Pamela E. Peterson

Frequency of prenatal loss in a macaque breeding colony

An accurate knowledge of the historical incidence of prenatal loss is essential for management of breeding colonies and for performing developmental toxicity studies in nonhuman primates. Data from the California Regional Primate Research Center indoor (timed‐mated) and outdoor (random‐mated) colonies of rhesus, cynomolgus, and bonnet macaques (Macaca mulatta, M. fascicularis, and M. radiata) were evaluated for a 10 year breeding period from 1984 to 1993.

Pathogenesis of retinoic acid-induced ear malformations in a primate model

13-cis retinoic acid (RA) is a causative agent for human/monkey retinoic acid embryopathy (RAE), in which the most common type of malformation is microtia or anotia. In the present study, malformed ears of monkey fetuses exposed to RA during early embryogenesis were analyzed and revealed a subtype of defects., i.e., apparent duplication of the external/middle ear. A part of the posterior auricle appeared to be ectopically formed in the anterior auricular region or in the region posterior to the auricle. Additionally, there was duplication of the zygomatic arch, malleus, and incus. In order to characterize possible pathogenetic events underlying these malformations, embryos at selected stages were collected after dosing dams with RA at 5 mg/kg/day during gestational days 12-27. Cellular retinoic acid binding protein I whole-mount immunostaining showed that RA induced specific alterations in the migration of cranial neural crest cells (NCC). NCC en route to the second pharyngeal arch were bifurcated, and some of these NCC migrated abnormally into the first and/or third arches, which may underlie external ear duplication. Scanning electron microscopy and neurofilament immunostaining provided evidence that there was partial duplication of trigeminal nerve/ganglion following RA insult. The duplication of NCC neuronal derivatives in the first pharyngeal arch is consistent with duplication of NCC mesenchymal components (zygomatic arch, malleus, and incus). Therefore, RA-induced alterations in cranial NCC migration patterns are likely to be a pathogenetic event underlying ear malformations (including duplication) of RAE in monkeys.

The nonhuman primate as a model of developmental immunotoxicity

Macaques are well suited for preclinical testing of biopharmaceutics due to reproductive and developmental similarities with humans. In order to characterize ontogeny of the immune system in this model, we studied lymphocyte and antigen-presenting cell populations in developing lymphoid tissues of rhesus macaque fetuses during the second and third trimesters [gestation days (GD) 75–145, term 165 days]. Systemic lymphoid tissues (thymus, spleen and lymph nodes, and intestinal tissue) were examined for morphology and cell surface markers by immunohisto-chemistry. Lymphocytes were further characterized by flow cytometry for differentiation markers. Splenic tissue from early second trimester fetuses was populated mainly by CD20+ B cells while the thymus contained large numbers of CD3+ T cells. In the late second trimester (day 80), appro-ximately equal populations of B and T cells were present in both tissues and numerous dendritic cells (p55+) were present in the intestinal lamina propria. By the second trimester, the rhesus macaque fetal lymphoid system is well developed. Analysis of lymphoid organs from retinoic acid-treated fetuses indicated that the T-cell (thymus)-dependent compartment of the spleen white pulp in specimens with thymic aplasia showed a reduction in size and proportion of CD3+ T cells compared to controls. Our findings indicate that RA-induced thymic defects result in disrupted development of the splenic T-cell-dependent compartment.

Frequency of spontaneous congenital defects in rhesus and cynomolgus macaques.

Abstract: This paper summarizes the spontaneous incidence of congenital defects in the rhesus and cynomolgus macaque colonies (Macaca mulatta and M. fascicularis) at the California Regional Primate Research Center. The computerized database used in this analysis included fetuses, term infants, juveniles, and adults that underwent a necropsy procedure over a 14‐year period (1983–1996). The calculated malformation rates were 0.9% (40/4,390) and 0.3% (3/965) for the rhesus and cynomolgus monkey, respectively. Most of the observed malformations in both species affected the musculoskeletal and the cardiovascular systems, while a smaller number of defects were observed in the gastrointestinal, urogenital, endocrine, and central nervous systems. Inbreeding did not contribute to the spontaneous malformation incidence and there was no predilection for sex (male vs. female) or housing (indoors vs. outdoors) among the malformed cases. This spontaneous malformation database in our macaque colony aids in the interpretation of defects that occur in an experimental study as well as in the ongoing assessment of a healthy nonhuman primate breeding colony.

Nonhuman primates : their role in assessing developmental effects of immunomodulatory agents

There are close physiologic similarities between humans and macaques that make them well suited for preclinical testing of biopharmaceutics. These include menstrual cycles of similar length and hormonal control, comparable cellular and endocrine processes of implantation, and similar time-tables of prenatal development. Three teratogenic agents have induced abnormal development of the macaque thymus that is a key organ in the development of the fetal immune system. Embryonic exposure to triamcinolone acetonide, a potent corticosteroid, during critical periods of thymus development caused marked hypoplasia, depletion of thymic lymphocytes, and reduction of epithelial elements. Aplasia and hypoplasia of the thymus were a distinct feature of the “retinoid syndrome” in cynomolgus macaques following exposure to 13-cis-retinoic acid (Accutane) in early pregnancy, the time of neural crest migration. Experimentally induced zinc deficiency in rhesus macaques from conception to 1-year of age caused severe alterations in immunocompetence. More recent studies have shown that the levels of IgG and IgA in cervicovaginal lavages of the rhesus macaque exhibit specific temporal patterns during the normal menstrual cycle. Taken together, theses data suggest that several macaque species are appropriate animal models for preclinical safety assessment of immunomodulatory drugs. Current teratology protocols in these models may require slight modifications to adequately assess the safety of these biologics.

Vitamin A teratogenicity and risk assessment in the macaque retinoid model.

Studies were performed in the cynomolgus monkey (Macaca fascicularis) to provide risk assessment information on safe dose levels of Vitamin A during human pregnancy. Vitamin A palmitate was orally administered at 7500 IU/kg (2.25 mg/kg) to 80 000 IU/kg (24 mg/kg) body weight during early pregnancy (gestation day [GD] 16-27). The results indicated a dose-related increase in exposure (AUC) to retinyl esters and retinoic acids (RA) (all-trans-RA, all-trans-4-oxo-RA, 13-cis-RA, 13-cis-4-oxo-RA). There was also a dose-related increase in abortion and malformation that affected typical retinoid target tissues in the embryo, including the craniofacial region, heart, and thymus. The NOAEL and LOAEL for structural malformations were 7500 IU/kg and 20 000 IU/kg (6 mg/kg), respectively. A companion study involving oral administration of 13-cis-RA during the same gestational period established the NOAEL for malformations at 0.5 mg/kg/day, which is close to the human therapeutic dose range (0.5 to 1.5 mg/kg/day) associated with retinoid embryopathy. Based on the known similarities in teratogenic susceptibility to 13-cis-RA, the monkey NOAEL for Vitamin A (7500 IU/kg) was used to estimate safe levels of this nutrient in humans applying a safety factor of 10. This approach yielded safe levels of Vitamin A during human pregnancy in the range of approximately 25 000 to 37 000 IU/day.

Analysis of hindbrain neural crest migration in the long-tailed monkey (Macaca fascicularis)

Neural crest cells make a substantial contribution to normal craniofacial development. Despite advances made in identifying migrating neural crest cells in avian embryos and, more recently, rodent embryos, knowledge of crest cell migration in primates has been limited to what was obtained by conventional morphological techniques. In order to determine the degree to which the nonhuman primate fits the mammalian pattern, we studied the features of putative neural crest cell migration in the hindbrain of the long-tailed monkey (Macaca fascicularis) embryo. Cranial crest cells were identified on the basis of reported distributional and morphological criteria as well as by immunocytochemical detection of the neural cell adhesion molecule (N-CAM) that labels a subpopulation of these cells. The persistent labeling of a sufficient number of crest cells with antibodies to N-CAM following their exit from the rostral, preotic and post-otic regions of the hindbrain facilitated tracking them along subectodermal pathways to their respective destinations in the first, second and third pharyngeal arches. Peroxidase immunocytochemistry was also employed to localize laminin and collagen-IV in neuroepithelial basement membranes. At stage 10 (8–11 somites), crest emigration occurred in areas of unfused neural folds through focal disruptions in the neuroepithelial basement membrane in both the rostral and pre-otic regions, although there was little evidence of crest migration in the post-otic hindbrain. By stage 11 (16–17 somites), the neural folds were fused (pre- and post-otic hindbrain) or in the process of fusing (rostral hindbrain), yet crest cell emigration was apparent in all three areas through discontinuities in the basement membrane. Emigration was essentially complete at stage 12 (21 somites) as indicated by nearly continuous cranial neural tube basement membranes. At this stage the pre-ganglia (trigeminal, facioacoustic and glossopharyngeal) were consistently stained with N-CAM. The current study has provided new information on mammalian neural crest in a well-established experimental model for normal and abnormal human development, including its use as a model for the retinoic acid syndrome. In this regard, the current results provide the basis for probing the mechanisms of retinoid embryopathy which may involve perturbation of hindbrain neural crest development.

Distribution of Extracellular Matrix Components during Early Embryonic Development in the Macaque

The distribution of fibronectin (FN), laminin (LM), hyaluronic acid (HA) and chondroitin sulfate (CS) were examined by peroxidase immunocytochemistry in long-tailed monkey embryos during the period of neural tube and notochord formation (stages 8-11). Reactivity for each component in the neuroepithelial basement membrane (BM) increased with advancing development. Discontinuous staining was observed in areas of epithelial-to-mesenchymal transformation, i.e. in dorsolateral sites of neural crest emigration and in the axial region of the primitive streak. The BM forming around the developing notochord also showed increased reactivity for FN, LM, HA and CS between stages 8 and 11. No staining occurred within the notochord. Stage-related increases in reactivity in the mesenchymal matrix was particularly notable for FN and HA which were ubiquitous throughout the mesoderm by stage 11. The results of this study are consistent with the proposed role of these components in maintaining epithelial integrity and providing a permissive substrate for cell migration during development. The observed temporal and regional staining patterns suggest that these glycoproteins and glycosaminoglycans are important morphogenetic factors in the macaque.

Retinoid metabolism and transplacental pharmacokinetics in the cynomolgus monkey following a nonteratogenic dosing regimen with all-trans-retinoic acid

Retinoids often exhibit a complex metabolic pattern and differential transplacental kinetics, which make it difficult to pinpoint the proximate compound responsible for the observed teratogenic effect. We have therefore studied the pharmacokinetics and metabolism of all-trans-retinoic acid (all-trans-RA) in cynomolgus monkeys following application of a nonteratogenic dosing regimen and compared the results with corresponding data from a previous study with a teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. All-trans-RA was administered to pregnant cynomolgus monkeys (Macaca fascicularis) by nasogastric intubation at a dose of 5 mg/kg body wt once daily from gestational day (GD) 16 to 26 and twice daily at 8-h intervals from GD 27 to 31. Examination of the fetuses of four dams on GD 100 +/- 2 showed no embryotoxic or teratogenic effects of the applied dosing regimen (Experiment 1). Maternal plasma retinoid pharmacokinetics on GD 16, 26, and 31 as well as embryonic retinoid profiles after the last drug administration on GD 31 were determined in thirteen further dams (Experiment 2). All-trans-RA reached much lower plasma concentrations after the last two treatments on GD 31 than after the first one on GD 16 and the eleventh one on GD 26 (0-24-h area-under-the-concentration-time-curve (AUC) values: 104 +/- 59 ng x h/ml (after the last treatment on GD 31), 189 +/- 110 (GD 16) and 393 +/- 305 ng x h/ml (GD 26). The predominant plasma metabolites of all-trans-RA were its beta-glucuronide and the beta-glucuronide of all-trans-4-oxo-RA. Both of these retinoids accumulated in the plasma during the period of treatment and displayed AUC values 5- to 30-fold higher than those of all-trans-RA. Embryonic concentrations of all-trans-RA were not increased over endogenous levels after the last administration on GD 31 when plasma concentrations were low. To evaluate the placental transport of all-trans-RA in the presence of high plasma concentrations, a further experiment was performed, in which a single dose of all-trans-RA (10 mg/kg body wt) was given to four pregnant monkeys on GD 31, and plasma pharmacokinetics as well as embryonic concentrations of retinoids at 4 h post-treatment were determined (Experiment 3). This dosing schedule yielded high plasma concentrations of all-trans-RA, while embryonic concentrations were about 40% of plasma levels. Based on the plasma AUC values on GDs 16 and 26 obtained in Experiment 2 and the degree of placental transfer, as determined on GD 31 in the presence of high plasma levels in Experiment 3, we estimated embryonic AUC values for the 24-h period following the nonteratogenic doses on GDs 16 and 26 in Experiment 2. These AUC values were similarly high to the embryonic AUC value of all-trans-RA obtained after application of the teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. In addition, plasma AUC values of all-trans-RA were 2- to 7-fold higher after all-trans-RA administration (present study) than after dosing with the teratogenic dose of 13-cis-RA. These results strengthen our recent suggestion that the teratogenic effects induced in cynomolgus monkeys by 13-cis-RA treatment cannot solely result from the action of all-trans-RA, but may involve 13-cis-RA and 13-cis-4-oxo-RA, which could act directly or function as transport vehicle.

Two cases of digital defects in Macaca mulatta infants and a survey of the literature

Although congenital digital defects, particularly polydactyly, have been reported frequently in humans, their occurrence in rhesus macaques is relatively rare. We observed two cases of spontaneous digital defects in male rhesus monkey infants recently born at the California Regional Primate Research Center. One infant exhibited bilateral postaxial polydactyly and the other infant had bilateral oligodactyly with unilateral phalangeal duplication. In this report, we present the clinical/pathological details of these cases as well as discuss the embryology of normal and abnormal limb development. We will also summarize a variety of spontaneous and experimentally induced digital defects that have been reported in several nonhuman primate species.