Central nervous system malformations induced by triamcinolone acetonide in nonhuman primates: pathology

Triamcinolone acetonide (TAC) was administered to pregnant macaques (Macaca mulatta [15] and M. radiata [7]) during gestational days (GD) 23 to 41 using various dosing schedules. A daily dose of 10 mg/kg is approximately equal to 100 x the human dose equivalent. The brains of the fetuses and infants were studied grossly and histologically. All cases displayed either the mild form of the TAC-induced syndrome (craniofacial dysmorphia, cranium bifidum occultum, meningocele, and mild distortion of the midbrain) or the more severe form (occipital encephalocele, hydrocephalus, severe distortion of the midbrain or midbrain "beaking," shunting of cerebrospinal fluid, and craniofacial malformations). The dysmorphology was dose-related, with severity increasing at higher doses or with increased numbers of treatments. Individual cases were assessed for the severity of the syndrome by comparison of like components between groups. The lesions observed were morphologically comparable to those described in spontaneous human cases; the TAC-induced occipital encephaloceles were associated with brainstem and cerebellar abnormalities, and, with the less severe form of the syndrome, brainstem abnormalities were occasionally present, with occipital meningoceles. Controversy exists concerning the significance and temporal development of the midbrain changes. However, the associated alteration in aqueduct conformation may have been responsible for functional compromise and ensuing hydrocephalus.     

Morphogenesis of the respiratory bronchiole in rhesus monkey lungs

The epithelium of the respiratory bronchiole in the adult rhesus monkey consists of two populations: a pseudostratified epithelium with basal, mucous goblet, and ciliated cells located near the pulmonary artery (PA); and a simple cuboidal epithelium composed only of nonciliated bronchiolar epithelial (or Clara) cells in areas away from the PA. This study describes the pattern of differentiation of these two epithelial populations, and their relationship to the PA and to the time of appearance of alveoli in the respiratory bronchiole of the rhesus monkey during the period of 90-125 days gestational age (DGA). These events were related to changes in the adjacent parenchyma. Dissected airways of infusion-fixed, critical-point-dried lungs were evaluated by scanning microscopy followed by light microscopy of the same airways. At 54% of gestation (90 DGA), the distal airway was lined by a mixture of ciliated and nonciliated cells. By 67% of gestation (110 DGA), the ciliated cells were confined to the epithelium over the PA. The underlying connective tissue initially was cellular containing few fibers but was fibrous by 76% of gestation (125 DGA). Alveolarization began near the most distal cartilage at 57% of gestation (95 DGA), the same period at which secondary septation occurred in the distal acinus. Thus, alveolarization occurred simultaneously in two centers: 1) the proximal centriacinar region in the vicinity of the most distal cartilage and 2) the distal lung parenchyma. The duration of centriacinar alveolarization was short, approximately 5 days.     

Diethylstilbestrol-induced cervical and vaginal adenosis using the neonatal mouse model

The relevance of diethylstilbestrol (DES) administration to neonatal mice as a model for human pathology attributed to the use of DES in high-risk pregnancies has been investigated, particularly with respect to cervical and vaginal changes in female offspring. Neonatal DES treatment of mice results in tonic pituitary gonadotropin release and continuous estrogen secretion by the ovary. Studies were designed to determine the effect of this altered ovarian endocrine activity on cervical and vaginal histopathology. Ovariectomy of DES-treated mice, with or without estradiol replacement, did not eliminate the lesions, nor did estrogen and progesterone administered in a regimen intended to mimic estrous cycle changes. Induction of the constant estrus state by neonatal estradiol benzoate or testosterone propionate administration or by exposure to constant light did not produce the type of vaginal or cervical changes seen in DES mice. Thus, altered ovarian function is apparently not required for the vaginal and cervical changes appearing in later life. A role for endogenous (or exogenous) ovarian hormones in the developmental progression toward normality is suggested.     

Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. Experimental verification on unilamellar vesicles and lipid/alpha-lactalbumin complexes

Various models for the analysis of time-dependent fluorescence anisotropy measurements were evaluated. The discussion was based on the analysis of pulsed experiments with 1,6-diphenyl-1,3,5-hexatriene embedded in small unilamellar vesicles of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine and in dimyristoylphosphatidylcholine/alpha-lactalbumin complexes. It was shown that a recently proposed model (Van der Meer, W., H. Pottel, W. Herreman, M. Ameloot, H. Hendrickx, H. Schröder, 1984, Biophys. J., 46:515-523) described the data better than did the earlier suggested cone model (Kinosita K., Jr., S. Kawato, and A. Ikegami, 1977, Biophys. J., 20:289-305). This permitted the use of the new model for the estimation of the second- and fourth-rank order parameters on nonoriented systems. The results indicated that a fraction of the probes was oriented perpendicularly to the preferred direction of the lipids. An increase of the rotational correlation times of the fluorescent probe and a higher order of its environment were detected after the interaction of alpha-lactalbumin with the dimyristoylphosphatidylcholine vesicles at acidic pH at 24.2 degrees C.     

Monitoring ovulation and implantation in the cynomolgus macaque (Macaca fascicularis) through evaluations of urinary estrone conjugates and progesterone metabolites: a technique for the routine evaluation of reproductive parameters

Investigations were undertaken to determine the applicability of recently reported specific radioimmunoassays for urinary estrone conjugates and progesterone metabolites for monitoring ovarian function in the cynomolgus macaque (Macaca fasciularis) and other macaque species. Mean estrone conjugate measurements appear to accurately reflect the preovulatory estrogen peak in both conceptive (n = 5) and nonconceptive (n = 6) cycles, as well as to indicate early pregnancy through increases which are significantly elevated by Day + 15 (p less than 0.049) post estrone conjugates peak. The mean luteal phase levels of these progesterone metabolites are significantly elevated by Day + 14 (p less than 0.012) in conceptive cycles when compared to the mean values for nonconceptive cycles.     

Craniofacial and central nervous system malformations induced by triamcinolone acetonide in nonhuman primates: II. Craniofacial pathogenesis

This study further defines the craniofacial malformations induced by triamcinolone acetonide in the rhesus monkey. Ten timed-mated pregnant rhesus monkeys (Macaca mulatta) received intramuscular injections of 10 mg/kg TAC on days 23, 25, 27, 29, and 31 of gestation. Results of previous experiments with rhesus and bonnet monkeys and baboons indicated that specific craniofacial and brain malformations could be induced with TAC during this period of pregnancy (Hendrickx et al., '80). Stage-matched TAC-treated and control embryos (stages 17-18 and 22) and age-matched TAC-treated and control fetuses (50, 60, and 70 days gestation) were removed by hysterotomy. Stage 17-18 TAC embryos appeared grossly normal but histologic evaluation revealed a shortened anlage of the posterior cranial base. Stage 22 TAC embryos and all TAC fetuses exhibited craniofacial dysmorphia and encephalocele. The developing sphenoid was the earliest affected and most severely malformed bone. Its defects included reduced anterioposterior and transverse dimensions, reduced orbitosphenoid and alisphenoid, abnormal pituitary fossa, and reduced dorsum and tuberculum sellae. In addition, shortening of the posterior cranial base and decreased cranial base angle was a consistent finding in the treated embryos and fetuses. Decreased ossification and remodeling in the facial bones and abnormal position due to the malformed sphenoid occurred.     

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.     

Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus.

An animal model for the heterosexual transmission of human immunodeficiency virus (HIV) was developed by the application of simian immunodeficiency virus (SIV) onto the genital mucosas of both mature and immature, male and female rhesus macaques. Virus preparations were infused into the vaginal vaults or the urethras (males) of the animals through a soft plastic pediatric nasogastric feeding tube. The macaques that were infected by this route (six males and nine females) developed SIV-specific antibodies, and SIV was isolated from peripheral mononuclear cells of all seropositive animals. One male and one female infected by this route developed severe acquired immunodeficiency syndrome-like disease with retroviral giant-cell pneumonia. As few as two inoculations of cell-free SIV containing 50 50% tissue culture infective doses induced persistent viremia. Cell-free virus preparations were capable of producing infection by the genital route. Much higher doses of virus were required to transmit SIV by this route than are required for transmission by intravenous inoculation. Thus, it appears that the mucous membranes of the genital tract act as a barrier to SIV infection. Spermatozoa and seminal plasma were not required for the genital transmission of SIV. Rarely, SIV was recovered from mononuclear cells in semen and vaginal secretions. The SIV-rhesus macaque model is suitable for assessing the role of cofactors in heterosexual transmission of HIV and will be useful for testing the effectiveness of spermicides, pharmacologic agents, and vaccines in preventing the heterosexual transmission of HIV.     

Development of the definitive kidney in the cynomolgus monkey (Macaca fascicularis)

Abstract: Formation of the definitive kidney in Macaca fascicularis embryos was investigated using light and electron microscopy. Appearance of the definitive kidney at stage 14 was indicated by the ureteric bud invading the metanephrogenic blastema. Glomerular capillaries originate from the connective tissue that surrounds the developing renal vesicle. At 46–100 days gestational age the more developed glomeruli show thinning of the capillary endothelium, thickening of the basal membrane, and presence of pedicels, suggesting a capability of renal function.     

The influence of polyalcohols and carbohydrates on the thermostability of alpha-amylase

The influence of polyhydric alcohols and carbohydrates on the thermostability, i.e., the heat inactivation kinetics, of Bacillus licheniformis alpha-amylase was studied in the temperature range 96 degrees to 130 degrees C. High concentrations (from 9 to 60 weight percent) of glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing additive concentration. Statements about stabilization should, however, be specified carefully with respect to temperature, because E(A) is mostly altered likewise. For dissolved enzyme E(A) was almost always decreased in the presence of polyol or carbohydrate, whereas for immobilized enzyme it was augmented in each studied instance. The inactivation of dissolved enzyme can, in all the studied cases, be adequately described as a first-order process. Immobilized enzyme, however, shows biphasic then first-order inactivation kinetics, depending on the additive concentration and temperature.