Atherosclerosis induced by infection with Marek's disease herpesvirus in chickens.

BACKGROUND: This research was suggested after crystals that we observed in herpesvirus-infected cell cultures were identified as cholesterol. Other reports and the development of defined reagents led us to select the use of Marek's disease herpesvirus (MDV) infection of chickens to demonstrate a potential role of herpesviruses in the pathogenesis of atherosclerosis. Available for our use were a clone-purified strain of MDV of known virulence, genetically selected, specific pathogen-free chickens, and appropriate isolation facilities to design controlled experiments to fulfill Koch's postulates. METHODS AND RESULTS: Experiments were performed to test the roles of both MDV and dietary cholesterol in atherosclerosis. The birds were examined 7 months after MDV infection with and without cholesterol feeding for gross and microscopic arterial lesions. Atherosclerotic lesions were found only in infected normocholesterolemic or hypercholesterolemic birds. The character and distribution of these lesions closely resembled those found in the chronic human arterial disease. Atherosclerotic lesions were not found in uninfected birds even if the birds were hypercholesterolemic. CONCLUSIONS: Evidence was obtained from other experiments that after MDV infection, cholesterol and cholesteryl esters accumulated in cell cultures and in atherosclerotic lesions. These changes were associated with altered enzymatic activity of the cholesterol synthesis cycle. Immunization with turkey herpesvirus vaccine or SB-1 vaccine prevented atherosclerotic lesions.

A new solid-phase support for oligonucleotide synthesis.

An alternative solid support for oligonucleotide synthesis was developed by coupling a polymer colloid to a modified polyethylene filter disc. The functions on the polymer colloid not used for attachment to the surface were derivatized with a Jeffamine diamine and loaded with appropriate deoxynucleoside succinates. The performance of this support system was evaluated and compared to existing resins.

Tissue tropism of three cloacal isolates and Massachusetts strain of infectious bronchitis virus.

Three virus isolates (ECV-1, -2, and -3) recovered from cloacae of chickens in flocks that experienced drops in egg production were identified as infectious bronchitis virus (IBV), based on characteristic embryo lesions, chloroform sensitivity, coronavirus morphology, and serology. Because these isolates were recovered from the cloacae of the hens, their tissue tropism was compared with the prototype strain of IBV, Massachusetts-41 (M-41), in experimentally inoculated chickens. During the 39-day period postinoculation (PI), virus isolation was attempted from digestive and respiratory tracts, kidney, and cloacal swabs. ECV-1, ECV-2, and M-41 were more frequently recovered from the cecal tonsils than from other tissues. ECV-1, ECV-3, and M-41 were also recovered from kidney for up to 39 days PI. ECV-2 and ECV-3 had a limited distribution in respiratory tissues, being isolated only sporadically from trachea, bronchus, and lung. Surprisingly, ECV-2 was isolated from esophagus at 2, 16, 30, and 39 days PI; otherwise, its distribution in other tissues was sporadic. Results confirmed that IBV, including M-41, can infect a variety of tissues and that some isolates may be recovered frequently from digestive tract tissues, particularly from the cecal tonsils.

Cerebral blood flow evaluation of arteriovenous malformations with stable xenon CT.

Twenty patients with supratentorial arteriovenous malformations (AVMs) were evaluated with angiography, conventional CT, and stable xenon CT to determine cerebral blood flow. Contralateral and ipsilateral regions of interest relative to the AVM were evaluated from cerebral blood flow maps and correlated with angiography. A significant decrease in cerebral blood flow was observed in the ipsilateral cortical gray matter adjacent to the AVM relative to the corresponding contralateral cortex (mean difference = 9.52 ml/100 g/min, p less than .01). The larger AVMs (greater than 8 cm3) were associated with a more marked decrease with a mean difference of 12.22 ml/100 g/min (p less than .02). Regions of interest were also chosen on the basis of angiographic findings, which suggested areas of decreased flow. Comparison of these areas with analogous contralateral areas also showed a significant decline in cerebral blood flow (mean difference = 8.86 ml/100 g/min); this decline was greater with larger AVMs (volume greater than 8 cm3), which had a mean difference of 11.38 ml/100 g/min (p less than .01). Our correlative study enabled us to pinpoint the regions most likely to have reduced flow from an AVM.

Changes in melanin granules in the fox due to coat color mutations.

Hair samples from 11 different coat color phenotypes of the fox (Vulpes vulpes) were examined microscopically to determine the effects of several mutations on melanin granule color and distribution. Standard silver (b/b) causes the production of eumelanin rather than the phaeomelanin produced in wild-type red fox. Fromm brown (bf/bf) and Collicott brown (cb/cb) change the shape of the granules and convert eumelanin to brown and dark brown, respectively. The color dilution of Eastern Pearl (pe/pe) and Mansfield Pearl (pm/pm) is caused by clumping of granules in specific manners. Hairs from animals expressing more than one mutant gene, such as Amber (b/b pe/pe bf/bf), show the color and distribution of granules expected from interactions of independent loci.

Indications of immunodepression in chickens infected with various strains of Marek's disease virus.

The effect of infection by various strains of Marek's disease virus (MDV) on the immune function of 3-week-old chickens was examined. MDV strains of low (CU-2, RB-7) and high (RB-3, MD-5, and MD-11) pathogenicity were compared with prototype JM-10 strain of moderate pathogenicity. Mortality, whole body weight, relative weights of lymphoid organs, histopathology, humoral antibody responses to thymus-dependent and -independent antigens, and in vitro lymphocyte responses to mitogen stimulation were investigated at 1, 2, and 3 weeks postinfection. MDV strains of high pathogenicity significantly depressed responses at 3 weeks postinfection, seeming to indicate the ability of these viruses to induce severe immunodepression. However, the fact that the moderately pathogenic and even some of the low-pathogenicity strains induced immunodepression suggests that other viral mechanisms are also important in its determination.

Stereotactic heavy-particle irradiation of intracranial arteriovenous malformations.

Stereotactic irradiation appears to be effective in causing partial or complete thrombosis of AVM that are not surgically resectable. Use of heavy particles generated in a cyclotron allows better spatial definition and dose distribution than do other methods, allowing larger AVM to be treated. From these preliminary results, it is evident that heavy-particle irradiation therapy, like proton beam therapy, does not offer protection from recurrent hemorrhage for at least 12 months, nor is it devoid of major complications; it does offer a noninvasive mode of therapy for AVM that are difficult to treat surgically, however.

Virus-induced atherosclerosis. Herpesvirus infection alters aortic cholesterol metabolism and accumulation.

Infection of normocholesterolemic, specific-pathogen-free chickens with Marek's disease herpesvirus (MDV) has been shown histologically to lead to chronic atherosclerosis like that in humans. The development of herpesvirus-induced atherosclerosis in vivo and the presence of specific Marek's antigen within aortic cells suggested that MDV infection may modify lipid metabolism and lead to significant lipid accumulation. Experiments reported herein were designed to determine the types and quantity of lipid present in aortas from MDV-infected and uninfected chickens between 2 and 8 months of age following infection and assess one possible mechanism of lipid accumulation by evaluating the effect of MDV infection on aortic cholesterol and cholesteryl ester (CE) metabolism. Chromatographic-fluorometric analyses indicated that at 4 and 8 months of age after MDV inoculation, MDV-infected animals had a significant (P less than 0.05) two-fold to threefold increase in total aortic lipid accumulation characterized by significant increases in cholesterol, CE, triacylglycerol, and phospholipid as compared with aortas from uninfected animals. At 8 months of age, similar increases in aortic lipid accumulation were observed in MDV-infected animals as compared with those animals vaccinated with turkey herpesvirus and later challenged with MDV. CE synthetic activity was increased significantly by 50% at 4 months of age in the MDV-infected group as compared with the uninfected group, which could explain the initial increase in CE accumulation. By 8 months of age, the authors also observed a twofold increase in CE synthetic activity and a 30% and 80% reduction in lysosomal and cytoplasmic CE hydrolytic activities, respectively, in aortas of MDV-infected chickens as compared to controls. Moreover, infection with MDV blocked the activation of cytoplasmic CE hydrolytic activity by dibutyryl cyclic AMP or exogenous cyclic AMP-dependent protein kinase. Taken together, these results suggest that lipid accretion in aortas of MDV-infected chickens results, in part, from alterations in cholesterol/CE metabolism during early stages of the disease. These findings support the hypothesis that human atherosclerosis may result from specific herpesvirus infection which can alter lipid metabolism and lead to lipid accretion.

Current status of bioassays in genetic toxicology--the dominant lethal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The term dominant lethal may be defined as death of the heterozygote arising through multiple chromosomal breaks. The assay is generally conducted by treating male animals, usually mice or rats, acutely (1 dose), subacutely (5 doses), or over the entire period of spermatogenesis. Animals treated acutely or subacutely are mated at weekly intervals to females for a sufficient number of weeks to cover the period of spermatogenesis. Those treated for the entire spermatogenic cycle are mated for 1 or 2 successive weeks at the termination of treatment. Females usually are killed at 14 days of pregnancy and examined for the number of total implantations in the uterus, the number of implantations classified as early deaths, and, in some cases, the number of corpora lutea. The category of early death is the most significant index of dominant lethality. A total of 249 papers were reviewed and 140 chemicals were evaluated. Of the 140 chemicals, 65 were positive by the criteria used by the Work Group in evaluating each publication. The category of "positive" includes those responses of a borderline nature. 99 chemicals were declared negative. There is considerable overlap of chemicals in both categories, which accounts for the incongruity in the total number of chemicals tested and the number considered positive and negative. A total of 44 animal carcinogens have been tested in the dominant lethal assay, 26 of which were positive and 18 negative for a correlation of 59%. The role of the assay should be that of confirming positive results from lower tier chromosomal aberration-detecting systems (confirming in the sense of indicating the ability of the chemical to penetrate gonadal tissue and to produce cytogenetic damage). The dominant lethal assay should not be used as a risk assessment method.

Altered cholesteryl ester cycle is associated with lipid accumulation in herpesvirus-infected arterial smooth muscle cells.

We describe herein the effects of Marek's disease herpesvirus (MDV) on cholesterol and cholesteryl ester metabolism in cultured chicken arterial smooth muscle cells. Infection of arterial smooth muscle cells from specific pathogen-free chickens with MDV, but not a virus control, herpesvirus of turkeys led to a 7-10-fold increase in the accumulation of free and esterified cholesterol and a 2-fold increase in phospholipids. The cellular lipid changes observed in the MDV-infected arterial smooth muscle cells resulted, in part, from the following: decreased low-density lipoprotein-cholesteryl ester hydrolysis due to decreased lysosomal (acid) cholesteryl ester hydrolytic activity; increased de novo synthesis of cholesterol; decreased excretion of free cholesterol; and, both increased cholesteryl ester synthetic activity and decreased cytoplasmic (neutral) cholesteryl ester hydrolytic activity which resulted in increased incorporation of oleic acid into cholesteryl ester. Other changes noted in the MDV-infected cells as compared to uninfected cells included a 2-fold increase in both total protein synthesis and lysosomal and microsomal marker enzyme activities. These alterations in lipid and protein metabolism in MDV-infected arterial smooth muscle cells may explain in part our in vivo findings that herpesvirus (MDV) infection of specific pathogen-free chickens fed a normocholesterolemic diet will induce arterial thickening and lipid accumulation resembling human atherosclerosis.

Pathogenesis of infection with attenuated Marek's disease virus strains.

Recently, attenuated Marek's disease virus (MDV) became of renewed interest as a component in bi- or polyvalent vaccines. The effect of attenuation on the pathogenesis of infection was investigated. Cloned preparations of the JM-16, BC-1A and RB-1B strains of MDV were attenuated by serial passage in chick kidney cells or chicken embryo fibroblasts. Subclones were obtained from the JM-16 strain at passage (p) 26 (JM-16d) and 50 (JM-16a, b and c). The passage level at which each virus became attenuated was dependent on the virus strain. The highly oncogenic RB-1B strain was still oncogenic after 37 passages, while JM-was already attenuated at p. 27. In ovo infection of high passage JM-16 and RB-1B (p 54 and 55) demonstrated the presence of residual pathogenicity. Attenuated virus failed to induce the early cytolytic infection which is characteristic for the pathogenesis of infection with oncogenic MDV. Low levels of lymphocyte-associated viraemia could be detected after infection with all attenuated viruses except with the subclone JM-16a. This virus was, however, able to induce moderate protection against challenge and antibodies were detectable, suggesting that cells other than lymphocytes became infected. The pathogenesis after in ovo infection with attenuated virus was similar to that after infection of chicks. The in vivo data suggested that attenuation reduced the efficiency of infection of, or virus replication in, lymphocytes. A markedly reduced ability to establish in vitro infection of lymphocytes by exposure to heavily infected lymphocytes was observed, and this supports this hypothesis. The altered characteristic of attenuated virus to infect lymphocytes in vivo or in vitro was not caused by the selection of temperature sensitive or thymidine kinase negative mutants.

Orthotopic ovarian transplantation in young and aged CBA mice.

Reproductive and hormonal changes associated with CBA mice which were orthotopically transplanted with ovaries from young or aged CBA mice were studied. Reproductive decline was defined by the number of mice mating and the number of implantation sites and resorptions in pregnant mice. A high percentage of resorptions was observed in aged mice receiving ovaries from young mice. An increase in the number of resorptions and a decrease in the number of implantation sites was observed in young mice transplanted with aged ovaries. Concentrations of LH, FSH and Prl were analyzed by radioimmunoassay from plasma of pregnant and nonpregnant transplanted mice, ovariectomized and sham-operated controls. Young ovariectomized mice had elevated concentrations of LH and FSH when compared to sham-operated controls. No differences were noted between the aged ovariectomized mice although the gonadotrophin levels in these animals were higher than those in comparable young groups. The highest LH and lowest FSH concentrations were found in young to young transplanted mice. The Prl concentrations in transplanted mice did not vary between groups. These data suggest that the hypothalamic-hypophyseal complex is functioning adequately in all transplanted groups and age-related changes in the ovary and uterus are responsible for embryonic mortality.

Field trials with a bivalent vaccine (HVT and SB-1) against Marek's disease.

White leghorn chickens on five farms were given a bivalent Marek's disease (MD) vaccine consisting of turkey herpesvirus (HVT) and SB-1 (a nononcogenic MD virus); other chickens received only HVT. The farms had histories of "vaccination failures," presumably owing to an exceptionally virulent challenge MD virus. The bivalent vaccine uniformly protected chickens better than HVT alone between 12 and 16-20 weeks of age, when serious MD losses occurred. During that period, total mortality in groups given both viruses ranged from 0.39 to 1.26% (mean 0.86%), whereas that in HVT-vaccinated groups not exposed to SB-1 varied from 1.92 to 7.44% (mean 3.43%). Chickens in pens or rows with close contact to those given bivalent vaccine also had low MD mortality rates (0.46-1.06%, mean 0.77%), probably from the spread of SB-1.

Herpesvirus-induced atherosclerosis in chickens.

Repeated experiments have established that infection with Marek's disease herpesvirus (MDV) leads to atherosclerosis in specific pathogen free (SPF) normocholesterolemic chickens. Neither normocholesterolemic nor hypercholesterolemic uninfected SPF chickens develop this disease. The MDV-induced arterial disease is remarkably similar to chronic human atherosclerosis. Cholesterol and saturated cholesteryl esters accumulated in cultured arterial smooth muscle cells (SMC) infected with MDV. Similar preliminary observations were made in vivo. These findings suggest that MDV-induced alteration of SMC lipid metabolism is of major importance in the pathogenesis of MDV-induced atherosclerosis. In addition, immunization with turkey herpesvirus, used commercially to prevent MDV-induced tumors in chickens, also protected against MDV-induced atherosclerosis. This animal model has introduced important new dimensions and tools in atherosclerosis research: a defined etiologic agent (MDV) that causes atherosclerosis in a defined animal of known genetic susceptibility to the etiologic agent. With these tools, important mechanisms in the pathogenesis of atherosclerosis may be established in a relatively short period of time. Further, this animal model should be considered important in other models of atherosclerosis research because herpesvirus infections are ubiquitous in these animals. Finally, because humans are widely and persistently infected with up to five herpesviruses, these studies may lead to the understanding and eventual control of human atherosclerosis.

Post-meiotic cell mediation of behavior in progeny of male rats treated with cyclophosphamide.

Transmission of the effects of paternal pre-fertilization exposure to a genotoxic drug being manifested in the F1 progeny is well established using the traditional rodent dominant lethal protocol [7]. More recently, the induction of genotoxic effects in the F1 progeny following chronic paternal exposure prior to fertilization has been described in rats using behavioral testing as an endpoint [1]. In our initial study, male F344 adult rats were treated chronically with 10 mg/kg cyclophosphamide (CP) or saline for 5 weeks (5 days of daily treatment and 2 days of rest). The present study describes the behavior of progeny whose fathers were treated with a single intraperitoneal injection of 10 mg/kg CP and mated at different times post-injection. This design showed that post-meiotic germ cells are the most sensitive to the effects of CP as seen by behavioral testing of the F1 progeny.