Design and rationale of the randomized trial of comprehensive lifestyle modification, optimal pharmacological treatment and utilizing PET imaging for quantifying and managing stable coronary artery disease (the CENTURY study).

BACKGROUND: The literature reports no randomized trial in chronic coronary artery disease (CAD) of a comprehensive management strategy integrating intense lifestyle management, maximal medical treatment to specific goals and high precision quantitative cardiac positron emission tomography (PET) for identifying high mortality risk patients needing essential invasive procedures. We hypothesize that this comprehensive strategy achieves greater risk factor reduction, lower major adverse cardiovascular events and fewer invasive procedures than standard practice. METHODS: The CENTURY Study (NCT00756379) is a randomized-controlled-trial study in patients with stable or at high risk for CAD. Patients are randomized to standard of care (Standard group) or intense comprehensive lifestyle-medical treatment to targets and PET guided interventions (Comprehensive group). Comprehensive Group patients are regularly consulted by the CENTURY team implementing diet/lifestyle/exercise program and medical treatment to target risk modification. Cardiac PET at baseline, 24-, and 60-months quantify the physiologic severity of CAD and guide interventions in the Comprehensive group while patients and referring physicians of the Standard group are blinded to PET results. The primary end-point is the CENTURY risk score reduction during 5 years follow-up. The secondary endpoint is a composite of death, non-fatal myocardial infarction, stroke, and coronary revascularization. CONCLUSIONS: The CENTURY Study is the first study in stable CAD to test the incremental benefit of a comprehensive strategy integrating intense lifestyle modification, medical treatment to specific goals, and high-precision quantitative myocardial perfusion imaging to guide revascularization. A total of 1028 patients have been randomized, and the 5 years follow-up will conclude in 2022.

Organizational Risk: "Muddling Through" 40 Years of Research.

The recognition that organizations are a part of adverse outcomes has become commonplace in risk research. Social organization is a key theme in relation to risk minimization through institutional control and monitoring, and in how organizations are connected to society's perceptions of risk (beyond outcomes). The article reviews progress made in research on organizational risk over the last four decades and the contributions made to the field by fieldwork and descriptive approaches, understanding risk as partly determined by organizational context. A key issue for risk analysis is to figure out what these insights mean for risk professionals, such as while developing assessment methodologies and management approaches. Analysis of the literature shows that what to model if organizational factors are to be included in risk assessments remains as big a question as how to model. Integrating fieldwork and descriptive approaches for analyzing organizational risk, accidents, and safety is argued to be a main task for the risk analysis community.

Bond financing in volatile times.

A competitive landscape for providers and changing market conditions require an understanding of key capital sources: tax-exempt bonds remain an attractive capital source. Credit enhancement for bonds is more expensive and more difficult to find than it was in years past. Direct bond purchases by commercial banks mitigate the traditional risks.

Permeability of the rhesus monkey oocyte membrane to water and common cryoprotectants.

Successful cryopreservation of oocytes of the rhesus monkey (Macaca mulatta) would facilitate the use of this valuable animal model in research on reproduction and development, while providing a stepping stone towards human oocyte cryopreservation and the conservation of endangered primate species. To enable rational design of cryopreservation techniques for rhesus monkey oocytes, we have determined their osmotic and permeability characteristics in the presence of dimethylsulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PROH), three widely used cryoprotectants. Using nonlinear regression to fit a membrane transport model to measurements of dynamic cell volume changes, we estimated the hydraulic conductivity (L(p)) and cryoprotectant permeability (P(s)) of mature and immature oocytes at 23.5 degrees C. Mature oocyte membranes were most permeable to PROH (P(s) = 0.56 +/- 0.05 microm/sec) and least permeable to DMSO (P(s) = 0.24 +/- 0.02 microm/sec); the permeability to EG was 0.34 +/- 0.07 microm/sec. In the absence of penetrating cryoprotectants, mature oocytes had L(p) = 0.55 +/- 0.05 microm/min/atm, whereas the hydraulic conductivity increased to 1.01 +/- 0.10, 0.61 +/- 0.07, or 0.86 +/- 0.06 microm/min/atm when mature oocytes were exposed to DMSO, EG, or PROH, respectively. The osmotically inactive volume (V(b)) in mature oocytes was 19.7 +/- 2.4% of the isotonic cell volume. The only statistically significant difference between mature and immature oocytes was a larger hydraulic conductivity in immature oocytes that were exposed to DMSO. The biophysical parameters measured in this study were used to demonstrate the design of cryoprotectant loading and dilution protocols by computer-aided optimization.

Varespladib (A-002), a secretory phospholipase A2 inhibitor, reduces atherosclerosis and aneurysm formation in ApoE-/- mice.

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE mouse models.

Chimpanzee (Pan troglodytes) spermatozoa osmotic tolerance and cryoprotectant permeability characteristics.

Osmotic properties of chimpanzee spermatozoa were studied at 22 degrees C. An electronic particle counter was used to determine the isosmotic cell volume, and the volume response after exposure to four commonly used cryoprotectants: dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol. The data were analyzed to determine the hydraulic conductivity and the permeability coefficients for the four cryoprotectants. The osmotically inactive volume fraction was determined using a Boyle van't Hoff plot of cells exposed to sodium chloride solutions. A computer-assisted semen analysis system was used to determine the osmotic tolerance of chimp spermatozoa, as well as the effects of a one-step addition and removal of 1 M permeating cryoprotectant on sperm motility. The isosmotic volume of chimpanzee sperm is 27.7 microm3. The osmotically inactive cell fraction is 69%. Hydraulic conductivity was higher in the presence of ethylene glycol: 4.09 +/- 0.76 (mean +/- SEM) and propylene glycol: 3.91 +/- 0.71 as compared to dimethyl sulfoxide: 3.49 +/- 0.79 and glycerol: 2.83 +/- 0.40 microm/min per atmosphere. The permeability of chimpanzee sperm in ethylene glycol (2.18 +/- 0.40 x 10(-3) cm/min) and propylene glycol (1.75 +/- 0.17 x 10(-3) cm/min) was higher than in glycerol (1.42 +/- 0.12 x 10(-3) cm/min) and dimethyl sulfoxide (0.82 +/- 0.015 x 10(-3) cm/min). Although chimpanzee sperm tolerated osmotic stress in the range of 169-400 mOsm very well, loss of motility was observed as the solution concentrations diverged from isosmotic condition. Exposure to the four cryoprotectants at 1 M did not cause a significant reduction in sperm motility. This information on membrane permeability characteristics and cryoprotectant tolerance will aid in designing more reliable cryopreservation protocols for chimpanzee sperm.

Germline transmission in transgenic Huntington's disease monkeys.

Transgenic nonhuman primate models are an increasingly popular model for neurologic and neurodegenerative disease because their brain functions and neural anatomies closely resemble those of humans. Transgenic Huntington's disease monkeys (HD monkeys) developed clinical features similar to those seen in HD patients, making the monkeys suitable for a preclinical study of HD. However, until HD monkey colonies can be readily expanded, their use in preclinical studies will be limited. In the present study, we confirmed germline transmission of the mutant huntingtin (mHTT) transgene in both embryonic stem cells generated from three male HD monkey founders (F0) and in second-generation offspring (F1) produced via artificial insemination by using intrauterine insemination technique. A total of five offspring were produced from 15 females that were inseminated by intrauterine insemination using semen collected from the three HD founders (5 of 15, 33%). Thus far, sperm collected from the HD founder (rHD8) has led to two F1 transgenic HD monkeys with germline transmission rate at 100% (2 of 2). mHTT expression was confirmed by quantitative real-time polymerase chain reaction using skin fibroblasts from the F1 HD monkeys and induced pluripotent stem cells established from one of the F1 HD monkeys (rHD8-2). Here, we report the stable germline transmission and expression of the mHTT transgene in HD monkeys, which suggest possible expansion of HD monkey colonies for preclinical and biomedical research studies.

Leptin administration increases nocturnal concentrations of luteinizing hormone and growth hormone in juvenile female rhesus monkeys.

The importance of leptin in regulating sexual maturation is supported by data showing that deletions of the leptin gene or alterations in the leptin receptor result in infertility. However, attempts to define a role for leptin in normal puberty have produced equivocal results, leading to the conclusion that, if leptin is involved in puberty, its role is permissive and not obligatory. To better define the importance of leptin in primate puberty, the present study tested the hypothesis that a premature elevation in nocturnal leptin concentrations would accelerate indices of puberty, including nocturnal LH secretion in female rhesus monkeys (Macaca mulatta). Juvenile, gonadally intact females were treated daily with leptin (n = 6; 30 micro g/kg, sc at 1700 h) from 12-30 months of age and were compared with age-matched control females (n = 13). Chronic elevation in peripheral concentrations of leptin increased serum levels of both daytime and nighttime bioactive LH at a significantly younger age compared with control females. The earlier rise in LH in leptin-treated females was associated with an earlier increase in serum estradiol and occurrence of menarche. Despite this effect of leptin, nocturnal serum LH was significantly higher at each age assessed in non-leptin-treated ovariectomized controls (n = 6). In addition, leptin increased skeletal lengths and maturity that were associated with significantly higher serum levels of nocturnal GH and daytime IGF-I. Although body weights were not consistently affected by treatment, body mass index, as an index of body fat, was consistently lower in leptin-treated females. Taken together, these data indicate that the chronic elevation in serum leptin concentrations advances the nocturnal increase in serum LH as well as other parameters of female puberty. Furthermore, the observation that nocturnal LH was higher in age-matched, agonadal females compared with the leptin-treated females suggests that the nongonadal drive to LH secretion is operative in female macaques as early as 14 months of age, suggesting that the effect of leptin on puberty in female primates may involve a diminution in gonadal negative feedback suppression of LH secretion. Such a role would suggest that leptin is permissive yet critical for advancing female puberty.

Improved methods for freeze preservation of chimpanzee sperm.

Freeze preservation of human and nonhuman semen has been used effectively for a number of years; however, the application of freezing to preserve nonhuman primate sperm has been less successful. This study compares five freeze methods and various concentrations of the cryoprotectants glycerol and dimethylsulfoxide (DMSO) for cryopreservation of chimpanzee (Pan troglodytes) sperm. The different methods were compared using quantitative analysis of sperm function and, by an indirect measure of fertilizing capacity, the denuded hamster oocyte penetration assay (SPA). The best results were obtained using an extender comprising Ham's F10 medium containing 15% heat inactivated human cord serum and 15.6% glycerol. Semen extended 1 to 1 by this method, for a final glycerol concentration of 7.8%, and frozen using a computer programmed freezer maintained approximately 65% of its original viability and up to 70% of its initial motility. The extended semen was used to initiate pregnancy in two female chimpanzees.

Improved method for artificial insemination in the great apes.

Artificial insemination in the great apes has not achieved its potential as a tool in maintenance of the endangered captive population. Three factors can influence the success rate of artificial insemination: sperm preparation, site of insemination, and timing of insemination. We have tried to optimize methods regarding these three steps. A modified method for insemination is described which has resulted in a 21% success rate (six term pregnancies from 29 inseminations) in the chimpanzee and which has successfully initiated a pregnancy in a gorilla.

Alterations in primate sperm motility with maturation and during exposure to theophylline.

Micropuncture was used to collect pure suspensions of sperm from the caput and cauda regions of chimpanzee epididymides, which were analyzed with a Motion Analysis VP-110. Sperm recovered from the caput region showed no forward motility. Incubation of these sperm with cauda epididymal fluid affected motility in 62%-90% of the sperm. Dilution of cauda sperm into buffer containing >50 mM theophylline resulted in immediate initiation of progressive forward motility. Although this motility was maintained by at least 50% of the sperm for over 5 hr, these "activated" caput sperm did not penetrate zona-free hamster ova. These data show that sperm from the caput epididymis of the chimpanzee have the capacity for normal motility but do not have the capacity to bind to and penetrate an ovum. Cauda epididymal chimpanzee sperm were motile at the time of recovery and this motility was maintained for over 5 hr. These sperm penetrated both hamster zona-free ova and intact chimpanzee ova. These data show that sperm from the cauda epididymis of the chimpanzee have the capacity for normal motility and also have the capacity to bind to and penetrate an ovum. This is the first use of computer assisted analysis to quantify motility in maturing nonhuman primate sperm.

Selected endocrine parameters of the adult male chimpanzee.

This study provides baseline data on reproductive endocrine parameters of the male chimpanzee. A colony group of 47 male chimpanzees were classified by age as juvenile, ages 4-6 years (n = 7); adolescent, ages 7-9 (n = 9); and adult, ages 10-33 years (n = 31). Blood samples (n = 112) obtained from these animals [juveniles (n = ll), adolescents (n = 25), and adults (n = 76)] were analyzed for testosterone (T), luteinizing hormone (LH), and follicle stimulating hormone (FSH). There was a significant increase (mean ± SE) in T (ng/ml) between the juvenile (0.2 ± 0.1) and adolescent group (2.4 ± 0.3) and between the adolescent (2.4 ± 0.3) and the 10-14‒year-old adult age group (4.2 ± 0.2). T peaked at 15-24 years of age and then declined. There was a significant difference in T between animals aged 20-24 years (5.3 ± 0.4) and 25-29 years (3.0 ± 0.4). There was no significant change in serum LH (mlU/ml) with age. Serum FSH (mlU/ml) increased significantly between 4-6 years of age (90.0 ± 11.6) and 10-14 years of age (120.5 ± 8.3), plateaued between 10 and 19 years of age and was significantly lower in males older than 20 years (92.6 ± 4.2). Blood samples (n = 5) obtained from an experimental group of six adult male chimpanzees, ages 10 to 15 years, were analyzed for T, LH, and FSH, Pituitary response to challenge with exogenous GnRH and to hypotha-lamic stimulation with NMA also was assessed. T, LH, and FSH in the experimental group did not differ significantly from animals of equivalent age in the colony group. Challenge with 50, 100, 200, and 500µg GnRH stimulated LH release. The response was not directly dose related. Challenge with 3 and 6 mg/kg NMA stimulated LH release. The response was dose related. © 1993 Wiley-Liss, Inc.

Validation of radioimmunoassays for LH and FSH in the sooty mangabey (Cercocebus atys): Characterization of LH and the response to GnRH.

Heterologous radioimmunoassays (RIA) for macaque LH and FSH were validated for the measurement of these hormones in the sooty mangabey and mangabey pituitary LH was characterized relative to rhesus monkey LH. Dilutions of a pituitary mangabey extract and a partially purified preparation of mangabey LH ran parallel to a rhesus monkey standard (LER 1909-2) in the ovine-ovine (o-o) LH assay but showed some deviation from parallelism in the rhesus monkey FSH assay. The LH potency of the mangabey extract and standard were six and 190 times more potent, respectively, than LER 1909-2 in the LH RIA. Mangabey LH was estimated to have a molecular weight of 40,000-42,000 daltons vs 35,000-38,000 daltons for rhesus LH on Sephadex G-100 chromatography. Plasma levels of radioimmunoreactive LH, FSH, and testosterone were assayed before and after a bolus administration of 25, 50, or 100 µg synthetic go-nadotropin releasing hormone (GnRH) to adult male mangabeys. A significant increase in serum levels of LH was seen within 30 min with levels more than fourfold higher than the basal level of LH after administration of 100 µg GnRH. However, no consistent increases in plasma FSH values were detected. The integrated mean LH response above preinjection levels following 25, 50, or 100 µg GnRH was dose related. Serum levels of testosterone were also elevated after administration of GnRH, but peak concentrations of testosterone lagged behind peak levels of LH by approximately 30 min. These studies indicate that the heterologous RIAs may be used for measuring gonadotropins in the mangabey and that the male mangabey is apparently more sensitive to GnRH than the rhesus monkey.

Semen characteristics of the adult male chimpanzee (Pan troglodytes).

The chimpanzee, because of its similarities to the human, is especially valuable in studies of reproductive function. However, relatively little is known about the physiology of reproduction in the adult male chimpanzee. This study provides, for five adult male chimpanzees, baseline values for testicular volume without and with pressure and for cellular and biochemical characteristics of ejaculates collected by artificial vagina (AV). There was no correlation between body weight and testicular volume measured without or with pressure. The ratios of mean testicular volume without and with pressure were not statistically different among animals. Statistical analysis of penetration of denuded hamster oocytes by ejaculated chimpanzee sperm revealed no correlations between sperm count and percentage of eggs penetrated. There was significant variability in concentrations of protein and fructose and in activities of alpha-glucosidase and acid phosphatase among samples from different animals. Computer-assisted motion analysis (CAMA) of sperm provided baseline data on motion parameters necessary for future evaluation of this technique for semen analysis in the chimpanzee. The level of demonstrated inter-animal variation mandates use of each animal as its own control for studies on normal and altered reproductive function. © 1993 Wiley-Liss, Inc.

Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue.

Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. Mouse arteries were dissected and placed in chloroform-methanol without tissue grinding. Extracts underwent hydrolysis of cholesteryl esters and derivatization of cholesterol followed by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We demonstrated that FC and CE could be quantitatively extracted without tissue grinding and that lipid extraction simultaneously worked for tissue fixation. Delipidated tissues can be embedded in paraffin, sectioned, and stained. Microscopic images obtained from delipidated arteries have not revealed any structural alterations. Delipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE(-/-) mice, LC/MS/MS revealed early antiatherosclerotic effects of dual PPARalpha,gamma agonist LY465606 in brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in CE and FC accumulation in atherosclerotic lesions was associated with the reduction of lesion size. Thus, a combination of LC/MS/MS measurements of CE and FC followed by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.