Mouse H-2k-restricted cytotoxic T cells recognize antigenic determinants in both the HA1 and HA2 subunits of the influenza A/PR/8/34 hemagglutinin.

We have constructed two chimeric influenza hemagglutinin (HA) genes in which the HA1 and HA2 subunits of the HA molecule have been interchanged between influenza A/PR/8/34 (H1 subtype) and A/NT/60/68 (H3 subtype). These genes were used to construct recombinant vaccinia viruses that expressed intact chimeric HA. These recombinant viruses were used to test whether murine CTL recognize antigenic determinants in either the HA1, HA2, or both subunits. We found that both subunits of the HA molecule contain determinants for CTL. This implies that CTL have, at least in part, separate antigenic determinants from B lymphocytes, which recognize mainly epitopes within the HA1 subunit.

Dispersal of rabbit lung into individual viable cells: a new model for the study of lung metabolism.

This new enzymatic method disperses rabbit lung into morphologically intact, viable individual cells. The scattered cells constitute more than 50 percent of the original tissue. At least 90 percent of the cells exclude trypan blue from the nucleus. The dispersed lung cells consumed 6.2 microliters of oxygen per hour per milligram, dry weight. They incorporated [1-(14)C]palmitate into lecithin.

Primates.

Nonhuman primates demonstrate marked similarities to humans in almost all aspects of their anatomy, endocrinology, and physiology. These similarities underlie the value of these animals for appropriate studies in neurobiology, immunology, pathology, reproductive biology, teratology, neonatology, endocrinology, cardiology, and psychology. Investigations with nonhuman primates has made, and continues to make, significant contributions to biomedical and behavioral research. This review provides an overview of basic and applied studies for which primates are appropriate subjects and a summary of the advantages and problems of using nonhuman primates in research.

LMP2+ proteasomes are required for the presentation of specific antigens to cytotoxic T lymphocytes.

BACKGROUND: Major histocompatibility complex (MHC) class I molecules present short peptides generated by intracellular protein degradation to cytotoxic T lymphocytes (CTL). The multisubunit, non-lysosomal proteinases known as proteasomes have been implicated in the generation of these peptides. Two interferon-gamma (IFN-gamma)-inducible proteasome subunits, LMP2 and LMP7, are encoded within the MHC gene cluster in a region associated with antigen presentation. The incorporation of these LMP subunits into proteasomes may alter their activity so as to favour the generation of peptides able to bind to MHC class I molecules. It has been difficult, however, to demonstrate a specific requirement for LMP2 or LMP7 in the presentation of peptide epitopes to CTL. RESULTS: We describe a T-cell lymphoma, termed SP3, that displays a novel selective defect in MHC class I-restricted presentation of influenza virus antigens. Of the MHC-encoded genes implicated in the class I pathway, only LMP2 is underexpressed in SP3 cells. Expression of IFN-gamma in transfected SP3 cells simultaneously restores LMP2 expression and antigen presentation to CTL. Expression of antisense-LMP2 mRNA in these IFN-gamma-transfected cells selectively represses antigen recognition and the induction of surface class I MHC expression. Moreover, the expression of this antisense-LMP2 mRNA in L929 fibroblast cells, which constitutively express LMP2 and have no presentation defect, blocks the presentation of the same influenza virus antigens that SP3 cells are defective in presenting. CONCLUSIONS: Our results show that the LMP2 proteasome subunit can directly influence both MHC class I-restricted antigen presentation and class I surface expression.

Demonstration and characterization of estrogen receptor in chimpanzee sex skin: correlation between nuclear receptor levels and degree of swelling.

The presence of cytosol and nuclear estrogen receptors has been demonstrated in chimpanzee sex skin. Both receptors sedimented at approximately 4S in sucrose gradients containing 0.6 M KCl. Both had a steroid specificity and high affinity for estrogen that conform to an estrogen receptor. The absence of such receptors in the pigmented skin surrounding the sex skin and in the abdominal skin indicates their discrete localization in the sex skin. While the cytosol receptor remained low (less than 10 fmol/mg cytosol protein), the nuclear estrogen receptor showed a fluctuation during the menstrual cycle, attaining the highest level during the late follicular phase (90.4 +/- 8.4 fmol/mg nuclear extract protein; nearly 3-4 times the level during the early follicular and luteal phases). When ovariectomized animals were treated with mestranol, the concentration of nuclear estrogen receptor increased from below detection to 74 fmol/mg nuclear extract protein. An increase in serum progesterone during the luteal phase, despite concurrently elevated serum estradiol levels, was associated with a reduction of the nuclear estrogen receptor as was the administration of progesterone and mestranol to estrogen-primed ovariectomized animals. Changes in the concentration of the nuclear estrogen receptor were positively correlated with changes in the degree of the sexual swelling. These results strongly suggest that estrogen controls the sexual swelling by acting through specific receptors in the sex skin, and that counteraction by progesterone of estrogen stimulation of the sexual swelling is effected through a reduction in nuclear estrogen receptors.

Metabolism of 1 alpha-hydroxyvitamin D2 to activated dihydroxyvitamin D 2 metabolites decreases endogenous 1 alpha, 25-dihydroxyvitamin D 3 in rats and monkeys.

The vitamin D analog 1 alpha-hydroxyvitamin D2 (1 alpha-OHD2) is under development for the treatment of secondary hyperparathyroidism and metabolic bone disease. This analog is metabolized in vivo to the natural active dihydroxylated metabolite of vitamin D2, 1 alpha,25-dihydroxyvitamin D2 [1 alpha,25-(OH)2D2]. To study the metabolism of this analog, an assay involving HPLC separation and purification of metabolites followed by RRA with the vitamin D receptor was developed to quantitate the active metabolites of the analog and the endogenous active metabolite of vitamin D3, 1 alpha,25-(OH)2D3, from the same blood sample. This assay was used to determine blood levels of active dihydroxylated vitamin D compounds in rats and monkeys treated with oral 1 alpha-OHD2. As the circulating 1 alpha,25-(OH)2D2 level increased dose dependently in these rats and monkeys, a concomitant decrease in the endogenous 1 alpha,25-(OH)2D3 was observed. In rats orally administered more than 2.5 micrograms 1 alpha-OHD2/kg.day, a second active metabolite of 1 alpha-OHD2, 1 alpha,24-(OH)2D2, was detected in concentrations similar to those of 1 alpha,25-(OH)2D2. These results indicate that the regulatory control of endogenous vitamin D metabolism as well as analog metabolism must be considered when assessing the therapeutic potential of a vitamin D analog.

A potential primate model for bone loss resulting from medical oophorectomy or menopause.

This study examined the potential use of the GnRH agonist-treated female monkey as a model for bone loss after medical oophorectomy or the onset of menopause in women. Three female rhesus monkeys (13-16 yr of age) were treated continuously for 10 months with 25 micrograms/day GnRH agonist using osmotic minipumps. All three animals exhibited normal menstrual cycles before treatment. Within 5 weeks of the beginning of GnRH agonist treatment, serum progesterone and estradiol concentrations had fallen to low values and did not rise significantly during the remaining treatment period. The decline in ovarian steroidogenesis was correlated with a reduction in bone mineral density (BMD; bone mineral content/bone width) of the caudal vertebrae and humerus. The reduction of BMD of the caudal vertebrae occurred gradually. The downward trend was evident during the first 3 treatment months, but did not fall significantly below pretreatment levels until 9 months of GnRN agonist treatment. The overall decline in BMD for the caudal vertebrae was approximately 14% after 9 months of GnRH agonist treatment. The measured decline in BMD of the humorous was 11%. Serum osteocalcin levels rose more than 10-fold above pretreatment values between 4 and 7 months of GnRH agonist treatment before declining to levels that approached pretreatment concentrations between 8 and 10 months of treatment. Menstrual cycles were reinitiated within 4 weeks after the termination of treatment, as shown by luteal phase increases in serum progesterone concentrations. BMD of the humerus and caudal vertebrae remained subnormal 2 months posttreatment, but by 5 months had recovered to near-pretreatment values. These data suggest that ovarian hormone deprivation induced by GnRH agonist administration is associated with a decline in BMD in female monkeys, and that this animal model may be an excellent model for postmenopausal bone loss or bone reduction resulting from medical oophorectomy. The GnRH agonist-treated monkey also has the potential to be developed as a model for type I postmenopausal osteoporosis.

Blockade of neonatal activation of the pituitary-testicular axis: effect on peripubertal luteinizing hormone and testosterone secretion and on testicular development in male monkeys.

The objective of this study was to examine the effect of blockade of neonatal activation of the pituitary-testicular axis, using a GnRH agonist, on sexual development in male rhesus monkeys. Monkeys were treated with either a GnRH agonist (10 micrograms/day; n = 8) or vehicle (n = 9) for 112 days using osmotic minipumps beginning at 10-13 days of age. In control monkeys serum LH and testosterone concentrations during the first 3 postnatal months were similar to those in adults; they then declined to very low levels. GnRH agonist administration caused an immediate and precipitous decline in serum LH and testosterone concentrations to very low levels, and both remained low throughout the rest of the agonist administration period. Neither group had any significant elevation in serum LH or testosterone concentrations during the next 2 yr. In the control monkeys serum LH and testosterone began to rise during the third year, with a rapid increase occurring during the fall coincident with the breeding season. This peripubertal rise of LH and testosterone secretion was associated with rapid enlargement of the testes and the appearance of sperm in ejaculates. The monkeys who had received GnRH agonist had subnormal serum LH and testosterone increases, and testicular enlargement was also attenuated compared to that in the control animals during the third year of life. Semen samples were recovered from only 50% of the GnRH agonist-treated monkeys during this period, and the sperm count per ejaculate was suppressed. The serum LH responses of the GnRH agonist-treated monkeys to an iv bolus dose of GnRH (5 micrograms/kg BW) during the third year were normal. These results suggest that the induction of reversible hypogonadotropin-hypogonadism in neonatal male monkeys alters subsequent testicular development and peripubertal endocrine changes. Thus, neonatal activation of the pituitary-testicular axis may be a critical developmental event in the process of sexual development in male primates.

Influence of continuous gonadotropin-releasing hormone (GnRH) agonist treatment on luteinizing hormone and testosterone secretion, the response to GnRH, and the testicular response to human chorionic gonadotropin in male rhesus monkeys.

Five adult male rhesus monkeys were continuously infused for 56 days with 25 micrograms/day of GnRH agonist (Wy-40972; Ag) using an implanted osmotic pump. Bioassayable serum levels of LH were elevated 8-fold on the second day of Ag treatment and then declined precipitously to below pretreatment levels by day 15. Serum levels of testosterone (T) changed similarly during Ag treatment, except that the fall in serum LH levels preceded the decline in serum levels of T by at least 2 days. Ag administration also eliminated the diurnal variation in serum LH and T. GnRH administration (50 micrograms) induced a 13- to 20-fold rise in serum LH and a 3- to 7-fold increase in serum T in control monkeys. After 4 weeks of Ag administration , none of the animals responded to GnRH. Both control and experimental monkeys had a rise in serum T in response to hCG after 7 weeks of Ag treatment. Basal levels of LH and T returned to normal by 12 days posttreatment, and the serum LH and T responses to GnRH were normal 19 days posttreatment. These results indicate that 1) continuous administration of Ag is an effective method of inducing antiferility effects in male rhesus monkeys; 2) pituitary desensitization is a major factor involved in Ag-induced gonadal dysfunction in this species; and 3) the method of administration may be the critical factor in determining the effectiveness of GnRH agonists.

Treatment of endometriosis in monkeys: effectiveness of continuous infusion of a gonadotropin-releasing hormone agonist compared to treatment with a progestational steroid.

The use of a GnRH agonist or a progestational steroid (levonorgestrel) for the treatment of endometriosis in monkeys was compared. Four monkeys with spontaneous endometriosis were treated for 6 months with a continuous infusion of a GnRH agonist (25 micrograms/day). Five animals with surgically induced endometriosis were treated with the same agonist for 3 months. An additional group of five monkeys with surgically induced endometriosis was treated orally with levonorgestrel (1 mg/kg X day), while a final group of four monkeys served as untreated controls. During agonist treatment, the four monkeys with spontaneous endometriosis gained body weight and had a greater than 80% decline in cyst size (representing a decline in secretory activity). Monkeys with surgically induced endometriosis had almost total resolution of endometrial lesions during agonist treatment, which was maintained throughout a 4-month posttreatment period. After initial stimulation at the onset of the GnRH agonist infusion, serum LH, FSH, estradiol, and progesterone levels decreased to near the levels of detection, where they remained until treatment was terminated. In comparison, levonorgestrel reduced endometrial lesion size, but the monkeys did not resume normal cycles as early as those treated with the agonist. Levonorgestrel-treated monkeys had normal serum LH and FSH levels, but low serum estradiol and progesterone levels. The results of this study indicate that either continuous infusion of a GnRH agonist or administration of levonorgestrel is effective for treating endometriosis in monkeys. The hormonal data suggest that the GnRH agonist acts at the level of the hypothalamus and pituitary, whereas levonorgestrel acts at the ovarian level.

Effect of diet on oxidation of 17 beta-estradiol in vivo.

The effect of a high fat, low carbohydrate, low protein diet on the in vivo oxidation of 17 beta-estradiol was studied using radiometric methods. Five male chimpanzees were fed a normal (13%) fat diet or a high (65%) fat diet for 8 weeks. After a 4-week rest period, the animals were fed the alternative diet. The mean percent oxidation of 16 alpha-[3H]estradiol-17 beta 24 h after injection was 3.8 +/- 1.3% (+/- SD) on the normal diet vs. 18.4 +/- 4.7% on the high fat diet (P less than 0.01). In contrast, the mean percent oxidation of 2-[3H]estradiol 24 h after injection was 31.6 +/- 3.8% (+/- SD) on the normal diet vs. 20.0 +/- 3.5% on the high fat diet (P less than 0.05). These results suggest that the oxidation of 17 beta-estradiol to estriols relative to that to catechol estrogens is increased by a high fat diet.

Influence of simultaneous gonadotropin-releasing hormone agonist and testosterone treatment on spermatogenesis and potential fertilizing capacity in male monkeys.

We examined the effect of continuous sc infusion of a GnRH agonist (Ag) and testosterone (T) supplementation on spermatogenesis and the potential fertilizing capacity of sperm in 15 rhesus monkeys. The monkeys were divided into 3 groups of 5 animals each. Groups 1 and 2 received 25 micrograms/day Ag for 44 weeks. Group 2 also received T replacement therapy [sufficient to maintain serum T values within the normal range; 4-5 ng/mL (13.9-17.3 nmol/L)]. Group 3 received a low dose of the Ag (5 micrograms/day for 24 weeks and then 10 micrograms/day for 20 weeks) to prolong the oligospermic phase before the onset of azoospermia. In groups 1 and 3, there was an initial transient (1 week) rise in serum LH and T levels which then fell below pretreatment value where they remained throughout the treatment period. The serum LH and T levels were lower in the group treated with 25 micrograms/day Ag. Similar changes in serum LH levels occurred in group 2, but T supplementation maintained serum T in the physiological range. The decline in serum LH levels was associated with reduced sensitivity to GnRH, since the responses of serum LH and T to GnRH were either abolished or greatly reduced in the 2 groups treated with 25 micrograms/day Ag and were less than 50% of the pre-Ag responses in monkeys treated with 5-10 micrograms/day Ag. Four of five monkeys treated with 25 micrograms/day Ag alone became azoospermic within 21 weeks. All five animals receiving Ag and T supplementation became azoospermic (mean time to onset, 12.6 weeks). Four of five monkeys treated with 5-10 micrograms/day Ag also had azoospermic ejaculates during the late treatment and early recovery period. Sperm counts recovered to the pretreatment levels in most monkeys by 10 weeks of the recovery period. The quality of semen samples taken from oligospermic monkeys was greatly reduced. The percentage of motile and percentage of live sperm per ejaculate, the net negative surface charge on sperm, and the scores of sperm in the hamster oocyte penetration test were subnormal. T supplementation did not improve these measures of semen quality. Testicular biopsies taken at the end of the Ag administration period from monkeys given 25 micrograms/day Ag showed diffuse atrophy of the seminiferous tubules, which contained primarily Sertoli cells and a few spermatogonia and spermatocytes, but no spermatids. The tubular atrophy and the suppression of spermatogenesis did not appear to be influenced by T replacement.(ABSTRACT TRUNCATED AT 400 WORDS)

Preservation of bone mass in hypogonadal female monkeys with recombinant human growth hormone administration.

This study examined the effect of human recombinant GH supplementation on bone loss in female monkeys made hypogonadal with GnRH agonist (GnRH-Ag). Animals were randomly assigned to three treatment groups: vehicle, GnRH-Ag, and GnRH-Ag and GH. After an initial 5-month pretreatment period during which all animals were maintained on a normal monkey chow diet containing a high level of calcium (1%) animals were maintained on a normal monkey chow diet containing a high level of calcium (1%), animals were shifted to a lower calcium diet (0.1%) for 4 to 5 months before the beginning of treatment and were maintained on this diet throughout the remainder of the study. Monkeys were treated continuously for 10 months with 25 micrograms/day GnRH-Ag or vehicle. GH was administered by im injection three times per week at a dose of 100 micrograms/kg body wt/day. Animals treated with GnRH-Ag were amenorrheic throughout the treatment period, and serum estradiol and progesterone levels were below minimum levels of detection. Vehicle-treated animals continued to cycle throughout the study. Monkeys treated with GnRH-Ag alone showed a significant decline (12%) in bone mineral density (BMD) of the lumbar spine. BMD was reduced below pretreatment levels from 6 months of GnRH-Ag treatment through 3 months post treatment in this group. GH supplementation reduced the decline of BMD in GnRH-Ag-treated monkeys. BMD did not change significantly with time in the GH-supplemented group. BMD values in GH supplemented animals between 5 and 10 months of the treatment period exceeded levels in animals treated with GnRH-Ag alone, but BMD levels during this interval were lower than in the vehicle-treated group. In the vehicle-treated group, there was small, but significant, increase in BMD over the course of the study. Serum osteocalcin concentrations were elevated above pretreatment values after 6 and 9 months of GnRH-Ag treatment alone or with GH supplementation, but did not change in vehicle-treated animals. GH also increased serum insulin-like growth factor 1 levels. In response to the lower calcium diet, serum PTH levels increased approximately 200% in vehicle-treated monkeys and animals treated with GnRH-Ag alone. GH attenuated this increase in serum PTH. The data indicate that the level of calcium in the diet of adult monkeys can be reduced more than 10-fold without affecting lumbar BMD provided ovarian function is normal, but if animals are made hypogonadal with a GnRH-Ag, bone mass declines.(ABSTRACT TRUNCATED AT 400 WORDS)

Neonatal treatment of male monkeys with a gonadotropin-releasing hormone agonist alters differentiation of central nervous system centers that regulate sexual and skeletal development.

Male rhesus monkeys treated continuously with a GnRH agonist for the first 4 months of postnatal life exhibited a delay in the onset of puberty and an attenuated peripubertal rise in testosterone (T) secretion. The objectives of the current study were to determine whether these effects on sexual development were permanent and whether the hypothalamic-pituitary-testicular axis was functioning normally in these animals as adults. Neonatal GnRH agonist treatment delays but does not permanently block sexual maturation in male monkeys. Treated animals that did not show a pubertal rise in serum T during the breeding season of their 4th year exhibited a seasonal but subnormal elevation of serum T during the subsequent breeding season. Growth of the skeleton was diminished as evidenced by shorter adult crown-rump, tibia, and femur length and reduced bone mineral density of the humerus and lumbar spine. The magnitude of the serum LH and T response to iv pulses of GnRH [50 ng/kg body weight (BW)] and naloxone (1 mg/kg BW) did not differ between control and treated animals during the nonbreeding or breeding season at 6 yr of age. Conversely, treated animals showed a subnormal serum LH and T response to N-methyl-D,L-aspartic acid (5 mg/kg BW iv) during the nonbreeding season. These data suggest that adult monkeys treated neonatally with a GnRH agonist exhibit subnormal sensitivity of the central nervous system to one or more excitatory amino acids (e.g. aspartate or glutamate). Thus, abolishing neonatal activation of the pituitary-testicular axis with a GnRH agonist may permanently alter differentiation of central nervous system centers that are either involved in GnRH secretion or govern this process.

Inhibin-B in the male rhesus monkey: impact of neonatal gonadotropin-releasing hormone antagonist treatment and sexual development.

We examined the effect of reversibly suppressing pituitary-testicular function during the neonatal period on developmental changes in inhibin-B and FSH secretion in male rhesus monkeys. Infants were treated with either vehicle, a GnRH antagonist (Ant) or the Ant and androgen (Ant/And) for the first 4 postnatal months, and the effects on serum inhibin-B and FSH were monitored during the neonatal and peripubertal periods. In neonates, Ant or Ant/And treatment lowered both serum FSH and inhibin-B levels. By 12 months of age, inhibin-B concentrations no longer differed across treatment groups. A major increase in inhibin-B occurred between 27-36 months of age (late prepubertal period) in all groups, but levels were lower at 33 and 36 months of age in Ant/And-treated animals than in controls. These differences most likely were related to fewer Ant/And-treated animals achieving sexual maturity during their fourth year of life. Regardless of treatment, inhibin-B levels were higher in those that were destined to become mature (in year 4) than in those that were not. During the late prepubertal period, serum inhibin-B was positively correlated with age and testicular volume, but not with serum LH or testosterone. After this period (39-52 months of age), inhibin-B no longer correlated with these parameters. FSH levels were near or below detection limits in most peripubertal animals, but FSH was detectable in fewer samples from control than treated animals. The data suggest that inhibin-B secretion in the neonate is driven by gonadotropin secretion, but during the juvenile hiatus in gonadotropin secretion, the monkey testis continues to produce substantial amounts of this hormone.

Neonatal treatment with luteinizing hormone-releasing hormone analogs alters peripheral lymphocyte subsets and cellular and humorally mediated immune responses in juvenile and adult male monkeys.

We examined the effect of treatment with a LH-releasing hormone (LHRH) agonist (Ag), antagonist (Ant), or Ant and androgen (Ant/And) for the first 4 months of postnatal life on lymphocyte subsets and cellular and humorally mediated immune responses in juvenile and adult male monkeys. We also determined the effect of 9 weeks of Ant treatment on lymphocyte subsets in adult male monkeys. Adult male monkeys that had been treated neonatally with an Ag had increased levels of CD8-positive (CD8+) T-cells and reduced levels of B-cells compared to vehicle-treated controls. Lymphocytes from these animals also showed an elevated proliferative response to a variety of mitogens compared to cells from control animals. Antibody production in response to tetanus toxoid was normal in treated animals. Other neonates treated with Ant/And exhibited subnormal levels of lymphocytes, CD8+ T-cells, and B-cells at 4 months of age. Similar changes, but of lesser magnitude, were observed in animals treated with Ant alone. At 6 months of age, lymphocytes from both groups of Ant-treated monkeys exhibited an above normal proliferative response to streptolysin-O, but not to other mitogens. At 18 months of age, animals treated with Ant alone produced more antitetanus antibody in response to a tetanus toxoid booster than the controls or Ant/And-treated animals. Ant treatment was without major effect on lymphocyte subsets in adult monkeys. Serum LH and testosterone levels declined, and there was a small but significant increase in B-cells, lymphocytes expressing the interleukin-2 receptor, and the CD4+/CD8+ T-cell ratio during treatment, but these parameters normalized during the posttreatment period. The data suggest that chronic neonatal treatment with an Ag or Ant alters the development of immune system responses in male primates. The significance of these changes and their impact on the ability of these animals to respond to pathogenic agents is under investigation.

Effects of age and sex on bone density in the rhesus monkey.

Normative data for bone density of cortical and trabecular bone in the rhesus monkeys is described in the present study. Changes of bone density (g/cm2) for the humerus, the third lumbar vertebra, and the eighth caudal vertebra of the rhesus monkey show differences due to age and sex of the subjects (males n = 57; females n = 49). In general, bone density increased with age and then reached a plateau at approximately 3 to 4 years in all bones measured. In the humerus, older females (greater than 30 years) had a significantly lower bone density than females of 4 to 24 years, while bone density in older males did not decrease. In the vertebrae, some evidence of advanced age-related decreases in bone density was found in both sexes. These results indicate that the rhesus monkey shows a natural pattern of change in bone mineralization which parallels that seen in humans. The physiological similarity between the rhesus monkey and human further suggests a potential role for this species in the future investigation of osteoporosis.

Effect of neonatal treatment with a gonadotropin releasing hormone antagonist on developmental changes in circulating lymphocyte subsets: a longitudinal study in male rhesus monkeys.

We have examined changes in circulating lymphocyte subsets from the neonatal period until adulthood (4 months until 5.5 years of age) in male rhesus monkeys, and the impact of neonatal treatment with a GnRH antagonist (Ant) or Ant and androgen (Ant/And) on these parameters. Absolute numbers of lymphocytes, B cells, total T lymphocytes, and CD4+ T cells decreased, neutrophils increased, and CD8+ T cells did not change with age. WBC counts increased between 4 mo and 2 years of age and then fell to neonatal levels over the next two years. The decline of CD4 + T cells in association with stable CD8+ T cell levels resulted in an age-related decrease in the CD4+/CD8+ T cell ratio. At 4 months of age, WBC's, lymphocytes, total T cells, CD8+ T cells and B cells were lower in Ant- and Ant/And-treated animals compared to controls. With the exception of WBC counts, these values had normalized by 2 years of age. Reduced WBC levels in treated animals persisted through adulthood. CD4+ T cell levels tended to be lower in Ant-treated and higher in Ant/And-treated animals than in controls at 4 months of age. CD4+ T cells remained lower in Ant- than in Ant/And-treated animals at most ages. The higher CD4 + T cell counts in Ant/And-treated animals resulted in an elevated CD4 + /CD8 + T cell ratio that persisted until the onset of year 5. During years 5 and 6, seasonal fluctuations in WBC's and neutrophils were observed with counts being higher in the breeding (fall) than in the nonbreeding (summer) season. The data document that developmental changes in circulating immune cells in the rhesus monkey are qualitatively similar to those reported in humans, and provide further evidence that neonatal treatment of male rhesus monkeys with Ant or Ant/And may alter early programming of the immune system.

Sexual maturation in male rhesus monkeys: importance of neonatal testosterone exposure and social rank.

In a 5-year longitudinal study, we examined the effect of disrupting the neonatal activity of the pituitary-testicular axis on the sexual development of male rhesus monkeys. Animals in a social group under natural lighting conditions were treated with a GnRH antagonist (antide), antide and androgen, or both vehicles, from birth until 4 months of age. In antide-treated neonates, serum LH and testosterone were near or below the limits of detection throughout the neonatal period. Antide + androgen-treated neonates had subnormal serum LH, but above normal testosterone concentrations during the treatment period. From 6 to 36 months of age, serum LH and testosterone were near or below the limits of detection. Ten of 12 control animals reached puberty during the breeding season of their 4th year, compared with five of 10 antide- and three of eight antide + androgen-treated animals. Although matriline rank was balanced across treatment groups at birth, a disruption within the social group during year 2 resulted in a marginally lower social ranking of the two treated groups compared with the controls. More high (78%) than low (22%) ranking animals reached puberty during year 4. During the breeding season of that year, serum LH, testosterone and testicular volume were positively correlated with social rank. Thus the lower social rank of treated animals may have contributed to the subnormal numbers of these animals reaching puberty during year 4. However, of those animals achieving puberty during year 4, the pattern of peripubertal changes in serum testosterone and testicular volume differed between control and antide-treated animals. The results appear to suggest that the disruption of normal activity of the neonatal pituitary--testicular axis retarded sexual development, but that social rank is a key regulatory factor in setting the timing of sexual maturation in male rhesus monkeys. The effect of neonatal treatment with antide and low social rank on sexual development could not be reversed by neonatal exposure to greater than normal concentrations of androgen.

Leptin and thyroxine during sexual development in male monkeys: effect of neonatal gonadotropin-releasing hormone antagonist treatment and delayed puberty on the developmental pattern of leptin and thyroxine secretion.

OBJECTIVE: Neonatal treatment of male monkeys with a gonadotropin-releasing hormone antagonist (Ant) increased the incidence of delayed puberty. Using blood samples that had been collected from monkeys with normal or delayed puberty, we assessed the potential involvement of leptin and thyroxine (T4) in sexual development. DESIGN AND METHODS: Monkeys were treated from birth until 4 months of age with vehicle, Ant or Ant/androgen and blood samples were drawn from 10 to 62 months of age. RESULTS: Serum leptin and total T4 concentrations declined in parallel throughout adolescence in all treatment groups. There was no transient rise in leptin before or in association with the onset of puberty. Also, leptin did not differ during the peripubertal period between animals experiencing puberty at that time versus those in which puberty was being delayed. Neonates treated with Ant either alone or with androgen replacement had higher leptin levels than controls throughout development. While leptin exhibited no significant changes during the peripubertal period, T4 values increased and declined in parallel with the peripubertal changes in hypothalamic-pituitary-testicular activity. CONCLUSIONS: These data do not support the concept that a transient rise in leptin triggers the onset of puberty in male monkeys. However, the disruption of neonatal activity of the pituitary-testicular axis alters the developmental pattern of leptin. The changes in T4 levels during the peripubertal period suggest that thyroid status may be a significant contributor to the process of sexual development in the male monkey and that peripubertal changes in secretion of this hormone may serve as an effective physiological response during a critical period of elevated energy expenditure.