Osteoarthritic changes in vervet monkey knees correlate with meniscus degradation and increased matrix metalloproteinase and cytokine secretion.

OBJECTIVE: Meniscus injury increases osteoarthritis risk but its pathobiology in osteoarthritis is unclear. We hypothesized that older adult vervet monkeys would exhibit knee osteoarthritic changes and the degenerative menisci from these animals would secrete matrix metalloproteinases (MMPs) and pro-inflammatory cytokines that contribute to the development of osteoarthritis. DESIGN: In a cross sectional analysis of healthy young adult (9-12 years) and old (19-26 years) adult female vervet monkeys, knees were evaluated in vivo with computed tomography (CT) imaging, and joint tissues were morphologically graded at necropsy. Meniscus explants were subsequently cultured to evaluate meniscal MMP and cytokine secretion. RESULTS: CT images revealed significant bony osteoarthritic changes in 80% of older monkeys which included increases in osteophyte number and meniscal calcification. Meniscus and cartilage degradation scores were greater in the older monkeys and were positively correlated (r > 0.7). Menisci from older animals exhibiting osteoarthritic changes secreted significantly more MMP-1, MMP-3, and MMP-8 than healthy menisci from younger monkeys. Older menisci without significant osteoarthritic changes secreted more IL-7 than healthy young menisci while older osteoarthritic menisci secreted more IL-7 and granulocyte-macrophage colony-stimulating factor than healthy older menisci. CONCLUSIONS: Aged vervets develop naturally occurring knee osteoarthritis that includes involvement of the meniscus. Degenerative menisci secreted markedly increased amounts of matrix-degrading enzymes and inflammatory cytokines. These factors would be expected to act on the meniscus tissue and local joint tissues and may ultimately promote osteoarthritis development. These finding also suggest vervet monkeys are a useful animal model for studying the progression of osteoarthritis.

Chronic stress, metabolism, and metabolic syndrome.

The prevalence of obesity has rapidly escalated and now represents a major public health concern. Although genetic associations with obesity and related metabolic disorders such as diabetes and cardiovascular disease have been identified, together they account for a small proportion of the incidence of disease. Environmental influences such as chronic stress, behavioral and metabolic disturbances, dietary deficiency, and infection have now emerged as contributors to the development of metabolic disease. Although epidemiological data suggest strong associations between chronic stress exposure and metabolic disease, the etiological mechanisms responsible remain unclear. Mechanistic studies of the influence of chronic social stress are now being conducted in both rodent and nonhuman primate models, and phenotypic results are consistent with those in humans. The advantage of these models is that potential neural mechanisms may be examined and interventions to treat or prevent disease may be developed and tested. Further, circadian disruption and metabolic conditions such as diabetes mellitus could increase susceptibility to other stressors or serve as a stressor itself. Here, we review data from leading investigators discussing the interrelationship between chronic stress and development of metabolic disorders.

Liquid chromatographic assay of warfarin: similarity of warfarin half-lives in human subjects.

A high pressure liquid chromatographic assay was developed to measure warfarin concentrations in biological fluids. Twelve healthy, unrelated volunteers received a single oral dose of warfarin (0.75 mg per kilogram of body weight). The mean plasma warfarin half-life was 36.3 +/- 3.5 hours by liquid chromatography but 55.9 +/- 8.4 hours by a currently used fluorimetric assay that fails to separate warfarin from its metabolites. Interindividual variation was greater and each half-life longer by the fluorimetric than by the chromatographic procedure. Warfarin shows less interindividual variation than that observed for other drugs primarily metabolized by hepatic microsomal mixed function oxidases. Advantages of specificity, rapidity, sensitivity, accuracy, and simplicity recommend liquid chromatography in the development of other drug assays.

Estrogen and progesterone replacement therapy reduces low density lipoprotein accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys.

The effect of estrogen and progesterone replacement therapy on the initiating events in atherogenesis was studied in surgically postmenopausal cynomolgus monkeys. Monkeys were ovariectomized and divided randomly into two groups, one receiving 17 beta-estradiol and cyclic progesterone treatment (n = 9) and ovariectomized controls receiving no hormone replacement therapy (n = 8). The monkeys were fed a moderately atherogenic diet for 18 wk to accelerate the early pathogenic processes but not to be of sufficient duration to produce grossly visible atherosclerotic lesions. Sex hormone replacement therapy decreased the accumulation of LDL and products of LDL degradation in the coronary arteries by greater than 70% while having no significant effect on plasma lipid, lipoprotein, or apoprotein concentrations. Arterial intimal lesions were small with no difference between groups. The reduction in arterial LDL metabolism occurred very early in the pathogenesis of atherosclerosis and was independent of indices of endothelial cell injury, such as enhanced endothelial cell turnover or leukocyte adhesion to the endothelium. Results of this study suggest that one mechanism by which sex hormone treatment inhibits the initiation of atherosclerosis is a direct effect at the level of the arterial wall by suppressing the uptake and/or degradation of LDL.

Social subordination stress, behavior, and central monoaminergic function in female cynomolgus monkeys.

BACKGROUND: Social subordination in female cynomolgus monkeys is stressful and activates the hypothalamic-pituitary-adrenal axis. In a previous experiment behavioral depression was observed in a subset of subordinates. METHODS: In the experiment reported here behavioral and physiological indicators of stress were evaluated in dominant and subordinate female cynomolgus monkeys, and brain dopaminergic activity was assessed, as reflected in the prolactin response to haloperidol, a dopamine2 (D2) receptor antagonist. RESULTS: Subordinates were aggressed more, spent more time in fearful scanning of the social environment, spent less time as the recipients of the active affiliative behavior of being groomed, had more variable heart rates in response to a novel environment, and were hypercortisolemic compared to dominants. Prolactin responses to haloperidol challenge were lower in subordinates than dominants, an observation consistent with the hypothesis that subordinate females have decreased D2 receptor function. CONCLUSIONS: These observations suggest that social subordination is stressful and may alter brain dopaminergic function in primates. The neurophysiological characteristics of social subordinates may contribute to their susceptibility to depression.

Behavior and physiology of social stress and depression in female cynomolgus monkeys.

The hypothesis that social subordination is stressful, and results in a depressive response in some individuals, was examined in socially housed female cynomolgus monkeys. Social status was manipulated such that half of the previously subordinate females became dominant and half of the previously dominant females became subordinate. Current subordinates hypersecreted cortisol, were insensitive to negative feedback, and had suppressed reproductive function. Current subordinates received more aggression, engaged in less affiliation, and spent more time alone than dominants. Furthermore, they spent more time fearfully scanning the social environment and displayed more behavioral depression than dominants. Current subordinates with a history of social subordination were preferentially susceptible to a behavioral depression response. The results of this experiment suggest that the stress of social subordination causes hypothalamic-pituitary-adrenal and ovarian dysfunction, and support the hypothesis that chronic, low-intensity social stress may result in depression in susceptible individuals.

Steroid regulation of tryptophan hydroxylase protein in the dorsal raphe of macaques.

BACKGROUND: Tryptophan hydroxylase (TPH) is the rate-limiting enzyme for the synthesis of serotonin, and serotonin is a pivotal neurotransmitter in the regulation of mood, affective behavior, pituitary hormone secretion, and numerous autonomic functions. We previously demonstrated that estradiol (E) and progesterone (P) increase TPH mRNA levels in the dorsal raphe of macaques. METHODS: This study employed western blotting and densitometric quantitation to determine whether the changes observed at the level of gene expression were manifested by changes in TPH protein expression and whether modified estrogens or progestins had actions similar to the native ligands. In addition, the effect of the antiestrogen tamoxifen was examined. Ovariectomized (ovx) rhesus and cynomolgus macaques were untreated or treated with E, P, E+P, equine estrogens (EE), medroxyprogesterone (MPA), EE+MPA, or tamoxifen. The dorsal raphe region was subjected to Western analysis. RESULTS: E treatment for 28 days increased TPH protein mass four to six fold over ovariectomized controls. Addition of P to the E regimen or treatment with P for 28 days after E priming did not alter TPH from E treatment alone. Treatment of ovx macaques with a low dose of P caused a two-fold increase in TPH protein. Treatment of ovariectomized macaques for 30 months with EE alone or MPA alone significantly increased TPH protein; however, unlike P, the addition of MPA to the EE regimen blocked the stimulatory effect of EE. Tamoxifen treatment significantly reduced TPH protein compared to EE and ovariectomized control animals. CONCLUSION: The stimulatory effect of E and P on TPH protein in the dorsal raphe of macaques correlates with the previously observed effect at the level of mRNA expression. P had no effect on the stimulatory action of E, whereas MPA blocked the stimulatory effect of EE. Tamoxifen acted as a potent antiestrogen on TPH protein expression. If TPH protein mass influences serotonin synthesis, then these steroids will impact many autonomic systems that are regulated by serotonin.

Temporal variations in acetaminophen and phenacetin half-life in man.

Acetaminophen (APAP) and phenacetin mean plasma half-lives were approximately 15% longer in normal male volunteers at 6 A.M. than at 2 P.M. as determined after oral administration of each drug at 6 A.M. and 2 P.M. Concentrations of plasma 11-hydroxycorticoids (11-OHCS) in these volunteers were approximately 42% higher at 6 A.M. than at 2 P.M. The mean apparent volume of distribution (aVd) of APAP decreased by approximately 13% from 6 A.M. to 2 P.M., whereas the mean metabolic clearance rate (MCR) of APAP did not change significantly. Neither the mean aVd nor the mean MCR of phenacetin changed significantly from 6 A.M. to 2 P.M. Measurement of urinary metabolites showed no alteration in APAP glucuronidation in the 8-hr period following APAP administration at 6 A.M. or 2 P.M., but indicated that after phenacetin administration 10% more of the dose was oxidatively metabolized to APAP during a 24-hr period beginning at 2 P.M. than at 6 A.M. Within each subject, plasma acetaminophen or phenacetin half-life at 2 P.M. or 6 A.M. was highly reproducible, but large interindividual variations occurred in the extent of temporal variation during this period.

Diurnal rhythms of aminopyrine metabolism: failure to sleep deprivation to affect them.

After a single oral dose of aminopyrine (9 mg/kg), mean salivary aminopyrine half-lives (t 1/2s) and metabolic clearance rates in 12 normal male volunteers exhibited diurnal variations. Salivary aminopyrine t 1/2s were approximately 50% longer at 8 P.M. (2.1 +/- 0.7 hr) than at 8 A.M. (1.4 +/- 0.3 hr). Mean aminopyrine metabolic clearance rates decreased 20% from 8 A.M. (418.2 +/- 152.0 ml/min) to 8 P.M. (335.3 +/- 107.6 ml/min). There were large interindividual variations in the magnitude of these diurnal changes in aminopyrine t 1/2 and metabolic clearance rates. There were nonsignificant changes in mean aminopyrine apparent volumes of distribution (aVd) which increased only slightly from 53.1 +/- 20.6 L at 8 A.M. to 59.7 +/- 26.5 L at 8 P.M. There were diurnal variations in plasma 11-hydroxycorticosteroids (11-OHCS), mean values of which decreased 60% from 16.2 +/- 4.2 microgram/100 ml at 8 A.M. to 6.5 +/- 1.9 microgram/100 ml at 8 P.M. Sleeplessness for 24 or 48 hr under the conditions of this experiment failed to affect diurnal rhythms in aminopyrine metabolism or plasma 11-OHCS concentrations.

Sex differences in absorption kinetics of sodium salicylate.

Sodium salicylate in aqueous solution (9 mg/kg) was given by oral and intravenous routes to normal male and female subjects. Because the bioavailability of salicylate was complete, salicylate was given orally in all subsequent experiments. There were sex differences in time required to attain peak salicylate concentration (tmax), but not in maximum plasma salicylate concentration (Cmax). There were no sex differences in apparent volume of distribution, plasma salicylate clearance, or area under the concentration-time curve. In female subjects, tmax tended to reach a nadir at the middle of the menstrual cycle, when gastric emptying time is shortest, whereas Cmax remained relatively unchanged throughout the menstrual cycle. Equilibrium dialysis studies on the binding of sodium salicylate and of 14C-racemic warfarin to plasma from 25 normal male and 25 normal female subjects of similar age disclosed no sex differences either in the extent of binding of these drugs or in serum albumin concentration. The possibility of sex differences in rates of gastrointestinal absorption of other drug should be investigated.

Failure of hydrocortisone to alter acutely antipyrine disposition.

The disposition of intravenously administered antipyrine in single doses of 40 mg/kg was studied in 3 dogs at 7:00 A.M. and 3 wk later at 7:00 P.M. Our purpose was to determine whether hydrocortisone differentially affected antipyrine disposition according to a circadian pattern. After each intravenous dose of antipyrine, saline was infused for 3 hr followed by intravenous hydrocortisone (2 mg/15 kg loading dose; 2 mg/15 mg/hr infusion for 3 hr). This regimen of hydrocortisone failed at either 7:00 A.M. or 7:00 P.M. to alter acutely antipyrine decay curves. Furthermore, in 4 normal male volunteers, no inflection in the salivary antipyrine decay curve occurred when hydrocortisone was injected 8 hr after antipyrine. In the human volunteers an intravenous injection of 3 mg was followed immediately by an additional 6 hr of continuous hydrocortisone infusion at 3 mg/hr. These experiments show that hydrocortisone alters the pharmacokinetics of previously administered antipyrine in neither dogs nor man.

Antipyrine and warfarin disposition in a patient with idiopathic hypoalbuminemia.

In a subject with the rare condition of idiopathic hypoalbuminemia, antipyrine and warfarin disposition was investigated to determine whether, in an otherwise healthy subject, low albumin concentrations affect the kinetics of these prototypic drugs. Because antipyrine is negligibly bound to albumin its disposition would be expected to be normal, but because at usual therapeutic doses warfarin is 99% bound to albumin, warfarin elimination would be expected to be accelerated in idiopathic hypoalbuminemia. In our patient antipyrine disposition was normal, whereas warfarin clearance was increased and plasma warfarin half-life reduced. The apparent volume of distribution of warfarin was within normal range. Warfarin binding to the patient's plasma was decreased; the free fraction of warfarin in plasma was correspondingly elevated. Albumin isolated from the patient and purified exhibited kinetic values for warfarin binding. Caution is necessary in extending these results to other drugs and other patients with idiopathic hypoalbuminemia since the validity of such extrapolations depends on the extent to which the drug investigated is normally bound to albumin and also on the magnitude of the particular patient's hypoalbuminemia at the precise time of study.

Altered plasma half-lives of antipyrine, propylthiouracil, and methimazole in thyroid dysfunction.

In normal, nonmedicated volunteers and in patients with thyroid disorders the plasma half-lives of antipyrine, propylthiouracil, and methimazole were determined after single oral doses. The plasma half-liver plus or minus S.D. of antipyrine, propylthiouracil, and methimazole were 11.9 plus or minus 1.4 hr, 6.7 plus or minus 1.0 hr, and 9.3 plus or minus 1.4 hr, respectively, in normal volunteers, but were shortened to 7.7 plus or minus 1.2 hr, 4.3 plus or minus 0.7 hr, and 6.9 plus or minus 0.6 hr, respectively, in hyperthyroid patients. In hypothyroid patients the plasma half-lives of these drugs were prolonged to 26.4 plus or minus 4.0 hr, 24.7 plus or minus 34.5 hr, and 13.6 plus or minus 4.8 hr, respectively. Return to the euthyroid state restored plasma half-lives to or toward normal. Alterations in plasma drug half-lives during thyroid dysfunction appear to result mainly from accelerated hepatic microsomal drug metabolism in hyperthyroidism and retarded drug biotransformation during hypothyroidism.

High levels of methylxanthines in chocolate do not alter theobromine disposition.

Theobromine disposition was measured twice in 12 normal men, once after 14 days of abstention from all methylxanthines and once after 1 week of theobromine (6 mg/kg/day) in the form of dark chocolate. Mean theobromine t 1/2, apparent volume of distribution, and clearance after abstinence from all methylxanthines were 10.0 hours, 0.76 L/kg, and 0.88 ml/min/kg. High daily doses of chocolate for 1 week did not change these values. After subjects abstained from methylxanthines, urinary radioactivity over 72 hours after a single, oral dose of [8-14C]theobromine consisted of 42% 7-methylxanthine, 20% 3-methylxanthine, 18% theobromine, 10% 7-methyluric acid, and 10% 6-amino-5[N-methylformylamino]-1-methyluracil. A week of daily theobromine consumption in the form of dark chocolate also did not alter this urinary profile of theobromine and its metabolites. Although these results might appear to differ from other reports of inhibition of theobromine elimination after five consecutive daily doses of theobromine in aqueous suspensions, both the rate and extent of absorption of theobromine in chocolate were less than that of theobromine in solution. Relative bioavailability of theobromine in chocolate was 80% that of theobromine in solution. This reinforces the fundamental principle that both the metabolic and the therapeutic consequences of a particular chemical can differ when that chemical is given in the pure compared with the dietary form.

Postprandial glucose and insulin responses to various snacks of equivalent carbohydrate content in normal subjects.

To evaluate glucose and insulin responses after ingestion of snacks, we gave healthy, nondiabetic male subjects carbohydrate equivalent (25 g) snacks or isocaloric (265 kcal) snack meals in a random crossover design. Individual snacks composed of either a milk chocolate bar, granola bar, chocolate milk, peanut butter cups, yogurt, or potato chips produced similar glucose response curves. Plasma glucose concentrations were lower (p less than or equal to 0.05) at 30 and 60 min postprandially than after a corresponding oral glucose challenge. In contrast, insulin responses to the snacks exhibited a two-fold variation in peak values. Isocaloric snack meals of cereal-milk, cheese sandwich-milk, and peanut butter sandwich-chocolate milk produced glucose and insulin responses similar to individual snacks. Although glucose concentrations at 60 min fell somewhat below baseline values after each snack, clinical hypoglycemia was not evident. These data clearly indicate a similarity in glycemic response among normal individuals consuming a variety of common snacks.

Digestibility of cocoa butter and corn oil in human subjects: a preliminary study.

The comparative absorption of cocoa butter (25.5% C16:0, 34.4% C18:0, 34.4% C18:1, 3.4% C18:2) and corn oil (11.4% C16:0, 2.0% C18:0, 26.4% C18:1, 60.0% C18:2) was assessed in six healthy male subjects. During 3-d experimental diet periods, free-living subjects consumed either cocoa butter or corn oil as virtually the sole source of dietary fat, provided at 40% of the total energy intake in the form of specially formulated cookies. Fat absorption was determined by quantifying total fecal lipid excretion over the 3-d period. Total fecal lipid and fecal fatty acids were determined. The percentage of fat excreted was significantly higher (p less than or equal to 0.001) when subjects consumed the cocoa butter (10.8 +/- 3.2%) vs the corn oil (3.5 +/- 1.0%) diet. These results indicate that the digestibility of cocoa butter is significantly less than corn oil and may explain, in part, previous reports of a neutral effect of dietary cocoa butter on plasma cholesterol concentrations.

Social deprivation and coronary artery atherosclerosis in female cynomolgus monkeys.

Plasma lipid concentrations and coronary artery atherosclerosis extent were compared in a retrospective study of female cynomolgus monkeys consuming a moderately atherogenic diet and housed in single cages or social groups. There was no difference between single caged and socially housed monkeys in plasma lipid concentrations. However, females housed in single cages had significantly more coronary artery atherosclerosis than those housed in social groups. It has been found previously that socially subordinate females have more extensive coronary artery atherosclerosis than social dominants, and that subordinates spend more time alone than dominants. Subsequent analyses of the data presented here revealed that single caged monkeys had significantly more coronary artery atherosclerosis than socially dominant, but not socially subordinate, monkeys. Characteristics of single cage housing which could be disease promoting include restraint and social isolation. These findings should be considered preliminary, and serve as a basis for further study.

Effects of chronic social separation on cardiovascular disease risk factors in female cynomolgus monkeys.

A lack of social support is associated with increased risk of coronary heart disease morbidity and mortality in human beings. Similarly, chronic social separation (single cage housing) potentiates atherosclerosis in female monkeys. Under the hypothesis that autonomic arousal and/or ovarian impairment may mediate this effect (as both are associated with increased atherosclerosis), heart rate and luteal phase plasma progesterone concentrations were measured in 12 female cynomolgus monkeys that were first socially housed, then individually housed, and finally returned to their original social groups. Afternoon heart rates increased during social separation compared to the social groupings (P < 0.001). Increased heart rates could not be explained by activity levels, which were lower during social separation than in social groupings (P < 0.001). Ovarian function (i.e. luteal-phase progesterone concentrations) was not influenced by housing condition. Single caging reduced the extent of social signaling, even though animals were in visual and auditory contact. Rates of affiliative behaviors increased and time spent alone decreased in post-reunion social groups compared to pre-separation social groups (P's < 0.01). The results indicate that chronic social separation in this group-living species may exacerbate atherosclerosis via altered autonomic activity, as evidenced by higher heart rates during social separation.

Behavioral and neurobiological effects of estrogen replacement therapy and a history of triphasic oral contraceptive exposure.

The effects of contraceptive steroids and estrogen replacement therapy on behavior and neuroendocrine function were evaluated in adult female cynomolgus monkeys. During the 'premenopausal' phase of the experiment, the animals were assigned to either treatment with a triphasic oral contraceptive (OC) for 24 months or the untreated control group. The monkeys were then ovariectomized and half of each of the premenopausal groups were randomly assigned to either treatment with conjugated equine estrogens (ERT) or the untreated control group for 12 months (the 'postmenopausal' phase). All evaluations were completed during the postmenopausal phase of the experiment. Both types of exogenous steroid treatments appeared to increase cardiovascular and hypothalamic-pituitary-adrenal responses to stress in socially dominant but not socially subordinate females. A history of triphasic OC administration increased contact aggression received, and reduced the prolactin response to fenfluramine, suggesting reduced serotonergic activity, for at least a year following the cessation of triphasic OC treatment.

Triphasic oral contraceptive treatment alters the behavior and neurobiology of female cynomolgus monkeys.

Oral contraceptives (OCs) are the most widely prescribed and effective of the reversible contraceptive methods. In addition to inhibiting ovulation, OCs alter central nervous system function in women; however, methodological problems have prevented clear human studies. Thus, in this experiment we investigated the effects of OC treatment on behavior, hypothalamic- pituitary-adrenal axis function and the central nervous system in 75 adult female cynomolgus monkeys (Macaca fascicularis) housed in social groups of four to five monkeys per pen. Monkey social groups were randomly divided into either a control or an OC treatment group which was administered a clinically prescribed OC (Triphasil(R), levonorgestrel and ethinyl estradiol tablets) for 2 years. OC treatment increased the frequency of contact aggression received, time spent in locomotion, and sitting close to another animal, and decreased time spent fearfully scanning. OC treatment decreased heart rate, increased activity levels, and increased baseline cortisol concentrations and the cortisol response to adrenocorticotropin compared to control animals. OC treatment decreased the prolactin response to fenfluramine suggesting decreased serotonergic activity. These results suggest that this triphasic OC disrupts social behavior, hypothalamic-pituitary-adrenal axis regulation and the underlying central nervous system function.