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Background Synaptic degeneration and accumulation of amyloid β-peptides (Aβ) are hallmarks of the Alzheimer diseased brain. Aβ is synaptotoxic and produced by sequential cleavage of the amyloid precursor protein (APP) by the β-secretase BACE1 and by γ-secretase. If APP is instead cleaved by the α-secretase ADAM10, Aβ will not be generated. Although BACE1 is considered to be a presynaptic protein and ADAM10 has been reported to mainly localize to the postsynaptic density, we have previously shown that both ADAM10 and BACE1 are highly enriched in synaptic vesicles of rat brain and mouse primary hippocampal neurons. Results Here, using brightfield proximity ligation assay, we expanded our previous result in primary neurons and investigated the in situ synaptic localization of ADAM10 and BACE1 in rat and human adult brain using both pre- and postsynaptic markers. We found that ADAM10 and BACE1 were in close proximity with both the presynaptic marker synaptophysin and the postsynaptic marker PSD-95. The substrate APP was also detected both pre- and postsynaptically. Subcellular fractionation confirmed that ADAM10 and BACE1 are enriched to a similar degree in synaptic vesicles and as well as in the postsynaptic density. Conclusions We show that the α-secretase ADAM10 and the β-secretase BACE1 are located in both the pre- and postsynaptic compartments in intact brain sections. These findings increase our understanding of the regulation of APP processing, thereby facilitating development of more specific treatment strategies.

The toxic amyloid β-peptide (Aβ) is a key player in Alzheimer Disease (AD) pathogenesis and selective inhibition of the production of this peptide is sought for. Aβ is produced by the sequential cleavage of the Aβ precursor protein (APP) by β-secretase (to yield APP-C-terminal fragment β (APP-CTFβ) and soluble APPβ (sAPPβ)) and γ-secretase (to yield Aβ). We reasoned that proteins that associate with γ-secretase are likely to regulate Aβ production and to be targets of pharmaceutical interventions and therefore performed a pull-down assay to screen for such proteins in rat brain. Interestingly, one of the purified proteins was potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), which has been shown to be involved in epilepsy. We found that silencing of HCN2 resulted in decreased secreted Aβ levels. To further investigate the mechanism behind this reduction, we also determined the levels of full-length APP, sAPP and APP-CTF species after silencing of HCN2. A marked reduction in sAPP and APP-CTF, as well as glycosylated APP levels was detected. Decreased Aβ, sAPP and APP-CTF levels were also detected after treatment with the HCN2 inhibitor ZD7288. These results indicate that the effect on Aβ levels after HCN2 silencing or inhibition is due to altered APP maturation or processing by β-secretase rather than a direct effect on γ-secretase. However, HCN2 and γ-secretase were found to be in close proximity, as evident by proximity ligation assay and immunoprecipitation. In summary, our results indicate that silencing or inhibition of HCN2 affects APP processing and thereby could serve as a potential treatment strategy.

Many attempts have been made to identify laboratory tests that are predictive of sperm fertility, both to improve the quality of stallion semen doses for artificial insemination (AI) and to identify potential breeding sires if no fertility data are available. Sperm quality at the stud is mostly evaluated by assessing subjective motility, although this parameter can be poorly indicative of fertility. Sperm morphology and chromatin integrity in Swedish stallions are correlated to pregnancy rate after AI. Because single layer centrifugation (SLC) selects for spermatozoa with normal morphology and good chromatin, retrospective analysis was carried out to investigate whether sperm yield after SLC is linked to potential fertility. Commercial semen doses for AI from 24 stallions (five stallions with four ejaculates each, 19 stallions with three ejaculates each; n = 77) obtained during the breeding season were cooled, and sent overnight to the Swedish University of Agricultural Sciences in an insulated box for evaluation, with other doses being sent to studs for commercial AI. On arrival at Swedish University of Agricultural Sciences, the semen was used for SLC and also for evaluation of sperm motility, membrane integrity, chromatin integrity, and morphology. The seasonal pregnancy rates for each stallion were available. The yield of progressively motile spermatozoa after SLC (calculated as a proportion of the initial load) was found to be highly correlated with pregnancy rate (r = 0.75; P0.001). Chromatin damage was highly negatively correlated with pregnancy rate (r = -0.69; P0.001). Pregnancy rate was also correlated with membrane integrity (r = 0.58; P0.01), progressive motility (r = 0.63; P0.01), and normal morphology (r = 0.45; P0.05). In conclusion, these preliminary results show that sperm yield after SLC is related to the potential fertility of the original ejaculate, and could be an alternative indicator of stallion fertility if breeding data are not available. Single layer centrifugation is fast (30 minutes) and does not require expensive equipment, whereas other assays require a flow cytometer and/or specialist skills. An additional option could be to transport semen doses to a laboratory for SLC if the stud personnel do not want to perform the procedure themselves.

The stochastic effects in the lung of inhaled, insoluble particles of alpha- and beta-emitting particles and low-linear energy transfer (LET) thoracic irradiation were compared in rats using data from previously conducted studies. Male and female F344 rats were exposed briefly by nasal inhalation to relatively insoluble aerosols of CeO(2) or PuO(2) to achieve a range of four lung burdens. The mean lifetime beta doses to the lung were 3.6 + or - 1.3 Gy, 6.8 + or - 1.7 Gy, 12 + or - 4.5 Gy, and 37 + or - 5.9 Gy. The mean lifetime alpha doses to the lung were 0.06 + or - 0.03 Gy, 0.95 + or - 0.46 Gy, 3.7 + or - 1.6 Gy, and 12 + or - 2.4 Gy. Additional rats were exposed to fractionated thoracic doses of x rays given on 10 successive working days. The lifetime doses to the lung were 3.3 Gy, 5.7 Gy, 11 Gy, and 38 Gy. Appropriate sham controls were included in each group and all groups were observed for their life spans. Lung neoplasms were found after all exposures, with the incidence increasing with radiation dose. Rats exposed to PuO(2) had the highest incidence, 94% in the group with a dose of 12 Gy. The incidence in the groups exposed to inhaled CeO(2) or fractionated thoracic x-irradiation was not significantly different. The incidence of lung tumors in the PuO(2) groups was 21 times higher than that of the groups exposed to the lower LET radiations. These results support a radiation-weighting factor of 20, as recommended by ICRP 60.

Synaptic degeneration and accumulation of the neurotoxic amyloid β-peptide (Aβ) in the brain are hallmarks of Alzheimer disease. Aβ is produced by sequential cleavage of its precursor protein, APP, by the β-secretase BACE1 and γ-secretase. However, Aβ generation is precluded if APP is cleaved by the α-secretase ADAM10 instead of BACE1. We have previously shown that Aβ can be produced locally at the synapse. To study the synaptic localization of the APP processing enzymes we used western blotting to demonstrate that, compared to total brain homogenate, ADAM10 and BACE1 were greatly enriched in synaptic vesicles isolated from rat brain using controlled-pore glass chromatography, whereas Presenilin1 was the only enriched component of the γ-secretase complex. Moreover, we detected ADAM10 activity in synaptic vesicles and enrichment of the intermediate APP-C-terminal fractions (APP-CTFs). We confirmed the western blotting findings using in situ proximity ligation assay to demonstrate close proximity of ADAM10 and BACE1 with the synaptic vesicle marker synaptophysin in intact mouse primary hippocampal neurons. In contrast, only sparse co-localization of active γ-secretase and synaptophysin was detected. These results indicate that the first step of APP processing occurs in synaptic vesicles whereas the final step is more likely to take place elsewhere. This article is protected by copyright. All rights reserved. (Less)

The decrease in foaling rates after artificial insemination with cooled semen warrants the search for new predictors of fertility. The objectives were to investigate levels of naturally occurring reactive oxygen species (ROS) in cooled, stored stallion semen doses for artificial insemination (AI), and their relationship with parameters of semen quality and with pregnancy rate. Semen was collected from warmblood stallions (n=15) and used to prepare commercial semen doses for AI. Sperm quality was evaluated after cooled transport to the laboratory overnight. The results were correlated with observed foaling and pregnancy rates. Hydroethidine and dichlorodihydrofluorescein diacetate were used as indicators for the ROS superoxide and hydrogen peroxide, respectively. Sperm morphology, motility, plasma membrane integrity and chromatin integrity were also evaluated. These variables were correlated with each other and with pregnancy rates. We found a high inter-individual variation in the ROS levels between stallions. The proportion of live, hydrogen peroxide-negative spermatozoa was correlated with progressive motility, whereas live hydrogen peroxide-negative spermatozoa and chromatin damage were negatively correlated, indicating that low levels of hydrogen peroxide were correlated with good chromatin integrity. The percentage of dead hydrogen peroxide-positive sperm was negatively related to the foaling rate. The negative relationships were stronger when combining results from both assays for ROS. These results for stored semen samples indicate that high individual variation exists for superoxide and hydrogen peroxide measurements, and that ROS status can influence sperm quality. Thus, ROS may be some of the factors influencing fertility. Moreover, combinations of ROS variables improved the correlation with fertility, indicating the usefulness of including these variables in a future model for prediction of the fertility of a semen sample.

Blood concentrations of doxo- and epirubicin were studied in mice after i.v. or i.p. administration under normal and hypothermic conditions. The animals either were pretreated i.p. with chlorpromazine at 15 mg/kg and allowed to cool to a rectal temperature of 28 degrees C or were given saline i.p. with their rectal temperature remaining at 37 degrees C. The anthracyclines were 14-14C-labeled and were given at a dose of 0.85 mg/kg. Blood samples were taken at 5, 15, and 25 min and 2, 6, 24, and 48 hours after injection and were analyzed by liquid scintillation counting. The blood concentration related to time was similar for the two anthracyclines. The peak concentration was highest for i.v. administration and was higher for the hypothermic groups. The peak concentration and the area under the curve were highest under hypothermic conditions. The terminal half-life was longer after i.p. administration. The ratio calculated for the blood concentration under hypothermic/normothermic conditions over time was substantially increased after i.p. administration, the increase being most pronounced for epirubicin. The pharmacokinetic characteristics found might be related to the anthracycline toxicity encountered in tumor-inoculated mice treated at different body temperatures.

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease. The molecular mechanism underlying this degeneration is not fully elucidated but one key player appears to be the synaptotoxic amyloid β-peptide (Aβ). The exact localization of the production of Aβ and the mechanisms whereby Aβ is released remain elusive. We have earlier shown that Aβ can be produced in crude synaptic vesicle fractions and it has been reported that increased synaptic activity results in increased secreted but decreased intracellular Aβ levels. Therefore, we considered whether Aβ could be produced in synaptic vesicles and/or released through the same mechanisms as neurotransmitters in synaptic vesicle exocytosis. Small amounts of Aβ were found to be produced in pure synaptic vesicle preparations. We also studied the release of glutamate and Aβ from rat cortical nerve terminals (synaptosomes). We found that large amounts of Aβ were secreted from non-stimulated synaptosomes, from which glutamate was not released. On the contrary, we could not detect any differences in Aβ release between non-stimulated synaptosomes and synaptosomes stimulated with KCl or 4-aminopyridine, whereas glutamate release was readily inducible in this system. To conclude, our results indicate that the major release mechanism of Aβ from isolated nerve terminals differs from the synaptic release of glutamate and that the activity-dependent increase of secreted Aβ, reported by several groups using intact cells, is likely dependent on post-synaptic events, trafficking and/or protein synthesis mechanisms.

The inflammatory cell composition and the expression of major histocompatibility complex (MHC) antigens in the central nervous system (CNS) of 13 cats with a spontaneous, Borna disease-like meningoencephalomyelitis (staggering disease) was investigated by immunohistochemistry with a panel of monoclonal and polyclonal antibodies. T lymphocytes were the predominating inflammatory cells within the adventitial space. CD4+ T cells were more abundant than CD8+ T cells. Scattered IgG-, IgA- and IgM-containing cells were found in the adventitial space and surrounding neuropil, often adjacent to neurons. There was a markedly increased MHC class II expression in cells morphologically resembling microglia. In several cats, Borna disease virus specific antigen was detected, but only in a few cells, mainly of macrophage character. Our findings indicate a long-standing inflammatory reaction in the CNS of cats with staggering disease, possibly triggered and sustained by a persistent viral infection.

Summary In 24 cats (Uppsala, Sweden) with neurological signs of “staggering disease” and typical neuropathology, 44% had Borna disease virus (BDV)-specific antibodies. In 173 cat sera (Berlin, Germany) of animals with unknown record, 7% were BDV positive. Out of 24 cats with undefined neurological disorders, 13% were BDV positive. Similarities in staggering disease of cats and Borna disease of horses and sheep suggest related etiological agents.

The lung neoplasms induced in rats by inhaled, internally deposited 144CeO2 were described and classified using histologic criteria. F344 rats were exposed once or repeatedly by inhalation to 144CeO2 and observed for their life span. There was significant life shortening only in those rats with the highest radiation doses. In these rats, there was a high percentage of squamous cell carcinomas of the lung, as well as much lower percentages of adenocarcinomas of the lung, hemangiosarcomas of the lung, and pleural mesotheliomas. At lower doses, adenocarcinomas were the most predominant tumor. These adenocarcinomas were subdivided based on their histologic pattern: alveolar, papillary, tubular, or undifferentiated. Neither the mode of exposure (single or repeated) nor the sex of the rat influenced the lung tumor incidence or tumor type. The lung neoplasms induced by this beta-emitting radionuclide are similar in nature to those induced by alpha-emitting radionuclides deposited in the lung in rats. However, the radiation-induced squamous cell carcinomas of the lung differ from those induced by heavy particle loads of nonradioactive compounds. The radiation-induced squamous cell carcinomas occur in higher incidence and have a more malignant behavior than those induced by heavy particle loads.

The acute lethality of various cytotoxic drugs (doxorubicin, vinblastine and nitrogen mustard) and long-term survival in a syngenic mouse-tumour system were studied at normal body temperature and at 28 degrees C induced by chlorpromazine. Chlorpromazine-induced hypothermia itself neither caused acute toxicity nor influenced long-term survival. Doxorubicin (15 mg/kg) and nitrogen mustard (6 mg/kg) lethality was reduced at decreased temperature. The median survival time increased significantly from 35 days in normothermic to 52 days in hypothermic doxorubicin-treated mice. With nitrogen mustard, no increase in long-term survival was seen in the hypothermic group. The acute lethality of vinblastine was enhanced by hypothermia and the long-term tumour survival was unaffected. Hypothermia or possibly chlorpromazine considerably modulates drug toxicity and possibly anti-tumoural activity.

A spontaneous neurological disease in cats characterized by behavioural and motor disturbances was reported in Sweden by Kronevi et al (1974). Generally, the animals showed no gross pathological lesions. Detailed neuro-pathological investigation revealed mononuclear perivascular cuffing and gliosis throughout the brain and spinal cord consistent with a non-suppurative meningoencephalomyelitis. After this first report, the disease has become recognized in different parts of Sweden, preferably Uppland and the area around Lake Mälaren, and is referred to as “staggering disease” of cats. The clinical manifestation of the disease includes hindleg ataxia and paresis (Fig. 1), inability to retract the claws (Fig. 2), mental changes, anorexia, increased salivation, hypersensitivity to sound and light, hyperesthesia, impaired vision and seizures (Kronevi et al 1974, Ström et al 1992). Despite treatment with antimicrobial drugs and corticosteroids most cats deteriorate and die or have to be euthanised after 1-4 weeks of illness.

As a portion of a study to examine how chronic cigarette smoke exposure might alter the risk of lung tumors from inhaled 239puO2 in rats, the effects of smoke exposure on alpha-particle lung dosimetry over the life-span of exposed rats were determined. Male and female rats were exposed to inhaled 239PuO2 alone or in combination with cigarette smoke. Animals exposed to filtered air alone served as controls for the smoke exposure. Whole-body exposure to mainstream smoke diluted to concentrations of either 100 or 250 mg total particulate matter m(-3)(LCS or HCS, respectively) began at 6 wk of age and continued for 6 h d(-1), 5d wk(-1), for 30 mo. A single, pernasal, acute exposure to 239PuO2 was given to all rats (control, LCS and HCS) at 12 wk of age. Exposure to cigarette smoke caused decreased body weight gains in a concentration dependent manner. Lung-to-body weight ratios were increased in smoke-exposed rats. Rats exposed to cigarette smoke before the 239PuO2 exposure deposited less 239Pu in the lung than did controls. Except for male rats exposed to LCS, exposure to smoke retarded the clearance of 239Pu from the lung compared to control rats through study termination at 870 d after 239PuO2 exposure. Radiation doses to lungs were calculated by sex and by exposure group for rats on study for at least 360 d using modeled body weight changes, lung-to-body weight ratios, and standard dosimetric calculations. For both sexes, estimated lifetime radiation doses from the time of 239PuO2 exposure to death were 3.8 Gy, 4.4 Gy, or 6.7 Gy for the control, LCS, or HCS exposure groups, respectively. Assuming an approximately linear dose-response relationship between radiation dose and lung neoplasm incidence, approximate increases of 20% or 80% in tumor incidence over controls would be expected in rats exposed to 239PuO2 and LCS or 239PuO2 and HCS, respectively.

This study was conducted to examine the carcinogenic effects of inhaled beta-particle-emitting radionuclides, particularly in lower dose regions in which there were substantial uncertainties associated with available information. A total of 2751 F344/N rats (1358 males and 1393 females) approximately 12 weeks of age at exposure were used. Of these, 1059 rats were exposed to aerosols of 144CeO2 to achieve mean desired initial lung burdens (ILBs) of 18 kBq (low level), 247 rats to achieve mean ILBs of 60 kBq (medium level) and 381 rats to achieve mean ILBs of 180 kBq (high level). Control rats (total of 1064) were exposed to aerosols of stable CeO2. Based on the 95% confidence intervals of the median survival times and the cumulative survival curves, there were no significant differences in the survival of groups of female and male exposed rats relative to controls. The mean lifetime beta-particle doses to the lungs of the rats in the four groups were: low level, 3.6 +/- 1.3 (+/-SD) Gy; medium level, 12 +/- 4.5 Gy; and high level, 37 +/- 5.9 Gy. The crude incidence of lung neoplasms increased linearly with increasing doses to the lungs (controls, 0.57%; low level, 2.0%; medium level, 6.1%; and high level, 19%). The estimated linear risk coefficients for lung neoplasms per unit of dose to the lung were not significantly different for the three dose levels studied. The risk coefficient at the lower level was 39 +/- 14 (+/-SE) excess lung neoplasms per 10(4) rat Gy; at the medium level the risk was 47 +/- 12; and at the higher level the risk was 50 +/- 9.0. The relationship of beta-particle dose to the lung and the crude incidence of lung neoplasms was described adequately by a linear function. We concluded that the risk of lung neoplasms in rats per unit of radiation dose did not increase with decreasing mean beta-particle dose to the lung over the range of 3.6 to 37 Gy. The weighted average of these three values was 47 +/- 6.4 (+/-SE) excess lung neoplasms per 10(4) rat Gy. To extend the risk coefficients for lung neoplasms to lower doses by experimentation will require much larger numbers of rats than used in this study.

Pulmonary carcinogenesis was compared in female F344 and Wistar rats after inhalation of high-fired 239PuO2. Plutonium particle aggregation, as determined by quantitative light and scanning electron microscopic autoradiography, was greater for the F344 strain than for the Wistar strain. The median survival times were similar in control and low-dose (0.8-1.0 Gy) groups of both strains, but were significantly decreased in the high-dose (34-37 Gy) groups of both strains. Squamous metaplasia was not found in control or low-dose groups of either strain, but was found in 62-65% of high-dose groups of both strains. Adenomatous metaplasia was considerably higher in control and low-dose groups of F344 rats than in Wistar rats. A total of 87 lung tumors were found in 140 exposed F344 rats and 46 lung tumors in 176 exposed Wistar rats. The incidence of lung tumors in F344 rats was 1.7% in controls, 20% in the low-dose group and 82% in the high-dose group. The incidence of lung tumors in Wistar rats was 0.1% in controls, nil in the low-dose group and 68% in the high-dose group. About half of all lung tumors in both strains were considered to be the primary cause of death. The median survival times of rats of both strains in the high-dose groups that died with lung tumors were greater compared with rats in these groups that died without lung tumors. In contrast, these differences did not occur among rats in the low-dose groups. The absolute risk was 1900 lung tumors per 10(4) Rat-Gy for F344 rats receiving low doses and nil for Wistar rats receiving low doses, but about 210 lung tumors per 10(4) Rat-Gy for high-dose groups of both strains. The adenomatous tumor phenotype predominated in the F344 strain, while the squamous tumor phenotype predominated in the Wistar strain. Risk of squamous tumors was similar for both strains. Overall, the F344 strain appears to be more "sensitive" than the Wistar strain to formation of lung tumors at low to moderate doses from inhaled 239PuO2 due mostly to an increased incidence of adenomatous phenotype tumors.

To study the long-term biological effects of repeated inhalation exposure to 239PuO2, 84-day-old rats were exposed to aerosols of 239PuO2 to re-establish desired 239Pu lung burdens of 26, 80 or 250 Bq every other month for 1 year (seven exposures). Other rats were exposed once at 84 or 450 days of age to achieve desired initial lung burdens of 30, 90, 280 or 850 Bq. The incidences of lung tumors were not significantly different (Fisher's exact test; P0.05) in groups of rats with similar lifetime mean alpha-particle doses to the lungs of 0.90 +/- 0.39 to 4.4 +/- 1.8 (+/- SD) Gy, whether exposed once or repeatedly. Among rats with mean alpha-particle doses of 12 +/- 2.4 to 10 +/- 2.1 Gy to the lungs after single or repeated exposures, respectively, the crude incidence of lung tumors was significantly less (Fisher's exact test; P0.05) in the rats exposed repeatedly. Times to death of rats with lung tumors were compared among groups with similar alpha-particle doses to the lungs after single or repeated exposure to 239PuO2. Those that died with lung tumors after repeated exposures died at times similar to (Mantel-Cox statistic; P0.05) or later than (Mantel-Cox statistic; P0.05) those for 84-day-old rats exposed once. The risk of lung tumors in rats per unit dose to the lungs was less in the rats exposed repeatedly than in those exposed once. It was concluded that alpha-particle doses to the lung of rats exposed repeatedly to aerosols of 239PuO2 were not more carcinogenic and possibly were less carcinogenic than the dose after a single inhalation exposure when rats with similar lifetime alpha-particle doses to the lungs were compared. The relative biological effectiveness in rats of the alpha-particle dose to the lungs from inhaled 239PuO2 relative to beta-particle doses to the lungs from inhaled 144CeO2 was 21 +/- 3.

Light microscopy, morphometry, and cytokinetic techniques were used to examine the dynamics of plutonium-induced pulmonary proliferative lesions and neoplasms in rats at several intervals to 450 days after inhalation exposure to aerosols of 239PuO2. Maximal increases in alveolar and bronchiolar epithelial cell labeling were seen at 30 days; decreasing subsequently, the levels remained elevated above control indices. Focal proliferative epithelial lesions developed in the lung by 180 days and before the onset of pulmonary neoplasms. Pulmonary neoplasms, predominantly adenocarcinomas and squamous cell carcinomas, were initially observed at 308 days. The proliferative lesions progressed through a succession of morphological changes leading to the development of neoplasms. The volume density (fraction) and epithelial surface area of foci of alveolar epithelial hyperplasia increased progressively between 180 and 450 days after exposure, in contrast to the other proliferative lesions. We conclude that plutonium-induced pulmonary neoplasms develop through a succession of focal proliferative lesions that represent developmental preneoplastic lesions. Progressive increases in volume and epithelial surface area of the alveolar epithelial hyperplasias suggest that they may be more at risk for neoplastic transformation than the other histological types of proliferative foci.

To develop a better understanding of the influence of cumulative radiation dose and dose rate to the lungs on the biological responses to inhaled radionuclides, several studies are in progress at this institute in which laboratory animals have been exposed once or repeatedly to aerosols of insoluble particles containing 144Ce or 239Pu. In the study reported here, F344 rats were exposed repeatedly to aerosols of 144CeO2 beginning at 94 days of age to reestablish desired lung burdens of 1.9, 9.2, 46, or 230 kBq of 144Ce every 60 days for 1 year (seven exposures). Other 94-day-old rats were exposed once to achieve similar desired initial lung burdens of 144Ce. Older rats were exposed once to achieve desired initial lung burdens of 46 or 230 kBq when 500 days of age, the age of the repeatedly exposed rats when exposed for the last time. Control rats were either unexposed, sham-exposed once or repeatedly, or exposed once or repeatedly to stable CeO2. Approximately equal numbers of male and female rats were used. The cumulative beta-radiation doses to the lungs, liver, and skeleton of rats exposed repeatedly were similar to those of rats with similar total lung burdens of 144Ce from a single inhalation exposure. The average beta-radiation dose rate to the lungs of the rats exposed repeatedly was about one-fifth of that in rats with similar total lung burdens after a single exposure.

Groups of 94-day-old F344/Crl rats were exposed repeatedly to aerosols of 144CeO2 to reestablish desired lung burdens of 1.9, 9.2, 46, or 230 kBq of 144Ce every 60 days for 1 year (seven exposures). Other 94-day-old rats were exposed once to achieve similar desired initial lung burdens of 144Ce. Older rats were exposed once to achieve desired initial lung burdens of 46 or 230 kBq when 500 days of age, the same age at which rats had the last of the repeated exposures. Control rats were either unexposed, sham-exposed once or repeatedly, or exposed once or repeatedly to stable CeO2. Approximately equal numbers of male and female rats were used. The median survival time and cumulative percentage survival curves were significantly decreased only in male and female rats exposed repeatedly to reestablish a 230-kBq lung burden and among the 94-day-old male rats exposed once to achieve a 230-kBq lung burden of 144Ce. The crude incidences of primary lung cancers (well described by a single Weibull distribution function), time to death with lung tumors, and risk of lung cancer per unit of beta-radiation dose to the lungs were correlated with the cumulative beta-radiation dose rather than the rate at which the dose was accumulated. A linear function, 70 (+/- 7.3) + -0.15 (+/- 0.056) x dose (+/- SD), adequately described the excess numbers of rats with lung cancers over a beta-radiation dose range to the lungs of 6.8 to 250 Gy for two groups of rats with the highest doses to the lungs after a single exposure and for two groups with the highest doses after repeated exposure.

A spontaneous neurological disease in cats characterized by behavioural and motor disturbances was investigated by clinical, morphological and immunological methods. Neuropathological examination showed a marked inflammatory reaction in the cerebral leptomeninges and the grey matter of the brain. In the white matter, the reaction was moderate. The changes consisted of perivascular cuffing by mononuclear cells and neuronal damage. The brain stem (thalamus, mesencephalon, caudal colliculus) was most severely affected. The spinal cord and its leptomeninges were involved to a lesser degree. The histopathological picture as well as the laboratory findings suggests a viral cause of the disease. The morphology of the disease and serological as well as immunohistochemical results indicate that this disorder is different from previously known feline viral encephalitides.

An ethanol extract of the fruits of Solanum tuberosum was partitioned between water and petroleum ether. The aqueous fraction had contractive effect on the isolated guinea-pig ileum. Chromatography of this fraction on Sephadex LH-20 and silica gel was monitored by the guinea-pig ileum test and resulted in isolation of serotonin. HPLC analysis of the content of serotonin in fruits, leaves and tubers of the plant, showed that the fruits and leaves contained 7.5 micrograms and 2 micrograms, respectively, of serotonin/g fresh weight. In agreement with previous observations, no serotonin was detected in the tubers.

We investigated the modifying effects of preexisting, bleomycin-induced pulmonary fibrosis on the deposition, retention, and biological effects of inhaled 239PuO2 in the rat. Among rats exposed to similar airborne concentrations of 239PuO2, initial lung burdens of 239Pu per kilogram body mass were similar whether or not pulmonary fibrosis was present. However, clearance of 239Pu from the lungs was significantly decreased in the rats with preexisting pulmonary fibrosis. The incidence of lung lesions (epithelial hyperplasia, diffuse macrophage increases and aggregation, and loose and dense connective tissue) was significantly greater among rats with preexisting pulmonary fibrosis than among the exposed controls. Rats with preexisting fibrosis had shorter life spans than 239PuO2-exposed control rats. When groups of rats with similar alpha doses to the lungs were compared, the incidences of neoplastic lesions in the lung, the times to death of rats with lung neoplasms, and the risk of lung tumors per unit of alpha dose to the lungs in rats with or without pulmonary fibrosis were similar. The results of this study suggest that humans with uncomplicated pulmonary fibrosis may not be more sensitive to the carcinogenic effects of inhaled 239PuO2 than are individuals with normal lungs, assuming that the total alpha dosesmore » to the lungs are similar.« less

Male Syrian hamsters were scheduled to be exposed by inhalation approximately every 60 days for 1 year (7 exposures) to aerosols of 239PuO2 beginning at 84 days of age. Other hamsters were exposed once when 84 or 320 days of age. Plutonium-239 deposited in the lungs by the repeated inhalation exposures was cleared from the lungs at a rate similar to that following a single inhalation exposure. The incidence of radiation pneumonitis, bronchiolar epithelial hyperplasia, and alveolar squamous metaplasia were the only lesions that were related to radiation dose. Only two primary lung tumours were found among the hamsters exposed to 239PuO2. No primary lung tumours were found in the control hamsters. It was concluded that the incidence of lung tumours was not increased by the protraction of the alpha radiation dose to the lungs from repeated inhalation exposure.

The effect of preexisting pulmonary disease on the deposition and retention of inhaled relatively insoluble radiolabeled particles was studied. Two dogs were exposed to 500 ppm sulfur dioxide 2 h/day, 5 days/week for 16 weeks and two dogs were sham-exposed to filtered air for the same period. Dogs exposed to sulfur dioxide developed chronic bronchitis during this exposure period. After the bronchitis had been established for 10 weeks, all 4 dogs were exposed by inhalation to 1.7 micron aerodynamic diameter 134Cs-labeled fused aluminosilicate particles (FAP) and the exposures to SO2 were discontinued. The bronchitis resolved within 4 weeks after SO2 was discontinued. Dogs were periodically whole-body counted and excreta were periodically collected and counted over a 385-day period following the 134Cs exposure. Deposition of the [134Cs]FAP in the pulmonary region averaged 42% for control dogs and 16% for dogs exposed to SO2; deposition in extrapulmonary regions of the respiratory tract averaged 58% for control dogs and 84% for dogs exposed to SO2. Although deposition patterns were different, early and long-term clearance pathways and rates were the same for all dogs, as indicated by analysis of whole body, excreta, and tissues for their burdens of 134Cs.

The fractional deposition of cigarette smoke particles in the respiratory tracts of rats was studied. Male and female rats were conditioned in nose-only exposure tubes 25 min/day for 2 days, exposed to cigarette smoke at mass concentrations of 95 or 341 mg/m3, 25 min/day for 3 days, and then exposed to smoke at mass concentrations of 212 and 657 mg/m3, 25 min/day for 5 days. Mainstream cigarette smoke was generated by a modified Walton smoking machine from two 1R3 research cigarettes burned sequentially for each exposure. Deposition studies were conducted by placing the rats in plethysmograph tubes to allow respiratory minute volume measurements during exposure, then exposing them to [14C]dotriacontane-labeled cigarette smoke at mass concentrations of 202 or 624 mg/m3 for 25 min, using the same smoking machine. Size distribution, real-time concentration, and 14C activity of the smoke particles were determined using a multijet Mercer impactor, a real-time aerosol monitor, and filter samples, respectively. Immediately after the exposure, the rats were terminated to determine the distribution of the 14C. Individual lung lobes, trachea and lobar bronchi, head, larynx, kidneys, liver, gastrointestinal (GI) tract, blood, and depleted carcass of each rat were analyzed for 14C content. Results showed that the GI tract contained 16-31% of the total activity, indicating significant clearance from the large airways and nose to the GI tract during the exposure and during the 10-15 min between the cessation of the exposure and the removal of the organs. Total deposition of the inhaled 14C activity was 20.1 +/- 1.6% for both exposure concentrations. The intrapulmonary deposition fractions (lung lobes plus airways below the lobar bronchi) were 12.4 +/- 0.9 and 15.9 +/- 1.4% for concentrations of 202 and 624 mg/m3, respectively, suggesting a slight enhancement in upper airway deposition for animals exposed to the higher smoke concentration.

This study was done to produce a model of chronic bronchitis. Twelve beagle dogs were exposed to 500 ppm sulfur dioxide (SO2) for 2 h/d, 5 d/wk for 21 wk and 4 dogs were sham-exposed to filtered ambient air for the same period. Exposure effects were evaluated by periodically examining the dogs using chest radiographs, pulmonary function, tracheal mucous clearance; and the cellular and soluble components of bronchopulmonary lavage fluids. Dogs were serially sacrificed after 13 and 21 wk of exposure and after 6 and 14 wk of recovery. Clinical signs produced in the SO2-exposed dogs included mucoid nasal discharge, productive cough, moist rales on auscultation, tonsilitis, and conjunctivitis. Chest radiographs revealed mild peribronchiolar thickening. Histopathology, tracheal mucous clearance measurements, and lavage cytology were consistent with a diagnosis of chronic bronchitis. It is concluded that repeated exposure to 500 ppm SO2 for 21 wk produced chronic bronchitis in the beagle dog. Complete recovery occurred within 5 wk following cessation of SO2 exposure.

Lundgren , D. L. (University of Utah, Salt Lake City), B. D. Thorpe, and C. D. Haskell . Infectious diseases in wild animals in Utah. VI. Experimental infection of birds with Rickettsia rickettsii . J. Bacteriol. 91: 963–966. 1966.—Chickens, pigeons, pheasants, sparrow hawks, red-tailed hawks, ravens, magpies, and a marsh hawk were inoculated with Rickettsia rickettsii , the etiological agent of Rocky Mountain spotted fever. The development and persistence of complement-fixing (CF) antibodies and rickettsemias were tested for in these birds. Rickettsiae were recovered from the blood of a number of birds up to the 16th day after inoculation, whereas only the pigeon was found to develop high CF antibody titers. It was concluded that certain species of birds have the potential of contributing to the dissemination of R. rickettsii in nature, and that the CF test is generally unsuitable for serological diagnosis of this organism in birds.

Summary The results of a five-year survey for Q fever in wild animals and livestock in the Great Salt Lake Desert of Utah have been presented. Complement-fixing antibodies to Coxiella burnetii were demonstrated in the sera of 19 wild mammalian species, and in cattle sera. The Q fever rickettsiae were recovered from 15 wild mammalian species, one sheep, and also from ticks, fleas, lice, and mites. A number of birds tested were found to contain neither detectable Q fever antibody nor rickettsiae. The incidence of isolations and seropositives reached a relatively high peak in certain western Utah areas during 1960, indicating an epizootic in the fauna of the area. The relationship of previously reported experimental data to survey results is discussed.

A total of 1484 rectal, nose and throat swabs from 734 dogs were tested for human enteroviruses. Six isolations of coxsackievirus B1 were obtained; two from Beagle dogs, two from Dalmation dogs and two from mongrel dogs. The relationship of these viruses in dogs to the epidemiology of the virus is unknown. An untyped adeno-like virus was recovered from a mongrel dog. Eleven adenoviruses, three polioviruses and one coxsackievirus were recovered from 632 swabs from 559 humans in a comparative study.

Fifty-four coyote pups 1 to 2 months of age and 18 pups 6 to 7 months of age, divided into groups of six each, were inoculated with 10-fold serial dilutions of Venezuelan equine encephalomyelitis (VEE) virus. Blood and serum collected from these animals after inoculation were tested for viremia and antibody, respectively. In nearly all of the young pups signs of illness developed. Virus was recovered from six of 13 1- to 2- month-old pups that died between the 2nd and 18th day after inoculation. There were no signs of illness in any of the older pups other than temporary loss of appetitie, and none succumbed. There was a complete remission of signs of illness in all animals that survived. Infected animals had viremia, which persisted for 3 to 5 days, and complement-fixing, hemagglutination-inhibiting, and neutralizing antibody. The concentration of virus in the blood was great enough and of sufficient duration to permit mosquitoes to obtain an infected blood-meal. We concluded that because of its low population density the young coyote would not have a major role in the cycle of VEE in nature, although conditions for its acting as a host in enzootic areas may exist.

The effects of chronic beta-radiation from inhaled /sup 144/CeO/sub 2/ that resulted in beta-radiation doses of 16,000 to 19,000 rads to the lung during 8- to 20-wk intervals had little or no effect on the influenza virus concentration or hemagglutinin titers in the lungs of virus inoculated mice when compared with virus inoculated control mice. There was, however, a slight effect in suppressirg hemagglutination inhibitirg antibody formation. Lung weights, lung lesions and mortality of mice inoculated with influenza virus at 8, 12 and 20 wk after inhaling /sup 144/CeO/sub 2/ were reported. Increased mortality occurred at 8 wk, increases in all three parameters at 12 wk and increases in lung weights and lung lesions were observed at 20 wk. Mice that inhaled /sup 144/ CeO/sub 3/ and were inoculated with virus had significant histopathological changes. These were most marked in the mice infected with virus at 20 wk postexposure in which squamous cell differentiation was a prominent finding progressing in one case to a superficial resemblance to epidermoid carcinoma. (auth)

To determine the long-term biological effects of protracted alpha irradiation of the lung, 84-day-old C57BL/6J mice were repeatedly exposed by inhalation to aerosols of 239PuO2 every other month for up to six exposures in 10 months to reestablish lung burdens of 20, 90, or 460 Bq. Other mice were exposed only once when either 84 or 460 days of age to achieve desired initial lung burdens of 20, 90, 460, or 2300 Bq. Suitable control groups were maintained. Groups of mice with similar cumulative alpha doses to the lung had 3.4 to 4.4 times greater incidence of pulmonary tumors (adenomas and adenocarcinomas) when the dose to the lung was protracted by the repeated inhalation exposures compared to mice that received a single inhalation exposure. Excess pulmonary tumors per unit dose to the lung were also greater in groups of repeatedly exposed mice compared to those exposed only once. Repeatedly exposed mice also died earlier with pulmonary tumors than did those exposed once. It appears that protraction of an alpha dose to lungs increases the carcinogenic risk of inhaled 239PuO2 in mice.

The dose of cyclophosphamide that permits the colonization of the nasopharynx with Pseudomonas aeruginosa and the survival of the animal was determined in mice. This dose, 100 mg/kg of cyclophosphamide, allowed P aeruginosa to colonize but not invade mouse nasal epithelium. Mice treated with 100 mg/kg cyclophosphamide were exposed to aerosol of 35S-labeled P aeruginosa to study clearance. Results indicated that this dose of cyclophosphamide suppressed both the pulmonary clearance of viable P aeruginosa (in situ killing) and the clearance of radiolabeled P aeruginosa (mechanical clearance).

Summary Studies were conducted on the subcutaneous and intraperitoneal susceptibility to Brucella abortus, B. suis, B. melitensis, and B. neotomae of laboratory mice, guinea pigs, and rabbits and of 12 species of wildlife. These wild mammals included: deer mice (Peromyscus maniculatus), pinyon mice (Peromyscus truei), montane meadow mice (Microtus montanus), desert wood rats (Neotoma lepida), Ord and chisel-toothed kangaroo rats (Dipodomys ordii and Dipodomys microps), white-tailed antelope squirrels (Ammospermophilus leucurus), and black-tailed jack rabbits (Lepus californicus). The mice were generally found to be relatively susceptible to these bacteria, while the rats, lagomorphs, and the squirrels were more resistant. The persistence of serum agglutinins and viable organisms in the tissues of certain of these animals was determined up to 24 months after infection. All four bacterial species caused chronic infection in the animals, but no evidence was obtained to show that they pass the organisms in their excreta or other body wastes. Bacteremia was found to occur regularly in many of the animals during the early stages of infection, and the possibility of transmission by ectoparasites was noted. Mule deer (Odocoileus hemionus) and sheep were found to be relatively resistant to locally isolated strains of B. neotomae.