Protection against 7, 12-dimethylbenz[a]anthracene-induced rat mammary carcinoma by infection with mouse xenotropic type C virus.

A single ip inoculation of female, outbred Sprague-Dawley rats with a viable mouse xenotropic type C virus significantly reduced the incidence and/or retarded the development of mammary carcinoma induced by 7, 12-dimethylbenz[a]anthracene administered orally 7 days after virus. Although infectious virus could not be isolated from organs of infected rats, high titers of circulating and tumor-associated antibodies were detected against the viral internal core protein p30, and a low-grade antibody response to intact virus or envelope glycoprotein was found. Moreover, a cell-mediated immune response, measured by lymphocyte transformation, was detected with the use of intact virus but not with p30 antigen. No immunity developed after a single inoculation of UV-inactivated virus. These data indicated that inoculation of adult individuals of heterologous species with viable xenotropic mouse type C virus resulted in the rapid disappearance of infectious virus from the recipient, followed by the development of both humoral and cellular immunity to virion constituents. These events led, by unknown mechanisms, to the effective retardation of chemical carcinogenesis when infection preceded carcinogen administration.

Spontaneous adrenal tumors in the aged, ovariectomized NIH swiss mouse without enhanced retrovirus expression.

A high incidence of adrenal tumors was observed in aged female NIH Swiss mice which had been ovariectomized at 2 to 4 weeks of age but not in nonovariectomized controls. Although tumors weighing more than 1 g were not infrequent in the oldest (> 24 months) animals, adrenal glands did not appear macroscopically abnormal before the age of 18 months. Histologically, however, focal or diffuse abnormalities were found in essentially every gland examined from mice over 12 months of age, including glands of normal size. Since the NIH Swiss mouse has been shown to contain an endogenous xenotropic virus whose expression is under hormonal control, the adrenal tumors were examined in detail for evidence of abnormal viral expression. We were unable, by a variety of techniques, to demonstrate elevated expression of type C virus in these adrenal tumors.

Alteration of the syncytium-forming property of XC cells by productive Moloney leukemia virus infection.

The effect of productive murine leukemia virus (MuLV) infection of the syncytium-forming property of XC cells was studied MuLV-Moloney-infected XC cells, designated XC(M), initially went through a period of spontaneous syncytium formation. The syncytia then disappeared, and XC(M) cells continued to propagate and produce both infectious MuLV and MuLV group-specific antigens. However, XC(M) cells became refractory to syncytium formation that was induced by Kirsten, Friend, Rauscher, and Moloney strains of murine oncornaviruses. These data suggest that XC(M) cells lost their syncytium-forming ability as a result of productive MuLV infection.

In vitro comparisons of SENCAR and BALB/c primary epidermal cells.

Grafting experiments show that the enhanced sensitivity of the SENCAR mouse to skin carcinogenesis by initiation and promotion is a property of the skin itself, suggesting the usefulness of in vitro studies to elucidate the mechanism. Such studies have indicated that cultured epidermal cells of SENCAR mice and the resistant BALB/c strain are remarkably similar in a variety of respects. DNA repair and carcinogen binding are quantitatively similar in cultured cells of SENCAR and more resistant mouse strains. Epidermal Langerhans cell (LC) number and LC-mediated functions were indistinguishable in SENCAR and BALB/c mice. Primary epidermal cells cultured in the presence of various concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid, epidermal growth factor (EGF), hydrocortisone, or fluocinolone acetonide failed to reveal differences in growth between BALB/c and SENCAR cells. Cells from these animals bound comparable amounts of EGF with similar kinetics, and the modulation of this binding by TPA and retinoic acid was indistinguishable between strains. Spontaneous expression of infectious, endogenous xenotropic type C RNA virus at very low levels could be demonstrated in primary BALB/c epidermal cells and both BALB/c and SENCAR epidermal lines resistant to Ca2+-induced terminal differentiation. The number of foci of initiated cells after exposure to carcinogens in vivo or in vitro did not differ significantly between SENCAR and BALB/c, suggesting that SENCAR sensitivity is primarily to promotion. However, there are qualitative differences between SENCAR and BALB/c foci. The appearance of foci of cells resistant to terminal differentiation in untreated SENCAR cultures supports the evidence from in vivo studies for the existence of a constitutively initiated cell population in SENCAR mouse skin.

Disparate response of encephalomyocarditis virus and MM virus to interferon in JLS-V9R cells.

JLS-V9R cells, a Balb/c mouse bone marrow cell line chronically infected with Rauscher leukemia virus, were treated with mouse interferon and inoculated with several different lytic viruses. Relatively low interferon concentrations protected the cells against Sindbis virus, vesicular stomatitis virus and MM virus. In contrast, encephalomyocarditis virus replication was inhibited by less than 1 log even with an interferon concentration of 1000 U/ml. These findings provide further evidence that interferon-induced antiviral effects are mediated through multiple mechanisms and demonstrate that even viruses which are classified within the same family (MM and encephalomyocarditis virus) can exhibit differential interferon sensitivities.

Suppression by phenobarbital of ethionine-induced hepatocellular carcinoma formation and hepatic S-adenosylethionine levels.

An 18-month carcinogenicity study was conducted in male weanling F344 rats (28/group) to examine the effects of the simultaneous feeding of selected concentrations of ethionine and 0.05% phenobarbital in a normal chow diet. The effects of a 1-6-week feeding of phenobarbital and ethionine on the hepatic levels of the related metabolites S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylethionine were also examined. Ethionine at 0.3% or 0.1% induced hepatocellular carcinoma (HCCa) at incidences of 90% (19/21) and 89% (24/27), respectively. Adding phenobarbital to the 0.1% ethionine diet reduced the incidence of HCCa to 36% (10/28) and reduced the number of liver tumor-associated deaths occurring prior to terminal sacrifice from 10/27 to 1/28. No hepatic tumors were observed in rats fed 0, 0.003, 0.01, or 0.03% ethionine. Phenobarbital alone or combined with 0.03% ethionine produced no hepatic tumors. Dietary ethionine at 0.1% reduced the intracellular hepatic level of S-adenosylmethionine to <50% of that seen in control rats. Phenobarbital alone had little effect on either S-adenosylmethionine or S-adenosylhomocysteine levels. The combination of phenobarbital and 0.1% ethionine led to increases in the hepatic levels of S-adenosylmethionine of 40-60% after 3 and 6 weeks of feeding, compared to those seen in rats receiving 0.1% ethionine alone. Ethionine feeding resulted in high levels of S-adenosylethionine in the livers. Combining phenobarbital with ethionine in the diet led to 30-50% reductions in hepatic S-adenosylethionine content. The results indicate that phenobarbital inhibits hepatocarcinogenesis by ethionine, that ethionine may cause HCCa via methyl group insufficiency, and that at levels of < or =0.03% ethionine did not show evidence of tumorigenicity.

RNA-directed DNA polymerase activity of reticuloendotheliosis virus: characterization of the endogenous and exogenous reactions.

Reticuloendotheliosis virus (REV) contains an endogenously instructed, RNA-directed DNA polymerase activity. Both the endogenous and exogenous DNA polymerase activities exhibited up to 10-fold greater activity at the optimum concentration of manganous ion (0.025 mM for exogenous; 0.25 mM for endogenous) than at any concentration of magnesium ion. Antiserum to the DNA polymerase of an REV group virus (spleen necrosis virus) inhibited both endogenous and exogenous DNA polymerase activity of REV, whereas antiserum to the Rous sarcoma virus (Rous-associated virus-0) [RSV(RAV-0)]DNA polymerase did not. The DNA product of the endogenous reaction is associated with the high-molecular-weight RNA of REV and anneals with REV RNA but not with RNA from Rous sarcoma virus.

Replication of Western equine encephalomyelitis virus. II. Cytoplasmic structure involved in the synthesis and development of the virions.

Analysis of the cytoplasmic fraction of chick embryo cells during the exponential phase of Western equine encephalomyelitis (WEE) virus growth showed that the viral ribonucleic acid (RNA) labeled by a short pulse with (3)H-uridine was associated with a structure which sedimented in sucrose density gradients with a coefficient of 65S. The RNA extracted from this structure sedimented in sucrose density gradients at 26S. After a longer period of exposure to (3)H-uridine, the radio-active viral RNA was associated with a structure which sedimented in sucrose density gradients as would materials with coefficients of about 140S. The 140S structure contained viral RNA and viral protein. It was shown that the 140S structures are not virus-induced polysomes. The 140S structure contained predominantly the 40S type of viral RNA and some 26S type. Electrophoretic analysis of the disrupted virion revealed that at least two proteins (types I and II) were present in the purified virion. Only type II protein was present in the 140S structure. Unlike the virion, the 140S structure did not contain any lipid which could be detected by the incorporation of (14)C-choline. These data suggest that the 140S structure represents the internal nucleoprotein part of the virion. The rate of appearance of labeled virus lags behind that of the formation of the 140S structure in infected cells. Pulse-chase experiments with (3)H-leucine suggest that the 140S structure may represent a precursor to the virus particle. The results are discussed in terms of the maturation of WEE virus in the infected cells.

Rapid sensitive assay for interferons based on the inhibition of MM virus nucleic acid synthesis.

A method for assaying mouse interferon based on the inhibition of viral ribonucleic acid (RNA) synthesis was devised. The amount of MM virus and RNA synthesized in interferon-treated L-cell cultures was determined by measuring the amount of (3)H-uridine converted into a trichloroacetic acid-insoluble form after treatment of the infected cultures with 2.5 mug of actinomycin D per ml. The amount of RNA synthesized was inversely related to the concentration of interferon used for treatment. A linear dose-response regression curve was obtained by plotting the log of the amount of RNA made, expressed as a percentage of the control, versus the log of the reciprocal of the interferon dilution. A unit of interferon was defined as that concentration which inhibited nucleic acid synthesis by 50% (INAS(50)). The concentration of mouse interferon could be determined within 24 hr. This assay method, on the average, was approximately half as sensitive as the method which measured the 50% reduction of MM virus plaque number (PDD(50)-MM method), but was, on the average, almost 1.7 times as sensitive as the PDD(50)-VSV method. It averaged approximately 20 times the sensitivity of the methods which used as end points the 70% reduction in yield of MM virus or the complete inhibition of cytopathic effect by MM virus. The reproducibility of the INAS(50) technique was tested in two ways. (i) Four independent assays of an interferon specimen were performed with replicate cultures. The standard deviation was 11.2% of the mean titer. (ii) On different dates, one interferon specimen was assayed seven times and another was assayed four times. The standard deviations were 21.5 and 26.6% of the respective mean titers.

Effect of Estrogen and Other Steroids on MM Virus Infection in Mice.

The effect of several steroid hormones on the susceptibility of mice to infection with MM virus was studied. Estrone, cortisone, and hydrocortisone increased mortality, whereas progesterone, prednisolone, and testosterone had no effect. The viral infection-enhancing (VIE) activity of estrone was maximal when the hormone was given 24 to 72 hr prior to viral inoculation. Less VIE activity was seen when the estrone was administered at the same time as the virus, and hormone treatment 24 hr after inoculation had no significant effect on mortality. Virus was found in the blood 24 hr before it appeared in the brain, regardless of estrone treatment. However, viremia was demonstrated in estrone-treated mice 48 hr before it occurred in control animals. There was no significant difference in the respective titers reached in the sera or the brains of the two groups.

Evidence that detergent-reactivated interferons are not renatured.

The sedimentation rate of human leukocyte interferon (HLIF) reactivated from sodium dodecyl sulphate (SDS) solution was studied by glycerol gradient centrifugation and compared to that of native HLIF. Reactivated HLIF consistently sedimented faster than native HLIF, indicating that full recovery of antiviral activity does not require renaturation of the entire interferon molecule.

Selecting a treatment for primary varicose veins.

The treatment of varicose veins includes injection/compression sclerotherapy and surgical stripping or ligation or both. Surgery appears to be favoured when the saphenous system is involved or when the patient is 35 to 64 years old or presents with ankle edema or flare. On the other hand, sclerotherapy has been found to be more effective in patients with dilated superficial veins or incompetent perforating veins in the lower legs and to be more acceptable and less expensive than surgical treatment.

PIP: At present 3 treatment alternatives for primary varicose veins are available: surgical stripping and ligation, injection/compression sclerotherapy, and a combination of the 2. At least 4 factors contribute to the decision of whether to treat the patient: pregnancy, obesity, oral contraceptive (OC) use, and age. Since varicose veins in pregnant women may later recede, only palliative treatment is recommended before delivery. Deep vein thrombosis may develop as a result of OC use during varicose vein treatment, leading some to advise OC discontinuation. Numerous clinical trials have endeavored to examine the relative effectiveness of treatment methods currently in use. The only randomized trial to evaluate all 3 treatment options over a 3-year period found that surgical stripping was significantly more effective than a combination of ligation and sclerotherapy, and that the combination was significantly more effective than sclerotherapy alone. Surgery appears to be the recommended treatment when the saphenous system is involved; surgery is also preferred for patients 35-64 years of age and for those presenting with signs of ankle edema and flare. Sclerotherapy seems to be more effective for dilated superficial veins and incompetent perforating veins in the lower leg. In addition, sclerotherapy is the most acceptable and least expensive method for the patient. The prevalence of primary varicose veins has been estimated at 20% in Europe and North America, with a female:male ratio of 5:1.

S-adenosylmethionine, S-adenosylhomocysteine and DNA methylation levels in the liver of rats fed methapyrilene and analogs.

The antihistamine methapyrilene (hydrochloride) and four close structural analogs, methaphenilene, methafurylene, thenyldiamine and clorothen, were given to rats at a concentration of 0.1% in drinking water for 34 weeks. Only methapyrilene produced notable histopathological changes in the liver, bile duct hyperplasia and focal cellular change. Methapyrilene produced an early and persistent elevation in the ratio of S-adenosylmethionine to S-adenosylhomocysteine, which was 2.8 times the control levels at 34 weeks; none of the other antihistamines produced so high a ratio or altered the ratio as early. Methapyrilene, but not the other antihistamines, produced a significant increase in the methylation of liver DNA at 20 and 34 weeks, as measured by the level of 5-methyldeoxycytidine. The increase in deoxycytosine methylation is so far the only detected effect of the carcinogen methapyrilene on DNA which is absent in rats treated with its non-carcinogenic analogs.

Transformation of cells in human bone tumor cultures.

Two cell lines from tumors of 16 patients with osteosarcoma and two cell lines from 5 patients with giant cell tumor of bone showed conversion of cell type after 2 to 13 months in culture. Transformed cells of epithelial like morphology appeared in small focal areas and rapidly overgrew nontransformed cells. These cells were characterized by rapid growth, loss of contact inhibition, and growth in soft agar. Attempts to demonstrate virus(es) by electron microscopy, treatment with chemicals, or by inoculation of human cell lines were thus far unsuccessful. Antigens not present in parental cultures were observed in the transformed cells by fixed immunofluorescence test with sera of 13 and 20 osteosarcoma patients and 3 of 8 patients with giant cell tumor of bone. Absorption of positive sera with transformed cells of either osteosarcoma or giant cell tumor removed the reaction but not with absorption with heterophile material or mycoplasma. Presence of group-specific-like antigen (gs-3) in the transformed cells (but not in parent cultures) was shown by immunofluorescence. Fluids of transformed cultures contained heavy RNA similar to that of oncornaviruses. These findings suggest the presence of viral information in some human bone tumors.

Virus retrieval studies in human neoplasia.

Short- and long-term co-cultures of 49 cases of human osteosarcoma cells with bone marrow or peripheral blood cells of patients with different types of leukemia were studied. Morphological changes were observed in 7 of 13 long-term co-cultures resembling those induced by RNA tumor viruses. The changes were accompanied by appearance of cytoplasmic antigen as shown by fixed immunofluorescence test with sera from patients with osteosarcoma, leukemia, and of some apparently normal blood donors. Absorption with Forssman-like substances, whole human embryo cells or osteosarcoma cells demonstrated the reaction to be due to tumor antigen(s) in co-culture cells showing morphological changes. Electron microscopy showed a few type C virus particles in one co-culture. Cell-free filtrates of fluid from the transformed co-cultures induced morphological changes in 1 of 4 human embryo cultures. Uninoculated embryo cultures or those inoculated with filtrates from parental sarcoma or leukemia cultures showed no morphological changes. Human embryo cell cultures treated with fluid from parental leukemic bone marrow but not from parental sarcoma cultures showed appearance of cytoplasmic antigen by immunofluorescence test with sera of osteosarcoma and leukemia patients and of some apparently normal blood donors. Transformed human co-cultures showed the cytoplasmic antigen with 28 of 48 sera of osteosarcoma and leukemia patients tested, after absorption with Forssman-like material, human embryo, and mycoplasma suspensions. Fourteen of 49 sera of normal donors were also positive with the transformed co-cultures. Similar results were obtained in an earlier series of experiments with human embryonic cultures transformed by fluid from different osteosarcoma-leukemia co-cultures when examined by fixed immunofluorescence tests with sera of patients with osteosarcoma and leukemia. In 2 whole human embryo cell cultures showing morphological changes high molecular weight RNA was found, similar to that of RNA animal tumor viruses and in one of the cultures transient reverse transcriptase was detected.

Interferon-inducing characteristics of MM virus.

Interferon induction by MM virus in mice and in L cells was studied. In mice the virus readily induced interferon. The time of appearance was dose-dependent. A large virus dose induced interferon by 4 hr, whereas a small dose resulted in interferon production which paralleled virus replication 24 hr after infection. In L cells the interferon-inducing capacity of the virus was rapidly destroyed by ultraviolet light irradiation. Heating (56 C) of the virus, on the other hand, greatly increased its ability to induce interferon. Interferon production could also be increased by prior treatment of the cells with homologous interferon (priming). The increase in interferon production after priming was dependent on the concentration of interferon used for priming, the length of interferon treatment, and the multiplicity of infection. It is suggested that MM virus might be useful for the further study of the mechanisms involved in the production and action of interferon.

Assay of chick interferons by the inhibition of viral ribonucleic acid synthesis.

A method for assaying chick interferons by their inhibition of viral ribonucleic acid synthesis was devised and evaluated. The technique yielded results faster and had more flexibility than other methods with similar sensitivity and reproducibility.

Influence of interferon on the synthesis of virus particles in oncornavirus carrier cell lines. IV. Relevance to the potential application of interferon in natural infectious diseases.

That interferon reduced the release of C-type oncornavirus particles by chronically infected mouse cells was shown by radiolabeling of the particles with uridine or amino acids and by determination of particle-associated reverse transcriptase. The number of released particles, as determined by direct electron microscopic enumeration, was reduced to a lesser extent. In contrast, interferon failed to affect the number of budding particles and caused a slight increase in the number of completed particles present in the microspace contiguous to the cell membranes. A working hypothesis is that, in the presence of interferon, C-type particle assembly and release are slowed but not arrested; sizable numbers of particles continue to be assembled and released. Some of these particles may be defective in one or more proteins, such as reverse transcriptase or proteins necessary for final release. These in vitro data justify speculation that, in vivo, interferon may be expected to reduce tissue damage due to antigen-antibody complex formation, but not damage due to sytolytic immune attack on cells carrying the antigens.