Evaluation of bendectin embryotoxicity in nonhuman primates: I. Ventricular septal defects in prenatal macaques and baboon

Cynomolgus monkeys, rhesus monkeys and baboons were administered 10 to 40 times the human dose equivalent of Bendectin throughout the major period of organogenesis (22(+/-3)-50 days of gestation). In animals examined prenatally (100 +/- 2 days gestation) the total incidence of ventricular septal defects (VSD) was 40% in cynomolgus monkeys, 18% in rhesus monkeys, and 23% in baboons. The majority of VSD involved the muscular portion of the septum. No dose response was evident and there were no other cardiac or extracardiac defects found except for one baboon fetus with multiple defects. No defects were observed in cynomolgus monkeys administered Bendectin for 4-day periods between 22 and 41 days of gestation. There was no association of Bendectin treatment with any noncardiac defect. In cynomolgus and rhesus monkeys examined at term there was one mitral valve defect and no incidence of VSD. The increased incidence of VSD observed prenatally in all three species and the absence of defects in macaques at term suggests a delay in closure of the ventricular septum in treated animals. The Bendectin-treated monkey may be a suitable model for the study of the pathogenesis of VSD and the mechanism of spontaneous closure of the defect.     

Embryotoxicity of sex steroidal hormone combinations in nonhuman primates: I. Norethisterone acetate + ethinylestradiol and progesterone + estradiol benzoate (Macaca mulatta, Macaca fascicularis, and Papio cynocephalus)

Two sex steroid hormone combinations which have been used clinically as tests for detection of early pregnancy were examined for embryotoxic effects in macaques and baboons. Norethisterone acetate and ethinyl estradiol (NEA + EE) were orally administered to rhesus and cynomolgus monkeys and baboons at dosages ranging from one to 1,000 times the human dose equivalent (HDE) during days 20-50 of pregnancy. Progesterone and estradiol benzoate (P + EB) were delivered by two to six intramuscular injections to rhesus and cynomolgus monkeys between gestational days 20 and 35 at 0.1-25 X HDE. Fetuses were examined following cesarean section at 100 +/- 2 days (NEA + EE) or at term (P + EB). The results showed increased embryolethality over controls at 100-1,000 X HDE (NEA + EE) and at 10 and 25 X HDE (P + EB). Besides growth retardation, isolated cases of minor nongenital malformations were observed only in cynomolgus monkeys following treatment with both hormone combinations mainly at embryolethal dose levels and were considered spontaneous in nature. Virilization of female cynomolgus fetuses following NEA + EE treatment was manifested as two cases of clitoral enlargement in the 300 X HDE group and two cases of increased anogenital distance with reduced vaginal opening in the 1,000 X HDE group. The highest dose of NEA + EE was also maternally toxic, as two maternal deaths occurred at the end of the treatment period. One dead female cynomolgus fetus exposed to P + EB (10 X HDE) also exhibited masculinized external genitalia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Examination of bone chemical composition in osteoporosis using fluorescence-assisted synchrotron infrared microspectroscopy.

Although it is clear that osteoporosis is associated with a reduction in bone mass and a fragile skeleton, it is not understood whether the chemical composition of osteoporotic bone is different from normal bone. In this study, cynomolgus monkeys (Macaca fascicularis) were administered fluorochrome labels at one and two years after ovariectomy (Ovx) or Sham ovariectomy (intact), that were taken up into newly remodeled bone. Using fluorescence-assisted synchrotron infrared microspectroscopy, the chemical composition of bone from intact versus Ovx monkeys has been compared. Results from overall composition distributions (labeled + non-labeled bone) reveal similar carbonate/protein and phosphate/protein ratios, but increased acid phosphate content and different collagen structure in the Ovx animals. Analysis of the fluorochrome-labeled bone indicates similar degrees of mineralization in bone remodeled after one year, but decreased mineralization in Ovx bone remodeled two years after surgery. Thus, bone from monkeys with osteoporosis can be characterized as having abnormal collagen structure and reduced rates of mineralization. Coupled with factors such as trabecular architecture and bone shape and size, these ultrastructural factors may play a contributing role in the increased bone fragility in osteoporosis.     

Use of biochemical markers to monitor changes in bone turnover in cynomolgus monkeys

The ovariectomized old cynomolgus monkey is a recognized model of human osteoporosis, and the same species can be used for the assessment of the efficacy and potential toxicity of agents intended to prevent or treat osteoporosis. Several assays have been developed that can measure the same biochemical markers of bone turnover as are used in human patients for the diagnosis and treatment follow-up of bone-related diseases, including osteoporosis. The aim of the present study was to describe the results obtained with these assays in normal control monkeys, their variations with age and sex, and their sensitivity in monitoring the bone turnover induced by ovariectomy in old skeletally mature cynomolgus monkeys. Seven old cynomolgus monkeys were bilaterally ovariectomized and 13 age-matched monkeys were sham-operated. Bone mineral density and biochemical markers were measured before and at regular intervals after surgery for up to 20 months. Total alkaline phosphatase (total ALP), bone-specific alkaline phosphatase isoenzyme (bone ALP) and osteocalcin (OC) were highly correlated to the decrease in bone mineral density (BMD) induced by ovariectomy. Deoxypyridinoline (DPD) measured by enzyme-linked immunoassay was insensitive to the bone resorption induced by ovariectomy, but cross-linked N-telopeptide (NTX-I) was higher in ovariectomized monkeys than in control monkeys. These results demonstrate that reliable biochemical parameters are available to adequately monitor and provide insight into osteoclastic bone resorption and osteoblastic bone formation, the two components of bone turnover in this animal model, and can thus be used to assess the efficacy and toxicity of potential therapeutic agents.     

Use of biochemical markers to monitor changes in bone turnover in cynomolgus monkeys.

The ovariectomized old cynomolgus monkey is a recognized model of human osteoporosis, and the same species can be used for the assessment of the efficacy and potential toxicity of agents intended to prevent or treat osteoporosis. Several assays have been developed that can measure the same biochemical markers of bone turnover as are used in human patients for the diagnosis and treatment follow-up of bone-related diseases, including osteoporosis. The aim of the present study was to describe the results obtained with these assays in normal control monkeys, their variations with age and sex, and their sensitivity in monitoring the bone turnover induced by ovariectomy in old skeletally mature cynomolgus monkeys. Seven old cynomolgus monkeys were bilaterally ovariectomized and 13 age-matched monkeys were sham-operated. Bone mineral density and biochemical markers were measured before and at regular intervals after surgery for up to 20 months. Total alkaline phosphatase (total ALP), bone-specific alkaline phosphatase isoenzyme (bone ALP) and osteocalcin (OC) were highly correlated to the decrease in bone mineral density (BMD) induced by ovariectomy. Deoxypyridinoline (DPD) measured by enzyme-linked immunoassay was insensitive to the bone resorption induced by ovariectomy, but cross-linked N-telopeptide (NTX-I) was higher in ovariectomized monkeys than in control monkeys. These results demonstrate that reliable biochemical parameters are available to adequately monitor and provide insight into osteoclastic bone resorption and osteoblastic bone formation, the two components of bone turnover in this animal model, and can thus be used to assess the efficacy and toxicity of potential therapeutic agents.     

Effectiveness of zona pellucida protein ZPB as an immunocontraceptive antigen

Immunization of female mammals with native zona pellucida (ZP) proteins is known to cause infertility. Since each human ZP protein is now available as a purified recombinant protein, is it possible to compare the immunocontraceptive potential of each ZP protein. A breeding study was conducted in cynomolgus monkeys (Macaca fasicularis) after immunization with recombinant human ZP (rhZP) proteins (ZPA, ZPB, ZPC) separately and in combinations. This study demonstrated that immunization with recombinant human ZPB (rhZPB) protein caused cynomolgus monkeys to become infertile for 9-35 months. A second study was conducted in baboons (Papio cynocephalus), which yielded a similar result. The baboons immunized with rhZPB became infertile for 9 to > 20 months. During the time of maximum antibody titre, some animals experienced disruption of the menstrual cycle, but eventually all of the animals resumed normal menstrual cycles. Control animals and animals immunized with other rhZP proteins all became pregnant before any of the rhZPB-treated animals. This is the first study in which a recombinant ZP protein has consistently induced infertility in a primate without permanent disruption of the normal menstrual cycle.     

A comparative study of the adrenergic nerves to the anterior eye segment of some primates

Summary The distribution of adrenergic terminals to the anterior eye segment of humans, Cynomolgue monkeys, squirrel monkeys, owl monkeys, Cebus monkeys, vervets, tamarins, and baboons has been investigated. The cornea is normally devoid of adrenergic terminals, except in a plexus near the limbus. The trabecular meshwork contains varying numbers of adrenergic terminals: usually none in Cynomolgus monkeys, patas monkeys, vervets, and humans, although fibres have very rarely been observed in Cynomolgus monkeys, vervets, and humans; a few in owl monkeys, squirrel monkeys, and tamarins; and moderate numbers in Cebus monkeys and baboons. From the evidence, however, it seems premature to presume an adrenergic innervation of the trabecular mechanism regulating the outflow resistance. The dilatator pupillae is regularly supplied with numerous adrenergic terminals and in the iris stroma there is probably an adrenergic innervation of the melanophores. The sphincter pupillae regularly contains adrenergic terminals with notable species differences; most fibres occur in baboons and fewest in humans, with the remaining species forming a middle class. The ciliary processes in all species contain a moderate number of adrenergic terminals, presumably primarily associated with the epithelium. Intraepithelial adrenergic terminals have been observed on the pars plana of the ciliary body of humans, Cebus monkeys, vervets, baboons, and patas monkeys. The ciliary muscle of baboons and Cynomolgus monkeys contains numerous adrenergic terminals. Moderate numbers occur in Cebus monkeys and vervets, and still less in (in falling order) tamarins, squirrel monkeys, humans, and patas monkeys.     

Accuracy and precision of digital volume correlation in quantifying displacements and strains in trabecular bone

Strain measurement is an essential tool in the study of trabecular bone structure-function relationships. Digital volume correlation (DVC) is a measurement technique that quantifies strains throughout the interior of a specimen, rather than simply those on the surface. DVC relies on tracking the movement of microstructural features, and as such, the accuracy and precision of this technique may depend on trabecular structure. This study quantified displacement and strain measurement errors in six types of trabecular bone that spanned a wide range of volume fraction and trabecular architecture. Accuracy and precision were compared across bone type and also across three DVC methods. Both simulated and real displacement fields were analyzed using micro-computed tomography images of specimens from the bovine distal femur, bovine proximal tibia, rabbit distal femur, rabbit proximal tibia, rabbit vertebra, and human vertebra. Differences as large as three-fold in accuracy and precision of the displacements and strains were found among DVC methods and among bone types. The displacement precision and the strain accuracy and precision were correlated with measures of trabecular structure such as structural model index. These results demonstrate that the performance of the DVC technique can depend on trabecular structure. Across all bone types, the displacement and strain errors ranged 1.86-3.39 microm and 345-794 microepsilon, respectively. For specimens from the human vertebra and bovine distal femur, the measurement errors were approximately 20 times smaller than the yield strain. In these cases, DVC is a viable technique for measuring pre- and post-yield strains throughout trabecular bone specimens and the trabecular compartment of whole bones.     

Detection of trabecular bone microdamage by micro-computed tomography

Microdamage is an important component of bone quality and affects bone remodeling. Improved techniques to assess microdamage without the need for histological sectioning would provide insight into the role of microdamage in trabecular bone strength by allowing the spatial distribution of damage within the trabecular microstructure to be measured. Nineteen cylindrical trabecular bone specimens were prepared and assigned to two groups. The specimens in group I were damaged to 3% compressive strain and labeled with BaSO(4). Group II was not loaded, but was labeled with BaSO(4). Micro-computed tomography (Micro-CT) images of the specimens were obtained at 10 microm resolution. The median intensity of the treated bone tissue was compared between groups. Thresholding was also used to measure the damaged area fraction in the micro-CT scans. The histologically measured damaged area fraction, the median CT intensity, and the micro-CT measured damaged area fraction were all higher in the loaded group than in the unloaded group, indicating that the micro-CT images could differentiate the damaged specimen group from the unloaded specimens. The histologically measured damaged area fraction was positively correlated with the micro-CT measured damaged area fraction and with the median CT intensity of the bone, indicating that the micro-CT images can detect microdamage in trabecular bone with sufficient accuracy to differentiate damage levels between samples. This technique provides a means to non-invasively assess the three-dimensional distribution of microdamage within trabecular bone test specimens and could be used to gain insight into the role of trabecular architecture in microdamage formation.     

Peripheral aromatization: studies on controlling factors

Using constant infusions of 3H-labeled androgens and 14C-labeled estrogens with measurements of radiolabeled estrogens in blood and/or urine we have carried out studies on the peripheral aromatization of androgens in humans, nonhuman primates, sheep, and rabbits. In the human, aromatization is increased in women as they become postmenopausal, although the mechanism remains uncertain. In humans and cynomolgus monkeys the administration of ACTH and/or glucocorticoids does not increase peripheral aromatization, but results in a slight decrease in the aromatization of androstenedione. The administration of l-thyroxine to cynomolgus monkeys increases peripheral aromatization of androstenedione from basal, 1.16 +/- 0.15%, to 1.71 +/- 0.14% probably due to increased tissue blood flow. The aromatization of testosterone is not affected, probably due to an increase in sex hormone-binding globulin. Peripheral aromatization occurs to a similar degree in humans, rhesus and cynomolgus monkeys, and baboons, but is much lower in sheep and rabbits. The compound 10-(2-propynyl)-estr-4-ene-3,17-dione is an effective inhibitor of the peripheral aromatization of both androstenedione and testosterone.     

Effects of aging on bone mineral content and bone biomarkers in female cynomolgus monkeys.

Age-related changes in bone mineral content and bone biomarkers were assessed over the complete lifespan of female cynomolgus monkeys. The bone mass of the lumbar spine increased linearly from birth to about 2.5 years of age, and this increase gradually slowed thereafter until a peak bone mass was achieved at 9 years of age. The bone mass stabilized after 9 years of age, showing no sign of further reduction with age. In contrast with the significant increase in bone mass before 2.5 years of age, significant decreases occurred in the serum concentrations of the following bone formation markers: intact osteocalcin, bone-specific alkaline phosphatase and amino-terminal propeptide of type I procollagen, but the serum concentration of carboxy-terminal propeptide of type I procollagen did not change significantly throughout the entire lifespan. Concerning the bone resorption markers, the levels of tartrate-resistant acid phosphatase fluctuated throughout the entire lifespan. The skeleton of an aging female monkey undergoes changes similar to those observed in senescent humans, but did not undergo the menopausal changes seen in women. The use of female cynomolgus monkeys to model human skeletal interventions should therefore be undertaken with consideration of the similarities and differences between cynomolgus monkeys and humans.     

Comparative analysis of natural killer cell activity, lymphoproliferation and lymphocyte surface antigen expression in nonhuman primates housed at the CIRMF Primate Center, Gabon

Six different species of nonhuman primates housed at the CIRMF Primate Center, cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), mandrills (Mandrillus sphinx), vervets (Cercopithecus aethiops pygerythrus), chimpanzees (Pan troglodyte) and baboons (Papio hamadryas), were evaluated for their natural killer cell activity and for the ability of their peripheral blood mononuclear cells to proliferate in response to known mitogens (concanavalin A, phytohemagglutinin and staphylococcal enterotoxin A) and to react with a panel of mouse monoclonal antibodies directed against human leukocyte surface antigens. Basic information on normal immune functions in these primates is important because of their use as experimental animal models for the study of human diseases such as acquired immunodeficiency syndrome (AIDS), hepatitis, loiasis and malaria.     

Movement of cynomolgus and rhesus monkey spermatozoa collected from the lower female reproductive tract

Postcoital (pc) cervical mucus was collected in 73 menstrual cycles of cynomolgus monkeys and in 43 cycles of rhesus monkeys at 2,6,10,30 hr pc. Videomicrography was used to analyze sperm numbers and movement in the mucus. Both cynomolgus and rhesus monkeys had comparable populations of motile sperm in the mucus at 2 hr pc. However, by 6 hr pc, cervical mucus from cynomolgus monkeys contained twice as many total sperm and motile sperm as mucus from rhesus monkeys (P <.05). Mean swimming speeds of the free-swimming cervical sperm were similar for the two species at this time. No motile sperm were recovered in mucus from rhesus monkeys at 30 hr pc. In cynomolgus monkeys, however, 14 of the 26 animals examined at 30 hr pc had motile sperm in their mucus. These sperm exhibited lower percent molility, percent free-swimming sperm, and swimming speed than those sperm observed at 6 hr pc. Uterine sperm were collected by transcervical or transuterine aspiration from cynomolgus monkeys. In the transcervical technique, sperm were successfully obtained in four of nine animals examined at 6 hr and in four of five animals at 30 hr pc. The percentage of motile sperm in the uterine fluid was high, 82% ± 4%, and the swimming speeds (86 ± 2μm/sec) were higher than those observed in cervical mucus. Approximately 5–10% of the uterine sperm exhibited swimming motions similar to the hyperactivated motility seen in most mammals. These findings indicate that the sperm cervical mucus interaction in vivo in cynomolgus monkeys has more similarities to the human situation than does the interaction in rhesus monkeys.     

Rib biopsy technique for cortical bone evaluation in rhesus monkeys (Macaca mulatta)

Old World primates are often studied to model human skeletal physiology. An important advantage of monkeys over other animal models (i.e., rodents) is the presence of cortical bone Haversian remodeling. Seventy-five female rhesus monkeys (Macaca mulatta) were subjected to bone biopsy. With monkeys in lateral decubitus position, the tenth rib was surgically exposed and freed from periosteum by use of careful sharp and blunt dissection. The rib section was resected, using bone cutters, and the surgical wound was closed. This procedure was repeated for the contralateral rib at a later time point in 65 monkeys. There was no mortality or appreciable morbidity. The bone specimens were (mean +/- SD) 2.50 +/- 0.25 cm long, with 5.5 +/- 1.0 mm2 total cross-sectional area. They were adequate for histologic, immunohistochemical, and quantitative histomorphometric examinations. Prevalence of pneumothorax was approximately 8.0% for the 140 procedures. This complication was immediately and successfully corrected by insertion of a small thoracic tube, evacuation of pneumothorax, and closure of the incision. This well-tolerated, repeatable procedure yields excellent specimens for performance of cortical bone histologic examination without euthanasia, allowing longitudinal evaluation.     

印度尼西亚源食蟹猴干扰素-γ基因的克隆和序列分析

目的获得印度尼西亚食蟹猴的干扰素-γ基因,为常用实验猕猴干扰素-γ的基因工程生产奠定基础。方法根据GenBank上公布的恒河猴干扰素-γ基因序列设计特异性引物,从印度尼西亚食蟹猴的外周血液中分离单核淋巴细胞,利用Trizol试剂,提取淋巴细胞的总RNA,通过RT-PCR的方法获得干扰素-γ基因片段,并对该片段进行克隆、鉴定和序列分析。结果扩增到一498bp的目的片段,经序列测定证实为印度尼西亚食蟹猴的干扰素-γ基因,与恒河猴、人及狒狒的干扰素-γ基因相比,同源性分别为100%、96%、99%。结论常用的两种实验猕猴食蟹猴与恒河猴的干扰素-γ基因完全相同。     

A global relationship between trabecular bone morphology and homogenized elastic properties.

An alternative concept of the relationship between morphological and elastic properties of trabecular bone is presented and applied to human tissue from several anatomical locations using a digital approach. The three-dimensional morphology of trabecular bone was assessed with a microcomputed tomography system and the method of directed secants as well as the star volume procedure were used to compute mean intercept length (MIL) and average bone length (ABL) of 4 mm cubic specimens. Assuming isotropic elastic properties for the trabecular tissue, the general elastic tensors of the bone specimens were determined using the homogenization method and the closest orthotropic tensors were calculated with an optimization algorithm. The assumption of orthotropy for trabecular bone was found to improve with specimen size and hold within 6.1 percent for a 4 mm cube size. A strong global relationship (r2 = 0.95) was obtained between fabric and the orthotropic elastic tensor with a minimal set of five constants. Mean intercept length and average bone length provided an equivalent power of prediction. These results support the hypothesis that the elastic properties of human trabecular bone from an arbitrary anatomical location can be estimated from an approximation of the anisotropic morphology and a prior knowledge of tissue properties.     

On the influence of mechanical conditions in osteochondral defect healing

Despite the introduction of new surgical techniques, the treatment of cartilage defects remains challenging. Delay or complete failure of cartilage healing is associated with problems in biological regeneration. The influence of mechanical conditions on this process, however, remains unevaluated. Osteochondral defects were generated on the left femoral condyle in 18 Yucatan minipigs. After 4, 6 and 12 weeks the defect filling, trabecular orientation and bone density were compared to the intact contralateral side. The mechanical straining during this period was then analyzed using an adaptive finite element technique. Histologically, the osteochondral defects showed bone resorption at the base and bone formation from the circumference. At 12 weeks, the macroscopically healed specimens showed fibrous cartilage formation, a minimally organized trabecular structure and increased trabecular volume fraction compared to the controls (p < 0.002). The amount of cancellous, cartilagineous, and fibrous tissue and the defect size as measured in histomorphometric analysis for the three time points (4, 6 and 12 weeks) was comparable in magnitude to that predicted by finite element analysis. The simulated osteochondral healing process was not fully capable of re-establishing a hyaline-like cartilage layer. The correlation between simulation and histology allows identification of mechanical factors that appear to have a larger impact on the healing of osteochondral defects than previously considered.     

Application of homogenization theory to the study of trabecular bone mechanics.

It is generally accepted that the strength and stiffness of trabecular bone is strongly affected by trabecular microstructure. It has also been hypothesized that stress induced adaptation of trabecular bone is affected by trabecular tissue level stress and/or strain. At this time, however, there is no generally accepted (or easily accomplished) technique for predicting the effect of microstructure on trabecular bone apparent stiffness and strength or estimating tissue level stress or strain. In this paper, a recently developed mechanics theory specifically designed to analyze microstructured materials, called the homogenization theory, is presented and applied to analyze trabecular bone mechanics. Using the homogenization theory it is possible to perform microstructural and continuum analyses separately and then combine them in a systematic manner. Stiffness predictions from two different microstructural models of trabecular bone show reasonable agreement with experimental results, depending on metaphyseal region, (R2 greater than 0.5 for proximal humerus specimens, R2 less than 0.5 for distal femur and proximal tibia specimens). Estimates of both microstructural strain energy density (SED) and apparent SED show that there are large differences (up to 30 times) between apparent SED (as calculated by standard continuum finite element analyses) and the maximum microstructural or tissue SED. Furthermore, a strut and spherical void microstructure gave very different estimates of maximum tissue SED for the same bone volume fraction (BV/TV). The estimates from the spherical void microstructure are between 2 and 20 times greater than the strut microstructure at 10-20% BV/TV.     

Macaca fascicularis, a nonpermissive host for the human filarial parasite Loa loa

The ability of the filarial nematode Loa loa to infect 2 species of primates was studied. The primate species selected were closely related to species known to be susceptible. A mandrill (Mandrillus sphinx) and 6 cynomolgus monkeys (Macaca fascularis) were infected by subcutaneous injection of third-stage larvae of human L. loa from Gabon. The mandrill developed microfilaremia with an estimated prepatent period of 147 days, but microfilariae were not detected in any of the cynomolgus monkeys. Thus, mandrills appear permissive to human L. loa, whereas cynomolgus monkeys are not. Serum antibody responses were examined on western blots of adult L. loa antigens. Preinfection sera from all animals gave no reactions, but, after infection, sera from cynomolgus monkeys reacted more intensely and with more antigens than mandrill sera. Antibodies were still detectable in cynomolgus monkeys 15 mo postinfection. These reactions were compared with those found using human infection sera. Reactions with the cynomolgus monkey sera resembled those found with resistant endemic and amicrofilaremic human sera.