Synergy among responding lymphoid cells in the one-way mixed lymphocyte reaction. Interaction between two types of thymus-dependent cells.

The present studies have shown that two subpopulations of thymus-dependent lymphocytes may act synergistically in the mixed lymphocyte reaction (MLR) in the mouse. One subpopulation was well represented in the young adult thymus and the other in lymph nodes. For optimum synergy, both populations must be allogeneic to the stimulator cells. Pretreatment of either population with mitomycin-C abolished synergy. Anti-theta serum abolished both MLR responding and synergizing activities of lymphoid cells. The two thymus-dependent subpopulations were both present in the spleen, and displayed different migratory patterns when injected into irradiated mice: one population went to spleens of the irradiated mice, the other to lymph nodes. The effects of anti-thymocyte serum on the MLR and upon synergy were assessed. While minor differences exist and are herein described, our overall results strongly suggest that in our experiments with synergy in MLR, we may be dealing with the same T(1)- and T(2)-cell subpopulations described by Cantor and Asofsky and coworkers (1, 2, 4, 5, 14) as displaying synergy in the graft-vs.-host reaction.

Increased incidence of lymphoma after injections of mice with cells differing at weak histocompatibility loci.

Newborns of two congenic strains of mice, in which the incidence of leukemia is low, differing only at the weak H-1 locus, were. injected at birth with adult spleen cells of the parent and of the congenic strain. A marked increase in the incidence of lymphoma ensued in those mice of both strains injected with cells from the other strain. The experiment lends some support to the idea that transplantation immunologic mechanisms may play a role in the genesis of lymphomas.

Dietary restriction in mice beginning at 1 year of age: effect on life-span and spontaneous cancer incidence.

Lifelong dietary restriction beginning at 3 to 6 weeks of age in rodents is known to decelerate the rate of aging, increase mean and maximum life-spans, and inhibit the occurrence of many spontaneous cancers. Little is known about the effects of dietary restriction started in middle age. In the experiments now reported the food intake of 12- to 13-month-old mice of two long-lived strains was restricted by using nutrient-enriched diets in accordance with the concept of "undernutrition without malnutrition." The mice on the restricted diet averaged 10 to 20 percent increases in mean and maximum survival times compared to the control mice. Spontaneous lymphoma was inhibited by the food restriction.

Dietary restriction retards age-related loss of gamma crystallins in the mouse lens.

The soluble crystallins in lenses from diet-restricted and control mice of diverse ages (2, 11, or 30 months) were studied by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results obtained with both methods suggest that dietary restriction decelerates age-related loss of soluble gamma crystallins.

Changes in plasma lipids and lipoproteins in humans during a 2-year period of dietary restriction in Biosphere 2.

BACKGROUND: A cohort study was performed of 8 people sealed inside Biosphere 2 to evaluate the effects of dietary restriction in humans on lipid and lipoprotein levels and the relationship of these levels to energy, fat, and protein content of the diet, and body weight, weight change, and energy expenditure. METHODS: Eight healthy people aged 27 to 67 years, 4 women and 4 men, were sealed inside Biosphere 2 from September 26, 1991, to September 26, 1993, the longest sustained period in an "isolated confined environment" on record. They were studied throughout confinement and for more than 2 years after their exit and return to an ad libitum diet. Food available was severely restricted during most of the 2-year period inside Biosphere 2. High work output was maintained and food quality remained high, resulting in prolonged restriction of energy intake without malnutrition. RESULTS: Fasting plasma cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol levels; HDL subfraction distribution; dietary energy, fat, and protein content intake; and height, weight, weight change, and energy expenditure were measured. Total plasma cholesterol and triglyceride levels decreased 30% and 45%, respectively. The HDL and low-density lipoprotein levels also decreased and, in some participants, levels of HDL2 subfractions were increased. Multivariate analysis showed that the major cause of these changes was energy restriction. CONCLUSIONS: Energy restriction was the major factor leading to low lipid and lipoprotein levels. Energy restriction with adequate nutrition of young and middle-aged people may substantially reduce risk for atherosclerosis and consequent coronary artery and cerebrovascular disease.

Age-related decrease of growth hormone and prolactin gene expression in the mouse pituitary.

The effects of aging on pituitary GH, PRL, and alpha-tubulin messenger RNA (mRNA) levels were measured in 3-, 12-, and 27-month-old male C57BL/6J mice by dot-blot hybridization. The amount of GH and PRL mRNA in the pituitary deceased dramatically with age. However, total poly(A+) RNA (mRNA), as measured by hybridization with radioactively labeled oligo-(dT), was not altered during aging. In addition, there were no age-related changes in the level of alpha-tubulin mRNA. Thus, the effects of aging on GH and PRL mRNA levels are specific; the levels of the majority of cellular mRNAs are not altered with age. GH and mRNA levels decreased 35% between 3 and 12 months (P less than 0.05) and a total of 75% after 27 months (P less than 0.01). PRL mRNA levels decreased 65% between 12 and 27 months (P less than 0.01), although there was no significant decrease before 12 months. Whereas T3 is the most potent regulator of GH gene expression, we did not detect any significant age-related change in serum T3 levels. These results suggest that factors other than T3 play a role in the age-related decline in GH and PRL gene expression.

Histocompatibility antigens (HLA) and patterns of cognitive loss in dementia of the Alzheimer type.

Recent data suggest that a proportion of patients with primary neuronal degeneration of the Alzheimer type have the HLA-B7 antigen. However, the possibility that other differences might exist between patients with and without the marker has not been examined. We tested a range of cognitive skills in Alzheimer patients with and without the HLA-B7 to examine whether those individuals with the HLA marker would exhibit a profile of cognitive loss different from those without it. Our results indicate that patients with HLA-B7 antigens had selective attentional scores that were significantly lower than Alzheimer patients without the antigen. Neither group was significantly different in either memory capacity or retrieval from short-term and long-term memory. The data support the hypothesis that there may be more than one disorder in what is now referred to as dementia of the Alzheimer type.

Failure of dietary restriction to retard age-related neurochemical changes in mice.

Age-sensitive neurochemical measures and estrous cyclicity were studied in female mice from the long-lived C3B10F1 strain fed either a control diet or subjected to dietary restriction (DR) from 3 weeks of age. Striatal dopaminergic D2 receptor density decreased by 25% from 9-10 months to 28-30 months of age in the control group. This decline was uninfluenced by DR. Anterior pituitary dopamine + dihydroxyphenylacetic acid content increased by 2.5 fold with age in the control group but DR failed to oppose this age-related change. In contrast to DR's lack of influence on these two neurochemical measures were findings on estrous cyclicity. Although mice on DR did not display estrous cycles, cyclicity was rapidly initiated when these mice were switched to the control diet at 12 and even at 22 months of age. Thus, limited aspects of neuroendocrine aging were retarded by DR in this long-lived mouse model.

Structure and regulation of the mouse GRP78 (BiP) promoter by glucose and calcium ionophore.

Dietary calorie restriction, also termed energy restriction, increases mean and maximum life span, reduces the incidence of tumors and increases the mean age of onset of diseases and tumors in every animal tested. Because life-span is genetically determined, we are studying the mechanisms by which energy restriction regulates the expression of genes. We found that energy restriction reduces hepatic glucose-regulated protein-78 (GRP78) and protein-94 mRNA levels by 2-3-fold in mice [Spindler et al., J. Nutr. 20 (1990) 1412-1417]. To investigate this down-regulation, we have cloned the mouse GRP78 promoter (pGRP78) and studied its regulation by glucose. The mouse pGRP78 and the previously cloned rat promoter mediate responsiveness to glucose deprivation, as well as to the calcium ionophore A23187. These studies are the first demonstration that cis-elements in the pGRP78 mediate responsiveness to glucose deprivation.

Energy metabolism after 2 y of energy restriction: the biosphere 2 experiment.

BACKGROUND: An adaptive decrease in energy expenditure (EE) in response to 6 mo of severely restricted energy intake was shown in a classic semistarvation study-the Minnesota experiment. OBJECTIVE: Our objective was to examine whether such adaptation also occurs in response to less severe but sustained energy restriction. DESIGN: Body composition, 1-wk total EE (TEE), 24-h sedentary EE, and spontaneous physical activity were measured in 8 healthy subjects (4 men and 4 women) at the end of a 2-y confinement inside Biosphere 2. Unexpectedly, the food supply was markedly restricted during most of the confinement and all subjects experienced a marked, sustained weight loss (9.1 +/- 6.6 kg; P: < 0.001) from the low-energy (7000-11000 kJ/d), low-fat (9% of energy), but nutrient-dense, diet they consumed. RESULTS: The TEE inside Biosphere 2, assessed 3 wk before exit, averaged 10700 +/- 560 kJ/d (n = 8). Within 1 wk after exit, the adjusted 24-h EE and spontaneous physical activity were lower in the biospherians (n = 5) than in 152 control subjects (6% and 45%, respectively; both P: < 0.01). Six months after exit and return to an ad libitum diet, body weight had increased to preentry levels; however, adjusted 24-h EE and spontaneous physical activity were still significantly lower than in control subjects. CONCLUSIONS: In lean humans, an adaptive decrease in EE appears to occur not only in states of life-threatening undernutrition, but also in response to less severe energy restriction sustained over several years.

Transgenic mice expressing a truncated Peromyscus leucopus TNF-alpha gene manifest an arthritis resembling ankylosing spondylitis.

Several studies have implicated tumor necrosis factor-alpha (TNF-alpha) in autoimmune diseases, such as rheumatoid arthritis (RA). To elucidate further the role of TNF-alpha in inflammatory arthritis, we generated transgenic mice harboring a truncated Peromyscus leucopus TNF-alpha (Pe-TNF) gene. An arthritic phenotype closely resembling human ankylosing spondylitis was observed only in transgenic lines expressing the Pe-TNF transgene at the mRNA level. We characterized the arthritic phenotype in detail by radiographic and histologic techniques. It consisted of severe axial skeletal kyphosis and ankylosis, accompanied by an inflammatory and fibrotic process at the end plates and enthesis. Peripheral joint lesions were absent in mice expressing the P. leucopus TNF-alpha gene, in contrast to the RA-like phenotype described in transgenic mice expressing a truncated human TNF-alpha gene. The Pe-TNF transgenic mouse model provides a unique opportunity to explore potential mechanisms whereby TNF-alpha may initiate an autoimmune arthritis resembling ankylosing spondylitis.

Multigene families, histocompatibility systems, transformation, meiosis, stem cells, and DNA repair.

Aging is probably not directly traceable to changes along the whole genome, but to a small portion thereof. The main histocompatibility complex appears to be one among the postulated sets of multigene families responsible. The immortality of transformed cells, the germ line, and possibly certain pluripotential stem cells may suggest common qualitative and/or quantitative differences in DNA repair mechanisms between these cell populations and committed, normal cell populations. A relationship between HLA and at least two diseases showing defective DNA-repair suggests that the same chromosome carrying the main histocompatibility complex may control some repair processes. The correspondence of variation in lifespans in different mouse strains with the DNA repair capabilities and degrees of autoimmune susceptibility of the same strains lends further support to the idea that DNA repair, immune dysfunction and aging in higher animals may be intimately related.

Modulatory effect of nicotinamide on unscheduled DNA synthesis in lymphocytes from young and old mice.

We studied the DNA repair capacity, as measured by unscheduled DNA synthesis, of resting lymphocytes from a long-lived strain of mouse after UV irradiation. Lymphocytes from old mice showed a lower level of repair than lymphocytes from young mice. After in vitro treatment with nicotinamide, a precursor of cellular NAD+, the level of UV-induced DNA repair increased in resting lymphocytes from both young and old mice. This effect was more dramatic in old mice, which showed a twofold relative increase in repair. Nicotinamide at a concentration of 0.5-5 mM did not inhibit the proliferation of concanavalin A (Con A) stimulated mouse lymphocytes; on the contrary, 3-amino benzamide, a potent poly(ADP-ribose)polymerase inhibitor, strongly affected the lymphocyte responsiveness to Con A. Nicotinamide did not significantly increase the UV-induced DNA repair in lymphocytes stimulated to proliferate by Con A. However, Con A activated, but non-proliferating (hydroxyurea-treated) lymphocytes from old mice displayed a level of DNA repair similar to that of lymphocytes from young animals. These results suggest that one of the limiting factors affecting the DNA repair activity of resting lymphocytes from old mice is the level of intracellular NAD+.

Proliferative potential and DNA repair in lymphocytes from short-lived and long-lived strains of mice, relation to aging.

The replicative capacity and DNA repair ability of spleen lymphocytes of young and old mice from short-lived and long-lived strains were studied. DNA repair after both UV- and gamma-induced damage was investigated. Proliferation after Con A decreased as a function of age in both mouse strains and paralleled an age-associated decline in repair of DNA damage induced by either UV or gamma-irradiation. Compared to the long-lived, the short-lived strain displayed an earlier impairment of both proliferative and repair potentials. DNA repair after gamma-induced damage only occurred if lymphocytes were stimulated to proliferate. Resting lymphocytes appeared unable to repair strand breaks. By contrast, DNA repair of UV-induced damage showed two components: one was dependent on the cell proliferative state, the other was not. Both components were stimulated or induced by mitogen. Resting lymphocytes were able to perform an appreciable amount of repair after UV irradiation. Our results suggest that resting or post-mitotic cells possess a greater possibility to regulate repair of UV-induced than gamma-induced damage. We speculate that it may be the level of this proliferation-independent, but mitogen inducible form of repair which correlates with maximum life-spans between species, thereby explaining why repair of UV- but not gamma-induced damage reveals such a correlation.

Autoimmunity, histocompatibility, and aging.

The immunologic theory of aging proposes that the normal process of aging in man and all animals is pathogenetically related to faulty immunological processes and may be analogous to a type of autoimmune phenomena ultimately involving all body tissues. It may be said that the sharply increased incidence in elderly humans of the autoimmune and immunodeficiency "diseases of age" are thought to be greatly potentiated by the age-related decline in immune surveillance mechanisms particularly involving self/non-self discriminatory abilities. The major histocompatibility complex has emerged as a complex of "supergenes" coding for antigens whose ultimate biological function may be to serve as recognition units allowing lymphocytes to recognize self from non-self on an immunological basis. Also, recent data are consistent with our supposition that differences in age-specific peaks of various immune functional parameters in genetically homozygous mice may be influenced by genes linked to the major histocompatibility complex. These differences may account, at least in part, for the highly strain-dependent, age-specific incidence of certain diseases, including autoimmune and malignant diseases in the mouse. Heightened susceptibility to develop a particular disease in a susceptible animal occurs when a certain balance is reached between the interplay of immune functional parameters which mature, differentiate, or decline at different rates in the same animal. The age-specificity of this balance may be under partial control of H-2 or HLA-linked genes.

A mathematical model of physiological processes and its application to the study of aging.

The behavior of a physiological system which, after displacement, returns by homeostatic mechanisms to its original condition can be described by a simple differential equation in which the "recovery time" is a parameter. Two such systems, which influence one another, can be linked mathematically by the use of "coupling" or "feedback" coefficients. These concepts are the basis for many mathematical models of physiological behavior, and we describe the general nature of such models. Next, we introduce the concept of a "fatal limit" for the displacement of a physiological system, and show how measures of such limits can be included in mathematical models. We show how the numerical values of such limits depend on the values of other system parameters, i.e., recovery times and coupling coefficients, and suggest ways of measuring all these parameters experimentally, for example by monitoring changes induced by X-irradiation. Next, we discuss age-related changes in these parameters, and show how the parameters of mortality statistics, such as the famous Gompertz parameters, can be derived from experimentally measurable changes. Concepts of onset-of-aging, critical or fatal limits, equilibrium value (homeostasis), recovery times and coupling constants are involved. Illustrations are given using published data from mouse and rat populations. We believe that this method of deriving survival patterns from model that is experimentally testable is unique.

Cyclic nucleotide levels in resting and mitogen-stimulated spleen cell suspensions from young and old mice.

The levels of cyclic adenosine 3', 5'-monophosphate (cAMP) in suspensions of unstimulated spleen cells from tumor-free 30-month old (C57BL/10Sn X C3H/HeDiSn)F1 hybrid mice averaged only 14% of that of 6-month old mice. By contrast, the level of cyclic guanosine 3', 5'-monophosphate (cGMP) in spleen cell suspensions from old mice was about 270% that of young mice. The cAMP/cGMP ratio for the unstimulated (resting) state showed a decline by 30 months to about 5% of its 6-month value. Cyclic nucleotide levels were also measured in cell suspensions from old and young mice at intervals over a two hour period following in vitro stimulation with the plant mitogens phytohemagglutinin, concanavalin-A and pokeweed mitogen. Quantitative and in some instances qualitative differences in responses were noted. These results might conceivably reflect either age-related changes in the splenic lymphoid cell subpopulations or intrinsic cellular alterations or both. It is unlikely that changes of this degree could be wholly explained by population shifts. An imbalance in cyclic nucleotide levels in both resting and stimulated lymphoid cells in older animals might contribute to the immune dysfunction known to occur with normal aging.

Disulfide bonds and the structure of the chromatin complex in relation to aging.

Triton X-100 washed nuclei from livers of young-adult and old mice were digested with micrococcal nuclease and pelleted. Supernatants (1SF) were saved and the pellets lysed in a hypotonic EDTA buffer. A second supernatant (2SF) and a final pellet (P) were obtained by recentrifugation (7000 g, 7 minutes). The 1SF/2SF ratio, which has been shown to be an index of the transcriptionally active to inactive chromatin ratio, was lower in older mice. The fraction relatively resistant to solubilization by the nuclease (P) was found by isopycnic sucrose gradient centrifugation to be in a more compatc, condensed state when prepared from older mice. Higher amounts of heavy density chromatin were obtained from nuclei of old than young mice by hypotonic lysis plus minimal mechanical shearing. 2-Mercaptoethanol (2ME) treatment brought the density of the material of P from old mice back to the levels of young mice. In both age groups 2ME decreased the densities of mechanically sheared chromatin as well as of the whole Triton X-100 washed nuclei. In nuclease digestion experiments treatment of the nuclei from both age groups with S-S reducing agents increased the release of DNA from P into the supernatants. The results are consistent with S-S bonds being involved in the condensed structure of chromatin in young and old mice and in the shift of the chromatin complex towards a more compact, condensed state in old age.

Proliferative and cytotoxic immune functions in aging mice. III. Exogenous interleukin-2 rich supernatant only partially restores alloreactivity in vitro.

The ability of exogenous interleukin-2 (IL-2) rich supernatant to restore the defective T cell mediated immune functions of spleen cells from aged C57BL/6 mice was analyzed. Addition of IL-2 rich supernatant to allogeneic mixed lymphocyte cultures (MLC) resulted in an increase in the proliferative response of spleen cells from both young and old mice. The MLC response of cells from old mice was, however, not restored to the level of proliferation seen with splenocytes from young animals. In studying the generation of specific T cell suppressor function, it was found that IL-2 rich supernatant enhanced this function only for spleen cells from those aged animals which demonstrated a defective response in its absence. The response of these mice was thereby restored to the normal level. The response of cells from young control animals and aged mice with normal suppressor activity was not affected by the addition of IL-2 rich supernatant. We conclude that decreased IL-2 production constitutes a functionally important aspect, but is by no means the only defect in the immune response of aged mice. The results also suggest that responsiveness to IL-2 is less affected by age than lymphokine production.

Specific inhibition of pituitary prolactin production by energy restriction in C3H/SHN female mice.

Pituitaries were excised from control (C; 95 kcal/week) or energy restricted (ER; 48 kcal/week) female mice of 2, 3, 7, and 18 months of age. The total RNA and relative actin mRNA amounts in the pituitary were significantly greater in C than in ER mice both at 7 and 18 months. Prolactin (PRL) mRNA, standardized with actin mRNA, was significantly less in ER mice of 7 (50%) and 18 (51%) months of age than in age-matched controls, suggestive of specific inhibition of PRL mRNA transcription. Pituitary RNA and actin mRNA increased from 7 to 18 months in C mice but not in ER mice. Similarly, mean pituitary volumes increased between 2 and 18 months in C mice but not in ER mice. PRL mRNA, standardized with actin mRNA, did not change in either C or ER mice 7-18 months of age. All examined C mice of 3, 7, and 18 months of age had estrous cycles but none of the ER mice of the same ages. After 1 month of ER, the pituitary volumes and serum insulin concentrations in 2-month-old female mice were reduced. Thus net reduction of PRL mRNA per pituitary by ER is attributable to decreases in pituitary size and specific inhibition of PRL production, both of which may be due to low estrogen and insulin levels.