Evaluation of tuberculin testing and measles prophylaxis procedures used in rhesus macaque quarantine/conditioning protocols.

Twenty-three of 103 adult rhesus macaques (Macaca mulatta) entering NIH holding facilities with no history of measles vaccination or infection, no titer to rubeola virus, a minimum of four negative results of intrapalpebral tuberculosis tests, and negative for Herpesvirus simiae and type D retroviruses were selected to evaluate the adequacy of commonly used quarantine/conditioning protocol procedures. One month after sensitization by subcutaneous inoculation with 100 mg of killed Mycobacterium tuberculosis in oil, an intrapalpebral tuberculosis test was administered in the right eyelid. All animals had reactions that ranged from grade II to grade V. The animals were then randomly allotted to three groups. Ten animals were inoculated with a rubeola-containing veterinary vaccine (VET), 10 were inoculated with a human measles vaccine routinely used in macaque quarantine procedures (HUM), and 3 were used as unvaccinated controls. Intradermal tuberculosis tests were administered in the left eyelid and the skin of the abdomen at vaccination (day 0), and subsequent abdominal skin tests were performed on days 5, 14, and 28. In addition, intrapalpebral tests were conducted on day 28. A higher response in the rubeola antibody enzyme-linked immunosorbent assay (ELISA) optical density (OD) results was observed in the VET-inoculated group at 14 days after inoculation. More significantly, two members of the HUM-vaccinated group had negative ELISA results after a single dose of vaccine. Three other members of the HUM-inoculated group had ELISA results that were near the OD cutoff value (0.15) and were retested by the measles indirect fluorescent antibody (IFA) test.(ABSTRACT TRUNCATED AT 250 WORDS)

A controlled seroprevalence survey of primate handlers for evidence of asymptomatic herpes B virus infection.

Herpes B virus (BV) is a common cause of recurring mucocutaneous infections in monkeys of the genus Macaca. Like its human counterpart, herpes simplex virus (HSV), BV establishes lifelong latency and can be reactivated from infected monkeys symptomatically or asymptomatically. Incidental infection of humans handling BV-shedding monkeys can result in fatal meningoencephalitis. To determine whether humans exposed to infected monkeys can acquire asymptomatic BV infections, 480 subjects were evaluated in a controlled seroprevalence study. Sera from 321 primate handlers, including many with repeated injuries inflicted by Macaca monkeys, and 159 people never exposed to monkeys were tested in blinded fashion by both competition ELISA and Western blot to determine the prevalence of BV and HSV seropositivity. Although 293 persons proved positive for HSV antibodies, no primate handlers or control subjects showed BV-specific antibody responses. There is no serologic evidence that BV causes asymptomatic infections in humans.

Excretion rates and metabolites of oestradiol and progesterone in baboon (Papio cynocephalus cynocephalus) faeces.

Two unanaesthetized female yellow baboons (Papio cynocephalus cynocephalus) were infused (i.v.) with [3H]oestradiol and two with [3H]progesterone, early in the follicular phases of their cycles. One month later, the two females infused with [3H]oestradiol were simultaneously infused with [14C]progesterone and [3H]dehydroepiandrosterone. All urine and faeces were collected for 96 h after infusion. The proportion of steroid excreted in faeces (versus urine) was 10.0% for oestradiol and 40% for progesterone. Peak excretion in urine occurred 4.5 h after infusion. Peak excretion in faeces occurred an average of 36.4 h after infusion, with remarkable consistency between steroids. Eighty per cent of faecal oestradiol and progesterone metabolites were excreted as free (rather than conjugated) steroids. Simply boiling (20 min) the dried faecal sample in 90% ethanol proved to be the most rapid and efficient means of extracting these steroid metabolites. High pressure liquid chromatography and immunoreactivity studies revealed that oestradiol was excreted in faeces as oestradiol (36%), oestrone (44%) and a conjugated metabolite that co-eluted with oestrone sulfate (20%). Progesterone was excreted as eight different free forms, only a minor portion of which was progesterone, and what appeared to be a conjugated metabolite that co-eluted with pregnanediol-glucuronide (20%). The free progesterone metabolites were identified by gas-chromatography-mass-spectrometry as epimers of 5-pregnane-3-diol and 5-pregnane-3-ol-one. These data suggest that currently available immunoassays for free oestradiol and oestrone should adequately characterize faecal oestrogen profiles in baboons. However, high variability in crossreactivities of various progesterone antisera to progesterone metabolites in baboons makes antiserum selection a more serious concern in attempts to quantify faecal progestogen dynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine profile of catch-up growth in the cynomolgus monkey.

We studied catch-up growth following withdrawal of glucocorticoid administration in seven intact prepubertal cynomolgus monkeys. To reduce stress during blood sampling, a vascular access port was implanted subcutaneously in each animal for the duration of the study. After a baseline observation period of 50 weeks, the monkeys received injections of dexamethasone at a dose of 100 micrograms.kg-1.day-1 for 15 weeks. Growth velocity was monitored every 3 weeks by measuring lower leg length and body weight. Spontaneous serum growth hormone (GH) concentrations and GH levels after insulin and L-dopa stimulation, as well as serum insulin-like growth factor I (IGF-I) and plasma thyrotropin, triiodothyronine and thyroxine, were measured during the study. Differences between animals were analyzed by repeated measures analysis of variance and Student's paired t-test. Mean +/- SEM growth velocity (mm/3 weeks) decreased from 0.90 +/- 0.08 during the baseline period to 0.29 +/- 0.07 (p < 0.001) during the period of growth retardation, and increased to 1.23 +/- 0.2 (p < 0.001) during the period of catch-up growth. Spontaneous GH and peak GH levels following insulin did not show any significant variation during the study. Peak GH during the L-dopa test decreased from 15.4 +/- 3.2 micrograms/l during the baseline period to 6.2 +/- 2.4 micrograms/l during the period of growth retardation (p < 0.05), and increased to 23.0 +/- 5.9 micrograms/l during the period of catch-up growth (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dietary fibre on faecal steroid measurements in baboons (Papio cynocephalus cynocephalus).

A study was conducted in captive baboons to determine (i) the impact of cereal dietary fibre on faecal progestogen excretion, and (ii) whether means of controlling dietary effects could be identified. Blood was collected on 3 days per week and faeces on 5 days per week from four unanesthetized cyclic female baboons, consecutively fed three diets of 5, 10 and 20% fibre for 90 days per diet. A 2 day lag time was detected before progesterone in the blood appeared in the faeces, regardless of diet (mean correlation was 0.62, P = 0.002). Increased dietary fibre had a negative effect on progestogen excretion (P < 0.004). Correspondence between blood and faecal progestogens was consistently greatest and the effect of dietary fibre least when faecal progestogens were expressed g-1 dry faeces. Several means of indexing faecal steroid excretion rates were examined including dehydroepiandrosterone (DHEA) and a number of byproducts of cholesterol metabolism. The cholesterol metabolite, cholestanone, was positively correlated with dietary fibre (r = 0.27; P < 0.04). Multiplying faecal progestogen concentration by the cholestanone g-1 dry faeces concentration increased the correlation between serum and cholestanone-indexed faecal progestogens (r = 0.78, P = 0.0001) compared with nonindexed progestogens (r = 0.71, P = 0.0001). We conclude that expressing faecal progestogens g-1 dry faeces may be sufficient and the most cost-effective method for controlling for most dietary effects when the objective is monitoring longitudinal endocrine status in baboons. However, it may be appropriate to express faecal progestogens by cholestanone concentrations when increased precision is needed to overcome the effects of profound variations in dietary fibre.

Metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the patas monkey: pharmacokinetics and characterization of glucuronide metabolites.

The metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined in the patas monkey, in order to provide further information about NNK metabolic pathways in primates. Female patas monkeys were given i.v. injections of [5-3H]NNK, and metabolites in serum and urine were analyzed by HPLC. Metabolism by alpha-hydroxylation of NNK was rapid and extensive, and the products of this pathway, 4-hydroxy-4-(3-pyridyl)butyric acid and 4-oxo-4-(3-pyridyl) butyric acid, accounted for a relatively large proportion of serum and urinary metabolites at all time points. This is significant because the formation of these products is associated with modification of DNA by NNK. The other major metabolic pathway was carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which detected both unconjugated and diastereomeric O-glucuronides. One of these glucuronides had been previously identified in rat urine, but the other diastereomer, which was the more prevalent of the two in serum and urine, had not been observed in studies of NNK metabolism in rodents. It was characterized by its spectral properties, by enzymatic hydrolysis to NNAL, and by derivatization of the released NNAL enantiomer with (R)-(+)-alpha-methylbenzylisocyanate. The two NNAL glucuronides accounted for 15-20% of the urinary metabolites in monkeys given 0.1 micrograms/kg NNK, which is similar to a smoker's dose, suggesting their use as dosimeters of NNK exposure in humans. Pharmacokinetic parameters were consistent with those observed in previous studies of nitrosamines, and varied predictably with body weight of five species. The results of this study have provided new insights relevant to assessing human metabolism of NNK.

Effects of pituitary-testicular axis suppression in utero and during the early neonatal period with a long-acting luteinizing hormone-releasing hormone analog on genital development, somatic growth, and bone density in male cynomolgus monkeys in the first 6 months of life.

The specific role of late fetal and early neonatal gonadotropins and/or sex steroids on genital development, linear growth, and bone mass accretion remains unclear. To investigate this, we attempted to selectively suppress pituitary-testicular activation from midgestation through early infancy with a long-acting LHRH agonist (LHRHA), D-Trp6,Pro9-NEt-LHRH, in microspheres. The agonist was injected sc on days 72-81 in utero, on day 1 of life, and 3 months postnatally in male cynomolgus monkeys. Control animals were treated with placebo. We then examined the consequences of such an intervention in the first 6 months of life. In the LHRHA-treated animals, marked suppression of plasma testosterone and gonadotropin levels were evident in the first 3 months of life compared to control values. The mean testicular volumes of the LHRHA group were significantly lower at birth and in the first 2 months of life than those of the placebo group (P less than 0.05). However, by 4 months of age, the mean testicular volumes of the two groups were comparable. Similarly, the mean stretched phallic lengths of the LHRH approximately A group were significantly lower than those of the placebo group throughout the first 6 months of life (P less than 0.05). By contrast, LHRHA treatment had no effect on somatic growth, as mean body weights, total body lengths, and trunk lengths of the two groups were similar over the first 6 months of life. Mean bone widths and densities of the distal third of the left radius and the left midfemur were similar in the two groups at 1 and 6 months of life. We conclude that pituitary-testicular axis suppression with a long-acting LHRHA in utero and during early infancy results in markedly stunted penile and testicular growth without affecting general somatic growth and bone density of appendicular cortical bone in the cynomolgus monkey in the first 6 months of life. Thus, an intact fetal and neonatal pituitary-testicular axis is critical for normal genital growth. However, the sex steroid requirement for maintenance of bone mineral content of appendicular cortical bone may be lower than that necessary for normal genital development.

Growth hormone (GH) responses to the hexapeptide GH-releasing peptide and GH-releasing hormone (GHRH) in the cynomolgus macaque: evidence for non-GHRH-mediated responses.

GH-releasing peptide (GHRP; His-D-Trp-Ala-Trp-D-Phe-Lys-NH2), a hexapeptide derived from enkephalin, has been shown to have GH-releasing activity in man and several animal species. To characterize the GHRP dose-response curve and compare it with that of GH-releasing hormone [GHRH-(1-44)NH2], six unanesthetized young adult cynomolgus macaques were tested with a range of iv doses of GHRP or GHRH in random order. Animals were fitted with vests and tethers. Blood samples were obtained before and at 15-min intervals after the administration of drugs. Doses ranged from 0.03-3 mg/kg for GHRP and from 1-30 micrograms/kg for GHRH. The dose-response curves for the two peptides were not parallel. GHRP had lower potency, but evoked a much higher peak GH response than GHRH (greater than 55 vs. 12 micrograms/L). Because one of the proposed mechanisms of action of GHRP is the inhibition of somatostatin (SS), we tested the effects of propranolol, which inhibits SS, on the GH responses to GHRH and GHRP. Propranolol was given at a dose of 14 micrograms/kg, iv, 10 min before the injection of saline, GHRH (10 micrograms/kg), or GHRP (1 mg/kg). GH responses to propranolol alone did not differ from those to placebo (peak, 6 +/- 2 vs. 8 +/- 2 micrograms/L). However, propranolol pretreatment doubled the GH responses to both GHRH and GHRP compared with those to GHRH or GHRP alone 28 +/- 5 micrograms/L vs. 14 +/- 5 (P less than 0.05) and 54 +/- 2 vs. 25 +/- 6 micrograms/L (P less than 0.001), respectively]. These results show that GHRP causes a potent dose-dependent release of GH in this primate species. Since GHRP can produce a greater maximal GH response than GHRH, mechanisms other than release of endogenous GHRH must be involved.

Structure-kinetic relationships of choriogonadotropin and related molecules.

To assess how profound differences in carbohydrate and/or polypeptide structures affect parameters of plasma disappearance of glycoprotein hormones, we calculated and compared the initial volume of distribution, rate constants, and metabolic clearance rates of several highly purified human choriogonadotropin (hCG) analogues in monkeys. hCG, deglycosylated hCG, desialylated hCG, or core fragment of hCG-beta purified from pregnancy urine (beta-core) was administered as a rapid intravenous injection to adult male cynomolgus monkeys (n = 3/group). The metabolic clearance rates of deglycosylated hCG, beta-core fragment, and desialylated hCG were increased 15-, 47-, and 152-fold, respectively, over that of hCG. Their corresponding initial volumes of distribution, however, remained essentially unchanged compared with that of hCG and approximated the estimated plasma volume. In contrast, the fast and slow rate constants of plasma disappearance of the hCG analogues were increased as much as 18- and 23-fold, respectively, relative to those of hCG. These studies of structure-kinetic relationships in primates show that major carbohydrate and polypeptide modifications of a glycoprotein hormone cause profound changes in the rate constants of the disappearance curves without changes in the initial volume of distribution.

Stimulation of testosterone production in the cynomolgus monkey in vivo by deglycosylated and desialylated human choriogonadotropin.

Modifications of carbohydrate structures of hCG, such as deglycosylation or desialylation, have been shown to reduce the biological activity of the hormone derivatives in vivo. We posed the question of whether deglycosylated hCG (dg-hCG) and desialylated hCG (ds-hCG) would behave as agonists at the LH/CG receptor in the primate in vivo, as this would bear on their potential clinical utility as LH/CG agonists or antagonists. Thus, we administered large doses (approximately 3 nmol) of highly purified dg-hCG, ds-hCG, hCG, or normal saline as a rapid iv injection to adult male cynomolgus monkeys (n = 3/group). Mean areas under the curves of plasma T over the first 6 h achieved with dg-hCG and ds-hCG were about 5-fold, significantly (P less than 0.05) greater than that in the saline controls and not significantly (P greater than 0.05) different from that in hCG-injected animals. Despite comparable plasma T responses in the first 6 h, mean plasma concentrations of ds-hCG, dg-hCG, and hCG differed dramatically among the groups. Plasma ds-hCG and dg-hCG levels were undetectable by 15 and 180 min, respectively, while the mean plasma hCG level was more than 2.10 nmol/L at 360 min. These data indicate that 1) dg-hCG is a full agonist at the LH/CG receptor in the primate in vivo, despite having minimal intrinsic activity in the rat Leydig cell adenyl cyclase assay and being able to near-completely antagonize hCG action therein; and 2) ds-hCG is a full agonist in the monkey in vivo, capable of stimulating a full testicular response over 6 h, despite being cleared from the circulation in 15 min. We conclude that the signal transduction system at the monkey LH/CG receptor is capable of achieving full steroidogenesis despite dramatically shortened exposure to stimulus or exposure to a stimulus with markedly reduced adenyl cyclase-stimulating activity in vitro.