Morphometrical study of physical growth of laboratory-bred cynomolgus monkeys aged from zero to 9 years

The relative growth was examined in laboratory-bred cynomolgus monkeys (Macaca fascicularis) aged from zero to 9 years. The principal component analysis and multivariate allometry were applied to the biometrical data. As the result of the principal component analysis, the cumulative contribution ratio of the 1 st (PC 1) and 2 nd (PC 2) principal components accounted for 99.0 percent. According to the eigen vector values of each morphological site (14 sites), PC 1 was acceptable not only as a size factor but also as a shape factor of the morphological growth changes from birth to the 3rd year of life in both sexes. PC 2 was acceptable as a shape factor of the morphological differences between sexes after the 3rd year of life. As the result of the multivariate allometric analysis applied to the same data, proximate sites of the trunk showed relatively high growth rates compared to distal ones of the trunk. The head, limbs and arms grew slowly in contrast to the trunk. Thus, we could demonstrate the relative growth of cynomolgus monkeys morphometrically.     

Analysis of infant growth curves using multivariate adaptive splines

In this paper, we study the effect of cocaine use by a pregnant woman on the growth of her infant after birth. Using a data set from a retrospective study, we found that cocaine use was a marginally significant contributor to the infant growth as measured by bodyweight. From a statistical point of view, the data represent a common, though complex, structure that has received little attention in the statistical literature. To analyze these data, we adopt and further enhance an approach developed recently called MASAL (multivariate adaptive splines for the analysis of longitudinal data). In addition to the fitting of growth curves, we demonstrate particularly how to explore and estimate the underlying covariance structures for the longitudinal data that were collected from a rather irregular schedule.     

Effect of thyroid hormone on epidermal growth factor-like immunoreactivity and growth velocity in cynomolgus monkeys

To examine the potential role of epidermal growth factor (EGF) in mediating the effects of thyroid hormone on linear growth, we measured serum EGF levels by RIA in cynomolgus monkeys before and during methimazole-induced hypothyroidism, and after 9 weeks of T4 replacement at different doses. Ten castrated prepubertal monkeys were rendered hypothyroid by methimazole (0.0125% in drinking water for 12 weeks). Methimazole was continued, and T4 was then administered for 9-week intervals. Six weeks elapsed between successive T4 doses. The sequence of different T4 doses for each animal was random. Serum EGF level was measured at baseline and at the end of each treatment period with a newly developed RIA using a polyclonal antiserum against human recombinant EGF. Serum EGF level correlated significantly with the level of serum thyroxine but not with serum triiodothyronine, over the thyroxine dosage range of 1-4 micrograms/kg/day (r = 0.41, p less than 0.005). Lower-leg growth rate correlated significantly with serum EGF level over this same thyroxine dosage range (r = 0.41, p less than 0.005). These data are consistent with the hypothesis that EGF may mediate some of the effects of thyroid hormone on skeletal growth.     

Non-invasive analysis of gallbladder bile composition in cynomolgus monkeys using in vivo 1H magnetic resonance spectroscopy

The purpose of the present study was (i) to establish a modality for non-invasively probing bile composition in cynomolgus monkeys and (ii) to ascertain the variability in biliary metabolism by repeatedly assessing gallbladder bile in situ. Localised in vivo (1)H magnetic resonance spectroscopy (MRS) provided high-resolution spectra of gallbladder bile that allowed for the first time different species of bile acids, their taurine and glycine conjugates, and phospholipids to be identified and quantified in situ. A combined cross-sectional and longitudinal study of bile composition was conducted over 4 weeks in monkeys kept under standardised nutritional conditions. All biles were composed of the same major constituents. Bile acids contributed 267+/-47 micromol/ml whereof cholate, deoxycholate and chenodeoxycholate were the most abundant primary bile acids. Bile acid conjugation reached an extent of 100%. However, the actual quantitative contributions of different bile constituents varied distinctly. Correlation analysis revealed that intra-individual variability (r=0.77+/-0.03) was significantly (p<0.01) smaller than inter-individual variability (r=0.68+/-0.01), thus purporting the notion that bile composition is a hallmark of individual metabolism. Extension of quantitative bile analysis by in vivo (1)H-MRS to pathological states will provide a rapid and non-invasive modality for monitoring an important, yet elusive compartment of cholesterol and lipid metabolism.     

Left ventricular volume and function in cynomolgus monkeys using real-time three-dimensional echocardiography

BACKGROUND: The purpose of this study was to investigate the feasibility of evaluating cardiac function by real time three-dimensional (RT3D) echocardiography in isoflurane-anesthetized male cynomolgus monkeys. Additionally differences between inhibitory effects of beta-blockers and a Ca channel blocker on left ventricular (LV) function were examined. METHODS AND RESULTS: End-diastolic volume (EDV), end-systolic volume (ESV) and ejection fraction (EF) in the control (without any drug effect) were not significantly changed by repetitive measurement at a 30-day interval. Propranolol and metoprolol (0.1 and 0.3 mg/kg/10 minutes, i.v.) caused a dose-dependent increase in ESV, but little effect on EDV, resulting in a decrease in EF. Verapamil (0.1 and 0.3 mg/kg/10 minutes, i.v.) increased both EDV and ESV, but decreased EF was noted at 0.3 mg/kg. CONCLUSIONS: These results demonstrate the feasibility of RT3D echocardiography in providing reproducible estimations of LV volume and EF in monkeys when evaluating drugs that may affect cardiac function.     

Body temperature of newborn cynomolgus monkeys

This report dealt with the change of body temperature (rectal temperature) in the newborn cynomolgus monkeys (Macaca fascicularis) with a view to take it as an index for their health conditions. The body temperatures of 183 newborn babies which were well cared for by their mothers was 33.0 to 37.7 degrees C about 10 hr after birth. On the other hand, the body temperatures of 21 newborn babies which were not well cared for by their mothers was very low, ranging from 24.1 to 34.8 degrees C. In five newborn monkeys which were well cared for, the body temperature averaged about 36 degrees C just after birth and then declined rapidly by 32 to 33 degrees C at 40 to 50 minutes after birth. Then it gradually began to rise, reaching 36 to 37 degrees C at 180 to 240 min after birth. In the other four newborn monkeys which were delivered by Caesarean section, the temperature was 37 to 38 degrees C just after birth. Then it decreased to 29 to 32 degrees C at 120 minutes after birth when the newborns remained singly in a cage without warming.     

Dust mite-induced asthma in cynomolgus monkeys.

Animal models exhibiting high homology with humans at the genetic and pathophysiological levels will facilitate identification and validation of gene targets underlying asthma. In the present study, a nonhuman primate model of allergic asthma was developed by sensitizing cynomolgus monkeys to dust mite antigen. Sensitization elevated allergen-specific serum IgE and IgG levels, and peripheral blood mononuclear cells isolated from sensitized animals released IL-4, IL-5, and IL-10, but not IFN-gamma. Aerosolized allergen decreased dynamic compliance and induced airway inflammation and hyperresponsiveness to aerosolized histamine. Albuterol and dexamethasone inhibited the airway constriction and allergen-induced inflammation, respectively. Airway wall remodeling that included goblet cell hyperplasia, basement membrane thickening, and smooth muscle hypertrophy was particularly evident in neonatally sensitized animals. In contrast to animals sensitized as adults, neonatally sensitized animals exhibited increased sensitivity to adenosine and larger allergen-induced changes in airway resistance and dynamic compliance. These results demonstrate that sensitization of cynomolgus monkeys with dust mite induces asthmalike symptoms, some of which may be dependent on age at the time of sensitization.     

Serum hormone levels in pregnant cynomolgus monkeys

Serum levels of estradiol (E2), progesterone (P), prolactin (Prl), monkey chorionic gonadotropin (mCG), dehydroepiandrosterone sulfate (DHEAS), and cortisol (C) in pregnant cynomolgus monkeys are described. mCG, E2, and Prl patterns resembled those of pregnant rhesus monkeys. P patterns differed from data from the literature. DHEAS patterns did not follow E2 patterns. C levels did not change during pregnancy.     

Iliac biopsy for histomorphometric analysis of trabecular bone in cynomolgus monkeys and baboons

A humane, repeatable surgical technique was developed to harvest trabecular bone for histomorphometry from nonhuman primates. Using a direct surgical approach to the iliac crest, bone specimens with a mean cross sectional area of trabecular bone plus marrow (AT) of 12.02 and 18.07 mm2 were harvested from cynomolgus monkeys (Macaca fascicularis) and baboons (Papio anubis) respectively. The method provides bone samples with little artifact or bone dust contamination, and easy orientation and reproducibility of the plane of section. None of the cynomolgus monkeys were affected adversely by the surgery, but the baboons were quieter than normal for 12-24 hours afterward. The technique offers excellent biopsy specimens, repeatability and a minimum of stress to the subjects.     

Heterotopic autotransplantation of ovarian cortex in cynomolgus monkeys

In recent years, removal of ova or ovaries before chemotherapy or radiation therapy has been investigated in young female cancer patients to avoid the adverse effects of treatment. Orthotopic autotransplantation of ovarian cortex has advantages such as easy collection of ova and the possibility of spontaneous pregnancy. Although children have been born after successful orthotopic autotransplantation into the residual ovaries, some patients cannot undergo this procedure such as those who need bilateral ovariectomy or pelvic radiation therapy, therefore it is still necessary to investigate suitable heterotopic autotransplantation sites. The present study was performed in primates (cynomolgus monkeys) with the objective of determining the optimum site for heterotopic autotransplantation of ovarian cortex to enhance the clinical application of this method. The retroperitoneal iliac fossa and omentum were selected as sites for heterotopic autotransplantation. Two cynomolgus monkeys were subjected to laparotomy under anesthesia. After resection of the bilateral adnexae, the ovaries were cut into 0.5 cm cubes that were transplanted. Blood levels of follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone were monitored, and monkeys with a regular estrus cycle underwent superovulation and egg collection. In both monkeys studied, recovery of a regular estrus cycle was confirmed after heterotopic autotransplantation of ovarian tissue. MII phase ova were successfully collected from tissues transplanted into the retroperitoneal iliac fossa or omentum. Development to the early blastocyst stage was confirmed after microfertilization. We established an appropriate method of heterotopic autotransplantation using ovarian cortex into the retroperitoneal iliac fossa or omentum in primates.     

Polymorphism in the mitochondrial DNA of cynomolgus monkeys

Variations in the mitochondrial DNA of a total of 150 cynomolgus monkeys (Macaca fascicularis) from Indonesia, the Philippines, and Malaysia were studied using a restriction endonuclease, EcoRI. Three distinct patterns were detected and they were denoted as morph 1, 2, and 3. The Malaysian population proved to be significantly different from the remaining two populations in the distributions of the three EcoRI morphs.     

The development of range of action in infant cynomolgus monkeys (Macaca fascicularis) reared by restrained mothers

In order to study the effects of the mothers' range of action on the development of their infants' range of action during the first year of life, mother cynomolgus monkeys were restrained in their range of action by penning them in a separation cage within the large cage of the harem group they belonged to. The infants, however, could leave their mothers' separation cage. The control group consisted of infants growing up with unrestrained mothers in the same group. It appeared that infants of restrained mothers were initially retarded in the development of their range of action but at the end of the first year they did not differ anymore from infants of unrestrained mothers. It is concluded that the maternal range of action only temporarily affects the development of the infant's range of action.     

Suppressor macrophages in lymphocyte proliferation of cynomolgus monkeys

The present study demonstrated the presence of cells belonging to monocyte/macrophage lineage which suppressed mitogen-induced blastogenesis of peripheral blood lymphocytes in cynomolgus monkeys. Depletion of adherent or phagocytic cells from peripheral mononuclear cells caused a substantial increase in the blastogenic response of cynomolgus monkey lymphocytes whereas the same treatment led to marked reduction rather than enhancement in human lymphocyte blastogenesis. Addition of thioglycollate-elicited peritoneal exudate adherent cells as macrophages suppressed the blastogenic response of nonadherent lymphocytes in a dose-dependent manner. The suppressive effect was observed not only in autologous but also in allogeneic macrophages to the responder lymphocytes. Treatment of macrophages with silica, carrageenan or freezing-thawing reduced their suppressive effect but there was no reduction with mitomycin C or indomethacin. No suppressive activity was detected in the cell-free supernatant of macrophages cultured in the presence or absence of mitogens for up to 4 days. From these findings, it appeared that monocyte/macrophage lineage might be responsible for the observed suppressive effect on mitogen-induced blastogenesis of cynomolgus monkey lymphocytes.     

Epigenetic changes with dietary soy in cynomolgus monkeys

Nutritional interventions are important alternatives for reducing the prevalence of many chronic diseases. Soy is a good source of protein that contains isoflavones, including genistein and daidzein, and may alter the risk of obesity, Type 2 diabetes, osteoporosis, cardiovascular disease, and reproductive cancers. We have shown previously in nonhuman primates that soy protein containing isoflavones leads to improved body weight, insulin sensitivity, lipid profiles, and atherosclerosis compared to protein without soy isoflavones (casein), and does not increase the risk of cancer. Since genistein has been shown to alter DNA methylation, we compared the methylation profiles of cynomolgus monkeys, from multiple tissues, eating two high-fat, typical American diets (TAD) with similar macronutrient contents, with or without soy protein. DNA methylation status was successfully determined for 80.6% of the probes in at least one tissue using Illumina's HumanMethylation27 BeadChip. Overall methylation increased in liver and muscle tissue when monkeys switched from the TAD-soy to the TAD-casein diets. Genes involved in epigenetic processes, specifically homeobox genes (HOXA5, HOXA11, and HOXB1), and ABCG5 were among those that changed between diets. These data support the use of the HumanMethylation27 BeadChip in cynomolgus monkeys and identify epigenetic changes associated with dietary interventions with soy protein that may potentially affect the etiology of complex diseases.     

Evidence for morphine-induced galactorrhea in male cynomolgus monkeys

We evaluated the effect of a daily injection of morphine hydrochloride on galactorrhea in male cynomolgus monkeys. Three groups of three monkeys (nine total) were used. The treatment schedule was separated into three periods: pre-treatment, treatment, and post-treatment. Each group of monkeys was subcutaneously injected daily with 1.5, 3.0, or 6.0 mg/kg (monkey weight) of morphine for 74–130 days, respectively, during the treatment period, and with saline during the pre-treatment and post-treatment periods. We then measured the prolactin (PRL) and testosterone (T) levels from weekly blood samples that were taken 20 hours after injection. No statistically significant differences in either the PRL or T level were detected throughout the treatment period. However, monkeys treated with 3.0 and 6.0 mg/kg/day showed a decrease in T level and an increase in PRL level during the early post-treatment period. Seven of the nine monkeys produced a milk-like secretion from their mammary gland (a symptom of galactorrhea) during the treatment and post-treatment periods. For several months of post-treatment period (average 6.75 months), we monitored the time-course changes in PRL and T levels in all monkeys for 10 hours after a single injection of morphine at the same dose given during the treatment period. Morphine induced a sudden increase in the PRL level (peaked within 30 minutes) and a gradual decrease in the T level (leveled off within 6.5–10 hours), and then returned to basal levels. These results indicate that morphine does not cause a long-term effect on hormonal changes and that a morphine-induced transient rise in PRL levels accompanied by a decrease in T levels can induce spontaneous galactorrhea in male cynomolgus monkeys.     

Comparative pharmacokinetics of frusemide in female rhesus monkeys, cynomolgus monkeys and baboons

1. The pharmacokinetics of frusemide have been compared in 3 non-human primate species after single intravenous dose of 3 mg/kg of the drug. 2. Peak mean plasma concentrations of frusemide were 31.6, 33.6, 43.6 micrograms/ml in the rhesus monkey, cynomolgus monkey and baboon respectively, and concentrations declined with a half-life of about 20 min. 3. There were no notable differences in the pharmacokinetic parameters estimated from either a one-compartment or two-compartment open model. 4. There were statistically significant species-related differences in clearance, half-lives and volumes of distribution adjusted for bodyweight. 5. The pharmacokinetics of frusemide in the cynomolgus monkey are closer to those in man than are those in the rhesus monkey, the baboon or other commonly used laboratory animal species.     

Immune suppression in cynomolgus monkeys by XPro9523

The CTLA4-Ig fusion proteins abatacept and belatacept are clinically proven immunosuppressants used for rheumatoid arthritis and renal transplant, respectively. Given that both biologics are typically administered chronically by infusion, a need exists for a next-generation CTLA4-Ig with more convenient dosing. We used structure-based protein engineering to optimize the affinity of existing CTLA4-Ig therapeutics for the ligands CD80 and CD86, and for the neonatal Fc receptor, FcRn. From a rationally designed library, we identified four substitutions that enhanced binding to human CD80 and CD86. Coupled with two IgG1 Fc substitutions that enhanced binding to human FcRn, these changes comprise the novel CTLA4-Ig fusion protein, XPro9523. Compared with abatacept, XPro9523 demonstrated 5.9-fold, 23-fold, and 12-fold increased binding to CD80, CD86, and FcRn, respectively; compared with belatacept, CD80, CD86, and FcRn binding increased 1.5-fold, 7.7-fold, and 11-fold, respectively. XPro9523 and belatacept suppressed human T cell proliferation and IL-2 production more potently than abatacept. XPro9523 also suppressed inflammation in the mouse collagen-induced arthritis model. In cynomolgus monkeys, XPro9523 saturated CD80 and CD86 more effectively than abatacept and belatacept, potently inhibited IgM and IgG immunization responses, and demonstrated longer half-life. Pharmacokinetic modeling of its increased potency and persistence suggests that, in humans, XPro9523 may demonstrate superior efficacy and dosing convenience compared with abatacept and belatacept.