify the previously reported J-shaped relationships between the incidence of ischemic cardiovascular disease and BP levels after treatment. A long-term prospective study of all causes of mortality and cerebrovascular and cardiovascular mortality has been conducted in Ohasama, Japan, since 1987. Cox proportional hazard model after adjusting for age and sex demonstrated that during a 5-year follow-up period involving 893 of the subjects aged 50 years and over in this cohort, those with the lowest quintile of ambulatory BP levels exhibited a significantly high hazard ratio of cardiovascular as well as all causes of mortality. During the same follow-up period in 1,226 subjects aged 50 years and over, those with the lowest and highest quintiles of home BP levels demonstrated a significantly high hazard ratio of cardiovascular as well as all causes of mortality (i.e., J-shaped relationship). The amplitude of the fall in nocturnal BP obtained by ambulatory BP monitoring correlated positively with the severity of silent cerebrovascular lesions in elderly women, but not in elderly men. These results suggest the significantly high risk associated with low BP levels, which can be determined only by ambulatory and home BP measurements, but not by casual BP measurements.

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A cross-sectional community survey using home blood pressure measurements was performed in northern Japan to estimate the prevalence of definite hypertension, white coat hypertension and the success of blood pressure control in patients receiving antihypertensive drugs. A total of 1334 subjects (mean age +/- SD, 53.8 +/- 17.3 years; 8-91 years) participated in the screening and home blood pressure measurement program. They measured blood pressure at home at least 3 times (mean measurement frequency, 20.8 +/- 8.3 times). Of these 1334 subjects, 314 (65.1 +/- 8.9 years) were taking drugs (treated group) while 1020 (50.3 +/- 17.8 years) were not (untreated group). The WHO criteria were used to categorize screening blood pressure. Criteria for diagnosis of hypertension by home blood pressure measurements were as follows: definitely hypertensive (systolic blood pressure > or = 144 mmHg and/or diastolic blood pressure > or = 89 mmHg) and normotensive (104 < systolic blood pressure < or = 131 mmHg and 60 < diastolic blood pressure < or = 79 mmHg). Of the 1018 subjects identified as normotensive on screening measurements, home measurements indicated that 73 (7.2%) were hypertensive and 765 (74.7%) were normotensive or lower. Of the 112 subjects identified as hypertensive on screening measurements, home measurements showed that 42 (37.5%) were hypertensive and 30 (26.8%) were normotensive or lower. Of the 314 treated subjects, 45 (14.3%) were identified as hypertensive by screening measurements and 88 (28.0%) as hypertensive by home measurements. Only 20 (44.4%) of the former 45 subjects were also defined as definitely hypertensive by home measurements. Of the 1020 untreated subjects, 67 (6.6%) were hypertensive by screening measurements and 84 (8.2%) by home measurements. Only 22 (32.8%) of the former 67 subjects were classified as hypertensive by home measurements. Of the 67 untreated subjects identified as hypertensive by screening measurements, 20 (29.9%) were normotensive or lower by home measurements, suggesting that these subjects were "white coat" hypertensives. The study first confirmed based on the large community data that there are large discrepancies between screening (casual) blood pressure and home blood pressure measurements for recognition of hypertension and normotension. Determination of blood pressure levels by home blood pressure measurements may predict prognosis of hypertension differently from that by screening blood pressure measurements. Further prospectively study is needed to validate the prognostic value of home blood pressure measurements.

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The use of ELISA for the potency test of inactivated rabies vaccines for animal use was evaluated. Sandwich ELISA was developed, using monoclonal antibodies which enabled to recognize the glycoprotein (G-protein) of rabies virus and showed a protective effect in mice. Soluble G-protein which displays an ELISA activity but a low immunogenicity was removed by gel filtration before ELISA was carried out. The ELISA titres of the first fraction of vaccines separated by gel filtration were correlated with the neutralizing antibody titres of vaccinated guinea-pigs. Our results suggest that the ELISA titres of the first fraction of vaccines separated by gel filtration reflected the protective G-protein contents for the in vitro potency test of rabies vaccines compared with the current in vivo test.

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UNLABELLED: Increased clinical use of 123I-labeled 15-(p-iodophenyl)-3-(R,S)-methyl- pentadecanoic acid ([123I]BMIPP) revealed discordance between BMIPP uptake and that of perfusion agents, which was inexplicable due to the uncertainty of its myocardial metabolism. This study clarifies the metabolic fate of BMIPP and its relation to substrates in isolated rat hearts. METHODS: Rat hearts were perfused with 5 mmole/liter HEPES buffer containing various energy substrates and 1% bovine serum albumin. The buffer was recirculated for 4 hr after bolus injection of [123I]BMIPP. Heart time-activity curves were monitored externally. After perfusion, the radioactivity in the heart and recirculated buffer was measured. The metabolites in the buffer were then extracted and analyzed by HPLC and TLC. RESULTS: when 0.4 mmole/liter oleate was the energy substrate, more than eight radioactive BMIPP metabolites were detected. The metabolites in the coronary effluent depended on the energy substrate in the buffer. The radioactivity in the heart at the end of the perfusion period was significantly higher when 0.4 mmole/liter oleate (28.0% +/- 1.2% ID/g, mean +/- s.e.m.) or 10 mmole/liter glucose with 25 U/liter insulin (43.9% +/- 2.2% ID/g) were the substrates compared to when 5 mmole/liter acetate (8.5% +/- 0.4% ID/g) or 0.4 mmole/liter cold BMIPP (6.2% +/- 0.3% ID/g) were the substrates. The distribution of metabolites suggests that oleate stimulated both alpha and beta oxidations, whereas glucose with insulin inhibited both. Acetate also stimulated alpha oxidation but not beta oxidation. Cold BMIPP strongly inhibited both alpha- and beta-oxidations, and little alpha oxidation occurred compared to beta-oxidation. CONCLUSION: These results suggest that [123I]BMIPP is metabolized in the myocardium and the metabolism is closely related to myocardial carbohydrate utilization.

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We investigated the role of natural killer (NK) cells in the development of autoantibody production in which (C57BL/6 x DBA/2) F1 (BDF1) hybrid mice were injected intravenously with spleen cells (SC) from parental DBA/2 mice (treated BDF1 mice). Treated BDF1 mice began to show an increase in serum anti-dsDNA antibody 2 weeks after injection, while the NK activity of their SC transiently increased initially in the first 1 to 2 weeks after injection, but subsequently decreased dramatically. Flow cytometric analysis suggested that this sequential change in NK activity correlated with the absolute number of host-derived NK1.1+ cells in SC from treated BDF1 mice. We demonstrated that the level of anti-dsDNA in serum is directly influenced by the level of NK activity in treated BDF1 mice. Depletion of NK cells by administration of anti-NK1.1 mAb accelerated the development of autoantibody production, whereas augmentation of NK activity by administration of poly-(I:C) inhibited the development of autoantibody production. This inhibitory effect of poly(I:C) was abolished by prior depletion of NK cells. Interestingly, suppression of autoantibody production was seen only when poly(I:C) was administrated within 1 week after injection of parental SC. Last, we demonstrate that adoptive transfer of interleukin-2 (IL-2)-activated NK cells had a protective effect against the development of autoantibody production. These findings imply that NK cells may have a protective role in lupus-like disease especially in its early stage. In addition, it opens up the possibility that adoptive immunotherapy with IL-2-activated NK cells can delay or even prevent the development of autoimmune disease.

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One of the attenuated and genetically recombinant modified-live viral (MLV) vaccine strains currently used contains a deletion in its glycoprotein III (gIII) gene, while prototypic wild-type pseudorabies (WT-PR) viruses contain an intact gIII gene. A polymerase chain reaction (PCR) system differentiating, based on this difference, between the vaccine virus and prototypic WT-PR viruses was investigated. This PCR system utilized two consecutive stages. Primers for the first-stage PCR were designed so as to amplify of DNA fragments lengths in respect to the vaccine and WT-PR viruses. The second-stage PCR amplification for improving the sensitivity and specificity and for confirming of the sites deleted from the first-stage PCR products produced an all-or-none result: internal DNA fragments were derived from only WT-PR viruses but not from the vaccine virus. These PCR-amplified fragment length polymorphisms clearly distinguished the vaccine virus from WT-PR viruses. The vaccine and WT-PR viruses in mixtures were each identified in this PCR system. This PCR system may permit rapid and sensitive detection of PR viral gIII gene, analysis of the genotype of PR virus isolates, and also examination of the isolates for purity and identity.

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A method for detecting wild-type canine parvovirus (CPV) strains which contaminate vaccines for dogs has been developed by PCR. PCR primers which distinguish vaccine strains from the most common, recent strains of wild-type CPV in many countries, including Japan and the United States, were developed. This PCR is based on the differences in nucleotide sequences which determine the two antigenic types of this virus. CPV vaccine strains derived from antigenically old-type virus prevalent in former times were not detected by PCR with differential primers. Detection sensitivity of PCR was 100- to 10,000-fold higher than that of the culture method in Crandell feline kidney cells.

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We analyzed metabolites of 123I-BMIPP in blood and urine using rats, rabbits and human, while human samples were obtained from normal volunteers of Phase I clinical study. We estimated metabolic pathway of 123I-BMIPP as a myocardial metabolic imaging agent. Radioactivity accumulated in heart after administration gradually decreased and was mainly excreted to bladder via kidneys. The main radioactive component in blood was 123I-PIPA for any species and the urinary components were metabolic conjugates of 123I-PIPA. As results of these studies, we considered that 123I-BMIPP was metabolized to 123I-PIPA by alpha-oxidation process for the first step, follow by beta-oxidation process, then 123I-PIPA was released to blood from tissues. Moreover, 123I-PIPA in blood was conjugated with other compounds and excreted to the bladder.

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