Clarence E. Chrisp

Treating activated CD4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice.

Human antigen-specific CD4+ T cells become autoreactive after treatment with various DNA methylation inhibitors, including 5-azacytidine, procainamide, and hydralazine. This suggests a mechanism that could contribute to the development of some forms of autoimmunity. In this report we have asked whether T cells treated with DNA methylation inhibitors can induce autoimmunity. Murine CD4+ T cells were treated with 5-azacytidine or procainamide and were shown to respond to syngeneic antigen-presenting cells, similar to CD4+ human T cell clones treated with these drugs. Functional characterization demonstrated that cells treated with either drug spontaneously lysed syngeneic macrophages and secreted IL-4, IL-6, and IFN-gamma. Adoptive transfer of 5-azacytidine- or procainamide-treated cells into unirradiated syngeneic recipients induced an immune complex glomerulonephritis and IgG anti-DNA and antihistone antibodies. These experiments demonstrate that T cells treated with either of two distinct DNA methyltransferase inhibitors are sufficient to induce a lupus-like disease. It is possible that the lysis of macrophages, together with the release of cytokines promoting B cell differentiation, contributes to the autoantibody production and immune complex deposition. These results suggest that environmental agents that inhibit DNA methylation could interact with T cells in vivo to produce a lupus-like illness, a mechanism that could have relevance to drug-induced and idiopathic lupus.

Pathologic characterization of brown Norway, brown Norway x Fischer 344, and Fischer 344 x brown Norway rats with relation to age.

Abstract The rat is a common laboratory animal utilized in a variety of investigations including experimental gerontology. Gerontologic investigations can be compromised when the differences observed when comparing young and old animals are actually differences between normal and disease states. It is of critical interest to know the pathology of the animals being studied and to understand the impact of these disease processes on the parameters being measured. The incidence and average age of occurrence for lesions have been characterized and are reported here for one inbred (Brown Norway) and two hybrid strains (Brown Norway × Fischer 344 and Fischer 344 × Brown Norway) of rat. Total lesion incidence functions as a biomarker of aging for all of the strains examined (p ≤ .00001). These three genotypes have significantly lower incidence of several major pathologic processes (including glomerulonephritis, retinal atrophy, and leukemia) than do the Fischer 344 and the Wistar rats, two commonly utilized strains. Additionally, the BN and F344 × BN F1 hybrid attain 50% mortality at 130 and 146 weeks of age, respectively, which is significantly greater than the 103 weeks for the F344 rat. It is hoped that access to basic information on these three rat genotypes will increase their utilization by the community of gerontologic scientists.

Mutagenicity of airborne particles

The physical and chemical properties of airborne particles are important for the interpretation of their potential biologic significance as genotoxic hazards. For polydisperse particle size distributions, the smallest, most respirable particles are generally the most mutagenic. Particulate collection for testing purposes should be designed to reduce artifact formation and allow condensation of mutagenic compounds. Other critical factors such as UV irradiation, wind direction, chemical reactivity, humidity, sample storage, and temperature of combustion are important. Application of chemical extraction methods and subsequent class fractionation techniques influence the observed mutagenic activity. Particles from urban air, coal fly ash, automobile and diesel exhaust, agricultural burning and welding fumes contain primarily direct-acting mutagens. Cigarette smoke condensate, smoke from charred meat and protein pyrolysates, kerosene soot and cigarette smoke condensates contain primarily mutagens which require metabolic activation. Fractionation coupled with mutagenicity testing indicates that the most potent mutagens are found in the acidic fractions of urban air, coal fly ash, and automobile diesel exhaust, whereas mutagens in rice straw smoke and cigarette smoke condensate are found primarily in the basic fractions. The interaction of the many chemical compounds in complex mixtures from airborne particles is likely to be important in determining mutagenic or comutagenic potentials. Because the mode of exposure is generally frequent and prolonged, the presence of tumor-promoting agents in complex mixtures may be a major factor in evaluation of the carcinogenic potential of airborne particles.

Effects of hepatic arterial yttrium 90 glass microspheres in dogs

A 22‐μm glass microsphere called TheraSphere (Theragenics Corp., Atlanta, GA) has been developed in which yttrium 89 oxide is incorporated into the glass matrix and is activated by neutron bombardment to form the beta‐emitting isotope yttrium 90 (Y 90) before using the spheres as radiotherapeutic vehicles. The injection of up to 12 times (on a liver weight basis) the anticipated human dose of nonradioactive TheraSphere into the hepatic arteries of dogs was well tolerated and produced clinically silent alterations within centrolobular areas. The hepatic arterial (HA) injection of radioactive TheraSphere also produced portal changes similar to those observed in humans after external beam therapy. While the extent of damage increased with the delivered dose, radiation exposures in excess of 30,000 cGy did not cause total hepatic necrosis and were compatible with survival. No microspheres distributed to the bone marrow and absolutely no myelosuppression was encountered in any animal. Proposed hepatic exposures to humans of 5000 to 10,000 cGy by means of these microspheres, therefore, would appear to be feasible and tolerable. Radiotherapeutic microsphere administration preceded by regional infusion of a radiosensitizing agent and/or immediately following the redistribution of blood flow toward intrahepatic tumor by vasoactive agents can potentially yield a synergistic, highly selective attack on tumors confined to the liver.

Early castration reduces prostatic carcinogenesis in transgenic mice

OBJECTIVES To test the hypothesis that transgenic mouse models of prostate cancer could be useful for testing chemoprevention strategies by evaluating the effects of early castration on prostate carcinogenesis in TRAMP mice. Human prostate cancer, unlike other cancers, requires androgens for oncogenesis yet acquires partial androgen independence in the castrated milieu. This paradigm is the basis for an ongoing clinical trial using selective androgen deprivation for prostate cancer chemoprevention. However, preclinical correlates for hormonal prevention or other chemoprevention strategies of prostate cancer have not previously been demonstrated in autochthonous models of prostate carcinogenesis. METHODS Magnetic resonance imaging was used to longitudinally measure prostate growth in castrated and noncastrated TRAMP mice, and mice were prospectively examined for the onset of advanced, palpable prostate cancer. Modulation of androgen-responsive oncogene expression, as well as oncogene expression in refractory cancers, was evaluated by Western blot. RESULTS Early castration significantly reduced prostate tumor growth as measured by magnetic resonance imaging and improved cancer-free survival. Prevention of prostate cancer development in these mice was associated with durable suppression of androgen-responsive oncogene expression (T-antigen expression not detectable by Western blot); prostate cancers refractory to the hormonal prevention strategy demonstrated androgen-independent oncogene expression. CONCLUSIONS These findings suggest that carcinogenesis related to androgen-responsive oncogene expression can be prevented in some cases by hormonal manipulation and that transgenic TRAMP mice are useful for the preclinical evaluation of hormonal and possibly other strategies of prostate cancer chemoprevention.

Exotic mice as models for aging research: Polemic and prospectus

Most gerontological research using rodent models employs inbred strains, or F1 hybrids derived from them, rather than populations of genetically heterogeneous individuals. This study presents the argument that reliance on genetically homogeneous rodents, though sanctioned by tradition, may not be ideal for many sorts of investigations, and that use of heterogeneous mice and rats would allow researchers to reach robust conclusions that were less likely to reflect strain-specific idiosyncrasies. Segregating stocks, bred by backcross, F2 cross, or four-way cross procedures, would be an improvement over inbred and F1 stocks, providing inexpensive, arbitrarily large, and reproducible populations of genetically diverse test subjects. These stocks would not, however, recapture allelic variations that are likely to have been lost when wild-trapped mice and rats are selected inadvertently over dozens of generations for breeding success in laboratory conditions. Development of specific pathogen free stocks from wild-trapped progenitors particularly from populations selected for relevant evolutionary history and physiological characteristics, may be of great value for analysis of aging and late-life pathophysiology.

CD4 memory T cell levels predict life span in genetically heterogeneous mice.

Aging leads to changes in the relative proportions of several functionally distinct T cell subsets, including increases in the proportions of memory cells in the CD4 and CD8 subsets and in the proportion of T cells expressing the multiple‐drug resistance pump P‐glycoprotein. To see whether individual differences in T cell subset levels predict life span, we measured the levels of five age‐sensitive T cell subsets, at 8 and again at 18 months of age, in the peripheral blood of genetically heterogeneous mice bred as the progeny of CB6F1 females and C3D2F1 males. The strongest immunological predictor of life span in univariate regression analyses was the proportion of CD4 memory cells measured at 18 months of age (P= 0.003). CD4 memory cell levels remained strongly correlated with life span (P< 0.0003) in a multiple regression analysis after adjustment for sex. The proportion of CD4 cells expressing P‐glycoprotein was also correlated with life span (P<0.01), but only in male mice. Weaker relationships were observed between life span and 8‐month tests of CD8 memory and CD8 P‐glycoprotein levels, for CD4 naive cells at 18 months, and for the change in CD4 naive cells between 8 and 18 months of age; these were, however, near the margin of statistical significance and could reflect chance relationships. The relationship between CD4 memory cell levels and life span was similarly strong regardless of the cause of death in mice whose death was attributable to lymphoma, fibrosarcoma, mammary carcinoma, and other forms of terminal pathology. Additional work is needed to discriminate between two hypotheses: 1) that high levels of CD4 memory cell themselves predispose to disease and early death, particularly from neoplasia; or 2) that accumulation of CD4 memory cells is a biomarker of some underlying process—perhaps accelerated aging—that itself leads to early mortality.—Miller, R. A., Chrisp, C., Galecki, A. CD4 memory T cell levels predict life span in genetically heterogeneous mice. FASEB J. 11, 775–783 (1997)

Mouse (Mus musculus) stocks derived from tropical islands: new models for genetic analysis of life‐history traits

Abstract Founder effects, together with access to unoccupied ecological niches, may allow rodent populations on isolated islands to evolve constellations of life‐history traits that distinguish them from their mainland relatives, for example in body size, litter size, and longevity. In particular, low intrinsic mortality risks on islands with reduced predator numbers and not subject to harsh winter climates may in principle support the development of stocks with extended longevity. Conversely, the conditions under which laboratory rodents are typically bred are thought to select for genotypes that produce large, rapidly maturing races with high early reproductive rates but diminished longevity. To test these ideas, and to generate new mouse stocks suitable for genetic and molecular analysis of the processes that time life‐history events, we have developed specific pathogen‐free stocks from mice trapped from three distinct populations: the U.S. mainland (Idaho) and the tropical Pacific islands Majuro and Pohnpei. Mice from all three locations were found to be shorter and lighter, to have smaller litters, and to have higher faecal corticosterone levels than mice of a genetically heterogeneous stock derived from four common laboratory inbred strains. Among the wild‐derived stocks, mice from Pohnpei and Majuro were significantly lighter and shorter than Idaho‐derived animals, even in populations kept from birth under identical housing conditions. Litter size and reproductive success rates did not differ significantly among the three wild‐derived stocks. Although further work will be needed to see if, as predicted, the wild‐derived stocks differ from one another and from laboratory mice in longevity, these stocks provide useful tools for genetic dissection of factors that regulate body size and reproductive success.

T Cell Subset Patterns That Predict Resistance to Spontaneous Lymphoma, Mammary Adenocarcinoma, and Fibrosarcoma in Mice

Aging leads to changes in the proportion of several T cell subsets in peripheral blood, but it is not yet known whether these changes have prognostic significance for late-life diseases. To examine this question, levels of T cell subsets were measured at 8 and 18 mo of age in the peripheral blood of mice of a genetically heterogeneous stock, and the mice were then subsequently evaluated for life span and for cause of death. The results indicate that mice whose T cell subset patterns look like those of old mice tend to die at earlier ages, regardless of the specific cause of death. At 18 mo, 39% of the variance within the set of seven measured subsets could be combined statistically into a single number, whose correlation with individual subsets suggested that it could be interpreted as an index of immunological aging. T cell subset pattern, as represented by this index, was a predictor of life span in mice dying of lymphoma, fibrosarcoma, mammary adenocarcinoma, or of all other causes considered together. Even as early as 8 mo of age, T cell subset patterns are significant predictors of all three forms of cancer, although at this age the association is stronger in mated female mice than in virgin mice. These results support two controversial hypotheses, which are not mutually exclusive: 1) early immune senescence might predispose to early death from cancer and 2) differences in aging rate, as monitored by tests of immune status, might accelerate or decelerate a wide range of late life neoplastic diseases.

Lifespan and Lesions in Genetically Heterogeneous (Four-way Cross) Mice: A New Model for Aging Research

Genetically heterogeneous animal models provide many advantages for research on aging but have been used infrequently. We present here lifespan and lesion data from a study of mice bred as a cross between (AKR/J x DBA/2J)F1 females and (C57BL/6J x SJL/J)F1 males. In such a four-way cross population, each mouse is genetically unique, but replicate populations of essentially similar genetic structure can be generated quickly, at low cost, and of arbitrary size from commercially available, genetically stable hybrid parents. We employed a protocol in which mice judged to be severely ill were euthanatized to obtain tissue in optimal condition for necropsy, and we were able to infer a likely cause of illness in 42 of 44 animals. Malignant lymphoma, including al least four histopathologically distinct subtypes, was the most common cause and was also a frequent incidental finding in mice dying of other causes. Neoplastic disease, benign or malignant, was the sole or a contributing cause of illness in 90% of the mice for which a cause could plausibly be assigned. A wide range of lethal and nonlethal degenerative lesions was also noted. The coefficient of variation for lifespan in these genetically heterogeneous mice was 26%, similar to that seen in analyses of recombinant inbred mouse lines. Baseline lifespan and pathology data on four-way cross mice is a useful prelude to the exploitation of this rodent model in tests of genetic and mechanistic hypotheses about aging.